Du Yi-Chen, Wang Si-Yuan, Wang Ya-Xin, Ma Jia-Yi, Wang Dong-Xia, Tang An-Na, Kong De-Ming
State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Centre for Analytical Sciences, College of Chemistry, Nankai University, Tianjin, 300071, PR China.
State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Centre for Analytical Sciences, College of Chemistry, Nankai University, Tianjin, 300071, PR China.
Biosens Bioelectron. 2021 Jan 1;171:112734. doi: 10.1016/j.bios.2020.112734. Epub 2020 Oct 14.
A simple and highly sensitive biosensing strategy was reported by cascading terminal deoxynucleotidyl transferase (TdT)-catalyzed substrate extension and CRISPR-Cas12a -catalyzed short-stranded DNA probe cleavage. Such a strategy, which is named as TdT-combined CRISPR-Cas12a amplification, gives excellent signal amplification capability due to the synergy of two amplification steps, and thus shows great promise in the design of various biosensors. Based on this strategy, two representative biosensors were developed by simply adjusting the DNA substrate design. High signal amplification efficiency and nearly zero background endowed the biosensors with extraordinary high sensitivity. By utilizing these two biosensors, ultrasensitive detection of uracil-DNA glycosylase (UDG) and T4 polynucleotide kinase (T4 PNK) was achieved with the detection limit as low as 5 × 10 U/mL and 1 × 10 U/mL, respectively. The proposed UDG-sensing platform was also demonstrated to work well for the UDG activity detection in cancer cells as well as UDG screening and inhibitory capability evaluation, thus showing a great potential in clinical diagnosis and biomedical research.
通过级联末端脱氧核苷酸转移酶（TdT）催化的底物延伸和CRISPR-Cas12a催化的短链DNA探针切割，报道了一种简单且高度灵敏的生物传感策略。这种被命名为TdT联合CRISPR-Cas12a扩增的策略，由于两个扩增步骤的协同作用，具有出色的信号放大能力，因此在各种生物传感器的设计中显示出巨大潜力。基于此策略，通过简单调整DNA底物设计开发了两种代表性生物传感器。高信号放大效率和近乎零的背景使生物传感器具有极高的灵敏度。利用这两种生物传感器，实现了对尿嘧啶-DNA糖基化酶（UDG）和T4多核苷酸激酶（T4 PNK）的超灵敏检测，检测限分别低至5×10 U/mL和1×10 U/mL。所提出的UDG传感平台还被证明在癌细胞中的UDG活性检测以及UDG筛选和抑制能力评估方面表现良好，从而在临床诊断和生物医学研究中显示出巨大潜力。
