Probing the recognition specificity of a protein molecularly imprinted polymer using force spectroscopy

Molecularly imprinted polymers (MIPs) are synthetic receptors obtained by polymerization using molecular templates. We have synthesized MIP films (co-polymers of acrylamide and different acrylic acid-based cross-linkers) with specific binding sites for cytochrome c, which were imprinted in the bulk or in the surface. The binding specificity of the polymers was studied at the macroscale by equilibrium binding experiments with fluorescein-labeled cytochrome c. Imprinting factors of up to 4.1 were obtained that were a function of the cross-linker used and the degree of cross-linking. We have then employed, for the first time, AFM force spectroscopy to directly measure the force of interaction of the protein with the synthetic receptor sites obtained by molecular imprinting. The polymer surfaces were scanned with AFM cantilevers carrying covalently attached cytochrome c molecules, giving raise to specific binding events with binding forces between 85 and 95 pN. Control cantilevers without cytochrome c or with covalently attached bovine serum albumin, as well as non-imprinted control polymers, did not yield specific binding events. We believe that these results demonstrate the great potential of force spectroscopy for the characterization of molecularly imprinted polymers.