The overall goal of this procedure is to produce oligomeric clusters of gold nanoparticles, which are:highly stable, easily derivatized, and have well-controlled diameters via the reduction of chloroauric acid with sodium thiocyanate. The particles that we've been looking at today are interesting particles because their sizes are so well-controlled, and as a result of that, they can be used to look at the permeability of membranes to particles of different sizes. Since they are very stable in biological solutions, this is a very powerful property of the particle.
The main advantage of this method is that it allows well-controlled synthesis of high-quality gold oligoclusters under bench-top conditions, and is easily performed with standard lab equipment and a limited number of reagents. After preparing all necessary solutions as indicated in the text protocol, begin delay-time synthesis of gold oligoclusters by adding 7 milliliters of the Borax solution to a clean 125 milliliter glass bottle containing 59.5 milliliters of water under stirring. Next, add 2.8 milliliters of the gold chloride solution to the bottle, which begins the delay-time that determines the size of the oligoclusters.
Add 700 microliters of the sodium thiocyanate solution to the bottle at the end of the delay period. Continue stirring it overnight to complete synthesis of the oligoclusters. While attempting this procedure, it's important to rapidly add gold chloride and sodium thiocyanate solution to all the reagent at larger volumes of this solution, using wide-mouth dispensers.
For add-on growth, combine 10 milliliters of the gold oligoclusters with 60 milliliters of the hydroxylated gold chloride solution. Next, add 900 microliters of the sodium thiocyanate solution with vigorous stirring. Allow the mixture to stir overnight, until the reaction is complete.
Prior to derivatization, add 70 milliliters of the crude oligoclusters to a 30 kilodalton cut-off centrifugal filter, and spin for 15 minutes at 3, 000 times g. Recover the ree-tin tape by inverting the filter, and centrifuging for 3 minutes at 500 times g. Then, measure the recovered volume with a micro-pipette.
Next, add a volume of GSH equal to one-ninth of the recovered volume of the oligoclusters. Allow the reaction to sit at room temperature for no more than 10 minutes. Dilute the derivatized oligoclusters into 50 milliliters of Dulbecco's phosphate buffered saline to halt the reaction.
Then, apply the derivatized oligoclusters to a 30 kilodalton cut-off centrifugal filter, and spin for 15 minutes at 3, 000 times g. Finally, recover the ree-tin tape by inverting the filter, and spinning for three minutes at 500 times g. Store the glutathione derivatized oligoclusters at four degrees Celsius.
To analyze the crude oligoclusters by gel electrophoresis, mix them at a ratio of two-to-one with loading buffer. Then, load 30 microliters of the oligoclusters onto a precast polyacrylamide gradient gel. Run the gel with tris glycine buffer for 26 minutes at 200 volts.
For gel electrophoresis of derivatized oligoclusters, first dilute them at a ratio of one-to-three with water. Next, dilute the clusters further at a ratio of two-to-one with loading buffer. Load 10 microliters of the oligocluster mixture onto a precast polyacrylamide gradient gel, and then run the gel with tris glycine buffer for 26 minutes at 200 volts.
To analyze by transmission electron microscopy, wash 20 microliters of concentrated oligoclusters with 0.5 milliliters of water, and load them onto a 0.5 millileter, 30 kilodalton cut-off centrifugal filter. Spin at 14, 000 times g for 10 minutes. Next, remove the filtrate from the tube, and resuspend the ray-tah-date in 0.5 milliliters of water.
Repeat the wash twice more, and then dilute the oligoclusters 500-fold with water. After glow discharging your carbon coated grid, deposit 0.6 microliters of the oligoclusters onto the grid, and allow them to air-dry for 10 minutes. Finally, visualize the oligoclusters using 100, 000 x magnification at 80 kilovolts in the transmission electron microscope.
The size of the gold oligoclusters, synthesized using the delay-time and add-on procedures, were analyzed by gel electrophoresis. Lanes two through four of the gel showed that the cluster size increases as the delay-time becomes longer. Lanes five through eight confirm that the size of the clusters also increases with the amount of hydroxylated gold used during the add-on procedure.
Transmission electron microscopy was used to determine the diameters of the oligocluster particles. Diameters were calculated by taking measurements from images of 50 nanometer by 50 nanometer grid areas containing the particles. The relationship between particle sized delay time and add-on amounts of hydroxylated gold were graphed.
These data were then converted into predictive equations for the particle diameters for each procedure. The method of making the gold clusters, I am sure you have talked about, is dependent on the hydroxylation of gold chloride in alkaline solution. So that, the more hydroxylation is, the less likely it is to generate a cluster of gold.
My new book here is the first interesting to me, because it's the first time that I realize the time element of exposure to alkaline could be used to control the size of the clusters. When performed correctly, gold oliclusters of sizes from three to 70 nanometers can be synthesized in less than a day. The resulting nanoparticles are stable in physiological buffers and plasma, making them suitable for in-view experiments.
The gold oligoclusters described here have the additional benefit of being able to be concentrated without prior derivatization, thus allowing the expanse of the rootization agents to be used in smaller volumes.
