Abstract
Since glycoproteins have become increasingly recognized as key players in a wide variety of disease processes, there is an increasing need for advanced affinity materials for highly selective glycoprotein binding. Herein, for the first time, a surface-initiated controlled radical polymerization is integrated with supramolecular templating and molecular imprinting to yield highly reproducible synthetic recognition sites on surfaces with dissociation constants (KD) in the low micromolar range for target glycoproteins and minimal binding to nontarget glycoproteins. Importantly, it is shown that the synthetic strategy has a remarkable ability to distinguish the glycosylated and nonglycosylated forms of the same glycoprotein, with a >5-fold difference in binding affinity. The precise control over the polymer film thickness and positioning of multiple carbohydrate receptors plays a crucial role in achieving an enhanced affinity and selectivity. The generated functional materials of unprecedented glycoprotein recognition performance open up a wealth of opportunities in the biotechnological and biomedical fields.
Highlights
In the context of creating highly selective imprinted glycoprotein cavities on surfaces, strategies that are amenable to a three-dimensional (3D) display of the carbohydrate receptors are appealing since they can more closely create a sterically and chemically complementary cavity to the natural 3D glycan structure. With this proviso in mind, we demonstrate a surface imprinting methodology that meets these criteria by relying on the precomplexation of boronic acid (BA)-based carbohydrate receptors with the target glycoprotein and atom transfer radical polymerization (ATRP), which enables precise control over the surface macromolecular structure and functionality (Figure 1).[25]
The trend in thickness growth over time is dependent on the ATRP conditions, which affect the polymerization rate and cessation of polymerization.[35−37] These results demonstrate that this system provides elegant control over the thicknesses of the polymer layer and can be used to reliably grow surfaceconfined ultrathin molecularly imprinted polymer (MIP) films
The unprecedented performance relies on the recognition capabilities of the surface-confined imprinted cavities that are dictated by the well-defined pattern of multiple BAs within the cavity that are sterically complementary to the unique molecular structure of the glycan on the target glycoprotein
Summary
Glycoproteins make up the majority of human proteins, with many having a close association with disease progression.[1−3] the recognition and quantification of glycoproteins are of paramount importance for a variety of research purposes and practical applications, including life sciences and medical research, clinical diagnostics, medical devices, and imaging.[4−7] major challenges remain regarding their specific recognition and in particular how the structural features of both the protein and glycan parts of the glycoprotein can be simultaneously recognized to maximize selectivity.[8,9]. Our modular strategy involves four main steps: (1) functionalization of a gold surface with an ATRP initiatorterminated SAM; (2) formation of a high order BA/ glycoprotein complex using protein compatible conditions; (3) surface-initiated highly controlled ATRP polymerization in the presence of the preformed BA/glycoprotein complexes to create glycoprotein glycan-specific 3D interaction sites within ultrathin, imprinted polymer films; (4) following formation of the well-controlled molecular cavities, the glycoprotein template can be removed by washing with an elution buffer due to the reversible nature of the BA/diol interactions.[24,26] This strategy provides the ability to achieve surface binding sites, which are complementary to the glycoprotein template in their size and shape and to the very specific orientation and sugar sequence of the glycoprotein glycan This latter recognition mode is facilitated by the initial generation of a BA/glycoprotein complex (step 2), in which the spatial arrangement of the multiple BA receptors in the complex is preserved upon surface incorporation via surface-initiated ATRP polymerization (step 3). The chosen control proteins provide a range of sizes, charges, and percentages of glycosylation (Table S1) to give a representative selection of different properties that can contribute to the binding response
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