TITLE: Synthesis of Silver Nanoparticles
AUTHORS: Raphael D. Ayivi, Bukola Adesanmi, Eric S McLamore, Sherine O. Obare

[DESCRIPTION]
This protocol describes the synthesis of silver nanoparticles for colorimetric sensing of environmental samples and for other biochemical applications. The protocol requires 110 minutes to complete (excluding working solution prep the protocol requires 65 min).

  


[BEFORE_START]
Be sure to wear appropriate safety PPE throughout (lab coat, gloves, eyewear).
Electronic or physical lab notebook may be used throughout
See experimental plan guide for tips on planning your work

[GUIDELINES]
This protocol describes the synthesis of silver nanoparticles for colorimetric sensing of environmental samples and for other biochemical applications. The steps for the process are based on Brown et al (REF 1).; 

The steps for the process are based on Brown et al 1; summarized in Figure 1
  


[STEPS]
SECTION: SECTION 1) Preparation
1. Prepare stabilizer solution (0.01 M Sodium citrate; Na3C6H5O7) 
Prepare a glass beaker with 50 ml of deionized water and label
Weigh 0.1290 g of trisodium citrate on scale
Dissolve sodium citrate powder in prepared glass beaker with deionized water
Place magnetic stir bar in beaker and place on a stir plate
Stir for 15 minutes at 150-200 rpm
After stirring, inspect the beaker to ensure dissolution of the solute 
The trisodium citrate serves as the stabilizer and is ready to be used for particle synthesis

SECTION: SECTION 1) Preparation
2. Prepare precursor solution (0.01 M Silver nitrate; AgNO3)

Prepare a glass beaker with 50 ml of deionized water and label
Weigh 0.085 g of silver nitrate on scale
Dissolve powder in prepared glass beaker with deionized water
Place magnetic stir bar in beaker and place on a stir plate
Stir for 15 minutes at 150-200 rpm
After stirring, inspect the beaker to ensure dissolution of the solute 
The silver nitrate is the precursor and is ready to be used for particle synthesis
SECTION: SECTION 1) Preparation
3. Prepare reducing agent solution : (0.01 M Sodium borohydride (NaBH4)

Prepare a sealable container with 50 ml of 0.1N NaOH and label. Place container in the chemical hood 
 

Cool the NaOH solution in an ice bath under the chemical hood (cool to approximately 0 to 5°C)
Working under a chemical hood, weigh 0.020 g of sodium borohydride on scale
Immediately return sodium borohydride powder to proper storage system (away from moisture or water, stored under inert gas in a flammable containment unit)
Dissolve sodium borohydride powder in cooled 0.1N NaOH and label
Seal the container with the sodium borohydride
Place magnetic stir bar in beaker and place on a stir plate
Stir for 15 minutes at 150-200 rpm
After stirring, inspect the beaker to ensure dissolution of the solute 
The sodium borohydride serves as the reducing agent and is ready to be used for particle synthesis
Store the sealed container at 4°C until used (best practice is to place in a secondary spill container, particularly if sealed container is glass) 
 

SECTION: SECTION 2) Particle synthesis
4. Combine stabilizer and precursor solutions

In an Erlenmeyer flask, add 18.5ml of deionized water 
Add a magnetic stir bar to the flask
Pipette 0.5 ml of 0.01M sodium citrate to the flask
Pipette 0.5 ml of 0.01M silver nitrate to the flask
The resulting mixture in the flask should be gently stirred for 3 minutes at a temperature of 10°C. The stir rate should be the lowest stirring rate that maintains constant agitation
SECTION: SECTION 2) Particle synthesis
5. Add reducing agent solution to mixture
 

Slowly pipette 0.5 ml of 0.01M NaBH4 to the solution to the flask containing the reaction mixture with a stir rate no higher than 50 rpm.
Slowly pipette 0.5 ml of 0.01M NaBH4 to the solution to the flask containing the reaction mixture with a stir rate no higher than 50 rpm. 
 
Seal Erlenmeyer flask after 0.5 ml has been transferred
Under a chemical hood, carefully transfer solution to container with lid and label as AgNP solution
Wrap container in aluminum foil
Transfer AgNP solution from flask to container and seal 
SECTION: SECTION 3) Particle analysis (post reaction)
6. Qualitative analysis (color)

The first step of qualitative analysis is visual inspection of the solution color
The color of the reaction mixture (gold or yellow) confirms the formation of AgNP and completion of the synthesis reaction.
Analysis of polychromatic visible (VIS) color is an important quality control mechanism in particle synthesis, and may be carried out manually or using instrumentation (Fig 1)

 
A representative example of color analysis is shown in Fig 2
Photographs before (Fig 2A) and after (Fig 2C) reaction are shown. Color analysis via a mobile phone app are also shown for before (Fig 2B) and after (Fig 2D) the reaction.
 
  
SECTION: SECTION 3) Particle analysis (post reaction)
7. Quantitative analysis (UV-VIS)
If required, turn on the computer for the Agilent UV-VIS Cary-60 instrument and then turn on the instrument and wait until the LED stops blinking.
Open the Agilent software and wait until the instrument fully initializes.
To select experimental parameters, select Setup in the Agilent software.
Select wavelength range if desired (default is 190-1100 nm) 
Click the baseline tab, select “Baseline correction” and select OK.
Click on “Zero” for lamp intensity correction. When performing this procedure, the sample compartment has to be empty.
Click on “Baseline”. 
Fill an empty cuvette with DI water (blank) and another cuvette with the silver nanoparticle (AgNP) sample
Wipe the blank cuvette with a Kimwipe and inspect the surface for any debris
Place the blank cuvette in the sample compartment.
Close the lid of the chamber 
Click OK to scan the baseline
Remove the blank 
Wipe the sample cuvette with a Kimwipe 
Insert the sample cuvette into the holder
Click Start to run the scan
When finished with measurement, select Finish
If processing multiple samples with the same parameters, select OK after each run. 
If processing a large batch of samples (>20), scan a blank cuvette every 7 samples as a quality control (QC) procedure
After the scan is complete, save the file under a unique name (see data management section)
After finishing the experiments, close the software.
Leave both the instrument and the computer on.
Representative UV-VIS absorbance for the AgNP solution using an excitation wavelength of 190nm is shown in Fig 3 
A clear peak at 395nm is observed for the AgNP solution prepared using this protocol (no custom emission filters or signal processing was performed).

  
 
SECTION: SECTION 3) Particle analysis (post reaction)
8. Quantitative analysis (fluorescence)
If required, turn on the Horiba Fluoro-Max 4 Fluorometerinstrument and wait for the software and hardware to initialize
Open the Horiba software 
 
Choose the “Experiment Menu” Button
Choose “Spectra”, 
then “Emission”
Click the Experiment File field, and enter a new file name or select a previously saved file (see data management section) 
Verify that experimental parameters are correct
Prepare a blank sample in a cuvette and the AgNP solution
 
Wipe the blank cuvette with a Kimwipe and inspect the surface for any debris
Place the blank cuvette in the sample compartment. 
Close the lid of the chamber
Click “Run” to scan the baseline
Enter a name for the project, or browse for an existing project, then click OK 
Open the lid and remove the blank sample from the compartment
Wipe the sample cuvette with a Kimwipe 
Insert the sample cuvette into the compartment
Close the lid and then click “Run”
When finished with measurement, select Finish
If processing multiple samples with the same parameters, select Ok after each run. 
If processing a large batch of samples (>20), scan a blank cuvette every 7 samples as a quality control (QC) procedure
After the scan is complete, save the file under a unique name (see data management section)
After finishing the experiments, close the software.
A representative fluorescence emission for the AgNP solution using an excitation wavelength of 250nm is shown in Fig 4.
No custom emission filters or signal processing was performed
A bimodal peak contains distinct peaks at 357nm and 370 nm, and an isolated smaller peak at 504nm are common.

  

SECTION: SECTION 5) Cleanup
9. Clean up space and dispose of waste

Turn off magnetic stir plate and all equipment used
Wrap storage vials in aluminum foil 
Store solution(s) in the dark at 4°C.
Dispose of used chemicals according to the lab safety plan
 


Wash all glassware with mild detergent and warm water. 
Clean up the chemical hood 
Rinse used pipette tips or other materials used for handing reducing agents (collecting rinse in waste jar)
Dispose of rinsed tips per solid waste handling procedures (without this rinse step, used materials contain residue)



SECTION: SECTION 6) Data management
10. File naming: For saving all files from spectrometer, use the following file structure or similar structure as appropriate for each laboratory:
                
            AgNP_(instrument name)_(date of experiment)_(iniatials of researchers).txt
 

File storage: Store all spec data in the Desktop folder connected to the instrument using the naming procedure above.

Backup files: At least once per year, ensure that the folder is backed up on the lab external hard drive.