Chemical Engineering Laboratory 1

Lab report

Experimental number : 6th experiment

(Localized Surface Plasmon Resonance Biosensor)
Name :
Student number :
 [Experiment 6] Localized Surface Plasmon Resonance Biosensor

1. Experimental Procedure

* Synthesis of Gold Nanoparticles

(1) TA made aqua regia that is 3:1 composition of HCl : HNO3. Make sure that

hydrochloric acid and nitric acid is extremely dangerous chemical, so it should be

made on chemical hood.

(2) Then, they put the aqua regia into 3-neck round bottom flask. Put the magnetic

stirring bar to stirring solution easily. In this process, aqua regia is extremely

dangerous, and it have possibility of explosion. Therefore, soaking process is

proceeded in opened vessel like the below picture. After soaking process is done,

clean the 3-neck flask with DI water couple times. They mentioned that soaking

process goes more than 15 minutes, so there are enough time to impurities

dissolved on it. The meaning of this process is get perfectly clean vessel to make

good quality of gold nanoparticles.

 [Figure 1] Aqua regia soaking process (opened vessel) and Cleaning process

(3) To make gold nanoparticle, 2mL of 50mM HAuCl4 solution added on three neck

flask that is cleaned by process (2). Additionally, add 98mL of Millipore water on it to

make the HAuCl4 concentration to 1mM. Also, millipore water is used to reduce the

impurities as possible. In this process, several errors occurred. They mentioned that

the concentration of HAuCl4 is small, so they can’t weigh the exact weight of

HAuCl4. Also, some HAuCl4 was remained on the weighing paper. Finally, HAuCl4

solution is not perfectly transferred into 3-neck flask. To sum up, concentration of

HAuCl4 solution is not same as 1mM in this experiment.

[Figure 2] Weighing HAuCl4 (left) and making solution (right)

(4) Then, they connected the condenser and stopper on 3-neck flask. Set the

temperature of heating mantle at 120℃ and put the 3-neck flask on it. Reflux occurs

when the solution is stirred. They mentioned that some leaking occurred in this

process. Also, they found that water is not circulated well in the condenser at the end

of experiment. So, reflux process might be done not well.

(5) When reflux is observed, they remove stopper and add 10mL of 38.8mM sodium
citrate. If experiment goes well, the color should change to deep red. But they said

that red color was not existed, so the perfect gold nanoparticle was not formed. This

process is done for about 1 hour.

From the paper1, the reason why put sodium citrate is that it acts as a reducing

agent and coordinating agent. Carbonyl groups in the sodium citrate stabilize the

nanoparticle. Reducing occurred in this reaction, and gold particle tends to gather.

[Figure 3] The pictures of process (5)

(6) After 1 hour of refluxing process done, they turn off the heating mantle and let

them cool at room temperature during stirring. Ideally, ~13nm diameter of gold

nanoparticle is formed in this process. But several errors occurred in this experiment,

so I think they didn’t get that good quality of gold nanoparticles.

(7) Finally, by using UV-vis spectrometer, absorbance of made gold nanoparticle

solution is measured. The absorbance vs wavelength plot is presented on

experimental result.

* Preparation of Thrombin Binding Aptamer-Conjugated AuNPs

These processes are done by TA, and there are no things that describe how

1
The role of tannic acid and sodium citrate in the synthesis of silver nanoparticles
experiment goes. But I can assume that AuNPs formed well in this process because

the colors of solution showed deep red color.

[Figure 4] Thrombin injection process (color of AuNPs solution is red)

* Preparation of Thrombin Binding Aptamer-Conjugated AuNPs

(1) 10μL of Thrombin is added to DNA conjugated AuNPs solution and incubate it for

30 minutes. In this process, to compare the effect of concentration difference, 4

different concentration of AuNPs solution are added.

(2) Then, measure the absorbance of it by using UV-vis spectrometer. The result is

presented on experimental result.

2. Experimental Result

By using excel, I plotted the bare gold nanoparticle’s absorbance according to

wavelength as follows.
 [Figure 5] Bare AuNPs plot between absorbance and wavelength

Also, the absorbance of 4 different concentration of AuNPs solution were measured.

The result is as follows.

[Figure 6] AuNPs’ absorbance at 4 different concentration

The absorbance difference was very slight, so four different graph looks like almost

same. It is hard to identify the concentration difference, so I collected 10 absorbance

value that are biggest than other in each graph.

 [Figure 7] Comparison of peak absorbance value

The peak value of each concentration shows slight difference, but it shows some

coherent tendency. From the theory, more absorbance will be observed at higher

concentration. Therefore, I can assume that the concentration comparison is as

follows.

target 4>target 3>target 2>target 1

Target 4 have highest concentration, and target 1 have lowest concentration.

Now, we can get the ∆ λ max of each concentration. First, in bare AuNPs, absorbance

value is highest at 515nm. When thrombin is added on it, absorbance values are

highest at 521nm, 521.5nm, 521.5nm, 521.5nm (target 1, target 2, target 3, target 4).

∆ λ max ( target 1 ) =521nm−515 nm=6 nm

∆ λ max ( target 2,3,4 )=521.5 nm−515 nm=6.5 nm

3. Discussion

From the uploaded video, group 6 mentioned several errors at last part of video.

First, they mentioned that soaking process goes more than 15 minutes. But I don’t

think this is error. The meaning of soaking process is cleaning apparatus perfectly as

possible. Aqua regia dissolves almost everything, and the soaking process goes

more and more, the possibility of remaining impurities goes less. So, this event does

not affect the result badly.

Second error is occurred on weighing and transferring solution. Group 6 said that

small amount of HAuCl4 powder were not put on the solution, their weighing was not

perfect, and some solution was left during transfer the solution. All of these errors are
related with concentration of solution. Ideally, 1mM of HAuCl4 solution will be made,

but I think their solution’s concentration is less than 1mM. These can affect the

amount and quality of AuNPs. These kinds of error are inevitable but try to pay

attention as possible on it to get a good result.

Third, some leaking occurred during reflux process. Leaking reduces the amount of

AuNPs generation. Changing amount of AuNPs can affect the sensing performance

that detects something adsorb on it. To prevent this problem, the connection part of

condenser should be strong enough. Using grease is highly recommended, because

grease can endure high temperature.

Last, they found that condenser did not work well after experiment done. It means

that reflux does not work on this experiment. I think this is why they can’t observe the

red wine color which means AuNPs produced. Reflux prevents the solvent turn into

vapor and fly away, as a result it increases the amounts of product. Reflux is

essential to get desired compound. If reflux does not act properly, there is no

guarantee that AuNPs produced. To prevent this error, check the several apparatus

(in this case, condenser) works well before experiment start.

4. Summary

In metallic nanoparticle, localized surface plasmon resonance (LSPR) occurs. When

LSPR occurs, electromagnetic field is created and light extinction occurs. By using

this property, we can use metallic nanoparticle as a sensor. If something adsorbs on

it, light extinction might be changed. To check this point, this experiment was

proceeded. The notable point of this experiment is reducing impurities as possible to

get a good result. Specially, aqua regia and Millipore water were used. Finally, we

can check that different peak shift occurred at different concentration which is
correspond to the theory.

5. Reference

[Experimental Procedure] “Chemical Engineering Laboratory I”, Sungkyunkwan

University School of Chemical Engineering p.27-32

[Experimental Procedure] “The role of tannic acid and sodium citrate in the synthesis

of silver nanoparticles”, Katarzyna Soliwoda, Emilia Tomaszewska, Ewelina Socha

[Experimental Procedure] “화학백과” – Naver 지식백과