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WO2021173865A2 - Compounds for detection of viral pathogens - Google Patents

Compounds for detection of viral pathogens Download PDF

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Publication number
WO2021173865A2
WO2021173865A2 PCT/US2021/019720 US2021019720W WO2021173865A2 WO 2021173865 A2 WO2021173865 A2 WO 2021173865A2 US 2021019720 W US2021019720 W US 2021019720W WO 2021173865 A2 WO2021173865 A2 WO 2021173865A2
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WO
WIPO (PCT)
Prior art keywords
biosensor
sample
bond
group
amino acid
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PCT/US2021/019720
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French (fr)
Other versions
WO2021173865A3 (en
Inventor
Satish C. AGRAWAL
Prakash Rai
Michael P. Filosa
Peter Tzuyu WEN
Robb J. Osinski
Michael G. Horner
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Bambu Vault Llc
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Publication of WO2021173865A2 publication Critical patent/WO2021173865A2/en
Publication of WO2021173865A3 publication Critical patent/WO2021173865A3/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus

Definitions

  • the present invention relates to a novel compounds and methods for detecting pathogens in a subject and on surfaces.
  • Rationally designed libraries are devised for the detection of viruses using screening tools that are well established in the field.
  • a system for detecting SARS-CoV pathogen in a sample is disclosed.
  • 2019-nCoV also known as COVID-19
  • SARS-CoV-2 coronavirus disease caused by 2019-nCoV
  • SARS-CoV-2 The Center for Disease Control and Prevention (CDC) of United States believes that symptoms of SARS-CoV-2 may appear in as few as 2 days or up to 14 days after exposure. This is based on what has previously been seen as the incubation period of MERS viruses.
  • Common human coronaviruses including types 229E, NL63, OC43, and HKU1, usually cause mild to moderate upper-respiratory tract illnesses, like the common cold. Most people get infected with these viruses at some point in their lives. These illnesses usually only last for a short amount of time. Symptoms may include runny nose, headache, cough, sore throat, fever and a general feeling of being unwell.
  • MERS-CoV and SARS-CoV-1 Two other human coronaviruses, MERS-CoV and SARS-CoV-1 have been known to frequently cause severe symptoms. MERS symptoms usually include fever, cough, and shortness of breath which often progress to pneumonia. About 3 or 4 out of every 10 patients reported with MERS have died. MERS cases continue to occur, primarily in the Arabian Peninsula. SARS symptoms often included fever, chills, and body aches which usually progressed to pneumonia. No human cases of SARS-CoV-1 have been reported anywhere in the world since 2004. Symptoms of SARS-CoV-2 are similar to those of other coronaviruses.
  • the CDC released a test kit, called the Centers for Disease Control and Prevention (CDC) 2019-Novel Coronavirus (SARS-CoV-2) Real-Time Reverse Transcriptase (RT)-PCR Diagnostic Panel (CDC SARS-CoV-2 Real Time RT-PCR), which is designed for use with an existing RT-PCR testing instrument that is commonly used to test for seasonal influenza.
  • the test is intended for use with upper and lower respiratory specimens collected from people who meet the CDC criteria for SARS-CoV-2 testing.
  • the test uses a technology that can provide results in four hours from initial sample processing to result.
  • Many PCR-based test kits have received emergency use authorization (EUA) from the FDA and provide results in a few hours.
  • EUA emergency use authorization
  • HAI hospital-acquired infections
  • CAI community-acquired infections
  • HCWs healthcare workers
  • both the patient and the HCW need to be protected from contracting or transmitting hospital-acquired infections.
  • a quick and inexpensive way to identify hospital beds, equipment’s instruments, health care workers, patients and/or even visitors that may be carrying pathogens like the coronavirus would be highly beneficial in stopping their spread.
  • Fig. 1 shows the chemical structure of a FRET biosensor described in this disclosure.
  • Fig. 2 is a graph showing comparison of cross-reactivity with PLpro from SARS-CoV-2 and enzymes from other sources.
  • FIG. 3 schematically illustrates the components of the SARS-CoV pathogen detection system operably connected as described in this disclosure.
  • Fig. 4 shows the structure of (N-Boc-LRGG-N)2-rhodamine 110, a biosensor described in this disclosure.
  • Fig. 5 shows kinetic scan data from a 96-well plate showing enzyme reactivity with substrate for (i) negative control, (ii) positive control, and (iii) positive and negative patient samples.
  • Fig. 6a shows kinetic scan data of a negative control sample from Well A1 in Fig. 5. This data demonstrates a stable, average signal of 143 RFU with a maximum random oscillation of about 12 RFU.
  • Fig. 6b shows kinetic scan data of a PCR-confirmed negative clinical sample from Well A8 in Fig. 5. This data demonstrates a stable, average signal of 180 RFU with a maximum random oscillation of about 13 RFU.
  • Fig. 6c shows kinetic scan data from a PCR-confirmed positive clinical sample from Well E5 in Fig. 5. A strong signal is observed that starts at 500 RFU and rises to 2500 RFU over the course of 1 hour. The fluorescence continues to increase for hours (data not shown).
  • Fig. 6d shows kinetic scan data from a PCR-confirmed positive clinical sample from Well E3 in Fig XI . While the fluorescence from this sample was less than that in Fig. 6c, it was high enough to clearly observe the kinetic differences from the responses of the negative control and negative clinical sample. The maximum signal reached was 525 RFU from a starting value of about 250 RFU.
  • Fig. 7 show end point scan data for a set of control and clinical samples.
  • Fig. 8a shows a 96-well plate with samples that have been incubated with Z-RLRGG- AMC substrate (top four rows) and with (NFh-LRGG ⁇ Rhodamine (bottom four rows) when illuminated with a “black light” flashlight emitting at 395 nm.
  • Fig 8b shows visible color development in samples shown in Fig. 4a. Color development could only be observed in the samples containing the Rhodamine product, since the AMC product has very low visible absorbance.
  • Fig. 9a shows 1 hour end point emission spectra of positive and negative samples treated with DABCYL-KVRLQSK-DANSYL, showing the fluorescence of the free DANSYL in positive samples.
  • Fig. 9b shows visible fluorescence of samples treated with DABCYL-KVRLQSK- DANSYL in comparison to those treated with the PLpro substrate, (NH2-LRGG)2Rhodamine. In both cases, positive samples were clearly differentiated from negative samples that showed no fluorescence.
  • Fig. 10a shows the effect of a PLpro inhibitor on the kinetic curve for fluorescence generated by enzyme reaction with the Z-RLRGG-AMC substrate.
  • Fig. 10b shows PLpro inhibition at higher concentrations of the inhibitor.
  • Fig. 10c shows PLpro inhibition in a clinical sample. Note the higher concentration of inhibitor required as compared to Fig. 10b.
  • Fig. 11 is a photograph of a preferred configuration of a tongue scraper.
  • Fig. 12a schematically illustrates a simple fluorogenic peptide substrate.
  • Fig. 12b schematically illustrates an internally quenched fluorogenic peptide substrate.
  • This disclosure provides novel biosensors that can detect specific viruses like coronaviruses. This disclosure also describes formulations and compositions using the biosensors, and well as methods for detecting infection by viruses using the biosensors.
  • biosensors may comprise unnatural amino acids, including the D-enantiomers of the naturally occurring L-forms, and non-proteinogenic amino acids listed in Table 2.
  • Unnatural amino acids may also include modifications of natural amino acids following protein biosynthesis.
  • Post-translational modification may include chemical changes such as phosphorylation, carbonylation, glycosylation, acylation, notrosylation, and other similar reactions.
  • Ac refers to an acetyl (CH3C(0)-) group
  • Sue refers to a succinyl (H0C(0)CH2CH2C(0)-) group
  • Cbz also referred to herein as “Z” refers to a carboxybenzoyl group
  • Fmoc refers to a fluorenylmethoxycarbonyl group
  • Boc refers to a tert-butyloxycarbonyl group
  • alkyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, generally having from one to ten carbon atoms (e.g., (Cl-lO)alkyl or Cl-10 alkyl).
  • a numerical range such as “1 to 10” refers to each integer in the given range - e.g., “1 to 10 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms, although the definition is also intended to cover the occurrence of the term “alkyl” where no numerical range is specifically designated.
  • alkyl examples include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl, /c/V-butyl, isopentyl, and n-pentyl, neopentyl, hexyl, septyl, octyl, nonyl and decyl.
  • the alkyl moiety may be attached to the rest of the molecule by a single bond, such as for example, methyl (Me), ethyl (Et), n-propyl (Pr), 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl) and 3-methylhexyl.
  • an alkyl group is optionally substituted by one or more of substituents which are independently heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -ORa, -SRa, -N(Ra)2, where each Ra is independently hydrogen, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.
  • substituents which are independently heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy
  • aryl refers to a benzene ring or to a fused benzene ring system, for example anthracene, phenanthrene, or naphthalene ring systems.
  • aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl, indenyl, azulenyl, fluorenyl, anthracenyl, phenanthrenyl, tetrahydronaphthyl, indanyl, phenanthridinyl and the like.
  • aryl also includes each possible positional isomer of an aromatic hydrocarbon radical, such as in 1 -naphthyl, 2-naphthyl, 5-tetrahydronaphthyl, 6- tetrahydronaphthyl, 1 -phenanthridinyl, 2-phenanthridinyl, 3 -phenanthridinyl, 4-phenanthridinyl, 7-phenanthridinyl, 8-phenanthridinyl, 9-phenanthridinyl and 10-phenanthridinyl and the like.
  • One preferred aryl group is phenyl.
  • halogen refers to fluorine, chlorine, bromine, or iodine.
  • haloalkyl refers to an alkyl group, as defined herein that is substituted with at least one halogen.
  • branched or straight chained “haloalkyl” groups useful in the present invention include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, and t-butyl substituted independently with one or more halogens, e.g., fluoro, chloro, bromo, and iodo.
  • haloalkyl should be interpreted to include such substituents such as -CF3, -CH2-CH2-F, -CH2-CF3, and the like.
  • alkoxy refers to a group -ORa, where Ra is alkyl as herein defined.
  • cyano refers to a group -CN.
  • Esters of the compounds of the present invention are independently selected from the group of (1) carboxylic acid esters obtained by esterification of the hydroxy groups, in which the non-carbonyl moiety of the carboxylic acid portion of the ester grouping is selected from straight or branched chain alkyl (for example, ethyl, n-propyl, t-butyl, or n-butyl), alkoxyalkyl (for example, methoxymethyl), aralkyl (for example, benzyl), aryloxyalkyl (for example, phenoxymethyl), aryl (for example, phenyl optionally substituted by, for example, halogen, Cl -4 alkyl, or Cl -4 alkoxy or amino); (2) sulfonate esters, such as alkyl- or aralkyl sulfonyl (for example, methanesulfonyl); (3) amino acid esters (for example, L-valyl or L-isoleuc
  • the phosphate esters may be further esterified by, for example, a Cl -20 alcohol or reactive derivative thereof, or by a 2, 3-di (C6-24) acyl glycerol.
  • any alkyl moiety present advantageously contains from 1 to 18 carbon atoms, particularly from 1 to 6 carbon atoms, more particularly from 1 to 4 carbon atoms.
  • Any cycloalkyl moiety present in such esters advantageously contains from 3 to 6 carbon atoms.
  • Any aryl moiety present in such esters advantageously comprises a phenyl group.
  • Ethers of the compounds of the present invention include, but are not limited to methyl, ethyl, butyl and the like.
  • Alkylaryl refers to an -(alkyl)aryl radical where aryl and alkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for aryl and alkyl respectively.
  • Alkylhetaryl refers to an -(alkyl)hetaryl radical where hetaryl and alkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for aryl and alkyl respectively.
  • Alkylheterocycloalkyl refers to an -(alkyl) heterocyclyl radical where alkyl and heterocycloalkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for heterocycloalkyl and alkyl respectively.
  • alkene refers to a group of at least two carbon atoms and at least one carbon-carbon double bond
  • alkyne refers to a group of at least two carbon atoms and at least one carbon-carbon triple bond.
  • the alkyl moiety may be branched, straight chain, or cyclic.
  • Alkenyl refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond, and having from two to ten carbon atoms (i.e., (C2-10)alkenyl or C2-10 alkenyl).
  • a numerical range such as “2 to 10” refers to each integer in the given range - e.g., “2 to 10 carbon atoms” means that the alkenyl group may consist of 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms.
  • the alkenyl moiety may be attached to the rest of the molecule by a single bond, such as for example, ethenyl (i.e., vinyl), prop-l-enyl (i.e., allyl), but-l-enyl, pent-l-enyl and penta-l,4-dienyl.
  • an alkenyl group is optionally substituted by one or more substituents which are independently alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, - ORa, -SRa, -OC(0)-Ra, -N(Ra) 2 , -C(0)Ra, -C(0)ORa, -OC(0)N(Ra) 2 , -C(0)N(Ra) 2 , - N(Ra)C(0)ORa, -N(Ra)C(0)Ra, -N(Ra)C(0)N(Ra) 2 , N (Ra)C(0)ORa, -N(Ra)C(0)Ra, -N(Ra)C(0)N(Ra)
  • Alkenyl-cycloalkyl refers to an -(alkenyl)cycloalkyl radical where alkenyl and cycloalkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for alkenyl and cycloalkyl respectively.
  • Alkynyl refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one triple bond, having from two to ten carbon atoms (i.e., (C2-10)alkynyl or C2-10 alkynyl).
  • a numerical range such as “2 to 10” refers to each integer in the given range - e.g., “2 to 10 carbon atoms” means that the alkynyl group may consist of 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms.
  • alkynyl may be attached to the rest of the molecule by a single bond, for example, ethynyl, propynyl, butynyl, pentynyl and hexynyl.
  • an alkynyl group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -ORa, -SRa, -OC(0)-Ra, - N(Ra) 2 , -C(0)Ra, -C(0)ORa, -OC(0)N(Ra) 2 , -C(0)N(Ra) 2 , -N(Ra)
  • Alkynyl-cycloalkyl refers to an -(alkynyl)cycloalkyl radical where alkynyl and cycloalkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for alkynyl and cycloalkyl respectively.
  • Cycloalkyl refers to a monocyclic or polycyclic radical that contains only carbon and hydrogen, and may be saturated, or partially unsaturated. Cycloalkyl groups include groups having from 3 to 10 ring atoms (i.e. (C3-10)cycloalkyl or C3-10 cycloalkyl). Whenever it appears herein, a numerical range such as “3 to 10” refers to each integer in the given range - e.g., “3 to 10 carbon atoms” means that the cycloalkyl group may consist of 3 carbon atoms, etc., up to and including 10 carbon atoms.
  • cycloalkyl groups include, but are not limited to the following moieties: cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl, and the like.
  • a cycloalkyl group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -ORa, -SRa, -OC(0)-Ra, - N(Ra) 2 , -C(0)Ra, -C(0)ORa, -OC(0)N(Ra) 2 , -C(0)N(Ra) 2 , -N(Ra)C(0)ORa, - N(Ra)C(0)Ra, -N(Ra)C(0)ORa, - N(Ra)C(0)Ra, -N(Ra)C(0)N(Ra) 2 , N (Ra)C (NR
  • Cycloalkyl-alkenyl refers to a -(cycloalkyl)alkenyl radical where cycloalkyl and alkenyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for cycloalkyl and alkenyl, respectively.
  • Cycloalkyl-heterocycloalkyl refers to a -(cycloalkyl)heterocycloalkyl radical where cycloalkyl and heterocycloalkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for cycloalkyl and heterocycloalkyl, respectively.
  • Cycloalkyl-heteroaryl refers to a -(cycloalkyl)heteroaryl radical where cycloalkyl and heteroaryl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for cycloalkyl and heteroaryl, respectively.
  • alkoxy refers to the group -O-alkyl, including from 1 to 8 carbon atoms of a straight, branched, cyclic configuration and combinations thereof attached to the parent structure through an oxygen. Examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy and cyclohexyloxy. “Lower alkoxy” refers to alkoxy groups containing one to six carbons.
  • substituted alkoxy refers to alkoxy wherein the alkyl constituent is substituted (i.e., -0-(substituted alkyl)).
  • the alkyl moiety of an alkoxy group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroaryl alkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -ORa, -SRa, -OC(0)-Ra, -N(Ra)2, -C(0)Ra, -C(0)ORa, -OC(0)N(Ra)2, - C(0)N(Ra) 2 , -N(Ra)C(0)ORa, -
  • “Amino” or “amine” refers to a -N(Ra)2 radical group, where each Ra is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, unless stated otherwise specifically in the specification.
  • a -N(Ra)2 group has two Ra substituents other than hydrogen, they can be combined with the nitrogen atom to form a 4-, 5-, 6- or 7-membered ring.
  • -N(Ra)2 is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl.
  • an amino group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -ORa, -SRa, -0C(0)-Ra, - N(Ra) 2 , -C(0)Ra, -C(0)0Ra, -0C(0)N(Ra) 2 , -C(0)N(Ra) 2 , -N(Ra)C(0)0Ra, - N(Ra)C(0)Ra, -N(Ra)C(0)0Ra, -N
  • substituted amino also refers to N-oxides of the groups -NHRa, and NRaRa each as described above. N-oxides can be prepared by treatment of the corresponding amino group with, for example, hydrogen peroxide or m-chloroperoxybenzoic acid.
  • Amide or “amido” refers to a chemical moiety with formula -C(0)N(R) 2 or -NHC(0)R, where R is selected from the group of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon), and heteroalicyclic (bonded through a ring carbon), each of which moiety may itself be optionally substituted.
  • R is selected from the group of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon), and heteroalicyclic (bonded through a ring carbon), each of which moiety may itself be optionally substituted.
  • the two R of -N(R) 2 of the amide may optionally be taken together with the nitrogen to which it is attached to form a 4-, 5-, 6- or 7- membered ring.
  • an amido group is optionally substituted independently by one or more of the substituents as described herein for alkyl, cycloalkyl, aryl, heteroaryl, or heterocycloalkyl.
  • An amide may be an amino acid or a peptide molecule attached to a compound disclosed herein, thereby forming a prodrug.
  • the procedures and specific groups to make such amides are known to those of skill in the art and can readily be found in seminal sources such as Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, N.Y., 1999, which is incorporated herein by reference in its entirety.
  • absorption generally refers to the process of matter taking up exogenous energy and transforming the state of that matter to a higher electronic state when interacting with an exogenous source described herein.
  • the process of absorption leads to an attenuation in the intensity of the exogenous energy.
  • chromophore refers to a molecule or a part of a molecule responsible for its color. Color arises when a molecule absorbs certain wavelengths of visible light and transmits or reflects others. A molecule having an energy difference between two different molecular electronic states falling within the range of the visible spectrum may absorb visible light and thus be aptly characterized as a chromophore. Visible light incident on a chromophore may be absorbed thus exciting an electron from a ground state molecular state into an excited state molecular state.
  • “Isomers” are different compounds that have the same molecular formula. “Stereoisomers” are isomers that differ only in the way the atoms are arranged in space - i.e., having a different stereochemical configuration. “Enantiomers” are a pair of stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a “racemic” mixture. The term “( ⁇ )” is used to designate a racemic mixture where appropriate. “Diastereoisomers” are stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other.
  • the absolute stereochemistry is specified according to the Cahn- Ingold-Prelog R-S system.
  • the stereochemistry at each chiral carbon can be specified by either (R) or (S).
  • Resolved compounds whose absolute configuration is unknown can be designated (+) or (-) depending on the direction (dextro- or levorotatory) which they rotate plane polarized light at the wavelength of the sodium D line.
  • Certain of the compounds described herein contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined, in terms of absolute stereochemistry, as (R) or (S).
  • enantiomerically enriched and “non-racemic,” as used herein, refer to compositions in which the percent by weight of one enantiomer is greater than the amount of that one enantiomer in a control mixture of the racemic composition (e.g., greater than 1 : 1 by weight).
  • an enantiomerically enriched preparation of the (S)-enantiomer means a preparation of the compound having greater than 50 % by weight of the (S)-enantiomer relative to the (R)-enantiomer, such as at least 55 % by weight, such as at least 60 % by weight, such as at least 65 % by weight, such as at least 70 % by weight, such as at least 75 % by weight, or such as at least 80 % by weight.
  • the enrichment can be significantly greater than 80 % by weight, providing a “substantially enantiomerically enriched” or a “substantially non-racemic” preparation, which refers to preparations of compositions that have at least 85 % by weight of one enantiomer relative to other enantiomer, such as at least 90% by weight, or such as at least 95 % by weight.
  • enantiomerically pure or “substantially enantiomerically pure” refers to a composition that comprises at least 98 % of a single enantiomer and less than 2 % of the opposite enantiomer.
  • the enantiomerically enriched preparation of the (S)-enantiomer may include, for example, 52 %, 54 %, 56 %, 58 %, 60 %, 62 %, 65 %, 70 %, 73 %, 76 %, 80 %, 84 %, 88 %, 90 %, 93 %, 95 %, or 98 % by weight of the (S)-enantiomer relative to the (R)-enantiomer.
  • “Moiety” or “fragment” refers to a specific segment or functional group of a molecule. Chemical moieties are often recognized chemical entities embedded in or appended to a molecule.
  • “Tautomers” are structurally distinct isomers that interconvert by tautomerization. “Tautomerization” is a form of isomerization and includes prototropic or proton-shift tautomerization, which is considered a subset of acid-base chemistry. “Prototropic tautomerization” or “proton-shift tautomerization” involves the migration of a proton accompanied by changes in bond order, often the interchange of a single bond with an adjacent double bond. Where tautomerization is possible (e.g., in solution), a chemical equilibrium of tautomers can be reached. An example of tautomerization is keto-enol tautomerization.
  • keto-enol tautomerization is the interconversion of pentane-2, 4-di one and 4- hydroxypent-3-en-2-one tautomers.
  • Another example of tautomerization is phenol-keto tautomerization.
  • a specific example of phenol-keto tautomerization is the interconversion of pyridin-4-ol and pyridin-4(lH)-one tautomers.
  • “Substituted” means that the referenced group may have attached one or more additional groups, radicals or moieties individually and independently selected from, for example, acyl, alkyl, alkylaryl, cycloalkyl, aralkyl, aryl, carbohydrate, carbonate, heteroaryl, heterocycloalkyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, ester, thiocarbonyl, isocyanato, thiocyanato, isothiocyanato, nitro, oxo, perhaloalkyl, perfluoroalkyl, phosphate, silyl, sulfmyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea, and amino, including mono- and di-substituted amino groups, and protected derivative
  • substituents themselves may be substituted, for example, a cycloalkyl substituent may itself have a halide substituent at one or more of its ring carbons.
  • optionally substituted means optional substitution with the specified groups, radicals or moieties.
  • substituted or “optionally substituted” refer to chemical moieties, wherein one or more hydrogen atoms may be replaced by a halogen atom, a -NIL ⁇ , -SH, -NO2 or -OH group, or by an alkyl, alkenyl, alkanoyl, heteroalkyl, aryl, heteroaryl, cycloalkyl or heterocycle group as defined herein.
  • the last-mentioned groups may be optionally substituted.
  • FRET fluorescence resonance energy transfer which is the transfer of the excited energy of a donor to an acceptor without the emission of light.
  • body geography refers to the region of the body from which a sample is obtained.
  • sample geography include, but are not limited to, the mouth, the nose, upper respiratory tract and the lower respiratory tract.
  • a specific or general sample method may be deployed to obtain a sample from a particular body geography.
  • swab samples may be obtained from the mouth (such as oral or buccal swab samples) or the nose (nasal or nasopharyngeal swab samples) or feces and any other suitable human body geography.
  • the sample method or geography is distinguished from the device used to obtain the sample. That is, a buccal swab sample is obtained from mouth geography and is distinguished from the swab (device) used to collect the sample which further may deploy specific or general methods for collection.
  • Coronaviruses are relatively large viruses containing a single-stranded positive- sense RNA genome encapsulated within a membrane envelope. (Liu, et ak, ACS Cent. Sci. 2020, 6, 315-331, incorporated herein in its entirety). While little is currently known about the SARS- COV 2 virus, an examination of the genome sequence shows strong homology with its more well-studied cousin, SARS-CoV.
  • the betacoronavirus genome encodes for three nonstructural proteins, including RNA-dependent RNA polymerase and two enzymes, or more specifically proteases that are critical to its replication process, namely the coronavirus main proteinase (3CLpro) and the papain-like protease (PLpro).
  • 3CLpro coronavirus main proteinase
  • PLpro papain-like protease
  • the viral RNA is translated by the host translational machinery to produce a polyprotein that comprises the full compliment of effector proteins necessary to support viral reproduction and assembly, including these non-structural proteins.
  • 3CLpro and PLpro are cysteine proteases. These proteases are critically important in cleaving the initially translated polyprotein into effector proteins.
  • the 3CLpro enzyme has been found to cleave 11 sites in the viral polyprotein. These cleavage sites in the substrate are similar, in that the sequences targeted by the enzyme comprise at least four amino acid residues ending in LQ- on the C-terminus side of the peptide scission. Specificity on the N-terminus side of the break is a preference for S or A, with examples of N and G also known (Grum-Tokars, et ah, Virus Res. 2008, 733, 63-73).
  • the biosensor has a Formula (1) D-FG, wherein FG is a peptide comprising at least four amino acid residues that terminate in the sequence -LQ- or -LQS-.
  • FG is a peptide comprising four to six amino acid residues that terminate in the sequence -LQ- or -LQS- and include at least one unnatural amino acid residue (c) i.e. - (yLQ- or (-c LQS-) to the left of the -L.
  • FG is a peptide comprising six to eight amino acid residues that terminate in the sequence -LQ- or -LQS- and include at least one unnatural amino acid residue (c) i.e. - (yLQ- or (-c LQS-) to the left of the -L.
  • FG is a peptide comprising ten to twelve amino acid residues that terminate in the sequence -LQ- or -LQS- and include at least one unnatural amino acid residue (c) i.e. - (yLQ- or (-c LQS-) to the left of the -L.
  • c unnatural amino acid residue
  • longer peptide may provide better specificity by providing a longer substrate for the enzyme to target, thereby requiring a greater sequence alignment between the FG peptide and the enzyme’s natural substrate.
  • more amino acid residues may be added while keeping the consensus sequence.
  • the PLpro enzyme has been found to target four residue sequences ending in - GG- on the C-terminus (Baez-Santos, et al.) .
  • the five sites targeted in vivo are specifically -LRGG-, -LKGG-, and -LNGG- sequences.
  • the N-terminus requires little amino acid specificity, since this terminus is represented by -A-, -K-, or the e-amino group of -K- in the natural substrate.
  • the biosensor has a Formula (1) D-FG, wherein FG is a peptide comprising at least four amino acid residues that terminate in the sequence -GG-.
  • FG is a peptide comprising four to six amino acid residues that terminate in the sequence -GG- and include at least one unnatural amino acid residue (c) i.e. - (-c GG-) to the left of the -L. In some embodiments, FG is a peptide comprising six to eight amino acid residues that terminate in the sequence -GG- and include at least one unnatural amino acid residue (c) i.e.
  • FG is a peptide comprising ten to twelve amino acid residues that terminate in the sequence -GG- and include at least one unnatural amino acid residue (c) i.e. - (-c GG-) to the left of the -L.
  • c unnatural amino acid residue
  • longer peptide may provide better specificity by providing a longer substrate for the enzyme to target, thereby requiring a greater sequence alignment between the FG peptide and the enzyme’s natural substrate. Thus, more amino acid residues may be added while keeping the consensus sequence.
  • the selectivity of the biosensor may be further improved by selecting a substrate that is specific to the selected enzyme.
  • selectivity of the biosensor can be improved by selecting an amino acid sequence for FG that can specifically act as a substrate for the selected enzyme and not for any other enzymes.
  • the sensitivity of such a biosensor depends on the amount of enzyme produced by a host cell after the target virus infects the host cell.
  • selecting FG that is specific to an enzyme that is produced in relatively larger quantities in the infected host cell may improve the sensitivity of the biosensor.
  • the biosensor is responsive to enzymes produced by human host cells that are encoded by a target virus.
  • the biosensor may have a Formula (I) D-FG, wherein FG is a peptide having an amino acid sequence that selectively provides a substrate for the enzyme that is encoded by the target virus.
  • Selectivity of the biosensor can thus, be improved by first selecting a suitable enzyme that is specific to the target virus and does not have significant sequence identity with, or activity similar to, enzymes produced by pathogens other than the target virus.
  • the enzyme encoded by the target virus is a protease encoded by the viruses.
  • the enzymes encoded by the target virus comprise proteases that are necessary for viral replications.
  • the enzyme encoded by the target virus is a serine protease. In some embodiments, the enzyme encoded by the target virus is a microbial cysteine protease. In some embodiments, the enzyme encoded by the target virus is a microbial enzyme. In some embodiments, the enzyme encoded by the target virus is selected from the group of 3CLpro and PLpro encoded by coronavirus. In some embodiments, the enzyme encoded by the target virus is 3CLpro encoded by coronavirus. In some embodiments, the enzyme encoded by the target virus is PLpro encoded by coronavirus.
  • this disclosure provides a biosensor for the detection of a coronavirus such as, for example, SARS-CoV-1, SARS-CoV-2, or MERS-CoV, the biosensor having a Formula (1) D-FG, wherein: (i) FG comprises a material responsive to an enzyme encoded by the coronavirus such as, for example, SARS-CoV-1, SARS-CoV-2, or MERS-CoV; and (ii) D is a spectroscopic probe, wherein FG is conjugated to the spectroscopic probe D via a covalent bond, wherein FG masks the activity of the spectroscopic probe D, wherein the enzyme causes the cleavage of the FG to release the spectroscopic probe D, resulting in a detectable optical response; and wherein the enzyme is present in a virus-infected host cell and is released outside for access by the biosensor when the host cell is ruptured.
  • FG comprises a material responsive to an enzyme encoded by the coronavirus such as, for
  • the covalent bond is selected from the group of -CO-0-, -CO-NH- , -SO2-O-, -SO2-NH-, -SO-O-, or -SO-NH-.
  • the design features of the disclosed biosensors have two main components: (1) a novel spectroscopic probe composed of a chromophore, (2) a material that selectively responds to a specific type of enzyme encoded by the viruses, wherein the enzyme is present in a virus- infected host cell and is released outside for access by the biosensor when the host cell is ruptured.
  • the enzyme ecoded by the virus cleaves the biosensor to release the spectroscopic probe and causes the spectroscopic probe to produce detectable optical responses.
  • the spectroscopic probe can be any group that, upon fragmentation of D-FG, produces a change in an optical response.
  • the spectroscopic probe D is selected from the group of a FRET donor/acceptor pair, coumarins, phenothiazines, phenoxazines, fluoresceins, rhodols, or rhodamines.
  • the optical response produced by the biosensor is a color change from colored state to colorless state.
  • the optical response produced by the biosensor is a color change from colorless state to colored state.
  • the optical response produced by the biosensor is a change from non- fluorescent state to fluorescent state.
  • the spectroscopic probe in the biosensor is a fluorogenic chromophore that can produce a fluorescence response upon degradation of the biosensor by the enzyme.
  • the fluorophore in the biosensor comprises coumarin, rhodamine, or fluorescein.
  • the fluorophore comprises coumarin.
  • the fluorophore in the biosensor comprises rhodamine.
  • the rhodamine is selected from the rhodamine 110, rhodamine B, Rhodamine 6G, Rhodamine GreenTM (Rho G), tetramethylrhodamine, or 5-carboxy-X-rhodamine.
  • the biosensor comprises the fluorophore covalently attached to the material that is a fragment of a substrate of SARS-CoV PLpro or SARS-CoV 3CLpro. In some embodiments, the biosensor comprises the fluorophore covalently attached to the material that is a fragment of a substrate of SARS-CoV PLpro encoded by a coronavirus. In some embodiments, the biosensor comprises the fluorophore covalently attached to the material that is a fragment of a substrate of SARS-CoV 3CLpro encoded by a coronavirus. In some embodiments, the biosensor comprises the fluorophore covalently attached to a peptide selected from the peptides disclosed in Table 5 below.
  • the biosensor comprises the fluorophore covalently attached to ubiquitin. In some embodiments, the biosensor comprises 7-amino-3- methylcoumarin (AMC) as the fluorophore covalently attached to ubiquitin.
  • AMC 7-amino-3- methylcoumarin
  • the biosensor comprises the fluorophore covalently attached to ubiquitin or ISG15 protein.
  • the biosensor comprises AMC as the fluorophore covalently attached to ubiquitin and the biochemical factor is SARS-CoV-1 PLpro or SARS-CoV-2 PLpro.
  • the biosensor comprises AMC as the fluorophore covalently attached to ISG15 protein and the biochemical factor is SARS-CoV-1 PLpro or SARS-CoV-2 PLpro.
  • the spectroscopic probe comprises at least one fluorophore.
  • the fluorophore may be any fluorescent and luminescent probes for biological activity that is known in the art.
  • the fluorophore is selected from the group of 5-carboxytetramethylrhodamine (TAMRA), EDANS, DANSYL, DMACA, FITC, ICG, AMCA-x, Marina Blue, PyMPO, Lucifer Yellow, Mca, Trp, Rho G, rhodamine 6G, rhodamine B, rhodamine 110, 5-carboxy-X-rhodamine, Bodipy493, Bodipy TMR-x, Bodipy630, Cyanine 5 (Cy5), Cyanine 5.5 (Cy5.5), and Cyanine7.5 (Cy7.5), rhodamine, fluorescein, boron- dipyrromethene (BODIPY), fluorescein substitute
  • TAMRA 5-carboxytetramethylr
  • the fluorophore display a high quantum yield. Long-wavelength (excitation and emission) fluorophores are preferred because of less interference from other absorbing species. It is preferred that the fluorophore should not exhibit significant sensitivity to pH change or to non-specific quenching by metal ions or other species.
  • the spectroscopic probe D comprises a chromophore and a fluorophore as a FRET donor/acceptor pair, wherein the fluorophore is selected from the group consisting of coumarins, fluoresceins, rhodols, and rhodamines, and the chromophore is selected from the group consisting of 4-aminobenzoyl, tryptophan, coumarins, and fluoresceins, and wherein the fluorophore and the chromophore are each independently attached to a different portion of the FG.
  • the fluorophore is selected from the group consisting of coumarins, fluoresceins, rhodols, and rhodamines
  • the chromophore is selected from the group consisting of 4-aminobenzoyl, tryptophan, coumarins, and fluoresceins
  • the spectroscopic probe D comprises a fluorophore and a quencher as a FRET donor/acceptor pair, wherein the fluorophore is selected from the group of coumarins, fluoresceins, rhodols, or rhodamines; and the quencher is selected from the group of 2,4-dinitrophenyl, para-nitroaniline, 4-nitro-phenylalanine, dimethylaminophenylazo)benzoyl, 4- (4-diethylaminophenylazo)-benzenesulfonyl, QSY7, or QSY21, wherein the fluorophore and the quencher each is attached to a different portion of the FG,
  • the spectroscopic probe D has chemical moiety selected from the group of Formulae (2)-(7), wherein:
  • U is O or N or C
  • W is O, N, S, SiMe 2 or -CH 2-;
  • Z is -NR 9 R 10 , -NH-A-, or -0-CH2-PhA, where A is a fragmentable group FG;
  • R 1 , R 2 , R 3 , R 4 are each independently selected from the group of H, substituted and unsubstituted Cl -Cl 2 alkyl group, substituted and unsubstituted Cl -Cl 2 alkenyl group, substituted and unsubstituted Cl -Cl 2 alkynyl group, and substituted and unsubstituted aryl group;
  • R 5 , R 6 , R 7 , and R 8 is each independently selected from the group of H, Cl, F, Br, CN, NO2, -NR 9 R 10 , C1-C6 alkyl group, and C1-C6 alkoxyl group;
  • R 9 and R 10 is a substituent each independently selected from the group of H, substituted and unsubstituted Cl -Cl 2 alkyl group, substituted and unsubstituted Cl -Cl 2 alkenyl group, substituted and unsubstituted Cl -Cl 2 alkynyl group, substituted and unsubstituted aryl group, fluoroalkyl, substituted and unsubstituted carbocyclyl, substituted and unsubstituted carbocyclylalkyl, substituted and unsubstituted aralkyl, substituted and unsubstituted heterocycloalkyl, substituted and unsubstituted heterocycloalkylalkyl, substituted and unsubstituted heteroaryl, and substituted and unsubstituted heteroarylalkyl; and
  • R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , and R 17 is each independently selected from the group of H, Cl, F, Br, CN, NO2, -NR 9 R 10 , C1-C6 alkyl group, and C1-C6 alkoxyl group.
  • the spectroscopic probe D of Formula (2) is a fragment selected from the fragments represented by Formula (
  • the spectroscopic probe D of Formula (5) is a fragment selected from Formula ( , Formula Formula (5)
  • the FG comprises a fragment derived from a chemical inhibitor of the enzyme encoded by the target virus, or a peptide derived from a substrate of the enzyme encoded by the target virus.
  • the FG to be bonded to D of Formulae (2)-(7) is a peptide derived from a substrate for a 3CLpro enzyme encoded by a coronavirus, and the peptide has at least four amino acid residues, e.g., 4 to 6 amino acid residues, 6 to 8 amino acid residues, 8-10 amino acid residues, 10-12 amino acid residues, 12-14 amino acid residues or 14-16 amino acid residues and a portion of the peptide has the sequence -LQ- and Q residue is at the peptide terminus, wherein the spectroscopic probe D is bound to the terminal Q residue.
  • the FG to be bonded to D of Formulae (2)-(7) is a peptide derived from a substrate for a PLpro enzyme encoded by a coronavirus, and the peptide has at least four amino acid residues, e.g., 4 to 6 amino acid residues, 6 to 8 amino acid residues, 8-10 amino acid residues, 10-12 amino acid residues, 12-14 amino acid residues or 14-16 amino acid residues and a portion of the peptide has the sequence -GG- and G residue is at the peptide terminus, wherein the spectroscopic probe D is bound to the terminal G residue.
  • peptide fragment may be further substitured by groups commonly used for functional group protection in peptide synthesis or chemistry.
  • groups may be, but are not limited to, Ac, Cbz, Fmoc, and Boc.
  • the FG is VRLQS when the FG is a peptide derived from a substrate for a 3CLpro enzyme encoded by SARS-CoV-1, SARS-CoV-2, or MERS-CoV.
  • the FG is VRLQ when the FG is a peptide derived from a substrate for a 3CLpro enzyme encoded by SARS-CoV-1, SARS-CoV-2, or MERS-CoV.
  • the FG is LRGG or RLRGG when the FG is a peptide derived from a substrate for a PLpro enzyme encoded by SARS-CoV-1, SARS-CoV-2, or MERS-CoV.
  • the biosensor comprises LRGG conjugated to 7-amino-4- methylcoumarin.
  • the biosensor comprises LRGG conjugated to Rhodamine 110.
  • the biosensor comprises LRGG conjugated to fluorescein. In some embodiments, the biosensor comprises VRLQ conjugated to 7-amino-4- methylcoumarin. In some embodiments, the biosensor comprises VRLQ conjugated to Rhodamine 110. In some embodiments, the biosensor comprises VRLQ conjugated to fluorescein.
  • the biosensor comprises LRGG conjugated to 7-amino-4- methylcoumarin via glycine residue at C-terminus. In some embodiments, the biosensor comprises LRGG conjugated to Rhodamine 110 via glycine residue at C-terminus. In some embodiments, the biosensor comprises LRGG conjugated to fluorescein via glycine residue at C- terminus. In some embodiments, the biosensor comprises VRLQ conjugated to 7-amino-4- methylcoumarin via glutamine residue at C-terminus. In some embodiments, the biosensor comprises VRLQ conjugated to Rhodamine 110 via glutamine residue at C-terminus. In some embodiments, the biosensor comprises VRLQ conjugated to fluorescein via glutamine residue at C-terminus.
  • Both 3CLpro and PLpro are cysteine proteases. Because both proteases are vital to the virus for replication and controlling the host cell, they are viable targets for antiviral agents and diagnostics. Over the last two decades, much of the research in drugging SARS-CoV has focused on the development of small molecule, peptide, and peptidomimetic inhibitors of 3CLpro and PLpro. These are shown in Table 3 below and represent the microbial protease inhibitors of 3CLpro and PLpro encoded by coronavirus from which the fragmentable group FG derives. The compounds containing the fragmentable group FG bind to the targeted enzymes (e.g., 3CLPro and PLpro) encoded by coronavirus.
  • the targeted enzymes e.g., 3CLPro and PLpro
  • the fragmentable group FG may include peptide sequences that are recognized as a substrate of the targeted enzyme wherein the binding of the fragmentable group is enhanced by association with multiple binding domains in the targeted enzyme.
  • a peptide sequence terminating in -LRGG at the C-terminus may be linked to a D while the N-terminus is linked to an inhibitor that prefers binding at a site other than the active site, thereby increasing the binding of the molecule.
  • Table 3 Inhibitor binding to proteases produced by coronaviruses a. inhibition constant against SARS-CoV 3CLpro.
  • Table 4 Chromogenic Sensor Molecules of Formula (1) for Detection of Coronaviruses.
  • the R group for the compounds disclosed in Table 4 is selected from the group of H, substituted and unsubstituted C1-C12 alkyl group, substituted and unsubstituted C1-C12 alkenyl group, substituted and unsubstituted C1-C12 alkynyl group, and substituted and unsubstituted aryl group.
  • the R group for the compounds disclosed in Table 4 is selected from the group of H, substituted and unsubstituted C1-C 6 alkyl group, substituted and unsubstituted C1-C 6 alkenyl group, substituted and unsubstituted C1-C 6 alkynyl group, and substituted and unsubstituted aryl group. In some embodiments, the R group for the compounds disclosed in Table 4 is selected from the group of H, substituted and unsubstituted C1-C4 alkyl group, and substituted and unsubstituted aryl group. In some embodiments, the R group for the compounds disclosed in Table 4 is substituted and unsubstituted C1-C4 alkyl group. In some embodiments, the R group for the compounds disclosed in Table 4 is selected from the group of methyl group and ethyl group.
  • 3CLpro and PLpro are two proteases that process the polypeptide translation from the genomic RNA to either structural or non-structural protein components for replication and packaging of new generation viruses.
  • PLpro also serves as a deubiquitinase that function to deubiquitinate host cell proteins such as interferon factor 3 (IRF3) as well as to inactivate the pathway for nuclear factor k-light-chain-enhancer of activated B cells (NF-KB).
  • IRF3 interferon factor 3
  • coronaviruses are enveloped positive-strand RNA viruses that replicate in the cytoplasm of infected cells. Coronavirus replication involves complex replication machineries including RNA genomes (27 to 32 kb), and viral proteins from the host’s antiviral defense mechanism.
  • SARS-CoV papain-like protease SARS-CoV PLpro
  • SARS-CoV 3C-like protease SARS-CoV 3 Cipro
  • SARS-CoV PLpro (nsp3) has an essential role during the formation of virus replication complexes via its insertion into host membranes and its numerous interactions with other nsps (See Baez-Santos et al. supra).
  • SARS-CoV PLpro is a key enzyme in the pathogensis of SARS-CoV.
  • PLpro enzymatic activities include processing of the viral polyprotein, deubiquitination (removal of ubiquitin), and delSGlation (the removal of ISG15) from host-cell proteins (See Mesecar et al., X-ray Structural and Biological Evaluation of a Series of Potent and Highly Selective Inhibitors of Human Coronavirus Papain-like Proteases, J. Med. Chem., 2014, vol. 57, pp. 2393-2412).
  • SARS-CoV PLpro is reported to cleave the consensus sequence LXGG (Barretto et al., The Papain-like Protease of Severe Acite Respiratory Syndrome Coronavirus Has Deubiquitinating Activity, J. Virology, 2005, pp. 15189-15198).
  • SARS-CoV PLpro 1541-2204 is reported to cleave the synthetic 12-mer peptide (ERELNGGAPIKS) derived from the viral proteins nspl/nsp2 and nsp2/nsp 3 (See Barretto et al. supra).
  • SARS-CoV-2 PLpro 1568-1882 is reported as the new class of papain-like protease encoded by SARS-CoV-2 (See Shanker et al., Whole Genome Sequence Analysis and Homology Modelling of a 3C Like Peptidase and aNon- Structural Protein 3 of the SARS-CoV-2 Shows Protein Ligand Interaction with an Aza-Peptide and a Noncovalent Lead Inhibitor with Possible Antiviral Properties. ChemRxiv. Preprint. 2020, https://doi.org/10.26434/chemrxiv.11846943.v7). SARS-CoV-2 nsp3 is mapped within ORFlab sequence 818-2763.
  • the nsp3 encoded by SARS-CoV-2 differs from nsp 3 encoded by SARS- CoV-1 at position 1010 (192 in the nsp3 protein). Regarding the amino acid at position 1010 (192 position of nsp3 protein), the SARS-CoV-2 nsp3 has proline, whereas SARS-CoV-1 nsp3 has non-polar amino acid isoleucine.
  • the destabilizing mutation in nsp3 suggests a potential mechanism differentiating SARS-CoV-2 from SARS-CoV-1 (Angeletti et al., COVID-2019: The role of the nsp2 and nsp 3 in its pathogenesis, J. Medical Virology, 2020, pp. 1-5; the viral nucleic acid sequence for SARS-CoV-2 can be found at GISAID archive h Up s : //www. gl sai d . org/) .
  • the coronaviruses are enveloped positive-strand RNA viruses that replicate in the cytoplasm of infected cells and specifically that two virally encoded cysteine proteases, the SARS-CoV papain-like protease (SARS-CoV PLpro) and the SARS-CoV 3C-like protease (SARS-CoV 3 Cipro) catalyze their own release from the polyprotein encoded by the viral genome (See Baez-Santos et al.). Since the production of these two proteases occurs inside the host cell, when testing for presence of these enzymes they are much more likely to be found in the host cell (intracellular) than extracellular.
  • SARS-CoV papain-like protease SARS-CoV PLpro
  • SARS-CoV 3C-like protease SARS-CoV 3 Cipro
  • virus replication models indicate that as new virus particle copies are made, each generating a new copy of the two proteases, upon full assembly of the new virus particle (+ve strand RNA together with nucleocapsid protein) it is expelled from the cell by exocytosis (Astuti, et al., Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): An overview of viral structure and host response, Diabetes Metab Syndr. 2020 July-August; 14(4): 407-412; published online 2020 Apr 18.doi: 10.1016/j .dsx.2020.04.020).
  • SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2
  • this disclosure provides for a molecular test to detect proteases coded in the virus genome and created when virus infects a host cell. It is expected that the majority of the virally encoded enzymes will be preserved in intact cells or in secreted exosomes that must be disrupted in order to assay their contents.
  • the test to detect proteases is performed on biological cell samples that have been lysed.
  • the samples upon lysing, may have a concentration of the target enzyme in a range from about 1 pM to 10 nM.
  • the concentartion of the target enzyme in the sample may be less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM or less than about 5 pM.
  • the concentration of the target enzyme in the sample may be greater than about 1 pM, greater than about 5 pM, greater than about 10 pM, greater than about 20 pM, greater than about 30 pM, greater than about 40 pM, greater than about 50 pM, greater than about 60 pM, greater than about 70 pM, greater than about 80 pM, greater than about 90 pM, greater than about 100 pM, greater than about 150 pM, greater than about 200 pM, greater than about 250 pM, greater than about 300 pM, greater than about 350 pM, greater than about 400 pM, greater than about 450 pM, greater than about 500 pM, greater than about 550 pM, greater than about 600 pM, greater than about 650 pM, greater than about 700 pM, greater than about 750 pM, greater than about 800 pM, greater than about 850 pM, greater than about 900 pM, greater than about 1 pM, greater than about 5
  • the concentration of the target enzyme is in a range from about 1 pM to about 5 pM, about 5 pM to about 10 pM, about 10 pM to about 20 pM, about 20 pM to about 50 pM, about 50 pM to about 75 pM, about 75 pM to about 100 pM, about 100 pM to about 150 pM, about 150 pM to about 200 pM, about 200 pM to about 500 pM, about 500 pM to about 1 nM, about 1 nM to about 2 nM, about 2 nM to about 5 nM, about 5 nM to about 7.5 nM, about 7.5 nM to about 10 nM, about 1 pM to about 10 pM, about 10 pM to about 50 pM, about 10 pM to about 100 pM, about 50 pM to about 250 pM, about 50 pM to about 500 pM, about 100 pM, about 1 pM to
  • this disclosure provides a biosensor for the detection of viruses having a formula (1) D-FG, wherein (i) FG comprising a material responsive to an enzyme encoded by a target coronavirus such as, for example, those selected from the group of SARS-CoV-1, SARS- CoV-2, or MERS-CoV; and (ii) D is a spectroscopic probe, wherein the material FG is conjugated to the spectroscopic probe D via a covalent bond, wherein the FG masks the activity of the spectroscopic probe D, wherein the enzyme causes the cleavage of the FG to release the spectroscopic probe D, resulting in a detectable optical response; and wherein the enzyme is present in a virus-infected host cell.
  • FG comprising a material responsive to an enzyme encoded by a target coronavirus such as, for example, those selected from the group of SARS-CoV-1, SARS- CoV-2, or MERS-CoV
  • D is a spectroscopic
  • the covalent bond is selected from the group of -C0-0-, -CO-NH- , -SO2-O-, -SO2-NH-, -SO-O-, or -SO-NH-.
  • the enzyme encoded by the target virus is a protease encoded by the viruses.
  • the enzymes encoded by the target virus comprise proteases that are necessary for viral replications.
  • the enzyme encoded by the target virus is a serine protease.
  • the enzyme encoded by the target virus is a microbial cysteine protease.
  • the enzyme encoded by the target virus is a microbial enzyme.
  • the enzyme encoded by the target virus is selected from the group of 3CLpro and PLpro encoded by coronavirus.
  • the enzyme encoded by the target virus is 3CLpro encoded by coronavirus.
  • the enzyme encoded by the target virus is PLpro encoded by coronavirus.
  • the biosensor is designed to be a colorless material which upon attack by an enzyme on the material triggers the release of the latent leuco dye which then spontaneously forms colored dye.
  • This enzymatically triggered release of a latent leuco dye by proper design can be made to selectively produce a strong color in the presence of a pathogenic virus which produces an enzyme capable of attacking the material.
  • the material is a fragment of the small molecule, peptide, or peptidomimetic inhibitors of 3CLpro and PLpro such as the biosensors disclosed in Table 4 which are sensitive to the protease encoded by coronavirus. Upon reaction with the protease, the material is designed to release the leuco-chromophore which then forms a strong color indicating the presence of coronavirus.
  • This biosensor design is not limited to protease encoding pathogens.
  • the protease may be from human host that is important for virus replication.
  • Peptidases which cleave specific proteins can also be incorporated into this design to produce color specific to an overexpressed peptidase.
  • the material is a substrate of protease encoded by human coronaviruses. In some embodiments, the material is a peptide derived from the substrate of protease encoded by human coronaviruses.
  • the human coronaviruses may include SARS-CoV-1, MERS-CoV, or SARS-CoV-2. In some embodiments, the human coronaviruses is SARS-CoV-1. In some embodiments, the human coronaviruses is MERS-CoV. In some embodiments, the human coronavirus is SARS-CoV-2.
  • the substrate of protease encoded by human coronaviruses comprises ubiquitin or interferon- stimulated gene encoded (ISG15) protein and the biochemical factor comprise SARS-CoV-1 PLpro or SARS-CoV-2 PLpro.
  • ISG15 interferon- stimulated gene encoded
  • ubiquitin refers to a small (8.6 kDa) regulatory protein found in most tissues of eukaryotic organisms. Key features include its C-terminal tail and the 7 lysine residues. The addition of ubiquitin to a substrate protein is called ubiquitination. Ubiquitination can mark them for degradation via the proteasome.
  • ISG15 protein refers to a 17 kDa secreted protein that in humans encoded by the ISG15 gene and is the first ubiquitin-like modifier identified, and is similar to a ubiquitin linear dimer.
  • the main cellular function of the protein is ISGylation, its covalent addition to cytoplasmic and nuclear proteins, similar to ubiquitination. It is also known as UCRP (ubiquitin cross-reactive protein) since it contains 2 tandem ubiquitin homology domains and is cross-reactive with ubiquitin antibodies.
  • the peptide is selected from the peptide sequence disclosed in Table 5.
  • the material is conjugated to the spectroscopic probe via the C- terminus or N-terminus of the peptide sequence of the peptide as disclosed in Table 5.
  • the material is a peptide derived from the substrate of 3CLpro encoded by human coronaviruses SARS CoV-1.
  • the peptide is selected from the group of Leu, Val, Val-Leu, Val-Phe, Ala-Val-Leu, Ser-Val-Phe, Val-Thr-Phe-Gln, Val-Thr-Leu-Gln, Val-Thr-Ala-Gln, Glu-Ser-Ala-Thr-Leu-Gln-Ser-Gly-Leu-Ala-Lys-Ser, Thr- Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met, and Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe- Arg-Lys-Met.
  • the material comprises Val-Leu, Val-Phe, Ala-Val-Leu, and Ser-Val-Phe. In some embodiments, the material comprises Glu-Ser-Ala-Thr-Leu-Gln-Ser- Gly-Leu-Ala-Lys-Ser. In some embodiments, the material comprises Thr-Ser-Ala-Val-Leu-Gln- Ser-Gly-Phe-Arg-Lys-Met. In some embodiments, the material is conjugated to the spectroscopic probe via the C-terminus or N-terminus of the peptide sequence of peptide derived from the substrate of 3CLpro encoded by human coronaviruses SARS CoV 1 as described herein. In some embodiments, the material comprises at least one glutamine residue.
  • the material comprises one or more unnatural amino acids such as those described in Table 2.
  • the material is Ac-Abu-Tle-Leu-Gln or Ac- Thz-Tle-Leu-Gln.
  • the one or more unnatural amino acids are positioned relatively closer to the N-terminus of the peptide as compared to the C-terminus of the peptide.
  • the latent leuco dye is bound to the material via a labile bond susceptible to enzymatic degradation.
  • the labile bond is selected from the group of an ester bond, an amide bond, an imine bond, an acetal bond, a ketal bond, and combinations thereof.
  • an ester bond may provide improved sensitivity since it can be relatively easy to cleave an ester bond.
  • an amide bond may provide for a more stable bond between the material and the spectroscopic probe, and thereby provide a relatively more selective biosensor.
  • the biochemical factor encoded by the virus is a reducing agent.
  • the reducing agent encoded by the virus is glutathione.
  • the material is derived from the substrate of a microbial peptidase. In some embodiments, the material is derived from the substrate of a microbial protease.
  • the pathogen diagnostic bio-sensor operates by using the endogenous pathogen-encoded cysteine protease activity of a certain SARS-CoV protease to cleave a spectroscopic probe from the biosensor of the invention comprising (i) a material responsive to SARS-CoV protease, and (ii) a latent leuco dye or FRET donor/accept pair as a spectroscopic probe, wherein the material is conjugated to the spectroscopic probe, wherein the material masks the activity of the spectroscopic probe, wherein the enzyme encoded by the target virus causes the cleavage of the material to release the latent spectroscopic probe and result in a detectable optical response.
  • the material is derived from the substrate of the SARS-CoV protease (e.g., nsp3 is the substrate and SARS-CoV papain like protease is the enzyme). Only the SARS-CoV protease can cleave the spectroscopic probe from the biosensor. Thus, the properly chosen material derived from the substrate peptide sequence for the SARS-CoV protease in the biosensor is responsive to the appropriate SARS-CoV protease activity and can be used to detect a SARS-CoV pathogen of interest.
  • the D is a pair of fluorophore/chromophore and the FG is a peptide derived from a substrate for 3CLpro encoded by a coronavirus.
  • the fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C-terminus
  • the chromophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N-terminus.
  • the FG peptide has at least five amino acid residues and a portion of the peptide has the sequence -LQS-. In some embodiments, the FG peptide has 4 to 6 amino acid residues, 6 to 8 amino acid residues, 8 to 10 amino acid residues, 10 to 12 amino acid residues, 12 to 14 amino acid residues, or 14-16 amino acid residues. In some embodiments, the FG peptide includes at least one unnatural amino acid residue. In some embodiments, the unnatural amino acid residue is relatively closer to the N-terminus of the FG peptide.
  • the D is a pair of fluorophore/chromophore and the FG is a peptide derived from a substrate for a 3CLpro enzyme encoded by a coronavirus.
  • the fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N-terminus
  • the chromophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C-terminus.
  • the FG peptide has at least five amino acid residues and a portion of the peptide has the sequence -LQS-. In some embodiments, the FG peptide has 4 to 6 amino acid residues, 6 to 8 amino acid residues, 8 to 10 amino acid residues, 10 to 12 amino acid residues, 12 to 14 amino acid residues, or 14-16 amino acid residues. In some embodiments, the FG peptide includes at least one unnatural amino acid residue. In some embodiments, the unnatural amino acid residue is relatively closer to the N-terminus of the FG peptide.
  • the D is the FRET fluorophore/quencher and the FG is a peptide derived from a substrate for a 3CLpro enzyme encoded by a coronavirus.
  • the fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C- terminus
  • the quencher is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N-terminus.
  • the FG peptide has at least five amino acid residues and a portion of the peptide has the sequence -LQS-. In some embodiments, the FG peptide has 4 to 6 amino acid residues, 6 to 8 amino acid residues, 8 to 10 amino acid residues, 10 to 12 amino acid residues,
  • the FG peptide includes at least one unnatural amino acid residue. In some embodiments, the unnatural amino acid residue is relatively closer to the N-terminus of the FG peptide.
  • the D is the FRET fluorophore/quencher and the FG is a peptide derived from a substrate for a 3CLpro enzyme encoded by a coronavirus.
  • the fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N- terminus
  • the quencher is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C-terminus.
  • the FG peptide has at least five amino acid residues and a portion of the peptide has the sequence -LQS-. In some embodiments, the FG peptide has 4 to 6 amino acid residues, 6 to 8 amino acid residues, 8 to 10 amino acid residues, 10 to 12 amino acid residues,
  • the FG peptide includes at least one unnatural amino acid residue. In some embodiments, the unnatural amino acid residue is relatively closer to the N-terminus of the FG peptide.
  • the D is a pair of fluorophore/chromophore and the FG is a peptide derived from a substrate for a PLpro enzyme encoded by a coronavirus.
  • the fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C- terminus
  • the chromophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N-terminus.
  • the FG peptide has at least four amino acid residues and a portion of the peptide has the sequence -GG-. In some embodiments, the FG peptide has 4 to 6 amino acid residues, 6 to 8 amino acid residues, 8 to 10 amino acid residues, 10 to 12 amino acid residues, 12 to 14 amino acid residues, or 14-16 amino acid residues. In some embodiments, the FG peptide includes at least one unnatural amino acid residue. In some embodiments, the unnatural amino acid residue is relatively closer to the N-terminus of the FG peptide.
  • the D is a pair of fluorophore/chromophore and the FG is a peptide derived from a substrate for a PLpro enzyme encoded by a coronavirus.
  • the fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N- terminus
  • the chromophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C-terminus.
  • the FG peptide has at least four amino acid residues and a portion of the peptide has the sequence -GG-. In some embodiments, the FG peptide has 4 to 6 amino acid residues, 6 to 8 amino acid residues, 8 to 10 amino acid residues, 10 to 12 amino acid residues, 12 to 14 amino acid residues, or 14-16 amino acid residues. In some embodiments, the FG peptide includes at least one unnatural amino acid residue. In some embodiments, the unnatural amino acid residue is relatively closer to the N-terminus of the FG peptide.
  • the D is the FRET fluorophore/quencher and the FG is a peptide derived from a substrate for a PLpro enzyme encoded by a coronavirus.
  • the fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N- terminus
  • the quencher is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C-terminus.
  • the FG peptide has at least four amino acid residues and a portion of the peptide has the sequence -GG-. In some embodiments, the FG peptide has 4 to 6 amino acid residues, 6 to 8 amino acid residues, 8 to 10 amino acid residues, 10 to 12 amino acid residues, 12 to 14 amino acid residues, or 14-16 amino acid residues. In some embodiments, the FG peptide includes at least one unnatural amino acid residue. In some embodiments, the unnatural amino acid residue is relatively closer to the N-terminus of the FG peptide.
  • the D is the FRET fluorophore/quencher and the FG is a peptide derived from a substrate for a PLpro enzyme encoded by a coronavirus.
  • the fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C- terminus
  • the quencher is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N-terminus.
  • the FG peptide has at least five amino acid residues and a portion of the peptide has the sequence -GG-. In some embodiments, the FG peptide has 4 to 6 amino acid residues, 6 to 8 amino acid residues, 8 to 10 amino acid residues, 10 to 12 amino acid residues,
  • the rhodamine is Rhodamine GreenTM having a succinimidyl ester attached to the 5- or 6 position of the rhodamine molecule as in the following formula
  • the rhodamine is rhodamine
  • the fluorogenic chromophore is chemically linked to the peptide derived from the substrate of the virus encoded enzyme via a reactive functional group on the side chain of an amino acid residue in the peptide sequence or the peptide terminal groups (C -terminus or N-terminus), and the fluorogenic chromophore has reactive functional groups including an amine group (-NH2), a carboxyl group (-COOH), a hydroxyl group (-OH), sulfonyl group (-SO2CI), succinimidyl ester, or a thiol group (-SH).
  • the chemical linkage between the fluorogenic chromophore and the peptide derived from the substrate of the virus encoded enzyme comprises an amide bond formed by NHS/EDC chemistry.
  • Rhodamine GreenTM may be chemically linked to the carboxyl group on the C-terminus of LRGG- via an ester bond by the reaction of the -OH of the carboxyl group of the C-terminus glycine with the succinimidyl ester at the 5- or 6- position of the Rhodamine GreenTM molecule (See chemical formula above).
  • the spectroscopic probe is a fluorescent chromophore that may be excited by energy transfer from a first chromophore that is preferably excited by a first optical radiation. Following the cleavage of the biosensor by the enzyme, the fluorescent chromophore is no longer nearby the first chromophore, leading to loss of the fluorescent signal from the fluorescent chromophore.
  • the biosensor comprises a fluorophore and a quencher as a FRET donor/acceptor pair, wherein the fluorophore and the quencher each is attached to a different portion of the material moiety, and the fluorescence of the fluorophore is quenched.
  • the fluorophore is attached at one end of the material and the quencher is attached at the opposite end, e.g., the biosensor has the formula: fluorophore donor-material-quencher acceptor.
  • the fluorophore are on opposite sides of a cleavage site targeted by the enzyme.
  • the fluorophore and the quencher are within about 30 Angstroms of each other.
  • the enzymes encoded by the coronavirus e.g., SARS-CoV PLpro and SARS-CoV 3CLpro
  • the enzyme degradation of the biosensor results in fluorophore-quencher separation and strong fluorescence emission.
  • the distance between the donor and the acceptor in the FRET pair ranges from 1 nm to 10 nm.
  • the emission spectrum of the donor overlaps with the absorption spectrum of the acceptor in the FRET pair.
  • the fluorophore has an excitation wavelength ranging from 280 nm to 490 nm. In some embodiments, the fluorophore has an excitation wavelength ranging from 340 nm to 360 nm.
  • the fluorophore has an excitation peak wavelength selected from the group of 280 nm, 320 nm, 325 nm, 340 nm, 342 nm, 350 nm, 430 nm, or 490 nm. In some embodiments, the fluorophore has an emission wavelength that ranges from 360 nm to 562 nm.
  • the fluorophore has an emission wavelength that ranges from 440 nm to 450 nm. In some embodiments, the fluorophore has a peak emission wavelength selected from the group of 360 nm, 392 nm, 420 nm, 465 nm, 490 nm, 520 nm, or 562 nm.
  • the donor of the FRET pair is a fluorophore selected from the group of coumarin, fluorescein, rhodamine, xanthene, BODIPY® (boron-dipyrromethene),
  • Alexa Fluor® sulfonated derivative of coumarin, rhodamine, xanthene or cyanine dye
  • EDANS 5-[(2-Aminoethyl) amino] naphthalene- 1 -sulfonic acid
  • cyanine dye sulfonated derivative of coumarin, rhodamine, xanthene or cyanine dye
  • the fluorescent dye is (2-aminobenzoyl or anthraniloyl) (Abz), (N-Methyl-anthraniloyl) (N-Me- Abz), (5-(Dimethylamino)naphthalene-l-sulfonyl) (Dansyl), (7-Dimethylaminocoumarin-4- acetate) (DMACA), fluorescein isothiocyanate (FITC), indocyanine green (ICG), AMCA-x, Marina Blue, PyMPO, (6-Amino-2,3-dihydro-l,3-dioxo-2-hydrazinocarbonylamino-lH- benz[d,e]isoquinoline-5,8-disulfonic acid) (Lucifer Yellow), ((7-Methoxycoumarin-4-yl)acetyl) (Mca), tryptophan (Trp), Rhodamine GreenTM (Rho G), r
  • the fluorescence quencher (acceptor) of the FRET pair may include 4-(4-dimethylaminophenylazo)benzoyl (Dabcyl), 2,4-dinitrophenyl (Dnp), para- nitroaniline (pNA), 4-nitro-benzyloxycarbonyl (4-Nitro-Z), N-(9- ⁇ 2-[(4- ⁇ [(2,5-dioxopyrrolidin- l-yl)oxy]carbonyl ⁇ piperidin-l-yl)sulfonyl]phenyl ⁇ -6-[methyl(phenyl)amino]-3H-xanthen-3- ylidene)-N-methylanilinium (QSY7), (2,5-dioxopyrrolidin-l-yl) l-[2-[3-(l,3-dihydroisoindol-2- ium-2-ylidene)-6-(l,3
  • the biosensor comprises rhodamine /coumarin, rhodamine 6G/fluorescein, rhodamine 6G/FITC, Rho G/QSY7, TMR/BHQ1, Cy5/QSY21, CY5.5/QSY21, EDANS/ Dabcyl as FRET donor/acceptor pair.
  • the biosensor comprises rhodamine 6G/fluorescein, rhodamine 6G/FITC, Rho G/QSY7 as FRET donor/acceptor pair.
  • the biosensor comprises the FRET donor/acceptor pair each covalently attached to the material that is a fragment of a substrate of SARS-CoV-1 PLpro and SARS-CoV-2 PLpro encoded by a coronavirus. In some embodiments, the biosensor comprises the FRET donor/acceptor pair each covalently attached to the material that is a fragment of a substrate of SARS-CoV-1 3CLpro and SARS-CoV-2 3CLpro encoded by a coronavirus.
  • the fragment of the substrate of SARS-CoV-1 PLpro and SARS-CoV-2PLpro or SARS-CoV-1 3CLpro and SARS-CoV-23CLpro comprises a peptide selected from the peptides disclosed in Table 5 below.
  • the substrate may be a simple fluorogenic peptide (SFP), the mechanism of which is schematically shown in FIG. 12A.
  • SFPs are typically relatively short peptides, and generally are expected to have low catalytic efficiencies for reactions with target proteases.
  • the substrate may be an internally quenched fluorogenic peptide (IQFP), the mechanism of which is schematically shown in FIG. 12B.
  • IQFPs typically are expected to have relatively higher catalytic efficiencies for reactions with target proteases.
  • a peptide sequence particularly that of an IQFP, can have higher sensitivity as well as selectivity for specific protease detection.
  • Table 6 includes some examples of SFP and IQFP peptides of various sequence lengths with at least one unnatural amino acid.
  • the donor and the acceptor of the FRET pair in the biosensor each is independently attached to the C-terminus or N-terminus of a peptide derived from the substrate of SARS-CoV-1 PLpro and SARS-CoV-2 PLpro or SARS-CoV-1 3CLpro and SARS-CoV-2 3CLpro selected from those disclosed in Table 5 below.
  • the peptide is derived from the substrate of SARS-CoV-1 PLpro and SARS-CoV-2 PLpro and includes a sequence selected from LXGG, RLRGG, LRGG,
  • the peptide is derived from the substrate of SARS-CoV-1 3CLpro and SARS-CoV-23CLpro and includes a sequence selected from VRLQ, PRLQ, VKLQ, VSLQ, AVLQ, VTFQ, LQSAG, VRLQS, PRLQS, VVRLQ, VVVLQ, VAVLQ, TSAVLQ, SGVTFQ, TSDVLQ, TNAVLQ, SGFRK, SGFRR, GKFKK, GKFRK, VARLQ, and VPRLQ.
  • the peptide is derived from an unnatural substrate of SARS-CoV- 1 3CLpro and SARS-CoV-2 3CLpro.
  • the peptide comprises a sequence of four residues given by X1-X2-LQ-, wherein Xi is selected from Abu, V, A, Tie, Me, Tba, Thz, Aph,and Phg, preferably Abu, Thz; and X2 is selected from Tie, D-Phe, D-Tyr, Om, Har, Dab,
  • the peptides comprises a sequence of 4 to 6 amino acid residues including the sequence given by X1-X2-LQ-, 6 to 8 amino acid residues including the sequence given by X1-X2-LQ-, 8 to 10 amino acid residues including the sequence given by X1-X2-LQ-, 10 to 12 amino acid residues including the sequence given by X1-X2-LQ-, 12 to 14 amino acid residues including the sequence given by X1-X2-LQ-, or 14 to 16 amino acid residues including the sequence given by X1-X2-LQ-.
  • the sequence given by X1-X2-LQ- is at the C terminus, while for IQHPs, the sequence given by X1-X2-LQ- is positioned such that the unnatural amino acid residues are closer to the N- terminus of the peptide.
  • the peptide is derived from an unnatural substrate of SARS-CoV- 1 PLpro and SARS-CoV-2 PLpro.
  • the peptide comprises a sequence of at least four residues given by X1-X2-GG-, wherein Xi is selected from hTyr, hPhe, hPhe(4F), hPhe(4CF3), Abu, Abu(Bth), Me, Phg, Tba, or Tie, and X2 is selected from Dap, Dab, Lys, Orn, hArg, 4-Pip, Aph, or 2-Pal.
  • biosensor comprises ACC-Gly-hTyr-DAP- Gly-Gly-Gly-Thr-Glu-Pro-Gly-Lys(dnp)-Lys-MTR, ACC-Gly-hTyr-DAP-Gly-Gly-Ala-Ile-Thr- Lys(dnp)-Lys-MTR, ACC-Gly-hPhe-DAP-Gly-Gly-Gly-Thr-Glu-Pro-Gly-Lys(dnp)-Lys-MTR, or ACC-Gly-hTyr-Phe(guan)-Gly-Gly-Gly-Thr-Glu-Pro-Gly-Lys(dnp)-Lys-MTR, wherein R is H-, CH3CO-, H0C(0)-(CH2)m-C0-, or H(-0-CH2CH2)p-, and wherein n and m are independently 1-4 and p is 2-100.
  • the peptides comprises a sequence of 4 to 6 amino acid residues including the sequence given by X1-X2-GG-, 6 to 8 amino acid residues including the sequence given by X1-X2-GG-, 8 to 10 amino acid residues including the sequence given by X1-X2-GG-, 10 to 12 amino acid residues including the sequence given by X1-X2-GG-, 12 to 14 amino acid residues including the sequence given by X1-X2-GG-, or 14 to 16 amino acid residues including the sequence given by X1-X2-GG-.
  • the sequence given by X1-X2-GG- is at the C terminus, while for IQHPs, the sequence given by X1-X2-GG- is positioned such that the unnatural amino acid residues are closer to the N-terminus of the peptide.
  • the biosensor comprises rhodamine 6G/fluorescein, rhodamine 6G/FITC, or Rho G/QSY7 attached to a peptide derived from the substrate of SARS-CoV-1 PLpro and SARS-CoV-2 PLpro and includes a sequence selected from LXGG, RLRGG, LRGG, LKGG, RLKGG, MRGG, IRGG, MKGG, IKGG, LSGG, LYGG, LNGG, LDGG, LQGG, LEGG, RLEGG, LHGG, LQGG, LTGG, SLKGG, and KKAG.
  • the biosensor comprises rhodamine 6G/fluorescein, rhodamine 6G/FITC, or Rho G/QSY7 attached to a consensus peptide sequence LXGG that is recognized by SARS-CoV-1 PLpro and SARS- CoV-2 PLpro.
  • the biosensor comprises rhodamine 6G/fluorescein, rhodamine 6G/FITC, or Rho G/QSY7 attached to a synthetic 12-mer peptide (ERELNGGAPIKS) derived from the nspl/nsp2 and nsp2/nsp3 sequences that are recognized by SARS-CoV-1 PLpro and SARS-CoV-2 PLpro 1541-2204.
  • the biosensor comprises rhodamine 6G/fluorescein attached to a consensus peptide sequence LXGG that is recognized by SARS-CoV-1 PLpro and SARS-CoV-2 PLpro.
  • the biosensor comprises rhodamine 6G/fluorescein attached to a synthetic 12-mer peptide (ERELNGGAPIKS) derived from the nspl/nsp2 and nsp2/nsp3 sequences that are recognized by SARS-CoV-1 PLpro and SARS-CoV-2 PLpro 1541-2204.
  • ERELNGGAPIKS synthetic 12-mer peptide
  • the biosensor comprises rhodamine 6G/fluorescein, or Rho G/QSY7 attached to a peptide derived from the substrate of SARS-CoV-1 3CLpro and SARS- CoV-2 3CLpro and includes a sequence selected from VRLQ, PRLQ, VKLQ, VSLQ, AVLQ, VTFQ, LQSAG, VRLQS, PRLQS, VVRLQ, VVVLQ, VAVLQ, TSAVLQ, SGVTFQ, TSDVLQ, TNAVLQ, SGFRK, SGFRR, GKFKK, GKFRK, VARLQ, and VPRLQ.
  • the biosensor comprises rhodamine 6G/fluorescein, rhodamine 6G/FITC, or Rho G/QSY7 attached to a consensus peptide sequence LQSAG that is recognized by SARS-CoV-1 3CLpro and SARS-CoV-2 3CLpro.
  • the biosensor comprises rhodamine 6G/fluorescein, rhodamine 6G/FITC, or Rho G/QSY7 attached to a synthetic 10-mer peptide (AVLQSGFRKK) derived from the natural non- structural proteins that are recognized by SARS- CoV-1 3CLpro and SARS-CoV-2 3CLpro.
  • the biosensor comprises rhodamine 6G/fluorescein attached to a consensus peptide sequence LQSAG that is recognized by SARS-CoV-1 3CLpro and SARS-CoV-2 3CLpro. In some embodiments, the biosensor comprises rhodamine 6G/fluorescein attached to a consensus peptide sequence LQSAG that is recognized by SARS-CoV-1 3CLpro and SARS-CoV-2 3CLpro. In some embodiments, the biosensor comprises rhodamine 6G/fluorescein attached to a synthetic 10-mer peptide (AVLQSGFRKK) derived from the natural non- structural proteins that are recognized by SARS- CoV-1 3CLpro and SARS-CoV-2 3CLpro.
  • AOLQSGFRKK synthetic 10-mer peptide
  • the peptide is derived from an unnatural substrate of SARS-CoV- 1 3CLpro and SARS-CoV-23CLpro.
  • the peptide comprises a sequence of at least four residues given by X1-X2-X3-Q-, wherein Xi is selected from Abu, Val, Ala, Tie,
  • Tba, Phg, Me, and other small, aliphatic amino acids; X2 is selected from Tie, D-Phe, D-Tyr,
  • the FRET donor/acceptor pair are chemically linked to the peptide derived from the substrate of the virus encoded enzyme via a reactive functional group on the side chain of an amino acid residue in the peptide sequence or the peptide terminal groups (C-terminus or N-terminus), and the FRET donor/acceptor pair have reactive functional groups including an amine group (-NH2), a carboxyl group (-COOH), a hydroxyl group (-OH), sulfonyl group (-SO2CI), succinimidyl ester, or a thiol group (-SH).
  • the chemical linkage between the FRET donor/acceptor pair and the peptide derived from the substrate of the virus encoded enzyme comprises an amide bond formed by MTS/EDC chemistry.
  • EDANS is chemically linked to the 1st glutamic acid on N- terminus via an amide bond formed between the amine group as highlighted in the square of EDANS formula the carboxylic acid on the side chain of the 1st glutamic acid residue.
  • Dabcyl is chemically linked to the 11th lysine residue of the C-termus via an amide bond formed between the amine group on the side chain of lysine residue and the carboxylic acid as highlighted in the square of DABCYL formula
  • SFP SFP
  • SFP SFP
  • SFP SFP
  • SFP SFP
  • b Ac-Gly-Ala-Gly-hPhe-Dap-Gly-Gly- ACC
  • SFP Ac-Ala-Gly-Thr-Thz-Tle-Leu-Gln-ACC
  • SFP SFP
  • c Ac-Gly-Ala-Trp-hTyr-Dap-Gly-Gly- ACC
  • SFP SFP
  • Lys(dnp)-NH2 Lys(dnp)-NH2 (IQFP)
  • IQFP Lys(dnp)-NH2
  • b ACC-Gly- hTyr-DAP -Gly-Gly-Gly-Thr-Glu- b.
  • Lys(dnp)-Lys-NH2 Arg-Lys(dnp)-NH2 (IQFP)
  • b ACC-Gly- hTyr-Dap -Gly-Gly-Gly-Thr-Glu- b.
  • IQFP IQFP
  • IQFP IQFP
  • b ACC-Gly- hTyr-Dap -Gly-Gly-Gly-Thr-Glu-
  • b ACC-Gly -Thz -Tle-Leu-Gln-Ser-Gly -Phe-
  • 16-mer a ACC-Gly- hTyr-Dap -Gly-Gly-Ala-Ile-Thr- a. ACC-Gly -Abu-Tle-Leu-Gln-Ser-Gly-Phe- Arg- Val-Thr-Phe-Gly-Lys(dnp)-Lys-Lys-NH2 Arg-Ser-Ala-Val-Lys(dnp)-Arg-Lys-Lys-NH2 (IQFP) (IQFP) b. ACC-Gly- hTyr-Dap -Gly-Gly-Gly-Thr-Glu- b.
  • hTyr can be replaced with hPhe while for 3CLpro sequences, Abu can be replaced by Thz.
  • the non-natural amino acid Dap may be replaced by Dab.
  • Lys(dnp)- A modified Lysine amino acid derivative with quencher, dnp or dinitrophenyl which is the preferred quencher for ACC. Used in IQFP
  • this disclosure provides a biosensor having Formula (15) A-Y-LSP, wherein A is derived from a peptidic substrate for or a chemical inhibitor of an enzyme encoded by SARS-CoV-1, SARS-CoV-2, or MERS-CoV, LSP is a spectroscopic probe chosen from a group consisting of coumarins, rhodamines, rhodols, fluoresceins, xanthenes, phenothiazines, and phenoxazines; Y is a bond, NRa, O, or S; Ra is H or Ci- 6 alkyl; wherein LSP is capable of changing color to provide a detectable signal after exposure to the enzyme encoded by SARS- CoV-1, SARS-CoV-2, or MERS-CoV.
  • A is derived from a peptidic substrate for or a chemical inhibitor of an enzyme encoded by SARS-CoV-1, SARS-CoV-2, or MERS-CoV
  • LSP
  • the biosensor of Formula (15) has the A selected from the group LSP selected from the group of a spectroscopic probe having Formula (
  • Y is a bond, N, O, or S
  • U is O or N
  • W is O, N, S or CH 2 ,
  • R 14 , and R 15 are each independently selected from the group of H, substituted and unsubstituted C1-C12 alkyl group, substituted and unsubstituted C1-C12 alkenyl group, substituted and unsubstituted C1-C12 alkynyl group, and substituted and unsubstituted aryl group;
  • R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , and R 22 is each independently selected from the group of H, Cl, F, Br, CN, NO2, -NR 14 R 15 , C1-C6 alkyl group, and C1-C6 alkoxyl group;
  • R 23 , R 24 , R 25 , R 26 and R 27 are each independently selected from the group of H, C1-C2 alkyl group, C1-C2 fluoroalkyl group; and wherein when A is the peptide derived from the substrate of the enzyme encoded by SARS-CoV-1, SARS-CoV-2, or MERS-CoV, LSP is chemically linked to A via Y to a reactive functional group on the side chain of an amino acid residue, or to the reactive functional group on any one of the C-terminus or N-terminum amino acid residue of the peptide.
  • LSP is chemically linked to the reactive functional group via Y on the side chain of an amino acid residue in the peptide sequence and the reactive functional group is selected from an amine group, an amide group, a carboxyl group, a hydroxyl group, a thiol group, and a sulfhydryl group.
  • LSP is chemically linked to any one of the C-terminus or N-terminum group of the peptide via Y and the reactive functional group on any one of the C-terminus or N-terminum amino acid residue is selected from an amine group, an amide group, a carboxyl group, a hydroxyl group, a thiol group, and a sulfhydryl group.
  • D of Formula (15) is a leuco colorless dye component having
  • the colorless leuco dye component of Formula (19) is the colorless leuco dye component of Formula (19)
  • the biosensor of Formula (19) is defined when Z is or - O-CFh-Ph.
  • the biosensor of Formula (15) is defined when R 14 , R 15 are each independently selected from the group of methyl and ethyl group.
  • the biosensor of Formula (15) is defined when Y is a bond or N and LSP is selected from the group of Formula ( Formula (9) Formula (11) , Formula (13) g y ,
  • the biosensor of Formula (15) is defined when Y is a bond or N, and A is selected from the group cyan colored state, Formula (17) , Formula (23) or Formula (24)
  • the biosensor of Formula (15) is defined when Y is a bond or N, A is selected from the group
  • the biosensor of Formula (15) is defined when Y is a bond or N, A is selected from the group
  • the biosensor of Formula (15) is defined when Y is a bond or N, A is selected from the group
  • the biosensor of Formula (15) is defined when Y is a bond or N, A
  • the biosensor of Formula (15) is defined when Y is a bond or N, A [0185] In some embodiments, the biosensor of Formula (15) is defined when Y is a bond or N, A
  • the biosensor of Formula (15) is defined when Y is a bond or N, A is selected from the group
  • the biosensor of Formula (15) is defined when Y is a bond or N, and A is selected from the group
  • the biosensor of Formula (15) is defined when Y is a bond or N, and A is selected from the group
  • the biosensor of Formula (15) is defined when Y is a bond or N, and A is selected from the group
  • the biosensor of Formula (15) is defined when Y is a bond or N, and A is selected from the group
  • the biosensor of Formula (15) is defined when Y is a bond or N, A is selected from the group
  • the biosensor of Formula (15) is defined when Y is a bond or N, A is selected from the group
  • the biosensor of Formula (15) is defined when Y is a bond or N, A is selected from the group and LSP is selected from the group of Formula , , having a cyan colored
  • A is a peptide derived from the substrate of coronavirus 3CLpro and A is selected from the group of Leu, Val, Val-Leu, Val-Phe, Ala-Val-Leu, Ser-Val-Phe, Val- Thr-Phe-Gln, Val-Thr-Leu-Gln, Val-Thr-Ala-Gln, Glu-Ser-Ala-Thr-Leu-Gln-Ser-Gly-Leu-Ala- Lys-Ser, Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys, and Thr-Ser-Ala-Val-Leu-Gln-Ser- Gly-Phe-Arg-Lys-Met.
  • A comprises Val-Leu, Val-Phe, Ala-Val-Leu, and Ser-Val-Phe. In some embodiments, A comprises Glu-Ser-Ala-Thr-Leu-Gln-Ser-Gly-Leu-Ala- Lys-Ser. In some embodiments, A comprises Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys- Met.
  • A is a tetrapeptide, pentapeptide, or hexapeptide that is recognized as a substrate for cleavage by either a PLpro or 3CLpro enzyme encoded by a virus.
  • A is a peptide derived from the substrate of coronavirus 3CLpro or PLpro and A is selected from a peptide disclosed in Table 5. In some embodiments, A is a peptide derived from the substrate of coronavirus PLpro and A is selected from LXGG,
  • A is a peptide derived from the substrate of coronavirus 3CLpro and A is selected from VRLQ, PRLQ, VKLQ, VSLQ, AVLQ, VTFQ, LQSAG, VRLQS, PRLQS, VVRLQ, VVVLQ, VAVLQ, TSAVLQ, SGVTFQ, TSDVLQ, TNAVLQ, SGFRK, SGFRR, GKFKK, GKFRK, VARLQ, and VPRLQ.
  • A is an unnatural peptide 25 comprising at least four residues, wherein R 23 and R 24 are H or alkyl, R 25 is an alkyl amine, R 26 is -(CH2)n-Ar, and R 27 is H-, CH3CO-, FhN-(CFl2)m-CC ) -, or H(-0-CH2CH2)p-, and wherein n and m are independently 1-4 and p is 2-100.
  • A is an unnatural peptide selected from Ac-hTyr-Dap-Gly-Gly, Arg-hTyr-Dap-Gly-Gly, Ac-hPhe-Dap-Gly-Gly, Ac-hPhe(4F)-Dap-Gly-Gly, and Ac- hPhe(4CF3)-Dap-Gly-Gly, Ac-Abu(Bth)-Dap-Gly-Gly, Ac-Abu(Bth)-Dab-Gly-Gly, Ac-hTyr- Lys-Gly-Gly, Ac-Abu(Bth)-Lys-Gly-Gly, Ac-Abu-Tle-Leu-Gln, Ac-hPhe-Lys-Gly-Gly, Ac- hTyr-hTyr-Gly-Gly, hPhe-Dap-Gly-Gly, Suc-hP
  • A is an unnatural peptide comprising at least four residues represented by the sequence -Gln-R28-R29-R3o, wherein R28 is Leu, 2-Abz, 3-Abz, 4-N02-Phe, b- Ala, Dht, hLeu, Ilu, or Met; R29 is Tie, D-Phe, D-Tyr, Om, hArg, Dab, Dht, Lys, D-Phg, D-Trp, Arg, or Met(0)2; and R30 is a small aliphatic residue such as Abu, Val, Ala, Tie, wherein the N- terminus of the sequence is bound to H-, CH3CO-, HOC(0)-(CH2)m-CO-, or Fh-O-CFLCFhV, and wherein n and m are independently 1-4 and p is 2-100.
  • A is a peptide derived from the substrate of SARS-CoV PLpro and A is ubiquitin or ISG15 protein.
  • the SARS-Cov encoding PLpro is SARS-CoV-1 or SARS-CoV-2 PLpro.
  • the coronavirus is SARS-CoV-1. In some embodiments, the coronavirus is SARS-CoV-2.
  • A is a peptide derived from the substrate of coronavirus 3CLpro selected from the group of Leu, Val, Val -Leu, Val-Phe, Ala-Val-Leu, Ser-Val-Phe, Leu-X-Gly- Gly, Val-Thr-Phe-Gln, Val-Thr-Leu-Gln, Val-Thr-Ala-Gln, Glu-Ser-Ala-Thr-Leu-Gln-Ser-Gly- Leu-Ala-Lys-Ser, Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys, Glu-Arg-Glu-Leu-Asn-Gly- Gly-Ala-Pro-Ile-Lys-Ser, and Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met; and LSP is
  • the biosensor of Formula (15) has A is Thr-Ser-Ala-Val-Leu-Gln-
  • the biosensor of Formula (15) has A is a peptide having a sequence selected from the group of LQSAG, VRLQS, or PRLQS, and D selected from the group of Formula o , Formula (22) (Et ,Me) , Formula (23) attached to D via Y at Q residue.
  • the biosensor is a molecule comprising two fragmentable groups, represented by A-Y-D-Y-B wherein D is a spectroscopic probe, Y is an O, N, or S atom, and A and B are selected from of a natural or unnatural peptide
  • a and B are the same. In some embodiments, A and B differ. In some embodiments, A is selected from groups that are substrates (or derived from substrates, inhibitors or therapeutics) for PLpro described herein and B is selected from peptides that are substrates (or derived from substrates, inhibitors or therapeutics) for 3CLpro described herein.
  • the biosensor where the leaving groups A and B may be separately cleaved by PLpro and by 3CLpro enzymes.
  • A can be cleaved by PLpro and B can be cleaved by 3CLpro, or A can be cleaved by 3CLpro and B can be cleaved be PLpro.
  • indoaniline dyes can be produced by the oxidative coupling of p-phenylene diamines with phenols.
  • US Pat. No. 4,681,841 teaches an assay for enzymes based upon such color-forming chemistry.
  • the biosensor is provided by a combination of any of the compounds and color couplers known in the art such as, for example, those described in US Pat. No. 4,681,841, which is incorporated herein by reference in its entirety.
  • the enzyme is encoded by SARS-CoV-2. In some embodiments, the enzyme is encoded by SARS-CoV-1. In some embodiments, the enzyme is encoded by MERS-CoV. In some embodiments, the enzyme is virally encoded cysteine protease. In some embodiments, the enzyme is papain like virus protease encoded by SARS-Cov-1, SARS-CoV-2 or MERS-CoV. In some embodiments, the enzyme is SARS-CoV-1 PLpro, or SARS-CoV-1 3CLpro. In some embodiments, the enzyme is SARS-CoV-2 PLpro, or SARS-CoV-23CLpro.
  • the enzyme is MERS-CoV-1 3CLpro, SARS-CoV-2 3CLpro or MERS-CoV 3CLpro. In some embodiments, the enzyme is MERS-CoV-1 PLpro, SARS-CoV-2 PLpro or MERS-CoV PLpro.
  • the library of biosensors is based on a platform containing other leuco dyes including but not limited to, spiropyran, quinone, thiazine, phenazine, oxazine, pthalide-type, triarylmethanes, fluoran, and tetrazoliums.
  • leuco dyes including but not limited to, spiropyran, quinone, thiazine, phenazine, oxazine, pthalide-type, triarylmethanes, fluoran, and tetrazoliums.
  • the library of biosensors is based on a platform containing naturally occurring dyes including but not limited to, curcumins, hypericin, carotenes, anthocynanins, and any other phytochemical dyes.
  • the library of biosensors is based on a platform containing synthetic dyes that may not be leuco dyes for e.g. azo dyes, coumarins, xanthenes, phthalides and azomethine dyes.
  • Screening such library of biosensors will enable us to identify one or more molecules that can be used as a biosensor for sensitive and specific detection of certain viruses.
  • this disclosure provides a composition containing a biosensor for the detection of viruses having a Formula (1) D-FG or Formula (15) A-Y-LSP as described above, wherein (i) FG or A comprising a material responsive to an enzyme encoded by a target virus selected from the group of SARS-CoV-1, SARS-CoV-2, or MERS-CoV; and (ii) D or LSP is a spectroscopic probe, wherein the FG or A masks the activity of the spectroscopic probe D or LSP, wherein the enzyme causes the cleavage of the FG or A to release the spectroscopic probe D or LSP, resulting in a detectable optical response; and wherein the enzyme is present in a virus-infected host cell and is released outside for access by the biosensor when the host cell is ruptured.
  • D-FG or Formula (15) A-Y-LSP as described above, wherein (i) FG or A comprising a material responsive to an enzyme encoded by a target virus selected from the group of
  • the enzyme encoded by the target virus is a protease.
  • the enzymes encoded by the target virus comprise proteases that are necessary for viral replications.
  • the enzyme encoded by the target virus is a serine protease.
  • the enzyme encoded by the target virus is a microbial cysteine protease.
  • the enzyme encoded by the target virus is selected from the group of 3CLpro and PLpro encoded by coronavirus.
  • the enzyme encoded by the target virus is 3CLpro encoded by coronavirus.
  • the optical response is a color change within the visible region of the electromagnetic spectrum. In some embodiments, the optical response is fluorescence.
  • the two or more colors of the two or more biosensors are selected to give a mixed color having sufficient difference such that each of the two colors are visually discemable by naked eye.
  • the released spectroscopic probe gives a discrete blue color having the absorption wavelength in the visible light spectrum ranging from 400 nm to 500 nm. In some embodiments, the released spectroscopic probe gives a discrete red color having the absorption wavelength in the visible light spectrum ranging from 400 nm to 600 nm.
  • the present invention is related to formulations containing a colorimetric biosensor useful for the detection of viruses like coronaviruses using visible color-change technology.
  • the target enzymes are produced inside infected host cells.
  • the enzyme in order to enable the reaction of the substrate included in the biosensor of the present disclosure with a target enzyme, the enzyme must be released from the infected cell into the fluid comprising a biosensor.
  • the release of the enzyme may be the result of chemophysical action, as in for example the action of a detergent or a surfactant to lyse cells.
  • the enzyme may be released as a result of mechanical action such as produced by the action of a homogenizer or microfluidizer.
  • Methods of cell lysis have been reviewed (Islam, et al., Micromachines 2017, 8, 83-119). The cell lysis may be performed prior to the introduction of the biosensor, or it can be performed upon introduction of the biosensor, such as may be initiated by a detergent in a diagnostic composition.
  • a diagnostic composition may comprise a biosensor as described herein and a carrier.
  • the carrier comprises an organic solvent that is compatible with biological samples.
  • the carrier comprises a biological buffer.
  • the carrier comprises a surfactant or a detergent.
  • the diagnostic composition may comprise two or more biosensors, wherein each sensor independently has a masked spectroscopic probe giving a discrete optical response after release.
  • the independent optical response is a fluorescent response to produce emission at multiple wavelengths.
  • the optical response is a color formation (optical response is from a colorless state to color state).
  • the two or more colors of the two or more biosensors are selected to give a mixed color having sufficient difference such that each of the two colors are visually discemable by naked eye.
  • the two or more biosensors are responsive to different enzymes.
  • a first biosensor is responsive to PLpro enzyme and a second biosensor is responsive to 3CLpro enzyme.
  • a first biosensor is responsive to PLpro enzyme, a second biosensor is responsive to 3CLpro enzyme and a third biosensor is responsive to a human enzyme.
  • the diagnostic composition may comprise two or more biosensors, wherein each sensor independently has a spectroscopic probe exhibiting a unique color and produces a detectable optical response from colored state to colorless state.
  • the two or more colors of the two or more biosensors are selected to give a mixed color that has sufficient difference such that each of the two colors are visually discernable by naked eye.
  • the diagnostic composition comprises three different biosensors each having a cyan, magenta and yellow color probe.
  • the mixture of the three biosensors gives black color. If the biosensor having cyan color is released by the enzyme encoded by the virus, then the diagnostic composition would produce red color. Similarly, if the biosensor having magenta color is released by the enzyme encoded by the virus, then the diagnostic composition would produce green color. Further, if the biosensor having yellow color is released by the enzyme encoded by the virus, then the diagnostic composition would produce blue color. If any two of the color biosensors are activated by the enzymes encoded by the virus, then the diagnostic composition would produce a color change from black to the remaining subtractive primary color i.e. either cyan, magenta or yellow.
  • the diagnostic composition further incorporates an enzyme inhibitor.
  • the inhibitor is a broad-spectrum deubiquitinase (DUB) inhibitor.
  • the broad spectrum DUB inhibitor is 2,6-Diamino-3,5- dithiocyanopyridine.
  • the inhibitor comprises a dihydropyrrole.
  • the DUB inhibitor is a tricyclic heterocyclic compound.
  • the DUB inhibitor is PX-478.
  • the DUB inhibitor comprises 6-amino- pyrimidine.
  • the inhibitor is a proteasome inhibitor.
  • the proteosome inhibitor is MG132 (Cbz-Leu-Leu-Leucinal).
  • the proteosome inhibitor is b-AP15.
  • the proteosome inhibitor comprises azepan-4-one.
  • the inhibitor is a viral 3C protease inhibitor. In some embodiments, the inhibitor is a human rhinovirus 3C protease inhibitor. In some embodiments, the inhibitor is a human enterovirus 3C protease inhibitor. In some embodiments, the 3C protease inhibitor is SG85. In some embodiments the 3C protease inhibitor is luteoloside.
  • the inhibitor is an inhibitor of ubiquitin C-terminal hydrolase (UCH). In some embodiments, the inhibitor is an inhibitor of UCH-L1. In some embodiments, the inhibitor is an inhibitor of UCH-L3. In some embodiments, the inhibitor is an inhibitor of UCH-L5. In some embodiments, the inhibitor is 4,5,6,7-tetrachloro-l,3-indanedione (TCID). In some embodiments, the inhibitor is an acylated oxime isatin derivative.
  • UCH ubiquitin C-terminal hydrolase
  • the biosensor further comprises a solid support, wherein the fragment component is bound to the solid support via a covalent bond or via electrostatic interaction.
  • the biosensor further comprises a solid support, wherein the spectroscopic probe is covalently bound to the solid support.
  • the solid support is selected from the group of a particle, fiber, a microgel, a wound dressing, a catheter, a membrane, a resin, a sponge, a sheet, a suture, an implant scaffold, a stent, a swab, a hydrogel, a film, a patch, a tape, a woven fabric, and a nonwoven fabric.
  • the biosensor is rendered onto a paper or plastic film strip comprising both lysing agent and biosensor composition for placement onto a tongue or mouth and then observing for a color change.
  • the solid support is a paper impregnated with a biosensor.
  • the paper impregnated with a biosensor is prepared by treating a paper substrate with a biosensor solution in a solvent such as a volatile solvent including dichloromethane, ethylacetate, hexane, methanol, ethanol, or non-volatile solvent including polyethylene glycol, or glycerol.
  • the solid support is a hydrogel or microgel impregnated with a biosensor solution in a solvent such as water, aqueous solution of methanol, aqueous solution of ethanol, polyethylene glycol, or glycerol.
  • a solvent such as water, aqueous solution of methanol, aqueous solution of ethanol, polyethylene glycol, or glycerol.
  • the solid support is a woven fabric or a nonwoven fabric impregnated with a biosensor solution in a solvent such as volatile solvent including dichloromethane, ethylacetate, hexane, methanol, ethanol, or non-volatile solvent including polyethylene glycol, or glycerol.
  • a solvent such as volatile solvent including dichloromethane, ethylacetate, hexane, methanol, ethanol, or non-volatile solvent including polyethylene glycol, or glycerol.
  • the solid support is a microgel comprising a dendritic polymer.
  • Dendrimers are also classified by generation, which refers to the number of repeated branching cycles that are performed during its synthesis. For example, if a dendrimer is made by convergent synthesis, and the branching reactions are performed onto the core molecule three times, the resulting dendrimer is considered a third generation dendrimer (G3). Each successive generation results in a dendrimer roughly twice the molecular weight of the previous generation. Higher generation dendrimers also have more exposed functional groups on the surface, which can later be used to customize the dendrimer for a given application.
  • the dendritic polymer is selected from hyperbranched PEG dendrimers, PEG core dendrimers, hyperbranched polyglycerol dendrimers, hyperbranched polylysine dendrimers, hyperbranched polyesters, alkyne-terminated dendrimers, amine terminated PEG-core dendrimers, azide terminated dendrimers, 2,2-bis(methylol)propionic acid (bis-MPA) dendrimers, carboxylic acid terminated dendrimers, Poly(amidoamine) (PAMAM) dendrimers, polyethylenimine dendrimers (PEI), and combinations thereof.
  • hyperbranched PEG dendrimers PEG core dendrimers
  • hyperbranched polyglycerol dendrimers hyperbranched polylysine dendrimers
  • hyperbranched polyesters alkyne-terminated dendrimers
  • amine terminated PEG-core dendrimers alkyne-terminated dendrimers
  • bis-MPA
  • the dendritic polymer has reactive surface group available for biosensor conjugation selected from the group of 8 surface groups, 16 surface groups, 32 surface groups, 64 surface groups, and 128 surface groups.
  • the dendrimer is a polyester bis-MPA dendrimer (tert-butylic acid protected amine core, 8 alkyne end groups, G3, branching units bis-MPA).
  • These alkyne- functionalized dendrimers can be readily functionalized using either copper (I)-catalyzed alkyne- azide cycloaddition (CuAAC), strain-promoted alkyne-azide cycloaddition (SPAAC), or thiol- yne click reactions.
  • the amine-functionalized core can be readily used in EDC or DCC coupling reactions (after Boc deprotection) with carbonyl-containing compounds to yield highly functionalized materials for a variety of biomedical applications.
  • the biosensor as disclosed herein is modified with an azide group and is conjugated with the G3 polyester bis-MPA dendrimer via click chemistry.
  • the dendritic polymer is selected from the group of bis-MPA hyperbranched PEG10k-OH dendrimer (10K PEG core, pseudo generation 2, bis-MPA branching units, 8 surface hydroxyl groups, Mw 10696 Da), bis-MPA hyperbranched PEGlOk- OH dendrimer (10K PEG core, pseudo generation 3, bis-MPA branching units, 16 surface hydroxyl groups, Mw 11643 Da), bis-MPA hyperbranched PEG20k-OH dendrimer (20K PEG core, pseudo generation 2, bis-MPA branching units, 8 surface hydroxyl groups, Mw 20759 Da), bis-MPA hyperbranched PEG20k-OH dendrimer (20K PEG core, pseudo generation 3, bis-MPA branching units, 16 surface hydroxyl groups, Mw 21688 Da), bis-MPA hyperbranched PEG6k- OH dendrimer (6K PEG core, pseudo generation 4, bis-MPA branching units, 32 surface hydroxy
  • the dendritic polymer is selected from the group of G2 polylysine, G3 polylysine, G4 polylysine, G5 polylysine, and G6 polylysine.
  • a spacer can link the biosensor to the dendrimer.
  • the spacer links biosensor and dendrimer via an amide bond formed by NHS/EDC chemistry.
  • the spacer links biosensor and the dendrimer via a disulfide (S- S) bond.
  • the spacer links biosensor and the dendrimer via triazoles formed by copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC), or strain-promoted alkyne- azide cycloaddition (SPAAC), or thiol-yne click reactions.
  • the biosensor comprises 3CLpro or PLpro enzyme inhibitor fragment -(amino-(spacer)x)y-dendrimer or dendrimer-(spacer)z-3CLpro or PLpro enzyme inhibitor fragment, wherein the spacer has from 2 to 50 atoms, x, y and z are integers from 5 to 15.
  • the bond joining the spacer and the 3CLpro or PLpro enzyme inhibitor fragment is a degradable bond selected from the group of an ester bond, an amide bond, an imine bond, an acetal bond, a ketal bond, and combinations thereof.
  • the spacer is selected from the group of polyethylene glycol having 2-50 repeating units, e-maleimidocaproic acid, para-aminobenzyloxy carbamate, and combinations thereof.
  • the spacer comprises polyamino acid having 2-30 amino acid residues.
  • the spacer comprises a linear polylysine, or polyglutamine.
  • the concentration of the biosensor provided in the diagnostic compositions of the invention is less than, for example, 100 %, 90 %, 80 %, 70 %, 60 %, 50 %, 40 %, 30 %, 20 %, 19 %, 18 %, 17 %, 16 %, 15 %, 14 %, 13 %, 12 %, 11 %, 10 %, 9 %, 8 %, 7 %, 6 %, 5 %, 4 %, 3 %, 2 %, 1 %, 0.5 %, 0.4 %, 0.3 %, 0.2 %, 0.1 %, 0.09 %, 0.08 %, 0.07 %, 0.06 %, 0.05 %, 0.04 %, 0.03 %, 0.02 %, 0.01 %, 0.009 %, 0.008 %, 0.007 %, 0.006 %, 0.005 %, 0.004 %, 0.003 %,
  • the concentration of the biosensor provided in the diagnostic compositions of the invention is independently greater than 90 %, 80 %, 70 %, 60 %, 50 %, 40 %, 30 %, 20 %, 19.75 %, 19.50 %, 19.25 %, 19 %, 18.75 %, 18.50 %, 18.25 % 18 %, 17.75 %,
  • the concentration of the biosensor provided in the diagnotsic compositions of the invention is independently in the range from about 0.0001 % to about 50 %, about 0.001 % to about 40 %, about 0.01 % to about 30 %, about 0.02 % to about 29 %, about 0.03 % to about 28 %, about 0.04 % to about 27 %, about 0.05 % to about 26 %, about 0.06 % to about 25 %, about 0.07 % to about 24 %, about 0.08 % to about 23 %, about 0.09 % to about 22 %, about 0.1 % to about 21 %, about 0.2 % to about 20 %, about 0.3 % to about 19 %, about 0.4 % to about 18 %, about 0.5 % to about 17 %, about 0.6 % to about 16 %, about 0.7 % to about 15 %, about 0.8 % to about 14 %, about 0.9 % to about 12 %
  • the concentration of the biosensor provided in the diagnostic compositions of the invention is independently in the range from about 0.001 % to about 10 %, about 0.01 % to about 5 %, about 0.02 % to about 4.5 %, about 0.03 % to about 4 %, about 0.04 % to about 3.5 %, about 0.05 % to about 3 %, about 0.06 % to about 2.5 %, about 0.07 % to about 2 %, about 0.08 % to about 1.5 %, about 0.09 % to about 1 %, about 0.1 % to about 0.9 % w/w, w/v or v/v.
  • sample refers to any substance containing or presumed to contain one or more SARS-CoV pathogen including, but not limited to, clinical samples (e.g., tissue or fluid isolated from one or more subjects or individuals), in vitro cell culture constituents, environmental samples, and the like. Samples may be obtained from any body geography known to exhibit the presence of virally infected cells, including, but not limited to, the mouth, the nose, the upper and the lower respiratory tract. Exemplary sample types include blood, plasma, serum, feces, bronchoalveolar lavage, nasal samples, oral samples, NP cavity samples, OP area samples, oral buccal samples, tongue scrape samples, urine, synovial fluid, mucus, or sputum.
  • clinical samples e.g., tissue or fluid isolated from one or more subjects or individuals
  • samples may be obtained from any body geography known to exhibit the presence of virally infected cells, including, but not limited to, the mouth, the nose, the upper and the lower respiratory tract.
  • Exemplary sample types include blood, plasma, serum,
  • the sample is selected from the group of NP swab samples, nasal swab samples, oral swab samples, sublingual swab samples, parotid duct opening swab samples, OP swab samples, buccal swab samples, or tongue scrape samples, an aerosol sample collected in a respiratory mask, masticated oral sample and the like.
  • a biological sample (5) provided by a patient for analysis is placed into the sample tube (4).
  • the sample is a tongue scrape sample.
  • the sample is a saliva sample.
  • the sample is a sputum sample.
  • the sample is a scraping (2) from buccal mucosa.
  • the sample is a nasal swab sample.
  • this disclosure provides a device for sample collection such as a respiratory mask used for collecting aerosolized droplets containing virus, cells, and enzymes.
  • the mask is aN95 respirator mask.
  • the respiratory mask has been used by a patient or a health care provider.
  • the respiratory mask has been only exposed in the open air for a ceratin amount of time (e.g., 2 hours, 4 hours, 8 hours, one day, two days, three days, or more); in these instances, the respiratory mask can be used to identify the presence of viruses where the respiratory mask has been exposed in the open air.
  • Some embodiments disclose a method for collecting samples from the mouth by mastication using a masticant such as a gum or other agent.
  • a masticant such as a gum or other agent.
  • the masticant agent can have moieties to enable retention of cells and/or virus particles
  • the sample in the sample tube is held for a period of time to perform the inactivation of a pathogen. In some embodiments, the inactivation time is at least five minutes. In some embodiments, the sample tubes of multiple patients are maintained in a sample tray (6). In some embodiments, the sample tray holds at least 10 tubes.
  • this disclosure provides a method for detecting the presence or absence of a viruses in a sample from a human subject.
  • the sample from the human subject is selected from the group of blood, plasma, serum, feces, bronchoalveolar lavage, nasal swab sample, oral swab sample, sublingual swab samples, parotid duct opening swab samples, nasopharyngeal (NP) swab sample, oropharyngeal (OP) swab sample, buccal swab sample, tongue scrape sample, urine, synovial fluid, mucus, or sputum.
  • the sample is a masticated oral sample.
  • the method of collection uses a swab device, such as a buccal swab, a nasal swab, an OP swab, a NP swab, or the like.
  • the sample is collected with a respiratory mask
  • the sample is collected using a masticating device, such as a chewing gum.
  • a tongue scrape sample can be obtained by scraping the tongue of a subject using a tongue scraper such as, for example, the one illustrated in FIG. 11. The debris collected in the tongue scraper upon scraping the tongue of the subject is then collected, e.g., a vial or a tube for further processing.
  • this disclosure provides a method for detecting the presence or absence of viruses in a sample selected from the group of blood, plasma, serum, feces, bronchoalveolar lavage, nasal swab sample, oral swab sample, sublingual swab samples, parotid duct opening swab samples, NP swab sample, OP swab sample, buccal swab sample, tongue scrape, mucus, an aerosol sample collected in a respiratory mask, a masticated oral sample, or sputum comprising the steps of: (1) obtaining the sample from a patient and placing the sample in a container, (2) adding the biosensor or diagnostic composition as disclosed herein to the sample in the container, (3) incubating the biosensor or diagnostic composition with the sample, (3) observing the absence or presence of an optical response in the sample, wherein the presence of the optical response indicates the presence of the viruses, wherein the enzyme encoded by the target virus causes the clea
  • the sample is an aerosol sample that has been collected in a respiratory mask.
  • the mask is aN95 respirator mask.
  • the sample is a masticated oral sample.
  • the sample is masticated gum.
  • this disclosure provides a method for detecting the presence or absence of viruses in a sputum sample comprising the steps of: (1) obtaining the sputum sample from a patient and placing the sputum sample in a container, (2) adding any one of herein described composition containing the biosensor or diagnostic composition to the sputum sample in the container, (3) incubating the biosensor or diagnostic composition with the sputum sample in the container, (3) observing the absence or presence of an optical response in the sputum sample, wherein the presence of the optical response indicates the presence of viruses, wherein the enzyme encoded by the target virus causes the cleavage of the material to release the spectroscopic probe, resulting in a detectable optical response.
  • the composition may include a lysis buffer to enable or facilitate release of the target enzyme from the infected host cells so that the substrate of the biosensor is accessible to the target enzyme.
  • the process of collecting the sputum sample by the patient comprising the following steps: (1) opening a sample tube; (2) the patient produces the sputum (3) the patient spits the sputum into the sample tube and this is repeated until there is enough sputum to cover the bottom of the tube (e.g., about 2 mL); (4) screwing the cap on the sample tube tightly so it does not leak; (5) labeling the sample tube and sealing the sample in a plastic bag for testing with the biosensor or diagnostic composition.
  • the composition may include a lysis buffer to enable or facilitate release of the target enzyme from the infected host cells so that the substrate of the biosensor is accessible to the target enzyme.
  • the sputum sample is split into several fractions and placed into several tubes for parallel testing including incubation with the biosensor or diagnostic composition disclosed herein.
  • DNA test by conventional nucleic acid amplification (PCR) test may also performed in parallel. The testing results from different tests are compared to confirm the presence of the viruses (e.g., coronavirus).
  • viruses e.g., coronavirus
  • the system includes at least one container holding the biosensor as diagnostic agent.
  • the system optionally further includes at least one thermal modulator operably connected to the container to modulate temperature in the container, and/or at least one material transfer component (e.g., an automated pipette, etc.) that transfers fluid to and/or from the container (e.g., for performing one or more enzyme digestion reactions in the container, etc.).
  • at least one thermal modulator operably connected to the container to modulate temperature in the container
  • at least one material transfer component e.g., an automated pipette, etc.
  • the sample container is selected from glass tube, plastic tube, an array of tubes on a rack, or biological assay plate such as 6-, 12-, 24-, 48-, 96-, 384-, 1536-well microplate.
  • the sample container is selected from glass tube, plastic tube, an array of tubes on a rack as illustrated in FIG. 3, and the like.
  • the tubes are capped with screw cap, or septum.
  • the tubes have a conical bottom.
  • the tubes have a round bottom.
  • the plastic tubes may be comprised of, but are not limited to, polypropylene or polyurethane.
  • detection of infection makes use of enzymes that are encoded by the virus and produced in infected host cells.
  • samples should be from a human body region that has high likelihood of having infected cells. It has been found that tongue samples have a greater number of infected epithelial cells.
  • a sample is collected from a tongue by brushing or scraping.
  • samples are collected by tongue scraping.
  • tongue scraping is performed with a device such as shown in Fig. 11.
  • the tongue scraper may have a shape of an L-beam, such that the edges of the L-beam scrape the tongue laterally to collect debris from the surface of the tongue.
  • the tongue scraper may be made of a suitable polymer such as, for example, polyethylene, or polypropylene, or a metal such as, for example stainless steel.
  • the tongue scraper is a single-use disposable unit that is individually packaged in a sterile packaging.
  • the tongue scraper can be a reused following sterilization.
  • the tongue scraper is made from a material that can withstand various sterilization techniques such as, for example steam sterilization or UV sterilization, without degradation in material properties.
  • various sterilization techniques such as, for example steam sterilization or UV sterilization, without degradation in material properties.
  • Other shapes and materials for the tongue scraper are contemplated within the scope of the present disclosure.
  • the sample is deactivated with a lysis buffer containing a surfactant including a non-ion and non-denaturing surfactant.
  • the sample is deactivated with a lysis buffer like TRIzol, AVL, RLT, MagMAX, and easyMAG to rupture the cells and inactivate viruses for increasing sensitivity of detection.
  • the tube further contains a lysis buffer comprising Triton X-100, Digitonin, Tween-20, etc. at different % (0.05-10%) in a buffered saline solution containing TCEP/DTT and glycerol.
  • the lysis buffer comprises the non-ion and non-denaturing surfactant including, without limitation to the specific species described herein, Triton X-100, Digitonin, Tween-20 at different % (0.05-10%) in a buffered saline solution containing TCEP/DTT and glycerol.
  • the inactivating agent is a surfactant.
  • the surfactant is a nonionic surfactant.
  • the surfactant is Triton-X.
  • the surfactant is a polysorbate surfactant.
  • the surfactant is a polyoxyethylene surfactant.
  • the inactivating agent is digitonin.
  • the sample is obtained from the patient from any body geography and placed into a container, and the sample is incubated in the container with a diagnostic composition comprising a biosensor.
  • this disclosure provides a method for detecting the presence or absence of a viruses in a sample comprising the steps of: (1) providing the composition containing the biosensor as disclosed herein, (2) incubating the composition with the sample, (3) observing the absence or presence of an optical response in the sample, wherein the presence of the optical response indicates the presence of the viruses, wherein the enzyme encoded by the target virus causes the cleavage of the material to release the spectroscopic probe, resulting in a detectable optical response.
  • the composition may include a lysis buffer to enable or facilitate release of the target enzyme from the infected host cells so that the substrate of the biosensor is accessible to the target enzyme.
  • additional enzyme can be added to the sample prior to the addition of the biosensor to enhance the sensitivity of the test. Since a minimum amount of enzyme may be required to detect an optical change in a given period of time, the additional enzyme in the sample provides a means to detect enzyme from the sample at a lower concentration. With such an enhanced sample, the rate of change of the optical change (fluorescence or absorption) can be compared to a blank comparative sample containing only the enzyme and the biosensor, with none of the biological component. An increase in the rate over the blank comparative sample is interpreted as the presence of enzyme in the biological specimen, with the amount of increase providing information about the degree of infection represented by the sample.
  • the additional enzyme is 3CLpro.
  • the additional enzyme is PLpro.
  • the additional enzyme is a generic protease that has been demonstrated to convert the biosensor. In some embodiments, more than one type of additional enzyme is added.
  • this disclosure provides a method for detecting the presence or absence of a viruses in a sample comprising the steps of: (1) providing the biosensor or diagnostic composition as disclosed herein, (2) incubating the biosensor or diagnostic composition with the sample, (3) observing the absence or presence of an optical response in the sample, wherein the presence of the optical response indicates the presence of the viruses, wherein the enzyme encoded by the target virus causes the cleavage of the material to release the spectroscopic probe, resulting in a detectable optical response.
  • the composition may include a lysis buffer to enable or facilitate release of the target enzyme from the infected host cells so that the substrate of the biosensor is accessible to the target enzyme.
  • the detection method for viruses uses three different biosensors responsive to three different enzymes, wherein the first population of biosensors have a material only responsive to 3CLpro, the second population of biosensors have a material only responsive to PLpro, and the third population of biosensors have a material only responsive to serine protease.
  • this disclosure provides a method for detecting the presence or absence of viruses in a sample comprising the steps of: (1) obtaining the sample from a patient and placing the sample in a tube, (2) adding the biosensor as disclosed herein to the sample in the tube, (3) incubating the biosensor with the sample, (4) observing the absence or presence of an optical response in the sample, wherein the presence of the optical response indicates the presence of the viruses, wherein the enzyme encoded by the target virus causes the cleavage of the material to release the spectroscopic probe, resulting in a detectable optical response.
  • the incubating the biosensor with the same may further include contacting the sample a lysis buffer during incubation to enable or facilitate release of the target enzyme from the infected host cells so that the substrate of the biosensor is accessible to the target enzyme.
  • infectious nature can be identified by replication of virus in a host medium, such as a biological medium containing cells such as Escherichia Coli.
  • infection level can be quantified by a method comprising (1) collecting a sample; (2) splitting the sample contents into at least two portions; (3) inactivating the first portion by lysis as described above; (4) incubating the lysed first portion with a diagnostic composition as described above;
  • the system includes one or more biosensors containing the biosensors as described herein.
  • the system also includes at least one detector (e.g., a spectrometer) that detects the optical response resulting from activity of a target enzyme upon the biosensor.
  • the target enzyme may be the endogenous SARS-CoV pathogen protease.
  • the at least one detector may include naked eye, camera, a spectrometer, or any other suitable instrument that can measure an optical response such as, for example, a fluorescence spectroscope, a spectrophotometer, a fluorimeter, a colorimetric spectrophotometer, a photometer, a camera, a microplate reader, or a combination thereof.
  • the system also includes at least one controller operably connected to the detector. The controller includes one or more instructions sets that correlate the optical response detected by the detector with a presence of SARS-CoV in the sample.
  • Detectors are structured to detect detectable signals produced, e.g., in or proximal to another component of the system (e.g., in container, on a solid support, etc.).
  • Suitable optical response detectors systems may include, e.g., a fluorescence spectroscope, a spectrophotometer, a fluorimeter, a colorimetric spectrophotometer, a photometer, a camera, a microplate reader, and the like.
  • Detectors optionally monitor one or a plurality of optical response from upstream and/or downstream of the performance of, e.g., a given assay step. For example, the detector optionally monitors a plurality of optical responses, which correspond in position to "real time" results.
  • the systems of the present invention include multiple detectors.
  • the optical response is qualitative and can be observed by naked eye.
  • the optical response is a quantifiable optical property.
  • the quantification of the optical response can be achieved using colorimetric spectrophotometers.
  • the quantification of the optical response can be achieved using a fluorimeter.
  • the optical response is a change in the optical absorption of the sample.
  • the change in optical absorption is observed as a visual color change.
  • the change in optical absorption is a quantifiable change detected with a spectrophotometer.
  • the change in optical absorption is detected over the wavelength range of 400-700 nm.
  • the change in optical absorption is detected over the wavelength range of 400-500 nm.
  • the change in optical absorption is detected over the wavelength range of 500-600 nm.
  • the change in optical absorption is detected over the wavelength range of 600-700 nm.
  • the change in optical absorption is detected at infrared wavelengths in the range of 700-1100 nm.
  • the optical response is a fluorescence emission.
  • the fluorescence emission is detected visually.
  • the fluorescence emission is detected as a quantifiable signal from a fluorimeter.
  • the fluorescence emission is detected over the wavelength range of 400-700 nm.
  • the fluorescence emission is detected over the wavelength range of 400- 500 nm. In some embodiments, the fluorescence emission is detected over the wavelength range of 500-600 nm. In some embodiments, the fluorescence emission is detected over the wavelength range of 600-700 nm. In some embodiments, the fluorescence emission is detected at infrared wavelengths in the range of 700-1100 nm.
  • the precision of the quantifiable fluorescence signal can be maximixed by (i) identifying the spectrum of excitation and fluorescence spectra of the fluorophore to be detected in the sample, (ii) identifying the excitation and fluorescence spectra of other fluorescent materials in the sample, (iii) choosing an excitation wavelength that can preferably excite the fluorophore to be detected and minimally excite the other fluorescent materials, and (ii) choosing a wavelength for detecting the fluorescence in a spectral window that is preferred for the fluorescence to be detected and which minimizes the capture of undesired fluorescence.
  • Isotropic optical scattering in a sample can affect the characteristics of optical absorption or fluorescence as a result of increasing the effective path length of excitation rays.
  • samples that exhibit different amounts of turbidity can behave as if the concentration of the absorbing species is greater than the true concentration. For example the appearance of color in a turbid absorptive sample will be greater than in a non-scattering sample. Similarly, a turbid fluorescent sample will show a higher fluorescent signal than a non-scattering sample.
  • optical data can be normalized for the sample turbidity using the measurement of optical density in a non-absorbing region, for example, at a wavelength of 600 nm or greater.
  • the quantified optical response is normalized for sample turbidity.
  • the optical response is normalized by the optical density at 600 nm.
  • this disclosure provides method for the high-throughput (HTS) measurement of optical properties of clinical samples that have been lysed and reacted with the herein described biosensors and placed into the wells of a multiwell plate, wherein the optical properties are measured using a multi-well plate reader.
  • an optical fiber within a fiber bundle containing no corrective optics between the fiber ends and the well plate bottom illuminates the sample in order to induce fluorescence, and multiple fibers collect emission radiation and transmit it to a fluorescence detector such as a spectrometer.
  • the HTS measurement of optical properties involves a light scattering illumination source with detection fibers located in either the same bundle containing the fluorescence monitoring fibers or an independent light scattering detection bundle for the measurement of static and/or dynamic light scattering.
  • the HTS measurement of optical properties involves the measurement of phase analysis light scattering (PALS).
  • PALS enables the measurement of multiple optical properties of a clinical sample being made simultaneously or in succession.
  • the methods for the HTS measurement of optical properties described herein directly measure the samples in the multiwell plates. The high-throughput nature of the measurements permits the rapid screening of the individual sample, as well as reducing the sample volume required.
  • Standard multiwell plates have 96, 384, or 1536 wells, with each well containing a discrete sample, and all wells, under common operational conditions, may be tested in a single data collection run.
  • the use of these multiwell plates obviates the need for labor as compared to the conventional scintillation vial based method, which requires the cleaning and drying of individual scintillation vials after each measurement.
  • the individual well in these multiwell plates generally has very low volume, e.g ., commercially available multiwell plate based measurement instruments are capable of measurements with sample volumes of 1 pL or less.
  • sample volumes are of great benefits when one has a limited amount of sample available for the measurements, particularly when compared to the 300 pL or larger sized measurement volumes often required by other measurement techniques, such as flow-through fluorescence monitoring and flow-through multiangle light scattering (MALS).
  • MALS flow-through fluorescence monitoring and flow-through multiangle light scattering
  • Additional benefits for the multiwall plate reader based HTS optical property measurement methods include the ability to automate the measurement of between 1 and over 1500 samples with little or no human intervention after the sample is prepared and introduced into the plate for analysis.
  • the high-throughput apparatus for optical property measurement comprises a biological assay plate reader.
  • the high-throughput apparatus comprises a multipipette dispenser.
  • the high-throughput apparatus comprises a robotic multipipette dispenser.
  • Multiwell plates can be used with various optical analysis techniques, most commonly absorbance measurements performed as light is scanned across a plate and the transmitted light is measured by a detector system placed on the opposite side of the plate to the incident light, permitting, thereby a measurement of the absorbance of light by the sample contained in each individual well as, for example, the method described by A. J. Russell and C. Calvert in U.S. Pat. No. 4,810,096, the contents are incorporated herein by reference by its entirety. Measurements of absorbance enables the calculation of the concentration of the sample contained in each individual well. The changes in absorbance over time provide information about reaction rates. As described previously, measurement of optical density in spectroscopic regions lacking absorbance provides information about sample turbidity, and as a result is a measurement of cellular concentration.
  • Multi-well plates used in plate reader systems are produced in transparent plastic, or in opaque white or black forms.
  • White plates can lead to more efficient capture of fluorescent signals, but can introduce noise from background light and crosstalk between wells.
  • Black plates lead to lower overall signal, but lower overall noise that can arise from background light and crosstalk between wells.
  • use of an opaque white multi-well plate is preferred.
  • the use of a black multi-well plate is preferred.
  • An advantage of the use of a multi-well plate reader is the parallelism of data capture on a large number of samples at the same time.
  • the throughput of a system using a plate reader can be significantly increased by preparing and incubating prepared plates outside of the reader.
  • a baseline scan can be performed immediately after preparation of a plate, followed by 60 minutes or more of incubation while the desired chemical reactions proceed, followed in turn by an end point reading of the incubated plate. During this incubation period, other plates can be read, either for baseline scans of for enpoint scans. In this way, the analysis of a large number of samples carried on a number of separate plates can be multiplexed in time.
  • each plate can be used to assay 96 samples.
  • each plate can be used to assay 384 samples.
  • each plate can be used to assayl536 samples.
  • the biological plate reader is a Biotek SynergyTM HT Multi- mode Microplate Reader (a single-channel absorbance, fluorescence, or luminescence microplate reader; fluorescence l ranges include: standard PMT (excitation 300 nm to 650 nm and emission 300 nm to 700 nm); operated using BioTek’s Gen5TM or KC4TM PC Data Analysis Software; injector models dispense to 6-, 12-, 48-, and 96-well microplates; read 6-, 12-, 24-, 48-, 96-, and 384-well microplates).
  • the biological plate reader is a VICTOR® Nivo Multimode Microplate Reader by PerkinElmer (operated using VICTOR® Nivo GxP Software), an EnSightTM Multimode Microplate Reader by PerkinElmer (cell-imaging system, operated using KaleidoTM Data Acquisition and Analysis Software), or an EnVision Multimode Microplate Reader by PerkinElmer (configured for 1536-well plate).
  • this disclosure provides methods of detection of viral infection that can be performed in a non-clinical environment.
  • a sample can be collected and prepared by mixing with a biosensor formulation and other adjuvants and evaluated visually for an optical response.
  • the optical response is a change in visual absorption.
  • the optical response is a fluorescent emission.
  • the optical response is fluorescent
  • it can be generated by using an appropriate wavelength illuminating device such as, but not limited to, an LED device, and the optical response can be observed visually.
  • the LED device is a UV or blue LED flashlight.
  • the fluorescent optical response is generated by illumination in a device comprising an LED and a sample holder, wherein the observation of fluorescence occurs at approximately 90° to the excitation of the sample.
  • the optical response is detected with a digital camera.
  • the digital camera is a camera on a cellular phone.
  • the cell phone app should have features that prevent fraud (deliberate false negative results)
  • test may deploy reusable vials that hold the reagent and/or solid tablet form and a tongue sample collection device like the L-beam tongue scraper (Fig. 10) that can be sterilized for reuse.
  • test capacity is almost limitless.
  • the test can be deployed also in self reporting on a daily basis for enabling businesses and offices to operate at full capacity
  • this invention provides a system for detecting infection by a coronavirus pathogen in a human subject.
  • the system can be performed as a self-test by an individual.
  • the system comprises: a computer or computer readable medium, sample container, a pathogen diagnostic assay utilizing the biosensors described herein, a controller, a detector coupled to an output of the computer or computer readable medium.
  • the sample container is selected from glass tube or vial, or a plastic tube or vial.
  • the sample container can be sterilized for reuse.
  • the container is capped with a screw cap or a septum.
  • the container has a conical bottom.
  • the container has a round bottom.
  • the plastic tube or vial comprises polypropylene or polyurethane.
  • the sample container includes a lysis buffer solution.
  • the system optionally includes a sample collection device.
  • the sample collection device may be a nasal swab, an oral swab, a buccal swab, or a tongue scraper. It is preferred that the method of collection is chosen such that the collection can be individually performed without assistance.
  • the system include one or more biosensors and biosensor compositions as described herein. In some embodiments, the system include one or more biosensors containing the biosensors as described herein. In some embodiments, the system includes at least one container holding the biosensor as diagnostic agent.
  • the system optionally includes an illuminator that can be used to generate a fluorescent signal.
  • the illuminator is an LED device.
  • the illuminator is filtered light from a cell phone flashlight.
  • the cell phone is used to detect optical signals generated by changes in absorbance. In other embodiments cell phone is used to generate and detect fluorescence changes
  • the system includes a cell phone that performs multiple functions excitation source, optical signal detector, system controller (sequencing test steps), patient registration, store and report data.
  • the cell phone may embody steps to prevent fraudulent data collection by invalidating test.
  • the cell phone embodies steps of data capture and reporting required to file claims to entities responsible for reimbursement and payment for the system performing and reporting test results.
  • the cell phone used has been modified to include an additional light source (beyond normally present) that is a light source with a filter, narrow band light source for excitation.
  • an additional light source that is a light source with a filter, narrow band light source for excitation.
  • the cell phone used has been modified with a filter so the detector captures region around peak emission
  • the system includes a detector that detects the optical response resulted from the endogenous SARS-CoV pathogen protease activity upon the biosensor.
  • the detector is a camera.
  • the camera is a camera of a cell phone.
  • the camera includes an optical filter to reject light used for illumination from a captured fluorescent optical response.
  • the system includes at least one controller operably connected to the detector.
  • the controller is a cell phone.
  • the controller includes one or more instructions sets that correlate the optical response detected by the detector with a presence of SARS-CoV in the sample.
  • the instructions include a look-up table used with the detector data correlate the optical response detected by the detector with a presence of SARS-CoV in the sample.
  • the instructions include a registration by the user to enable the test.
  • this disclosure provides a method for detecting infection by coronavirus in a human subject, wherein the method comprises the steps:
  • step (d) exposing the inactivated sample in step (c) to a biosensor described herein to produce an optical response
  • step (e) detecting the optical response generated in step (d) with a detector, wherein the detector is configured to process the optical response into a data output, and the detector is configured to communicate the data output to a computer or computer readable medium,
  • step (f) saving the data output labeled with the patient information in step (e) on the computer or computer readable medium
  • the method using the system described reduces the need for trained medical personnel to perform the testing, and provides the ability to perform the test at home.
  • the method does not require the use of advanced equipment and eliminates process bottlenecks with regard to loading and unloading multiple samples. Since many human subjects may simultaneously be testing themselves with this system, the system incorporates massive parallelism to enable testing of millions of subjects in a short period of time.
  • an efficient system of reusable hardware associated with the test such as reusable test tubes and tongue scrapers
  • mass population testing with high frequency of testing is impossible to deploy due to supply constraints of non-reusable testing hardware.
  • the system creates massive parallelism by eliminating the sequential sample loading bottleneck of institutional laboratories relying instruments to run their tests.
  • this invention provides a system for detecting infection by SARS- CoV pathogens in a multiple human subjects.
  • the system comprises: a computer or computer readable medium, sample container, a substrate container, a pathogen diagnostic assay utilizing the biosensors described herein, a controller, a detector coupled to an output of the computer or computer readable medium.
  • FIG. 2 schematically illustrates the components of the SARS-CoV pathogen detection system operably connected.
  • the system may further include a computer or computer readable medium with a data set that relates to patient information (e.g., patient demographic information) and optical response imputed from the detector.
  • the computer or computer readable medium further includes a location tracking device coupled to an output of the computer or computer readable medium. The location tracking device accepts instructions from the computer or computer readable medium, which instructs the location tracking activity corresponding to the detection the presence of optical response.
  • the systems may include controllers that are operably connected to one or more components (e.g., detectors, thermal modulator, fluid transfer components, etc.) of the system to control operation of the components.
  • the controller may be included either as separate or integral system components that are utilized, e.g., to receive data from detectors, to effect and/or regulate temperature in the containers, to effect and/or regulate fluid flow to or from selected containers, or the like.
  • Controllers and/or other system components is/are optionally coupled to an appropriately programmed processor, computer, digital device, or other information appliance (e.g., including an analog to digital or digital to analog converter as needed), which functions to instruct the operation of these instruments in accordance with preprogrammed or user input instructions, receive data and information from these instruments, and interpret, manipulate and report this information to the user.
  • Suitable controllers are generally known in the art and are available from various commercial sources.
  • Any controller or computer optionally includes a monitor, which is often a cathode ray tube ("CRT") display, a flat panel display (e.g., active matrix liquid crystal display, liquid crystal display, etc.), or others.
  • Computer circuitry is often placed in a box, which includes numerous integrated circuit chips, such as a microprocessor, memory, interface circuits, and others.
  • the box also optionally includes a hard disk drive, a floppy disk drive, a high capacity removable drive such as a writeable CD-ROM, and other common peripheral elements.
  • Inputting devices such as a keyboard or mouse optionally provide for input from a user.
  • the computer typically includes appropriate software for receiving user instructions, either in the form of user input into a set of parameter fields, e.g., in a GUI, or in the form of preprogrammed instructions, e.g., preprogrammed for a variety of different specific operations.
  • the software then converts these instructions to appropriate language for instructing the operation of one or more controllers to carry out the desired operation.
  • the computer may then communicate with the sensor/detectors included within the system to receive the data.
  • the computer may be hardwired to the sensors/detectors or may communicate with the sensors/detectors wirelessly. In some embodiments, the computer may communicate with the sensors/detectors over a network such as, for example, a WAN, a LAN, the Internet using a suitable communication protocol.
  • the computer need not be in the same location as the sensors/detectors.
  • the computer interprets the data, and either provides it in a user understood format, or uses that data to initiate further controller instructions, in accordance with the programming, e.g., such as initiating the location tracking activity.
  • the software may be operating softwares including BioTek’s Gen5TM or KC4TM PC Data Analysis Software in microplate reader for data collection and analysis, PerkinElmer’ s VICTOR® Nivo GxP Software in microplate reader for data collection and analysis, or KaleidoTM Data Acquisition and Analysis Software in microplate reader for cell-imaging, or any other commercially available or proprietary application operable on a mobile device such as, for example, an App.
  • the computer may include: a processor on a mobile device, a PC, Power PC, a Unix-based working station, or other common commercially available computer, which is known to one of skill in the art.
  • Standard desktop applications such as word processing software (e.g., Microsoft WordTM or Corel WordPerfectTM) and database software (e.g., spreadsheet software such as Microsoft ExcelTM, Corel Quattro ProTM, or database programs such as Microsoft AccessTM or ParadoxTM) can be adapted to the present invention.
  • Software for performing, e.g., initiating location tracking is optionally constructed by one of skilled artisan using a standard programming language such as Visual basic, Fortran, Basic, Java, or the like.
  • the computer may be a cell phone.
  • the invention is optionally implemented in hardware and/or software. In some embodiments, different aspects of the invention are implemented in either client-side logic or server-side logic.
  • the components of the system may be embodied in a media program component (e.g., a fixed media component) containing logic instructions and/or data that, when loaded into an appropriately configured computing device, cause that device to perform according to the invention.
  • a media program component e.g., a fixed media component
  • the fixed media containing logic instructions may be delivered to a viewer on a fixed media for physically loading into a viewer's computer or a fixed media containing logic instructions may reside on a remote server that a viewer accesses through a communication medium in order to download a program component.
  • a patient can self-identify for testing by scanning or otherwise providing a code.
  • the code can be provided through a paper document, such as a doctor’s form or promotional literature, or may be provided through a website.
  • the patient initiates registration in a database that will track the analysis. This database entry can include information such as phone number and email address.
  • the code may be scanned using a software application that connects the patient with a network database.
  • the database assigns a unique patient code for each patient registration.
  • the software application also captures GPS data that is used to provide the patient with location information for testing, including directions and approximate wait time.
  • the testing may be performed at a hospital, a clinic, a drive-through testing site, or at home.
  • capped sample collection tubes (1) that are marked, labeled, or otherwise individually identified are provided that contain an inactivating agent.
  • the label is a QR code that can be read by a code scanner, cell phone, or other reader.
  • the label is an RFID device that can be detected and read by an RFID reader.
  • sample derived from a subject refers to a sample obtained from the subject (e.g., human patients suspected of having SARS-CoV infections, etc.), whether or not that sample undergoes one or more processing steps (e.g., cell lysis, debris removal, stabilization, etc.) prior to analysis.
  • processing steps e.g., cell lysis, debris removal, stabilization, etc.
  • samples can be derived from subjects by scraping, venipuncture, swabbing, biopsy, or other techniques known in the art.
  • the sample label information is associated with previously stored patient information from a database.
  • the database can be managed locally on a mobile device to associate patient information with the individualized label of the sample tube. This information may be subsequently uploaded to a network database, such as a cloud-based database. Alternatively, a wireless connection can be used to directly populate a network database with such data.
  • the local or network database can be used for further management of data associated with the analysis of the sample.
  • the invention provides a method to analyze a biological sample by placing the sample in contact with an analysis reagent.
  • the analysis reagent is a solid formulation.
  • the analysis reagent is provided in a cap that can be placed on the sample tube (3).
  • the analysis reagent can be injected into the sample tube.
  • the analysis reagent is injected through a diaphragm or septum on the sample tube cap.
  • the presence of pathogen is shown by the observation of an optical signal.
  • the optical signal can be observed as a qualitative visual signal as a color change that indicates the presence of the pathogen.
  • the optical signal may be detected by a quantitative spectral response from a device or instrument.
  • the optical signal may be a fluorescence signal.
  • the optical signal can be detected using the camera of a cell phone or other mobile electronic device.
  • a signal difference in the rate of change of color formation can be used to quantify a level of infection in a patient.
  • a rate of change is determined by comparison of sample color in photographs that are separated by known times.
  • a rate of change is determined from a video recording.
  • the viruses are coronavirus. In some embodiments, the viruses are selected from the group of SARS-CoV-2, SARS-CoV-1, or MERS-CoV. In some embodiments, the virus is SARS-CoV-2. In some embodiments, the virus is SARS-CoV. In some embodiments, the virus is MERS-CoV.
  • this disclosure provides a system for detecting SARS-CoV pathogen in a sample from a human subject, wherein the system comprises: a computer or computer readable medium, sample container, a biosensor as described herein, a controller, and a detector coupled to an input to the computer or computer readable medium.
  • the detector is structured to detect an optical response produced by the SARS-CoV pathogen diagnostic assay.
  • the detector is selected from the group of a fluorescence spectroscope, a spectrophotometer, a fluorimeter, a colorimetric spectrophotometer, a photometer, or a camera.
  • the computer comprises a software for receiving user instructions in the form of user input, wherein the software is configured to convert the user instructions to control the operation of the controller.
  • the computer is configured to receive data from the detector.
  • the computer is a cell phone.
  • the sample container is selected from a glass tube, a plastic tube, or an array of tubes on a rack.
  • the SARS-CoV pathogen diagnostic assay system is contacted with a sample from a subject to perform a diagnostic test according to any of the herein described the methods.
  • the sample from the human subject is selected from the group of blood, plasma, serum, feces, bronchoalveolar lavage, nasal swab sample, oral swab sample, NP swab sample, OP swab sample, buccal swab sample, tongue scrape, urine, synovial fluid, mucus, an aerosol sample collected in a respiratory mask, masticated oral sample or sputum.
  • this disclosure provides a method for detecting coronavirus in a sample from a human subject, wherein the method comprises the steps:
  • step (d) exposing the inactivated sample in step (c) to a biosensor described herein to produce an optical response
  • step (e) detecting the optical response generated in step (d) with a detector, wherein the detector is configured to process the optical response into a data output, and the detector is configured to communicate the data output to a computer or computer readable medium; and wherein the computer or computer readable medium is configured to generate a label for the data output received with the patient information received by the registration process in step (a),
  • step (f) saving the data output labeled with the patient information in step (e) on the computer or computer readable medium
  • step (g) generating a report to the human subject for the data output labeled with the patient information in step (e).
  • the coronavirus is SARS-CoV-2, SARS-CoV-1, or MERS-CoV.
  • the computer or computer readable medium used in the SARS- CoV pathogen method is a cell phone.
  • the sample from the human subject is selected from the group of blood, plasma, serum, feces, bronchoalveolar lavage, nasal swab sample, oral swab sample, NP swab sample, OP swab sample, buccal swab sample, tongue scrape, urine, synovial fluid, mucus, an aerosol sample collected in a respiratory mask, a masticated oral sample, or sputum.
  • the sample is selected from the group of NP swab sample, nasal swab sample, oral swab sample, OP swab sample, buccal swab sample, or tongue scrape.
  • the sample is a sputum sample. In some embodiments, the sample is a NP swab sample or OP swab sample. In some embodiments, the sample is a NP swab sample. In some embodiments, the sample is a OP swab sample. In some embodiments, the sample is bronchoalveolar lavage. In some embodiments, the sample is an aerosol sample that has been collected in a respiratory mask. In some embodiments, the sample is a masticated oral sample.
  • the sample is an aerosol sample that has been collected in a respiratory mask.
  • the mask is aN95 respirator mask.
  • the detector is selected from the group of a fluorescence spectroscope, a spectrophotometer, a fluorimeter, a colorimetric spectrophotometer, a photometer, or a camera.
  • the detector is a camera embedded within a cell phone.
  • the chemical agent in step (c) comprises a surfactant.
  • this disclosure provides a kit for detecting the presence of viruses in a sample of a human subject and/or for the determination of antiviral drug susceptibility of a pathogen and the kit include: biosensors or composition thereof as described herein, cotton swabs, sample tubes with screw cap, lysis buffer in a bottle like TRIzol, AVL, RLT, MagMAX, and an instruction sheet providing instructions to the user to collect the sample into the sample tube, condition the sample with the lysis buffer, register the patient information to a registering system, and a diagnostic assay procedure for conducting the diagnostic test upon the sample with the biosensor or compositions thereof.
  • FIG. 3 illustrates the parking lot professional workflow chart comprising the following components of the pathogen detection process.
  • samples are collected using tongue scrapers, but any method of sample collection as described herein can be used.
  • Step 1 of FIG. 3 shows a set of 50 commercially available samples tubes placed in a packaging box.
  • Each test tube is identified by a unique QR label, a standard cap and can be pre filled with inactivating solution.
  • This QR code will match the database so there is no risk to counterfeiting.
  • This QR code will be used to tag each patient to the sample in the tube.
  • the inactivating agent can be pre-filled by contract manufacturer with fill and finish facility.
  • Step 2 of FIG. 3 shows a set of 50 commercially available tongue scrapers (or any other sample collection modality) packed in a large box with each in its own sterile packaging.
  • Step 3 of FIG. 3 shows a set of 50 proprietary substrate papers stored in screen on cap. This is supplied by the substrate supplier. Given the substrate is extremely hydrophobic, it needs be stored in its own airtight packaging.
  • Step 4 of FIG. 3 shows that a HCS picks a sample collection tube labeled with QR code from the packaging box of Step 1 when a patient arrives.
  • the QR code on the tube will be tagged to the patient by the digital app.
  • Step 5 of FIG. 3 shows that a patient picks a package containing a tongue scraper from the packaging box of Step 2. Patient conducts the sample collection with the scraper. When the sample collection is complete, the patient drops the tongue scraper into the collection test tube held in the hand of the HCS. Alternatively, to avoid risk of contamination, the patient is given the collection tube and places the tongue scrape sample in the collection tube.
  • Step 6 of FIG. 3 shows that the QR code labeled sample collection tube with closed cap is placed on a large “Inactivation Tray” to be batched.
  • Each patient sample collection takes 1 to 2 minutes, each tray can be filled between 25 minutes to 50 minutes for a tray capable of holding 25 tubes. To assure the last sample collected had ample time for inactivation, an extra 10 minutes will expire before moving to step 7.
  • Step 7 of FIG. 3 shows that cap for each sample collection tube in the Inactivation tray will be swapped with a cap containing substrate.
  • the sample on the tray with the longest elapse of time from the time point of collection is tested first. Screwing the substrate contain cap onto the sample collection tube will deploy the paper substrate onto the liquid in the sample.
  • Step 8 of FIG. 3 shows that the assay process takes 30 seconds or less for each test tube for a total of 12 minutes for 50 samples collected.
  • Step 9 of FIG. 3 shows that the results will be available after 70 to 80 minutes since the collection of the first sample of this batch.
  • the throughput of the test is about 6 minutes per sample for either positive or negative.
  • the Abbott Now ID test takes 5 minutes for positive result and 13 minutes for negative result confirmation, not including setup time in between test. However, the Abbott test gives a throughput of 8 minute per sample at best.
  • Step 1 The patient scans a QR code from the promotional material and the Patient downloads the Now AwareTM App.
  • Step 2. 2(a) The patient selects to register for a test on the App.
  • the app will use GPS data to suggest the nearest testing location.
  • the patient selects a preferred location.
  • 2(b) The patient registers testing by entering basic information including their phone number so that they can receive test result on the app.
  • 2 (c) The patient requests to schedule for testing. This may not be necessary if GPS information is used at the testing site to give approximate wait time at the location based on number of patients waiting at the location. This way, it is first come first serve. It avoids the complexity of scheduling. If the test throughput is fast, each patient can be processed, including sample collection, in less than 2 minutes. 2(d) The patient finishes test registration and is assigned a unique patient code.
  • This patient code will be a QR code displayed on the phone when patient is ready to be processed by the HCS at the test site. 2(e).
  • the App plays a video of the entire testing process including video instruction of how to do tongue scrape and how to put the swab in the collection tube.
  • Step 3 When the patient reaches the testing center, the QR code will be displayed for processing.
  • Step 1 When the patient arrives the tent, the app will display a unique QR code that identifies the patient.
  • Step 2 The HCS’s App will scan the QR code from patient’s App. It will display the patient registration information. HCS can verbally confirm patient by name.
  • Step3 The HCS “sample collector” will take a collection tube out of the box of tubes (#1). HCS will scan the QR code on the collection tube. This will tag the patient to the collection tube.
  • Step 4 The HCS will take out a tongue scrapper package from (#2) and give it to the patient in the car.
  • Step 5 The patient will take out the tongue scrapper (#5) from the package and follow the video instruction from the app to collect the tongue scrape sample.
  • Step 6 When a tongue scrape sample is collected, the HCS gives the patient the sample collection tube with the cap already removed so that the patient inserts the tongue scrapper into the tube, and then the HCS gives the patient the cap so it is screwed onto the tube. This is to prevent contamination of sample because it is handled completely by the patient and reduces infection risk to HCS.
  • the HCS removes the cap from the sample collection tube and asks the patient to insert the tongue scrape sample in the tube, and the HCS puts the cap back on the tube. This is to avoid the dexterity needed for the patient to handle the collection tube.
  • This patient processing should take no more than 1 to 2 minutes for each patient.
  • Step 7 The HCS places the completed sample collection tube onto “Inactivation Tray” (#6). This will continue until the tray is filled, or whenever the HCS feels there is sufficient number of samples collected for the batch collection. Upon a batch is collected, the HCS sample collector will move the Inactivation Tray (#6) to a workstation designated for a HCS to process the samples. The HCS sample collector will take an empty Inactivation Tray (#6) to start a new batch of sample collection.
  • Step 8 Upon receiving last sample for the batch (#6), the testing process will not start until there was sufficient time for sample inactivation of the last sample placed into the tray. This is to assure all samples in the tray have completed inactivation and lysis of cells in the last collected sample. However, this workflow can be improved by starting the testing process (#7) of the first sample that was collected in the batch and then the subsequent sample collected.
  • Step 9 The HCS “sample processor”, will take a cap containing the substrate paper from box of caps (#3).
  • the HCS “sample processor” will swap cap on top of each collected sample with cap containing the substrate on paper and deploy the substrate into the collection tube (#7). This process will continue until the entire batch in “Result Tray” (#8) has received the substrate.
  • the “sample processor” can wait for the period of time that assures the last sample with substrate had sufficient time for the catalytic reaction. This workflow can also be improved by examining right away the test result of the first sample in the batch that received substrate and then the subsequent sample in the batch.
  • Step 10 For each test result, the HCS “sample processor” will scan the QR code on the test tube that is linked to the patient. The App will ask the “sample processor” to input the test result based on the color change. Alternatively, the App will scan the QR code and determine the test result based on the color spectrum observed by the camera to completely automate the result and associating it to the patient.
  • Step 11 The Now AwareTM digital network will notify the test result to the patient on the app.
  • the app will assist the patient with the necessary procedures to follow based on the test result.
  • the app will offer e-Commerce to connect service providers for the patient.
  • the app will notify the patient to get a confirmation test in 5 days. This is important because true confirmation of requires testing at the front end and back end of the 5 day window between infection and virus production.
  • Step 12 The NowAwareTM database will generate daily test result in a standard reporting format to the Center For Disease Control and Prevention (CDC) of United States. Direct data connectivity to CDC reporting can be established on the backend of the NowAware ecosystemTM.
  • compositions of this invention may be made by various methods known in the art. Such methods include those of the following examples, as well as the methods specifically exemplified below. Modifications of such methods that involve techniques commonly practiced in the art of sensors and particle technology may be used.
  • a solution containing 700 mM Z-RLRGG-7-amido-4 methylcoumarin and 10% dimethylsulfoxide in 90% 50 mM HEPES buffer with 150 mM sodium chloride, 2.5 mM dithiothreitol and 0.1 mg/mL bovine serum albumin was prepared.
  • To 1 ml of this solution was added 2 pL of 1 ImM SARS-CoV-2-PLPro solution (Boston Biochem) and the combination was thoroughly mixed. Initial fluorescence was recorded, and the solution was incubated at room temperature for 30 minutes, monitoring for fluorescence at 10 minute intervals. Reaction was detectable as a low intensity fluorescence at 441 nm (380 nm excitation) that increased linearly with time.
  • Example 2 Preparation of Ac-hPhe-Dap-Gly-Gly-ACC.
  • Fmoc-Rink Amide AM resin (lmmol, 1 equivalent) was prepared by swelling with dry DMF (15 mL) for 30 min. The DMF was then drained, and the Fmoc protecting group removed by treatment with 20% piperidine in DMF (10 mL). The solution part was removed, and the resin was washed with dry DMF. The resin was treated a final time with 20% piperidine in DMF (10 mL), and the solution part was removed. The resin was then washed five times with 10 ml dry DMF, each time shaking for 2 minutes before removing the wash solution. Positive Kaiser test proved that free amine group was present on the resin.
  • the Fmoc-ACA-resin was treated with 20% piperidine in DMF (10 mL) for 20 min. The solution part was removed, and the resin was washed with dry DMF. The resin was treated a final time with 20% piperidine in DMF (10 mL) for 20 min, and the solution part was removed. The resin was then washed five times with 10 ml dry DMF, each time shaking for 2 minutes before removing the wash solution. Kaiser test showed show faint color of the bead after heating for 5 min at 100 °C.
  • the Fmoc-Gly-Gly-ACA-resin was treated with 20% piperidine in DMF (10 mL) for 20 min. The solution was removed, and the resin was washed with dry DMF. The resin was treated a final time with 20% piperidine in DMF (10 mL) for 20 min, and the solution part was removed. The resin was then washed five times with 10 ml dry DMF, each time shaking for 2 minutes before removing the wash solution. Kaiser test showed strong purple color of the bead.
  • the Fmoc-Dap(Boc)-Gly-Gly-ACA-resin was treated with 20% piperidine in DMF (10 mL) for 20 min. The solution part was removed, and the resin was washed with dry DMF. The resin was treated a final time with 20% piperidine in DMF (10 mL) for 20 min, and the solution part was removed. The resin was then washed five times with 10 ml dry DMF, each time shaking for 2 minutes before removing the wash solution. Kaiser test showed strong purple color of the bead.
  • N-Boc-LRGG-OH Four equivalents of N-Boc-LRGG-OH was treated with 4 equivalents of 1 ⁇ (3 ⁇ di m ethylaminopropyl)-3 -eth lcarhodi imide in DMF with 0.4 equivalents of 4- dimethylaminopyridine to prepare an activated peptide. This material was then treated with one equivalent of rhodamine 110 over 2 days to yield (N-Boc-LRGG-N)2-rhodamine (See FIG. 4 for chemical structure).
  • EDC (1.34 g, 7.0 mmol) was added and stirred for 5 min.
  • (NH2-GG)2Rhodamine*HBR (900 mg, 1.25 mmol) was dissolved in DMF/Py (5 mL) and added to the reaction mixture. Stirred at the same temperature for 2 h and the stirred at RT overnight. The reaction was stopped by adding DEE (180 mL) and the mixture was centrifuged (6000 rpm, 10 min).
  • EDC 563 mg, 2.93 mmol
  • (NH2-LR(pbf)GG)2Rhodamine 400 mg, 0.245 mmol) was dissolved in DMF/Py (5 mL) and added to the reaction mixture. Stirred at the same temperature for 2 hours and the stirred at RT for 1 hour.
  • the reaction was stopped by adding DEE (160 mL) and the mixture was centrifuged (6000 rpm, 10 min). The supernatant was discarded and the solid was dissolved in DCM (10 mL), precipitated by adding DEE (80 mL) and centrifuged again. This step was repeated two more times. The solid was then dissolved in DCM (200 mL) and washed with water (200 mL). The aqueous layer was extracted with more DCM (200 mL). The organic layer was washed with water (100 mL x 1) and brine (100 mL).
  • peptide-chromophore conjugate substrate Z-RLRGG-AMC was purchased from BA Chem (Switzerland).
  • a stock solution of the substrate was prepared from a 10% DMSO solution of the chromophore and a mixture of 50 mM HEPES, pH 7.5, 0.1 mg/ml bovine serum albumin [BSA], 150 mM NaCl, and 2.5 mM DTT and 0.5% Triton X-100 to give a concentration of 5 mg/ml of substrate solution.
  • Spectral Scan a spectral reading after 1 hour to confirm the presence of the product of the substrate/enzyme reaction
  • Endpoint Scan a single point reading of fluorescence intensity after 60minutes.
  • a known positive (by PCR) clinical sample with equivalent amount of substrate added [0401] A known positive (by PCR) clinical sample with equivalent amount of substrate added. [0402] A known negative (by PCR) clinical sample with the same amount of substrate added.
  • Example 4a Kinetic scans of controls and clinical samples.
  • the kinetic graphs of each test reveal strong discrimination of samples showing a flat line versus line with positive slope after one hour of reaction of substrate with each sample.
  • All flat line samples are either controls or patient samples that tested negative by PCR.
  • the curves with steep positive slope are positive signals from either positive controls or PCR-positive samples while the curves with a gentle (but positive) slope still exhibit significant discrimination relative to control.
  • the positive signal read shows a minimum signal-to-noise ratio of 10 and slope error of about 10%.
  • Example 4b End point scan of control and clinical samples.
  • End Point Scans were obtained by preparing samples for analysis as before and incubating the samples containing the biosensors for 1 hour before reading the end point fluorescence. Results are shown in Fig. 7 and in Table 7. Fluorescence from positive samples show a significant difference from that of negative controls, discrimination of positive and negative assignments for samples of unknown PCR results.
  • Table 5 shows calculated test sensitivity and specificity fortesting of clinical samples using the NFb-LRGG-AMC substrate. Sensitivity is calculated as 1-(FN/TP). Specificity is calculated as TN/(TN+FP) Table 7. Preliminary sensitivity and specificity forNP (48 samples) and Tongue Scrapings (43 Samples)
  • TP true positive
  • FP False Positive
  • TN True Negative
  • FN False Negative
  • T True
  • F False
  • PPV Positive Predictive Value
  • NPV Negative Predictive Value
  • NP Nasopharyngeal
  • ETT Endotracheal Tube
  • PCR Polymerase Chain Reaction
  • Example 4c Clinical samples treated with (NH2-LRGG)2Rhodamine substrate
  • a FRET -based substrate was evaluated against SARS-CoV-2 positive and negative samples.
  • This substrate DABCYL-KVRLQSK-DANSYL, contains the FRET donor and quencher separated by the VRLQS sequence recognized by 3CLpro from SARS-CoV-1 and SARS-CoV-2.
  • DANSYL is the absorber and latent fluorophore (“donor”)
  • DABCYL is the fluorescence quencher (“acceptor”).
  • donor absorber and latent fluorophore
  • acceptor the fluorescence quencher
  • fluorescence from the DANSYL is quenched by the DABCYL moiety.
  • the donor and acceptor can separate by much more than the Forster distance, revealing the fluorescence of the donor to be captured by the plate reader or observed visually.
  • Samples were prepared as described previously, substituting a FRET substrate for the NH2-RLRGG-AMC substrate in some cases. Samples were incubated for 1 hour before end point observation for spectra using the plate reader (Fig. 9a), followed by observation of visible fluorescence (Fig. 9b).
  • Example 4e Inhibition of fluorescence generation by PLpro inhibitor
  • PL pro inhibitor Cftefrtsal Stroeitjre CAS bio ' 10&3C7G-14-4 [0413] The PLpro inhibitor showed partial inhibition of purified enzyme activity at a 50 mM concentration as shown in Fig. 10a. A concentration-dependent inhibition of PLpro reaction with RLRGG-AMC was observed and is shown in Fig. 10b. Almost complete inhibition was observed using an inhibitor concentration of 0.75mM.
  • Samples were collected using several different collection methods, with tongues being scraped either once or twice. Samples were prepared and analyzed as in Example 3. To normalize the fluorescence intensity from the samples, O ⁇ oo was measured and the relative fluorescence units was divided by the O ⁇ oo. Raw data and results of the normalization are shown below. For each collection instrument and the site on the tongue that was sampled, the normalized RFU numbers were in a similar range. For example, with the plastic knife, when the tongue was sampled either once or twice, the normalized RFUs were in the range of 15,000 to 16,000 RFU per unit of O ⁇ oo. A range of absorbances for linear correlation for absorbance versus cell number can be established and used for normalization of fluorescence intensity.
  • Table 8 Normalized fluorescence analysis of tongue scrape samples.
  • Example 6 Comparison of nasopharyngeal RT-PCR with tongue scrape droplet digital PCR (ddPCR) and fluorescent biosensor detection of SARS-CoV-2 in clinical samples.
  • RNA quantity was measured using Invitrogen Qubit High-Sensitivity RNA assay #Q32852.
  • Single digital droplet PCR was performed on each sample using IDT CDC RUO assay for viral genes N1 and N2 #10006713. Results from ddPCR are below. The numbers represent the calculated total copy number of each gene, N1 and N2. Results in relative fluorescence units (RFUs) seen on the plate reader from the rapid enzyme activity detection test (Now Aware Rapid Test) are also shown for comparison.
  • REUs relative fluorescence units

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Abstract

The disclosure provides compositions and methods of use thereof to detect pathogenic viruses like coronavirus.

Description

COMPOUNDS FOR DETECTION OF VIRAL PATHOGENS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to US Provisional Application No. 62/981,408, filed on February 25, 2020, US Provisional Application No. 63/012,779, filed on April 2, 2020, US Provisional Application No. 63/014,476, filed on April 23, 2020, US Provisional Application No. 63/035,438, filed on June 5, 2020, and US Provisional Application No. 63/037,239, filed on June 10, 2020, each of which is incorporated herein by reference in its entirety for all purposes.
FIELD OF INVENTION
[0002] The present invention relates to a novel compounds and methods for detecting pathogens in a subject and on surfaces. Rationally designed libraries are devised for the detection of viruses using screening tools that are well established in the field. A system for detecting SARS-CoV pathogen in a sample is disclosed.
BACKGROUND OF THE INVENTION
[0003] Reports about coronavirus disease caused by 2019-nCoV (also known as COVID-19), renamed as SARS-CoV-2, began rising at the end of 2019 with 41 instances in Wuhan, a city in China with a population of 11 million. Today there are over 95 million cases with more than 2,000,000 deaths worldwide, with cases being reported in over 210 countries or territories.
[0004] In order to contain this pandemic, healthcare workers need to rapidly and precisely detect new coronavirus cases so that patients can get essential medical care and spread can be halted.
[0005] The Center for Disease Control and Prevention (CDC) of United States believes that symptoms of SARS-CoV-2 may appear in as few as 2 days or up to 14 days after exposure. This is based on what has previously been seen as the incubation period of MERS viruses. Common human coronaviruses, including types 229E, NL63, OC43, and HKU1, usually cause mild to moderate upper-respiratory tract illnesses, like the common cold. Most people get infected with these viruses at some point in their lives. These illnesses usually only last for a short amount of time. Symptoms may include runny nose, headache, cough, sore throat, fever and a general feeling of being unwell. Unfortunately, these symptoms are fairly similar to that of the common flu that infects a lot of people every winter. Human coronaviruses can sometimes cause lower- respiratory tract illnesses, such as pneumonia or bronchitis. This is more common in people with cardiopulmonary disease, people with weakened immune systems, infants, and older adults.
[0006] Two other human coronaviruses, MERS-CoV and SARS-CoV-1 have been known to frequently cause severe symptoms. MERS symptoms usually include fever, cough, and shortness of breath which often progress to pneumonia. About 3 or 4 out of every 10 patients reported with MERS have died. MERS cases continue to occur, primarily in the Arabian Peninsula. SARS symptoms often included fever, chills, and body aches which usually progressed to pneumonia. No human cases of SARS-CoV-1 have been reported anywhere in the world since 2004. Symptoms of SARS-CoV-2 are similar to those of other coronaviruses.
[0007] The CDC released a test kit, called the Centers for Disease Control and Prevention (CDC) 2019-Novel Coronavirus (SARS-CoV-2) Real-Time Reverse Transcriptase (RT)-PCR Diagnostic Panel (CDC SARS-CoV-2 Real Time RT-PCR), which is designed for use with an existing RT-PCR testing instrument that is commonly used to test for seasonal influenza. The test is intended for use with upper and lower respiratory specimens collected from people who meet the CDC criteria for SARS-CoV-2 testing. The test uses a technology that can provide results in four hours from initial sample processing to result. Many PCR-based test kits have received emergency use authorization (EUA) from the FDA and provide results in a few hours.
[0008] Unfortunately, the 4-hour diagnostic test is not fast enough to contain the spread of the virus. Cases of SARS-CoV-2 spread outside mainland China through people who visited the country and then traveled to other countries, in many cases by individuals who appeared to be asymptomatic. A faster test that can detect SARS-CoV-2 in minutes will help contain this crisis by allowing people who are truly infected by SARS-CoV-2 and not the regular flu to be quickly quarantined. For example, at international airports or at quarantine site borders, a rapid test that does not need extensive procedures and equipment’s could be very useful to screen all travelers for this virus in minutes instead of hours.
[0009] An additional complication in the detection and management of outbreaks of SARS CoV- 2 infection is the relatively long period of 3-8 days between an individual’s initial infection and the onset of symptoms. During this time, the person can infect a host of other people. Furthermore, PCR testing fails to identify presence of virus for the first 3-5 days of infection. Moreover, because PCR testing requires extensive facilities as well as technicians with particular expertise, it is desirable to have a test that can be performed at the point of care by a lay person with minimal instructions. Therefore, new testing methods to identify viral load in asymptomatic individuals in a point of care setting that are sufficiently sensitive to detect the target proteins such as the two proteases nsp3 and nsp5 at concentrations present within the first 3-5 days are desired.
[0010] The ability to detect deadly infections in minutes instead of the several hours it currently takes will dramatically improve the management of hospital-acquired infections (HAI) and community-acquired infections (CAI). After an outbreak like the SARS-CoV-2 , affected patients are isolated and medical staff wear protective gear until it is safe. Usually the isolation will last for as long as the patient is in the hospital which adds to the healthcare costs. Rapid detection and disinfection of equipment, instruments, hospital beds, railings, and all the other hospital associated areas would help reduce the spread of infections and the associated costs. Novel molecules can be rationally designed for rapid detection of viruses. Using rationally designed libraries for screening against viruses can yield such molecules with excellent sensitivity and specificity.
[0011] The importance of detection within health care settings is exemplified by experience with transmission of HAI in hospitals. For decades, hospitals have worked to get doctors, nurses and other health care workers to wash their hands and prevent the spread of germs. However, a recent study (https://academic.oup.com/cid/article/69/ll/1837/5445425) suggests they should expand those efforts to their patients, too. In the study, 14 percent of 399 hospital patients tested had superbugs on their hands or nostrils very early in their hospital stay, the research finds. And nearly a third of tests for such germs on objects that patients commonly touch in their rooms, such as the nurse call button, came back positive. An additional 6 percent of the patients who didn’t have multidrug-resistant organisms, or MDROs, on their hands at the start of their hospitalization tested positive for them on their hands later in their stay. One-fifth of the objects tested in their rooms had persistent microbes on them, too. Further, they note that health care workers’ hands are still the primary mode of microbe transmission to patients. Within the hospital, healthcare workers (HCWs) are often exposed to infections. Any transmissible disease can occur in the hospital setting and may affect HCWs. HCWs are not only at risk of acquiring infections but also of being a source of infection to patients, especially for infections such as COVID-19 (https://doi.org/10.1016/ S1473-3099(20)30527-2). Therefore, both the patient and the HCW need to be protected from contracting or transmitting hospital-acquired infections. A quick and inexpensive way to identify hospital beds, equipment’s instruments, health care workers, patients and/or even visitors that may be carrying pathogens like the coronavirus would be highly beneficial in stopping their spread.
[0012] In addition to spread within healthcare mileau, a major source of community spread can be mass gatherings, including sporting events and celebrations, and even schools and workplaces. In the case of COVID-19 infections, transmission can occur from asymptomatic carriers several days before the onset of symptoms associated with the disease, emphasizing the importance of early detection. For example, a corporate meeting at Biogen in Boston in March, 2020, led to the direct infection of 99 participants, but may have contributed to infection in about 20,000 people over four counties in Massachusetts.
[0013] Therefore, there is a need for early and rapid detection of viruses like coronaviruses. The present invention provides a solution to meet such need.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Fig. 1 shows the chemical structure of a FRET biosensor described in this disclosure.
[0015] Fig. 2 is a graph showing comparison of cross-reactivity with PLpro from SARS-CoV-2 and enzymes from other sources.
[0016] Fig. 3 schematically illustrates the components of the SARS-CoV pathogen detection system operably connected as described in this disclosure.
[0017] Fig. 4 shows the structure of (N-Boc-LRGG-N)2-rhodamine 110, a biosensor described in this disclosure.
[0018] Fig. 5 shows kinetic scan data from a 96-well plate showing enzyme reactivity with substrate for (i) negative control, (ii) positive control, and (iii) positive and negative patient samples. [0019] Fig. 6a shows kinetic scan data of a negative control sample from Well A1 in Fig. 5. This data demonstrates a stable, average signal of 143 RFU with a maximum random oscillation of about 12 RFU.
[0020] Fig. 6b shows kinetic scan data of a PCR-confirmed negative clinical sample from Well A8 in Fig. 5. This data demonstrates a stable, average signal of 180 RFU with a maximum random oscillation of about 13 RFU.
[0021] Fig. 6c shows kinetic scan data from a PCR-confirmed positive clinical sample from Well E5 in Fig. 5. A strong signal is observed that starts at 500 RFU and rises to 2500 RFU over the course of 1 hour. The fluorescence continues to increase for hours (data not shown).
[0022] Fig. 6d shows kinetic scan data from a PCR-confirmed positive clinical sample from Well E3 in Fig XI . While the fluorescence from this sample was less than that in Fig. 6c, it was high enough to clearly observe the kinetic differences from the responses of the negative control and negative clinical sample. The maximum signal reached was 525 RFU from a starting value of about 250 RFU.
[0023] Fig. 7 show end point scan data for a set of control and clinical samples.
[0024] Fig. 8a shows a 96-well plate with samples that have been incubated with Z-RLRGG- AMC substrate (top four rows) and with (NFh-LRGG^Rhodamine (bottom four rows) when illuminated with a “black light” flashlight emitting at 395 nm.
[0025] Fig 8b shows visible color development in samples shown in Fig. 4a. Color development could only be observed in the samples containing the Rhodamine product, since the AMC product has very low visible absorbance.
[0026] Fig. 9a shows 1 hour end point emission spectra of positive and negative samples treated with DABCYL-KVRLQSK-DANSYL, showing the fluorescence of the free DANSYL in positive samples.
[0027] Fig. 9b shows visible fluorescence of samples treated with DABCYL-KVRLQSK- DANSYL in comparison to those treated with the PLpro substrate, (NH2-LRGG)2Rhodamine. In both cases, positive samples were clearly differentiated from negative samples that showed no fluorescence. [0028] Fig. 10a shows the effect of a PLpro inhibitor on the kinetic curve for fluorescence generated by enzyme reaction with the Z-RLRGG-AMC substrate.
[0029] Fig. 10b shows PLpro inhibition at higher concentrations of the inhibitor.
[0030] Fig. 10c shows PLpro inhibition in a clinical sample. Note the higher concentration of inhibitor required as compared to Fig. 10b.
[0031] Fig. 11 is a photograph of a preferred configuration of a tongue scraper.
[0032] Fig. 12a schematically illustrates a simple fluorogenic peptide substrate.
[0033] Fig. 12b schematically illustrates an internally quenched fluorogenic peptide substrate.
DETAILED DESCRIPTION OF THE INVENTION
[0034] This disclosure provides novel biosensors that can detect specific viruses like coronaviruses. This disclosure also describes formulations and compositions using the biosensors, and well as methods for detecting infection by viruses using the biosensors.
Definitions
[0035] As used in the preceding sections and throughout the rest of this specification, unless defined otherwise, all the technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference in their entireties.
[0036] [0001] When ranges are used herein to describe, for example, physical or chemical properties such as molecular weight or chemical formulae, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. Use of the term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary from, for example, between 1% and 15% of the stated number or numerical range. The term “comprising” (and related terms such as “comprise” or “comprises” or “having” or “including”) includes those embodiments such as, for example, an embodiment of any composition of matter, method or process that “consist of’ or “consist essentially of’ the described features. As used herein and in the claims, the term “comprising” is inclusive or open-ended and does not exclude additional unrecited elements, compositional components, or method steps.
[0037] Amino acids are represented in three-letter code or one letter code, as illustrated in the Table 1 below. In some embodiments, biosensors may comprise unnatural amino acids, including the D-enantiomers of the naturally occurring L-forms, and non-proteinogenic amino acids listed in Table 2.
Table 1
Figure imgf000009_0001
Figure imgf000010_0001
Table 2
Figure imgf000010_0002
Figure imgf000011_0001
[0038] Unnatural amino acids may also include modifications of natural amino acids following protein biosynthesis. Post-translational modification may include chemical changes such as phosphorylation, carbonylation, glycosylation, acylation, notrosylation, and other similar reactions.
[0039] As used herein, “Ac” refers to an acetyl (CH3C(0)-) group, “Sue” refers to a succinyl (H0C(0)CH2CH2C(0)-) group, “Cbz” (also referred to herein as “Z”) refers to a carboxybenzoyl group, “Fmoc” refers to a fluorenylmethoxycarbonyl group, and “Boc” refers to a tert-butyloxycarbonyl group,
[0040] As used, herein the term “alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, generally having from one to ten carbon atoms (e.g., (Cl-lO)alkyl or Cl-10 alkyl). Whenever it appears herein, a numerical range such as “1 to 10” refers to each integer in the given range - e.g., “1 to 10 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms, although the definition is also intended to cover the occurrence of the term “alkyl” where no numerical range is specifically designated. Examples of “alkyl” as used herein include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl, /c/V-butyl, isopentyl, and n-pentyl, neopentyl, hexyl, septyl, octyl, nonyl and decyl. The alkyl moiety may be attached to the rest of the molecule by a single bond, such as for example, methyl (Me), ethyl (Et), n-propyl (Pr), 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl) and 3-methylhexyl. Unless stated otherwise specifically in the specification, an alkyl group is optionally substituted by one or more of substituents which are independently heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -ORa, -SRa, -N(Ra)2, where each Ra is independently hydrogen, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.
[0041] As used herein, the term “aryl” refers to a benzene ring or to a fused benzene ring system, for example anthracene, phenanthrene, or naphthalene ring systems. Examples of “aryl” groups include, but are not limited to, phenyl, biphenyl, naphthyl, indenyl, azulenyl, fluorenyl, anthracenyl, phenanthrenyl, tetrahydronaphthyl, indanyl, phenanthridinyl and the like. Unless otherwise indicated, the term “aryl” also includes each possible positional isomer of an aromatic hydrocarbon radical, such as in 1 -naphthyl, 2-naphthyl, 5-tetrahydronaphthyl, 6- tetrahydronaphthyl, 1 -phenanthridinyl, 2-phenanthridinyl, 3 -phenanthridinyl, 4-phenanthridinyl, 7-phenanthridinyl, 8-phenanthridinyl, 9-phenanthridinyl and 10-phenanthridinyl and the like.
One preferred aryl group is phenyl.
[0042] As used herein the term “halogen” refers to fluorine, chlorine, bromine, or iodine.
[0043] As used herein the term “haloalkyl” refers to an alkyl group, as defined herein that is substituted with at least one halogen. Examples of branched or straight chained “haloalkyl” groups useful in the present invention include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, and t-butyl substituted independently with one or more halogens, e.g., fluoro, chloro, bromo, and iodo. The term “haloalkyl” should be interpreted to include such substituents such as -CF3, -CH2-CH2-F, -CH2-CF3, and the like.
[0044] As used herein, the term “oxo” refers to a group =0.
[0045] As used herein the term “alkoxy” refers to a group -ORa, where Ra is alkyl as herein defined.
[0046] As used herein the term “cyano” refers to a group -CN.
[0047] As used herein throughout the present specification, the phrase “optionally substituted” or variations thereof denote an optional substitution, including multiple degrees of substitution, with one or more substituent group, preferably one or two. The phrase should not be interpreted to be imprecise or duplicative of substitution patterns herein described or depicted specifically. [0048] Esters of the compounds of the present invention are independently selected from the group of (1) carboxylic acid esters obtained by esterification of the hydroxy groups, in which the non-carbonyl moiety of the carboxylic acid portion of the ester grouping is selected from straight or branched chain alkyl (for example, ethyl, n-propyl, t-butyl, or n-butyl), alkoxyalkyl (for example, methoxymethyl), aralkyl (for example, benzyl), aryloxyalkyl (for example, phenoxymethyl), aryl (for example, phenyl optionally substituted by, for example, halogen, Cl -4 alkyl, or Cl -4 alkoxy or amino); (2) sulfonate esters, such as alkyl- or aralkyl sulfonyl (for example, methanesulfonyl); (3) amino acid esters (for example, L-valyl or L-isoleucyl); (4) phosphonate esters, and (5) mono-, di- or triphosphate esters. The phosphate esters may be further esterified by, for example, a Cl -20 alcohol or reactive derivative thereof, or by a 2, 3-di (C6-24) acyl glycerol. In such esters, unless otherwise specified, any alkyl moiety present advantageously contains from 1 to 18 carbon atoms, particularly from 1 to 6 carbon atoms, more particularly from 1 to 4 carbon atoms. Any cycloalkyl moiety present in such esters advantageously contains from 3 to 6 carbon atoms. Any aryl moiety present in such esters advantageously comprises a phenyl group.
[0049] Ethers of the compounds of the present invention include, but are not limited to methyl, ethyl, butyl and the like.
[0050] “Alkylaryl” refers to an -(alkyl)aryl radical where aryl and alkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for aryl and alkyl respectively.
[0051] “Alkylhetaryl” refers to an -(alkyl)hetaryl radical where hetaryl and alkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for aryl and alkyl respectively.
[0052] “Alkylheterocycloalkyl” refers to an -(alkyl) heterocyclyl radical where alkyl and heterocycloalkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for heterocycloalkyl and alkyl respectively.
[0053] An “alkene” moiety refers to a group of at least two carbon atoms and at least one carbon-carbon double bond, and an “alkyne” moiety refers to a group of at least two carbon atoms and at least one carbon-carbon triple bond. The alkyl moiety, whether saturated or unsaturated, may be branched, straight chain, or cyclic. [0054] “Alkenyl” refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond, and having from two to ten carbon atoms (i.e., (C2-10)alkenyl or C2-10 alkenyl). Whenever it appears herein, a numerical range such as “2 to 10” refers to each integer in the given range - e.g., “2 to 10 carbon atoms” means that the alkenyl group may consist of 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms. The alkenyl moiety may be attached to the rest of the molecule by a single bond, such as for example, ethenyl (i.e., vinyl), prop-l-enyl (i.e., allyl), but-l-enyl, pent-l-enyl and penta-l,4-dienyl. Unless stated otherwise specifically in the specification, an alkenyl group is optionally substituted by one or more substituents which are independently alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, - ORa, -SRa, -OC(0)-Ra, -N(Ra)2, -C(0)Ra, -C(0)ORa, -OC(0)N(Ra)2, -C(0)N(Ra)2, - N(Ra)C(0)ORa, -N(Ra)C(0)Ra, -N(Ra)C(0)N(Ra)2, N (Ra)C (NRa)N (Ra)2, -N(Ra)S(0)tRa (where t is 1 or 2), -S(0)tORa (where t is 1 or 2), -S(0)tN(Ra)2 (where t is 1 or 2), or P03(Ra)2, where each Ra is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.
[0055] “Alkenyl-cycloalkyl” refers to an -(alkenyl)cycloalkyl radical where alkenyl and cycloalkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for alkenyl and cycloalkyl respectively.
[0056] “Alkynyl” refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one triple bond, having from two to ten carbon atoms (i.e., (C2-10)alkynyl or C2-10 alkynyl). Whenever it appears herein, a numerical range such as “2 to 10” refers to each integer in the given range - e.g., “2 to 10 carbon atoms” means that the alkynyl group may consist of 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms. The alkynyl may be attached to the rest of the molecule by a single bond, for example, ethynyl, propynyl, butynyl, pentynyl and hexynyl. Unless stated otherwise specifically in the specification, an alkynyl group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -ORa, -SRa, -OC(0)-Ra, - N(Ra)2, -C(0)Ra, -C(0)ORa, -OC(0)N(Ra)2, -C(0)N(Ra)2, -N(Ra)C(0)ORa, - N(Ra)C(0)Ra, -N(Ra)C(0)N(Ra)2, N (Ra)C (NRa)N (Ra)2, -N(Ra)S(0)tRa (where t is 1 or 2), - S(0)tORa (where t is 1 or 2), -S(0)tN(Ra)2 (where t is 1 or 2), or P03(Ra)2, where each Ra is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.
[0057] “Alkynyl-cycloalkyl” refers to an -(alkynyl)cycloalkyl radical where alkynyl and cycloalkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for alkynyl and cycloalkyl respectively.
[0058] “Carboxaldehyde” refers to a -(C=0)H radical.
[0059] “Carboxyl” refers to a -(C=0)OH radical.
[0060] “Cycloalkyl” refers to a monocyclic or polycyclic radical that contains only carbon and hydrogen, and may be saturated, or partially unsaturated. Cycloalkyl groups include groups having from 3 to 10 ring atoms (i.e. (C3-10)cycloalkyl or C3-10 cycloalkyl). Whenever it appears herein, a numerical range such as “3 to 10” refers to each integer in the given range - e.g., “3 to 10 carbon atoms” means that the cycloalkyl group may consist of 3 carbon atoms, etc., up to and including 10 carbon atoms. Illustrative examples of cycloalkyl groups include, but are not limited to the following moieties: cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl, and the like. Unless stated otherwise specifically in the specification, a cycloalkyl group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -ORa, -SRa, -OC(0)-Ra, - N(Ra)2, -C(0)Ra, -C(0)ORa, -OC(0)N(Ra)2, -C(0)N(Ra)2, -N(Ra)C(0)ORa, - N(Ra)C(0)Ra, -N(Ra)C(0)N(Ra)2, N (Ra)C (NRa)N (Ra)2, -N(Ra)S(0)tRa (where t is 1 or 2), - S(0)tORa (where t is 1 or 2), -S(0)tN(Ra)2 (where t is 1 or 2), or PCb(Ra)2, where each Ra is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.
[0061] “Cycloalkyl-alkenyl” refers to a -(cycloalkyl)alkenyl radical where cycloalkyl and alkenyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for cycloalkyl and alkenyl, respectively. [0062] “Cycloalkyl-heterocycloalkyl” refers to a -(cycloalkyl)heterocycloalkyl radical where cycloalkyl and heterocycloalkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for cycloalkyl and heterocycloalkyl, respectively.
[0063] “Cycloalkyl-heteroaryl” refers to a -(cycloalkyl)heteroaryl radical where cycloalkyl and heteroaryl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for cycloalkyl and heteroaryl, respectively.
[0064] The term “alkoxy” refers to the group -O-alkyl, including from 1 to 8 carbon atoms of a straight, branched, cyclic configuration and combinations thereof attached to the parent structure through an oxygen. Examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy and cyclohexyloxy. “Lower alkoxy” refers to alkoxy groups containing one to six carbons.
[0065] The term “substituted alkoxy” refers to alkoxy wherein the alkyl constituent is substituted (i.e., -0-(substituted alkyl)). Unless stated otherwise specifically in the specification, the alkyl moiety of an alkoxy group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroaryl alkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -ORa, -SRa, -OC(0)-Ra, -N(Ra)2, -C(0)Ra, -C(0)ORa, -OC(0)N(Ra)2, - C(0)N(Ra)2, -N(Ra)C(0)ORa, -N(Ra)C(0)Ra, -N(Ra)C(0)N(Ra)2, N (Ra)C (NRa)N (Ra)2, - N(Ra)S(0)tRa (where t is 1 or 2), -S(0)tORa (where t is 1 or 2), -S(0)tN(Ra)2 (where t is 1 or 2), or P03(Ra)2, where each Ra is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.
[0066] “Amino” or “amine” refers to a -N(Ra)2 radical group, where each Ra is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, unless stated otherwise specifically in the specification. When a -N(Ra)2 group has two Ra substituents other than hydrogen, they can be combined with the nitrogen atom to form a 4-, 5-, 6- or 7-membered ring. For example, -N(Ra)2 is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. Unless stated otherwise specifically in the specification, an amino group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -ORa, -SRa, -0C(0)-Ra, - N(Ra)2, -C(0)Ra, -C(0)0Ra, -0C(0)N(Ra)2, -C(0)N(Ra)2, -N(Ra)C(0)0Ra, - N(Ra)C(0)Ra, -N(Ra)C(0)N(Ra)2, N (Ra)C (NRa)N (Ra)2, -N(Ra)S(0)tRa (where t is 1 or 2), - S(0)tORa (where t is 1 or 2), -S(0)tN(Ra)2 (where t is 1 or 2), or PCh(Ra)2, where each Ra is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.
[0067] The term “substituted amino” also refers to N-oxides of the groups -NHRa, and NRaRa each as described above. N-oxides can be prepared by treatment of the corresponding amino group with, for example, hydrogen peroxide or m-chloroperoxybenzoic acid.
[0068] “Amide” or “amido” refers to a chemical moiety with formula -C(0)N(R)2 or -NHC(0)R, where R is selected from the group of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon), and heteroalicyclic (bonded through a ring carbon), each of which moiety may itself be optionally substituted. The two R of -N(R)2 of the amide may optionally be taken together with the nitrogen to which it is attached to form a 4-, 5-, 6- or 7- membered ring. Unless stated otherwise specifically in the specification, an amido group is optionally substituted independently by one or more of the substituents as described herein for alkyl, cycloalkyl, aryl, heteroaryl, or heterocycloalkyl. An amide may be an amino acid or a peptide molecule attached to a compound disclosed herein, thereby forming a prodrug. The procedures and specific groups to make such amides are known to those of skill in the art and can readily be found in seminal sources such as Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, N.Y., 1999, which is incorporated herein by reference in its entirety.
[0069] The term “absorption” as used herein, generally refers to the process of matter taking up exogenous energy and transforming the state of that matter to a higher electronic state when interacting with an exogenous source described herein. The process of absorption leads to an attenuation in the intensity of the exogenous energy.
[0070] As used herein, the term "chromophore" refers to a molecule or a part of a molecule responsible for its color. Color arises when a molecule absorbs certain wavelengths of visible light and transmits or reflects others. A molecule having an energy difference between two different molecular electronic states falling within the range of the visible spectrum may absorb visible light and thus be aptly characterized as a chromophore. Visible light incident on a chromophore may be absorbed thus exciting an electron from a ground state molecular state into an excited state molecular state.
[0071] “Isomers” are different compounds that have the same molecular formula. “Stereoisomers” are isomers that differ only in the way the atoms are arranged in space - i.e., having a different stereochemical configuration. “Enantiomers” are a pair of stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a “racemic” mixture. The term “(±)” is used to designate a racemic mixture where appropriate. “Diastereoisomers” are stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other. The absolute stereochemistry is specified according to the Cahn- Ingold-Prelog R-S system. When a compound is a pure enantiomer the stereochemistry at each chiral carbon can be specified by either (R) or (S). Resolved compounds whose absolute configuration is unknown can be designated (+) or (-) depending on the direction (dextro- or levorotatory) which they rotate plane polarized light at the wavelength of the sodium D line. Certain of the compounds described herein contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined, in terms of absolute stereochemistry, as (R) or (S). The present chemical entities, pharmaceutical compositions and methods are meant to include all such possible isomers, including racemic mixtures, optically pure forms and intermediate mixtures. Optically active (R)- and (S)-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefmic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.
[0072] The terms “enantiomerically enriched” and “non-racemic,” as used herein, refer to compositions in which the percent by weight of one enantiomer is greater than the amount of that one enantiomer in a control mixture of the racemic composition (e.g., greater than 1 : 1 by weight). For example, an enantiomerically enriched preparation of the (S)-enantiomer, means a preparation of the compound having greater than 50 % by weight of the (S)-enantiomer relative to the (R)-enantiomer, such as at least 55 % by weight, such as at least 60 % by weight, such as at least 65 % by weight, such as at least 70 % by weight, such as at least 75 % by weight, or such as at least 80 % by weight. In some embodiments, the enrichment can be significantly greater than 80 % by weight, providing a “substantially enantiomerically enriched” or a “substantially non-racemic” preparation, which refers to preparations of compositions that have at least 85 % by weight of one enantiomer relative to other enantiomer, such as at least 90% by weight, or such as at least 95 % by weight. The terms “enantiomerically pure” or “substantially enantiomerically pure” refers to a composition that comprises at least 98 % of a single enantiomer and less than 2 % of the opposite enantiomer. In some embodiments, the enantiomerically enriched preparation of the (S)-enantiomer may include, for example, 52 %, 54 %, 56 %, 58 %, 60 %, 62 %, 65 %, 70 %, 73 %, 76 %, 80 %, 84 %, 88 %, 90 %, 93 %, 95 %, or 98 % by weight of the (S)-enantiomer relative to the (R)-enantiomer.
[0073] “Moiety” or “fragment” refers to a specific segment or functional group of a molecule. Chemical moieties are often recognized chemical entities embedded in or appended to a molecule.
[0074] “Tautomers” are structurally distinct isomers that interconvert by tautomerization. “Tautomerization” is a form of isomerization and includes prototropic or proton-shift tautomerization, which is considered a subset of acid-base chemistry. “Prototropic tautomerization” or “proton-shift tautomerization” involves the migration of a proton accompanied by changes in bond order, often the interchange of a single bond with an adjacent double bond. Where tautomerization is possible (e.g., in solution), a chemical equilibrium of tautomers can be reached. An example of tautomerization is keto-enol tautomerization. A specific example of keto-enol tautomerization is the interconversion of pentane-2, 4-di one and 4- hydroxypent-3-en-2-one tautomers. Another example of tautomerization is phenol-keto tautomerization. A specific example of phenol-keto tautomerization is the interconversion of pyridin-4-ol and pyridin-4(lH)-one tautomers.
[0075] “Substituted” means that the referenced group may have attached one or more additional groups, radicals or moieties individually and independently selected from, for example, acyl, alkyl, alkylaryl, cycloalkyl, aralkyl, aryl, carbohydrate, carbonate, heteroaryl, heterocycloalkyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, ester, thiocarbonyl, isocyanato, thiocyanato, isothiocyanato, nitro, oxo, perhaloalkyl, perfluoroalkyl, phosphate, silyl, sulfmyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea, and amino, including mono- and di-substituted amino groups, and protected derivatives thereof. The substituents themselves may be substituted, for example, a cycloalkyl substituent may itself have a halide substituent at one or more of its ring carbons. The term “optionally substituted” means optional substitution with the specified groups, radicals or moieties.
[0076] The terms “substituted” or “optionally substituted” refer to chemical moieties, wherein one or more hydrogen atoms may be replaced by a halogen atom, a -NIL·, -SH, -NO2 or -OH group, or by an alkyl, alkenyl, alkanoyl, heteroalkyl, aryl, heteroaryl, cycloalkyl or heterocycle group as defined herein. The last-mentioned groups may be optionally substituted.
[0077] Unless otherwise stated, the chemical structures depicted herein are intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds where one or more hydrogen atoms is replaced by deuterium or tritium, or wherein one or more carbon atoms is replaced by 13C- or 14C-enriched carbons, are within the scope of this invention.
[0078] The term “FRET” as used herein refers to fluorescence resonance energy transfer which is the transfer of the excited energy of a donor to an acceptor without the emission of light.
[0079] The term “body geography” as used herein refers to the region of the body from which a sample is obtained. Examples of sample geography include, but are not limited to, the mouth, the nose, upper respiratory tract and the lower respiratory tract. A specific or general sample method may be deployed to obtain a sample from a particular body geography. For example, swab samples may be obtained from the mouth (such as oral or buccal swab samples) or the nose (nasal or nasopharyngeal swab samples) or feces and any other suitable human body geography. As used herein, the sample method or geography is distinguished from the device used to obtain the sample. That is, a buccal swab sample is obtained from mouth geography and is distinguished from the swab (device) used to collect the sample which further may deploy specific or general methods for collection.
Biological basis
[0080] Coronaviruses (CoVs) are relatively large viruses containing a single-stranded positive- sense RNA genome encapsulated within a membrane envelope. (Liu, et ak, ACS Cent. Sci. 2020, 6, 315-331, incorporated herein in its entirety). While little is currently known about the SARS- COV 2 virus, an examination of the genome sequence shows strong homology with its more well-studied cousin, SARS-CoV. In addition to the glycosylated spike protein, the betacoronavirus genome encodes for three nonstructural proteins, including RNA-dependent RNA polymerase and two enzymes, or more specifically proteases that are critical to its replication process, namely the coronavirus main proteinase (3CLpro) and the papain-like protease (PLpro). After gaining entry to a host cell, the viral RNA is translated by the host translational machinery to produce a polyprotein that comprises the full compliment of effector proteins necessary to support viral reproduction and assembly, including these non-structural proteins. Both 3CLpro and PLpro are cysteine proteases. These proteases are critically important in cleaving the initially translated polyprotein into effector proteins.
[0081] Of particular interest is the fact that the 3Clpro protease of SARS-CoV-2 shares over 95% sequence similarity with its cousin, SARS-CoV even though the genome for this enzyme is only 79% similar. Likewise, the PLpro genome is only 83% similar between these two viruses, while the translated enzymes share the same active site.
[0082] The 3CLpro enzyme has been found to cleave 11 sites in the viral polyprotein. These cleavage sites in the substrate are similar, in that the sequences targeted by the enzyme comprise at least four amino acid residues ending in LQ- on the C-terminus side of the peptide scission. Specificity on the N-terminus side of the break is a preference for S or A, with examples of N and G also known (Grum-Tokars, et ah, Virus Res. 2008, 733, 63-73). In some embodiments, the biosensor has a Formula (1) D-FG, wherein FG is a peptide comprising at least four amino acid residues that terminate in the sequence -LQ- or -LQS-. In some embodiments, FG is a peptide comprising four to six amino acid residues that terminate in the sequence -LQ- or -LQS- and include at least one unnatural amino acid residue (c) i.e. - (yLQ- or (-c LQS-) to the left of the -L. In some embodiments, FG is a peptide comprising six to eight amino acid residues that terminate in the sequence -LQ- or -LQS- and include at least one unnatural amino acid residue (c) i.e. - (yLQ- or (-c LQS-) to the left of the -L. In some embodiments, FG is a peptide comprising ten to twelve amino acid residues that terminate in the sequence -LQ- or -LQS- and include at least one unnatural amino acid residue (c) i.e. - (yLQ- or (-c LQS-) to the left of the -L. Without wishing to be bound by theory, longer peptide may provide better specificity by providing a longer substrate for the enzyme to target, thereby requiring a greater sequence alignment between the FG peptide and the enzyme’s natural substrate. Thus, more amino acid residues may be added while keeping the consensus sequence.
[0083] Likewise, the PLpro enzyme has been found to target four residue sequences ending in - GG- on the C-terminus (Baez-Santos, et al.) . In particular, the five sites targeted in vivo are specifically -LRGG-, -LKGG-, and -LNGG- sequences. The N-terminus requires little amino acid specificity, since this terminus is represented by -A-, -K-, or the e-amino group of -K- in the natural substrate. In some embodiments, the biosensor has a Formula (1) D-FG, wherein FG is a peptide comprising at least four amino acid residues that terminate in the sequence -GG-. In some embodiments, FG is a peptide comprising four to six amino acid residues that terminate in the sequence -GG- and include at least one unnatural amino acid residue (c) i.e. - (-c GG-) to the left of the -L. In some embodiments, FG is a peptide comprising six to eight amino acid residues that terminate in the sequence -GG- and include at least one unnatural amino acid residue (c) i.e.
- (-c GG-) to the left of the -L. In some embodiments, FG is a peptide comprising ten to twelve amino acid residues that terminate in the sequence -GG- and include at least one unnatural amino acid residue (c) i.e. - (-c GG-) to the left of the -L. Without wishing to be bound by theory, longer peptide may provide better specificity by providing a longer substrate for the enzyme to target, thereby requiring a greater sequence alignment between the FG peptide and the enzyme’s natural substrate. Thus, more amino acid residues may be added while keeping the consensus sequence.
[0084] The selectivity of the biosensor may be further improved by selecting a substrate that is specific to the selected enzyme. Thus, in a biosensor having a Formula (I) D-FG, wherein FG is a peptide, selectivity of the biosensor can be improved by selecting an amino acid sequence for FG that can specifically act as a substrate for the selected enzyme and not for any other enzymes.
[0085] Without wishing to be bound by theory, the sensitivity of such a biosensor depends on the amount of enzyme produced by a host cell after the target virus infects the host cell. Thus, selecting FG that is specific to an enzyme that is produced in relatively larger quantities in the infected host cell may improve the sensitivity of the biosensor.
[0086] Thus, in an aspect of the present disclosure, the biosensor is responsive to enzymes produced by human host cells that are encoded by a target virus. The biosensor may have a Formula (I) D-FG, wherein FG is a peptide having an amino acid sequence that selectively provides a substrate for the enzyme that is encoded by the target virus. Selectivity of the biosensor can thus, be improved by first selecting a suitable enzyme that is specific to the target virus and does not have significant sequence identity with, or activity similar to, enzymes produced by pathogens other than the target virus. Thus, in some embodiments, the enzyme encoded by the target virus is a protease encoded by the viruses. In some embodiments, the enzymes encoded by the target virus comprise proteases that are necessary for viral replications. In some embodiments, the enzyme encoded by the target virus is a serine protease. In some embodiments, the enzyme encoded by the target virus is a microbial cysteine protease. In some embodiments, the enzyme encoded by the target virus is a microbial enzyme. In some embodiments, the enzyme encoded by the target virus is selected from the group of 3CLpro and PLpro encoded by coronavirus. In some embodiments, the enzyme encoded by the target virus is 3CLpro encoded by coronavirus. In some embodiments, the enzyme encoded by the target virus is PLpro encoded by coronavirus.
Biosensor structure and composition
[0087] In an embodiment, this disclosure provides a biosensor for the detection of a coronavirus such as, for example, SARS-CoV-1, SARS-CoV-2, or MERS-CoV, the biosensor having a Formula (1) D-FG, wherein: (i) FG comprises a material responsive to an enzyme encoded by the coronavirus such as, for example, SARS-CoV-1, SARS-CoV-2, or MERS-CoV; and (ii) D is a spectroscopic probe, wherein FG is conjugated to the spectroscopic probe D via a covalent bond, wherein FG masks the activity of the spectroscopic probe D, wherein the enzyme causes the cleavage of the FG to release the spectroscopic probe D, resulting in a detectable optical response; and wherein the enzyme is present in a virus-infected host cell and is released outside for access by the biosensor when the host cell is ruptured.
[0088] In some embodiments, the covalent bond is selected from the group of -CO-0-, -CO-NH- , -SO2-O-, -SO2-NH-, -SO-O-, or -SO-NH-.
[0089] The design features of the disclosed biosensors have two main components: (1) a novel spectroscopic probe composed of a chromophore, (2) a material that selectively responds to a specific type of enzyme encoded by the viruses, wherein the enzyme is present in a virus- infected host cell and is released outside for access by the biosensor when the host cell is ruptured. The enzyme ecoded by the virus cleaves the biosensor to release the spectroscopic probe and causes the spectroscopic probe to produce detectable optical responses.
[0090] The spectroscopic probe can be any group that, upon fragmentation of D-FG, produces a change in an optical response. In some embodiments, the spectroscopic probe D is selected from the group of a FRET donor/acceptor pair, coumarins, phenothiazines, phenoxazines, fluoresceins, rhodols, or rhodamines. In some embodiments, the optical response produced by the biosensor is a color change from colored state to colorless state. In some embodiments, the optical response produced by the biosensor is a color change from colorless state to colored state. In some embodiments, the optical response produced by the biosensor is a change from non- fluorescent state to fluorescent state.
[0091] In some embodiments, the spectroscopic probe in the biosensor is a fluorogenic chromophore that can produce a fluorescence response upon degradation of the biosensor by the enzyme. In some embodiments, the fluorophore in the biosensor comprises coumarin, rhodamine, or fluorescein. In some embodiments, the fluorophore comprises coumarin. In some embodiments, the fluorophore in the biosensor comprises rhodamine. In some embodiments, the rhodamine is selected from the rhodamine 110, rhodamine B, Rhodamine 6G, Rhodamine Green™ (Rho G), tetramethylrhodamine, or 5-carboxy-X-rhodamine.
[0092] In some embodiments,
Figure imgf000024_0001
[0093] In some embodiments, the biosensor comprises the fluorophore covalently attached to the material that is a fragment of a substrate of SARS-CoV PLpro or SARS-CoV 3CLpro. In some embodiments, the biosensor comprises the fluorophore covalently attached to the material that is a fragment of a substrate of SARS-CoV PLpro encoded by a coronavirus. In some embodiments, the biosensor comprises the fluorophore covalently attached to the material that is a fragment of a substrate of SARS-CoV 3CLpro encoded by a coronavirus. In some embodiments, the biosensor comprises the fluorophore covalently attached to a peptide selected from the peptides disclosed in Table 5 below. In some embodiments, the biosensor comprises the fluorophore covalently attached to ubiquitin. In some embodiments, the biosensor comprises 7-amino-3- methylcoumarin (AMC) as the fluorophore covalently attached to ubiquitin.
[0094] In some embodiments, the biosensor comprises the fluorophore covalently attached to ubiquitin or ISG15 protein. In some embodiments, the biosensor comprises AMC as the fluorophore covalently attached to ubiquitin and the biochemical factor is SARS-CoV-1 PLpro or SARS-CoV-2 PLpro. In some embodiments, the biosensor comprises AMC as the fluorophore covalently attached to ISG15 protein and the biochemical factor is SARS-CoV-1 PLpro or SARS-CoV-2 PLpro.
[0095] In some embodiments, the spectroscopic probe comprises at least one fluorophore. In some embodiments, the fluorophore may be any fluorescent and luminescent probes for biological activity that is known in the art. In some embodiments, the fluorophore is selected from the group of 5-carboxytetramethylrhodamine (TAMRA), EDANS, DANSYL, DMACA, FITC, ICG, AMCA-x, Marina Blue, PyMPO, Lucifer Yellow, Mca, Trp, Rho G, rhodamine 6G, rhodamine B, rhodamine 110, 5-carboxy-X-rhodamine, Bodipy493, Bodipy TMR-x, Bodipy630, Cyanine 5 (Cy5), Cyanine 5.5 (Cy5.5), and Cyanine7.5 (Cy7.5), rhodamine, fluorescein, boron- dipyrromethene (BODIPY), fluorescein substitutes (Alexa Fluor® dye, Oregon Green® dye), 6- carboxyfluorescein (FAM, excitation range of 460-500 nm), and combinations thereof.
[0096] When choosing a fluorophore, it is preferable that the fluorophore display a high quantum yield. Long-wavelength (excitation and emission) fluorophores are preferred because of less interference from other absorbing species. It is preferred that the fluorophore should not exhibit significant sensitivity to pH change or to non-specific quenching by metal ions or other species.
[0097] In some embodiments, the spectroscopic probe D comprises a chromophore and a fluorophore as a FRET donor/acceptor pair, wherein the fluorophore is selected from the group consisting of coumarins, fluoresceins, rhodols, and rhodamines, and the chromophore is selected from the group consisting of 4-aminobenzoyl, tryptophan, coumarins, and fluoresceins, and wherein the fluorophore and the chromophore are each independently attached to a different portion of the FG.
[0098] In some embodiments, the spectroscopic probe D comprises a fluorophore and a quencher as a FRET donor/acceptor pair, wherein the fluorophore is selected from the group of coumarins, fluoresceins, rhodols, or rhodamines; and the quencher is selected from the group of 2,4-dinitrophenyl, para-nitroaniline, 4-nitro-phenylalanine, dimethylaminophenylazo)benzoyl, 4- (4-diethylaminophenylazo)-benzenesulfonyl, QSY7, or QSY21, wherein the fluorophore and the quencher each is attached to a different portion of the FG,
[0099] In some embodiments the spectroscopic probe D has chemical moiety selected from the group of Formulae (2)-(7), wherein:
Formula (
Figure imgf000026_0001
Figure imgf000026_0002
Formula
Figure imgf000026_0003
U is O or N or C;
V is -0-C(=0)-0-, -0-, or -NH-C(=0)-0-; W is O, N, S, SiMe2 or -CH 2-;
Z is -NR9R10 , -NH-A-, or -0-CH2-PhA, where A is a fragmentable group FG;
R1, R2, R3, R4 are each independently selected from the group of H, substituted and unsubstituted Cl -Cl 2 alkyl group, substituted and unsubstituted Cl -Cl 2 alkenyl group, substituted and unsubstituted Cl -Cl 2 alkynyl group, and substituted and unsubstituted aryl group;
R5, R6, R7, and R8 is each independently selected from the group of H, Cl, F, Br, CN, NO2, -NR9R10, C1-C6 alkyl group, and C1-C6 alkoxyl group;
R9 and R10 is a substituent each independently selected from the group of H, substituted and unsubstituted Cl -Cl 2 alkyl group, substituted and unsubstituted Cl -Cl 2 alkenyl group, substituted and unsubstituted Cl -Cl 2 alkynyl group, substituted and unsubstituted aryl group, fluoroalkyl, substituted and unsubstituted carbocyclyl, substituted and unsubstituted carbocyclylalkyl, substituted and unsubstituted aralkyl, substituted and unsubstituted heterocycloalkyl, substituted and unsubstituted heterocycloalkylalkyl, substituted and unsubstituted heteroaryl, and substituted and unsubstituted heteroarylalkyl; and
R11, R12, R13, R14, R15, R16, and R17 is each independently selected from the group of H, Cl, F, Br, CN, NO2, -NR9R10, C1-C6 alkyl group, and C1-C6 alkoxyl group.
[0100] In some embodiments, the spectroscopic probe D of Formula (2) is a fragment selected from the fragments represented by Formula (
Figure imgf000027_0001
[0101] In some embodiments, the spectroscopic probe D of Formula (5) is a fragment selected from Formula (
Figure imgf000027_0002
, Formula
Figure imgf000027_0003
Formula
Figure imgf000028_0001
[0102] In some embodiments, the FG comprises a fragment derived from a chemical inhibitor of the enzyme encoded by the target virus, or a peptide derived from a substrate of the enzyme encoded by the target virus.
[0103] In some embodiments, the FG to be bonded to D of Formulae (2)-(7) is a peptide derived from a substrate for a 3CLpro enzyme encoded by a coronavirus, and the peptide has at least four amino acid residues, e.g., 4 to 6 amino acid residues, 6 to 8 amino acid residues, 8-10 amino acid residues, 10-12 amino acid residues, 12-14 amino acid residues or 14-16 amino acid residues and a portion of the peptide has the sequence -LQ- and Q residue is at the peptide terminus, wherein the spectroscopic probe D is bound to the terminal Q residue.
[0104] In some embodiments, the FG to be bonded to D of Formulae (2)-(7) is a peptide derived from a substrate for a PLpro enzyme encoded by a coronavirus, and the peptide has at least four amino acid residues, e.g., 4 to 6 amino acid residues, 6 to 8 amino acid residues, 8-10 amino acid residues, 10-12 amino acid residues, 12-14 amino acid residues or 14-16 amino acid residues and a portion of the peptide has the sequence -GG- and G residue is at the peptide terminus, wherein the spectroscopic probe D is bound to the terminal G residue.
[0105] It is understood that the peptide fragment may be further substitured by groups commonly used for functional group protection in peptide synthesis or chemistry. Such groups may be, but are not limited to, Ac, Cbz, Fmoc, and Boc.
[0106] In some embodiments, the FG is VRLQS when the FG is a peptide derived from a substrate for a 3CLpro enzyme encoded by SARS-CoV-1, SARS-CoV-2, or MERS-CoV.
[0107] In some embodiments, the FG is VRLQ when the FG is a peptide derived from a substrate for a 3CLpro enzyme encoded by SARS-CoV-1, SARS-CoV-2, or MERS-CoV. [0108] In some embodiments, the FG is LRGG or RLRGG when the FG is a peptide derived from a substrate for a PLpro enzyme encoded by SARS-CoV-1, SARS-CoV-2, or MERS-CoV. [0109] In some embodiments, the biosensor comprises LRGG conjugated to 7-amino-4- methylcoumarin. In some embodiments, the biosensor comprises LRGG conjugated to Rhodamine 110. In some embodiments, the biosensor comprises LRGG conjugated to fluorescein. In some embodiments, the biosensor comprises VRLQ conjugated to 7-amino-4- methylcoumarin. In some embodiments, the biosensor comprises VRLQ conjugated to Rhodamine 110. In some embodiments, the biosensor comprises VRLQ conjugated to fluorescein.
[0110] In some embodiments, the biosensor comprises LRGG conjugated to 7-amino-4- methylcoumarin via glycine residue at C-terminus. In some embodiments, the biosensor comprises LRGG conjugated to Rhodamine 110 via glycine residue at C-terminus. In some embodiments, the biosensor comprises LRGG conjugated to fluorescein via glycine residue at C- terminus. In some embodiments, the biosensor comprises VRLQ conjugated to 7-amino-4- methylcoumarin via glutamine residue at C-terminus. In some embodiments, the biosensor comprises VRLQ conjugated to Rhodamine 110 via glutamine residue at C-terminus. In some embodiments, the biosensor comprises VRLQ conjugated to fluorescein via glutamine residue at C-terminus.
[0111] Both 3CLpro and PLpro are cysteine proteases. Because both proteases are vital to the virus for replication and controlling the host cell, they are viable targets for antiviral agents and diagnostics. Over the last two decades, much of the research in drugging SARS-CoV has focused on the development of small molecule, peptide, and peptidomimetic inhibitors of 3CLpro and PLpro. These are shown in Table 3 below and represent the microbial protease inhibitors of 3CLpro and PLpro encoded by coronavirus from which the fragmentable group FG derives. The compounds containing the fragmentable group FG bind to the targeted enzymes (e.g., 3CLPro and PLpro) encoded by coronavirus. Examples of such compounds, based on Formula (1) above, are shown in Table 4. It should be noted that useful biosensors will position the spectroscopic probe D to be released near the functionally important cysteine residue in the enzyme active site. Furthermore, the fragmentable group FG may include peptide sequences that are recognized as a substrate of the targeted enzyme wherein the binding of the fragmentable group is enhanced by association with multiple binding domains in the targeted enzyme. For example, a peptide sequence terminating in -LRGG at the C-terminus may be linked to a D while the N-terminus is linked to an inhibitor that prefers binding at a site other than the active site, thereby increasing the binding of the molecule.
Table 3: Inhibitor binding to proteases produced by coronaviruses
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
a. inhibition constant against SARS-CoV 3CLpro.
Table 4: Chromogenic Sensor Molecules of Formula (1) for Detection of Coronaviruses.
Figure imgf000033_0001
Figure imgf000034_0001
[0112] In some embodiments, the R group for the compounds disclosed in Table 4 is selected from the group of H, substituted and unsubstituted C1-C12 alkyl group, substituted and unsubstituted C1-C12 alkenyl group, substituted and unsubstituted C1-C12 alkynyl group, and substituted and unsubstituted aryl group. In some embodiments, the R group for the compounds disclosed in Table 4 is selected from the group of H, substituted and unsubstituted C1-C6 alkyl group, substituted and unsubstituted C1-C6 alkenyl group, substituted and unsubstituted C1-C6 alkynyl group, and substituted and unsubstituted aryl group. In some embodiments, the R group for the compounds disclosed in Table 4 is selected from the group of H, substituted and unsubstituted C1-C4 alkyl group, and substituted and unsubstituted aryl group. In some embodiments, the R group for the compounds disclosed in Table 4 is substituted and unsubstituted C1-C4 alkyl group. In some embodiments, the R group for the compounds disclosed in Table 4 is selected from the group of methyl group and ethyl group.
[0113] 3CLpro and PLpro are two proteases that process the polypeptide translation from the genomic RNA to either structural or non-structural protein components for replication and packaging of new generation viruses. PLpro also serves as a deubiquitinase that function to deubiquitinate host cell proteins such as interferon factor 3 (IRF3) as well as to inactivate the pathway for nuclear factor k-light-chain-enhancer of activated B cells (NF-KB).
Biosensors for Detection of Viruses.
[0114] The coronaviruses are enveloped positive-strand RNA viruses that replicate in the cytoplasm of infected cells. Coronavirus replication involves complex replication machineries including RNA genomes (27 to 32 kb), and viral proteins from the host’s antiviral defense mechanism. Two virally encoded cysteine proteases, the SARS-CoV papain-like protease (SARS-CoV PLpro) and the SARS-CoV 3C-like protease (SARS-CoV 3 Cipro) catalyze their own release from the polyprotein encoded by the viral genome (See Baez-Santos et ah, The SARS-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds, Antiviral Research, 2015, vol. 115, pp. 21-38).
[0115] Processing two viral polyproteins by SARS-CoV PLpro and SARS-CoV 3Clpro is required for the release and maturation of 16 viral proteins (nonstructural proteins or nsps) nsps 1-16 by specifically recognizing the sequence Leu-Gln*-Ser-Ala-Gly (* marks the cleavage site)
, which is responsible for directing the replication and transcription of the viral genome. However, no human protease with similar cleavage specificity is known (See Kumar et ah, In silico identification and docking-based drug repurposing against the main protease of SARS- CoV-2, causative agent of SARS-CoV-2 , April 2020, Preprint, DOL 10.26434/chemrxiv.12049590).
[0116] The establishment of viral replication sites is initiated by the recruitment of replicase proteins to host membrane mediated by several viral transmembrane domain-containing proteins such as nsp3, nsp4 and nsp6. SARS-CoV PLpro (nsp3) has an essential role during the formation of virus replication complexes via its insertion into host membranes and its numerous interactions with other nsps (See Baez-Santos et al. supra). In addition, SARS-CoV PLpro is a key enzyme in the pathogensis of SARS-CoV. The well established roles of PLpro enzymatic activities include processing of the viral polyprotein, deubiquitination (removal of ubiquitin), and delSGlation (the removal of ISG15) from host-cell proteins (See Mesecar et al., X-ray Structural and Biological Evaluation of a Series of Potent and Highly Selective Inhibitors of Human Coronavirus Papain-like Proteases, J. Med. Chem., 2014, vol. 57, pp. 2393-2412).
[0117] Further, SARS-CoV PLpro is reported to cleave the consensus sequence LXGG (Barretto et al., The Papain-like Protease of Severe Acite Respiratory Syndrome Coronavirus Has Deubiquitinating Activity, J. Virology, 2005, pp. 15189-15198). SARS-CoV PLpro 1541-2204 is reported to cleave the synthetic 12-mer peptide (ERELNGGAPIKS) derived from the viral proteins nspl/nsp2 and nsp2/nsp 3 (See Barretto et al. supra). SARS-CoV-2 PLpro 1568-1882 is reported as the new class of papain-like protease encoded by SARS-CoV-2 (See Shanker et al., Whole Genome Sequence Analysis and Homology Modelling of a 3C Like Peptidase and aNon- Structural Protein 3 of the SARS-CoV-2 Shows Protein Ligand Interaction with an Aza-Peptide and a Noncovalent Lead Inhibitor with Possible Antiviral Properties. ChemRxiv. Preprint. 2020, https://doi.org/10.26434/chemrxiv.11846943.v7). SARS-CoV-2 nsp3 is mapped within ORFlab sequence 818-2763. The nsp3 encoded by SARS-CoV-2 differs from nsp 3 encoded by SARS- CoV-1 at position 1010 (192 in the nsp3 protein). Regarding the amino acid at position 1010 (192 position of nsp3 protein), the SARS-CoV-2 nsp3 has proline, whereas SARS-CoV-1 nsp3 has non-polar amino acid isoleucine. The destabilizing mutation in nsp3 suggests a potential mechanism differentiating SARS-CoV-2 from SARS-CoV-1 (Angeletti et al., COVID-2019: The role of the nsp2 and nsp 3 in its pathogenesis, J. Medical Virology, 2020, pp. 1-5; the viral nucleic acid sequence for SARS-CoV-2 can be found at GISAID archive h Up s : //www. gl sai d . org/) .
[0118] As discussed elsewhere herein, the coronaviruses are enveloped positive-strand RNA viruses that replicate in the cytoplasm of infected cells and specifically that two virally encoded cysteine proteases, the SARS-CoV papain-like protease (SARS-CoV PLpro) and the SARS-CoV 3C-like protease (SARS-CoV 3 Cipro) catalyze their own release from the polyprotein encoded by the viral genome (See Baez-Santos et al.). Since the production of these two proteases occurs inside the host cell, when testing for presence of these enzymes they are much more likely to be found in the host cell (intracellular) than extracellular. Some of the virus replication models indicate that as new virus particle copies are made, each generating a new copy of the two proteases, upon full assembly of the new virus particle (+ve strand RNA together with nucleocapsid protein) it is expelled from the cell by exocytosis (Astuti, et al., Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): An overview of viral structure and host response, Diabetes Metab Syndr. 2020 July-August; 14(4): 407-412; published online 2020 Apr 18.doi: 10.1016/j .dsx.2020.04.020).
[0119] In an embodiment, this disclosure provides for a molecular test to detect proteases coded in the virus genome and created when virus infects a host cell. It is expected that the majority of the virally encoded enzymes will be preserved in intact cells or in secreted exosomes that must be disrupted in order to assay their contents. In a preferred embodiment, the test to detect proteases is performed on biological cell samples that have been lysed.
[0120] In some embodiments, the samples, upon lysing, may have a concentration of the target enzyme in a range from about 1 pM to 10 nM. For example, the concentartion of the target enzyme in the sample may be less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM or less than about 5 pM.
[0121] In some embodiments, the concentration of the target enzyme in the sample may be greater than about 1 pM, greater than about 5 pM, greater than about 10 pM, greater than about 20 pM, greater than about 30 pM, greater than about 40 pM, greater than about 50 pM, greater than about 60 pM, greater than about 70 pM, greater than about 80 pM, greater than about 90 pM, greater than about 100 pM, greater than about 150 pM, greater than about 200 pM, greater than about 250 pM, greater than about 300 pM, greater than about 350 pM, greater than about 400 pM, greater than about 450 pM, greater than about 500 pM, greater than about 550 pM, greater than about 600 pM, greater than about 650 pM, greater than about 700 pM, greater than about 750 pM, greater than about 800 pM, greater than about 850 pM, greater than about 900 pM, greater than about 1 nM, greater than about 2 nM, greater than about 3 nM, greater than about 4 nM, greater than about 5 nM, greater than about 6 nM, greater than about 7 nM, greater than about 8 nM, or greater than about 9 nM. [0122] In some embodiments, the concentration of the target enzyme is in a range from about 1 pM to about 5 pM, about 5 pM to about 10 pM, about 10 pM to about 20 pM, about 20 pM to about 50 pM, about 50 pM to about 75 pM, about 75 pM to about 100 pM, about 100 pM to about 150 pM, about 150 pM to about 200 pM, about 200 pM to about 500 pM, about 500 pM to about 1 nM, about 1 nM to about 2 nM, about 2 nM to about 5 nM, about 5 nM to about 7.5 nM, about 7.5 nM to about 10 nM, about 1 pM to about 10 pM, about 10 pM to about 50 pM, about 10 pM to about 100 pM, about 50 pM to about 250 pM, about 50 pM to about 500 pM, about 100 pM to about 250 pM, about 100 pM to about 500 pM, about 100 pM to about 750 pM, about 100 pM to about 1 nM, about 250 pM to about 500 pM, about 250 pM to about 750 pM, about 250 pM to about 1 nM, about 500 pM to about 750 pM, about 500 pM to about 1 nM, about 500 pM to about 2.5 nM, about 1 nM to about 1.1 nM, about 1 nM to about 1.5 nM, about 1 nM to about 2.5 nM, about 1 nM to about 5 nM, about 1 nM to about 7.5 nM, about 1 nM to about 10 nM, about 1 nM to about 2 nM, about 1 nM to about 3 nM, about 1 nM to about 4 nM, about 1 nM to about 5 nM, about 1 nM to about 6 nM, about 1 nM to about 7 nM, about 1 nM to about 8 nM, about 1 nM to about 9 nM, about 1 nM to about 10 nM, about 2 nM to about 2.5 nM, about 2 nM to about 3 nM, about 3 nM to about 4 nM, about 4 nM to about 5 nM, about 5 nM to about 6 nM, about 6 nM to about 7 nM, about 7 nM to about 8 nM, about 8 nM to about 9 nM, or about 9 nM to about 10 nM, or another range between any two of these ranges.
[0123] In an embodiment, this disclosure provides a biosensor for the detection of viruses having a formula (1) D-FG, wherein (i) FG comprising a material responsive to an enzyme encoded by a target coronavirus such as, for example, those selected from the group of SARS-CoV-1, SARS- CoV-2, or MERS-CoV; and (ii) D is a spectroscopic probe, wherein the material FG is conjugated to the spectroscopic probe D via a covalent bond, wherein the FG masks the activity of the spectroscopic probe D, wherein the enzyme causes the cleavage of the FG to release the spectroscopic probe D, resulting in a detectable optical response; and wherein the enzyme is present in a virus-infected host cell.
[0124] In some embodiments, the covalent bond is selected from the group of -C0-0-, -CO-NH- , -SO2-O-, -SO2-NH-, -SO-O-, or -SO-NH-.
[0125] In some embodiments, the enzyme encoded by the target virus is a protease encoded by the viruses. In some embodiments, the enzymes encoded by the target virus comprise proteases that are necessary for viral replications. In some embodiments, the enzyme encoded by the target virus is a serine protease. In some embodiments, the enzyme encoded by the target virus is a microbial cysteine protease. In some embodiments, the enzyme encoded by the target virus is a microbial enzyme. In some embodiments, the enzyme encoded by the target virus is selected from the group of 3CLpro and PLpro encoded by coronavirus. In some embodiments, the enzyme encoded by the target virus is 3CLpro encoded by coronavirus. In some embodiments, the enzyme encoded by the target virus is PLpro encoded by coronavirus.
[0126] In some embodiments, the biosensor is designed to be a colorless material which upon attack by an enzyme on the material triggers the release of the latent leuco dye which then spontaneously forms colored dye. This enzymatically triggered release of a latent leuco dye by proper design can be made to selectively produce a strong color in the presence of a pathogenic virus which produces an enzyme capable of attacking the material. One primary example is when the material is a fragment of the small molecule, peptide, or peptidomimetic inhibitors of 3CLpro and PLpro such as the biosensors disclosed in Table 4 which are sensitive to the protease encoded by coronavirus. Upon reaction with the protease, the material is designed to release the leuco-chromophore which then forms a strong color indicating the presence of coronavirus.
[0127] This biosensor design is not limited to protease encoding pathogens. The protease may be from human host that is important for virus replication. Peptidases which cleave specific proteins can also be incorporated into this design to produce color specific to an overexpressed peptidase.
[0128] In some embodiments, the material is a substrate of protease encoded by human coronaviruses. In some embodiments, the material is a peptide derived from the substrate of protease encoded by human coronaviruses. In some embodiments, the human coronaviruses may include SARS-CoV-1, MERS-CoV, or SARS-CoV-2. In some embodiments, the human coronaviruses is SARS-CoV-1. In some embodiments, the human coronaviruses is MERS-CoV. In some embodiments, the human coronavirus is SARS-CoV-2. In some embodiments, the substrate of protease encoded by human coronaviruses comprises ubiquitin or interferon- stimulated gene encoded (ISG15) protein and the biochemical factor comprise SARS-CoV-1 PLpro or SARS-CoV-2 PLpro.
[0129] As used herein, the term “ubiquitin” refers to a small (8.6 kDa) regulatory protein found in most tissues of eukaryotic organisms. Key features include its C-terminal tail and the 7 lysine residues. The addition of ubiquitin to a substrate protein is called ubiquitination. Ubiquitination can mark them for degradation via the proteasome.
[0130] As used herein, the term “ISG15 protein” refers to a 17 kDa secreted protein that in humans encoded by the ISG15 gene and is the first ubiquitin-like modifier identified, and is similar to a ubiquitin linear dimer. The main cellular function of the protein is ISGylation, its covalent addition to cytoplasmic and nuclear proteins, similar to ubiquitination. It is also known as UCRP (ubiquitin cross-reactive protein) since it contains 2 tandem ubiquitin homology domains and is cross-reactive with ubiquitin antibodies.
[0131] In some embodiments, the peptide is selected from the peptide sequence disclosed in Table 5.
[0132] In some embodiments, the material is conjugated to the spectroscopic probe via the C- terminus or N-terminus of the peptide sequence of the peptide as disclosed in Table 5.
[0133] In some embodiments, the material is a peptide derived from the substrate of 3CLpro encoded by human coronaviruses SARS CoV-1. In some embodiments, the peptide is selected from the group of Leu, Val, Val-Leu, Val-Phe, Ala-Val-Leu, Ser-Val-Phe, Val-Thr-Phe-Gln, Val-Thr-Leu-Gln, Val-Thr-Ala-Gln, Glu-Ser-Ala-Thr-Leu-Gln-Ser-Gly-Leu-Ala-Lys-Ser, Thr- Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met, and Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe- Arg-Lys-Met. In some embodiments, the material comprises Val-Leu, Val-Phe, Ala-Val-Leu, and Ser-Val-Phe. In some embodiments, the material comprises Glu-Ser-Ala-Thr-Leu-Gln-Ser- Gly-Leu-Ala-Lys-Ser. In some embodiments, the material comprises Thr-Ser-Ala-Val-Leu-Gln- Ser-Gly-Phe-Arg-Lys-Met. In some embodiments, the material is conjugated to the spectroscopic probe via the C-terminus or N-terminus of the peptide sequence of peptide derived from the substrate of 3CLpro encoded by human coronaviruses SARS CoV 1 as described herein. In some embodiments, the material comprises at least one glutamine residue.
[0134] In some embodiments, the material comprises one or more unnatural amino acids such as those described in Table 2. In some embodiments, the material is Ac-Abu-Tle-Leu-Gln or Ac- Thz-Tle-Leu-Gln. In some embodiments, the one or more unnatural amino acids are positioned relatively closer to the N-terminus of the peptide as compared to the C-terminus of the peptide. [0135] In some embodiments, the latent leuco dye is bound to the material via a labile bond susceptible to enzymatic degradation. In some embodiments, the labile bond is selected from the group of an ester bond, an amide bond, an imine bond, an acetal bond, a ketal bond, and combinations thereof. Without wishing to be bound by theory, an ester bond may provide improved sensitivity since it can be relatively easy to cleave an ester bond. In contrast, an amide bond, for example, may provide for a more stable bond between the material and the spectroscopic probe, and thereby provide a relatively more selective biosensor.
[0136] In some embodiments, the biochemical factor encoded by the virus is a reducing agent. In some embodiments, the reducing agent encoded by the virus is glutathione.
[0137] In some embodiments, the material is derived from the substrate of a microbial peptidase. In some embodiments, the material is derived from the substrate of a microbial protease.
[0138] The pathogen diagnostic bio-sensor operates by using the endogenous pathogen-encoded cysteine protease activity of a certain SARS-CoV protease to cleave a spectroscopic probe from the biosensor of the invention comprising (i) a material responsive to SARS-CoV protease, and (ii) a latent leuco dye or FRET donor/accept pair as a spectroscopic probe, wherein the material is conjugated to the spectroscopic probe, wherein the material masks the activity of the spectroscopic probe, wherein the enzyme encoded by the target virus causes the cleavage of the material to release the latent spectroscopic probe and result in a detectable optical response. The material is derived from the substrate of the SARS-CoV protease (e.g., nsp3 is the substrate and SARS-CoV papain like protease is the enzyme). Only the SARS-CoV protease can cleave the spectroscopic probe from the biosensor. Thus, the properly chosen material derived from the substrate peptide sequence for the SARS-CoV protease in the biosensor is responsive to the appropriate SARS-CoV protease activity and can be used to detect a SARS-CoV pathogen of interest.
1A. FRET-Responsive Fluorophore Labeled Biosensor
[0139] In some embodiments, the D is a pair of fluorophore/chromophore and the FG is a peptide derived from a substrate for 3CLpro encoded by a coronavirus. The fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C-terminus, and the chromophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N-terminus. The FG peptide has at least five amino acid residues and a portion of the peptide has the sequence -LQS-. In some embodiments, the FG peptide has 4 to 6 amino acid residues, 6 to 8 amino acid residues, 8 to 10 amino acid residues, 10 to 12 amino acid residues, 12 to 14 amino acid residues, or 14-16 amino acid residues. In some embodiments, the FG peptide includes at least one unnatural amino acid residue. In some embodiments, the unnatural amino acid residue is relatively closer to the N-terminus of the FG peptide.
[0140] In some embodiments, the D is a pair of fluorophore/chromophore and the FG is a peptide derived from a substrate for a 3CLpro enzyme encoded by a coronavirus. The fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N-terminus, and the chromophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C-terminus. The FG peptide has at least five amino acid residues and a portion of the peptide has the sequence -LQS-. In some embodiments, the FG peptide has 4 to 6 amino acid residues, 6 to 8 amino acid residues, 8 to 10 amino acid residues, 10 to 12 amino acid residues, 12 to 14 amino acid residues, or 14-16 amino acid residues. In some embodiments, the FG peptide includes at least one unnatural amino acid residue. In some embodiments, the unnatural amino acid residue is relatively closer to the N-terminus of the FG peptide.
[0141] In some embodiments, the D is the FRET fluorophore/quencher and the FG is a peptide derived from a substrate for a 3CLpro enzyme encoded by a coronavirus. The fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C- terminus, and the quencher is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N-terminus. The FG peptide has at least five amino acid residues and a portion of the peptide has the sequence -LQS-. In some embodiments, the FG peptide has 4 to 6 amino acid residues, 6 to 8 amino acid residues, 8 to 10 amino acid residues, 10 to 12 amino acid residues,
12 to 14 amino acid residues, or 14-16 amino acid residues. In some embodiments, the FG peptide includes at least one unnatural amino acid residue. In some embodiments, the unnatural amino acid residue is relatively closer to the N-terminus of the FG peptide.
[0142] In some embodiments, the D is the FRET fluorophore/quencher and the FG is a peptide derived from a substrate for a 3CLpro enzyme encoded by a coronavirus. The fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N- terminus, and the quencher is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C-terminus. The FG peptide has at least five amino acid residues and a portion of the peptide has the sequence -LQS-. In some embodiments, the FG peptide has 4 to 6 amino acid residues, 6 to 8 amino acid residues, 8 to 10 amino acid residues, 10 to 12 amino acid residues,
12 to 14 amino acid residues, or 14-16 amino acid residues. In some embodiments, the FG peptide includes at least one unnatural amino acid residue. In some embodiments, the unnatural amino acid residue is relatively closer to the N-terminus of the FG peptide.
[0143] In some embodiments, the D is a pair of fluorophore/chromophore and the FG is a peptide derived from a substrate for a PLpro enzyme encoded by a coronavirus. The fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C- terminus, and the chromophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N-terminus. The FG peptide has at least four amino acid residues and a portion of the peptide has the sequence -GG-. In some embodiments, the FG peptide has 4 to 6 amino acid residues, 6 to 8 amino acid residues, 8 to 10 amino acid residues, 10 to 12 amino acid residues, 12 to 14 amino acid residues, or 14-16 amino acid residues. In some embodiments, the FG peptide includes at least one unnatural amino acid residue. In some embodiments, the unnatural amino acid residue is relatively closer to the N-terminus of the FG peptide.
[0144] In some embodiments, the D is a pair of fluorophore/chromophore and the FG is a peptide derived from a substrate for a PLpro enzyme encoded by a coronavirus. The fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N- terminus, and the chromophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C-terminus. The FG peptide has at least four amino acid residues and a portion of the peptide has the sequence -GG-. In some embodiments, the FG peptide has 4 to 6 amino acid residues, 6 to 8 amino acid residues, 8 to 10 amino acid residues, 10 to 12 amino acid residues, 12 to 14 amino acid residues, or 14-16 amino acid residues. In some embodiments, the FG peptide includes at least one unnatural amino acid residue. In some embodiments, the unnatural amino acid residue is relatively closer to the N-terminus of the FG peptide.
[0145] In some embodiments, the D is the FRET fluorophore/quencher and the FG is a peptide derived from a substrate for a PLpro enzyme encoded by a coronavirus. The fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N- terminus, and the quencher is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C-terminus. The FG peptide has at least four amino acid residues and a portion of the peptide has the sequence -GG-. In some embodiments, the FG peptide has 4 to 6 amino acid residues, 6 to 8 amino acid residues, 8 to 10 amino acid residues, 10 to 12 amino acid residues, 12 to 14 amino acid residues, or 14-16 amino acid residues. In some embodiments, the FG peptide includes at least one unnatural amino acid residue. In some embodiments, the unnatural amino acid residue is relatively closer to the N-terminus of the FG peptide.
[0146] In some embodiments, the D is the FRET fluorophore/quencher and the FG is a peptide derived from a substrate for a PLpro enzyme encoded by a coronavirus. The fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C- terminus, and the quencher is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N-terminus. The FG peptide has at least five amino acid residues and a portion of the peptide has the sequence -GG-. In some embodiments, the FG peptide has 4 to 6 amino acid residues, 6 to 8 amino acid residues, 8 to 10 amino acid residues, 10 to 12 amino acid residues,
12 to 14 amino acid residues, or 14-16 amino acid residues. In some embodiments, the FG peptide includes at least one unnatural amino acid residue. In some embodiments, the unnatural amino acid residue is relatively closer to the N-terminus of the FG peptide. [0147] In some embodiments, the rhodamine is Rhodamine Green™ having a succinimidyl ester attached to the 5- or 6 position of the rhodamine molecule as in the following formula
Figure imgf000045_0001
, wherein the reactive succinimidyl ester is for reacting with a reactive amine group on the material.
[0148] In some embodiments, the rhodamine is rhodamine
Figure imgf000045_0002
[0149] In some embodiments, for any of the herein described biosensors, the fluorogenic chromophore is chemically linked to the peptide derived from the substrate of the virus encoded enzyme via a reactive functional group on the side chain of an amino acid residue in the peptide sequence or the peptide terminal groups (C -terminus or N-terminus), and the fluorogenic chromophore has reactive functional groups including an amine group (-NH2), a carboxyl group (-COOH), a hydroxyl group (-OH), sulfonyl group (-SO2CI), succinimidyl ester, or a thiol group (-SH). In some embodiments, the reactive functional group on the side chain of an amino acid residue in the peptide sequence and the peptide terminal groups of the peptide are independently selected from an amine group (-NH2), an amide group (-C(=0)-NH2), a carboxyl group (- COOH), a hydroxyl group (-OH), a thiol group (-SH), and a sulfhydryl group (-S-S-). In some embodiments, the chemical linkage between the fluorogenic chromophore and the peptide derived from the substrate of the virus encoded enzyme comprises an amide bond formed by NHS/EDC chemistry.
[0150] For example, Rhodamine Green™ may be chemically linked to the carboxyl group on the C-terminus of LRGG- via an ester bond by the reaction of the -OH of the carboxyl group of the C-terminus glycine with the succinimidyl ester at the 5- or 6- position of the Rhodamine Green™ molecule (See chemical formula above).
[0151] In some embodiments, the spectroscopic probe is a fluorescent chromophore that may be excited by energy transfer from a first chromophore that is preferably excited by a first optical radiation. Following the cleavage of the biosensor by the enzyme, the fluorescent chromophore is no longer nearby the first chromophore, leading to loss of the fluorescent signal from the fluorescent chromophore.
[0152] In some embodiments, the biosensor comprises a fluorophore and a quencher as a FRET donor/acceptor pair, wherein the fluorophore and the quencher each is attached to a different portion of the material moiety, and the fluorescence of the fluorophore is quenched. In some embodiments, the fluorophore is attached at one end of the material and the quencher is attached at the opposite end, e.g., the biosensor has the formula: fluorophore donor-material-quencher acceptor. In some embodiments, the fluorophore are on opposite sides of a cleavage site targeted by the enzyme. In a preferred embodiment, the fluorophore and the quencher are within about 30 Angstroms of each other. After the biosensor is contacted with samples containing the coronavirus, the enzymes encoded by the coronavirus (e.g., SARS-CoV PLpro and SARS-CoV 3CLpro) cleaves the biosensor to release any one of the fluorophore or the quencher. The enzyme degradation of the biosensor results in fluorophore-quencher separation and strong fluorescence emission.
[0153] In some embodiments, the distance between the donor and the acceptor in the FRET pair ranges from 1 nm to 10 nm. In some embodiments, the emission spectrum of the donor overlaps with the absorption spectrum of the acceptor in the FRET pair. In some embodiments, the fluorophore has an excitation wavelength ranging from 280 nm to 490 nm. In some embodiments, the fluorophore has an excitation wavelength ranging from 340 nm to 360 nm. In some embodiments, the fluorophore has an excitation peak wavelength selected from the group of 280 nm, 320 nm, 325 nm, 340 nm, 342 nm, 350 nm, 430 nm, or 490 nm. In some embodiments, the fluorophore has an emission wavelength that ranges from 360 nm to 562 nm.
In some embodiments, the fluorophore has an emission wavelength that ranges from 440 nm to 450 nm. In some embodiments, the fluorophore has a peak emission wavelength selected from the group of 360 nm, 392 nm, 420 nm, 465 nm, 490 nm, 520 nm, or 562 nm. [0154] In some embodiments, the donor of the FRET pair is a fluorophore selected from the group of coumarin, fluorescein, rhodamine, xanthene, BODIPY® (boron-dipyrromethene),
Alexa Fluor® (sulfonated derivative of coumarin, rhodamine, xanthene or cyanine dye), EDANS (5-[(2-Aminoethyl) amino] naphthalene- 1 -sulfonic acid), or cyanine dye. In some embodiments, the fluorescent dye is (2-aminobenzoyl or anthraniloyl) (Abz), (N-Methyl-anthraniloyl) (N-Me- Abz), (5-(Dimethylamino)naphthalene-l-sulfonyl) (Dansyl), (7-Dimethylaminocoumarin-4- acetate) (DMACA), fluorescein isothiocyanate (FITC), indocyanine green (ICG), AMCA-x, Marina Blue, PyMPO, (6-Amino-2,3-dihydro-l,3-dioxo-2-hydrazinocarbonylamino-lH- benz[d,e]isoquinoline-5,8-disulfonic acid) (Lucifer Yellow), ((7-Methoxycoumarin-4-yl)acetyl) (Mca), tryptophan (Trp), Rhodamine Green™ (Rho G), rhodamine 6G, rhodamine B, rhodamine 110, tetramethylrhodamine, 5-carboxy-X-rhodamine, Bodipy493, Bodipy TMR-x, Bodipy630, Cyanine 5 (Cy5), Cyanine 5.5 (Cy5.5), and Cyanine7.5 (Cy7.5).
[0155] In some embodiments, the fluorescence quencher (acceptor) of the FRET pair may include 4-(4-dimethylaminophenylazo)benzoyl (Dabcyl), 2,4-dinitrophenyl (Dnp), para- nitroaniline (pNA), 4-nitro-benzyloxycarbonyl (4-Nitro-Z), N-(9-{2-[(4-{[(2,5-dioxopyrrolidin- l-yl)oxy]carbonyl}piperidin-l-yl)sulfonyl]phenyl}-6-[methyl(phenyl)amino]-3H-xanthen-3- ylidene)-N-methylanilinium (QSY7), (2,5-dioxopyrrolidin-l-yl) l-[2-[3-(l,3-dihydroisoindol-2- ium-2-ylidene)-6-(l,3-dihydroisoindol-2-yl)xanthen-9-yl]phenyl]sulfonylpiperidine-4- carboxylate;chloride (QSY21), (2,5-dioxopyrrolidin-l-yl) 2-[4-[(4-nitro-2,l,3-benzoxadiazol-7- yl)amino]phenyl]acetate (QSY35), 4-((4-((E)-(2-methoxy-5-methyl-4-((E)-(4-methyl-2- nitrophenyl)diazenyl)phenyl)diazenyl)phenyl)(methyl)amino)butanoic acid (BHQ1), fluorescein, or FITC.
[0156] In some embodiments, the biosensor comprises rhodamine /coumarin, rhodamine 6G/fluorescein, rhodamine 6G/FITC, Rho G/QSY7, TMR/BHQ1, Cy5/QSY21, CY5.5/QSY21, EDANS/ Dabcyl as FRET donor/acceptor pair. In some embodiments, the biosensor comprises rhodamine 6G/fluorescein, rhodamine 6G/FITC, Rho G/QSY7 as FRET donor/acceptor pair.
[0157] In some embodiments, the biosensor comprises the FRET donor/acceptor pair each covalently attached to the material that is a fragment of a substrate of SARS-CoV-1 PLpro and SARS-CoV-2 PLpro encoded by a coronavirus. In some embodiments, the biosensor comprises the FRET donor/acceptor pair each covalently attached to the material that is a fragment of a substrate of SARS-CoV-1 3CLpro and SARS-CoV-2 3CLpro encoded by a coronavirus. In some embodiments, the fragment of the substrate of SARS-CoV-1 PLpro and SARS-CoV-2PLpro or SARS-CoV-1 3CLpro and SARS-CoV-23CLpro comprises a peptide selected from the peptides disclosed in Table 5 below.
[0158] In some embodiments, the substrate may be a simple fluorogenic peptide (SFP), the mechanism of which is schematically shown in FIG. 12A. SFPs are typically relatively short peptides, and generally are expected to have low catalytic efficiencies for reactions with target proteases.
[0159] In some embodiments, the substrate may be an internally quenched fluorogenic peptide (IQFP), the mechanism of which is schematically shown in FIG. 12B. IQFPs typically are expected to have relatively higher catalytic efficiencies for reactions with target proteases.
[0160] Without wishing to be bound by theory, addition of specific unnatural amino acid residues in the peptide sequences of substrates can reduce cross-reactivity with non-target enzymes. Thus, a peptide sequence, particularly that of an IQFP, can have higher sensitivity as well as selectivity for specific protease detection. Table 6 includes some examples of SFP and IQFP peptides of various sequence lengths with at least one unnatural amino acid.
[0161] In some embodiments, the donor and the acceptor of the FRET pair in the biosensor each is independently attached to the C-terminus or N-terminus of a peptide derived from the substrate of SARS-CoV-1 PLpro and SARS-CoV-2 PLpro or SARS-CoV-1 3CLpro and SARS-CoV-2 3CLpro selected from those disclosed in Table 5 below.
[0162] In some embodiments, the peptide is derived from the substrate of SARS-CoV-1 PLpro and SARS-CoV-2 PLpro and includes a sequence selected from LXGG, RLRGG, LRGG,
LKGG, RLKGG, MRGG, IRGG, MKGG, IKGG, LSGG, LYGG, LNGG, LDGG, LQGG, LEGG, RLEGG, LHGG, LQGG, LTGG, SLKGG, and KKAG.
[0163] In some embodiments, the peptide is derived from the substrate of SARS-CoV-1 3CLpro and SARS-CoV-23CLpro and includes a sequence selected from VRLQ, PRLQ, VKLQ, VSLQ, AVLQ, VTFQ, LQSAG, VRLQS, PRLQS, VVRLQ, VVVLQ, VAVLQ, TSAVLQ, SGVTFQ, TSDVLQ, TNAVLQ, SGFRK, SGFRR, GKFKK, GKFRK, VARLQ, and VPRLQ. [0164] In some embodiments, the peptide is derived from an unnatural substrate of SARS-CoV- 1 3CLpro and SARS-CoV-2 3CLpro. In some embodiments, the peptide comprises a sequence of four residues given by X1-X2-LQ-, wherein Xi is selected from Abu, V, A, Tie, Me, Tba, Thz, Aph,and Phg, preferably Abu, Thz; and X2 is selected from Tie, D-Phe, D-Tyr, Om, Har, Dab,
K, D-Phg, D-Trp, and R, preferably Tie. In some embodiments, the peptides comprises a sequence of 4 to 6 amino acid residues including the sequence given by X1-X2-LQ-, 6 to 8 amino acid residues including the sequence given by X1-X2-LQ-, 8 to 10 amino acid residues including the sequence given by X1-X2-LQ-, 10 to 12 amino acid residues including the sequence given by X1-X2-LQ-, 12 to 14 amino acid residues including the sequence given by X1-X2-LQ-, or 14 to 16 amino acid residues including the sequence given by X1-X2-LQ-. Further, if the peptide is SFP, the sequence given by X1-X2-LQ- is at the C terminus, while for IQHPs, the sequence given by X1-X2-LQ- is positioned such that the unnatural amino acid residues are closer to the N- terminus of the peptide.
[0165] In some embodiments, the peptide is derived from an unnatural substrate of SARS-CoV- 1 PLpro and SARS-CoV-2 PLpro. In some embodiments, the peptide comprises a sequence of at least four residues given by X1-X2-GG-, wherein Xi is selected from hTyr, hPhe, hPhe(4F), hPhe(4CF3), Abu, Abu(Bth), Me, Phg, Tba, or Tie, and X2 is selected from Dap, Dab, Lys, Orn, hArg, 4-Pip, Aph, or 2-Pal. In some embodiments, biosensor comprises ACC-Gly-hTyr-DAP- Gly-Gly-Gly-Thr-Glu-Pro-Gly-Lys(dnp)-Lys-MTR, ACC-Gly-hTyr-DAP-Gly-Gly-Ala-Ile-Thr- Lys(dnp)-Lys-MTR, ACC-Gly-hPhe-DAP-Gly-Gly-Gly-Thr-Glu-Pro-Gly-Lys(dnp)-Lys-MTR, or ACC-Gly-hTyr-Phe(guan)-Gly-Gly-Gly-Thr-Glu-Pro-Gly-Lys(dnp)-Lys-MTR, wherein R is H-, CH3CO-, H0C(0)-(CH2)m-C0-, or H(-0-CH2CH2)p-, and wherein n and m are independently 1-4 and p is 2-100. In some embodiments, the peptides comprises a sequence of 4 to 6 amino acid residues including the sequence given by X1-X2-GG-, 6 to 8 amino acid residues including the sequence given by X1-X2-GG-, 8 to 10 amino acid residues including the sequence given by X1-X2-GG-, 10 to 12 amino acid residues including the sequence given by X1-X2-GG-, 12 to 14 amino acid residues including the sequence given by X1-X2-GG-, or 14 to 16 amino acid residues including the sequence given by X1-X2-GG-. Further, if the peptide is SFP, the sequence given by X1-X2-GG- is at the C terminus, while for IQHPs, the sequence given by X1-X2-GG- is positioned such that the unnatural amino acid residues are closer to the N-terminus of the peptide. [0166] In some embodiments, the biosensor comprises rhodamine 6G/fluorescein, rhodamine 6G/FITC, or Rho G/QSY7 attached to a peptide derived from the substrate of SARS-CoV-1 PLpro and SARS-CoV-2 PLpro and includes a sequence selected from LXGG, RLRGG, LRGG, LKGG, RLKGG, MRGG, IRGG, MKGG, IKGG, LSGG, LYGG, LNGG, LDGG, LQGG, LEGG, RLEGG, LHGG, LQGG, LTGG, SLKGG, and KKAG. In some embodiments, the biosensor comprises rhodamine 6G/fluorescein, rhodamine 6G/FITC, or Rho G/QSY7 attached to a consensus peptide sequence LXGG that is recognized by SARS-CoV-1 PLpro and SARS- CoV-2 PLpro. In some embodiments, the biosensor comprises rhodamine 6G/fluorescein, rhodamine 6G/FITC, or Rho G/QSY7 attached to a synthetic 12-mer peptide (ERELNGGAPIKS) derived from the nspl/nsp2 and nsp2/nsp3 sequences that are recognized by SARS-CoV-1 PLpro and SARS-CoV-2 PLpro 1541-2204. In some embodiments, the biosensor comprises rhodamine 6G/fluorescein attached to a consensus peptide sequence LXGG that is recognized by SARS-CoV-1 PLpro and SARS-CoV-2 PLpro. In some embodiments, the biosensor comprises rhodamine 6G/fluorescein attached to a synthetic 12-mer peptide (ERELNGGAPIKS) derived from the nspl/nsp2 and nsp2/nsp3 sequences that are recognized by SARS-CoV-1 PLpro and SARS-CoV-2 PLpro 1541-2204.
[0167] In some embodiments, the biosensor comprises rhodamine 6G/fluorescein, or Rho G/QSY7 attached to a peptide derived from the substrate of SARS-CoV-1 3CLpro and SARS- CoV-2 3CLpro and includes a sequence selected from VRLQ, PRLQ, VKLQ, VSLQ, AVLQ, VTFQ, LQSAG, VRLQS, PRLQS, VVRLQ, VVVLQ, VAVLQ, TSAVLQ, SGVTFQ, TSDVLQ, TNAVLQ, SGFRK, SGFRR, GKFKK, GKFRK, VARLQ, and VPRLQ. In some embodiments, the biosensor comprises rhodamine 6G/fluorescein, rhodamine 6G/FITC, or Rho G/QSY7 attached to a consensus peptide sequence LQSAG that is recognized by SARS-CoV-1 3CLpro and SARS-CoV-2 3CLpro. In some embodiments, the biosensor comprises rhodamine 6G/fluorescein, rhodamine 6G/FITC, or Rho G/QSY7 attached to a synthetic 10-mer peptide (AVLQSGFRKK) derived from the natural non- structural proteins that are recognized by SARS- CoV-1 3CLpro and SARS-CoV-2 3CLpro. In some embodiments, the biosensor comprises rhodamine 6G/fluorescein attached to a consensus peptide sequence LQSAG that is recognized by SARS-CoV-1 3CLpro and SARS-CoV-2 3CLpro. In some embodiments, the biosensor comprises rhodamine 6G/fluorescein attached to a consensus peptide sequence LQSAG that is recognized by SARS-CoV-1 3CLpro and SARS-CoV-2 3CLpro. In some embodiments, the biosensor comprises rhodamine 6G/fluorescein attached to a synthetic 10-mer peptide (AVLQSGFRKK) derived from the natural non- structural proteins that are recognized by SARS- CoV-1 3CLpro and SARS-CoV-2 3CLpro.
[0168] In some embodiments, the peptide is derived from an unnatural substrate of SARS-CoV- 1 3CLpro and SARS-CoV-23CLpro. In some embodiments, the peptide comprises a sequence of at least four residues given by X1-X2-X3-Q-, wherein Xi is selected from Abu, Val, Ala, Tie,
Tba, Phg, Me, and other small, aliphatic amino acids; X2 is selected from Tie, D-Phe, D-Tyr,
Orn, hArg, Dab, Dap, Dht, Lys, D-Phg, D-Trp, Arg, and Met(0)2; and X3 is selected from Leu, 2-Abz, 3-Abz, (4-N02)Phe, b-Ala, Dht, hLeu, Met, and He.
[0169] In some embodiments, for any of the herein described FRET based biosensors, the FRET donor/acceptor pair are chemically linked to the peptide derived from the substrate of the virus encoded enzyme via a reactive functional group on the side chain of an amino acid residue in the peptide sequence or the peptide terminal groups (C-terminus or N-terminus), and the FRET donor/acceptor pair have reactive functional groups including an amine group (-NH2), a carboxyl group (-COOH), a hydroxyl group (-OH), sulfonyl group (-SO2CI), succinimidyl ester, or a thiol group (-SH). In some embodiments, the reactive functional group on the side chain of an amino acid residue in the peptide sequence and the peptide terminal groups of the peptide are independently selected from an amine group (-NH2), an amide group (-C(=0)-NH2), a carboxyl group (-COOH), a hydroxyl group (-OH), a thiol group (-SH), and a sulfhydryl group (-S-S-). In some embodiments, the chemical linkage between the FRET donor/acceptor pair and the peptide derived from the substrate of the virus encoded enzyme comprises an amide bond formed by MTS/EDC chemistry.
[0170] For example, for a biosensor composed of FRET donor/acceptor pair EDANS/ Dabcyl and the synthetic 12-mer peptide Glu-Ser-Ala-Thr-Leu-Gln-Ser-Gly-Leu-Ala-Lys-Ser and has the chemical linkages as in the formula (E-EDANS)RELNGGAPI(K-DABCYL)S (See FIG. 1 for chemical structure). In this case, EDANS is chemically linked to the 1st glutamic acid on N- terminus via an amide bond formed between the amine group as highlighted in the square of EDANS formula
Figure imgf000052_0001
the carboxylic acid on the side chain of the 1st glutamic acid residue. Dabcyl is chemically linked to the 11th lysine residue of the C-termus via an amide bond formed between the amine group on the side chain of lysine residue and the carboxylic acid as highlighted in the square of DABCYL formula
Figure imgf000052_0002
Table 5. Peptide Sequences Derived From the Substrate of Protease Encoded by the Human Coronavirus
Figure imgf000052_0003
Figure imgf000053_0001
Figure imgf000054_0001
Table 6. Examples of peptide sequences of different lengths and including unnatural amino acids that can be used in the biosensor.
# Length PLpro (SFP or IQFP) 3CLpro (SFP or IQFP) of sequence
5-mer a. Ac-Gly-hTyr-Dap-Gly-Gly- ACC (SFP) a. Ac-Val-Abu-Tle-Leu-Gln-ACC (SFP) b. Ac-Gly-hPhe-Dap-Gly-Gly- ACC (SFP) b. Ac-Thr-Thz-Tle-Leu-Gln-ACC (SFP) c. Ac-Trp-hTyr-Dap-Gly-Gly- ACC (SFP) c. Ac-Gly-Abu-Tle-Leu-Gln-ACC (SFP)
6-mer a. Ac-Ala-Gly-hTyr-Dap-Gly-Gly- ACC (SFP) a. Ac-Gly-Val-Abu-Tle-Leu-Gln-ACC (SFP) b. Ac-Ala-Gly-hPhe-Dap-Gly-Gly- ACC (SFP) b. Ac-Gly-Thr-Thz-Tle-Leu-Gln-ACC (SFP) c. Ac-Ala-Trp-hTyr-Dap-Gly-Gly- ACC (SFP) c. Ac-Gly-Gly-Abu-Tle-Leu-Gln-ACC (SFP)
7-mer a. Ac-Gly-Ala-Gly-hTyr-Dap-Gly-Gly- ACC a. Ac-Ala-Gly-Val-Abu-Tle-Leu-Gln-ACC
(SFP) (SFP) b. Ac-Gly-Ala-Gly-hPhe-Dap-Gly-Gly- ACC b. Ac-Ala-Gly-Thr-Thz-Tle-Leu-Gln-ACC (SFP) (SFP) c. Ac-Gly-Ala-Trp-hTyr-Dap-Gly-Gly- ACC c. Ac-Ala-Gly-Gly-Abu-Tle-Leu-Gln-ACC (SFP) (SFP)
8-mer a. Ac-Gly-Gly-Ala-Gly- hTyr-Dap -Gly-Gly- a. Ac-Gly-Ala-Gly-Val-Abu-Tle-Leu-Gln-
ACC (SFP) ACC (SFP) b. Ac-Gly-Gly-Ala-Gly- hTyr-DAP -Gly-Gly- b. Ac-Gly-Ala-Gly-Thr-Thz-Tle-Leu-Gln- ACC (SFP) ACC (SFP) c. ACC-Gly- hTyr-Dap -Gly-Gly-Ala-Ile- c. ACC-Gly -Abu-Tle-Leu-Gln-Ser-Gly - Lys(dnp)-NH2 (IQFP) Lys(dnp)-NH2 (IQFP) d. ACC-Gly- hTyr-DAP -Gly-Gly-Gly-Thr- d. ACC-Gly -Thz -Tle-Leu-Gln-Ser-Gly - Lys(dnp)-NH2 (IQFP) Lys(dnp)-NH2 (IQFP)
9-mer a. ACC-Gly- hTyr-Dap -Gly-Gly-Ala-Ile-Thr- a. ACC-Gly -Abu-Tle-Leu-Gln-Ser-Gly-Phe-
Lys(dnp)-NH2 (IQFP) Lys(dnp)-NH2 (IQFP) b. ACC-Gly- hTyr-DAP -Gly-Gly-Gly-Thr-Glu- b. ACC-Gly -Thz -Tle-Leu-Gln-Ser-Gly -Phe- Lys(dnp)-NH2 (IQFP) Lys(dnp)-NH2 (IQFP)
10-mer a. ACC-Gly- hTyr-Dap -Gly-Gly-Ala-Ile-Thr- a. ACC-Gly -Abu-Tle-Leu-Gln-Ser-Gly-Phe-
Lys(dnp)-Lys-NH2 (IQFP) Arg-Lys(dnp)-NH2 (IQFP) b. ACC-Gly- hTyr-Dap -Gly-Gly-Gly-Thr-Glu- b. ACC-Gly-Thz-Tle-Leu-Gln-Ser-Gly-Phe- Lys(dnp)-Lys-NH2 (IQFP) Arg-Lys(dnp)-NH2 (IQFP)
11-mer a. ACC-Gly- hTyr-Dap -Gly-Gly-Ala-Ile-Thr- a. ACC-Gly -Abu-Tle-Leu-Gln-Ser-Gly -Phe- Arg-Lys(dnp)-Lys-NH2 (IQFP) Arg-Lys(dnp)-Lys-NH2 (IQFP) b. ACC-Gly- hTyr-Dap -Gly-Gly-Gly-Thr-Glu- b. ACC-Gly-Thz-Tle-Leu-Gln-Ser-Gly-Phe-
Pro-Lys(dnp)-Lys-NH2 (IQFP) Arg-Lys(dnp)-Lys-NH2 (IQFP) c. ACC-Gly- hTyr-Dap -Gly-Gly-Ala-Ile-Thr- c. ACC-Gly -Abu-Tle-Leu-Gln-Ser-Gly -Phe- Arg-Lys(dnp)-Arg-NH2 (IQFP) Arg-Lys(dnp)-Arg-NH2 (IQFP) d. ACC-Gly- hTyr-Dap -Gly-Gly-Gly-Thr-Glu- d. ACC-Gly-Thz-Tle-Leu-Gln-Ser-Gly-Phe-
Pro-Lys(dnp)-Arg-NH2 (IQFP) Arg-Lys(dnp)-Arg-NH2 (IQFP)
12-mer a. ACC-Gly- hTyr-Dap -Gly-Gly-Ala-Ile-Thr- a. ACC-Gly -Abu-Tle-Leu-Gln-Ser-Gly-Phe- Arg- Val-Lys(dnp)-Lys-NH2 (IQFP) Arg-Ser-Ala-Lys(dnp)-NH2 (IQFP) b. ACC-Gly- hTyr-Dap -Gly-Gly-Gly-Thr-Glu- b. ACC-Gly-Thz-Tle-Leu-Gln-Ser-Gly-Phe- Pro-Gly-Lys(dnp)-Lys-NH2 (IQFP) Arg-Ser-Ala-Lys(dnp)-NH2 (IQFP)
13-mer a. ACC-Gly- hTyr-Dap -Gly-Gly-Ala-Ile-Thr- a. ACC-Gly -Abu-Tle-Leu-Gln-Ser-Gly-Phe-
Arg- Val-Thr-Lys(dnp)-Lys-NH2 (IQFP) Arg-Ser-Ala-Val-Lys(dnp)-NH2 (IQFP) b. ACC-Gly- hTyr-Dap -Gly-Gly-Gly-Thr-Glu- b. ACC-Gly-Thz-Tle-Leu-Gln-Ser-Gly-Phe-
Pro-Gly-Gly-Lys(dnp)-Lys-NH2 (IQFP) Arg-Ser-Ala-Val-Lys(dnp)-NH2 (IQFP)
14-mer a. ACC-Gly- hTyr-Dap -Gly-Gly-Ala-Ile-Thr- a. ACC-Gly -Abu-Tle-Leu-Gln-Ser-Gly -Phe- Arg- Val-Thr-Phe-Lys(dnp)-Lys-NH2 (IQFP) Arg-Ser-Ala-Val-Lys(dnp)-Arg-NH2 (IQFP) b. ACC-Gly- hTyr-Dap -Gly-Gly-Gly-Thr-Glu- b. ACC-Gly-Thz-Tle-Leu-Gln-Ser-Gly-Phe- Pro-Gly-Gly-Arg-Lys(dnp)-Lys-NH2 (IQFP) Arg-Ser-Ala-Val-Lys(dnp)-Arg-NH2 (IQFP)
15-mer a. ACC-Gly- hTyr-Dap -Gly-Gly-Ala-Ile-Thr- a. ACC-Gly-Abu-Tle-Leu-Gln-Ser-Gly-Phe- Arg- Val-Thr-Phe-Gly-Lys(dnp)-Lys-NH2 Arg-Ser-Ala-Val-Lys(dnp)-Arg-Lys-NH2
(IQFP) (IQFP) b. ACC-Gly- hTyr-Dap -Gly-Gly-Gly-Thr-Glu- b. ACC-Gly -Thz -Tle-Leu-Gln-Ser-Gly -Phe-
Pro-Gly-Gly-Arg-Ser-Lys(dnp)-Lys-NH2 Arg-Ser-Ala-Val-Lys(dnp)-Arg-Lys-NH2
(IQFP) (IQFP)
16-mer a. ACC-Gly- hTyr-Dap -Gly-Gly-Ala-Ile-Thr- a. ACC-Gly -Abu-Tle-Leu-Gln-Ser-Gly-Phe- Arg- Val-Thr-Phe-Gly-Lys(dnp)-Lys-Lys-NH2 Arg-Ser-Ala-Val-Lys(dnp)-Arg-Lys-Lys-NH2 (IQFP) (IQFP) b. ACC-Gly- hTyr-Dap -Gly-Gly-Gly-Thr-Glu- b. ACC-Gly-Thz-Tle-Leu-Gln-Ser-Gly-Phe- Pro-Gly-Gly-Arg-Ser-Lys(dnp)-Lys-Lys-NH2 Arg-Ser-Ala-Val-Lys(dnp)-Arg-Lys-Lys -NH2
(IQFP) (IQFP)
Ac - Acetylation, usually at the N-terminus.
ACC - 7-amino-4-carbamoylmethylcoumarin - a fluorophore that can be replaced by another fluorophore, attached at C-terminus for SFP and N-terminus for IQFP. Amino Acid - Non-natural Amino Acids that can be substituted by other non-natural amino acids of similar type (size, charge/polarity, side chains etc). For example, in peptide sequences for PLpro, hTyr can be replaced with hPhe while for 3CLpro sequences, Abu can be replaced by Thz. In either the PLpro or 3CLpro reactive sequences the non-natural amino acid Dap may be replaced by Dab.
Lys(dnp)- A modified Lysine amino acid derivative with quencher, dnp or dinitrophenyl which is the preferred quencher for ACC. Used in IQFP
SFP- Simple Fluorogenic Peptide
IQFP- Internally Quenched Fluorogenic peptide
IB. Colorimetric Biosensor
[0171] In an embodiment, this disclosure provides a biosensor having Formula (15) A-Y-LSP, wherein A is derived from a peptidic substrate for or a chemical inhibitor of an enzyme encoded by SARS-CoV-1, SARS-CoV-2, or MERS-CoV, LSP is a spectroscopic probe chosen from a group consisting of coumarins, rhodamines, rhodols, fluoresceins, xanthenes, phenothiazines, and phenoxazines; Y is a bond, NRa, O, or S; Ra is H or Ci-6alkyl; wherein LSP is capable of changing color to provide a detectable signal after exposure to the enzyme encoded by SARS- CoV-1, SARS-CoV-2, or MERS-CoV.
[0172] In some embodiments, the biosensor of Formula (15) has the A selected from the group
Figure imgf000056_0001
LSP selected from the group of a spectroscopic probe having Formula (
Figure imgf000057_0001
Formula
Figure imgf000057_0002
having a cyan colored state, Formula (17)
Figure imgf000057_0004
Y is a bond, N, O, or S;
U is O or N;
V is -0-C(=0)-0-, -0-, or -NH-C(=0)-0-; W is O, N, S or CH2,
Figure imgf000057_0003
R14, and R15 are each independently selected from the group of H, substituted and unsubstituted C1-C12 alkyl group, substituted and unsubstituted C1-C12 alkenyl group, substituted and unsubstituted C1-C12 alkynyl group, and substituted and unsubstituted aryl group;
R16, R17, R18, R19, R20, R21, and R22 is each independently selected from the group of H, Cl, F, Br, CN, NO2, -NR14R15, C1-C6 alkyl group, and C1-C6 alkoxyl group; R23, R24, R25, R26 and R27 are each independently selected from the group of H, C1-C2 alkyl group, C1-C2 fluoroalkyl group; and wherein when A is the peptide derived from the substrate of the enzyme encoded by SARS-CoV-1, SARS-CoV-2, or MERS-CoV, LSP is chemically linked to A via Y to a reactive functional group on the side chain of an amino acid residue, or to the reactive functional group on any one of the C-terminus or N-terminum amino acid residue of the peptide.
[0173] In some embodiments, the reactive functional group on the side chain of an amino acid residue in the peptide sequence and the reactive functional group on any one of the C-terminus or N-terminum amino acid residue are independently selected from an amine group (-NH2), an amide group (-C(=0)-NH2), a carboxyl group (-COOH), a hydroxyl group (-OH), a thiol group (- SH), and a sulfhydryl group (-S-S-). In some embodiments, LSP is chemically linked to the reactive functional group via Y on the side chain of an amino acid residue in the peptide sequence and the reactive functional group is selected from an amine group, an amide group, a carboxyl group, a hydroxyl group, a thiol group, and a sulfhydryl group. In some embodiments, LSP is chemically linked to any one of the C-terminus or N-terminum group of the peptide via Y and the reactive functional group on any one of the C-terminus or N-terminum amino acid residue is selected from an amine group, an amide group, a carboxyl group, a hydroxyl group, a thiol group, and a sulfhydryl group. For example, a biosensor A-Y-LSP is defined when A is LRGG- and LSP is linked to A via C-terminus glycine forming an amide bond with Y=N.
[0174] In some embodiments, D of Formula (15) is a leuco colorless dye component having
Figure imgf000058_0001
[0175] In some embodiments, the colorless leuco dye component of Formula (19)
Figure imgf000058_0002
Figure imgf000059_0004
Y
[0176] In some embodiments, the biosensor of Formula (19) is defined when Z is
Figure imgf000059_0001
or - O-CFh-Ph.
[0177] In some embodiments, the biosensor of Formula (15) is defined when R14, R15 are each independently selected from the group of methyl and ethyl group.
[0178] In some embodiments, the biosensor of Formula (15) is defined when Y is a bond or N and LSP is selected from the group of Formula (
Figure imgf000059_0002
Formula (9)
Figure imgf000059_0005
Formula (11)
Figure imgf000059_0003
, Formula (13)
Figure imgf000060_0002
g y ,
Figure imgf000060_0003
[0179] In some embodiments, the biosensor of Formula (15) is defined when Y is a bond or N, and A is selected from the group
Figure imgf000060_0001
Figure imgf000060_0004
Figure imgf000061_0001
cyan colored state, Formula (17)
Figure imgf000061_0004
, Formula (23)
Figure imgf000061_0002
or Formula (24)
Figure imgf000061_0005
[0180] In some embodiments, the biosensor of Formula (15) is defined when Y is a bond or N, A is selected from the group
Figure imgf000061_0003
Figure imgf000062_0001
[0181] In some embodiments, the biosensor of Formula (15) is defined when Y is a bond or N, A is selected from the group
Figure imgf000062_0002
Figure imgf000062_0003
Figure imgf000063_0001
[0182] In some embodiments, the biosensor of Formula (15) is defined when Y is a bond or N, A is selected from the group
Figure imgf000063_0002
Figure imgf000063_0003
[0183] In some embodiments, the biosensor of Formula (15) is defined when Y is a bond or N, A
Figure imgf000063_0004
Figure imgf000064_0001
[0184] In some embodiments, the biosensor of Formula (15) is defined when Y is a bond or N, A
Figure imgf000064_0002
[0185] In some embodiments, the biosensor of Formula (15) is defined when Y is a bond or N, A
Figure imgf000065_0001
[0186] In some embodiments, the biosensor of Formula (15) is defined when Y is a bond or N, A is selected from the group
Figure imgf000065_0002
Figure imgf000066_0001
[0187] In some embodiments, the biosensor of Formula (15) is defined when Y is a bond or N, and A is selected from the group
Figure imgf000066_0002
Figure imgf000066_0003
Figure imgf000067_0001
[0188] In some embodiments, the biosensor of Formula (15) is defined when Y is a bond or N, and A is selected from the group
Figure imgf000067_0002
Figure imgf000067_0003
[0189] In some embodiments, the biosensor of Formula (15) is defined when Y is a bond or N, and A is selected from the group
Figure imgf000067_0004
Figure imgf000068_0001
[0190] In some embodiments, the biosensor of Formula (15) is defined when Y is a bond or N, and A is selected from the group
Figure imgf000068_0002
Figure imgf000068_0003
Figure imgf000069_0001
[0191] In some embodiments, the biosensor of Formula (15) is defined when Y is a bond or N, A is selected from the group
Figure imgf000069_0002
Figure imgf000069_0003
[0192] In some embodiments, the biosensor of Formula (15) is defined when Y is a bond or N, A is selected from the group
Figure imgf000069_0004
Figure imgf000070_0001
[0193] In some embodiments, the biosensor of Formula (15) is defined when Y is a bond or N, A is selected from the group
Figure imgf000070_0002
and LSP is selected from the group of Formula
Figure imgf000070_0003
, ,
Figure imgf000071_0001
having a cyan colored
Figure imgf000071_0002
[0194] In some embodiments, A is a peptide derived from the substrate of coronavirus 3CLpro and A is selected from the group of Leu, Val, Val-Leu, Val-Phe, Ala-Val-Leu, Ser-Val-Phe, Val- Thr-Phe-Gln, Val-Thr-Leu-Gln, Val-Thr-Ala-Gln, Glu-Ser-Ala-Thr-Leu-Gln-Ser-Gly-Leu-Ala- Lys-Ser, Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys, and Thr-Ser-Ala-Val-Leu-Gln-Ser- Gly-Phe-Arg-Lys-Met. In some embodiments, A comprises Val-Leu, Val-Phe, Ala-Val-Leu, and Ser-Val-Phe. In some embodiments, A comprises Glu-Ser-Ala-Thr-Leu-Gln-Ser-Gly-Leu-Ala- Lys-Ser. In some embodiments, A comprises Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys- Met.
[0195] In some embodiments, A is a tetrapeptide, pentapeptide, or hexapeptide that is recognized as a substrate for cleavage by either a PLpro or 3CLpro enzyme encoded by a virus.
[0196] In some embodiments, A is a peptide derived from the substrate of coronavirus 3CLpro or PLpro and A is selected from a peptide disclosed in Table 5. In some embodiments, A is a peptide derived from the substrate of coronavirus PLpro and A is selected from LXGG,
RLRGG, LRGG, LKGG, RLKGG, MRGG, IRGG, MKGG, IKGG, LSGG, LYGG, LNGG,
LDGG, LQGG, LEGG, RLEGG, LHGG, LQGG, LTGG, SLKGG, and KKAG. In some embodiments, A is a peptide derived from the substrate of coronavirus 3CLpro and A is selected from VRLQ, PRLQ, VKLQ, VSLQ, AVLQ, VTFQ, LQSAG, VRLQS, PRLQS, VVRLQ, VVVLQ, VAVLQ, TSAVLQ, SGVTFQ, TSDVLQ, TNAVLQ, SGFRK, SGFRR, GKFKK, GKFRK, VARLQ, and VPRLQ.
[0197] In some embodiments, A is an unnatural peptide 25
Figure imgf000072_0001
comprising at least four residues, wherein R23 and R24 are H or alkyl, R25 is an alkyl amine, R26 is -(CH2)n-Ar, and R27 is H-, CH3CO-, FhN-(CFl2)m-CC)-, or H(-0-CH2CH2)p-, and wherein n and m are independently 1-4 and p is 2-100.
[0198]
[0199] In some embodiments, A is an unnatural peptide selected from Ac-hTyr-Dap-Gly-Gly, Arg-hTyr-Dap-Gly-Gly, Ac-hPhe-Dap-Gly-Gly, Ac-hPhe(4F)-Dap-Gly-Gly, and Ac- hPhe(4CF3)-Dap-Gly-Gly, Ac-Abu(Bth)-Dap-Gly-Gly, Ac-Abu(Bth)-Dab-Gly-Gly, Ac-hTyr- Lys-Gly-Gly, Ac-Abu(Bth)-Lys-Gly-Gly, Ac-Abu-Tle-Leu-Gln, Ac-hPhe-Lys-Gly-Gly, Ac- hTyr-hTyr-Gly-Gly, hPhe-Dap-Gly-Gly, Suc-hPhe-Dap-Gly-Gly, hPhe(4CF3)-Dap-Gly-Gly, Suc-hPhe(4CF3)-Dap-Gly-Gly, Suc-hTyr-hTyr-Gly-Gly,
[0200] In some embodiments, A is an unnatural peptide comprising at least four residues represented by the sequence -Gln-R28-R29-R3o, wherein R28 is Leu, 2-Abz, 3-Abz, 4-N02-Phe, b- Ala, Dht, hLeu, Ilu, or Met; R29 is Tie, D-Phe, D-Tyr, Om, hArg, Dab, Dht, Lys, D-Phg, D-Trp, Arg, or Met(0)2; and R30 is a small aliphatic residue such as Abu, Val, Ala, Tie, wherein the N- terminus of the sequence is bound to H-, CH3CO-, HOC(0)-(CH2)m-CO-, or Fh-O-CFLCFhV, and wherein n and m are independently 1-4 and p is 2-100.
[0201] In some embodiments, A is a peptide derived from the substrate of SARS-CoV PLpro and A is ubiquitin or ISG15 protein. In some embodiments, the SARS-Cov encoding PLpro is SARS-CoV-1 or SARS-CoV-2 PLpro. In some embodiments, the coronavirus is SARS-CoV-1. In some embodiments, the coronavirus is SARS-CoV-2.
[0202] In some embodiments, A is a peptide derived from the substrate of coronavirus 3CLpro selected from the group of Leu, Val, Val -Leu, Val-Phe, Ala-Val-Leu, Ser-Val-Phe, Leu-X-Gly- Gly, Val-Thr-Phe-Gln, Val-Thr-Leu-Gln, Val-Thr-Ala-Gln, Glu-Ser-Ala-Thr-Leu-Gln-Ser-Gly- Leu-Ala-Lys-Ser, Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys, Glu-Arg-Glu-Leu-Asn-Gly- Gly-Ala-Pro-Ile-Lys-Ser, and Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met; and LSP is
Figure imgf000073_0001
Figure imgf000073_0002
(CH3, CF3) or Formula (24) (CH3, CF3)
[0203] In some embodiments, the biosensor of Formula (15) has A is Thr-Ser-Ala-Val-Leu-Gln-
Ser-Gly-Phe-Arg-Lys-Met, and D selected from the group of Formula (7) Formula
Figure imgf000073_0003
Figure imgf000074_0003
Figure imgf000074_0004
22)
Figure imgf000074_0001
[0204] In some embodiments, the biosensor of Formula (15) has A is a peptide having a sequence selected from the group of LQSAG, VRLQS, or PRLQS, and D selected from the group of Formula
Figure imgf000074_0002
Figure imgf000075_0003
o , Formula (22) (Et ,Me) , Formula (23)
Figure imgf000075_0001
attached to D via Y at Q residue.
[0205] In some embodiments, the biosensor of Formula (15) of which A is a peptide having the sequence LXGG-, and X is selected from the group of R, K, S, Y, N, D, O, E, FI, or T, and LSP is selected from the group of Formula
Figure imgf000075_0002
Figure imgf000076_0001
Figure imgf000076_0003
(CHj, CFJ) or Formula (24) (CH3, CF3) wherein A is bonded to D via Y at the terminal G residue.
[0206] In some embodiments, the biosensor is a molecule comprising two fragmentable groups, represented by A-Y-D-Y-B wherein D is a spectroscopic probe, Y is an O, N, or S atom, and A and B are selected from of a natural or unnatural peptide
Figure imgf000076_0002
Figure imgf000077_0001
embodiments, A and B are the same. In some embodiments, A and B differ. In some embodiments, A is selected from groups that are substrates (or derived from substrates, inhibitors or therapeutics) for PLpro described herein and B is selected from peptides that are substrates (or derived from substrates, inhibitors or therapeutics) for 3CLpro described herein.
[0207] In an example, the biosensor
Figure imgf000077_0002
where the leaving groups A and B may be separately cleaved by PLpro and by 3CLpro enzymes. For example, A can be cleaved by PLpro and B can be cleaved by 3CLpro, or A can be cleaved by 3CLpro and B can be cleaved be PLpro.
[0208] It is well known in the chemical literature that strongly colored materials can be produced by coupling of two colorless or weakly colored molecules. For example, indoaniline dyes can be produced by the oxidative coupling of p-phenylene diamines with phenols.
Figure imgf000077_0003
[0209] US Pat. No. 4,681,841 teaches an assay for enzymes based upon such color-forming chemistry. In some embodiments, the biosensor is provided by a combination of any of the compounds and color couplers known in the art such as, for example, those described in US Pat. No. 4,681,841, which is incorporated herein by reference in its entirety.
[0210] In some embodiments, the enzyme is encoded by SARS-CoV-2. In some embodiments, the enzyme is encoded by SARS-CoV-1. In some embodiments, the enzyme is encoded by MERS-CoV. In some embodiments, the enzyme is virally encoded cysteine protease. In some embodiments, the enzyme is papain like virus protease encoded by SARS-Cov-1, SARS-CoV-2 or MERS-CoV. In some embodiments, the enzyme is SARS-CoV-1 PLpro, or SARS-CoV-1 3CLpro. In some embodiments, the enzyme is SARS-CoV-2 PLpro, or SARS-CoV-23CLpro. In some embodiments, the enzyme is MERS-CoV-1 3CLpro, SARS-CoV-2 3CLpro or MERS-CoV 3CLpro. In some embodiments, the enzyme is MERS-CoV-1 PLpro, SARS-CoV-2 PLpro or MERS-CoV PLpro.
[0211] In some embodiments the library of biosensors is based on a platform containing other leuco dyes including but not limited to, spiropyran, quinone, thiazine, phenazine, oxazine, pthalide-type, triarylmethanes, fluoran, and tetrazoliums.
[0212] In some embodiments the library of biosensors is based on a platform containing naturally occurring dyes including but not limited to, curcumins, hypericin, carotenes, anthocynanins, and any other phytochemical dyes.
[0213] In some embodiments the library of biosensors is based on a platform containing synthetic dyes that may not be leuco dyes for e.g. azo dyes, coumarins, xanthenes, phthalides and azomethine dyes.
[0214] Screening such library of biosensors will enable us to identify one or more molecules that can be used as a biosensor for sensitive and specific detection of certain viruses.
[0215] In one aspect, this disclosure provides a composition containing a biosensor for the detection of viruses having a Formula (1) D-FG or Formula (15) A-Y-LSP as described above, wherein (i) FG or A comprising a material responsive to an enzyme encoded by a target virus selected from the group of SARS-CoV-1, SARS-CoV-2, or MERS-CoV; and (ii) D or LSP is a spectroscopic probe, wherein the FG or A masks the activity of the spectroscopic probe D or LSP, wherein the enzyme causes the cleavage of the FG or A to release the spectroscopic probe D or LSP, resulting in a detectable optical response; and wherein the enzyme is present in a virus-infected host cell and is released outside for access by the biosensor when the host cell is ruptured.
[0216] In some embodiments, the enzyme encoded by the target virus is a protease. In some embodiments, the enzymes encoded by the target virus comprise proteases that are necessary for viral replications. In some embodiments, the enzyme encoded by the target virus is a serine protease. In some embodiments, the enzyme encoded by the target virus is a microbial cysteine protease. In some embodiments, the enzyme encoded by the target virus is selected from the group of 3CLpro and PLpro encoded by coronavirus. In some embodiments, the enzyme encoded by the target virus is 3CLpro encoded by coronavirus.
[0217] In some embodiments, the optical response is a color change within the visible region of the electromagnetic spectrum. In some embodiments, the optical response is fluorescence.
[0218] In some embodiments, the two or more colors of the two or more biosensors are selected to give a mixed color having sufficient difference such that each of the two colors are visually discemable by naked eye.
[0219] In some embodiments, the released spectroscopic probe gives a discrete blue color having the absorption wavelength in the visible light spectrum ranging from 400 nm to 500 nm. In some embodiments, the released spectroscopic probe gives a discrete red color having the absorption wavelength in the visible light spectrum ranging from 400 nm to 600 nm.
Formulation
[0220] In some aspects of the invention, the present invention is related to formulations containing a colorimetric biosensor useful for the detection of viruses like coronaviruses using visible color-change technology.
[0221] As has been discussed elsewhere herein, the target enzymes are produced inside infected host cells. Thus, in order to enable the reaction of the substrate included in the biosensor of the present disclosure with a target enzyme, the enzyme must be released from the infected cell into the fluid comprising a biosensor. The release of the enzyme may be the result of chemophysical action, as in for example the action of a detergent or a surfactant to lyse cells. Similarly, the enzyme may be released as a result of mechanical action such as produced by the action of a homogenizer or microfluidizer. Methods of cell lysis have been reviewed (Islam, et al., Micromachines 2017, 8, 83-119). The cell lysis may be performed prior to the introduction of the biosensor, or it can be performed upon introduction of the biosensor, such as may be initiated by a detergent in a diagnostic composition.
[0222] In some embodiments, a diagnostic composition may comprise a biosensor as described herein and a carrier. In some embodiments, the carrier comprises an organic solvent that is compatible with biological samples. In some embodiments, the carrier comprises a biological buffer. In some embodiments, the carrier comprises a surfactant or a detergent.
[0223] In the case of COVID-19 infection, both PLpro and 3CLpro enzymes must be present for viral replication. Identifying samples containing both enzymes would limit false positive detection resulting from a response of a deubiquitinating enzyme other than PLpro, since such a false detection would not show a concurrent response from 3CLpro. In some cases, identifying a unique response from a normal human enzyme helps to assure that sufficient human cells are collected for a valid test, thereby eliminating a false negative response due to insufficient material to analyze. In some embodiments, the diagnostic composition may comprise two or more biosensors, wherein each sensor independently has a masked spectroscopic probe giving a discrete optical response after release. In some embodiments, the independent optical response is a fluorescent response to produce emission at multiple wavelengths. In some embodiments, the optical response is a color formation (optical response is from a colorless state to color state). In some embodiments, the two or more colors of the two or more biosensors are selected to give a mixed color having sufficient difference such that each of the two colors are visually discemable by naked eye. In some embodiments, the two or more biosensors are responsive to different enzymes. In some embodiments, a first biosensor is responsive to PLpro enzyme and a second biosensor is responsive to 3CLpro enzyme. In some embodiments, a first biosensor is responsive to PLpro enzyme, a second biosensor is responsive to 3CLpro enzyme and a third biosensor is responsive to a human enzyme.
[0224] In some embodiments, the diagnostic composition may comprise two or more biosensors, wherein each sensor independently has a spectroscopic probe exhibiting a unique color and produces a detectable optical response from colored state to colorless state. In some embodiments, the two or more colors of the two or more biosensors are selected to give a mixed color that has sufficient difference such that each of the two colors are visually discernable by naked eye.
[0225] For example, the diagnostic composition comprises three different biosensors each having a cyan, magenta and yellow color probe. The mixture of the three biosensors gives black color. If the biosensor having cyan color is released by the enzyme encoded by the virus, then the diagnostic composition would produce red color. Similarly, if the biosensor having magenta color is released by the enzyme encoded by the virus, then the diagnostic composition would produce green color. Further, if the biosensor having yellow color is released by the enzyme encoded by the virus, then the diagnostic composition would produce blue color. If any two of the color biosensors are activated by the enzymes encoded by the virus, then the diagnostic composition would produce a color change from black to the remaining subtractive primary color i.e. either cyan, magenta or yellow.
[0226] In some embodiments, the diagnostic composition further incorporates an enzyme inhibitor. In some embodiments, the inhibitor is a broad-spectrum deubiquitinase (DUB) inhibitor. In some embodiments the broad spectrum DUB inhibitor is 2,6-Diamino-3,5- dithiocyanopyridine. In some embodiments, the inhibitor comprises a dihydropyrrole. In some embodiments, the DUB inhibitor is a tricyclic heterocyclic compound. In some embodiments, the DUB inhibitor is PX-478. In some embodiments, the DUB inhibitor comprises 6-amino- pyrimidine.
[0227] In some embodiments, the inhibitor is a proteasome inhibitor. In some embodiments the proteosome inhibitor is MG132 (Cbz-Leu-Leu-Leucinal). In some embodiments, the proteosome inhibitor is b-AP15. In some embodiments, the proteosome inhibitor comprises azepan-4-one.
[0228] In some embodiments, the inhibitor is a viral 3C protease inhibitor. In some embodiments, the inhibitor is a human rhinovirus 3C protease inhibitor. In some embodiments, the inhibitor is a human enterovirus 3C protease inhibitor. In some embodiments, the 3C protease inhibitor is SG85. In some embodiments the 3C protease inhibitor is luteoloside.
[0229] In some embodiments, the inhibitor is an inhibitor of ubiquitin C-terminal hydrolase (UCH). In some embodiments, the inhibitor is an inhibitor of UCH-L1. In some embodiments, the inhibitor is an inhibitor of UCH-L3. In some embodiments, the inhibitor is an inhibitor of UCH-L5. In some embodiments, the inhibitor is 4,5,6,7-tetrachloro-l,3-indanedione (TCID). In some embodiments, the inhibitor is an acylated oxime isatin derivative.
[0230] In some embodiments, the biosensor further comprises a solid support, wherein the fragment component is bound to the solid support via a covalent bond or via electrostatic interaction.
[0231] In some embodiments, the biosensor further comprises a solid support, wherein the spectroscopic probe is covalently bound to the solid support.
[0232] In some embodiments, the solid support is selected from the group of a particle, fiber, a microgel, a wound dressing, a catheter, a membrane, a resin, a sponge, a sheet, a suture, an implant scaffold, a stent, a swab, a hydrogel, a film, a patch, a tape, a woven fabric, and a nonwoven fabric.
[0233] In some embodiments, the biosensor is rendered onto a paper or plastic film strip comprising both lysing agent and biosensor composition for placement onto a tongue or mouth and then observing for a color change.
[0234] In some embodiments, the solid support is a paper impregnated with a biosensor. In some embodiments, the paper impregnated with a biosensor is prepared by treating a paper substrate with a biosensor solution in a solvent such as a volatile solvent including dichloromethane, ethylacetate, hexane, methanol, ethanol, or non-volatile solvent including polyethylene glycol, or glycerol.
[0235] In some embodiments, the solid support is a hydrogel or microgel impregnated with a biosensor solution in a solvent such as water, aqueous solution of methanol, aqueous solution of ethanol, polyethylene glycol, or glycerol.
[0236] In some embodiments, the solid support is a woven fabric or a nonwoven fabric impregnated with a biosensor solution in a solvent such as volatile solvent including dichloromethane, ethylacetate, hexane, methanol, ethanol, or non-volatile solvent including polyethylene glycol, or glycerol.
[0237] In some embodiments, the solid support is a microgel comprising a dendritic polymer. Dendrimers are also classified by generation, which refers to the number of repeated branching cycles that are performed during its synthesis. For example, if a dendrimer is made by convergent synthesis, and the branching reactions are performed onto the core molecule three times, the resulting dendrimer is considered a third generation dendrimer (G3). Each successive generation results in a dendrimer roughly twice the molecular weight of the previous generation. Higher generation dendrimers also have more exposed functional groups on the surface, which can later be used to customize the dendrimer for a given application.
[0238] In some embodiments, the dendritic polymer is selected from hyperbranched PEG dendrimers, PEG core dendrimers, hyperbranched polyglycerol dendrimers, hyperbranched polylysine dendrimers, hyperbranched polyesters, alkyne-terminated dendrimers, amine terminated PEG-core dendrimers, azide terminated dendrimers, 2,2-bis(methylol)propionic acid (bis-MPA) dendrimers, carboxylic acid terminated dendrimers, Poly(amidoamine) (PAMAM) dendrimers, polyethylenimine dendrimers (PEI), and combinations thereof.
[0239] In some embodiments, the dendritic polymer has reactive surface group available for biosensor conjugation selected from the group of 8 surface groups, 16 surface groups, 32 surface groups, 64 surface groups, and 128 surface groups. In some embodiments, the reactive surface groups carried by the dendritic polymer is selected from the group of (-CH=CH2), ethynyl group (-CºC-), azide group (-N3), vinyl dimethyl sulfone group, hydroxyl group (-OH), thiol group (- SH), amine group (-NH2), aldehyde group (-CHO), carboxylic acid group (-COOH), and combinations thereof.
[0240] In some embodiments, the dendrimer is a polyester bis-MPA dendrimer (tert-butylic acid protected amine core, 8 alkyne end groups, G3, branching units bis-MPA). These alkyne- functionalized dendrimers can be readily functionalized using either copper (I)-catalyzed alkyne- azide cycloaddition (CuAAC), strain-promoted alkyne-azide cycloaddition (SPAAC), or thiol- yne click reactions. Additionally, the amine-functionalized core can be readily used in EDC or DCC coupling reactions (after Boc deprotection) with carbonyl-containing compounds to yield highly functionalized materials for a variety of biomedical applications. In some embodiments, the biosensor as disclosed herein is modified with an azide group and is conjugated with the G3 polyester bis-MPA dendrimer via click chemistry.
[0241] In some embodiments, the dendritic polymer is selected from the group of bis-MPA hyperbranched PEG10k-OH dendrimer (10K PEG core, pseudo generation 2, bis-MPA branching units, 8 surface hydroxyl groups, Mw 10696 Da), bis-MPA hyperbranched PEGlOk- OH dendrimer (10K PEG core, pseudo generation 3, bis-MPA branching units, 16 surface hydroxyl groups, Mw 11643 Da), bis-MPA hyperbranched PEG20k-OH dendrimer (20K PEG core, pseudo generation 2, bis-MPA branching units, 8 surface hydroxyl groups, Mw 20759 Da), bis-MPA hyperbranched PEG20k-OH dendrimer (20K PEG core, pseudo generation 3, bis-MPA branching units, 16 surface hydroxyl groups, Mw 21688 Da), bis-MPA hyperbranched PEG6k- OH dendrimer (6K PEG core, pseudo generation 4, bis-MPA branching units, 32 surface hydroxyl groups, Mw 9480 Da), and combinations thereof. In some embodiments, the bis-MPA hyperbranched dendrimer forms microparticle or microgel.
[0242] In some embodiments, the dendritic polymer is selected from the group of G2 polylysine, G3 polylysine, G4 polylysine, G5 polylysine, and G6 polylysine.
[0243] In some embodiments, a spacer can link the biosensor to the dendrimer. In some embodiments, the spacer links biosensor and dendrimer via an amide bond formed by NHS/EDC chemistry. In some embodiments, the spacer links biosensor and the dendrimer via a disulfide (S- S) bond. In some embodiments, the spacer links biosensor and the dendrimer via triazoles formed by copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC), or strain-promoted alkyne- azide cycloaddition (SPAAC), or thiol-yne click reactions.
[0244] In some embodiments, the biosensor comprises 3CLpro or PLpro enzyme inhibitor fragment -(amino-(spacer)x)y-dendrimer or dendrimer-(spacer)z-3CLpro or PLpro enzyme inhibitor fragment, wherein the spacer has from 2 to 50 atoms, x, y and z are integers from 5 to 15.
[0245] In some embodiments, the bond joining the spacer and the 3CLpro or PLpro enzyme inhibitor fragment is a degradable bond selected from the group of an ester bond, an amide bond, an imine bond, an acetal bond, a ketal bond, and combinations thereof.
[0246] In some embodiments, the spacer is selected from the group of polyethylene glycol having 2-50 repeating units, e-maleimidocaproic acid, para-aminobenzyloxy carbamate, and combinations thereof. In some embodiments, the spacer comprises polyamino acid having 2-30 amino acid residues. In some embodiments, the spacer comprises a linear polylysine, or polyglutamine. [0247] In some embodiments, the concentration of the biosensor provided in the diagnostic compositions of the invention is less than, for example, 100 %, 90 %, 80 %, 70 %, 60 %, 50 %, 40 %, 30 %, 20 %, 19 %, 18 %, 17 %, 16 %, 15 %, 14 %, 13 %, 12 %, 11 %, 10 %, 9 %, 8 %, 7 %, 6 %, 5 %, 4 %, 3 %, 2 %, 1 %, 0.5 %, 0.4 %, 0.3 %, 0.2 %, 0.1 %, 0.09 %, 0.08 %, 0.07 %, 0.06 %, 0.05 %, 0.04 %, 0.03 %, 0.02 %, 0.01 %, 0.009 %, 0.008 %, 0.007 %, 0.006 %, 0.005 %, 0.004 %, 0.003 %, 0.002 %, 0.001 %, 0.0009 %, 0.0008 %, 0.0007 %, 0.0006 %, 0.0005 %, 0.0004 %, 0.0003 %, 0.0002 % or 0.0001 % w/w, w/v or v/v.
[0248] In some embodiments, the concentration of the biosensor provided in the diagnostic compositions of the invention is independently greater than 90 %, 80 %, 70 %, 60 %, 50 %, 40 %, 30 %, 20 %, 19.75 %, 19.50 %, 19.25 %, 19 %, 18.75 %, 18.50 %, 18.25 % 18 %, 17.75 %,
17.50 %, 17.25 %, 17 %, 16.75 %, 16.50 %, 16.25 %, 16 %, 15.75 %, 15.50 %, 15.25 % 15 %,
14.75 %, 14.50 %, 14.25 %, 14 %, 13.75 %, 13.50 %, 13.25 % 13 %, 12.75 %, 12.50 %, 12.25 %
12 %, 11.75 %, 11.50 %, 11.25 % 11 %, 10.75 %, 10.50 %, 10.25 %,10 %, 9.75 %, 9.50 %, 9.25
%, 9 %, 8.75 %, 8.50 %, 8.25 %, 8 %, 7.75 %, 7.50 %, 7.25 %, 7 %, 6.75 %, 6.50 %, 6.25 %, 6 %, 5.75 %, 5.50 %, 5.25 %, 5 %, 4.75 %, 4.50 %, 4.25 %, 4 %, 3.75 %, 3.50 %, 3.25 %, 3%,
2.75 %, 2.50 %, 2.25 %, 2 %, 1.75 %, 1.50 %, 1.25 %, 1.0 %, 0.5 %, 0.4 %, 0.3 %, 0.2 %, 0.1 %, 0.09 %, 0.08 %, 0.07 %, 0.06 %, 0.05 %, 0.04 %, 0.03 %, 0.02 %, 0.01 %, 0.009 %, 0.008 %, 0.007 %, 0.006 %, 0.005 %, 0.004 %, 0.003 %, 0.002 %, 0.001 %, 0.0009 %, 0.0008 %, 0.0007 %, 0.0006 %, 0.0005 %, 0.0004 %, 0.0003 %, 0.0002 %, or 0.0001 % w/w, w/v, or v/v.
[0249] In some embodiments, the concentration of the biosensor provided in the diagnotsic compositions of the invention is independently in the range from about 0.0001 % to about 50 %, about 0.001 % to about 40 %, about 0.01 % to about 30 %, about 0.02 % to about 29 %, about 0.03 % to about 28 %, about 0.04 % to about 27 %, about 0.05 % to about 26 %, about 0.06 % to about 25 %, about 0.07 % to about 24 %, about 0.08 % to about 23 %, about 0.09 % to about 22 %, about 0.1 % to about 21 %, about 0.2 % to about 20 %, about 0.3 % to about 19 %, about 0.4 % to about 18 %, about 0.5 % to about 17 %, about 0.6 % to about 16 %, about 0.7 % to about 15 %, about 0.8 % to about 14 %, about 0.9 % to about 12 %, or about 1 % to about 10% w/w, w/v or v/v.
[0250] In some embodiments, the concentration of the biosensor provided in the diagnostic compositions of the invention is independently in the range from about 0.001 % to about 10 %, about 0.01 % to about 5 %, about 0.02 % to about 4.5 %, about 0.03 % to about 4 %, about 0.04 % to about 3.5 %, about 0.05 % to about 3 %, about 0.06 % to about 2.5 %, about 0.07 % to about 2 %, about 0.08 % to about 1.5 %, about 0.09 % to about 1 %, about 0.1 % to about 0.9 % w/w, w/v or v/v.
Methods of Detecting Viruses
Virus detection
Samples and Methods of Sample Collection and Preparation
[0251] As used herein, the term "sample" refers to any substance containing or presumed to contain one or more SARS-CoV pathogen including, but not limited to, clinical samples (e.g., tissue or fluid isolated from one or more subjects or individuals), in vitro cell culture constituents, environmental samples, and the like. Samples may be obtained from any body geography known to exhibit the presence of virally infected cells, including, but not limited to, the mouth, the nose, the upper and the lower respiratory tract. Exemplary sample types include blood, plasma, serum, feces, bronchoalveolar lavage, nasal samples, oral samples, NP cavity samples, OP area samples, oral buccal samples, tongue scrape samples, urine, synovial fluid, mucus, or sputum. In some embodiments, the sample is selected from the group of NP swab samples, nasal swab samples, oral swab samples, sublingual swab samples, parotid duct opening swab samples, OP swab samples, buccal swab samples, or tongue scrape samples, an aerosol sample collected in a respiratory mask, masticated oral sample and the like.
[0252] In an embodiment, a biological sample (5) provided by a patient for analysis is placed into the sample tube (4). In some embodiments, the sample is a tongue scrape sample. In some embodiments, the sample is a saliva sample. In some embodiments, the sample is a sputum sample. In some embodiments, the sample is a scraping (2) from buccal mucosa. In some embodiments, the sample is a nasal swab sample.
[0253] In some embodiments, this disclosure provides a device for sample collection such as a respiratory mask used for collecting aerosolized droplets containing virus, cells, and enzymes. In some embodiments, the mask is aN95 respirator mask. In some embodiments, the respiratory mask has been used by a patient or a health care provider. In some embodiments, the respiratory mask has been only exposed in the open air for a ceratin amount of time (e.g., 2 hours, 4 hours, 8 hours, one day, two days, three days, or more); in these instances, the respiratory mask can be used to identify the presence of viruses where the respiratory mask has been exposed in the open air.
[0254] Some embodiments disclose a method for collecting samples from the mouth by mastication using a masticant such as a gum or other agent. The masticant agent can have moieties to enable retention of cells and/or virus particles
[0255] In some embodiments, the sample in the sample tube is held for a period of time to perform the inactivation of a pathogen. In some embodiments, the inactivation time is at least five minutes. In some embodiments, the sample tubes of multiple patients are maintained in a sample tray (6). In some embodiments, the sample tray holds at least 10 tubes.
[0256] In some embodiments, this disclosure provides a method for detecting the presence or absence of a viruses in a sample from a human subject. In some embodiments, the sample from the human subject is selected from the group of blood, plasma, serum, feces, bronchoalveolar lavage, nasal swab sample, oral swab sample, sublingual swab samples, parotid duct opening swab samples, nasopharyngeal (NP) swab sample, oropharyngeal (OP) swab sample, buccal swab sample, tongue scrape sample, urine, synovial fluid, mucus, or sputum. In some embodiments, the sample is a masticated oral sample.
[0257] In sme embodiments, the method of collection uses a swab device, such as a buccal swab, a nasal swab, an OP swab, a NP swab, or the like. In some embodiments, the sample is collected with a respiratory mask, In some embodiments, the sample is collected using a masticating device, such as a chewing gum. In some embodiments, a tongue scrape sample can be obtained by scraping the tongue of a subject using a tongue scraper such as, for example, the one illustrated in FIG. 11. The debris collected in the tongue scraper upon scraping the tongue of the subject is then collected, e.g., a vial or a tube for further processing.
[0258] In some embodiments, this disclosure provides a method for detecting the presence or absence of viruses in a sample selected from the group of blood, plasma, serum, feces, bronchoalveolar lavage, nasal swab sample, oral swab sample, sublingual swab samples, parotid duct opening swab samples, NP swab sample, OP swab sample, buccal swab sample, tongue scrape, mucus, an aerosol sample collected in a respiratory mask, a masticated oral sample, or sputum comprising the steps of: (1) obtaining the sample from a patient and placing the sample in a container, (2) adding the biosensor or diagnostic composition as disclosed herein to the sample in the container, (3) incubating the biosensor or diagnostic composition with the sample, (3) observing the absence or presence of an optical response in the sample, wherein the presence of the optical response indicates the presence of the viruses, wherein the enzyme encoded by the target virus causes the cleavage of the material to release the spectroscopic probe, resulting in a detectable optical response. The composition may include a lysis buffer to enable or facilitate release of the target enzyme from the infected host cells so that the substrate of the biosensor is accessible to the target enzyme.
[0259] In some embodiments, the sample is an aerosol sample that has been collected in a respiratory mask. In some embodiments, the mask is aN95 respirator mask. In some embodiments, the sample is a masticated oral sample. In some embodiments, the sample is masticated gum.
[0260] In some embodiments, this disclosure provides a method for detecting the presence or absence of viruses in a sputum sample comprising the steps of: (1) obtaining the sputum sample from a patient and placing the sputum sample in a container, (2) adding any one of herein described composition containing the biosensor or diagnostic composition to the sputum sample in the container, (3) incubating the biosensor or diagnostic composition with the sputum sample in the container, (3) observing the absence or presence of an optical response in the sputum sample, wherein the presence of the optical response indicates the presence of viruses, wherein the enzyme encoded by the target virus causes the cleavage of the material to release the spectroscopic probe, resulting in a detectable optical response. The composition may include a lysis buffer to enable or facilitate release of the target enzyme from the infected host cells so that the substrate of the biosensor is accessible to the target enzyme.
[0261] In some embodiments, the process of collecting the sputum sample by the patient comprising the following steps: (1) opening a sample tube; (2) the patient produces the sputum (3) the patient spits the sputum into the sample tube and this is repeated until there is enough sputum to cover the bottom of the tube (e.g., about 2 mL); (4) screwing the cap on the sample tube tightly so it does not leak; (5) labeling the sample tube and sealing the sample in a plastic bag for testing with the biosensor or diagnostic composition. The composition may include a lysis buffer to enable or facilitate release of the target enzyme from the infected host cells so that the substrate of the biosensor is accessible to the target enzyme.
[0262] In some embodiments, the sputum sample is split into several fractions and placed into several tubes for parallel testing including incubation with the biosensor or diagnostic composition disclosed herein. DNA test by conventional nucleic acid amplification (PCR) test may also performed in parallel. The testing results from different tests are compared to confirm the presence of the viruses (e.g., coronavirus).
[0263] In some embodiments, the system includes at least one container holding the biosensor as diagnostic agent. In some embodiments, the system optionally further includes at least one thermal modulator operably connected to the container to modulate temperature in the container, and/or at least one material transfer component (e.g., an automated pipette, etc.) that transfers fluid to and/or from the container (e.g., for performing one or more enzyme digestion reactions in the container, etc.).
[0264] In some embodiments, the sample container is selected from glass tube, plastic tube, an array of tubes on a rack, or biological assay plate such as 6-, 12-, 24-, 48-, 96-, 384-, 1536-well microplate.
[0265] In some embodiments, the sample container is selected from glass tube, plastic tube, an array of tubes on a rack as illustrated in FIG. 3, and the like. In some embodiments, the tubes are capped with screw cap, or septum. In some embodiments, the tubes have a conical bottom. In some embodiments, the tubes have a round bottom. In some embodiments, the plastic tubes may be comprised of, but are not limited to, polypropylene or polyurethane.
[0266] In this disclosure, detection of infection makes use of enzymes that are encoded by the virus and produced in infected host cells. To optimally detect the presence of infected cells, samples should be from a human body region that has high likelihood of having infected cells. It has been found that tongue samples have a greater number of infected epithelial cells.
[0267] In some embodiments, a sample is collected from a tongue by brushing or scraping. In a preferred method, samples are collected by tongue scraping. In a preferred method, tongue scraping is performed with a device such as shown in Fig. 11. In some embodiments, the tongue scraper may have a shape of an L-beam, such that the edges of the L-beam scrape the tongue laterally to collect debris from the surface of the tongue. The tongue scraper may be made of a suitable polymer such as, for example, polyethylene, or polypropylene, or a metal such as, for example stainless steel. In some embodiments, the tongue scraper is a single-use disposable unit that is individually packaged in a sterile packaging. In some embodiments, the tongue scraper can be a reused following sterilization. In such embodiments, the tongue scraper is made from a material that can withstand various sterilization techniques such as, for example steam sterilization or UV sterilization, without degradation in material properties. Other shapes and materials for the tongue scraper are contemplated within the scope of the present disclosure.
Sample Enzyme Extraction
[0268] In some embodiments, the sample is deactivated with a lysis buffer containing a surfactant including a non-ion and non-denaturing surfactant. In some embodiments, the sample is deactivated with a lysis buffer like TRIzol, AVL, RLT, MagMAX, and easyMAG to rupture the cells and inactivate viruses for increasing sensitivity of detection. In some embodiments, the tube further contains a lysis buffer comprising Triton X-100, Digitonin, Tween-20, etc. at different % (0.05-10%) in a buffered saline solution containing TCEP/DTT and glycerol. In some embodiments, the lysis buffer comprises the non-ion and non-denaturing surfactant including, without limitation to the specific species described herein, Triton X-100, Digitonin, Tween-20 at different % (0.05-10%) in a buffered saline solution containing TCEP/DTT and glycerol.
[0269] In some embodiments, the inactivating agent is a surfactant. In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the surfactant is Triton-X. In some embodiments, the surfactant is a polysorbate surfactant. In some embodiments, the surfactant is a polyoxyethylene surfactant. In some embodiments, the inactivating agent is digitonin.
[0270] In some embodiments, the sample is obtained from the patient from any body geography and placed into a container, and the sample is incubated in the container with a diagnostic composition comprising a biosensor. In some embodiments, this disclosure provides a method for detecting the presence or absence of a viruses in a sample comprising the steps of: (1) providing the composition containing the biosensor as disclosed herein, (2) incubating the composition with the sample, (3) observing the absence or presence of an optical response in the sample, wherein the presence of the optical response indicates the presence of the viruses, wherein the enzyme encoded by the target virus causes the cleavage of the material to release the spectroscopic probe, resulting in a detectable optical response. The composition may include a lysis buffer to enable or facilitate release of the target enzyme from the infected host cells so that the substrate of the biosensor is accessible to the target enzyme.
[0271] In some embodiments, additional enzyme can be added to the sample prior to the addition of the biosensor to enhance the sensitivity of the test. Since a minimum amount of enzyme may be required to detect an optical change in a given period of time, the additional enzyme in the sample provides a means to detect enzyme from the sample at a lower concentration. With such an enhanced sample, the rate of change of the optical change (fluorescence or absorption) can be compared to a blank comparative sample containing only the enzyme and the biosensor, with none of the biological component. An increase in the rate over the blank comparative sample is interpreted as the presence of enzyme in the biological specimen, with the amount of increase providing information about the degree of infection represented by the sample. In some embodiments, the additional enzyme is 3CLpro. In some embodiments, the additional enzyme is PLpro. In some embodiments, the additional enzyme is a generic protease that has been demonstrated to convert the biosensor. In some embodiments, more than one type of additional enzyme is added.
[0272] In some embodiments, this disclosure provides a method for detecting the presence or absence of a viruses in a sample comprising the steps of: (1) providing the biosensor or diagnostic composition as disclosed herein, (2) incubating the biosensor or diagnostic composition with the sample, (3) observing the absence or presence of an optical response in the sample, wherein the presence of the optical response indicates the presence of the viruses, wherein the enzyme encoded by the target virus causes the cleavage of the material to release the spectroscopic probe, resulting in a detectable optical response. The composition may include a lysis buffer to enable or facilitate release of the target enzyme from the infected host cells so that the substrate of the biosensor is accessible to the target enzyme.
[0273] In some embodiments, the detection method for viruses uses three different biosensors responsive to three different enzymes, wherein the first population of biosensors have a material only responsive to 3CLpro, the second population of biosensors have a material only responsive to PLpro, and the third population of biosensors have a material only responsive to serine protease.
[0274] In some embodiments, this disclosure provides a method for detecting the presence or absence of viruses in a sample comprising the steps of: (1) obtaining the sample from a patient and placing the sample in a tube, (2) adding the biosensor as disclosed herein to the sample in the tube, (3) incubating the biosensor with the sample, (4) observing the absence or presence of an optical response in the sample, wherein the presence of the optical response indicates the presence of the viruses, wherein the enzyme encoded by the target virus causes the cleavage of the material to release the spectroscopic probe, resulting in a detectable optical response. The incubating the biosensor with the same may further include contacting the sample a lysis buffer during incubation to enable or facilitate release of the target enzyme from the infected host cells so that the substrate of the biosensor is accessible to the target enzyme.
Methods of Detecting Virus State
[0275] In some cases, it is useful to identify that viruses identified by this method continue to be infectious. The infectious nature can be identified by replication of virus in a host medium, such as a biological medium containing cells such as Escherichia Coli. In some embodiments, infection level can be quantified by a method comprising (1) collecting a sample; (2) splitting the sample contents into at least two portions; (3) inactivating the first portion by lysis as described above; (4) incubating the lysed first portion with a diagnostic composition as described above;
(5) observing the presence of an optical response as described above; (6) incubating a second portion with a sacrificial host medium for at least 24 hours; (6) inactivating the second portion, (7) incubating the lysed second portion with a diagnostic composition; (8) observing an optical response; and (9) comparing the optical response of the first portion with that of the second portion.
[0276]
Methods of Optical Detection
[0277] In some embodiments, the system includes one or more biosensors containing the biosensors as described herein. The system also includes at least one detector (e.g., a spectrometer) that detects the optical response resulting from activity of a target enzyme upon the biosensor. For example, the target enzyme may be the endogenous SARS-CoV pathogen protease. The at least one detector may include naked eye, camera, a spectrometer, or any other suitable instrument that can measure an optical response such as, for example, a fluorescence spectroscope, a spectrophotometer, a fluorimeter, a colorimetric spectrophotometer, a photometer, a camera, a microplate reader, or a combination thereof. In addition, the system also includes at least one controller operably connected to the detector. The controller includes one or more instructions sets that correlate the optical response detected by the detector with a presence of SARS-CoV in the sample.
[0278] Detectors are structured to detect detectable signals produced, e.g., in or proximal to another component of the system (e.g., in container, on a solid support, etc.). Suitable optical response detectors systems may include, e.g., a fluorescence spectroscope, a spectrophotometer, a fluorimeter, a colorimetric spectrophotometer, a photometer, a camera, a microplate reader, and the like. Detectors optionally monitor one or a plurality of optical response from upstream and/or downstream of the performance of, e.g., a given assay step. For example, the detector optionally monitors a plurality of optical responses, which correspond in position to "real time" results. Optionally, the systems of the present invention include multiple detectors.
[0279] In some embodiments, the optical response is qualitative and can be observed by naked eye. In some embodiments, the optical response is a quantifiable optical property. In some embodiments, the quantification of the optical response can be achieved using colorimetric spectrophotometers. In some embodiments, the quantification of the optical response can be achieved using a fluorimeter.
[0280] In some embodiments, the optical response is a change in the optical absorption of the sample. In some embodiments, the change in optical absorption is observed as a visual color change. In some embodiments, the change in optical absorption is a quantifiable change detected with a spectrophotometer. In some embodiments, the change in optical absorption is detected over the wavelength range of 400-700 nm. In some embodiments, the change in optical absorption is detected over the wavelength range of 400-500 nm. In some embodiments, the change in optical absorption is detected over the wavelength range of 500-600 nm. In some embodiments, the change in optical absorption is detected over the wavelength range of 600-700 nm. In some embodiments, the change in optical absorption is detected at infrared wavelengths in the range of 700-1100 nm.
[0281] In some embodiments, the optical response is a fluorescence emission. In some embodiments, the fluorescence emission is detected visually. In some embodiments, the fluorescence emission is detected as a quantifiable signal from a fluorimeter. In some embodiments, the fluorescence emission is detected over the wavelength range of 400-700 nm.
In some embodiments, the fluorescence emission is detected over the wavelength range of 400- 500 nm. In some embodiments, the fluorescence emission is detected over the wavelength range of 500-600 nm. In some embodiments, the fluorescence emission is detected over the wavelength range of 600-700 nm. In some embodiments, the fluorescence emission is detected at infrared wavelengths in the range of 700-1100 nm.
[0282] The observation of a fluorescence emission requires optical excitation of the released spectroscopic probe. In some cases, the absorption spectrum of the substrate overlaps that of the released spectroscopic probe. If an excitation wavelength is chosen that excites both species, then the resulting fluorescence emission can include a contribution from the substrate that reduces the precision of measurement of the emission from the released probe. By carefully choosing an excitation wavelength, excitation bandwidth, and wavelength for detection of the probe emission, the contribution of emission from the original substrate can be minimized. As a result, the signal-to-noise ratio of the desired signal can be maximized even while the signal itself is not maximized. In some embodiments, the precision of the quantifiable fluorescence signal can be maximixed by (i) identifying the spectrum of excitation and fluorescence spectra of the fluorophore to be detected in the sample, (ii) identifying the excitation and fluorescence spectra of other fluorescent materials in the sample, (iii) choosing an excitation wavelength that can preferably excite the fluorophore to be detected and minimally excite the other fluorescent materials, and (ii) choosing a wavelength for detecting the fluorescence in a spectral window that is preferred for the fluorescence to be detected and which minimizes the capture of undesired fluorescence.
[0283] Isotropic optical scattering in a sample can affect the characteristics of optical absorption or fluorescence as a result of increasing the effective path length of excitation rays. As a consequence, samples that exhibit different amounts of turbidity can behave as if the concentration of the absorbing species is greater than the true concentration. For example the appearance of color in a turbid absorptive sample will be greater than in a non-scattering sample. Similarly, a turbid fluorescent sample will show a higher fluorescent signal than a non-scattering sample.
[0284] It is well established that the turbidity of fluid samples of biological media can be used to assess cellular counts through optical density measurements in spectroscopic regions lacking optical absobance. Due to differences in cell size and inherent scattering properties, it is crucial that calibration curves be established for such use. Likewise, the turbidity of cellular samples which have been lysed is known to decrease, although scattering in such samples is not eliminated due to the presence of remaining insoluble biogenic material. As a result, the original cellular concentration of a sample can be quantified, even for samples comprising only lysed cellular material. For accurate quantification of spectroscopic data arising from turbid biological samples, optical data can be normalized for the sample turbidity using the measurement of optical density in a non-absorbing region, for example, at a wavelength of 600 nm or greater. As a result, such normalized spectroscopic data can be correlated with viral load of the biological sample. In some embodiments, the quantified optical response is normalized for sample turbidity. In some embodiments, the optical response is normalized by the optical density at 600 nm.
[0285] In some embodiments, this disclosure provides method for the high-throughput (HTS) measurement of optical properties of clinical samples that have been lysed and reacted with the herein described biosensors and placed into the wells of a multiwell plate, wherein the optical properties are measured using a multi-well plate reader. In some embodiments, an optical fiber within a fiber bundle containing no corrective optics between the fiber ends and the well plate bottom illuminates the sample in order to induce fluorescence, and multiple fibers collect emission radiation and transmit it to a fluorescence detector such as a spectrometer. In some embodiments, the HTS measurement of optical properties involves a light scattering illumination source with detection fibers located in either the same bundle containing the fluorescence monitoring fibers or an independent light scattering detection bundle for the measurement of static and/or dynamic light scattering. In some embodiments, the HTS measurement of optical properties involves the measurement of phase analysis light scattering (PALS). PALS enables the measurement of multiple optical properties of a clinical sample being made simultaneously or in succession. The methods for the HTS measurement of optical properties described herein directly measure the samples in the multiwell plates. The high-throughput nature of the measurements permits the rapid screening of the individual sample, as well as reducing the sample volume required. Standard multiwell plates have 96, 384, or 1536 wells, with each well containing a discrete sample, and all wells, under common operational conditions, may be tested in a single data collection run. In addition, the use of these multiwell plates obviates the need for labor as compared to the conventional scintillation vial based method, which requires the cleaning and drying of individual scintillation vials after each measurement. The individual well in these multiwell plates generally has very low volume, e.g ., commercially available multiwell plate based measurement instruments are capable of measurements with sample volumes of 1 pL or less. These low sample volumes are of great benefits when one has a limited amount of sample available for the measurements, particularly when compared to the 300 pL or larger sized measurement volumes often required by other measurement techniques, such as flow-through fluorescence monitoring and flow-through multiangle light scattering (MALS). Additional benefits for the multiwall plate reader based HTS optical property measurement methods include the ability to automate the measurement of between 1 and over 1500 samples with little or no human intervention after the sample is prepared and introduced into the plate for analysis.
Further labor saving benefits can be achieved by automated sample preparation robots such as the Freedom EVO® series produced by Tecan (Tecan Trading AG, Switzerland). In some embodiments, the high-throughput apparatus for optical property measurement comprises a biological assay plate reader. In some embodiments, the high-throughput apparatus comprises a multipipette dispenser. In some embodiments, the high-throughput apparatus comprises a robotic multipipette dispenser.
[0286] Multiwell plates can be used with various optical analysis techniques, most commonly absorbance measurements performed as light is scanned across a plate and the transmitted light is measured by a detector system placed on the opposite side of the plate to the incident light, permitting, thereby a measurement of the absorbance of light by the sample contained in each individual well as, for example, the method described by A. J. Russell and C. Calvert in U.S. Pat. No. 4,810,096, the contents are incorporated herein by reference by its entirety. Measurements of absorbance enables the calculation of the concentration of the sample contained in each individual well. The changes in absorbance over time provide information about reaction rates. As described previously, measurement of optical density in spectroscopic regions lacking absorbance provides information about sample turbidity, and as a result is a measurement of cellular concentration.
[0287] In addition to the existing systems for measuring fluorescence from a multiwell plate, improvements to the optics and light collection and rejection systems have also been reported. For example, U.S. Pat. No. 7,595,881 by S. W. Leonard, et. al, describes a useful optical system placing a shadow disc within the path of emission radiation collected and directed, in free space, by a mirror located below the well plate. By careful positioning and alignment of the optical elements, the shadow disc absorbs light scattered by the meniscus of the sample cell, the remaining radiation is then focused with an aspheric lens onto a detector. The optical systems described by Leonard method improves the overall signal-to-noise of the collected light. Further, a simplified system that maximizes the signal to noise ratio while minimizing stray light and permitting the high-throughput analysis enabled by multiwell plates has also been made possible. Multi-well plates used in plate reader systems are produced in transparent plastic, or in opaque white or black forms. White plates can lead to more efficient capture of fluorescent signals, but can introduce noise from background light and crosstalk between wells. Black plates lead to lower overall signal, but lower overall noise that can arise from background light and crosstalk between wells. In some embodiments, use of an opaque white multi-well plate is preferred. In some embodiments, the use of a black multi-well plate is preferred.
[0288] An advantage of the use of a multi-well plate reader is the parallelism of data capture on a large number of samples at the same time. When kinetic data about the evolution of a spectroscopic response is not required, the throughput of a system using a plate reader can be significantly increased by preparing and incubating prepared plates outside of the reader. A baseline scan can be performed immediately after preparation of a plate, followed by 60 minutes or more of incubation while the desired chemical reactions proceed, followed in turn by an end point reading of the incubated plate. During this incubation period, other plates can be read, either for baseline scans of for enpoint scans. In this way, the analysis of a large number of samples carried on a number of separate plates can be multiplexed in time. For example, if 10 minutes are required to read a plate, then 9,210 samples (including controls) can be run on six plates over the course of two hours. In an embodiment, multi-well plate samples are incubated outside of the plate reader prior to an end point scan. [0289] In some embodiments, the optical response is detected with a biological plate reader. Patient samples that have been inactivated and treated with the biosensor may be distributed into wells of an assay plate for cell culture. The absorbance and/or fluorescence of each sample well can be individually recorded and the presence and level of infection determined. In some embodiments, each plate can be used to assay 96 samples. In some embodiments, each plate can be used to assay 384 samples. In some embodiments, each plate can be used to assayl536 samples. In some embodiments, the biological plate reader is a Biotek Synergy™ HT Multi- mode Microplate Reader (a single-channel absorbance, fluorescence, or luminescence microplate reader; fluorescence l ranges include: standard PMT (excitation 300 nm to 650 nm and emission 300 nm to 700 nm); operated using BioTek’s Gen5™ or KC4™ PC Data Analysis Software; injector models dispense to 6-, 12-, 48-, and 96-well microplates; read 6-, 12-, 24-, 48-, 96-, and 384-well microplates). In some embodiments, the biological plate reader is a VICTOR® Nivo Multimode Microplate Reader by PerkinElmer (operated using VICTOR® Nivo GxP Software), an EnSight™ Multimode Microplate Reader by PerkinElmer (cell-imaging system, operated using Kaleido™ Data Acquisition and Analysis Software), or an EnVision Multimode Microplate Reader by PerkinElmer (configured for 1536-well plate).
[0290] In some embodiments, this disclosure provides methods of detection of viral infection that can be performed in a non-clinical environment. In some embodiments, a sample can be collected and prepared by mixing with a biosensor formulation and other adjuvants and evaluated visually for an optical response. In some embodiments, the optical response is a change in visual absorption. In some embodiments, the optical response is a fluorescent emission.
[0291] In some embodiments, wherein the optical response is fluorescent, it can be generated by using an appropriate wavelength illuminating device such as, but not limited to, an LED device, and the optical response can be observed visually. In some embodiments, the LED device is a UV or blue LED flashlight. In some embodiments, the fluorescent optical response is generated by illumination in a device comprising an LED and a sample holder, wherein the observation of fluorescence occurs at approximately 90° to the excitation of the sample. In some embodiments, the optical response is detected with a digital camera. In some embodiments, the digital camera is a camera on a cellular phone. Pathogen Detection System Mega- scale Test Concept
[0292] The need for increasing testing capacity from the current (-1.5M (US) per week) to 10 to 50 fold is being contemplated. Before that can occur one needs to understand the factors preventing this from happening. Although almost all of the focus is on the specific test cycle time, which ranges from 10 minutes to 4 hours, the bottleneck is in sample collection and sample loading. For sample loading, today, medical personnel are required for sample collection centered on nasopaharyngeal, oropharyngeal. While some attempts have been made at having patients take their own samples and send them to a centralized processing centre. Beyond the sample transportation time, loading and processing through a machine will still be a bottleneck to reach levels of 50 million and higher tests per week. Hence there is a need for a test with the below listed characteristics.
[0293] In an embodiment:
• a test wherein people can not only collect their own sample painlessly, effortlessly, and accurately, as in foolproof (such as using a tongue scraper), whereby there is visible confirmation of actual sample collected by the device, but also have prepackaged reagents in the form of a kit , and by simple mixing execute the test and observe the result.
• With simple mixing conduct the test in the reagent vial, wait a preset time, generally less than an hour and be able to determine a positive test by visual observation of a color change
• Record the result by capturing an image with the cell phone camera
• Cell phone can also be used for subject registration, recording and reporting of data
• It is preferred that the cell phone app should have features that prevent fraud (deliberate false negative results)
• When tests are conducted at mega levels, sample collection devices such as swabs could also become supply bottlenecks and prevent execution, To eliminate these type of bottlenecks the test may deploy reusable vials that hold the reagent and/or solid tablet form and a tongue sample collection device like the L-beam tongue scraper (Fig. 10) that can be sterilized for reuse.
• All materials should be easily scalable
[0294] With the above concept, test capacity is almost limitless. The test can be deployed also in self reporting on a daily basis for enabling businesses and offices to operate at full capacity
[0295] In an embodiment for a mega capacity coronavirus test method,
• Comprising a test individual taking a dropper and collect a premeasured amount of reagent A (Triton X- 100-Cell Lysing Agent) via a dropper to lyse the cell
• Squeezes the same into a vial (reusable) containing premeasured amount of buffer solution
• Waits for a set amount of time for up to an hour
• Observes a distinct color change for determination of positive test (with no color change test is negative)
• Captures an image by cell phone camera for verification of test and reporting.
[0296] In some embodiments, this invention provides a system for detecting infection by a coronavirus pathogen in a human subject. In some embodiments, the system can be performed as a self-test by an individual. In some embodiments, the system comprises: a computer or computer readable medium, sample container, a pathogen diagnostic assay utilizing the biosensors described herein, a controller, a detector coupled to an output of the computer or computer readable medium.
[0297] In some embodiments, the sample container is selected from glass tube or vial, or a plastic tube or vial. In some embodiments, the sample container can be sterilized for reuse. In some embodiments, the container is capped with a screw cap or a septum. In some embodiments, the container has a conical bottom. In some embodiments, the container has a round bottom. In some embodiments, the plastic tube or vial comprises polypropylene or polyurethane. In some embodiments, the sample container includes a lysis buffer solution.
[0298] In some embodiments, the system optionally includes a sample collection device. By way of example, the sample collection device may be a nasal swab, an oral swab, a buccal swab, or a tongue scraper. It is preferred that the method of collection is chosen such that the collection can be individually performed without assistance.
[0299] In some embodiments, the system include one or more biosensors and biosensor compositions as described herein. In some embodiments, the system include one or more biosensors containing the biosensors as described herein. In some embodiments, the system includes at least one container holding the biosensor as diagnostic agent.
[0300] In some embodiments, the system optionally includes an illuminator that can be used to generate a fluorescent signal. In some embodiments, the illuminator is an LED device. In some embodiments, the illuminator is filtered light from a cell phone flashlight.
[0301] In some embodiments, the cell phone is used to detect optical signals generated by changes in absorbance. In other embodiments cell phone is used to generate and detect fluorescence changes
[0302] In some embodiments, the system includes a cell phone that performs multiple functions excitation source, optical signal detector, system controller (sequencing test steps), patient registration, store and report data. In some embodiments, the cell phone may embody steps to prevent fraudulent data collection by invalidating test. In some embodiments, the cell phone embodies steps of data capture and reporting required to file claims to entities responsible for reimbursement and payment for the system performing and reporting test results.
[0303] In some embodiments, the cell phone used has been modified to include an additional light source (beyond normally present) that is a light source with a filter, narrow band light source for excitation.
[0304] In some embodiments the cell phone used has been modified with a filter so the detector captures region around peak emission
[0305] In some embodiments, the system includes a detector that detects the optical response resulted from the endogenous SARS-CoV pathogen protease activity upon the biosensor. In some embodiments, the detector is a camera. In some embodiments, the camera is a camera of a cell phone. In some embodiments, the camera includes an optical filter to reject light used for illumination from a captured fluorescent optical response. In some embodiments, the system includes at least one controller operably connected to the detector. In some embodiments, the controller is a cell phone. In some embodiments, the controller includes one or more instructions sets that correlate the optical response detected by the detector with a presence of SARS-CoV in the sample. In some embodiments, the instructions include a look-up table used with the detector data correlate the optical response detected by the detector with a presence of SARS-CoV in the sample. In some embodiments, the instructions include a registration by the user to enable the test.
[0306] In some embodiments, this disclosure provides a method for detecting infection by coronavirus in a human subject, wherein the method comprises the steps:
(a) registering the test by the human subject including insurance eligibility verification,
(b) generating a sample by the human subject with a sample collection device, or by providing a sample of sputum,
(c) conditioning the sample from the human subject by lysis with the lysis buffer to inactivate the coronavirus,
(d) exposing the inactivated sample in step (c) to a biosensor described herein to produce an optical response,
(e) detecting the optical response generated in step (d) with a detector, wherein the detector is configured to process the optical response into a data output, and the detector is configured to communicate the data output to a computer or computer readable medium,
(f) saving the data output labeled with the patient information in step (e) on the computer or computer readable medium,
(g) processing the data according to the instructions, and
(h) generating a report to the human subject for the data output.
(i) generating claims to entities for reimbursement and payment for obtaining the test results.
[0307] The method using the system described reduces the need for trained medical personnel to perform the testing, and provides the ability to perform the test at home. The method does not require the use of advanced equipment and eliminates process bottlenecks with regard to loading and unloading multiple samples. Since many human subjects may simultaneously be testing themselves with this system, the system incorporates massive parallelism to enable testing of millions of subjects in a short period of time. By deploying an efficient system of reusable hardware associated with the test such as reusable test tubes and tongue scrapers, there are no limitations to the number of times or the frequency the system can run the test other than periodic replenishment of reagents that are consumed by the test. Without these embodiments, mass population testing with high frequency of testing is impossible to deploy due to supply constraints of non-reusable testing hardware. Also, because each test subject performs the task of sample loading in order to conduct the test, the system creates massive parallelism by eliminating the sequential sample loading bottleneck of institutional laboratories relying instruments to run their tests.
Coronavirus Detection System
[0308] In some embodiments, this invention provides a system for detecting infection by SARS- CoV pathogens in a multiple human subjects. In some embodiments, the system comprises: a computer or computer readable medium, sample container, a substrate container, a pathogen diagnostic assay utilizing the biosensors described herein, a controller, a detector coupled to an output of the computer or computer readable medium. FIG. 2 schematically illustrates the components of the SARS-CoV pathogen detection system operably connected.
[0309] The system may further include a computer or computer readable medium with a data set that relates to patient information (e.g., patient demographic information) and optical response imputed from the detector. Typically, the computer or computer readable medium further includes a location tracking device coupled to an output of the computer or computer readable medium. The location tracking device accepts instructions from the computer or computer readable medium, which instructs the location tracking activity corresponding to the detection the presence of optical response.
[0310] In some embodiments, the systems may include controllers that are operably connected to one or more components (e.g., detectors, thermal modulator, fluid transfer components, etc.) of the system to control operation of the components. In some embodiments, the controller may be included either as separate or integral system components that are utilized, e.g., to receive data from detectors, to effect and/or regulate temperature in the containers, to effect and/or regulate fluid flow to or from selected containers, or the like. Controllers and/or other system components is/are optionally coupled to an appropriately programmed processor, computer, digital device, or other information appliance (e.g., including an analog to digital or digital to analog converter as needed), which functions to instruct the operation of these instruments in accordance with preprogrammed or user input instructions, receive data and information from these instruments, and interpret, manipulate and report this information to the user. Suitable controllers are generally known in the art and are available from various commercial sources.
[0311] Any controller or computer optionally includes a monitor, which is often a cathode ray tube ("CRT") display, a flat panel display (e.g., active matrix liquid crystal display, liquid crystal display, etc.), or others. Computer circuitry is often placed in a box, which includes numerous integrated circuit chips, such as a microprocessor, memory, interface circuits, and others. The box also optionally includes a hard disk drive, a floppy disk drive, a high capacity removable drive such as a writeable CD-ROM, and other common peripheral elements. Inputting devices such as a keyboard or mouse optionally provide for input from a user.
[0312] The computer typically includes appropriate software for receiving user instructions, either in the form of user input into a set of parameter fields, e.g., in a GUI, or in the form of preprogrammed instructions, e.g., preprogrammed for a variety of different specific operations. The software then converts these instructions to appropriate language for instructing the operation of one or more controllers to carry out the desired operation. The computer may then communicate with the sensor/detectors included within the system to receive the data. The computer may be hardwired to the sensors/detectors or may communicate with the sensors/detectors wirelessly. In some embodiments, the computer may communicate with the sensors/detectors over a network such as, for example, a WAN, a LAN, the Internet using a suitable communication protocol. Accordingly, the computer need not be in the same location as the sensors/detectors. , The computer then interprets the data, and either provides it in a user understood format, or uses that data to initiate further controller instructions, in accordance with the programming, e.g., such as initiating the location tracking activity. For example, the software may be operating softwares including BioTek’s Gen5™ or KC4™ PC Data Analysis Software in microplate reader for data collection and analysis, PerkinElmer’ s VICTOR® Nivo GxP Software in microplate reader for data collection and analysis, or Kaleido™ Data Acquisition and Analysis Software in microplate reader for cell-imaging, or any other commercially available or proprietary application operable on a mobile device such as, for example, an App.
[0313] In some embodiments, the computer may include: a processor on a mobile device, a PC, Power PC, a Unix-based working station, or other common commercially available computer, which is known to one of skill in the art. Standard desktop applications such as word processing software (e.g., Microsoft Word™ or Corel WordPerfect™) and database software (e.g., spreadsheet software such as Microsoft Excel™, Corel Quattro Pro™, or database programs such as Microsoft Access™ or Paradox™) can be adapted to the present invention. Software for performing, e.g., initiating location tracking is optionally constructed by one of skilled artisan using a standard programming language such as Visual basic, Fortran, Basic, Java, or the like. In some embodiments, the computer may be a cell phone.
[0314] In some embodiments, the invention is optionally implemented in hardware and/or software. In some embodiments, different aspects of the invention are implemented in either client-side logic or server-side logic. In some embodiments, the components of the system may be embodied in a media program component (e.g., a fixed media component) containing logic instructions and/or data that, when loaded into an appropriately configured computing device, cause that device to perform according to the invention. As used herein, the fixed media containing logic instructions may be delivered to a viewer on a fixed media for physically loading into a viewer's computer or a fixed media containing logic instructions may reside on a remote server that a viewer accesses through a communication medium in order to download a program component.
[0315] A patient can self-identify for testing by scanning or otherwise providing a code. The code can be provided through a paper document, such as a doctor’s form or promotional literature, or may be provided through a website. By providing the code, the patient initiates registration in a database that will track the analysis. This database entry can include information such as phone number and email address. In an embodiment, the code may be scanned using a software application that connects the patient with a network database. The database assigns a unique patient code for each patient registration. In an embodiment, the software application also captures GPS data that is used to provide the patient with location information for testing, including directions and approximate wait time. [0316] The testing may be performed at a hospital, a clinic, a drive-through testing site, or at home. At the point of testing, a sample from a patient can be collected. In an embodiment, capped sample collection tubes (1) that are marked, labeled, or otherwise individually identified are provided that contain an inactivating agent. In an embodiment, the label is a QR code that can be read by a code scanner, cell phone, or other reader. In an embodiment, the label is an RFID device that can be detected and read by an RFID reader.
[0317] The phrase "sample derived from a subject" refers to a sample obtained from the subject (e.g., human patients suspected of having SARS-CoV infections, etc.), whether or not that sample undergoes one or more processing steps (e.g., cell lysis, debris removal, stabilization, etc.) prior to analysis. To illustrate, samples can be derived from subjects by scraping, venipuncture, swabbing, biopsy, or other techniques known in the art.
[0318] In an embodiment, the sample label information is associated with previously stored patient information from a database. The database can be managed locally on a mobile device to associate patient information with the individualized label of the sample tube. This information may be subsequently uploaded to a network database, such as a cloud-based database. Alternatively, a wireless connection can be used to directly populate a network database with such data. The local or network database can be used for further management of data associated with the analysis of the sample.
[0319] The invention provides a method to analyze a biological sample by placing the sample in contact with an analysis reagent. In some embodiments, the analysis reagent is a solid formulation. In some embodiments, the analysis reagent is provided in a cap that can be placed on the sample tube (3).
[0320] In some embodiments, the analysis reagent can be injected into the sample tube. In some embodiments, the analysis reagent is injected through a diaphragm or septum on the sample tube cap.
[0321] The presence of pathogen is shown by the observation of an optical signal. In some embodiments, the optical signal can be observed as a qualitative visual signal as a color change that indicates the presence of the pathogen. In some embodiments, the optical signal may be detected by a quantitative spectral response from a device or instrument. In some embodiments, the optical signal may be a fluorescence signal. In some cases, the optical signal can be detected using the camera of a cell phone or other mobile electronic device. A signal difference in the rate of change of color formation can be used to quantify a level of infection in a patient. In some embodiments, a rate of change is determined by comparison of sample color in photographs that are separated by known times. In some embodiments, a rate of change is determined from a video recording.
[0322] In some embodiments, the viruses are coronavirus. In some embodiments, the viruses are selected from the group of SARS-CoV-2, SARS-CoV-1, or MERS-CoV. In some embodiments, the virus is SARS-CoV-2. In some embodiments, the virus is SARS-CoV. In some embodiments, the virus is MERS-CoV.
[0323] In some embodiments, this disclosure provides a system for detecting SARS-CoV pathogen in a sample from a human subject, wherein the system comprises: a computer or computer readable medium, sample container, a biosensor as described herein, a controller, and a detector coupled to an input to the computer or computer readable medium.
[0324] In some embodiments, the detector is structured to detect an optical response produced by the SARS-CoV pathogen diagnostic assay.
[0325] In some embodiments, the detector is selected from the group of a fluorescence spectroscope, a spectrophotometer, a fluorimeter, a colorimetric spectrophotometer, a photometer, or a camera.
[0326] In some embodiments, the computer comprises a software for receiving user instructions in the form of user input, wherein the software is configured to convert the user instructions to control the operation of the controller.
[0327] In some embodiments, the computer is configured to receive data from the detector.
[0328] In some embodiments, the computer is a cell phone.
[0329] In some embodiments, the sample container is selected from a glass tube, a plastic tube, or an array of tubes on a rack.
[0330] In some embodiments, the SARS-CoV pathogen diagnostic assay system is contacted with a sample from a subject to perform a diagnostic test according to any of the herein described the methods. [0331] In some embodiments, the sample from the human subject is selected from the group of blood, plasma, serum, feces, bronchoalveolar lavage, nasal swab sample, oral swab sample, NP swab sample, OP swab sample, buccal swab sample, tongue scrape, urine, synovial fluid, mucus, an aerosol sample collected in a respiratory mask, masticated oral sample or sputum.
Coronavirus detection steps
[0332] In some embodiments, this disclosure provides a method for detecting coronavirus in a sample from a human subject, wherein the method comprises the steps:
(a) receiving patient information by a registration process,
(b) obtaining a sample from the human subject,
(c) conditioning the sample from the human subject by lysis with a chemical agent to inactivate the coronavirus,
(d) exposing the inactivated sample in step (c) to a biosensor described herein to produce an optical response,
(e) detecting the optical response generated in step (d) with a detector, wherein the detector is configured to process the optical response into a data output, and the detector is configured to communicate the data output to a computer or computer readable medium; and wherein the computer or computer readable medium is configured to generate a label for the data output received with the patient information received by the registration process in step (a),
(f) saving the data output labeled with the patient information in step (e) on the computer or computer readable medium, and
(g) generating a report to the human subject for the data output labeled with the patient information in step (e).
[0333] In some embodiments, the coronavirus is SARS-CoV-2, SARS-CoV-1, or MERS-CoV.
[0334] In some embodiments, the computer or computer readable medium used in the SARS- CoV pathogen method is a cell phone.
[0335] In some embodiments, the sample from the human subject is selected from the group of blood, plasma, serum, feces, bronchoalveolar lavage, nasal swab sample, oral swab sample, NP swab sample, OP swab sample, buccal swab sample, tongue scrape, urine, synovial fluid, mucus, an aerosol sample collected in a respiratory mask, a masticated oral sample, or sputum. In some embodiments, the sample is selected from the group of NP swab sample, nasal swab sample, oral swab sample, OP swab sample, buccal swab sample, or tongue scrape. In some embodiments, the sample is a sputum sample. In some embodiments, the sample is a NP swab sample or OP swab sample. In some embodiments, the sample is a NP swab sample. In some embodiments, the sample is a OP swab sample. In some embodiments, the sample is bronchoalveolar lavage. In some embodiments, the sample is an aerosol sample that has been collected in a respiratory mask. In some embodiments, the sample is a masticated oral sample.
In some embodiments, the sample is an aerosol sample that has been collected in a respiratory mask. In some embodiments, the mask is aN95 respirator mask.
[0336] In some embodiments, the detector is selected from the group of a fluorescence spectroscope, a spectrophotometer, a fluorimeter, a colorimetric spectrophotometer, a photometer, or a camera.
[0337] In some embodiments, the detector is a camera embedded within a cell phone.
[0338] In some embodiments, the chemical agent in step (c) comprises a surfactant.
[0339] In some embodiments, this disclosure provides a kit for detecting the presence of viruses in a sample of a human subject and/or for the determination of antiviral drug susceptibility of a pathogen and the kit include: biosensors or composition thereof as described herein, cotton swabs, sample tubes with screw cap, lysis buffer in a bottle like TRIzol, AVL, RLT, MagMAX, and an instruction sheet providing instructions to the user to collect the sample into the sample tube, condition the sample with the lysis buffer, register the patient information to a registering system, and a diagnostic assay procedure for conducting the diagnostic test upon the sample with the biosensor or compositions thereof.
Workflow chart
[0340] FIG. 3 illustrates the parking lot professional workflow chart comprising the following components of the pathogen detection process. In this example, samples are collected using tongue scrapers, but any method of sample collection as described herein can be used.
[0341] Step 1 of FIG. 3 shows a set of 50 commercially available samples tubes placed in a packaging box. Each test tube is identified by a unique QR label, a standard cap and can be pre filled with inactivating solution. This QR code will match the database so there is no risk to counterfeiting. This QR code will be used to tag each patient to the sample in the tube. There are service providers that can automate the labeling on the test tubes. There is no issue of reading the QR code on the round tube from a mobile device. The inactivating agent can be pre-filled by contract manufacturer with fill and finish facility.
[0342] Step 2 of FIG. 3 shows a set of 50 commercially available tongue scrapers (or any other sample collection modality) packed in a large box with each in its own sterile packaging.
[0343] Step 3 of FIG. 3 shows a set of 50 proprietary substrate papers stored in screen on cap. This is supplied by the substrate supplier. Given the substrate is extremely hydrophobic, it needs be stored in its own airtight packaging.
[0344] Step 4 of FIG. 3 shows that a HCS picks a sample collection tube labeled with QR code from the packaging box of Step 1 when a patient arrives. The QR code on the tube will be tagged to the patient by the digital app.
[0345] Step 5 of FIG. 3 shows that a patient picks a package containing a tongue scraper from the packaging box of Step 2. Patient conducts the sample collection with the scraper. When the sample collection is complete, the patient drops the tongue scraper into the collection test tube held in the hand of the HCS. Alternatively, to avoid risk of contamination, the patient is given the collection tube and places the tongue scrape sample in the collection tube.
[0346] Step 6 of FIG. 3 shows that the QR code labeled sample collection tube with closed cap is placed on a large “Inactivation Tray” to be batched. Each patient sample collection takes 1 to 2 minutes, each tray can be filled between 25 minutes to 50 minutes for a tray capable of holding 25 tubes. To assure the last sample collected had ample time for inactivation, an extra 10 minutes will expire before moving to step 7.
[0347] Step 7 of FIG. 3 shows that cap for each sample collection tube in the Inactivation tray will be swapped with a cap containing substrate. The sample on the tray with the longest elapse of time from the time point of collection is tested first. Screwing the substrate contain cap onto the sample collection tube will deploy the paper substrate onto the liquid in the sample.
[0348] Step 8 of FIG. 3 shows that the assay process takes 30 seconds or less for each test tube for a total of 12 minutes for 50 samples collected. [0349] Step 9 of FIG. 3 shows that the results will be available after 70 to 80 minutes since the collection of the first sample of this batch. The throughput of the test is about 6 minutes per sample for either positive or negative. By comparison, the Abbott Now ID test takes 5 minutes for positive result and 13 minutes for negative result confirmation, not including setup time in between test. However, the Abbott test gives a throughput of 8 minute per sample at best.
Digital application work flow
[0350] Digital app workflow of patient (The numbers in this section is not directly related to the numbers in FIG. 3):
[0351] Step 1. The patient scans a QR code from the promotional material and the Patient downloads the Now Aware™ App.
[0352] Step 2. 2(a). The patient selects to register for a test on the App. The app will use GPS data to suggest the nearest testing location. The patient selects a preferred location. 2(b). The patient registers testing by entering basic information including their phone number so that they can receive test result on the app. 2 (c). The patient requests to schedule for testing. This may not be necessary if GPS information is used at the testing site to give approximate wait time at the location based on number of patients waiting at the location. This way, it is first come first serve. It avoids the complexity of scheduling. If the test throughput is fast, each patient can be processed, including sample collection, in less than 2 minutes. 2(d) The patient finishes test registration and is assigned a unique patient code. This patient code will be a QR code displayed on the phone when patient is ready to be processed by the HCS at the test site. 2(e). The App plays a video of the entire testing process including video instruction of how to do tongue scrape and how to put the swab in the collection tube.
[0353] Step 3. When the patient reaches the testing center, the QR code will be displayed for processing.
Digital App workflow for the HCS
[0354] (The numbers below is in direct reference to the numbers in the diagram). In this example, samples are collected using tongue scrapers, but any method of sample collection as described herein can be used. [0355] Step 1. When the patient arrives the tent, the app will display a unique QR code that identifies the patient.
[0356] Step 2. The HCS’s App will scan the QR code from patient’s App. It will display the patient registration information. HCS can verbally confirm patient by name.
[0357] Step3. The HCS “sample collector” will take a collection tube out of the box of tubes (#1). HCS will scan the QR code on the collection tube. This will tag the patient to the collection tube.
[0358] Step 4. The HCS will take out a tongue scrapper package from (#2) and give it to the patient in the car.
[0359] Step 5. The patient will take out the tongue scrapper (#5) from the package and follow the video instruction from the app to collect the tongue scrape sample.
[0360] Step 6. When a tongue scrape sample is collected, the HCS gives the patient the sample collection tube with the cap already removed so that the patient inserts the tongue scrapper into the tube, and then the HCS gives the patient the cap so it is screwed onto the tube. This is to prevent contamination of sample because it is handled completely by the patient and reduces infection risk to HCS. Alternatively, the HCS removes the cap from the sample collection tube and asks the patient to insert the tongue scrape sample in the tube, and the HCS puts the cap back on the tube. This is to avoid the dexterity needed for the patient to handle the collection tube.
This patient processing should take no more than 1 to 2 minutes for each patient.
[0361] Step 7. The HCS places the completed sample collection tube onto “Inactivation Tray” (#6). This will continue until the tray is filled, or whenever the HCS feels there is sufficient number of samples collected for the batch collection. Upon a batch is collected, the HCS sample collector will move the Inactivation Tray (#6) to a workstation designated for a HCS to process the samples. The HCS sample collector will take an empty Inactivation Tray (#6) to start a new batch of sample collection.
[0362] Step 8. Upon receiving last sample for the batch (#6), the testing process will not start until there was sufficient time for sample inactivation of the last sample placed into the tray. This is to assure all samples in the tray have completed inactivation and lysis of cells in the last collected sample. However, this workflow can be improved by starting the testing process (#7) of the first sample that was collected in the batch and then the subsequent sample collected.
[0363] Step 9. The HCS “sample processor”, will take a cap containing the substrate paper from box of caps (#3). The HCS “sample processor” will swap cap on top of each collected sample with cap containing the substrate on paper and deploy the substrate into the collection tube (#7). This process will continue until the entire batch in “Result Tray” (#8) has received the substrate. The “sample processor” can wait for the period of time that assures the last sample with substrate had sufficient time for the catalytic reaction. This workflow can also be improved by examining right away the test result of the first sample in the batch that received substrate and then the subsequent sample in the batch.
[0364] Step 10. For each test result, the HCS “sample processor” will scan the QR code on the test tube that is linked to the patient. The App will ask the “sample processor” to input the test result based on the color change. Alternatively, the App will scan the QR code and determine the test result based on the color spectrum observed by the camera to completely automate the result and associating it to the patient.
[0365] Step 11. The Now Aware™ digital network will notify the test result to the patient on the app. The app will assist the patient with the necessary procedures to follow based on the test result. For patients with positive result, the app will offer e-Commerce to connect service providers for the patient. For patients with negative result, the app will notify the patient to get a confirmation test in 5 days. This is important because true confirmation of requires testing at the front end and back end of the 5 day window between infection and virus production.
[0366] Step 12. The NowAware™ database will generate daily test result in a standard reporting format to the Center For Disease Control and Prevention (CDC) of United States. Direct data connectivity to CDC reporting can be established on the backend of the NowAware ecosystem™. EXAMPLES
[0367] The embodiments encompassed herein are now described with reference to the following examples. These examples are provided for the purpose of illustration only and the disclosure encompassed herein should in no way be construed as being limited to these examples, but rather should be construed to encompass any and all variations which become evident as a result of the teachings provided herein.
General Procedures
[0368] The compositions of this invention may be made by various methods known in the art. Such methods include those of the following examples, as well as the methods specifically exemplified below. Modifications of such methods that involve techniques commonly practiced in the art of sensors and particle technology may be used.
[0369] As used herein the symbols and conventions used in these processes, schemes and examples are consistent with those used in the contemporary scientific literature, for example, the Journal of the American Chemical Society or the Journal of Biological Chemistry.
[0370] The following examples describe the invention in further detail, with reference to specific embodiments. These are representative embodiments of the invention which are provided for illustrative purposes only, and which should not be regarded as limiting the invention in any way.
Example 1. Degradation of Fluorophore modified peptide by Cysteine Protease
[0371] A solution containing 700 mM Z-RLRGG-7-amido-4 methylcoumarin and 10% dimethylsulfoxide in 90% 50 mM HEPES buffer with 150 mM sodium chloride, 2.5 mM dithiothreitol and 0.1 mg/mL bovine serum albumin was prepared. To 1 ml of this solution was added 2 pL of 1 ImM SARS-CoV-2-PLPro solution (Boston Biochem) and the combination was thoroughly mixed. Initial fluorescence was recorded, and the solution was incubated at room temperature for 30 minutes, monitoring for fluorescence at 10 minute intervals. Reaction was detectable as a low intensity fluorescence at 441 nm (380 nm excitation) that increased linearly with time. Example 2. Preparation of Ac-hPhe-Dap-Gly-Gly-ACC.
[0372] Fmoc-Rink Amide AM resin (lmmol, 1 equivalent) was prepared by swelling with dry DMF (15 mL) for 30 min. The DMF was then drained, and the Fmoc protecting group removed by treatment with 20% piperidine in DMF (10 mL). The solution part was removed, and the resin was washed with dry DMF. The resin was treated a final time with 20% piperidine in DMF (10 mL), and the solution part was removed. The resin was then washed five times with 10 ml dry DMF, each time shaking for 2 minutes before removing the wash solution. Positive Kaiser test proved that free amine group was present on the resin.
[0373] A solution of Fmoc-ACA-OH (2.0 equiv.), HOBt (2.0 equiv.) in DMF (10 mL) was added to the flask containing the resin followed by addition of DICI (2.0 equiv.). It was then shaken overnight (24 h) at room temperature. The resin was then washed 5 times with 10 ml aliquots of dry DMF, each time shaking for 2 minutes before draining the wash solution. Remaining free amine groups, if any, were reacted with 1 : 1 of Ac20:DMF (50 mL) for 2h. The resin was then washed 5 times with dry DMF, each time shaking for 2 minutes before draining the wash solution to produce Fmoc-ACA-resin. Kaiser test was negative indicating the absence of free amines.
[0374] The Fmoc-ACA-resin was treated with 20% piperidine in DMF (10 mL) for 20 min. The solution part was removed, and the resin was washed with dry DMF. The resin was treated a final time with 20% piperidine in DMF (10 mL) for 20 min, and the solution part was removed. The resin was then washed five times with 10 ml dry DMF, each time shaking for 2 minutes before removing the wash solution. Kaiser test showed show faint color of the bead after heating for 5 min at 100 °C.
[0375] A solution of Fmoc-Gly-Gly-OH (5.0 equiv.), HATU (5.0 equiv.), and 2,4,6-collidine (5.0 equiv.) in DMF (10 mL) was prepared and added to the resin. It was then shaken overnight (24 h) at room temperature. The solution was then drained from the resin, and the resin was washed five times with 10 ml aliquots of dry DMF, each time shaking for 2 min before draining out the solvent to produce Fmoc-Gly-Gly-ACA-resin. Kaiser test was negative.
[0376] The Fmoc-Gly-Gly-ACA-resin was treated with 20% piperidine in DMF (10 mL) for 20 min. The solution was removed, and the resin was washed with dry DMF. The resin was treated a final time with 20% piperidine in DMF (10 mL) for 20 min, and the solution part was removed. The resin was then washed five times with 10 ml dry DMF, each time shaking for 2 minutes before removing the wash solution. Kaiser test showed strong purple color of the bead.
[0377] A solution of Fmoc-Dap(Boc)-OH (2.0 equiv.), HOBt (2.0 equiv.) and DICI (2.0 equiv.) was prepared and added to the resin. It was then shaken for overnight (24 h) at room temperature. The solution was then removed, and the resin was then washed five times with 10 ml aliquots of dry DMF, each time shaking for 2 min before draining out the solvent to produce Fmoc-Dap(Boc)-Gly-Gly-ACA-resin. Kaiser test was negative.
[0378] The Fmoc-Dap(Boc)-Gly-Gly-ACA-resin was treated with 20% piperidine in DMF (10 mL) for 20 min. The solution part was removed, and the resin was washed with dry DMF. The resin was treated a final time with 20% piperidine in DMF (10 mL) for 20 min, and the solution part was removed. The resin was then washed five times with 10 ml dry DMF, each time shaking for 2 minutes before removing the wash solution. Kaiser test showed strong purple color of the bead.
[0379] A solution of Fmoc-hPhe-OH (3.0 equiv.), HOBt (3.0 equiv.) and DICI (3.0 equiv.) was prepared in DMF (10 mL) and added to the resin. It was then shaken for overnight (24 h) at room temperature. The solution was then removed, and the resin was then washed five times with 10 ml aliquots of dry DMF, each time shaking for 2 min before draining out the solvent to produce Fmoc-hPhe-Dap(Boc)-Gly-Gly-ACA-resin. Kaiser test was negative.
[0380] The Fmoc-hPhe-Dap(Boc)-Gly-Gly-ACA-resin was treated with 20% piperidine in DMF (10 mL) for 20 min. The solution part was removed, and the resin was washed with dry DMF. The resin was treated a final time with 20% piperidine in DMF (10 mL) for 20 min, and the solution part was removed. The resin was then washed five times with 10 ml dry DMF, each time shaking for 2 minutes before removing the wash solution. Kaiser test showed strong purple color of the bead.
[0381] The H-hPhe-Dap(Boc)-Gly-Gly-ACA-resin was treated with 10 ml of 1:1 of Ac20:DMF for 1 hour, and the solution removed. The resin was washed once with 10 ml dry DMF, followed by a second treatment with 10 ml of 1 : 1 of Ac20:DMF for 1 hour. The resin was then washed five times with 10 ml dry DMF, each time shaking for 2 minutes before removing the wash solution. Kaiser test was negative. Finally, the resin was washed five times with 10 ml aliquots of diethyl ether (DEE), and then dried under high vacuum for 2 hours.
[0382] The Ac-hPhe-Dap(Boc)-Gly-Gly-ACA-resin was treated two times (10 mL x 2) for one hour per treatment with 95:2.5:2.5 of TFA:H20:TIPS. The solution was drained into two 50 mL centrifuge tubes (10 mL each) and DEE (40 mL) was added to each, and the product was centrifuged (7000 rpm c 5 min). The supernatant was discarded, and the off-white solid was washed 3 more times with DEE (30 mL, 7000 rpm x 5 min). The solid was then dried overnight in at high vacuum. The off-white crude solid of Ac-hPhe-Dap-GG-ACC was obtained in 90% overall yield.
[0383] The product was then purified by flash chromatography using Combiflash® with a reverse phase C-18, 30 g column using water: acetonitrile solvent system. The product in the fractions was recovered by lyophilization, to give a yield of -50% based on starting resin. The product structure of Ac-hPhe-Dap-GG-ACC was confirmed by MS analysis.
Example 3. Preparation of fluorophore-modified peptide
Example 3a. Synthesis of (N-Boc-LRGG)2Rhodamine (see FIG. 4)
[0384] Four equivalents of N-Boc-LRGG-OH was treated with 4 equivalents of 1~(3~ di m ethylaminopropyl)-3 -eth lcarhodi imide in DMF with 0.4 equivalents of 4- dimethylaminopyridine to prepare an activated peptide. This material was then treated with one equivalent of rhodamine 110 over 2 days to yield (N-Boc-LRGG-N)2-rhodamine (See FIG. 4 for chemical structure).
Example 3b. Synthesis of (NFh-LRGG^Rhodamine
[0385] CbzGG-COOH (5.32 g, 20 mmol) was dissolved in dry DMF/Py = 1:1 (80 mL) and cooled in an ice/water bath (-4 °C) while stirring under N2. To this solution, EDC (3.48 g, 18.2 mmol) was added and stirred for 5 min. Rhodamine 110 (262 mg, 0.71 mmol) was dissolved in DMF/pyridine (5 mL) and added to the reaction mixture. Stirred at the same temperature for 2 h and the stirred at RT for 48 h. The reaction was stopped by adding diethyl ether (DEE) (280 mL) and the mixture was centrifuged (6000 rpm, 10 min). The supernatant was discarded and the gel like solid was suspended in methanol (20 mL), precipitated by adding DEE (280 mL) and centrifuged again. This step was repeated two more times. The solid was then suspended in dichloromethane (DCM) (200 mL) and washed with water (100 mL). The aqueous layer was extracted with more DCM (200 mL). The organic later was washed with water (100 mL x 2) and brine (100 mL). The organic layer was dried over NaiSCL and the solvent was evaporated to obtain a brown color solid. The product was then purified through Combiflash™ to yield (Cbz- GG)2Rhodamine.
[0386] (CbzGG)2Rhodamine (1.0 g, 1.2 mmol) was suspended in HBr (33% in AcOH, 30 mL) and stirred at RT for 2 h. The product was precipitated with DEE (80 mL) and centrifuged (6000 rpm, 10 min). The supernatant was discarded and th solid was dissolved in MeOH (3 mL), precipitated with DEE (40 mL) and centrifuged. This step was repeated once more. The solid was then suspended in DEE (30 mL) and centrifuged. The solid was then dried in vacuum oven to obtain (NH2-GG)2Rhodamine*2HBr as an orange-brown solid (1.0 g, quant.).
[0387] Fmoc-Arg(pbf)-OH (4.06 mg, 6.25 mmol) was dissolved in dry DMF/Py = 1:1 (40 mL) and cooled in an ice/water bath (~4 °C) while stirring under N2. To this solution, EDC (1.34 g, 7.0 mmol) was added and stirred for 5 min. (NH2-GG)2Rhodamine*HBR (900 mg, 1.25 mmol) was dissolved in DMF/Py (5 mL) and added to the reaction mixture. Stirred at the same temperature for 2 h and the stirred at RT overnight. The reaction was stopped by adding DEE (180 mL) and the mixture was centrifuged (6000 rpm, 10 min). The supernatant was discarded and the solid was dissolved in in methanol/DCM (10 mL), precipitated by adding DEE (70 mL) and centrifuged again. This step was repeated two more times. The solid was then suspended in DEE (60 mL) and centrifuged. The solid was then suspended in DCM (200 mL) and washed with water (200 mL). The aqueous layer was extracted with more DCM (200 mL). The organic layer was washed with water (200 mL c 1) and brine (50 mL). The organic layer was dried over Na2S04 and the solvent was evaporated to obtain a off-white color solid. The product (Fmoc- R(pbf)GG)2Rhodamine was then purified through Combiflash™ (1.3 g, 57%).
[0388] (Fmoc-R(pbf)GG)2Rhodamine (1.3 g) was dissolved in DCM (10 mL) and piperidine (10 mL) was added. Stirred at RT for 10 min. Much of the solvent was evaporated and to the rest, DEE (80 mL) was added. The mixture was centrifuged (6000 rpm, 10 min) and the solid was suspended in MeOH (5 mL) and more DEE was added and centrifuged again. The soilid was resuspended in MeOH/DCM (5 mL) precipitated with DEE and centrifuged. This step was repeated once more. To the solid was added DEE (40 mL) and centrifuged again. The final solid was dried in vacuum oven to yield the product (NH2-R(pbf)GG)2Rhodamine in an off-white solid (992 mg, quant.)
[0389] Fmoc-Leu-OH (2.37 mg, 6.7 mmol) was dissolved in dry DMF/Py = 1:1 (30 mL) and cooled in an ice/water bath (~4 °C) while stirring under N2. To this solution, EDC (1.54 g, 8.0 mmol) was added and stirred for 5 min. (NH2-R(pbf)GG)2Rh (900 mg, 1.25 mmol) was dissolved in DMF/Py (10 mL) and added to the reaction mixture. Stirred at the same temperature for 2 h and the stirred at RT for 1 h. The reaction was stopped by adding DEE (300 mL) and the mixture was centrifuged (6000 rpm, 10 min). The supernatant was discarded and the solid was dissolved in in DCM (10 mL), precipitated by adding DEE (80 mL) and centrifuged again. This step was repeated two more times. The solid was then suspended in DEE (60 mL) and centrifuged. The solid was then suspended in DCM (200 mL) and washed with water (100 mL). The aqueous layer was extracted with more DCM (200 mL). The organic layer was washed with water (150 mL x 2) and brine (50 mL). The organic layer was dried over Na2S04 and the solvent was evaporated and dried in vacuum oven to obtain (Fmoc-LR(pbf)GG)2Rhodamine as an off- white color solid (1.2 g, 85%).
[0390] (Fmoc-LR(pbf)GG)2Rhodamine (1.1 g) was dissolved in DCM (10 mL) and piperidine (10 mL) was added. Stirred at RT for 10 min. Much of the solvent was evaporated and to the rest, DEE (160 mL) was added. The mixture was centrifuged (6000 rpm, 10 min) and the solid was suspended in MeOH (20 mL) and more DEE (160 mL) was added and centrifuged again. The soilid was resuspended in MeOH (10 mL) precipitated with DEE (80 mL) and centrifuged. This step was repeated once more. To the solid was added DEE (40 mL) and centrifuged again. The final solid was dried in vacuum oven to yield (NH2-LR(pbf)GG)2Rhodamine as an off-white solid (815 mg, 94%)
[0391] (NH2-LR(pbf)GG)2Rhodamine (100 mg) was dissolved in 2 ml of 95% TFA/H2O and stirred at RT for 2 h. The reaction was stopped by adding DEE (35 mL). The mixture was centrifuged (6000 rpm, 10 min). The solid was dissolved in MeOH (2 mL) and DEE (40 mL) was added and centrifuged. This step was repeated once more. The solid was suspended in DEE (30 mL) and centrifuged. Repeated once more. The solid was then dried in the vacuum oven to yield the fully deprotected (NH2-LRGG)2Rhodamine as a light orange solid (70 mg, quant.) Example 3c. Synthesis of (NH2-RLRGG)2Rhodamine
[0392] Fmoc-Arg(pbf)-OH (1.58 g, 2.45 mmol) was dissolved in dry DMF/Py = 1:1 (15 mL) and cooled in an ice/water bath (~4 °C) while stirring under N2. To this solution, EDC (563 mg, 2.93 mmol) was added and stirred for 5 min. (NH2-LR(pbf)GG)2Rhodamine (400 mg, 0.245 mmol) was dissolved in DMF/Py (5 mL) and added to the reaction mixture. Stirred at the same temperature for 2 hours and the stirred at RT for 1 hour. The reaction was stopped by adding DEE (160 mL) and the mixture was centrifuged (6000 rpm, 10 min). The supernatant was discarded and the solid was dissolved in DCM (10 mL), precipitated by adding DEE (80 mL) and centrifuged again. This step was repeated two more times. The solid was then dissolved in DCM (200 mL) and washed with water (200 mL). The aqueous layer was extracted with more DCM (200 mL). The organic layer was washed with water (100 mL x 1) and brine (100 mL).
The organic layer was dried over Na2S04 and the solvent was evaporated to obtain a off-white color solid. The product (Fmoc-R(pbf)LR(pbf)GG)2Rhodamine was then purified through Combiflash™ (0.4 g).
[0393] (Fmoc-R(pbf)LR(pbf)GG)2Rhodamine (350 mg) was dissolved in DCM (10 mL) and piperidine (10 mL) was added. Stirred at RT for 10 min. Much of the solvent was evaporated and to the rest, DEE (80 mL) was added. The mixture was centrifuged (6000 rpm, 10 min) and the solid was suspended in MeOH (5 mL) and more DEE (40 mL) was added and centrifuged again. This step was repeated twice more. To the solid was added DEE (40 mL) and centrifuged again. This process was repeated once more, and the final solid was dried in vacuum oven to yield (NH2-R(pbf)LR(pbf)GG)2Rhodamine as an off-white solid (243 mg).
[0394] (NH2-R(pbf)LR(pbf)GG)2Rhodamine (230 mg) was dissolved in 2 ml of 95% TFA/H2O and stirred at RT for 4 hours. The reaction was stopped by adding DEE (40 mL). The mixture was centrifuged (6000 rpm, 10 min). The solid was dissolved in MeOH (5 mL) and DEE (40 mL) was added and centrifuged. This step was repeated once more. The solid was suspended in DEE (40 mL) and centrifuged. This process was repeated once more. The solid was then dried in the vacuum oven to yield the fully deprotected (NH2-RLRGG)2Rhodamine as a light orange solid (116 mg). Example 4. Clinical Samples tested using RLRGG-AMC (PLpro substrate) on a multi-well plate reader
[0395] All spectroscopic evaluations for this example were performed on a Biotek Synergy Neo2 Multi-mode Microplate Reader using a 96-well Falcon black plate with a clear bottom . The peptide-chromophore conjugate substrate Z-RLRGG-AMC was purchased from BA Chem (Switzerland). A stock solution of the substrate was prepared from a 10% DMSO solution of the chromophore and a mixture of 50 mM HEPES, pH 7.5, 0.1 mg/ml bovine serum albumin [BSA], 150 mM NaCl, and 2.5 mM DTT and 0.5% Triton X-100 to give a concentration of 5 mg/ml of substrate solution.
[0396] Clinical samples that had been previously tested for the presence of virus by polymerase chain reaction test (PCR) were treated with a transport medium comprising 50 mM HEPES, pH 7.5, 0.1 mg/ml bovine serum albumin [BSA], 150 mM NaCl, and 2.5 mM DTT and 0.5% Triton X-100 prior to shipment to the laboratory for analysis. Shipment occurred over the course of about 24 hours. Upon receipt, the clinical samples were immediately tested using RLRGG-AMC. Cells were each loaded with 90 microliters of sample and 10 microliters of stock solution of RLRGG-AMC (as described above). The each well on the plate can be read on the Synergy Neo 2 microplate reader with a 395nm excitation and 440nm emission any of using three scan modes:
• Kinetics Scan: readings at 1 -minute intervals over a 1-hour time period to obtain a kinetic plot;
• Spectral Scan: a spectral reading after 1 hour to confirm the presence of the product of the substrate/enzyme reaction; and
• Endpoint Scan: a single point reading of fluorescence intensity after 60minutes.
[0397] The conditions used for fluorescence evaluation provided information about the amount of free AMC that had been released for the RLRGG peptide fragment. Each plate that was read included both internal and external control samples. Internal controls included:
[0398] The substrate alone with no enzyme added (“Negative Control”).
[0399] The equivalent amount of the substrate with 100 nM of PLpro enzyme (“Positive Control”). [0400] External controls included:
[0401] A known positive (by PCR) clinical sample with equivalent amount of substrate added. [0402] A known negative (by PCR) clinical sample with the same amount of substrate added.
Example 4a. Kinetic scans of controls and clinical samples.
[0403] Representative results of kinetic scans are shown in Fig. 5 and Fig. 6a-d. In the kinetics scan of negative samples and controls the fluorescence intensity did not change over time, while the positive control and COVID-19 positive samples showed a steady increase in fluorescence emission intensity over time. It could be demonstrated that the slope of the kinetic curves was a function of the enzyme concentration (not shown). All samples with kinetics curves with a positive slope are therefore considered to be “positive” for the presence of the virus-encoded enzyme, while those that are flat are interpreted as “negative” for the enzyme.
[0404] In particular, the kinetic graphs of each test reveal strong discrimination of samples showing a flat line versus line with positive slope after one hour of reaction of substrate with each sample. All flat line samples are either controls or patient samples that tested negative by PCR. The curves with steep positive slope are positive signals from either positive controls or PCR-positive samples while the curves with a gentle (but positive) slope still exhibit significant discrimination relative to control. The positive signal read shows a minimum signal-to-noise ratio of 10 and slope error of about 10%.
Example 4b. End point scan of control and clinical samples.
[0405] End Point Scans were obtained by preparing samples for analysis as before and incubating the samples containing the biosensors for 1 hour before reading the end point fluorescence. Results are shown in Fig. 7 and in Table 7. Fluorescence from positive samples show a significant difference from that of negative controls, discrimination of positive and negative assignments for samples of unknown PCR results.
[0406] Table 5 shows calculated test sensitivity and specificity fortesting of clinical samples using the NFb-LRGG-AMC substrate. Sensitivity is calculated as 1-(FN/TP). Specificity is calculated as TN/(TN+FP) Table 7. Preliminary sensitivity and specificity forNP (48 samples) and Tongue Scrapings (43 Samples)
Figure imgf000123_0001
TP: true positive; FP: False Positive; TN: True Negative; FN: False Negative;T: True; F: False; PPV: Positive Predictive Value; NPV: Negative Predictive Value; NP: Nasopharyngeal; ETT: Endotracheal Tube; PCR: Polymerase Chain Reaction
[0407] Our data is revealing tongue swabs to be better samples for viral load. For tongue we have a sensitivity of 95% and specificity of 42%. Specificity is biased low because of very small number of control negative samples. Having close to 100 controlled negative samples will bring specificity to about 90%. Hence the false positives are not likely true positives. This was confirmed using an ultrasensitive PCR tool called droplet digital PCR or ddPCR. All 17 “false” positives on the REaD test, tested positive for SARS-CoV-2 using ddPCR.
Example 4c. Clinical samples treated with (NH2-LRGG)2Rhodamine substrate
[0408] Samples were prepared as described above, replacing the NH2-RLRGG-AMC substrate in some cases with the (NH2-LRGG)2Rhodamine substrate produced in Example 2b. A concentration of 1 mg/ml of (NH2-LRGG)2Rhodamine was used in 100 microliters of total solution per well. Theses samples were read using an excitation of 498 nm ± 5nm with emission at 535nm ± 10 nm. Much like the NH2-RLRGG-AMC, a steady increase in fluorescence intensity signal was seen in the positive control (PLpro enzyme) and PCR confirmed SARS- CoV-2 positive samples. No change (within error) was seen in the no enzyme (negative) control and the negative clinical sample. A visible color change was observable in room light in the positive samples and controls (Fig. 8a). The visible fluorescence signal from released Rhodamine was observed using flashlight emitting at 395 nm (Fig. 8b). Example 4d. Clinical samples treated with 3CLpro FRET substrates
[0409] A FRET -based substrate was evaluated against SARS-CoV-2 positive and negative samples. This substrate, DABCYL-KVRLQSK-DANSYL, contains the FRET donor and quencher separated by the VRLQS sequence recognized by 3CLpro from SARS-CoV-1 and SARS-CoV-2.
[0410] In this substrate, DANSYL is the absorber and latent fluorophore (“donor”), and DABCYL is the fluorescence quencher (“acceptor”). In the intact sequence, fluorescence from the DANSYL is quenched by the DABCYL moiety. Upon cleavage of the Q-S bond by the 3CLpro in the positive clinical samples, the donor and acceptor can separate by much more than the Forster distance, revealing the fluorescence of the donor to be captured by the plate reader or observed visually.
[0411] Samples were prepared as described previously, substituting a FRET substrate for the NH2-RLRGG-AMC substrate in some cases. Samples were incubated for 1 hour before end point observation for spectra using the plate reader (Fig. 9a), followed by observation of visible fluorescence (Fig. 9b).
Example 4e. Inhibition of fluorescence generation by PLpro inhibitor
[0412] Clinical samples and samples containing authenic SAR.S-CoV-2 PLpro enzyme were tested with the NFh-LRGG-AMC substrate in the presence of various concentrations of a known inhibitor of this enzyme, shown below. This small molecule is a potent inhibitor of PLpro form SARS-CoV-1 with an IC50 of 2.6mM. It inhibits PLpro from SARS-CoV-2 with an IC50 of 5mM and an EC50 of 21 mM.
Figure imgf000124_0001
PL pro inhibitor Cftefrtsal Stroeitjre CAS bio ' 10&3C7G-14-4 [0413] The PLpro inhibitor showed partial inhibition of purified enzyme activity at a 50 mM concentration as shown in Fig. 10a. A concentration-dependent inhibition of PLpro reaction with RLRGG-AMC was observed and is shown in Fig. 10b. Almost complete inhibition was observed using an inhibitor concentration of 0.75mM.
[0414] A similar trend was observed using a clinical sample that was positive for COVID-19, as shown in Fig. 10c. Much higher concentrations of the inhibitor were required to inhibit enzyme activity in the clinical samples as compared to the purified PLpro enzyme.
Example 5. Normalization of fluorescence signal intensity.
[0415] Samples were collected using several different collection methods, with tongues being scraped either once or twice. Samples were prepared and analyzed as in Example 3. To normalize the fluorescence intensity from the samples, Oϋόoo was measured and the relative fluorescence units was divided by the Oϋόoo. Raw data and results of the normalization are shown below. For each collection instrument and the site on the tongue that was sampled, the normalized RFU numbers were in a similar range. For example, with the plastic knife, when the tongue was sampled either once or twice, the normalized RFUs were in the range of 15,000 to 16,000 RFU per unit of Oϋόoo. A range of absorbances for linear correlation for absorbance versus cell number can be established and used for normalization of fluorescence intensity.
Table 8. Normalized fluorescence analysis of tongue scrape samples.
Figure imgf000125_0001
Figure imgf000126_0001
Example 6. Comparison of nasopharyngeal RT-PCR with tongue scrape droplet digital PCR (ddPCR) and fluorescent biosensor detection of SARS-CoV-2 in clinical samples.
[0416] Six clinical samples, three of which tested positive and three that tested negative on PCR were tested using ddPCR, which is more sensitive that PCR. All six samples had tested positive using RLRGG-AMC as the substrate on the BioTek Neo2 plate reader using the rapid enzyme activity detection test. Copies of viral N1 and N2 gene were tested for using the Zymo SARS- CoV-2 detection kit on a droplet digital PCR (ddPCR). Samples were tested as is as well as after centrifugation and pelleting of cells. Viral load was quantified in the supernatant and in the pellets of the cells after they were lysed (intracellular load).
[0417] The following steps were used for quantification. First, the viral RNA was extracted from entire samples (supernatant or lysed cells from the pellets) using the Zymo Quick-RNA Viral Kit #R1035. Next, the RNA quantity was measured using Invitrogen Qubit High-Sensitivity RNA assay #Q32852. RNA was then converted to complementary DNA (cDNA) using Invitrogen Superscript IV reverse transcriptase (SSIV) #18090010 and oligoDt primer and random hexamers. Single digital droplet PCR (ddPCR) was performed on each sample using IDT CDC RUO assay for viral genes N1 and N2 #10006713. Results from ddPCR are below. The numbers represent the calculated total copy number of each gene, N1 and N2. Results in relative fluorescence units (RFUs) seen on the plate reader from the rapid enzyme activity detection test (Now Aware Rapid Test) are also shown for comparison.
[0418] While the concepts of the present technology have been particularly shown and described above with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art, that various changes in form and detail can be made without departing from the spirit and scope of the concepts described herein. It is to be understood that features from any one embodiment described herein may be combined with features of any other embodiment described herein to form another embodiment of the invention.
Table 9. Comparison of nasopharyngeal RT-PCR with tongue scrape droplet digital PCR (ddPCR) and fluorescent biosensor detection.
Figure imgf000127_0001
ddPCR digital droplet PCR, NP Nasopharyngeal, RFU relative fluorescence units

Claims

We Claim:
1. A biosensor for the detection of a coronavirus having Formula (1) D-FG, wherein FG comprises a material responsive to an enzyme encoded by the coronavirus; D is a spectroscopic probe; wherein FG is conjugated to the spectroscopic probe D via a covalent bond; wherein FG masks the activity of the spectroscopic probe D; wherein the enzyme causes the cleavage of the FG to release the spectroscopic probe D, resulting in a detectable optical response; and wherein the enzyme is present in a virus-infected host cell and is released outside for access by the biosensor when the host cell is ruptured.
2. The biosensor of Claim 1, wherein the spectroscopic probe D is selected from the group consisting of a FRET donor/acceptor pair, coumarins, phenothiazines, phenoxazines, fluoresceins, rhodols, and rhodamines.
3. The biosensor of Claim 1, wherein the covalent bond is selected from the group consisting of -C0-0-, -CO-NH-, -SO2-O-, -SO2-NH-, -SO-O-, and -SO-NH-.
4. The biosensor of Claim 1, wherein the spectroscopic probe D comprises a chromophore or a fluorophore as a FRET donor/acceptor pair, wherein the fluorophore is selected from the group consisting of coumarins, fluoresceins, rhodols, and rhodamines, and the chromophore is selected from the group consisting of 4-aminobenzoyl, tryptophan, coumarins, and fluoresceins, and wherein the fluorophore and the chromophore are each independently attached to a different portion of the FG.
5. The biosensor of Claim 1, wherein the spectroscopic probe D comprises a fluorophore and a quencher as a FRET donor/acceptor pair, wherein the fluorophore is selected from the group consisting of coumarins, fluoresceins, rhodols, and rhodamines; and the acceptor is selected from the group consisting of 2,4-dinitrophenyl, para-nitroaniline, 4-nitro-phenylalanine, di methyl ami nophenyl azojbenzoyl, 4-(4-diethylaminophenylazo)-benzenesulfonyl, QSY7, or QSY21, wherein the fluorophore and the acceptor each is attached to a different portion of the FG.
6. The biosensor of Claim 1, wherein the spectroscopic probe D comprising a moiety selected from the group of Formulae (2)-(7), wherein:
Figure imgf000129_0001
Formula
Figure imgf000129_0002
U is O or N;or C
V is -0-C(=0)-0-, -0-, or -NH-C(=0)-0-; W is O, N, S or -CH 2-;
Z is -NR9R10, -O-CFb-Ph;
Y is a bond, N, O, or S;
R1, R2, R3, R4 are each independently selected from the group of H, substituted and unsubstituted Cl -Cl 2 alkyl group, substituted and unsubstituted Cl -Cl 2 alkenyl group, substituted and unsubstituted Cl -Cl 2 alkynyl group, and substituted and unsubstituted aryl group; R5, R6, R7, and R8 is each independently selected from the group of H, Cl, F, Br, CN, NO2, -NR9R10, C1-C6 alkyl group, and C1-C6 alkoxyl group;
R9 and R10 is a substituent each independently selected from the group of H, substituted and unsubstituted Cl -Cl 2 alkyl group, substituted and unsubstituted Cl -Cl 2 alkenyl group, substituted and unsubstituted Cl -Cl 2 alkynyl group, substituted and unsubstituted aryl group, fluoroalkyl, substituted and unsubstituted carbocyclyl, substituted and unsubstituted carbocyclylalkyl, substituted and unsubstituted aralkyl, substituted and unsubstituted heterocycloalkyl, substituted and unsubstituted heterocycloalkylalkyl, substituted and unsubstituted heteroaryl, and substituted and unsubstituted heteroarylalkyl; and
R11, R12, R13, R14, R15, R16, and R17 is each independently selected from the group of H, Cl, F, Br, CN, NO2, -NR9R10, C1-C6 alkyl group, and C1-C6 alkoxyl group.
7. The biosensor of Claim 6, wherein the spectroscopic probe D comprises a moiety represented by Formula
Figure imgf000130_0001
8. The biosensor of Claim 6, wherein the spectroscopic probe D of Formula (5) comprises a
Figure imgf000130_0002
Formula
Figure imgf000131_0002
, Formula (
Figure imgf000131_0001
Formula
Figure imgf000131_0003
9. The biosensor of Claims 1-8, wherein the FG comprises a moiety derived from a chemical inhibitor of the enzyme encoded the coronavirus, or a peptide derived from a substrate of the enzyme encoded by the coronavirus.
10. The biosensor of Claim 4, wherein the FG comprises a peptide derived from a substrate for 3CLpro encoded by the coronavirus, and the fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C-terminus, and the chromophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N- terminus; wherein the peptide comprises at least five amino acid residues and comprises the sequence -LQS-.
11. The biosensor of Claim 10, wherein the FG is a peptide derived from a substrate for a 3CLpro enzyme encoded by a coronavirus, and the fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N-terminus, and the chromophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C-terminus;, wherein the peptide has at least five amino acid residues and a portion of the peptide has the sequence -LQS-.
12. The biosensor of Claim 5, wherein the FG is a peptide derived from a substrate for a 3CLpro enzyme encoded by the coronavirus, and the fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C-terminus, and the quencher is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N-terminus, wherein the peptide comprises at least five amino acid residues and comprises the sequence - LQS-.
13. The biosensor of Claim 5, wherein the FG is a peptide derived from a substrate for a 3CLpro enzyme encoded by the coronavirus, and the fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N-terminus, and the quencher is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C-terminus, wherein the peptide comprises at least five amino acid residues and comprises the sequence - LQS-.
14. The biosensor of Claim 4, wherein the FG is a peptide derived from a substrate for a PLpro enzyme encoded by the coronavirus, and the fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C-terminus, and the chromophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N- terminus; wherein the peptide comprises at least four amino acid residues and comprises the sequence -GG-.
15. The biosensor of Claim 4, wherein the FG is a peptide derived from a substrate for a PLpro enzyme encoded by the coronavirus, and the fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N-terminus, and the chromophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C- terminus; wherein the peptide comprises at least four amino acid residues and comprises the sequence -GG-.
16. The biosensor of claim 5, wherein the FG is a peptide derived from a substrate for a PLpro enzyme encoded by the coronavirus, and the fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N-terminus, and the quencher is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C-terminus; wherein the peptide comprises at least four amino acid residues and comprises the sequence - GG-.
17. The biosensor of claim 5, wherein the FG is a peptide derived from a substrate for a PLpro enzyme encoded by the coronavirus, and the fluorophore is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at C-terminus, and the quencher is bound to the FG via an ester bond, an amide bond, an urea bond, a carbonate bond, or a carbamate bond formed between a reactive functional group at the amino acid residues at N-terminus; wherein the peptide comprises at least five amino acid residues and comprises the sequence - GG-.
18. The biosensor of Claim 6, wherein the FG is a peptide derived from a substrate for a 3CLpro enzyme encoded by the coronavirus, and the peptide comprises at least four amino acid residues and comprises the sequence -LQ- and Q residue is at the peptide terminus, wherein the spectroscopic probe D is bound to the terminal Q residue.
19. The biosensor of Claim 6, wherein the FG is a peptide derived from a substrate for a PLpro enzyme encoded by the coronavirus, and the peptide comprises at least four amino acid residues and comprises the sequence -GG- and G residue is at the peptide terminus, wherein the spectroscopic probe D is bound to the terminal G residue.
20. The biosensor of any one of Claims 10-13, wherein the FG is VRLQS.
21. The biosensor of Claim 18, wherein the FG is VRLQ.
22. The biosensor of any one of Claims 14-17 and 19, wherein the FG is LRGG or RLRGG.
23. The biosensor of any one of Claims 18-19, wherein the biosensor comprises LRGG conjugated to 7-amino-4-methylcoumarin.
24. The biosensor of any one of Claims 18-19, wherein the biosensor comprises LRGG conjugated to Rhodamine 110.
25. The biosensor of any one of Claims 18-19, wherein the biosensor comprises LRGG conjugated to fluorescein.
26. The biosensor of any one of Claims 18-19, wherein the biosensor comprises VRLQ conjugated to 7-amino-4-methylcoumarin.
27. The biosensor of any one of Claims 18-19, wherein the biosensor comprises VRLQ conjugated to Rhodamine 110.
28. The biosensor of any one of Claimsl8-19, wherein the biosensor comprises VRLQ conjugated to fluorescein.
29. A biosensor having Formula (15) A-Y-LSP, wherein A is derived from a peptidic substrate for or a chemical inhibitor of an enzyme encoded by the coronavirus, LSP is a spectroscopic probe chosen from a group consisting of coumarins, rhodamines, rhodols, fluoresceins, xanthenes, phenothiazines, and phenoxazines; Y is a bond, NRa, O, or S; Ra is H or Ci- 6alkyl; wherein LSP is capable of changing color to provide a detectable signal after exposure to the enzyme encoded by the coronavirus.
30. The biosensor of claim 1, wherein the material comprises at least one unnatural amino acid.
31. The biosensor of claim 30, wherein the material is Ac-Abu-Tle-Leu-Gln or Ac-Thz-Tle-Leu- Gln.
32. The biosensor of claim 30, wherein peptide is derived from an unnatural substrate of SARS- CoV-1 3CLpro and SARS-CoV-2 3CLpro given by X1-X2-LG-, wherein Xi is selected from Abu, V, A, Tie, Me, Tba, Aph,and Phg; and X2 is selected from Tie, D-Phe, D-Tyr, Om,
Har, Dab, K, D-Phg, D-Trp, and R.
Figure imgf000134_0001
Figure imgf000135_0001
LSP is selected from the group of a spectroscopic probe having Formula (
Figure imgf000135_0002
Formula
Figure imgf000135_0003
having a cyan colored state, Formula (17)
Figure imgf000135_0005
of Formula (18) Rl 155 , Formula (19) R , Formula (20)
Figure imgf000135_0004
R 25 R 24 3 or Formula (21) R25 R24
Y is a bond, N, O, or S; and U is O or N;
V is -0-C(=0)-0-, -0-, or -NH-C(=0)-0-; W is O, N, S or CH2,
Figure imgf000136_0001
R14, and R15 are each independently selected from the group of H, substituted and unsubstituted C1-C12 alkyl group, substituted and unsubstituted C1-C12 alkenyl group, substituted and unsubstituted C1-C12 alkynyl group, and substituted and unsubstituted aryl group;
R16, R17, R18, R19, R20, R21, and R22 is each independently selected from the group of H, Cl, F, Br, CN, NO2, -NR14R15, C1-C6 alkyl group, and C1-C6 alkoxyl group;
R23, R24, R25, R26 and R27 are each independently selected from the group of H, C1-C2 alkyl group, C1-C2 fluoroalkyl group; and wherein when A is the peptide derived from the substrate of the enzyme encoded by the coronavirus, LSP is chemically linked to A via Y to a reactive functional group on the side chain of an amino acid residue, or to the reactive functional group on any one of the C-terminus or N- terminum amino acid residue of the peptide.
34. The biosensor of Claim 33, wherein Z is
Figure imgf000136_0002
, or -O-CFh-Ph.
35. The biosensor of Claim 33, wherein R14, R15 are each independently methyl or ethyl group.
36. The biosensor of Claim 33, wherein Y is a bond or N, and LSP is selected from the group consisting of Formula (
Figure imgf000136_0004
, Formula (
Figure imgf000136_0003
, Formula (10)
Figure imgf000136_0005
, Formula (12)
Figure imgf000137_0002
O , Formula (22) (Et ,Me) , Formula (23)
Figure imgf000137_0001
37. The biosensor of Claim 33, wherein Y is a bond or N, and A is selected from the group of
Figure imgf000137_0003
Figure imgf000138_0001
38. The biosensor of Claim 33, wherein Y is a bond or N, and A is selected from the group of
Figure imgf000139_0001
39. The biosensor of Claim 33, wherein Y is a bond or N, and A is selected from the group of
Figure imgf000139_0002
Figure imgf000140_0001
Figure imgf000141_0001
41. The biosensor of Claim 33, wherein the biosensor of Formula (15) is defined when Y is a bond or N, and A is selected from the group
Figure imgf000141_0002
Figure imgf000141_0003
42. The biosensor of Claim 33, wherein Y is a bond or N, and A is selected from the group of
Figure imgf000141_0004
Figure imgf000142_0001
43. The biosensor of Claim 33, wherein Y is a bond or N, and A is selected from the group of
Figure imgf000142_0002
44. The biosensor of Claim 33, wherein Y is a bond or N, and A is selected from the group of
Figure imgf000143_0001
45. The biosensor of Claim 33, wherein Y is a bond or N, and A is selected from the group of
Figure imgf000143_0002
Figure imgf000144_0001
46. The biosensor of Claim 33, wherein Y is a bond or N, and A is selected from the group of
Figure imgf000144_0002
Figure imgf000145_0001
47. The biosensor of Claim 33, wherein Y is a bond or N, and LSP is selected from the group of
Figure imgf000145_0002
48. The biosensor of Claim 33, wherein Y is a bond or N, and A is selected from the group of
Figure imgf000146_0001
49. The biosensor of Claim 33, wherein Y is a bond or N, and A is selected from the group of
Figure imgf000146_0003
Figure imgf000146_0002
, , Formula (13)
Figure imgf000147_0001
50. The biosensor of Claim 33, wherein A is a peptide derived from the substrate of coronavirus 3CLpro and A is selected from the group of Leu, Val, Val-Leu, Val-Phe, Ala-Val-Leu, Ser- Val-Phe, Val-Thr-Phe-Gln, Val-Thr-Leu-Gln, Val-Thr-Ala-Gln, Glu-Ser-Ala-Thr-Leu-Gln- Ser-Gly-Leu-Ala-Lys-Ser, Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys, and Thr-Ser- Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met.
51. The biosensor of Claim 33, wherein A comprises Val-Leu, Val-Phe, Ala-Val-Leu, and Ser- Val-Phe.
52. The biosensor of Claim 33, wherein A comprises Glu-Ser-Ala-Thr-Leu-Gln-Ser-Gly-Leu- Ala-Lys-Ser.
53. The biosensor of claim 33, wherein A comprises Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe- Arg-Lys-Met.
54. The biosensor of Claim 33, wherein Y is a bond or N; A is a peptide derived from the substrate of coronavirus 3CLpro and A is selected from the group of ubiquitin, ISG15 protein, Leu, Val, Val-Leu, Val-Phe, Ala-Val-Leu, Ser-Val-Phe, Val-Thr-Phe-Gln, Val-Thr- Leu-Gln, Val-Thr-Ala-Gln, Glu-Ser-Ala-Thr-Leu-Gln-Ser-Gly-Leu-Ala-Lys-Ser, Thr-Ser- Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys, Glu-Arg-Glu-Leu-Asn-Gly-Gly-Ala-Pro-Ile-Lys- Ser, and Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met; and LSP is selected from the
Figure imgf000148_0002
, ,
Figure imgf000148_0003
o , Formula (22) (Et ,Me) , Formula (23)
Figure imgf000148_0001
55. The biosensor of Claim 33, wherein Y is a bond or N, and A is Thr-Ser-Ala-Val-Leu-Gln-
Ser-Gly-Phe-Arg-Lys-Met, and LSP is selected from the group of Formula (
Figure imgf000149_0001
Figure imgf000149_0002
Figure imgf000149_0003
56. A biosensor comprising two fragmentable groups, represented by A-Y-D-Y-B wherein Y is an O, N, or S atom, and A and B are selected from
Figure imgf000150_0001
Figure imgf000150_0002
57. A biosensor of claim 56 wherein A and B are different and are responsive to different enzymes.
58. A biosensor comprising a amino phenol or a phenylenediamine color developer released by a viral protease to an oxidizable form and oxidatively reacted with a color coupler, the color coupler comprising phenols, anilines, beta-acetoacetanilides, or heterocylic couplers.
59. A diagnostic composition comprising at least one biosensor of claim 1 and a carrier.
60. A diagnostic composition comprising at least two biosensors of claim 1 wherein a first biosensor is responsive to PLpro enzyme and a second biosensor is responsive to 3CLpro enzyme.
61. A diagnostic composition of either of claims 59 or 60, wherein the carrier comprises an organic solvent that is compatible with biological samples.
62. A diagnostic composition of either of claims 59 or 60, wherein the carrier comprises a biological buffer.
63. A diagnostic composition of either of claims 59 or 60, wherein the carrier comprises a surfactant.
64. A diagnostic composition of either of claims 59 or 60 additionally comprising an enzyme inhibitor.
65. A diagnostic composition of claim 64, wherein the inhibitor is a broad spectrum deubiquitinase inhibitor.
66. A diagnostic composition of claim 65, wherein the inhibitor is 2,6-Diamino-3,5- dithiocyanopyridine.
67. A diagnostic composition of claim 65, wherein the inhibitor comprises a dihydropyrrole.
68. A diagnostic composition of claim 65, wherein the inhibitor is a tricyclic heterocyclic compound.
69. A diagnostic composition of claim 65, wherein the inhibitor is PX-478.
70. A diagnostic composition of claim 65, wherein the inhibitor comprises 6-amino-pyrimidine.
71. A diagnostic composition of claim 64, wherein the inhibitor is a proteasome inhibitor.
72. A diagnostic composition of claim 71, wherein the inhibitor is MG132 (Cbz-Leu-Leu- Leucinal).
73. A diagnostic composition of claim 71, wherein the inhibitor is b-AP15.
74. A diagnostic composition of claim 71, wherein the inhibitor comprises azepan-4-one.
75. A diagnostic composition of claim 64, wherein the inhibitor is a viral 3C protease inhibitor.
76. A diagnostic composition of claim 75, wherein the inhibitor is a human rhinovirus 3C protease inhibitor.
77. A diagnostic composition of claim 75, wherein the inhibitor is a human enterovirus 3C protease inhibitor.
78. A diagnostic composition of claim 75, wherein the inhibitor is SG85.
79. A diagnostic composition of claim 75, wherein the inhibitor is luteoloside.
80. A diagnostic composition of claim 64, wherein the inhibitor is an inhibitor of ubiquitin C- terminal hydrolase (UCH).
81. A diagnostic composition of claim 80, wherein the inhibitor is an inhibitor of UCH-L1.
82. A diagnostic composition of claim 80, wherein the inhibitor is an inhibitor of UCH-L3.
83. A diagnostic composition of claim 80, wherein the inhibitor is an inhibitor of UCH-L5.
84. A diagnostic composition of claim 80, wherein the inhibitor is 4,5,6,7-tetrachloro-l,3- indanedione (TCID).
85. A diagnostic composition of claim 80, wherein the inhibitor is an acylated oxime isatin derivative.
86. A method for detecting the presence or absence of SARS-CoV-2, SARS-Cov-1, or MERS- Cov in a sample comprising the steps of: (1) obtaining a sample from a patient and placing the sample in a tube, (2) adding the biosensor of any one of Claims 1-46 to the sample in the tube, (3) incubating the biosensor with the sample, (3) observing the absence or presence of an optical response in the sample, wherein the presence of the optical response indicates the presence of viruses, wherein the biochemical factor causes degradation of the material to release the spectroscopic probe, resulting in a detectable optical response.
87. The method of Claim 60, wherein the sample is selected from the group of blood, plasma, serum, feces, bronchoalveolar lavage, nasal swab sample, oral swab sample, a sublingual swab sample, a sample from the paipilla of the parotid duct, NP swab sample, OP swab sample, buccal swab sample, tongue scrape, urine, synovial fluid, an aerosol sample that has been collected in a respiratory mask, a masticated oral sample, mucus, or sputum.
88. The method of Claim 60, wherein the sample is an aerosol sample that has been collected in a respiratory mask.
89. The method of Claim 62, wherein the mask is a N95 respirator mask.
90. The method of claim 60, wherein the sample is a masticated oral sample.
91. The method of Claim 64, wherein the samle is a masticated gum.
92. The method of claim 60, wherein the sample is deactivated with a lysis buffer containing a surfactant.
93. The method of claim 66, wherein the lysis buffer is selected from the group of TRIzol, AVL, RLT, MagMAX, and easyMAG.
94. The method of claim 66, wherein the lysis buffer comprises Triton X-100, Digitonin, or Tween-20.
95. The method of claim 60, wherein the sample is obtained from the patient by a mouth or nasal swab and the swab is incubated into a tube containing a diagnostic composition comprising a biosensor.
96. The method of claim 60, wherein the optical response is a change in optical absorption of the sample.
97. The method of claim 70, wherein the optical response is observed a a visible color change.
98. The method of claim 70, wherein the change in optical absorption is a quantifiable change detected with a spectrophotometer.
99. The method of claim 60, wherein the optical response is a fluorescence emission.
100. The method of claim 72, wherein the fluorescence emission is detected visually.
101. The method of claim 72, wherein the fluorescence emission is detected as a quantifiable signal from a fluorimeter.
102. The method of claim 72 or 75, wherein the quantifiable optical response is normalized for sample turbidity.
103. The method of claim 76, wherein the optical response is normalized by optical density at 600 nm.
104. The method of claim 60, wherein a high-throughput (HTS) measurement of optical properties of clinical samples that have been lysed and reacted with the herein described biosensors and placed into the wells of a multiwell plate is performed with a multi-well plate reader.
105. The method of claim 78, wherein the optical response is normalized for sample turbitity.
106. The method of claim 60, wherein the optical response is generated by illumination with an LED device and observed visually.
107. The method of claim 80, wherein the optical response is a fluorescence response that is generated by illumination in a device comprising an LED and a sample holder, wherein the observation of fluorescence occurs at approximately 90° to the excitation of the sample.
108. The method of claim 60, wherein the optical response is detected with a digital camera.
109. The method of claim 82, wherein the digital camera is a camera on a cell phone.
110. A method for quantifying infection level of a coronavirus in a sample comprising the steps of: (1) collecting a sample; (2) splitting the sample contents into at least two portions; (3) inactivating the first portion by lysis as described above; (4) incubating the lysed first portion with a diagnostic composition as described above; (5) observing the presence of an optical response as described above; (6) incubating a second portion with a sacrificial host medium for at least 24 hours; (6) inactivating the second portion, (7) incubating the lysed second portion with a diagnostic composition; (8) observing an optical response; and (9) comparing the optical response of the first portion with that of the second portion.
111. A system for detecting SARS-CoV pathogen in a sample from a human subject, wherein the system comprises: a computer or computer readable medium, sample container, the biosensors of any one of the claims 1-46, a controller, and a detector coupled to an input to the computer or computer readable medium.
112. The system of Claim 85, wherein the detector is selected from the group of a fluorescence spectroscope, a spectrophotometer, a fluorimeter, a colorimetric spectrophotometer, a photometer, a biological plate reader, or a camera.
113. The system of Claim 85, wherein the computer comprises a software for receiving user instructions in the form of user inputs, wherein the software is configured to convert the user instructions to control the operation of the controller.
114. The system of Claim 85, wherein the computer is configured to receive data from the detector.
115. The system of Claim 85, wherein the computer is a cell phone.
116. The system of Claim 85, wherein the sample container is selected from glass tube, plastic tube, an array of tubes on a rack, or a biological assay plate.
117. The system of Claim 85, wherein the sample from the human subject is selected from the group of blood, plasma, serum, feces, bronchoalveolar lavage, nasal swab sample, oral swab sample, a sublingual swab sample, a sample from the paipilla of the parotid duct, NP swab sample, OP swab sample, buccal swab sample, tongue scrape, urine, synovial fluid, mucus, an aerosol sample collected in a respiratory mask, a masticated oral sample, or sputum.
118. A method for detecting coronavirus in a sample from a human subject, wherein the method comprising the steps:
(a) receiving patient information by a registration process,
(b) obtaining a sample from the human subject, wherein such sample comprises cells infected by the coronavirus;
(c) inactivating the sample from the human subject to release by lysis one or more enzymes encoded by the coronavirus;
(d) exposing the inactivated sample in step (c) to a biosensor of any one of claims 1-46 to produce an optical response;
(e) detecting the optical response generated in step (d) with a detector, wherein the detector is configured to process the optical response into a data output, and the detector is configured to communicate the data output to a computer or computer readable medium; and wherein the computer or computer readable medium is configured to generate a label for the data output received with the patient information received by the registration process in step (a),
(f) saving the data output labeled with the patient information in step (e) on the computer or computer readable medium, and
(g) generating a report to the human subject for the data output labeled with the patient information in step (e).
119. The method of Claim 93, wherein the coronavirus is SARS-CoV-2, SARS-Cov-1, or MERS-Cov-1.
120. The method of Claim 93, wherein the computer or computer readable medium is a cell phone.
121. The method of Claim 93, wherein the sample from the human subject is selected from the group of blood, plasma, serum, feces, bronchoalveolar lavage, nasopharyngeal swab sample, nasal swab sample, oral swab sample, a sublingual swab sample, a sample from the paipilla of the parotid duct, NP swab sample, OP swab sample, buccal swab sample, tongue scrape, urine, synovial fluid, mucus, an aerosol sample collected in a respiratory mask, a masticated oral sample, or sputum.
122. The method of Claim 93, wherein the detector is selected from the group of a fluorescence spectroscope, a spectrophotometer, a fluorimeter, a transilluminator, a black light, a colorimetric spectrophotometer, a photometer, a biological plate reader, or a camera.
123. The method of Claim 93, wherein the detector is a camera embedded within a cell phone.
124. A method for detecting coronavirus in a sample from a human subject, wherein the method comprising the steps:
(a) registering the test by the human subject,
(b) generating a sample by the human subject with a sample collection device, or by providing a sample of sputum,
(c) conditioning the sample from the human subject by lysis with the lysis buffer to inactivate the coronavirus,
(d) exposing the inactivated sample in step (c) to a biosensor described herein to produce an optical response, (e) detecting the optical response generated in step (d) with a detector, wherein the detector is configured to process the optical response into a data output, and the detector is configured to communicate the data output to a computer or computer readable medium,
(f) saving the data output labeled with the patient information in step (e) on the computer or computer readable medium,
(g) processing the data according to the instructions, and
(h) generating a report to the human subject for the data output.
125. A kit for detecting the presence of viruses in a sample of a human subject comprising a biosensor of Claims 1-55, cotton swabs, sample tubes with screw cap, a lysis buffer, and an instruction sheet providing instructions to the human subject to collect the sample into the sample tube and condition the sample with the lysis buffer, register the patient information to a registering system, and a diagnostic assay procedure for conducting the diagnostic test upon exposing the sample with the biosensor.
126. The kit of Claim 101, wherein the lysis buffer comprises a nonionic surfactant, TRIzol, AVL, RLT, MagMAX, or combinations thereof.
127. The kit of Claim 101, wherein the sample is a sputum sample, blood, plasma, serum, feces, bronchoalveolar lavage, a nasal swab sample, an oral swab sample, a sublingual swab sample, a sample from the paipilla of the parotid duct, a nasopharyngeal swab sample, an oropharyngeal swab sample, a buccal swab sample, a tongue scrape, mucus, an aerosol sample collected in a respiratory mask, or a masticated oral sample.
128. A tongue scraper having a shape of an L-beam, wherein the edges of the L-beam scrape the tongue laterally to collect debris from the surface of the tongue.
129. The diagnostic composition of claim 59 further comprising a lysis buffer.
130. The diagnostic composition of claim 130, wherein the lysis buffer comprises a nonionic surfactant, TRIzol, AVL, RLT, MagMAX, or combinations thereof.
131. The method of claim 86, wherein incubating the biosensor with the sample comprises adding a lysis buffer to the sample during incubation so as to enable or facilitate release of the enzyme from the virus-infected host cell.
132. The biosensor of claim 1, wherein FG comprises a peptide having at least 4 amino acid residues.
133. The biosensor of claim 1, wherein FG comprises a peptide having 4-6 amino acid residues.
134. The biosensor of claim 1, wherein FG comprises a peptide having 6-8 amino acid residues.
135. The biosensor of claim 1, wherein FG comprises a peptide having 8-10 amino acid residues.
136. The biosensor of claim 1, wherein FG comprises a peptide having 10-12 amino acid residues.
137. The biosensor of claim 1, wherein FG comprises a peptide having 12-14 amino acid residues.
138. The biosensor of claim 1, wherein FG comprises a peptide having 14-16 amino acid residues.
139. A biosensor for detection of pathogens having Formula (1) D-FG, wherein
FG comprises at least one unnatural amino acid residue and is responsive to a target enzyme encoded by the pathogen,
D is a spectroscopic probe conjugated to FG by a covalent bond that is cleaveable by target enzyme so as to release D from FG,
FG, while bonded to D, masks activity of D and release of D from FG results in a detectable optical response, wherein the target enzyme is released outside a pathogen-infected host cell when the host cell is ruptured.
140. The biosensor of claim 139, wherein the at least one unnatural amino acid residue is relatively closer to the N-terminus of FG.
141. The biosensor of claim 139, wherein the at least one unnatural amino acid reside is selected from the group consisting of the unnatural amino acid residues listed in Table 2.
142. The biosensor of claim 139, wherein the pathogen is a coronavirus.
143. The biosensor of claim 139, wherein D comprises a chromophore or a fluorophore as a FRET donor/acceptor pair, wherein the fluorophore is selected from the group consisting of coumarins, fluoresceins, rhodols, and rhodamines, and the chromophore is selected from the group consisting of 4-aminobenzoyl, tryptophan, coumarins, and fluoresceins, and wherein the fluorophore and the chromophore are each independently attached to a different portion of the FG.
144. The biosensor of claim 139, wherein FG is an internally fluorogenic peptide.
145. The biosensor of claim 139, wherein FG is a simple fluorogenic peptide.
146. The biosensor of claim 139, wherein FG is selected from the peptides listed in Table 5.
147. The biosensor of claim 139, wherein FG comprises a peptide derived from a substrate for a PLpro enzyme encoded by SARS-CoV-1, SARS-CoV-2, or MERS-CoV.
148. The biosensor of claim 139, wherein FG comprises a peptide derived from a substrate for a 3CLpro enzyme encoded by SARS-CoV-1, SARS-CoV-2, or MERS-CoV.
149. The biosensor of claim 139, wherein FG comprises a peptide listed in Table 5.
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