This protocol addresses the batch-to-batch reproducibility of sulfonate and amphiphilic gold nanoparticles that our laboratory uses in experiments with cells, viruses, and proteins. This technique addresses the inorganic contaminants common to this type of synthesis. It also introduces checks and balances after each step to ensure the reproducibility of amphiphilic gold nanoparticles.
This technique is laborious and requires patience. The level of difficulty depends on scale, so start at a small scale, and familiarize yourself with each instrument and step before scaling up. First, add 11-bromo-1-undecene, sodium sulfite, and benzyltriethylammonium bromide to 200 milliliters of methanol and 450 milliliters of deionized water, in a one liter round bottom flask.
Reflux the reaction mixture at 102 degrees Celsius for 48 hours until the solution becomes colorless. After workup, suspend the isolated white powder in 400 milliliters of methanol in a round bottom flask. Using a filtering flask and borosilicate filter, filter the solution to remove the methanol insoluble inorganic byproducts.
Next, dissolve approximately 30 grams of sodium undec-10-enesulfonate in 500 milliliters of methanol, in a one-liter round bottom flask. Add 2.6 times excess of thioacetic acid to the solution, and stir it in front of a 250 watt UV lamp overnight. Once the reaction is complete, evaporate the methanol completely.
Dry the powder under vacuum, and then disperse it in diethyl ether. Filter the mixture and wash the solid product with diethyl ether to remove any excess thioacetic acid until no more colored substances appear in the diethyl ether supernatant. After drying the solid under high vacuum, dissolve it in methanol, yielding a yellow to orange solution.
Next, add three grams of carbon black to the solution and mix vigorously. Then, filter the mixture through Celite, covering 2/3 of a fluted filter paper. To synthesize MUS, combine approximately 400 milliliters of one molar hydrochloric acid and 35 grams of sodium 11-acetylthio-undecanesulfonate in a one liter round bottom flask.
Reflux the mixture at 102 degrees Celsius for 12 hours to cleave the thioacetate group and obtain a thiol. On the following day, transfer the product to a two liter round bottom flask. To keep the solution acidic and prevent crystallization of inorganic salts, add 200 milliliters of one molar sodium hydroxide, and 400 milliliters of deionized water to the flask to give a final volume of one liter.
Store the clear solution at four degrees Celsius overnight to crystallize the product as fine solids that are viscous when wet. On the following day, decant the clear supernatant. Then, transfer the product to 50 milliliter centrifuge tubes and centrifuge for five minutes at 4000 times g.
Following centrifugation, decant the supernatant into another round bottom flask. Transfer the white pellets to centrifuge tubes, and dry under high vacuum to obtain methanol-soluble MUS in about 30%yield. Weigh 177.2 milligrams of gold(III)chloride trihydrate in a small glass vial.
Following this, dissolve 87 milligrams of MUS in 10 milliliters of methanol, in a 20 milliliter glass vial. Sonicate the solution in an ultrasonic bath until no solid material is visible, to ensure complete dissolution. Add 26 microliters of 1-Octanethiol to the methanol solution, and agitate it to mix the ligands.
Add 500 milligrams of sodium borohydride to 100 milliliters of ethanol in a 250 milliliter round bottom flask. Stir vigorously until the solution is clear. Dissolve the gold salt in 100 milliliters of ethanol in a 500 milliliter round bottom flask and stir at 800 RPM until the gold salt dissolves completely.
Next, add the ligand solution to the reaction mixture. Wait 15 minutes for the formation of the gold-thiolate complex, which is indicated by a color change from translucent yellow to turbid yellow. Add the previously prepared sodium borohydride solution drop-wise to the reaction mixture, using a separatory funnel.
Adjust the interval time of the drops so that the addition of sodium borohydride takes about an hour. Once the sodium borohydride addition is complete, remove the addition funnel and allow the reaction mixture to stir for another hour. Then, remove the magnetic stir bar using a magnet placed on the outside of the round bottom flask.
Store the reaction mixture at four degrees Celsius overnight to precipitate the nanoparticles. After decanting the supernatant ethanol, transfer the remaining precipitant to 50 milliliter centrifuge tubes and centrifuge for three minutes at 4000 times g. After centrifugation, decant the supernatant.
Disperse the nanoparticles again with ethanol by vortexing. Then, centrifuge the samples again. Dry the nanoparticles under vacuum to remove the residual ethanol.
To clean the nanoparticles from free hydrophilic ligands, dissolve the precipitates in 15 milliliters of deionized water, and transfer the solutions to centrifuge tubes with filtration membranes of 30 kilodalton cutoff molecular weight. Concentrate the nanoparticle solutions by centrifugation for five minutes at 4000 times g. Following centrifugation, add 15 milliliters of deionized water, and centrifuge to concentrate again.
To turn the nanoparticles into a manageable powder, freeze-dry the remaining aqueous solution. To characterize the nanoparticles by ligand ratio, prepare a 150 milligram per milliliter methanol-d4 solution of iodine. Add 600 microliters of the solution to approximately five milligrams of nanoparticles in a glass vial to etch the nanoparticles.
Wrap the cap of the vial with paraffin film and sonicate it in an ultrasonic bath for 20 minutes. Then, transfer the solution to an NMR tube and acquire a proton NMR spectrum with 32 scans. The MUS synthesis is shown here.
The proton NMR spectra of the product of each step are represented here. The synthesis workflow of the binary MUS Octanethiol amphiphilic gold nanoparticles is described here. Prior to characterization, the cleanliness of the nanoparticles from unbound free ligands was monitored by proton NMR.
The size distribution of the nanoparticles was characterized by TEM, which shows that the average diameter is 2.4 nanometers pointing to approximately 18.08 nanometers squared of surface area, and 7.23 nanometers cubed of volume per particle. Localized surface plasma and resonance absorption was measured by acquiring UV-vis spectra. Representative proton NMR spectra with peak assignments and integration for determining the ligand ratio in the iodine-etched nanoparticles are shown here.
The NMR spectrum of the nanoparticles showed that the ratio of MUS to Octanethiol is 85 to 15. The surface coverage of the nanoparticles was examined by TGA. Based on TGA data, ligand density can be estimated to be 4.8 ligands per nanometer squared.
The stoichiometric ratios versus the NMR ratios of Octanethiol resulting from various syntheses are compared here. The most important things to remember in this procedure are to, on one side, the removal of inorganic impurities while preparing MUS ligands, and on the other side, the workup of the nanoparticles. This procedure is amenable to various combinations of ligands, but make sure to always characterize each batch individually.
One question this procedure can answer, for example, is to what extent the ligand ratio corresponds to the stoichiometry found on the surface of the nanoparticles. Scaling up the production of the ligand and proving the batch-to-batch reproducibility of the nanoparticles, has allowed us to tackle important questions related to biology and medicine. For example, we have established the virucidal properties of these nanoparticles.
Please, work carefully when using the UV lamp, and always wear protective gloves when handling liquid nitrogen. Always follow chemical safety rules.
