Colloidal gold has high kinetic stability, and its self-aggregation is extremely slow when the stability factor is not damaged, and it can be left for several years without agglomeration. The factors affecting stability mainly include electrolytes, sol concentration, temperature, and non-electrolytes.
The gold sol must have a small amount of electrolyte as a stabilizer, but the concentration should not be too high. The high concentration of the hydrophilic non-electrolyte can peel off the hydrated film outside the colloidal particles to agglomerate. A small amount of high molecular substances promote the sol condensation, but a certain amount of high molecular substances can increase the stability of the sol, such as protein, glucose, PEG20000, etc. have a good stabilizing effect.
When the gold sol adsorbs the protein, the stability of the sol changes with the pH of the solution, and this change depends on the isoelectric point of the adsorbed protein, such as ConA, peroxidase, etc., when the pH is low, it remains stable, and the pH is raised. It is unstable and becomes stable when it is close to the isoelectric point or slightly higher. The labeled colloidal gold solution may use 0.2 to 0.5 mg/ml PEG 20000 as a stabilizer. It is effective for storage for several months at 4~10 °C, and should not be frozen. Condensation of varying degrees may occur during storage and may be removed by centrifugation.
Precautions for preparing high quality colloidal gold
(1) The glassware must be thoroughly cleaned, preferably a siliconized glassware, or a colloidal gold-stabilized glassware prepared in the first time, and then rinsed with double distilled water. Otherwise, the stability of the gold particles after the binding and activation of the biomacromolecules to the gold particles is affected, and the gold particles of the expected size cannot be obtained.
(2) Reagents, water quality and environment: The preparation of the agent must be kept strictly pure. All reagents must be prepared by using double or distilled water and deionized, or the reagents should be ultrafiltered or micro before use. A pore filter (0.45 μm) was filtered to remove the polymer and other impurities that might be mixed therein. Chloroauric acid is very easy to absorb moisture and has strong corrosiveness to metals. Metallic spoons cannot be used to avoid contact with the balance scale. Its 1% aqueous solution can be stable for several months at 4 °C. The experimental water is generally double distilled water. The dust particles in the laboratory should be minimized, otherwise the results of the experiment will be lack of repeatability.
The gold particles are easily adsorbed on the electrodes to block them, so the pH of the gold solution cannot be measured with a pH electrode. In order to make the pH of the solution not change, a buffer system with sufficient buffer capacity should be used, generally using citrate phosphate (pH 3 ~ 5.8), Tris-HCL (pH 5.8 ~ 8.3) and sodium borate (pH 8.5). ~10.3) and other buffer systems. However, care should be taken not to allow the gold sol to self-coagulate if the buffer concentration is too high.
(3) The pH of the colloidal gold solution is preferably neutral (pH 7.2).
(4) The quality of chloroauric acid is superior and the impurities are small. It is best to import.
(5) The chloroauric acid is formulated into a 1% aqueous solution which is stable for several months at 4 ° C. Since chloroauric acid is easily deliquescent, it is preferable to dissolve the entire small package at one time during preparation.
Application of colloidal gold technology
1. Application in immunology
(1) Application of colloidal gold at the level of electron microscope
The earliest research on the application of electron microscopy in colloidal gold is the fastest growing and the most widely used. The biggest advantage is that it can be double or multi-labeled by applying different sized particles or binding enzyme labels. Colloidal gold with a diameter of 3 to 15 nm can be used as a marker for electron microscopy levels. 3 to 15 nm of colloidal gold is mostly used for the detection of single antigen particles, while the diameter of 15 nm is mostly used to detect infected cells with a large amount.
Colloidal gold is used in the study of electron microscopy levels, including:
1 Observation of cell surface antigen in cell suspension or monolayer culture.
2 Detection of intracellular antigens in monolayer culture.
3 Detection of tissue antigens.
The application of gold markers at the level of electron microscopy includes: pre-embedding staining, post-embedding staining, immunonegative staining, double labeling techniques, and in situ hybridization techniques.
The experiment proves that the method has small sample dosage, fast detection speed, obvious contrast, simple operation, high sensitivity and specificity, and can be used for both antigen detection and antibody detection. Therefore, it can be applied to both scientific research and diagnosis.
(2) Application of colloidal gold at the level of light microscope
Colloidal gold can also be used as a marker at the level of light microscopy, replacing traditional fluorescein, enzymes and the like. Various cell smears and slices can be applied. Mainly used:
1 Detect membrane surface antigen of cell suspension or cultured monolayer cells with monoclonal antibody or antiserum.
2 detecting the intracellular antigen of cultured monolayer cells,
3 Detection of antigen in tissue or sub-thin sections.
Colloidal gold is used in the study of light microscopy level to compensate for the inevitable background of other markers and the interference of internal enzyme activity.
(3) Application of colloidal gold in flow cytometry:
The use of fluorescein-labeled antibodies to analyze cell surface antigens by flow cytometry is one of the important techniques in immunology research. However, since the spectra of different fluoresceins overlap each other, it is difficult to distinguish different markers, so it is necessary to find a non-fluorescein marker for flow cytometry. This allows several markers to be made simultaneously. The marker must be able to change the scattering angle, and colloidal gold can significantly change the red laser scattering angle and thus can be used as one of the markers of flow cytometry.
(4) Agglutination test: The monodisperse immunogold sol is a clear and transparent solution whose color varies with the size of the sol particles. When it reacts specifically with the corresponding antigen or antibody, it condenses, and the sol particles are extremely enlarged, and light scattering As a result of the change, the particles will also settle, the color of the solution will become lighter or even become colorless, and this principle can be applied qualitatively or quantitatively to the immune response.
(5) Immunoblotting: Immunoblotting is a relatively new immunochemical technique. The protein was separated by polyacrylamide gel electrophoresis, and the resulting band was transferred to a nitrocellulose membrane and quantified by enzyme immunoassay (or immunofluorescence, RIA).
Immunocolloidal gold can also be used for the quantification of this method. After the transferred nitrocellulose membrane is incubated with a specific antibody, it is incubated with the staphylococcal sensitized colloidal gold, and the excess colloidal gold is thoroughly washed away, and the color of the colloidal gold particles on the membrane can be measured. Specific antigen in the sample.
The use of gold particles can catalyze the reduction of silver ions into metallic silver. Silver developer can enhance the visibility of gold particles, and the sensitivity of the measurement can be greatly improved. The detection limit can be as low as 0.1 ng. This immunogold and silver staining method has been applied. It is becoming more and more widespread.
Because the colloidal gold immunoblotting technique is simple, rapid, and has a relatively high sensitivity, it has great potential for clinical immunodiagnosis.
(6) Application of colloidal gold at the naked eye level
Colloidal gold replaces the traditional three major markers for immunoassay at the naked eye level. In addition to the characteristics of colloidal gold itself, there are the following advantages:
1 reagent and sample dosage is very small, the sample size can be as low as 1 ~ 2ul;
2 No need for γ-counter, fluorescence microscope, enzyme label detector and other valuable instruments, more suitable for field applications;
3 no harmful substances such as radioisotopes and o-phenylenediamine are involved;
4 experimental results can be stored for a long time;
5 time is greatly shortened, and the detection speed is improved.
In the gold standard process, no covalent bond is formed, which is physical adsorption at a certain ion concentration. Therefore, almost all macromolecular substances can be labeled with gold, and the activity of macromolecular substances does not change after labeling. The experimental results show that the sensitivity of colloidal gold can reach the level of ELISA. When combined with silver staining, the sensitivity of the detection is greatly increased.
2. Application in immunodiagnostic technology
Colloidal gold labeling technology is simple and sensitive, and the method is sensitive and specific. It does not need to use radioisotopes, or enzyme chromogenic substrates with potential carcinogens, and no fluorescence microscope. It has a wide range of applications, except for application to light microscopy. Or electron microscopy immunohistochemistry, more widely used in various liquid immunoassays and solid phase immunoassays and flow cytometry.
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