A Homogeneous Label-Free Electrochemical microRNA Biosensor Coupling With G-Triplex/Methylene Blue Complex and λ-Exonuclease-Assisted Recycling Amplification.
A Homogeneous Label-Free Electrochemical microRNA Biosensor Coupling With G-Triplex/Methylene Blue Complex and λ-Exonuclease-Assisted Recycling Amplification.
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DOI：
10.3389/fchem.2021.753253
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发表时间：
2021
影响因子：
5.5
通讯作者：
Zhang DW
中科院分区：
文献类型：
--
作者：
Meng Y;Chen F;Jiang M;Guo Q;Wang Y;Wang J;Zhang DW
以G-三链体/亚甲基蓝（G3/MB）复合物为信号发生器，λ-外切核酸酶（λ-Exo）辅助扩增，构建了一种新型的均相无标记电化学生物传感器，用于microRNA（miRNA）的超灵敏检测。为了实现均相电化学分析的微型化，设计了一种集成微电极。利用G3能特异性结合MB并降低其扩散电流的特性，设计了一种单链的功能性DNA发夹结构作为生物识别探针。该探针由G3、阻断G3的8个碱基和靶miRNA的互补序列组成。在这里，我们选择了miRNA 141-一种潜在的前列腺癌诊断生物标志物作为模型靶标。miRNA 141的存在可以与探针DNA杂交形成具有5′-磷酸化末端的双链结构。然后采用λ-Exo从5′端开始消化单核苷酸，导致G3部分和miRNA 141的释放。释放的miRNA可以与另一种探针杂交以触发循环过程，而释放的G3因此可以与MB相互作用以引起可检测的扩散电流降低。所提出的策略显示出16 fM的低检测限和优异的特异性来区分单碱基错配。将该传感器应用于稀释的人血清样品中miRNA 141的检测，表明该传感器在真实的样品的核酸检测中具有很大的应用潜力。
A novel homogeneous label-free electrochemical biosensor using G-triplex/methylene blue (G3/MB) complex as the signal generator together with an amplification assisted by the λ-exonuclease (λ-Exo) has been successfully constructed for ultrasensitive microRNA (miRNA) detection. An integrated microelectrode was designed to realize the miniaturization of the homogeneous electrochemical assay. Taking advantage of G3, that can specifically bind with MB and decrease its diffusion current, a single-stranded functional DNA hairpin structure was designed as the bio-recognition probe. The probe consisted of G3, eight bases to block G3, and the complementary sequences of the target miRNA. Here we chose miRNA141—a potentially diagnostic biomarker of prostate cancer as the model target. The presence of miRNA141 could hybridize with the probe DNA to form a double-stranded structure with a 5′-phosphorylated terminus. Then λ-Exo was adopted to digest mononucleotides from the 5′-end, leading to the release of G3 part and miRNA141. The released miRNA could hybridize with another probe to trigger the cycling process, while the released G3 could therefore interact with MB to cause a detectable decrease of diffusion current. The proposed strategy showed a low detection limit of 16 fM and an excellent specificity to discriminate single-base mismatches. Furthermore, this sensor was applied to detect miRNA141 from diluted human serum samples, indicating that it has great potential in the application of nucleic acid detection in real samples.
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