User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/28

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  • Au/ADA samples made on 2012/11/27 were ran on UV-vis spectrophotometer and Atomic Absorption Spectrometer, results were analyzed.
  • Au/ADA samples were resuspended in a range of Tris buffers to test resuspension based on concentration of buffer and pH.

Procedure for Running Au/ADA samples on UV-vis Spectrophotometer

  • 3mL of Au/ADA samples with the following mole ratios were analyzed using UV-vis spectrophotometer.
  • 3mL of distilled water was run on UV-vis under spectrum method followed by test samples with above mole ratios. The absorbance obtained from wavelength range 200nm to 800nm were used as base line correction for the Au/ADA samples.
  • Both reference and test samples were loaded into quartz cuvette with 10.0mm pathlength.

Results for Running Au/ADA samples on UV-vis Spectrophotometer

  • The Au/ADA samples made with dialyzed ADA proteins all appeared purple with purple fiber formation at the bottom of tubes. A picture of the samples after 4 hours of heating is shown in picture below:

Au ADA samples after dialysis.jpg

  • The result indicated that the lack of salt in samples might have an affect on nanoparticle aggregation formation. The lack of salt in solution might lead to ADA proteins to wrap around gold in solution at a lower temperature. The formed gold nanoparticles might have different isoelectric points that engages protein aggregations.
  • While the fiber formation can be due to lack of salt in solution that encouraged nanoparticle aggregation, the fiber formation might also be affected by the usage of plastic falcon tubes during the reaction.
  • The absorbance obtained from UV-vis spectrophotometer were plotted on a graph for all Au/ADA samples. The graph was plotted with absorbance versus wavelength scanning from 200nm to 800nm shown below:

UV-Vis Spectra of AuADA.JPG

  • From the graph above, it can be shown that there the trend for all Au/ADA samples appeared similar, and that there were no significant difference in gold concentration between each Au/ADA samples solutions. This might indicate that the ratio between Au and dialyzed ADA in solution does not determine gold nanoparticle formation.
  • Comparing the all samples, including water, at a specific wavelength:525nm, it can be seen that there were no significant difference between water and all Au/ADA samples. A data table showing the samples run and absorbance at 525nm is shown below:
Au/ADA samples Absorbance at 525nm
0 0.039
60 0.041
70 0.039
80 0.04
90 0.037
100 0.038
110 0.037
120 0.04
130 0.038
140 0.037
150 0.036
Standard Deviation: 0.001566699
  • A graph is made with absorbance of each sample at wavelength 525nm versus water and all Au/ADA samples:

Water and Au ADA.jpg

  • From the graph above, it can be concluded that there was no significant differences between the absorbance between water samples and each Au/ADA samples due to low standard deviation. This indicates that there is no relationship between the mole ratios of Au/ADA in solution and the gold nanoparticle formation in solution.
  • The data can be compared against the Au/ADA solution made on 2012/11/27. While the Au/ADA before dialysis appeared to have influence between the mole ratio of gold to ADA and concentration of gold nanoparticles in solution, the Au/ADA samples after dialysis does not. This can be explained by the presence of salt in solution that encouraged gold nanoparticle formation but can stabilize the protein nanoparticles to prevent nanoparticles from aggregating.
  • Furthermore, the solutions for Au/ADA samples before dialysis did not appear purple, while solutions for Au/ADA samples after dialysis formed purple solutions and purple fibers. This can also be explained by the presence of salt in solution. The presence of salt might interact with gold nanoparticles in a way that encouraged gold nanoparticle configuration that does not absorbce at 525nm. The absence of salt in solution might allow gold nanoparticle to adapt another confirmation that does absorb at 525nm.

Procedure for Running Au/ADA samples on Atomic Absorption Spectrometer

  • The same sample of Au/ADA were run on Atomic Absorption Spectrometer.
  • Atomic Absorption Spectrometer was calibrated by running HCl with gold at the following parts per million:
  • HCl was ran to for blank, and water was ran to set base line.
  • Au/ADA samples were run, and the sample tubing was raised with distilled water after each sample.

Results for Running Au/ADA samples on Atomic Absorption Spectrometer

  • The HCl with gold for calibration made up a calibration curve for establishing the concentration of Au/ADA samples. The absorbance measured for HCl with gold were listed in the following table:
HCl with gold[ppm] Absorbance
5 0.1125
8 0.1604
10 0.2012
15 0.278
20 0.358
25 0.4342
30 0.5077
40 0.6435
  • A graph plotting HCl to gold in parts per million versus absorbance is shown below:

Absorbance gold HCl ppm.png

  • The equation on graph was used to calculate the concentration of gold in Au/ADA samples. The following equation was used:
  Absorbance = 0.0153 * Concentration + 0.045
  • Absorbance for all Au/ADA samples were taken. Below is a table listing the absorbance of each Au/ADA samples. Concentration of gold in parts per million is also listed after converting from absorbance to concentration using the formula above:
Au/ADA mole ratios Absorbance(Abs.) Concentration[ppm]
60 0.0657 1.3572
70 0.041 -0.2608
80 0.1011 3.6762
90 0.0292 -1.0337
100 0.0246 -1.331
110 0.0259 -1.2499
120 0.0246 -1.3351
130 0.0222 -1.4923
140 0.0264 -1.2172
150 0.0229 -1.4464
  • A graph was made with concentration in parts per million of gold versus the Au/ADA samples in mole ratios.

Concentration versus Au ADA mole ratios.png

  • From the graph above, it can be seen that the concentration calculated were below zero. This can indicate that the spectrometer was not properly calibrated. Furthermore, a reference sample with water was not run for this set of data, thus the data cannot be readjusted. Despite the negative concentration, it can be seen that the gold concentration in Au/ADA samples remain relatively the same.
  • There was a high peak at 80 Au/ADA, but it was due to the accidental intake of fibers instead of solutions. With the data point at 80Au/ADA excluded, it can be concluded that there is no relationship between mole ratio of Au/ADA and gold concentration in solutions.

Procedure for Au/ADA Resuspension in Tris Buffer

  • Au/ADA samples ranging from 60 to 110 were used to test resuspension of fibers using Tris buffer of different concentration and pH.
  • 100mM of Tris buffer made on 2012/09/04 were diluted on 2012/09/05 to100uM, 10uM, and 1uM at pH 10.0 and pH 8.0 were used to resuspension.
  • Au/ADA samples with mole ratios ranging from 60 to 110 have about 7.5mL left in Falcon tube after Atomic Absorption spectrometer testing. 1mL of tris buffer of varying concentrations and pH were added into each sample to make up a 1:7.5 ratio of tris buffer to Au/ADA sample.
  • Table listing Tris buffer used and the pH for each Au/ADA samples were listed below:
Mole ratio of Au/ADA Concentration of Tris buffer used[uM] pH of Tris buffer
60 1 10
70 10 10
80 100 10
90 1 8
100 10 8
110 100 8
  • The samples were pipetted up and down for a couple of times for mixing, and let sit at room temperature.

Results for Au/ADA Resuspension in Tris Buffer

  • All solutions were successfully resuspended after the addition of Tris buffer. The solution after resuspension appeared as purple homogenous mixture and are clear in color. A picture taken after resuspension of all resuspended Au/ADA samples is shown below:

Au ADA after resuspension.jpg

  • The color for solution for 110 Au/ADA were clearer than other Au/ADA samples because the solution was lighter in color before resuspension.
  • The amount of time it took for each solution to resuspend was recorded in table below:
Mole ratio of Au/ADA Concentration of Tris buffer used[uM] pH of Tris buffer Time for resuspension[sec]
60 1 10 3
70 10 10 4
80 100 10 4
90 1 8 7
100 10 8 6
110 100 8 6
  • From table above, it can be concluded that resuspension happened faster in solutions with Tris buffer pH 10.0 then solutions with Tris buffer pH 8.0.
  • It was observed that resuspension time needed was must shorter in Tris buffer with pH 10.0 than in Tris buffer with pH 8.0. Possible reason for this result can be due to the high pH introduced by Tris buffer at pH 10.0 is much higher than the isoelectric point in ADA protein. This allows proteins to become negatively charged faster. Ions in buffer with stronger ionic strength can dissociate in solution and break apart the nanoparticle aggregation by interacting with the net negative charge on protein.
  • On the other side, at pH 8.0, less ADA proteins make the transition in time in order to take on a whole negative charge. As a result, not all ions would be able to form charge-charge interaction with the proteins, and in total delaying the process for protein aggregation breakdown, slowing down the time for complete resuspension.