Method for using detection microspheres to measure concentration of target molecules in sample, computer-readable medium, and analysis device

Abstract

Provided in the present application are a method for using detection microspheres to measure the concentration of target molecules in a sample, a computer-readable medium, and an analysis device. The method for measuring the concentration of target molecules comprises: providing micropores; sealing the micropores such that the micropores are fluidly isolated from one another; obtaining the total number of micropores; acquiring first fluorescence pictures according to a pre-determined time interval; obtaining the sum of brightness of the micropores of each of at least two first fluorescence pictures; acquiring at least one difference between the sums of the brightness of the micropores of the first fluorescence pictures or the average value of a plurality of differences therebetween; dividing the at least one difference or the average value of the plurality of differences by the total number of micropores, so as to obtain the average increment of brightness values, and using same as a characteristic value; obtaining a standard curve of characteristic values versus concentration; and determining the concentration of the target molecules on the basis of the standard curve. In the method of the present application, there is no need to determine whether each microsphere is connected to a target molecule, thereby simplifying the complexity of the method, and maintaining high measurement precision.

Description

Method, computer-readable medium, and analytical device for determining target molecule concentration in a sample using detection microspheres

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application filed with the China Patent Office on October 24, 2023, with application number 202311383238.2 and invention name “Method for determining the concentration of target molecules in a sample using detection microspheres, computer-readable medium and analytical equipment”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application relates to a method for determining the concentration of a target molecule in a biological sample, in particular a method for determining the concentration of a target molecule at very low concentrations. The present application also relates to a computer-readable medium storing instructions for executing the method and a corresponding analysis device. Background Art

[0004] The ability to measure low-abundance analytes from biological samples is crucial for several fields, including clinical diagnostics. Many protein and nucleic acid diagnostic biomarkers exist at extremely low concentrations, requiring analytical methods with very low detection limits.

[0005] However, for target molecules with concentrations in the picomolar (pM), femtomolar (fM), attomolar (aM), or even megamolar (zM) range, accurate concentration determination is extremely challenging. One known solution utilizes antibody-labeled microspheres, adding thousands to millions of microspheres to each sample to capture the target molecules in the sample and labeling the target molecules with enzyme reporter molecules. This solution disperses the microspheres into spatially isolated wells and calculates the average number of enzymes on a single microsphere based on a Poisson distribution. This solution assumes that at low concentrations, the vast majority of microspheres are not coupled to enzyme molecules ("off" spheres). Therefore, the average number of enzymes on a single microsphere is characterized by the ratio of microspheres coupled to enzyme molecules ("on" spheres) to all microspheres. This algorithm is also known as a digital algorithm. However, at high concentrations, most microspheres are bound to more than one enzyme molecule, and the digital algorithm is no longer applicable. Therefore, the average number of enzymes on a single microsphere is characterized by calculating the ratio of the average fluorescence intensity of the well containing microspheres coupled to the fluorescence intensity of a single enzyme. This algorithm is also known as an analog algorithm. In practical applications, 70% of the "on" balls are used as the threshold. Below 70%, the digital algorithm is used, and above 70%, the analog algorithm is used.

[0006] However, the smooth transition between digital and analog algorithms at 70% or other thresholds relies on a perfect Poisson distribution of molecules on the microspheres. The Poisson distribution assumes that all molecules and microspheres are identical, that molecules bind randomly to the microspheres without bias, that binding affinity remains constant despite concentration changes, and that the detected intensity is uniform. However, this is not achievable in reality. Various factors can lead to non-random distribution of microsphere-molecule binding or heterogeneous detection signals, including, but not limited to, uniformity of enzyme and substrate concentrations, uniformity of pore volume, variations between instruments, non-uniformity of excitation light, and non-uniformity of imaging quality. Indeed, recent reports have confirmed this discontinuity between analog and digital algorithms.

[0007] In view of this, it is necessary to overcome the defects in the prior art and provide a new concentration determination method so that it has measurement continuity in all concentration ranges.

[0008] Summary of the Invention

[0009] One aspect of the present application provides a method for determining the concentration of a target molecule in a sample using detection microspheres, comprising the following steps: (a) providing micropores, wherein a portion of the micropores contains a substrate and a detection microsphere, and another portion of the micropores contains a substrate but does not contain a detection microsphere, and the surface of at least one of the detection microspheres contains a sandwich structure formed by a first ligand-target molecule-reporter molecule, and the reporter molecule contains a catalyst capable of catalyzing the substrate to emit a first fluorescence; (b) sealing the micropores to isolate the fluid between each micropore; (c) obtaining a microsphere image of at least a portion of the detection microspheres, and counting the micropores containing microspheres in a selected area to obtain the total number of micropores; (d) obtaining N first fluorescence images corresponding to the first fluorescence at predetermined time intervals, where N is greater than or equal to an integer greater than 2; (e) aligning at least two first fluorescence images with the microsphere image, obtaining the micropore brightness value of each micropore containing microspheres in the selected area in each of the at least two first fluorescence images, and summing the micropore brightness values ​​of each first fluorescence image to obtain the total micropore brightness of each first fluorescence image; (f) obtaining at least one difference or an average of multiple differences between the total micropore brightness of the at least two first fluorescence images; (g) dividing the at least one difference or the average of the multiple differences by the total number of micropores to obtain an average increase in brightness value as a characteristic value; (h) obtaining a standard curve of characteristic value versus concentration; and (i) determining the concentration of the target molecule in the sample based on the standard curve and the characteristic value of the target molecule.

[0010] In some embodiments, in step (f): when N is an even number, the first difference is obtained by subtracting the sum of the micropore brightness values ​​of the Nth first fluorescence picture from the sum of the micropore brightness values ​​of the N / 2th first fluorescence picture, the second difference is obtained by subtracting the sum of the micropore brightness values ​​of the (N / 2-1)th first fluorescence picture from the sum of the micropore brightness values ​​of the N-1th first fluorescence picture, and so on, until the N / 2th difference between the sum of the micropore brightness values ​​of the N / 2+1th first fluorescence picture and the sum of the micropore brightness values ​​of the 1st first fluorescence picture is obtained.

[0011] In some embodiments, in step (f): when N is an odd number, the first difference is obtained by subtracting the sum of the micropore brightness values ​​of the (N+1) / 2th first fluorescence picture from the sum of the micropore brightness values ​​of the Nth first fluorescence picture, the second difference is obtained by subtracting the sum of the micropore brightness values ​​of the (N+1) / 2-1th first fluorescence picture from the sum of the micropore brightness values ​​of the N-1th first fluorescence picture, and so on, until the (N-1) / 2th difference between the sum of the micropore brightness values ​​of the (N+1) / 2+1th first fluorescence picture and the sum of the micropore brightness values ​​of the 2nd first fluorescence picture is obtained.

[0012] In some embodiments, in step (f): when N is an odd number, the first difference is obtained by subtracting the sum of the micropore brightness values ​​of the (N-1) / 2th first fluorescence picture from the sum of the micropore brightness values ​​of the (N-1) / 2th first fluorescence picture, the second difference is obtained by subtracting the sum of the micropore brightness values ​​of the (N-1) / 2-1th first fluorescence picture from the sum of the micropore brightness values ​​of the (N-2)th first fluorescence picture, and so on, until the (N-1) / 2th difference between the sum of the micropore brightness values ​​of the (N-1) / 2+1th first fluorescence picture and the sum of the micropore brightness values ​​of the 1st first fluorescence picture is obtained.

[0013] In some embodiments, step (a) comprises heating the substrate. For example, in some embodiments, the substrate is heated to 30-40° C. and maintained for 30 minutes.

[0014] In some embodiments, in steps (c) and / or (d), an LED light source is used to illuminate the microwells to obtain corresponding images. For example, in some embodiments, the power of the LED light source is 0.1W to 10W.

[0015] In some embodiments, providing a microwell in step (a) comprises providing a microwell in a channel of a microfluidic device.

[0016] In some embodiments, the sealing in step (b) comprises sealing with oil.

[0017] In some embodiments, step (d) further comprises pre-processing the first fluorescence image to exclude abnormal light spots. For example, in some embodiments, the pre-processing comprises target detection based on deep learning.

[0018] In some embodiments, in step (c), the detection microspheres contain a fluorescent dye, and the fluorescent dye is excited to cause the detection microspheres to emit a second fluorescence, thereby obtaining the microsphere image, wherein the second fluorescence is different from the first fluorescence.

[0019] In some embodiments, in step (c), the selected area is the entire area of ​​the microsphere image.

[0020] In some embodiments, in step (e), the microwell brightness value of each microwell is the sum of brightness values ​​of individual microwells displayed as 9×9 pixels or the average brightness value of individual microwells displayed as 9×9 pixels.

[0021] In some embodiments, prior to step (e), a correction algorithm is applied to the first fluorescence image. For example, the correction algorithm is used to correct for differences in microwell images introduced by aberrations between the central field of view and the peripheral field of view of the optical system, thereby obtaining a fluorescence image that is closer to the actual sample. For example, in some embodiments, the correction algorithm includes: (i) for the central region of the image, obtaining a 9×9 pixel light intensity distribution matrix Ii (i is an integer corresponding to the microwell number) for each microwell, dividing Ii by the maximum element value of the matrix to obtain a normalized light intensity distribution matrix IIi, averaging all elements of the normalized light intensity distribution matrix IIi for all microwells in the region to obtain Ia, which serves as the correction matrix for the light intensity distribution of each microwell; and (ii) for the peripheral region of the image, obtaining a 9×9 pixel light intensity distribution matrix IIIj (j is an integer corresponding to the microwell number) for each microwell, obtaining the maximum intensity within each 9×9 pixel region, and multiplying the maximum intensity by the correction matrix Ia to obtain the accurate light intensity distribution for each microwell, thereby correcting the peripheral region and obtaining a new image as the first fluorescence image.

[0022] In some embodiments, step (a) is performed at any constant temperature between 30°C and 37°C.

[0023] In some embodiments, in step (a), the reporter molecule is composed of a second ligand that specifically binds to the target molecule and the catalyst, the second ligand is different from the first ligand, and step (a) includes: (i) providing a sample that may contain a target molecule; (ii) adding detection microspheres, the surface of which is modified with the first ligand that specifically binds to the target molecule; (iii) adding a reporter molecule; (iv) adding a substrate; (v) introducing the detection microspheres into the microwells; wherein the order of execution of the steps is: i / ii / iii-iv-v or i / ii / iii / v-iv, wherein " / " indicates that the previous and next steps are interchangeable, and "-" indicates that the previous and next steps are executed in the order shown.

[0024] In some embodiments, in step (a), the reporter molecule consists of a first part and a second part that are independent of each other, the first part includes a second ligand that specifically binds to the target molecule and a first affinity element, the second part includes a second affinity element and the catalyst, the first affinity element and the second affinity element can be connected together through affinity, the second ligand is different from the first ligand, and step (a) includes: (i) providing a sample that may contain a target molecule; (ii) adding a detection microsphere, the surface of which is modified with the first ligand that specifically binds to the target molecule; (iii) adding the first part of the reporter molecule; (iv) adding the second part of the reporter molecule; (v) adding a substrate; (vi) introducing the detection microsphere into the microwell; wherein the order of execution of the steps is: i / ii / iii / iv-v-vi or i / ii / iii / iv / vi-v, wherein " / " indicates that the previous and next steps are interchangeable, and "-" indicates that the previous and next steps are executed in the order shown.

[0025] In some embodiments, in step (a), the reporter molecule is composed of a second ligand that specifically binds to the target molecule and the catalyst, the surface of the detection microsphere is modified with a first affinity element, the first ligand is coupled to the second affinity element, and the first ligand is bound to the first affinity element through the second affinity element. Step (a) includes: (i) providing a sample that may contain a target molecule; (ii) adding detection microspheres; (iii) adding the first ligand coupled to the second affinity element; (iv) adding the reporter molecule; (v) adding the substrate; (vi) introducing the detection microsphere into the microwell; wherein the order of execution of the steps is: i / ii / iii / iv-v-vi or i / ii / iii / iv / vi-v, wherein " / " indicates that the previous and next steps are interchangeable, and "-" indicates that the previous and next steps are executed in the order shown.

[0026] In some embodiments, in step (a), the reporter molecule is composed of a first part and a second part that are independent of each other, the first part includes a second ligand that specifically binds to the target molecule and a first affinity element, the second part includes a second affinity element and the catalyst, the first affinity element and the second affinity element can be connected together by affinity, the second ligand is different from the first ligand, the surface of the detection microsphere is modified with a third affinity element, the first ligand is coupled to a fourth affinity element, the first ligand is bound to the third affinity element via the fourth affinity element, the third affinity element and the fourth affinity element can be connected together by affinity, and step (a) includes: (i) providing a (ii) adding a sample that can contain a target molecule; (ii) adding a detection microsphere, a first ligand coupled to a fourth affinity element, and adding a blocking agent when there is a possibility of cross-reaction between the first and second affinity elements and the third and fourth affinity elements, wherein the blocking agent blocks the third affinity element, and removing excess blocking agent before proceeding to the next step; (iii) adding the first part of the reporter molecule; (iv) adding the second part of the reporter molecule; (v) adding a substrate; (vi) introducing the detection microsphere into the microwell; wherein the order of execution of the steps is: i / ii / iii / iv-v-vi or i / ii / iii / iv / vi-v, wherein " / " indicates that the previous and next steps are interchangeable, and "-" indicates that the previous and next steps are executed in the order shown.

[0027] In some embodiments, one or more washing steps are included between any two, three, four, or all of steps (i) to (vi).

[0028] In some embodiments, the first ligand and the second ligand are different antibodies raised against the target molecule.

[0029] In some embodiments, (a) the first affinity element is one of biotin and streptavidin, and the second affinity element is the other of biotin and streptavidin; or (b) the first affinity element is one of biotin and avidin, and the second affinity element is the other of biotin and avidin.

[0030] In some embodiments, the combination of the first affinity element and the second affinity element, and the combination of the third affinity element and the fourth affinity element, are independently selected from: (a) biotin and streptavidin; and (b) biotin and avidin.

[0031] In some embodiments, the catalyst is β-galactosidase and the substrate is resorufin-β-D-galactopyranoside.

[0032] In some embodiments, the step of introducing the detection microspheres into the microwells includes driving the microspheres repeatedly through the microwells to increase the probability of the microspheres landing in the microwells. In some embodiments, the step of introducing the detection microspheres into the microwells further includes positioning a magnet below the microwells to attract the microspheres into the microwells via magnetic force.

[0033] In some embodiments, in step (d), the predetermined time interval is from 10 seconds to 5 minutes.

[0034] In some embodiments, in step (c), white light is used to illuminate at least a portion of the detection microspheres to obtain the microsphere image.

[0035] In some embodiments, the concentration of the target molecule is 0.1 zM to 100 pM.

[0036] In some embodiments, the target molecule is a protein or a nucleic acid.

[0037] In some embodiments, the microspheres are polymer microspheres or magnetic beads.

[0038] In some embodiments, the method does not include individually counting microspheres to which target molecules are attached.

[0039] In some embodiments, the target molecules include multiple types, the detection microspheres also include corresponding multiple types, and the reporter molecules also include corresponding multiple types.

[0040] In some embodiments, the volume of each microwell is between 1 femtoliter and 1 picoliter.

[0041] Another aspect of the present application provides a computer-readable medium having computer-readable instructions stored thereon, and when the computer-readable instructions are executed, any one of the methods described in the present application is performed.

[0042] Another aspect of the present application provides an analytical device comprising a computer control system and a microfluidic device, wherein the computer control system comprises the computer readable medium described in the present application.

[0043] In some embodiments, the microfluidic device comprises a channel comprising a plurality of microwells, each microwell being configured to accommodate one or more detection microspheres.

[0044] In some embodiments, the volume of each microwell is between 1 femtoliter and 1 picoliter.

[0045] The method provided in this application does not require determining whether each microsphere is connected to a target molecule (i.e., "activated" or "on"), which simplifies the complexity of the method while maintaining high detection accuracy. In addition, the method provided in this application has excellent continuity in all measurement concentration ranges, and the vast majority of data CV is below 20%, which solves the data discontinuity problem caused by the use of analog algorithms and digital algorithms at high and low concentrations in the prior art. In addition, for samples of different multiple concentrations, the characteristic values ​​obtained by the algorithm have greater discrimination in certain intervals and higher resolution for sample concentrations, thereby further improving the detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] This application will be described in more detail with reference to the accompanying drawings. It should be noted that the illustrated scheme is only a representative example of an embodiment of the present application, and in order to more clearly illustrate the details of the exemplary embodiment, the elements in the drawings are not drawn in proportion to the actual size, the number of actual elements may vary, the relative positional relationship of the actual elements is basically consistent with the illustration, and some elements are not shown. In the case of multiple embodiments, when one or more features described in the previous embodiment can also be applied to another embodiment, for the sake of brevity, these reusable features will not be repeated in the subsequent one or more embodiments, and the subsequent one or more embodiments should be understood to have described these reusable features unless otherwise stated. After reading this application, those skilled in the art will realize that one or more features shown in one figure can be combined with one or more features in another figure to construct one or more alternative embodiments not specifically shown in the drawings, and these alternative embodiments also constitute part of this application.

[0047] FIG1 is a schematic flow chart of a method according to one embodiment of the present application.

[0048] FIG2 shows a fluorescence image of microspheres obtained by exciting a fluorescent dye according to an embodiment of the present application and a partial schematic diagram thereof.

[0049] FIG3 shows a plurality of first fluorescence images and images after fluorescence subtraction obtained in one embodiment.

[0050] FIG4 shows a standard curve of characteristic value versus concentration obtained according to one embodiment.

[0051] FIG5 shows a comparison of the CV values ​​of the characteristic values ​​obtained by the digital algorithm and the method of the present application. DETAILED DESCRIPTION

[0052] The exemplary embodiments of the present application are described in detail below with reference to the accompanying drawings. It should be understood that the scope of the present application is not limited to the disclosed embodiments, and that those skilled in the art, after reading the contents disclosed herein and based on the inspiration of the present application, may modify and change these exemplary embodiments without inventive effort, and such modifications and changes are intended to be included within the scope outlined by the appended claims.

[0053] Figure 1 shows a schematic flow diagram of an embodiment of the method according to the present application. Method 100 is a method for determining the concentration of a target molecule, which is generally contained in a sample (e.g., a biological sample). The method is used to detect whether the target molecule is present in the sample, and if so, at what concentration the target molecule is present. Therefore, method 100 can be used for qualitative and quantitative determination of target molecules. The target molecule can be a chemical or biological molecule, including but not limited to a protein molecule, such as a cytokine (such as IL-12, IL-6, etc.) or an antibody (such as PD-1 antibody, etc.); and a nucleic acid molecule, such as DNA or RNA. Other suitable target molecules are, for example, antigen molecules (such as Aβ1-42), gene fragments or fusion proteins. The biological sample can be, for example, blood, serum, plasma, urine, saliva, tissue fluid or other liquid derived from a human or animal body, or a culture medium, cell or tissue processing fluid (such as a tissue homogenate or cell lysate) derived from a laboratory. These biological samples may or may not contain target molecules.

[0054] Method 100 begins at step 102, which provides microwells, wherein a portion of the microwells contains a substrate and a detection microsphere, while another portion of the microwells contains a substrate but does not contain a detection microsphere, and the surface of at least one of the detection microspheres contains a sandwich structure formed by a first ligand-target molecule-reporter molecule, and the reporter molecule contains a catalyst that can catalyze the substrate to emit a first fluorescence.

[0055] In step 102, providing micropores can be achieved by providing a microfluidic device comprising an inlet and an outlet and a channel for fluid communication between the inlet and the outlet. The channel can be provided with a plurality of micropores on its sides or bottom, for example, hundreds to tens of thousands of micropores, each micropore being sized to accommodate one or more microspheres, thereby spatially isolating the microspheres from each other. For example, the volume of each micropore can be from about 1 femtoliter to about 1 picoliter. In the present application, a portion of the micropores contains a substrate and detection microspheres, while another portion of the micropores contains a substrate but no detection microspheres. Thus, some micropores do not contain microspheres, while other micropores contain one or more microspheres. In the micropores containing microspheres, the microspheres may or may not be coupled to the target molecule. The method of the present application does not involve individually counting microspheres coupled to the target molecule, as described in detail below. In other embodiments, each micropore is sized to accommodate only one microsphere, so that some micropores do not contain microspheres, while other micropores contain only one microsphere. The detection microspheres can be any microspheres commonly used in the art, such as polymer microspheres or magnetic beads. Examples of polymer microspheres can be found in the composite microspheres described in CN111318238 B. As an example, the detection microspheres are coupled or encoded with a fluorescent dye, such as CY5 fluorescent dye.

[0056] In the present application, in the micropore, the surface of at least one of the detection microspheres comprises a sandwich structure formed by a first ligand-target molecule-reporter molecule, and the reporter molecule comprises a catalyst that can catalyze the substrate to emit a first fluorescence. The first ligand is, for example, a ligand that specifically binds to the target molecule, such as an antibody against the target molecule, and the first ligand is directly or indirectly coupled to the surface of the detection microsphere. In the present application, the reporter molecule can specifically bind to the target molecule and is directly or indirectly coupled with a catalyst that can catalyze the substrate to emit a first fluorescence. For example, the reporter molecule comprises a second ligand that specifically binds to the target molecule, and the second ligand is, for example, an antibody against the target molecule that binds to a different epitope of the target molecule than the first ligand. Therefore, a "sandwich" structure (or sandwich structure) is formed between the first ligand, the target molecule and the reporter molecule (second ligand), in which the target molecule is sandwiched between the first ligand and the reporter molecule.

[0057] There are many ways to form the sandwich structure. In some embodiments, the reporter molecule is composed of a second ligand that specifically binds to the target molecule and the catalyst, and the second ligand is different from the first ligand. Step 102 may include: (i) providing a sample that may contain the target molecule; (ii) adding a detection microsphere, the surface of which is modified with the first ligand that specifically binds to the target molecule; (iii) adding a reporter molecule; (iv) adding a substrate; and (v) introducing the detection microsphere into the microwell. For the above steps, the execution order may be i / ii / iii-iv-v or i / ii / iii / v-iv, where " / " indicates that the previous and next steps are interchangeable, and "-" indicates that the previous and next steps are executed in the order shown. For example, in the sequence i / ii / iii-iv-v, the order of steps (i), (ii) and (iii) can be interchanged at will, and after these three steps are completed, steps (iv) and (v) are performed in sequence.

[0058] In other embodiments, the reporter molecule is composed of a first part and a second part that are independent of each other, the first part includes a second ligand that specifically binds to the target molecule and a first affinity element, the second part includes a second affinity element and the catalyst, the first affinity element and the second affinity element can be connected together through affinity, and the second ligand is different from the first ligand. Step 102 may include: (i) providing a sample that may contain the target molecule; (ii) adding a detection microsphere, the surface of which is modified with the first ligand that specifically binds to the target molecule; (iii) adding the first part of the reporter molecule; (iv) adding the second part of the reporter molecule; (v) adding a substrate; and (vi) introducing the detection microsphere into the microwell. For the above steps, the execution order can be i / ii / iii / iv-v-vi or i / ii / iii / iv / vi-v, where " / " indicates that the previous and next steps are interchangeable and "-" indicates that the previous and next steps are executed in the order shown.

[0059] In other embodiments, the reporter molecule is composed of a second ligand that specifically binds to the target molecule and the catalyst, the surface of the detection microsphere is modified with a first affinity element, the first ligand is coupled to the second affinity element, and the first ligand binds to the first affinity element via the second affinity element. Step 102 may include: (i) providing a sample that may contain the target molecule; (ii) adding the detection microsphere; (iii) adding the first ligand coupled to the second affinity element; (iv) adding the reporter molecule; (v) adding the substrate; and (vi) introducing the detection microsphere into the microwell. The above steps may be performed in the following order: i / ii / iii / iv-v-vi or i / ii / iii / iv / vi-v, where " / " indicates that the previous and next steps are interchangeable and "-" indicates that the previous and next steps are performed in the order shown.

[0060] In other embodiments, the reporter molecule is composed of a first part and a second part that are independent of each other, the first part includes a second ligand that specifically binds to the target molecule and a first affinity element, the second part includes a second affinity element and the catalyst, the first affinity element and the second affinity element can be connected together by affinity, the second ligand is different from the first ligand, the surface of the detection microsphere is modified with a third affinity element, the first ligand is coupled to a fourth affinity element, the first ligand binds to the third affinity element through the fourth affinity element, and the third affinity element and the fourth affinity element can be connected together by affinity, step 102 may include: (i) providing a sample that may contain the target molecule; (ii) adding the detection microsphere and the first ligand coupled to the fourth affinity element, and when there is a possibility of cross-reaction between the first and second affinity elements and the third and fourth affinity elements, adding a blocking agent, the blocking agent blocks the third affinity element, and removing excess blocking agent before proceeding to the next step; (iii) adding the first part of the reporter molecule; (iv) adding the second part of the reporter molecule; (v) adding a substrate; and (vi) introducing the detection microsphere into the microwell. For the above steps, the execution order can be: i / ii / iii / iv-v-vi or i / ii / iii / iv / vi-v, where " / " indicates that the previous and next steps are interchangeable, and "-" indicates that the previous and next steps are executed in the order shown.

[0061] In any of the above embodiments, one or more washing steps are included between any two, three, four or all of steps (i) to (v) or steps (i) to (vi). In some embodiments, one or more washing steps may be performed between each step (i) to (v) or steps (i) to (vi). In some embodiments, one or more washing steps may be performed between two of the steps (i) to (v) or steps (i) to (vi). The washing step may be performed by introducing a buffer solution (e.g., PBS or saline) that is compatible with the sample to wash the microspheres and / or micropores to remove components that are non-specifically bound to or not bound to the ligand coupled to the microspheres and / or non-specifically bound to or not bound to the reporter molecules, so as to exclude as much as possible the interference caused by components other than non-specific binding or target molecules in the determination. For example, in the case of using magnetic beads, after the magnetic beads are mixed with the sample, a magnetic field may be applied to fix the magnetic beads (thereby fixing the target molecule that specifically binds to the first ligand on the magnetic beads), and PBS buffer may be introduced to remove components in the sample that are not specifically bound to the ligand on the magnetic beads (e.g., non-target molecule proteins in plasma).

[0062] In any of the above embodiments, the first ligand and the second ligand may be different antibodies against the target molecule. For example, when the target molecule is IL-15, the first ligand is a first antibody against IL-15, and the second ligand is a second antibody against IL-15, where the first antibody and the second antibody bind to different epitopes of IL-15.

[0063] In any of the above embodiments, the first affinity element may be one of biotin and streptavidin, and the second affinity element is the other of biotin and streptavidin. In any of the above embodiments, the first affinity element is one of biotin and avidin, and the second affinity element is the other of biotin and avidin. In any of the above embodiments, the third affinity element may be one of biotin and streptavidin, and the fourth affinity element is the other of biotin and streptavidin. In any of the above embodiments, the third affinity element is one of biotin and avidin, and the fourth affinity element is the other of biotin and avidin. In one embodiment, the combination of the first affinity element and the second affinity element, and the combination of the third affinity element and the fourth affinity element, are independently selected from: (a) biotin and streptavidin; and (b) biotin and avidin.

[0064] In some embodiments, the blocking agent is an avidin blocking agent, such as bovine serum albumin (BSA) or polyethylene glycol (PEG) coupled to biotin. In some embodiments, the blocking agent is a biotin blocking agent, such as BSA or PEG coupled to avidin or streptavidin.

[0065] In step 102, the catalyst is β-galactosidase and the substrate is resorufin-β-D-galactopyranoside (CAS No.: 95079-19-9). As described above, other suitable catalyst and substrate pairs include a catalyst being β-galactosidase and a substrate being fluorescein-di-β-D-galactopyranoside (FDG, CAS No.: 17817-20-8), or a catalyst being β-galactosidase and a substrate being 4-methylumbelliferyl-β-D-galactoside (CAS No.: 6160-78-7), etc.

[0066] In the presence of a target molecule, the catalyst to which the reporter molecule is connected catalyzes the substrate to emit fluorescence. Within a certain range, the greater the concentration of the connected reporter molecule (that is, the greater the concentration of the target molecule), the more substrate is catalyzed, and thus the intensity and / or brightness of the fluorescence is greater. When the reporter molecule reaches saturation concentration, in the case of an excess of substrate, the fluorescence brightness of the microspheres reaches a maximum value. In one embodiment, due to differences in micropore size, in order to ensure uniform substrate concentration, given that the substrate is stored at 2-8°C, aggregation may occur, and the substrate is heated at 30 to 40°C (e.g., 35 to 37°C, e.g., 35°C, 36°C, or 37°C) for 30 minutes before use to ensure that the substrate is fully dissolved.

[0067] In addition, in some embodiments, considering that the catalytic efficiency of individual enzymes in different microwells of the same batch of enzymes in the same experiment is inconsistent, it is ensured that the enzyme catalyzed reaction is carried out under any constant temperature condition of 30°C to 37°C (e.g., 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C or 37°C).

[0068] In other embodiments, the present application is committed to improving the hole-falling rate of microspheres, ensuring sufficient sampling base, and mitigating the deviation caused by individual enzyme differences by averaging. In one example, the step of introducing the detection microspheres into the micropores includes, by external force pushing (such as suction microspheres), driving the microspheres to pass through the micropores repeatedly, increasing the hole-falling probability of the microspheres. In another example, the microspheres are driven repeatedly through the micropores by the external force, and a magnet is set below the micropores.

[0069] In step 104, the micropores are sealed to isolate the fluid between each micropore. Sealing can include sealing each of the individual micropores to isolate the fluid from each other, so that the buffer in the micropores and the microspheres are isolated from each other to shield fluorescence contamination between the micropores / microspheres as much as possible. In some embodiments, sealing can include introducing a sealing oil into the microfluidic device, which, on the one hand, removes residual microspheres and / or reporter molecules in the channel and, on the other hand, seals each micropore due to its immiscibility with the buffer in the micropores. Suitable sealing oils can be mineral oil, fluorinated oil, or silicone oil.

[0070] In method 100, step 106 includes obtaining a microsphere image of at least a portion of the detection microspheres and counting the microwells containing microspheres in the selected area to obtain the total number of microwells. In one embodiment, the detection microspheres are encoded or coupled with a fluorescent dye, which can be excited to emit fluorescence. The fluorescent signal can be collected by, for example, a fluorescence camera to form a fluorescence image for locating the fluorescent microspheres. For example, a laser light source is used to excite the fluorescent dye (such as CY5 fluorescent dye) to obtain a microsphere fluorescence image, and each microwell containing the microspheres is located based on the fluorescence image. In another embodiment, an LED light source can be used to excite the fluorescent dye (such as CY5 fluorescent dye) to obtain a microsphere fluorescence image, and each microsphere is located based on the microsphere fluorescence image. For example, the power of the LED light source can be 0.1 to 10W, such as 0.1 to 5W or 1 to 5W, such as 1W, 2W, 3W, 4W or 5W. In addition, as an option, a commercial large numerical aperture NA 0.28, achromatic objective lens can be used.

[0071] In another embodiment, the detection microspheres are not encoded or conjugated with fluorescent dyes, white light is used to illuminate the microspheres, a conventional camera is used to obtain an image of the microspheres, and each microwell containing the microspheres is located based on the image of the microspheres.

[0072] In some embodiments, the CY5 fluorescence image can be Gaussian blurred to analyze its contour and obtain the coordinate position of each microwell. In some embodiments, the processing of the CY5 fluorescence image can be implemented using computer software, such as OpenCV 2.0 and above. For convenience, in the case of detecting microspheres coupled or encoded with fluorescent dyes, the fluorescence emitted by the microspheres upon excitation is referred to as the second fluorescence, while the fluorescence emitted by the reporter molecule catalyzed substrate is referred to as the first fluorescence, which is different from the first fluorescence.

[0073] Part A of Figure 2 shows a CY5 fluorescence image obtained by the above method, which covers 80% to 90% of all micropores. In other embodiments, the image can cover 100% of all micropores. In other embodiments, the image can cover less than 80% of all micropores. Part B of Figure 2 is a partial enlargement of the dotted portion in Part A of Figure 2. A white bright spot indicates the presence of microspheres (i.e., the corresponding micropores, the same below), and the position where the bright spot is not shown indicates that there are no microspheres in the corresponding micropores. Part C of Figure 2 is a further partial enlargement of Part B of Figure 2.

[0074] In the present application, software is used to count microwells, regardless of whether the microspheres in the microwells are coupled to target molecules. In some embodiments, the microwells containing microspheres are counted across the entire area of ​​a microsphere image (e.g., a fluorescent microsphere image). In other embodiments, the microwells containing microspheres are counted across 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the area of ​​the microsphere image.

[0075] In step 108, N first fluorescence images corresponding to the first fluorescence are acquired at predetermined time intervals, where N is an integer greater than or equal to 2, for example, an integer between 2 and 1000. This time interval can be determined based on the typical reaction rate of the catalyst and substrate. Slower reaction rates generally require longer times, while faster reaction rates require shorter times. In some embodiments, a catalyst with higher catalytic efficiency can be optionally used, thereby shortening the entire testing process. In some embodiments, the predetermined time interval can be, for example, 10 seconds to 5 minutes, for example, 30 seconds to 3 minutes, 40 seconds to 2 minutes, 50 seconds to 90 seconds, or 60 seconds to 90 seconds. In alternative embodiments, the predetermined time interval is 30 seconds to 90 seconds, for example, 30 seconds, 40 seconds, 50 seconds, 60 seconds, 70 seconds, 80 seconds, or 90 seconds. In other embodiments, longer or shorter time intervals may be used. In the present application, the number of first fluorescence images can be reasonably determined as needed. Preliminary experiments can be conducted to determine the number of images that best meets the requirements. The number of pictures is usually considered in conjunction with the predetermined time interval. In addition, the number of fluorescence pictures is also designed based on the fluorescence growth curve corresponding to the first fluorescence. In some embodiments, the present application obtains 2 to 1000 first fluorescence pictures, such as 100 to 1000, 500 to 1000, 10 to 100, 20 to 50, 50 to 100, 2 to 50, 2 to 20, 2 to 10, 5 to 10, or more or fewer first fluorescence pictures. In an embodiment of the present application, the number of first pictures obtained can be an odd number or an even number, but an even number is optional. In a specific embodiment, 10 first fluorescence pictures are obtained at a time interval of 1 minute within 10 minutes. In some embodiments, step 108 may further include preprocessing the first fluorescence picture to exclude abnormal light spots, such as abnormal light spots caused by bubbles, impurities, etc. The preprocessing may include target detection based on deep learning.

[0076] In some embodiments, the order of steps 106 and 108 may be reversed. For example, the method may proceed to step 104 first, then to step 108, and then to step 106 before starting step 110.

[0077] In step 110, at least two first fluorescence images are aligned with the microsphere image, and the microwell brightness value for each microwell containing a microsphere in the selected region in each of the at least two first fluorescence images is obtained. The microwell brightness values ​​for each first fluorescence image are summed to obtain the total microwell brightness for each first fluorescence image. Those skilled in the art will appreciate that the microwell brightness value obtained from the microwell position is formed by the fluorescence emitted by all catalyzed substrates within the single microwell where the microsphere is located.

[0078] Part A of Figure 3 is a maximum pixel magnification of a single micropore (bright spot). In Part A of Figure 3, a single micropore is displayed with 9×9 pixels. It is anticipated that using cameras with different resolutions can cause a single micropore to be displayed with higher or lower pixels. In one embodiment of the present application, for a single micropore, the brightness average of 9×9 pixels is used as its fluorescence brightness. For example, for the single micropore shown in Part A of Figure 3, the sum of the brightness values ​​of 9×9 pixels is divided by 81 to obtain the brightness average of the single micropore. The same method can be used to obtain the brightness average of all micropores containing microspheres in the selected area of ​​the image. For example, for all micropores containing microspheres in the selected area, the brightness average of each micropore containing microspheres is calculated according to the above method, and then the brightness averages of all micropores containing microspheres in the selected area are summed and divided by the number of all micropores containing microspheres in the selected area to obtain the average brightness value of the micropores containing microspheres in the selected area. In other embodiments of the present application, for a single microwell, the sum of the brightness values ​​of 9×9 pixels is used as its fluorescence brightness. In alternative embodiments of the present application, the selected area covers the entire area of ​​the image. In other embodiments, the selected area may occupy at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more of the entire area of ​​the image. In other embodiments, the selected area may occupy less than 50%, less than 40%, less than 30%, or even less of the entire area of ​​the image.

[0079] In an optional embodiment, before step 110, a correction algorithm is applied to the entire first fluorescence image to correct the micropore image difference introduced by the aberration between the central field of view and the edge field of view of the optical system, so as to obtain a fluorescence image closer to the real sample. For example, the correction algorithm may include: (i) selecting a central area of ​​the first fluorescence image, obtaining a 9×9 pixel light intensity distribution matrix Ii (i is an integer corresponding to the microwell number; the matrix size is 9×9) for each microwell, dividing Ii by the maximum element value of the matrix to obtain a normalized light intensity distribution matrix IIi, averaging all elements of the normalized light intensity distribution matrix IIi of all microwells in the area (i.e., averaging all elements of all normalized light intensity distribution matrices IIi together) to obtain Ia (the matrix size is 9×9, and the matrix elements are all the average values ​​obtained previously), which is used as the correction matrix for the light intensity distribution of each microwell; and (ii) for the edge area of ​​the first fluorescence image, obtaining a 9×9 pixel light intensity distribution matrix IIIj (j is an integer corresponding to the microwell number; the matrix size is 9×9) for each microwell, obtaining the maximum intensity value in each 9×9 pixel area, multiplying the maximum intensity value by the correction matrix Ia to obtain the accurate light intensity distribution of each microwell, thereby correcting the edge area and obtaining a new image as the first fluorescence image. In some embodiments, the selected central region may be a region of 100×100 microwells with the central coordinate as the center; in some embodiments, the selected edge region may be a region extending one-fifth of the way from the four edges of the first fluorescent image to the center. In some embodiments, the selected central region may be a region of 100×100 microwells with the central coordinate as the center, and the selected edge region may be a region extending one-fifth of the way from the four edges of the first fluorescent image to the center.

[0080] In addition, commercial high-concentration solid fluorescent slices can be used as calibration plates, and the solid fluorescent slice images can be corrected algorithmically to further correct for any unevenness in the illumination light path. For example, the algorithmic correction steps are as follows: First, appropriately adjust the illumination light intensity of the illumination light path to obtain an unexposed fluorescence image of the high-concentration solid fluorescent slice under the illumination light path, and obtain the grayscale matrix I1 of the unexposed fluorescence image; then find the maximum grayscale value MAXI1 of the grayscale matrix I1, and divide the maximum grayscale value MAXI1 by each element value of the grayscale matrix I1 to obtain a new matrix I2, which is the correction matrix; for each first fluorescence image, perform a dot product with the correction matrix I2 to obtain a uniformly corrected first fluorescence image.

[0081] In one embodiment, in step 110, it is important to note that the microsphere image and at least two first fluorescence images should be aligned so that the analysis area in the first fluorescence image is the same as the analysis area in the microsphere image. After step 110, multiple fluorescence brightness values ​​are obtained for each microwell position, each corresponding to a first fluorescence image. For example, each microwell position may have 10 fluorescence brightness values. For some microwell positions, each fluorescence brightness value is the same or substantially the same. For other microwell positions, each fluorescence brightness value is different and increases over time. The magnitude of the fluorescence brightness value that increases over time may be the same or different for different microwell positions.

[0082] In step 110, the fluorescence brightness values ​​of all microwells containing microspheres in at least two first fluorescence images are summed to obtain the total microwell brightness of each first fluorescence image. The total microwell brightness can be obtained by adding the fluorescence brightness values ​​of each microwell containing microspheres.

[0083] In step 112, at least one difference between the sums of the brightness of the microspheres in the first fluorescence images is obtained, or an average of multiple differences between the sums of the brightness of the microspheres in the first fluorescence images is obtained when N is greater than 2. When N is an even number, a first difference is obtained by subtracting the sum of the brightness of the micropores in the N / 2th first fluorescence image from the sum of the brightness of the micropores in the Nth first fluorescence image, a second difference is obtained by subtracting the sum of the brightness of the micropores in the (N / 2-1)th first fluorescence image from the sum of the brightness of the micropores in the N-1th first fluorescence image, and so on, until the N / 2th difference between the sum of the brightness of the micropores in the N / 2+1th first fluorescence image and the sum of the brightness of the micropores in the 1st first fluorescence image is obtained. For example, when step 108 collects 10 (N=10) first fluorescence images, in step 110, for each microwell containing microspheres, the sum of the microwell brightness values ​​of the 10th, 9th, 8th, 7th, and 6th images is subtracted from the sum of the microwell brightness values ​​of the 5th, 4th, 3rd, 2nd, and 1st images, respectively, to obtain five difference values. The above images are numbered in the order of the time of acquisition. Part B of Figure 3 shows the 5th image obtained in one embodiment, and Part C of Figure 3 shows the 10th image obtained in the same embodiment. The two images have the same field of view and both show hundreds of microwells containing microspheres. Part D of Figure 3 shows the image obtained by subtracting the image in Part C of Figure 3 from the image in Part B of Figure 3. The sum of the microwell brightness values ​​in Part D of Figure 3 is the first difference value obtained by subtracting the sum of the microwell brightness values ​​of the 10th image from the sum of the microwell brightness values ​​of the 5th image.

[0084] When N is an odd number, the first difference value can be obtained by subtracting the sum of the micropore brightness values ​​of the (N+1) / 2th first fluorescence image from the sum of the micropore brightness values ​​of the Nth first fluorescence image. The second difference value can be obtained by subtracting the sum of the micropore brightness values ​​of the (N+1) / 2-1th first fluorescence image from the sum of the micropore brightness values ​​of the (N-1th) first fluorescence image. This is repeated until the (N-1) / 2th difference value between the sum of the micropore brightness values ​​of the (N+1) / 2+1th first fluorescence image and the sum of the micropore brightness values ​​of the 2nd first fluorescence image is obtained. For example, when 11 (N=11) first fluorescence images are collected in step 108, in step 110, the sum of the micropore brightness values ​​of the 6th, 5th, 4th, 3rd, and 2nd images are subtracted from the sum of the micropore brightness values ​​of the 11th, 10th, 9th, 8th, and 7th images, respectively, to obtain five difference values. The above images are numbered in the order of their collection time. In other embodiments, step 110 may be performed using a greater or lesser number of first fluorescence images.

[0085] Alternatively, when N is an odd number, the first difference value may be obtained by subtracting the sum of the micropore brightness values ​​of the (N-1) / 2th first fluorescence image from the sum of the micropore brightness values ​​of the (N-1) / 2th first fluorescence image. The second difference value may be obtained by subtracting the sum of the micropore brightness values ​​of the (N-1) / 2-1th first fluorescence image from the sum of the micropore brightness values ​​of the (N-2th) first fluorescence image. This process is repeated until the (N-1) / 2th difference value between the sum of the micropore brightness values ​​of the (N-1) / 2+1th first fluorescence image and the sum of the micropore brightness values ​​of the 1st first fluorescence image is obtained. For example, when 11 (N=11) first fluorescence images are collected in step 108, in step 110, the sum of the micropore brightness values ​​of the 5th, 4th, 3rd, 2nd, and 1st images may be subtracted from the sum of the micropore brightness values ​​of the 10th, 9th, 8th, 7th, and 6th images, respectively, to obtain five difference values.

[0086] Then, in step 114 , at least one difference value obtained in step 112 , or an average value of multiple differences when more than one difference value is obtained, is divided by the total number of micropores obtained in step 106 to obtain an average increase in brightness value as a characteristic value.

[0087] In other embodiments, the brightness value of each microwell containing a microsphere may be calculated for only a local region in at least two first fluorescence images. For example, the brightness value of each microwell containing a microsphere may be calculated for only 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the region in the first fluorescence image. However, it should be noted that in this case, the difference value or average difference value obtained in step 112 must be divided by the total number of microwells counted in the microsphere image for the corresponding local region to obtain the characteristic value. For example, if the brightness value of each microwell containing a microsphere is calculated for 50% of the region in the microsphere image to obtain the total brightness of the microwells in the corresponding image, and a difference value is obtained based on this difference value in step 112, the difference value or average difference value should be divided by the total number of microwells counted in the microsphere image for the corresponding 50% region to obtain the characteristic value.

[0088] In step 116, a standard curve of the characteristic value versus concentration is obtained. The standard curve can be established in advance using target molecules or other molecules with known concentrations according to the method described in steps 102 to 114.

[0089] After obtaining the standard curve, step 118 is performed to determine the concentration of the target molecule in the sample based on the standard curve and the characteristic value of the target molecule. In some embodiments, the concentration of the target molecule in the sample is at an extremely low level, such as 0.1zM to 100pM, such as 1zM to 100fM, or even lower.

[0090] According to the method described in method 100, a standard curve was established using 6 different concentrations of Aβ1-42 molecule samples. The characteristic values ​​and concentrations are shown in Table 1 below.

[0091] Table 1. Aβ1-42 test results (6 concentrations, unit: pg / mL, 8 replicates per well)

[0092] Table 2. Evaluation of the characteristic values ​​of the standard curve

[0093] The standard curve is established with 6 gradually increasing concentrations, 8 replicates for each concentration, and a protein molecule (such as Aβ1-42) is used as the target molecule. In the above embodiment, the coefficient of variation (CV) of the data of the standard curve is less than 15%, except for the group (S0) with a concentration of 0. In the above embodiment, the two adjacent concentrations have obvious distinction, and the ratio is above 1.5. Figure 4 shows the standard curve of characteristic value versus concentration obtained according to this embodiment. It can be seen that the characteristic value is proportional to the concentration, and the higher the concentration, the larger the characteristic value. In addition, the range of characteristic values ​​in the standard curve established by this method is larger, which increases the sensitivity of the signal response; and it has good continuity at high and low concentrations.

[0094] In another embodiment, the eigenvalue algorithm provided by the present application was tested using samples of different concentrations. The results are shown in Table 3.

[0095] Table 3. Test results of the present method for samples with different concentrations

[0096] From the results in Table 3, it can be seen that the characteristic value repeatability of the method of the present application is good, and the characteristic value CV of the method of the present application is within 25%, among which the characteristic value CVs of the last three concentration samples are all within 15%.

[0097] FIG5 is a scatter plot showing the relationship between the CV of characteristic values ​​obtained by the digital algorithm and the method of the present application, based on some test data (not shown). As can be seen from the scatter plot, for the test results of the blood sample (Part A) and the calibrator (Part B), the CV of the characteristic value of the digital algorithm is close to the CV of the characteristic value of the method of the present application. The majority of the scatter points of the characteristic value CV of the two algorithms are located in the region of <20%. For the very few scatter points with CV >20%, the CV of the characteristic value obtained by the method of the present application is superior to the CV of the characteristic value obtained by the digital algorithm.

[0098] In some embodiments, the method of the present application can simultaneously analyze two or more different types of target molecules that may be present in a sample, such as IL-12 and IL-10. In such an embodiment, the method can use different types of microspheres and different types of fluorescent dyes corresponding to different types of target molecules, as well as different types of reporter molecules and different types of substrate and enzyme combinations, to identify each microsphere and reporter molecule via different fluorescence channels. The method for preparing two different fluorescently encoded microspheres can be found in CN 111218498 A.

[0099] Another aspect of the present application provides a computer-readable medium having computer-readable instructions stored thereon, wherein the computer-readable instructions, when executed, perform any of the methods described above in the present application. The computer-readable medium may include a removable medium that is a packaged medium including a magnetic disk (including a floppy disk), an optical disk (including a CD-ROM (Compact Disc Read Only Memory) and a DVD (Digital Versatile Disc)), a magneto-optical disk (including an MD (Mini Disk)), or a semiconductor memory.

[0100] Another aspect of the present application provides an analytical device comprising a computer control system and a microfluidic device, wherein the computer control system comprises the computer readable medium of the present application.

[0101] In one embodiment, the microfluidic device comprises a channel comprising a plurality of microwells, each microwell being configured to accommodate at most one microsphere. In some embodiments, the volume of each microwell is between 1 femtoliter and 1 picoliter. The analytical device is configured to perform any of the methods described herein.

[0102] The above are all representative examples of the embodiments of the present application and are provided for illustrative purposes only. The present application anticipates that one or more technical features used in one embodiment may be added to another embodiment to form an improved or alternative embodiment without violating the purpose of the embodiment. Similarly, one or more technical features used in one embodiment may be omitted or replaced to form an alternative or simplified embodiment without violating the purpose of the embodiment. In addition, one or more technical features used in one embodiment may be combined with one or more technical features in another embodiment to form an improved or alternative embodiment without violating the purpose of the embodiment. The present application is intended to include all of the above improved, alternative, and simplified technical solutions.

Claims

1. A method for determining the concentration of a target molecule in a sample using a detection microsphere, comprising the following steps: (a) providing microwells, wherein a portion of the microwells contains a substrate and a detection microsphere, and another portion of the microwells contains a substrate but does not contain a detection microsphere, and a surface of at least one of the detection microspheres contains a sandwich structure formed by a first ligand-target molecule-reporter molecule, and the reporter molecule contains a catalyst that can catalyze the substrate to emit a first fluorescence; (b) sealing the microwells to isolate the fluid between each microwell; (c) obtaining a microsphere image of at least a portion of the detection microspheres, and counting the micropores containing the microspheres in the selected area to obtain a total number of micropores; (d) acquiring N first fluorescence images corresponding to the first fluorescence at predetermined time intervals, where N is an integer greater than or equal to 2; (e) aligning at least two first fluorescent images with the microsphere image, obtaining the micropore brightness value of each micropore containing microspheres in the selected area in each of the at least two first fluorescent images, and summing the micropore brightness values ​​of each first fluorescent image to obtain the sum of the micropore brightness of each first fluorescent image; (f) obtaining at least one difference value or an average value of multiple differences between the sums of the micropore brightness of the at least two first fluorescent images; (g) dividing the at least one difference or the average of the plurality of differences by the total number of micropores to obtain an average increase in brightness as a characteristic value; (h) obtaining a standard curve of characteristic value versus concentration; and (i) determining the concentration of the target molecule in the sample according to the standard curve and the characteristic value of the target molecule.

2. The method according to claim 1, wherein in step (f): (1) When N is an even number, the first difference is obtained by subtracting the sum of the micropore brightness values ​​of the Nth first fluorescent picture from the sum of the micropore brightness values ​​of the N / 2th first fluorescent picture, and the second difference is obtained by subtracting the sum of the micropore brightness values ​​of the (N / 2-1)th first fluorescent picture from the sum of the micropore brightness values ​​of the N-1th first fluorescent picture, and so on, until the N / 2th difference between the sum of the micropore brightness values ​​of the N / 2+1th first fluorescent picture and the sum of the micropore brightness values ​​of the 1st first fluorescent picture is obtained; (2) When N is an odd number, the first difference is obtained by subtracting the sum of the micropore brightness values ​​of the (N+1) / 2th first fluorescent picture from the sum of the micropore brightness values ​​of the Nth first fluorescent picture, and the second difference is obtained by subtracting the sum of the micropore brightness values ​​of the (N+1) / 2-1th first fluorescent picture from the sum of the micropore brightness values ​​of the (N-1th) first fluorescent picture, and so on, until the (N-1) / 2th difference between the sum of the micropore brightness values ​​of the (N+1) / 2+1th first fluorescent picture and the sum of the micropore brightness values ​​of the 2nd first fluorescent picture is obtained; or (3) When N is an odd number, the first difference is obtained by subtracting the sum of the micropore brightness values ​​of the (N-1) / 2th first fluorescent picture from the sum of the micropore brightness values ​​of the (N-1) / 2th first fluorescent picture, and the second difference is obtained by subtracting the sum of the micropore brightness values ​​of the (N-1) / 2-1th first fluorescent picture from the sum of the micropore brightness values ​​of the (N-2)th first fluorescent picture, and so on, until the (N-1) / 2th difference between the sum of the micropore brightness values ​​of the (N-1) / 2+1th first fluorescent picture and the sum of the micropore brightness values ​​of the 1st first fluorescent picture is obtained.

3. The method of claim 1, wherein step (a) comprises heating the substrate.

4. The method according to claim 3, wherein the substrate is heated to 30-40°C and maintained for 30 minutes.

5. The method according to claim 1, wherein in step (c) and / or (d), an LED light source is used to illuminate the microhole to obtain a corresponding image.

6. The method according to claim 5, wherein the power of the LED light source is 0.1W to 10W.

7. The method of claim 1, wherein providing the microwell in step (a) comprises providing the microwell in a channel of a microfluidic device.

8. The method of claim 1, wherein the sealing in step (b) comprises sealing with oil.

9. The method according to claim 1, wherein step (d) further comprises pre-processing the first fluorescent image to exclude abnormal light spots.

10. The method of claim 9, wherein the preprocessing comprises deep learning based object detection.

11. The method according to claim 1, wherein in step (c), the detection microspheres contain a fluorescent dye, and the fluorescent dye is excited to make the detection microspheres emit a second fluorescence to obtain the microsphere image, and the second fluorescence is different from the first fluorescence.

12. The method according to claim 1, wherein in step (c), the selected area is the entire area of ​​the microsphere image.

13. The method according to claim 1, wherein in step (e), the micropore brightness value of each micropore is the sum of brightness values ​​of individual micropores displayed by 9×9 pixels or the average brightness of individual micropores displayed by 9×9 pixels.

14. The method of claim 1, wherein prior to step (e), a correction algorithm is applied to the first fluorescence image.

15. The method of claim 14, wherein the correction algorithm comprises: (i) For the central area of ​​the image, obtain the light intensity distribution matrix Ii of 9×9 pixels for each microwell, where i is an integer and corresponds to the microwell number, divide Ii by the maximum element value of the matrix to obtain the normalized light intensity distribution matrix IIi, average all elements of the normalized light intensity distribution matrix IIi of all microwells in the area to obtain Ia, which is used as the correction matrix for the light intensity distribution of each microwell; and (ii) For the edge area of ​​the image, obtain the light intensity distribution matrix IIIj of 9×9 pixels for each micropore, where j is an integer and corresponds to the micropore number, obtain the maximum intensity value in each 9×9 pixel area, and use the Ia correction matrix to multiply the intensity maximum value as the accurate light intensity distribution of each micropore, so as to correct the edge area and obtain a new image as the first fluorescent image.

16. The method according to claim 1, wherein step (a) is carried out under any constant temperature condition between 30 and 37°C.

17. The method according to claim 1, wherein in step (a), the reporter molecule consists of a second ligand that specifically binds to the target molecule and the catalyst, the second ligand is different from the first ligand, and step (a) comprises: (i) providing a sample that may contain a target molecule; (ii) adding detection microspheres, the surface of which is modified with the first ligand that specifically binds to the target molecule; (iii) adding reporter molecules; (iv) adding a substrate; (v) introducing the detection microspheres into the microwells; The order of step execution is: i / ii / iii-iv-v or i / ii / iii / v-iv, where " / " indicates that the previous and next steps are interchangeable, and "-" indicates that the previous and next steps are executed in the order shown.

18. The method according to claim 1, wherein in step (a), the reporter molecule consists of a first part and a second part that are independent of each other, the first part includes a second ligand that specifically binds to the target molecule and a first affinity element, the second part includes a second affinity element and the catalyst, the first affinity element and the second affinity element can be connected together by affinity, the second ligand is different from the first ligand, and step (a) comprises: (i) providing a sample that may contain a target molecule; (ii) adding detection microspheres, the surface of which is modified with the first ligand that specifically binds to the target molecule; (iii) adding the first part of the reporter molecule; (iv) adding said second part of a reporter molecule; (v) adding a substrate; (vi) introducing the detection microspheres into the microwells; The order of step execution is: i / ii / iii / iv-v-vi or i / ii / iii / iv / vi-v, where " / " indicates that the previous and next steps are interchangeable, and "-" indicates that the previous and next steps are executed in the order shown.

19. The method according to claim 1, wherein in step (a), the reporter molecule is composed of a second ligand that specifically binds to a target molecule and the catalyst, the surface of the detection microsphere is modified with a first affinity element, the first ligand is coupled with a second affinity element, and the first ligand is bound to the first affinity element via the second affinity element, and step (a) comprises: (i) providing a sample that may contain a target molecule; (ii) adding detection microspheres; (iii) adding a first ligand coupled to a second affinity element; (iv) adding reporter molecules; (v) adding a substrate; (vi) introducing the detection microspheres into the microwells; The order of step execution is: i / ii / iii / iv-v-vi or i / ii / iii / iv / vi-v, where " / " indicates that the previous and next steps are interchangeable, and "-" indicates that the previous and next steps are executed in the order shown.

20. The method according to claim 1, wherein in step (a), the reporter molecule consists of a first part and a second part which are independent of each other, the first part includes a second ligand and a first affinity element which specifically bind to the target molecule, the second part includes a second affinity element and the catalyst, the first affinity element and the second affinity element can be connected together by affinity, the second ligand is different from the first ligand, the surface of the detection microsphere is modified with a third affinity element, the first ligand is coupled with a fourth affinity element, the first ligand is bound to the third affinity element via the fourth affinity element, the third affinity element and the fourth affinity element can be connected together by affinity, and step (a) comprises: (i) providing a sample that may contain a target molecule; (ii) adding detection microspheres, the first ligand coupled to the fourth affinity element, and when there is a possibility of cross-reaction between the first and second affinity elements and the third and fourth affinity elements, adding a blocking agent, wherein the blocking agent blocks the third affinity element, and removing excess blocking agent before proceeding to the next step; (iii) adding the first part of the reporter molecule; (iv) adding said second part of a reporter molecule; (v) adding a substrate; (vi) introducing the detection microspheres into the microwells; The order of step execution is: i / ii / iii / iv-v-vi or i / ii / iii / iv / vi-v, where " / " indicates that the previous and next steps are interchangeable, and "-" indicates that the previous and next steps are executed in the order shown.

21. The method of claim 17, wherein one or more washing steps are included between any two, three, four or all of steps (i) to (v).

22. The method according to any one of claims 18, 19 or 20, wherein one or more washing steps are included between any two, three, four or all of steps (i) to (vi).

23. The method of any one of claims 17, 18, 19 or 20, wherein the first ligand and the second ligand are different antibodies against the target molecule.

24. The method according to any one of claims 17 or 18, wherein: (a) the first affinity element is one of biotin and streptavidin, and the second affinity element is the other of biotin and streptavidin; or (b) The first affinity element is one of biotin and avidin, and the second affinity element is the other of biotin and avidin.

25. The method of claim 20, wherein the combination of the first affinity element and the second affinity element, and the combination of the third affinity element and the fourth affinity element are independently selected from: (a) biotin and streptavidin; and (b) Biotin and avidin.

26. The method of any one of claims 1, 17, 18, 19 or 20, wherein the catalyst is β-galactosidase and the substrate is resorufin-β-D-galactopyranoside.

27. The method according to any one of claims 17, 18, 19 or 20, wherein the step of introducing the detection microspheres into the microwells comprises: The microspheres are driven to pass through the micropores repeatedly to increase the probability of the microspheres falling into the micropores.

28. The method according to claim 27, wherein the step of introducing the detection microspheres into the microwells further comprises disposing a magnet under the microwells to attract the microspheres into the wells by magnetic force.

29. The method of claim 1, wherein in step (d), the predetermined time interval is from 10 seconds to 5 minutes.

30. The method of claim 1, wherein in step (c), white light is used to illuminate at least a portion of the detection microspheres to obtain the microsphere image.

31. The method of claim 1, wherein the concentration of the target molecule is 0.1 zM to 100 pM.

32. The method of claim 1, wherein the target molecule is a protein or a nucleic acid.

33. The method of claim 1, wherein the microspheres are polymer microspheres or magnetic beads.

34. The method of claim 1, wherein the method does not comprise individually counting microspheres to which target molecules are attached.

35. The method of claim 1, wherein the target molecule comprises a plurality of types, the detection microspheres also comprise a corresponding plurality of types, and the reporter molecules also comprise a corresponding plurality of types.

36. The method of claim 1, wherein the volume of each microwell is between 1 femtoliter and 1 picoliter.

37. A computer-readable medium having computer-readable instructions stored thereon, wherein the computer-readable instructions, when executed, perform the method of any one of claims 1 to 36.

38. An analytical device comprising a computer control system and a microfluidic device, wherein the computer control system comprises the computer readable medium of claim 37.

39. The analytical device according to claim 38, wherein the microfluidic device comprises a channel, the channel comprising a plurality of microwells, each microwell being used to accommodate one or more detection microspheres.

40. The analytical device of claim 38, wherein the volume of each microwell is 1 femtoliter to 1 picoliter.

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