Hyperphosphorylation at tyrosine is commonly observed in tumor proteomes and, hence, specific phosphoproteins or phosphopeptides could serve as markers useful for cancer diagnostics and therapeutics. The analysis of such targets is, however, a challenging task, because of their commonly low abundance and the lack of robust and effective preconcentration techniques. As a robust alternative to the commonly used immunoaffinity techniques that rely on phosphotyrosine(pTyr)-specific antibodies, we have developed an epitope-imprinting strategy that leads to a synthetic pTyr-selective imprinted polymer receptor. The binding site incorporates two monourea ligands placed by preorganization around a pTyr dianion template. The tight binding site displayed good binding affinities for the pTyr template, in the range of that observed for corresponding antibodies, and a clear preference for pTyr over phosphoserine (pSer). In further analogy to the antibodies, the imprinted polymer was capable of capturing short tyrosine phosphorylated peptides in the presence of an excess of their non-phosphorylated counterparts or peptides phosphorylated at serine.
A set of polymers has been imprinted with (−)-ephedrine at six different temperatures,
ranging from −30 to +80 °C. Polymer affinity and specificity were observed to be strongly dependent on
the polymerization temperature. The experimental results suggest that the polymer is able to “memorize”
the temperature used in the polymerization process in a manner similar to previously documented MIP
“memory” effects for the template and polymerization solvent. In a study of the effect of temperature on
retention and selectivity in HPLC (using the MIP as a column packing), a clear gradient change in the
Van't Hoff plots was observed at 20−30 °C. This indicates a transition in binding mechanism from
exothermic at higher temperatures to endothermic at lower temperatures. These results, supported by
the evidence of template-induced MIP swelling, are interpreted in terms of desolvation and conformational
changes in the polymers induced by the interaction with the template.
A set of polymers was imprinted with (-)-ephedrine using two different initiators. A chemometrics approach was used to optimise experiments aimed at analysis of the interplay of parameters such as polymerisation time, temperature and percentage of initiator. The results presented demonstrate the importance of keeping the right balance between these various parameters of polymerisation conditions. It is shown that enhancing one single parameter such as polymer rigidity does not necessarily improve polymer performance. In general it could be concluded that MIPs should be synthesised over a long period of time using low concentration of initiator and low temperature. The best selectivity was achieved for polymers prepared by photo-initiation with 2,2-dimethoxy-2-phenylacetophenone as initiator.
A set of polymers was imprinted with (−)-ephedrine using thermal and UV initiation. The
performance of the synthesized materials was studied by HPLC. It was shown that the polymer morphology
and enantioselective properties are dependent on the polymerization conditions and time of the reaction.
The binding mechanism of synthesized polymers was studied using Van't Hoff analysis. The results of
testing strongly indicate that the polymer receptor structure is shaped during the initial phase of polymer
gelation. The present study shows the importance of controlling experimental conditions in the MIP
synthesis and highlights potential problems expected during scaling up of MIP production.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.