Electrochemistry and Optical Spectroscopy Center of Excellence, Department of Chemistry, Faculty of Science, Chulalongkorn University, Pathumwan, Bangkok, 10330, Thailand.
Department of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta 4259 B-57, Yokohama, 226-8501, Japan.
Biosens Bioelectron. 2023 Feb 1;221:114352. doi: 10.1016/j.bios.2022.114352. Epub 2022 May 11.
Nucleic acid biosensors for point-of-care (POC) diagnostic applications are highly desirable. The ability to detect DNA and RNA in a simple, rapid, affordable and portable format leads to a range of important applications for early screening in the field of disease monitoring and management. Herein, we report the development of an isothermal, label-free electrochemical biosensor that was designed on the basis of target-driven MNAzyme cleavage activity. Hybridization with HPV mRNA, a model nucleic acid target, activated MNAzyme and initiated the cleavage of immobilized hairpin substrates, leading to changes in the electrochemical signal. Under optimal conditions, a detection limit of 2.6 pM was obtained with an incubation time of 60 min. Furthermore, an artificial chaperone-enhanced MNAzyme (ACEzyme) system was integrated to an electrochemical biosensor for the first time. The analytical performance of the biosensor was enhanced, and the detection time was significantly reduced by the addition of PLL-g-Dex, which exhibits nucleic acid chaperone-like activity. A detection limit of 0.88 pM was obtained with a threefold decrease in incubation time without prior amplification. The proposed biosensing platform shows the advantages of simple fabrication and operation, good selectivity in the presence of single-base mismatch, and excellent versatility in a complex mixture of total RNA. We believe that this isothermal, label-free, and protein-free nucleic acid analysis platform could provide foundations for the further development of a universal nucleic acid biosensing platform for clinical application.
用于即时（POC）诊断应用的核酸生物传感器是非常需要的。能够以简单、快速、经济和便携的格式检测 DNA 和 RNA，为疾病监测和管理领域的早期筛查带来了一系列重要的应用。在此，我们报告了一种基于目标驱动的 MNAzyme 切割活性设计的等温、无标记电化学生物传感器的开发。与 HPV mRNA（一种模型核酸靶标）杂交激活了 MNAzyme 并启动了固定化发夹底物的切割，导致电化学信号发生变化。在最佳条件下，孵育 60 分钟后可获得 2.6 pM 的检测限。此外，首次将人工伴侣增强的 MNAzyme（ACEzyme）系统集成到电化学生物传感器中。通过添加 PLL-g-Dex，该生物传感器的分析性能得到了增强，检测时间显著缩短，PLL-g-Dex 具有核酸伴侣样活性。在不进行预先扩增的情况下，孵育时间减少了三倍，检测限达到 0.88 pM。所提出的生物传感平台具有简单的制造和操作优势，在存在单碱基错配的情况下具有良好的选择性，并且在复杂的总 RNA 混合物中具有出色的多功能性。我们相信，这种等温、无标记和无蛋白的核酸分析平台可以为临床应用的通用核酸生物传感平台的进一步发展提供基础。
