Miniaturized DyP Peroxidase-Based Biosensor for On-Site Open Air Use

Abstract

A dye-decolorizing peroxidase (DyP)-based electrochemical biosensor for hydrogen peroxide (H₂O₂) is developed in miniaturized, disposable, and user-friendly configuration. Wild type and variant DyPs are immobilized on self-assembled monolayer (SAM)-coated and nanostructure-modified screen-printed electrodes (SPEs) to ensure biocompatibility and increase the enzyme loading and hence the biosensor sensitivity. The structure of the enzymes attached to gold and silver nanoparticle (AuNP and AgNP)-modified carbon- and gold-based SPEs (C-SPE and Au-SPE) is monitored by resonance Raman spectroscopy and their electrocatalytic performance toward H₂O₂ by electrochemistry. Among the tested configurations, the wild type DyP/SAM/AuNP/C-SPE construct shows a superior performance, with a linear response range to H₂O₂ from 30 to 475 μM, a sensitivity of 234 ± 9 mA·M^–1·cm^–2, and a limit of detection (LOD) of 3.5 μM, measured in open air conditions. The device is suitable for single-use, on-site measurements of H₂O₂ in air-exposed samples such as physiological fluids. The herein developed biosensor shows a high potential for customization for the detection of other small hydroperoxide substrates and additional improvement of selected traits (e.g., sensitivity, thermal stability, and substrate inhibition properties) due to the easy and well-established production and genetic manipulation of the employed biocatalyst, DyP.