基于二硫化钨和核酸外切酶Ⅲ的荧光法高灵敏检测血液中汞离子  

Highly Sensitive Fluorometric Hg^(2+) Biosensor Based on WS_2 and Exonuclease Ⅲ-assisted Signal Amplification

作　　者：耿琴[1] Geng Qin(Department of Pharmacy, Zhongnan Hospital of Wuhan University, Wuhan 430071 , China)

机构地区：[1]武汉大学中南医院药学部,武汉430071

出　　处：《中国药师》2019年第4期778-780,共3页China Pharmacist

摘　　要：目的:将信号放大策略用于荧光法实现血液中低浓度汞离子的检测。方法:将汞离子加入到富含胸腺嘧啶且3’端荧光标记的单链DNA溶液中,汞离子介导该特异序列形成分子内的双链结构,且该双链DNA具有3’凹端,因此能够被核酸外切酶Ⅲ水解并释放出荧光标记的单核苷酸,当加入二硫化钨(WS_2)后,荧光标记的单核苷酸不能被二硫化钨吸附从而荧光信号明显增加。对WS_2和ExoⅢ的量进行优化考察,对方法的线性、检测限、特异性等性能进行验证。3个不同浓度汞离子在血浆样品中的加样回收率也进行验证。结果:汞离子在1～30 nmol·L^(-1)和30～250 nmol·L^(-1)范围内线性关系良好,r分别为0.993 6和0.995 2,检测限为0.1 nmol·L^(-1),加样回收率分别为110.0%,116.0%,90.9%,RSD分别为16.5%,18.7%,17.8%(n=6)。结论:建立的荧光法可以实现血液中低含量汞离子的检测,为临床上检测汞离子提供一种新方法。Objective: To establish a fluorometric biosensor with signal amplification for the highly sensitive detection of Hg^2+ in human plasma. Methods: Hg2+ was added to the solution containing thymine-rich single-stranded( ss-) DNA labeled with a fluorescence tag. The double-stranded( ds-) DNA with a recessed 3-terminus in the mediation of Hg^2+ was formed. ExonucleaseⅢ( ExoⅢ)could catalyze the stepwise removal of mononucleotides from the 3-hydroxyl ends of ds DNA. The cleaved FAM-labeled mononucleotides fragments didn’t adsorb on the surface of WS2 resulting in the significant increase of fluorescence intensity. The contents of WS2 and ExoⅢ were optimized. In the optimal experiment conditions,the linearity,detection limit and specificity of the method were validated.The average recovery was also validated after the pretreatment of human plasma. Results: The fluorometric biosensor had achieved high selectivity and very low detection limit down to 0. 1 nmol·L^-1. The linear range was from 1 to 30 nmol·L^-1 and from 30 to 250 nmol·L^-1,and the regression coefficient was 0. 993 6 and 0. 995 2,respectively. The average recovery was 110. 0%,116. 0% and 90. 9%,and the RSD was 16. 5%,18. 7% and 17. 8%( n = 6),respectively. Conclusion: The established fluorometric biosensor can realize the detection of the low content of Hg^2+ in human plasma. It can provide a new method for the detection of Hg2+ in clinics.

关 键 词：荧光法 二硫化钨 核酸外切酶Ⅲ 汞离子 血浆 

分 类 号：R917[医药卫生—药物分析学]