Methods, computer-readable media, and analytical devices for detecting concentrations of target molecules in a sample using detection microspheres

Abstract

The present application provides a method, a computer readable medium and an analysis device for determining the concentration of a target molecule in a sample using detection microspheres, wherein the method for determining the concentration of the target molecule comprises: providing microwells; sealing the microwells to make each microwell fluidically isolated; obtaining the total number of microwells; obtaining a first fluorescent image at a predetermined time interval; obtaining the total brightness of microwells of at least two first fluorescent images; obtaining the average of at least one difference or a plurality of differences between the total brightness of microwells of the first fluorescent images; dividing the average of the at least one difference or the plurality of differences by the total number of microwells to obtain the average increase of brightness value as a characteristic value; obtaining a standard curve of the characteristic value versus the concentration; and determining the concentration of the target molecule according to the standard curve. The method of the present application does not need to determine whether each microsphere is connected with the target molecule, simplifies the complexity of the method, and at the same time maintains high detection accuracy.

Description

[0001] This application is a divisional application of the invention patent application with application number 202311383238.2, application date October 24, 2023, entitled "Method for determining the concentration of target molecules in a sample using detection microspheres, computer-readable medium and analytical device". Technical Field

[0002] This invention relates to a method for determining the concentration of a target molecule in a biological sample, particularly a method for determining the concentration of a target molecule at extremely low concentrations. The invention also relates to a computer-readable medium storing instructions for performing the method, and a corresponding analytical apparatus. Background Technology

[0003] 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.

[0004] However, accurate concentration determination is extremely challenging for target molecules at concentrations in the picomolar (pM), femtomolar (fM), amolar (aM), and even mesomolar (zM) ranges. One known solution utilizes antibody-labeled microspheres, adding thousands to millions of microspheres to each sample to capture the target molecules, and labeling the target molecules with enzyme reporter molecules. This approach disperses the microspheres into spatially isolated wells and calculates the average number of enzymes on a single microsphere based on a Poisson distribution. This approach assumes that at low concentrations, the vast majority of microspheres are not coupled with enzyme molecules (“off” spheres), so the average number of enzymes on a single microsphere is characterized by the proportion of microspheres coupled with enzyme molecules (“on” spheres) to all microspheres; this algorithm is also known as a numerical algorithm. However, at high concentrations, most microspheres bind more than one enzyme molecule, rendering the numerical algorithm inapplicable. 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 enzyme-coupled microspheres to the fluorescence intensity of a single enzyme; this algorithm is also known as a simulation algorithm. In practical applications, 70% of the "on" balls are used as the threshold. When the percentage is below 70%, a digital algorithm is used, and when it is above 70%, an analog algorithm is used.

[0005] 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, molecules bind randomly on the microspheres without bias, binding affinity remains constant with concentration changes, and the detected intensity is uniform. However, this is unrealistic. Various factors in practice can cause the binding of microspheres and molecules to be non-random or result in non-uniform detection signals, including but not limited to the uniformity of enzyme and substrate concentrations, the uniformity of well volume, differences between instruments, non-uniformity of excitation light, and non-uniformity of imaging quality. In fact, recent reports have confirmed the discontinuity between analog and digital algorithms.

[0006] In view of this, it is necessary to overcome the shortcomings of the existing technology and provide a new concentration determination method that has measurement continuity across all concentration ranges. Summary of the Invention

[0007] One aspect of the present invention 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, while another portion of the micropores contains a substrate but not a detection microsphere, wherein the surface of at least one of the detection microspheres comprises a sandwich structure formed by a first ligand-target molecule-reporter molecule, the reporter molecule comprising a catalyst capable of catalyzing the substrate to emit a first fluorescence; (b) sealing the micropores to fluidly isolate each micropore; (c) obtaining microsphere images of at least a portion of the detection microspheres and counting the micropores containing microspheres in selected areas 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 greater than or equal to... (e) Align at least two first fluorescence images with microsphere images, obtain the micropore brightness value of each micropore containing microspheres in the selected region in each of the at least two first fluorescence images, and sum the micropore brightness values ​​of each first fluorescence image to obtain the total micropore brightness of each first fluorescence image; (f) Obtain at least one difference or the average of multiple differences between the total micropore brightness of the at least two first fluorescence images; (g) Divide the at least one difference or the average of the multiple differences by the total number of micropores to obtain the average increase in brightness value as a feature value; (h) Obtain a standard curve of feature value versus concentration; and (i) Determine the concentration of the target molecule in the sample according to the standard curve and the feature value of the target molecule.

[0008] In some implementations, in step (f): when N is an even number, the sum of the micropore brightness values ​​of the Nth first fluorescence image is subtracted from the sum of the micropore brightness values ​​of the N / 2th first fluorescence image to obtain a first difference; the sum of the micropore brightness values ​​of the (N-1)th first fluorescence image is subtracted from the sum of the micropore brightness values ​​of the (N / 2-1)th first fluorescence image to obtain a second difference; and so on, until the N / 2th difference between the sum of the micropore brightness values ​​of the N / 2+1th first fluorescence image and the sum of the micropore brightness values ​​of the 1st first fluorescence image is obtained.

[0009] In some embodiments, in step (f): when N is odd, the sum of the micropore brightness values ​​of the Nth first fluorescence image is subtracted from the sum of the micropore brightness values ​​of the (N+1) / 2th first fluorescence image to obtain a first difference; the sum of the micropore brightness values ​​of the (N-1)th first fluorescence image is subtracted from the sum of the micropore brightness values ​​of the (N+1) / 2-1th first fluorescence image to obtain a second difference, 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 image and the sum of the micropore brightness values ​​of the 2nd first fluorescence image is obtained.

[0010] In some implementations, in step (f): when N is odd, the sum of the micropore brightness values ​​of the (N-1)th first fluorescent image is subtracted from the sum of the micropore brightness values ​​of the (N-1) / 2th first fluorescent image to obtain a first difference; the sum of the micropore brightness values ​​of the (N-2)th first fluorescent image is subtracted from the sum of the micropore brightness values ​​of the (N-1) / 2-1th first fluorescent image to obtain a second difference, 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 image and the sum of the micropore brightness values ​​of the first first fluorescent image is obtained.

[0011] In some embodiments, step (a) includes heating the substrate. For example, in some embodiments, the substrate is heated to 30–40°C and maintained for 30 minutes.

[0012] In some embodiments, in steps (c) and / or (d), an LED light source is used to illuminate the micropores to obtain a corresponding image. For example, in some embodiments, the power of the LED light source is 0.1W to 10W.

[0013] In some embodiments, providing micropores in step (a) includes providing micropores located in channels of the microfluidic device.

[0014] In some embodiments, the sealing in step (b) includes sealing with oil.

[0015] In some embodiments, step (d) further includes preprocessing the first fluorescence image to exclude anomalous spots. For example, in some embodiments, the preprocessing includes deep learning-based target detection.

[0016] In some embodiments, in step (c), the detection microsphere contains a fluorescent dye, and the fluorescent dye is excited to cause the detection microsphere to emit a second fluorescence to obtain an image of the microsphere, the second fluorescence being different from the first fluorescence.

[0017] In some implementations, in step (c), the selected area is the entire area of ​​the microsphere image.

[0018] In some implementations, in step (e), the micro-orifice brightness value of each micro-orifice is either the sum of the brightness values ​​of a single micro-orifice displayed in 9×9 pixels or the average brightness value of a single micro-orifice displayed in 9×9 pixels.

[0019] In some embodiments, a correction algorithm is applied to the first fluorescence image prior to step (e). For example, the correction algorithm is used to correct the micro-aperture image differences introduced by aberrations between the central and peripheral fields of view of the optical system, thereby obtaining a fluorescence image closer to the real 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 for each micro-aperture (i is an integer corresponding to the micro-aperture number), 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 matrices IIi of all micro-apertures in the region to obtain Ia, which is used as the correction matrix for the light intensity distribution of each micro-aperture; and (ii) for the edge region of the image, obtaining a 9×9 pixel light intensity distribution matrix IIIj for each micro-aperture (j is an integer corresponding to the micro-aperture number), obtaining the maximum intensity value within each 9×9 pixel region, multiplying the Ia correction matrix by the maximum intensity value to obtain the accurate light intensity distribution of each micro-aperture, thereby correcting the edge region and obtaining a new image as the first fluorescence image.

[0020] In some embodiments, step (a) is performed under any isothermal conditions between 30 and 37°C.

[0021] In some embodiments, in step (a), the reporter molecule consists of a second ligand that specifically binds to the target molecule and the catalyst, the second ligand being different from the first ligand, and step (a) includes: (i) providing a sample that may contain the 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 reporter molecule; (iv) adding a substrate; and (v) introducing the detection microspheres into microwells; wherein the order of the steps is i / ii / iii-iv-v or i / ii / iii / v-iv, where “ / ” indicates that the steps before and after are interchangeable, and “-” indicates that the steps before and after are performed in the order shown.

[0022] In some embodiments, in step (a), the reporter molecule comprises 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 are linked together by an affinity interaction. The second ligand is different from the first ligand. Step (a) includes: (i) providing a sample that may contain the target molecule; (ii) adding detection microspheres whose surface 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 microspheres into microwells. The order of the steps is i / ii / iii / iv-v-vi or i / ii / iii / iv / vi-v, where " / " indicates that the steps before and after are interchangeable, and "-" indicates that the steps before and after are performed in the order shown.

[0023] In some embodiments, in step (a), the reporter molecule consists 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 with a second affinity element, the first ligand binds to the first affinity element through the second affinity element, and step (a) includes: (i) providing a sample that may contain the target molecule; (ii) adding the detection microsphere; (iii) adding the first ligand coupled with the second affinity element; (iv) adding the reporter molecule; (v) adding the substrate; and (vi) introducing the detection microsphere into a microwell; wherein the order of execution of the steps is i / ii / iii / iv-v-vi or i / ii / iii / iv / vi-v, where “ / ” indicates that the steps before and after are interchangeable, and “-” indicates that the steps before and after are executed in the order shown.

[0024] In some embodiments, in step (a), the reporter molecule comprises a first part and a second part that are independent of each other. The first part includes a second ligand and a first affinity element that 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 are linked together by an affinity interaction. 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 binds to the third affinity element through the fourth affinity element. The third affinity element and the fourth affinity element are linked together by an affinity interaction. Step (a) includes: (i) providing a... (ii) Adding a sample containing the target molecule; (ii) Adding detection microspheres, a first ligand coupled with a fourth affinity element, and adding a blocking agent to block the third affinity element if cross-reactivity is possible between the first and second affinity elements and the third and fourth affinity elements, removing excess blocking agent before proceeding to the next step; (iii) Adding the first portion of the reporter molecule; (iv) Adding the second portion of the reporter molecule; (v) Adding the substrate; (vi) Introducing the detection microspheres into the microwells; wherein the order of the steps is i / ii / iii / iv-v-vi or i / ii / iii / iv / vi-v, where “ / ” indicates that the steps before and after are interchangeable, and “-” indicates that the steps before and after are performed in the order shown.

[0025] In some implementations, one or more washing steps are included between any two, three, four or all of steps (i) to (vi).

[0026] In some implementations, the first ligand and the second ligand are different antibodies against the target molecule.

[0027] 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.

[0028] 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.

[0029] In some embodiments, the catalyst is β-galactosidase and the substrate is halogen-β-D-galactopyranoside.

[0030] In some embodiments, the step of introducing the detection microsphere into the micropore includes: driving the microsphere repeatedly through the micropore to increase the probability of the microsphere falling into the micropore. In some embodiments, the step of introducing the detection microsphere into the micropore further includes placing a magnet below the micropore to attract the microsphere into the micropore by magnetic force.

[0031] In some implementations, in step (d), the predetermined time interval is 10 seconds to 5 minutes.

[0032] In some implementations, in step (c), white light is used to illuminate at least a portion of the detected microspheres to obtain an image of the microspheres.

[0033] In some embodiments, the concentration of the target molecule is from 0.1 oz M to 100 p M.

[0034] In some implementations, the target molecule is a protein or nucleic acid.

[0035] In some embodiments, the microspheres are polymer microspheres or magnetic beads.

[0036] In some embodiments, the method does not include counting the microspheres individually connected to the target molecule.

[0037] In some embodiments, the target molecule includes multiple types, the detection microspheres also include multiple corresponding types, and the reporter molecule also includes multiple corresponding types.

[0038] Another aspect of the present invention provides a computer-readable medium having computer-readable instructions stored thereon, which, when executed, perform any of the methods described in the present invention.

[0039] Another aspect of the present invention provides an analytical apparatus comprising a computer control system and a microfluidic device, wherein the computer control system comprises the computer-readable medium described in this invention.

[0040] In some embodiments, the microfluidic device includes a channel comprising a plurality of micropores, each micropore being used to accommodate one or more detection microspheres.

[0041] In some implementations, the volume of each micropore is 1 fl liter to 1 picoliter.

[0042] The method provided by this invention eliminates the need to determine whether each microsphere is connected to a target molecule (i.e., "activated" or "on"), simplifying the method's complexity while maintaining high detection accuracy. Furthermore, the method exhibits excellent continuity across all measured concentration ranges, with the vast majority of data having a CV below 20%, resolving the data discontinuity issue caused by using analog and digital algorithms at high and low concentrations respectively in existing technologies. Moreover, for samples with different concentration multiples, the feature values ​​obtained by this algorithm show greater discriminative power in certain intervals, resulting in higher sample concentration resolution and further improving detection sensitivity. Attached Figure Description

[0043] This invention will be described in more detail with reference to the accompanying drawings. It should be noted that the illustrated schemes are merely representative examples of embodiments of the invention, and to more clearly illustrate the details of exemplary embodiments, the elements in the drawings are not drawn to scale; the number of actual elements may vary, the relative positions of the actual elements remain substantially consistent with the illustrations, and some elements are not shown. In cases where multiple embodiments exist, when one or more features described in previous embodiments are also applicable to another embodiment, for the sake of brevity, these reusable features will not be repeated in subsequent embodiments. These subsequent embodiments should be understood as having described these reusable features, unless otherwise stated. Those skilled in the art will recognize upon reading this invention 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 a part of this invention.

[0044] Figure 1 This is a flowchart illustrating a method according to an embodiment of the present invention.

[0045] Figure 2 This image shows a fluorescence image of microspheres obtained by exciting a fluorescent dye according to an embodiment of the present invention, and a partial schematic diagram thereof.

[0046] Figure 3 Multiple first fluorescence images and fluorescence subtraction images obtained in one embodiment are shown.

[0047] Figure 4 A standard curve of characteristic values ​​versus concentration obtained according to one embodiment is shown.

[0048] Figure 5 The comparison shows the CV values ​​of the feature values ​​obtained by the numerical algorithm and the method of this invention. Detailed Implementation

[0049] The exemplary embodiments of the present invention are described in detail below with reference to the accompanying drawings. It should be understood that the scope of the present invention is not limited to the disclosed embodiments. Those skilled in the art, after reading the disclosure of this invention, can modify and vary these exemplary embodiments based on the teachings of the present invention, without any inventive effort. Such modifications and variations are intended to be included within the scope outlined in the appended claims.

[0050] Figure 1 A schematic flowchart of one embodiment of the method according to the present invention is shown. Method 100 is a method for determining the concentration of a target molecule, which is typically contained in a sample (e.g., a biological sample). This method is used to detect the presence of the target molecule in the sample, and if present, at what concentration. Therefore, method 100 can be used for the qualitative and quantitative determination of the target molecule. The target molecule can be a chemical or biological molecule, including but not limited to protein molecules, such as cytokines (e.g., IL-12, IL-6, etc.) or antibodies (e.g., PD-1 antibodies); and nucleic acid molecules, such as DNA or RNA. Other suitable target molecules are, for example, antigen molecules (e.g., Aβ1-42), gene fragments, or fusion proteins. The biological sample can be, for example, blood, serum, plasma, urine, saliva, tissue fluid, or other fluids derived from a human or animal body, or it can be a laboratory culture medium, cell or tissue processing fluid (e.g., tissue homogenate or cell lysate). These biological samples may or may not contain the target molecule.

[0051] Method 100 begins at step 102, which provides micropores, wherein a portion of the micropores contains a substrate and a detection microsphere, while another portion of the micropores contains a substrate but not 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, the reporter molecule containing a catalyst capable of catalyzing the substrate to emit a first fluorescence.

[0052] In step 102, providing micropores can be achieved by providing a microfluidic device including an inlet and an outlet, and a channel fluidly communicating between the inlet and outlet. This channel may have a plurality of micropores, such as hundreds to tens of thousands, disposed on its sides or bottom, each micropore being sized to accommodate one or more microspheres, thus spatially isolating the microspheres from each other. For example, the volume of each micropore may be about 1 femtoliter to about 1 picoliter. In this invention, some micropores contain both substrate and detection microspheres, while others contain substrate but not detection microspheres. Therefore, some micropores do not contain microspheres, while others contain one or more microspheres. In the micropores containing microspheres, the microspheres may or may not be coupled with a target molecule. The method of this invention does not involve individually counting microspheres coupled with target molecules, as detailed later. In other embodiments, each micropore is sized to accommodate only one microsphere, thus some micropores do not contain microspheres, while others 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 composite microspheres in CN111318238 B. As an example, the detection microspheres are coupled with or encoded with fluorescent dyes, such as CY5 fluorescent dyes.

[0053] In this invention, within the micropores, the surface of at least one of the detection microspheres comprises a sandwich structure formed by a first ligand, a target molecule, and a reporter molecule, wherein the reporter molecule comprises a catalyst capable of catalyzing the emission of a first fluorescence from the substrate. The first ligand is, for example, a ligand that specifically binds to the target molecule, such as an antibody against the target molecule, and is directly or indirectly coupled to the surface of the detection microsphere. In this invention, the reporter molecule is capable of specifically binding to the target molecule and is directly or indirectly coupled to a catalyst capable of catalyzing the emission of a first fluorescence from the substrate. For example, the reporter molecule comprises a second ligand that specifically binds to the target molecule, such as an antibody against the target molecule, which binds to a different epitope of the target molecule than the first ligand. Thus, a "sandwich" structure (or sandwich structure) is formed between the first ligand, the target molecule, and the reporter molecule (second ligand), wherein the target molecule is sandwiched between the first ligand and the reporter molecule.

[0054] There are several ways to form the sandwich structure. In some embodiments, the reporter molecule consists of a second ligand that specifically binds to the target molecule and the catalyst, the second ligand being different from the first ligand. Step 102 may include: (i) providing a sample that may contain the 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 reporter molecule; (iv) adding a substrate; and (v) introducing the detection microspheres into micropores. The execution order of the above steps may be i / ii / iii-iv-v or i / ii / iii / v-iv, where " / " indicates that the steps before and after are interchangeable, and "-" indicates that the steps before and after are performed 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 in any way, and steps (iv) and (v) are performed sequentially after these three steps are completed.

[0055] In other embodiments, the reporter molecule comprises 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 linked together by an affinity interaction. 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 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 the second part of the reporter molecule; (v) adding a substrate; and (vi) introducing the detection microspheres into microwells. The execution order of the above steps may be i / ii / iii / iv-v-vi or i / ii / iii / iv / vi-v, where " / " indicates that the steps before and after are interchangeable, and "-" indicates that the steps before and after are executed in the order shown.

[0056] In other embodiments, the reporter molecule consists 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, and the first ligand is coupled with a second affinity element. The first ligand binds to the first affinity element through 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 with the second affinity element; (iv) adding the reporter molecule; (v) adding the substrate; and (vi) introducing the detection microsphere into a microwell. The execution order of the above steps may be i / ii / iii / iv-v-vi or i / ii / iii / iv / vi-v, where " / " indicates that the steps before and after are interchangeable, and "-" indicates that the steps before and after are executed in the indicated order.

[0057] In other embodiments, the reporter molecule comprises 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 linked 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 binds to the third affinity element through the fourth affinity element. The third affinity element and the fourth affinity element can be linked together by affinity. Step 102 may include: (i) providing a sample that may contain the target molecule; (ii) adding the detection microsphere, the first ligand coupled with the fourth affinity element, and adding a blocking agent when there is a possibility of cross-reactivity between the first and second affinity elements and the third and fourth affinity elements, the blocking agent blocking 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 a microwell. The execution order of the above steps can be: i / ii / iii / iv-v-vi or i / ii / iii / iv / vi-v, where " / " indicates that the steps before and after are interchangeable, and "-" indicates that the steps before and after are executed in the order shown.

[0058] 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 of steps (i) to (v) or steps (i) to (vi). In some embodiments, one or more washing steps may be performed between two of steps (i) to (v) or steps (i) to (vi). The washing steps may involve washing the microspheres and / or microwells by introducing a sample-compatible buffer (e.g., PBS or physiological saline) to remove components that are not specifically bound to or unbound by the ligands coupled to the microspheres and / or non-specifically bound or unbound reporter molecules, thereby minimizing interference from components other than non-specifically bound or target molecules. For example, when using magnetic beads, after mixing the magnetic beads with the sample, the beads may be immobilized by applying a magnetic field (thus immobilizing the target molecules specifically bound to the first ligand on the magnetic beads), and components in the sample that are not specifically bound to the ligands on the magnetic beads (e.g., non-target molecular proteins in plasma) may be removed by introducing PBS buffer.

[0059] In any of the above embodiments, the first ligand and the second ligand can 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. The first antibody and the second antibody bind to different epitopes of IL-15.

[0060] In any of the above embodiments, the first affinity element may be one of biotin and streptavidin, and the second affinity element may be the other of biotin and streptavidin. In any of the above embodiments, the first affinity element may be one of biotin and avidin, and the second affinity element may be 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 may be the other of biotin and streptavidin. In any of the above embodiments, the third affinity element may be one of biotin and avidin, and the fourth affinity element may be 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.

[0061] In some embodiments, the blocking agent is an avidin blocking agent, such as bovine serum albumin (BSA) conjugated with biotin or polyethylene glycol (PEG). In some embodiments, the blocking agent is a biotin blocking agent, such as BSA or PEG conjugated with avidin or streptavidin.

[0062] In step 102, the catalyst is β-galactosidase, and the substrate is halogen-β-D-galactopyranoside (CAS No.: 95079-19-9). As mentioned above, other suitable catalyst and substrate pairs include catalysts that are β-galactosidase and substrates that are fluorescein-di-β-D-galactopyranoside (FDG, CAS No.: 17817-20-8) or catalysts that are β-galactosidase and substrates that are 4-methylumbelliferone-β-D-galactopyranoside (CAS No.: 6160-78-7), etc.

[0063] In the presence of the target molecule, the catalyst attached to the reporter molecule catalyzes the substrate to emit fluorescence. Within a certain range, the higher the concentration of the attached reporter molecule (i.e., the higher the concentration of the target molecule), the more substrate is catalyzed, resulting in greater fluorescence intensity and / or brightness. When the reporter molecule reaches a saturation concentration, the fluorescence brightness of the microspheres reaches its maximum in the presence of excess substrate. In one embodiment, due to variations in micropore size, to ensure a uniform substrate concentration, and considering the potential aggregation of substrates stored at 2-8°C, the substrate is heated at 30-40°C (e.g., 35-37°C, such as 35°C, 36°C, or 37°C) for 30 minutes before use to ensure complete substrate dissolution, given the potential aggregation of substrates stored at 2-8°C.

[0064] In addition, in some embodiments, considering that the catalytic efficiency of individual enzymes in different micropores may vary in the same batch of enzymes in the same experiment, the enzyme catalytic reaction is carried out under any isothermal condition from 30°C to 37°C (e.g., 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C or 37°C).

[0065] In other embodiments, the present invention aims to improve the microsphere settling rate, ensure a sufficient sampling base, and reduce bias caused by individual enzyme differences through averaging. In one example, the step of introducing the detection microspheres into the microwells includes driving the microspheres repeatedly through the microwells by external force (e.g., suction of the microspheres), increasing the probability of the microspheres settling into the microwells. In another example, the external force drives the microspheres repeatedly through the microwells, and a magnet is placed below the microwells.

[0066] In step 104, the microwells are sealed to fluidly isolate each microwell. Sealing may include sealing each individual microwell described above, thereby fluidly isolating each microwell, thus isolating the buffer solution within the microwell and the microspheres from each other to shield against fluorescent contamination between microwells / microspheres as much as possible. In some embodiments, sealing may include introducing a sealing oil into the microfluidic device, which, on the one hand, removes residual microspheres and / or reporter molecules from the channels, and on the other hand, seals the individual microwells due to its immiscibility with the buffer solution in the microwells. Suitable sealing oils may be mineral oil, fluorinated oil, or silicone oil.

[0067] In method 100, step 106 includes obtaining a microsphere image of at least a portion of the detected microspheres and counting the micropores containing the microspheres within the selected area to obtain the total number of micropores. In one embodiment, the detected microspheres are coded or coupled with a fluorescent dye that can be excited to emit fluorescence. The fluorescence signal can be acquired by, for example, a fluorescence camera to form a fluorescence image, which is used to locate the fluorescent microspheres. For example, a laser light source can be used to excite the fluorescent dye (such as CY5 fluorescent dye) to obtain a microsphere fluorescence image, and each micropore containing the microsphere can be 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 can be located based on the microsphere fluorescence image. For example, the power of the LED light source can be 0.1 to 10 W, such as 0.1 to 5 W or 1 to 5 W, such as 1 W, 2 W, 3 W, 4 W or 5 W. Alternatively, a commercially available large numerical aperture (NA) 0.28 achromatic objective lens can be used.

[0068] In another embodiment, the detection microspheres are either not coded or coupled with fluorescent dyes. The microspheres are illuminated with white light, and images of the microspheres are obtained using a conventional camera. Based on these images, each micropore containing the microspheres is located.

[0069] In some implementations, the CY5 fluorescence image can be Gaussian blurred to analyze its contour and obtain the coordinates of each micropore. In some embodiments, the processing of the CY5 fluorescence image can be implemented using computer software, such as OpenCV 2.0 and above. For ease of description, in the case of detecting microsphere-coupled or encoded fluorescent dyes, the fluorescence emitted by the dye upon excitation is referred to as the second fluorescence, while the fluorescence emitted by the reporter molecule catalytic substrate is referred to as the first fluorescence; the second fluorescence is different from the first fluorescence.

[0070] Figure 2 Part A shows a CY5 fluorescence image obtained by the above method, which covers 80% to 90% of all micropores. In other embodiments, the image may cover 100% of all micropores. In other embodiments, the image may cover less than 80% of all micropores. Figure 2 Part B is Figure 2 The magnified view of the dashed area in Part A shows that a white bright spot indicates the presence of microspheres (i.e., the corresponding micropores, the same below), while the location where no bright spot is shown indicates that the corresponding micropore does not contain microspheres. Figure 2 Part C is Figure 2 A further enlarged view of part B.

[0071] In this invention, software is used to count the micropores, regardless of whether the microspheres within the micropores are coupled with target molecules. In some embodiments, the micropores containing microspheres are counted across the entire area of ​​a microsphere image (e.g., a microsphere fluorescence image). In other embodiments, the micropores containing microspheres are counted across 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the area of ​​the microsphere image.

[0072] 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 from 2 to 1000. This time interval can be determined based on the typical reaction rate between the catalyst and the substrate; slower reaction rates typically require longer times, while faster reaction rates require shorter times. In some embodiments, a catalyst with high catalytic efficiency is preferably used, thus shortening the overall testing process. In some embodiments, the predetermined time interval can be, for example, 10 seconds to 5 minutes, such as 30 seconds to 3 minutes, 40 seconds to 2 minutes, 50 seconds to 90 seconds, or 60 seconds to 90 seconds. In a preferred embodiment, 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 can be used. In this invention, the number of first fluorescence images can be reasonably determined as needed. Preliminary experiments can be conducted to determine the optimal number of images. The number of images is typically considered in conjunction with the predetermined time interval, and the number of fluorescence images is also designed based on the fluorescence growth curve of the corresponding first fluorescence. In some embodiments, the present invention acquires 2 to 1000 first fluorescence images, for example, 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 images. In embodiments of the present invention, the number of first images acquired can be odd or even, but is preferably even. In a specific embodiment, 10 first fluorescence images are acquired at 1-minute time intervals within 10 minutes. In some embodiments, step 108 may further include preprocessing the first fluorescence images to remove abnormal spots, such as abnormal spots caused by bubbles, impurities, etc. The preprocessing may include target detection based on deep learning.

[0073] In some embodiments, the order of steps 106 and 108 can be reversed. For example, after the method performs step 104, step 108 is performed first, and step 106 is performed before starting step 110.

[0074] In step 110, at least two first fluorescence images and microsphere images are aligned. The micropore brightness value of each micropore containing a microsphere within the selected region is obtained in each of the at least two first fluorescence images, and the micropore brightness values ​​in each first fluorescence image are summed to obtain the total micropore brightness of each first fluorescence image. Those skilled in the art will understand that the micropore brightness value obtained from the micropore location is formed by the fluorescence emitted by all the catalyzed substrates within a single micropore containing the microsphere.

[0075] Figure 3 Part A is a magnified view of the largest pixels of a single micro-aperture (bright spot). Figure 3 In Part A, a single microaperture is displayed with 9×9 pixels. It is anticipated that using cameras of different resolutions could allow a single microaperture to be displayed with higher or lower pixel counts. In one embodiment of the invention, for a single microaperture, the average brightness of 9×9 pixels is used as its fluorescence brightness. For example, for... Figure 3 The average brightness of a single micropore, as shown in Part A, is obtained by dividing the sum of the brightness values ​​of 9×9 pixels by 81. The average brightness of all micropores containing microspheres within a selected area of ​​the image can be obtained using the same method. For example, for all micropores containing microspheres within a selected area, the average brightness of each micropore containing microspheres is calculated using the method described above. Then, the average brightness of all micropores containing microspheres within the selected area is summed and divided by the number of micropores containing microspheres within the selected area to obtain the average brightness value of the micropores containing microspheres within the selected area. In other embodiments of the invention, the fluorescence brightness of a single micropore is the sum of the brightness values ​​of 9×9 pixels. In a preferred embodiment of the invention, 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 less of the entire area of ​​the image.

[0076] In a preferred embodiment, prior to step 110, a correction algorithm is applied to the entire first fluorescence image to correct the micropore image differences introduced by aberrations between the central and edge fields of view of the optical system, thereby obtaining a fluorescence image that is closer to the real sample. For example, the correction algorithm may include: (i) selecting the central region of the first fluorescence image, obtaining a 9×9 pixel light intensity distribution matrix Ii for each micropore (i is an integer, corresponding to the micropore number; the matrix size is 9×9), 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 micropores in the region (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 above), and using this as the correction matrix for the light intensity distribution of each micropore; and (ii) for the edge region of the first fluorescence image, obtaining a 9×9 pixel light intensity distribution matrix IIIj for each micropore (j is an integer, corresponding to the micropore number; the matrix size is 9×9), obtaining the maximum intensity value in each 9×9 pixel region, multiplying the Ia correction matrix by the maximum intensity value to obtain the accurate light intensity distribution of each micropore, thereby correcting the edge region and obtaining a new image as the first fluorescence image. In some embodiments, the selected central region may be a region of 100×100 micropores centered at the center coordinates; in some embodiments, the selected edge region may be a region extending one-fifth of the distance from the four edges of the first fluorescence image towards the center. In some embodiments, the selected central region may be a region of 100×100 micropores centered at the center coordinates, and the selected edge region may be a region extending one-fifth of the distance from the four edges of the first fluorescence image towards the center.

[0077] Furthermore, commercially available high-concentration solid fluorescent slices can be used as calibration plates. Algorithms can be used to correct the solid fluorescent slice images, further correcting non-uniformity in the illumination path. For example, the algorithm calibration steps are as follows: First, appropriately adjust the illumination intensity of the illumination path to obtain an unexposed fluorescence image of the high-concentration solid fluorescent slice under the illumination path, obtaining the grayscale matrix I1 of this unexposed fluorescence image; 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 calibration matrix; for each first fluorescence image, multiplying it by the calibration matrix I2 yields a uniformly calibrated first fluorescence image.

[0078] 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 analytical region in the first fluorescence image is the same as the analytical region in the microsphere image. After step 110, multiple fluorescence brightness values ​​are obtained for each microwell location, with each fluorescence brightness value corresponding to one first fluorescence image; for example, each microwell location may have 10 fluorescence brightness values. For some microwell locations, each fluorescence brightness value is the same or substantially the same, while for others, each fluorescence brightness value is different and increases over time. For different microwell locations, the magnitude of the increase in fluorescence brightness value over time may be the same or different.

[0079] In step 110, the fluorescence brightness values ​​of all micropores containing microspheres in at least two first fluorescence images are summed to obtain the total brightness of the micropores in each first fluorescence image. The total brightness of the microspheres can be obtained by adding the fluorescence brightness values ​​of each micropore containing microspheres.

[0080] In step 112, at least one difference between the sums of the microsphere brightness values ​​of the first fluorescence images is obtained, or when N is greater than 2, the average of multiple differences between the sums of the microsphere brightness values ​​of the first fluorescence images is obtained. When N is even, the sum of the micropore brightness values ​​of the Nth first fluorescence image is subtracted from the sum of the micropore brightness values ​​of the N / 2th first fluorescence image to obtain a first difference; the sum of the micropore brightness values ​​of the (N-1)th first fluorescence image is subtracted from the sum of the micropore brightness values ​​of the (N / 2-1)th first fluorescence image to obtain a second difference, and so on, until the N / 2 difference between the sum of the micropore brightness values ​​of the N / 2+1th first fluorescence image and the sum of the micropore brightness values ​​of the 1st first fluorescence image is obtained. For example, when 10 (N=10) first fluorescence images are acquired in step 108, in step 110, for each micropore containing microspheres, the sum of the micropore brightness values ​​in images 10, 9, 8, 7, and 6 is subtracted from the sum of the micropore brightness values ​​in images 5, 4, 3, 2, and 1, respectively, to obtain 5 differences. The images are numbered according to the acquisition time sequence. Figure 3 Part B shows the fifth image obtained in one embodiment. Figure 3 Section C shows the 10th image obtained in the same embodiment. Both images have the same field of view and show hundreds of micropores containing microspheres. Figure 3 Part D shows that Figure 3 The image in part C and Figure 3 The image obtained by subtracting the images in part B. Figure 3 The sum of the micro-aperture brightness values ​​in part D is the first difference obtained by subtracting the sum of the micro-aperture brightness values ​​in the 5th image from the sum of the micro-aperture brightness values ​​in the 10th image.

[0081] When N is odd, the sum of the micro-well brightness values ​​of the Nth first fluorescence image is subtracted from the sum of the micro-well brightness values ​​of the (N+1) / 2th first fluorescence image to obtain the first difference. The sum of the micro-well brightness values ​​of the (N-1)th first fluorescence image is subtracted from the sum of the micro-well brightness values ​​of the (N+1) / 2-1th first fluorescence image to obtain the second difference, and so on, until the (N-1) / 2 difference between the sum of the micro-well brightness values ​​of the (N+1) / 2+1th first fluorescence image and the sum of the micro-well brightness values ​​of the 2nd first fluorescence image is obtained. For example, if 11 (N=11) first fluorescence images were acquired in step 108, in step 110, the sum of the micro-well brightness values ​​of images 11, 10, 9, 8, and 7 is subtracted from the sum of the micro-well brightness values ​​of images 6, 5, 4, 3, and 2, respectively, to obtain 5 differences. The images are numbered according to the acquisition time sequence. In other embodiments, step 110 may be performed using more or fewer first fluorescence images.

[0082] Alternatively, when N is odd, the sum of the micro-well brightness values ​​of the (N-1) / 2th first fluorescence image can be subtracted from the sum of the micro-well brightness values ​​of the (N-1)th first fluorescence image to obtain the first difference. The sum of the micro-well brightness values ​​of the (N-2)th first fluorescence image can be subtracted from the sum of the micro-well brightness values ​​of the (N-1) / 2-1th first fluorescence image to obtain the second difference, and so on, until the (N-1) / 2 / 2 difference between the sum of the micro-well brightness values ​​of the (N-1) / 2+1th first fluorescence image and the sum of the micro-well brightness values ​​of the first 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 micro-well brightness values ​​of the 10th, 9th, 8th, 7th, and 6th images can be subtracted from the sum of the micro-well brightness values ​​of the 5th, 4th, 3rd, 2nd, and 1st images, respectively, to obtain 5 differences.

[0083] Subsequently, in step 114, at least one difference obtained in step 112, or when more than one difference is obtained, the average of the multiple differences, divided by the total number of micropores obtained in step 106, is used to obtain the average increase in brightness value as a feature value.

[0084] In other embodiments, the brightness value of each micropore location containing a microsphere can be calculated only for local regions in at least two first fluorescence images. For example, the brightness value of each micropore location containing a microsphere can be calculated only for 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the regions in the first fluorescence images. However, it should be noted that in this case, the difference or average difference obtained in step 112 needs to be divided by the total number of micropores counted in the corresponding local region in the microsphere image to obtain the feature value. For example, when the brightness value of each micropore location containing a microsphere is calculated for 50% of the region in the microsphere image to obtain the sum of the micropore brightness of the corresponding image, and the difference is obtained in step 112 based on this, the difference or average difference should be divided by the total number of micropores counted in the corresponding 50% region in the microsphere image to obtain the feature value.

[0085] In step 116, a standard curve of the above-mentioned characteristic values ​​versus concentration is obtained. This standard curve can be pre-established using target molecules or other molecules at known concentrations according to the methods described in steps 102 to 114.

[0086] 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, for example, 0.1 oz M to 100 p M, for example, 1 oz M to 100 f M, or even lower.

[0087] According to the method described in Method 100, standard curves were established using six different concentrations of Aβ1-42 molecular samples. The characteristic values ​​and concentrations are shown in Table 1 below.

[0088] Table 1. Test results of Aβ1-42 (standard curve with 6 concentrations, unit pg / mL, 8 replicates per well)

[0089]

[0090]

[0091] Table 2. Evaluation of the eigenvalues ​​of the standard curve

[0092] concentration Eigenvalues Sn+1 / Sn S0 0 3.8819 - S1 0.75 5.9605 1.54 S2 1.5 10.5690 1.77 S3 3 20.9243 1.98 S4 6 60.9896 2.91 S5 12 260.5080 4.27 S5 / S0 67.11

[0093] The standard curve was constructed using six progressively increasing concentrations, with eight replicates for each concentration, and protein molecules (such as Aβ1-42) as the target molecules. In the above embodiments, the coefficient of variation (CV) of the standard curves, except for the group with a concentration of 0 (S0), was below 15%. In the above embodiments, there was a clear distinction between two adjacent concentrations, with a ratio greater than 1.5. Figure 4A standard curve of eigenvalues ​​versus concentration obtained according to this embodiment is shown. It can be seen that the eigenvalues ​​are directly proportional to the concentration; the higher the concentration, the larger the eigenvalues. Furthermore, the standard curve established by this method has a wider range of eigenvalues, increasing the sensitivity of the signal response; and it exhibits good continuity at both high and low concentrations.

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

[0095] Table 3. Test results of samples with different concentrations using the method of this invention.

[0096]

[0097] As can be seen from the results in Table 3, the characteristic value repeatability of the method of the present invention is good, and the characteristic value CV of the method of the present invention is within 25%, of which the characteristic value CV of the last three concentration samples is within 15%.

[0098] Figure 5 A scatter plot showing the relationship between the CV values ​​of the digital algorithm and the method of this invention is presented based on some test data (not listed). The scatter plot shows that for the test results of blood samples (part A) and calibrators (part B), the CV values ​​of the digital algorithm and the method of this invention are close. Most of the scatter plots of the CV values ​​of both algorithms fall within the <20% region. For a very small number of scatter plots where the CV is >20%, the CV value of the eigenvalue obtained by the method of this invention is superior to that obtained by the digital algorithm.

[0099] In some embodiments, the method of the present invention can simultaneously analyze two or more different types of target molecules, such as IL-12 and IL-10, that may be present in a sample. In such embodiments, the method can use different types of microspheres corresponding to different types of target molecules, different types of fluorescent dyes, and different types of reporter molecules and different types of substrate-enzyme combinations to identify each microsphere and reporter molecule via different fluorescence channels. A method for preparing two different fluorescently encoded microspheres can be found in CN 111218498A.

[0100] Another aspect of the invention provides a computer-readable medium having computer-readable instructions stored thereon, which, when executed, perform any of the methods described above. The computer-readable medium may include a removable medium as a package medium for a disk (including a floppy disk), an optical disk (including a CD-ROM (Optical Disc Read-Only Memory) and a DVD (Digital Universal Optical Disk)), a magneto-optical disk (including an MD (Minimum Disk Drive)) or a semiconductor memory.

[0101] Another aspect of the present invention provides an analytical apparatus comprising a computer control system and a microfluidic device, wherein the computer control system includes the computer-readable medium of the present invention.

[0102] In one embodiment, the microfluidic device includes a channel comprising a plurality of micropores, each micropore for accommodating up to one microsphere. In some embodiments, the volume of each micropore is from 1 femtoliter to 1 picoliter. The analytical device is configured to perform any of the methods described in this invention.

[0103] The above descriptions are representative examples of embodiments of the present invention and are provided for illustrative purposes only. The present invention contemplates that one or more technical features used in one embodiment can be added to another embodiment to form improved or alternative embodiments without departing from the purpose of the embodiment. Similarly, one or more technical features used in one embodiment can be omitted or replaced without departing from the purpose of the embodiment to form alternative or simplified embodiments. Furthermore, one or more technical features used in one embodiment can be combined with one or more technical features in another embodiment without departing from the purpose of the embodiment to form improved or alternative embodiments. The present invention is intended to include all of the above-mentioned improved, alternative, and simplified technical solutions.

Claims

1. 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, while another portion of the micropores contains a substrate but not a detection microsphere, each micropore being used to contain at most one microsphere, at least one of the detection microspheres having a surface comprising a sandwich structure formed of a first ligand-target molecule-reporter molecule, the reporter molecule comprising a catalyst capable of catalyzing the substrate to emit a first fluorescence; (b) Seal the micropores to isolate the fluid between each micropore; (c) Obtain microsphere images of at least a portion of the detected microspheres, and count the micropores containing microspheres within the selected area to obtain the total number of micropores; (d) At predetermined time intervals, acquire N first fluorescence images corresponding to the first fluorescence, where N is an integer greater than or equal to 2; (e) Align at least two first fluorescence images with microsphere images, obtain the micropore brightness value of each micropore containing microspheres in the selected area in each of the at least two first fluorescence images, and sum the micropore brightness values ​​of each first fluorescence image to obtain the total micropore brightness of each first fluorescence image. (f) Obtain at least one difference or the average of multiple differences between the sum of the micropore brightness of the at least two first fluorescence images; (g) The average increase in brightness value is obtained by dividing the at least one difference or the average of the multiple differences by the total number of micropores, and is used as a feature value; (h) Obtain the standard curve of characteristic values ​​versus concentration; and (i) Determine the concentration of the target molecule in the sample based on 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 even, the sum of the micro-hole brightness values ​​of the Nth first fluorescence image is subtracted from the sum of the micro-hole brightness values ​​of the N / 2th first fluorescence image to obtain the first difference. The sum of the micro-hole brightness values ​​of the N-1th first fluorescence image is subtracted from the sum of the micro-hole brightness values ​​of the (N / 2-1)th first fluorescence image to obtain the second difference. This process is repeated until the N / 2th difference between the sum of the micro-hole brightness values ​​of the N / 2+1th first fluorescence image and the sum of the micro-hole brightness values ​​of the 1st first fluorescence image is obtained. (2) When N is odd, subtract 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 to obtain the first difference; subtract 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-1)th first fluorescence image to obtain the second difference, 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 image and the sum of the micropore brightness values ​​of the 2nd first fluorescence image is obtained; or (3) When N is odd, the sum of the micropore brightness values ​​of the (N-1) / 2th first fluorescence image is subtracted from the sum of the micropore brightness values ​​of the (N-1)th first fluorescence image to obtain the first difference. The sum of the micropore brightness values ​​of the (N-2)th first fluorescence image is subtracted from the sum of the micropore brightness values ​​of the (N-1) / 2-1th first fluorescence image to obtain the second difference. This process is repeated until the (N-1) / 2th difference 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 first first fluorescence image 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 micropores 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 micropores in step (a) comprises providing micropores located in channels of a microfluidic device.

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

9. The method of claim 1, wherein step (d) further comprises preprocessing the first fluorescence image to exclude abnormal spots.

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

11. The method of claim 1, wherein in step (c), the detection microsphere comprises a fluorescent dye, and the fluorescent dye is excited to cause the detection microsphere to emit a second fluorescence to obtain an image of the microsphere, the second fluorescence being different from the first fluorescence.

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

13. The method according to claim 1, wherein in step (e), the micro-aperture brightness value of each micro-aperture is the sum of the brightness values ​​of a single micro-aperture displayed in 9×9 pixels or the average brightness value of a single micro-aperture displayed in 9×9 pixels.

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

15. The method of claim 14, wherein the correction algorithm comprises: (i) For the central region of the image, obtain the light intensity distribution matrix Ii of each microhole (9×9 pixels), where i is an integer and corresponds to the microhole number. Divide Ii by the maximum element value of the matrix to obtain the normalized light intensity distribution matrix IIi. Take the average of all elements of the normalized light intensity distribution matrix IIi of all microholes in the region to obtain Ia, which is used as the correction matrix for the light intensity distribution of each microhole. and (ii) For the edge region of the image, obtain the light intensity distribution matrix IIIj of each micro-hole (9×9 pixels), where j is an integer and corresponds to the micro-hole number. Obtain the maximum intensity value within each 9×9 pixel region, and multiply the maximum intensity value by the Ia correction matrix to obtain the accurate light intensity distribution of each micro-hole. Correct the edge region in this way and obtain a new image as the first fluorescence image.

16. The method according to claim 1, wherein step (a) is performed under any isothermal conditions between 30 and 37°C.

17. The method of claim 1, wherein in step (a), the reporter molecule comprises a second ligand that specifically binds to the target molecule and the catalyst, the second ligand being different from the first ligand, and step (a) comprises: (i) Provide a sample that may contain the target molecule; (ii) Add detection microspheres, the surface of which is modified with the first ligand that specifically binds to the target molecule; (iii) Add reporter molecules; (iv) Add the substrate; (v) Introduce the detection microspheres into the micropores; The execution order of the steps is i / ii / iii-iv-v or i / ii / iii / v-iv, where " / " indicates that the steps before and after it can be interchanged, and "-" indicates that the steps before and after it are executed in the order shown.

18. The method of claim 1, wherein in step (a), the reporter molecule comprises a first portion and a second portion that are independent of each other, the first portion comprising a second ligand and a first affinity element that specifically bind to a target molecule, the second portion comprising a second affinity element and the catalyst, the first affinity element and the second affinity element being able to be linked together by an affinity interaction, the second ligand being different from the first ligand, and step (a) comprising: (i) Provide a sample that may contain the target molecule; (ii) Add detection microspheres, the surface of which is modified with the first ligand that specifically binds to the target molecule; (iii) Add the first part of the reporter molecule; (iv) Add the second part of the reporter molecule; (v) Add substrate; (vi) Introduce the detection microspheres into the micropores; The execution order of the steps is i / ii / iii / iv-v-vi or i / ii / iii / iv / vi-v, where " / " indicates that the steps before and after it can be interchanged, and "-" indicates that the steps before and after it are executed in the order shown.

19. The method according to claim 1, wherein in step (a), the reporter molecule comprises 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 with a second affinity element, the first ligand binds to the first affinity element through the second affinity element, and step (a) comprises: (i) Provide a sample that may contain the target molecule; (ii) Add detection microspheres; (iii) Adding a first ligand coupled with a second affinity element; (iv) Add reporter molecules; (v) Add substrate; (vi) Introduce the detection microspheres into the micropores; The execution order of the steps is i / ii / iii / iv-v-vi or i / ii / iii / iv / vi-v, where " / " indicates that the steps before and after it can be interchanged, and "-" indicates that the steps before and after it are executed in the order shown.

20. The method of claim 1, wherein in step (a), the reporter molecule comprises a first part and a second part that are independent of each other, the first part comprising a second ligand and a first affinity element that specifically bind to a target molecule, the second part comprising a second affinity element and the catalyst, the first affinity element and the second affinity element being able to be linked together by an affinity interaction, the second ligand being different from the first ligand, the surface of the detection microsphere being modified with a third affinity element, the first ligand being coupled with a fourth affinity element, the first ligand binding the third affinity element through the fourth affinity element, the third affinity element and the fourth affinity element being able to be linked together by an affinity interaction, and step (a) comprising: (i) Provide a sample that may contain the target molecule; (ii) Add detection microspheres, a first ligand coupled with a fourth affinity element, and add 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, the blocking agent blocking the third affinity element, and remove excess blocking agent before proceeding to the next step; (iii) Add the first part of the reporter molecule; (iv) Add the second part of the reporter molecule; (v) Add substrate; (vi) Introduce the detection microspheres into the micropores; The execution order of the steps is i / ii / iii / iv-v-vi or i / ii / iii / iv / vi-v, where " / " indicates that the steps before and after it can be interchanged, and "-" indicates that the steps before and after it 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 according to 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 claim 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 according to any one of claims 1, 17, 18, 19 or 20, wherein the catalyst is β-galactosidase and the substrate is halogen-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 micropores comprises: The microspheres are driven to repeatedly pass through the micropores, increasing the probability of the microspheres falling into the pores.

28. The method of claim 27, wherein the step of introducing the detection microsphere into the micropore further comprises placing a magnet below the micropore to attract the microsphere into the micropore by magnetic force.

29. The method according to 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 the at least a portion of the detected microspheres to obtain an image of the microspheres.

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

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

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

34. The method of claim 1, wherein the method does not include individually counting the microspheres connected to the target molecule.

35. The method according to claim 1, wherein the target molecule includes multiple types, the detection microspheres also include multiple corresponding types, and the reporter molecule also includes multiple corresponding types.

36. A computer-readable medium having stored thereon computer-readable instructions, which, when executed, perform the method according to any one of claims 1 to 35.

37. An analytical apparatus comprising a computer control system and a microfluidic device, wherein the computer control system comprises the computer-readable medium of claim 36.

38. The analytical apparatus of claim 37, wherein the volume of each microwell is 1 femtoliter to 1 picoliter.

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