Method for detecting non-protein small molecules and application

Abstract

The invention belongs to the technical field of detection, and particularly relates to a method for detecting non-protein small molecules and application, the method comprises the following steps: providing functional microspheres, the functional microspheres comprise microspheres, molecular arms and non-protein small molecules, the non-protein small molecules are combined to the microspheres through the molecular arms, and optionally, the non-protein small molecules are combined to the microspheres through the molecular arms; the molecular arm is connected with the microsphere through an avidin ligand; performing a competitive reaction on an object to be detected, the non-protein small molecules combined on the functionalized microspheres and a quantum dot labeled detection antibody, and forming a microsphere array by the functionalized microspheres before or after the competitive reaction; and detecting quantum dot signals in the microsphere array, and obtaining the concentration and / or content of the non-protein small molecules in the to-be-detected object based on a detection result. The method provided by the invention is beneficial to reducing the detection limit and / or shortening the detection time.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of detection, and particularly relates to a method for detecting non-protein small molecules and application. BACKGROUND

[0002] Small molecule new pollutants represented by antibiotics pose a threat to ecology and human health due to biological toxicity, environmental persistence and biological accumulation, and therefore it is necessary to detect their presence in water and other environments for targeted treatment. However, because small molecule new pollutants exist in trace amounts (ng / L) in the environment, and their migration and transformation are complex, detection is difficult.

[0003] Existing detection technologies have obvious limitations when detecting trace small molecule substances: online solid-phase extraction-liquid chromatography tandem mass spectrometry is accurate, but it takes a long time to analyze a single sample and the cost is high, making it difficult to support high-throughput research; electrochemical sensors are portable and have fast response, but the signal specificity is insufficient due to the overlap of the oxidation and reduction potentials of substances in complex matrices, making it impossible to accurately trace the source; Raman spectroscopy can theoretically detect multiple targets simultaneously, but the intrinsic Raman cross-section of trace substances is weak, the detection limit is much higher than required after being interfered by impurities, and the single-point focusing mode is complex to operate, making it unsuitable for high-throughput scenarios; enzyme-linked immunosorbent assay (ELISA) has high throughput and low cost, but the detection limit cannot meet the needs of trace detection, and it takes a long time, and the ELISA method relies on the colorimetric absorbance of HRP enzyme and the calibration deviation caused by the fluctuation of enzyme activity between batches, making it limited in high-throughput trace antibiotic detection scenarios. Therefore, existing detection technologies cannot meet the needs of high-throughput detection of trace small molecule substances in multiple scenarios. SUMMARY

[0004] Embodiments of the present application provide a method for detecting non-protein small molecules and application, which can at least partially solve at least one of the above-mentioned or other shortcomings in the prior art.

[0005] The first aspect of the present application provides a method for detecting non-protein small molecules, comprising: providing functionalized microspheres, the functionalized microspheres comprising microspheres, molecular arms, and non-protein small molecules, wherein the non-protein small molecules are bound to the microspheres through the molecular arms, and optionally, the molecular arms and the microspheres are connected through an avidin-based ligand, making the analyte, the non-protein small molecules bound on the functionalized microspheres, and quantum dot-labeled detection antibodies compete in a reaction, wherein the functionalized microspheres form a microsphere array before or after the competition reaction; detecting the quantum dot signal in the microsphere array, and obtaining the concentration of non-protein small molecules in the analyte based on the detection result.

[0006] The application at least includes the following beneficial effects: in the method of the application, non-protein small molecules are combined to microspheres through molecular arms, which is conducive to improving the detection accuracy; the application connects non-protein small molecules to microspheres, which can improve the reaction rate; the application makes the non-protein small molecules in the test substance and the non-protein small molecules combined on the functionalized microspheres compete with the quantum dot labeled detection antibody, and the concentration of non-protein small molecules in the test substance is indirectly obtained by detecting the quantum dot signal of the functionalized microspheres, which avoids the limitation of small molecular weight and few epitopes of non-protein small molecules, and is conducive to improving the detection accuracy; the method of the application makes the functionalized microspheres form a microsphere array, and the concentration of non-protein small molecules in the test substance is ultimately obtained by detecting the quantum dot signal in the microsphere array, which is conducive to reducing the detection limit and improving the detection accuracy, and further realizes the accurate detection of trace small molecule substances.

[0007] In some embodiments, the providing functionalized microspheres comprises combining the microspheres with the non-protein small molecules through the avidin-like ligand and the molecular arm, the molecular arm comprising a biotin-like substance and a linear carbon chain connected thereto, thereby facilitating the stability of the combination of the microspheres and the non-protein small molecules. The avidin-like ligand is connected with one or more molecular arms, preferably 1, 2, 3 or 4 molecular arms, thereby facilitating the stability of the reaction.

[0008] In some embodiments, the providing functionalized microspheres comprises: combining the microspheres with the avidin-like ligand; combining the molecular arm with the non-protein small molecule; connecting the avidin-like ligand with the molecular arm.

[0009] In some embodiments, the avidin-like ligand comprises one or more of avidin, streptavidin, neutravidin. Thereby, the stability of the combination of the microspheres and the non-protein small molecules is facilitated.

[0010] In some embodiments, the linear carbon chain comprises one or more of C3-C 20 alkane, polyethylene glycol, aromatic ring. Thereby, the shielding effect of the microspheres on the non-protein small molecules is reduced or eliminated, thereby facilitating the improvement of the detection accuracy.

[0011] In some embodiments, the biotin-like substance comprises one or more of biotin, molecularly imprinted polymer, aptamer, click chemistry pair, active group-containing biotin derivative. Thereby, the stability of the combination of the microspheres and the non-protein small molecules is facilitated.

[0012] In some embodiments, the molecularly imprinted polymer comprises a MIP layer synthesized with the non-protein small molecule as a template.

[0013] In some embodiments, the nucleic acid aptamer comprises one or both of a teicoplanin aptamer and a kanamycin aptamer.

[0014] In some embodiments, the click chemistry pair comprises one or more of azide- cycloalkyne, tetrazine-trans-cyclooctene, cyclooctyne-azide.

[0015] In some embodiments, the active group-containing biotin derivative comprises one or more of NHS-biotin, biotin hydrazide, maleimide biotin, click chemistry biotin, long chain biotin, and photosensitive biotin.

[0016] In some embodiments, the detection method first causes the analyte, the non-protein small molecule bound on the functionalized microspheres, and the quantum dot-labeled detection antibody to compete, to obtain a mixed system; and then causes the functionalized microspheres in the mixed system to form a microsphere array on a microfluidic chip. In this way, the efficiency of the competition reaction is improved, thereby shortening the detection time, and improving the detection accuracy and reducing the detection limit.

[0017] In some embodiments, the detection method first causes the functionalized microspheres to form a microsphere array on a microfluidic chip; and then applies the analyte and the quantum dot-labeled detection antibody to the microsphere array, so that the analyte, the non-protein small molecule bound on the functionalized microspheres, and the quantum dot-labeled detection antibody compete. In this way, the detection accuracy is improved, and the detection limit is reduced.

[0018] In some embodiments, the microfluidic chip comprises a digital ELISA chip, which is provided with a sample adding area, a detection area, a flow channel, and a micro-trap. The flow channel extends from the sample adding area to the detection area. The micro-trap is arranged in the flow channel in the detection area and is arrayed in the detection area. The micro-trap is used to fix the functionalized microspheres. In this way, the formation of the microsphere array is facilitated, and the detection accuracy is improved.

[0019] In some embodiments, after the microsphere array is formed, the method further comprises cleaning the microsphere array with an eluent. The eluent can comprise an organic solvent.

[0020] In some embodiments, some or all of the key parameters in the method described in the present application can be determined by using the CRITIC weight method. For example, the number of microspheres, the spacing of microspheres in the microsphere array, the concentration and usage amount of the quantum dot-labeled detection antibody, the type of organic solvent, the concentration of the organic solvent in the eluent, the usage amount of the eluent, and the flow rate of the eluent can be determined by using the CRITIC weight method.

[0021] As an example, the method for determining the optimal concentration of the organic solvent in combination with the CRITIC weight method comprises the following steps before the cleaning: (a) selecting an organic solvent, so that the quantum dot signal obtained by the detection method for the standard sample containing the non-protein small molecule is greater than 0.98; 2 R (b) determining the optimal concentration of the organic solvent selected in step (a) in combination with the CRITIC weight method, taking the quantitative detection range, sensitivity, detection limit and precision of the detection method as evaluation indexes; and (c) preparing an eluent by mixing the organic solvent selected in step (a) with water, and using the eluent for the cleaning, wherein the concentration of the organic solvent in the eluent is the optimal concentration. Thus, the method described in the present application optimizes the method by the CRITIC weight method, and finds the optimal concentration of the organic solvent, so as to reduce the detection limit and shorten the detection time.

[0022] In some embodiments, the non-protein small molecule comprises one or more of a drug, an endocrine disruptor, and a persistent organic substance.

[0023] In some embodiments, the drug comprises an antibiotic, an antiepileptic drug, an antiviral drug, and an antifungal drug.

[0024] In some embodiments, the endocrine disruptor comprises one or both of bisphenol A and phthalate.

[0025] In some embodiments, the persistent organic substance comprises a polyfluoroalkyl compound; optionally, the polyfluoroalkyl compound comprises a perfluoroalkyl compound.

[0026] In some embodiments, the detection limit of the detection method is 0.5 ng / L-500 ng / L.

[0027] In some embodiments, the detection time of the detection method is less than or equal to 20 min, and further, the detection time is 8 min-20 min.

[0028] The second aspect of the present application provides an application of the method of the first aspect of the present application in drug detection, environmental water detection, sewage detection, and food detection.

[0029] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0030] Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The accompanying drawings are included to provide a description of the preferred embodiments, and are not intended to limit the scope of the application. Moreover, in the drawings, like reference numerals designate like parts throughout the various figures. In the drawings: Figure 1 is a schematic diagram of the overall structure of the digital ELISA chip of the embodiments of the present application; Figure 2 is a schematic diagram of the micro-well structure of the detection zone of the digital ELISA chip of the embodiments of the present application; Figure 3 is a schematic diagram of the functionalized microspheres prepared in Example 1 of the present application; Figure 4 is a Logistic curve obtained from the number of bright spots of Parallel Test 1 of Example 1 of the present application; Figure 5 is a Logistic curve obtained from the number of bright spots of Parallel Test 2 of Example 1 of the present application; Figure 6 is a Logistic curve obtained from the number of bright spots of Parallel Test 3 of Example 1 of the present application; Figure 7 is a Logistic curve obtained from the ratio of the average number of bright spots of Example 1 of the present application; Figure 8 is a Logistic curve obtained from the average number of bright spots of Example 2 of the present application; Figure 9 is a Logistic curve obtained from the ratio of the average number of bright spots of Example 2 of the present application; Figure 10 is a standard curve obtained from Comparative Example 2 of the present application.

[0031] Explanation of Reference Signs: 100 sample addition zone; 200 communication groove; 300 detection zone; 301 micro-well structure; 302 microsphere card slot; 303 flow channel; 304 functionalized microspheres; 400 waste liquid collection zone. DETAILED DESCRIPTION

[0032] Hereinafter, the embodiments of the method for detecting small molecules of the present application are specifically disclosed with appropriate reference to the accompanying drawings. However, there can be cases where unnecessary detailed descriptions are omitted. For example, there can be cases where detailed descriptions of matters well known in the art, repeated descriptions of substantially identical structures are omitted. This is to avoid the following description from becoming unnecessarily lengthy and to facilitate understanding by those of ordinary skill in the art. Furthermore, the accompanying drawings and the following description are provided so that those of ordinary skill in the art can fully understand the present application, and are not intended to limit the subject matter recited in the claims.

[0033] The ranges disclosed herein are intended to be shorthand for a range of values from the lower limit to the upper limit of the range. The range is defined as being inclusive of the recited endpoints to the exclusion of any other number that can be exactly determined as falling between the recited lower limit and the recited upper limit. Such ranges can be combined, i.e., any lower limit, of a range or ranges, can be combined with any upper limit, of a range or ranges, to create a new range. For example, if a range of 60-120 and a range of 80-110 are listed as exemplary ranges for a particular parameter, it is understood that a range of 60-110 and a range of 80-120 are also contemplated. Further, if a minimum range value of 1 and 2 are listed, and if a maximum range value of 3, 4, and 5 are listed, then the following ranges are all contemplated: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In this application, the use of a numerical range "a-b" to describe a variable refers to the shorthand means for describing all individual real numbers that fall within the range of a to b, where a and b are both real numbers, and the range is inclusive of the endpoints a and b. For example, the numerical range "0-5" means that all real numbers between 0 and 5 have been listed herein, and "0-5" is merely a shorthand means for listing these numerical combinations. Also, when a parameter is stated to be an integer > 2, it is equivalent to disclose that the parameter is, for example, an integer 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0034] As used herein, the term "comprising" is to be interpreted as inclusive rather than exclusive and does not exclude additional, unrecited elements or steps. Specifically, when used in the description and the claims, the term "comprising" and variations thereof mean including, but not limited to, the listed features, steps or components. Such terms do not exclude the presence of additional features, steps or components.

[0035] If not specifically explained, all the embodiments and optional embodiments of the present application can be combined with each other to form new technical solutions.

[0036] If not specifically explained, all the technical features and optional technical features of the present application can be combined with each other to form new technical solutions.

[0037] If not specifically explained, all the steps of the present application can be performed in sequence or randomly, and preferably in sequence.

[0038] The first aspect of the present application provides a method for detecting a non-protein small molecule, comprising: providing a functionalized microsphere, the functionalized microsphere comprising a microsphere, a molecular arm, and a non-protein small molecule, wherein the non-protein small molecule is bound to the microsphere via the molecular arm, optionally, the molecular arm and the microsphere are connected via an avidin-like ligand; The non-protein small molecules in the sample, the non-protein small molecules bound on the functionalized microspheres compete with the quantum dot labeled detection antibodies, wherein the functionalized microspheres form a microsphere array before or after the competition reaction; The quantum dot signals in the microsphere array are detected, and the concentration of the non-protein small molecules in the sample is obtained based on the detection results.

[0039] The present application at least includes the following beneficial effects: (1) In the method of the present application, the non-protein small molecules are bound to the microspheres through molecular arms, which are beneficial to reduce or eliminate the shielding effect of the microspheres on the non-protein small molecules, so that the non-protein small molecules are more easily combined with the detection antibodies under the premise of being combined with the magnetic beads, which is beneficial to improve the detection accuracy; (2) The non-protein small molecules usually have small molecular weight and single antigen epitope, and it is difficult to combine two antibodies at the same time if direct detection (such as "capture - label" double antibody sandwich method, etc.) is used, and the present application makes the non-protein small molecules in the sample and the non-protein small molecules bound on the functionalized microspheres compete with the quantum dot labeled detection antibodies, and then the concentration of the non-protein small molecules in the sample is indirectly obtained by detecting the quantum dot signals of the functionalized microspheres, which avoids the limitation of small molecular weight and few epitopes of non-protein small molecules, and is beneficial to improve the detection accuracy; (3) The traditional ELISA method is to mix the sample liquid with the antibody working liquid to compete with the antigen fixed on the surface of the cylindrical bottom layer of the solid carrier to react, and the contact efficiency of the antigen on the surface of the solid carrier with the antibody is low, and the present application binds the non-protein small molecules to be detected on the surface of the microspheres, so that the non-protein small molecules in the sample and the non-protein small molecules bound on the functionalized microspheres compete with the quantum dot labeled detection antibodies, which can improve the contact efficiency and thus improve the reaction rate and shorten the detection time; (4) The method of the present application forms a microsphere array of the functionalized microspheres before or after the competition reaction, and the concentration of the non-protein small molecules in the sample is finally obtained by detecting the quantum dot signals in the microsphere array, which can reduce or avoid the signal overlapping interference caused by the aggregation of the microspheres, which is beneficial to accurately detect the quantum dot signals on the microspheres, and the high fluorescence intensity characteristics of the quantum dots are beneficial to reduce the detection limit and improve the detection accuracy, and thus realize the accurate detection of trace small molecule substances.

[0040] In some embodiments, the detection method first makes the analyte, the non-protein small molecule bound on the functionalized microspheres, and the quantum dot-labeled detection antibody compete in a reaction to obtain a mixed system; and then makes the functionalized microspheres in the mixed system form a microsphere array on a microfluidic chip. In this way, in the competition reaction, the non-protein small molecule connected on the functionalized microspheres, the non-protein small molecule in the analyte, and the detection antibody can be mixed and reacted quickly, which is conducive to improving the efficiency of the competition reaction; in addition, the microspheres form a microsphere array on the microfluidic chip, which is conducive to reducing or avoiding the influence of microsphere agglomeration on the detection result, thereby being conducive to improving the detection precision and reducing the detection limit.

[0041] In some embodiments, the detection method first makes the functionalized microspheres form a microsphere array on a microfluidic chip; and then applies the analyte and the quantum dot-labeled detection antibody to the microsphere array, so that the analyte, the non-protein small molecule bound on the functionalized microspheres, and the quantum dot-labeled detection antibody compete in a reaction. In this way, the microspheres form a microsphere array on the microfluidic chip, which is conducive to reducing or avoiding the influence of microsphere agglomeration on the detection result, thereby being conducive to improving the detection precision and reducing the detection limit.

[0042] The method described in the present application is suitable for the detection of various non-protein small molecules.

[0043] As an example, the non-protein small molecule can include one or more of a drug, an endocrine disruptor, and a persistent organic substance. As an example, the drug includes one or more of an antibiotic, an anti-epileptic drug, an anti-viral drug, and an anti-fungal drug. As an example, the endocrine disruptor includes one or both of bisphenol A and phthalate. As an example, the persistent organic substance includes a polyfluoroalkyl compound; optionally, the polyfluoroalkyl compound includes a perfluoroalkyl compound.

[0044] As an example, the non-protein small molecule can include one or more of 3-methyl-quinoline-2-carboxylic acid, abamectin, abendazole, amikacin, amoxicillin, ampicillin, azithromycin, penicillin, a β-lactam antibiotic, cefadroxil, ciprofloxacin, chloramphenicol, chloφromazine, chlortetracycline, ciprofloxacin, colistin, doramectin, doxycycline, enrofloxacin, erythromycin, florfenicol, fluroquinolone, furazolidone, gentamicin, isoniazid, kanamycin, lincomycin, melamine, metronidazole, natamycin, nitrofurazone, nitrofurantoin, furacilin, oxytetracycline, pyrrolnitrin, quinolone, ribavirin, sarafloxacin, spectinomycin, streptomycin, sulfadiazine, sulfamerazine, sulfamethazine, sulfamethoxazole, sulfamonomethoxine, sulfathalidine, sulfonamides, sulfquinoxaline, tetracycline, thiamphenicol, tilmicosin, trimethoprim, tylosin, and vancomycin.

[0045] The detection method described in the present application is beneficial to reduce the detection limit, for example, the detection limit is 0.5 ng / L-500 ng / L, for example, specifically can be 0.5 ng / L, 0.8 ng / L, 1 ng / L, 10 ng / L, 50 ng / L, 100 ng / L, 200 ng / L, 500 ng / L, etc., or a range between any two of the above values.

[0046] The detection method described in the present application is beneficial to shorten the detection time, for example, the detection time is less than or equal to 20 min, further, the detection time is 8 min-20 min.

[0047] In some embodiments, the providing functionalized microspheres comprises binding the microspheres with the non-protein small molecules through the avidin-like ligand and the molecular arm, the molecular arm comprises a biotin-like substance and a linear carbon chain connected thereto, thereby, the stability of the binding of the microspheres with the non-protein small molecules is improved. The avidin-like ligand is connected with one or more molecular arms, preferably, 1, 2, 3 or 4 molecular arms, thereby, at least one detection antibody can be connected on one microsphere, when there is a target substance in the to-be-tested liquid, the signal of the microspheres decreases more strongly, which is beneficial to improve the stability of the reaction.

[0048] In some embodiments, the providing functionalized microspheres comprises: binding the microspheres with the avidin-like ligand; binding the molecular arm with the non-protein small molecules; connecting the avidin-like ligand with the molecular arm. More specifically, the microspheres can be bound with the avidin-like ligand; the linear carbon chain at one end of the molecular arm is bound with the non-protein small molecules; the avidin-like ligand is connected with the biotin-like substance at the other end of the molecular arm. Thus, the connection of the microspheres with the molecular arm is realized through the avidin-like ligand and the biotin-like substance, the connection of the microspheres with the non-protein small molecules can be realized, and since the binding of the avidin-like ligand and the biotin-like ligand has a low dissociation coefficient, thereby, the stability of the connection of the microspheres with the non-protein small molecules is improved.

[0049] In the present application, the linear carbon chain functions to increase the distance between the non-protein small molecule and the microspheres, to reduce or eliminate the shielding effect of the microspheres in the functionalized microspheres on the non-protein small molecule, and to reduce or eliminate the adverse effect of the microspheres on the binding of the non-protein small molecule and the antibody in the competition reaction, thereby improving the detection accuracy. In the present application, the linear carbon chain refers to a molecular chain segment including carbon atoms and generally in a chain shape, and the present application does not limit whether the linear carbon chain has branches, whether the linear carbon chain further includes other atoms, or the length of the linear carbon chain, as long as the above functions can be achieved. As an example, the linear carbon chain can include an alkane, which can be a straight-chain alkane or an alkane including branched chains, for example, the alkane can be C3-C 20 As an example, the linear carbon chain can include a polyethylene glycol, and the present application does not limit the degree of polymerization of the polyethylene glycol. As an example, the linear carbon chain can also include an aromatic ring chain, which includes a continuous molecular skeleton structure formed by one or more aromatic rings (aromatic rings) directly connected by a covalent bond or connected by a short carbon chain (such as a methylene group, a vinyl group, etc.), and the present application does not limit the specific length of the aromatic ring chain.

[0050] In some embodiments, the biotin-like substance includes one or more of biotin, a molecularly imprinted polymer, an aptamer, a click chemistry pair, and a biotin derivative including an active group. Thereby, the stability of the connection between the biotin-like substance and the avidin-like ligand is improved, and the stability of the connection between the microspheres and the non-protein small molecule is improved.

[0051] In some embodiments, the molecularly imprinted polymer includes a MIP layer synthesized with the non-protein small molecule as a template.

[0052] In some embodiments, the aptamer includes one or both of a teicoplanin aptamer and a kanamycin aptamer.

[0053] In some embodiments, the click chemistry pair includes one or more of an azide-cycloalkyne, a tetrazine-trans-cyclooctene, and a cyclooctyne-azide.

[0054] In some embodiments, the biotin derivative including an active group includes one or more of an NHS-biotin, a biotin hydrazide, a maleimide biotin, a click chemistry biotin, a long-chain biotin, and a photosensitive biotin.

[0055] In some embodiments, the avidin-like ligand includes one or more of avidin, streptavidin, and neutravidin. Thereby, the stability of the connection between the avidin-like ligand and the biotin-like substance is improved, and the stability of the connection between the microspheres and the non-protein small molecule is improved.

[0056] In the present application, the matching relationship between the avidin-like ligand and the biotin-like substance is not limited, as long as the stable connection between the microspheres and the non-protein small molecules can be achieved. As an example, when the avidin-like ligand is streptavidin, the biotin-like substance is preferably biotin, so that the stable connection between the two can be achieved, thereby improving the stability of the connection between the microspheres and the non-protein small molecules.

[0057] The present application does not limit the specific structure of the microfluidic chip, as long as the functionalized microspheres before or after the competition reaction can form a microsphere array. As an example, the microfluidic chip can include a digital ELISA chip, which is provided with a sample adding area, a detection area, a flow channel and a micro-trap, the flow channel extending from the sample adding area to the detection area; the micro-trap is arranged in the flow channel in the detection area and is arrayed in the detection area, and the micro-trap is used for fixing the functionalized microspheres. Thus, it is conducive to the formation of the microsphere array and the improvement of the detection accuracy.

[0058] As an example, reference is made to the accompanying drawings Figures 1-2 , the specific digital ELISA chip can include: a sample adding area 100, which is communicated with the detection area 300 inside the chip through a communication groove 200, and the solution or suspension sample is added to the sample adding area 100, and the sample can flow into the detection area 300 through the communication groove 200; a detection area 300, which includes a plurality of arrayed micro-trap structures 301, the micro-trap structure 301 includes a plurality of microsphere clamping grooves 302 and a flow channel 303 arranged on each periphery of the microsphere clamping groove 302, the height of the microsphere clamping groove 302 is 1 times the diameter of the microsphere, and the length of the four peripheries of the microsphere clamping groove is 1.2 1.5 times, the height of the flow channel is 1 / 2 of the diameter of the microspheres, each microsphere card slot 302 can capture and fix one functionalized microsphere 304, the distance between each microsphere card slot 302 can be adjusted according to actual needs. If the functionalized microspheres 304 are fixed first and then subjected to a competition reaction, after the test sample and the detection antibody pass through the flow channel, the non-protein small molecules in the test sample compete with the non-protein small molecules connected to the functionalized microspheres 304 and the detection antibody for a reaction, wherein the detection antibody bound to the non-protein small molecules connected to the functionalized microspheres 304 is fixed in the detection area 300, the quantum dot label on the fixed detection antibody forms an array signal in the detection area 300, and the remaining reagents flow into the waste liquid collection area 400 through the flow channel 303; if the functionalized microspheres 304 are fixed after the competition reaction, the detection area 300 can form an array signal after the microspheres are fixed, and the remaining reagents flow into the waste liquid collection area 400 through the flow channel 303; and a waste liquid collection area 400, which is used to collect the liquid flowing out of the detection area 300.

[0059] The present application does not limit the method of forming the microsphere array. As an example, the microsphere array can be formed by the following method: using microspheres with a diameter of 3 um, diluting with a diluent to obtain a suspension with a final concentration of 1 mg / ml; using an automatic sample applicator to draw 1.5 ul of the suspension and then adding it to the detection area 300 of the ELISA chip, so that the microspheres are uniformly dispersed and fall into the micro-trap structure 301 of the detection area 300, after the microspheres are precipitated for 5 min, washing the excess diluent with a cleaning solution, and using an oven to dry the cleaning solution at 60°C, thereby forming a microsphere array in the detection area 300 of the chip.

[0060] In some embodiments, after forming the microsphere array, the microsphere array is further washed with an eluent. The eluent can contain an organic solvent.

[0061] In some embodiments, part or all of the key parameters in the method of the present application can be determined by using the CRITIC weight method, for example, the number of microspheres, the spacing of microspheres in the microsphere array, the concentration and amount of quantum dot-labeled detection antibody, the type of organic solvent, the concentration of organic solvent in the eluent, the amount of eluent, and the flow rate of the eluent can be determined by using the CRITIC weight method.

[0062] As an example, the method for determining the optimal concentration of the organic solvent by using the CRITIC weight method includes the following steps before the cleaning: (a) selecting an organic solvent such that the quantum dot signal obtained by the detection method for a standard sample containing the non-protein small molecules and the standard curve of the concentration of the quantum dot signal has an R 2 greater than 0.98. (b) using the quantitative detection range, sensitivity, limit of detection and precision of the detection method as evaluation indexes, and combining the CRITIC weight method to determine the optimal concentration of the organic solvent selected in step (a); and (c) using the organic solvent selected in step (a) to prepare an eluent with water for the cleaning, and the concentration of the organic solvent in the eluent is the optimal concentration. The method described in the present application optimizes the method by the CRITIC weight method, and finds the optimal concentration of the organic solvent, so as to reduce the detection limit and / or shorten the detection time.

[0063] The present application creatively combines the quantitative detection range, sensitivity, limit of detection and precision of the competitive digital microfluidic immunoassay (i.e. the detection method described in the present application) to comprehensively and accurately evaluate the influence of the optimized conditions on the method by using the four parameters for multidimensional consideration when performing the conditional experiment. Compared with optimizing a certain condition by evaluating the parallelism of the curve or using traditional evaluation indexes (such as IC 50 ), the present method can rely on data for judgment, and is more scientific and accurate, which provides theoretical guidance for using the method described in the present application for detection, and provides an important guarantee for the accuracy and reliability of the qualitative and quantitative results of the non-protein small molecules such as antibiotics in the sample to be detected.

[0064] In some embodiments, the sample to be detected includes a water sample, and when determining the optimal concentration in step (b), the water properties and the differences of the dissolved substances in the water sample can be further considered to adjust the concentration of the organic solvent in the eluent. When selecting the organic solvent and its concentration in the eluent, the type and concentration of the organic solvent can also be appropriately adjusted according to the water properties of the water sample to be detected and the differences of the dissolved substances in the water. The present application innovatively considers the quantitative detection range, sensitivity, limit of detection and precision of the competitive digital microfluidic immunoassay, optimizes and screens the concentration of the organic solvent by combining the CRITIC weight method, and can provide improved detection limit and sensitivity for different water bodies and different substances.

[0065] In some embodiments, the step (b) comprises: (i) preparing a plurality of standard solutions containing the non-protein small molecules with a concentration gradient and a series of standard solutions containing the organic solvent selected in step (a) with a concentration gradient; (ii) detecting each group of standard solutions of non-protein small molecules with concentration gradient according to the detection method described in the present application, respectively, and using the different concentrations of organic solvent-containing solutions described above to elute in the elution step, respectively, to finally obtain the standard curve of the quantum dot signal and concentration of each group of standard solutions containing the non-protein small molecules, and calculate the quantitative detection interval, sensitivity, detection limit and precision value corresponding to each of the standard curves; and (iii) taking the normalized dimensionless values of the quantitative detection interval, sensitivity, detection limit and precision as evaluation indexes, using the CRITIC weight method for weighting, obtaining the weight corresponding to each evaluation index, and taking the concentration of the organic solvent in the standard aqueous solution corresponding to the minimum value of the sum of the values of each evaluation index multiplied by the corresponding weight as the optimal concentration.

[0066] In some embodiments, the step (b) further comprises: selecting the proportion interval of the organic solvent and water according to the water quality of the water sample; and preparing the series of standard solutions with concentration gradient containing the organic solvent selected in step (a) within the proportion interval.

[0067] In some embodiments, the proportion interval of the organic solvent and water is in the range of 1%-50% in terms of volume percentage of the organic solvent.

[0068] In some embodiments, for clean water bodies such as drinking water, surface water, groundwater, seawater, atmospheric water and glacial water, the proportion interval of the organic solvent and water is in the range of 1%-10% in terms of volume percentage of the organic solvent.

[0069] In some embodiments, for contaminated water bodies such as domestic sewage, industrial wastewater, agricultural wastewater and medical wastewater, the proportion interval of the organic solvent and water is in the range of 10%-50% in terms of volume percentage of the organic solvent.

[0070] In some embodiments, the standard curve is fitted to the following equation (1): (1) In equation (1), x is the concentration of the non-protein small molecule; y is the percentage absorbance value, bright dot number or bright dot number ratio value corresponding to x; A1 is the asymptote estimate on the curve; A2 is the asymptote estimate under the curve; x0 is the half maximal inhibitory concentration IC 50 ; and p is the slope corresponding to the position of x0 on the standard curve.

[0071] In some embodiments, the fitting of the standard curve can use computer software such as Origin.

[0072] In some embodiments, the sensitivity of the competitive digital microfluidic immunoassay is represented by the slope at the position of the half maximal inhibitory concentration (IC 50 ) on the standard curve.

[0073] IC 50 value represents the concentration of the compound required to achieve 50% inhibition. Generally, the lower the IC 50 value, the stronger the inhibitory effect of the compound, i.e., the higher the sensitivity of the compound to the biological process. The present application innovatively uses the slope at the position of the IC 50 value as the sensitivity, which can better provide more comprehensive and dynamic information. Compared to the IC 50 point, the slope can better reflect the significant change in the reaction caused by a small increase in the concentration in the curve, and better represent the sensitivity of the competitive digital microfluidic immunoassay to the detection of the target compound.

[0074] In some embodiments, the normalized dimensionless value of the sensitivity is calculated by the following equation (2): (2) In equation (2), represents the reciprocal of the slope at the position of the half maximal inhibitory concentration IC 50 value on the standard curve; is the average value of the IC for each standard curve; is the minimum value of the IC for each standard curve; is the maximum value of the IC for each standard curve; represents the normalized dimensionless value of the sensitivity.

[0075] In the present application, in order to conveniently and quickly process data, the quantitative detection interval is mapped to the 0-1 range for processing, the dimensional expression is changed into a dimensionless expression, and comparison and weighting are facilitated. In some embodiments, the normalized dimensionless value of the quantitative detection interval is calculated by the following equations (3)-(6): Upper limit of the quantitative detection interval (3) Lower limit of the quantitative detection interval (4) Quantitative detection interval (5) Normalized dimensionless value of the quantitative detection interval (6) In equations (3)-(6), is the left end point of the quantitative detection interval; is the right end point of the quantitative detection interval; is 80%; is 20%; represents the width of the quantitative detection interval; is the average of corresponding to each standard curve; is the minimum of corresponding to each standard curve; is the maximum of corresponding to each standard curve; represents the normalized dimensionless value of the quantitative detection interval; and are the same as defined above.

[0076] In some embodiments, the normalized dimensionless value of the limit of detection is calculated by the following equations (7)-(10): (7) (8) (9) (10) In equations (7)-(10), is the percentage absorbance value or bright point number corresponding to the limit of detection; is the average percentage absorbance value or bright point number obtained by repeated measurements of the standard aqueous solution with a concentration of 0 of the non-protein small molecule; 3s represents 3 times the standard deviation; is the binding rate corresponding to the limit of detection; represents the limit of detection; represents the normalized dimensionless value of the limit of detection; represents the average of corresponding to each standard curve; represents the minimum of corresponding to each standard curve; represents the maximum of corresponding to each standard curve; , , and are the same as defined above.

[0077] In some embodiments, the normalized dimensionless value of the precision is calculated by the following equations (11) and (12): (11) (12) In equations (11) and (12), n represents the number of repeated measurements of the standard aqueous solution of each gradient concentration of the non-protein small molecule; Ci represents the concentration obtained in the ith measurement; Cn represents the average of the concentrations obtained in n measurements; S represents the standard deviation; RSDi represents the relative standard deviation, representing the precision; RSDn represents the normalized dimensionless value of the precision; RSDavg represents the average of the RSDs corresponding to the respective standard curves; RSDmin represents the minimum value in the RSDs corresponding to the respective standard curves; and RSDmax represents the maximum value in the RSDs corresponding to the respective standard curves.

[0078] The CRITIC weighting method is an objective weighting method. The idea is to use two indicators, namely the contrast intensity and the conflict indicator. The contrast intensity is represented by the standard deviation, and the greater the data standard deviation, the greater the fluctuation and the higher the weight. The conflict is represented by the correlation coefficient, and the greater the correlation coefficient value between the indicators, the smaller the conflict, and the lower the weight. When calculating the weight, the contrast intensity and the conflict indicator are multiplied and normalized, and the final weight is obtained.

[0079] In some embodiments, in the method of the present application, the process of weighting using the CRITIC weighting method includes: (1) forming an m-row and 4-column matrix X of 4 evaluation indicators (i.e., the normalized dimensionless values of the quantitative detection interval, the sensitivity, the detection limit, and the precision), where m is the number of concentration gradients of the organic solvent, and the elements X ij Xij represents the value of the jth column of the evaluation indicator corresponding to the ith row of the gradient concentration of the organic solvent, i.e., the value of the jth column of the ith row of the evaluation indicator of the matrix X; Taking 5 groups of samples to be evaluated (corresponding to the gradient concentrations of the organic solvent, for example, 0, 1%, 2%, 4%, and 8%, respectively) as an example, the 4 evaluation indicators form the original indicator data matrix as follows: .

[0080] (2) Calculate the objective weight corresponding to each evaluation indicator according to the following equations (13)-(19): (13) (14) (15) (16) (17) (18) (19) wherein, in equations (13)-(19), represents the value of the evaluation index in the i-th row and j-th column of the matrix X; is the average value of the evaluation index of the i-th row; is the average value of the evaluation index of the j-th column; S j is the standard deviation of the evaluation index of the j-th column, indicating the index variability; is the standard deviation of the value of the i-th row; is the standard deviation of the value of the j-th column; represents the covariance of the matrix X; represents the correlation coefficient between the i-th row and j-th column of the matrix X; represents the index conflict of the j-th column evaluation index; C j represents the information amount of the j-th column evaluation index; and W j represents the objective weight of the j-th column evaluation index; and (3) multiplying the four evaluation indexes of each row of the matrix X by the corresponding objective weight respectively, and then summing, taking the gradient concentration of the organic solvent corresponding to the minimum sum as the optimal concentration.

[0081] In some embodiments, the organic solvent mentioned in the method of the present application is selected from methanol, acetonitrile, dimethyl sulfoxide, etc. In some embodiments, the selected organic solvent is methanol. When selecting the solvent, factors such as its compatibility with reactants and products, solubility, polarity, toxicity and environmental friendliness, and more importantly its influence on the detection of competitive digital microfluidic immunoassay antibodies should be considered.

[0082] In some embodiments, before detecting the water body sample, a filter membrane, for example, a filter membrane with a size of 0.45 μm, is used for filtration.

[0083] In some embodiments, before detecting the water body sample, the water body sample is pretreated to remove metal ions therein, for example, by adding 0.1% formic acid (500 μL) to adjust its pH value to 2-3, and adding 0.5% of ethylenediaminetetraacetic acid disodium salt EDTA to complex metal ions.

[0084] In the method of the present application, an ELISA kit can be used, and the kit can be selected from kits suitable for various antibiotic class new pollutants and other non-protein small molecules in the water environment.

[0085] The detection method provided by the application is simple to operate, and is an accurate, reliable, rapid and specific detection method, which can be used for rapid screening of trace non-protein small molecules in a large number of samples. The method described in the application can realize simultaneous enrichment and detection of various non-protein small molecules such as antibiotics in drug analysis samples, sewage, underground water, surface water and other different water qualities, and provides a cost-reducing and efficiency-increasing, sensitive and accurate detection method for medical analysis, life health, sewage treatment plants, drinking water plants and environmental monitoring stations in multiple environments and multiple places.

[0086] The second aspect of the application provides an application of the method described in the first aspect of the application in drug detection, environmental water detection, sewage detection and food detection.

[0087] Hereinafter, the embodiments of the application will be described. The embodiments described below are exemplary and are only used to explain the application, and cannot be understood as a limitation of the application. If the specific technology or condition is not specified in the embodiments, the technology or condition described in the literature in the art or according to the product instruction is used. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be obtained by purchase.

[0088] Example 1

[0089] A method for detecting tetracycline in water, comprising the following steps: 1. Preparing quantum dot labeled detection antibody 1.1 Quantum dot hydroxyl activation (operation in the dark) (1) Take 100 μL CdSe quantum dot (QD) solution (solvent: deionized water; solid content 10 mg / mL, Zeta potential about -40 mV), add 400 μL pH 6.0 phosphate buffer (PBS), and mix gently; the total volume of the system is 500 μL, and the final concentration of quantum dots is 1.6 μM; (2) Add 10 μL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) solution (50 mg / mL, prepared with ultrapure water) to the above solution, vortex mix, and the final concentration of EDC is 1 mg / mL; (3) Add 10 μL N-hydroxysulfosuccinimide (Sulfo-NHS) solution (25 mg / mL, prepared with ultrapure water), vortex mix, and the final concentration of Sulfo-NHS is 0.5 mg / mL; (4) Place the mixed solution at room temperature (25°C), and vortex oscillate for 15 min to complete the carboxyl activation of QD surface.

[0090] 1.2 Antibody coupling reaction

[0091] (1) Take 50 μg of tetracycline antibody (i.e. 25 μL of antibody stock solution with a concentration of 2 mg / mL), add 175 μL of PBS with a pH of 7.4 for dilution, so that the total volume of the system is 200 μL, and the final concentration of the antibody is 0.25 mg / mL.

[0092] (2) Take the activated QD solution in 1.1 and add it to the antibody solution, control the molar ratio of QD to antibody (i.e. control the labeling density) to be 1:20, vortex for 10 s to fully mix; (3) Place the mixed solution in a 37°C dark condition, incubate in a water bath shaker at a speed of 200 rpm for 60 min.

[0093] 1.3 Purification and blocking

[0094] (1) Transfer the coupling reaction solution to an ultrafiltration centrifuge tube with a molecular weight cut-off of 100 kDa; (2) Place it in a 4°C centrifuge, centrifuge at a speed of 14000 x g for 15 min, discard the supernatant (remove free antibodies); (3) Add 1 mL of PBS with a pH of 7.4 to resuspend the centrifuge tube (i.e. resuspend, disperse and dissolve the material precipitated after centrifugation with a solution, so that it forms a uniform suspension or solution), repeat the centrifugation 3 times; (4) After the last centrifugation, retain the sediment at the bottom of the tube, add 200 μL of 1wt% bovine serum albumin (BSA) solution and resuspend; (5) Place the resuspended solution in an ice bath for ultrasonic dispersion: set the power to 40 W, work for 2 s, interval for 5 s, total processing time for 3 min; (6) After ultrasonic dispersion, place the solution in a room temperature condition, block in the dark for 120 min.

[0095] 1.4 Preservation and quality control

[0096] (1) After blocking is completed, dilute the solution to a working concentration of 10 nM with a preservation buffer (PBS containing 0.1wt% BSA and 0.05wt% sodium azide); (2) Dispense into brown centrifuge tubes, store in the dark at 4°C, with a validity period of 6 months.

[0097] (3) Quality control detection: Fluorescence spectrum analysis: confirm that the emission peak is at 605 nm and there is no shift; Dynamic light scattering (DLS) detection: the particle size of the complex meets 25±3 nm (single peak distribution); ELISA activity detection: confirm the half-effective concentration (EC50) of the antibody to be less than 10 nM. ) Change from naked antibody < 20%.

[0098] 2. Preparation of biotinylated tetracycline (Direct method: aminoalkylated tetracycline -> NHS-biotin coupling)

[0099] (1) Dissolve tetracycline hydrochloride and AHA (6-aminohexanoic acid) in 5 mL of anhydrous tetrahydrofuran (THF) and cool to 0°C in an ice bath under nitrogen protection; (2) Prepare a THF solution containing DCC (dicyclohexyl carbodiimide) and DMAP (4-dimethylamino pyridine) 5 mL; slowly add the DCC / DMAP THF solution to the mixture obtained in step 2.1 using a pipette or a dropping funnel, and continuously monitor the temperature during the addition to ensure that the system temperature is < 5°C; (3) After the addition is complete, remove the ice bath and place the reaction vessel in a 25°C environment, and stir the reaction under dark conditions for 24 h; (4) After the reaction is complete, transfer the mixture to a centrifuge tube and centrifuge at 8000 rpm for 10 min at room temperature (25°C), and discard the precipitate (the precipitate is the reaction byproduct dicyclohexyl urea, DCU); (5) Collect the supernatant after centrifugation and concentrate by rotary evaporation at 40°C; after concentration, add 20 mL of pre-cooled diethyl ether (temperature 0-4°C) to the residue and stir to precipitate the solid; (6) Filter the mixture obtained in step (5) and collect the filtered solid (crude product), and wash the crude product with a diethyl ether / ethanol mixture (volume ratio 9:1) 3 times to obtain biotinylated tetracycline.

[0100] 3. Preparation of streptavidin-coated microspheres

[0101] 3.1 Activating microspheres

[0102] (1) Take 1 mL of a carboxyl microsphere suspension (containing 10 mg of 2 μm diameter silica microspheres) and wash with 0.1 M, pH 5.5 2-(N-morpholino)ethanesulfonic acid (MES) buffer 2 times; (2) Resuspend the washed microspheres in 1.5 mL of 0.1 M, pH 5.5 MES buffer; (3) Add 50 μL of an EDC solution (50 mg / mL) and 50 μL of a Sulfo-NHS solution (25 mg / mL) to the above suspension and rotate at 20 rpm for 15 min at 4°C.

[0103] 3.2 SA coupling

[0104] (1) Magnetic separation of the above-activated microsphere suspension, discard the supernatant; resuspend the microspheres in 1 mL of streptavidin (SA) solution (1 mg / mL, dissolved in PBS buffer at pH 7.4); (2) Place at 25°C, rotate at 30 rpm for 2 h.

[0105] 3.3 Blocking and washing

[0106] (1) Add 100 μL of 1 M ethanolamine solution (pH 8.5) to the coupling reaction solution obtained in 3.2, shake at room temperature for 30 min (quench the residual active groups on the surface of the microspheres); (2) Magnetic separation and discard the supernatant, wash the microspheres with PBS buffer 3 times; (3) Resuspend the washed microspheres in 2 mL of blocking buffer (0.5wt% BSA + 1wt% glycine PBS buffer, pH 7.4), block at 37°C for 60 min.

[0107] 3.4 Storage conditions

[0108] (1) After blocking is complete, magnetic separation is performed to discard the blocking buffer, and the microspheres are resuspended in PBS buffer containing 0.1wt% Na and 0.1wt% BSA, and stored at 4°C in the dark.

[0109] (2) Concentration calibration: quantitative calibration by turbidity method, adjusted to 5 mg SA microspheres / mL.

[0110] 4. Preparation of functionalized microspheres

[0111] (1) Take 200 μL of streptavidin microsphere suspension (concentration 5 mg / mL), place in a centrifuge tube (PBS buffer, pH=7.4) and wash; (2) Resuspend the washed streptavidin microspheres in 1 mL of biotinylated tetracycline solution (0.5 μg / mL, prepared with PBS), and place at 37°C for 45 min of shaking and binding; (3) Centrifugal separation (1600 rpm, 5 min) to discard the supernatant, and collect the microspheres; (4) Wash the microspheres with PBST (0.05wt% Tween-20 in PBS) 3 times; (5) Resuspend the washed microspheres in PBS (e.g., resuspend in 200 μL PBS to restore the concentration to 5 mg / mL) to obtain a functionalized microsphere suspension (concentration of 1 mg / mL). The structure of the functionalized microspheres prepared in this example is referenced. Figure 3 .

[0112] 5. Competitive Response

[0113] (1) Add 15 μL of the test sample, 20 μL of quantum dot-labeled detection antibody and 15,000 functionalized microspheres to the reaction vessel and incubate at 37°C for 4 min to carry out a competitive reaction; (2) After the reaction is complete, add 200 μL PBS (pH=6.0) to wash and separate the microspheres. The purpose is to wash away the free test sample and quantum dot antibody to obtain the functionalized microspheres after the reaction. (3) The washed microspheres were resuspended in PBS (resuspended in 200 μL PBS to restore the concentration to 5 mg / mL) to obtain the functionalized microsphere suspension (concentration of 1 mg / mL).

[0114] 6. Forming a microsphere array

[0115] 6.1 Structure of Digital ELISA Chip

[0116] For the specific structure of the digital ELISA chip used in this embodiment, please refer to the attached document. Figures 1-2 Specifically, it includes a sample application area 100, a detection area 300, and a waste liquid collection area 400. The sample application area 100 is connected to the chip's interior. The detection area 300 includes several arrayed micro-well structures 301, each micro-well structure 301 comprising several microsphere slots 302 and flow channels 303 disposed on the periphery of each microsphere slot 302. The microsphere slots 302 have a height of 4 μm and a width of 4.5 μm, and the flow channels have a height of 2 μm. The waste liquid collection area 400 is connected to the detection area and is used to collect the liquid flowing out of the detection area 300.

[0117] 6.2 Fabrication of Microsphere Arrays

[0118] 1.5 μL of the functionalized microsphere suspension obtained in step 5 was extracted using an automated spotting machine and added to the detection area 300 of the ELISA chip, so that the microspheres were evenly dispersed and fell into the micro-trap structure 301 of the detection area 300. After the microspheres settled for 5 minutes, excess diluent was rinsed with cleaning solution, and the cleaning solution was dried in an oven at 60°C, thus forming a microsphere array in the detection area 300 of the chip.

[0119] 7. Screening for optimal conditions for organic solvents: (1) Selection of organic solvent: methanol and ethanol were added to different groups of PBS solution with pH 7.2 and concentration of 2 mM, respectively, as eluent for parallel experiments, and the organic solvent was selected according to the method described above, and finally methanol was selected as the organic solvent added in the eluent of this example; (2) Selection of organic solvent concentration: methanol was added to multiple groups of PBS solution with pH 7.4 and concentration of 2 mM according to the volume ratio of 0%, 1%, 2%, 4%, 8%, 10%, 20%, 40%, and 50%, respectively, to prepare eluent with gradient concentration of methanol, and the concentration of methanol in the eluent was selected according to the method described above, and finally the volume concentration of methanol was 1%.

[0120] 8. Cleaning and detection

[0121] (1) Using the organic solvent selected in step 7, prepare the eluent according to the optimal concentration determined in step 7, and use the eluent to clean the microsphere array on the microfluidic chip in step 6; (2) Place the cleaned microfluidic chip on the inverted fluorescence microscope stage, excite with 405 nm laser, and collect quantum dot signals through 605 / 40 nm emission filter; (3) Use a 20x objective lens (NA=0.75) to perform Z-stack imaging (layer thickness 0.8 μm, imaging range: from 2 μm below the bottom of the microsphere to 2 μm above the top) with an EMCCD exposure time of 200 ms; (4) Import the image into the imaging software, identify the fluorescent bright spots based on the OpenCV algorithm, and count the quantum dot signal integral intensity of each microsphere.

[0122] 9. Calculation

[0123] 9.1 Establishment of standard curve

[0124] (1) Standard detection: Prepare a series of concentrations of tetracycline standard solution: 0, 0.05 ppb, 0.1 ppb, 0.25 ppb, 0.5 ppb, 1, 5 ppb, 25 ppb, 100 ppb (1 ppb = 1000 ng / L), and detect according to the above steps 1-8, record the number of bright spots corresponding to each concentration, and detect the standard at each concentration multiple times (1-3 are three parallel experiments), and the number of bright spots corresponding to each concentration, the average number of bright spots and the ratio of bright spots (i.e. the ratio of each average bright spot to the bright spot of concentration 0) are shown in Table 1: Table 1

[0125] (2) Fitting Logistic curve: referring to equation (1) above, a four-parameter Logistic equation was used to fit the standard concentration (x, unit: ppb) and the number of bright spots (y), equation (1):

[0126] Wherein: x is the concentration of the non-protein small molecule; y is the corresponding percentage absorbance value; A1 is the estimated value of the asymptote of the curve; A2 is the estimated value of the asymptote below the curve; x0 is the half-inhibitory concentration IC 50 , that is, the tetracycline concentration corresponding to the signal response value falling to (A1+A2) / 2; p is the slope of the position corresponding to x0 on the standard curve (the larger the p, the steeper the curve near x0, and the higher the sensitivity). The Logistic curve obtained according to the data and average value of each parallel experiment in the present embodiment is shown in Figures 4-7 .

[0127] (3) Goodness of fit verification: the determination coefficient R 2 of the Logistic curve was calculated 2 , and it was verified that the R 2 of the present embodiment could reach more than 0.99, which met R LOD >0.98, and the goodness of fit was good, indicating that the results obtained by the method disclosed in the present application were relatively accurate. Moreover, it can also be seen from Figures 4-7 that the fitting curves of each parallel experiment and the average value in the present embodiment were basically consistent, proving that the repeatability of the method was good.

[0128] 9.2 Calculation of key detection parameters (calculated according to the average value)

[0129] (1) Sensitivity: referring to the above method and Figure 7 , the sensitivity value of the present embodiment was about 2.30.

[0130] (2) Limit of detection (LOD): referring to the above method, Table 1 and Figure 7 , the limit of detection LOD of the method in the present embodiment was calculated, and it was obtained that x LOD =0.31 ppb.

[0131] (3) Quantitative detection interval: referring to the above method, Table 1 and Figure 7 , it was calculated that the quantitative detection interval of the present embodiment was [0.38 ppb-1.02 ppb].

[0132] (4) Precision: according to the data of the parallel experiment of the standard with a concentration of 0.5 ppb, the relative standard deviation RSD of the signal response value was calculated, RSD=100%×(s / AV), wherein s is the standard deviation of 3 repeated detections, and AV is the average value of the signals of 3 detections, and the requirement is RSD≤15%. It was calculated that the RSD of the present embodiment met the requirement of ≤15%.

[0133] In this embodiment, the method described in this application is used to detect a plurality of tetracycline standard solutions, and the standard curve is obtained by fitting the Logistic equation. After verifying the detection parameters, it is proved that the method described in this application can ensure the stability of detection (RSD≤15%), and the sensitivity (x0=0.60ppb) and the detection limit (0.31ppb) are good, and the quantitative interval covers the trace concentration range, which basically meets the analysis requirements of trace non-protein small molecules in water. Moreover, using the method described in this embodiment, the detection sample, quantum dot labeled detection antibody and functionalized microspheres only need to be incubated for 4 min, and the detection time of one sample is not more than 15 min, which can meet the high-throughput detection requirements.

[0134] In addition, after obtaining the standard curve, if you want to continue to detect the concentration of the unknown sample, you only need to operate according to the above steps 1-7 to obtain the signal response value y 待测 of the detection sample, and then put it into the above Logistic equation (i.e. the standard curve) to calculate the corresponding tetracycline concentration x 待测 of the detection sample. If y 待测 exceeds the above determined quantitative detection interval, the detection sample needs to be diluted or concentrated and then detected and calculated according to the above method.

[0135] Example 2

[0136] The eluent used in this embodiment is a 2 mM PBS solution with a pH of 7.4, to which 1% (volume ratio) methanol is added.

[0137] Prepare a series of tetracycline standard solution with different concentrations: 0, 0.05ppb, 0.1ppb, 0.25ppb, 0.5ppb, 1, 5ppb, 25ppb, 100ppb, and detect them according to the method described in Example 1. Record the corresponding bright dot number of each concentration of tetracycline standard solution. Each concentration of standard sample is detected multiple times (1-3 are three parallel experiments), and the bright dot number, average bright dot number and bright dot ratio (i.e. the ratio of each average bright dot number to the bright dot number of the concentration of 0) corresponding to each concentration are shown in Table 2: Table 2

[0138] According to the average bright dot number data in Table 2, the Logistic curve is fitted according to the method of Example 1 (as shown in Figure 8 ). In addition, in order to reduce the influence of the difference between different parallel test data on the final result, this embodiment also fits the standard curve according to the bright dot ratio in Table 2 (as shown in Figure 9 ), so as to normalize the difference between the absolute values of different parallel experiments.

[0139] In combination with Table 2 and Figures 8-9 The method of Example 1 was used to obtain the R 2 > 0.99, meeting the reliability requirements, and the sensitivity was about 2.31, the detection limit was about 0.02 ppb, the quantitative detection interval was about [0.32 ppb-1.08 ppb], and the precision RSD was ≤15%. The above results show that the method described in the application can ensure the detection stability, and the sensitivity and the detection limit are good, the quantitative interval covers the trace concentration range, and basically meets the analysis requirements of trace non-protein small molecules in water samples.

[0140] Example 3

[0141] The method of step 2 of this example is different from that of Example 1, and the order of steps 5 and 6 is different, and the other steps and conditions are the same.

[0142] Steps 2, 5 and 6 of this example are as follows: 2. Preparation of biotinylated tetracycline (1) Dissolve the aminoalkylated tetracycline (Tetracycline-6-AHA) in 2 mL of anhydrous dimethyl sulfoxide (DMSO), add long-chain biotin-NHS ester (NHS-LC-Biotin) and triethylamine (TEA), and stir under 25°C light-free conditions for 12 h; (2) After the reaction is completed, the reaction solution is dropped into 20 mL of ice-cold anhydrous ether (0-4°C) to precipitate; (3) Centrifuge the reaction system (5000 rpm for 5 min), discard the supernatant, and collect the bottom precipitate; purify the precipitate by Sephadex LH-20 column chromatography (eluent: methanol / PBS (7:3 v / v), flow rate: 1 mL / min, collect the main peak, and detect UV 360 nm); freeze-dry the purified product to obtain biotinylated tetracycline, and store it in a light-free environment at-20°C.

[0143] 5. Formation of microsphere array

[0144] 5.1 Structure of digital ELISA chip

[0145] The digital ELISA chip used in this example is the same as that of Example 1.

[0146] 5.2 Preparation of microsphere array

[0147] Using an automatic spotter, 1.5 μL of the functionalized microspheres suspension prepared in step 4 was drawn and added to the detection area 300 of the ELISA chip, so that the microspheres were uniformly dispersed and fell into the micro-well structures 301 of the detection area 300, after the microspheres were precipitated for 5 min, the excess diluent was washed away with a cleaning solution, and the cleaning solution was dried in an oven at 60°C, thereby forming a microsphere array in the detection area 300 of the chip.

[0148] 6. Competition reaction

[0149] (1) 15 μL of the sample to be tested and 20 μL of the quantum dot-labeled detection antibody were added to the sample addition area 100 of the ELISA chip, and the two were driven to flow into the microsphere array in the detection area 300, and incubated at 37°C for 8 min, so that the non-protein small molecules connected to the microspheres competed with the non-protein small molecules in the sample to be tested and the detection antibody; (2) After the reaction was completed, 200 μL of PBS (pH = 6.0) was continuously added from the sample addition area 100 to wash away the free sample to be tested and detection antibody in the detection area 300.

[0150] According to the detection and calculation method of Example 1, the R 2 of the Logistic equation fitted in this example can also be above 0.99, which meets the R 2 > 0.98. Moreover, the sensitivity, detection limit, quantitative range, and precision of this example are similar to those of Example 1, which can ensure the stability of the detection, and the sensitivity and detection limit perform well, the quantitative range covers the trace concentration range, which can basically meet the analysis requirements of trace non-protein small molecules in water. Moreover, using the method described in this example, the sample to be tested, the quantum dot-labeled detection antibody, and the functionalized microspheres only need to be incubated for 8 min, and the entire detection time is not more than 20 min, which can meet the high-throughput detection requirements.

[0151] Comparative Example 1: Detection of tetracycline residues based on imprinted microspheres

[0152] This comparative example provides a method for detecting tetracycline residues based on imprinted microspheres, which uses molecularly imprinted polymers (MIPs) as artificial recognition elements to establish an enzyme-linked immunosorbent assay (ELISA) method: using tetracycline (TC) as a template molecule, methacrylic acid (MAA) as a functional monomer, and ethylene glycol dimethacrylate (EGDMA) as a crosslinking agent, MIP microspheres are prepared by the precipitation polymerization method; after the MIP microspheres are coated in the microwell plate wells, a molecularly imprinted ELISA (MI-ELISA) method based on the direct competition between free TC and horseradish peroxidase (HRP)-labeled TC in a heterogeneous phase is established.

[0153] This comparative example specifically includes the following steps: 1. Preparation of tetracycline imprinted microspheres (MIP microspheres) by precipitation method (1) Dissolve template molecule: dissolve template molecule TC (0.5 mmol) in 50 mL acetonitrile, and place in a 180 mm x 30 mm (inner diameter) quartz cuvette; (2) Add functional monomer and incubate: add functional monomer MAA (2 mmol, 0.170 mL), and incubate at 4°C for 24 h; (3) Add crosslinking agent and initiator: add crosslinking agent EGDMA (10 mmol, 1.886 mL) and initiator azobisisobutyronitrile AIBN (0.365 mmol, 60 mg) in steps, pour the mixed solution into an ultrasonic treatment tank, and ultrasonically treat for 5 min, and blow with nitrogen for 5 min; (4) Ultraviolet light-induced polymerization: after sealing the tube in a nitrogen atmosphere, irradiate the mixture with ultraviolet light (λ = 365 nm, 6 W) in ice water (0°C) for 48 h; after the reaction is completed, collect the generated particles by centrifugation (6000 rpm, 5 min); (5) Washing and drying: wash the particles with methanol containing 10% acetic acid (V / V) by centrifugation (6000 rpm, 5 min) until no template is detected from the washing solvent by spectral measurement; wash the polymer particles with acetone again, and dry in a vacuum chamber to obtain MIP microspheres.

[0154] The above functional monomer can also use 4-vinylpyridine (4-VP, 2 mmol, 0.225 mL) or acrylamide (AM, 2 mmol, 142.157 mg).

[0155] 2. Synthesis of non-imprinted reference polymer (NIP)

[0156] The preparation method of NIP is the same as that of the above MIP microspheres, and the only difference is that no template TC is added.

[0157] 3. Preparation of tetracycline conjugate (HRP-TC)

[0158] (1) Dissolve HRP: weigh 45 mg of HRP sample, and dissolve in 13 mL of 0.05 mo / L carbonate buffer (pH = 9.6) at room temperature; (2) Dissolve TC: weigh 10 mg of TC, and dissolve in 1 mL of 1 mmol / L sodium hydroxide (NaOH) solution; (3) Mix the reaction: take 50 μL of the above TC solution and add to the above HRP buffer solution, mix, add 15 μL of 25 wt% glutaraldehyde solution dropwise while stirring magnetically, and incubate for 30 min; (4) Adjusting pH and continuing reaction: after adjusting pH to 9.6 with 1 mmol / L hydrochloric acid (HCl), continuing reaction for 2 h; (5) Dialsis purification: dialysing the combined product in 0.01 mol / L PBS (pH=7.4) for 5 days, removing impurities by the retention of semi-permeable membrane, retaining the "HRP-TC" combined product with large molecular weight, and finally obtaining purified tetracycline combined product; (6) Determining combined rate: determining HRP-TC combined rate by ultraviolet spectrophotometer, so as to obtain actual coupling efficiency of HRP and TC, and excluding interference of free components on detection results.

[0159] 4. Molecular imprinting ELISA (MI-ELISA) detection process

[0160] 4.1 Micro-hole plate coating

[0161] (1) Diluting MIP microspheres and NIP microspheres to 1 mg / mL with 0.05 mol / L carbonate buffer (pH=9.6) respectively; (2) Adding 100 μL MIP microsphere suspension to each hole of MIP group, and adding 100 μL NIP microsphere suspension to each hole of NIP control group, coating at 4°C overnight; (3) Discarding coating liquid, washing 3 times with PBST washing liquid (containing 0.05% Tween-20, PBS, pH=7.4) for 3 min each time, and patting dry residual liquid.

[0162] 4.2 Blocking

[0163] Adding 200 μL 1% BSA (dissolved in PBS) to each hole, blocking at 37°C for 1 h, removing blocking liquid, and washing 3 times with PBST, and patting dry.

[0164] 4.3 Competition reaction

[0165] (1) Preparing TC standard with series of concentrations: 0, 100 μg / L, 500 μg / L, 1000 μg / L, 2000 μg / L, 3200 μg / L (diluted with PBS); (2) Mixing the above TC standard with HRP-TC combined product according to volume ratio 1:1 (50 μL sample+50 μL HRP-TC), and vortexing to mix; (3) Adding 100 μL mixed liquid to each hole of MIP group and NIP control group, and incubating at 37°C for 30 min (free TC competes with HRP-TC to combine with the binding sites of MIP / NIP microspheres).

[0166] 4.4 Washing and color development

[0167] (1) Discard the reaction solution, wash with PBST for 5 times, 3 min each time, and completely remove the unbound HRP-TC; (2) Add 100 μL TMB color developing solution (substrate) to each well, and incubate at 37°C for 15 min in the dark; (3) Add 50 μL 2 mol / L S Stop the reaction and mix gently.

[0168] 4.5 Signal detection

[0169] Determine the absorbance (OD value) of each well of the MIP group and the NIP control group at 450 nm by using an enzyme label instrument, and record the data.

[0170] 5. Calibration curve establishment and performance verification

[0171] 5.1 Calibration curve establishment

[0172] The standard curve is fitted according to the method of Reference Example 1, and it is verified that the method of the present comparison example has a good linear relationship in the range of 100 μg / L-3200 μg / L, and the regression coefficient R²=0.999.

[0173] 5.2 Verification of key performance indicators

[0174] (1) NIP specificity verification: calculate the OD value ratio (OD NIP / OD MIP ) of the NIP control group and the MIP group under the same TC concentration, and it is verified that the ratio is ≤15%, indicating that the non-specific adsorption of NIP to TC is extremely low, and the binding of MIP mainly depends on the specific imprinting site; (2) Detection limit (LOD): determine the OD value of the MIP group of 20 blank samples (not containing TC), calculate the average value (yblank) and the standard deviation (Sblank), and the LOD=100 μg / L (corresponding to the concentration when the signal threshold value yblank-3Sblank); (3) Precision: repeat the detection of 500 μg / L and 2000 μg / L TC standard samples for 6 times, calculate the relative standard deviation (RSD), and it is verified that the intra-day RSD≤15% and the inter-day RSD≤20%; (4) Detection time: the overall process from sample addition to result output takes ≥50 min (not including MIP preparation and binder purification time).

[0175] The difference between the method of the present comparison example and the method provided in the present application is that the molecular arm structure is not used, and from the above detection results, it can be seen that the detection limit of the present comparison example is 100 μg / L (i.e. 10 5ng / L), which is significantly higher than the limit of detection of the embodiments of the present application. Moreover, the reaction time of the comparative example is long (the overall process from sample addition to result output takes ≥50 min), which is much longer than the detection time of the embodiments of the present application (≤20 min). Therefore, the method provided by the present application can reduce the detection limit and detection time of non-protein small molecules such as tetracycline.

[0176] Comparative Example 2: Biotinylated competitive ELISA for detecting tetracycline residues

[0177] The comparative example provides a biotin-avidin mediated competitive enzyme-linked immunosorbent assay (ELISA) for detecting tetracycline (TC) residues in honey and the like, and the specific steps are as follows: 1. Reagent preparation and optimization 1.1 Preparation of standard and buffer TC standard: prepare a series of TC standard solutions (0, 1.52 μg / L, 15.2 μg / L, 152 μg / L) with PBS-EDTA analysis buffer at pH = 7.2; Blocking solution: 1% bovine serum albumin (BSA) in PBS; Washing solution: 0.05% Tween-20 in PBS (PBST); Substrate solution: 3,3',5,5'-tetramethylbenzidine (TMB) color developing solution; Termination solution: 2 mol / L S .

[0178] 1.2 Optimization of key reagents

[0179] Coating antigen: ovalbumin-tetracycline conjugate (OVA-TC conjugate), the optimized concentration is 5 mg / L; Primary antibody: tetracycline monoclonal antibody, the optimized dilution is 1:1000 (final concentration 9.98 mg / L); Secondary antibody-biotin conjugate: goat anti-mouse IgG-biotin, the optimized concentration is 0.5 mg / L; Avidin-HRP: the optimized concentration is 0.4 mg / L.

[0180] 2. Detection process

[0181] 2.1 Coating

[0182] Add 100 μL OVA-TC conjugate (5 mg / L) to each well, and coat at 4°C for 3 h; discard the coating solution, wash with PBST for 3 times (330 μL each time, wash at room temperature for 3 min), and pat dry the residual liquid.

[0183] 2.2 Blocking

[0184] Add 300 μL blocking solution to each well, block at room temperature (about 25°C) for 30 min; discard the blocking solution, wash with PBST for 3 times, and pat dry.

[0185] 2.3 Competition reaction

[0186] Add 50 μL TC standard and 50 μL primary antibody solution (1:1000 dilution) to each well, incubate at room temperature for 1 h (free TC competes with coated OVA-TC to bind the primary antibody); after the reaction, discard the reaction solution, wash with PBST for 3 times, and pat dry.

[0187] 2.4 Signal amplification and color development

[0188] (1) Add 100 μL secondary antibody-biotin conjugate (0.5 mg / L) to each well, incubate at room temperature for 1 h; then discard the reaction solution, wash with PBST for 3 times, and pat dry; (2) Add 100 μL avidin-HRP (0.4 mg / L) to each well, incubate at room temperature for 30 min; then discard the reaction solution, wash with PBST for 5 times (to completely remove free HRP), and pat dry; (3) Add 100 μL substrate (TMB color developing solution) to each well, incubate at room temperature for 10 min in the dark; (4) Add 50 μL stop solution (2 M sulfuric acid) to each well, shake well, and the reaction solution changes from blue to yellow; then use the enzyme label E-max to measure the absorbance (OD value) at 450 nm wavelength.

[0189] 3. Performance verification and calibration curve

[0190] 3.1 Calibration curve establishment

[0191] The standard curve of absorbance and Log(TC) was fitted according to the method of Reference Example 1, and the results are shown in Figure 10 ( Figure 10 The lower left straight line graph is an enlarged view of the part with a larger slope in the curve graph, and the dynamic range is 1.52 μg / L-152 μg / L, and the R² of the regression equation is 0.998.

[0192] 3.2 Key performance indicators

[0193] Limit of detection (LOD): 0.19 μg / L (based on the OD value of 20 blank samples, y 空白 -3S 空白 corresponding to the concentration); Limit of quantification (LLOQ): 0.38 μg / L (corresponding to the concentration with a signal-to-noise ratio of ≥10); Precision: 6 repeated detections of 15.2 μg / L and 76 μg / L standard samples, intra-day RSD ≤8%, inter-day RSD ≤12%; Cross-reactivity: ≤3% cross-reactivity with structural analogues such as oxytetracycline and aureomycin, good specificity; Recovery: 95%-101% recovery of the honey sample (spiked concentration 5 μg / L-100 μg / L); Detection time: single detection time ≥120 min (not including reagent preparation time) from sample addition to result output.

[0194] 4. Differences between the method of the present application and comparative examples

[0195] The method used in the comparative example relies on microwell plate immobilization of OVA-TC conjugate, and realizes detection through "free TC-coated TC competition binding of primary antibody", and the signal is read by HRP catalyzed TMB color development, which is easily affected by enzyme activity fluctuations; while the method provided in the present application uses functionalized microsphere array to immobilize TC, and quantum dot labeled antibody competition reaction, which has better signal stability. In addition, although the method of the comparative example has a lower LOD (0.19 μg / L, i.e. 190 ng / L), its detection time is as long as 2 h, which is much longer than the method provided in the present application (≤20 min), and is not suitable for high-throughput detection, and cannot meet the demand for rapid detection of ng / L level trace amounts in environmental water.

[0196] It should be noted that the present application is not limited to the above embodiments. The above embodiments are only examples, and embodiments having the same technical idea and playing the same role and effect within the scope of the technical solutions of the present application are all included in the technical scope of the present application. In addition, within the scope of the main idea of the present application, various modifications of the embodiments that can be thought of by those skilled in the art, and other ways constructed by combining part of the constituent elements of the embodiments are also included in the scope of the present application.

Claims

1. A method of detecting a non-proteinaceous small molecule, characterized in that, The application provides a method for detecting non-protein small molecules in a sample, comprising: providing functionalized microspheres, the functionalized microspheres comprising microspheres, molecular arms and non-protein small molecules, wherein the non-protein small molecules are bound to the microspheres through the molecular arms, and the molecular arms are connected to the microspheres through an affinity ligand; making the non-protein small molecules in the sample and the non-protein small molecules bound to the functionalized microspheres compete with a quantum dot-labeled detection antibody, wherein the functionalized microspheres are formed into a microsphere array before or after the competition; and detecting quantum dot signals in the microsphere array, and obtaining the content of the non-protein small molecules in the sample based on the detection results.

2. The method of claim 1, wherein, The providing of the functionalized microspheres comprises binding the microspheres to the non-protein small molecules through the affinity ligand and the molecular arms. The affinity ligand is connected to one or more molecular arms, preferably 1, 2, 3 or 4 molecular arms. The molecular arms comprise a biotin substance and a linear carbon chain connected thereto. Preferably, the providing of the functionalized microspheres comprises: binding the microspheres to the affinity ligand; binding the molecular arms to the non-protein small molecules; and connecting the affinity ligand to the molecular arms.

3. The method of claim 2, wherein, One or more of the following conditions are met: The affinity ligand comprises one or more of avidin, streptavidin and neutravidin. said linear carbon chain comprises one or more of C3-C 20 alkanes, polyethylene glycol, one or more of aromatic rings in the chain; The biotin substance comprises one or more of biotin, a molecularly imprinted polymer, an aptamer, a click chemistry pair and a biotin derivative containing an active group. Optionally, the molecularly imprinted polymer comprises a MIP layer synthesized by taking the non-protein small molecules as a template. Optionally, the aptamer comprises one or both of a teicoplanin aptamer and a kanamycin aptamer. Optionally, the click chemistry pair comprises one or more of azido-cycloalkyne, tetrazine-trans-cyclooctene and cyclooctyne-azido. Optionally, the biotin derivative containing an active group comprises one or more of NHS-biotin, biotin hydrazide, maleimide biotin, click chemistry biotin, long-chain biotin and photosensitive biotin.

4. The method of claim 1, wherein, The non-protein small molecules in the sample and the non-protein small molecules bound to the functionalized microspheres are made to compete with a quantum dot-labeled detection antibody to obtain a mixed system, and then the functionalized microspheres in the mixed system are formed into a microsphere array on a microfluidic chip.

5. The method of claim 1, wherein, The functionalized microspheres are first formed into a microsphere array on a microfluidic chip, and then the sample and a quantum dot-labeled detection antibody are applied to the microsphere array, so that the non-protein small molecules in the sample and the non-protein small molecules bound to the functionalized microspheres compete with the quantum dot-labeled detection antibody.

6. The method according to claim 4 or 5, characterized in that, The microfluidic chip comprises a digital ELISA chip, and the digital ELISA chip is provided with a sample adding area, a detection area, a flow channel and microtraps, the flow channel extends from the sample adding area to the detection area, and the microtraps are arranged in the flow channel in the detection area and are arrayed in the detection area, and the microtraps are used for fixing the microspheres and / or the functionalized microspheres.

7. The method according to any one of claims 1 to 5, characterized in that, After the microsphere array is formed, the microsphere array is further cleaned with an eluent. The application further provides a kit for detecting non-protein small molecules in a sample, comprising: the functionalized microspheres; and the quantum dot-labeled detection antibody. The kit further comprises a microfluidic chip, and the microfluidic chip comprises a digital ELISA chip, and the digital ELISA chip is provided with a sample adding area, a detection area, a flow channel and microtraps, the flow channel extends from the sample adding area to the detection area, and the microtraps are arranged in the flow channel in the detection area and are arrayed in the detection area, and the microtraps are used for fixing the microspheres and / or the functionalized microspheres. Optionally, the eluent comprises an organic solvent, and the cleaning further comprises: (a) selecting an organic solvent such that the quantum dot signal obtained by the detection method for a standard comprising the non-proteinaceous small molecule as a function of concentration has an R 2 greater than 0.98; (b) determining the optimal concentration of the selected organic solvent in step (a) by using the quantitative detection range, sensitivity, limit of detection and precision of the detection method as evaluation indexes, and combining with the CRITIC weight method; and (c) preparing the eluent by using the selected organic solvent in step (a) and water, wherein the concentration of the organic solvent in the eluent is the optimal concentration.

8. The method according to any one of claims 1 to 5, characterized in that, The non-protein small molecules comprise one or more of drugs, endocrine disruptors, and persistent organic compounds; Optionally, the drugs comprise one or more of antibiotics, antiepileptic drugs, antiviral drugs, and antifungal drugs; Optionally, the endocrine disruptors comprise one or both of bisphenol A and phthalate esters. Optionally, the persistent organic compounds comprise polyfluoroalkyl compounds; and optionally, the polyfluoroalkyl compounds comprise perfluoroalkyl compounds.

9. The method according to any one of claims 1 to 5, characterized in that, The limit of detection is 0.5 ng / L-500 ng / L; and / or, the detection time is less than or equal to 20 min, optionally 8 min-20 min.

10. Use of the method of any one of claims 1-9 in drug detection, environmental water detection, sewage detection, and food detection.

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