Luminol-Zn2+-KIO4系統之化學發光性質探討與應用

Abstract

Luminol is a common chemiluminescence（CL）reagent. It emits light at 425 nm. The chemiluminescence which caused by luminol is usually carried out in alkaline solution and in the presence of an oxidant and a catalyst. In this study, we use KIO4 as oxidant and Zn2+ as catalyst to construct a luminol - Zn2+ - KIO4 CL system, utilizing flow-injection analysis to detect the CL signal. The effects of pH, concentration of reagents (luminol, Zn2+, KIO4) and modes of reagent mixing on CL intensity were investigated and optimized. In this system, the optimal conditions are: pH = 13.0, [luminol] = 10 μM, [Zn2+] = 400 μM, [KIO4] = 0.5 mM, double flow channels （flow rate fixed at 5 mL/min）. In the radical-scavenging studies, we added the selective scavengers of reactive oxygen species (ROS), such as O2･－, 1O2 and ･OH scavengers into the reaction system. The result showed a decreasing CL intensity for all scavengers, suggesting that theses radicals participated in the CL reaction. By using this CL system, we tested 49 biomolecules and organic compounds for their effect on the CL emission. It was found that compounds bearing a dihydroxybenzene and catecholamine structure, exhibited best inhibition on the CL intensity. After that, we selected 15 compounds to investigate in depth their best inhibition on the CL peak. They are common antioxidants, which have an ability to eliminate free radicals, thereby causing a decrease in CL emission. We then calculated the detection limits and IC50 of these antioxidants by measuring their extent of inhibition on the signal. The determination of dihydroxybenzenes such as hydroquinone (LOD: 2.6 nM, IC50: 0.028 μM), catechol (LOD: 3.5 nM, IC50: 0.034 μM), resorcinol (LOD: 133.5 nM, IC50: 2.13 μM), and catecholamines such as L-dopa (LOD: 12.9 nM, IC50: 0.093 μM), dopamine (LOD: 3.8 nM, IC50: 0.06 μM), norepinephrine (LOD: 28.9 nM, IC50: 0.35 μM), epinephrine (LOD: 72.1 nM, IC50: 1.02 μM), and other phenolic compounds such as normetanephrine (LOD: 28.4 nM, IC50: 0.29 μM), chlorogenic acid (LOD: 36.6 nM, IC50: 0.49 μM), homovanillic acid (LOD: 67.9 nM, IC50: 1.03 μM), catechin (LOD: 49.3 nM, IC50: 0.51 μM), octopamine (LOD: 63.9 nM, IC50: 0.89 μM), synephrine (LOD: 95.9 nM, IC50, 1.77 μM), sesamol (LOD: 82 nM，IC50: 1.33 μM), and amino acid - cysteine (LOD: 418.1 nM, IC50: 3.32 μM). In order to attain better sensitivity, we perfomed an inhibition-optimized experiment. By adjustment of the regent concentration, optimal conditions for greatest suppression of signal were obtained, and applied to the determination of hydroquinone and catechol. The detection limits of hydroquinone and catechol were 1.13 nM and 2.63 nM, respectively. In order to figure out what role Zn2+ plays in CL reaction, EDTA was added in the sample at injection site. We accidentally found that the signal is enhanced. The emission spectrum of luminol - Zn2+ - KIO4 system showed a maximum at 425 nm. So the CL is caused by luminol, not by other emitter. When the dissolved oxygen was removed from the solution by purging with nitrogen, the CL intensity decreased by 40%. This might indicated that two CL pathways are required to account for CL emission. One is that KIO4 interacted with dissolved oxygen in water to produce reactive oxygen species, and the generated ROS may then react with luminol to induce CL emission. The other is that luminol is oxidized by KIO4 directly. Both pathways were catalysed by Zn2+ . Based on these results, we proposed the mechanism of luminol - Zn2+ - KIO4 CL system.