Truly Immobilization-Free Diffusivity-Mediated Photoelectrochemical Biosensing Strategy for Facile and Highly Sensitive MicroRNA Assay
Truly Immobilization-Free Diffusivity-Mediated Photoelectrochemical Biosensing Strategy for Facile and Highly Sensitive MicroRNA Assay
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真正的免固定扩散介导的光电化学生物传感策略，用于简便且高灵敏度的 microRNA 测定
DOI：
10.1021/acs.analchem.8b02523
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发表时间：
2018-08-07
影响因子：
7.4
通讯作者：
Li, Feng
中科院分区：
文献类型：
--
作者：
Hou, Ting;Xu, Ningning;Li, Feng
在传统的光电化学（PEC）分析中，光活性材料通常被固定在电极表面，这种固定过程繁琐耗时，并且也难以制备具有良好重现性的电极。为了规避这些限制，我们在这里提出了一种真正的无固定扩散介导的 PEC 生物传感策略，用于 microRNA 测定，使用溶液中的亚甲基蓝（MB）作为光活性探针，并使用未改性的氧化铟锡（ITO）玻璃作为工作电极。目标microRNA与MB标记的单链DNA探针（MB-DNA）的杂交触发T7核酸外切酶（T7 Exo）对MB-DNA的消化，从而产生MB标记的单核苷酸，然后释放的目标microRNA启动后续的循环过程并产生大量的MB标记的单核苷酸。由于MB-DNA和MB标记的单核苷酸之间的扩散率差异，与MB-DNA相比，MB标记的单核苷酸的光电流信号显着增加。因此，通过这种“信号开启”模式和 T7 Exo 促进信号放大，可以轻松实现简便且高度灵敏的免固定化 PEC microRNA 测定，检测限低至 27 aM。此外，该策略表现出优异的特异性，并成功应用于检测血清样品中掺入的 microRNA。由于所有反应都在均相溶液中进行，并且不需要对电极进行修饰，因此这种PEC生物传感策略具有简单、快速和重现性好的优点。更重要的是，它提供了设计真正免固定化PEC生物传感系统的新颖概念，并显示出在生物分析和生化研究中应用的潜力。
In conventional photoelectrochemical (PEC) analysis, photoactive materials are usually immobilized on electrode surfaces, and such immobilization procedures are tedious and time-consuming, and it is also difficult to prepare electrodes with good reproducibility. To circumvent such limitations, we propose here a truly immobilization-free diffusivity-mediated PEC bionsensing strategy for microRNA assay, using methylene blue (MB) in solution as the photoactive probe, and nonmodified indium tin oxide (ITO) glass as the working electrode. The hybridization between the target microRNA and the MB-labeled single-stranded DNA probe (MB-DNA) triggers the digestion of MB-DNA by T7 exonuclease (T7 Exo), thus to generate MB-labeled mononucleotide, and then the released target microRNA initiates the subsequent cycling processes and generates a large amount of MB-labeled mononucleotides. Due to the diffusivity difference between MB-DNAs and MB-labeled mononucleotides, significantly increased photocurrent signal is observed for MB-labeled mononucleotides as compared to that of MB-DNAs. Therefore, via this "signal-on" mode and the T7 Exo facilitated signal amplification, a facile and highly sensitive immobilization-free PEC microRNA assay is readily realized, with a detection limit down to 27 aM. Moreover, this strategy exhibits excellent specificity and is successfully applied in detecting microRNA spiked in serum samples. Since all the reactions take place in homogeneous solutions and no electrode modification is needed, this PEC biosensing strategy exhibits the advantages of simplicity, rapidness, and good reproducibility. More significantly, it provides a novel concept to design truly immobilization-free PEC biosensing systems, and shows potential to be applied in bioanalysis and biochemical research.