Direct and sensitive determination of trypsin in human urine using a water-soluble signaling fluorescent molecularly imprinted polymer nanoprobe

A molecularly imprinted polymer (MIP) for biosensing trypsin is prepared by incorporating a benzamidine-based signaling fluorescent monomer during its synthesis. Binding to trypsin results in a 100-fold fluorescence enhancement when the MIP is excited with UV light. The assay can be performed by simply mixing a small amount of water-soluble MIP (100 μg) and trypsin together in a quartz cuvette, followed by a direct read-out on a spectrofluorimeter. There is no need to separate the free from the bound trypsin. The limit of quantification is 50 nM in phosphate buffer and 210 nM in urine. The aqueous MIP is prepared by a solid-phase synthesis method on glass-beads functionalized with a metal chelate to immobilize trypsin via its surface histidine. The active site of the enzyme is left free for binding to the benzamidine moiety of the fluorescent monomer, resulting in a sensitive MIP for probing the enzyme's active site. Incorporation of a thermoresponsive monomer, N-isopropyacrylamide, in the polymerization mixture, yields thermoresponsive MIP nanoparticles that are released from the support by a simple temperature change, generating template-free polymers. The MIPs are endowed with improved binding site homogeneity since all binding sites have the same orientation. Their size is ∼70 nm and the dissociation constant (Kd) of the MIP-trypsin complex is 237 nM, with little or no cross-reactivity with other proteins, including serine proteases.