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

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[[Image:Oct_16,_Exper_biochem_kinetics_HRPAUNP's.png]]
[[Image:Oct_16,_Exper_biochem_kinetics_HRPAUNP's.png]]
*Based on the relatively static graph, or due to the lack in a shift in the spectra overtime shows that the HRP linked to the AuNP's is inactive.
*Based on the relatively static graph, or due to the lack in a shift in the spectra overtime shows that the HRP linked to the AuNP's is inactive.
*This inactivity is most likely explained by the  method of synthesis. If the heat was too high while preparing the HRP-link nanoparticles it could have denatured the protein.
*This inactivity is most likely explained by the  method of synthesis. If the heat was too high while preparing the HRP-linked nanoparticles it could have denatured the protein.


===UV-Vis Spectra of AU-NP's Currently Synthesized===
===UV-Vis Spectra of AU-NP's Currently Synthesized===

Revision as of 19:25, 21 October 2013

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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
  • Put in oven at 80 degrees Celsius for 3-4 hours.
    • Note the nanoparticles were placed in the fridge afterward to prevent the formation of nanoparticle fibers.

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

HRP AU-NP's

  • This graph shows the catalytic activity of of HRP-linked AuNP's in relation to luminol.

  • Based on the relatively static graph, or due to the lack in a shift in the spectra overtime shows that the HRP linked to the AuNP's is inactive.
  • This inactivity is most likely explained by the method of synthesis. If the heat was too high while preparing the HRP-linked nanoparticles it could have denatured the protein.

UV-Vis Spectra of AU-NP's Currently Synthesized

  • The spectra represented below are going to be used to compare the different nanoparticles synthesized in lab. Note that the BSA nanoparticles aren't shown here because they had to be remade in lab last class due to the previous batch solidifying.

  • Peak for Citrate AuNP's: 523nm
  • Peak for HRP-AuNP's: 526nm
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