High-sensitive and rapid electrochemical detection of miRNA-31 in saliva using Cas12a-based 3D nano-harvester with improved trans-cleavage efficiency

• Published in: Talanta (Oxford) Vol. 266; no. Pt 2; p. 125066
• Main Authors: Wu, Yu, Pei, Jingwen, Li, Yi, Wang, Guobin, Li, Lan, Liu, Jinbo, Tian, Gang
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2024
• Subjects: CRISPR/Cas12a; Electrochemical biosensor; MiRNA testing; Nanomachine; Electrochemical biosensor; MiRNA testing; Nanomachine; CRISPR/Cas12a
• ISSN: 0039-9140; 1873-3573; 1873-3573
• DOI: 10.1016/j.talanta.2023.125066

Salivary miRNA-31 is a reliable diagnostic marker for early-stage oral squamous cell carcinoma (OSCC), but accurate detection of miRNA-31 in saliva samples is a challenge because of its low level and high sequence homology. The CRISPR/Cas12a system has the exceptional potential to enable simple nucleic acid analysis but suffers from low speed and sensitivity. To achieve rapid and high-sensitive detection of miRNA-31 using the CRISPR/Cas12a system, a Cas12a-based nano-harvester activated by a polymerase-driven DNA walker, named as dual 3D nanorobots, was developed. The target walked rapidly on the surface of DNA hairpin-modified magnetic nanoparticles driven by DNA polymerase, generating numerous double-strand DNA (dsDNA). Then, the Cas12a bound to the generated dsDNA for activating its trans-cleavage activity, forming 3D nano-harvester. Subsequently, the harvester cut and released methylene blue-labeled DNA hairpins immobilized on the sensing interface, leading to the change in electrochemical signal. We found that the trans-cleavage activity of the harvester was higher than the conventional CRISPR/Cas12a system. The developed dual 3D nanorobots could enable rapid (detection time within 60 min), high-sensitive (detection limit of femtomolar), and specific analysis of miRNA-31 in saliva samples. Thus, our established electrochemical biosensing strategy has great potential for early diagnosis of OSCC. [Display omitted] •Compared with conventional CRISPR/Cas12a, the Cas12-based nano-harvester has higher trans-cleavage efficiency.•The Cas12-based nano-harvester combined with ISAR-based 3D DNA walker is developed.•The developed dual 3D nanorobots enable rapid, ultrasensitive, and specific analysis of miRNA-31 in saliva samples.