User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/10/02

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Horseradish Peroxidase Assay Absorbance

Goals

  • Run a HPR assay that varies the concentration of Gold nanoparticle solution (AuNP/BSa)

Procedure

  • We followed the HRP Protocol File:3760-Protocol HRP AbsorbanceAssay.pdf and pay particular attention to what the other group( File:HRP Assays.pdf had done two weeks ago in regards to this same experiment.
  • The previous group had already tested to see how varying the concentrations of aminoantipyrine and hydrogen perioxide had affected the rate of the reaction after the addition of the HRP. As a result, we wanted to rest what effect the addition of the gold nanoparticles had on that reaction.
  1. We made .2 M sodium phosphate at a pH of 7 ( we made 60mL)
  2. 0.0017 M of hydrogen peroxide was made by adding 1 mL of 30 % hydrogen peroxide to 100 ml

distilled water. It was then further diluted by taking 1mL of the solution in 50mL of 0.2 M sodium phosphate buffer.

  1. 0.0025 M 4-Aminoantipyrine with 0.17 M phenol:
    • Because the iodophenol was immiscible in water, it had to be dissolved in DMSO. It was dissolved in 1mL of DMSO to make a concentration of 700μM. The 25mg of aminoantipyrine was dissolved in 50mL of water.
  1. 1mg of HRP was dissolved in 1mL of distilled water.

Based on the other groups tests, we decided to keep all our variables constant except for the percent by volume of the AuNP added into each cuvet.The Gold Nanoparticle were made in a 1% citrate solution rather than in BSa. We decided that between the two separate trials, we would vary the concentration of the AAP because on the other groups results, it seems that the concentrations 156.25 mM and 312 mM were the most linear.

Because of changes in calculations and incorrect data results from the UV-Vis instrument, The spectra that we run today did not react in the manner in which we were looking for. However, please look at Mary's Notebook to see the spectras and analysis collected on this day.