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

Abstract Cell-free biosensor systems offer a promising platform for portable diagnostics. However, most employ fluorescent reporter proteins that require complex instrumentation and can be affected by photo-bleaching and auto-fluorescence, limiting translatability. Electrochemical reporters do not suffer from these drawbacks. Here, we evaluate horseradish peroxidase (HRP) as a redox enzyme reporter for cell-free biosensor systems. HRP was synthesized in an E. coli cell-free transcription-translation system supplemented with hemin, calcium acetate, and commercial disulfide bond enhancers. The electrochemical detection of its activity was established by chronoamperometry, with H2O2 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 3-fold current increase in the presence of anhydrotetracycline. These results support HRP as an electrochemical reporter for cell-free biosensors, offering a practical alternative to optical reporters for future use in handheld analytical devices. Competing Interest Statement The authors have declared no competing interest.