WO2024118685A1

WO2024118685A1 - Analyte detection and quantification by discrete enumeration of particle complexes

Info

Publication number: WO2024118685A1
Application number: PCT/US2023/081470
Authority: WO
Inventors: Yong Qin Chen; George C. BRITTAIN
Original assignee: Genotix Biotechnologies, Inc.
Priority date: 2022-11-28
Filing date: 2023-11-28
Publication date: 2024-06-06

Abstract

Described herein are systems and methods for the discrete detection and quanti fication of target analytes in a sample based oonn their binding by two or mmoorree particles to form analyte-linked particle complexes. The analyte-linked particle complexes can be di f ferentiated and enumerated versus unbound singlet particles, and from each other, based on the unique physical characteristics of the particles utili zed. In some embodiments of the current invention, this may involve one type of analyte, while in other embodiments it may involve multiple di f ferent types of analytes, either individually or in analyte complexes

Description

Title of Invention

ANALYTE DETECTION AND QUANTIFICATION BY DISCRETE ENUMERATION OF PARTICLE COMPLEXES

Technical Field

The present disclosure relates generally to the technical field of analyzing and quantifying biological analytes and, more particularly, to systems and methods therefor that provide improved accuracy and sensitivity, greater dynamic range, and simplified work flow.

Background Art

This patent application incorporates by reference Published Patent Cooperation Treaty ("PCT") International Application Number WO 2021/087006 Al as though fully set forth here.

High-sensitivity analytical measurements are fundamental to modern science and medicine. Protein and other small-particle measurements are also broadly essential to biomedical research and medical diagnostics. However, most biomedical assays lack the sensitivity and precision to accurately measure single analytes, particularly within complex sample matrices. Most protein and small-particle assays are formatted around bulk analyses, and often require signal amplification. While bulk analyses can and do provide useful information regarding the system overall, they generally always have fundamental limitations that prevent their ability to identify and quantify multiple characteristics of analytes or analyte subpopulations. This issue can be particularly problematic with complex samples, such as serum, urine or saliva, where the concentrations of target proteins and other analytes are extremely low, there are many non-target analytes present at concentrations that are several orders of magnitude higher than the target analyte, and yet the precision and accuracy of the data or diagnostic readout is essential.

Published Patent Cooperation Treaty ("PCT") International Application Number WO 2021/087006 Al, discloses a fully digital method for measuring analyte concentrations in biological samples at the single-molecule level. In essence, two types of particles coded with distinct physical characteristics, termed capture and detection particles, are separately surface-labeled with unique reagents capable of binding to epitopes at different locations of the same target analyte. These particles are mixed with a sample containing target analytes and incubated for a certain amount of time to form analyte-linked capture- and detection-particle complexes. The concentration of the target analyte is then directly correlated to the number of discretely enumerated particle complexes. Using well-characterized particles as detectors, the disclosed assay minimizes the noise associated with the signal amplification commonly practiced in other types of biological assays. Through the discrete enumeration of particle complexes and non-complexed singlet particles, the assay may also be performed without the wash steps, thus further improving the assay reproducibility. Despite these advantages, however, it was found that the incubation time required for the formation of capture- and detection-particle complexes in the disclosed assay is much longer than corresponding incubation times for other types of assays involving molecule-to-particle interactions. Clearly, such an extended incubation time significantly limits the application scope of the disclosed assay, particularly in clinical and other time-sensitive situations.

As a result, it would be advantageous to develop a system and method that maintain the merits of the particle-counting assay that was disclosed in the Published PCT Patent Application, yet with a significantly reduced assay incubation time.

Disclosure of Invention

This invention relates to systems and methods to reduce the incubation time for, and to improve the efficiency of, analyte-linked particle-complex formation in digital assays based on interactions between particles in biological solutions. In the primary embodiment of the current invention, discloses a system and a method for detecting and enumerating one type of target analytes in a sample at the single-analyte level. The system consists of two distinguished groups of particles: capture particles and detection particles. The capture particles are conjugated with an analyte-specific reagent that has a specific affinity to a certain binding site or epitope of the target analyte, while the detection particles are conjugated with another analyte-specific reagent that has a specific affinity to a secondary binding site or epitope of the same target analyte. The capture and detection particles are utilized by mixing them with a sample containing the target analyte, and then incubating the mixture for a certain amount of time sufficient to form measurable analyte-linked particle complexes. However, because the analytes are typically much smaller than the particles, their collision frequency with the particles is much higher than the particle-particle collision frequency. Consequently, while a significant number of analytes may be captured by the particles during the incubation, few particles with captured analyte will encounter their counterpart detection particle due to the relatively low particle-particle collision frequency. To increase the particle-particle collision frequency, the mixture is then subjected to a "particle-concentration enhancement" process.

In one of the embodiments of the current invention, the particle-concentration enhancement process may involve centrifugation. The centrifugal force, or angular velocity, the centrifugation time, and the total mixture volume are carefully adjusted such that all of the capture and detection particles are thrown by centrifugal force to near the wall of the sample container, without significant pellet formation. As a result, the local particle concentration, and thus the particle-particle collision frequency, will be significantly enhanced, enabling the rapid formation of analyte-linked capture- and detection-particle complexes. If the concentration of the target analyte in the sample mixture is significantly lower than the concentration of the capture and detection particles, then the complexes will be mostly particle doublets, consisting of a single capture particle linked to a single detection particle through a single target analyte. The relative number of the detected doublets, normalized to the total number of capture particles, should be a linear function of the analyte concentration. By analyzing the sample mixture in a manner that enables the discrete detection, differentiation and enumeration of the analyte-linked particle doublets versus the non-analyte-bound, henceforth referred to as unbound, singlet particles, the concentration of the target analyte in the sample may be accurately determined. At higher target-analyte concentrations, higher-order particle complexes containing multiple analytes may form. By analyzing the sample mixture in a manner that enables the discrete detection, differentiation and enumeration of all analyte-linked particle multiplets versus unbound singlet particles, such as with the use of a nonlinear least squares optimization algorithm, the concentration of the target analyte in the sample may again be accurately determined.

In an alternative embodiment of the current invention, a filtering device that contains a membrane or a filter with the pore size smaller than the particle diameters may be used to remove the majority of solvent in the sample-particle mixture, and, therefore, significantly increase the particle concentration and consequently speed up the particle-particle collision frequency. A filtering device could be a centrifugal device, a multi-well filter plate, or a custom-designed microfluidic device. The filtration of solvent may be achieved by gravitational flow, centrifugation, vacuum suction, or positive air pressure.

In another alternative embodiment of the current invention, the procedure is divided into multiple steps. As an example of a two-step process, the analyte is first bound to the capture particles and then the detection particles are added to form complexes with the analyte-bound capture particles. In step 1, the capture particles are mixed with the sample at a relatively low concentration and incubated for a period of time sufficient to capture a detectable fraction of the analyte within the sample, for example, 30 minutes or 1 hour. In step 2, a greater quantity of detection particles, for example, 10 times the concentration of the capture particles, are then added to the sample and capture-particle mixture. By using a lower capture-particle concentration in step 1, the average number of analytes captured per particle can be maximized and kept above the detection limit of the assay. Meanwhile, using a higher detection-particle concentration in step 2 significantly increases the collision frequency between the detection particles and the analyte- linked capture particles, therefore substantially reduce the necessary incubation time. The concentrations of both capture particles and detection particles may be varied and selected according to the system requirements. For example, a much lower concentration of capture particles may be used in the first step of the procedure to achieve better assay sensitivity. In some systems, more than two steps may be performed, such as when additional reagents or signaldevelopment steps are required. After completion of the intermediate steps, the detection particles are then added in a final step and the resulting mixture is incubated or processed as necessary.

In yet another embodiment of the current invention, the reaction buffers for eacnh step of the procedure may be different. For example, the reaction buffer in step 1 of the procedure may be different in chemical and biological composition from the reaction buffer in step 2, such that the buffer in step 1 is optimized to maximize the binding of analytes to the capture particles, while the buffer in step 2 is optimized to enhance the particle-particle interactions. A similar approach may be used for any procedural adaptation of the current invention, including but not limited to simple incubation, centrifugation, or filtration.

It should be pointed out that the analyte specificity of the reagents discussed in this invention should not be taken literally. The capture particles in the current invention may be conjugated to a collection of reagents, C, and the detection particles conjugated to another collection of reagents, D, with a portion of C targeting a group of analytes, A, and a portion of D targeting either A or a subgroup of A. It should also be noted here that C and D may be identical, or partially overlapping, or totally different.

In the preceding embodiments, as well as in the following embodiments, of the current invention, it should be apparent to those skilled in the art that the particular nomenclature for which a particle is referenced, such as capture or detection particle, is to simplify the explanation. In fact, both particles can be interchanged in both form and identity, and the system can instead be considered to comprise two or more groups of particles (e.g., Group 1, Group 2, etc.) that are capable of forming analyte-linked particle complexes that are discretely distinguishable from unbound singlet particles and from each other.

In another embodiment of the current invention, a system and a method are disclosed for multiplexed assays to simultaneously detect and enumerate multiple types of target analytes in a sample at the single-analyte level. The system comprises two distinct groups of particles: capture particles and detection particles, with the capture particles further divided into subgroups, wherein each subgroup of capture particles is uniquely labeled with certain physical characteristics. The capture particles are each conjugated with an analyte-specific reagent that has a specific affinity to a certain binding site or epitope of a particular type of target analyte. The detection particles are each conjugated with one of a different set of analyte-specific reagents that has a specific affinity to a secondary binding site or epitope of their respective type of target analyte. When a sample containing target analytes is mixed with the capture and detection particles, multiple groups of analyte-linked particle complexes may form, each associated with one type of analyte and the corresponding detection particles and subgroup of capture particles. By differentiating and grouping according to the capture-particle labels, multiple types of analytes in a sample can be simultaneously analyzed, with each type of analyte analyzed in the same way as proposed in the primary embodiment of the current invention. For those skilled in the art, it should be apparent that the multiplexed-assay embodiment of the current invention may also be implemented by differentially labeling the detection particles or both the capture and detection particles.

In a related embodiment of the current invention, the previously disclosed multiplexed-assay embodiment may be combined with one or more conventional analog assays. For example, one subgroup of analyte-specific detection particles

- corresponding to one subgroup of labeled capture particles

- may be replaced by analyte-specific molecular probes, such as the analyte-specific detection reagents directly conjugated with fluorescent molecules. As a result, the concentration of the corresponding analyte will be extracted from the mean fluorescence intensity of the analyte-linked particle-and- molecular-probe sandwiches, instead of from the enumeration of analyte-linked particle complexes. One possible application of such a combined multiplexed assay is to simultaneously measure, in one sample, low-abundance analytes using analyte- linked particle complexes, and high-abundance analytes using analyte-linked particle-and-molecular-probe sandwiches. It should be apparent to those skilled in the art that this example can be extended to assays that combine multiple types of particle-con ugated and molecular detection reagents.

In a different embodiment of the current invention, a system and a method are disclosed for studying analyte-analyte interactions in a sample at the single-analyte level. The system consists of two distinct groups of particles: capture particles and detection particles. The capture particles are conjugated with an analyte-specific reagent that has a specific affinity to a certain binding site or epitope of a primary target analyte. The detection particles are conjugated with an analyte-specific reagent that has a specific affinity to a certain binding site or epitope of a secondary target analyte. When a sample containing the two analytes is mixed with the capture and detection particles, analyte-linked particle complexes may form due to interaction between the two different target analytes. The analyte complex may, therefore, be analyzed in the same way as proposed in the primary embodiment of the current invention. In some embodiments of the current invention, another group of detection particles specific to the primary target analyte may also be introduced to simultaneously measure its concentration, and consequently the fractional occupancy. In other embodiments of the current invention, one or more of the target analytes may be directly or indirectly bound or conjugated to the capture and/or detection particles without intermediating capture and/or detection reagents, or the secondary target analyte may actually be a capture and/or detection reagent. It should be apparent to those skilled in the art that the analyte-analyte-interaction-assay embodiment of the current invention may also be multiplexed, as discussed in the previous multiplexed-assay embodiments of the current invention.

While the disclosed embodiments of the current invention focus on measuring the concentration of analytes or analyte complexes through the discrete detection and enumeration of analyte-linked particle complexes, it should be apparent to those skilled in the art that they can be easily implemented as kinetics and/or dynamics assays to study analyte-reagent or analyte-analyte interactions, including changes due to the introduction of a modulating agent, such as drugs, pharmaceuticals, proteins, kinases, transcription factors, peptides, sugars, oligosaccharides, polysaccharides, nucleic acids, lipids, detergents, hormones, growth factors, cytokines, chemokines, activators, inhibitors, small-molecule activators, small-molecule inhibitors, other modulators, and/or combinations or complexes thereof. Such assays may be used for optimizing pharmaceutical development, determining drug efficacies and selection, elucidating on-target versus off- target responses, identifying effective concentrations in different experimental and physiological conditions, elucidating pharmacodynamics, mapping signaling and metabolic pathways, optimizing antibody manufacturing, and many other research and/or development applications.

The capture and detection particles, as well as the analyte-linked particle complexes discussed in the embodiments of the current invention, may be distinctively characterized using any particle-counting techniques that leverage their distinguishable size, mass, chemical, optical, electrical, magnetic, radioisotopic, and/or biological properties. For example, particles with unique optical properties, such as light scattering, absorption and/or fluorescence, may be distinctively counted using multi-parameter particle counters, such as flow cytometers, or imaging or laser-scanning microscopes. Particles with unique sizes may be distinctively counted using impedance-based particle counters. Alternatively, any measurement technique may be utilized that enables the extraction, from a sample, of the distinctive counts of the capture and detection particles, as well as the analyte-linked particle complexes discussed in the various embodiments of the current invention.

In these embodiments of the current invention, analyte refers to any test molecule or particle of interest, including, but not limited to: proteins, kinases, transcription factors, antibodies, receptors, peptides, cytokines, chemokines, sugars, oligosaccharides, polysaccharides, DNA, RNA, oligonucleotides, polynucleotides, nucleotide complexes, single-stranded nucleic acid sequences, double-stranded nucleic acid sequences, natural polymers, synthetic polymers, lipids, detergents, hormones, growth factors, micelles, liposomes, lipoproteins, extracellular vesicles, exosomes, oncosomes, viruses, viruslike particles, cells, cell fragments, natural particles, synthetic particles, synthetic compounds, plant-derived compounds, animal-derived compounds, chemicals, drugs, pharmaceuticals, activators, inhibitors, small-molecule activators, small-molecule inhibitors, modulators, and/or combinations or complexes thereof. In some embodiments of the current invention, the analytes may be targeted to bind to the capture and/or detection particles by cognate capture and/or detection reagents that are conjugated to the particle surface, while in other embodiments the analytes may be directly or indirectly bound or conjugated to the capture and/or detection particles without using intermediating capture and/or detection reagents, such as by use of covalent bonding or affinity tags. The various embodiments of the current invention are not intended to be limited to any particular analyte.

In these embodiments of the current invention, the capture and detection reagents may be anything that binds to a site or epitope of the target analytes, including, but not limited to: antibodies, binding proteins, peptides, polypeptides, protein complexes, sugars, oligosaccharides, polysaccharides, DNA, RNA, oligonucleotides, polynucleotides, nucleotide complexes, single-stranded nucleic acid sequences, double-stranded nucleic acid sequences, aptamers, natural polymers, synthetic polymers, pharmaceuticals, drugs, lipids, detergents, micelles, liposomes, lipoproteins, extracellular vesicles, exosomes, oncosomes, viruses, virus-like particles, cells, cell fragments, chemicals, etc. In some embodiments of the current invention, one or more of the capture and/or detection reagents may function as target analytes, particularly in analyte- reagent- and/or analyte-analyte-interaction assays. In some embodiments of the current invention, the binding may be specific, and in other embodiments the binding may be intentionally nonspecific. In some embodiments of the current invention, the capture and/or detection reagents may be directly conjugated to the capture and/or detection particles, while in other embodiments the capture and/or detection reagents may be indirectly conjugated to the capture and/or detection particles, such as by use of affinity tags. The various embodiments of the current invention are not intended to be limited to any particular capture or detection reagent.

In these embodiments of the current invention, affinity tags refer to any molecule or element with affinity to, or that can be identified and more generally targeted by, a second molecule or binding partner, and can be used to bring two components together into a complex when differentially conjugated to the pair of components. Such affinity tags include, but are not limited to: biotin, streptavidin, avidin, neutravidin, hemagglutinin, poly-histidine, maltose-binding protein, myc, glutathione-s-transferase, FLAG, protein A, protein G, protein L, DNA, RNA, oligonucleotides, polynucleotides, single-stranded nucleic acid sequences, double-stranded nucleic acid sequences, etc. In other embodiments of the current invention, if there is a difference in species origin for a particular pair of capture and/or detection reagents, or some general difference that allows for differentiation between the reagents, for example mouse vs. rabbit antibodies, IgG vs. IgM, IgGl vs. IgG3, etc., then these differences may also be targeted by species-, class- or isotype-specific targeting reagents, such as an anti-mouse- IgG3-specific antibody, conjugated directly or indirectly to the capture and/or detection particles, functioning similar to affinity tags. In this context, and for the embodiments of the current invention, species-, class- and isotype-specific targeting reagents and/or antibodies are included in the definition of affinity tags and/or reagents. In some embodiments of the current invention, affinity tags may be used to conjugate the capture and/or detection reagents to the capture particles, the detection particles, both particles, or neither particle. In other embodiments of the current invention, affinity tags may be used to specifically or non- specifically bind the analyte directly to the capture and/or detection particles, in which scenario a particular capture and/or detection reagent may not be necessary. The various embodiments of the current invention are not intended to be limited to any particular affinity tag or reagent.

Finally, in these embodiments of the current invention, the capture and detection particles may be composed of any inorganic, organic or biological material, or composite of materials, including, but not limited to: polystyrene, silica, glass, metals, magnets, proteins, peptides, polypeptides, protein complexes, sugars, oligosaccharides, polysaccharides, DNA, RNA, oligonucleotides, polynucleotides, nucleotide complexes, single-stranded nucleic acid sequences, doublestranded nucleic acid sequences, aptamers, natural polymers, synthetic polymers, lipids, detergents, micelles, liposomes, lipoproteins, extracellular vesicles, exosomes, oncosomes, viruses, virus-like particles, cells, cell fragments, etc. The capture and detection particles may also be labeled with certain unique physical, chemical and/or biological characteristics. Furthermore, the capture and detection particles may be of any size, shape, or material uniformity, as long as they can be discretely detected and enumerated. In some embodiments of the current invention, the particles will be Inm to lOGjam in diameter. In other embodiments of the current invention, the particles will be 2nm to lOjim in diameter. In yet other embodiments of the current invention, the particles will be 5nm to 2|um in diameter. In the preferred embodiment of the current invention, the particles will be lOnm to Ijim in diameter.

Brief Description of Drawings

FIG. 1 is a schematic representing the primary embodiment of the analyte-linked particle complexes described in the current invention.

FIG. 2 are schematics representing various particles and analyte-linked particle complexes that may form in a singleanalyte system.

FIG. 3 are schematics representing analyte-linked particle complexes that may form in a multiplexed assay of the current invention.

FIG. 4 shows an analyte complex formed between two moieties, with one bonded to a capture particle and the other to a detection particle.

FIG. 5 shows some possible data that may be extracted from a system of the current invention based on the detection of single-particle light scatter and fluorescence.

FIG. 6 shows some possible data that may be extracted from a system of the current invention based on the detection of single-particle fluorescence, using fluorescently labeled capture and detection particles.

FIG. 7 shows some empirical 2D-fluorescence-histogram and enumeration results obtained from a particular implementation of the current invention, using fluorescent capture and detection particles at similar concentrations to analyze Low, Intermediate, and High concentrations of human PSA.

FIG. 8 shows examples of a gating grid being used to discretely analyze the various analyte-linked particle multiplets formed in 2 fluorescence dimensions, as obtained from a particular implementation of the current invention, using fluorescent capture and detection particles at similar concentrations to analyze Low, Intermediate, and High concentrations of human PSA.

FIG. 9 shows some possible data that may be extracted from a multiplexed assay of the current invention based on the detection of single-particle light scatter and fluorescence.

FIG. 10 illustrates some differences between the data that may be extracted from the current invention and from some prior art, comparing the enumeration of particles and analyte-linked particle complexes versus analog signal detection.

FIG. 11 illustrates the different linear ranges of analyte-linked particle doublets vs. higher-order analyte- linked particle complexes, which are shifted to higher analyte concentration ranges.

FIG. 12 illustrates the major difference between the current invention and the prior art, comparing the use of particle-con ugated reagents versus molecular reagents.

FIG. 13 illustrates the linear dynamic range of the proposed digital particle assay of the current invention. As shown, after appropriately taking into account of the effects of steric hindrance, the DAP, or detected analytes per particle, is directly proportional to the total analyte concentration in the sample across the entire measurement range, from femtograms per milliliter to nanograms per milliliter.

Best Mode for Carrying Out the Invention

The long incubation time required for the formation of analyte-linked particle complexes may be explained by the diffusion theory of particles in liquid. In order to form a capture- and detection-particle complex, the two particles must be in proximity of each other. In other words, the rate of complex formation will be limited by the collision frequency. For simplicity, we may assume that the capture particles and the detection particles are of a similar size. According to diffusion theory, the collision frequency for any given particle with the other particles in a mixture at concentration, C, is:

where k_B is the Boltzmann constant, T the temperature, and ju. the viscosity of the liquid. According to the equation, the collision frequency is 4.8 collisions per hour at a particle concentration of 100,000 per microliter in room temperature water. It may be tempting to significantly increase the particle concentrations to increase the collision frequency, however the sensitivity of a particle-counting assay is ultimately determined by the capability of the system to differentiate the analyte-linked particle complexes from the false positives due to non-specific binding. At an analyte concentration of 1 femtogram per milliliter, there may be only about 20 analyte molecules per microliter. At 100,000 particles per microliter, even if all of the analytes are captured, the positive rate is only 0.0002. At such a small positive rate, any effort to increase the collision frequency by further increasing the particle concentration will risk increasing the false-positive counts.

In the current invention, systems and methods are proposed for the discrete detection and quantification of individual analytes in a sample. More specifically, this invention relates to the binding of target analytes by two particles, a primary analyte-specific capture particle and a secondary analyte-specific detection particle, forming analyte-linked particle complexes. In some embodiments of the current invention, one type of analyte may be targeted by one set of capture and detection particles, while in other embodiments multiple types of analytes may be simultaneously targeted using different subgroups of capture and/or detection particles, each uniquely labeled with certain distinguishable physical characteristics. According to one embodiment of the current invention, individual target analytes are first bound by a capture particle, and then bound by a detection particle. In some embodiments of the current invention, the capture and detection particles may be added to the sample simultaneously, while in other embodiments the sample may be added to a solution containing capture and detection particles in a sequential process. It should be clear to those skilled in the art that no specific sequence or order in mixing a sample with the particles is critical to the current invention. Analysis of a sample mixture in a manner that enables the discrete detection, differentiation and enumeration of the analyte- linked particle complexes versus unbound singlet particles, together with differentiating any particle ssubgroups, can then be used to accurately determine the concentration of one or more types of target analytes in a sample. In the preferred embodiment of the current invention, the particles and particle complexes will be analyzed by a multi-parameter particle counter, such as a flow cytometer, or an imaging or laserscanning microscope. Analyte-linked particle doublets represent the discrete detection of a single analyte, which can be directly counted to give the total number of analytes per volume analyzed. Higher-order particle multiplets discretely shift the fluorescence, light-scatter, and/or other optical signature to greater intensities, representing the discrete inclusion of additional analytes. The total number of analytes counted per volume analyzed can, therefore, be derived from the summation of the number of each type of particle complex multiplied by the corresponding number of analytes contained in each complex. In other words, if N_a is the total number of analytes detected per volume analyzed, and N_m. the number of detected particle complexes containing m analytes, then:

N_α = Σ_m (m x N_m) (1)

It should be clear to those skilled in the art that by normalizing, or dividing both sides of Equation (1) by the total number of detected capture particles in a given experiment, the results will be independent of the volume analyzed, and thus an explicit measure of the analyte concentration. Denoting the normalized total number of detected analytes per capture particle, DAP, we then have:

DAP = _m(m x n_m)) (2) where is the detected particle complexes

containing m analytes, N_m, normalized to the total number of detected capture particles, N_cap. Steric hindrance may slow down the formation of higher-order particle complexes, Such steric-hindrance effects may be included in Equation (2) using the steric-hindrance parameters, S_m

In one embodiment of the current invention, the steric- hindrance parameters, S_m, may be simplified to 2 parameters: one related to the onset, and the other to the magnitude of hindrance. In other words, S_m is a step function that equals 1 if m is less than the onset parameter; otherwise it is equal to a fixed value greater than 1. In another embodiment of the current invention, the steric hindrance may be accounted for by a sigmoid function, such that it is near 1 for small m, and gradually increases as m increases. It should be apparent to anyone skilled in the art that other types of functions with similar mathematical properties may also be used to describe the steric effect. For any given function, the exact values of the steric-hindrance parameters may then be optimized by minimizing the root-mean-square differences between DAP and a linear response function for the analyte concentration. FIG. 13 displays a typical example obtained this way with an optimized step function. As shown, with consideration of the steric hindrance, the DAP is linear to the analyte concentration across the entire measurement range, from femtograms per milliliter to nanograms per milliliter.

In the primary embodiment of the current invention, the system and method pertain to the discrete detection and quantification of one type of analyte within a liquid sample matrix. As shown in Figure 1, this is accomplished by binding the analyte 101 to a primary capture particle 102 and a secondary detection particle 103, forming an analyte-linked particle doublet 104. The analyte specificity of the capture particle 102 is provided by the specificity of the analytespecific capture reagent 105, which can be conjugated to the particle by a direct or indirect bond 106, such as by a covalent bond or an affinity tag, including, but not limited to: biotin, streptavidin, hemagglutinin, poly-histidine, glutathione-s-transferase, etc. Similarly, the specificity of the detection particle 103 is provided by the specificity of the analyte-specific detection reagent 107, which can be conjugated to the particle by a direct or indirect bond 106. A representative capture or detection reagent for protein analytes would be an analyte-specific antibody, which has the characteristic Y-shape depicted in multiple FIG.s of the current invention; however, the system is not intended to be limited to any particular type of capture or detection reagent. Fundamentally, the capture or detection reagents can be any molecules or entities that bind to the target analytes, specifically or nonspecifically. The capture and detection reagents would preferably bind to the analytes at two different binding sites, shown as 108 and 109 in FIG. 1, although in some cases the experimental objective may be to analyze the competition for two or more binding reagents to a particular binding site. In some embodiments of the current invention, the capture and/or detection particles may be each targeted to bind to one specific type of analyte, while in other embodiments the capture and/or detection particles may be intended to bind to a mixture of different types of analytes, specifically or nonspecifically. In the latter case, the specificity of the assay will depend upon the specificity of the detection reagent or reagents.

In the FIG.s of the current invention, the capture 102 and detection particles 103 are each depicted with only one or several capture 105 or detection reagents 107 conjugated to their surface in order to simplify the explanation of the particular embodiment of the current invention. However, in reality, there could be hundreds or thousands of capture or detection reagents conjugated to each particle, with no particular maximum or minimum number intended. The precise number will depend on the size of the capture or detection reagents; the size and, thus, surface area of the capture or detection particles; the concentration of the capture or detection reagents used for particle manufacturing; the addition of alternative reagents or materials into the manufacturing reaction mixture in order to alter the specific molar ratio of the capture or detection reagents and, thus, compete with them for conjugation to the surface of the capture or detection particles; and other details of the specific manufacturing protocols and procedures. For those skilled in the art, these constraints would be apparent, and it would be clear that the particles in these FIG.s are simplified for the purpose of explanation. While it may be theoretically possible to conjugate particles with only one copy of a particular reagent, this would actually be a big limitation to their functionality in most assays, as it would significantly decrease the probability that an analyte would appropriately contact a capture or detection particle at the location of a capture or detection reagent during any particular collision that occurs between the analytes and the particles in a sample mixture in any given time frame. In contrast, a higher reagent density on the particles means a higher local reagent concentration, which will favor the analyte-reagent bond formation in an equilibrium reaction comparable to a molecular reagent of equivalent concentration that is evenly distributed in bulk.

While the primary embodiment of the current invention describes the formation of analyte-linked particle doublets, each comprising a pair of capture and detection particles bonded together by a single analyte, FIG. 2 illustrates a variety of additional possibilities that may form or remain in the reaction mixture depending on the concentration ratio of the analytes to the particles. First, as shown in FIG. 2A, if the capture 102 and/or detection particles 103 do not bind to an analyte 101, particularly if the concentration of the analyte 101 is significantly lower than the concentrations of the capture and/or detection particles, then there will be residual unbound singlet capture 102 and/or detection particles 103 in the sample mixture. At low analyte concentrations, with sufficient analyte-binding affinity and an excess capture and detection particles, almost all of the target analytes will be present in the form of analyte-linked particle doublets 104, as shown in FIG. 2B. When the concentration of the target analytes increases, higher-order analyte-linked particle multiplets will form. FIG. 2C illustrates an analyte-linked particle triplet 201; FIG. 2D an analyte-linked particle quadruplet 202; and, FIG. 2E an analyte-linked particle quintuplet 203. For those skilled in the art, it should be apparent that other types of analyte-linked particle complexes are possible as well. Using a multi-parameter particle counter capable of resolving these particles and analyte-linked particle complexes from each other, the number of each type of the particles and particle complexes can be easily measured, and the total number of target analytes contained in the sample can be readily extracted from such a measurement, as discussed previously.

Another embodiment of the current invention would be a multiplexed assay simultaneously targeting multiple different analytes in a complex sample mixture. As shown in FIG. 3, three different types of analytes 101-1, 101-2, and 101-3 in a sample are bonded separately to their corresponding capture particles 102-1, 102-2, or 102-3 and detection particles 103-1, 103-2, or 103-3, each one conjugated to a specific capture reagent 105-1, 105-2, or 105-3 or detection reagent 107-1, 107- 2, or 107-3 with specificity to a primary 108-1, 108-2, or 108- 3 or secondary binding site 109-1, 109-2, or 109-3 on their respective target analyte 101-1, 101-2, or 101-3. Each capture particle 102-1, 102-2, or 102-3, representing a subgroup of capture particles, is labeled with a unique hash, symbolizing some unique physical characteristics and/or labels, such as size and/or optical properties (e.g., intensities and/or wavelengths of absorption, fluorescence and/or light scattering). A multi-parameter particle counter, capable of resolving the particles according to their corresponding labels, could then be used to differentiate and group the particles and analyte-linked particle complexes into subgroups, each corresponding to a specific type of target analyte in the sample mixture. Consequently, each subgroup of capture particles and the corresponding analyte-linked particle complexes can be simultaneously analyzed in the same way as proposed in the primary embodiment of the current invention, enabling the simultaneous measurement of the concentrations of all three types of target analytes.

It should be apparent to those skilled in the art that the discussion revolving around FIG. 3 is for illustrative purpose only. The multiplexed-assay embodiment of the current invention is not intended to be limited to any particular format of multiplexed detection, any type of target analyte, or any particular number of different target analytes that are simultaneously analyzed. In some embodiments, this invention may be used to analyze 1 to 1000 different target analytes simultaneously. In other embodiments, this invention may be used to analyze 1 to 100 different target analytes simultaneously. In the preferred embodiment, this invention would be used to analyze 1 to 50 different target analytes simultaneously. Increasing the number of multiplexed target analytes may reduce the throughput and dynamic range proportionally, but these can both be counterbalanced and increased by improving the throughput of the instrumentation, or by increasing the acquisition time.

Another embodiment of the current invention is aimed at the study of analyte-analyte interactions. FIG. 4 illustrates one exemplary embodiment of the current invention wherein the capture particle 102 is targeted to a binding site 108-1 on a primary analyte 101-1, and the detection particle 103 is ttargeted to a binding site 108-2 on a secondary analyte 101-2, with the primary analyte 101-1 and secondary analyte 101-2 bound together in an analyte complex 401. A modulating agent 402 is also included in the FIG. to indicate that the interaction between the two moieties 101-1 and 101-2 of the analyte complex 401 may be actively modulated, inhibited or enhanced. In the present embodiment of the current invention, the observation of capture-and-detection-particle complexes 403 represents the binding of the two analytes 101-1 and 101-2 that form the analyte complex 401, and the number of complexes 401 can be enumerated similar to as described in the primary embodiment of the current invention. Further, using a variation of the multiplexed assays proposed in the previous embodiments of the current invention, detection particles labeled with different physical characteristics may be introduced to target the primary analyte 101-1 in order to simultaneously measure its concentration. Such a multiplexed assay would enable the accurate measurement of the fractional occupancy of the secondary analyte 101-2 bound to the primary analyte 101-1 at the single-analyte level. In some cases, the binding of multiple capture and/or detection particles to an analyte complex may be used to identify the presence of multiple copies of the same analyte within the complex. It should be apparent to those skilled in the art that the proposed embodiment of the current invention can be used to study, at the single-analyte level, the binding affinities and/or kinetics of two or more analytes that bind together and form analyte complexes, for example, due to the introduction of particular pharmaceuticals, drugs, or other associationmodulating molecules or particles of interest. Such assays may be used to optimize pharmaceutical development, determine drug efficacies and selection, elucidate on-target versus off-target responses, identify effective concentrations in different experimental and physiological conditions, elucidate pharmacodynamics, map signaling and metabolic pathways, optimize antibody manufacturing, and many other research and/or development applications. These examples are not exhaustive, and the current invention is not intended to be limited to any particular format of analyte-complex detection or analysis, any type of target analyte, or any particular number of different target analytes that are simultaneously analyzed.

The unbound particles and analyte-linked particle complexes prepared using the systems and methods of the current invention can be analyzed in any manner that enables the discrete detection, differentiation and enumeration of particle complexes versus singlet particles. In some embodiments of the current invention, these methods may include, but are not limited to: imaging or laser-scanning microscopy, resistive- pulse sensing, and flow cytometry. In other embodiments of the current invention, the combination of signals and/or differences in physical properties produced by the proximity of the capture and detection particles in analyte-linked particle complexes can allow for bulk analyses. For example, the size difference between unbound particles and the various analyte-linked particle complexes may be differentiated using centrifuges or size-selective filters, or even dynamic light scattering or nanoparticle tracking analysis. Energy transfer between the capture and detection particles in analyte-linked particle complexes may be interrogated using spectroscopic methods. The list is certainly not intended to be exhaustive. Bulk analyses, however, will not provide the precision and accuracy of particle-by-particle counting and analyses. In the preferred embodiment of the current invention, the particles and analyte-linked particle complexes will be discretely detected, differentiated and enumerated using a multi-parameter particle counter similar to a flow cytometer, or an imaging or laser-scanning microscope.

FIG. 5 shows some possible data that may be extracted from a system of the current invention based on the detection of single-particle light scatter and fluorescence, using a multiparameter particle counter. In this case, the capture particles are fluorescently labeled, while the detection particles are non-fluorescent. Consequently, in FIG. 5A, the analyte-linked particle complexes 104, 201, 202, and 203 and unbound capture particles 102 are clearly differentiated from unbound detection particles 103 according to their difference in fluorescence intensity. As shown, the fluorescence intensities of the unbound capture particles 102 and all analyte-linked particle complexes 104, 201, 202, and 203 are above the fluorescence threshold 501, while the fluorescence intensity of the unbound detection particles 103 is below the threshold 501. In addition, the formation of analyte-linked capture-and-detection-particle complexes causes a discrete shift in the light-scatter intensity, dependent on the number of particles bound. FIG. 5B shows a possible histogram plot. After exclusion of the unbound detection particles 103, which are below the fluorescence threshold 501, the number of unbound capture particles 102 and analyte-linked particle complexes 104, 201, 202, and 203, containing correspondingly 0, 1, 2, 3 or 4 detection particles, and thus analytes, can be easily resolved and enumerated. FIG. 6 shows some possible data that may be extracted from a system of the current invention based on the detection of multi-color single-particle fluorescence, using capture particles that are labeled with a fluorescent color (Fluorescence 1) different from that of detection particles (Fluorescence 2). Consequently, as shown schematically in FIG. 6A, the unbound capture particles 102 and analyte-linked particle complexes 104, 201, 202, and 203 can be clearly differentiated from unbound detection particles 103 according to their difference in the intensity of Fluorescence 1. Meanwhile the formation of analyte-linked capture-and- detection-particle complexes causes a discrete shift in the intensity of Fluorescence 2, dependent on the number of particles bound. FIG. 6B shows a possible histogram plot. After exclusion of unbound detection particles 103 with a fluorescence intensity below the threshold 501 in the Fluorescence-1 channel, the number of unbound capture particles 102 and analyte-linked particle complexes 104, 201, 202, or 203 containing 0, 1, 2, 3 or 4 detection particles, and thus analytes, can be easily resolved and enumerated based on their distinct intensities in Fluorescence 2. Again, FIG. 5 and FIG. 6 are for illustration purposes only. As stated previously, the types of analyte-linked particle complexes observed in an actual embodiment of the current invention would depend on the relative concentrations of the analytes to the particles.

If both the capture and detection particles are fluorescently labeled, then the resulting analyte-linked particle complexes may produce a fractal pattern in the two fluorescent dimensions that are used for labeling. FIG. 7 demonstrates some actual preliminary results displaying this phenomena when labeling human prostate-specific antigen (PSA). FIG. 7A, FIG. 7B and FIG. 7C represent Low, Intermediate, and High concentrations of PSA, respectively. As seen in FIG. 7A, when the concentration of PSA was low, the capture 102 and detection particles 103 formed analyte-linked particle doublets 104 when bound to PSA, as normal. However, as the concentration of PSA was increased in FIG. 7B and FIG. 7C, higher-order analyte-linked particle complexes were discretely resolved in both of the fluorescence dimensions used. In this case, the analyte-linked particle multiplets in the 2 dimensions, such as 201-1 and 201-2, or 202-1, 202-2 and 202-3, comprise different particle combinations, yet the number of analytes bound per analyte-linked particle complex of the same particle multiple would be equivalent regardless of the particular bead combination. For example, whether an analyte- linked particle triplet is composed of lx Particle 1 + 2x Particle 2 (201-1), or vice versa (201-2), both of the analyte- linked particle triplets represent 2 captured analytes. In the magnified inset of FIG. 7C, 202-1, 202-2 and 202-3 each represent analyte-linked particle quadruplets, 203-1 and 203-2 represent two potential combinations of analyte-linked particle quintuplets, and 701 represents one potential combination of an analyte-linked particle sextuplet. These specific particle complexes are only provided as examples and are not intended to be an exhaustive list of the many combinations that can be formed. Because all of the populations are discretely resolved, each population can be directly enumerated, which can then be used to calculate the analyte concentration either mathematically, statistically, or by comparison to a standard curve. Furthermore, the natural generation of multiple data points at each concentration by the current invention, unlike many conventional assays that generate only one data point at a given analyte concentration, enables more accurate measurements. An empirical example of the gating to enumerate the various populations can be seen in FIG. 8, where FIG. 8A, FIG. 8B, FIG. 8C and FIG. 8D represent Control, Low, Intermediate, and High PSA concentrations, respectively. In these histograms, the analyte-linked particle doublets are located at the bottom-left corner of the grid, while the higher-order analyte-linked particle complexes increase in their particle multiple as they progress upward and to the right.

Similar data analyses may be applied to multiplexed assays of the current invention. FIG. 9 demonstrates one exemplary embodiment of the current invention, where the fluorescently labeled capture particles are used to simultaneously identify and analyze multiple types of analytes. Specifically, as shown in FIG. 9A, the capture particles are divided into subgroups Al, A2, ..., D4 and D5. Each subgroup, Al, A2, ..., D4 or D5, of capture particles is labeled with a unique combination of fluorescent dyes, Fluorescence 1 and Fluorescence 2, with distinct fluorescence intensities, and is conjugated to a specific reagent targeting a specific type of analyte. For example, the capture particles in subgroup DI have the lowest intensity in Fluorescence 1 and the lowest intensity in Fluorescence 2; the capture particles in subgroup A5 have the highest intensity in both Fluorescence 1 and Fluorescence 2; and so on. FIG. 9B, FIG. 9C and FIG. 9D show hypothetically that, once the unbound particles and analyte-linked particle complexes are differentiated and grouped into subgroups according to the fluorescent labels of the capture particles, as illustrated in FIG. 9A, then, for each type of analyte, the number of analyte-linked particle complexes containing 0, 1, 2 or more detection particles (102, 104, 201, 202 and 203), and thus analytes, can be easily resolved and enumerated based on the corresponding histograms of light-scatter intensity. The system discussed in this exemplary embodiment is not intended to be limited to any particular number or variety of fluorophores or spectral labels. In some embodiments of the current invention, more than two fluorescence channels may be used for analyte labels. In other embodiments, a variety of fluorescence, scatter and/or other optical or spectral properties may be used as analyte labels. Further, the detection particles may also be fluorescently labeled, for example, using colors different from the capture-particle labels.

The ability to discretely count analyte-linked particle complexes and unbound particles is one major characteristic that differentiates the current invention from prior art that ultimately rely on analog signal analyses. FIG. 10 highlights schematically the difference in data that may be extracted from experiments using the two approaches. FIG. 10A, FIG. 10B and FIG. IOC are from the current invention, while FIG. 10D, FIG. 10E and FIG. 10F are an analog system from the prior art. The analyte concentrations increase from left to right in both cases. As shown, for the current invention, the number of analyte-linked capture-and-detection-particle doublets (104 in FIG. 10A) increases as the concentration of analyte increases; then, at intermediate analyte concentrations, the number of analyte-linked particle triplets (201 in FIG. 10B) starts to increase; and finally, at higher analyte concentrations, higher-order analyte-linked particle multiplets are formed (FIG. 10C). However, in all cases, from FIG. 10A to FIG. 10C, the signals (104, 201, 202, and 203) are always clearly resolved from the background unbound particles 102 and 103. On the other hand, for a prior art that relies on the measurement of average intensities to extract analyte concentrations, the signal (1001) is poorly resolved from the background (1002) at lower analyte concentrations (FIG. 10D) and saturates at higher concentrations (FIG. 10F).

Furthermore, as demonstrated in FIG. 11, as the analyte concentration increases, the higher-order analyte-linked particle complexes also have a linear range that is shifted from the initial linear range of the analyte-linked particle doublets. In FIG. 11, analyte-linked particle doublets 104 have an initial linear range that spans several decades, while a particular intermediate-sized analyte-linked particle complex 1101 begins to form at analyte concentrations that are several decades higher and has a linear range that also extends several decades higher than that of the analyte-linked particle doublets 104. Likewise, a particular large-sized analyte- linked particle complex 1102 only begins to form at analyte concentrations that are several decades higher than that required for the intermediate analyte-linked particle complexes 1101, and has a linear range that extends even higher. Because the data that result from an assay of this invention produce many discrete data points, each with their own characteristic population distributions and aggregation behaviors, these data points provide additional characteristics for determining analyte concentrations, and improve the accuracy and reliability of the assay. For example, one common problem with immunoassays that generate only one data point at a given analyte concentration, once they reach the saturation point for the assay, is the hook or prozone effect, where the addition of further analyte inhibits and actually reduces proper antibody binding or complex formation. This leads to uncertainty as to whether any individual result is located on the increasing or decreasing side of the assay signal vs. concentration curve, and requires either an additional step after the assay is complete, where more sample is added and it is then reanalyzed, or additional dilution points to be read in order to determine if the signal increases or decreases for the additional data point (s). As demonstrated in FIG. 11, with our primary focus being on the analyte-linked particle doublets in the discrete particle-complex range, higher concentrations simply shift the linear range to proximal higher-order analyte- linked particle complexes, so that multiple data points can be separately quantified and the combination used to accurately determine the analyte concentration.

Unlike the prior art that are based on analog signals and molecular reagents, in the current invention, each analyte- linked capture-and-detection-particle doublet that is enumerated represents a single analyte or analyte complex. The dynamic range of an assay of the current invention is, thus, directly proportional to the total number of particles counted during an experiment. For example, if 10⁶ particles are analyzed, then the nominal dynamic range would be 6 decades. This would only take minutes to acquire on a basic multiparameter particle counter, such as a flow cytometer, or an imaging or laser-scanning microscope. In practice, when higher-order particle complexes are taken into consideration, the range could be extended by another decade or more even with the same number of counted particles. In addition, since the dynamic range is proportional to the total number of events, it could be extended even further by extending the sample acquisition time. For example, acquiring the sample for 10 minutes rather than 1 minute would proportionally increase the dynamic range by another decade. As discussed in the previous embodiments of the current invention, the practical dynamic range for the current invention may be further expanded using a multiplexed assay that combines particle-conjugated and molecular detection reagents in order to measure low- and high- abundance analytes, respectively.

FIG. 12 illustrates another major difference between the current invention and prior art. To accurately measure analyte concentrations, it is common to use reagent concentrations in excess of the target-analyte concentrations in a sample, such that the formation of analyte-reagent complexes is favored in the binding equilibrium. As shown in FIG. 12A, in assays based on prior art, at least one of the reagents is in molecular form (107). Consequently, irrespective of how small the sample volume is, the ubiquitous unbound molecular reagent 107 must be removed from the sample through careful washing prior to the final measurement. Washing not only consumes time and resources, but it also breaks the equilibrium, resulting in the loss of analyte-reagent complexes and biased results. Conversely, in the current invention, both the capture and the detection reagent are conjugated to labeled particles. Without relying on enzymatic or other forms of signal amplification, the analyte-linked particle doublets 104 and higher-order particle multiplets provide a sensitive and reliable probe for their corresponding analyte or analyte complex at the singleanalyte level. In addition, since the analyte-linked particle complexes can be readily differentiated from unbound particles 102 and 103 by a multi-parameter particle counter, such as the flow cytometer illustrated schematically in FIG. 12B, no wash is needed to remove the unbound particles prior to the final measurement. Indeed, simultaneously enumerating the unbound particles enables the concentration of the analyte to be precisely determined using only a fraction of the total sample volume, and provides important statistical and performancereliability information. Consequently, the binding equilibrium for the assay can be maintained throughout the entire process.

Although several exemplary embodiments of the current invention have been described in some detail, in light of the above teaching, it will be apparent to those skilled in the art that many modifications and variations of the described embodiments are possible without departing from the principles and concepts of the inventions as set forth in the claims.

Claims

The Claims What is claimed is:

Claim 1. A system for the simultaneous detection and quantification of biological analytes, comprising at least one subsystem, wherein each subsystem consists of: a. A set of target analytes, wherein each target analyte consists of a single component with multiple binding sites, or multiple components, wherein each component contains one or more binding sites; b. A set of capture particles, wherein each capture particle is capable of binding to one binding site of the target analyte; c. A set of detection particles, wherein each detection particle is capable of binding to another binding site of the target analyte.

Claim 2. The system of claim 1, wherein the capture particles and/or detection particles are labeled with unique physical characteristics.

Claim 3. The system of claim 2, wherein particle complexes can form, comprising capture particles and detection particles linked together by one or more of their corresponding target analytes.

Claim 4. The system of claim 3, wherein, in each subsystem, the analyte-linked particle complexes and unbound particles can be discretely differentiated and enumerated .

Claim 5. The system of Claim 4, wherein target analyte refers to any substance whose chemical or biological properties are being identified and/or measured; and, wherein particle refers to any small localized object to which several physical, chemical and/or biological properties, such as diameter, charge and/or material composition, can be ascribed.

Claim 6. The system of Claim 4, wherein the target analyte may bind to a reagent that is conjugated to a capture or detection particle either covalently or using an affinity tag.

Claim 7. The system of claim 6, wherein the reagent may be any substance that binds specifically or nonspecifically to a site of the target analyte. Reagents include, but are not limited to: antibodies, binding proteins, peptides, polypeptides, protein complexes, sugars, oligosaccharides, polysaccharides, DNA, RNA, oligonucleotides, polynucleotides, nucleotide complexes, single-stranded nucleic acid sequences, double-stranded nucleic acid sequences, aptamers, natural polymers, synthetic polymers, pharmaceuticals, drugs, lipids, detergents, micelles, liposomes, lipoproteins, extracellular vesicles, exosomes, oncosomes, viruses, virus-like particles, cells, cell fragments, and chemicals.

Claim 8. The system of claim 6, wherein the affinity tag refers to any molecule or entity with affinity to, or that can be identified and more generally targeted by, a second molecule or binding partner, and can be used to bring two components, such as a reagent and a particle, together into a complex when differentially conjugated to the pair of components. Affinity tags include, but are not limited to: biotin, streptavidin, avidin, neutravidin, hemagglutinin, poly-histidine, maltose-binding protein, myc, glutathione-s-transferase, FLA.G, protein A, protein G, protein L, DNA, RNA, oligonucleotides, polynucleotides, single-stranded nucleic acid sequences, double-stranded nucleic acid sequences, species-specific antibodies, class-specific antibodies, and isotype-specific antibodies .

Claim 9. The system of Claim 4, wherein a particle may bind directly to an analyte due to conjugating physical, chemical and/or biological properties between the binding partners, including, but not limited to: shape, charge, chemical bonding and/or biological affinities .

Claim 10. A system of claim 4, wherein the detection particles in certain subsystems may be replaced with molecular probes .

Claim 11. The system of claim 10, wherein the concentrations of the analytes in the subsystems containing molecular probes are measured in accordance with the average number of analyte-linked molecular probes on labeled capture particles.

Claim 12. A system of claim 4, wherein, in certain subsystems, modulating agents may be introduced to modulate, inhibit or enhance the binding affinities among the analytes and coupling reagents, or among the components of analyte complexes.

Claim 13. The system of Claim 4, wherein the subsystems will be differentiated from each other, and the various analyte-linked particle complexes and unbound particles within each subsystem will be discretely detected, differentiated and enumerated, all simultaneously, by any method.

Claim 14. The system of Claim 4, wherein the subsystems will be differentiated from each other, and the various analyte-linked particle complexes and unbound particles within each subsystem will be discretely detected, differentiated and enumerated, all simultaneously, by their optical, electronic, electromagnetic, fluorescent, radioisotopic, chemical, mass, size, affinity, material composition and/or density signatures, as well as logical combinations of these signatures.

Claim 15. The system of Claim 4, wherein the subsystems will be differentiated from each other, and the various analyte-linked particle complexes and unbound particles within each subsystem will be discretely detected, differentiated and enumerated, all simultaneously, using a multi-parameter particle counter, such as a flow cytometer, or an imaging or laser-scanning microscope.

Claim 16. The system of Claim 4, wherein the resulting data from the discrete detection, differentiation and enumeration of a single subsystem will be used to determine the analyte concentration.

Claim 17. The system of Claim 4, wherein the resulting data from the discrete detection, differentiation and enumeration of multiple different subsystems will be collectively used to determine the analyte concentration .

Claim 18. A method for the simultaneous detection and quantification of biological analytes, comprising at least one subsystem, wherein each subsystem consists of: d. A set of target analytes, wherein each target analyte consists of a single component with multiple binding sites, or multiple components, wherein each component contains one or more binding sites; e. A set of capture particles, wherein each capture particle is capable of binding to one binding site of the target analyte; f. A set of detection particles, wherein each detection particle is capable of binding to another binding site of the target analyte.

Claim 19. The method of claim 18, wherein the capture particles and/or detection particles are labeled with unique physical characteristics.

Claim 20. The method of claim 19, wherein particle complexes can form, comprising capture particles and detection particles linked together by one or more of their corresponding target analytes.

Claim 21. The method of claim 20, wherein, in each subsystem, the analyte-linked particle complexes and unbound particles can be discretely differentiated and enumerated .

Claim 22. The method of Claim 21, wherein target analyte refers to any substance whose chemical or biological properties are being identified and/or measured; and, wherein particle refers to any small localized object to which several physical, chemical and/or biological properties, such as diameter, charge and/or material composition, can be ascribed.

Claim 23. The method of Claim 21, wherein the target analyte may bind to a reagent that is conjugated to a capture or detection particle either covalently or using an affinity tag.

Claim 24. The method of claim 23, wherein the reagent may be any substance that binds specifically or nonspecifically to a site of the target analyte. Reagents include, but are not limited to: antibodies, binding proteins, peptides, polypeptides, protein complexes, sugars, oligosaccharides, polysaccharides, DNA, RNA, oligonucleotides, polynucleotides, nucleotide complexes, single-stranded nucleic acid sequences, double-stranded nucleic acid sequences, aptamers, natural polymers, synthetic polymers, pharmaceuticals, drugs, lipids, detergents, micelles, liposomes, lipoproteins, extracellular vesicles, exosomes, oncosomes, viruses, virus-like particles, cells, cell fragments, and chemicals.

Claim 25. The method of claim 23, wherein the affinity tag refers to any molecule or entity with affinity to, or that can be identified and more generally targeted by, a second molecule or binding partner, and can be used to bring two components, such as a reagent and a particle, together into a complex when differentially conjugated to the pair of components. Affinity tags include, but are not limited to: biotin, streptavidin, avidin, neutravidin, hemagglutinin, poly-histidine, maltose-binding protein, myc, glutathione-s-transf erase, FLAG, protein A, protein G, protein L, DNA, RNA, oligonucleotides, polynucleotides, single-stranded nucleic acid sequences, double-stranded nucleic acid sequences, spec ies-spe cific antibodies, class-specific antibodies, and isotype-specific antibodies .

Claim 26. The method of Claim 21, wherein a particle may bind directly to an analyte due to conjugating physical, chemical and/or biological properties between the binding partners, including, but not limited to: shape, charge, chemical bonding and/or biological affinities .

Claim 27. A method of claim 21, wherein the detection particles in certain subsystems may be replaced with molecular probes.

Claim 28. The method of claim 27, wherein the concentrations of the analytes in the subsystems containing molecular probes are measured in accordance with the average number of analyte-linked molecular probes on labeled capture particles.

Claim 29. A method of claim 21, wherein, in certain subsystems, modulating agents may be introduced to modulate, inhibit or enhance the binding affinities among the analytes and coupling reagents, or among the components of analyte complexes.

Claim 30. The method of Claim 21, wherein the subsystems will be differentiated from each other, and the various analyte-linked particle complexes and unbound particles within each subsystem will be discretely detected, differentiated and enumerated, all simultaneously, by any method.

Claim 31. The method of Claim 21, wherein the subsystems will be differentiated from each other, and the various analyte-linked particle complexes and unbound particles within each subsystem will be discretely detected, differentiated and enumerated, all simultaneously, by their optical, electronic, electromagnetic, fluorescent, radioisotopic, chemical, mass, size, affinity, material composition and/or density signatures, aass well aass logical combinations of these signatures.

Claim 32. The method of Claim 21, wherein the subsystems will be differentiated from each other, and the various analyte-linked particle complexes and unbound particles within each subsystem will be discretely detected, differentiated and enumerated, all simultaneously, using a multi-parameter particle counter, such as a flow cytometer, or an imaging or laser-scanning microscope.

Claim 33. The method of Claim 21, wherein the resulting data from the discrete detection, differentiation and enumeration of a single subsystem will be used to determine the analyte concentration.

Claim 34. The method of Claim 21, wherein the resulting data from the discrete detection, differentiation and enumeration of multiple different subsystems will be collectively used to determine the analyte concentration .