Single-molecule assays for ultra-high-sensitivity detection of biomolecules

Abstract

Provided herein are assays that provide digital measurements for detecting proteins and other biomolecules, for example, at low to mid-attomolar concentrations.

Description

[Technical Field]

[0001] Claim of priority This application claims priority to U.S. Provisional Patent Application No. 63 / 042,596, filed on 23 June 2020, and U.S. Provisional Patent Application No. 63 / 076,833, filed on 10 September 2020. All of the foregoing is incorporated herein by reference.

[0002] Provided herein are improved single-molecule assays that offer digital measurement methods for detecting proteins and other biomolecules at low to moderate atomolecular concentrations. [Background technology]

[0003] The ability to accurately measure extremely low levels of biomolecules, such as proteins, nucleic acids, and metabolites, is essential for a wide range of clinical and environmental applications, including disease diagnosis, drug delivery, pathogen detection in food, toxin detection in the environment, and bioprocess control. Since numerous promising biomarkers exist in available biological fluids at levels far below the detection limits of current laboratory methods, ultra-high-sensitivity measurement techniques are particularly important in clinical diagnosis. 1 Digital assay methods, such as digital enzyme-linked immunosorbent assay (ELISA), offer up to 1000-fold improvements in measurement sensitivity compared to traditionally used analytical techniques, such as conventional ELISA. 2~5 However, the sensitivity of digital measurement techniques remains insufficient for many diagnostic applications, particularly for measuring disease-related proteins. For example, while several protein biomarkers for neurological disorders have been shown to be upregulated in cerebrospinal fluid, the highly invasive lumbar puncture required for these measurements makes screening individuals for early disease detection impractical. 6~9 Since only a small fraction of brain-derived proteins cross the blood-brain barrier, highly sensitive technologies that can detect and identify rare protein biomarkers through simple blood tests are crucial in addressing this unmet diagnostic need.10~12 Improving analytical sensitivity is a major challenge in other diseases where rapid point-of-care (POC) diagnosis is crucial for effective medical practice, but readily available biofuels, such as saliva or urine, are required. These biofuels contain only small amounts of serum components and require ultra-high-sensitivity techniques for protein biomarker detection.

[0004] One major obstacle to increasing sensitivity in digital ELISA is low sample collection efficiency. While digital ELISA utilizes single-molecule counting to improve measurement sensitivity, low sample collection efficiency limits the number of target molecules that can be counted. Poisson noise from counting a single event at very low target concentrations...

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[0007] Existing digital ELISA methods utilize microwells or water-in-oil droplets to isolate individual beads containing a single target protein molecule. 2、5、13~15The most advanced digital ELISA currently available is Single Molecule Arrays (Simoa), which captures a single target molecule on antibody-coated paramagnetic beads and isolates individual beads into femtoliter-sized microwells for single-molecule counting. 2 Each bead contains zero or one captured target molecule, and a large excess of beads is used to ensure digital measurements follow a Poisson distribution, exceeding the number of target molecules in the sample. Each captured molecule is labeled with a biotinylated detection antibody so as to form an immune complex sandwich that is then labeled with the enzyme conjugate streptavidin-β-galactosidase (SβG). Subsequently, the beads are loaded with a fluorescence-generating enzyme substrate into microwells, each capable of fitting up to one bead. The microwells are sealed with oil, and high local concentrations of fluorescence are catalytically generated in each well containing beads harboring SβG molecules. The number of target molecules is then measured by counting the "on" and "off" wells.

[0008] While Simoa can achieve subfemtomole detection limits and is the current absolute standard for ultra-sensitive protein detection, its sensitivity is limited by low sample collection efficiency. Only about 5% of the total number of beads can be loaded into the microwells by gravity and analyzed. 16 In the recently developed Simoa instrument, the HD-X Analyzer, external magnetic force is used for bead loading, but the percentage of beads analyzed remains at approximately 5%. Other methods to improve bead loading are being explored, including electric field directional bead loading, hydrophobic-in-hydrophobic microwell arrays, and digital microfluidics. 14、15、17~19While these methods have increased bead loading efficiency, their demonstrated improvement in digital immunoassay sensitivity has remained limited. Furthermore, complex fabrication methods and workflows limit the use of these techniques in POC applications. Another strategy for improving sample collection efficiency in digital bioassays is encapsulating beads into water-in-oil droplets. Digital droplet-based immunoassays have demonstrated bead loading efficiency up to 60% and shown sensitivity improvements similar to or up to one order of magnitude higher than current Simoa technology. 5、13 While droplet microfluidic systems are well established for a variety of applications, the need for highly controlled high-throughput droplet generation introduces additional fabrication and processing steps that add further complexity when integrating into POC systems. Furthermore, a significant proportion of droplets do not contain beads but should be imaged, so improving the image processing volume remains another challenge towards POC implementation. SUMMARY OF THE INVENTION

[0009] <000007Measuring very low levels of biomolecules, including proteins and nucleic acids, remains a significant challenge in many clinical diagnostic applications due to insufficient sensitivity. While digital assays such as Single Molecule Arrays (Simoa) or digital ELISA have made significant progress in sensitivity, numerous promising disease biomarkers still exist in available biofluids at levels below the detection limits of these techniques. Described herein is a highly sensitive digital ELISA platform that addresses the aforementioned challenges. The extremely simple reading process and improved cost-effectiveness of the method, in some embodiments requiring only a glass slide for bead loading and a simple optical device for signal reading, facilitates integration into point-of-care systems. The platform can achieve atomolecular detection limits that are up to 25-fold higher in sensitivity than current techniques (e.g., Simoa and digital ELISA). As proof of concept, we demonstrated the ability of the method to measure previously undetectable levels of Brachyury, a tissue biomarker for chordoma, a rare form of bone cancer, in plasma. This enhances the sensitivity and simplicity of the method, providing a platform for biomarker discovery and POC diagnostic development.

[0010] Another exemplary method involves using a gel to encapsulate the beads in a single layer so that imaging can be performed without the beads moving; all reactions take place in solution, followed by gel polymerization and imaging around the beads.

[0011] Thus, provided herein is a method for detecting a biomolecule in a sample. The method includes preparing a solution containing the sample; contacting the solution with a plurality of beads comprising a capture moiety that binds to the biomolecule under conditions and for a time sufficient for the biomolecule in the sample to bind to the capture moiety; contacting the solution with a binding moiety (e.g., sequentially or simultaneously with the capture moiety) that enables the generation of an on-bead non-diffusive detectable signal sufficient to enable detection of each bead that has bound a biomolecule and carries a target molecule, and then generating an amplified signal; optionally immobilizing the beads in a monolayer; and detecting the signal.

[0012] In some embodiments, immobilizing the beads includes drop-casting a solution containing the beads onto a slide or catalyzing the gelation of the solution.

[0013] In some embodiments, the method includes contacting the solution with a signal amplification moiety that binds to the binding moiety.

[0014] In some embodiments, the signal amplification moiety includes an enzyme or branched DNA.

[0015] In some embodiments, detecting the signal includes imaging the beads to detect a fluorescence or other signal. In some embodiments, the beads are immobilized in a monolayer and single z-section imaging may be used; in embodiments where the beads are not in a monolayer, the method may include imaging different z-sections.

[0016] In some embodiments, the method includes determining the number and / or percentage of beads comprising the bead-biomolecule complex.

[0017] In some embodiments, the beads include a polymer, metal, metal oxide, semiconductor, and / or semiconductor oxide. <着

[0018] In some embodiments, the detectable signal is generated by rolling circle amplification followed by hybridization with a complementary fluorescently labeled DNA probe; tyramide signal amplification (TSA); hybridization chain reaction; enzyme-catalyzed proximity labeling (PL) polymerization; polymerization-based signal amplification; or magnetic bead quantum dot immunoassay.

[0019] In some embodiments, the detectable signal is generated by a pre-amplified signal, such as a labeled polymer or nanoparticles.

[0020] In some embodiments, before a signal is detected, the beads are drop-cast onto a surface and dried to form a film, for example, or a solution is applied to or in contact with the surface to catalyze gelation.

[0021] In some embodiments, the surface is a slide, tip, or flow cell.

[0022] In some embodiments, catalyzing the gelation of the solution involves mixing fibrinogen and / or thrombin; fibrin; cellulose; collagen; gelatin; agarose; hyaluronic acid; polyhydroxyethyl methacrylate (poly(HEMA)); polyethylene glycol (PEG); or acrylamide into the solution.

[0023] In some embodiments, the solution comprises a polymer selected from fibrinogen and / or thrombin; fibrin; cellulose; collagen; gelatin; agarose; hyaluronic acid; polyhydroxyethyl methacrylate (poly(HEMA)); polyethylene glycol (PEG); or acrylamide; and the method comprises catalyzing the gelation of the polymer.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the invention pertains. Methods and materials are described herein for use in the invention; other suitable methods and materials known in the art may also be used. Materials, methods and examples are illustrative and not intended to limit. All publications, patent applications, patents, sequences, database entries and other references mentioned herein are incorporated in their entirety by reference. In case of any conflict, this specification, including definitions, shall prevail.

[0025] Other features and advantages of the present invention will become apparent from the following detailed description and drawings, as well as from the claims. [Brief explanation of the drawing]

[0026] [Figure 1] This is a schematic diagram of an exemplary drop-cast single-molecule assay. In the formation of a single immunocomplex sandwich on antibody-coated paramagnetic beads and labeling with a streptavidin-DNA conjugate, rolling circle amplification (RCA) is performed on the beads to generate long concatemers attached to each immunocomplex. A fluorescently labeled DNA probe is hybridized to the concatemers during RCA to produce a localized fluorescent signal on the beads containing the complete immunocomplex sandwich. After RCA, the beads are concentrated, drop-cast onto a glass slide, and dried to form a monolayer film. The single target molecule is counted by fluorescence imaging of the drop-cast film and by counting "on" and "off" beads. [Figure 2A-G]These are images of the film. (A) Representative photograph and (B) bright-field image of the drop-cast bead film on a glass slide. Approximately 2000-2500 beads are analyzed in each frame. Scale bar = 100 μm. (C-E) Representative images of "on" and "off" dye-encoded beads in the drop-cast film: bead fluorescence (488 nm; C), ATTO 647N probe (647 nm; D), and merge (E). Gray arrows indicate "on" beads. Scale bar = 10 μm. (F-G) Representative histograms of the maximum fluorescence intensity values ​​(subtracted from the background fluorescence intensity values ​​in the image) on each bead for 0 fM IL-1β (F) and 10 fM IL-1β (G) samples. A normal distribution was fitted to the fluorescence intensity values, and the cutoff for "on" vs. "off" beads was defined as above 5 times the standard deviation from the mean. [Figure 3A-F] This graph shows a comparison of the assay sensitivity of this method and conventional Simoa. (A) Calibration curves for human IL-10 of this method and (B) conventional Simoa. The dashed line shows the calculated limit of detection (LOD). (C) Comparison of signal-to-background ratio between this method and conventional Simoa over the IL-10 calibration curve range. (D) Calibration curves for human IL-1β of this method and (E) conventional Simoa. (F) Comparison of signal-to-background ratio between this method and conventional Simoa over the IL-1β calibration curve range. [Figure 4A-B] This graph shows the effect of sample collection efficiency on measurement accuracy and sensitivity. (A) Measured CV of background signal and (B) Calculated LOD for a randomly selected subset of beads imaged using the calibration curve for this method for IL-10. The percentage of analyzed beads represents the percentage of all assay beads. Each point represents the mean of four different randomly selected subsets of beads. [Figure 5A-F]This graph shows the measurement of Bracuri in plasma. Calibration curves for human Bracuri: (A) the present method and (B) the conventional Simoa method. The dashed line shows the calculated limit of detection (LOD). (C) Comparison of the signal-to-background ratio between the present method and conventional Simoa over the calibration curve range. (D-E) Mean enzyme (AEB) and mean molecular weight (AMB) values ​​per bead measured by conventional Simoa and the present method in chordoma patient plasma samples (D) and commercially available plasma and serum samples (E), respectively. The brown line shows the assay LOD. (F) Concentrations measured in chordoma, chondrosarcoma, and commercially available plasma and serum samples using the present method and conventional Simoa. Measurements below the LOD were set to zero. [Figure 6A-B] This figure shows the validation of the human IL-10 dSimoa assay in pooled human saliva. (A) Recovery rate of recombinant human IL-10 protein added to 4-fold diluted pooled human saliva. (B) IL-10 concentration measured in series diluted samples of pooled human saliva. [Figure 7A-B] This figure shows the validation of the human Braquili dSimoa assay in human plasma and serum. (A) Recovery rates of recombinant human Braquili protein added to individual 8-fold diluted commercial human serum samples and pooled human plasma. (B) Braquili concentrations measured in series diluted samples of pooled human plasma. [Figure 8A-C] This graph shows the conventional Simoa assays performed at different SβG concentrations and incubation times for (A) human IL-10 (150 pM SβG, 5 minutes), (B) human IL-1β (150 pM SβG, 5 minutes), and (C) human Bracuri (300 pM SβG, 15 minutes). The gray dashed line shows the LOD calculated for each assay. The LOD values ​​for each assay were 575 aM, 2.77 fM, and 1.48 fM for IL-10, IL-1β, and Bracuri, respectively. [Figure 9A-C]This is a schematic diagram of CARD-dELISA. A. Target protein molecules are captured by antibody-coded beads, and the protein molecules are then labeled with biotinylated detection antibody and streptavidin-poly-HRP. In the on-beads enzymatic signal amplification step, the beads are incubated with tyramide-Alexa Fluor 488. In the presence of hydrogen peroxide, HRP catalyzes radical formation of the tyramide molecule, which then forms a covalent bond with a phenol residue on a nearby protein. Only beads containing a complete immunocomplex are labeled with the tyramide-Alexa Fluor 488 reagent. B. To encapsulate the beads in fibrin hydrogel for imaging, the beads are placed on a glass slide in a silicone isolation well, and then a solution of fibrinogen and thrombin is added to the bead array. The beads are immobilized in fibrin hydrogel formed in situ. C. The immobilized bead array is imaged using a fluorescence microscope to perform single-molecule counting. The beads are identified in bright-field imaging, and the bead intensity from fluorescence imaging is used to determine whether the beads are "on" or "off". [Figure 10A-B] This figure shows bead immobilization in fibrin hydrogel. A. Each isolation well (7mm x 7mm x 2mm) contains one sample with beads immobilized in fibrin hydrogel. B. Bright-field image (10x magnification) of several hundred beads (small black dots) in the fibrin hydrogel. This image represents a small area of ​​the entire fibrin hydrogel in a single isolation well. The entire isolation well can be captured in approximately 20-25 images at 10x magnification. Scale bar = 100 μm. [Figure 11A-E]This figure shows image analysis and single-molecule counting. A-C. Representative microscopic images showing the target subregion containing "on" and "off" beads. (A.) Images showing the beads (bright-field image) and (B.) the fluorescence intensity of the tyramide Alexa Fluor 488 reagent (488 nm fluorescence image) are overlaid with (C.) a gray arrow indicating the "on" beads. Scale bar = 10 μm. (D.) Representative histograms of bead fluorescence intensity for 0 fM and (E.) 50 fM IL-6. The cutoff between "off" and "on" is indicated by a gray square in each histogram. [Figure 12] This graph shows the IL-6 calibration curve using CARD-dELISA. Each data point on the curve represents the mean of two sets of measurements. Error bars represent the standard deviation of the two sets of measurements. Inset: Comparison of saliva samples measured by CARD-dELISA and conventional Simoa. Data points represent the mean of two sets of measurements, and error bars represent the standard deviation of the two sets of measurements. The dotted line represents the exact correlation between the two methods. Spearman's correlation coefficient is 1.00. [Figure 13] This graph shows the IL-6 calibration curve created using the conventional Simoa method. The data points represent the average of two sets of measurements. [Modes for carrying out the invention]

[0027] Minimally invasive quantitative and ultra-sensitive detection of protein biomarkers in biological fluids, such as blood or saliva, has the potential to revolutionize medical diagnostics, including early disease diagnosis, treatment monitoring, and disease recurrence monitoring. Technologies such as digital enzyme-linked immunosorbent assay (ELISA) and single-molecule array (Simoa) are used to analyze proteins (Rissin et al., Nat. Biotechnol. 2010, 28 (6), 595-599; Leirs et al., Anal. Chem. 2016, 88 (17), 8450-8458; Cohen et al., Chem. Rev. 2019, 119 (1), 293-321), nucleic acids (Song et al., Anal. Chem. 2013, 85 (3), 1932-1939; Cohen et al., Nucleic Acids Res. 2017, 45 (14), e137-e137) and other biologically relevant small molecules (Wang et al., J. Am. Chem. Soc. 2018, 140 (51)). Ultra-sensitive detection of low-content biomolecules, such as microwell arrays (Rondelez et al., Nat. Biotechnol. 2005, 23 (3), 361-365; Rissin et al., Nano Lett. 2006, 6 (3), 520-523; Cohen and Walt, Annu. Rev. Anal. Chem. 2017, 10 (1), 345-363) or microfluidic droplets (Kim et al., Lab. Chip 2012, 12 (23), 4986; Witters et al., Lab. Chip 2013, 13 (11)), This is made possible by isolating and counting individual molecules (2047; Yelleswarapu et al., Proc. Natl. Acad. Sci. 2019, 116 (10), 4489-4495; Cohen et al., ACS Nano 2020, 14, 8, 9491-9501).

[0028] Ultra-high sensitivity protein detection can be achieved in Simoa as follows: First, individual protein molecules are captured on antibody-coated paramagnetic beads. An excess number of beads is used compared to the number of protein molecules to ensure that each bead either does not bind to a protein molecule or binds to one protein molecule. The bound protein molecules are then labeled with a biotinylation detection antibody and a streptavidin-conjugate enzyme. Finally, the beads are resuspended in a solution containing a fluorescent enzyme substrate and loaded into a microwell array. The array is sealed with oil, and localized concentrations of the fluorescent product are produced only in wells containing complete immunocomplexes. Single-molecule counting is performed by counting the active wells, calculating the percentage of "on" beads relative to the total number of beads, which is then converted to average enzyme per bead (AEB) to create a calibration curve.In recent years, Simoa has been used in neurological and neurodegenerative diseases (Mattsson et al., JAMA Neurol. 2017, 74 (5), 557; Shahim et al., JAMA Neurol. 2014, 71 (6), 684; Gill et al., Neurology 2018, 91 (15), e1385-e1389; Ng et al., Clin. Transl. Neurol. 2019, 6 (3), 615-619), oncology (Wilson et al., Clin. Chem. 2011, 57 (12), 1712-1721; Shi et al., Nature 2019, 569 (7754), 131-135; Olsen et al., J. Immunol. Methods 2018, 459, Simoa has been used in numerous clinical applications, including (63-69) and infectious diseases (Leirs et al., 2016, supra; J. Clin. Microbiol. 2018, 56 (8); Anderson et al., Clin. Infect. Dis. 2018, 67 (1), 137-140; Ahmad et al., Sci. Transl. Med. 2019, 11 (515), eaaw8287) (Wu et al., Crit. Rev. Clin. Lab. Sci. 2020, 57 (4), 270-290). Simoa can be used to detect proteins at femtomole (fg / mL) or subfemtomole concentrations.

[0029] The inventors have developed an innovative and simple single-molecule measurement platform capable of detecting low to moderate atomol protein concentrations. By addressing the inefficiency challenges of sampling rare target molecules in digital ELISA methods, the inventors have enhanced sensitivity by up to 25 times compared to the current Simoa technology, which is the current absolute standard for ultra-sensitive protein detection. The atomol detection limit (LOD) achieved by this method represents an increase of more than 10,000 times in sensitivity compared to conventional immunoassays. The localization of the non-diffusive, amplified signal to each bead eliminates the need for signal compartmentalization into microwells or droplets.

[0030] In some embodiments, the method involves direct drop casting of all beads onto a surface, for example, a slide, for rapid drying and formation of a monolayer film, or immobilization of beads in a layer of hydrogel. One exemplary method uses on-bead signal generation for single-molecule counting in combination with bead drop casting onto a monolayer film. One embodiment of these methods is referred to herein as dSimoa. In addition, provided herein is a method that uses a method for tyramide signal amplification (TSA), catalytic reporter deposition (CARD) for on-bead signal generation (Figure 9A), followed by immobilization of beads onto a fibrin hydrogel (Figure 9B) and imaging for single-molecule counting (Figure 9C). Some embodiments of this method are referred herein as CARD digital ELISA (CARD-dELISA). By localizing a non-diffusible fluorescence signal to each bead containing the target molecule, this platform not only eliminates the need for bead loading into microwells or droplets for signal compartmentalization, but also allows for the analysis of significantly more beads for improved sample collection efficiency, thereby enhancing sensitivity.

[0031] This simplified method ensures that an average of 40–50% of all assay beads are analyzed, an 8–10-fold increase over the approximately 5% sampling efficiency of current Simoa techniques. At low sample concentrations, especially with capture efficiencies well below 100% (approximately 1–3% across all capture and labeling steps in the assay developed in this study), improved sampling is crucial to minimize the Poisson noise CV associated with the measurement. While some beads are lost during the washing step or excluded from analysis if they overlap or aggregate, experimental results showed that analyzing 20% ​​of all assay beads improved the LOD by approximately an order of magnitude, with a slight further improvement in measured CV and LOD due to the higher number of beads analyzed. The significant improvement in sampling efficiency of this method also allows for the use of fewer assay beads compared to conventional Simoa, increasing the proportion of "on" beads and the resulting signal-to-background ratio. Further improvements in sensitivity can be achieved by using affinity reagents with low dissociation constants and reducing nonspecific binding between the affinity reagents and the streptavidin-DNA label. With the development of better affinity reagents and methods that reduce nonspecific binding, this method may be able to detect zeptomolecular proteins down to their concentration.

[0032] With its atomolar sensitivity, this method has the potential to pave the way for the discovery of novel biomarkers and biological mechanisms underlying various diseases. As proof of principle, the inventors demonstrated that this method can measure low concentrations of the T-box family transcription factor Braquili in plasma samples from chordoma patients, which were previously undetectable by current Simoa techniques. While Braquili has been shown to be highly overexpressed in chordoma tumors, its levels in plasma have not been evaluated. 26~30 Because diagnosing chordoma requires invasive needle or incision biopsy to the skull base or spine, blood-based tests offer a significantly lower-risk diagnostic procedure and may facilitate early diagnosis of chordoma. 31、32Although our measurements were performed only in a small sample cohort, the significant improvement in the detectability of Braculi in plasma samples from chordoma patients using this method opens up new possibilities for promising blood tests and the discovery of novel biological mechanisms. Achieving an order of magnitude or greater improvement in sensitivity using this method also has significant implications for the discovery of novel blood-based biomarkers for numerous other types of cancer and neurological disorders. Diagnostic blood tests for neurodegenerative diseases such as Alzheimer's and Parkinson's disease have proven particularly important for widespread screening and early diagnosis, which are currently very difficult due to the need for highly invasive lumbar punctures. In many cases where biomarker levels are only detectable after clear disease progression, enhanced sensitivity of this method can accelerate early disease diagnosis for improved health outcomes.

[0033] Importantly, this method also increases the ease of reading digital bioassay signals, and in further development, it could be integrated into a POC platform, potentially addressing the challenge of low sensitivity in current POC diagnostics. While increased sample collection efficiency for enhanced sensitivity in digital immunoassays has been demonstrated in bead droplet arrays and droplet digital ELISA methods, these methods introduce additional complexity into the fabrication and processing steps. 13、14In contrast, the digital reading process for this method requires only a slide glass for bead loading and a simple optical device, without the need for additional materials or complex equipment. Furthermore, the dropcasting process is extremely simple and rapid. The dropcast method simplifies the single-molecule detection reading process and increases cost-effectiveness compared to current microwell-based or droplet-based digital ELISA methods. The enhanced sensitivity of this method enables the detection of various previously unmeasured biomarkers in readily available biological fluids, such as saliva and urine, where they exist at very low concentrations. An additional interesting aspect of this method is the long-term signal stability on the dropcast film, which increases flexibility in the assay process. For example, in material-limited environments where suitable optical devices are not readily available, the dropcast film can be easily transported to imaging and analysis facilities without signal loss for at least one month.

[0034] This method can be adapted for POC applications, including integration into microfluidic devices for sample preparation and the incorporation of portable imaging. For example, the sample preparation workflow can be automated into a microfluidic system, and combined with the single-molecule resolution and high sample collection efficiency of this method, it may be possible to reduce assay time while still achieving high sensitivity for detecting low-content biomarkers that are currently undetectable by existing POC platforms. Furthermore, the rapid dynamics of RCA and the reduced diffusion distance in small microfluidic reaction volumes can shorten the time for each sample preparation step, including RCA. In the examples described below, RCA was performed for 1 hour, but a detectable signal was observable after 15 minutes. The RCA signal amplification time can be further reduced by increasing the spatial density of the fluorescent label on each concatemer. Several automated and streamlined microfluidic-based methods have been developed for bead-based immunoassays, and this sample preparation step can be integrated into an automated microfluidic platform. 5、33~36In addition, numerous portable fluorescence imaging platforms have been developed that include smartphone accessories for imaging single fluorescent nanoparticles and RCA products, and can be easily adapted to this method. 5、37~41 In some embodiments, multiple frames may be used to fully capture each dropcast film, and even shorter imaging times may be used, for example, by using an automated handheld reader or wide-angle camera. Furthermore, as supported by sampling analysis of results using this method, only about 20% of the total assay beads may need to be imaged to achieve near maximum sensitivity. The integration of this method into an ultra-sensitive, portable, and automated platform can be used to facilitate widespread screening, early detection, and monitoring of numerous diseases, including infectious diseases such as the recent SARS-CoV-2 pandemic and tuberculosis, traumatic brain injury, and myocardial infarction.

[0035] Current versions of digital ELISA have a simple workflow and enable ultra-sensitive quantification of proteins in biological fluids. In some embodiments, such as CARD-dELISA, tyramide signal amplification is used for on-bead enzymatic signal generation.

[0036] This method may include bead encapsulation in fibrin hydrogel and imaging for single-molecule counting. CARD-dELISA exhibits good sensitivity and dynamic range, demonstrating that it is a simpler but more robust alternative to conventional Simoa. Furthermore, CARD-dELISA eliminates the need for several expensive instruments and consumables required in conventional Simoa, demonstrating its suitability for integration into point-of-care digital ELISA platforms. Future research will focus on integrated sample handling (i.e., target capture, labeling, and on-bead signal amplification). 13、32、33This would include developing a combined point-of-care CARD-dELISA platform that includes bead imaging and data analysis. For bead imaging, the inventors incorporate a miniature microscope module into a platform having sensitivity for measuring micro and nanoscale objects. The microscope module includes an LED light source, appropriate filters and lenses, and imaging is performed using a smartphone camera. 34~36 or compact CMOS 37 It is performed using [method name]. Furthermore, the inventors continue to optimize and improve CARD-dELISA to increase assay sensitivity and reduce total assay time. Currently, CARD-dELISA is about one-tenth the sensitivity of conventional Simo, but automating CARD-dELISA into a fully integrated point-of-care device will likely reduce the coefficient of variation (CV) of background signals, thereby improving assay sensitivity. When integrated into a point-of-care device, CARD-dELISA is a promising platform for triage testing or early diagnosis of diseases such as TB, sepsis, or mild traumatic brain injury.

[0037] Assay method In this method, a sample in solution is brought into contact with multiple beads conjugated with a capture portion that binds to the biomolecule of interest, under conditions that allow the sample to bind to the beads in order to form a bead-biomolecular complex. Once the complex is formed, the method involves bringing the biomolecule into contact with a second capture portion, which allows for the detection of each bead containing the target molecule and then the generation of an amplified, non-diffusible, detectable signal on the beads, such as a fluorescent signal. The method may include the removal of unbound beads.

[0038] The method then includes immobilizing the beads, for example, by drop-casting a solution containing the beads onto a surface to form a film (e.g., formation of a thin film by evaporation of the solution following a drop of the solution onto a flat surface), or by catalyzing the gelation of the solution. Examples of surfaces include slides, tips, or flow cells, which are applied to detection by imaging. Finally, the method includes detecting a signal originating from the beads and, optionally, determining the number and / or percentage of beads containing bead-biomolecular complexes.

[0039] Generally, samples are kept unprocessed before imaging; for example, they are not subdivided or partitioned into individual wells before imaging. This method eliminates the need for partitioning the signal into microwells or droplets.

[0040] sample As used herein, the term “sample” refers to material being tested for the presence of biomolecules of a target marker using the method of the present invention, and includes, in particular, tissue, whole blood, plasma, serum, urine, sweat, saliva, breath, exosomes or exosome-like microvesicles (U.S. Patent No. 8,901,284), lymph, feces, cerebrospinal fluid, ascites, bronchoalveolar lavage fluid, pleural fluid, semen, sputum, papillary aspirate, postoperative seroma, or wound drainage fluid. The type of sample used may vary depending on the characteristics of the biological marker being tested and the clinical situation in which the method is used. Various methods for identifying and / or isolating and / or purifying biological markers derived from samples are well known in the art. “Isolated” or “purified” biological markers are substantially free from cytomaterial or other contaminants derived from the cell or tissue source from which the biological marker originates, i.e., they have been partially or completely altered or removed from their natural state through human intervention. For example, nucleic acids contained in a sample are initially isolated according to standard methods, such as using soluble enzymes or chemical solutions, or isolated by nucleic acid-binding resin according to the manufacturer's instructions.

[0041] beads This method involves the use of micro or nanoparticle beads conjugated to a capture portion that binds to a desired biomolecule. Micro and / or nanoparticles (e.g., microbeads) may be made from a variety of materials. In general, any polymeric or plastic material may be used to create microparticles, microbeads or nanoparticles, such as polystyrene and polyethylene. In some embodiments, microparticles may be formed from biocompatible polymer materials, such as polyacrylic acid, polymethacrylic acid, and / or polyamides.

[0042] In certain embodiments, metallic, 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 may be fabricated and used in a multitude of sizes. For example, single-crystal iron oxide nanoparticles (MION) and crosslinked iron oxide (CLIO) particles may be used. In some embodiments, the beads are paramagnetic. Suitable beads include, but are not limited to, magnetic beads (e.g., paramagnetic beads), plastic beads, ceramic beads, glass beads, silica beads, polystyrene beads, methylstyrene beads, acrylic polymer beads, carbon graphited beads, titanium dioxide beads, latex or crosslinked dextran, e.g., SEPHAROSE beads, cellulose beads, nylon beads, crosslinked micelles, and TEFLON® beads. In some embodiments, spherical beads are used, but non-spherical or irregularly shaped beads may also be used.

[0043] In some embodiments, the beads are: "Ultra-sensitive detection of molecules on single molecule arrays," D. Duffy, E. Ferrell, J. Randall, D. Rissin, D. Walt. U.S. Patent No. 8,222,047, July 17, 2012; "Methods and arrays for target analyte detection and determination of target analyte concentration in solution," DMRissin, DRWalt. U.S. Patent No. 8,460,879, June 11, 2013; "Methods and arrays for target analyte detection and determination of reaction components that affect a reaction," David Walt, David Rissin, Hans-Heiner Gorris. U.S. Patent No. 8,492,098, July 23, 2013; "Ultra-sensitive detection of molecules on single molecule arrays," David C. Duffy, Evan Ferrell, Jeffrey D. Randall, David M. Rissin, David R. Walt. U.S. Patent No. 8,846,415, September 30, 2014; "Ultra-sensitive detection of molecules or particles using beads or other capture objects," DCDuffy, DMRissin, DRWalt, D. Fournier, C. Kan. Quanterix Corporation. U.S. Patent No. 9,310,360, April 12, 2016; "Methods and arrays for target analyte detection and determination of target analyte concentration in solution," DRWalt, DMRissin.As described in U.S. Patent No. 9,395,359, July 19, 2016; or “Ultra-sensitive detection of molecules or particles using beads or other capture objects,” DCDuffy, DMRissin, DRWalt, D. Fournier, C. Kan. Quanterix Corporation, U.S. Patent No. 9,482,662, November 1, 2016; or as described in International Publication No. 2020037130.

[0044] Capture and joining parts The beads are coated, for example, conjugated, with a capture site that binds to the target biomolecule. Additionally, the binding site is used to detect the beads bound to the biomolecule and amplify the signal derived from them.

[0045] In some embodiments, the capture or binding portion is an aptamer that binds to an antibody or its antigen-binding portion or to a biomolecule, for example, a protein or peptide. In some embodiments, the capture or binding portion is an oligonucleotide complementary to a portion of the nucleic acid of interest. In some embodiments, the capture or binding portion is the ligand-binding portion of a protein, for example, a receptor, and the biomolecule is a molecule such as a hormone.

[0046] The capture or binding portion can specifically bind to the capture portion or the target analyte, or otherwise specifically associate with it. The capture or binding portion can conjugate, capture, adhere to, bind to, or attach to the capture portion. For example, in some embodiments, the capture or binding portion is an antibody (e.g., full-length antibody (e.g., IgG, IgA, IgD, IgE, or IgM antibody) or antigen-binding antibody fragment (e.g., scFv, Fv, dAb, Fab, Fab', Fab'2, F(ab')2, Fd, Fv, or Feb)), an aptamer, an antibody mimetic (e.g., afibody, affin, affimer, afitin, alphabody, antikalin, avimer, DARPin, fynomer, Kunitz domain peptide, monobody, or 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. For example, in some embodiments, the capture or binding component binds to the Fc region of the antibody.

[0047] In some embodiments, the method includes the use of a capture portion that binds to a biomolecule; and a binding portion that binds to the capture portion. In some embodiments, the method includes the use of a capture portion that binds to a biomolecule; a first binding portion that binds to the capture portion; and a second binding portion or signal amplification portion that binds to the first binding portion. One or more of the capture portion, binding portion, or second binding portion / signal amplification portion may contain or generate a detectable label. For example, in some embodiments, the binding portion contains biotin, and the second binding portion / signal amplification portion is a streptavidin-labeled horseradish peroxidase (HRP) enzyme that binds before signal generation. The sample may be in contact with the capture and binding portions simultaneously (e.g., in the same solution) or sequentially, for example, in contact with the capture portion and then with the binding portion. The method may include removing all unbound reagents, e.g., beads, capture and / or binding portions, before detection.

[0048] In some embodiments, the capture or binding portion is described in "Ultra-sensitive detection of molecules on single molecule arrays," D. Duffy, E. Ferrell, J. Randall, D. Rissin, D. Walt. U.S. Patent No. 8,222,047, July 17, 2012; "Methods and arrays for target analyte detection and determination of target analyte concentration in solution," DMRissin, DRWalt. U.S. Patent No. 8,460,879, June 11, 2013; "Methods and arrays for target analyte detection and determination of reaction components that affect a reaction," David Walt, David Rissin, Hans-Heiner Gorris. U.S. Patent No. 8,492,098, July 23, 2013; "Ultra-sensitive detection of molecules on single molecule arrays," David C. Duffy, Evan Ferrell, Jeffrey D. Randall, David M. Rissin, David R. Walt. U.S. Patent No. 8,846,415, September 30, 2014; "Ultra-sensitive detection of molecules or particles using beads or other capture objects," DC Duffy, D Rissin, D Walt, D. Fournier, C. Kan. Quanterix Corporation. U.S. Patent No. 9,310,360, April 12, 2016; "Methods and arrays for target analyte detection and determination of target analyte concentration in solution," D Walt, DMRissin. U.S. Patent No. 9,395,359, July 19, 2016; or “Ultra-sensitive detection of molecules or particles using beads or other capture objects,” DC Duffy, DMRissin, DRWalt, D. Fournier, C. Kan. Quanterix Corporation. U.S. Patent No. 9,482,662, November 1, 2016; or as described in International Publication No. 2020037130.

[0049] Biomolecules In some embodiments, the biomolecule of interest is a protein, peptide, nucleic acid, virus, cell surface molecule, metabolite, or small molecule.

[0050] The term "biomolecule" means any atom, molecule, ion, molecular ion, compound, particle, cell, virus, complex, or fragment thereof that is detected, measured, quantified, or evaluated. The target analyte may be contained in a sample (e.g., a liquid sample (e.g., a biological or environmental sample)). Exemplary target analytes, non-limitingly, include small molecules (e.g., organic compounds, steroids, hormones, haptens, biogenic amines, antibiotics, mycotoxins, organic contaminants, nucleotides, amino acids, monosaccharides, or secondary metabolites), proteins (including glycoproteins or prions), nucleic acids (e.g., modified nucleic acids or miRNAs), polysaccharides, lipids, extracellular vesicles, glycans, toxins, fatty acids, cells, gases, therapeutic agents, organisms (e.g., pathogens), or viruses. The target analyte may be naturally occurring or synthetic. In some embodiments, the target analyte is interferon, e.g., interferon g (IFN g). In some embodiments, the target analyte is an interleukin, for example, interleukin-2 (IL-2).

[0051] The terms “nucleic acid” and “polynucleotide” are interchangeable herein and refer to nucleotide monomers linked by at least two covalent bonds. The terms include, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), their hybrids, and mixtures thereof. While nucleotides are typically linked by phosphate diester bonds in nucleic acids, the term “nucleic acid” also includes nucleic acid analogs having other types of linkages or skeletons (e.g., phosphorothioates, phosphoramides, dithiophosphates, O-methylphosphoamides, morpholino, locked nucleic acid (LNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), and peptide nucleic acid (PNA) linkages or skeletons). Nucleic acids may be single-stranded, double-stranded, or contain portions of both single-stranded and double-stranded sequences. Nucleic acids may contain any combination of deoxyribonucleic acid and ribonucleotides, as well as any combination of bases, including, for example, adenine, thymine, cytosine, guanine, uracil, and modified or non-standard bases.

[0052] In this specification, “protein” means at least two amino acids linked by a covalent bond, and includes proteins, polypeptides, oligopeptides, and peptides. Proteins may consist of naturally occurring amino acids and peptide bonds or synthetic peptide-mimicking structures. Thus, “amino acid” or “peptide residue” as used herein means both naturally occurring and synthetic amino acids. For example, homophenylalanine, citrulline, and norleucine are considered amino acids for the purposes of this invention. The side chain may be in either an (R) or (S) configuration. In some embodiments, the amino acid is in either an (S) or L configuration. When side chains that are not naturally occurring are used, non-amino acid substituents may be used, for example, to prevent or slow down degradation in vivo. The term “part” 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 an active polypeptide) containing fewer amino acids than the full-length or reference polypeptide (e.g., about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% fewer amino acids).

[0053] The term “small molecule,” as used herein, means any molecule having a molecular weight less than 5000 Da. For example, in some embodiments, small molecules are organic compounds, steroids, hormones, haptens, biogenic amines, antibiotics, mycotoxins, cyanotoxins, nitro compounds, drug residues, pesticide residues, organic contaminants, nucleotides, amino acids, monosaccharides, or secondary metabolites. See also International Publication No. 2020037130.

[0054] Detection method This method involves on-bead signal amplification for single-molecule signal generation. The signal may be any detectable signal, such as an optically detectable label such as fluorescence, chemiluminescence, or colorimetric analysis, or other labels, such as gold beads or other labels detectable by non-optical assays (e.g., using surface plasmon resonance or other methods). In some embodiments, the method uses rolling circle amplification of concatemers; the generated DNA concatemers attached to each immune complex may be hybridized to a number of complementary fluorescently labeled DNA probes for visualization. In these methods, sensitivity may be adjusted by increasing (higher sensitivity) or decreasing (lower sensitivity) the RCA time.

[0055] Other nucleic acid amplification methods may also be used, e.g., hybridization chain reactions, enzyme-catalyzed proximity labeling (PL) polymerization (see, e.g., Branon et al., Nat Biotechnol. 2018 Oct;36(9):880-887); polymerization-based signal amplification (e.g., visible light-induced polymerization, e.g., as described in Badu-Tawiah et al., Lab Chip, 2015, 15, 655); magnetic bead quantum dot immunoassay (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). Branched DNA may also be used.

[0056] Alternatively, the method involves using tyramide signal amplification (TSA). TSA, also known as catalytic reporter deposition (CARD), is a highly sensitive method that enables the detection of biomolecules present at low concentrations. TSA is used in immunohistochemistry and in situ hybridization experiments and in digital ELISA (Akama et al., Anal. Chem. 2016, 88 (14), 7123-7129). In TSA, HRP (e.g., bound to a second binding site) then catalyzes the conversion of labeled tyramide to a reactive radical that produces a high-density detectable signal by fetching a nearby tyrosine residue.

[0057] Other amplification chemistry methods may also be used, for example, branched DNA assays (bDNA), as described in Dunbar and Das, J Clin Virol. 2019 Jun; 115: 18-31.

[0058] In some embodiments, the method uses labeled polymers or nanoparticles as the binding site; e.g., Tang et al., Analyst, 2013, 138, 981-990; Hansen et al., Anal Bioanal Chem. 2008 Sep; 392(1-2): 167-175; Wu et al., Chem 2, 760-790, June 8, 2017; Skaland et al., Applied immunohistochemistry & molecular morphology: AIMM / official publication of the Society for Applied Immunohistochemistry 18(1):90-6 (2009); Gormley et al., Nano Lett. 2014, 14, 11, See 6368-6373 (radicals generated by either an enzyme or a metal ion are polymerized to form a polymer enclosing multiple gold nanoparticles (AuNPs)); this includes contacting a pre-amplified signal such as dye-loaded polymer nanoparticles (e.g., as described in Melnychuk and Klymchenko, J. Am. Chem. Soc. 2018, 140, 34, 10856-10865).

[0059] Suitable imaging or other methods may be used for detectable labels for selection. In some embodiments, the beads are immobilized in a single layer and single z-section imaging may be used; in embodiments where the beads are not in a single layer, the method may include imaging different z-sections.

[0060] Hydrogel In some embodiments, the method includes gelling a layer of hydrogel to immobilize individual beads before signal detection. Methods for catalyzing the gelation of hydrogels are known in the art. Hydrogels may include, for example, fibrin, fibrinogen, cellulose, collagen, gelatin, agarose and hyaluronic acid, or synthetic hydrogels such as polyhydroxyethyl methacrylate (poly(HEMA)), polyethylene glycol (PEG), or acrylamide. See, for example, Ahmed, J Adv Res. 2015 Mar;6(2):105-21.

[0061] kit Similarly, provided herein are kits for use in the methods described herein, for example, including beads and reagents described herein. [Examples]

[0062] The present invention is further described in the following embodiments, which do not limit the scope of the invention as described in the claims.

[0063] [Example 1] Ultra-high sensitivity detection of atomized protein concentration using a drop-cast single-molecule assay. The inventors have developed a simple, ultra-high-sensitivity single-molecule detection platform that offers up to 25-fold increased sensitivity compared to current state-of-the-art digital ELISA technologies. By improving the sampling of rare target molecules, this method enables protein detection in the attomolecular range, thereby opening up opportunities for a wide range of promising disease biomarkers that were previously unmeasurable. Importantly, this method simplifies the digital assay readout process, making it more suitable for future integration into point-of-care (POC) systems. The platform is simpler and more sensitive than previously developed Simoa assays and can be easily adapted to measure other disease-related biomolecules, including microRNAs and small molecules. 42、43By measuring biomolecules at extremely low concentrations that cannot be detected by current methods, this method provides a platform for ultra-high-sensitivity detection that can accelerate early disease diagnosis.

[0064] method Materials. All antibodies, recombinant proteins, and DNA sequences used in this study are listed in the supplementary information. DNA primers, templates, and probes were obtained from Integrated DNA Technologies or MilliporeSigma. Conjugation and assay buffers, as well as dye-encoded carboxylated 2.7 μm paramagnetic beads (Homebrew Multiplex Beads 488), were purchased from Quanterix Corporation.

[0065] Preparation of antibody-coated capture beads. For each target, the capture antibody was exchanged for bead conjugation buffer (Quanterix) using a 50K Amicon centrifuge filter (0.5 mL, MilliporeSigma). 500 μL of bead conjugation buffer was added to the antibody solution in the filter, and centrifugation was continued at 14,000 x g for 5 minutes. The eluent was discarded, and the process was repeated twice. The exchanged antibody was recovered by inverting the filter into a new tube, centrifugation at 1000 x g for 2 minutes, followed by rinsing with 50 μL of bead conjugation buffer and a second centrifugation at 1000 x g for 2 minutes. Antibody concentrations were measured using a NanoDrop spectrophotometer, and the antibodies were diluted to 0.5 mg / mL (IL-10), 0.3 mg / mL (Braquilibrium), or 0.2 mg / mL (IL-1β) in bead conjugation buffer for subsequent bead coupling. 2.8 × 10 8Each dye-encoded paramagnetic bead was washed three times with 200 μL of bead washing buffer (bead conjugation buffer) and twice with 200 μL of bead conjugation buffer, and then resuspended in 190 μL of cold bead conjugation buffer. Next, 1 mg vial of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (Thermo Fisher Scientific) was reconstituted into 100 μL of cold bead conjugation buffer, and 10 μL was immediately added to the beads. The beads were activated for 30 minutes with shaking. After activation, the beads were washed with 200 μL of cold bead conjugation buffer, resuspended in 200 μL of capture antibody solution, and left on a shaker for 2 hours for antibody coupling. Subsequently, the antibody-coupled beads were washed twice with 200 μL of bead washing buffer and blocked with 200 μL of bead blocking buffer (Quanterix) for 30 minutes with shaking. After blocking, the beads were washed with 200 μL of bead washing buffer, followed by 200 μL of bead diluent (Quanterix), before resuspending them in 200 μL of bead diluent. For IL-1β, the EDC activation and antibody coupling steps were performed at 4°C, using 4.2 × 10⁶ beads. 8 The assay was performed using 9 μL of EDC for bead activation and 300 μL of 0.2 mg / mL antibody for conjugation. A Beckman Coulter Z1 Particle Counter was used to count the beads, which were stored at 4°C for subsequent use in the assay.

[0066] Preparation of streptavidin-DNA conjugates. The RCA template (MilliporeSigma) was initially annealed to the 5' azide-modified primer (Integrated DNA Technologies) by heating a solution of 45 μL of 100 μM primer, 54 μL of 100 μM template, and 26.6 μL of 5xNEBNext® Quick Ligation reaction buffer (New England Biolabs) at 95°C for 2 minutes and then slowly cooling to room temperature over 90 minutes. Next, 7.5 μL of T4 DNA ligase (2,000,000 units / mL, New England Biolabs) was added to the template, and the reaction was ligated by incubation at room temperature for 3 hours. The ligated reaction was then buffer-exchanged in PBS containing 1 mM EDTA using a Zeba® spin desalting column (7K MWCO, Thermo Fisher Scientific). Streptavidin (Biolegend 280302) was buffered with phosphate-buffered saline (PBS) using a 10K Amicon centrifuge filter (0.5 mL, MilliporeSigma), followed by the same buffer exchange procedure described above for the capture antibody, which was then diluted to 1 mg / mL in PBS. Dibenzocyclooctin-PEG4-N-hydroxysuccinimidyl ester (DBCO-PEG4-NHS, 1 mg, MilliporeSigma) was dissolved in 200 μL of dimethyl sulfoxide and added to the buffered streptavidin in a 20-fold molar excess. The conjugation reaction was incubated at room temperature for 30 minutes and then purified using a 10K Amicon centrifuge filter. The conjugated streptavidin was washed with PBS containing 1 mM EDTA by centrifugation at 14,000xg for 5 minutes five times, followed by centrifugation at 14,000xg for 15 minutes. Next, the purified DBCO-conjugated streptavidin was recovered by inverting the filter and centrifuging at 1000xg for 2 minutes. The annealed primer template was added to the DBCO-conjugated streptavidin in a 2x molar excess, and the conjugation reaction was allowed to proceed overnight at 4°C.Next, the streptavidin DNA conjugate was stored at -80°C in aliquots containing 0.1% bovine serum albumin (BSA), 5 mM EDTA, and 0.02% sodium azide without further purification.

[0067] Drop-cast single-molecule assays. All dSimoa assays were performed in 96-well plates (Greiner Bio-One, 655096). Antibody-coated beads, recombinant proteins, and biotinylated detection antibodies were diluted to the desired concentrations in sample diluent (Quanterix). The detection antibody and streptavidin-DNA concentrations for each assay are described in the accompanying information. For each assay, 10 μL of antibody-coated beads (100,000 total beads) and 10 μL of biotinylated detection antibody were added to 100 μL of protein sample. The plates were then sealed and shaken for 1 hour for immunocomplex formation. The beads were washed six times using a BioTek 405 TS microplate washer with System Wash Buffer 1 (Quanterix), and then resuspended in 100 μL of streptavidin-DNA conjugate diluted in sample diluent containing 5 mM ethylenediaminetetraacetic acid (EDTA). The plates were shaken for 15 minutes for streptavidin-DNA labeling of the immune complexes, and then washed eight times using a microplate washer with System Wash Buffer 1. After washing, the beads were transferred to a new 96-well plate and washed additionally with 200 μL of System Wash Buffer 1 before resuspending in 60 μL of RCA solution. The RCA solution consisted of 0.5 mM deoxynucleotide mix (New England Biolabs), 0.33 U / uL phi29 DNA polymerase (Lucigen), 0.2 mg / mL BSA, 1 nM ATTO 647N labeled DNA probe (Integrated DNA Technologies), and 0.1% Tween-20 in a reaction buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM (NH4)2SO4, and 10 mM MgCl2. Dithiothreitol (DTT) was removed from the manufacturer's phi29 polymerase solution using a Zeba® spin desalting column (7K MWCO, Thermo Fisher Scientific). RCA was performed at 37°C for 1 hour with plate shaking, and then 150 μL of PBS containing 5 mM EDTA was added to each sample to stop the RCA reaction.The beads were then washed twice with 200 μL of dropcast buffer (50 mM Tris-HCl, 50 mM NaCl, 0.1% Tween-20, 0.5% BSA) and concentrated to 10–15 μL before resuspending and dropcasting onto glass slides by manual pipetting. The dropcast beads were dried for 10–15 minutes to form a single-layer film.

[0068] For saliva samples, pooled human saliva (BioIVT) was centrifuged at 13,150xg for 20 minutes at 4°C. In dilution linearity experiments, the desired volume of supernatant was sequentially diluted 2 to 32 times in sample diluents containing a protease inhibitor (Halt® Protease Inhibitor Cocktail, Thermo Fisher Scientific). For addition and recovery experiments, recombinant human IL-10 protein was added to 4-fold diluted saliva samples at concentrations of 100, 10, and 1 fM.

[0069] Plasma samples from chordoma patients were obtained from Dr. Sandro Santagata and Dr. Keith Ligon (Brigham and Women's Hospital), centrifuged at 2000xg for 10 minutes at 4°C, and the supernatant was aliquoted to avoid freeze-thaw cycles. Commercially available plasma and serum samples were obtained from BioIVT. All samples were diluted 8-fold in sample diluent for analysis.

[0070] Imaging and Analysis. Brightfield and fluorescence images of dropcast bead films were acquired using a scientific CMOS camera (ORCA-Flash4.0 LT+, Hamamatsu) and an Olympus IX81 inverted microscope with a 10x objective lens. Fluorescence images obtained with a GFP filter (1-second exposure) were used to position the dye-encoded beads, and fluorescence images obtained with a Cy5 filter (1-second exposure) were used to identify "on" versus "off" beads. Commercial software (cellSens) was used to control the stage and camera. Brightfield and fluorescence images were acquired for each frame, and multiple frames were acquired to capture the entire dropcast film excluding the film edges. Approximately 20–25 frames were acquired per dropcast film with an average total imaging time of approximately 15 minutes.

[0071] Image analysis was performed in MATLAB. Beads were initially positioned in the 488 nm fluorescence image using a disk-type morphological structuring element with top-hat filtering to compensate for uneven illumination. Overlapping or aggregated beads were separated by watershed segmentation, and all remaining aggregated beads were removed by size cutoff. The maximum signal intensity of each identified bead was determined in the corresponding Cy5 fluorescence image, which had initially undergone top-hat filtering to compensate for uneven illumination. A Gaussian distribution was fitted to the bead fluorescence intensities, and the cutoff intensity value for "on" vs. "off" beads was determined as being above 5 times the standard deviation from the mean of the distribution. Thus, all beads with intensities exceeding the cutoff value were counted as "on" beads. The bead proportion was calculated as the total number of "on" beads per total number of beads, and the mean molecular weight (AMB) per bead was subsequently calculated from a Poisson distribution.

[0072] The calibration curve was fitted in GraphPad Prism using 4-parameter logistic (4PL) fitting and used to determine the concentration of the unknown sample. The R-squared value of the calibration curve fit was... 2 The values ​​can be found in the supplementary information. Except for the dilution linearity and addition / recovery assays performed in two series, all measurements were performed in 3-4 series. The limit of detection (LOD) for each assay was calculated as the concentration corresponding to being above three times the standard deviation from the background AEB.

[0073] Simoa assay. The conventional Simoa assay was performed on an HD-X Analyzer (Quanterix) using the same antibody-coated capture beads (500,000 beads per assay) and biotinylated detection antibody at the same concentration as in the corresponding dSimoa assay, with a sample volume of 100 μL. Streptavidin-β-galactosidase (SβG) concentrate (Quanterix) was diluted to the desired concentration in SβG diluent (Quanterix). The same incubation time of 1 hour was used for the antibody capture step to incubate the beads, sample, and detection antibody for immunocomplex sandwich formation. For each target, two assay conditions were performed: one assay using the same SβG concentration and incubation time as the corresponding dSimoa assay, and one assay using the standard SβG concentration and incubation time used in HD-X (150 pM SβG, 5 minutes). The beads, detection antibody, and SβG were placed in a plastic bottle (Quanterix), the sample was added to a 96-well plate, and all of these were loaded into the HD-X Analyzer. The enzyme substrate (resorphin β-D-galactopyranoside), wash buffer 1, wash buffer 2, and Simoa sealing oil were loaded into the HD-X Analyzer according to the manufacturer's instructions. All assay steps, image analysis, and calculation of average enzyme per bead (AEB) were automated as previously detailed. 13 .

[0074] result Development of drop-cast single-molecule assays To enable bead drop casting for counting "on" and "off" beads, the inventors first developed a strategy for generating a localized signal on each bead harboring a complete immunocomplex sandwich. Rolling circle amplification (RCA), an isothermal DNA amplification method based on the progressive action of polymerase around a circular DNA template, generates long concatemers of DNA repeats to provide rapid and strong signal amplification. Since RCA has been successfully used for the detection of individual protein-protein complexes and nucleic acids, the inventors hypothesized that RCA could enable the detection of single immunocomplex sandwiches trapped on beads. 20~23 RCA is performed on immune complexes on beads isolated in a microwell array to enable multiplex protein detection. 24 To incorporate RCA into our single-molecule detection platform, we labeled each immune complex sandwich using RCA primers annealed to a circular DNA template (Figure 1). After performing RCA, the generated DNA concatemers attached to each immune complex can be hybridized with a number of complementary fluorescently labeled DNA probes for visualization.

[0075] The dSimoa method utilizes the same target capture step as conventional Simoa, in which antibody-coated paramagnetic beads are incubated with the sample and biotinylated detection antibody to form an immunocomplex sandwich. However, instead of labeling the immunocomplex sandwich with streptavidin-β-galactosidase (SβG), streptavidin conjugated to a pre-annealed primer template pair is used to label the immunocomplex sandwich. RCA is then performed at 37°C for each labeled immunocomplex sandwich for signal amplification. Furthermore, a fluorescently labeled DNA probe is added to the RCA reaction for in situ hybridization. After the RCA reaction, the beads are washed, concentrated, drop-cast onto glass slides, and dried to form a monolayer film for imaging. The inventors used streptavidin-DNA conjugates for all dSimoa assays because preliminary attempts using detection antibody-DNA conjugates directly for immunocomplex formation followed by RCA resulted in high background signal (data not described).

[0076] To evaluate signal amplification and bead distribution in dropcast films, the inventors used dSimoa to detect interleukin-1 beta (IL-1β) as a model analyte, using the same capture and detection antibody pair used in previously validated Simoa assays. Because salt crystal formation from the dropcast buffer can interfere with bright-field bead identification, fluorescent dye-encoded beads (488 nm) were used to facilitate bead identification in the dropcast film for analysis. Using 100,000 assay beads and approximately 15 μL of dropcast volume, the dropcast bead film exhibited minimal bead aggregation and a high, uniform bead density across the film (Figures 2A-B). Furthermore, the dropcast process was rapid, with a 15 μL dropcast volume drying to a film 12-15 mm in diameter within 15 minutes. The presence of the target analyte captured on the beads was indicated by a fluorescent signal covering all or part of the beads (Figures 2C-E). Because the presence of aggregated beads in image analysis can affect the accuracy of the calculated percentage of "on" beads, bead aggregates of two or more beads, which constitute approximately 20-25% of the beads in each film, were separated by watershed segmentation in the image analysis algorithm, and any remaining bead aggregates were excluded from the analysis via a size threshold. Representative histograms of the maximum fluorescence intensity for all beads imaged in the dropcast film show a wide range of "on" bead signal intensities due to the broad size distribution of concatemers generated by RCA (Figures 2F-G). The number of "on" and "off" beads in each dropcast film was calculated by fitting a normal distribution to the maximum fluorescence intensity of each bead and assigning a threshold for "on" beads to be greater than 5 times the standard deviation from the mean. The average target molecule (AMB) per bead was determined using the Poisson distribution equation, similar to the average enzyme (AEB) per bead calculated in conventional Simoa. 25 .

[0077] By simply transferring the entire volume of beads to a glass slide, the inventors can image and analyze, on average, 40–50% of the total number of assay beads, with the majority of the remaining beads either lost during the washing or transfer step or excluded from analysis due to aggregate formation. Thus, the sampling efficiency in dSimoa shows a significant improvement of over 5% of the beads analyzed using current Simoa techniques. In addition to eliminating the need for microwells, dSimoa can use far fewer beads for target capture due to the increased percentage of beads that can be analyzed, thus improving the sampling of rare target molecules while minimizing Poisson noise. Current Simoa techniques use 500,000 beads, while dSimoa uses 100,000 beads. The reduction in the number of beads can increase the signal against background due to more "on" beads and thus a higher AMB compared to the total number of beads. Furthermore, in the dry state, the fluorescence signal remains highly stable in the drop-cast film, with no decrease in the measured AMB value even after one month (Table 2).

[0078] Digital detection of proteins using dSimoa To evaluate the sensitivity of dSimoa, the inventors created calibration curves for two human cytokines, IL-1β and interleukin-10 (IL-10), using the same antibody pairs previously used in corresponding Simoa assays. These dSimoa assays achieved low to moderate atomolecular limits of detection (LOD) for IL-10 and IL-1β, respectively, demonstrating 25-fold and 15-fold improvements in sensitivity compared to the corresponding conventional Simoa assays (Figures 3A-F; Table 1). The limit of quantification (LOQ), calculated as above 10 times the standard deviation from the background (blank AMB or AEB), was also improved by an order of magnitude in the dSimoa assays compared to conventional Simoa assays. By substantially increasing the percentage of beads that can be analyzed, dSimoa enhances the sampling efficiency of low-content molecules and allows for the use of far fewer beads. In addition, a one-fifth reduction in the number of beads increases the signal against the background, contributing to a significant enhancement in sensitivity (Figures 3C, F).

[0079] In addition to improving the signal against background noise, the inventors hypothesized that increased sampling efficiency of captured target molecules would also help achieve lower LODs using dSimoa by reducing measurement inaccuracies due to Poisson noise. To determine whether the inventors' experimental results supported this hypothesis, they randomly selected a subset of beads analyzed in the IL-10 calibration curve and varied the percentage of all assay beads analyzed to determine the LOD and coefficient of variation (CV) of the background measurements (Figures 4A-B). When only a small number of beads (less than 10% of all beads) were analyzed, the inaccuracy of the background measurements was very high, with a CV of over 20%, corresponding to a low LOD, as predicted from Poisson sampling noise. Furthermore, when a low percentage of beads were analyzed, there was high variability in the LODs obtained in different random samplings. These observations thus demonstrate the important role of sampling efficiency in the accuracy and sensitivity of digital measurements. The LOD value calculated when more than 20% of the beads were analyzed did not increase further, suggesting that near-maximum sensitivity can be achieved by imaging at least 20% of the assay beads.

[0080] To validate the performance of dSimoa in biological fluids, the inventors conducted addition and recovery experiments for IL-10 in human saliva. Recovery rates of recombinant human IL-10 protein at various concentrations added to pooled human saliva ranged from 76% to 122%, demonstrating that dSimoa can reliably detect the protein in saliva (Figure 6A). Furthermore, dSimoa measurements of IL-10 in series dilutions of human saliva showed linear dilution and minimal interference from the salivary matrix in the dSimoa assay (Figure 6B). The dSimoa assay also demonstrated high measurement accuracy with a CV well below 10% across all salivary samples.

[0081] To investigate the potential diagnostic utility of improved sensitivity of dSimoa, we developed a dSimoa assay for Brachyuris, a T-box transcription factor strongly associated with chordoma, primary bone cancer in the spine or skull base. 26 Elevated levels of Braculi expression have been found in chord tumors, but to the best of our knowledge, there are no reports on the measurement of Braculi in plasma. 27~30 Calibration curves generated for Braquili by the dSimoa and conventional Simoa assays yielded LODs of 244.6 aM and 841.4 aM, respectively (Figures 5A-B). This dSimoa assay only showed a 3-fold improvement in sensitivity compared to the conventional Simoa assay. The relatively small improvement in LOD may be due to a smaller increase in signal relative to background compared to the conventional Simoa assay (Figure 5C). Since reducing the total number of assay beads also reduces the capture antibody concentration, the degree of improvement in signal relative to background from a reduction in the number of assay beads may be smaller for antibodies with lower binding affinity, which may result in a decrease in capture efficiency despite a higher ratio of target molecules to beads. To validate the performance of the dSimoa assay in plasma and serum matrices, we performed addition and recovery experiments and obtained recovery rates of at least 65-70% with most measured CVs below 10% in the majority of the added plasma and serum samples (Figure 7A). The inventors further verified the accuracy of the dSimoa assay in plasma by confirming acceptable dilution linearity (Figure 7B).

[0082] Finally, the inventors compared the ability of dSimoa and conventional Simoa to detect endogenous Braculi in several chordoma patient plasma samples as well as commercially available plasma and serum samples from healthy donors. Conventional Simoa measurements were below its LOD for all six chordoma patient samples, while dSimoa was able to measure detectable levels of Braculi in all six samples (Figure 5D), demonstrating that even a relatively small improvement in sensitivity can be sufficient to measure a clinically important biomarker. The inventors also examined one chondrosarcoma patient sample that was undetectable by conventional Simoa but detectable by dSimoa. Of the six commercially available plasma and serum samples, Braculi was detectable in one sample using conventional Simoa and in three samples using dSimoa (Figure 5E). Notably, in a large number of samples, the concentrations measured by dSimoa were in the low femtomole range and exceeded the calculated LOD of the conventional Simoa assay, while the measurements of these samples with conventional Simoa remained below that LOD (Figure 5F). However, among the AEBs below the conventional Simoa LOD, higher AEB values ​​generally correlated with higher AMB values ​​and concentrations measured in the dSimoa assay. Furthermore, at higher sample concentrations, dSimoa and conventional Simoa yielded similar measured concentrations. The superior performance of dSimoa in plasma and serum at low concentrations may be due to several factors, including improved LOD and better sample collection efficiency in dSimoa, which can increase the precision of measurements, especially at low concentrations. Another possibility is that dSimoa performs more accurately than conventional Simoa in plasma and serum matrices, with higher signal and recovery against background in plasma and serum, using one-fifth fewer beads. In addition, conventional Simoa uses larger enzyme labels that may exhibit even higher nonspecific binding than the smaller oligonucleotide labels used in dSimoa.Ultimately, the larger volume of washing solution used in the dSimoa assay compared to the conventional Simoa assay may further reduce interference from plasma and serum components.

[0083] [Table 1] LOD and LOQ values ​​were calculated as being above 3 times and 10 times the standard deviation from the background, respectively. The LOD value reported by the corresponding Quanterix Simoa assay was calculated as being above 2.5 times the standard deviation from the background.

[0084] [Table 2]

[0085] [Table 3]

[0086] [Table 4]

[0087] [Table 5]

[0088] [Table 6]

[0089] References for Example 1

[0090] [Table 7-1]

[0091] [Table 7-2]

[0092] [Table 7-3]

[0093] [Table 7-4]

[0094] [Table 7-5]

[0095] [Example 2] Simplification of digital enzyme-coupled immunosorbent assays using tyramide signal amplification and fibrin hydrogels. method Materials. All materials were obtained and used according to the manufacturer's instructions unless otherwise specified below. The IL-6 protein standard (#206-IL-010) and antibodies (capture #MAB206 and detection #BAF206) were purchased from R&D Systems.

[0096] Preparation of antibody-coated capture beads. The IL-6 antibody was first buffer-changed to remove the storage buffer. 0.13 mg of antibody was added to a 50K Amicon Ultra-0.5 mL Centrifugal Filter (MilliporeSigma). Bead conjugation buffer (Quanterix Corp.) was added to the filter up to a volume of 500 μL. The filter device was centrifuged at 14,000 × g for 5 minutes. After centrifugation, the effluent was discarded and additional bead conjugation buffer was added to the filter (total volume 500 μL). The centrifugation process was repeated two more times. The filter was then inverted into a new tube and centrifuged at 1000 × g for 2 minutes. The concentration of the recovered antibody was measured using NanoDrop One (ThermoFisher). The buffer-changed antibody was diluted to 0.5 mg / mL using bead conjugation buffer. 2.8 × 10⁶ beads were prepared to make conjugation beads. 8 Quanterix 647nm dye-encoded carboxylated paramagnetic beads (2.7 μm) were washed three times with bead washing buffer (Quanterix) and three times with bead conjugation buffer, and then resuspended in 190 μL of bead conjugation buffer. Before use, 1 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was dissolved in 100 μL of bead conjugation buffer. After washing the beads, 10 μL of EDC was added to the beads, and the beads were stirred on a rotator for 30 minutes. After bead activation with EDC, the beads were washed once with bead conjugation buffer and then resuspended in 200 μL of 0.5 mg / mL capture antibody solution. The beads were stirred on a rotator for 2 hours. After conjugation, the beads were washed twice with bead washing buffer and then blocked with BSA in 200 μL of bead blocking buffer (Quanterix) for 30 minutes. Finally, the antibody-conjugate beads were washed with bead washing buffer and bead diluent (Quanterix), and resuspended in 200 μL of bead diluent. The beads were counted using a Beckman Coulter Z1 Particle Counter and stored at 4°C.

[0097] CARD digital ELISA. To create a calibration curve using CARD-dELISA, a three-step assay was performed to capture and label target proteins on beads, followed by an on-bead signal amplification step. In the first step, IL-6 protein standards were serially diluted in Homebrew sample diluent (Quanterix), and 100 μL of each calibration standard was added to a low-binding 96-well plate (Greiner Bio-One). IL-6 capture beads (10 μL, at 20,000 beads / μL) and 10 μL of biotinylated IL-6 detection antibody (final concentration 0.3 μg / mL) were also added to the 96-well plate. The plate was incubated for 1 hour with shaking, then washed three times with System Wash Buffer 1 (Quanterix). After the final wash cycle, the remaining wash buffer was removed, and the beads were resuspended in 100 μL of sample diluent. In the second step, 10 μL of 5 μg / mL streptavidin-poly-HRP (Thermo Scientific Pierce) was added to each sample. The plate was incubated for 10 minutes with shaking, and then washed three times with System Wash Buffer 1. After the final washing cycle, the remaining wash buffer was removed. In the third step, on-bead tyramide signal amplification was performed using a modified protocol with the Alexa Fluor 488 Tyramide SuperBoost Kit (ThermoFisher Scientific). Specifically, the working solution was prepared by mixing 1.6 mL of 1X reaction buffer with 16 μL of 1X hydrogen peroxide solution and 16 μL of Alexa Fluor 488-tyramide reagent. The beads were then resuspended in 200 μL of the tyramide working solution. The plate was incubated for 1 hour without shaking. After labeling, 50 μL of a 1:11 diluted reaction stop solution (SuperBoost Kit) was added to each bead suspension, and the plate was incubated for 2 minutes with shaking. Next, the plate was washed six times with System Wash Buffer 1 containing a 1:11 diluted reaction stop solution. After the final wash cycle, the beads were resuspended in 30 μL of 1×PBS. The beads were then added to a silicon isolation well on a glass slide (Electron Microscopy Sciences).To prepare the fibrin hydrogel, equal volumes of 10 mg / mL of fibrinogen (derived from bovine plasma, IS type, MilliporeSigma) in 1×PBS and 1.25 U / mL of thrombin (derived from bovine plasma, MilliporeSigma) in 1×PBS were mixed. To dissolve the fibrinogen in PBS, the solution was heated to 37°C before use. After mixing the hydrogel reagents, 50 μL of the mixture was added to each isolation well, and the hydrogel was allowed to form for 15 minutes before imaging.

[0098] Imaging and Analysis. Brightfield and fluorescence images of the hydrogel-immobilized bead array were acquired using an Olympus IX81 inverted microscope at 10x magnification with an OCRA-Flash 4.0 LT+ CMOS camera (Hamamatsu). CellSens software was used to manage the microscope stage and acquire images. Brightfield images used to identify bead positions were acquired with an exposure time of 20 ms. Fluorescence images using a GFP filter cube, used to identify "on" and "off" beads, were acquired with an exposure time of 1 second.

[0099] Image analysis was performed using a custom MATLAB algorithm. Brightfield images were processed by computer interpolation (making beads brighter and the background darker), filtering with a top-hat filter to correct for uneven illumination, and converting the binary images. Fluorescence images were also filtered with a top-hat filter to correct for uneven illumination. Beads were positioned in the brightfield images using a disk-type morphological structuring element. The signal intensity of each bead was measured from the corresponding GFP fluorescence image by calculating the intensity of the top quarter within the bead region. The cutoff value between "off" and "on" beads was determined by fitting the distribution of bead intensities in a blank (0 fM) standard to a normal distribution, and setting the cutoff intensity to above 3 times the standard deviation from the mean for low-concentration samples (<50 fM, cutoff value approximately 90) and above 4 times the standard deviation from the mean for high-concentration samples (≧50 fM, cutoff value approximately 100). The proportion of "on" beads was calculated by dividing the number of "on" beads by the total number of beads.

[0100] Calibration curves for AEB versus concentration were fitted to 4-parameter logistic (4PL) regression in GraphPad Prism version 8.3.0. The 4PL-fitted curve was used to determine unknown IL-6 concentrations in saliva samples. All measurements were performed in pairs.

[0101] Saliva sample analysis. Pooled saliva samples were purchased from BioIVT and stored at -80°C until use. Saliva was centrifuged at 13,150 × g for 20 minutes at 4°C. After centrifugation, the supernatant was removed, and the saliva samples were diluted to 25X for CARD-dELISA analysis and to 8X for Simoa analysis.

[0102] Simoa assay. The conventional Simoa assay was performed using an HD-1 Analyzer (Quanterix). Capture beads, detection antibody, and streptavidin-β-galactosidase (SβG) solution were placed in reagent bottles and loaded into the instrument. For calibration curves, sequentially diluted IL-6 protein standards and diluted saliva samples were pipetteed into 96-well plates (Quanterix) and loaded into the instrument. SβG substrate resolphin β-D-galactopyranoside, system wash buffer 1, system wash buffer 2, and Simoa sealing oil were obtained from the Quanterix and loaded into the instrument according to the manufacturer's instructions. Standards and samples were processed using a standard 3-step assay. Image analysis and AEB calculation were performed automatically by the onboard Simoa software.

[0103] result The inventors have attempted to develop a simplified digital ELISA format that reduces the need for expensive equipment and sophisticated microfluidics or robotics. By performing a signal amplification step in which fluorophores are directly conjugated to beads, the inventors eliminate the need for compartmentalizing beads in microwell arrays or microfluidic droplets. Instead, the signal amplification step occurs in bulk solution, such as in the same reaction chamber as the target protein capture and labeling step. Furthermore, this method does not require the expensive engineering and robotics required for bead loading into microwell arrays. CARD-dELISA has a simplified method for bead imaging used for single-molecule counting by immobilizing beads in inexpensive fibrin hydrogel. In this format, beads are placed on a glass slide and immobilized in a fibrin hydrogel layer formed in situ. After image acquisition, the beads are positioned and a MATLAB algorithm is used to measure their fluorescence intensity for single-molecule counting. As proof of concept, the inventors created a calibration curve for interleukin-6 (IL-6) and measured the IL-6 levels in saliva samples.

[0104] The first step in developing CARD-dELISA was to establish and optimize a method for on-bead enzyme amplification for single-molecule signal generation. The inventors use TSA, which is commonly used in immunohistochemistry and in situ hybridization experiments. Other researchers have also reported on the use of TSA for digital ELISA. 27The inventors improved upon previous literature reports by reducing the number of steps in the immunoassay (reduced from a 5-step assay to a 3-step assay), which is an important consideration when implementing digital ELISA in point-of-care devices. The assay format of CARD-dELISA is summarized in Figure 9A. Antibody-coated capture beads (200,000) are added to the sample to enable capture of target protein molecules. Similar to conventional Simoa, the inventors use a large number of beads compared to the number of target protein molecules. This ensures that the assay follows a Poisson distribution, where the majority of beads do not bind to the target molecules, and only a small percentage of beads bind to a single target molecule. After protein capture, the target molecules are labeled with a biotinylated detection antibody and streptavidin-poly-HRP (a streptavidin-conjugate polymer containing several horseradish peroxidase molecules) to form a complete enzyme-labeled immunocomplex. Next, the beads are resuspended in a solution containing hydrogen peroxide and a tyramide fluorophore conjugate (tyramide Alexa Fluor 488) for the signal generation step. In the presence of hydrogen peroxide, HRP catalytically converts tyramide to a radical intermediate. This tyramide radical forms covalent bonds with other aromatic rings near the HRP molecule, nearby proteins on the beads, and tyrosine residues on antibodies. Upon completion of this step, beads with complete immunocomplexes are labeled with numerous fluorescent dyes attached by feed bonds. This completes the on-bead signal generation step and enables subsequent single-molecule counting. The inventors did not observe any detectable cross-labeling between beads after the tyramide labeling step. This is supported by the observation that, as predicted in this assay format following a Poisson distribution, only a small number of beads have a detectable fluorescent signal at low protein concentrations. Furthermore, in the tyramide labeling step, the inventors use a dilute bead solution to prevent the tyramide radical from diffusing to other beads during the lifetime of the radical intermediate. 27 .

[0105] The second step in developing CARD-dELISA was to establish a method for bead immobilization for imaging and single-molecule counting. Since the amplified enzyme signal is already conjugated to the beads in a preceding step of the assay, the inventors are not limited by the need to compartmentalize the beads in microwells or droplets for enzyme amplification. The inventors used fibrin hydrogel for bead immobilization (Figure 9B). The fibrin hydrogel is formed when thrombin enzymatically polymerizes fibrinogen into a fibrin hydrogel network. 28 Synthetic fibrin hydrogels are often used for applications including cell encapsulation and tissue engineering, and can be readily formed in situ. 29~31 Embedding beads in fibrin hydrogel is a quick and simple method for immobilizing beads for imaging. Figure 10A shows several bead samples embossed in fibrin hydrogel. To immobilize the beads in fibrin hydrogel, the bead solution is first drop-cast onto a glass slide inside a silicon isolation well (7mm x 7mm x 2mm). A mixture of fibrinogen and thrombin solutions is immediately added to the isolation well. The hydrogel forms in approximately 15 minutes, and the beads are trapped in the fibrin polymer network. Figure 10B shows a bright-field image of several hundred beads in fibrin hydrogel. This image is approximately 100 μm x 100 μm and represents a small area of ​​the bead array. The entire bead array can be captured in approximately 20-25 images at 10x magnification. Because the beads are relatively large (2.7 μm in diameter) and settle rapidly from the solution, the inventors observed that the beads are mainly located in the same z-plane (i.e., the interface between the glass slide and the fibrin hydrogel). In addition to being quick and simple, bead encapsulation in fibrin hydrogel is also a less expensive option compared to microwell arrays. This cost reduction is crucial for developing an inexpensive point-of-care digital ELISA platform.

[0106] The final step in CARD-dELISA was to develop a method for single-molecule counting. Bright-field and fluorescence images of the bead array were captured using an inverted fluorescence microscope, and the images were then analyzed using a MATLAB algorithm to perform single-molecule counting. Examples of bright-field images (Figure 11A) and 488 nm fluorescence images (Figure 11B) of a small area of ​​interest are shown. The bright-field image was used to locate each bead, and the fluorescence image was used to identify beads with deposited tyramide Alexa Fluor 488 dye from the signal amplification step. By superimposing the two images (Figure 11C), the inventors observed that two of the eight beads in the image were "on" (gray arrows). The position of each bead was automatically determined by the MATLAB algorithm, and the corresponding fluorescence intensity of each bead was calculated. For all samples, the fluorescence intensity of all beads was plotted as a histogram; the 0 fM IL-6 standard is plotted in Figure 11D, and the 50 fM IL-6 standard is plotted in Figure 11E. The histogram for the blank sample in Figure 11D is fitted to a normal distribution, and the cutoff for "on" versus "off" beads is set above four times the standard deviation from the mean fluorescence intensity of the blank (gray squares in Figures 11D and 11E). AEB is calculated by dividing the number of "on" beads by the total number of beads. The inventors analyze approximately 50,000 to 60,000 beads per sample (i.e., about 30% of the beads), which is an improvement in the percentage of beads analyzed compared to the conventional Simoa, which analyzes only about 5% of the beads.

[0107] As proof of concept, the inventors used IL-6 as a model protein to generate a calibration curve using CARD-dELISA, plotted in Figure 12; the AEB values ​​for the calibration curve are shown in Table 7. As expected, at low protein concentrations, most beads do not bind to IL-6 (small AEB values). As the IL-6 concentration increases, the number of beads that bind to IL-6 increases. The inventors also created an IL-6 calibration curve using a standard Simoa assay on an HD-1 Analyzer (Figure 13). Both curves were fitted to a four-parameter logistic (4PL) regression used to estimate the LOD and LOQ for each assay (Table 8). CARD-dELISA yielded a wide dynamic range and an LOD of 1.36 fM for IL-6. Finally, the inventors measured IL-6 in commercially available saliva samples using CARD-dELISA and compared it with conventional Simoa results to confirm that CARD-dELISA can be used to reliably detect the protein in biological fluids. The results from both assays are reported in Table 9 and plotted in the inset in Figure 12. We observed sufficient agreement between the two methods (Spearman correlation coefficient is 1.00), demonstrating that CARD-dELISA can reliably detect IL-6 in saliva. Furthermore, like Simoa, CARD-dELISA allows for the use of highly diluted samples. Saliva samples analyzed by CARD-dELISA were diluted 25X. The use of high sample dilution ratios reduces nonspecific adsorption and thus improves the accuracy of the assay. In addition, CARD-dELISA requires only 10 μL of saliva, meaning that protein biomarkers can be measured from small sample volumes.

[0108] [Table 8]

[0109] [Table 9]

[0110] [Table 10]

[0111] References for Example 2

[0112] [Table 11-1]

[0113] [Table 11-2]

[0114] [Table 11-3]

[0115] [Table 11-4]

[0116] Other Embodiments Although the present invention has been described in conjunction with its detailed description, it is understood that the foregoing description is intended to be illustrative and not intended to limit the scope of the invention as defined by the appended claims. Other aspects, advantages and modifications are within the scope of the following claims. The inventions described in the original claims of this application are listed below. [Invention 1] A method for detecting biomolecules in a sample, Prepare a solution containing the aforementioned sample; The solution is brought into contact with a plurality of beads, each containing the capture portion that binds to the biomolecules in the sample, under conditions and for a sufficient amount of time for the capture portion to bind to the biomolecules; The solution is brought into contact with a binding site that allows for the generation of a non-diffusible, detectable signal on each bead, sufficient to bind to the biomolecule and enable the detection of each bead containing the target molecule, and then an amplified signal is generated; The aforementioned beads are optionally fixed in a single layer; and To detect the aforementioned signal The method, including the method described above. [Invention 2] The method according to Invention 1, wherein immobilizing the beads includes drop-casting the solution containing the beads onto a slide or catalyzing the gelation of the solution. [Invention 3] The method according to invention 1 or 2, further comprising bringing the solution into contact with a signal amplification portion that is coupled to the bonding portion. [Invention 4] The method according to Invention 3, wherein the signal amplification portion comprises an enzyme or branched DNA. [Invention 5] The method according to any one of inventions 1 to 3, wherein detecting the signal includes imaging the beads to detect fluorescence or other signals. [Invention 6] The method according to any one of inventions 1 to 4, further comprising determining the number and / or percentage of beads containing a bead-biomolecular complex. [Invention 7] The method according to Invention 1, wherein the beads include a polymer, a metal, a metal oxide, a semiconductor and / or a semiconductor oxide. [Invention 8] The method according to Invention 1, wherein the detectable signal is generated by rolling circle amplification followed by hybridization with a complementary fluorescently labeled DNA probe; tyramide signal amplification (TSA); hybridization chain reaction; enzyme-catalyzed near-field labeling (PL) polymerization; polymerization-based signal amplification; or magnetic bead quantum dot immunoassay. [Invention 9] The method according to Invention 1, wherein the detectable signal is generated by a pre-amplified signal. [Invention 10] The method according to invention 8, wherein the pre-amplified signal is a labeled polymer or nanoparticles. [Invention 11] The method according to Invention 1, wherein the beads are drop-cast onto the surface and dried before the signal is detected. [Invention 12] The method according to Invention 1, wherein the solution is applied to or comes into contact with a surface before the signal is detected, thereby catalyzing gelation. [Invention 13] The method according to invention 11 or 12, wherein the surface is a slide, tip, or flow cell. [Invention 14] The method according to Invention 1, wherein catalyzing the gelation of the solution involves mixing fibrinogen and / or thrombin; fibrin; cellulose; collagen; gelatin; agarose; hyaluronic acid; polyhydroxyethyl methacrylate (poly(HEMA)); polyethylene glycol (PEG); or acrylamide into the solution. [Invention 15] The method according to Invention 1, wherein the solution comprises fibrinogen and / or a polymer selected from thrombin; fibrin; cellulose; collagen; gelatin; agarose; hyaluronic acid; polyhydroxyethyl methacrylate (poly(HEMA)); polyethylene glycol (PEG); or acrylamide; and comprises catalyzing gelation to the polymer.

Claims

1. A method for detecting biomolecules in a sample, Prepare a solution containing the aforementioned sample; The solution is brought into contact with a plurality of beads, each containing the capture portion that binds to the biomolecules in the sample, under conditions and for a sufficient amount of time for the capture portion to bind to the biomolecules; The solution is brought into contact with a binding site that binds to the biomolecule and enables the generation of a non-diffusible, detectable signal on each bead containing the target molecule, thereby enabling detection of each bead, and then an amplified signal is generated; Immobilizing the aforementioned beads in a single layer; and To detect the aforementioned signal Includes, The detectable signal is generated by rolling circle amplification followed by hybridization with a complementary fluorescently labeled DNA probe; tyramide signal amplification (TSA); hybridization chain reaction; enzyme-catalyzed near-field labeling (PL) polymerization; polymerization-based signal amplification; or magnetic bead quantum dot immunoassay. The immobilization of the beads includes drop-casting the solution containing the beads onto a slide, or catalyzing the gelation of the solution containing the beads. The aforementioned method.

2. The method according to claim 1, further comprising bringing the solution into contact with a signal amplification portion that is bonded to the bonding portion.

3. The method according to claim 2, wherein the signal amplification portion comprises an enzyme or branched DNA.

4. The method according to claim 1 or 2, wherein detecting the signal includes imaging the beads to detect fluorescence or other signals.

5. The method according to any one of claims 1 to 4, further comprising determining the number and / or percentage of beads containing a bead-biomolecular complex.

6. The method according to claim 1, wherein the beads include a polymer, a metal, a metal oxide, a semiconductor and / or a semiconductor oxide.

7. The method according to claim 1, wherein the detectable signal is generated by rolling circle amplification followed by hybridization with a complementary fluorescently labeled DNA probe.

8. The method according to claim 1, wherein the detectable signal is generated by a pre-amplified signal.

9. The method according to claim 8, wherein the pre-amplified signal is a labeled polymer or nanoparticles.

10. The method according to claim 1, wherein the beads are drop-cast onto the surface and dried before the signal is detected.

11. The method according to claim 1, wherein the solution is applied to or comes into contact with a surface before the signal is detected, and gelation is catalyzed.

12. The method according to claim 10 or 11, wherein the surface is a slide, tip, or flow cell.

13. The method according to claim 1, wherein catalyzing the gelation of the solution containing the beads involves mixing fibrinogen and / or thrombin; fibrin; cellulose; collagen; gelatin; agarose; hyaluronic acid; polyhydroxyethyl methacrylate (poly(HEMA)); polyethylene glycol (PEG); or acrylamide into the solution.

14. The method according to claim 1, wherein the solution containing the beads further comprises a polymer selected from fibrinogen and / or thrombin; fibrin; cellulose; collagen; gelatin; agarose; hyaluronic acid; polyhydroxyethyl methacrylate (poly(HEMA)); polyethylene glycol (PEG); or acrylamide, and the method comprises catalyzing the gelation of the polymer.

Images