College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Normal University , Jinan, Shandong 250014, P. R. China.
Anal Chem. 2014 Apr 15;86(8):4008-15. doi: 10.1021/ac500426b. Epub 2014 Apr 2.
Homogenous electrochemical biosensor has attracted substantial attention owing to its simplicity, rapid response, and improved recognition efficiency compared with heterogeneous biosensor, but the relatively low detection sensitivity and the limited detection analytes prohibit its potential applications. To address these issues, herein, a simple, rapid, isothermal, and ultrasensitive homogeneous electrochemical DNA biosensing platform for target DNA and protein detection has been developed on the basis of an exonuclease III (Exo III)-aided autocatalytic target recycling strategy. A ferrocene-labeled hairpin probe (HP1) is ingeniously designed, which contains a protruding DNA fragment at 3'-termini as the recognition unit for target DNA. Also, the DNA fragment that could be used as secondary target analogue was introduced, but it was caged in the stem region of HP1. In the presence of target DNA, its recognition with the protruding fragment of HP1 triggered the Exo III cleavage process, accompanied with the target recycling and autonomous generation of secondary target analogues. This accordingly resulted into the autonomous accumulation of ferrocene-labeled mononucleotide, inducing a distinct increase in the electrochemical signal owing to its elevated diffusivity toward indium tin oxide (ITO) electrode surface. The autocatalytic biosensing system was further extended for protein detection by advising an aptamer hairpin switch with the use of thrombin as a model analyte. The current developed autocatalytic and homogeneous strategy provided an ultrasensitive electrochemical detection of DNA and thrombin down to the 0.1 and 5 pM level, respectively, with a high selectivity. It should be further used as a general autocatalytic and homogeneous strategy toward the detection of a wide spectrum of analytes and may be associated with more analytical techniques. Thus, it holds great potential for the development of ultrasensitive biosensing platform for the applications in bioanalysis, disease diagnostics, and clinical biomedicine.
均相电化学生物传感器因其与异相生物传感器相比具有简单、快速响应和提高的识别效率而受到广泛关注，但相对较低的检测灵敏度和有限的检测分析物限制了其潜在应用。为了解决这些问题，本文基于外切酶 III（Exo III）辅助的自动催化目标循环策略，开发了一种用于目标 DNA 和蛋白质检测的简单、快速、等温、超灵敏的均相电化学生物传感器平台。巧妙设计了一种带有二茂铁标记的发夹探针 (HP1)，其 3'末端包含一个突出的 DNA 片段作为目标 DNA 的识别单元。此外，还引入了可作为二级目标类似物的 DNA 片段，但它被包含在 HP1 的茎区。在存在目标 DNA 的情况下，其与 HP1 的突出片段的识别触发 Exo III 切割过程，伴随着目标的循环和自动生成二级目标类似物。这相应导致了带有二茂铁标记的单核苷酸的自动积累，由于其向氧化铟锡 (ITO) 电极表面的扩散率升高，导致电化学信号明显增加。通过使用凝血酶作为模型分析物，建议使用适体发夹开关，将自动催化生物传感器系统进一步扩展用于蛋白质检测。所开发的自动催化和均相策略分别对 DNA 和凝血酶进行了超灵敏的电化学检测，检测下限低至 0.1 和 5 pM，具有高选择性。它应该进一步作为一种通用的自动催化和均相策略，用于检测广泛的分析物，并可能与更多的分析技术相关联。因此，它在生物分析、疾病诊断和临床生物医学中的超灵敏生物传感平台的发展中具有很大的潜力。
