HK1191063A

HK1191063A - Nmr systems and methods for the detection of analytes

Info

Publication number: HK1191063A
Application number: HK14104249.5A
Authority: HK
Inventors: Thomas Jay Lowery; Mark John Audeh; Matthew Blanco; James Franklin Chepin; Vasiliki Demas; Rahul Dhanda; Marilyn Lee Fritzemeier; Isaac Koh; Sonia Kumar; Lori Anne Neely; Brian Mozeleski; Daniella Lynn Plourde; Charles William Rittershaus; Parris Wellman
Original assignee: T2 Biosystems, Inc.
Priority date: 2010-10-22
Filing date: 2011-10-19
Publication date: 2014-07-18

Description

Nuclear magnetic resonance system and method for detecting an analyte

Technical Field

The present invention relates to assays and devices for detecting analytes, and the use of such assays and devices in disease treatment and diagnosis.

Background

Magnetic sensors have been designed to detect molecular interactions in a variety of media, including biological fluids, food products, soil samples, and other media. Upon target binding, these sensors cause a change in the properties of adjacent water molecules (or any solvent molecules with free hydrogen) in the sample, which can be detected using magnetic resonance (NMR/MRI) techniques. Thus, by using these sensors in a liquid sample, it is possible to detect the presence and possibly determine the amount of analytes (small molecules, DNA, RNA, proteins, carbohydrates, organisms, metabolites and pathogens (e.g. viruses)) at very low concentrations using magnetic sensors.

Generally, the magnetic sensor is bound or linked to a predetermined molecule thereofThe target to form clustered (aggregated) magnetic particles. It is believed that when magnetic particles are assembled into clusters and the effective cross-sectional area becomes larger (and the number density of the clusters becomes smaller), the interaction with water or other solvent molecules is altered, resulting in a measured relaxation rate (e.g., T @) ₂、T₁、T₂ ^*) Variations in magnetic susceptibility (susceptability), precession frequency (frequency of progression), and other physical variations. Furthermore, cluster formation can be designed to be reversible (e.g., using temperature changes, chemical cleavage, pH changes, etc.), whereby "forward" or "reverse" (competitive and inhibition) assays can be developed for the detection of specific analytes. Forward (aggregation) and reverse (deaggregation) types of assays can be used to detect a wide variety of biologically relevant materials. The MRS (magnetic resonance switch) phenomenon has been described previously (see U.S. patent publication No. 20090029392).

Many diagnostic assays require sensitivity in the picomolar or sub-picomolar range. Current detection of infectious agents, nucleic acids, small molecules, biological warfare agents and microorganisms, as well as molecular targets (biomarkers) or combinations of molecules and immunoassay targets typically requires the preparation of a sample in advance, the time to analyze the sample, and a single detection of each individual analyte. There is a need for a fast and commercially viable NMR-based analyte detection device suitable for use with magnetic nano-sensors having four unique features and properties: (1) little to no sample preparation, (2) multiplex detection across multiple molecular types, (3) rapid extraction of diagnostic information, and (4) accurate information for immediate clinical decision-making.

Disclosure of Invention

The invention features systems and methods for detecting an analyte.

The invention features a method for detecting the presence of an analyte in a liquid sample, the method including: (a) contacting the solution with magnetic particles to prepare a solution containing 1X 10⁶To 1X 10¹³A (e.g., 1 × 10)⁶To 1X 10⁸、1×10⁷To 1X 10⁸、1×10⁷To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹³A) magnetic particles/ml of liquid sample, wherein the magnetic particles have an average diameter of 150 nm to 699 nm (e.g., 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, or 500 to 699 nm), 1 x 10 per particle⁸To 1X 10¹² mM^-1s^-1(e.g., 1X 10)⁸To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹² mM^-1s^-1) T of₂Relaxivity, and binding moieties on their surface; the role of these binding moieties is to alter the aggregation of magnetic particles in the presence of an analyte or multivalent binding agent; (b) placing a liquid sample in a device, the device comprising a support defining a well containing the liquid sample comprising magnetic particles, a multivalent binding agent, and an analyte, and having a radio frequency coil disposed around the well, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using one or more magnets and an RF pulse sequence; (c) exposing the sample to a bias magnetic field and a sequence of RF pulses; (d) measuring the signal after step (c); and (e) detecting the analyte based on the results of step (d). In certain embodiments, the magnetic particles are substantially monodisperse; exhibits non-specific reversibility in the absence of analyte and multivalent binding agent; and/or the magnetic particles further comprise a surface modified with a blocking agent (blocking agent) selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidases, and amine-carrying moieties (e.g., aminopolyethylene glycol, glycine, ethylenediamine, or aminodextran). In particular embodiments, the liquid sample further comprises a buffer, 0.1% to 3% (w/w) (e.g., 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%, 0.8% to 2%, or 1.5% To 3% (w/w)) albumin, 0.01% to 0.5% (e.g., 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5%) nonionic surfactant, or a combination thereof. In other embodiments, the magnetic particles comprise a surface modified with 40 μ g to 100 μ g (e.g., 40 μ g to 60 μ g, 50 μ g to 70 μ g, 60 μ g to 80 μ g, or 80 μ g to 100 μ g) of one or more proteins per milligram of magnetic particles. The liquid sample may comprise a multivalent binding agent that carries a plurality of analytes conjugated to a polymeric scaffold. For example, the analyte may be creatinine, the liquid sample may comprise a multivalent binding agent carrying a plurality of creatinine conjugates, and the magnetic particles may comprise a surface modified with creatinine antibodies. In another embodiment, the analyte may be tacrolimus, the liquid sample may include a multivalent binding agent carrying a plurality of tacrolimus conjugates, and the magnetic particles may include a surface modified with tacrolimus antibodies. In a particular embodiment of the method, step (d) comprises measuring T of the liquid sample₂A relaxation response, and wherein increasing agglomeration in the liquid sample results in an observed T of the sample ₂The relaxation rate increases. In certain embodiments, the analyte is a target nucleic acid (e.g., a target nucleic acid extracted from a leukocyte or a pathogen).

The invention features a method for detecting the presence of an analyte in a liquid sample, the method including: (a) contacting the solution with magnetic particles to prepare a solution containing 1X 10⁶To 1X 10¹³A (e.g., 1 × 10)⁶To 1X 10⁸、1×10⁷To 1X 10⁸、1×10⁷To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹³Magnetic particles/ml liquid sample, wherein the magnetic particles have an average diameter of 700 nm to 1200 nm (e.g., 700 to 850, 800 to 950, 900 to 1050, or 1000 to 1200 nm), 1 x 10 per particle⁹To 1X 10¹² mM^-1s^-1(e.g., 1X 10)⁹To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹² mM^-1s^-1) T of₂Relaxivity and have binding moieties on their surface, the role of which is to alter the aggregation of magnetic particles in the presence of an analyte; (b) placing a liquid sample in a device comprising a support defining a well containing the liquid sample comprising magnetic particles, a multivalent binding agent, and an analyte, and having a radio frequency coil disposed around the well, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using one or more magnets and an RF pulse sequence; (c) exposing the sample to a bias magnetic field and a sequence of RF pulses; (d) measuring the signal after step (c); and (e) detecting the presence or concentration of the analyte based on the results of step (d). In certain embodiments, the magnetic particles are substantially monodisperse; exhibits non-specific reversibility in the absence of analyte and multivalent binding agent; and/or the magnetic particles further comprise a surface modified with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidases, and amine-bearing moieties (e.g., aminopolyethylene glycol, glycine, ethylenediamine, or aminodextran). In particular embodiments, the liquid sample further comprises: a buffer, 0.1% to 3% (w/w) (e.g., 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (w/w)) albumin, 0.01% to 0.5% (e.g., 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5%) nonionic surfactant, or a combination thereof. In other embodiments, the magnetic particles comprise a surface modified with 40 μ g to 100 μ g (e.g., 40 μ g to 60 μ g, 50 μ g to 70 μ g, 60 μ g to 80 μ g, or 80 μ g to 100 μ g) of one or more proteins per milligram of magnetic particles. The liquid sample may comprise a multivalent binding agent carrying a plurality of analytes conjugated to a polymeric scaffold. For example, the analyte may be creatinine, the liquid sample may comprise a multivalent binding agent carrying a plurality of creatinine conjugates, and the magnetic particles may comprise a surface modified with creatinine antibodies. In another embodiment, the analyte may be The liquid sample may comprise a multivalent binding agent carrying a plurality of tacrolimus conjugates, and the magnetic particles may comprise a surface modified with tacrolimus antibodies. In a particular embodiment of the method of the invention, step (d) comprises measuring T of the liquid sample₂A relaxation response, and wherein increasing agglomeration in the liquid sample results in an observed T of the sample₂The relaxation rate is improved. In certain embodiments, the analyte is a target nucleic acid (e.g., a target nucleic acid extracted from a leukocyte or a pathogen).

The invention further features a method for detecting the presence of a pathogen in a whole blood sample, the method including: (a) providing a whole blood sample from a subject; (b) mixing a whole blood sample with a red blood cell lysing agent solution to prepare lysed red blood cells; (c) after step (b), centrifuging the sample to form a supernatant and a pellet, discarding some or all of the supernatant, and resuspending the pellet to form an extract, optionally washing the pellet (e.g., with TE buffer) and optionally repeating step (c) before resuspending the pellet; (d) lysing cells in the extract to form a lysate; (e) placing the lysate of step (d) in a detection tube and amplifying the target nucleic acid in the lysate to form an amplified lysate solution comprising the target nucleic acid, wherein the target nucleic acid is characteristic of the pathogen to be detected; (f) after step (e), adding 1X 10 to the test tube ⁶To 1X 10¹³Magnetic particles/ml amplified lysate solution (e.g., 1X 10)⁶To 1X 10⁸、1×10⁷To 1X 10⁸、1×10⁷To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹³Magnetic particles/ml), wherein the magnetic particles have an average diameter of 700 nm to 1200 nm (e.g., 700 to 850, 800 to 950, 900 to 1050, or 1000 to 1200 nm) and binding moieties on their surface that function to alter aggregation of the magnetic particles in the presence of a target nucleic acid or multivalent binding agent; (g) placing the detection tube in a device comprising a chamber defined for containing magnetic particles and a targetA support for a well of a detection tube for nucleic acids and having a radio frequency coil disposed around the well, the radio frequency coil configured to detect a signal generated by exposing a liquid sample to a bias magnetic field generated by one or more magnets and an RF pulse sequence; (h) exposing the sample to a bias magnetic field and a sequence of RF pulses; (i) after step (h), measuring a signal from the detector tube; and (j) detecting the pathogen based on the results of step (i). In certain embodiments, steps (a) through (i) are completed within 4 hours (e.g., within 3.5 hours, 3.0 hours, 2.5 hours, 2 hours, 1.5 hours, or 1 hour). In another embodiment, step (i) is performed without any prior purification of the amplified lysate solution (i.e., without fractionation of the lysate solution after formation). In particular embodiments, step (c) comprises washing the precipitate prior to resuspending the precipitate to form the extract. In a specific embodiment, step (d) comprises mixing the extract with beads to form a mixture and stirring the mixture to form a lysate. The magnetic particles may comprise one or more populations having first and second probes conjugated to their surfaces, the first probe acting to bind to a first segment of the target nucleic acid and the second probe acting to bind to a second segment of the target nucleic acid, wherein the magnetic particles form aggregates in the presence of the target nucleic acid. Alternatively, the assay may be a disaggregation assay, wherein the magnetic particles comprise a first population having a first binding moiety on their surface and a second population having a second binding moiety on their surface, and a multivalent binding moiety comprising a first probe and a second probe, the first probe acting to bind to the first binding moiety and the second probe acting to bind to the second binding moiety, the binding moiety and multivalent binding moiety acting to alter aggregation of the magnetic particles in the presence of the target nucleic acid. In certain embodiments, the magnetic particles are substantially monodisperse; exhibits non-specific reversibility in the absence of analyte and multivalent binding agent; and/or the magnetic particles further comprise a surface modified with a blocking agent selected from the group consisting of albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidases and amine-carrying moieties (e.g., aminopolyethylene glycol, glycine, ethylenediamine, or aminodextran). In particular embodiments, the lysate further comprises: a buffer, 0.1% to 3% (w/w) (e.g., 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (w/w)) albumin, 0.01% to 0.5% (e.g., 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5%) nonionic surfactant, or a combination thereof. In other embodiments, the magnetic particles comprise a surface modified with 40 μ g to 100 μ g (e.g., 40 μ g to 60 μ g, 50 μ g to 70 μ g, 60 μ g to 80 μ g, or 80 μ g to 100 μ g) of one or more proteins per milligram of magnetic particles. The lysate may comprise a multivalent binding agent that carries a plurality of analytes conjugated to a polymeric scaffold.

The invention features a method for detecting the presence of a target nucleic acid in a whole blood sample, the method including: (a) providing one or more cells from a whole blood sample from a subject; (b) lysing the cells to form a lysate; (c) amplifying the target nucleic acid in the lysate to form an amplified lysate solution comprising the target nucleic acid; (d) after step (c), adding the amplified lysate solution and 1X 10 to a detection tube ⁶To 1X 10¹³Magnetic particles/ml of amplified lysate solution, wherein the magnetic particles have an average diameter of 700 nm to 1200 nm and binding moieties on their surface, the role of which binding moieties is to alter the aggregation of the magnetic particles in the presence of a target nucleic acid or multivalent binding agent; (e) placing a detection tube in a device comprising a support defining a well for receiving the detection tube comprising magnetic particles and a target nucleic acid, and having a radio frequency coil disposed around the well, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using one or more magnets and an RF pulse sequence; (f) exposing the sample to a bias magnetic field and a sequence of RF pulses; (h) after step (f), measuring a signal from the detector tube; and (i) detecting the target nucleic acid based on the result of step (h). In particular embodiments, the target nucleic acid is purified prior to step (d). In a specific embodiment, step (b) comprises contacting the extract with beadsMixing to form a mixture and stirring the mixture to form a lysate. The magnetic particles may comprise one or more populations having first and second probes conjugated to their surfaces, the first probe acting to bind to a first segment of the target nucleic acid and the second probe acting to bind to a second segment of the target nucleic acid, wherein the magnetic particles form aggregates in the presence of the target nucleic acid. Alternatively, the assay may be a disaggregation assay, wherein the magnetic particles comprise a first population having a first binding moiety on their surface and a second population having a second binding moiety on their surface, and a multivalent binding moiety comprising a first probe and a second probe, the first probe acting to bind to the first binding moiety and the second probe acting to bind to the second binding moiety, the binding moiety and multivalent binding moiety acting to alter aggregation of the magnetic particles in the presence of the target nucleic acid. In certain embodiments, the magnetic particles are substantially monodisperse; exhibits non-specific reversibility in the absence of analyte and multivalent binding agent; and/or the magnetic particles further comprise a surface modified with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidases, and amine-bearing moieties (e.g., aminopolyethylene glycol, glycine, ethylenediamine, or aminodextran). In particular embodiments, the lysate further comprises: a buffer, 0.1% to 3% (w/w) (e.g., 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (w/w)) albumin, 0.01% to 0.5% (e.g., 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5%) nonionic surfactant, or a combination thereof. In other embodiments, the magnetic particles optionally comprise a surface modified with 40 μ g to 100 μ g (e.g., 40 μ g to 60 μ g, 50 μ g to 70 μ g, 60 μ g to 80 μ g, or 80 μ g to 100 μ g) of one or more proteins per milligram of magnetic particles. The lysate may comprise a multivalent binding agent that carries a plurality of analytes conjugated to a polymeric scaffold.

The invention further features a method for detecting the presence of a target nucleic acid in a whole blood sample, the method including: (a) lifting deviceAn extract for preparation by the process of: lysing red blood cells in a whole blood sample from a subject, centrifuging the sample to form a supernatant and a pellet, discarding some or all of the supernatant, and resuspending the pellet to form an extract, optionally washing the pellet (e.g., with TE buffer) prior to resuspending the pellet and optionally repeating the centrifuging, discarding, and washing of step (a); (b) lysing cells in the extract to form a lysate; (c) placing the lysate of step (b) in a detection tube and amplifying the nucleic acid in the detection tube to form an amplified lysate solution comprising 40% (w/w) to 95% (w/w) (e.g., 40 to 60%, 60 to 80%, or 85 to 95% (w/w)) of the target nucleic acid and 5% (w/w) to 60% (w/w) (e.g., 5 to 20%, 20 to 40%, or 40 to 60% (w/w)) of the non-target nucleic acid; (d) after step (c), adding 1X 10 to the test tube⁶To 1X 10¹³Magnetic particles/ml of amplified lysate solution, wherein the magnetic particles have an average diameter of 700 nm to 1200 nm and binding moieties on their surface, the role of which binding moieties is to alter the aggregation of the magnetic particles in the presence of a target nucleic acid or multivalent binding agent; (e) placing a detection tube in a device comprising a support defining a well for receiving the detection tube comprising magnetic particles and a target nucleic acid, and having a radio frequency coil disposed around the well, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using one or more magnets and an RF pulse sequence; (f) exposing the sample to a bias magnetic field and a sequence of RF pulses; (g) after step (f), measuring a signal from the detector tube; and (h) detecting the target nucleic acid based on the result of step (g), wherein step (g) is performed without any prior purification of the amplified lysate solution. In a specific embodiment, step (b) comprises mixing the extract with beads to form a mixture and stirring the mixture to form a lysate. The magnetic particles may comprise one or more populations having first and second probes conjugated to their surfaces, the first probe acting to bind to a first segment of the target nucleic acid and the second probe acting to bind to a second segment of the target nucleic acid, wherein the magnetic particles form a poly-mer in the presence of the target nucleic acid To be collected. Alternatively, the assay may be a disaggregation assay, wherein the magnetic particles comprise a first population having a first binding moiety on their surface and a second population having a second binding moiety on their surface, and a multivalent binding moiety comprising a first probe and a second probe, the first probe acting to bind to the first binding moiety and the second probe acting to bind to the second binding moiety, the binding moiety and multivalent binding moiety acting to alter aggregation of the magnetic particles in the presence of the target nucleic acid. In certain embodiments, the magnetic particles are substantially monodisperse; exhibits non-specific reversibility in the absence of analyte and multivalent binding agent; and/or the magnetic particles further comprise a surface modified with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidases, and amine-bearing moieties (e.g., aminopolyethylene glycol, glycine, ethylenediamine, or aminodextran). In particular embodiments, the lysate further comprises: a buffer, 0.1% to 3% (w/w) (e.g., 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (w/w)) albumin, 0.01% to 0.5% (e.g., 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5%) nonionic surfactant, or a combination thereof. In other embodiments, the magnetic particles comprise a surface modified with 40 μ g to 100 μ g (e.g., 40 μ g to 60 μ g, 50 μ g to 70 μ g, 60 μ g to 80 μ g, or 80 μ g to 100 μ g) of one or more proteins per milligram of magnetic particles. The lysate may comprise a multivalent binding agent that carries a plurality of analytes conjugated to a polymeric scaffold.

The invention features a method for detecting the presence of a candida species in a liquid sample, the method including: (a) lysing candida cells in a liquid sample; (b) amplifying a nucleic acid to be detected in the presence of a forward primer and a reverse primer to form a solution comprising candida amplicons, each primer being generic to a plurality of candida species; (c) contacting the solution with magnetic particles to prepare a solution containing 1X 10⁶To 1X 10¹³A (e.g., 1 × 10)⁶To 1X 10⁸、1×10⁷To 1X 10⁸、1×10⁷To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹³Magnetic particles/ml liquid sample, wherein the magnetic particles have an average diameter of 700 nm to 1200 nm (e.g., 700 to 850, 800 to 950, 900 to 1050, or 1000 to 1200 nm), 1 x 10 per particle⁹To 1X 10¹² mM^-1s^-1(e.g., 1X 10)⁸To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹² mM^-1s^-1) T of₂Relaxivity, and binding moieties on their surface that act to alter the aggregation of magnetic particles in the presence of candida amplicons or multivalent binding agents; (d) placing a liquid sample in a device comprising a holder defining an aperture for receiving the liquid sample comprising magnetic particles and candida amplicons, and having a radio frequency coil disposed around the aperture, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using one or more magnets and an RF pulse sequence; (e) exposing the sample to a bias magnetic field and a sequence of RF pulses; (f) after step (e), measuring the signal; and (g) determining whether a candida species is present in the sample based on the results of step (f). In certain embodiments, the magnetic particles are substantially monodisperse; exhibits non-specific reversibility in the absence of analyte and multivalent binding agent; and/or the magnetic particles further comprise a surface modified with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidases, and amine-bearing moieties (e.g., aminopolyethylene glycol, glycine, ethylenediamine, or aminodextran). In particular embodiments, the liquid sample further comprises: a buffer, 0.1% to 3% (w/w) (e.g., 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (w/w)) albumin, 0.01% to 0.5% (e.g., 0.01% to 0.05%, 0.05% to 3% (w/w)) albumin 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5%) of a nonionic surfactant, or a combination thereof. In other embodiments, the magnetic particles comprise a surface modified with 40 μ g to 100 μ g (e.g., 40 μ g to 60 μ g, 50 μ g to 70 μ g, 60 μ g to 80 μ g, or 80 μ g to 100 μ g) of one or more proteins per milligram of magnetic particles. The liquid sample may comprise a multivalent binding agent carrying a plurality of analytes conjugated to a polymeric scaffold. The forward primer may comprise, for example: sequence 5'-GGC ATG CCT GTT TGA GCG TC-3' (SEQ ID NO. 1). Reverse primers may include, for example: sequence 5'-GCT TAT TGA TAT GCT TAA GTT CAG CGG GT-3' (SEQ ID number 2). In certain embodiments, (i) the candida species is candida albicans, the first probe comprises oligonucleotide sequence 5'-ACC CAG CGG TTT GAG GGA GAA AC-3' (SEQ ID No.3), and the second probe comprises oligonucleotide sequence 5'-AAA GTT TGA AGA TAT ACG TGG TGG ACG TTA-3' (SEQ ID number 4); (ii) the candida species is candida krusei, and the first probe and the second probe comprise oligonucleotide sequences selected from the group consisting of: 5'-CGC ACG CGC AAG ATG GAA ACG-3' (SEQ ID NO.5), 5'-AAG TTC AGC GGG TAT TCC TAC CT-3' (SEQ ID NO.6), and 5'-AGC TTT TTG TTG TCT CGC AAC ACT CGC-3' (SEQ ID NO. 15); (iii) the candida species is candida glabrata, and the first probe comprises an oligonucleotide sequence: 5'-CTA CCA AAC ACA ATG TGT TTG AGA AG-3' (SEQ ID NO.7), and the second probe comprises the oligonucleotide sequence: 5'-CCT GAT TTG AGG TCA AAC TTA AAG ACG TCT G-3' (SEQ ID NO. 8); and (iv) the candida species is candida parapsilosis or candida tropicalis, and the first probe and the second probe comprise oligonucleotide sequences selected from the group consisting of: 5 '-AGT CCT ACC TGA TTT GAG GTCNitIndAA-3' (SEQ ID NO.9), 5 '-CCG NitIndGG GTT TGA GGG AGA AAT-3' (SEQ ID NO.10), AAA GTT ATG AAATAA ATT GTG GTG GCC ACT AGC (SEQ ID NO.16), ACC CGG GGGTTT GAG GGA GAA A (SEQ ID NO.17), AGT CCT ACC TGA TTT GAG GTC GAA (SEQ ID NO.18) and CCG AGG GTT TGA GGG AGA AAT (SEQ ID NO. 19). In certain embodiments, steps (a) through (h) are within 4 hours (e.g., at 3.5 hours, 3.0 hours, 2.5 hours, 2 hours, 1.5 hours, or 1 hour) Or in a shorter time). In a specific embodiment, the magnetic particles comprise two populations, a first population carrying a first probe on its surface and a second population carrying a second probe on its surface. In another embodiment, the magnetic particles are a single population carrying both the first and second probes on the surface of the magnetic particles. The magnetic particles may comprise one or more populations of first probes and second probes conjugated to their surfaces, the first probes being operative to bind to a first segment of a candida amplicon and the second probes being operative to bind to a second segment of the candida amplicon, wherein the magnetic particles form aggregates in the presence of the target nucleic acid. Alternatively, the assay may be a disaggregation assay, wherein the magnetic particles comprise a first population having a first binding moiety on their surface and a second population having a second binding moiety on their surface, and a multivalent binding moiety comprising a first probe and a second probe, the first probe acting to bind to the first binding moiety and the second probe acting to bind to the second binding moiety, the binding moiety and multivalent binding moiety acting to alter aggregation of the magnetic particles in the presence of a candida amplicon. In particular embodiments, the method can result in: (i) coefficient of variation for T2 values of less than 20% in candida positive samples; (ii) at least 95% correct detection at less than or equal to 5 cells/mL in a sample spiked into 50 individual healthy patient blood samples; (iii) at least 95% correct detection at less than or equal to 5 cells/mL in a sample spiked into 50 individual unhealthy patient blood samples; and/or (iv) greater than or equal to 80% correct detection in a clinically positive patient sample (i.e., determined to be candida positive using other techniques such as cell culture) starting with 2 mL of blood.

The invention features a method for detecting the presence of a candida species in a whole blood sample, the method comprising: (a) providing an extract prepared by lysing red blood cells in a whole blood sample from a subject; (b) centrifuging the sample to form a supernatant and a precipitate, discarding some or all of the supernatant; (c) washing the precipitate by mixing the precipitate with a buffer (e.g., with TE buffer), agitating the sample (e.g., by vortexing), centrifuging the sample to form a supernatant and a precipitate, discarding some or all of the supernatant; (d) optionally repeating steps (b) and (c); (e) bead beating the pellet in the presence of a buffer (e.g., TE buffer) to form a lysate; (f) centrifuging the sample to form a supernatant containing the lysate; (g) amplifying the nucleic acid in the lysate of step (f) to form candida amplicons; and (h) detecting the presence of candida amplicons, wherein the method can produce: (i) at least 95% correct detection at less than or equal to 5 cells/mL in a sample spiked into 50 individual healthy patient blood samples; (ii) at least 95% correct detection at less than or equal to 5 cells/mL in a sample spiked into 50 individual unhealthy patient blood samples; and/or (iii) greater than or equal to 80% correct detection in a clinically positive patient sample (i.e., candida positive as determined by cell culture) starting with 2 mL of the blood of step (a).

The invention features a method for detecting the presence of a pathogen in a whole blood sample, the method including the steps of: (a) providing 0.05 to 4.0 mL of a whole blood sample (e.g., 0.05 to 0.25, 0.25 to 0.5, 0.25 to 0.75, 0.4 to 0.8, 0.5 to 0.75, 0.6 to 0.9, 0.65 to 1.25, 1.25 to 2.5, 2.5 to 3.5, or 3.0 to 4.0 mL of whole blood); (b) placing an aliquot of the sample of step (a) in a container and amplifying the target nucleic acid in the sample to form an amplified solution comprising the target nucleic acid, wherein the target nucleic acid is characteristic of the pathogen to be detected; (c) placing the amplified liquid sample in a detection device; (d) detecting a pathogen based on the results of step (c), wherein the pathogen is selected from bacteria and fungi, and wherein the method is capable of detecting a concentration of the pathogen of 10 cells/mL (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 cells/mL) in the whole blood sample. The detection device may detect the pathogen by performing optical, fluorescent, mass, density, magnetic, chromatographic and/or electrochemical measurements on the amplified liquid sample. In certain embodiments, steps (a) through (d) are completed within 3 hours (e.g., within 3.2, 2.9, 2.7, 2.5, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, or 1.5 hours or 1 hour). In other embodiments, step (c) is performed without any prior purification of the amplified solution, and/or the liquid sample of step (c) comprises whole blood proteins and non-target oligonucleotides. In certain embodiments, the pathogen is selected from bacteria and fungi. The pathogen may be any bacterial or fungal pathogen described herein.

The invention further features a method for detecting the presence of a pathogen in a whole blood sample, the method including the steps of: (a) providing a whole blood sample from a subject; (b) mixing a 0.05 to 4.0 mL whole blood sample (e.g., 0.05 to 0.25, 0.25 to 0.5, 0.25 to 0.75, 0.4 to 0.8, 0.5 to 0.75, 0.6 to 0.9, 0.65 to 1.25, 1.25 to 2.5, 2.5 to 3.5, or 3.0 to 4.0 mL of whole blood) with a red blood cell lysing agent solution to prepare lysed red blood cells; (c) after step (b), centrifuging the sample to form a supernatant and a pellet, discarding some or all of the supernatant, and resuspending the pellet to form an extract, optionally washing the pellet (e.g., with TE buffer) prior to resuspending the pellet and optionally repeating step (c); (d) lysing cells of the extract to form a lysate; (e) placing the lysate of step (d) in a vessel and amplifying the target nucleic acid in the lysate to form an amplified lysate solution comprising the target nucleic acid, wherein the target nucleic acid is characteristic of the pathogen to be detected; (f) after step (e), the amplified lysate solution is mixed with 1X 10⁶To 1X 10¹³A (e.g., 1 × 10)⁶To 1X 10⁸、1×10⁷To 1X 10 ⁸、1×10⁷To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹³Magnetic particles/ml) magnetic particles/ml amplified lysate solution, wherein the magnetic particles have an average diameter of 150 nm to 1200 nm (e.g., 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, 500 to 700 nm, 700 to 850, 800 to 950, 900 to 1050, or 1000 to 1200 nm), 1 x 10 per particle, to form a liquid sample⁸To 1X 10¹² mM^-1s^-1(e.g., 1X 10)⁸To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹² mM^-1s^-1) T of₂Relaxivity, and binding moieties on their surface that function to alter the aggregation of magnetic particles in the presence of a target nucleic acid or multivalent binding agent; (g) placing a liquid sample in a device comprising a support defining a well for receiving a detection tube comprising magnetic particles and target nucleic acids, and having a radio frequency coil disposed around the well, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using one or more magnets and an RF pulse sequence; (h) exposing the sample to a bias magnetic field and a sequence of RF pulses; (i) after step (h), measuring a signal from the liquid sample; and (j) detecting a pathogen based on the results of step (i), wherein the pathogen is selected from bacteria and fungi, and wherein the method is capable of detecting a concentration of the pathogen of 10 cells/mL (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 cells/mL) in the whole blood sample. In certain embodiments, steps (a) through (i) are completed within 3 hours (e.g., within 3.2, 2.9, 2.7, 2.5, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, or 1 hour or less). In other embodiments, step (i) is performed without any prior purification of the amplified lysate solution, and/or the liquid sample of step (i) comprises whole blood proteins and non-target oligonucleotides. In certain embodiments, the pathogen is selected from bacteria and fungi. The pathogen may be any bacterial or fungal pathogen described herein. In particular embodiments, the method can measure a pathogen concentration of 10 cells/mL in a whole blood sample with a coefficient of variation of less than 15% (e.g., less than 10 cells/mL for a coefficient of variation of 15%, 10%, 7.5%, or 5%, or less than 25 cells/mL for a coefficient of variation of 15%, 10%, 7.5%, or 5%, or less than 50 cells/mL for a coefficient of variation of 15%, 10%, 7.5%, or 5%, or less than 1% 100 cells/mL for a coefficient of variation of 5%, 10%, 7.5%, or 5%). In certain embodiments, the magnetic particles are substantially monodisperse; exhibits non-specific reversibility in the absence of analyte and multivalent binding agent; and/or the magnetic particles further comprise a surface modified with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidases, and amine-bearing moieties (e.g., aminopolyethylene glycol, glycine, ethylenediamine, or aminodextran). In particular embodiments, the liquid sample further comprises: a buffer, 0.1% to 3% (w/w) (e.g., 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (w/w)) albumin, 0.01% to 0.5% (e.g., 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5%) nonionic surfactant, or a combination thereof. In other embodiments, the magnetic particles comprise a surface modified with 40 μ g to 100 μ g (e.g., 40 μ g to 60 μ g, 50 μ g to 70 μ g, 60 μ g to 80 μ g, or 80 μ g to 100 μ g) of one or more proteins per milligram of magnetic particles. The liquid sample may comprise a multivalent binding agent carrying a plurality of analytes conjugated to a polymeric scaffold. The method for monitoring may include any of the magnetically assisted agglomeration methods described herein. The magnetic particles may comprise one or more populations having first and second probes conjugated to their surfaces, the first probe acting to bind to a first segment of the target nucleic acid and the second probe acting to bind to a second segment of the target nucleic acid, wherein the magnetic particles form aggregates in the presence of the target nucleic acid. Alternatively, the assay may be a disaggregation assay, wherein the magnetic particles comprise a first population having a first binding moiety on their surface and a second population having a second binding moiety on their surface, and a multivalent binding moiety comprising a first probe and a second probe, the first probe acting to bind to the first binding moiety and the second probe acting to bind to the second binding moiety, the binding moiety and multivalent binding moiety acting to alter aggregation of the magnetic particles in the presence of the target nucleic acid.

The invention further features a method for detecting a diseaseA method of poisoning the presence in a whole blood sample, the method comprising the steps of: (a) providing a plasma sample from a subject; (b) mixing 0.05 to 4.0 mL (e.g., 0.05 to 0.25, 0.25 to 0.5, 0.25 to 0.75, 0.4 to 0.8, 0.5 to 0.75, 0.6 to 0.9, 0.65 to 1.25, 1.25 to 2.5, 2.5 to 3.5, or 3.0 to 4.0 mL of whole blood) of the plasma sample with a lysing agent to prepare a mixture comprising lysed virus; (c) placing the mixture of step (b) in a vessel and amplifying the target nucleic acid in the filtrate to form an amplified filtrate solution comprising the target nucleic acid, wherein the target nucleic acid is characteristic of the virus to be detected; (d) after step (c), the amplified filtrate solution is mixed with 1X 10⁶To 1X 10¹³A (e.g., 1 × 10)⁶To 1X 10⁸、1×10⁷To 1X 10⁸、1×10⁷To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹³Magnetic particles/ml) magnetic particles/ml amplified filtrate solution, wherein the magnetic particles have an average diameter of 150 nm to 1200 nm (e.g., 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, 500 to 700 nm, 700 to 850, 800 to 950, 900 to 1050, or 1000 to 1200 nm), 1 × 10 per particle, to form a liquid sample ⁸To 1X 10¹² mM^-1s^-1(e.g., 1X 10)⁸To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹² mM^-1s^-1) T of₂Relaxivity, and binding moieties on their surface that function to alter the aggregation of magnetic particles in the presence of a target nucleic acid or multivalent binding agent; (e) placing a liquid sample in a device comprising a support defining a well for receiving a detection tube comprising magnetic particles and target nucleic acids, and having a radio frequency coil disposed around the well, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using one or more magnets and an RF pulse sequence; (f) exposing the liquid sampleIn a bias magnetic field and RF pulse sequence; (g) after step (f), measuring a signal from the liquid sample; and (h) detecting a virus based on the results of step (g), wherein the method is capable of detecting less than 100 copies of the virus (e.g., less than 80, 70, 60, 50, 40, 30, 20, or 10 copies) in a whole blood sample. In certain embodiments, steps (a) through (g) are completed within 3 hours (e.g., within 3.2, 2.9, 2.7, 2.5, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5 hours, or 1 hour or less). The virus may be any viral pathogen described herein. In certain embodiments, the magnetic particles are substantially monodisperse; exhibits non-specific reversibility in the absence of analyte and multivalent binding agent; and/or the magnetic particles further comprise a surface modified with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidases, and amine-bearing moieties (e.g., aminopolyethylene glycol, glycine, ethylenediamine, or aminodextran). In particular embodiments, the liquid sample further comprises: a buffer, 0.1% to 3% (w/w) (e.g., 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (w/w)) albumin, 0.01% to 0.5% (e.g., 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5%) nonionic surfactant, or a combination thereof. In other embodiments, the magnetic particles comprise a surface modified with 40 μ g to 100 μ g (e.g., 40 μ g to 60 μ g, 50 μ g to 70 μ g, 60 μ g to 80 μ g, or 80 μ g to 100 μ g) of one or more proteins per milligram of magnetic particles. The liquid sample may comprise a multivalent binding agent carrying a plurality of analytes conjugated to a polymeric scaffold. The method for monitoring may include any of the magnetically assisted agglomeration methods described herein. The magnetic particles may comprise one or more populations having first and second probes conjugated to their surfaces, the first probe acting to bind to a first segment of the target nucleic acid and the second probe acting to bind to a second segment of the target nucleic acid, wherein the magnetic particles form aggregates in the presence of the target nucleic acid. Alternatively, the assay may be a disaggregation assay, wherein the magnetic particles comprise a first magnetic particle having a first magnetic domain on their surface A first population of binding moieties and a second population having a second binding moiety on their surface, and a multivalent binding moiety comprising a first probe and a second probe, the first probe being operative to bind to the first binding moiety and the second probe being operative to bind to the second binding moiety, the binding moiety and multivalent binding moiety being operative to alter aggregation of magnetic particles in the presence of the target nucleic acid.

In any of the systems and methods of performing PCR amplification of the present invention, the PCR method can be real-time PCR that quantifies the amount of target nucleic acid present in the sample.

The invention further features a method of quantifying a target nucleic acid molecule in a sample by amplifying the target nucleic acid molecule in an amplification reaction mixture in a detection tube (e.g., using PCR or isothermal amplification) resulting in the formation of an amplicon corresponding to the target nucleic acid molecule. In this method, the amplification reaction mixture comprises: (1) a target nucleic acid molecule, (2) an amplification primer specific for the target nucleic acid molecule, and (3) a superparamagnetic particle. In this method, amplification is performed in a device comprising a support defining a well for receiving a detection tube comprising superparamagnetic particles and target nucleic acid molecules and having a radio frequency coil disposed around the well; the radio frequency coil is configured to detect a signal generated by exposing the sample to a bias magnetic field generated by one or more magnets and a sequence of RF pulses. The amplification of this method comprises the following steps:

(a) Performing one or more amplification cycles;

(b) exposing the amplification reaction mixture or an aliquot thereof to conditions that allow aggregation or disaggregation of the superparamagnetic particles,

(c) exposing the sample to a bias magnetic field and a sequence of RF pulses;

(d) after step (c), measuring a signal from the detector tube;

(e) repeating steps (a) - (d) until a desired amount of amplification is obtained; and

(f) quantifying the amplicons present in the respective amplification cycles based on the results of step (d).

In this method, the initial number of target nucleic acid molecules in the sample is determined based on the determined number of amplicons in each amplification cycle.

In any of the foregoing methods for quantifying a target nucleic acid molecule, the detection tube can remain sealed throughout the amplification reaction. The superparamagnetic particles of these methods may have a diameter of more or less than 100 nm (e.g., a diameter of 30 nm).

Additionally, in any of the foregoing methods of quantifying a target nucleic acid molecule, the method can further comprise applying a magnetic field to the detection tube after measuring a signal from the detection tube, thereby attracting the superparamagnetic particles to the sides of the detection tube, and releasing the magnetic field after completing one or more additional amplification cycles.

In addition, in any of the foregoing methods for quantifying a target nucleic acid molecule, the sample can, for example, comprise no isolated nucleic acid molecules prior to step (a) (e.g., the sample can be whole blood or comprise no target nucleic acid molecules prior to step (a)).

The invention features a method of monitoring one or more analytes in a fluid sample derived from a patient for the purpose of diagnosing, controlling or treating a medical condition of the patient; the method comprises the following steps: (a) will be 1 × 10⁶To 1X 10¹³A (e.g., 1 × 10)⁶To 1X 10⁸、1×10⁷To 1X 10⁸、1×10⁷To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹³One) magnetic particles/ml of liquid sample, wherein the magnetic particles have an average diameter of 150 nm to 1200 nm (e.g., 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, 500 to 700 nm, 700 to 850, 800 to 950, 900 to 1050, or 1000 to 1200 nm) and 1 x 10 per particle is mixed with the liquid sample⁸To 1X 10¹² mM^-1s^-1(e.g., 1X 10)⁸To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹² mM^-1s^-1) T of₂Relaxivity, and wherein the magnetic particles have binding moieties on their surface, the role of these binding moieties being to alter the specific aggregation of the magnetic particles in the presence of one or more analytes or multivalent binding agents; (b) placing a liquid sample in a device, the device comprising a support defining an aperture for receiving a liquid sample comprising magnetic particles and one or more analytes, and having a radio frequency coil disposed around the aperture, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using one or more magnets and an RF pulse sequence; (c) exposing the sample to a bias magnetic field and a sequence of RF pulses; (d) after step (c), measuring the signal; (e) monitoring one or more analytes based on the results of step (d); and (f) using the results of step (e) to diagnose, control or treat the medical condition. In one embodiment, the one or more analytes comprise creatinine. In another embodiment, the patient is immunocompromised and the one or more analytes comprise an analyte selected from the group consisting of pathogen-related analytes, antibiotics, antifungal agents, and antiviral agents (e.g., the one or more analytes may comprise candida species, tacrolimus, fluconazole, and/or creatinine). In yet another embodiment, the patient has cancer and the one or more analytes are selected from the group consisting of an anti-cancer agent and a genetic marker present in cancer cells. The patient may have or be at risk of infection and the one or more analytes comprise an analyte selected from the group consisting of a pathogen-associated analyte, an antibiotic, an antifungal agent, and an antiviral agent. The patient may have immune inflammation and the one or more analytes comprise an analyte selected from the group consisting of an anti-inflammatory agent and TNF-a. The patient may have a heart disease and the one or more analytes may comprise cardiac markers. The patient may have HIV/AIDS and one or more The analyte may comprise CD3, viral load and AZT. In certain embodiments, the method is for monitoring liver function in a patient, and wherein the one or more analytes are selected from the group consisting of albumin, aspartate transaminase, alanine transaminase, alkaline phosphatase, gamma-glutamyl transpeptidase, bilirubin, alpha-fetoprotein, lactase dehydrogenase, mitochondrial antibodies, and cytochrome P450. For example, the one or more analytes include a cytochrome P450 polymorphism, and the ability of the patient to metabolize the drug is evaluated. The method may include identifying the patient as a weak metabolizer, a normal metabolizer, a moderate metabolizer, or a very fast metabolizer. The method can be used to determine an appropriate dose of a therapeutic agent in a patient by: (i) administering a therapeutic agent to a patient; (ii) (ii) obtaining a sample comprising the therapeutic agent or a metabolite thereof from the patient after step (i); (iii) contacting the sample with magnetic particles and exposing the sample to a bias magnetic field and a sequence of RF pulses and detecting a signal generated by the sample; and (iv) determining the concentration of the therapeutic agent or metabolite thereof based on the results of step (iii). The therapeutic agent can be an anti-cancer agent, an antibiotic, an anti-fungal agent, or any of the therapeutic agents described herein. In any of the above monitoring methods, the monitoring may be intermittent (e.g., periodic) monitoring, or continuous monitoring. In certain embodiments, the magnetic particles are substantially monodisperse; exhibits non-specific reversibility in the absence of analyte and multivalent binding agent; and/or the magnetic particles further comprise a surface modified with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidases, and amine-bearing moieties (e.g., aminopolyethylene glycol, glycine, ethylenediamine, or aminodextran). In particular embodiments, the liquid sample further comprises: a buffer, 0.1% to 3% (w/w) (e.g., 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (w/w)) albumin, 0.01% to 0.5% (e.g., 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5%) nonionic surfactant, or a combination thereof. In other embodiments, the magnetic particles comprise particles that are used at 40 μ g to 100 μ g (e.g., 40 μ g to 60 μ g, 50 μ g to 70 μ g) μ g, 60 μ g to 80 μ g, or 80 μ g to 100 μ g) of one or more proteins per milligram of magnetic particles. The liquid sample may comprise a multivalent binding agent carrying a plurality of analytes conjugated to a polymeric scaffold. The method for monitoring may include any of the magnetically assisted agglomeration methods described herein.

The invention features a method of diagnosing sepsis in a subject, the method including (a) obtaining a liquid sample derived from blood of a patient; (b) by mixing 1 × 10⁶To 1X 10¹³A (e.g., 1 × 10)⁶To 1X 10⁸、1×10⁷To 1X 10⁸、1×10⁷To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹³Magnetic particles/ml of liquid sample with a portion of the liquid sample to prepare a first assay sample, wherein the magnetic particles have an average diameter of 150 nm to 1200 nm (e.g., 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, 500 to 700 nm, 700 to 850, 800 to 950, 900 to 1050, or 1000 to 1200 nm) and 1 x 10 per particle⁸To 1X 10¹² mM^-1s^-1(e.g., 1X 10)⁸To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹² mM^-1s^-1) T of₂(ii) a relaxivity, and wherein the magnetic particles have binding moieties on their surface which function to alter the specific aggregation of the magnetic particles in the presence of one or more pathogen-associated analytes or multivalent binding agents; (c) by mixing 1 × 10 ⁶To 1X 10¹³A (e.g., 1 × 10)⁶To 1X 10⁸、1×10⁷To 1X 10⁸、1×10⁷To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹³Second) magnetic particles/ml liquid sample is mixed with a portion of the liquid sample to prepare a secondDetermining a sample, wherein the magnetic particles have an average diameter of 150 nm to 1200 nm (e.g., 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, 500 to 700 nm, 700 to 850, 800 to 950, 900 to 1050, or 1000 to 1200 nm) and 1 x 10 per particle⁸To 1X 10¹² mM^-1s^-1(e.g., 1X 10)⁸To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹² mM^-1s^-1) T of₂Relaxivity and wherein the magnetic particles have on their surface a binding moiety which functions to alter the specific aggregation of the magnetic particles in the presence of one or more analytes characteristic of sepsis selected from the group consisting of GRO-alpha, high mobility group protein B1(HMBG-1), IL-1 receptor antagonist, IL-1B, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, macrophage inflammatory protein (MIP-1), macrophage Migration Inhibitory Factor (MIF), osteopontin, RANTES (stimulated modulation of activation, normal T cell expression and secretion; or CCL5), TNF-alpha, C-reactive protein (CRP), CD64, monocyte chemotactic protein 1(MCP-1), Adenosine deaminase binding protein (ABP-26), Inducible Nitric Oxide Synthase (iNOS), lipopolysaccharide binding protein, and procalcitonin; (d) placing each assay sample in a device comprising a support defining an aperture for receiving a liquid sample comprising magnetic particles and one or more analytes, and having a radio frequency coil disposed around the aperture, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using one or more magnets and an RF pulse sequence; (e) exposing each assay sample to a bias magnetic field and a sequence of RF pulses; (f) after step (e), measuring the signal generated by the first assay sample and the signal generated by the second assay sample; (g) monitoring the first assay sample for one or more analytes and monitoring the second assay sample for one or more analytes based on the results of step (f); and (h) using the results of step (g) to diagnose the subject. In one embodiment, the first assay sample The one or more pathogen-associated analytes of the article are derived from a sepsis-associated pathogen selected from the group consisting of acinetobacter baumannii, Aspergillus fumigatus (Aspergillus fumigatus), Bacteroides fragilis (Bacteroides fragilis), Bacteroides fragilis (b. fragilis), blaSHV, Burkholderia cepacia (Burkholderia cepacia), campylobacter jejuni/campylobacter coli, candida guilliermondii, candida albicans, candida glabrata (c. glabrata), candida krusei, candida lucitae (c. lucitanium), candida parapsilosis, candida tropicalis, Clostridium perfringens (Clostridium peinin), Coagulase-negative staphylococcus (coagregative Staph), Enterobacter aerogenes (Enterobacter aelogens), Enterobacter cloacae, Enterobacter coeruleus, escherichia coli, and lactobacillus plantarum, candida albicans, escherichia coli, candida albicans, candida aeta, candida albicans, candida albicans, c Klebsiella pneumoniae (K. pneumniae), Listeria monocytogenes, Mec A gene (MRSA), Morganella morgana, Neisseria meningitidis, Neisseria species other than Neisseria meningitidis (Neisseria spp. non-menitididis), Prevotella buccae (Prevotella buccae), Prevotella intermedia (P. intermedia), Prevotella melanogenesis (P. melanogenesis), Propionibacterium acnes, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella enterica, Serratia marcescens, Staphylococcus aureus, Staphylococcus haemolyticus (S. haemolyticus), Stenotrophomonas maltophilia (dy), Staphylococcus aureus (S. maltophilia), Streptococcus stenotrophis (S. Stenotrophomonas), Streptococcus stenotrophis (S. lactis), Streptococcus stenotrophis, Streptococcus lactis, Streptococcus lacticola, Streptococcus, streptococcus mutans, streptococcus pneumoniae, streptococcus pyogenes and streptococcus sanguis (s. sanguinis). The one or more pathogen-associated analytes can be derived from a therapeutically resistant bacterial strain, such as a penicillin-resistant, methicillin-resistant, quinolone-resistant, macrolide-resistant, and/or vancomycin-resistant bacterial strain (e.g., resistant to Methicillin-resistant staphylococcus aureus or vancomycin-resistant enterococcus). In certain embodiments, the one or more analytes of the second assay sample are selected from the group consisting of GRO-alpha, high mobility group box protein B1(HMBG-1), IL-1 receptor antagonist, IL-1B, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, macrophage inflammatory protein (MIP-1), macrophage Migration Inhibitory Factor (MIF), osteopontin, RANTES (stimulated activity regulation, normal T cell expression and secretion; or CCL5), TNF-alpha, C-reactive protein (CRP), CD64, and monocyte chemotactic protein 1 (MCP-1). In a specific embodiment, the method further comprises preparing a third assay sample to monitor the concentration of the antiviral, antibiotic or antifungal agent circulating in the bloodstream of the subject. In certain embodiments, the subject may be an immunocompromised subject, or a subject at risk of becoming immunocompromised. In any of the above monitoring methods, the monitoring may be intermittent (e.g., periodic) monitoring or continuous detection. In certain embodiments, the magnetic particles are substantially monodisperse; exhibits non-specific reversibility in the absence of analyte and multivalent binding agent; and/or the magnetic particles further comprise a surface modified with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidases, and amine-bearing moieties (e.g., aminopolyethylene glycol, glycine, ethylenediamine, or aminodextran). In particular embodiments, the liquid sample further comprises a buffer, 0.1% to 3% (w/w) (e.g., 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (w/w)) albumin, 0.01% to 0.5% (e.g., 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5%) non-ionic surfactant, or a combination thereof. In other embodiments, the magnetic particles comprise a surface modified with 40 μ g to 100 μ g (e.g., 40 μ g to 60 μ g, 50 μ g to 70 μ g, 60 μ g to 80 μ g, or 80 μ g to 100 μ g) of one or more proteins per milligram of magnetic particles. The liquid sample may comprise a multivalent binding agent carrying a plurality of analytes conjugated to a polymeric scaffold. The method for monitoring may comprise any of the magnetically assisted agglomeration methods described herein 。

The invention further features a method of monitoring one or more analytes in a liquid sample derived from a patient in order to diagnose, control or treat sepsis or SIRS in the patient, the method comprising: (a) will be 1 × 10⁶To 1X 10¹³A (e.g., 1 × 10)⁶To 1X 10⁸、1×10⁷To 1X 10⁸、1×10⁷To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹³One) magnetic particles/ml of liquid sample, wherein the magnetic particles have an average diameter of 150 nm to 1200 nm (e.g., 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, 500 to 700 nm, 700 to 850, 800 to 950, 900 to 1050, or 1000 to 1200 nm) and 1 x 10 per particle is mixed with the liquid sample⁸To 1X 10¹² mM^-1s^-1(e.g., 1X 10)⁸To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹² mM^-1s^-1) T of₂Relaxivity, and wherein the magnetic particles have binding moieties on their surface, the role of these binding moieties being to alter the specific aggregation of the magnetic particles in the presence of one or more analytes or multivalent binding agents; (b) placing the liquid sample in a device comprising a holder defining an aperture for receiving a liquid sample comprising magnetic particles and one or more analytes, and having a radio frequency coil disposed around the aperture, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using one or more magnets and an RF pulse sequence; (c) exposing the sample to a bias magnetic field and a sequence of RF pulses; (d) after step (c), measuring the signal; (e) monitoring one or more analytes based on the results of step (d); and (f) using the results of step (e) to diagnose, control or treat sepsis or SIRS. The method can include (i) monitoring a pathogen-associated analyte, and (ii) monitoring Determining a second analyte characteristic of sepsis, the second analyte selected from the group consisting of GRO-alpha, high mobility group box protein B1(HMBG-1), IL-1 receptor antagonist, IL-1B, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, macrophage inflammatory protein (MIP-1), macrophage Migration Inhibitory Factor (MIF), osteopontin, RANTES (regulated by activation, normal T cell expression and secretion; or CCL5), TNF- α, C-reactive protein (CRP), CD64, monocyte chemotactic protein 1(MCP-1), adenosine deaminase binding protein (ABP-26), Inducible Nitric Oxide Synthase (iNOS), lipopolysaccharide binding protein, and procalcitonin. In certain embodiments, the pathogen-associated analyte is derived from a sepsis-associated pathogen selected from the group consisting of acinetobacter baumannii, aspergillus fumigatus, bacteroides fragilis, blaSHV, bockholderia cepacia, campylobacter jejuni/campylobacter coli, candida guilliermondii, candida albicans, candida glabrata, candida krusei, candida ruchii, candida parapsilosis, candida tropicalis, clostridium perfringens, coagulase-negative staphylococcus, enterobacter aerogenes, enterobacter cloacae, enterobacteriaceae, enterococcus faecalis, enterococcus faecium, escherichia coli, haemophilus influenzae, aureobacter aureofaciens, klebsiella oxytoca, klebsiella pneumoniae, listeria monocytogenes, Mec a gene (MRSA), Morganella morganganganganana morganana, neisseria meningitidis, neisseria species other than neisseria, neisseria species, and combinations thereof, Prevotella buccae, Prevotella intermedia, Prevotella melanogenes, Propionibacterium acnes, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella enterica, Serratia marcescens, Staphylococcus aureus, Staphylococcus hemolyticus, stenotrophomonas maltophilia, Staphylococcus saprophyticus, stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus bovis, Streptococcus dysgalactiae, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus sanguis. The pathogen-associated analyte may be derived from a bacterial strain resistant to treatment, such as a penicillin-, methicillin-, quinolone-, macrolide-and/or vancomycin-resistant bacterial strain (e.g., methicillin-resistant staphylococcus aureus or vancomycin-resistant bacterial strain Enterococcus archaeus). In particular embodiments, the second analyte is selected from GRO-alpha, high mobility group box protein B1(HMBG-1), IL-1 receptor antagonist, IL-1B, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, macrophage inflammatory protein (MIP-1), macrophage Migration Inhibitory Factor (MIF), osteopontin, RANTES (stimulated regulation, normal T cell expression and secretion; or CCL5), TNF-alpha, C-reactive protein (CRP), CD64, and monocyte chemotactic protein 1 (MCP-1). In a specific embodiment, the method further comprises preparing a third assay sample to monitor the concentration of the antiviral, antibiotic, or antifungal agent circulating in the bloodstream of the subject. In certain embodiments, the subject may be an immunocompromised subject, or a subject at risk of transitioning to immunocompromisation. In any of the above monitoring methods, the monitoring may be intermittent (e.g., periodic) monitoring, or continuous monitoring. In certain embodiments, the magnetic particles are substantially monodisperse; exhibits non-specific reversibility in the absence of analyte and multivalent binding agent; and/or the magnetic particles further comprise a surface modified with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidases, and amine-bearing moieties (e.g., aminopolyethylene glycol, glycine, ethylenediamine, or aminodextran). In particular embodiments, the liquid sample further comprises: a buffer, 0.1% to 3% (w/w) (e.g., 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (w/w)) albumin, 0.01% to 0.5% (e.g., 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5%) nonionic surfactant, or a combination thereof. In other embodiments, the magnetic particles comprise a surface modified with 40 μ g to 100 μ g (e.g., 40 μ g to 60 μ g, 50 μ g to 70 μ g, 60 μ g to 80 μ g, or 80 μ g to 100 μ g) of one or more proteins per milligram of magnetic particles. The liquid sample may comprise a multivalent binding agent carrying a plurality of analytes conjugated to a polymeric scaffold. The method for monitoring may include any of the magnetically assisted agglomeration methods described herein.

The invention further features a system for detecting one or more analytes, the system comprising: (a) a first unit comprising (a1) a permanent magnet defining a magnetic field; (a2) a holder defining an aperture for receiving a liquid sample containing magnetic particles and one or more analytes, and having a radio frequency coil disposed around the aperture, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using a permanent magnet and an RF pulse sequence; and (a3) one or more electrical components coupled to the radio frequency coil, the electrical components configured to amplify, condition, transmit, and/or digitize the signal; and (b) a second unit comprising a removable cartridge sized to facilitate insertion and removal from the system, wherein the removable cartridge is a modular cartridge comprising: (i) a reagent module for containing one or more assay reagents; and (ii) a detection module comprising a detection chamber for containing a liquid sample comprising magnetic particles and one or more analytes, wherein the reagent module and the detection module are assemblable into a modular cartridge prior to use, and wherein the detection chamber is removable from the modular cartridge. The modular cartridge may further comprise an inlet module, wherein the inlet module, the reagent module and the detection module may be assembled into the modular cartridge prior to use, and wherein the inlet module is sterilizable. In certain embodiments, the system further comprises a system computer having a processor for executing the assay protocol and storing assay data, and wherein the removable cartridge further comprises (i) a readable label displaying the analyte to be detected, (ii) a readable label displaying the assay protocol to be executed, (iii) a readable label displaying a patient identification number, (iv) a readable label displaying the location of the assay reagent contained in the cartridge, or (v) a readable label containing instructions for a programmable processor. The system may include: a cartridge unit, a stirring unit, a centrifuge, or any other system component described herein.

The invention further features a system for detecting one or more analytes, the system comprising: (a) a disposable sample holder defining an aperture for receiving a liquid sample and having a radio frequency coil received within the disposable sample holder and disposed about the aperture, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a biasing magnetic field generated using a permanent magnet and an RF pulse sequence, wherein the disposable sample holder includes one or more fusible links; and (b) an MR reader comprising (b1) a permanent magnet defining a magnetic field; (b2) an RF pulse sequence and detection coil; (b3) one or more electrical components coupled to the radio frequency coil, the electrical components configured to amplify, condition, transmit, and/or digitize the signal; and (b4) one or more electrical components coupled to the fuse link and configured to apply an excess current to the fuse link, thereby causing the link to open and render the coil inoperable after a predetermined operational life. In certain embodiments, an electrical component connected to the radio frequency coil is inductively coupled (inductively coupled) to the radio frequency coil.

The invention features a system for detecting creatinine, tacrolimus, and candida, the system including: (a) a first unit comprising (a1) a permanent magnet defining a magnetic field; (a2) a holder defining an aperture for receiving a liquid sample comprising magnetic particles and creatinine, tacrolimus, and candida, and having a radio frequency coil disposed around the aperture, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using a permanent magnet and an RF pulse sequence; and (a3) an electrical component coupled to the radio frequency coil, the electrical component configured to amplify, condition, transmit, and/or digitize the signal; and (b) a second unit comprising a removable cartridge sized to facilitate insertion and removal from the system, wherein the removable cartridge is a modular cartridge comprising: (i) a plurality of reagent modules for holding one or more assay reagents; and (ii) comprising a detection chamber for containing a liquid sample comprising magnetic particles and creatinine, tacrolimus and candidaA plurality of detection modules, wherein the plurality of reagent modules comprises (i) a first population of magnetic particles having an average diameter of 150 nm to 699 nm (e.g., 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, or 500 to 699 nm), 1 x 10 of each particle ⁸To 1X 10¹² mM^-1s^-1(e.g., 1X 10)⁸To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹² mM^-1s^-1) T of₂Relaxivity, and creatinine antibodies conjugated to their surface; (ii) a multivalent binding agent bearing a plurality of creatinine conjugates designed to form aggregates with a first population of magnetic particles in the absence of creatinine; (iii) a second population of magnetic particles having an average diameter of 150 nm to 699 nm (e.g., 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, or 500 to 699 nm), 1 × 10 per particle⁸To 1X 10¹² mM^-1s^-1(e.g., 1X 10)⁸To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹² mM^-1s^-1) T of₂Relaxivity, and tacrolimus antibodies conjugated to their surface; (iv) a multivalent binding agent carrying a plurality of tacrolimus conjugates designed to form aggregates with a second population of magnetic particles in the absence of tacrolimus; (v) a third population of magnetic particles having an average diameter of 700 nm to 1200 nm (e.g., 700 to 850, 800 to 950, 900 to 1050, or 1000 to 1200 nm), 1 x 10 per particle⁹To 1X 10¹² mM^-1s^-1(e.g., 1X 10)⁸To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹² mM^-1s^-1) T of₂Relaxivity, and having a conjugation selected to form aggregates in the presence of candida nucleic acids A first probe and a second probe on their surfaces, the first probe being operative to bind to a first segment of a candida nucleic acid and the second probe being operative to bind to a second segment of the candida nucleic acid. In certain embodiments, the magnetic particles are substantially monodisperse; exhibits non-specific reversibility in the absence of analyte and multivalent binding agent; and/or the magnetic particles further comprise a surface modified with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidases, and amine-bearing moieties (e.g., aminopolyethylene glycol, glycine, ethylenediamine, or aminodextran). In particular embodiments, the liquid sample further comprises: a buffer, 0.1% to 3% (w/w) (e.g., 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (w/w)) albumin, 0.01% to 0.5% (e.g., 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5%) nonionic surfactant, or a combination thereof. In other embodiments, the magnetic particles comprise a surface modified with 40 μ g to 100 μ g (e.g., 40 μ g to 60 μ g, 50 μ g to 70 μ g, 60 μ g to 80 μ g, or 80 μ g to 100 μ g) of one or more proteins per milligram of magnetic particles. The liquid sample may comprise a multivalent binding agent carrying a plurality of analytes conjugated to a polymeric scaffold. In another embodiment, the liquid sample comprises 1 × 10 ⁶To 1X 10¹³A (e.g., 1 × 10)⁶To 1X 10⁸、1×10⁷To 1X 10⁸、1×10⁷To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹³One) magnetic particles per ml liquid sample.

The invention features a method for measuring the concentration of creatinine in a liquid sample, the method including: (a) contacting the solution with (i) magnetic particles and (ii) a multivalent binding agent bearing multiple creatinine conjugates designed to form aggregates with the magnetic particles in the absence of creatinine to produce a composition comprising 1 x 10⁶To 1X 10¹³A (e.g., 1 × 10)⁶To 1X 10⁸、1×10⁷To 1X 10⁸、1×10⁷To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹³A) magnetic particles/ml of liquid sample, wherein the magnetic particles have an average diameter of 150 nm to 1200 nm (e.g., 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, 500 to 700 nm, 700 to 850, 800 to 950, 900 to 1050, or 1000 to 1200 nm), 1 x 10 per particle⁸To 1X 10¹² mM^-1s^-1(e.g., 1X 10)⁸To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹² mM^-1s^-1) T of₂Relaxivity, and creatinine antibodies conjugated to their surface; (b) placing a liquid sample in a device, the device comprising a support defining an aperture for receiving the liquid sample comprising magnetic particles, a multivalent binding agent, and creatinine, and having a radio frequency coil disposed around the aperture, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using one or more magnets and an RF pulse sequence; (c) exposing the sample to a bias magnetic field and a sequence of RF pulses; (d) measuring the signal after step (c); and (e) determining the concentration of creatinine in the liquid sample based on the results of step (d). In certain embodiments, the magnetic particles are substantially monodisperse; exhibits non-specific reversibility in the absence of analyte and multivalent binding agent; and/or the magnetic particles further comprise a surface modified with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidases, and amine-bearing moieties (e.g., aminopolyethylene glycol, glycine, ethylenediamine, or aminodextran). In particular embodiments, the liquid sample further comprises a buffer, 0.1% to 3% (w/w) (e.g., 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (w/w)) albumin, 0.01% to 0.5% (e.g., 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2 % to 0.4%, alternatively 0.3% to 0.5%) of a nonionic surfactant, or a combination thereof. In other embodiments, the magnetic particles comprise a surface modified with 40 μ g to 100 μ g (e.g., 40 μ g to 60 μ g, 50 μ g to 70 μ g, 60 μ g to 80 μ g, or 80 μ g to 100 μ g) of one or more proteins per milligram of magnetic particles. The liquid sample may comprise a multivalent binding agent carrying a plurality of analytes conjugated to a polymeric scaffold.

The invention features a multivalent binding agent that includes 2 or more creatinine moieties covalently bound to a scaffold. In certain embodiments, the multivalent binding agent is a compound of formula (I):

(A)_n-(B) (I)

wherein (A) is

(B) Is a polymer scaffold covalently bound to each (a), m is an integer from 2 to 10, and n is an integer from 2 to 50.

The invention is characterized in that⁶To 1X 10¹³A (e.g., 1 × 10)⁶To 1X 10⁸、1×10⁷To 1X 10⁸、1×10⁷To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹³Magnetic particles/ml solution, wherein the magnetic particles have an average diameter of 150 nm to 600 nm (e.g., 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, or 500 to 600 nm), 1 x 10 per particle ⁸To 1X 10¹² mM^-1s^-1(e.g., 1X 10)⁸To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹² mM^-1s^-1) T of₂Relaxivity, and a surface carrying a creatinine conjugate (a), wherein (a) is selected from:

and m is an integer of 2 to 10.

The invention also relates to a composition comprising 1 x 10⁶To 1X 10¹³A (e.g., 1 × 10)⁶To 1X 10⁸、1×10⁷To 1X 10⁸、1×10⁷To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹³Magnetic particles/ml solution, wherein the magnetic particles have an average diameter of 150 nm to 600 nm (e.g., 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, or 500 to 600 nm), 1 x 10 per particle⁸To 1X 10¹² mM^-1s^-1(e.g., 1X 10)⁸To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹² mM^-1s^-1) T of₂Relaxivity, and a surface carrying an antibody with affinity for a creatinine conjugate (e.g., a creatinine conjugate described herein).

The invention further features a method for measuring the concentration of tacrolimus in a liquid sample, the method comprising: (a) contacting the solution with (i) magnetic particles and (ii) a multivalent binding agent carrying a plurality of tacrolimus conjugates designed to form aggregates with the magnetic particles in the absence of tacrolimus to prepare a solution comprising 1 x 10 ⁶To 1X 10¹³A (e.g., 1 × 10)⁶To 1X 10⁸、1×10⁷To 1X 10⁸、1×10⁷To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹³A) magnetic particles/ml of liquid sample, wherein the magnetic particles have an average diameter of 150 nm to 1200 nm (e.g., 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, 500 to 700 nm, 700 to 850, 800 to 950, 900 to 1050, or 1000 to 1200 nm), 1 x 10 per particle⁸To 1X 10¹² mM^-1s^-1(e.g., 1X 10)⁸To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹² mM^-1s^-1) T of₂Relaxivity, and tacrolimus antibodies conjugated to their surface; (b) placing a liquid sample in a device comprising a support defining an aperture for receiving the liquid sample comprising magnetic particles, a multivalent binding agent, and tacrolimus, and having a radio frequency coil disposed around the aperture, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using one or more magnets and a sequence of RF pulses; (c) exposing the sample to a bias magnetic field and a sequence of RF pulses; (d) measuring the signal after step (c); and (e) determining the concentration of tacrolimus in the liquid sample based on the results of step (d). In certain embodiments, the magnetic particles are substantially monodisperse; exhibits non-specific reversibility in the absence of analyte and multivalent binding agent; and/or the magnetic particles further comprise a surface modified with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidases, and amine-bearing moieties (e.g., aminopolyethylene glycol, glycine, ethylenediamine, or aminodextran). In particular embodiments, the liquid sample further comprises: a buffer, 0.1% to 3% (w/w) (e.g., 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (w/w)) albumin, 0.01% to 0.5% (e.g., 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5%) nonionic surfactant, or a combination thereof. In other embodiments, the magnetic particles include 40 μ g to 100 μ g (e.g., 40 μ g to 60 μ g, 50 μ g to 70 μ g, 60 μ g to 80 μ g, or 80 μ g to 100 μ g) of one or more proteins per milligram of magnetic particle modified surface. The liquid sample may comprise a multivalent binding agent carrying a plurality of analytes conjugated to a polymeric scaffold.

The invention features a multivalent binding agent comprising 2 or more tacrolimus moieties comprising a structurally similar compound to which a tacrolimus metabolite or antibody described herein has an affinity for covalent binding to a scaffold. In certain embodiments, the multivalent binding agent is a compound of formula (II):

(A)_n-(B) (II)

wherein (A) is

(B) Is a polymer scaffold covalently bound to each (a), and n is an integer from 2 to 50.

The invention is characterized in that⁶To 1X 10¹³A (e.g., 1 × 10)⁶To 1X 10⁸、1×10⁷To 1X 10⁸、1×10⁷To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹³Magnetic particles/ml solution, wherein the magnetic particles have an average diameter of 150 nm to 600 nm (e.g., 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, or 500 to 600 nm), 1 x 10 per particle⁸To 1X 10 ¹² mM^-1s^-1(e.g., 1X 10)⁸To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹² mM^-1s^-1) T of₂Relaxation rate, and surface carrying antibodies with affinity for tacrolimus conjugates:

wherein (B) is a polymer scaffold.

In one embodiment of any of the above solutions, (i) the magnetic particles are substantially monodisperse; (ii) the magnetic particles show non-specific reversibility in plasma; (iii) the magnetic particles further comprise a surface modified with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidases, and amine-bearing moieties (e.g., aminopolyethylene glycol, glycine, ethylenediamine, or aminodextran); (iv) the liquid sample further comprises: a buffer, 0.1% to 3% (w/w) (e.g., 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (w/w)) albumin, 0.01% to 0.5% (e.g., 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5%) nonionic surfactant, or a combination thereof; and/or (iv) the magnetic particles comprise a surface modified with 40 μ g to 100 μ g (e.g., 40 μ g to 60 μ g, 50 μ g to 70 μ g, 60 μ g to 80 μ g, or 80 μ g to 100 μ g) of one or more proteins per milligram of magnetic particles. The solution may be used in any of the systems or methods described herein.

The invention features a removable cartridge sized to be inserted into and removed from a system of the invention, wherein the removable cartridge contains one or more chambers for housing a plurality of reagent modules for housing one or more assay reagents, wherein the reagent modules comprise: (i) for accommodating a 1 x 10⁶To 1X 10¹³A (e.g., 1 × 10)⁶To 1X 10⁸、1×10⁷To 1X 10⁸、1×10⁷To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹³A chamber of magnetic particles having an average diameter of 100 nm to 699 nm (e.g., 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, or 500 to 699 nm), 1 x 10 per particle⁸To 1X 10¹² mM^-1s^-1(e.g., 1X 10)⁸To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹² mM^-1s^-1) T of₂Relaxivity, and binding moieties on their surface, which function to alter the specific aggregation of magnetic particles in the presence of one or more analytes or multivalent binding agents; and (ii) a chamber for containing a buffer. In a related aspect, the invention features a removable cartridge sized to be inserted into and removed from the system of the invention, wherein the removable cartridge contains one or more chambers for containing a plurality of reagent modules for containing one or more assay reagents, wherein the reagent modules include (i) a housing for containing 1 x 10 assay reagents ⁶To 1X 10¹³A (e.g., 1 × 10)⁶To 1X 10⁸、1×10⁷To 1X 10⁸、1×10⁷To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹³A chamber of magnetic particles having an average diameter of 700 nm to 1200 nm (e.g., 700 to 850, 800 to 950, 900 to 1050, or 1000 to 1200 nm), 1 x 10 per particle⁹To 1X 10¹² mM^-1s^-1(e.g., 1X 10)⁹To 1X 10¹⁰、1×10⁹To 1X 10¹¹Or 1X 10¹⁰To 1X 10¹² mM^-1s^-1) T of₂Relaxivity, and oligonucleotide binding moieties on their surface, which function to alter the specific aggregation of magnetic particles in the presence of one or more analytes; and (ii) for accommodating buffersA chamber for flushing the liquid. The magnetic particles may be any magnetic particle described herein, and may be modified with any binding moiety described herein in order to detect any analyte described herein. In a specific embodiment of the removable cartridge, the magnetic particles and the buffer are jointly contained in a single chamber within the cartridge. In other embodiments, the buffer comprises 0.1% to 3% (w/w) albumin, 0.01% to 0.5% non-ionic surfactant, lysing agent, or a combination thereof. The removable cartridge may further comprise a chamber containing beads for lysing cells; a chamber comprising a polymerase; and/or a chamber comprising a primer.

The invention features a removable cartridge sized to be inserted into and removed from a system of the invention, wherein the removable cartridge contains one or more chambers for containing a plurality of reagent modules for containing one or more assay reagents, wherein the reagent modules include (i) a housing for containing 1 x 10 assay reagents⁸To 1X 10¹⁰A chamber of magnetic particles having an average diameter of 100 nm to 350 nm, 5 x 10 per particle⁸To 1X 10¹⁰ mM^-1s^-1T of₂Relaxivity, and binding moieties (e.g., antibodies, conjugated analytes) on their surface that function to alter the specific aggregation of magnetic particles in the presence of one or more analytes or multivalent binding agents; and (ii) a chamber for holding a buffer comprising 0.1% to 3% (w/w) (e.g., 0.1% to 0.5%, 0.3% to 0.7%, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (w/w)) albumin, 0.01% to 0.5% (e.g., 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5%) non-ionic surfactant, or a combination thereof. In one embodiment, the magnetic particles and the buffer are jointly contained in a single chamber within the cartridge.

In any of the systems, kits, cartridges, and methods of the invention, the liquid sample can comprise 1 x10⁸To 1X 10¹⁰Magnetic particles having an average diameter of 100 nm to 350 nm, each particle5X 10 of son⁸To 1X 10¹⁰ mM^-1s^-1T of₂Relaxivity, and binding moieties (e.g., antibodies, conjugated analytes) on their surface that act to alter the specific aggregation of magnetic particles in the presence of one or more analytes or multivalent binding agents.

In any of the systems, kits, cartridges, and methods for detecting any analyte in a whole blood sample of the present invention, the rupturing of red blood cells can be performed using a red blood cell lysing agent (i.e., a lysis buffer, or a non-ionic detergent). Erythrocyte lysis buffers that may be used in the methods of the invention include, but are not limited to: isotonic ammonium chloride solution (optionally containing carbonate buffer and/or EDTA) and hypotonic solutions. Alternatively, the red blood cell lysing agent can be an aqueous solution of a non-ionic detergent (e.g., nonylphenoxypolyethoxyethanol (NP-40), polyoxyethylene octylphenol ether (Triton-X100), Brij-58, or related non-ionic surfactants, and mixtures thereof). The red blood cell lysing agent disrupts at least a portion of the red blood cells, thereby enabling a majority of certain components of the whole blood (e.g., certain whole blood proteins) to be separated from the white blood cells, yeast cells, and/or bacterial cells present in the whole blood sample (e.g., as a supernatant after centrifugation). After red blood cell lysis and centrifugation, the formed pellet is reconstituted to form an extract.

The methods, kits, cartridges, and systems of the invention can be constructed as a predetermined list (panel) for detecting pathogen-associated analytes. For example, the list may be a list of candida fungi configured to individually detect three or more of candida guilliermondii, candida albicans, candida glabrata, candida krusei, candida ruchii, candida parapsilosis, and candida tropicalis. In another embodiment, the list may be a list of bacteria configured for individually detecting three or more of coagulase-negative staphylococcus, enterococcus faecalis, enterococcus faecium, pseudomonas aeruginosa, staphylococcus aureus, and escherichia coli. In a particular embodiment, the list may be a viral list configured for individually detecting Cytomegalovirus (CMV), epstein-barr virus, BK virus, hepatitis b virus, hepatitis c virus, Herpes Simplex Virus (HSV), HSV1, HSV2, Respiratory Syncytial Virus (RSV), influenza virus; three or more of influenza a virus, influenza a virus subtype H1, influenza a virus subtype H3, influenza B virus, human herpesvirus 6, human herpesvirus 8, human metapneumovirus (hMPV), rhinovirus, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, and adenovirus. The list may be a list of bacteria configured for individually detecting three or more of escherichia coli, cos (coagulase negative staphylococcus), pseudomonas aeruginosa, staphylococcus aureus, enterococcus faecium, enterococcus faecalis, and klebsiella pneumoniae. The list may be a list of bacteria configured for individually detecting three or more of aspergillus fumigatus and aspergillus flavus. The list may be a list of bacteria configured for individually detecting three or more of acinetobacter baumannii, enterobacter aerogenes, enterobacter cloacae, torpedo-leucobacter aucklandi, proteus mirabilis, serratia marcescens, staphylococcus haemolyticus, xanthomonas maltophilia, streptococcus agalactiae, streptococcus mitis, streptococcus pneumoniae, and streptococcus pyogenes. The list may be a meningitis list configured for individually detecting three or more of streptococcus pneumoniae, haemophilus influenzae, neisseria meningitidis, HSV1, HSV2, enteroviruses, listeria, escherichia coli, group B streptococci. This list may be configured to individually detect three or more of neisseria gonorrhoeae, staphylococcus aureus, streptococcus pyogenes, cos and borrelia burgdorferi. The list may be configured to detect three or more of clostridium difficile, toxin a and toxin B individually. The list may be a pneumonia list constructed for individually detecting three or more of streptococcus pneumoniae, MRSA, legionella, chlamydia pneumoniae, and mycoplasma pneumoniae. The list can be constructed to individually detect three or more of the therapeutic-resistant mutants selected from mecA, vanA, vanB, NDM-1, KPC and VIM. This list may be constructed for individually detecting three or more of haemophilus influenzae, neisseria gonorrhoeae, helicobacter pylori, campylobacter, brucella, legionella and stenotrophomonas maltophilia. The list can be constructed to detect the total viral load caused by CMV, EBV, BK virus, HIV, HBV and HCV. This list can be constructed for detecting fungal and/or bacterial loads. The sample may be measured using and with reference to a standard curve, or the viral load may be determined using some other method of quantifying the pathogen in the sample. Quantitative measurement methods may include real-time PCR, competitive PCR (ratio of two competing signals), or other methods mentioned herein. This list can be constructed for detecting an immune response in a subject by monitoring PCT, MCP-1, CRP, GRO-alpha, high mobility group protein B1(HMBG-1), IL-1 receptor antagonist, IL-1B, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, macrophage inflammatory protein (MIP-1), macrophage Migration Inhibitory Factor (MIF), osteopontin, RANTES (stimulated regulation, normal T cell expression and secretion; or CCL5), Th1, Th17, and/or TNF-alpha. This list can be constructed for individual detection of three or more of the genera Escherichia (Ehrlichea), Mycobacterium, syphilis, Borrelia burgdorferi, Cryptococcus, Histoplasma and Blastomyces. The list may be a list of influenza viruses configured for individually detecting three or more of influenza a, influenza B, RSV, parainfluenza, metapneumovirus, rhinovirus, and adenovirus.

The methods, kits, cartridges, and systems of the invention can be configured to reduce sample-to-sample variability by determining magnetic resonance signals before and after hybridization (hybridization). Addition of derivatized nanoparticles to the sample to enhance aggregation (clustering) prior to performing the method can provide a baseline, internal T₂Signals that can be subtracted after analyte-derivatized particle binding and aggregation or used to adjust T₂A signal. This method can also be used to determine or control inter-kit variability.

The terms "aggregation", "agglomeration" and "aggregation" are used interchangeably in the context of the magnetic particles described herein, and denote that 2 or more magnetic particles are bound to each other, e.g. via a multivalent analyte, multimeric form of an analyte, an antibody, a nucleic acid molecule, or other binding molecule or entity. In some cases, the agglomeration of the magnetic particles is reversible.

"analyte" means a substance or component of a sample to be analyzed. Exemplary analytes include one or more of the following: proteins, peptides, polypeptides, amino acids, nucleic acids, oligonucleotides, RNA, DNA, antibodies, carbohydrates, polysaccharides, glucose, lipids, gases (e.g., oxygen or carbon dioxide), electrolytes (e.g., sodium, potassium, chloride, bicarbonate, BUN, magnesium, phosphate, calcium, ammonia, lactate), lipoproteins, cholesterol, fatty acids, glycoproteins, proteoglycans, lipopolysaccharides, cell surface markers (e.g., CD3, CD4, CD8, IL2R, or CD35), cytoplasmic markers (e.g., CD4/CD8 or CD 4/viral load), therapeutic agents, metabolites of therapeutic agents, markers for detecting weapons (e.g., chemical or biological weapons), organisms, pathogens, pathogen byproducts, parasites (e.g., protozoa or mold), protists, fungi (e.g., yeast or mold), bacteria, nucleic acids, and/or nucleic acids, Actinomycetes, cells (e.g., whole cells, tumor cells, stem cells, leukocytes, T cells (e.g., showing CD3, CD4, CD8, IL2R, CD35, or other surface markers), or another cell identified with one or more specific markers), viruses, prions, plant components, plant by-products, algae by-products, plant growth hormones, pesticides, artificial toxins, environmental toxins, oil components, and components derived from the foregoing. The term "small molecule" as used herein refers to a drug, medicine, medicament, or other chemically synthesized compound intended for therapeutic use in humans. The term "biological" as used herein refers to a substance that is obtained from a biological source and is not synthetic, intended for human therapeutic use. A "biomarker" is a biological substance that can be used as an indicator of a particular disease state or a particular physiological state of an organism, typically a protein or other natural compound measured in a bodily fluid, the concentration of which reflects the presence or severity or stage of the disease state or disorder, can be used to monitor the progress of treatment of a disease or condition or disorder treatment, or can be used as a surrogate measure of clinical outcome or development. The term "metabolic biomarker" as used herein refers to a substance, molecule or compound synthesized or obtained by a biological pathway for determining the hepatic or renal function status of a patient or subject. The term "genotyping" as used herein refers to the ability to determine genetic differences in a particular gene that may or may not affect the phenotype of the particular gene. The term "phenotype" as used herein refers to the biological expression (metabolic or physiological) of a protein as defined by its genotype. The term "gene expression profiling" as used herein refers to the ability to determine the rate or amount of production of a gene product or the activity of gene transcription in a particular tissue in a temporal or spatial manner. The term "proteomic analysis" as used herein refers to a protein map or protein array used to identify the major differences in proteins or peptides in normal and diseased tissues. Other exemplary analytes are described herein. The term "analyte" also includes the components of such a sample: the direct product of the biochemical method of amplification of the initial target analyte, e.g., the product of a nucleic acid amplification reaction.

An "isolated" nucleic acid molecule refers to a nucleic acid molecule removed from its naturally occurring environment. For example, a naturally occurring nucleic acid molecule present in the genome of a cell or as part of a gene bank is not isolated, but the same molecule isolated from the remainder of the genome as a result of, for example, a cloning event, amplification or enrichment is "isolated". Typically, an isolated nucleic acid molecule is free of the nucleic acid region (e.g., coding region) immediately adjacent to the nucleic acid molecule at the 5 'or 3' end of the naturally occurring genome. Such an isolated nucleic acid molecule may be part of a vector or composition and may still be isolated in that such a vector or composition is not part of its natural environment.

"attached" as used herein means attached or associated by covalent bonds, non-covalent bonds, and/or by van der waals forces, hydrogen bonding, and/or other intermolecular forces.

The term "magnetic particles" refers to particles comprising materials of high positive susceptibility, such as paramagnetic compounds, superparamagnetic compounds, and magnetite, gamma-iron oxide, or metallic iron.

As used herein, "non-specific reversibility" refers to colloidal stability in a liquid sample and robustness of magnetic particles against non-specific aggregation, and can be determined by subjecting the particles to predetermined assay conditions in the absence of a specific aggregating moiety (i.e., an analyte or an aggregating agent). For example, the magnetic field can be made uniform at 0.45T (defined as <5000 ppm) of magnetic particles at 37 ℃ for 3 minutes before and after the incubation₂Value to determine the non-specific reversibility. T before and after the magnetic particles are subjected to predetermined measurement conditions₂Magnetic particles are considered to have non-specific reversibility if the difference in value varies by less than 10% (e.g., varies by less than 9%, 8%, 6%, 4%, 3%, 2%, or 1%). If the difference is greater than 10%, the particles exhibit irreversibility in the buffers, diluents, and matrices tested, and it may be desirable to treat the particle and matrix properties (e.g., coating and buffer formulations) to prepare a system in which the particles have non-specific reversibility. In another embodiment, T of the magnetic particle solution can be measured before and after incubation in a gradient magnetic field of 1 Gauss/mm-10000 Gauss/mm₂The test is applied with the value.

The term "NMR relaxation rate" as used herein refers to the measurement in a sample of any one of: t is₁、T₂、T₁/T₂Mixing, T_1rho、T_2rhoAnd T₂ ^*. The systems and methods of the present invention are designed to produce an NMR relaxation rate that is indicative of the presence or absence of an analyte in a liquid sample. In some casesThe NMR relaxation rate characterizes the amount of analyte present in the liquid sample.

The term "T" as used herein ₁/T₂Mixing means mixing T₁And T₂Any detection method of measurement incorporation. E.g. T₁/T₂The value of the blend may be obtained by combining two or more different T₁And T₂The ratio or difference between the measurements is combined to obtain a composite signal. T is₁/T₂The mixing may be, for example, by using a pulse sequence (where T is₁And T₂Alternately measured or acquired in an interleaved manner). Furthermore, T₁/T₂The mixed signal may be obtained using a pulse sequence, the pulse sequence measurement being represented by T₁And T₂Relaxation rate or relaxation rate of mechanism.

"pathogen" means an agent that causes its host to develop a disease or illness, such as an organism or infectious particle capable of causing a disease in another organism, including but not limited to bacteria, viruses, protozoa, prions, yeasts and fungi, or a pathogen byproduct. A "pathogen byproduct" is a biological substance produced by a pathogen that may be harmful to the host or that stimulates an excessive host immune response, such as one or more pathogen antigens, metabolites, enzymes, biological substances, or toxins.

By "pathogen-associated analyte" is meant an analyte that is characteristic of the presence of a pathogen (e.g., bacteria, fungi, or viruses) in a sample. The pathogen-associated analyte can be a specific substance derived from the pathogen (e.g., a protein, nucleic acid, lipid, polysaccharide, or any other substance produced by the pathogen) or a mixture derived from the pathogen (e.g., a whole cell, or a whole virus). In some cases, pathogen-associated analytes are selected to characterize the genus, species, or specific strain of the pathogen being detected. Alternatively, pathogen-associated analytes are selected to determine characteristics of the pathogen, such as resistance to a particular treatment. For example, the pathogen-associated analyte may be a gene that characterizes vancomycin resistance in many different bacterial species, such as the Van a gene or the Van B gene.

A "pulse sequence" or "RF pulse sequence" denotes one or more radio frequency pulses to be applied to a sample and is designed to measure, for example, certain NMR relaxation rates, such as spin echo sequences. The pulse sequence may also include signal acquisition after one or more pulses to minimize noise and improve the accuracy of the generated signal values.

The term "signal" as used herein refers to NMR relaxation rates, frequency shifts, sensitivity measurements, diffusion measurements, or related measurements.

As used herein, "size" of the magnetic particles refers to the average diameter of the mixture of magnetic particles as measured by microscopy, light scattering, or other methods.

The term "substantially monodisperse" as used herein refers to a mixture of magnetic particles having a particle size distribution polydispersity (as determined by the shape of the particle size distribution curve in light scattering measurements). Particle distribution curves having a FWHM (full width at half maximum) less than 25% of the peak position are considered to be substantially monodisperse. In addition, only 1 peak should be observed in the light scattering experiment and the peak position should be within 1 standard deviation of a known population of monodisperse particles.

"T per particle ₂The "relaxation rate" represents the average T per particle in a population of magnetic particles₂The relaxation rate.

As used herein, "unfractionated" refers to an assay in which no components in the sample under test are removed after the addition of magnetic particles to the sample and prior to NMR relaxation measurements.

It is contemplated that the claimed units, systems, methods, and processes of the invention include variations and modifications that may be made using the information of the embodiments described herein. Throughout the description of the specification, if units and systems are described as having, containing, or containing specific components (ingredients), or if processes and methods are described as having, containing, or containing specific steps, it is additionally contemplated that units and systems of the present invention exist that consist essentially of or consist of the recited components (ingredients), and that processes and methods according to the present invention exist that consist essentially of or consist of the recited processing steps. It should be understood that the order of steps or order for performing certain operations is not essential, unless otherwise specified, so long as the invention remains operable. Further, 2 or more steps or operations may be performed simultaneously in many cases.

Other features and advantages of the invention will become apparent based upon the following detailed description, the accompanying drawings, and the claims.

Drawings

FIG. 1A is a schematic diagram of an NMR unit for detecting the signal response of a sample to an RF pulse sequence, according to an illustrative embodiment of the invention.

FIG. 1B depicts a typical coil configuration around a sample tube for measuring relaxation signals in a 20 μ L sample.

FIGS. 2A-2E illustrate the geometry of a microcoil that may be used for NMR (for excitation and/or detection) according to an illustrative embodiment of the invention; designs include, but are not limited to: non-closed solenoid coils (fig. 2A), planar coils (fig. 2B), MEMS solenoid coils (fig. 2C), MEMS Helmholz coils (fig. 2D), and saddle coils (fig. 2E). Three-dimensional lithographic coil fabrication (dimensional lithographic coil fabrication) of well characterized coils for MR detection has also been established and can be used for these purposes Demas et al, "Electronic characterization of lithographic patterned coils for high sensitivity NMR detection" J-Mag Reson 200: 56(2009).

Fig. 3A is a graph depicting an aggregation assay of the present invention. The magnetic particles (dots) are coated with a binding agent (i.e., antibody, oligonucleotide, etc.) to facilitate separation The analyte or multivalent binding agent forms an aggregate in the presence of the analyte or multivalent binding agent. The dotted circle represents a diffusion sphere or at T₂The fraction of the total fluid volume through which solution molecules can pass by diffusion during the measurement (the specific path traveled by the water molecules is random and this figure is not drawn to scale). Concentration (right hand side) of interference-reducing water T₂Part of the sample of microscopic magnetic inhomogeneity of the signal, resulting in T₂An increase in relaxation.

FIG. 3B is a graph depicting a polydispersion model, and shows T when particles form clusters of a particular size₂A transition (transition) occurs between two points on the curve. The response curve will be linear for analyte addition but non-linear for the volume fraction of the cluster, as the particles transition between state 1 and state 2. The slope of the response curve is directly proportional to the sensitivity of the assay.

FIGS. 4A-4C are graphs depicting different assay formats for assays of the invention. Fig. 4A depicts an agglomerated sandwich immunoassay in which two populations of magnetic particles are designed to bind to 2 different epitopes of an analyte. Fig. 4B depicts a competitive immunoassay in which an analyte in a liquid sample binds to a multivalent binding agent (multivalent antibody), thereby inhibiting aggregation. Fig. 4C shows a hybridization-mediated agglomeration assay in which two populations of particles are designed to bind to first and second portions of a nucleic acid target, respectively.

Fig. 5 shows the modular cartridge concept in sections that can be packed and stored separately. The concept is designed, for example, such that the inlet module (shown raised, with inverted single-use vacuum blood collection tubes affixed) can be sterilized while the module containing the reagents in the middle is not sterilized. This allows the reagent-containing part to be the only part that is frozen.

Fig. 6A-6F depict a single use evacuated blood collection tube inlet module. Fig. 6A shows the disposable vacuum blood collection tube in an inverted position after the user has removed the cap from it and placed the cartridge on it. Figure 6B shows the cartridge right side turned to the upper blood will flow out of the disposable vacuum blood collection tube and into the sample loading area of the molding path. The foil seal may be the bottom side of the channel, thereby forming a low cost molded part with a closed channel. Figure 6C is a cross-sectional view showing the venting tube allowing air to enter the vial as blood exits and fills the sample region. Fig. 6D-6F show an inlet module for aliquot sampling of samples, designed to interface with uncapped single-use evacuated blood collection tubes, and designed to aliquot sample two sample volumes that can be used to perform, for example, candida assays. The inlet module has two hard plastic parts that are ultrasonically welded together and sealed with foil to form a network of channels, allowing flow channels to form in the first well and then spill into the second sample well. The soft disposable vacuum blood collection tube sealing member is used for sealing the disposable vacuum blood collection tube. It has one port for sample flow and a gas permeable port that allows flow to occur.

Fig. 7A is a graph depicting the components of the creatinine competitive assay of example 6. Magnetic particles modified with creatinine were used in combination with creatinine antibodies to form an aggregation system. Creatinine present in the liquid sample competes with the magnetic particles for antibodies, resulting in a decrease in aggregation as the concentration of creatinine increases. T of hydrogen nuclei in water molecules with liquid sample₂Change in relaxation rate, change in aggregation was observed. T by observation of a liquid sample₂The relaxation rate is compared to a standard curve to determine the concentration of creatinine.

Fig. 7B is a diagram depicting the architecture of the tacrolimus competition assay of example 9.

Fig. 7C is a diagram depicting the candida agglomeration sandwich assay architecture of example 10.

Figures 8A-8C are a series of graphs showing response curves for creatinine competition assays. FIG. 8A is a view showing T for observation₂Standard Curve of the Creatinine Competition assay of example 6, with relaxivity correlated to the concentration of creatinine in a liquid sampleFigure (a). FIG. 8B shows T for creatinine-modified particles with 2 different antibody formulations₂And (6) responding. Preparation 1 was prepared beforehand (with aggregated antibody) and preparation 2 was prepared purified (without aggregated antibody). FIG. 8C shows T of creatinine-modified particles with unaggregated antibody, biotinylated antibody, and finely multimerized antibody ₂In response, and the increased agglomeration capacity of the multivalent agglomerating agent was confirmed.

FIG. 9 is a graph showing T of a liquid sample to be observed₂Graph of a standard curve for the tacrolimus competition assay of example 9 correlating relaxation rate with tacrolimus concentration in the liquid sample.

Figure 10 is a graph depicting a creatinine inhibition curve (see example 7) using antibody-coated particles and an amino-dextran-creatinine multivalent binding agent to induce aggregation by competing with any target analyte (creatinine) present in the sample to cause particle aggregation. The binding agent used was 40kDa dextran with approximately 10 creatinine/dextran molecules.

FIG. 11 is a graph depicting the ability of Tac-dextran conjugates to accumulate by performing a titration (see example 8). As observed, the increase in molecular weight of Tac-glucan leads to T₂The signal is improved.

FIG. 12 is a graph depicting the ability of Tac-dextran conjugates to accumulate by performing a titration (see example 8). As observed, higher substitutions improved T₂A signal.

FIG. 13 is a graph depicting the ability of Tac-BSA conjugate to accumulate by performing a titration similar to that used for the Tac-dextran conjugate (see example 8). As observed, the aggregation performance varied with the tacrolimus substitution ratio.

FIG. 14 is a graph depicting T for detection of anti-biotin antibodies (as described in example 1) in blood and PBS matrices using prepared magnetic particles₂Graph of the measurement results.

FIG. 15 is a graph depicting T's for detection of anti-biotin antibodies (as described in examples 8 and 9) using prepared magnetic particles with (open circles) and without (closed circles) protein blocking (protein blocks)₂Graph of the measurement results.

FIG. 16 is a graph depicting T for detection of anti-biotin antibodies (as described in example 2) using magnetic particles prepared with BSA blocking (black filled diamonds, squares, triangles) or FSG blocking (light grey X and circles)₂Graph of the measurement results.

Fig. 17A and 17B are schematic illustrations of provided particle coatings.

FIGS. 18A-18B depict T for detection of biotin in the competitive assay format described in example 4₂The results of the measurement. Fig. 18A depicts the results of the experiment in buffer, while fig. 18B depicts the results of the experiment in lysed blood.

Fig. 19A is a table and 19B is a graph depicting the reproducibility of candida measurements over an 8 day period, respectively, according to the method of the present invention. To determine the reproducibility of the T2 measurements on candida albicans infected human whole blood, we performed an 8-day study in which the same donor spiked and amplified samples were hybridized to superparamagnetic particles (n =3) each day and the resulting T2 values were recorded (see example 13). The accuracy of the analysis in batches is shown in FIG. 19A and is generally tight (light) with all measured CV's being less than 12%. The reproducibility of the observations over the 8-day period is shown in fig. 19B (mean T2 values measured from the same donor spiked and amplified samples over the 8-day period +/-95% confidence interval), and CVs were less than 10% over the entire candida concentration range, and less than 6% for the negative control.

FIG. 20 is a scheme describing a workflow for detecting bacterial or fungal pathogens in a whole blood sample (see example 14 and example 17).

Fig. 21A and 21B are graphs depicting results from donor samples. FIG. 21A is a graph depicting the results obtained from 16 experiments designed to evaluate the assay performance of 6 different donor blood samples spiked with a range of Candida albicans cells (see example 13). Each data point is the mean +/-95% confidence interval (n = 48). At the lowest concentration detected (10 cells/mL), we failed to detect candida albicans within 37% of the time (6 out of 16 experiments); whereas candida albicans was detected at 100 cells/mL in 100% of the time. This indicates that the assay can be reliably detected at candida albicans concentrations greater than or equal to 100 cells/mL, and without major performance inhibition introduced by the donor blood sample. FIG. 21B is a graph depicting results obtained from 7 experiments designed to evaluate the assay performance of 6 different donor blood samples spiked with a range of Candida krusei cells (see example 13). Each data point is the mean +/-95% confidence interval (n = 21). We did not detect at 10 cells/mL in any experimental batch, but at 100 cells/mL in all experimental batches. This indicates that the LOD is between 10 and 100 cells/mL.

FIG. 22 is a dot plot showing T2 measurements of 5 Candida albicans clinical isolates spiked into 400 μ L whole blood at a concentration of 0 to 1E4 cells/mL. The results plotted are mean +/-1 SD. These data indicate that while the absolute T2 values obtained were spread among the different isolates, all values at 50 cells/mL exceeded those of the candida-free control (3 replicates from 20 independent assays, for a total of 60 different aggregation reactions).

FIGS. 23A and 23B are ROC plots of the T2 results generated at 10 cells/mL (FIG. 23A) and 50 cells/mL (FIG. 23B). The area under the curve was 0.72 at 10 cells/mL (95CI =0.56 to 0.88) and 0.98 at 50 cells/mL (95CI =0.95 to 1.001). The area under the curve is often used to quantify diagnostic accuracy; in this case we were able to distinguish candidemia patients with 10 cells/mL or 50 cells/mL infection from patients without candidemia. The area under the curve at 10 cells/mL was 0.72, which means if T is performed in randomly selected people with candidemia of 10 cells/mL infection level₂As determined, then there is a 72% probability that their T2 value will be higher than that of a person without candidemia. The clinical accuracy of this assay was much higher at 50 cells/mL, with an area under the curve of 0.98. Again, T is shown to be 98% of the time in people with this level of infectious candidemia ₂The assay will yield a value higher than samples from patients without candidemia. See example 13.

Figure 24 is a graph depicting the sensitivity of an assay using standard thermal cycling (reporting time of about 3 hours) and a process that combines annealing/extension steps (reporting time of about 2 hours, 13 minutes). Combining the annealing step and the extension step in the thermal cycling reduced the total assay TAT to 2.25 hours and did not affect assay sensitivity.

FIG. 25 is a T depicting PCR cycles₂Graph of signal change (see example 14). These results show that the methods and systems of the invention can be used to perform real-time PCR and provide quantitative information about the amount of target nucleic acid present in a sample.

FIG. 26 is a series of photographs showing a simple magnetic separator/PCR block insert.

Fig. 27 is an image showing the amount of DNA produced by amplifying: (1) 100 copies of the candida albicans genome amplified in the presence of 3 'and 5' candida albicans single probe nanoparticles; these particles were held on the side walls during PCR using a magnetic field, (2) 100 copies of the Candida albicans genome amplified without nanoparticles, and (3) 100 copies of the Candida albicans genome amplified in the presence of 3 'and 5' Candida albicans single probe nanoparticles; no magnetic field.

FIGS. 28A-28E are schematic illustrations of sample tubes containing magnetizable metal foam (shaded), magnetic particles (circles), and immobilized portions of analyte (triangles). A magnetizable metal foam (e.g. made of nickel) may be inserted into the catheter and immobilized by exposure to heat, which shrinks the catheter around the metal foam, thereby forming a tight seal. Then, a sample containing magnetic particles and an analyte was introduced at one end of the catheter (fig. 28A). Next, the catheter is exposed to a magnet (fig. 28B), and the magnetic particles are attracted to the metal foam and magnetically bound within its pores or crevices. The average diameter of the pores in the metal foam is, for example, between 100 and 1000 microns. Analyte molecules may be transported to the metal foam by binding to the magnetic particles, or a fluid may be driven through the metal foam to reach the bound magnetic particles. When bound in the metal foam, the magnetic particles have enhanced interactions, as they are now confined and closer to other magnetic particles, and cluster formation is enhanced. The metal foam is then demagnetized (fig. 28C), i.e. the magnetic field of the metal foam becomes negligible. The majority of the magnetic particle and analyte cluster complexes still remained in the metal foam because the diffusion of the magnetic particle clusters was relatively low, although the analyte partially naturally diffused into and out of the metal foam (fig. 28D). Alternatively, the magnetizable metal foam (hollow cylinder) floats freely in the sample tube with magnetic particles (circles) and analyte (star). The magnetization and demagnetization of the free-floating metal foam serves to increase the rate of aggregate formation.

Fig. 29 is a table depicting T2MR results for 32 clinical samples, which showed 14 samples as candida positive. This test identified 4 samples containing candida krusei or candida glabrata, 7 samples containing candida albicans or candida tropicalis, and 3 samples containing candida parapsilosis. The solid black line indicates the decision threshold (T2=128 msec) (see example 16).

Detailed Description

The invention features systems, devices, and methods for rapid detection of an analyte or determination of the concentration of an analyte in a sample. The system and method of the present invention uses: magnetic particles, NMR unit, optionally one or more incubation stations at different temperatures, optionally one or more vortex mixers (vortexer), optionally one or more centrifuges, optionally a flow control station, optionally a robotic system, and optionally one or more modular cartridges. The systems, devices, and methods of the invention can be used to assay biological samples (e.g., blood, sweat, tears, urine, saliva, semen, serum, plasma, cerebrospinal fluid (CSF), stool, vaginal fluid or tissue, sputum, nasopharyngeal aspirate or swab-removed nasopharyngeal specimen, tears, mucus, or swab-removed epithelial specimen (swab-removed buccal specimen), tissue, organ, bone, tooth, or tumor, etc.). Alternatively, the systems, devices and methods of the present invention are for monitoring environmental conditions (e.g., plant growth hormones, pesticides, artificial or environmental toxins, nucleic acid sequences important for insect resistance/susceptibility, algae and algae by-products); as part of a biological blowdown procedure, for growing plants or animals, or identifying environmental hazards. Similarly, the systems, devices and methods of the present invention are for detecting and monitoring biological warfare or biological warfare agents, such as ricin, salmonella typhimurium, botulinum toxin, aflatoxins, mycotoxins, Francisella tularesis, smallpox, anthrax, and the like.

Magnetic particles can be coated with binding moieties (i.e., antibodies, oligonucleotides, aptamers, etc.) to form aggregates in the presence of an analyte or multivalent binding agent. Aggregation-reducing interference solvent T₂Part of the sample of microscopic magnetic inhomogeneity of the signal, resulting in T₂Increase in relaxation (see fig. 3).

T₂The measurement is a single measurement of all spins in a population of measurements typically lasting 1-10 seconds, which duration enables the solvent to travel hundreds of microns, which is a long distance relative to microscopic non-uniformities in the liquid sample. Each solvent molecule takes a volume of sample from the liquid sample, and T₂The signal is the average (net total signal) of all (nuclear spins) on the solvent molecules in the sample; in other words, T₂The measurement is a net measurement of the entire environment in which the solvent molecules are located, and is an average measurement of all microscopic inhomogeneities in the sample.

Observed T of solvent molecules in liquid samples₂The relaxation rate is determined by the magnetic particles, which form a high magnetic dipole moment in the presence of a magnetic field. Observed T of liquid sample in absence of magnetic particles₂The relaxation rate is generally longer (i.e., T)₂(Water) = approximately 2000 ms, T₂(blood) = about 1500 ms). As the particle concentration increases, the microscopic inhomogeneities in the sample increase, and solvent diffusion (through these microscopic inhomogeneities) leads to an increase in spin decoherence and T ₂The value decreases. Observed T₂The values depend in a non-linear manner on the particle concentration and on the relaxation rate parameter of each particle.

In the aggregation assay of the invention, the number of magnetic particles and the number of aggregated particles (if present) remain constant during the assay. When these particles are aggregated, the spatial distribution of the particles changes. Aggregation changes the average "experience" of the solvent molecules, as aggregation facilitates the localization of particles into clusters rather than obtaining a more uniform distribution of particles. At high degrees of aggregation, many solvent molecules do not experience the microscopic inhomogeneities caused by the magnetic particles and T₂Value close to T of solvent₂The value is obtained. The observed T increases as the fraction of magnetic particles aggregated in the liquid sample₂The values are the average values of a heterogeneous suspension of aggregated and individual (non-aggregated) magnetic particles. The assay of the invention is designed to use aggregation to increase T₂The value change is maximized to improve the sensitivity of the assay to differences in analyte presence and analyte concentration.

There are two ways of particle agglomeration (registers) and T₂Based on particle size (see fig. 3B, the boundary is typically about 100 nm particle size). For any given liquid sample measurement, the particle count of magnetic particles having a particle size of 250 nm may be about 1X 10 ⁷The particle count of the magnetic particles having a particle size of 30 nm may be about 1X 10¹³. This is because smaller particles have a lower relaxation rate per particle (for the same type of material), resulting in inherent sensitivity aspectsAnd (4) the defect. In a typical assay of the invention, the magnetic particles are selected such that T₂The value increases as the fraction of aggregated particles increases.

The assays of the invention can be designed to alter T in the presence of an analyte₂In the direction of (see fig. 4A-4C). For example, the assay may be an agglomerated sandwich immunoassay in which two populations of magnetic particles bind to different epitopes of the analyte (see fig. 4A); a competitive assay, wherein the analyte competes with a multivalent binding agent to inhibit aggregation of the magnetic particles (see fig. 4B); or hybridization-mediated agglomeration, in which two populations of magnetic particles are bound to the first and second portions of the oligonucleotide (see fig. 4C). Other forms of competition may include binding when two particle binding moieties bind without an agglomerant (e.g., the DNA oligonucleotide is designed such that two nanoparticles have two different oligonucleotides and they can anneal (anneal) together, and the analyte or amplicon or target DNA competes or interferes with np apposition when heated).

Other formats for performing the assays of the invention may be used, for example: (i) the target sample may be incubated in the presence of magnetic particles that have been modified with a binding moiety specific for the target analyte and the multivalent binding agent, binding of the analyte to the magnetic particles in an inhibition assay hindering agglomeration of the magnetic particles with the multivalent binding agent; (ii) the target sample may be incubated in the presence of magnetic particles that have been modified with binding moieties specific for the target analyte and multivalent binding agent, the analyte is exposed to pre-formed aggregates of multivalent binding agent and magnetic particles in a disaggregation assay, and the analyte displaces multivalent binding agent to reduce aggregation in the liquid sample; or (iii) the target sample may be incubated in the presence of magnetic particles that have been modified with a binding moiety specific for the target analyte and the target analyte itself forms a self-assembled single population of magnetic particles, the presence of binding of the analyte to the magnetic particles in the inhibition or disaggregation assay hindering self-aggregation of the magnetic particles; or (iv) the target sample may be incubated in the presence of a soluble aggregation agent and magnetic particles modified with the analyte or analyte analogue, the presence of binding of the analyte to the soluble aggregation agent hindering aggregation of the particles in the inhibition assay.

In the case of multivalent binding agents (aggregators), the analytes are attached to a carrier (e.g., a simple synthetic scaffold, or a larger carrier protein or polysaccharide such as BSA (bovine serum albumin), transferrin, or dextran).

Magnetic particles

The magnetic particles described herein include, for example, those described in U.S. patent No. 7,564,245 and U.S. patent application publication No. 2003-0092029, the contents of which are incorporated herein by reference. These magnetic particles are typically in the form of conjugates, i.e. magnetic particles having one or more binding moieties (e.g. oligonucleotides, nucleic acids, polypeptides, or polysaccharides) attached thereto. The binding moiety causes a specific interaction with the target analyte. The binding moiety specifically binds to a selected target analyte, such as a nucleic acid, polypeptide, or polysaccharide. In some cases, the binding results in aggregation of the conjugate, resulting in a change, such as a decrease (e.g., in the case of smaller magnetic particles) or an increase (e.g., in the case of larger magnetic particles), in the spin-spin relaxation time (T2) of the adjacent aqueous proton (or proton in the non-aqueous solvent) in the aqueous solution. Alternatively, the analyte binds to preformed aggregates (e.g., aggregates formed by multivalent binding agents and magnetic particles) in a competitive disaggregation assay, or competes with multivalent binding agents for binding moieties on magnetic particles (i.e., inhibits formation of aggregates in the presence of the analyte) in an inhibition assay.

The conjugates have a high relaxivity because of the superparamagnetic properties of the iron, metal oxide or other ferrous compound (ferro) or ferrimagnetic nanomaterial of these conjugates. Iron, cobalt and nickel compounds and their alloys, rare earth elements (e.g., gadolinium), and certain intermetallic compounds (e.g., gold and vanadium) are ferromagnetic substances and thus can be used to make superparamagnetic particles. The magnetic particles may be monodisperse (single crystal/per magnetic particle of a magnetic material such as a metal oxide (e.g. superparamagnetic iron oxide)) or polydisperse (e.g. crystals/per magnetic particle). The magnetic metal oxide may also contain cobalt, magnesium, zinc, or mixtures of these metals with iron. Important characteristics and factors of magnetic particles that can be used to make conjugates include: (i) high relaxivity, i.e. a strong influence on the relaxation of water (or other solvent), (ii) functional groups to which the binding moieties can be covalently bound, (iii) low non-specific binding of the interacting moieties to the magnetic particles and/or (iv) stability in solution, i.e. the magnetic particles remain suspended in solution without precipitation and/or the nps retain their ability to be applied in the described method (i.e. the nps have a shelf life).

The magnetic particles may be attached to the binding moiety via a functional group. In some embodiments, the magnetic particles may be partially bound to a polymer comprising selected functional groups to increase the non-specific reversibility of the magnetic particles. The polymer may be a synthetic polymer such as, but not limited to, polyethylene glycol or silane, a natural polymer, or a derivative of a synthetic or natural polymer, or a combination thereof. The polymer may be hydrophilic. In some embodiments, the polymer "coating" is not a continuous film around the magnetic metal oxide, but rather is a "mesh" or "cloud" of extended polymer chains connected to and surrounding the metal oxide. The polymer may comprise polysaccharides and derivatives thereof, including dextran, pullulan, carboxydextran, carboxymethyl dextran and/or reduced carboxymethyl dextran. The metal oxide may be a group of one or more crystals in contact with each other or individually bound or surrounded by a polymer.

Alternatively, the magnetic particles may be combined with a non-polymer functional group composition. Methods for the synthesis of stable, functionalized magnetic particles without bound polymer are described, for example, in Halbreich et al, Biochimie,80:379 (1998).

The magnetic particles typically comprise single crystals and polycrystals of metal oxides having a diameter of about 1-25 nm, for example about 3-10 nm or about 5 nm, per crystal. The magnetic particles may also comprise a polymer component in the form of a core and/or a coating (e.g. about 5 to 20 nm thick or more). The total size of the magnetic particles may be, for example, 20 to 50 nm, 50 to 200 nm, 100 to 300 nm, 250 to 500 nm, 400 to 600 nm, 500 to 750 nm, 700 to 1,200 nm, 1,000 to 1,500 nm, or 1,500 to 2,000 nm.

Magnetic particles can be prepared in a variety of ways. Preferably, the magnetic particles have functional groups that attach the magnetic particles to the binding moiety. For example, carboxyl-functionalized magnetic particles can be prepared according to the method of Gorman (see PCT publication WO 00/61191). In this method, reduced Carboxymethyl (CM) dextran is synthesized from commercially available dextran. Carboxymethyl dextran was mixed with iron salts and then neutralized with ammonium hydroxide. The resulting carboxyl-functionalized magnetic particles can be used to conjugate amino-functionalized oligonucleotides. Carboxyl-functionalized magnetic particles can also be made from polysaccharide-coated magnetic particles by reaction with bromoacetic acid or chloroacetic acid in a strong base to attach carboxyl groups. In addition, carboxyl-functionalized particles can be made from amino-functionalized magnetic particles by converting amino groups to carboxyl groups using a reagent (e.g., succinic anhydride or maleic anhydride).

The size of the magnetic particles can be controlled by adjusting the reaction conditions, for example, using low temperatures during the neutralization of iron salts with bases, as described in U.S. patent No. 5,262,176. Uniformly sized materials can also be made by classifying the particles using centrifugation, ultrafiltration, or gel filtration, for example as described in U.S. patent No. 5,492,814.

Magnetic particles can also be synthesized according to the method of Molday (Molday, R.S. and D.MacKenzie, "immunological magnetic iron-dextran reagents for the labeling and magnetic separation of cells", J.Immunol. Methods,52:353(1982)), and treated with periodate to form aldehyde groups. The aldehyde-containing magnetic particles can then be reacted with a diamine (e.g., ethylenediamine or hexamethylenediamine) to form a schiff base, followed by reduction with sodium borohydride or sodium cyanoborohydride.

Dextran-coated magnetic particles can be produced andand crosslinked with epichlorohydrin. The added ammonia reacts with the epoxy group to produce an amine group, see Hogemann, D.et al, "Improvement of MRI probes to allow effective detection of gene expression," bioconjugate.chem., 11:941(2000), and Josephson et al, "High-efficiency intracellular labeling with novel superior-peptide conjugates," bioconjugate.chem., 10:186 (1999). This material is known as crosslinked iron oxide or "CLIO" and when functionalized with an amine is known as amine-CLIO or NH ₂-CLIO. Carboxyl-functionalized magnetic particles can be converted to amino-functionalized magnetic particles by using water-soluble carbodiimides and diamines, such as ethylenediamine or hexamethylenediamine.

The magnetic particles may be formed from a ferromagnetic liquid (i.e., a stable colloidal suspension of magnetic particles). For example, the magnetic particles may be a composite comprising a plurality of metal oxide crystals having a size of several tens of nanometers and dispersed in a liquid containing a surfactant, which adsorbs to these particles and stabilizes them, or by precipitation in an alkaline medium of a metal ion solution. Suitable ferrofluids are sold by the company Liquids Research, Inc. under the index WHKS1S9(A, B or C), which is a magnet (Fe) comprising a diameter of 10 nm₃O₄) The water-based ferrofluid of (a); WHJS1(A, B or C) which is a magnet (Fe) with a diameter of 10 nm₃O₄) An isoparaffin-based ferrofluid of the particles; and BKS25 glucan which is a polysaccharide containing magnetite (Fe) having a diameter of 9 nm₃O₄) A water-based ferrofluid of particles stabilized with dextran. Other suitable ferrofluids for use in the present systems and methods are oleic acid stabilized ferrofluids available from Ademtech, which comprise about 70% by weight alpha-Fe ₂O₃Particles (diameter about 10 nm), 15 wt% octane and 15 wt% oleic acid.

These magnetic particles are typically composites comprising a plurality of metal oxide crystals and an organic matrix, and have a surface modified with functional groups (i.e., amine or carboxyl groups) that attach binding moieties to the surface of the magnetic particles. For example, magnetic particles useful in the methods of the invention include those sold by Dynal, Seradyn, Kisker, Miltenyi Biotec, Chemicell, Anvil, Biopal, Estapor, Genovis, Thermo Fisher Scientific, JSR micro, Invitrogen, and Ademtech, as well as those described in U.S. patent nos. 4,101,435, 4,452,773, 5,204,457, 5,262,176, 5,424,419, 6,165,378, 6,866,838, 7,001,589, and 7,217,457, each of which is incorporated herein by reference.

Avidin or streptavidin may be attached to magnetic particles for use with biotinylated binding moieties (e.g., oligonucleotides or polypeptides) (see, e.g., Shen et al, "magnetic labeled polypeptide receptors after to microorganisms cells," bioconjugate. chem.,7:311 (1996)). Similarly, biotin may be attached to the magnetic particles for binding moieties labeled with avidin. Alternatively, the binding moiety is covalently attached to the surface of the magnetic particle; these particles can be modified with IgG molecules; these particles can be modified with anti-His antibodies; alternatively, these particles can be modified with His-tagged FAb.

Low molecular weight materials can be separated from the magnetic particles prior to use using ultrafiltration, dialysis, magnetic separation, or other means. For example, unreacted binding moieties and linking agents can be separated from the magnetic particle conjugates by magnetic separation or size exclusion chromatography. In some cases, the magnetic particles may be sized to produce a mixture of particles having a particular size range and average diameter.

For certain assays requiring high sensitivity, T is used₂Analyte detection for relaxation assays may require the selection of suitable particles to achieve sufficiently sensitive analyte-induced agglomeration. Particles containing multiple superparamagnetic iron oxide cores (5-15 nm in diameter) within a single larger polymer matrix or ferrofluid assembly (assembly) (100 nm-1200 nm overall diameter, e.g. particles with 100 nm, 200 nm, 250 nm, 300 nm, 500 nm, 800 nm or 1000 nm average diameter) may be used, either by using higher magnetic moment materials or particles with higher densityAnd/or particles with a higher iron content to achieve a higher sensitivity. Without being bound by theory, it is hypothesized that the much larger number of iron atoms per particle results in these types of particles providing a sensitivity gain of greater than 100 x, which is believed to result in an increase in sensitivity (due to the reduced number of particles present in the assay solution and the higher amount of superparamagnetic iron that is likely to be affected by each aggregation event).

The relaxation rate and particle size of each particle is a useful term for selecting the best particle for high sensitivity assays. Ideally, this term would be as large as possible. The relaxation rate of each particle is the T measured for each particle pair₂A measure of the impact of the value. The larger this number, the more T that is given₂The smaller the number of particles required for the response. In addition, decreasing the concentration of particles in the reaction solution can increase the analytical sensitivity of the assay. The relaxation rate of each particle may be a more useful parameter, since the iron density and relaxation rate may vary between different magnetic particles depending on the composition used to make the particles (see table 1). The relaxation rate of each particle is proportional to the saturation magnetization of the superparamagnetic material.

TABLE 1

Bell power Diameter (Hydroynamic Diameter) (nm)	Number of metal atoms per particle	Relaxation Rate per particle (mM)^-1s^-1)
			10-30	1.0E +03 to 1.0E +06	1.0E +6 to 1.0E +11
10-50	8.0E +02 to 4.0E +04	1.0E +04 to 4.0E +06
			10-50	1.0E +04 to 5.0E +05	1.0E +06 to 1.0E +08
50-100	1.0E +04 to 1.0E +07	1.0E +06 to 1.0E +09
			100-200	5.0E +06 to 5.0E +07	5.0E +08 to 8.0E +09
200-300	1.0E +07 to 1.0E +08	3.0E +09 to 1.0E +10
			300-500	5.0E +07 to 1.0E +09	7.0E +09 to 5.0E +10
500-800	1.0E +08 to 4.1E +09	1.0E +10 to 5.0E +11
			800-1000	5.0E +08 to 5.0E +09	5.0E +10 to 5.0E +11
1000-1200	1.0E +09 to 7.0E +09	1.0E +11 to 1.0E +12

The base particles used in the systems and methods of the present invention can be any of the commercially available particles shown in table 2.

TABLE 2

Directory number	Source/description	Diameter (μm)
				Kisker
MAv-1	Polystyrene, avidin coated magnet particles	1.0-1.9
			PMSt-0.6	Polystyrene, streptavidin coated magnet particles	0.5-0.69
PMSt-0.7	Polystyrene, streptavidin coated magnet particles	0.7-0.9
			PMSt-1.0	The amount of polystyrene to be used is such that,streptavidin-coated magnet particles	1.0-1.4
PMB-1	Polystyrene, magnet particles with biotin covalently bound to BSA coating	1.0-1.9
			PMP-200	Dextran-based, uncoated, pure (plain)	0.2
PMP-1000	Dextran-based, uncoated, pure	0.10
			PMP-1300	Dextran-based, uncoated, pure	0.13
PMP-2500	Dextran-based, uncoated, pure	0.25
			PMN-1300	Based on dextran, NH₂-coated	0.13
PMN-2500	Based on dextran, NH₂-coated	0.25
			PMC-1000	Based on dextran, COOH-coating	0.10
PMC-1300	Based on dextran, COOH-coating	0.13
			PMC-2500	Based on dextran, COOH-coating	0.25
PMAV-1300	Based on dextran, avidin coated	0.13
			PMAV-2500	Based on dextran, avidin coated	0.25
PMSA-1000	Based on dextran, streptavidin-coated	0.1
			PMSA-1300	Based on dextran, streptavidin-coated	0.13
PMSA-2500	Based on dextran, streptavidin-coated	0.25
			PMB-1000	Based on dextran, biotin-coated	0.1
PMB-1300	Based on dextran, biotin-coated	0.13
			PMB-2500	Based on dextran, biotin-coated	0.25
PMPA-1000	Dextran-based, protein A coated	0.1
			PMPA-1300	Dextran-based, protein A coated	0.13
PMPA-2500	Dextran-based, protein A coated	0.25
			PMC-0.1	Based on dextran, COOH-functionalised	0.1-0.4
PMC-0.4	Based on dextran, COOH-functionalised	0.4-0.7
			PMC-0.7	Based on dextran, COOH-functionalised	0.7-0.9
PMC-1.0	Based on dextran, COOH-functionalised	1.0-1.4
			PMN-1.0	Based on dextran, NH₂Functionalized	1.0-1.4
PMC-0.1	Based on dextran, COOH-functionalised	0.1-0.4
				Accurate Chemical
ADM01020	Carboxyl functionalization	0.2
			ADM01030	Carboxyl functionalization	0.3
ADM02020	Carboxyl functionalization	0.2
			ADM02133	High carboxyl functionalization	0.3
ADM02150	Carboxyl functionalization	0.5
			ADM02220	Very high amino functionalization	0.2
ADM02230	Very high amino functionalization	0.3
			ADM02250	Carboxyl functionalization	0.5
ADM02030	High carboxyl functionalization	0.3
			ADM02110	High carboxyl functionalization	0.1
ADM02120	Very high carboxyl functionalization	0.2
			ADM02130	Very high carboxyl functionalization	0.3
ADM02252	Carboxyl functionalization	0.5
			ADM03120	Streptavidin functionalization	0.2
ADM03121	Streptavidin functionalization	0.2
				Chemicell
1201-5 1	Si-(CH₂)₃-COOH	0.5
			1201-5 1	Si-(CH₂)₃-COOH	0.75
1201-5 1	Si-(CH₂)₃-COOH	1.0
			1202-5 1	Si-(CH₂)₃-SO₃H	0.5
1202-5 1	Si-(CH₂)₃-SO₃H	0.75
			1202-5 1	Si-(CH₂)₃-SO₃H	1.0
1205-1	Si-(CH₂)₃-PO₃H₂	0.5
			1205-1	Si-(CH₂)₃-PO₃H₂	0.75
1205-1	Si-(CH₂)₃-PO₃H₂	1.0
				Estapor
M1-130/12	Carboxylated polystyrene	0.7-1.3
			M1-180/12	Carboxylated polystyrene	0.9-1.3
M1-180/20	Carboxylated divinylbenzene	0.8-1.2
			M1-050/20	Carboxylated polystyrene	0.5-0.7
M1-070/40	Carboxylated polystyrene	0.7-1.3
			M1-070/60	Carboxylated polystyrene	0.7-1.3
M1-020/50	Carboxylated polystyrene	0.16-0.24
			M1-030/40	Carboxylated polystyrene	0.3-0.5
	Genovis
			AMI-25	Glucan	80-150
	Thermo Fisher
			4515-2105	Carboxylated modified (MG-CM)	1.0
7815-2104	Neutravidin (MG-NA)	1.0
			5915-2104	Streptavidin (MG-SA)	1.0
2415-2105	Modification of carboxyl groupSex (MG-CM)	1.0
			4415-2105	Carboxylated modified (MG-CM)	1.0
	JSR micro
			MB100	Carboxylated	1.1
	Invitrogen
			354-01	Carboxylated	1
355-00	Tosyl activated (Tosylactivated)	1
			650-11	Carboxylated	1
655-11	Tosyl activated	1
				Biopal
M02Q05	Amino-activated	1.5
			M02Q05	Biotin activated	1.5
M02Q05	Streptavidin-activated	1.5

The magnetic particles used in the systems and methods of the invention may have a ring power diameter of 10 nm to 1200 nm and contain an average of 18 x 10²To 1X 10¹⁰Metal atoms/particles, and having 1 × 10 of each particle⁴To 1X 10¹³ mM^-1s^-1The relaxation rate of (2). The magnetic particles used in the systems and methods of the present invention may be of any design, composite, or source as described above, and may be further modified to function as a magnetic resonance switch in the manner described herein.

In addition to the relaxation rate of each particle, several other practical problems have to be addressed in the selection and design of magnetic particles in order to achieve high analytical sensitivity assays.

For example, in order to maximize the iron content and relaxation rate per particle, it may be desirable to use large particles (i.e., 1000 nm or greater). However, we have observed that particles of this size tend to settle out of solution quickly. We have observed that particle sedimentation generally does not interfere with the assay if the size of the magnetic particles is kept below 500 nm. When particles larger than 500 nm are used in the assay or if smaller particles with a high density are used, the sedimentation is monitored and the sedimentation pair T is determined ₂The effect of the measurement. We have found that magnetic particles of size about 100-300 nm are ideal for stability in terms of sedimentation, even after functionalization (increasing the ring power diameter by about 50-300 nm), and provide high sensitivity due to high relaxivity per particle. The particle density clearly plays a role in buoyancy. Therefore, the relative density of the solution to the particles plays an important role in the sedimentation of the particles. Thus, one possible solution to this problem is to use buoyant magnetic particles (i.e., hollow particles, or particles containing both a low density framework and a high density metal oxide). Sedimentation may influence T₂Detection, therefore, the solution additive can be used to change the ratio of particle to solution density. If there is a significant amount of precipitation of the superparamagnetic material from the measured liquid volume, T₂Detection may be affected by sedimentation. Sedimentation can be assessed by diluting the particles to a concentration such that the absorbance of UV-V at 410 nm is between 0.6 and 0.8 absorbance units and then monitoring the absorbance for 90 minutes. If sedimentation occurs, the difference between the initial absorbance and the endpoint absorbance divided by the initial absorbance will be greater than 5%. If the% sedimentation exceeds 5%, the particles are generally unsuitable for use in assays requiring high analytical sensitivity. The magnetic particles used in the assay of the invention may be, but are not limited to, non-settling magnetic particles. High sedimentation means difficult handling and can cause repeatability problems.

For magnetic particles having a particle size of 100 nm or greater, the plurality of superparamagnetic iron oxide crystals, which typically comprise a particle core, produce a net dipole moment in the presence of an external magnetic field, i.e. the dipole moment is a force sufficient to overcome brownian motion. The reversibility of non-specificity is a measure of colloidal stability and robustness against non-specific aggregation. By measuring at a uniform magnetic field (defined as<5000 ppm) T of the particle solution before and after incubation₂Values to assess non-specific reversibility. For particles with 0.01 mM Fe concentration, the starting T₂The value is typically 200 ms. If before and after incubation in a homogeneous magnetic field T₂The difference in values was less than 20 ms, and these samples were considered reversible. Furthermore, 10% are such that T is the starting T₂The measurement can reflect a threshold value at which the concentration of particles is determined. If the difference is greater than 10%, then the particles exhibit irreversibility in the buffer, diluent, and matrix being tested. The non-specific reversibility of the magnetic particles can be altered in the manner described herein. For example, colloidal stability and robustness against non-specific aggregation can be affected by the surface characteristics of the particles, binding moieties, assay buffers, matrices, and assay processing conditions. The maintenance of colloidal stability and resistance to nonspecific binding can be altered by binding chemistry, blocking methods, buffer modification, and/or changes in assay processing conditions.

We have observed that a very important property for a reliable and reproducible determination is the monodispersity of the particle size distribution of the magnetic particles used, a difference being observed between the polydispersed particles after coating and the monodisperse particles before coating. Polydisperse batches of magnetic particles may lack repeatability and lose sensitivity. Polydisperse samples also present problems in obtaining uniform coatings. For certain highly sensitive assays, it is desirable that the particle size distribution of the magnetic particles be substantially monodisperse (i.e., have a polydispersity index of less than about 0.8-0.9). Alternatively, the assay of the invention may be designed to accommodate the use of polydisperse magnetic particles.

It is assumed that the assay of the present invention requires monitoring for changes in the aggregation state of the agglomeration assay, and that measuring changes in aggregation may require a large number of aggregated particles (e.g., it is generally recognized that>1-10%) the total number of particles in the assay should be minimized in order to be able to obtain the highest sensitivity. However, a sufficient number of particles must be present to allow the use of T₂The dynamic range is detected. We have found that the highest sensitivity (i.e. in inhibition assays) is observed when the number of magnetic particles (or molar equivalents) is approximately of the same order as the number of analytes (or molar equivalents) to be detected and the number of multivalent binding agents (or molar equivalents) employed.

For protein samples, it may also be desirable to modify the surface of the magnetic particles to reduce non-specific binding of background proteins to the magnetic particles. Non-specific binding of background proteins to the particles may cause or prevent particle aggregation, resulting in false signals and/or lack of false signals. For example, in some cases, the magnetic particle surface may include a blocking agent covalently attached to the magnetic particle surface that reduces non-specific binding of background proteins. There are a variety of agents that can be used to achieve the desired effect, and in some cases, it is the combination of a variety of agents that is optimal (see Table 3; exemplary particles, coatings, and binding moieties).

TABLE 3

。

We have therefore found that protein blocking may be required to obtain assay activity and sensitivity, especially in protein samples (e.g. plasma samples or whole blood samples), which is comparable to the results in non-protein buffer samples. Some common protein blocking agents that may be used in the provided formulations include, for example: bovine Serum Albumin (BSA), Fish Skin Gelatin (FSG), Bovine Gamma Globulin (BGG), lysozyme, casein, peptidase, or skim milk powder. In certain embodiments, the magnetic particle coating comprises BSA or FSG. In other embodiments, the combination of coatings is a combination of those exemplary coatings listed in table 3.

In addition, non-specific binding may be due to lipids or other non-protein molecules in the biological sample. For non-protein mediated non-specific binding, the particle repulsion can be increased by selecting a change in pH and buffer ionic strength, but not enough to limit the results of the expected interaction.

Assay reagent

The assay of the invention may comprise reagents for reducing non-specific binding to magnetic particles. For example, the assay may include one or more proteins (e.g., albumin (e.g., human or bovine albumin), fish skin gelatin, lysozyme, or transferrin); low molecular weight (<500 daltons) amines (e.g., amino acids, glycine, ethylamine, or mercaptoethanolamine); and/or water-soluble nonionic surfactants (e.g., polyethylene glycol, Tween 20, Tween 80, Pluronic, or Igepoal).

The surfactant may be selected from a wide variety of water-soluble nonionic surfactants, including the surfactants commonly sold under the trade name IGEPAL by the company GAF. IGEPAL liquid nonionic surfactants are polyethylene glycol p-isooctylphenyl ether compounds and are available in a variety of molecular weight characterizations, such as IGEPAL CA720, IGEPAL CA630, and IGEPAL CA 890. Other suitable nonionic surfactants include those sold under the trade name TETRONIC909 by BASF Wyandotte. This material is a tetrafunctional block copolymer surfactant terminated with primary hydroxyl groups. Suitable nonionic surfactants are also available from VISTA ALPHONIC under the VISTA ALPHONIC trade name, VISTA chemical company, and such materials are nonionic biodegradable ethoxylates obtained from blends of linear primary alcohols of various molecular weights. The surfactant may also be selected from poloxamers (e.g., polyoxyethylene-polyoxypropylene block copolymers (e.g., the products sold under the tradenames Synperonic PE series (ICI), Pluronic series (BASF), Suronic, Monolan, Pluracane and Plurodac)), polysorbate surfactants (e.g., Tween 20(PEG-20 sorbitan monolaurate), and glycols (e.g., ethylene glycol and propylene glycol).

By selecting such a nonionic surfactant, an appropriate amount of detergency can be provided for the assay without adversely affecting the assay reaction. In particular, surfactants may be included in the reaction mixture to inhibit non-specific interactions between the various components of the aggregation assay of the invention. The nonionic surfactant is usually added to the former liquid sample in an amount of 0.01% (w/w) to 5% (w/w).

The nonionic surfactant can also be used with one or more proteins (e.g., albumin, fish skin gelatin, lysozyme, or transferrin) previously added to the liquid sample in an amount of 0.01% (w/w) to 5% (w/w).

Furthermore, the assay, method and cartridge unit of the present invention may comprise: other suitable buffer components (e.g., Tris base, selected to provide a pH of about 7.8 to 8.2 in the reaction environment); and chelating agents for scavenging cations (e.g., disodium EDTA, ethylenediaminetetraacetic acid (EDTA), citric acid, tartaric acid, glucuronic acid, glyconic acid, or suitable salts thereof).

Binding moieties

In general, a binding moiety is a synthetic or natural molecule that specifically binds or is linked to (e.g., covalently or non-covalently binds to) or hybridizes to a target molecule or other binding moiety (or, in certain embodiments, to aggregation-causing molecules). For example, the binding moiety may be an antibody directed to an antigen, or any protein-protein interaction. Alternatively, the binding moiety may be a polysaccharide that binds to a corresponding target or a synthetic oligonucleotide that hybridizes to a particular complementary nucleic acid target. In certain embodiments, a binding moiety may be designed or selected to act as a substrate for a target molecule (e.g., an enzyme in solution) when bound to another binding moiety.

Binding moieties include, for example, oligonucleotide binding moieties (DNA, RNA, or substituted or derivatized nucleotide substitutions), polypeptide binding moieties, antibody binding moieties, aptamers, and polysaccharide binding moieties.

Oligonucleotide binding moieties

In certain embodiments, the binding moiety is an oligonucleotide attached/linked to the magnetic particle (e.g., a functional group attached/linked to the magnetic particle at the 3 'or 5' end) by a single bond (e.g., a covalent bond) using any of a variety of chemical methods. Such binding moieties can be used in the systems, devices, and methods of the invention to detect mutations (e.g., SNPs, translocations, large deletions, small deletions, insertions, substitutions) or to monitor gene expression (e.g., the presence of expression, or changes in gene expression levels, monitoring RNA transcription), or CHP assays that characterize the presence of a pathogen, disease state, or disease progression.

Chemical synthesis can be used to construct the oligonucleotide binding moiety. Double-stranded DNA binding moieties can be constructed by enzymatic ligation reactions (enzymatic ligation reactions) using procedures known in the art. For example, nucleic acids (e.g., oligonucleotides) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to enhance the biological stability of the molecules or to enhance the physical stability of the duplex formed between complementary strands, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Nucleic acids can also be produced biologically using expression vectors into which the nucleic acids are subcloned.

One method uses at least two populations of oligonucleotide magnetic particles, each having a strong effect on relaxation of water (or other solvent). When the oligonucleotide-magnetic particle conjugates react with the target oligonucleotide, they form aggregates (e.g., clusters of magnetic particles). After standing for a long time (e.g., overnight at room temperature), these aggregates form large clusters (micron-sized clusters). With the method of the invention, large clusters can be formed more rapidly by multiple cycles of magnetically assisted agglomeration. Magnetic resonance is used to determine the relaxation properties of the solvent, which are altered when a mixture of magnetic oligonucleotide magnetic particles reacts with a target nucleic acid to form aggregates.

Certain embodiments use a mixture of at least two types of magnetic metal oxide magnetic particles, each having a particular oligonucleotide sequence, and each having more than one copy of the oligonucleotide attached (e.g., covalently attached to each magnetic particle). For example, an assay protocol may include: a mixture of each population of oligonucleotide-magnetic particle conjugates is prepared and reacted with a target nucleic acid. Alternatively, the oligonucleotide-magnetic particle conjugates can be reacted with the target in a sequential manner. Certain embodiments are characterized by: magnetic resonance is used to detect the reaction of the oligonucleotide-magnetic particle conjugate with the target nucleic acid. When the target is present, the dispersed conjugate self-assembles to form small aggregates.

For example, the oligonucleotide binding moiety may be attached to the metal oxide by covalent attachment to a functionalized polymer, or to a non-polymeric surface functionalized metal oxide. In the latter method, magnetic particles can be synthesized according to the method of Albrecht et al, Biochimie,80:379 (1998). Dimercaptosuccinic acid is conjugated to iron oxide and provides a carboxyl functionality.

In certain embodiments, the oligonucleotides can be attached to the magnetic particles using functionalized polymers bound to metal oxides. In some embodiments, the polymer is hydrophilic. In certain embodiments, the conjugates are made using an oligonucleotide having a terminal amino, thiol, or phosphate group and a superparamagnetic ferromagnetic oxide particle carrying an amino or carboxyl group on a hydrophilic polymer. There are several methods for the synthesis of carboxyl-and amino-derivatized magnetic particles.

In one embodiment, the oligonucleotide is attached to the particle via a ligand-protein binding interaction (e.g., biotin-streptavidin), wherein the ligand is covalently attached to the oligonucleotide and the protein is covalently attached to the particle, or vice versa. This method can achieve faster reagent preparation.

Other forms of oligonucleotides may be used. For example, aptamers are single-stranded RNA or DNA oligonucleotides 15 to 60 bases in length that form intramolecular interactions in solution that fold linear nucleic acid molecules into a three-dimensional complex that can then bind to a specific molecular target with high affinity; often have equilibrium constants in the range of 1 pM to 1 nM, similar to some monoclonal antibody-antigen interactions. Aptamers can specifically bind to other nucleic acid molecules, proteins, small organic compounds, small molecules and cells (organisms or pathogens).

Polypeptide binding moieties

In certain embodiments, a binding moiety is a polypeptide (i.e., a protein, polypeptide, or peptide) that is attached by a single covalent bond using any of a variety of chemical methods in a manner that does not affect the biological activity of the polypeptide. In one embodiment, attachment is accomplished using the sulfhydryl group of a single reactive cysteine residue, which modification does not affect the biological activity of the polypeptide. In this regard, the use of a linear polypeptide having a cysteine at the C-terminus or N-terminus provides a single sulfhydryl group in a manner similar to that of an alkanethiol (alkanethiol) which provides a sulfhydryl group at the 3 'or 5' end of an oligonucleotide. Similar bifunctional conjugation reagents (e.g. SPDP) that react with the amino group of the magnetic particle and the thiol group of the polypeptide can be used for any binding moiety that carries a thiol group. The types of polypeptides used as binding moieties can be antibodies, antibody fragments, and natural and synthetic polypeptide sequences. Peptide binding moieties have binding partners, i.e., molecules to which they selectively bind.

The use of peptides as binding moieties offers several advantages. For example, the polypeptide may be designed to have unique reactive residues at sites remote from those required for biological activity for attachment to magnetic particles. The reactive residue may be a cysteine thiol group, an N-terminal amino group, a C-terminal carboxyl group, or a carboxyl group of aspartic acid or glutamic acid, etc. A single reactive residue on the peptide is used to ensure a unique attachment site. These design principles may be applied to chemically synthesized peptides or biologically produced polypeptides.

The binding moiety may also contain an amino acid sequence derived from a naturally occurring (wild-type) polypeptide or protein. For example, the native polypeptide can be a hormone (e.g., cytokine, growth factor), a serum protein, a viral protein (e.g., hemagglutinin), an extracellular matrix protein, a lectin, or an exo-functional region of a cell surface protein. Another example is a ligand binding protein, such as streptavidin or avidin (which binds biotin). Generally, the binding moiety-magnetic particles formed are used to measure the presence of an analyte that reacts with the binding moiety in a test medium.

In addition, polypeptide binding moieties can be used in general reagent construction, wherein the target (e.g., small molecule, ligand, or binding partner) of the binding moiety is pre-linked to a target analyte to form a labeled analyte that induces aggregation in the presence of polypeptide-modified particles.

Examples of protein hormones that can be used as binding moieties include, but are not limited to: platelet Derived Growth Factor (PDGF) that binds to the PDGF receptor; insulin-like growth factor-I and-II (Igf) that bind to Igf receptors; nerve Growth Factor (NGF) that binds to NGF receptors; fibroblast Growth Factor (FGF) bound to FGF receptors (e.g., aFGF and bFGF); epidermal Growth Factor (EGF) that binds to EGF receptors; transforming growth factors (TGF, e.g., TGF-alpha and TGF-beta) that bind to TGF receptors; erythropoietin that binds to an erythropoietin receptor; growth hormone that binds to growth hormone receptors (e.g., human growth hormone); and proinsulin, insulin, A-chain insulin and B-chain insulin all bound to the insulin receptor.

The receptor binding moieties are useful for detecting and imaging receptors that accumulate on the surface of cells. Useful outer functional regions include: notch proteins, delta proteins, integrins, cadherins and other cell adhesion molecules.

Antibody binding moieties

Other polypeptide binding portions include immunoglobulin binding portions that comprise at least one immunoglobulin domain and typically at least two such domains. "immunoglobulin domain" refers to a domain of an antibody molecule, such as a variable or constant region. "immunoglobulin superfamily domain" refers to a domain that has a three-dimensional structure related to an immunoglobulin domain, but is derived from a non-immunoglobulin molecule. Immunoglobulin domains and immunoglobulin superfamily domains typically include two β -sheets (β -sheets) formed by approximately 7 β -strands, as well as conserved disulfide bonds (see, e.g., Williams and Barclay, ann. rev immunol.,6:381 (1988)). Proteins comprising domains of Ig superfamily domains include: t cell receptor, CD4, Platelet Derived Growth Factor Receptor (PDGFR) and intercellular adhesion molecule (ICAM).

One type of immunoglobulin binding moiety is an antibody. The term "antibody" as used herein refers to full-length, double-stranded immunoglobulin molecules, as well as antigen-binding proteins and fragments thereof, including synthetic variants. A typical antibody comprises two heavy (H) variable regions (abbreviated herein as VH), and two light (L) variable regions (abbreviated herein as VL). The VH and VL regions may be further subdivided into highly variable regions, termed "complementarity determining regions" (CDRs), interspersed with more conserved regions termed "framework regions" (FRs). The extent of the framework regions and CDRs has been precisely defined (see Kabat, E.A. et al (1991) Sequences of Proteins of Immunological Interest, fifth edition, U.S. Department of Health and Human Services, NIH Publication No.91-3242, and Chothia et al, J.mol.biol.,196:901 (1987)). Each VH and VL is composed of three CDRs and four FRs, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 are arranged from the amino terminus to the carboxyl terminus.

Antibodies may also include constant regions that are part of a light chain or a heavy chain. The light chain may include a kappa or lambda-constant region gene (referred to as CL) at the COOH-terminus. The heavy chain may comprise, for example, a gamma-constant region (IgG1, IgG2, IgG3, IgG 4; encoding about 330 amino acids). The gamma-constant region may include, for example, CH1, CH2, and CH 3. The term "full length antibody" refers to a protein comprising one polypeptide comprising VL and CL and a second polypeptide comprising VH, CH1, CH2, and CH 3.

The term "antigen-binding fragment" as used herein refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to a target. Examples of antigen-binding fragments include, but are not limited to: (i) fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains; (ii) f (ab')₂A fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) f consisting of VH and CH1 domainsd, a fragment; (iv) (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment consisting of the VH domain (Ward et al, Nature 341:544 (1989)); and (vi) an isolated Complementarity Determining Region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by synthetic linkers that allow them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single chain Fv (scFv); see, e.g., Bird et al, Science 242: 423 (1988); and Huston et al, Proc. Natl. Acad. Sci. USA,85:5879 (1988)). Such single chain antibodies are also encompassed within the term "antigen-binding fragment".

Single domain antibodies (sdabs, nanobodies) are antibody fragments consisting of a single monomeric variable antibody domain, and can also be used in the systems and methods of the invention. Like an intact antibody, an sdAb is capable of selectively binding to a specific antigen. Because the molecular weight is only 12-15 kDa, single domain antibodies are much smaller than the common antibodies consisting of two heavy protein chains and two light chains (150-160 kDa), and even smaller than Fab fragments (about 50 kDa, one light chain and half a heavy chain) and single chain variable fragments (about 25 kDa, two variable domains, one from the light chain and one from the heavy chain).

Polysaccharide binding moieties

In certain embodiments, the binding moiety is a polysaccharide attached to a functional group on the magnetic particle at one of its ends by a single bond (e.g., a covalent bond), for example, using any of a variety of chemical methods. The polysaccharide may be synthetic or natural. Monosaccharides, disaccharides, trisaccharides and polysaccharides may be used as binding moieties. When bound to magnetic particles is used with suitable attachment chemistry, these binding moieties include, for example, glycosides, N-glycosylamines, O-acyl derivatives, O-methyl derivatives, osazones, sugar alcohols, sugar acids, sugar phosphates.

One way to achieve attachment is to conjugate avidin to the magnetic particles and to make the avidin-magnetic particlesThe seed is reacted with a commercially available biotinylated polysaccharide to form a polysaccharide-magnetic particle conjugate. For example, sialylated Lewis based polysaccharides (sialyl Lewis based polysaccharides) are commercially available as biotinylation reagents and will react with avidin-CLIO (see Syntesome, Gesellschaft fur medizinische Biochemie mbH.). Sialylated Lewis tetrasaccharide (Sle)^x) A protein called selectin, which is present on the surface of granulocytes and functions as part of the inflammatory cascade for the recruitment of granulocytes.

Other targeting moieties include: a non-protein element, such as a glycosyl modification (e.g., a Lewis antigen) or another non-protein organic molecule. Another approach is to covalently conjugate the protein to magnetic particles.

Another feature of the method includes: any of the above-described binding moieties is used to identify specific cell types for hematological or histopathological studies (e.g., CD4/CD3 cell counts and circulating tumor cells).

Multivalent binding agents

The assays of the invention may comprise multivalent binding agents that (i) carry a plurality of analytes linked to a carrier (e.g., a simple synthetic scaffold, or a larger carrier protein or polysaccharide such as BSA, transferrin, or dextran) or carry a plurality of epitopes for binding to, for example, two or more populations of magnetic particles to form aggregates.

Where multivalent binding agents are used, multiple analytes may be attached to a carrier (e.g., a simple synthetic scaffold, or a larger carrier protein or polysaccharide such as BSA, transferrin, or dextran). Alternatively, the multivalent binding agent may be a nucleic acid designed to bind to two or more populations of magnetic particles. Such multivalent binding agents function as aggregants, and the assay structure is characterized by competition between the analyte being detected and the multivalent binding agent (e.g., in an inhibition assay, a competition assay, or a disaggregation assay).

Functional groups present in the analyte may be used to form covalent bonds with the support. Alternatively, the analyte can be derivatized to provide a linker (i.e., a spacer that separates the analyte from the carrier in the conjugate) that terminates in a functional group (i.e., an alcohol, amine, carboxyl, sulfhydryl, or phosphate group) that is used to form a covalent bond with the carrier.

Covalent attachment of the analyte to the support may be effected using a linker comprising a reactive moiety capable of reacting with the analyte and such functional groups present in the support. For example, a hydroxyl group of the analyte may react with a carboxyl group of the linker or an activated derivative thereof to form an ester linking the two.

Examples of moieties capable of reacting with a thiol group include: XCH₂A-haloacetyl compound of the type CO- (wherein X = Br, Cl or I), XCH₂CO-shows specific reactivity towards thiol groups, but can also be used to modify imidazolyl, thioether, phenol and amino groups as described by Gurd, Methods enzymol.11:532 (1967). N-maleimide derivatives are also considered selective for thiol groups, but may additionally be used for conjugation to amino groups under certain conditions. Reagents that introduce a thiol group by amino conversion, such as 2-iminothiolane (2-iminothiolane) (Traut et al, Biochemistry 12:3266(1973)), can be considered as thiol reagents if the linkage occurs through the formation of a disulfide bridge.

Examples of reactive moieties capable of reacting with an amino group include, for example: an alkylating agent and an acylating agent. Representative alkylating agents include: (i) alpha-haloacetyl compound showing specificity for amino groups in the absence of reactive thiol groups and belonging to the group of XCH₂CO- (where X = Cl, Br or I), for example as described by Wong, Biochemistry 24:5337 (1979); (ii) n-maleimide derivatives which can react with amino groups using a Michael type reaction or by acylation (by addition to a cyclic carbonyl group), as described, for example, by Smyth et al, J.Am.chem.Soc.82: 4600(1960) and biochem.J.91:589 (1964); (iii) a halogenated aromatic hydrocarbon, Such as reactive nitrohalogenated aromatic compounds; (iv) halogenated hydrocarbons, such as those produced by McKenzie et al, j.protein chem.7: 581 (1988); (v) aldehydes and ketones capable of forming schiff bases with amino groups, the addition products formed being stabilized, usually by reduction to give stable amines; (vi) epoxide derivatives, such as epichlorohydrin and dioxirane, which can react with amino, mercapto or phenolic hydroxyl groups; (vii) chlorine-containing derivatives of s-triazines (s-triazines) which are very reactive towards nucleophiles such as amino, mercapto and hydroxyl groups; (viii) aziridines based on the s-triazine compounds detailed above, as described, for example, by Ross, j.adv.cancer res.2:1(1954), react with nucleophiles (e.g., amino groups) by ring opening; (ix) diethyl squarates (squaric acid diethyl esters), such as titeze, chem. be.124: 1215 (1991); and (x) α -haloalkyl ethers, which are alkylating agents that are more reactive than the commonly used haloalkanes due to activation by an ether oxygen atom, as described by Benneche et al, eur.j.med.chem.28:463 (1993).

Representative amino-reactive acylating agents include: (i) isocyanates and isothiocyanates, especially aromatic derivatives, which form stable urea and thiourea derivatives, respectively; (ii) sulfonyl chlorides, which have been described by Herzig et al, Biopolymers 2:349 (1964); (iii) acid halides; (iv) active esters, such as nitrophenyl esters or N-hydroxysuccinimidyl esters (N-hydroxysuccinimidyl esters); (v) anhydrides, such as mixed, symmetrical, or N-carboxylic anhydrides; (vi) other useful reagents for amide bond formation are described, for example, by M. Bodansky, Principles of Peptide Synthesis, Springer-Verlag, 1984; (vii) acyl azides, for example, where the azide group is generated from a preformed hydrazide derivative using sodium nitrite, as described by Wetz et al, anal. biochem.58:347 (1974); and (viii) imidates which form stable amidines upon reaction with amino groups, as described, for example, by Hunter and Ludwig, j.am.chem.soc.84:3491 (1962). Aldehydes and ketones can react with amines to form schiff bases, which can be advantageously stabilized by reductive amination. Alkoxyamino moieties readily react with ketones and aldehydes to give stable alkoxyamines (alkoxamines), as described, for example, by Webb et al, Bioconjugate chem.1:96 (1990). Examples of reactive moieties capable of reacting with a carboxyl group include diazo compounds such as diazoacetates and diazoacetamides which react with high specificity to produce an ester group, such as those described by Herriot, adv. 169 (1947). Carboxyl modifying reagents such as carbodiimides, which react via O-acylurea formation followed by amide bond formation, may also be used.

It will be appreciated that functional groups in the analyte and/or support may be converted to other functional groups prior to reaction if desired, for example to confer additional reactivity or selectivity. If desired, so-called zero-length linkers, including direct covalent attachment of the reactive chemical group of the analyte to the reactive chemical group of the support without the introduction of other linking materials, may be used in the present invention. Most commonly, however, the linker will comprise two or more reactive moieties linked by a spacer element, as described above. The presence of such a spacer allows the bifunctional linker to react with the analyte and a specific functional group within the carrier, thereby forming a covalent link between the two. The reactive moieties in the linker may be the same (homobifunctional linker) or different (heterobifunctional linker, or heteromultifunctional linker if several different reactive moieties are present), thereby providing a diversity of potential reagents that can lead to covalent attachment between the analyte and the support. The spacer element in the linker is typically composed of straight or branched chains and may include C_1-10Alkyl, 1 to 10-atom heteroalkyl, C _2-10Olefin, C_2-10Alkyne, C_5-10Aryl, a ring system of 3 to 10 atoms, or- (CH)₂CH₂O)_nCH₂CH₂- (wherein n is 1 to 4). Typically, one multivalent binding agent will comprise 2, 3, 4, 5, 6, 7, 8, 15, 50, or 100 (e.g., 3 to 100, 3 to 30, 4 to 25, or 6 to 20) conjugated analytes. Multivalent binding agents are typically 10 kDa to 200 kDa in size and may bePrepared in the manner described in the examples.

Analyte

Embodiments of the invention include devices, systems, and/or methods for detecting and/or measuring the concentration of one or more analytes (e.g., proteins, peptides, enzymes, polypeptides, amino acids, nucleic acids, oligonucleotides, therapeutic agents, metabolites of therapeutic agents, RNA, DNA, circulating DNA (e.g., from a cell, tumor, pathogen, or fetus), antibodies, organisms, viruses, bacteria, carbohydrates, polysaccharides, glucose, lipids, gases (e.g., oxygen and/or carbon dioxide), electrolytes (e.g., sodium, potassium, chloride, bicarbonate, BUN, magnesium, phosphate, calcium, ammonia, and/or lactate), chemical molecules in general (creatinine, glucose), lipoproteins, cholesterol, fatty acids, glycoproteins, proteoglycans, and/or lipopolysaccharides) in a sample. The analyte may include the identification of a cell or a particular cell type. The analyte(s) may include one or more bioactive substances and/or metabolite(s), marker(s), and/or other indicator(s) of bioactive substances. The biologically active substance may be described as a single entity or a combination of entities. The term "biologically active substance" includes, but is not limited to: a drug; a vitamin; a mineral supplement; substances for the treatment, prevention, diagnosis, cure or alleviation of diseases or conditions; or substances that affect body structure or function; or a prodrug that becomes biologically active or active upon exposure to a predetermined physiological environment; or a biotoxic agent, such as a biotoxic agent for biological warfare, including organisms such as anthrax, ebola (ebola), salmonella typhimurium, marburg virus, plague, cholera, Francisella tularensis (harp's disease), brucellosis, Q fever, borliviia hemorrhagic fever, coccidioidomycosis, melidososis, melioidosis, shigella, rocky mountain spotted fever, typhus, parrot fever, yellow fever, japanese encephalitis, rift fever, and smallpox; naturally occurring toxins that may be used as weapons include ricin, aflatoxin, SEB, botulinum toxin, saxitoxin, and many mycotoxins. Analytes may also include organisms such as Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis, coagulase-negative staphylococci, enterococcus faecalis, enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Acinetobacter baumannii, Aspergillus fumigatus, Bacteroides fragilis, blasHV, Burkholderia cepacia, Campylobacter jejuni/Colostridia, Candida guilliermondii, Candida ruchii, Clostridium perfringens, Enterobacter aerogenes, Enterobacter cloacae, Enterobacter enterobacteriaceae, Haemophilus influenzae, gold bacterium, Klebsiella oxytoca, Listeria monocytogenes, bacteria carrying the Mec A gene (MRSA), Morganella morganganganganganganana, Neisseria meningitidis, Neisseria species other than Neisseria, Neisseria, Prevotella buccae, Prevotella intermedia, Prevotella melanogenes, Propionibacterium acnes, Proteus mirabilis, Proteus vulgaris, Salmonella enterica, Serratia marcescens, Staphylococcus haemolyticus, Staphylococcus maltophilia, Staphylococcus saprophila saprophyllus, stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus bovis, Streptococcus dysgalactiae, Streptococcus mitis, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus sanguis, Van A gene, Van B gene. Analytes can also include viral organisms, such as dsDNA viruses (e.g., adenovirus, herpesvirus, poxvirus); ssDNA virus (+) sense DNA (e.g., parvovirus); dsRNA viruses (e.g., reoviruses); (+) ssRNA virus (+) sense RNA (e.g., picornavirus, togavirus); (-) ssRNA virus (-) sense RNA (e.g., orthomyxovirus, baculovirus); ssRNA-RT viral (+) sense RNA (e.g., retrovirus) with DNA intermediates in the life cycle; and dsDNA-RT viruses (e.g., hepadnavirus).

Opportunistic infections that can be detected using the systems and methods of the present invention include, but are not limited to: fungal, viral, bacterial, protozoan infections, such as: (1) fungal infections, such as those caused by candida species (resistant and non-resistant strains), candida albicans, candida krusei, candida glabrata, and aspergillus fumigatus; (2) gram-negative infections, such as those caused by E.coli, stenotrophomonas maltophilia, Klebsiella pneumoniae/Klebsiella oxytoca, and Pseudomonas aeruginosa; and (3) gram-positive infections, such as those caused by staphylococcus species, staphylococcus aureus, streptococcus pneumoniae, enterococcus species (enterococcus faecalis and enterococcus faecium). The infection may be an infection by coagulase-negative staphylococci, corynebacterium species, clostridium species, morganella morganii, pneumocystis jie (formerly pneumocystis carinii), f. hominis, streptococcus pyogenes, pseudomonas aeruginosa, polyomavirus JC polyomavirus (a virus causing progressive multifocal leukoencephalopathy), acinetobacter baumannii, toxoplasma gondii, cytomegalovirus, aspergillus species, kaposi's sarcoma, cryptosporidium species, cryptococcus neoformans and histoplasma capsulatum.

Non-limiting examples of a broad class of analytes that can be detected using the devices, systems, and methods of the present invention include, but are not limited to, the following classes of therapeutic agents: anabolic, antacid, antiasthmatic, anticholesterolemic and lipid-lowering agents (anti-lipid agents), anticoagulants, anticonvulsants, antidiarrheal, antiemetic, anti-infective, anti-inflammatory agents, antimanic agents, antineoplastics, antiobesity agents, antipyretics and analgesics, antispasmodics, antithrombotic agents, anti-uricemic agents, antianginal agents, antihistamines, antitussives, appetite suppressants, biologicals, cerebral vasodilators, coronary vasodilators, decongestants, diuretics, diagnostic agents, erythropoietics, expectorants, gastrointestinal sedatives, hyperglycemic agents, hypnotics, hypoglycemic agents, ion exchange resins, laxatives, mineral supplements, mucolytic agents, neuromuscular agents, peripheral vasodilators, psychotropics, sedatives, stimulants, thyroid and antithyroid agents, Uterine relaxants, vitamins and prodrugs.

Apparatus, system and method for utilizing the present inventionThe bioactive substances detected by the method include, but are not limited to, drugs for gastrointestinal tract or digestive system, such as antacids, reflux inhibitors, antiflatulents, antihyperaminergics, proton pump inhibitors, H ₂-receptor antagonists, cytoprotective agents, prostaglandin analogues, laxatives, antispasmodics, anti-diarrheals, bile acid sequestrants and opioids; drugs for the cardiovascular system, such as β -receptor blockers, calcium channel blockers, diuretics, cardiac glycosides, antiarrhythmics, nitrates, antianginals, vasoconstrictors, vasodilators, peripheral activators, ACE inhibitors, angiotensin receptor blockers, α -blockers, anticoagulants, heparin, HSGAGs, antiplatelet agents, fibrinolytic agents, antihemophilic factors, hemostats, hypolipidemics, and statins; drugs for the central nervous system such as hypnotics, anesthetics, antipsychotics, antidepressants, antiemetics, anticonvulsants, antiepileptics, anxiolytics, barbiturates, dyskinesias, stimulants, benzodiazepines, cyclic pyrrolidones, dopamine antagonists, antihistamines, cholinergics, anticholinergics, emetics, cannabinoids, 5-HT antagonists; drugs for pain and/or consciousness, such as non-steroidal anti-inflammatory drugs, opioids and drugs (orphans) such as acetaminophen, tricyclic antidepressants and anticonvulsants; drugs for musculoskeletal disorders, such as non-steroidal anti-inflammatory drugs, muscle relaxants, neuromuscular drugs, anticholinesterase drugs; drugs for the eye, such as adrenergic neuron blockers, astringents, ocular lubricants, local anesthetics, sympathomimetics, parasympathetic blockers, mydriatics, cycloplegics, antibiotics, topical antibiotics, sulfonamides, aminoglycosides, fluoroquinolones, antivirals, antifungals, imidazoles, polyenes, nonsteroidal anti-inflammatory drugs, corticosteroids, mast cell inhibitors, adrenergic agonists, beta-blockers, carbonic anhydrase inhibitors/hypertonics (hyperosmotics), cholinergics, miotics, parasympathomimetics, prostaglandin agonists/inhibitors, nitroglycerin; for ear, nose and oropharynx For example, sympathomimetics, antihistamines, anticholinergics, nonsteroidal anti-inflammatory drugs, steroidal drugs, antiseptics, local anesthetics, antifungals, cerumen (cerumenoyltics); drugs for the respiratory system, such as bronchodilators, non-steroidal anti-inflammatory drugs, anti-allergic drugs, antitussives, mucolytics, decongestants, corticosteroids, beta-receptor antagonists, anticholinergics, steroidal drugs; drugs used for endocrine problems such as androgens, antiandrogens, gonadotrophins, corticosteroids, growth hormones, insulin, antidiabetics, thyroid hormones, antithyroid agents, calcitonin, bisphosphonates (diphosphonates), and vasopressin analogues; drugs for the reproductive system or urinary system such as antifungal agents, basifying agents, quinolones, antibiotics, cholinergic agents, anticholinergic agents, anticholinesterase agents, antispasmodics, 5-alpha reductase inhibitors, selective alpha-1 blockers and sildenafil; drugs for contraception, such as oral contraceptives, spermicides, and long-acting contraceptives; drugs for obstetrics and gynecology, such as non-steroidal anti-inflammatory drugs, anticholinergics, hemostatics, antifibrinolytics, hormone replacement therapy, bone regulators, beta-receptor agonists, follicle stimulating hormone, luteinizing hormone, LHRH total cis-octadeca-6, 9, 12-trienoic acid, gonadotropin release inhibitors, progesterone, dopamine agonists, estradiol, prostaglandins, gonadotropin releasing hormone, clomiphene, tamoxifen, and diethylstilbestrol; medicaments for the skin, such as emollients, antipruritics, antifungals, disinfectants, sarcoptides, pediculicides, tar products, vitamin a derivatives, vitamin D analogs, keratolytics, abrasives, systemic antibiotics, topical antibiotics, hormones, desludging agents, exudate absorbents, fibrinolytic agents, proteolytic agents, sunscreens, antiperspirants, and corticosteroids; drugs for infection and infestation, e.g., antibiotics, antifungal agents, anti-leprosy agents, anti-tuberculosis agents, anti-malarial agents, anthelmintics, anti-amoebic agents, antiviral agents, antiprotozoal agents, and antisera; drugs for the immune system, e.g. vaccines, immunoglobulins, immunosuppressants, interferons A monoclonal antibody; drugs for allergic diseases, for example, antiallergic drugs, antihistamines and non-steroidal anti-inflammatory drugs; drugs for nutrition, such as tonics, iron preparations, electrolytes, vitamins, antiobesity drugs, protein assimilation drugs, hematogenic drugs, and food drugs; drugs for neoplastic diseases, such as cytotoxic drugs, sex hormones, aromatase inhibitors, somatostatin inhibitors, recombinant interleukins, G-CSF and erythropoietin; agents for diagnosis, such as contrast agents; and drugs for cancer (anticancer agents).

Bioactive substances that can be detected using the devices, systems and methods of the invention include, but are not limited to: hematologic agents such as anti-anemic agents, hematopoietic anti-anemic agents, blood clotting agents, anticoagulants, hemostatic blood clotting agents, platelet inhibitor blood clotting agents, thrombolytic enzyme blood clotting agents, and blood volume expanders; anticoagulant, heparin, HSGAG, antiplatelet drug, fibrinolytic agent, antihemophilic factor, hemostatic. Examples of antithrombotic agents (e.g., thrombolytic, anticoagulant, and antiplatelet agents) that can be detected using the devices, systems, and methods of the present invention include: vitamin K antagonists such as nitryl coumarin, chlorindione, dicumarol, benzindenone, dicumarol ethyl ester, coumarine, phenindione, thiocoumarin and warfarin; heparins (platelet aggregation inhibitors), such as antithrombin III, bemiparin (bemiparin), dalteparin (dalteparin), danaparin, enoxaparin, heparin, nadroparin, parnaparin (parnaparin), reviparin, sulodexide and tinzaparin (tinzaparin); other platelet aggregation inhibitors, such as abciximab, acetylsalicylic acid (aspirin), alopecrine, beraprost, ditozole, carbapenem calcium, clocrolimus, clopidogrel, dipyridamole, epoprostenol, eptifibatide, indobufen, iloprost, picotamide, prasugrel, ticlopidine, tirofiban, treprostinil (treprostinil), and triflusal; enzymes such as alteplase, ancrod, anistreplase, plasmin, tegaserod alpha (drotrecogin alfa), plasmin, procein C, reteplase, sarepratpase, streptokinase, tenecteplase and urokinase; direct thrombin inhibitors such as argatroban, bivalirudin, desipramine, lepirudin, melagatran and ximelagatran; other antithrombotic agents such as dabigatran, defibrotide, dermatan sulphate, fondaparinux (fondaparinux), and revapraxiban, among others; and other drugs such as citrate, EDTA and oxalate.

Other biologically active substances that can be detected using the devices, systems and methods of the invention include those mentioned in Basic and Clinical pharmacy (LANGE Basic Science), Katzung and Katzung, ISBN 0071410929, McGraw-Hill Medical, 9 th edition (2003).

Medical conditions

The methods of the invention may be used to monitor one or more analytes in the diagnosis, control and/or treatment of any of a wide range of medical conditions. Various types of medical conditions include, for example, painful conditions; disorders of changes in body temperature (e.g., fever); disorders of nervous system dysfunction (e.g., syncope, myalgia, dyskinesia, numbness, loss of sensation, delirium, dementia, memory loss, or sleep disorders); disorders of the eyes, ears, nose and throat; disorders of circulatory and/or respiratory system function (e.g., dyspnea, pulmonary edema, cough, hemoptysis, hypertension, myocardial infarction, hypoxia, cyanosis, cardiovascular collapse, congestive heart failure, edema, or shock); disorders of gastrointestinal function (e.g., dysphagia, diarrhea, constipation, gastrointestinal bleeding, jaundice, ascites, dyspepsia, nausea, vomiting); disorders of renal and urinary tract function (e.g., acidosis, alkalosis, fluid and electrolyte imbalance, azotemia, or urinary abnormalities); disorders of sexual function and reproduction (e.g., erectile dysfunction, irregular menstruation, hirsutism, virilization, infertility, pregnancy-related disorders, and standard measures); skin disorders (e.g., eczema, psoriasis, acne, rosacea, skin infections, immunological skin disorders, or photoallergic); hematological disorders (e.g., hematology); genetic disorders (e.g., genetic disorders); disorders of drug response (e.g., adverse drug reactions); and nutritional disorders (e.g., obesity, eating disorders, or nutritional assessment). Other medical areas to which embodiments of the invention may be applied include: oncology (e.g., a tumor, a malignancy, angiogenesis, a paraneoplastic syndrome, or a neoplastic emergency); hematology (e.g., anemia, hemoglobinopathy, megaloblastic anemia, hemolytic anemia, aplastic anemia, myelodysplasia, bone marrow failure, polycythemia vera, myeloproliferative disorders, acute myeloid leukemia, chronic myeloid leukemia, lymphoid malignancies, plasmacytosis, transfusion biology (transfusion biology), or transplantation); hemostasis (e.g., disorders of coagulation and thrombosis, or disorders of platelets and vessel walls); and infectious diseases (e.g., sepsis, septic shock, fever of unknown origin, endocarditis, bites, burns, osteomyelitis, abscesses, food poisoning, pelvic inflammatory disease, bacteria (e.g., gram-positive, gram-negative, promiscuous (nocardia, actinomyces, mixed type), mycobacteria (mycobacteral), spirochetes, rickettsiae, or mycoplasma), chlamydia, viral (DNA, RNA), fungal and algal infections, protozoal and helminthic infections, endocrine disorders, nutritional disorders, metabolic disorders.

Other medical conditions and/or fields to which embodiments of the invention may be applied include: harrison's Principles of Internal Medicine, Kasper et al, ISBN 0071402357, McGraw-Hill Professional, 16 th edition (2004) and Robbins Basic Pathology, edited by Kumar, Cotran and Robbins, ISBN1416025340, Elsevier, 7 th edition (2005).

Medical tests (e.g., blood tests, urine tests, and/or other human or animal tissue tests) that can be performed using various embodiments of the invention described herein include, for example, general chemical tests (e.g., analytes including albumin, blood urea nitrogen, calcium, creatinine, magnesium, phosphorus, total protein, and/or uric acid); electrolyte detection (e.g., analytes including sodium, potassium, chloride, and/or carbon dioxide); diabetes detection (e.g., analytes including glucose, hemoglobin A1C, and/or microalbumin); lipid detection (e.g., analytes including apolipoprotein a1, apolipoprotein B, cholesterol, triglycerides, low density lipoprotein cholesterol, and/or high density lipoprotein cholesterol); nutritional assessment (e.g., analytes including albumin, prealbumin, transferrin, retinol binding protein, alpha 1-acid glycoprotein, and/or ferritin); liver detection (e.g., analytes including alanine transaminase, albumin, alkaline phosphatase, aspartate transaminase, direct bilirubin, gamma glutamyltransferase, lactate dehydrogenase, immunoglobulin a, immunoglobulin G, immunoglobulin M, prealbumin, total bilirubin, and/or total protein); cardiac assays (e.g., analytes including apolipoprotein a1, apolipoprotein B, cardiac troponin-1, creatine kinase MB isozyme, high sensitivity CRP, mass creatine kinase MB isozyme myoglobin (mass production kinase MB isozyme myoglobin), and/or N-terminal brain natriuretic peptide precursor (N-terminal pro-brain natural peptide)); detection of anemia (e.g., analytes including ferritin, folate, homocysteine, haptoglobin, iron, soluble transferrin receptor, total iron binding capacity, transferrin, and/or vitamin B12); pancreatic detection (e.g., analytes including amylase and/or lipase); renal disease (e.g., analytes including albumin, alpha l-microglobulin, alpha 2-macroglobulin, beta 2-microglobulin, cystatin C, retinol binding protein, and/or transferrin); bone detection (e.g., analytes including alkaline phosphatase, calcium, and/or phosphorus); cancer marker monitoring (e.g., analytes including total PSA); thyroid detection (e.g., analytes including free thyroxine, free triiodothyronine, thyroxine, thyroid stimulating hormone, and/or triiodothyronine); fertility assays (e.g., analytes including beta-human chorionic gonadotropin); therapeutic drug monitoring (e.g., analytes including carbamazepine, digoxin, digitoxin, gentamicin, lidocaine, lithium, N-acetyl procainamide, phenobarbital, phenytoin, procainamide, theophylline, tobramycin, valproic acid, and/or vancomycin); immunosuppressive drugs (e.g., analytes including cyclosporine a, sirolimus, and/or tacrolimus); detection of complement activity and/or autoimmune disease (e.g., analytes including C3 complement, C4 complement, C1 inhibitors, C-reactive protein, and/or rheumatoid factor); polyclonal/monoclonal gammopathy (e.g., analytes including immunoglobulin a, immunoglobulin G, immunoglobulin M, 1G light chain (kappa and/or lambda types), 1,2, 3, and/or 4 subtypes of immunoglobulin G); detection of infectious diseases (e.g., analytes including antistreptolysin O); detection of inflammatory diseases (e.g., analytes including alpha 1-acid glycoprotein, alpha 1-antitrypsin, ceruloplasmin, C-reactive protein and/or haptoglobin); allergy detection (e.g., analytes including immunoglobulin E); urine protein detection (e.g., analytes including α 1-microglobulin, immunoglobulin G, 1G light chain (kappa and/or lambda type), microalbumin, and/or urine protein/cerebrospinal protein); detection of protein-CSF (e.g., analytes including immunoglobulin G and/or urine protein/cerebrospinal protein); toxicity detection (e.g., analytes including serum acetaminophen, serum barbiturates, serum benzodiazepines, serum salicylate, serum tricyclic antidepressants, and/or urinary ethanol); and/or detection of drug abuse (e.g., analytes including amphetamine, cocaine, barbiturates, benzodiazepines, a hallucinogen (ecstasy), methadone, opioids, phencyclidine, tetrahydrocannabinoids, propoxyphene, and/or methadone). Specific cancer markers that can be detected using the methods, devices, cartridges, and kits of the invention include, but are not limited to: 17-beta-hydroxysteroid dehydrogenase type 1, Abl interactor 2 (Abl interactor 2), actin-related protein 2/3 complex subunit 1A, albumin, aldolase A, alkaline phosphatase, placental type, alpha 1 antitrypsin, alpha-1-acid glycoprotein 1, alpha-2-HS-glycoprotein, alpha-lactalbumin, alpha-2-macroglobulin, alpha-fetoprotein (AFP), angiogenin ribonuclease RNase A family 5, angiogenin 1, angiogenin 2, antigen recognized by monoclonal antibody Ki-67, anti-leukocyte protease 1 (Antileukaproteinase 1) (SLPI), apolipoprotein A1, ATP7B, beta 2-microglobulin, B-cell CLL/lymphoma 2, BCL 2-associated X protein, BRCA1, B-cell CLL 1, BRCA2, BrMS1, Butyrate-induced transcript 1 (Butyrate-induced transcript 1), CA15.3/CA27-29, carcinoma antigen 125, carcinoma antigen 15.3, carcinoma antigen 19.9, carcinoma antigen 602, carcinoma antigen 72-4/TAG-72, carcinoma-associated galactosyltransferase antigen, carcinoma-associated serum antigen (CASA), carcinoembryonic antigen (CEA), catenin beta 1, cathepsin D, cathepsin member 8, CC chemokine 4(HCC-4), CCL21 (small induced cytokine A21 (small induced cytokine A21)), CCL5, CD15, CD24, CD34, CD44, cytokinin kinase 5, ceruloplasmin, cervical carcinoma 1 protooncogene protein p40, c-Ets 36, chaperonin (charoconination) containing TCP1, subunit 1, subunit 3, small chemokine (CCL-induced cytokine A8938), MIP-1-beta), chemokine ligand 5, chitinase-3-like protein 1(YKL-40), Chloride intracellular channel 4 (Chloride intracellular channel 4) (CLIC4), chorionic gonadotropin beta chain, Claudin-3 (Claudin-3), Claudin-4, clusterin, factor II (prothrombin), factor III, factor XIIIa chain, factor XIIIb chain, collagen Ic-terminal peptide, colony stimulating factor 2, colony stimulating factor 3, complement component 3, c-reactive protein, Creatinine Kinase Brain (CKB), CTD phosphatase-like (CTD small phosphatase-like), cyclin-like kinase 6(CDK6), cyclin-dependent kinase inhibitor 1(p21), cyclin-dependent kinase inhibitor 1 (CDK-3), and the like, Cyclooxygenase-1, cytochrome c oxidase Va, cytochrome c-1, desmin, dystrophin-1, CD105 lymphocyte antigen (Endoglin), endothelin 1, Epidermal Growth Factor Receptor (EGFR), Epidermal Growth Factor (EGF), erythropoietin, E-selectin, EST translocation variant 4(EST 4), inducer of Extracellular Matrix Metalloproteinase (EMMPRIN), ferritin H, ferritin L, fibroblast growth factor 2, fibronectin, Fit-3 ligand, fluorodeoxyglucose-PET with CA125 (FDG-PET), Fms-related tyrosine kinase 1(VEGFR-1), GADD45A, bipotent protein (Geminin), Glyphosate N-acetyltransferase (Glyphosate N-acetyltransferase), epithelin-Granulin precursor (Granulin-epiheperlin precusor) (GEP), Growth differentiation factor 15, haptoglobin 1, haptoglobin-a-subunit, HE4 (human epididymis protein), Her2, HER2-neu, hK10, hK11, hK13, hK6, hK7, hK8, HLA class II Do beta, hLMH1, hLMH2, HNF-1 beta, human chorionic gonadotropin-beta subunit, human chorionic gonadotropin (hCG), IGFBP-2, IL-2R alpha (soluble interleukin 2 receptor alpha), immunoglobulin, immunosuppressive acidic protein (lAP), indoleamine 2, 3-dioxygenase, insulin-like growth factor binding protein 1, insulin-like growth factor binding protein 2, insulin-like growth factor binding protein 3, integrin alpha-V, integrin alpha V beta 6, intercellular adhesion molecule, interferon alpha 1, interleukin 1 alpha-V beta 6, and human interferon alpha-V, Interleukin 1 beta, Interleukin 10, Interleukin 12A, Interleukin 16, Inter-alpha-trypsin inhibitor fragments, kallikrein 8, keratin 18, keratin, type I cytoskeleton 19 (cytokeratin 19), Kit ligand, KRAS, Lactoferrin, laminin-beta 3, leptin-selectin, luteinizing hormone releasing hormone receptor, Mac-2 binding protein 90k, macrophage colony stimulating factor, macrophage migration inhibitory factor, breast serum antigen (Mammary serum antigen), mammaglobin B (Mammoglobin B), M-CAM, MIR21, Mesothelin (Mesothelin), MMP3, mucin-type glycoprotein antigen, myosin X, nerve growth factor beta, nerve growth factor-1 (Netrin-1), Neuroendocrine protein-55 (Neuroendocrine protein-55), Neutrophil defensin 1, neutrophil defensin 3, Nm 23-H1, nonmetaplastic cell protein 2 (nonmetastatic cell protein 2), non-metastatic cell 1 protein (NM23A), O-acyltransferase domain-containing 2 (O-acyltransferase domain linking 2), OVX1, OX40, P53, paraoxonase 2, Pcaf, P-glycoprotein, phosphoribosylaminoimidazole carboxylase (Phosphoribosylimidazole carboxylase), platelet-derived growth factor receptor alpha, platelet-derived growth factor receptor beta, platelet endothelial cell adhesion molecule (PEPCE-1), platelet factor 4, zone-associated plasma protein-A, gestagen, Procol-lys 1,2 oxoglutate 5-digixyg 3, Procol-lys 1,2 oxoglutate 5-digygog 1, Progesteron (PR 32), Progesteron P94, Progesteron-2, Progesteron-receptor, Progesteron 5-D2, Progesteron, prostate Specific Antigen (PSA), Prostatin (Prostatin), protein kinase C-binding protein 1, p-selectin, pyrroline-5-carboxylate reductase 1, Regulator of G protein signaling 12 (Regulator of G protein signaling 12), endoplasmic reticulum calcium binding protein (Reticulocalbin), S-100 alpha chain, S-adenosylhomocysteine hydrolase, Serum amyloid A protein (Serum amyloid A protein), 7 transmembrane domain proteins (derived transmembrane domain protein), sex determinant Y-box-4, Sialyl SSEA-1 (Sialyl SSEA-1), small inducible cytokine A18(CCL18, MlP-4), small inducible cytokine A2(CCL2), small inducible cytokine A3(CCL3) (macrophage inflammatory protein 1-alpha, small inducible cytokine B5(CX 5), Somatostatin, growth hormone growth factor, squamous cell carcinoma antigen 1, squamous cell carcinoma antigen 2, steroid hormone receptor, survivin, syndecan-1, Synuclein-gamma (Synuclein gamma), tetranectin 9 (Tetrasplanin 9), TGF-alpha, Thymidine Phosphorylase (TP), thyroglobulin (Tg), tissue metalloproteinase 2 inhibitor, tissue specific transplantation antigen P35B, tissue plasminogen activator (tPA), topoisomerase II, transferrin receptor P90 CD71 (Transferring receptor P90 CD 71), Transforming growth factor alpha (transforminggrowth factor alpha), Transforming growth factor beta 1, translocase of the outer mitochondrial membrane, Transthyretin (Transthyretin), fragment of Transthyretin (trophoblastin), tropomyosin 1 alpha chain (alpha-tropomyosin), Trypsin, tubulin beta 2, tubulin beta 3, tumor necrosis factor (ligand) superfamily member 5(CD154), tumor necrosis factor (ligand) superfamily member 6(Fas ligand), tumor necrosis factor alpha, tumor necrosis factor receptor p75/p55, tumor necrosis factor receptor superfamily member 6(Fas), tumor necrosis factor receptor-related protein 1, tumor protein p53, ubiquitin-conjugating enzyme E2C (ubiquitin tig enz), hemangiostatin (uAS), vascular Endothelial Growth Factor (VEGF), vascular smooth muscle growth promoting factor (VSGPIF-Spondin), VEGF (165) b, V-erb-b2, vitamin D binding protein, vitamin K dependent protein C, vitronectin, von Wilms disease factor, Wilms tumor 1(WT-1), WW domain binding protein 11, X box binding protein-1, and YKL-40. See Polanski et al, Biomark instruments, 1:1 (2006); cherneva et al, Biotechnol. & Biotechnol. EQ.21/2007/2: 145 (2007); Alaoui-Jamali et al, j.zhejiang Science B7: 411 (2006); basil et al, Cancer Res.66:2953 (2006); suh et al, Expert rev. mol. diagn.10:1069 (2010); and Diamandis, e.p., Molecular and Cellular Proteomics 3:367 (2004).

Other analytes that can be detected using the devices, systems and methods of the invention include those mentioned in the text book by Tietz "Clinical Chemistry and Molecular Diagnostics," Burtis, Ashwood and Bruns, ISBN 0721601898, Elsevier, 4 th edition (2006).

The methods, kits, cartridges, and systems of the invention can be configured to detect a predetermined list of analyte combinations that can be used to understand a medical condition of a subject. For example, the combined list may include: detection of pathogens, therapeutic agents for treating suspected pathogen(s), and potential biomarkers to monitor the pharmacological course of treatment (efficacy or pharmacokinetics), or to monitor the presence of pathogens or pathogen byproducts. Furthermore, the following list of disease treatments, which are constructed for detection, can be envisaged: disease or disease biomarkers, levels or concentrations of therapeutic agents used to treat suspected diseases, potential biomarkers to monitor the pharmacologic course (efficacy or pharmacokinetics) of treatment, and general chemical or other physiological markers of disease or therapeutic efficacy. Thus, a list of analyte detections may be used to inform and form appropriate medical decisions.

For example, the systems and methods of the present invention may be used to monitor immunocompromised subjects following allogeneic transplantation. In transplant subjects receiving solid organs, bone marrow, hematopoietic stem cells or other allogenic donations, there is a need to monitor immune status, organ function, and, if desired, to quickly and accurately determine opportunistic infections. For example, it is desirable to monitor creatinine and tacrolimus levels from the same blood sample from a subject, as monitoring of drug concentration and renal function can aid and guide physicians in optimal post-transplant treatment. Optimizing therapy is a close balance that prevents rejection but also ensures immune function against opportunistic infections and overall results in increased subject compliance with immunosuppressive therapy. Most transplant recipients die from transplant rejection, graft versus host disease, or opportunistic infections. In the first two cases, the immunosuppressive agent can prevent or inhibit rejection. However, clinical control is challenging if there is a potential infection in the subject. As a specific example, heart and lung transplant subjects exhibiting an unknown cause of fever enter a health care facility. Subjects were started using broad spectrum antibiotics until culture results were known. If the disease is worsening and the culture shows a particular infection (e.g., candida), a particular antifungal agent, fluconazole, may be administered to a known subject. However, this antifungal agent can alter the level of immunosuppressive agents (tacrolimus) administered to almost all allograft recipients. After testing for tacrolimus and creatinine levels, physicians discontinued using tacrolimus, believing that fluconazole will defeat the infection and in a rapid manner. Under this protocol, the subject's condition will worsen if the candida species is resistant to fluconazole and then the subject begins to use the appropriate antifungal agent. However, because tacrolimus will be discontinued, immunosuppressive therapy is not controlled and the subject will become unresponsive to any other therapy and may subsequently die. Thus, if there is a test that simultaneously monitors creatinine (renal function) and tacrolimus blood levels and accurately confirms opportunistic infections, the subject can be rescued.

The system and method of the present invention may include: multiple single detection methods without sample preparation; an automated system for determining a determinant of drug level, toxicity or adverse reaction; and pathogen identification plays an important role in immunocompromised subject settings. For example, it contains: (1) magnetic particles with creatinine-specific antibodies modified on their surface, (2) magnetic particles with tacrolimus-specific antibodies on their surface, and (3) magnetic particles with specific nucleic acid probes for identifying the pathogen species, cartridges with inlets or wells can be used to quickly determine and provide clinical control values for a given transplant subject. Opportunistic infections that can be monitored in such subjects, as well as in any other patient population at risk of infection, include, but are not limited to: fungi; candida (drug-resistant and non-drug-resistant strains); gram-negative bacterial infections (e.g., escherichia coli, stenotrophomonas maltophilia, klebsiella pneumoniae/klebsiella oxytoca, or pseudomonas aeruginosa); and gram-positive bacterial infections (e.g., Staphylococcus species: Staphylococcus aureus, Streptococcus pneumoniae, enterococcus faecalis, and enterococcus faecium). Other opportunistic infections that can be monitored include: coagulase-negative staphylococci, corynebacterium species, clostridium species and morganella morganii, as well as viral organisms such as CMV, BKV, EBC, HHV-6, HIV, HCV, HBV and HAV.

The systems and methods of the present invention may also be used to monitor and diagnose cancer patients as part of a multiplex diagnostic test. One particular type of cancer (colorectal cancer) has shown positive promise in the individualized medical treatment of specific solid tumors. The pharmacogenetic signatures can be used to optimize the treatment of colorectal and other cancers. Significant individual genetic variation exists in the metabolism of 5-FU, capecitabine, irinotecan and oxaliplatin, which affects the toxicity and efficacy of these drugs. Examples of genetic markers include UGT1a1 x 28, which leads to conjugation of the reduction of SN-38 (an active metabolite of irinotecan), resulting in an increased incidence of adverse effects, particularly neutropenia. To a lesser extent, increased 5-FU toxicity was predicted from DPYD x 2A. Polymorphisms in an indefinite series of repeat regions in the thymidylate synthase enhancer region, along with the single nucleotide polymorphism C > G, can be expected to give poor response to 5-FU. The efficacy of oxaliplatin is influenced by polymorphisms in the components of the DNA repair system (e.g., ERCC1 and XRCC 1). Polymorphic changes in endothelial growth factor receptor may predict the efficacy of cetuximab. Furthermore, polymorphisms in the immunoglobulin G fragment C receptor may reduce antibody-dependent cell-mediated cytotoxicity of cetuximab. Polymorphic changes in the VEGF gene and the hypoxia inducible factor 1 alpha gene are also thought to lead to differences in therapeutic outcomes. Thus, the identification of such polymorphisms in a subject can be used to aid a physician in making a treatment decision. For example, PCR-based genetic tests have been developed to assist physicians in making therapeutic treatment decisions for subjects with non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and gastric cancer. Expression of ERCC1, TS, EGFR, RRM1, VEGFR2, HER2 and detection of mutations in KRAS, EGFR and BRAF may help physicians determine the optimal treatment options. However, these PCR assays cannot be performed on-site, and therefore the sample must be delivered to a laboratory that is not on-site. Frequently, biopsies are taken of these solid tumors and FFPE (formalin fixed paraffin embedded (tissue)) samples are prepared. The system and method of the present invention can be used without the need for a 5-7 day turnaround time to obtain data and information and to use the fixed samples required by prior methods. The systems and methods of the present invention can provide a single platform for analyzing samples without sample preparation, for multiple analyte types, such as in cancer chemotherapy drugs, and genotyping, toxicity, and efficacy markers can drastically alter the practice of personalized medicine and provide rapid, accurate diagnostic testing.

The systems and methods of the present invention may also be used to monitor and diagnose neurological disorders such as dementia (loss of cognitive ability in a previously unimpaired person) and other forms of cognitive impairment. Dementia can be broadly classified into two types: cortical dementia and subcortical dementia. Cortical dementias include: alzheimer's disease, vascular dementia (also known as multi-infarct dementia), including bevengege's disease, dementia with lewy bodies (DLB), alcohol-induced persistent dementia, korsakoff's syndrome, weirnike's encephalopathy, frontotemporal lobar degeneration (FTLD), including pick's disease, frontotemporal dementia (or frontotemporal variant FTLD), lewy body dementia (or temporary variant frontotemporal variant FTLD), progressive non-fluent aphasia, creutzfeldt-jakob disease, dementia pugilistica, abnormal vascular network of the floor of the brain, thebestia (often mistakenly recognized as cancer), retrocortical atrophy or Benson's syndrome. Subcortical dementia includes dementia due to Huntington's disease, dementia due to hypothyroidism, dementia due to Parkinson's disease, dementia due to vitamin B1 deficiency, dementia due to vitamin B12 deficiency, dementia due to folate deficiency, dementia due to syphilis, dementia due to subdural hematoma, dementia due to hypercalcemia, dementia due to hypoglycemia, AIDS dementia complex, pseudodementia (major depressive episode with overt cognitive symptoms), substance-induced persistent dementia (associated with psychoactive drug use and previous absinthism), dementia due to multiple etiologies, dementia due to other general medical conditions (i.e., end-stage renal failure, cardiovascular disease, etc.), or dementia not specifically prescribed (for use in cases where specific criteria are not met). Alzheimer's disease is a common form of dementia. Because dementia is fundamentally associated with many neurodegenerative diseases, the ability to detect these proteins as disease biomarkers, along with drug or drug metabolite levels, in a single platform will assist physicians in adjusting dosages, changing regimens, or monitoring disease progression in general. These tests are currently performed off-site at a location remote from the subject and caregiver. Thus, in order to have the ability to monitor drug levels and biomarkers in the same detection system, in-situ detection would provide great advantages in the alleviation and treatment of disease. The method of the invention can be a single detection method, automated system, with multiple, no sample preparation, for determining drug levels, toxicity or adverse reaction determinants, and potential biomarkers for disease development. For example, a cartridge with an inlet or well containing (1) magnetic particles with protein dementia-specific biomarker antibodies modified on their surface, (2) magnetic particles with specific antibodies on their surface, and (3) magnetic particles with nucleic acid-specific probes for identifying protein expression levels can be used to quickly determine and provide clinical control values for a given dementia subject.

The systems and methods of the present invention may also be used to monitor and diagnose infectious diseases in a multiplex, automated, sample-free preparation system. Examples of pathogens that may be detected using the devices, systems and methods of the present invention include, for example: candida species (resistant and non-resistant strains), such as candida albicans, candida glabrata, candida krusei, candida tropicalis, and candida parapsilosis; aspergillus fumigatus; escherichia coli, stenotrophomonas maltophilia, Klebsiella pneumoniae/Klebsiella oxytoca, Pseudomonas aeruginosa; staphylococcus species (e.g., staphylococcus aureus or streptococcus pneumoniae); enterococcus faecalis, enterococcus faecium, coagulase-negative staphylococcal species, corynebacterium species, clostridium species, morganella morganii, pneumocystis jeikeium (formerly pneumocystis carinii), f. hominis, streptococcus pyogenes, pseudomonas aeruginosa, polyomavirus JC polyomavirus (a virus causing progressive multifocal leukoencephalopathy), acinetobacter baumannii, toxoplasma gondii, cytomegalovirus, aspergillus species, kaposi's sarcoma, cryptosporidium, cryptococcus neoformans, histoplasma capsulatum, and other bacteria, yeasts, fungi, viruses, prions, molds, actinomycetes, animals, parasites, protists, and helminth infectious organisms.

The systems and methods of the invention can be used to identify and monitor the pathogenesis of a disease in a subject, to select therapeutic interventions, and to monitor the effectiveness of selected therapies. For example, for patients infected with or at risk of viral infection, the systems and methods of the present invention may be used to determine infectious virus, viral load, and monitor white blood cell counts and/or biomarkers indicative of infection status. The identification of the virus can be used to select the appropriate treatment. The treatment regimen may also be correlated with the circulating concentration of antiviral agent and viral load by monitoring therapeutic intervention (e.g., a particular antiviral agent) to ensure that the patient is responsive to treatment.

The systems and methods of the invention can be used to monitor a subject for viral infection, for example, using a viral list configured to detect Cytomegalovirus (CMV), epstein-barr virus, BK virus, hepatitis b virus, hepatitis c virus, Herpes Simplex Virus (HSV), HSV1, HSV2, Respiratory Syncytial Virus (RSV), influenza virus; influenza A, influenza A subtype H1, influenza A subtype H3, influenza B, human herpesvirus 6, human herpesvirus 8, human metapneumovirus (hMPV), rhinovirus, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, and adenovirus. The methods of the invention may be used to monitor suitable therapy for a subject infected with a virus (e.g., abacavir, acyclovir, adefovir, amantadine, amprenavir, azapridine, Arbidol, Atazanavir (Atazanavir), Atripla, Boceprevir (Boceprevir), cidofovir, cobivir (Combivir), dirura vir (Darunavir), delavirdine, dideoxyinosine, docosanol, edexuridine (edoudine), efavirenz, emtricitabine, enviviride (Enfuvirtide), entecavir, famciclovir, fomivirsen, Fosamprenavir (Fosamprenavir), foscarnet, Fosfonenet, ganciclovir, Imibacitabine (Ibacitadine), inosine (isopropyl), iovir, quinavir, indinavir, an I, interferon beta I, interferon beta interferon, interferon beta type I, interferon beta interferon, interferon type II, interferon beta interferon, and combinations thereof, Lopinavir, lovirin, Maraviroc (Maraviroc), moroxydine, metisazone, nelfinavir, nevirapine, Nexavir, nucleoside analogs, oseltamivir (Tamiflu), PEG interferon alpha-2 a, penciclovir, Peramivir (Peramivir), Pleconaril (Pleconaril), podophyllotoxin, Raltegravir (Raltegravir), reverse transcriptase inhibitors, ribavirin, rimantadine, ritonavir, Pyramidine, saquinavir, stavudine, tea tree oil, tenofovir dipivoxil, Tipranavir (Tipranavir), trifluridine, trovirvir (trimavir), trodamide, terluda (Truvada), acyclovir (Valtrex), valganciclovir, vicrovirc, acyclonidine, valacitrexadine, viraadenosine, valtreviravirine (Vizavirzavir), valtrevir, Vizavir (Vizavir), Vizavir (Vizavirzavir), or zidine); and monitoring the circulating concentration of the therapeutic agent administered to the subject.

The system and method of the present invention may also be used to monitor HIV/AIDS patients. Detection of HIV RNA is often performed when a clinician suspects an acute infection (e.g., in a subject reported to have recent dangerous behavior associated with symptoms and signs of acute retroviral syndrome). Diagnosis of acute HIV infection is supported by high levels of HIV RNA detected in plasma using sensitive amplification detection (PCR, bDNA or NASBA), together with negative or undefined HIV antibody detection. Monitoring the viral load, drug levels, CD4 cell count and toxicity profile of HIV/AIDS subjects in a single platform diagnostic method would provide significant advantages to the subjects. The systems and methods of the invention can be used in multiplex, non-sample preparation, single detection methods, and automated systems to determine drug levels, toxicity or adverse reaction determinants, and potential biomarkers for disease development. For example, cartridges with inlets or wells containing (1) magnetic particles with CD4 cell-specific antibodies modified on their surface, (2) magnetic particles with toxic biomarker-specific antibodies on their surface, and (3) magnetic particles with nucleic acid-specific probes for identifying viral load levels can be used to quickly determine and provide clinical control values for a given HIV/AIDS subject.

The systems and methods of the invention can also be used to monitor and diagnose immune disorders (e.g., crohn's disease, ileitis, enteritis, inflammatory bowel disease, irritable bowel syndrome, ulcerative colitis, and non-gastrointestinal immune disorders) in a subject. The development of relatively recent genetically engineered agents has made it possible to radically alter the treatment of immune diseases, and Infliximab (Remicade), also known as Infliximab (Infliximab), an anti-TNF antibody, has been introduced as a novel type of treatment with high potency, rapid onset of action, long-lasting efficacy, and improved tolerance. However, these agents are expensive and at least one third of the patients with indications fail to show any useful response. It is therefore clearly important to find a means of predicting patients who will respond and anticipating relapse. TNF polymorphisms also appear to affect the nature and frequency of parenteral manifestations of Inflammatory Bowel Disease (IBD). Several large control trials have shown that infliximab plays a role in the treatment of moderate to severe crohn's disease patients as well as fistulizing crohn's disease. Small studies have shown a possible correlation between poor response to infliximab and increased activated NF- κ B mucosal levels, homozygosity for polymorphism of exon 6 of TNFR2 (genotype Arg196Arg), positivity of perinuclear anti-neutrophil cytoplasmic antibodies (ANCA), and the presence of increased numbers of activated lamina propria monocytes producing interferon- γ and TNF- α. Thus, monitoring TNF- α and the toxic class of crohn's disease patients in a single platform diagnostic method would be of significant advantage. The methods of the invention can be multiplex, no sample preparation, single detection methods and automated systems to determine drug levels, toxicity or adverse reaction determinants, and potential biomarkers for disease development. For example, cartridges with inlets or wells containing (1) magnetic particles with anti-TNF-alpha specific antibodies modified on their surface, (2) magnetic particles with toxic biomarker specific antibodies on their surface, and (3) magnetic particles with specific probes for identifying markers of disease progression can be used to rapidly determine and provide clinical control values for a given crohn's disease or IBD patient.

The systems and methods of the present invention may also be used to monitor and diagnose infectious diseases and inflammation in a multiplex automated non-sample preparation system. Such systems and methods may be used to monitor, for example, bacteremia, sepsis, and/or Systemic Inflammatory Response Syndrome (SIRS). Early diagnosis is clinically important because this type of infection, if left untreated, can lead to organ dysfunction, hypoperfusion, hypotension, refractory (septic) shock/SIRS shock and/or Multiple Organ Dysfunction Syndrome (MODS). With respect to typical patients, many bacterial or fungal infections are the result of culture upon entry into a medical facility and are referred to as hospital-associated infections (HAIs), also known as nosocomial or nosocomial infections in hospitals. The most common types of bacteria infecting immunocompromised patients are the gram-positive bacteria MRSA (methicillin-resistant staphylococcus aureus) and the gram-negative bacteria helicobacter. Although there are antibacterial drugs that can treat diseases caused by gram-positive MRSA, there are currently less effective drugs against acinetobacter. Common pathogens in bloodstream infections are: coagulase-negative staphylococci, enterococci and staphylococcus aureus. In addition, candida albicans and pathogens of pneumonia (e.g., pseudomonas aeruginosa, staphylococcus aureus, klebsiella pneumoniae, and haemophilus influenzae) cause many infections. Pathogens of urinary tract infections include: escherichia coli, candida albicans, and pseudomonas aeruginosa. In addition, gram-negative intestinal organisms are common in urinary tract infections. Surgical site infections include: staphylococcus aureus, pseudomonas aeruginosa, and coagulase-negative staphylococcus. The infectious agent may be selected from, but is not limited to: pathogens associated with sepsis, such as Acinetobacter baumannii, Aspergillus fumigatus, Bacteroides fragilis, blasHV, Borkholderia cepacia, Campylobacter jejuni/Campylobacter coli, Candida guilliermondii, Candida albicans, Candida glabrata, Candida krusei, Candida ruchii, Candida parapsilosis, Candida tropicalis, Clostridium perfringens, coagulase-negative staphylococci, Enterobacter aerogenes, Enterobacter cloacae, Enterobacteriaceae, enterococcus faecalis, enterococcus faecium, Escherichia coli, Haemophilus influenzae, aureobacter aureofaciens, Klebsiella pneumoniae, Listeria monocytogenes, Mec A gene (MRSA), Morganella morganganca, Neisseria meningitidis, Neisseria non-meningitidis species of Neisseria meningitidis, Prevotella bucca, Prevotella intermedia, Melanogonia melanogenesis, Prevotella, Propionibacterium acnes, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella enterica, Serratia marcescens, Staphylococcus aureus, Staphylococcus hemolyticus, stenotrophomonas maltophilia, Staphylococcus saprophyticus, stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus bovis, Streptococcus dysgalactiae, Streptococcus mitis, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus sanguis; or any other infectious agent described herein. In certain instances, the methods and systems will be designed to determine whether an infectious agent carries a Van a gene or a Van B gene that is characteristic of vancomycin resistance; methicillin-resistant mecA, NDM-1 and ESBL, which are more generally resistant to beta-lactam antibiotics.

Sepsis or septic shock is a serious medical condition characterized by a systemic inflammatory state (systemic inflammatory response syndrome or SIRS) and in the presence of a known or suspected infection. Sepsis is defined as SIRS in the presence of infection, septic shock is defined as sepsis with refractory arterial overpressure or hypoperfusion abnormalities (albeit with adequate fluid resuscitation), and severe sepsis is defined as sepsis with organ dysfunction, hypoperfusion, or hypotension. Many studies have examined the value of combining currently available markers, and thus the platform has been described as being capable of determining the levels of multiple factors, such as: GRO-alpha, high mobility group protein B1(HMBG-1), IL-1 receptor antagonist, IL-1B, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, macrophage inflammatory protein (MIP-1), macrophage Migration Inhibitory Factor (MIF), osteopontin, RANTES (stimulated modulation, normal T cell expression and secretion; or CCL5), TNF-alpha, C-reactive protein (CRP), CD64, and monocyte chemotactic protein 1 (MCP-1). In addition, the systems and methods can be designed to monitor certain proteins that characterize sepsis, such as adenosine deaminase binding protein (ABP-26), Inducible Nitric Oxide Synthase (iNOS), Lipopolysaccharide Binding Protein (LBP), and Procalcitonin (PCT). Thus, this platform reduces the experience regimen, and/or the use of non-specific/general antibacterial agents that may or may not target specific pathogens and/or potential systemic dysfunction in a given patient. This platform enables rapid and correct diagnosis, which means effective treatment, providing critical information for physician decision making and reducing morbidity and mortality.

In order to determine whether a patient is septic or not, the presence of the pathogen must be distinguished. In order to treat patients most effectively, it is important to initiate appropriate treatment earliest. Antimicrobial and other treatments for sepsis rely on the classification of the pathogen at multiple levels, including the identification of the pathogen as (1) a bacterium, virus, fungus, parasite, or other; (2) gram positive, gram negative, yeast or mold; (3) seed growing; and (4) sensitivity.

Each of these levels of specificity shortens the time to begin appropriate therapy, and each step below the route will narrow the therapeutic range to the most specific group. Without absolute sensitivity data, empirical methods of care rely on available information (at any level) about the pathogen and patterns of pathogen frequency and susceptibility propensity in the hospital of another healthcare unit. Thus, certain types of pathogens are often considered pathogenic until more data exists to improve the pairing of pathogens with treatments. In particular, these targets fall into the ESKAPE category (which is a range of drug-resistant pathogens) and the SPACE category (which is a group of highly virulent pathogens requiring patient isolation).

In addition to identifying pathogens in these multiple sample types (blood, tissue, urine, etc.), another method of distinguishing symptomatic patients (e.g., patients with systemic inflammatory syndrome or SIRS) from sepsis patients is to identify infected patients by using biomarkers that establish correlations, either individually or using indices. In cases where infection is not detected due to interference of antimicrobial therapy with diagnosis, control of the immune system of the treatment, or these biomarkers, which may be various types of analytes (cytokines, metabolites, DNA, RNA/gene expression, etc.), will indicate infection and thus sepsis.

To generate diagnostic information needed for the presence of infection and some level of species discrimination, a list may be: (i) gram-positive clusters (e.g., staphylococcus aureus and CoNS (coagulase-negative staphylococci)); (ii) gram-positive chains/pairs (e.g., streptococcus species, streptococcus mitis, pneumococcus species, agalactia (agalactie) species, pyogenes (pyogenenes) species, enterococcus species (enterococcus faecium, enterococcus faecalis); (iii) gram-negative bacilli (e.g., escherichia coli, proteus species, klebsiella species, serratia species, acinetobacter species, stenotrophomonas species); (iv) SPACE (e.g., Serratia species, Pseudomonas species, Acinetobacter species, Citrobacter species, Enterobacter species); (v) pseudomonas (e.g., pseudomonas species); (vi) ESKAPE (enterococcus faecium, staphylococcus aureus, klebsiella species, acinetobacter species, pseudomonas species, enterobacteria species); and (vii) whole bacteria (all bacterial species).

This list should be used for a full range of fungal tests. These types represent the information required for effective intervention with appropriate therapeutic agents, given that each healthcare unit will have an empirically derived approach based on positive responses to gram-positive bacteria, gram-negative bacteria, and the like. The species identified in each type represent those that fit in each title but are not comprehensive. In addition, the whole bacterial signature is included to encompass any species not encompassed by diagnostic methods for various types. Moreover, although not entirely complete, combinations of the individual results if included as described above will also give an indication of such species. Type-based cross-referencing of positives and negatives allows a method of excluding pathways to probabilistically identify species.

The systems and methods of the present invention may also be used to monitor and diagnose heart disease, such as myocardial infarction, in a subject. Myocardial markers or myocardial enzymes are proteins that leak out of damaged cardiomyocytes and are used to assess myocardial damage. Cardiac markers include, but are not limited to: the enzymes SGOT, LDH, the MB subtype of creatine kinase and cardiac troponin (T and I). Thus, in acute situations, monitoring troponin I and T and potentially other biomarkers of myocardial ischemia in addition to drug treatment and toxicity profile in a single platform diagnostic approach would be of significant advantage. The systems and methods of the invention can be used to provide multiple, no sample preparation, single detection methods and automated systems for determining drug levels, toxicity or adverse reaction determinants, and potential biomarkers for disease progression. For example, a cartridge with an inlet or well containing (1) magnetic particles with anti-troponin I or troponin T specific antibodies modified on their surface, (2) magnetic particles with toxic biomarker specific antibodies on their surface and (3) magnetic particles with specific probes for identifying markers of disease progression can be used to rapidly determine and provide clinical control values for a given myocardial infarction patient.

One or more porous cartridges may be configured for use in the systems and methods of the present invention; the cartridge may be prepared with at least one whole blood sample from a patient; magnetic particles for detecting each analyte to be detected (small molecule(s); metabolite(s) of small molecule(s); metabolic biomarker(s), such as those described in the list of liver functions); and dilution and washing buffers. Liver function testing is performed on serum or plasma samples of patients, and clinical biochemical laboratory blood analysis provides critical data about the condition of a patient's liver. The "liver function list" is a blood test in which low or high levels of one or more enzymes may indicate liver disease or liver damage. For example, the liver function list may include one or more of the following analyte detection assays: one or more small molecules; one or more metabolites of one or more small molecules; biological, metabolic biomarkers; genotyping \ gene expression profiling; and proteomics analysis.

The liver function list may include an analysis of one or more of the following proteins in a biological sample of a patient or subject: (1) albumin (the major component of total liver protein; the remainder is known as globulin; albumin must be present at 3.9 to 5.0 g/dL, hypoalbuminemia indicates malnutrition, lower protein catabolism, cirrhosis or nephrotic syndrome); (2) aspartate Aminotransferase (AST) (also known as serum glutamate oxaloacetate transaminase or aspartate aminotransferase, is an enzyme in liver parenchymal cells and is typically 10 to 34 IU/L; elevated levels are indicators of acute liver injury); (3) alanine Aminotransferase (ALT) (also known as glutamate pyruvate transaminase or alanine aminotransferase, an enzyme present in liver cells at levels between 8 and 37 IU/L; elevated levels are indicators of viral hepatitis acute liver injury or acetaminophen overdose; the ratio of AST to ALT is used to differentiate between causes of liver injury); (4) alkaline phosphatase (ALP) (an enzyme present in cells lining the bile ducts of the liver; normal range 44 to 147 IU/L and elevated levels in the case of invasive liver disease, intrahepatic cholestasis or large bile duct obstruction); (5) gamma-glutamyl transpeptidase (GGT) (a more sensitive marker of biliary obstructive lesions than ALP, very specific for liver; standard range 0 to 51 IU/L; GGT can be elevated in both acute and chronic alcoholism; GGT can be used to detect isolated causes of ALP elevation); (6) total Bilirubin (TBIL) (an increase in total bilirubin can lead to jaundice and can be attributed to cirrhosis, viral hepatitis, hemolytic anemia, or internal bleeding); (7) direct bilirubin; (8) prothrombin time (PTT) (changes in clotting time can be caused by hepatocyte injury and bile flow obstruction); (9) alpha-fetoprotein detection (elevated levels indicate hepatitis or cancer); (10) a lactate dehydrogenase; and (11) mitochondrial antibodies (which if present may indicate chronic active hepatitis, primary biliary cirrhosis, or other autoimmune disease). The above-described proteins in the liver function list may be analyzed using the systems and methods of the present invention.

Another liver function list may include: genotyping of cytochrome P450 enzymes. Cytochrome P450 genotyping assays are used to determine how a patient or subject metabolizes a drug. The results of cytochrome P450 detection can be used to classify individuals into four main types:

(i) for weak metabolism. Metabolism of certain drugs is slower than normal levels, the drugs will have longer half-lives and may increase the likelihood of causing adverse reactions.

(ii) Normal metabolizing. The drugs will be metabolized at an average rate and thus is an indication of the benefit from treatment and fewer adverse effects compared to other individuals who do not metabolize these particular drugs.

(iii) And those with moderate metabolism. The drug may or may not be metabolized at the average rate. At least one gene involved in drug metabolism is suspected to be dysfunctional. There is then a tendency to metabolize certain drugs differently.

(iv) Ultra-fast metabolizing. The drug is metabolized faster and more efficiently than average. Because the metabolic rate is higher than average, some drugs are activated faster than normal and are excreted faster, and the drug may not have the desired potency.

Currently, genotyping of the genes responsible for these enzymes in the human population has shown that polymorphic differences in these enzymes can lead to changes in the efficacy and toxicity of some drugs. Genotyping requires a sample of cells representing the genome of a patient or subject and the purpose of this analysis is to determine genetic differences among these clinically important genes.

Possible liver metabolizing enzymes may be part of the list of liver functions, including but not limited to: CYP2C19, CYP2D6, CYP2C9, CYP2C19, CYP1a2, NAT2, DPD, UGT1a1, 5 HTT.

The present invention features a multiplex analysis of a single blood sample from a patient (e.g., a single blood draw, or any other type of patient sample described herein) to determine (a) liver enzyme status and (b) genotypes of key metabolic enzymes, which can then be used to design drug treatment regimens for optimal therapeutic care using the systems and methods of the present invention.

The systems and methods of the present invention may include one or more multi-well cartridges prepared with at least one whole blood sample from a patient; magnetic particles for detecting each analyte to be detected; an analyte antibody; a multivalent binding agent; and/or dilution and wash buffers for multiplex assays as described above.

Nephrotoxicity

Nephrotoxicity is a common adverse reaction with exogenous compounds, and early rapid detection of early stages of nephrotoxicity can help to make medical decisions. Early reports of nephrotoxicity tests suggested that increased mRNA expression of certain genes could be monitored. However, other reports suggest that markers of renal toxicity can be detected in urine. These markers include: kim-1, lipocalin-2, neutrophil gelatinase-associated lipocalin (NGAL), timp-1, clusterin, osteopontin, vimentin, and heme oxygenase 1 (HO-1). More broadly, detection of DNA, heavy metal ions or BUN levels in urine can be useful clinical information. Thus, the methods and utilities of the present invention also include the ability to detect these markers of renal toxicity. Optionally, the list of liver functions may also include one or two biomarkers of nephrotoxicity, and vice versa.

Amplification and detection of nucleic acids from complex samples

The systems and methods of the invention can include amplification-based nucleic acid detection assays (e.g., for diagnostic, forensic, and environmental analysis) performed starting from complex samples.

Sample preparation must also remove or provide resistance to common PCR inhibitors found in complex samples (e.g., body fluids, soil, or other complex environments). Common inhibitors include those identified in Wilson, appl.environ.microbiol.,63:3741(1997), and the like. Inhibitors generally work by preventing cell lysis, degradation, or masking of target nucleic acid, and/or inhibition of polymerase activity. The presence of 0.1% blood in the reaction inhibited the most commonly used polymerase (Taq). More recently, mutant Taq polymerases resistant to common inhibitors found in blood and soil (e.g., hemoglobin and/or humic acid) have been engineered (Kermekchiev et al, nuclear.acid.res., 37 (5): e40, (2009)). The manufacturer's introduction showed that these mutations could be amplified directly from up to 20% of the blood. Although there is resistance due to mutation, accurate real-time PCR detection is complicated due to fluorescence quenching observed in the presence of blood samples (Kermekchiev et al, nuclear.

Polymerase chain reaction amplification of DNA or cDNA is a proven and proven method; however, as noted above, the polymerase is measured by reagents contained in the crude sample (including, but not limited to, commonly used anticoagulants and hemoglobinWhite) is suppressed. Recently, mutant Taq polymerases resistant to common inhibitors found in blood and soil have been engineered. Polymerases currently commercially available, e.g. HemoKlenaTaq^TM(New England BioLabs, Inc., Ipswich, MA) and OmniTaq^TMAnd OmniKlenaQ^TM(DNA Polymerase Technology, Inc., St. Louis, MO) are mutant (e.g., N-terminal truncation and/or point mutations) Taq polymerases that are capable of amplifying DNA in the presence of up to 10%, 20%, or 25% whole blood, depending on the product and reaction conditions (see, e.g., Kermekchiev et al, nucleic acid Res.31:6139 (2003); and Kermekchiev et al, nucleic acid Res.37: e40 (2009); and see U.S. Pat. No. 7,462,475). Furthermore, Phusion's blood direct PCR kit (Finnzymes Oy, Espoo, finland) comprises a unique fusion DNA polymerase engineered to contain a double stranded DNA binding domain which allows amplification under conditions which are normally inhibitory to conventional polymerases (e.g., Taq or Pfu) and allows DNA amplification in the presence of up to about 40% whole blood under certain reaction conditions. See Wang et al, Nuc. acids Res.32:1197 (2004); and see U.S. patent nos. 5,352,778 and 5,500,363. In addition, Kapa Blood PCR mixtures (Kapa Biosystems, Woburn, Mass.) provide a genetically engineered DNA polymerase that allows direct amplification of whole Blood to be performed at reaction volumes up to about 20% under certain reaction conditions. Despite these breakthroughs, direct optical detection of the amplicons produced by existing methods is not possible because fluorescence, absorbance and other light-based methods produce signals that are quenched by the presence of blood. See Kermekchiev et al, nucleic.acid. res.,37: e40 (2009).

We have found that complex samples (e.g. whole blood) can be directly amplified using about 5%, about 10%, about 20%, about 25%, about 30%, about 25%, about 40% and about 45% or more of whole blood in the amplification reaction, and that the formed amplicons can be detected directly from the amplification reaction using Magnetic Resonance (MR) relaxation measurements after addition of conjugated magnetic particles bound to oligonucleotides complementary to the target nucleic acid sequences. Alternatively, the magnetic properties may be manipulated prior to amplificationParticles are added to the sample. Thus, a method is provided for nucleic acid amplification for complex dirty samples, hybridizing the formed amplicons to paramagnetic particles, followed by direct detection of the hybridized magnetic particle conjugates and target amplicons using a magnetic particle-based detection system. In particular embodiments, measurement is by MR relaxation (e.g., T₂、T₁T1/T2 hybrid, T₂ ^*Etc.) to directly detect the hybridized magnetic particle conjugates and amplicons. Also provided are methods that are dynamic, such that the initial nucleic acid copy number within a sample is quantified (e.g., sampling and nucleic acid detection at a predetermined number of cycles, comparison of endogenous internal control nucleic acids, use of exogenous spiked, homologous competitive control nucleic acids).

The term "amplification" or derivatives thereof, as used herein, refers to one or more methods known in the art for replicating a target or template nucleic acid, thereby increasing the copy number of a selected nucleic acid sequence. Amplification may be exponential or linear. The target or template nucleic acid can be DNA or RNA. The sequences amplified in this way constitute "amplified regions" or "amplicons". One skilled in the art can readily design primer probes to target a particular template nucleic acid sequence. In certain preferred embodiments, the amplicons formed are short, thereby allowing rapid cycling and copy generation. The size of the amplicon can be varied as desired to provide the ability to distinguish between target and non-target nucleic acids. For example, the amplicon can be less than about 1,000 nucleotides in length. Ideally, the amplicon is 100 to 500 (e.g., 100 to 200, 150 to 250, 300 to 400, 350 to 450, or 400 to 500) nucleotides in length.

Although the exemplary methods described below relate to amplification using the polymerase chain reaction ("PCR"), many other methods are known in the art of nucleic acid amplification (e.g., isothermal methods, rolling circle methods, etc.). Those skilled in the art will appreciate that these other methods may be used in place of, or in addition to, the PCR method. See, e.g., Saiki "Amplification of Genomic DNA" in PCR Protocols, edited by Innis et al, Academic Press, San Diego, Calif., pages 13-20 (1990); wharam et al, Nucleic Acids Res.29: e54 (2001); hafner et al, Biotechniques,30:852 (2001). Other amplification methods suitable for use in the methods of the invention include, for example: polymerase Chain Reaction (PCR) method, reverse transcription PCR (RT-PCR), Ligase Chain Reaction (LCR), transcription-based amplification system (TAS), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA) method, Strand Displacement Amplification (SDA) method, loop-mediated isothermal amplification (LAMP) method, isothermal and chimeric primer-primed nucleic acid amplification (ICAN) method, and smart amplification System (SMAP) method. These and other methods are well known in the art and may be adapted for use in conjunction with the provided methods for detecting amplified nucleic acids.

The PCR method is a technique for making many copies of a specific template DNA sequence. PCR methods are disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, each of which is incorporated herein by reference. A set of primers complementary to the template DNA is designed and the regions flanked by the primers are amplified with a DNA polymerase in a reaction comprising multiple amplification cycles. Each amplification cycle includes initial denaturation, and up to 50 annealing cycles, strand extension (or elongation), and strand separation (denaturation). In each cycle of the reaction, the DNA sequence between the primers is copied. Primers can bind to the copied DNA as well as the original template sequence, so the total number of copies increases exponentially over time. PCR can be performed according to the method described by Whelan et al (Journal of Clinical Microbiology,33:556 (1995)). Various modified PCR methods are available and are well known in the art. Various improvements such as: the "RT-PCR" method (in which DNA is synthesized from RNA using reverse transcriptase before PCR is performed) and the "TaqMan PCR" method (in which only specific alleles are amplified and detected using fluorescently labeled TaqMan probes and Taq DNA polymerase) are known to those skilled in the art. RT-PCR and variants thereof have been described, for example, in U.S. patent nos. 5,804,383, 5,407,800, 5,322,770, and 5,310,652, and the references described herein, which are incorporated herein by reference; and TaqMan PCR and related reagents for use in this method are described, for example, in U.S. patent nos. 5,210,015, 5,876,930, 5,538,848, 6,030,787 and 6,258,569, the contents of which are incorporated herein by reference.

LCR is a similar to PCR DNA amplification method, but its use of four primers instead of two primers and the use of ligase to join or join two DNA fragments. Amplification can be performed in a thermal cycler (e.g., LCx from Abbott Labs, North Chicago, IL). The method may be performed, for example, according to the Journal of Clinical Microbiology 36 of Moore et al: 1028 (1998). LCR processes and variants thereof are described, for example, in european patent application publication No. EP0320308 and U.S. patent No. 5,427,930, each of which is incorporated herein by reference.

The TAS method is a method for specifically amplifying a target RNA, in which a transcript is obtained from a template RNA using a cDNA synthesis step and an RNA transcription step. In the cDNA synthesis step, a sequence recognized by a DNA-dependent RNA polymerase (i.e., a polymerase binding sequence or PBS) is inserted downstream of the cDNA copy of the target or marker sequence to be amplified using a two-domain oligonucleotide primer. In a second step, RNA polymerase is used to synthesize multiple copies of RNA from the cDNA template. Amplification using TAS requires only a few cycles, since DNA-dependent RNA transcription can produce 10-1000 copies of each copy of the cDNA template. TAS may be performed according to the method of KWoh et al PNAS 86:1173 (1989). The TAS method has been described, for example, in International patent application publication WO 1988/010315, the contents of which are incorporated herein by reference.

Transcription-mediated amplification (TMA) is a transcription-based isothermal amplification reaction; the isothermal amplification reaction utilizes RNA transcription by RNA polymerase and DNA transcription by reverse transcriptase to produce RNA amplicons from the target nucleic acids. The advantage of TMA is that 100 to 1000 copies of amplicon can be produced per amplification cycle, as opposed to PCR or LCR methods which produce only 2 copies per cycle. TMAs have been described, for example, in U.S. patent No. 5,399,491, the contents of which are incorporated herein by reference. NASBA is a transcription-based method for specifically amplifying target RNA from an RNA or DNA template. NASBA is a method for the continuous amplification of nucleic acids in a single mixture at one temperature. A transcript is obtained from a template RNA using a DNA-dependent RNA polymerase using a forward primer having the same sequence as the target RNA and a reverse primer having a sequence complementary to the target RNA on the 3 'side and a promoter sequence recognizing T7 RNA polymerase on the 5' side. The transcripts were further synthesized using the resulting transcripts as templates. The method may be performed in accordance with Heim et al, Nucleic Acids Res., 26:2250 (1998). The NASBA method has been described in U.S. patent No. 5,130,238, which is incorporated herein by reference.

The SDA method is an isothermal nucleic acid amplification method in which a target DNA is amplified using a DNA strand substituted with a strand synthesized by a strand-substituting DNA polymerase (lacking 5 '- > 3' exonuclease activity) through a single-strand nick generated by a restriction enzyme as a template for the next replication. Primers containing restriction sites anneal to the template, and amplification primers anneal to the 5' adjacent sequences (nicks are formed). Amplification was initiated at a fixed temperature. The newly synthesized DNA strand is cut with restriction enzymes and polymerase amplification begins again, displacing the newly synthesized strand. SDA can be performed according to the method of Walker et al, PNAS,89:392 (1992). The SDA method has been described in U.S. patent nos. 5,455,166 and 5,457,027, the contents of which are incorporated herein by reference.

The LAMP method is an isothermal amplification method in which a loop is always formed at the 3' end of a synthesized DNA, a primer is annealed in the loop, and specific amplification of a target DNA is performed isothermally. LAMP can be performed according to the method in Nagamine et al, Clinical chemistry.47:1742 (2001). LAMP methods have been described in U.S. patent nos. 6,410,278, 6,974,670, and 7,175,985, each of which is incorporated herein by reference.

The ICAN method is a non-isothermal (anisothermal) amplification method in which specific amplification of target DNA is performed isothermally by a strand displacement reaction, a template exchange reaction and a nick introduction reaction using chimeric primers comprising RNA-DNA, and DNA polymerase having strand displacement activity and ribonuclease H. The protein can be obtained according to Mukai et al, j. biochem.142: 273(2007) for ICAN. The ICAN process has been described in U.S. Pat. No. 6,951,722, which is incorporated herein by reference.

The SMAP (MITANI) method is a method in which a target nucleic acid is synthesized continuously under isothermal conditions using a primer set comprising two types of primers and DNA or RNA as a template. The first primer included in the primer set includes, in the 3 ' end region thereof, a sequence (Ac ') hybridizable with the sequence (a) in the 3 ' end region of the target nucleic acid sequence, and on the 5 ' side of the above sequence (Ac '), a sequence (B ') hybridizable with the sequence (Bc) complementary to the sequence (B) on the 5 ' side of the above sequence (a) present in the above target nucleic acid sequence. The second primer includes, in its 3 'end region, a sequence (Cc') hybridizable to the sequence (C) in the 3 'end region of the sequence complementary to the above-mentioned target nucleic acid sequence, and a loop back sequence (D-Dc') comprising two nucleic acid sequences hybridizable to each other on the same strand on the 5 'side of the above-mentioned sequence (Cc'). SMAP can be performed according to the method of Mitani et al, nat. methods,4(3):257 (2007). The SMAP process has been described in U.S. patent application publication Nos. 2006/0160084, 2007/0190531, and 2009/0042197, the contents of each of which are incorporated herein by reference.

The amplification reaction can be designed to produce a specific type of amplification product, e.g., double stranded; single-stranded; a duplex with a 3 'or 5' overhang; or a double stranded nucleic acid with chemical ligands at the 5 'and 3' ends. The amplified PCR product can be detected by the following method: (i) hybridizing the amplification product to a magnetic particle-bound complementary oligonucleotide, wherein two different oligonucleotides are used to hybridize to the amplification product such that the nucleic acid functions as an inter-particle junction (inter-particle) that promotes particle agglomeration; (ii) hybridization-mediated detection, in which the DNA of the amplified product must first be denatured; (iii) a hybridization-mediated detection, wherein particles are hybridized to the 5 'and 3' overhangs of the amplification product; (iv) chemical or biochemical ligands that allow the particles to bind to the ends of the amplification products, such as streptavidin-functionalized particles that bind to biotin-functionalized amplification products.

The systems and methods of the invention can be used to perform real-time PCR and provide quantitative information about the amount of target nucleic acid present in a sample (see fig. 25 and example 14). Methods for performing quantitative real-time PCR are provided in the literature (see, e.g., RT-PCR protocols, methods in Molecular Biology, Vol.193, Joe O' Connell, ed. Totowa, NJ: Humana Press, pp. 2002,378. ISBN 0-89603-875-0.). Example 14 describes the use of the method of the invention in real-time PCR analysis of whole blood samples.

The system and method of the present invention can be used to perform real-time PCR directly in opaque samples (e.g., whole blood) using nanomagnetic particles modified with capture probes and magnetic separation. The use of real-time PCR enables quantitative analysis of a target nucleic acid without opening the reaction tube after the PCR reaction has been started.

Previous work showed that in some cases the presence of particles in the PCR reaction can inhibit PCR. With respect to these inhibitory particles, it is contemplated that the particles may be pulled to one side of the tube (or other location within the vessel) during the PCR reaction so that they remain out of solution. Various methods can be used to release the particles back into suspension to allow them to hybridize to the PCR products and then pull them out of solution.

In certain embodiments, the invention features the use of enzymes compatible with whole blood, such as NEB Hemoklentaq, DNAP Omniklentaq, Kapa Biosystems hemorrhenase, Thermo-Fisher Finnzymes Phusion enzyme.

The invention also features quantitative asymmetric PCR. In any of the real-time PCR methods of the invention, the method may comprise the steps of: (i) sampling a whole blood sample into the prepared PCR reaction mixed solution (PCR mastermix) containing the superparamagnetic particles; (ii) before the first PCR cycle, the tube is closed until the PCR cycle is completed; (iii) loading the tube onto a thermal cycler; (iv) performing "n" cycles of standard PCR thermal cycling; (v) performing a T2 test (the exact duration and step of this change depends on the biochemistry and particle design methods described below); and (vi) repeating steps (iv) and (v) until sufficient T2 readings have been obtained for accurate quantitative analysis of the initial target concentration.

The above method may be used for any of the following types of detection of aggregation or disaggregation described herein, comprising:

。

various impurities and components of whole blood can be inhibitory to polymerase and primer annealing. These inhibitors can lead to the generation of false positives and low sensitivity. To reduce the occurrence of false positives and low sensitivity, when amplifying and detecting nucleic acids in complex samples, it is desirable to use thermostable polymerases that are not inhibited by whole blood samples (see, e.g., U.S. Pat. No. 7,462,475) and include one or more internal PCR assay controls (see Rosenstruus et al, J.Clin Microbiol.36:191(1998) and Hoofar et al, J.Clin. Microbiol.42:1863 (2004)). For example, to ensure successful amplification and detection of a clinical sample, the assay may include an internal standard nucleic acid containing a primer binding region identical to the target sequence. As shown in the examples, the target nucleic acid and the internal control are selected such that they each have a unique probe binding region that distinguishes the internal standard from the target nucleic acid.

Kinetics of the reaction

The reaction of magnetic particles with a particular analyte to form aggregates can be used to generate a diagnostic signal in the assays of the invention. In many cases, the reaction mixture is incubated for a time sufficient to form aggregates. The methods, kits, cartridges, and devices of the invention may be constructed to reduce the amount of time required to capture a particular analyte, or to generate aggregates of magnetic particles. While changing the total concentration of magnetic particles would seem to be a simple and straightforward method to increase the aggregation rate, this method is complicated by the following reasons: (i) non-specific aggregation, which can increase with high magnetic particle concentrations, and (ii) the need to produce observable signal changes (i.e., changes in relaxation signals) in response to aggregation in the presence of low concentrations of analyte. The reaction kinetics may be improved, for example, by mechanically induced aggregation, by acoustically induced aggregation, by ultrasonically induced aggregation, by electrostatically induced aggregation, or by binding magnetic particles (e.g., by exposing nanoparticles to a magnetic field, using a porous membrane, using a magnetizable metal foam, or centrifugation) in a portion of the liquid sample.

NMR unit

A system for carrying out the method of the invention may comprise one or more NMR units. FIG. 1A is a schematic diagram 100 of an NMR system for detecting the signal response of a liquid sample to a suitable RF pulse sequence. The bias magnet 102 establishes a bias magnetic field Bb104 through the sample 106. The magnetic particles are in a liquid or lyophilized state in a cartridge until the liquid sample 106 is introduced into the well 108, prior to introduction of the sample well 108 (the term "well" as used herein includes any indentations, conduits, containers, or stents), or the magnetic particles may be added to the sample 106 prior to introducing the liquid sample into the well 108. The radio frequency coil 110 and the radio frequency oscillator 112 provide RF excitation at a Larmor frequency that is a linear function of the bias magnetic field Bb. In one embodiment, the radio frequency coil 110 is wrapped around the sample well 108. Excitation of RF causes an unbalanced distribution of spins of water protons (or free protons in a non-aqueous solvent). When the RF excitation is turned off, the protons "relax" to their initial state and emit an RF signal that can be used to extract information about the presence and concentration of the analyte. The coil 110 functions as an RF antenna and detects signals that probe different properties of the material, such as T, based on the applied RF pulse sequence ₂And (6) relaxation. For the case of some techniques, the signal of interest is spin-spin relaxation (typically 10-2000 milliseconds) and is referred to as T₂And (6) relaxation. The RF signal from the coil 110 is amplified 114 and processed to determine T in response to excitation in the bias magnetic field Bb₂(decay time). Well 108 may be a small capillary or other tube containing nanoliters to microliters of sample containing the analyteAnd a coil of appropriate size wound around it (see fig. 1B). The coil is typically wound around the sample and sized according to the sample volume. For example, but not limited to, for a sample having a volume of about 10 ml, a solenoid coil having a length of about 50 mm and a diameter of 10 to 20 mm may be used; for samples having a volume of about 40 μ l, a solenoid coil having a length of about 6 to 7 mm and a diameter of 3.5 to 4 mm may be used; for samples having a volume of about 0.1 nl, a solenoid coil having a length of about 20 μm and a diameter of about 10 μm may be used. Alternatively, the coil around or near the aperture may be constructed as shown in any of fig. 2A-2E. The NMR system may also contain multiple radio frequency coils for multi-purpose detection. In certain embodiments, the radio frequency coil has a conical shape with dimensions of 6 mm x 2 mm.

Fig. 2A-2E show exemplary micro NMR coil (radio frequency coil) designs. Fig. 2A shows a wound solenoid microcoil 200 of about 100 μm in length, however coils having lengths of 200 μm, 500 μm, or up to 1000 μm are contemplated. Fig. 2B shows a "planar" coil 202 having a diameter of about 1000 μm (the coil is not truly planar because the coil has a finite thickness). Fig. 2C shows a MEMS solenoid coil 204 defining a volume of approximately 0.02 μ L. Fig. 2D shows a schematic diagram of the MEMS Helmholz coil 206 configuration and fig. 2E shows a schematic diagram of the saddle coil 220 configuration.

Wound solenoid microcoils 200 for conventional NMR detection are described in Seeber et al, "Design and testing of high sensitivity micro-receiver coil apparatus for nuclear magnetic resonance and imaging," Ohio State University, Columbus, Ohio. Planar microcoils 202 for conventional NMR detection are described in Massin et al, "High Q factor RF planar for micro-scale NMR spectroscopy, Sensors and Actuators A97-98,280-288 (2002). Helmholtz coil construction 206 features a hole 208 for holding the sample, a top Si layer 210, a bottom Si layer 212, and a placed metal coil 214. An example of a Helmholtz coil configuration 206 for conventional NMR detection is described in Syms et al, "MEMS Helmholtz Coils for Magnetic Resonance Spectroscopy," Journal of Micromechanics and micromeching15 (2005) S1-S9.

The NMR unit includes a magnet (i.e., a superconducting magnet, an electromagnet, or a permanent magnet). The design of the magnet may be open or partially closed, ranging from a U-shaped or C-shaped magnet, to a magnet with three or four posts, to a fully enclosed magnet with a small opening for sample placement. The trade-off is the accessibility of the magnet and the "sweet spot" of mechanical stability (mechanical stability may be a problem in case a high field homogeneity is desired). For example, the NMR unit may include one or more permanent magnets of cylindrical shape and made of SmCo, NdFeB, or other low field permanent magnets that provide a magnetic field in the range of about 0.5 to about 1.5T (i.e., suitable SmCo and NdFeB permanent magnets are commercially available from Neomax, Osaka, Japan). For purposes of illustration and not limitation, such a permanent magnet may be a dipole/box Permanent Magnet (PM) assembly, or a Hallbach design (see Demas et al, Concepts Magn respon Part a 34A: 48 (2009)). The NMR unit may include, but is not limited to, a centrally located permanent magnet having a magnetic field uniformity of about 20-30 ppm and a strength of about 0.5T at the 40 μ L sweet spot. This magnetic field homogeneity allows for cheaper magnets to be used (smaller fine-tuning assembly/shimming), allows for the movement of ferromagnetic or conductive objects (these have less influence and therefore require less shielding) in stray fields without affecting the measurement of the assay (relaxation measurements and correlation measurements do not require fields of high homogeneity) in systems that are less subject to fluctuations (e.g. temperature drift, mechanical stability over time-indeed any influence is too small to be observed).

The coil configuration may be selected or adapted for implementation of the micro NMR-MRS technique, as different coil configurations provide different performance characteristics. For example, the geometry of each of these coils has different performance and field localization. The planar coil 202 has an RF field perpendicular to the plane of the coil. Solenoid coil 200 has an RF field that is directed down the coil axis, and Helmholtz coil 206 has an RF field that intersects the two rectangular coils 214. Helmholtz 206 and saddle coil 220 have a transverse magnetic field that will allow permanent magnet bias fields to be placed above and below the hole. Helmholtz coil 206 and saddle coil 220 may be the most efficient for chip design, while solenoid coil 200 may be the most efficient when the sample and MRS magnetic particles are contained in a microtube.

The micro NMR device may be fabricated by winding or printing a coil or by Micro Electro Mechanical System (MEMS) semiconductor fabrication techniques. For example, the diameter of the winding or printing coil/sample well module may be about 100 μm, or 1 cm or more. For example, a MEMS unit or chip (so named because it is fabricated in a semiconductor process as a die on a wafer) may have a coil with a feature size of about 10 μm to about 1000 μm. The wound or printed coil/sample hole configuration is referred to herein as a module, and the MEMS version is referred to herein as a chip. For example, the liquid sample 108 may be contained in a tube (e.g., a capillary tube, a pipette, or a microtube) having a coil wound thereon, or it may be contained in a well on a chip having a radio frequency coil around the well. Alternatively, the sample is positioned to flow through a tube, capillary, or cavity near the radio frequency coil.

The basic components of an NMR unit include electrical elements such as tuned RF circuits within the magnetic field, including the MR sensor, receiver and transmitter electronics (which may include preamplifiers, amplifiers and protection circuits), data acquisition components, a pulse programmer and a pulse generator.

A system containing an NMR unit with a radio frequency coil and a microwell containing the magnetic particle sensor described herein can be designed for detection and/or concentration measurement of a particular analyte(s) of interest by modeling the particle aggregation phenomenon and modeling the RF-NMR signal chain. For example, particle aggregation can be characterized by physical properties (including, e.g., affinity, relative size and concentration effects)Experiments were performed on the analyte/magnetic particle system (interesting). In addition, experiments can be performed to characterize the NMR signal(s) (T₂、T₁、T₂*、T_2rho、T_1rhoAnd/or other signal characteristics such as T1/T2 mixed signals, and may also include, but is not limited to, diffusion, sensitivity, frequency) as a function of particle aggregation or depletion and magnetic particle characteristics. Signal characteristics specific to MRS (magnetic resonance switching) phenomena in a given system may be used to increase detection sensitivity and/or improve performance.

The NMR system may include a chip having radio frequency coil(s) and micro-machined (micro-machined) electronics thereon. For example, a chip may be surface micromachined to build a structure on top of a substrate. In the case where the structure is built on top of the substrate rather than inside, the properties of the substrate are not as important as in bulk micromachining, and the expensive silicon wafers used in bulk micromachining can be replaced with less expensive materials such as glass or plastic. However, alternative embodiments may include bulk micromachined chips. Surface micromachining typically starts with a wafer or other substrate and grows multiple layers on top. These layers are selectively etched using photolithography and wet etching involving an acid or dry etching involving ionized gas or plasma. Dry etching may combine chemical etching with physical etching or ion bombardment of the material. The surface microfabrication may comprise a number of layers as desired.

In some cases, an inexpensive radio frequency coil may be integrated into the disposable cartridge and be a disposable component. The coil may be placed in a manner that allows electrical contact with circuitry on a fixed NMR setup, or may be inductively coupled to the circuitry.

When the relaxation measure is T₂In this case, accuracy and repeatability (precision) will be a function of the temperature stability of the sample with respect to: calibration, stability of the assay, Signal-to-noise ratio (S/N), pulse for refocusingThe burst sequence (e.g., CPMG, BIRD, Tango, etc.), and signal processing factors such as signal conditioning (e.g., amplification, conditioning, and/or digitization of the echo signals), time/frequency domain translation, and the signal processing algorithms employed. The signal-to-noise ratio is a function of the bias magnetic field (Bb), sample volume, fill factor, coil geometry, coil Q-factor (Q-factor), electronics bandwidth, amplifier noise, and temperature.

In order to understand the required T₂Precision of measurement, the response curve of the upcoming (at hand) assay should be observed and the desired precision in determining the analyte concentration is related to the measurable value (e.g., in some cases T₂Value) is correlated. Then, an appropriate error estimate (error budget) can be formed.

For example, to obtain a 10-fold improvement in the detection limit of 0.02 ng/mL of troponin (a 10-fold increase in sensitivity), a delta-T of less than about 5.6 milliseconds must be achieved₂From a conventional (non-MRS-measured) T of about 100 milliseconds ₂The values are distinguished. The minimum signal-to-noise ratio (S/N) needs to be about 20 to detect this difference.

The NMR unit used in the system and method of the invention may be that described in U.S. patent No. 7,564,245, the contents of which are incorporated herein by reference.

The NMR unit of the invention may comprise a small probe for a portable magnetic resonance relaxometer as described in PCT publication WO09/061481, which is incorporated herein by reference.

The system of the invention may be implantable or partially implantable in a subject. For example, the NMR unit of the invention may comprise an implantable radio frequency coil and optionally an implantable magnet as described in PCT publications WO09/085214 and WO08/057578, each of which is incorporated herein by reference.

The system of the present invention may comprise a polymeric sample container for partially or completely reducing the effect of NMR signals associated with the sample container on nuclear magnetic resonance parameters of the liquid sample, as described in PCT publication WO09/045354, the contents of which are incorporated herein by reference.

The system of the present invention may include a disposable cartridge for an MR reader configured to perform a predetermined number of measurements (i.e., designed for a limited number of uses). The disposable cartridge may include 0, some, or all of the elements in the RF detection coil (i.e., so that the MR reader lacks the detection coil). For example, the disposable cartridge may include a "read" coil for RF detection that is inductively coupled to a "pick-up" coil present in the MR reader. When the sample container is within the MR reader, it is in close proximity to the pick-up coil and can be used to measure the NMR signal. Alternatively, the disposable cartridge comprises a radio frequency coil for RF detection, which is electrically connected to the MR reader after insertion into the sample container. Thus, when the sample container is inserted into the MR reader, a suitable electrical connection is established to allow detection. The number of times each disposable cartridge can be used can be controlled by interrupting the fusible link included in the circuitry within the disposable cartridge or between the disposable cartridge and the MR reader. After a disposable sample holder is used to detect NMR relaxation in a sample, the instrument can be configured to apply an excessive amount of current to the fusable link, causing the link to break, rendering the coil inoperable. Optionally, multiple fusible links operating in parallel may be used, each connected to a pick-up on the system and each individually disconnected from each use until all are disconnected and the disposable sample rack is rendered inoperable.

Cartridge unit

A system for performing the method of the present invention may include one or more cartridge units to provide a convenient method for placing all assay reagents and consumables on the system. For example, the system may be customized to perform a particular function, or adapted to perform more than one function (e.g., by a variable cartridge unit containing an array of microwells having customized magnetic particles contained therein). The system may comprise a replaceable and/or interchangeable cartridge containing an array of magnetic particle pre-loaded wells and designed for detection and/or concentration measurement of specific analytes. Alternatively, the system may be used with different cartridges, each designed for detection and/or concentration measurement of different analytes, or configured with separate cartridge modules for reagents and detection for a given assay. The cartridge may be sized to facilitate insertion into and ejection from a housing for preparation of a liquid sample to be transferred to other units in the system (i.e., a magnetically assisted agglomeration unit, or an NMR unit). The cartridge unit itself could potentially interface directly with the operator station and the MR reader(s). The cartridge unit may be a modular cartridge with an inlet module that may be sterilized independently of the reagent module.

For the processing of biological samples (e.g., blood samples), there are many conflicting requirements in cartridge design, including the need for sterility of the inlet module to prevent cross-contamination and false positive test results, and the need for reagents included in packages that cannot be readily sterilized using standard terminal sterilization techniques (e.g., irradiation). The inlet module for sample specimen sampling may be designed to interface with uncapped single use vacuum blood collection tubes (vacutainers tubes), and to sample twice the volume of sample available for, e.g., candida assays (see fig. 7D-7F). Disposable vacuum blood collection tubes allow for partial or complete filling. The inlet module has two hard plastic parts that are ultrasonically welded together and foil sealed to form a network of channels, allowing a flow path to be formed from the first well to the second sample well. The soft disposable vacuum blood collection tube sealing member is a seal for a disposable vacuum blood collection tube, and includes a port through which a sample flows and an exhaust port. To overcome the flow resistance created by disposable evacuated blood collection tubes once loaded and inverted, some hydrostatic pressure is required. Each time a sample is removed from a sample well, the well will be replenished with effluent from a single-use evacuated blood collection tube.

Modular cartridges may provide a simple means for controlling cross-contamination during certain assays, including but not limited to dispensing PCR products into detection aliquots. In addition, the modular cartridge may be compatible with automated liquid dispensing and provides a method of holding very small volumes of reagents for long periods of time (over 1 year). Finally, pre-dispensing these reagents allows concentration and volume accuracy to be set by the manufacturing process and provides for more convenient instrument use as it requires pipetting with much less precision.

The modular cartridge of the present invention is a cartridge that is separated into modules that can be packaged and sterilized separately if desired. They may also be handled and stored separately if, for example, the reagent module requires refrigeration but the detection module does not. Fig. 5 shows a representative cartridge with an inlet module, a reagent module, and a detection module snapped together. In this embodiment, the inlet module is packaged separately in sterile packaging, and the reagent module and the detection module are pre-assembled and packaged together.

During storage, the reagent module may be stored in a refrigerator while the inlet module may be stored in a dry reservoir. This provides additional advantages: only a very small amount of refrigerator or freezer space is required to store many assays. In use, the operator will remove the detection module and open the packaging, if the assay is required, to prevent contamination by skin flora using aseptic techniques. Then, remove the disposable vacuum blood collection tube cap, and the inverted inlet module is placed on the blood collection tube, as shown in figure 6A. This module has been designed to be easily shaped with a single pulling tool (as shown in fig. 6B and 6C), and the top and bottom of the cartridge are sealed with foil to prevent contamination and close the channel. Once the tube is resealed with the inlet module, the right side of the assembly is up and snapped over the remainder of the cartridge. The access port includes a well with overflow that allows use of sample tubes with between 2 to 6ml of blood and still provides an iso-depth interface for system automation. This is accomplished by spillage, wherein blood that overflows the sampling hole falls only into the cartridge body, thereby preventing contamination.

Alternatively, the modular cartridge is designed for multiple tests. The challenge in multiplex assays is the combination of multiple assays with incompatible assay bottom requirements (i.e., different incubation times and/or temperatures) on one cartridge. The cartridge may be characterized by 2 main components: (i) a reagent module containing all individual reagents required for the full assay list, and (ii) a detection module. The detection module contains only the components of the cartridge that perform the incubation, and can perform one or several assays if desired. The detection module may comprise two detection chambers for one assay, the first detection chamber being a control and the second detection chamber being for a sample. This cartridge format can be expandable because other analytes can be added by including reagents and other detection modules.

Operation of the module begins when the user inserts all or a portion of the cartridge into the instrument. The instrument performs assay initiation, sampling of the assay into a separate detection chamber. The individual detection chambers are then separated from the test strips and from each other and run individually through the system. Because the reagent module is separated and discarded, there is a minimum possible sample unit passing through the instrument, thereby saving space inside the instrument. By dispensing each assay into its respective unit, different incubation times and temperatures are possible because each multiplex assay is physically separated from each other and each sample is handled separately.

The cartridge unit of the present invention may comprise one or more populations of magnetic particles, either as a liquid suspension or as dried magnetic particles to be reconstituted before use. For example, a cartridge unit of the present invention may comprise a cartridge containing 1 x 10⁶To 1X 10¹³A (e.g., 1 × 10)⁶To 1X 10⁸、1×10⁷To 1X 10⁹、1×10⁸To 1X 10¹⁰、1×10⁹To 1X 10¹¹、1×10¹⁰To 1X 10¹²、1×10¹¹To 1X 10¹³Or 1X 10⁷To 5X 10⁸Individual magnetic particles) for determining the magnetic particles of a single liquid sample.

System for controlling a power supply

A system for performing the method of the invention may comprise one or more NMR units, a kit unit, and a stirring unit (e.g., to disrupt non-specific magnetic particle interactions and to redistribute magnetic particles into a liquid sample, or to simply stir the sample tube to thoroughly mix assay reagents, e.g., sonicate, vortex, shake, or an ultrasonic station for mixing one or more liquid samples). Such a system may also include other components for performing the automated assays of the present invention, such as: a PCR unit for oligonucleotide detection; a centrifuge; a robotic arm for transporting the liquid sample between units within the system; one or more incubation units; a fluid transfer unit (i.e., a pipetting device) for mixing assay reagents with a biological sample to form a liquid sample; a computer having a programmable processor for storing data, processing data, and for controlling activation and deactivation of various units according to one or more preset schemes; and a cartridge insertion system for delivering the pre-filled cartridge to the system, optionally with instructions of a computer displaying the reagents and protocol to be used in conjunction with the cartridge.

The system of the present invention may provide an efficient means for high throughput and real-time detection of analytes present in a bodily fluid from a subject. The detection method can be used in a wide variety of situations including, but not limited to: identification and/or quantitative analysis of analytes associated with a particular biological process, physiological condition, disorder or stage of a disorder. Thus, these systems have broad applications in, for example, drug screening, disease diagnosis, pedigree classification, parental and forensic identification, disease onset and recurrence, individual response to treatment (as compared to population basis), and monitoring of treatment. The devices and systems are also particularly useful for accelerating preclinical and clinical phases of therapeutic drug development, improving patient compliance, monitoring ADR associated with prescription drugs, forming personalized medicine, home-to-home or prescription-based exotic device (outsouring) blood testing, and monitoring therapeutics according to regulatory approval. These devices and systems may provide a flexible system for individualized medical treatment. The system of the present invention may be varied or interchanged with instructions or schemes of the programmable processor of the system to perform a wide variety of assays as described herein. The system of the present invention provides many of the advantages of a laboratory setup contained in a bench top or smaller sized automated instrument.

The system of the invention can be used to simultaneously determine analytes present in the same liquid sample over a wide range of concentrations, and can be used to monitor the rate of change of analyte concentration and/or PD or PK marker concentration over a period of time in a single subject, or to perform a trend analysis of the concentrations or PD or PK markers (whether they are concentrations of drugs or their metabolites). For example, if glucose is the analyte of interest, the concentration of glucose in the sample at a given time and the rate of change of the glucose concentration over a given time period may be highly useful in predicting and avoiding, for example, hypoglycemic events. Thus, data generated using the fluidic devices and systems can be used to perform trend analysis of analyte concentrations in a subject. For example, a patient may be provided with multiple cartridge units for detecting multiple analytes at predetermined times. The subject may, for example, use different cartridge units on different days of the week. In some embodiments, software in the system is designed to recognize an identifier on the kit that instructs the system computer to run a particular protocol to run the assay and/or process the data. The protocol in the system may be updated via an external interface (e.g., a USB drive or ethernet connection) or, in some embodiments, the entire protocol may be recorded in a bar code connected to the cartridge. The protocol may be optimized by prompting the user for various inputs (i.e., to change the dilution of the sample, the amount of reagent provided to the liquid sample, change the incubation time or magnetically assisted aggregation time, or change the NMR relaxation acquisition parameters), as desired. In the case of a multiplex array configured for detection of target nucleic acids, the assay may include multiplex PCR to generate different amplicons, followed by serial detection of the different reactions. The multiplex assay optionally includes a logic array in which targets are set by a binary search to reduce the number of assays required (e.g., gram positive or negative results in different species-based detection to be performed for only one or the other set).

The system of the present invention can perform a variety of assays regardless of the analyte being detected from the bodily fluid sample. The protocol depending on the identification of the cartridge unit being used may be stored in the system computer. In some embodiments, the cartridge unit has an Identifier (ID) or barcode (1D or 2D) on the card that is detected or read by the system computer that is specific to the assay or patient or subject specific information that is subsequently needed to provide analytical information (e.g., calibration curve, protocol, previous analyte concentration or level) to track or access. The cartridge unit identifier is used to select a protocol stored in the system computer, or to confirm the location of various assay reagents in the cartridge unit, if desired. The protocol to be run on the system may include instructions for the controller that the system executes the protocol (including but not limited to the particular assay to be run and the detection method to be performed). Once the system has performed the assay, data indicative of the analyte in the biological sample is generated and transmitted to the communication assembly where it can be transmitted to an external device for processing (including, but not limited to, calculating the concentration of the analyte in the sample) or processed by the system computer and the results presented on a display readout device.

For example, the identifier may be a bar code identifier having a series of black and white lines that may be read by a bar code reader (or another type of detector) upon insertion into the cartridge unit. Other identifiers may be used, such as a series of alphanumeric values, colors, bumps, RFID, or any other identifier that may be located on the cartridge unit and detected or read by the system computer. The detector may also be an LED that emits light that can interact with an identifier of the reflected light and be measured by the system computer to determine the identity of a particular cartridge unit. In some embodiments, the system includes a storage or memory device having a cartridge unit or detector for transmitting information to a system computer.

Thus, the system of the present invention may include an operating program to perform different assays, and a cartridge that is coded to: (i) reporting to the operating program which pre-programmed assay to employ; (ii) reporting the configuration of the cartridge to an operating program; (iii) informing an operating system of the order of steps used to perform the assay; (iv) telling the system which preprogrammed routine to use; (v) prompting the user to input certain measured variables; (vi) recording a patient identification number (which may also be included on a single use evacuated blood collection tube containing a blood sample); (vii) record certain cartridge information (i.e., lot #, calibration data, measurements on the cartridge, analytical data ranges, expiration dates, storage requirements, characteristics of acceptable samples); or (viii) report assay upgrades or revisions to the operating program (i.e., so that updated versions of the assay will only appear on the cartridge upgrade rather than being reported to a larger, more expensive system).

The system of the present invention may include one or more fluid transfer units configured for attachment to a robotic arm. The fluid transfer unit may be a pipette, such as an air displacement, liquid withdrawal (liquid backed), or syringe pipette. For example, the fluid transfer unit may also include a motor coupled to a programmable processor of the system computer that can move the plurality of heads based on a protocol from the programmable processor. Thus, the programmable processor of the system may include instructions or commands and may operate the fluid transfer unit according to the instructions to transfer the liquid sample by retracting (for aspirating liquid) or extending (for expelling liquid) the piston to the closed gas chamber. For example, the volume and speed of movement of the moving air can be precisely controlled using a programmable processor. Mixing the sample (or reagent) with the diluent (or other reagent) can be achieved by pumping the components to be mixed into a common tube, and then pumping a large portion of the mixed liquid volume up and down into the tip repeatedly. The dried reagent can be dissolved in the tube in a similar manner.

The system may comprise one or more incubation units for heating the liquid sample and/or for controlling the assay temperature. Heat may be used to determine the incubation step of the reaction to facilitate the reaction and shorten the duration of the incubation step required. The system may include a heat block configured to receive the liquid sample at a predetermined temperature for a predetermined time. The heating block may be configured to receive a plurality of samples.

The system temperature can be carefully regulated. For example, the system includes a housing (casting) maintained at a predetermined temperature (i.e., 37 ℃) using stirred temperature-controlled air. The waste heat from each unit will exceed the heat that can be passively dissipated by a simple enclosure through conduction and convection to air. To eliminate waste heat, the system may include two chambers separated by an insulating floor. The upper chamber contains those parts of the components (components) required for the manipulation and measurement of liquid samples, while the lower chamber contains the heating elements of the individual cells (e.g., motor for centrifugation, motor for agitation of the cells, electronics for each individual cell, and heating block for incubation of the cells). The lower plate is then opened and forced air cooling is used to remove heat from the system.

The MR unit may require a more closely controlled temperature (e.g., ± 0.1 ℃) and may therefore optionally include a separate enclosure into which air heated at a predetermined temperature is blown. The housing may include an opening through which the liquid sample is inserted and removed and from which heated air is allowed to escape.

A full disclosure and description of how to implement the devices, systems, and methods described herein is provided to one of ordinary skill in the art by providing the following examples; these examples are intended to be purely illustrative of the invention and are not intended to limit the scope of what the inventors regard as their invention.

Example 1 preparation of coated particles

Briefly, 1 mg of essentially monodisperse carboxylated magnetic particles were washed and resuspended in 100. mu.l of activation buffer, 10mM MES. Mu.l of 10mg/ml 10kDa amino-dextran (Invitrogen) was added to the activation buffer and incubated for 5 minutes at room temperature on a rotator. To conjugate the carboxyl group to the amine on dextran, 30. mu.l of 10mg/ml 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride (EDC) was added and incubated for 2 hours at room temperature on a rotator. The particles were eluted from free dextran using magnetic separation (3 × 1 mL PBS) and then resuspended in 1 mL PBS. Mu.l of a 100 mM solution of Sulfo-NHS-biotin (Sulfo-NHS-biotin) (Invitrogen) was used to modify the amino groups on the dextran surface with biotin. After incubation for 30 minutes, the particles were washed 3 times with 1 ml of activation buffer. Next, 100. mu.l of protein block (protein block) of 0.5 mg/ml Bovine Serum Albumin (BSA) (Sigma) and 30. mu.l of 10mg/ml EDC (Sigma) were added and incubated overnight. The prepared particles were washed 3 times in 1 ml PBS and resuspended to the desired concentration.

For analyte detection, the prepared particles synthesized using this protocol have been shown at T ₂Similar results were obtained in the assay, regardless of whether the sample included buffer or 20% lysed blood (see fig. 14). The prepared variant, in which the pre-biotinylated aminodextran is directly conjugated to the particles in one step, also leads to T in blood and buffer samples₂Similar behavior of the assay.

Example 2 evaluation of particles prepared with and without protein blocking

Briefly, in an avidin titration T₂In the assay, biotin-modified amino-dextran magnetic particles prepared according to the method described in example 1 were assayed in PBS and 20% lysed blood samples.

The measurement was performed according to the following procedure. 50 μ L of matrix (PBS or 20% lysed blood sample), 50 μ L of varying concentrations of anti-biotin antibody, 50 μ L of 1.0 μ g/ml secondary antibody (secondary antibody) were added to a 5 mm NMR tube. Then, 150. mu.L of 0.02 mM Fe particles were added to each tube (i.e., 2.7X 10)⁸Particles/tube). These samples were then vortexed for 4 seconds and incubated in a 37 ℃ heat block for 2 minutes. Then, each sample was vortexed again for 4 seconds and incubated in a 37 ℃ heat block for an additional 1 minute. After incubation, each sample was placed in a Bruker Minispec for 10 minutes (under magnetic field). After 10 minutes, the sample was removed from the magnet, vortexed for 4 seconds, and incubated in a 37 ℃ heat block for 5 minutes. After 5 minutes, each sample was vortexed again and incubated in a 37 ℃ heat block for an additional 1 minute. T was obtained using the Bruker Minispec program and the following parameters ₂The value: 1, scanning; the gain is 75; tau is 0.25; 3500 echo chain; 4500 parts of total echo chain; and false echo (Dummy echo): 2. Δ T was calculated as follows₂The value: t is₂-(T₂)₀And these results are shown in fig. 14.

Synthetic particles with protein blocking AXN4 achieved almost the same performance in blood and buffer (fig. 14). The graph depicted in fig. 15 compares the particles prepared with (open circles) and without (filled circles) the protein blocking step. We have therefore found that protein blocking may be required in the blood matrix to achieve similar functions.

Other protein blockages, including but not limited to fish skin gelatin, have also been successful. Particles were prepared according to the above method except that Fish Skin Gelatin (FSG) was used instead of BSA as the protein blocking. The graph shown in FIG. 16 shows T₂Results of assays (as described above) the assays used antibody titrations with BSA blocked particles and comparisons with FSG. The data show little or no difference between the two protein blocking methods (see FIG. 16). However, BSA has proven to be a more reliable blocking.

Example 3 determination of the amount of dextran coating

It has been found that attempts to increase the dextran coating density on the particles reduces the functionality of the prepared particles in the blood. The preparation of particles described in example 1 above (showing nearly equal buffer/blood performance) used 10 x excess dextran based on a space-filling model to determine the amount of dextran included in the coating experiment. In an attempt to functionalize the particles with higher fidelity, increasing the dextran coating to 1000-10000 × excess dextran in the coating experiment produced particles with thicker dextran coatings, which produced a reduced response in blood compared to buffer. We conclude that medium density dextran with protein blocking is useful for producing T in the presence of blood samples ₂It may be desirable to have a particle coating that performs well in the assay (see fig. 17A and 17B).

Example 4 detection of Small molecule analytes in Whole blood samples

Buffer/analyte preparation: preparing a solution of 0.1 percent BSA and 0.1 percent Tween in 1 multiplied by PBS: 10 wt% Tween 20 solution. Briefly, solutions of Tween @in1X PBS were prepared. 500 mL of 0.2% Tween solutions were prepared by adding 10 mL of 10% Tween into 490 mL of 1X PBS. A 2 wt% BSA solution was prepared in 1x PBS solution. A0.2% solution of BSA solution was prepared by adding 50 mL of 2% BSA in PBS to 450 mL of 1 XPBS. The dilutions were combined to prepare a final volume of 1L and a final buffer concentration of a solution of 0.1% BSA, 0.1% Tween in 1. times. PBS.

PEG-FITC-biotin analyte: 100 μ l of a 0.5 mM solution was prepared from 1mM Tris HCl. Mu.l PEG FITC biotin was mixed with 40. mu.l 0.5 mM Tris HCl and incubated for 15 minutes at room temperature. After 15 minutes, 70. mu.l of PEG-FITC-biotin dissolved in 0.5 mM Tris HCl was added to 630. mu.l of 0.1% Tween @, to prepare 100. mu.M stock solutions. The stock solution was mixed vigorously by vortexing. 200 μ l of 100 μ M solution was added to 900 μ l of 0.1% Tween @tomake 20,000 nM analyte. 10-fold dilutions were made down to 0.02 nM.

The process is as follows:

mu.l of the appropriate analyte and 50. mu.l of the 1:5 lysed blood matrix were transferred directly into a 5 mm NMR tube using a pipette. The sample was vortexed for 4 seconds. Mu.l of primary anti-biotin antibody (0.18. mu.g/ml, diluted in 0.1% Tween 20, 0.1% BSA, 1 XPBS) was added, followed by incubation at 37 ℃ for 15 minutes. After 15 minutes, 50. mu.l of 3.0. mu.g/ml secondary anti-mouse antibody (diluted in 0.1% Tween, 0.1% BSA, 1 XPBS) and 150. mu.l of 0.02 mM Fe particles (2.7X 10)⁸Particles/tube) was added to the NMR tube. The sample was then vortexed for 4 seconds and incubated at 37 ℃ for 5 minutes. The sample was placed in a Bruker Minispec for 10 minutes (under magnetic field). After 10 minutes, the sample was removed from the magnet and incubated for a further l5 minutes. The sample was vortexed again for 4 seconds and incubated for an additional 1 minute. T was obtained using the Bruker Minispec program and the following parameters₂The value: 1, scanning; the gain is 75; tau is 0.25; echo chain: 3500, a table top; 4500 parts of total echo chain; and false echo: 2.

example 5: synthesis of antibody-modified particles

Aminodextran-coated magnetic particles prepared as described in example 1 can be further functionalized with antibodies via SMCC-SATA linkages (SMCC =4- [ N-maleimidomethyl ] cyclohexane-1-carboxylic acid succinimidyl ester; SATA = N-succinimidyl-S-acetylthioacetate). The carboxylated magnetic particles were first conjugated to 10kDa aminodextran using EDC chemistry as described above. The dextran-coated particles were further modified with an excess of sulfo-SMCC to provide maleimide functionality. Antibodies were modified with SATA linkers (amines that bind primarily to the antibody). SATA linkages are controlled to minimize over-functionalization of the antibody, which can result in cross-linking of the particles or a decrease in affinity of the antibody. Upon deacetylation, the SATA linker exposes a thiol functional group, which can be used to directly attach to maleimide functionalized particles (forming a thioether bond). The amount of antibody conjugated to each particle can be measured using the BCA protein assay (Pierce). Linkers that provide similar functionality to SATA, such as SPDP (N-succinimidyl 3- [ 2-pyridyldithio ] propionate), have been successfully used.

Antibody-coated magnetic particles can also be prepared using the chemistry described above, but using direct covalent bonding to the matrix carboxylated particles. In some cases, it may be desirable to add another coating (e.g., dextran or a blocking agent) to the particle surface. Similar chemistry can be used for aminodextran with alternative coatings (e.g., PEG or BSA).

Example 6 creatinine assay

Briefly, the assay comprises the steps of: the target sample is incubated in the presence of magnetic particles that have been modified with creatinine attached to the surface of the magnetic particles. The creatinine-modified magnetic particles are designed to aggregate in the presence of creatinine antibodies. Each creatinine-modified magnetic particle and creatinine antibody is added to a liquid sample containing creatinine that competes with the magnetic particle for the creatinine antibody. Thus, binding of creatinine to the antibody hinders aggregation of the magnetic particles, and low levels of creatinine are identified by the formation of aggregates. These aggregates alter the spin-spin relaxation rate and T of the sample when exposed to a magnetic field₂The change in relaxation time (the change in measured magnetic resonance signal from the surrounding water molecules) can be directly related to the presence and/or concentration of the analyte in the target sample.

Creatinine antibodies

In the antibody generation procedure to create creatinine, a modified creatinine molecule (COOH-creatinine) was designed and conjugated to transferrin for immunization of BALB-C and AJ mice.

34 clones were generated that produced stable antibodies. These clones were derived from BALB-C (splenocytes) (n =17) or AJ mice (n = 17). These two genetically distinct mouse strains were selected because their genetic differences in the immune system are known. Standards and selection methods were developed for screening and identifying the best monoclonal antibodies for use in this assay. Antibody selection methods include screening for binding to BSA-creatinine (by ELISA), antibody affinity/sensitivity/specificity (by ELISA competition assays using free creatinine and potential interferents), determining the ability of the antibody to conjugate to magnetic particles, and determination of the binding affinity of the antibody to magnetic particles at T₂Function in magnetic relaxation switch assays.

Using the established antibody selection criteria outlined above, seven monoclonal antibodies were identified and selected as potential candidates in the assay.

Creatinine-coated magnetic particles

The essentially monodisperse carboxylated magnetic particles were washed and resuspended in 100 μ l of conjugation buffer (50 mM MES, pH = 4.75). sulfo-NHS (55. mu. mol in 200. mu.l MES buffer) was added and the mixture was vortexed. To this mixture was added EDC (33.5. mu. mol in 200. mu.l MES buffer). The solution was briefly vortexed, placed on an upright cylinder mixer (end over end mixer) at room temperature for 1 hour, settled, and the supernatant removed. To the solid formed, 1 mL of 1% BSA dissolved in PBS was added, and the mixture was vortexed again and placed on an upright cylinder mixer at room temperature for 15-18 hours. The particles were allowed to settle and the supernatant removed.

BSA coated particles were suspended in 0.5 mL PBS-0.01% T20(10 mM phosphate buffer, pH =7.4, 150 mM NaCl, containing 0.01% Tween 20). Unreacted carboxyl groups were treated with methyl-PEG 4-amine (20. mu.l of a 10% v/v solution in DMSO) as a blocking agent. The mixture was vortexed and placed on a vertical cylinder mixer at room temperature for 8 hours. The resulting BSA coated particles were washed repeatedly with 0.5 mL PBS-0.01% T20.

COOH-creatinine (66. mu. mol), EDC (140. mu. mol) and NHS (260. mu. mol) were mixed with 300. mu.l of dry DMSO to form a slurry, which became clear at the end of the reaction. BSA-coated particles were suspended in 0.5 mL PBS-0.01% T20(pH =8), followed by addition of activated COOH-creatinine solution. The resulting mixture was vortexed and placed on a vertical cylinder mixer at room temperature for 4 hours. The particles formed were sonicated using 1:15 and 1:30 DMSO: PBS-0.01% T20 (vol/vol) was washed 3 times each. These particles were then washed 3 times with PBS-0.01% T20, respectively, under sonication. These particles were resuspended in PBS-0.1% T20(pH =8), and a solution of 2 mg NHS-PEG 2K in 200 μ l PBS-0.01% T20 was added. The mixture was placed on a vertical cylinder mixer at room temperature for 12-20 hours. These particles were then washed 3 times with PBS-0.01% T20, respectively, under sonication to generate creatinine-conjugated magnetic particles with sequential (sequential) BSA, creatinine coating, PEG cap (cap), and blocking.

These creatinine-coated particles were resuspended in assay buffer (100 mM glycine (pH =9.0), 150 mM NaCl, 1% BSA, 0.05% ProClin @, and 0.05% Tween @).

Creatinine assay protocol was performed using creatinine-conjugated particles and soluble creatinine antibodies, and generation/completion of utilizing T₂And (4) detecting the signal. The structure of the creatinine competition assay is shown in fig. 7A.

Where indicated, the solutions of magnetic particles, antibodies and liquid samples were diluted with assay buffer comprising 100mM Tris (pH =7.0), 800 mM NaCl, 1% BSA, 0.1% Tween @, and 0.05% ProClin @.

Creatinine-coated magnetic particles were diluted to 0.4 mM Fe (5.48X 10) in assay buffer⁹Particles/ml) were vortexed thoroughly and equilibrated at 4-8 ℃ for 24 h.

An anti-creatinine mouse monoclonal antibody (described above) was used as a multivalent binding agent for creatinine-conjugated magnetic particles. The antibody was diluted to a concentration of 0.8. mu.g/ml in assay buffer and vortexed thoroughly.

Each sample and calibrator (calibretors) were diluted as follows: 1 part of sample: 3 parts assay buffer. The upper limit of determination was about 4 mg/dL creatinine. For samples with predicted creatinine levels >4 mg/dL, additional sample dilutions were performed (using 1 initial diluted sample: 4 assay buffer).

Each of the pre-diluted sample, assay buffer, magnetic particles and antibody solution was vortexed. 10 μ L of each solution was added to the tube, and the tube was vortexed for 5 seconds.

The tubes were then magnetically assisted agglomerated in a gradient magnetic field for 12 minutes, incubated at 37 ℃ for 5 minutes, and placed in an MR reader (T)₂MR with 2200 Fluke temperature controller with NDxlient software 0.9.14.1/hardware version 0.4.13 Build 2, firmware version 0.4.13 Build 0) to measure the T of the sample₂Relaxation rate, and the T of the sample₂The relaxation rate was compared to a standard curve (see fig. 8A) to determine the concentration of creatinine in the liquid sample.

Properties of modified creatinine antibodies

Different creatinine antibodies were tested in this assay to determine the effect of the antibodies on aggregation. We observed that the performance of the creatinine antibodies when combined with creatinine-coated magnetic particles varied in their performance characteristics (see fig. 8B). SDS-PAGE gel analysis of 2 preparations showed a significant increase in aggregation in preparation 1, which is believed to be due to an increase in creatinine binding valency for this antibody, which aggregates due to its purification process. For comparison, we multimerized another creatinine monoclonal antibody (14HO3) by biotinylating the antibody and multimerizing the antibody in the presence of streptavidin. Monomeric, biotinylated monomeric and multimeric forms were then detected with creatinine-coated magnetic particles to evaluate Increased valence to T₂The effect of time. These results are shown in fig. 8C, which indicates that at much lower concentrations than non-multimerized antibodies, multimerized antibodies formed clusters. Increased valency of particle aggregation was also observed with IgM antibodies.

Example 7 magnetic particles coated with creatinine antibody

An alternative assay configuration is used, the assay comprising the steps of: subjecting a target sample to (i) magnetic particles that have been modified with a creatinine antibody; and (ii) incubating in the presence of a multivalent binding agent comprising a plurality of creatinine conjugates. The magnetic particles are designed to aggregate in the presence of multivalent binding agents, but aggregation is inhibited by competition with creatinine in the liquid sample. Thus, binding of creatinine to antibody-coated particles prevents aggregation of magnetic particles in the presence of multivalent binders, and low levels of creatinine are identified by the formation of aggregates. These aggregates alter the spin-spin relaxation rate of the sample when exposed to a magnetic field, and T₂The change in relaxation time (the change in measured magnetic resonance signal from the surrounding water molecules) can be directly related to the presence and/or concentration of the analyte in the target sample.

The essentially monodisperse carboxylated magnetic particles were washed, resuspended in 300 μ l conjugation buffer (50 mM MES, pH =4.75) and sulfo-NHS (46 μmol) EDC (25 μmol) was added to these particles. The solution was briefly vortexed and placed on a vertical cylinder mixer at room temperature for 1 hour. Activated particles were washed with mL PBS-0.01% T20 and resuspended in 1 mL 10% (w/v) amine-PEG-amine dissolved in PBS-0.01% T20. The mixture was vortexed and placed on an upright drum mixer at room temperature for 2 hours, then washed 3 times with PBS-0.01% T20.

As an alternative chemistry, BSA may replace amine-PEG-amine. BSA-coated magnetic particles were prepared in the manner described in the section describing creatinine-coated magnetic particles in example 6.

These particles were resuspended in 260. mu.l PBS-0.01% T20 and reacted with 198. mu.l sulfoSMCC (5 mg/mL solution in PBS-0.01% T20). The solution was briefly vortexed and placed on a vertical cylinder mixer at room temperature for 1 hour, then washed 3 times with 10mM EDTA in PBS-0.01% T20 to make SMCC coated particles.

SATA-tagged antibodies were prepared by mixing SATA (30 nmol in DMSO) with antibody (2nmol in PBS, pH = 7.4). The solution was placed on a vertical cylinder mixer at room temperature for 1 hour. Prior to use, the blocked thiol group on the SATA-tagged antibody was deprotected as follows: the treatment was performed with deacetylation buffer (0.5M hydroxylamine hydrochloride (pH =7.4), 10mM phosphate, 150 mM sodium chloride, 10mM EDTA) for 1 hour, and purification was performed through a desalting column using PBS containing 10mM EDTA.

As an alternative to SATA, SPDP-labeled antibodies may be used. SPDP-labeled antibodies were prepared by adding SPDP (10mmol in DMSO) and antibody (2nmol in PBS, pH = 7.4). The solution was incubated at room temperature for 1 hour and purified by passing through a desalting column. The disulfide bonds of SPDP on the SPDP-labeled antibody were cleaved in the reaction using 5mM mercaptoethylamine and incubated for 10 minutes at ambient temperature. Before use, the disulfide-cleaved SPDP-labeled antibody is purified by passing through a desalting column.

The PEG-or BSA-coated SMCC-functionalized particles were mixed with deacetylated SATA-modified antibody, placed on an upright cylinder mixer overnight at room temperature, washed 3 times with PBS-0.05% Tween 80, and resuspended in PBS-0.01% T20 containing 10mM EDTA. Adding a blocking agent (m-PEG-SH 2K), placing the solution on an upright cylinder mixer for 2 hours, washing the solution for 2 times with PBS-0.05 percent Tween 80, and suspending the solution in PBS-0.05 percent Tween 80, 1 percent BSA and 0.05 percent ProClin to prepare the antibody-coated magnetic particles.

SMCC-functionalized BSA coated particles were mixed with disulfide-cleaved SPDP labeled antibody and placed on a vertical cylinder mixer for 2 hours at room temperature, washed 2 times with PBS-0.01% Tween 20, 10mM EDTA, and resuspended in PBS, 0.01% T20, and 10mM EDTA. The blocking agent m-PEG-SH 2K (1 micromolar) was added and the solution was placed on a vertical cylinder mixer for 2 hours. The second capping reagent, N-ethylmaleimide (5. mu. mol), was added. The particles were mixed for 15 minutes, washed twice with PBS-0.01% Tween 20, resuspended in 100mM Tris, 0.05% Tween 80, 1% BSA and 0.05% ProClin with pH =9 to make antibody coated magnetic particles.

The procedure outlined above can be used for creatinine antibodies, or creatinine antibodies can be conjugated directly to the surface of the carboxylated magnetic particles using EDC conjugation.

Creatinine multivalent binding agent

COOH-creatinine was conjugated to 3 amino-dextran compounds (Invitrogen company; dextran with molecular weights of 10k, 40k and 70k, having 6.5, 12 and 24 amino groups per molecule, respectively) and BSA by EDC conjugation. The BSA-creatinine and amino-dextran-creatinine multivalent binding agents formed were used in the competitive inhibition assay described above. A degree of substitution of 2-30 creatinine per dextran moiety was achieved. An example of a creatinine inhibition curve is shown in fig. 10. The binding agent used was 40kDa dextran with about 10 creatinine per dextran molecule.

EXAMPLE 8 preparation of tacrolimus multivalent binding agent

Tacrolimus conjugates were prepared using dextran and BSA. The olefin metathesis (olefin metathesis reaction) of FK-506 was carried out using Grubbs second generation catalyst in the presence of 4-vinylbenzoic acid as shown in scheme 1 below. The crude product mixture was purified by normal phase silica gel chromatography.

Dextran conjugates

Three different molecular weights of amino-dextran each with different amino substitutions were used to prepare dextran-tacrolimus conjugates.

2.78 mL of EDC solution (40 mg/mL EDC hydrochloride) was mixed with 2.78 mL of sulfo-NHS solution (64 mg/mL sulfo-NHS) with stirring. To this mixture was added 0.96 mL of a solution of tacrolimus-acid derivative (C21) (28.8 mg/mL in DMSO), and the contents were stirred at room temperature for 30 minutes to form an activated tacrolimus-acid derivative (activated Tac solution 4.6 mM). The activated tacrolimus is used immediately.

Each amino-dextran polymer was dissolved in 100 mM sodium phosphate buffer (pH =8.0) to prepare a 9.5 mg/mL stock solution.

The activated Tac solution was added dropwise to the stock solution of amino-dextran at the ratio listed in the table below with stirring at room temperature. Each reaction was stirred vigorously for at least 2 hours.

TABLE 4

Reaction of	Molecular weight of aminodextran	Ratio of amine to Tac	Aminodextran (μ L)	Volume of Tac (μ L)	Estimated Tac glucan molar ratio
						1	70K	1:0.2	1000	70.8	Not detected
2	70K	1:0.4	1000	141.6	Not detected
						3	70K	1:0.8	1000	283.2	4.1
4	70K	1:1.6	1000	566.4	Not detected
						5	70K	1:3.2	1000	1132.8	Not detected
6	70K	1:5	1000	1770	15.8
						7	10K	1:0.8	1000	283	1.0
8	10K	1:5	1000	1766	2.2
						9	40K	1:0.8	1000	287	2.0
10	40K	1:5	1000	1793	8.2

The formed Tac-dextran conjugate was purified by 5-step series dialysis of each reaction product (1 st, 15% (v/v) aqueous DMSO solution; 2 nd, 10% (v/v) aqueous methanol solution; 3 rd to 5 th, high purity water; each step for at least 2 hours; 10K molecular weight amino-dextran using 3,500 MWCO dialysis membrane, and 40K and 70K amino-dextran using 7K MWCO dialysis membrane).

After purification, each sample was lyophilized and the dry weight was determined. The multivalent binding agent is reconstituted prior to use.

After reconstitution, the tacrolimus substitution ratio was estimated based on the absorbance at 254 nm.

Experiments were performed to determine what size of dextran provides the best agglomeration performance. Briefly, 10 μ L of 10% MeOH, 1% BSA dissolved in PBS (pH =6.3) buffer, 20 μ L of dextran Tac aggregates, 10K, 40K, 70K (at different concentrations), and 10 μ L of anti-tacrolimus coated magnetic particles (0.2 mM Fe) were added to a 200 μ L PCR tube (2.7 × 10 Fe)⁹Particles/tube). Vortex the sample with a plate mixer at 2000 rpm for 2 minutes, preheat at 37 ℃ for 15 minutes in an incubation station, expose to side and bottom magnets for 1 minute each, repeat 6 cycles, vortex again at 2000 rpm for 2 minutes, incubate for 5 minutes in a 37 ℃ incubator containing a heating block of the PCR tube design, and read the T in an MR reader₂. The data indicate that changes in the molecular weight gain/substitution ratio of dextran Tac may result in improved T₂The signal (see fig. 11). In addition, higher substitutions also resulted in improved response (see fig. 12).

BSA conjugates

BSA-tacrolimus conjugates were prepared at different degrees of tacrolimus substitution.

34.5 μ L of NHS solution (66.664 mg/mL in acetonitrile) was mixed with 552 μ L of EDC (6.481 mg/mL in 50 mM MES (pH = 4.7) with stirring. mu.L of this EDC NHS mixture was added dropwise to 220.8. mu.L of a solution of tacrolimus-acid derivative (C21) (33.33 mg/mL in acetonitrile), and the contents were stirred at room temperature for 1 hour to form an activated tacrolimus-acid derivative. The activated tacrolimus is used immediately.

BSA was dissolved in phosphate buffered saline and acetonitrile to form a solution containing 5mg/mL BSA in 40% acetonitrile.

The activated Tac solution was added dropwise to the BSA solution at the ratios listed in the table below with stirring at room temperature. Each reaction was stirred vigorously for at least 2 hours.

TABLE 5

Reaction of	Ratio of Tac to BSA	BSA(μL)	Volume of Tac (μ L)
				1	5:1	1000	35
2	10:1	1000	70
				3	20:1	1000	140
4	30:1	1000	210
				5	50:1	1000	350

The Tac-BSA conjugate formed was purified using a PD10 size exclusion chromatography column pre-equilibrated with 40% acetonitrile. The eluate was collected in 1 mL fractions and the absorbance at 280 nm was monitored to identify BSA containing fractions.

The BSA containing fractions were combined and acetonitrile was removed under vacuum.

The aggregation capacity of the Tac-BSA conjugate was evaluated by performing a titration in a manner similar to that used for the dextran-tacrolimus conjugate. As observed, the agglomeration performance varied with the Tac substitution ratio (see FIG. 13).

Example 9 tacrolimus competitive assay protocol (structure of antibody on particle).

Tacrolimus assays using anti-tacrolimus antibody conjugated particles and BSA-tacrolimus multivalent binders were developed and detected using an MR reader (see example 6). This assay is designed to detect a whole blood sample that has been extracted to release tacrolimus from red blood cells and binding proteins (extraction of a hydrophobic analyte from a sample can be achieved, for example, using the method described in us patent 5,135,875). The structure of the tacrolimus competition assay is depicted in fig. 7B.

Where indicated, the solutions of magnetic particles and multivalent binder were diluted with assay buffer containing 100 mM glycine (pH =9), 0.05% Tween 80, 1% BSA, 150 mM NaCl, 0.1% CHAPS.

Using sequential aminated coatings (PEG or BSA), antibody covalent attachment, PEG caps and PEG/protein blocking (as described in the examples above)Modifying the base particles having COOH functional groups. Antibody-coated magnetic particles were diluted to 0.4 mM Fe (5.48X 10) in assay buffer⁹Particles/ml) and vortexed thoroughly.

Multivalent binders were formed from COOH-modified tacrolimus covalently conjugated to BSA (as described in example 8). The multivalent binding agent was diluted to 0.02 μ g/ml in assay buffer and vortexed thoroughly.

The extracted sample solution (10. mu.L) was mixed with the magnetic particle solution (10. mu.L), vortexed for 5 seconds, and incubated at 37 ℃ for 15 minutes. To this mixture was added 20 μ L of multivalent binding agent, and the resulting mixture was vortexed for 5 seconds and incubated at 37 ℃ for 5 minutes.

Several samples were prepared as described above. All samples were subjected to magnetic assisted agglomeration for 1 minute in a gradient magnetic field. All samples were then placed in a pan removed from the magnetic field. Each sample was vortexed for at least 5 seconds and returned to the pan. Again, magnetic assisted agglomeration was performed for 1 minute on all samples followed by vortexing. This process was repeated 12 times for each sample.

The sample was incubated at 37 ℃ for 5 minutes and placed in an MR reader (see example 6) to measure the T of the sample₂Relaxation rate and the T of the sample₂The relaxation rate was compared to a standard curve (see fig. 9) to determine the tacrolimus concentration in the liquid sample.

Example 10 Candida assay

In assays for candida, magnetic particles of two pools (pools) were used for detection of each candida species. In the first library, species-specific capture oligonucleotide probes are conjugated to magnetic particles. In the second library, another species-specific capture oligonucleotide probe is conjugated to a magnetic particle. After hybridization, the two particles will hybridize to two different species-specific sequences separated by about 10 to 100 nucleotides within the sense strand of the target nucleic acid. (alternatively, two capture oligonucleotides may be conjugated to particles of a single pool, thereby forming separate particles with specificity for both the first and second regions). The oligonucleotide-modified magnetic particles are designed to aggregate in the presence of nucleic acid molecules from a particular candida species. Thus, unlike inhibition assays for creatinine and tacrolimus, candida assays are characterized by an increase in aggregation in the presence of a targeted candida nucleic acid molecule. The structure of the hybridization-mediated aggregation assay is shown in fig. 7C.

Carboxylated magnetic particles were used for candida assays. Magnetic particles are conjugated to oligonucleotide capture probes to form oligonucleotide-particle conjugates. For each target amplicon, two populations of oligonucleotide-particle conjugates were prepared. Oligonucleotide-particle conjugates are prepared using standard EDC chemistry between aminated oligonucleotides and carboxylated particles, or optionally by conjugating biotin-TEG modified oligonucleotides to streptavidin particles. The conjugation reaction is typically carried out at a particle concentration of 1% solids.

After conjugation, functionalized oligonucleotide density was measured by the following method: hybridizing Cy 5-labeled complement to the particles, washing the particles three times to remove non-hybridized oligonucleotides; elution was carried out by heating to 95 ℃ for 5 minutes. The amount of Cy 5-labeled oligonucleotide was quantitatively analyzed by fluorescence spectroscopy.

The conjugation reaction was carried out overnight at 37 ℃ under continuous mixing conditions using a rocker or roller (roller). (ii) contacting the formed particle conjugate: washing twice with 1 reaction volume of Millipore water; washing twice (at 37 ℃ for 5 min) with 1 reaction volume of 0.1M imidazole (pH 6.0); three washes with 1 reaction volume of 0.1M sodium bicarbonate (5 min at 37 ℃); then, two washes (30 min at 65 ℃) with 1 reaction volume of 0.1M sodium bicarbonate were performed. The particle conjugates formed were stored at 1% solids in TE (pH =8), 0.1% Tween @ 20.

The list of detected candida species includes candida albicans, candida glabrata, candida krusei, candida tropicalis, and candida parapsilosis. The sequences were amplified using universal primers that recognize highly conserved sequences within candida. The capture oligonucleotide is designed to recognize and hybridize to a species-specific region within the amplicon.

First, an aliquot of a blood sample is lysed as follows:

(i) the whole blood sample is mixed with an excess (1.25 ×, 1.5 ×, or 2 ×) volume of ammonium chloride hypotonic lysis solution. Addition of the lysis solution destroys all RBCs, but not WBCs, yeast or bacterial cells. The material of the cells was centrifuged at 9000 rpm for 5 minutes and the lysate was removed. Reconstitute intact cells with 100 μ Ι TE (tris EDTA, pH =8) to a final volume of approximately 100 μ Ι; and

(ii) to approximately 100. mu.l of sample 120 mg of 0.5 mm beads were added. The sample was stirred at approximately 3K rpm for 3 minutes, thereby forming a lysate.

Then, by adding the lysate to the inclusion nucleotides; buffer (5 mM (NH)₄)SO₄、3.5 mM MgCl₂6% glycerol, 60 mM zwitterionic buffer (pH =8.7) (at 25 ℃); primers (forward primer 4 × excess (300 mM forward; 0.75mM reverse) to allow formation of asymmetric single strands in the final product); and a thermostable polymerase (HemoKlenaTaq (New England Biolabs)) and an aliquot of approximately 50. mu.l of the lysate is PCR amplified. After an initial incubation of 3 minutes at 95 ℃, the mixture was subjected to PCR cycles: annealing at 62 ℃; stretching at 68 ℃; and the circulation is carried out for 95-40 times. Note that: there is a 6 ℃ difference in annealing and extension temperatures; annealing and extension can be combined into a single step to reduce the total amplification-out reporting time.

The PCR amplicons now ready for detection are mixed with the two populations of particles in a sandwich assay.

PCR primers and capture probes useful for candida assays are provided in table 6 below.

TABLE 6

PCR primer
		Pan Candida-PCR forward primer	GGC ATG CCT GTT TGA GCG TC(SEQ ID NO.1)
Pan Candida-PCR reverse primer	GCT TAT TGA TAT GCT TAA GTT CAG CGG GT(SEQ ID NO.2)
		Capture probe
Candida albicans probe #1	ACC CAG CGG TTT GAG GGA GAA AC(SEQ ID NO.3)
		Candida albicans probe #2	AAA GTT TGA AGA TAT ACG TGG TGG ACG TTA(SEQ ID NO. 4)
Candida krusei Probe #1	CGC ACG CGC AAG ATG GAA ACG(SEQ ID NO.5)
		Candida krusei probe #2	AAG TTC AGC GGG TAT TCC TAC CT(SEQ ID NO.6)
Candida krusei probe	AGC TTT TTG TTG TCT CGC AAC ACT CGC(SEQ ID NO.15)
		Candida glabrata Probe #1	CTA CCA AAC ACA ATG TGT TTG AGA AG(SEQ ID NO.7)
Candida glabrata Probe #2	CCT GAT TTG AGG TCA AAC TTA AAG ACG TCT G(SEQ ID NO.8)
		Candida parapsilosis/Candida tropicalis Probe #1	AGT CCT ACC TGA TTT GAG GTC NitInd¹AA(SEQ ID NO.9)
Candida parapsilosis/Candida tropicalis Probe #2	CCG NitInd¹GG GTT TGA GGG AGA AAT(SEQ ID NO.10)
		Candida tropicalis	AAA GTT ATG AAATAA ATT GTG GTG GCC ACT AGC(SEQ ID NO.16)
Candida tropicalis	ACC CGG GGGTTT GAG GGA GAA A(SEQ ID NO.17)
		Candida parapsilosis	AGT CCT ACC TGA TTT GAG GTC GAA (SEQ ID NO.18)
Candida parapsilosis	CCG AGG GTT TGA GGG AGA AAT (SEQ ID NO.19)
		Inhibition control 5'	GG AAT AAT ACG CCG ACC AGC TTG CAC TA (SEQ ID NO. 20)
Inhibition control 3'	GGT TGT CGA AGG ATC TAT TTC AGT ATG ATG CAG(SEQ ID NO. 21)

1. NitInd is a 5', 5-nitroindole, a base that can anneal with any of the 4 DNA bases

2. Note that the oligonucleotide Ts is added as a spacer.

Optionally, the assay is performed in the presence of a control sequence and magnetic particles modified with probes (for confirming the presence of the control sequence).

Example 11 non-agglomeration process.

This method has been demonstrated using amino silane (aminosilane) -treated nickel metal foam modified with anti-creatinine antibodies with 400 μm pores and shown to specifically bind creatinine-derivatized magnetic particles. Washing 1 cm by incubation in 2M HCL for 1 hour at room temperature ²The piece of nickel metal foam (Recemat RCM-Ni-4753.016) of (a) was thoroughly washed with deionized water and dried at 100 ℃ for 2 hours. The nickel foam was then treated with a solution of 2% 3-aminopropyltriethoxysilane in acetone overnight at room temperature. Then, the nickel metal foam was sufficiently washed with deionized water and dried at 100 ℃ for 2 hours. The amino silane treated nickel metal foam was treated with a 2% solution of glutaraldehyde in water at room temperature for 2 hours and washed thoroughly with deionized water. Next, the metal foam was exposed to 100. mu.g/ml anti-creatinine antibody (14H03) (see example 6) in PBS overnight, washed extensively with PBS, and treated with Surmodics Stabilguard to stabilize and block non-specific binding. Carefully without damaging the foam structureNext, 2mm was cut with a new razor²A block of derivatized metal foam of (a). A piece of derivatized metal foam was placed into 20. mu.l of assay buffer (100 mM glycine (pH =9.0), 150 mM NaCl, 1% BSA, 0.05% ProClin @, and 0.05% Tween @) in a PCR tube. Add 20. mu.l of control particles (0.2mM Fe) (not to be bound to metal foam ABX1-11) to the tube to a final volume of 40 ul and a final particle concentration of 0.1 mM Fe (1X 10) ⁶To 1X 10⁸Particles/tube). Separate PCR tubes with only particles and buffer but no metal foam were also prepared. The PCR tube containing the derivatized metal foam and control particles was subjected to magnetic assisted agglomeration for 1 minute in a gradient magnetic field, then contacted with a handheld demagnetizer for another 1 minute, removed from contact with the handheld demagnetizer, subjected to magnetic assisted agglomeration for another 1 minute, and vortexed (three magnetic exposures for 1 minute). Mu.l of sample was removed from both PCR tubes, heated to 37 ℃ in a grant block heater (grant block heater) for 5 minutes and read out for T using an MR reader₂Values (see example 6). T from sample (no foam)₂The read-out was 39.2 and the sample read-out from the foam-containing PCR tube was 45.1, showing a low level of particle consumption due to NSB. The derivatized metal foam was demagnetized, vortexed, and washed in assay buffer. At a final particle concentration of 0.1m MFe. They were placed in a new PCR tube (final particle concentration 0.1 mMFe) with 20. mu.l of assay buffer and 20. mu.l of AACr2-3-4 particles derivatized with creatinine. Pairs of PCR tubes without derivatized metal foam were also set up in control experiments. The PCR tube with metal foam was cycled twice by magnetic assisted agglomeration only as a control experiment (three 1 minute exposures with demagnetization after each exposure, final vortexing). Samples of 30. mu.l were removed from both tubes, heated to 37 ℃ for 5 minutes and then read in an MR reader. The sample readout from the PCR tube with derivatized metal foam was 41.5 and the sample readout from the PCR tube with derivatized metal foam with anti-creatinine antibody was 324.2, thus showing a specific junction of the appropriate creatinine-derivatized magnetic particles from the water volume read by the MR reader Make/consume.

Example 12 detection of Single nucleotide polymorphisms

There are many whereby T₂Measuring the method which can detect single nucleotide polymorphism.

The simplest application would include the discrimination of mismatches using thermophilic DNA ligase (Tth ligase). This assay would require lysis of the sample material followed by DNA shearing. Adapters may be ligated to the sheared DNA if universal amplification of genomic DNA is desired. SNPs will be detected using engineered superparamagnetic particle-bound capture probes flanking the SNPs such that the 5 'end of the 3' aminated capture probe will be fully complementary to one specific SNP allele, and subsequent treatment with Tth ligase will result in ligation of the two particle-bound capture probes. Thus, the connection will lock the particles into a conglomerate state. In cases where genomic DNA amplification is not desired (due to the risk of amplification bias), repeated cycles of melting (melt) hybridization will result in signal amplification. The same 5 ' aminated capture probe can be used in all cases, while 3 ' aminated probes can be formed to generate 4 different libraries (A, G, C, or T) at the 5 ' end. Detection will require that the sample be divided into 4 pools to determine which nucleotide(s) are present at the polymorphic site within a particular individual. For example, strong T in G detector tubes ₂Switches (switch) would only indicate that the individual is homozygous for G at a particular sequence position, while switches at G and A would indicate that the individual is heterozygous for G and A at that particular SNP site. The advantage of this approach is that Tth polymerase has been shown to have superior discrimination even in discriminating G-T mismatches (specific allowed mismatches and also most common) (1: 200 fold (relative to the correct complement)). Although the restriction of the nucleotide sequence of a known polymorphic site has been performed in the past by using a ligase detection reaction and an oligonucleotide ligase assay, amplification is required before or after the ligation reaction; as specified hereinIn the examples, the increase in size of the formed agglomerate particle complexes and thus the measured relaxation time (T) may be caused by a linking reaction₂) To amplify the signal.

A modification of this procedure may include hybridization of particle-bound capture probes (which flank hybridization of biotinylated probes). When a perfectly complementary duplex is formed by hybridization of the particle-bound probe, the ligase will covalently bind the biotin probe to the magnetic particle. Repeating the cycle of thermal denaturation again, and subsequent annealing and ligation, should result in a high proportion of long biotinylated oligonucleotides on the surface of the magnetic particles. A wash will be performed to remove any free probes followed by the addition of a second streptavidin-labeled superparamagnetic particle. Only when the biotinylated probe is attached to the surface of the first particle does the agglomeration occur.

Hybridization discrimination methods may also be employed. In this example, aminated oligonucleotide complements adjacent to known SNPs will be generated. These aminated oligonucleotides can be used to derivatize the surface of 96-well plates (1 SNP detection reaction per well). The genomic DNA is then sheared, ligated to adaptors, and amplified asymmetrically. This amplified genomic DNA was then applied to an array and short biotinylated SNP detection probes. The amplified genomic DNA will hybridize to the well-bound capture probes and then the SNP detection probes will bind to the tethered genomic DNA. Washing was performed to remove free SNP detection probes. Streptavidin (SA) magnetic particles were then added to each well. To remove free SA particles, a further wash would be required. T is₂Detection can be performed directly in the wells by adding biotinylated superparamagnetic particles to produce surface-tethered agglomerated particles, or SA magnetic particles can be eluted from each well on the array and incubated in a detection reaction using biotinylated magnetic particles.

Finally, a primer extension reaction can be coupled to T₂Detection to identify which nucleotide is present at the polymorphic site. In this assay, a pool of dideoxynucleotides, one of which Nucleotide/library with biotin (i.e., ddA, ddT, dd biotin-C, and/or ddG). Superparamagnetic particles carrying a capture probe whose last base is adjacent to the SNP after hybridization will be used.

Sheared genomic DNA was isolated and incubated in four separate primer extension reactions. The exoDNA polymerase will then catalyze the addition of dideoxy complementary to the nucleotides present in the SNP. If a thermophilic polymerase is used to ensure that most of the capture probes on the particles will be extended, the reaction can be cycled again. Magnetic separation will be performed, followed by washing of the particles and incubation with streptavidin superparamagnetic particles. Aggregation will then occur, which is proportional to the extent of the biotinylated capture probes on the surface of the first particles. If two dideoxy pools produce T₂Gain in (i.e., promoting particle agglomeration), then the patient will be heterozygous. If only one bank generates an increased T₂Then the patient will be homozygous.

The last method for detecting SNPs employs allele-specific PCR primers, wherein the 3' end of the primer contains the SNP. Because stringent amplification conditions are used, PCR amplification will be affected, producing little or no product if the target sequence is not perfectly complementary to the primer. In general, multiple forward primers (one perfectly complementary to each allele) and a single reverse primer will be designed. Amplicons were detected as follows: two or more capture probe-bound superparamagnetic particles are utilized to cause a hybridization-based agglomeration reaction. One advantage of this method is that it utilizes T on PCR in crude samples ₂Part of the work that has been done, and will only produce primers designed to include known SNPs. One disadvantage of this method is that newly generated SNP positions cannot be determined.

Another method that can be employed is relying solely on the discrimination ability of particle-particle cross-links due to hybridization to short nucleic acid targets. Mismatches in base pairs of oligonucleotides have been shown to significantly alter the aggregation state of particles and measured T₂Signal (due to single base)Reduced hybridization efficiency due to the presence of group mismatches).

Example 13 diagnosis of Candida

The test was performed over a 45 day time course. Candida albicans and Candida krusei reference strains, as well as Candida albicans clinical isolates, were cultured and maintained for the duration of the study.

Materials:

candida albicans and candida krusei nanoparticles: for each species, two populations of particles were generated, these particles being covalently conjugated to oligonucleotides complementary to species-specific sequences within the ITS2 region (see example 10). These particles were stored at 4-8 ℃ in TE (pH =8), 0.1% Tween, and diluted to 0.097 mM Fe in DNA hybridization buffer immediately prior to use.

Candida strains: the list was performed using candida albicans reference strain MYA 2876(GenBank FN652297.1), candida krusei reference strain 24210(GenBank AY939808.1) and candida albicans clinical isolates. The five Candida albicans isolates used were cultured in YPD at room temperature. Individual colonies were selected, washed 3 times with PBS, and then quantified using a hemocytometer to prepare whole blood spikes (whole blood spikes). These samples were stored as frozen glycerol stocks at-80 ℃.

Human whole blood: whole blood was collected from healthy donors, treated with K2EDTA, and spiked with washed serially diluted candida cells (at concentrations ranging from 1E5 to 5 cells/mL). Cell spiking prepared in fresh blood was stored at-20 ℃.

Erythrocyte lysis buffer: hypotonic lysis buffer containing 10 mM potassium bicarbonate, 155 mM ammonium chloride and 0.1 mM EDTA was filter sterilized and stored at room temperature prior to use. Alternatively, a red blood cell lysing agent, such as a non-ionic detergent (e.g., a mixture of Triton-X100 and igepal, or Brij-58) may be used.

PCR reaction mixture: reaction mixtures containing buffer, nucleotides, primers and enzyme (20. mu.L 5 × reaction buffer, 22. mu.L water, 2. mu.L 10 mM dNTPs, 3. mu.L of 10. mu.M forward primer, 3. mu.L of 2.5. mu.M reverse primer, 10. mu.L HemoKlenaq and 40. mu.L of bead beating lysate (bead beaten lysate)) were prepared and stored at room temperature.

Particle hybridization reaction mixture: a reaction mix consisting of the nanoparticle conjugate, salt, surfactant and formamide was prepared just prior to use (78. mu.L formamide, 78. mu.L 20 XSSC, 88.3. mu.L 1 XTE + 0.1% Tween, 7.5. mu.L CP 1-3 ', and 8.2. mu.L CP 3-5').

Glass beads (0.5 mm) used in mechanical lysis of candida were washed in acid and autoclaved and stored at room temperature before use.

PCR protocol:

a general protocol for a workflow for detecting pathogens (e.g., candida) in whole blood samples is shown in fig. 20. The scheme is as follows: (i) the sample spiked with human whole blood was allowed to warm to room temperature (approximately 30 minutes); (ii) 1 mL of red blood cell lysis buffer was sampled in aliquots into each tube; (iii) centrifuge each tube at 9000 g for 5 minutes and discard lysed blood; (iv) 100 μ L of 0.2 micron filtered TE was sampled equally into each tube; (v) add 120 mg of acid washed glass beads to each tube; (vi) each tube was vortexed at maximum speed (approximately 3000 rpm) for 3 minutes; (vii) sampling 50 μ L of the lysed sample in aliquots into tubes containing the PCR reaction mix; (viii) the PCR reaction was cycled as follows: (initial denaturation: 95 ℃ C. for 3 minutes; 30-40 cycles at 95 ℃ C. for 20 seconds; 30-40 cycles at 62 ℃ C. for 30 seconds; 30-40 cycles at 68 ℃ C. for 20 seconds; final extension: 68 ℃ C. for 10 minutes; final immersion: 4 ℃ C.); (ix) simple centrifugation of each sample after thermal cycling to form a precipitate bloody stasis; (x) 5 μ L of the particle reaction mixture was sampled equally into the tube (for every 10 μ L of amplified sample); (xi) Filling the resulting mixture with Mixing and denaturing the sample at 95 ℃ for 3 minutes; (xii) Hybridization of the sample was performed at 60 ℃ for 1 hour under mild stirring; (xiii) The sample was then diluted to 150 uL with particle dilution buffer and equilibrated to 37 ℃ in a heating block for 1 min; and (xiv) by T₂The MR reader measures the T of the sample₂。

The result of the detection

Reproducibility of the candida albicans detection in human whole blood: to determine T of human Whole blood against Candida albicans infection₂Repeatability of measurements, we performed an eight-day study in which the same donor spike and amplified samples were hybridized to superparamagnetic particles (n =3) each day and the resulting T recorded₂The value is obtained.

The accuracy of the analysis in batches is shown in FIG. 19A and is generally tight with all measured CV's being less than 12%. The reproducibility of the observations over the 8-day period is shown in fig. 19B, with CV less than 10% and CV less than 6% for the negative control over the candida concentration range. Two-tailed Student's T-test was applied to determine whether the difference between the mean of mock candida infected blood (10 cells/mL) and healthy donor blood was significant. The resulting P values were less than 0.0001, confirming that these differences were statistically significant.

Effect of sample matrix on candida albicans and candida krusei detection and reproducibility: a range of candida albicans or candida krusei cells (1E5 cells/mL to 0 cells/mL) were spiked into healthy blood from 6 donors. 16 independent experiments were performed from blood spiked with Candida albicans. Each experiment consisted of PCR amplification of blood from 1E5 to 0 cells/mL spiked, with three replicates of T for each amplification reaction₂Detection experiment; thus for Candida albicans, a total of 48T's were recorded at each concentration tested₂Values (see fig. 21A). At the lowest concentration tested (10 cells/mL), we failed to detect candida albicans within 37% of the time (6 out of 16 experiments); whereas candida albicans was detected at 100 cells/mL in 100% of the time. This indicates that the LOD of Candida albicans is higher than that of Candida albicans10 cells/mL but less than 100 cells/mL. More concentrations will be tested between 10 CFU to 100 cells/mL to better define LOD; however, we have not expected to observe any significant matrix effects on assay performance. This is represented by T₂The measured CV was confirmed: 12.6% at 1E5 cells/mL, 13.7% at 1E4 cells/mL, 15% at 1E3 cells/mL, 18% at 1E2 cells/mL and 6% at 0 cells/mL in 6 donor blood. This indicates that the assay can be reliably detected at candida albicans concentrations greater than or equal to 100 cells/mL, and that the donor blood sample has no significant effect on performance.

The same experiment was performed using a reference strain of candida krusei. In this case, 7 independent experiments were performed, as the remaining spiked blood was preserved for blood culture analysis. We did not detect at 10 cells/mL in any experimental batch, but at 100 cells/mL in all experimental batches. This suggests a LOD of between 10 cells/mL to 100 cells/mL. Again, a titration of cell concentration between 100 cells/mL to 10 cells/mL would be required in order to better define the LOD. The measured CV over the entire concentration range was: 10.5% at 1E5 cells/mL, 9% at 1E4 cells/mL, 12% at 1E3 cells/mL, 20% at 1E2 cells/mL, 6.4% at 10 cells/mL, 5.2% at 0 cells/mL. These results are shown in fig. 21B.

Preliminary determination of detection limits: five clinical isolates of candida albicans were spiked into 6 different donor blood samples at concentrations of 1E4, 1E3, 5E2, 1E2, 50, 10, 5 and 0 cells/mL. Each isolate was spiked into a minimum of two different donor blood samples. Through T₂Measurements the amplification reaction was detected and the results are plotted in figure 22. It is important to note that no data was removed for reasons within this study. We could not detect candida albicans at 5 cells/mL or 10 cells/mL within 50% of the time; however, Candida albicans was detected at 50 cells/mL over 95% of the time. These data were generated using different clinical isolates; each isolate Contain varying numbers of rDNA repeats, and the number of these repeats can vary up to 4-fold between strains (i.e., about 50 genes (units) to 200 genes). Since the input target copy number will vary slightly from strain to strain and certainly from species to species, the absolute T observed at very low cell numbers (i.e., 10 cells/mL)₂There will be a slight difference between the values. Based on our very preliminary study, the data suggests a cut-off of 10 cells/mL; however this determination cannot be done in the absence of the final formulation of the reagent and the instrument/cartridge. This suggests that it would be difficult to define the C5-C95 interval, since each reaction contained only 4 cells at 10 cells/mL. For this input volume of blood, titration at cell numbers below this becomes difficult. The number of responses that would contain 0 cells at 10 cells/mL was calculated using poisson distribution, indicating that only 2% of the responses would contain no cells; however at 5 cells/mL, 13% of the responses will be free of candida cells, and at 2 cells/mL, approximately 37% of the responses will be free of candida cells. To increase the detection sensitivity to 95% at 10 cells/mL, we can increase the amount of lysate added to the PCR reaction from 40. mu.L to 50. mu.L and the amount of patient blood from 400. mu.L/reaction to 800. mu.L/reaction.

Preliminary determination of sensitivity/specificity: initially, input candida colony forming units were quantitatively analyzed using a hemocytometer; however, in this case the operator counted the budding daughter cells as individual cells. Because our data is reported in colony forming units/mL rather than cells/mL, the budding should not be quantified. Due to this error, there were fewer candida cells/mL at various spike concentrations and our sensitivity (at 10 cells/mL) was only 90% while our specificity was 100%. At 25 cells/mL or higher, we observed 100% sensitivity and 100% specificity. In all cases, blood culture vials inoculated with candida cells by day 8 were positive for blood culture. It should be noted that the default setting for blood culture was 5 days of incubation; however we need to extend this incubation time as many of our inocula require >5 days of incubation. As an example, table 7 shows the time from inoculation to culture positivity recorded for four different clinical isolates of candida albicans inoculated into blood culture.

Tp to be performed on 800. mu.L aliquots of whole blood samples from these spiked samples ₂The measurement results are shown in Table 8. In all cases we were able to detect at 25 cells/mL or higher, whereas we were unable to detect clinical isolate C3 at 12 cells/mL. It is important to note that in this particular method comparison experiment, CFU was quantified using a hemocytometer rather than a coulter counter. A total of 51 blood culture flasks were inoculated with clinical isolates of candida albicans quantified by a hemocytometer, and 35 negative blood culture flasks were included in the experiment. The results for inocula greater than 25 cells/mL are shown in the tabulation of table 8.

TABLE 7.4 time to blood culture Positive results for different clinical isolates of Candida albicans

Candida albicans isolate	100 CFU/mL	25 CFU/mL	12 CFU/mL	0.0	0.0
						C1	1161 hours +/-12	161 hours +/-12	161 hours +/-12	192 hours	192 hours
C2	40 hours +/-12	65 hours +/-12	47.5 hours	192 hours	192 hours
						C3	69.5 hours	161 hours +/-12	161 hours +/-12	192 hours	192 hours
C4	40 hours +/-12	43 hours	47.5 hours	192 hours	192 hours

Note that: all blood culture negative vials were negative and discarded on day 8.

TABLE 8 PCR amplification and T on blood samples from the preculture in vitro spiking shown above ₂T obtained after detection₂Value (measurement time of about 3 hours)

Candida albicans isolate	100 CFU/mL	25 CFU/mL	12 CFU/mL	0.0	0.0
						C1	739.0	409.0	632.5	112.7	112.8
C2	983.2	1014.5	977.6	117.4	114.8
						C3	912.7	510.5	113.3	116.2	112.0
C4	807.6	741.2	665.2	119.1	115.9

T2 values (expressed in milliseconds) are mean values (n = 3) and CV for repeated measurements is less than 10%.

TABLE 9 tabulation for calculation of sensitivity/specificity at >25 cells/mL Candida albicans

Positive for	51 (true positive)	0 (false positive)	51 (true positive + false positive)
				Negative of	0 (false negative)	35 (true negative)	35 (false negative and true negative)
Total of	51 (true positive + false negative)	35 (false positive and true negative)	86 (negative)

Estimated sensitivity =100 × [ true positive/(true positive + false negative) ] =100% (95% confidence interval =93% to 100%)

Estimated specificity =100 × [ true negative/(false positive + true negative ] =100% (95% confidence interval =90% to 100%).

Standardization of the CFU quantitative analysis improves the sensitivity and reproducibility of our assays. Preliminary results from 27 blood culture flasks are shown in table 10. These preliminary results indicate that we have 100% sensitivity and specificity at 10 cells/mL or higher. Furthermore we have started a method comparison using candida tropicalis. Preliminary results (from 36 vials) are shown in table 11. These results indicate that for C.krusei we have a sensitivity/specificity of 88%/100% at 10 cells/mL or higher and a sensitivity/100% specificity of 100% at 33 cells/mL or higher. Another important variation, established prior to new blood culture consistent comparisons (blood culture consensus composites), is the use of multi-probe particles. In this case, T of Candida albicans detection ₂The aggregation reaction was performed using Candida albicans (albicans)/Candida parapsilosis (parapsilosis)/Candida tropicalis (tropicalis) multi-functional particles, and Candida krusei (glabrata)/Candida krusei (krusei) multi-functional particles.

TABLE 10 tabulation for calculation of sensitivity/specificity at >10 cells/mL Candida albicans

Positive for	18 (true positive)	0 (false positive)	18 (true positive + false positive)
				Negative of	0 (false negative)	6 (true negative)	6 (false negative + true negative)
Total of	18 (true positive + false negative)	6 (false positive and true negative)	24 (negative)

Estimated sensitivity =100 × [ true positive/(true positive + false negative) ] =100% (95% confidence interval =81.4% to 100%)

Estimated specificity =100 × [ true negative/(false positive + true negative ] =100% (95% confidence interval =54% to 100%).

TABLE 11 tabulation for calculation of sensitivity/specificity at >10 cells/mL C.krusei

Positive for	24 (true positive)	0 (false positive)	24 (true positive + false positive)
				Negative of	3 (false negative)	9 (true negative)	12 (false negative + true negative)
Total of	27 (true positive + false negative)	9 (false positive and true negative)	36 (negative)

Estimated sensitivity =100 × [ true positive/(true positive + false negative) ] =89% (95% confidence interval =71 to 98%)

Estimated specificity =100 × [ true negative/(false positive + true negative ] =100% (95% confidence interval =66 to 100%).

Preliminary assessment of clinical accuracy: clinical accuracy is defined as the ability to discriminate between two or more clinical states (e.g., candidemia versus candidemia). Receiver Operating Characteristic (ROC) graphs describe the performance of the assay, graphically illustrating the relationship between sensitivity (true positive score) and specificity (true negative score). The clinical accuracy (sensitivity/specificity pair) is shown for the entire range of decision levels. Using the data generated from whole blood samples spiked with 10 cells/mL and 50 cells/mL clinical isolates, two ROC plots were generated and are shown in fig. 23A and 23B. The area under the curve is often used to quantify the diagnostic accuracy; in this case we have the ability to discriminate between infected candidemia patients of 10 cells/mL or 50 cells/mL and patients without candidemia. At 10 cells/mL, the area under the curve is 0.72, which means that if T is performed in randomly selected people with candidemia (at an infection level of 10 cells/mL) ₂As determined, there is a 72% chance of their T₂The value will be higher than in people without candidemia. The detection is nearBed accuracy was much higher than 50 cells/mL and area under the curve was 0.98. Again, T is shown to be in humans with candidemia (at this level of infection)₂The assay will give a value over 98% of the time that is higher than samples from patients without candidemia. This is excellent clinical accuracy for an infection level of 50 cells/mL. ROC plots were not prepared for samples of 100 cells/mL or higher, as this area would be translated to 100% clinical diagnostic accuracy. The final clinical accuracy was determined from the actual patient samples on the clinical platform.

And (3) detecting the report time: the preliminary measurement steps with estimated time are: (i) hypotonic lysis/centrifugation/bead beating (8 min); (ii) PCR (120 minutes); (iii) hybridizing amplicons to the particles (30 min); (iv) magnetic force assisted agglomeration (10 min) in a uniform magnetic field; and (v) transfer and read (10 seconds). The measured treatment time was estimated to be about 178 minutes (about 3 hours), excluding the time for reagent and equipment preparation. This is a workflow for quantification; however, we have shown that the following improved workflow with shorter PCR and hybridization steps does not achieve the same detection sensitivity (see fig. 24) (although for some candida species (i.e., candida glabrata) a reduction in the amount of amplicon produced, and therefore a smaller δ T between diseased and normal ₂): (i) hypotonic lysis/centrifugation/bead beating (8 min); (ii) PCR (70 min); (iii) hybridizing amplicons to the particles (30 min); (iv) magnetic force assisted agglomeration (10 min) in a uniform magnetic field; and (v) transfer and reading (10 seconds). The modified workflow produced a TAT of 133 minutes or 2 hours and 13 minutes (and this without switching to a faster thermal cycler).

Conclusion

This test shows that the T-based for candidemia of the invention₂Having the following matrix: (i) detecting Candida albicans in whole blood in the range of 5 to 1E5 cells/mL (5-log); (ii) detecting candida krusei in whole blood ranging from 10 cells/mL to 1E5 cells/mL; (iii) sensitivity/specificity was 100%/100%, in>25 cellsWhen the volume is/mL; (iv) concentration of>Diagnostic accuracy greater than 98% at 50 cells/mL; (v) assay compatibility with whole blood (12 different donor blood samples were used, no significant matrix effect was observed); (vi) t is₂Repeatability of the measurement (less than 12% in the same day, less than 13% in 8 days); and (vii) reducing the total assay reporting time to 2 hours and 3 minutes.

We have examined higher input volumes of human blood and found that efficient hypotonic lysis can be achieved with these larger blood volumes; there was also an increase in reproducibility of the assay at 10 cells/mL.

Contamination was observed in 2 samples of 50 titrations. To reduce contamination problems, the PCR step may be separated from the detection step. Furthermore, chemical/biochemical methods can be used to render amplicons non-amplifiable. For example, uracil can be incorporated into the PCR product, and a pre-PCR incubation can be performed using uracil N-transglucosylase.

Advantages of the systems and methods of the present invention include the ability to assay a whole blood sample without the need to separate proteins and non-target nucleic acids from the sample. Because no loss of target nucleic acid is incurred through DNA purification (e.g., >10 × loss of sensitivity by running Qiagen columns after lysis and before amplification; and whole blood interference optical detection methods are used at concentrations above 1%), sample-to-sample variation and bias (which can be caused by DNA purification) is minimized and sensitivity is maximized.

More than 10% of septic shock patients are candida carriers; candida is the third most prevalent pathogen following staphylococcus aureus and escherichia coli, and there is approximately a 50% mortality rate in patients with septic shock infected with candida. Candida is the fourth leading cause of hospital-acquired infections. Rapid identification of these patients is critical to the selection of an appropriate treatment regimen.

Example 14 Virus assay

CMV genomic DNA was spiked into CMV-free healthy donor blood samples, and 40. mu.L of the spiked blood was sampled in aliquots to a total volume of 100. mu.L of PCR reactions. Amplification was performed using a thermophilic DNA polymerase compatible with whole blood (T2 Biosystems, Lexington, MA) and exemplary universal primers designed as follows: the 24 mer end-C6 linker-CMV specific sequence, the exact sequence being as follows:

5'-CAT GAT CTG CTG GAG TCT GAC GTT A-3' (SEQ ID NO.11, Universal tail Probe #1)

5'-GCA GAT CTC CTC AAT GCG GCG-3' (SEQ ID NO.12, Universal tail Probe #2)

5'-CGT GCC ACC GCA GAT AGT AAG-3' (SEQ ID NO.13, CMV US8 forward primer)

5'-GAA TAC AGA CAC TTA GAG CTC GGG-3' (SEQ ID NO.14, CMV US8 reverse primer)

The primers are designed such that the capture probe (i.e. the nucleic acid modifying the magnetic particle) will anneal to a 10mer region (the 10 mers are different at the 5 'or 3' end). The final primer concentration in the reaction tube was 300 nM and the PCR reaction mix included 5mM (NH)₄)₂SO₄、3.5 mM MgCl₂6% glycerol, 60 mM zwitterionic buffer (pH = 8.7). 5 individual sample reaction tubes were provided. The cycling PCR reaction was performed as follows: initial denaturation at 95 ℃ for 3 min, each cycle consisting of: 20 seconds at 95 ℃; 30 seconds at 55 ℃; and 68 ℃ for 20 seconds. At 30, 33, 36, 39 and 42 cycles, the reaction tube was removed and maintained at 4 ℃. Once all samples were prepared, 5 μ Ι _ of the particle reaction mix (6 x SSC, 30% formamide, 0.1% Tween) was aliquoted into tubes for every 10 μ Ι _ of amplified sample; the resulting mixture was mixed well and denaturation of the sample was carried out at 95 ℃ for 3 minutes; hybridization of the sample was performed at 45 ℃ for 1 hour under mild stirring; the samples were then diluted to 150 μ L with particle dilution buffer (PBS, 0.1% Tween, 0.1% BSA), magnetic assisted agglomeration was performed in a uniform magnetic field for 10 minutes, and equilibrated to 37 ℃ in a heating block for 1 minute; by T ₂MR reader measures T for each of 5 individual samples₂Relaxation time (see fig. 25).

These primers are designed to allow annealing of magnetic particles modified with capture probes to 10mer regions (the 10 mers are different at the 5 'or 3' end) providing particles with a generic structure for aggregation with specific amplification primers.

The results provided in FIG. 25 indicate that the methods and systems of the present invention can be used to perform real-time PCR and provide quantitative information about the amount of target nucleic acid present in a whole blood sample.

Example 15 real-time PCR

The previous results show that the generation of amplicons is suppressed when particles are present in the PCR reaction. We assume that moving the particles to the side of the reaction tube during thermal cycling will allow for amplicon generation. Simple magnetic separator/PCR block inserts (fig. 26) were designed to hold the nanoparticles on the sidewalls during the PCR reaction, thus minimizing interference and particle exposure to PCR reaction components. After removal of the magnetic field, the particles can be completely resuspended in the reaction mixture.

In one experiment, we tested the rate at which particles can be bound to the side of the tube and returned to solution. In this experiment, 100. mu.L of a mixture of Candida albicans (3 'and 5') particles in 1XTE (approximately 150 milliseconds with no aggregated T2 baseline) was subjected to three aggregation/deaggregation steps at 95 ℃. The following protocol was then performed: (i) vortexed, incubated at 37 ℃ for 1 minute, and T2 measured; (ii) heating at 95 ℃ for 5 minutes on magnetic PCR inserts; (iii) incubation at 37 ℃ for 1 minute, measuring T2; (iv) vortex for 15 seconds, incubate for 1 minute at 37 ℃ and measure T2; and (v) diverting to step (ii). The results of this experiment are shown in table 12 below.

TABLE 12

。

As shown in table 12, fully reversible nanoparticle aggregation was shown at 95 ℃ when the magnetic separator was used for detection. The particles were stable at 95 ℃ for at least 3 aggregation/deaggregation cycles.

We next examined the PCR efficiency in the presence of nanoparticles in the reaction solution. PCR was performed under 2 conditions: (1) completely dispersing the nano particles in the solution; and (2) concentrating the nanoparticles on the side wall of the PCR detection tube by using the magnetic insert.

Three PCR reactions were set up using Candida albicans genomic DNA as the starting material (with nanoparticles concentrated on the detection tube wall; completely dispersed in solution; and no nanoparticles). Successful target DNA amplification was confirmed by gel electrophoresis. Capture-probe modified Seramag particles were used.

An asymmetric (4:1) PCR reaction was set up using a pre-prepared PCR mix and 100 copies of genomic Candida albicans DNA as starting materials. A mixed solution of candida albicans capture particles dissolved in 1xTE (3 'and 5') was added to reactions (1) and (3) (baseline approximately 150 msec). Control reaction (2) had no added nanoparticles (fig. 27).

During PCR, no difference in PCR product formation was observed when the nanoparticles were present in solution (dispersed in solution or concentrated on the side wall of the detection tube using a magnetic field). Thus, nanoparticles modified with capture probes do not interfere with PCR. As confirmed by gel electrophoresis, considerable amounts of product are produced in the reaction with and without nanoparticles present in the solution. In addition, the concentration of magnetic nanoparticles on the side wall of the detection tube during the PCR process has no effect on the PCR.

Example 16 Candida assay and clinical data

A rapid, accurate and reproducible molecular diagnostic assay was developed for the detection of 5 candida species directly in human whole blood with a limit of detection (LOD) of 10 cells/mL and a time to outcome of less than 2 hours. The clinical performance of this assay was determined using 32 blinded clinical specimens, and in this study we observed 100% positive and 100% negative agreement with blood cultures, while accurately identifying the causative candida species within 100% of the candidemia patient samples. We further applied this assay to blood samples drawn from candida positive patients and observed a decrease in candida detection consistent with the time course of antifungal therapy. This diagnostic method is rapid, amenable to automation, and provides clinicians with the opportunity to detect multiple human pathogens within complex biological samples.

Magnetic Resonance relaxation measuring instrument (Magnetic Resonance relaxation Relaxometer)

Compact Magnetic Resonance (MR) systems are designed and manufactured for accurate T2 relaxation measurements in order to perform predetermined measurements under the conditions. The system was maintained at 37 ℃ by temperature control and contained a samarium cobalt permanent magnet at about 0.5T (corresponding to an operating proton frequency of 22-24 MHz). All standard MR components: the rf probe, low noise preamplifier and transmitter electronics, spectrophotometer board, and temperature control hardware are packaged in the system. The system uses a standard AC power input and connects to an external computer via ethernet. A user-friendly graphical user interface allows a user to set experimental parameters.

The system has been designed to receive samples in standard 0.2 ml PCR tubes. The accuracy of the measurement of the applicable sample volume is improved by optimizing the electronics and coils, allowing us to implement a single scan run to run less than 0.1% CV in T2. Inter-instrument variability of less than 2%: with minimal tolerance requirements on system components and without calibration.

Nanoparticle sensor conjugation and characterization

800 nm of carboxylated iron oxide superparamagnetic particles (said particles consisting of a number of iron oxide nanocrystals embedded in a polymer matrix containing a total particle size of 800 nm) were conjugated to aminated DNA oligonucleotides using standard carbodiimide chemistry methods (see Demas et al, New J. Phys.13:1 (2011)). The DNA-derivatized nanoparticles were stored at 4 ℃ in 1 XTTris-EDTA (pH8), 0.1% Tween-20. The iron concentration of the nanoparticle conjugates was measured by dissolving the particles with 6M HCl followed by addition of hydroxylamine hydrochloride and 1, 10O-phenanthroline followed by spectrophotometric detection as described in Owen et al, J Immunol Methods,73:41 (1984). Then, the oligonucleotide-derivatized particles were tested for functional properties by: the aggregation reaction caused by hybridization was performed in sodium phosphate hybridization buffer 4 XSSPE (600 mM NaCl, 40 mM sodium phosphate, 4mM EDTA) using a diluted synthetic oligonucleotide target identical in sequence to the fungal ITS2 sequence from these 5 different Candida species. Reversibility of the agglomeration reaction was confirmed by subjecting the agglomerated reactants to a thermal denaturation step at 95 ℃, performing a T2 measurement, repeating the hybridization at 60 ℃, followed by performing a second T2 measurement.

PCR primer and nanoparticle capture probe design

The universal Pan candida PCR primers were designed to be complementary to the 5.8S and 26S rRNA sequences that amplify the region of the intervening transcribed spacer 2 (ITS 2) of the candida genome. A pair of oligonucleotide capture probes are designed to be complementary to nested sequences at the 5 'and 3' ends, respectively, of the asymmetrically amplified PCR product. The capture probe hybridized to the 5 'end of the amplicon is 3' aminated, while the capture probe hybridized to the 3 'end of the amplicon is 5' aminated. A poly-T linker (n =9 to 24) was added between the amino group and the first nucleotide base of the capture probe sequence. HPLC purified PCR primers and capture probes were obtained from IDT Technologies (Coralville, IA).

Inhibition control design

The PCR inhibition control was designed to co-amplify with candida species and monitor the factors that inhibit PCR amplification within the whole blood sample. The synthetic template was designed to contain 30 nucleotide flanking sequences identical in sequence to the 5.8S and 26S regions of the Candida rRNA operon. The internal sequence within this template is composed of random, scrambled Candida albicans amplicons. The capture probe was designed to be complementary to the excessively amplified strand in an asymmetric candida PCR reaction. Synthetic oligonucleotide ultramers were obtained from IDT (Coralville, IA), which was identical in sequence to the inhibition control. The oligonucleotides were annealed in 2 XSSC at a concentration of 5. mu.M using standard methods and cloned into HindII/EcoRV digested pBR322(NEB, Ipshich, MA). Transformation was performed in electrically-induced (electrocompetant) E.coli K12 cells by electroporation of 1. mu.L of the ligation reaction, and the transformants were placed on Luria Bertani (LB) agar plates containing 100. mu.g/mL ampicillin. Two ampicillin-resistant colonies were selected and cultured in 2 mL of LB ampicillin media. Plasmid minipreps (mini-prep) were performed followed by restriction endonuclease gene mapping to confirm that the clones contained the correct insert. Sanger dideoxy sequencing (SeqWright, houston, TX) was then performed to confirm successful control clones, and mass preparation of DNA was performed on clones carrying the correct insert (maxi-preps). Titration of the inhibition controls was performed in the presence of increasing concentrations of all 5 candida species to determine the lowest concentration of inhibition controls that could be reproducibly detected. Confirmation of inhibition of the function of the control was confirmed by performing a PCR reaction in the presence of a titration of known PCR interferents (SDS, heparin, ethanol) and showed that amplification of the control was inhibited.

Sample preparation for candida culture and in vitro spiking

MYA-2876, ATCC 2001, ATCC 24210, ATCC 66029 and ATCC 22019 are Candida albicans, Candida glabrata, Candida krusei, Candida tropicalis, and Candida parapsilosis laboratory reference strains (ATCC, Manassas, VA) used to prepare whole blood samples for in vitro spiking. The yeast were cultured on yeast peptone glucose agar plates (YPD) and incubated at 25 ℃. Individual colonies were selected and suspended in Phosphate Buffered Saline (PBS). Each species was verified by ITS2 sequencing at Accugenix (Newark, Delaware). The cells were then centrifuged at low speed (3000 g, 2 min) and washed three times with fresh PBS. An aliquot of the PBS washed cells was then diluted in ISOTON II diluent (Beckman Coulter, break, CA) in 20 mL Accuvette and the cells were quantified on a Multisizer 4 Coulter granulometer (Beckman Coulter, break, CA) according to the manufacturer's instructions. The cells were then serially diluted to a concentration in the range of 500 to 5 cells/100 μ Ι _ of PBS buffer. Fresh healthy donor blood drawn by sterile collection in K2EDTA disposable vacuum blood collection tubes (BD Diagnostics, Franklin Lakes, NJ) was obtained from promedxx. Typically, 5 ml of human blood is spiked with 100. mu.L of a quantified amount of Candida cells. Samples of the whole blood spike were then immediately used for the assay.

Whole blood PCR

Erythrocyte lysis was performed in 1 mL of whole blood samples using the previously described methods (see Bramley et al, Biochimica et Biophysica Acta (BBA) -biomembrans, 241:752(1971) and Wessels JM, Biochim Biophys Acta, 2:178(1973)), followed by low speed centrifugation, removal and discarding of the supernatant. Then 100 uL of Tris EDTA (TE) buffer (pH =8.0) containing 1500 copies of the inhibition control was added to the harvested pellet and the suspension was subjected to mechanical lysis (see Garver et al, appl. Microbiol., 1959.7: 318 (1959); Hamilton et al, appl. Microbiol.,10:577(1962); and Ranhand, J.M., appl. Microbiol.,28:66 (1974)). Then 50. mu.L of lysate was added to 50. mu.L of asymmetric PCR reaction mixture containing deoxynucleotides, PCR primers and a thermophilic DNA polymerase compatible with whole blood (T2 Biosystems, Lexington, Mass.). The thermal cycling was performed using the following cycle parameters: heat denaturation at 95 ℃ for 5 minutes, 40 cycles consisting of a 30 second heat denaturation step at 95 ℃, an annealing step at 62 ℃ for 20 seconds, an extension step at 68 ℃ for 30 seconds, and a final extension at 68 ℃ for 10 minutes.

Determination of aggregation by hybridization

15 microliters of the resulting amplification reaction was sampled equally into 0.2 mL thin-walled PCR tubes and incubated in sodium phosphate hybridization buffer (4 x SSPE) (containing paired oligonucleotide-derivatized nanoparticles at a final iron concentration of 0.2 mM iron per reaction). The hybridization reactions were incubated at 95 ℃ for 3 minutes followed by 60 ℃ for 30 minutes in a shaking incubator (vortex, LabNet International) set at a stirring speed of 1000 rpm. The hybridized sample was then placed in a 37 ℃ heat block to equilibrate the temperature to that of the MR reader over a 3 minute period. Each sample was then subjected to 5 second vortexing steps (3000 rpm) and inserted into an MR reader for T2 measurements.

Candida patient sample collection protocol

Blood sample discards (Blood specific discards) drawn in K2EDTA single-use vacuum Blood collection tubes (BDs) on the same day as the samples drawn for Blood culture (T =0) were obtained from clinical hematology laboratories at Massachusetts General Hospital (MGH) or houston university hospital. Each sample was collected and classified by category according to patients with blood culture positive results. Samples were stored at-80 ℃ in original single use vacuum blood collection tubes and the blinded specimen collections were shipped on dry ice overnight to T2 Biosystems. Clinical sample collection protocols were reviewed by the appropriate human research committee.

Statistical analysis

For each species, the limit of detection is determined by using a probabilistic unit model. For each species, a 90% level of detection and 95% confidence interval were calculated. Each raw T2 signal was converted to T2 (milliseconds) throughout the assay background. SAS v.9.1.3(Cary, NC) was used for statistical calculations for detection limit analysis, spiked sample to culture consistency, sensitivity and specificity in clinical samples, and serial assays to measure candida clearance.

Consistency of Candida T2MR assay with blood culture

The current gold standard for candida diagnostics is blood culture. In vitro spiked healthy donor whole blood samples were prepared using laboratory reference strains of candida albicans and candida krusei at concentrations of 0, 33 and 100 cells/mL, as well as clinical isolates of candida albicans. Pediatric BACTEC blood culture vials (BACTEC Peas Plus/F vials, Beckton Dickenson) were inoculated with an aliquot of a sample of the in vitro spike evaluated by T2 MR. In all cases, the blood culture vials inoculated with candida cells on day 8 were blood culture positive. In total, 133 blood culture flasks were inoculated with either 90 candida spiked blood samples (33 cells/mL inoculum) or 43 negative blood samples. 98% positive and 100% negative concordance were observed between T2MR and blood culture.

Clinical specimen data

Samples of K2 EDTA whole blood patients were obtained to test the clinical performance of the T2MR candida assay. These patients exhibited symptoms of sepsis, and blood was withdrawn for culture. Blood samples were stored at 4 ℃ in a hematology laboratory and were selected for T2MR (if the results were candida blood culture positive, bacteremic blood culture positive, or blood culture negative) to better represent the range of samples that would be tested on the platform. 14 samples were from candidemia patients, 8 samples were from bacteremia patients, and 10 samples were from blood culture negative patients. Fig. 29 shows the measured T2 values for all 32 patient samples. A single PCR reaction was performed using 1 mL of each sample. 750 copies of the internal suppression control were added to each PCR reaction. In candida negative samples, the mean Internal Control (IC) signal was 279 ms and CV in 18 candida negative specimens was 25%. In all cases the IC signal was not below the decision threshold (128 ms, 5-fold standard deviation added to the mean T2 value measured in candida negative detection reactions), indicating that all negatives were true negatives and that no inhibitory substance was present in the whole blood sample. The detection reaction is multiplexed, based on the IDSA guidelines, reporting three results as follows: positive for candida albicans or candida tropicalis; positive for candida krusei or candida glabrata; and positive for candida parapsilosis. The average T2 measured in the candida negative samples was 114 ms, the CV for these measurements was 2.4%, and the decision threshold (calculated by adding the standard deviation measured in the 5-fold candida negative detection reaction to the average T2 value measured in the candida negative samples) was 128 ms. In candida positive samples, IC signal is suppressed due to competition of amplification reagents. In the case of candida albicans, some cross-reactivity was observed when detected with candida parapsilosis particles (e.g., patient sample No. 3), however this signal was not significantly above the limit (20 ms) and did not result in differences in antifungal therapy, as both candida albicans and candida parapsilosis were sensitive to fluconazole.

T2MR successfully identified 14 samples of candida albicans, candida parapsilosis, or candida krusei that were confirmed positive by blood culture followed by Vitek 2 biochemical card. Furthermore, the assay is specific for candida species, as bacteremic patient samples with e.coli, enterococcus species, staphylococcus aureus, klebsiella pneumoniae, coagulase-negative staphylococci, or alpha-hemolytic streptococci remain negative.

Serial samples taken from 2 patients showing symptoms of candidemia (e.g. persistent fever after receiving antibiotics) were tested to show the usefulness of the assay in monitoring candida clearance. On the same day that blood was drawn for blood culture, blood was drawn for T2 MR. The monitor cultures of patient a were then withdrawn over a period of 9 days and the monitor cultures of patient B were withdrawn over a period of 5 days. Figure 3 shows the results obtained for two patients using the T2MR method. A blood draw (t =0) was performed for the culture on patient a, who was diagnosed with candidemia and who was administered micafungin (candida glabrata) intravenously after the day of blood culture (t = 1). Whole blood samples were tested at T =0, T =3, T =7, T =8, and T =9 days using T2 MR. The T2MR values obtained were 320 ms (T =0), 467 ms (T = 3), 284 ms (T = 7), 245 ms (T = 8) and 17 ms (below the limit) (T = 9). Subsequent blood cultures drawn on days 3 and 8 took 24 and 48 hours to reach culture positivity. A series of serial samples were obtained from patient B. Candida albicans was correctly detected on day 0 with T2MR (T2=426 ms). Blood cultures became positive on day 2 (identified with candida albicans later). After 1 day of micafungin administration to the patient, a steep decline was found in candida albicans T2MR (T2=169 ms) and no monitorable candida albicans was observed after 3 or more days of starting antifungal treatment. All the tests were completed in a total processing time of 2 hours using a rapid block PCR thermocycler and a three-step thermal cycling program that did not optimize the speed.

Conclusion

We have developed and validated a whole blood T2MR candida assay capable of detecting 5 clinically important candida species that takes advantage of non-optical detection to rule out analyte purification and thus enable faster reporting times and more reproducible results. Asymmetric PCR was used to specifically amplify the ITS2 region of the candida genome directly in whole blood to obtain clinically relevant detection sensitivity. Develop T₂A detection method wherein oligonucleotide-derivatized nanoparticles of two pools are hybridized to each end of a single-stranded amplicon. These amplicons thus act as interparticle tethers and cause nanoparticle agglomeration that results in a measurable and reproducible change in the spin-spin relaxation (T2) of protons in water molecules. We further constructed and implemented an internal inhibition control to monitor PCR inhibitors that may be present in patient samples.

The assay was evaluated using reference and clinical isolates quantified by a coulter sizer and spiked into healthy donor whole blood. Measurement of assay reproducibility over a 10 day period using blood spiked with candida albicans (same sample, same operator, same instrument), we observed a CV of less than 12.8% (n =30) over the entire dynamic response range (0 to 1E5 cells/mL). The analytical sensitivity and detection limit of < 10 cells/mL were measured in Candida albicans, Candida tropicalis, Candida krusei and Candida parapsilosis, and >10 cells/mL were detected in Candida glabrata (92.5% detected at 10 cells/mL). Although not proven, one possible reason for the higher LoD observed in candida glabrata may be a reduction in rDNA operon copy number in candida glabrata compared to other suspected candida species, since candida glabrata is known to exist in nature in a haploid form while other candida species exist in a diploid form. High agreement with candida diagnostic gold standards: 98% positive and 100% negative concordance were observed for the 133 individual spiked candida albicans and candida krusei samples. It should be noted that the time to reach the results of the Candida test of T2 was 2 hours, whereas the time to reach blood culture positivity for Candida albicans was typically 2 days, and the time to reach blood culture positivity for Candida krusei was typically about 1 day (18-24 hours).

32 clinical samples were similar to the blood culture results. The measured T2 was above the limit established based on the five-fold standard deviation plus the mean of the T2 values measured in candida negative samples. In this case, the threshold is 128 ms (n = 54). In all cases we did not observe inhibition of the PCR reaction, as internal controls were detected in all 32 reactions and the IC signal observed was reduced in candida positive patients and CV was 25% in candida negative specimens (n =18) (mean T2 value of 279 ms). The assay was highly specific for candida detection, as no cross-reactivity was observed in any bacteremia sample (n = 8). Candida positive samples were accurately identified and pathogenic candida species were accurately identified, all within 2 hours of time to reach response.

The potential of this assay to provide a rapid measure of candida clearance following administration of antifungal therapy has also been demonstrated. Samples from 2 groups of patients were taken and subjected to T2MR (fig. 3). With the antifungal agent administered on day 1, a moderate to high T2 signal for candida glabrata was observed on days 0 and 3 in patient a. In the following days, a decrease in candida glabrata signal was observed, with no detectable candida glabrata after 8 days of antifungal treatment. Patient B measured a strong candida albicans signal on day 0, a sharp drop in T2 signal was observed 1 day after antifungal administration (delta T2 of 306 ms), and was not detectable 2 days after antifungal treatment. Although preliminary, this data suggests that this assay can be used to monitor treatment efficacy and candida clearance in a real-time manner.

In summary, we have developed a sensitive and specific assay for diagnosing candidemia caused by the five most commonly encountered candida species. Early clinical results were encouraging and showed that rapid diagnosis and species identification was achievable, not only facilitating early treatment with appropriate antifungal agents, but also providing a means of monitoring candida clearance. We expect this nanoparticle-based T2MR method to be widely applicable for diagnosis of infectious diseases in a variety of specimen types and pathogens.

Example 17 tacrolimus assay Using Fab

Tacrolimus assays are homogeneous competitive immunoassays which are performed using EDTA whole blood samples extracted to release tacrolimus from red blood cells and binding proteins. Key elements of this assay are the improvement of high affinity tacrolimus antibodies, reliable extraction methods, and buffer systems selected to promote specific aggregation and minimize non-specific aggregation. This version of the assay uses a recombinant monovalent Fab with high affinity for tacrolimus.

The tacrolimus assay was evaluated using whole blood calibrators, commercially available whole blood controls, spiked samples, and patient samples.

The assay reagent comprises: (a)244 nm particles conjugated with sequential BSA and monovalent Fab antibodies and blocked with mPEG-thiol + NEM (particles diluted to 0.2 mM Fe in assay buffer); (b) tacrolimus to BSA input ratio of 10:1C22 modified tacrolimus conjugated to BSA (diluted to 600 ng/ml in assay buffer); (c) assay buffer of 100 mM glycine (pH =9.0), 1% BSA, 0.05% Tween 80, 150 mM NaCl, and 0.05% Proclin; and (d)70% MeOH, 60 mM ZnSO dissolved in dH20₄The extraction reagent of (1).

Whole blood calibrators were prepared using a 1 mg/ml Sigma FK506 stock solution (in 100% MeOH). EDTA whole blood was spiked with tacrolimus solution at various levels. Spiked blood was incubated at 37 ℃ with gentle mixing and then stored overnight at 4 ℃ before specimen sampling and freezing. Target levels were 0, 1, 2, 5, 10, 20, 50, 100 and 250 ng/ml tacrolimus. The calibrant was provided to an external laboratory for assignment using an Architect tacrolimus assay. These samples were measured by mass spectrometry. The results show a correlation of the theoretical value with the actual assigned value of 0.9998.

Quality control consisted of 3 levels of UTAK immunosuppressive Matrix Controls (UTAK immunosuppressive Matrix Controls). Patient samples were obtained from transplant patients receiving tacrolimus treatment.

The detection scheme is as follows:

(i) all samples, calibrators, QC and reagents were allowed to equilibrate to room temperature and mixed by gentle inversion.

(ii) 200 μ L of the sample, calibrator or QC material was transferred to a 1.5 mL microcentrifuge tube (microfuge tube) with a pipette. Add 200 uL of extraction reagent and vortex for 30 seconds. The sample was incubated at room temperature for 2 minutes and centrifuged at 10,000 rpm for 5 minutes. The clean supernatant was transferred to a clean tube and a 2.5 x dilution was prepared using assay buffer.

(iii) mu.L of the diluted extract and 10. mu.L of the diluted particles were transferred into a reaction tube with a pipette, vortexed, and incubated at 37 ℃ for 15 minutes. mu.L of BSA-tac conjugate was transferred into the reaction tube with a pipette, vortexed, and incubated at 37 ℃ for 15 minutes. The samples were subjected to 6 cycles (12 min) of magnetically assisted agglomeration in a gradient magnetic field. Vortex mix, incubate 5 minutes at 37 ℃ and read in a T2 reader at 37 ℃.

For each test run, triplicate tests were performed on the calibrator (6 runs total). Each run was fitted with a 5PL model using GraphPad Prism 5 for Windows (version 5.02, GraphPad Software, San Diego California USA). 0 calibrator was input as 0.01 ng/ml and used for the curve model. The resulting calibration curve (running calibration) was used to back-calculate the tacrolimus concentration for all calibrators, whole blood spikes, QC and patient samples included in the run.

In addition, Master calibration curves were obtained using fitted data throughout the 3-day study (n =18) for each calibrator. All samples were back-calculated using the Master curve and the resulting tacrolimus levels were compared to those obtained using the running calibration.

A reproducible list of 13 members (9 calibrators, 3 controls and 1 spiked whole blood sample) was tested in triplicate for a total of 3 days, with 2 runs per day, and a total of 18 replicates. The calibrator was stored at-80 ℃ for the duration of the study, while the control and whole blood spikes were stored at 4-8 ℃.

The sample concentration was predicted using a running calibration curve, and a master curve in GraphPrism. Intra-batch (Within-run) precision, intra-day precision, inter-day precision and total precision were calculated by ANOVA using MiniTab 15.

Data predicted using a running calibration method showed a total inaccuracy (imperception) of < 25% CV over a tacrolimus concentration range of about 3-210 ng/ml.

Analytical sensitivity was calculated using the 2SD method. The standard deviation of 18 replicate tests of 0 calibrator was determined. Tacrolimus levels at maximum T2 (upper asymptote of curve fit) -2SD were then calculated and concentrations were predicted using the Master calibration curve. The assay sensitivity was 0.8 ng/ml.

During tacrolimus antibody formation and screening, antibody specificity was evaluated against 5 tacrolimus metabolites. ELISA inhibition of 5 affinity matured clones using each of the 5 metabolites and 7 clones with additional affinity maturation by cross cloning and comparison to free tacrolimus. Data for two of the cross-clones and the murine monoclonal RUO antibodies of the state of the art are shown below. The only observed cross-reactivity was a slight reactivity to 15-O-demethylated metabolites.

A summary of the performance measurements for tacrolimus is presented in the table below.

Table 13.

。

Example 18 preparation of nanoparticles for nucleic acid analyte detection.

Preparation of single probe particles: before use, 800 nm of carboxylated iron oxide superparamagnetic particles (said particles consisting of a number of iron oxide nanocrystals embedded in a polymer matrix comprising a total particle size of 800 nm) (see Demas et al, New j. phys.13:1(2011)) are washed with magnetic tracks. Magnetic particles were resuspended in 66 μ L nuclease-free water, 20 μ L250 mM MES buffer (pH =6) and 4 μ L aminated probe (obtained from IDT) at a concentration of 1 mM per mg of particles to be prepared. A3 'aminated probe particles and 5' aminated probe particles (for example, probes for Candida parapsilosis) were prepared. The probe was added to the particles and the suspension was vortexed using a vortexer equipped with a foam holder holding a tube. The vortex was set at a speed that kept the particles in sufficient suspension without any splashing. N-ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was then dissolved in water and immediately added to the vortexed particle-probe mixture. The tube was then closed and incubated for 2 hours at 37 ℃ in an incubator with rotation. The tube was then placed in the track and the reaction solution was removed. These particles were washed with a series of detergents (125 μ L/mg particles) as follows: water; water; 0.1M imidazole (pH =6.0), incubated at 37 ℃ for 5 minutes with rotation; water; 0.1M sodium bicarbonate (pH =8.0), incubated at 37 ℃ for 5 minutes with rotation; and (3) water. These particles were then heat stressed in 0.1M sodium bicarbonate (pH =8.0) for 1 hour at 60-65 ℃ under rotation. After heat stress, the bicarbonate was removed by placing the tube in the track. These particles were then resuspended in storage buffer (Tris-EDTA, 0.1% Tween 20) and vortexed. The storage buffer was removed and the final 100 μ l of storage buffer was added to the particle preparation. These particles were stored at 2-8 ℃, validated using an iron assay to determine the iron concentration of these particles, and detected against the target nucleic acid (e.g., candida parapsilosis ITS2 oligonucleotide titration). In the candida assay, these particles were diluted in 8 x SSPE supplemented with 0.09% sodium azide (as preservative).

Preparation of dual probe particles: for the preparation of dual probe particles, the procedure is the same as described above, except that the same volume of the second probe (e.g., 3 'aminated candida albicans) and the first probe (e.g., 3' aminated candida tropicalis) are mixed prior to addition to the magnetic particles. Similarly, the same volume of 5' aminated probe was mixed before addition to the magnetic particles.

Example 19 Candida assay improvements

The detection limit of the candida assay of example 16 was improved by washing the precipitate. 2.0 mL of whole blood was mixed with 100. mu.L of TRAX red blood cell lysis buffer (i.e., a mixture of nonylphenoxy-polyethoxyethanol (NP-40) and polyoxyethylene 4-octylphenol ether (Triton-X100)) and incubated for about 5 minutes. The sample was centrifuged at 6000 g for 5 minutes and the resulting supernatant was removed and discarded. To wash the pellet, the pellet was mixed with 200 μ L Tris EDTA (TE) buffer (pH = 8.0) and vortexed. The sample was centrifuged again at 6000 g for 5 minutes and the resulting supernatant was removed and discarded. After the washing step, the pellet is mixed with 100 μ L TE buffer and bead beating is performed with vigorous stirring (e.g., using 0.5 mm glass beads, 0.1 mm silica beads, 0.7 mm silica beads, or a mixture of different sized beads, for example). The sample was centrifuged again. Then 50. mu.L of the resulting lysate was added to 50. mu.L of an asymmetric PCR reaction mixture containing deoxynucleotides, PCR primers and a thermophilic DNA polymerase compatible with whole blood (T2 Biosystems, Lexington, Mass.). Thermal cycling and hybridization induced aggregation assays were performed in the manner described in example 16 to generate T2 values characteristic of the presence of candida in blood samples. The assay can yield (i) a coefficient of variation of the T2 value of less than 20% in candida positive samples; (ii) at least 95% correct detection at less than or equal to 5 cells/mL in a sample spiked into 50 individual healthy patient blood samples; (iii) at least 95% correct detection at less than or equal to 5 cells/mL in a sample spiked into 50 individual unhealthy patient blood samples; and/or (iv) greater than or equal to 80% correct detection in a clinically positive patient sample (i.e., determined to be candida positive using other techniques such as cell culture) starting with 2 mL of blood.

This application claims the preferred rights to U.S. application serial No. 12/910, 594 filed on 22/10/2010 and claims the benefits of U.S. provisional patent application No. 61/414,141 filed on 16/2010, U.S. provisional patent application No. 61/418,465 filed on 1/12/2010 and U.S. provisional patent application No. 61/497,374 filed on 15/6/2011, the respective contents of which are incorporated herein by reference.

Other embodiments

All publications, patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains: which are known or customary practice in the art to which this invention pertains and which may be applied to the key features enumerated above and fall within the scope of the claims.

Other embodiments are within the scope of the following claims.

What is claimed is the subject matter of the claims.

Claims

1. A method for detecting the presence of an analyte in a liquid sample, the method comprising:

(a) contacting the solution with magnetic particles to prepare a solution containing 1X 10⁶To 1X 10¹³A liquid sample of magnetic particles/ml liquid sample, wherein the magnetic particles have an average diameter of 150 nm to 699 nm, 1 x 10 per particle⁸To 1X 10¹² mM^-1s^-1T of₂Relaxivity, and binding moieties on their surface which function as binding moieties on the analyte or multivalent junctionChanging the aggregation of the magnetic particles in the presence of a binding agent;

(b) placing the liquid sample in a device comprising a support defining a well containing the liquid sample comprising magnetic particles, multivalent binding agent, and analyte, and having a radio frequency coil disposed around the well, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using one or more magnets and an RF pulse sequence;

(c) exposing the sample to a bias magnetic field and a sequence of RF pulses;

(d) measuring the signal after step (c); and

(e) detecting the analyte based on the results of step (d).

2. A method for detecting the presence of an analyte in a liquid sample, the method comprising:

(a) Contacting the solution with magnetic particles to prepare a solution containing 1X 10⁶To 1X 10¹³A liquid sample of magnetic particles/ml liquid sample, wherein the magnetic particles have an average diameter of 700 nm to 1200 nm, 1 x 10 per particle⁹To 1X 10¹² mM^-1s^-1T of₂Relaxivity, and binding moieties on their surface, the role of which is to alter the aggregation of magnetic particles in the presence of an analyte;

(c) exposing the sample to a bias magnetic field and a sequence of RF pulses;

(d) measuring the signal after step (c); and

(e) detecting the presence or concentration of the analyte based on the results of step (d).

3. The method of claim 1 or 2, wherein the magnetic particles are substantially monodisperse.

4. The method of claim 1 or 2, wherein the magnetic particles exhibit non-specific reversibility in the absence of analyte and multivalent binding agent.

5. The method of claim 1 or 2, wherein step (d) comprises measuring T of the liquid sample₂A relaxation response, and wherein increasing agglomeration in the liquid sample results in an observed T of the sample₂The relaxation rate increases.

6. The method of claim 1 or 2, wherein the analyte is a target nucleic acid.

7. The method of claim 6, wherein the target nucleic acid is extracted from a leukocyte.

8. The method of claim 6, wherein the target nucleic acid is extracted from a pathogen.

9. A method for detecting the presence of a pathogen in a whole blood sample, the method comprising:

(a) providing a whole blood sample from a subject;

(b) mixing a whole blood sample with a red blood cell lysing agent to prepare lysed red blood cells;

(c) after step (b), centrifuging the sample to form a supernatant and a pellet, discarding some or all of the supernatant, and resuspending the pellet to form an extract, optionally washing the pellet before resuspending the pellet and optionally repeating step (c);

(d) lysing cells in the extract to form a lysate;

(e) placing the lysate of step (d) in a detection tube and amplifying the target nucleic acid in the lysate to form an amplified lysate solution comprising the target nucleic acid, wherein the target nucleic acid is characteristic of the pathogen to be detected;

(f) After step (e), adding 1X 10 to the test tube⁶To 1X 10¹³Magnetic particles/ml of amplified lysate solution, wherein the magnetic particles have an average diameter of 700 nm to 1200 nm and binding moieties on their surface, the role of which binding moieties is to alter the aggregation of the magnetic particles in the presence of a target nucleic acid or multivalent binding agent;

(g) placing a detection tube in a device comprising a support defining a well for receiving the detection tube comprising magnetic particles and a target nucleic acid, and having a radio frequency coil disposed around the well, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using one or more magnets and an RF pulse sequence;

(h) exposing the sample to a bias magnetic field and a sequence of RF pulses;

(i) after step (h), measuring a signal from the detector tube; and

(j) (ii) detecting the pathogen based on the results of step (i).

10. The method of claim 9, wherein steps (a) through (i) are completed within 3 hours.

11. The method of claim 9, wherein step (i) is performed without any prior purification of the amplified lysate solution.

12. A method for detecting the presence of a target nucleic acid in a whole blood sample, the method comprising:

(a) providing one or more cells from a whole blood sample from a subject;

(b) lysing the cells to form a lysate;

(c) amplifying the target nucleic acid in the lysate to form an amplified lysate solution comprising the target nucleic acid;

(d) after step (c), adding the amplified lysate solution and 1X 10 to a detection tube⁶To 1X 10¹³Magnetic particles/ml of amplified lysate solution, wherein the magnetic particles have an average diameter of 700 nm to 1200 nm and binding moieties on their surface, the role of which binding moieties is to alter the aggregation of the magnetic particles in the presence of a target nucleic acid or multivalent binding agent;

(e) placing a detection tube in a device comprising a support defining a well for receiving the detection tube comprising magnetic particles and a target nucleic acid, and having a radio frequency coil disposed around the well, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using one or more magnets and an RF pulse sequence;

(f) exposing the sample to a bias magnetic field and a sequence of RF pulses;

(h) after step (f), measuring a signal from the detector tube; and

(i) Detecting the target nucleic acid based on the result of step (h).

13. The method of claim 12, wherein the target nucleic acid is purified prior to step (d).

14. A method for detecting the presence of a target nucleic acid in a whole blood sample, the method comprising:

(a) providing an extract prepared by the process of: lysing red blood cells in a whole blood sample from a subject, centrifuging the sample to form a supernatant and a pellet, discarding some or all of the supernatant, and resuspending the pellet to form an extract, optionally washing the pellet prior to resuspending the pellet and optionally repeating the centrifuging, discarding, and resuspending steps;

(b) lysing cells in the extract to form a lysate;

(c) placing the lysate of step (b) in a detection tube and amplifying the nucleic acids in the detection tube to form an amplified lysate solution comprising 40% (w/w) to 95% (w/w) target nucleic acid and 5% (w/w) to 60% (w/w) non-target nucleic acid;

(d) after step (c), adding 1X 10 to the test tube⁶To 1X 10¹³Magnetic particles/ml of amplified lysate solution, wherein the magnetic particles have an average diameter of 700 nm to 1200 nm and binding moieties on their surface, the role of which binding moieties is to alter the aggregation of the magnetic particles in the presence of a target nucleic acid or multivalent binding agent;

(f) exposing the sample to a bias magnetic field and a sequence of RF pulses;

(g) after step (f), measuring a signal from the detector tube; and

(h) detecting the target nucleic acid based on the result of step (g), wherein step (g) is performed without any prior purification of the amplified lysate solution.

15. The method of claim 9, 12 or 14, wherein step (b) comprises mixing the extract with beads to form a mixture and stirring the mixture to form a lysate.

16. The method of claims 9 to 15, wherein the magnetic particles comprise one or more populations having first and second probes conjugated to their surfaces, the first probe acting to bind to a first segment of the target nucleic acid and the second probe acting to bind to a second segment of the target nucleic acid, wherein the magnetic particles form aggregates in the presence of the target nucleic acid.

17. The method of claims 9 to 15, wherein the magnetic particles comprise a first population having a first binding moiety on their surface and a second population having a second binding moiety on their surface, and the multivalent binding moiety comprising a first probe and a second probe, the first probe acting to bind to the first binding moiety and the second probe acting to bind to the second binding moiety, the binding moieties and multivalent binding moieties acting to alter aggregation of magnetic particles in the presence of the target nucleic acid.

18. A method for detecting the presence of candida species in a liquid sample, the method comprising:

(a) lysing candida cells in a liquid sample;

(b) amplifying a nucleic acid to be detected in the presence of a forward primer and a reverse primer to form a solution comprising candida amplicons, each primer being generic to a plurality of candida species;

(c) contacting the solution with magnetic particles to prepare a solution containing 1X 10⁶To 1X 10¹³A liquid sample of magnetic particles/ml liquid sample, wherein the magnetic particles have an average diameter of 700 nm to 1200 nm, 1 x 10 per particle⁹To 1X 10¹² mM^-1s^-1T of₂Relaxivity, and binding moieties on their surface that act to alter the aggregation of magnetic particles in the presence of candida amplicons or multivalent binding agents;

(d) Placing a liquid sample in a device comprising a holder defining an aperture for receiving the liquid sample comprising magnetic particles and candida amplicons, and having a radio frequency coil disposed around the aperture, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using one or more magnets and an RF pulse sequence;

(e) exposing the sample to a bias magnetic field and a sequence of RF pulses;

(f) after step (e), measuring the signal; and

(g) determining whether a candida species is present in the sample based on the results of step (f).

19. The method of claim 18, wherein the forward primer comprises oligonucleotide sequence 5'-GGC ATG CCT GTT TGA GCG TC-3'.

20. The method of claim 18, wherein the reverse primer comprises oligonucleotide sequence 5'-GCT TAT TGA TAT GCT TAA GTT CAG CGG GT-3'.

21. The method of claim 18, wherein the candida species is candida albicans, and wherein the first probe comprises an oligonucleotide sequence:

5’-ACC CAG CGG TTT GAG GGA GAA AC-3’，

and the second probe comprises the oligonucleotide sequence:

5’-AAA GTT TGA AGA TAT ACG TGG TGG ACG TTA-3’。

22. the method of claim 18, wherein the candida species is candida krusei, and wherein the first probe and the second probe comprise oligonucleotide sequences selected from the group consisting of:

5’-CGC ACG CGC AAG ATG GAA ACG-3’，

5'-AAG TTC AGC GGG TAT TCC TAC CT-3', and

5’-AGC TTT TTG TTG TCT CGC AAC ACT CGC-3’。

23. the method of claim 18, wherein the candida species is candida glabrata, and wherein the first probe comprises an oligonucleotide sequence:

5’-CTA CCA AAC ACA ATG TGT TTG AGA AG-3’，

and the second probe comprises the oligonucleotide sequence:

5’-CCT GAT TTG AGG TCA AAC TTA AAG ACG TCT G-3’。

24. the method of claim 18, wherein the candida species is candida parapsilosis or candida tropicalis, and wherein the first probe and the second probe comprise oligonucleotide sequences selected from the group consisting of:

5’-AGT CCT ACC TGA TTT GAG GTCNitIndAA-3’，

5’-CCG NitIndGG GTT TGA GGG AGA AAT-3’，

5’-AAA GTT ATG AAATAA ATT GTG GTG GCC ACT AGC-3’，

5’-ACC CGG GGGTTT GAG GGA GAA A-3’，

5'-AGT CCT ACC TGA TTT GAG GTC GAA-3', and

5’-CCG AGG GTT TGA GGG AGA AAT-3’。

25. the method of any one of claims 18-24, wherein steps (a) through (h) are completed within 3 hours.

26. The method of any one of claims 18 to 24, wherein the magnetic particles comprise two populations, a first population carrying a first probe on its surface and a second population carrying a second probe on its surface.

27. The method of any one of claims 18 to 24, wherein the magnetic particles comprise one or more populations of first and second probes conjugated to their surfaces, the first probe acting to bind to a first segment of a candida amplicon and the second probe acting to bind to a second segment of the candida amplicon, wherein the magnetic particles form aggregates in the presence of the candida amplicon.

28. The method of any one of claims 18 to 24, wherein the magnetic particles comprise a first population having a first binding moiety on their surface and a second population having a second binding moiety on their surface, and the multivalent binding moiety comprises a first probe and a second probe, the first probe acting to bind to the first binding moiety and the second probe acting to bind to the second binding moiety, the binding moiety and multivalent binding moiety acting to alter aggregation of magnetic particles in the presence of a candida amplicon.

29. A method for detecting the presence of a pathogen in a whole blood sample, the method comprising:

(a) providing a 0.05 to 4.0 mL whole blood sample from a subject;

(b) placing an aliquot of the sample of step (a) in a container and amplifying the target nucleic acid in the sample to form an amplified solution comprising the target nucleic acid, wherein the target nucleic acid is characteristic of the pathogen to be detected;

(c) placing the amplified liquid sample in a detection device; and

(d) detecting a pathogen based on the results of step (c),

wherein the pathogen is selected from the group consisting of bacteria and fungi, and wherein the method is capable of detecting a pathogen concentration of 10 cells/mL in a whole blood sample.

30. The method of claim 29, wherein the detection device detects pathogens by performing optical, fluorescence, mass, density, magnetic, chromatographic, and/or electrochemical measurements on the amplified liquid sample.

31. The method of claim 29, wherein steps (a) through (d) are completed within 3 hours.

32. The method of claim 29, wherein step (b) or (c) is performed without any prior purification of the amplified solution.

33. The method of claim 29, wherein the liquid sample of step (c) comprises whole blood proteins and non-target oligonucleotides.

34. The method of claim 29, wherein the pathogen is selected from the group consisting of bacteria and fungi.

35. A method for detecting the presence of a pathogen in a whole blood sample, the method comprising:

(a) providing a whole blood sample from a subject;

(b) mixing 0.05 to 4.0 mL of a whole blood sample with a red blood cell lysing agent solution to prepare lysed red blood cells;

(c) after step (b), centrifuging the sample to form a supernatant and a precipitate, discarding some or all of the supernatant, and resuspending the precipitate to form an extract, optionally washing the precipitate before resuspending the precipitate and optionally repeating step (c);

(d) Lysing cells of the extract to form a lysate;

(e) placing the lysate of step (d) in a vessel and amplifying the target nucleic acid in the lysate to form an amplified lysate solution comprising the target nucleic acid, wherein the target nucleic acid is characteristic of the pathogen to be detected;

(f) after step (e), the amplified lysate solution is mixed with 1X 10⁶To 1X 10¹³Mixing magnetic particles/ml of the amplified lysate solution to form a liquid sample, wherein the magnetic particles have an average diameter of 150 nm to 1200 nm, 1 x 10 per particle⁸To 1X 10¹² mM^-1s^-1T of₂Relaxivity, and binding moieties on their surface that function to alter the aggregation of magnetic particles in the presence of a target nucleic acid or multivalent binding agent;

(g) placing a liquid sample in a device comprising a support defining a well for receiving a detection tube comprising magnetic particles and target nucleic acids, and having a radio frequency coil disposed around the well, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using one or more magnets and an RF pulse sequence;

(h) exposing the sample to a bias magnetic field and a sequence of RF pulses;

(i) after step (h), measuring a signal from the liquid sample; and

(j) (ii) detecting a pathogen based on the results of step (i),

36. The method of claim 35, wherein steps (a) through (i) are completed within 3 hours.

37. The method of claim 35, wherein step (i) is performed without any prior purification of the amplified lysate solution.

38. The method of claim 35, wherein the liquid sample of step (i) comprises whole blood proteins and non-target oligonucleotides.

39. The method of claim 35, wherein the pathogen is selected from the group consisting of bacteria and fungi.

40. The method of claim 35, wherein the method is capable of measuring a pathogen concentration of 10 cells/mL in a whole blood sample with a coefficient of variation of less than 15%.

41. A method for detecting the presence of a virus in a whole blood sample, the method comprising:

(a) providing a plasma sample from a subject;

(b) mixing 0.05 to 4.0 mL of the plasma sample with a lysing agent to prepare a mixture comprising lysed virus;

(c) placing the mixture (b) in a vessel and amplifying the target nucleic acid in the filtrate to form an amplified filtrate solution comprising the target nucleic acid, wherein the target nucleic acid is characteristic of the virus to be detected;

(d) After step (c), the amplified filtrate solution is mixed with 1X 10⁶To 1X 10¹³Mixing magnetic particles/ml of the amplified filtrate solution to form a liquid sample, wherein the magnetic particles have an average diameter of 150 nm to 1200 nm, 1 × 10 per particle⁸To 1X 10¹² mM^-1s^-1T of₂Relaxivity, and binding moieties on their surface that function to alter the aggregation of magnetic particles in the presence of a target nucleic acid or multivalent binding agent;

(e) placing a liquid sample in a device comprising a support defining a well for receiving a detection tube comprising magnetic particles and target nucleic acids, and having a radio frequency coil disposed around the well, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using one or more magnets and an RF pulse sequence;

(f) exposing the liquid sample to a bias magnetic field and a sequence of RF pulses;

(g) after step (f), measuring a signal from the liquid sample; and

(h) detecting a virus based on the result of step (g),

wherein the method is capable of detecting less than 100 copies of the virus in a whole blood sample.

42. The method of claim 41, wherein steps (a) through (g) are completed within 3 hours.

43. The method of any one of claims 35 to 42, wherein the magnetic particles comprise one or more populations having first and second probes conjugated to their surfaces, the first probe acting to bind to a first segment of the target nucleic acid and the second probe acting to bind to a second segment of the target nucleic acid, wherein the magnetic particles form aggregates in the presence of the target nucleic acid.

44. The method of any one of claims 35 to 42, wherein the magnetic particles comprise a first population having a first binding moiety on their surface and a second population having a second binding moiety on their surface, and the multivalent binding moiety comprises a first probe and a second probe, the first probe acting to bind to the first binding moiety and the second probe acting to bind to the second binding moiety, the binding moieties and multivalent binding moieties acting to alter aggregation of magnetic particles in the presence of the target nucleic acid.

45. A method of monitoring one or more analytes in a liquid sample derived from a patient for the purpose of diagnosing, controlling or treating a medical condition of the patient, the method comprising:

(a) will be 1 × 10 ⁶To 1X 10¹³Mixing magnetic particles/ml of liquid sample with the liquid sample, wherein the magnetic particles have an average diameter of 150 nm to 1200 nm, and 1 × 10 per particle⁸To 1X 10¹² mM^-1s^-1T of₂Relaxivity, and wherein the magnetic particles have binding moieties on their surface, the role of these binding moieties being to alter the specific aggregation of the magnetic particles in the presence of one or more analytes or multivalent binding agents;

(b) placing a liquid sample in a device, the device comprising a support defining an aperture for receiving a liquid sample comprising magnetic particles and one or more analytes, and having a radio frequency coil disposed around the aperture, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using one or more magnets and an RF pulse sequence;

(c) exposing the sample to a bias magnetic field and a sequence of RF pulses;

(d) after step (c), measuring the signal;

(e) monitoring one or more analytes based on the results of step (d); and

(f) using the results of step (e) to diagnose, control or treat the medical condition.

46. The method of claim 45, wherein the patient is immunocompromised and the one or more analytes comprise an analyte selected from the group consisting of pathogen-related analytes, antibiotics, antifungal agents, and antiviral agents.

47. The method of claim 46, wherein the one or more analytes comprise Candida species, tacrolimus, fluconazole, and creatinine.

48. The method of claim 45, wherein the patient has cancer and the one or more analytes are selected from an anti-cancer agent and a genetic marker present in cancer cells.

49. The method of claim 45, wherein the patient has or is at risk of infection and the one or more analytes comprise an analyte selected from the group consisting of a pathogen-associated analyte, an antibiotic, an antifungal, and an antiviral.

50. The method of claim 45, wherein the patient has immune inflammation and the one or more analytes comprise an analyte selected from the group consisting of an anti-inflammatory agent and TNF- α.

51. The method of claim 45, wherein the patient has a heart disease and the one or more analytes comprise a cardiac marker.

52. The method of claim 45, wherein the method is for monitoring liver function in a patient, and wherein the one or more analytes are selected from the group consisting of albumin, aspartate transaminase, alanine transaminase, alkaline phosphatase, gamma-glutamyl transpeptidase, bilirubin, alpha-fetoprotein, lactase dehydrogenase, mitochondrial antibodies, and cytochrome P450.

53. The method of claim 45, wherein the method is for determining an appropriate dose of a therapeutic agent in a patient, the method further comprising:

(i) administering a therapeutic agent to a patient;

(ii) (ii) obtaining a sample comprising the therapeutic agent or a metabolite thereof from the patient after step (i);

(iii) contacting the sample with magnetic particles and exposing the sample to a bias magnetic field and an RF pulse sequence and detecting a signal generated by the sample; and

(iv) (iv) determining the concentration of the therapeutic agent or a metabolite thereof based on the results of step (iii).

54. The method of claim 53, wherein the therapeutic agent is an anti-cancer agent, an antibiotic, or an anti-fungal agent.

55. The method of claim 45 or 53, wherein the monitoring is intermittent.

56. The method of claim 45 or 53, wherein the monitoring is continuous.

57. A method of diagnosing sepsis in a subject, the method comprising

(a) Obtaining a liquid sample derived from a patient's blood;

(b) by mixing 1 × 10⁶To 1X 10¹³Mixing magnetic particles/ml of the liquid sample with a portion of the liquid sample to prepare a first assay sample, wherein the magnetic particles have an average diameter of 150 nm to 1200 nm and a diameter of 1 × 10 per particle ⁸To 1X 10¹² mM^-1s^-1T of₂Relaxivity and wherein the magnetic particles have binding moieties on their surface, the role of said binding moieties being in one or more diseasesAltering specific aggregation of magnetic particles in the presence of a pathogen-associated analyte or multivalent binding agent;

(c) by mixing 1 × 10⁶To 1X 10¹³Mixing magnetic particles/ml of the liquid sample with a portion of the liquid sample to prepare a second assay sample, wherein the magnetic particles have an average diameter of 150 nm to 1200 nm and a diameter of 1 × 10 per particle⁸To 1X 10¹² mM^-1s^-1T of₂Relaxivity and wherein the magnetic particles have on their surface a binding moiety which functions to alter the specific aggregation of the magnetic particles in the presence of one or more analytes characteristic of sepsis selected from the group consisting of GRO-alpha, high mobility group protein B1(HMBG-1), IL-1 receptor antagonist, IL-1B, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, macrophage inflammatory protein (MIP-1), macrophage Migration Inhibitory Factor (MIF), osteopontin, RANTES (stimulated modulation of activation, normal T cell expression and secretion; or CCL5), TNF-alpha, C-reactive protein (CRP), CD64, monocyte chemotactic protein 1(MCP-1), Adenosine deaminase binding protein (ABP-26), Inducible Nitric Oxide Synthase (iNOS), lipopolysaccharide binding protein, and procalcitonin;

(d) Placing each assay sample in a device comprising a support defining an aperture for receiving a liquid sample comprising magnetic particles and one or more analytes, and having a radio frequency coil disposed around the aperture, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using one or more magnets and an RF pulse sequence;

(e) exposing each assay sample to a bias magnetic field and a sequence of RF pulses;

(f) after step (e), measuring the signal generated by the first assay sample and the signal generated by the second assay sample;

(g) monitoring the first assay sample for one or more analytes and monitoring the second assay sample for one or more analytes based on the results of step (f); and

(h) diagnosing the subject using the results of step (g).

58. The method of claim 57, wherein the one or more pathogen-associated analytes of the first assay sample are derived from a sepsis-associated pathogen selected from the group consisting of Acinetobacter baumannii, Aspergillus fumigatus, Bacteroides fragilis, blasHV, Borkholderia cepacia, Campylobacter jejuni/Coleobacter coli, Candida guillieri, Candida albicans, Candida glabrata, Candida krusei, Candida ruxiensis, Candida parapsilosis, Candida tropicalis, Clostridium perfringens, coagulase-negative staphylococci, Enterobacter aerogenes, Enterobacter cloacae, Enterobacteriaceae, enterococcus faecalis, enterococcus faecium, Escherichia coli, Haemophilus influenzae, gold plaque, Klebsiella pneumoniae, Listeria monocytogenes, Mec A gene (MRSA), Morganella morgana, Neisseria meningitidis, Neisseria species other than Neisseria meningitidis, Prevotella buccina, Prevotella intermedia, Prevotella melanogenes, Propionibacterium acnes, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella enterica, Serratia marcescens, Staphylococcus aureus, Staphylococcus haemolyticus, stenotrophomonas maltophilia, Staphylococcus saprophyticus, stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus bovis, Streptococcus dysgalactiae, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus sanguis.

59. The method of claim 57, wherein the one or more pathogen-associated analytes of the first assay sample are derived from a therapeutic-resistant bacterial strain.

60. The method of claim 59, wherein said one or more pathogen-associated analytes are derived from penicillin-resistant, methicillin-resistant, quinolone-resistant, macrolide-resistant, and/or vancomycin-resistant bacterial strains.

61. The method of claim 60, wherein the one or more pathogen-associated analytes are derived from methicillin-resistant Staphylococcus aureus or vancomycin-resistant enterococci.

62. The method of any one of claims 57-61, wherein the one or more analytes of the second assay sample are selected from GRO-a, high mobility group box protein B1(HMBG-1), IL-1 receptor antagonists, IL-1B, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, macrophage inflammatory protein (MIP-1), macrophage Migration Inhibitory Factor (MIF), osteopontin, TES (stimulated modulation, normal T cell expression and secretion; or CCL5), TNF-a, C-reactive protein (CRP), CD64, and monocyte chemotactic protein 1 (MCP-1).

63. The method of claim 57, wherein the method further comprises preparing a third assay sample to monitor the concentration of an antiviral, antibiotic or antifungal agent circulating in the bloodstream of the subject.

64. The method of claim 57, wherein the patient is immunocompromised.

65. A method of monitoring one or more analytes in a liquid sample derived from a patient for the diagnosis, control or treatment of sepsis or SIRS in the patient, the method comprising:

(a) will be 1 × 10⁶To 1X 10¹³Mixing magnetic particles/ml of liquid sample with the liquid sample, wherein the magnetic particles have an average diameter of 150 nm to 1200 nm and a diameter of 1 × 10 per particle⁸To 1X 10¹² mM^-1s^-1T of₂Relaxivity, and wherein the magnetic particles have binding moieties on their surface, the role of these binding moieties being to alter the specific aggregation of the magnetic particles in the presence of one or more analytes or multivalent binding agents;

(b) placing the liquid sample in a device comprising a holder defining an aperture for receiving a liquid sample comprising magnetic particles and one or more analytes, and having a radio frequency coil disposed around the aperture, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using one or more magnets and an RF pulse sequence;

(c) exposing the sample to a bias magnetic field and a sequence of RF pulses;

(d) After step (c), measuring the signal;

(e) monitoring one or more analytes based on the results of step (d); and

(f) using the results of step (e) to diagnose, control or treat sepsis or SIRS.

66. The method of claim 65, comprising (i) monitoring a pathogen-associated analyte, and (ii) monitoring a second analyte that is characteristic of sepsis, the second analyte being selected from GRO- α, high mobility group protein B1(HMBG-1), IL-1 receptor antagonist, IL-1B, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, macrophage inflammatory protein (MIP-1), macrophage Migration Inhibitory Factor (MIF), osteopontin, RANTES (stimulated regulation, normal T cell expression and secretion), or CCL5), TNF- α, C-reactive protein (CRP), CD64, monocyte chemotactic protein 1(MCP-1), adenosine deaminase binding protein (ABP-26), Inducible Nitric Oxide Synthase (iNOS), lipopolysaccharide binding protein, and procalcitonin.

67. The method of claim 66, wherein the pathogen-associated analyte is derived from a sepsis-associated pathogen selected from Acinetobacter baumannii, Aspergillus fumigatus, Bacteroides fragilis, blaSVV, Borkholderia cepacia, Campylobacter jejuni/Campylobacter coli, Candida guilliermondii, Candida albicans, Candida glabrata, Candida krusei, Candida ruxiensis, Candida parapsilosis, Candida tropicalis, Clostridium perfringens, coagulase-negative staphylococci, Enterobacterium aerogenes, Enterobacter cloacae, Enterobacteriaceae, enterococcus faecalis, enterococcus faecium, Escherichia coli, Haemophilus influenzae, gold bacterium, Klebsiella oxytoca, Klebsiella pneumoniae, Listeria monocytogenes, Mec A gene (MRSA), Morganella morganana, Neisseria meningitidis, Neisseria meningitidis, and Neissa, Neisseria species other than Neisseria meningitidis, Prevotella buccae, Prevotella intermedia, Prevotella melanogenes, Propionibacterium acnes, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella enterica, Serratia marcescens, Staphylococcus aureus, Staphylococcus haemolyticus, stenotrophomonas maltophilia, Staphylococcus saprophyticus, stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus bovis, Streptococcus dysgalactiae, Streptococcus mitis, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus sanguis.

68. The method of claim 66, wherein the pathogen-associated analyte is derived from a bacterial strain that is resistant to treatment.

69. The method of claim 71, wherein said pathogen-associated analyte is derived from a penicillin-resistant, methicillin-resistant, quinolone-resistant, macrolide-resistant, and/or vancomycin-resistant bacterial strain.

70. The method of claim 72, wherein the pathogen-associated analyte is derived from methicillin-resistant Staphylococcus aureus or vancomycin-resistant enterococci.

71. The method of any one of claims 66-70, wherein the second analyte is selected from GRO-alpha, high mobility group box protein B1(HMBG-1), IL-1 receptor antagonist, IL-1B, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, macrophage inflammatory protein (MIP-1), macrophage Migration Inhibitory Factor (MIF), osteopontin, RANTES (regulated activation, normal T cell expression and secretion; or CCL5), TNF-alpha, C-reactive protein (CRP), CD64, and monocyte chemotactic protein 1 (MCP-1).

72. The method of claim 66, wherein the method further comprises monitoring the concentration of an antiviral, antibiotic or antifungal agent circulating in the bloodstream of the subject.

73. The method of claim 65, wherein the patient is immunocompromised.

74. A system for detecting one or more analytes, the system comprising:

(a) a first unit comprising (a1) a permanent magnet defining a magnetic field; (a2) a holder defining an aperture for receiving a liquid sample containing magnetic particles and one or more analytes, and having a radio frequency coil disposed around the aperture, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a bias magnetic field generated using a permanent magnet and an RF pulse sequence; and (a3) one or more electrical components coupled to the radio frequency coil, the electrical components configured to amplify, condition, transmit, and/or digitize the signal; and

(b) a second unit comprising a removable cartridge sized to facilitate insertion and removal from the system, wherein the removable cartridge is a modular cartridge comprising: (i) a reagent module for containing one or more assay reagents; and (ii) a detection module comprising a detection chamber for containing a liquid sample comprising magnetic particles and one or more analytes,

Wherein the reagent module and the detection module are assemblable into the modular cartridge prior to use, and wherein the detection chamber is removable from the modular cartridge.

75. The system of claim 74, wherein the modular cartridge further comprises an inlet module, wherein the inlet module, reagent module, and detection module are assembled into the modular cartridge prior to use, and wherein the inlet module is sterilizable.

76. The system of claim 74, wherein the system further comprises a system computer having a processor for executing the assay protocol and storing assay data, and wherein the removable cartridge further comprises (i) a readable label displaying the analyte to be detected, (ii) a readable label displaying the assay protocol to be executed, (iii) a readable label displaying a patient identification number, (iv) a readable label displaying the location of the assay reagent contained in the cartridge, or (v) a readable label containing instructions for a programmable processor.

77. A system for detecting one or more analytes, the system comprising:

(a) a disposable sample holder defining an aperture for receiving a liquid sample and having a radio frequency coil received within the disposable sample holder and disposed about the aperture, the radio frequency coil configured to detect a signal generated by exposing the liquid sample to a biasing magnetic field generated by a permanent magnet and a sequence of RF pulses, wherein the disposable sample holder comprises one or more fusible links; and

(b) An MR reader comprising (b1) a permanent magnet defining a magnetic field; (b2) an RF pulse sequence and detection coil; (b3) one or more electrical components coupled to the radio frequency coil, the electrical components configured to amplify, condition, transmit, and/or digitize the signal; and (b4) one or more electrical components coupled to the fuse link and configured to apply an excess current to the fuse link, thereby causing the link to open and render the coil inoperable after a predetermined operational life.

78. The system of claim 77, wherein the electrical component connected to the radio frequency coil is inductively coupled to the radio frequency coil.

79. A removable cartridge sized to facilitate insertion into and removal from the system of the present invention, wherein the removable cartridge comprises one or more chambers for housing a plurality of reagent modules for housing one or more assay reagents, wherein the reagent modules comprise: (i) for accommodating a 1 x 10⁶To 1X 10¹³A chamber of magnetic particles having an average diameter of 100 nm to 699 nm, 1X 10 per particle⁸To 1X 10¹² mM^-1s^-1T of₂Relaxivity, and binding moieties on their surface which function to alter the specific aggregation of magnetic particles in the presence of one or more analytes or multivalent binding agents; and (ii) a chamber for containing a buffer.

80. A removable cartridge sized to facilitate insertion and removal from the system of the present invention, wherein the removable cartridge comprises one or more chambers for housing a plurality of reagent modules for housing one or more assay reagents, wherein a reagent module comprises (i) a housing for housing 1 x 10 assay reagents⁶To 1X 10¹³A chamber of magnetic particles having an average diameter of 700 nm to 1200 nm, 1X 10 per particle⁹To 1X 10¹² mM^-1s^-1T of₂Relaxivity, and oligonucleotide binding moieties on their surface, which function to alter the specific aggregation of magnetic particles in the presence of one or more analytes; and (ii) a chamber for containing a buffer.

81. The removable cartridge of claim 79 or 80, wherein the magnetic particles and the buffer are housed together in a single chamber within the cartridge.

82. The removable cartridge of claim 79 or 80, wherein the buffer comprises 0.1% to 3% (w/w) albumin, 0.01% to 0.5% non-ionic surfactant, lysing agent, or a combination thereof.

83. The removable cartridge of claim 79 or 80, further comprising a chamber containing beads for lysing cells.

84. The removable cartridge of claim 79 or 80, further comprising a chamber containing a polymerase.

85. The removable cartridge of claim 79 or 80, further comprising a chamber comprising one or more primers.