Qiao Shan-Peng, Liu Zhen-Ni, Li Hai-Chao, He Xin, Pan Hong, Gao Yu-Zhou
Department of Changchun Institute of Engineering Technology, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Science (CAS), 3333 Shengbei Street, Changchun, 130052, Jilin, P. R. China.
Department of Jilin City Institute of Biological Products, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Science (CAS), 1228 Songjiangnan Road, Jilin, 132013, Jilin, P. R. China.
Chembiochem. 2021 Jun 2;22(11):1974-1984. doi: 10.1002/cbic.202100054. Epub 2021 Apr 14.
The clustered regularly interspaced short palindromic repeats (CRISPR) technology has been widely applied for nucleic acid detection because of its high specificity. By using the highly specific and irreversible bond between HaloTag and its alkane chlorine ligand, we modified dCas9 (deactivated CRISPR/Cas9) with biotin as a biosensor to detect nucleic acids. The CRISPR biosensor was facilely prepared to adequately maintain its DNA-recognition capability. Furthermore, by coupling biolayer interferometry (BLI) with the CRISPR biosensor, a real-time, sensitive, and rapid digital system called CRISPR-BLI was established for the detection of double-stranded DNA. The CRISPR biosensor immobilised on the biolayer could recruit the target DNA onto the biosensor surface and change its optical thickness, resulting in a shift in the interference pattern and responding signal of the BLI. The CRISPR-BLI system was further applied to detect the ALP gene of Escherichia coli DH5α combined with a polymerase chain reaction, which demonstrated a linear range from 20 to 20 000 pg and a low detection limit (1.34 pg). The CRISPR-BLI system is a promising approach for rapid and sensitive detection of target DNA analytes.
成簇规律间隔短回文重复序列（CRISPR）技术因其高特异性已被广泛应用于核酸检测。通过利用卤代标签与其烷烃氯配体之间高度特异性且不可逆的键合，我们用生物素修饰失活的CRISPR/Cas9（dCas9）作为生物传感器来检测核酸。该CRISPR生物传感器制备简便，能充分保持其DNA识别能力。此外，通过将生物层干涉术（BLI）与CRISPR生物传感器耦合，建立了一种名为CRISPR-BLI的实时、灵敏且快速的数字系统用于双链DNA检测。固定在生物层上的CRISPR生物传感器可将靶DNA招募到生物传感器表面并改变其光学厚度，导致干涉图谱和BLI响应信号发生偏移。CRISPR-BLI系统进一步与聚合酶链反应结合用于检测大肠杆菌DH5α的碱性磷酸酶（ALP）基因，其线性范围为20至20000 pg，检测限低（1.34 pg）。CRISPR-BLI系统是一种用于快速灵敏检测靶DNA分析物的有前景的方法。
