Method for visually detecting antigen

Abstract

The invention provides a method for visually detecting an antigen. The method comprises the steps that a visual sensor is provided and comprises a transparent reaction container, and a first antibody capable of being specifically combined with an antigen is fixed to the bottom of the reaction container; adding a to-be-detected sample into the reaction container for reaction, and performing elution for the first time after the reaction is completed; adding a second antibody capable of being specifically bound with the antigen into the reaction container for reaction, coupling the second antibody with catalase, and performing secondary elution after the reaction is completed; a hydrogen peroxide solution is added into the reaction container for a reaction, and in the reaction process, a metal nanoparticle solution is added for a mixed reaction; and after the reaction is completed, irradiating the reaction container with laser, collecting the color R / G value of the reaction container and / or temperature signals before and after irradiation, and calculating the content of the antigen in the sample to be detected according to the standard curve. The method is simple and convenient to operate and high in detection accuracy.

Description

Technical Field

[0001] The present application relates to the field of biochemical analysis technology, and in particular to a method for visually detecting antigens. Background Art

[0002] Microcystins (MCs) are a class of cyclic heptapeptide hepatotoxins produced by cyanobacteria with multiple isomers. Among them, microcystin LR (MC-LR) is the most widely distributed and most toxic biotoxin in the microcystin family. Due to the strong stability, low volatility, acid, alkali and high temperature resistance of MC-LR's ring structure, it cannot be degraded by some common protein or peptide hydrolases. It can maintain a high stability in water and is widely distributed in freshwater environments around the world. It has strong toxicity to the liver, nervous system and kidneys. When MC-LR enters the human body, it is absorbed into the blood and then transported to various organs. As the concentration accumulates, it can cause chronic poisoning, resulting in symptoms such as nausea, dizziness, and vomiting, posing a serious threat to human health. my country stipulates that the content of MC-LR in drinking water must not exceed 1.0μg / L. Therefore, rapid detection of MC-LR in freshwater sources is crucial to ensure people's health.

[0003] At present, the methods for detecting MC-LR mainly include electrochemical immunosensor analysis, optical immunosensor analysis, colorimetric analysis and chromatographic immunosensor analysis. However, these methods respond to the concentration of microcystin LR through a single signal, which is prone to false positive and false negative phenomena, affecting the accuracy of the test results. Summary of the Invention

[0004] In view of the problems of the existing technology, the present application provides a method for visually detecting antigens, which is simple and convenient to operate and has high detection accuracy.

[0005] The method for visually detecting antigens in this application comprises the following steps in sequence:

[0006] Step S100, providing a visual sensor, wherein the visual sensor comprises a transparent reaction container, and a first antibody capable of specifically binding to the antigen is fixed at the bottom of the reaction container;

[0007] Step S200, adding the sample to be tested into the reaction container to react, and performing the first elution after the reaction is completed;

[0008] Step S300, adding a second antibody that can specifically bind to the antigen to the reaction container for reaction, wherein the second antibody is coupled with catalase, and performing a second elution after the reaction is completed;

[0009] Step S400, adding hydrogen peroxide solution to the reaction container for reaction, and during the reaction process, adding metal nanoparticle solution for mixing reaction;

[0010] Step S500: After the reaction is completed, the reaction container is irradiated with a laser, and the color R / G value of the reaction container and / or the temperature signal before and after irradiation are collected; a standard curve of the color R / G value and the antigen concentration, and / or a standard curve of the temperature signal and the antigen concentration are constructed, and the content of the antigen in the sample to be tested is calculated according to the standard curve.

[0011] Several optional methods are also provided below, but they are not intended to be additional limitations on the above-mentioned overall solution. They are merely further supplements or optimizations. Under the premise that there are no technical or logical contradictions, each optional method can be combined separately for the above-mentioned overall solution, or multiple optional methods can be combined.

[0012] Optionally, the concentration of the hydrogen peroxide solution is 2-4 mM, preferably 3 mM.

[0013] Optionally, the hydrogen peroxide solution is added and reacted for 2 to 4 minutes before adding the metal nanoparticle solution.

[0014] Optionally, the reaction and mixing reaction temperatures in step S400 are both 36-38° C., preferably 37° C., and the total reaction time is 15-17 min, preferably 16 min.

[0015] Optionally, the concentration of the metal nanoparticle solution is 1 to 30 nM, preferably 10 to 20 nM.

[0016] Optionally, step S400 includes: adjusting the pH of the reaction system and adding ferrous ions (Fe 2+ ) solution. If there is hydrogen peroxide, it will 2+ and H + The HO· and ·OOH generated under the catalysis of HO can oxidize and etch metal nanoparticles.

[0017] Optionally, the pH is 1 to 3, preferably 1.

[0018] Optionally, the concentration of the ferrous ion solution is 2-5 mM, preferably 4 mM.

[0019] Optionally, the metal nanoparticles are gold nanorods or gold nanocones.

[0020] Optionally, the characteristic peak of the gold nanorods or gold nanocones is greater than 800 nm. Optionally, the volume ratio of the hydrogen peroxide solution to the metal nanoparticle solution is 1:2 to 4, preferably 1:3.

[0021] Optionally, the laser irradiates the reaction container, and the reaction container is photographed using a device to collect the color R / G value, and / or measure the temperature signal before and after irradiation.

[0022] Optionally, the device is a smart phone and / or a portable infrared thermometer.

[0023] Optionally, the antigen is microcystin LR (MC-LR).

[0024] Optionally, the visual sensor is an ELISA plate, wherein the bottom of the reaction wells in the ELISA plate is fixed with a MC-LR primary antibody (primary antibody).

[0025] Optionally, the first elution and the second elution are performed using a PBS buffer having a pH of 7.4.

[0026] Compared to existing technologies, the detection method provided in this application leverages the LSPR and photothermal effects of Au NRs, enabling the simultaneous measurement of both temperature and color signals, improving detection accuracy. Combined with a portable device, it enables simple identification and results, with a minimum detection limit of 0.0010 μg / L, effectively meeting the requirements for quantitative determination of MC-LR in water samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The structure and detection principle diagram of the visual sensor in this application;

[0028] Figure 2A UV-visible spectra of Au NRs at different hydrogen peroxide concentrations;

[0029] Figure 2B Transmission electron microscopy characterization of Au NRs in different H2O2 concentration solutions; among them, B1 is 0mM, B2 is 0.1mM, B3 is 0.5mM, and B4 is 0.75mM;

[0030] Figure 2C Temperature characterization diagram of Au NRs under different hydrogen peroxide concentrations;

[0031] Figure 3A The results of the color change and temperature difference of the solution under different concentrations of MC-LR are shown in the figure;

[0032] Figure 3B It is the standard curve of R / G value and temperature difference change with MC-LR concentration;

[0033] Figure 4A The results of color change and temperature difference change of water samples spiked with different concentrations are shown in the figure;

[0034] Figure 4BThe bar graph shows the R / G value and temperature difference of spiked water samples at different concentrations;

[0035] Figure 4C This is the result diagram of the color change and temperature difference of water samples in different environments;

[0036] Figure 4D This is a bar graph showing the color changes and temperature differences of water samples in different environments. DETAILED DESCRIPTION

[0037] The technical solution described in this application is further described below in conjunction with specific implementation methods, but this application is not limited thereto.

[0038] Preparation Example 1

[0039] 1.1 Preparation method

[0040] (1) Pipette 125 μL of 10 mM chloroauric acid (HAuCl4) into 5.0 mL of 100 mM cetyltrimethylammonium bromide (CTAB). Mix well, then quickly add 300 μL of 10 mM freshly prepared sodium glacial borohydride (NaBH4). Stir vigorously for 2 min. At this time, the color of the solution changes from yellow to dark brown. Slowly reduce the speed, remove the magnetic stirrer, and place it in a 30°C water bath for 1.5 to 2 h to obtain a gold seed solution.

[0041] (2) Pipette 5.0 mL of 10 mM HAuCl4 into 100 mL of 100 mM CTAB, mix well, add 900 μL of 10 mM silver nitrate (AgNO3), and then quickly add 570 μL of 100 mM ascorbic acid (VC). Stir vigorously (1000 r / s) for 30 s, then add 240 μL of gold seed solution, stir evenly, remove the magnetic stirrer, and let it stand in a 30 ° C water bath overnight to obtain gold nanorods (Au NRs) solution.

[0042] 1.2 Structural characterization

[0043] The spectral characterization was performed using UV-visible spectrometer, such as Figure 2A As shown in Figure 2, Au NRs have two UV-visible absorption peaks, namely the lateral absorption peak at about 520nm and the longitudinal absorption peak at about 850nm (red line). In addition, the morphology of Au NRs was characterized by transmission electron microscopy (TEM). Figure 2B As shown in the figure, the Au NRs are rod-shaped with a longitudinal length of about 43 nm (B1) and an aspect ratio of 6:1. This proves the successful synthesis of Au NRs.

[0044] 1.3 Physical and chemical properties

[0045] (1) 5.0 mL of the above Au NRs solution was centrifuged at 9000 rpm for 10 min, the supernatant was removed, and the precipitate was dissolved in 500 μL of 0.4 mM CTAB and concentrated 10 times.

[0046] (2) Add 60 μL of concentrated gold nanorod solution to each of the eight reaction wells of a blank ELISA plate, and then quickly add 10 μL of 2 M hydrochloric acid (HCl) and 10 μL of 0.4 mM ferrous sulfate (FeSO4) in that order. Finally, add 20 μL of hydrogen peroxide (H2O2) of different concentrations, namely 0 mM, 0.01 mM, 0.1 mM, 0.5 mM, 0.75 mM, 1.0 mM, 2.0 mM and 5.0 mM, to the corresponding ELISA plate. Incubate at room temperature for 15 min, and observe the color change of the solution in each reaction well.

[0047] like Figure 2A As shown, when the H2O2 solution is continuously improved, the Fe 2+ and H + The concentrations of HO· and ·OOH generated under the catalysis of Au NRs also gradually increased, and the degree of oxidation etching of Au NRs also gradually deepened. From the UV-visible spectrum of Au NRs, it can be found that the longitudinal absorption peak of Au NRs gradually blue-shifted, and the color gradually changed from brown-red to yellow.

[0048] The changes of Au NRs can be further confirmed by TEM characterization. The sizes of Au NRs are also different under different H2O2 concentration solutions, such as Figure 2B As shown, 0mM (B1, ~43nm), 0.1mM (B2, ~30nm), 0.5mM (B3, ~22nm), 0.75mM (B3, ~15nm).

[0049] In addition, the photothermal intensity of Au NRs of different sizes is different. As the concentration of H2O2 solution gradually increases, the length of Au NRs gradually becomes shorter, and the heat generated by them under the action of 808nm laser is less ( Figure 2C ). It can be seen that changes in the concentration of hydrogen peroxide will cause changes in two types of visual signals: color and temperature.

[0050] Preparation Example 2

[0051] (1) Pipette 5.0 μL of 4.8 mg / mL MC-LR secondary antibody (purchased from Shenzhen Kejie Industrial Development Co., Ltd.), mix with 490 μL of 10 mM phosphate-buffered saline (PBS) buffer and 5 μL of 1 mg / mL polyethylene glycol (PEG), and incubate at 25°C for 30 min.

[0052] (2) Place the protein desalting centrifugal column in a 15 mL centrifuge tube and centrifuge at 1000 × g (RCF) for 2 min to remove the storage solution in the centrifuge tube;

[0053] (3) Slowly add 1 mL of PBS buffer to the column, centrifuge at 1000 × g (RCF) for 2 min, and then remove the storage solution in the centrifuge tube. Repeat three times, then add 500 μL of the above antibody mixture and 40 μL of PBS buffer, centrifuge at 1000 × g (RCF) for 2 min, retain the solution in the centrifuge tube and add 500 μL of 0.50 mM catalase, and react in a refrigerator at 4°C for 2 h to obtain the catalase-conjugated MC-LR secondary antibody.

[0054] Example 1

[0055] (1) 200 μL of MC-LR standard solution of different concentrations (0, 0.0010, 0.010, 0.10, 1.0, 10 μg / L) were respectively added to the reaction wells of an ELISA plate (purchased from Shanghai ELISA Biotechnology Co., Ltd., 96T). The bottom of each reaction well was fixed with MC-LR primary antibody. The plate was placed in a 37°C constant temperature mixer. After reacting for 20 minutes, the solution was poured out and the ELISA plate was filled with PBS buffer for washing. After standing for 1 minute, the solution was poured out and the residual liquid in the kit was gently tapped on filter paper. The washing was repeated three times.

[0056] (2) Add 50 μL of catalase-conjugated MC-LR secondary antibody to each reaction well, place in a 37°C constant temperature mixer, react for 20 min, then discard the solution, and fill each reaction well with PBS buffer for washing. Let it stand for 1 min and then discard. Repeat the washing three times;

[0057] (3) Then, 20 μL of 3.0 mM hydrogen peroxide (H2O2) solution was added to each reaction well. After standing for 3 min, 10 μL of 2 M HCl solution, 10 μL of 4 mM ferrous sulfate (FeSO4), and 60 μL of the Au NRs solution prepared in Preparation Example 1 were added in sequence and reacted for 13 min.

[0058] (4) After the reaction is completed, a smartphone is used to take a photo and read the RGB. At the same time, an infrared thermometer is used to record the temperature change under the action of an 808 nm laser. A standard curve of the color R / G value and the MC-LR antigen concentration and a standard curve of the temperature signal and the MC-LR antigen concentration are constructed, where ΔT is the temperature difference of the reactor before and after laser irradiation.

[0059] like Figure 3AAs shown in the figure, as the concentration of MC-LR continues to increase, more antibodies modified with catalase are left in the reaction tank. In the presence of catalase, the subsequently added hydrogen peroxide will be decomposed into oxygen and water, and the resulting free radicals will be fewer, and the oxidation etching of Au NR will be weaker. The temperature signal gradually increases, and the color (R / G value) signal gradually decreases. The two signal intensities show a good linear relationship with the logarithm of the MC-LR concentration, that is: Y R / G =-0.159X+0.725, correlation coefficient R 2 =0.972; Y ΔT =0.526X+3.63, correlation coefficient R 2 =0.982; the minimum detection limit can reach 0.0010μg / L, which can meet the quantitative determination of MC-LR in water samples, such as Figure 3B shown.

[0060] Example 2

[0061] (1) Pipette 200 μL of spiked water samples with different concentrations (1.0, 0.10, 0.010, and 0 μg / L) and water samples to be tested from different environments, numbered ZJ, GZ, SD, and HN, and add them to the reaction wells of the ELISA plate. The bottom of each reaction well is fixed with the MC-LR primary antibody. Place it in a 37°C constant temperature mixer. After reacting for 20 minutes, pour out the solution and fill each reaction well with PBS buffer for washing. After standing for 1 minute, pour out the solution and gently tap the residual liquid in the kit on filter paper. Repeat the washing three times.

[0062] (2) Add 50 μL of catalase-conjugated MC-LR secondary antibody to each reaction well, place in a 37°C constant temperature mixer, react for 20 min, then discard the solution, and fill each reaction well with PBS buffer for washing. Let it stand for 1 min and then discard. Repeat the washing three times;

[0063] (3) Then, 20 μL of 3.0 mM H2O2 solution was added to each reaction well. After standing for 3 min, 10 μL of 2 M HCl, 10 μL of 4 mM FeSO4, and 60 μL of Au NRs were added in sequence and reacted for 13 min.

[0064] (4) After the reaction was completed, RGB and temperature signals were obtained using a smartphone and an infrared thermometer under the action of an 808 nm laser, respectively, and the RGB and temperature signals were substituted into the standard curve in Example 1 to calculate the content of MC-LR in each environmental water sample.

[0065] The results are as follows Figure 4A 、 Figure 4BAs shown in the figure, with the continuous increase of spike concentration, the color of the solution gradually changes from pink to green, the R / G value gradually decreases and the temperature difference change gradually increases.

[0066] Substituting the two signal intensities into the corresponding linear equations, we can obtain that the recovery rate of the temperature signal of this method is 90%-103%, and the recovery rate of the color signal (R / G value) is 91%-105%, as shown in Figure 1. The color and temperature changes of the water samples under different environments after reaction are different ( Figure 4C 、 Figure 4D ). This demonstrates that the proposed method has good reliability and has the potential to be applied to the on-site rapid detection of MC-LR in environmental water samples.

[0067] Table 1 Test results of spiked water samples

[0068]

[0069] As shown in Table 2, compared with the standard ELISA method, the deviation of the detection results of the present application method is smaller, indicating that the present application method is reliable and has high accuracy.

[0070] Table 2 Concentration test results of water samples under different environments

[0071]

[0072] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present application. It should be noted that a person of ordinary skill in the art may make various modifications and improvements without departing from the spirit of the present application, and these modifications and improvements fall within the scope of protection of the present application. Therefore, the scope of protection of the present application shall be determined by the appended claims.

Claims

1. A method for visually detecting antigens, characterized in that: The method includes the following steps: Step S100, providing a visual sensor, wherein the visual sensor comprises a transparent reaction container, and a first antibody capable of specifically binding to the antigen is fixed at the bottom of the reaction container; Step S200, adding the sample to be tested into the reaction container to react, and performing the first elution after the reaction is completed; Step S300, adding a second antibody that can specifically bind to the antigen to the reaction container for reaction, wherein the second antibody is coupled with catalase, and performing a second elution after the reaction is completed; Step S400, adding hydrogen peroxide solution to the reaction container for reaction, and during the reaction process, adding metal nanoparticle solution for mixing reaction; Step S500: After the reaction is completed, the reaction container is irradiated with a laser, and the color R / G value of the reaction container and / or the temperature signal before and after irradiation are collected; a standard curve of the color R / G value and the antigen concentration, and / or a standard curve of the temperature signal and the antigen concentration are constructed, and the content of the antigen in the sample to be tested is calculated according to the standard curve.

2. The method according to claim 1, characterized in that The concentration of the hydrogen peroxide solution is 2-4 mM.

3. The method according to claim 2, characterized in that After adding the hydrogen peroxide solution and reacting for 2 to 4 minutes, the metal nanoparticle solution is added.

4. The method according to claim 2, characterized in that The concentration of the metal nanoparticle solution is 1-30 nM.

5. The method according to claim 4, characterized in that The volume ratio of the hydrogen peroxide solution to the metal nanoparticle solution is 1:2-4.

6. The method according to claim 1, characterized in that Step S400 includes: adjusting the pH of the reaction system and adding a ferrous ion solution during the reaction, and then adding the metal nanoparticle solution.

7. The method according to claim 6, characterized in that The pH is 1 to 3; The concentration of the ferrous ion solution is 2-5 mM.

8. The method according to claim 1, characterized in that The metal nanoparticles are gold nanorods or gold nanocones.

9. The method according to claim 8, characterized in that The characteristic peak of the gold nanorod or gold nanocone is greater than 800 nm.

10. The method according to claim 1, characterized in that The antigen is microcystin LR.

Images