Applications of nanoporous gold monoliths as substrates for the capture and release of lectins and glycoproteins.

摘要

Nanoporous gold (np-Au) monoliths are a free-standing nanostructured material with typical pore dimensions in the tens of nanometers range. The microstructure of np-Au resembles those of macroporous monolithic materials being used in chromatographic separations. The surfaces of np-Au monoliths were modified via flow methods with different ligands to develop affinity substrates for separations. A carbohydrate-modified np-Au monolith was prepared by immobilizing thiolated saccharides and further used to separate lectins. The np-Au monolith surface was also functionalized with self-assembled monolayers (SAMs) of alpha-lipoic acid (LA) followed by activation of carboxyl terminal groups to create amine reactive esters. Concanavalin A (Con A) was then covalently immobilized to develop a substrate for extraction of glycoprotein from a mixture. Likewise, aminophenylboronic acid was immobilized to develop a substrate that was tested for pH-dependent capture and release of cis-diol containing molecules. Preservation of SAMs and immobilized ligands were possibly due to the in situ surface modification of np-Au monoliths that limited the possible damage and degradation of molecules on the surface.;Selectivity of the developed substrates was enhanced by capping the unreacted functional groups or by incorporation of protein resistant spacers to limit the non-specific adsorption of unwanted molecules. The loading and surface coverage of molecules on np-Au monolith surface were determined by thermogravimetric analysis (TGA) and by an in situ solution depletion method. TGA was able to quantify the amount of loading based from the mass loss after the pyrolysis of modified np-Au monoliths. The in situ solution depletion method estimates the amount of loading by the difference in the initial and final concentration of a circulating solution monitored by a UV detector.;This research aims to introduce np-Au monolith as an addition to the materials being used as substrates in chromatographic separation and extraction. The chemical stability, simple but reproducible preparation, high surface-to-volume ratio and availability of wide variety of Au surface functionalization are the features of np-Au monolith that could complement the limitations of the existing materials used in separations. The focus of this research is on the separation of lectins and glycoproteins, which is an important step towards an effective glycan analysis in glycomics.