Electrochemical characterization of globotriose-containing self-assembled monolayers on nanoporous gold and their binding of soybean agglutinin

Abstract

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.

Figure 1

Cyclic voltammogram of the bare and modified electrodes in 10 mM K₃[Fe(CN)₆]. CV scans were performed in 10 mM K₃[Fe(CN)₆] at a scan rate of 100 mV sec⁻¹ in a three electrode cell arrangement, using gold wires as working electrode, Ag|AgCl as a reference electrode and platinum wire as a counter electrode. Figure 1a shows CVs on a bare gold wire electrode (GW, solid line), and also for a gold wire electrode modified with an SAM of Gb3-C₈-SH (dotted line). Figure 1b shows CV data on gold wire electrodes modified with mixed SAMs of 1:5 Gb3-C₈-SH:HO-PEG₂-SH (long dash line), 1:5 Gb3-C₈-SH:OCT (dash dot dash dot line), and the SAM of Gb3-C₈-SH (dotted line). The ratio referred to is the solution molar ratio from which the SAM was prepared. Figure 1c shows CV data for bare NPG (solid line), SAM of Gb3-C₈-SH (dotted line), and 1:5 mixed SAMs of Gb3-C₈-SH:HO-PEG₂-SH (long dash line), and SAM of 1:5 Gb3-C₈-SH:OCT (dash dot dash dot line). Figure 1d is an expanded view of the CV for the mixed SAM of 1:5 Gb3-C₈-SH:OCT on NPG.

Figure 2

Nyquist plots for the modified gold wire and NPG electrodes before and after exposure to soybean agglutinin (SBA). Figure 2a shows data for SAM of Gb3-C₈-SH (solid line), Gb3-C₈-SH after incubation with 2 μM SBA (dotted line), 1:5 mixed SAM of Gb3-C₈-SH:OCT before (long dash line), and after (short dashed line) incubation with 2 μM SBA on gold wire. Figure 2b shows data for the 1:5 mixed SAM of Gb3-C₈-SH:OCT SAM on NPG before (long dashed line), and after (dotted line) incubation with 2 μM SBA. The analogous Nyquist plots for the Gb3-C₈-SH SAMs on NPG before (solid line) and after (dotted line) incubation with 2 μM SBA are shown in the inset of Figure 2b. Figure 2c shows data for the 1:5 mixed SAM of Gb3-C₈-SH: HO-PEG₂-SH SAM on gold wire before (solid line) and after (dotted line) incubation with 2 μM SBA. Figure 2d shows data for the 1:5 mixed SAM of Gb3-C₈-SH: HO-PEG₂-SH SAM on NPG before (solid line) and after (dotted line) incubation with 2 μM SBA.

Figure 3

Binding kinetics for soybean agglutinin (SBA) to mixed SAMs containing Gb3-C₈-SH as measured by the increase in charge transfer resistance. The binding kinetics study was performed by incubating modified electrodes with 0.5 μM SBA for the given time interval, rinsing and then conducting EIS measurements. Figure 3a shows data for binding of SBA to Gb3-C₈-SH in mixed SAMs of 1:5 Gb3-C₈-SH: HO-PEG₂-SH on gold wire electrodes (circles) and on NPG electrodes (triangles). Similar data for mixed SAMs of 1:5 Gb3-C8-SH:OCT are shown on gold wire (circles) and on NPG (triangles) in Figure 3b.

Figure 4

Binding isotherms for the interaction of soybean agglutinin (SBA) with Gb3-C₈-SH in mixed SAMs as measured using the increase in charge transfer resistance. Figure 4a shows data for mixed SAMs of 1:5 Gb3-C₈-SH: HO-PEG₂-SH on gold wire electrodes (circles) and on NPG electrodes (triangles). Figure 4b shows data for mixed SAMs of 1:5 Gb3-C8-SH:OCT on gold wire (circles) and on NPG (triangles).

Figure 5

Soybean agglutinin (SBA) binding to the SAM surfaces. 5(a) and 5(c) show AFM topographic images of NPG modified with of mixed SAMs of 1:5 Gb3-C₈-SH:HO-PEG₂-SH and 1:5 Gb3-C₈-SH:OCT, respectively. A higher SBA coverage was observed on the mixed component SAMs surface containing OCT (5d), as compared to the SAM containing HO-PEG₂-SH (5b). The AFM topographic images are were taken over a 500 nm × 500 nm area.

Scheme 1

Retrosynthetic analysis for the synthesis of α-D-Gal-(1→4)-β-D-Gal-(1→4)-D-Glc-mercaptooctane (Gb3-C₈-SH) (compound 1).