Terminal deoxynucleotidyl transferase combined CRISPR-Cas12a amplification strategy for ultrasensitive detection of uracil-DNA glycosylase with zero background

• Published in: Biosensors & bioelectronics Vol. 171; p. 112734
• Main Authors: Du, Yi-Chen, Wang, Si-Yuan, Wang, Ya-Xin, Ma, Jia-Yi, Wang, Dong-Xia, Tang, An-Na, Kong, De-Ming
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 01.01.2021
• Subjects: Amplification; Biosensing Techniques; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR-Cas Systems; CRISPR-Cas12a; DNA Nucleotidylexotransferase; Uracil-DNA Glycosidase - genetics; Uracil-DNA Glycosidase - metabolism; Uracil-DNA glycosylase (UDG); Zero background; Amplification; CRISPR-Cas12a; Zero background; Uracil-DNA glycosylase (UDG)
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2020.112734

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 × 10−6 U/mL and 1 × 10−4 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. •A novel signal amplification strategy was reported for biosensor design.•Two biosensors targeting at uracil-DNA glycosylase and polynucleotide kinase were designed.•Powerful signal amplification and zero background confer the biosensors with extraordinarily high sensitivity.•The developed biosensors work well for complex biological samples.•The developed sensing platforms can also be used for enzyme inhibitor screening and inhibitory capability evaluation.