User:Carly M. Montanero/Notebook/CHEM-571/2013/10/16: Difference between revisions

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==Objective==
To test the activity of our HRP-NPs for the catalytic conversion of luminol.
==Procedure==
==Procedure==
*Luminol --> final concentration such that A(330)< 1.
'''Creating the Luminol-H<sub>2</sub>O<sub>2</sub> Solution'''
*Hydrogen peroxide --> final concentration to be 50x luminol concentration
# Add 969.61 μL of the luminol stock and 1.598 mL of the hydrogen peroxide stock to a 10 mL volumetric flask.
*HRP --> 60:1 Au:HRP solutions, 100 μL
# Dilute the solution to 10 mL with buffer.
*Buffer --> add up to 3 mL
 
'''Enzyme Kinetics Measurement'''
# Add 2 mL of the luminol-H<sub>2</sub>O<sub>2</sub> solution to a cuvette.
# Start kinetics measurement:
:* 1 ms integration
:* 10 scan average
:* Set "Save the first available scan every" to 15 seconds
:* Set "Stop after this amount of time" to 10 minutes
:* Set "File Type" to Tab Delimited
:* Just before 1 minute, add 1 mL of 60:1 Au:HRP solution.


In cuvette, luminol, buffer, and hydrogen peroxide
'''BSA-AuNP Synthesis'''
* Start kinetics by adding HRP
# Add 1.00 mL of 2.33 mM gold solution (HAuCL<sub>4</sub>) to a 10 mL volumetric flask.
# Add 1.73 mL BSA (90x more gold than BSA) to the volumetric flask.
# Dilute the solution up to 10 mL with deionized water.
# Transfer the solution to a test tube and cap with aluminum foil.
# Heat in the oven at 80°C for four hours.
# Transfer the solution to a plastic falcon tube.


*What percent of HRP is still active?
'''Remaking the Solution of Gold and HRP'''
# For 60:1 Au:HRP solution, 2.56 mL HRP stock and 850.34 μL of gold stock were added to a 10 mL volumetric flask. The solution was diluted up to 10 mL with deionized water.
# The 10 mL solution of gold and HRP was placed into a test tube.
# The test tube was placed in the oven at 80°C for four hours.


Control:
==Figures==
*experiment with BSA-AuNPs and citrate-AuNPs
[[Image:Cmj_HRPKineticsUV-Vis_10.16.2013.png ]]
:* what concentration of AuNPs about the same (use absorption spectra)
*Note: Spectra overlap for a total elapsed time of 19 minutes.
[[Image:Cmj_HRPKinetics_520overtime_10.16.2013.png]]


==Data==
==Notes==
A1V1 = A2V2
* The buffer was 5.1 mM Tris at pH 8.
(0.3)(100 μL)=A2(3 mL)
* The stock concentration of luminol was 1.46 mM.
A2 = ?
* The stock concentration of hydrogen peroxide was 44.29 mM.
* The stock concentration of gold (HAuCL<sub>4</sub>)was 2.33 mM.
* The stock concentration of BSA was 15 μM.
* The stock solution of gold was 2.94 mM.
* The stock solution of HRP was 16.27 μM.


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Revision as of 05:08, 29 October 2013

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Objective

To test the activity of our HRP-NPs for the catalytic conversion of luminol.

Procedure

Creating the Luminol-H2O2 Solution

  1. Add 969.61 μL of the luminol stock and 1.598 mL of the hydrogen peroxide stock to a 10 mL volumetric flask.
  2. Dilute the solution to 10 mL with buffer.

Enzyme Kinetics Measurement

  1. Add 2 mL of the luminol-H2O2 solution to a cuvette.
  2. Start kinetics measurement:
  • 1 ms integration
  • 10 scan average
  • Set "Save the first available scan every" to 15 seconds
  • Set "Stop after this amount of time" to 10 minutes
  • Set "File Type" to Tab Delimited
  • Just before 1 minute, add 1 mL of 60:1 Au:HRP solution.

BSA-AuNP Synthesis

  1. Add 1.00 mL of 2.33 mM gold solution (HAuCL4) to a 10 mL volumetric flask.
  2. Add 1.73 mL BSA (90x more gold than BSA) to the volumetric flask.
  3. Dilute the solution up to 10 mL with deionized water.
  4. Transfer the solution to a test tube and cap with aluminum foil.
  5. Heat in the oven at 80°C for four hours.
  6. Transfer the solution to a plastic falcon tube.

Remaking the Solution of Gold and HRP

  1. For 60:1 Au:HRP solution, 2.56 mL HRP stock and 850.34 μL of gold stock were added to a 10 mL volumetric flask. The solution was diluted up to 10 mL with deionized water.
  2. The 10 mL solution of gold and HRP was placed into a test tube.
  3. The test tube was placed in the oven at 80°C for four hours.

Figures

  • Note: Spectra overlap for a total elapsed time of 19 minutes.

Notes

  • The buffer was 5.1 mM Tris at pH 8.
  • The stock concentration of luminol was 1.46 mM.
  • The stock concentration of hydrogen peroxide was 44.29 mM.
  • The stock concentration of gold (HAuCL4)was 2.33 mM.
  • The stock concentration of BSA was 15 μM.
  • The stock solution of gold was 2.94 mM.
  • The stock solution of HRP was 16.27 μM.


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