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

Abstract

The invention provides a method for preparing a colloidal gold solution through a reduction method, the colloidal gold solution, a reagent strip and a kit, the colloidal gold solution is prepared through a reaction of a tetrachloroauric acid solution and a reducing agent, and the particle size of colloidal gold particles in the prepared colloidal gold solution is 80-90 nanometers; the colloidal gold solution has a maximum absorption peak when a spectrum at the wave band of 200-800 nm is scanned by using an ultraviolet spectrophotometer, the wavelength of the maximum absorption peak is 549-552 nm, the peak is symmetrical and has no impure 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 reduction method has the advantages of large particle size, stable solution and good dispersity, and can be stored for a long time.

Description

Technical Field

[0001] The invention relates to the field of biological detection, and in particular to a method for preparing a colloidal gold solution by a reduction method, and a solution, a reagent strip and a reagent kit thereof. Background Art

[0002] Colloidal gold is also called gold solution. The structure of colloidal gold particles consists of a gold core and a double ion layer surrounded by an outer layer, that is, the inner layer of negative ions AuCl2- tightly surrounds the surface of the gold core and the outer layer of positive ions H + Dispersed in colloidal gold solution, it presents a good monodisperse state. Using different types and doses of reducing agents to reduce chloroauric acid solutions of different concentrations can synthesize colloidal gold nanoparticles of different particle sizes and morphologies, 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 chloroauric acid with a reducing agent. At the beginning 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 size of the microcrystals increases until all the chloroauric acid is reduced. The type of reducing agent and the concentration of the components determine the ratio of nucleation and growth, and thus the final particle size. Gold sols can be made in the laboratory and are typically stable solutions with particle sizes between 2 and 40 nanometers, depending on the type and concentration of the reducing agent.

[0004] Research reports show that commonly used reducing agents include trisodium citrate, sodium borohydride, ascorbic acid, tannic acid, white phosphorus, hydroquinone, hydroxylamine, etc. In 2001, Jana NR and Gearheart L reported the synthesis of large-particle colloidal gold by seed growth method. By controlling the addition ratio of seeds, reducing agents and chloroauric acid, the particle size of the synthesized colloidal gold can be controlled. In the process of synthesizing colloidal gold by seed growth method, layer-by-layer growth can more effectively avoid the possibility of secondary nucleation than primary growth. Compared with the original direct reduction method, the colloidal gold synthesized by this method has uniform particle size, nearly spherical shape and good monodispersity. However, the colloidal gold synthesized by this method is more complicated and not easy to industrialize.

[0005] At present, the particle size of the colloidal gold prepared by the reducing agent reduction method for industrialization is generally 10-60nm, which is relatively small; the colloidal gold solution prepared with a particle size of more than 60nm has different gold particle sizes in the obtained colloidal gold solution, and the uniformity of the entire colloidal gold solution is very poor, and it is unstable and easy to precipitate, which cannot be used for the preparation of colloidal gold test strips and is not suitable for industrialization. Other reported composite microspheres prepared by derivatization with silica, polyacrylamide, etc. have a particle size between 100nm-300nm, but this type of synthesis method is relatively complicated, and the postscript labeling method is also relatively complicated. It is not suitable for industrialization. In addition, the method of using large-particle colloidal gold derivatized with carboxyl or amino groups such as hydroquinone and oleylamine has a complex synthesis process, and harmful by-products may be produced during the reaction process, which is even more unfavorable for industrialization for immunochromatographic test strips. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a method for preparing a colloidal gold solution by a reduction method. The method prepares the colloidal gold solution by reacting a tetrachloroauric acid solution with a reducing agent. The colloidal gold particles in the prepared colloidal gold solution have a particle size of 80 to 90 nanometers. The colloidal gold solution has a maximum absorption peak when scanning a 200nm-800nm ​​band spectrum with an ultraviolet spectrophotometer. The wavelength of the maximum absorption peak is 549 to 552nm, and the peaks are symmetrical without impurity peaks. 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 comprises heating and stirring a tetrachloroauric acid solution and a trisodium citrate solution to react, then adding a polyvinyl pyrrolidone solution, and immediately adding a polyvinyl alcohol solution after the reaction solution turns into a dark wine red color, continuing to heat the reaction, and finally cooling to obtain the colloidal gold solution.

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

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

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

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

[0013] In one embodiment, a colloidal gold immunochromatographic test strip is provided, wherein the colloidal gold immunochromatographic test strip is prepared by the above-mentioned colloidal gold solution.

[0014] In one embodiment, a colloidal gold immunochromatographic test paper kit is provided, wherein the colloidal gold immunochromatographic test paper kit comprises the above-mentioned colloidal gold immunochromatographic test paper strip.

[0015] The present invention also uses a combination of reagents to prepare colloidal gold with a particle size of 80-90 nm based on the theoretical basis of the reduction method. Colloidal gold with a large particle size can greatly improve the sensitivity of the immunochromatographic test strip.

[0016] Compared with the colloidal gold solution prepared by the reduction method in the prior art, the colloidal gold solution prepared by the reduction method in the present invention has a large colloidal gold particle size, a stable solution and good dispersibility, and can be stored for a long time; the gold particle size in the stable colloidal gold solution of the present invention is 80-90nm; the existing colloidal gold solution prepared by the reduction method can be used as a stable colloidal gold solution for labeling, but the particle size of the gold particles is generally about 40nm. When the colloidal gold solution with a particle size of 80-90nm is prepared by the prior method, the particle size is uneven, the solution is unstable, and cannot be stored for a long time, and the concentration of the colloidal gold particles in the solution is low.

[0017] The density of the colloidal gold prepared by the method is greater than that of the colloidal gold prepared by other methods, and the gold particle size is 80-90nm, which is significantly higher than the existing gold particle size of 40nm, so that the sensitivity of the reagent strip prepared by the present invention is significantly higher than the sensitivity of the existing reagent strip; at the same time, the OD value of the stable colloidal gold solution of the present invention is greater than the OD value of the colloidal gold solution prepared by the existing reduction method, indicating that the density of the colloidal gold prepared by the method is greater than that of the colloidal gold prepared by the existing method, further increasing the sensitivity of the reagent strip.

[0018] When the reduction method of the present invention is used to prepare colloidal gold with large particle size, PVP is added as a reaction stabilizer, so that the violent reaction of trisodium citrate reduction is changed into a stable slow reaction at a certain time and temperature; after adding a certain amount of PVA within a certain range, the reaction is promoted to proceed further, so that the small gold particles are further aggregated into gold particles with a particle size of 80-90nm, forming a colloidal gold solution with a stable particle size of 80nm-90nm, and the two cooperate with each other to prepare the colloidal gold solution of the present invention. Therefore, the solution finally prepared by the method of the present invention has good stability, uniformity, high sensitivity and specificity. PVP is also added in the existing colloidal gold preparation, which is mainly added after the colloidal gold solution is prepared to keep the colloidal gold solution stable, which is completely different from the use of PVP in the method of the present invention to achieve the technical purpose. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

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

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

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

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

[0024] Figure 5 This is the ultraviolet scanning 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 scanning spectrum of colloidal gold solution No. 1 in Example 2 of the present invention;

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

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

[0029] In order to enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below in conjunction with embodiments. Obviously, the described embodiments are only part of the embodiments of this application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of this application.

[0030] Example 1. Preparation of colloidal gold solution under different conditions (I)

[0031] 1. Preparation of colloidal gold

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

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

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

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

[0036] 5. Place 6 0.2L Erlenmeyer flasks on a magnetic stirrer with heating function, numbered 0-5; add 0.1L ultrapure water to each Erlenmeyer flask. Turn on the magnetic stirrer and heat.

[0037] 6. When the temperature of the aqueous solution in each conical flask is heated to 95-100°C, add 0.1 mL of the above chloroauric acid aqueous solution into the conical flask. Turn on the magnetic stirrer and set the stirrer speed to the maximum speed. At this time, the aqueous solution in the conical flask is yellow.

[0038] 7. Start timing after adding, heat for another 30s-60s, and then quickly add 0.15mL of trisodium citrate aqueous solution into the above flask.

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

[0040] 9. At this time, the aqueous solution in the conical flask is purple-black at first, then turns purple, and finally turns dark wine red. Immediately add 0 mL 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 of 10% PVA solution to flask 0, flask 1, flask 2, flask 3, flask 4, flask 5, respectively. Continue stirring for 10 minutes.

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

[0042] 11. The reaction-completed solution was transferred to another magnetic stirrer and cooled to room temperature in a water bath to obtain colloidal gold solutions No. 0-5 corresponding to the flask numbers above.

[0043] 2. Detection

[0044] After the prepared colloidal gold solution is cooled, 1 mL of the solution is taken and then scanned with a micro-ultraviolet spectrophotometer (Nanodrop type) in the 200nm-800nm ​​band. The scanning graph is detailed in Figure 1-Figure 6 . The colloidal gold solution No. 0 prepared by this method has a maximum absorption peak at 528nm, and the OD value is 1.007, without any impurity peaks; that is, the colloidal gold has a particle size of about 31nm. The colloidal gold solution No. 1 has a maximum absorption peak at 535nm, and its particle size is about 47nm. The colloidal gold solution No. 2 has a maximum absorption peak at 549nm, and the peaks are symmetrical, without impurity peaks, the OD value at the maximum peak is 1.39, and the particle size is about 80nm. The colloidal gold solution No. 3 has a maximum absorption peak at 552, and the peaks are symmetrical, without impurity peaks, and the OD value at the maximum peak is 1.51, that is, the particle size is about 90nm. The scanning spectra of solution No. 4 and solution No. 5 have maximum peaks, but the spectra are asymmetrical. The curves of the scanning spectra are messy.

[0045] The above experimental data show that when 0.75 mL of 0.01 g / mL PVP is added to the above reaction system, and 0.3 mL-0.5 mL (No. 2, No. 3 reaction bottles) of 0.1 g / mL PVA solution is added, 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 the colloidal gold in the prepared solution is uniform, and there is a maximum absorption peak, and its OD value is ≥1.0, reflecting that the concentration of colloidal gold particles contained 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 antibodies to prepare colloidal gold test strips. The judgment criteria for colloidal gold solution are as follows: (1) The maximum peak of its scanning spectrum should be symmetrical, and the OD at the maximum peak should be ≥1.0; (2) Based on the statistical calculation formula Y=0.4271X+514.56, the particle size of the colloidal gold particles in the colloidal gold solution with the measured OD value is calculated.

[0046] Without adding 0.1 g / mL PVA solution, the colloidal gold prepared has uniform particle size, but its particle size is less than 80 nm. Figures 4 and 5 show that during the preparation of the colloidal gold solution, the state of the colloidal gold particles is disordered, and the content of the colloidal gold particles is very low, that is, the reaction yield is low.

[0047] Example 2. Preparation of colloidal gold solution under different conditions (II)

[0048] 1. Preparation of colloidal gold

[0049] This example 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. As in Example 1, 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 were prepared.

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

[0052] 3. When the temperature of the aqueous solution in each conical flask is heated to 95-100°C, add 0.1 mL of the above chloroauric acid aqueous solution into the conical flask. Turn on the magnetic stirrer and set the stirrer speed to the maximum speed. At this time, the aqueous solution in the conical flask is yellow.

[0053] 4. Start timing after adding, heat for another 30s-60s, and then quickly add 0.15mL of trisodium citrate aqueous solution into the above flask.

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

[0055] 6. The aqueous solution in the conical flask is purple-black at first, then purple, and finally dark wine red. Immediately add 0.3 mL of 0.1 g / mL PVA solution to flask No. 1 and flask No. 2 respectively. Continue stirring for 10 minutes.

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

[0057] 8. The reaction-completed solution was transferred to another magnetic stirrer and cooled to room temperature in a water bath to obtain colloidal gold solutions No. 1 and No. 2 corresponding to the flask numbers above.

[0058] 2. Test results

[0059] The UV scans of colloidal gold solutions No. 1 and No. 2 are shown in Figure 7 and Figure 8 ; Adding 0.3mL of 0.01g / mL PVP, the maximum absorption peak of colloidal gold is at 538nm, and the particle size is about 54nm. Adding 1mL of 0.01g / mL PVP, the maximum absorption peak of colloidal gold is at 539nm, and the particle size is about 57nm.

[0060] The experimental data of Example 1 and Example 2 show that only when 0.01g / mL PVP and 0.1g / mL PVA are added in a certain ratio, a colloidal gold solution with a particle size of 80nm-90nm can be obtained. Simply reducing the amount of PVP by 0.01g / mL or increasing the amount of PVP by 1% cannot obtain a colloidal gold solution with a particle size of 80nm-90nm. It shows that the ratio of the amount of 1% PVP added to the amount of 0.1g / mL PVA plays a decisive role in the stable reaction to obtain 80nm-90nm.

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

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

[0063] 1. Prepare 0.01% purified aqueous solution of chloroauric acid by conventional reduction method. Then take 1L of the solution into a conical flask. Place it on a magnetic stirrer and heat it to 100℃ and stir.

[0064] 2. Add 20mL-4.23mL of 1% purified sodium citrate trisodium aqueous solution to the above solution to obtain a colloidal gold solution of 10nm-90nm. The comparison table of its particle size and the amount of sodium citrate trisodium added 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-visible spectrum of 90.5 nm colloidal gold prepared by this method is shown in Fig. 9 : The peak of colloidal gold is relatively wide, indicating that the uniformity of the particle size is poor. At the same time, the OD value at its maximum absorption peak is relatively small, which is 0.419; this indicates that the yield of the prepared colloidal gold particles is low. This large-particle colloidal gold is not suitable for industrial production.

[0068] Example 4 Comparison of the stability of colloidal gold solutions prepared by different methods

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

[0070] Table 2

[0071] Number of days Principal Gold Solution gold solution 0 months No sedimentation, deep purple No precipitation, dark purple 1 month No sedimentation, deep purple No precipitation, dark purple 3 months No sedimentation, deep purple There is precipitation, the solution is transparent dark purple 6 months No sedimentation, deep purple There is precipitation, and the supernatant is close to water color 9 months No sedimentation, deep purple There is precipitation, the supernatant is close to water color 12 months Slight precipitation, no precipitation after shaking There is precipitation, clear water color,

[0072] The above data show that the stability and uniformity of the colloidal gold solution prepared by the patented method 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 in Example 1 (the colloidal gold solution of the present invention) and the colloidal gold solution with a particle size of 41 nm prepared by the conventional method in Example 3 (a conventional colloidal gold stable solution, which has a stable particle size, good particle size uniformity, and a large OD value at the maximum absorption peak) were prepared. Then, the colloidal gold solution of the present invention and the conventional colloidal gold stable solution were used to prepare a kit according to the following method; and used for subsequent tests.

[0076] 2. Labeling of HP-labeled antibodies

[0077] This technology uses a direct labeling method. That is, at room temperature, the commercially available anti-HP antibody is directly reacted with the colloidal gold solution, and then centrifuged to obtain the required gold label. The specific method is as follows:

[0078] (1) Measure the colloidal gold solution according to the amount of antibody per 100 parts of colloidal gold solution, for example: take 100 ml of colloidal gold solution and put it into a beaker.

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

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

[0081] Preparation of Tris buffer: weigh 2.2 parts of Tris per 100 parts of water and put it in a beaker, add 9 parts of water and then adjust the pH to 7.4 with 6M hydrochloric acid solution, and then make up to the required volume with purified water.

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

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

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

[0085] (7) The precipitate is redissolved with 1 part of 2 mM Tris buffer at pH 7.4 to obtain the labeled gold-labeled HP antibody.

[0086] 3. Preparation of Sample Pad

[0087] 3.1 Preparation of sample pad treatment solution

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

[0089] (2) Add 0.3 parts of ethylenediaminetetraacetate into the beaker and continue stirring;

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

[0093] (6) Add 0.5 parts of Tween 20 per 10,000 parts of 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 dilute to the required volume with purified water.

[0095] (8) Keep aside at room temperature.

[0096] 3.2 Preparation of sample pad

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

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

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

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

[0101] 4. Preparation of Sheets

[0102] (1) Paste the nitrocellulose membrane onto 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 2 μl / cm per piece.

[0105] Quality control line: Dilute goat anti-mouse IgG to 0.5 mg / ml with 0.01 M PB, and add 2 μl / cm

[0106] The amount of the quality control line is prepared by the amount of

[0107] Coating: Coat the solution onto nitrocellulose membrane using a stripping agent.

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

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

[0110] 5. Preparation of Gold Label

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

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

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

[0114] (4) The dried gold label strip is sealed in a ziplock bag and then stored in a dry environment away from light.

[0115] 6. Assembly of the reagent card

[0116] (1) Cut the prepared gold label into small strips with a length of 300 mm and a width of 8 mm, and stick them on the lower edge of the nitrocellulose membrane of the sheet, pressing 1-2 mm over the nitrocellulose membrane;

[0117] (2) Cut the sample pad into strips 300 mm long and 20 mm wide and stick them on the lower end of the sheet; the lower edge of the sample pad should extend 3 mm beyond the lower edge of the sheet, and the upper edge of the sample pad should extend beyond the white edge of the gold label strip.

[0118] (3) Cut the absorbent filter paper into strips 300 mm long and 17 mm 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 2 mm.

[0119] (4) Press the attached reagent card tightly and then cut it into small strips with a width of 3.0 mm using a cutting machine.

[0120] 7. Assembly of the Kit

[0121] The test strips are assembled into the cartridge, and the cartridge is tightly closed to obtain a test kit, namely, the colloidal gold test kit of this example and the conventional colloidal gold test kit.

[0122] 8. Preparation of sample diluent

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

[0124] 9. Testing

[0125] The collected stool samples were used to conduct comparative tests on the kit prepared from the colloidal gold solution of the present invention, the kit prepared from the conventional colloidal gold stabilized solution, and the commercially available kit. The test methods and results are as follows. Fecal sample detection method:

[0126] Use the sampling stick to pick up 0.05g-0.5g of feces (i.e., feces the size of a soybean), put it into the sample diluent in the feces collection tube, mix well, and let it stand for 1 minute. Add the solution dropwise into the sample well of each kit, and observe the results after 10 minutes. The results are shown in Table 3.

[0127] Table 3 Test results

[0128]

[0129]

[0130] Note: In the table, “-” indicates negative, “- / +” indicates weak positive, “+” indicates positive, “++” indicates moderately positive, “+++” indicates strongly positive, and “++++” indicates extremely strongly positive.

[0131] The above data show that the colloidal gold particles prepared by the present invention are applied to the test kit, and the test kit prepared has higher detection sensitivity.

[0132] It should be understood that the disclosed invention is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the terminology used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0133] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. A method for preparing a colloidal gold solution by a reduction method, wherein the colloidal gold solution is prepared by reacting a tetrachloroauric acid solution with a reducing agent, wherein: The colloidal gold particles in the prepared colloidal gold solution have a particle size of 80 to 90 nanometers. The colloidal gold solution has a maximum absorption peak when scanning a 200nm-800nm ​​band spectrum with an ultraviolet spectrophotometer. The wavelength of the maximum absorption peak is 549 to 552nm, and the peaks are symmetrical without mixed peaks. 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 method comprises heating and stirring a tetrachloroauric acid solution and a trisodium citrate solution for reaction, then adding a polyvinyl pyrrolidone solution, and immediately adding a polyvinyl alcohol solution after the reaction solution turns into a dark wine red color, continuing the heating reaction, and finally cooling to obtain the colloidal gold solution.

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

20.

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

4.

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

20.

7. A colloidal gold solution, characterized in that: The colloidal gold solution is prepared by the method described in any one of claims 1-6.

8. A colloidal gold immunochromatographic test strip, characterized in that: The colloidal gold immunochromatographic test strip is prepared by the colloidal gold solution described in claim 7.

9. A colloidal gold immunochromatographic test paper kit, characterized in that: The colloidal gold immunochromatographic test paper box comprises the colloidal gold immunochromatographic test strip according to claim 8.

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