Objective
- Obtain kinetic UV/Vis spectra for HRP assay at wavelength 510.
Procedure
- Procedure, and most concentrations will be the same as HRP assays done by another group1 in |AU's Biomaterial Design Lab.
- The following modifications were made:
- Varying concentrations of gold nanoparticles will be added.
- The HRP concentration was increased from 1.5μM to 2.3μM because it did not produce a peak at first.
- For six cuvettes to have 1.7mM hydrogen peroxide, 18μM iodophenol, 156.25μM AAP and 1.5μM and 2.3μM HRP, the following volumes of the respective stock solutions (made 10/02) were added:
- 750μL 30% hydrogen peroxide solution
- 10μL .17M iodophenol solution
- 35μL 2.5mM AAP solution
- 15 μL 23μM HRP solution first, which did not produce a peak
- The rest were done with 22μL 23μM HRP solution
Amount of gold and water used in each cuvette
| AuNP solution (μL) |
water (μL)
|
| 0 |
630
|
| 20 |
610
|
| 40 |
590
|
| 60 |
570
|
| 80 |
550
|
| 100 |
530
|
- The amount of stock solution needed of each reagant was calculated as such:
- M1V1=M2V2
- .023mM HRP stock * .001L = .0023mM HRP * volume needed
- 22μL HRP stock needed
Data
Discussion
- The more nanoparticles added, the more stretched out the curve and the slower the reaction. Although the trend isn't perfect, the sets with 80μL and 100μL begin to slope up much later, and reach a much lower absorption over the 3 minute trial span.
References
Names Removed. Horseradish Peroxidase Assays. File:HRP Assays.pdf
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Project name
