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Precautions for the preparation of colloidal gold and its application

wallpapers Tech 2020-03-23

Gold Colloidal Solution has a very high dynamic stability. When the stability factor is not damaged, it will condense very slowly. It can be left for several years without agglomeration. The main factors affecting stability are electrolyte, sol concentration, temperature, non-electrolyte, and so on.

Gold sol must have a small amount of electrolyte as a stabilizer, but the concentration should not be too high. High-concentration hydrophilic non-electrolyte can peel off the hydrated film outside the colloidal particles to make them aggregate. A small amount of high-molecular substance promotes the aggregation of the sol, but a certain amount of high-molecular substance can increase the stability of the sol. For example, the addition of protein, glucose, PEG20000. etc. has a good stabilizing effect.

After the gold sol adsorbs protein, the stability of the sol changes with the pH of the solution, and this change depend on the isoelectric point of the adsorbed protein, such as ConA, peroxidase, etc., when the pH is low, it remains stable and increases the pH. It appears unstable and becomes stable again near the isoelectric point or slightly higher. The labelled colloidal gold solution can use 0.2-0.5 mg/ml PEG20000 as a stabilizer. It is effective to store at 4 ～ 10 ℃ for several months, and should not be frozen. Coagulation may occur to varying degrees during storage and can be removed by centrifugation.

的 Precautions for preparing high-quality colloidal gold

(1) Glassware must be thoroughly cleaned, preferably siliconized glassware, or glassware stabilized with colloidal gold for the first time and rinsed with double distilled water before use. Otherwise, it will affect the stability of gold particles after the binding of biomacromolecules to gold particles and activation, and gold particles of expected size cannot be obtained.

(2) Reagents, water quality and environment: the preparation of the reagents must be strictly pure, and all reagents must be prepared with double or triple distilled water and deionized, or the prepared reagents are subjected to ultrafiltration or microfiltration The pore filter (0.45 μm) is filtered to remove the polymer and other impurities that may be mixed in. Chloroauric acid is extremely easy to absorb moisture and has strong corrosiveness to metals. Do not use metal spoons to avoid contact with the balance scale. It's 1% aqueous solution is stable for several months at 4 ° C. Experimental water is usually double-distilled water. The number of dust particles in the laboratory should be minimized, otherwise, the results of the experiment will lack repeatability.

The gold particles are easily adsorbed on the electrode to block it, so the pH value of the gold solution cannot be measured with a pH electrode. In order to prevent the pH of the solution from changing, a buffer system with a sufficiently large buffer capacity should be selected. Generally, citrate phosphate (pH 3 to 5.8), Tris-HCL (pH 5.8 to 8.3), and sodium borate hydroxide (pH 8.5) are used. ~ 10.3) and other buffer systems. However, it should be noted that the concentration of the buffer solution should not be too high to cause the gold sol to self-coagulate.

(3) The pH of the colloidal gold solution is preferably neutral (pH 7.2).

(4) The quality requirements of chloroauric acid are high, and the impurities are few. It is best imported.

5 (5) Chloroauric acid can be stable for several months at 4 ° C with a 1% aqueous solution. Since chloroauric acid is prone to deliquescent, it is best to dissolve the entire small package at one time when preparing.

应用 ApplicationApplication of colloidal gold technology

1. Application in immunology

(1) Application of colloidal gold at electron microscope level

The research on the ApplicationApplication of colloidal gold to the electron microscope was the earliest, the fastest, and the most widely used. The biggest advantage is that it can be used for double or multiple labelling by using different size particles or binding enzyme labels. Colloidal gold with a diameter of 3-15nm can be used as a marker for the level of the electron microscope. Colloidal gold from 3 to 15 nm is mostly used for the Detection of a single antigen particle, and 15 nm diameter is mostly used for the Detection of a large number of infected cells.

Colloidal gold is used for the research of electron microscope level, mainly 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 antigen.

The ApplicationApplication of gold labelling at the level of electron microscope includes the following methods: pre-embedding staining, post-embedding staining, negative immunostaining, double labelling technology and in situ hybridization technology.

Experiments show that the method has a small sample amount, 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 used for scientific research and diagnosis.

(2) Application of colloidal gold at the level of the light microscope

Plutonium colloidal gold can also be used as a light microscope level marker to replace traditional fluorescein and enzymes. Various cell smears and sections can be applied. Mainly used:

① Use monogram 或 antibody or antiserum to detect the cell surface antigen of the cell suspension or cultured monolayer cells.

② detecting intracellular antigen of cultured monolayer cells,

③ Detection of antigens in tissues or thin sub-sections.

Gold Colloidal Solution is used to study the level of a light microscope, which can make up for the shortcomings of other markers such as the unavoidably high background and interference of internal enzyme activity.

(3) Application of colloidal gold in flow cytometry:

Using fluorescein-labelled antibodies to analyze cell surface antigens by flow cytometry is one of the important techniques in immunological research. However, because the spectra of different fluorescein overlap each other, it is difficult to distinguish different labels, so it is necessary to find a non-fluorescein label for flow cytometry. This allows several markings to be performed simultaneously. The marker must be able to change the scattering angle. Colloidal gold can significantly change the red laser scattering angle, so it can be used as one of the markers for flow cytometry.

(4) Agglutination test: The monodispersed immunogold sol is a clear and transparent solution, and its colour changes with the size of the soil particles. When it specifically reacts with the corresponding antigen or antibody, agglutination occurs, the soil particles increase extremely, and light scattering. With this change, the particles will settle, and the colour of the solution will become pale or even colourless. This principle can be applied qualitatively or quantitatively to the immune response.

(5) Immunoblotting: Immunoblotting is a newer immunochemical technique. The proteins were separated by polyacrylamide gel electrophoresis, and the resulting bands were transferred to a nitrocellulose membrane, and then quantified by enzyme immunoassay (or immunofluorescence, RIA).

Tritium 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 colloidal gold sensitized by Staphylococcus A protein, and the excess colloidal gold is thoroughly washed away. The colour depth of the colloidal gold particles on the membrane can be measured—specific antigen in the sample.

Utilizing the principle that gold particles can catalyze the reduction of silver ions to metallic silver, the use of a silver developer to enhance the visibility of gold particles can greatly increase the sensitivity of the assay, and the detection limit can be as low as 0.1ng. This immunogold staining method has been applied Widening.

Since the colloidal gold immunoblotting technique is simple, fast, and has 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 and is used in naked eye immunoassay. In addition to the characteristics of colloidal gold, it also has the following advantages:

① The amount of reagents and samples is extremely small, and the sample amount can be as low as 1 ~ 2ul;

② No need for expensive instruments such as γ-counter, fluorescence microscope, enzyme-labelled detector, etc., more suitable for field applications;

③No harmful substances such as radioisotopes and o-phenylenediamine are involved;

④ The experimental results can be stored for a long time;

⑤The time is greatly shortened, and the detection speed is improved.

In the process of gold labelling, no covalent bond is formed, which is physical adsorption at a certain ion concentration. Therefore, almost all macromolecular substances can be labelled with gold, and the activity of macromolecular substances does not change after labelling. The experimental results show that the sensitivity of colloidal gold can reach the level of ELISA. When combined with silver staining, the detection sensitivity is greatly improved.

2. Application in immunodiagnostic technology

Colloidal gold labelling technology is easy to prepare, sensitive, and specific. It does not require the use of radioisotopes or enzyme-producing substrates with potential carcinogens, nor does it require fluorescence microscopy. It has a wide range of applications, except for light microscopy. In addition to the immunohistochemical method of electron microscopy, it is more widely used in various liquid-phase immunoassays, solid-phase immunoassays, and flow cytometry.

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