February 18th
Goals
- Make graph showing max peak over time
- Making HRP nanoparticles using dopamine method
- Run UV vis on HRP nanoparticles to see if there is any difference after having it run a week later
- Run UV vis on HRP supernatant
- We want to see there is free HRP in the solution and that all of the HRP did not get spun down
- make hemoglobin nanoparticles using the alpha method
Procedures
Other things to do
- take aliquot of 10^-8M HRP nanoparticles and put in freezer
- Protein Gold Nanoparticles
- I already have my 10^-6M Hemoglobin solution made
- Take 1ml of the solution and add to 9ml of nanoparticles...take 100ul and add to 9.9mls of gold to make solutions
- one solution was 10^-7M protein in nanoparticles. The other was 10^-8M.
Making HRP nanoparticles
- First I made the buffer solution. The pH was 5. I made it using 0.0003 moles of acetic acid 1M and 0.0006 moles of potassium acetate
- add 300ul of acid and 0.059g of base
- Going to make 10^-6M HRP solution in the buffer
- HRP molecular mass is 44kDa or 44000g/mol
- we then filled up 100ml volumetric flask
- Take 1ml of the solution and add to 9ml of nanoparticles...take 100ul and add to 9.9mls of gold to make solutions
- one solution was 10^-7M protein in nanoparticles. The other was 10^-8
measure activity of HRP nanoparticles
- HRP-AuNP complex solution from the alpha method and the polydopamine-coated AuNPs 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 AuNPs were stored at 4 degrees Celsius
- The resuspended HRP-AuNP 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
- Stock solution of OPD with a concentration of 1mg/mL
- Stock solution of H2O2 113uL
Notes
- HRP was 10^-8 M added to nanoparticles
- when I made the new HRP particles, there was definitely some wierd impurities present...this is important to note if the HRP doesn't work
Reflections
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