User:Melissa Novy/Notebook/CHEM-571/2012/10/24

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Objectives

  • Optimize the HRP-luminol chemiluminescence assay.
    • Carbonate buffer, luminol, HRP, and H2O2

solutions made on 2012/10/23 were used.

Assay Solution Concentrations and Volumes

' Volume and Stock Concentration Added to Cuvette ' ' ' Volume Added to Cuvette [µL] '
Trial Luminol H2O2 4-Iodophenol HRP Water Carbonate Buffer
1 454 µL at 0.564 mM 112.7 µL at 5 mM 13.2 µL at 18 mM 33 µL at 9.6 µM 1033 µL x
2 454 µL at 0.564 mM 112.7 µL at 5 mM 13.2 µL at 18 mM 33 µL at 9.6 µM 1033 µL x
3 454 µL at 0.564 mM 112.7 µL at 5 mM 13.2 µL at 18 mM 33 µL at 9.6 µM 2000 µL x
4 454 µL at 0.564 mM 112.7 µL at 5 mM 13.2 µL at 18 mM 66 µL at 0.96 µM 2000 µL x
5 454 µL at 0.564 mM 112.7 µL at 5 mM 13.2 µL at 18 mM 33 µL at 2.3 µM x 1000 µL
6 454 µL at 0.564 mM 112.7 µL at 5 mM 13.2 µL at 18 mM 16.5 µL at 2.3 µM x 1000 µL
7 454 µL at 0.564 mM 112.7 µL at 5 mM 13.2 µL at 18 mM 16.5 µL at 0.96 µM x 1000 µL
8 454 µL at 0.564 mM 112.7 µL at 5 mM 13.2 µL at 18 mM 16.5 µL at 2.3 µM x 2000 µL
  • Note that the concentration of the carbonate buffer was ? M and pH 8.5.
  • Trials 5 through 8 used another HRP solution that was diluted with carbonate buffer instead of deionized water.
    • 25 μL of 9.6 μM HRP in water was added to 75 μL of carbonate buffer to produce a solution of 2.3 μM HRP in carbonate buffer.
    • 417.4 μL of this 2.3 μM HRP solution in carbonate buffer was then added to 582.6 μL of carbonate buffer to produce a solution of 0.96 μM HRP in carbonate buffer.
  • Trials 1 through 4 were diluted with water, while trials 5 through 8 were diluted with carbonate buffer.

Emission Spectra and Data Analysis

  • Please refer to Dhea Patel's entry for an emission spectrum taken at 425 nm of trial 6 of the chemiluminescence assay.
  • Note that all trials were conducted over a time period of 200 s in a darkened room. The cuvette containing all of the components of the reaction solution, with the exception of the 5 mM H2O2 solution, were placed in the sample cell. The detector was activated, and then the 5 mM H2O2 solution was pipetted into the cuvette to begin the reaction.
  • The time difference between the activation of the detector and the start of the reaction can be seen in the spectrum of trial 6. It appears that luminescence was first registered by the instrument at about 8 seconds. However, the intensity quickly decreased over a period of about 7 seconds and returned to a near-baseline intensity.
  • The reason for using the spectrum of trial 6 is that it produced the longest-lasting luminescence of all trials. Therefore, it must be assumed that diluting assay solutions with carbonate buffer and using HRP diluted in carbonate buffer at a relatively lower concentration than before will produce more useful results.
  • If further tests are conducted, it may be advantageous to dilute the assay solutions with carbonate buffer at higher and lower concentrations.