Characterization of Micropatterned Lipid Membranes on a Gold Surface by Surface Plasmon Resonance Imaging and Electrochemical Signaling of a Pore-Forming Protein

Abstract

We report the fabrication and characterization of a micropatterned membrane electrode for electrochemical signaling of a bacterial pore-forming toxin, Streptolysin O (SLO) from S. pyogenes. Microcontact printing of an alkylthiol monolayer was used to fabricate an array template, onto which cholesterol-containing DMPC vesicles were fused to form lipid layer structures. The construction of the supported membranes, including pattern transfer and vesicle fusion, was characterized by in-situ surface plasmon resonance (SPR) imaging and electrochemistry. Quantitative analysis of the resulting membrane by using SPR angular shift measurements indicates that the membranes in the hydrophilic pockets have an average thickness of 8.2 +/- 0.4 nm. Together with fluorescence microscopy studies, the results suggest that this could be a mixed lipid assembly that may consist of a bilayer, vesicle fragments, and lipid junctions. The voltammetric response of the redox probe ferrocene carboxylic acid (FCA) was measured to quantify the toxin action on the supported membrane. The electrochemical measurements indicate that fusion of vesicles on the template blocked the access of FCA, whereas the injection of SLO toxin restored the redox response. The anodic peak current of FCA was found to increase with toxin concentration until a plateau was reached at 40 HU/mL. The method is highly sensitive such that 0.1 HU/mL of SLO (1.25 pM) can yield a well-defined response. In addition, it eliminates the need for a highly insulating layer in membrane sensing, which opens up new avenues in developing novel sensing interfaces for membrane-targeting proteins and peptides.

Electrochemical Surface Plasmon Resonance Spectroscopy at Bilayered Silver/Gold Films

“…31,32 Imaging SPR had also been used in tandem with voltammetry to characterize micropatterned lipid membranes. 33 Tao and co-workers combined cyclic voltammetry with a multiwavelength SPR to determine conformational and electronic changes of cytochrome c. 34 Our group quantified the orientation changes of ferrocenyl alkanethiol self-assembled monolayers (SAMs) upon electrochemical oxidation of the ferrocene termini. 35 To alleviate the effect of an applied potential on the SPR response, we electrochemically induced local structural changes with redox species generated at a scanning electrochemical microscopic tip electrode and simultaneously determined such changes using a highly sensitive SPR.…”

In situ imaging of micropatterned phospholipid membranes by surface plasmon fluorescence microscopy

“…SPR-imaging (SPM) has been used to monitor interactions of bio-related molecules [13][14][15][16][17]. However, SPM can detect only the difference in thickness of layers (films) at the interface.…”

Application of thin metal film elements in bioanalysis

“…Gold electrodes are preferable for good chemical stability and high affinity to thiol compounds allowing simple surface modification. Surface formation of self-assembled monolayer with a suitable end group (amine, carboxyl) allows consecutive immobilization of selected reagents such as aptamers and dendrimers [12][13][14][15][16], proteins [17], nucleic acids [18] and other species [19]. Further, electrochemical modifications allow selective labeling of closely packed electrodes with sensing biomolecules [20][21][22][23].…”