Outcomes of the project, so far.
The design elements of MIP recognition, able to recognize and bind specific peptide sequences (epitopes) of proteins mainly involved in activating autoimmune reactions in people with celiac disease, has been achieved by means of in silico search in the protein databases of toxic, allergenic, gluten peptide sequences. Immunogenic epitopes were selected to play the role of templates for imprinting. Investigated epitopes were: PQQPFPQQ (8-mer), PFPQPQLPY (9-mer), PQPQLPYPQ (9-mer), and PYPQPQLPY (9-mer). These sequences have been used as templates in the further step of preparing the MIP recognition elements.
The MIP composition has been optimized by quantum-chemical computation, so to define the type of monomer to form stable interaction with the peptide epitopes and the stechiometry to be used in the polymerization. Both functional monomers and cross-linking monomers were chosen from the existing 'library' of monomers based on the bis(2,2’-bithienyl)methane derivatives. Quantum-chemistry calculations were performed in order to optimize the structures of the complexes between the gluten epitope templates and the functional monomers. The most stable complexes were selected for the polymerization.
The imprinting with the toxic gluten epitope (PQQPFPQQ) was performed by electrodeposition directly on the surfaces of chosen transduction structures, Pt electrodes and Au-glass extended gates of field effect transistors (EG-FETs). The characterization of the MIP films by surface investigating techniques, such as AFM, XPS, PM-IRRAS was then performed. Procedures for template removal from the MIP networks were investigated and optimized.
With focus on the sensor performance, both the peptide template and peptide interferents with aminoacid substituents or fully non-related sequences of peptides were tested for their response on the gluten sensor, finally entire gluten proteins as well as gluten extracts from semola, from rice, from whey were also tested. The selectivity of the gluten sensor was proven even to the level of discrimitating the single aminoacid substitution respect to the template sequence. The sensitivity of the gluten sensor was within the range required by actual regulations. The validation of the fabricated chemical sensor included checking its selective response to the gluten epitope both towards the model samples and to the real samples. Model samples involved the digestion of gluten in pepsin, and the separation of the peptides into fractions (HPLC fingerprinting). All the fractions were tested with EG-FETs. For a comparison commercially available ELISA gluten kits were utilized. ELISA and gluten sensor data were in correlation showing the gluten sensor so far responded with adequate selectivity and with the sensitivity required by actual regulations of allergen free foods.
