Ge Lei, Wang Wenxiao, Sun Ximei, Hou Ting, Li Feng
College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University , Qingdao 266109, People's Republic of China.
Anal Chem. 2016 Feb 16;88(4):2212-9. doi: 10.1021/acs.analchem.5b03844. Epub 2016 Feb 3.
As is well-known, graphene shows a remarkable difference in affinity toward nonstructured single-stranded (ss) DNA and double-stranded (ds) DNA. This property makes it popular to prepare DNA-based optical sensors. In this work, taking this unique property of graphene in combination with the sensitive electrochemical transducer, we report a novel affinity-mediated homogeneous electrochemical aptasensor using graphene modified glassy carbon electrode (GCE) as the sensing platform. In this approach, the specific aptamer-target recognition is converted into an ultrasensitive electrochemical signal output with the aid of a novel T7 exonuclease (T7Exo)-assisted target-analog recycling amplification strategy, in which the ingeniously designed methylene blue (MB)-labeled hairpin DNA reporters are digested in the presence of target and, then, converted to numerous MB-labeled long ssDNAs. The distinct difference in differential pulse voltammetry response between the designed hairpin reporters and the generated long ssDNAs on the graphene/GCE allows ultrasensitive detection of target biomolecules. Herein, the design and working principle of this homogeneous electrochemical aptasensor were elucidated, and the working conditions were optimized. The gel electrophoresis results further demonstrate that the designed T7Exo-assisted target-analog recycling amplification strategy can work well. This electrochemical aptasensor realizes the detection of biomolecule in a homogeneous solution without immobilization of any bioprobe on electrode surface. Moreover, this versatile homogeneous electrochemical sensing system was used for the determination of biomolecules in real serum samples with satisfying results.
众所周知，石墨烯对非结构化单链（ss）DNA和双链（ds）DNA的亲和力存在显著差异。这一特性使得制备基于DNA的光学传感器很受欢迎。在本工作中，结合石墨烯的这一独特性质与灵敏的电化学传感器，我们报道了一种新型的亲和力介导的均相电化学适体传感器，该传感器以石墨烯修饰玻碳电极（GCE）作为传感平台。在这种方法中，借助一种新型的T7核酸外切酶（T7Exo）辅助的靶标类似物循环扩增策略，将特异性适体-靶标识别转化为超灵敏的电化学信号输出，其中巧妙设计的亚甲基蓝（MB）标记的发夹DNA报告分子在靶标存在时被消化，然后转化为大量MB标记的长单链DNA。石墨烯/GCE上设计的发夹报告分子与生成的长单链DNA在差分脉冲伏安法响应上的明显差异使得能够超灵敏地检测靶标生物分子。在此，阐明了这种均相电化学适体传感器的设计和工作原理，并对工作条件进行了优化。凝胶电泳结果进一步证明所设计的T7Exo辅助的靶标类似物循环扩增策略能够很好地发挥作用。这种电化学适体传感器实现了在均相溶液中检测生物分子，而无需在电极表面固定任何生物探针。此外，这种通用的均相电化学传感系统用于测定实际血清样品中的生物分子，结果令人满意。
