期刊
BIOSENSORS & BIOELECTRONICS
卷 178, 期 -, 页码 -出版社
ELSEVIER ADVANCED TECHNOLOGY
DOI: 10.1016/j.bios.2021.113027
关键词
CRISPR/Cas technology; On-site testing; Nucleic acid diagnostics; Catalytic hairpin; DNA circuit; Electrochemical analysis
类别
资金
- National Natural Science Foundation of China [21804023]
- Science and Technology Program of Guangzhou, China [2019050001]
- Scientific Research Projects of Guangdong Province, China [2019QN01Y725]
- Recruitment Program of Global Experts, China
- Natural Science Foundation of Guangdong Province, China [2018A030313319]
- Scientific Research Projects of Guangzhou, China [201805010002]
- Fundamental Research Funds for the Provincial Universities of Zhejiang [RFA2020008]
- Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) [421356369]
The study introduces a dual signal amplification strategy using the CRISPR/Cas13a system and CHDC for rapid and accurate detection of low expression level RNAs in early-stage cancer. The reusable biosensor platform showed high specificity and sensitivity in distinguishing early-stage lung cancer patients from healthy subjects.
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 mu L. 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.
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