Fabrication of Carbohydrate Microarrays on Gold Surfaces:  Direct Attachment of Nonderivatized Oligosaccharides to Hydrazide-Modified Self-Assembled Monolayers

Abstract

This paper describes a new and simple microarray platform for presenting multiple nonderivatized oligosaccharides to protein targets, with utility for mapping carbohydrate-protein recognition events. The approach is based on the creation of a hydrazide-derivatized, self-assembled monolayer on a gold surface in a single or two-step procedure, for efficient and selectively oriented anchoring of oligosaccharide probes via their reducing ends, with detection using fluorescence detection of bound proteins. The biggest hurdles in employing gold-based substrate for fluorescence-based microarray detection include fluorescence quenching and nonspecific surface adsorption of proteins. We found that the quenching effect could be minimized by introducing a omega-thiolated fatty acid (C16) self-assembled monolayer between the gold surface and hydrazide groups, followed by detection involving three successive binding protein layers covering the gold surface. In addition, an effective blocking scheme involving poly(ethylene glycol) aldehyde and bovine serum albumin was employed to reduce nonspecific protein adsorption to the chip surface. As proof of principle, we demonstrate here that sulfated oligosaccharide probes from heparin can be effectively and covalently attached without prior derivatization onto the hydrazide-modified, self-assembled monolayer on gold-coated slide surfaces in a microarray format. This platform is used to assess binding of specific heparin-binding protein targets at very high sensitivity, and we also demonstrate that the approach can be extended to nonsulfated sugars. Direct attachment of nonderivatized sugar probes on the chip is advantageous since it avoids the need for laborious prederivatization and cleanup steps. This versatile fluorescence microarray platform provides a facile approach for interrogating multiple carbohydrate-protein interactions in a high-throughput manner and has potential as a common gold surface platform for other diverse interrogations by MALDI-MS, surface plasmon resonance, and quartz crystal microbalances.

Fabrication of Carbohydrate Surfaces by Using Nonderivatised Oligosaccharides, and their Application to Measuring the Assembly of Sugar–Protein Complexes

“…[25][26][27] Indeed, in our previous studies, we demonstrated that glycoarrays can be prepared by using hydrazide-modified surfaces on which nonspecific binding is reduced by the use of PEG-aldehyde blocking agent. [28] However, surface characterisation of these arrays has not been performed. Therefore, a short-chain PEG with a reducing end to react with unreacted hydrazide was employed as a blocking agent (Figure 2).…”

Automated Motif Discovery from Glycan Array Data

“…However, the glycan determinants recognized by individual GBPs are just beginning to be understood (Cummings, 2009), and is a major quest of modern glycomics research. The exploration of GBP interactions with glycans on microarrays of defined glycan structures represents a high-throughput method for exploring glycan-binding specificities (Blixt et al, 2004;Feizi and Chai, 2004;Fukui et al, 2002;Powell et al, 2009;Rillahan and Paulson, 2011;Song et al, 2008Song et al, , 2009Tateno et al, 2008;Willats et al, 2002;Zhi et al, 2006). Such information is important in identifying potential ligands for a GBP and developing hypotheses about their roles in GBP function.…”

Carbohydrate Coated Polymer Particles: Preparation and Protein‐binding Studies

“…Therefore investigators have made great efforts to devise the novel routes and materials which can directly attach the unmodified carbohydrates. Coupling the carbohydrates with the aromatic amino groups, 5 aminooxy groups 33,34 and hydrazide groups 33,35 by reductive amination or with the phthalimide chromophores 36,37 functionalized surfaces under photocatalysis were the two reported effective routes to achieve this goal. As the reductive amination can be easily operated and suitable for various reducing carbohydrates, it was preferred by a number of researchers.…”