User:Helen L. Slucher/Notebook/CHEM-572/2014/02/11

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Objective

  • Remake the dopamine addition from 02/05
  • Test the activity of citrate-HRP-GNPs using O2 binding assays

Procedure

Activity of citrate-HRP-GNPs

  • 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 GNPs
  • We also are going to run UV Vis of the samples to see how the absorption spectra changes over time

Dopamine Coating

  1. Dissolve dopamine in 100mL Tris Buffer to have a 10mM solution at pH=8.5
  2. Mix 17mL of GNP solution with 17mL of the dopamine solution
  3. Stir under air at room temperature for 1 hour
  4. Purify by centrifuging and then redisperse in water.
  5. Filtrate through syringe filters with cellulose acetate membranes with pore size of 1um
  6. Make 2 solutions.
    1. to be stored over night on lab bench
    2. to be stored over night in the refrigerator

Data

  • The GNPs with HRP were functional
  • In the 10^-11 dilution, we observed a color change from deep red initially to a much lighter red
  • In our calculations we decided to use the 10^-11 dilution and water as our blank, so we could subtract protein from water.

Protein, OBD, H202

  • For each sample run in UV-Vis there were:
  1. 0.225mL HRP
  2. 2.25mL OBD stock
  3. 0.75mL hydrogen peroxide stock



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