Nmr systems and methods for the rapid detection of analytes

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

• (a) contacting a solution with magnetic particles to produce a liquid sample comprising from 1×106 to 1×1013 magnetic particles per milliliter of the liquid sample, wherein the magnetic particles have a mean diameter of from 150 nm to 950 nm, a T2 relaxivity per particle of from 1×108 to 1×1012 mM−1s−1, and have binding moieties on their surface, the binding moieties operative to alter an aggregation of the magnetic particles in the presence of the analyte;
• (b) placing the liquid sample in a device, the device comprising a support defining a well holding the liquid sample comprising the magnetic particles, the multivalent binding agent, and the analyte, and having an RF coil disposed about the well, the RF coil configured to detect a signal produced by exposing the liquid sample to a bias magnetic field created using one or more magnets and an RF pulse sequence;
• (c) exposing the sample to a bias magnetic field and an RF pulse sequence;
• (d) following step (c), measuring the signal;
• and
• (e) on the basis of the result of step (d), detecting the presence or concentration of an analyte.

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 the whole blood sample with an erythrocyte lysis agent to produce disrupted red blood cells;
• (c) following 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 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 detection tube and amplifying a 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) following step (e), adding to the detection tube from 1×106 to 1×1013 magnetic particles per milliliter of the amplified lysate solution, wherein the magnetic particles have a mean diameter of from 700 nm to 1200 nm and binding moieties on their surface, the binding moieties operative to alter aggregation of the magnetic particles in the presence of the target nucleic acid or a multivalent binding agent;
• (g) placing the detection tube in a device, the device comprising a support defining a well for holding the detection tube comprising the magnetic particles and the target nucleic acid, and having an RF coil disposed about the well, the RF coil configured to detect a signal produced by exposing the liquid sample to a bias magnetic field created using one or more magnets and an RF pulse sequence;
• (h) exposing the sample to a bias magnetic field and an RF pulse sequence;
• (i) following step (h), measuring the signal from the detection tube;
• and
• (j) on the basis of the result of step (i), detecting the pathogen.

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 said cells to form a lysate;
• (c) amplifying a target nucleic acid in the lysate to form an amplified lysate solution comprising the target nucleic acid;
• (d) following step (c), adding to a detection tube the amplified lysate solution and from 1×106 to 1×1013 magnetic particles per milliliter of the amplified lysate solution, wherein the magnetic particles have a mean diameter of from 700 nm to 1200 nm and binding moieties on their surface, the binding moieties operative to alter aggregation of the magnetic particles in the presence of the target nucleic acid or a multivalent binding agent;
• (e) placing the detection tube in a device, the device comprising a support defining a well for holding the detection tube comprising the magnetic particles and the target nucleic acid, and having an RF coil disposed about the well, the RF coil configured to detect a signal produced by exposing the liquid sample to a bias magnetic field created using one or more magnets and an RF pulse sequence;
• (f) exposing the sample to a bias magnetic field and an RF pulse sequence;
• (h) following step (f), measuring the signal from the detection tube;
• and
• (i) on the basis of the result of step (h), detecting the target nucleic acid.

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

• (a) providing an extract produced by lysing the 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 nucleic acids therein to form an amplified lysate solution comprising from 40% (w/w) to 95% (w/w) the target nucleic acid and from 5% (w/w) to 60% (w/w) nontarget nucleic acid;
• (d) following step (c), adding to the detection tube from 1×106 to 1×1013 magnetic particles per milliliter of the amplified lysate solution, wherein the magnetic particles have a mean diameter of from 700 nm to 1200 nm and binding moieties on their surface, the binding moieties operative to alter aggregation of the magnetic particles in the presence of the target nucleic acid or a multivalent binding agent;
• (e) placing the detection tube in a device, the device comprising a support defining a well for holding the detection tube comprising the magnetic particles and the target nucleic acid, and having an RF coil disposed about the well, the RF coil configured to detect a signal produced by exposing the liquid sample to a bias magnetic field created using one or more magnets and an RF pulse sequence;
• (f) exposing the sample to a bias magnetic field and an RF pulse sequence;
• (g) following step (f), measuring the signal from the detection tube;
• and
• (h) on the basis of the result of step (g), detecting the target nucleic acid, wherein step (g) is carried out without any prior purification of the amplified lysate solution.

A method for detecting the presence of a Candida species in a liquid sample, the method comprising:

• (a) lysing the Candida cells in the liquid sample;
• (b) amplifying a nucleic acid to be detected in the presence of a forward primer and a reverse primer, each of which is universal to multiple Candida species to form a solution comprising a Candida amplicon;
• (c) contacting the solution with magnetic particles to produce a liquid sample comprising from 1×106 to 1×1013 magnetic particles per milliliter of the liquid sample, wherein the magnetic particles have a mean diameter of from 700 nm to 1200 nm, and binding moieties on their surface, the binding moieties operative to alter aggregation of the magnetic particles in the presence of the Candida amplicon or a multivalent binding agent;
• (d) placing the liquid sample in a device, the device comprising a support defining a well for holding the liquid sample comprising the magnetic particles and the Candida amplicon, and having an RF coil disposed about the well, the RF coil configured to detect a signal produced by exposing the liquid sample to a bias magnetic field created using one or more magnets and an RF pulse sequence;
• (e) exposing the sample to a bias magnetic field and an RF pulse sequence;
• (f) following step (e), measuring the signal;
• and
• (g) on the basis of the result of step (f), determining whether the Candida species was present in the sample.

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

• (a) providing from 0.05 to 4.0 mL of the whole blood sample from a subject;
• (b) placing an aliquot of the sample of step (a) in a container and amplifying a 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 detecting device;
• and
• (d) on the basis of the result of step (c), detecting the pathogen,
• wherein the method is capable of detecting a pathogen concentration of at least 3 cells/mL in the whole blood sample.

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 from 0.05 to 4.0 mL of the whole blood sample with an erythrocyte lysis agent to produce disrupted red blood cells;
• (c) following 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 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 container and amplifying a 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) following step (e), mixing the amplified lysate solution with from 1×106 to 1×1013 magnetic particles per milliliter of the amplified lysate solution to form a liquid sample, wherein the magnetic particles have a mean diameter of from 150 nm to 1200 nm, a T2 relaxivity per particle of from 1×108 to 1×1012 mM−1s−1, and binding moieties on their surface, the binding moieties operative to alter aggregation of the magnetic particles in the presence of the target nucleic acid or a multivalent binding agent;
• (g) placing the liquid sample in a device, the device comprising a support defining a well for holding the detection tube comprising the magnetic particles and the target nucleic acid, and having an RF coil disposed about the well, the RF coil configured to detect a signal produced by exposing the liquid sample to a bias magnetic field created using one or more magnets and an RF pulse sequence;
• (h) exposing the sample to a bias magnetic field and an RF pulse sequence;
• (i) following step (h), measuring the signal from the liquid sample;
• and
• (j) on the basis of the result of step (i), detecting the pathogen,
• wherein the pathogen is selected from bacteria and fungi, and wherein the method is capable of detecting a pathogen concentration of 10 cells/mL in the whole blood sample.

A method of quantifying a target nucleic acid molecule in a sample, said method comprising amplifying the target nucleic acid molecule in an amplification reaction mixture in a detection tube resulting in the production of amplicons corresponding to the target nucleic acid molecule, wherein said amplification reaction mixture comprises:

• (1) a target nucleic acid molecule;
• and
• (2) amplification primers specific for said target nucleic acid molecule;
• wherein said amplification is performed in a device, the device comprising a support defining a well for holding the detection tube comprising the superparamagnetic particles and the target nucleic acid molecule, and having an RF coil disposed about the well, the RF coil configured to detect a signal produced by exposing the sample to a bias magnetic field created using one or more magnets and an RF pulse sequence;
• and said amplification comprising the following steps:
• (a) performing one or more cycles of amplification;
• (b) exposing said amplification reaction mixture to superparamagnetic particles under conditions permitting the aggregation or disaggregation of said superparamagnetic particles;
• (c) exposing the sample to a bias magnetic field and an RF pulse sequence;
• (d) following step (c), measuring the signal from the detection tube;
• (e) repeating steps (a)-(d) until a desired amount of amplification is obtained;
• and
• (f) on the basis of the result of step (d), quantifying the amplicons present at the corresponding cycle of amplification;
• wherein the initial quantity of target nucleic acid molecule in said sample is determined based on the quantity of amplicons determined at each cycle of said amplification.

A method of monitoring one or more analytes in a liquid sample derived from a patient for the diagnosis, management, or treatment of a medical condition in a patient, the method comprising:

• (a) combining with the liquid sample from 1×106 to 1×1013 magnetic particles per milliliter of the liquid sample, wherein the magnetic particles have a mean diameter of from 150 nm to 1200 nm and a T2 relaxivity per particle of from 1×108 to 1×1012 mM−1s−1, and wherein the magnetic particles have binding moieties on their surfaces, the binding moieties operative to alter the specific aggregation of the magnetic particles in the presence of the one or more analytes or a multivalent binding agent;
• (b) placing the liquid sample in a device, the device comprising a support defining a well for holding the liquid sample comprising the magnetic particles and the one or more analytes, and having an RF coil disposed about the well, the RF coil configured to detect a signal produced by exposing the liquid sample to a bias magnetic field created using one or more magnets and an RF pulse sequence;
• (c) exposing the sample to the bias magnetic field and the RF pulse sequence;
• (d) following step (c), measuring the signal;
• (e) on the basis of the result of step (d), monitoring the one or more analytes;
• and
• (f) using the result of step (e) to diagnose, manage, or treat the medical condition.

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

• (a) obtaining a liquid sample derived from the blood of a patient;
• (b) preparing a first assay sample by combining with a portion of the liquid sample from 1×106 to 1×1013 magnetic particles per milliliter of the liquid sample, wherein the magnetic particles have a mean diameter of from 150 nm to 1200 nm and a T2 relaxivity per particle of from 1×108 to 1×1012 mM−1s−1, and wherein the magnetic particles have binding moieties on their surfaces, the binding moieties operative to alter the specific aggregation of the magnetic particles in the presence of one or more pathogen-associated analytes or a multivalent binding agent;
• (c) preparing a second assay sample by combining with a portion of the liquid sample from 1×106 to 1×1013 magnetic particles per milliliter of the liquid sample, wherein the magnetic particles have a mean diameter of from 150 nm to 1200 nm and a T2 relaxivity per particle of from 1×108 to 1×1012 mM−1s−1, and wherein the magnetic particles have binding moieties on their surfaces, the binding moieties operative to alter the specific aggregation of the magnetic particles in the presence of one or more analytes characteristic of sepsis selected from GRO-alpha, High mobility group-box 1 protein (HMBG-1), IL-1 receptor, 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 on activation, normal T-cell expressed and secreted;
• or CCL5), TNF-α, C-reactive protein (CRP), CD64, monocyte chemotactic protein 1 (MCP-1), adenosine deaminase binding protein (ABP-26), inducible nitric oxide synthetase (iNOS), lipopolysaccharide binding protein, and procalcitonin;
• (d) placing each assay sample in a device, the device comprising a support defining a well for holding the liquid sample comprising the magnetic particles and the one or more analytes, and having an RF coil disposed about the well, the RF coil configured to detect a signal produced by exposing the liquid sample to a bias magnetic field created using one or more magnets and an RF pulse sequence;
• (e) exposing each assay sample to the bias magnetic field and the RF pulse sequence;
• (f) following step (e), measuring the signal produced by the first assay sample and the signal produced by the second assay sample;
• (g) on the basis of the result of step (f), monitoring the one or more analytes of the first assay sample and monitoring the one or more analytes of the second assay sample;
• and
• (h) using the results of step (g) to diagnose the subject.

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

• (a) combining with the liquid sample from 1×106 to 1×1013 magnetic particles per milliliter of the liquid sample, wherein the magnetic particles have a mean diameter of from 150 nm to 1200 nm and a T2 relaxivity per particle of from 1×108 to 1×1012 mM−1s−1, and wherein the magnetic particles have binding moieties on their surfaces, the binding moieties operative to alter the specific aggregation of the magnetic particles in the presence of the one or more analytes or a multivalent binding agent;
• (b) placing the liquid sample in a device, the device comprising a support defining a well for holding the liquid sample comprising the magnetic particles and the one or more analytes, and having an RF coil disposed about the well, the RF coil configured to detect a signal produced by exposing the liquid sample to a bias magnetic field created using one or more magnets and an RF pulse sequence;
• (c) exposing the sample to the bias magnetic field and the RF pulse sequence;
• (d) following step (c), measuring the signal;
• (e) on the basis of the result of step (d), monitoring the one or more analytes;
• and
• (f) using the result of step (e) to diagnose, manage, or treat the sepsis or SIRS.

A system for the detection of one or more analytes, the system comprising:

• (a) a first unit comprising (a1) a permanent magnet defining a magnetic field;
• (a2) a support defining a well for holding a liquid sample comprising magnetic particles and the one or more analytes and having an RF coil disposed about the well, the RF coil configured to detect a signal produced by exposing the liquid sample to a bias magnetic field created using the permanent magnet and an RF pulse sequence;
• and (a3) one or more electrical elements in communication with the RF coil, the electrical elements configured to amplify, rectify, transmit, and/or digitize the signal;
• and
• (b) a second unit comprising a removable cartridge sized to facilitate insertion into and removal from the system, wherein the removable cartridge is a modular cartridge comprising (i) a reagent module for holding one or more assay reagents;
• and (ii) a detection module comprising a detection chamber for holding a liquid sample comprising the magnetic particles and the one or more analytes,
• wherein the reagent module and the detection module can be assembled into the modular cartridge prior to use, and wherein the detection chamber is removable from the modular cartridge.