11.1 Introduction

The first example of an enzymatic biofuel cell (EBFC) using enzymes as catalysts was proposed in 1964 [1]. This concept just slowly evolved over two decades until new achievements in enzyme wiring and new nanotechnological approaches, initially developed for biosensing, led to an impressively growing interest in biological energy production in the year 2000 [2].

The principle of EBFCs is similar to that of classic fuel cells, which is based on a catalytic fuel‐oxidizing anode and a catalytic oxidizer‐reducing cathode. The difference lies in the nature of the catalysts used, which are of biological origin in the case of biofuel cells [3], contrary to abiotic fuel cells where principally noble‐metal‐based catalysts or alloys are used. Compared with conventional fuel cells, EBFCs are safe due to the enzyme reactions that can operate under mild conditions such as room temperature, atmospheric pressure, and neutral pH. Additionally, EBFCs can be used for several biologically related reductants (fuels) as electron donors such as sugars, alcohols, amines, organic acids, and hydrogen at the anode side. On the cathode side, O₂ or H₂O₂ is mostly used as an electron acceptor. Such advantages of EBFCs lead to a large variety of potential applications. One promising application of glucose EBFCs is the power supply of implanted medical devices such as pacemakers, sensors, or actuators as actually glucose is the “fuel” and oxygen is the oxidizer in living organisms. There are many reviews about implantable power generators using biological and abiotic catalysts [4–8]. In the highly complex media of body fluids, abiotic catalysts have the disadvantage of insufficient selectivity toward glucose oxidation and oxygen reduction and its inhibition by various compounds, and these catalysts generally show low efficiency at neutral pH. In contrast, such conditions are ideal for optimal operation of enzymes, but the drawback here is the insufficient lifetime of these catalysts. Up to date, no competitive setup exists that can replace the currently used lithium batteries.

Another promising sector for glucose fuel cell application is the power supply of low‐power‐consuming portable devices. Taking into account the limited time of operational (bio)catalysis, one‐time‐use devices such as sensors can be focused [9, 10].

Despite the remaining issues keeping this research field at the academic level, tremendous progresses were achieved in terms of power output that increased by factor 1000 from microwatts to milliwatts for individual glucose biofuel cells [11–13]. The challenges of enzyme wiring are now better understood leading to these improvements. In fact, dependent on the nature and catalytic center of each enzyme used either for the electrocatalytic oxidation of glucose or the electrocatalytic reduction of oxygen, the concept of efficient electron transfers from or to the electrode has to be adjusted. There are two common ways to transfer electrons to or from the catalytic centers of enzymes. The first and generally envisioned type is the direct regeneration of the enzyme by the electrode. This so‐called direct electron transfer (DET) allows achieving optimal cell voltages and transfer rate kinetics but strongly depends on the position of the redox center in or on the enzyme.

DET reactions between proteins (enzymes) and electrodes have been extensively studied from the viewpoints of both understanding the fundamental features and for applications as biosensors and biofuel cells. The first DET reactions of proteins were achieved by Eddowes and Hill [14], Yeh and Kuwana [15], and Niki et al. [16], in which the reversible cyclic voltammetric responses of cytochrome c on gold electrode coated with 4,4′‐bipyridyl and tin‐doped indium oxide electrode, and cytochrome c ₃ on mercury electrode, were reported. Before the achievement of the first DET reactions, it was impossible to observe a voltammetric response based on DET reaction of a protein because of extremely slow DET reaction of a protein even though an electron transfer protein in respiratory chain in mitochondrion was used. During the period of late 1970s to early 1980s, key concept for successful DET was based on specific electrode surface structures [17]. The electrode surface was functionalized to inhibit adsorptive surface denaturation of proteins and adsorption of passivating impurities. Additionally, the functionalized electrode may control other factors such as orientation.

Among functional electrodes, so‐called promoter‐modified electrodes were very convenient to use within several years [18, 19]. Promoters or electron‐transfer promoters stand for functions that accelerate DET kinetics but are themselves electrochemically inactive at the potentials of interest [17]. Especially chemisorption of organothiols on gold or silver is a powerful approach to functionalize electrode surfaces. Using bis(4‐pyridyl)disulfide‐modified gold as a promoter‐functionalized electrode, the first “promoted” DET reactions of cytochrome c were performed by Taniguchi et al. [20]. Such chemisorption functionalization of organothiols on gold or silver has been extensively developed using SAM (self‐assembled monolayer) techniques. During late 1980s to early 2000s, the surface structure of SAM‐modified single‐crystal gold with atomically flat surface has been well analyzed and understood at molecular scale using, for example, electrochemical reductive desorption method [21], in situ surface‐enhanced IR adsorption spectroscopy [22], X‐ray photoelectron spectroscopy [23], or in situ electrochemical scanning tunneling microscopy [24]. At this stage, the understanding of the design of the effective electrode surface structure for fast DET rates with enzymes resulted from comprehensive investigations at molecular scale [19, 25, 26].

Lipid‐membrane‐modified electrodesare also efficient examples for promoted DET with enzymes. In this regard, lipids are not only biological surfactants that are usually components of biomembranes but also artificial surfactants. A lipid membrane structure can be formed artificially on solid surfaces. Enzyme can be adsorbed on the lipid membrane surface or be embedded within, which has a stabilizing effect for enzymes [27]. A typical achievement for promoted DET using lipid‐membrane‐modified electrodes was demonstrated in the case of cytochrome c oxidase. Cytochrome c oxidase (or complex IV) has a huge molecular weight (204 kDa) and catalyzes the final step in the mitochondrial electron transfer chain and is regarded as one of the major regulation sites for oxidative phosphorylation. Immobilized cytochrome c oxidase on lipid‐ membrane‐modified electrodes showed promoted DET with reduced cytochrome c. With this setup, the final step of the model in the mitochondrion could be artificially reproduced [28].

These approaches were developed for the DET reactions of redox proteins at the early stage, but these achievements remained at a fundamental level.

Redox enzymes are generally large molecules of 20–800 kDa in mass. The average hydrodynamic diameters are 50 to several hundred Ångstrom [29]. Therefore, DET is difficult to achieve, as in many cases, the redox center of enzymes is deeply buried within the protein shell. The electron transfer rate is exponentially dependent on the distance between the redox‐active centers as predicted by the Marcus theory [30]. Therefore, in order to shorten the electron transfer distance between the active center of the enzyme and the electrode, surface functionalization of an electrode to control the orientation might be an essential or suitable approach.

When no DET can be achieved, redox molecules with appropriate redox kinetics and diffusion capacities to shuttle the electrons via mediated electron transfer (MET) are necessary. A mediator can be organic molecules or metal complexes, which may have access to a redox‐active center located deeply inside the protein molecule. MET is still a powerful method and is used in biosensors employing enzymes that do not satisfyingly exchange electrons with the electrodes. The cornerstone of commercial glucose biosensors is based on MET. In general, MET‐based EBFCs show higher electric power density in comparison with DET‐based EBFCs even when a reduced cell voltage has to be accepted [31]. This reduced cell voltage accounts for the electrocatalytic reaction occurring at the redox potential of the redox mediator that requires additional overpotential in order to provide a sufficient driving force for the establishment of a fast MET. The principles of these electron transfer types are depicted in Figure 11.1.

A further advantage of MET is the possibility to establish electronic communication in solution, whereas DET is a purely surface‐dependent phenomenon. In this context, highly porous conductive nanostructures became the material of choice for efficient wiring of enzymes. Carbon nanotubes (CNTs) have very favored properties as electrode material for electron transfer with enzymes due to their nanowire structures enabling naturally close contact to the redox centers of some enzymes [12]. CNTs can further be shaped to give freestanding electrodes, which facilitates the integration on electronic devices [32]. Another promising approach is to use nanostructured carbon materials with controlled pores as electrodes. Mesoporous carbons with narrow pore size distributions could be tailored to the target enzymes while providing high specific surface area. These carbons were investigated as enzyme supports with an increase in the total amount of electrochemically active enzymes. The enzymes could be stabilized by encapsulating them in the pores of the support, thus preventing their loss from the support and their aggregation, or degradation of their molecular structure [33]. The enzyme‐support interactions can be adjusted by the pore characteristics, including the pore structure and morphology, and by surface chemical characteristics, such as hydrophobic/hydrophilic interactions, electrostatic interactions, and hydrogen bonding. Among these factors, the pore size is an important parameter affecting enzyme immobilization [34].

Regarding the constant improvements in glucose biofuel cell performances, molecular technology became an important tool not only in terms of mediator design but also for oriented immobilization of enzymes, thus enabling ET. The different principles using molecular technology for improved performances of biocatalytic anodes and cathodes in glucose biofuel cell setups are described and illustrated with relevant examples.

11.2 Molecular Approaches for Enzymatic Electrocatalytic Oxidation of Glucose

The performance of EBFCs can be limited by mass transport of fuels, catalytic activity, or electron transfer rate. If the electron transfer is the rate‐limiting process, molecular design of mediators can improve the performance of the cell and is of particular interest for bioanodes. The choice of the mediators can increase electron transfer rate; therefore, MET is generally faster by several orders of magnitude than DET. In contrast, the resulting cell potential (ΔE _cell) is reduced by the electron‐mediating process (Figure 11.1). This implies that redox potential of a mediator is essentially important to obtain high power densities. Furthermore, the aim of the design and conception of glucose‐oxidizing bioanodes is the fast and repeatable regeneration (oxidation) of the enzyme by the electrode material, which is often in competition with its natural process.

In the case of glucose oxidase (GOx), the enzyme is regenerated by oxygen in a two‐electron reduction producing hydrogen peroxide [35]. GOx is a flavoprotein that catalyzes oxidation of β‐D‐glucose utilizing molecular oxygen as the electron acceptor. GOx is a homodimeric enzyme, with a noncovalently but tightly bound FAD molecule at the active site. This catalytic center is surrounded by a vast glycosylated protein shell (total mass ∼160 kDa), which is responsible for its outstanding stabilities and activity over several pH values. GOx resists furthermore to chemical functionalization that even tagged enzymes could be commercialized. The disadvantage of such protected catalytic sites is that DET with electrodes is very hard to achieve as the tunneling distance is too high [36]. Several approaches were described to achieve DET with GOx. One strategy consisted in the deglycosylation of the enzyme, which presumably decreases the distance between the active site and the electrode surface [37]. Another biotechnological pathway was the production of FAD less GOx. This nonactive apo‐enzyme could be successfully reactivated and wired on FAD‐functionalized nanostructured conductors. Xiao et al. first proposed in 2003 this approach by the reconstitution of an apo‐GOx using FAD‐modified gold nanoparticles. The thus reactivated enzyme showed a sevenfold improved electron transfer turnover rate compared with the natural electron transfer rate to oxygen [38]. One year later, Patolsky et al. attached FAD to CNTs enabling the wiring of apo‐GOx and showed a sixfold improved electron transfer turnover rate [39]. The challenge to obtain DET with native commercially available GOx was successfully overcome by Zebda et al. [40]. Bioanodes were obtained by the compression of a CNT/GOx mixture. However, the low yield of wired enzymes had to be compensated by their quantity and the enzyme catalase had to be added to reduce the high amount of produced hydrogen peroxide by unwired GOx.

MET might be a more appropriate way to wire GOx even when a related reduction of the cell voltage has to be taken into account. The Heller group first described a MET with GOx using an osmium‐based redox polymer [41]. As the redox potential of osmium can be adjusted by the ligand, more appropriate osmium redox hydrogels were synthesized targeting glucose biofuel cell applications [42–44]. These hydrogels were successfully combined with CNT microfibers [45] or CNT‐based textiles [46], reaching power output of 2 mW cm⁻².

Besides metal complexes, pure organic redox molecules showed to be promising alternatives due to clearly enhanced stabilities of the biocatalytic electrodes. Reuillard et al. used naphthoquinone in GOx/CNT pellets and obtained a 14‐fold increase of catalytic current compared with the DET setup mentioned earlier [31] while accepting an increase in the open‐circuit potential and consequently a cell voltage loss of ∼200 mV of the constructed glucose biofuel cell. The most common strategy to wire glucose‐oxidizing enzymes with quinones or Os hydrogels is depicted in Figure 11.2.

Quinones are becoming more popular as mediators in bioanodes. Therefore, quinones are excellent mediators for other glucose‐oxidizing enzymes such as glucose dehydrogenases (GDH) as the redox potential of quinones can be tailored to suit the desirable value by changing the substituents [47, 48]. In addition, heteroatoms or π‐conjugated systems connected to quinone frameworks can enhance the interaction with carbon‐based electrode surfaces [47]. The advantage of GDHs over GOx is that no oxygen is reduced to hydrogen peroxide, which is generally considered a negative effect. The regeneration of these enzymes is assured by mediators, which can especially be designed for biofuel cell applications [49]. There are three main classes of GDHs that are related to their respective cofactors. These cofactors are pyrroloquinoline quinone (PQQ), nicotine adenine dinucleotide (NAD) with or without phosphate (P), and flavin–adenine–dinucleotide (FAD).

The soluble group of pyrroloquinoline quinone–glucose dehydrogenase (PQQ‐GDH) is composed of two identic subunits each with strongly bound PQQ and three calcium ions [50]. Tanne et al. functionalized CNT layers on gold electrodes with PQQ and coupled covalently a reconstituted apo‐GDH. The authors could thus achieve MET via the intermittent PQQ layer [51]. Few years ago, the same research team realized DET with this reconstituted apo‐GDH by using poly(3‐aminobenzoic acid‐co‐2‐methoxyaniline‐5‐sulfonic acid) – PABMSA, a sulfonated polyaniline that seems to form favored interactions with PQQ‐GDH for DET [52, 53]. DET could also be achieved via simple amide coupling using 1‐pyrenebutanoic acid succinimidyl ester with PQQ‐GDH π‐stacked on a CNT buckypaper [54]. The authors explained this electron transfer phenomenon by the porous 3D structure of buckypaper where the distance of embedded enzymes to the CNT matrix is on average short enough for DET. This phenomenon was also observed by Ivnitski et al. [55]. An operational glucose biofuel cell based on bioelectrocatalytic buckypaper electrodes could successfully be inserted in a snail [54], in clams [56], onto exposed rat cremaster tissue [57], and in lobsters [58].

NAD‐dependent GDH is composed of four identic subunits where the cofactor NAD is not confined in the protein structure [59]. In order to achieve electron transfer from the enzyme, this cofactor has to be included together with a catalyst that re‐oxidizes the generated NADH and a redox mediator. Even when many compounds are necessary for this setup, the overpotential of NAD for glucose oxidation seems to be interesting enough to face the issues of multimolecular engineering. Furthermore, there is a wide series of NAD‐dependent enzymes that can be used as anodic catalyst of the oxidation of biofuels such as glucose [60], alcohols [61], and L‐lactate [62]. Therefore, once the optimal strategy is determined for the regeneration of NAD, it can serve for many different biofuel cell applications. Lalaoui et al. studied different setups for the electrocatalytic oxidation of NADH using multiwalled carbon nanotube (MWCNT) defects, immobilized diaphorases, and immobilized ruthenium complexes [63] where the ruthenium‐based compound provided the best compromise between current density and overpotential. Another original example for a molecular approach to oxidize NADH was proposed by Giroud et al. They used a dithiobis (nitrobenzoic acid)‐modified pyrene derivative for this purpose and used this molecule at the same time for oriented immobilization of multicopper enzymes at the cathode [64]. However, for these and other examples [65], NAD has to be dissolved in solution to obtain the reported performances, which is quite inconvenient for practical applications. To circumvent this issue, Sakai et al. proposed subsequent deposition of poly‐L‐lysine as the cationic ground layer followed by the enzyme NAD‐GDH, NAD, diaphorase for efficient NADH oxidation, the mediator 2‐methyl‐1,4‐naphthoquinone, and finally polyacrylic acid to form a polyion complex with the ground layer tightly fixing the different components in between [66]. With this design, a power density of 1.4 ± 0.24 mW cm⁻² at 0.3 V with an open‐circuit voltage of 0.8 V could be obtained. Based on this principle, an original glucose biofuel cell design was proposed combining two fuel cells that were connected in parallel and could provide doubled power outputs. Another strategy involved the immobilization of NADH on CNT‐based electrodes using modified pyrene derivatives [67] or polymers [68].

The complexity to incorporate cofactors in a biofuel cell design guided the research to another, here, FAD‐dependent GDH. The active site for glucose oxidation of this enzyme is similar to that of GOx, but the enzyme is independent of oxygen and therefore needs artificial electron mediators [69]. Zafar et al. tested several FAD‐dependent GDH for glucose biofuel cell applications [60]. FAD GHDs from Glomerella cingulate, a recombinant form expressed in Pichia pastoris, and the commercially available glycosylated enzyme from Aspergillus sp. were wired with the osmium redox polymer [Os(4,4′‐dimethyl‐2,2′‐bipyridine)₂ (PVI)₁₀ Cl]⁺ on graphite electrodes. All of them showed excellent performances for electrocatalytic oxidation of glucose in terms of current density, selectivity, and turnover. Nonetheless, the deglycosylated form of FAD‐GDH from G. cingulate provided higher catalytic currents due to the reduced size of the enzyme, which enabled higher densities of immobilized enzymes. FAD‐GDH from Aspergillus terreus was co‐immobilized with a PVI‐Os(2,2‐bipyridine)₂Cl derivative on a glassy carbon (GC) electrodes. The steady‐state catalytic current for glucose oxidation was 2.6 mA cm⁻² at pH 7 and 25 °C. This value increased 1.6‐fold after oxidative deglycosylation of the enzyme [70]. MgO‐templated porous carbon electrode further coated with the deglycosylated FAD‐GDH and PVI‐Os(bipyridine)₂Cl showed a 33‐fold increase in glucose oxidation current density (ca. 100 mA cm⁻² at pH 7 phosphate buffer containing 0.5 M glucose, at 25 °C) compared with that of the flat electrode [71]. FAD‐GDH‐hydrogel‐modified porous carbon electrode showed exceptionally long‐term stability by caging effect of porous carbon scaffold and shrinking effect by increasing the phosphate buffer concentration [72]. Hexacyanoferrate ([Fe(CN)₆]³⁻; ferricyanide) was used as a redox mediator for commercially available FAD‐GDH‐based blood glucose sensor strips because of its high solubility in water, low cost, and high stability. The reactivity between FAD‐GDH and ferricyanide is quite low, leading to a bimolecular rate constant to be as low as 10³ M⁻¹ s⁻¹ in phosphate buffer (pH 7.0) at room temperature [73]. The rate constant for FAD‐GDH toward quinones and organic redox dyes, such as phenothiazines, was approximately 2.5 orders of magnitude higher than that for GOx [74]. The difference suggests that the electron transfer kinetics is determined by the potential difference (the driving force of electron transfer), as well as the electron transfer distance between the redox‐active site of the mediator and the FAD, affected by steric or chemical interactions. Naphthoquinone‐based hydrogels have also been successfully used for FAD‐GDH entrapment and MET wiring, reaching catalytic glucose oxidation currents of 2 mA cm⁻², accompanied with onset potentials of −0.13 V vs Ag/AgCl [48].

11.3 Molecular Designs for Enhanced Electron Transfers with Oxygen‐Reducing Enzymes

Most enzymes used for the bioelectrocatalytic reduction of oxygen into water are from the multicopper enzyme family where laccases and bilirubin oxidases (BOD) can be considered enzymes of choice. They are composed of two distinct redox centers where a trinuclear 2 and 3 type (T₂/T₃) center reduces oxygen to water in a four‐electron process and a mononuclear 1 type (T₁) center usually oxidizes its natural substrate (in general phenolic compounds) in a one‐electron process and supplies the T₂/T₃ center with the harvested electrons [75]. These multicopper enzymes are generally smaller than the ones for glucose oxidation and the electron transfer is more evident to achieve as the active sites are more accessible.

Several strategies were proposed to wire laccases via MET and DET [76]. Os‐based redox polymers are again famous examples for efficient electron shuttling from the electrode to the enzyme [77], but the molecule 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid) (ABTS) became the mediator for laccases of choice due to its appropriate redox potential, electron transfer rates, and stability [78]. Furthermore, ABTS retains entirely these beneficial properties after chemical modifications for its immobilization [79]. By modifying ABTS with two pyrene groups, this mediator could also act as a crosslinking agent and reinforce buckypapers leading to freestanding redox‐active electrodes [80]. Recently, an original approach has involved the design of chimeric protein based on a prion domain and a rubredoxin domain. These proteins are able to form self‐assembled amyloid nanofibers [81]. Thanks to the rubredoxin domain, these protein nanofibers were not only able to entrap enzymes but were also able to trigger MET with entrapped laccases. This type of bioassembly represents a promising alternative in the design of versatile “all‐protein” bioelectrodes.

An original alternative to “all immobilized” component setup was proposed with these mediator‐functionalized buckypapers as higher current densities were obtained when laccase was dissolved in solution. With this configuration, the biological catalysts can easily be replaced when the catalytic activity decreases [82].

Besides the highly efficient approaches for MET for laccases, the fact that the active centers are close to the protein surface motivates to achieve the generally preferred DET. Furthermore, laccase from Trametes versicolor is particularly suitable for optimized DET wiring as the protein structure provides a hydrophobic domain close to the T₁ center. The F. A. Armstrong group first discovered the possibility of oriented immobilization of this laccase via hydrophobic interactions on anthracene‐modified surfaces, thus enabling DET for the electrocatalytic reduction of oxygen [83]. Several examples followed reporting the immobilization, orientation, and wiring of laccase using polyaromatic hydrocarbons such as anthracene [84], naphthalene [85], pyrene [86], or anthraquinone [87] derivatives attached to carbon surfaces and therein mainly CNTs. Lalaoui et al. studied this phenomenon in more detail and calculated the binding energy of the anthraquinone representing the polyaromatic compounds and adamantane, a saturated hydrocarbon [88]. The calculations were accompanied by electrochemical and quartz crystal microbalance (QCM) experiments. The modeling revealed a higher binding energy (−15.4 ± 1.8 kcal mol⁻¹) for anthraquinone than for adamantane (−7.8 ± 1.5 kcal mol⁻¹), but the experiments showed higher catalytic currents for adamantane (2.13 mA cm⁻² at 0.55 V vs SCE) than for anthraquinone (0.74 mA cm⁻² at 0.52 V vs SCE) both, immobilized of CNTs. It was concluded that anthraquinone tends to form π‐stacking interactions with the CNT walls and this leads to a lower amount of available anchor groups and thus to a lower surface coverage of oriented enzymes. This π‐stacking issue for polyaromatic substances was already evoked 2 years before and qualitatively studied by Bourourou et al. [87]. The same group also adapted this strategy for the covalent modification of reduced graphene oxide. This nanomaterial showed lower DET properties compared with CNTs but exhibited strong π‐stacking interactions with CNT films [89]. Another strategy also involved the specific modification of laccase mutants with pyrene groups, localized at the vicinity of the T₁ center [90]. These enzymes were successfully immobilized on CNT electrodes and gold nanoparticles modified with β‐cyclodextrin groups, showing efficient DET at both nanomaterials. In addition to this latter work, several works have underlined the excellent properties of gold nanoparticles in terms of DET toward oriented laccases, either via supramolecular [90] or via covalent interactions [91].

BOD from Myrothecium verrucaria is a promising alternative to laccase as its highest catalytic activity is in the neutral range [92–94]. Similar to other multicopper enzymes, BOD consists of a single subunit with four redox‐active Cu atoms (T₁ and T₂/T₃), but the substrate binding site is hydrophilic and is incompatible with the oriented immobilization and wiring strategies for laccase [95]. Nonetheless, the strong interaction with its substrate bilirubin allows site‐specific immobilization of this enzyme, enabling efficient DET [95–97]. More efficient performances were obtained using the protoporphyrin IX, a mimic of bilirubin [98]. It was concluded via several parallel experiments that the presence of carboxylates mainly contributes to the oriented immobilization of BOD via both electrostatic interactions and favorable dipolar moment of the enzyme. Based on these results, Lalaoui et al. proposed a simplified and less expensive approach by functionalization of CNTs via covalent diazonium grafting with carboxynaphthyls [99]. A scheme of oriented wiring of BOD and laccase is presented in Figure 11.3. Tsujimura's group previously reported DET reaction of BOD using pore‐size‐controlled mesoporous carbons, including carbon gel and MgO‐templated carbon [100, 101]. By using MgO template with pore diameters of 38 nm, the DET catalytic current density was found to be 6 mA cm⁻² with an electrode rotation rate of 8000 rpm at pH 5, 25 °C, with O₂ saturation. To further improve the current production efficiency, a three‐dimensional (3D) hierarchical pore structure was fabricated using a MgO‐templated porous carbon produced from two MgO templates with sizes of 40 and 150 nm [102]. The macropores improve mass transfer inside the carbon material, and the mesopores improve the electron transfer efficiency of the enzyme by surrounding the enzyme with carbon. The electrode showed 13 mA cm⁻² of oxygen reduction current at pH 5 without any further surface modification.

11.4 Conclusion and Future Perspectives

EBFCs have unique features compared with other energy harvesters and batteries, including the potential for miniaturization, high theoretical power densities, and high biocompatibility. Considerable efforts have been made to develop EBFCs as novel power sources that are cost–effective, environmentally friendly, and readily available in order to drive implantable, epidermal, or wearable (bio)electronic devices, as well as the ubiquitous sensor‐node systems required for technologies related to the Internet of things (IoT). Potential applications of EBFCs are illustrated in Figure 11.4. However, EBFC technology is still at an early stage of development, with many fundamental scientific and engineering problems that have still to be resolved. Two critical issues related to EBFCs are their short lifetimes and poor power densities; the number of electroactive enzymes available for a reaction is limited because enzymes on electrode surfaces are generally unstable, and there is a large barrier to electron transfer between enzyme‐active sites and electrode surface. Usually, a trade‐off is attempted between the output power density and lifetime, as it is highly challenging to improve both simultaneously. Considering these inherent drawbacks, there are two possibilities for the application of EBFCs, with the first focusing on disposable products and the second on longer‐lasting devices that require less power.

The advantage of EBFCs for primary battery‐like single‐use disposable applications is the easy disposal of used EBFCs, as they are basically composed of enzymes and carbon electrodes, both of which can be made using ecofriendly, biodegradable materials. Primary batteries are usually used as portable chargers, which need to be recycled. The present requirement of powering electronic devices such as wireless communication tools anytime and anywhere will increase considerably in the future. To compare with existing primary batteries, the output performance as well as the price competitiveness of EBFCs should be improved. EBFCs can also meet the demands of powering wearable or epidermal electronic devices because of their high safety, as they lack strongly basic electrolytes and metal packages. Additionally, EBFCs that operate using sweat, urine, tear, saliva, or blood are better suited to disposable use. Such disposable energy devices would open up new routes for applications such as epidermal healthcare electronics, contact lens or mouth guard‐type sensing devices, self‐powered urine/blood glucose monitoring devices, and communication tools for emergencies [103].

Low‐power but longer‐lasting devices could power implantable medical devices such as pacemakers or neurostimulators using glucose and oxygen in the body as fuels. The simple structure of EBFCs would allow for straightforward miniaturization, reducing the burden imposed on a patient. However, improving the stabilities of enzymes that have been immobilized on electrode surfaces still presents a significant challenge for the development of EBFCs with lifetimes greater than 1 year.

It should be noted that another possible application that makes the best use of EBFCs could be single‐use disposable self‐powered biosensors, which require low amounts of electricity for transmitting and amplifying sensor signals. To achieve this, it will be necessary to design cell configurations that allow the output power to depend on the fuel concentration.

The most feasible fuel for EBFCs is glucose due to its chemical stability, safety, and accessibility. Although the number of electrons available from glucose oxidation is limited to two in the present technology, this should be increased by developing novel biomimetic (bioinspired), enzymatic, or abiotic electrocatalyst cascade systems. The capacities of EBFCs could exceed those of lithium ion batteries or other secondary batteries if 24 electrons could be taken from one glucose molecule. Although the output power density depends on the kinetics of the enzymes, exceptionally high capacities (energy densities) would open up new applications for EBFCs not only as fuel cells but also as primary batteries (Figure 11.4).

Much research still focuses on monosaccharides such as glucose, but stable and abundant polysaccharides, including paper, cotton, leaves and stems of plants, and starch, can be utilized in combination with specific hydrolases. On the other hand, alcohols, carbohydrates, or organic acids of low molecular weight can be considered as alternative anodic fuels to increase energy density. Recently, lactic acid from sweat has gathered considerable attention as an energy source for epidermal or wearable power devices [62]. Oxygen is most widely used as a final electron acceptor in enzymatic biocathodes. Although it is the most abundant and accessible oxidant, the delivery of oxygen to enzymes can be the rate‐limiting factor in some systems, especially in implantable EBFCs, due to its low solubility in solution. Alternative oxidants that have high formal potentials as well as that are stable, of low cost, and safe should be developed.

A promising approach to overcoming both the short lifetimes and low power densities of EBFCs and allowing them to reach practical applications is to use nanostructure‐controlled porous carbon materials as an electrode. However, enzymes in confined nanospaces have yet to be elucidated with respect to electrochemical and biological enzymatic reactions, as well as the 3D structural changes in the enzymes. A combination of more efficient electron transfer technology by modification of the microscopic interface between the enzyme and the porous carbon materials with macro–meso hierarchical structures would be helpful for achieving higher and more stable current outputs with lower amounts of enzymes, contributing to a practical advancement in fuel cell technology. Eliminating diffusional mediators by facilitating efficient electron transfer between conductive nanostructured materials and dehydrogenases could improve EBFC stability, which would be beneficial for the development of implantable EBFCs.

Molecular technology, protein engineering, nanostructured materials, hydrogels, and polymers have all been used to enhance the kinetics of electron transfer between enzyme‐active sites and electrode surfaces, as well as the stability of the three‐dimensional structures of enzymes inside electrode nanospaces, thereby improving the performance, stability, and durability of EBFCs. The ultimate goal for an EBFC with both high output power and stability is application to a ubiquitous wireless power supply for any electronic devices that require electricity.

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