Electrochemical biosensor for ultrasensitive thrombin detection based on aptamer-modulated transcriptional inhibition of cell-free synthetic biology

In this study, a novel strategy integrating aptamer-gated cell-free synthetic biology with electrochemical analysis has been developed for Thrombin detection (using Thrombin as a model target). A DNA template embedded with a target-specific aptamer is designed as a "molecular switch". Target binding induces a conformational change in the aptamer, creating steric hindrance for T7 RNA polymerase and thereby inhibiting transcription of the RNA reporter strand. The transcribed RNA can hybridize with methylene blue (MB)-labeled DNA probes immobilized on the electrode surface, causing electroactive species to move away from the electrode surface and resulting in a relatively low electrochemical signal. Conversely, a reduced amount of transcribed RNA leads to an increased electrochemical signal. Under optimized conditions, the biosensor exhibits a linear range of 0.06 nM - 6 μM and a limit of detection (LOD) of 0.032 nM (S/N = 3). The sensor demonstrates excellent specificity against interfering proteins. Spike-recovery tests in 20-fold diluted human serum yield recoveries of 95.35% - 105.20% with RSD values of 2.32% - 4.90%, confirming its anti-interference ability in complex biological matrices. This methodological innovation realizes sensitive and specific detection of Thrombin and provides a generalizable strategy for cell-free electrochemical analysis of protein targets. By replacing the aptamer sequence, the platform can be extended to diverse proteins, opening new avenues for the application of cell-free synthetic biology in biosensing and clinical diagnostics.