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WO2025022389A1 - Détection spécifique et sans étiquette de petites molécules cibles à l'aide de transistors biologiques - Google Patents

Détection spécifique et sans étiquette de petites molécules cibles à l'aide de transistors biologiques Download PDF

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Publication number
WO2025022389A1
WO2025022389A1 PCT/IL2024/050724 IL2024050724W WO2025022389A1 WO 2025022389 A1 WO2025022389 A1 WO 2025022389A1 IL 2024050724 W IL2024050724 W IL 2024050724W WO 2025022389 A1 WO2025022389 A1 WO 2025022389A1
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bio
sample
transistor
molecule
affinity
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Gil Shalev
Rakefet SAMUELI
Shubham BABBAR
Offer Erez
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Mor Research Applications Ltd
BG Negev Technologies and Applications Ltd
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Mor Research Applications Ltd
BG Negev Technologies and Applications Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/403—Cells and electrode assemblies
    • G01N27/414—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
    • G01N27/4145—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS specially adapted for biomolecules, e.g. gate electrode with immobilised receptors

Definitions

  • the present disclosure relates to bio-transistors for sensing target molecules. More specifically, the present disclosure provides systems and methods for determining the presence and/or quantity of molecules, specifically of small molecules, particularly, chemical warfare agents or steroid hormones.
  • Bhattacharyya I. M. et al. A new approach towards the Debye length challenge for specific and label-free biological sensing based on field-effect transistors. Nanoscale 14, 2837-2847 (2022). 8. Bhattacharyya, I. M. el al. A New Approach toward the Realization of Specific and Label- Free Biological Sensing Based on Field-Effect Devices. Adv Electron Mater 8, (2022).
  • Transistor-based sensing has been suggested for specific, label-free, real-time, low cost and quantitative biological and chemical sensing from ultra small samples with high selectivity and sensitivity [1].
  • FET-based biological sensing is based on surface-bound recognition layer composed of receptor molecules with high affinity, selectivity and specificity toward the biomolecular target.
  • FET-based chemical sensing is mostly realized with a surface-bound semipermeable membrane to provide the specificity toward ions other than H + .
  • FET-based sensing has important implications and application in various fields ranging from medical diagnostics, homeland security, food industry, agriculture, and others.
  • This biosensor includes a semiconductor active region; a sensing region configured to contact a fluid; and multiple electrodes that comprise decoupling electrodes and additional electrodes.
  • the decoupling electrodes may be configured, wherein operating in a first mode, to prevent a formation of a top conductive channel within the semiconductor active region; and the additional electrodes are configured, wherein operating in the first mode, to independently control (i) one or more properties of one or more other conductive channels formed within the semiconductor active region, and (ii) a Debye length at an interface between the sensing region and the fluid [2].
  • SPR Surface plasmon resonance
  • ELISA enzyme-linked immunosorbent assay
  • BioFETs Chemiresistors and Field-effect biosensors (bioFETs), of various novel nanostructures and materials, have been suggested for specific and label-free sensing of small molecules [Nakatsuka et al. Science (1979) 362, 319-324 (2016)].
  • bioFETs Field-effect biosensors
  • the competitive format ‘displacement mode’) is applied in many cases to overcome the challenge of detecting the changes induced by binding small, sometimes neutral, molecules to the binding sited on the antibody surface [3].
  • bioFETs Field-effect biosensors
  • bioFETs are promising candidates for specific, label-free, and real-time sensing, and specifically for POC and home-care medical diagnostics.
  • the underlying bioFET sensing mechanism is charge introduction and/or charge redistribution, at the sensing area due to the capture of target molecules by the surface-bound receptor molecules.
  • bioFETs based on silicon nanowires were reported for label-free sensing of small molecules.
  • McAlpine et al reported sensing of small molecules via the conformational modification and charge redistribution of the receptor molecule [4] introduced by the small molecule.
  • Different receptor molecules have been employed in these works ranging from chemical receptors, peptides/large proteins, and DNA strands.
  • Organophosphate (OP) sensing stems from the pressing need to address environmental, health, and security challenges. OPs are extensively used in agriculture to control pests and enhance crop yields. The annual use of organophosphorus pesticides, reaching several hundred thousand tons, and their low decomposition rates lead to accumulation in the environment, rivers, groundwater, and food sources, posing risks to human and animal health [Dhamu, et. al. ECS Sensors Plus 2, (2023)]. Moreover, hazardous phased-out and prohibited organophosphorus pesticides can enter the environment through damaged packaging during prolonged storage [Andrianova et al. Electroanalysis 28, (2016)]. Furthermore, OPs are employed in chemical warfare agents, posing significant threats to national security and public safety [Kumar, D. et al.
  • the nerve agent Sarin exemplifies the lethal nature of organophosphorus compounds, and has been employed by terrorists, underscoring the urgent need for inexpensive portable sensors capable of real-time detection and quantification of OP residues with high sensitivity and specificity.
  • OPs are measured using various analytical techniques, including chromatography, spectroscopy, and immunoassays.
  • Chromatographic methods such as gas chromatography (GC) and high-performance liquid chromatography (HPLC), offer high sensitivity and selectivity but often require extensive sample preparation and specialized equipment [Kumaran, A. et al. Microchemical Journal 178, 107420 (2022)].
  • Analytical techniques like mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy, provide detailed structural information but are less suitable for rapid on-site analysis [Kumar, P., et al. Biosens Bioelectron 70, (2015)].
  • Immunoassays such as enzyme-linked immunosorbent assays (ELISA) and lateral flow assays (LFAs) offer rapid and cost-effective screening but may lack specificity and sensitivity. Additionally, fluorescent-based detection methods have emerged as promising alternatives for OP sensing.
  • ELISA enzyme-linked immunosorbent assays
  • LFAs lateral flow assays
  • nanomaterials such as graphene and carbon nanotubes (CNT) improves sensor performance by increasing surface area and facilitating charge transport [5].
  • CNT carbon nanotubes
  • SW single wall
  • HFIPP Hexafluoroisopropanolphenyl- 1 -pyrenebutyramide
  • DMMP dimethyl methylphosphonate
  • SWNT-FET sensors with p-hexafluoroisopropanol phenyl (HFIPPH) as the active material, enhancing sensitivity and selectivity for DMMP detection through efficient accumulation of DMMP vapors via hydrogen bonding [5].
  • HFIPPH p-hexafluoroisopropanol phenyl
  • MNChem The Meta-Nano-Channel Chemically-modified FET (MNChem) is a derivation of the recently introduced MNC bioFET reported for real-time, specific, and label-free sensing of biological interactions and activities [7, 8].
  • Estriol is a small neutral chemical molecule and is one of three markers measured during mid-gestation for the Down syndrome risk calculation and screening. Estriol secretion is increased by 1000 times during pregnancy due to placental synthesis reaching at 37 weeks onwards a concentration of 80-130 pg/ml [Mesiano et al. 256-284 (Elsevier, 2019)]. The increase in plasma estriol concentration is much higher than that of estradiol or estrone which increase only 100 times more during pregnancy [Blackburn et al. Elsevier Health Sciences, 2014].
  • the concentrations of free estriol in maternal plasma during pregnancy are considered as a marker for placental health, and low concentrations of multiple of median (MoM) are related to chromosomal abnormalities such as trisomy 21 and trisomy 18, fetal anomalies [Haddow et al. The New England Journal of Medicine. 327, 588-593 (1992)] and placental disorders such as preeclampsia [Shu, C. et al. Drug Des Devel Ther. 15, 2543-2550 (2021)]. Following the CO VID pandemic, there is an increased demand for POC testing, that will allow the patients to perform diagnostic testing at their home, rather than in the clinics or laboratory centres.
  • Estriol is a member of the estrogen family.
  • Estrogens are structurally related hydrophobic molecules, sharing a similar ring sterol backbone that is decorated with varying hydroxyl and ketone groups. Specifically, estrone (El) has a single hydroxyl and a single ketone group, while other estrogens display only hydroxyl groups, two in estradiol (E2), three in estriol (E3), and four in estetrol (E4). Compared with small molecules, antibodies have a wider selectivity and by large present superior specificity [C. Rader et al. Trends Biotechnol. 2014, 32, 186]. Typically, flat surfaces accommodate the interaction with large protein regions, grooves enable peptide binding, and deep pockets support interactions with small molecules and haptens [R. M.
  • the first aspect of the present disclosure relates to a bio-transistor system comprising at least one transistor unit and a control unit.
  • the transistor unit comprises (i) at least one channel, (ii) source and drain electrodes, (iii) at least one gate electrode and (iv) at least one active region located in proximity to the channel region and carrying at least one affinity moiety. Each affinity moiety is specific for a target molecule.
  • the at least one active region is configured for accepting at least one sample.
  • the transistor unit also comprises (v) at least one additional electrode positioned to be in electrical contact with the sample.
  • the control system comprises at least one processor and memory circuitry. The control system is configured and operable for performing one or more measurements of the sample.
  • Each measurement comprises maintaining a selected electric potential on the at least one additional electrode and determining current transmission profile through the at least one channel with respect to potential variation of the at least one gate electrode.
  • the control unit thereby configured to determine data on the presence and/or quantity of one or more target molecules in the sample.
  • the affinity moieties of the disclosed bio-transistor are protein receptors that recognize and bind a small molecule target.
  • Another aspect of the present disclosure relates to a battery comprising two or more of the bio-transistor system as defined above.
  • Another aspect of the present disclosure related to a method for determining presence and/or quantity of at least one target molecule in at least one sample. The method comprising: (a) contacting the at least one sample with a bio-transistor having an active region carrying at least one affinity moiety, or a battery comprising at least two of the biotransistors.
  • each affinity moiety is specific for a target molecule; (b) performing one or more measurements, each measurement comprising: applying a selected electric potential on the sample, and determining current transmission profile through a channel of the bio-transistor with respect to potential variation of at least one gate electrode of the bio-transistor; and (c) processing data on the current transmission through the channel for one or more selected gate potential and one or more selected electric potential values applied on the sample and determining presence and/or quantity of the one or more target molecules in accordance with pre-stored calibration data.
  • Another aspect of the current disclosure is a diagnostic method for determining a physiological and/or environmental condition or state of a subject and/or a media and/or a habitat.
  • the method comprising: (a) contacting the at least one sample with a bio-transistor having an active region carrying at least one affinity moiety, or a battery comprising at least two of said bio-transistors.
  • Each affinity moiety is specific for a target molecule; (b) performing one or more measurements, each measurement comprising: applying a selected electric potential on the sample, and determining current transmission profile through a channel of the bio-transistor with respect to potential variation of at least one gate electrode of the bio-transistor; (c) processing data on the current transmission through the channel for one or more selected gate potential and one or more selected electric potential values applied on the sample; (d) determining presence and/or quantity of the one or more target molecules in accordance with pre-stored calibration data, thereby obtaining a target molecule value for the sample; and (e) determining that the subject and/or media and/or habitat display the physiological and/or environmental condition or state, if the at least one target molecule value obtained for the sample in step (d), is positive with respect to a reference target molecule value pre-determined for the physiological and/or environmental condition or state, or with respect to a target molecule value determined for at least one control sample.
  • Another aspect of the present disclosure is a screening method for identifying a compound that modulates the interaction of an affinity moiety with a target molecule in at least one sample.
  • the method comprising: (i) contacting the at least one sample with a bio-transistor system, in the presence and the absence of at least one candidate compound.
  • the biotransistor having an active region carrying at least one affinity moiety, each affinity moiety is specific for a target molecule; (ii) performing one or more measurements for each sample, each measurement comprising: applying a selected electric potential on the sample, and determining current transmission profile through a channel of the bio-transistor with respect to potential variation of at least one gate electrode of the bio-transistor; and (iii) processing data on the current transmission through the channel for one or more selected gate potential and one or more selected electric potential values applied on the sample; and (iv) determining presence and/or quantity of the one or more target molecules in accordance with pre-stored calibration data, thereby determining a target molecule value for the sample in the presence of the candidate compound, and a target molecule value in the absence of the candidate compound; (v) determining that the candidate compound is a modulator of the interaction between the affinity moiety and the target molecule, if the target molecule value obtained for the sample in the presence of the candidate compound is different from the target molecule value obtained in the absence of
  • the diagnostic kit comprising: (a) at least one bio-transistor system, and optionally, at least on of: (b) at least one control sample; and (c) at least one therapeutic agent.
  • the bio-transistor system comprises at least one transistor unit and a control system.
  • the transistor unit comprising: (i) at least one channel; (ii) source and drain electrodes; (iii) at least one gate electrode; (iv) at least one active region located in proximity to the channel region and carrying at least one affinity moiety, each affinity moiety is specific for a target molecule.
  • the at least one active region is configured for accepting at least one sample; and (v) at least one additional electrode positioned to be in electrical contact with the sample.
  • the control system comprising at least one processor and memory circuitry.
  • the control system is configured and operable for performing one or more measurements of the sample, wherein each measurement comprises maintaining a selected electric potential on the at least one additional electrode and determining current transmission profile through the at least one channel with respect to potential variation of said the least one gate electrode.
  • the control unit thereby configured to determine on the presence and/or quantity of one or more target molecules in the sample.
  • Figure 1 schematically exemplifies a bio-transistor system according to some embodiments of the present disclosure.
  • Figure 2 schematically exemplifies a bio-transistor system formed from a plurality of transistor units according to some embodiments of the present disclosure.
  • Figure 3 exemplifies method actions for determining presence and/or quantity of one or more target molecules according to some embodiments of the present disclosure.
  • FIG. 4A-4B IDS-VGL curves for selected VGF values performed for MNC biosensor biofunctionalized with HFIP receptor molecules (for PBS solution)
  • Fig. 4A Illustrations of the MNChem, an optical image of the MNChem chip with electrical connections, and the employed surface functionalization strategy.
  • Fig. 4B IDS-VGL for selected VGF values and aminophenyl-HFIP-MNChem. The measurements are performed in 0.5 pL drops of 0.1 mM PBS. The data points reflect an average of 30 measurements, and the error bars are the respective standard deviations (see inset). The corresponding second derivatives are presented. The excitations of the various channels are indicated.
  • Figure 5A-5B IDS-VGL curves for selected VGF values performed for MNC biosensor biofunctionalized with HFIP receptor molecules (for PBS solution spiked with DCNP)
  • Fig. 5B Control non-specific measurements for: 1 ) DCNP introduction to an unmodified MNChem, 2) DCNP introduction to APTMS-modified MNChem, 3) Methanol (MeOH) introduction to aminophenyl-HFIP-MNChem, and 4) tryptophan introduction to aminophenyl-HFIP-MNChem.
  • the selected target concentrations are provided in the legend. The data points and the error bars follow the convention in Fig. 5A.
  • the figure presents Table 1 that shows a summary for the sensing performance of the proposed aminophenyl-HFIP-MNChem towards DCNP, specifically, sensing of a small molecule using a receptor molecule.
  • FIGS. 8A-8B IDS-VGL curves for selected VGF values performed for MNC biosensor biofunctionalized with anti-estriol antibodies (for diluted plasma)
  • Fig. 8A An optical image of the MNC biochip, a 3D illustration of the MNC bioFET, and a cross-section of the MNC bioFET midway between source and drain.
  • Fig. 8B IDS-VGL curves for selected VGF values performed with 0.5 pF drops of 1: 100 diluted plasma.
  • the MNC biosensor is unmodified. Each data point is an average of 51 measurements (17 drops, each drop is measured 3 times), and the error bars are the corresponding standard deviations (see inset).
  • Figure 9A-9D IDS-VGL curves for selected VGF values performed for unmodified MNC BioFET and MNC BioFET biofunctionalized with anti-estriol antibodies
  • Fig. 9A An illustration showing the process of Si/SiCh surface biofunctionalization.
  • Fig. 9B Contact angle and ellipsometry measurements of Si/SiCh samples post various modification steps.
  • Fig. 9C EIS measurements for the various modification steps.
  • Fig. 9D IDS-VGL curves for various VGF values for unmodified MNC BioFET and MNC BioFET biofunctionalized with anti-estriol antibodies.
  • the data points and the error bars reflect the averages and standard deviations of 16 measurements (4 drops each measured 4 times).
  • FIGS. 10A-10E IDS-VGL curves for selected VGF values performed for MNC BioFET biofunctionalized with anti-estriol antibodies
  • Fig. 10A IDS-VGL for selected VGF values for MNC bioFET modified with anti-estriol antibodies. The measurements are performed in 0.5 pL drops of 1: 100 diluted plasma. The data points reflect an average of 51 measurements (Total of 17 drops each measured 3 times), and the error bars are the respective standard deviations (see inset).
  • Fig. 10C Left: IDS-VGL for VGI - 0.5 V for 0.5 pL drops of 1:100 diluted plasma spiked with estriol concentration from 1 fg/ml - 10 pg/ml.
  • the sensing area of the MNC bioFET is biofunctionalized with anti-estriol antibodies.
  • the IDS-VGL curves are presented on both linear and logarithmic y-axis scales. Each data point is the average of 4 measurements performed for each drop, and the error bars are the corresponding standard deviations (see inset).
  • the channel configuration is also indicated.
  • Fig. 11A iNormaiized curves for estriol introduced to an unmodified bioFET.
  • Fig. 11B INormaiized curves for estriol introduced to a MNC bioFET modified with APTMS.
  • Fig. 11C Estrone molecules introduced to MNC bioFET modified with anti-estriol antibodies.
  • Fig. HD Estradiol molecules introduced to an MNC bioFET modified with anti-estriol antibodies.
  • the figure shows an illustration of the suggested sensing mechanism.
  • the figure presents Table 2 that show the dependency of the sensitivity, linearity, dynamic range and LOD on channel configuration for the specific and label-free sensing of estriol in 0.5 pL drops of 1: 100 diluted plasma with the MNC bioFET.
  • IDS-VGL curves for selected VGF values measured for MNC biosensor biofunctionalized with anti-AFP antibodies Each data point is an average of 64 measurements (16 drops, each drop is measured 4 times), and the error bars are the corresponding standard deviations (see inset).
  • Figure 16A-16D Process of Si/SiO surface biofunctionalization with anti-AFP antibodies
  • Fig. 16A An illustration showing the process of Si/SiCh surface biofunctionalization. The corresponding contact angle measurements are also shown.
  • Fig. 16B Ellipsometry measurements and contact angle measurements of Si/SiCh samples post various modification steps.
  • Fig. 16C EIS measurements showing the real and imaginary capacitances for the various modification steps.
  • Fig. 16D IDS-VGL for various VGF values for unmodified MNC device and biofunctionalized MNC biosensor.
  • the data points and the error bars reflect the averages and standard deviations of 12 measurements (3 drops each measured 4 times).
  • FIG. 17A- 17C IDS-VGL for selected VGF values for MNC biosensor modified with anti-AFP molecules
  • Fig. 17A IDS-VGL for selected VGF values for MNC biosensor modified with anti-AFP molecules. The measurements are performed in 1 : 100 diluted serum. The data points reflect an average of 42 measurements (Total of 14 drops each measured 3 times), and the error bars are the respective standard deviations (see inset).
  • Fig. 17B The second derivatives of the curves presented in Figure 17 A where a peak reflects the excitation of a conducting channel. The dependence of channel excitation on gates’ voltage configuration is shown.
  • AFP introduced to an unmodified MNC biosensor Fig. 17C(i)
  • AFP introduced to an MNC biosensor modified with APTMS Fig. 17C(ii)
  • hCG molecules introduced to an MNC biosensor modified with anti-AFP antibodies Fig. 17C(iii)
  • PSA molecules introduced to an MNC biosensor modified with anti-AFP antibodies Fig. 17C(iv)
  • Fig. 18B The extracted Readout corresponding to the curves presented in Figure 18A.
  • the insets show the corresponding error bars.
  • the labels at the top of the graphs indicate the corresponding conducting channels.
  • Fig. 18C An illustration showing one possible mechanism for the Readout polarity switch induced by the double layer electric field.
  • Fig. 19A Readout calibration curves. The illustrations on the right reflect the channel configurations. The vertical dashed grey lines indicate the calibration threshold.
  • the figure presents Table 3, that shows a summary of the MNC biosensor sensing performance for specific and label-free detection of AFP.
  • the present disclosure provides bio-transistor systems, methods and kits thereof, for effective detection of a target molecule in a sample, using an interaction based on the affinity between small molecules and proteineous molecules. More specifically, the present disclosure effectively demonstrates bio-transistor systems and methods for detecting small molecule targets using proteineous affinity moieties, for example, antibodies or receptors.
  • the diagnostic bio-transistor systems and method provided herein demonstrate effective detection of synthetic small molecules (e.g., organophosphates), and natural small molecules (e.g., steroid molecules such as estriol) that serves as a marker for placental health, in environmental samples, as well as in biological samples, specifically, blood and serum.
  • the current disclosure employs an MNChem chemically modified with a recognition layer composed of 2-(4- Aminophenyl)- 1, 1,1, 3,3,3- hexafluoro-2-propanol (aminophenyl-HFIP) receptors for specific and label-free sensing of the Organophosphate (OP) simulant Diethyl cyanophosphonate (DCNP).
  • OP Organophosphate
  • DCNP Diethyl cyanophosphonate
  • HFIP forms robust hydrogen bonds with target analytes, enhancing their selective capture and interaction with the sensor surface [Amara, et al. Macromolecules 38, (2005)]. Additionally, HFIP's compatibility with sensor fabrication processes and its ability to covalently bind onto sensor surface make it a preferred choice for enhancing sensor performance in OP detection applications [Amara, et al. Macromolecules 38, (2005)]. Importantly, the optimization of hydrogen-bond acidity and basicity through the electronwithdrawing effect of fluorine atoms in HFIP efficiently prevents self-association of HFIP substituents [Grate, J. W., et al. Anal.
  • DCNP selected target molecule
  • VX Sarin or venomous agent X
  • the MNC platform aims to address the inherent challenge of local gating induced by the biological or chemical interactions distributed non-uniformly at the device sensing area. This is in stark contrast with the global gating provided by conventional metal-gate transistors.
  • the MNC method allows an electrostatic tuning of drain-source current IDS) such as to optimally couple IDS electrodynamics with the electrostatics of the specific interactions at the sensing area [7].
  • the MNChem sensor is fabricated in a CMOS process which ensures optimal electrical performance in terms of noise and amplification.
  • the current work employs the MNChem sensor, modified with an aminophenyl-HFIP recognition layer, for real-time label-free and specific detection of DCNP in 0.5 pL drops of 0.1 mM PBS pH7.4. Calibration curves, characterization of surface modification, and extensive control measurements are provided.
  • estriol is one of three markers measured during mid-gestation for the Down syndrome risk calculation and screening. Estriol secretion is increased by 1000 times during pregnancy due to placental synthesis reaching at 37 weeks onwards a concentration of 80-130 pg/ml. The increase in plasma estriol concentration is much higher than that of estradiol or estrone which increase only 100 times more during pregnancy.
  • concentrations of free estriol in maternal plasma during pregnancy are considered as a marker for placental health, and low concentrations of multiple of median are related to chromosomal abnormalities such as trisomy 21 and trisomy 18, fetal anomalies and placental disorders such as preeclampsia.
  • chromosomal abnormalities such as trisomy 21 and trisomy 18, fetal anomalies and placental disorders such as preeclampsia.
  • estriol along with beta HCG and alfa fetoprotein can be an excellent POC test, that if available, will increase maternal compliance for aneuploidy midgestation screening and as a surrogate marker for potential placental complications.
  • the MNC bioFET provides a tunable channel configuration to enable optimal coupling between the electrostatics of the biological interactions and the electrodynamics of the conducting readout current [7].
  • the MNC biosensor is fabricated in a CMOS process which entails high-end bioFETs operating at the thermodynamic limit in terms of noise levels and amplification and coupled with low-cost and low power. Moreover, the ultimate miniaturization permitted by current microelectronic industry paves the way to unparalleled multiplexing in ultra-small samples.
  • the recognition layer is composed of anti-estriol antibodies to provide high specificity toward estriol compared with the other members of the estrogen family.
  • Antibody-antigen binding is non-covalent and is mediated by complementarity in the binding interface charge and shape.
  • flat surfaces accommodate the interaction with large protein regions, grooves enable peptide binding, and deep pockets support interactions with small molecules and haptens.
  • the inventors employ the MNC bioFET to the specific and label-free sensing of estriol from ultra-small samples of 0.5 pL drops of 1: 100 diluted plasma.
  • estriol Despite the small size and the electrical neutrality of estriol, realtime is demonstrated, specific, and label-free sensing of estriol with excellent linearity, sensitivity, dynamic range extending to ten orders of magnitude in estriol concentration and limit-of-detection of 1 fg mF -1 . Extensive control and non-specific measurements are reported including the response to other neutral members of the estrogen family.
  • bio-transistor system configured and operable for detection of presence and/or quantity of one or more selected molecules.
  • An exemplary configuration of bio-transistor system 100 is illustrated in Fig. 1.
  • the system includes at least one transistor unit 110 including source 120 and drain 130 regions associated with respective source 124 and drain 134 electrodes, and a channel region 140 between them.
  • the transistor unit 110 also includes one or more gate electrodes 144, located and configured to apply selected electric field on the channel 140 region to thereby enable switching of the transistor unit 110 by affecting charge carriers within the channel 140.
  • the source 120, drain 130 and channel 140 regions are placed on a back insulator layer 160 and covered by a top insulator 170.
  • the at least one gate electrode may be a side (e.g., lateral) gate electrode with respect to the channel region 140, alternatively or additionally, the at least one gate electrode may be positioned between one of the sources and drain electrodes 124 and 134 and the active region 150.
  • the active region 150 is located on top of the channel region 140, electrically insulated from the channel region 1 40 by the top insulator 170.
  • the at least one gate electrode 144 is generally separated from the channel region 140 by a gate insulator, which may be a portion of top insulator 170.
  • the transistor unit 110 may be formed of one or more semiconductor materials such as silicon, having selected doped regions with selected p-type or n-type doping. .
  • the active layer defining the source 120 and drain 130 regions and the channel region 140 between them may be formed of silicon semiconductor having varying p- and n-type doping regions.
  • the transistor unit 110 further includes an active region 150 located in vicinity (e.g., above) a region of the channel 140 and separated from the channel, e.g., by top insulator 170.
  • the active region 150 is formed of a region of top insulator 170, which is modified to carry selected one or more types of binding molecules, or affinity moieties, selected to bind one or more selected target molecules. This configuration provides the bio-transistor system 100 with capabilities for detection of the selected target molecules.
  • the transistor unit 110 is configured to accept a liquid sample 50, e.g., a liquid drop, positioned on, or in contact with, the active region 150.
  • the active region 150 may be surrounded by edge 152 configured to limit flow of liquid sample and prevent contact of the sample with electrodes of the unit 110 such as gate electrodes 144.
  • the edge 152 may be formed of any electrically insulating material and may be a part of top insulator 170.
  • Transistor unit 110 may also include at least one additional electrode 154 positioned to be in electric contact with the liquid sample 50, the additional electrode 154 is also referred to as quasi-reference electrode or sample electrode.
  • transistor unit 110 may be formed of silicone including n- and p-doped silicon regions forming the channel region 140 as well as source 120 and drain 130 regions.
  • Top 170 and bottom 160 insulators may be formed of silicon oxide layers. It should be noted that in some other embodiments, the transistor may be formed of selected one or more other semiconductor materials as the case may be.
  • Bio-transistor system 100 may also include a control system 500.
  • Control system 500 may include one or more processor and memory circuitries (PMC) 510 and may also include an electric circuit 52 configured to selectively apply electric potential and/or transmit electric current through the electrodes of transistor unit 110 to enable bio-transistor system 100 to determine data on presence and/or quantity of target molecules in the sample 50.
  • PMC processor and memory circuitries
  • PMC 510 may include one or more processors and memory, and may include, or be associated with, an I/O interface for receiving input data and for transmitting output instructions and/or data for operation of the bio-transistor system 100.
  • PMC 510 is operatively connected to the I/O interface and is configured to provide processing necessary for operation of the bio-transistor as described herein.
  • PMC 510 is configured to execute one or more functional operations in accordance with computer readable instructions implemented on a computer readable medium (e.g., non-transitory memory) being pre-stored at the memory of PMC 510 or at a separate computer readable medium.
  • a computer readable medium e.g., non-transitory memory
  • PMC 510 may operate the electric circuit 520 for selectively providing electric potential to the electrodes of transistor unit 110, and for determining data on current flow between the source electrode 124 and drain electrode 134.
  • control unit 500 is configured and operable for performing one or more measurements of the sample 50 to determine data on presence and/or quantity of target molecules in the sample 50.
  • each of the one or more measurements may include operation of the control system to maintain a selected electric potential VGF on electrode 154 to maintain a selected potential on the sample 50.
  • the control system 500 may operate for determining current transmission profile between the source 124 and drain 134 electrodes with respect to varying potential VG on at least one gate electrode 144. For example, this may be done by applying a selected potential between the source 124 and drain 134 electrodes and determining level of current transmission between the source 120 and drain 130 regions through the channel 140 with respect to potential VG applied to the at least one gate electrode 144.
  • each of the one or more measurements may be performed using a different selected potential VGF applied on the sample 50.
  • each measurement may include collecting data on current to gate potential (LVG) characteristics of the transistor unit 110 for given sample potential VGF.
  • LVG current to gate potential
  • control system 500 may be configured to perform a selected number of one or more, or two or more measurements, using different sample potentials VGF, and to store collected data on current for different gate potential values for each measurement.
  • This measurement data, indicative of transistor unit 110 I-VG characteristics for one or more different sample potential values is indicative of presence and/or quantity of the target molecules in the sample 50.
  • control system 500 may include pre-stored data, e.g., pre-stored at the memory of PMC 510, or stored in a remote location accessible view network communication, for calibrating transistor unit 110 characteristics to data on presence and/or quantity of target molecules in the sample 50.
  • the pre stored data may be indicative of I- VG characteristics of the transistor unit 110 as a function of presence and/or quantity of the target molecules for one or more given sample potentials VGF. Additionally, or alternatively, the pre stored data may be indicative of a relation between current transmission through the channel as a function of sample potential VGF, for one or more given gate voltage VG values.
  • the control system 500 may operate to generate output signal indicative of the data and provide the output signal via I/O interface to be presented to an operator using user interface and/or for use in further processing.
  • the bio-transistor system 100 utilizes at least one affinity moiety (also referred to herein as selected affinity moieties) placed (adsorbed, attached) on an active region 150 located in vicinity to the channel region 140.
  • the active region 150 may be separated from semiconductor region of the channel region 140 by an electric insulating layer 170, such that electrostatic changes at the active region 150 may apply electric fields affecting the channel region 140.
  • the present disclosure may also provide a bio-transistor system 100 including a plurality of transistor units, each carrying a selected different type of affinity moieties, selected to interact with one or more target molecules, including e.g., one or more different target molecules. It should be understood that in some embodiments, each of the affinity moieties is specific for one target molecule. Thus, in some embodiments the disclosed bio transistor system may comprise at least one affinity moiety, and in some embodiments, a plurality of affinity moieties that are specific for one or more target molecule.
  • each of the affinity moieties is specific for one target molecule, but for each target molecule it is possible to have more than one either identical or different affinity moiety.
  • the disclosed bio transistor system may comprise different affinity moieties specific for more than one target molecule, wherein each of the affinity moiety is specific for one target molecule.
  • Fig. 2 exemplifying a bio-transistor array configuration of system 100.
  • the system includes a selected number of transistor units 110a to 1 lOn, each having source and drain regions 120 and 130 and a channel region between them, and active region 150 carrying the selected affinity moieties, gate electrode 144, and an additional electrode (not shown) positioned to be in contact with a sample positioned on the active region 150.
  • the plurality of transistor units 110a- 11 On are connected to a control system 500 and are independently operated for determining data on presence and/or quantity of the respective target molecules as described herein.
  • the bio-transistor system 100 may include a sample channel configured for receiving a liquid sample and channeling portions of the liquid samples to different active regions of a plurality of transistor units as illustrated in Fig. 2.
  • the channel may include a selected number of electrical insulating valves configured to close prior to performing measurements on the samples.
  • Fig. 3 exemplifies a method for determining presence and/or quantity of at least one type of target molecules according to some embodiments of the present disclosure in a way of a block diagram.
  • the method includes providing selected affinity moieties in contact (e.g., adsorbed, attached) with an active region 3010 of one or more transistor units.
  • the bio-transistor unit may be treated with the selected affinity moieties during production and provided for use in determining presence and/or quantity of the selected target molecules.
  • the method includes contacting a sample with the active region of the one or more transistor units 3020 and operating the transistor unit to perform one or more measurements 3030.
  • each measurement includes maintaining a sample potential 3032, using one or more additional electrodes being in electric contact with the sample, and determining current-voltage characteristics of the transistor unit 3034.
  • the method may include a selected number of measurements, typically using different one or more potentials applied to the sample.
  • the method includes processing the measurement data and determining data on presence and/or quantity of the target molecules 3040.
  • the processing may generally include using pre-provided calibration data indicative of variation in transistor current-voltage characteristics for given amounts of the target molecules in the sample.
  • the system and method of the present disclosure may utilize predetermined calibration data, determined for given activity region 150 carrying given amount of affinity moieties of selected type.
  • the calibration data may be in the form of a multi-dimensional calibration data including data on variation of the source-drain current with respect to different gate potentials VG and to different sample potentials VGF.
  • variation of the sample potential VGF may be used as a parameter for determining presence and/or quantity of the target molecules.
  • variation of the sample potential VGF may provide for varying detection range for difference ranges of quantity of the target molecule, where for each value of sample potential, variation of gate potential VG and the relation between gate potential and source-drain current provides the calibration for quantity of target molecules in the sample.
  • each measurement includes maintaining sample potential VGF at a selected value and determining source-drain current for selected range of gate potentials VG.
  • the different measurements utilize different values of the sample potential VGF.
  • the measurement may also utilize a selected, generally constant, source-drain voltage to eliminate, or at least significantly reduce effects of the potential variation on the interaction between the affinity moieties and target molecules.
  • variation of sample potential VGF may affect concentration of the target molecules within the sample, e.g., forming layers of different concentrations of the target molecules, thereby limiting detection accuracy.
  • the present technique may utilize a selected number of different sample potential VGF values, and respective calibration data.
  • the system and method of the present disclosure provide for determining presence and/or quantity of selected target molecules within a sample.
  • the present technique utilizes calibration data, determined in accordance with known concentrations of the target molecules.
  • the calibration data may be determined in accordance with amount and/or density of affinity moieties associated with the active region 150.
  • variation in source-drain current, associated with gate potential VG, and sample potential VGF may be associated with concentration of charge carrier in the channel region 140.
  • variation of gate potential VG may affect size and/or shape of the channel region 140 and accordingly affect current transmission through the channel.
  • An exemplary structure of transistor unit suitable for affecting channel shape and dimensions by variation of gate potential VG is described in US 2023/0022648 incorporated herein for reference with respect to configurations of the transistor unit [2].
  • the first aspect of the present disclosure relates to a bio-transistor system comprising at least one transistor unit and a control unit.
  • the transistor unit comprises (i) at least one channel, (ii) source and drain electrodes, (iii) at least one gate electrode and (iv) at least one active region located in proximity to the channel region and carrying at least one affinity moiety (also referred to herein as selected affinity moieties).
  • each affinity moiety is specific for a target molecule.
  • each affinity moiety is specific for a target molecule.
  • each of the affinity moieties is specific for one target molecule.
  • the active region may carry two or more different affinity moieties, each specific for one target molecule.
  • the at least one active region is configured for accepting at least one sample.
  • the transistor unit also comprises (v) at least one additional electrode positioned to be in electrical contact with the sample (e.g., a liquid sample).
  • the control system comprises at least one processor and memory circuitry. The control system is configured and operable for performing one or more measurements of the sample. Each measurement comprises maintaining a selected electric potential on the at least one additional electrode and determining current transmission profile through the at least one channel with respect to potential variation of the at least one gate electrode. The control unit thereby configured to determine data on the presence and/or quantity of the at least one target molecules in the sample.
  • control system is configured to performed two or more measurements utilizing two or more different selected electric potentials applied to the at least one additional electrode.
  • control system comprises pre-stored calibration data comprising data on electric transmission through the channel for given gate electrode potential with respect to one or more selected electric potentials applied to the at least one additional electrode.
  • control system comprises pre-stored calibration data comprising data on electric transmission trough the channel with respect to variation of the gate electrode potential.
  • the pre-stored calibration data may be generated from different concentrations of the target molecule in a sample. More specifically, the pre-stored calibration data may be generated from at least one concentration of target molecule. Still further, the pre-stored calibration data may be generated from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000 or more different concentrations of the target molecule in the sample. It should be noted that different concentrations of the target molecule may be also affected from the different dilutions of the samples.
  • the bio-transistor system comprising a plurality of two or more transistor units comprising respective plurality of two or more active regions carrying two or more different or identical types of affinity moieties.
  • the active region is separated from the channel region by an electrical insulator layer.
  • the at least one gate electrode is electrically insulated from the active region.
  • the control system comprises at least one electrical circuit coupled vie electrical connection to the transistor unit and configured to provide selected electric potentials to electrodes of the transistor unit.
  • the at least one affinity moiety comprises at least one of: an amino acid-based molecule, a nucleic acid-based molecule, a small molecule, a carbohydrate-based molecule, a lipid-based molecule or any combination thereof. The affinity moiety specifically binds, either directly or indirectly, the at least one target molecule in the sample.
  • the affinity moiety comprises, or is derived from a component of an affinity pair.
  • affinity moiety or “affinity molecule” as used herein, or “affinity pair” (as used herein after), refers to a corresponding binding couple biomolecule partners.
  • affinity or “binding affinity” is the strength of the binding interaction between a binding biomolecule to its binding partner or target (e.g., a protein, DNA or small molecule). Binding affinity is typically measured and reported by the equilibrium dissociation constant (KD), which is used to evaluate and rank order strengths of bimolecular interactions. The smaller the KD value, the greater the binding affinity of the binding molecule for its target.
  • KD equilibrium dissociation constant
  • Binding affinity is influenced by non-covalent intermolecular interactions such as hydrogen bonding, electrostatic interactions, and hydrophobic and van der Waals forces between the two molecules.
  • binding affinity between a binding molecule and its target molecule may be affected by the presence of other molecules.
  • the affinity pair comprises at least one of: receptor-ligand, antibody-antigen, enzyme-substrate, aptamer- aptamer target, or any combination thereof. It should be noted that each affinity moiety is specific for one target molecule. In some embodiments, the affinity moiety and the target molecule recognized by the affinity moiety form together an affinity pair.
  • the term "specific” and "specifically bind" is as described herein after.
  • the affinity molecule specifically binds the at least one target molecule in the sample.
  • the target molecule comprises at least one of: an amino acid-based molecule, a nucleic acid-based molecule, a small molecule, a carbohydrate-based molecule, a lipid-based molecule or any combination thereof.
  • the affinity molecule, and/or the target molecule and/or the candidate compound disclosed herein after in connection with the screening methods may be amino acid-based, a nucleic acid-based, a small molecule-based, a carbohydrate-based and/or a lipid-based.
  • the term "-based" as used herein refers to the at least one affinity moiety or molecule, and/or to the target molecule, which is composed of the building block specified prior to "-based".
  • amino acid-based affinity molecule/s and/or target molecule/s are affinity molecule/s and/or target molecule/s, which are composed of amino acids, such as proteins, peptides, glycoproteins, lipoproteins, etc.
  • nucleic acidbased affinity molecules and/or target molecule/s are affinity molecule/s and/or target molecule/s, which are composed of nucleic acids such as polynucleotides, aptamers, etc.
  • Small molecule-based affinity molecule/s and/or target molecule/s are affinity molecule/s and/or target molecule/s, which are composed of small molecules, which are low molecular weight organic compound, having a molecular weight lower than 900 Daltons.
  • Carbohydrate-based affinity molecules are affinity molecules which are composed of carbohydrates such as monosaccharides, disaccharides, oligosaccharides, and polysaccharide as well as proteoglycans, glycoproteins, etc.
  • lipid-based molecules are affinity molecule/s and/or target molecule/s, which are composed of lipids such as fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, proteolipids, and others.
  • lipids such as fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, proteolipids, and others.
  • the at least one affinity moiety and/or the at least one target molecule of the disclosed bio transistor systems and methods may be, or may comprise a small molecule.
  • a small molecule in the context of the present disclosure refers to a low molecular weight organic compound, having a molecular weight lower than 900 Daltons, and in some embodiments less than about 2 kilodaltons (kDa) in mass. In some embodiments, the small molecule is less than about 1.5 kDa, or less than about 1 kDa. In some embodiments, the small molecule is less than about 900 daltons (Da), 800 Da 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, or 100 Da.
  • a small molecule has a mass of at least 50 Da.
  • a small molecule is non-polymeric.
  • a small molecule is not an amino acid.
  • a small molecule is not a nucleotide.
  • a small molecule is not a saccharide.
  • a small molecule contains multiple carbon-carbon bonds and can comprise one or more heteroatoms and/ or one or more functional groups important for structural interaction with proteins (e.g., hydrogen bonding), e.g., an amine, carbonyl, hydroxyl, or carboxyl group, and in some embodiments at least two functional groups.
  • Small molecules often comprise one or more cyclic carbon or heterocyclic structures and/or aromatic or polyaromatic structures, optionally substituted with one or more of the above functional groups.
  • crystalline and amorphous forms of those compounds including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (including anhydrates), conformational polymorphs, and amorphous forms of the compounds, as well as mixtures thereof.
  • Crystal form or "polymorph,” as used herein include all crystalline and amorphous forms of a small molecule, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (including anhydrates), conformational polymorphs, and amorphous forms, as well as mixtures thereof, unless a particular crystalline or amorphous form is referred to.
  • small molecule may include pharmaceutically acceptable forms of the recited compounds, including chelates, non- covalent complexes, prodrugs, and mixtures thereof. Further and in accordance with the preset disclosure, the term “small molecule” includes also pharmaceutically acceptable forms of a particular molecule and as such the term small molecule also encompasses pharmaceutically acceptable salts.
  • the at least one affinity moiety and/or the at least one target molecule of the disclosed bio transistor systems and methods may be, or may comprise an amino acid-based molecule.
  • An "amino acid-based molecule”, as used herein refers to a molecule that is primarily composed of amino acids or derivatives of amino acids. Amino acids are organic compounds that contain both an amino group (-NH2) and a carboxyl group (-COOH), along with a unique side chain specific to each amino acid.
  • Amino-acid based molecules in accordance with the present disclosure encompass various structures including peptides, that are short chains of amino acids linked by peptide bonds (typically consist of 2 to 50 amino acids), polypeptides, that are intermediate-length chains of amino acids that can fold into specific structures and may function independently or as part of a larger protein complex and proteins, that are composed of long chains of amino acids, typically consisting of 50 or more amino acids, and fold into specific three- dimensional structures that determine their function in biological processes.
  • An example for an affinity moiety-target pair composed of a protein include, but is not limited to the antibody-antigen, receptor-ligand and peptide aptamers and targets thereof.
  • amino-acid-based molecule as used in the present disclosure further encompasses amino acid derivatives, specifically, molecules derived from amino acids through chemical modifications.
  • the at least one affinity moiety and/or the at least one target molecule of the disclosed bio transistor systems and methods may be, or may comprise a lipid-based molecule.
  • a lipid-based molecule is a chemical compound primarily composed of lipids, which are a diverse group of hydrophobic or amphipathic organic molecules. Lipids are insoluble in water but soluble in nonpolar solvents. It should be understood that the present disclosure encompasses any affinity moiety and/or target molecule composed of or comprising a lipid-based molecule, of any type, for example, triglycerides, that consist of three fatty acids attached to a glycerol backbone, phospholipids, that have two fatty acids and a phosphate group attached to glycerol, steroids, that have a structure based on a carbon skeleton with four fused rings (e.g., Cholesterol), glycolipids, that contain a carbohydrate group attached to a lipid, and fatty Acids, that are carboxylic acids with a long hydrocarbon chain, which can be saturated (no double bonds) or unsaturated (one or more double bonds).
  • triglycerides that consist of three
  • the at least one affinity moiety and/or the at least one target molecule of the disclosed bio transistor systems and methods may be, or may comprise a carbohydrate-based molecule.
  • a carbohydrate-based molecule as used herein, is a chemical compound that primarily consists of carbohydrate components, which are organic molecules made up of carbon (C), hydrogen (H), and oxygen (O) atoms, usually with the hydrogen and oxygen atoms in a ratio of 2: 1, as in water.
  • Carbohydrates are one of the four main classes of biomolecules and can be simple sugars (monosaccharides like glucose and fructose), double sugars (disaccharides like sucrose and lactose), or complex carbohydrates (polysaccharides like starch, cellulose, and glycogen).
  • the at least one affinity moiety and/or the at least one target molecule of the disclosed bio transistor systems and methods may be, or may comprise a nucleic acid molecule.
  • nucleic acid molecule as used herein relates to a complex organic substance that forms the basis of genetic material in all living organisms.
  • each nucleotide is composed of three components: a phosphate group, a five-carbon sugar (either ribose in RNA or deoxyribose in DNA), and a nitrogenous base.
  • a phosphate group a five-carbon sugar (either ribose in RNA or deoxyribose in DNA)
  • a nitrogenous base There are four main types of nitrogenous bases in DNA (adenine [A], thymine [T], cytosine [C], and guanine [G]) and in RNA (adenine [A], uracil [U], cytosine [C], and guanine [G]).
  • the nucleotides are linked together by covalent bonds between the phosphate group of one nucleotide and the sugar of the next, forming a sugar-phosphate backbone with protruding nitrogenous bases.
  • the present disclosure encompasses Deoxyribonucleic Acid (DNA) molecules, Ribonucleic Acid (RNA), any combinations thereof or any derivatives thereof. It should be understood that the target molecule is specifically recognized and bound by the affinity moiety.
  • the target molecule comprises, or is derived from a component of an affinity pair.
  • the affinity pair comprises at least one of: receptor-ligand, antibody-an antigen, enzyme-substrate, aptamer-aptamer target, or any combination thereof.
  • target molecule specifically interacts with the affinity moiety.
  • target molecule refers to at least one specific molecule in a sample for which determining its presence and/or quantity is desired. This target molecule will be the corresponding partner of the affinity moiety. It should be understood that a target molecule as used herein, further encompasses a single molecule or alternatively two or more molecules, subunits, complexes, conjugates and the like, forming a specific recognition partner recognized and bound by the specific affinity moiety of the disclosed bio-transistor systems.
  • the present bio transistor systems and the detection methods disclosed herein are based on the specific recognition and binding of the at least one affinity moiety and the target molecule, that form together an affinity pair.
  • the affinity pair in accordance with the present disclosure may comprise at least one amino acid-based molecule and at least one small molecule thereby forming together an affinity pair.
  • the affinity moiety may be or may comprise an amino acid-based molecule.
  • the target molecule may be or may comprise at least one small molecule.
  • Affinity pair the comprise amino acid-based and small molecule components may be in accordance with some embodiments an antibody-antigen pair, specifically, when the antigen is a small molecule, forming the antigen recognized by the specific antibody.
  • an optional affinity pair may be a receptor-ligand affinity pair.
  • Other affinity pairs, specifically, enzyme-substrate, aptamer-aptamer target, or any combination thereof, are also optional in accordance with the present disclosure.
  • the target molecule is a ligand composed of a small molecule and the corresponding receptor for such ligand is an amino acid-based receptor serving as the affinity moiety.
  • an affinity pair, for the affinity moiety and target molecule may be a receptor-ligand binding pair, specifically, where the affinity moiety is or comprises a receptor where the target molecule is the ligand or vice versa.
  • a receptor-ligand affinity pair refers to the specific and often high-affinity interaction between a receptor (a protein molecule usually located on the cell surface or within a cell) and a ligand (a molecule that binds to the receptor).
  • each receptor typically binds to a particular ligand or a group of structurally similar ligands, thereby forming the receptor-ligand complex, that is detected and quantified by the disclosed bio transistor systems and methods, and often triggers a conformational change in the receptor, initiating a cascade of intracellular events.
  • Example that may be useful in the present disclosure may include hormone receptors, neurotransmitter receptors, and the like.
  • Receptors are chemical structures, composed of protein, that receive and transduce signals that may be integrated into biological systems. These signals are typically chemical messengers which bind to a receptor and produce physiological responses such as change in the electrical activity of a cell. Receptor proteins can be classified by their location.
  • Cell surface receptors also known as transmembrane receptors, include ligand-gated ion channels, G protein-coupled receptors, and enzyme-linked hormone receptors. Intracellular receptors are those found inside the cell and include cytoplasmic receptors and nuclear receptors.
  • a molecule that binds to a receptor is called a "ligand” and can be a protein, peptide , or another small molecule.
  • a non-limiting embodiment for a receptor-ligand pair is provided by the present disclosure that use the receptor molecule HFIP as the affinity moiety in the disclosed bio transistor systems, that specifically recognizes and binds the small molecule target organophosphate molecule DCNP.
  • an affinity pair composed of a small molecule and an amino-acid based molecule may be an Antibody-antigen pair.
  • An "antibody Ab)" also known as an “immunoglobulin (Ig)"
  • Ig immunoglobulin
  • the antibody recognizes a unique molecule called an "antigen”.
  • Each tip of the "Y” of an antibody contains a paratope (analogous to a lock) that is specific for one particular epitope (analogous to a key) on an antigen, allowing these two structures to bind together with precision.
  • antibody as used herein, means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen.
  • CDR complementarity determining region
  • the term “antibody” includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM).
  • Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains, CHI, CH2 and CH3.
  • Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the light chain constant region comprises one domain (CL1).
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • antigen-binding domains that can be used in the context of the present invention include antibodies, antigen-binding portions of antibodies, peptides that specifically interact with a particular antigen (e.g., peptibodies), receptor molecules that specifically interact with a particular antigen, proteins comprising a ligand-binding portion of a receptor that specifically binds a particular antigen or antigen-binding scaffolds.
  • the antigen binding domains in accordance with the invention may recognize and bind a specific antigen or epitope.
  • binding specificity specifically binds to an antigen
  • binding reaction which is determinative of the presence of the epitope in a heterogeneous population of proteins and other biologies.
  • epitope is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody.
  • Epitopes or "antigenic determinants” usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics, as in the estriol molecule used according to some embodiments as the target molecule.
  • an "antigen-binding domain” can comprise or consist of an antibody or antigen-binding fragment of an antibody.
  • antigen-binding fragment of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.
  • Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains.
  • DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized.
  • the DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
  • antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR)).
  • CDR complementarity determining region
  • engineered molecules such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment,” as used herein.
  • An antigen-binding fragment of an antibody will typically comprise at least one variable domain.
  • the variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences.
  • the VH and VL domains may be situated relative to one another in any suitable arrangement.
  • the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers.
  • the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.
  • an affinity pair applicable for the present disclosure is an Enzyme-substrate pair.
  • Enzymes differ from most other catalysts by being much more specific. An enzyme's specificity comes from its unique three-dimensional structure. Enzymes are usually much larger than their substrates. Sizes range from just 62 amino acid residues, for the monomer of 4-oxalocrotonate tautomerase, to over 2,500 residues in the animal fatty acid synthase. Only a small portion of their structure (around 2-4 amino acids) is directly involved in catalysis: the catalytic site. This catalytic site is located next to one or more binding sites where residues orient the substrates. The catalytic site and binding site together compose the enzyme's active site. The remaining majority of the enzyme structure serves to maintain the precise orientation and dynamics of the active site.
  • Enzyme structures may also contain allosteric sites where the binding of a small molecule causes a conformational change that increases or decreases activity. Enzymes must bind their substrates before they can catalyze any chemical reaction. Specificity is achieved by binding pockets with complementary shape, charge and hydrophilic/hydrophobic characteristics to the substrates. Enzymes can therefore distinguish between very similar substrate molecules to be chemoselective, regioselective and stereospecific.
  • the enzyme component of the enzyme-substrate affinity pair can be a whole enzyme, and/or any fragments thereof, such as the enzyme's active site or the enzyme's binding site as long as it's binding capability is maintained.
  • affinity pair may a nucleic acid sequence and a proteineous factor.
  • the affinity pair may be aptamer-target affinity pair.
  • “Aptamers” are short sequences of artificial DNA, RNA, XNA, or peptide that bind a specific target molecule, or family of target molecules. They exhibit a range of affinities (KD in the pM to pM range), and are sometimes classified as "chemical antibodies” or "antibody mimics".
  • the nucleic acid-based structure of aptamers which are mostly oligonucleotides, is very different from the amino acid-based structure of antibodies, which are proteins. Aptamers are usually obtained by selection from a large random sequence library, using methods well known in the art, such as SELEX and/or Molinex.
  • an affinity pair for the affinity moiety and target molecule, may be a proteineous factor-specific nucleic acid binding site bindingpair.
  • a proteineous factor-specific nucleic acid binding site refers to a specific sequence or structure within a nucleic acid molecule (DNA or RNA) that is recognized and bound by a particular protein factor, that may be a protein, often a regulatory protein such as a transcription factor, RNA-binding protein, or enzyme, that interacts with nucleic acids.
  • a particular protein factor that may be a protein, often a regulatory protein such as a transcription factor, RNA-binding protein, or enzyme, that interacts with nucleic acids.
  • proteins are proteins involved in various cellular processes, including gene expression, replication, and RNA processing.
  • the nucleic acid binding site that may be used in the present disclosure as the affinity moiety, is a specific region on a nucleic acid (DNA or RNA) where the protein binds.
  • the binding site is typically a specific sequence of nucleotides or a particular structural motif that the protein recognizes and interacts with.
  • an affinity pair for the affinity moiety and target molecule, may be an aptamer-aptamer target binding pair. More specifically, an aptamer- aptamer target binding pair, as used herein, refers to the specific and high-affinity interaction between an aptamer and its corresponding target molecule. Aptamers according to some embodiments, are short, single-stranded DNA or RNA molecules that can fold into unique three-dimensional structures, allowing them to bind selectively to a wide range of targets, including proteins, small molecules, ions, and even whole cells.
  • the affinity moiety of the disclosed bio transistor systems is, composed of, or comprising, at least one aptamer, for example, a nucleic acid-based aptamer.
  • the target molecule recognized by such affinity moiety may be a protein, small molecule, ion, and even a whole cell.
  • aptamer-target binding is mediated by non-covalent interactions, including hydrogen bonds, electrostatic interactions, Van der Waals forces, and sometimes hydrophobic interactions.
  • the affinity moiety and the specific target molecule may form an affinity pair composed of an enzyme- substrate. More specifically, "affinity pair” highlights the complementary nature of the enzyme and substrate, where the enzyme's active site is specifically shaped to recognize and bind its substrate, resulting in a high-affinity interaction and high specificity. This interaction is central to the enzyme's catalytic function and involves specificity, that is meant herein that each enzyme binds to a particular substrate or a group of closely related substrates, affinity, that relates to the strength of the binding between an enzyme and its substrate is referred to as affinity. High-affinity binding ensures that the substrate is efficiently captured by the enzyme.
  • the formation of the enzyme-substrate complex is referred to herein as binding of the target molecule to the affinity moiety that is detected and/or quantified by the bio transistor systems and methods of the present disclosure. Formation of the enzyme-substrate complex is followed by catalysis and release of the products from the enzyme's active site.
  • the at least one affinity pair is receptor-ligand affinity pair.
  • the at least one affinity moiety and the at least one target molecule are derived from a receptor-ligand binding pair.
  • the affinity moiety is derived from a receptor molecule. Accordingly, the target is derived from a ligand for the receptor molecule.
  • the affinity moiety is derived from a ligand molecule. Accordingly, the target is derived from a receptor for the ligand molecule.
  • the target recognition moiety that is also referred to herein as an affinity moiety, comprise in some embodiments an amino acid-based molecule (e.g., receptor or an antibody) that specifically recognizes and binds the target small molecule.
  • "Specifically binds" as used herein refers to an affinity moiety which interacts with a specific target molecule, while avoiding interactions with other molecules. Examples of moieties with specific binding capabilities include for example, receptors, antibodies, nucleic acid binding sites, aptamers, as well as enzymes.
  • the target recognition component also referred to herein as the affinity moiety, specifically, the antibody or the receptor, specifically binds the target molecule in any stoichiometric ratio.
  • one target recognition component binds at least one, at least two, at least three, at least four, at least five, at least ten, at least hundred, at least thousand and even more, target molecules.
  • the targetrecognition component specifically, binding site, comprises more than one moiety, wherein the plurality of moieties may be the same and/or may be different (e.g. different binding sites, etc).
  • the target molecule is at least one ligand comprising a small molecule chemical substance
  • the affinity moiety comprises an amino acid-based receptor molecule for the ligand
  • the chemical substance is toxic for at least one eukaryotic organism.
  • the eukaryotic organism is at least one organism of the biological kingdom Animalia or of the biological kingdom Plantae.
  • the eukaryotic organism of the biological kingdom Animalia may be an insect, avian, fish and a mammalian subject.
  • the chemical substance is organophosphate.
  • the organophosphate comprises at least one of pesticides, herbicides, and/or chemical warfare agents.
  • the at least one organophosphate is diethylcyanophosphonate (DCNP).
  • Diethylcyanophosphonate (DCNP) is an organophosphorus compound with the chemical formula (CiHsO ⁇ P OjCN. It is used in organic synthesis, particularly in the formation of phosphonate esters.
  • DCNP consists of a phosphonate group (P(0)(0R)2) attached to a cyano group (CN). The two ethoxy groups (C2H5O) are bonded to the phosphorus atom, having a molecular weight of 151.11 g/mol.
  • the receptor is hexafluoroisopropanol (HFIP).
  • the affinity moiety of the disclosed bio-transistor systems may comprise HFIP or any functional fragment thereof.
  • Hexafluoroisopropanol HFIP
  • HFIP Hexafluoroisopropanol
  • CFs ⁇ CHOH fluorinated alcohol with the chemical formula (CFs ⁇ CHOH). It is widely used in organic chemistry and materials science due to its unique properties.
  • HFIP consists of a central isopropanol structure where all six hydrogen atoms on the carbon backbone are replaced with fluorine atoms, giving it the chemical formula (CFs ⁇ CHOH, having a molecular weight of 168.04 g/mol.
  • Hexafluoroisopropanol can act as a receptor for diethylcyanophosphonate (DCNP) through non-covalent interactions, primarily hydrogen bonding and possibly dipole interactions.
  • HFIP has a hydroxyl group that can form strong hydrogen bonds with the electronegative atoms in DCNP, such as the oxygen atoms in the ethoxy groups and the nitrogen atom in the cyano group. This hydrogen bonding can facilitate the formation of a stable complex between HFIP and DCNP.
  • the at least one sample is a biological sample and/or an environmental sample.
  • a “sample” as used herein may be any biological and/or environmental sample. More specifically, an “Environmental sample” refers to any sample derived from any media or material in the environment. The most common environmental samples are air, water, soil, biological materials, and wastes (liquids, solids or sludges) such as sewage. Environmental sampling is typically performed to determine the presence of hazardous materials in any media or material, including indoor or outdoor air, soil, groundwater, surface water or building materials. Still further, in some embodiments, a sample may comprise any food product or any byproduct derived from any industrial activity or facility, or any water reservoir that may be contaminated by the disclosed organophosphates.
  • the sample may be a "biological sample", specifically, a biological material collected from living and/or deceased organisms and/or living and/or dried out plants or their environment.
  • biological samples including biofluids, tissue, cells and other.
  • Biological samples can be obtained from the body via several different methods such as excretion (e.g. urine), secretion (e.g. breast milk) or extraction (e.g. blood).
  • Non limiting examples of biological samples include blood, bile, bone marrow aspirate, breast milk/mammary gland milk, Cerebral Spinal Fluid (CSF), feces, plasma, saliva, semen, serum, sputum, sweat, as well as oral, nasal and vaginal fluids (typically collected using a swab), and synovial fluid, tears and urine.
  • Biological samples include also cells such as epithelial cells, fibroblasts, immune cells (e.g.
  • T cell T cell, B cells, NK cells etc.
  • PBMCs peripheral blood mononuclear cells
  • RBCs red blood cells
  • buffy coat bone marrow mononuclear cells
  • dissociated tumor cells mesenchymal stem cells, myoblasts, hepatocytes, etc as well as tissues.
  • Cell samples are collected and isolated from either tissue samples, biofluid samples, or biopsy samples. Depending on the cell type, specific cell isolation protocols are required in order to obtain the purified cell sample.
  • Biological material collected from plants may be for example any sample derived from the leaf, roots, stems, nectar, seeds and/or fruits of any plant.
  • the sample is an environmental sample, particularly where the target molecule is an organophosphate molecule, for example, DCNP.
  • the target molecule is an organophosphate molecule, for example, DCNP.
  • the at least one affinity pair of the bio-transistor system of the present disclosure is an antigen-antibody affinity pair, where the antigen in this pair is a small molecule target antigen recognized by the amino-acid based affinity moiety that is composed of an antibody specific for the small molecule target.
  • the target is at least one antigen comprising a small molecule steroid compound.
  • the affinity moiety of the disclosed biotransistor system comprises an antibody specific for the antigen, specifically, the small molecule steroid compound.
  • the target molecule is, or comprises a small molecule steroid compound.
  • the target small molecule steroid compound is a molecule naturally produced by at least one eukaryotic organism.
  • the target small molecule steroid compound is of the estrogen family. In yet some further specific embodiments, the small molecule steroid compound is Estriol.
  • Estriol is a naturally occurring estrogen and one of the three main estrogens produced by the human body, specifically, the fetus and placenta, alongside estradiol and estrone, having the chemical formula C18H24O3, a molecular weight of 288.38 g/mol, and IUPAC Name (8R,9S,13S,14S,16R,17S)-13-Methyl- 6, 7, 8, 9, 11 , 12, 14, 15, 16, 17-decahydrocyclopenta[a]phenanthrene-3, 16, 17-triol.
  • Formula I disclose the chemical structure of estriol.
  • Estriol has three hydroxyl groups located at positions 3, 16, and 17 on the steroid backbone, making it a triol.
  • Estriol is synthesized primarily in the placenta during pregnancy. It is a metabolite of estrone and estradiol, both of which are precursors in the biosynthetic pathway. Estriol is the dominant estrogen during pregnancy. Its levels increase significantly as pregnancy progresses and are considered an indicator of fetal well-being and placental function. Low levels can be associated with chromosomal abnormalities.
  • the affinity moiety of the bio-transistor system disclosed herein is an antibody molecule.
  • the affinity moiety is or comprises at least one anti-Estriol antibody or any functional fragments thereof.
  • the at least one sample is a biological sample and/or environmental sample.
  • the disclosed bio-transistor system is configured for determining the presence and/or the quantity of the estriol in a sample, specifically, a biological sample.
  • such sample may be a blood and/or serum sample.
  • the sample is a serum sample obtained from a pregnant female subject.
  • the disclosed bio transistor system may be useful in screening for compounds that may modulate the interaction between the two components of the binding pair, specifically, any molecule that decreases or alternatively decreases the interaction between the affinity moiety and the target molecule.
  • the sample may further comprise a candidate compound that is evaluated for its ability to change the interaction (e.g., binding) of the affinity moiety (e.g., a receptor and/or n antibody), and its specific target (e.g., a ligand or a small molecule antigen).
  • a candidate compound may be any molecule.
  • the at least one sample further comprises at least one candidate compound that modulates the interaction between the affinity moiety and the target molecule.
  • the candidate compound comprises at least one of: an amino acid-based molecule, a nucleic acid-based molecule, a small molecule, a carbohydrate-based molecule, a lipid-based molecule, or any combination thereof.
  • the disclosed screening method is particularly applicable for screening of a compound that inhibits the interaction of the affinity moiety with the target molecule.
  • a compound that "inhibits the interaction” as used herein refers to a compound that reduces, prevents, blocks, impedes, suppresses, prevents, hinders and/or restricts interaction between the at least one affinity moiety and the target molecule either partially, or completely. For example, any inhibition of between about 10-50%, 51-80%, 81-99% or even, of 100% of the interaction or binding between the affinity moiety and the target molecule.
  • Another aspect of the present disclosure relates to a battery comprising two or more of the bio-transistor systems as defined above.
  • the battery of the present disclosure comprises two or more biotransistor systems. In some embodiments, one of these at least two bio-transistor systems comprise the estriol- anti-estriol antibody as defined above. In yet some further embodiments, the at least one additional bio-transistor system of the disclosed battery may comprise at least one affinity moiety for at least one additional maternal serum marker.
  • a maternal serum marker is a substance found in a pregnant woman's blood that can provide important information about the health and development of the fetus, as well as the mother's health during pregnancy. These markers are typically proteins, hormones, or other molecules that are produced by the placenta, the fetus, or the mother's body in response to pregnancy. Maternal serum markers are used in prenatal screening tests to assess the risk of certain fetal conditions, such as chromosomal abnormalities (e.g., Down syndrome), neural tube defects, and other genetic or developmental disorders. They can also help monitor the health of the pregnancy and the risk of complications such as preeclampsia. Common maternal serum markers encompassed by the present disclosure include, but are not limited to AFP, hCG, Estriol (uE3), Inhibin A, PAPP-A (Pregnancy- Associated Plasma Protein A), and the like.
  • the present disclosure provides a battery of bio transistor systems comprising:
  • At least one bio-transistor system comprising at least one affinity moiety specific for Estriol.
  • the affinity moiety is or comprises at least one anti- Estriol antibody.
  • the disclosed battery further comprises at least one of:
  • the affinity moiety is or comprises at least one anti-AFP antibody.
  • the disclosed battery comprises additionally, or alternatively, (c), at least one bio-transistor system comprising at least one affinity moiety specific for human chorionic gonadotropin (hCG).
  • hCG human chorionic gonadotropin
  • the affinity moiety is, or comprises at least one anti- hCG antibody.
  • Alpha-fetoprotein is a glycoprotein produced primarily by the fetal liver, yolk sac, and gastrointestinal tract. It is a major plasma protein in the developing fetus and plays a role similar to that of serum albumin in adults. AFP is also produced in certain pathological conditions in adults, making it a valuable biomarker in clinical diagnostics. AFP is a single-chain glycoprotein composed of approximately 590 amino acids. The protein has carbohydrate moieties attached to specific asparagine residues, contributing to its stability and solubility. AFP contains three homologous domains, each contributing to its overall function and binding properties. These domains are involved in the transport and binding of various ligands, such as fatty acids and bilirubin. AFP binds and transports various molecules, including fatty acids, bilirubin, and steroids, and is thus essential for fetal development.
  • AFP refers to the human AFP, that comprises the amino acid sequence as denoted by UniProt accession number P02771.
  • the human AFP as used herein comprises the amino acid sequence as denoted by SEQ ID NO: 1, or any homologs or variants thereof.
  • Human Chorionic Gonadotropin (hCG) as used herein is a hormone produced by the placenta. Abnormal levels can indicate issues like multiple pregnancies or risk of chromosomal abnormalities.
  • hCG refers to the human hCG, that comprises the amino acid sequence as denoted by UniProt accession number P01215.
  • the human hCG as used herein comprises the amino acid sequence as denoted by SEQ ID NO: 2, or any homologs or variants thereof.
  • the present disclosure provides in some aspects thereof, a battery of bio transistors useful for monitoring pregnancy and female health.
  • Another aspect of the present disclosure related to a method for determining presence and/or quantity of at least one target molecule in at least one sample.
  • the method comprising: in step (a), contacting the at least one sample with a bio-transistor having an active region carrying at least one affinity moieties, or a battery comprising at least two of the bio-transistors.
  • each affinity moiety is specific for a target molecule.
  • the one or more affinity moieties are selected moieties.
  • the bio transistor or battery used by the disclosed methods may comprise one or more different or similar or identical affinity moieties.
  • each one of the at least one affinity moiety is specific for one target molecule.
  • step (b) performing one or more measurements, each measurement comprising: applying a selected electric potential on the sample, and determining current transmission profile through a channel of the bio-transistor with respect to potential variation of at least one gate electrode of the bio-transistor; and (c) processing data on the current transmission through the channel for one or more selected gate potential and one or more selected electric potential values applied on the sample and determining presence and/or quantity of the one or more or the at least one target molecules in accordance with pre-stored calibration data.
  • the bio-transistor used by the method comprising: (i) at least one channel; (ii) source and drain electrodes; (iii) at least one gate electrode; and (iv) at least one additional electrode positioned to be in electrical contact with the sample.
  • the at least one active region is located in proximity to the channel region, separated from the channel region by an electrically insulating layer.
  • the control unit thereby configured to determine data on presence and/or quantity of one or more target molecules in the sample.
  • the method comprising performing two or more measurements, wherein each measurement comprises applying respective selected different potential on the sample.
  • bio-transistor used in the disclosed methods is as defined by the present disclosure, herein above.
  • the methods and systems of the present disclosure may be applicable for any organism of the biological kingdom Animalia.
  • such organism may be any unicellular or multicellular invertebrate or vertebrate organism.
  • invertebrates may be organisms of the Phylum Porifera - Sponges, the Phylum Cnidaria - Jellyfish, hydras, sea anemones, corals, the Phylum Ctenophora - Comb jellies, the Phylum Platyhelminthes - Flatworms, the Phylum Mollusca - Molluscs, the Phylum Arthropoda - Arthropods, the Phylum Annelida - Segmented worms like earthworm and the Phylum Echinodermata - Echinoderms.
  • the methods of the present disclosure may be applicable for any vertebrate organism, specifically, any organism derived from any of the vertebrates groups that include Fish, Amphibians, Reptiles, Birds and Mammals (e.g., Marsupials, Primates, Rodents and Cetaceans).
  • the methods of the present disclosure may be applicable for a mammal (specifically, at least one of a human, Cattle, rodent, domestic pig (swine, hog), sheep, horse, goat, alpaca, lama and Camels).
  • Vertebrates comprise all species of animals within the subphylum Vertebrata (chordates with backbones).
  • the animals of the vertebrates group include Fish, Amphibians, Reptiles, Birds and Mammals (e.g., Marsupials, Primates, Rodents and Cetaceans).
  • Vertebrates represent the overwhelming majority of the phylum Chordata, with currently about 66,000 species described. Vertebrates include the jawless fish and the jawed vertebrates, which include the cartilaginous fish (sharks, rays, and ratfish) and the bony fish. Still further, in some embodiments, the disclosed bio transistor systems and methods may be applicable for samples obtained from any one of a human or non-human mammal, an avian, an insect, a fish, an amphibian, a reptile, a crustacean, a crab, a lobster, a snail, a clam, an octopus, a starfish, a sea-urchin, jellyfish ⁇ and worms.
  • the subject of the present disclosure may be a mammal.
  • such mammalian organisms may include any member of the mammalian nineteen orders, specifically, Order Artiodactyla (even-toed hoofed animals), Order Carnivora (meat-eaters), Order Cetacea (whales and purpoises), Order Chiroptera (bats), Order Dermoptera (colugos or flying lemurs), Order Edentata (toothless mammals), Order Hyracoidae (hyraxes, desserties), Order Insectivora (insect- eaters), Order Lagomorpha (pikas, hares, and rabbits), Order Marsupialia (pouched animals), Order Monotremata (egg-laying mammals), Order Perissodactyla (odd-toed hoofed animals), Order Pholidata, Order Pinnipedia (seals and walruses), Order Primates (primates), Order
  • the present disclosure may be applicable for any organism of the order primates. More specifically, primates are divided into two distinct suborders, the first is the strepsirrhines that includes lemurs, galagos, and lorisids. The second is haplorhines - that includes tarsier, monkey, and ape clades, the last of these including humans.
  • the present disclosure may be applicable for any organism of the subfamily Homininae, that includes the hylobatidae (gibbons) and the hominidae that includes ponqunae (orangutans) and homininae [gorillini (gorilla) and hominini ((panina(chimpanzees) and hominina (humans))].
  • a subject as disclosed herein relates to a human subject.
  • the human subject may be of any sex, ethnic group, age or physical or mental condition.
  • the methods of the present disclosure may be applicable for a mammal that may be at least one of a Cattle, domestic pig (swine, hog), sheep, horse, goat, alpaca, lama and Camels.
  • the subject the present disclosure as well as the methods disclosed herein above offer great economic advantage for any industrial or agricultural use of animals, specifically, livestock.
  • the present disclosure may be applicable for mammalian livestock, specifically those used for meat, milk and leather industries.
  • Livestock are domesticated animals raised in an agricultural setting to produce labor and commodities such as meat, eggs, milk, fur, leather, and wool.
  • the term includes but is not limited to Cattle, sheep, domestic pig (swine, hog), horse, goat, alpaca, lama and Camels.
  • cattle applicable in the meat and milk industry, as well as in the leather industry.
  • the subject of the present disclosure may be Cattle, colloquially cows, that are the most common type of large, domesticated ungulates, that belong to the Bovidae family.
  • the organism applicable in the methods of the present disclosure may be avian organisms.
  • the present disclosure may be suitable for birds. More specifically, domesticated and undomesticated birds are also suitable organisms for the present disclosure.
  • the disclosed bio transistor systems and methods may be applicable for samples obtained from organism of the biological kingdom Plantae that may be a dioecious plant, specifically, a plant presenting biparental reproduction.
  • the plant may be of the family Cannabaceae, specifically, any one of Cannabis (hemp, marijuana) and Humulus (hops).
  • the plant of the family Cannabaceae may be Cannabis (hemp, marijuana).
  • the plant of the family Cannabaceae may be Humulus (hops).
  • any plants are applicable in the present disclosure, for example, any model plants such as, Arabidopsis, Tobacco, Solanum licopersicum, Solanum tuberosum.
  • Canola, Cereals (Corn wheat, Barley), rice, sugarcane, Beet, Cotton, Banana, Cassava, sweet potato, lentils, chickpea, peas, Soy, nuts, peanuts, Lemna, Apple, may be applicable in the present disclosure.
  • a non-comprehensive list of useful annual and perennial, domesticated or wild, monocotyledonous or dicotyledonous land plant or Algae - i.e unicellular or multicellular algae including diatoms, microalgae, ulva, nori, gracilaria
  • applicable in accordance with the present disclosure may include but are not limited to crops, ornamentals, herbs (i.e., labiacea such as sage, basil and mint, or lemon grass, chives), grasses (i.e., lawn and biofuel grasses and animal feed grasses), cereals (i.e., rice, wheat, rye, oats, corn), legumes (i.e.
  • Crucifera i.e., oilseed rape, mustard, brassicas, cauliflower, radish
  • Sesame the monocot Aspargales (i.e. onion, garlic, leek, asparagus, vanilla, lilies, tulips, narcissus), Myrtacea (i.e., Eucalyptus, pomegranate, guava), Subtropical fruit trees (i.e. Avocado, Mango, Litchi, papaya), Citrus (i.e. orange, lemon, grapefruit), Rosacea (i.e. apple, cherry, plum, almond, roses), berry-plants (i.e.
  • grapes mulberries, blueberries, raspberry, strawberry
  • nut trees i.e. macademia, hazelnut, pecan, walnut, chestnuts, brazil nut, cashew
  • palms i.e., oil-palm, coconut and dates
  • evergreen coniferous or deciduous trees, woody species.
  • the disclosed methods are applicable for eukaryotic organism that may be at least one organism of the biological kingdom Animalia or of the biological kingdom Plantae.
  • the eukaryotic organism of the biological kingdom Animalia may be an insect, avian, fish and a mammalian subject.
  • the bio transistor system used by the disclosed method is based on a receptor-ligand binding pair that comprise at least one ligand comprising a small molecule chemical substance, and at least one affinity moiety comprising an amino acidbased receptor molecule for the ligand.
  • the chemical substance used herein as the target molecule is a small molecule toxic for at least one eukaryotic organism.
  • the toxic small molecule chemical substance is at least one organophosphate.
  • the comprise at least one of pesticides, herbicides, and/or chemical warfare agents.
  • the at least one organophosphate is diethylcyanophosphonate (DCNP).
  • the receptor is hexafluoroisopropanol (HFIP).
  • the at least one sample is a biological sample and/or environmental sample.
  • the present disclosure provides methods particularly useful for detecting, quantifying and/or monitoring organophosphate contamination in a sample, specifically, an environmental sample.
  • the disclosed bio transistor systems and methods provide a sensitive and specific tool for detecting organophosphates in a sample, specifically a an enviromental sample. In some embodiments, the disclosed bio transistor systems and methods detect organophosphate such as DCNP in a sample in an amount of about Ipg/ml to about 100 ug/ml.
  • the present disclosure provides methods for determining the presence and/or the quantity of a target molecule produced naturally in an organism.
  • any of the eukaryotic organisms disclosed by the present disclosure specifically, at least one mammalian organism.
  • the disclosed methods are particularly applicable for determining the presence and/or the quantity of a target molecule that is at least one antigen comprising a small molecule steroid compound.
  • the at least one affinity moiety of the bio-transistor systems and/or at least one battery thereof used by the disclosed methods may comprise an antibody specific for such antigen.
  • at least one antibody specific for the target small molecule steroid compound may comprise an antibody specific for such antigen.
  • the small molecule steroid compound used as a target by the disclosed methods is a small molecule steroid compound naturally produced by at least one eukaryotic organism. Specifically, at least one small molecule steroid compound produced by a human subject.
  • the small molecule steroid compound is of the estrogen family. In some specific embodiments, the small molecule steroid compound is Estriol. Accordingly, the at last one affinity moiety of the biotransistor system used in the disclosed methods is at least one antibody, specifically, at least one anti-Estriol antibody or any functional fragments thereof. In yet some further embodiments, the disclosed methods are applicable for determining the presence and/or quantity of at least one small molecule steroid compound, specifically of estriol in a sample, specifically a biological sample. In some specific embodiments the biological sample may be a blood sample and/or a serum sample. In yet some further embodiments, the sample is a serum sample obtained from a pregnant female subject.
  • the disclosed bio transistor systems and methods provide a sensitive and specific tool for detecting Estriol in a sample, specifically a biological sample such as serum sample. In some embodiments, the disclosed bio transistor systems and methods detect Estriol in a serum sample in an amount of about Ifg/ml to about 10 ug/ml.
  • the present disclosure provides methods for determining the presence and/or quantity of at least one maternal serum marker.
  • the maternal serum marker comprises estriol, as well as at least one additional maternal serum marker.
  • the present disclosure provides methods using a battery of bio transistor systems comprising:
  • At least one bio-transistor system comprising at least one affinity moiety specific for Estriol.
  • the affinity moiety is or comprises at least one anti- Estriol antibody.
  • the disclosed battery further comprises at least one of:
  • the affinity moiety is or comprises at least one anti-AFP antibody.
  • the disclosed battery comprises additionally, or alternatively, (c), at least one bio-transistor system comprising at least one affinity moiety specific for human chorionic gonadotropin (hCG).
  • hCG human chorionic gonadotropin
  • the affinity moiety is, or comprises at least one anti- hCG antibody.
  • the present disclosure thus provides effective methods for determining and monitoring pregnant female subjects during pregnancy.
  • Another aspect of the current disclosure is a diagnostic method for determining a physiological and/or environmental condition or state of a subject and/or a media and/or a habitat.
  • the method comprising: (a) contacting the at least one sample with a bio-transistor having an active region at least one affinity moiety, or a battery comprising at least two of said bio-transistors.
  • each affinity moiety is specific for a target molecule.
  • the active region is modified to include the disclosed affinity moieties.
  • step (b) performing one or more measurements, each measurement comprising: applying a selected electric potential on the sample, and determining current transmission profile through a channel of the bio-transistor with respect to potential variation of at least one gate electrode of the bio-transistor.
  • step (c) involves processing data on the current transmission through the channel for one or more selected gate potential and one or more selected electric potential values applied on the sample.
  • step (d) determining presence and/or quantity of the one or more target molecules in accordance with pre-stored calibration data, thereby obtaining a target molecule value for the sample.
  • step (e) determining that the subject and/or media and/or habitat display the physiological and/or environmental condition or state, if the at least one target molecule value obtained for the sample in step (d), is positive or negative with respect to a reference target molecule value pre-determined for the physiological and/or environmental condition or state, or with respect to a target molecule value determined for at least one control sample.
  • bio-transistor system used by the diagnostic methods is as defined by the present disclosure herein above.
  • the physiological state and/or condition of a subject comprises pathological condition/s and/or health condi tion/s in the subject.
  • the physiological state and/or condition includes the nerve system, the respiratory system, the skin, the metabolic state, behavioral and/or mental state and/or organ failure, tissue damage, etc.
  • the bio-transistor system and/or battery used by the disclosed diagnostic methods may comprise at least one affinity moiety comprising HFIP. More specifically, the affinity moiety of the bio-transistor system used by the methods is specific for the target DCNP. It should be understood that the at least one affinity moiety used by the disclosed methods is or comprises HFIP that is a receptor molecule for the organophosphate DCNP.
  • the method is for the diagnosis of organophosphate/s contamination in a media and/or habitat.
  • the disclosed methods are applicable for the diagnosis of a pathological condition in a subject, caused by, or associated with exposure to a media and/or habitat contaminated by the organophosphate/s. specifically, any pathologic disorder caused by exposure to DCNP.
  • Organophosphate is a chemical compound that contains a phosphate group bonded to an organic moiety.
  • Organophosphates are widely used in various industries, including agriculture, medicine, and chemical manufacturing.
  • Organophosphates are commonly used as insecticides. Examples include malathion, parathion, and chlorpyrifos.
  • Organophosphates act by inhibiting acetylcholinesterase, an enzyme essential for nerve function in insects, leading to their death.
  • organophosphates such as sarin, tabun, and VX, are highly toxic and used as chemical warfare agents. They also inhibit acetylcholinesterase, causing severe neurological effects in humans.
  • Organophosphates are employed as plasticizers, flame retardants, and lubricants in various industrial processes.
  • Organophosphates inhibit acetylcholinesterase, an enzyme that breaks down acetylcholine, a neurotransmitter. This inhibition leads to an accumulation of acetylcholine in synapses, causing continuous stimulation of muscles, glands, and central nervous system pathways.
  • pathologic disorders caused by exposure to organophosphates include acute toxicity, as well as chronic exposure. More specifically, symptoms of acute organophosphate poisoning include headache, dizziness, nausea, vomiting, abdominal pain, muscle weakness, and respiratory distress. Severe cases can lead to convulsions, coma, and death. Still further, chronic exposure include long-term exposure to low levels of organophosphates can result in neurological and cognitive impairments, including memory loss, mood changes, and peripheral neuropathy.
  • the present disclosure provides diagnostic methods based on detection and determination of the quantity of estriol. Accordingly, the disclosed diagnostic methods use the bio-transistor systems and/or battery that comprise at least one affinity moiety specific for estriol. In some embodiments, such affinity moiety is or comprises at least one anti-estriol antibody. In yet some further embodiments, step (d) of the disclosed diagnostic methods results in obtaining the estriol value for a maternal serum sample. Thus, in some embodiments, the detection and/or quantification of the estriol levels by the disclosed bio-transistor system, is of a diagnostic value.
  • a level of the target estriol determined by the disclosed bio-transistor systems is indicative of at least one genetic disorder associated with chromosomal abnormalities.
  • chromosomal abnormalities comprise at least one trisomy.
  • low levels of estriol, as compared to a standard level is indicative of a trisomy 21, for example, Down Syndrome (DS), and/or a trisomy 18, for example, Edward's Syndrome.
  • Standard estriol levels for non-pregnant adults are 80-130 fg/mL, for pregnant women levels can be much higher and vary depending on the stage of pregnancy.
  • estriol secretion is increased by 1000 times during pregnancy due to placental synthesis reaching at 37 weeks onwards a concentration of 80-130 pg/ml.
  • the present disclosure provides diagnostic methods that display a profile of maternal serum markers. More specifically, in some embodiments the diagnostic methods of the present disclosure may use a battery of two or more of the biotransistor systems disclosed herein. In some specific embodiments, the two or more of the bio-transistor systems of the battery used by the disclosed diagnostic methods may comprise, in addition to a bio-transistor system comprising estriol specific antibody, also at least one bio-transistor system comprising at least one affinity moiety for at least one additional maternal serum marker. According to such embodiments, the battery used by the disclosed diagnostic methods may comprise:
  • the affinity moiety comprises anti-Estriol antibody.
  • the battery used by the disclosed diagnostic methods may further comprise at least one of: (ii), at least one bio-transistor system comprising at least one affinity moiety specific for alphafetoprotein (AFP). In some embodiments, the affinity moiety comprises anti-AFP antibody.
  • the battery used by the disclosed diagnostic methods may comprise additionally, or alternatively, (iii), at least one bio-transistor system comprising at least one affinity moiety specific for human chorionic gonadotropin (hCG). In some embodiments, the affinity moiety may comprise anti- hCG antibody.
  • step (d) of the disclosed diagnostic methods results in obtaining the estriol value for at least one maternal serum sample, and optionally of at least one of, the AFP value and/or the hCG value of the at least one maternal serum sample.
  • the bio-transistor system of the present disclosure is specifically designed for detecting and/or quantifying a target molecule that is the Alpha-fetoprotein (AFP).
  • AFP Alpha-fetoprotein
  • the at least one affinity moiety carried by the active region of the bio-transistor system of the present disclosure comprises, is, or is derived from, at least one antibody specific for AFP.
  • the detection and/or quantification of the estriol and at least one of AFP and hGC levels by the disclosed bio-transistor systems and battery is of a diagnostic value. More specifically, in some embodiments, a level of the target estriol and at least one of AFP and hGC determined by the disclosed bio-transistor systems, that is below a standard level (also referred to herein as a "negative"), is indicative of at least one genetic disorder associated with chromosomal abnormalities. In some specific embodiments, such chromosomal abnormalities comprise at least one trisomy.
  • low levels of estriol, and of AFP and/or hGC, as compared to a standard level is indicative of a trisomy 21, for example, Down Syndrome (DS), and/or a trisomy 18, for example, Edward's Syndrome.
  • Standard AFP levels for non-pregnant adults are less than 10 ng/mL, for pregnant women levels can be much higher and vary depending on the stage of pregnancy.
  • Standard Estriol levels for non- pregnant adults are less than 150 fg/mL, for pregnant women levels can be much higher and vary depending on the stage of pregnancy.
  • the highest sensitivity of 77.56 iNormaii d per dec for a dynamic range of Ifg/ml to lOpg/ml with LOD of 1 fg/ml.
  • the disclosed methods involve in the first step determination of the level of specific target molecule to obtain the value for each target in the sample, as will be elaborated herein after.
  • the next step involves determination if the expression value is positive or negative. It should be understood that determination of a "positive” or alternatively “negative” value of the target molecule levels and/or amount with respect to a standard value or a control value may involve in some embodiments comparison of the value determined for the quantity and/or mount) of the target molecule of the examined sample as obtained in step (d), with the amount/level value of the target obtained for a control sample, or from any established or predetermined value of the target molecule level and/or amount and/or quantity (e.g., a standard value) obtained from a known control (either healthy controls or of subjects suffering from a pathological disorder).
  • "positive” is meant a value that is higher, increased, elevated, overexpressed in about 5% to 100% or more, specifically, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, when compared to the amount/level/quantity value of the reference and/or the standard value of a healthy control, any other suitable control or any other predetermined standard.
  • a "negative” value in some embodiments may be a reduced, low, non-existing or lack of expression of a target molecule in about 5% to 100% or more, specifically, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, when compared to the value of the amount of the target molecule in a healthy control, any other suitable control or any other predetermined standard.
  • “healthy controls” or “healthy population” may refer to a population of subjects that does not suffer from a disease of interest or refer to a population before appearance of a disease of interest.
  • the value of the target molecule in a control population refers to a baseline level of the target molecule of a healthy population or to a baseline level of the target molecule before appearance of a disease in a studied population.
  • a “healthy control” or “control” may refer to the to a baseline level of the target molecule before appearance of a disease in a specific subject.
  • the subject is classified as a subject that display a specific disorder as indicated herein for each of the target molecules.
  • a “reference value” or a “Standard” or a “predetermined standard” or a “predetermined reference value” as used herein, denotes either a single standard value or a plurality of standards with which the level of at least one of the target molecule/s from the tested sample is compared.
  • the standards may be provided, for example, in the form of discrete numeric values or in the form of a comparative curve prepared on the basis of such standards (standard curve).
  • the method of the invention involves comparing the values of the amount and/or quantity of the target molecule/s determined for the tested sample with predetermined standard values or cutoff values, or alternatively, with the values of at least one control sample.
  • comparing denotes any examination of the level and/or amount and/or quantity values obtained in the samples disclosed herein as detailed throughout in order to discover similarities or differences between at least two different samples. It should be noted that in some embodiments, comparing according to the present disclosure encompasses the possibility to use a computer-based approach.
  • Step (a) of the disclosed methods involves the action of contacting the bio transistors of the present disclosure with the examined sample.
  • the term "contacting” means to bring, put or incubates together.
  • a first component e.g., affinity moiety
  • a second component specifically, the target molecule when the two components are brought or put together, e.g., by touching them to each other or combining them.
  • the term "contacting” includes all measures or steps which allow interaction between the at least one of the affinity moieties of at least one of the target molecules.
  • the contacting is performed in a manner so that the at least one of affinity moieties of at least one of the target molecules, can interact with or bind to the target molecule in the tested sample.
  • the binding will preferably be non-covalent, reversible binding, e.g., binding via salt bridges, hydrogen bonds, hydrophobic interactions or a combination thereof.
  • the diagnostic methods disclosed herein are applicable for the diagnosis of neural tube defects and/or genetic abnormalities in a fetus.
  • chromosomal abnormalities or genetic abnormalities refer to changes in the normal structure or number of chromosomes in cells, which can lead to developmental and health issues. These abnormalities can be classified into several types, specifically, aneuploidy and structural abnormalities, each with specific characteristics and potential impacts. More specifically, Aneuploidy, relate to the presence of an extra chromosome or the absence of a chromosome. Examples include trisomy and monosomy. More specifically, Trisomy, having three copies of a chromosome instead of the usual two. Common trisomies include Trisomy 21 (Down syndrome), Trisomy 18 (Edwards syndrome), and Trisomy 13 (Patau syndrome). Monosomy, having only one copy of a chromosome instead of two, for example, Turner syndrome (Monosomy X), where an individual has only one X chromosome.
  • Turner syndrome Monosomy X
  • Structural abnormalities involve changes in the structure of chromosomes, and include deletions, where a portion of the chromosome is missing or deleted (e.g., Cri-du-chat syndrome, caused by a deletion on chromosome 5); duplications, where a portion of the chromosome is duplicated, resulting in extra genetic material; translocations, where segment of one chromosome is transferred to another chromosome; inversions, where a chromosome segment breaks off, flips around, and reattaches, changing the order of the genes, and rings, where a chromosome forms a ring structure due to deletions in telomeres, causing the ends to fuse.
  • deletions where a portion of the chromosome is missing or deleted (e.g., Cri-du-chat syndrome, caused by a deletion on chromosome 5); duplications, where a portion of the chromosome is duplicated, resulting in extra genetic material; translocations, where segment of one chromos
  • Another aspect of the present disclosure is a screening method for identifying a compound that modulates the interaction of an affinity moiety with a target molecule in at least one sample.
  • the method comprising: (i) contacting the at least one sample with a bio-transistor system, in the presence and the absence of at least one candidate compound.
  • the biotransistor having an active region (also referred to herein in some embodiments as a modified active region) carrying selected at least one type of affinity moieties; (ii) performing one or more measurements for each sample, each measurement comprising: applying a selected electric potential on the sample, and determining current transmission profile through a channel of the bio-transistor with respect to potential variation of at least one gate electrode of the bio-transistor; and (iii) processing data on the current transmission through the channel for one or more selected gate potential and one or more selected electric potential values applied on the sample; and (iv) determining presence and/or quantity of the one or more target molecules in accordance with pre-stored calibration data, thereby determining a target molecule value for the sample in the presence of the candidate compound, and a target molecule value in the absence of the candidate compound; and (v) determining that the candidate compound is a modulator of the interaction between the affinity moiety and the target molecule, if the target molecule value obtained for the sample in the presence of the candidate compound is different from
  • the bio-transistor system is as defined above.
  • the disclosed screening method is particularly applicable for screening of a compound that inhibits the interaction of the affinity moiety with the target molecule.
  • a compound that "inhibits the interaction" as used herein refers to a compound that reduces, prevents, blocks, impedes, suppresses, prevents, hinders and/or restricts interaction between the at least one affinity moiety and the target molecule either partially, or completely. For example, any inhibition of between about 10-50%, 51-80%, 81-99% or even, of 100% of the interaction or binding between the affinity moiety and the target molecule.
  • the present disclosure further provides personalized therapeutic methods that comprise a diagnostic step using the bio-transistor systems and methods of the present disclosure, followed by administration of a therapeutic compound, for example, any compound that inhibits damage caused by acute or chronic exposure to at least one organophosphate compound.
  • a therapeutic compound for example, any compound that inhibits damage caused by acute or chronic exposure to at least one organophosphate compound.
  • treat means preventing, ameliorating or delaying the onset of one or more clinical indications of disease activity in a subject having a pathologic disorder.
  • Treatment refers to therapeutic treatment. Those in need of treatment are subjects suffering from a pathologic disorder. Specifically, providing a "preventive treatment” (to prevent) or a “prophylactic treatment” is acting in a protective manner, to defend against or prevent something, especially a condition or disease.
  • treatment or prevention refers to the complete range of therapeutically positive effects of administrating to a subject including inhibition, reduction of, alleviation of, and relief from, an immune-related condition and illness, immune-related symptoms or undesired side effects or immune-related disorders. More specifically, treatment or prevention of relapse or recurrence of the disease, includes the prevention or postponement of development of the disease, prevention or postponement of development of symptoms and/or a reduction in the severity of such symptoms that will or are expected to develop. These further include ameliorating existing symptoms, preventing- additional symptoms and ameliorating or preventing the underlying metabolic causes of symptoms.
  • the terms “inhibition”, “moderation”, “reduction”, “decrease” or “attenuation” as referred to herein, relate to the retardation, restraining or reduction of a process by any one of about 1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85% about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, 100% or more.
  • percentage values such as, for example, 10%, 50%, 120%, 500%, etc., are interchangeable with "fold change” values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively.
  • amelioration as referred to herein, relates to a decrease in the symptoms, and improvement in a subject's condition brought about by the methods according to the present disclosure, wherein said improvement may be manifested in the forms of inhibition of pathologic processes associated with the immune-related disorders described herein, a significant reduction in their magnitude, or an improvement in a diseased subject physiological state.
  • inhibitor and all variations of this term is intended to encompass the restriction or prohibition of the progress and exacerbation of pathologic symptoms or a pathologic process progress, said pathologic process symptoms or process are associated with.
  • delay means the slowing of the progress and/or exacerbation of a disorder associated with the immune-related disorders and their symptoms slowing their progress, further exacerbation or development, so as to appear later than in the absence of the treatment according to the present disclosure.
  • the diagnostic kit comprising: (a) at least one bio-transistor system, and optionally, at least on of: (b) at least one control sample; and (c) at least one therapeutic agent.
  • the bio-transistor system comprises at least one transistor unit and a control system.
  • the transistor unit comprising: (i) at least one channel; (ii) source and drain electrodes; (iii) at least one gate electrode; (iv) at least one active region located in proximity to the channel region and carrying at least one affinity moiety. It should be understood that each affinity moiety is specific for a target molecule.
  • the at least one active region is configured for accepting at least one sample; and (v) at least one additional electrode positioned to be in electrical contact with the sample.
  • the control system comprising at least one processor and memory circuitry.
  • the control system is configured and operable for performing one or more measurements of the sample, wherein each measurement comprises maintaining a selected electric potential on the at least one additional electrode and determining current transmission profile through the at least one channel with respect to potential variation of said the least one gate electrode.
  • the control unit thereby configured to determine on the presence and/or quantity of one or more target molecules in the sample.
  • the kit is adapted for performing any of the methods of the present disclosure as described herein above.
  • the kit provided by the present disclosure is adapted for performing a method for the diagnosis of organophosphate/s contamination in a media and/or habitat and/or for the diagnosis of a pathological condition in a subject caused by exposure to a media and/or habitat contaminated by the organophosphate/s.
  • the bio-transistor system provided with the disclosed kits comprise as the at least one affinity moiety at least one receptor for at least one organophosphate, specifically, HFIP that is a receptor molecule for the organophosphate DCNP.
  • the present disclosure provides a kit adapted for performing a method for the diagnosis of neural tube defects and/or genetic abnormalities in a fetus.
  • the bio-transistor system provided with the disclosed kits comprise as the at least one affinity moiety at least one antibody that specifically recognizes at least one maternal serum marker, for example, estriol.
  • the disclosed kits may comprise at least one bio-transistor systems that comprise at least one affinity moiety specific for at least one additional maternal serum marker.
  • a kit of the present disclosure may comprise according to some embodiments, bio-transistor systems comprising affinity moiety for estriol, and bio-transistor system comprising at least one affinity moiety specific for AFP and/or at least one affinity moiety specific for hCG, specifically, anti-AFP antibodies, and/or anti- hCG antibodies.
  • composition or method may include additional ingredients and/or steps, and/or parts, but only if the additional ingredients and/or steps do not materially alter the basic and novel characteristics of the claimed composition or method.
  • word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
  • the silicon FET device is realized with a silicon-on-insulator (SOI) technology.
  • the thickness of the SOI device layer in 145 nm and the thickness of the buried oxide is 400 nm.
  • the n-type device layer is doped with 10 17 cm' 3 , the lateral p-type regions and the n-type source and drain regions are all degenerated.
  • the distance between the lateral p-type region define the width of the channel volume, and the distance between the source and drain define the length of the channel volume (10 pm).
  • Standard CMOS cobalt-silicide is performed for all silicon regions contacting the aluminum lines to ensure ohmic contacts with low contact resistance.
  • the sensing area is composed of 5.5 nm thermal SiCh.
  • the entire chip is passivated with 1 pm SiCh except the sensing area and the contacting pads.
  • the aminophenyl-HFIP recognition layer is first developed on 1 cm X 1 cm substrates of silicon decorated with 10 nm SiO2.
  • the samples are thoroughly cleaned using a 2-minute sonication and 1 -minute rotation in ethyl acetate, acetone, 2-propanol, and deionized (DI) water, successively.
  • the surfaces are activated using 4-minute immersion in a solution 4:1 H2SO4:H2O2 (piranha), followed by DI rinse and dried with N2.
  • the samples are incubated in a 0.1% v:v solution of 3 -aminopropyl trimethoxysilane (APTMS) in methanol for three hours.
  • API 3 -aminopropyl trimethoxysilane
  • the samples are sonicated and rinsed several times to remove excess of APTMS, and hydrolysed for 24 hours followed by 1 hour bake at 120°C.
  • the samples are immersed in 0.5% glutaraldehyde for 40 minutes and washed with a 10 rnM PBS followed by drying with N2.
  • the samples are incubated in aminophenyl-HFIP (B24048, Thermo Scientific) solution for 12 h at 4°C (0.1% aminophenyl-HFIP in dimethylformamide (DMF)), cleaned with 10 mM PBS solution and dried with N2.
  • the samples are characterized with electrochemical impedance spectroscopy (EIS) by Palmsens 4, Palmsens Inc.
  • the EIS is performed in a three-electrode 0.8 mL electrochemical cell filled with 0.1 mM PBS pH7.4. Conductive carbon tape is applied at the backside of the substrates which function as the working electrodes.
  • the counter electrode is made of platinum (SEC-C Pt Gauze, ALS Japan), and an Ag/AgCl reference electrode is used (RE- 1BP, ALS Japan).
  • Ellipsometer Alpha-SE Ellipsometer, J.A. Woollam
  • contact angle tensiometer Model OCA 20, dataphysics
  • the MNC device is modified into an MNC bioFET as the active sensing area is biofunctionalized with anti-estriol antibodies.
  • the process is developed using 2 cm X 2 cm silicon samples decorated with 10 nm SiCh layer. Subsequently, the same process is applied to the MNC devices.
  • the sample is thoroughly cleaned using ethyl acetate, acetone, 2-propanol, and deionized (DI) water. The samples are treated with each of these for 2 minutes in a sonicator and rotated for 1 minute.
  • Surface activation (the formation surface hydroxyls) is performed using a piranha solution (4: 1 FhSO ⁇ FhCfe) for 4 minutes.
  • the sample After rinsing with DI water and drying with an N2 gun, the sample is incubated in a 0.1% v:v solution of 3-aminopropyltrimethoxysilane (APTMS) in methanol for 3 hours. Excess APTMS is removed using sonication and rinsing with methanol in multiple cycles. The sample is transferred into DI water for 24 hours to facilitate hydrolysis to promote the extension of amine moieties away from the surface [Zhu, M., et al. Langmuir 28, 416-423 (2012]. To stabilize the covalent bonds between the silane molecules and the oxide surface, the sample is placed in an oven for 1 hour at a temperature of 120°C. This helps to ensure the durability and stability of the bonds.
  • APTMS 3-aminopropyltrimethoxysilane
  • the next step involves the immobilization of antiestriol antibodies on the SiCh surface.
  • the sample is immersed in a 0.5% glutaraldehyde (GA) crosslinker solution, providing an anchor for the antibodies.
  • the sample is incubated for 12 hours in a solution containing 1 pg/ml of anti-estriol (abl50407, Abeam) in 10 mM TBS buffer at pH 7.4, maintaining a temperature of 4°C.
  • This strategy is based on the known principle that antibodies are typically immobilized to aldehyde-terminated surfaces by the reaction between the amine moieties of the antibody and the aldehyde group.
  • the different biofunctionalization steps are characterised by spectroscopic ellipsometry (Alpha-SE Ellipsometer, J.A. Woollam), electrochemical impedance spectroscopy (EIS) (Palmsens 4, Palmsens Inc.), and contact angle tensiometer (Model OCA 20, dataphysics).
  • EIS electrochemical impedance spectroscopy
  • the EIS measurements are conducted in a 0.1 mM PBS buffer, pH of 7.4.
  • the sample is positioned within a three-electrode electrochemical cell (0.8 mL).
  • the back side of the silicon working electrode is scratched and contacted with conductive carbon paint.
  • a Pt wire (SEC-C Pt Gauze working electrode, ALS Japan) serves as the counter electrode, and an Ag/AgCl electrode serves as the reference electrode (RE- IBP, ALS Japan).
  • RE- IBP reference electrode
  • the MNC decorated with devices were biofunctionalized and transformed into MNC biosensors.
  • the biofunctionalization was first developed on plain silicon samples (2 X 2 cm) 5 nm of SiO2.
  • the various involved biofunctionalization steps were characterized by electrochemical impedance spectroscopy (EIS) (Palmsens 4, Palmsens Inc.), contact angle tensiometer (Model OCA 20, dataphysics), and spectroscopic ellipsometry (J.A. Woollam Alpha-SE Ellipsometer).
  • EIS electrochemical impedance spectroscopy
  • the samples were successively cleaned with ethyl acetate, acetone, and 2- propanol for 2 min in a bath sonicator for each cleaning step.
  • the cleaning was followed by surface activation with piranha for 4 minutes (4: 1 ratio of H2SO4:H2O2), DI water wash and N2 drying.
  • the next step was surface chemical modification with 3- aminopropyltrimethoxysilane (APTMS) linker molecules.
  • APTMS 3- aminopropyltrimethoxysilane
  • the samples were incubated in APTMS solution (0.1% v:v in methanol) for 3 hours, followed by three cycles of 3 minutes each of sonication in methanol.
  • the samples were hydrolyzed by 24 hours incubation in DI water, dried and placed in an oven for 1 hour at 120°C [M. Zhu, M. Z. Lerum, W. Chen, Langmuir 2012, 28, 416-423. DOI 10.1021/la203638g].
  • the APTMS was modified with glutaraldehyde (GA) (SAB4501531- 100UG, Sigma Aldrich). Finally, the samples were incubated for 12 hours in 1 pg/ml solution (10 mM PBS, pH 7.4) of anti-AFP (Abeam, AB-ab3980).
  • the MNC chips were biofunctionalized in a similar manner. The various involved biofunctionalization steps were characterized by electrochemical impedance spectroscopy (EIS) (Palmsens 4, Palmsens Inc.), contact angle tensiometry (Model OCA 20, dataphysics), and spectroscopic ellipsometry (J.A. Woollam Alpha-SE ellipsometer). The contact angle of anti-AFP modified chip was 52° ⁇ 0.5° and the measured thickness of the anti-AFP layer was 2.2 ⁇ 0.8 nm.
  • EIS electrochemical impedance spectroscopy
  • the contact angle of anti-AFP modified chip was 52° ⁇ 0.5° and the measured thickness of the anti-
  • the electrical measurements are performed on a probe station where the MNChem electrical pads are contacted with needles secured to micro-positioners connected to source measuring units (SMUs) of the B1500 Semiconductor Parametric Analyzer (Keysight Ltd.). 0.5 pL drops of 0.1 mM PBS pH7.4 are drop cast on the MNChem.
  • the drop potential is set by an Ag/Ag + quasi-reference electrode formed from a commercial reference electrode (012171 RE-7, ALS Co., Ltd) where the glass holder is removed and the Ag wire is painted with an Ag/AgCl ink (011464, ALS Co., Ltd).
  • the quasi-reference electrode is secured to a micro-positioner and electrically connected to an additional B1500 SMU.
  • the sensing measurements are performed with respect to the DCNP target molecule (CAS no. 2942-58-7, TCI). Electrical measurements in neutral solutions (for estriol).
  • the electrical measurements of the MNC devices are performed on a probe station.
  • the chip metal pads are contacted with probe needles to establish connections with the source measuring units (SMUs) (B1500 semiconductor parameter analyzer, Keysight Ltd).
  • SMUs source measuring units
  • a pipette is used to manually introduce a 0.5 L drop of 0.1 mM pH 7.4 phosphate-buffered saline (PBS) to the MNC sensing area.
  • the solution gate is realized by physically contacting the drop with a quasi-reference electrode, which comprises an Ag/Ag+ reference electrode (012171 RE-7, ALS Co., Ltd). In this configuration, the glass holder is removed to expose the Ag wire, which is coated with an Ag/AgCl ink (011464, ALS Co., Ltd).
  • An additional B1500 SMU is connected to the quasi-reference electrode to enable potential determination of the solution (VGF).
  • the quasi-reference electrode is securely mounted on a probe manipulator, ensuring stability during the measurements.
  • the drop maintains its integrity for a period of 2-3 minutes before evaporation, offering a sufficient time window for conducting different current-voltage (LV) measurements.
  • LV current-voltage
  • the measurements in a neutral solution are exclusively carried out during the biofunctionalization process of the MNC devices. These measurements serve to validate the functionality of the devices after modification.
  • the remarkable repeatability observed in both unmodified MNC devices and biofunctionalized MNC biosensors underscores the buffer capacity of the small solution drops.
  • it demonstrates the stability of the quasi-reference electrode, accounting for possible variations between successive drops.
  • Sensing measurements commence with the preparation of a 0.5 pL drop of 1:100 diluted plasma with a Tris-buffered saline (TBS) 0.1 mM pH 7.4 solution.
  • TBS Tris-buffered saline
  • the plasma samples are obtained from the Kaplan Medical Center blood bank.
  • the 1: 100 diluted plasma is spiked with estriol molecules (E1253, Sigma-Aldrich).
  • estriol molecules E1253, Sigma-Aldrich
  • a two-step process is employed. Initially, estriol is dissolved in ethanol, where it exhibits solubility, and afterwards this estriol solution is added to the diluted plasma. This approach enables the preparation of estriol concentrations ranging from 1 fg/ml to 10 pg/ml. Importantly, the final solutions all contain the same amount of ethanol (1%).
  • the 1: 100 diluted plasma drop is measured as described earlier for the neutral solution drop. To establish and validate repeatability, a series of successive I-V measurements are performed for each drop. After completing the measurements, the 1:100 diluted plasma drop is carefully collected using a clean-room wipe. To proceed with the next set of measurements, a fresh 1: 100 diluted plasma drop with a higher estriol concentration is introduced onto the MNC sensing area, and the same measurement procedure is repeated. This process is iterated to ensure reliable data collection for various estriol concentrations.
  • the non-specific measurements involve introducing estriol to an unmodified MNC device and exposing an APTMS -modified MNC biosensor to estriol.
  • MNC biosensors modified with anti-estriol antibodies are subjected to the introduction of estrone (E9750, Sigma-Aldrich) and estradiol (E1024, Sigma- Aldrich), both are members of the estrogen family and differ very little from the estriol.
  • concentrations of the small molecules were 10 fg/ml, 10 pg/ml, 10 ng/ml, and 10 pg/ml.
  • the MNC devices were electrically measured on a probe station with probe needles contacting the chip metal pads to source measuring units (SMUs) of the B1500 semiconductor parameter analyzer by Keysight Ltd.
  • SMUs source measuring units
  • a neutral solution of 0.1 mM pH 7.4 phosphate-buffered saline (PBS) with a volume of 0.5 pL was manually applied with a pipette to the MNC sensing area.
  • the drop was electrically contacted with a quasi-reference electrode made of an Ag/Ag + reference electrode (012171 RE-7, ALS Co., Ltd) where the glass holder was removed by exposing the Ag wire which was coated with an Ag/AgCl ink for reference electrode (011464, ALS Co., Ltd).
  • the quasi-reference electrode (VG ) was connected to an additional B1500 SMU for determining potential of the solution.
  • the quasi-reference electrode was mounted on a probe manipulator.
  • the drop lifetime before evaporation is 2-3 minutes, which provides a time window for the various current-voltage (LV) measurements.
  • the I-V measurements were repeated during the drop lifetime, and the measurements were performed for successive applied drops in order to confirm and establish repeatability.
  • the measurements in neutral solution were performed solely during the process of MNC device biofunctionalization in order to validate device functionality post modification. The excellent repeatability of unmodified MNC devices and biofunctionalized MNC biosensors ensure the buffer capacity of the small solution drops [R. E. G. Van Hal, et al.
  • the ALP sensing measurements were performed with 0.5 pL drops of 1: 100 diluted serum obtained from Kaplan Medical Center blood bank. The dilution of the serum was performed with Tris-Buffered saline (TBS) 0.1 mM pH 7.4. Mixtures of diluted serum spiked with different concentrations of AFP (Abeam, ab 114216) were prepared. The serum drop was biased in the same manner described for the neutral solution drop. The stability of the quasireference electrode with respect to drop-to-drop variations of the diluted serum [L. R. F. Allen J.
  • hCG human chorionic gonadotropin
  • PSA prostate specific antigen
  • AFP is a glycoprotein which consists of a polypeptide chain with 591 amino acids and a carbohydrate chain with a molecular mass of -68.8 kDa.
  • concentrations of the biomarkers were 10 ng/ml, 100 ng/ml, and 1 pg/ml corresponding with the highest AFP concentrations (equivalent to 105pM, 1.05nM and 10.5nM, respectively) considered in this work for the specific measurements.
  • the excellent repeatability of the non-specific measurements also removes the concern of pH fluctuations due to the presence of biomolecules in the drops [R. E. G. Van Hal, et al. Sensors Actuators B 1995, 24-25, 201].
  • FIG 4A presents the suggested MNChem sensor. Details of the MNChem are provided in the experimental procedures section. Briefly, the MNChem sensing methodology is based on electrostatic management of both the MNChem solid phase and solution phase to maximize the coupling between the chemical events and IDS, as was already demonstrated for biosensing [8]. This approach is enabled by the presence of multiple gates. IDS is gated by four gates: backgate (VGB) which is grounded in the current work, two lateral gates ( VGL), and a front solution gate (quasi-reference electrode) which also governs the solution potential (VGF)- The MNChem conducting channel IDS is composed of electrons majority carriers present in the channel volume ( Figure 4A).
  • the width of the channel is determined by p-n junctions serving as lateral gates (VGL) reverse biasing of the junctions increases the depletions inside the channel volume and therefore decreases the width of IDS-
  • the solution potential is determined by the quasi-reference electrode (VGF) which also affects the top region of IDS-
  • VGL lateral gates
  • IDS-VGL curves are measured for various VGF values. Such measurements allow the excitations of various channel configurations, differing in shape, size, and location within the channel volume, as well as allowing for different conditions at the sensing area double layer as determined by VGF (i.e. different distributions of ion concentration pH level and electric field) [8].
  • the channel volume is composed of three main channels: top, middle and bottom, as illustrated in the cross-section of Figure 4A.
  • the excitation and the size of each channel depends on the respective gating conditions.
  • Figure 4A also schematically illustrates the process of surface chemical modifications performed for the tethering of the aminophenyl-HFIP receptor molecules on the MNChem sensing area.
  • the sensing area is activated with piranha, (3- Aminopropyl) trimethoxysilane (APTMS) molecules are tethered to the surface, and the aminophenyl-HFIP receptor molecules are tethered to the APTMS molecules with a glutaraldehyde (GA) linker.
  • the different biofunctionalization steps are characterised by spectroscopic ellipsometry (Alpha-SE Ellipsometer, J.A. Woollam), electrochemical impedance spectroscopy (EIS) (Palmsens 4, Palmsens Inc.), and contact angle tensiometer (Model OCA 20, dataphysics).
  • the EIS measurements are conducted in a 0.1 mM PBS buffer, pH of 7.4.
  • the thickness of each layer is measured by ellipsometry and is fixed for the measurement of the successive layer.
  • the mean square error (MSE) is presented as well.
  • the SiO2 thickness is ⁇ 6 nm (Jaw model), per fabrication.
  • the APTMS layer is ⁇ lnm (B-spline model), a bit higher than the expected APTMS monolayer for an APTMS molecule length of ⁇ 5 A chain length, with 1 A per bond.
  • the HFIP thickness is ⁇ 1 nm (B-spline model).
  • the contact angle is measured at three locations for each sample, two measurements at each location, with a total of 6 measurements.
  • the measured contact angle value for SiCh is 59.08° ⁇ 0.73°, for piranha- treated surfaces is 24.5° ⁇ 3.43°, APTMS 35.85° ⁇ 0.42° and HFIP 65.14° ⁇ 3.07°.
  • a clear shift toward hydrophilicity is measured post activation and contact angle values for APTMS and receptor molecules are consistent with previous reports.
  • EIS measurements are performed in a 10 mM pH 7.4 PBS solution for unmodified SiCh (SiCh), SiCh modified with APTMS post hydrolysis (APTMS), and SiCh biofunctionalized with HFIP.
  • Figure 4B presents IDS-VGL for various values of VGF for MNChem modified with aminophenyl-HFIP (aminophenyl-HFIP-MNChem).
  • the measurements are performed in 0.5 pl drops of 0.1 mM PBS pH 7.4.
  • Each data point is the average of 30 data measurements (10 drops, each drop measured 3 times) and the error bars are the corresponding standard deviations (see inset).
  • the excellent repeatability of the aminophenyl-HFIP-MNChem ensures stability of both the sensing area and the quasi-reference electrode VGF with respect to drop-to-drop pH and potential variations.
  • Figure 4B also presents the corresponding extracted 2 nd derivatives of the IDS-VGL curves where each peak reflects the excitation of a channel [7]. Note that the bottom channel is not excited for the selected range of VGF and VGL values. Hence, IDS persists either as a middle channel, top channel, or combination of both. Finally, the excitation voltage (threshold voltage) is higher for lower VGL and VGF values, and each combination of these determines a unique IDS of a distinct shape, size and location in the channel volume.
  • the excitation voltage threshold voltage
  • FIG. 5A presents IDS- VGL curves, for various VGF values, following the introduction of Diethyl cyanophosphonate (DCNP) target molecules to the aminophenyl-HFIP-MNChem.
  • the sensing is performed in 0.5 pL drops of 0.1 mM PBS solution spiked with DCNP concentrations ranging from 100 fg/ml to 100 pg/ml. Each drop contains a different DCNP concentration, and the application performed from the lowest to highest DCNP concentrations.
  • DCNP Diethyl cyanophosphonate
  • INormaiized (IDCNP - IPBS) / IPBS
  • IDCNP IDS measured for a certain DCNP concentration
  • IPBS the baseline IDS measured for PBS containing no DCNP.
  • IDCNP IDS measured for a certain DCNP concentration
  • IDCNP IDS measured for a certain DCNP concentration
  • IPBS the baseline IDS measured for PBS containing no DCNP.
  • the monotonous increase of IDS and INormaiized with increasing DCNP concentrations is evident and reflects the overall accumulation of positive charge at the aminophenyl-HFIP-MNChem sensing area upon binding of the DCNP.
  • the channel configuration, following Figure 4B, is also indicated at the top of the INormaiized curves which reflect the dependency of INormaiized on the selection of VGL and VGF values.
  • Figure 5B presents the control measurements performed to quantify the non-specific signal. To this end four sets of measurements are performed: 1) the introduction of DCNP to an unmodified MNChem, 2) the introduction of DCNP to APTMS -modified MNChem, 3) the introduction of methanol (MeOH) aminophenyl-HFIP-MNChem, and 4) the introduction of tryptophan to aminophenyl-HFIP-MNChem. Both MeOH and tryptophan are polar and can bind the aminophenyl-HFIP layer via hydrogen bonding. As the nonspecific response of the above is very limited and not visible in the IDS-VGL curves, only the respective INormaiized curves are provided for the IDS-VGL curves and INormaiized vs. target molecule concentration.
  • Figure 6 presents calibration curves, that is the dependency of iNormaii ⁇ d on DCNP concentrations for the selected VGL and VGF values.
  • the calibration curves reflect the specific and label-free sensing of DCNP as the non-specific measurements are quantitively accounted for in the following manner: A population of measurements performed for a single drop at a given VGF and VGL values is represented by (Av.) ⁇ (o), where Av. is the average of the drop measurements and o is the standard deviation. Specific sensing is concluded once two criteria are satisfied: 1) The difference between successive drops of Figure 5A must be greater than the average difference between the identical PBS drops of Figure 4B. 2) The difference between successive drops of Figure 5A must be greater than the difference between the respective drops of Figure 5B (control non-specific measurements).
  • Figure 5B The measurements of Figure 5B are performed for four concentrations of target molecules (100 fg/ml, 100 pg/ml, 100 ng/ml, and 100 pg/ml).
  • the selected concentrations of Figure 5B for the comparison with Figure 5A are the smallest ones which are still higher than the relevant Figure 5A concentrations.
  • VGF the channel configuration
  • VGF governs both channel configuration as well as the conditions at the sensing area double layer in terms of pH, ion concentration and electric field distributions [Bhattacharyya et al. ACS Sens 5, (2020].
  • the dependency of hydrogen bonding on pH, ion concentration and electric field, for values characteristic of solid-solution double layer are reported to be negligible [Bhattacharyya et al. ACS Sens 5, (2020)].
  • Figure 7 discloses Table 1 that shows a summary for the sensing performance of the proposed aminophenyl-HFIP-MNChem towards DCNP. Only channels (determined by VGL, VFG values) with linearity equal to or greater than 0.97 and a dynamic range equal to or greater than 8 orders of magnitude, in DCNP concentrations, are selected for presentation. The performance is concluded with an LOD of 100 fg/ml, a dynamic range of 9-10 orders of magnitude and with excellent linearity and sensitivity.
  • the current work demonstrates specific and label-free sensing of DCNP in 0.5 pL drops of buffer solution with the MNChem modified with aminophenyl-HFIP.
  • the MNChem provides excellent sensing performance as compared with the state-of-the-art.
  • the MNChem is fabricated in a CMOS process implying potential integration of various CMOS technologies on a single chip (embedded) providing possibilities for independent field-deployed units for OP monitoring.
  • the MNChem allows a unique platform for electrochemical sensing performed in ultra-small volumes for real-time sensing.
  • Figure 8A shows an optical image of the MNC biochip, a three-dimensional (3D) illustration of the MNC bioFET, and an MNC bioFET cross-section midway between source and drain.
  • the optical image shows the needle-probe contacting strategy, and the electrical contacting of the 1 : 100 diluted plasma drop with a quasi-reference electrode.
  • the MNC bioFET source-drain current IDS is composed of electron majority carriers running between the source and drain (VDS)-
  • the bioFET is equipped with six gates.
  • the backgate (VGB) and the two transverse gates (Vai, Van) are not employed in this work.
  • the MNC bioFET is protected by a SiCF passivation layer except for the sensing area and the electrical pads. Every combination of VGF and VGL results in the formation of different depletion areas within the channel volume, determining IDS shape, size, and location, as shown in the cross-section. Therefore, unlike the conventional MOSFET (metal-oxide-silicon FET), where gating predominately impacts the inversion layer carrier density, the MNC bioFET defines a distinct IDS shape, size, and location for each data point in an IDS-VGL curve. In general, the channel volume accommodates three distinct channels: the back channel, middle channel, and top channel ( Figure 8A cross-section); the shape, size and location of each channel depends on the gating configuration.
  • MOSFET metal-oxide-silicon FET
  • the primary motivation for the MNC bioFET is to tailor IDS to optimally couple with the unknown distributions of receptor-target molecule complexes at the sensing area.
  • the MNC bioFET design enables the effective translation of non-uniform sensing area potential, induced by biological complexes, into above noise level variations in IDS-
  • Figure 8B shows IDS-VGL curves for various VGF values.
  • the measurements are conducted for 0.5 pL drops of 1: 100 diluted plasma using an unmodified MNC device.
  • Each curve represents an average of 17 drops, with three measurements performed for each drop, resulting in a total of 51 measurements (see Experiments procedures section).
  • the inset provides a closer view of a single data point, where an error bar reflects the standard deviation of 51 measurements (the error bars are not visible in the IDS-VGL curves as the distributions of measurements are very tight).
  • the curves reveal the outstanding repeatability and robustness of an unmodified MNC device.
  • the repeatability alleviates concerns regarding: 1) drop-to-drop pH fluctuations: the measurements are performed in ambient, and the presence of the user can potentially affect the pH of the drop due to exhalation of CO2, for example [B. M. Loweet al, Analyst 2017, 142, 4173]. Such an effect could result in drop-to-drop variations and lack of repeatability. 2) The measurements are performed in 1: 100 diluted plasma with a background protein concentration (primarily albumin and globulins) of 600-800 pg/mL. The application of consecutive drops can result in non-specific adsorption of background proteins (surface biofouling) that will directly affect the repeatability. The excellent repeatability removes this concern. 3) Quasireference electrodes are more susceptible to potential variations compared with standard reference electrodes.
  • the excellent repeatability removes the concern of quasi-reference electrode potential variations due to swapping of drops [A. J. Bard et al. Wiley, Hoboken, New Jersey, USA 2000]. It also removes the concern of potential variations due to nonspecific adsorption of background proteins on the electrode surface.
  • Figure 9A presents the key steps in the biofunctionalization of the sensing area (see Experimental procedures).
  • piranha is used for the activation of the Si/SiCh sensing area.
  • the activation is followed by chemical modification with APTMS linker molecules followed by hydrolysis.
  • the last step entails the binding of the anti-estriol antibodies to the SiCh-bound APTMS molecules using GA linkers.
  • the surface biofunctionalization is developed and physically characterized on 2 cm X 2 cm Si/SiCb samples.
  • Figure 9B shows the contact angle and ellipsometry measurements performed for unmodified SiCh (SiCh), post piranha activation (Activated), post APTMS modification and hydrolysis (APTMS), and post binding of the anti-estriol antibodies (anti-estriol).
  • the contact angle of each sample is measured at four different locations, three measurements are performed at every location, amounting to a total of 12 measurements.
  • the data points represent the average values, and the error bars signify the corresponding standard deviations.
  • the measured contact angle value for SiCh is 55.60° ⁇ 1.48°, for piranha-treated surfaces is 19.9° ⁇ 1.83°, APTMS 68.85° ⁇ 2.96° and anti-estriol 46.61° ⁇ 3.79°.
  • the thickness of the SiO2 layer is about 10 nm (Jaw model), as expected.
  • the thickness of the APTMS layer is approximately 1.65 nm (B-spine model), slightly higher than the anticipated value for a monolayer based on the length of the APTMS molecule (around 5 A chain length, assuming 1 A per bond) [Ron, I. et al. J Am Chem Soc 132, 4131-4140 (2010].
  • the thickness of the antibody layer is 4.65 nm (B-spine model) which suggests that the antibodies might be oriented toward the surface.
  • FIG. 9C presents EIS measurements performed for unmodified SiCh (SiCh), SiCh modified with APTMS post hydrolysis (APTMS), and modified with anti-estriol antibodies (anti-estriol).
  • the curves present the relationships between the imaginary capacitance (C") and the real capacitance (C).
  • the measurements are performed in 0.1 mM pH 7.4 PBS solution. For repeatability, every sample is measured, the electrodes and the solution are removed from the EIS cell, a new solution is introduced, the electrodes are reconnected, and another measurement is performed. This procedure is repeated three times for each sample (indicated as 1 st , 2 nd and 3 rd in the legend). The impact of the modifications is reflected by three distinct curves. Noticeably, the surface modifications lead to a gradual increase in capacitance suggesting that the mechanism behind the capacitance increase is related to the increase in the double layer dielectric constant.
  • Figure 9D shows IDS-VGL curves for unmodified MNC bioFET and MNC bioFET modified with anti-estriol antibodies.
  • the measurements are performed in 0.5 pl drops of 0.1 mM 7.4 pH PBS solution.
  • the data points represent the average of 4 measured drops, where each drop is measured 4 times, removed with a clean-room wet towel, and a new drop is applied and measured.
  • the error bars, enlarged in the inset, indicate the corresponding standard deviations.
  • the small standard deviations demonstrate the excellent repeatability in 0.1 mM 7.4 pH PBS solution which ensures the stability of the quasireference electrode under possible drop-to-drop variations and pH fluctuations.
  • FIG 10A presents IDS- VGL curves for various VGF values of an MNC bioFET modified with anti-estriol antibodies.
  • the measurements are conducted in 0.5 pl drops of 1:100 diluted plasma.
  • the data points represent the average values of 17 measured drops.
  • Each drop is measured 3 times, removed with a clean-room wet towel, and a new drop is applied and measured.
  • the error bars shown in the inset, indicate the corresponding standard deviations.
  • the biofunctionalized MNC bioFET exhibits a remarkable repeatability and robustness in 1: 100 diluted plasma, thereby ensuring the stability of the MNC bioFET and the quasi-reference electrode with respect to possible variations as discussed in Figure 8B.
  • the IDS decrease after biofunctionalization with anti-estriol antibodies indicates an overall negative charge at the double layer induced by surface reorganization upon the biofunctionalization.
  • Figure 10B presents the second derivatives of the IDS-VGL curves presented in Figure 10A, where a peak reflects the excitation of a channel [8].
  • VGF 0 and 0.5 V and for negative VGL
  • the top and bottom channels are excited (no off-state), and the middle channel is excited with increasing VGL-
  • VGL For negative values of VGF and VGL, all channels are closed, and as VGL increases the bottom channel is excited first followed by the middle channel. Note how the peaks associated with the bottom and middle channels appear for higher VGL as VGF gets smaller (the lower is VGF the more depleted is the channel volume and higher VGL values are needed to trigger the channels).
  • 0.5 pL drops of 1 100 diluted plasma spiked with estriol concentrations ranging from 1 fg/ml to 10 pg/ml are successively introduced and measured from smallest to highest estriol concentrations. Each drop (i.e., a different estriol concentration) is measured 4 times, removed with a clean-room wet towel, a new drop with one order of magnitude higher estriol concentration is applied and measured (See Experimental procedures).
  • measurements are performed to quantify the extent of the non-specific signal.
  • the following measurements are performed: 1) the introduction of estriol to an unmodified MNC bioFET, 2) the introduction of estriol to an MNC bioFET modified with APTMS post hydrolysis, and 3) the introduction of other members of the estrogen family (estrone and estradiol) to MNC bioFETs biofunctionalized with anti-estriol antibodies.
  • 4 concentrations of the relevant target analyte are selected: 10 fg/ml, 10 pg/ml, 10 ng/ml and 10 pg/ml.
  • Figure 11A shows iNormaii d curves for selected VGF values for estriol introduced to an unmodified MNC bioFET.
  • the goal of this measurement is to quantify the non-specific signal attributed to estriol molecules physically adsorbed on the sensing area and/or on the surface of the quasi-reference electrode.
  • Two observations are in place: 1) the iNormaii ⁇ d values are considerably smaller than the iNormaii d values presented in Figure 10C-10E, and 2) the lack of monotonous IDS increase with increasing estriol concentration, in contrast with the monotonous IDS increase for MNC bioFET biofunctionalized with anti-estriol antibodies (Figure 10C-10E).
  • a similar response is measured for the MNC bioFET modified with APTMS ( Figure 11B).
  • the small non-specific signals imply that the introduction of high concentrations of estriol to the 0.5 pL drops of 1: 100 diluted plasma does not produce significant pH variations.
  • estrone and estradiol are presented in Figure 11C and Figure 11D, respectively.
  • estrone presented below as formula #2
  • estradiol presented below as formula #3
  • the estriol presented below as formula #1
  • the estrone presents a carbonyl instead of the hydroxyl of the estriol.
  • both estrone and estradiol are non-specific with respect to the anti- estriol antibody.
  • the extracted specific sensing performance accounts for possible drop-to-drop pH and potential variations, non-specific adsorption on the sensing area and/or the quasi-reference electrode, interactions between the anti-estriol antibodies and non-specific target molecules, pH variations due to the introduction of target molecules to the 0.5 pL drops of diluted plasma, and natural drop-to-drop variations associated with the experimental set-up.
  • Figure 12 presents calibration curves, iNormaii ⁇ d vs. estriol concentrations at selected channel configurations (i.e., selected VGF and VGL values). The calibration curves are generated after the above steps 1 and 2 are performed, and therefore these calibration curves represent specific sensing performance. Unlike Figure 10 and Figure 11, the calibration curves are presented for all the VGF values considered in the current work.
  • Estriol sensing is challenging due to the need for high specificity and selectivity, coupled with the need to generate sufficient electrostatic perturbation notwithstanding the estriol charge neutrality and small size.
  • Fab antigen-binding fragments
  • CDR complementarity determining regions
  • Aromatic residues that surround the antigen binding site contribute to ligand binding owing to their large hydrophobic surfaces and the ability to form hydrogen bonds.
  • DB3 antiprogesterone antibody
  • a Trp residue occludes the binding site to form a “closed” conformation in an unbound state, whereas binding induces the orientation of the Trp to an “open” position. This is mediated by the rotation of Trp towards the D ring of the sterol. Further orientation of sterols in the binding pocket is driven by the formation of a hydrogen bond with the keto group of the sterol rings.
  • FIG. 13 Suggested conformational changes are provided in Figure 13 and can include: Structural changes in the antibody CDR (Figure 13 item 1), structural changes in the crystallizable region fragment (Fc) ( Figure 13 item 2), rearrangements may include a change in the angles between the heavy chains and/or light chains ( Figure 13 item 3), CDR movements that affect the opening of pocket binding sites, side chain rearrangements ( Figure 13 item 4), or movement of the constant domains ( Figure 13 item 5).
  • binding of small molecules such as steroids is expected to produce only mild structural and chemical changes and therefore pose challenges in identification and quantification of binding events by the antibody [M. M. Al Qaraghuli, et al. Front. Mol. Biosci. 2021, 8.].
  • antigen binding to recombinant anti-testosterone Fab induced mild structural changes in the variable domains ( Figure 13 item 6) that resulted in tighter packing of the testosterone with the antibody [J. Valjakka, A. et al. J. Biol. Chem. 2002, 277, 44021].
  • Anti estradiol antibodies showed no major conformational change upon ligand binding [C. Monnet, et al. J. Mol.Biol.
  • An additional or alternative sensing mechanism can be related to the perturbation of the antibody charge distribution induced by the binding of the estriol [W. Shao et al. ACS Appl. Mater. Interfaces 2023, 75] . However, this is expected to be a limited effect due to the small size of the estriol.
  • estriol binding may induce conformational changes in the antibody, either by formation of new contact sites, adjustment of positions, or stabilization of regions, thereby inducing signal transduction by the sensor.
  • the positions of the polar groups complexed with the antibody, and the rearrangement in water molecules at the binding site may induce charge alterations that may be detected by the sensor.
  • both these mechanisms are expected to yield limited electrostatic perturbation to conclude a measurable sensing signal.
  • the MNC bioFET provides excellent label-free and specific estriol sensing performance.
  • Figure 14 presents Table 2 that summarizes the MNC bioFET real-time, specific, and label-free estriol sensing performance in terms of sensitivity, linearity, dynamic range, and limit-of-detection (LOD).
  • the selected threshold values for linearity and dynamic range are 0.97 and at least 4 orders of magnitude, respectively (which also applies for the calibration curves presented in Figure 12).
  • the LOD for all considered channels is 1 fg/ml which is calculated in accordance with IUPAC definition [The IUPAC Compendium of Chemical Terminology. (International Union of Pure and Applied Chemistry (IUPAC), 2019). doi:10.1351/goldbook].
  • Piece-wise linearity is not considered in conventional labelled sensing methods, such as ELISA, where a sensing signal is generated regardless of the molecular arrangement at the sensing area.
  • ELISA electrospray ionization-sensitive electrospray ionization-sensitive electrospray ionization-sensitive electrospray ionization-sensitive electrospray ionization-sensitive electrospray ionization-sensitive electrosprays
  • Figure 15 shows the dependency of IDS on VGL for different VGF values measured with a 0.5 pL drop of 1: 100 diluted serum for an unmodified MNC device. Each curve was an average of 16 drops and each drop was measured 4 times with a total of 64 measurements. The inset shows a magnification of the data points showing the errors bar reflecting the standard deviations of the 64 measurements. The data presents an excellent repeatability of an unmodified MNC device in 1: 100 diluted serum. The repeatability removes the concern of drop-to-drop pH fluctuations [Hal et al. Sensors and Actuators B 24-25, 201-205 (1995)], and establishes the potential stability of the quasi-reference electrode under possible drop-to-drop variations [Allen J. Bard, Electrochemical Methods: Fundamentals and Applications, 2nd Edition, John Wiley and Sons Inc., 2008]. The dependency of IDS on VGL and VGF is detailed below in Figure 17 for MNC biofunctionalized with AFP- antibodies.
  • Figure 16A illustrates the main steps towards sensing area biofunctionalization with anti- AFP.
  • the Si/SiCh sensing area was activated with piranha followed by surface chemical modification with APTMS linker molecules, and final surface-tethering of the anti-AFP antibodies (see Experimental procedure).
  • Figure 16A also presents the corresponding contact angle measurements.
  • the surface modifications were developed and characterized using 1 cm x 1 cm samples of silicon substrates decorated with 5 nm SiCF ( Figure 16B-16C).
  • Figure 16B presents contact angle measurements for Si/SiCh samples post piranha activation, post APTMS modification and surface biofunctionalization with anti-AFP antibodies.
  • Figure 16B also shows ellipsometry measurements performed on the same Si/SiCh samples. First, the SiCF sample is measured and the 5 nm SiCF thickness is validated. Afterwards, the sample is measured post APTMS modification where the SiCL is fixed and the APTMS thickness is fitted.
  • the sample is measured post surface biofunctionalization with anti-AFP antibodies where both the SiCL and the APTMS thicknesses are fixed to the measured values and the antibody layer is fitted.
  • the mean square error (MSE) for all measurements is smaller than 2.
  • the thicknesses presented in Figure 16C reflect the expected values of APTMS and the antibody layer [I. M. Bhattacharyya, et al. Nanoscale 2022, 14, 2837; K. Bierbaum, et al. Langmuir 1995, 11, 512; I. M. Bhattacharyya, et al. Adv. Electron. Mater. 2022, 2200399].
  • Figure 16C presents the EIS measurements of the imaginary capacitance (C”) vs.
  • Figure 16D presents an excellent robustness and repeatability of a modified MNC device in 0.1 mM 7.4 pH PBS solution, as well as asserts the stability of the quasi-reference electrode under possible drop-to-drop variations [L. R. F. Allen J. Bard, Electrochemical Methods: Fundamentals and Applications, 2nd Edition, John Wiley And Sons Inc., 2008]. Finally, the repeatability addresses the concern of possible drop-to-drop pH fluctuations [R. E. G. Van Hal, J. C. T. Eijkel, P. Bergveld, Sensors Actuators B 1995, 24-25, 201].
  • FIG. 17A presents IDS-VGL for the selected 6 VGF values of MNC biosensor modified with anti-AFP measured in 1:100 diluted serum.
  • the contact angle of anti-AFP modified chip is 52° ⁇ 0.5° and 2.2 ⁇ 0.8 nm is the measured thickness of the anti-AFP layer.
  • the data points and the error bars reflect the averages and standard deviations, respectively, of 14 measured drops where each drop is measured 3 times. Note the excellent repeatability and robustness of the biofunctionalized MNC biosensor in 1:100 diluted serum. Furthermore, the repeatability reflects the buffering capability of the 0.5 pL drops of 1: 100 diluted serum [R. E. G. Van Hal, J. C. T. Eijkel, P. Bergveld, Sensors Actuators B 1995, 24-25, 201], as well as the stability of the quasi-reference electrode under possible drop-to-drop variations [L. R. F. Allen J. Bard, Electrochemical Methods: Fundamentals and Applications, 2nd Edition, John Wiley And Sons Inc., 2008].
  • Figure 17B presents the corresponding second derivatives of the curves presented in Figure 17A where each peak represents the excitation of a conducting channel [I. M. Bhattacharyya, et al. Nanoscale 2022, 14, 2837; A. Ortiz-Conde, et al. Microelectron. Reliab. 2002, 42, 583].
  • a conducting channel I. M. Bhattacharyya, et al. Nanoscale 2022, 14, 2837; A. Ortiz-Conde, et al. Microelectron. Reliab. 2002, 42, 583.
  • FIG. 17B illustrates the excitation sequence of the 3 channels for the various applied voltages.
  • each combination of VGL and VGF induces the formation of IDS of a different shape and size.
  • the measurements were performed in 1:100 diluted serum, which implies protein background concentrations of 600-800 pg/ml (primarily albumin and globulins). More control measurements are provided in Figure 17C.
  • Figure 17C(i)-17C(iv) show the nonspecific response of the MNC biosensor for the introduction of AFP to an unmodified MNC biosensor, the introduction of AFP to an APTMS-modified MNC biosensor, and for the introduction of PSA and hCG to anti-AFP-modified MNC biosensors.
  • the measurements are performed for 0.5 pL drops of 1: 100 diluted serum spiked with the respective target molecule concentration.
  • the target molecule concentrations for hCG are 0.27 nM, 2.70 nM and 27.0 nM and for PSA those are 0.35 nM, 3.50 nM and 35.0 nM in accordance with the 3 highest concentrations selected for AFP-specific sensing (0.11 nM, 1.05 nM and 10.5 nM).
  • the negligible non-specific signals and the excellent repeatability remove the concerns of: 1) a sensing signal due to serum pH fluctuations induced by the presence of biomolecules [R. E. G. Van Hal, J. C. T. Eijkel, P. Bergveld, Sensors Actuators B 1995, 24-25, 201], 2) a sensing signal originating from physical adsorption of target molecules on the quasi-reference electrode, 3) a sensing signal due to effect of drop-to-drop variations on the quasi-reference electrode potential [L. R. F. Allen J. Bard, Electrochemical Methods: Fundamentals and Applications, 2nd Edition, John Wiley And Sons Inc., 2008], and 4) a sensing signal due to non-specific adsorption of target molecules on the MNC biosensor sensing area.
  • FIG. 18A shows IDS-VGL curves for the 6 selected VGF values, on both linear and logarithmic scales, and for all 10 AFP concentrations.
  • the following procedure was followed. First, it is ensured that the variation between one drop to the second (variation between successive AFP concentrations) is not due to the natural variation between two successive drops.
  • the IDS difference between two adjacent concentrations, for each VGF and VGL values is required to be higher than the average difference between the 14 drops of Figure 17A.
  • the IDS difference between a given AFP concentration and the baseline (diluted serum without AFP), for each VGF and VGL values is required to be higher than the corresponding non-specific measurements presented in Figure 17C.
  • sensing signals originating from pH fluctuations induced by the target molecules or drop-to-drop variation in pH [R. E. G. Van Hal, J. C. T. Eijkel, P.
  • Figure 17B shows the Readout curves corresponding to the IDS-VGL curves of Figure 17A.
  • the Readout was calculated as (IDS AFP -I DS basehne )/I D s baselme , where IDS AFP is the measured IDS for a given AFP concentration and for set values of VGF and VGL, and i DS baselme i s the IDS measured for 1 : 100 diluted serum without AFP spiking.
  • the x-axis scales were adjusted to ensure that values reflecting a null IDS (noise level) are not considered.
  • the relevant excited channels are also marked in Figure 17B (T for top channel, M for middle channel, and B for back channel).
  • the switch in Readout polarity suggests that the four gates do not only affect the transduction of the sensing area potential into IDS, but also affects the interactions themselves, refereed to as active sensing. In other words, if the total charge distribution of the complexes at the sensing area is positive, then the Readout must be positive and cannot take negative values.
  • a switch in Readout polarity suggests that the biological interactions at the sensing area are affected by the gating configuration (active sensing).
  • the effect of VGL on biological interactions does not seem probable as VGL produces horizontal electric fields in the silicon device layer.
  • VGF affects the double layer (which is also affected by the biological entities and interactions) adjacent to the sensing area.
  • Figure 18C illustrates one possible mechanism to induce a switch in Readout polarity which relates to the direction of the double layer electric field determined by VGF- Such a switch in the polarity of the electric field can affect the orientation of the biological complexes and to generate either positive or negative Readout.
  • a switch in the polarity of the electric field can affect the orientation of the biological complexes and to generate either positive or negative Readout.
  • the effect of the double layer electric field on the orientation of the target molecules also depends on the density of the surface-bound complexes, as presented in Figure 18C.
  • Active sensing with the MNC biosensor is provided by the solution potential which affects the biological interactions indirectly by determining the conditions at the double layer. Still, more research is needed to underpin the mechanism by which VGF affects the biological interactions at the sensing area.
  • FIG 19A shows the Readout calibration curves for the considered channel configurations.
  • the illustrations on the right of the calibration curves show the crosssections, midway between the source and drain, of the various channels visualizing the size, shape, and location of the corresponding channels.
  • each line pattern represents a calibration curve obtained for a different channel configuration.
  • dashed vertical grey lines indicate the ‘calibration threshold’ of the calibration curves which marks the onset of the linear region of the dynamic range.
  • VGL determines the sensitivity (the slope of the linear region) and lower VGL generates higher sensitivity. This directly reflects the effect of channel configuration on sensing performance.
  • active sensing is allowed as VGF affects the calibration threshold value.
  • Figure 19B(ii) shows the lack of dependency of the calibration threshold value on VGL- This lack of dependency is expected as VGL affects solely the electron charge carriers in the silicon, and its effect on the potential of the solid-biological interface is negligible.
  • the dependency of the calibration threshold on VGF is expected, as VGF determines N+0 and the double layer conditions in terms of pH, ionic strength and electric field, each of these can potentially affect the ligand-receptor interaction.
  • FIG. 20 A summary of the sensing performance is provided in Figure 20 that discloses Table 3, which presents the dependency of the sensitivity, linearity (R 2 ), dynamic range, and LOD on channel configuration.
  • the sensitivity which is the slope of the linear fit in units of Readout per decade of concentration (%dec -1 ), and the LOD, defined as the lowest concentration with an IDS greater than the average IDS plus three standard deviations of the background diluted serum, are extracted in accordance with the IUPAC conventions [M. Nic, J. Jirat, et al. IUPAC Compendium of Chemical Terminology: Gold Book, IUPAC, Research Triagle Park, NC, 2.1.0., 2009].
  • the requirements for the presented performances are a dynamic range of at least four orders of magnitude, and R 2 higher or equal to 0.96.
  • the dependency of the sensing performance on channel configuration is shown.
  • the criteria for channel configuration selection are 7? 2 >0.96 and a dynamic range of at least 4 orders of magnitude.
  • the LOD and the dynamic range are extracted in accordance with the IUPAC definition [M. Nic, et al. IUPAC Compendium of Chemical Terminology: Gold Book, IUPAC, Research Triagle Park, NC, 2.1.0., 2009]
  • 105fM, 1.05pM, 10.5pM, 105pM, 1.05nM, and 10.5nM AFP corresponds to lOpg/ml, lOOpg/ml, Ing/ml, lOng/ml, lOOng/ml and Ipg/ml AFP, respectively.

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Abstract

La présente divulgation concerne des bio-transistors pour détecter des molécules cibles. Plus particulièrement, la présente divulgation concerne des systèmes et des procédés pour déterminer la présence et/ou la quantité de molécules cibles, en particulier, de petites molécules cibles, telles que des composés organophosphorés ou en variante, produit naturellement des molécules telles que l'estriol, dans un échantillon biologique, ce qui permet de fournir un diagnostic et une surveillance de conditions associées aux molécules cibles.
PCT/IL2024/050724 2023-07-23 2024-07-23 Détection spécifique et sans étiquette de petites molécules cibles à l'aide de transistors biologiques Pending WO2025022389A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170131267A1 (en) * 2012-01-23 2017-05-11 Ohio State Innovation Foundation Devices and methods for the rapid and accurate detection of analytes
US20230022648A1 (en) * 2021-07-14 2023-01-26 Tower Semiconductor Ltd. Biosensor having a fluid compartment

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170131267A1 (en) * 2012-01-23 2017-05-11 Ohio State Innovation Foundation Devices and methods for the rapid and accurate detection of analytes
US20230022648A1 (en) * 2021-07-14 2023-01-26 Tower Semiconductor Ltd. Biosensor having a fluid compartment

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