User:Melissa Novy/Notebook/CHEM-571/2012/10/09

Objectives

• Analyze Au/BSA solutions made on 2012/10/03 with atomic absorption.
• Calculate activity of HRP from the UV-vis data obtained from the HRP-AAP assay on 2012/09/18 and 2012/09/19.

Atomic Absorption of Au/BSA Solutions

AAS Protocol

• The AA was calibrated with standard solutions at concentrations of 5, 8, 10, 15, 20, 25, 30, and 40 ppm made by Dr. Miller on 2012/10/08 and blanked with 1 N HCl.
• Deionized water was run through the AA between samples.

• Au/BSA solutions containing fibers at Au:BSA mole ratios of 120 through 170 were filtered with dry 0.45 μM pore-size nylon filter paper into a clean, dry flask. The filtrate was then decanted into a Falcon tube for testing.
  • Note that the fibers on the filter paper were not washed. The fibers on the filter paper were dark purple in color.
  • Colored solutions at Au:BSA mole ratios of 120, 128, and 130 that contained fibers produced clear filtrate after being filtered and the AA detected that they contained little to no gold (please refer to Table 1). This indicates that either the gold was of large enough size to be filtered by the filter paper, or that the pores in the filter paper were blocked with fibers and thus did not filter the gold. It would be incorrect to assume that the solutions did not contain any free gold, as literature states that the purple color of the solutions before filtering is due to the optical properties of gold nanoparticles.

• Table 1: Change in Au Concentration (ppm) of Solutions of Au/BSA

• Note that as negative concentrations of Au are experimentally impossible, the values are likely due to improper zeroing of the AAS. For the purposes of data analysis, it will be assumed that the concentration of Au in those solutions is zero.
• According to Table 1, Au/BSA solutions at mole ratios of 60, 80, 100, 132, 133, and 134 contained Au. These solutions likely successfully formed AuNPs, corroborated by visual observation of their purple color. The remainder of the solutions likely did not form AuNPs, as no Au was detected by the AAS. Thus, it can be assumed that BSA encapsulated the Au added to the solutions and thereby prevented Au from being present in solution. However, it is also possible that filtration removed AuNPs from solution, as in the case of solutions of Au:BSA mole ratios of 136 through 170.

• Table 2: Change in Moles of Au of Solutions of Au/BSA

Activity of HRP Based on the HRP-AAP Assay

• Note that data obtained on 2012/09/18 was used to calculate the activity of HRP.
• The ratio of the change in time to the change in absorbance was calculated to be the rate of the reaction. The inverse of the rate was plotted against the inverse of the concentration of whichever reactant was varied, either AAP or H₂O₂.

• Note that the concentration of H₂O₂ was fixed at 1.7 mM.

• Note that the concentration of AAP was fixed at 2.5 mM.