WO2015195404A1

WO2015195404A1 - Methods for detection of an analyte by movement of tethered microparticles

Info

Publication number: WO2015195404A1
Application number: PCT/US2015/034815
Authority: WO
Inventors: Jonathan E. Silver; Keir C. NEUMAN
Original assignee: The United States Of America, As Represented By The Secretary, Department Of Health & Human Services
Priority date: 2014-06-20
Filing date: 2015-06-09
Publication date: 2015-12-23
Also published as: WO2015195404A8

Abstract

An immune sandwich assay in which read-out is based on counting micron-size beads tethered by polymeric molecules (such as nucleic acid) is described. The motion of tethered beads in flow is recognizable with a low-power optical microscope and distinguishable from that of beads that stick non-specifically to the flow cell surface. Analyte molecules are captured efficiently by capture beads and labeled efficiently by labeled detection antibody. The assay disclosed herein is sensitive enough for the detection of single molecules and/or the detection of very low concentrations of analyte in a sample.

Description

METHODS FOR DETECTION OF AN ANALYTE BY MOVEMENT OF TETHERED

MICROPARTICLES

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 62/015,122, filed

June 20, 2014, which is herein incorporated by reference in its entirety.

FIELD

This disclosure concerns methods for detecting very low concentrations of analyte in a sample. This disclosure further concerns methods for optical detection of an analyte based on movement of tethered microparticles.

BACKGROUND

Over the past few decades several methods have been developed that can detect very low concentrations of analyte molecules. For immunoassays, these methods often involve immune capture of analyte on a solid phase followed by binding of a labeled, second antibody (forming an "immune sandwich") and sensitive detection of the label. Transduction methods for label detection include total internal reflection-fluorescence microscopy (Yanagida et al, Nat Cell Biol 2: 168-172, 2000; Jain et ah, Nature 473:484-488, 2011), mechanical oscillators sensitive to the mass of single gold nanoparticle labels (Burg et ah, Nature 446:1066-1069, 2007), field effect transistors (Liu and Guo, NPG Asia Materials 4:e23, 2012), whispering gallery mode optical sensors (Vollmer and Arnold, Nat Methods 5:591-596, 2008), magnetic sensors sensitive to micrometer or nanometer sized magnetic particles (Osterfeld et al, Proc Natl Acad Sci USA 105:20637-20640, 2008;

Mulvaney et al., Biosens and Bioelectron 23: 191-200, 2007), and enzyme-based assays with enhanced sensitivity resulting from confinement of enzyme products to micron-sized chambers

(Rissin et al., Nat Biotechnol 28:595-599, 2010). The enhanced sensitivity of these methods often requires expensive, cumbersome detection equipment.

Tethered bead technology was initially developed to study biophysical properties of DNA molecules and enzymes that act on DNA, at the single molecule level (Schafer et al. , Nature 352:444-448, 1991; Cluzel et al, Science 271:792-794, 1996; Larsen et al, Physical Review E 55: 1794-1797, 1997; Bustamante et al, Nature 421:423-427, 2003; Abbondanzieri et al, Nature 438:460-465, 2005; Kim et al, Nat Methods 4:397-399, 2007; Neuman and Nagy, Nat Methods 5:491-505, 2008). In a typical experimental set-up, DNA molecules of known length are attached via one end to a surface in a flow cell. The other end of the DNA is attached to a micron-sized bead that can be pulled to stretch the DNA. Pulling can be effected via liquid flow (drag force on the bead), laser trapping, or magnetic force if the bead is paramagnetic. Tethered beads are easily identified in light microscopy by their characteristic motion - they exhibit damped Brownian motion in the absence of external force, and translation by a predictable distance in the presence of an external force. In single-molecule studies of DNA, the tether length is usually measured very precisely to provide quantitative information about DNA conformation and elasticity.

SUMMARY

Methods, flow cells and kits for detecting and/or quantitating an analyte in a sample are disclosed. Detection and quantitation methods are based on optical detection of flow characteristics of tethered microparticles.

Provided is a method for detection and/or quantitation of an analyte in a sample. In some embodiments the method includes providing a flow cell comprising a flow cell surface; providing a plurality of elongated tether molecules, such as substantially or completely linear polymeric molecules, each having a first terminus and a second terminus, wherein the first terminus of each polymeric molecule is capable of binding to the flow cell surface; providing microparticles conjugated to a binding moiety that binds a first binding site on the analyte; providing a bridging moiety that binds a second binding site on the analyte and binds the second terminus of the polymeric molecules; contacting the microparticles conjugated to the binding moiety with the sample under conditions sufficient to allow binding of the binding moiety to analyte present in the sample to form analyte-bound microparticles; contacting the analyte-bound microparticles, the flow cell, the plurality of polymeric molecules and the bridging moiety to form tethered microparticles; and detecting the tethered microparticles by virtue of a characteristic of their motion that identifies them as tethered to the flow cell surface via the polymeric molecules.

Also provided are flow cells for the detection and/or quantitation of an analyte in a sample. In some embodiments, the flow cells include a flow cell surface; a plurality of elongated tether molecules, such as substantially or completely linear polymeric molecules, each having a first terminus and a second terminus, wherein the first terminus of each polymeric molecule is bound to the flow cell surface; and a bridging moiety bound to the second terminus of each polymeric molecule, wherein the bridging moiety specifically binds a first binding site of the analyte.

Further provided are kits for the detection and/or quantitation of an analyte in a sample. In some embodiments, the kits include a flow cell comprising a flow cell surface; a plurality of elongated tether molecules, such as substantially or completely linear polymeric molecules, each having a first terminus and a second terminus, wherein the first terminus of each polymeric molecule is capable of binding to the flow cell surface; microparticles conjugated to a binding moiety that specifically binds a first binding site on the analyte; and a bridging moiety that specifically binds a second binding site on the analyte and binds the second terminus of the polymeric molecules.

The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic showing a typical configuration of tethered bead in a single- molecule pulling experiment. FIG. IB is a schematic showing an attachment configuration in an immune- sandwich tethered bead assay. FIG 1C depicts a flow cell assembly.

FIG. 2 is a schematic of an ELISA assay to determine efficiency of capturing prostate specific antigen (PSA), detection antibody and streptavidin. Row 1, ELISA assay of a sample with a known concentration of biotin-labeled PSA. Row 2, ELISA assay of supernatant from the Row 1 assay. Reduction in ELISA signal indicates how much biotin-labeled PSA is captured by anti-PSA beads. Row 3, ELISA assay of unlabeled PSA at the same concentration as PSA in Row 1, followed by biotin-labeled detection antibody. Comparing ELISA signal in Row 4 vs. Row 1 indicates how efficiently biotin-detection antibody is captured by bound PSA.

FIG. 3 is a diagram of a tethered bead with tether stretched by flow to the right, for calculation of tether length x as a function of drag force Fdrag. Bead of radius r is attached to a flow cell surface via a single DNA tether. Tension in DNA F_T increases until it balances the drag force on the bead. When the bead is stationary in flow, the height h of the tether attachment point is such that torque from shear flow (clockwise arrow) balances that from off-center tether attachment (counter-clockwise arrow) and F_T COS(([>) = Fdrag. Angles φ and κ are defined in the diagram. A relation between force and extension from the worm-like chain model of DNA and fluid dynamics predictions of force and torque on a sphere as a function of flow rate for a sphere resting on a surface allow all variables to be determined as a function of lateral flow rate (see Example 3).

DETAILED DESCRIPTION

I. Abbreviations

ELISA enzyme-linked immunosorbent assay

PDMS polydimethylsiloxane PNA peptide nucleic acid

PSA prostate specific antigen

WB wash buffer

XNA xeno-nucleic acid

II. Terms and Methods

Unless otherwise noted, technical terms are used according to conventional usage.

Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632- 02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

Addressable: Molecules at addressable locations refers to molecules located in discrete and defined regions, such as in discrete and defined regions of a solid support.

Analyte: A target molecule to be detected, quantified and/or analyzed.

Antibody: A protein (or protein complex) that includes one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

The basic immunoglobulin (antibody) structural unit is generally a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" (about 50-70 kDa) chain. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms "variable light chain" (V_L) and "variable heavy chain" (V_H) refer, respectively, to these light and heavy chains.

As used herein, the term "antibodies" includes intact immunoglobulins as well as a number of well-characterized fragments. For instance, Fabs, Fvs, and single-chain Fvs (scFvs) that bind to target protein (or epitope within a protein or fusion protein) would also be specific binding agents for that protein (or epitope). These antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')₂, the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab')₂, a dimer of two Fab' fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single chain antibody, a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Methods of making these fragments are routine (see, for example, Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999).

Antibodies for use in the methods of this disclosure can be monoclonal or polyclonal, and for example specifically bind a target such as the target antigen. Merely by way of example, monoclonal antibodies can be prepared from murine hybridomas according to the classical method of Kohler and Milstein {Nature 256:495-97, 1975) or derivative methods thereof. Detailed procedures for monoclonal antibody production are described in Harlow and Lane, Using

Antibodies: A Laboratory Manual, CSHL, New York, 1999.

A "capture antibody" is an antibody that specifically binds a target analyte (such as a target antigen) and is capable of separating the target analyte from other components in a sample. For example, a capture antibody can be affixed to a solid surface {e.g. an ELISA plate) or a microparticle to provide a means for physical separation of the target molecule. A "detection antibody" is an antibody that specifically binds a target analyte (such as an antigen) and is labeled {e.g. with a fluorophore, radioisotope, hapten or enzyme) to provide a means for detection. Capture and detection antibodies are used together in immune sandwich assays, such as sandwich ELISAs. When used in such assays, the capture and detection antibodies must bind different, non- overlapping epitopes of the target antigen.

Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. Antigen-specific: As used herein, an "antigen-specific" antibody is an antibody that was elicited (produced and/or activated) in response to a particular antigen. An "antigen- specific" antibody is capable of binding to the antigen, typically with high affinity.

Aptamer: A small nucleic acid or peptide that specifically binds a target molecule.

Avidin/Streptavidin: The extraordinary affinity of avidin for biotin allows biotin- containing molecules in a complex mixture to be discretely bound with avidin. Avidin is a glycoprotein found in the egg white and tissues of birds, reptiles and amphibia. It contains four identical subunits having a combined mass of 67,000-68,000 daltons. Each subunit consists of 128 amino acids and binds one molecule of biotin. Extensive chemical modification has little effect on the activity of avidin, making it especially useful for protein purification.

Another biotin-binding protein is streptavidin, which is isolated from Streptomyces avidinii and has a mass of 60,000 daltons. In contrast to avidin, streptavidin has no carbohydrate and has a mildly acidic pi of 5.5. Another version of avidin is NEUTRAVIDIN™ Biotin Binding Protein (available from Pierce Biotechnology) with a mass of approximately 60,000 daltons.

The avidin-biotin complex is the strongest known non-covalent interaction (Ka = 10¹⁵ M^"1) between a protein and ligand. The bond formation between biotin and avidin is very rapid, and once formed, is unaffected by extremes of pH, temperature, organic solvents and other denaturing agents.

Although examples disclosed herein use streptavidin as a specific binding agent, the streptavidin could be substituted with other types of avidin. The term "avidin" is meant to refer to avidin, streptavidin and other forms of avidin (such as derivatives or analogs thereof) that have similar biotin binding characteristics. Analogs or derivatives of avidin/streptavidin include, but are not limited to, nitro-streptavidin, non-glycosylated avidin, N-acyl avidins (such as N-acetyl, N- phthalyl and N-succinyl avidin), and the commercial products EXTRA VIDIN™ (Sigma- Aldrich), Neutralite Avidin (SouthernBiotech) and CaptAvidin (Invitrogen). Additional avidin/streptavidin analogs and derivatives are known in the art (see, for example, U.S. Patent No. 5,973,124 and U.S.

Patent Application Publication Nos. US 2004/0191832; US 2007/0105162; and US 2008/0255004).

Biomarker: A measurable substance in an organism, the presence of which is indicative of disease, infection, environmental exposure or the like. Biomarkers can be used to measure the presence of disease, the progress of disease and/or the effects of treatment. As one example, prostate specific antigen (PSA) is a biomarker for cancer of prostate. Biomarkers can be, for example, proteins, carbohydrates, lipids, and nucleic acid molecules.

Biotin: A molecule (also known as vitamin H or vitamin B₇) that binds with high affinity to avidin and streptavidin. Biotin is often used to label nucleic acids and proteins for subsequent detection by avidin or streptavidin linked to a detectable label, such as a fluorescent or enzymatic reporter molecule. Biotinylation of a molecule (such as an antibody or other protein sample) is routinely achieved in the art by reacting a free carboxyl group on biotin with an amine group on a protein, such as an amine group found in an antibody or protein analyte/analog. Unless indicated otherwise, the term "biotin" includes derivatives or analogs that participate in a binding reaction with avidin. Biotin analogs and derivatives include, but are not limited to, N-hydroxysuccinimide- iminobiotin (NHS-iminobiotin), amino or sulfhydryl derivatives of 2-iminobiotin, amidobiotin, desthiobiotin, biotin sulfone, caproylamidobiotin and biocytin, biotinyl-e-aminocaproic acid-N- hydroxysuccinimide ester, sulfo-succinimide-iminobiotin, biotinbromoacetylhydrazide, p- diazobenzoyl biocytin, 3-(N-maleimidopropionyl) biocytin, 6-(6- biotinamidohexanamido)hexanoate and 2-biotinamidoethanethiol. Biotin derivatives are also commercially available, such as DSB-X™ Biotin (Invitrogen). Additional biotin analogs and derivatives are known in the art (see, for example, U.S. Patent No. 5,168,049; U.S. Patent

Application Publication Nos. 2004/0024197, 2001/0016343, and 2005/0048012; and PCT

Publication No. WO 1995/007466).

Biotin binding protein: A protein that binds biotin with sufficiently great affinity for an intended purpose. Examples of biotin binding proteins are well known in the art, and include avidin, streptavidin, NEUTRAVIDIN™, and monoclonal antibodies or receptor molecules that specifically bind biotin. In the context of this disclosure, streptavidin could be replaced with any other biotin-binding proteins, or a combination of biotin binding proteins.

Binding moiety: In the context of the present disclosure, a "binding moiety" is a molecule or molecular complex that specifically binds a target analyte, such as a target antigen. Binding moieties include, but are not limited to, antibodies and aptamers.

Bridging moiety: In the context of the present disclosure, a "bridging moiety" is a molecule or molecular complex comprising one or more components. The bridging moieties of the methods, flow cells and kits described herein are capable of simultaneously binding an analyte (such as an analyte of an analyte-bound microparticle) and a polymeric molecule (such as a terminus of a polymeric molecule bound to a flow cell surface).

Brownian motion: The random motion of particles suspended in a fluid.

Chemical modification (of a nucleic acid): Refers to any non-naturally occurring chemical alteration of a nucleic acid molecule. Exemplary chemical modifications include but are not limited to modified internucleoside linkages, modified sugar moieties and modified bases. Conjugated: Refers to two molecules that are bonded together, for example by covalent bonds. An example of a conjugate is a molecule (such as avidin/streptavidin) conjugated to a detectable label, such as a fluorophore, to form a detection substrate.

Contacting: Placement in direct physical association; includes both in solid and liquid form. As used herein, "contacting" is used interchangeably with "exposed."

Control: A reference standard, for example a positive control or negative control. A positive control is known to provide a positive test result. A negative control is known to provide a negative test result. However, the reference standard can be a theoretical or computed result, for example a result obtained in a population.

Digoxigenin: A steroid found exclusively in the flowers and leaves of the plants Digitalis purpurea, Digitalis orientalis and Digitalis lanata (foxgloves), where it is attached to sugars to form glycosides. Digoxigenin is commonly used in biotechnology applications, such as

immunoassays. For example, digoxigenin can be conjugated to a biomolecule (such as a protein or nucleic acid) and then detected using an anti-digoxigenin antibody.

Epitope: An antigenic determinant. Epitopes are particular chemical groups or contiguous or non-contiguous peptide sequences on a molecule that are antigenic, that is, that elicit a specific immune response. An antibody binds a particular antigenic epitope based on the three dimensional structure of the antibody and the matching (or cognate) epitope.

Infectious agent: An organism that causes disease or illness to its host. Infectious agents include, for example, bacteria, viruses, fungi, protozoa and parasites. Infectious agents are also referred to as pathogens.

Microparticle: A particle that is generally about 0.01 to about 1000 microns in diameter. Microparticles include microspheres (spherical microparticles), beads, or the like with a surface suitable for binding (e.g., suitable for binding an antibody). For example, a microparticle can be a microsphere with a carboxylated surface. In some embodiments, the microparticles are polymeric microparticles (a microparticle made up of repeating subunits of a particular substance or substances). In some examples, the polymeric microparticles are polystyrene microparticles, such as a polystyrene microparticle with a carboxylated surface. In other examples, the microparticles are magnetic beads. Suitable magnetic beads are well known in the art and include, but are not limited to, functional magnetic beads (e.g., beads of 1 or 5 microns) from Bioclone Inc. (San Diego, CA), DYNAL™ Dynabeads™ (Invitrogen, Carlsbad, CA) and MAGNALINK™ magnetic beads from Solulink (San Diego, CA). In the context of the present disclosure, an "analyte-bound microparticle" is a microparticle having at least one analyte molecule bound to the binding moiety to which the microparticle is conjugated. A "tethered microparticle" is a microparticle bound indirectly to a solid surface (e.g. , the surface of a flow cell) via a polymeric molecule.

Nanoparticle: A particle about 10 to about 100 nanometers (nm) in diameter.

Polymer: A natural or synthetic substance made up of repeating units, such as a macromolecule comprising repeating monomers. Polymeric molecules include, but are not limited to nucleic acid molecules, such as DNA, RNA, peptide nucleic acid (PNA), xeno-nucleic acid (XNA) and combinations thereof, protein polymers (such as microtubules or collagen), or synthetic linear polymers. A nucleic acid polymer can be either single- stranded or double- stranded. A

"linear polymeric molecule" is a substantially or completely non-branching, non-cyclic polymeric molecule, such as a non-branching, non-cyclic nucleic acid molecule (e.g. DND or RNA). In some examples of the linear molecules, the bond angle between all or substantially all of the atoms is about 180°.

Sample: Refers to any biological or environmental sample. A biological sample is a sample obtained from a subject (such as a human or veterinary subject). In particular examples, the biological sample is a fluid sample. Biological samples from a subject include, but are not limited to, serum, blood, plasma, urine, saliva, cerebral spinal fluid (CSF) or other bodily fluid.

Specific binding partner: A member of a pair of molecules that interact by means of specific, non-covalent interactions that depend on the three-dimensional structures of the molecules involved. Exemplary pairs of specific binding partners include antigen/antibody, hapten/antibody, ligand/receptor, nucleic acid strand/complementary nucleic acid strand, substrate/enzyme, inhibitor/enzyme, carbohydrate/lectin, biotin/avidin (such as biotin/streptavidin), and virus/cellular receptor.

Synthetic: Produced by artificial means in a laboratory, for example a synthetic nucleic acid can be chemically synthesized in a laboratory.

Tether molecule: A flexible molecular tether that is secured to a substrate surface, or is capable of being secured to a substrate surface. An example of such a molecule is a polymeric molecule, for example a linear or substantially linear polymeric molecule.

Tumor antigen: An antigen produced in tumor cells that can stimulate tumor- specific immune responses. Exemplary tumor antigens include, but are not limited to, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen 125 (CA- 125), MUC-1, epithelial tumor antigen (ETA), TAG-72, immature laminin receptor, HPV E6/E7, BING-4, calcium-activated chloride channel 2, RAGE- 1, MAGE-1, MAGE-2, tyrosinase, Cyclin-B l, 9D7, Ep-CAM, EphA3, Her2/Neu, telomerase, mesothelin, SAP-1, survivin, NY-ESO- 1, Melan-A/MART- 1, glycoprotein (gp) 75, gpl00/pmell7, β-catenin, POTE, PRAME, MUM-1, WT- 1, PR- 1 BAGE family, CAGE family, GAGE family, MAGE family, SAGE family, XAGE family, TRP-1, TRP-2, MC1R, PSA, CDK4, BRCAl/2, CML66, fibronectin, MART-2, p53, Ras, TGF- RII and MUC1.

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. "Comprising A or B" means including A, or B, or A and B. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are

incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

III. Overview of Several Embodiments

Disclosed herein is a highly sensitive method for the detection of an analyte in a sample based on optical detection of the motion of micron-sized beads attached via bridging moieties (such as immune sandwiches) to the ends of single polymeric molecules (such as nucleic acid molecules), forming "tethered microparticles." Tethered microparticles can be detected with a low- magnification imaging system and inexpensive camera. The methods disclosed herein can be used to detect any analyte that can bind two different antibodies or aptamers simultaneously (e.g. the analyte must have two distinct epitopes for antibody binding). Furthermore, because each tethered bead is attached via one (or very few) analyte molecules, detection of single analyte molecules is feasible. Detection of very low concentrations of analyte is particularly important in infectious disease diagnosis, cancer diagnosis, monitoring treatment in infectious disease or cancer, and screening blood products. Also provided herein are flow cells and kits for the detection and/or quantitation of an analyte in a sample.

A. Methods for detecting and quantitating analytes

Provided herein are methods for the detection and/or quantitation of analytes in a sample. The methods include providing a flow cell comprising a flow cell surface; providing a plurality of tether molecules each having a first terminus and a second terminus, wherein the first terminus of each tether molecule is capable of binding to the flow cell surface; providing microparticles conjugated to a binding moiety that binds a first binding site on the analyte; providing a bridging moiety that binds a second binding site on the analyte and binds the second terminus of the tether molecules; contacting the microparticles conjugated to the binding moiety with the sample under conditions sufficient to allow binding of the binding moiety to analyte present in the sample to form analyte-bound microparticles; contacting the analyte-bound microparticles, the flow cell, the tether molecules and the bridging moiety to form tethered microparticles; and detecting the tethered microparticles by virtue of a characteristic of their motion that identifies them as tethered to the flow cell surface via the tether molecules.

In some embodiments, contacting the analyte-bound microparticles, the flow cell, the plurality of tether molecules and the bridging moiety includes contacting the analyte-bound microparticles with the bridging moiety under conditions sufficient for the bridging moiety to bind the second binding site on the analyte to form microparticle complexes; contacting the

microparticle complexes with the tether molecules under conditions sufficient for the bridging moiety to bind the second terminus of the tether molecules to form microparticle-polymer complexes; and introducing the microparticle-polymer complexes into the flow cell under conditions sufficient to allow binding of the first terminus of each tether molecule to the flow cell surface to form tethered microparticles.

In other embodiments, contacting the analyte-bound microparticles, the flow cell, the plurality of tether molecules and the bridging moiety includes contacting the analyte-bound microparticles with the bridging moiety under conditions sufficient for the bridging moiety to bind the second binding site on the analyte to form microparticle complexes; introducing the plurality of tether molecules into the flow cell under conditions sufficient to allow binding of the first terminus of each tether molecule to the flow cell surface; and introducing the microparticle complexes into the flow cell under conditions sufficient to allow binding of the bridging moiety to the second terminus of each tether molecule to form tethered microparticles.

In other embodiments, contacting the analyte-bound microparticles, the flow cell, the plurality of tether molecules and the bridging moiety includes introducing the plurality of tether molecules into the flow cell under conditions sufficient to allow binding of the first terminus of each tether molecule to the flow cell surface; introducing the bridging moiety into the flow cell under conditions sufficient to allow binding to the second terminus of each tether molecule; and introducing the analyte-bound microparticles into the flow cell under conditions sufficient to allow binding of the bridging moiety to the second binding site of the analyte to form tethered microparticles. In yet other embodiments, contacting the analyte-bound microparticles, the flow cell, the plurality of tether molecules and the bridging moiety includes contacting the analyte-bound microparticles with a first component of the bridging moiety to form microparticle complexes; introducing the tether molecules into the flow cell under conditions sufficient to allow binding of the first terminus of each tether molecule to the flow cell surface; introducing a second component of the bridging moiety into the flow cell under conditions sufficient to allow binding to the second terminus of each tether molecule; and introducing the microparticle complexes into the flow cell under conditions sufficient to allow binding of the first component of the bridging moiety to the second component of the bridging moiety to form tethered microparticles.

In some examples, the bridging moiety comprises an antibody that specifically binds the second binding site on the analyte.

In some examples, the bridging moiety includes a first component and a second component, wherein the first component comprises an antibody that specifically binds the second binding site on the analyte, wherein the antibody is labeled with a first specific binding partner; and the second component comprises a second specific binding partner that binds the first specific binding partner. In particular examples, the second terminus of each tether molecule is labeled with the first specific binding partner and binds the bridging moiety via binding to the second specific binding partner. In specific non-limiting examples, the first specific binding partner comprises biotin and the second specific binding partner comprises avidin. In particular instances, the second specific binding partner comprises a nanoparticle coated with avidin.

In some embodiments of the methods, the first terminus of each tether molecule is conjugated to a hapten and the flow cell surface is coated with an antibody specific for the hapten, or a specific binding partner for the hapten, to enable binding of each tether molecule to the flow cell surface. In some examples, the hapten is digoxigenin and the antibody is an anti-digoxigenin antibody. In other examples, the hapten is biotin and the specific binding partner is avidin.

In some embodiments, the microparticle is detected as being tethered to the flow cell surface if the microparticle moves under force more than a threshold distance that distinguishes tethered microparticles from microparticles non-specifically bound to the flow cell. The threshold distance is less than or equal to the stretched out length of the polymer under force. In some examples, the threshold distance is about the length of the microparticle diameter. In particular examples in which the tether molecules are nucleic acid molecules, such as DNA molecules, the stretched out length is the length predicted by the worm-like chain model (see Example 3 and FIG. 3). In specific non-limiting examples, the polymer is double- stranded DNA about 17 kb in length and the stretched out length is less than or equal to about 5 μιη. In this example, a microparticle is detected as being tethered to the flow cell surface if the microparticle moves more than one microparticle diameter and less than five microparticle diameters in the flow cell.

In other embodiments, a microparticle is detected as being tethered to the flow cell surface by measuring Brownian motion over time. In this embodiment, the microparticles are not subject to a force (i.e. a force is not applied to the flow cell to subject the microparticles to flow).

Brownian motion is recorded over time and one can predict the distance a tethered particle (versus non-tethered particle) will move based on the properties of the polymer.

In some embodiments, the methods further include quantitating the analyte in the sample by determining the number of microparticles tethered to the flow cell surface.

In some embodiments, the tethered microparticles are detected optically, such as by using transmitted light or dark field illumination. However, any suitable optical detection method is contemplated herein. For example, if the microparticles are fluorescent, fluorescence microscopy could be used. In other examples, total internal reflection microscopy or phase-contrast

microscopy are used for detection of tethered microparticles.

In some embodiments, the method allows detection of more than one analyte in a single flow cell. For example, tether molecules whose second termini have binding moieties for different analytes can be attached to the flow cell in different locations (i.e. the tether molecules are bound to the flow cell surface at addressable locations), the location of tethers then indicating which analytes have been captured. In other embodiments, detection of multiple analytes is achieved by using a unique tether length for each analyte, the different tether lengths being distinguishable

microscopically.

In some embodiments, the tether molecules are about 100 nm to about 20 μιη in length. In some examples, the tether molecules are about 1 to about 15 μιη in length, about 2 to about 10 μιη in length, or about 4 to about 6 μιη in length. In specific examples, the tether molecules are about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 μπι in length.

In some embodiments, the tether molecules are polymeric molecules, such as nucleic acid molecules, for example DNA, RNA, PNA or XNA molecules (or combinations thereof). In some examples, the nucleic acid molecules include one or more chemical modifications, such as to increase stability and/or increase nuclease resistance. In some examples, the nucleic acid molecules are synthetic nucleic acid molecules, and may be single- stranded or double- stranded. In some examples, the nucleic acid molecules are about 1 kb to about 70 kb in length, such as about 10 kb to about 50 kb in length, or about 15 kb to about 25 kb. In specific non-limiting examples, the nucleic acid molecules are about 17 kb in length. In particular examples, the nucleic acid is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 kb in length.

In some embodiments, the bridging moiety comprises a nucleic acid aptamer that binds to the second binding site on the analyte and binds to the second terminus of the nucleic acid molecules via a single- stranded region complementary to the second terminus of the nucleic acid molecules.

In some embodiments of the methods, the tether molecules are spatially positioned to be detectable as individual molecules.

In an alternative embodiment for detecting and/or quantitating an analyte in a sample, the method includes providing a flow cell in which a flow cell surface provides a plurality of linear nucleic acid molecules each having a first terminus and a second terminus, wherein the first terminus of each nucleic acid molecule is bound to the flow cell surface and the second terminus of each nucleic acid molecule is bound to a first specific binding partner; providing microparticles conjugated to a capture antibody that specifically binds a first epitope of the analyte; contacting the microparticles conjugated to the capture antibody with the sample under conditions sufficient to allow binding of the capture antibody to analyte present in the sample to form analyte-bound microparticles; contacting the analyte-bound microparticles with a detection antibody that specifically binds a second epitope of the analyte to form an immune complex, wherein the detection antibody is conjugated to a second specific binding partner; introducing the immune complex into the flow cell under conditions sufficient to allow binding of the first specific binding partner to the second specific binding partner to form tethered microparticles; and detecting the tethered microparticles, thereby detecting the analyte in the sample.

In another alternative embodiment for detecting and/or quantitating an analyte in a sample, the method includes providing a flow cell in which a flow cell surface provides a plurality of linear nucleic acid molecules each having a first terminus and a second terminus, wherein the first terminus of each nucleic acid molecule is bound to the flow cell surface and the second terminus of each nucleic acid molecule is bound to a detection antibody that specifically binds a first epitope of the analyte; providing microparticles conjugated to a capture antibody that specifically binds a second epitope of the analyte; contacting the microparticles conjugated to the capture antibody with the sample under conditions sufficient to allow binding of the capture antibody to analyte present in the sample to form analyte-bound microparticles; introducing the analyte-bound microparticles into the flow cell under conditions sufficient to allow binding of the detection antibody to the second epitope of the analyte to form a tethered microparticles; and detecting the tethered microparticles, thereby detecting the analyte in the sample. In another alternative embodiment for detecting and/or quantitating an analyte in a sample, the method includes providing a flow cell in which a flow cell surface presents a plurality of linear nucleic acid molecules each having a first terminus and a second terminus, wherein the first terminus of each nucleic acid molecule is bound to the flow cell surface; providing microparticles conjugated to a capture antibody that specifically binds a first epitope of the analyte; contacting the microparticles conjugated to the capture antibody with the sample under conditions sufficient to allow binding of the capture antibody to analyte present in the sample to form analyte-bound microparticles; providing a detection antibody that specifically binds a second epitope of the analyte; introducing the detection antibody into the flow cell under conditions sufficient to allow binding of the detection antibody to the second terminus of the nucleic acid molecules; introducing the analyte-bound microparticles into the flow cell under conditions sufficient to allow binding of the analyte-bound microparticles to the detection antibody to form tethered microparticles; and detecting the tethered microparticles, thereby detecting and/or quantitating the analyte in the sample.

In some examples, the second terminus of each nucleic acid molecule is bound to a first specific binding partner and the detection antibody is conjugated to a second specific binding partner, and the method includes introducing the detection antibody into the flow cell under conditions sufficient to allow binding of the first specific binding partner to the second specific binding partner; and introducing the analyte-bound microparticles into the flow cell under conditions sufficient to allow binding of the analyte-bound microparticles to the detection antibody to form tethered immune complexes; and detecting the tethered immune complexes, thereby detecting and/or quantitating the analyte in the sample.

In other examples, the second terminus of each nucleic acid molecule is bound to a first specific binding partner and the detection antibody is conjugated to a second specific binding partner, and the method includes contacting the analyte-bound microparticles with the detection antibody to form immune complexes; introducing the immune complexes into the flow cell under conditions sufficient to allow binding of the first specific binding partner to the second specific binding partner to form tethered immune complexes; and detecting the tethered immune complexes, thereby detecting and/or quantitating the analyte in the sample. In specific examples, the first and second specific binding partners are selected from avidin and biotin. In other specific examples, the first and second specific binding partners are selected from a hapten and an antibody specific for the hapten.

In other examples, the second terminus of each nucleic acid molecule is bound to the detection antibody that specifically binds a second epitope of the analyte, and the method includes introducing the analyte-bound microparticles into the flow cell under conditions sufficient to allow binding of the detection antibody to the second epitope of the analyte to form a tethered immune complex; and detecting the tethered immune complex, thereby detecting and/or quantitating the analyte in the sample. In specific examples, the second terminus of each nucleic acid molecule is directly bound to the detection antibody. In other specific examples, the second terminus of each nucleic acid molecule is indirectly bound to the detection antibody. For example, the second terminus of each nucleic acid molecule is bound to a first specific binding partner and the detection antibody is bound to a second specific binding partner, and binding of the first specific binding partner to the second specific binding partner indirectly binds the second terminus of each nucleic acid molecule to the detection antibody. In non-limiting examples, the first and second specific binding partners are selected from avidin and biotin, or the first and second specific binding partners are selected from a hapten and an antibody specific for the hapten.

In some embodiments, the methods further include detecting the tethered microparticles by flow characteristics of the microparticle. For example, the microparticle is detected as being tethered to the flow cell surface if the microparticle moves under force more than a threshold distance that distinguishes tethered microparticles from microparticles non- specifically bound to the flow cell. The threshold distance is less than or equal to the stretched out length of the nucleic acid (or other polymer) under force. In some examples, the threshold distance is about the length of the microparticle diameter. In particular examples in which the polymeric molecules are nucleic acid molecules, such as DNA molecules, the stretched out length is the length predicted by the worm-like chain model (see Example 3 and FIG. 3). In specific non-limiting examples, the polymer is double- stranded DNA about 17 kb in length and the stretched out length is less than or equal to about 5 μιη. In this example, a microparticle is detected as being tethered to the flow cell surface if the microparticle moves more than one microparticle diameter and less than five microparticle diameters in the flow cell.

Brownian motion is recorded over time and one can predict the distance a tethered particle (versus non-tethered particle) will move based on the properties of the nucleic acid (or other polymer).

In some embodiments, the tethered microparticles are detected optically, such as by using transmitted light or dark field illumination. However, any suitable optical detection method is contemplated herein. For example, if the microparticles are fluorescent, fluorescence microscopy could be used. In other examples, total internal reflection microscopy or phase-contrast microscopy are used for detection of tethered microparticles.

microscopically.

In some embodiments, the nucleic acid molecules are DNA molecules or RNA molecules. In some examples, the nucleic acid molecules include one or more chemical modifications, such as to increase stability and/or increase nuclease resistance. In some examples, the nucleic acid molecules are synthetic nucleic acid molecules.

In some examples, the nucleic acid molecules are about 1 kb to about 70 kb in length, such as about 10 kb to about 50 kb in length, or about 15 kb to about 25 kb. In specific non-limiting examples, the nucleic acid molecules are about 17 kb in length. In particular examples, the nucleic acid is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 kb in length.

In some examples, the nucleic acid molecules are about 100 nm to about 20 μιη in length. In some examples, the nucleic acid molecules are about 1 to about 15 μιη in length, about 2 to about 10 μιη in length, or about 4 to about 6 μιη in length. In specific examples, the nucleic acid molecules are about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 μηι in length.

In some embodiments, a hapten is conjugated to the first terminus of each nucleic acid molecule and the flow cell surface is coated with an antibody specific for the hapten, or a specific binding partner for the hapten, to enable binding of each nucleic acid molecule to the flow cell surface. In some examples, the hapten is digoxigenin and the antibody is an anti-digoxigenin antibody. In non-limiting examples, the hapten is biotin and the specific binding partner is avidin. The nucleic acids (or other polymeric molecule) can be bound to the flow cell surface using any means known in the art.

In some embodiments, the linear nucleic acid molecules are spatially positioned to be detectable as individual molecules. In some embodiments of the methods disclosed herein, the microparticles are denser than water. In some examples, the microparticles are magnetic beads.

In some embodiments, the microparticles are about 1 to about 10 μιη in diameter. In some examples, the microparticles are about 2 to about 5 μιη, such as about 2.8 μιη in diameter. In specific examples, the microparticles are about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 μιη in diameter.

In some embodiments of the methods disclosed herein, the analyte is a disease-specific biomarker. In some examples, the biomarker is a protein biomarker. However, the analyte need not be a protein. The disclosed methods are capable of detecting any analyte that has at least two distinct binding sites. The analyte can be, for example, a carbohydrate or a lipid, or any molecule that is recognizable in a sandwich immunoassay.

In some examples, the protein biomarker is a tumor antigen. In specific examples, the tumor antigen is prostate-specific antigen. Other tumor antigens include, but are not limited to AFP, CEA, CA-125, MUC-1, ETA, TAG-72, immature laminin receptor, HPV E6/E7, BING-4, calcium-activated chloride channel 2, RAGE-1, MAGE-1, MAGE-2, tyrosinase, Cyclin-Bl, 9D7, Ep-CAM, EphA3, Her2/Neu, telomerase, mesothelin, SAP-1, survivin, NY-ESO-1, Melan- A/MART-1, gp75, gpl00/pmell7, β-catenin, POTE, PRAME, MUM-1, WT-1, PR-1 BAGE family, CAGE family, GAGE family, MAGE family, SAGE family, XAGE family, TRP-1, TRP-2, MC1R, CDK4, BRCAl/2, CML66, fibronectin, MART-2, p53, Ras, TGF- RII and MUC1.

In other examples, the protein biomarker is an antigen from an infectious agent, such as a virus, bacterium, fungus, protozoan or nematode.

In some examples, the disease is cancer, an infectious disease, a cardiovascular disease, a renal disease, a neurological disorder, a neurodegenerative disease, a pulmonary disease, an autoimmune disease, an inflammatory disease, a hematologic disease, a liver disease, osteoarthritis, ischemia-reperfusion injury or a prion disease.

The cancer can be a hematologic cancer or a solid tumor. Examples of hematological tumors include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,

myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplasia syndrome, hairy cell leukemia and myelodysplasia.

Examples of solid tumors, include fibrosarcoma, myxosarcoma, liposarcoma,

chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma and retinoblastoma).

Cardiovascular diseases include, but are not limited to coronary artery disease,

cardiomyopathy, hypertension, heart failure, myocarditis, endocarditis, cerebrovascular disease, peripheral artery disease, congenital heart disease and rheumatic heart disease.

Neurological disorders include, but are not limited to, brain damage, brain dysfunction, spinal cord disorders, peripheral neuropathy, cranial nerve disorder, autonomic nerve disorder, seizure disorders (such as epilepsy), sleep disorders (such as narcolepsy), migraines, and stroke.

Neurodegenerative diseases include, but are not limited to, Alzheimer's disease,

Parkinson's disease, Huntington's disease, ALS, multiple sclerosis, Lewy body dementia, vascular dementia, progressive supranuclear palsy, corticobasal degeneration, multiple system atrophy and frontotemporal dementia.

Examples of pulmonary disease include cystic fibrosis, emphysema, asthma, sarcoidosis, chronic bronchitis, bronchopulmonary dysplasia, pulmonary fibrosis, pneumonia and adult respiratory distress syndrome.

Exemplary autoimmune diseases include, but are not limited to, Addison's disease, ALS, celiac disease, Chagas disease, Chrohn's disease, diabetes mellitus type 1, Graves' disease,

Guillain-Barre syndrome, Hashimoto's thyroiditis, lupus, multiple sclerosis, myasthenia gravis, pernicious anemia, rheumatoid arthritis, Sjogren's syndrome and ulcerative colitis.

Inflammatory diseases include systemic inflammatory response syndrome (SIRS), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), multi-organ failure (MOF), ischemia reperfusion injury (IRI) and adverse drug reaction (ADRS).

Exemplary hematologic diseases include hemoglobinopathies (such as sickle-cell disease, thalassemia and methemoglobinemia), anemias, myeloproliferative disorders, coagulopathies (such as thrombocytosis, disseminated intravascular coagulation, hemophilia, Von Willebrand disease) and platelet disorders. Liver diseases include, for example, hepatitis, alcoholic liver disease, cirrhosis and fatty liver disease.

Prion diseases (or transmissible spongiform encephalopathies) include, but are not limited to the human prion diseases Creutzfeldt-Jakob disease, kuru, fatal familial insomnia and

Gerstman ---Straus sler---Scheinker syndrome; and animal prion diseases, such as scrapie

(sheep/goats), bovine spongiform encephalopathy (cattle), feline spongiform encephalopat y (cats) and chronic wasting disease (elk/deer).

In some embodiments of the methods disclosed herein, the sample is a serum, blood, plasma, urine, saliva or cerebral spinal fluid (CSF) sample.

B. Flow cells and kits for detecting and quantitating analytes

Further provided herein are flow cells for detecting and/or quantitating analytes in a sample. The flow cells include a flow cell surface; a plurality of tethers, such as linear polymeric molecules, each having a first terminus and a second terminus, wherein the first terminus of each tether molecule is bound to the flow cell surface; and a bridging moiety bound to the second terminus of each tether molecule, wherein the bridging moiety specifically binds a first binding site of the analyte.

In some embodiments, the bridging moiety comprises a detection antibody or an aptamer. In some examples, the detection antibody or aptamer is directly bound to the second terminus of each tether molecule. In other examples, the detection antibody or aptamer is indirectly bound to the second terminus of each tether molecule. In specific examples, the second terminus of each tether molecule is bound to a first specific binding partner and the detection antibody or aptamer is bound to a second specific binding partner, and binding of the first specific binding partner to the second specific binding partner indirectly binds the second terminus of each tether molecule to the detection antibody or aptamer. In non-limiting examples, the first and second specific binding partners are selected from avidin and biotin.

In some embodiments, the tether molecules are bound to the flow cell surface at addressable locations.

In some embodiments, the tethers are polymeric molecules, such as nucleic acid molecules, for example DNA or RNA molecules. In some examples, the nucleic acid molecules include one or more chemical modifications, such as modifications to increase nuclease resistance.

In an alternative embodiment, the flow cell includes a flow cell surface; a plurality of linear nucleic acid molecules, each having a first terminus and a second terminus, wherein the first terminus of each nucleic acid molecule is bound to the flow cell surface; and a detection antibody that specifically binds a first epitope of the analyte, wherein the detection antibody is bound to the second terminus of each nucleic acid molecule. In some examples, the detection antibody is directly bound to the second terminus of each nucleic acid molecule. In some examples, the detection antibody is indirectly bound to the second terminus of each nucleic acid molecule.

In some examples, the second terminus of each nucleic acid molecule is bound to a first specific binding partner and the detection antibody is bound to a second specific binding partner, and binding of the first specific binding partner to the second specific binding partner indirectly binds the second terminus of each nucleic acid molecule to the detection antibody. In specific examples, the first and second specific binding partners are selected from avidin and biotin. In other specific examples, the first and second specific binding partners are selected from a hapten and an antibody specific for the hapten.

Also provided herein are kits for detecting and/or quantitating analytes in a sample. In some embodiments, the kit includes a flow cell described herein and a microparticle conjugated to a capture antibody that specifically binds a second binding site on the analyte.

In other embodiments, the kit includes a flow cell comprising a flow cell surface; a plurality of tethers, such as linear polymeric molecules, each having a first terminus and a second terminus, wherein the first terminus of each tether molecule is capable of binding to the flow cell surface; microparticles conjugated to a binding moiety that specifically binds a first binding site on the analyte; and a bridging moiety that specifically binds a second binding site on the analyte and binds the second terminus of the tether molecules. In some examples, the first terminus of each tether molecule is bound to the flow cell surface. In some examples, the second terminus of each nucleic acid molecule is bound to the bridging moiety. In some examples, the bridging moiety is bound to the second binding site on the analyte.

In some embodiments of the kits, the tether molecules are bound to the flow cell surface at addressable locations.

In some embodiments of the kits, the tethers are polymeric molecules, such as nucleic acid molecules, for example DNA molecules or RNA molecules.

In an alternative embodiment, the kit includes a microparticle conjugated to a capture antibody that specifically binds a first epitope of the analyte; a detection antibody that specifically binds a second epitope of the analyte; and a flow cell in which a flow cell surface presents a plurality of linear nucleic acid molecules each having a first terminus and a second terminus, wherein the first terminus of each nucleic acid molecule is bound to the flow cell surface.

In some examples, the second terminus of each nucleic acid molecule is bound to the detection antibody. In other examples, the second terminus of each nucleic acid molecule is bound to a first specific binding partner and the detection antibody is conjugated to a second specific binding partner. The first and second specific binding partners can be, for example avidin and biotin, or a hapten and an antibody specific for the hapten.

In some embodiments of the flow cells and kits disclosed herein, the linear polymeric molecules (such as linear nucleic acid molecules) are spatially positioned to be detectable as individual molecules.

The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.

EXAMPLES

Example 1: Materials and Methods

This example describes the materials and methods for the studies described in Example 2.

Reagents

Flow cells were made by attaching coverslips to microscope slides via approximately 100 μιη thick gaskets made of double-sided sticky tape (3M Company, 300SLE adhesive) (FIG. 1C). The coverslips had two approximately 1mm holes drilled by bombardment with 50 μιη A10₂ particles using a Comco Inc. MicroBlaster. Nylon adaptors (McMaster Carr #91145A133) were attached over the holes with epoxy glue or melted PARAFILM™ to provide inflow and outflow ports. Slides and coverslips were cleaned by sonication in 15% KOH in ethanol and deionized water before assembling into flow cells. Alternatively, flow cells were made by bonding air- plasma treated polydimethylsiloxane (PDMS) slabs to microscope slides; the PDMS slabs were cast over scotch tape strips to form shallow channels approximately 40 mm x 2 mm x 0.1 mm; inflow and outflow ports were formed by puncturing the PDMS with syringe needles. Flow cell volumes were about 30 μΐ.

DNA for tethers was prepared from a 17 kb plasmid derived from pBluescript (Stratagene). The linearized plasmid was labeled with digoxigenin at one end (for attachment to flow cell surfaces via anti-digoxigenin antibody), and biotin at the other end (for attachment to capture beads via streptavidin), as follows:

The plasmid was linearized with restriction enzyme Bsal, which cuts the plasmid once leaving overhangs 5'GTTT— and 5'AAAC— · The GTTT overhang was filled in with biotin-dATP using Klenow DNA polymerase lacking 3'-exonuclease activity (New England Biolabs) followed by column purification (Qiagen) to remove unincorporated biotin-dATP. This should attach a maximum of three biotins at the end of each DNA. Since a streptavidin molecule binds four biotins, each streptavidin bound to biotin at the end of a DNA would have at least one unoccupied site capable of binding an additional biotin. The AAAC overhang at the other end of the DNA was then filled in with dGTP and digoxigenin-dUTP (Roche Applied Science) and unincorporated nucleotides removed as above.

Flow cells were filled with 0.1 μΜ polyclonal sheep antibody to digoxigenin (Roche, #11333089001) in phosphate buffered saline (PBS) and incubated for approximately 1 hour to non- specifically adsorb anti-digoxigenin antibody to the flow cell surfaces. Flow cell surfaces were then passivated by washing with wash buffer (WB; 0.3% bovine serum albumin, 0.5 mM EDTA, 0.01% Tween-20, and 0.02% sodium azide in PBS). 10⁸-10⁹ molecules of biotin/digoxigenin-end labeled DNA were introduced into the flow cell and incubated for 2-4 hours to attach to the anti- digoxigenin antibodies. Unattached DNA was removed by washing with WB. Streptavidin (20 nM in WB) was added to bind biotin at the end of the DNAs, and unattached streptavidin was removed by washing with approximately 20 flow cell volumes of WB under gravity flow.

Capture beads were made by covalently attaching anti-PSA monoclonal antibody to 2.8 μηι- paramagnetic beads using hydrazine-aldehyde chemistry (Solulink). Anti-PSA monoclonal antibody (BiosPacific #8311) was modified by covalent attachment of hydrazine-nicotinamide to primary amino groups. The hydrazine-derivatized antibody was incubated with 4-fluorobenzene- derivatized paramagnetic beads (MAGNALINK™ beads, Solulink) according to the

manufacturer's instructions. Efficiency of antibody attachment to beads was estimated

spectroscopically by measuring the antibody (protein) concentration before and after incubation (about 90% of antibody attached to beads, equivalent to approximately 10⁶ antibody

molecules/bead). Detection antibody (BiosPacific, monoclonal 8301) was biotinylated using NHS- PEG₄ biotin (Thermo Scientific #21329). For analytic purposes, a batch of PSA analyte was biotinylated in the same way.

Enzyme-linked immunoassay (ELISA)

The efficiency with which capture beads bound low concentrations of PSA, detection antibody and streptavidin was estimated by ELISA, using streptavidin-peroxidase and Sure Blue enzyme substrate (KPL Inc.). Bead-bound streptavidin-peroxidase was determined from light- absorption of enzyme-modified substrate using a NANODROP™ spectrophotometer. Optical detection of tethered beads

Flow cells were imaged with 5x or lOx objectives using transmitted light or dark field illumination. Movies of bead motion were taken at 7.5 frames per second with a Thorlabs CCD camera (model DC310-C) or an Imaging source CMOS camera (model DMK 72AUC02) while fluid was pushed alternately to the left and right for approximately 1-2 seconds in each direction using a syringe and tubing connected to one of the ports of a flow cell. Flow rate was adjusted manually while watching the motion of free beads. Beads were deemed tethered if they moved more than 1 bead diameter and less than 5 bead diameters in each direction under flow that was maintained in a given direction for a few seconds. Free beads moved »5 bead diameters in each direction under these conditions.

Example 2: Tethered-bead immune sandwich assay

This example describes a tethered-bead immune assay for detection of analytes at very low concentrations in a sample.

Tethered bead immune sandwich assay

Anti-PSA capture beads (10⁵) were incubated with 1 nM PSA in 50 μΐ WB for 1 hour on a rotary mixer. At 10⁶ antibody molecules/bead (see Example 1), the capture antibody concentration was nominally 3 nM. Since this is greater than the PSA concentration and 10 times the reported dissociation constant of the antibody for PSA (K_D= 0.1 nM, BiosPacific), most PSA molecules in the incubation mixture should be captured on beads at equilibrium. The beads were pulled to the side of the tube using a magnet, washed and incubated with 50 μΐ of 1 nM biotinylated detection antibody for 1 hour. The capture and detection antibodies bind to different epitopes on PSA and do not interfere with each other's binding (Ferguson et ah, Clin Chem 42:675-684, 1996). Since the detection antibody was in excess over the potentially captured analyte, and its concentration was approximately lOOx the K_D of its binding to PSA (0.01 nM), almost all of the captured PSA molecules should bind detection antibody at equilibrium. After washing, the beads were introduced into flow cells coated with DNA strands that had streptavidin at their distal ends (see Example 1). Beads were introduced by pipetting about 10⁵ beads suspended in about 1 flow cell volume of WB into the inflow port of a flow cell from which excess WB had been drained, and the flow cell was inverted several times to mix the added bead suspension with WB retained in the flow cell. Flow cells were not further washed, so unattached beads remained in the flow cell. About 50 μΐ of WB was added to entry and exit port reservoirs and a syringe and tubing filled with air was attached to one of the ports. Gentle pushing and pulling on the syringe plunger caused fluid in the reservoirs to flow back and forth. Bead motion was observed using an inverted microscope with a 10X objective and dark field illumination. When beads had been exposed to 1 nM PSA, most of the beads appeared tethered, moving approximately 12 μιη (judged from an ocular calibration ruler) as fluid flowed back and forth. By contrast, unattached beads moved approximately 120 μιη before reversing direction with the flow. Almost all beads from a control experiment in which PSA was omitted were unattached, as expected.

The expected travel distance for tethered beads was calculated using a model (see Example 3) that takes into account DNA length in base pairs, DNA extension as a function of drag force based on the worm-like chain model (Bustamante et ah, Science 265:1599-1600, 1994), and drag force as a function of fluid velocity (Goldman et ah, Nature 473:484-488, 1967). The calculation determines the position of DNA attachment on the bead surface that balances drag force and torque when a tether is extended by flow. The model predicts a center-to-center bead travel distance of 12 μιη for fluid flow of 120 μιη/s around a tethered bead. The fluid flow speed was estimated from the speed of unattached beads in the same focal plane (height above the flow cell surface) as tethered beads, and was approximately 100-120 μητ/s. The agreement between observed and predicted tether length and flow speed provides strong support that the tether structures are as depicted in FIG. 1.

A few beads moved less than 1 bead diameter during flow. These probably represent beads that have attached to the surface non-specifically due to incomplete passivation of the bead and flow cell surfaces. One bead showed characteristic tethered motion even though beads were not exposed to PSA; such tethers could result from detection antibody sticking non-specifically to a capture bead, or a capture bead sticking non-specifically to DNA. There were also a few beads that moved greater than 1 bead diameter but less than 12 μιη. These shorter- than-expected tether lengths could result from beads being attached via more than one DNA, or DNA sticking to glass or bead surfaces at positions internally along the DNA. There were also some bead aggregates, which result from non-specific interactions since they are seen in beads not exposed to PSA.

The paramagnetic capture beads contained iron, are denser that water, and settle to the lower surface of flow cells by gravity within minutes. Tethers also formed within minutes, with no noticeable increase in the number of tethers after about 5 minutes.

Limits of detection

When this experiment was performed with 10-fold serial dilutions of PSA, the number of tethered beads was maximal at about 10 pM and decreased as PSA concentration decreased, with a approximately 1 pM lower limit of detection (Table 1). At higher concentrations of analyte, the number of tethers might be limited by the density of capture DNAs on the sensor surface. At 1 pM PSA, the 50 μΐ sample volume contained approximately 300 PSA molecules/bead. Since only about 3% of beads formed tethers at this concentration despite potentially hundreds of PSA molecules per bead, the efficiency of various steps in the assay was investigated.

Table 1. Average # (+ sd) of tethered, stuck, and total beads per 5x field of view*

5 fields of view averaged for 100 pM sample, 20 fields of view for other samples

Efficiency of bead capture of PSA, detection antibody, and streptavidin

An enzyme-linked immune sandwich assay was used with anti-PSA beads as the solid phase to estimate the efficiency of capturing PSA and detection antibody, as shown schematically in FIG. 2. In the first experiment, PSA that had been labeled with biotin was used so that a single binding step would capture biotin on beads. It was estimated how efficiently anti-PSA beads bound PSA by measuring how completely anti-PSA beads removed biotinylated PSA from an incubation mixture, as illustrated in the first two rows in FIG. 2. When 10⁵ capture beads were incubated with 10⁸ biotinylated PSA molecules in 50 μΐ (3 pM) for 1.5 hours (sample 1), essentially all of the biotin was captured as determined by incubating the supernatant of these beads with fresh capture beads (sample 2). In the representative experiment reported in Table 2, the OD452 was 0.170 for beads incubated with biotin-PSA (sample 1) versus 0.044 for beads incubated with the supernatant (sample 2), and 0.052 for control beads incubated with WB without PSA in the first step (sample 3). Thus, it was estimated that the first step captured approximately 75% or more of the target PSA molecules. Table 2. Enzyme-linked immunoassay to monitor efficiency of capture of analyte, detection antibody and streptavidin-peroxidase^*

# anti-PSA # biotin-anti-PSA #SA-perox

Sample beads Target molecule molecules added molecules added OD452

1 2 x l0⁵ 2 x lO⁸ bio-PSA None 4 x 10⁹ 0.170

Supernatant of

2 2 x l0⁵ #1 after 1.5h None 4 x 10⁹ 0.044 3 2 x 10⁵ None None 4 x 10⁹ 0.052 4 2 x l0⁵ 2 x l0⁸ PSA 2 x 10¹⁰ 4 x 10⁹ 0.181

OD452 as a function of the number of SA-peroxidase molecules in solution

5 2 x 10⁸ 0.764

6 2 x 10⁷ 0.189

7 2 x 10⁶ 0.036

Assay volumes were 44 μΐ; SA-perox = streptavidin-peroxidase

To estimate the efficiency of binding of detection antibody, the amount of biotin-labeled detection antibody captured by beads that had been incubated with 3 pM unlabeled PSA was compared to the amount of biotin-labeled PSA captured by beads incubated with 3 pM biotin- labeled PSA (sample 4 in FIG. 2). The ELISA signals were similar (OD452 0.170 vs. 0.181, sample 1 vs. sample 4, Table 2), indicating that most of the unlabeled PSA molecules captured on beads bound detection antibody. To be more quantitative, the number of biotins per labeled antibody (about 8) and per labeled PSA molecule (about 3) was estimated using a HABA-avidin reagent (Sigma #H2153). Based on these numbers, it was estimated that 37% of captured PSA molecules bound detection antibody under the described conditions. This estimate is conservative in the sense that if biotin-labeled PSA was captured less efficiently than plain PSA, or if steric interference prevented SA-peroxidase from binding all the biotins on the detection antibody, one would estimate that more than 37% of PSA molecules bound detection antibody. This implies that a 1 pM PSA test sample would lead to at least approximately 0.37*0.75*300 = 80 biotinylated detection antibody molecules/bead.

To estimate the efficiency with which biotin on capture beads bound streptavidin, the ELISA signal from beads that captured biotin-PSA was compared to the signal from serially diluted streptavidin-peroxidase in solution. In the above experiment, 2 x 10⁷ streptavidin-peroxidase molecules in solution resulted in an OD452 of 0.189 (Table 2, sample 6). Since this is comparable to the ELISA signal generated when approximately 7.5 x 10⁷-10⁸molecules of biotinylated-PSA were captured on beads (Table 2, sample 1), it was estimated that approximately 20% of biotin- labeled PSA molecules captured on beads bound streptavidin-peroxidase. The streptavidin- peroxidase concentration in these experiments was 30 pM.

The above experiments show that PSA analyte and detection antibody are captured efficiently, as are free streptavidin-peroxidase molecules. However, only a few percent of beads estimated to have bound at least 80 molecules of biotinylated detection antibody (those exposed to 1 pM PSA) formed tethers (Table 1), suggesting that these beads bind relatively inefficiently to streptavidin at the ends of tethered DNA.

Discussion

The correspondence between observed and predicted travel distance for many tethered beads in the system described herein provides strong evidence that binding via single analyte molecules to single DNA molecules is sufficient to form tethers. This conclusion is consistent with a large body of literature in single-molecule biophysics using DNA-tethered beads.

If the affinity of streptavidin on DNA for biotin on beads was the same as its affinity in solution, one would expect almost all beads with at least one biotinylated detection antibody to form tethers at equilibrium. This follows from the fact that the concentration of streptavidin on DNA in the vicinity of beads should be much greater than the solution K_D. The concentration of DNA-bound streptavidin in the vicinity of beads in flow cells is not known precisely but can be estimated as follows. The beads settle by gravity to the surface. For beads that settle "on top of a DNA, the local concentration of streptavidin is one molecule per volume of a hemisphere with radius equal to a DNA length = 1 molecule/250 μιη³ = 7 pM. The local streptavidin concentration can also be estimated from the number of DNAs per unit area of surface divided by the maximal length (height) of a DNA. The number of DNAs on the surface must be equal to or greater than the number of tethers that formed when streptavidin-coated beads were introduced into flow cells containing biotin-dig-labeled DNAs. This number was approximately 10⁶ tethers/flow cell. 10⁶ DNAs in a volume = flow cell area (3 cm²) x 1 DNA length (about 5 μιη) corresponds to a concentration approximately 1 pM. Both of these concentration estimates are well over the solution K_D of biotin- streptavidin (about 1 fM) and roughly equal to the concentration of streptavidin-peroxidase that bound efficiently to beads in the ELISA experiments. This suggests that streptavidin at the end of DNA is less accessible to beads than free streptavidin.

Streptavidin at the ends of long DNA molecules may be less accessible due to "masking" from negative charge or steric effects of adjacent DNA. Raising the salt concentration to 1M NaCl to shield charge did not lead to significantly more tethers. Steric interference from adjacent DNA likely increases with DNA length. For example, if the DNA is modeled as a "blob" made up of n, statistically independent segments, capture by biotin on a bead might require the streptavidin- labeled DNA segment to be closer to the bead that any other DNA segment, in which case binding efficiency would decrease proportional to 1/n, i.e. inversely with DNA length. This suggests efficiency might be improved by shortening the DNA. However, shortening the DNA makes it more difficult to distinguish tethered beads from non-specifically stuck beads.

Another possible contributor to inefficient tether formation is reduced capture rate as the number of biotins/bead decreases. For molecules in solution, reducing the area of absorption from the entire surface of a sphere to a single, small absorbing patch reduces the absorption rate by the ratio of the radius of the patch to that of the sphere (Berg, H.C. 1993. Random Walks in Biology p.30. Princeton University Press). For a 5 nm antibody and 2.8 μιη bead, this would be about 500- fold. In the system described herein, 2.8 μιη beads completely coated with streptavidin (about 10⁶molecules/bead, Dynal, M280 beads) formed tethers with DNAs on flow cell surfaces without noticeable delay upon settling to the surface (i.e. < minutes), which sets an upper limit to delay from this phenomenon of several hours. However, an increase in the number of tethers after overnight incubation was not observed. If time to bind DNA is limiting, it should decrease proportionally to bead radius, but smaller beads are more difficult to detect in low magnification, large field-of-view microscopy.

Other approaches to increasing efficiency of tether formation include increasing the concentration of biotin or streptavidin (e.g. putting multiple biotins on each DNA, using aggregated forms of streptavidin rather than molecular streptavidin, using higher concentrations of DNA, or incubating DNA with beads in suspension before introduction into flow cells).

The tethered bead transduction method has several potential advantages compared to previously reported, highly sensitive methods. First, it is inherently a single molecule counting method since beads can be tethered via single analyte molecules. Second, the method allows one to disregard signal from beads or molecules sticking non-specifically to flow cell surfaces as these do not lead to tethers of the expected length. Non-specific sticking of reporter molecules to the sensor surface limits the sensitivity of fluorescence-based sensors since there is no way to distinguish fluorescent reporter molecules stuck non-specifically to sensor surfaces from those stuck to analyte molecules captured on the sensor surface. Non-specific sticking to DNA does pose a problem for the tethered bead method but the surface area of the DNA is much less than the surface area of the flow sensor, so non-specific sticking to DNA is quantitatively less of a problem. In the

experiments disclosed herein, non-specific sticking in the absence of analyte led to approximately 30 tethers per 10⁵ beads. This imposes a floor on the lowest detectable concentration of approximately 100 molecules/50 μΐ sample, or about 3aM. A third potential advantage of the tethered bead method is that detection of tethered beads does not require advanced technology - only ~5x magnification, simple illumination, and a low cost video camera. The rapid development of inexpensive consumer CMOS cameras with greater than 10M pixels of about 1 micron pixel size promises to make detection of micron- size beads easier, and potentially achievable with a cellphone camera.

Example 3: Quantitative model of tether attachment geometry and tether extension as a function of flow rate

Liquid flow around a tethered bead exerts a drag force on the bead in the direction of flow and also a torque because the liquid flows faster over the portion of the bead farther from the glass surface (Poiseuille flow). Fluid dynamics theory predicts how the force and torque near a surface are modified compared to their bulk values. For example, Goldman et al. {Nature 473:484-488, 1967) predict that stationary spherical beads touching a surface experience a drag force

and torque

where η is the fluid viscosity, r the radius of the bead, v the fluid velocity at a height r above the surface, a = 1.7, and β = 0.94. Thus, the ratio, R, of the torque to r*drag force = (2/3)(β/ ) = 0.37. The DNA tether must exert a force and a torque equal and opposite to that of the shear flow at the trajectory end points where tethered beads are stationary. The position of the attachment point on a bead (see FIG. 3) determines the component of tether force in the direction of flow and the torque exerted by the DNA tether:

Force balance: FTCOS(([>)= Fd_rag = όπηΓν (A. l)

Torque balance: ΓΡτ8Ϊη(φ+π/2-2κ) = r*Fd_ragR (A.2)

where F_T is the tension in the DNA and φ and κ are angles defined in FIG. 3. Geometry determines a relationship between φ and K:

xsin^) = r(l+cos(2K)) (A.3)

(1), (2) and (3) can be combined to yield:

The center-to-center travel distance, D_cc, for a tethered bead can be expressed as:

Dec = 2(χοοβ(φ) + rsin(2K)) (A.5)

The well-validated worm- like chain model of DNA predicts a relationship between tension and relative extension of a piece of DNA. To good approximation (Bustamante et ah, Science

265: 1599-1600, 1994): F_Tp/k_BT = (l/4)(l-(x/L))-² -(1/4) + (x/L) (A.6)

where p is the persistence length of DNA (about 50 nm), 1¾ is Boltzmann's constant, T is temperature in degrees Kelvin, and L is the "fully stretched out" length of the DNA, approximately 0.3 nm/base pair.

In the experiment described herein, D_cc = 12 μιη was observed; r =1.4 μιη, L = 5.1 μιη, p = 50 nm, and I _BT = 4*10^"21J. If one considers R =0.37 as given from fluid dynamics, the four equations (A3), (A4), (A5) and (A6) have four remaining variables φ, κ, F_T, and x, which are therefore all determined; φ = 16°, 2κ = 95°, FT = 5.8N, and x = 4.8μιη. From (Al), Fd_rag = 5.6 pN is calculated. If one takes cc=1.7, then v is predicted from (Al): for η = 10^"3Ns/m², one finds v = 125 μητ/s. But v is also estimated from the observed motion of free beads, v₀bs = 100 - 120 μιη/s. The agreement between the predicted and observed v provides an independent test of the model.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

1. A method for detection and/or quantitation of an analyte in a sample, comprising: providing a flow cell comprising a flow cell surface;

providing a plurality of tether molecules each having a first terminus and a second terminus, wherein the first terminus of each tether molecule is capable of binding to the flow cell surface; providing microparticles conjugated to a binding moiety that binds a first binding site on the analyte;

providing a bridging moiety that binds a second binding site on the analyte and binds the second terminus of the tether molecules;

contacting the microparticles conjugated to the binding moiety with the sample under conditions sufficient to allow binding of the binding moiety to analyte present in the sample to form analyte-bound microparticles;

contacting the analyte-bound microparticles, the flow cell, the plurality of tether molecules and the bridging moiety to form tethered microparticles; and

detecting the tethered microparticles by virtue of a characteristic of their motion that identifies them as tethered to the flow cell surface via the tether molecules, thereby detecting and/or quantitating the analyte in the sample.

2. The method of claim 1, wherein contacting the analyte-bound microparticles, the flow cell, the plurality of tether molecules and the bridging moiety, comprises:

contacting the analyte-bound microparticles with the bridging moiety under conditions sufficient for the bridging moiety to bind the second binding site on the analyte to form

microparticle complexes;

contacting the microparticle complexes with the plurality of tether molecules under conditions sufficient for the bridging moiety to bind the second terminus of the tether molecules to form microparticle-polymer complexes; and

introducing the microparticle-polymer complexes into the flow cell under conditions sufficient to allow binding of the first terminus of each tether molecule to the flow cell surface to form tethered microparticles.

3. The method of claim 1, wherein contacting the analyte-bound microparticles, the flow cell, the plurality of tether molecules and the bridging moiety, comprises: contacting the analyte-bound microparticles with the bridging moiety under conditions sufficient for the bridging moiety to bind the second binding site on the analyte to form

microparticle complexes;

introducing the plurality of tether molecules into the flow cell under conditions sufficient to allow binding of the first terminus of each tether molecule to the flow cell surface; and

introducing the microparticle complexes into the flow cell under conditions sufficient to allow binding of the bridging moiety to the second terminus of each tether molecule to form tethered microparticles.

4. The method of claim 1, wherein contacting the analyte-bound microparticles, the flow cell, the plurality of tether molecules and the bridging moiety, comprises:

introducing the plurality of tether molecules into the flow cell under conditions sufficient to allow binding of the first terminus of each tether molecule to the flow cell surface;

introducing the bridging moiety into the flow cell under conditions sufficient to allow binding to the second terminus of each tether molecule; and

introducing the analyte-bound microparticles into the flow cell under conditions sufficient to allow binding of the bridging moiety to the second binding site of the analyte to form tethered microparticles.

5. The method of claim 1, wherein contacting the analyte-bound microparticles, the flow cell, the plurality of tether molecules and the bridging moiety, comprises:

contacting the analyte-bound microparticles with a first component of the bridging moiety to form microparticle complexes;

introducing a second component of the bridging moiety into the flow cell under conditions sufficient to allow binding to the second terminus of each tether molecule; and

introducing the microparticle complexes into the flow cell under conditions sufficient to allow binding of the first component of the bridging moiety to the second component of the bridging moiety to form tethered microparticles.

6. The method of any one of claims 1-5, wherein the bridging moiety comprises an antibody that specifically binds the second binding site on the analyte.

7. The method of any one of claims 1-6, wherein the bridging moiety comprises a first component and a second component, wherein:

the first component comprises an antibody that specifically binds the second binding site on the analyte, wherein the antibody is labeled with a first specific binding partner; and

the second component comprises a second specific binding partner that binds the first specific binding partner.

8. The method of claim 7, wherein the second terminus of each tether molecule is labeled with the first specific binding partner and binds the bridging moiety via binding to the second specific binding partner.

9. The method of claim 7 or claim 8, wherein the first specific binding partner comprises biotin and the second specific binding partner comprises avidin.

10. The method of claim 9, wherein the second specific binding partner comprises a nanoparticle coated with avidin.

11. The method of any one of claims 1-10, wherein the first terminus of each tether molecule is conjugated to a hapten and the flow cell surface is coated with an antibody specific for the hapten, or a specific binding partner for the hapten, to enable binding of each tether molecule to the flow cell surface.

12. The method of claim 11, wherein the hapten is digoxigenin and the antibody is an anti-digoxigenin antibody.

13. The method of claim 11, wherein the hapten is biotin and the specific binding partner is avidin.

14. The method of any one of claims 1-13, wherein a microparticle is detected as being tethered to the flow cell surface if the microparticle moves more than one microparticle diameter and less than five microparticle diameters in the flow cell.

15. The method of any one of claims 1-13, wherein a microparticle is detected as being tethered to the flow cell surface by measuring Brownian motion over time.

16. The method of any one of claims 1-15, further comprising quantitating the analyte in the sample by determining the number of microparticles tethered to the flow cell surface.

17. The method of any one of claims 1-16, wherein the tethered microparticles are detected optically.

18. The method of claim 17, wherein the tethered microparticles are detected optically using transmitted light or dark field illumination.

19. The method of any one of claims 1-18, wherein the tether molecules are bound to the flow cell surface at addressable locations.

20. The method of any one of claims 1-19, wherein the tether molecules are about 100 nm to about 20 μιη in length.

21. The method of any one of claims 1-20, wherein the tether molecules are linear polymeric molecules.

22. The method of claim 21, wherein the linear polymeric molecules are nucleic acid molecules.

23. The method of claim 22, wherein the nucleic acid molecules are DNA molecules.

24. The method of claim 22, wherein the nucleic acid molecules are RNA molecules.

25. The method of any one of claims 22-24, wherein the nucleic acid molecules are chemically modified.

26. The method of any one of claims 22-25, wherein the nucleic acid molecules are synthetic nucleic acid molecules.

27. The method of any one of claims 22-26, wherein the nucleic acid molecules are about 1 kb to about 70 kb in length.

28. The method of claim 27, wherein the nucleic acid molecules are about 10 kb to about 50 kb in length.

29. The method of any one of claims 22-28, wherein the bridging moiety comprises a nucleic acid aptamer that binds to the second binding site on the analyte and binds to the second terminus of the nucleic acid molecules via a single-stranded region complementary to the second terminus of the nucleic acid molecules.

30. The method of any one of claims 1-29, wherein the microparticles are denser than water.

31. The method of claim 30, wherein the microparticles are magnetic beads.

32. The method of any one of claims 1-31, wherein the microparticles are about 1 to about 10 microns in diameter.

33. The method of claim 32, wherein the microparticles are about 2 to about 5 microns in diameter.

34. The method of claim 33, wherein the microparticles are about 2.8 microns in diameter.

35. The method of any one of claims 1-34, wherein the analyte is a disease-specific biomarker.

36. The method of claim 35, wherein the biomarker is a protein biomarker.

37. The method of claim 36, wherein the protein biomarker is a tumor antigen.

38. The method of claim 37, wherein the tumor antigen is pro state- specific antigen.

39. The method of claim 36, wherein the protein biomarker is an antigen from an infectious agent.

40. The method of claim 39, wherein the infectious agent is a virus, bacterium, fungus, protozoan or nematode.

41. The method of claim 35, wherein the disease is cancer, an infectious disease, a cardiovascular disease, a renal disease, a neurological disorder, a neurodegenerative disease, a pulmonary disease, an autoimmune disease, an inflammatory disease, a hematologic disease, a liver disease, osteoarthritis or ischemia-reperfusion injury.

42. The method of any one of claims 1-41, wherein the sample is a serum, blood, plasma, urine, saliva or cerebral spinal fluid (CSF) sample.

43. A method for detection and/or quantitation of an analyte in a sample, comprising: providing a flow cell in which a flow cell surface presents a plurality of linear nucleic acid molecules each having a first terminus and a second terminus, wherein the first terminus of each nucleic acid molecule is bound to the flow cell surface;

providing microparticles conjugated to a capture antibody that specifically binds a first epitope of the analyte;

contacting the microparticles conjugated to the capture antibody with the sample under conditions sufficient to allow binding of the capture antibody to analyte present in the sample to form analyte-bound microparticles;

providing a detection antibody that specifically binds a second epitope of the analyte;

introducing the detection antibody into the flow cell under conditions sufficient to allow binding of the detection antibody to the second terminus of the nucleic acid molecules;

introducing the analyte-bound microparticles into the flow cell under conditions sufficient to allow binding of the analyte-bound microparticles to the detection antibody to form tethered immune complexes; and

detecting the tethered immune complexes, thereby detecting and/or quantitating the analyte in the sample.

44. A method for detection and/or quantitation of an analyte in a sample, comprising: providing a flow cell in which a flow cell surface provides a plurality of linear nucleic acid molecules each having a first terminus and a second terminus, wherein the first terminus of each nucleic acid molecule is bound to the flow cell surface and the second terminus of each nucleic acid molecule is bound to a first specific binding partner;

contacting the analyte-bound microparticles with a detection antibody that specifically binds a second epitope of the analyte to form an immune complex, wherein the detection antibody is conjugated to a second specific binding partner;

introducing the immune complex into the flow cell under conditions sufficient to allow binding of the first specific binding partner to the second specific binding partner to form a tethered immune complex; and

detecting the tethered immune complex, thereby detecting the analyte in the sample.

45. A method for detection and/or quantitation of an analyte in a sample, comprising: providing a flow cell in which a flow cell surface provides a plurality of linear nucleic acid molecules each having a first terminus and a second terminus, wherein the first terminus of each nucleic acid molecule is bound to the flow cell surface and the second terminus of each nucleic acid molecule is bound to a detection antibody that specifically binds a first epitope of the analyte; providing microparticles conjugated to a capture antibody that specifically binds a second epitope of the analyte;

introducing the analyte-bound microparticles into the flow cell under conditions sufficient to allow binding of the detection antibody to the second epitope of the analyte to form a tethered immune complex; and

46. A flow cell for detecting and/or quantitating an analyte in a sample, comprising: a flow cell surface;

a plurality of tether molecules, each having a first terminus and a second terminus, wherein the first terminus of each tether molecule is bound to the flow cell surface; and a bridging moiety bound to the second terminus of each tether molecule, wherein the bridging moiety specifically binds a first binding site of the analyte.

47. The flow cell of claim 46, wherein the bridging moiety comprises a detection antibody.

48. The flow cell of claim 47, wherein the detection antibody is directly bound to the second terminus of each tether molecule.

49. The flow cell of claim 47, wherein the detection antibody is indirectly bound to the second terminus of each tether molecule.

50. The flow cell of claim 49, wherein the second terminus of each tether molecule is bound to a first specific binding partner and the detection antibody is bound to a second specific binding partner, and binding of the first specific binding partner to the second specific binding partner indirectly binds the second terminus of each tether molecule to the detection antibody.

51. The flow cell of claim 50, wherein the first and second specific binding partners are selected from avidin and biotin.

52. The flow cell of any one of claims 46-51, wherein the tether molecules are bound to the flow cell surface at addressable locations.

53. The flow cell of any one of claims 46-52, wherein the tether molecules are linear polymeric molecules.

54. The flow cell of claim 53, wherein the linear polymeric molecules are nucleic acid molecules.

55. A kit comprising the flow cell of any one of claims 46-54, and a microparticle conjugated to a capture antibody that specifically binds a second binding site on the analyte.

56. A kit for detecting and/or quantitating an analyte in a sample, comprising:

a flow cell comprising a flow cell surface;

a plurality of tether molecules each having a first terminus and a second terminus, wherein the first terminus of each tether molecule is capable of binding to the flow cell surface;

microparticles conjugated to a binding moiety that specifically binds a first binding site on the analyte; and

a bridging moiety that specifically binds a second binding site on the analyte and binds the second terminus of the tether molecules.

57. The kit of claim 56, wherein the first terminus of each tether molecule is bound to the flow cell surface.

58. The kit of claim 56 or claim 57, wherein the second terminus of each tether molecule is bound to the bridging moiety.

59. The kit of claim 56 or claim 57, wherein the bridging moiety is bound to the second binding site on the analyte.

60. The kit of any one of claims 54-57, wherein the tether molecules are bound to the flow cell surface at addressable locations.

61. The kit of any one of claims 56-60, wherein the tether molecules are linear polymeric molecules.

62. The kit of claim 61, wherein the linear polymeric molecules are nucleic acid molecules.

63. The method, flow cell or kit of any of the preceding claims, wherein the linear nucleic acid molecules are spatially positioned to be detectable as individual molecules.