Natural deep eutectic solvents in combination with ultrasonic energy as a green approach for solubilisation of proteins: application to gluten determination by immunoassay.

In this work, a fast and miniaturised procedure based on the use of a natural deep eutectic solvent (NADES) in combination with ultrasound-assisted extraction (UAE) has been proposed for gluten determination by a commercial enzyme-linked immunosorbent assay (ELISA). Fourteen NADESs were prepared by combining two natural primary metabolites and water. Studies on NADES viscosity and gluten solubilisation in NADESs and ethanol-water solutions (for comparison purposes) were carried out. Different strategies for speeding-up gluten solubilisation in NADESs were evaluated: dilution, temperature and sonication by a cup-horn sonoreactor. Diluted fructose-citric acid NADES and sonication were finally selected for gluten solubilisation. Solubilised proteins were characterized by electrophoresis and molecular fluorescence. The proposed procedure was also assessed in real samples, especially ultrasound time. Kit solvents (including reducing agents) were replaced by NADES, and hence, a reassessing of immunoassay system was necessary. Samples with and without gluten as well as recovery tests were used for this purpose. Recoveries were in the range of 79-106% and the repeatability, expressed as relative standard deviation, was better than 15%.