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bioRxiv · 10.64898/2025.12.17.694927

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

Ruhela, A.·Liyanagedera, S. B. W.·Laohakunakorn, N.·Marland, J. R. K.
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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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=46 SRC="FIGDIR/small/694927v1_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@1b39294org.highwire.dtl.DTLVardef@5c3578org.highwire.dtl.DTLVardef@1ba827forg.highwire.dtl.DTLVardef@1cd8bd1_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Ruhela, A., Liyanagedera, S. B. W., Laohakunakorn, N., Marland, J. R. K.. 2025-12-17. Horseradish peroxidase as an electrochemical reporter protein for cell-free biosensors. https://doi.org/10.64898/2025.12.17.694927

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