TRmax 4aFnrGOxA6

A different method to electrically wire a redox enzyme by a molecular electroactive relay unit involved the use of molecular rotaxane architectures [49]. Rotaxanes are supramolecular architectures that include an interlocked molecular ring on a molecular wire that is stoppered at its two ends by bulky molecular components [50, 51]. The rotaxanes are usually prepared by the formation of an affinity complex between the ring and a molecular site associated with the wire followed by a chemical reaction

Fig. 3.8 (legend see page 49)

at the wire ends that stopper the ring on the wire, and this leads to a noncovalent stable supramolecular structure. Ingenious rotaxane structures were developed in solutions [52-54] and on surfaces [55], and the signal-controlled translocations of the ring-component to distinct specific positions on the molecular wire were demonstrated using photonic [56], electrochemical [57] or chemical [58] stimuli. An electrically contacted enzyme-stoppered rotaxane that includes a threaded electron relay in the rotaxane structure was synthesized [49], Figure 3.8A. A monolayer consisting of chains that include the fcis-iminophenylene n-donor unit was synthesized on the Au-surface by a sequence of condensation reactions that included 1,4-phthaldialdehyde and 1,4-diaminobenzene. The monolayer-functionalized electrode was then interacted with fcis-(paraquat)phenylene (16) to form the supramolecular n -donor-acceptor complex, and the resulting supramolecular assembly was in situ stoppered with the amino-FAD cofactor unit, (14). The surface reconstitution of apo-GOx on the FAD sites yielded the surface-reconstituted, aligned, electrically contacted enzyme-electrode. Microgravimetric quartz - crystal-microbalance measurements indicated a surface coverage of the enzyme that corresponds to 2 x 10-12 mol cm 2 on the electrode surface. Figure 3.8B shows the cyclic voltammograms corresponding to the bioelectrocatalyzed oxidation of different concentrations of glucose. The bioelectrocatalyzed oxidation of glucose is observed at -0.4 V versus SCE, the lowest potential ever observed for glucose oxidation. The potential at which glucose oxidation proceeds in this system is only ca 100 mV more positive than the redox potential of FAD associated with the enzyme. Figure 3.8B inset, shows the derived calibration curve that shows the amperometric responses of the nano-engineered enzyme-electrode at variable concentrations of glucose. The turnover rate of electron transfer in the system was estimated to be ca 450 s-1. The very low potential for the oxidation of glucose in the system has enormous significance for the development of biofuel cells of high power output (see Section 3.4). The effective electrical contacting of the redox enzyme in the rotaxane structure is attributed to the dynamic freedom of the electron relay in the system. The bioelectrocatalyzed oxidation of glucose results in the ET from the FAD cofactor to the threaded bipyridinium relay in the rotaxane configuration, Figure 3.8A. The reduction of the relay perturbs the donor-acceptor complex, and the reduced relay freely moves on the molecular wire leading to its oxidation at the electrode surface, and the reorganization of the oxidized relay at the n -donor site. This system reveals a rigid electrically contacted enzyme configuration with the advantages of a dynamically free mediated ET transfer by the electroactive rotaxane shuttle.

A different approach to electrically contact reconstituted flavoenzymes on electrodes involved the use of the conductive redox-polymer polyaniline as a redox mediator for the electrical contacting of the enzyme with the electrode [59]. Aniline was

Fig. 3.8 (A) Assembly of a rotaxane-based reconstituted glucose oxidase electrode. (B) Cyclic voltammograms corresponding to the bioelectrocatalyzed oxidation of different concentrations of glucose by the GOx-reconstituted electrode in the rotaxane structure: (a) 0 mM, (b) 5 mM, (c) 10mM, (d) 20 mM, (e) 30 mM, (f) 50 mM, (g) 80 mM. inset: Calibration curve derived from the cyclic voltammograms at -0.1 V (vs SCE).

Fig. 3.9 Assembly of an electrically contacted polyaniline-reconstituted glucose oxidase electrode.

electropolymerized on Au-electrodes in the presence of polyacrylic acid. Polymerization yielded a polyaniline film with entangled polyacrylic acid chains. In contrast to a bare polyaniline film that reveals redox activities only in acidic aqueous solutions, the polyaniline/polyacrylic acid composite film was reported [60] to be electrochemically active in neutral aqueous solutions, and thus the polymer film could be coupled to bioelectrocatalyzed transformations. The enzyme-electrode was constructed as depicted in Figure 3.9. The amino-FAD semi-synthetic cofactor (14) was covalently linked to the polyacrylic acid chains entangled in the polymer film, and apo-glucose oxidase was reconstituted onto the FAD units to generate the integrated enzyme-electrode. In the specific construction assembled on the electrode, the polyaniline/polyacrylic acid film exhibited a thickness of ca 90 nm and the surface coverage of the FAD units and of the reconstituted GOx components was estimated

Fig. 3.10 Cyclic voltammograms corresponding to the bioelectrocatalyzed oxidation of variable concentrations of glucose by the integrated, electrically contacted polyaniline-reconstituted glucose oxidase electrode. Glucose concentrations correspond to (a) 0 mM, (b) 5 mM, (c) 10 mM, (d) 20 mM, (e) 35 mM, (f) 50 Mm.

Fig. 3.10 Cyclic voltammograms corresponding to the bioelectrocatalyzed oxidation of variable concentrations of glucose by the integrated, electrically contacted polyaniline-reconstituted glucose oxidase electrode. Glucose concentrations correspond to (a) 0 mM, (b) 5 mM, (c) 10 mM, (d) 20 mM, (e) 35 mM, (f) 50 Mm.

to be 2 x 10-11 mol cm 2 and 3 x 10-12 mol cm 2 respectively. The resulting enzyme-electrode revealed bioelectrocatalytic activities toward the oxidation of glucose, Figure 3.10. Knowing the enzyme content in the film and using the saturated current density observed in the system, i = 0.3 mA cm-2, the maximum ET turnover rate for the biocatalyst was estimated to be TRmax ^ 1000 s-1. In a control experiment, the lysine residues of native GOx, with its naturally implanted FAD cofactor, were directly coupled to the polyacrylic acid chains of the polyaniline/polyacrylic acid film. The resulting electrode revealed very poor bioelectrocatalytic activities, and the maximum current output was ca 100-fold lower. These results highlight the importance of alignment of the biocatalyst in respect to the redox-active polymer film. The electrostatic attraction of the polyacrylic acid chains to the oxidized polyaniline simultaneously attracts the aligned tethered enzyme components, thus enabling the mediated ET from the active site through the oxidized polyaniline to the electrode, Figure 3.9. This mediated ET electrically contacts the biocatalyst toward the biocatalyzed oxidation ofglucose.

The system was further characterized by in situ electrochemical/SPR measurements [59], and SPR spectroscopy was introduced as a means to follow bioelectrocatalytic transformations. Surface plasmon resonance spectroscopy is a useful method for the

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