User:Melissa Novy/Notebook/CHEM-571/2012/11/13

Objectives

• Analyze Au-HRP solutions with UV-vis spectroscopy.
• Analyze solutions of Au-HRP and Au-lysozyme with AAS.
• Conduct ADA activity assay on ADA fractions using adenosine.

ADA Activity Assay

• 50 mL of 0.05 M sodium phosphate buffer was made with deionized water. The pH of the solution was adjusted to 7.4 using 3 drops of 12 M HCl.

• 5 mL of 0.1 mM adenosine was made.
  • 100 mg of adenosine was dissolved in 1 mL of 0.05 M phosphate buffer, giving a solution of 0.00373 M adenosine.
  • 0.134 mL of 0.00373 M adenosine was diluted to a final volume of 5 mL with 0.05 M phosphate buffer, giving a solution of 0.1 mM adenosine.

  (0.001 g)/(268.02 g/mol) = 3.73×10-6 mol
  (3.73×10-6 mol)/0.001 L = 0.00373 M
  (0.00373 M)(V1)=(5 mL)(0.1×10-3 M)
  V1 = 0.134 mL

UV-Vis Analysis of Au-HRP Solutions

• The Au-HRP solutions were made on 2012/11/07 and the spectra of the solutions were obtained over 200 to 800 nm in quartz cuvettes. Please refer to Dhea Patel's entry for a photograph of the solutions.

• Peaks at around 300 and 520 nm were visible. The peak at 520 nm indicates the presence of GNPs in the 50, 100, and 150 mole ratio Au:HRP solutions.
• The peak at around 300 nm was present in the spectra of the 250, 300, 350, 400, and 450 mole ratio Au:HRP solutions. It is possible that this peak is due to the relatively higher concentration of HAuCl₄ in these solutions, based on literature. Note that the literature indicated that a peak at around 287 nm is present in the spectra of aqueous HAuCl₄.

Atomic Absorption of Au-Lysozyme and Au-HRP Solutions

• The Au-lysozyme solutions were made on 2012/10/31.
• The Au-HRP solutions were made on 2012/11/07.

• Gold was detected in solutions at mole ratios of Au-lys from 20 to 60. The 60 Au-lys solution had the highest concentration of gold. This agrees with UV-vis data obtained on 2012/11/06. However, no peak or a slight peak was visible on the spectrum of the 20 Au-lys solution. This is corroborated by the fact that the AAS detected that the solution contained 0.0057 ppm Au.
• The 70 through 130 AuLys solutions displayed the highest percent change in [Au], likely because the GNPs were aggregated with the lysozyme fibers. The 20 AuLys solution displayed the second highest percent change [Au] probably due to a procedural error. The detection limit of the AAS is between 2 and 4 ppm Au, and the amount of Au initially added to this solution was around 3 ppm. Therefore, it can be hypothesized that the AAS was unable to detect the actual [Au] in this solution.
• It can be assumed that the fibers formed in the rest of the Au-lys solutions at mole ratios from 70 to 130 encapsulated the gold, such that there was no free gold or gold nanoparticles in solution. Theoretically, the gold concentration in these solutions was zero, but the AAS registered negative concentrations due to the fact that it was blanked with 1 N HCl, as opposed to deionized water.

• The concentration of Au in ppm increased as the mole ratio of Au:HRP increased. However, there was the greatest percent change in [Au] in ppm in the 10 AuHRP solution. However, as the limit of detection of the AAS for Au is around 2-4 ppm Au, the AAS was likely unable to accurately detect the actual concentration of Au in this solution. The lowest percent change in Au ppm was observed for the 200 AuHRP solution, so it is likely that this mole ratio is the best at producing GNPs relative to the amount of Au added before the reaction between Au and HRP.