School of Environment and Natural Resources, Renmin University of China, Beijing, 100872, China.
State Key Laboratory of NBC Protection for Civilian, Beijing, 102205, China.
Biosens Bioelectron. 2023 Apr 1;225:115109. doi: 10.1016/j.bios.2023.115109. Epub 2023 Jan 28.
Although CRISPR-based biosensors for pathogenic detection are highly specific, they not sensitive enough and nucleic acid amplification is generally required to improve their sensitivity. However, this allows only binary operations and significantly limits practical applications. Here, a CRISPR/Cas12a-powered Evanescent wAve fluorescence nanobiosensing plaTform (CREAT) was developed for ultrasensitive nucleic acid amplification-free quantitative detection of pathogens with multiple signal enhancements. In addition to collateral cleavage amplification of the CRISPR/Cas12a system, we constructed nanophotonic structure-based evanescent wave fluorescence enhancement, Mg or DNA-mediated fluorescence enhancement, and air-displacement fluorescence enhancement strategies for ultrasensitive detection of Staphylococcus aureus (S. aureus). Especially, the fluorescence signal detected by CREAT can be significantly enhanced by adding a simple air displacement step, thus improving detection sensitivity. This nanobiosensor detected real samples containing S. aureus, with a detection limit of 592 CFU/mL and 13.2 CFU/mL in 45 min and 90 min, respectively, which are comparable to those of RT-qPCR. This paves a new way for simple, rapid, sensitive, robust, and flexible on-site detection of S. aureus as well as other pathogens.
尽管基于 CRISPR 的生物传感器在病原体检测方面具有高度特异性，但它们的灵敏度还不够高，通常需要核酸扩增来提高其灵敏度。然而，这仅允许进行二进制操作，并且显著限制了实际应用。在这里，我们开发了一种基于 CRISPR/Cas12a 的渐逝波荧光纳米生物传感平台（CREAT），用于通过多种信号增强进行无核酸扩增的超灵敏病原体核酸定量检测。除了 CRISPR/Cas12a 系统的旁链切割扩增外，我们还构建了基于纳米光子结构的渐逝波荧光增强、Mg 或 DNA 介导的荧光增强以及空气置换荧光增强策略，用于超灵敏检测金黄色葡萄球菌（S. aureus）。特别是，通过添加简单的空气置换步骤，可显著增强 CREAT 检测到的荧光信号，从而提高检测灵敏度。该纳米生物传感器可检测含有金黄色葡萄球菌的实际样本，在 45 分钟和 90 分钟时，检测限分别为 592 CFU/mL 和 13.2 CFU/mL，与 RT-qPCR 相当。这为金黄色葡萄球菌以及其他病原体的简单、快速、灵敏、稳健和灵活的现场检测开辟了新途径。
