Preparation method and application of large-particle colloidal gold

Abstract

The invention discloses a preparation method and application of large-particle colloidal gold, and the method comprises the following steps: (1) pouring double distilled water into a container, adding a gold chloride solution, and stirring at the rotating speed of 600-800rpm / min for 3-7min; (2) adding ascorbic acid with the concentration of 0.8-1.2 M, and continuously stirring for 15-25 minutes; (3) adding a trisodium citrate solution, heating and stirring until boiling, continuously stirring for 3-7 minutes, stopping heating, standing and cooling to obtain a colloidal gold solution; and (4) after the colloidal gold solution is cooled, pouring the colloidal gold solution into a clean brown light-proof bottle, ensuring that the volume of the obtained colloidal gold solution is consistent with the volume of the initial double distilled water in the step (1), adding Proclin300 into the colloidal gold solution, detecting an absorption peak and absorbance by using a spectrophotometer, and preserving in a dark place.

Description

Technical Field

[0001] The invention belongs to the technical field of immunonano-gold, and in particular relates to a preparation method and application of large-particle colloidal gold. Background Art

[0002] Immune colloidal gold technique (GICT) originated in the 1870s. It is an immunolabeling technique based on colloidal gold as a tracer marker for antigen-antibody applications. As a conventional technical means of immunoassay, it is widely used in scientific research, technology application and other fields because of its convenience, speed, no need for professionals and instruments, and intuitive results. After decades of development, it has become an indispensable tool for biomedical testing with its fast, simple, accurate and low-cost characteristics. The classic colloidal gold preparation technology uses sodium citrate reduction to prepare nano-gold below 40nm and a concentration of one ten-thousandth. Colloidal gold above 40nm mostly uses the seed generation method. First, a small-particle colloidal gold is prepared by reduction. The colloidal gold is used as a seed crystal. The production of the seed crystal is controlled by adjusting the amount of reducing agent and gold chlorate as well as the rotation speed and temperature, and finally the colloidal gold of the required particle size is prepared.

[0003] The colloidal gold prepared by the classic sodium citrate reduction method has uniform particle size, simple labeling, and a wide range of practical applications. However, due to its small particle size, it is difficult to meet the needs of high-sensitivity detection projects. Although the seed generation method can prepare large-particle colloidal gold, the preparation process is complex and unstable, and protein labeling is difficult. It has very high requirements for personnel and equipment and is difficult to mass produce. Therefore, it is of great significance to prepare an immune colloidal gold with a simple process, stable labeling, and the ability to significantly improve production efficiency. Summary of the invention

[0004] The present invention aims to overcome the deficiencies of the prior art and provide a preparation method and application of large-particle colloidal gold.

[0005] In order to achieve the above object, the technical solution provided by the present invention is:

[0006] The preparation method of the large-particle colloidal gold comprises the following steps:

[0007] (1) pouring double distilled water into a container, adding a gold chloride solution, and stirring at a speed of 600-800 rpm / min for 3-7 min; the mass percentage concentration of gold chloride in the gold chloride solution is 8-12%, and after the gold chloride solution is added to the double distilled water, the mass percentage concentration of gold chloride in the entire system is 0.08-0.12‰;

[0008] (2) adding ascorbic acid at a concentration of 0.8-1.2 M and continuing stirring for 15-25 min; the volume ratio of ascorbic acid to double distilled water is (200-300) μL:1000 mL;

[0009] (3) adding trisodium citrate solution, heating and stirring until boiling, continuing stirring for 3-7 minutes, stopping heating, standing and cooling, and obtaining a colloidal gold solution; the volume ratio of the trisodium citrate solution to double distilled water is (400-600) μL:1000 mL; the mass percentage concentration of the trisodium citrate in the trisodium citrate solution is 0.8-1.2%;

[0010] (4) After the colloidal gold solution is cooled, pour it into a clean brown light-proof bottle, ensuring that the volume of the obtained colloidal gold solution is consistent with the volume of the initial double distilled water in step (1), add Proclin300 to the colloidal gold solution to make the volume percentage concentration of Proclin300 be 0.008-0.012%, detect the absorption peak and absorbance with a spectrophotometer, and then store it in a dark place.

[0011] Preferably, in step (1), stirring is performed at a speed of 500 rpm / min for 5 min; the mass percentage concentration of gold chloride in the gold chloride solution is 10%, and after the gold chloride solution is added to deionized water, the mass percentage concentration of gold chloride in the entire system is 0.2‰.

[0012] Preferably, step (2) is to add ascorbic acid at a concentration of 1 M and continue stirring for 20 min; the volume ratio of ascorbic acid to double distilled water is 250 μL:1000 mL.

[0013] Preferably, step (3) is to add trisodium citrate solution, heat and stir until boiling, continue stirring for 5 minutes, stop heating, let stand and cool, and obtain colloidal gold solution; the volume ratio of the trisodium citrate solution to double distilled water is 500μL:1000mL; the mass percentage concentration of the trisodium citrate in the trisodium citrate solution is 1%.

[0014] Preferably, in step (4), the volume percentage concentration of Proclin 300 in the colloidal gold solution is 0.01%.

[0015] Preferably, the particle size of the colloidal gold particles in the obtained colloidal gold solution is 80 nm.

[0016] The colloidal gold prepared by the above-mentioned method for preparing large-particle colloidal gold can be used to prepare a test strip for detecting Alzheimer's disease-related protein Aβ1-42.

[0017] The present invention will be further described below:

[0018] The present invention adopts double reducing agents, ascorbic acid is used for the preparation of large colloidal gold particles, and trisodium citrate is both a reducing agent and can stabilize the colloidal gold solution. The temperature and speed are strictly controlled by the reactor, and the volume does not change during the reaction process, and the preparation process is stable and controllable. The present invention can prepare stable, uniform large colloidal gold particles, the preparation method is simple and efficient, and the concentration is about twice that of conventional colloidal gold, that is, the production efficiency is doubled (see Figure 1 , Figure 2 The prepared colloidal gold labeled antibody has good reproducibility and its sensitivity is about 8 times higher than that of colloidal gold with conventional particle size ( Figure 3-Figure 7 The preparation method is stable, and the prepared colloidal gold has a good antibody labeling effect after being stored at room temperature for one month ( Figure 8 and Fig. 9 ).

[0019] Table 1: Colloidal gold absorbance

[0020] Colloidal gold particle size Absorption peak Absorbance Small (20nm) 522nm 0.983 Medium (29nm) 528nm 1.101 Large (79nm) 568nm 2.030

[0021] Note: The concentration of large-particle colloidal gold in the colloidal gold solution prepared by the present invention is 0.2‰, and the absorbance is about twice that of the conventional preparation method, that is, the production efficiency is increased by about 1 times.

[0022] In conclusion, the method of the present invention can prepare large-particle (80 nm) immunocolloidal gold with high concentration (0.2‰), uniform particle size and stability, which can not only meet the high sensitivity requirements of project detection, but also improve production efficiency to a large extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Colloidal gold color changes with different particle sizes (orange-red for small particles, dark red for regular particles, and earthy color for large particles);

[0024] Figure 2 : Spectral scans of different particle sizes (from left to right: small particle size absorption peak 522nm; conventional particle size absorption peak 528nm, large particle size absorption peak 568nm. According to He Xin et al.'s "Preparation and Particle Size Control of Nano-Colloidal Gold for Immunoassay", the relationship between absorption peak and colloidal particle size is calculated, Y = 0.786X + 505.53, corresponding to colloidal gold particle sizes of 20nm, 29nm and 79nm respectively);

[0025] Figure 3 :Repeatability test of low-value quality control products;

[0026] Figure 4 :Repeatability testing of high-value quality control products;

[0027] Figure 5 : 20nm small particle size colloidal gold detection sensitivity (sensitivity about 0.5ug / mL);

[0028] Figure 6 : 29nm conventional particle size colloidal gold detection sensitivity (sensitivity about 0.5ug / mL);

[0029] Figure 7 : 80nm colloidal gold detection sensitivity (sensitivity about 62.5ng / mL);

[0030] Figure 8 : Detection of low-value quality control products after colloidal gold storage for 1 month (left: colloidal gold prepared now, right: colloidal gold stored for 1 month after preparation);

[0031] Fig. 9 : Testing of high-value quality control products after colloidal gold was stored for one month (left: colloidal gold was prepared now, right: colloidal gold was stored for one month after preparation). DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0033] Example 1

[0034] (I) Preparation of large colloidal gold particles:

[0035] 1) Measure 1000 mL of double distilled water, pour it into a clean round-bottom flask and assemble a gold-flask;

[0036] 2) Adjust the speed to 700 rpm / min, add 2 mL of 10% gold chloride solution to make the gold chloride concentration 0.2‰, and stir for 5 minutes;

[0037] 3) Add 250uL of 1M ascorbic acid and continue stirring for 20min;

[0038] 4) Add 500uL of 1% trisodium citrate solution and start heating and stirring;

[0039] 5) After the solution boils, continue heating and stirring for 5 minutes, then stop heating and wait for the steam in the condenser to fall back;

[0040] 6) After cooling, pour the colloidal gold solution back into a clean brown light-proof bottle and make sure the volume is still 1000 mL; add Proclin 300 to make its concentration 0.01%, detect the absorption peak and absorbance with a spectrophotometer, and store at room temperature away from light.

[0041] (II) Application of large-particle colloidal gold in detecting Alzheimer's disease-related protein Aβ1-42

[0042] Alzheimer's disease (AD) is the most common form of dementia, accounting for 60-70% of cases. It is a degenerative disease of the central nervous system that mainly occurs in the elderly or pre-elderly, and the risk of disease increases with age. Studies have found that the typical histopathological changes of Alzheimer's disease are amyloid deposition and neurofibrillary tangles in the brain. A large amount of research evidence shows that changes in Aβ1-42 in cerebrospinal fluid and blood are closely related to the progression of AD.

[0043] According to the colloidal gold prepared by the present invention, a test strip for preparing Aβ1-42 is prepared: the colloidal gold-labeled capture antibody (Aβ42 N-terminal antibody) is prepared into a binding pad, and the detection antibody (Aβ42 C-terminal antibody) is coated in the NC membrane detection area (T line), and the quality control antibody sheep anti-mouse IgG is coated in the quality control area (C line). When the sample contains Aβ1-42, it first binds to the colloidal gold-capture antibody complex labeled with colloidal gold to form a colloidal gold-antibody-Aβ1-42 complex. When the complex migrates to the T line area, Aβ1-42 binds to the detection antibody, thereby retaining part of the colloidal gold particles in the T line to form color. Since the C line is coated with sheep anti-mouse IgG, regardless of whether the T line is colored or not, the colloidal gold-antibody-Aβ1-42 complex will bind to the C line to form color.

[0044] Capture antibody labeled with colloidal gold:

[0045] 1) Measure the colloidal gold solution: Take 100 mL of colloidal gold solution in a 200 mL beaker (with a rotor).

[0046] 2) Add capture antibody: add 2.2 mL 0.1 M K2CO3 while stirring at 200 rpm, continue stirring for 5 min, add capture antibody dropwise to a final concentration of 10 ug / mL, and keep stirring at 200 rpm for 30 min.

[0047] 3) Add BSA solution: add 1 mL of 20% BSA to make the final concentration 0.2%, and stir at 200 rpm for 30 min.

[0048] 4) Centrifugation: 5000 rpm, 4°C, centrifuge for 15 min. After centrifugation, aspirate the white supernatant as much as possible, but do not aspirate the precipitate.

[0049] 5) Redissolve: 10% trehalose, 0.1% S9, 1% BSA, 0.05% Proclin300, 10 mM Tris, pH 8.5. Directly reconstitute and make up the volume to 10% of the original volume.

[0050] Conjugate pad preparation:

[0051] 1) Solution preparation: 0.7% casein, 0.2% Tween-20, 0.05% ProClin300.

[0052] 2) Spreading liquid: Take glass fiber 8964 (300*250mm), spread the binding pad treatment liquid evenly on the glass fiber at 45mL / sheet using a pipette, and then spread it evenly with a coating rod.

[0053] 3) Drying: 37°C, 17±1 hours, with the bonding pad being uniform, dry, and moderately soft and hard.

[0054] 4) Gold spraying: The labeled colloidal gold solution is sprayed with gold at a rate of 10ul / cm using an XYZ three-dimensional large plane film spraying instrument.

[0055] 5) Drying: 37°C, 17±1 hours, with the bonding pad being uniform, dry, and moderately soft and hard.

[0056] Sample pad preparation:

[0057] 1) Solution preparation: 2% BSA, 1% PVP10, 1% S9, 6% sucrose, 0.5% casein, 0.05% ProClin300, 10 mM Tris-base, pH 8.6.

[0058] 2) Spreading liquid: Take glass fiber SB08 (300*200mm), spread the sample pad treatment liquid evenly on the glass fiber at 42mL / sheet using a pipette, and then spread it evenly with a coating rod.

[0059] 3) Drying: 37°C, 17±1 hours, with the sample pad being uniform, dry and hard.

[0060] Film cutting:

[0061] Line C: 10 mM PB, 1% trehalose, 0.3 mg / mL goat anti-mouse IgG

[0062] T line: 10 mM PBS, 1% trehalose, 1 mg / mL QK03

[0063] Streak the membrane at 1.5ul / cm and dry at 37℃ for 17±1 hours.

[0064] Assembly:

[0065] Assemble the colloidal gold card in the order of sample pad, conjugate pad, NC membrane, and absorbent pad. After assembly, add 0.5 g of desiccant to complete the sealing and store in a moisture-proof cabinet.

[0066] Test (results see Figure 7 and Table 2):

[0067] 1) Use normal saline to prepare Aβ42 standard (1 mg / mL) to the final concentration of 2 ug / mL (500X), 1 ug / mL (1000X), 500 ng / mL (2000X), 250 ng / mL (4000X), 125 ng / mL (8000X), 62.5 ng / mL (16000X), 31.25 ng / mL (32000X).

[0068] 2) Equilibrate the test strip to room temperature, open the aluminum foil bag, and vertically add 100ul Aβ1-42 standard.

[0069] 3) Judge the result with naked eyes within 10-15 minutes.

[0070] 4) If both the C line and the T line are colored, it is positive; if the C line is colored and the T line is not colored, it is negative; if the C line is not colored and the T line is colored or not colored, it is an invalid test strip.

[0071] Table 2

[0072]

Claims

1. A method for preparing large-particle colloidal gold, characterized in that: The preparation method of the large-particle colloidal gold comprises the following steps: (1) pouring double distilled water into a container, adding a gold chloride solution, and stirring at a speed of 600-800 rpm / min for 3-7 minutes; the mass percentage concentration of gold chloride in the gold chloride solution is 8-12%, and after the gold chloride solution is added to the double distilled water, the mass percentage concentration of gold chloride in the entire system is 0.08-0.12‰; (2) adding ascorbic acid at a concentration of 0.8-1.2 M and continuing stirring for 15-25 min; the volume ratio of ascorbic acid to double distilled water is (200-300) μL:1000 mL; (3) adding trisodium citrate solution, heating and stirring until boiling, continuing stirring for 3-7 minutes, then stopping heating and standing to cool, to obtain a colloidal gold solution; the volume ratio of the trisodium citrate solution to double distilled water is (400-600) μL:1000 mL; the mass percentage concentration of the trisodium citrate in the trisodium citrate solution is 0.8-1.2%; (4) After the colloidal gold solution has cooled, pour it into a clean brown light-proof bottle, ensuring that the volume of the resulting colloidal gold solution is consistent with the volume of the initial double-distilled water in step (1), add Proclin 300 to the colloidal gold solution to make the volume percentage concentration of Proclin 300 0.008-0.012%, detect the absorption peak and absorbance with a spectrophotometer, and then store it in a dark place.

2. The method for preparing large-particle colloidal gold according to claim 1, characterized in that: In step (1), stirring is performed at a speed of 500 rpm / min for 5 minutes; the mass percentage concentration of gold chloride in the gold chloride solution is 10%, and after the gold chloride solution is added to double distilled water, the mass percentage concentration of gold chloride in the entire system is 0.2‰.

3. The method for preparing large-particle colloidal gold according to claim 1, characterized in that: Step (2) is to add ascorbic acid at a concentration of 1 M and continue stirring for 20 minutes; the volume ratio of ascorbic acid to double distilled water is 250 μL:1000 mL.

4. The method for preparing large-particle colloidal gold according to claim 1, characterized in that: Step (3) is to add trisodium citrate solution, heat and stir until boiling, continue stirring for 5 minutes, stop heating, let stand and cool, and obtain colloidal gold solution; the volume ratio of the trisodium citrate solution to double distilled water is 500 μL:1000 mL; the mass percentage concentration of the trisodium citrate in the trisodium citrate solution is 1%.

5. The method for preparing large-particle colloidal gold according to claim 1, characterized in that: In step (4), the volume percentage concentration of Proclin300 in the colloidal gold solution is 0.01%.

6. The method for preparing large-particle colloidal gold according to any one of claims 1 to 5, characterized in that: The particle size of the colloidal gold particles in the obtained colloidal gold solution is 80 nm.

7. Use of the colloidal gold prepared by the method for preparing large-particle colloidal gold as claimed in claim 6 in preparing a test strip for detecting Alzheimer's disease-related protein Aβ1-42.

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