In the present work we have established glycocalyx models to investigate the binding interactions of an important class of lectins that can interact with multiple components of the glycocalyx: the AB5 bacterial toxin family of proteins that is responsible for several diarrhoeal diseases. AB5 toxins have a quaternary structure consisting of 5 subunits of a glycan-binding protein that arrange into a doughnutshaped pentamer (B5), and an A-subunit that is enzymatically active and toxic to the host cell (Figure ). A prominent example of AB5 toxins is cholera toxin (CT), secreted by Vibrio cholerae, which is the cause of life-threating diarrhoea in the world's longest pandemic. To recognize and enter its intestinal epithelial and endothelial host cells, CT first binds the cell glycocalyx through the B-subunit (CTB). CTB has two sets of binding sites that recognise distinct glycans: the canonical binding site is located on the base of the protein and recognizes the oligosaccharide portion of ganglioside GM1 with high specificity and affinity; the noncanonical binding site is located on the lateral face of CTB and binds more weakly, with Kd in the millimolar range, to histo-blood group antigens Lewis y (Le y ) and Lewis x (Le x ) (Figure ). On the other hand, shiga toxin (STx) is an AB5 toxin secreted by Shigella dysenteriae and some strains of Escherichia coli such as O157:H7. Infection causes food poisoning, resulting in abdominal pain, watery diarrhoea, haemorrhagic colitis and hemolytic uremic syndrome. The B-subunit pentamer of Shiga Toxin (STxB) has three sets of glycan binding sites per protomer (total 3  5 = 15) located on the base of the protein, all of which recognize the glycosphingolipid Gb3 (and in some protein subtypes, also the glycosphingolipid Gb4). While the multi-valent interaction has a Kd in the nanomolar range, individual Gb3 oligosaccharides bind with varying affinity from 1.5 to >15 mM depending on the binding site(Figure ). An open question currently is how the multiple binding sites on AB5 toxins conspire for selective recognition of their host cells, and to facilitate cell entry. The arrangement of the glycolipid-binding sites on the pentameric faces of CTB and STxB appear optimal for interaction with multiple glycolipids in the cell membrane, but what about other potential interactions that could occur higher in the glycocalyx? The arrangement of the lower affinity Le x or Le y binding sites around the periphery of CTB might be better disposed for multivalent interactions with a 3D arrangement of glycans attached to glycoproteins. But could having similarly low affinity binding sites arranged on the flat surface of StxB also allow efficient multivalent interactions with a 3D glycocalyx? How strongly does the binding of AB5 toxins depend on the density of their glycan binders in the glycocalyx? To address these questions, we describe the synthesis of glycopolymers based on an HA backbone with mucin-like densities of pendant glycans, and with a defined degree of substitution (DS). We incorporated a biotin (and alternatively, a polyhistidine) anchorage tag at one end of the polymers to attach them to a surface for the construction of glycocalyx models with defined composition and molecular organization (Figure ). After characterization on a surface, we describe their use to investigate their capacity for superselective (i.e., sharply glycan-density dependent) binding with CTB and STxB.