WO2010039179A1 - Détection ultra-sensible de molécules ou d'enzymes - Google Patents

Détection ultra-sensible de molécules ou d'enzymes Download PDF

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Publication number
WO2010039179A1
WO2010039179A1 PCT/US2009/005248 US2009005248W WO2010039179A1 WO 2010039179 A1 WO2010039179 A1 WO 2010039179A1 US 2009005248 W US2009005248 W US 2009005248W WO 2010039179 A1 WO2010039179 A1 WO 2010039179A1
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WIPO (PCT)
Prior art keywords
substrate
particles
dissociated species
molecules
reaction vessels
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PCT/US2009/005248
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English (en)
Inventor
David C. Duffy
Evan Ferrell
Jeffrey D. Randall
David M. Rissin
David R. Walt
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Quanterix Corporation
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Publication date
Priority claimed from US12/236,490 external-priority patent/US20100075355A1/en
Priority claimed from US12/236,486 external-priority patent/US8222047B2/en
Priority claimed from US12/236,488 external-priority patent/US20100075439A1/en
Application filed by Quanterix Corporation filed Critical Quanterix Corporation
Publication of WO2010039179A1 publication Critical patent/WO2010039179A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

Definitions

  • the present invention relates to systems and methods for detecting analyte molecules or particles in a fluid sample and in some cases, determining a measure of the concentration of the molecules or particles in the fluid sample.
  • these known processes include enzyme-linked immunosorbent assays (ELISA) for amplifying the signal in antibody-based assays, as well as the polymerase chain reaction (PCR) for amplifying target DNA strands in DNA-based assays.
  • ELISA enzyme-linked immunosorbent assays
  • PCR polymerase chain reaction
  • a more sensitive but indirect protein target amplification technique, called immunoPCR makes use of oligonucleotide markers, which can subsequently be amplified using PCR and detected using a DNA hybridization assay
  • One disadvantage of typical known methods and/or systems for accurately detecting and, optionally, quantifying low concentrations of a particular analyte in solution is that they are based on ensemble responses in which many analyte molecules give rise to the measured signal. Most detection schemes require that a large number of molecules are present in the ensemble for the aggregate signal to be above the detection threshold. This disadvantage limits the sensitivity of most detection techniques and the dynamic range (i.e., the range of concentrations that can be detected). Many of the known methods and techniques are further plagued with problems of non-specific binding, which is the binding of analyte molecules/particles to be detected or reporter species non-specifically to sites other than those expected. This leads to an increase in the background signal, and therefore limits the lowest concentration that may be accurately or reproducibly detected.
  • the present invention relates to systems and methods for detecting analyte molecules or particles in a fluid sample and in some cases, determining a measure of the concentration of the molecules or particles in the fluid sample.
  • the subject matter of the present invention involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles.
  • the invention is directed towards a method.
  • a method of detecting analyte molecules or particles in a fluid sample comprises exposing a substrate comprising a plurality of capture components to a sample comprising a plurality of analyte molecules or particles, so that analyte molecules or particles associate with capture components to form a plurality of complexes, each complex being immobilized with respect to the substrate and comprising at least one capture component and at least one analyte molecule or particle, dissociating at least a portion of each complex to form a plurality of dissociated species, which are not immobilized with respect to the substrate, partitioning the plurality of dissociated species across a plurality of reaction vessels, and determining the presence or absence of a dissociated species in at least one reaction vessel.
  • a system for detecting analyte molecules or particles in a fluid sample comprises a first substrate comprising a plurality of capture components able to become specifically immobilized with respect to a plurality of analyte molecules or particles of a sample, such that the analyte molecules or particles associate with capture components to form a plurality of complexes, wherein the complexes are susceptible to being dissociated from the first substrate by a dissociating agent to form a plurality of dissociated species, and a second substrate comprising an array comprising a plurality of reaction vessels, the reaction vessels being configured to receive and contain dissociated species therein.
  • a method for determining a measure of the concentration of analyte molecules or particles in a fluid sample comprises capturing a plurality of analyte molecules or particles on a first substrate, releasing a plurality of molecules or particles from the first substrate, detecting molecules or particles released from the first substrate on or within a second substrate comprising a plurality of reaction vessels, and determining a measure of the concentration of the analyte molecules or particles in the fluid sample based on the detection of molecules or particles released from the first substrate on or within the second substrate.
  • a method for determining a measure of the concentration of the analyte molecules or particles in a fluid sample comprises capturing a plurality of analyte molecules or particles on a first substrate, releasing a plurality of molecules or particles from the first substrate, detecting molecules or particles released from the first substrate on or within a second substrate, and determining a measure of the concentration of the analyte molecules or particles in the fluid sample based on the detection of molecules or particles released from the first substrate, wherein the true concentration of the analyte molecules or particles in the fluid sample is less than about 50 x 10 "15 molar, and wherein the measure of the concentration determined in the determining act differs from the true concentration by no greater than 10%.
  • a method for determining a measure of the concentration of analyte molecules or particles in a fluid sample comprises capturing a plurality of analyte molecules or particles on a first substrate, releasing a plurality of molecules or particles from the first substrate, immobilizing at least one particle comprising a binding partner for the molecule or particle released from the first substrate to each of at least a fraction of the plurality of molecules or particles released from the first substrate to form a plurality of complexes, detecting the plurality of complexes formed in the immobilizing act, and determining a measure of the concentration of the analyte molecules or particles in the fluid sample based on the detection of the complexes.
  • a method for determining a measure of the concentration of analyte molecules or particles in a fluid sample comprises, capturing a plurality of analyte molecules or particles on a first substrate by immobilizing the plurality of analyte molecules or particles on the first substrate, releasing a plurality of immobilized molecules or particles from the first substrate, wherein the number of molecules or particles released is related to the number of analyte molecules or particles captured, detecting molecules or particles released from the first substrate on or within a second substrate, wherein at least a fraction of the molecules or particles released from the first substrate become immobilized with respect to the second substrate, and wherein the number of molecules or particles detected on or within the second substrate is no greater than the number of molecules or particles which are released from the first substrate, and determining a measure of the concentration of the analyte molecules or particles in the fluid sample based on the detection of molecules or particles released from the first substrate.
  • a method for determining a measure of the concentration of analyte molecules or particles in a fluid sample comprises capturing a plurality of analyte molecules or particles on a first substrate by immobilizing the plurality of analyte molecules or particles on the first substrate, releasing a plurality of immobilized molecules or particles from the first substrate, wherein the number of molecules or particles released is related to the number of analyte molecules or particles captured, immobilizing at least one first binding ligand with respect to each of at least a fraction of the plurality of analyte molecules or particles or to each of at least a fraction of the plurality of molecules or particles released from the first substrate, capturing at least a fraction of the plurality of molecules or particles released from the first substrate on or within a second substrate, exposing the second substrate to a plurality of precursor labeling agent molecules, wherein the plurality of precursor labeling agent molecules are converted to a plurality of labeling agent molecules upon exposure to first binding ligands, and
  • a method of detecting analyte molecules or particles in a fluid sample comprises exposing a substrate comprising a plurality of capture components to a sample comprising a plurality of analyte molecules or particles, so that analyte molecules or particles associate with capture components to form a plurality of complexes, each complex being immobilized with respect to the substrate and comprising at least one capture component and at least one analyte molecule or particle, dissociating at least a portion of each complex to form a plurality of dissociated species, which are not immobilized with respect to the substrate, and detecting at least some of the plurality of the dissociated species substantially simultaneously, wherein substantially all of the detected dissociated species are spatially separated with respect to the other detected dissociated species during detection, such that detection is able to resolve individual dissociated species of the plurality of dissociated species.
  • a method of detecting analyte molecules or particles in a fluid sample comprises exposing a substrate comprising a plurality of capture components to a sample comprising a plurality of analyte molecules or particles, so that analyte molecules or particles associate with capture components to form a plurality of complexes, each complex being immobilized with respect to the substrate and comprising at least one capture component and at least one analyte molecule or particle, exposing the plurality of complexes to a reducing agent to cause at least a portion of at least some of the plurality of complexes to dissociate from the substrate to form a plurality of dissociated species, which are not immobilized with respect to the substrate, and determining the presence of and/or a measurement of the amount or concentration of the analyte molecules or particles in the fluid sample based at least in part on detecting at least some of the dissociated species.
  • a system for detecting analyte molecules or particles in a fluid sample comprises a first substrate comprising a plurality of capture components able to become specifically immobilized with respect to a plurality of analyte molecules or particles in the fluid sample, such that the analyte molecules or particles associate with capture components to form a plurality of complexes, wherein at least a portion of at least some of the complexes is susceptible to being dissociated from the first substrate by exposure to a reducing agent, to form a plurality of dissociated species, a second substrate configured to receive and contain dissociated species therein or thereon, and a detector configured to detect dissociated species in or on the second substrate.
  • a method for determining a measure of the concentration of analyte molecules or particles in a fluid sample comprises capturing a plurality of analyte molecules or particles on a substrate, causing at least a portion of at least some of the plurality of molecules or particles to dissociate from the substrate by exposing the plurality of molecules or particles to a reducing agent, thereby forming a plurality of dissociated species, detecting at least a some of the dissociated species, and determining a measure of the concentration of the analyte molecules or particles in the fluid sample based on the detection of at least some of the plurality of dissociated species.
  • a method for determining a measure of the concentration of analyte molecules or particles in a fluid sample comprises capturing a plurality of analyte molecules or particles on a first substrate by immobilizing the plurality of analyte molecules or particles on the first substrate, exposing the first substrate to a reducing agent to cause a plurality of dissociated species to be released from the substrate, detecting at least some of the plurality of dissociated species, wherein the number of dissociated species detected is no greater than the number of dissociated species released from the substrate in the exposing step and is also no greater than the number of analyte molecules or particles captured on the first substrate in the capturing step, and determining a measure of the concentration of the analyte molecules or particles in the fluid sample based on the detection of dissociated species.
  • a method of detecting analyte molecules or particles in a fluid sample comprises exposing a substrate comprising a plurality of capture components to a sample comprising a plurality of analyte molecules or particles, so that analyte molecules or particles associate with capture components to form a plurality of complexes, each complex being immobilized with respect to the substrate and comprising at least one capture component, at least one analyte molecule or particle, and at least one disulfide linkage, cleaving at least some of the disulfide linkages to cause at least a portion of at least some of the plurality of complexes to dissociate from the substrate to form a plurality of dissociated species, which are not immobilized with respect to the substrate, and determining the presence of and/or a measurement of the amount or concentration of the analyte molecules or particles in the fluid sample based at least in part on detecting a least some of the dissociated species.
  • a method of detecting analyte molecules or particles in a fluid sample comprises exposing a substrate comprising a plurality of capture components to a sample comprising a plurality of analyte molecules or particles, so that analyte molecules or particles associate with capture components to form a plurality of complexes, each complex being immobilized with respect to the substrate and comprising at least one capture component and at least one analyte molecule or particle, exposing the plurality of complexes to a reducing agent to cause at least a portion of at least some of the plurality of complexes to dissociate from the substrate to form a plurality of dissociated species, which are not immobilized with respect to the substrate, and detecting at least some of the plurality of the dissociated species substantially simultaneously, wherein each of the detected dissociated species are spatially separated with respect to the other detected dissociated species during detection, such that detection is able to resolve individual dissociated species of the plurality of dissociated species
  • a method of detecting analyte molecules or particles in a fluid sample comprises exposing a substrate comprising a plurality of capture components to a sample comprising a plurality of analyte molecules or particles, so that analyte molecules or particles associate with capture components to form a plurality of complexes, each complex being immobilized with respect to the substrate and comprising at least one capture component, at least one analyte molecule or particle, and at least one enzymatic component, exposing the substrate to a dissociating agent, which has essentially no specific affinity for the capture components, to cause at least a portion of at least some of the plurality of complexes to dissociate from the substrate to form a plurality of dissociated species, which are not immobilized with respect to the substrate and wherein at least some of the dissociated species each comprise at least a portion of the enzymatic component, and determining the presence of and/or a measurement of the amount or concentration of the analyte molecules
  • a method for determining a measure of the concentration of analyte molecules or particles in a fluid sample comprises exposing a substrate comprising a plurality of capture components to a sample comprising a plurality of analyte molecules or particles, so that analyte molecules or particles associate with capture components to form a plurality of complexes, each complex being immobilized with respect to the substrate and comprising at least one capture component and at least one analyte molecule or particle, exposing the plurality of complexes to a plurality of binding ligands comprising an enzymatic component, wherein at least one binding ligand is immobilized with respect to at least some of the complexes, exposing the plurality of complexes to a dissociating agent, which has essentially no specific affinity for the capture components, to dissociate at least a portion of at least some of the plurality of molecules or particles from the first substrate, thereby forming a plurality of dissociated species, wherein at least some
  • a method of detecting analyte molecules or particles in a fluid sample comprises exposing a substrate comprising a plurality of capture components to a sample comprising a plurality of analyte molecules or particles, so that analyte molecules or particles associate with capture components to form a plurality of complexes, each complex being immobilized with respect to the substrate and comprising at least one capture component, at least one analyte molecule or particle, and at least one enzymatic component, exposing the substrate to a dissociating agent to cause at least a portion of at least some of the plurality of complexes to dissociate from the substrate to form a plurality of dissociated species, which are not immobilized with respect to the substrate and wherein at least some of the dissociated species each comprise at least a portion of the enzymatic component, and detecting a plurality of the dissociated species substantially simultaneously, wherein each of the detected dissociated species are spatially separated with respect to the other detected dis
  • FIG. 1 is a schematic flow diagram depicting one embodiment of steps (A-E) for performing one method of the present invention
  • FIG. 2 is a schematic flow diagram depicting a sequence of steps (A-E) of one embodiment of one method of the present invention
  • FIG. 3 is a schematic flow diagram depicting embodiments (A-C) of how an immobilized complex may dissociate from a substrate according to certain embodiments;
  • FIG. 4 is a schematic flow diagram illustrating immobilized complexes comprising cleavable linkages and cleaving of the cleavable linkages, according to various embodiments (A-C);
  • FIG. 5 is a schematic flow diagram depicting steps (A-E) of one embodiment of one method of the present invention
  • FIG. 6 depicts a second substrate comprising a plurality of capture components with which a plurality of dissociated species may become associated as shown by steps (A-B), according to one embodiment;
  • FIG. 7 is a schematic flow diagram depicting steps (A-E) of one embodiment of one method of the present invention.
  • FIG. 8 is a schematic flow diagram depicting steps (A-E) of one method of the present invention, according to one embodiment
  • FIG. 9 is a schematic flow diagram depicting steps (A-D) of one embodiment of one method of the present invention.
  • FIGS. 10A- 1OC show non-limiting examples of substrates that may be used according to certain embodiments of the present invention
  • FIG. 11 is a schematic flow diagram depicting an embodiment of a method (steps A-D) for the formation of a plurality of reaction vessels through mating of a substrate and a sealing component and depicting examples of the size (E, F) of a sealing component relative to a substrate
  • FIG. 12 shows side view cross-section schematics showing an etched fiber optic bundle that forms an array of micro wells (steps A-D), according to one embodiment of the present invention, and depicting the localization of a capture component within a reaction vessel (steps E-G), according to some embodiments of the present invention;
  • FIG. 13A is a photocopy of a photomicrograph of an entire fiber optic array, with an inset showing a close-up of the bundle, according to one embodiment of the present invention
  • FIG. 13B is a photocopy of an atomic force microscope (AFM) image of a portion of an etched surface of a fiber optic array, according to one embodiment of the present invention
  • FIG. 14A depicts an experimental set-up for detection using light, according to one embodiment of the present invention
  • FIG. 14B shows a fiber optic array that has been sealed with a sealing component, according to one embodiment
  • FIG. 15A is a schematic flow diagram depicting a method of indirectly detecting a dissociated species and/or a molecule or particle released from a substrate, according to some embodiments;
  • FIG. 15B is a schematic flow diagram depicting a method of indirectly detecting a dissociated species and/or a molecule or particle released from a substrate, according to some embodiments;
  • FIG. 16 is a schematic flow diagram depicting steps (A-B) of one embodiment of one method of the present invention in which dissociated species are immobilized with respect to particles prior to partitioning across a plurality of reaction vessels;
  • FIG. 17A and 17B show non-limiting examples of a system employing an optical detection system of the present invention according to some embodiments
  • FIG. 18 is a schematic block diagram showing a system employing a fiber optic assembly with an optical detection system according to an embodiment of the invention
  • FIG. 19 shows a graph of a schematic calibration curve of a form that may be used to determine the concentration of an analyte molecule or particle in a fluid sample, according to some embodiments of the present invention
  • FIG. 2OA shows a plot of the percent active wells against the concentration of TNF-alpha in serum samples, according to one example of the present invention
  • FIG. 2OB shows a magnified view of the lower end of the graph in FIG. 2OA
  • FIG. 21 shows a plot of the percent active wells against concentration of prostate specific antigen (PSA) in serum samples, in one embodiment of the present invention
  • FIG. 22 shows a plot of the percent active wells against concentration of interleukin-6 (IL-6) in serum samples, in one embodiment of the present invention
  • FIG. 23 shows a plot of the percent active wells against concentration of vascular endothelial growth factor (VEGF) in serum samples, in one embodiment of the present invention
  • FIG. 24 is a graph depicting the effects of the concentration of beta- mercaptoethanol on the release of dissociated species, in one embodiment of the invention.
  • FIG. 25 is a graph depicting the effects of removing beta-mercaptoethanol before detection of the dissociated species on an array, in one embodiment of the invention
  • FIG. 26 is a graph depicting results of an example of the optimization of non- specific binding, according to one method of the present invention
  • FIG. 27 is a graph depicting results of the detection of tumor necrosis factor- alpha (TNF-alpha) using a method/system of the present invention, according to one embodiment
  • FIG. 28 is a graph depicting results of the detection of TNF-alpha using a standardized ELISA assay
  • FIG. 29 is a graph depicting results of the detection of TNF-alpha using one embodiment of a method of the present invention, wherein the first substrate comprised beads and the beads were modified with antibody (Ab) stabilized with bovine serum albumin (BSA);
  • FIG. 30 is a graph depicting results of an assay involving the immobilization of
  • FIG. 31 is a graph depicting of an assay using a 96-well plate as the first substrate, followed by rebinding of a plurality of dissociated species to a biotin array, according to one embodiment of the present invention
  • FIG. 32 is a graph depicting the results of method according to an embodiment of the present invention, wherein one of the binding ligands of the assay comprised a photocleavable linkage;
  • FIG. 33 is a graph depicting the detection of TNF-alpha using a standardized ELISA assay
  • FIG. 34 is a graph depicting the detection of TNF-alpha using a method/system of the present invention, according to one embodiment.
  • the present invention relates to systems and methods for the detection and/or quantification of analyte molecules, particles (such as, for example, cells, cell organelles and other biological or non-biological particulates) and the like, in a sample.
  • the subject matter of the present invention involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles. It should be understood, that while much of the discussion below is directed to analyte molecules, this is by way of example only, and other materials may be detected and/or quantified, for example, analyte particles. Particular examples of analytes will be discussed more below.
  • Non-specific binding is the binding or association in a non-specific fashion of one component of an assay with another component of the assay with which it is not desirable that it interact. For example, association, binding, or immobilization of a binding ligand with the material forming a reaction vessel wall as opposed to with an analyte molecule or particle to which it has binding specificity. Non-specific binding may lead to false positive signals. Non-specific binding may not only affect the accuracy of the assay measurement, but may also limit the lowest level of detection.
  • methods and/or systems of the present invention may allow for the detection and/or quantification of analyte molecules in a sample at a lower detection limit as compared to typical current technologies.
  • certain embodiments of the methods and/or systems of the present invention may also allow for the detection and/or quantification of analyte molecules in certain samples that have previously been undetected and/or unquantif ⁇ able in such samples because of the very low concentration in which they are present.
  • Capture-and-release systems and methods of certain embodiments of present invention which utilize a first substrate and a second substrate also can provide additional benefits as compared to a basic capture assay that employs only a single substrate.
  • the first substrate may be configured to provide a greater surface area, greater volumetric capacity, and/or increased number/density of capture components as compared to an assay employing only one substrate. That is, the surface area and/or volumetric capacity of the first substrate may be greater than the substrate used in an assay employing only one substrate such that the analyte molecules may be more effectively, rapidly and/or thoroughly immobilized with respect to the first substrate versus immobilization of the target analyte in the sample directly on a single substrate where detection also occurs.
  • the required exposure time of the substrate to the analyte molecules may also be decreased in some cases using an assay of the present invention due to an increase in surface area and/or volumetric capacity of the substrate (e.g., by increasing binding kinetics).
  • the methods for detection and/or quantifying analyte molecules in a sample comprise a capture-and-release protocol, wherein the analyte molecules are captured on a substrate and form a plurality of complexes associated with the substrate.
  • the substrate may be optionally exposed to additional reaction components such as at least one binding ligand, as discussed more below.
  • At least a portion of the plurality of complexes may be dissociated or otherwise released from the substrate, for example, by exposing the complexes to a dissociating agent (e.g., a reducing agent) and/or by cleaving a cleavable linkage (e.g., a disulfide linkage or a photocleavable linkage) comprising at least a portion of the complexes dissociated.
  • a dissociable/dissociated complex may comprise an enzymatic component, which in certain embodiments may be used to facilitate dissociation and/or detection of the dissociated complex.
  • At least a portion of the plurality of dissociated complexes may be detected and may be used to determine the presence of and/or quantify the analyte molecules in the sample.
  • the dissociated complexes may be detected either directly or indirectly, as explained in more detail below.
  • FIG. 1 An assay method according to some embodiments of the present invention is illustrated in FIG. 1.
  • a first substrate 200 is exposed to a sample comprising a plurality of analyte molecules 202 (step (A)). At least a portion of the analyte molecules associate with the first substrate 200 to form a plurality of immobilized complexes 204, however, some analyte molecules 206 may not associate with the first substrate (step (B)). At least a portion of at least some of the immobilized complexes are dissociated from the substrate to form a plurality of dissociated species 210 (step (C)). The dissociated species, in the present example are not immobilized with respect to the first substrate.
  • the immobilized complexes 208 remain associated with the first substrate.
  • the plurality of dissociated species 210 are exposed to a second substrate 212 (step (D)), and at least some of the dissociated species associate with the second substrate 212 (step (E)).
  • the plurality of dissociated species that associate with the second substrate can then be detected and/or quantified and, thereby, the analyte molecules in the sample can be detected and/or quantified.
  • At least one of the first substrate and/or second substrate may comprise a plurality of capture components with which the analyte molecules and/or dissociated species may associate.
  • at least a portion of at least some of the immobilized complexes dissociate from the substrate to form a plurality of dissociated species, wherein, depending on the particular design of the assay as explained below, the dissociated species may or may not comprise any portion of an analyte molecule and/or capture component.
  • the substrate may be additionally exposed to a plurality of at least one type of binding ligand such that at least one binding ligand associates with at least a portion of the plurality of complexes, either before, after or essentially simultaneously with the immobilization of the complex on the substrate.
  • a plurality of at least one type of binding ligand such that at least one binding ligand associates with at least a portion of the plurality of complexes, either before, after or essentially simultaneously with the immobilization of the complex on the substrate.
  • FIG. 2 comprising a plurality of capture components 4 (step (A)) is exposed to plurality of analyte molecules 6, as indicated by arrow 5.
  • Analyte molecules 6 associate with one of the plurality of capture components 4 (step (B)).
  • the substrate is then exposed to an optional binding ligand 10, as indicated by arrow 9, such that a binding ligand associates with at least a portion of the analyte molecules associated with the substrate and form a plurality of immobilized complexes 8 (step (C)).
  • a binding ligand associates with at least a portion of the analyte molecules associated with the substrate and form a plurality of immobilized complexes 8 (step (C)).
  • At least a portion of some of the plurality of immobilized complexes may be dissociated to form a plurality of dissociated species 12, as indicated by arrow 11 (step (D)).
  • the dissociated species may or may not comprise a portion of an analyte molecule, a capture component and/or a binding ligand.
  • a portion 12 of each of the binding ligands were dissociated.
  • At least a portion of the plurality of dissociated species may be quantified and/or detected, as indicated by arrow 13
  • the invention provides methods for detecting and/or quantifying a plurality of dissociated species.
  • the method may comprise partitioning the plurality of dissociated species across a plurality of reaction vessels, such that at least some of the reaction vessels contain at least one or, in certain cases, one dissociated species and at least some of the reaction vessels contain no dissociated species.
  • the plurality of dissociated species may be partitioned across a plurality of reaction vessels, such that a statistically significant fraction of the reaction vessels contains at least one or, in certain cases, one dissociated species and a statistically significant fraction of reaction vessels contain no dissociated species.
  • the presence or absence of a dissociated species in a reaction vessel may be determined to determine the number of reaction vessels which contain zero, one, or multiple dissociated species.
  • the number of reaction vessels containing various quantities of dissociated species thusly determined may then be used to quantify the dissociated species, and additionally may be used to determine a measure of the concentration of the analyte molecules in the original fluid sample.
  • the foregoing methods can be performed using a number of different assay formats, different reaction conditions, and/or detection systems in different embodiments of the invention.
  • the reaction vessels may be adapted to capture the dissociated species of interest.
  • the reaction vessel may comprise a microwell and a dissociated species capture component (also referred to as a detection capture component) for capturing the dissociated species, as discussed more below.
  • the present inventions involve exposing a substrate comprising a plurality of capture components to a plurality of analyte molecules.
  • a "capture component”, as used herein, is any molecule, other chemical/biological entity or solid support modification disposed upon a solid support that can be used to specifically attach, bind or otherwise capture a target molecule or particle (e.g., an analyte molecule or dissociated species), such that the target molecule/particle becomes immobilized with respect to the capture component and solid substrate.
  • immobilized means captured, attached, bound, or affixed so as to prevent dissociation or loss of the target molecule/particle, but does not require absolute immobility with respect to either the capture component or the solid substrate.
  • Capture components which are useful or potentially useful for practicing certain aspects and embodiments of the invention are discussed in more detail below.
  • At least some of the analyte molecules upon exposure to the substrate comprising a plurality of capture components, can become immobilized with respect to a capture component, thereby forming a plurality of immobilized complexes.
  • substantially all of the plurality of analyte molecules may become immobilized with respect to capture components such that essentially each of the plurality of immobilized complexes comprises a capture component and an analyte molecule.
  • the substrate on or in which the plurality of immobilized complexes is immobilized may comprise a microtiter plate, a plurality of beads (e.g., magnetic beads), or any other substrate that, in certain embodiments, comprises a plurality of capture components, as discussed more below
  • the substrate comprising the immobilized complexes may be exposed to at least one additional reaction component, for example, at least a first type of binding ligand.
  • binding ligand is any molecule, particle, or the like which specifically binds to or otherwise specifically associates with an analyte molecule, immobilized complex and/or dissociated species or another molecule or particle bound to or otherwise associated with the analyte molecule, immobilized complex and/or dissociated species (e.g., another binding ligand).
  • the binding ligand can convert a precursor labeling agent molecule to a labeling agent, as discussed more below. More than one type of binding ligand may be employed in any given assay method, for example, a first type of binding ligand and a second type of binding ligand.
  • the first binding ligand is able to associate with an analyte molecule and the second binding ligand is able to associate with the first binding ligand.
  • the substrate is exposed to a plurality of types of binding ligand, at least some of the plurality of immobilized complexes may additionally comprise, in some cases, at least one of each type of binding ligand.
  • the binding ligand can be exposed to the substrate after capture of the analyte molecule so that the binding ligand binds to the immobilized complex.
  • the binding ligand may become associated with the analyte molecule to form a complex followed by capture of the complex by the substrate to form the immobilized complex.
  • the binding ligand may bind to the dissociated species formed upon release of the immobilized complex, or portion thereof, from the substrate.
  • a plurality of dissociated species may be formed by dissociating at least a portion of at least some of the plurality of immobilized complexes.
  • a plurality of molecules or particles may be released from the substrate. Any or each of the plurality of dissociated species (e.g., molecules or particles released from a substrate) may comprise any portion of an immobilized complex.
  • the dissociated species may comprise (i) at least a portion of a binding ligand, (ii) at least a portion of a binding ligand and at least a portion of the analyte molecule, (iii) at least a portion of a binding ligand, at least a portion of an analyte molecule and at least a portion of a capture component, or (iv) essentially all of the immobilized complex.
  • Non-limiting examples of how a dissociated species may be released from the substrate are depicted in FIG. 3.
  • a portion 68 of a binding ligand 64 is dissociated from immobilized complex 58, which comprises capture component 60, analyte molecule 62, and binding ligand 64.
  • the remainder 66 of binding ligand 64 remains associated with analyte molecule 62.
  • Scheme (B) shows the formation of a dissociated species 74 comprising a portion 72 of the analyte molecule 62 and binding ligand 64.
  • the remainder 70 of the analyte molecule 62 remains associated with capture component 60.
  • Scheme (C) shows the formation of a dissociated species 80 comprising binding ligand 64, analyte molecule 60, and a portion 76 of capture component 60, while the remainder 78 of capture component 60 remains attached to the substrate.
  • the plurality of immobilized complexes may be dissociated in certain embodiments by exposure to a dissociating agent.
  • a "dissociating agent,” as used herein, is any agent that causes at least a portion of an immobilized complex to dissociate, thereby forming a dissociated species.
  • the dissociating agent may be a chemical dissociating agent, electromagnetic radiation, a change in pH, a change in temperature, a change in ionic strength, or the like.
  • the dissociating agent is a chemical dissociating agent that interacts with a portion of an immobilized complex, thereby causing a portion of the immobilized complex to dissociate.
  • the dissociated species may comprise a detectable component or a reactive component (e.g., an enzymatic component) that is able to interact with a precursor labeling agent molecule (e.g., an enzymatic substrate) to produce a labeling agent.
  • a detectable component or a reactive component e.g., an enzymatic component
  • a precursor labeling agent molecule e.g., an enzymatic substrate
  • the immobilized complex comprises a cleavable linkage.
  • a "cleavable linkage,” as used herein, is linkage that is able to be readily (i.e. under conditions not detrimental to the integrity of other portions of the immobilized complex) and selectively cleaved upon exposure to a dissociating agent.
  • the cleavable linkage upon cleavage by exposure to a dissociating agent forms the dissociated species.
  • a cleavable linkage which can be cleaved using beta- mercaptoethanol, is a disulfide linkage. Cleavable linkages and corresponding dissociating agents that can cause the cleavable linkage to cleave are discussed in more detail below.
  • a cleavable linkage may be present in any portion of an immobilized complex.
  • FIG. 4, panel (A) depicts an immobilized complex 90 wherein the capture component 92 comprises a cleavable linkage 94. Cleaving the cleavable linkage forms the dissociated species 96 comprising the binding ligand 64, the analyte molecule 62 and a portion 97 of the capture component, wherein the complementary portion 98 of the capture component remains attached to the substrate.
  • FIG. 4, panel (B) depicts an immobilized complex 100 wherein analyte molecule 102 comprises a cleavable linkage.
  • FIG. 4 depicts immobilized complex 110 wherein binding ligand 112 comprises cleavable linkage 114.
  • Cleaving the cleavable linkage forms the dissociated species 116 comprising a portion of the binding ligand wherein the complementary portion 117 of the binding ligand, the analyte molecule 62, and the capture component 60 form a portion 118, which remains immobilized with respect to the substrate.
  • at least some of the plurality of dissociated species released from a substrate are partitioned across a plurality of reaction sites, such as, for example, a plurality of reaction vessels (e.g., in an array format).
  • the plurality of reaction vessels may be formed in or of any suitable material, and in some cases, the reaction vessels can be sealed or may be formed upon the mating of a substrate with a sealing component, as discussed in more detail below.
  • the partitioning of the dissociated species is performed such that at least some (e.g., a statistically significant fraction) of the reaction vessels comprise at least one or, in certain cases, one dissociated species and at least some (e.g., a statistically significant fraction) of the reaction vessel comprise no dissociated species.
  • the detectable species may be quantified in certain embodiments, thereby allowing for the detection and quantification of the analyte molecule in the fluid sample by techniques described in more detail below.
  • the plurality of reactions vessels may comprise detection capture components, wherein the detection capture components are molecules or other entities able to specifically bind at least one dissociated species.
  • the plurality of dissociated species may be detected in the reaction vessels, either indirectly or directly, as discussed more below.
  • Certain methods of the present invention may be useful for characterizing analyte molecules in a sample.
  • the methods may be useful for detecting and/or quantifying analyte molecules in a fluid sample which is suspected of containing at least one analyte molecule, since, as explained in more detail below, the number of reaction vessels which contain one or more of the dissociated species can be correlated to the concentration of analyte molecules in the fluid sample (e.g., the number of reaction vessels which comprise a dissociated species can be related to a measure of the concentration of the analyte molecules in the sample).
  • Certain embodiments of present invention thus can provide a measure of the concentration of analyte molecules in a fluid sample based on the proportion of reaction vessels which contain a dissociated species. Specific methods and calculations of how to quantify analyte molecules in a fluid sample using embodiments of the invention are discussed more below.
  • Exemplary Basic Assay Formats The inventive assays may be carried our according to a very wide variety of basic protocols and formats. The particular format chosen can be based on the nature of the analyte molecules and the availability and properties of binding partners of the analyte as well as other factors. Several exemplary basic formats were discussed previously in the context of the discussion of FIGS. 1-4. As would be apparent to those skilled in the art with the benefit of the teachings provided by the present disclosure, the invention may alternatively be performed according to protocols/formats not specifically described in the specific, exemplary embodiments illustrated in this detailed description, but which do not require undue burden or experimentation to practice.
  • a method for determining a measure of the concentration of analyte molecules or particles in a fluid sample comprises capturing a plurality of analyte molecules or particles (e.g., though association with a capture component) on a first substrate (e.g., beads, microtiter plate, etc.). A plurality of molecules or particles is released (e.g., dissociated) from the first substrate. At least a portion of the molecules or particles released (dissociated species) from the first substrate are detected on or within a second substrate, which in certain embodiments comprises a plurality of reaction vessels (e.g., an etched end of a fiber optic bundle - see below). A measure of the concentration of the analyte molecules or particles in the initial fluid sample can then be determined based on a measure of quantification of the molecules or particles on or within the second substrate that were released from the first substrate.
  • a first substrate e.g., beads, microtiter plate, etc.
  • a plurality of molecules or particles is released (
  • a method for determining a measure of the concentration of analyte molecules or particles in a fluid sample comprises first capturing a plurality of analyte molecules or particles on a first substrate then releasing a plurality of molecules or particles (disassociated species) from the first substrate (e.g., immobilizing analyte molecules or particles with respect to a capture component on a first substrate to form a plurality of complexes and then releasing at least a portion of some of the complexes formed).
  • At least one binding ligand comprising a binding partner for the molecule or particle released from the first substrate is immobilized with respect to each of at least a fraction of the plurality of molecules or particles released from the first substrate to form a plurality of complexes.
  • at least one of the binding ligands may advantageously comprise a particle to facilitate or enhance the performance of the assay, for example by one or more of: increasing the degree of capture of and/or enhancing the binding kinetics with the dissociated species; facilitating transfer of the disassociated species from the first substrate to the second substrate; and/or facilitating detection of the disassociated species on or within the second substrate.
  • the plurality of complexes released from the first substrate can then be detected and quantified (e.g., by partitioning the plurality of dissociated species comprising particles across the plurality of reaction vessels), and a measure of the concentration of the analyte molecules or particles in the fluid sample may be determined based on the quantification of the dissociated species.
  • a plurality of analyte molecules or particles is captured on a first substrate 230 to form a plurality of immobilized complexes 232 (step (A)). At least a portion of some of the immobilized complexes are dissociated from the first substrate 230 and form a plurality of dissociated species 236 (step (B)).
  • the plurality of dissociated species may be formed by exposing the immobilized complexes to a reducing agent and/or by causing a cleavable linker located in the immobilized complex to cleave.
  • a portion of the immobilized complexes 234 may remain associated with the first substrate 230.
  • the plurality of dissociated species may be detected and/or quantified, in some cases, by partitioning at least a portion of the plurality of dissociated species across a plurality of reaction vessels (step (C)).
  • a measure of the concentration of the analyte molecules or particles in the fluid sample may be determined based on the quantification of dissociated species.
  • the plurality of dissociated species may be exposed to a plurality of binding ligands.
  • At least one binding ligand 240 comprising a binding partner for the molecule or particle released from the first substrate may be immobilized with respect to at least a fraction of the plurality of dissociated species 236 from the first substrate to form a plurality of complexes 244 forming a two-component dissociated species (step (D)).
  • An excess of binding ligands 246 may be present in some cases.
  • the plurality of two-component dissociated species 244 may then be detected and/or quantified, in some cases, by partitioning at least a portion of the plurality of complexes across a plurality of reaction vessels 242 (step (E)).
  • the plurality of binding ligands 246 that do not associate with a dissociated species may also be partitioned across the plurality of reaction vessels 242.
  • a measure of the concentration of the analyte molecules or particles in the fluid sample may be determined based on the quantification of the complexes comprising a binding ligand and a dissociated species.
  • a method for determining a measure of the concentration of analyte molecules or particles in a fluid sample may be performed as described in any of the methods above with at least a fraction of the molecules or particles released from the first substrate (disassociated species) becoming immobilized with respect to the second substrate.
  • the second substrate may comprise a plurality of capture components with which the dissociated species may become associated.
  • second substrate 250 comprising a plurality of capture components 252 may be exposed to a plurality of dissociated species 254 (step (A)). At least a portion of dissociated species 254 may associate with capture components 252 on second substrate 250 to form complexes 256 (step (B)).
  • the plurality of molecules may be released from the first substrate by exposure to a dissociating agent.
  • a substrate comprising a plurality of capture components may be exposed to a sample comprising a plurality of analyte molecules or particles, such that analyte molecules or particles associate with capture components to form a plurality of complexes, which are immobilized with respect to the substrate.
  • Each of the immobilized complexes may comprise at least one capture component and at least one analyte molecule or particle.
  • Exposure of the plurality of immobilized complexes to a reducing agent causes at least a portion of at least some of the plurality of immobilized complexes to dissociate from the substrate to form a plurality of dissociated species. At least some of the dissociated species may be detected to determine the presence of and/or a measurement of the amount or concentration of the analyte molecules or particles in the fluid sample, as discussed more herein.
  • the reducing agent may or may not be removed form the solution comprising the dissociated species prior to detection of the dissociated species, as discussed more herein.
  • the dissociating agent is a reducing agent (e.g., beta- mercaptoethanol).
  • the dissociating agent has essentially no specific affinity for the capture components. That is, the dissociating agent does not bring about release of the dissociating species by interacting with the capture component and employing competitive binding to release the analyte molecule that associated with the capture component.
  • First substrate 215 comprising a plurality of capture components 216 is exposed to a plurality of analyte molecules 217 (step (A)). At least some of the analyte molecules 217 associate with a capture component 216 on first substrate 215 to form a plurality of immobilized complexes 218 (step (B)).
  • the first substrate is then exposed to a dissociating agent (e.g., a reducing agent) 219 and at least a portion of the immobilized complexes are dissociated to form a plurality of dissociated species 220 (step (C)).
  • the complementary portion 221 of the immobilized complexes may remain associated with first substrate 215.
  • the plurality of dissociated species 220 are then exposed to second substrate 222 comprising a plurality of capture components 223 which are able to specifically bind the dissociated species (step (D)). At least some the dissociated species 220 are immobilized with respect to a capture component 223 (step (E)) and at least some of the dissociated species may be detected to determine the presence of and/or a measurement of the amount or concentration of the analyte molecules or particles in the fluid sample.
  • the plurality of dissociated species may be formed by cleavage of cleavable linkages.
  • each of the immobilized complexes may comprise at least one cleavable linkage (e.g., a disulfide linkage).
  • the cleavable linkage may located in a capture component, analyte molecule or a binding ligand and may be cleaved to form a plurality of dissociated species, for example, see FIG. 4 as discussed more herein.
  • the cleavable linkage is a disulfide linkage which may, in some cases, be cleaved by exposure of the immobilized complexes to a reducing agent.
  • an immobilized complex comprises an enzymatic component. That is, at least one of the capture component, the analyte molecule or any additional components of the immobilized complex (e.g., binding ligand(s)) comprises an enzymatic component.
  • the enzymatic component may be located in the portion of the immobilized complex which is dissociated from the first substrate to form a dissociated species.
  • FIG. 8 illustrate an exemplary embodiment of an assay wherein the binding ligand comprises a moiety (e.g., an enzymatic component), as discussed more herein.
  • the protocol may include the use of at least one binding ligand, at least a portion of which comprises at least a portion of the dissociated species transferred from the first substrate to the second substrate (e.g., the binding ligand may be immobilized prior to release or following release of the molecules or particles from the first substrate).
  • the binding ligand comprises a cleavable linkage (e.g., a disulfide linkage) and/or is dissociated from the first substrate by exposure to a reducing agent.
  • at least one binding ligand comprises an enzymatic component.
  • the binding ligand(s), or at least the portions thereof forming at least a portion of the dissociated species transferred from the first substrate to the second substrate may further comprise a moiety (e.g., an enzymatic component or enzyme substrate) able to convert a precursor labeling agent molecule (e.g., an enzymatic substrate) into a labeling agent (e.g., a detectable product).
  • a moiety e.g., an enzymatic component or enzyme substrate
  • the second substrate may be exposed to a plurality of precursor labeling agent molecules, wherein the plurality of precursor labeling agent molecules are converted to a plurality of labeling agent molecules upon exposure to a binding ligand.
  • a measure of the concentration of the analyte molecules or particles in the fluid sample can then be determined based on the measurement of the labeling agent molecules on or within the second substrate. For example, as shown in FIG. 8, a plurality of analyte molecules are immobilized with respect to first substrate 262 to form a plurality of immobilized complexes 260 (step (A)). First substrate 262 is exposed to a plurality of binding ligands 264 which associate with immobilized complexes 260 (step (B)).
  • a portion of at least some of binding ligands 264 are dissociated from the first substrate to form a plurality of dissociated species 268 (step (C)) (e.g., by exposure to a dissociating agent and/or by cleavage of a cleavable linker).
  • Each dissociated species 268 comprises a moiety 266 which is able to convert a precursor labeling agent molecule into a labeling agent.
  • the plurality of dissociated species 266 is immobilized with respect to second substrate 270 (step (D)).
  • the second substrate is exposed to a plurality of precursor labeling agent molecules 272, at least some of which are converted to a labeling agent molecules 274 upon exposure to moiety 268 of dissociated species 266, as indicated by arrow 276 (step (E)).
  • a plurality of analyte molecules are immobilized with respect to first substrate 282 to form a plurality of immobilized complexes 280 (step (A)).
  • At least a portion of at least some of the immobilized complexes are dissociated from first substrate 282 to form a plurality of dissociated species 284 (step (B)) (e.g., by exposure to a dissociating agent and/or by cleavage of a cleavable linker).
  • a plurality of dissociated species 284 may be exposed to a plurality of binding ligands 286 (step (C)).
  • Binding ligands 286 may associated with at least some of the dissociated species 284 to form a plurality of complexes 288 forming a two- component dissociated species (i.e. binding ligand 286 and species 284) (step (D)).
  • binding ligand 286 may comprise a moiety 290 that is able to convert a precursor labeling agent molecule into a labeling agent.
  • detection and/or quantification of the dissociated species may be carried out according to the methods discussed herein.
  • substantially all of the detected dissociated species that are applied to the second substrate become spatially separated with respect to the other detected dissociated species during detection (e.g., by being located in separate and distinct areas of the second substrate, such as separate reaction vessels), so that detection of the dissociated species is able to resolve individual dissociated species (i.e. individual molecules or particles) of the plurality of dissociated species.
  • the ability to achieve spatially separated species may aid in the determination of a measure of the concentration of analyte molecules in a fluid sample and/or the detection of a plurality of species substantially simultaneously.
  • the ability to spatially separate a plurality of dissociated species may permit low numbers of dissociated species to be detected and quantified, thereby enabling measurement of very low concentrations of analyte molecules in a fluid sample to be tested.
  • a system comprising a first substrate comprising a plurality of capture components and a second substrate configured to receive and contain dissociated species therein or thereon.
  • the system can additionally comprise a detector configured to detect dissociated species in or on the second substrate.
  • the plurality of capture components may be able to become specifically immobilized with respect to a plurality of analyte molecules or particles in the fluid sample, such that the analyte molecules or particles associate with capture components to form a plurality of immobilized complexes. At least a portion of at least some of the complexes is susceptible to being dissociated from the first substrate by exposure to a reducing agent, to form a plurality of dissociated species.
  • the systems and/or methods of the present invention can be employed using precursor labeling agent molecules, wherein the precursor labeling agent molecules are converted to labeling agent molecules which are insoluble in the liquid substantially surrounding the labeling agent molecules (e.g., the liquid in the reaction vessel) and/or which become immobilized with respect to the second substrate (e.g., within the reaction vessel).
  • the systems and/or methods of the present invention may be used in combination with the systems and/or methods as described in commonly owned U.S. Patent Application Serial No. 12/236,484, filed September 23, 2008, entitled "High Sensitivity Determination of the Concentration of Analyte Molecules or Particles in a Fluid Sample" by Duffy, et al.
  • a system for detecting analyte molecules or particles in a fluid sample may comprise a first substrate comprising a plurality of capture components configured for specifically immobilizing the analyte molecules or particles with respect to the substrate to form a plurality of immobilized complexes.
  • the complexes may be configured to be susceptible to being dissociated from the first substrate by a dissociating agent, e.g., by cleaving of a cleavable linker to form a plurality of dissociated species.
  • the system may further comprise a second substrate comprising an array comprising a plurality of reaction vessels. The reaction vessels may be configured to receive and contain dissociated species therein.
  • a system for detecting analyte molecules or particles in a fluid sample may comprise a first substrate comprising a plurality of capture components able to become specifically immobilized with respect to a plurality of analyte molecules or particles in the fluid sample, such that the analyte molecules or particles associate with capture components to form a plurality of immobilized complexes. At least a portion of the some of the immobilized complexes, in this embodiment, may be susceptible to being dissociated from the first substrate by exposure to a reducing agent, to form a plurality of dissociated species.
  • the system may also comprise a second substrate configured to receive and contain dissociated species therein or thereon and a detector configured to detect dissociated species in or on the second substrate.
  • the first substrate may comprise a plurality of beads 900 (e.g., magnetic beads), a planar surface 902, or a microtiter plate 904, as shown in FIGS. 10A- 1OC, respectively, and as discussed in more detail herein.
  • Second substrates may comprise a plurality of reaction vessels are also discussed in more detail herein.
  • the plurality of reaction vessels may be formed in the end of a fiber optic bundle.
  • the volume of each of the reactions vessels of the second substrate may be between about 10 attoliters and about 100 picoliters or between about 10 femtoliters and about 100 femtoliters, in certain embodiments, as discussed previously.
  • the plurality of reaction vessels may comprise a plurality of dissociated species capture components to which dissociated species are able to become specifically immobilized.
  • the second substrate may, in some embodiments, further comprise a sealing component configured for sealing the plurality of reaction vessels.
  • the second substrate comprises a substantially planar surface and the sealing component comprises a plurality of microwells, such that a plurality of reaction vessels is formed upon the mating of at least a portion of the planar surface with at least a portion of the sealing component comprising the plurality of microwells.
  • the plurality of reaction vessels may be formed upon the mating of at least a portion of a sealing component and at least a portion of the second substrate, as shown in FIGS. 1 IA-I IF and as discussed in more detail herein.
  • the surfaces of a substrate may, as mentioned previously, incorporate at least one type of capture component.
  • a capture component is any molecule, other chemical/biological entity or solid support modification disposed upon a solid support that can be used to specifically attach, bind or otherwise capture a target molecule or particle (e.g., an analyte molecule or dissociated species), such that the target molecule/particle becomes immobilized with respect to the capture component and solid substrate.
  • the capture component allows the attachment of a molecule, particle, or complex to a solid support (that is, a surface of a substrate) for the purposes of immobilization, detection, quantification, and/or other analysis of the molecule, particle, or complex.
  • a capture component is used in the present invention, in some cases, to immobilize an analyte molecule with respect to the first substrate.
  • a capture component may comprise a cleavable linkage, as discussed more below.
  • the composition of the capture component will depend on the composition of the analyte molecule (or dissociated species).
  • Capture components for a wide variety of target molecules are known or can be readily found using known techniques.
  • the capture components may comprise proteins, particularly antibodies or fragments thereof (e.g., antigen-binding fragments (Fabs), Fab' fragments, pepsin fragments, F(ab') 2 fragments, full-length polyclonal or monoclonal antibodies, antibody- like fragments, etc.), other proteins, such as receptor proteins, Protein A, Protein C, etc., or small molecules.
  • capture components for proteins comprise peptides.
  • suitable capture components may include enzyme substrates and enzyme inhibitors.
  • the capture component may comprise a phosphate-binding agent.
  • the phosphate-binding agent may comprise metal-ion affinity media such as those describe in U.S. Patent No. 7,070,921 and U.S. Patent Application No. 20060121544.
  • the capture component may be a complementary nucleic acid.
  • the target molecule may be a nucleic acid binding protein and the capture component may be a single-stranded or double-stranded nucleic acid; alternatively, the capture component may be a nucleic acid-binding protein when the target molecule is a single or double stranded nucleic acid.
  • nucleic acid "aptamers" may be developed for capturing virtually any target molecule.
  • any molecule that can specifically associate with a target molecule of interest may potentially be used as a capture component.
  • a capture component include, for example, antibodies, lectins, and selectins.
  • suitable target molecule e.g., analyte molecule or dissociated species
  • suitable target molecule/capture component pairs can include, but are not limited to, antibodies/antigens, receptors/ligands, proteins/nucleic acid, nucleic acids/nucleic acids, enzymes/substrates and/or inhibitors, carbohydrates (including glycoproteins and glycolipids)/lectins and/or selectins, proteins/proteins, proteins/small molecules; small molecules/small molecules, etc.
  • the capture components are portions (particularly the extracellular portions) of cell surface receptors that are known to multimerize, such as the growth hormone receptor, glucose transporters (particularly GLUT 4 receptor), and T-cell receptors and the target analytes are one or more receptor target ligands.
  • the capture component may be attached to a binding surface (for example, the surface of a microwell or of a microbead) via a linkage, which may comprise any moiety, functionalization, or modification of the binding surface and/or capture component that facilitates the attachment of the capture component to the surface.
  • the linkage between the capture component and the surface may comprise one or more chemical or physical (e.g., non-specific attachment via van der Waals forces, hydrogen bonding, electrostatic interactions, hydrophobic/hydrophilic interactions; etc.) bonds and/or chemical linkers providing such bond(s).
  • the capture component comprises a capture extender component.
  • the capture component comprises a first portion that binds the analyte molecule and a second portion that can be used for attachment to the binding surface.
  • the substrate surface may also comprise a protective or passivating layer that can reduce or minimize non-specific attachment of non-capture components (e.g., analyte molecules, binding ligands, dissociated species) to the binding surface during the assay which may lead to false positive signals during detection or to loss of signal.
  • non-capture components e.g., analyte molecules, binding ligands, dissociated species
  • passivating layers include, but are not limited to: polymers, such as poly(ethylene glycol), that repel the non-specific binding of proteins; naturally occurring proteins with this property, such as serum albumin and casein; surfactants, e.g. zwitterionic surfactants, such as sulfobetaines; naturally occurring long-chain lipids; and nucleic acids, such as salmon sperm DNA.
  • the method of attachment of the capture component to the substrate surface depends of the type of linkage employed and may potentially be accomplished by a wide variety of suitable coupling chemistries/techniques known to those of ordinary skill in the art.
  • the particular means of attachment selected will depend on the material characteristics of the substrate surface and the nature of the capture component.
  • the capture components may be attached to the substrate surface through the use of reactive functional groups on each.
  • the functional groups are chemical functionalities. That is, the binding surface may be derivatized such that a chemical functionality is presented at the binding surface which can react with a chemical functionality on the capture component resulting in attachment.
  • Examples of functional groups for attachment that may be useful include, but are not limited to, amino groups, carboxy groups, epoxide groups, maleimide groups, oxo groups and thiol groups.
  • Functional groups can be attached, either directly or through the use of a linker, the combination of which is sometimes referred to herein as a "crosslinker.”
  • Crosslinkers are known in the art; for example, homo-or hetero- bifunctional crosslinkers as are well known (e.g., see 1994 Pierce Chemical. Company catalog, technical section on crosslinkers, pages 155-200, or "Bioconjugate Techniques" by Greg T. Hermanson, Academic Press, 1996).
  • Non-limiting example of crosslinkers include alkyl groups (including substituted alkyl groups and alkyl groups containing heteroatom moieties), esters, amide, amine, epoxy groups and ethylene glycol and derivatives.
  • a linker may also be a sulfone group, forming a sulfonamide.
  • the functional group is a light-activated functional group. That is, the functional group can be activated by light to attach the capture component to the substrate surface.
  • the functional group can be activated by light to attach the capture component to the substrate surface.
  • One example is PhotoLinkTM technology available from SurModics, Inc. in Eden Prairie, MN.
  • the substrate may comprise streptavidin-coated surfaces and the capture component may be biotinylated. Exposure of the capture component to the streptavidin-coated surfaces can cause association of the capture component with the surface by interaction between the biotin component and streptavidin.
  • attachment of the capture component to the binding surface may be effected without covalently modifying the binding surface of a substrate.
  • the attachment functionality can be added to the binding surface by using a linker that has both a functional group reactive with the capture component and a group that has binding affinity for the binding surface.
  • a linker comprises a protein capable of binding or sticking to the binding surface; for example, in one such embodiment, the linker is serum albumin with free amine groups on its surface.
  • a second linker crosslinker
  • crosslinker can then be added to attach the amine groups of the albumin to the capture component (e.g., to carboxy groups).
  • the analyte molecule may be captured on the binding surface of a substrate using a capture component attached via chemical crosslinking in the following manner.
  • the binding surface is derivatized with a functional group, such as, an amine group.
  • a crosslinker and the capture component are placed in contact with the binding surface such that one end of the crosslinker attaches to the amine group and the capture component attaches to the other end of the crosslinker.
  • capture components comprising proteins, lectins, nucleic acids, small organic molecules, carbohydrates can be attached.
  • Proteinaceous capture components any number of techniques may be used to attach a proteinaceous capture component to a wide variety of solid surfaces.
  • Protein or “proteinaceous” in this context includes proteins, polypeptides, peptides, including, for example, enzymes and antibodies.
  • a wide variety of techniques are known to add reactive moieties to proteins, for example, the method outlined in U.S. Patent No. 5,620,850.
  • the attachment of proteins to surfaces is known, for example, see Heller, Ace. Chem. Res. 23:128 (1990), and many other similar references.
  • the capture component may comprise Fab' fragments.
  • the use of Fab' fragments as opposed to whole antibodies may help reduce non-specific binding between the capture component and the binding ligand.
  • the Fc region of a capture component (or binding ligand) may be removed (e.g., proteolytically).
  • an enzyme may be used to remove the Fc region (e.g., pepsin, which may produce F(ab') 2 fragments and papain, which may produce Fab fragments).
  • the capture component may be attached to a binding surface using amines or may be modified with biotin (e.g., using NHS-biotin) to facilitate binding to an avidin or streptavidin coated substrate surface.
  • F(ab') 2 fragments may be subjected to a chemical reduction treatment (e.g., by exposure to 2- mercaptoethylamine) to, in some cases, form two thiol-bearing Fab' fragments. These thiol-bearing fragments can then be attached via reaction with a Michael acceptor such as maleimide.
  • the Fab' fragments may then be treated with a reagent (e.g., maleimide-biotin) to attach at least one biotin moiety (i.e., biotinylated) to facilitate attachment to streptavidin-coated surfaces as described above.
  • a reagent e.g., maleimide-biotin
  • biotin moiety i.e., biotinylated
  • nucleic acids as the capture component, for example for when the analyte molecule is a nucleic acid or a nucleic acid binding protein, or when the it is desired that the capture component serve as an aptamer for binding a protein, as is well known in the art.
  • each binding surface of a substrate comprises a plurality of capture components.
  • the plurality of capture components may be distributed randomly on the binding surface like a "lawn."
  • the capture components may be spatially segregated into distinct region(s) and distributed in any desired fashion.
  • Binding between the capture component and the analyte molecule is specific, e.g., as when the capture component and the analyte molecule are complementary parts of a binding pair.
  • the capture component binds both specifically and directly to the analyte molecule.
  • specifically bind or “binding specificity,” it is meant that the capture component binds the analyte molecule with specificity sufficient to differentiate between the analyte molecule and other components or contaminants of the test sample.
  • the capture component may be an antibody that binds specifically to some portion of an analyte molecule (e.g., an antigen).
  • the antibody can be any antibody capable of binding specifically to an analyte molecule of interest.
  • appropriate antibodies include, but are not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to as antibody mimetics), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and fragments of each, respectively.
  • the analyte molecule may be an antibody and the capture component may be an antigen.
  • an analyte particle is a biological cell (e.g., mammalian, avian, reptilian, other vertebrate, insect, yeast, bacterial, cell, etc.)
  • the capture component may be a ligand having specific affinity for a cell surface antigen (e.g., a cell surface receptor).
  • the capture component is an adhesion molecule receptor or portion thereof, which has binding specificity for a cell adhesion molecule expressed on the surface of a target cell type. In use, the adhesion molecule receptor binds with an adhesion molecule on the extracellular surface of the target cell, thereby immobilizing or capturing the cell.
  • the capture component is fibronectin, which has specificity for, for example, analyte particles comprising neural cells.
  • analyte molecules using capture components for which binding to analyte molecules/dissociated species that is not highly specific.
  • capture components such as, for example, a panel of different binding ligands, and detection of any particular analyte molecule/dissociated species is determined via a "signature" of binding to this panel of binding ligands, similar to the manner in which "electronic noses" work. This may find particular utility in the detection of certain small molecule analytes.
  • the binding affinity between analyte molecules and capture components should be sufficient to remain bound under the conditions of the assay, including wash steps to remove molecules or particles that are non-specifically bound.
  • the binding constant of the analyte molecule or dissociated species to its complementary capture component may be between at least about 10 4 and about 10 6 M "1 , at least about 10 5 and about 10 9 M "1 , at least about 10 7 and about 10 9 M "1 , greater than about 10 9 M '1 , or the like.
  • the capture component is chosen to be able to bind to a corresponding binding partner associated with or attached to the analyte molecule or dissociated species.
  • the capture component according to one embodiment is a chemical crosslinker as described above able to bind to proteins generally.
  • every protein molecule in a fluid sample comprises an analyte molecule that attaches to such a chemical crosslinker.
  • the capture component comprises streptavidin, which binds with high affinity to biotin, and thus captures any analyte molecules to which biotin has been attached.
  • the capture component may be biotin, and streptavidin may be attached to or associated with the analyte molecules such that the analyte molecules can be captured by the biotin.
  • the binding surfaces of a substrate may be functionalized with capture components in the following manner.
  • the surface of a substrate e.g., one end of a fiber optic bundle, a plurality of microbeads, etc.
  • the binding surfaces of the substrate are derivatized with a chemical functionality as described above.
  • the capture component may be added, which binds to and is immobilized by the chemical functionality.
  • FIG. 12 A specific embodiment is depicted in FIG. 12, in which the binding surface comprises an array of micro wells functionalized with biotin.
  • an array of microwells 130 in this non-limiting example is formed at the end of a fiber optic bundle 126.
  • the binding surface of the microwells 130 are first modified (e.g., with aminopropyl silane), as indicated by arrow 127, which may be bound to both the core 131 and cladding 132 surfaces of the end of the fiber bundle 126, as shown in FIG. 12, panel (B).
  • the capture component should be present only within the microwells, the external surfaces of the fiber optic bundle, such as the external surfaces 133 of the cladding 132, should not be modified.
  • chemical functionalities may be removed from the external cladding surface 133 to avoid attachment of a capture component in this region.
  • treated binding surface 128 may be removed from the external cladding portion 133, e.g., by polishing the tip of the fiber optical bundle (e.g., for 10 seconds with 0.3 um lapping film), thereby removing the topmost layer of the cladding in this region, thereby removing the added binding moieties.
  • the capture component can be attached, as indicated by arrow 131 and shown in FIG. 12, panel (D).
  • the surface is treated with aminopropyl silane and the capture component comprises biotin or is labeled with biotin.
  • the capture component comprises biotin or is labeled with biotin.
  • panel (D) a capture component comprising biotin succinimidyl ester 136 is attached to the amino groups of treated surface 128 of the microwells 130.
  • the modification with aminopropyl silane is effective in this example because NHS-biotin attaches to an amino-silanized binding surface 128.
  • a capture component 146 may be localized directly on the surface of the micro well 130, which may contain an optional seal 138, on a microparticle 134 contained within the microwell 130 (FIG. 12, panel (F)), and/or on the seal 138 of the microwell 130 (FIG. 12, panel (G)). Additional locations where a capture component may be immobilized and additional substrates that may be used for one or both of capturing an analyte molecule or a dissociated species according to certain methods of the invention are discussed more below.
  • an immobilized complex may comprise a capture component and at least two analyte molecules.
  • a single analyte molecule may become immobilized with respect to two or more capture components (either of the same or differing types) such that an immobilized complex comprises at least two capture component and an analyte molecule.
  • At least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 99%, or the like, of analyte molecules in the fluid sample exposed to the binding substrate become immobilized with respect to at least one capture component.
  • the method and systems of the present invention comprise a first substrate and a second substrate.
  • the first substrate comprises a plurality of capture component with specificity for target analyte molecules (or particles) in a fluid sample to be tested
  • the second substrate comprises a plurality of capture component with specificity for dissociated species released from the first substrate.
  • the second substrate in certain embodiments comprises the substrate utilized for detection and quantization and to facilitate this comprises a plurality of reaction vessels in which a plurality of dissociated species (or molecules or particles) released from first substrate are partitioned across or received and contained in.
  • the first substrate may be fabricated from any suitable material, for example, plastics, polysaccharides, nylon or nitrocellulose, composite materials, ceramics, silica or silica-based materials, carbon, metals, inorganic glasses, and a variety of other polymers.
  • suitable materials for example, plastics, polysaccharides, nylon or nitrocellulose, composite materials, ceramics, silica or silica-based materials, carbon, metals, inorganic glasses, and a variety of other polymers.
  • Non-limiting examples of potentially suitable configurations include of beads (e.g., magnetic beads), microtiter plates, slides, membranes, plates, flasks, dipsticks, optical fibers, or the like.
  • the first substrate may comprise a planar surface 902, for example, as shown in FIG. 1OB.
  • the first substrate comprises a plurality of beads 900 (FIG. 10A).
  • the beads may each comprise a plurality of capture components by which a plurality of analyte molecules may be immobilized.
  • the use of beads or other large surface area formats as the first substrate may increase the speed and/or efficiency in which a plurality of analyte molecules are immobilized with respect to the substrate.
  • the solution comprising the plurality of analyte molecules and beads may be agitated (e.g., stirred) and may increase the rate of exposure of the substrate (e.g., bead) to an analyte molecule, as compared to a first substrate comprising a different configuration (e.g., a microtiter plate).
  • the beads may be magnetic beads. The magnetic property of the beads may help in separating the beads from a solution (e.g., comprising a plurality of dissociated species) and/or during washing step(s) (e.g., to remove excess fluid sample, binding ligands, and/or dissociated species).
  • a solution e.g., comprising a plurality of dissociated species
  • washing step(s) e.g., to remove excess fluid sample, binding ligands, and/or dissociated species.
  • Potentially suitable beads, including magnetic beads are available from a number of commercial suppliers.
  • the diameter of the beads may be greater than about 0.1 um, greater than about 1 um, greater than about 10 um, greater than about 100 um, greater than about 1 mm, or the like. In other embodiments, the diameter of the beads may be between about 0.1 um and about 100 um, between about 1 um and about 100 um, between about 10 um and about 100 um, between about 0.1 um and about 1 mm, between about 10 um and about 10 mm, between about 0.1 um and about 10 um, or the like.
  • the first substrate comprises a microtiter plate 904, wherein each well 906 of the plate comprises a plurality of capture components.
  • the volume of each well may be greater than about 1 nL, greater than about 10 nL, greater than about 100 nL, greater than about 1 uL, greater than about 10 uL, greater than about 100 uL, greater than about 1 mL, greater than about 2 mL, or the like.
  • the volume of each well may be between about 1 nL and about 2 mL, between about 10 nL and about 1 mL, between about 100 nL and about 100 uL (microliter), between about 10 uL and about 1 mL, between about 100 uL and about 1 mL, or the like.
  • the number of wells in the microtiter plate may be any appropriate number for example about 6, about 24, about 96, about 384, about 1536, or the like.
  • the number of wells may be between about 5 and about 2000, about 10 and about 1000, about 20 and about 500, about 50 and about 100, or the like.
  • the microtiter plate may be a 96-well plate (FIG. 10C). Microtiter plates may be purchased from many commercial sources.
  • the second substrate is employed for the detection and/or quantification of a plurality of dissociated species (e.g., molecules or particles released from the first substrate) and comprises a plurality of small volume reaction vessels.
  • the second substrate in certain embodiments, is configured to receive and contain dissociated species therein or thereon (e.g., within wells comprising capture components having specificity for the dissociated species, or within wells without such capture components but including means for sealing of the wells of the substrate - e.g., through use of a sealing component (see below), etc.).
  • the plurality of dissociated complexes can be partitioned across a plurality of reaction vessels of the second substrate (e.g., configured as an array of reaction vessels), in some cases, to facilitate determination of a measure of the concentration of dissociated species and thereby a measure of the concentration of analyte molecules in a fluid sample by means discussed in further detail below and in the examples.
  • the plurality of reaction vessels may be formed through the mating of the second substrate and a sealing component, wherein at least one of the second substrate and the sealing component comprises a plurality of microwells.
  • a sealing component e.g., as shown in FIG. 12, panels (E-G)
  • an array comprises a plurality of depressions in a first surface of the second substrate (e.g., a support material).
  • the sealing component may comprise a second surface with the same or different topology as the first surface may be brought into contact with the first surface to create a plurality of sealed reaction vessels. Either the first surface or the second surface may be fabricated from a compliant material to aid in sealing.
  • the sealing component may be capable of contacting the exterior surface of an array of microwells (e.g., the cladding of a fiber optic bundle as shown in FIG. 12) such that each reaction vessel thus formed is sealed or isolated such that the contents of each reaction vessel cannot escape the reaction vessel.
  • the sealing component may be a silicone elastomer gasket that may be placed against an array of microwells with application of uniform pressure across the entire substrate.
  • the reaction vessels may be sealed after the addition of a dissociated species and, optionally, a precursor labeling agent molecule to facilitate detection of the dissociated species.
  • a reaction to produce the detectable labeling agent molecule can proceed within the sealed reaction vessels, thereby producing a detectable amount of a labeling agent molecule that is retained in the reaction vessel for detection purposes.
  • the plurality of reaction vessels formed on a planar second substrate upon the mating of at least a portion of a sealing component comprising a plurality of microwells and at least a portion of the planar substrate e.g., see FIG. 1 IB below.
  • panel (A) shows a surface comprising a plurality of microwells 139, which have been exposed to a fluid sample 141, and a sealing component 143.
  • Sealing component 143 in this example comprises a substantially planar bottom surface. Mating of the microwell containing surface 139 with sealing component 143 forms a plurality of sealed reaction vessels 145. The areas between the reaction vessels 148, may be modified to aid in the formation of a tight seal between the reaction vessels.
  • a second embodiment is shown in FIG. 11, panel (B), in which sealing component 162 comprising a plurality of microwells 163 is mated with a substantially planar surface 158 which has been exposed to fluid sample 162, thereby forming a plurality of reaction vessels 164.
  • substrate surface 166 comprising a plurality of microwells 167 is mated with sealing component 170 also comprising a plurality of microwells 171.
  • the microwells in the substrate and the microwells in the sealing components are substantially aligned so each reaction vessel 172 formed comprises a portion of the microwell from the sealing component and a portion of a microwell from the substrate.
  • panel (D) the microwells are not aligned such that each reaction vessel comprises either a microwell from the sealing component 173 or a microwell from the substrate 175.
  • the sealing component may be essentially the same size as the substrate or may be different in size. In some cases, the sealing component is approximately the same size as the substrate and mates with substantially the entire surface of the substrate. In other cases, as depicted in FIG. 11, panel (E), the sealing component 176 is smaller than the substrate 174 and the sealing component only mates with a portion 178 of the substrate. In yet another embodiment, as depicted in FIG. 11, panel (F), the sealing component 182 is larger than the substrate 180, and only a portion 184 of the sealing component mates with the substrate 180. In some embodiments, the reaction vessels may all have approximately the same volume. In other embodiments, the reaction vessels may be of differing volumes.
  • the volume of each individual reaction vessel can range for different embodiments from attoliters or smaller to nanoliters or larger depending upon the nature of dissociated species, the detection technique and equipment employed, and the expected concentration of the dissociated species in the fluid applied to the second substrate for analysis.
  • the size of the reaction vessel may be selected such that at the concentration of interest, between zero and ten molecules/particles of a dissociated species would be statistically expected to be found in each reaction vessel.
  • the volume of the reaction vessel is selected such that at the concentration of interest, either zero or one molecules/particles of the dissociated species would be statistically expected to be found in each reaction vessel.
  • the reaction vessels may have a volume between about 1 femtoliter and about 1 picoliter, between about 10 femtoliters and about 100 femtoliters, between about 10 attoliters and about 50 picoliters, between about 1 picoliter and about 50 picoliters, between about 1 femtoliter and about 1 picoliter, between about 30 femtoliters and about 60 femtoliters, or the like.
  • the reaction vessels have a volume of less than about 1 picoliter, less than about 500 femtoliters, less than about 100 femtoliters, less than about 50 femtoliters, less than about 1 femtoliter, or the like. In some cases, the reaction vessels have a volume of about 10 femtoliters, about 20 femtoliters, about 30 femtoliters, about 40 femtoliters, about 50 femtoliters, about 60 femtoliters, about 70 femtoliters, about 80 femtoliters, about 90 femtoliters, about 100 femtoliters, or the like.
  • the number of reaction vessels in the array will depend on the composition and end use of the array. Arrays containing from about 2 to many billions of reaction vessels can be made by utilizing a variety of techniques and materials. Increasing the number of reaction vessels in the array can be used to increase the dynamic range of an assay or to allow multiple samples or multiple types of dissociated species to be assayed in parallel. Generally, the array will comprise between one thousand and one million reaction vessels per sample to be analyzed. In some cases, the array will comprise greater than one million reaction vessels.
  • the array will comprise between about 1,000 and about 50,000, between about 1,000 and about 1,000,000, between about 1,000 and about 10,000, between about 10,000 and about 100,000, between about 100,000 and about 1 ,000,000, between about 1 ,000 and about 100,000, between about 50,000 and about 100,000, between about 20,000 and about 80,000, between about 30,000 and about 70,000, between about 40,000 and about 60,000, or about 50,000, or the like, reaction vessels.
  • the array of reaction vessels may be arranged on a substantially planar surface or in a non-planar three-dimensional arrangement.
  • the reaction vessels may be arrayed in a regular pattern or may be randomly distributed.
  • the array is a regular pattern of sites on a substantially planar surface permitting the sites to be addressed in the X-Y coordinate plane.
  • the reaction vessels are formed in a solid material.
  • the number of potentially suitable materials in which the reaction vessels can be formed is very large, and includes, but is not limited to, glass (including modified and/or functionalized glass), plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon ® , polysaccharides, nylon or nitrocellulose, etc.), elastomers (such as poly(dimethyl siloxane) and poly urethanes), composite materials, ceramics, silica or silica-based materials (including silicon and modified silicon), carbon, metals, optical fiber bundles, or the like.
  • the substrate material may be selected to allow for optical detection without appreciable autofluorescence.
  • the reaction vessels may be formed in a flexible material.
  • Individual reaction vessels may contain a binding surface.
  • the binding surface may comprise essentially the entirety or only a portion of the interior surface of the reaction vessel or may be on the surface of another material or object that is confined within the reaction vessel, such as, for example, a bead, or a particle (for example, a micro-particle or a nanoparticle).
  • a microwell in a surface may be formed using a variety of techniques known in the art, including, but not limited to, photolithography, stamping techniques, molding techniques, etching techniques, or the like. As will be appreciated by those of the ordinary skill in the art, the technique used will depend on the composition and shape of the supporting material and the size and number of reaction vessels.
  • an array of reaction vessels is formed by creating microwells on one end of a fiber optic bundle and utilizing a planar compliant surface as a sealing component.
  • an array of reaction vessels in the end of a fiber optic bundle may be formed as follows. First, an array of microwells is etched into the end of a polished fiber optic bundle. Techniques and materials for forming and etching a fiber optic bundle are known to those of ordinary skill in the art. For example, the diameter of the optical fibers, the presence, size and composition of core and cladding regions of the fiber, and the depth and specificity of the etch may be varied by the etching technique chosen so that microwells of the desired volume may be formed.
  • the etching process creates microwells by preferentially etching the core material of the individual glass fibers in the bundle such that each well is approximately aligned with a single fiber and isolated from adjacent wells by the cladding material.
  • Potential advantages of the fiber optic array format is that it can produce thousands to millions of reaction vessels without complicated microfabrication procedures and that it can provide the ability to observe and optically address many reaction vessels simultaneously.
  • An example of an etched fiber optic array is shown in FIGS. 13 A and 13B.
  • FIG. 13 A shows a fiber optic array that has been etched to form a plurality or reaction vessels.
  • the wells have a diameter of approximately 4.5 um and a volume of about 46 fL.
  • FIG. 13B shows an AFM image of a portion of the fiber optic array of FIG. 13 A.
  • Each microwell may be aligned with an optical fiber in the bundle so that the fiber optic bundle can carry both excitation and emission light to and from the wells, enabling remote interrogation of the well contents.
  • an array of optical fibers may provide the capability for simultaneous or non-simultaneous excitation of molecules in adjacent vessels, without signal "cross-talk" between fibers. That is, excitation light transmitted in one fiber does not escape to a neighboring fiber.
  • the physical alterations to a fiber optic may be made as taught in U.S. Patents 6,023,540, 6,327,410, and 6,858,394. Any one or more of the surface of the glass microwells, the surface of the sealing component, or particles within microwells can be functionalized in certain embodiments to create binding surface(s).
  • the equivalent structures can be fabricated using other methods that do not utilize the ends of an optical fiber bundle as a substrate.
  • the array may be a spotted, printed or photolithographically fabricated substrate produced by techniques known in the art; see for example WO95/25116; WO95/35505; PCT US98/09163; U.S. Patent Nos. 5,700,637, 5,807,522, 5,445,934, 6,406,845, and
  • the array may be produced using molding and/or etching techniques as will be known to those of ordinary skill in the art.
  • the present invention provides a system equipped with a mechanical platform that applies a sealing component to a substrate.
  • the platform may be positioned beneath a stage on the system.
  • the sealing component may be mated with the array.
  • the sealing component may be sandwiched between a flat surface (such as, for example, a microscope slide) and the array of reaction vessels using uniform pressure applied by the mechanical platform.
  • FIG. 14A A non-limiting embodiment is illustrated in FIG. 14A.
  • a sealing component 300 is placed on top of mechanical platform 302.
  • the fluid sample 304 is placed on top of the sealing component 300.
  • the mechanical platform is moved upwards towards the array 306 (e.g., fiber optic array) such that uniform pressure is applied.
  • the sealing component 300 forms a tight seal with the array 306.
  • varying pressure may be applied to the sealing component to form a tight seal between the sealing component and the array.
  • the system may also comprise additional components 312 that may be utilized to analyze the array (e.g., microscope, computer, etc.) as discussed more herein.
  • the first and/or second substrate to which the analyte molecules/dissociated species are immobilized may be subjected to at least one washing step.
  • a substrate may be washed after exposing the substrate to one or more solutions comprising analyte molecules, binding ligands, precursor labeling agent molecules, or dissociated species.
  • the wash step(s) may be used to wash away any analyte molecules or non-analyte molecules, any binding ligands or non- binding ligands, any precursor labeling agent molecules or non-precursor labeling agent molecules, or any dissociated species or non-dissociated species that are not immobilized with respect to the substrate.
  • the wash step(s) may be performed by any suitable technique known to those of ordinary skill in the art, for example, by submersion of the substrate in a wash solution, flushing the substrate with a wash solution, etc.
  • the wash solution is selected so that it does not cause appreciable change to the configuration of the substrate surface and/or does not disrupt an interaction between at least two components of the assay (e.g., a capture component and an analyte molecule/dissociated species).
  • the wash solution may be a solution that is selected to chemically interact with one or more components of the substrate.
  • a wash step may be performed at any appropriate time point during the inventive methods (e.g., after exposure of the substrate to a reagent or after immobilization of and agent with respect to a substrate) during a method of the present invention.
  • analyte molecules and particles may be detected and, optionally, quantified using methods and systems of the present invention; basically, any analyte molecule or particle that is able to be made to become immobilized with respect to (e.g., by binding) a capture component can be potentially investigated using the invention.
  • Certain more specific targets of potential interest that may comprise an analyte molecule or particle are mentioned below. The list below is exemplary and non-limiting.
  • the analyte molecule may be an enzyme.
  • enzymes include, an oxidoreductase, transferase, kinase, hydrolase, lyase, isomerase, ligase, and the like.
  • Additional examples of enzymes include, but are not limited to, polymerases, cathepsins, calpains, amino-transferases such as, for example, AST and ALT, proteases such as, for example, caspases, nucleotide cyclases, transferases, lipases, enzymes associated with heart attacks, and the like.
  • appropriate target enzymes include viral or bacterial polymerases and other such enzymes, including viral or bacterial proteases, or the like.
  • the analyte molecule or particle may comprise an enzymatic component.
  • the analyte particle can be a cell having an enzyme or enzymatic component present on its extracellular surface.
  • the analyte particle is a cell having no enzymatic component on its surface.
  • Such a cell is typically identified using an indirect assaying method described below.
  • a non-limiting example of an enzymatic component is beta-galactosidase.
  • the analyte molecule may be a biomolecule.
  • biomolecules include hormones, antibodies, cytokines, proteins, nucleic acids, lipids, carbohydrates, lipids cellular membrane antigens and receptors
  • Non-limiting embodiments of proteins include peptides, polypeptides, protein fragments, protein complexes, fusion proteins, recombinant proteins, phosphoproteins, glycoproteins, lipoproteins, or the like.
  • suitable protein analyte molecules include, but are not limited to, immunoglobulins, hormones, growth factors, cytokines (many of which serve as ligands for cellular receptors), cancer markers, etc.
  • the analyte molecule may be a host-translationally modified protein (e.g., phosphorylation, methylation, glycosylation) and the capture component may be an antibody specific to a post-translational modification.
  • Modified proteins may be captured with capture components comprising a multiplicity of specific antibodies and then the captured proteins may be further bound to a binding ligand comprising a secondary antibody with specificity to a post-translational modification.
  • modified proteins may be captured with capture components comprising an antibody specific for a post-translational modification and then the captured proteins may be further bound to binding ligands comprising antibodies specific to each modified protein.
  • the analyte molecule is a nucleic acid.
  • a nucleic acid may be captured with a complementary nucleic acid fragment (e.g., an oligonucleotide) and then optionally subsequently labeled with a binding ligand comprising a different complementary oligonucleotide.
  • a complementary nucleic acid fragment e.g., an oligonucleotide
  • Suitable analyte molecules and particles include, but are not limited to small molecules (including organic compounds and inorganic compounds), environmental pollutants (including pesticides, insecticides, toxins, etc.), therapeutic molecules (including therapeutic and abused drugs, antibiotics, etc.), biomolecules (including hormones, cytokines, proteins, nucleic acids, lipids, carbohydrates, cellular membrane antigens and receptors (neural, hormonal, nutrient, and cell surface receptors) or their ligands, etc), whole cells (including prokaryotic (such as pathogenic bacteria) and eukaryotic cells, including mammalian tumor cells), viruses (including retroviruses, herpesviruses, adenoviruses, lentiviruses, etc.), spores, etc.
  • small molecules including organic compounds and inorganic compounds
  • environmental pollutants including pesticides, insecticides, toxins, etc.
  • therapeutic molecules including therapeutic and abused drugs, antibiotics, etc.
  • biomolecules including hormones, cytokines
  • the fluid sample comprising or suspected of comprising and analyte molecule or particle may be derived from any suitable source.
  • the sample may comprise a liquid, fluent particulate solid, fluid suspension of solid particles, supercritical fluid and/or gas.
  • the analyte molecule or particle may be separated or purified from its source prior to determination; however, in certain embodiments, an untreated sample containing the analyte molecule or particle may be tested directly.
  • the source of the analyte molecule may be synthetic (e.g., produced in a laboratory), the environment (e.g., air, soil, etc.), a mammal, an animal, a plant, or any combination thereof.
  • the source of an analyte molecule is a human bodily substance (e.g., blood, serum, plasma, urine, saliva, tissue, organ, or the like).
  • Binding ligands may be selected from any suitable molecule, particle, or the like, as discussed more below, able to associate with an analyte molecule and/or to associate with another binding ligand. For example, when only a first binding ligand is employed, a first binding ligand may associate with an analyte molecule. In another example, when a first binding ligand and a second binding ligand are employed, a first binding ligand may associate with an analyte molecule and a second binding ligand may become immobilized with respect to the analyte molecule by becoming associated with the first binding ligand. Some non-limiting examples of potentially suitable binding ligands are discussed more below.
  • binding ligands can comprise a component that is able to facilitate detection, either directly or indirectly.
  • a component may facilitate indirect detection, for example, by converting a precursor labeling agent molecule to a labeling agent molecule (e.g., an agent that is detected in an assay).
  • the binding ligand may comprise an enzymatic component (e.g., horseradish peroxidase, beta-galactosidase, etc).
  • a first binding ligand may or may not be used in conjunction with additional (e.g., second, etc.) binding ligands, examples of which are discussed herein.
  • a "precursor labeling agent” is any molecule, particle, or the like, that can be converted to a labeling agent molecule upon exposure to a suitable converting agent.
  • a converting agent may, in certain embodiments, comprise at least a portion of at least one of a capture component, an analyte, and/or a binding ligand.
  • the converting agent comprises at least a portion of the dissociated species released from the first substrate and transferred to the second substrate for detection.
  • a "labeling agent” is any molecule, particle, or the like, that facilitates detection, by acting as the detected entity, using a chosen detection technique.
  • binding ligands may be selected such that are able to function in the following manner.
  • the first binding ligand may associate directly with an analyte molecule. In other cases, if both a first and a second binding ligand are employed, the first binding ligand may associate with an analyte molecule and comprise a component which is able to interact with the second binding ligand. Certain assays may also comprise additional components (e.g., a third binding ligand that can interact with the second binding ligand, first binding, or analyte). In certain embodiments, any one or more of the binding ligands may comprise at least a portion thereof able to convert a precursor labeling agent into a labeling agent molecule. In some cases, an assay component may be able to associate with more than one other assay component, for example, a third binding ligand may associate with both a first and a second binding ligand.
  • additional components e.g., a third binding ligand that can interact with the second binding ligand, first binding, or analyte.
  • a first binding ligand may be employed alone or in conjunction with a second (and a third, etc.) binding ligand.
  • a first binding ligand may be selected (e.g., in the case when only a first and a second binding ligand is employed) such that it may associate with an analyte molecule and be able to interact with the second binding ligand (e.g., such that the first binding ligand is associated with the second binding ligand).
  • the substrate (e.g., the substrate in/on which the plurality of analyte molecules are immobilized) may be exposed to a plurality of first binding ligands such that a first binding ligand associates with substantially all of the plurality of analyte molecules or particles to form a plurality of immobilized complexes.
  • the substrate may also be exposed to a plurality of second binding ligands such that the second binding ligand associates with substantially all of the plurality of first binding ligands which are associated with an analyte molecule or particle to form a plurality of immobilized complexes.
  • the substrate may be additionally exposed to a plurality of third binding ligands such that a third binding ligand associates with substantially all of the second binding ligands which are associated with a first binding ligand which are associated with an analyte molecule or particle to form at least a portion of the plurality of immobilized complexes.
  • substantially all refers to at least greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, or more, of a molecule/particle/species of a specific type.
  • a binding ligand (e.g., a first, second and/or third, etc. binding ligand) may comprise a cleavable linker (e.g., disulfide).
  • at least one of the binding ligands may comprise biotin.
  • binding ligands of the present invention may be functionalized to reduce non-specific binding, e.g., direct binding or association of a binding ligand to another component (e.g., capture component, surface of a substrate, etc.) of the assay, where such binding is undesired.
  • the binding ligand may be functionalized with a large steric molecule and/or particle which prevent the binding ligand from approaching and/or associating with the substrate, another component, etc.
  • the binding ligand may be functionalized with poly(ethylene glycol) groups or particles, poly(ethylene oxide) groups or particles (e.g., particles which are protein resistant such as beads coated in poly(ethylene glycol), DNA, or zwitterionic lipids).
  • a plurality of dissociated species is formed during the assay.
  • a "dissociated species,” as used herein, is a molecule, complex, or particle (which, as previously noted, includes biological cells and sub cellular organelles) at least a portion of which has been released/dissociated from a molecule, complex, particle, surface, etc., to which it was immobilized.
  • the dissociated species may form by causing an association (e.g., bond, attachment, etc.) to cleave, thereby releasing the dissociated species.
  • a molecule or particle may be released from a substrate in certain instances without causing cleavage of a bond (e.g., by physical means); such released molecules or particles may still be referred to herein as dissociated species.
  • the plurality of dissociated species may be formed by the dissociation of at least a portion of a plurality of complexes immobilized with respect to a substrate.
  • Each immobilized complex in most cases, will comprise an analyte molecule, a capture component, and, in some cases, at least one binding ligand.
  • each immobilized complex may additionally comprise at least one cleavable linker (e.g., a disulfide linker) and/or an enzymatic component.
  • a dissociated species may comprise any portion of the previously immobilized complex (e.g., at least a portion of any one or more of the analyte molecule, the capture component and/or the at least one binding ligand).
  • at least one or each of the plurality of dissociated species may comprise at least a portion of a capture component, analyte molecule, and/or binding ligand(s) (e.g., first, second, third, etc., binding ligands).
  • the number of dissociated species released from the substrate may be, but need not be, substantially equal to the total number of analyte molecules or particles in the fluid sample and/or immobilized on the substrate.
  • the ratio of immobilized analyte molecules to dissociated species may be about 1 :1, about 2:1, about 5:1, about 10:1, about 100: 1 , about 1000: 1 , about 10000: 1 , or the like. In some cases, the ratio is greater than about 1 : 1 but does not exceed 10:1, greater than about 10:1 but does not exceed 100:1, greater than about 100:1 but does not exceed 1000:1, greater than about 1000:1 but does not exceed 10000:1, greater than about 10000:1 but does not exceed 1,000,000:1, or greater than about 1,000,000:1 in certain embodiments.
  • the number of the plurality of immobilized molecules or particles released is approximately equal to, is directly proportional to, is about one half of, is about one quarter of, is about one tenth of, is about one hundredth of, is about one thousandth of, is about one ten-thousandth of, or is about one hundred- thousandth of, the number of the plurality of analyte molecules or particles captured.
  • a dissociated species in some cases, may comprise a component which is able to be detected, either indirectly or directly (i.e., may comprise a labeling agent molecule and/or is capable of converting a precursor labeling agent molecule to a labeling agent molecule).
  • the presence of a detectable component on a dissociated species can allow for the detection of the dissociated species, thereby allowing for the determination and/or quantification of the dissociated species.
  • the determination and/or quantification of the dissociated species can be related to the detection and/or quantification of the analyte molecule in a fluid sample, as discussed more below.
  • the detectable component is an enzymatic component.
  • At least a portion of at least some of the immobilized complexes may be susceptible to being dissociated from a substrate by exposure to a dissociating agent, thereby forming a plurality of dissociated species.
  • dissociating agents include chemical dissociating agents, electromagnetic radiation, a change in the pH, a change in the temperature, etc.
  • exposing the plurality of immobilized complexes to a reducing agent causes at least a portion of at least some of the plurality of complexes to dissociate from the substrate to form a plurality of dissociated species.
  • the dissociating agent may have essentially no specific affinity for the capture components. That is, the dissociating agent does not cause the dissociating species to form by interacting with the capture component and employing competitive binding to release the analyte molecule that associated with the capture component.
  • the dissociated species may comprise an enzymatic component.
  • each of the plurality of immobilized complexes may comprise a cleavable linkage and the plurality of dissociated species may be formed by cleaving at least some of the cleavable linkages.
  • a dissociating agent may be employed in conjunction with a cleavable linkage, wherein exposure of the cleavable linkage to the dissociating agent causes the cleavable linkage to cleave and a dissociated species to form.
  • a cleavable linkage is a linkage that is able to be cleaved upon exposure to a dissociating agent.
  • the cleavable linkage may be present in any portion of the immobilized complex (e.g., the analyte molecule, the binding ligand (e.g., first, second or third, etc. binding ligand), the capture component, etc.).
  • the cleavable linkage may be a linkage that is specifically cleaved, that is, a linkage that can be cleaved without altering or damaging any other portion of the complex not being cleaved and which relatively uniformly alters the molecule in a reproducible manner.
  • a cleavable linkage may be selected depending on various parameters including desired size, solubility, ability to cleave or not cleave the linkage under certain conditions, the type of cleavage method to be used, etc.
  • a cleavable linkage may be homobifunctional (e.g., contain two or more identical leaving groups) or heterobifunctional (e.g., contain two or more different leaving groups).
  • the cleavable linkage may be a photocleavable linkage and the plurality of dissociated species may be formed by exposing the substrate to electromagnetic radiation (e.g., ultraviolet light).
  • electromagnetic radiation e.g., ultraviolet light
  • the photocleavable linkage may be cleaved at a wavelength of light that does not damage the molecule being released, for example, in the ultraviolet to visible range.
  • photocleavable linkages include linkages containing o-nitrobenzyl, desyl, trans-o-cinnamoyl, m-nitrophenyl, benzylsulfonyl groups and others.
  • the cleavable linkage may be a chemically cleavable linkage (i.e., susceptible to cleavage upon exposure to a chemical dissociating agent)
  • chemically cleavable linkages which may be cleaved by a chemical dissociating agent include a disulfide, which can be cleaved with reducing agents; a diol, which can be cleaved with periodates; a diazo bond, which can be cleaved with dithionates; an ester, which can be cleaved with hydroxylamines; a sulfone, which can be cleaved with bases, and others known in the art, as discussed more below.
  • the concentration of dissociating agent used to bring about dissociation will depend on the type of cleavable linkage and dissociating agent employed as well as other assay conditions.
  • the appropriate concentration and conditions to bring about a desired degree of dissociation may be selected in particular applications using routine screening tests and optimization.
  • the immobilized complexes may be exposed to a chemical dissociating agent in a concentration of between about 10 mM and about 100 mM, between about 1 mM and about 1 M, between about 0.1 M and about 1 M, etc.
  • the plurality of dissociated species is formed by exposing the substrate to a chemical dissociating agent.
  • the chemical dissociating agent may comprise at least one of a pH altering agent, an ionic strength altering agent, a denaturing agent, a reducing agent, a reactive agent, or an enzyme.
  • the chemical dissociating agent may comprise beta-mercaptoethanol.
  • the plurality of complexes may be exposed to the chemical dissociating agent thereby forming a plurality of dissociated species.
  • a chemical dissociating agent is particularly suited to cleave a particular cleavable linkage. Non limiting combinations of chemical dissociating agents and cleavable linkages will now be discussed in more detail.
  • the chemical dissociating agent is a reducing agent.
  • a reducing agent is a compound or complex that chemically reduces another chemical species susceptible to chemical reduction which can, in certain cases, lead to scission of the species.
  • the immobilized complex may be reduced by the reducing agent and at least a portion of the complex may be dissociated by the reducing agent to form a dissociated species.
  • Non- limiting examples of reducing agents that may be used to cleave a cleavable linkage include sodium borohydride, beta-mercaptoethanol, cysteine, tris(2-carboxyethyl) phosphine, zinc and dilute acid, triphenylphosphine (Ph 3 P) and water, dithiothreitol (DTT), and others known in the art.
  • a chemical dissociating agent may be substantially removed from the solution comprising the plurality of dissociated species prior to further manipulation of the plurality of dissociated species (e.g., partitioning of the plurality of dissociated species across a plurality of reaction vessels).
  • the chemical dissociating agent may be removed by filtration through a material to which the chemical dissociating agent may bind, by precipitation, or the like.
  • the chemical dissociated may not be substantially removed prior to further manipulation of the solution comprising the plurality of dissociated species.
  • each immobilized complex may comprise a disulfide bond, some of which may be cleaved to form a plurality of dissociated species.
  • each immobilized complex may comprise at least one capture component, at least one analyte molecule or particle, and at least one disulfide linkage. At least some of the disulfide linkages may be cleaved to form a plurality of dissociated species (e.g., which are not immobilized with respect to the substrate).
  • a non-limiting reagent that may be used for the formation of a disulfide linkage is pyridyl disulfide-containing heterobifunctionals (e.g., SDPD, N- succinimidyl-pyridyldithioproprionate and SMPT, 4-succinimidyloxycarbonyl- ⁇ -methyl- ⁇ - (2-pyridyldithio)toluene).
  • Disulfide bonds may be cleaved, for example, through hydrogenolysis by a reducing agent (e.g., beta-mercaptoethanol, dithiothreitol, dithioerythritol, cysteine, tris(2-carboxytheyl) phosphine, and/or butanethiol) or through a disulfide interchange process with a compound containing one or more free sulfhydryls.
  • a reducing agent e.g., beta-mercaptoethanol, dithiothreitol, dithioerythritol, cysteine, tris(2-carboxytheyl) phosphine, and/or butanethiol
  • a disulfide linkage may be cleaved in certain embodiments by exposing the linkage to a reducing agent.
  • the reduction will yield unconjugated molecules, one or both of which may contain a portion of the disulfide linkage, and in some cases, a free sulfhydryl (e.g., - SH) will be formed on one or both the ends of the cleaved linkage.
  • a free sulfhydryl e.g., - SH
  • a binding ligand may comprise a disulfide linkage.
  • the disulfide group may be cleaved by exposure of an immobilized complex comprising the binding ligand to beta-mercaptoethanol.
  • a capture component may comprise a disulfide linkage that may be cleaved by exposure to beta-mercaptoethanol and/or another reducing agent.
  • the cleavable linkage may be a diol or a glycol.
  • a diol or glycol linkage may be incorporated into a portion of the immobilized complex using methods that are known to those skilled in the art (e.g., inclusion of a tartaric acid residue).
  • the glycol or diol linkage may be cleaved by oxidation.
  • the immobilized complex comprising a diol or a glycol cleavable linkage may be exposed to a reagent such as sodium periodate or palladium acetate (Pd(OAc) 2 ), and the linkage may be cleaved to form aldehydes.
  • Pd(OAc) 2 palladium acetate
  • X and Y are components of the immobilized complex relative to the glycol linkage.
  • the cleavable linkage may be a diazo bond.
  • a diazo bond may be incorporated into a portion of the immobilized complexes using methods that are known to those skilled in the art (e.g., coupling between an arendiazonium cation and a phenol). For non-limiting examples see March, J.; Advanced Organic Chemistry, New York, John Wiley and Sons, 1992, pages 591-592, 638, etc.
  • a diazo linkage may be cleaved upon exposure to a chemical dissociating agent such as dithionite, zinc metal, and sodium hydrosulfite (Na 2 S 2 O 4 ) to form primary amines.
  • Equation 3 A non-limiting example of the cleavage of a diazo linkage is shown in Equation 3 : ⁇ N ⁇ N ⁇ - Y ⁇ X-NH 2 + H 2 N-Y (3) wherein X and Y are components of the immobilized complex relative to the diazo linkage.
  • the linkage may be an ester or a thioester.
  • An ester or a thioester linkage may be cleaved upon exposure to a nucleophile, such as hydroxylamine. The cleavage of an ester with hydroxylamine may result in the formation of an amide derivative on one side of the cleaved crosslinker and a hydroxyl group on the other side, as shown in Equation 4.
  • the linkage may be a sulfone linkage.
  • Sulfone linkages that may be incorporated into a complex include 4,4'-difluoro-3,3'- dinitrodiphenyl sulfone (DFDNPS) and bis(2-[succinimidooxycarbonyloxy]ethyl) sulfone (BSOCOES).
  • DFDNPS 4,4'-difluoro-3,3'- dinitrodiphenyl sulfone
  • BSOCOES bis(2-[succinimidooxycarbonyloxy]ethyl) sulfone
  • the sulfone linkage may be cleaved by hydrolysis under basic conditions, such as in the presence of sodium phosphate. The cleavage of a sulfone group is shown in Equation 5:
  • X and Y are components of the immobilized complex relative to the sulfone linkage.
  • the cleavable linkage may be an enzymatically cleavable linkage.
  • a protease may be used to cleave a cleavable functional group having a suitable recognition sequence for the protease.
  • Particularly useful proteases are endopeptidases such as factor Xa, tobacco etch virus (TEV) protease, trypsin, chymotrypsin, Staphylococcus aureus protease, submaxillaris protease, and others.
  • TEV tobacco etch virus
  • trypsin trypsin
  • chymotrypsin chymotrypsin
  • Staphylococcus aureus protease submaxillaris protease
  • the protease can be selected based on the incorporation of a particular cleavable recognition sequence as a functional group. Other considerations for selecting a protease include the presence or absence of a recognition sequence in the molecule being captured and released.
  • the cleavable linkage may be a pH-cleavable linkage and comprise pH-labile bonds.
  • ketals may be labile (e.g., cleaved) in acidic environments (e.g., pH less than 7) and form a diol and a ketone.
  • acetals may be labile (e.g., cleaved ) in acidic environments (e.g., pH less than 7) and form a diol and an aldehyde.
  • imines or iminiums may be labile in acidic environments (e.g., pH less than7) and form an amine and an aldehyde or a ketone, respectively.
  • a cleavable linkage may be cleaved in basic conditions (e.g., sulfone linkage).
  • a method of the present invention may comprise releasing a plurality of molecules or particles from the first substrate.
  • the plurality of molecules or particles released from the first substrate may comprise at least a portion of an analyte molecule or particle captured on the first substrate or may be free of any portion of an analyte molecule or particle captured on the first substrate.
  • a plurality of molecules or particles may be released by exposing the substrate to electromagnetic radiation (e.g., ultraviolet light).
  • a plurality of molecules or particles may be released by exposing the substrate to a chemical dissociating agent (e.g., beta- mercaptoethanol).
  • a plurality of molecules or particles may be released by physical manipulations (e.g., scraping the substrate, agitation, etc.).
  • the plurality of dissociated species may be detected and/or quantified, and the detection and/or quantification may be related to the presence and, optionally, quantity and/or concentration of analyte molecules/particles in the sample being tested.
  • the plurality of dissociated species may be detected and/or quantified by partitioning the plurality of dissociated species across a plurality of reaction vessels (e.g., in an array). In certain embodiments, the partitioning may occur such that at least some (e.g., a statistically significant fraction) of the reaction vessels comprise at least one or, in certain cases, one dissociated species and at least some (e.g., a statistically significant fraction) of the reaction vessel comprises no dissociated species.
  • a detector may be configured to detect dissociated species in or on a substrate (e.g., an array of reaction vessels).
  • the number of dissociated species detected on or within the second substrate may be directly proportional to the number of dissociated species released from the second substrate. In other embodiments, the number of dissociated species detected on or within the second substrate may be approximately equal to, less than about one half, less than about one quarter, less than about one tenth, less than about one hundredth, less than about one thousandth, less than about one ten- thousandth, or the like, of the number of the plurality of dissociated species dissociated from the first substrate.
  • the number of dissociated species detected is no greater than the number of dissociated species released and/or the number of dissociated species is no greater than the number of analyte molecules or particles associated with a first substrate.
  • the number of dissociated species detected (e.g., on a second substrate) may be approximately equal to, no greater than about three quarters of, no greater than about one half of, no greater than about one quarter of, no greater than about one tenth of, no greater than about one hundredth of, no greater than about one thousandth of, no greater than about ten thousandth of, or no greater than about one hundred thousandth of, the number of analyte molecules or particles associated (e.g., captured) with a first substrate and/or the number of dissociated species released (e.g., from a first substrate).
  • the dissociated species may be detected and/or quantified directly or indirectly.
  • the dissociated species itself comprises a molecule or moiety that may be directly interrogated and detected, for example, a fluorophore.
  • a reaction vessel that comprises such a dissociated species will therefore emit a signal upon interrogation of the reaction vessel.
  • the indirect approach can include, for example, exposing dissociated species comprising at least one precursor labeling agent molecule to a suitable converting agent to produce a detectable labeling agent molecule.
  • the dissociated species may comprise a converting agent and may be exposed within the reaction vessel to a precursor labeling agent molecule to produce a detectable labeling agent molecule.
  • At least one precursor labeling agent molecule is converted to a labeling agent molecule when contained in a reaction vessel comprising a dissociated species.
  • the presence or absence of a dissociated species in a reaction vessel may then be determined by determining the presence or absence of a labeling agent molecule in the reaction vessel.
  • the dissociated species may comprise an enzymatic component and the precursor labeling agent molecule may be a chromogenic, fluorogenic, or chemiluminescent enzymatic precursor labeling agent molecule. Exposure of the precursor labeling agent molecule to the enzymatic component may convert the precursor labeling agent molecule to a chromogenic, fluorogenic, of chemiluminescent labeling agent molecule that may be detected.
  • the indirect approach can include, for example, the situation where the dissociated species does not have intrinsic activity to convert a precursor labeling agent molecule to a labeling agent molecule and is not detectable itself. Therefore, the dissociated species may be exposed to at least one dissociated species binding ligand which may associate with the dissociated species, thus forming a complex comprising the dissociated species and the dissociated species binding ligand.
  • the dissociated species binding ligand may comprise a component that is able to convert a precursor labeling agent molecule into a labeling agent molecule or may comprise a precursor labeling agent molecule able to be converted by exposure to a converting agent.
  • the presence of the labeling agent molecule can be determined to determine whether a dissociated species is present in a reaction vessel.
  • a dissociated species comprises an enzymatic component
  • enzymatic precursor labeling agent molecules can be added to the reaction vessel whereupon the enzymatic component converts the precursor labeling agent molecule into a chromogenic, fluorogenic, or chemiluminescent labeling agent molecule and permits the detection of the dissociated species.
  • FIGS Two non-limiting examples of indirect detection of a dissociated species are illustrated in FIGS.
  • a reaction vessel 30 which comprises a detection capture component 32.
  • Dissociated species 34 is associated with detection capture component 34.
  • the reaction vessel is exposed to precursor labeling agent molecule 36, which upon exposure to dissociated species 34 carrying a converting agent, converts to a labeling agent molecule 38, as indicated by arrow 37.
  • a reaction vessel 40 is provided which comprises a detection capture component 42.
  • Dissociated species 44 is associated with the detection capture component 42.
  • a dissociated species binding ligand 46 is provided which associates with dissociated species 44.
  • the reaction vessels is exposed to precursor labeling agent molecule 48 which upon exposure to dissociated species binding ligand 44 converts to labeling agent molecule 50, as indicated by arrow 49.
  • a dissociated species may comprise an enzymatic component (e.g., from a binding ligand).
  • the precursor labeling agent molecule may be an enzymatic label, for example, a chromogenic, fluorogenic, or chemiluminescent enzymatic precursor labeling agent molecule, that upon contact with the enzymatic component, converts to a labeling agent molecule.
  • the chromogenic, fluorogenic, or chemiluminescent enzymatic precursor labeling agent molecule is provided in an amount sufficient to contact every dissociated species which was partitioned across a plurality of reaction vessels.
  • the enzymatic component may comprise beta-galactosidase or horseradish peroxidase.
  • chromogenic, fluorogenic, or chemiluminescent enzymatic precursor labeling agent molecules may be selected for conversion by many different enzymes.
  • any known chromogenic, fluorogenic, or chemiluminescent enzyme precursor labeling agent molecule capable of producing a labeling agent molecule in a reaction with a particular enzyme can potentially be used in the present invention as the precursor labeling agent molecule in embodiments where the dissociated species comprises an enzymatic component (or a dissociated species binding ligand comprises an enzymatic component).
  • the plurality of reaction vessels may additionally comprise at least one dissociated species capture component.
  • a "dissociated species capture component” is a capture component that specifically binds to or otherwise captures a dissociated species, such that the dissociated species is immobilized during the assay.
  • the dissociated species capture component allows the attachment of a dissociated species to a solid support (that is, the surface of a microwell, a sealing component or a nanoparticle) for the purposes of detection, quantification, or other analysis.
  • a method of the present invention will comprise the step of immobilizing at least one of the plurality of dissociated species with respect to the at least one dissociated species capture component.
  • at least some of the plurality of dissociated species may be immobilized with respect to at least one particle comprising at least one binding partner for the dissociated species, thereby forming a plurality of complexes.
  • a plurality of particles comprising a binding partner may be provided, in some cases, such that one or zero dissociated species released from the substrate associates with each binding partner of at least a fraction of the plurality of particles comprising a binding partner.
  • the particles may have an average diameter of less than about 100 urn, less than about 50 um, less than about 10 um, less than about 1 um, less than about 500 nm, less than about 100 nm, less than about 10 nm, less than about 1, or the like.
  • the particles may be provided such that there is greater than about 10, greater than about 100, greater than about 1000, greater than about 10,000, greater than about 100,000, greater than about 1,000,000, or the like, particles provided for every dissociated species released from the substrate.
  • the plurality of dissociated species may be immobilized with respect to a plurality of particles prior to partitioning across a plurality of reaction vessels.
  • the plurality of dissociated species may be exposed to a plurality of particles such that either one or no dissociated species are immobilized with respect to a single particle.
  • the particles may comprise a plurality of binding partners (e.g., biotin) with respect to which a dissociated species may be immobilized, thereby forming a plurality of particle-containing complexes.
  • the particle- containing complexes may be partitioned across a plurality of reaction vessels such that at least some (e.g., a statistically significant fraction) of the plurality of reaction vessels contain zero particle-containing complexes and at least some (e.g., a statistically significant fraction) of the plurality of reaction vessels contain only one or at least one particle-containing complex.
  • the reaction vessels which comprise a particle-containing complex may then be determined, and the methods describe herein may be used to detect and/or quantify the dissociated species and analyte molecules.
  • the number or fraction of the plurality of reaction vessels that contain a particle-containing complex may be related to a measure of the concentration of analyte molecules or particles in the fluid sample.
  • the particle-containing complexes may be detected directly or indirectly using methods as discussed herein (e.g., electromagnetic radiation, microscopy, etc.).
  • Each reaction vessel may comprise at least about 10 particles, at least about 100 particles, at least about 500 particles, at least about 1000 particles, at least about 5000 particles, at least about 10000 particles, or the like, wherein in some reaction vessels, at least one particle may comprise a complex (e.g., a particle-containing complex).
  • Each reaction vessel may comprise approximately the same or differing number of particles as the reaction vessels which it is adjacent to.
  • a non-limiting embodiment of the above assay method is depicted in FIG. 16.
  • a plurality of particles 330 are exposed to a plurality of dissociated species such that at least some of each of the plurality of dissociated species 332 are immobilized with respect to a particles 330 to form a plurality of dissociated species in the form of particle- containing complexes 334 (step (A)).
  • the plurality of particles and particle-containing complexes may be partitioned across a plurality of reaction vessels 336 such that at least some of the plurality of reaction vessels contain only one or at least one particle- containing complex 338 and at least some of the plurality of reaction vessels will contain zero particle-containing complexes 340 (step (B)).
  • at least a fraction of the number of dissociated species may be detected substantially simultaneously.
  • substantially simultaneously when used in conjunction with detection, as used herein, refers to detection of the species/molecules/particles of interest at approximately the same time, as opposed to sequentially detected.
  • a plurality of species/molecules/particles may be detected substantially simultaneously using various techniques, including optical techniques (e.g., CCD detector).
  • optical techniques e.g., CCD detector.
  • each of the detected species/molecules/particles e.g., dissociated species
  • resolving individual molecules/particles of a plurality of molecules/particles that are partitioned across a plurality of reaction vessels such that each reaction vessel contains zero or only one species/molecule/particle involves detecting molecules/particles that are spatially separated.
  • at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% of all species/molecules/particles are spatially separated with respect to other species/molecules/particles during detection.
  • the reaction vessels may be interrogated using a variety of techniques, including techniques known to those of ordinary skill in the art.
  • reaction vessels are optically interrogated.
  • the reaction vessels exhibiting changes in their optical signature may be identified by a conventional optical train and optical detection system.
  • optical filters designed for a particular wavelength may be employed for optical interrogation of the reaction vessels.
  • the plurality of reaction vessels of the present invention is formed directly as part of a fiber optic bundle.
  • the array of reaction vessels of the present invention can be used in conjunction with an optical detection system such as the system described in U.S. Publication No. 20030027126.
  • the array of reaction vessels of the present invention is formed in one end of a fiber optic assembly comprising a fiber optic bundle constructed of clad fibers so that light does not mix between fibers.
  • FIG. 17A shows a non-limiting example of a system of the present invention according to some embodiments.
  • the system comprises a light source 252, excitation filter 454, dichromatic mirror 458, emission filter 460, objective 470, and second substrate 472. Light 453 given off from light source 452 is passed through excitation filter 454.
  • the light reflects off dichromatic mirror 458, passes through objective 470 and shines on second substrate 472.
  • stray light 464 may be reduced by a stray light reducing function 468, such as an iris or aperture.
  • Light 471 emitted from the second substrate passes through objective 472 and emission filter 460 and is observed.
  • the system may comprise additional components (e.g., additional filters, mirrors, magnification devices, etc.) , as needed for particular applications, as would be understood by those of ordinary skill in the art.
  • the optical detection system of U.S. Publication No. 20030027126 operates as follows. Light returning from the distal end of the fiber optic bundle is passed by the attachment to a magnification changer which enables adjustment of the image size of the fiber's proximal or distal end. Light passing through the magnification changer is then shuttered and filtered by a second wheel. The light then is imaged on a charge coupled device (CCD) camera. A computer executes imaging processing software to process the information from the CCD camera and also optionally controls the first and second shutter and filter wheels. As depicted in U.S. Publication No. 20030027126, the proximal end of the bundle is received by a z-translation stage and x-y micropositioner.
  • CCD charge coupled device
  • FIG. 18 shows a schematic block diagram of a system employing a fiber optic assembly 400 with an optical detection system.
  • the fiber optic assembly 400 comprises a fiber optic bundle or array 402 that is constructed from clad fibers so that light does not mix between fibers.
  • An array or system, 400 is attached to the bundle's distal end 412, with the proximal end 414 being received by a z-translation stage 416 and x-y micropositioner 418. These two components act in concert to properly position the proximal end 414 of the bundle 402 for a microscope objective lens 420.
  • Light collected by the objective lens 420 is passed to a reflected light fluorescence attachment with three pointer cube slider 422.
  • the attachment 422 allows insertion of light from a 75 Waft Xe lamp 424 through the objective lens 420 to be coupled into the fiber bundle 402.
  • the light from the source 424 is condensed by condensing lens 426, then filtered and/or shuttered by filter and shutter wheel 428, and subsequently passes through a ND filter slide 430.
  • Light returning from the distal end 412 of the bundle 402 is passed by the attachment 422 to a magnification changer 432 which enables adjustment of the image size of the fiber's proximal or distal end.
  • Light passing through the magnification changer 432 is then shuttered and filtered by a second wheel 434.
  • the light is then imaged on a charge coupled device (CCD) camera 436.
  • a computer 438 executes imaging processing software to process the information from the CCD camera 436 and also possibly control the first and second shutter and filter wheels 428, 434.
  • CCD charge coupled device
  • the array of reaction vessels of the present invention may be integral with or attached to the distal end of the fiber optic bundle using a variety of compatible processes.
  • microwells are formed at the center of each individual fiber of the fiber optic bundle and the microwells may or may not be sealed.
  • Each optical fiber of the fiber optic bundle may convey light from the single micro well formed at the center of the fiber's distal end. This feature enables the interrogation of the optical signature of individual reaction vessels to identify reactions/contents in each microwell. Consequently, by imaging the end of the bundle onto the CCD array, the optical signatures of the reaction vessels are individually interrogatable and may be detected substantially simultaneously.
  • the plurality of reaction vessels may be sealed, for example, through the mating of the second substrate and a sealing component.
  • the sealing of the reaction vessels may be such that the contents of each reaction vessel cannot escape the reaction vessel.
  • the reaction vessels may be sealed after the addition of a dissociated species and, optionally, a precursor labeling agent molecule to facilitate detection of the dissociated species.
  • a reaction to produce the detectable labeling agent molecule can proceed within the sealed reaction vessels, thereby producing a detectable amount of a labeling agent molecule that is retained in the reaction vessel for detection purposes.
  • the methods, systems, and/or devices may be used to determine the presence and/or a measure of the concentration of a plurality of analyte molecules (or particles) in a fluid sample based at least in part on detecting and/or quantifying at least some of a plurality of dissociated species (or particles or molecules released from a substrate on which the analyte molecules/particles are captured). In some cases, there is a correlation between the percentage of reaction vessels containing one or more dissociated species and the quantity/concentration of analyte molecules in the fluid sample.
  • the quantification method of the present invention may allow for a determination of a measure of the concentration of analyte molecules in a fluid sample based on the percentage of reaction vessels that contain a dissociated species released from a substrate.
  • concentration of the analyte molecules in a fluid sample will be linearly proportional to the quantification of the dissociated species.
  • the measure of concentration of the analyte molecules in a fluid sample will be related to the determination and/or quantification of the dissociated species by a non-linear relationship.
  • the measure of the concentration of analyte molecules in a fluid sample will be determined using a calibration curve. Methods to determine a measure of the concentration of analyte molecules in a fluid sample are discussed more below.
  • Certain embodiments of present invention are distinguished by the ability to detect and/or quantify low numbers/concentrations of dissociated species and may be well suited to determine a measure of the concentration of analyte molecules in a fluid sample containing very low concentrations of the analyte. It is currently believed that this ability may be achieved by spatially isolating individual or small numbers of dissociated species, for example, as partitioned across an array of reaction vessels, and then detecting their presence in the reaction vessels.
  • the presence of a dissociated species in a reaction vessel can be counted in a binary fashion (e.g., zero when a dissociated species is absent; one when a dissociated species is present), for example by determining the presence of a detectable molecule or particle in a reaction vessel that contains at least one dissociated species.
  • the plurality of dissociated species may be partitioned such that at least some of the reaction vessels contain no dissociates species and at least some of reaction vessels contain at least one or, in certain cases, only one dissociates species.
  • the plurality of dissociates species may be partitioned such that a statistically significant fraction of the reaction vessels contain no dissociates species and a statistically significant fraction of reaction vessels contain at least one dissociates species.
  • the plurality of dissociated species may be partitioned such that a statistically significant fraction of the reaction vessels contain no dissociated species and a statistically significant fraction of reaction vessels contain only one dissociated species.
  • the number of the plurality of reaction vessels and/or fraction of the plurality of reaction vessels that contain or do not contain a dissociated species may be determined.
  • the number and/or fraction of the plurality of reaction vessels that contain a dissociated species can be related to the concentration of analyte molecules or particles in the sample.
  • a measure of the concentration of analyte molecules or particles in the fluid sample is determined based on the determination of the number and/or fraction of the plurality of reaction vessels that contain a dissociated species.
  • the measure of the concentration of the analyte molecules or particles in the fluid sample is determined at least in part by comparison of a measured parameter to a calibration standard and/or by a Poisson and/or Gaussian distribution analysis of the number or fraction of the plurality of reaction vessels that contain a dissociated species, as discussed more below.
  • a "statistically significant fraction" of the reaction vessels that contain a specified quantity of dissociated species is defined as the minimum number of reaction vessels that can be reproducibly determined to contain a dissociates species with a particular system of detection (i.e., substantially similar results are obtained for multiple essentially identical fluid samples comprising the dissociates species) and that is above the background noise (e.g., non-specific binding) that is determined when carrying out the assay with a sample that does not contain any analyte molecules or particles, divided by the total number of reaction vessels.
  • the statistically significant fraction may be experimentally determined for a certain assay type and equipment set up (e.g., for each dissociates species determined, each binding ligand, etc).
  • the percentage of reaction vessels (e.g., the statistically significant fraction) which comprises only one or at least one dissociates species is less than about 10%, less than about 5%, less than about 1%, less than about 0.5%, or less than about 0.1% of the total reaction vessels. In some cases, the percentage of reaction vessels which do not contain an dissociates species is at least about 20%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, or at least about 95%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater, of the total number of reaction vessels.
  • a measure of the concentration of analyte molecules or particles in the fluid sample may be determined at least in part by comparison of a measured parameter to a calibration standard.
  • the fraction of reaction vessels that comprise a dissociated species may be calibrated against a calibration curve to determine a measure of the concentration of the analyte molecule in the fluid sample.
  • the calibration curve may be produced by completing the assay with a plurality of standardized samples of known concentration under the conditions used to analyze the test samples. A reading may be taken for the signal related to the detection/quantification of the dissociated species for each standardized sample, therefore allowing for the formation of a calibration curve relating the detection of the dissociated species with a known concentration of the analyte molecule.
  • the assay may then be completed on a sample comprising the analyte molecule in an unknown concentration, and the detection of the dissociated species from this assay may be plotted on the calibration curve, therefore determining a measure of the concentration of the analyte molecule in the fluid sample.
  • four standardized fluid samples comprising an analyte molecule in varying concentration (w, x, y, and z) are provided.
  • An assay e.g., immobilizing the analyte molecules, dissociating a plurality of dissociated species, detecting at least a portion of the dissociated species
  • a value corresponding to the detection of the dissociated species (b, c, d, and e) may be determined.
  • a plot is produced of the values related to detection of the dissociated species (b, c, d, and e) versus the concentration of the standardized samples (w, x, y, and z), as depicted in FIG. 19.
  • the assay may be then be carried out under substantially identical conditions on a fluid sample comprising an analyte molecule of unknown concentration t, wherein the resulting value related to detection of the dissociated species is f.
  • This value may be plotted on the graph and a measure of the unknown concentration of the target analyte in the fluid sample may be interpolated from the values of the standardized samples.
  • the calibration curve may have a limit of detection, wherein the limit of detection is the lowest concentration of analyte molecules in a fluid sample that may be accurately determined.
  • the r 2 value of the calibration curve may be greater than about 0.5, greater than about 0.75, greater than about 0.8, greater than about 0.9, greater than about 0.95, greater than about 0.97, greater than about 0.98, greater than about 0.99, greater than about 0.9999, or about 1.
  • the concentration of analyte molecules in the fluid sample may be determined by comparison to a calibration curve using a system comprising a computer.
  • the computer may comprise software that may use the data collected to produce the calibration curve and/or a reading of the measured concentration of the analyte molecules in the fluid sample.
  • a fluorescence image of an array comprising the dissociated species partitioned across the array e.g., second substrate
  • image analysis software e.g., IP Lab, BD
  • the analysis software may automatically calculate the number of reaction vessels that have fluorescence intensity over the background intensity and produce a number indicative of the total number of reaction vessel which comprises a fluorescent intensity over the background intensity (e.g., a number that correlates to the number of reaction vessels which comprise a dissociated species).
  • the number of reaction vessels which comprise fluorescence intensity over the background intensity may be divided by the total number of reaction vessels to give a number correlating to the fraction of reaction vessels which comprise a dissociated species.
  • the active well fraction may be compared to a calibration curve to determine a measure of the concentration of analyte molecules in the fluid sample.
  • the number of dissociated species that are detected may or may not be approximately equal to the number of analyte molecules in the fluid sample.
  • the ratio of analyte molecules to dissociated species that are detected may be about 1 :1, about 2:1, about 5:1, about 10:1, about 100:1, about 1000:1, about 10000:1, or the like. In some cases, the ratio is greater than about 1:1, greater than about 10:1, great than about 100:1, greater than about 1000:1, greater than about 10000:1, or the like. Additionally, the number of dissociated species that are detected may or may not be approximately equal to the total number of dissociated species released from the substrate.
  • the ratio of dissociated species dissociated to dissociated species that are detected may be about 1 :1, about 2:1, about 5:1, about 10:1, about 100:1, about 1000:1, about 10000:1, or the like. In some cases, the ratio is greater than about 1:1, greater than about 10:1, great than about 100:1, greater than about 1000:1, greater than about 10000: 1 , or the like.
  • the number of molecules or particles detected on or within the second substrate is less than about one half, less than about one quarter, less than about one tenth, less than about one hundredth, less than about one thousandth, less than about one ten-thousandth, less than about one hundred- thousandth of the number of the plurality of molecules or particles released from the first substrate.
  • the concentration of analyte molecules or particles in the fluid sample that may be substantially accurately determined is less than about 5000 fM, less than about 3000 fM, less than about 2000 fM, less than about 1000 fM, less than about 500 fM, less than about 300 fM, less than about 200 fM, less than about 100 fM, less than about 50 fM, less than about 25 fM, less than about 10 fM, less than about 5 fM, less than about 2 fM, less than about 1 fM, less than about 0.5 fM, less than about 0.1 fM, or the like.
  • the concentration of analyte molecules or particles in the fluid sample that may be substantially accurately determined is between about 5000 fM and about 0.1 fM, between about 3000 fM and about 0.1 fM, between about 1000 fM and about 0.1 fM, between about 1000 fM and about 1 fM, between about 100 fM and about 1 fM, between about 100 fM and about 0.1 fM, or the like.
  • the concentration of analyte molecules or particles in a fluid sample may be considered to be substantially accurately determined if the measured concentration of the analyte molecules or particles in the fluid sample is within about 10% of the actual (e.g., true) concentration of the analyte molecules or particles in the fluid sample.
  • the measured concentration of the analyte molecules or particles in the fluid sample may be within about 5%, within about 4%, within about 3%, within about 2%, within about 1%, within about 0.5%, within about 0.4%, within about 0.3%, within about 0.2% or within about 0.1%, of the actual concentration of the analyte molecules or particles in the fluid sample.
  • the measure of the concentration determined differs from the true (e.g., actual) concentration by no greater than about 20%, no greater than about 15%, no greater than 10%, no greater than 5%, no greater than 4%, no greater than 3%, no greater than 2%, no greater than 1%, or no greater than 0.5%.
  • the accuracy of the assay method may be determined, in some embodiments, by determining the concentration of analyte molecules in a fluid sample of a known concentration using the selected assay method.
  • the quantification method is believed to be driven in part by the fact that the number and volume of reaction vessels employed govern the dynamic range of concentrations that can be determined. That is, based on the number and volume of the reaction vessels in an array of the present invention, an estimate can be made of the range of concentrations of detected molecules in the solution partitioned across the vessels that allows for a measure of the concentration to be determined using certain methods of the present invention.
  • a solution having a concentration of approximately 4 x 10 " M dissociated species in a fluid sample will yield, on average, one dissociated species per reaction vessel.
  • distributing a fluid sample having a dissociated species concentration within the appropriate range into an array of reaction vessels will not result in the distribution of exactly one dissociated species per each reaction vessel; statistically, some vessels will have multiple dissociated species while others will have no dissociated species.
  • the ratio of vessels containing one or more dissociated species to the number of vessels containing no dissociated species is high, the data may be fit to a Gaussian distribution.
  • the Poisson distribution may be applied to the data. This limiting distribution may be used to calculate the probability of rare events occurring in a large number of trials. For example, based on Poisson statistics, for a concentration of approximately 4 x 10 " M, a distribution between zero and five dissociated species per container is predicted, with the most probable values being zero and one. Equation 1 can be used to determine the probability of observing events based on the expected average number of events per trial, ⁇ :
  • the array of the present invention can be used to determine the concentration of a dissociated species by a simple digital readout system (e.g., "counting" of active wells) as described herein in combination with a suitable calibration.
  • the quantification method of the present invention can be performed as follows.
  • the method employs a digital readout system (also referred to as a "binary readout system”) that involves first detecting the dissociated species in the plurality of reaction vessels by any detection method as described herein.
  • the number of reaction vessels which comprise a dissociated species is then counted and a percentage of the total number of reaction vessels which comprise a dissociate species is calculated. That is, utilization of a yes or no response, in conjunction with the high- density array of reaction vessels, permits the digital readout of bulk concentrations of dissociated species.
  • this readout is accomplished by counting the number of reaction vessels containing at least one labeling agent molecule, with the resulting number of reaction vessels comprising a labeling agent molecule corresponding to the number of reaction vessels comprising a dissociated species.
  • the ratio of dissociated species to reaction vessels may be at least about 1 :100, at least about 1 : 1000, at least about 1 : 10,000, as the large number of reaction vessels provides a statistically significant signal even at this low ratio.
  • the quantification method of the present invention may only be limited by the number of individual reaction vessels that can be fabricated and interrogated.
  • expanding the number of reaction vessels may increase both the dynamic range and the sensitivity of the assay.
  • increasing the number of reaction vessels by a factor often may decrease the ratio of dissociated species to reaction vessels by a factor often, thereby increasing the dynamic range and/or sensitivity of the assay.
  • an array will comprise between about 1,000 and about 50,000, between about 1,000 and about 1,000,000, between about 1,000 and about 10,000, between about 10,000 and about 100,000, between about 100,000 and about 1,000,000, between about 1,000 and about 100,000, between about 50,000 and about 100,000, between about 20,000 and about 80,000, between about 30,000 and about 70,000, between about 40,000 and about 60,000, or about 50,000, or the like, reaction vessels.
  • accuracy of this technique may be compromised above and below the thresholds of the dynamic range. For example, as the concentration of the dissociated species goes below the lower limit of the dynamic range, the number of dissociated species may be too low to observe a sufficient number of occupied wells.
  • the number of reaction vessels could be decreased in order to make sure that at least some (e.g., a statistically significant number) of them are occupied by a dissociated species, the volume of the reaction vessels could be increased, and/or the sample tested could be concentrated. Results for extremely dilute concentrations may have large relative errors associated with them, due to the very small number of reaction vessels that are expected to show activity. In other cases, the ultimate upper limit to this technique may occur when 100% of the reaction vessels contain at least one dissociated species. At this limit, discrimination between two solutions of high dissociated species concentrations may not be feasible.
  • reaction vessels could be used, and/or the volume of each reaction vessel could be reduced, and/or the concentration of the sample could be reduced, e.g., through serial dilutions.
  • the probability that any well contains two or more dissociated species is very small and the number of dissociated species closely matches the number of occupied reaction vessels. Between 20% occupied and 100% occupied, an increasing number of wells may contain more than one dissociated species, however Gaussian statistics can still be used to correlate occupancy fraction with concentration with reasonable accuracy until the occupancy fraction approaches 100%.
  • the practical dynamic range of the method may be increased in several ways.
  • the sample and/or solution carrying the dissociated species may be diluted by a factor of 10 or more. Both the solution comprising the dissociated species and the diluted solution comprising the dissociated species may be assayed concurrently using the method of this invention.
  • the dynamic ranges of the two assays may overlap, but be offset by the dilution factor, hence extending the dynamic range.
  • multiple arrays of reaction vessels can be used, each array having reaction vessels with differing volumes, differing binding surface areas, or differing density and/or type of capture components on the binding surface.
  • These configurations can be constructed as either distinct arrays or as one large array with distinct sub-arrays with varying characteristics. Since the probability of a dissociated species being detected in a given reaction vessel can be related to volume, binding surface area, and capture component density, the sub-arrays may be designed to provide different sensitivity ranges. Thus, with such configurations the effective range of the combined array may be extended.
  • less than about 20% of the total number of the plurality of reaction vessels will contain at least one dissociated species (i.e. at least 80% of reaction vessels will be free of dissociated species). Under such circumstances, the number of reaction vessels containing at least one dissociated species will typically fall within the linear range of a Poisson distribution. In another embodiment, more than about 20% but less than about 60% of the total number of reaction vessels contains at least one dissociated species. Under such circumstances, the number of reaction vessels containing at least one dissociated species will typically fall within the non-linear range of a Poisson distribution.
  • more than about 60% but less than about 95% of the total number of reaction vessels contains at least one dissociated species. Under such circumstances, the number of reaction vessels containing at least one dissociated species will typically fall within the highly non-linear range of a Poisson distribution. In embodiments where greater than about 60% of the total number of reaction vessel contains at least one dissociated species, it may be desirable to decrease the percentage of reaction vessels which comprise at least one dissociated species (e.g., to less than about 20%). This may be accomplished using any suitable technique discussed herein, for example, diluting the fluid comprising the dissociated species and/or increasing the number of reaction vessels.
  • less than about 1%, less than about 5%, less than about 10%, less than about 20%, less than about 40%, less than about 60%, less than about 80%, less than about 90%, less than about 95%, or less than about 99% of the total number of the plurality of reaction vessels will contain at least one dissociated species. In certain embodiments, more than about 1%, more than about 5%, more than about 10%, more than about 20%, more than about 40%, more than about 60%, more than about 80%, more than about 90%, more than about 95%, or more than about 99% of the total number of the plurality of reaction vessels will contain no dissociated species.
  • the invention provides a method of determining the concentration of the dissociated species in a fluid, the method comprising dividing the fluid containing the dissociated species into a plurality of second, smaller fluid samples of essentially equal volume so that at least some (e.g., a statistically significant fraction) of the second, smaller fluid samples contain either no dissociated species or a single dissociated species; determining the presence or absence of a dissociated species in each of the second, smaller fluid samples so as to identify the number of second, smaller fluid samples that contain a dissociated species; and determining the concentration of dissociated species in the fluid sample to be tested from the number of second, smaller samples that contain the dissociated species.
  • the present invention provides a method for determining the concentration of dissociated species in a fluid, the method comprising the partitioning at least a portion of the dissociated species in the fluid across a plurality of reaction vessels so that a statistically significant fraction of the reaction vessels contain a dissociated species and a statistically significant fraction of the reaction vessels contain no dissociated species; determining the presence or absence of a dissociated species in each reaction vessel to identify the number of reaction vessels that contain a dissociated species and/or to identify the number of reaction vessels that contain no dissociated species; and determining the concentration of dissociated species in the fluid at least in part from the number of reaction vessels that do or do not contain a dissociated species.
  • the concentration of dissociated species in the fluid sample is determined at least in part using a calibration curve, a Poisson distribution analysis and/or a Gaussian distribution of the number of reaction vessels that contain at least one or one dissociates species. In other embodiments, the concentration of dissociated species in the fluid sample is determined at least in part by a Gaussian distribution analysis of the number of reaction vessels that contain a dissociated species.
  • the present invention provides a method of determining the concentration of dissociated species in a fluid, the method comprising exposing the fluid to a plurality of reaction vessels under conditions so that at least one dissociated species is captured in at least some of the reaction vessels, wherein each reaction vessel comprises a microwell and an optional sealing component and each reaction vessel defines a binding surface that has a capture component immobilized thereon; determining the presence or absence of a dissociated species in each reaction vessel so as to identify the number of reaction vessels that contain a dissociated species and/or the number of reaction vessels that do not contain a dissociated species; and determining the concentration of dissociated species in the fluid sample to be tested from the number of reaction vessels that contain and/or do not contain a dissociated species.
  • At least about 99%, at least about 95%, at least about 90%, at least about 80%, at least about 50%, at least about 20%, at least about 10% at least about 5%, at least about 1%, and the like of the reaction vessels contain either zero or one dissociated species.
  • the concentration of dissociated species in the fluid sample is determined at least in part using a calibration curve, a Poisson distribution analysis and/or a Gaussian distribution analysis of the number of reaction vessels that contain at least one or one dissociated species.
  • the present invention provides a method of determining the concentration of dissociated species in a fluid sample to be tested, the method comprising partitioning at least a portion of the dissociated species in the fluid into a plurality of reaction vessels, so that, for substantially all of the reaction vessels, each reaction vessel contains either no dissociated species or a single dissociated species; determining the presence or absence of a dissociated species in a plurality of reaction vessels to provide a fraction of the interrogated reaction vessels that contain a dissociated species; and determining the concentration of dissociated species in the fluid from the fraction of interrogated reaction vessels that contain a dissociated species.
  • the present invention provides a method of determining the concentration of dissociated species in a fluid, the method comprising partitioning the fluid into a plurality of second, smaller fluid samples of equal volume so that at least some of the second, smaller fluid samples contain either a single dissociated species or no dissociated species, (b) determining the presence or absence of a dissociated species in at least a subset of the second samples so as to identify the fraction of second samples in the subset that contain a dissociated species; and determining the concentration of dissociated species in the sample to be tested from the fraction of second samples of the subset that contain the dissociated species.
  • a method of the present invention may be used for the detection of dissociated species in a fluid.
  • a method of detecting dissociated species in a fluid may comprise providing a fluid containing the dissociated species and an array, the array comprising a plurality of reaction vessels; contacting the array with the fluid such that the ratio of the number of dissociated species in the fluid contacted with the array to the number of reaction vessels in the array is less than 1 :1; and determining the number of reaction vessels which contain a dissociated species.
  • the ratio of the number of dissociated species in the fluid contacted with the array to the number of reaction vessels in the array is less than about 1 :5, less than about 1:10, less than about 1:100, or less than about 1:500.
  • a method of detecting dissociated species in a fluid comprises providing a fluid and an array, the fluid comprising at least one dissociated species at a first concentration, the array comprising a plurality of reaction vessels; diluting the fluid to create a diluted fluid, wherein the diluted fluid comprises the dissociated species at a second concentration,; contacting the array with the diluted fluid such that the ratio of dissociated species to the total number of reaction vessels in the array is between 1 : 1 and 1 :500; and determining the number of vessels of said array which contain a dissociated species.
  • the ratio is less than about 1 :1, less than about 1 :5, less than about 1 :10, less than about 1 : 100, or less than about 1 :500.
  • Optical fiber bundles were purchased from Schott North America, Inc (Southbridge, MA).
  • the core glass comprised barium, lanthanum, boron, silica, and aluminum.
  • the refractive index of the core was 1.694, and the density was 4.23 g/cc.
  • the cladding glass comprised silica, lead, potassium, sodium, and aluminum.
  • the refractive index of the clading was 1.559, and the density was 3.04 g/cc.
  • the fiber array was a bundle of 50,000 individual fibers, each with a core diameter of 4.5 ⁇ m and the center-to-center spacing of the cores was 8 ⁇ m.
  • Non-reinforced gloss silicone sheeting was obtained from Specialty Manufacturing Inc. (Saginaw, MI). Hydrochloric acid, 3-aminopropyl trimethoxysilane, anhydrous ethanol, molecular biology grade Tween®20, and N,N-Dimethyl formamide were all obtained from Sigma-Aldrich (Saint Louis, MO). Phosphate buffered saline, BlockerTM BSA (10%), Zeba Desalt spin columns, NHS-ss-biotin and NHS-LC-biotin were obtained from Pierce (Rockford, IL).
  • Resorufin-beta-D-galactopyranoside, streptavidin-beta-galactosidase, and 1 urn functionalized magnetic microspheres were purchased from Invitrogen (Carlsbad, CA). Monoclonal anti-human TNF-alpha capture antibody, polyclonal anti-human TNF-alpha detection antibody, and recombinant human TNF-alpha were purchased from R&D systems, Inc (Minneapolis, MN). The fiber polisher and polishing consumables were purchased from Allied High Tech Products, Inc. (Rancho Dominguez, California).
  • Example 2 The example outlines the preparation of 1 urn magnetic bead functionalized with
  • TNF-alpha capture antibody 20 uL of amine-reactive bead stock was washed three times in 0.1 M sodium borate coating buffer at pH 9.5 using a microtube magnetic separation device (BioMag® Tube Separator; Polysciences Inc., Warrington, PA). 500 ug TNF-alpha capture antibody was dissolved in 183.5 uL of sodium borate coating buffer. 104 uL of 3 M ammonium sulfate was added to the antibody solution. 135 uL of the resulting antibody solution was added to the cleaned 20 uL bead aliquot and mixed at 37 0 C for 24 hours.
  • TNF-alpha detection antibody binding ligand
  • TNF-alpha detection antibody binding ligand
  • 100 ug of TNF-alpha detection antibody R&D Systems, AF-210-NA
  • 100 ug of TNF-alpha detection antibody R&D Systems, AF-210-NA
  • 164 uL of PBS pH 7.4 buffer was added to a tared vial containing 1 mg sulfo- NHS-ss-biotin, yielding a 10 mM stock solution.
  • 1.34 uL of the 10 mM stock solution was added to the 100 uL antibody solution, equating to a 20-fold molar excess of the biotin label.
  • the antibody/NHS-ss-biotin solution was mixed for 1 hr at room temperature.
  • the antibody-ss-biotin conjugate was purified using a Zeba Desalt Spin Column. The purification was performed according to the Pierce protocol for product #89882.
  • the purified conjugate was diluted up to 2 mL, making a 50 ug/mL stock solution. 50 uL aliquots of this 50 ug/mL solution were stored at -20 °C for later use.
  • Example 6 The following is an example of the cleaving of a photo-cleavable linkage in a TNF-alpha magnetic bead containing an immobilized immunocomplex.
  • a 96 well white Nunc Maxisorb plate on which were immobilized complete immunocomplexes, prepared similarly as described in the above Examples, except comprising photo-cleavable biotin linkages as opposed to disulfide cleavable biotin linkage on the detection antibody (binding ligand) were washed three times in 150 uL of 5X PBS containing 0.1% Tween®20 for 2 minutes with 500 RPM mixing on a VWR plate shaker each time. The final wash volume was removed and 50 uL of IX PBS solution was added to the wells.
  • the plate was placed ⁇ 5 cm from the UV light source (365 nm black light bulbs, 15 watt) for 30 to 60 minutes to activate the UV photo-cleavage of the biotin linkage. Photo- cleaved samples were transferred to a fresh plate following separation from the Nunc plate.
  • UV light source 365 nm black light bulbs, 15 watt
  • Example 7 The following is an example of the preparation of biotinylated array for capture of released reporter molecules (dissociated species).
  • Optical fiber bundles approximately 5 cm long were polished on a polishing machine using 30, 9, and 1 micron-sized diamond lapping films.
  • the polished fiber bundles were chemically etched in a 0.025 M HCl solution for 115 seconds, and then immediately submerged into water to quench the reaction.
  • the etched fibers were sonicated for 5 seconds in water and washed in water for 5 min. The fibers were then dried under vacuum and exposed to air plasma for 5 minutes to clean and activate the glass surface.
  • the arrays were silanized for 30 minutes in a 2% silanization solution that was prepared by mixing 950 uL of anhydrous ethanol, 50 uL of water, and 20 uL of 3-aminopropyl trimethoxysilane, and then washed with anhydrous ethanol for 10 min and dried under nitrogen.
  • the amine silanized arrays were then biotinylated using the following procedure.
  • Example 9 The following is an example of instrumental set-up that may be employed to analyze the fiber optic arrays prepared as described in Example 8.
  • the set-up is similar to that as described above in FIG. 17A and is also pictured in FIG. 17B.
  • the imaging system used for collecting the data integrated an Olympus BX-61 microscope system with a Cooke Corporation Sensicam QE CCD camera, 474.
  • the BX-61 microscope is an upright microscope specifically designed for fluorescence detection.
  • 10x and 2Ox UMPlanFl objectives (e.g., 470), which have a NA of 0.3 and 0.5, respectively, were installed on the BX-61 microscope.
  • Wavelength specific filter sets are held in a cube, which is stored in a rotating wheel within the microscope.
  • the BX-61 can hold 6 cubes for easy access to wavelengths specific for a number of fluorophores.
  • Filter sets were purchased from Chroma Technology Corporation and are compatible with the cubes on the BX-61.
  • the resorufin filter cube (Chroma Technology filter set #41010) was used for all of the enzyme amplification experiments described herein. This filter set uses a HQ577/10x excition filter, a 585 long-pass dichroic mirror, and a HQ620/60m emission filter.
  • a 6-axis mechanical platform was constructed beneath the microscope stage, which was used to house a non-reinforced silicone gasket material (sealing component) to be applied to the fiber optic array.
  • Arrays were mounted on the microscope stage using a fiber array holder fabricated to secure and position the fiber array on the stage.
  • a droplet of beta-galactosidase substrate (RDG) precursor labeling agent molecule was placed on the silicone gasket material, and put into contact with the distal end of the fiber array.
  • the mechanical platform was used to move the silicone sheet into contact with the distal end of the etched optical fiber array, creating an array of isolated femtoliter reaction vessels.
  • the number of wells that bound an enzyme molecule carried by the dissociated species correlated to the bulk dissociated species concentration, which, in turn, correlated with the concentration of TNF analyte in the initial fluid sample applied to the magnetic bead substrate.
  • the following example outlines a standard procedure for a complete assay according to one embodiment of the present invention.
  • the following procedure is a basic protocol that may be completed for any suitable sample comprising a plurality of analyte molecules, and the assay components (e.g., binding ligands, capture components, etc.) may be selected according to the methods described herein. This protocol or portions of it was followed when performing the assays discussed in Examples 11-14.
  • Chemical and Materials The following solutions should be prepared:
  • IX PBS, pH 7.5 The solution should be 0.2 um filtered and may be diluted from 1OX PBS (Omnipure cat#6506). To prepare, mix 10OmL 1OX PBS and 90OmL Milli-Q water to form 1 liter total. The solution may be stored at room temperature.
  • wash Buffer The solution comprises 5X PBS + 0.1% Tween®20 and was 0.2 um filtered. To prepare, mix 250 uL 100% Tween®20 and 125 mL Ultrapure Water and
  • the solution may be stored at room temperature for up to one week
  • the solution comprises 10% BSA + 0.1% Tween®20. To prepare mix 5 g BSA, 45 mL IX PBS and 50 uL 100% Tween®20 to form 50 mL total. The solution may be stored at 4 0 C for up to one week
  • Dilution Buffer The solution comprises 0.5% BSA + 0.1% Tween®20. To prepare mix 2.5 mL 10% BSA, 47.5 mL IX PBS, 47.5 uL 100% Tween®20 to form 50 mL total. The solution should be discarded after completing the assay.
  • the solution comprises IX PBS + 1 mM MgCl 2 .
  • the solution should be stored at room temperature.
  • Preparation of the 96 well plate 1. Obtain coupled beads (e.g., MyOne Beads (Invitrogen) prebound with ⁇ TNF Ab BAF210 (R&D Systems)).
  • the beads may be at 40 ug Ab/mg Dynabeads. Briefly vortex beads at approximately setting 8 until resuspended. Pipette up and down if necessary. Sonicate for approximately 10 seconds.
  • TNF-alpha Place plate on bar magnet and wait 30 seconds. Pour Dilution Buffer into a reagent reservoir. Using multichannel, remove all liquid from each well. Remove plate from bar magnet. Using multichannel, add 100 uL of Dilution buffer to each well. Pipette up and down vigorously 15 times to resuspend beads. Cover plate with hard plastic lid. Incubate on plate shaker at 500 RPM for 2 minutes. Repeat wash 2x more for a total of 3 washes. Capture TNF-alpha on plate: 1. Dilute TNF-alpha (R&D systems 210-TA/CF) to appropriate concentration(s) in dilution buffer
  • Capture antigen Aliquot 100 uL/well of TNF-alpha solution or dilution buffer only (for NSB wells) with single channel pipette. Cover plate with hard plastic lid. Incubate on plate shaker at 500RPM for 1 hour. After 1 hour, beads will have partially settled. Pipette up and down vigorously 15 times to resuspend beads. Continue to incubate for 1 additional hour.
  • Wash Plate Place plate on bar magnet and wait 30 seconds. Pour Wash Buffer into a reagent reservoir. Using multichannel remove all liquid from each well. Remove plate from bar magnet. Using multichannel, add 100 uL of wash buffer to each well. Pipette up and down vigorously 15 times to resuspend beads. Cover plate with hard plastic lid. Incubate on plate shaker at 500 RPM for 2 minutes. Repeat wash 2x more for a total of 3 washes. Sandwich TNF-alpha on beads using Biotin-labeled TNF-alpha Detection Antibody
  • TNF-alpha detection Antibody Dilute releasable Biotin-labeled TNF-alpha detection Antibody (R&D systems B AF210 - modified with Sulfosuccinimidyl 2-(biotinamido)-ethyl- 1 , 3 -dithiopropionate (ThermoFisher Scientific)) in dilution buffer to 0.3 ug/mL. In a 15 mL tube add and vortex to combine: 42 uL TNF-alpha Detection Antibody (50 ug/mL Stock) 7 mL Detection Ab buffer to form 7 mL total at 0.3 ug/mL TNF-alpha Antibody (2 nM) 2.
  • Sandwich antigen by binding Detection antibody in each well Dump TNF- alpha Antibody solution into a reagent reservoir. Aliquot 100 ⁇ L/well with multichannel pipette. Seal Plate with clear plastic lid. Incubate on plate shaker at 500RPM for 1 hour.
  • Wash Plate Place plate on bar magnet and wait 30 seconds. Using multichannel remove all liquid from each well. Remove plate from bar magnet. Using multichannel, add lOOuL of wash buffer to each well. Pipette up and down vigorously 15 times to resuspend beads. Cover plate with hard plastic lid. Incubate on plate shaker at 500RPM for 2 minutes. Repeat wash 2x more for a total of 3 washes. Label TNF-alpha Detection Antibody with signal amplification enzyme (Streptavidin- ⁇ - galactosidase)
  • SA- ⁇ -gal Dilute Streptavidin labeled TNF-alpha detection ⁇ -galactosidase (SA- ⁇ -gal) in dilution buffer to 500 pM: In a 15 mL tube add and vortex to combine 3.1 uL SA- ⁇ -gal (1.6 uM Stock), 10 mL Dilution buffer, to form 10 mL total of 500 pM SA- ⁇ -gal
  • Wash Plate Place plate on bar magnet and wait 30 seconds. Using multichannel remove all liquid from each well. Remove plate from bar magnet. Using multichannel, add lOOuL of wash buffer to each well. Pipette up and down vigorously 15 times to resuspend beads. Cover plate with hard plastic lid. Incubate on plate shaker at 500RPM for 5 minutes. Repeat wash 3x more for a total of 4 washes.
  • Transfer Beads Place plate on bar magnet and wait 30 seconds. Using multichannel remove all liquid from each well. Obtain a second 96-well plate. Add 75 uL of wash buffer to each well of the original plate. Transfer entire volume from each well to the corresponding well of the second plate. Repeat previous steps to ensure complete bead transfer. Cleave detection Ab
  • Wash Plate Place plate on bar magnet and wait 30 seconds. Using multichannel remove all liquid from each well. Remove plate from bar magnet. Using multichannel, add lOOuL of wash buffer to each well. Pipette up and down vigorously 15 times to resuspend beads. Cover plate with hard plastic lid. Incubate on plate shaker at 500 RPM for 2 minutes. Repeat wash 2x more for a total of 3 washes. 2. BME/EDTA Incubation: Dilute Beta Mercaptoethanol (BME) to 1OmM in PBS. Add EDTA to 1OmM final concentration. 0.2 uM-filter the solution. Using multichannel remove all liquid from each well.
  • BME Beta Mercaptoethanol
  • Example 11 The following is an example of the quantification of Tumor Necrosis Factor alpha (TNF ⁇ ) using the protocol of Example 10.
  • Magnetic beads coated in a monoclonal antibody to human TNF were prepared as described in Example 10. Solutions were then made containing human TNF at concentrations from 0 fM to 1000 fM spiked into bovine serum. The magnetic beads and TNF serum solutions were then combined in a microtiter plate to give final concentrations of magnetic beads of approximately 1 ug/well. The beads and serum were mixed at 37 0 C for 1 hour. A magnet was then applied to the plate to separate the magnetic beads and serum, and the serum was removed.
  • the beads were then subjected to cycles of washing, exposure to biotinylated detection antibody (first binding ligand), exposure to streptavidin-beta-galactosidase (second binding ligand), and magnetic separation as described in Example 10.
  • the immunocomplex was then released from the beads by adding 100 ul of 10 mM beta- mercaptoethanol to each well of the plate. Solutions containing the dissociated species were then incubated with biotin functionalized fiber arrays to capture the released immunocomplexes. Single dissociated complexes were then detected by fiuorescently imaging single beta-galactosidase molecules in the wells of the fiber arrays as described above in Examples.
  • FIG. 2OA shows a plot of % active wells against concentration of TNF in the serum samples.
  • FIG. 2OB shows a magnified view of the lower end of the graph in FIG. 2OA.
  • Table 1 shows the tabulated limit of detection (LOD) for the methods presented in FIGS. 20-23 as compared to a standard enzyme-linked immunosorbent assay (ELISA) using essentially similar reagents;
  • PSA Prostate Specific Antigen
  • Magnetic beads coated in a monoclonal antibody to human PSA were prepared using the method described above Example 10. Solutions were then made containing human PSA at concentrations from 0 fM to 200 fM spiked into bovine serum. The magnetic beads and PSA serum solutions were then combined in a microtiter plate to give final concentrations of magnetic beads of 0.7 ug/well. The beads and serum were mixed at 25 0 C for 1 hour. The samples were then processed as for TNF in Example 11 and the % active wells arising from the released single enzymes was determined.
  • FIG. 21 shows a plot of % active wells against concentration of PSA in the serum samples.
  • the limit of detection for PSA is 2 fM, compared to 2000 fM for a standard ELISA using the same reagents (Table 1).
  • Example 13 The following is an example of the quantification of Interleukin 6 (IL-6)
  • Magnetic beads coated in a monoclonal antibody to human IL-6 were created using the method described above in Example 10. Solutions were then made containing human IL-6 at concentrations from 0 fM to 20 fM spiked into bovine serum. The magnetic beads and IL-6 serum solutions were then combined in a microtiter plate to give final concentrations of magnetic beads of 0.5 ug/well. The beads and serum were mixed at 25 0 C for 1 hour. The samples were then processed as for TNF in Example 11 and the % active wells arising from the released single enzymes was determined. FIG. 22 shows a plot of % active wells against concentration of IL-6 in the serum samples. The limit of detection for IL-6 is 0.2 fM, compared to 400 fM for a standard ELISA using the same reagents (Table 1).
  • Example 14 The following is an example of the quantification of Vascular Endothelial
  • VEGF Growth Factor
  • Magnetic beads coated in a monoclonal antibody to human VEGF were created using the method described above in Example 10. Solutions were then made containing human VEGF at concentrations from 0 fM to 1000 fM spiked into bovine serum. The magnetic beads and VEGF serum solutions were then combined in a microtiter plate to give final concentrations of magnetic beads of 0.5 ug/well. The beads and serum were mixed at 25 0 C for 1 hour. The samples were then processed as for TNF in Example 11 and the % active wells arising from the released single enzymes was determined.
  • FIG. 23 shows a plot of % active wells against concentration of VEGF in the serum samples. The limit of detection for VEGF is 0.5 fM, compared to 500 fM for a standard ELISA using the same reagents (Table 1 ).
  • FIG. 24 describes the effect of the concentration of beta-mercaptoethanol on release of dissociated species, in one embodiment of the invention.
  • concentration of beta-mercaptoethanol (bME) used to release SbG was optimized for signal-to- background to enhance selectivity for specifically bound molecules.
  • the released SbG was detected on single molecule array fiber bundles.
  • 5 mM bME was determined to yield the best results based on signal-to-background ((ii)/(i)).
  • FIG. 25 describes the effect of removing beta-mercaptoethanol before detection of the dissociated species on an array.
  • beta-mercaptoethanol may adversely affect the spectrum of the fluorescent product and/or may reduce the enzymatic activity of SbG.
  • Beta-mercaptoethanol may be removed from the assay, in some cases, for example by either purifying the solution of SbG using a filtration column to remove the bME or by capturing the SbG on fiber arrays and washing the bME away.
  • immunocomplexes of TNF-alpha were formed and released by bME as described in Example 11. The solution containing the dissociated complexes were (left) passed over a ZEBA column to remove the bME or (right) were left untreated.
  • Non-specific binding can be a major limitation on the sensitivity of an immunoassay.
  • FIG. 26 shows that method of the present, in this embodiment, can be optimized to result in non-specific binding of less than about ⁇ 0.1%. In some cases, the non-specific binding was reduced to less than about 10 wells in an 11,000- well image. In this example, when the immunocomplexes were formed directly on the fiber array (i.e., a one-step assay rather than a two-step, "capture-and-release,” assay), non-specific binding was typically observed to be approximately 2%.
  • FIG. 27 shows results for the detection of TNF-alpha using the method described in Example 11.
  • detection limits in single digit femtomolar ranges could be obtained as compared to 10 3 femtomolar (e.g.1250 fM) using the substantially the same reagents on a traditional ELISA plate assay (see FIG. 28 , Maxisorb ELISA 96well plate, same reagents and incubation times as used in comparative assay, detection on a Tecan M200 plate reader).
  • FIG. 29 shows a similar assay performed according to an embodiment of the inventive methods for the detection of TNF-alpha where the beads were modified with capture components comprising anti- TNF Ab stabilized with bovine serum albumin.
  • FIG. 30 shows results for the immobilization of Fab' fragments of an anti-TNF monoclonal antibody (MAB610) generated using enzymatic degradation and purification as the capture components immobilized on beads.
  • MAB610 anti-TNF monoclonal antibody
  • the graph shows a comparison of detection of TNF using MAB610 on beads versus Fab' of MAB610 on beads where part of the immunocomplex comprising SbG was released with 10 mM bME.
  • higher signals but also higher NSB backgrounds were observed when using Fab' fragments.
  • FIG. 31 shows a graph of results obtained using the plate as the first substrate. A substantially greater sensitivity was observed as compared to results obtainable using traditional ELISA methods, but less sensitivity was observed as compare to instances wherein the analyte molecules were captured on beads.
  • FIG. 32 shows the results for a method of the present invention, wherein one of the binding ligands of the assay comprised a photocleavable linkage.
  • the immobilized complexes were formed on an ELISA 96-well plate as the first substrate.
  • the plate was exposed to UV radiation, and a plurality of dissociated species was formed.
  • the dissociated species were then detected/quantified using methods described herein.
  • the graph depicts the plate reader fluorescence for three samples: i) no target analyte present, ii) target analyte present and the binding ligand does not comprise a photocleavable linker and iii) target analyte present and the binding ligand comprises a photocleavable linker.
  • a Maxisorb ELISA 96-well plate (Nunc) was employed.
  • the ELISA plate was functionalized with monoclonal anti-TNF capture components.
  • the substrate was then exposed to varying concentrations of TNF-alpha.
  • the substrate was additionally exposed to a first binding ligand (polyclonal biotinylated anti-TNF antibody) and a second binding ligand (streptavidin-b-galactosidase).
  • the conversion of substrate of b-galactosidase was detected on Tecan M200 plate reader. In this example, the limit of detection was approximately 1250 fM (see FIG. 33).
  • magnetic beads were provided which comprised monoclonal anti-TNF capture components.
  • the beads were exposed to varying concentrations of TNF-alpha.
  • the beads were additionally exposed to polyclonal disulfide-containing biotinylated anti-TNF antibody and streptavidin-b- galactosidase (SbG). At least a portion of the SbG was released from the beads using 10 mM beta-mercaptoethanol.
  • the released complexes comprising SbG were captured on a fiber bundle well array comprising biotin capture components.
  • Each reaction vessel was exposed to at least one precursor labeling agent molecule comprising resorufin-beta-D- galactopyranoside (RDG) that was converted to fluorescent resorufin upon exposure to SbG.
  • RDG resorufin-beta-D- galactopyranoside
  • the presence of at least one labeling agent molecules formed via the conversion of the precursor labeling agent molecule by the b-galactosidase of the SbG in the reaction vessels was detected using a fluorescence imaging microscope as described in Example 9. In this embodiment, the limit of detection was less than 5 fM (see FIG. 34).

Abstract

L'invention concerne des systèmes et des procédés pour détecter des molécules ou des particules d'analyte dans un échantillon fluidique, et dans certains cas, pour déterminer une mesure de concentration desdites molécules ou particules dans ledit échantillon fluidique. Des procédés selon l'invention consistent à immobiliser plusieurs molécules ou particules d'analyte pour former plusieurs complexes, à libérer au moins une partie de certains de ces complexes, à déterminer au moins une partie des complexes libérés, et/ou à déterminer une mesure de la concentration des molécules ou des particules d'analyte dans un échantillon fluidique.
PCT/US2009/005248 2008-09-23 2009-09-22 Détection ultra-sensible de molécules ou d'enzymes WO2010039179A1 (fr)

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US12/236,488 2008-09-23
US12/236,486 2008-09-23
US12/236,490 US20100075355A1 (en) 2008-09-23 2008-09-23 Ultra-sensitive detection of enzymes by capture-and-release followed by quantification
US12/236,486 US8222047B2 (en) 2008-09-23 2008-09-23 Ultra-sensitive detection of molecules on single molecule arrays
US12/236,490 2008-09-23
US12/236,488 US20100075439A1 (en) 2008-09-23 2008-09-23 Ultra-sensitive detection of molecules by capture-and-release using reducing agents followed by quantification

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WO2021211754A2 (fr) 2020-04-15 2021-10-21 Quanterix Corporation Procédés et systèmes liés à des dosages hautement sensibles et à l'administration d'objets de capture

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