Bioactive substance detection method based on digital fluorescence immunoadsorption and application thereof

Abstract

The invention provides a bioactive substance detection method based on digital fluorescence immunoadsorption and application thereof, and belongs to the technical field of biological detection. According to the invention, bioactive substances are detected based on a digital fluorescence immunoadsorption technology, a plate-type platform is adopted, and the bioactive substances are subjected to fluorescence amplification and digital coding by using the digital fluorescence immunoadsorption technology; furthermore, by virtue of a fluorescence microscopic imaging system (without a liquid path detection module) and in combination with image recognition and data analysis software, the bioactive substances can be detected at a single-molecule level, so that the cost and the operation difficulty can be effectively reduced while medium and high throughput is maintained, and single-molecule detection of various bioactive substances can be realized. A liquid path detection module is not needed, the equipment complexity is reduced, meanwhile, the economical efficiency is improved, the method has remarkable advantages in the aspects of cost, flux, operation, sensitivity and the like, and the huge in-vitro diagnosis market application prospect is shown.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a biologically active substance detection method based on digital fluorescent immunoadsorption and an application thereof. Background Art

[0002] Bioactive substances are biologically active compounds that come from organisms and have an impact on life phenomena. Bioactive substances are usually characterized by trace amounts, high efficiency, and complexity, and they play a key regulatory role in organisms. Since the concentrations of these substances are often very low, traditional detection methods often have difficulty accurately and sensitively detecting their presence and changes. Therefore, a high-precision and high-sensitivity detection technology is needed to meet the detection needs of bioactive substances.

[0003] Single-molecule immunoassay technology can capture single target molecules in nanospace, thereby achieving ultra-sensitive detection, breaking through the limitations of traditional immunoassay technology and providing new means and methods for early diagnosis of diseases, treatment monitoring, and biomarker research. However, at present, the market already has single-molecule immunoassay technology, which requires biochips and supporting imaging equipment that requires liquid detection modules. Representative companies include Quanterix (simoa system), Merk (SMC system), Changsha Caike, etc., and also require the use of special supporting reagents. The technology is relatively costly, the application scenarios are limited, and the operation is complicated, which is not conducive to large-scale promotion and application.

[0004] Therefore, it is crucial to develop a detection method with high accuracy, simple methodology, convenient operation, medium to high throughput, flexible application scenarios, and easy market promotion. Summary of the invention

[0005] The purpose of the present invention is to provide a method for detecting bioactive substances based on digital fluorescent immunoadsorption and its application, which can solve the problems of high cost, limited application scenarios and great difficulty in promotion of single molecule detection kits and equipment in the prior art.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for detecting biologically active substances based on digital fluorescent immunoadsorption, comprising the following steps:

[0008] (1) The plate-type platform adsorbs the capture substances corresponding to the bioactive substances;

[0009] (2) The fluorescent particles adsorb the detection substances corresponding to the bioactive substances;

[0010] (3) Capture of bioactive substances;

[0011] (4) Fluorescence amplification and digital encoding of captured bioactive substances;

[0012] (5) Use a fluorescence microscopy system to digitally image the fluorescently amplified and digitally encoded bioactive substances, and combine image recognition and data processing software to generate analysis results.

[0013] Preferably, in step (1), the plate-type platform is a plate-type platform with a transparent base; the adsorption temperature is 2 to 8° C., and the adsorption time is 15 to 20 hours.

[0014] Preferably, in step (2), the step of adsorbing the detection substance corresponding to the biologically active substance by the fluorescent particles is:

[0015] A. Dilute the fluorescent particle aqueous dispersion with fluorescent particle coating buffer, centrifuge, remove the supernatant, and repeat the centrifugation step; after the centrifugation is completed, discard the supernatant, re-add the fluorescent particle coating buffer for dilution, and then add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride ultrapure aqueous solution and N-hydroxysuccinimide ultrapure aqueous solution in sequence to react; after the reaction is completed, centrifuge and discard the supernatant;

[0016] B. Take the precipitate and continue to disperse it in 1 mL of fluorescent particle coating buffer. After centrifugation and discarding the supernatant, the precipitate is dispersed with fluorescent particle coating buffer. After mixing, add fluorescent particles to adsorb the corresponding detection substances of the bioactive substances to react. After the reaction is completed, add blocking solution to block.

[0017] Preferably, in step (2), the fluorescent particle coating buffer consists of 10 mol / L tris(hydroxymethylaminomethane) and 0.05%wt ProClin 300 ultrapure aqueous solution; the concentration of the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride ultrapure aqueous solution is 8-12 mg / mL, and the concentration of the N-hydroxysuccinimide ultrapure aqueous solution is 8-12 mg / mL; the volume ratio of the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride ultrapure aqueous solution to the N-hydroxysuccinimide ultrapure aqueous solution is 1-2:1-2; and the blocking solution consists of 5 mol / L boric acid, 0.05%wt Tween-20, 1%wt bovine serum albumin, 5%wt trehalose and 0.1%wt ProClin 300 ultrapure aqueous solution.

[0018] Preferably, in step (2) A, the reaction temperature is 35-38°C, the reaction time is 1-2h, and the reaction conditions are dark conditions; in step B, the reaction temperature is 35-38°C, the reaction time is 2-3h, and the reaction conditions are dark conditions.

[0019] Preferably, in step (3), the step of capturing the bioactive substance comprises:

[0020] Adding the standard substance of the bioactive substance, the quality control substance and the sample to be tested to the plate platform on which the capture substance corresponding to the bioactive substance is adsorbed in step (1), placing it on a shaker, reacting, washing and drying;

[0021] Preferably, in step (4), the steps of fluorescence amplification and digital encoding of the captured bioactive substances include:

[0022] In step (3), the fluorescent particles adsorbed with the detection substance corresponding to the bioactive substance prepared in step (2) are added to the plate platform for capturing the bioactive substance, and reacted, washed and dried.

[0023] Preferably, in step (4), the reaction temperature is 35-38° C., the reaction time is 1-2 h, and the reaction conditions are dark conditions.

[0024] Preferably, in step (5), the step of using a fluorescence microscopy imaging system to digitally image the fluorescence-amplified and digitally encoded biologically active substance, combined with image recognition and data processing software, to generate analysis results comprises:

[0025] The plate platform after the reaction in step (4) is placed on the sample stage of the fluorescence microscope system, and digital imaging is performed by using the transmission or reflection light path through the objective lens and CCD; the imaging result is input into the image recognition and data processing software, and a standard curve is drawn according to the standard sample of the bioactive substance, and the standard curve equation is generated. The signals of the quality control product and the sample to be tested corresponding to the bioactive substance are substituted into the equation to calculate the content of the bioactive substance in the quality control product and the body fluid sample.

[0026] Preferably, in step (5), the objective lens is a 20x, 40x or 50x objective lens; the image recognition and data processing software includes: imagej, excel, origin and MATLAB.

[0027] The present invention also provides an application of the above-mentioned biologically active substance detection method based on digital fluorescent immunoadsorption in the detection of biologically active substances.

[0028] The beneficial effects of the present invention compared with the prior art are:

[0029] (1) The present invention detects bioactive substances based on digital fluorescence immunosorbent technology, adopts a plate-type platform, and uses digital fluorescence immunosorbent technology to perform fluorescence amplification and digital encoding on bioactive substances; further, through a fluorescence microscopy imaging system (liquid-free detection module) combined with image recognition and data analysis software, bioactive substances can be detected at the single molecule level, while maintaining medium and high throughput, the cost and operation difficulty can be effectively reduced, and single molecule detection of multiple bioactive substances (such as IL6, Aβ40, Aβ42, P-tau181) can be achieved, and the detection limit is less than 1pg / mL. The protein (IL6, Aβ40, Aβ42, P-tau181) detection standard curve drawn has a good linear relationship, R 2 All are greater than 0.99.

[0030] (2) The present invention does not have a liquid path detection module, which reduces the complexity of the equipment and improves its economic efficiency. It has significant advantages in terms of cost, throughput, operation, sensitivity, etc., and shows huge application prospects in the in vitro diagnostic market. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0032] Figure 1 It is a schematic diagram of the basic principle of detecting biologically active substances based on digital fluorescent immunosorbent technology of the present invention;

[0033] Figure 2 The interleukin 6 (IL6) detection standard curve of the biologically active substance detection method based on digital fluorescent immunoadsorption in Example 1 of the present invention;

[0034] Figure 3 The standard curve for detecting human β-amyloid protein 40 (Aβ40) according to the method for detecting biologically active substances based on digital fluorescence immunosorbent in Example 2 of the present invention;

[0035] Figure 4 The standard curve for detecting beta-amyloid protein 42 (Aβ42) of the method for detecting biologically active substances based on digital fluorescent immunosorbent according to Example 3 of the present invention;

[0036] Figure 5 This is the standard curve for detecting human phosphorylated tau protein 181 (P-tau181) in the method for detecting biologically active substances based on digital fluorescent immunosorbent according to Example 4 of the present invention. DETAILED DESCRIPTION

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0040] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0041] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0042] The present invention provides a method for detecting biologically active substances based on digital fluorescent immunoadsorption (eg Figure 1 As shown), comprising the following steps:

[0043] (1) The plate-type platform adsorbs the capture substances corresponding to the bioactive substances;

[0044] Wherein, the plate-type platform is a plate-type platform with a transparent base; the adsorption temperature is 2 to 8°C, more preferably 8°C, and the adsorption time is 15 to 20 hours, more preferably 16 hours.

[0045] (2) Detection substances corresponding to the bioactive substances adsorbed by fluorescent particles:

[0046] A. Dilute the fluorescent particle dispersion with fluorescent particle coating buffer, centrifuge, remove the supernatant, and repeat the centrifugation step; after the centrifugation is completed, discard the supernatant, re-add the fluorescent particle coating buffer for dilution, and then add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride ultrapure aqueous solution and N-hydroxysuccinimide ultrapure aqueous solution in sequence to react; after the reaction is completed, centrifuge and discard the supernatant;

[0047] B. Take the precipitate and continue to disperse it in 1 mL of fluorescent particle coating buffer. After centrifugation and discarding the supernatant, the precipitate is dispersed with fluorescent particle coating buffer. After mixing, add fluorescent particles to adsorb the corresponding detection substances of the bioactive substances to react. After the reaction is completed, add blocking solution to block.

[0048] Among them, the concentration of the ultrapure aqueous solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 8-12 mg / mL, more preferably 10 mg / mL; the concentration of the ultrapure aqueous solution of N-hydroxysuccinimide is 8-12 mg / mL, more preferably 10 mg / mL; the volume ratio of the ultrapure aqueous solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to the ultrapure aqueous solution of N-hydroxysuccinimide is 1-2:1-2, more preferably 1:1; the blocking solution is composed of 5 mol / L boric acid, 0.05%wt Tween-20, 1%wt bovine serum albumin, 5%wt trehalose and 0.1%wt ProClin 300 ultrapure aqueous solution.

[0049] The reaction temperature in step (2) A is 35-38° C., more preferably 37° C.; the reaction time is 1-2 h, more preferably 1 h; and the reaction conditions are dark conditions.

[0050] The reaction temperature in step (2) B is 35-38° C., more preferably 37° C.; the reaction time is 2-3 h, more preferably 2 h; and the reaction conditions are dark conditions.

[0051] (3) Capturing the bioactive substance: adding the bioactive substance standard, quality control substance and the sample to be tested to the plate platform on which the capture substance corresponding to the bioactive substance is adsorbed in step (1), placing it on a shaker, reacting, washing and drying;

[0052] The reaction temperature is 35-38°C, more preferably 37°C; the reaction time is 1-2h, more preferably 1h; and the reaction conditions are dark conditions.

[0053] (4) Fluorescence amplification and digital coding of captured bioactive substances: adding the fluorescent particles adsorbed with the detection substances corresponding to the bioactive substances prepared in step (2) to the plate platform for capturing the bioactive substances in step (3), reacting, washing, and drying;

[0054] The reaction temperature is 35-38°C, more preferably 37°C; the reaction time is 1-2h, more preferably 1h; and the reaction conditions are dark conditions.

[0055] (5) Using a fluorescence microscopy imaging system to digitally image the fluorescently amplified and digitally encoded bioactive substances, and combining image recognition and data processing software to generate analysis results: placing the plate platform after the reaction in step (4) on the sample stage of the fluorescence microscopy system, and using an objective lens in combination with a CCD to perform digital imaging using a transmission or reflection light path; inputting the imaging results into the image recognition and data processing software, drawing a standard curve based on the standard sample of the bioactive substance, generating a standard curve equation, substituting the signals of the quality control product and the sample to be tested corresponding to the bioactive substance into the equation, and calculating the content of the bioactive substance in the quality control product and the body fluid sample.

[0056] Wherein, in step (5), the objective lens is a 20x, 40x or 50x objective lens, more preferably a 20x objective lens; the image recognition and data processing software includes: imagej, excel, origin and MATLAB.

[0057] The following examples of the present invention are used as reagents:

[0058] ELISA plate washing buffer: prepare an ultrapure aqueous solution containing 0.05 mol / L sodium carbonate, 0.05 mol / L sodium bicarbonate and 0.05%wt Tween-20.

[0059] ELISA plate coating buffer: prepare an ultrapure aqueous solution containing 0.05 mol / L sodium carbonate and 0.05 mol / L sodium bicarbonate.

[0060] ELISA plate blocking buffer: a solution containing 0.05 M disodium hydrogen phosphate, 0.05 M sodium hydrogen phosphate, 1% wt bovine serum albumin and 0.1% wt ProClin 300 ultrapure aqueous solution.

[0061] Fluorescent particle coating buffer: a solution containing 10 mol / L tris(hydroxymethyl)aminomethane and 0.05% wt ProClin 300 ultrapure aqueous solution.

[0062] Fluorescent particle blocking solution: a solution containing 5 mol / L boric acid, 0.05% wt Tween-20, 1% wt bovine serum albumin, 5% wt trehalose and 0.1% wt ProClin 300 ultrapure aqueous solution.

[0063] Phosphate buffered saline cleaning solution: prepare a phosphate buffered saline solution containing 0.05%wt Tween-20.

[0064] Example 1

[0065] Example 1 of the present invention provides a method for detecting IL6 based on a biologically active substance detection method using digital fluorescent immunoadsorption, and the specific steps are as follows:

[0066] The capture substance (Aladdin) corresponding to IL6 was diluted to 2 μg / mL using ELISA plate coating buffer.

[0067] Add 100 μL of the capture substance dilution corresponding to IL6 to the sample well of the 96-base transparent ELISA plate, react in an 8°C refrigerator for 16 hours, then wash 5 times with ELISA plate washing buffer, add 150 μL / well blocking solution, block at 37°C for 2 hours, wash 5 times with ELISA plate washing buffer and dry for use.

[0068] The fluorescent particle dispersion with a fluorescent particle content of 1% wt was diluted 20 times with the fluorescent particle coating buffer, ultrasonically mixed, centrifuged at 15000 rpm for 15 min, the supernatant was removed, and the solution was dispersed into 1 mL of fluorescent particle coating buffer, ultrasonically mixed, centrifuged at 15000 rpm for 15 min, the supernatant was removed, and the solution was dispersed into 1.5 mL of fluorescent particle coating buffer. Then, 15 μL of 10 mg / mL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride ultrapure aqueous solution was added, vortexed, and then 15 μL of 10 mg / mL N-hydroxysuccinimide ultrapure aqueous solution was added, vortexed. In the dark, the mixture was placed on a shaker at 37°C for 1 h. Then, centrifuge at 15000rpm for 15min, remove the supernatant, continue to disperse in 1mL fluorescent particle coating buffer, ultrasonically mix, centrifuge at 15000rpm for 15min, remove the supernatant, continue to disperse in 0.75mL fluorescent particle coating buffer, and ultrasonically mix. Then add 0.25mL of the detection substance (Aladdin) corresponding to IL6 at a concentration of 0.3mg / mL, vortex mix, place on a shaker in the dark, and react at 37℃ for 2h. Then add 0.5mL of fluorescent particle blocking solution, vortex mix, place on a shaker in the dark, and react at 37℃ for 1h. Then centrifuge at 15000rpm for 15min, remove the supernatant, continue to disperse in 1mL fluorescent particle coating buffer, ultrasonically mix, continue to centrifuge at 15000rpm for 15min, remove the supernatant, and disperse in 0.5mL fluorescent particle preservation buffer. Store at 2℃ in the dark for standby use.

[0069] Add 45 μL of IL6 standard, quality control and test samples into the sample wells of the 96-base transparent ELISA plate coated with the capture substance, where the concentrations of IL6 standard are 21, 7, 2.3, 0.76, 0.25, and 0.028 pg / mL, and place on a shaker at 37°C for 1 hour; wash 5 times with phosphate washing buffer, and set aside after drying.

[0070] Add fluorescent particles coated with the detection substance diluted 20 times with fluorescent particle storage solution to the sample wells of the 96-base transparent ELISA plate that captures IL6, and place on a shaker at 37°C for 1 hour under dark conditions; wash 5 times with phosphate washing buffer, and set aside after drying.

[0071] The 96-base transparent ELISA plate was placed on the sample stage of the fluorescence microscope system, and digital imaging was performed using a 20x objective lens with a CCD and a reflected light path. Further, the image recognition and data processing software imagej, excel, origin and MATLAB were used to draw a standard curve based on the standard sample of the bioactive substance and generate a standard curve equation. The quality control product and body fluid sample signals corresponding to IL6 were further substituted into the equation to calculate the IL6 content in the quality control product and body fluid sample. The results are as follows: Figure 2 shown.

[0072] Figure 2 The results showed that the concentration of IL6 standard had a good correlation with the signal value through four-parameter fitting, R 2 It is 0.997, and the detection limit is 0.028pg / mL.

[0073] Example 2

[0074] Example 2 of the present invention provides a method for detecting Aβ40 based on a biologically active substance detection method using digital fluorescent immunosorbent, and the specific steps are as follows:

[0075] The capture substance corresponding to Aβ40 was diluted to 1.5 μg / mL using the ELISA plate coating buffer. 100 μL of the capture substance dilution corresponding to Aβ40 was added to the sample well of the 96-base transparent ELISA plate, reacted in a 2°C refrigerator for 16 hours, and then washed 5 times with the ELISA plate washing buffer, dried and set aside.

[0076] The fluorescent particle dispersion with a solid content of 1%wt was diluted 20 times with the fluorescent particle coating buffer, ultrasonically mixed, centrifuged at 15000rpm for 15min, the supernatant was removed, and the solution was dispersed into 1mL of fluorescent particle coating buffer, ultrasonically mixed, centrifuged at 15000rpm for 15min, the supernatant was removed, and the solution was dispersed into 1.5mL of fluorescent particle coating buffer. Then, 15μL of 10mg / mL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride ultrapure aqueous solution was added, vortexed, and then 15μL of 10mg / mL N-hydroxysuccinimide ultrapure aqueous solution was added, vortexed. In the dark, the mixture was placed on a shaker at 37°C for 1h. Then, centrifuge at 15000rpm for 15min, remove the supernatant, continue to disperse in 1mL fluorescent particle coating buffer, ultrasonically mix, centrifuge at 15000rpm for 15min, remove the supernatant, continue to disperse in 0.75mL fluorescent particle coating buffer, ultrasonically mix. Then add 0.25mL of the detection substance (Aladdin) corresponding to Aβ40 with a concentration of 0.1mg / mL, vortex mix, place on a shaker in the dark, and react at 37℃ for 2h. Then add 0.5mL of fluorescent particle blocking solution, vortex mix, place on a shaker in the dark, and react at 37℃ for 1h. Then centrifuge at 15000rpm for 15min, remove the supernatant, continue to disperse in 1mL fluorescent particle coating buffer, ultrasonically mix, continue to centrifuge at 15000rpm for 15min, remove the supernatant, and disperse in 0.5mL fluorescent particle preservation buffer. Store at 2℃ in the dark for later use.

[0077] Add 45 μL of Aβ40 standards, quality control substances and test samples into the sample wells of the 96-base transparent ELISA plate coated with the capture substance, where the concentrations of Aβ40 standards are 111, 37, 12.3, 4.1, 1.4, and 0.46 pg / mL, and place on a shaker at 37°C for 1 hour; wash 5 times with phosphate washing buffer, and set aside after drying.

[0078] Add fluorescent particles coated with the detection substance diluted 20 times with fluorescent particle storage solution into the sample wells of the 96-base transparent ELISA plate for capturing Aβ40, and place on a shaker at 37°C for 1 hour under dark conditions; wash 5 times with phosphate washing buffer, and set aside after drying.

[0079] The 96-substrate transparent ELISA plate was placed on the sample stage of the fluorescence microscope system, and digital imaging was performed using a 20x objective lens with a CCD and a reflected light path. Further, the standard curve was drawn based on the standard sample of the bioactive substance using image recognition and data processing software imagej, excel, origin, and MATLAB to generate the standard curve equation. The quality control product and body fluid sample signals corresponding to Aβ40 were further substituted into the equation to calculate the Aβ40 content in the quality control product and body fluid sample, as shown in Figure 3 shown.

[0080] Figure 3 The results showed that the concentration of Aβ40 standard had a good correlation with the signal value through four-parameter fitting, R 2 It is 0.997, and the detection limit is 0.46pg / mL.

[0081] Example 3

[0082] Example 2 of the present invention provides a method for detecting Aβ42 based on a biologically active substance detection method using digital fluorescent immunosorbent, and the specific steps are as follows:

[0083] The capture substance (Aladdin) corresponding to Aβ42 was diluted to 3 μg / mL using the ELISA plate coating buffer. 100 μL of the capture substance dilution corresponding to Aβ42 was added to the sample well of the 96-base transparent ELISA plate, reacted in a 2°C refrigerator for 16 hours, and then washed 5 times with the ELISA plate washing buffer, dried and set aside.

[0084] The fluorescent particle dispersion with a solid content of 1%wt was diluted 20 times with the fluorescent particle coating buffer, ultrasonically mixed, centrifuged at 15000rpm for 15min, the supernatant was removed, and the solution was dispersed into 1mL of fluorescent particle coating buffer, ultrasonically mixed, centrifuged at 15000rpm for 15min, the supernatant was removed, and the solution was dispersed into 1.5mL of fluorescent particle coating buffer. Then, 15μL of 10mg / mL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride ultrapure aqueous solution was added, vortexed, and then 15μL of 10mg / mL N-hydroxysuccinimide ultrapure aqueous solution was added, vortexed. In the dark, the mixture was placed on a shaker at 37°C for 1h. Then, centrifuge at 15000rpm for 15min, remove the supernatant, continue to disperse in 1mL fluorescent particle coating buffer, ultrasonically mix, centrifuge at 15000rpm for 15min, remove the supernatant, continue to disperse in 0.75mL fluorescent particle coating buffer, and ultrasonically mix. Then add 0.25mL of the detection substance (Aladdin) corresponding to Aβ42 with a concentration of 0.3mg / mL, vortex mix, place on a shaker in the dark, and react at 37℃ for 2h. Then add 0.5mL of fluorescent particle blocking solution, vortex mix, place on a shaker in the dark, and react at 37℃ for 1h. Then centrifuge at 15000rpm for 15min, remove the supernatant, continue to disperse in 1mL fluorescent particle coating buffer, ultrasonically mix, continue to centrifuge at 15000rpm for 15min, remove the supernatant, and disperse in 0.5mL fluorescent particle preservation buffer. Store at 8℃ in the dark for later use.

[0085] Add 45 μL of Aβ40 standards, quality control materials and test samples into the sample wells of the 96-base transparent ELISA plate coated with the capture material, where the concentrations of Aβ42 standards are 111, 37, 12.3, 4.1, and 0.46 pg / mL, and place on a shaker at 37°C for 1 hour; wash 5 times with phosphate washing buffer, and set aside after drying.

[0086] Add fluorescent particles coated with the detection substance diluted 20 times with fluorescent particle storage solution into the sample wells of the 96-base transparent ELISA plate for capturing Aβ42, and place on a shaker at 37°C for 1 hour under dark conditions; wash 5 times with phosphate washing buffer, and set aside after drying.

[0087] The 96-base transparent ELISA plate was placed on the sample stage of the fluorescence microscope system, and digital imaging was performed using a 20x objective lens with a CCD and a reflected light path. Further, the standard curve was drawn based on the standard sample of the bioactive substance using image recognition and data processing software imagej, excel, origin and MATLAB to generate the standard curve equation. The quality control product and body fluid sample signals corresponding to Aβ42 were further substituted into the equation to calculate the Aβ42 content in the quality control product and body fluid sample, as shown in Figure 4 shown.

[0088] Figure 4 The results showed that the concentration of Aβ42 standard had a good correlation with the signal value through four-parameter fitting, R 2 It is 0.997, and the detection limit is 0.46pg / mL.

[0089] Example 4

[0090] Example 2 of the present invention provides a method for detecting P-tau181 based on a biologically active substance detection method using digital fluorescent immunosorbent, and the specific steps are as follows:

[0091] The capture substance (Aladdin) corresponding to P-tau181 was diluted to 2.5 μg / mL using the ELISA plate coating buffer. 100 μL of the capture substance dilution corresponding to P-tau181 was added to the sample well of the 96-base transparent ELISA plate, reacted in a 6°C refrigerator for 16 hours, and then washed 5 times with the ELISA plate washing buffer, dried and set aside.

[0092] The fluorescent particle dispersion with a solid content of 1%wt was diluted 20 times with the fluorescent particle coating buffer, ultrasonically mixed, centrifuged at 15000rpm for 15min, the supernatant was removed, and the solution was dispersed into 1mL of fluorescent particle coating buffer, ultrasonically mixed, centrifuged at 15000rpm for 15min, the supernatant was removed, and the solution was dispersed into 1.5mL of fluorescent particle coating buffer. Then, 15μL of 10mg / mL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride ultrapure aqueous solution was added, vortexed, and then 15μL of 10mg / mL N-hydroxysuccinimide ultrapure aqueous solution was added, vortexed. In the dark, the mixture was placed on a shaker at 37°C for 1h. Then, centrifuge at 15000rpm for 15min, remove the supernatant, continue to disperse into 1mL fluorescent particle coating buffer, ultrasonically mix, centrifuge at 15000rpm for 15min, remove the supernatant, continue to disperse into 0.75mL fluorescent particle coating buffer, ultrasonically mix. Then add 0.25mL of the detection substance (Aladdin) corresponding to P-tau181 with a concentration of 0.1-0.3mg / mL, vortex mix, place on a shaker in the dark, and react at 37℃ for 2h. Then add 0.5mL of fluorescent particle blocking solution, vortex mix, place on a shaker in the dark, and react at 37℃ for 1h. Then centrifuge at 15000rpm for 15min, remove the supernatant, continue to disperse into 1mL fluorescent particle coating buffer, ultrasonically mix, continue to centrifuge at 15000rpm for 15min, remove the supernatant, and disperse into 0.5mL fluorescent particle preservation buffer. Store at 2 to 8℃ in the dark for standby use.

[0093] Add 45 μL of P-tau181 standards, quality control products and test samples into the sample wells of the 96-base transparent ELISA plate coated with the capture substance, where the concentrations of P-tau181 standards are 111, 37, 12.3, 1.4, 0.46, and 0.15 pg / mL, and place on a shaker at 37°C for 1 hour; wash 5 times with phosphate washing buffer, and set aside after drying.

[0094] Add fluorescent particles coated with the detection substance diluted 20 times with fluorescent particle storage solution to the sample wells of the 96-base transparent ELISA plate for capturing P-tau181, and place on a shaker at 37°C for 1 hour under dark conditions; wash 5 times with phosphate washing buffer, and set aside after drying.

[0095] The 96-substrate transparent ELISA plate was placed on the sample stage of the fluorescence microscope system, and digital imaging was performed using a 20x objective lens with a CCD and a reflected light path. Further, the image recognition and data processing software imagej, excel, origin and MATLAB were used to draw a standard curve based on the standard sample of the bioactive substance and generate a standard curve equation. The signals of the quality control and body fluid samples corresponding to P-tau181 were further substituted into the equation to calculate the P-tau181 content in the quality control and body fluid samples, as shown in Figure 5 shown.

[0096] Figure 5 The results showed that the concentration of P-tau181 standard had a good correlation with the signal value through four-parameter fitting, R 2 It is 0.996, and the detection limit is 0.15pg / mL.

[0097] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting biologically active substances based on digital fluorescent immunoadsorption, characterized in that: The steps include: (1) The plate-type platform adsorbs the capture substances corresponding to the bioactive substances; (2) The fluorescent particles adsorb the detection substances corresponding to the bioactive substances; (3) Capture of bioactive substances; (4) Fluorescence amplification and digital encoding of captured bioactive substances; (5) Use a fluorescence microscopy system to digitally image the fluorescently amplified and digitally encoded bioactive substances, and combine image recognition and data processing software to generate analysis results.

2. The method for detecting biologically active substances based on digital fluorescent immunoadsorption according to claim 1, characterized in that: In step (1), the plate-type platform is a plate-type platform with a transparent base; the adsorption temperature is 2 to 8° C., and the adsorption time is 15 to 20 hours.

3. The method for detecting biologically active substances based on digital fluorescent immunoadsorption according to claim 1, characterized in that: In step (2), the step of adsorbing the detection substance corresponding to the biologically active substance by the fluorescent particles is as follows: A. Dilute the aqueous dispersion of fluorescent particles with fluorescent particle coating buffer, centrifuge, remove the supernatant, and repeat the centrifugation step; after the centrifugation is completed, discard the supernatant, re-add the fluorescent particle coating buffer for dilution, and then add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride ultrapure aqueous solution and N-hydroxysuccinimide ultrapure aqueous solution in sequence to react; after the reaction is completed, centrifuge and discard the supernatant; B. Take the precipitate and continue to disperse it in 1 mL of fluorescent particle coating buffer. After centrifugation and discarding the supernatant, the precipitate is dispersed with fluorescent particle coating buffer. After mixing, add fluorescent particles to adsorb the corresponding detection substances of the bioactive substances to react. After the reaction is completed, add blocking solution to block.

4. The method for detecting biologically active substances based on digital fluorescent immunoadsorption according to claim 3, characterized in that: In step (2), the fluorescent particle coating buffer is composed of 10 mol / L tris(hydroxymethylaminomethane) and 0.05%wt ProClin 300 ultrapure aqueous solution; the concentration of the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride ultrapure aqueous solution is 8-12 mg / mL, and the concentration of the N-hydroxysuccinimide ultrapure aqueous solution is 8-12 mg / mL; the volume ratio of the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride ultrapure aqueous solution to the N-hydroxysuccinimide ultrapure aqueous solution is 1-2:1-2; the blocking solution is composed of 5 mol / L boric acid, 0.05%wt Tween-20, 1%wt bovine serum albumin, 5%wt trehalose and 0.1%wt ProClin 300 ultrapure aqueous solution; In step (2) A, the reaction temperature is 35-38° C., the reaction time is 1-2 h, and the reaction condition is dark; in step B, the reaction temperature is 35-38° C., the reaction time is 2-3 h, and the reaction condition is dark.

5. The method for detecting biologically active substances based on digital fluorescent immunoadsorption according to claim 1, characterized in that: In step (3), the step of capturing the bioactive substance comprises: The standard substance of the bioactive substance, the quality control substance and the sample to be tested are added to the plate platform on which the capture substance corresponding to the bioactive substance is adsorbed in step (1), and placed on a shaker, reacted, washed and dried.

6. The method for detecting biologically active substances based on digital fluorescent immunoadsorption according to claim 1, characterized in that: In step (4), the steps of fluorescence amplification and digital encoding of the captured bioactive substances include: In step (3), the fluorescent particles adsorbed with the detection substance corresponding to the bioactive substance prepared in step (2) are added to the plate platform for capturing the bioactive substance, and reacted, washed and dried.

7. The method for detecting biologically active substances based on digital fluorescent immunoadsorption according to claim 6, characterized in that: In step (4), the reaction temperature is 35-38° C., the reaction time is 1-2 h, and the reaction condition is dark.

8. The method for detecting biologically active substances based on digital fluorescent immunoadsorption according to claim 1, characterized in that: In step (5), the steps of using a fluorescence microscopy imaging system to digitally image the fluorescence-amplified and digitally encoded biologically active substances and combining image recognition and data processing software to generate analysis results include: The plate platform after the reaction in step (4) is placed on the sample stage of the fluorescence microscope system, and digital imaging is performed by using the transmission or reflection light path through the objective lens and CCD; the imaging result is input into the image recognition and data processing software, and a standard curve is drawn according to the standard sample of the bioactive substance, and the standard curve equation is generated. The signals of the quality control product and the sample to be tested corresponding to the bioactive substance are substituted into the equation to calculate the content of the bioactive substance in the quality control product and the body fluid sample.

9. The method for detecting biologically active substances based on digital fluorescent immunoadsorption according to claim 8, characterized in that: In step (5), the objective lens is a 20x, 40x or 50x objective lens; the image recognition and data processing software includes: imagej, excel, origin and MATLAB.

10. Use of the method for detecting biologically active substances based on digital fluorescent immunoadsorption according to any one of claims 1 to 9 in detecting biologically active substances.

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