Key Laboratory of Luminescence Analysis and Molecular Sensing (Southwest University), Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing, 400715, PR China.
Key Laboratory of Luminescence Analysis and Molecular Sensing (Southwest University), Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing, 400715, PR China.
Biosens Bioelectron. 2022 Jan 1;195:113662. doi: 10.1016/j.bios.2021.113662. Epub 2021 Sep 22.
Herein, the sequence-specific short-stranded biomarker DNA (hDNA, 21-nt) is acted as targeting out-primer to implement the loop-mediated isothermal amplification for releasing hydrogen ions (LAMP-H). Using LAMP-H as signaling transducer, we report a highly sensitive electrochemical ratiometric biosensor for hDNA with minimized background signal, which is achieved via magnetic separation using AuNPs-modified FeO (Au@FeO) as micro-reactor. In Au@FeO, a double-stranded complex of a pH-responsible strand (I*) and a substrate strand (S*) is bound via Au-N bonds, where the treatment with LAMP-H leads to I* folding into i-motif conformation and S* dehybridization. The S* further hybridizes a catalytic strand (C*) to assemble Mg-DNAzymes that are cleaved by Mg, releasing C* for repeated formation and robust nicking of Mg-DNAzymes. The resultant output fuel strands (F*) are introduced in a modified electrode to drive the strand displacement of two hairpins individually labeled with two electron mediators. Through F*-mediated recycled amplification, the ratio of their electrochemical currents changed in opposite is highly sensitive to the varied hDNA down to 2.1 fM. By integrating LAMP-H-stimulated i-motif switching with Mg-DNAzyme cleavage, this logic transduction of LAMP-H(i-motif/Mg-DNAzyme)F* efficiently minimizes the inherent background of traditional LAMP-based assays. Resultantly, our electrochemical ratiometric strategy would be applicable to diverse short-stranded DNAs or even RNAs as targeting primers of LAMP.
在此，序列特异性短链生物标志物 DNA（hDNA，21 个核苷酸）被用作靶向外引物，以实现释放氢离子的环介导等温扩增（LAMP-H）。我们利用 LAMP-H 作为信号转导子，报告了一种基于电化学比率法的高灵敏度生物传感器，用于检测 hDNA，其背景信号最小化，这是通过使用 AuNPs 修饰的 FeO（Au@FeO）作为微反应器进行磁性分离来实现的。在 Au@FeO 中，通过 Au-N 键结合 pH 响应链（I*）和底物链（S*）的双链复合物，用 LAMP-H 处理会导致 I折叠成 i- 发夹构象并使 S去杂交。S进一步与催化链（C）杂交以组装 Mg-DNAzyme，Mg 切割这些酶，释放 C以进行重复形成和 Mg-DNAzyme 的强烈缺口。所得输出燃料链（F）被引入修饰电极中，以驱动两个发夹各自单独标记有两个电子介体的链位移。通过 F*-介导的循环扩增，其电化学电流的比率变化与变化的 hDNA 呈高度敏感关系，低至 2.1 fM。通过整合 LAMP-H 刺激的 i- 发夹开关与 Mg-DNAzyme 切割，LAMP-H(i-motif/Mg-DNAzyme)F* 的这种逻辑转导有效地最小化了传统 LAMP 基测定法的固有背景。因此，我们的电化学比率策略可适用于各种短链 DNA 甚至 RNA，作为 LAMP 的靶向引物。
