Selective glycoprotein detection through covalent templating and allosteric click-imprinting

Abstract

Many glycoproteins are intimately linked to the onset and progression of numerous heritable or acquired diseases of humans, including cancer. Indeed the recognition of specific glycoproteins remains a significant challenge in analytical method and diagnostic development. Herein, a hierarchical bottom-up route exploiting reversible covalent interactions with boronic acids and so-called click chemistry for the fabrication of glycoprotein selective surfaces that surmount current antibody constraints is described. The self-assembled and imprinted surfaces, containing specific glycoprotein molecular recognition nanocavities, confer high binding affinities, nanomolar sensitivity, exceptional glycoprotein specificity and selectivity with as high as 30 fold selectivity for prostate specific antigen (PSA) over other glycoproteins. This synthetic, robust and highly selective recognition platform can be used in complex biological media and be recycled multiple times with no performance decrement.

Fig. 1. Experimental design for formation of surface restricted click-imprinted binding sites for glycoproteins.

Fig. 2. SPR sensorgram traces performed with PSA-imprinted surfaces on the SPR chip and different concentrations of (a) PSA and (b) RNAse B flowed over the surface. (c) SPR responses at equilibrium against the concentration of injected protein, PSA, lysozyme, α-1-acid glycoprotein (α1-AGP), RNAse B, bovine serum albumin (BSA), α-1-antitrypsin (α1-AT) and horseradish peroxidase (HRP), from which dissociation constants (K _d) have been obtained.

Fig. 3. (a) SPR responses at equilibrium obtained from RNAse B-imprinted surfaces against the concentration of injected protein, from which dissociation constants (K _d) have been obtained. (b and c) SPR responses at equilibrium obtained from RNAse B-imprinted surfaces, which were prepared in the (b) presence and (c) absence of BA carbohydrate receptors, against the concentration of injected RNAse B and A proteins. (d) SPR responses at equilibrium obtained from pre-conditioned RNAse B-imprinted surfaces against the % (w/w) of RNAse B in 0.5% serum solution. (e) SPR responses at equilibrium from 10 SPR cycles (as shown in the inset), which were performed using the RNAse B-imprinted surface.