Direct PCR-free electrochemical biosensing of plant-food derived nucleic acids in genomic DNA extracts. Application to the determination of the key allergen Sola l 7 in tomato seeds

• Autor(a) principal: Pereira-Barros, Magda A.
• Data de Publicação: 2019
• Outros Autores: Barroso, M. Fátima, Martín-Pedraza, Laura, Vargas, Eva, Benedé, Sara, Villalba, Mayte, Rocha, João M., Campuzano, Susana, Pingarrón, José M.
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.22/16004
• topic: Sola l 7 allergen coding-sequences; Sandwich hybridization; AbRNA/DNA; Screen-printed electrodes; Amperometry; Genomic DNA extract

A novel and disposable electrochemical biosensor for PCR-free and selective detection of Sola l 7, a non-specific lipid transfer protein (nsLTP) found in tomato seeds associated to severe symptoms of tomato-allergic patients, is reported in this work. The methodology involves the formation of DNA/RNA heterohybrids by sandwich hybridization of a specific fragment of the Sola l 7 allergen coding sequence with appropriate RNA probes designed and described for the first time in this work. Labeling was carried out with commercial antibodies specific to the heteroduplexes and secondary antibodies conjugated with HRP onto the surface of magnetic beads. Amperometric transduction was performed upon magnetic capture of the resulting magnetic bioconjugates on screen-printed electrodes using the system H2O2/HQ. A comparison of the sandwich hybridization format with a direct approach as well as between different labeling strategies was performed. The LOD value achieved was 0.2 pM (5 amol in 25 μL). The biosensor was successfully applied to the selective analysis of the targeted Sola l 7 specific region directly in just 100 ng of non-fragmented denatured genomic DNA extracted from tomato seeds.