Method for preparing colloidal gold solution by reduction method, and solution, reagent strip and kit thereof

Abstract

The present application provides a method for preparing a colloidal gold solution by a reduction method, and a solution, a reagent strip and a kit thereof, wherein the colloidal gold solution is prepared by reacting a tetrachloroauric acid solution with a reducing agent, the particle size of the colloidal gold particles in the prepared colloidal gold solution is 80-90 nm, the colloidal gold solution has a maximum absorption peak when scanned by a UV spectrophotometer in a 200-800 nm wave band spectrum, the wavelength of the maximum absorption peak is 549-552 nm, the peak is symmetrical, there is no impurity peak, and the OD value at the maximum peak is not less than 1.39. Compared with the existing colloidal gold solution prepared by the reduction method, the colloidal gold solution prepared by the method has a large particle size, is stable and has good dispersibility, and can be stored for a long time.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and in particular to a method for preparing colloidal gold solutions by reduction, as well as solutions, reagent strips, and kits thereof. Background Technology

[0002] Colloidal gold, also known as gold sol, consists of a gold core and an outer biionic layer. The inner layer is composed of AuCl₂⁻ negative ions tightly surrounding the gold core surface, while the outer layer contains H⁺ positive ions. + Dispersed in colloidal gold solution, it exhibits a good monodisperse state. By using different types and dosages of reducing agents to reduce chloroauric acid solutions of different concentrations, colloidal gold nanoparticles of different sizes and morphologies can be synthesized. That is, the particle size of colloidal gold nanoparticles can be adjusted by changing the type, content, and substrate concentration of the reducing agent.

[0003] Gold sols are produced by boiling a solution of tetrachloroauric acid with a reducing agent. At the start of the reduction process, gold atoms are released from the chloroauric acid. The gold atoms aggregate to form microcrystals. As more chloroauric acid is reduced, the microcrystals increase in size until all the chloroauric acid is reduced. The type of reducing agent and the concentration of its components determine the nucleation and growth ratio, thus determining the final particle size. Gold sols can be manufactured in the laboratory, typically as stable solutions with particle sizes between 2 and 40 nanometers, depending on the type and concentration of the reducing agent.

[0004] Studies have reported that commonly used reducing agents include trisodium citrate, sodium borohydride, ascorbic acid, tannic acid, white phosphorus, hydroquinone, and hydroxylamine. In 2001, Jana NR and Gearheart L reported a seed-grown method for synthesizing large-particle colloidal gold. By controlling the proportions of seeds, reducing agents, and chloroauric acid, the particle size of the synthesized colloidal gold can be controlled. In the seed-grown method, layer-by-layer growth more effectively avoids the possibility of secondary nucleation compared to single-stage growth. Compared to the original direct reduction method, this method produces colloidal gold with uniform particle size, near-spherical shape, and good monodispersity. However, this type of method for synthesizing colloidal gold is relatively complex and not easily industrialized.

[0005] Currently, colloidal gold prepared by reducing agents and suitable for industrial application typically has a particle size of 10-60 nm, which is relatively small. Colloidal gold solutions with a particle size exceeding 60 nm result in inconsistent gold particle sizes within the solution, exhibiting poor homogeneity, instability, and a tendency to precipitate, making them unsuitable for colloidal gold test strips and thus unsuitable for industrialization. Other reported methods, such as derivatization with silica or polyacrylamide, produce composite microspheres with particle sizes between 100 nm and 300 nm; however, these synthetic methods are complex, as are the subsequent labeling methods, making them unsuitable for industrialization. Furthermore, methods using hydroquinone, oleylamine, or similar derivatizations of carboxyl or amino groups to produce large-particle colloidal gold have complex synthetic processes and may generate harmful byproducts, further hindering their industrial application in immunochromatographic test strips. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a method for preparing colloidal gold solution via a reduction method. The method involves reacting tetrachloroauric acid solution with a reducing agent to obtain the colloidal gold solution. The colloidal gold particles in the prepared solution have a particle size of 80–90 nanometers. When the colloidal gold solution is scanned with a UV spectrophotometer in the 200 nm–800 nm wavelength range, it exhibits a maximum absorption peak with a wavelength between 549 and 552 nm. The peak is symmetrical, free of impurities, and the OD value at the maximum peak is not less than 1.39.

[0007] In one embodiment, the OD value at the maximum peak is 1.39-1.51.

[0008] In one embodiment, the method includes heating and stirring a tetrachloroauric acid solution and a trisodium citrate solution to react, then adding a polyvinylpyrrolidone solution, and once the reaction solution turns a deep wine red color, immediately adding a polyvinyl alcohol solution, continuing to heat the reaction, and finally cooling to obtain the colloidal gold solution.

[0009] In one embodiment, the mass ratio of added polyvinylpyrrolidone to added polyvinyl alcohol is 1:4 to 3:20.

[0010] In one embodiment, the mass ratio of added polyvinylpyrrolidone to added polyvinyl alcohol is 1:4.

[0011] In one embodiment, the mass ratio of added polyvinylpyrrolidone to added polyvinyl alcohol is 3:20.

[0012] In one embodiment, a colloidal gold solution is provided, which is prepared by the method described above.

[0013] In one embodiment, a colloidal gold immunochromatographic test strip is provided, which is prepared using the colloidal gold solution described above.

[0014] In one embodiment, a colloidal gold immunochromatographic test kit is provided, the colloidal gold immunochromatographic test kit comprising the aforementioned colloidal gold immunochromatographic test strip.

[0015] This invention, based on the theory of reduction methods, uses a combination of reagents to prepare colloidal gold with a particle size of 80-90 nm. Large-particle-size colloidal gold can significantly improve the sensitivity of immunochromatographic test strips.

[0016] Compared with existing colloidal gold solutions prepared by reduction methods, the colloidal gold solution prepared by this invention has a larger colloidal gold particle size, more stable solution, better dispersibility, and can be stored for a long time. The gold particle size in the stable colloidal gold solution of this invention is 80-90 nm. Existing colloidal gold solutions prepared by reduction methods can be used to label stable colloidal gold solutions, but their gold particle size is generally around 40 nm. When colloidal gold solutions with a particle size of 80-90 nm are prepared by existing methods, the particle size is uneven, the solution is unstable, cannot be stored for a long time, and the concentration of colloidal gold particles in the solution is low.

[0017] The colloidal gold prepared by this method has a higher density than colloidal gold prepared by other methods. The gold particle size is 80-90 nm, which is significantly larger than the existing gold particle size of 40 nm. This results in the reagent strips prepared by this invention having significantly higher sensitivity than existing reagent strips. At the same time, the OD value of the stable colloidal gold solution of this invention is larger than that of colloidal gold solutions prepared by existing reduction methods, indicating that the colloidal gold prepared by this method has a higher density than colloidal gold prepared by existing methods, further increasing the sensitivity of the reagent strips.

[0018] In the reduction method of this invention for preparing large-particle colloidal gold, PVP is added as a reaction stabilizer, transforming the vigorous reduction reaction of trisodium citrate into a stable, slow reaction under certain time and temperature conditions. The addition of a certain amount of PVA further promotes the reaction, causing the small gold particles to aggregate into gold particles with a diameter of 80-90 nm, forming a colloidal gold solution with a stable particle size of 80-90 nm. The two work together to prepare the colloidal gold solution of this invention. Therefore, the solution finally prepared by the method of this invention has good stability, homogeneity, high sensitivity, and specificity. Existing colloidal gold preparation methods also add PVP, mainly after the colloidal gold solution is prepared to maintain its stability, which is completely different from the method of this invention that uses PVP to achieve the technical objective. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is the ultraviolet scan spectrum of colloidal gold solution No. 0 in Example 1 of the present invention;

[0021] Figure 2 This is the ultraviolet scan spectrum of colloidal gold solution No. 1 in Example 1 of the present invention;

[0022] Figure 3 This is the ultraviolet scan spectrum of colloidal gold solution No. 2 in Example 1 of the present invention;

[0023] Figure 4 This is the ultraviolet scan spectrum of colloidal gold solution No. 3 in Example 1 of the present invention;

[0024] Figure 5 This is the ultraviolet scan spectrum of colloidal gold solution No. 4 in Example 1 of the present invention;

[0025] Figure 6 This is the ultraviolet scanning spectrum of colloidal gold solution No. 5 in Example 1 of the present invention;

[0026] Figure 7 This is the ultraviolet scan spectrum of colloidal gold solution No. 1 in Example 2 of the present invention;

[0027] Figure 8 This is the ultraviolet scan spectrum of colloidal gold solution No. 2 in Example 2 of the present invention;

[0028] Figure 9 This is the ultraviolet scanning spectrum of a colloidal gold solution with a particle size of 90.5 nanometers in Example 3 of the present invention. Detailed Implementation

[0029] To enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this application.

[0030] Example 1. Preparation of colloidal gold solutions under different conditions (Part 1)

[0031] I. Preparation of Colloidal Gold

[0032] 1. Weigh tetrachloroauric acid into a graduated tube at a rate of 0.4 g / mL, and then add ultrapure water to prepare 10 mL of chloroauric acid aqueous solution.

[0033] 2. Weigh 0.2 g / mL of trisodium citrate into a graduated tube, then add ultrapure water to prepare a 5 mL aqueous solution of trisodium citrate.

[0034] 3. Weigh polyvinyl alcohol (PVA, molecular weight 120,000 to 150,000) into a graduated tube at a rate of 0.1 g / mL, then add 9.5 mL of ultrapure water and 0.5 mL of ethanol; prepare a 10 mL solution of 0.1 g / mL PVA.

[0035] 4. Weigh polyvinylpyrrolidone (PVP10, average molecular weight 10,000) into a graduated tube at a rate of 0.01 g / mL, then add 10 mL of ultrapure water to prepare a 0.01 g / mL PVP aqueous solution.

[0036] 5. Place six 0.2-liter Erlenmeyer flasks on a magnetic stirrer with a heating function, numbered 0-5. Add 0.1L of ultrapure water to each flask. Turn on the magnetic stirrer and heat.

[0037] 6. When the aqueous solution in each Erlenmeyer flask reaches 95-100℃, add 0.1 mL of the above-mentioned chloroauric acid aqueous solution to the Erlenmeyer flask. Turn on the magnetic stirrer and set the stirrer speed to the maximum. At this time, the aqueous solution in the Erlenmeyer flask will turn yellow.

[0038] 7. After adding the ingredients, start timing and heat for another 30-60 seconds. Then, quickly add 0.15 mL of trisodium citrate aqueous solution to each of the above flasks.

[0039] 8. Continue heating and stirring for 6 minutes, and then quickly add 0.75 mL of PVP aqueous solution to each of the above flasks.

[0040] 9. At this point, the aqueous solution in the Erlenmeyer flask is initially purplish-black, then turns purple, and finally becomes a deep wine-red. Immediately add 0 mL of 10% PVA solution to flask 0, 0.15 mL to flask 1, 0.3 mL to flask 2, 0.45 mL to flask 3, 0.75 mL to flask 4, and 1 mL to flask 5. Continue stirring for 10 minutes.

[0041] 10. After 10 minutes, stop heating; continue stirring the solution in the flask for 20 minutes.

[0042] 11. Transfer the reacted solution to another magnetic stirrer and cool it to room temperature in a water bath. Obtain the colloidal gold solutions numbered 0-5 as described above.

[0043] II. Testing

[0044] After the prepared colloidal gold solution has cooled, take 1 mL of the solution and scan the 200 nm–800 nm wavelength range using a micro-ultraviolet spectrophotometer (Nanodrop type). See the detailed scan diagram below. Figures 1-6 The colloidal gold solution No. 0 prepared by this method has a maximum absorption peak at 528 nm with an OD value of 1.007 and no impurity peaks; this indicates colloidal gold with a particle size of approximately 31 nm. Colloidal gold solution No. 1 has a maximum absorption peak at 535 nm, with a particle size of approximately 47 nm. Colloidal gold solution No. 2 has a maximum absorption peak at 549 nm, with symmetrical peaks and no impurity peaks; the OD value at the maximum peak is 1.39, and the particle size is approximately 80 nm. Colloidal gold solution No. 3 has a maximum absorption peak at 552 nm, with symmetrical peaks and no impurity peaks; the OD value at the maximum peak is 1.51, indicating a particle size of approximately 90 nm. Solutions No. 4 and No. 5 show maximum peaks in their scan spectra, but the spectra are asymmetrical. The scan spectra are cluttered.

[0045] The above experimental data show that when 0.01 g / mL PVP 0.75 mL and 0.3 mL-0.5 mL (reaction flasks 2 and 3) of 0.1 g / mL PVA solution are added to the above reaction system, the particle size of the obtained colloidal gold solution is between 80 nm and 90 nm, and its scanning spectrum is smooth and symmetrical. That is, the particle size of colloidal gold in the prepared solution is uniform, and there is a maximum absorption peak. Its OD value is ≥1.0, which reflects that the concentration of colloidal gold particles in a unit of colloidal gold aqueous solution is high, that is, the reaction yield is high. In this case, colloidal gold solution can be used for the industrialization of labeling antibody preparation of colloidal gold test strips. The criteria for judging colloidal gold solution are: (1) the maximum peak of its scanning spectrum should be symmetrical, and the OD at the maximum peak should be ≥1.0; (2) the particle size of colloidal gold particles in the colloidal gold solution with the measured OD value is calculated based on the statistical calculation formula Y=0.4271X+514.56.

[0046] Without the addition of 0.1 g / mL PVA solution, the prepared colloidal gold, although uniform in particle size, has a particle size of less than 80 nm. Figures 4 and 5 show that the colloidal gold particles in the prepared solution are in a disordered state and have a very low content, indicating a low reaction yield.

[0047] Example 2. Preparation of colloidal gold solutions under different conditions (Part 2)

[0048] I. Preparation of Colloidal Gold

[0049] This embodiment is based on the preparation method of Example 1, and explores the effect of adding 0.3 mL and 1 mL of 0.01 g / mL PVP on the preparation of 80 nm-90 nm colloidal gold.

[0050] 1. Prepare 0.4 g / mL chloroauric acid aqueous solution, 0.2 g / mL trisodium citrate aqueous solution, 0.1 g / mL PVA aqueous solution and 0.01 g / mL PVP aqueous solution as in Example 1.

[0051] 2. Place two 0.2-liter Erlenmeyer flasks on a magnetic stirrer with a heating function, numbered 1 and 2. Add 0.1L of ultrapure water to each flask. Turn on the magnetic stirrer and heat.

[0052] 3. When the aqueous solution in each Erlenmeyer flask reaches 95-100℃, add 0.1 mL of the above-mentioned chloroauric acid aqueous solution to the Erlenmeyer flask. Turn on the magnetic stirrer and set the stirrer speed to the maximum. At this time, the aqueous solution in the Erlenmeyer flask will turn yellow.

[0053] 4. After adding the ingredients, start timing and heat for another 30-60 seconds. Then, quickly add 0.15 mL of trisodium citrate aqueous solution to each of the above flasks.

[0054] 5. Continue heating and stirring for 6 minutes. Quickly add 0.3 mL of PVP aqueous solution to flask No. 1 and 1 mL to flask No. 2.

[0055] 6. At this point, the aqueous solution in the Erlenmeyer flask is first purplish-black, then turns purple, and finally becomes a deep wine red. Immediately add 0.3 mL of 0.1 g / mL PVA solution to both flasks 1 and 2. Continue stirring for 10 minutes.

[0056] 7. After 10 minutes, stop heating; continue stirring the solution in the flask for 20 minutes.

[0057] 8. Transfer the reacted solution to another magnetic stirrer and cool it to room temperature in a water bath. Obtain the colloidal gold solutions No. 1 and No. 2 corresponding to the flask numbers above.

[0058] II. Test Results

[0059] The UV scan images of colloidal gold solutions 1 and 2 are shown below. Figure 7 and Figure 8 Adding 0.3 mL of 0.01 g / mL PVP resulted in a colloidal gold with a maximum absorption peak at 538 nm and a particle size of approximately 54 nm. Adding 1 mL of 0.01 g / mL PVP resulted in a colloidal gold with a maximum absorption peak at 539 nm and a particle size of approximately 57 nm.

[0060] The experimental data from Examples 1 and 2 show that a solution of colloidal gold with a particle size of 80nm-90nm is only obtained when 0.01g / mL PVP and 0.1g / mL PVA are added in a specific ratio. Simply reducing the amount of PVP by 0.01g / mL or increasing the amount of PVP by 1% does not yield a colloidal gold solution with a particle size of 80nm-90nm. This indicates that the ratio of 1% PVP to 0.1g / mL PVA plays a decisive role in the stable yield of 80nm-90nm particles.

[0061] Analysis of the results from Examples 1 and 2 shows that when the mass ratio of added PVP to PVA is between 1:4 and 3:20, a colloidal gold solution with a particle size of 80nm-90nm can be prepared. The colloidal gold has a uniform particle size and a maximum absorption peak with an OD value ≥1.39.

[0062] Example 3. Comparison with colloidal gold prepared by conventional reduction methods

[0063] 1. Prepare a 0.01% purified aqueous solution of chloroauric acid using conventional reduction methods. Then, take 1 L of this solution and place it in an Erlenmeyer flask. Heat the flask to 100°C and stir on a magnetic stirrer.

[0064] 2. Adding 20 mL to 4.23 mL of 1% trisodium citrate purified aqueous solution to the above solution yields a colloidal gold solution with a particle size of 10 nm to 90 nm. A comparison of particle size with the added trisodium citrate is shown in Table 1 below.

[0065] Table 1

[0066] 1% trisodium citrate added Corresponding particle size 20mL 16nm 15mL 24nm 10mL 41nm 6mL 71nm 4.2mL 90.5nm

[0067] The UV-Vis spectrum of the 90.5 nm colloidal gold prepared by this method is shown in [reference needed]. Figure 9 The colloidal gold exhibits a wide peak, indicating poor particle size uniformity. Furthermore, the OD value at its maximum absorption peak is relatively small, at 0.419, suggesting a low yield of the prepared colloidal gold particles. Such large-particle-size colloidal gold is not suitable for industrial production.

[0068] Example 4: Stability Comparison of Colloidal Gold Solutions Prepared by Different Methods

[0069] The colloidal gold solution No. 3 (original gold solution) from Example 1 and the colloidal gold solution with a particle size of 90.5 nm prepared by conventional methods in Example 3 (normal gold solution) were placed in refrigerators at 2-8°C for 1 month, 3 months, 6 months, 9 months, and 12 months, respectively. At each of these time points, the sedimentation of the solutions was observed, and the results are shown in Table 2.

[0070] Table 2

[0071] Number of days Gold solution gold solution 0 months No sedimentation, deep purplish-red No sediment, deep purple 1 month No sedimentation, deep purplish-red No sediment, deep purple 3 months No sedimentation, deep purplish-red There is a precipitate, and the solution is a transparent dark purple. 6 months No sedimentation, deep purplish-red There is sediment; the supernatant is close to the color of water. 9 months No sedimentation, deep purplish-red There is sediment; the supernatant is close to the color of water. 12 months There was a slight sediment, but after shaking, there was no sediment. There is sediment, and the water is clear.

[0072] The above data demonstrate that the stability and uniformity of the colloidal gold solution prepared by the method of this patent are significantly better than those prepared by conventional methods.

[0073] Example 5: Application of Colloidal Gold Solutions Prepared by Different Methods

[0074] 1. Preparation of colloidal gold

[0075] The colloidal gold solution No. 3 from Example 1 (the colloidal gold solution of this invention) and the colloidal gold solution with a particle size of 41 nm prepared by conventional methods in Example 3 (a conventional colloidal gold stable solution; this particle size solution is stable, has good particle size uniformity, and a large OD value at the maximum absorption peak) were used to prepare kits for subsequent testing. Kits were then prepared using the colloidal gold solution of this invention and the conventional colloidal gold stable solution, respectively, according to the methods described below.

[0076] 2. Labeling of HP-labeled antibodies

[0077] This technique employs a direct labeling method. Specifically, at room temperature, a commercially available anti-HP antibody is directly reacted with a colloidal gold solution, followed by centrifugation to obtain the desired gold-labeled product. The specific method is as follows:

[0078] (1) Measure the colloidal gold solution according to the amount of one antibody per 100 parts of colloidal gold solution, such as: take 100ml of colloidal gold solution into a beaker.

[0079] (2) Adjust the pH of the colloidal gold solution to 8.0 with 0.1M potassium carbonate solution and stir it on a magnetic stirrer.

[0080] (3) Dilute the HP-labeled antibody to 1 mg / ml with 2 mM pH 7.4 Tris buffer, and then add 0.8 ml of the diluted protein solution to the colloidal gold solution while stirring.

[0081] Preparation of Tris buffer: Weigh out Tris according to the ratio of 2.2 parts Tris per 100 parts water and place it in a beaker. Add 9 parts water and adjust the pH to 7.4 with 6M hydrochloric acid solution. Then, bring the volume to the required level with purified water.

[0082] (4) Stir on a magnetic stirrer for 25 minutes;

[0083] (5) Add 8.5% BSA aqueous solution to 100ml of the above solution at a rate of 24μl / ml, and continue stirring for 45 minutes.

[0084] (6) Transfer the above-reacted solution to a centrifuge tube; centrifuge at 4°C at a speed of 8000-15000 rpm / min, for example, 12000 rpm / min, for 35 minutes; discard the supernatant.

[0085] (7) The precipitate was reconstituted with Tris buffer containing 1 part of 2mM pH 7.4 per 100 parts to obtain the labeled gold-labeled HP antibody.

[0086] 3. Preparation of the sample pad

[0087] 3.1 Preparation of Sample Pad Treatment Solution

[0088] (1) Weigh out borax at a rate of 4 parts per 100 parts and put it into a beaker. Then add 80 parts of water to the beaker and place the beaker on a magnetic stirrer to stir.

[0089] (2) Add 0.3 parts of ethylenediaminetetraacetic acid salt to the above beaker and continue stirring;

[0090] (3) Add 0.1 to 1 part of sodium alkylphenol polyoxyethylene ether sulfosuccinate monoester to the above solution and continue stirring;

[0091] (4) Add 1 to 1.5 parts of propylene oxide-ethylene oxide-vinyl diamine copolymer (commercially available) to the above solution and continue stirring;

[0092] (5) Add 2 parts of Proclin 300 per 10,000 parts to the above solution and continue stirring;

[0093] (6) Add 0.5 parts of Tween20 per 10,000 parts to the above solution and continue stirring.

[0094] (7) After the solution is completely dissolved and clarified, adjust the pH of the solution to 8.2 with hydrochloric acid or sodium hydroxide, and then make up to the required volume with purified water.

[0095] (8) Keep at room temperature.

[0096] 3.2 Preparation of sample pad

[0097] (1) Prepare a commercially available fiberglass mat (length*width: 300mm*254mm);

[0098] (2) Soak the glass fiber pad at a rate of 1 pad per 100 samples; first, soak the glass fiber pad in solution 2.4.1 for 2 minutes, then remove it and gently filter out excess water with a glass rod. Turn it over and soak the glass fiber pad in the solution again for 2 minutes, then gently filter out excess water with a glass rod. The sample pad is now obtained.

[0099] (3) Place the obtained sample pad in a 37℃ oven and dry it overnight (17-20 hours).

[0100] (4) After drying, the sample pad should be sealed and stored in a dry environment.

[0101] 4. Sheet preparation

[0102] (1) Adhere the nitrocellulose membrane to the PVC base plate;

[0103] (2) Preparation of coating solution:

[0104] Test line: Dilute the HP-coated antibody to 1 mg / ml with 0.01 M PB, and prepare the test line at a rate of 2 μl / cm per piece.

[0105] Quality control line: Dilute goat anti-mouse IgG to 0.5 mg / ml with 0.01 M PB, at a rate of 2 μl / cm² per tablet.

[0106] The quantity is used to prepare the quantity for the quality control line.

[0107] Coating: The solution is coated onto a nitrocellulose membrane using a coating agent.

[0108] (3) Place the coated sheet in a 37°C oven and dry it overnight.

[0109] (4) The dried sheets should be stored in a sealed container in a dry place.

[0110] 5. Preparation of gold bar

[0111] (1) Dilute the gold-labeled solution prepared in step 2.3 with 0.01M PB buffer containing 1 part bovine serum albumin and 20 parts sucrose per 100 parts. The dilution ratio is 46 parts gold-labeled solution and 54 parts of the above-mentioned 0.01M PB solution per 100 parts of solution.

[0112] (2) The diluted solution was sprayed with gold labels using a gold labeling machine at a rate of 60 μl per label. The base pad for spraying was a glass fiber pad (length * width: 300 mm * 84 mm).

[0113] (3) After spraying, place the gold strip in a 37°C oven and dry it overnight.

[0114] (4) The dried gold label strips are sealed in a self-sealing bag and then stored in a dry environment away from light.

[0115] 6. Assembly of reagent cards

[0116] (1) Cut the prepared gold strip into strips 300mm long and 8mm wide, and paste them onto the lower edge of the nitrocellulose membrane of the sheet, pressing them over the nitrocellulose membrane by 1-2mm;

[0117] (2) Cut the sample pad into strips 300mm long and 20mm wide and stick them to the bottom of the sheet; the bottom edge of the sample pad extends 3mm beyond the bottom edge of the sheet and the top edge of the sample pad extends beyond the white edge of the gold strip.

[0118] (3) Cut the absorbent filter paper into strips 300mm long and 17mm wide. Align the upper edge of the absorbent filter paper with the upper edge of the sheet, and press the lower edge over the nitrocellulose membrane by about 2mm.

[0119] (4) Press the pasted reagent card firmly. Then cut it into strips 3.0 mm wide using a cutter.

[0120] 7. Assembly of the reagent kit

[0121] Assemble the test strips into the cartridge, close the cartridge tightly, and you will obtain the reagent kit. That is: the colloidal gold reagent kit in this example and the standard colloidal gold reagent kit.

[0122] 8. Preparation of sample diluent

[0123] Prepare a solution by adding 0.6 parts sodium dihydrogen phosphate, 8 parts sodium chloride, 0.2 parts potassium dihydrogen phosphate, and 2 parts Proclin 300 per 10,000 parts of water, and adjust the pH of the solution to 8.0.

[0124] 9. Test

[0125] The reagent kit prepared with the colloidal gold solution of this invention, the reagent kit prepared with a conventional colloidal gold stable solution, and a commercially available reagent kit were compared and tested using collected fecal samples. The test methods and results are as follows. Fecal sample detection method:

[0126] Using a sampling swab, collect 0.05-0.5g of feces (the size of a soybean) and place it into the sample diluent in the feces collection tube. Mix well and let stand for 1 minute. Add the solution dropwise into the sample wells of each kit and observe the results after 10 minutes. The results are shown in Table 3.

[0127] Table 3 Detection Results

[0128]

[0129]

[0130] Note: In the table, "-" indicates negative, "- / +" indicates weak positive, "+" indicates positive, "++" indicates moderate positive, "+++" indicates strong positive, and "++++" indicates extremely strong positive.

[0131] The above data demonstrate that when the colloidal gold particles prepared using this invention are applied to a reagent kit, the resulting kit exhibits higher detection sensitivity.

[0132] It should be understood that the disclosed invention is not limited to the specific methods, schemes, and substances described, as these are all subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0133] Those skilled in the art will also recognize, or be able to identify, many equivalents of the specific embodiments of the invention described herein using no more than conventional experiments. These equivalents are also included in the appended claims.

Claims

1. A method for preparing colloidal gold solution by reduction, wherein the colloidal gold solution is prepared by reacting tetrachloroauric acid solution with a reducing agent, characterized in that, The method includes heating and stirring tetrachloroauric acid solution and trisodium citrate solution to react, then adding polyvinylpyrrolidone solution. After the reaction solution turns deep wine red, polyvinyl alcohol solution is immediately added, and the reaction is continued to be heated. Finally, the mixture is cooled to obtain the colloidal gold solution. The mass ratio of polyvinylpyrrolidone to polyvinyl alcohol added is 1:4-3:

20. The colloidal gold particles in the prepared colloidal gold solution have a particle size of 80-90 nanometers. When the colloidal gold solution is scanned with a UV spectrophotometer in the 200nm-800nm ​​band, it has a maximum absorption peak with a wavelength of 549-552nm. The peak is symmetrical, without impurity peaks, and the OD value at the maximum peak is not less than 1.

39.

2. The method according to claim 1, characterized in that, The OD value at the maximum peak is 1.39-1.

51.

3. The method according to claim 1, characterized in that, The mass ratio of added polyvinylpyrrolidone to added polyvinyl alcohol is 1:

4.

4. The method according to claim 1, characterized in that, The mass ratio of added polyvinylpyrrolidone to added polyvinyl alcohol is 3:

20.

5. A colloidal gold solution, characterized in that, The colloidal gold solution is prepared by the method described in any one of claims 1-4.

6. A colloidal gold immunochromatographic test strip, characterized in that, The colloidal gold immunochromatographic test strip is prepared using the colloidal gold solution described in claim 5.

7. A colloidal gold immunochromatographic test kit, characterized in that, The colloidal gold immunochromatographic test kit includes the colloidal gold immunochromatographic test strip as described in claim 6.

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