Early stress detection in forest trees using a nanobody-functionalized electrochemical biosensor for ascorbate peroxidase

• Successful isolation of monoclonal antibodies against APX from a nanobody pre-immune library. • Expression of application-ready fusion constructs formed by nanobodies and fluorescent proteins. • Demonstration of anti-APX cross-reactivity towards pea and poplar antigens. • Preparation of a point-of-care biosensor for APX quantification. Forest environments are exposed to multiple stressful factors of both abiotic and biotic nature such as heavy metal contamination, drought, or pest infestations which may lead to their massive decline. We designed a comprehensive approach for isolating, producing and functionalizing reagents suitable for the affordable detection of forest plant stress biomarkers with the aim to provide quantitative data to assess plant stress fluctuation and, possibly, to design mitigation strategies. We first optimized a panning protocol to recover nanobodies targeting shared sequences that could cross-react with both Pisum sativum and Populus nigra ascorbate peroxidase (APX). After their production as recombinant constructs and their extensive biophysical and biochemical characterization, such reagents were exploited as the immunocapture element of an electrochemical biosensor conceived as a potential point-of-care device. Such biosensor could detect both pea and poplar APX in leaf extracts and could be used to clearly discriminate between control and heavy metal-stressed poplar plants based on their APX activity, even before the appearance of any phenotypic symptom. The combination of fast and inexpensive reagent production with the development of portable diagnostics opens the opportunity for large-scale, on-site surveys of forest trees.