Department of Chemistry and Biochemistry, University of Maryland Baltimore County (UMBC) 1000 Hilltop Circle, Baltimore, Maryland 21250, United States.
Anal Chem. 2014 Jan 21;86(2):1131-7. doi: 10.1021/ac4029054. Epub 2014 Jan 9.
Folding-based electrochemical sensors utilizing structure-switching aptamers are specific, selective, sensitive, and widely applicable to the detection of a variety of target analytes. The specificity is achieved by the binding properties of an electrode-bound RNA or DNA aptamer biorecognition element. Signaling in this class of sensors arises from changes in electron transfer efficiency upon target-induced changes in the conformation/flexibility of the aptamer probe. These changes can be readily monitored electrochemically. Because of this signaling mechanism, there are several approaches to maximizing the analytical attributes (i.e., sensitivity, limit of detection, and observed binding affinity) of the aptamer sensor. Here, we present a systematic study of several approaches, including electrochemical interrogation parameters and biomolecular engineering of the aptamer sequence, to develop a sensor for the detection of aminoglycoside antibiotics. Specifically, through a combination of optimizing the electrochemical signal and engineering the parent 26-nucleotide RNA aptamer sequence to undergo larger conformation changes, we develop several improved sensors. These sensors exhibit binding affinities ranging from 220 nM to 42 μM, as much as a 100-fold improved limit of detection in comparison to previously reported sensors, and a variety of linear ranges including the therapeutic window for tobramycin. These data demonstrate that rational engineering of the aptamer structure to create large conformation changes upon target binding leads to improved sensor performance. We believe that the sensor design guidelines outlined here represent a general strategy for developing new aptamer folding-based electrochemical sensors.
基于构象变化适体的折叠式电化学传感器具有特异性、选择性、灵敏性，并且广泛适用于多种目标分析物的检测。其特异性是通过电极结合的 RNA 或 DNA 适体生物识别元件的结合特性来实现的。在这类传感器中，信号源于目标诱导适体探针构象/柔韧性变化时电子转移效率的变化。这些变化可以通过电化学方法进行监测。由于这种信号机制，有几种方法可以最大限度地提高适体传感器的分析特性（即灵敏度、检测限和观察到的结合亲和力）。在这里，我们系统地研究了几种方法，包括电化学检测参数和适体序列的生物分子工程，以开发用于检测氨基糖苷类抗生素的传感器。具体来说，通过优化电化学信号和对 26 个核苷酸 RNA 适体序列进行工程改造以产生更大的构象变化相结合，我们开发了几种改进的传感器。这些传感器的结合亲和力范围为 220 nM 至 42 μM，与之前报道的传感器相比，检测限提高了约 100 倍，并且具有多种线性范围，包括妥布霉素的治疗窗。这些数据表明，通过合理设计适体结构以在结合目标时产生大的构象变化，可提高传感器的性能。我们相信，这里概述的传感器设计准则代表了开发新的基于适体折叠的电化学传感器的一般策略。
