- Make GNPs with hemoglobin
- Determine if the HRP GNPs are active
Monitoring Activity of HRP-GNP complexes
- HRP-GNP complex solution from the alpha method and the polydopamine-coated GNPs were placed in high velocity tubes.
- The nano particle solutions were spun at 18,000rpm for 3 hours.
- The supernatants were discarded and the pellets were resuspended in water.
- The polydopamine-coated GNPs were stored at 4 degrees Celsius
- The resuspended HRP-GNP complex solutions were analyzed for activity using UV-Vis
- The nano particles were added to hydrogen peroxide and OPD (Ophenylenediamine)
- Active HRP catalyzes the reaction of Ophenylenediamine with H2O2 to produce 2,3-Diaminophenazine
- OPD showed no changed after 30min, 60min, 90min, and 150min
- The HRP GNPs were active; activity increased with time
UV-Vis spectra were recorded for solutions over a period of time
- One solution contained peroxide and OPD only
- One solution contained peroxide, OPD and the HRP-GNP
The formation of 2,3-Diaminophenazine will produce a peak between 350 and 400 nm.
The spectra of both solution were compared over time to determine whether or not there were active HRP molecule with the GNPs.
- The results showed that the solution containing the HRP-GNPs had a significantly faster rate of formation for 2,3-Diaminophenazine.
This result confirms the presence of active HRP molecule in the GNP solution. The next step is determining whether or not these active HRP
molecules are attaches to the nano particles.