Copper deposition enhanced lateral flow immunoassay detection kit, detection method and application

Abstract

The invention relates to the field of biological detection, in particular to a copper deposition enhanced lateral flow immunoassay detection kit, a detection method and application. The detection method comprises the following steps: firstly, obtaining a gold-labeled antibody; mixing a sample to be detected with the gold-labeled antibody to form an antigen-antibody complex solution, and dropping the antigen-antibody complex solution onto the lateral flow immunoassay test strip for color development; immersing the lateral flow immunoassay test strip dripped with the antigen-antibody complex solution into a mixed solution containing CuSO4 and ascorbic acid, and adding sodium dichloroisocyanurate for reaction; and quantitatively analyzing the concentration of the target analyte according to the developing intensity on the test strip. The invention also relates to a lateral flow immunodetection kit and application thereof. According to the detection method, detection can be realized in a complex matrix sample, and detection can be realized in a low-concentration target analyte.

Description

Technical Field

[0001] The invention relates to the field of biological detection, and in particular to a copper deposition enhanced lateral flow immunoassay detection method. Background Art

[0002] Lateral flow immunoassay (LFIA) has been widely used in food safety testing and environmental monitoring due to its rapid, portable and easy-to-operate characteristics. However, the detection sensitivity of traditional LFIA technology is limited, especially in the detection of low-concentration target analytes, which is difficult to meet the actual application needs. In recent years, in order to improve the detection performance of LFIA, many researchers have tried to introduce signal enhancement materials based on gold nanoparticles (AuNPs) into LFIA detection. However, the sensitivity improvement of these methods is limited, and they are usually accompanied by problems such as complex operation and high cost, which further limits the detection application. Therefore, there is an urgent need for a new signal enhancement strategy that is highly sensitive, easy to operate and suitable for complex matrix samples. Summary of the invention

[0003] In view of the above-mentioned deficiencies of the prior art, the present invention provides a copper deposition enhanced lateral flow immunoassay detection method, which can detect complex matrix samples and detect low-concentration target analytes.

[0004] The present invention provides a copper deposition enhanced lateral flow immunoassay detection method, comprising the following steps: Step 1: Add borate buffer to the AuNPs solution, stir and react at room temperature, then add 0.1 mg / mL goat anti-human IgG antibody and 10% polyvinyl pyrrolidone, continue to stir and react, then add 1% bovine serum albumin, and continue to stir at room temperature. After the reaction is completed, centrifuge the solution and discard the supernatant to obtain the gold-labeled antibody; Step 2: Mix the sample to be tested with the gold-labeled antibody described in step 1 to form an antigen-antibody complex solution, and drop the antigen-antibody complex solution onto a lateral flow immunoassay test strip for color development; Step 3: Immerse the lateral flow immunoassay test strip dripped with the antigen-antibody complex solution in step 2 in a mixed solution containing CuSO4 and ascorbic acid AA, add 0.01~1 M sodium dichloroisocyanurate DCCNa, and react at 15~45°C; quantitatively analyze the concentration of the target analyte based on the color intensity on the test strip.

[0005] Preferably, the mixed solution containing CuSO4 and ascorbic acid AA in step 3 is specifically prepared by using 0.1~0.5 M propionic acid buffer solution to prepare 25~300 mM of the mixed solution containing CuSO4 and ascorbic acid AA, the pH of the mixed solution is 3~5.5, and the volume ratio of CuSO4 and ascorbic acid AA is 1:0.5~1:5.

[0006] Preferably, the volume ratio of the mixed solution to the sodium dichloroisocyanurate DCCNa is 20:1 to 100:1.

[0007] Preferably, the propionic acid buffer solution comprises propionic acid and sodium propionate, and the volume ratio of the two is 1:9 to 9:1.

[0008] Preferably, the preparation method of the AuNPs gold nanoparticle solution in step 1 is: mixing a sodium citrate solution and a HAuCl4 solution for reaction, then sequentially adding the sodium citrate solution and the HAuCl4 solution, repeating the addition 6 to 16 times, with a time interval of 1 to 3 minutes, reacting for 30 minutes and then cooling to room temperature to obtain an AuNPs solution, wherein the particle size of the AuNPs gold nanoparticles in the AuNPs solution is 30 to 45 nm; The mixed reaction of the sodium citrate solution and the HAuCl4 solution is specifically as follows: the sodium citrate solution is heated vigorously to the beginning of boiling, and the HAuCl4 solution is added, the reaction temperature is controlled at 90-100°C, the reaction time is 5-20 minutes, and the volume ratio of the sodium citrate solution to the HAuCl4 solution is 200:1-50:1.

[0009] Preferably, the ratio of the AuNPs gold nanoparticle solution, borate buffer, goat anti-human IgG antibody, polyvinyl pyrrolidone, and bovine serum albumin in step 1 is: 1-5 mL: 50-200 μL: 1-20 μL: 1-10 μL: 50-500 μL.

[0010] The present invention also provides a lateral flow immunoassay kit for the above detection method, the kit comprising a lateral flow immunoassay test strip, the detection line of the lateral flow immunoassay test strip is coated with goat anti-human IgG antibody, and the control line is coated with mouse anti-goat IgG antibody.

[0011] The present invention also provides an application of a lateral flow immunoassay kit for detecting target analytes in food safety detection and environmental monitoring, wherein the target analytes are pathogen antigens, toxins or biomarkers present in low concentrations.

[0012] Preferably, it is used to detect human IgG in fish serum.

[0013] Preferably, it is used to detect human IgG in rabbit serum.

[0014] The present invention is beneficial in that: 1. Significantly improved sensitivity: The present invention significantly enhances the copper deposition signal by introducing the propionic acid buffer + DCCNa system, thereby increasing the detection sensitivity by 3-5 orders of magnitude compared to the LIFA test strip based on AuNPs.

[0015] 2. Easy operation: The copper deposition reaction conditions are mild and the operation is simple. Efficient detection can be achieved without complex instruments or heating reactions.

[0016] 3. Strong applicability: The present invention can achieve efficient detection in complex biological matrices such as fish serum and rabbit serum, and is not significantly affected by interference from the sample matrix.

[0017] 4. High accuracy: The detection recovery rate is over 85%, and the relative standard deviation (RSD) is less than 5%, indicating that the test results are highly accurate and repeatable.

[0018] 5. Low cost: Compared with traditional enhancement technologies (such as quantum dots or high-end fluorescent labels), copper deposition enhancement strategies have lower costs and do not require additional complex equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Flow chart of copper deposition enhancement test strip. (a) Flow chart of copper deposition enhancement test strip in comparative example 1; (b) Flow chart of copper deposition enhancement test strip in example 1.

[0020] Figure 2 The AuNPs in the embodiment are characterized in (a) low-magnification TEM image, the scale bar is 50 nm; (b) high-magnification TEM image, the scale bar is 100 nm.

[0021] Figure 3 These are the relevant pictures and UV-visible absorption spectra of AuNPs gold nanoparticles and gold-labeled antibodies.

[0022] Figure 4 The copper deposition results of Comparative Example 1 and Example 1 under different concentrations of human IgG are shown in FIG. The red asterisk (*) indicates the detection limit visible to the naked eye.

[0023] Figure 5 The calibration curves of copper deposition color development in Comparative Example 1 and Example 1. The inset shows the linear calibration curve of Example 1.

[0024] Figure 6 It is the copper deposition reaction in which DCCNa enhances the color development of copper deposition.

[0025] Figure 7 These are the UV-visible absorption spectra of copper deposition reactions in three systems.

[0026] Figure 8 These are photos of copper deposition of human IgG at different concentrations in Application Examples 1 and 2. DETAILED DESCRIPTION

[0027] Example 1 A copper deposition enhanced lateral flow immunoassay detection method comprises the following steps: Step 1: Add 150 mL of 2.2 mM sodium citrate solution to a three-necked round-bottom flask, stir vigorously and heat until boiling begins, then add 1 mL of HAuCl4 (25 mM). The color of the solution changes from yellow to blue-gray and then to soft pink. After the gold seed is synthesized, cool the reaction immediately until the solution temperature reaches 90°C. Then, inject 1 ml of sodium citrate (60 mM) and 1 ml of HAuCl4 solution (25 mM) in sequence (time interval 2 min). Repeat 6 to 16 times. After reacting for 30 min, cool to room temperature to obtain a 30 to 45 nm AuNPs gold nanoparticle solution; Figure 2 is the characterization of AuNPs. Figure 2 (a) is a low-magnification TEM image with a scale bar of 50 nm, showing the overall distribution of AuNPs. It can be seen that the AuNPs are evenly distributed and not aggregated. Figure 2 (b) is a high-magnification TEM image with a scale of 100 nm, showing the morphology and size characteristics of AuNPs. It can be seen that the AuNPs are spherical as a whole and are about 45 nm in size.

[0028] Step 2: Add borate buffer to 1 mL of AuNPs solution described in step 1 and stir at room temperature for 15 minutes. Subsequently, add 10 μL of 0.1 mg / mL goat anti-human IgG (Fab-specific) antibody and 1 μL of 10% polyvinyl pyrrolidone and react for 30 minutes under the same conditions. Next, add 100 μL of 1% bovine serum albumin to block nonspecific binding sites and continue stirring at room temperature for 20 minutes. After the reaction is completed, centrifuge the solution, discard the supernatant, and resuspend the precipitate in borate buffer to obtain the gold-labeled antibody; Figure 3 These are the relevant pictures and UV-visible absorption spectra of AuNPs gold nanoparticles and gold-labeled antibodies. It can be seen that the color of AuNPs gold nanoparticles is wine red, the color of gold-labeled antibodies is red-black, the absorption peak of AuNPs gold nanoparticles is 520 nm, and the absorption peak of gold-labeled antibodies is 525 nm. Compared with the absorption peak of AuNPs gold nanoparticles, there is a slight red shift, indicating that the gold-labeled antibodies are adsorbed on the surface of AuNPs gold nanoparticles.

[0029] Step 3: The detection line of the lateral flow immunoassay test strip is coated with specific capture antibodies, and the control line is coated with mouse anti-sheep antibodies. Subsequently, the sample pad, conjugate pad, NC membrane and absorbent pad are assembled on the PVC backing plate in sequence, with adjacent components overlapping by about 2 mm. After assembly, it is cut into test strips with a width of 4 mm. The sample to be tested is mixed with the gold-labeled antibody described in step 2 to form an antigen-antibody complex solution, and the antigen-antibody complex solution is dropped onto the lateral flow immunoassay test strip for color development.

[0030] Step 4: Immerse the lateral flow immunoassay test strip dripped with the antigen-antibody complex solution in step 3 into a mixture containing CuSO4 and AA (125 mM CuSO4 and AA are prepared with a pH = 4.4 propionic acid buffer solution to stabilize the formation of the copper complex), add 0.01~1 M sodium dichloroisocyanurate DCCNa, and react at room temperature for 10 minutes; use AA and DCCNa to 2+ Reduced to elemental copper and deposited on the test line and control line area of ​​the test strip; according to the color intensity on the test strip, the concentration of the target analyte is quantitatively analyzed, and the detection of human IgG with a minimum concentration of 50 pg / mL can be achieved. The copper deposition color signal is linearly related to the human IgG concentration in the range of 50-500 pg / mL. 100 μL of DCCNa is added to the copper deposition reaction solution to reduce copper and increase the active sites on the surface of the gold nanoparticles on the test strip to promote accurate copper deposition. The copper deposition signal is used to enhance the detection sensitivity of human IgG, such as Figure 4 As shown in (b), the detection limit can reach 50 pg / mL.

[0031] The propionate ions ionized by sodium propionate NaPr in water are complexed with copper ions to increase the dissolution of copper ions. The propionic acid buffer solution can effectively stabilize the pH value in the reaction environment and increase the solubility of copper ions in the solution. The hypochlorite produced by the hydrolysis of DCCNa has strong oxidizing properties and can promote the reduction of copper ions to copper elements. At the same time, propionate is difficult to be oxidized by hypochlorite. Formula I is a reaction formula for the formation of a complex between copper ions and propionate ions. This reaction can promote the dissolution of copper sulfate in water, so that more copper ions in the solution participate in the copper deposition reaction.

[0032]

[0033] Example 2 A copper deposition enhanced lateral flow immunoassay detection method is the same as Example 1, except that sodium dichloroisocyanurate DCCNa is not added in step 4.

[0034] Step 4 is as follows: immerse the lateral flow immunoassay test strip dripped with the antigen-antibody complex solution in step 3 into a mixture containing CuSO4 and AA (125 mM CuSO4 and AA are prepared with a pH = 4.4 propionic acid buffer solution to stabilize the formation of the copper complex); quantitatively analyze the concentration of the target analyte according to the color intensity on the test strip, and the lowest concentration that can be achieved is 10 4 pg / mL human IgG detection.

[0035] Comparative Example 1 A lateral flow immunoassay detection method, which is the same as Example 1, except that in step 4, deionized water is used to prepare CuSO4 and AA, the two solutions are mixed and then inserted into a test strip, which is placed in a 37°C water bath for reaction for 10 minutes and then taken out. Figure 1 As shown in (a) in the figure, the color of the mixed solution changes from blue to light red, and the test strip shows poor sensitivity enhancement, such as Figure 4 As shown in (a), the detection of human IgG with a minimum concentration of 10 ng / mL can be achieved, and the detection limit of copper deposition enhancement in Example 1 is increased by 200 times compared with that in Comparative Example 1. Figure 1 As shown in (b), the color of the solution in Example 1 quickly changes from light green to dark red, and the test strip shows excellent sensitivity enhancement.

[0036] like Figure 5 As shown in (a) in FIG. 1 , the calibration curve of copper deposition color development in Comparative Example 1. Figure 5 As shown in (b), the calibration curve of copper deposition color development in Example 1. The inset shows the linear calibration curve under Example 1 of the present invention, which has good linearity (R 2 = 0.989).

[0037] Figure 6 This is a study on the effect of DCCNa on the color enhancement of copper deposition. From left to right, the copper deposition reactions in the deionized water + DCCNa system of Comparative Example 1, the propionic acid buffer system of Example 2, and the propionic acid buffer + DCCNa system of Example 1 are respectively shown. It can be seen that the color change of the deionized water + DCCNa system is very small during the reaction, and the reaction is very slow. However, the color changes of the propionic acid buffer system and the propionic acid buffer + DCCNa system are very large, especially the propionic acid buffer + DCCNa system shows obvious copper element color (deep red).

[0038] Figure 7 The UV-visible absorption spectra of copper deposition reaction under three systems (deionized water + DCCNa system in comparative example 1, propionic acid buffer system in Example 2 of the present invention, and propionic acid buffer + DCCNa system in Example 1 of the present invention). It can be seen that the UV absorption values ​​under the propionic acid buffer system and the propionic acid buffer + DCCNa system are significantly higher than the absorption value under the deionized water + DCCNa system, among which the UV absorption value under the propionic acid buffer + DCCNa system is the highest, indicating that its copper deposition reaction is the most thorough, which is the result of the joint action of the propionic acid buffer and DCCNa.

[0039] Experiments show that under room temperature conditions, the use of the propionic acid buffer + DCCNa system provided by the present invention can significantly enhance the copper deposition signal.

[0040] Application Example 1: Detection of Human IgG in Fish Serum 1. Sample preparation: Fish serum was spiked with different concentrations of human IgG (0 to 10 5 pg / mL); 2. Detection operation: perform copper deposition detection according to the method of Example 1; 3. Result analysis: The test results show that under the influence of fish serum matrix, the copper deposition color recovery rate remains between 90 and 125%, with high reproducibility.

[0041] Application Example 2: Detection of Human IgG in Rabbit Serum 1. Sample preparation: Rabbit serum was spiked with different concentrations of human IgG (0 to 10 5 pg / mL); 2. Detection operation: perform copper deposition detection according to the method of Example 1; 3. Result analysis: The test results showed that under the influence of rabbit serum matrix, the copper deposition color recovery rate remained between 85% and 120%, with high reproducibility.

[0042] Table 1 shows the content of human IgG in the samples of Application Example 1 and Application Example 2. It can be seen that the recovery rate is between 85% and 125%, and the RSD is less than 6%.

[0043] Table 1 Content of human IgG in samples of Application Example 1 and Application Example 2

[0044] Figure 8 The following are photos of copper deposition of human IgG at different concentrations in Application Examples 1 and 2. To evaluate the color stability, three experiments were repeated in fish serum and rabbit serum for each concentration. 5 pg / mL), the color intensity of the T line showed high consistency in different serum backgrounds, with little color difference. This shows that the copper deposition color enhancement method not only significantly improves the detection sensitivity, but also has good color stability, which is suitable for reliable detection of IgG protein in actual samples.

[0045] The present invention discloses a copper deposition enhanced lateral flow immunoassay detection method, which is suitable for the detection of target analytes (such as human IgG) in complex matrices (such as fish serum and rabbit serum), and can also be extended to the detection of other biological molecules (such as pathogen antigens, toxins, etc.). The present invention can be summarized in other specific forms that do not violate the spirit or main features of the present invention. Therefore, no matter from which point of view, the above embodiments of the present invention can only be regarded as an explanation of the present invention and cannot limit the present invention. The claims indicate the scope of the present invention, while the above description does not indicate the scope of the present invention. Therefore, any changes within the meaning and scope equivalent to the claims of the present invention should be considered to be included in the scope of the claims of the present invention.

Claims

1. A copper deposition enhanced lateral flow immunoassay detection method, characterized in that: The following steps are involved: Step 1: Add borate buffer to the AuNPs gold nanoparticle solution, stir and react at room temperature, then add 0.1 mg / mL goat anti-human IgG antibody and 10% polyvinyl pyrrolidone, continue to stir and react, then add 1% bovine serum albumin, and continue to stir at room temperature. After the reaction is completed, centrifuge the solution and discard the supernatant to obtain the gold-labeled antibody; Step 2: Mix the sample to be tested with the gold-labeled antibody described in step 1 to form an antigen-antibody complex solution, and drop the antigen-antibody complex solution onto a lateral flow immunoassay test strip for color development; Step 3: Immerse the lateral flow immunoassay test strip dripped with the antigen-antibody complex solution in step 2 in a mixed solution containing CuSO4 and ascorbic acid AA, add 0.01~1 M sodium dichloroisocyanurate DCCNa, and react at 15~45°C; quantitatively analyze the concentration of the target analyte based on the color intensity on the test strip.

2. The detection method according to claim 1, characterized in that The mixed solution containing CuSO4 and ascorbic acid AA in step 3 is specifically prepared by using 0.1~0.5 M propionic acid buffer solution to prepare 25~300 mM of the mixed solution containing CuSO4 and ascorbic acid AA, the pH of the mixed solution is 3~5.5, and the volume ratio of CuSO4 and ascorbic acid AA is 1:0.5~1:

5.

3. The detection method according to claim 2, characterized in that The volume ratio of the mixed solution to the sodium dichloroisocyanurate DCCNa is 20:1-100:

1.

4. The detection method according to claim 2, characterized in that The propionic acid buffer solution comprises propionic acid and sodium propionate, and the volume ratio of the two is 1:9-9:

1.

5. The detection method according to claim 3, characterized in that: The preparation method of the AuNPs gold nanoparticle solution in step 1 is: mixing a sodium citrate solution and a HAuCl4 solution for reaction, then sequentially adding the sodium citrate solution and the HAuCl4 solution, repeating the addition 6 to 16 times, with a time interval of 1 to 3 minutes, reacting for 30 minutes and then cooling to room temperature to obtain an AuNPs solution, wherein the particle size of the AuNPs gold nanoparticles in the AuNPs solution is 30 to 45 nm; The mixed reaction of the sodium citrate solution and the HAuCl4 solution is specifically as follows: the sodium citrate solution is heated vigorously to the beginning of boiling, and the HAuCl4 solution is added, the reaction temperature is controlled at 90-100°C, the reaction time is 5-20 minutes, and the volume ratio of the sodium citrate solution to the HAuCl4 solution is 200:1-50:

1.

6. The detection method according to claim 1, characterized in that The ratio of the AuNPs gold nanoparticle solution, borate buffer, goat anti-human IgG antibody, polyvinyl pyrrolidone, and bovine serum albumin in step 1 is: 1~5 mL: 50~200 μL: 1~20 μL: 1~10 μL: 50~500 μL.

7. A lateral flow immunoassay kit using the detection method of claim 1, characterized in that: The kit comprises a lateral flow immunoassay test strip, wherein the detection line of the lateral flow immunoassay test strip is coated with goat anti-human IgG antibody, and the control line is coated with mouse anti-goat IgG antibody.

8. A use of the lateral flow immunoassay kit as claimed in claim 7, characterized in that: Used for detection of target analytes in food safety testing and environmental monitoring, wherein the target analytes are pathogen antigens, toxins or biomarkers present in low concentrations.

9. Use of the lateral flow immunoassay kit according to claim 8, characterized in that: For detection of human IgG in fish serum.

10. Use of the lateral flow immunoassay kit according to claim 8, characterized in that: For detection of human IgG in rabbit serum.

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