User:Zachary I. Mendel/Notebook/Zacks Notebook/2014/02/18

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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
        • added 0.0044g of HRP
      • 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

  1. HRP-AuNP complex solution from the alpha method and the polydopamine-coated AuNPs were placed in high velocity tubes.
  2. The nano particle solutions were spun at 18,000rpm for 3 hours.
  3. The supernatants were discarded and the pellets were resuspended in water.
  4. The polydopamine-coated AuNPs were stored at 4 degrees Celsius
  5. The resuspended HRP-AuNP complex solutions were analyzed for activity using UV-Vis
    1. The nano particles were added to hydrogen peroxide and OPD (Ophenylenediamine)
    2. Active HRP catalyzes the reaction of Ophenylenediamine with H2O2 to produce 2,3-Diaminophenazine

      1. Stock solution of OPD with a concentration of 1mg/mL
      2. 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

Data

Alpha HRP Pellet
Alpha HRP Pellet
This graph shows absorbance increases in the HRP Pellet over time


Alpha HRP supernatant Image:HRP Supernatant.png
This graph shows absorbance increasing slightly in the HRP supernatant over time

Control. OPD and H202 only
Image:CONTROL HRP ALPHA.png
This graph reveals what happens to absorbance over time when no HRP nanoparticles are present
Here there is only hydrogen peroxide and OPD


Reflections


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