CN117871418A

CN117871418A - Method, computer readable medium and analysis device for determining concentration of target molecules in sample using detection microspheres

Info

Publication number: CN117871418A
Application number: CN202410281858.3A
Authority: CN
Inventors: 请求不公布姓名
Original assignee: Caike Suzhou Biotechnology Co ltd
Current assignee: Caike Suzhou Biotechnology Co ltd
Priority date: 2024-03-13
Filing date: 2024-03-13
Publication date: 2024-04-12

Abstract

The present invention provides a method, computer readable medium and analysis device for determining the concentration of a target molecule in a sample using a detection microsphere, wherein the method for determining the concentration of the target molecule comprises: providing a micropore; sealing the microwells to fluidly isolate each microwell from the other microwells; obtaining the total number of micropores; acquiring a first fluorescent picture according to a preset time interval; obtaining a micropore brightness value; calculating an enzyme catalysis efficiency value based on the microwell brightness value, summing the enzyme catalysis efficiency values of all microwells containing microspheres, dividing the sum by the total number of microwells, and taking the sum as a characteristic value; obtaining a standard curve of the characteristic value to the concentration; determining the concentration of the target molecule in the sample according to a standard curve. The method of the invention does not need to judge whether each microsphere is connected with a target molecule, simplifies the complexity of the method and maintains high detection accuracy.

Description

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

Technical Field

The present invention relates to a method for determining the concentration of a target molecule in a biological sample, in particular a concentration of a target molecule at very low concentrations. The invention also relates to a computer readable medium storing instructions for performing the method and a corresponding analysis device.

Background

Being able to measure low abundance analytes from biological samples is very important for several fields including clinical diagnostics. Many proteins and nucleic acid diagnostic biomarkers are present in very low concentrations, which requires that their analytical methods have very low detection limits.

However, accurate concentration determination is very challenging for target molecules at concentrations of picomolar (pM), femtomolar (fM), attomolar (aM) and even equimolar (zM). One known solution utilizes antibody-labeled microspheres, adding thousands to millions of microspheres per sample to capture target molecules in the sample, and labeling the target molecules with enzyme reporter molecules. This scheme, which disperses the microspheres into spatially isolated wells and calculates the average number of enzymes on individual microspheres based on poisson distribution, assumes that enzyme molecules are not coupled to the vast majority of microspheres at low concentrations ("off" spheres), so the average number of enzymes on individual microspheres is characterized by the ratio of microspheres coupled with enzyme molecules ("on" spheres) to all microspheres, an algorithm also known as a digital algorithm. However, at high concentrations, where most microspheres have more than 1 enzyme molecule bound to them, the numerical algorithm is no longer applicable, and thus in turn the average number of enzymes on a single microsphere is characterized by calculating the ratio of the average fluorescence intensity of the pores containing microspheres coupled with enzyme molecules to the fluorescence intensity of the single enzyme, this algorithm is also known as a simulation algorithm. In practical applications, 70% of the "on" spheres are used as thresholds, with digital algorithms being used below 70% and analog algorithms being used above 70%.

However, the smooth engagement of digital and analog algorithms at 70% or other thresholds depends on the perfect poisson distribution of the molecules on the microspheres. Poisson distribution assumes that all molecules and microspheres are identical, that the molecules bind randomly on the microspheres, that there is no bias, that the binding affinity remains unchanged at varying concentrations, and that the intensity detected is uniform. However, this is not possible in practice where various factors may lead to non-random distribution of the binding of the microspheres and molecules or non-uniformity of the detection signal, including, but not limited to, uniformity of enzyme and substrate concentrations, uniformity of pore volume, differences between instruments, non-uniformity of excitation light, non-uniformity of imaging quality, etc. Indeed, recent reports have demonstrated discontinuities between analog and digital algorithms.

In view of the above, it is desirable to provide a new concentration measurement method that overcomes the drawbacks of the prior art and provides measurement continuity over all concentration ranges.

Disclosure of Invention

In one aspect, the invention provides a method for determining the concentration of a target molecule in a sample using a detection microsphere, comprising the steps of: (a) Providing microwells, wherein in one portion of microwells a substrate and a detection microsphere are contained, and in another portion of microwells a substrate is contained but no detection microsphere is contained, at least one of the surfaces of the detection microsphere comprising a sandwich structure formed by a first ligand-target molecule-reporter molecule, the reporter molecule comprising a catalyst capable of catalyzing the emission of a first fluorescence by the substrate; (b) sealing the microwells to fluidly isolate each microwell from the other microwells; (c) Obtaining a microsphere image of at least a portion of the detection microspheres and counting the microwells containing the microspheres in the selected area to obtain a total number of microwells; (d) Acquiring N first fluorescent pictures corresponding to the first fluorescent light according to a preset time interval, wherein N is an integer greater than or equal to 2; (e) Aligning at least two first fluorescent pictures with the microsphere pictures, and acquiring the micropore brightness value of each micropore containing the microsphere in the selected area in each of the at least two first fluorescent pictures; (f) Calculating an enzyme catalysis efficiency value for each microwell containing microspheres based on the microwell brightness value, summing the enzyme catalysis efficiency values of all microwells containing microspheres, dividing by the total number of microwells, as a characteristic value; (g) obtaining a standard curve of characteristic value versus concentration; and (h) determining 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, wherein said providing a microwell in step (a) comprises providing a microwell located in a channel of a microfluidic device. In some embodiments, wherein step (a) comprises placing the microspheres into microwells in a channel of a microfluidic device such that each microwell has at most one microsphere.

In some embodiments, wherein in step (b), the sealing comprises sealing with oil.

In some embodiments, step (d) further comprises pre-processing the first fluorescent image to exclude abnormal light spots. In some embodiments, wherein the preprocessing comprises deep learning based target detection.

In some embodiments, wherein in step (c), the detection microsphere comprises a fluorescent dye that is excited to cause the detection microsphere to emit a second fluorescence to obtain the microsphere image, the second fluorescence being different from the first fluorescence.

In some embodiments, in step (c), the selected region is the entire region of the microsphere image.

In some embodiments, wherein in step (e), the microwell luminance value is the sum of the luminance values of the individual microwells displayed in 9 x 9 pixels or the average of the luminance values of the individual microwells displayed in 9 x 9 pixels.

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

In some embodiments, wherein in step (f), the enzyme catalytic efficiency value is calculated from one or more of an average rate of increase of the microwell intensity value, a fitted curve characteristic value, a microwell intensity value to peak time, a ratio of microwell intensity value to maximum fluorescence intensity value after a predetermined reaction time, and an average slope of the increase curve of the microwell intensity value.

In some embodiments, wherein in step (f), the enzyme catalytic efficiency value is calculated by:

(f1) For each microwell containing microspheres, taking an average of at least one difference or a plurality of differences between microwell luminance values of the at least two first fluorescent pictures, wherein

(f 1 a) when N is even, subtracting the micropore brightness value of the N/2 th first fluorescent picture from the micropore brightness value of the N/1 st first fluorescent picture to obtain a first difference value, subtracting the micropore brightness value of the (N/2-1) th first fluorescent picture from the micropore brightness value of the N/1 st first fluorescent picture to obtain a second difference value, and so on until the N/2 difference value between the micropore brightness value of the N/2+1 th first fluorescent picture and the micropore brightness value of the 1 st first fluorescent picture is obtained;

(f 1 b) when N is an odd number, subtracting the (n+1)/2 th micropore brightness value of the first fluorescent picture from the (n+1)/2 th micropore brightness value of the first fluorescent picture to obtain a first difference value, subtracting the (n+1)/2-1 th micropore brightness value of the first fluorescent picture from the (N-1) th micropore brightness value of the first fluorescent picture to obtain a second difference value, and so on until the (N-1)/2 difference value between the (n+1)/2+1 th micropore brightness value of the first fluorescent picture and the (N-1)/2 nd micropore brightness value of the 2 nd first fluorescent picture is obtained; or (b)

(f 1 c) when N is an odd number, subtracting the (N-1)/2 th micropore brightness value of the first fluorescent picture from the (N-1) th micropore brightness value of the first fluorescent picture to obtain a first difference value, subtracting the (N-1)/2-1 th micropore brightness value of the first fluorescent picture from the (N-2 th micropore brightness value of the first fluorescent picture to obtain a second difference value, and so on until the (N-1)/2+1 th micropore brightness value of the first fluorescent picture and the (N-1)/2 nd difference value of the (1 st) th micropore brightness value of the first fluorescent picture are obtained;

(f2) Calculating a brightness value increase rate, taking the brightness value increase rate as an enzyme catalysis efficiency value of the microwell, wherein:

(f 2 a) for the case of step (f 1 a), calculating the luminance value increase rate by dividing the at least one difference value or an average value of the plurality of difference values by the time elapsed from the N/2 th picture to the N-th picture;

(f 2 b) for the case of step (f 1 b), calculating the luminance value increase rate by dividing the at least one difference value or an average value of the plurality of difference values by the time elapsed from (n+1)/2 th picture to nth picture; or (b)

(f 2 c) for the case of step (f 1 c), calculating the luminance value increase rate by dividing the at least one difference value or the average value of the plurality of difference values by the time elapsed from (N-1)/2 th picture to (N-1) th picture.

(f1) Obtaining a predetermined maximum fluorescence brightness value for a single microwell comprising a microsphere with the target molecule and the enzyme;

(f2) For each microwell containing microspheres, obtaining at least one difference between microwell luminance values of the at least two first fluorescent pictures divided by the maximum fluorescent luminance value to obtain at least one luminance difference ratio, wherein

(f 2 a) when N is even, subtracting the micropore brightness value of the N/2 th first fluorescent picture from the micropore brightness value of the N/1 st first fluorescent picture to obtain a first difference value, subtracting the micropore brightness value of the (N/2-1) th first fluorescent picture from the micropore brightness value of the N/1 st first fluorescent picture to obtain a second difference value, and so on until the N/2 difference value between the micropore brightness value of the N/2+1 th first fluorescent picture and the micropore brightness value of the 1 st first fluorescent picture is obtained;

(f 2 b) when N is an odd number, subtracting the (n+1)/2 th micropore brightness value of the first fluorescent picture from the (n+1)/2 th micropore brightness value of the first fluorescent picture to obtain a first difference value, subtracting the (n+1)/2-1 th micropore brightness value of the first fluorescent picture from the (N-1) th micropore brightness value of the first fluorescent picture to obtain a second difference value, and so on until the (N-1)/2 difference value between the (n+1)/2+1 th micropore brightness value of the first fluorescent picture and the (N-1)/2 nd micropore brightness value of the 2 nd first fluorescent picture is obtained; or (b)

(f 2 c) when N is an odd number, subtracting the (N-1)/2 th micropore brightness value of the first fluorescent picture from the (N-1) th micropore brightness value of the first fluorescent picture to obtain a first difference value, subtracting the (N-1)/2-1 th micropore brightness value of the first fluorescent picture from the (N-2 th micropore brightness value of the first fluorescent picture to obtain a second difference value, and so on until the (N-1)/2+1 th micropore brightness value of the first fluorescent picture and the (N-1)/2 nd difference value of the (1 st) th micropore brightness value of the first fluorescent picture are obtained;

and

(f3) For each microwell containing microspheres, fitting a growth curve of the plurality of luminance difference ratios of the microwell by Langmuir equation i=ka/(1+a x), wherein I is the luminance difference ratio, x is the residual substrate content (characterized by the difference between the maximum fluorescent luminance value and the microwell luminance value at the corresponding time divided by the maximum fluorescent luminance value), k is the maximum theoretical reaction rate of the enzyme and the substrate divided by the predetermined time interval described in step (d), to calculate a characteristic parameter a, which is the enzyme catalytic efficiency value of the microwell.

In some embodiments, in step (a), the reporter molecule consists of a second ligand that specifically binds to a target molecule and the catalyst, the second ligand being different from the first ligand, and step (a) comprises: (i) providing a sample that may contain a target molecule; (ii) Adding detection microspheres, wherein the surfaces of the detection microspheres are modified with the first ligands which are specifically combined with target molecules; (iii) adding a reporter; (iv) adding a substrate; (v) introducing detection microspheres into the microwells; the execution sequence of the steps is as follows: i/ii/iii-iv-v or i/ii/iii/v-iv, wherein "/" means that the steps preceding and following thereof are interchangeable, and "-" means that the steps preceding and following thereof are performed in the order shown.

In some embodiments, in step (a), the reporter molecule consists of a first moiety and a second moiety independent of each other, the first moiety comprising a second ligand that specifically binds to the target molecule and a first affinity element, the second moiety comprising a second affinity element and the catalyst, the first affinity element and the second affinity element being capable of being linked together by an affinity interaction, the second ligand being different from the first ligand, and step (a) comprises: (i) providing a sample that may contain a target molecule; (ii) Adding detection microspheres, wherein the surfaces of the detection microspheres are modified with the first ligands which are specifically combined with target molecules; (iii) adding the first portion of a reporter; (iv) adding said second portion of a reporter; (v) adding a substrate; (vi) introducing detection microspheres into the microwells; the execution sequence of the steps is as follows: i/ii/iii/iv-v-vi or i/ii/iii/iv/vi-v, wherein "/" means that the steps preceding and following thereof are interchangeable and "-" means that the steps preceding and following thereof are performed in the order shown.

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, 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 a reporter; (v) adding a substrate; (vi) introducing detection microspheres into the microwells; the execution sequence of the steps is as follows: i/ii/iii/iv-v-vi or i/ii/iii/iv/vi-v, wherein "/" means that the steps preceding and following thereof are interchangeable and "-" means that the steps preceding and following thereof are performed in the order shown.

In some embodiments, in step (a), the reporter molecule consists of a first moiety and a second moiety independent of each other, the first moiety comprising a second ligand that specifically binds to the target molecule and a first affinity element, the second moiety comprising a second affinity element and the catalyst, the first affinity element and the second affinity element being capable of being linked together by an affinity, 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 being bound to the third affinity element via the fourth affinity element, the third affinity element and the fourth affinity element being capable of being linked together by an affinity, and step (a) comprises: (i) providing a sample that may contain a target molecule; (ii) Adding a detection microsphere, a first ligand coupled with a fourth affinity element, and adding a blocking agent when cross reaction is possible 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 the excessive blocking agent before proceeding to the next step; (iii) adding the first portion of a reporter; (iv) adding said second portion of a reporter; (v) adding a substrate; (vi) introducing detection microspheres into the microwells; the execution sequence of the steps is as follows: i/ii/iii/iv-v-vi or i/ii/iii/iv/vi-v, wherein "/" means that the steps preceding and following thereof are interchangeable and "-" means that the steps preceding and following thereof are performed in the order shown.

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

In some embodiments, the first ligand and the second ligand are different antibodies to the target molecule.

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.

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 the group consisting of: (a) biotin and streptavidin; and (b) biotin and avidin.

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

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

In some embodiments, wherein the target molecule is a protein or a nucleic acid.

In some embodiments, wherein the microsphere is a polymeric microsphere or a magnetic bead.

In some embodiments, wherein the method does not include separately counting the microspheres to which the target molecules are attached.

In some embodiments, wherein the target molecule comprises a plurality of types, the detection microsphere also comprises a corresponding plurality of types, and the reporter also comprises a corresponding plurality of types.

Another aspect of the invention provides a computer readable medium having stored thereon computer readable instructions which when executed perform any of the methods of the invention described above.

In yet another aspect, the invention provides an analytical device comprising a computer control system and a microfluidic device, wherein the computer control system comprises any one of the computer readable media described above.

In some embodiments, wherein the microfluidic device comprises a channel comprising a plurality of microwells, each microwell for receiving at most one microsphere.

In some embodiments, each microwell has a volume of about 1 angstrom to about 100 picoliters, e.g., about 1 angstrom to about 1 picoliter, about 1 angstrom to about 100 femtoliters, about 1 angstrom to about 1 femtoliter, or about 1 femtoliter to about 1 picoliter.

The method provided by the invention does not need to judge whether each microsphere is connected with a target molecule (namely 'activation' or 'on'), simplifies the complexity of the method, and simultaneously maintains high detection accuracy. In addition, the method provided by the invention has excellent continuity in all measured concentration ranges, and the data CV is below 20%, so that the problem of data discontinuity caused by respectively using an analog algorithm and a digital algorithm at high concentration and low concentration in the prior art is solved. In addition, for samples with different times of concentration, the characteristic value obtained by the algorithm is more differentiated in certain intervals, and the resolution ratio for the concentration of the samples is higher, so that the detection sensitivity is further improved.

Drawings

The invention will be described in more detail with reference to the accompanying drawings. It is noted that the illustrated embodiments are merely representative examples of the embodiments of the present invention, and that elements in the drawings are not drawn to scale such as actual dimensions, the number of actual elements may vary, the relative positional relationship of the actual elements is substantially consistent with the illustration, and some elements are not shown in order to more clearly illustrate the details of the exemplary embodiments. Where multiple embodiments exist, while one or more features described in the previous embodiments may also be applied to another embodiment, for brevity, the latter embodiment or embodiments will not be described in further detail as having described such features, unless otherwise indicated. Those skilled in the art will appreciate upon reading the present disclosure that one or more features illustrated in one drawing may be combined with one or more features in another drawing to construct one or more alternative embodiments not specifically illustrated in the drawings, which also form a part of the present disclosure.

FIG. 1 is a schematic flow chart of the method of the present invention.

FIG. 2 shows a fluorescent photograph of a microsphere obtained by exciting a fluorescent dye according to one embodiment of the present invention and a partial schematic view thereof.

Fig. 3 shows a 9 x 9 pixel map of a single microwell according to one embodiment of the invention.

FIG. 4 shows a schematic flow chart of a method for calculating an enzyme catalytic efficiency value from microwell luminance values, according to one embodiment of the invention.

FIG. 5 shows a schematic flow chart of a method for calculating an enzyme catalytic efficiency value from a microwell luminance value, according to another embodiment of the invention.

FIG. 6 shows a schematic flow chart of a method for calculating an enzyme catalytic efficiency value from microwell luminance values, in accordance with yet another embodiment of the invention.

FIG. 7 shows a plot of the ratio of the difference in fluorescence brightness values fitted according to the method shown in FIG. 6.

Detailed Description

Exemplary embodiments of the present invention are described in detail below with reference to the accompanying drawings. It is to be understood that the scope of the present invention is not limited to the disclosed embodiments, and that modifications and variations of the exemplary embodiments may be made by those skilled in the art in light of the present disclosure without undue effort and are intended to be included within the scope of the appended claims.

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

The method 100 begins at step 102 by providing microwells in which a substrate and a detection microsphere are contained in one portion of the microwells and the substrate is contained in another portion of the microwells but the detection microsphere is not contained, at least one of the detection microsphere surfaces comprising a sandwich structure formed by a first ligand-target molecule-reporter molecule, the reporter molecule comprising a catalyst capable of catalyzing the emission of a first fluorescence from the substrate.

In step 102, providing microwells may be accomplished by providing a microfluidic device comprising an inlet and an outlet and a channel in fluid communication with the inlet and the outlet. The channel may be provided with a plurality of microwells, e.g., millions to tens of thousands of microwells, on either side or bottom thereof, each microwell being sized to receive one or more microspheres, thereby spatially isolating the microspheres from each other. In the present invention, the substrate and the detection microsphere are contained in one portion of the microwells, while the substrate is contained in another portion of the microwells but the detection microsphere is not contained. Thus, some microwells do not contain microspheres, while other microwells contain one or more microspheres. In microwells containing microspheres, the microspheres may or may not be coupled with target molecules. The method of the present invention does not involve the separate counting of microspheres coupled with target molecules, as described in detail below. In other embodiments, for example, the volume of each microwell can be about 1 angstrom to about 100 picoliters, such as about 1 angstrom to about 1 picoliter, about 1 angstrom to about 100 femtoliters, about 1 angstrom to about 1 femtoliter, or about 1 femtoliter to about 1 picoliter. For example, by selecting the appropriate microsphere volume and pore size such that each pore can only accommodate one microsphere, some of the pores are free of microspheres, while others contain only one microsphere. The detection microsphere may be any microsphere commonly used in the art, such as a polymeric microsphere or a magnetic bead. Examples of polymeric microspheres can be found in composite microspheres in CN 111318238B. As an example, the detection microsphere has a fluorescent dye coupled or encoded thereon, such as a CY5 fluorescent dye.

In the present invention, in the microwells, 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 emission of a first fluorescence from a substrate. The first ligand is, for example, a ligand that specifically binds to the target molecule, e.g., an antibody directed against the target molecule, and the first ligand is coupled directly or indirectly to the surface of the detection microsphere. In the present invention the reporter molecule is capable of specifically binding to said target molecule and is directly or indirectly coupled to a catalyst capable of catalyzing the emission of a first fluorescence from a substrate. For example, the reporter molecule comprises a second ligand that specifically binds to the target molecule, e.g., an antibody against the target molecule that binds to a different epitope of the target molecule than the first ligand. Thus, a "sandwich" structure (or sandwich) is formed between the first ligand, the target molecule and the reporter molecule (second ligand), wherein the target molecule is sandwiched by the first ligand and the reporter molecule.

There are a number of ways to form the sandwich structure. In some embodiments, the reporter 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 a target molecule; (ii) Adding detection microspheres, wherein the surfaces of the detection microspheres are modified with the first ligands which are specifically combined with target molecules; (iii) adding a reporter; (iv) adding a substrate; (v) introducing the detection microspheres into the microwells. For the above steps, the order of execution may be i/ii/iii-iv-v or i/ii/iii/v-iv, where "/" means that the steps preceding and following are interchangeable and "-" means that the steps preceding and following are executed in the order shown. For example, in the order i/ii/iii-iv-v, the order of steps (i), (ii) and (iii) may be any reversed, and steps (iv) and (v) may be performed sequentially after the three steps are completed.

In other embodiments, the reporter molecule is composed of a first moiety and a second moiety that are independent of each other, the first moiety comprising a second ligand that specifically binds to the target molecule and a first affinity element, the second moiety comprising a second affinity element and the catalyst, the first affinity element and the second affinity element being capable of being linked together by affinity, the second ligand being different from the first ligand, step 102 may comprise: (i) providing a sample that may contain a target molecule; (ii) Adding detection microspheres, wherein the surfaces of the detection microspheres are modified with the first ligands which are specifically combined with target molecules; (iii) adding the first portion of a reporter; (iv) adding said second portion of a reporter; (v) adding a substrate; (vi) introducing detection microspheres into the microwells. For the above steps, the order of execution may be i/ii/iii/iv-v-vi or i/ii/iii/iv/vi-v, where "/" means that the steps before and after are interchangeable, and "-" means that the steps before and after are executed in the order shown.

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, the first ligand is coupled with a second affinity element, and the first ligand binds to the first affinity element through the second affinity element, step 102 may comprise: (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 a reporter; (v) adding a substrate; (vi) introducing detection microspheres into the microwells. For the above steps, the execution sequence may be: i/ii/iii/iv-v-vi or i/ii/iii/iv/vi-v, wherein "/" means that the steps preceding and following thereof are interchangeable and "-" means that the steps preceding and following thereof are performed in the order shown.

In other embodiments, the reporter molecule is composed of a first portion and a second portion that are independent of each other, the first portion including a second ligand that specifically binds to the target molecule and a first affinity element, the second portion including a second affinity element and the catalyst, the first affinity element and the second affinity element being capable of being linked together by affinity, 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 being bound to the third affinity element via the fourth affinity element, the third affinity element and the fourth affinity element being capable of being linked together by affinity, step 102 may include: (i) providing a sample that may contain a target molecule; (ii) Adding a detection microsphere, a first ligand coupled with a fourth affinity element, and adding a blocking agent when cross reaction is possible 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 the excessive blocking agent before proceeding to the next step; (iii) adding the first portion of a reporter; (iv) adding said second portion of a reporter; (v) adding a substrate; (vi) introducing detection microspheres into the microwells. For the above steps, the execution sequence may be: i/ii/iii/iv-v-vi or i/ii/iii/iv/vi-v, wherein "/" means that the steps preceding and following thereof are interchangeable and "-" means that the steps preceding and following thereof are performed in the order shown.

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) through (v) or steps (i) through (vi). In some embodiments, one or more washing steps may be performed between steps (i) to (v) or two of steps (i) to (vi). The washing step may be performed by introducing a buffer (e.g., PBS or physiological saline) compatible with the sample to wash the microspheres and/or microwells to remove non-specifically bound or unbound components of the ligand coupled to the microspheres and/or non-specifically bound or unbound reporter molecules, so as to eliminate as much as possible interference of the assay by components other than non-specifically bound or target molecules. For example, in the case of using magnetic beads, the magnetic beads (and thus the target molecules that specifically bind to the first ligands on the magnetic beads) may be immobilized by applying a magnetic field after the magnetic beads are mixed with the sample, and components of the sample that do not specifically bind to the ligands on the magnetic beads (e.g., non-target molecule proteins in plasma) may be removed by introducing PBS buffer.

In any of the above embodiments, the first ligand and the second ligand may be different antibodies to the target molecule. For example, where the target molecule is an IL-15 molecule, the first ligand is a first antibody directed against IL-15 and the second ligand is a second antibody directed against IL-15, the first and second antibodies binding different epitopes of IL-15.

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 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 with the second affinity element, and the combination of the third affinity element with the fourth affinity element, are independently selected from the group consisting of: (a) biotin and streptavidin; and (b) biotin and avidin.

In some embodiments, the blocking agent is an avidin blocking agent, such as biotin-conjugated Bovine Serum Albumin (BSA) or polyethylene glycol (PEG). In some embodiments, the blocking agent is a biotin blocking agent, such as BSA or PEG coupled to avidin or streptavidin.

In step 102, the catalyst is beta-galactosidase and the substrate is resorufin-beta-D-galactopyranoside (CAS number 95079-19-9). As previously described, other suitable catalyst and substrate pairs include catalysts that are beta-galactosidase, which is fluorescein-di-beta-D-galactopyranoside (FDG, CAS number: 17817-20-8) or catalysts that are beta-galactosidase, which is 4-methylumbelliferone-beta-D-galactoside (CAS number: 6160-78-7), and the like.

In the presence of the target molecule, the catalyst to which the reporter molecule is attached catalyzes a substrate to fluoresce. Within a certain range, the greater the concentration of the attached reporter molecule (i.e., the greater the concentration of the target molecule), the more substrate is catalyzed and the greater the intensity and/or brightness of the fluorescence. When the reporter reaches the saturation concentration, the fluorescent brightness of the microsphere reaches a maximum value in the presence of an excess of substrate.

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

In the method 100, step 106 includes obtaining a microsphere image of at least a portion of the detected microspheres and counting the microwells containing the microspheres in the selected area to obtain a total number of microwells. In one embodiment, the detection microsphere is encoded or coupled with a fluorescent dye that can be excited to emit fluorescence, and a fluorescent signal can be acquired by, for example, a fluorescent camera to form a fluorescent image that is used to locate the fluorescent microsphere, for example, exciting the fluorescent dye (e.g., CY5 fluorescent dye) using a laser light source to obtain a fluorescent image of the microsphere, and locating each microwell containing the microsphere based on the fluorescent image.

In another embodiment, the detection microsphere is not encoded or coupled with a fluorescent dye, the microsphere is illuminated with white light, a picture of the microsphere is obtained by a conventional camera, and each microwell containing the microsphere is located based on the picture of the microsphere.

In some embodiments, the CY5 fluorescence image may be analyzed for its contour by gaussian blur processing and the coordinate position of each microwell obtained. In some embodiments, the processing of CY5 fluorescence pictures may be implemented by computer software, such as OpenCV 2.0 and above. For convenience of description herein, in the case of detecting microsphere coupling or encoding fluorescent dye, the fluorescence emitted by excitation thereof is referred to as second fluorescence, while the fluorescence emitted by the reporter catalytic substrate is referred to as first fluorescence, which is different from the first fluorescence.

Part a of fig. 2 shows a CY5 fluorescence picture obtained by the above method, which covers 80% to 90% of all microwells. In other embodiments, the picture may be made to cover 100% of all microwells. In other embodiments, the picture may be made to cover less than 80% of all microwells. Part B of fig. 2 is a partial enlarged view of the dotted line part in part a of fig. 2, and one white bright spot indicates the presence of a microsphere (i.e., corresponding microwell, the same applies below), and the position where no bright spot is shown indicates the absence of a microsphere in the corresponding microwell. Part C of fig. 2 is a further enlarged partial view of part B of fig. 2.

In the present invention, microwells are counted using software, regardless of whether the microspheres in the microwells are coupled with target molecules. In some embodiments, microwells containing microspheres are counted for the entire area of a microsphere image (e.g., microsphere fluorescence image). In other embodiments, microwells comprising microspheres are counted for 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the area of the microsphere image.

In step 108, N first fluorescence pictures 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. The time interval may be determined based on the typical reaction rates of the catalyst and substrate, with slower reaction rates generally requiring longer times and faster reaction rates requiring shorter times. In some embodiments, it is preferred to use a catalyst that is more catalytically efficient, thus shortening the overall testing process. In some embodiments, the predetermined time interval may 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, such as 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 invention, the number of the first fluorescent pictures can be reasonably determined according to the needs. A pre-experiment may be performed to determine the number of pictures that best meets the requirements. The number of pictures is typically considered in connection with the predetermined time interval, and the number of fluorescent pictures is also designed based on the fluorescence growth curve corresponding to the first fluorescence. In some embodiments, the present invention obtains 2 to 1000 first fluorescent pictures, 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 less first fluorescent pictures. In the embodiment of the present invention, the number of the acquired first pictures may be odd or even, but is preferably even. In a specific embodiment, 10 first fluorescence pictures are obtained at 1 minute intervals within 10 minutes. In some embodiments, step 108 may further include pre-processing the first fluorescent image to exclude abnormal light spots, such as abnormal light spots due to bubbles, impurities, and the like. The preprocessing may include deep learning based target detection.

In some embodiments, the order of steps 106 and 108 may be reversed. For example, after the method proceeds to step 104, step 108 is first performed and step 106 is performed before step 110 is started.

In step 110, at least two first fluorescent images are aligned with the microsphere images, and a microwell brightness value of each microwell containing the microsphere in the selected area in each of the at least two first fluorescent images is obtained. Those skilled in the art will appreciate that the microwell intensity obtained from the microwells is formed by fluorescence emitted by all catalyzed substrates together within the single microwell in which the microsphere resides, and thus microwell intensity values are also referred to herein as fluorescence intensity values or microwell fluorescence intensity values, and these terms are used interchangeably herein.

Fig. 3 is a maximum pixel magnification of a single microwell (bright spot) in the first fluorescent image. In fig. 3, a single microwell is shown in 9×9 pixels. It is contemplated that using cameras of different resolutions may allow a single microwell to be displayed with higher or lower pixels. In one embodiment of the present invention, for a single microwell, the average luminance value of 9×9 pixels is taken as its microwell luminance value. For example, for a single microwell shown in fig. 3, the sum of the luminance values of 9×9 pixels is divided by 81 to obtain the luminance average value of the single microwell. In other embodiments of the present invention, for a single microwell, the sum of the luminance values of 9×9 pixels is taken as its microwell luminance value. In a preferred embodiment of the invention, the selected area covers the entire area of the picture. In other embodiments, the selected region may occupy at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more of the entire region of the picture. In other embodiments, the selected region may occupy less than 50%, less than 40%, less than 30% or less of the entire region of the picture.

In one embodiment, in step 110, it is noted that the microsphere image and at least two first fluorescent images are aligned such that the analysis area in the first fluorescent image is the same as the analysis area of the microsphere image. After step 110, each microwell obtains a corresponding plurality of microwell luminance values, each microwell luminance value corresponding to one first fluorescent image, e.g., each microwell location may have 10 microwell luminance values. For some of the microwells, each microwell luminance value is the same or substantially the same, and for other microwells, each microwell luminance value is different and increases over time. The magnitudes of the luminance values of the microwells that increase over time are the same or different for different microwells.

Subsequently, in step 112, for each microsphere-containing microwell, an enzyme catalytic efficiency value is calculated based on the microwell brightness value, and the enzyme catalytic efficiency values of all microsphere-containing microwells are summed, divided by the total number of microwells, as a characteristic value.

In step 114, a standard curve of the characteristic value versus concentration is obtained. The standard curve may be previously established with known concentrations of the target molecule or other molecules according to the methods described in steps 102 to 112.

After the standard curve is obtained, step 116 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 is at an extremely low level, e.g., 0.1 zM to 100 pM, e.g., 1 zM to 100 fM, or even lower, in the sample.

In step 112, various methods for calculating the enzyme catalytic efficiency value according to the microwell brightness value can be used, for example, calculation can be performed according to the average increase rate of the microwell brightness value, the characteristic value of the fitted curve, the time for reaching the peak of the microwell brightness value, the ratio of the microwell brightness value to the maximum fluorescence brightness value after the predetermined reaction time, the slope of the average increase curve of the microwell brightness value, and the like. In some embodiments, the calculation method of the peak reaching time of the micropore brightness value is that a relation curve of the micropore brightness value and time is drawn, the increasing trend of the micropore brightness value along with time is obtained, a specific percentage of the micropore brightness value actually reaching the maximum fluorescence brightness value can be defined as a peak reaching time point, or according to a fitting curve, a specific percentage of the micropore brightness value reaching the maximum fluorescence brightness value is defined as a peak reaching time point, and in some embodiments, the percentage can be 90% of the maximum fluorescence brightness value. And obtaining the ratio of the micropore brightness value to the maximum fluorescence brightness value after the preset reaction time, namely the preset reaction time. The average growth curve slope of the brightness of the micro-holes is the average growth slope used, that is, the average growth slope in the preset interval time, and may be the average growth slope in any interval time. Two different calculation methods of the average growth rate of the microporous luminance value and the fitting curve characteristic value are shown in detail below, and those skilled in the art will appreciate that more calculation methods can be devised based on the present disclosure.

In some embodiments, for each microwell containing microspheres, taking an average of at least one difference or a plurality of differences between microwell luminance values of the at least two first fluorescent pictures, and calculating a rate of increase in luminance value based on the average of the at least one difference or the plurality of differences, the rate of increase being taken as an enzyme catalytic efficiency value for that microwell.

For example, as shown in FIG. 4, a method 200 of calculating an enzyme catalytic efficiency value is shown, where N is an even number. The method 200 begins at step 210 with subtracting the microwell luminance value of the nth first fluorescent picture from the microwell luminance value of the nth/2 th first fluorescent picture to obtain a first difference value, subtracting the microwell luminance value of the (N/2-1) th first fluorescent picture from the microwell luminance value of the nth-1 th first fluorescent picture to obtain a second difference value, and so on until the nth/2+1 th difference value between the microwell luminance value of the nth/2+1 th first fluorescent picture and the microwell luminance value of the 1 st first fluorescent picture is obtained.

For example, when 10 (n=10) first fluorescent pictures are acquired in step 108, in step 210, the microwell luminance values of the 10 th, 9 th, 8 th, 7 th and 6 th pictures are subtracted from the microwell luminance values of the 5 th, 4 th, 3 th, 2 nd and 1 th pictures, respectively, to obtain 5 microwell luminance differences. The pictures are numbered according to the time sequence of collection. In other embodiments, step 210 may be performed using a greater or lesser number of first fluorescence pictures. In some cases, step 210 may be performed using only two first fluorescence pictures to obtain a difference.

In step 220, the first difference, the second difference, …, the N/2 difference are summed and divided by N/2 to obtain a difference average. In the example of n=10 described above, the obtained 5 difference sums are divided by 5 to obtain a difference average value. For another example, at n=20, 10 fluorescence luminance differences will be obtained in step 210, and an average of the 10 fluorescence luminance differences will be obtained in step 220.

Finally, in step 230, the average value of the difference is divided by the time elapsed from the nth/2 th picture to the nth picture, and the increase rate of the brightness value is calculated and is used as the enzyme catalytic efficiency value of the microwell. For example, in the above-described example of n=10, the average value of the fluorescence luminance difference values obtained in step 220 is divided by the time elapsed from the 5 th (n=10) th picture to the 10 th (n=10) th first fluorescence picture, and in the case where the predetermined interval time is 60 seconds, for example, the elapsed time is set to 5 minutes or 300 seconds. In some embodiments, the rate of increase of the luminance value may be in seconds ^-1 Or divide into ^-1 。

The standard curve obtained by the method of step 100 was performed with the enzyme catalytic efficiency values of the microsphere positions calculated by the method 200, and the target molecule concentrations in a plurality of experimental samples were measured. The procedure was repeated 2 or 3 times, and the characteristic values and concentrations are shown in Table 1 below.

TABLE 1 determination of target molecule concentration for test samples

。

As shown in FIG. 5, a method 200' of calculating an enzyme catalytic efficiency value is shown, where N is an odd number. The method 200 'begins at step 210', for each microwell containing microspheres, subtracting the microwell luminance value of the (n+1)/2 th first fluorescent picture from the microwell luminance value of the nth first fluorescent picture to obtain a first difference value, subtracting the microwell luminance value of the (n+1)/2-1 th first fluorescent picture from the microwell luminance value of the nth-1 th first fluorescent picture to obtain a second difference value, and so on until the (N-1)/2 difference value between the microwell luminance value of the (n+1)/2+1 th first fluorescent picture and the microwell luminance value of the 2 nd first fluorescent picture is obtained.

For example, when 11 (n=11) first fluorescent pictures are acquired in step 108, in step 210', the microwell luminance values of the 11 th, 10, 9, 8, and 7 th pictures are subtracted from the microwell luminance values of the 6 th, 5 th, 4 th, 3 th, and 2 nd pictures, respectively, for each microsphere position, to obtain 5 fluorescent luminance differences. The pictures are numbered according to the time sequence of collection.

Thereafter, in step 220', the first difference, the second difference, …, the (N-1)/2 difference is summed and divided by (N-1)/2 to obtain a difference average. In the above-described example of n=11, the obtained 5 difference sums are divided by 5 to obtain a difference average value.

Finally, in step 230', the increase rate of the brightness value is calculated by dividing the average value by the time elapsed from the (n+1)/2 th picture to the nth picture, and the increase rate is used as the enzyme catalytic efficiency value of the microwell.

In other embodiments, when N is an odd number, the N-th picture may be discarded, and the luminance value increase rate may be calculated from the N-1 th picture as the enzyme catalytic efficiency value of the microwell. And will not be described in detail herein.

In other embodiments, calculating an enzyme catalytic efficiency value based on the microwell brightness value for each microwell comprising microspheres may be performed by the method 300. As shown in FIG. 6, step 300 begins with step 310 in which a pre-determined maximum fluorescence intensity value for a single microwell containing microspheres with target molecules and the enzyme is obtained. As previously described, the maximum fluorescence intensity value can be obtained when the target and reporter molecules bound by the microspheres in the microwells reach saturation concentration, which can be obtained by pre-experiments or by empirical values. In one embodiment, the predetermined maximum fluorescence brightness value may be an average of a plurality of microwell maximum fluorescence brightness values. The maximum fluorescence intensity value of an individual microwell depends at least in part on the type and concentration of fluorescent molecules generated after the substrate is catalyzed. In one embodiment, the measured maximum fluorescence brightness value is 6×10e5. In other embodiments, the measured maximum fluorescence brightness may be higher or lower, for example between 1×10e3 and 1×10e7.

Subsequently, in step 320, for each microwell containing microspheres, at least one difference between the microwell luminance values of the at least two first fluorescent pictures is obtained, divided by the maximum fluorescent luminance value to obtain at least one luminance difference ratio. In the present invention, the plurality of microwell luminance values is derived from the microwell luminance values corresponding to each of the first fluorescent pictures determined in step 110. Therefore, the number of the plurality of micro-hole brightness values is less than or equal to the number N of acquired first fluorescent pictures. In some embodiments, the plurality of microwell luminance values is the N microwell luminance values. Thus, in step 320, no more than (N-1) luminance difference ratio values are obtained. In some embodiments, the luminance difference ratio is less than 1, e.g., 0.1 to 1, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, or any value therebetween.

Next, in step 330, for each microwell containing microspheres, fitting a growth curve of the plurality of luminance difference ratios of the microwell by Langmuir equation i=kχ/(1+aχ), wherein I is the luminance difference ratio, x is the residual substrate content (characterized by the difference between the maximum fluorescent luminance value and the microwell luminance value at the corresponding time divided by the maximum fluorescent luminance value), and k is the maximum theoretical reaction rate of the enzyme and the substrate divided by the predetermined time interval in step 108, to calculate a characteristic parameter a, which is the enzyme catalytic efficiency value of the microwell. In some embodiments, the coefficient k may be a non-zero coefficient, step 320 obtaining 9 luminance difference ratio values altogether, and in step 330, fitting a growth curve of the luminance difference ratio values by Langmuir equation with the 9 luminance difference ratio values and their corresponding residual substrate contents (characterized by the difference between the maximum fluorescence luminance value and the microwell luminance value at the corresponding time divided by the maximum fluorescence luminance value). In one embodiment, the coefficient k is set to 1 and the growth curve fitted by the method 300 is shown in FIG. 7. FIG. 7 shows the luminance difference ratio of individual microwells with increasing residual substrate content for different individual microwells. In fig. 7, the abscissa indicates the content of the single microporous residual substrate; the ordinate indicates the ratio of the luminance differences of the individual microwells. In addition, the maximum theoretical reaction rate of the enzyme and the substrate may be obtained beforehand by experience or actual measurement.

The standard curve obtained by the method of step 100 was performed with the enzyme catalytic efficiency values of the microsphere positions calculated by the method 300, and the concentration of the target molecule in one experimental sample was determined. The procedure was repeated 3 times with the characteristic values shown in Table 2 below and a concentration of 12 pg/mL.

TABLE 2 determination of target molecule concentration for test samples

In some embodiments, the methods of the invention can simultaneously analyze a sample for two or more different types of target molecules, such as IL-12 and IL-10, that may be present. In such embodiments, the method uses different types of microspheres and different types of first fluorescent dyes corresponding to different types of target molecules, and different types of reporter molecules and different types of second fluorescent dyes, identifying each microsphere and reporter via different fluorescent channels. Two different methods of preparing fluorescent encoded microspheres can be seen in CN 111218498A.

Another aspect of the invention provides a computer readable medium having stored thereon computer readable instructions which when executed perform any of the methods of the invention described above. The computer readable medium may include a removable medium as a package 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 disc)), or a semiconductor memory.

Another aspect of the invention provides an analytical device comprising a computer control system and a microfluidic device, wherein the computer control system comprises a computer readable medium of the invention.

In one embodiment, the microfluidic device comprises a channel comprising a plurality of microwells, each microwell for receiving at most one microsphere. In some embodiments, each microwell has a volume of 1 femtoliter to 1 picoliter. The analysis device is configured to perform any of the methods described herein.

The foregoing is a representative example of embodiments of the present invention and is provided for illustrative purposes only. The present invention contemplates that one or more features used in one embodiment can be added to another embodiment to form an improved or alternative embodiment without departing from the purpose of the embodiment. Likewise, one or more features used in one embodiment may be omitted or substituted without departing from the purpose of the embodiment to form a substituted or simplified embodiment. Furthermore, one or more features used in one embodiment may be combined with one or more features of another embodiment to form improved or alternative embodiments without departing from the purpose of the embodiments. The present invention is intended to include all such improved, alternative, and simplified embodiments.

Claims

1. A method for determining the concentration of a target molecule in a sample using a detection microsphere, comprising the steps of:

(a) Providing microwells, wherein in one portion of microwells a substrate and a detection microsphere are contained, and in another portion of microwells a substrate is contained but no detection microsphere is contained, at least one of the surfaces of the detection microsphere comprising a sandwich structure formed by a first ligand-target molecule-reporter molecule, the reporter molecule comprising a catalyst capable of catalyzing the emission of a first fluorescence by the substrate;

(b) Sealing the microwells to fluidly isolate each microwell from the other microwells;

(c) Obtaining a microsphere image of at least a portion of the detection microspheres and counting the microwells containing the microspheres in the selected area to obtain a total number of microwells;

(d) Acquiring N first fluorescent pictures corresponding to the first fluorescent light according to a preset time interval, wherein N is an integer greater than or equal to 2;

(e) Aligning at least two first fluorescent pictures with the microsphere pictures, and acquiring the micropore brightness value of each micropore containing the microsphere in the selected area in each of the at least two first fluorescent pictures;

(f) Calculating an enzyme catalysis efficiency value for each microwell containing microspheres based on the microwell brightness value, summing the enzyme catalysis efficiency values of all microwells containing microspheres, dividing by the total number of microwells, as a characteristic value;

(g) Obtaining a standard curve of the characteristic value to the concentration; and

(h) And determining the concentration of the target molecules in the sample according to the standard curve and the characteristic value of the target molecules.

2. The method of claim 1, wherein the providing of the microwells in step (a) comprises providing microwells located in channels of a microfluidic device.

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

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

5. The method of claim 4, wherein the preprocessing comprises deep learning based target detection.

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

7. The method of claim 1, wherein in step (c) the selected area is the full area of the microsphere image.

8. The method of claim 1, wherein in step (e), the microwell luminance value is a sum of luminance values of individual microwells displayed in 9 x 9 pixels or a luminance average of individual microwells displayed in 9 x 9 pixels.

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

10. The method of claim 1, wherein in step (f), the enzyme catalytic efficiency value is calculated from one or more of an average rate of increase of the microwell intensity value, a fitted curve characteristic value, a microwell intensity value to peak time, a ratio of microwell intensity value to maximum fluorescent intensity value after a predetermined reaction time, and an average slope of the increase curve of microwell intensity value.

11. The method of claim 10, wherein in step (f) the enzyme catalytic efficiency value is calculated by:

12. The method of claim 10, wherein in step (f) the enzyme catalytic efficiency value is calculated by:

(f1) Obtaining a predetermined maximum fluorescence brightness value for a single microwell comprising a microsphere with a target molecule and the enzyme;

(f 2 c) when N is an odd number, subtracting the (N-1)/2 th micropore brightness value of the first fluorescent picture from the (N-1) th micropore brightness value of the first fluorescent picture to obtain a first difference value, subtracting the (N-1)/2-1 th micropore brightness value of the first fluorescent picture from the (N-2 th micropore brightness value of the first fluorescent picture to obtain a second difference value, and so on until the (N-1)/2+1 th micropore brightness value of the first fluorescent picture and the (N-1)/2 nd difference value of the (1 st) th micropore brightness value of the first fluorescent picture are obtained; and

(f3) Fitting a growth curve of the luminance difference ratios of the microwells to each microwell containing microspheres by Langmuir equation i=ka x/(1+a x), wherein I is the luminance difference ratio, x is the residual substrate content and is characterized by dividing the difference between the maximum fluorescent luminance value and the microwell luminance value at the corresponding moment by the maximum fluorescent luminance value, k is the maximum theoretical reaction rate of the enzyme and the substrate divided by the predetermined time interval described in step (d), to calculate a characteristic parameter a, which is the enzyme catalytic efficiency value of the microwell.

13. The method of claim 1, wherein in step (a), the reporter molecule consists of a second ligand that specifically binds to a target molecule and the catalyst, the second ligand being different from the first ligand, and step (a) comprises:

(i) Providing a sample that may contain a target molecule;

(ii) Adding detection microspheres, wherein the surfaces of the detection microspheres are modified with the first ligands which are specifically combined with target molecules;

(iii) Adding a reporter molecule;

(iv) Adding a substrate;

(v) Introducing the detection microsphere into the microwell;

the execution sequence of the steps is as follows: i/ii/iii-iv-v or i/ii/iii/v-iv, wherein "/" means that the steps preceding and following thereof are interchangeable, and "-" means that the steps preceding and following thereof are performed in the order shown.

14. The method of claim 1, wherein in step (a) the reporter molecule consists of a first moiety and a second moiety independent of each other, the first moiety comprising a second ligand that specifically binds to the target molecule and a first affinity element, the second moiety comprising a second affinity element and the catalyst, the first affinity element and the second affinity element being capable of being linked together by an affinity interaction, the second ligand being different from the first ligand, and step (a) comprises:

(i) Providing a sample that may contain a target molecule;

(iii) Adding the first portion of the reporter;

(iv) Adding said second portion of the reporter;

(v) Adding a substrate;

(vi) Introducing the detection microsphere into the microwell;

the execution sequence of the steps is as follows: i/ii/iii/iv-v-vi or i/ii/iii/iv/vi-v, wherein "/" means that the steps preceding and following thereof are interchangeable and "-" means that the steps preceding and following thereof are performed in the order shown.

15. The method of 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 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, 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 a reporter molecule;

(v) Adding a substrate;

(vi) Introducing the detection microsphere into the microwell;

16. The method of claim 1, wherein in step (a), the reporter molecule consists of a first moiety and a second moiety independent of each other, the first moiety comprising a second ligand that specifically binds to the target molecule and a first affinity element, the second moiety comprising a second affinity element and the catalyst, the first affinity element and the second affinity element being capable of being linked together by an affinity, 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 being bound to the third affinity element by the fourth affinity element, the third affinity element and the fourth affinity element being capable of being linked together by an affinity, and step (a) comprises:

(i) Providing a sample that may contain a target molecule;

(ii) Adding a detection microsphere, a first ligand coupled with a fourth affinity element, and adding a blocking agent when cross reaction is possible 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 the excessive blocking agent before proceeding to the next step;

(iii) Adding the first portion of the reporter;

(iv) Adding said second portion of the reporter;

(v) Adding a substrate;

(vi) Introducing the detection microsphere into the microwell;

17. The method of claim 13, wherein one or more washing steps are included between any two, three, four or all of steps (i) through (v).

18. The method of any one of claims 14, 15 or 16, wherein any two, three, four or all of steps (i) to (vi) comprise one or more washing steps therebetween.

19. The method of any one of claims 13, 14, 15 or 16, wherein the first ligand and the second ligand are different antibodies to the target molecule.

20. The method according to claim 14 or 15, 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 alternatively

(b) The first affinity element is one of biotin and avidin and the second affinity element is the other of biotin and avidin.

21. The method of claim 16, 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 the group consisting of:

(a) Biotin and streptavidin; and

(b) Biotin and avidin.

22. The method of any one of claims 1, 13, 14, 15 or 16, wherein the catalyst is β -galactosidase and the substrate is resorufin- β -D-galactopyranoside.

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

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

25. The method of claim 1, wherein the detection microsphere is a polymeric microsphere or a magnetic bead.

26. The method of claim 1, wherein the method does not comprise separately counting the detection microspheres attached to the target molecules.

27. The method of claim 1, wherein the target molecule comprises a plurality of types, the detection microsphere also comprises a corresponding plurality of types, and the reporter also comprises a corresponding plurality of types.

28. A computer readable medium having stored thereon computer readable instructions which when executed perform the method of any of claims 1 to 27.

29. An analysis device comprising a computer control system and a microfluidic device, wherein the computer control system comprises the computer readable medium of claim 28.

30. The analytical device of claim 29, wherein the microfluidic device comprises a channel comprising a plurality of microwells, each microwell for receiving at most one microsphere.

31. The analytical device of claim 30, wherein the volume of each microwell is from 1 angstrom to 100 picoliters.