ABSTRACT
The initial idea for constructing electrode-supported bilayers containing cytochrome c oxidase involved the use of Langmuir-Blodgett procedures. John K. Cullison started this work about ten years ago, but this approach failed to produce stable responses. Cullison sought alternative methods for achieving stable responses and devised an approach that combined self-assembly chemistry with deoxycholate dialysis used to prepare vesicles containing cytochrome c oxidase. Cullison used this cholate dialysis procedure to prepare stable bilayer membranes containing the oxidase on gold electrodes functionalized with submonolayers of octadecyl mercaptan [66]. The octadecyl mercaptan submonolayer, which is covalently bonded to the gold surface through thiol chemistry, anchors the bilayer to the electrode by becoming incorporated into the lipid membrane. Voltammetric data showed direct electron transfer between the oxidase and the gold electrode, and spectroelectrochemical data verified that the enzyme could both reduce and oxi-dize cytochrome c in solution [66]. However, only about one in eight electrodes showed this behavior, as reported in this paper. We now attribute this to a lack of control of the surface coverage of octadecyl mercaptan on gold. Although thiol monolayer structures
can be reliably prepared with substantial reaction times (i.e., 12 hours [67]), consistently forming submonolayer coverages of octadecyl mer-captan on gold quartz crystal microbalance (QCM) electrodes from dilute solution was not possible in this laboratory (see below). As discussed below, the thiol self-assembly reaction can be better controlled at gold QCM electrodes that have been coated with 1.6 monolayers of electrodeposited silver [68]. The electrodeposited silver masks chromium at the surface of the gold QCM electrodes. Chromium is used as an adhesion layer between the quartz and gold. Controlling the surface coverage of octadecyl mercaptan was found to be a critical step in the reliable implementation of John Cullison’s method. The dialysis step for constructing bilayer membranes containing cytochrome c oxidase was also refined. The original dialysis conditions were taken from the literature for reconstituting cytochrome c oxidase into vesicles in solution [64]. Bilayers were formed by placing gold electrodes modified with about 0.5 monolayer of thiol into dialysis tubing for an electrode preparation time of five to six days. In this work, a simple, small-volume, dual-chambered cell was constructed, and dialysis was complete in 12 hours. This cell design, which is shown in Figure 9 [69], is just another variation on the design developed in Ted Kuwana’s laboratory thirty years ago [8]. Additionally, the electrochemical behavior of the immobilized oxidase can be evaluated using this procedure without further handling of the modified electrode.
