User:Melissa Novy/Notebook/CHEM-571/2012/11/14

Objectives

• Make Au-ADA solutions at mole ratios of Au:ADA from 60 to 150.
• Make Au-HRP solutions at mole ratios of Au:HRP from 60 to 150.
  • Analyze the concentration of HRP stock solution with UV-vis.
  • The molar absorptivity coefficient of HRP at 403 nm is 1.02×10⁵ M^-1 cm^-1.
• Dialyze fractions of ADA made on 2012/10/03 and 2012/11/06.

Au-ADA Solutions

• Ten solutions at Au-ADA mole ratios of 60, 70, 80, 90, 100, 110, 120, 130, 140, and 150 were made.
• Please refer to Dhea Patel's entry for theoretical and actual calculations of stock solutions, and volumes of stock solutions and deionized H₂O added to each solution.
• Note that ADA made on 2012/10/03 was used to make the solutions. The solution of HAuCl₄ was made on 2012/09/05.

Au-HRP Solutions

• Ten solutions at Au-HRP mole ratios of 60, 70, 80, 90, 100, 110, 120, 130, 140, and 150 were made.
• Please refer to Dhea Patel's entry for theoretical and actual calculations of stock solutions, and volumes of stock solutions and deionized H₂O added to each solution.
• Note that 130, 140, 150 Au-HRP solutions were recalculated to account for a new stock solution at different concentration of HRP.
  • The concentration of the new HRP stock solution was calculated from its absorbance at 403 nm and its molar absorptivity coefficient.
  • Its concentration was calculated to be 28.5 μM.

  Abs.: 2.1
  Path length (b): 1 cm
  Molar absorptivity coefficient (ε) = 1.02×105 M-1 cm-1
  Concentration (c) = 2.1/(1 cm)(1.02×105 M-1 cm-1) = 28.5 μM

• The solution of HAuCl₄ was made on 2012/09/05.

ADA Activity Assay with Adenosine

• Frac 1,3 of ADA, made on 2012/11/06, was used. Its concentration was 10.35 μM, as determined by Bradford assay.
• It was diluted to 1 ng/μL, then to 0.1 ng/μL using phosphate buffer, prepared on 2012/11/13.
  • Please refer to the above entry for phosphate buffer calculations and actual concentrations. The pH of the phosphate buffer was adjusted to 7.4 with three drops of 12 M HCl.

  Initial concentration of ADA: 10.35 μM in 1 mL of solution
  (10.35×10-6)(0.001 mL) = 1.035×10-8 mol ADA
  (1.035×10-8 mol)(42000 g/mol) = 0.0004347 g ADA/mL
  (0.0004347 g ADA/mL)(1 mL/1000 μL)(109 ng/1 g) = 434.7 ng/μL

  (434.7 ng/μL)(V1) = (1 ng/μL)(1000 μL)
  V1 = 2.3 μL
  (1 ng/μL)(V1) = (0.1 ng/μL)(1000 μL)
  V1 = 100 μL

References

R&D Systems ADA Activity Assay
AU Biomaterials Design Lab ADA Activity Assay