Evolution of molecularly imprinted polymer nanoparticles as antibody mimics through the characterization of the molecular recognition process by single-molecule microscopy – MIPTIME

The aim of MIPTIME is to detect and characterize binding events of single ‘antigen’ molecules to single nanoparticles of molecularly imprinted polymer (MIP)-based synthetic antibody mimics. While the properties of MIPs have been studied at the macroscopic level, the molecular proof through the characterization of single binding events is still missing. Within MIPTIME we will set a new experimental framework to study molecular interactions in MIPs and, more generally, in biomimetic nanomaterials. For this purpose, we will use fluorescently-labeled MIP nanoparticles (MIP-NPs) with oriented binding sites, synthesized using an innovative solid-phase synthesis approach, where antigen molecules are immobilized on a solid support in an orientated way. These specifically-developed MIP-NPs will enable us to answer fundamental questions regarding the number of binding sites in a MIP, their orientation homogeneity, the dynamics of single molecule binding events related to antigen size, and the functionality of bivalent and bi-specific MIPs. To this aim, we will use super-resolved fluorescence lifetime imaging microscopy at the single molecule level with ultimate spatial and temporal resolution thanks to an innovative method recently developed within the consortium. This will allow the guided evolution of nanometer-sized MIPs towards 'ideal' molecular biomimics.