Department of Chemistry and Biochemistry , Auburn University , Auburn , Alabama 36849 , United States.
Anal Chem. 2018 Mar 6;90(5):3584-3591. doi: 10.1021/acs.analchem.8b00036. Epub 2018 Feb 9.
Electrochemical bioanalytical sensors with oligonucleotide transducer molecules have been recently extended for quantifying a wide range of biomolecules, from small drugs to large proteins. Short DNA or RNA strands have gained attention recently due to the existence of circulating oligonucleotides in human blood, yet challenges remain for adequately sensing these targets at electrode surfaces. In this work, we have developed a quantitative electrochemical method which uses target-induced proximity of a single-branched DNA structure to drive hybridization at an electrode surface, with readout by square-wave voltammetry (SWV). Using custom instrumentation, we first show that precise control of temperature can provide both electrochemical signal amplification and background signal depreciation in SWV readout of small oligonucleotides. Next, we thoroughly compared 25 different combinations of binding energies by their signal-to-background ratios and differences. These data served as a guide to select the optimal parameters of binding energy, SWV frequency, and assay temperature. Finally, the influence of experimental workflow on the sensitivity and limit of detection (LOD) of the sensor is demonstrated. This study highlights the importance of precisely controlling temperature and SWV frequency in DNA-driven assays on electrode surfaces while also presenting a novel instrumental design for fine-tuning of such systems.
电化学生物分析传感器与寡核苷酸转导分子最近已被扩展用于定量分析各种生物分子，从小型药物到大中型蛋白质。由于人类血液中存在循环寡核苷酸，短 DNA 或 RNA 链最近受到关注，但在电极表面充分感应这些靶标仍然存在挑战。在这项工作中，我们开发了一种定量电化学方法，该方法使用单链 DNA 结构的目标诱导接近来驱动电极表面的杂交，通过方波伏安法 (SWV) 进行读出。使用定制仪器，我们首先表明，温度的精确控制可以在小寡核苷酸的 SWV 读出中提供电化学信号放大和背景信号衰减。接下来，我们通过信号与背景的比值和差异彻底比较了 25 种不同的结合能组合。这些数据为选择最佳结合能、SWV 频率和分析温度的参数提供了指导。最后，还证明了实验工作流程对传感器的灵敏度和检测限 (LOD) 的影响。这项研究强调了在电极表面的 DNA 驱动分析中精确控制温度和 SWV 频率的重要性，同时还提出了一种新颖的仪器设计，用于微调此类系统。
