Ferrocenylhexyl- and ferrocenylpropyl-modified linear poly(ethylenimine) (Fc-C6-LPEI, Fc-C3-LPEI) were used with periodate-modified glucose oxidase (p-GOX) in the layer-by-layer assembly of enzymatic bioanodes on gold. Fc-C6-LPEI/p-GOX and Fc-C3-LPEI/p-GOX films of 16 bilayers were capable of generating up to 381 ± 3 and 1417 ± 63 μA cm(-2), respectively, in response to glucose. These responses are greater than those of analogous bioanodes fabricated using conventional cross-linking techniques and are extremely high for planar, low surface area, single-enzyme electrodes. (Fc-C3-LPEI/p-GOX)8 films generated 86 ± 3 μW cm(-2) at pH 7.0 and 149 ± 7 μW cm(-2) at pH 5.0, when poised against an air-breathing platinum cathode in a compartment-less biofuel cell. An increase in power output with decreasing pH was shown to be a result of increases in the platinum cathode performance, indicating it is the rate-limiting electrode in the biofuel cells. The effect of fabrication wash time on the buildup of material at the electrode's surface was probed using cyclic voltammetry (CV) and constant potential amperometry. The use of electrochemical techniques as a diagnostic tool for studying the material deposition process is discussed. CV peak separation (ΔE), surface coverage of the electroactive ferrocene (ΓFc), and amperometric sensitivity of the enzyme to glucose (Jmax), studied as a function of numbers of bilayers, showed that physisorption of materials onto the surface results from initial patchy deposition, rather than in distinctly uniform layers.

Herein, both electrostatic and covalent layer-by-layer assembly were used for the construction of multicomposite thin films using a ferrocene-modified linear poly(ethylenimine) redox polymer (Fc-C6-LPEI) as the cationic polyelectrolye, and poly(acrylic acid) (PAA), poly(glutamic acid) (PGA), or glucose oxidase (GOX) as the negative polyelectrolyte. The assembly of the multilayer films was characterized by cyclic voltammetry (CV), UV/Vis spectroscopy, and ellipsometry with the enzymatic response of the films containing GOX being characterized via constant potential amperometry. CV measurements suggested that the successful buildup of multilayer films was dependent upon the nature of the anionic polyelectrolyte used. Electrostatic assembly of films composed of Fc-C6-LPEI and either PAA or PGA produced large oxidation peak current densities of 630 and 670 μA cm(-2), respectively, during cyclic voltammetry. Increased measured absorbance by UV/Vis spectroscopy and increased measured film thicknesses (400-600 nm) by ellipsometry provided additional evidence of successful film formation. In contrast, the films incorporating GOX that were electrostatically assembled surprisingly produced significantly lower electrochemical responses (12 μA cm(-2)), low absorbance values, and reduced film thicknesses (~15 nm), and glucose electro-oxidation current densities less than 1 μA cm(-2), which all suggested unstable or minimal film formation. Subsequently, we developed a covalent layer-by-layer approach to fabricate films of Fc-C6-LPEI/GOX by covalently linking the amine groups of Fc-C6-LPEI to the aldehyde groups of periodate-oxidized glucose oxidase. Covalent assembly of the Fc-C6-LPEI/GOX films produced oxidation peak current densities during cyclic voltammetry of 40 μA cm(-2) and glucose electro-oxidation current densities of 220 μA cm(-2). These films also showed an increase in their thicknesses (~140 nm) relative to the electrostatic GOX films. For the films containing either PAA or PGA, the pH of the polymer solutions used for construction was found to have a significant effect on the response of the multilayer films, and the electrochemical response of the Fc-C6-LPEI/PAA, Fc-C6-LPEI/PGA, or covalently assembled Fc-C6-LPEI/GOX films could be tuned by varying the number of bilayers (n=1-16) in the film. These results are important because this is the first report of the use of the novel Fc-C6-LPEI redox polymer in the successful development of multicomposite layer-by-layer films. The electrochemical response achieved with the covalently assembled Fc-C6-LPEI/GOX films demonstrates that this redox polymer and layer-by-layer assembly technique can be used for possible biosensor and biofuel applications, and the success of multiple anionic polyelectrolytes could lead to additional applications with other enzyme systems.