Department of Chemical and Environmental Engineering, University of California, Riverside, CA, USA.
Biological Mimetics, Inc., 124 Byte Drive, Frederick, MD 21702, USA.
Lab Chip. 2023 Dec 20;24(1):47-55. doi: 10.1039/d3lc00780d.
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.
CRISPR 技术因其出色的灵敏度和特异性而被广泛应用于病原体检测。尽管最近的研究已经探讨了高纵横比微结构在增强生化应用方面的潜力，但它们在基于 CRISPR 的检测中的应用相对较少。在这项研究中，我们开发了一种基于 FRET 的生物传感器，结合高纵横比微结构和 Cas12a 介导的转切割，用于检测 HPV 16 DNA 片段。值得注意的是，我们的结果表明，具有更高密度的微柱具有更优越的分子结合能力，从而将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开发的 FRET 测定的有效性。还开发了一种简单有效的方法来减轻微流控设备中气泡的产生，这是此类设备中生化反应的关键问题。总的来说，这项工作介绍了一种使用微柱进行基于 CRISPR 的病毒检测的新方法，并为优化微流控设备中的生化反应提供了有价值的见解。
