Study on Enhancement Principle and Stabilization for the Luminol-H2O2-HRP Chemiluminescence System

PLoS One. 2015 Jul 8;10(7):e0131193. doi: 10.1371/journal.pone.0131193. eCollection 2015.

Abstract

A luminol-H2O2-HRP chemiluminescence system with high relative luminescent intensity (RLU) and long stabilization time was investigated. First, the comparative study on the enhancement effect of ten compounds as enhancers to the luminol-H2O2-HRP chemiluminescence system was carried out, and the results showed that 4-(imidazol-1-yl)phenol (4-IMP), 4-iodophenol (4-IOP), 4-bromophenol (4-BOP) and 4-hydroxy-4'-iodobiphenyl (HIOP) had the best performance. Based on the experiment, the four enhancers were dissolved in acetone, acetonitrile, methanol, and dimethylformamide (DMF) with various concentrations, the results indicated that 4-IMP, 4-IOP, 4-BOP and HIOP dissolved in DMF with the concentrations of 0.2%, 3.2%, 1.6% and 3.2% could get the highest RLU values. Subsequently, the influences of pH, ionic strength, HRP, 4-IMP, 4-IOP, 4-BOP, HIOP, H2O2 and luminol on the stabilization of the luminol-H2O2-HRP chemiluminescence system were studied, and we found that pH value, ionic strength, 4-IMP, 4-IOP, 4-BOP, HIOP, H2O2 and luminol have little influence on luminescent stabilization, while HRP has a great influence. In different ranges of HRP concentration, different enhancers should be selected. When the concentration is within the range of 0~6 ng/mL, 4-IMP should be selected. When the concentration of HRP ranges from 6 to 25 ng/mL, 4-IOP was the best choice. And when the concentration is within the range of 25~80 ng/mL, HIOP should be selected as the enhancer. Finally, the three well-performing chemiluminescent enhanced solutions (CESs) have been further optimized according to the three enhancers (4-IMP, 4-IOP and HIOP) in their utilized HRP concentration ranges.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetone / chemistry
  • Acetonitriles / chemistry
  • Biphenyl Compounds / chemistry
  • Buffers
  • Dimethylformamide / chemistry
  • Horseradish Peroxidase / chemistry*
  • Hydrogen Peroxide / chemistry*
  • Hydrogen-Ion Concentration
  • Imidazoles / chemistry
  • Iodobenzenes / chemistry
  • Kinetics
  • Luminescence*
  • Luminescent Measurements / instrumentation
  • Luminescent Measurements / methods*
  • Luminol / chemistry*
  • Methanol / chemistry
  • Organic Chemicals / chemistry
  • Phenols / chemistry
  • Piperidines / chemistry
  • Solvents / chemistry

Substances

  • 4-hydroxy-4'-iodobiphenyl
  • Acetonitriles
  • Biphenyl Compounds
  • Buffers
  • Imidazoles
  • Iodobenzenes
  • Organic Chemicals
  • Phenols
  • Piperidines
  • Solvents
  • Acetone
  • 4-(1H-imidazol-4-ylmethyl)piperidine
  • 4-iodophenol
  • Luminol
  • Dimethylformamide
  • Hydrogen Peroxide
  • Horseradish Peroxidase
  • 4-bromophenol
  • Methanol
  • acetonitrile

Grants and funding

This study was supported by International Science & Technology Cooperation Program of China (No. 2012DFA31140), the Chinese Public Interest Industrial Science & Technology Project (201203094) and National Natural Science Foundation of China (No. 31201371). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.