Gold Nanoparticle Synthesis

Summary

A protocol for synthesizing ~12 nm diameter gold nanoparticles (Au nanoparticles) in an organic solvent is presented. The gold nanoparticles are capped with oleylamine ligands to prevent agglomeration. The gold nanoparticles are soluble in organic solvents such as toluene.

Abstract

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of tetrachloroauric acid in 3.0 g (3.7 mmol, 3.6 mL) of oleylamine (technical grade) and 3.0 mL of toluene into a boiling solution of 5.1 g (6.4 mmol, 8.7 mL) of oleylamine in 147 mL of toluene. While boiling and mixing the reaction solution for 2 hours, the color of the reaction mixture changed from clear, to light yellow, to light pink, and then slowly to dark red. The heat was then turned off, and the solution was allowed to gradually cool down to room temperature for 1 hour. The gold nanoparticles were then collected and separated from the solution using a centrifuge and washed three times; by vortexing and dispersing the gold nanoparticles in 10 mL portions of toluene, and then precipitating the gold nanoparticles by adding 40 mL portions of methanol and spinning them in a centrifuge. The solution was then decanted to remove any remaining byproducts and unreacted starting materials. Drying the gold nanoparticles in a vacuum environment produced a solid black pellet; which could be stored for long periods of time (up to one year) for later use, and then redissolved in organic solvents such as toluene.

Introduction

Gold nanoparticles are an interesting and useful class of nanomaterials that are the subject of many research studies and applications; such as biology¹, medicine², nanotechnology³, and electronic devices⁴. Scientific research on gold nanoparticles dates back to as early as 1857, when Michael Faraday performed foundational studies on the synthesis and properties of gold nanoparticles⁵. The two primary "bottom up" techniques for synthesizing gold nanoparticles are the citrate reduction method^6,7,8 and the organic two-phase synthesis method^9,10. The "Turkevich" citrate reduction method produces fairly monodisperse gold nanoparticles under 20 nm in diameter, but the polydispersity increases for gold nanoparticles above 20 nm in diameter; whereas the "Brust-Schiffrin" two-phase method uses sulfur/thiol ligand-stabilization to produce gold nanoparticles up to ~10 nm in diameter¹¹. Gold nanoparticle solutions that are pre-synthesized using these methods are commercially available. For applications where large volumes, high monodispersity, and large diameters of gold nanoparticles are not necessary, it may be sufficient to purchase and use these pre-synthesized gold nanoparticles from suppliers. However, gold nanoparticles that are stored in solution, such as many of those that are commercially available, may degrade over time as nanoparticles begin to agglomerate and form clusters. Alternatively, for large-scale applications, long-term projects in which gold nanoparticles need to be used frequently or over a long period of time, or in which there are more stringent requirements for the monodispersity and size of the gold nanoparticles, it may be desirable to perform the gold nanoparticle synthesis oneself. By performing the gold nanoparticle synthesis process, one has the opportunity to potentially control various synthesis parameters such as the amount of gold nanoparticles that are produced, the diameter of the gold nanoparticles, the monodispersity of the gold nanoparticles, and the molecules used as the capping ligands. Furthermore, such gold nanoparticles can be stored as solid pellets in a dry environment, helping to preserve the gold nanoparticles so that they can be used at a later time, up to a year later, with minimal degradation in quality. There is also the potential for cost savings and the reduction of waste by fabricating gold nanoparticles in larger volumes and then storing them in a dry state so that they last longer. Overall, synthesizing gold nanoparticles oneself provides compelling advantages that may not be feasible with commercially available gold nanoparticles.

In order to realize the many advantages that are possible with gold nanoparticle synthesis, a process is presented herein for synthesizing gold nanoparticles. The gold nanoparticle synthesis process that is described is a modified version of a process that was developed by Hiramatsu and Osterloh¹². Gold nanoparticles are typically synthesized with a diameter of ~12 nm using this synthesis process. The primary chemical reagents that are used to perform the gold nanoparticle synthesis process are tetrachloroauric acid (HAuCl₄), oleylamine, and toluene. A nitrogen glovebox is used to provide an inert dry environment for the gold nanoparticle synthesis process, because tetrachloroauric acid is sensitive to water/humidity. The gold nanoparticles are encapsulated with oleylamine ligand molecules to prevent the gold nanoparticles from agglomerating in solution. At the end of the synthesis process, the gold nanoparticles are dried out in a vacuum environment so that they can be stored and preserved in a dry state for later use, up to one year later. When the gold nanoparticles are ready to be used, they can be resuspended into solution in organic solvents such as toluene.

Protocol

Chemical Amounts:
​NOTE: To obtain the appropriate chemical amounts for the nanoparticle synthesis, take the initial amounts found on the "Nanoparticle Synthesis" sheet (on the 2nd page of the supporting information from the Osterloh research article¹²), and multiply the amount of all doses by 3, with some slight modifications. Table 1 shows the chemical amounts that are needed for the injection solution, boiling solution, washing/purification solutions, and gold etchant solution.

Cleaning and Preparation for Gold Nanoparticle Synthesis Process (Day 1)
NOTE: The following steps can be completed on the first day of the synthesis process.

1. Things to Check and Ensure Before Preparing for the Gold Nanoparticle Synthesis

CAUTION: Ensure that the pre-synthesis cleaning and preparation are performed in the fume hood and acid wet bench while wearing personal protective equipment (PPE) such as nitrile gloves, safety glasses/goggles, and a lab coat while using the fume hood; and while additionally wearing chemical gloves, a chemical gown, a face shield, and goggles while using the acid wet bench.

1. Ensure that a nitrogen glove box is available, in which to perform the solvent/reagent preparations and synthesis/chemical reaction process.
   NOTE: If a nitrogen glove box is not available, a fume hood can be used instead (possibly with a Schlenk line), although the inert atmosphere in the nitrogen glove box should produce higher quality nanoparticles by preserving the purity of the tetrachloroauric acid (HAuCl₄). The gold nanoparticle injection solution that contains the tetrachloroauric acid should be prepared in an inert atmosphere or nitrogen glove box if possible.
2. Ensure that a stand with a clamp is located in the nitrogen glove box, to hold and support the condenser tube during the gold nanoparticle synthesis process.
   NOTE: This stand with clamp will also allow the condenser tube to be lifted up and suspended over the reaction vessel while the toluene, tetrachloroauric acid and oleylamine solution is injected into the reaction vessel.
3. Ensure that the heater with the magnetic stirrer and circular concave receptacle with a fiberglass lining (for holding and supporting the reaction vessel sphere, and for heating the reaction vessel and for rotating the magnetic stirrer bar) is located in the nitrogen glove box.
4. Ensure that there are two rubber hoses (for connecting the condenser tube to the water inlet/outlet ports) located inside the nitrogen glove box.
5. Ensure that a microbalance that is capable of milligram (mg) resolution is located in the nitrogen glove box.
6. Ensure that there are enough chemical reagents and solvents for the cleaning and synthesis process (see Table 1).
   NOTE: It is best to use fresh/new high-purity (≥99.8%) toluene and methanol which have never been opened or exposed to air/water. It is also best to use fresh/new tetrachloroauric acid (HAuCl₄) which is stored in the fridge and never opened until it is transferred to the nitrogen glove box. The tetrachloroauric acid should not be exposed to air or water/humidity at any time, should only be opened in the nitrogen glove box, and should be stored in the nitrogen glove box after opening it in the nitrogen glove box. It is preferable to use new oleylamine, and the oleylamine should also be stored in the nitrogen glove box. Tetrachloroauric acid and oleylamine that are brand new or less than 1 year old should produce better results.
7. Ensure that there are plastic bags, XL nitrile gloves, cleanroom wipes, and aluminum foil in the nitrogen glove box.

2. Clean the Chemical Reaction Glassware (Before Gold Nanoparticle Synthesis)

CAUTION: Gold etchant TFA and aqua regia are corrosive. Wear the necessary personal protective equipment (PPE) such as chemical gloves, chemical gown, goggles, and face shield. Only handle the corrosive solution in an acid wet bench while wearing the necessary PPE.

1. In the acid wet bench, place the glass reaction vessel with the condenser tube attached to it into a 600 mL beaker for support, and rest the side of the condenser tube against the sidewall of the acid wet bench for further support.
2. Clean the chemical reaction glassware (condenser tube, reaction vessel, glass pipette) and magnetic stir bar by pouring ~150 mL of the gold etchant TFA solution and ~150 mL of DI water (1:1 mixture) into the condenser tube and reaction vessel glassware. Place the magnetic stir bar and long glass graduated pipette into the condenser tube and allow the gold etchant TFA bath to sit and clean the glassware for 30 minutes.
   NOTE: Supplementary Figure 1 shows the chemical reaction glassware being cleaned with gold etchant.
3. After 30 minutes, separate the glassware to crack the seal between the condenser tube and the reaction vessel to collect all the gold etchant solution into the reaction vessel, and pour the used gold etchant solution into a 400 mL beaker in the acid wet bench.
   NOTE: The gold etchant solution will be reused later to clean the chemical reaction glassware after the synthesis process is over.
4. Still in the acid wet bench, wash the chemical reaction glassware and magnetic stir bar 3-4 times with DI water to flush out the remaining gold etchant solution, and then allow the chemical reaction glassware and magnetic stir bar to sit in a DI water bath for an additional 30 minutes.
5. After 30 minutes of sitting in a DI water bath, empty out the water and use the DI water gun to wash the water down the acid wet bench drain. Blow the glassware dry with the nitrogen gun.
6. In the fume hood, clean the chemical reaction glassware (condenser tube, reaction vessel, glass pipette) and magnetic stir bar by rinsing with acetone, methanol, and isopropanol; then blow dry the glassware with nitrogen. Discard the dirty solvents into a flammable waste bottle.
7. In the acid wet bench, clean the chemical reaction glassware and magnetic stir bar with DI water, then blow dry the glassware with nitrogen.
8. In the fume hood, clean the chemical reaction glassware and magnetic stir bar with toluene, then blow dry the glassware with nitrogen. Discard the dirty toluene solution into a flammable waste bottle.
9. Cover the chemical reaction glassware (condenser tube, reaction vessel, glass pipette) and magnetic stir bar with aluminum foil (especially the openings/ports of the glassware) to keep the glassware clean. Poke a couple small holes into the aluminum foil with tweezers, to allow for water to evaporate from the glassware.

3. Clean the Other Glassware and Synthesis Supplies

1. In the fume hood, clean the other glassware (e.g., 400 mL glass beaker, 5 mL small graduated glass cylinder, two non-aqueous 20 mL glass vials with PTFE-lined caps), and supplies (e.g., metal spatula/scoopula, tweezers) with acetone, methanol or isopropanol, and DI water; then blow dry the other glassware and supplies with nitrogen. Discard the dirty solvents into a flammable waste bottle.
2. If there is any residue visible on the glassware or supplies, wipe them down with a cleanroom wipe or wash with soap and acetone/isopropanol until the residue disappears. Then rinse them with acetone, methanol, and isopropanol solvents again, and then blow the glassware dry with nitrogen.
3. In the fume hood, clean the other glassware and supplies with toluene; then blow dry the other glassware and supplies with nitrogen. Discard the dirty toluene solution into a flammable waste bottle.
4. In the fume hood, clean the 50 mL conical centrifuge tubes with acetone, methanol or isopropanol, and toluene; then blow dry them with nitrogen.
5. Cover the other glassware and supplies with aluminum foil, especially the openings/ports of the glassware, to keep the glassware clean. Poke a couple small holes into the aluminum foil with tweezers, to allow for water to evaporate from the glassware. Ensure that the caps are on the 50 mL centrifuge tubes.
6. Clean the rubber pipette bulb with valves by wiping it with a cleanroom wipe with isopropanol, then use the valves to suck up some isopropanol (e.g., while squirting some into it from an isopropanol squeeze bottle) into the bulb and squirt out the isopropanol into a flammable waste bottle. Ensure that there is no residue on the bulb. Blow dry the bulb with nitrogen and cover it with aluminum foil.
   ​NOTE: Supplementary Figure 2 shows the glassware and supplies after being cleaned.

4. Transfer the Chemicals, Glassware and Supplies into the Nitrogen Glove Box

1. Use a fresh pair of XL nitrile gloves over the glove box gloves for handling items and chemicals inside the nitrogen glove box.
2. Put the new chemical bottles (toluene and methanol) into the nitrogen glove box (by transferring them into the loadlock and pumping down to remove the ambient air with the vacuum pump, then purging the loadlock with nitrogen). Ensure that there is also a flammable waste bottle for used/dirty toluene in the nitrogen glove box.
3. Ensure that the tetrachloroauric acid (HAuCl₄) and the oleylamine are also in the nitrogen glove box, where they are stored to prevent exposure to oxygen and water/humidity.
4. Place the chemical reaction glassware (condenser tube, reaction vessel, glass pipette), magnetic stir bar, 50 mL conical centrifuge tubes, and other glassware (e.g., 400 mL glass beaker, 5 mL small graduated glass cylinder, two non-aqueous 20 mL glass vials with PTFE-lined caps) and other supplies (e.g., micropipette, new clean micropipette tips in a plastic bag, metal spatula/scoopula, tweezers, valved pipette bulb) in the glove box loadlock. Close the loadlock door, pump down the loadlock to vacuum, leave them under vacuum for 2 minutes, purge the loadlock with nitrogen, and then transfer/place the items inside the nitrogen glove box.
   NOTE: Any residual water and solvents should have evaporated in the loadlock while pumping it down to vacuum, before purging the loadlock with nitrogen.
5. After transferring the items inside the nitrogen glove box, use another layer of aluminum foil to cover up the items (especially the glassware) that are covered with aluminum foil with holes in the foil, to cover the holes and prevent the items from getting dirty inside the nitrogen glove box.
6. Leave the clean items in the nitrogen glove box overnight, with the nitrogen circulating, to remove and filter out any residual water/moisture/humidity from within the nitrogen glove box.

Gold Nanoparticle Synthesis Process (Day 2)
​NOTE: The following steps can be completed on the second day of the synthesis process.

5. Set Up and Clean the Chemical Reaction Glassware & Supplies in the Nitrogen Glove Box

1. Begin setting up and cleaning the chemical reaction glassware and supplies in the nitrogen glove box. Inside the nitrogen glove box, place the glass reaction vessel on top of the fiberglass mesh receptacle on top of the heater/stirrer, and place the condenser tube over the glass reaction vessel, supporting the condenser tube with the stand with the clamps.
   NOTE: Supplementary Figure 3 shows the gold nanoparticle synthesis experimental setup.
2. Ensure that the magnetic stir bar is inside the glass reaction vessel. Pour ~200 mL of toluene into the glass reaction vessel. Place the glass reaction vessel with ~200 mL of toluene onto the stirring heating mantle and lower the glass condenser tube into the reaction vessel.
3. Connect the two hoses inside the nitrogen glove box to the water inlet and outlet ports of the condenser tube.
4. Outside the nitrogen glove box, place the end of the water outlet drainage hose into the drainage reservoir/sink in the adjacent fume hood. Use a clamp or tape to hold the hose and keep the hose oriented down into the drain.
5. Connect the water supply inlet hose to the water supply line on the adjacent fume hood.
6. Slowly turn on and monitor the water to ensure it is gently flowing up through the outer chamber of the condenser tube. Adjust the water flow as necessary by slightly opening/closing the water valve.
7. Allow water to flow through the inlet port on the bottom of the condenser tube, up the condenser tube, and out the outlet port on the top of the condenser tube.
8. Ensure that there are no large air bubbles in the water supply and ensure that the hoses are mechanically stable.
   NOTE: When boiling solutions in the chemical reaction vessel, slowly flow some water from the bottom of the condenser tube, up through the condenser tube vessel outer chamber, to the top of the condenser tube so that water slowly drains out through the drainage hose. This slow but continuous water flow will cool the condenser tube and assist with condensing and recollecting the boiled vapor.
9. Ensure that water is gently flowing through the condenser tube to cool it.
10. Continuously flow fresh nitrogen into the nitrogen glove box to purge the glove box. Continuously ventilate the nitrogen glove box by pulling a slight vacuum on the nitrogen glove box so that nitrogen and toluene vapor is pumped out of the glove box.
    NOTE: Pull a slight vacuum on the nitrogen glove box by slightly opening the equalization valve between the nitrogen glove box and the loadlock while pulling vacuum on the loadlock. Do not fully open the equalization valve or the vacuum level and nitrogen flow will be too high. Flow just enough nitrogen to continuously flush out and ventilate the toluene/chemical vapor in the glove box over time. The vacuum exhaust line should be vented into a fume hood.
11. Start heating and stirring the toluene with the magnetic stirrer on the stirring and heating mantle. Allow the toluene to approach a gentle boil. Do not approach or exceed the flash point temperature of toluene; reduce the heat when it starts to boil.
12. Allow the toluene to boil and evaporate for 30 minutes with the magnetic stir bar stirring to clean the reaction glassware (reaction vessel and condenser tube).
    ​NOTE: The evaporated toluene will cool and condense in the condenser tube, and drip back down into the reaction vessel.
13. After 30 minutes, turn off the heater and magnetic stirrer, and allow the toluene to cool down for several minutes, until the toluene stops evaporating and condensing inside the reaction vessel.
14. After the toluene cools down, carefully lift up the condenser tube and suspend it above the reaction vessel by supporting it using the stand with clamps. Make sure to tighten the clamp and support the condenser tube properly, as it could be unstable.
15. Pour the toluene from the reaction vessel into the 400 mL glass beaker. Be careful to not accidentally pour out the magnetic stir bar. Place the reaction vessel back on the heating and stirring mantle.
16. Swirl the toluene around in the 400 mL glass beaker to clean the beaker. Pour out and discard the dirty/used toluene into the flammable waste bottle. Clean the 400 mL glass beaker again with some fresh toluene, and then discard the used toluene into the flammable waste bottle.

6. Toluene & Oleylamine Boiling Solution Preparation

CAUTION: Oleylamine is toxic and corrosive, so handle it carefully. If handling oleylamine outside the nitrogen glove box, wear the necessary personal protective equipment (PPE) such as chemical gloves, chemical gown, goggles, and face shield. If handling oleylamine inside the nitrogen glove box, make sure to cover the glove box gloves with new/clean XL nitrile gloves. Be careful to not accidentally spill the oleylamine. Some cleanroom wipes can be put down on the lab bench surface inside the glove box to help absorb any small spills.

1. Inside the nitrogen glove box, make a boiling solution of 147 mL (~150 mL) of toluene and 8.7 mL (~9 mL) of oleylamine in the reaction vessel.
   1. Use the 400 mL glass beaker to measure the 147 mL (~150 mL) of toluene. Pour the 147 mL (~150 mL) of toluene from the glass beaker into the reaction vessel.
   2. Use the 5 mL small glass graduated cylinder to carefully measure the 8.7 mL (~9 mL) of oleylamine. First carefully measure and pour 4 mL, and then 4.7 mL, of oleylamine from the small glass graduated cylinder into the reaction vessel.
2. Carefully lower the condenser tube down into the glass reaction vessel again.
3. Ensure that water is gently flowing through the outer chamber of the condenser tube to cool, condense, and collect the toluene and oleylamine vapor.
4. Heat and stir the oleylamine and toluene solution in the reaction vessel and allow the solution to approach a slow/gentle boil (using the stirring and heating mantle, with the magnetic stir bar rotating to mix the solution). Once the oleylamine and toluene solution reaches a gentle boil, turn the heat down a little bit so it is boiling slowly. Do not approach or exceed the flash point of toluene.

7. Tetrachloroauric Acid, Oleylamine & Toluene Injection Solution Preparation

1. Begin preparing the injection solution (150 mg tetrachloroauric acid, 3.6 mL oleylamine, 3.0 mL toluene).
2. Ensure that the tetrachloroauric acid is fresh or hasn't been exposed to air, water, moisture, or humidity. Remove the laboratory film or seal that is protecting the tetrachloroauric acid from air and moisture.
   NOTE: The tetrachloroauric acid is very sensitive to water/moisture/humidity. Every effort should be made to prevent exposing the tetrachloroauric acid powder to air/water. The tetrachloroauric acid comes in a sealed pouch and new container vessels are sealed with wax to prevent water vapor from getting into new vessels. A new batch of tetrachloroauric acid costs ~$100, but it should last a year if not exposed to water vapor. Store new unopened batches of tetrachloroauric acid in the fridge. Transfer a new unopened batch of tetrachloroauric acid to the nitrogen glove box prior to opening it. Only open a new container of tetrachloroauric acid in the nitrogen glove box, when the humidity has reached an appropriately low and stable level (less than 0.8% relative humidity). Store the tetrachloroauric acid in the nitrogen glove box after opening it. After opening the tetrachloroauric acid, wrap laboratory film around the lid of the container to help with sealing the container and to prevent water vapor and contaminants from getting into the container.
3. In the nitrogen glove box, place one of the two non-aqueous 20 mL glass vials with the PTFE-lined caps on the microbalance/scale and remove the PTFE-lined cap.
4. Make sure to "re-zero" or "tare" the microbalance with the 20 mL glass vial on the scale before beginning to weigh out the tetrachloroauric acid powder.
5. In the nitrogen glove box, use the small metal spatula to deposit tetrachloroauric acid powder from the container into the 20 mL glass vial on the microbalance, to a measured weight of 150 mg of tetrachloroauric acid powder.
6. Remove the PTFE-lined cap from the other non-aqueous 20 mL glass vial (the empty one that is not currently on the microbalance).
   CAUTION: Oleylamine is toxic and corrosive, so handle it carefully.
7. Use the 5 mL small glass graduated cylinder to measure 3.6 mL of oleylamine. Carefully pour the 3.6 mL of oleylamine from the 5 mL small glass graduated cylinder into the 20 mL glass vial without the tetrachloroauric acid.
8. Carefully pour and measure 3.0 mL of toluene into the 5 mL small glass graduated cylinder. Carefully pour the 3.0 mL of toluene from the 5 mL small glass graduated cylinder into the 20 mL glass vial with the oleylamine.
   NOTE: If too much toluene is poured into the graduated glass cylinder, the excess solvent can be poured into the flammable waste bottle. It is best to use the small 5 mL graduated glass cylinder for measuring the oleylamine and toluene. Be careful to not spill the oleylamine, as it is corrosive and toxic.
9. Screw the PTFE-lined cap back onto the 20 mL glass vial with the oleylamine and toluene inside. Shake and swirl the closed glass vial to mix the oleylamine and toluene solution together.
10. Open the 20 mL solution glass vial. Carefully pour the ~150 mg of tetrachloroauric acid powder into the glass vial with the oleylamine and toluene solution.
11. Screw the PTFE-lined caps back onto the glass vials. Shake and swirl the closed glass vial with the tetrachloroauric acid, oleylamine and toluene to mix the solution together. Keep shaking the solution, and ensure that it is mixed thoroughly.
    NOTE: The tetrachloroauric acid, oleylamine and toluene injection solution should turn dark red or purple after shaking and mixing it, as shown in Supplementary Figure 4.

8. Injection of the Tetrachloroauric Acid, Oleylamine & Toluene Solution into the Vessel

1. Ensure that water is slowly flowing into the bottom of the condenser tube, and up out the top of the condenser tube. Adjust the water flow as necessary by carefully opening/closing the water valve.
2. Ensure that the oleylamine and toluene solution in the glass reaction vessel is at a gentle boil, with some toluene and oleylamine evaporating into the condenser tube. Ensure that the magnetic stirrer is on.
3. Raise the condenser tube above the reaction vessel, using the stand with clamps to support the glassware. Ensure that there is enough room and clearance to inject the tetrachloroauric acid, oleylamine, and toluene solution into the reaction vessel.
4. Remove the long graduated glass pipette from the aluminum foil (which was protecting the pipette to keep it clean) and attach the rubber bulb with valves to the pipette. Ensure familiarity with operating the rubber bulb with valves to suck up and squirt out a solution with the long graduated glass pipette before using it.
5. Shake the closed 20 mL non-aqueous glass vial with the PTFE-lined cap with the tetrachloroauric acid, oleylamine, and toluene injection solution and ensure it is well-mixed. Open the 20 mL glass vial with the injection solution by removing the cap.
6. Press on the upper valve while squeezing the rubber bulb to deflate the rubber bulb. Carefully place the tip of the long graduated glass pipette into the 20 mL glass vial with the tetrachloroauric acid, oleylamine, and toluene injection solution.
7. Gently press the lower valve on the rubber bulb connected to the long graduated glass pipette to slowly draw up all of the tetrachloroauric acid, oleylamine, and toluene injection solution into the glass pipette.
   NOTE: Supplementary Figure 5 shows the injection solution being drawn into the long graduated glass pipette with the rubber bulb with valves just before injecting the solution into the reaction vessel. It may be beneficial to practice operating the long graduated glass pipette with the bulb with valves (e.g., with some toluene) before actually drawing up and injecting the tetrachloroauric acid, oleylamine, and toluene solution.
8. Carefully place the tip of the glass pipette into the opening of the reaction vessel, and quickly inject the tetrachloroauric acid, oleylamine, and toluene injection solution into the boiling solution of oleylamine and toluene in the reaction vessel.
   NOTE: The solution color should initially change from red to yellow to white within about a minute, as gold nanoparticles begin to nucleate and grow.
9. Use the clamp on the stand to lower the condenser tube back down into the reaction vessel.
10. Heat the gold nanoparticle chemical reaction solution at a gentle boil for 2 hours.
    NOTE: The toluene vapor from the boiling solution should condense in the tube and drip back down into the reaction vessel. Over several minutes, the color of the reaction mixture should then change from white to yellow to light pink and then to red as the gold nanoparticles grow larger. Over the course of 1-2 hours, the color of the reaction mixture should gradually change from light red to deep red/purple.
11. After 2 hours of heating the reaction solution, turn off the heater.
    NOTE: At this point, the solution can either be allowed to cool down naturally to room temperature, or the solution can be immediately quenched by adding ~100 mL of methanol into the solution. The best-known practice as of now is to allow the solution to cool down naturally rather than quenching the solution right away.
12. Allow the solution to cool down naturally to room temperature for 1 hour (recommended); or quench the gold nanoparticle solution immediately with 100 mL of methanol (not recommended).

9. Quenching the Reaction with Methanol After Cooling the Gold Nanoparticle Solution

1. Ensure that the heater has been turned off, and the solution has cooled down.
2. Stop flowing water through the condenser tube. Carefully remove the water drainage hose from the sink/drain in the adjacent fume hood and connect it to the vacuum port in the fume hood.
3. Pull vacuum on the drainage hose to suck away the water in the condenser tube and the drainage hose. Carefully remove the condenser tube from the stand with the clamp and lay it horizontally in the nitrogen glove box.
   NOTE: The vacuum that is being pulled on the glass condenser tube should evaporate the water within the condenser tube.
4. In the nitrogen glove box, pour ~35 mL of methanol into each of the 50 mL conical centrifuge tubes (quantity 12).
   NOTE: Methanol will be used to remove unreacted reagents and byproducts from the synthesis process, in order to clean and wash the gold nanoparticles. The 50 mL centrifuge tubes should be held upright in test tube racks.
5. Pour the gold nanoparticle solution in equal volumes (~12 mL) into each of the 50 mL centrifuge tubes (quantity 12) with methanol. Be careful to not accidentally pour out the magnetic stir bar while pouring the gold nanoparticle solution into each centrifuge tube.
   NOTE: Supplementary Figure 6 shows ~12 mL of gold nanoparticle solution being poured into each of the 50 mL conical centrifuge tubes with methanol. After pouring ~12 mL of gold nanoparticle solution into each of the 50 mL conical centrifuge tubes with ~35 mL of methanol, each centrifuge tube should have ~47 mL of solution (slightly below the 50 mL mark).
6. Distribute any remaining gold nanoparticle solution evenly between the centrifuge tubes.
7. Screw the caps onto the 50 mL centrifuge tubes to close them and tighten the caps.
8. Disconnect the inlet and outlet hoses from the glass condenser tube, connect the inlet and outlet hoses together by feeding one into the other, and then wrap the connection of the tubes with laboratory film to seal the connection. Turn off the vacuum that is being pulled on the hoses.
   NOTE: The tubes are connected and sealed to prevent water or water vapor from accidentally getting into the nitrogen glove box.
9. Remove the 50 mL conical centrifuge tubes with the gold nanoparticle solution and methanol from the nitrogen glove box through the load lock. Also remove the methanol bottle and toluene bottle from the nitrogen glove box. Place them in the adjacent fume hood.
10. Also remove the glass reaction vessel, the magnetic stir bar, the glass condenser tube, the long glass graduated pipette, and the rubber bulb with valves from the nitrogen glove box through the load lock. Place them in the adjacent fume hood.
11. Label the top of each 50 mL centrifuge tube on the caps with a sample number (e.g., 1, 2, 3, 4, …) to keep track of the different samples.
    NOTE: After removing the gold nanoparticle solution and glassware/supplies, the nitrogen glove box should continue to be ventilated for several hours or overnight by flowing fresh nitrogen into the glovebox while pulling a slight vacuum to flush out and ventilate the toluene/oleylamine vapor. The vacuum exhaust line should be vented into a fume hood. The nitrogen glovebox should also be regenerated with regeneration gas to remove moisture/solvents from the filtration system. Some nitrogen gloveboxes may also come with a solvent trap, which helps with removing solvent vapors.

10. Washing and Purifying the Gold Nanoparticles with Toluene and Methanol

NOTE: Each 50 mL centrifuge tube with gold nanoparticles will be washed and purified with 10 mL of toluene and 40 mL of methanol 3 times, cleaning the gold nanoparticles in batches of 6 centrifuge tubes at a time. The centrifuge tubes should have an equal amount of gold nanoparticle solution and should be equally weighted and balanced.

1. Place 6 of the 50 mL centrifuge tubes with gold nanoparticle solution into the centrifuge.
2. Close the lid of the centrifuge, and enter the following settings for spinning the gold nanoparticles:
   RPM: 2328
   RCF: 1000
   Time: 5 minutes
3. Start spinning 6 of the 12 conical centrifuge tubes with the gold nanoparticle solution and methanol in the centrifuge.
4. After the first 6 centrifuge tubes with gold nanoparticles are done spinning, gently remove the tubes from the centrifuge. Be careful to not disturb the gold nanoparticle pellets while placing the centrifuge tubes in the tube racks.
   NOTE: Supplementary Figure 7 shows how the gold nanoparticle solution should appear in the 50 mL conical centrifuge tubes after centrifugation. The centrifugal force will pull down the gold nanoparticles in solution and separate them from the methanol and toluene. The gold nanoparticles will precipitate into pellets at the bottom of each centrifuge tube. The supernatant methanol/toluene solution will appear to be clear/transparent above the dark gold nanoparticle pellets, indicating that centrifugation has precipitated the gold nanoparticles from solution.
5. Place the last 6 of the 12 conical centrifuge tubes with the gold nanoparticle solution and methanol into the centrifuge. Close the lid of the centrifuge and enter the same centrifuge settings as before. Start spinning the tubes in the centrifuge.
6. After the last 6 centrifuge tubes are done spinning, gently remove the tubes from the centrifuge. Be careful to not disturb the gold nanoparticle pellets while placing the centrifuge tubes in the tube racks.
7. Carefully carry all of the centrifuge tubes with the gold nanoparticles over to the fume hood and try not to disturb or agitate them during transport.
8. Slowly and gently pour out the waste methanol into a flammable waste vessel/beaker. Be careful to not disturb and to not pour out or lose the black gold nanoparticle pellets at the bottom of the centrifuge tubes.
   NOTE: The first methanol rinse cycle is now complete.
9. Begin the second methanol rinse cycle by pouring ~10 mL of fresh toluene into each of the conical centrifuge tubes with black nanoparticle pellets in the fume hood. Screw the caps back on to close the 50 mL centrifuge tubes.
10. Vortex each of the 50 mL centrifuge tubes until the black liquid/precipitate/gold nanoparticles are resuspended and dispersed in the 10 mL toluene solution, and the solution looks cloudy/dark. Check the bottom of each centrifuge tube to ensure that most of the black residue (gold nanoparticles) has been resuspended into solution.
    NOTE: Supplementary Figure 8 shows the centrifuge tubes with gold nanoparticle solution and toluene being vortexed and resuspended. Vortexing is much better and gentler on the gold nanoparticles than sonicating the gold nanoparticles. Do not sonicate the gold nanoparticles as sonication could strip off the oleylamine ligands from the gold nanoparticles and cause aggregation and sedimentation of the gold nanoparticles.
    NOTE: Supplementary Figure 9 shows how the gold nanoparticle solution should appear when the gold nanoparticles are resuspended into solution by vortexing each gold nanoparticle pellet with ~10 mL of toluene.
11. Pour ~40 mL of fresh methanol into each of the conical centrifuge tubes with toluene and nanoparticles, so that with the 10 mL of toluene that is already in each centrifuge tube, there is a total of ~50 mL of solution in each 50 mL centrifuge tube. Screw the caps back onto the 50 mL centrifuge tubes to close the tubes, and ensure that the caps are on tight.
12. Place the centrifuge tubes into the centrifuge. Spin the centrifuge tubes in the centrifuge to collect the gold nanoparticles into a pellet at the bottom of each tube, 6 centrifuge tubes at a time. Use the same centrifuge settings as before (RCF 1000, 5 minutes).
13. After the centrifuge stops, gently take out the tubes with the nanoparticles, and then carefully take them to the fume hood (try not to disturb or agitate them during transport). Carefully pour out the waste toluene and methanol into the flammable waste vessel/beaker.
    NOTE: The second methanol rinse cycle is now complete.
14. For the third and final rinse cycle, follow the same process as before for vortexing in 10 mL of toluene, cleaning in 40 mL of methanol, centrifugation, and carefully pouring out the toluene/methanol solvent. Ensure that the gold nanoparticles in each of the 50 mL centrifuge tubes get resuspended with toluene and washed with methanol 3 times.

11. Drying the Gold Nanoparticles

NOTE: After the gold nanoparticles in the 50 mL centrifuge tubes have been washed 3 separate times, and the toluene and methanol has been poured out for the last time, the gold nanoparticles need to be dried to evaporate the remaining solvent. There are two ways to dry the gold nanoparticles and evaporate the solvent:

1. Option 1 - Nitrogen Gun (not recommended):
   1. Use a nitrogen gun or valve in the fume hood to gently blow dry the centrifuge tubes containing the black pellets of gold nanoparticles at the bottom of the tubes.
   2. Take care to not use too much nitrogen pressure, or the fragile gold nanoparticle pellets may get dislodged.
      NOTE: Drying the gold nanoparticles with the nitrogen gun is not ideal because it could cause the gold nanoparticle pellets to get damaged/lost.
2. Option 2 - Vacuum Drying (recommended):
   1. Loosen the caps on the 50 mL centrifuge tubes with gold nanoparticle pellets so that the tubes are still covered, but solvent can evaporate and escape from inside the tubes.
   2. Place the rack of tubes with gold nanoparticles inside the vacuum load lock of the nitrogen glove box. Close and seal the outer load lock door and open the valve to the vacuum pump to start pulling vacuum on the load lock.
   3. Pump down to about half the gauge pressure (~-15 inHg) to evaporate the solvent and dry the nanoparticles.
   4. Leave the gold nanoparticles in the load lock at a moderate vacuum pressure (half-gauge, ~-15 inHg) for ~5 minutes. Do not pump down to a lower pressure and do not leave in vacuum for too long, or the oleylamine ligands may get detached.
   5. After the gold nanoparticles have been under vacuum for a few minutes to dry the gold nanoparticles and evaporate the remaining solvent, purge the load lock with nitrogen until the load lock reaches atmospheric pressure.
   6. Remove the 50 mL centrifuge tubes with gold nanoparticles from the load lock and inspect the dryness of the gold nanoparticle pellets in the fume hood.
      NOTE: Supplementary Figure 10 shows how a dried gold nanoparticle pellet at the bottom of a 50 mL conical centrifuge tube should look after vacuum drying it. If there is still some solvent inside the 50 mL conical centrifuge tube, the gold nanoparticles need to be dried further to evaporate the remaining solvent. Vacuum drying is the preferred method for drying because it is less likely to damage or lose the gold nanoparticle pellet, compared to more aggressive methods such as nitrogen gun drying. If a vacuum load lock is not available, or if preferred, the gold nanoparticles may also be dried in a vacuum desiccator.
3. After the gold nanoparticle pellets are dry, screw the caps tightly back onto the centrifuge tubes.
4. Wrap laboratory film around the tightly closed caps to seal the centrifuge tubes with the gold nanoparticle pellets inside.
5. Label the 50 mL centrifuge tubes with gold nanoparticle precipitate pellets with an appropriately descriptive label, such as "Dried Au NP" and the date (e.g., 9-28-2020).
6. Place the sealed centrifuge tubes with dried gold nanoparticle pellets inside a 2 °C - 8 °C fridge. Use a tray or 50 mL conical centrifuge tube racks to hold the tubes upright.
   NOTE: Supplementary Figure 11 shows the centrifuge tubes capped, wrapped with laboratory film, labeled, and stored in a 2 °C - 8 °C fridge. Each centrifuge tube can be stored in the fridge until it is used to make a solution of resuspended gold nanoparticles.

12. Clean the Chemical Reaction Glassware (After Gold Nanoparticle Synthesis)

CAUTION: Gold etchant TFA and aqua regia are corrosive. Wear the necessary personal protective equipment (PPE) such as chemical gloves, chemical gown, goggles, and face shield. Only handle the corrosive solution in an acid wet bench while wearing the necessary PPE.

1. In the fume hood, clean the glass reaction vessel with acetone and swirl the acetone around in the glass reaction vessel to wash away the residual gold nanoparticle solution, then dump the dirty acetone into a dirty solvent collection beaker and discard the dirty solvent into a flammable waste bottle.
2. In the acid wet bench, place the glass reaction vessel with the condenser tube attached to it into a 600 mL beaker for support, and rest the side of the condenser tube against the sidewall of the acid wet bench for further support.
3. Clean the chemical reaction glassware (condenser tube, reaction vessel, glass pipette) and magnetic stir bar by pouring the used ~300 mL gold etchant TFA solution (which was saved earlier and set aside for reuse) that was mixed 1:1 with DI water into the condenser tube and reaction vessel glassware. Place the magnetic stir bar and long glass graduated pipette into the condenser tube. Fill up the condenser tube with DI water as necessary to top it off and allow the gold etchant TFA bath to sit and clean the glassware for 30 minutes.
4. After 30 minutes, crack the seal between the condenser tube and the reaction vessel to collect all the gold etchant solution into the reaction vessel, and pour the used gold etchant solution into the 400 mL beaker. Pour the gold etchant solution into the chemical waste bottle for used gold etchant solution.
5. Still in the acid wet bench, wash the chemical reaction glassware and magnetic stir bar 3-4 times with DI water to flush out the remaining gold etchant solution, and then allow the chemical reaction glassware and magnetic stir bar to sit in a DI water bath for an additional 30 minutes.
6. After 30 minutes of sitting in a DI water bath, empty out the water and use the DI water gun to wash the water down the acid wet bench drain. Rinse with acetone and then blow the glassware dry with the nitrogen gun.

Results

Figure 1 shows how the gold nanoparticle synthesis chemical reaction mixture solution (tetrachloroauric acid, oleylamine, and toluene) should gradually change color over the course of several minutes as it initially boils in the reaction vessel; from clear, to light yellow (left image), to light pink (center image), to light red (right image). The changing color of the solution is an indication of the changing size of the gold nanoparticles as they begin to nucleate and grow larger over...

Discussion

Performing the gold nanoparticle synthesis protocol as presented above should produce gold nanoparticles with ~12 nm diameter and fairly high monodispersity (± 2 nm). However, there are some critical steps and process parameters that can be adjusted to potentially change the size/diameter and monodispersity/polydispersity of the gold nanoparticles. For example, after injecting the precursor solution into the reaction vessel and allowing the tetrachloroauric acid, oleylamine, and toluene solution to boil for two hour...

Materials

Name	Company	Catalog Number	Comments
50 mL Conical Centrifuge Tubes with Plastic Caps (Quantity: 12)	Ted Pella, Inc.	12942	used for cleaning/storing gold nanoparticle solution/precipitate (it's best to use 12 tubes, to allow the gold nanoparticles from the synthesis process to last up to one year (e.g., 1 tube per month))
Acetone	Sigma-Aldrich	270725-2L	solvent for cleaning glassware/tubes
Acid Wet Bench	N/A	N/A	for cleaning chemical reaction glassware/supplies with gold etchant solution (part of wet chemical lab facilities)
Aluminum Foil	Reynolds	B08K3S7NG1	for covering glassware after cleaning it to keep it clean
Burette Clamps	Fisher Scientific	05-769-20	for holding the condenser tube and reaction vessel during the synthesis process (located in the nitrogen glove box)
Centrifuge (with 50 mL Conical Centrifuge Tube Rotor/Adapter)	ELMI	CM-7S	for spinning the gold nanoparticles in solution and precipitating/collecting them at the bottom of the 50 mL conical centrifuge tubes
DI Water	Millipore	Milli-Q Direct	deionized water
Fume Hood	N/A	N/A	for cleaning laboratory glassware and supplies with solvents (part of wet chemical lab facilities)
Glass Beaker (600 mL)	Ted Pella, Inc.	17327	for holding reaction vessel, condenser tube, glass pipette, and magnetic stir bar during cleaning with gold etchant and then with water
Glass Beakers (400 mL) (Quantity: 2)	Ted Pella, Inc.	17309	for measuring toluene and gold etchant
Glass Graduated Cylinder (5 mL)	Fisher Scientific	08-550A	for measuring toluene and oleylamine for injection
Glass Graduated Pipette (10 mL)	Fisher Scientific	13-690-126	used with the rubber bulb with valves to inject the gold nanoparticle precursor solution into the reaction vessel
Gold Etchant TFA	Sigma-Aldrich	651818-500ML	(with potassium iodide) for cleaning reaction vessel, condenser tube, magnetic stir bar, glass pipette [alternatively, use Aqua Regia]
Isopropanol	Sigma-Aldrich	34863-2L	solvent for cleaning glassware/tubes
Liebig Condenser Tube (~500 mm) (24/40)	Fisher Scientific	07-721C	condenser tube, attaches to glass reaction vessel
Magnetic Stirring Bar	Fisher Scientific	14-513-51	for stirring reaction solution during the synthesis process
Methanol (≥99.9%)	Sigma-Aldrich	34860-2L-R	new, ≥99.9% purity (for washing gold nanoparticles after synthesis)
Microbalance (mg resolution)	Accuris Instruments	W3200-120	for weighing tetrachloroauric acid powder (located in the nitrogen glove box)
Micropipette (1000 µL)	Fisher Scientific	FBE01000	for measuring and dispensing liquid chemicals such as oleylamine and toluene (if using micropipette instead of graduated cylinder for measurement)
Micropipette Tips (1000 µL)	USA Scientific	1111-2831	for measuring and dispensing liquid chemicals such as oleylamine and toluene (if using micropipette instead of graduated cylinder for measurement)
Nitrile Gloves	Ted Pella, Inc.	81853	personal protective equipment (PPE), for protection, and for keeping nitrogren glove box gloves clean
Nitrogen Glove Box	M. Braun	LABstar pro	for performing gold nanoparticle synthesis in a dry and inert environment
Non-Aqueous 20 mL Glass Vials with PTFE-Lined Caps (Quantity: 2)	Fisher Scientific	03-375-25	for weighing tetrachloroauric acid powder and mixing with oleylamine and toluene to make injection solution
Oleylamine (Technical Grade, 70%)	Sigma-Aldrich	O7805-100G	technical grade, 70%, preferably new, stored in the nitrogen glove box
Parafilm M Sealing Film (2 in. x 250 ft)	Sigma-Aldrich	P7543	for sealing the gold nanoparticles in the 50 mL centrifuge tubes after the synthesis process is over
Round Bottom Flask (250 mL) (24/40)	Wilmad-LabGlass	LG-7291-234	glass reaction vessel, attaches to condenser tube
Rubber Bulb with Valves (Rubber Bulb-Type Safety Pipet Filler)	Fisher Scientific	13-681-50	used with the long graduated glass pipette to inject the gold nanoparticle precursor solution into the reaction vessel
Rubber Hoses (PVC Tubes) (Quantity: 2)	Fisher Scientific	14-169-7D	for connecting the condenser tube to water inlet/outlet ports
Stainless Steel Spatula	Ted Pella, Inc.	13590-1	for scooping tetrachloroauric acid powder from small container
Stand (Base with Rod)	Fisher Scientific	12-000-102	for holding the condenser tube and reaction vessel during the synthesis process (located in the nitrogen glove box)
Stirring Heating Mantle (250 mL)	Fisher Scientific	NC1089133	for holding and supporting reaction vessel sphere, while heating with magnetic stirrer rotating the magnetic stirrer bar
Tetrachloroauric(III) Acid (HAuCl4) (≥99.9%)	Sigma-Aldrich	520918-1G	preferably new or never opened, ≥99.9% purity, stored in fridge, then opened only in the nitrogen glove box, never exposed to air/water/humidity
Texwipes / Kimwipes / Cleanroom Wipes	Texwipe	TX8939	for miscellaneous cleaning and surface protection
Toluene (≥99.8%)	Sigma-Aldrich	244511-2L	new, anhydrous, ≥99.8% purity
Tweezers	Ted Pella, Inc.	5371-7TI	for poking small holes in aluminum foil, and for removing Parafilm
Vortexer	Cole-Parmer	EW-04750-51	for vortexing the gold nanoparticles in toluene in 50 mL conical centrifuge tubes to resuspend the gold nanoparticles into the toluene solution