Evidence of Positive Cooperativity in the Binding Behavior of Molecularly Imprinted Polymers Prepared by Solid Phase Polymerization Synthesis

Nanosized molecularly imprinted polymers (nanoMIPs) and polyclonal antibodies are assumed to share the same binding behavior due to the presence of mutually independent binding sites and characterized by a continuous distribution of the binding affinity with respect to an average value. This gives rise to a convex Scatchard plot and is well described by the Langmuir–Freundlich (Sips) isotherm. Nevertheless, some preliminary experimental indications suggest the possibility of a different binding behavior, characterized by positive cooperativity. Since this behavior would have significant repercussions on the practical use of nanoMIPs, the binding behavior of a nanoMIP with molecular recognition properties toward ciprofloxacin has been thoroughly investigated. Repeated equilibrium binding experiments on nanoMIPs of different cross-linking degrees (2%, 10%, and 20% molar of methylene-bis-acrylamide as cross-linker, respectively) allowed to accurately determine the binding isotherms over a wide range of ligand concentrations. The consequent statistical treatment of the data showed the clear prevalence of binding isotherm models consistent with positive cooperativity (heterogeneity parameter n > 1 and concave Scatchard plot) when compared to other alternative binding models considered. Moreover, the exponential heterogeneity parameter does not correlate with the degree of cross-linking of the nanoMIPs, suggesting that the origin of positive cooperativity could be due not only to the degree of stiffness of the nanopolymer but also to the structural proximity of the binding sites, as shown by further experimental data obtained from nanoMIPs prepared with rabbit γ-globulins, a much bulkier template than ciprofloxacin.