User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/13

Purpose

• Au/HRP solution were run under UV-vis spectrometer from 200nm to 800nm to obtain spectrum of supernatant
• Au/HRP and Au/ADA solutions were run using Atomic Absorption spectrometer to test the concentration of gold present in solution supernatant
• Adenosine stock solution was prepared to perform test to calculate ADA enzymatic activity

Procedure on UV-vis spectrometry of Au/HRP

• Uv-vis spectrometer was set to scan Au/HRP samples made on 2012/11/07 from 200nm to 800nm
• 3mL of distilled water was loaded into quartz cuvette with 10.0mm pathlength and placed into base line standard
• 3mL of supernatant from following ratios of Au/HRP samples made from 2012/11/07 were loaded into quartz cuvette with 10.0mm pathlength separately to obtain spectrum.

    10 - 50 - 100 - 150 - 200 - 250 - 300 - 350 - 400 - 450

• The supernatant fro Au/HRP solutions were carefully pipetted out without including fibers in the samples to make sure more accurate measurement.
• Spectrum were run from 200nm and 800nm, and results were collected and plotted and shown in Data section below.

Data on UV-vis spectrometry of Au/HRP

• Graph of Absorbance versus wavelength scanned were shown below. All ratios of Au/HRP solutions were tested and plotted on the following graph:

• According to the graph, the ratio of Au/HRP with the highest absorbance is 100 Au/HRP. There is a linear relationship between absorbance obtained and the concentration of Au/HRP gold nanoparticles present in samples. This means that Au/HRP has the highest amount of gold nanoparticles formed in the supernatant.
• Also according to the graph, 100Au/HRP yields the highest concentration of gold nanoparticles in solution, while the rest of ratios tested yields not as high. At ratios around 100Au/HRP: 50Au/HRP and 150Au/HRP, the concentrations of gold nanoparticles were decreasing. This result suggests a more in depth test with smaller increments between the range of ratios 50Au/HRP and 150Au/HRP to determine the exact ratio of Au/HRP that yields the maximum concentration gold nanoparticle in solution. With this information, one can predict the ratio of gold to HRP at which the gold nanoparticles in solution starts to form fibers.
• The absorbance at 525nm were looked at separately to show a better relationship between absorbance and different ratios of Au/HRP. The wavelength 525nm was choosen because this wavelength best measures the amount of gold nanoparticles in solution according to Bakshi, et al, a paper that investigates protein films made using gold nanoparticles formed between gold and BSA.
• A table of absorbance at different ratios of Au/HRP was listed below:

• The data points were graphed on a separate graph to show the absorbance of solutions as ratios of Au/HRP change from 10 to 450. A graph of Absorbance at 525nm versus Ratios of Au/HRP ranging from 10 to 450 is shown below:

• From the graph above, it can be seen that the concentration of gold nanoparticles in supernatant increases drastically from 10Au/HRP and peaked at 100Au/HRP. Then the concentration of gold nanoparticles decreases about 20% around 100Au/HRP to 200Au/HRP, then decreases back to 0 absorbance starting at 300Au/HRP.
• Thus, it can be predicted that under 85°C, the gold nanoparticles forms in solution until 100Au/HRP, then fibers starts to form after 100Au/HRP. All gold nanoparticles aggregate and form fibers when the ratio of Au to HRP reaches 300.
• It was concluded to test ratios between 50 to 150 in smaller increments for more accurate conclusion on when protein aggregates form.

Procedure on Atomic Absorption Spectrometry of Au/lysozyme and Au/HRP solutions

• 8 of HCl samples with the concentrations below were run to set up calibration curve:

  5ppm - 8ppm - 10ppm - 15ppm - 20ppm - 25ppm - 30ppm - 40ppm

• The absorbance obtained were graphed with absorption versus concentration of HCl. The slope of the plot was used to calculate the concentration of gold in Au/HRP and Au/lysozyme solutions. Because the absorption data and graph were not saved during experimental process, the results cannot be displayed in Data section.
• Supernatant of Au/HRP samples made on 2012/11/07 with a ratio ranging from 10 to 450 were run using Atomic Absorption Spectrometry.
• Supernatant of Au/lysozyme samples made on 2012/10/31 with a ratio ranging from 20 to 130 were also run using Atomic Absorption Spectrometry.

Data on Atomic Absorption Spectrometry of Au/ADA and Au/HRP solutions