β-環糊精增強過碘酸鉀氧化Luminol之化學發光及應用

Abstract

本研究主要是利用停止流儀（Stopped-flow spectrometry）發展以β-環糊精（β-cyclodextrin）增強過碘酸鉀（KIO4）氧化luminol的化學發光系統。探討影響化學發光的因素，包括pH值、反應物（luminol、過碘酸鉀和環糊精）濃度等等，找出最佳化條件。實驗中亦發現pH值的控制對於訊號再現性的影響非常重要。於是選擇在pH 12.0的強鹼溶液下，添加5 mM β-環糊精增強0.05 mM luminol和0.2 mM過碘酸鉀的化學發光強度約190倍左右。若比較不同腔體大小的α-或γ-環糊精，則增強效果比β-環糊精降低約6倍左右，推測是形成可以穩定化學發光中間態的β-環糊精包絡物所致，因此利用檢測物與化學發光中間態彼此競爭形成β-環糊精包絡物而抑制訊號的機制，可應用於檢測抗氧化劑，例如：trolox、BHA、類黃酮（flavonoids）等。其中，trolox在1.5 x 10-8∼2.5 x 10-7 M內以指數形式呈現快速且明顯的抑制效果，對於抑制化學發光訊號強度一半之濃度（IC50值）達0.05 μM；另外，亦可區分類黃酮的抗氧化能力在不同的氫氧取代基位置，具有不同的抑制效果。最後，此系統亦嘗試應用於非抗氧化劑的檢測，如螢光標記物－TNS，得到IC50值為19.9 μM，推論TNS和luminol共同競爭β-環糊精的疏水性腔體導致訊號降低。未來，將持續對於系統的機制作更進一步的探討，以及增加其應用性。

In this study, a novel chemiluminescence (CL) method based on the luminol-KIO4-β-cyclodextrin(β-CD) system has been developed. The stopped-flow spectrometry was used to measure the CL produced from the oxidation of luminol by KIO4 in alkaline condition (pH 12.0). Various factors, including pH, concentration of reagents (luminol, KIO4, and β-CD), and temperature, that affect the CL intensity were also discussed. The results indicate that maintaining a constant pH is very important for the reproducibility of the CL signal. In addition, the CL intensity was enhanced about 190-fold by the presence of 5 mM β-CD in 0.05 mM luminol and 0.2 mM KIO4. α– and γ–CD caused much weaker enhancement (about 1/6) as compared to β-CD, suggesting that cavity of correct size was required. It’s proposed that the enhancement is due to the stabilization of CL intermediate by the formation of β-CD inclusion complex. Therefore, the inhibition of CL intensity by the competition between analytes and luminol intermediate to form the β-CD inclusion complex was employed to determine the antioxidants, such as trolox, butylated hydroxyanisole (BHA), and flavonoids. The trolox quenched the CL intensity exponentially in the range of 1.5×10-8~2.5×10-7M, and the half-inhibition concentration (IC50) was 0.05 μM. On the other hand, the antioxidant activities of different flavonoids can be distinguished by their abilities in CL inhibition. Furthermore, the CL method was also applied to determine a non-antioxidant, such as fluorescence probe－TNS, and the IC50 value was 19.9 μM. In the future, we will try to explain the mechanism of the CL system and to develop more applications.