Luminol-Based Enhanced Chemiluminescence Assay for Quantification of Peroxidase and Hydrogen Peroxide in Aqueous Solutions: Effect of Reagent pH and Ionic Strength

Luminol-Based Enhanced Chemiluminescence Assay for Quantification of Peroxidase and Hydrogen Peroxide in Aqueous Solutions: Effect of Reagent pH and Ionic Strength- Oct 2010

Alok Bhandari, Wongee Kim, and Keith Hohn- Iowa State University, Kansas State University
The effect of reagent pH and ionic strength was evaluated on the horseradish peroxidase (HRP)-luminol-p-iodophenol enhanced
chemiluminescence assay. This assay was optimized for HRP and H2O2 quantification during enzyme-mediated remediation of groundwater or wastewater contaminated with phenolic chemicals. The maximum chemiluminescence intensity (ICLmax) and total area under the chemiluminescence intensity profiles were measured as a function of p-iodophenol concentration, reagent solution pH, and reagent ionic strength. ICLmax values were optimum at pH 8.5 and increased linearly with reagent ionic strength. Optimum chemiluminescence enhancement was produced at a p-iodophenol concentration of 0.3 mM under the reaction conditions. ICLmax values were linearly correlated with HRP and H2O2 concentrations within the ranges of 0.1–1 activity units/mL and 0.1–1 mM, respectively. Results indicate that the HRP-luminol-p-iodohenol enhanced chemiluminescence assay has a potential to be used for quantification of HRP activity and H2O2 concentration in aqueous solutions encountered in groundwater remediation or wastewater treatment scenarios. … Experiments were first conducted for baseline reaction conditions defined by pH 8.5, 0.5-mM luminol, 0.1-mM -iodophenol, 2 AU/mL HRP, 5-mM , and 50-mM ionic strength. Chemiluminescence was measured using an EPP Miniature Fiber Optic Spectrometer from StellarNet.