基于滚环扩增及串联G-四链体-血红素DNA酶的高灵敏“Turn-on”型Hg^(2+)传感器研究  

Fabrication of “Turn-On” Sensor for Sensitive Detection of Hg^(2+) Based on Rolling Circle Amplification and Tandem G-quadruplex-hemin DNAzymes

作　　者：张何[1] 王青 傅昕[1] 赵智粮 ZHANG He;WANG Qing;FU Xin;ZHAO Zhi-Liang(School of Chemistry and Chemical Engineering,Hunan Institute of Engineering,Xiangtan 411104,China)

机构地区：[1]湖南工程学院化学化工学院,湘潭411104

出　　处：《分析化学》2018年第10期1644-1651,共8页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(No.21005067);湖南省教育厅资助科研项目(Nos.17A044;16B060)资助~~

摘　　要：以聚苯乙烯微球为载体,利用滚环放大技术,发展了一种以串联G-四链体-血红素DNA酶催化及T-Hg^(2+)-T特异识别为基础的"Turn-on"型Hg^(2+)高灵敏生物传感器,用于尿液样本中Hg^(2+)的高效检测。通过链霉亲和素和生物素的特异性结合,将富T生物素化Hg^(2+)捕获探针固定至微球表面,当Hg^(2+)存在时,通过形成T-Hg^(2+)-T结构将含有G-四链体互补序列的环化单链DNA序列捕获至微球表面,滚环扩增后在微球表面产生大量包含串联G-四链体的DNA序列。当氯化血红素(Hemin)插入G-四链体后,形成具有增强催化活性的G-四链体-hemin DNA酶,可催化ABTS和H_2O_2反应形成ABTS^(·+),在420 nm处具有最大吸收。考察了多种因素对检测体系的影响,在最优实验条件下,此方法对Hg^(2+)的线性检测范围为0.4~100 pmol/L,检出限为0.3pmol/L(S/N=3),回归方程为△A_(420 nm)=0.1+0.0019C_(Hg^(2+))(pmol/L)。当共存离子大量存在时,传感器对Hg^(2+)仍然具有高的选择性。应用于尿液样品中Hg^(2+)检测,加标回收率为94.0%~106.0%,相对标准偏差(RSD)为1.4%~2.6%。此方法具有良好的选择性、灵敏度及抗干扰能力,可用于复杂样品中Hg^(2+)的检测。Based on the tandem G-quadruplex-hemin DNAzymes for catalytic signal amplification and T-Hg^2+-T structure for specific recognition,a highly sensitive"Turn-on"biosensor for detection of mercury ion(Hg^2+)was developed on the polystyrene microbeads to achieve the detection of Hg^2+in urine samples.Through the specific binding of streptavidin and biotin,the T-rich biotinylated Hg^2+capture probe was immobilized on the surface of the microspheres.When Hg^2+was present in reaction system,the T-rich Hg^2+capture probe could capture the cyclic DNA containing G-quadruplex complementary fragments to the surface of beads by forming T-Hg^2+-T structure.Through rolling circle amplification,a large number of tandem G-quadruplex forming sequences were amplified.When hemin was inserted into the G-quadruplex,a G-quadruplex-hemin DNAzyme with enhanced catalytic activity was formed,which could catalyze the oxidation of ABTS to ABTS·+under the condition of H2O2.Finally,the absorbance difference at 420 nm was detected by UV-vis spectrophotometer.Under the optimal experimental conditions,the linear range for detection of Hg^2+was 0.4 pmol/L-100 pmol/L with the detection limit of 0.3 pmol/L,and the regression equation wasΔA 420 nm=0.1+0.0019 C Hg^2+(pmol/L).This Hg^2+sensing system had excellent selectivity for Hg^2+against other possible competing ions.The fabricated sensor was applied to detection of Hg^2+in urine samples,the relative standard deviation and the recovery were 1.4%-2.6%and 94.0%-106.0%,respectively.The sensor had good selectivity,sensitivity,anti-interference ability and reliable detection capability for complex practical samples.

关 键 词：G-四链体-血红素DNA酶 滚环放大 汞离子 尿液分析 比色分析 

分 类 号：O657.3[理学—分析化学] R446.1[理学—化学]