Photo-Click Immobilization on Quartz Crystal Microbalance Sensors for Selective Carbohydrate−Protein Interaction Analyses

Abstract

A photo-click method based on azide photoligation and Cu-catalyzed azide-alkyne cycloaddition has been evaluated for the immobilization of carbohydrates to polymeric materials. The biomolecular recognition properties of the materials have been investigated with regard to applicable polymeric substrates and selectivity of protein binding. The method was used to functionalize a range of polymeric surfaces (polystyrene, polyacrylamide, poly(ethylene glycol), poly(2-ethyl-2-oxazoline) and polypropene) with various carbohydrate structures (based on α-Dmannose, β-D-galactose and N-acetyl-β-D-glucosamine). The functionalized surfaces were evaluated in real-time studies of protein-carbohydrate interactions using a quartz crystal microbalance flow-through system with a series of different carbohydrate-binding proteins (lectins). The method proved to be robust and versatile, resulting in a range of efficient sensors showing high and predictable protein selectivities.

Photogenerated lectin sensors produced by thiol-ene/yne photo-click chemistry in aqueous solution

“…These lectins were chosen based on their binding specificities towards the chosen carbohydrates, their sizes and physical properties. RCA-I and Con A thus display similar molecular weights and pI values, (Bhattacharyya and Brewer 1990; Norberg et al 2011; Ready et al 1984; Sweeney et al 1997) leading to similar behavior towards the surfaces in the QCM setup. Furthermore, two different types of carbohydrate thiols were utilized to investigate the versatility of the method as well as the potential implications of different divalent ligands on protein binding: 1-thio-carbohydrates 17 – 18 and thioethyl-linked carbohydrates 4 and 8 .…”

“…The photo-click immobilization method presented in Figure 1 is based on initial photochemical insertion of perfluorophenyl azides (PFPAs) into the polymeric material, as previously reported, (Norberg et al 2011; Norberg et al 2009a) and subsequent amide coupling to produce the corresponding active materials. In the present case, the polystyrene surfaces were functionalized with triethyleneglycol-linked alkynes or alkenes in a two-step procedure, starting with spincoating of PFPA-NHS structure 15 from an ethanol solution, and subsequent nitrene-mediated photoligation under UV-irradiation at 254 nm.…”

Sensing lectin–glycan interactions using lectin super-microarrays and glycans labeled with dye-doped silica nanoparticles

“…Compared with other techniques for assessing glycan-lectin interactions, such as nuclear magnetic resonance (NMR) (Johnson and Pinto 2004), ELISA (Gervay and McReynolds 1996), ITC (Dam and Brewer 2002), agglutination (Knibbs et al 1993), equilibrium dialysis (Cho and Cummings 1996), spectrophotometry (Polizzotti et al 2007), fluorescence competition assay (Wang et al 2010b), SPR (Deng et al 2011; Tyagi et al 2010), carbohydrate microarrays (Pei et al 2007), dynamic light scattering (DLS) (Wang et al 2011c), and quartz-crystal microbalance (QCM) (Norberg et al 2011; Norberg et al 2009), the present method is high-throughput and affords assessment of an apparent affinity constant from simple mixing and incubation experiments on a single slide. For each assay on the super-microarray, only ~0.3 μg of glycan is needed to synthesize the corresponding FSNP-labeled glycan.…”

Stereocontrolled 1-S-glycosylation and comparative binding studies of photoprobe-thiosaccharide conjugates with their O-linked analogs

“…Several methods to study carbohydrate–protein interactions can be employed to address this difference, e.g., isothermal titration calorimetry (ITC) [44], hemagglutination inhibition [40], precipitation inhibition [45], equilibrium dialysis [46], spectrophotometry [47], enzyme-linked immunosorbent assay (ELISA) [48], fluorescence assay [49,50], nuclear magnetic resonance (NMR) [51], surface plasmon resonance (SPR) [41,52], quartz-crystal microbalance (QCM) [53,54], carbohydrate microarrays [55], and scanning probe microscopy (SPM) [56]. Of these, two major methods are especially advantageous: ITC and solid-phase binding techniques, the combination of which results in elucidation of overall binding performances.…”