Horseradish peroxidase as an electrochemical reporter protein for cell-free biosensors

Abstract Cell-free biosensor systems offer a promising platform for portable diagnostics. Here, we evaluate electrochemical readout from these systems, using horseradish peroxidase (HRP) as a redox enzyme reporter. HRP was synthesized in an Escherichia coli cell-free transcription-translation system supplemented with hemin, calcium acetate, and commercial disulfide bond enhancers. Electrochemical detection of its activity was established by chronoamperometry, with hydrogen peroxide as a substrate and tetramethylbenzidine as a redox mediator. Cell-free expressed HRP produced a strong steady-state current compared to a catalytically inactive mutant and a no-template control. Kinetic analysis showed a Km for the cell-free expressed HRP close to that of the native enzyme. To explore the potential of HRP as an electrochemical reporter, we placed it under the control of a tetracycline-responsive regulatory promoter and demonstrated a 2.5-fold current increase in the presence of anhydrotetracycline. These results support HRP as an electrochemical reporter for cell-free biosensors, offering a complementary alternative to existing optical reporters for future use in handheld analytical devices.