Institute of Molecular Materials Chemistry and Technology, School of Chemistry and Chemical Engineering, Jiangxi University of Science and Technology, Ganzhou, 341000, PR China; State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, PR China.
Institute of Molecular Materials Chemistry and Technology, School of Chemistry and Chemical Engineering, Jiangxi University of Science and Technology, Ganzhou, 341000, PR China.
Anal Chim Acta. 2022 Jan 15;1190:339266. doi: 10.1016/j.aca.2021.339266. Epub 2021 Nov 10.
Diseases caused by bacteria pose great challenges to human health. The key to reduce disease transmission and mortality is to develop accurate and rapid methods for the detection and identification of bacteria. Herein, a rapid bacteria 16S rDNA electrochemical sensor based on target-triggered hairpin self-assembly and tripedal DNA walker (TD walker) amplification strategy was constructed. Specific variable region of 16S rDNA fragment of bacteria was used as biomarker. The target-triggered hairpin self-assembly strategy was used to prepare a TD walker. The hairpin DNA probes labeled with ferrocene (F) were designed and modified on surface of electrode. The "legs" of TD walker hybridized with three hairpin probes and opened their hairpin structures. Exo III enzyme recognised hybrid duplexes and selectively digest hairpin probes. The "legs" of TD walker was released and hybridized with the other three hairpin probes. In this way, the enzyme drived the walkers to walk along electrode interface, until hairpin DNA probes were all removed from the electrode, the F was far away from electrode interface. A significantly current reduction signal was obtained and bacteria were detected by recording this response. This strategy was low-cost and scalable, it could continuously recycle low-concentration targets, thus enhanced the detection sensitivity. As the proof-of-concept work, the electrochemical sensor was utilized as detector. The limit of detection (LOD) of detecting Staphylococcus aureus (S. aureus) was 20 CFU mL and detection time was less than 3 h. It was expected to be widely used in clinical early diagnosis.
细菌引起的疾病对人类健康构成了巨大挑战。减少疾病传播和死亡率的关键是开发准确、快速的细菌检测和鉴定方法。本文构建了一种基于靶标触发发夹自组装和三足 DNA walker（TD walker）扩增策略的快速细菌 16S rDNA 电化学传感器。以细菌 16S rDNA 片段的特异性可变区为生物标志物。采用靶标触发发夹自组装策略制备 TD walker。设计并修饰了带有二茂铁（F）的发夹 DNA 探针。TD walker 的“腿”与三个发夹探针杂交并打开其发夹结构。外切酶 III 识别杂交双链体并选择性地消化发夹探针。TD walker 的“腿”被释放并与其他三个发夹探针杂交。通过这种方式，酶驱动 walker 沿着电极界面行走，直到所有发夹 DNA 探针都从电极上被去除，F 远离电极界面。通过记录该响应获得了显著的电流减少信号，并通过该信号检测细菌。该策略具有成本效益高和可扩展的特点，它可以连续循环低浓度的靶标，从而提高了检测灵敏度。作为概念验证工作，电化学传感器被用作检测器。检测金黄色葡萄球菌（S. aureus）的检测限（LOD）为 20 CFU mL，检测时间小于 3 h。预计它将在临床早期诊断中得到广泛应用。
