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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

Publication . Pereira-Barros, Magda A.; Barroso, M. Fátima; Martín-Pedraza, Laura; Vargas, Eva; Benedé, Sara; Villalba, Mayte; Rocha, João M.; Campuzano, Susana; Pingarrón, José M.

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.

2019-05-07Journal article

Open access

The carboxylic acid transporters Jen1 and Jen2 affect the architecture and fluconazole susceptibility of Candida albicans biofilm in the presence of lactate

Publication . Alves, Rosana; Mota, Sandra; Silva, Sónia; F. Rodrigues, Célia; P. Brown, Alistair J.; Henriques, Mariana; Casal, Margarida; Paiva, Sandra

Candida albicans has the ability to adapt to different host niches, often glucose-limited but rich in alternative carbon sources. In these glucose-poor microenvironments, this pathogen expresses JEN1 and JEN2 genes, encoding carboxylate transporters, which are important in the early stages of infection. This work investigated how host microenvironments, in particular acidic containing lactic acid, affect C. albicans biofilm formation and antifungal drug resistance. Multiple components of the extracellular matrix were also analysed, including their impact on antifungal drug resistance, and the involvement of both Jen1 and Jen2 in this process. The results show that growth on lactate affects biofilm formation, morphology and susceptibility to fluconazole and that both Jen1 and Jen2 might play a role in these processes. These results support the view that the adaptation of Candida cells to the carbon source present in the host niches affects their pathogenicity.

2017Journal article

Open access