Development of innovative extraction and detection methods for gluten based on deep eutectic solvents and aptamers

Celiac disease is an autoimmune disease, affecting about 1% of the global population which is triggered by gluten. Currently the only effective therapy available is a strict gluten-free diet. Hence, people affected by celiac disease can eat naturally gluten-free products or food specifically produced gluten-free. The term “gluten” refers to the entire protein component of wheat, while gliadin is the alcohol-soluble fraction of gluten that contains a bulk of the toxic components. These kinds of dietetic foods are regulated by the “Codex Alimentarius standard for foods for special dietary use for persons intolerant to gluten”, that sets a limit value of 20 mg/kg of gluten in these products. The aim of this thesis was the development of an analytical method for the reliable detection of gliadin in food. Regardless of all the advances that have occurred so far, the development of gluten quantification methods still encounters significant difficulties derived from the identity of the allergen, the lack of an effective and universal extraction method and the availability of few receptors of sufficient affinity and selectivity in order to obtain a reliable analytical method. On the other hand, considering that the use of deep eutectic solvents (DESs) as extractants is an expanding field as well as the use of aptamers as recognition elements. We combined these two aspects to bring advances in gluten quantification in food. For this purpose, the suitability of two DESs, ethaline and reline, to extract gliadin (50% content of gluten) was evaluated employing a commercially available ELISA kit. Moreover, we performed the selection and characterization of new ssDNA aptamers as biological receptors for gliadin in DESs. We used SELEX, which is a universal and iterative process, where an initial degenerated library of oligonucleotides is challenged against the analyte, to select sequences with the highest affinity. We took a step further by demonstrating the viability of SELEX in a green extraction solvent for the first time, thus providing a method for obtaining aptamers able to recognize non-soluble and poorly water-soluble molecules or species prone to aggregation in aqueous solutions. Lastly, we developed a competitive assay sufficiently sensitive and selective to be applied to the determination of gluten in foods labeled "gluten-free". The competitive assay confirmed the usefulness of the selected aptamer for the direct detection of gluten in ethaline extracts, without extra-dilutions. It is important to highlight that this approach allows gluten extraction and its quantification in ethaline without any dilution of the sample. Therefore, it allows to reach very low quantification limits.