User:Megan L. Channell/Notebook/Horseradish/2013/10/16: Difference between revisions
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|style="background-color: #EEE"|[[Image:BDLlogo_notext_lr.png|128px]]<span style="font-size:22px;"> Biomaterials Design Lab</span> | |||
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html> </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}} | |||
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==Objective== | |||
To test the activity of our HRP-NPs for the catalytic conversion of luminol. | |||
==Procedure== | |||
'''Creating the Luminol-H<sub>2</sub>O<sub>2</sub> Solution''' | |||
# 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. | |||
'''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. | |||
'''BSA-AuNP Synthesis''' | |||
# 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. | |||
'''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. | |||
==Data== | |||
[[Image:Cmj_HRPKineticsUV-Vis_10.16.2013.png ]] | |||
*Note: Spectra overlap for a total elapsed time of 19 minutes. | |||
[[Image:Cmj_HRPKinetics_520overtime_10.16.2013.png]] | |||
==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 (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. | |||
[[Category:CHEM571]] | |||
[[Category:horseradish peroxidase]] | |||
[[Category:luminol]] | |||
Revision as of 14:52, 20 October 2013
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