Development of fructosyl valine binding polymers by covalent imprinting

Molecularly imprinted polymers (MIPs) against fructosyl valine (Fru-Val), the N-terminal constituent of hemoglobin A1c β-chains, were prepared by cross-linking of β-d-Fru-Val-O-bis(4-vinylphenylboronate) with an excess of ethylene glycol dimethacrylate (EDMA) or trimethylolpropane trimethacrylate (TRIM). Control MIPs were prepared in analogy by cross-linking the corresponding vinylphenylboronate esters of fructose and pinacol. After template extraction batch rebinding studies were performed using different pH values and buffer compositions. The Fru-Val imprinted TRIM cross-linked polymer binds about 1.4 times more Fru-Val than the fructose imprinted polymer and 2.7 times more Fru-Val than pinacol imprinted polymer. The highest imprinting effect was obtained in 100 mM sodium carbonate/10% methanol (pH 11.4). The TRIM cross-linked Fru-Val imprinted polymer showed a better specificity than the EDMA cross-linked polymer. The binding of valine was very low. Thermo gravimetric analysis indicated that the generated Fru-Val imprinted polymer has high thermo stability. No change in binding was observed after incubation of the polymers in buffer at 80 °C for 36 h. Since the functional group of the polymers (phenyl boronic acid) targets the sugar part of Fru-Val the imprint technique used should also be applicable for the development of MIPs against other glycated amino acids and peptides.