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

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==Procedure==
==Procedure==
'''Creating the Luminol-H<sub>2</sub>O<sub>2</sub> Solution'''
'''Creating the Luminol-H<sub>2</sub>O<sub>2</sub> Solution'''
# Add 1.293 mL of the luminol stock and 2.131 mL of the hydrogen peroxide stock to a 10 mL volumetric flask.  
# Add 969.61 μL of the luminol stock and 1.598 mL of the hydrogen peroxide stock to a 10 mL volumetric flask.  
# Dilute the solution to 10 mL with buffer.
# Dilute the solution to 10 mL with buffer.


'''Enzyme Kinetics Measurement'''
'''Enzyme Kinetics Measurement'''
# Add 3 mL of the luminol-H<sub>2</sub>O<sub>2</sub> solution to a cuvette.
# Add 2 mL of the luminol-H<sub>2</sub>O<sub>2</sub> solution to a cuvette.
# Start kinetics measurement:
# Start kinetics measurement:
:* 1 ms integration
:* 1 ms integration
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:* Set "Stop after this amount of time" to 10 minutes
:* Set "Stop after this amount of time" to 10 minutes
:* Set "File Type" to Tab Delimited
:* Set "File Type" to Tab Delimited
:* Just before 1 minute, add 100 μL of 60:1 Au:HRP solution.  
:* Just before 1 minute, add 1 mL of 60:1 Au:HRP solution.
 
 
*What percent of HRP is still active?
 
Control:
*experiment with BSA-AuNPs and citrate-AuNPs
:* what concentration of AuNPs about the same (use absorption spectra)
 
 


==Notes==
==Notes==

Revision as of 11:37, 16 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.

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.


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