A rapid on-site quantitative detection method for biomarkers based on lateral flow immunochromatography and smartphone

Abstract

This invention provides a rapid on-site quantitative detection method for biomarkers based on lateral flow immunochromatography and a smartphone, belonging to the field of biodetection technology. This method utilizes a smartphone camera combined with a macro lens featuring built-in LED illumination to construct a portable reader. The reader is used to photograph the T-line region of the lateral flow immunochromatography test strip. The image is processed and analyzed via a smartphone application to obtain the colorimetric value of the T-line. Based on the colorimetric value and a pre-established calibration curve, the concentration of the biomarker in the sample is calculated, and the results are finally displayed on the smartphone application interface. This method features high sensitivity, good repeatability, high accuracy, simple operation, and low cost, and can be widely applied in fields such as disease diagnosis, food safety, and environmental monitoring, providing a new technical solution for the rapid quantitative detection of biomarkers.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and specifically relates to a method for rapid on-site quantitative detection of biomarkers based on lateral flow immunochromatography and a smart phone. Background Art

[0002] Lateral flow immunoassay (LFIA) is a fast, simple and inexpensive immunoassay method that is widely used in disease diagnosis, food safety, environmental monitoring and other fields. Traditional LFIA methods can usually only perform qualitative analysis and cannot provide quantitative information on biomarkers. With the rapid development of smartphone technology, smartphone-based bioassay technology has also received widespread attention. Combining smartphones with LFIA technology can achieve on-site rapid quantitative detection of biomarkers, providing a more accurate tool for disease diagnosis and monitoring.

[0003] At present, most smartphone-based LFIA quantitative detection methods use image analysis technology. For example, Haoyang Tong et al. developed a lateral flow immunoassay (LFIA) platform based on artificial intelligence (AI)-assisted colorimetric polydopamine nanoparticles (PDA) [1] The platform was used to test for SARS-COV-2 neutralizing antibodies (SARS-COV-2NAb), achieving a detection limit of 160ng / mL and a quantitative detection range of 625-10000ng / mL. However, the detection limit achieved by this technology is 160ng / mL, which has met the requirements for distinguishing vaccinated and unvaccinated individuals, but still has the technical defect of low sensitivity. [1] .

[0004] In the prior art, the optimization direction of the quantitative detection method based on smartphones is mostly focused on the structural optimization of the camera device. For example, Chinese patent CN104569379B provides a structurally optimized smartphone rapid immunochromatographic test strip matching detection device, but there is no actual experimental data to support the detection effect of the device.

[0005] The existing technology of lateral flow immunochromatography analysis based on smartphones has the following problems: low sensitivity, difficulty in detecting low-concentration biomarkers; poor repeatability and insufficient stability of test results; low accuracy and inconsistency with laboratory quantitative test results.

[0006] Therefore, there is an urgent need to provide a smartphone-based rapid quantitative detection method for biomarkers, which has high sensitivity, good repeatability, and high accuracy, and can meet the needs of on-site rapid quantitative detection of different biomarkers.

[0007] [1]Tong H, Cao C, You M, et al. Artificial intelligence-assisted colorimetric lateral flow immunoassay for sensitive and quantitative detection of SARS-COV-2 neutralizing antibody. Biosens Bioelectron. 2022; 213:114449. Summary of the invention

[0008] In order to solve the above problems, the present invention provides a method for rapid on-site quantitative detection of biomarkers based on lateral flow immunochromatography and smart phones.

[0009] Specifically, the detection method comprises the following steps:

[0010] S1: Detection of SARS-COV-2 neutralizing antibodies using lateral flow immunochromatographic test strips;

[0011] The width of the lateral flow immunochromatographic test strip is 3 mm;

[0012] The lateral flow immunochromatographic test strip comprises: an RBD protein colloidal gold marker binding pad and an NC coating membrane;

[0013] The preparation method of the RBD protein colloidal gold marker conjugate pad is as follows: a RBD protein colloidal gold marker solution with an RBD protein concentration of 0.4-0.8 g / L is evenly sprayed on the conjugate pad according to the parameters of 1-3 μL / cm and each length of 25-35 cm;

[0014] Use a film spraying instrument to continuously and evenly draw C lines and T lines on the NC membrane, where the T line is the RBD coating solution;

[0015] The RBD coating solution includes: 1-5 mg / mL RBD protein, 0.001-0.1 mmol / L phosphate buffer with a pH of 7.4, 0.5-1.5 g / L NaCl, 35-80 g / L trehalose, 0.1-1.5 mL / L PC300, and 0.5-1.5 g / L sodium azide;

[0016] S2: Signal detection:

[0017] The camera of a smartphone is combined with a macro lens with built-in LED light fill light function to construct a portable reader, and the specific position of the T line of the lateral flow immunochromatography test strip is photographed in close proximity; the focal length of the macro lens with built-in LED light fill light function is 5.5-7mm; the specific position refers to a specific area of ​​0.2mm×1.6mm-0.4mm×2.0mm 0.2mm upward from the intersection of the center line of the T line and the lower end horizontal line.

[0018] S3: Result analysis:

[0019] Results analysis via smartphone app:

[0020] The smart phone application comprises: an image acquisition module, an image processing module, a concentration calculation module and a result display module.

[0021] Further specifically, the C line in S1 is a sheep anti-chicken IgY coating solution, which includes: 0.3-1.2 mg / mL sheep anti-chicken IgY, 0.001-0.1 mmol / L phosphate buffer with a pH of 7.4, 0.5-1.5 g / L NaCl, 35-80 g / L trehalose, 0.1-1.5 mL / L PC300, and 0.5-1.5 g / L sodium azide.

[0022] Specifically, the preparation method of the RBD protein colloidal gold marker solution in S1 is as follows: 7-10 mL of colloidal gold solution is added with 0.05-0.2% K 2 CO 3 The solution adjusts the pH of the colloidal gold solution to 10.5±0.1, stirs and mixes, measures 4-8 mg of RBD protein and adds it to the colloidal gold solution, stirs and mixes, and reacts at room temperature; adds blocking solution and continues to stir, collects the precipitate by refrigerated centrifugation, adds 2-5 mL of colloidal gold marker diluent, and then adds 0.4-0.6 g of sucrose, and stores at 2-8°C for later use.

[0023] More specifically, in S1, the lateral flow immunochromatography test strip comprises: a PVC base plate and a sample pad, an RBD protein colloidal gold marker binding pad, a chicken IgY colloidal gold marker binding pad, an NC coating membrane and a water absorbent pad which are arranged thereon in an overlapping manner.

[0024] More specifically, the preparation method of the chicken IgY colloidal gold marker conjugate pad is as follows: a chicken IgY colloidal gold marker solution having a chicken IgY protein concentration of 0.4-0.8 g / L is evenly sprayed on the conjugate pad according to the parameters of 1-3 μL / cm and each strip length of 25-35 cm;

[0025] More specifically, the preparation method of the chicken IgY colloidal gold marker solution is as follows: 7-10 mL of colloidal gold solution is taken, and 0.05-0.2% K 2 CO 3 The solution adjusts the pH of the colloidal gold solution to 10.5±0.1, measures 4-8 mg of chicken IgY protein and adds it to the colloidal gold solution, stirs and mixes, and reacts at room temperature; the blocking solution continues to be stirred, and the precipitate is collected by refrigerated centrifugation, 2-5 mL of colloidal gold marker diluent is added, and then 0.4-0.6 g of sucrose is added, and the solution is stored at 2-8°C for later use.

[0026] More specifically, the sample pad treatment solution is formulated as follows: 0.001-0.05 mmol / L boric acid buffer at pH 9.0, 0.05-0.5 mg / mL ACE2 protein, 15-30 g / L casein, 5-15 g / L PVP-10, 0.5-1.5 g / L NaCl, 1-3 mL / L Tween-20, 1-5 g / L EDTANa 2 , 0.3-0.8mL / L PC300, 0.5-1.5g / L sodium azide, 0.2-0.8mg / mL rabbit anti-human erythrocyte antibody.

[0027] More specifically, in S3, the image acquisition module is used to obtain an image of a specific position of the T line of the lateral flow immunochromatography test strip.

[0028] More specifically, in S3, the image processing module is used to process and analyze the T-line image to obtain the chromaticity value of the T-line.

[0029] More specifically, in S3, the concentration calculation module calculates the concentration value of the biomarker in the sample by comparing the colorimetric value with a pre-established calibration curve.

[0030] In some optional specific embodiments of the present invention, the establishment of the calibration curve comprises the following steps:

[0031] 1) Measure the chromaticity value of the T line;

[0032] 2) Use a standard of known concentration to prepare a series of samples with a concentration gradient, and measure the chromaticity value of the T line according to the above steps. Draw a calibration curve with concentration as the horizontal axis and T line chromaticity value as the vertical axis.

[0033] More specifically, in S3, the concentration calculation module displays the calculated biomarker concentration value on an application program interface.

[0034] Compared with the prior art, the present invention has the following advantages:

[0035] The method of the present invention has high sensitivity, good repeatability, high accuracy, simple operation and low cost, and can be widely used in disease diagnosis, food safety, environmental monitoring and other fields, providing a new technical solution for the rapid quantitative detection of biomarkers. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the structure of the immune test strip used in Example 1, wherein 1) is a sample pad, 2) is an RBD protein colloidal gold marker conjugate pad, 3) is a chicken IgY colloidal gold marker conjugate pad, 4) is a cut absorbent pad, 5) is an NC coating membrane, and 6) is a PVC plate.

[0037] Figure 2 This is the image acquisition method in Example 1.

[0038] Figure 3 This is a picture of the chromaticity detection result when the method in Example 1 detects samples of different concentrations. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0040] Table 1 Reagents

[0041]

[0042]

[0043] Table 2 Instruments

[0044] name model Manufacturer Film Spraying Instrument HM3035 Shanghai Gold Label Strip cutting machine ZQ3500 Shanghai Gold Label Shell Press YK1225 Shanghai Gold Label Sealing Machine 770 Wuhan Youshengkai Dehumidifier HR-1506D Shanghai Shimei Electrical Appliances

[0045] Example 1

[0046] Lateral flow immunochromatographic test strips for detecting SARS-COV-2 neutralizing antibodies:

[0047] 1. Preparation method of lateral flow immunochromatography test strips:

[0048] 1) Preparation of colloidal gold label solution

[0049] RBD protein colloidal gold label solution: Take 8.5 mL of colloidal gold solution and add 0.1% K 2 CO 3The solution was adjusted to pH 10.5 with colloidal gold solution, stirred for 10 min, 6 mg of RBD protein was added to the colloidal gold solution, stirred, and reacted at room temperature for 20 min under stirring; 0.2 mL of blocking solution was added and stirring was continued for 20 min, refrigerated centrifugation (7000 g, 4 ° C, 25 min), the precipitate was collected, 2.5 mL of colloidal gold marker diluent was added, and then 0.6 g of sucrose was added and stored at 2-8 ° C for later use.

[0050] Chicken IgY colloidal gold labeling solution: Take 8.5 mL of colloidal gold solution and add 0.1% K 2 CO 3 The solution was adjusted to pH 10.5 with colloidal gold solution, stirred for 10 min, 6 mg chicken IgY protein was added to the colloidal gold solution, stirred, and reacted at room temperature for 20 min under stirring; 0.2 mL of blocking solution was added and stirring was continued for 20 min, refrigerated centrifugation (7000 g, 4 ° C, 25 min), the precipitate was collected, 2.5 mL of colloidal gold marker diluent was added, and 0.6 g of sucrose was added, and the solution was stored at 2-8 ° C for later use.

[0051] 2) Preparation of colloidal gold label conjugate pad

[0052] Clean the pipeline of the film spraying instrument. The cleaning steps are as follows: ultrapure water cleaning 10 times, alcohol cleaning 10 times, ultrapure water cleaning 10 times, 0.01mol / L TRIS cleaning 10 times; set the parameters so that the spray volume is 2μL / cm.

[0053] The RBD protein colloidal gold marker and the chicken IgY colloidal gold marker were sprayed evenly on the binding pad at 2 μL / cm and each strip was 30 cm long. The evenly sprayed binding pad was placed flat on the gauze and dried in a forced air drying oven. The drying oven temperature was 37°C, the relative humidity was ≤20%, and the drying time was 72 hours.

[0054] 3) Sample pad preparation

[0055] Sample pad treatment solution composition: 0.01mmol / L boric acid buffer with a pH of 9.0, 0.1mg / mL ACE2 protein, 20g / L casein, 10g / L PVP-10, 0.9g / L NaCl, 2mL / L Tween-20, 3g / L EDTANa 2 , 0.5mL / LPC300, 0.9g / L sodium azide, 0.4mg / mL rabbit anti-human erythrocyte antibody;

[0056] Take the sample pad treatment solution and put it in a soaking tank. Soak the SB08 glass fiber in the soaking tank for 10 minutes. Gently shake the soaking tank.

[0057] Apply evenly. Place the evenly coated glass fiber flat on the gauze and place it in the drying room in a forced air drying oven for drying. The drying oven temperature is 37°C, the relative humidity is ≤20%, and the drying time is 20 hours.

[0058] 4) Coating membrane preparation

[0059] Sheep anti-chicken IgY coating solution: 0.75mg / mL sheep anti-chicken IgY, 0.01mmol / L pH 7.4 phosphate buffer, 0.9g / L NaCl, 50g / L trehalose, 0.5mL / L PC300, 0.9g / L sodium azide.

[0060] RBD coating solution: 3 mg / mL RBD protein, 0.01 mmol / L phosphate buffer with a pH of 7.4, 0.9 g / L NaCl, 50 g / L trehalose, 0.5 mL / L PC300, and 0.9 g / L sodium azide.

[0061] Clean the pipeline of the film spraying instrument. The cleaning steps are as follows: ultrapure water cleaning 10 times, alcohol cleaning 10 times, ultrapure water cleaning 10 times, 0.01mol / L PB cleaning 10 times; set the parameters so that the spray volume of 1# pump is 0.8μL / cm, and the spray volume of 4# pump is 1.0μL / cm.

[0062] According to the standard operating procedures of the film spray gold instrument, the No. 1 pump is placed with the quality control line sheep anti-chicken IgY coating solution, the No. 4 pump is placed with the RBD coating solution, the No. 1 and No. 4 marking pens are 4mm apart, and the film spray gold instrument is used to continuously and evenly draw lines (coating) on ​​the NC membrane. Monitor the situation of each coating membrane line, and mark the uneven sections with a signature pen. The coated NC membrane is placed horizontally on the grid with the coating surface facing up, and then placed in the electric heating blast drying oven in the drying room, requiring a temperature of 37°C, a relative humidity of ≤20%, and drying for 24 hours.

[0063] according to Figure 1 Assemble as shown, the distance between the T line and the sample hole is 24mm.

[0064] 2. Sample testing

[0065] 1) Place the assembled test strips on a level, dry surface.

[0066] 2) Use a disposable plastic pipette to drop 25 μL of blood sample vertically into the sample well, then add 2 drops of sample diluent and read the T line test results 10 minutes later.

[0067] 3. Data Analysis

[0068] The camera of a smart phone is combined with a macro lens with built-in LED light fill function to construct a portable reader, and the specific position of the T line of the lateral flow immunochromatography test strip is fitted and photographed. The focal length of the macro lens with built-in LED light fill function is 6mm; the fitting and photographing of the specific position of the T line of the lateral flow immunochromatography test strip refers to fitting and photographing a specific area of ​​0.3mm×1.8mm 0.2mm above the intersection of the center line of the T line and the lower end horizontal line. The image acquisition method is as follows Figure 2 shown.

[0069] The images captured by the smartphone camera are processed and analyzed by the smartphone application:

[0070] The application automatically calculates the concentration value of the biomarker in the sample based on the calibration curve pre-established based on the chromaticity value of the T line, and displays the calculated biomarker concentration value on the smartphone application interface.

[0071] Example 2

[0072] 1. Preparation method of lateral flow immunochromatography test strips:

[0073] 1) Preparation of colloidal gold label solution

[0074] RBD protein colloidal gold label solution: Take 10 mL of colloidal gold solution and add 0.1% K 2 CO 3 The solution was adjusted to pH 10.5 with colloidal gold solution, stirred for 10 min, 8 mg of RBD protein was added to the colloidal gold solution, stirred, and reacted at room temperature for 20 min under stirring; 0.2 mL of blocking solution was added and continued to stir for 20 min, refrigerated centrifugation (7000 g, 4 ° C, 25 min), the precipitate was collected, 5 mL of colloidal gold marker diluent was added, and then 0.6 g of sucrose was added and stored at 2-8 ° C for later use.

[0075] Chicken IgY colloidal gold labeling solution: Take 10 mL of colloidal gold solution and add 0.1% K 2 CO 3 The solution was adjusted to pH 10.5 with colloidal gold solution, stirred for 10 min, 8 mg chicken IgY protein was added to the colloidal gold solution, stirred, and reacted at room temperature for 20 min under stirring; 0.2 mL of blocking solution was added and continued to stir for 20 min, refrigerated centrifugation (7000 g, 4 ° C, 25 min), the precipitate was collected, 5 mL of colloidal gold marker diluent was added, and then 0.6 g of sucrose was added, and stored at 2-8 ° C for later use.

[0076] 2) Preparation of colloidal gold label conjugate pad

[0077] Clean the pipeline of the film spraying instrument. The cleaning steps are as follows: ultrapure water cleaning 10 times, alcohol cleaning 10 times, ultrapure water cleaning 10 times, 0.01mol / L TRIS cleaning 10 times; set the parameters so that the spray volume is 3μL / cm.

[0078] The RBD protein colloidal gold marker and the chicken IgY colloidal gold marker were sprayed evenly on the binding pad at 3 μL / cm and each strip was 30 cm long. The evenly sprayed binding pad was placed flat on the gauze and dried in a forced air drying oven. The drying oven temperature was 37°C, the relative humidity was ≤20%, and the drying time was 72 hours.

[0079] 3) Sample pad preparation

[0080] Sample pad treatment solution composition: 0.05mmol / L boric acid buffer with a pH of 9.0, 0.5mg / mL ACE2 protein, 30g / L casein, 15g / L PVP-10, 1.5g / L NaCl, 3mL / L Tween-20, 5g / L EDTANa 2 , 0.8mL / LPC300, 1.5g / L sodium azide, 0.8mg / mL rabbit anti-human erythrocyte antibody;

[0081] Take the sample pad treatment solution and put it in a soaking tank. Soak the SB08 glass fiber in the soaking tank for 10 minutes. Gently shake the soaking tank.

[0082] Apply evenly. Place the evenly coated glass fiber flat on the gauze and place it in the drying room in a forced air drying oven for drying. The drying oven temperature is 37°C, the relative humidity is ≤20%, and the drying time is 20 hours.

[0083] 4) Coating membrane preparation

[0084] Sheep anti-chicken IgY coating solution: 1.2mg / mL sheep anti-chicken IgY, 0.1mmol / L pH 7.4 phosphate buffer, 1.5g / L NaCl, 80g / L trehalose, 1.5mL / L PC300, 1.5g / L sodium azide.

[0085] RBD coating solution: 5 mg / mL RBD protein, 0.1 mmol / L phosphate buffer with a pH of 7.4, 1.5 g / L NaCl, 80 g / L trehalose, 1.5 mL / L PC300, and 1.5 g / L sodium azide.

[0086] Clean the pipeline of the film spraying instrument. The cleaning steps are as follows: ultrapure water cleaning 10 times, alcohol cleaning 10 times, ultrapure water cleaning 10 times, 0.01mol / L PB cleaning 10 times; set the parameters so that the spray volume of 1# pump is 0.8μL / cm, and the spray volume of 4# pump is 1.0μL / cm.

[0087] According to the standard operating procedures of the film spray gold instrument, the No. 1 pump is placed with the quality control line sheep anti-chicken IgY coating solution, the No. 4 pump is placed with the RBD coating solution, the No. 1 and No. 4 marking pens are 4mm apart, and the film spray gold instrument is used to continuously and evenly draw lines (coating) on ​​the NC membrane. Monitor the situation of each coating membrane line, and mark the uneven sections with a signature pen. The coated NC membrane is placed horizontally on the grid with the coating surface facing up, and then placed in the electric heating blast drying oven in the drying room, requiring a temperature of 37°C, a relative humidity of ≤20%, and drying for 24 hours.

[0088] according to Figure 1 Assemble as shown, the distance between the T line and the sample hole is 24mm.

[0089] The difference between this comparative example and embodiment 1 is that when shooting the T-line image, the focal length of the macro lens is different, and the specific position of the shot T-line image is different.

[0090] The camera of a smartphone is combined with a macro lens with built-in LED light fill light function to construct a portable reader, and the specific position of the T line of the lateral flow immunochromatography test strip is photographed by fitting. The focal length of the macro lens with built-in LED light fill light function is 5.5mm; the fitting and photographing of the specific position of the T line of the lateral flow immunochromatography test strip refers to fitting and photographing a specific area of ​​0.2mm×1.6mm 0.2mm upward from the intersection of the center line of the T line and the lower end horizontal line.

[0091] Example 3

[0092] 1. Preparation method of lateral flow immunochromatography test strips:

[0093] 1) Preparation of colloidal gold label solution

[0094] RBD protein colloidal gold label solution: Take 7 mL of colloidal gold solution and add 0.1% K 2 CO 3 The solution was adjusted to pH 10.5 with colloidal gold solution, stirred for 10 min, 4 mg of RBD protein was added to the colloidal gold solution, stirred, and reacted at room temperature for 20 min under stirring; 0.2 mL of blocking solution was added and continued to stir for 20 min, refrigerated centrifugation (7000 g, 4 ° C, 25 min), the precipitate was collected, 2 mL of colloidal gold marker diluent was added, and then 0.4 g of sucrose was added and stored at 2-8 ° C for later use.

[0095] Chicken IgY colloidal gold labeling solution: Take 7 mL of colloidal gold solution and add 0.1% K 2 CO 3The solution was adjusted to pH 10.5 with colloidal gold solution, stirred for 10 min, 4 mg chicken IgY protein was added to the colloidal gold solution, stirred, and reacted at room temperature for 20 min under stirring; 0.2 mL of blocking solution was added and continued to stir for 20 min, refrigerated centrifugation (7000 g, 4 ° C, 25 min), the precipitate was collected, 2 mL of colloidal gold marker diluent was added, and then 0.4 g of sucrose was added, and stored at 2-8 ° C for later use.

[0096] 2) Preparation of colloidal gold label conjugate pad

[0097] Clean the pipeline of the film spray metallization instrument. The cleaning steps are as follows: ultrapure water cleaning 10 times, alcohol cleaning 10 times, ultrapure water cleaning 10 times, 0.01mol / L TRIS cleaning 10 times; set the parameters so that the spray volume is 1μL / cm.

[0098] The RBD protein colloidal gold marker and the chicken IgY colloidal gold marker were sprayed evenly on the binding pad at 1 μL / cm, each strip was 30 cm long, and the evenly sprayed binding pad was placed flat on the gauze and placed in a forced air drying oven for drying. The drying oven temperature was 37°C, the relative humidity was ≤20%, and the drying time was 72 hours.

[0099] 3) Sample pad preparation

[0100] Sample pad treatment solution composition: 0.001mmol / L boric acid buffer with a pH of 9.0, 0.05mg / mL ACE2 protein, 15g / L casein, 5g / L PVP-10, 0.5g / L NaCl, 1mL / L Tween-20, 1g / L EDTANa 2 , 0.3mL / LPC300, 0.5g / L sodium azide, 0.2mg / mL rabbit anti-human erythrocyte antibody;

[0101] Take the sample pad treatment solution and put it in a soaking tank. Soak the SB08 glass fiber in the soaking tank for 10 minutes. Gently shake the soaking tank.

[0102] Apply evenly. Place the evenly coated glass fiber flat on the gauze and place it in the drying room in a forced air drying oven for drying. The drying oven temperature is 37°C, the relative humidity is ≤20%, and the drying time is 20 hours.

[0103] 4) Coating membrane preparation

[0104] Sheep anti-chicken IgY coating solution: 0.3 mg / mL sheep anti-chicken IgY, 0.001 mmol / L pH 7.4 phosphate buffer, 0.5 g / L NaCl, 35 g / L trehalose, 0.1 mL / L PC300, 0.5 g / L sodium azide.

[0105] RBD coating solution: 1 mg / mL RBD protein, 0.001 mmol / L phosphate buffer with a pH of 7.4, 0.5 g / L NaCl, 35 g / L trehalose, 0.1 mL / L PC300, and 0.5 g / L sodium azide.

[0106] Clean the pipeline of the film spraying instrument. The cleaning steps are as follows: ultrapure water cleaning 10 times, alcohol cleaning 10 times, ultrapure water cleaning 10 times, 0.01mol / L PB cleaning 10 times; set the parameters so that the spray volume of 1# pump is 0.8μL / cm, and the spray volume of 4# pump is 1.0μL / cm.

[0107] According to the standard operating procedures of the film spray gold instrument, the No. 1 pump is placed with the quality control line sheep anti-chicken IgY coating solution, the No. 4 pump is placed with the RBD coating solution, the No. 1 and No. 4 marking pens are 4mm apart, and the film spray gold instrument is used to continuously and evenly draw lines (coating) on ​​the NC membrane. Monitor the situation of each coating membrane line, and mark the uneven sections with a signature pen. The coated NC membrane is placed horizontally on the grid with the coating surface facing up, and then placed in the electric heating blast drying oven in the drying room, requiring a temperature of 37°C, a relative humidity of ≤20%, and drying for 24 hours.

[0108] according to Figure 1 Assemble as shown, the distance between the T line and the sample hole is 24mm.

[0109] The difference between this embodiment and the first embodiment is that when the T-line image is photographed, the focal length of the macro lens is different, and the specific position of the photographed T-line image is different.

[0110] The camera of a smartphone is combined with a macro lens with built-in LED light fill light function to construct a portable reader, and the specific position of the T line of the lateral flow immunochromatography test strip is photographed by fitting. The focal length of the macro lens with built-in LED light fill light function is 7mm; the fitting and photographing of the specific position of the T line of the lateral flow immunochromatography test strip refers to fitting and photographing a specific area of ​​0.4mm×2.0mm 0.2mm above the intersection of the center line of the T line and the lower end horizontal line.

[0111] Comparative Example 1

[0112] The difference between this comparative example and Example 1 is that when shooting the T-line image, a macro lens with a built-in LED light fill light function is not used.

[0113] Use the camera of a smartphone to directly fit and photograph the specific position of the T line of the lateral flow immunochromatography test strip; the said fitting and photographing the specific position of the T line of the lateral flow immunochromatography test strip refers to fitting and photographing a specific area of ​​0.3mm×1.8mm 0.2mm upward from the intersection of the center line of the T line and the lower end horizontal line.

[0114] Comparative Example 2

[0115] The difference between this comparative example and Example 1 is that the specific position area of ​​the captured T-line image is different.

[0116] The camera of a smartphone is combined with a macro lens with a built-in LED light fill light function to construct a portable reader, and the specific position of the T line of the lateral flow immunochromatography test strip is photographed by fitting. The focal length of the macro lens with a built-in LED light fill light function is 6 mm; the fitting and photographing of the specific position of the T line of the lateral flow immunochromatography test strip refers to fitting and photographing a specific area of ​​0.15 mm×1.0 mm 0.2 mm upward from the intersection of the center line of the T line and the lower end horizontal line.

[0117] Comparative Example 3

[0118] The difference between this comparative example and Example 1 is that the specific position area of ​​the captured T-line image is different.

[0119] The camera of a smartphone is combined with a macro lens with built-in LED light fill light function to construct a portable reader, and the specific position of the T line of the lateral flow immunochromatography test strip is photographed by fitting. The focal length of the macro lens with built-in LED light fill light function is 6mm; the fitting and photographing of the specific position of the T line of the lateral flow immunochromatography test strip refers to fitting and photographing a specific area of ​​0.6mm×3.0mm 0.2mm above the intersection of the center line of the T line and the lower end horizontal line.

[0120] Experimental Example 1 Linear Correlation Coefficient R 2 test

[0121] Take the SARS-COV-2NAb lateral flow immunochromatographic test strips and test the seven concentration gradients of the standard. Each gradient concentration is tested 3 times and the average is taken. The average of the measured values ​​and the theoretical concentration of the standard are regressed. The X-axis represents the theoretical concentration and the Y-axis represents the mean of the measured value. The general linear correlation coefficient R 2 ≥0.9900 is considered to have good linearity.

[0122] The results are shown in Table 1:

[0123] Table 1 Linear correlation coefficient R 2 Test Results

[0124] Example Concentration range <![CDATA[R 2 ]]> Example 1 625-40000ng / mL 0.9989 Example 2 625-40000ng / mL 0.9930 Example 3 625-40000ng / mL 0.9936 Comparative Example 1 625-40000ng / mL 0.9850 Comparative Example 2 625-40000ng / mL 0.9864 Comparative Example 3 625-40000ng / mL 0.9838

[0125] The results show that the on-site rapid quantitative detection method of biomarkers based on lateral flow immunochromatography and smartphones provided in Examples 1-3 of the present invention has good linearity in the range of 625-40000 ng / mL when applied to SARS-COV-2 NAb detection. The chromaticity diagram of the method in Example 1 when detecting samples of different concentrations is as follows: Figure 3 shown.

[0126] Experimental Example 2 Limit of Detection (LOD) and Limit of Quantification (LOQ)

[0127] Take the SARS-COV-2NAb lateral flow immunochromatographic test strip, and estimate the minimum detection limit (LOD) and minimum quantification limit (LOQ) by repeating the blank solution test 10 times to obtain the blank value b and standard deviation s. The calculation formula is: LOD = b + 3s, LOQ = b + 10s. The lower the LOD and LOQ values, the higher the sensitivity of the method.

[0128] The results are shown in Table 2:

[0129] Table 2 Test results of the limit of detection (LOD) and limit of quantification (LOQ)

[0130] Example LOD(ng / mL) LOQ (ng / mL) Example 1 35 75 Example 2 39 84 Example 3 42 94 Comparative Example 1 64 123 Comparative Example 2 44 101 Comparative Example 3 46 103

[0131] The results showed that the on-site rapid quantitative detection methods of biomarkers based on lateral flow immunochromatography and smartphones provided in Examples 1-3 of the present invention had lower LOD and LOQ when applied to SARS-COV-2NAb detection, indicating that the methods described in the present invention have higher sensitivity.

[0132] By comparing Example 1 and Comparative Example 1, it can be seen that when the acquisition method of the T-line image of the portable reader is changed, the LOD and LOQ measurement results are increased and the sensitivity is reduced; by comparing Example 1 and Comparative Examples 2-3, it can be seen that when the specific area of ​​T-line image acquisition is changed, the LOD and LOQ measurement results are increased and the sensitivity is reduced.

[0133] Example 3 Precision

[0134] The same SARS-COV-2NAb sample (625-40000ng / mL) was continuously extracted 20 times and measured according to the above method. The mean value, standard deviation (SD) and coefficient of variation (CV) of the measured value were calculated. CV = (standard deviation / mean value) × 100%. The results are shown in Table 3. CV is usually used to measure the precision of a measurement method. The smaller the CV value, the better the precision of the result of the measurement method. For clinical immunology test items, a method precision of CV less than 5% is generally recognized to be acceptable.

[0135] Table 3 Repeatability test results

[0136] Example Average value (ng / mL) Standard Deviation (SD) Precision (CV) Example 1 5048 95 1.89％ Example 2 5009 224 4.46％ Example 3 5044 174 3.45％ Comparative Example 1 4984 381 7.64％ Comparative Example 2 5055 334 6.61％ Comparative Example 3 5062 294 5.81％

[0137] The results show that the CV values ​​of the methods provided in Examples 1-3 of the present invention are 1.89-4.46%, indicating that the method of the present invention has good precision.

[0138] By comparing Example 1 and Comparative Example 1, it can be seen that when the acquisition method of the T-line image of the portable reader is changed, the coefficient of variation (CV) of the sample detection is greater than 5% and the precision is reduced; by comparing Example 1 and Comparative Examples 2-3, it can be seen that when the specific area of ​​T-line image acquisition is changed, the coefficient of variation (CV) of the sample detection is greater than 5% and the precision is reduced.

[0139] Experimental Example 4 Accuracy

[0140] The same SARS-COV-2NAb quality control product (7225±72ng / mL) was drawn 20 times continuously and measured according to the above method. The average value and relative deviation of the measured value were calculated. The results are shown in Table 4. For clinical immunology test items, the accuracy of the method with a relative deviation of less than ±10% is generally recognized to be acceptable.

[0141] Table 4 Accuracy test results

[0142]

[0143]

[0144] The results show that the relative deviations of the methods provided in Examples 1-3 of the present invention are 3.20-7.18%, all less than 10%, indicating that the methods of the present invention have good accuracy.

[0145] Comparing Example 1 with Comparative Example 1, it can be seen that when the acquisition method of the T-line image of the portable reader is changed, the relative deviation of the quality control product detection is greater than 10%; comparing Example 1 with Comparative Examples 2-3, it can be seen that when the specific area of ​​T-line image acquisition is changed, the relative deviation of the detection concentration of the quality control product is greater than 10%.

[0146] The experimental results of the above embodiments and comparative examples show that the on-site rapid quantitative detection method of biomarkers based on lateral flow immunochromatography and smartphones provided by the present invention has the advantages of high sensitivity, good repeatability and high accuracy, and has significant advantages over the existing technology.

[0147] The above is a further description of the present invention in conjunction with specific embodiments, but these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

Claims

1. A method for rapid on-site quantitative detection of biomarkers based on lateral flow immunochromatography and smartphones, characterized in that: The following steps are involved: S1: Detection of SARS-COV-2 neutralizing antibodies using lateral flow immunochromatographic test strips; The width of the lateral flow immunochromatographic test strip is 2-4 mm; The lateral flow immunochromatographic test strip comprises: an RBD protein colloidal gold marker binding pad and an NC coating membrane; The preparation method of the RBD protein colloidal gold marker conjugate pad is as follows: a RBD protein colloidal gold marker solution with an RBD protein concentration of 0.4-0.8 g / L is evenly sprayed on the conjugate pad according to the parameters of 1-3 μL / cm and each length of 25-35 cm; The preparation method of the NC coating film is: using a film-scribing gold spraying instrument to continuously and evenly draw C lines and T lines on the NC film, wherein the T line is the RBD coating solution; The RBD coating solution includes: 1-5 mg / mL RBD protein, 0.001-0.1 mmol / L phosphate buffer with a pH of 7.4, 0.5-1.5 g / L NaCl, 35-80 g / L trehalose, 0.1-1.5 mL / L PC300, and 0.5-1.5 g / L sodium azide; S2: Signal detection: The camera of a smart phone is combined with a macro lens with built-in LED light fill function to construct a portable reader, and the specific position of the T line of the lateral flow immunochromatography test strip is fitted and photographed; the focal length of the macro lens with built-in LED light fill function is 5.5-7mm; the specific position refers to the specific area of ​​0.2mm×1.6mm-0.4mm×2.0mm 0.2mm upward from the intersection of the center line of the T line and the lower end horizontal line, and the photograph is taken; S3: Result analysis: Results analysis via smartphone app: The smart phone application comprises: an image acquisition module, an image processing module, a concentration calculation module and a result display module.

2. The method for rapid on-site quantitative detection of biomarkers according to claim 1, characterized in that: The C line is a sheep anti-chicken IgY coating solution, which includes: 0.3-1.2 mg / mL sheep anti-chicken IgY, 0.001-0.1 mmol / L phosphate buffer with a pH of 7.4, 0.5-1.5 g / L NaCl, 35-80 g / L trehalose, 0.1-1.5 mL / L PC300, and 0.5-1.5 g / L sodium azide.

3. The method for rapid on-site quantitative detection of biomarkers according to claim 1, characterized in that: The preparation method of the RBD protein colloidal gold marker solution is as follows: taking 7-10 mL of colloidal gold solution, adjusting the pH of the colloidal gold solution to 10.5±0.1 with a K2CO3 solution, stirring and mixing, measuring 4-8 mg of RBD protein and adding it to the colloidal gold solution, stirring and mixing, and reacting at room temperature; adding a blocking solution and continuing to stir, collecting the precipitate by frozen centrifugation, adding 2-5 mL of colloidal gold marker diluent, and then adding 0.4-0.6 g of sucrose, and storing at 2-8°C for later use.

4. The method for rapid on-site quantitative detection of biomarkers according to claim 1, characterized in that: In S1, the lateral flow immunochromatography test strip comprises: a PVC base plate and a sample pad, an RBD protein colloidal gold marker binding pad, a chicken IgY colloidal gold marker binding pad, an NC coating membrane and a water absorbent pad which are sequentially overlapped and arranged thereon.

5. The method for rapid on-site quantitative detection of biomarkers according to claim 4, characterized in that: The treatment solution formula of the sample pad is: 0.001-0.05mmol / L boric acid buffer with a pH of 9.0, 0.05-0.5mg / mL ACE2 protein, 15-30g / L casein, 5-15g / L PVP-10, 0.5-1.5g / L NaCl, 1-3mL / L Tween-20, 1-5g / LEDTANa2, 0.3-0.8mL / L PC300, 0.5-1.5g / L sodium azide, and 0.2-0.8mg / mL rabbit anti-human red blood cell antibody.

6. The method for rapid on-site quantitative detection of biomarkers according to claim 1, characterized in that: In S3, the image acquisition module is used to obtain an image of a specific position of the T line of the lateral flow immunochromatography test strip.

7. The method for rapid on-site quantitative detection of biomarkers according to claim 1, characterized in that: In S3, the image processing module is used to process and analyze the T-line image to obtain the chromaticity value of the T-line.

8. The method for rapid on-site quantitative detection of biomarkers according to claim 1, characterized in that: In S3, the concentration calculation module calculates the concentration value of the biomarker in the sample by comparing the colorimetric value with a pre-established calibration curve.

9. The method for rapid on-site quantitative detection of biomarkers according to claim 8, characterized in that: The establishment of the calibration curve comprises the following steps: 1) Measure the chromaticity value of the T line; 2) Use a standard of known concentration to prepare a series of samples with a concentration gradient, and measure the chromaticity value of the T line according to the above steps. Draw a calibration curve with concentration as the horizontal axis and T line chromaticity value as the vertical axis.

10. The method for rapid on-site quantitative detection of biomarkers according to claim 1, characterized in that: In S3, the concentration calculation module displays the calculated biomarker concentration value on an application program interface.

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