User:Matthew R Skorski/Notebook/471 - Exp BioChem/2016/02/10

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Objective

Today's objective was to analyze the samples synthesized on February 9 and to make more fiber samples to be analyzed next week. However, our objective was not exactly met. We found numerous errors with our synthesis and analysis, so we spent today trying to improve our techniques for next week. We also collected fluorescence measurements of our samples from February 9. Finally, we made a more concentrated stock of acetylsalicylic acid so that the volume of acetylsalicylic acid we would need to add to our samples would be negligible.

Protocol

We made a stock solution of 20049µM acetylsalicylic acid:

  • 1.806g acetylsalicylic acid
  • Diluted to 500mL in DI water in a 500mL flask
  • The molecular weight of acetylsalicylic acid is 180.16g/mol
  • We heated the solution on a hot plate with a magnetic stir bar in order to help the acetylsalicylic acid dissolve

Fluorescence

We used an excitation wavelength of 280nm.

We used 200µL of each sample and blank to take a fluorescence measurement. Between each sample or blank, we cleaned out the cuvette with HCl, DI water, and methanol.

Data, Analysis, and Observations of Samples Synthesized on February 9, 2016

We had a number of errors with our synthesis from yesterday and our measurements today:

  • We did the math wrong for our samples synthesized yesterday. By adding an extra 198.02µL of DI water to our samples to keep the volume constant, the final concentration of acetylsalicylic acid would actually be just slightly less than 200, 100, or 50µM.
  • However, we forgot to actually add the water or acetylsalicylic acid after taking the samples out of the oven before we took fluorescence and UV-Vis measurements. As a result, our "after" samples did not contain any acetylsalicylic acid, and our "before" samples actually would have a concentration of either 200, 100, or 50µM acetylsalicylic acid.
  • Unfortunately, between 200-500µL of water evaporated from the samples on average while they were in the oven. As a result, the concentration of lysozyme AuNP, and acetylsalicylic acid in the samples were no longer precise and varied between samples.
    • In the future, we will cap the eppindorff tubes rather than covering them with tin foil in order to prevent evaporation. We will also measure the mass of the samples just before and just after taking them out of the oven in case any water is lost so that we can determine exactly how much water is lost. (The only problem with this technique would be if gasses are formed and released from the samples during formation of fibers. Then, water would not be the only substance lost. We could potentially use GCMS to determine whether other substances were lost from the sample during synthesis. However, we are confident that capping the samples will prevent loss of anything from the samples. Measuring the mass of the samples is more to prove this hypothesis.)
  • The fluorescence measurements for the samples showed a fluorescence of acetylsalicylic acid that was too high to measure. We believe this might have been due to a few reasons:
    • Evaporation of water, which increased the concentration of acetylsalicylic acid in the samples
    • The gold in the solution may have amplified the fluorescence of the acetylsalicylic acid, lysozyme, and gold
    • Fluorescence from AuNP and lysozyme: both of these substances fluoresce at a similar wavelength of light as the aspirin. We considered creating AuNP and lysozyme blanks so that we could subtract the blanks out to determine fluorescence caused by the acetylsalicylic acid alone. However, we quickly realized that this would not be possible. We had observed (by looking at our samples after taking them out of the oven) that the samples that contained acetylsalicylic acid seemed to form fibers more consistently than the samples that did not have acetylsalicylic acid. This observation led us to believe that higher acidity may be necessary to consistently form fibers, since acetylsalicylic acid is acidic. As a result, we would not actually know the concentration of AuNP or lysozyme that remained in the supernatant, so we would not be able to correct for the interference caused by these substances. Instead, we decided that we would use HPLC and UV-Vis absorbance spectroscopy in the future in order to analyze the supernatants in the future. Fluorescence did not seem to be a useful tool to measure the substances in the samples. (Note that the UV-Vis absorbance spectrophotometer that we are using is not the most precise instrument, which is another reason why we need HPLC to back up our findings.)


After discussing all of these errors, we decided to continue taking fluorescence and UV-Vis measurements of all of the samples anyway. The data would most likely be irrelevant, since our synthesis did not proceed correctly and our fluorescence measurements were too high to be useful in determining concentrations of acetylsalicylic acid. However, we still wanted to collect data before discarding the samples.


Figure 1: Fluorescence Intensity of Samples and Blanks

The above figure shows the fluorescence intensity of each of the samples and blanks as a function of the wavelength light at which they fluoresce. Since the samples were done in triplicate, their fluorescence intensities were averaged. The legend shows which line on the plot corresponds to which sample or blank.



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