Biomarker multi-joint detection method based on single wavelength fluorescence coding

Abstract

The invention provides a biomarker multi-joint detection method based on single-wavelength fluorescence coding, and belongs to the technical field of biomarker detection. The method comprises the following steps of: performing fluorescence amplification and digital coding on a biomarker by using processed single-wavelength fluorescent particles through a sample adding area of a sample in an arrayed coating plate type platform; a plurality of biomarkers in a sample can be further quantitatively analyzed through a fluorescence microscopic imaging system (non-flow fluorescence detection and a multi-fluorescence channel module) in combination with image recognition and data analysis software; the multiplex detection technology has the advantages of low cost, simplicity in use, medium and high throughput and the like, and shows huge advantages in the field of biotechnology.

Description

Technical Field

[0001] This invention relates to the field of biomarker detection technology, and in particular to a method for multiple detection of biomarkers based on single-wavelength fluorescence encoding. Background Technology

[0002] Current biomarker multi-detection technologies face numerous limitations in practical applications. These technologies rely on a variety of magnetic beads with different fluorescence emission characteristics, each bead corresponding to a specific detection target, which places extremely high demands on the research and development of these magnetic beads.

[0003] Meanwhile, the supporting analytical equipment must be equipped with a flow cytometry fluorescence detection module and multi-fluorescence channel detection function to achieve simultaneous analysis of multiple biomarkers. However, both the preparation of special magnetic beads and the development of multifunctional analytical equipment face significant technical challenges and high research and development costs.

[0004] From an operational perspective, the entire testing process is relatively complex due to the steps involved in processing multi-component magnetic beads and calibrating and interpreting multi-channel signals, requiring a high level of professional expertise from operators. These factors combined not only increase the barriers to technology promotion but also limit its widespread adoption in grassroots medical institutions and other settings, significantly restricting the market application of this technology.

[0005] Therefore, it is essential to provide a multi-inspection technology that is simple to operate and conducive to large-scale application. Summary of the Invention

[0006] The purpose of this invention is to provide a method for multiple detection of biomarkers based on single-wavelength fluorescence encoding. This method has the advantages of low cost, simple use, medium to high throughput, and multiple detection, which is conducive to the large-scale application in the biological diagnostics market.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for multiple detection of biomarkers based on single-wavelength fluorescence encoding, comprising the following steps: (1) Prepare a single-wavelength fluorescent particle dispersion, a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, and an N-hydroxysuccinimide solution; (2) Dilute the concentration of the capture antibody corresponding to the biomarker to obtain the diluent of the capture biomarker, and add the diluent to the plate platform in an array layout to obtain the plate platform coated with the biomarker; (3) Couple the single-wavelength fluorescent particle dispersion, centrifuge, remove the supernatant, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and N-hydroxysuccinimide solution in sequence, react, centrifuge, remove the supernatant, couple again, centrifuge, add the analyte solution containing the biomarker, block the reaction, centrifuge, remove the supernatant, continue coupling, centrifuge, remove the supernatant, and obtain the analyte sample containing the biomarker; (4) Add the standard containing the biomarker, the test sample containing the biomarker, or the quality control containing the biomarker to the plate platform coated with the biomarker, react, use a monochrome fluorescence module for digital imaging, plot a standard curve, and calculate the concentration of the biomarker contained in the quality control solution and the test sample.

[0008] Preferably, the preparation method of the single-wavelength fluorescent particle dispersion in step (1) is as follows: dissolve tris(hydroxymethyl)aminomethane, sodium chloride, Tween-20, bovine serum albumin, trehalose and Proclin 300 in water to obtain the dispersion; the mass-volume ratio of tris(hydroxymethyl)aminomethane, sodium chloride, Tween-20, bovine serum albumin, trehalose, Proclin 300 and water is 60~61g: 85~90g: 240~260µL: 0.5~1.5g: 4~6g: 90~110mg: 4~6mL.

[0009] Preferably, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution in step (1) is prepared by dissolving 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride in a single-wavelength fluorescent particle coupling buffer. The N-hydroxysuccinimide solution is prepared by dissolving N-hydroxysuccinimide in a single-wavelength fluorescent particle coupling buffer. The single-wavelength fluorescent particle coupling buffer is prepared by dissolving 2-morpholinoethanesulfonic acid and Proclin300 in water and adjusting the pH to 6.2-6.4.

[0010] Preferably, the mass-to-volume ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to single-wavelength fluorescent particle coupling buffer is 90-100 mg: 9-10 ml; the mass-to-volume ratio of N-hydroxysuccinimide to single-wavelength fluorescent particle coupling buffer is 90-100 mg: 9-10 ml; and the mass-to-volume ratio of 2-morpholine ethanesulfonic acid, Proclin 300, and water is 19-20 g: 50-55 mg: 70-80 ml.

[0011] Preferably, the coupling in step (3) uses a single-wavelength fluorescent particle coupling buffer.

[0012] Preferably, the concentration of the diluent for capturing biomarkers in step (2) is 2~3µg / ml; the array layout is a triangular array; the plate platform includes a 96-well transparent substrate enzyme labeling plate, a 384-well transparent substrate enzyme labeling plate, a transparent glass slide or a transparent plastic sheet.

[0013] Preferably, the centrifugal force in step (3) is 1900~2100g and the centrifugation time is 8~12min.

[0014] Preferably, the blocking reaction in step (3) uses a single-wavelength fluorescent particle blocking solution; the single-wavelength fluorescent particle blocking solution is prepared by dissolving boric acid, sodium tetraborate decahydrate, Tween-20, bovine serum albumin and ethanolamine in water; the mass-volume ratio of boric acid, sodium tetraborate decahydrate, Tween-20, bovine serum albumin, ethanolamine and water is 30~32g: 425~430mg: 480~520µl: 1.8~2.2g: 460~500mg: 90~100ml.

[0015] Preferably, the reaction temperature in step (4) is 35~40℃, and the reaction time is 0.8~1.2h; the digital imaging method is transmission or reflection.

[0016] Preferably, the biomarkers include IL-6, IL-8, and IL-10.

[0017] The beneficial effects of this invention compared to the prior art are as follows: This invention employs a biomarker multiplex detection method that, by combining a plate platform, retains the operational habits of traditional enzyme-linked immunosorbent assay (ELISA). Through fluorescence encoding and digital magnification, digital imaging is performed using a monochromatic fluorescence microscopy system, combined with digital analysis, enabling the detection of multiple biomarkers within a single sample application area on the plate platform. This reduces reagent and sample volume and repetitive operational steps. This method offers advantages such as low cost, ease of use, medium-to-high throughput, and multiplex detection, making it highly suitable for large-scale application in the biodiagnostics market.

[0018] The biomarker multi-detection method provided by this invention can detect multiple proteins (IL-6, IL-8, IL-10) in a single sample. The standard curves for protein (IL-6, IL-8, IL-10) detection generated by the method provided by this invention exhibit excellent linearity, R0. 2 All are greater than 0.99. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 A schematic diagram illustrating the basic principle of the detection method provided by this invention; Figure 2 The results are fluorescence microscopy images of the IL-6, IL-8 and IL-10 arrays in a single well of an ELISA plate. Figure 3 Digital imaging and analysis results of a microscopic system for detecting human interleukin-6 (IL-6); Figure 4 The standard curve for human interleukin-6 (IL-6); Figure 5 Digital imaging and analysis results of a microscopic system for detecting human interleukin-8 (IL-8); Figure 6 The standard curve for human interleukin-8 (IL-8); Figure 7 Digital imaging and analysis results of a microscopic system for detecting human interleukin-10 (IL-10); Figure 8 The standard curve for human interleukin-10 (IL-10); Figure 9 This is a schematic diagram of the sample loading layout for a comparative enzyme-linked immunosorbent assay (ELISA) plate. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] This invention provides a method for multiple detection of biomarkers based on single-wavelength fluorescence encoding, comprising the following steps: (1) Prepare a single-wavelength fluorescent particle dispersion, a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, and an N-hydroxysuccinimide solution; (2) Dilute the concentration of the capture antibody corresponding to the biomarker to obtain the diluent of the capture biomarker, and add the diluent to the plate platform in an array layout to obtain the plate platform coated with the biomarker; (3) Couple the single-wavelength fluorescent particle dispersion, centrifuge, remove the supernatant, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and N-hydroxysuccinimide solution in sequence, react, centrifuge, remove the supernatant, couple again, centrifuge, add the analyte solution containing the biomarker, block the reaction, centrifuge, remove the supernatant, continue coupling, centrifuge, remove the supernatant, and obtain the analyte sample containing the biomarker; (4) Add the standard containing the biomarker, the test sample containing the biomarker, or the quality control containing the biomarker to the plate platform coated with the biomarker, react, use a monochrome fluorescence module for digital imaging, plot a standard curve, and calculate the concentration of the biomarker contained in the quality control solution and the test sample.

[0027] In this invention, the preferred method for preparing the single-wavelength fluorescent particle dispersion in step (1) is to dissolve tris(hydroxymethyl)aminomethane, sodium chloride, Tween-20, bovine serum albumin, trehalose, and Proclin 300 in water; the preferred mass-to-volume ratio of tris(hydroxymethyl)aminomethane, sodium chloride, Tween-20, bovine serum albumin, trehalose, Proclin 300, and water is 60~61g:85~90g:240~260µL:0.5~1.5g:4~6g:90~110mg:4~6mL, and more preferably 60.4~60.8g:8 The preferred concentrations are: 6~88g: 245~255µL: 0.8~1.2g: 4.5~5.5g: 95~105mg: 4.5~5.5mL, and more preferably 60.6g: 87g: 250µL: 1.0g: 5g: 100mg: 5mL; the preferred method for preparing the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is to dissolve 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride in a single-wavelength fluorescent particle coupling buffer; the preferred method for preparing the N-hydroxysuccinimide solution is to dissolve N-hydroxysuccinimide in a single-wavelength fluorescent particle coupling buffer. Succinimide is dissolved in a single-wavelength fluorescent particle coupling buffer; the preferred method for preparing the single-wavelength fluorescent particle coupling buffer is to dissolve 2-morpholine ethanesulfonic acid and Proclin 300 in water and adjust the pH to 6.2-6.4; the pH is further preferably 6.3; the preferred mass-to-volume ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to the single-wavelength fluorescent particle coupling buffer is 90-100 mg: 9-10 ml, further preferably 94-98 mg: 9.4-9.8 ml, and even more preferably 95 mg: 9.5 ml. ml; the preferred mass-to-volume ratio of N-hydroxysuccinimide to single-wavelength fluorescent particle coupling buffer is 90-100 mg: 9-10 ml, more preferably 94-98 mg: 9.4-9.8 ml, and even more preferably 95 mg: 9.5 ml; the preferred mass-to-volume ratio of 2-morpholine ethanesulfonic acid, Proclin300 to water is 19-20 g: 50-55 mg: 70-80 ml, more preferably 19.4-19.8 g: 52-54 mg: 74-78 ml, and even more preferably 19.5 g: 53 mg: 75 ml.

[0028] In this invention, the concentration of the diluent for capturing biomarkers in step (2) is preferably 2~3µg / ml, more preferably 2.4~2.8µg / ml, and even more preferably 2.5µg / ml; the array layout is preferably a triangular array; when adding the diluent, it is preferred to use a spotting device to place 1µl of the diluent according to the three vertices of the triangle and add it clockwise; the plate platform preferably includes a 96-well transparent substrate enzyme labeling plate, a 384-well transparent substrate enzyme labeling plate, a transparent glass slide or a transparent plastic sheet; the material of the plate platform is preferably glass, polystyrene, polymethyl methacrylate or polysiloxane.

[0029] In this invention, the coupling in step (3) preferably uses a single-wavelength fluorescent particle coupling buffer; the volume ratio of the single-wavelength fluorescent particle dispersion to the single-wavelength fluorescent particle coupling buffer is preferably 140~160µl:2~4ml, more preferably 150µl:3ml; the mass-volume concentration of the single-wavelength fluorescent particle dispersion is preferably 8~12mg / ml, more preferably 10mg / ml; the ultrasonic treatment is preferably performed before centrifugation, and the ultrasonic time is preferably 2~4min, more preferably 3min; the centrifugal force is preferably 1900~2100g, more preferably 1950~2050g, and even more preferably 2000g; the centrifugation time is preferably 8~12min, more preferably 9~11min, and even more preferably 10min; after the re-coupling, the product obtained after centrifugation is preferably divided into 3 portions, and a single-wavelength fluorescent particle coupling buffer is added to each portion. Wavelength fluorescent particle coupling buffer; after adding the analyte solution containing the biomarker, a shaker reaction is preferably performed, the temperature of which is preferably 35~40℃, more preferably 36~38℃, and even more preferably 37℃; the shaker reaction time is preferably 1.5~2.5h, more preferably 2h; a single-wavelength fluorescent particle blocking solution is preferably used for the blocking reaction; the single-wavelength fluorescent particle blocking solution is preferably prepared by dissolving boric acid, sodium tetraborate decahydrate, Tween-20, bovine serum albumin, and ethanolamine in water; the mass-volume ratio of boric acid, sodium tetraborate decahydrate, Tween-20, bovine serum albumin, ethanolamine, and water is preferably 30~32g:425~430mg:480~520µl:1.8~2.2g:460~500mg:90~100ml, more preferably 31g:427mg:500µl:2g:480mg:95ml.

[0030] In this invention, the preferred method for adding the biomarker-containing standard in step (4) is as follows: IL-6, IL-8, and IL-10 standards are mixed to obtain five mixed standards, which are then added to a plate platform, reacted on a shaker, washed, dried, diluted with a single-wavelength fluorescent particle preservation solution, and added to the plate platform coated with the biomarker. The preferred temperature for the shaker reaction is 35-40°C, more preferably 36-38°C, and even more preferably 37°C. The preferred reaction time is 0.8-1.2 h, more preferably... The washing time is selected as 0.9~1.1h, more preferably 1h; the washing process preferably uses phosphate buffered washing solution; the phosphate buffered washing solution is prepared by dissolving sodium hydrogen phosphate, sodium dihydrogen phosphate and Tween-20 in water; the preferred mass-to-volume ratio of sodium hydrogen phosphate, sodium dihydrogen phosphate, Tween-20 and water is 3.2~3.5g:6.30~6.35g:4~6ml:0.8~1.2L, more preferably 3.4g:6.32g:5ml:1L; the single-wavelength fluorescent particle preservation solution The preferred preparation method is as follows: dissolve tris(hydroxymethyl)aminomethane, sodium chloride, Tween-20, bovine serum albumin, trehalose, and Proclin 300 in water; the preferred mass-to-volume ratio of tris(hydroxymethyl)aminomethane, sodium chloride, Tween-20, bovine serum albumin, trehalose, Proclin 300, and water is 60-61 g: 85-90 g: 240-260 µl: 0.8-1.2 g: 4-6 g: 90-110 mg: 4-6 ml, and more preferably 60.6 g: 87.7 g: 250 µl: 1 g The reaction mixture is 5g:100mg:5ml; the preferred reaction temperature is 35~40℃, more preferably 36~38℃, and even more preferably 37℃; the preferred reaction time is 0.8~1.2h, more preferably 0.9~1.1h, and even more preferably 1h; after the reaction, the sample is preferably washed 4~6 times with phosphate buffer and patted dry; more preferably, it is washed 5 times; the preferred digital imaging method is transmission or reflection; the preferred methods for digital imaging are 20x, 40x, and 50x objectives.

[0031] In this invention, the biomarkers preferably include IL-6, IL-8 and IL-10.

[0032] Example 1

[0033] A method for detecting multiple biomarkers IL-6, IL-8, and IL-10 based on single-wavelength fluorescence encoding, comprising the following steps: (1) Dissolve 60.6g of tris(hydroxymethyl)aminomethane, 87.7g of sodium chloride, 250µL of Tween-20, 1g of bovine serum albumin, 5g of trehalose, and 100mg of Proclin 300 in 5mL of water to obtain a single-wavelength fluorescent particle dispersion; dissolve 19.5g of 2-morpholinoethanesulfonic acid and 52.5mg of Proclin 300 in 80mL of water, adjust the pH to 6.3 with 280mg / mL KOH ultrapure water, and bring the volume to 100mL with ultrapure water to obtain a single-wavelength fluorescent particle coupling buffer; dissolve 100mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride in 10mL of single-wavelength fluorescent particle coupling buffer to obtain a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution; dissolve 100mg of... N-hydroxysuccinimide was dissolved in 10 ml of single-wavelength fluorescent particle coupling buffer to obtain an N-hydroxysuccinimide solution; 1.59 g of sodium carbonate and 2.94 g of sodium bicarbonate were placed in a beaker, and ultrapure water was added to bring the volume to 1 L. The mixture was stirred thoroughly to dissolve and obtain a plate-type platform coating buffer; 3.40 g of sodium hydrogen phosphate, 6.32 g of sodium dihydrogen phosphate, 20.0 g of bovine serum albumin and 1.05 g of Proclin 300 were placed in a beaker, and ultrapure water was added to bring the volume to 1 L. The mixture was stirred thoroughly to dissolve and obtain a plate-type platform blocking buffer. (2) Dilute the capture antibodies corresponding to IL6, IL8 and IL10 to 2µg / ml using plate platform coating buffer to obtain the capture biomarker dilution. Use a spotting device to add 1µl of the dilution in a clockwise direction to the three vertices of a triangle to form an array of capture substances, and obtain an ELISA plate coated with IL-6, IL-8 and IL-10. Incubate at 4℃ for 12h. Wash the plate 5 times with washing buffer (add 1.59g sodium carbonate, 2.94g sodium bicarbonate and 5mL Tween-20 to a beaker, add ultrapure water to make up to 1L, stir thoroughly to dissolve) and pat dry. Then add plate platform blocking buffer, place on a shaker, incubate at 37℃ for 1h, wash the plate 5 times with washing buffer, pat dry and set aside. (3) Add 150µl of a single-wavelength fluorescent particle dispersion with a concentration of 10mg / ml to 3ml of single-wavelength fluorescent particle coupling buffer, sonicate for 3min, centrifuge at 2000g for 10min, remove the supernatant, add 3ml of single-wavelength fluorescent particle coupling buffer, sonicate for 3min, centrifuge at 2000g for 10min, remove the supernatant, add 3ml of single-wavelength fluorescent particle coupling buffer, sonicate for 3min, and then add 30µl of single-wavelength fluorescent particle coupling buffer in sequence. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and N-hydroxysuccinimide solution were sonicated for 1 min, reacted on a shaker at 37°C for 30 min, centrifuged at 2000g for 10 min, the supernatant was discarded, and 3 ml of single-wavelength fluorescent particle coupling buffer was added. The obtained product was divided into 3 aliquots, sonicated for 3 min, centrifuged at 2000g for 10 min, and the supernatant was discarded. 750 µl of single-wavelength fluorescent particle coupling buffer was added to each aliquot, and the mixture was sonicated for 3 min. Then, 250 µl of analyte solutions containing IL-6, IL-8, and IL-10 (0.2 mg / ml concentrations) were added respectively. The mixture was vortexed for 1 min, mixed, and reacted on a shaker at 37°C in the dark for 2 h. Add 500µl of single-wavelength fluorescent particle blocking buffer (31g boric acid, 427mg sodium tetraborate decahydrate, 500µl Tween-20, 2.0g bovine serum albumin, and 480mg ethanolamine dissolved in 100ml water), vortex for 1min, mix well, react at 37℃ in the dark on a shaker for 1h, centrifuge at 2000g for 10min, discard the supernatant, add 1ml of single-wavelength fluorescent particle coupling buffer, sonicate for 3min, centrifuge at 2000g for 10min, discard the supernatant, and obtain the test sample containing the biomarker; add 500µl of single-wavelength fluorescent particle preservation buffer (60.6g tris(hydroxymethyl)aminomethane, 87.7g sodium chloride, 250µL Tween-20, 1g bovine serum albumin, 5.00g trehalose, and 100mg Proclin 300 dissolved in 5mL ultrapure water), and store at 4℃ for later use; (4) Mix the standard solutions of IL-6, IL-8 and IL-10 to obtain five 45µl mixed standards. Add the five mixed standards to different wells of the microplate (i.e., add one mixed standard to each well). Incubate at 37℃ for 1 h with shaking. Wash five times with 300µl phosphate buffer (dissolve 3.4g sodium hydrogen phosphate, 6.32g sodium dihydrogen phosphate and 5ml Tween-20 in 1L water). Pat dry to obtain IL-6 standards with concentrations of 2000, 222, 24, 2.7 and 0 pg / mL, IL-8 standards with concentrations of 1000, 333, 37, 4.1 and 0 pg / mL, and IL-10 standards with concentrations of 7500, 833, 277, 10.3 and 0 pg / mL. Dilute 20 times with single-wavelength fluorescent particle preservation solution and add 50µl of each to the coating. In an ELISA plate containing IL-6, IL-8, and IL-10, either the test sample or the quality control sample containing IL-6, IL-8, and IL-10 is added to the IL-6, IL-8, and IL-10-coated ELISA plate. The plate is incubated at 37°C for 1 hour, washed five times with 300 µl of phosphate buffer, and then patted dry. The ELISA plate is then placed on the sample stage of a fluorescence microscopy system. Using 20x, 40x, and 50x objectives and a monochromatic fluorescence module, digital imaging of the arrayed coated area within each well is performed via transmission or reflection. A standard curve correlation equation is established using counting and data processing software, and a standard curve is plotted. The signal values ​​of the quality control sample and the test sample are substituted into the standard curve equation to calculate the concentrations of IL-6, IL-8, and IL-10 in the quality control solution and the test sample.

[0034] The counting and data processing software results showed that, through four-parameter fitting, the concentrations of IL-6, IL-8, and IL-10 standards had a good correlation with the signal values, R0 2 The values ​​are 0.999, 0.998, and 0.996, respectively.

[0035] Example 2

[0036] A method for detecting multiple biomarkers IL-6, IL-8, and IL-10 based on single-wavelength fluorescence encoding, comprising the following steps: (1) Dissolve 60g of tris(hydroxymethyl)aminomethane, 85g of sodium chloride, 240µL of Tween-20, 0.5g of bovine serum albumin, 4g of trehalose, and 90mg of Proclin 300 in 4mL of water to obtain a single-wavelength fluorescent particle dispersion; dissolve 19g of 2-morpholinoethanesulfonic acid and 50mg of Proclin 300 in 70mL of water and adjust the pH to 6.2 to obtain a single-wavelength fluorescent particle coupling buffer; dissolve 90mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride in 9mL of the single-wavelength fluorescent particle coupling buffer to obtain a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution; dissolve 90mg of... N-hydroxysuccinimide was dissolved in 9 ml of single-wavelength fluorescent particle coupling buffer to obtain an N-hydroxysuccinimide solution; 1.59 g of sodium carbonate and 2.94 g of sodium bicarbonate were placed in a beaker, and ultrapure water was added to bring the volume to 1 L. The mixture was stirred thoroughly to dissolve and obtain a plate-type platform coating buffer; 3.40 g of sodium hydrogen phosphate, 6.32 g of sodium dihydrogen phosphate, 20.0 g of bovine serum albumin and 1.05 g of Proclin 300 were placed in a beaker, and ultrapure water was added to bring the volume to 1 L. The mixture was stirred thoroughly to dissolve and obtain a plate-type platform blocking buffer. (2) Dilute the capture antibodies corresponding to IL6, IL8 and IL10 to 2.5 µg / ml using plate platform coating buffer to obtain the capture biomarker dilution. Use a spotting device to add 1 µl of the dilution in a clockwise direction to the three vertices of a triangle to form an array of capture substances, and obtain an ELISA plate coated with IL-6, IL-8 and IL-10. Incubate at 4℃ for 12 h, wash the plate 5 times with washing buffer (add 1.59 g sodium carbonate, 2.94 g sodium bicarbonate and 5 mL Tween-20 to a beaker, add ultrapure water to make up to 1 L, stir thoroughly to dissolve) and blot dry. Then add plate platform blocking buffer, place on a shaker, incubate at 37℃ for 1 h, wash the plate 5 times with washing buffer, blot dry and set aside. (3) Add 140µl of a single-wavelength fluorescent particle dispersion with a concentration of 8mg / ml to 2ml of single-wavelength fluorescent particle coupling buffer, sonicate for 2min, centrifuge at 1900g for 12min, remove the supernatant, add 2~4ml of single-wavelength fluorescent particle coupling buffer, sonicate for 2min, centrifuge at 1900g for 12min, remove the supernatant, add 2~4ml of single-wavelength fluorescent particle coupling buffer, sonicate for 2min, and then add 30µl of the mixture sequentially. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and N-hydroxysuccinimide solution were sonicated for 1 min, reacted on a shaker at 35°C for 35 min, centrifuged at 1900g for 12 min, the supernatant was discarded, and 2 ml of single-wavelength fluorescent particle coupling buffer was added. The obtained product was divided into 3 aliquots, sonicated for 2 min, centrifuged at 1900g for 12 min, the supernatant was discarded, and 750 µl of single-wavelength fluorescent particle coupling buffer was added to each aliquot. After sonication for 2 min, 250 µl of analyte solutions containing IL-6, IL-8, and IL-10 (0.2 mg / ml concentrations) were added respectively. The mixture was vortexed for 1 min, mixed, and reacted on a shaker at 35°C in the dark for 2.5 h. Prepare 500 µl of single-wavelength fluorescent particle blocking buffer (30 g boric acid, 425 mg sodium tetraborate decahydrate, 480 µl Tween-20, 1.8 g bovine serum albumin, and 460 mg ethanolamine dissolved in 90 mL of water), vortex for 1 min, mix well, and react in a shaker at 35 °C in the dark for 1.2 h. Centrifuge at 1900 g for 12 min, discard the supernatant, add 1 mL of single-wavelength fluorescent particle coupling buffer, sonicate for 2 min, centrifuge at 1900 g for 12 min, discard the supernatant, and obtain the test sample containing the biomarker; add 500 µl of single-wavelength fluorescent particle preservation buffer (60.6 g tris(hydroxymethyl)aminomethane, 87.7 g sodium chloride, 250 µL Tween-20, 1 g bovine serum albumin, 5.00 g trehalose, and 100 mg Proclin 300 dissolved in 5 mL of ultrapure water), and store at 4 °C for later use; (4) Mix the IL-6, IL-8 and IL-10 standards to obtain five 45µl mixed standards. Add the five mixed standards to different wells of the microplate (i.e., add one mixed standard to each well). Incubate at 35℃ with shaking for 1.2h. Wash five times with 300µl phosphate buffer (dissolve 3.2g sodium hydrogen phosphate, 6.30g sodium dihydrogen phosphate and 4ml Tween-20 in 0.8L water), pat dry, and obtain IL-6 standards with concentrations of 2000, 222, 24, 2.7 and 0 pg / mL, IL-8 standards with concentrations of 1000, 333, 37, 4.1 and 0 pg / mL, and IL-10 standards with concentrations of 7500, 833, 277, 10.3 and 0 pg / mL. Dilute 20 times with single-wavelength fluorescent particle preservation solution and add 50µl of each to the coating. In an ELISA plate containing IL-6, IL-8, and IL-10, either the test sample or the quality control sample containing IL-6, IL-8, and IL-10 is added to the IL-6, IL-8, and IL-10-coated ELISA plate. The plate is incubated at 35°C for 1.2 h, washed four times with 300 µl of phosphate buffer, and patted dry. The ELISA plate is then placed on the sample stage of a fluorescence microscopy system. Using 20x, 40x, and 50x objectives and a monochromatic fluorescence module, the arrayed coated area within each well is digitally imaged via transmission or reflection. A standard curve correlation equation is established using counting and data processing software, and a standard curve is plotted. The signal values ​​of the quality control sample and the test sample are substituted into the standard curve equation to calculate the concentrations of IL-6, IL-8, and IL-10 in the quality control solution and the test sample.

[0037] Example 3

[0038] A method for detecting multiple biomarkers IL-6, IL-8, and IL-10 based on single-wavelength fluorescence encoding, comprising the following steps: (1) Dissolve 61g of tris(hydroxymethyl)aminomethane, 90g of sodium chloride, 260µL of Tween-20, 1.5g of bovine serum albumin, 6g of trehalose, and 110mg of Proclin 300 in 6mL of water to obtain a single-wavelength fluorescent particle dispersion; dissolve 20g of 2-morpholinoethanesulfonic acid and 55mg of Proclin 300 in 75mL of water and adjust the pH to 6.4 to obtain a single-wavelength fluorescent particle coupling buffer; dissolve 95mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride in 9.5mL of single-wavelength fluorescent particle coupling buffer to obtain a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution; dissolve 95mg of... N-hydroxysuccinimide was dissolved in 9.5 ml of single-wavelength fluorescent particle coupling buffer to obtain an N-hydroxysuccinimide solution; 1.59 g of sodium carbonate and 2.94 g of sodium bicarbonate were placed in a beaker, and ultrapure water was added to bring the volume to 1 L. The mixture was stirred thoroughly to dissolve and obtain a plate-type platform coating buffer; 3.40 g of sodium hydrogen phosphate, 6.32 g of sodium dihydrogen phosphate, 20.0 g of bovine serum albumin and 1.05 g of Proclin 300 were placed in a beaker, and ultrapure water was added to bring the volume to 1 L. The mixture was stirred thoroughly to dissolve and obtain a plate-type platform blocking buffer. (2) Dilute the capture antibodies corresponding to IL6, IL8 and IL10 to 3µg / ml using plate platform coating buffer to obtain the capture biomarker dilution. Use a spotting device to add 1µl of the dilution in a clockwise direction to the three vertices of a triangle to form an array of capture substances, and obtain an ELISA plate coated with IL-6, IL-8 and IL-10. Incubate at 4℃ for 12h, wash the plate 5 times with washing buffer (add 1.59g sodium carbonate, 2.94g sodium bicarbonate and 5mL Tween-20 to a beaker, add ultrapure water to make up to 1L, stir thoroughly to dissolve) and blot dry. Then add plate platform blocking buffer, place on a shaker, incubate at 37℃ for 1h, wash the plate 5 times with washing buffer, blot dry and set aside. (3) Add 160µl of a single-wavelength fluorescent particle dispersion with a concentration of 12mg / ml to 4ml of single-wavelength fluorescent particle coupling buffer, sonicate for 4min, centrifuge at 2100g for 8min, remove the supernatant, add 4ml of single-wavelength fluorescent particle coupling buffer, sonicate for 4min, centrifuge at 2100g for 8min, remove the supernatant, add 4ml of single-wavelength fluorescent particle coupling buffer, sonicate for 4min, and then add 30µl of the mixture sequentially. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and N-hydroxysuccinimide solution were sonicated for 1 min, reacted on a shaker at 40°C for 25 min, centrifuged at 2100g for 8 min, the supernatant was discarded, and 4 ml of single-wavelength fluorescent particle coupling buffer was added. The obtained product was divided into 3 aliquots, sonicated for 4 min, centrifuged at 2100g for 8 min, the supernatant was discarded, and 750 µl of single-wavelength fluorescent particle coupling buffer was added to each aliquot. After sonication for 4 min, 250 µl of analyte solutions containing IL-6, IL-8, and IL-10 (0.2 mg / ml concentrations) were added respectively. The aliquots were vortexed for 1 min, mixed, and reacted on a shaker at 40°C in the dark for 1.5 h. Prepare 500 µl of single-wavelength fluorescent particle blocking buffer (32 g boric acid, 430 mg sodium tetraborate decahydrate, 520 µl Tween-20, 2.2 g bovine serum albumin, and 500 mg ethanolamine dissolved in 100 ml water), vortex for 1 min, mix well, react at 40 °C in the dark on a shaker for 0.8 h, centrifuge at 2100 g for 8 min, discard the supernatant, add 1 ml of single-wavelength fluorescent particle coupling buffer, sonicate for 4 min, centrifuge at 2100 g for 8 min, discard the supernatant to obtain the test sample containing the biomarker; add 500 µl of single-wavelength fluorescent particle preservation buffer (60.6 g tris(hydroxymethyl)aminomethane, 87.7 g sodium chloride, 250 µl Tween-20, 1 g bovine serum albumin, 5.00 g trehalose, and 100 mg Proclin 300 dissolved in 5 mL ultrapure water), and store at 4 °C for later use; (4) Mix the IL-6, IL-8 and IL-10 standards to obtain five 45µl mixed standards. Add the five mixed standards to different wells of the microplate (i.e., add one mixed standard to each well). Incubate at 40℃ on a shaker for 0.8h. Wash five times with 300µl phosphate buffer (dissolve 3.5g sodium hydrogen phosphate, 6.35g sodium dihydrogen phosphate and 6ml Tween-20 in 1.2L water), pat dry, and obtain IL-6 standards with concentrations of 2000, 222, 24, 2.7 and 0 pg / mL, IL-8 standards with concentrations of 1000, 333, 37, 4.1 and 0 pg / mL, and IL-10 standards with concentrations of 7500, 833, 277, 10.3 and 0 pg / mL. Dilute 20 times with single-wavelength fluorescent particle preservation solution and add 50µl of each to the coating. In an ELISA plate containing IL-6, IL-8, and IL-10, either the test sample or the quality control sample containing IL-6, IL-8, and IL-10 is added to the IL-6, IL-8, and IL-10-coated ELISA plate. The plate is incubated at 40°C for 0.8 h, washed six times with 300 µl phosphate buffer, and then patted dry. The ELISA plate is then placed on the sample stage of a fluorescence microscopy system. Using 20x, 40x, and 50x objectives and a monochromatic fluorescence module, the arrayed coated area within each well is digitally imaged via transmission or reflection. A standard curve correlation equation is established using counting and data processing software, and a standard curve is plotted. The signal values ​​of the quality control sample and the test sample are substituted into the standard curve equation to calculate the concentrations of IL-6, IL-8, and IL-10 in the quality control solution and the test sample.

[0039] Comparative Example 1 A triple detection method for cytokines IL-6, IL-8, and IL-10, comprising the following steps: (1) Solution preparation: Coupling buffer: 10 mM MES (pH 6.2 ± 0.05), containing 0.05% ProClin 300; EDC solution: 10 mg / mL, prepared with coupling buffer, and used immediately; NHS solution: 10 mg / mL, prepared with coupling buffer, and used immediately; Fluorescent microsphere blocking solution: borate buffer (5 mM boric acid, 11.2 mM sodium tetraborate decahydrate), 0.05% Tween-20 (pH 9.0±0.05), 1% BSA, 0.24% ethanolamine; Fluorescent microsphere washing solution: 50 mM Tris (pH 8.0 ± 0.05), 0.5% BSA, 0.05% Tween-20. 0.03% ProClin 300; Preservative solution for fluorescent microspheres: 25 mM Tris (pH 7.2 ± 0.05), 150 mM NaCl, 0.05% Tween-20, 1% BSA, 5% Trehalose, 0.1% ProClin 300.

[0040] (2) Activation of fluorescent microspheres A. Take 0.05 mL of fluorescent microsphere (1% solid content) suspension into a 2 mL centrifuge tube containing 1 mL of coupling buffer, sonicate to mix, centrifuge at 15℃ and 20000g for 10 min, and remove the supernatant; add another 1 mL of coupling buffer, sonicate to mix, centrifuge at 15℃ and 20000g for 10 min, and remove the supernatant. B. Take the precipitate from A, add 1 mL of coupling buffer, sonicate to mix, then add 3.5 μL of EDC solution, vortex to mix, then add 33 μL of NHS solution, sonicate to mix; place the centrifuge tube on a turntable, activate at 38℃, 40 r / min, in the dark for 30 min; after activation, centrifuge at 15℃, 20000g for 10 min, and remove the supernatant. C. Take the precipitate from B, add 1.5 mL of coupling buffer, sonicate to mix, centrifuge at 15℃ and 20000g for 10 min, remove the supernatant, add 1.5 mL of coupling buffer, and repeat the washing step once.

[0041] (3) Coupling of fluorescent microspheres with antibodies Add 0.75 mL of conjugation buffer and sonicate to mix. Add 50 μg of the antibodies to be labeled with cytokines IL-1β, IL-6, and IL-8 to 0.25 mL of conjugation buffer to prepare the conjugation solution. Add the antibody-containing conjugation solution to the mixed fluorescent microspheres and vortex to mix. Place the centrifuge tube on a turntable and conjugate at 38 °C, 40 r / min, in the dark for 2.5 h. (4) Blocking and preservation of fluorescent microspheres Add 0.5 mL of fluorescent microsphere blocking solution to the centrifuge tube and vortex to mix. Place the centrifuge tube on a turntable and seal at 37℃, 40 r / min, and in the dark for 1 h. Centrifuge at 15℃ and 20000 g for 10 min and remove the supernatant. Add 1.5 mL of fluorescent microsphere washing solution, sonicate to mix, and centrifuge at 15℃ and 20000 g for 10 min to remove the supernatant. Add 1.5 mL of fluorescent microsphere washing solution and repeat the washing step once. Finally, add 0.5 mL of fluorescent microsphere preservation solution (final concentration of fluorescent microspheres 1 mg / mL) to the centrifuge tube, sonicate to mix, and store at 2-8℃ in the dark for later use.

[0042] (5) Antibody-coated solid-phase detection plate A. Solution preparation Coating solution: Sodium carbonate (Na₂CO₃) 0.1 M, sodium bicarbonate (NaHCO₃) 0.1 M, pH 9.6; Blocking solution: 0.05-0.1M PBS (pH 7.4) + 1-2% BSA + 0.1% ProClin 300 + 0.1% protein protectant; Calibrator dilution solution: 0.1M PBS buffer, 1% protein (bovine serum albumin), 0.1% protein protectant, and 0.2% ProClin 300; Calibrator preparation: Prepare cytokine IL-6, IL-8, and IL-10 calibrators according to Table 1 using the above calibrator diluents.

[0043] Table 1 Preparation of calibrators Calibrator / quality control concentration / pg / ml IL-6 IL-8 IL-10 S0 0 0 0 S1 2.7 4.1 10.3 S2 24 37 277 S3 222 333 833 S4 2000 1000 7500 B. Antibody coating of the detection plate. Antibodies against cytokines L-6, IL-8, and IL-10 were taken at an antibody concentration of 0.5 μg / T, respectively. Figure 9 The polystyrene microplates were coated with 100 μL / well coating buffer at 9°C for 15 h. The plates were then washed 5 times with PBS + TWEEN (0.05 % wt) and patted dry. 150 μL / well blocking buffer was added and the plates were blocked at 37°C for 2 h. The plates were then washed 5 times with PBS + TWEEN (0.05 % wt) and patted dry. The plates were then placed in a desiccant bag, sealed, and stored for later use.

[0044] (6) Add the calibrator containing cytokines IL-6, IL-8 and IL-10 to the corresponding wells of the coated microplate, place it on a shaker, react at room temperature for 1 hour, wash 5 times with PBS + TWEEN (0.05 %wt) and pat dry.

[0045] (7) Dilute the corresponding sealed fluorescent microspheres 25 times with microsphere preservation solution, take 50 μL and add it to the well of the ELISA plate containing cytokines IL-6, IL-8 and IL-10. Place it on a shaker and react at room temperature for 1 h. Then wash it 5 times with PBS + TWEEN (0.05 % wt) and pat it dry.

[0046] (8) Place the above-mentioned enzyme-labeled plate on the sample stage of the fluorescence microscopy system, and use 20x, 40x and 50x objective lenses, monochromatic fluorescence module, and digital imaging of the arrayed coated area in a single well by transmission or reflection. Use counting and data processing software to establish the correlation equation of the standard curve, draw the standard curve, substitute the signal values ​​of the quality control and the test sample into the standard curve equation, and calculate the concentrations of IL-6, IL-8 and IL-10 contained in the quality control solution and the test sample.

[0047] In summary, compared to the comparative example, the embodiment reduced the sample size and operation time by 2 / 3, increased the detection throughput by more than 3 times, and reduced the operation steps and costs.

[0048] As can be seen from the above embodiments and comparative examples, the present invention provides a method for multiple biomarker detection based on single-wavelength fluorescence encoding. This method utilizes processed single-wavelength fluorescent particles to amplify the fluorescence and digitally encode biomarkers in the sample application area of ​​an arrayed coated plate platform. Furthermore, by using a fluorescence microscopy imaging system (flow-free fluorescence detection and multi-fluorescence channel module) combined with image recognition and data analysis software, multiple biomarkers in a single sample can be quantitatively analyzed. This multiple detection technology has advantages such as low cost, ease of use, and medium to high throughput, demonstrating significant advantages in the field of biotechnology.

[0049] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for multiple detection of biomarkers based on single-wavelength fluorescence encoding, characterized in that, Includes the following steps: (1) Prepare a single-wavelength fluorescent particle dispersion, a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, and an N-hydroxysuccinimide solution; (2) Dilute the concentration of the capture antibody corresponding to the biomarker to obtain the diluent of the capture biomarker, and add the diluent to the plate platform in an array layout to obtain the plate platform coated with the biomarker; (3) Couple the single-wavelength fluorescent particle dispersion, centrifuge, remove the supernatant, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and N-hydroxysuccinimide solution in sequence, react, centrifuge, remove the supernatant, couple again, centrifuge, add the analyte solution containing the biomarker, block the reaction, centrifuge, remove the supernatant, continue coupling, centrifuge, remove the supernatant, and obtain the analyte sample containing the biomarker; (4) Add the standard containing the biomarker, the test sample containing the biomarker, or the quality control containing the biomarker to the plate platform coated with the biomarker, react, use a monochrome fluorescence module for digital imaging, plot a standard curve, and calculate the concentration of the biomarker contained in the quality control solution and the test sample.

2. The method for multiple detection of biomarkers according to claim 1, characterized in that, The preparation method of the single-wavelength fluorescent particle dispersion in step (1) is as follows: dissolve tris(hydroxymethyl)aminomethane, sodium chloride, Tween-20, bovine serum albumin, trehalose, and Proclin 300 in water to obtain the dispersion; the mass-volume ratio of tris(hydroxymethyl)aminomethane, sodium chloride, Tween-20, bovine serum albumin, trehalose, Proclin 300, and water is 60~61g: 85~90g: 240~260µL: 0.5~1.5g: 4~6g: 90~110mg: 4~6mL.

3. The method for multiple detection of biomarkers according to claim 1, characterized in that, The preparation method of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution in step (1) is as follows: dissolve 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride in a single-wavelength fluorescent particle coupling buffer. The preparation method of the N-hydroxysuccinimide solution is as follows: dissolve N-hydroxysuccinimide in a single-wavelength fluorescent particle coupling buffer. The preparation method of the single-wavelength fluorescent particle coupling buffer is as follows: dissolve 2-morpholinoethanesulfonic acid and Proclin300 in water and adjust the pH to 6.2~6.

4.

4. The method for multiple detection of biomarkers according to claim 3, characterized in that, The mass-to-volume ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride to single-wavelength fluorescent particle coupling buffer is 90-100 mg: 9-10 ml; the mass-to-volume ratio of N-hydroxysuccinimide to single-wavelength fluorescent particle coupling buffer is 90-100 mg: 9-10 ml; and the mass-to-volume ratio of 2-morpholine ethanesulfonic acid, Proclin 300, and water is 19-20 g: 50-55 mg: 70-80 ml.

5. The method for multiple detection of biomarkers according to claim 3, characterized in that, In step (3), the coupling process uses single-wavelength fluorescent particle coupling buffer.

6. The method for multiple detection of biomarkers according to claim 1, characterized in that, The concentration of the diluent for capturing biomarkers in step (2) is 2~3µg / ml; the array layout is a triangular array; the plate platform includes a 96-well transparent base plate, a 384-well transparent base plate, a transparent glass slide or a transparent plastic sheet.

7. The method for multiple detection of biomarkers according to claim 1, characterized in that, In step (3), the centrifugal force is 1900~2100g and the centrifugation time is 8~12min.

8. The method for multiple detection of biomarkers according to claim 1, characterized in that, In step (3), a single-wavelength fluorescent particle blocking solution is used for the blocking reaction. The single-wavelength fluorescent particle blocking solution is prepared by dissolving boric acid, sodium tetraborate decahydrate, Tween-20, bovine serum albumin, and ethanolamine in water. The mass-volume ratio of boric acid, sodium tetraborate decahydrate, Tween-20, bovine serum albumin, ethanolamine, and water is 30~32g: 425~430mg: 480~520µl: 1.8~2.2g: 460~500mg: 90~100ml.

9. The method for multiple detection of biomarkers according to claim 1, characterized in that, The reaction temperature in step (4) is 35~40℃, and the reaction time is 0.8~1.2h; the digital imaging method is transmission or reflection.

10. The method for multiple detection of biomarkers according to claim 1, characterized in that, The biomarkers include IL-6, IL-8 and IL-10.

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