Selective capture of glycoproteins using lectin-modified nanoporous gold monolith

Abstract

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 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 × 1018 molecules m−2 and 1.85 × 1015 molecules m−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 ×1018 molecules m−2 and 1.32 × 1015 molecules m−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.

Use of monolithic supports for high‐throughput protein and peptide separation in proteomics

“…Taking into account this fact, the benefits with monoliths are more pronounced in enrichment of glycoproteins because they are usually high molecular weight proteins. Individual lectins or multiple lectins, were bound on different organic‐based monoliths since they offer a variety of chemistries suitable for immobilization . The limited sample capacity was a consequence of the relatively low surface area of organic‐based monoliths .…”

Electrochemical impedance spectroscopy study of carbohydrate-terminated alkanethiol monolayers on nanoporous gold: Implications for pore wetting

“…The use of SAMs with specific terminal functional groups is important, such as when these groups are activated and coupled to biomolecules for use in development of biosensors [18–20]. In a previous study using thermogravimetric analysis (TGA), we found that the coverage of an alkanethiol SAM within an np-Au monolith was close to that expected on the basis of packing geometry on flat Au [21, 22]. One recent study concerning electrochemical impedance spectroscopy (EIS) studies of alkanethiol SAMs on np-Au from octanethiol to tetradecanethiol (C8, C10, C12, and C14 chain lengths) reported that the charge transfer resistance varied gradually with chain length on np-Au but showed a steadier increase with chain length on flat gold [23].…”

“…Accessibility of electrolyte solutions to the interior of np-Au can be viewed as significant for the efficiency of immobilization of biomolecules such as antibodies [31–33] or oligonucleotides [25, 34], sensor response [1], loading for controlled release [10, 35], and substrate access and overall activity for immobilized enzymes [36]. Carbohydrate modified np-Au has been recently explored for applications in capture and elution of lectins [21], glycoprotein modified np-Au for competitive electrochemical immunoassay, and lectin-modified np-Au for capture and subsequent elution of glycoproteins [22]. …”

Sequence-Specific Electrical Purification of Nucleic Acids with Nanoporous Gold Electrodes

“…Owing to its catalytic properties, tunable morphology, microfabrication compatibility and excellent thiol-gold chemistry, np-Au is an emerging material for biosensing applications [20][21][22] . We recently demonstrated that np-Au electrochemical sensors exhibit excellent biofouling-resilience in the presence of complex media such as serum while preserving the sensitive DNA detection capabilities 23,24 .…”