Ultrasensitive sensing of T4 PNK phosphatase activity through establishing a novel transcription-based signal amplification platform

Signal amplification plays a significant role in nucleic acid analysis, especially in trace nucleic acid detection. Transcription-based signal amplification is a very important component in the field of signal amplification, and has received extensive attention depending on outstanding cyclic transcription ability. However, the low efficiency of transcription caused by limited template length usually restricts their further applications significantly. Herein, we developed a transcription-based signal amplification method with high sensitivity and selectivity. The approach we proposed couples the advantageous features of long template synthesis from phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP) with self-amplification from cell-free transcription system. The results demonstrate that this approach can be applied to quantify T4 Polynucleotide Kinase (T4 PNK) phosphatase activity in a broad range from 0.00001 to 0.01 U/mL, with a detection limit of 8.1 × 10-6 U/mL. Moreover, this device can be further employed as a superior signal amplification platform for the detection of other analytical targets after moderate modifications.