DOI: 10.1002/fuce.
200800115
Integrated Enzyme-Based Biofuel Cells–A
Review
I. Willner1*, Y.-M. Yan1, B. Willner1, R. Tel-Vered1
1
Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
Received November 18, 2008; accepted December 17, 2008
Abstract
Enzyme-based biofuel cells provide versatile means to gen-
erate electrical power from biomass or biofuel substrates,
and to use biological fluids as fuel-sources for the electrical
activation of implantable electronic medical devices, or pros-
thetic aids. This review addresses recent advances for
assembling biofuel cells based on integrated, electrically
contacted thin film-modified enzyme electrodes. Different
methods to electrically communicate the enzymes associated
with the anodes/cathodes of the biofuel cell elements are
presented. These include: (i) The reconstitution of apo-
enzymes on relay-cofactor monolayers assembled on electro-
des, or the crosslinking of cofactor-enzyme affinity com-
plexes assembled on electrodes. (ii) The immobilisation of
enzymes in redox-active hydrogels associated with electro-
The electrical contacting of redox enzymes with electrodes
is one of the most fundamental processes in bioelectrochemis-
try. Redox enzymes usually lack direct electron transfer com-
munication between their active redox centres and electrode
supports. This barrier for electron transfer (ET) is explained
by the Marcus electron transfer theory [1] that states that the
electron transfer rate between a donor and an acceptor pair is
given by Eq. 1, where d and do are the actual distance and the
Van der Waals distance separating the donor–acceptor pair,
respectively, DGo and k are the free energy change and the
reorganisation energy accompanying the electron transfer
(ET) process, respectively, and b is the electronic coupling
coefficient.
h i
ket a exp‰ b…d do †Š exp …DGo ‡k†2 =…4 RTk†
Realizing that the dimensions (diameters) of redox pro-
teins are in the range of 70–200 Å, and that the redox centres
are embedded in the protein matrices, the spatial separation
of the biocatalytic redox sites from the electrode prevents the
electrical contacting of the enzyme with the electrode [2]. Dif-
ferent methods to establish electrical communication between
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REVIEW
des. (iii) The use of nano-elements, such as carbon nano-
tubes, for the electrical contacting of the enzyme electrodes
comprising the biofuel cells. All three methods are imple-
mented for the electrical contacting of oxidases and dehy-
drogenases with electrodes acting as anodes of biofuel cells,
and for the electrical wiring of bilirubin oxidase, cytochrome
oxidase, and laccase with electrodes, that yield the cathode
units of the biofuel cells. Different methods to control the
biofuel cells, operation by external stimuli are discussed,
including the application of external magnetic fields, and
the electrochemical switching of the biofuel cell operation.
Keywords: Biofuel Cell, Carbon Nanotube, Enzyme, Electri-
cal Wiring, Monolayer, Magnetic Field, Redox Polymer
the redox centres of enzymes and electrodes were developed
in the past 25 years. These include, Figure 1, the use of diffu-
sional electron mediators that transport electrons between the
redox centres and the electrode, path (A) [3], the tethering of
redox-active relay units to the protein (on the periphery as
well as inner protein sites) to shorten the electron transfer dis-
tances and to mediate ET between the biocatalytic redox cen-
tres and the electrode, path (B) [4], and to immobilise the
redox enzymes in electroactive polymer matrices, and partic-
ularly, redox-active hydrogels, that transport the electrons
between the enzyme active sites and the electrodes by means
of flexible charge carrying redox-active segments associated
with the polymer matrices, path (C) [5]. Also, the reconstitu-
tion of apo-enzymes on relay/cofactor units associated with
(1) electrodes provided an effective means to electrically contact
redox enzymes with electrodes [6]. According to this para-
digm, Figure 1, path (d), the native cofactor is extracted from
the enzyme, and the reconstitution of the resulting enzyme
on the relay-cofactor dyad linked to the electrode yields a
–
[*] Corresponding author, e-mail: willnea@vms.huji.ac.il
© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 7

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Fig. 1 Schematic electrical contacting of redox enzymes with electrodes: (A) By the application of a diffusional electron transfer mediator. (B) By the
tethering of redox-relay units to the protein associated with the electrode. (C) By the immobilisation of the enzyme in a redox polymer associated with the
electrode. (D) By the reconstitution of an apo-enzyme on a relay-cofactor unit linked to the electrode.
structurally aligned protein on the electrode with the optimal
positioning of the relay unit for electrical contacting of the
enzyme redox centres with the electrode. The development of
the different methods to electrically contact redox enzymes
with electrodes led to tremendous efforts to use these
enzyme-functionalised electrodes as amperometric biosen-
sors. A series of review articles [7], and monographs [8],
described the different methods to immobilise, and electri-
cally contact, redox enzymes with electrode supports, and
addressed the various applications of the enzyme electrodes
as biosensors or bioelectronic devices.
An inter-related, tangential, research field that implements
the paradigms of electrical contacting of redox enzymes with
electrodes includes the development of biofuel cells. The
availability of energy-rich biomass or organic waste sub-
stances suggests that this chemical energy could be trans-
formed into electrical power. Indeed, early efforts have
applied microorganisms as living cells that convert biomass
or waste organic materials into products, such as hydrogen,
methanol or formic acids, and these products were used as
fuel substrates in conventional fuel cells [9–11]. Alternatively,
redox relay units were coupled to the electron transfer chain
operating in whole cells, and the resulting biocatalytically
generated reduced products and oxidised products were
coupled to membrane-bridged compartmentalised cells that
produced electrical power [12–14]. For example, a H2/O2 bio-
fuel cell that operates at ambient temperature and neutral pH
8 © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Willner et al.: Integrated Enzyme-Based Biofuel Cells–A Review
was reported [15]. The reduction of the N,N′-dimethyl-4,4′-
bipyridinium relay by hydrogen, using the hydrogenase
incorporated in Desulfovibrio Vulgaris as a biocatalyst, and the
subsequent transfer of electrons from the reduced relay to the
electrode, provided the fuel substrate for the oxidation pro-
cess in the anodic compartment. The concomitant oxidation
of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diso-
dium salt), ABTS2- to ABTS : through the bilirubin oxidase
four-electron reduction of O2 to water provided the reaction
in the cathodic compartment. The conversion of energy-rich
substrates into electrical power was also accomplished by
using isolated enzymes as catalysts that transform synthetic
redox relays into charge carriers that exchange electrons with
the electrode. For example [16], methanol was transformed to
CO2 by coupled three-enzyme biocatalytic transformations
that included the oxidation of methanol to formaldehyde, in
the presence of alcohol dehydrogenase, AlcDH, the further
oxidation of formaldehyde to formate, with aldehyde dehy-
drogenase, AldDH, and the subsequent oxidation of formate
to CO2, in the presence of formate dehydrogenase, for FDH.
All these three biocatalytic oxidative transformations resulted
in the reduced, 1,4-dihydronicotinamide adenine cofactor,
NADH, that reduced in the presence of Diaphorase, DI, N,N′-
dibenzyl-4,4′-bipyridinium, BV2+, to the respective radical
cation, Figure 2. This set of biocatalytic transformations pro-
vided the NADH fuel for the anodic processes by implement-
ing the mediator BV2+ as a charge carrier to the electrode. The
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Willner et al.: Integrated Enzyme-Based Biofuel Cells–A Review
Fig. 2 A CH3OH/O2, biofuel cell based on the enzymatic cascade consisting of the
NAD+-dependent enzymes, alcohol dehydrogenase (AlcDH), aldehyde dehydrogen-
ase (AldDH), and formate dehydrogenase (FDH). The anodic reaction involves the
diaphorase-mediated oxidation of NADH by BV2+ and the transfer of electrons to the
electrode by BV :.
‡
anode was coupled to a Pt electrode acting as a catalytic cath-
ode for O2 reduction, Figure 2. The advances achieved in the
tailoring of biofuel cells that use diffusional relays as electron
carriers of whole cell reactors, or pure enzyme assemblies
comprising the anodic and cathodic compartments, were
summarised in several excellent recent review articles [17,
18]. The advances in the development of integrated, electri-
cally contacted, enzyme electrodes introduced, however, new
dimensions to biofuel cell technology by providing the vision
to organise implantable, non-compartmentalised, biofuel cell
elements that use body fluids as the fuel source, and the
recent progress in the field was similarly addressed in the
review articles. The aim of this review article is to address the
methods of constructing the integrated anode and cathode
units of the biofuel cell elements. We emphasise the existing
problems in the development of biofuel cells, and discuss the
scientific methodologies that were implemented to resolve
these difficulties. Specifically, the development of methods to
manipulate surfaces with biomolecules at nanoscale preci-
sion, and the tailoring of enzyme-nanoscale objects as hybrid
systems, introduce new possibilities to couple biofuel cells
with the rapidly progressing field of nanobiotechnology. We
attempt to introduce this facet in this review article.
Figure 3 depicts the schematic configuration of an
enzyme-based biofuel cell. The anodic compartment consists
of an electrically contacted enzyme electrode that oxidises the
fuel substrate, while transferring electrons to the electrode.
The cathodic compartment includes an electrically contacted
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REVIEW
enzyme electrode that reduces the oxidiser, while
transferring electrons from the electrode to the oxi-
diser. The flow of electrons through the external cir-
cuit is accompanied by the transport of protons
through the electrolyte. Numerous enzymes, such
as oxidases or dehydrogenases, oxidise organic fuel
substrates. For example, oxidases oxidise sacchar-
ides (e.g. glucose), alcohols, or a-hydroxy acids (e.g.
lactate). Dehydrogenases, such as alcohol dehydro-
genase, lactate dehydrogenase, glucose dehydro-
genase or alanine dehydrogenase, oxidise alcohols,
lactate, glucose or alanine, respectively. Thus, the
different organic substrates may act as fuels in the
presence of the respective electrically wired enzyme
electrodes (anodes). The preferred oxidiser in the
biofuel cell is oxygen that undergoes the four-elec-
tron reduction to water, but H2O2 may also act as
the oxidiser [19]. The electrical power density, P, of
the biofuel cell is given by Eq. 2, where I is the cur-
rent density passing through the external circuit
and V is the voltage across the external resistor. The
maximum voltage extractable from the cell corre-
sponds to the difference in the thermodynamic
redox potentials of the redox enzymes that com-
prise the anode and cathode. The incorporation of
Fig. 3 Schematic configuration of a biofuel cell composed of enzymes, E1
and E2, electrically contacted to the anode and the cathode, respectively.
redox relays to electrically contact the redox centres of the
enzymes with the electrodes introduces, however, potential
drops that decrease the extractable power. Hence, the redox
potentials of the mediators should be as close as possible to
the thermodynamic potentials of the biocatalytic redox cen-
tres. The current generated by the biofuel cells is controlled
by the turnover rates (or ET contacting efficiencies) between
the electrodes and the fuel/oxidiser. The fact that the two
electrodes are coupled and operate cooperatively implies that
one of the electrodes is dominating the passage of currents
through the system (usually the O2-reduction process at the
cathode).
PˆI
V (2)
© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 9

The successful demonstration of a glucose/O2 non-com-
REVIEW
partmentalised biofuel cell [20], and the suggestion to imple-
ment the system as an implantable device that extracts electri-
cal power from body fluids (e.g. blood) [21], sparked the
scientific interest in the development of biofuel cells. Indeed,
tremendous progress was accomplished during the last dec-
ade in the construction of biofuel cell elements. Table 1 sum-
marises the schematic configurations of electrically contacted
enzyme electrodes that were implemented as anodes or cath-
odes for the assembly of integrated biofuel cells. The reconsti-
tution of biocatalysts on relay-cofactor units, the incorpora-
tion of the enzymes in redox polymer matrices, and the use of
nanoscale units (nanoparticles (NPs) or carbon nanotubes)
provide general methodologies to tailor the bioelectrocataly-
tic electrodes. The structure and operating features of the sys-
tems will be addressed in the present review article. Different
studies reported on the assembly of electrically contacted
enzyme electrodes by the direct adsorption of the biocatalyst
on the electrode materials. For example, laccase was electri-
cally wired by its direct immobilisation on graphite electrodes
Table 1 Different available methods for the assembly of biofuel cells: (I) The reconstitu-
tion of enzymes on the anode/cathode, (II) The incorporation of enzymes in redox
polymers associated with the anode/cathode (III) The electrical contacting of biocata-
lysts by nano-elements linked to the anode/cathode.
10 © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Willner et al.: Integrated Enzyme-Based Biofuel Cells–A Review
[22, 23], and biofuel cells were constructed by the use of elec-
trically contacted anodes and cathodes that included directly
immobilised biocatalysts on the electrode surfaces [24]. The
present review will address, however, the chemical tailoring
of nanostructured, electrically contacted enzyme electrodes in
monolayer or thin film configurations and their application in
the development of biofuel cells.
Monolayer-functionalised Enzyme Electrodes for
Biofuel Cells
An electrically contacted glucose oxidising anode was con-
structed by the reconstitution procedure, as outlined in Fig-
ure 4(A) [25]. Pyrroloquinoline quinone, (1), was assembled
on a Au electrode, and the semi-synthetic amino-FAD cofac-
tor (2) was covalently coupled to the PQQ that acted as ET
relay units. The reconstitution of apo-glucose oxidase, apo-
GOx, on the FAD sites resulted in the structurally aligned,
electrically contacted, GOx oxidising electrode. The turnover
rate of electrons between the redox centre of GOx
and the electrode was estimated to be 700 s–1. This
turnover rate is comparable to the rate of electron
transfer between the redox centre of the enzyme
and its native electron acceptor (O2). This property
enabled the activation of the bioelectrocatalysed
oxidation of glucose with no interference of O2 that
was reduced at the cathode. The O2-reduction cath-
ode was constructed by the primary assembly of the
cytochrome c, Cyt. c, hemoprotein cofactor on the
electrode, followed by the crosslinking of the affini-
ty complex generated between Cyt. c and cyto-
chrome oxidase, COx, on the electrode surface, Fig-
ure 4(B) [26]. The power density of the resulting
biofuel cell, Figure 4(C), corresponded to ca.
20 lW cm–2 at an external load of 0.9 kX. The low
power output was attributed to the inefficient ET
turnover rate at the O2-reducing cathode, ca. 20 s–1,
and to the low content of the electrically contacted
biocatalysts on the electrodes in the monolayer con-
figuration [20]. Other configurations of effectively
electrically contacted GOx anodes are depicted in
Figure 5. The linkage of the native flavin adenine
dinucleotide, FAD, to a PQQ monolayer-functiona-
lised electrode, by an aminophenyl boronic acid
bridge (3), followed by the reconstitution of apo-
GOx on the FAD cofactor, yielded an effective bioe-
lectrocatalytic anode for the oxidation of glucose
[27], (ET turnover rate of ca. 700 s–1), Figure 5(A). A
related approach to construct a bioelectrocatalytic
glucose-oxidising anode involved the assembly of a
supramolecular architecture, where the electrical
contacting between the redox site of the enzyme
and the electrode is achieved by the dynamic
shuttling of the electron carrier along a molecular
wire that links the enzyme with the electrode [28],
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Fig. 4 (A) Surface reconstitution of apo-glucose oxidase on a PQQ/FAD monolayer associated with a Au-electrode. Reprinted part with permission from
[25]. (B) Assembly of an integrated bioelectrocatalytic Cyt c/COx-electrode. Reprinted with permission from [26]. (C) Schematic configuration of a non-
compartmentalised biofuel cell employing glucose and O2 as fuel and oxidiser, and using PQQ/FAD/GOx and Cyt c/COx-functionalised Au electrodes
as biocatalytic anode and cathode, respectively. The I–V curve of the cell at various external resistances and the cell power output are depicted on the
right-hand side. Reproduced with permission from [20].

Figure 5(B). A semi-rotaxane supramolecular structure com- between (4) and the bis-aniline site, was stoppered by the
posed of the bipyridinium cyclophane, (4), threaded on a mo- FAD cofactor and was assembled on the electrode. The recon-
lecular wire and stabilised by p-donor–acceptor interactions stitution of apo-GOx on the FAD cofactor site yielded the

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Fig. 5 (A) Assembly of a PQQ/FAD monolayer on a Au electrode via a boronic acid bridge, and the reconstitution of apo-GOx on the FAD cofactor
sites. (B) The reconstitution of apo-glucose oxidase on an FAD cofactor that “stoppers” the cyclophane (4) on the molecular wire. The redox enzyme is
contacted with the electrode by means of the electrochemically shuttled redox unit along the wire.
electrically contacted enzyme, where the dynamic shuttling
of the cyclophane between the enzyme and the electrode, and
the accomanying ET provided the electrical contacting mech-
anism. The bioelectrocatalytic oxidation of glucose by the
functionalised electrode was observed at an onset potential of
–0.4 V versus SCE, close to the thermodynamic redox poten-
tial of the FAD site. The shuttling of the cyclophane from the
donor site to the electrode, and back, is stimulated by the
reduction of the cyclophane by the reduced FADH2 redox site
of the enzyme to the respective bis-radical cation. The
p-acceptor properties of the reduced cyclophane are depleted,
and hence, its dynamic translocation to the negatively biased
electrode is favored, due to the disruption of the stabilising
p-donor–acceptor interactions. The electrochemical oxidation
of the bis-bipyridinium cyclophane to the tetracation, (4),
regenerates the electron acceptor units, and its shuttling to
the p-donor site associated with the wire is favoured by the
supramolecular donor–acceptor interactions.
Electrically contacted enzyme electrodes consisting of
NAD(P)+-dependent dehydrogenases were also prepared by
the reconstitution paradigm [29]. The aminoethyl-NAD+
semi-synthetic cofactor was covalently linked to a pyrroloqui-
noline quinone, PQQ, monolayer. The subsequent formation
of an affinity complex between lactate dehydrogenase, LDH,
and NAD+, followed by the three-dimensional crosslinking of
the enzyme units, Figure 6, generated the electrically con-
tacted enzyme electrode. The bioelectrocatalysed oxidation of
lactate is accompanied by the reduction of NAD+ to NADH,
and the PQQ mediates the catalysed oxidation of NADH and
12 © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Willner et al.: Integrated Enzyme-Based Biofuel Cells–A Review
the transport of the electrons to the electrode [30]. This elec-
trode was coupled to the Cyt. c/COx O2-reduction cathode
(cf. Figure 6) to yield a biofuel cell. This biofuel cell was sug-
gested to act as a self-powered biosensor device [31].
Electrically Contacted Enzyme–Polymer
Composites for Biofuel Cells
The electrical contacting of redox enzymes by their incor-
poration in redox-active Os2+/3+ polypyridine or poly-bi-imi-
dazole complexes was extensively studied in the past two
decades, and different amperometric biosensors were devel-
oped by these “electrically wired” enzymes [5]. The thermo-
dynamic redox potentials of the Os2+/3+ can be tuned within a
wide range through the structures of the ligands. For an opti-
mal power density of the glucose/O2 biofuel cell, the anode
should operate at the lowest possible redox potential close to
that of the FAD redox site of glucose, whereas the biocatalytic
cathode should operate at the most positive potential. A glu-
cose oxidase (GOx) electrically wired enzyme–hydrogel
anode was prepared by the entrapment of GOx in poly[N-
vinylpyridine [Os(N,N′-dialkylated-2,2′-bimidazole)3]2+/3+,
(5) hydrogel, Figure 7(A). The polymer exhibits a rather nega-
tive redox potential of –0.195 V versus Ag/AgCl [32]. The
long alkyl chains tethering the Os2+/3+ complex to the
polymer backbone provide flexibility to the charge carrier
units, and an apparent electron diffusion coefficient of
6 × 10–6 cm2 s–1 was demonstrated in this hydrogel. The
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Fig. 6 Configuration of a self-powered biofuel cell-based biosensor composed of a PQQ-NAD+/LDH-functionalised anode utilising lactate-analyte as a
fuel, and a Cyt. c/COx-functionalised cathode utilising O2 as an oxidiser.

Fig. 7 (A) Structure of the redox polymer that electrically wires glucose oxidase on the anode of the glucose/O2 biofuel cell. The [Os(N,N’-dialkylated-
2,2’’ bi-imidazole)3]2+/3+ acts as an electron transfer mediator that connects the redox centre of the enzyme with the electrode (E = –0.195 V vs. Ag/
AgCl). (B) Polarisation of a 7 mm diameter, 2 cm long glucose electro-oxidising carbon fiber anode, made with the electrocatalyst formed by crosslinking
the electrostatic adduct of glucose oxidase and the “wire” of Figure 7(A). Quiescent solution, under air, 37.5 °C, phosphate buffer, 15 mM glucose,
1 mV s–1 scan rate. Reproduced from [17a] by permission of the Royal Society of Chemistry.

redox potential of the redox hydrogel and its charge transport
properties enabled the extraction of a current density corre-
sponding to 1.1 mA cm–2 at –0.1 V versus Ag/AgCl at 37 °C,
Figure 7(B).
Among the promising O2-reduction biocatalysts, the cop-
per-containing laccase and bilirubin oxidase should be men-
tioned. Laccases are non-specific oxidases that oxidise differ-
ent substrates, such as diphenols or aminophenols, and they
include a four-copper-ion redox-active domain. The ET path

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that leads to the four-electron reduction of O2 is depicted in
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Figure 8 [33]. The reduced state of the copper in the structure
includes a blue copper or T1 charge collection site, and a tri-
nuclear T2/T3 cluster domain, Figure 8. The activation of O2
proceeds in the four-copper ion cluster that yields a peroxo
bridged intermediate that is subsequently reduced by two
electrons to water. The redox potentials of copper oxidases
are dominated by the source of the proteins, and specifically,
the ligand environment of the T1 centre. Additional factors,
such as dipole orientations and hydrogen bonds in the vici-
nity of Cu ions, were, also, found to affect the redox poten-
tials of the copper oxidases [34]. Fungal laccases reveal a high
redox potential of 700 mV close to the thermodynamic redox
potential of O2, and thus, this class of biocatalysts seems to
act as a superior biocatalyst for the cathode of biofuel cells.
The optimal operation of the laccase at acidic pH value (ca.
pH = 5), and its sensitivity to added anions (chloride) are,
however, serious limitations. Bilirubin oxidase, BOD, prefer-
ably from Trachyderma Tsunode, reveals the advantages that it
operates at physiological pH values, pH = 7.2, and it tolerates
relatively high salt concentrations, 0.14 M NaCl [35]. Its redox
potential, ca. 0.45 V versus Ag/AgCl, implies its possible use
as a biocatalyst for the construction of the cathodes of biofuel
cells. Electrical wiring of laccase, Lac, or bilirubin oxidase,
BOD, and the biocatalytic reduction of O2 by these enzymes
was accomplished by using 2,2′-azino-bis(3-ethylbenzothia-
zoline-6-sulfonic acid), ABTS2-, (E = 0.44 V vs. Ag/AgCl) as
diffusional ET mediator [36, 37].
Integrated electrically contacted Lac or BOD O2-reduction
cathodes were prepared by the incorporation of the biocata-
Fig. 8 Molecular mechanism for the 4e–-reduction of O2 to H2O by the four-copper-ion cluster in laccase.
14 © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Willner et al.: Integrated Enzyme-Based Biofuel Cells–A Review
lysts in redox-active hydrogels. Lac was entrapped in a
poly (n-vinylimidazole)-[Os-2,2′,6′,2″-terpyridine-4,4′-dimeth-
yl-2,2′-bipyridine]2+/3+, (6), Figure 9(A) [38]. The onset cathod-
ic current of the biocatalytic integrated electrode was observed
at 0.65 V versus SCE with a maximum current value of
520 lA cm–2 at 0.55 V versus Ag/AgCl, at 37 °C. The extracted
current was limited by the concentration of dissolved O2
(0.2 mM), Figure 9(B). An improved bioelectrocatalytic cath-
ode for the reduction of O2 was constructed by the incorpora-
tion of bilirubin oxidase, BOD (from Trachyderma Tsunodae) in
the hydrogel poly [N-vinylimidazole-acrylamide-[Os-bis-
3,3′,3″,3⵮-tetrachloro-2,2′-bipyridine chloride]2+/3+, (7), Fig-
ure (9C) [39]. The electrocatalytic cathodic current at the wired
BOD electrode saturated at 0.35 V versus Ag/AgCl with a val-
ue of ca. 500 lA cm–2. The GOx wired electrode shown in Fig-
ure 7(A) and the BOD-electrically contacted electrode depicted
in Figure 9(B) were integrated as miniaturised biofuel cell con-
sisting of two 2 cm long, lm diameter, carbon fibers coated
with the respective biocatalysts [40]. The current observed
under the operating voltage of 0.52 V corresponded to 8.3 lA
(in a physiological buffer solution at 37 °C). These values
translate to a power output of 4.3 lW.
The construction of biofuel cells by the redox contacting of
biocatalysts and electrode surfaces has been extended by
applying different other fuel substrates, alternative enzymes
for the assembly of the anodes, and by using modified redox
polymeric hydrogels for the electrical contacting of the
enzymes. For example, glucose oxidase was substituted by
pyrroloquinoline quinone-dependent glucose dehydrogen-
ase, GDH, as the biocatalyst comprising the anode. Upon the
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Fig. 9 (A) Structure of the redox-active hydrogel poly(N-vinylimidazole)– [Os-2,2′,6′,2’’-terpyridine-4,4′-dimethyl-2,2′-bipyridine)2]2+/3+ used for the
electrical wiring of laccase associated with the cathode unit. (B) Polarisation curve corresponding to a 7 mm diameter, 2 cm long, O2-electroreducing
carbon fiber cathode, formed by crosslinking the electrostatic adduct of laccase from Coriolus Hirsutus and the “wire” of Figure 9(A). Quiescent solution,
under air, 37 °C, citrate buffer, 20 mM, pH 5, 1 mV s–1 scan rate. (C) Structure of the “wire” of bilirubin oxidase. The three polymer constituents are ran-
domly distributed. Reproduced from [17a] by permission of the Royal Society of Chemistry.

incorporation of GDH and bilirubin oxidase in the Os2+/3+
polypyridine hydrogels, a glucose/O2 biofuel cell with a
power output of 58 lW cm–2 was constructed [35]. Similarly,
cellobiose dehydrogenase, CDH, catalyses the electrochemi-
cal oxidation of different saccharides, such as glucose, lactose,
and cellobiose. Thus, different fuel substrates may be oxi-
dised at the CDH-modified anode. Upon incorporation of the
CDH in the Os2+/3+-polypyridine hydrogel, and using Pt
black as a cathode, a glucose/O2 biofuel cell operating at phy-
siological conditions with a power output of 157 lW cm–2
was constructed [41]. The tuning of the redox potentials of
the polymer hydrogels enabled the linking of enzymes to tai-
lored electrical contacting matrices. For example, a glucose
oxidase anode was constructed by the incorporation of GOx
into Os2+/3+ (4,4′-diamino-2,2′-bipyridine)poly(N-vinylimida-
zole), E = –0.11 V versus Ag/AgCl, and laccase was wired by
its incorporation in Os2+/3+(phenanthroline)poly(N-vinylimi-
dazole), E = 0.49 V versus Ag/AgCl. The 600 mV difference
in the potential between the GOx anode and the laccase cath-
ode led to a biofuel cell with a power output of 16 lW cm–2
under physiological conditions [42].
Nanotechnology and Biofuel Cells
The unique electronic properties of metal nanoparticles
(NPs) or of nanorods (NRs) or of carbon nanotubes (CNTs)
introduce new opportunities for the electrical contacting of
redox enzymes with electrodes. Indeed, electrical contacting
of redox enzymes, e.g. glucose oxidase [43], or glucose dehy-
drogenase [44], was accomplished by the reconstitution of the
respective apo-enzymes on FAD-modified Au NPs or pyrro-
loquinoline quinone, PQQ, functionalised Au NPs associated
with electrodes, respectively. Also, the reconstitution of apo-

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glucose oxidase on FAD cofactor units covalently linked to
REVIEW
the ends of CNTs associated with electrodes was reported to
yield the electrically contacted enzyme [45]. In all of these
NPs or CNTs/enzyme hybrid systems the bioelectrocatalysed
oxidation of glucose required, however, high overpotentials
that limited the use of the systems as active materials for bio-
fuel cells. The high surface area of the NPs or carbon nano-
tubes, together with their conductivity properties provide
nanometric platforms, to construct on these nanostructure
relay-cofactor cascades that electrically contact the redox sites
of enzymes with the electrodes, and thus, these biocatalytic
hybrid nanostructures may act as composite materials for the
preparation of electrodes for biofuel cell applications. Indeed,
the adsorption of enzymes on single-walled carbon nano-
tubes (SWCNTs) was used to directly electrically contact the
enzymes with the electrodes, and to prepare bioelectrocataly-
tic cathodes and anodes. The adsorption of laccase or biliru-
bin oxidase on CNTs led to effective cathodes for the four-
electron O2 reduction to H2O [46, 47]. Similarly, the hemopro-
tein cellobiose dehydrogenase was adsorbed on the CNTs,
and the resulting electrode was used as an anode for the bio-
catalytic oxidation of lactose [48]. In the presence of a Pt black
cathode, a lactose/O2 biofuel cell with a power output corre-
sponding to 32 lW cm–2 was fabricated.
Polycyclic aromatic compounds bind to CNT by p–p inter-
actions [49]. This feature was used to adsorb the redox active
Nile Blue ET mediator (8) onto CNTs, and to physically
adsorb the Nile Blue-functionalised CNTs onto a glassy car-
bon electrode [50]. Subsequent binding of the NAD+ or
NADP+ cofactors to the Nile Blue units by the carboxyphenyl
boronic acid bridging unit yielded the NAD(P)+ electrocataly-
tic regeneration system, Figure 10(A). That is, biocatalytically
generated NAD(P)H is oxidised by Nile Blue, that acts as an
electrocatalyst for the oxidation of NAD(P)H. The subse-
quently generated affinity complexes formed between the
NAD+ or NADP+ cofactors with alcohol dehydrogenase,
AlcDH, or glucose dehydrogenase, GluDH, respectively, fol-
lowed by the crosslinking of the enzyme units with glutaric
dialdehyde yielded integrated, electrically contacted, enzyme
electrodes [50]. The electrodes were implemented as ampero-
metric biosensors for alcohol and glucose. The electrocatalytic
anodic currents were observed at –0.15 V versus Ag/AgCl,
the redox potential of the Nile Blue mediator, suggesting the
potential use of these electrodes as anodes in a biofuel cell
element.
Also, the bilirubin oxidase was adsorbed on CNTs, and it
exhibited direct electrical contact with the electrode, Fig-
ure 10(B), and two redox waves, for the Cu centres embedded
in the enzyme were observed at 0.19 V and 0.42 V versus
Ag/AgCl [50]. The electrically contacted BOD electrode
biocatalysed the reduction of O2 at 0.5 V versus Ag/AgCl,
with a saturation current density that corresponded to ca.
39 lA cm–2. The CNTs-functionalised anodes consisting of
the wired NADP+/glucose dehydrogenase or the NAD+/
AlcDH matrix (cf. Figure 10(A)) were, then, coupled to the
electrically contacted BOD/CNTs cathode, to yield the inte-
16 © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Willner et al.: Integrated Enzyme-Based Biofuel Cells–A Review
grated biofuel cell, Figure 11(A). The current–voltage curves
and power outputs for the glucose/O2 and ethanol/O2 bio-
fuel cells are shown in Figure 11(B). The maximum power
outputs for the glucose-fueled or ethanol-fueled cells corre-
sponded to 24 and 48 lW cm–2, respectively [50].
Surface modification of pyrolytic graphite electrodes with
molecular promoter units was reported to tightly bind laccase
(from Pycnoporus Cinnabarinus) to the graphitic surface, and
to electrically wire the enzyme towards the electrocatalysed
reduction of O2 [23]. The pyrolytic graphite surface was mod-
ified with the anthracene-2-diazonium salt, and the anthra-
cene units were implemented as promoter units for the elec-
trical activation of laccase. The surface adsorbed laccase
revealed impressive stability and effective electrocatalytic
activities towards the reduction of O2 (ca. 550 lA cm–2 at
0.5 V vs. SHE). Although the precise mechanism for the
electrical contacting of laccase is not understood, this study
introduced an interesting concept for the future design of
modified surfaces that exhibit enhanced bioelectrocatalytic
functions.
Control of the Functions of Biofuel Cells by
External Signals
The control of the bioelectrocatalytic functions of enzyme
electrodes by external signals might enhance the performance
of the biofuel cells, or alternatively, could yield new func-
tions, such as signal-switchable biofuel cells. An external
magnetic field applied in a parallel direction on electrode sur-
faces was found to enhance electron transfer at the electrode–
solution interface. Theoretical studies demonstrated that
upon applying a parallel magnetic field to the electrode sur-
face, the kinetics of ET at the electrode is controlled by hydro-
dynamic convection, rather than by the diffusion, of the sub-
strates undergoing the ET. This physical phenomenon
resulted in a decrease in the diffusion double layer thickness,
and the currents were enhanced upon increasing the strength
of the external magnetic field [51]. These theoretical studies
were implemented to improve the power output of biofuel
cells [52]. The bioelectrocatalysed oxidation of glucose at
the PQQ-FAD glucose oxidase-reconstituted electrode (cf.
Figure 4A) was ca. two-fold enhanced upon the application
of external magnetic field of 0.92 T parallel to the electrode.
The effect of the external magnetic field on the power output
of a lactate/O2 biofuel cell element was demonstrated. An
electrically contacted lactate oxidation biocatalytic anode was
constructed by the integration of the NAD+-dependent lactate
dehydrogenase, LacDH, with the electrode, Figure 12(A). The
electrically contacted LacDH electrode was prepared by the
assembly of a PQQ/NAD+ monolayer on an electrode, fol-
lowed by the crosslinking of the affinity complex generated
between LacDH and the NAD+ cofactor. The cathode of the
biofuel cell consisted of the Cyt. c/COx integrated electrode
for the reduction of O2 (cf. Figure 4B). Figure 12 (B) shows the
effect of an external magnetic field on the cyclic voltammo-
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(A)

Fig. 10 (A) Assembly of the electrically contacted GDH-functionalised electrode by the surface crosslinking of an affinity complex of GDH with Nile Blue-
NADP+ associated with carbon nanotubes. Reproduced with permission from [50]. (B) Schematic structure of a biofuel cell cathode, assembled from a
bilirubin oxidase, BOD, electrically contacted with carbon nanotube-modified electrode.

grams corresponding to the bioelectrocatalysed oxidation of
lactate. The bioelectrocatalysed oxidation of lactate is
enhanced as the applied external magnetic field increases,
Figure 12(C). The power output of the lactate/O2 cell under a
magnetic field of 0.92 T is depicted in Figure 12 (D). A maxi-
mum power output corresponding to ca. 12 lW cm–2 at 1 kX
external resistance is observed under an applied magnetic
field of 0.92 T. This value should be compared to the power
output of ca. 4 lW cm–2, in the absence of an external mag-
netic field.
Electroswitchable and electro-tunable biofuel cells were
prepared by the assembly of electroswitchable conductive/

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 Willner et al.: Integrated Enzyme-Based Biofuel Cells–A Review
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Pcell / µW/cm-2
Pcell / µW/cm-2
Fig. 11 (A) Schematic configuration of a glucose/O2 biofuel cell based on bilirubin oxidase, BOD, and (I) glucose dehydrogenase, GDH, or, (II) alcohol
dehydrogenase, AlcDH, electrically contacted with CNT-modified electrodes. (B) Current–voltage curves (a) and power outputs (b) at different external
resistances for the glucose/O2 biofuel cell that employs (I) GDH, or, (II) AlcDH, and BOD electrically contacted bioelectrocatalytic electrodes. Reproduced
with permission from [50].

insulating films on the anode/cathode units of the biofuel cell
[53]. A polyacrylic acid film was electropolymerised onto Au
electrodes, and Cu2+ ions were incorporated in the polymer
films. The covalent attachment of polyethylene imine, PEI, to
the base Cu2+-polyacrylic acid films provided, then, the
anchoring matrix for the covalent attachment of the biocataly-
tic materials. The linkage of pyrroloquinoline quinone, PQQ,
to the PEI layer, followed by the binding of the amino-FAD
cofactor, (2), to the PQQ sites, and the reconstitution of apo-
glucose oxidase on the FAD cofactor units yielded the electro-
switchable anode, Figure 13(A). Similarly, the O2-reduction
cathode was assembled by the primary covalent attachment
of Cytochrome c, Cyt. c, to the PEI layer associated with the
Cu2+-PAA film, followed by the lateral crosslinking of the cy-
tochrome c/cytochrome oxidase (COx) affinity complex, Fig-
ure 13(B). Upon applying a bias potential that corresponded
to –0.5 V versus SCE, the Cu2+ ions in the PAA films were
reduced to Cu° metallic nanoclusters, resulting in the electri-
cal contacting of the biocatalysts associated with the anode
and cathode. Figure 14(A) and (B) show the open-circuit
potential of the biofuel cell in the presence of variable concen-
trations of glucose, indicating that the biocatalysts electrically
communicate with the electrodes. Upon the application of a
bias potential of +0.5 V versus SCE, the Cu° clusters were
reoxidised to the Cu2+ state, a process that transformed the
PAA matrices on the two electrodes into insulating films. This
blocked the electrical communication between the biocata-
lysts and the electrodes, resulting in the switching off of the
biofuel functions, Figure 14(C). The electro-switchable func-
tions of the biofuel cell were reversible, and upon the reduc-
tion of Cu2+ ions to Cu° clusters, the operation of the biofuel
was reactivated. The functions of the biofuel cell were found
to be controlled by the conductivity of the PAA film that was
activated by the electrochemically generated Cu° nanoclus-
ters. As the time interval for the reduction of the Cu2+ ions
was prolonged, the content of Cu° clusters in the PAA film
increased, resulting in enhanced electrical contacting of the
biofuel cell elements [53]. Figure 15(A) and (B) show the I–V
curves and the power outputs of the biofuel cell that includes
anode/cathode units with variable contents of Cu° clusters.

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Fig. 12 (A) The assembly of an integrated LDH monolayer-electrode by the crosslinking of an affinity complex formed between LDH and a PQQ/NAD+
monolayer-functionalised Au-electrode. (B) Cyclic voltammograms of the integrated crosslinked PQQ/NAD+/LDH electrode: (a) in the absence of lactate
and in the absence of magnetic field, (b) in the presence of lactate, 20 mM, and in the absence of magnetic field, (c) in the presence of lactate, 20 mM,
and in the presence of magnetic field, B = 0.92 T. (C) The dependence of the electrocatalytic current density on the magnetic flux density at lactate con-
centration of 20 mM. (D) The power density output generated by the biofuel cell: (a) in the absence of magnetic field, (b) in the presence of magnetic field,
B = 0.92 T. The biofuel cell operated upon pumping of a solution (flow rate 1 mL min–1) composed of 0.1 M TRIS-buffer, pH 7.0, containing CaCl2,
10 mM, lactate, 20 mM, and oxygen (the solution was equilibrated with air). Reprinted with permission from [52].

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 Willner et al.: Integrated Enzyme-Based Biofuel Cells–A Review
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Fig. 13 (A) Preparation and operation of the biocatalytic anode. Stepwise covalent binding of PQQ and N6-(2-aminoethyl)-flavin adenin dinucleotide to
the polymer-functionalised electrode followed by the reconstitution of apo-glucose oxidase, and reversible activation and deactivation of the biocatalytic
anode by the electrochemical reduction of the Cu2+-polymer film and the oxidation of the Cu0-polymer film, respectively. (B) Synthesis of a biocatalytic
cytochrome c (Cyt. c) modified electrode and the reversible activation and deactivation of the biocatalytic cathode by the electrochemical reduction of the
Cu2+-polymer film and the oxidation of the Cu0-polymer film, respectively.

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Fig. 14 Open-circuit voltage (Voc) at a variable concentrations of glucose injected into the biofuel cell device: (A) After the anode and cathode of the bio-
fuel cell were activated by the application of the potential corresponding to –0.5 V for 1,000 s. (B) Calibration plots of the glucose sensing when the bio-
fuel cell is activated (a) and deactivated (b). In all measurements, the glucose solution was equilibrated with air. (C) After the anode and cathode of the
biofuel cell were deactivated by the application of the potential of 0.5 V for 5 s. Arrows show the injection of glucose with the concentrations of:
(a) 2 mM, (b) 3 mM, (c) 8 mM, (d) 40 mM. The glucose samples of variable concentrations (50 lL) in 0.1 M TRIS-buffer, pH 7.0, were injected into the
flow stream (flow rate of 1 mL min–1). Reprinted with permission from [53].

Fig. 15 (A) Current density-voltage behaviour of the biofuel cell at different external load resistances. (B) Electrical power density extracted from the bio-
fuel cell at different external load resistances. Curves a–e show the biofuel cell output functions after the reductive potential of –0.5 V was applied to the
biocatalytic electrodes for different time intervals: (a) 200 s, (b) 400 s, (c) 600 s, (d) 800 s, and (e) 1,000 s. The measurements were performed in the
presence of 80 mM glucose solution saturated with air. Reprinted with permission from [53].

As the Cu° content increased, the electrical contacting of the
biocatalysts and the electrodes improved, resulting in higher
power output. The external triggering of biofuel cells might
be of specific value for the switchable activation of implanted
biofuel cells that use ingredients of body fluids (e.g. glucose
in blood) as fuels.
Conclusions and Perspectives
Since the first integrated enzyme-based biofuel cell was
demonstrated [20], substantial progress in the design and ap-
plications of biofuel cells was accomplished. While the origi-
nal integrated biofuel cells included electrically contacted bio-
catalysts in monolayer configurations that resulted in limited
electrical power, the second and third generations of biofuel
cells included the entrapment of the enzymes in redox-active
polymer hydrogels, or the immobilisation of the enzymes
on nano-objects, such as carbon nanotubes or NPs. These
approaches led to higher contents of the biocatalysts con-
tacted with the electrodes, and to enhanced values of the
power output. The power output of a miniaturised biofuel
cell (0.26 mm2 footprint of electrodes and reaction volume of

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0.0026 mm3) was reported to generate in a physiological, glu-
REVIEW
cose-enriched buffer solution (pH = 7.2, 0.14 M NaCl, 20 mM
phosphate, 30 mM glucose at 37 °C) a power of 4.4 lW under
continuous operation for six days [39, 40]. This power is ca.
1,000-fold higher than that of a zinc-air battery, and hence,
the use of the biofuel cell as an implantable battery seems fea-
sible. In fact, the glucose concentration and O2 content in tis-
sues should suffice to power miniaturised pumps, hearing
aids or heart pacemakers. Furthermore, the demonstration
that the biofuel cells may act as sensor devices powered by
human body fluids suggests that such miniaturised devices
could act as autonomous biosensor-transmitter devices that
transduce the local glucose concentrations for diabetes man-
agement, or alert the development of infections at wound sur-
geries.
The use of biofuel cells as electrical power resource has
substantial environmental advantages. Biofuel cells meet
nicely the “Green Chemistry” trend by providing a clean
technology for generating electrical power from non-toxic
natural materials, e.g. glucose. Indeed, commercial green bat-
teries are already in the market for powering computers or
cellular phones [54].
Albeit the impressive progress in the development of bio-
fuel cells, challenging issues, need to be resolved for their
abundant use as alternative electric power sources. Most of
the integrated biofuel cells use glucose as the fuel substrate.
The energy content in other organic substrates, such as alco-
hols or a-hydroxy acids is, however, substantially higher,
and their use as fuel substrates is certainly advantageous.
Enzymes for the oxidation of these substrates exist, e.g. alco-
hol oxidase, alcohol dehydrogenase, lactate oxidase or lactate
dehydrogenase. Thus, the assembly of such biofuel cells
seems feasible. The major challenge seems to be, however, the
increase of the power output of biofuel cells.
The increase in the power output can be achieved by sev-
eral complementary pathways that include: (i) The increase
of enzyme loadings on the electrodes. This will require devel-
oping matrices that effectively electrically contact densely
loaded biocatalysts with the electrodes. (ii) To enhance the
turnover rates between the biocatalysts and the electrodes.
This goal requires the parallel improvement of the anode and
cathode performances. While anodes with high turnover rates
are available, the electrical contacting of the O2 reduction bio-
catalysts is still inefficient. With the advances in the electrical
contacting of redox proteins by means of nanomaterials, such
as metallic NPs [43, 44], or carbon nanotubes [45], one may
envisage that the integration of the O2-reduction biocatalysts
with such nano-objects could improve the performances of
the cathodes. Alternatively, the genetic engineering of new
O2-reduction biocatalytic mutants could facilitate the electron
transfer between the copper oxidases and the electrodes. (iii)
To electrically contact the biocatalysts associated with the
anodes and cathodes at redox potentials close to the thermo-
dynamic redox potential of the enzymes. This strategy has
been already implemented successfully in designing different
enzyme electrodes. Several of these methods have, however,
22 © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Willner et al.: Integrated Enzyme-Based Biofuel Cells–A Review
only a fundamental scientific value due to the complexity in
assembling the systems, and difficulties in implementing the
principles for highly loaded enzyme matrices.
The efforts to control the biofuel cell performances by
external signals, such as an external magnetic field, or to tune
the power output by electrochemical means reveal new
venues to regulate the functions of biofuel cells.
Besides the use of biofuel cells as an electrical energy gen-
erator, other applications of these devices are desirable. For
example, the use of biofuel cells as transducers of logic gate
operations was recently suggested [55]. A new dimension in
biofuel cell technology could be, however, the design of
photo-biofuel cells, and particularly, solar-driven biofuel cells
[56]. Such systems are anticipated to yield electrical power
with the concomitant generation of a fuel product at the cath-
ode. This product could, then, act as a fuel substrate for regu-
lar biofuel cells. The photonic electrical wiring of enzymes
seems a challenging topic for the development of such biofuel
cells. In view of the recent advances in developing biofuel
cells, the future of this research topic seems to be bright. Joint
efforts of chemists, biologists, materials scientists and electri-
cal engineers are anticipated to resolve the challenging goals
existing ahead of us.
Acknowledgement
Our research on biofuel cells is supported by the BioMed-
Nano EC project.
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