Digital ELISA detection method to prevent fluorescence signal crosstalk

Abstract

The present invention particularly relates to a digital ELISA detection method for preventing fluorescence signal crosstalk, the method comprising: preparing a sample containing a target molecule to be detected, capturing the target molecule to be detected using magnetic beads, and forming a complex of "magnetic beads-target molecule to be detected-enzyme" by affinity reaction; transferring the sample formed with the complex to a microwell array chip; transferring a fluorescent substrate to the microwell array chip; adding an isolation liquid to the microwell array chip; waiting for the enzyme to perform an enzymatic reaction with the fluorescent substrate to generate fluorescent molecules; determining the number of microwells containing magnetic beads in the microwell array and the number of microwells in the microwell array whose fluorescent signals are higher than a threshold value; based on the above two quantities, and based on the number of magnetic beads in the sample and the probability that the target molecule to be detected is captured by the magnetic beads and further combined with the enzyme, determining the number of target molecules to be detected in the sample. The present invention improves the accuracy of distinguishing negative signals and positive signals.

Description

Technical Field

[0001] The invention relates to biological detection, and in particular to a digital ELISA detection method for preventing fluorescence signal crosstalk. Background Art

[0002] Currently, there are a variety of detection technologies on the market for the quantitative detection of low-concentration analytes.

[0003] One type uses highly sensitive optical instruments to detect single-molecule signals. Representative products include Merck's SMC TM (Single Molecule Counting) system, however, the optical path structure of this system is complex and expensive, the detection speed is slow, and the detection time is also very long for serial detection of microspheres.

[0004] Another type uses amplification technology to increase the number of target molecules to be detected in order to provide enough signal molecules. Representative products include Chimera's However, the detection method of this system is complicated and prone to false positive signals.

[0005] Simoa by Quanterix TM The Single-molecule Array (Single-molecule Array) system uses digital ELISA (Enzyme Linked Immunosorbent Assay) technology similar to digital PCR (Polymerase Chain Reaction). However, the background signal of a single reaction detection unit in this system is high, and the fluorescent substance generated in a single reaction detection unit will diffuse to adjacent reaction detection units, thereby generating fluorescent signal crosstalk and affecting the accuracy of distinguishing negative and positive signals.

[0006] Therefore, there is an urgent need for a digital ELISA detection method that can prevent fluorescence signal crosstalk, reduce the fluorescence signal background of the reaction system, and reduce the diffusion of the fluorescence signal within the reaction detection unit, thereby improving the accuracy of distinguishing negative and positive signals. Summary of the invention

[0007] In order to solve the above problems in the prior art, the present invention provides a digital ELISA detection method for preventing fluorescence signal crosstalk, reducing the fluorescence signal background of the reaction system and reducing the diffusion of the fluorescence signal in the reaction detection unit, thereby improving the accuracy of distinguishing negative signals and positive signals.

[0008] The present invention provides a digital ELISA detection method for preventing fluorescence signal crosstalk, the method comprising:

[0009] Step 1: prepare a sample, wherein the sample contains a target molecule to be detected, capture the target molecule to be detected in the sample using magnetic beads, and form a complex of "magnetic beads-target molecule to be detected-enzyme" through affinity reaction;

[0010] Step 2, transferring the sample formed with the complex into a microwell array chip, wherein the microwell array chip comprises a microwell array and a fluid chamber accommodating the microwell array, and each microwell in the microwell array is configured to accommodate only one magnetic bead;

[0011] Step 3, transferring the fluorescent substrate into the microwell array chip, wherein the enzyme can undergo an enzymatic reaction with the fluorescent substrate to generate fluorescent molecules;

[0012] Step 4, adding an isolation liquid into the microwell array chip to isolate all microwells in the microwell array from each other;

[0013] Step 5, waiting for the enzyme to perform an enzymatic reaction with the fluorescent substrate to generate the fluorescent molecule, wherein the fluorescence signal of the microwell where the magnetic beads that capture a single target molecule to be detected are located is higher than a threshold value, and the fluorescence signal of the microwell where the magnetic beads that capture zero target molecules to be detected are located is lower than a threshold value;

[0014] Step 6, determining the number of microwells in the microwell array containing the magnetic beads, and determining the number of microwells in the microwell array whose fluorescence signals are higher than a threshold value;

[0015] Step 7: Determine the number of target molecules to be detected in the sample based on the number of microwells in the microwell array containing the magnetic beads and the number of microwells in the microwell array whose fluorescence signals are higher than a threshold value, and based on the number of magnetic beads in the sample and the probability that the target molecules to be detected are captured by the magnetic beads and further bound to the enzyme.

[0016] In one embodiment of the present invention, the isolation liquid is fluorine oil, and the viscosity coefficient of the fluorine oil at 25° C. is in the range of 0.1 cSt to 12500 cSt.

[0017] In one embodiment of the present invention, the viscosity coefficient of the fluorine oil at 25° C. is in the range of 1 cSt to 5000 cSt.

[0018] In one embodiment of the present invention, the viscosity coefficient of the fluorine oil at 25° C. is 11.4 cSt, 451 cSt or 1366 cSt.

[0019] In one embodiment of the present invention, the isolation fluid is silicone oil, and the viscosity coefficient of the silicone oil at 25° C. is in the range of 10 cSt to 12500 cSt.

[0020] In one embodiment of the present invention, the viscosity coefficient of the silicone oil at 25° C. is in the range of 10 cSt to 5000 cSt.

[0021] In one embodiment of the present invention, the viscosity coefficient of the silicone oil at 25° C. is 350 cSt.

[0022] In one embodiment of the present invention, the number of the magnetic beads in the sample is determined based on counting using a particle counter, flow cytometry or a cell counting plate.

[0023] In one embodiment of the present invention, the ratio of the number of the magnetic beads in the sample to the number of the target molecules to be detected in the sample is greater than or equal to 5.

[0024] In one embodiment of the present invention, in step 6, bright field images and fluorescence images of one or more fields of view are taken of the microwell array, wherein the number of microwells in the microwell array containing the magnetic beads is determined based on the bright field images of the one or more fields of view, and the number of microwells in the microwell array whose fluorescence signals are higher than a threshold is determined based on the fluorescence images of the one or more fields of view.

[0025] In one embodiment of the present invention, in step 7, the following formula is used to determine the number of the target molecules to be detected in the sample: Wherein, M0 is the number of the target molecules to be detected in the sample, N0 is the number of the magnetic beads in the sample, M is the number of microwells in the microwell array whose fluorescence signals are higher than the threshold, N is the number of microwells in the microwell array containing the magnetic beads, and p is the probability that the target molecules to be detected are captured by the magnetic beads and further bound to the enzyme.

[0026] In one embodiment of the present invention, the ratio of the number of all microwells in the microwell array to the number of the magnetic beads in the sample is in the range of 0.1 to 10.

[0027] In one embodiment of the present invention, in step 2, the sample formed with the complex is transferred to the fluid chamber of the microwell array chip and the microwells of the microwell array by self-aspiration, centrifugation or pressure injection, and the excess magnetic beads in the fluid chamber are removed by centrifugation, and the magnetic beads in the microwells are retained.

[0028] In one embodiment of the present invention, in step 3, the fluorescent substrate is transferred to the fluid chamber of the microwell array chip and the microwells of the microwell array by self-absorption, centrifugation or pressure injection, and the excess fluorescent substrate in the fluid chamber is removed by centrifugation, and the fluorescent substrate in the microwell is retained.

[0029] In one embodiment of the present invention, in step 4, the isolation liquid is added to the fluid chamber of the microwell array chip by self-imbibition, centrifugation or pressure injection, and the magnetic beads and fluorescent substrate in the microwells of the microwell array are sealed and isolated.

[0030] In one embodiment of the present invention, the target molecule to be detected is a protein molecule to be detected.

[0031] As described above, the present invention has the following beneficial effects:

[0032] The present invention can absolutely quantify the target molecules to be detected in the sample by the number of sample distribution units clearly determined by counting methods such as a particle counter, flow cytometry or a cell counting plate, the number of reaction detection units determined by bright field images of one or more fields of view, the number of positive reaction detection units determined by fluorescent images of one or more fields of view, and the probability of the target molecules to be detected being captured by magnetic beads and further binding to the enzyme as a constant.

[0033] In the present invention, the number of magnetic beads in the sample determines the upper limit of the dynamic detection range. By changing the number of magnetic beads in the sample, the dynamic detection range can be accurately controlled. In theory, the upper limit of the dynamic detection range is less than 5 times the number of magnetic beads.

[0034] The present invention greatly reduces the fluorescence signal background of the reaction system through a two-step sample addition method, and greatly reduces the diffusion of the fluorescence signal in the reaction detection unit by optimizing the isolation liquid with a high viscosity coefficient, thereby improving the accuracy of distinguishing negative signals and positive signals. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a schematic diagram of the principle of a digital ELISA detection method for preventing fluorescence signal crosstalk according to an embodiment of the present invention;

[0036] Figure 2 is a schematic flow chart of a digital ELISA detection method for preventing fluorescence signal crosstalk according to an embodiment of the present invention;

[0037] Figure 3A is a fluorescence image obtained by a two-step loading method, and Figure 3B It is a fluorescence image obtained by one-step sample addition method;

[0038] Figure 4A is from Figure 3A The fluorescence signals of a certain column of microwells are extracted and calculated from the fluorescence image shown, and Figure 4B is from Figure 3B The fluorescence signals of a certain column of microwells are extracted and calculated from the fluorescence image shown;

[0039] Figure 5A1-Figure 5D2 These are the fluorescence images taken 1 minute and 5 minutes after isolation using different isolation solutions;

[0040] Figure 6A1-Figure 6B2 This is a schematic diagram of the change in fluorescence value of the positive micropores and the fluorescence value of the crosstalk micropores over time after isolation with different isolation fluids. DETAILED DESCRIPTION

[0041] Embodiments of the present invention are described below with reference to the accompanying drawings.

[0042] Exemplary embodiments will be described in detail herein, examples of which are shown in the accompanying drawings. When the following description refers to the drawings, the same numbers in different drawings represent the same or similar elements unless otherwise indicated. The implementations described in the following exemplary embodiments do not represent all implementations consistent with the present application. Instead, they are merely examples of devices and methods consistent with some aspects of the present application as detailed in the appended claims.

[0043] The terms used in this application are only for the purpose of describing specific embodiments, and are not intended to limit this application. The singular forms of "a", "said" and "the" used in this application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings. It should also be understood that the term "and / or" used in this article refers to and includes any or all possible combinations of one or more associated listed items. "Include" or "comprising" and similar words mean that the elements or objects appearing in front of "include" or "comprising" cover the elements or objects listed after "include" or "comprising" and their equivalents, and do not exclude other elements or objects.

[0044] Figure 1 is a schematic diagram of the principle of a digital ELISA detection method for preventing fluorescence signal crosstalk according to an embodiment of the present invention, and Figure 2 It is a schematic flow chart of a digital ELISA detection method for preventing fluorescence signal crosstalk according to an embodiment of the present invention.

[0045] like Figure 1 and Figure 2 As shown, the digital ELISA detection method for preventing fluorescence signal crosstalk includes:

[0046] In step 1 (S1), a sample is prepared, the sample contains a target molecule to be detected, the target molecule to be detected in the sample is captured using magnetic beads, and a complex of "magnetic beads-target molecule to be detected-enzyme" is formed through an affinity reaction.

[0047] Specifically, the target molecule to be detected can be a protein molecule to be detected, and the protein molecule to be detected can be derived from a liquid sample of the human body (blood, body fluid, tissue, etc.). More specifically, the protein molecule to be detected can be from the supernatant of serum, plasma, tissue homogenate or cell extract. Based on this, the present invention can accurately quantify ultra-low abundance protein molecules that are difficult to detect in normal people and disease patients by conventional methods, and develop new applications in the fields of early detection, companion diagnosis, and drug development of major diseases such as tumors, neurological diseases, infectious diseases, and immune inflammation.

[0048] Specifically, the magnetic beads may have a diameter of micrometer scale. As a sample distribution unit, the surface of the magnetic beads is modified with a capture antibody that can specifically bind to the target molecule to be detected, for example, to produce an antibody-antigen reaction with the protein molecule to be detected, thereby capturing the protein molecule to be detected.

[0049] The number of magnetic beads in the sample can be clearly determined based on counting methods such as a particle counter, flow cytometry or a cell counting plate. In addition, the uniformity of the magnetic beads in the sample can also be accurately controlled based on the above technology. Based on this, the present invention can absolutely quantify the target molecule to be detected in the sample based at least on the number of magnetic beads in the sample.

[0050] In addition, the number of magnetic beads in the sample should be much larger than the number of target molecules to be detected in the sample. Preferably, the ratio of the number of magnetic beads in the sample to the number of target molecules to be detected in the sample is greater than or equal to 5, so that the statistical distribution of the target molecules to be detected captured by the magnetic beads conforms to the Poisson distribution.

[0051] Theoretically, there are three possibilities for the number of target molecules to be detected captured by each magnetic bead: capturing zero target molecules to be detected, capturing a single target molecule to be detected, or capturing multiple target molecules to be detected. When the number of magnetic beads is large enough (for example, at least 5 times the number of target molecules to be detected), most of the magnetic beads only capture zero target molecules to be detected or a single target molecule to be detected, thereby achieving single-molecule fluorescence signal amplification as described below.

[0052] In the present invention, the number of magnetic beads in the sample determines the upper limit of the dynamic detection range. By changing the number of magnetic beads in the sample, the dynamic detection range can be accurately controlled. Theoretically, the upper limit of the dynamic detection range is less than 5 times the number of magnetic beads. For example, the number of magnetic beads in the sample is in the range of 100,000 to 10 million, so that the upper limit of the dynamic detection range is in the range of 500,000 to 50 million.

[0053] As an example, the capture antibody modified on the surface of the magnetic beads specifically captures the target molecule to be detected in the sample, and further connects the detection antibody and the enzyme, finally forming an immune complex of "magnetic beads-capture antibody-target molecule to be detected-detection antibody-enzyme", and the enzyme can react enzymatically with the fluorescent substrate to generate fluorescent molecules. For example, the magnetic beads can be connected with β-galactosidase through the above-mentioned double antibody sandwich reaction, and the fluorescent substrate can be non-fluorescent resorufin-β-galactoside (RGP), and β-galactosidase can catalyze the hydrolysis of non-fluorescent resorufin-β-galactoside (RGP) to generate resorufin molecules that can emit fluorescence.

[0054] At step 2 (S2), the sample with the complex formed is transferred to a microwell array chip, wherein the microwell array chip includes a microwell array and a fluid chamber accommodating the microwell array, and each microwell in the microwell array is configured to accommodate only one magnetic bead.

[0055] Specifically, the microwell array includes a plurality of microwells, each of which may be slightly larger than the size of a magnetic bead, so as to be configured to accommodate only one magnetic bead. The ratio of the number of all microwells in the microwell array to the number of magnetic beads in the sample is in the range of 0.1 to 10, so that as many magnetic beads as possible fall into the microwells. Figure 1 As shown, if the magnetic beads fall into the micropores, the micropores can be called effective micropores or reaction detection units. The number of reaction detection units (i.e., effective micropores) is not greater than the number of sample distribution units (i.e., magnetic beads). In theory, the closer the number of reaction detection units is to the number of sample distribution units, the higher the accuracy and resolution of digital detection.

[0056] For example, the microwell array includes 188,000 microwells, the microwells are circular microwells, the diameter of the microwells is 4 μm, the depth of the microwells is 4 μm, and the center distance between the microwells is 8 μm.

[0057] Specifically, under the first centrifugal condition, the sample with the complex formed is transferred to the micropore array chip, and the first centrifugal condition is, for example, 200rpm for 10 seconds, and the centrifugal force sends the sample into the fluid chamber of the micropore array chip. Then, let it stand for a specific time (for example, 2 minutes) until the sample settles into the micropores of the micropore array. Then, under the second centrifugal condition, the excess magnetic beads in the fluid chamber are removed, and the magnetic beads in the micropores are retained. The second centrifugal condition is, for example, 600rpm for 10 seconds. At this speed, the excess magnetic beads in the fluid chamber are separated, and the magnetic beads in the micropores are retained. It is understandable that the sample with the complex formed can also be transferred to the micropore array chip by self-priming or pressure injection.

[0058] At step 3 (S3), the fluorescent substrate is transferred into the microwell array chip.

[0059] Specifically, under the third centrifugal condition, the fluorescent substrate is transferred to the microporous array chip, and the third centrifugal condition is, for example, 200rpm for 10 seconds, and the centrifugal force sends the fluorescent substrate into the fluid chamber of the microporous array chip. The fluorescent substrate enters the micropores by means of fluid shear force or molecular diffusion. Then, under the fourth centrifugal condition, the excess fluorescent substrate in the fluid chamber is removed, and the fluorescent substrate in the micropores is retained. The fourth centrifugal condition is, for example, 600rpm for 10 seconds. At this speed, the excess fluorescent substrate in the fluid chamber is separated, and the fluorescent substrate in the micropores is retained. It is understandable that the fluorescent substrate can also be transferred to the microporous array chip by means of self-absorption or pressure injection.

[0060] At step 4 (S4), a spacer liquid is added into the microwell array chip to isolate all microwells in the microwell array from each other.

[0061] Specifically, the isolation liquid can be fluorine oil or silicone oil with a high viscosity coefficient. Under the fifth centrifugal condition, the isolation liquid is added to the fluid chamber of the micropore array chip, and the magnetic beads and fluorescent substrate in the micropores of the micropore array are sealed and isolated. The fifth centrifugal condition is, for example, 200rpm for 10 seconds. The centrifugal force sends the isolation liquid into the fluid chamber of the micropore array chip. The hydrophobic isolation liquid can fully infiltrate the surface of the micropore array, further remove the magnetic beads that have not fallen into the micropores, and isolate all the micropores from each other. The fluorescent molecules subsequently generated are difficult to diffuse, and the thermal stability of a single micropore is excellent. It is understandable that the isolation liquid can also be added to the fluid chamber of the micropore array chip by self-priming or pressure injection.

[0062] At step 5 (S5), wait for the enzyme to perform an enzymatic reaction with the fluorescent substrate to generate fluorescent molecules, wherein the fluorescence signal of the microwell where the magnetic beads that have captured a single target molecule to be detected are located is higher than the threshold, and the fluorescence signal of the microwell where the magnetic beads that have captured zero target molecules to be detected are located is lower than the threshold.

[0063] For example, wait for 1 minute at room temperature for, for example, β-galactosidase to catalyze the hydrolysis of non-fluorescent resorufin-β-galactoside (RGP) to generate fluorescent resorufin molecules. As described above, when the statistical distribution of the target molecules to be detected captured on the magnetic beads conforms to the Poisson distribution, most of the magnetic beads only capture zero or a single target molecule to be detected, thereby achieving single-molecule fluorescence signal amplification, that is, Figure 1As shown, the fluorescence signal of the microwell where the magnetic beads with a single target molecule to be detected are located is higher than the threshold, and the fluorescence signal of the microwell where the magnetic beads with zero target molecules to be detected are located is lower than the threshold. The microwells in the microwell array with a fluorescence signal higher than the threshold can be read as 1 ("positive"), and the microwells in the microwell array with a fluorescence signal lower than the threshold can be read as 0 ("negative").

[0064] At step 6 (S6), the number of microwells in the microwell array containing magnetic beads is determined, and the number of microwells in the microwell array having a fluorescence signal higher than a threshold value is determined.

[0065] In other words, step 6 determines the number of reaction detection units and the number of positive reaction detection units, respectively. Specifically, a bright field image (e.g., mercury lamp light source, and exposure time 50ms) and a fluorescent image (e.g., 577nm excitation, 620nm emission, and exposure time 600ms) of one or more fields of view (e.g., 35 fields of view) of the microwell array are taken, wherein, Figure 1 As shown, the number of microwells containing magnetic beads in the microwell array is determined based on the bright field images of one or more fields of view, and the number of microwells in the microwell array with fluorescence signals higher than a threshold is determined based on the fluorescence images of one or more fields of view.

[0066] At step 7 (S7), the number of target molecules to be detected in the sample is determined based on the number of microwells in the microwell array containing magnetic beads and the number of microwells in the microwell array whose fluorescence signals are higher than the threshold, and based on the number of magnetic beads in the sample and the probability that the target molecules to be detected are captured by the magnetic beads and further bound to the enzyme.

[0067] Specifically, the following formula is used to determine the number of target molecules to be detected in the sample: Wherein, M0 is the number of target molecules to be detected in the sample, N0 is the number of magnetic beads in the sample, M is the number of microwells in the microwell array whose fluorescence signals are higher than the threshold, N is the number of microwells in the microwell array containing magnetic beads, and p is the probability that the target molecules to be detected are captured by the magnetic beads and further bound to the enzyme (if the operation uniformity is high, the probability p is a constant less than 100%).

[0068] In other words, the present invention can absolutely quantify the target molecules to be detected in the sample by the number of sample distribution units clearly determined by counting methods such as a particle counter, flow cytometry or a cell counting plate, the number of reaction detection units determined by bright field images of one or more fields of view, the number of positive reaction detection units determined by fluorescent images of one or more fields of view, and the probability of the target molecules to be detected being captured by magnetic beads and further binding to the enzyme as a constant.

[0069] More specifically, the following formula is used to determine the concentration of the target molecules to be detected in the sample: c=M0m / V, where c is the concentration of the target molecules to be detected in the sample, M0 is the number of target molecules to be detected in the sample, m is the mass of a single target molecule to be detected in the sample, and V is the volume of the sample.

[0070] For example, the target molecule to be detected in the sample is interleukin-6 (IL-6), whose molecular weight is 21 kDa, and the volume V of the sample is 100 μL. The number N0 of magnetic beads (sample distribution units) in the sample determined by flow cytometry is 753,600, the number of all microwells in the microwell array is 188,000, the number N of microwells (reaction detection units) containing magnetic beads in the microwell array determined based on the bright field image of one or more fields of view is 121,000, the number M of microwells (positive reaction detection units) in the microwell array with fluorescence signals higher than the threshold value determined based on the fluorescence image of one or more fields of view is 5000, and the probability p that the target molecule to be detected is captured by the magnetic beads and further bound to the enzyme is 80%, then the number M0 of the target molecule to be detected in the sample is calculated using the following formula: The calculated value is 39753, and the concentration c of the target molecule to be detected is calculated as 13.9 fg / ml using the following formula c=M0m / V.

[0071] Example 1: Comparison of microwell signals between two-step sample addition method and one-step sample addition method

[0072] According to the above description, in the digital ELISA detection method for preventing fluorescence signal crosstalk according to an embodiment of the present invention, the sample and the fluorescent substrate that form the complex are transferred to the microwell array chip in different steps, and more specifically, the sample and the fluorescent substrate that form the complex are centrifuged and plated in batches, and this loading method is called a two-step loading method. In contrast, if the sample and the fluorescent substrate that form the complex are transferred to the microwell array chip together in the same step, and more specifically, the sample and the fluorescent substrate that form the complex are centrifuged and plated in a single time, then this loading method is called a one-step loading method.

[0073] In the one-step sample addition method, the magnetic beads and fluorescent substrate are pre-mixed and then added to the microwell array chip. The magnetic beads are left to settle in the microwells of the microwell array, and then an isolation liquid is added to isolate the microwells. In this process, the enzymatic reaction begins while the magnetic beads and the fluorescent substrate are mixed, and the generated fluorescent signal will diffuse in the entire reaction solution. When the isolation liquid isolates the magnetic beads and the fluorescent substrate, the enzyme in the positive reaction detection unit further catalyzes the fluorescent substrate to produce a stronger fluorescent signal, and the fluorescent signal in the negative reaction detection unit is the background signal generated by the enzyme-catalyzed reaction from the mixing of the enzyme and the fluorescent substrate to the isolation of the isolation liquid. In addition, the more enzyme molecules are connected to the magnetic beads, the stronger the background signal.

[0074] In the two-step addition method, since the magnetic beads are first added to the microwell array chip and left to settle, the fluorescent substrate is then added, and then the isolation liquid is immediately added for isolation, the reaction time for the enzyme to react with the fluorescent substrate to produce the background signal is greatly shortened. In the one-step addition method, the reaction time is about 3 minutes, while in the two-step addition method, the reaction time is about 20 seconds, thereby greatly reducing the fluorescent signal background of the reaction system.

[0075] As a simplified system for evaluating enzymatic reactions, in Example 1, biotin-linked magnetic beads (1,000,000) were reacted with a specific volume (100 μL) of streptavidin-β-galactosidase (100 fM, 1 pM, 10 pM) outside a microwell array chip. The reaction buffer solution was 1×PBS, and the reaction time was 30 minutes. After the reaction was completed, the chip was washed five times with 1×PBST washing solution, and then the operation inside the microwell array chip was performed according to the technical schemes of the two-step sample addition method and the one-step sample addition method, respectively.

[0076] As shown in Table 1 below, the negative micropore signal (i.e., background signal or background) in the one-step addition method was significantly increased, with an average value of 678 for the negative micropore signal at a SβG concentration of 100 fM, an average value of 1172 for the negative micropore signal at a SβG concentration of 1 pM, and an average value of 6410 for the negative micropore signal at a SβG concentration of 100 pM, and the high background value results in a low degree of differentiation between the signal and the background. The background increase in the two-step addition method was much lower, with an average value of 561 for the negative micropore signal at a SβG concentration of 100 fM, an average value of 1046 for the negative micropore signal at a SβG concentration of 1 pM, and an average value of 1662 for the negative micropore signal at a SβG concentration of 100 pM.

[0077]

[0078] Table 1. Comparison of microwell signals between two-step sample addition method and one-step sample addition method

[0079] Figure 3A This is the fluorescence image of the magnetic beads after the reaction with 10pM SβG concentration using the two-step sample addition method, and Figure 3B This is the fluorescence image obtained by using the one-step sample addition method after the magnetic beads reacted with 10pM SβG concentration. Figure 4A ImageJ software was used to Figure 3A The fluorescence signals of a certain column of microwells are extracted and calculated from the fluorescence image shown, and Figure 4B ImageJ software was used to Figure 3B The fluorescence signals of a certain column of microwells are extracted and calculated from the fluorescence image shown.

[0080] from Figure 3A-Figure 4BIt can be seen that the background of the one-step addition method is significantly increased, and the distinction between the signal and the background is reduced.

[0081] Example 2: Comparison of the isolation effects of isolation fluids with different viscosity coefficients

[0082] As a simplified system for evaluating enzymatic reactions, in Example 2, biotin-linked magnetic beads (1,000,000) were reacted with 100 fM streptavidin-β-galactosidase outside the microwell array chip. The reaction buffer solution was 1×PBS and the reaction time was 30 minutes. After the reaction was completed, it was washed five times with 1×PBST washing solution, and then the operation inside the microwell array chip was carried out according to the technical scheme of the two-step sample addition method, and isolation was performed using isolation fluids with different viscosity coefficients.

[0083] As shown in Table 2 below, the low viscosity coefficient fluoro oil No. 1 (viscosity coefficient at 25°C is 0.77 cSt) showed obvious dye diffusion phenomenon, while the high viscosity coefficient fluoro oil No. 2 (viscosity coefficient at 25°C is 11.4 cSt), No. 3 (viscosity coefficient at 25°C is 451 cSt) and No. 4 (viscosity coefficient at 25°C is 1366 cSt) fluoro oils did not show obvious dye diffusion phenomenon.

[0084] Similarly, the low viscosity coefficient silicone oil No. _#5 (viscosity coefficient at 25°C is 2.3 cSt) and No. _#6 (viscosity coefficient at 25°C is 6.6 cSt) both showed obvious dye diffusion phenomenon, while the high viscosity coefficient silicone oil No. _#7 (viscosity coefficient at 25°C is 350 cSt) did not show obvious dye diffusion phenomenon.

[0085] Serial number model Viscosity coefficient cSt@25℃ Fluorescent dye diffusion 1 No._#1 0.77 There is diffusion 2 No._#2 11.4 No diffusion 3 No._#3 451 No diffusion 4 No._#4 1366 No diffusion 5 No._#5 2.3 There is diffusion 6 No._#6 6.6 There is diffusion 7 No._#7 350 No diffusion

[0086] Table 2. Comparison of isolation effects of isolation fluids with different viscosity coefficients

[0087] More specifically, when the spacer liquid is fluorine oil, the viscosity coefficient of the fluorine oil at 25° C. is in the range of 0.1 cSt to 12500 cSt, preferably in the range of 1 cSt to 5000 cSt, and more preferably in the range of 10 cSt to 1500 cSt. When the spacer liquid is silicone oil, the viscosity coefficient of the silicone oil at 25° C. is in the range of 10 cSt to 12500 cSt, preferably in the range of 10 cSt to 5000 cSt, and more preferably in the range of 100 cSt to 500 cSt.

[0088] Figure 5A1 , Figure 5B1 , Fig.5C1 and Fig.5D1are the fluorescence images after 1 minute of isolation using isolation solutions No. 1, No. 2, No. 3, and No. 4, respectively, and Figure 5A2 , Figure 5B2 , Fig.5C2 and Fig.5D2 These are the fluorescence images taken 5 minutes after isolation using isolation solutions No. 1, No. 2, No. 3, and No. 4, respectively.

[0089] from Figure 5A1 and Figure 5A2 It can be seen that after isolation with the isolation fluid No. 1 with a low viscosity coefficient, the adjacent reaction detection units around the positive reaction detection unit have strong fluorescent signals, which seriously affect the judgment of positive micropores and negative micropores. At the same time, as the reaction time increases, the signals of some positive reaction detection units decrease (because the speed of dye diffusion is greater than the speed of enzymatic reaction to produce new fluorescent molecules), while the signals of the negative reaction detection units that are crosstalked continue to increase. Figure 5B1 and Fig.5D2 It can be seen that after isolation with the isolation liquids No. 2, No. 3 and No. 4 with high viscosity coefficients, there is no fluorescence signal enhancement around the positive reaction detection unit.

[0090] in addition, Fig.6A1 and Fig.6A2 is a schematic diagram of the change of the fluorescence value of the positive microwell and the fluorescence value of the crosstalk microwell over time after isolation with the isolation liquid No. _#1, and Fig.6B1 and Figure 6B2 This is a schematic diagram of the change in fluorescence value of the positive micropores and the fluorescence value of the crosstalk micropores over time after isolation using isolation liquid No. _#3.

[0091] from Fig.6A1 and Fig.6A2 It can be seen that after the isolation with the isolation fluid No. 1 with low viscosity coefficient, the fluorescence value of the crosstalk micropores increases rapidly. Fig.6B1 and Figure 6B2 It can be seen that after isolation with the isolation liquid No. 3 with a high viscosity coefficient, the fluorescence value of the crosstalk micropores did not increase significantly.

[0092] Although the present invention has been shown and described with reference to certain preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention.

Claims

1. A digital ELISA detection method for preventing fluorescence signal crosstalk, characterized in that: The method comprises: Step 1: prepare a sample containing a target molecule to be detected, capture the target molecule to be detected in the sample using magnetic beads, and form a complex of "magnetic beads-target molecule to be detected-enzyme" through affinity reaction; Step 2, transferring the sample formed with the complex into a microwell array chip, wherein the microwell array chip comprises a microwell array and a fluid chamber accommodating the microwell array, and each microwell in the microwell array is configured to accommodate only one magnetic bead; Step 3, transferring the fluorescent substrate into the microwell array chip, wherein the enzyme can undergo an enzymatic reaction with the fluorescent substrate to generate fluorescent molecules; Step 4, adding an isolation liquid into the microwell array chip to isolate all microwells in the microwell array from each other; Step 5, waiting for the enzyme to perform an enzymatic reaction with the fluorescent substrate to generate the fluorescent molecule, wherein the fluorescence signal of the microwell where the magnetic beads that capture a single target molecule to be detected are located is higher than a threshold value, and the fluorescence signal of the microwell where the magnetic beads that capture zero target molecules to be detected are located is lower than a threshold value; Step 6, determining the number of microwells in the microwell array containing the magnetic beads, and determining the number of microwells in the microwell array whose fluorescence signals are higher than a threshold value; Step 7: Determine the number of target molecules to be detected in the sample based on the number of microwells in the microwell array containing the magnetic beads and the number of microwells in the microwell array whose fluorescence signals are higher than a threshold value, and based on the number of magnetic beads in the sample and the probability that the target molecules to be detected are captured by the magnetic beads and further bound to the enzyme.

2. The method according to claim 1, characterized in that The isolation fluid is fluorine oil, and the viscosity coefficient of the fluorine oil at 25° C. is in the range of 0.1 cSt to 12500 cSt.

3. The method according to claim 2, characterized in that The viscosity coefficient of the fluorine oil at 25° C. is in the range of 1 cSt to 5000 cSt.

4. The method according to claim 3, characterized in that The viscosity coefficient of the fluorine oil at 25° C. is 11.4 cSt, 451 cSt or 1366 cSt.

5. The method according to claim 1, characterized in that The isolation fluid is silicone oil, and the viscosity coefficient of the silicone oil at 25° C. is in the range of 10 cSt to 12500 cSt.

6. The method according to claim 5, characterized in that The viscosity coefficient of the silicone oil at 25° C. is in the range of 10 cSt to 5000 cSt.

7. The method according to claim 6, characterized in that The viscosity coefficient of the silicone oil at 25° C. is 350 cSt.

8. The method according to claim 1, characterized in that In step 2, the sample formed with the complex is transferred to the fluid chamber of the microwell array chip and the microwells of the microwell array by self-aspiration, centrifugation or pressure injection, and the excess magnetic beads in the fluid chamber are removed by centrifugation, while the magnetic beads in the microwells are retained.

9. The method according to claim 1, characterized in that: In step 3, the fluorescent substrate is transferred to the fluid chamber of the microwell array chip and the microwells of the microwell array by self-absorption, centrifugation or pressure injection, and the excess fluorescent substrate in the fluid chamber is removed by centrifugation, and the fluorescent substrate in the microwell is retained.

10. The method according to claim 1, characterized in that In step 4, the isolation liquid is added into the fluid chamber of the microwell array chip by self-imbibition, centrifugation or pressure injection, and the magnetic beads and fluorescent substrate in the microwells of the microwell array are sealed and isolated.

11. The method according to claim 1, characterized in that: The target molecule to be detected is a protein molecule to be detected.

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