Single molecule assays for ultrasensitive detection of analytes

Abstract

The present invention provides an ultrasensitive method for the detection and quantification of target analytes in a sample. The method can be multiplexed to allow for simultaneous detection and quantification of multiple target analytes. The method can achieve attomolar detection limits. The present invention also provides related compositions and kits. The greatly simplified readout process and improved cost-effectiveness of the method of the present invention can facilitate potential integration into POC systems. In particular, the present invention provides a state-of-the-art digital ELISA technology, Molecules on Beads Signal Amplification for Individual Counting (MOSAIC), which achieves attomolar sensitivity and requires only a common testing platform, thus greatly increasing the availability of ultrasensitive protein detection.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 252,440, filed October 5, 2021, and U.S. Provisional Patent Application No. 63 / 341,540, filed May 13, 2022. The entire contents of each of the foregoing applications are incorporated herein by reference.

[0002] government support This invention was made with government support under EB029777 awarded by the National Institutes of Health. The Federal Government has certain rights in this invention. [Background technology]

[0003] 2. Background of the Invention The ability to accurately measure extremely low levels of molecules such as proteins, nucleic acids, and metabolites is essential for a wide range of clinical and environmental applications, including disease diagnosis, drug discovery, pathogen detection in food, environmental toxin detection, and bioprocess control. Ultrasensitive measurement techniques are particularly important in clinical diagnostics, as many potential biomarkers are present in available biological fluids at levels far below the detection limits of current laboratory methods (Cohen, L.; Walt, DR, Chemical Reviews 2019, 119 (1), 293-321). Digital assays such as digital enzyme-linked immunosorbent assays (ELISAs) have much improved measurement sensitivity, up to 1000 times, compared to traditional analytical techniques such as conventional ELISAs (Rissin, DM et al., Nature Biotechnology 2010, 28, 595;Rissin, DM; Walt, DR, Journal of the American Chemical Society 2006, 128 (19), 6286-6287;Rissin, DM; Walt, DR, Nano Letters 2006, 6 (3), 520-523;Yelleswarapu, V. et al., Proceedings of the National Academy of Sciences 2019, 116 (10), 4489).

[0004] However, the sensitivity of digital measurement techniques remains insufficient for many diagnostic applications, especially for the measurement of disease-related proteins. For example, several protein biomarkers for neurological disorders have been shown to be upregulated in cerebrospinal fluid, but their measurement requires highly invasive lumbar puncture, thus making it impractical to screen individuals for early disease detection (Robey, TT; Panegyres, PK, Future Neurology 2019, 14 (1), FNL6; Olsson, B. et al., The Lancet Neurology 2016, 15 (7), 673-684; Galasko, DR; Shaw, LM, Nature Reviews Neurology 2017, 13 (3), 131-132; Fortea, J. et al., The Lancet Neurology 2018, 17 (10), 860-869). Since only a small number of brain-derived proteins cross the blood-brain barrier and enter the circulation, highly sensitive techniques that can detect and identify rare protein biomarkers by a simple blood test are crucial to address this unmet diagnostic need (Hampel, H. et al., Nature Reviews Neurology 2018, 14 (11), 639-652;Simren, J. et al., Current Opinion in Neurobiology 2020, 61, 29-39;Parnetti, L et al., The Lancet Neurology 2019, 18 (6), 573-586). Improving analytical sensitivity is also essential for rapid point-of-care (POC) diagnosis to enable medical intervention, but is a major challenge in other diseases where readily available biofluids such as saliva or urine are required. These biofluids contain minimal serum components and require ultrasensitive techniques for protein biomarker detection.

[0005] One major barrier in aiming to increase sensitivity in digital ELISA is low sampling efficiency. Although digital ELISA methods utilize single molecule counting to improve measurement sensitivity, low sampling efficiency limits the number of target molecules counted. At very low target concentrations, the Poisson noise, √N, (where N is the number of molecules counted), originating from single event counting contributes significantly to measurement error. As an example, at a sampling efficiency of 5%, only 30 of 600 target molecules in 100 μL of 10 aM sample would be counted, even assuming perfect capture efficiency. The theoretical Poisson noise-related coefficient of variation (CV), 1 / √N, is 18% at this low sampling efficiency, and is much higher in practice when considering capture efficiencies far below 100% and experimental errors. This high measurement uncertainty therefore imposes a major limitation for the detection of rare molecules. Thus, increasing the sampling efficiency to count more target molecules can greatly improve measurement precision and sensitivity, but remains a challenge in digital ELISA.

[0006] Current digital ELISA methods utilize microwells or water-in-oil droplets to isolate individual beads carrying single target protein molecules (Rissin, DM et al. Nature Biotechnology 2010, 28, 595;Yelleswarapu, V. et al. Proceedings of the National Academy of Sciences 2019, 116 (10), 4489;Kim, SH et al., Lab on a Chip 2012, 12 (23), 4986-4991;Witters, D. et al. Lab on a Chip 2013, 13 (11), 2047-2054). The current state of the art for digital ELISA is the Single Molecule Array (Simoa), which captures single target molecules on antibody-coated paramagnetic beads and isolates individual beads in femtoliter-sized microwells for single molecule counting (Rissin, DM et al. Nature Biotechnology 2010, 28, 595). A large excess of beads over the number of target molecules in the sample is used to ensure digital measurements in which each bead has zero or one captured target molecule and follows a Poisson distribution. Each captured molecule is labeled with a biotinylated detection antibody to form an immune complex sandwich, followed by enzyme-conjugated streptavidin-β-galactosidase (SβG). The beads are then loaded into microwells, each of which can fit at most one bead, along with a fluorescent enzyme substrate. Upon sealing the microwells with oil, a high local concentration of fluorescent product is catalytically generated in each well containing beads carrying SβG molecules. Thus, the number of target molecules is determined by counting the "on" and "off" wells.

[0007] Although Simoa can achieve sub-femtomolar detection limits and is the current gold standard for ultrasensitive protein detection, its sensitivity is limited by low sampling efficiency: only about 5% of the total number of beads can be loaded into the microwells by gravity (or magnetic attraction in the case of the Quanterix HD-X Analyzer) and analyzed (Wilson, DH et al., Journal of Laboratory Automation 2015, 21 (4), 533-547). Other methods to improve bead loading have also been explored, including electric field-directed bead loading, hydrophobic-medium-hydrophilic microwell arrays, and digital microfluidics (Barbee, KD et al., Lab on a Chip 2009, 9 (22), 3268-3274; Decrop, D. et al., ACS Applied Materials & Interfaces 2017, 9 (12), 10418-10426; Decrop, D. et al., Analytical Chemistry 2016, 88 (17), 8596-8603). Although these methods have high bead loading efficiency, the demonstration of their improvement in digital immunoassay sensitivity remains limited. Magnetic meniscus sweeping, which uses a combination of hydrodynamic and magnetic forces to increase bead loading, has improved sensitivity, but as with other techniques, complex fabrication methods and workflows limit its use in POC applications (Kan, CW et al. Lab on a Chip 2020, 20 (12), 2122). Another strategy to improve sampling efficiency in digital bioassays is bead encapsulation in water-in-oil droplets.Digital droplet-based immunoassays have been shown to have up to 60% bead loading efficiency, demonstrating improved sensitivity comparable to or up to an order of magnitude higher than that of current Simoa technology (Yelleswarapu, V. et al. Proceedings of the National Academy of Sciences 2019, 116 (10), 4489). Although droplet microfluidic systems are well established for diverse applications, the need for highly controlled, high-throughput droplet generation introduces additional fabrication and processing steps that introduce higher complexity when integrating into POC systems. Furthermore, improving imaging throughput remains another challenge for POC implementation, as a significant fraction of droplets do not contain beads but still must be imaged. Thus, there remains a need in the art for sensitive and quantitative detection techniques that can be used to detect and measure the concentrations of analytes with high sampling efficiency and simple fabrication methods and workflows for point-of-care (POC) applications. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Cohen, L.; Walt, DR, Chemical Reviews 2019, 119 (1), 293-321 [Non-Patent Document 2] Rissin, DM et al., Nature Biotechnology 2010, 28, 595 [Non-Patent Document 3] Rissin, DMWalt, DR, Journal of the American Chemical Society 2006, 128 (19), 6286-6287 [Non-Patent Document 4] Rissin, DM; Walt, DR, Nano Letters 2006, 6 (3), 520-523 [Non-Patent Document 5] Yelleswarapu, V. et al., Proceedings of the National Academy of Sciences 2019, 116 (10), 4489 Summary of the Invention [Means for solving the problem]

[0009] Summary of the Invention Measurement of low levels of analytes, such as proteins and nucleic acids, is important for clinical diagnostic applications but remains challenging due to insufficient sensitivity. Ultrasensitive digital ELISA techniques, such as single molecule arrays (Simoa), have been developed to achieve detection of very low concentrations of disease-related proteins, but still face limitations such as large and complex instruments, high costs, and complex workflows, making their application in clinical practice impractical. Furthermore, there are still many potential disease biomarkers that exist below the detection limit of current digital detection methods.

[0010] An ultrasensitive detection method that addresses the above-mentioned problems is described herein. The greatly simplified readout process and improved cost-effectiveness of the method of the present invention can facilitate potential integration into POC systems. In particular, the present invention provides a state-of-the-art digital ELISA technology, Molecular On-Bead Signal Amplification for Individual Counting (MOSAIC), which achieves attomole sensitivity and requires only a common test platform, thus greatly increasing the availability of ultrasensitive protein detection. The present invention utilizes on-bead signal generation, and therefore does not require bead isolation into individual containers such as microwells or droplets for signal compartmentalization, eliminating the requirement for complex microfabrication or droplet generation.

[0011] Furthermore, the present invention focuses on the use of flow cytometry as a detection technique, which is readily available to many laboratories and can also be adapted to microfluidic systems at low cost, making the present invention more suitable for rapid integration into current testing platforms and point-of-care formats (Asghari, M. et al. Scientific Reports 2017, 7 (1) 12342). By integrating the rapid, high-throughput detection capabilities of flow cytometry with a method for localized signal amplification of single molecules, a low attomolar detection limit was obtained that is an order of magnitude better than the gold standard digital ELISA method, Simoa. Other digital measurement strategies with flow cytometry readout have been previously developed, but these methods are limited to femtomolar or higher detection limits, and attomolar sensitivity has not yet been demonstrated for protein detection. A key advantage of MOSAIC compared to previous digital ELISA methods is the much more rapid, automated - less than 1 minute per sample signal readout. Automation with 96-well plate sampling mode, already built into many benchtop flow cytometers, further provides a state-of-the-art workflow.

[0012] In addition to applying a rapid on-bead signal amplification strategy, the high sample collection efficiency of MOSAIC can be leveraged to systematically reduce the number of assay beads to enhance sensitivity to detect low attomolar protein concentrations. The improved sensitivity of MOSAIC enables the measurement of previously undetectable analytes in biofluids, such as low-abundance cytokines in saliva. Furthermore, MOSAIC extends the multiplexing capabilities of digital ELISA, as the number of bead types that can be analyzed within a single sample is no longer limited by the total number of microwells or other compartments. As an example of increased multiplexing in MOSAIC, eight protein targets were measured simultaneously with attomolar to low femtomolar sensitivity using small volumes of plasma.

[0013] The enhanced sensitivity, simplicity, and versatility of MOSAIC compared to current digital ELISA methods makes ultrasensitive protein detection widely available, providing a considerable advance toward the discovery of previously undetectable biomarkers for diverse clinical applications.

[0014] Thus, the present invention provides, in one aspect, a method for detecting a target analyte in a sample, the method comprising the steps of: (a) contacting a sample containing or suspected of containing the target analyte with a plurality of beads comprising a capture moiety that specifically binds the target analyte, under conditions and for a time sufficient for the target analyte in the sample to bind to the capture moiety, wherein a plurality of beads are associated with zero target analyte molecules; a plurality of beads are associated with one target analyte molecule; and at least about 20% of the beads are associated with either zero or one target analyte molecule; (b) contacting the product of step (a) with a detection moiety that binds to the target analyte, (c) contacting the product of step (b) with a signal amplification moiety that binds to the detection moiety to generate a detectable signal for each bead carrying the target analyte; and (d) detecting the target analyte in the sample by detecting the detectable signal by flow cytometry.

[0015] In some embodiments, the beads comprise magnetic beads, paramagnetic beads, non-magnetic beads, porous beads, or glass beads.

[0016] In some embodiments, the capture moiety comprises an antibody, an aptamer, an antibody mimetic, a polypeptide, a nucleic acid, a molecularly imprinted polymer, a receptor, a binding protein, or a small molecule.

[0017] In some embodiments, the detection moiety comprises an antibody, an aptamer, an antibody mimetic, a polypeptide, a nucleic acid, a molecularly imprinted polymer, a receptor, a binding protein, or a small molecule.

[0018] In some embodiments, the signal amplification moiety comprises an enzyme and / or a nucleic acid molecule.

[0019] In some embodiments, the detectable signal is generated by rolling circle amplification followed by hybridization with a complementary fluorescently labeled DNA probe; rolling circle transcription; hybridization chain reaction; loop-mediated isothermal amplification; radical polymerization; tyramide signal amplification (TSA); enzyme-catalyzed proximity labeling (PL) polymerization; labeling with a pre-amplified signal using a fluorescently labeled enzyme, nanoparticle or nucleic acid concatemer; polymerization-based signal amplification; or magnetic bead-quantum dot immunoassay.

[0020] In some embodiments, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the beads are associated with either 0 or 1 target analyte molecule.

[0021] In some embodiments, the detection moiety and the signal amplification moiety are directly linked.

[0022] In some embodiments, the detection moiety and the signal amplification moiety are linked by a non-covalent affinity binding pair, where the detection moiety is linked to a first member of the non-covalent affinity binding pair and the signal amplification moiety is linked to a second member of the non-covalent affinity binding pair.

[0023] In some embodiments, the non-covalent affinity binding pair is biotin-streptavidin, biotin-avidin, ligand-receptor, antigen-antibody, or antibody binding protein-antibody.

[0024] In some embodiments, beads containing capture moieties for target analytes differ from beads containing capture moieties for non-target analytes, optionally by having a different color, shape, or size.

[0025] In some embodiments, the method further comprises detecting the beads containing the capture moiety by flow cytometry.

[0026] In some embodiments, the method reduces cross-reactivity or non-specific binding. In some embodiments, the method reduces cross-reactivity or non-specific binding by detecting the beads and detectable signal by flow cytometry.

[0027] In some embodiments, the target analyte is a protein, a nucleic acid, a polysaccharide, a lipid, a cell, a fatty acid, a therapeutic agent, an organism, a virus, a toxin, a peptide, an oligosaccharide, a lipoprotein, a glycoprotein, a glycan, or a hormone.

[0028] In some embodiments, the sample comprises a biological sample, an environmental sample, or a synthetic material.

[0029] In some embodiments, the biological sample is (i) a bodily fluid selected from the group consisting of lymph, whole blood, plasma, serum, a blood fraction containing peripheral blood mononuclear cells, urine, saliva, semen, sweat, tears, synovial fluid, cerebrospinal fluid, feces, mucus, vaginal fluid, and spinal fluid, or (ii) breast tissue, liver tissue, pancreatic tissue, cervical tissue, lung tissue, kidney tissue, colon tissue, brain tissue, muscle tissue, synovial tissue, skin, hair follicle, bone marrow, tumor tissue, tissue lysate or homogenate, or organ lysate or homogenate.

[0030] In some embodiments, the biological sample is plasma.

[0031] In some embodiments, the biological sample is saliva.

[0032] In some embodiments, the method further comprises measuring the concentration of the target analyte in the sample, wherein the concentration of the target analyte in the sample is related to the level of the detectable signal.

[0033] In some embodiments, the method further comprises detecting or measuring the concentration of an additional target analyte in the sample.

[0034] In some embodiments, the additional target analytes include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more target analytes.

[0035] In some embodiments, the additional target analyte is a protein, a nucleic acid, a polysaccharide, a lipid, a cell, a fatty acid, a therapeutic agent, an organism, a virus, a toxin, a peptide, an oligosaccharide, a lipoprotein, a glycoprotein, a glycan, or a hormone.

[0036] In some embodiments, the method further comprises contacting the sample with: (i) a plurality of beads comprising additional capture moieties that specifically bind to additional target analytes; (ii) an additional detection moiety that binds to the additional target analytes; and (iii) an additional signal amplification moiety that binds to the additional detection moieties to generate additional detectable signals.

[0037] In another aspect, the invention provides a method of detecting a first target analyte and a second target analyte in a sample, the method comprising: (a) contacting a sample containing or suspected of containing a first target analyte and / or a second target analyte with (i) a plurality of first beads comprising a first capture moiety that specifically binds to the first target analyte and (ii) a plurality of second beads comprising a second capture moiety that specifically binds to the second target analyte, under conditions and for a time sufficient for the first target analyte in the sample to bind to the first capture moiety and for the second target analyte in the sample to bind to the second capture moiety, wherein a plurality of first beads are associated with zero first target analyte molecules; a plurality of first beads are associated with one first target analyte molecule; at least about 20% of the first beads are associated with zero or one first target analyte molecule; and a plurality of second beads are associated with zero second target analyte molecules; at least about 20% of the second beads are associated with 0 or 1 second target analyte molecule; (b) contacting the product of step (a) with (i) a first detection moiety that binds the first target analyte, and (ii) a second detection moiety that binds the second target analyte; (c) contacting the product of step (b) with (i) a first signal amplification moiety that binds to the first detection moiety that generates a first detectable signal for each bead carrying the first target analyte, and (ii) a second signal amplification moiety that binds to the second detection moiety that generates a second detectable signal for each bead carrying the second target analyte; and (d) detecting the first and second target analytes in the sample by detecting the first and second detectable signals by flow cytometry.

[0038] In some embodiments, the first detectable signal and the second detectable signal are different signals, optionally having different colors.

[0039] In some embodiments, the method further comprises detecting the first bead comprising the first capture moiety and the second bead comprising the second capture moiety by flow cytometry.

[0040] In some embodiments, the first bead comprising the first capture moiety and the second bead comprising the second capture moiety are different, optionally having different colors, shapes, or sizes.

[0041] In some embodiments, the method reduces cross-reactivity or non-specific binding. In some embodiments, the method reduces cross-reactivity or non-specific binding by detecting the beads and detectable signal by flow cytometry.

[0042] In some embodiments, the method has a detection limit of about 0.1 aM to about 1 mM.

[0043] In some embodiments, the detection limit is about 0.1 aM to about 1 mM, about 0.1 aM to about 1 μM, about 0.1 aM to about 1 nM, about 0.1 aM to about 1 pM, about 0.1 aM to about 1 fM, about 0.1 aM to about 900 aM, about 0.1 aM to about 800 aM, about 0.1 aM to about 700 aM, about 0.1 aM to about 600 aM, about 0.1 aM to about 500 aM, about 0.1 aM to about 400 aM, about 0.1 aM to about 300 aM, about 0.1 aM to about 200 aM, or about 0.1 aM to about 100 aM.

[0044] In some embodiments, the detection limit is about 1 fM, about 900 aM, about 800 aM, about 700 aM, about 600 aM, about 500 aM, about 400 aM, about 300 aM, about 200 aM, about 100 aM, about 90 aM, about 80 aM, about 70 aM, about 60 aM, about 50 aM, about 40 aM, about 30 aM, about 20 aM, about 10 aM, or about 1 aM, or about 0.1 aM.

[0045] In some embodiments, signal detection takes less than about 1 minute per sample, less than about 45 seconds per sample, or less than about 30 seconds per sample.

[0046] In some embodiments, the sample is contacted with about 2,000 to about 100,000 beads. In some embodiments, the sample is contacted with about 2,000 beads, about 5,000 beads, about 10,000 beads, about 20,000 beads, about 50,000 beads, or about 100,000 beads.

[0047] In some embodiments, the beads and sample are incubated for about 1 minute to about 48 hours, about 1 minute to about 10 hours, or about 1 hour to about 4 hours. In some embodiments, the beads and sample are incubated for about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours. The method of any one of claims 1 to 36.

[0048] Other features and advantages of the invention will become apparent from the following detailed description, the drawings, and the claims. [Brief description of the drawings]

[0049] [Figure 1] FIG. 1 depicts a schematic diagram of an exemplary single molecule assay of the present invention (Molecules on Beads Signal Amplification for Individual Counting (MOSAIC)). Single target molecules are first captured using paramagnetic beads coated with an excess number of antibodies, such that each bead carries zero or one target molecule according to a Poisson distribution. Upon formation of a single immune complex sandwich with a biotinylated detection antibody and labeling with a streptavidin-DNA conjugate, rolling circle amplification is performed to generate long DNA concatemers bound to each immune complex. Fluorescently labeled DNA probes are hybridized to the RCA products to allow counting of "on" and "off" beads by flow cytometry.

[0050] [Figure 2-1]2A-D depict the analytical sensitivity of the MOSAIC assay across different analytes and assay bead numbers. FIG. 2A provides calibration curves for the IL-10 MOSAIC assay using different assay bead numbers. Signal readings are shown in average molecules per bead (AMB). FIG. 2B depicts the effect of MOSAIC assay bead number on the limit of detection (LOD) and signal-to-background for IL-10 detection. LOD values ​​were calculated as 3 standard deviations above background AMB. Relative signal-to-background for each assay bead number was determined as the signal-to-background of a particular calibrator normalized to the signal-to-background of the same calibrator using 100,000 assay beads. FIG. 2C provides calibration curves for the corresponding IL-10 Simoa assay using 100,000 assay beads and 400,000 helper beads. FIG. 2D depicts the effect of MOSAIC assay bead number on the LOD and relative signal-to-background across additional analytes. All error bars represent the standard deviation of triplicates with six replicates performed on the blank. [Figure 2-2] Same as above.

[0051] [Figure 3-1]Figures 3A-D depict that increasing sampling efficiency improves analytical sensitivity and precision. Figures 3A-3B depict the detection limits for calibration curves generated from randomly sampled subsets of various bead numbers from the MOSAIC calibration curves for IL-10 (Figure 3A) and IFN-γ (Figure 3B). Each color indicates the starting total assay bead number. Each point represents the median of 100 randomly sampled subsets, and the error bars represent the interquartile range. Open circles indicate values ​​where the upper quartile had a positive infinite value due to the very high measurement coefficient of variation (CV) at very low bead subset sizes. Figures 3C-3D provide the measurement CV of the background signal for randomly sampled bead subsets of various bead numbers for IL-10 (Figure 3C) and IFN-γ (Figure 3D). [Figure 3-2] Same as above.

[0052] [Figure 4-1] Figures 4A-D depict the measurement of IFN-γ concentrations in human plasma and saliva using MOSAIC and Simoa. Figures 4A-4D provide the measured IFN-γ concentrations in 17 human plasma (Figure 4A, Figure 4B) and 26 saliva (Figure 4C, Figure 4D) samples between the MOSAIC and Simoa assays. The concentrations shown represent endogenous IFN-γ concentrations considering a 4-fold dilution factor, and the limit of detection (LOD) and lower limit of quantification (LLOQ) also reflect a 4-fold dilution factor. The Pearson correlation coefficients were 0.80 and 0.31 for plasma and saliva samples (among detectable values), respectively. The low correlation coefficient in saliva may be due to only a few detectable samples using Simoa as well as multiple samples with IFN-γ levels close to the LLOQ of Simoa or MOSAIC. The red dashed line indicates the LOD of the assay calculated as 3 standard deviations above the background (buffer only) AMB. Samples with measurements below the LOD of the assay were assigned a value equal to the LOD. Error bars represent the standard deviation of duplicate replicates. [Figure 4-2] Same as above.

[0053] [Figure 5-1] Figures 5A-D depict multiplexing with MOSAIC technology. Figure 5A depicts the correlation of cytokine measurements in human plasma between a 4-plex MOSAIC assay and a corresponding 4-plex Simoa assay. Concentrations, LOD, and LLOQ values ​​reflect the 4-fold dilution factor used in both assays. Samples with measurements below the LOD of the assay were assigned a value equal to the LOD. Figures 5B-5C provide the measured concentrations of eight protein analytes in human plasma (top) and saliva (bottom) using an 8-plex MOSAIC assay (Figure 5B) and two 4-plex Simoa assays (Figure 5C). Concentrations shown are the measured concentration values ​​in plasma and saliva samples diluted 16-fold and 8-fold, respectively. Measurements below the LOD of the assay were assigned a value equal to the LOD and are indicated by open circles. Figure 5D provides a schematic of multiplexing with MOSAIC. Beads coated with antibodies against different target analytes are coded by using different fluorescent dyes with different wavelengths, intensities, and / or by using multiple bead sizes. Upon capture of a single analyte molecule on each bead type, formation of a single immune complex sandwich, and labeling with streptavidin-DNA, rolling circle amplification is performed and the mixture of beads is analyzed by flow cytometry. After differentiating the beads by a series of gates in different fluorescent channels, the average molecules per bead for each bead type is determined from the intensity in the fluorescent channel corresponding to the probe color. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 5-4] Same as above.

[0054] [Figure 6-1] 6A-6E depict the calibration curves for MOSAIC assays performed for individual cytokines across different assay bead numbers. Error bars represent standard deviation of 3-6 replicates. [Figure 6-2] Same as above. [Figure 6-3] Same as above.

[0055] [Figure 7-1] 7A-7E depict the calibration curves for the Simoa assays performed for individual cytokines. The detection limit of the assay is indicated by the dashed red line. Error bars represent the standard deviation of triplicates and six blank replicates. [Figure 7-2] Same as above. [Figure 7-3] Same as above.

[0056] [Figure 8-1] 8A-8C depict MOSAIC assays performed with a 4 hour target capture time. FIGS. 8A-8B provide calibration curves for the MOSAIC assays of IL-10 (FIG. 8A) and IFN-γ (FIG. 8B) across different assay bead numbers with a 4 hour target capture time. Beads were incubated with samples for 4 hours, and detection antibody was added at the end of the target capture time. Error bars represent standard deviation of triplicates for each calibrator and six replicates for the blank. FIG. 8C lists the detection limit and lower limit of quantification for the MOSAIC assays of IL-10 and IFN-γ with a 4 hour target capture time. [Figure 8-2] Same as above.

[0057] [Figure 9-1]9A-9C depict MOSAIC and Simoa assays performed in human saliva. FIG. 9A-9B provide calibration curves for MOSAIC and Simoa assays of IFN-γ (FIG. 9A) and IL-12p70 (FIG. 9B) performed in saliva using StartingBlock™ Blocking Buffer (Thermo Fisher Scientific) as the dilution buffer. The limits of detection (LOD) of the assay for IFN-γ were 29.6 and 86.9 aM for MOSAIC and Simoa, respectively. The LOD of the assay for IL-12p70 was 44.2 and 304.3 aM for MOSAIC and Simoa, respectively. 10,000 assay beads were used for both MOSAIC assays, and 100,000 assay beads were used for the corresponding Simoa assays, along with 400,000 helper beads. Figure 9C depicts the measured IL-12p70 concentrations in 26 saliva samples using MOSAIC and Simoa. Concentrations and assay LODs, shown in dashed red lines, represent assay measurements multiplied by dilution factors. [Figure 9-2] Same as above.

[0058] [Figure 10-1] Figures 10A-10D depict the quadruple MOSAIC assay. Figures 10A-10B provide calibration curves for the quadruple MOSAIC (Figure 10A) and Simoa (Figure 10B) assays for IL-6, IL-1β, IL-10, and IFN-γ. Figure 10C describes the detection limits and lower limits of quantification for the quadruple MOSAIC and Simoa assays. Figure 10D provides graphs of the dropout curves for each analyte in the quadruple MOSAIC assay. Each graph shows the signal response of each bead for increasing amounts of individual analytes in the multiplex assay, with error bars representing the standard deviation of duplicate measurements. [Figure 10-2] Same as above. [Figure 10-3] Same as above.

[0059] [Figure 11-1] Figures 11A-11D depict representative calibration curves for an 8-plex MOSAIC assay and two 4-plex Simoa assays for IFN-γ, IL-1β, IL-5, IL-6, IL-10, IL-12p70, IL-18, and VEGF. Figures 11A-11B provide representative 8-plex MOSAIC assay calibration curves obtained with sample diluents used for plasma (Figure 11A) and saliva (Figure 11B) measurements. Figures 11C-11D provide corresponding 4-plex Simoa assay calibration curves obtained with sample diluents used for plasma (Figure 11C) and saliva (Figure 11D) measurements. Error bars represent standard deviation of triplicates with 6 replicates performed for the blank. [Figure 11-2] Same as above. [Figure 11-3] Same as above.

[0060] [Figure 12-1] Figure 12 depicts the dilution linearity of the 8-plex MOSAIC assay in three individual human plasma samples. The range of r2 values ​​from the linear regression is shown for each analyte. For plasma #1, the dilution linearity between 4-fold and 8-fold dilutions was weak, so the 4-fold dilution measurements were excluded from the linear regression. Therefore, a final dilution of 16-fold was selected for all reported plasma measurements to ensure consistent measurement accuracy. Error bars represent the standard deviation of duplicate measurements. [Figure 12-2] Same as above. [Figure 12-3] Same as above. [Figure 12-4] Same as above.

[0061] [Figure 13-1] Figure 13 depicts the dropout curves for each analyte in an 8-plex MOSAIC assay. Each graph shows the signal response of each bead for increasing amounts of an individual analyte, with error bars representing the standard deviation of duplicate measurements. [Figure 13-2] Same as above. [Figure 13-3] Same as above. [Figure 13-4] Same as above.

[0062] [Figure 14-1] Figures 14A-14B depict the correlation of measurements by MOSAIC and Simoa in plasma (Figure 14A) and saliva (Figure 14B) for each of the analytes in the 8-plex MOSAIC assay. Two separate 4-plex Simoa assays were performed as a comparison. The limit of detection (LOD) and lower limit of quantification (LLOQ) values ​​correspond to the effective LOD and LLOQ values ​​considering the 16-fold dilution factor in the plasma samples. The Pearson correlation coefficient, determined from the measured concentrations of all samples, is shown for each analyte. The weaker correlation between MOSAIC and Simoa for analytes whose concentrations were close to or below the LLOQ of either method may be due to several factors, including lower measurement precision close to the LLOQ or LOD. Although we validated the 8-plex MOSAIC assay by spike-and-recovery experiments and dilution linearity experiments, differences in matrix effects for the MOSAIC and Simoa assays may have contributed to the weaker correlation in the lower concentration range. Error bars represent the standard deviation of duplicate measurements. [Figure 14-2] Same as above.

[0063] [Figure 15-1]Figures 15A-15D depict representative gating strategies for identifying beads by flow cytometry for MOSAIC assay. Gating was performed using FlowJo™ software. Representative gates are shown for 488 multiplex beads (Quanterix Corp.). Figure 15A depicts the gating strategy where an initial forward vs. side scatter gate was applied to remove most of the debris. Figure 15B depicts the gating strategy where beads were further identified by the fluorescence channel. Figure 15C depicts the gating strategy where a final gate was applied to identify single beads. Figure 15D provides representative scatter plots and histograms of fluorescence intensity for different IL-10 concentrations at 20,000 assay beads using ATTO 647N-labeled DNA probes. Average molecules per bead (AMB) values ​​were then determined from the fluorescence intensity values ​​using Python as described in the Examples. [Figure 15-2] Same as above. [Figure 15-3] Same as above. Same as above.

[0064] [Figure 16-1] FIG. 16 depicts the dilution linearity of the 8-plex MOSAIC assay in three individual human saliva samples. The range of r2 values ​​from the linear regression is shown for each analyte. For saliva #1, the dilution linearity between 4-fold and 8-fold dilutions was weak, so the 4-fold dilution measurements were excluded from the linear regression. A final dilution of 8-fold was selected for all reported saliva measurements to ensure consistent measurement accuracy. Error bars represent the standard deviation of duplicate measurements. [Figure 16-2] Same as above. [Figure 16-3] Same as above. [Figure 16-4] Same as above.

[0065] [Figure 17-1]Figure 17 depicts the bead gating strategy used for the 8-plex MOSAIC assay. A series of gates in different fluorescence channels were used to distinguish bead types and remove any aggregates of different bead types. The gated populations highlighted in pink represent each bead type prior to further gating of single beads and subsequent analysis of fluorescence intensity in the probe channel. [Figure 17-2] Same as above. [Figure 17-3] Same as above.

[0066] [Figure 18] Figure 18 depicts the schematic diagram of both non-barcoded multiplex MOSAIC and barcoded multiplex MOSAIC formats. For barcoded multiplex MOSAIC format, a unique DNA template is conjugated to each detection antibody in multiplex MOSAIC assay and paired with the corresponding unique fluorescent dye-conjugated probe in RCA reaction. In contrast, in non-barcoded multiplex MOSAIC format, all detection antibodies are biotinylated and labeled with the same streptavidin-DNA conjugate and fluorescent probe, thus masking cross-reactive binding events.

[0067] [Figure 19] Figures 19A and 19B depict the dropout curves for increasing concentrations of each individual target protein for non-barcoded multiplex MOSAIC (Figure 19A) and barcoded multiplex MOSAIC (Figure 19B). Inclusion of only the correct capture bead-probe color pairs in barcoded multiplex MOSAIC allows for elimination of cross-reactive signals, thus providing a more accurate multiplex measurement. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] Detailed Description of the Invention The present invention provides methods and compositions for detecting or measuring the concentration of a target analyte.

[0069] Quantitative and ultrasensitive detection of protein biomarkers in minimally invasive biological fluids such as blood or saliva has the potential to revolutionize medical diagnostics with faster disease diagnosis, treatment, monitoring, and disease recurrence monitoring. Technologies such as digital enzyme-linked immunosorbent assay (ELISA) and single molecule array (Simoa) enable ultrasensitive detection of low-abundance analytes, including proteins, nucleic acids, and other biologically relevant small molecules. However, these technologies still face limitations such as large and complex instruments, high costs, and complex workflows, making their application in clinical practice impractical. Furthermore, there are still many potential disease biomarkers that exist below the detection limit of current digital detection methods.

[0070] The inventors of the present invention have developed an innovative, high-throughput, and highly accessible single molecule measurement platform capable of detecting low to mid-attomolar protein concentrations. By addressing the challenge of low efficiency in sampling rare target molecules in digital ELISA techniques, sensitivity was enhanced by more than 10-fold over the current Simoa technology, currently the gold standard for ultrasensitive protein detection. The attomolar limit of detection (LOD) achieved by the present method represents at least a 3-4 order of magnitude improvement compared to conventional immunoassays. By localizing the non-diffusible fluorescent signal to each bead carrying the target molecule, the platform not only obviates the need for bead loading into microwells or droplets for signal compartmentalization, but also allows significantly more beads to be analyzed for improved sampling efficiency, thereby enhancing sensitivity.

[0071] This simple technique allows for the analysis of about 50-60% of all beads on average, compared to the current Simoa technology's sampling efficiency of about 5%, which is about a 10-fold increase. At low sample concentrations, especially with capture efficiencies well below 100% (about 1-3% across all capture and labeling steps in the inventive assay developed in this work), improved sampling is essential to minimize Poisson noise-related measurement CVs. The significantly improved sampling efficiency of the inventive method also allows for the use of fewer assay beads compared to conventional Simoa, increasing the fraction of "on" beads and thereby increasing the signal to background. Further improvements in sensitivity can be achieved by using affinity reagents with lower dissociation constants and reducing non-specific binding of affinity reagents and streptavidin-DNA labels. Together with the development of better affinity reagents and methods to reduce non-specific binding, the inventive method can potentially detect down to zeptomolar protein concentrations. Together with attomolar sensitivity, the inventive method can pave the way for the search for new biomarkers and biological mechanisms underlying various diseases, such as tuberculosis.

[0072] The achievement of an order of magnitude or more improvement in sensitivity in the method of the present invention also holds important implications for the search for new blood-based biomarkers for many other cancer types and neurological disorders. Diagnostic blood tests for neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease will prove particularly essential for widespread screening and early diagnosis, which is currently very difficult due to the need for highly invasive lumbar puncture. In many cases where biomarker levels are only detectable after significant disease progression, the enhanced sensitivity of the method of the present invention can facilitate disease diagnosis at an early stage for improved health outcomes.

[0073] Importantly, the method of the present invention also increases the simplicity of digital bioassay signal readout, and upon further development, can potentially be integrated into POC platform, thus addressing the issue of low sensitivity in current POC diagnostics.Increasing sample collection efficiency for enhanced sensitivity in digital immunoassays has also been demonstrated in bead droplet arrays and droplet digital ELISA methods, but these methods bring additional complexity in fabrication and processing steps.In contrast, the readout process of the method of the present invention only requires flow cytometry, which is easily available in many laboratories and can also be adapted to microfluidic systems at low cost, making the present invention more suitable for rapid integration into current test platforms and point-of-care formats.

[0074] Moreover, another important advantage of the present invention compared to previous digital ELISA methods such as drop-cast single molecule assay is the much faster automated signal readout. Although drop-cast single molecule assay (dSimoa) enabled bead drop-casting methods to count single molecules captured on beads, these methods require long imaging times per sample to capture all beads across multiple fields of view, thus limiting throughput. In contrast, the present invention allows for much faster sample readout speeds. Signal readout takes less than about 1 minute per sample. Automation with 96-well plate sampling mode, which is already built into many benchtop flow cytometers, further provides a state-of-the-art workflow.

[0075] definition As used herein, the term "about" refers to a value within 10% above or below the stated value.

[0076] "Target analyte" refers to any atom, molecule, ion, molecular ion, compound, particle, cell, virus, complex, or fragment thereof to be detected, measured, quantified, or evaluated. The target analyte may be contained in a sample (e.g., a liquid sample (e.g., a biological sample or an environmental sample)). Exemplary target analytes include, but are not limited to, small molecules (e.g., organic compounds, steroids, hormones, haptens, biogenic amines, antibiotics, mycotoxins, organic pollutants, nucleotides, amino acids, monosaccharides, or secondary metabolites), proteins (glycoproteins or prions), nucleic acids, polysaccharides, lipids, fatty acids, cells, gases, therapeutic agents, organisms (e.g., pathogens), viruses, toxins, peptides, oligosaccharides, lipoproteins, glycoproteins, glycans, or hormones. Target analytes may be naturally occurring or synthetic.

[0077] As used herein, the term "small molecule" refers to any molecule having a molecular weight of less than 5000 Da. For example, in some embodiments, the small molecule is an organic compound, a steroid, a hormone, a hapten, a biogenic amine, an antibiotic, a mycotoxin, a cyanotoxin, a nitro compound, a drug residue, a pesticide residue, an organic pollutant, a nucleotide, an amino acid, a monosaccharide, or a secondary metabolite.

[0078] The terms "nucleic acid" and "polynucleotide", used interchangeably herein, refer to at least two covalently linked nucleotide monomers. The terms include, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), hybrids thereof, and mixtures thereof. Although nucleotides are typically linked in nucleic acids by phosphodiester bonds, the term "nucleic acid" also includes nucleic acid analogs having other types of linkages or backbones, such as phosphorothioate, phosphoramide, phosphorodithioate, O-methyl phosphoramidate, morpholino, locked nucleic acid (LNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), and peptide nucleic acid (PNA) linkages or backbones. Nucleic acids may be single-stranded, double-stranded, or contain portions of both single-stranded and double-stranded sequences. A nucleic acid may contain any combination of deoxyribonucleotides and ribonucleotides, and bases including, for example, adenine, thymine, cytosine, guanine, uracil, and modified or non-standard bases.

[0079] By "protein" herein is meant at least two covalently linked amino acids, including proteins, polypeptides, oligopeptides and peptides. Proteins may be composed of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures. Thus, as used herein, "amino acid" or "peptide residue" refers to both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and norleucine are considered amino acids for the purposes of the present invention. The side chains may be in the (R) or (S) configuration. In some embodiments, the amino acids are in the (S) or L-configuration. When non-naturally occurring side chains are used, non-amino acid substituents can be used, for example, to prevent or retard in vivo degradation. The term "portion" includes any region of a protein, such as a fragment (e.g., a cleavage product or recombinantly produced fragment) or an element or domain (e.g., a region of a polypeptide that has activity) that contains fewer amino acids (e.g., about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% fewer amino acids) than a full-length polypeptide or a reference polypeptide.

[0080] The terms "beads", "particles" and "microspheres", used interchangeably herein, refer to small individual particles. Suitable beads include, but are not limited to, paramagnetic beads, magnetic beads, non-magnetic beads, porous beads, plastic beads, ceramic beads, glass beads, polystyrene beads, methylstyrene beads, acrylic polymer beads, carbon graphite beads, titanium dioxide beads, latex or cross-linked dextran, such as SEPHAROSE beads, cellulose beads, nylon beads, cross-linked micelles, and TEFLON beads. In some embodiments, spherical beads are used, but it should be understood that non-spherical or irregularly shaped beads may also be used.

[0081] As used herein, the term "capture moiety" refers to any molecule, particle, etc. that can specifically bind to a target analyte. The capture moiety may be conjugated, captured, attached, bound, or immobilized to a bead. For example, in some embodiments, the capture moiety is an antibody (e.g., a full-length antibody (e.g., an IgG, IgA, IgD, IgE, or IgM antibody) or an antigen-binding antibody fragment (e.g., scFv, Fv, dAb, Fab, Fab', Fab'2, F(ab')2, Fd, Fv, or Feb)), an aptamer, an antibody mimic (e.g., an affibody, an affilin, an affimer, an affitin, an alphabody, an anticalin, an avimer, a DARPin, a finomer, a Kunitz domain peptide, a monobody, or a nanoCLAMP), an antibody IgG binding protein (e.g., Protein A, Protein G, Protein L, or recombinant Protein A / G), a polypeptide, a nucleic acid, or a small molecule.

[0082] As used herein, the term "detecting moiety" or "detection moiety" refers to any molecule, particle, etc. that can specifically bind to or otherwise associate with a target analyte, or another molecule (e.g., a capture moiety) that binds to or otherwise associates with a target analyte. For example, in some embodiments, the detection moiety is an antibody (e.g., a full-length antibody (e.g., an IgG, IgA, IgD, IgE, or IgM antibody) or an antigen-binding antibody fragment (e.g., an scFv, Fv, dAb, Fab, Fab', Fab'2, F(ab')2, Fd, Fv, or Feb)), an aptamer, an antibody mimetic (e.g., an affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, finomer, Kunitz domain peptide, monobody, or nanoCLAMP), a molecularly imprinted polymer, a receptor, a polypeptide, a nucleic acid, or a small molecule.

[0083] As used herein, the term "signal amplification moiety" refers to any molecule, particle, etc. that can specifically bind to or otherwise associate with a detection moiety and generate a detectable, e.g., amplified, signal that allows detection. The signal may be any detectable signal, e.g., an optically detectable label, e.g., fluorescent or chemiluminescent, or colorimetric label, detectable by flow cytometry or optical assay, or any other label, e.g., a gold bead or other label, detectable by non-optical assay (e.g., using surface plasmon resonance or other methods). In some embodiments, the signal amplification moiety includes an enzyme and / or a DNA molecule, e.g., a DNA primer and a template, e.g., a concatemer or long continuous DNA molecule containing multiple copies of the same DNA sequence linked in series. The signal amplification moiety further includes a probe with a detectable label, e.g., a fluorescently labeled DNA probe, that can hybridize to the generated concatemer.

[0084] A first moiety (e.g., a capture moiety) "specifically binds" (or grammatical variations thereof) to a second moiety if the first moiety (e.g., a target analyte) binds to the second moiety with sufficient specificity to distinguish between the second moiety and other components or contaminants of the test sample. The binding is generally sufficient to remain bound under the conditions of the assay, including wash steps to remove non-specific binding, although in some embodiments, i.e., for detecting low affinity binding partners, a wash step is not desirable. In some embodiments, the first moiety binds to the second moiety at a concentration of about 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M, 10 -14 M, 10 -15 The equilibrium dissociation constant (K D) which specifically binds to the second portion.

[0085] The term "cross-reactivity" refers to non-specific binding of a bead comprising a capture moiety for a target analyte with a non-target analyte, e.g., a different analyte, or a contaminant or other component of a test sample.

[0086] The term "non-covalent affinity binding pair" refers to a pair of molecules, e.g., a first member and a second member, that bind and form a non-covalent complex. Exemplary non-covalent affinity binding pairs include, but are not limited to, biotin-biotin binding protein (e.g., biotin-streptavidin and biotin-avidin), ligand-receptor, antigen-antibody or antigen-binding fragment, hapten-antihapten, immunoglobulin (Ig) binding protein-Ig, dioxigenin, SNAP tag, CLIP tag, or other complementary binding partners. Members of a non-covalent affinity binding pair may have any suitable binding affinity. For example, members of an affinity binding pair may have a binding affinity of about 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M, 10 -14 M, 10 -15 The equilibrium dissociation constant (K D Alternatively, binding may occur at Kd).

[0087] A "pathogen" is an agent that can cause disease or illness in its host, including, but not limited to, a virus (e.g., a parvovirus (e.g., adeno-associated virus (AAV)), a retrovirus (e.g., a lentivirus (e.g., human immunodeficiency virus (HIV))), a herpes virus, an adenovirus, etc.), a bacterium (e.g., Mycobacterium tuberculosis, or E. coli), a protozoan, a fungus, or a prion.

[0088] As used herein, "subject" refers to any animal. In one embodiment, the subject is a human. Other animals that can be subjects include, but are not limited to, non-human primates (e.g., monkeys, gorillas, and chimpanzees), domestic animals (e.g., horses, pigs, donkeys, goats, rabbits, sheep, cows, yaks, alpacas, and llamas), and companion animals (e.g., cats, lizards, snakes, dogs, fish, hamsters, guinea pigs, rats, mice, and birds).

[0089] As used herein, "biomarker" and "marker" refer interchangeably to an analyte (e.g., a small molecule, DNA, RNA, protein, carbohydrate, or glycolipid-based molecular marker) whose expression or presence in a subject's sample can be detected by the methods described herein and which is useful, for example, for determining a prognosis or for monitoring the responsiveness or sensitivity of a mammalian subject to a therapeutic agent.

[0090] As used herein, "biological sample" refers to any biological sample obtained from or derived from a subject, including bodily fluids, bodily tissues (e.g., tumor tissues), cells, or other sources. Bodily fluids are, for example, lymph, whole blood (including fresh, frozen, or dried and rehydrated), plasma (including fresh, frozen, or dried and rehydrated), serum (including fresh, frozen, or dried and rehydrated), blood fractions containing peripheral blood mononuclear cells, urine, saliva, semen, sweat, tears, synovial fluid, cerebrospinal fluid, feces, mucus, vaginal fluid, and spinal fluid. Samples also include breast tissue, liver tissue, pancreatic tissue, cervical tissue, kidney tissue, colon tissue, brain tissue, muscle tissue, synovial tissue, skin, hair follicles, bone marrow, tumor tissue, tissue lysates or homogenates, or organ lysates or homogenates. Methods for obtaining tissue biopsies and bodily fluids from mammals are well known in the art.

[0091] By "environmental sample" is meant any sample obtained from the environment, e.g., a water sample, a soil sample, an air sample, extraterrestrial material, etc. An environmental sample may contain biomolecules or organisms.

[0092] Methods of the Invention The present invention provides a method for detecting a target analyte in a sample. The method may comprise measuring the concentration of the target analyte in the sample.

[0093] In one aspect, the present invention provides a method for detecting a target analyte in a sample. The method includes the steps of: (a) contacting a sample containing or suspected to contain a target analyte with a plurality of beads comprising a capture moiety that specifically binds to the target analyte under conditions and for a time sufficient for the target analyte in the sample to bind to the capture moiety, where a plurality of beads are associated with zero target analyte molecules; a plurality of beads are associated with one target analyte molecule; at least about 20% of the beads are associated with either zero or one target analyte molecule; (b) contacting the product of step (a) with a detection moiety that binds to the target analyte; (c) contacting the product of step (b) with a signal amplification moiety that binds to the detection moiety to generate a detectable signal for each bead carrying the target analyte; and (d) detecting the target analyte in the sample by detecting the detectable signal by flow cytometry.

[0094] A sample containing or suspected to contain a target analyte is first contacted with a plurality of beads conjugated to a capture moiety capable of specifically binding to the analyte of interest under conditions that allow the analyte to bind to the bead to form a bead-analyte complex. Once the bead-analyte complex is formed, the method includes contacting the analyte with a detection moiety and a signal amplification moiety, and generating an on-bead non-diffusible detectable signal, e.g., a fluorescent signal, that allows detection of each bead carrying a single target analyte by flow cytometry. By localizing the signal on the bead, the method of the present invention eliminates the need for signal compartmentalization in a microwell or droplet.

[0095] In some embodiments, steps (a), (b), (c), or any combination thereof, of the methods of the present invention are performed sequentially or simultaneously.

[0096] In some embodiments, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the beads are associated with either 0 or 1 target analyte molecule.

[0097] The method of the present invention also includes measuring the concentration of the target analyte in the sample. In some embodiments, the concentration is proportional, e.g., directly proportional or inversely proportional, to the intensity level of at least one signal indicating the presence of the target analyte.

[0098] In some embodiments, the methods of the present invention comprise administering to the subject a therapeutically effective amount of a medicament at a concentration of from about 0 mM to about 5 mM, for example, from about 0.1 aM to about 5 mM, from about 0.1 aM to about 4 mM, from about 0.1 aM to about 3 mM, from about 0.1 aM to about 2 mM, from about 0.1 aM to about 1 mM, from about 0.1 aM to about 1 μM, from about 0.1 aM to about 1 nM, from about 0.1 aM to about 1 pM, or from about 0.1 aM to about 1 fM. , about 0.1 aM to about 900 aM, about 0.1 aM to about 800 aM, about 0.1 aM to about 700 aM, about 0.1 aM to about 600 aM, about 0.1 aM to about 500 aM, about 0.1 aM to about 400 aM, about 0.1 aM to about 300 aM, about 0.1 aM to about 200 aM, or about 0.1 aM to about 100 aM. In some embodiments, the detection limit of the methods of the invention is about 1 fM, about 900 aM, about 800 aM, about 700 aM, about 600 aM, about 500 aM, about 400 aM, about 300 aM, about 200 aM, about 100 aM, about 90 aM, about 80 aM, about 70 aM, about 60 aM, about 50 aM, about 40 aM, about 30 aM, about 20 aM, about 10 aM, about 1 aM, or about 0.1 aM.

[0099] The methods of the invention may further include detecting or measuring the concentration of one or more additional target analytes in the sample. In some embodiments, the methods may include detecting or measuring the concentration of about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 14, about 16, about 18, about 20 or more different target analytes.

[0100] In some embodiments, the target analyte is a small molecule, a protein, a nucleic acid, a polysaccharide, a lipid, a cell, a fatty acid, a therapeutic agent, an organism, a virus, a toxin, a peptide, an oligosaccharide, a lipoprotein, a glycoprotein, a glycan, or a hormone. The additional target analyte can be any suitable target analyte described herein or known in the art. In some embodiments, the method further comprises contacting the sample with (i) a plurality of beads having additional capture moieties that specifically bind to the additional target analytes; (ii) an additional detection moiety that binds to the additional target analyte, and (iii) an additional signal amplification moiety that binds to the additional detection moiety to generate an additional detectable signal.

[0101] In one aspect, the invention provides a method of detecting a first target analyte and a second target analyte in a sample, the method comprising: (a) contacting a sample containing or suspected of containing a first target analyte and / or a second target analyte with (i) a plurality of first beads comprising a first capture moiety that specifically binds to the first target analyte and (ii) a plurality of second beads comprising a second capture moiety that specifically binds to the second target analyte, under conditions and for a time sufficient for the first target analyte in the sample to bind to the first capture moiety and for the second target analyte in the sample to bind to the second capture moiety, wherein a plurality of first beads are associated with zero first target analyte molecules; a plurality of first beads are associated with one first target analyte molecule; at least about 20% of the first beads are associated with zero or one first target analyte molecule; and a plurality of second beads are associated with zero second target analyte molecules; the second beads are associated with one second target analyte molecule; at least about 20% of the second beads are associated with zero or one second target analyte molecule; (b) contacting the product of step (a) with (i) a first detection moiety that binds the first target analyte, and (ii) a second detection moiety that binds the second target analyte; (c) contacting the product of step (b) with (i) a first signal amplification moiety that binds to the first detection moiety that generates a first detectable signal for each bead carrying the first target analyte, and (ii) a second signal amplification moiety that binds to the second detection moiety that generates a second detectable signal for each bead carrying the second target analyte; and (d) detecting the first and second target analytes in the sample by detecting the first and second detectable signals by flow cytometry.

[0102] In another aspect, the invention provides a method of detecting a first target analyte, a second target analyte, and a third or subsequent target analyte in a sample, comprising the steps of: (a) contacting a sample containing or suspected of containing the first target analyte, the second target analyte, and / or the third or subsequent target analyte with (i) a plurality of first beads comprising a first capture moiety that specifically binds the first target analyte, (ii) a plurality of second beads comprising a second capture moiety that specifically binds the second target analyte, and (iii) a plurality of third or subsequent beads comprising a third or subsequent capture moiety that specifically binds the third or subsequent target analyte, under conditions and for a time sufficient for the first target analyte in the sample to bind to the first capture moiety, the second target analyte in the sample to bind to the second capture moiety, and the third or subsequent target analyte in the sample to bind to the third or subsequent capture moiety, wherein the plurality of first beads a plurality of first beads associated with 0 first target analyte molecules, a plurality of first beads associated with 1 first target analyte molecule, at least about 20% of the first beads associated with either 0 or 1 second target analyte molecule, a plurality of second beads associated with 0 second target analyte molecules, a plurality of second beads associated with 1 second target analyte molecule, at least about 20% of the second beads associated with either 0 or 1 second target analyte molecule, and a plurality of third beads associated with 0 third target analyte molecules. (b) contacting the product of step (a) with (i) a first detection moiety that binds the first target analyte, (ii) a second detection moiety that binds the second target analyte, and (iii) a third or subsequent detection moiety that binds the third or subsequent target analyte;(c) contacting the product of step (b) with (i) a first signal amplification portion that binds to a first detection portion to generate a first detectable signal for each bead carrying a first target analyte, (ii) a second signal amplification portion that binds to a second detection portion to generate a second detectable signal for each bead carrying a second target analyte, and (iii) a third or subsequent signal amplification portion that binds to a third or subsequent detection portion to generate a third or subsequent detectable signal for each bead carrying a third or subsequent target analyte; and (d) detecting the first detectable signal, the second detectable signal, and the third or subsequent detectable signal by flow cytometry, thereby detecting the first target analyte, the second target analyte, and the third or subsequent target analyte in the sample.

[0103] Any of the aforementioned methods may include providing a prognosis or diagnosis of a disease, such as tuberculosis, for a subject based on the concentration of one or more target analytes in the sample. Any of the aforementioned methods may include selecting a treatment for a patient based on the concentration of one or more target analytes in the sample. Any of the aforementioned methods may include treating a subject with a treatment based on the concentration of one or more target analytes in the sample. For example, in certain embodiments, the methods may be used to prognose or diagnose tuberculosis, a disease whose ability to be detected and diagnosed at an early stage is essential to prevent the development of active tuberculosis and the spread of the disease.

[0104] In some embodiments, the methods of the invention include detecting and / or quantifying one or more target analytes, such as one or more molecules expressed by Mycobacterium tuberculosis, in a sample. In other embodiments, the methods of the invention include prognosing or diagnosing tuberculosis for a subject based on the presence and / or concentration of one or more target analytes in the sample. The methods may also include selecting a therapy or treating the subject with a therapy based on the presence and / or concentration of one or more target analytes in the sample.

[0105] beads The method of the present invention involves the use of micro- or nanoparticle beads conjugated to a capture moiety that binds to the desired analyte. The beads can be made from a variety of materials. In general, any polymeric or plastic material can be used to create the microparticles, microbeads, or nanoparticles, including materials such as polystyrene and polyethylene. In some embodiments, the microparticles can be formed from biocompatible polymeric materials such as polyacrylates, polymethacrylates, and / or polyamides. In certain embodiments, metal, metal oxide, semiconductor, and / or semiconductor oxide micro- and / or nanoparticles formed from one or more of Au, Ag, Pt, Al, Cu, Ni, Fe, Cd, Se, Ge, Pd, Sn, iron oxide, TiO2, Al2O3, and SiO2 can be made and used in many sizes. For example, single crystal iron oxide nanoparticles (MIONs) and cross-linked iron oxide (CLIO) particles can be used. Any suitable beads can be used in the context of the present invention, including, but not limited to, magnetic beads, paramagnetic beads, non-magnetic beads, porous beads, plastic beads, ceramic beads, glass beads, polystyrene beads, methylstyrene beads, acrylic polymer beads, carbon graphite beads, titanium dioxide beads, latex or cross-linked dextran, such as SEPHAROSE beads, cellulose beads, nylon beads, cross-linked micelles, and TEFLON® beads. In certain embodiments, the beads are paramagnetic beads. In some embodiments, the beads may have different shapes, sizes, and / or colors. In some embodiments, beads that contain a capture moiety for one analyte have a different shape, size, and / or color than beads for a different analyte, so that the beads can be detected or identified, for example, by flow cytometry, to eliminate non-specific binding or cross-reactivity. In some embodiments, spherical beads are used, although non-spherical or irregularly shaped beads may also be used. In some embodiments, the beads contain or are conjugated to a detectable signal or label, such as a fluorescent, chemiluminescent, or colorimetric label.In some embodiments, the detectable signal or label can be detected by flow cytometry or optical assays, or any other detection method described herein or known in the art. The color of the label, e.g., fluorescent label, on the bead can be different for each different analyte to be detected.

[0106] In any of the above methods, the beads are magnetic beads (e.g., paramagnetic beads). In some embodiments, the beads have a size (e.g., diameter) of about 0.01 μm to about 10 μm, for example, about 0.01 μm, about 0.1 μm, about 0.2 μm, about 0.3 μm, about 0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about 0.9 μm, about 1 μm, about 1.5 μm, about 2 μm, about 2.5 μm, about 3 μm, about 3.5 μm, about 4 μm, about 4.5 μm, about 6 μm, about 6.5 μm, about 7 μm, about 7.5 μm, about 8 μm, about 8.5 μm, about 9 μm, about 9.5 μm, or about 10 μm. In some embodiments, the beads have a size of about 1 μm to about 5 μm, about 1 μm to about 4 μm, about 1 μm to about 3 μm, or about 1 μm to about 2 μm.

[0107] In any of the above-mentioned methods, the sample is mixed with about 1,000 to about 5,000,000 beads, for example, about 1000, about 2000, about 5000, about 10,000, about 20,000, about 30,000, about 40,000, about 50,000, about 60,000, about 70,000, about 80,000, about 90,000, about 100,000, about 20 The method may include contacting the beads with about 0,000, about 300,000, about 400,000, about 500,000, about 600,000, about 700,000, about 800,000, about 900,000, about 1 million, about 2 million, about 3 million, about 4 million, or about 5 million beads. In some embodiments, the method may include contacting the sample with about 2,000 to about 100,000 beads, about 10,000 to about 5,000,000 beads, about 10,000 to about 4,000,000 capture probes, about 10,000 to about 3,000,000 beads, about 10,000 to about 2,000,000 capture probes, about 10,000 to about 1,000,000 beads, about 10,000 to about 500,000 capture probes, about 10,000 to about 400,000 beads, about 10,000 to about 300,000 beads, about 10,000 to about 200,000 beads, or about 10,000 to about 100,000 beads. In some embodiments, the method may include contacting the sample with about 2,000 beads, about 5,000 beads, about 10,000 beads, about 20,000 beads, about 50,000 beads, or about 100,000 beads. In some embodiments, the method may include contacting the sample with about 20,000 beads.

[0108] The sensitivity and / or sampling efficiency of the method of the present invention can be adjusted based on the number of beads. In some embodiments, a decrease in the number of beads can improve the sensitivity of the method. In other embodiments, an increase in the number of beads can improve the sensitivity of the method. The number of beads used can also be optimized for each analyte to be detected.

[0109] Capture, detection and signal amplification moieties The beads for use in the method of the present invention are coated with, e.g., conjugated to, a capture moiety that can specifically bind to an analyte of interest. When the capture moiety binds to the target analyte, a bead-analyte complex is formed. A detection moiety and a signal amplification moiety are used to generate an on-bead, non-diffusible detectable signal for each bead that carries a single target analyte.

[0110] The capture and / or detection moiety can specifically bind or otherwise associate with the target analyte. Any suitable capture and / or detection moiety can be used in the context of the present invention. For example, in some embodiments, the capture and / or detection moiety can be an antibody, an aptamer, an antibody mimetic, a polypeptide, a nucleic acid, a molecularly imprinted polymer, a receptor, a binding protein, or a small molecule. The antibody can be a full-length antibody (e.g., an IgG, IgA, IgD, IgE, or IgM antibody) or an antigen-binding antibody fragment (e.g., an scFv, Fv, dAb, Fab, Fab', Fab'2, F(ab')2, Fd, Fv, or Feb). The antibody mimic can be an affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, finomer, Kunitz domain peptide, monobody, or nanoCLAMP.

[0111] In some embodiments, the capture and / or detection moiety is an antibody or its antigen-binding portion or an aptamer that binds to the analyte, for example, when the analyte is a protein or peptide.In some embodiments, the capture and / or detection moiety is an oligonucleotide that is complementary to a portion of the nucleic acid of interest.In some embodiments, the capture and / or detection moiety is a ligand-binding portion of a protein, for example, a receptor, when the analyte is a molecule such as a hormone.

[0112] The method further comprises an on-bead signal amplification step. Specifically, the method comprises contacting the sample with a signal amplification moiety that binds to the detection moiety and generates a detectable signal that allows detection of each bead carrying a single target analyte. The signal may be any detectable signal. In some embodiments, the signal comprises an optically detectable label, such as a fluorescent or chemiluminescent or colorimetric label, that can be detected by flow cytometry or optical assay.

[0113] In some embodiments, the signal amplification portion comprises an enzyme, e.g., phi29 DNA polymerase, and / or a nucleic acid molecule, e.g., a DNA primer and a template, e.g., a concatemer or a long continuous DNA molecule containing multiple copies of the same DNA sequence linked in series. The signal amplification portion further comprises a probe with a detectable label, e.g., a fluorescently labeled DNA probe or a ratiometric combination of fluorescently labeled DNA probes, that can hybridize to the generated concatemers. In some embodiments, the detectable label comprises different colors for different target analytes. In some embodiments, different target analytes can be detected by using a combination of different beads, e.g., beads with different colors, fluorescence intensities, shapes or sizes, and different detectable labels, e.g., labels with different colors or different ratios of colors, which can be distinguished by flow cytometry.

[0114] In some embodiments, the method reduces cross-reactivity or non-specific binding, for example, by detecting the beads and detectable signals by flow cytometry. In some embodiments, a unique pair of fluorescently labeled capture beads and DNA template-conjugated detection antibodies is used for each analyte. Each unique DNA template-conjugated antibody, when amplified during the RCA reaction, is labeled with a specific fluorescent dye-conjugated DNA probe or a ratiometric combination of dye-conjugated DNA probes. The color or ratiometric combination of colors of each probe can be identified using multiple detection channels in flow cytometry. Upon capture of a single analyte molecule on the bead, a mixture of unique primer-template sequence-conjugated detection antibodies is added, followed by washing and resuspension in the RCA reaction containing a mixture of fluorescent dye-conjugated DNA probes. Each analyte corresponds to a unique pair of (1) the color, fluorescence intensity, or size of the capture bead and (2) the color or color ratio of the fluorescent probe. As a result, only "correct" matched pairs of capture bead and probe signal are classified as "on" beads for each analyte, while "false" pairs of capture bead and probe color, i.e., cross-reactive binding events, are excluded from further analysis.

[0115] In some embodiments, detectable signals are generated by rolling circle amplification (RCA). For RCA, streptavidin-labeled or detection antibodies are conjugated with DNA primers and templates. The generated DNA concatemers bound to each bead-analyte complex can be hybridized with a number of complementary fluorescently labeled DNA probes for detection. In these methods, the sensitivity can be adjusted by increasing or decreasing the time of RCA.

[0116] Other nucleic acid amplification methods can be used to generate on-bead signals, such as hybridization chain reaction, loop-mediated isothermal amplification, radical polymerization, enzyme-catalyzed proximity labeling (PL) polymerization (see, e.g., Branon et al., Nat Biotechnol. 2018, 36(9):880-887); polymerization-based signal amplification (e.g., visible light-induced polymerization, as described, e.g., in Badu-Tawiah et al., Lab Chip, 2015, 15, 655); magnetic bead-quantum dot immunoassays (Kim et al., ACS Sens. 2017, 2, 6, 766-772); or immunosignal hybridization chain reaction (isHCR) (Lin et al., Nat Methods. 2018 Apr;15(4):275-278). A branched DNA assay, such as that described in Dunbar and Das, J Clin Virol. 2019; 115: 18-31, can also be used.

[0117] Alternatively, amplified on-bead signals can be generated using proximity-dependent labeling methods such as tyramide signal amplification (TSA), biotin ligase, and engineered ascorbate peroxidase. TSA, also called catalytic reporter deposition (CARD), is a highly sensitive method that allows for the detection of analytes present in low abundance. TSA has been used in immunohistochemistry and in situ hybridization experiments and for digital ELISA (Akama et al., Anal. Chem. 2016, 88 (14), 7123-7129). In TSA, HRP (e.g., conjugated to a second binding moiety) catalyzes the conversion of the labeled tyramide to a reactive radical, which then covalently binds to nearby tyrosine residues, generating a high-density detectable signal.

[0118] In some embodiments, the method includes contacting the detection moiety with a pre-amplified signal, e.g., a labeled polymer or nanoparticle or nucleic acid concatemer; see, e.g., Tang et al., Analyst, 2013,138, 981-990; Hansen et al., Anal Bioanal Chem. 2008; 392(1-2): 167-175; Wu et al., Chem 2017, 2, 760-790; Gormley et al., Nano Lett. 2014, 14, 11, 6368-6373 (polymerizing radicals generated by enzymes or metal ions to form polymers that entangle multiple gold nanoparticles (AuNPs)); Melnychuk and Klymchenko, J. Am. Chem. Soc. 2018, 140, 34, 10856-10865 (dye-loaded polymeric nanoparticles).

[0119] In some embodiments, the detection moiety is directly linked to the signal amplification moiety. In some embodiments, the detection moiety and the signal amplification moiety are indirectly linked, for example, by a non-covalent affinity binding pair, where the detection moiety is linked to a first member of the non-covalent affinity binding pair, and the signal amplification moiety is linked to a second member of the non-covalent affinity binding pair. In some embodiments, the non-covalent affinity binding pair is biotin-streptavidin, biotin-avidin, digoxigenin, SNAP tag, CLIP tag, ligand-receptor, antigen-antibody, or antibody binding protein-antibody, or any other complementary binding partner known in the art. In some embodiments, the detection moiety is bound to a biotin molecule, and the signal amplification moiety, for example, a DNA concatemer, is conjugated to a streptavidin molecule.

[0120] detection The present invention uses on-bead signal generation from a single captured target molecule that allows for signal molecule enumeration by flow cytometry. By localizing a non-diffusible fluorescent signal to each bead carrying a target molecule, this method allows for rapid enumeration of "on" and "off" beads by flow cytometry.

[0121] The present invention differs from past digital detection methods in several ways: (1) it does not require bead isolation into individual containers such as microwells or droplets for signal compartmentalization because the signal is localized to each bead, thus removing the requirement for complex microfabrication or droplet generation; (2) it increases the sampling efficiency of rare target molecules because it can count a greater percentage of beads than current Simoa technology (at least 30% compared to about 5%), thus improving the detection limit by about an order of magnitude; (3) flow cytometry is readily available to many laboratories and can also be adapted into microfluidic systems at low cost, making the present invention more suitable for rapid integration into current testing platforms and point-of-care formats.

[0122] Additionally, the present invention differs from other flow cytometry-based immunoassays, such as the Luminex assay: (1) it achieves digital detection by capturing a single target molecule on a bead, instead of multiple target molecules per bead as in current flow cytometry-based immunoassays, thus allowing for a much lower detection limit by several orders of magnitude; (2) it uses signal amplification to obtain a detectable signal for each individual target molecule, rather than relying on fluorescently labeled secondary antibodies, which require multiple target molecules per bead to generate a signal detectable by flow cytometry.

[0123] Another important advantage of the present invention compared to previous digital ELISA methods is the much faster, automated signal readout. Automation with the 96-well plate sampling mode already built into many benchtop flow cytometers further provides a state-of-the-art workflow.

[0124] In some embodiments, the signal detection takes less than about 5 minutes per sample. In some embodiments, the signal detection takes less than about 4 minutes per sample. In some embodiments, the signal detection takes less than about 3 minutes per sample. In some embodiments, the signal detection takes less than about 2 minutes per sample. In some embodiments, the signal detection takes less than about 1 minute per sample. In some embodiments, the signal detection takes less than about 30 seconds per sample.

[0125] The on-bead signal generated by the methods of the present invention can be detected and / or quantified, and the detection and / or quantification can be related to the presence, amount, and / or concentration of the target analyte in the sample being tested.

[0126] In some embodiments, the on-bead signal is detected by a flow cytometer, which is a device that feeds a particle suspension into a narrow tube, separates the suspended particles one by one, irradiates each particle with a laser, and detects and measures the fluorescent emission from the particles excited by the laser and the scattering of the laser by the particles.

[0127] The flow cytometer for use in the present invention is not particularly limited, and any conventional flow cytometer can be used as is.It has, for example, a laser source (e.g., He / Ne or argon) that emits light of a single wavelength in one or more detection regions.Laser sources that emit light at different wavelengths can be installed in different detection regions.Laser can be focused by using a beam shaping lens.

[0128] The beads are concentrated in a vertical line before being irradiated with the laser. In a traditional flow cytometer, a sheath fluid (e.g., IsoFlow sheath fluid (trade name, manufactured by Beckmann Coulter), Dako sheath fluid (trade name, manufactured by Dako Japan), etc.) and a sample solution containing the target analyte are introduced into a flow cell, where the sheath fluid and the sample solution form a laminar flow without intermixing with each other. During flow in the flow cell, the sample core is held between the sheath fluid, and the sample solution flows forming a sample core as it passes through the laser irradiation zone and is irradiated by the laser. However, Dean focusing, viscoelastic focusing, physical confinement, or other methods can also be used to align the beads.

[0129] In some embodiments, flow cytometer allows for the acquisition of detection data for a single target molecule.In other embodiments, flow cytometer allows for the simultaneous measurement of multiple targets in a single measurement operation.In some embodiments, one or more target analytes can be detected by using a combination of different beads, for example beads with different colors, shapes or sizes, and different detectable labels, for example labels with different colors, which can be distinguished by flow cytometry.

[0130] Flow cytometers for use in the present invention may be commercially available flow cytometers such as FACS Calibur (manufactured by Becton, Dickinson and Company), PERFLOW Ana (manufactured by The Furukawa Electric Co., Ltd.), EPICS ALTRA (manufactured by Beckmann Coulter), CyAn (manufactured by Dako Japan), or JSAN (manufactured by Bay bioscience Co., Ltd.). It is understood that "flow cytometer" also includes other devices that reproduce some or all of the characteristics of a flow cytometer sufficient to allow detection of beads and on-bead signals.

[0131] target analyte As will be appreciated by those skilled in the art, the method of the present invention can be used to detect and, if necessary, quantitate a large number of target analytes. Any suitable target analyte can be investigated using the method of the present invention. The target analytes listed below are provided as non-limiting examples. Target analytes may be naturally occurring or synthetic.

[0132] In some embodiments, the target analyte can be, but is not limited to, a protein (e.g., an antibody, a cytokine (e.g., an interleukin (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-7, IL-9, IL-10, IL-11, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, or IL-36), a lymphokine, a monokine, an interferon (IFN, e.g., IFN-beta and IFN-gamma), a colony stimulating factor (e.g., CSF, G-CSF, GM-CSF, etc.), a chemokine, a serotonin receptor agonist (SR-ARG), ... The protein may be a protein that is a cytoplasmic protein (e.g., a cytoplasmic protein ... The target analyte may be a molecule expressed by a bacterium, e.g., Mycobacterium tuberculosis, or E. coli) or a eukaryotic cell (e.g., a fungal cell or a human cell), including a tumor cell), a fatty acid, a glycoprotein, a biomolecule, a therapeutic agent (e.g., an antibody, a fusion protein (e.g., an Fc fusion protein), a cytokine, a soluble receptor, etc.), an organism (e.g., a pathogen), a virus (e.g., a parvovirus (e.g., an adeno-associated virus (AAV)), a retrovirus, a herpes virus, an adenovirus, a lentivirus, etc.), or a small molecule. In some embodiments, the target analyte may be a molecule expressed by a bacterium, e.g., Mycobacterium tuberculosis.In some embodiments, the target analyte may be post-translationally modified (eg, phosphorylated, methylated, glycosylated, ubiquitinylated, etc.).

[0133] In some embodiments, the target analyte has a molecular weight of at least about 1000 Da, e.g., at least about 1500 Da, at least about 2000 Da, at least about 2500 Da, at least about 3000 Da, at least about 3500 Da, at least about 4000 Da, at least about 4500 Da, or at least about 5000 Da.

[0134] In some embodiments, the target analyte may have one or more binding sites that can be recognized by the capture moiety and the detection moiety. The target analyte may be detected in a sandwich assay format based on the method of the present invention. In some embodiments, the bead may be associated with either 0 or 1 target analyte molecule, or the bead may be associated with one or more, for example, 1, 2, 3, 4, or 5 or more target analyte molecules.

[0135] Any of the methods described herein may further comprise detecting and optionally quantifying target analytes, for example in a multiplexed assay.For example, in some embodiments, the method may comprise detecting and optionally quantifying about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 14, about 16, about 18, about 20 or more different target analytes.In some embodiments, one or more target analytes can be detected by using a combination of different beads, for example beads having different colors, shapes or sizes, and different detectable labels, for example labels having different colors, which can be distinguished by flow cytometry.

[0136] In some embodiments, the target analyte may be post-translationally modified (eg, phosphorylated, methylated, glycosylated, ubiquitinylated, etc.).

[0137] In any of the aforementioned methods, the concentration of the target analyte in the sample is about 0 mM to about 5 mM, e.g., about 0.1 aM to about 5 mM, about 0.1 aM to about 4 mM, about 0.1 aM to about 3 mM, about 0.1 aM to about 2 mM, about 0.1 aM to about 1 mM, about 0.1 aM to about 1 μM, about 0.1 aM to about 1 nM, about 0.1 aM to about 1 pM, about 0.1 aM The range may be from about 0.1 aM to about 1 fM, from about 0.1 aM to about 900 aM, from about 0.1 aM to about 800 aM, from about 0.1 aM to about 700 aM, from about 0.1 aM to about 600 aM, from about 0.1 aM to about 500 aM, from about 0.1 aM to about 400 aM, from about 0.1 aM to about 300 aM, from about 0.1 aM to about 200 aM, or from about 0.1 aM to about 100 aM.

[0138] In any of the foregoing methods, the incubation times for the beads and the sample can be adjusted to enhance the signal-to-background ratio and analytical sensitivity. In some embodiments, the incubation can be performed for about 1 minute to about 48 hours, about 1 minute to about 10 hours, or about 1 hour to about 4 hours. In some embodiments, the incubation time is about minutes, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 40 hours, or about 48 hours.

[0139] In some embodiments, the beads and sample are incubated for about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours. In one embodiment, the incubation time is about 1 hour.

[0140] sample Any suitable sample can be used in the context of the present invention. For example, in some embodiments, the sample is a liquid sample (e.g., a biological sample or an environmental sample). Exemplary biological samples include, but are not limited to, bodily fluids, bodily tissues (e.g., tumor tissue), cells, or other sources. Exemplary bodily fluids include, but are not limited to, for example, lymph, whole blood (including fresh or frozen), plasma (including fresh or frozen), serum (including fresh or frozen), blood fractions containing peripheral blood mononuclear cells, urine, saliva, semen, sweat, tears, synovial fluid, cerebrospinal fluid, feces, mucus, vaginal fluid, and spinal fluid. Samples also include breast tissue, liver tissue, pancreatic tissue, cervical tissue, lung tissue, kidney tissue, colon tissue, brain tissue, muscle tissue, synovial tissue, skin, hair follicles, bone marrow, tumor tissue, tissue lysates or homogenates, and organ lysates or homogenates. Methods for obtaining tissue biopsies and bodily fluids from mammals are well known in the art. In other embodiments, the sample may be an environmental sample, such as a water sample, a soil sample, an air sample, extraterrestrial material, and the like.

[0141] The volume of the fluid sample to be analyzed can potentially be any amount within a range of volumes, depending on several factors, such as, for example, the number of capture probes used / available, the number of detection probes, etc. As non-limiting examples, the sample volume can be about 0.01 μl, about 0.1 μl, about 1 μl, about 5 μl, about 10 μl, about 100 μl, about 1 ml, about 5 ml, about 10 ml, etc. In some cases, the volume of the fluid sample is about 0.01 μl to about 10 ml, about 0.01 μl to about 1 ml, about 0.01 μl to about 100 μl, or about 0.1 μl to about 10 μl.

[0142] In some embodiments, the fluid sample may be diluted before use in the methods described herein. For example, in embodiments where the source of the analyte molecule is a bodily fluid (e.g., blood, plasma, or serum), the fluid may be diluted with a suitable solvent (e.g., a buffer such as PBS buffer). The fluid sample may be diluted about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, about 50-fold, about 100-fold, or more before use. The sample may be added to a solution containing multiple capture probes or detectable moieties, or multiple capture probes or detectable moieties may be added directly to the sample or as a solution.

[0143] composition The present invention provides a composition that can be used in the detection and, if necessary, quantification of target analytes in a sample.For example, the present invention provides a composition that includes: (a) a bead (e.g., paramagnetic bead) that is linked to a plurality of capture moieties (e.g., antibodies); (b) a detection moiety (e.g., antibodies), which is linked to a first member of a non-covalent affinity binding pair (e.g., biotin moiety); and (c) a signal amplification moiety (e.g., phi29 DNA polymerase, DNA primer, DNA concatemer, and fluorescently labeled probe), which is linked to a second member of a non-covalent affinity binding pair (e.g., streptavidin moiety), wherein the detection moiety binds to one of the target analytes captured by the capture moiety on the bead, and the signal amplification moiety binds to the detection moiety by the first member of the non-covalent affinity binding pair binding to the second member of the non-covalent affinity binding pair.

[0144] kit The present invention provides a kit for detecting or measuring the concentration of a target analyte in a fluid sample. The kit may, for example, include a plurality of beads (e.g., paramagnetic beads). The plurality of beads provided may have various properties and parameters, as described herein. The plurality of beads may be coated with a capture moiety that specifically binds to the target analyte. The plurality of beads may also be conjugated to a detectable signal or label, such as a fluorescent label, for detection. The kit may include a detection moiety that can specifically bind to the target molecule once it is captured by the capture moiety on the bead. The kit may further include a signal amplification moiety that can generate an on-bead non-diffusible signal that is detectable by flow cytometry. The capture moiety, detection moiety and signal amplification moiety provided may have various properties and parameters, as described herein.

[0145] The kit may also include a reaction vessel for collecting a sample by a detectable signal. The reaction vessel may be configured to receive and contain the beads in the sample.

[0146] The kits and articles of manufacture described herein can be configured, for example, in the Examples, to carry out any of the methods or assays described herein.

[0147] In some embodiments, the kit may include instructions for the use of the components described herein. That is, the kit may include, for example, a description of the use of the beads and reaction vessels for use with a system for determining a measure of the concentration of a target analyte(s) in a fluid sample. As used herein, "instructions" defines the components of instructions and / or promotions and may typically include written instructions on or associated with the packaging of the present invention. Instructions may also include any verbal or electronic instructions provided in any manner such that a user of the kit clearly recognizes that the instructions are associated with the kit. Additionally, the kit may include other components depending on the particular application, as described herein. EXAMPLES

[0148] The present invention will be more fully understood by referring to the following examples. However, they should not be interpreted as limiting the scope of the present invention. It is understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes may be suggested to those skilled in the art in this respect, and are included within the spirit and scope of this application and the scope of the appended claims.

[0149] Example 1 MOSAIC: High-throughput highly multiplexed digital protein detection with attomole sensitivity A major challenge in many clinical diagnostic applications is the measurement of low abundance proteins and other biomolecules in biological fluids such as plasma or saliva. The advent of digital technologies such as digital enzyme-linked immunosorbent assay (ELISA) has enabled a 1000-fold increase in sensitivity compared to traditional protein detection methods. However, current digital ELISA technologies still have insufficient sensitivity for biomarkers present below their detection limit and require specialized equipment or time-consuming workflows, limiting their widespread implementation. To address these challenges, we have developed a more sensitive, state-of-the-art, and rapid digital ELISA platform, Molecules on Beads Signal Amplification for Individual Counting (MOSAIC), which achieves a low attomolar detection limit with an order of magnitude increase in sensitivity compared to these other methods. MOSAIC uses a rapid, automatable flow cytometry readout that greatly increases throughput and is easily integrated into current testing platforms. Because MOSAIC provides high sampling efficiency for rare target molecules, the number of assay beads can be easily adjusted to enhance the signal to background ratio with high measurement precision. Furthermore, MOSAIC's solution-based signal readout expands the number of bead types that can be simultaneously analyzed for higher-order multiplexing at femtomolar or lower sensitivity, in contrast to microwell- or droplet-based digital methods. As a proof-of-principle demonstration, we applied MOSAIC to improve the detectability of low-abundance cytokines in saliva and the ultrasensitive multiplexed measurement of eight protein analytes in plasma. MOSAIC's attomole sensitivity, rapid throughput, and broad multiplexing capabilities provide a highly accessible and versatile ultrasensitive platform that can potentially facilitate protein biomarker discovery as well as diagnostic testing for diverse disease applications.

[0150] Development of the MOSAIC platform. To establish an ultrasensitive digital ELISA platform that exhibits rapid flow cytometric signal readout, we used a method to generate localized fluorescent signals from single target molecules captured on excess numbers of antibody-coated beads (Figure 1). 6 Upon formation of a single immune complex sandwich on a bead and labeling with streptavidin conjugated to the DNA primer-template pair, rolling circle amplification (RCA) is performed to form DNA concatemers bound to each immune complex sandwich. The incorporation of a fluorescently labeled DNA probe into the RCA reaction allows in situ hybridization to the concatemers, thus producing a strong fluorescent signal on each bead carrying the target molecule. As the amplified signal binds to each immune complex sandwich, the individual beads are subsequently analyzed by flow cytometry for counting of fluorescent "on" and "off" beads.

[0151] To assess the feasibility of single molecule counting using this readout, we first applied MOSAIC to detect interleukin-10 (IL-10), an anti-inflammatory cytokine with diverse roles across cancer, autoimmune diseases, and infectious diseases. 12、13 The "on" beads spanned a range of fluorescence intensities due to the heterogeneous size distribution of the RCA concatemers. A cutoff value for discriminating between "on" and "off" beads was determined by fitting a 2-mixture Gaussian model to the fluorescence intensity values ​​using expectation maximization. 14 At lower concentrations, nearly all beads fell into the lower ("off") Gaussian model, and beads were counted as "on" if they were higher than 5 standard deviations above the mean of this Gaussian model. At higher concentrations, labels were predicted for beads based on the likelihood they could be drawn from the "off" or "on" Gaussian models. Using this algorithm, we were able to consistently quantify the average molecules per bead (AMB), a measure of the fraction of "on" beads.

[0152] High sampling efficiency in MOSAIC improves sensitivity. Importantly, MOSAIC achieves high sampling efficiency, as almost all bead solutions can be analyzed by flow cytometry. Approximately 50-60% of the total initial assay beads are analyzed per sample, which corresponds to a more than 10-fold increase in sampling efficiency compared to Simoa. The analytical sensitivity of MOSAIC can be maximized by decreasing the number of total assay beads, increasing the target molecule to bead ratio, and thereby the signal to background ratio, while maintaining sufficient measurement precision. To exploit the possibility of enhancing sensitivity using lower bead numbers in MOSAIC, the number of assay beads was systematically varied, from 100,000 to 2,000 for IL-10. The signal to background ratio improved with decreasing bead number (Figure 2A). To quantify the improvement in the signal to background ratio, the signal to background ratio of the 0.2 fM calibrator was normalized to that of the 100,000 bead calibration curve in each calibration curve. This relative signal to background ratio increased as the number of beads was reduced, and a corresponding improvement in the limit of detection (LOD) was observed, enhancing the sensitivity by about one order of magnitude compared to 100,000 beads (Figure 2B). However, when the number of beads was further reduced to 2,000 beads, the analytical sensitivity did not improve further due to the increasing effect of Poisson noise on the measurement precision at very low bead numbers. Maximum sensitivity was achieved at 20,000 beads, with an LOD of 15.9 aM, representing a more than 12-fold enhancement in sensitivity compared to the corresponding Simoa assay (Figure 2C and Table 1). Standard Simoa assays typically use 500,000 total assay beads, but for a closer comparison with MOSAIC, 100,000 assay beads were used along with 400,000 helper beads for an improved signal to background ratio, despite fewer total beads being analyzed. Table 1. Limit of detection (LOD) and lower limit of quantification (LLOQ) values ​​of the optimized MOSAIC assay and the corresponding Simoa assay for various analytes. LOD and LLOQ values ​​are calculated as 3 and 10 standard deviations above background, respectively. [Table 1]

[0153] To assess whether reducing bead number could similarly enhance MOSAIC sensitivity for other analytes, the MOSAIC platform was extended to additional cytokines and similar changes in bead number for each analyte were performed (Figure 2D, Figures 6A-6E and Table 2). Improvements in signal to background upon reduction of bead number from 100,000 to 10,000 were also observed for interferon-gamma (IFN-γ), interleukin-8 (IL-8), and interleukin-12p70 (IL-12p70), with optimal sensitivity achieved at 10,000 beads for IFN-γ and IL-8. For IL-12p70, despite the increased signal to background, sensitivity remained similar across all bead numbers, although the lowest LOD of 5.8 aM was achieved using 100,000 assay beads. For IL-6 and IL-1β, there was a slight increase in signal to background upon decreasing bead number from 100,000. Consistent with this observation, the LOD did not improve appreciably with decreasing assay bead number. As expected from the lack of enhancement of signal over background, only a 3- to 5-fold improvement in sensitivity was observed compared to the corresponding Simoa assay for these analytes; these improvements can be attributed to increased sample collection efficiency. Our results show that MOSAIC achieves 3- to 12-fold higher sensitivity compared to Simoa, the current state-of-the-art for ultrasensitive protein detection (Table 1 and Figures 7A-7E). Due to the much higher sample collection efficiency of MOSAIC compared to standard digital ELISA, much smaller bead numbers up to 10,000 beads can be used to enhance signal over background for some analytes while maintaining sufficient measurement precision. The degree of signal over background improvement depends on the specific antibody pair, but the assay bead number can be easily optimized for each analyte. Table 2. Analytical sensitivity for all singleplex MOSAIC assays. Limits of detection (LOD) and lower limits of quantification (LLOQ) of MOSAIC assays performed for all cytokines across different assay bead numbers. [Table 2-1] [Table 2-2]

[0154] To test the reproducibility of the MOSAIC assay, the same calibration curve was measured on different days for some selected curves, and similar AMB values ​​and LODs were obtained. As a result, the average LOD of two separate calibration curves is reported for these curves. As a further confirmation of the reproducibility of these assays, the LOD obtained from the combination of all replicates was also evaluated between the separate curves in a single calibration curve, and values ​​similar to the reported average LOD values ​​were obtained.

[0155] Reducing the number of assay beads also reduces the number of capture antibody molecules present, so the capture rate may be slower. Therefore, we investigated whether a longer target capture time could further enhance the signal to background ratio and analytical sensitivity. However, for both IL-10 and IFN-γ, increasing the target capture time from 1 h to 4 h resulted in similar or slightly worse LODs (Figures 8A-8C). Therefore, for a more efficient workflow, a 1 h target capture time was used in subsequent experiments.

[0156] To determine the degree of sensitivity improvement resulting from improved sampling efficiency in MOSAIC, random sampling of subsets of beads was performed among the calibration curves obtained for IL-10 and IFN-γ to examine the effect of the number of beads analyzed on sensitivity and precision. Consistent with the high Poisson noise resulting from low event numbers, the LODs obtained when only a few hundred beads were analyzed were very high across all starting assay bead numbers (Figure 3A-B). For each subset size, random sampling was repeated 100 times, and high variation in the calculated LODs between replicate subsets was observed when 1,000 or fewer beads were analyzed, consistent with the low precision and reproducibility expected at these low sampling efficiencies. As the percentage of assay beads analyzed increases, analytical sensitivity improves considerably, with smaller increases in sensitivity as the number of beads analyzed increases beyond several thousand. Furthermore, the improvement in sensitivity with increasing sampling efficiency corresponded with a decrease in the measured coefficient of variation of the background signal (Figure 3C-3D). Thus, these results empirically support the important role of improved sampling efficiency in enhancing the sensitivity of MOSAIC.

[0157] MOSAIC improves the detectability of low-abundance analytes in biological fluids. We next exploited the performance of MOSAIC in biofluids to investigate whether its enhanced sensitivity could improve the detectability of low-abundance biomarkers. As a representative cytokine, IFN-γ was measured using both MOSAIC and the corresponding Simoa assay in a cohort of human plasma samples (Figures 4A-4B). IFN-γ concentrations measured by MOSAIC using 10,000 assay beads showed good correlation with those measured by Simoa, supporting the accuracy of the MOSAIC assay. Furthermore, although IFN-γ was generally detectable in plasma using both methods, one of 17 plasma samples remained undetectable by Simoa, whereas the more sensitive MOSAIC assay achieved 100% detectability.

[0158] To further evaluate whether the greater sensitivity of MOSAIC could improve the detectability of low-abundance analytes in biofluids, MOSAIC was applied to saliva. Because saliva contains minimal serum components filtered from blood to the salivary glands, many potential biomarkers are present at much lower levels in saliva than in blood, necessitating ultrasensitive techniques. As proof of principle, MOSAIC was used to measure levels of IFN-γ in a cohort of saliva samples. While IFN-γ was detectable in only 42% (11 / 26) of saliva samples using Simoa, the enhanced sensitivity of the low-bead MOSAIC assay improved the detectability of IFN-γ to over 65% (17 / 26) of saliva samples (Figures 4C-4D). All saliva samples with detectable IFN-γ levels using Simoa were detectable by MOSAIC, demonstrating positively correlated measurements. The wide variability between MOSAIC and Simoa measurements in saliva compared to plasma may be due to different matrix interference effects using the different assays. However, when spike-and-recovery experiments were performed in saliva, acceptable recoveries of 70-130% were observed (Table 3). The ability of MOSAIC to detect IFN-γ levels in some saliva samples that were undetectable by Simoa despite being above the LOD may be due not only to its enhanced signal-to-background and sensitivity at low bead numbers but also to a higher sample collection efficiency that increases measurement precision at low concentrations. Furthermore, the more thorough washes performed in MOSAIC compared to Simoa to minimize RCA products amplified from excess streptavidin-DNA may contribute to improved removal of interfering components. Table 3. Recovery of spiked recombinant human IFN-γ and IL-12p70 protein for MOSAIC assays performed on saliva from three individual human saliva samples. Saliva samples were diluted 4-fold with StartingBlock™ Blocking Buffer (Thermo Fisher Scientific). Recovery is reported as the mean ± standard deviation of duplicate determinations. [Table 3]

[0159] The ability of MOSAIC to detect attomolar concentrations of endogenous proteins that are undetectable by Simoa highlights its potential diagnostic utility for very low abundance biomarkers. Improved detection of an even lower abundance analyte in saliva, IL-12p70, was also achieved using MOSAIC, from 0% by Simoa to approximately 12%, or in 3 of 26 saliva samples (Figures 9A-9C). Although the sensitivity of MOSAIC remained insufficient to detect IL-12p70 in the majority of tested saliva samples, the results indicate that its enhanced sensitivity not only begins to reveal the "tip of the iceberg" of such rare analytes in saliva, but also highlights the need for even more sensitive methods.

[0160] MOSAIC multiplexing function. In addition to the enhanced sensitivity of MOSAIC, its on-bead signal generation strategy and flow cytometric readout can expand the multiplexing capabilities of digital ELISA. Current digital immunoassays are typically limited in the number of analytes that can be multiplexed, due in part to constraints on the total number of compartments for isolating individual targets. 15In contrast, MOSAIC's solution-based readout in principle allows for the analysis of an infinite number of bead types in one sample, with sensitivity and dynamic range easily tunable by the number of assay beads for each analyte. To validate MOSAIC's multiplexing capabilities, we first developed a multiplexed assay for IL-6, IL-1β, IL-10, and IFN-γ using beads coded to different fluorochromes. The assay showed moderate to high attomole sensitivity and acceptable recovery in human plasma, with little cross-reactivity in the concentration range of cytokines present in plasma (Table 4 and Figures 10A-10D). As further validation, its measurements in plasma correlated well with those of the corresponding four-plex Simoa assay, especially for analyte concentrations well above the LLOQ of both methods (Figure 5A). Table 4. Recovery of spiked recombinant proteins for the 4-plex MOSAIC assay reported in Figure 5 in human plasma at 4-fold dilutions. Recovery is reported as the mean ± standard deviation of duplicate determinations. [Table 4]

[0161] Once the ability of MOSAIC to measure multiple analytes simultaneously with high accuracy was established, we explored increasing the multiplexing versatility of MOSAIC by incorporating additional beads with different fluorescent dyes and intensities. As proof of principle, the MOSAIC assay was integrated into a previously unreported 8-plex assay with a digital ELISA method for the cytokines IL-6, IL-1β, IL-10, IFN-γ, IL-12p70, IL-5, and IL-18, as well as vascular endothelial growth factor (VEGF). High analytical sensitivity was maintained, with LOD values ​​ranging from mid-attomolar to low femtomolar concentrations (Figures 11A-11D and Tables 5A-5B). The assay was applied to human plasma using a 16-fold dilution factor to ensure acceptable recovery and consistent dilution linearity across all analytes in spike-and-recovery experiments (Tables 6A, 6B, and 7 and Figures 12 and 16). Importantly, there was little cross-reactivity across the measured concentration range (Figure 13). The exceptional sensitivity of MOSAIC further allows for the use of higher dilutions to minimize potential false signals due to cross-reactivity, in addition to enabling the collection of more clinical information from limited sample volumes. Using less than 15 μL of plasma across two replicates, these eight proteins were measured in a cohort of plasma samples and the measurements were compared to those of two quadruple Simoa assays. The measured concentrations correlated overall between the two methods for the analytes showing high detectability, further supporting the accuracy of the highly multiplexed MOSAIC assay (Figures 5B-5C and Figures 14A-B). Thus, MOSAIC allows for ultrasensitive multiplexed measurements of a broad panel of biomarkers using very small sample volumes. Table 5A. Limit of detection (LOD) and lower limit of quantification (LLOQ) values ​​for the 8-plex MOSAIC assay and the corresponding 4-plex Simoa assay in the sample diluent used for plasma measurements. Values ​​are reported as the median [range] of three calibration curves performed on separate days. LOD and LLOQ values ​​are calculated as 3 and 10 standard deviations above background, respectively. [Table 5-1] Table 5B. Limit of detection (LOD) and lower limit of quantification (LLOQ) values ​​for the 8-plex MOSAIC assay and the corresponding 4-plex Simoa assay in the sample diluent used for saliva measurements. LOD and LLOQ values ​​are calculated as 3 and 10 standard deviations above background, respectively. [Table 5-2] Table 6A. Recovery of spiked recombinant human proteins for the 8-plex MOSAIC assay in human plasma at 8-fold dilution. Recovery is reported as the mean ± standard deviation of duplicate determinations. [Table 6-1] Table 6B. Recovery of spiked recombinant human proteins for the 8-plex MOSAIC assay in human saliva at 8-fold dilution. Recovery is reported as the mean ± standard deviation of duplicate determinations. [Table 6-2] Table 7. Recovery of spiked recombinant human proteins for the 8-plex MOSAIC assay in human plasma at a dilution of 16. Recovery is reported as the mean ± standard deviation of duplicate determinations. [Table 7]

[0162] Consideration The ability to measure very low levels of protein biomarkers remains a significant challenge in clinical applications such as the diagnosis of cancer, infectious diseases, and neurodegenerative diseases. Although digital measurement techniques have enabled a thousand-fold increase in sensitivity, proteins at attomolar or lower concentrations are still not available, limiting efforts in biomarker discovery and early detection of diseases where biomarker levels may be very low in the early stages. One notable example is saliva, which is collected non-invasively and contains a variety of proteins with potential diagnostic value, but at much lower levels than in plasma, presenting a particular challenge to current digital ELISA techniques. Proof-of-principle measurements of IFN-γ and IL-12p70 in saliva highlight the potential utility of MOSAIC in uncovering an unknown area of ​​rare biomarker candidates. Importantly, the high sample collection efficiency of MOSAIC allowed us to reduce the number of assay beads to enhance the signal over background while maintaining sufficient measurement accuracy. Although reducing the number of beads improved sensitivity to different degrees for different analytes, the results indicate that the number of beads for each analyte can be easily adjusted according to the desired sensitivity and dynamic range required for a particular application.

[0163] Importantly, MOSAIC avoids the requirement for specialized equipment that has limited the widespread implementation of digital ELISA technology. By removing the need to isolate beads into individual compartments, MOSAIC enables single molecule counting using flow cytometry, thus turning digital ELISA into a current and widely available testing platform. A key advantage of MOSAIC compared to previous digital ELISA methods, including the recently developed drop-cast single molecule assay and droplet digital ELISA, is the much more rapid and automated - less than 1 minute signal readout per sample. The drop-cast single molecule assay (dSimoa) enables a simple bead drop-cast method for counting single molecules captured on beads, increasing sampling efficiency and analytical sensitivity by approximately 10-fold. However, these methods require long imaging times per sample to capture all beads across multiple fields of view, thus limiting throughput. Automation with 96-well plate sampling modes already built into many benchtop flow cytometers further provides a state-of-the-art workflow.

[0164] In addition to enhanced sensitivity and an accessible workflow, MOSAIC introduces increased multiplexing capabilities to digital ELISA. The ability to simultaneously measure multiple analytes in a single sample can accelerate sample throughput and biomarker signature discovery, and is particularly important in applications with limited sample volumes, such as neonatal saliva or finger-prick blood. Multiplexing in digital ELISA as well as recently developed ultrasensitive planar ELISA platforms have shown great utility in a variety of diagnostic applications. 16~18In contrast, the number of bead types that can be analyzed simultaneously in bead entrapment methods is limited by the total number of compartments and the low bead analysis efficiency. In contrast, the solution-based readout of MOSAIC removes this physical constraint on the total number of bead types that can be analyzed in a single sample. Because the sampling efficiency in MOSAIC is not affected by the total number of beads, the desired dynamic range of each analyte in a multiplexed MOSAIC assay is easily adjustable by the number of assay beads, and there is no upper limit on the number of beads or bead types in each sample. In addition to multiplexing by color and fluorescence intensity, the versatile flow cytometry readout offers the ability to discriminate beads by size, thus expanding the palette of multiplexable bead types. Although cross-reactivity remains a potential limitation in higher order multiplexed assays, the attomolar sensitivity of MOSAIC allows the use of higher dilutions while maintaining good target detectability, thus keeping measurements in a concentration range where cross-reactivity is minimal. Furthermore, smaller multiplexed panels can be used with signal readout as well as subsequent combination of all beads in a single sample for sequential target capture. 19 .

[0165] Despite the low attomole sensitivity of MOSAIC, further work is needed to achieve even higher sensitivity and detectability for rare analytes. Development and screening of higher affinity reagents could be used to achieve sub-attomole LODs. Furthermore, although the washing steps in the current MOSAIC workflow are automated using a microplate washer, further work will explore the integration of the entire assay into an automated liquid handling platform for improved measurement accuracy.

[0166] Taken together, by harnessing the power of on-bead signal amplification and the versatile capabilities of flow cytometry, MOSAIC offers an ultrasensitive protein detection method with attomole sensitivity, higher orders of multiplexing, and rapid high-throughput readout that is accessible to any laboratory with a flow cytometer.

[0167] method material All affinity reagents, recombinant proteins, and DNA oligos used in this study are listed below. Buffers and paramagnetic beads were purchased from Quanterix Corporation and Bangs Laboratories. Custom DNA oligos were purchased from Integrated DNA Technologies and MilliporeSigma. [Table 10-1] [Table 10-2]

[0168] Preparation of capture and labeling reagents. The capture antibody was buffer exchanged with Bead Conjugation Buffer (Quanterix) using a 50K Amicon Ultra-0.5mL centrifugal filter (MilliporeSigma). Buffer exchange was performed by adding Bead Conjugation Buffer to the antibody solution in the filter to 500 μL, followed by centrifugation at 14,000×g for 5 min three times, with 450 μL of Bead Conjugation Buffer added between centrifugation cycles. The buffer-exchanged antibody was recovered by inverting the filter in a new tube, centrifuging at 1000×g for 2 min, rinsing the filter with 50 μL of Bead Conjugation Buffer, and centrifuging once more at 1000×g for 2 min. The concentration of the buffer-exchanged antibody was then measured using a NanoDrop spectrophotometer. For each bead type, the beads were washed three times with 300 μL of Bead Wash Buffer (Quanterix) and twice with 300 μL of Bead Conjugation Buffer (Quanterix) before being resuspended in cold Bead Conjugation Buffer. The number of beads and conjugation conditions for each analyte are shown in the table below. A 1 mg vial of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (Thermo Fisher Scientific) was dissolved in 100 μL of cold Bead Conjugation Buffer and the desired volume was added to the beads. The beads were shaken for 30 minutes at either room temperature or 4°C. After EDC activation of the carboxyl groups on the beads, the beads were washed once with 300 μL of cold Bead Conjugation Buffer before being resuspended in the buffer-exchanged antibody solution. Antibody conjugation was performed by shaking the beads for 2 h at either room temperature or 4° C., followed by washing twice with 300 μL of Bead Wash Buffer. The antibody-coupled beads were then blocked with 300 μL of Bead Blocking Buffer (Quanterix) for 30 min at room temperature with shaking.After washing once each with 300 μL of Bead Wash Buffer and Bead Diluent (Quanterix), the beads were resuspended in Bead Diluent, counted using a Beckman Coulter Z1 Particle Counter, and stored at 4°C. Table 8. Coupling conditions for antibody-coated capture beads used in this example. For comparison with the 8-plex MOSAIC assay and Simoa, 4-plex assays were performed for IL-6, VEGF, IL-18, and IL-12p70 using 488, 647, 700, and 750 multiplex beads, respectively, from Quanterix. [Table 8]

[0169] The detection antibodies were obtained in biotinylated form, except for the IFN-γ detection antibody, which was obtained in unmodified form. The IFN-γ detection antibody was biotinylated by reconstituting it to 1 mg / mL in Biotinylation Reaction Buffer (Quanterix) and adding a 40-fold molar excess of NHS-PEG4-Biotin (Thermo Fisher Scientific) freshly dissolved in water. The biotinylation reaction was carried out for 30 min at room temperature, after which the biotinylated antibody was purified using a 50K Amicon Ultra-0.5 mL centrifugal filter. After five centrifugation cycles at 14,000×g for 5 min with the addition of 450 μL of Biotinylation Reaction Buffer between cycles, the purified antibody was collected by inversion of the filter in a new tube and centrifugation at 1000×g for 2 min. The filters were then rinsed with 50 μL of Biotinylation Reaction Buffer, followed by another centrifugation at 1000×g for 2 min, and the antibody concentration was quantified using a NanoDrop spectrophotometer.

[0170] Preparation of streptavidin-DNA conjugates. The 5' azido-modified primer was annealed to the DNA template for RCA by heating a solution of 33.8 μM primer and 40.6 μM template in NEBNext Quick Ligation Buffer (New England Biolabs) at 95°C for 2 minutes and cooling to room temperature for 90 minutes. Ligation was then performed by adding T4 DNA ligase and incubating at room temperature for 3 hours. The ligation reaction was then heated at 65°C for 10 minutes to inactivate the ligase and then cooled to room temperature. The ligation reaction was buffer exchanged into phosphate-buffered saline (PBS) containing 1 mM EDTA using 7K MWCO Zeba spin desalting columns (Thermo Fisher Scientific). For conjugation, streptavidin (Biolegend 280302) was buffer exchanged into PBS using a 10K Amicon Ultra-0.5mL centrifugal filter, incubated with a 20-fold molar excess of dibenzocyclooctyne-PEG4-N-hydroxysuccinimidyl ester (DBCO-PEG4-NHS, MilliporeSigma) for 30 min at room temperature, and purified using a 10K Amicon Ultra-0.5mL centrifugal filter in PBS containing 1 mM EDTA. A 2-fold molar excess of the ligated primer-template was then added to the DBCO-modified streptavidin and incubated overnight at 4°C. The conjugate was stored in aliquots at -80°C in PBS containing 5 mM EDTA, 0.1% BSA, and 0.02% sodium azide.

[0171] MOSAIC assay MOSAIC assays were performed in 96-well plates (Greiner Bio-One, 655096) with antibody-coated beads and detection antibodies diluted to the desired concentrations in Homebrew Sample Diluent (Quanterix). A sample volume of 100 μL was used with 10 μL of antibody-coated beads. For the two-step assay, 10 μL of detection antibody was added to each sample. The plate was sealed and shaken for 1 h for target capture, then washed with System Wash Buffer 1 (Quanterix) using a BioTek 405 TS Microplate Washer. For the three-step assay, 100 μL of detection antibody was added to the beads after the target capture and wash steps, followed by incubation for 10 min and further washing. The immune complex sandwich was then labeled with streptavidin-DNA by adding 100 μL of conjugate diluted in Sample Diluent with 5 mM EDTA to the beads and shaking for the desired time. The samples were then washed for eight cycles with System Wash Buffer 1, transferred to a new 96-well plate, washed once more with 200 μL of System Wash Buffer 1, and then resuspended in 60 μL of RCA reaction mixture. The RCA mixture consisted of 0.5 mM deoxynucleotide mix (New England Biolabs), 0.33 U / μL phi29 DNA polymerase (Lucigen), 0.2 mg / mL bovine serum albumin (BSA, New England Biolabs), 1 nM fluorescently labeled DNA probe (Integrated DNA Technologies), and 0.1% Tween®-20 in 50 mM Tris-HCl (pH 7.5), 10 mM (NH4)2SO4, and 10 mM MgCl2. ATTO-647N and ATTO-565 labeled DNA probes were used for single target and multiplex MOSAIC assays, respectively.Upon addition of the RCA mixture to each sample, the plate was shaken for 1 h at 37° C., after which 150 μL of PBS containing 0.1% Tween®-20 and 5 mM EDTA was added to stop the reaction. Samples were washed once with 200 μL of the same PBS-Tween®-EDTA buffer and resuspended in 100 μL of buffer supplemented with 0.1% BSA. Samples were measured using a CytoFlex LX flow cytometer (Beckman Coulter) equipped with six lasers in either tube or plate sampling mode. Bleach and buffer wells were included between different samples to minimize potential sample carryover. Multiplex MOSAIC assays were performed following the same protocol as singleplex MOSAIC assays, combining beads coded with different fluorochromes in the same sample.

[0172] Plasma and saliva samples were diluted in Sample Diluent or StartingBlock™ Blocking Buffer (Thermo Fisher Scientific), respectively, containing protease inhibitors (Halt™ Protease Inhibitor Cocktail, Thermo Fisher Scientific). Plasma samples were obtained from BioIVT and Mass General Brigham Biobank, and saliva samples were obtained from BioIVT. All human samples were anonymized, and the experiments were performed under Institutional Review Board approval by Mass General Brigham. All plasma and saliva samples were centrifuged at 2000×g for 10 min or 21000×g for 20 min, respectively, at 4° C., before dilution for measurement. Table 9. Assay conditions used for the MOSAIC and Simoa assays in this example. The same detection antibody concentrations and target capture times were used for each corresponding MOSAIC and Simoa assay. [Table 9]

[0173] Simoa assay All Simoa assays were performed on a HD-X Analyzer (Quanterix) with automated sample processing, image analysis, and calculation of average enzyme per bead (AEB). The same antibody-coated beads used in the singleplex MOSAIC assay were used for all Simoa assays, with the same detection antibody concentration as the MOSAIC assay. 100,000 antibody-coated beads and 400,000 helper (unconjugated) beads were used for each single Simoa assay, while 125,000 antibody-coated beads per analyte were used for each quadruple Simoa assay. Streptavidin-β-galactosidase (SβG) concentrate (Quanterix) was diluted with SβG Diluent (Quanterix) to the desired concentration for each assay. All assay reagents and consumables were loaded into the HD-X Analyzer according to the manufacturer's instructions.

[0174] Data analysis Flow cytometry data were first analyzed using FlowJo™ Software (Becton, Dickinson and Company); beads were identified using gates on forward scatter, side scatter, and bead fluorescence. Forward scatter was used to further gate on single beads (Figure 15A-D). Probe fluorescence intensity for each bead population was analyzed in Python using the packages jax, numpy, pandas, scikit-learn, and waltlabtools. Two Gaussian mixture models were fitted to the log-transformed fluorescence intensity for each well using expectation maximization. Two methods of counting "on" beads were calculated. In the first method, beads were assigned as "on" or "off" based on their predicted membership in one or the other Gaussian according to the expectation maximization algorithm. In the second method, beads were counted as "on" if they were at least 5 standard deviations above the mean of the lower ("off") Gaussian peak. When two peaks were identifiable, the first method gave better results, but when the number of "on" beads was very low, the higher Gaussian was more difficult to fit and therefore the second method gave a more reliable estimate. The metric used was therefore a weighted average of the two, with a sinusoidal weighting function: the weights in the first method were sin 4 The fraction of "on" beads is given by (πf / 2), where f is the fraction of "on" beads according to the first method (Gaussian mixture assignment). The fraction of "on" beads was then converted to the average number of analyte molecules bound per bead using Poisson statistics and mapped to concentration using a four-parameter logistic calibration curve. LOD and LLOQ were calculated to be 3 and 10 standard deviations above background, respectively, where a correction factor c4(n) was applied to unbiased estimates of the standard deviation. Pearson's correlation coefficients were calculated using GraphPad Prism.

[0175] A more detailed version of the method can be found in the Supplementary Methods section below.

[0176] Supplementary method Preparation of antibody-coated capture beads 1. Vortex the beads for 30 seconds and place them on a shaker. 2. If the antibody has been lyophilized, reconstitute in MES buffer to 0.2 mg / mL. 3. If the antibody is in solution, perform a buffer exchange. i. Measure antibody stock concentration or mass using a spectrophotometer or manufacturer's certificate of analysis. Calculate the volume of antibody stock solution that contains 80 mg of antibody. ii. Bring this volume up to 500 μL total volume with enough MES buffer and add to a clean 50K Amicon filter in an Amicon microcentrifuge tube. Centrifuge at 14,000×g for 5 minutes. Discard the flow-through and quickly add 450 μL of MES buffer to the Amicon filter. Centrifuge at 14,000×g for 5 minutes. Repeat one more time for a total of three 5 minute centrifugations. Discard the flow-through. Quickly invert the clean Amicon tube with the Amicon filter top side up. Centrifuge the tube with the inverted Amicon filter at 1,000 x g for 2 minutes. v. Rinse the filter membrane with 50 μL of MES buffer. Mix repeatedly. Centrifuge the tube with the inverted Amicon filter at 1,000 × g for 2 min. Measure antibody concentration using a spectrophotometer, blanking with MES buffer. Measure the volume of purified antibody with a pipette. Dilute the antibody to 0.2 mg / mL with MES buffer. 4. Wash the beads. i. Determine bead count using a particle size analyzer or manufacturer's certificate of analysis: 4.2 x 10 8 Calculate the volume of bead stock solution containing beads. ii. Transfer the volume of beads given in the table above into a test tube. Place the test tube on the magnetic separator. iii. Aspirate, remove from magnetic separation device, add 300 μL Bead Wash Buffer, vortex, pulse spin, and return to magnetic separation device. Repeat two more times with Bead Wash Buffer and two times with MES buffer. iv. Aspirate, remove from magnetic separator, add 300 μL MES buffer a third time, vortex and pulse spun, leaving beads in suspension. 5. Activate the beads. Carefully open one vial of i.EDC and slowly add 100 μL of MES buffer. Immediately replace the cap and vortex briefly until dissolved. ii. Add 9 pL of reconstituted EDC to the tube of beads, vortex and shake at 4 °C for 30 min. 6. Conjugate the antibody to the beads. i. Pulse spin, place beads on magnetic separation device, aspirate, remove from magnet, add 300 μL MES buffer, vortex, pulse spin, and return to magnetic separation device. ii. Aspirate, remove from magnetic separator, add achieved batch volume of buffer exchanged antibody, vortex. Shake for 2 hours at 4°C. 7. Block the beads. i. Pulse spin and place beads on magnetic separator. ii. Aspirate, remove from magnetic separation device, add 300 μL Bead Wash Buffer, vortex, pulse spin, and return to magnetic separation device. Repeat once. iii. Aspirate, remove from magnetic separator, add 300 μL of Bead Blocking Buffer, vortex, and shake at room temperature for 45 minutes. 8. Perform a final wash and resuspension. i. Pulse spin and place beads on magnetic separator. ii. Aspirate, remove from magnetic separation device, add 300 μL Bead Wash Buffer, vortex, pulse spin, and return to magnetic separation device. Repeat once. iii. Aspirate, remove from magnetic separation device, add 300 μL Bead Diluent, vortex, and pulse spin. iv. Determine the final bead count using a particle size analyzer. Note: Alternatively, 2.8×10 8 Bead coupling using starting beads can be performed at room temperature, with the EDC activation and antibody conjugation steps being performed at room temperature.

[0177] Biotinylation of detection antibodies 1. If the antibody is lyophilized, reconstitute in PBS to 1 mg / mL. 2. If the antibody is in solution, perform a buffer exchange. Measure the antibody stock concentration or mass using a spectrophotometer or manufacturer's certificate of analysis. Calculate the volume of antibody stock solution that contains 130 mg of antibody. ii. Bring this volume to 500 μL total volume with enough PBS and add to a clean 50K Amicon filter in an Amicon microcentrifuge tube. Centrifuge at 14,000×g for 5 minutes. Discard the flow-through and quickly add 450 μL of PBS to the Amicon filter. Centrifuge at 14,000×g for 5 minutes. Repeat one more time for a total of three 5 minute centrifugations. Discard the flow-through. Quickly invert the clean Amicon tube with the Amicon filter top side up. Centrifuge the tube with the inverted Amicon filter at 1,000 x g for 2 minutes. v. Rinse the filter membrane with 50 μL of PBS. Mix repeatedly. Centrifuge the tube with the inverted Amicon filter at 1,000 × g for 2 minutes. vi. Measure antibody concentration using a spectrophotometer, blanking with PBS. Measure the volume of purified antibody with a pipette. Dilute the antibody in PBS to 1 mg / mL. 3. Biotinylate the detection antibody. i. Reconstitute biotin to a stock concentration of 8.9 mM by adding 383 µL of deionized water. ii. Add 100 μL of antibody and 3 μL of biotin to a clean tube, vortex, pulse spin, and incubate at room temperature for 30 minutes. 4. Purify the biotinylated antibody. i. Transfer the contents of the biotinylation reaction tube to a clean Amicon filter in an Amicon microcentrifuge tube. Wash the biotinylation reaction tube with 400 μL of PBS and transfer it to the Amicon filter as well. Centrifuge at 14,000×g for 5 minutes. ii. Discard the flow-through and quickly add 450 μL of PBS to the Amicon filter. Centrifuge at 14,000×g for 5 minutes. Repeat three more times for a total of five 5-minute centrifugations. Discard the flow-through. Quickly invert the clean Amicon tube with the Amicon filter top side up. Centrifuge the tube with the inverted Amicon filter at 1,000 x g for 2 minutes. iv. Rinse the filter membrane with 50 μL of PBS. Mix repeatedly. Centrifuge the tube with the inverted Amicon filter at 1,000 × g for 2 minutes. v. Measure antibody concentration using a spectrophotometer, blanking with PBS.

[0178] Preparation of streptavidin-DNA conjugates Make a solution of 35.8 μM primer and 43.0 μM template in 1.1× Quick Ligation Buffer. 2. Heat the solution to 95° C. for 2 minutes and allow to cool to room temperature over 90 minutes. 3. Add T4 DNA ligase (6 µL of 2,000,000 U / mL per 100 µL reaction mixture) and incubate at room temperature for 3 hours. 4. Heat at 65°C for 10 minutes to inactivate the ligase, then cool to room temperature. 5. Buffer exchange into PBS containing 1 mM EDTA using Zeba spin desalting columns. 6. Using the molecular weights of the primer and template and the initial primer concentration in the ligation reaction, determine the buffer exchanged primer concentration by measuring the concentrations using a spectrophotometer. 7.Buffer exchange the streptavidin into PBS using a 10K Amicon filter and measure the concentration using a spectrophotometer. 8. Incubate streptavidin with a 20-fold molar excess of DBCO-PEG4-NHS, dissolved in dimethylsulfoxide at 5 mg / mL, for 30 minutes at room temperature. 9. Purify the DBCO-modified streptavidin using a 10K Amicon centrifugal filter in PBS containing 1 mM EDTA using four centrifugation cycles at 14,000 x g for 5 min and a final centrifugation cycle at 14,000 x g for 10 min. Measure the concentration using a spectrophotometer. A 10.2-fold molar excess of ligated primer-template (molar excess calculated relative to primer concentration determined in step 5) is added to the DBCO-modified streptavidin and incubated overnight at 4°C. 11. Store the conjugate in aliquots at -80°C in PBS containing 5mM EDTA, 0.1% BSA, and 0.02% sodium azide.

[0179] MOSAIC assay procedure For all washes on the plate washer, use System Wash Buffer 1, ensuring that the beads are held to the bottom of the plate by magnetic binding. Each wash cycle is 170 μL. 1. Vortex the beads for 30 seconds and dilute with Bead Diluent to the appropriate concentration to obtain the desired number of beads in 10 μL / sample. We used 2×10 6 It is recommended to start with 20,000 beads / sample, which is 100 beads / mL. For multiplexing, mix matching amounts of each bead type. 2. Perform serial dilutions of protein standards in Sample Diluent for the calibration curve. 3. Centrifuge the sample and dilute with sample diluent. We recommend: a. Plasma / serum: Centrifuge at 2,000 x g for 10 minutes and dilute 4 to 16 times. b. Saliva: Centrifuge at 21,000 x g for 20 minutes and dilute 4 to 16 times. c. Urine: Centrifuge at 10,000 x g for 5 minutes and dilute 1 to 2 times. 4. Add 100 μL of diluted sample (or calibrator) and 10 μL of beads to each well. 5. If performing the assay in step 2, dilute biotinylated detection antibody to 3.6 μg / mL. Add 10 μL of detection antibody to each well. 6. Seal the plate and incubate at room temperature for 1 hour with shaking at 650 rpm. 7. If performing a 3-step assay: i. Wash samples 3 times on a plate washer. ii. Dilute biotinylated detection antibody to 0.3 μg / mL. iii. Place plate on plate magnet to attract beads to one side of wells. Aspirate and add 100 μL of detection antibody to each well. iv. Seal the plate and incubate at room temperature for 10 minutes with shaking at 600 rpm. 8. Wash samples six times on the plate washer. 9. Place plate on plate magnet to attract beads to one side of wells. Aspirate and add 100 μL of streptavidin-DNA conjugate diluted to 300 pM (for 2-step assay) or 150 pM (for 3-step assay) in Sample Diluent containing 5 mM EDTA to each well. 10. Seal the plate and incubate at room temperature for 15 minutes (for the 2-step assay) or 10 minutes (for the 3-step assay) with shaking at 650 rpm. 11. Buffer exchange the phi29 polymerase into 10 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.1 mM EDTA, 0.5% Tween®-20, and 0.5% NP-40 using Zeba desalting columns to remove dithiothreitol. 12. Make the RCA reaction mixture: - 0.5mM dNTP mix - 0.33U / μL phi29 DNA polymerase - 0.2mg / mL BSA - 1 nM fluorescently labeled DNA probe - 0.1% Tween®-20 - 50mM Tris-HCl - 10mM Ammonium Sulfate - 10mM Magnesium Chloride 13. Wash samples 8 times on the plate washer. 14. Pipette mix to resuspend the beads and transfer samples to a new 96-well plate. 15. Place plate on plate magnet to attract beads to one side of wells. Aspirate and add 200 μL System Wash Buffer. Aspirate and resuspend in 60 μL RCA Reaction Mix. 16. Seal the plate and incubate at 37° C. for 1 hour with shaking at 650 rpm. 17. Add 150 μL of PBS with 0.1% Tween®-20 and 5 mM EDTA to each well to stop the reaction. Place plate on plate magnet to draw beads to one side of well and aspirate (aspirate to avoid complete dryness). Add 200 μL of the same PBS-Tween®-EDTA buffer, aspirate, and resuspend in 100 μL of buffer supplemented with 0.1% BSA. 18. Run samples on the flow cytometer within 24 hours. Bleach and buffer or water are run between different samples to minimize potential sample carryover. 19. Identify bead populations using flow cytometry analysis software. We recommend gating beads based on size / shape (forward and side scatter) and then fluorescence. 20. Separate on-beads from off-beads using gates in the flow cytometry analysis software or the waltlabtools.mosaic Python module. 21. Using software such as the waltlabtools Python package: i. Convert the fraction of on-beads for each sample to average molecules per bead (AMB): AMB = -log(1-f on ). ii. A four parameter logistic calibration curve is regressed onto the calibration concentrations and AMB. iii. Calculate the concentration of the samples using the calibration curve.

[0180] Incubation times; sample dilutions and diluents; assay protocols (2-step vs. 3-step); and concentrations of beads, detection antibodies, and streptavidin-DNA conjugates can be optimized for each assay or application. References 1 Rissin, DM et al. Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nature Biotechnology 28, 595-599, doi:10.1038 / nbt.1641 (2010). 2 Cohen, L. & Walt, DR Highly Sensitive and Multiplexed Protein Measurements. Chemical Reviews 119, 293-321, doi:10.1021 / acs.chemrev.8b00257 (2019). 3 Yelleswarapu, V. et al. Mobile platform for rapid sub-picogram-per-milliliter, multiplexed, digital droplet detection of proteins. Proceedings of the National Academy of Sciences 116, 4489, doi:10.1073 / pnas.1814110116 (2019). 4 Cohen, L. et al. Single Molecule Protein Detection with Attomolar Sensitivity Using Droplet Digital Enzyme-Linked Immunosorbent Assay. ACS Nano 14, 9491-9501, doi:10.1021 / acsnano.0c02378 (2020). 5 Chang, L. et al. Single molecule enzyme-linked immunosorbent assays: Theoretical considerations. Journal of Immunological Methods 378, 102-115, doi:https: / / doi.org / 10.1016 / j.jim.2012.02.011 (2012). 6 Wu, C., Garden, P. M. & Walt, D. R. Ultrasensitive Detection of Attomolar Protein Concentrations by Dropcast Single Molecule Assays. Journal of the American Chemical Society 142, 12314-12323, doi:10.1021 / jacs.0c04331 (2020). 7 Maley, A. M., Garden, P. M. & Walt, D. R. Simplified Digital Enzyme-Linked Immunosorbent Assay Using Tyramide Signal Amplification and Fibrin Hydrogels. ACS Sensors 5, 3037-3042, doi:10.1021 / acssensors.0c01661 (2020). 8 Kan, C. W. et al. Digital enzyme-linked immunosorbent assays with sub-attomolar detection limits based on low numbers of capture beads combined with high efficiency bead analysis. Lab on a Chip 20, 2122-2135, doi:10.1039 / D0LC00267D (2020). 9 Smith, L. D. et al. High-Fidelity Single Molecule Quantification in a Flow Cytometer Using Multiparametric Optical Analysis. ACS Nano 14, 2324-2335, doi:10.1021 / acsnano.9b09498 (2020). 10 Akama, K., Shirai, K. & Suzuki, S. Droplet-Free Digital Enzyme-Linked Immunosorbent Assay Based on a Tyramide Signal Amplification System. Analytical Chemistry 88, 7123-7129, doi:10.1021 / acs.analchem.6b01148 (2016). 11 Yang, K. S. et al. Extracellular Vesicle Analysis Allows for Identification of Invasive IPMN. Gastroenterology 160, 1345-1358.e1311, doi:10.1053 / j.gastro.2020.11.046 (2021). 12 Iyer, S. S. & Cheng, G. Role of interleukin 10 transcriptional regulation in inflammation and autoimmune disease. Crit Rev Immunol 32, 23-63, doi:10.1615 / critrevimmunol.v32.i1.30 (2012). 13 Moore, K. W., de Waal Malefyt, R., Coffman, R. L. & O’Garra, A. Interleukin-10 and the Interleukin-10 Receptor. Annual Review of Immunology 19, 683-765, doi:10.1146 / annurev.immunol.19.1.683 (2001). 14 Reynolds, D. in Encyclopedia of Biometrics (eds Stan Z. Li & Anil Jain) 659-663 (Springer US, 2009). 15 Rivnak, A. J. et al. A fully-automated, six-plex single molecule immunoassay for measuring cytokines in blood. Journal of Immunological Methods 424, 20-27, doi:https: / / doi.org / 10.1016 / j.jim.2015.04.017 (2015). 16 Ahmad, R. et al. A rapid triage test for active pulmonary tuberculosis in adult patients with persistent cough. Science Translational Medicine 11, eaaw8287, doi:10.1126 / scitranslmed.aaw8287 (2019). 17 Norman, M. et al. Ultra-Sensitive High-Resolution Profiling of Anti-SARS-CoV-2 Antibodies for Detecting Early Seroconversion in COVID-19 Patients. medRxiv, 2020.2004.2028.20083691, doi:10.1101 / 2020.04.28.20083691 (2020). 18 Tobos, C. I., Sheehan, A. J., Duffy, D. C. & Rissin, D. M. Customizable Multiplex Antibody Array Immunoassays with Attomolar Sensitivities. Analytical Chemistry 92, 5613-5619, doi:10.1021 / acs.analchem.0c00631 (2020). 19 Gilboa, T., Maley, A. M., Ogata, A. F., Wu, C. & Walt, D. R. Sequential Protein Capture in Multiplex Single Molecule Arrays: A Strategy for Eliminating Assay Cross-Reactivity. Advanced Healthcare Materials n / a, 2001111, doi:10.1002 / adhm.202001111 (2020).

[0181] (Example 2) Barcoded multiplex MOSAIC assay This example describes a barcoded multiplex MOSAIC assay. In the barcoded multiplex MOSAIC assay, a unique pair of fluorescent dye-coded capture beads and DNA template barcoded detection antibodies was used for each analyte (Figure 18). The capture beads were prepared as described herein, and the antibody-DNA conjugates were prepared as detailed below. The antibodies conjugated to each unique DNA template, when amplified during the RCA reaction, were labeled with a specific fluorescent dye-conjugated DNA probe or a ratiometric combination of dye-conjugated DNA probes. The color or ratiometric combination of colors of each probe can be distinguished using multiple detection channels in flow cytometry. Upon capture of a single analyte molecule on the bead, a mixture of detection antibodies conjugated to a unique primer-template sequence was added, followed by washing and resuspension in the RCA reaction containing a mixture of fluorescent dye-conjugated DNA probes. Each analyte corresponded to a unique pairing of (1) capture bead color, fluorescence intensity, or size and (2) fluorescent probe color or color ratio. As a result, only "correct" matched pairs of capture bead and probe signal were classified as "on" beads for each analyte, while "incorrect" pairs of capture bead and probe color, i.e., cross-reactive binding events, were excluded from further analysis. In contrast, in the non-barcoded multiplexed MOSAIC format, all detection antibodies were biotinylated and labeled with the same streptavidin-DNA conjugate and fluorescent probe.

[0182] Figures 19A and 19B depict the dropout curves for increasing concentrations of each individual target protein for non-barcoded multiplex MOSAIC (Figure 19A) and barcoded multiplex MOSAIC (Figure 19B). As shown by the figures, inclusion of only the correct capture bead-probe color pairs in the barcoded multiplex MOSAIC allows for the elimination of cross-reactive signals, thus providing a more accurate multiplex measurement.

[0183] Preparation of antibody-DNA conjugates For each detection antibody, the 5' azido-modified primer was annealed to its unique DNA template by heating a solution of 30 μM primer and 30.3 μM template in NEBNext Quick Ligation Buffer (New England Biolabs) at 95°C for 2 min and cooling to room temperature over 90 min. Ligation was then performed by adding T4 DNA ligase and incubating at room temperature for 2 h. The ligation reaction was buffer exchanged into phosphate-buffered saline (PBS) containing 1 mM EDTA using 7K MWCO Zeba spin desalting columns (Thermo Fisher Scientific). For conjugation, the detection antibody was reconstituted in PBS from lyophilized form or buffer exchanged into PBS using a 50K Amicon Ultra-0.5mL centrifugal filter and incubated with a 20-fold molar excess of dibenzocyclooctyne-PEG4-N-hydroxysuccinimidyl ester (DBCO-PEG4-NHS, MilliporeSigma) for 30 minutes at room temperature and purified using a 50K Amicon Ultra-0.5mL centrifugal filter in PBS containing 1 mM EDTA. A 2-fold molar excess of the ligated primer-template was then added to the DBCO modified antibody and incubated overnight at 4°C. The conjugate was stored in aliquots at -80°C in PBS containing 5 mM EDTA, 0.1% BSA, and 0.02% sodium azide.

Claims

1. 1. A method for detecting a target analyte in a sample, comprising: (a) contacting a sample containing or suspected of containing said target analyte with a plurality of beads comprising capture moieties that specifically bind to said target analyte under conditions and for a time sufficient for said target analyte in said sample to bind to said capture moieties; a plurality of said beads are associated with zero target analyte molecules; a plurality of said beads are associated with one target analyte molecule; at least about 20% of the beads are associated with either zero or one target analyte molecule; (b) contacting the product of step (a) with a detection moiety that binds to said target analyte; (c) contacting the product of step (b) with a signal amplification moiety that binds to the detection moiety to generate a detectable signal for each bead that carries the target analyte; and (d) detecting the target analyte in the sample by detecting the detectable signal by flow cytometry. A method comprising: (a) the beads comprise magnetic beads, paramagnetic beads, non-magnetic beads, porous beads, or glass beads; (b) the capture moiety comprises an antibody, an aptamer, an antibody mimetic, a polypeptide, a nucleic acid, a molecularly imprinted polymer, a receptor, or a small molecule; (c) the detection moiety comprises an antibody, an aptamer, an antibody mimetic, a polypeptide, a nucleic acid, a molecularly imprinted polymer, a receptor, a binding protein, or a small molecule; (d) the signal amplification portion comprises an enzyme and / or a nucleic acid molecule; (e) the detectable signal is generated by rolling circle amplification followed by hybridization with a complementary fluorescently labeled DNA probe; rolling circle transcription; hybridization chain reaction; loop-mediated isothermal amplification; radical polymerization; tyramide signal amplification (TSA); enzyme-catalyzed proximity labeling (PL) polymerization; labeling with a pre-amplified signal using a fluorescently labeled enzyme, nanoparticle, or nucleic acid concatemer; polymerization-based signal amplification; or magnetic bead-quantum dot immunoassay. (f) at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the beads are associated with either zero or one target analyte molecule; and / or (g) the beads containing the capture moieties for the target analytes are different from beads containing capture moieties for non-target analytes, and optionally have a different color, shape, or size; The method of claim 1. (a) the detection moiety and the signal amplification moiety are directly linked; or (b) the detection moiety and the signal amplification moiety are linked by a non-covalent affinity binding pair; the detection moiety is linked to a first member of the non-covalent affinity binding pair, the signal amplification moiety is linked to a second member of the non-covalent affinity binding pair, and optionally the non-covalent affinity binding pair is biotin-streptavidin, biotin-avidin, ligand-receptor, antigen-antibody, or antibody-binding protein-antibody; The method of claim 1. (a) further comprising the step of detecting the beads containing the capture moiety by flow cytometry; and / or (b) measuring the concentration of the target analyte in the sample, wherein the concentration of the target analyte in the sample is proportional to the level of the detectable signal; The method of claim 1.

5. The method of claim 1, wherein the method reduces cross-reactivity or non-specific binding, and, if necessary, the cross-reactivity or non-specific binding is reduced by detecting the beads and the detectable signal by flow cytometry.

6. 10. The method of claim 1, wherein the target analyte is a protein, nucleic acid, polysaccharide, lipid, cell, fatty acid, therapeutic agent, organism, virus, toxin, peptide, oligosaccharide, lipoprotein, glycoprotein, glycan, or hormone.

7. the sample comprises a biological sample, and optionally (a) the biological sample is a bodily fluid selected from the group consisting of lymph, whole blood, plasma, serum, a blood fraction containing peripheral blood mononuclear cells, urine, saliva, semen, sweat, tears, synovial fluid, cerebrospinal fluid, feces, mucus, vaginal fluid, and spinal fluid; or (b) the biological sample is breast tissue, liver tissue, pancreatic tissue, cervical tissue, lung tissue, kidney tissue, colon tissue, brain tissue, muscle tissue, synovial tissue, skin, hair follicle, bone marrow, tumor tissue, tissue lysate or homogenate, or organ lysate or homogenate; or (c) the biological sample is plasma; or (d) the biological sample is saliva; The method of claim 1.

8. 10. The method of claim 1, wherein steps (a), (b), (c), or any combination thereof, are performed sequentially or simultaneously. (a) further comprising the step of detecting or measuring the concentration of an additional target analyte in the sample, and optionally: the additional target analytes include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more target analytes; and / or the additional target analyte is a protein, nucleic acid, polysaccharide, lipid, cell, fatty acid, therapeutic agent, organism, virus, toxin, peptide, oligosaccharide, lipoprotein, glycoprotein, glycan, or hormone; and / or (b) contacting the sample with (i) a plurality of beads comprising an additional capture moiety that specifically binds to the additional target analyte; (ii) an additional detection moiety that binds to the additional target analyte; and (iii) an additional signal amplification moiety that binds to the additional detection moiety to generate an additional detectable signal. The method of claim 1.

10. 1. A method for detecting a first target analyte and a second target analyte in a sample, comprising: (a) contacting a sample containing or suspected of containing the first target analyte and / or the second target analyte with (i) a plurality of first beads comprising first capture moieties that specifically bind to the first target analyte, and (ii) a plurality of second beads comprising second capture moieties that specifically bind to the second target analyte, under conditions and for a time sufficient for the first target analyte in the sample to bind to the first capture moieties and for the second target analyte in the sample to bind to the second capture moieties; a plurality of the first beads are associated with zero first target analyte molecules; a plurality of the first beads are associated with one first target analyte molecule; at least about 20% of the first beads are associated with either zero or one first target analyte molecule; and a plurality of said second beads being associated with zero second target analyte molecules; a plurality of said second beads being associated with one second target analyte molecule; and at least about 20% of said second beads being associated with either zero or one second target analyte molecule; (b) contacting the product of step (a) with (i) a first detection moiety that binds to said first target analyte, and (ii) a second detection moiety that binds to said second target analyte; (c) contacting the product of step (b) with (i) a first signal amplification moiety that binds to the first detection moiety to generate a first detectable signal for each bead carrying the first target analyte, and (ii) a second signal amplification moiety that binds to the second detection moiety to generate a second detectable signal for each bead carrying the second target analyte; and (d) detecting the first target analyte and the second target analyte in the sample by detecting the first detectable signal and the second detectable signal by flow cytometry. A method comprising: (a) the first detectable signal and the second detectable signal are different signals and, optionally, have different colors; (b) the first bead containing the first capture moiety and the second bead containing the second capture moiety are different, optionally having different colors, shapes, or sizes; (c) the method further comprises detecting the first bead comprising the first capture moiety and the second bead comprising the second capture moiety by flow cytometry; and / or (d) the method reduces cross-reactivity or non-specific binding. The method of claim 10. (a) the method has a detection limit of about 0.1 aM to about 1 mM. (b) the detection limit is from about 0.1 aM to about 1 mM, from about 0.1 aM to about 1 μM, from about 0.1 aM to about 1 nM, from about 0.1 aM to about 1 pM, from about 0.1 aM to about 1 fM, from about 0.1 aM to about 900 aM, from about 0.1 aM to about 800 aM, from about 0.1 aM to about 700 aM, from about 0.1 aM to about 600 aM, from about 0.1 aM to about 500 aM, from about 0.1 aM to about 400 aM, from about 0.1 aM to about 300 aM, from about 0.1 aM to about 200 aM, or from about 0.1 aM to about 100 aM; and / or (c) the detection limit is about 1 fM, about 900 aM, about 800 aM, about 700 aM, about 600 aM, about 500 aM, about 400 aM, about 300 aM, about 200 aM, about 100 aM, about 90 aM, about 80 aM, about 70 aM, about 60 aM, about 50 aM, about 40 aM, about 30 aM, about 20 aM, about 10 aM, or about 1 aM, or about 0.1 aM; The method of claim 1 or 10.

13. 11. The method of claim 1 or 10, wherein the signal detection takes less than about 1 minute per sample, less than about 45 seconds per sample, or less than about 30 seconds per sample.

14. (a) the sample is contacted with about 2,000 to about 100,000 beads; and / or (b) the sample is contacted with about 2,000 beads, about 5,000 beads, about 10,000 beads, about 20,000 beads, about 50,000 beads, or about 100,000 beads; The method of claim 1 or 10.

15. (a) the beads and the sample are incubated for about 1 minute to about 48 hours, about 1 minute to about 10 hours, or about 1 hour to about 4 hours; and / or (b) the beads and the sample are incubated for about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours; The method of claim 1 or 10.

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