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

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(Atomic Absorption of Au-Lysozyme and Au-HRP Solutions)
 
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   '''V<sub>1</sub> = 0.134 mL'''
   '''V<sub>1</sub> = 0.134 mL'''
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==UV-Vis Analysis of Au-HRP Solutions==
 +
* The Au-HRP solutions were made on [[User:Dhea_Patel/Notebook/Experimental_Biological_Chemistry_Notebook/2012/11/07|2012/11/07]] and the spectra of the solutions were obtained over 200 to 800 nm in quartz cuvettes.  Please refer to [[User:Dhea_Patel/Notebook/Experimental_Biological_Chemistry_Notebook/2012/11/13|Dhea Patel's entry]] for a photograph of the solutions.
 +
[[Image:UVvis_of_AUHRP_.png]]
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* 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 HAuCl<sub>4</sub> in these solutions, based on [http://www.nanoscalereslett.com/content/pdf/1556-276X-5-124.pdf literature].  Note that the literature indicated that a peak at around 287 nm is present in the spectra of aqueous HAuCl<sub>4</sub>.
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==Atomic Absorption of Au-Lysozyme and Au-HRP Solutions==
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* The Au-lysozyme solutions were made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/10/31|2012/10/31]].
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* The Au-HRP solutions were made on [[User:Dhea_Patel/Notebook/Experimental_Biological_Chemistry_Notebook/2012/11/07|2012/11/07]].
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 +
[[Image:AAS_AuLys.JPG]]
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<br><br>
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[[Image:AAS_Percent_Change_Au.jpg]]
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<br><br>
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''Au-Lys Mole Ratio'''
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| align="center" style="background:#f0f0f0;"|'''Au [ppm]'''
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|-
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| 20||0.0057
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|-
 +
| 30||1.9388
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|-
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| 40||2.7586
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|-
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| 50||3.806
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|-
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| 60||4.5002
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|-
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| 70||-2.4599
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|-
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| 80||-2.4839
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|-
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| 100||-2.5856
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|-
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| 120||-2.6394
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|-
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| 130||-2.6454
|}
|}
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* 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 [[User:Melissa_Novy/Notebook/CHEM-571/2012/11/06|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.
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<br>
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[[Image:AAS_AuHRP.JPG]]
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<br><br>
 +
{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''Mole Ratio of Au to HRP'''
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| align="center" style="background:#f0f0f0;"|'''Au [ppm]'''
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|-
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| 10||-1.5443
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|-
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| 50||1.8191
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|-
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| 100||11.7714
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|-
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| 150||13.1419
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|-
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| 200||23.6748
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|-
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| 250||24.4588
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|-
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| 300||27.876
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|-
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| 350||25.4761
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|-
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| 400||35.1173
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|-
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| 450||25.6198
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|}
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<br>
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[[Image:AAS_AuHRP_Percent.jpg]]
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<br>
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{| {{table}}
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| align="center" style="background:#f0f0f0;"|'''HRP []'''
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| align="center" style="background:#f0f0f0;"|'''HAuCl4 []'''
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| align="center" style="background:#f0f0f0;"|'''HRP [uL]'''
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| align="center" style="background:#f0f0f0;"|'''HAuCl4 [uL]'''
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| align="center" style="background:#f0f0f0;"|'''Water [uL]'''
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| align="center" style="background:#f0f0f0;"|'''Mol Au [mol]'''
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| align="center" style="background:#f0f0f0;"|'''Mass Au [mg]'''
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| align="center" style="background:#f0f0f0;"|'''Initial [Au] [ppm]'''
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| align="center" style="background:#f0f0f0;"|'''Final [Au] [ppm]'''
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| align="center" style="background:#f0f0f0;"|'''Change in [Au] [ppm]'''
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| align="center" style="background:#f0f0f0;"|'''Percent Change in [Au] [%]'''
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|-
 +
| 1||10||280.7||7.62||7711.7||0.00000008||0.01575736||1.96967||-1.5443||3.51397||178.4039966
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|-
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| 1||50||280.7||38.1||7681.2||0.0000004||0.0787868||9.84835||1.8191||8.02925||81.52888555
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|-
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| 1||100||280.7||76.2||7643.1||0.0000008||0.1575736||19.6967||11.7714||7.9253||40.23668939
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|-
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| 1||150||280.7||114.3||7605.0||0.0000012||0.2363604||29.54505||13.1419||16.40315||55.51911403
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|-
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| 1||200||280.7||152.4||7566.9||0.0000016||0.3151472||39.3934||23.6748||15.7186||39.90160788
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|-
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| 1||250||280.7||190.5||7528.8||0.000002||0.393934||49.24175||24.4588||24.78295||50.32914143
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|-
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| 1||300||280.7||228.5||7490.7||0.0000024||0.4727208||59.0901||27.876||31.2141||52.82458483
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|-
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| 1||350||280.7||266.6||7452.7||0.0000028||0.5515076||68.93845||25.4761||43.46235||63.04515115
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|-
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| 1||400||280.7||304.7||7414.6||0.0000032||0.6302944||78.7868||35.1173||43.6695||55.42743201
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|-
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| 1||450||280.7||342.8||7376.5||0.0000036||0.7090812||88.63515||25.6198||63.01535||71.09521448
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|}
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<br>
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* 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.
 +
 +
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<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
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__NOTOC__
__NOTOC__

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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.

Image: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

Image:AAS_AuLys.JPG

Image:AAS_Percent_Change_Au.jpg

Au-Lys Mole Ratio Au [ppm]
200.0057
301.9388
402.7586
503.806
604.5002
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.


Image:AAS_AuHRP.JPG

Mole Ratio of Au to HRP Au [ppm]
10-1.5443
501.8191
10011.7714
15013.1419
20023.6748
25024.4588
30027.876
35025.4761
40035.1173
45025.6198


Image: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] [%]
110280.77.627711.70.000000080.015757361.96967-1.54433.51397178.4039966
150280.738.17681.20.00000040.07878689.848351.81918.0292581.52888555
1100280.776.27643.10.00000080.157573619.696711.77147.925340.23668939
1150280.7114.37605.00.00000120.236360429.5450513.141916.4031555.51911403
1200280.7152.47566.90.00000160.315147239.393423.674815.718639.90160788
1250280.7190.57528.80.0000020.39393449.2417524.458824.7829550.32914143
1300280.7228.57490.70.00000240.472720859.090127.87631.214152.82458483
1350280.7266.67452.70.00000280.551507668.9384525.476143.4623563.04515115
1400280.7304.77414.60.00000320.630294478.786835.117343.669555.42743201
1450280.7342.87376.50.00000360.709081288.6351525.619863.0153571.09521448


  • 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.



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