What is Colloidal Gold and Immunogold?

A. Properties and preparation of colloidal gold

(A) the structure of colloidal gold

Colloidal gold (colloidalgold) also known as gold sol (goldsol), is the gold salt is reduced to the original gold after the formation of gold particles suspension. Colloidal gold particles by a basic gold nucleus (atomic gold Au) and surrounded by a double ion layer, immediately attached to the surface of the gold nucleus is the inner layer of negative ions (AuC12-), the outer ion layer H + is dispersed in the solution between the colloid, in order to maintain the colloidal gold free in the suspension between the state of the solution.

Colloidal gold particles of the base gold nucleus is not the ideal round ball nucleus, smaller colloidal gold particles are basically round spherical, larger colloidal gold particles (generally refers to more than 30nm) more oval. In the electron microscope can observe the particle morphology of colloidal gold.

(B) the properties of colloidal gold

1 colloidal properties colloidal gold particle size in 1 ~ 100nm, tiny gold particles stable, uniformly, is a single dispersion state suspended in the liquid, become colloidal gold solution. Colloidal gold thus has a variety of properties of the colloid, especially the sensitivity to electrolytes. Electrolytes can destroy the colloidal gold particles of the periphery of the permanent hydration layer, thus breaking the stable state of the colloid, so that the dispersed single gold particles coalesce into large particles, and from the liquid precipitation down. Certain proteins and other macromolecular substances have protection colloidal gold, strengthen its stability role.

2, coloring tiny particles colloid is red, but different sizes of colloid coloring has some differences. The smallest colloidal gold (2 ~ 5nm) is orange, medium-sized colloidal gold (10 ~ 20nm) is burgundy, larger particles of colloidal gold (30 ~ 80nm) is purple-red. According to this characteristic, with the naked eye to observe the color of colloidal gold can roughly estimate the size of gold particles.

3, light absorption colloidal gold in the visible range has a single light absorption peak, the light absorption peak wavelength (λmax) in 510 ~ 550nm range, with the size of colloidal gold particles and change, large particles of colloidal gold λmax bias to the long wavelength, and vice versa, small particles of colloidal gold λmax is biased to the short wavelength, table 19-1 is part of the colloidal gold λmax.

(C) preparation of colloidal gold

1. Preparation method of colloidal gold preparation using reduction method. Chloroauric acid (HauC14) is the main raw material, commonly used reducing agents are sodium citrate, tannic acid, ascorbic acid, white phosphorus, sodium borohydride, etc.. According to the type of reducing agent and the strength of the reducing effect, can prepare 0.8nm ~ 150nm ranging colloidal gold. The most commonly used preparation method is citrate reduction method. Specific operation methods are as follows:

(1) will HauC14 first prepared into 0.01% aqueous solution, take 100ml heated to boiling.

(2) Add a certain amount of 1% trisodium citrate (Na3C6H5O7-2H2O) aqueous solution accurately under stirring.

(3) Continue to heat and boil for 15 min. at this time, it can be observed that the yellowish aqueous solution of chloroauric acid quickly turns gray after the addition of sodium citrate, and then turns black, and then gradually stabilizes into red. The whole process is about 2~3min.

(4) After cooling to room temperature, use distilled water to restore to the original volume.

With this method can prepare 16 ~ 147nm particle size of colloidal gold. The size of the gold particles depends on the amount of trisodium citrate added when preparing. Table 19-1 lists the preparation of four different particle size colloidal gold when the amount of trisodium citrate.

2. Notes

(1) Chloroauric acid is easy to deliquescence, should be dry, protected from light.

(2) Chloroauric acid is strongly corrosive to metal, therefore, when preparing aqueous solution of chloroauric acid, metal spoon should not be used for weighing chloroauric acid.

(3) for the preparation of colloidal gold distilled water should be double distilled water or triple distilled water, or high quality deionized water.

(4) is to prepare the colloidal gold glass container must be absolutely clean, should be used before the first acid wash and distilled water rinse. Preferably by the silicification process, silicification method can be used 5% dichloromethanesilane chloroform solution soaked for a few minutes, rinsed with distilled water and then dried.

(5) the identification and preservation of colloidal gold: the preparation of colloidal gold is not difficult, but to make good quality colloidal gold is not an easy task. Therefore, each time the good colloidal gold should be checked, the main inspection indicators are particle size, particle size uniformity and the absence of agglutination particles.

Naked eye observation is the most basic and the most simple and convenient method of calibration, but need some experience. Good colloidal gold should be clear and transparent, if the preparation of colloidal gold cloudy or liquid surface with floating matter, suggesting that the preparation of colloidal gold has more agglutination particles. In the daylight careful observation to compare the color of colloidal gold, can roughly estimate the size of the gold particles made. Of course, also available spectrophotometer scan λmax to estimate the particle size of gold particles. Junction preparation of colloidal gold best for electron microscopic observation, and select some representative for microphotography, can be more accurate determination of the average particle size of colloidal gold.

Colloidal gold in clean glassware can be stored for a long time, adding a little preservative (such as 0.02% NaN3) can be conducive to preservation. Preservation of improper bacterial growth or agglutination particles will be formed. A small amount of agglutination particles does not affect the future labeling of colloidal gold, when used to improve the efficiency of the labeling can be removed first low-speed centrifugation agglutination particles.

II. Characteristics and preparation of immunogold

(A) the characteristics of immunogold

Colloidal gold can be combined with proteins and other large molecules, in immunohistochemistry technology, the customary colloidal gold combined with protein complexes called gold probes. Used for immunoassay colloidal gold and immune active material (antigen or antibody) combined, this kind of colloidal gold combination often called immunogold complex, or referred to as immunogold (immunogold).

Colloidal gold and protein binding mechanism is still very clear, generally considered to be physical adsorption. Colloidal gold particles with a layer of surface negative charge, and the positive charge of the protein surface through electrostatic induction attached. Therefore, the environmental pH and ionic strength is the main factor affecting the adsorption, other factors such as the size of colloidal gold particles, the molecular weight of the protein and protein concentration will also affect the protein adsorption.

(B) Preparation of immunogold

1. Adjust the pH of the colloidal gold solution to the selected value with 0.2 mol/L K2CO3 or 0.1 mol/L HC1. In principle, you can choose the isoelectric point of the protein to be labeled, but also slightly alkaline. But usually the most suitable reaction pH often need to be determined by several trials.

When adjusting the pH of colloidal gold should be noted that colloidal gold will block the electrode of pH meter, not directly insert the electrode into the colloidal gold solution, it is advisable to use the final concentration of 0.15 polyethylene glycol (PEG, 20000) to stabilize the colloidal gold first, then the pH of colloidal gold.

2. Add 1/10 volume of suitable concentration of protein solution into colloidal gold solution and place the reaction at room temperature for 2~5min.

As salt components can affect the adsorption of colloidal gold to protein, and can make colloidal gold aggregation sink, so to be labeled protein solution if it contains high ion concentration, should be labeled before the first to low ionic strength of distilled water dialysis to salt.

3. Add a concentration of 0.2% PEG or BSA to saturate the free colloidal gold.

4. Centrifuge to remove the unbound protein from the supernatant. Centrifugal conditions depending on the particle size of colloidal gold particles vary: 5nm gold particles can be selected 40,000r/min centrifugal 1h; 8nm gold particles with 25,000r/min centrifugal 45min; 14nm gold particles with 25,000r/min centrifugal 30min, 40nm gold particles with 15,000r/min centrifugal 30min.

5. Lightly aspirate the supernatant. The precipitate was suspended with a buffer containing PEG or BSA, and then centrifuged after restoring the original volume. Wash this way 2 to 4 times. To completely remove the unbound protein.

6. The immunogold complex is finally prepared in diluent to a working concentration for storage. The diluent is usually a buffer solution with stabilizer added. The buffer solution is commonly used as neutral PBS or Tris buffer.