User:Alexander Cvitan/Notebook/Experimental Biological Chemistry Lab/2013/10/16

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Objective

  • To complete the procedure outlined by Dr.Hartings HERE.
  • Run UV-Vis on Citrate, BSA, HRP-Au nanoparticles. Run a water blank.
  • Run kinetic analysis on the 60:1 diluted HRP-AU nanoparticles.
  • Remake BSA nanoparticles. (The BSA nanoparticles made previously formed fibers, rendering them useless).

Procedure

Kinetics of HRP-AuNP's

Solutions Made

  • Luminol
    • Stock was 1.46mM
    • A 20 times dilution was made to make a final solution volume of .072mM. (This was done in order to observe an abs on UV-Vis less than 1).
    • .15ml of Luminol was used for the reaction.
  • Hydrogen Peroxide
    • Stock was 44.29mM
    • Was diluted to be 50 times the concentration of the Hydrogen Peroxide. Thus the concentration is, 3.65mM.
    • .247ml of hydrogen peroxide was used for the reaction.
  • HRP-AuNP's
    • Originally 100ul of the 60:1 diluted sample was used. This solution was made using Au [] of .25mM and a HRP [] of 4.167uM. This caused an extremely slow kinetic reaction.
    • This reaction was then repeated using 1ml of HRP-AuNP's.
  • For both kinetic runs luminol and hydrogen peroxide were added together. HRP-AuNP's were then added to get the reaction to start. To ensure that both runs were the same volume, the volume of each run was normalized to 3ml using buffer.

BSA Nanoparticle Synthesis

Procedure for Synthesis can be found HERE.

  • [HAuCl4] Stock- 2.33mM
  • [BSA] Stock- 15uM
  • Concentration of Gold must be 90 times the concentration of BSA.
  • In sum, we added 1.73ml of BSA and 1ml of HAuCl4 and diluted up to 10ml. This creates a molar ratio of 1:90

UV-Vis Runs

  • BSA Nanoparticles weren't run because they were ready before the end of class.
  • 1ml of Citrate-AuNP's were run with a concentration of .8349nM. This sample was a 10x dilution of the pure citrate nanoparticles synthesized.
  • 1ml of HRP-AuNP's were run. These a 10x dilution was taken of the 60:1 sample of HRP-Nanoparticles, essentially making the dilution, 600:1. Exact concentrations are known at this time because samples were just synthesized yesterday.
  • A water blank was also recorded.

Data

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