Yih Horng Tan

HPLC-Assisted Automated Oligosaccharide Synthesis

A standard HPLC was adapted to polymer supported oligosaccharide synthesis. Solution-based reagents are delivered using a software-controlled solvent delivery system. The reaction progress and completion can be monitored in real time using a standard UV detector. All steps of oligosaccharide assembly including loading, glycosylation, deprotection, and cleavage can be performed using this setup.

Characterization of protein immobilization on nanoporous gold using atomic force microscopy and scanning electron microscopy

Nanoporous gold (NPG), made by dealloying low carat gold alloys, is a relatively new nanomaterial finding application in catalysis, sensing, and as a support for biomolecules. NPG has attracted considerable interest due to its open bicontinuous structure, high surface-to-volume ratio, tunable porosity, chemical stability and biocompatibility. NPG also has the attractive feature of being able to be modified by self-assembled monolayers. Here we use scanning electron microscopy (SEM) and atomic force microscopy (AFM) to characterize a highly efficient approach for protein immobilization on NPG using N-hydroxysuccinimide (NHS) ester functionalized self-assembled monolayers on NPG with pore sizes in the range of tens of nanometres. Comparison of coupling under static versus flow conditions suggests that BSA (Bovine Serum Albumin) and IgG (Immunoglobulin G) can only be immobilized onto the interior surfaces of free standing NPG monoliths with good coverage under flow conditions. AFM is used to examine protein coverage on both the exterior and interior of protein modified NPG. Access to the interior surface of NPG for AFM imaging is achieved using a special procedure for cleaving NPG. AFM is also used to examine BSA immobilized on rough gold surfaces as a comparative study. In principle, the general approach described should be applicable to many enzymes, proteins and protein complexes since both pore sizes and functional groups present on the NPG surfaces are controllable.

Comparative Study of the Binding of Concanavalin A to Self-Assembled Monolayers Containing a Thiolated α-Mannoside on Flat Gold and on Nanoporous Gold

We have prepared SAMs containing 8-mercaptooctyl α-D-mannopyranoside either as a single component or in mixed SAMs with n-octanethiol on flat gold surfaces and on nanoporous gold. Electrochemical impedance spectroscopy showed that the mixed SAMs on flat gold surfaces showed the highest Con A binding, near 1:9 solution molar ratio of thiolated α-mannoside to n-octanethiol, whereas those on NPG showed the highest response at 1:19 solution molar ratio of thiolated α-mannoside to n-octanethiol. Atomic force microscopy was employed to image the monolayers and to image the bound Con A protein. Supplemental materials are available for this article. Go to the publishers online edition of Journal of Carbohydrate Chemistry to view the free supplemental file.

Selective capture of glycoproteins using lectin-modified nanoporous gold monolith.

The surface of nanoporous gold (np-Au) monoliths was modified via a flow method with the lectin Concanavalin A (Con A) to develop a substrate for separation and extraction of glycoproteins. Self-assembled monolayers (SAMs) of α-lipoic acid (LA) on the np-Au monoliths were prepared followed by activation of the terminal carboxyl groups to create amine reactive esters that were utilized in the immobilization of Con A. Thermogravimetric analysis (TGA) was used to determine the surface coverages of LA and Con A on np-Au monoliths which were found to be 1.31×10(18) and 1.85×10(15)moleculesm(-2), respectively. An in situ solution depletion method was developed that enabled surface coverage characterization without damaging the substrate and suggesting the possibility of regeneration. Using this method, the surface coverages of LA and Con A were found to be 0.989×10(18) and 1.32×10(15)moleculesm(-2), respectively. The selectivity of the Con A-modified np-Au monolith for the high mannose-containing glycoprotein ovalbumin (OVA) versus negative control non-glycosylated bovine serum albumin (BSA) was demonstrated by the difference in the ratio of the captured molecules to the immobilized Con A molecules, with OVA:Con A=2.3 and BSA:Con A=0.33. Extraction of OVA from a 1:3 mole ratio mixture with BSA was demonstrated by the greater amount of depletion of OVA concentration during the circulation with the developed substrate. A significant amount of captured OVA was eluted using α-methyl mannopyranoside as a competitive ligand. This work is motivated by the need to develop new materials for chromatographic separation and extraction substrates for use in preparative and analytical procedures in glycomics.

The influence of gold surface texture on microglia morphology and activation

Microglial cells play a critical role in the propagation of neuroinflammation in the central nervous system. Microglia sense and respond to environmental signals including chemical, physical and biological cues from the surrounding cell/tissue components. In this project, our goal was to examine the effects of surface texture on BV-2 microglia morphology and function by comparing flat and nanoporous gold (np-Au) surfaces to the more conventional glass. The biocompatibility of np-Au with microglia was evaluated using functional cell assays and high resolution imaging with scanning electron microscopy (SEM). Microglia seeded on glass, ultra-flat gold (UF-Au), ultra-thin (UT) np-Au and np-Au monolith were adherent to all surfaces and their viability was not compromised as assessed by multiple toxicity assays. SEM revealed detailed morphological characteristics of adherent microglia and indicated few dramatic changes as a result of the different surfaces. Microglia proliferation was hampered by np-Au monolith but less by UT np-Au and not at all on UF-Au or glass. Microglial activation, measured by tumor necrosis factor α (TNFα) production, was fully functional (and equivalent) on all gold surfaces compared to glass. The present findings should help further the understanding of basic microglia biology on textured surfaces and more fully evaluate np-Au as a multi-functional biocompatible material. The knowledge obtained and technology developed will have a significant impact in the fabrication of nanoelectronic devices, chemical sensor development, porous nanostructured materials for BioMEMs/NEMs integration, and functional biomaterial coatings for drug delivery.

Lectin–carbohydrate interactions on nanoporous gold monoliths

Monoliths of nanoporous gold (np-Au) were modified with self-assembled monolayers of octadecanethiol (C18-SH), 8-mercaptooctyl α-D-mannopyranoside (αMan-C8-SH), and 8-mercapto-3,6-dioxaoctanol (HO-PEG2-SH), and the loading was assessed using thermogravimetric analysis (TGA). Modification with mixed SAMs containing αMan-C8-SH (at a 0.20 mole fraction in the SAM forming solution) with either octanethiol or HO-PEG2-SH was also investigated. The np-Au monoliths modified with αMan-C8-SH bind the lectin Concanavalin A (Con A), and the additional mass due to bound protein was assessed using TGA analysis. A comparison of TGA traces measured before and after exposure of HO-PEG2-SH modified np-Au to Con A showed that the non-specific binding of Con A was minimal. In contrast, np-Au modified with octanethiol showed a significant mass loss due to non-specifically adsorbed Con A. A significant mass loss was also attributed to binding of Con A to bare np-Au monoliths. TGA revealed a mass loss due to the binding of Con A to np-Au monoliths modified with pure αMan-C8-SH. The use of mass losses determined by TGA to compare the binding of Con A to np-Au monoliths modified by mixed SAMs of αMan-C8-SH and either octanethiol or HO-PEG2-SH revealed that binding to mixed SAM modified surfaces is specific for the mixed SAMs with HO-PEG2-SH but shows a significant contribution from non-specific adsorption for the mixed SAMs with octanethiol. Minimal adsorption of immunoglobulin G (IgG) and peanut agglutinin (PNA) towards the mannoside modified np-Au monoliths was demonstrated. A greater mass loss was found for Con A bound onto the monolith than for either IgG or PNA, signifying that the mannose presenting SAMs in np-Au retain selectivity for Con A. TGA data also provide evidence that Con A bound to the αMan-C8-SH modified np-Au can be eluted by flowing a solution of methyl α-D-mannopyranoside through the structure. The presence of Con A proteins on the modified np-Au surface was also confirmed using atomic force microscopy (AFM). The results highlight the potential for application of carbohydrate modified np-Au monoliths to glycoscience and glycotechnology and demonstrate that they can be used for capture and release of carbohydrate binding proteins in significant quantities.

Surface-tethered iterative carbohydrate synthesis: a spacer study.

Comparative study of Surface-Tethered Iterative Carbohydrate Synthesis (STICS) using HPLC-assisted experimental setup clearly demonstrates benefits of using longer spacer-anchoring systems. The use of mixed self-assembled monolayers helps provide the required space for glycosylation reaction around the immobilized glycosyl acceptor. Both extension of the spacer length and using mixed self-assembled monolayers help promote the reaction, and the beneficial effects may include moving the glycosyl acceptor further out into solution and providing additional conformational flexibility. It is possible that surface-immobilized glycosyl acceptors with a longer spacer (C8-O-C8)-lipoic acid have a higher tendency to mimic a solution-phase reaction environment than acceptors with shorter spacers.

Electrochemical Characterization of Globotriose-Containing Self-Assembled Monolayers on Nanoporous Gold and their Binding of Soybean Agglutinin

Self-assembled monolayers (SAMs) of α-D-Gal-(1→4)-β-D-Gal-(1→4)-β-D-Glc-mercaptooctane (globotriose, Gb3-C8-SH) were prepared both as single-component SAMs and as mixed SAMs with either octanethiol (OCT) or 8-mercapto-3,6-dioxaoctanol (HO-PEG2-SH), on flat gold and on nanoporous gold (NPG) electrodes. The binding of soybean agglutinin (SBA) to the globotriose (Gb3) unit in the SAMs was then studied using electrochemical impedance spectroscopy (EIS), which is a label free method found to be quite sensitive to SAM composition and to the differences in SAM structure on NPG versus on flat Au. The affinity of SBA to the mixed SAM of HO-PEG2-SH and Gb3-C8-SH on NPG is found to be greater on NPG than on flat gold, and indicates a potential advantage for NPG as a substrate. The SAMs of HO-PEG2-SH were found to resist protein adsorption on either NPG or flat gold. The non-specific adsorption of SBA to OCT SAMs on flat Au was observed in EIS by the increase in charge transfer resistance; whereas, the increase seen on the NPG surface was smaller, and suggests that EIS measurements on NPG are less affected by non-specific protein adsorption. Atomic force microscopy (AFM) images of the SBA binding to mixed SAM of HO-PEG2-SH and Gb3-C8-SH on NPG showed a greater number of proteins on top of the OCT containing SAMs.

Reverse orthogonal strategy for oligosaccharide synthesis

Herein, we report the invention of a novel expeditious concept for oligosaccharide synthesis. Unlike the classic orthogonal strategy based on leaving groups, the reverse approach is based on orthogonal protecting groups, herein p-methoxybenzyl and 4-pentenoyl, which allows for efficient oligosaccharide assembly in the reverse direction.

Electrochemical impedance spectroscopy study of Concanavalin A binding to self-assembled monolayers of mannosides on gold wire electrodes

The interactions of the lectin Concanavalin A (Con A) with self-assembled monolayers (SAMs) of thiolated mono-, di-, and tri-mannosides were studied on the surface of gold wires using electrochemical impedance spectroscopy (EIS). The SAMs of mannosides were prepared either pure or along with thiolated triethylene glycol (TEG) at different molar ratios (1:1, 1:2, 1:4, 1:9, and 1:19) to better understand and optimize the interaction conditions. The charge-transfer resistance of the [Fe(CN)6]3-/4- redox probe was compared before and after the interaction at different concentrations of Con A to determine the equilibrium dissociation constant (Kd) and limit of detection (LOD). Values of Kd were found in the nanomolar range showing multivalent interactions between mannosides and Con A, and LOD was found ranging from 4-13 nM depending on the type of mannoside SAM used. Analysis using the Hill equation suggests negative cooperativity in the binding behavior. Peanut agglutinin was used as a negative control, and cyclic voltammetry was used to further support the experiments. We have found that neither the pure nor the widely dispersed monolayers of mannosides provide the conditions for optimal binding of Con A. The binding of Con A to these SAMs is sensitive to the molar ratio of the mannoside used to prepare the SAM and to the structure of the mannoside. A simple cleaning method has also been shown to regenerate the used gold wire electrodes. The results from these experiments contribute to the development of simple, cheap, selective, and sensitive EIS-based bioassays, especially for lectin-carbohydrate interactions.