A Biohydrogel-Enabled Microneedle Sensor for In Situ Monitoring of Reactive Oxygen Species in Plants

Abstract

This study introduces a plant sensor utilizing an array of microneedles to monitor hydrogen peroxide (H₂O₂) in tobacco and soybean plants under biotic stress response. The microneedle array features a biohydrogel layer composed of the natural biopolymer chitosan (Cs) and reduced graphene oxide (rGO), functionalized with horseradish peroxidase (HRP) (HRP/Cs-rGO). This HRP/Cs-rGO biohydrogel combines biocompatibility, hydrophilicity, porosity, and electron transfer ability, making it a suitable bioelectrode material for an electrochemical sensor. The sensor detects H₂O₂ through the catalytic reaction of the enzyme, either by direct attachment to the plant leaf with the inserted microneedle or by exposure to the solution extracted from plant parts such as leaves. Utilizing chronoamperometry, the sensor demonstrates high sensitivity of 14.7 μA/μM across a concentration range of 0.1–4500 μM with a low detection limit of 0.06 μM. The sensor enables rapid detection of H₂O₂ levels by exposing the sensor to extracted leaf solutions. For in situ measurements within the leaf, results are obtained in approximately 1 min, eliminating the need for sample preparation. H₂O₂ levels in leaves following bacterial pathogen inoculation are evaluated alongside results from qualitative histological staining and quantitative fluorescence-based Amplex Red Assay, validating the ability of the sensor to detect changes in H₂O₂ concentrations during plant defense responses. This sensor technology has the potential to function as a portable device for on-site measurement of reactive oxygen species in plants, providing a rapid and cost-effective solution for H₂O₂ quantification.