A copper deposition enhanced lateral flow immunoassay detection kit, detection method and application

Abstract

The present invention relates to the field of biological detection, and particularly to a copper deposition enhanced lateral flow immunoassay detection kit, a detection method and an application thereof. The detection method first obtains a gold-labeled antibody; mixes a sample to be detected with the gold-labeled antibody to form an antigen-antibody complex solution, and drops the antigen-antibody complex solution onto a lateral flow immunoassay test strip for color development; immerses the lateral flow immunoassay test strip dropped with the antigen-antibody complex solution into a mixed solution containing CuSO4 and ascorbic acid, adds sodium dichloroisocyanurate for reaction; and quantitatively analyzes the concentration of a target analyte according to the color development intensity on the test strip. The present invention also relates to a lateral flow immunoassay detection kit and its application. The detection method can achieve detection in complex matrix samples and detection of low-concentration target analytes.

Description

Technical Field

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

[0002] Lateral flow immunoassay (LFIA) technology has been widely used in food safety detection and environmental monitoring due to its characteristics of rapidity, portability, and simplicity of operation. However, the detection sensitivity of traditional LFIA technology is limited, especially in the detection of low-concentration target analytes, and it is difficult to meet the actual application requirements. In recent years, in order to improve the detection performance of LFIA, many researchers have tried to introduce signal enhancement material methods based on gold nanoparticles (AuNPs) into LFIA detection. However, the sensitivity improvement of these methods is limited, and usually accompanied by problems such as complex operation and high cost, which further limit the detection application. Therefore, there is an urgent need for a new signal enhancement strategy with high sensitivity, simple operation, and suitable for complex matrix samples. Summary of the Invention

[0003] Aiming at the above deficiencies of the prior art, the present invention provides a copper deposition enhanced lateral flow immunoassay detection method. This detection method can be used for detecting in complex matrix samples and detecting low-concentration target analytes.

[0004] The present invention provides a copper deposition enhanced lateral flow immunoassay detection method, including the following steps:

[0005] Step 1: Add borate buffer solution 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% polyvinylpyrrolidone, 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, discard the supernatant, and obtain the gold-labeled antibody;

[0006] Step 2: Mix the sample to be tested with the gold-labeled antibody obtained 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;

[0007] Step 3: Immerse the lateral flow immunoassay test strip dropped with the antigen-antibody complex solution in Step 2 into a mixed solution containing CuSO 4 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 according to the color development intensity on the test strip.

[0008] Preferably, the mixed solution containing CuSO 4The mixture with ascorbic acid AA is specifically: prepare a 25 - 300 mM mixture containing CuSO 4 and ascorbic acid AA with a 0.1 - 0.5 M propionic acid buffer solution. The pH of the mixture is 3 - 5.5, and the volume ratio of CuSO 4 to ascorbic acid AA is 1:0.5 - 1:5.

[0009] Preferably, the volume ratio of the mixture to sodium dichloroisocyanurate DCCNa is 20:1 - 100:1.

[0010] Preferably, the propionic acid buffer solution includes propionic acid and sodium propionate, and the volume ratio of the two is 1:9 - 9:1.

[0011] Preferably, the preparation method of the AuNPs gold nanoparticle solution in step 1 is: mix and react the sodium citrate solution and the HAuCl 4 solution, then add the sodium citrate solution and the HAuCl 4 solution in turn, repeat adding 6 - 16 times, with a time interval of 1 - 3 min. After reacting for 30 min, cool to room temperature to obtain the AuNPs solution. The particle size of the AuNPs gold nanoparticles in the AuNPs solution is 30 - 45 nm;

[0012] The mixing and reaction of the sodium citrate solution and the HAuCl 4 solution is specifically: when the sodium citrate solution is vigorously stirred and heated to the beginning of boiling, add the HAuCl 4 solution, control the reaction temperature at 90 - 100 °C, and the reaction time is 5 - 20 minutes. The volume ratio of the sodium citrate solution to the HAuCl 4 solution is 200:1 - 50:1.

[0013] Preferably, the ratio of the AuNPs gold nanoparticle solution, borate buffer solution, goat anti - human IgG antibody, polyvinylpyrrolidone, and bovine serum albumin in step 1 is: 1 - 5 mL: 50 - 200 μL: 1 - 20 μL: 1 - 10 μL: 50 - 500 μL.

[0014] The present invention also provides a lateral - flow immunoassay kit for the above - mentioned detection method. The kit includes a lateral - flow immunoassay strip. The detection line of the lateral - flow immunoassay strip is coated with goat anti - human IgG antibody, and the control line is coated with mouse anti - goat IgG antibody.

[0015] The present invention also provides an application of the lateral - flow immunoassay kit for detecting target analytes in food safety detection and environmental monitoring. The target analytes are pathogen antigens, toxins, or biomarkers present at low concentrations.

[0016] Preferably, it is used to detect human IgG in fish serum.

[0017] Preferably, it is used to detect human IgG in rabbit serum.

[0018] The advantages of the present invention are as follows:

[0019] 1. Significantly improve sensitivity: By introducing the propionic acid buffer + DCCNa system, the present invention significantly enhances the copper deposition signal, increasing the detection sensitivity by 3 - 5 orders of magnitude compared to the AuNPs-based LIFA test strip.

[0020] 2. Simple operation: The copper deposition reaction conditions are mild, and the operation is simple. High - efficiency detection can be achieved without complex instruments and without heating the reaction.

[0021] 3. Strong applicability: The present invention can achieve high - efficiency detection in complex biological matrices such as fish serum and rabbit serum, and is not significantly affected by sample matrix interference.

[0022] 4. High accuracy: The detection recovery rates all exceed 85%, and the relative standard deviation (RSD) is lower than 5%, indicating that the detection results have high accuracy and repeatability.

[0023] 5. Low cost: Compared with traditional enhancement techniques (such as quantum dots or high - end fluorescent labels), the copper deposition enhancement strategy has lower costs and does not require additional complex equipment. Description of the Drawings

[0024] Figure 1 It is the flow chart of the copper deposition enhanced test strip. (a) Flow chart of the copper deposition enhanced test strip in Comparative Example 1; (b) Flow chart of the copper deposition enhanced test strip in Example 1.

[0025] Figure 2 It is the characterization of AuNPs in the examples. (a) Low - magnification TEM image, scale bar is 50 nm; (b) High - magnification TEM image, scale bar is 100 nm.

[0026] Figure 3 It is the related pictures of AuNPs gold nanoparticles and gold - labeled antibodies and the ultraviolet - visible absorption spectrum.

[0027] Figure 4 It is the copper deposition results of Comparative Example 1 and Example 1 under different concentrations of human IgG. The red asterisk (*) represents the detection limit visible to the naked eye.

[0028] Figure 5 It is the calibration curve of copper deposition color development in Comparative Example 1 and Example 1. The inset shows the linear calibration curve of Example 1.

[0029] Figure 6It is a copper deposition reaction with the enhanced color development effect of DCCNa on copper deposition.

[0030] Figure 7 It is the ultraviolet-visible absorption spectrum of the copper deposition reaction under three systems.

[0031] Figure 8 It is the copper deposition color development photos of different concentrations of human IgG in Application Example 1 and Application Example 2. Detailed implementation manners Example 1

[0032] A copper deposition enhanced lateral flow immunoassay detection method includes the following steps:

[0033] Step 1: Add 150 mL of 2.2 mM sodium citrate solution into a three-necked round-bottom flask. When stirring vigorously and heating to the beginning of boiling, add 1 mL of HAuCl 4 (25 mM). The color of the solution changes from yellow to blue-gray and then to soft pink. After the synthesis of gold seeds, immediately cool the reaction until the solution temperature reaches 90 °C. Then, inject 1 mL of sodium citrate (60 mM) and 1 mL of HAuCl 4 solution (25 mM) successively (time interval 2 min). Repeat adding 6 - 16 times. After reacting for 30 min, cool to room temperature to obtain a 30 - 45 nm AuNPs gold nanoparticle solution; Figure 2 It is the characterization of AuNPs. Figure 2 In (a), it is a low-magnification TEM image with a scale of 50 nm, showing the overall distribution of AuNPs. It can be seen that the AuNPs are evenly distributed and do not aggregate; Figure 2 In (b), it 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 generally spherical with a size of about 45 nm.

[0034] Step 2: Add borate buffer to 1 mL of the AuNPs solution obtained 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% polyvinylpyrrolidone, and react under the same conditions for 30 minutes. Then, add 100 μL of 1% bovine serum albumin to block non-specific binding sites and continue to stir at room temperature for 20 minutes. After the reaction, centrifuge the solution, discard the supernatant, and resuspend the precipitate in borate buffer to obtain a gold-labeled antibody; Figure 3It is the relevant pictures of AuNPs gold nanoparticles and gold-labeled antibodies and the ultraviolet-visible absorption spectra. It can be seen that the color of AuNPs gold nanoparticles is wine red, and the color of gold-labeled antibodies is red-black. The absorption peak of AuNPs gold nanoparticles is at 520 nm, and the absorption peak of gold-labeled antibodies is at 525 nm. There is a slight red shift compared to the absorption peak of AuNPs gold nanoparticles, indicating that the gold-labeled antibodies are adsorbed on the surface of AuNPs gold nanoparticles.

[0035] Step 3: Coat the detection line of the lateral flow immunoassay strip with specific capture antibodies, and coat the control line with anti-sheep mouse antibodies. Subsequently, assemble the sample pad, conjugate pad, NC membrane, and absorbent pad onto the PVC backing board in sequence, with an overlap of approximately 2 mm between adjacent components. After assembly, cut the strip into strips with a width of 4 mm. Mix the sample to be tested with the gold-labeled antibodies described in Step 2 to form an antigen-antibody complex solution, and drop the antigen-antibody complex solution onto the lateral flow immunoassay strip for color development.

[0036] Step 4: Immerse the lateral flow immunoassay strip that has been dropped with the antigen-antibody complex solution in Step 3 into a mixture containing CuSO 4 and AA (prepare 125 mM of CuSO 4 and AA with a propionic acid buffer solution with pH = 4.4 to stabilize the formation of copper complexes), add 0.01 - 1 M of sodium dichloroisocyanurate DCCNa, and react at room temperature for 10 minutes; use AA and DCCNa to reduce Cu 2+ to elemental copper and deposit it in the detection line and control line areas of the strip; according to the color development intensity on the strip, quantitatively analyze the concentration of the target analyte. It can achieve the detection of human IgG with a minimum concentration of 50 pg / mL, and the copper deposition color development signal has a linear relationship with the human IgG concentration in the range of 50 - 500 pg / mL. Add 100 μL of DCCNa to the copper deposition reaction solution for the reduction of copper and to increase the active sites on the surface of the gold nanoparticles on the strip to promote precise copper deposition. The copper deposition signal is used to enhance the detection sensitivity of human IgG. As shown in (b) of Figure 4 , the detection limit can reach 50 pg / mL.

[0037] The propionate ions ionized by sodium propionate (NaPr) in water complex with copper ions, increasing 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. Formula I is the 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, making more copper ions in the solution participate in the copper deposition reaction.

[0038]

[0039] Example 2

[0040] A copper deposition enhanced lateral flow immunoassay detection method, which is the same as that in Example 1, except that sodium dichloroisocyanurate (DCCNa) is not added in step 4.

[0041] Step 4 is specifically as follows: Immerse the lateral flow immunoassay test strip dropped with the antigen-antibody complex solution in step 3 into a mixed solution containing CuSO 4 and AA (125 mM of CuSO 4 and AA are prepared with a propionic acid buffer solution with pH = 4.4 to stabilize the formation of copper complexes); according to the color development intensity on the test strip, quantitatively analyze the concentration of the target analyte, and the detection of human IgG with a minimum concentration of 10 4 pg / mL can be achieved. Comparative Example 1

[0042] A lateral flow immunoassay detection method, which is the same as that in Example 1, except that in step 4, CuSO 4 and AA are prepared with deionized water. After mixing the two solutions, insert the test strip, take it out after reacting in a 37°C water bath for 10 minutes. As shown in (a) of Figure 1 , the color of the mixed solution changes from blue to light red, and the test strip shows poor sensitivity enhancement. As shown in (a) of Figure 4 , the detection of human IgG with a minimum concentration of 10 ng / mL can be achieved. The detection limit enhanced by copper deposition in Example 1 is 200 times higher than that in Comparative Example 1. As shown in (b) of Figure 1 , the color of the solution in Example 1 quickly changes from colorless to light green and then to dark red, and the test strip shows excellent sensitivity enhancement.

[0043] As Figure 5 shown in (a), the calibration curve of copper deposition color development in Comparative Example 1. As shown in (b) of Figure 5 , 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) when the concentration of human IgG is 20 - 500 pg / mL.

[0044] Figure 6 is the study on the enhancement effect of DCCNa on copper deposition color development. From left to right, they respectively represent the copper deposition reactions in the deionized water + DCCNa system of Comparative Example 1, the propionic acid buffer solution system of Example 2, and the propionic acid buffer solution + DCCNa system of Example 1. It can be seen that the color change during the reaction in the deionized water + DCCNa system is very small and the reaction is very slow. While the color changes in the propionic acid buffer solution system and the propionic acid buffer solution + DCCNa system are very large, especially the propionic acid buffer solution + DCCNa system shows an obvious copper elemental color (dark red).

[0045] Figure 7 They are the ultraviolet-visible absorption spectra of copper deposition reactions under three systems (deionized water + DCCNa system in Comparative Example 1, propionate buffer system in Example 2 of the present invention, and propionate buffer + DCCNa system in Example 1 of the present invention). It can be seen that the ultraviolet absorption values under the propionate buffer system and the propionate buffer + DCCNa system are significantly higher than those under the deionized water + DCCNa system. Among them, the ultraviolet absorption value under the propionate buffer + DCCNa system is the highest, indicating that its copper deposition reaction is the most complete, which is the result of the combined action of propionate buffer and DCCNa.

[0046] Experiments show that under room temperature conditions, using the propionate buffer + DCCNa system provided by the present invention can significantly enhance the copper deposition signal.

[0047] Detection of human IgG in fish serum

[0048] 1. Sample preparation: Add human IgG at different concentrations (0 to 10 5 pg / mL) to fish serum;

[0049] 2. Detection operation: Perform copper deposition detection according to the method of Example 1;

[0050] 3. Result analysis: The detection results show that under the influence of the fish serum matrix, the color development recovery rate of copper deposition still remains between 90% and 125%, with high reproducibility.

[0051] Detection of human IgG in rabbit serum

[0052] 1. Sample preparation: Add human IgG at different concentrations (0 to 10 5 pg / mL) to rabbit serum;

[0053] 2. Detection operation: Perform copper deposition detection according to the method of Example 1;

[0054] 3. Result analysis: The detection results show that under the influence of the rabbit serum matrix, the color development recovery rate of copper deposition still remains between 85% and 120%, with high reproducibility.

[0055] 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%.

[0056] Table 1 Content table of human IgG in the samples of Application Example 1 and Application Example 2

[0057]

[0058] Figure 8It is the copper deposition color development photos of different concentrations of human IgG in Application Example 1 and Application Example 2. To evaluate the color development stability, each concentration was replicated three times in fish serum and rabbit serum. As can be seen from the figure, at four different concentrations (0, 200, 500, 10 5 pg / mL), the color development intensity of the T line showed high consistency in different serum backgrounds, with little color development difference. This indicates that the copper deposition color development enhancement method not only significantly improves the detection sensitivity but also has good color development stability, which is suitable for the reliable detection of IgG protein in actual samples.

[0059] A copper deposition enhanced lateral flow immunoassay detection method of the present invention is applicable to the detection of target analytes (such as human IgG) in complex matrices (such as fish serum, rabbit serum), and can also be extended to the detection of other biomolecules (such as pathogen antigens, toxins, etc.). The present invention can be outlined in other specific forms without departing from the spirit or main features of the present invention. Therefore, in any aspect, the above embodiments of the present invention should only be considered as illustrative of the present invention and not restrictive of the present invention. The claims point out the scope of the present invention, while the above description does not point out the scope of the present invention. Therefore, any change within the meaning and scope equivalent to the claims of the present invention should be considered as included within 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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