Luminol-Zn2+-KIO4系統之化學發光性質探討與應用

Abstract

Luminol是一種常用的化學發光試劑，在鹼性環境下與氧化劑及催化劑作用，產生425 nm左右的放光。本研究中，以KIO4 作為氧化劑、Zn2+ 作為催化劑建立一個luminol - Zn2+ - KIO4 化學發光系統，並利用流動注入分析法( flow-injection analysis )偵測此系統產生的光譜訊號。我們探討pH值、反應物（luminol、Zn2+、KIO4）濃度與混合的方式等因素對訊號強度的影響，並找出系統最佳化條件。系統最佳化條件為：pH = 13.0、[luminol] = 10 μM、[Zn2+] = 400 μM、[KIO4] = 0.5 mM，並且使用雙管流路配置（流速設定在5 mL/min)。 在自由基消滅劑的測試中，我們添加O2･－、1O2 與 ･OH等活性含氧物質( reactive oxygen species, ROS)的消滅劑，結果訊號都有下降的趨勢，推測這些自由基會參與本系統的化學發光反應。 利用此化學發光系統，我們初步篩檢49種生化有機分子，實驗結果顯示含有雙酚（dihydroxybenzene）結構、兒茶酚胺（catecholamine）結構的分子對訊號的抑制程度最大。接著我們對訊號抑制效果最好的十五種分子做深入的檢測，這些分子普遍為常見的抗氧化劑，它們能掃除自由基造成化學發光訊號下降，我們由訊號被抑制的程度推算出這些分子的偵測極限與IC50。如苯二酚化合物中的hydroquinone (偵測極限為2.6 nM，IC50為0.028 μM)、catechol (偵測極限為3.5 nM，IC50為0.034 μM)、resorcinol (偵測極限為133.5 nM，IC50為2.13 μM)；兒茶酚胺中的L-dopa (偵測極限為12.9 nM，IC50為0.093 μM)、dopamine (偵測極限為3.8 nM，IC50為0.06 μM)、norepinephrine (偵測極限為28.9 nM，IC50為0.35 μM)、epinephrine (偵測極限為72.1 nM，IC50為1.02 μM)；其他的酚類化合物如normetanephrine (偵測極限為28.4 nM，IC50為0.29 μM)、chlorogenic acid (偵測極限為36.6 nM，IC50為0.49 μM)、homovanillic acid (偵測極限為67.9 nM，IC50為1.03 μM)、catechin (偵測極限為49.3 nM，IC50為0.51 μM)、octopamine (偵測極限為63.9 nM，IC50為0.89 μM)、synephrine (偵測極限為95.9 nM，IC50為1.77 μM)、sesamol (偵測極限為82 nM，IC50為1.33 μM)；胺基酸中的cysteine (偵測極限為418.1 nM，IC50為3.32 μM)。為了獲得更好的檢測靈敏度，我們進行抑制最佳化實驗，找出對訊號抑制最多的試劑條件，應用在hydroquinone與catechol的偵測上，以此方法獲得了更低的偵測極限值，其偵測極限(LOD)分別為1.13 nM、2.63 nM。 為了了解Zn2+ 在系統中扮演的角色，我們在注入端添加EDTA，意外地發現訊號有增強的現象。我們掃描luminol - Zn2+ - KIO4系統的最大放光波長，發現在425 nm 附近有最大的放光強度，確定本系統的化學發光是由luminol所造成的，沒有新的放光物種產生。將試劑通入氮氣除去溶氧後化學發光訊號減弱約40 %，表示導致化學發光的原因有二種，第一是KIO4與溶氧反應產生活性含氧物質，活性含氧物質氧化luminol導致化學發光;第二是KIO4直接氧化luminol所造成，而且Zn2+ 的添加，能催化這兩種產生放光的途徑，藉此，我們推導出luminol - Zn2+ - KIO4化學發光系統的反應機制。

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.