To test the activity of our HRP-NPs for the catalytic conversion of luminol.
Creating the Luminol-H2O2 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-H2O2 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.
- Add 1.00 mL of 2.33 mM gold solution (HAuCL4) 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.
File:Cmj HRPKineticsUV-Vis 10.16.2013.png Note: Spectra overlap for a total elapsed time of 19 minutes.
- 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.