Graphene-gold colloid as an immunochromatographic test strip marker and a preparation method thereof

Abstract

The application belongs to the technical field of virus detection, and particularly relates to a graphene-gold colloid marker for an immunochromatography test strip and a preparation method thereof. In view of the problems of insufficient sensitivity and slow detection speed of the existing marker for the immunochromatography test strip, the application provides a preparation method of the graphene-gold colloid marker for the immunochromatography test strip, which comprises the following steps: a, after graphene powder is mixed with pyrene formic acid, methanol is added for reaction; b, after centrifugation, cleaning and drying, MES solution is added thereto, EDC and sNHS are dropped after 5-20 min, stirring is carried out at 50-100 rpm for 10-40 min, and centrifugation is carried out at 5000-15000 rpm for 10-40 min, then the precipitate is resuspended to prepare an aminated graphene solution; c, desalination is carried out by dialysis in a NaCl solution, and pure water is added; d, a gold colloid solution is prepared, the aminated graphene is mixed with the gold colloid solution for reaction for 20-60 min to obtain the graphene-gold colloid marker. The graphene-gold colloid marker prepared by the application has higher sensitivity in detection of pathogenic bacteria, and has faster detection speed.

Description

Technical Field

[0001] This invention belongs to the field of virus detection technology, specifically relating to an immunochromatographic test strip marker graphene-colloidal gold and its preparation method. Background Technology

[0002] Microbial contamination is a widespread problem in the food industry, especially acute poisoning incidents caused by pathogenic bacteria, which pose a significant threat to food safety. Rapid and accurate detection of pathogenic bacteria in food has always been a challenge, making the development of rapid microbial detection methods urgent. Current methods for pathogen detection suffer from low sensitivity and long detection times. Instrumental methods require extensive sample pretreatment, extensive training of professionals, and expensive equipment. While enzyme-linked immunosorbent assay (ELISA) offers high sensitivity, it is time-consuming and cannot be performed on-site. Colloidal gold immunochromatography, however, combines high sensitivity, convenience, and speed, making it particularly suitable for on-site screening and possessing significant application value.

[0003] Graphene has a two-dimensional crystal structure, composed of a single layer of carbon atoms arranged in an ordered six-membered ring pattern, which can extend infinitely in two-dimensional space. Within the carbon family, graphene can be considered the basic unit of other dimensional carbon-based materials, capable of forming carbon materials of different dimensions through different curling methods. For example, graphene can be encapsulated, curled, and stacked to form zero-dimensional fullerenes, three-dimensional carbon nanotubes, and three-dimensional graphite, respectively. Graphene possesses excellent mechanical properties, excellent electrical conductivity, excellent thermal conductivity, and a very high specific surface area.

[0004] Currently, there are no reports of graphene being used as a marker in immunochromatographic test strips. Summary of the Invention

[0005] The technical problem to be solved by this invention is that the existing immunochromatographic test strips have insufficient sensitivity of markers and slow detection speed.

[0006] The technical solution of this invention to solve the above-mentioned technical problems is as follows: a method for preparing graphene-colloidal gold as a marker for immunochromatographic test strips is provided. This method includes the following steps:

[0007] a. Graphene powder and pyrene carboxylic acid are mixed at a weight ratio of 1-2:0.5-1.5, then added to methanol and reacted for 1-8 hours to obtain carboxylated graphene.

[0008] b. Centrifuge the carboxylated graphene obtained in step a, wash and dry it to obtain carboxylated graphene powder. Add 30-70mM MES solution to it. After 5-20 min, add 1-3mM EDC and 4-6mM sNHS dropwise to react. Stir at 50-100 rpm for 10-40 min to obtain aminographene. Centrifuge at 5000-15000 rpm for 10-40 min. After resuspending the precipitate, prepare an aminographene solution of 0.1-0.3 mg / ml.

[0009] c. Dialyze the aminated graphene solution in NaCl solution to remove salt for 16-28 hours, add pure water, and adjust the pH to 8.0-9.5.

[0010] d. Prepare a colloidal gold solution with a concentration of 0.02-0.10 mg / ml and adjust its pH value to 8.0-9.5. Mix the aminographene from step c with the colloidal gold solution and react for 20-60 min to obtain the graphene-colloidal gold label.

[0011] In the preparation method of the graphene-colloidal gold marker for the immunochromatographic test strip, the weight ratio of graphene powder to pyrene carboxylic acid in step a is 1.5:1.

[0012] The amount of graphene incorporated plays a crucial role in the sensitivity of the detection results, but excessive graphene addition increases costs. This invention takes a comprehensive approach and finds that the best overall effect is achieved when the weight ratio of graphene powder to pyrene is 1.5:1.

[0013] In the preparation method of the graphene-colloidal gold marker for the immunochromatographic test strip, the graphene powder in step a has a particle size of 5-40 μm. Choosing this particle size results in a lower minimum visible concentration limit for graphene and avoids the problem of excessively large particle sizes hindering its movement on the immunochromatographic test strip, thus affecting the detection speed. This leads to a faster detection speed for the immunochromatographic test strip.

[0014] In the preparation method of the graphene-colloidal gold marker on the immunochromatographic test strip, the amount of graphene used in step a is 0.01-0.02% of the weight of methanol.

[0015] Furthermore, the methanol is analytical grade with a concentration of ≥99.5%.

[0016] In the preparation method of the graphene-colloidal gold marker for the immunochromatographic test strip, the centrifugation conditions in step b are centrifugation at 5000-15000 rpm for 15-30 min.

[0017] In the preparation method of the above-mentioned immunochromatographic test strip marker graphene-colloidal gold, the cleaning in step b is to clean with water and anhydrous ethanol in sequence.

[0018] In the preparation method of the above-mentioned immunochromatographic test strip marker graphene-colloidal gold, the drying in step b is to dry at 50-70℃ for 1-3 hours.

[0019] In the preparation method of the graphene-colloidal gold marker for the immunochromatographic test strip, step b describes MES as (2-(N-morpholine)ethanesulfonic acid monohydrate) with a pH of 6.0, EDC as (1-ethyl-(3-dimethylaminopropyl)carbodiimide), and sNHS as (sulfonated N-hydroxythiosuccinimide). MES can be a derivative of MES or other compounds containing both sulfonic acid and morpholine groups; EDC can be a derivative of MES; and sNHS can be a derivative such as N-hydroxysuccinimide-based biotin ester, N-hydroxysuccinimide ester bisacrididine, N-hydroxysuccinimide-α-naphthaleneacetic acid ester, etc.

[0020] In the preparation method of the graphene-colloidal gold marker for the immunochromatographic test strip, the concentration of EDC in step b is 2.08 mM and the concentration of sNHS is 5.10 mM.

[0021] In the preparation method of the above-mentioned immunochromatographic test strip marker graphene-colloidal gold, the mass ratio of the aminated graphene in step b to the mass ratio of the MES solution is 0.001%-0.01%.

[0022] In the preparation method of the graphene-colloidal gold marker for the above-mentioned immunochromatographic test strip, steps c and d, adjusting the pH value, are both performed using K2CO3. Preferably, the pH value is adjusted to 8.3.

[0023] In the preparation method of the graphene-colloidal gold marker on the immunochromatographic test strip, during step c, 50-100 mL of 5 mM NaCl solution is used for desalting every 1-3 mL of aminated graphene solution.

[0024] In the preparation method of the graphene-colloidal gold marker on the immunochromatographic test strip, in step c, during dialysis desalting, 50-100 mL of 5 mM NaCl solution is added three times in sequence for desalting, and finally pure water is added, and the pH value is adjusted to 8.0-9.5 with K2CO3.

[0025] In the preparation method of the above-mentioned immunochromatographic test strip marker graphene-colloidal gold, the volume ratio of the aminated graphene to the colloidal gold solution in step d is 1-3:1-3.

[0026] The beneficial effects of this invention are as follows:

[0027] This invention prepares an immunochromatographic test strip marker, namely graphene-colloidal gold. By first carboxylating and amylating graphene, and then reacting the amylated graphene with colloidal gold, the prepared graphene-colloidal gold has stronger sensitivity and faster detection speed for pathogenic bacteria. Detailed Implementation

[0028] This invention is the first to achieve the binding of graphene to gold-labeled antibodies. By first carboxylating and amylating the graphene, and then reacting it with colloidal gold, the minimum visible density of graphene can be significantly reduced, thereby improving the minimum antigen concentration for color development and increasing the sensitivity of antigen detection.

[0029] Normally, graphene lacks functional groups that can bind to other substances, making it difficult to bind to gold-labeled antibodies. This invention achieves the binding of graphene to gold-labeled antibodies through a special method: first carboxylation and then amination. The procedure is complex and challenging, but it represents a qualitative breakthrough.

[0030] Compared to graphene oxide, its minimum visible concentration is much higher, and the advantage of using graphene instead of graphene oxide is its higher sensitivity. After grafting amine groups onto graphene, its charge properties are changed, giving it an opposite charge to colloidal gold, thus enabling it to bind to colloidal gold through electrostatic adsorption.

[0031] The specific implementation of the present invention will be further explained and described below through examples, but this does not mean that the scope of protection of the present invention is limited to the scope described in the examples.

[0032] Example 1: Preparation of graphene-colloidal gold immunochromatographic test strip markers using the method of the present invention.

[0033] The specific steps are as follows:

[0034] (1) Graphene powder and pyrene carboxylic acid were reacted in methanol at a mass ratio of 1.5:1 for two hours to obtain carboxylated graphene.

[0035] (2) After centrifugation, the reaction solution was washed with water and ethanol and dried to obtain carboxylated graphene powder. 7 ml of 50 mM MES (pH 6.0) solution was added to the dried graphene powder. After 5 minutes, 1.5 ml of 2.10 mM EDC and 1.5 ml of 5.70 mM sNHS were added dropwise, and the mixture was stirred slowly for 30 minutes. The mixture was then centrifuged at 10,000 rpm for 30 minutes, and the precipitate was resuspended. A 0.1 mg / ml aminographene solution was prepared.

[0036] (3) Take 1 ml of 0.1 mg / ml aminographene and dialyze it in 50 ml of 5 mM NaCl solution to remove salt. Dialyze for 18 h, changing the solution three times during the process. When it reaches the pure water stage, adjust the pH to 8.0 with K2CO3.

[0037] (4) Take 1 ml of 50 μm / ml antibody and place it in 50 ml of 5 mM NaCl solution for dialyzing to remove salt. Dialyze for 18-24 h, changing the solution three times during the process. When reaching the pure water stage, adjust the pH to 9.0 with K2CO3.

[0038] (5) Take 1 ml of 0.05 mg / ml colloidal gold and adjust the pH of the colloidal gold solution to 9.0 with 0.1 mol / L K2CO3.

[0039] (6) Mix 2 ml of aminographene with 1 ml of 0.05 mg / ml colloidal gold and react for 40 minutes to obtain graphene-colloidal gold label.

[0040] The dialyzed SARS-CoV-2 antibody was mixed with the above solution to achieve an antibody concentration of 50 μg / 3 mL in colloidal gold. The reaction was allowed to proceed for 15 min, followed by the addition of 5% BSA for blocking until the final BSA concentration in the solution reached 1%. The reaction was allowed to continue for 20 min.

[0041] (7) Low-speed centrifugation: Centrifuge at 1200 rpm for 20 min, take the supernatant, discard the lower layer, and remove the gold particles that have aggregated in the binding reaction.

[0042] (8) Ultracentrifugation: Centrifuge at 12000 rpm for 30 min, remove the supernatant, and resuspend the precipitate in colloidal gold preservation solution.

[0043] (9) Prepare a colloidal gold-binding pad using a gold-labeled antibody, and then use a scribing instrument to draw the detection line and control line on the nitrocellulose membrane. Attach the sample absorption pad, colloidal gold-binding pad, reaction membrane, and absorbent pad sequentially to a PVC substrate. Assemble the immunochromatographic test strip.

[0044] Example 2: Preparation of graphene-colloidal gold immunochromatographic test strip markers using the method of the present invention.

[0045] Example 2 differs from Example 1 in that graphene powder and pyrene carboxylic acid were reacted in methanol at a mass ratio of 1.5:1 for 2.5 hours to obtain carboxylated graphene. The dried graphene powder was added to 7 ml of 30 mM MES (pH 6.0) solution. After 10 minutes, 1.5 ml of 1.00 mM EDC and 1.5 ml of 4.00 mM sNHS were added dropwise, and the mixture was stirred slowly for 20 minutes. Aminated graphene was then mixed with colloidal gold and reacted for 30 minutes. The volume of aminated graphene was 3 mL, and the concentration of colloidal gold was 0.04 mg / ml. The selected antigen and antibody were E. coli antigen-antibody pairs.

[0046] Example 3: Preparation of graphene-colloidal gold immunochromatographic test strip markers using the method of the present invention.

[0047] Example 2 differs from Example 1 in that graphene powder and pyrene carboxylic acid were reacted in methanol at a mass ratio of 1:1.5 for 3 hours to obtain carboxylated graphene. The dried graphene powder was added to 7 ml of 50 mM MES (pH 6.0) solution. After 15 minutes, 1.5 ml of 2.00 mM EDC and 1.5 ml of 6.00 mM sNHS were added dropwise, and the mixture was stirred slowly for 20 minutes. Aminated graphene was then mixed with colloidal gold and reacted for 20 minutes. The volume of aminated graphene was 2 mL, and the concentration of colloidal gold was 0.05 mg / ml. The selected antigen and antibody were Brucella antigen-antibody pairs.

[0048] Example 4: Preparation of graphene-colloidal gold immunochromatographic test strip markers using the method of the present invention.

[0049] Example 2 differs from Example 1 in that graphene powder and pyrene carboxylic acid were reacted in methanol at a mass ratio of 2:1.5 for 4 hours to obtain carboxylated graphene. The dried graphene powder was added to 7 ml of 40 mM MES (pH 6.0) solution, and after 25 minutes, 1.5 ml of 3.00 mM EDC and 1.5 ml of 5.00 mM sNHS were added dropwise, and the mixture was stirred slowly for 20 minutes. Aminated graphene was then mixed with colloidal gold and reacted for 40 minutes. The volume of aminated graphene was 1 mL, and the concentration of colloidal gold was 0.06 mg / ml. The selected antigen and antibody were African swine fever virus antigen-antibody pairs.

[0050] Comparative Example 1

[0051] The difference between Comparative Example 1 and Example 1 is that the colloidal gold was not bound to the amino-based graphene, but was directly used for antibody-labeled gold experiments before subsequent steps. The antigen and antibody used were the SARS-CoV-2 antigen-antibody pair.

[0052] Comparative Example 2

[0053] The difference between Comparative Example 2 and Example 1 is that the colloidal gold was not bound to the amino-based graphene, but was directly used for antibody-labeled gold assays before subsequent steps. The antigen and antibody used were E. coli antigen-antibody pairs.

[0054] Comparative Example 3

[0055] The difference between Comparative Example 3 and Example 1 is that the colloidal gold was not bound to the amino-based graphene, but was directly used for antibody-labeled gold assays before subsequent steps. The antigen and antibody used were Brucella antigen-antibody pairs.

[0056] Comparative Example 4

[0057] The difference between Comparative Example 4 and Example 1 is that the colloidal gold was not bound to the amino-based graphene, but was directly used for antibody-labeled gold assays before subsequent steps. The antigen and antibody used were African swine fever virus antigen-antibody pairs.

[0058] The sensitivity of the immunochromatographic test strips labeled with graphene-colloidal gold obtained in the examples and comparative examples was tested, and the results are as follows:

[0059] COVID-19 antigen standards with concentrations of 5, 10, 20, 40, 80, 160, 320, 640, and 1280 ng / mL were prepared using phosphate-buffered saline (PBS) (pH 7.4) or double-distilled water. 80–100 μL of each standard was loaded onto the immunochromatographic test strip in this embodiment. After a 5-minute reaction, the relative optical density of the T-line scan area was read using a strip reader. A standard curve was plotted with the percentage of relative optical density values ​​of different concentration standards compared to the blank standard on the ordinate and the commonly used logarithmic values ​​of different standard concentrations on the abscissa. Correlation regression analysis was performed to calculate the IC50 and limit of detection (LOD) of the test strip for the COVID-19 antigen. The regression equation for the COVID-19 antigen on this test strip was determined to be: y = -0.3162x + 0.9351, with a correlation coefficient of R² = 0.9921. Based on the regression equation, the IC50 of the test strip for COVID-19 antigen was calculated to be 54.1 ng / mL, and the limit of detection was 7.5 ng / mL, indicating that the immunochromatographic test strip has high sensitivity to COVID-19 antigen. Using the same method, the limit of detection of the immunochromatographic test strips in Examples 2-3 and Comparative Examples 1-4 was determined, and the results are shown in Table 1.

[0060] Table 1. Detection limits of immunochromatographic test strips obtained by different methods

[0061] Limit of detection (ng / mL) 7.5 14.7 8.9 15.1 Experimental Case Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Limit of detection (ng / mL) 46.1 68.5 54.9 63.2

[0062] This demonstrates that the sensitivity of virus detection has been effectively improved after using composite graphene.

Claims

1. A method for preparing graphene-colloidal gold as a marker for immunochromatographic test strips, characterized in that, Includes the following steps: a. Graphene powder and pyrene carboxylic acid are mixed at a weight ratio of 1-2:0.5-1.5, then added to methanol and reacted for 1-8 hours to obtain carboxylated graphene. b. Centrifuge the carboxylated graphene obtained in step a, wash and dry it to obtain carboxylated graphene powder. Add 30-70mM MES solution to it. After 5-20 min, add 1-3mM EDC and 4-6mM sNHS dropwise to react. Stir at 50-100 rpm for 10-40 min to obtain aminographene. Centrifuge at 5000-15000 rpm for 10-40 min. After resuspending the precipitate, prepare an aminographene solution of 0.1-0.3 mg / ml. c. Dialyze the aminated graphene solution in NaCl solution to remove salt for 16-28 hours, add pure water, and adjust the pH to 8.0-9.

5. d. Prepare a colloidal gold solution with a concentration of 0.02-0.10 mg / ml and adjust its pH value to 8.0-9.

5. Mix the aminographene from step c with the colloidal gold solution and react for 20-60 min to obtain the graphene-colloidal gold label.

2. The method for preparing the graphene-colloidal gold marker on the immunochromatographic test strip according to claim 1, characterized in that: The weight ratio of graphene powder to pyrene carboxylic acid in step a is 1.5:

1.

3. The method for preparing the graphene-colloidal gold marker on the immunochromatographic test strip according to claim 1, characterized in that: The graphene powder described in step a has a particle size of 5-40 μm.

4. The method for preparing the graphene-colloidal gold marker on the immunochromatographic test strip according to claim 1, characterized in that: The amount of graphene used in step a is 0.01-0.02% of the weight of methanol.

5. The method for preparing the graphene-colloidal gold marker on the immunochromatographic test strip according to claim 1, characterized in that: The centrifugation conditions described in step b are centrifugation at 5000-15000 rpm for 15-30 minutes.

6. The method for preparing the graphene-colloidal gold marker on the immunochromatographic test strip according to claim 1, characterized in that: The drying process described in step b involves drying at 50-70℃ for 1-3 hours.

7. The method for preparing the graphene-colloidal gold marker on the immunochromatographic test strip according to claim 1, characterized in that: The concentration of EDC in step b is 2.08 mM, and the concentration of sNHS is 5.10 mM.

8. The method for preparing the graphene-colloidal gold marker on the immunochromatographic test strip according to claim 1, characterized in that: In step b, the mass percentage of the amino-based graphene in the MES solution is 0.001%-0.01%.

9. The method for preparing the graphene-colloidal gold marker on the immunochromatographic test strip according to claim 1, characterized in that: In step c, during dialysis desalination, 50-100 mL of 5 mM NaCl solution is used to desalinate each 1-3 mL of aminated graphene solution.

10. The method for preparing the graphene-colloidal gold marker on the immunochromatographic test strip according to claim 1, characterized in that: The volume ratio of the aminated graphene to the colloidal gold solution in step d is 1-3:1-3.