基于T7 RNA聚合酶突变体的DNA信号输出型无细胞生物传感系统OA

Cell-free biosensing systems that perform signal reporting at the RNA level feature a sim-ple system architecture and rapid response.However,the poor stability and susceptibility to degrada-tion of RNA limit their application in complex environmental samples.This study proposes a novel cell-free biosensing strategy based on a paradigm shift of the signal carrier.By introducing the T7 RNA polymerase mutant QMF-M6,which efficiently utilizes dNTPs to synthesize single-stranded DNA,into a transcription regulation system centered on an allosteric transcription factor(aTF),we achieved the transition from an RNA signal carrier to a stable DNA reporter molecule,thereby signifi-cantly enhancing the robustness and anti-interference capability of the biosensing system.Experimen-tal results demonstrate that this platform exhibits excellent detection performance for tetracycline and copper ions,with limits of detection reaching 0.62 μmol/L and 26 nmol/L,respectively,and shows good applicability in spiked recovery tests for copper ions in environmental water samples.In summa-ry,this study proposes a DNA signal output strategy to overcome the instability of reporter systems,providing a feasible technical pathway for developing robust cell-free biosensing systems suitable for applications such as on-site monitoring and point-of-care testing.