- We have to redo the dopamine addition that we did last Wednesday
- We are going to test the activity of citrate-HRP-AuNPs using O2 binding assays
Activity of citrate-HRP-AuNPs
- We are going to use an assay provided by Dr. Hartings
- The assay uses a reagent that changes color when HRP is added in addition to hydrogen peroxide
- The hydrogen peroxide alone will change the color of the solution, so we will be comparing hydrogen peroxide alone with hydrogen peroxide and AuNPs
- We also are going to run UV Vis of the samples to see how the absorption spectra changes over time
- Dissolve dopamine in 100mL Tris Buffer to have a 10mM solution at pH=8.5
- Mix 17mL of AuNP solution with 17mL of the dopamine solution
- Stir under air at room temperature for 1 hour
- Purify by centrifuging and then redisperse in water.
- Filtrate through syringe filters with cellulose acetate membranes with pore size of 1um
- Make 2 solutions.
- to be stored over night on lab bench
- to be stored over night in the refrigerator
- the nanoparticles with HRP were functional
- In the 10^-11 dillution, we observed a color change from red initially to a much lighter solution
- In our calculations we decided to use teh 10^-11 and water as our blank, so we could subtract protein + water.
Protein, OBD, H202
- For each sample run in UV-Vis there was 0.225ml HRP, 2.25ml OBD stock and 0.75ml hydrogen peroxide stock
Today it seemed as if our nanoparticles with HRP were active. However, we forgot one key thing. It is possible that there were free HRP in the solution which could have resulted in the activity seen through UV Vis. Tomorrow we will need to centrifuge the solutions to take just the nanoparticles with protein attached to it, and determine confidently whether or not the proteins embedded on the nanoparticles are active.