A CRISPR/Cas13a-powered catalytic electrochemical biosensor for successive and highly sensitive RNA diagnostics

• Published in: Biosensors & bioelectronics Vol. 178; p. 113027
• Main Authors: Sheng, Yan, Zhang, Tenghua, Zhang, Shihong, Johnston, Midori, Zheng, Xiaohe, Shan, Yuanyue, Liu, Tong, Huang, Zena, Qian, Feiyang, Xie, Zihui, Ai, Yiru, Zhong, Hankang, Kuang, Tairong, Dincer, Can, Urban, Gerald Anton, Hu, Jiaming
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 15.04.2021
• Subjects: Biosensing Techniques; biosensors; blood serum; Carcinoma, Non-Small-Cell Lung; Catalytic hairpin; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR/Cas technology; detection limit; diagnostic techniques; DNA; DNA circuit; DNA, Catalytic; Electrochemical analysis; electrochemistry; Humans; lung neoplasms; Lung Neoplasms - diagnosis; Lung Neoplasms - genetics; Nucleic acid diagnostics; On-site testing; processing time; voltammetry; DNA circuit; Electrochemical analysis; On-site testing; Catalytic hairpin; Nucleic acid diagnostics; CRISPR/Cas technology
• DOI: 10.1016/j.bios.2021.113027
• ISSN: 0956-5663; 1873-4235

Rapid and specific quantitation of a variety of RNAs with low expression levels in early-stage cancer is highly desirable but remains a challenge. Here, we present a dual signal amplification strategy consisting of the CRISPR/Cas13a system and a catalytic hairpin DNA circuit (CHDC), integrated on a reusable electrochemical biosensor for rapid and accurate detection of RNAs. Signal amplification is accomplished through the unique combination of the CRISPR/Cas13a system with CHDC, achieving a limit of detection of 50 aM within a readout time of 6 min and an overall process time of 36 min, using a measuring volume of 10 μL. Enzymatic regeneration of the sensor surface and ratiometric correction of background signal allow up to 37 sequential RNA quantifications by square-wave voltammetry on a single biosensor chip without loss of sensitivity. The reusable biosensor platform could selectively (specificity = 0.952) and sensitively (sensitivity = 0.900) identify low expression RNA targets in human serum, distinguishing early-stage patients (n = 20) suffering from non-small-cell lung carcinoma (NSCLC) from healthy subjects (n = 30) and patients with benign lung disease (n = 12). Measurement of six NSCLC-related RNAs (miR-17, miR-155, TTF-1 mRNA, miR-19b, miR-210 and EGFR mRNA) shows the ability of the electrochemical CRISPR/CHDC system to be a fast, low-cost and highly accurate tool for early cancer diagnostics. [Display omitted] •An electrochemical biosensor based on dual isothermal amplification is developed.•Program the CRISPR/Cas13a system with catalytic hairpin DNA circuit to detect RNAs.•Enzymatic surface regeneration and ratiometric background correction are achieved.•Early-stage lung cancer patients are distinguished from healthy ones.