基于蛋白包覆金纳米簇的碱性蛋白酶荧光检测  

Fluorescence Detection of Alkaline Protease Based on Protein-Coated Gold Nanoclusters

作　　者：苏荣欣[1,2] 李伟[1] 齐崴[1,2] 何志敏[1] 

机构地区：[1]天津大学化工学院化学工程联合国家重点实验室,天津300072 [2]天津化学化工协同创新中心,天津300072

出　　处：《天津大学学报（自然科学与工程技术版）》2015年第7期620-624,共5页Journal of Tianjin University：Science and Technology

基　　金：国家重大科学仪器专项资助项目(2012YQ090194);国家科技支撑计划资助项目(2012BAD29B05)

摘　　要：设计开发了一种在碱性条件下由溶菌酶包覆合成并稳定的荧光金纳米簇,并利用它实现对碱性蛋白酶的快速灵敏检测.其检测原理是碱性蛋白酶将包覆和稳定金纳米簇的溶菌酶进行水解,造成金纳米簇的荧光下降. 通过关联荧光下降程度与碱性蛋白酶浓度的关系,可实现对碱性蛋白酶的定量检测. 考察了金纳米簇与碱性蛋白酶溶液体积比、反应温度和反应时间对检测灵敏性的影响规律. 结果表明：在金纳米簇与碱性蛋白酶溶液体积比为1∶9、反应温度为40,℃、反应时间为3,h 的条件下,检测效果最好;该检测方法的线性范围可达2～2,000,μg/mL（即酶活检测范围为4×10^-5～0.04 unit/mL）,检测限为0.1 μg/mL（即酶活检测限为2×10^-6 unit/mL）,且检测的专-性较好,有望应用于实际检测.A kind of gold nanoclusters (Au NCs) was prepared using lysozyme as a coating and stabilizing molecule. Using this Au NCs, a quick and sensitive assay for alkaline protease has been established. The detection was based on the hydrolysis activity of protease to the lysozyme which was used to coat and stabilize the Au NCs, which led to the decrease of their fluorescence intensity. Quantitative detection of alkaline protease can be realized based on the rela-tionship between the decrease of fluorescence intensity and the concentration of alkaline protease. The effects of vol-ume ratio of Au NCs to alkaline protease solution, reaction temperature and time on the detection sensitivity were in-vestigated. The optimal volume ratio of Au NCs to alkaline protease solution was 1∶9, while the optimal reaction temperature and reaction time were 40,℃ and 3,h, respectively. Under these conditions, the linear range of this method was from 2,μg/mL to 2,000,μg/mL(4×10-5-0.04,unit/mL) with a limit of detection of 0.1,μg/mL(2×10-6 unit/mL) and good selectivity. Therefore, this method shows its potential in the detection of alkaline protease in real samples.

关 键 词：碱性蛋白酶 溶菌酶 金纳米簇 荧光 

分 类 号：TQ925[轻工技术与工程—发酵工程]