Time-Resolved Fluorescence Spectroscopy of Molecularly Imprinted Nanoprobes as an Ultralow Detection Nanosensing Tool for Protein Contaminants

Abstract

Currently, optical sensors based on molecularly imprinted polymers (MIPs) have been attracting significant interest. MIP sensing relies on the combination of the MIP’s selective capability, which is conveyed to the polymeric material by a template-assisted synthesis, with optical techniques that offer exquisite sensitivity. In this work, we devised an MIP nanoparticle optical sensor for the ultralow detection of serum albumin through time-resolved fluorescence spectroscopy. The Fluo-nanoMIPs (∅~120 nm) were synthetized using fluorescein-O-methacrylate (0.1×, 1×, 10× mol:mol versus template) as an organic fluorescent reporter. The ability of 0.1× and 1×Fluo-nanoMIPs to bind albumin (15 fM–150 nM) was confirmed by fluorescence intensity analyses and isothermal titration calorimetry. The apparent dissociation constant (Kapp) was 30 pM. Conversely, the 10× fluorophore content did not enable monitoring binding. Then, the time-resolved fluorescence spectroscopy of the nanosensors was studied. The 1×Fluo-nanoMIPs showed a decrease in fluorescence lifetime upon binding to albumin (100 fM–150 nM), Kapp = 28 pM, linear dynamic range 3.0–83.5 pM, limit of detection (LOD) 1.26 pM. Selectivity was confirmed testing 1×Fluo-nanoMIPs against competitor proteins. Finally, as a proof of concept, the nanosensors demonstrated detection of the albumin (1.5 nM) spiked in wine samples, suggesting a possible scaling up of the method in monitoring allergens in wines.