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

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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.

UVvis of AUHRP .png

  • 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

AAS AuLys.JPG

AAS Percent Change Au.jpg

Au-Lys Mole Ratio Au [ppm]
20 0.0057
30 1.9388
40 2.7586
50 3.806
60 4.5002
70 -2.4599
80 -2.4839
100 -2.5856
120 -2.6394
130 -2.6454
  • 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.


AAS AuHRP.JPG

Mole Ratio of Au to HRP Au [ppm]
10 -1.5443
50 1.8191
100 11.7714
150 13.1419
200 23.6748
250 24.4588
300 27.876
350 25.4761
400 35.1173
450 25.6198


AAS AuHRP Percent.jpg

HRP [] HAuCl4 [] HRP [uL] HAuCl4 [uL] Water [uL] Mol Au [mol] Mass Au [mg] Initial [Au] [ppm] Final [Au] [ppm] Change in [Au] [ppm] Percent Change in [Au] [%]
1 10 280.7 7.62 7711.7 0.00000008 0.01575736 1.96967 -1.5443 3.51397 178.4039966
1 50 280.7 38.1 7681.2 0.0000004 0.0787868 9.84835 1.8191 8.02925 81.52888555
1 100 280.7 76.2 7643.1 0.0000008 0.1575736 19.6967 11.7714 7.9253 40.23668939
1 150 280.7 114.3 7605.0 0.0000012 0.2363604 29.54505 13.1419 16.40315 55.51911403
1 200 280.7 152.4 7566.9 0.0000016 0.3151472 39.3934 23.6748 15.7186 39.90160788
1 250 280.7 190.5 7528.8 0.000002 0.393934 49.24175 24.4588 24.78295 50.32914143
1 300 280.7 228.5 7490.7 0.0000024 0.4727208 59.0901 27.876 31.2141 52.82458483
1 350 280.7 266.6 7452.7 0.0000028 0.5515076 68.93845 25.4761 43.46235 63.04515115
1 400 280.7 304.7 7414.6 0.0000032 0.6302944 78.7868 35.1173 43.6695 55.42743201
1 450 280.7 342.8 7376.5 0.0000036 0.7090812 88.63515 25.6198 63.01535 71.09521448


  • 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.