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Abstract: 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.
Keywords: plant stress, scavengers, diagnostics, nanobodies, biosensors
Published in RUNG: 11.04.2025; Views: 240; Downloads: 2
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Keywords: comparative oncology, angiosarcoma, hemangiosarcoma, nanobodies
Published in RUNG: 18.03.2025; Views: 346; Downloads: 0
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Keywords: CDKL5, nanobodies, biopanning
Published in RUNG: 05.11.2024; Views: 814; Downloads: 0
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Abstract: Microcystis aeruginosa accumulation in freshwater poses a significant threat to aquatic organisms and human health. The toxicity of Cyanobacteria metabolites urges for the development of methods for their rapid and efficient detection but what is still almost completely missing is the availability of reagents for the quantification of M. aeruginosa cells in water to monitor the fluctuations of its population. In this study, nanobodies against cell surface antigens of the toxic Cyanobacteria M. aeruginosa were recovered bywhole-cell panning of a naive phage display library. Six unique sequences were identified and three of them sub-cloned and purified as fusion immunoreagents together with either green fluorescent protein or Avi-Tag to be used for diagnostics. Theirspecificity and sensitivity were evaluated by immunofluorescence, by fluorescent and colorimetric cell ELISA and by thermal lens spectrometry (TLS). No cross-reactivity with unrelated microalgae was detected, and both ELISA and TLS methods provided a linear range of detection of several logs. The limit-of-detection of TLS was as low as 1 cell/ml.
Keywords: cyanobacteria, nanobodies, phage display, thermal lens spectrometry, dissertations
Published in RUNG: 10.09.2024; Views: 1300; Downloads: 21
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Keywords: biosensors, nanobodies, C-reactive protein
Published in RUNG: 20.06.2024; Views: 1480; Downloads: 8
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