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
MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, Guangdong Provincial Key Laboratory of Laser Life Science, South China Normal University, Guangzhou, 510631, China; College of Biophotonics, South China Normal University, Guangzhou, 510631, China.
Department of Clinical Laboratory, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China.
Biosens Bioelectron. 2021 Apr 15;178:113027. doi: 10.1016/j.bios.2021.113027. Epub 2021 Jan 23.
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.
在早期癌症中对多种低表达水平的RNA进行快速、特异性定量分析是非常必要的，但仍然是一个挑战。在此，我们提出了一种双信号放大策略，该策略由CRISPR/Cas13a系统和催化发夹DNA电路（CHDC）组成，并集成在一个可重复使用的电化学生物传感器上，用于快速、准确地检测RNA。信号放大通过CRISPR/Cas13a系统与CHDC的独特组合来实现，使用10μL的测量体积，在6分钟的读出时间和36分钟的总处理时间内，检测限达到50 aM。传感器表面的酶促再生和背景信号的比率校正允许在单个生物传感器芯片上通过方波伏安法进行多达37次连续的RNA定量分析，而不会损失灵敏度。该可重复使用的生物传感器平台能够选择性地（特异性=0.952）和灵敏地（灵敏度=0.900）识别人类血清中的低表达RNA靶标，区分早期非小细胞肺癌（NSCLC）患者（n=20）与健康受试者（n=30）以及良性肺病患者（n=12）。对六种与NSCLC相关的RNA（miR-17、miR-155、TTF-1 mRNA、miR-19b、miR-210和EGFR mRNA）的测量表明，电化学CRISPR/CHDC系统有能力成为一种用于早期癌症诊断的快速、低成本且高度准确的工具。
