Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi'an 710119, China.
College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi'an 710119, China.
Analyst. 2022 May 30;147(11):2567-2574. doi: 10.1039/d2an00613h.
Due to the excellent activity of -cleavage and target recognition, the recently discovered CRISPR/Cas12a systems provide a promising opportunity for designing fluorescence biosensing. In the reported CRISPR/Cas12a-based biosensing platform, TaqMan probe is widely used as the reporter. However, the TaqMan probe provides single-wavelength fluorescence changes, which is easily influenced by various analyte-independent confounding factors to produce false-positive signals. In this study, a ratiometric fluorescent probe was designed to act as the reporter of a CRISPR/Cas12a-based system. As a proof-of-concept, fluorescein (FAM) and tetramethylrhodamine (TAMRA) were chosen as the two fluorescence dyes to label one short ssDNA at 5' and 3' ends, respectively, which was designed as one ratiometric fluorescent probe. When the ratiometric probe excites at 480 nm, duo to FRET effect, the probe emitted the 580 nm-fluorescence of TAMRA. The activated Cas12a can cleave the dual-labeled ssDNA, resulting in a decrease in the TAMRA's fluorescence and an increase in the FAM's fluorescence. This dual-response fluorescent probe can act as the reporter of the CRISPR/Cas12a-based biosensing platform. Compared with the classic TaqMan, CRISPR/Cas12a-based biosensing with the ratiometric probe as the reporter not only exhibited higher sensitivity but also could distinguish and avoid false positive signals.
由于 - 切割和靶标识别的优异活性，最近发现的 CRISPR/Cas12a 系统为设计荧光生物传感提供了一个有前途的机会。在报道的基于 CRISPR/Cas12a 的生物传感平台中，TaqMan 探针被广泛用作报告器。然而，TaqMan 探针提供单波长荧光变化，容易受到各种与分析物无关的混杂因素的影响，产生假阳性信号。在本研究中，设计了一种比率荧光探针作为基于 CRISPR/Cas12a 系统的报告器。作为概念验证，选择荧光素（FAM）和四甲基罗丹明（TAMRA）作为两种荧光染料，分别标记在 5'和 3'末端的一条短 ssDNA 上，设计成一种比率荧光探针。当比率探针在 480nm 激发时，由于 FRET 效应，探针发射出 TAMRA 的 580nm 荧光。激活的 Cas12a 可以切割双标记的 ssDNA，导致 TAMRA 的荧光减少和 FAM 的荧光增加。这种双响应荧光探针可以作为基于 CRISPR/Cas12a 的生物传感平台的报告器。与经典的 TaqMan 相比，基于 CRISPR/Cas12a 的生物传感使用比率探针作为报告器不仅表现出更高的灵敏度，而且还可以区分和避免假阳性信号。
