User:Michael F. Nagle/Notebook/Chem 571/2012/10/03: Difference between revisions

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==Objective==
==Objective==
* Obtain UV/Vis spectra for HRP assay with absorbance at 510nm plotted against time
* Obtain kinetic UV/Vis spectra for HRP assay at wavelength 510.
==Procedure==
==Procedure==
#Concentrations will be kept constant from HRP assays done by another group in [[AU_Biomaterials_Design_Lab||AU's Biomaterial Design Lab]] with nothing changed except for the addition of varying concentrations of gold nanoparticles. Their writeup can be found here: [[Image:HRP Assays.pdf]]. The concentration of HRP was raised by 150% because it did not produce a peak at first.
*Procedure, and most concentrations will be the same as HRP assays done by another group<sup>1</sup> in [[AU_Biomaterials_Design_Lab||AU's Biomaterial Design Lab]].
#For six cuvettes to have 1.7mM hydrogen peroxide, 18μM iodophenol, 156.25μM AAP and 2.3μM HRP, the following volumes of the respective stock solutions (made [[User:Michael_F._Nagle/Notebook/Chem_571/2012/10/02|10/02]]) were added:
*The following modifications were made:
**Varying concentrations of gold nanoparticles will be added.  
**The HRP concentration was increased from 1.5μM to 2.3μM because it did not produce a peak at first.
#For six cuvettes to have 1.7mM hydrogen peroxide, 18μM iodophenol, 156.25μM AAP and 1.5μM and 2.3μM HRP, the following volumes of the respective stock solutions (made [[User:Michael_F._Nagle/Notebook/Chem_571/2012/10/02|10/02]]) were added:
##750μL 30% hydrogen peroxide solution
##750μL 30% hydrogen peroxide solution
##10μL .17M iodophenol solution
##10μL .17M iodophenol solution
##35μL 2.5mM AAP solution
##35μL 2.5mM AAP solution
##22μL 23μM HRP solution<br><br>
##15 μL 23μM HRP solution first, which did not produce a peak
###The rest were done with 22μL 23μM HRP solution
{|
{|
|+ Amount of gold and water used in each cuvette
|+ Amount of gold and water used in each cuvette
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==Data==
==Data==
We are waiting for the spectra to be uploaded by the researcher who has it. It will be posted here once available, and reaction rates will be calculated.
[[Image:HRPwgold.png]]


*'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:49, 7 October 2012 (EDT)''':why don't you have it? it is not an excuse for it not being in your notebook.
 
==Discussion==
*The more nanoparticles added, the more stretched out the curve and the slower the reaction. Although the trend isn't perfect, the sets with 80μL and 100μL begin to slope up much later, and reach a much lower absorption over the 3 minute trial span.
==References==
Names Removed. Horseradish Peroxidase Assays. [[Image:HRP Assays.pdf]]


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Revision as of 22:42, 6 December 2012

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Objective

  • Obtain kinetic UV/Vis spectra for HRP assay at wavelength 510.

Procedure

  • Procedure, and most concentrations will be the same as HRP assays done by another group1 in |AU's Biomaterial Design Lab.
  • The following modifications were made:
    • Varying concentrations of gold nanoparticles will be added.
    • The HRP concentration was increased from 1.5μM to 2.3μM because it did not produce a peak at first.
  1. For six cuvettes to have 1.7mM hydrogen peroxide, 18μM iodophenol, 156.25μM AAP and 1.5μM and 2.3μM HRP, the following volumes of the respective stock solutions (made 10/02) were added:
    1. 750μL 30% hydrogen peroxide solution
    2. 10μL .17M iodophenol solution
    3. 35μL 2.5mM AAP solution
    4. 15 μL 23μM HRP solution first, which did not produce a peak
      1. The rest were done with 22μL 23μM HRP solution
Amount of gold and water used in each cuvette
AuNP solution (μL) water (μL)
0 630
20 610
40 590
60 570
80 550
100 530

    1. The amount of stock solution needed of each reagant was calculated as such:
    2. M1V1=M2V2
    3. .023mM HRP stock * .001L = .0023mM HRP * volume needed
    4. 22μL HRP stock needed

Data


Discussion

  • The more nanoparticles added, the more stretched out the curve and the slower the reaction. Although the trend isn't perfect, the sets with 80μL and 100μL begin to slope up much later, and reach a much lower absorption over the 3 minute trial span.

References

Names Removed. Horseradish Peroxidase Assays. File:HRP Assays.pdf


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