Yan Jiaxiang, Yin Bohan, Zhang Qin, Li Chuanqi, Chen Jiareng, Huang Yingying, Hao Jianhua, Yi Changqing, Zhang Yu, Wong Siu Hong Dexter, Yang Mo
Department of Biomedical Engineering, The Hong Kong Polytechnic University, Hong Kong, 999077, China.
Laboratory for Marine Drugs and Bioproducts, Qingdao Marine Science and Technology Center, Qingdao, 266237, China; School of Medicine and Pharmacy, Ocean University of China, Qingdao, 266003, China.
Biosens Bioelectron. 2025 Feb 15;270:116963. doi: 10.1016/j.bios.2024.116963. Epub 2024 Nov 19.
In this study, a CRISPR-Cas12a-mediated dual-mode upconversion luminescence/colorimetric nucleic acid biosensing platform is developed based on UCNP@SiO/CeO (UNSC) nanozyme. Here, UNSC is conjugated with single-stranded DNA (ssDNA) probes used as both peroxidase-like nanozyme and upconversion luminescence donors. When no target nucleic acid is present, ssDNA-conjugated UNSC attaches on magnetic graphene oxide (MGO) via pi-pi stacking force, resulting in upconversion luminescence quenching (OFF) and no color change after magnetic removal of nanozymes attached on the MGO. In the presence of target nucleic acid, Cas12a is specifically activated by targeted nucleic acid and indiscriminately cleaves the ssDNA probes on UNSCs. UNSCs then detach from the MGO surface due to the weakening of binding force, leading to upconversion luminescence recovery (ON) and colorimetric change due to the existence of free nanozyme in the 3,3',5,5'-tetramethyl-benzidine assay. As a proof-of-concept, this biosensing platform shows a limit of detection of around 320 fM in the upconversion luminescence mode and ∼28.4 pM in the colorimetric mode for nucleic acid detection, respectively. This UNSC nanozyme-based CRISPR-Cas12a dual-mode biosensing system also demonstrates high selectivity, good repeatability, and facile operation, which allows easy adaption to other nucleic acid-based detection only by redesigning the sequence of CRISPR RNA.
在本研究中，基于上转换纳米粒子@二氧化硅/二氧化铈（UNSC）纳米酶开发了一种CRISPR-Cas12a介导的双模式上转换发光/比色核酸生物传感平台。在此，UNSC与用作过氧化物酶样纳米酶和上转换发光供体的单链DNA（ssDNA）探针共轭。当不存在靶核酸时，与ssDNA共轭的UNSC通过π-π堆积力附着在磁性氧化石墨烯（MGO）上，导致上转换发光猝灭（关闭），并且在磁性去除附着在MGO上的纳米酶后没有颜色变化。在存在靶核酸的情况下，Cas12a被靶核酸特异性激活，并无差别地切割UNSCs上的ssDNA探针。由于结合力减弱，UNSCs随后从MGO表面脱离，导致上转换发光恢复（开启），并且由于在3,3',5,5'-四甲基联苯胺检测中存在游离纳米酶而发生比色变化。作为概念验证，该生物传感平台在上转换发光模式下对核酸检测的检测限约为320 fM，在比色模式下约为28.4 pM。这种基于UNSC纳米酶的CRISPR-Cas12a双模式生物传感系统还具有高选择性、良好的重复性和操作简便的特点，仅通过重新设计CRISPR RNA序列就可以轻松适用于其他基于核酸的检测。
