Universal Amplification-Free RNA Detection by Integrating CRISPR-Cas10 with Aptameric Graphene Field-Effect Transistor
Mingyuan Sun, Zhenxiao Yu, Shuai Wang, Jiaoyan Qiu, Yuzhen Huang, Xiaoshuang Chen, Yunhong Zhang, Chao Wang, Xue Zhang, Yanbo Liang, Hong Liu, Qunxin She, Yu Zhang, Han Lin, Yu Zhang, Han Lin
Abstract
Abstract Amplification-free, highly sensitive, and specific nucleic acid detection is crucial for health monitoring and diagnosis. The type III CRISPR-Cas10 system, which provides viral immunity through CRISPR-associated protein effectors, enables a new amplification-free nucleic acid diagnostic tool. In this study, we develop a CRISPR-graphene field-effect transistors (GFETs) biosensor by combining the type III CRISPR-Cas10 system with GFETs for direct nucleic acid detection. This biosensor exploits the target RNA-activated continuous ssDNA cleavage activity of the dCsm3 CRISPR-Cas10 effector and the high charge density of a hairpin DNA reporter on the GFET channel to achieve label-free, amplification-free, highly sensitive, and specific RNA detection. The CRISPR-GFET biosensor exhibits excellent performance in detecting medium-length RNAs and miRNAs, with detection limits at the aM level and a broad linear range of 10 −15 to 10 −11 M for RNAs and 10 −15 to 10 −9 M for miRNAs. It shows high sensitivity in throat swabs and serum samples, distinguishing between healthy individuals (N = 5) and breast cancer patients (N = 6) without the need for extraction, purification, or amplification. This platform mitigates risks associated with nucleic acid amplification and cross-contamination, making it a versatile and scalable diagnostic tool for molecular diagnostics in human health.