Lectin-carbohydrate interactions on nanoporous gold monoliths

Abstract

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.

Fig. 1

Thermogravimetric analysis of adsorption and assembly of 8-mercaptooctyl α-D-mannopyranoside onto representative np-Au monoliths of dimensions 8.0 mm ×8.0 mm ×0.25 mm. (A) Under static immobilization conditions, and (B) under flow-through conditions (6 mM in ethanol, 1.0 mL min⁻¹) at 2 (red), 4 (pink), 6 (blue), 8 (green) and 24 hour (orange) time points. TGA analysis was carried out by ramping the temperature at 5 °C min⁻¹ from room temperature to 600 °C. (C) Coverage versus time for static and (D) flow-through conditions, showing the adsorption kinetics of 8-mercaptooctyl α-D-mannopyranoside, in terms of molecules cm⁻² vs. time, onto np-Au. See text for mass loss at each time point in mg.

Fig. 2

Tapping mode-AFM topographs of the αMan-C₈-SH functionalized np-Au surface after adsorption of Concanavalin A. Top panels (A, B, and C) represent the amplitude images; lower panels represent the topographic images of the same areas as in top panels. The scan size is 1000 nm ×1000 nm for mannose passivated np-Au without exposure to Con A. AFM topographic images of mannose functionalized gold surface after exposure to 1.0 mg mL⁻¹ Con A (B and E, 1000 nm ×1000 nm scan size). Higher resolution AFM topographic images, 500 nm ×500 nm scan size, of the areas indicated by the boxes in C and F. Line profiles for the segments identified are shown in the panels underneath panels D, E, and F.

Fig. 3

Thermogravimetric analysis of Con A immobilized on np-Au monoliths modified with SAMs of 8-mercapto-3,6-dioxaoctanol (A), octanethiol (B), or 8-mercaptooctyl α-D-mannopyranoside (C). (A) A very small amount of Con A (red curve) was adsorbed onto the HO-PEG₂-SH (black curve) modified np-Au monoliths. (B) Non-specific adsorption of Con A (green curve) was observed on the hydrophobic C₈-SH SAM surface (blue curve), (C) a higher amount of Con A (pink curve is for 0.75 mg mL⁻¹, blue curve is for 1.00 mg mL⁻¹ of Con A) adsorbed onto the α-mannoside modified np-Au monolith (green curve) as expected due to the specific interactions between the lectin and carbohydrate. Experiments were carried out under flow-through conditions, the Con A solution (1.0 mg mL⁻¹) was allowed to pass through the np-Au for 3.5 hours at rate of 1.0 mL min⁻¹.

Fig. 4

Thermogravimetric analysis of Con A bound onto np-Au monolith modified with mixed SAMs of 8-mercapto-3,6-dioxaoctanol, octanethiol, and 8-mercaptooctyl α-D-mannopyranoside. (A) Specific interaction of Con A (green curve) with αMan-C₈-SH was observed for np-Au monoliths modified with mixed SAMs of αMan-C₈-SH and HO-PEG₂-SH (red curve). (B) A significant amount of Con A (black and blue curves) was non-specifically adsorbed onto np-Au modified with mixed SAMs of αMan-C₈-SH and octanethiol (orange curve).

Fig. 5

Specific protein adsorption onto mixed SAMs of HO-PEG₂-SH + αMan-C₈-SH. (A) The amount of Con A bound onto mixed SAMs of HO-PEG₂-SH + αMan-C₈-SH SAMs of solution mole fraction of αMan-C₈-SH of 0.05, 0.10, 0.20, 0.33, 0.50, 0.66 and 0.75, respectively, was investigated. The highest protein adsorption was observed for the 0.10 solution mole fraction. (B) Time dependent study of Con A adsorption onto mixed SAM (HO-PEG₂-SH + αMan-C₈-SH, 0.10 solution mole fraction of αMan-C₈-SH, 2.5 mM, ethanol) on np-Au. A gradual increase of Con A loading and saturation at longer incubation time was observed. The total Con A adsorbed on the surface on modified np-Au is 0.59 mg at 15 hours, and 0.66 mg at 24 hours. (C) Specific and non-specific interaction of IgG, PNA and Con A towards the HO-PEG₂-SH + αMan-C₈-SH mixed SAMs of 0.10 solution mole fraction αMan-C₈-SH. Blue bars represent the mass of the mixed SAMs before exposure to proteins. Red bars represent the additional mass of adsorbed protein.

Fig. 6

Elution of bound Con A from np-Au monoliths. (A) Con A immobilized via specific interactions with the mannoside moieties in the mixed SAMs of αMan-C₈-SH and HO-PEG₂-SH, was washed with methyl α-D-mannopyranoside. According to the TGA mass loss, approximately 0.1216 mg (at 400 °C) of bound Con A was eluted out from the np-Au monolith (red line). (B) A similar strategy was used to elute Con A bound to the mixed SAMs of αMan-C₈-SH and C₈-SH. Based on TGA analysis, 0.4414 mg (at 400 °C) of Con A were eluted off the np-Au monoliths. (C) and (D) Show bar charts, reflecting the amount of mixed SAM formed on np-Au monoliths, amount of Con A loss after TGA analysis, and amount of Con A eluted off the np-Au, at the indicated temperature.