TITLE: Gold nanoparticle synthesis
AUTHORS: Phuong H P Nguyen, Zhonglei He, Furong Tian, James Curtin, Julie Rose Mae Mondala

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol for citrate reduction methods to synthesize gold nanoparticles. Gold nanoparticles (GNP or AuNP) are sub-micrometre particles with various sizes ranging from 1nm to 8 µm created from gold. Besides, they also have different shapes, including spherical, sub-octahedral, octahedral, decahedral, nano triangles, etc. Due to their properties, GNPs have been used in many areas such as electronics, nanotechnology and biomedicine. In biomedical application, spherical and rod shapes are mainly used. Spherical usually is synthesized using a chemical reduction method, while Rod-shape uses a seed-mediated synthesis method. Different shapes and sizes of GNP could give various optical properties and cellular uptakes; by this, GNPs have broader use in modern biomedical applications both in vivo and in vitro. The citrate reduction method was initially introduced by Turkevich, where we use trisodium citrate as a reducing agent. The method widely studied in 1951, a substantial amount of important information regarding this system has been deposited in literature. It was demonstrated that GNP's size variation could readily be realized by controlling the concentration of sodium citrate. Absorbance, zeta potential and size of gold nanoparticles (GNPs) will be determined by using UV/Vis, Zetasizer and Scanning Transmission Electron Microscopy (STEM), respectively. </div></div>

[STEPS]
?. [Synthesis of AuNP by Citrate Reduction]
Dissolve   (HAuCl4 ) in   into a clean Duran bottle
[of Chloroauric acid]
[Distilled water]
HAuCl4 is corrosive so using glass to avoid contacting with metal
?. [Synthesis of AuNP by Citrate Reduction]
Heat with magnetic stirring and bring it to boiling. Keep the lid on loosely to remain evaporated water
?. [Synthesis of AuNP by Citrate Reduction]
Quickly add 5% (w/v)sodium citrate solution to final 3:5:1 molar ratio of citrate to Au3+
Sodium citrate solution is made by mixing   (Na3C6H5O) into
?. [Synthesis of AuNP by Citrate Reduction]
Keep heating and stirring until the colour changes
The colour should change from purple to wine red in   to   . When it is stable, turn off the heat and keep stirring for  .
?. [Synthesis of AuNP by Citrate Reduction]
Centrifugate gold colloid at  for  to concentrate the AuNPs stock solution to 2500µg/ml. Remove supernatant gently
10 g
?. [Characterization of AuNP]
AuNPs Absorbance Characterization by UV/Vis
The peak at around 550nm indicates the formation of Au particles
Use the absorbance ratio of Table S-2 to determine the particle size of AuNPs. If the initial concentration of gold (CAu in mol per litre) used to synthesis the particles are known, then using the equation below can provide a precise way to calculate particle size in the range of 5-50nmd = (Aspr(5.89×10-6) / C Auexp(C1))1/C2Use table S-3 to determine the concentration (c) in mol per litre from the absorbance (A) at 450nm. The equation would be used is:d = (Aspr(5.89×10-6) / C Auexp(C1))1/C2c= A450/ε450Tables are in the Supporting Information link:
?. [Characterization of AuNP]
Sample preparationAdding a few crystals into the bottom of the cuvetteFill with Distilled water until the markUse a pipette to mix the solution into a homogenous solution
Only touch the frosted window to avoid dirty the clear window. This is because the light will pass through the clear window when processing
?. [Characterization of AuNP]
Set up UV/VisTurn on the instrument and the computer Open the software called Carry UV Choose SCAN Click SET UP Go to the BASELINE tab. Click BASELINE CORRECTION then press OK
?. [Characterization of AuNP]
Blank preparationFill up two cuvettes with Distilled waterPlacing the cuvette into the Spectrometer  Close the lid
Before putting the cuvette into the machine, clean the clear side with tissue to make sure no fingerprint or any other residue left
?. [Characterization of AuNP]
SamplingUsing the blank sample as the controlset up the wavelength in a range from 350-800nm, a resolution of 1nm
?. [Characterization of AuNP]
AuNPs zeta potential Characterization by Zetasizer
?. [Characterization of AuNP]
Sample preparation
If the mean counts rate
?. [Characterization of AuNP]
MeasurementRemove two tips of the folded capillary cell (DTS 10701) and rise with Distilled water to eliminate contaminationLight shake and tap to clear the water out of the cellSlowly uptake   into the micropipetteInsert the micropipette tip into one of the two ports of the zeta cell and dispense the sample slowly and evenly Plug each end of two ports back with the plastic tipsWipe the cell before insert into the instrumentInsert the cell into the Zetasizer slowly Close the lidSlowly uptake  into the micropipetteInsert the micropipette tip into one of the two ports of the zeta cell and dispense the sample slowly and evenly Plug each end of two ports back with the plastic tipsWipe the cell before insert into the instrumentInsert the cell into the Zetasizer slowly Close the lidPlug each end of two ports back with the plastic tipsWipe the cell before insert into the instrumentInsert the cell into the Zetasizer slowly Close the lid
[solution]
make sure to have no bubbles along the way. If there are bubbles in the cell, gently tap the size of the cell
Two ports of the cell are facing left and right
?. [Characterization of AuNP]
SamplingOpen the Zetasizer softwareGo to SETTING and select zeta potential in the measurement typePut a sample name as “Gold nanoparticles”Choose water as in the DISPERSIONThe CELL TYPE is folded capillary cellFor GENERAL OPTION, select standard modelFor MEASUREMENT, select automaticInsert the number of measurement then choose OKClick START
Always check the phase and the frequency plot during each run to avoid sample degradation. If there is a change in the phase or the frequency plot, it indicates that sample degradation has occurred. In case there is a sample degradation, reducing the number of runs or the voltage can be used.
?. [Characterization of AuNP]
AuNPs size Characterization by Scanning Transmission Electron Microscopy (STEM)
?. [Characterization of AuNP]
Sample preparationUse micropipette to add  distilled water into Place the PCR tube into a beaker and put it in the Sonicator for to get a homogenous solutionTM grid is held by a tweezerDrop   using the micropipette onto TM grid Leave the droplet evaporates for around   The sample can go to the TM as soon as when it dryDrop  using the micropipette onto TM grid Leave the droplet evaporates for around
[distilled water]
[gold nanoparticles solution]
[solution]
Can leave under the light to speed up the process of evaporation
?. [Characterization of AuNP]
Set up and measuringRelease the clamp ease in the screws of the sample holderPlace the sample into the sample holderTighten the screws downPut the holder into the Microscope and wait for the Microscope to pump down the sample chamberAligning the Microscope and Inserting cameraMagnify the square of the grid that is interestedIncrease magnification to 40,000 time of the image would be good for measuring size distribution of the nanoparticles
Do not choose the regions that are dense as they are not good for size determination
?. [Characterization of AuNP]
Dilute AuNPs stock solution in water or culture medium in desired concentration for testing