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 25ng/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.
Highlights
ELISA is currently one of the most widely used methods in the field of analysis and detection [1, 2]
The research found that the ranking sequence of luminescence-enhancing effects for the 10 enhancers from least to greatest is: DIOP
We found that 4-IOP, 4-BOP, 2-IOP, 3-IOP, 4-PYP and 4-MYP could be dissolved in methyl alcohol, acetonitrile, acetone and DMF; 4-IMP can only be dissolved in DMF; 4-BIB and HIOP could be dissolved in acetone and DMF; and DIOP can’t be dissolved in the four organic solvents
Summary
ELISA is currently one of the most widely used methods in the field of analysis and detection [1, 2]. The colorimetric method, fluorescence and chemiluminescence (CL) and other detection methods all make use of the special quality of peroxidase with enzymatic activity to label the PLOS ONE | DOI:10.1371/journal.pone.0131193. Research has revealed that CL has higher sensitivity compared with other detection methods [3,4,5]. The chemiluminescence detection method is based on the fact that peroxidase will manifest catalytic oxidation and form luminol oxide under slightly alkaline conditions. Luminol oxide will promote the generation of the 3-amidogen-dimethyl phthalate ion in an excited state; it will emit light when it is returning to the ground state, and the maximum wavelength of the emitted light will be 425nm
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