Citation: ZHANG He, WANG Qing, FU Xin, ZHAO Zhi-Liang. Fabrication of "Turn-On" Sensor for Sensitive Detection of Hg2+ Based on Rolling Circle Amplification and Tandem G-quadruplex-hemin DNAzymes. Chinese Journal of Analytical Chemistry, 2018, 46(10): 1644-1651. doi: 10.11895/j.issn.0253-3820.181346 [复制]
Fabrication of "Turn-On" Sensor for Sensitive Detection of Hg2+ Based on Rolling Circle Amplification and Tandem G-quadruplex-hemin DNAzymes
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-Hg2+-T特异识别为基础的"Turn-on"型Hg2+高灵敏生物传感器，用于尿液样本中Hg2+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Hg2+捕获探针固定至微球表面，当Hg2+存在时，通过形成T-Hg2+-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H2O2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Hg2+的线性检测范围为0.4~100 pmol/L，检出限为0.3 pmol/L（S/N=3），回归方程为ΔA420 nm=0.1+0.0019CHg2+（pmol/L）。当共存离子大量存在时，传感器对Hg2+仍然具有高的选择性。应用于尿液样品中Hg2+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Hg2+的检测。
Based on the tandem G-quadruplex-hemin DNAzymes for catalytic signal amplification and T-Hg2+-T structure for specific recognition, a highly sensitive "Turn-on" biosensor for detection of mercury ion (Hg2+) was developed on the polystyrene microbeads to achieve the detection of Hg2+ in urine samples. Through the specific binding of streptavidin and biotin, the T-rich biotinylated Hg2+ capture probe was immobilized on the surface of the microspheres. When Hg2+ was present in reaction system, the T-rich Hg2+ capture probe could capture the cyclic DNA containing G-quadruplex complementary fragments to the surface of beads by forming T-Hg2+-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 Hg2+ was 0.4 pmol/L-100 pmol/L with the detection limit of 0.3 pmol/L, and the regression equation was ΔA420 nm=0.1+0.0019CHg2+ (pmol/L). This Hg2+ sensing system had excellent selectivity for Hg2+ against other possible competing ions. The fabricated sensor was applied to detection of Hg2+ 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.
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孔德明. 化学进展, 2011, 23(10): 2119-2131