Bao Mengdi, Dollery Stephen J, Yuqing Fnu, Tobin Gregory J, Du Ke
Department of Chemical and Environmental Engineering, University of California, Riverside, CA, USA.
Biological Mimetics, Inc. 124 Byte Drive, Frederick, MD 21702, United States.
bioRxiv. 2023 Aug 23:2023.08.23.554533. doi: 10.1101/2023.08.23.554533.
CRISPR technology has gained widespread adoption for pathogen detection due to its exceptional sensitivity and specificity. Although recent studies have investigated the potential of high-aspect-ratio microstructures in enhancing biochemical applications, their application in CRISPR-based detection has been relatively rare. In this study, we developed a FRET-based biosensor in combination with high-aspect-ratio microstructures and Cas12a-mediated trans-cleavage for detecting HPV 16 DNA fragments. Remarkably, our results show that micropillars with higher density exhibit superior molecular binding capabilities, leading to a tenfold increase in detection sensitivity. Furthermore, we investigated the effectiveness of two surface chemical treatment methods for enhancing the developed FRET assay. A simple and effective approach was also developed to mitigate bubble generation in microfluidic devices, a crucial issue in biochemical reactions within such devices. Overall, this work introduces a novel approach using micropillars for CRISPR-based viral detection and provides valuable insights into optimizing biochemical reactions within microfluidic devices.
由于其卓越的灵敏度和特异性，CRISPR技术已在病原体检测中得到广泛应用。尽管最近的研究探讨了高纵横比微结构在增强生化应用方面的潜力，但其在基于CRISPR的检测中的应用相对较少。在本研究中，我们开发了一种基于荧光共振能量转移（FRET）的生物传感器，结合高纵横比微结构和Cas12a介导的反式切割来检测HPV 16 DNA片段。值得注意的是，我们的结果表明，密度较高的微柱表现出卓越的分子结合能力，导致检测灵敏度提高了十倍。此外，我们研究了两种表面化学处理方法对增强所开发的FRET检测的有效性。还开发了一种简单有效的方法来减轻微流控装置中气泡的产生，这是此类装置中生化反应的一个关键问题。总体而言，这项工作引入了一种使用微柱进行基于CRISPR的病毒检测的新方法，并为优化微流控装置中的生化反应提供了有价值的见解。
