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一种基于刃天青还原的荧光方法对β-葡萄糖苷酶活性的检测 预览

Activity Determination of β-Glucosidase by a Turn-on Fluorescent Method Based on Reduction of Resazurin
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摘要 建立了一种简单的荧光分析方法实现了对β-葡萄糖苷酶的定量分析检测。其原理是底物2-O-β-D-葡萄糖基-L-抗坏血酸(AA-2βG)在β-葡萄糖苷酶的水解作用下生成抗坏血酸,后者将刃天青还原生成具有强烈荧光的试卤灵,通过溶液荧光强度的变化对β-葡萄糖苷酶活性进行检测。结果发现,抗坏血酸浓度在3~400μmol/L范围内与反应体系在585 nm处的荧光强度呈线性关系,相关系数r^2=0.981。并基于此实现了对β-葡萄糖苷酶活性的定量检测:β-葡萄糖苷酶的浓度在1~30 U/L范围内与溶液在585 nm处的荧光强度呈良好的线性关系,相关系数r^2=0.995,检出限为1.0 U/L。该方法操作简单,反应灵巧,检测范围较宽,可实现对β-葡萄糖苷酶活性的定量分析检测。 A simple fluorescence analytical method was established for the quantitation of activity of β-glucosidase.The synthetic 2-O-β-glucopyranosyl ascorbic acid(AA-2βG) was used as the enzymatic substrate.β-Glucosidase catalyzed the hydrolysis reaction and released ascorbic acid,which would reduce non-fluorescent resazurin to strongly fluorescent resorufin.Thus,β-glucosidase activity would be reflected by the fluorescent intensity variation of the system.Firstly,a validation assay was carried out to confirm the possibility to detect ascorbic acid taking advantage of reductive reaction.There was a linear relationship for the increased fluorescent intensity with the concentration of ascorbic acid in the range of 3-400 μmol/L with a correlation coefficient(r^2) of 0.981,while β-glucosidase activity was successfully determined by measuring fluorescent intensity at 585 nm.Results showed that there also existed a good linear relationship between the increased fluorescent intensity and concentration of β-glucosidase in range of 1-30 U/L,with a correlation coefficient(r^2) of 0.995 and a detection limit of 1.0 U/L.With the characteristics of simplicity,sensitivity and large detection range,the method could realize the quantitative detection on β-glucosidase activity.
作者 程鑫 张珩 马济美 CHENG Xin;ZHANG Heng;MA Ji-mei(College of Science,Huazhong Agricultural University,Wuhan 430070,China)
出处 《分析测试学报》 CAS CSCD 北大核心 2019年第3期360-363,共4页 Journal of Instrumental Analysis
基金 中央高校基本科研业务费专项资金(26622018JC011) 国家自然科学基金资助项目(21402056).
关键词 β-葡萄糖苷酶活性 刃天青 试卤灵 抗坏血酸 荧光 检测 β-glucosidase activity resazurin resorufin ascorbic acid fluorescence determination
作者简介 通讯作者:马济美,博士,副教授,研究方向:有机合成,E-mail:majimei@mail.hzau.edu.cn.
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