Enzyme-Based Biofuel Cells and Nanostructured Electrodes

T Sophie, AB Engel, D Cornu, and M Bechelany, European Institute of Membranes, Montpellier, France
© 2018 Elsevier Inc. All rights reserved.

Introduction 217
Enzyme-Based Biofuel Cells 217
Electrode Material 218
Porous Conductive Nanostructures 219
Porous Conductive Substrates 220
NPs Supported on Porous Templates 221
Limitations 222
Conclusion 222
Further Reading 222

Abbreviations
BOD Bilirubin oxidase
CF Carbon fiber
CNT Carbon nanotube
DET Direct electron transfer
EBFC Enzyme-based biofuel cell
GOD Glucose oxidase
MET Mediated electron transfer
NPs Nanoparticles

Introduction

Because of environmental concerns, numerous investigations have been made to develop alternative systems for energy production
that are capable of operating independently over prolonged periods of time. Since the last decades, enzyme-based biofuel cells
(BFCs) have received pronounced interest as energy-harvesting devices. Since the 1990s, these devices have known a great interest
appearing as an alternative to produce low power densities. EBFCs are a specific type of fuel cell that employs enzymes as catalysts,
in order to convert the chemical energy of renewable biofuels into electric energy through electrochemical reactions. Oxidative
enzymes are used for the oxidation of a substrate (fuel) occurring at the anode, and reductive enzymes are used for the reduction
of an oxidant (generally O2) at the cathode. Compared with conventional fuel cells that use metallic catalysts, the main advantages
of EBFCs are mild operating conditions, reaction selectivity, and diversity of clean fuels and products.
These devices can find potential applications for self-powered devices that do not require long-term operations such as portable
wireless consumer electronics and biomedical devices. The field of implanted EBFCs might, in the future, revolutionize how we
power implanted medical devices (artificial organs, pacemakers, hearing devices, etc.), since it would be ideal to use our own
metabolism to do so, instead of the lithium-ion batteries used nowadays. Ideally, implanted devices would take advantage of
the natural fuel substances found in the body and thus would continue to draw power for as long as the subject lives. These devices
could be implanted not only in human bodies but also in animals, which would guarantee electric power in, for example, military
missions, where power is not directly available.
However, EBFCs are limited by both short long-term stability and low power densities that restrict their potential applications.
As the electrode materials play an important role to overcome low current densities and mass transport limitations occurring during
the operation of these devices, nanostructured electrodes would be an important advantage to construct EBFCs. This article describes
recent advances in the applications of nanostructured materials with three-dimensional (3D) architectures for optimizing both
surface area and fluid delivery through the material (i.e., mass transport) for the development of efficient enzyme-based electrodes.

Enzyme-Based Biofuel Cells

EBFCs consist of two separated or coupled electrodes, an anode and a cathode. The general view of a typical EBFC is depicted in
Fig. 1. The enzymatic oxidation of fuel at an anode generates electrons that are transferred through an external circuit, to a cathode

217
218 Enzyme-Based Biofuel Cells and Nanostructured Electrodes

Fig. 1 Simplified view of enzyme-based biofuel cell based on the electric connection of redox enzymes, working from oxidation of fuel at the anode
and reduction of O2 at the cathode.

where O2 (as an oxidant) is enzymatically reduced into water with the help of protons resulting from the anodic reaction. The
electric current flows according to a difference of potential and the catalyzed reactions that generate power. In the actual interna-
tional context of renewable and low-environmental-impact energy source developments, EBFCs constitute a potential green energy
technology.
The research on these devices relies on the development of biocatalytically modified electrodes and enzyme reactions. Depend-
ing on the target fuels (glucose, H2, and alcohol), different enzymes are employed: glucose oxidase (GOD), hydrogenase, alcohol
and aldehyde dehydrogenase, and lactate oxidase, to cite some. The oxidant is usually oxygen, and the most common enzymes used
to efficiently reduce O2 into water are bilirubin oxidase (BOD) and laccase. The electric connection of the enzymes to the electrode
surface can be performed either by mediated electron transfer (MED) in the presence of a redox mediator or by direct electron
transfer (DET). The MED mechanism provides fast electron transfer and occurs in general at the low overpotential characteristic
of the redox mediator. In the case of DET, the advantage is the simplification in the construction of the bioelectrode and the absence
of mediators that are often health hazards and may affect the kinetics and thermodynamics of the electrochemical reaction.
However, the efficiency of the DET mechanism is strongly influenced by the enzyme structure and the proper orientation of the
redox center of the enzyme to perform direct wiring with the electrode surface.
Performance and operational stability of enzyme-based electrodes have been addressed to design electrocatalytic biomaterials
for bioenergy conversion, biosensing, and bioreactors. Strategies have been developed to improve the electronic wiring of the redox
enzymes through optimal physical or chemical methods in order to effectively incorporate enzymes into appropriate electrode
materials, considering their fragile 3D conformation. Physical methods are simpler and more efficient regarding enzyme immobi-
lization, and they are preferred to the methods involving the formation of chemical bonds. The resulting bioelectrodes are charac-
terized by electrochemical techniques like cyclic voltammetry, polarization curves, amperometry, and impedance measurements.
EBFCs generate power densities in the order of micro- to milliwatts, which is sufficient to supply small electronics.
However, the technology still needs scientific and technological advances to reach a maturity level required for industrial
developments. Research on the topic is therefore intense in order to overcome the existing drawbacks such as the limited stability
of the enzymatic catalysts over time and the low delivered power density caused by leakage, rearrangement, and/or denaturation of
the enzymes.

Electrode Material

The optimization of EBFCs is an extensive science that can focus on the design of the cell itself, the target fuel, the engineering of new
enzymes, the immobilization of such enzymes onto the electrodes, and the electrode material itself. This last feature is the focus of
the present work. The role of the material in the performance of enzyme-based electrodes is major: its connection with the immo-
bilized enzymes must be optimal for efficient bioelectrocatalytic activity. Some characteristics that the electrode material must
present are the following:

• High electric conductivitydto assure optimal flow of electrons

• High specific surfacedto assure enough space for a high amount of enzymes to be effectively connected to the electrode, which is
essential for higher delivered current
• Biocompatibilitydso that enzymes are not harmed
 Enzyme-Based Biofuel Cells and Nanostructured Electrodes 219

Fig. 2 Views of traditional dense metal electrodes, porous conductive substrates, and conductive nanomaterials (copyright being proceeded).

• Chemical stabilitydso that corrosion or other reactions do not take place at the working potential range in the electrolyte
solutions
• Appropriate mechanical strengthdso that electric contacts can be established properly and the operation can run smoothly
without the breaking of the electrode
For dense metal electrodes (Fig. 2), the delivered current densities are limited by the geometric surface area, the amount of
immobilized enzymes that are properly orientated to the electrode surface.
The performance of the electrodes are enhanced with the use of conductive nanomaterials (Fig. 2) ranging from carbon
nanotubes (CNTs), graphene, metallic nanoparticles (NPs), and nanowires, owing to (i) their high 3-D electroactive area that
increases the loading of enzymes and improves the orientation of the redox active centers of the enzyme and (ii) their nanoscale
size that allows a close proximity between the active sites of the enzymes and its surroundings, in order to achieve efficient electric
wiring with the electrode surface.
In addition, porous conductive substrates as host matrix (such as carbon paper, carbon fiber (CF) mats, carbon felt, buckypaper,
porous foams, and porous gold) are attractive thanks to the increased tortuous surface area that also contributes to (i) higher
enzyme loadings allowing for higher current densities, (ii) advantageous stability of the immobilized enzymes, and (iii) efficient
substrate diffusion into the material, especially in physiological media where the substrate concentrations are around 0.5 mM
and their diffusion coefficients are below 10 5 cm 2 s 1. It is known that the sum of these contributions can significantly increase
both the lifetime of the redox enzymes and the delivered power densities.
3D materials (like the ones shown in Fig. 2) are thus a great advantage to construct anodes and cathodes in EBFCs, and many
strategies have been proposed in the literature to develop such nanostructured electrodes. The use of conductive nanomaterials
combined with porous conductive substrates is one of these successful strategies.

Porous Conductive Nanostructures

Nanostructures ranging from single- or multiwalled CNTs, graphene, or metallic NPs, as shown in Fig. 2, are employed to construct
efficient enzyme-based electrodes in EBFCs. They have been largely used due to their high electronic, thermal, and mechanical
properties; their biocompatibility, their ease of functionalization; and their large surface area allowing immobilizing high amounts
of enzymes.
Generally, these nanostructures are randomly immobilized onto conductive substrates: they are dispersed in a liquid phase in
the absence or in the presence of surfactants, organic polymers, and biopolymers (for better dispersion) and then mixed by
ultrasonication to form a homogeneous colloidal suspension, which is drop casted at the electrode surface. Adsorption methods
based on hydrophobic interactions are usually employed for enzyme connexion. However, chemical modifications of the surface
of the nanostructures are also possible and can increase their dispersion in solution and their biocompatibility through coupling
with the amino and carboxylic groups of the enzymes. For example, sol–gel silica matrix incorporating CNTs can be used for enzyme
220 Enzyme-Based Biofuel Cells and Nanostructured Electrodes

encapsulation to construct enzyme electrodes in a glucose–oxygen biofuel cell to generate 120 mW cm 2 when operated at room
temperature.
However, two of the more efficient EBFCs reported so far are based on free-standing CNTs electrodes, obtained by
(i) a soft compression of a powder mixture of CNTs and enzymes to prepare stable disk electrodes applied in the construction of
a glucose/O2 BFC able to deliver a power density of 1.25 mW cm 2 thanks to the high enzyme loading,
(ii) the formation of a CNT forest consisting of long single-wall CNTs (1 mm length) incorporating a solution of enzymes, applied
in the construction of a fructose/O2 BFC able to deliver a power density of 1.8 mW cm 2.
Based on the literature, graphene can provide enzyme loading capacity and DET at comparable rates than those reported with CNTs.
Graphene-based enzymatic glucose/O2 biofuel cells, combining the enzymes GOD and laccase or BOD, deliver power densities
ranging from 20 to 520 mW cm 2. The first examples of devices were build from graphene sheets combined with sol-gel matrix
or conducting polymers. However, the recent implementation of more advanced configurations based on hybrid electrodes
combining graphene and CNT provide higher power density of 2.3 mW cm 2 due to the synergistic interaction between the two
forms of carbon.
The incorporation of noble metal NPs along with enzymes is also attractive for increasing the enzyme loading and the current
collected from enzyme-based electrodes. Having similar size as the enzymes, the incorporated metallic NPs can act as electron
carriers between the enzyme and the solid substrate, improving the bioelectrocatalytic process. Several techniques employed to
prepare 3D gold electrodes with controlled sizes are mainly based on the immobilization by drop casting, by layer-by-layer
methods, or in sol–gel matrix of already synthesized concentrated NPs colloids that are subsequently modified by redox enzymes.
Sugar/O2 EBFCs were accordingly constructed with gold(Au)NP-based bioanode and biocathode. The catalytic current varies with
the AuNP’s size, loading, and deposition procedure. As demonstrated for a H2/O2 biofuel cell, higher current densities are prefer-
entially reached with small AuNPs ( 20 nm), and the covalent grafting of the enzymes hydrogenase and BOD on the AuNPs
allowed generating a power density of 0.25 mW cm 2.
Furthermore, the synthesis in situ of the NPs on electrode surface presents the advantage of forming very stable metal bonding
with the support, with well-dispersed NPs, enabling to enhance the long-term stability of the enzymes. The modified electrodes are
usually obtained by

• electrochemical deposition,
• sputtering followed by dealloying,
• chemical reduction,
• seed-mediated growth of the NPs.

Porous Conductive Substrates

Mostly, employed materials used in EBFCs include gold and several forms of carbon owing to their biocompatibility, chemical
stability, and electric conductivity. The most frequently used approach in immobilizing enzymes into porous conductive matrix
is simple adsorption, and it depends, among others, on the pore size and charge interaction of the porous substrate and the enzyme.
The matching of the size of the pores and the molecular diameter of the enzymes is important to achieve good stability of adsorbed
enzymes, thus avoiding their leaching in solution. To prevent this problem, attractive interactions can be generated by the introduc-
tion of opposite charges on the surface of the pores (Fig. 3) compared with the surface charge of the enzymes. An example is the
functionalization of mesoporous materials with functional groups like carboxyl or amino groups.
Carbon felt commercial supports have properties such as low weight, high chemical resistance, high thermal stability, and ease of
handling and are employed in various electrochemical technologies. The thermal treatment of carbon felt by a gas flow containing
1% of oxygen at high temperature (1000 C) results in a porous carbon felt with upgraded properties in terms of surface area, pore
volume, and hydrophilicity (creation of negative charges at the surface), in comparison with commercial raw carbon felt. The

Fig. 3 Schematic surface charge interactions between porous material and enzyme. Reprinted with permission from Flexer, V., Brun, N., Courjean,
O., Backov, R., and Mano, N. (2011). Porous mediator-free enzyme carbonaceous electrodes obtained through integrative chemistry for biofuel cells.
Energy Environmental Science, 4, 2097–2106. Copyright 2010, Royal Society of Chemistry.
 Enzyme-Based Biofuel Cells and Nanostructured Electrodes 221

increase in the porosity allows for the generation of current densities in the range of 2.5 mA cm 2 owing to their ability to immo-
bilize a large amount of enzymes by electrostatic interactions in their intrananospaces.
Highly ordered macroporous gold electrodes with interconnected spherical voids have been prepared by gold electrodeposition
through polystyrene sphere templates. The immobilization of the redox enzymes laccase and GOD on such materials allows
increasing the power density by 10-folds compared to planar gold electrode.

NPs Supported on Porous Templates

The majority of BFCs are designed with electrode materials resulting from the incorporation of nanostructures into porous conduc-
tive templates. The resulting 3-D bioelectrocatalytic electrodes show synergistic effects, providing both (i) micropores to support the
immobilization of the enzymes and (ii) macropores to support the effective mass transport of fuel.
For instance, nanostructures combined with carbon nanofiber mats result in promising materials for efficient energy applica-
tions. The carbon nanofiber mats, prepared by electrospinning from a variety of precursor polymers, present also interesting
properties arising from the fibrous and porous morphology. The greatest advantage of electrospinning technique is the possibility
of obtaining randomly oriented or well-aligned fibers with very low mean diameter (in the range between some nanometers to
some micrometers) and long enough to form a free-standing mat of fibers (which is not possible to obtain from other nanofiber
fabrication techniques). Hierarchical structured materials can be obtained from electrospun CF mats by encapsulating multiwall
CNTs on the surface of the fibers or by growing well-dispersed AuNP’s in situ onto/into the fibers. As shown in Fig. 4, in the
case of AuNPs combined with CFs, two ranges of AuNP’s sizes are obtained with mean size of 215 nm on the fiber surface and
50 nm located inside the fibers. The catalytic performance of the resulting structured electrode has been tested as anode in a hybrid
glucose/O2 BFC, in combination with a cathode obtained from a mixture of BOD/multiwall CNTs entrapped inside the CF’s mat.
The system delivered a stable power density of 65 mW cm 2 during 3 weeks, the enhanced long-term stability being attributed to the
sum of the structural characteristic of both materials.
Numerous studies described 3D ordered macroporous gold electrodes functionalized with Au NPs. When the materials are used
to immobilize the enzymes glucose dehydrogenase and laccase, the resulting one-compartment glucose/O2 biofuel cell delivered
a maximum power density of 178 mW cm 2, which was 16 times larger than that of the BFC with the flat electrode under the
same conditions. Another example is the immobilization of AuNPs on Au microwires (diameter of 100 mm). This process leads
to the construction of 3-D nanostructured Au electrodes, increasing the geometric surface area a hundred times compared with
the bare Au microwires (without AuNPs). After the incubation of the electrodes in enzyme solutions of BOD and cellobiose
dehydrogenase, the resulting EBFC was able to operate in cerebrospinal fluid and in the brain of a rat, producing amounts of electric
power sufficient to drive a self-contained biodevice.
Promising candidates are micro/macro carbonaceous foam electrodes. These allow a considerable increase in enzyme loading,
which results thus in current increase and stabilization of the DET current from a few hours to several days, if compared with flat
electrodes. When the electrodes are modified by the enzyme BOD and tested toward the direct reduction of O2 into water, the
current delivered is increased 500-fold due to considerable increase in enzyme loading.
Another possibility to prepare hierarchical nano-/microporous architectures consists in coating a microporous carbon substrate
by the electrodeposition of a binary alloy like Au–Ag. A subsequent step consisting in preferential etching of Ag results in gold
reordering and formation of nanocauliflower-like structures. Compared with bare microporous carbon electrodes, the presence

Fig. 4 Scanning electron microscopy images of dispersed AuNPs directly grown on electrospun carbon fibers showing the presence of small and
large particles. Evolution of voltage and power density curves with time of a glucose/O2 BFC obtained from the assembly of dispersed AuNPs on
carbon fibers as the anode and BOD/MWCNT entrapment in carbon fibers as the cathode.
222 Enzyme-Based Biofuel Cells and Nanostructured Electrodes

of the additional nanoporous architecture greatly enhanced by 10 times the performance of a glucose/O2 EBFC working with the
enzyme glucose dehydrogenase.

Limitations

The benefit of electrode nanostructuration is usually estimated from the determination of the electroactive surface area by electro-
chemical methods like cyclic voltammetry and impedance spectroscopy. However, the current density generated by EBFCs with
nanostructured electrodes does not increase proportionally with the increase in electroactive surface area. As mentioned, two
possible reasons can explain this phenomenon:
(i) The size of the pores of the porous conductive substrate is too similar to the molecular diameter of the enzymes, and thus, the
lack of suitable porosity does not allow access of more immobilized enzymes.
(ii) The amount of immobilized enzymes is proportional to the substrate surface area, but the whole amount does not take part in
electric current because the enzymes are randomly oriented in relation to the surface or because they are denaturated or even too
deeply buried and not accessible by the molecules of substrates.
The discrimination between these factors is not straightforward from electrochemical measurements or titrations by activity assays,
due to the tortuous structure of the electrode materials. The estimation of the amount of actually immobilized enzymes is quite
complex and is still a challenge to overcome, in order to properly correlate the benefit of the nanostructuration of the electrodes
with the performance of the EBFCs. Besides, the pore sizes influence the mass transport of the substrate to the enzyme and the
exit of the products out of the surface layer. Clearly, when high amounts of enzymes fill the pores, the mass transport step can
become rate limiting.
Some of these limitations can thus potentially be overcome by the synthesis of materials with multiscale porosity and hierar-
chical structures, as mentioned previously, by developing efficient surface area for both optimal enzyme loadings and mass
transport.

Conclusion

The features of enzymatic BFCs are particularly appealing as sustainable energy sources in miniaturized and implantable
bioelectronics. The implementation of nanostructured electrodes in EBFCs enabled the development of novel surface architectures
for enzyme-based bioelectrodes. Various preparation methods allow the creation of original structures and morphologies. The high
surface area of the nanostructured electrodes has demonstrated their significant promise both as the electrode material and enzyme
immobilization matrix to fabricate bioanodes and biocathodes in EBFCs. The main benefits of the nanostructured materials are
achieving high enzyme loadings, allowing preferred orientation of the enzymes for DET mechanism, improved electron transfer
and mass transport into the pore structure. Successful examples have shown efficient EBFCs able to deliver power densities in
the order of mW cm 2, capable of powering small devices like watches and music players. Further researches are being held to
increase the electrochemical performance and long-term stability of BFCs, anticipating industrialization of this new type of energy
devices.

See also: Enzymatic Electrodes: Characteristics, Fabrication Methods, and Applications; Enzymatic Phosphorylation of Peptides and Proteins;
Implantable Enzyme-Based Biofuel Cells; Orientation-Oriented Adsorption and Immobilization of Redox Enzymes for Electrochemical Communication
With Electrodes.

Further Reading

Karimi, A.; Othman, A.; Uzunoglu, A.; Stanciu, L.; Andreescu, S. Graphene Based Enzymatic Bioelectrodes and Biofuel Cells. Nanoscale 2015, 7, 6909–6923.
Holzinger, M.; Le Goff, A.; Cosnier, S. Carbon Nanotube/Enzyme Biofuel Cells. Electrochim. Acta 2012, 82, 179–190.
De Poulpiquet, A.; Ciaccafava, A.; Lojou, E. New Trends in Enzyme Immobilization at Nanostructured Interfaces for Efficient Electrocatalysis in Biofuel Cells. Electrochim. Acta 2014,
126, 104–114.
Andoralov, V.; Falk, M.; Suyatin, D. B.; et al. Biofuel Cell Based on Microscale Nanostructured Electrodes With Inductive Coupling to Rat Brain Neurons. Sci. Rep. 2013, 3, 3270.
Wen, D.; Eychmüller, A. Enzymatic Biofuel Cells on Porous Nanostructures. Nanoscale 2016, 34, 4649–4661.
Flexer, V.; Brun, N.; Courjean, O.; Backov, R.; Mano, N. Porous Mediator-Free Enzyme Carbonaceous Electrodes Obtained Through Integrative Chemistry for Biofuel Cells. Energy
Environ. Sci. 2011, 4, 2097–2106.
Lim, J.; Malati, P.; Bonet, F.; Dunn, B. Nanostructured Sol–Gel Electrodes for Biofuel Cells. J. Electrochem. Soc. 2007, 154, A140–A145.
Miyake, T.; Yoshino, S.; Yamada, T.; Hata, K.; Nishizawa, M. Self-Regulating Enzyme-Nanotube Ensemble Films and Their Application as Flexible Electrodes for Biofuel Cells. J. Am.
Chem. Soc 2011, 133, 5129–5134.
Zebda, A.; Gondran, C.; Le Goff, A.; et al. Mediatorless High-Power Glucose Biofuel Cells Based on Compressed Carbon Nanotube-Enzyme Electrodes. Nat. Commun. 2011, 2, 370.
 Enzyme-Based Biofuel Cells and Nanostructured Electrodes 223

Monsalve, K.; Roger, M.; Gutierrez-Sanchez, C.; et al. Hydrogen Bioelectrooxidation on Gold Nanoparticle-Based Electrodes Modified by Aquifex Aeolicus Hydrogenase: Application
to Hydrogen/Oxygen Enzymatic Biofuel Cells. Bioelectrochemistry 2015, 106, 47–55.
Boland, S.; Leech, D. A Glucose/Oxygen Enzymatic Fuel Cell Based on Redox Polymer and Enzyme Immobilisation at Highly-Ordered Macroporous Gold Electrodes. Analyst 2012,
137, 113–117.
Sattayasamitsathit, S.; O’Mahony, A. M.; Xiao, X.; et al. Highly Ordered Tailored Three-Dimensional Hierarchical Nano: Microporous Gold-Carbon Architectures. J. Mater. Chem.
2012, 22, 11950–11956.
Deng, L.; Wang, F.; Chen, H.; et al. A Biofuel Cell With Enhanced Performance by Multilayer Biocatalyst Immobilized on Highly Ordered Macroporous Electrode. Biosens. Bioelectron.
2008, 24, 329–333.
Andoralov, V.; Falk, M.; Suyatin, D. B.; et al. Biofuel Cell Based on Microscale Nanostructured Electrodes With Inductive Coupling to Rat Brain Neurons. Sci. Rep. 2013, 3, 3270.