The development of new methods to facilitate direct electron transfer (DET) between enzymes and electrodes is of much interest because of the desire for stable biofuel cells that produce significant amounts of power. In this study, hydroxylated multiwalled carbon nanotubes (MWCNTs) were covalently modified with anthracene groups to help orient the active sites of laccase to allow for DET. The onset of the catalytic oxygen reduction current for these biocathodes occurred near the potential of the T1 active site of laccase, and optimized biocathodes produced background-subtracted current densities up to 140 μA/cm 2 . Potentiostatic and galvanostatic stability measurements of the biocathodes revealed losses of 25% and 30%, respectively, after 24 h of constant operation. Finally, the novel biocathodes were utilized in biofuel cells employing two different anodic enzymes. A compartmentalized cell using a mediated glucose oxidase anode produced an open circuit voltage of 0.819 ( 0.022 V, a maximum power density of 56.8 ((1.8) μW/cm 2 , and a maximum current density of 205.7 ((7.8) μA/cm 2 . A compartment-less cell using a DET fructose dehydrogenase anode produced an open circuit voltage of 0.707 ( 0.005 V, a maximum power density of 34.4 ((2.7) μW/cm 2 , and a maximum current density of 201.7 ((14.4) μA/cm 2 .

Enzymatic biofuel cells represent an emerging technology that can create electrical energy from biologically renewable catalysts and fuels. A wide variety of redox enzymes have been employed to create unique biofuel cells that can be used in applications such as implantable power sources, energy sources for small electronic devices, self-powered sensors, and bioelectrocatalytic logic gates. This review addresses the fundamental concepts necessary to understand the operating principles of biofuel cells, as well as recent advances in mediated electron transfer- and direct electron transfer-based biofuel cells, which have been developed to create bioelectrical devices that can produce significant power and remain stable for long periods.

Linear poly͑ethylenimine͒ ͑-͓CH 2 CH 2 NH͔ n -, LPEI͒ was modified by attachment of 3-͑dimethylferrocenyl͒propyl groups to ca. 17% of its nitrogen atoms ͑FcMe 2 -C 3 -LPEI͒ to form a new redox polymer for use as an anodic mediator in glucose/O 2 biofuel cells. The electrochemical properties of this polymer were compared to those of 3-ferrocenylpropyl-modified LPEI ͑Fc-C 3 -LPEI͒. When Fc-C 3 -LPEI or FcMe 2 -C 3 -LPEI was mixed with glucose oxidase and cross-linked with ethylene glycol diglycidyl ether to form hydrogels on planar, glassy carbon electrodes, limiting catalytic bioanodic current densities of up to ϳ2 mA/cm 2 at 37°C were produced. The use of dimethylferrocene moieties in place of ferrocene moieties lowered the E 1/2 of the films by 0.09 V and significantly increased electrochemical and operational stabilities. FcMe 2 -C 3 -LPEI was shown to be the more effective polymer for use in biofuel cells and, when coupled with a stationary O 2 cathode comprised of laccase and cross-linked poly͓͑vinylpyridine͒Os͑bipyridyl͒ 2 Cl 2+/3+ ͔ as a mediator, produced power densities of up to 56 W/cm 2 at 37°C. Power density increased to 146 W/cm 2 when a rotating biocathode was used. The stability of the biofuel cells constructed with FcMe 2 -C 3 -LPEI was higher than that of the cells using Fc-C 3 -LPEI.The development of fuel cells that can operate using biological catalysts and renewable fuels has gained recent attention. 1-4 Biofuel cells resemble traditional fuel cells in their fundamental operating principles ͑oxidation of a fuel to produce protons/reduction of oxygen to water͒ but differ greatly in other ways. Biofuel cells use renewable catalysts ͑microbes or enzymes͒ and are operated under mild conditions ͑usually 25 or 37°C, pH 5-7͒ relative to traditional fuel cells. The enzymes used in biofuel cells are extremely selective for their respective substrates, allowing for the removal of separator membranes and the operation of many biofuel cells in compartmentless containers. These properties make biofuel cells attractive as alternative energy sources for implantable electronic devices and other portable electronics.However, because biofuel cells use enzymes as catalysts, the stabilities of bioanodes and biocathodes can be fairly low and the highest power densities produced using single enzyme electrodes in compartmentless biofuel cells to date are only in the hundreds of microwatts per square centimeter. 5,6 In order to improve these power densities, some groups are working on complex enzyme cascades 7-9 to allow for complete oxidation of biofuels to CO 2 , and others are working with hybrid enzymatic/direct methanol fuel cells in order to increase the low power densities typically obtained from biofuel cells. 10,11 Still others are using innovative nanomaterials to enhance the connection between enzymes and electrode surfaces. [12][13][14][15][16] Because these systems are complex, are expensive, and/or use precious metal catalysts, there is a need for simple, low-cost, single enzyme bioelectrodes, especiall...

The effect of mediator distance from the polymer backbone on the redox behavior, electron transport, electrochemical stability, and electrical communication with redox enzymes was studied with novel linear poly(ethylenimine) redox polymers. To measure these effects, we synthesized two new ferrocene redox polymers having a three-carbon (Fc-C 3 -LPEI) and a six-carbon (Fc-C 6 -LPEI) spacer to complement our previous synthesized polymer (Fc-C 1 -LPEI) having a 1-carbon spacing. Increasing the spacer length to either three or six carbons resulted in a single peak redox behavior over the entire pH tested (3-11), which is in contrast with the multiwave redox behavior observed with a 1-carbon spacing under neutral or basic pH solutions. In addition increasing the spacer also resulted in a six-fold increase in the electrochemical stability of crosslinked redox polymer films. In contrast with previous reports of electron transport increasing with spacer length, we observed no correlation between mediator spacing and electron transport or electrical communication with the enzyme glucose oxidase. Surprisingly, the redox polymer (Fc-C 3 -LPEI) with the lowest electron transport produced the highest enzymatic response (>1 mA/cm 2 ), suggesting that other factors (e.g., polymer-enzyme complexation) were important. These results demonstrate how small changes in the redox polymer structure can affect its properties.

Amperometric biosensors for glucose and hydrogen peroxide have been built by immobilizing glucose oxidase (GOX) and horseradish peroxidase (HRP) in cross-linked films of ferrocene-modified linear poly(ethylenimine). At pH 7, the glucose sensors generated limiting catalytic current densities of 1.2 mA/cm2. These current densities are approximately 4 times higher than those with other ferrocene-based redox polymers and are comparable to the highest reported values for osmium-based redox polymers with GOX. Because of the high sensitivity of these films (73 nA/cm2.microM), glucose concentrations in the micromolar range could be detected. Similarly, sensors were constructed with HRP-generated current densities of 0.9 mA/cm2 under saturation conditions and sensitivities of 500 nA/cm2.microM. The results show that the ability of Fc-LPEI to effectively communicate with a variety of enzymes has potential applications in measuring low substrate concentrations in implantable biosensors and producing high current outputs in enzymatic biofuel cells.

The performance of immobilized enzyme systems is often limited by cofactor diffusion and regeneration. Here, we demonstrate an engineered enzyme capable of utilizing the minimal cofactor nicotinamide mononucleotide (NMN(+)) to address these limitations. Significant gains in performance are observed with NMN(+) in immobilized systems, despite a decreased turnover rate with the minimal cofactor.

Self-powered sensors are able to automatically signal the presence of a specific analyte without the aid of an external power source, making them useful as potential devices for batteryless sensing. Here, we present a self-powered enzymatic ethylenediaminetetraacetic acid (EDTA) sensor based on the inhibition and subsequent activation of glucose oxidase (GOx)-based bioelectrodes within the framework of a biofuel cell. Although EDTA is not redox-active, it is detected by the activation of a Cu(2+)-inhibited GOx bioanode in either a typical amperometric sensor (using a standard three-electrode setup) or in a self-powered sensor where the GOx bioanode is coupled to a platinum cathode. The sensors are able to detect concentrations of EDTA that correspond to the amount of Cu(2+) that is used to inhibit the enzymatic electrode. The self-powered sensor shows a greater than 10-fold increase in power output when it is activated by the presence of EDTA. This represents the first time that a non-redox-active analyte has been detected in a self-powered sensor that turns on in the presence of said analyte.

Laccase, a blue multicopper oxidoreductase enzyme, is a robust enzyme that catalyzes the reduction of oxygen to water and has been shown previously to perform improved direct electron transfer in a biocathode when mixed with anthracene-modified multi-walled carbon nanotubes. Previous cathode construction used crude laccase enzyme isolated as a brown cell extract powder containing both active and inactive proteins. Purification of this enzyme, yielding a blue solution, resulted in greatly improved enzyme activity and removed insulating protein that competed for docking space in this cathodic system. Cyclic voltammetry of the purified biocathodes showed a background subtracted limiting current density of 1.84 (±0.05) mA/cm 2 in a stationary air-saturated system. Galvanostatic and potentiostatic stability experiments show that the biocathode maintains up to 75% and 80% of the original voltage and current respectively over 24 hours of constant operation. Inclusion of the biocathode in a glucose/O 2 biofuel cell using a mediated glucose oxidase (GOx) anode produced maximum current and power densities of 1.28 (±0.18) mA/cm 2 and 281 (±50) μW/cm 2 at 25 • C and 1.80 (±0.06) mA/cm 2 and 381 (±33) μW/cm 2 at 37 • C, respectively. Enzymatic efficiency of this glucose/O 2 enzymatic fuel cell is among the highest reported for a glucose/O 2 enzymatic fuel cell.Laccase is an oxidoreductase enzyme from a class of multicopper oxidases (MCO) that catalyzes the four-electron reduction of molecular oxygen to water. Laccase has four copper atoms integrated into its two catalytic active sites: a tri-nuclear cluster responsible for the reduction of molecular oxygen, and a mononuclear Cu atom responsible for scavenging electrons from a variety of nonspecific aromatic substrates through one-electron oxidation and radical product formation. 1,2 Laccase is relatively thermostable and has a high turnover rate, making it an ideal target in the field of bioelectrocatalysis. 3,4 Biofuel cells allow for the harnessing of electrical energy that is available from a chemical reaction through the use of bioelectrocatalysts, and oxidoreductase enzymes are common bioelectrocatalysts considered for efficient energy conversion. For this purpose, a large amount of research effort has been put forth developing materials and methods to enhance the electrical connection of catalytic oxidoreductase enzyme active sites to electrode surfaces. 5-8 Two primary methods of electron transfer exist for connecting the enzyme active sites to a conductive electrode surface: mediated electron transfer (MET) and direct electron transfer (DET). MET focuses on using a reversible redox species as a shuttle for electrons from the active site of the enzyme to the electrode surface. This method is suitable for enzymes whose active sites are buried deep inside the insulating protein shell and are not very accessible to pass electrons directly to a conductive surface. Typically, MET employs a polymer matrix to immobilize the enzyme on the electrode surface while the mediator c...