基于双重脱氧核酶的超灵敏比色法检测铜离子  

Ultrasensitive and Colorimetric Detection of Copper(Ⅱ) Based on Dual-DNAzymes

作　　者：梅晓宏 田晶晶 张洋子 杜再慧 张媛 许文涛 MEI Xiao-Hong;TIAN Jing-Jing;ZHANG Yang-Zi;DU Zai-Hui;ZHANG-Yuan;XU Wen-Tao(College of Food Science and Nutritional Engineering,Beijing Advanced Innovation Center for Food Nutrition and Human Health,China Agricultural University,Beijing 100083,China;Key Laboratory of Safety Evaluation(Food)of Agricultural Genetically Modified Organisms, Ministry of Agriculture,China Agricultural University,Beijing 100083,China)

机构地区：[1]中国农业大学,食品科学与营养工程学院,北京食品营养与人类健康高精尖创新中心,北京100083 [2]农业部农业转基因生物安全评价(食用)重点实验室,北京100083

出　　处：《分析化学》2018年第12期1945-1952,共8页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(No.31671922)资助。

摘　　要：铜是生命的必需元素,是某些活性蛋白的辅基,而过量的铜具有一定毒性,可导致肝炎、胃肠炎、肺炎等病症。因此对Cu^(2+)的灵敏定量检测十分必要。本研究建立了一种基于双重脱氧核酶的比色生物传感器,用于灵敏、特异性定量检测Cu^(2+)。此生物传感器包含两种具有不同功能的脱氧核酶:Cu^(2+)依赖性的切割型脱氧核酶与G-四链体脱氧核酶。当待测物中存在Cu^(2+)时,Cu^(2+)依赖性的切割型脱氧核酶被切割,用于特异性信号识别;经末端转移酶诱导形成G-四链体脱氧核酶,用于"一对多"的信号放大与可视化信号输出。在"信号识别"与"信号输出"两步反应之间,利用指数扩增反应(Exponential amplification reaction,EXPAR),放大"信号识别"的反应信号。在最优条件下,可实现Cu^(2+)的灵敏的选择性检测:使用全波长酶标仪测定,比色法检出限为7.6 pmol/L;裸眼检出限为10 pmol/L。将此传感器用于自来水中Cu^(2+)的测定,裸眼检测范围为10～200 pmol/L,比色法加标回收率为96.3%～98.4%。此检测平台为特异性检测自来水中的Cu^(2+)提供了超灵敏、可视化的检测工具,具有良好的应用前景。Copper is an essential element for life and constitutes copper proteins,and attends life activities in human body.However,an excess of copper is poisonous,and can cause hepatitis,gastroenteritis and pneumonia.copper enters into biosphere from ores by weathering and erosion and exists in water in the form of a divalent copper ion.Therefore,it is necessary to detect copper ions in an ultrasensitive and quantitative way.Herein,a dual-functional DNAzymatic visual biosensor was developed,optimized and validated to detect divalent copper ions sensitively and selectively in tap water.In the method,dual DNAzymes with different functions were designed within one biosensor.One DNAzyme was Cu 2+-dependent cleaving DNAzyme,which could be cleaved by Cu 2+for signal recognition.The other one was G-quadruplex DNAzyme,which was induced by terminal deoxynucleotidyl transferase(TdT)for“one-to-multiplex”signal amplification and output.In the middle of two steps,the exponential amplification reaction(EXPAR)was applied to amplify the recognitive signal exponentially.Under optimized conditions,Cu 2+could be selectively observed with the naked eyes with the detection limit(LOD)of 10 pmol/L and with the microplate reader with LOD of 7.6 pmol/L in about 2 h.Furthermore,Cu 2+in the concentration range of 10 pmol/L-200 pmol/L in tap water samples could be discriminated by naked eyes.The platform provides an ultrasensitive and colorimetric detective tool for the analysis of trace Cu 2+in tap water with high specificity,and will have broad application prospect in many fields.

关 键 词：铜离子 脱氧核酶 末端脱氧核苷酸转移酶 等温扩增 

分 类 号：O657.3[理学—分析化学]