User:Moira M. Esson/Notebook/CHEM-571/2013/10/16

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(Catalytic activity testing)
Current revision (16:52, 21 October 2013) (view source)
(Catalytic activity testing)
 
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*No activity of the HRP was observed (see figure below).  
*No activity of the HRP was observed (see figure below).  
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Figure 1. Absorbance spectra of the catalytic activity of HRP-AuNPs
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[[Image:AbsorbancespectracatalyticactivityHRPAuNPs.png]]
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*None of the absorbance peaks changed throughout the anaylsis, indicating that HRP was rendered inactive by the incorporation of AuNPs. This may be due to the excessive heat applied to the protein during the incorporation. The activity of other proteins will be tested to determine if a new method of incorporation of AuNPs must be used in order to maintain the functionality of the protein.
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*Despite repreparing the luminol/HRP solutions multiple times, the strange, high absorbance peak at 300nm remained. This may indicate either HRP or luminol was prepared incorrectly.
==UV/vis of Citrate and BSA samples==
==UV/vis of Citrate and BSA samples==

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Objectives

  • Test the enzymatic/catalytic activity of HRP-AuNPs using luminol and UV/vis with the goal of determining the activity of HRP-AuNPs.
  • Calculate the absorbance prepared Citrate-Au solutions and BSA-Au solutions as standards (solutions were previously prepared on 08/28/2013


Catalytic activity testing

  • Catalytic activity was tested using UV/vis spectroscopy.
  • 3mL sample of luminol, H2O2 and buffer was placed in cuvette with micro-stir bar. After 1min., 100μL HRP-Au was added.
  • A spectrum was taken every 15 seconds for ten minutes.


Sample preparation

 Buffer:0.6140g Tris in 1L, pH set to 8 with HCl ---> 5.1mM
 Luminol:Dissolve 12.9mg luminol in 300uL of DMSO and add to 50mL buffer---> 1.46mM
 H2O2: 312uL 30% H2O2 into 100mL buffer ---> Should be 45mM


  • Preparation of HRP-AuNP samples:
    • General Description of the samples:
      • The final concentration of luminol is such that the absorbance at 333nm is less than one(this was confirmed using UV/vis)
      • The final concentration of hydrogen peroxide is fifty times that of the luminol.
      • Using the 60:1 [Au:HRP] solution, 100uL will be used
      • Add buffer so that the final concentration is 3mL
 Calculations for preparation of sample:
   Luminol stock solution preparation 5mL 7.26E-5 M luminol: 0.261mL luminol stock added to 5mL buffer.
   H2O2 stock preparation of 5mL 0.0038M(this is 50x more concentrated): 0.169mL stock in 5mL buffer.
  • Final 3mL solution of luminol, H2O2 and buffer was prepared by adding 1mL luminol, 1mL H2O2 and 1mL buffer.
  • Concentration of luminol was chosen based on previous luminol data on 10/01/2013.


Testing of HRP-AuNP activity

  • The first prepared sample of luminol and H2O2 already had completely oxidized luminol. A new solution was prepared.
  • No activity of the HRP was observed (see figure below).


Figure 1. Absorbance spectra of the catalytic activity of HRP-AuNPs
Image:AbsorbancespectracatalyticactivityHRPAuNPs.png

  • None of the absorbance peaks changed throughout the anaylsis, indicating that HRP was rendered inactive by the incorporation of AuNPs. This may be due to the excessive heat applied to the protein during the incorporation. The activity of other proteins will be tested to determine if a new method of incorporation of AuNPs must be used in order to maintain the functionality of the protein.
  • Despite repreparing the luminol/HRP solutions multiple times, the strange, high absorbance peak at 300nm remained. This may indicate either HRP or luminol was prepared incorrectly.

UV/vis of Citrate and BSA samples

  • A new BSA-Au sample was prepared because the protein had crashed out of solution. The BSA solution was prepared following the protocol described on 08/28/2013. The absorbance of the BSA solution will be determined during the next meeting.
 Preparation of BSA stock solution
  0.025g BSA in 25mL tris buffer(described above)-->15μM BSA
  • A 1/10 dilution of Citrate-Au solution was measured using UV/vis


Figure 2. Absorbance spectra of 1/10 dilution of Citrate-AuNP and 1/10 dilution of 30:1 HRP-Au solution




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