Search

A molecularly imprinted polymer (MIP) recognition system was devised for selective determination of an immunogenic gluten octamer epitope, PQQPFPQQ. For that, a thin MIP film was devised, guided by density functional theory calculations, and then synthesized to become the chemosensor recognition unit. Bis(bithiophene)-based cross-linking and functional monomers were used for this synthesis. An extended-gate field-effect transistor (EG-FET) was used as the transduction unit. The EG-FET gate surface was coated with the PQQPFPQQ-templated MIP film, by electropolymerization, to result in a complete chemosensor. X-ray photoelectron spectroscopy analysis confirmed the presence of the PQQPFPQQ epitope, and its removal from the MIP film. The chemosensor selectively discriminated between the octamer analyte and another peptide of the same number of amino acids but with two of them mismatched (PQQQFPPQ). The chemosensor was validated with respect to both the PQQPFPQQ analyte and a real gluten extract from semolina flour. It was capable to determine PQQPFPQQ in the concentration range of 0.5-45 ppm with the limit of detection (LOD) = 0.11 ppm. Moreover, it was capable of determining gluten in real samples in the concentration range of 4-25 ppm with LOD = 4 ppm, which is a value sufficient for discriminating between gluten-free and non-gluten-free food products. The gluten content in semolina flour determined with the chemosensor well correlated with that determined with a commercial ELISA gluten kit. The Langmuir, Freundlich, and Langmuir-Freundlich isotherms were fitted to the epitope sorption data. The sorption parameters determined from these isotherms indicated that the imprinted cavities were quite homogeneous and that the epitope analyte was chemisorbed in them.

The pool of peptides composing a protein allows for its distinctive identification in a process named fingerprint (FP) analysis. Here, the FP concept is used to develop a method for the rational preparation of molecularly imprinted polymers (MIPs) for protein recognition. The fingerprint imprinting (FIP) is based on the following: (1) the in silico cleavage of the protein sequence of interest with specific agents; (2) the screening of all the peptide sequences generated against the UniProtKB database in order to allow for the rational selection of distinctive and unique peptides (named as epitopes) of the target protein; (3) the selected epitopes are synthesized and used as templates for the molecular imprinting process. To prove the principle, NT-proBNP, a marker of the risk of cardiovascular events, was chosen as an example. The in silico analysis of the NT-proBNP sequence allowed us to individuate the peptide candidates, which were next used as templates for the preparation of NT-pro-BNP-specific FIPs and tested for their ability to bind the NT-proBNP peptides in complex samples. Results indicated an imprinting factor, IF, of ~10, a binding capacity of 0.5-2 mg/g, and the ability to rebind 40% of the template in a complex sample, composed of the whole digests of NT-proBNP.