PETN reductase-coupled alcohol-selective enzymes for dual-mode PETN amperometric sensing and power generation

Reliable on-site detection of pentaerythritol tetranitrate (PETN) remains challenging due to its low vapor pressure and limited suitability for direct electrochemical conversion. Here we report a PETN reductase (PETNR)-driven cascade strategy enabling dual-mode PETN detection and electrochemical decomposition, combining amperometric biosensing as the primary analytical mode and a proof-of-concept hybrid biofuel cell. Three enzyme bioelectrode architectures were developed on glassy carbon electrodes by covalent immobilization of PETNR with (1) NADP⁺-dependent alcohol dehydrogenase (ADH), (2) alcohol oxidase (AOx) with hydroquinone (HQ) mediation, and (3) AOx/horseradish peroxidase with Mendola blue as mediator to enable low-potential operation. The PETNR/ADH/GCE bioelectrode provided the highest analytical performance, exhibiting a linear response from 0.6 to 60 μM PETN, a limit of detection of 0.2 μM, and a high sensitivity of 0.16 ± 0.005 μA cm^-2·μM^-1. The AOx-coupled designs expanded operational flexibility, while mechanistic studies revealed that HQ can promote the formation of insulating polymeric deposits during PETN conversion; vibrational and solid-state NMR analyses supported a heterogeneous aromatic-aliphatic network formation pathway. The developed biosensor was validated on post-explosion soil extracts against HPLC, yielding PETN levels around 1.6 μM with deviations ≤7% and RSD <7%. In addition, a one-compartment Zn/PETNR-ADH-HQ hybrid biofuel cell generated PETN-dependent electrical output (OCV up to 0.878 V at 50 μM PETN in model solutions) and operated directly in contaminated soil, supporting the feasibility of a proof-of-concept self-powered configuration complementary to the primary amperometric sensing mode. Overall, the proposed PETNR-centered cascade strategy provides a versatile proof-of-concept platform for PETN detection with potential for future adaptation toward portable electrochemical devices.