Vietnam National Universities – HCMC

International University
School of Biomedical Engineering

Course of Biomaterials
Semester 1 2022-2023

LAB #5
METAL (SILVER NANOPARTICLES) & UV-VIS SPECTROSCOPY

I. Outline
 Overview of nanosilver particle.
 Overview of UV-Vis spectroscopy
 Experimental protocols.
 Report highlights.
II. Silver nanoparticles
Nanotechnology, which focuses on the development of nanostructure in the range of 1-100 nm,
has become increasingly prevalent and spans over various biomedical areas such as
nanomedicine (using nanodrug delivery system), diagnostics (using semiconductor
nanocrystals) and protective antimicrobial or antiviral therapeutics (using metal and metal
oxide nanoparticles). Besides its significance, toxicities of nanoparticles should also be
assessed carefully.
Silver nanoparticles (AgNPs) express unique antimicrobial property (Fig 1), which can serve
as a beneficial bioactive reagent loaded within wound dressing for optimal wound healing
process (Fig 2). It can also be applied as a conducting material for biosensor manufacturing.
There are many methods for AgNPs synthesis, which leads to particles with varying size,
morphology, stability, aggregation. In this lab session, chemical reduction method is applied.

Fig 1. Antibacterial mechanisms of silver nanoparticles

 Vietnam National Universities – HCMC
International University
School of Biomedical Engineering

Fig 2. Example of different silver nanoparticle-loading wound dressings

III. UV-Vis spectroscopy

UV-Vis spectroscopy is a sensitive and reliable method to detect the existence of metal
nanoparticles, because most of them exhibit a characteristic absorption peak owing to the
phenomenon of surface plasmon excitation. In other words, the free electrons within metal
nanoparticles can vibrate in resonance with the specific light wave and thus give the
corresponding absorption band. For example, AgNPs usually shows an absorption peak at
about 400 nm, while the peak shifts to about 525 nm for gold nanoparticles (Fig 3). Besides
the qualitative purpose, UV-Vis spectroscopy can also be used to quantitatively determine the
concentration of nanoparticles or other compounds based on Beer’s Law (relationship between
concentration and light intensity).

Fig 3. Example UV-Vis spectra of silver nanoparticles (A) and gold nanoparticles (B)
 Vietnam National Universities – HCMC
International University
School of Biomedical Engineering

The color of nanoparticle solution is complementary to the color that it absorbs (Fig 4). For
example, as AgNPs absorbs the wavelength of about 400 nm, the solution is observed to be
yellow at dilute concentrations or brown at higher concentrations.

Fig 4. Complementary color wheel

The schematic diagram of an ordinary absorption spectrophotometer is depicted in Fig 5. It
first uses a monochromator to filter and let a narrow range of wavelengths pass through the
sample. The transmitted photons then hit a photodetector (or photoresistor) that produces an
electric signal proportional to the amount of light detected. The computer processes these
signals to create a UV-Vis absorption spectrum.

Fig 5. The schematic diagram of an ordinary absorption spectrophotometer

IV. Experimental protocol

4.1. Synthesis of silver nanoparticles
 Vietnam National Universities – HCMC
International University
School of Biomedical Engineering

Step 1: Prepare 100 ml of stock silver nitrate (AgNO3) solution at 1000 ppm concentration
(corresponding to 0.1 % w/v) in dark condition.
Step 2: Prepare the following solutions
- 2 ml of trisodium citrate dihydrate (Na3C6H5O7.2H2O) aqueous solution at 3% w/v.
- 2.5 ml of tannic acid (C76H52O46) aqueous solution at 4% w/v.
Step 3: Prepare 50 ml of AgNO3 solution at 100 ppm by diluting the stock solution at step 1 in
dark condition. Repeat one more time. Label these solutions as “Sample 1” and “Sample 2”.
Step 4: Heat one sample at a time up to 100oC using microwave and then magnetic stirrer.
- For sample 1: Let the solution boil for 15 minutes.
- For sample 2: When the solution starts to boil, gently add trisodium citrate solution and
tannic acid solution (prepared in step 2), then let it boil for 15 minutes.
Step 5: Cool the sample to room temperature.
4.2. Measurement of UV-Vis spectra
Step 1: Sonicate the solution if necessary to disperse the particles.
Step 2: Dilute the solution depending on the color intensity of the solution (to ensure solution
transparency).
Step 3: Add 100 µL of the dilute solution into a 96-well plate.
Step 4: Measure the absorbance of the plate within the range of 350-500 nm using a microplate
reader (La1.210).

VI. Report highlights

 Introduction section should include brief information of:
+ Background of metal or metal oxide nanoparticles in general and silver nanoparticle
in particular, with more details regarding antibacterial mechanism and biomedical
applications.
+ Background of UV-Vis spectroscopy, focusing on its function to confirm the
existence of nanoparticles within the solution.
 Questions (for Result and Discussion section)
+ How do trisodium citrate and tannic acid contribute to the synthesis of silver
nanoparticles?
+ Why do we need to dilute the sample for UV-Vis spectra measurement? For an
unknown sample, how can we determine if it possesses a characteristic UV-Vis
absorption peak or not?