Preparation method and application of large-particle colloidal gold

Abstract

The application discloses a preparation method and application of large-particle colloidal gold, and 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 min; (2) adding ascorbic acid with a concentration of 0.8-1.2 M, and continuing to stir for 15-25 min; (3) adding a trisodium citrate solution, heating and stirring until boiling, continuing to stir for 3-7 min, then stopping heating, and cooling to obtain a colloidal gold solution; (4) after the colloidal gold solution is cooled, pouring 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), adding Proclin 300 to the colloidal gold solution, detecting an absorption peak and absorbance by using a spectrophotometer, and then storing in the dark.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of immune nanogold, and particularly relates to a preparation method and application of large-particle colloidal gold. BACKGROUND

[0002] Immune colloidal gold technique (GICT for short) originated in the 1970s of the 19th century, and is an immune labeling technique based on colloidal gold as a tracer marker applied to antigen-antibody. As a conventional technical means of immune detection, it has a wide application in scientific research, technical application and other fields due to its characteristics of convenience, quickness, no need for professional personnel and instruments, and intuitive results. After decades of development, it has become an indispensable tool for biomedical detection due to its characteristics of rapidness, simplicity, accuracy and low cost. The classic colloidal gold preparation technique adopts a sodium citrate reduction method to prepare nanogold with a concentration of one ten-thousandth and a particle size of less than 40 nm. Colloidal gold with a particle size of more than 40 nm is mostly prepared by a seed generation method, that is, small-particle-size colloidal gold is prepared by the reduction method, and the colloidal gold is used as a seed to control the production of the seed by adjusting the amount of the reducing agent and gold chlorate, the rotation speed and the temperature, so that colloidal gold with a desired particle size is finally prepared.

[0003] The colloidal gold prepared by the classic sodium citrate reduction method has uniform particle size, simple labeling and wide practical range, but it is difficult to meet the demand of high-sensitivity detection projects due to the small particle size. Although the seed generation method can prepare large-particle-size colloidal gold, the preparation process is complex and unstable, and it is difficult to label protein, so the personnel and equipment requirements are very high, and it is difficult to mass-produce. Therefore, it is of great significance to prepare immune colloidal gold with a simple process, stable labeling and greatly improved production efficiency. SUMMARY

[0004] The application 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-mentioned purpose, the technical scheme provided by the application is as follows.

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

[0007] (1) Pour double-distilled water into a container, and add a gold chloride solution, and stir at a rotation 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 whole system is 0.08-0.12 ‰;

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

[0009] (3) adding a trisodium citrate solution, heating and stirring until boiling, continuing to stir for 3-7min, then stopping heating, standing and cooling 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%;

[0010] (4) after the colloidal gold solution is cooled, pouring into a clean brown light-proof bottle, ensuring that the volume of the obtained colloidal gold solution is consistent with that of the initial double-distilled water in step (1), adding Proclin 300 to the colloidal gold solution so that the volume percentage concentration of the Proclin 300 is 0.008-0.012%, and then detecting the absorption peak and absorbance by a spectrophotometer and storing in the dark.

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

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

[0013] Preferably, in step (3), the trisodium citrate solution is added, heating and stirring until boiling, continuing to stir for 5min, then stopping heating, standing and cooling to obtain a 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%.

[0014] Preferably, in step (4), the volume percentage concentration of the 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 80nm.

[0016] The colloidal gold prepared by the above method of preparing large-particle colloidal gold can be used to prepare an Alzheimer's disease related protein Aβ1-42 test paper.

[0017] The present application is further described as follows:

[0018] The present application adopts double reducing agents, ascorbic acid is used for preparing large-particle colloidal gold, and trisodium citrate is both a reducing agent and can stabilize colloidal gold solution. The temperature and rotating speed are strictly controlled through a reaction kettle, and the volume does not change during the reaction process, so that the preparation process is stable and controllable. The present application can prepare stable, uniform-particle-size large-particle colloidal gold, and 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 and Table 1). The prepared colloidal gold-labeled antibody has good repeatability, and the sensitivity is about 8 times higher than that of conventional particle size colloidal gold ( Figures 3-7 ). The preparation method is stable, and the prepared colloidal gold has good labeling effect after being stored at room temperature for one month ( Figure 8 and Figure 9 ).

[0019] Table 1: Colloidal gold absorbance

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

[0021] Note: The concentration of large-particle colloidal gold in the colloidal gold solution prepared by the present application 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 summary, the method of the present application can prepare high-concentration (0.2‰), uniform-particle-size, stable large-particle (80 nm) immunocolloidal gold, which can meet the high sensitivity requirement of project detection and greatly improve the production efficiency in a large range. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 : Color change of colloidal gold with different particle sizes (orange for small particle size, deep red for conventional particle size, and mud color for large particle size);

[0024] Figure 2 : Spectral scanning of colloidal gold with different particle sizes (from left to right: small particle size absorption peak 522 nm; conventional particle size absorption peak 528 nm, large particle size absorption peak 568 nm. According to He Xin et al. Preparation and particle size control of nanometer colloidal gold for immunodetection, the relationship between absorption peak and colloidal particle size is calculated, Y = 0.786X + 505.53, and the corresponding colloidal gold particle sizes are 20 nm, 29 nm and 79 nm, respectively);

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

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

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

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

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

[0030] Figure 8 : Colloidal gold detection of low value quality control after storage for 1 month (left: colloidal gold prepared now, right: colloidal gold prepared and stored for 1 month);

[0031] Figure 9 : Colloidal gold detection of high value quality control after storage for 1 month (left: colloidal gold prepared now, right: colloidal gold prepared and stored for 1 month). DETAILED DESCRIPTION

[0032] The application will be further described below in combination with the drawings and examples.

[0033] Example 1

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

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

[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 min;

[0037] 3) Add 250 uL of 1 M ascorbic acid, continue to stir for 20 min;

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

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

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

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

[0042] Alzheimer's disease (AD), the most common form of dementia, can account for 60-70% of cases, is a degenerative disease of the central nervous system, 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 neuronal fiber tangles in the brain, and 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] The colloidal gold prepared according to the present application is used to prepare a test strip for detecting Aβ1-42: a colloidal gold-labeled capture antibody (Aβ42 N-terminal antibody) is prepared into a conjugate pad, a detection antibody (Aβ42 C-terminal antibody) is coated on the NC membrane detection area (T line), and a control antibody (goat anti-mouse IgG) is coated on the control area (C line). When the sample contains Aβ1-42, it first binds with the colloidal gold-labeled colloidal gold-capture antibody complex to form a colloidal gold-antibody-Aβ1-42 complex. When the complex migrates to the T line area, Aβ1-42 binds with the detection antibody, thereby trapping part of the colloidal gold particles on the T line to form a color development. The C line is coated with goat anti-mouse IgG, and regardless of whether the T line develops color or not, the colloidal gold-antibody-Aβ1-42 complex will bind with the C line to form a color development.

[0044] Colloidal gold-labeled capture antibody:

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

[0046] 2) Add capture antibody: under 200 rpm stirring, add 2.2 mL of 0.1M K2C03, 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 a final concentration of 0.2%, and keep stirring at 200 rpm for 30 min.

[0048] 4) Centrifugation: 5000 rpm, 4°C, centrifugation for 15 min, after centrifugation, try to suck as much white supernatant as possible, and do not suck the precipitate.

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

[0050] Conjugate pad preparation:

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

[0052] 2) Liquid spreading: Take glass fiber 8964 (300*250mm), evenly spread the binding pad treatment solution on the glass fiber with a pipette at 45mL per piece, and then evenly spread it flat with a coating rod.

[0053] 3) Drying: 37°C, 17±1 hours, with the standard of uniform texture, dry, soft and hard of the binding pad.

[0054] 4) Gold spraying: mark the colloidal gold solution, and spray gold with XYZ three-dimensional large plane film spraying instrument at 10ul / cm.

[0055] 5) Drying: 37°C, 17±1 hours, with the standard of uniform texture, dry, soft and hard of the binding pad.

[0056] Sample pad preparation:

[0057] 1) Liquid preparation: 2% BSA, 1% PVP10, 1% S9, 6% sucrose, 0.5% casein, 0.05% ProClin300, 10mM Tris-base, pH8.6.

[0058] 2) Liquid spreading: Take glass fiber SB08 (300*200mm), evenly spread the sample pad treatment solution on the glass fiber with a pipette at 42mL per piece, and then evenly spread it flat with a coating rod.

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

[0060] Film drawing:

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

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

[0063] Draw film at 1.5ul / cm, dry at 37°C for 17±1 hours.

[0064] Assembly:

[0065] Assemble the colloidal gold card in the order of sample pad, binding pad, NC membrane, and water absorption pad, and then add 0.5g desiccant to complete the sealing, and store in a moisture-proof cabinet.

[0066] Detection (results are shown in Figure 7 and Table 2):

[0067] 1) Prepare Aβ42 standard (1 mg / mL) in physiological saline to final concentrations 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 test strips to room temperature, open aluminum foil pouch, and add 100 ul of Aβ1-42 standard vertically.

[0069] 3) Read results visually after 10-15 min.

[0070] 4) Both C and T lines colored positive; C line colored, T line not colored negative; C line not colored, T line colored or not colored 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 includes the following steps: (1) Pour double-distilled water into a container, add gold chloride solution, and stir at 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 whole system is 0.08-0.12‰. (2) Add ascorbic acid with a concentration of 0.8-1.2M and continue stirring for 15-25 minutes; the volume ratio of the ascorbic acid to the double-distilled water is (200-300) μL: 1000mL; (3) Add trisodium citrate solution, heat and stir until boiling, continue stirring for 3-7 minutes, then stop heating and let stand to cool to obtain colloidal gold solution; the volume ratio of trisodium citrate solution to double-distilled water is (400-600) μL: 1000 mL; the mass percentage concentration of trisodium citrate in 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 obtained 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%. After detecting the absorption peak and absorbance with a spectrophotometer, store it in the dark.

2. The method for preparing large-particle colloidal gold as described in claim 1, characterized in that, In step (1), the mixture is stirred at 500 rpm 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 double-distilled water, the mass percentage concentration of gold chloride in the whole system is 0.2‰.

3. The method for preparing large-particle colloidal gold as described in claim 1, characterized in that, Step (2) involves adding 1M ascorbic acid and stirring for 20 minutes. The volume ratio of the ascorbic acid to the double-distilled water is 250 μL: 1000 mL.

4. The method for preparing large-particle colloidal gold as described in claim 1, characterized in that, Step (3) involves adding trisodium citrate solution, heating and stirring until boiling, continuing to stir for 5 minutes, stopping heating, and allowing it to stand and cool to obtain a 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 as described in 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 as described in 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. The application of the colloidal gold prepared by the method for preparing large-particle colloidal gold as described in claim 6 in the preparation of test strips for Alzheimer's disease-related protein Aβ1-42.

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