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Herein we report novel approaches to the molecular imprinting of proteins utilizing templates sizing around 10 nm and some 100 nm. The first step comprised synthesizing nanoparticles of molecularly imprinted polymers (MIP) towards bovine serum albumin (BSA) and characterizing them according to size and binding capacity. In a second step, they were utilized as templates. Quartz crystal microbalances (QCM) coated with MIP thin films based on BSA MIP nanoparticles lead to a two-fold increase in sensor responses, compared with the case of directly using the protein as the template. This also established that individual BSA molecules exhibit different "epitopes" for molecular imprinting on their outer surfaces. In light of this knowledge, a possible MIP-based biomimetic assay format was tested by exposing QCM coated with BSA MIP thin films to mixtures of BSA and imprinted and non-imprinted polymer (NIP) nanoparticles. At high protein concentrations (1000 ppm) measurements revealed aggregation behavior, i.e., BSA binding MIP NP onto the MIP surface. This increased sensor responses by more than 30% during proof of concept measurements. At lower a BSA concentration (500 ppm), thin films and particles revealed competitive behavior.

Combined Layer/Particle Approaches in Surface Molecular Imprinting of Proteins: Signal Enhancement and Competition