Biofuel cells based on direct enzyme–electrode contacts using PQQ

Biosensors and Bioelectronics 61 (2014) 631–638
Contents lists available at ScienceDirect
Biosensors and Bioelectronics
journal homepage: www.elsevier.com/locate/bios
Biofuel cells based on direct enzyme–electrode contacts using
PQQ-dependent glucose dehydrogenase/bilirubin oxidase and
modiﬁed carbon nanotube materials
V. Scherbahn a, M.T. Putze a, B. Dietzel b, T. Heinlein c, J.J. Schneider c, F. Lisdat a,n
a
Biosystems Technology, Technical University of Applied Sciences, 15745 Wildau, Germany
Institute for Thin Film and Microsensoric Technology, 14513 Teltow, Germany
c
Technical University Darmstadt, Eduard-Zintl-Institute for Inorganic and Physical Chemistry, 64287 Darmstadt, Germany
b
art ic l e i nf o
a b s t r a c t
Article history:
Received 26 February 2014
Received in revised form
6 May 2014
Accepted 10 May 2014
Available online 3 June 2014
Two types of carbon nanotube electrodes (1) buckypaper (BP) and (2) vertically aligned carbon
nanotubes (vaCNT) have been used for elaboration of glucose/O2 enzymatic fuel cells exploiting direct
electron transfer. For the anode pyrroloquinoline quinone dependent glucose dehydrogenase ((PQQ)
GDH) has been immobilized on [poly(3-aminobenzoic acid-co-2-methoxyaniline-5-sulfonic acid),
PABMSA]-modiﬁed electrodes. For the cathode bilirubin oxidase (BOD) has been immobilized on PQQmodiﬁed electrodes. PABMSA and PQQ act as promoter for enzyme bioelectrocatalysis. The voltammetric
characterization of each electrode shows current densities in the range of 0.7–1.3 mA/cm2.
The BP-based fuel cell exhibits maximal power density of about 107 mW/cm2 (at 490 mV). The vaCNTbased fuel cell achieves a maximal power density of 122 mW/cm2 (at 540 mV). Even after three days and
several runs of load a power density over 110 mW/cm2 is retained with the second system (10 mM
glucose). Due to a better power exhibition and an enhanced stability of the vaCNT-based fuel cells they
have been studied in human serum samples and a maximal power density of 41 mW/cm2 (390 mV) can
be achieved.
& 2014 Elsevier B.V. All rights reserved.
Keywords:
Enzymatic fuel cell
PQQ-dependent glucose dehydrogenase
Bilirubin oxidase
Buckypaper
Vertically aligned carbon nanotubes
1. Introduction
During the last decade enzymatic biofuel cells (EBFCs) have
become an interesting research topic particularly with respect to
their sustainability and potential application as power supply for
portable, implantable devices in medicine and biosensor systems
(Neto et al., 2010). Their stability, the generated power and the cell
voltage depend to a large extent on the choice of the enzymes for
anode and cathode reaction but also on the applied electrode
architecture. For the cathode multicopper enzymes such as bilirubin oxidase (BOD) (Brocato et al., 2012), laccases (Karaśkiewicz et
al., 2012) and ascorbate oxidases (Falk et al., 2012) are suitable
biocatalysts. For bioanodes, the application of different oxidizing
enzymes allows to harvest the energy out of diverse biofuels e.g.
glucose, fructose, cellobiose, alcohol or hydrogen. Thus, enzymes
such as glucose oxidase, nicotinamide adenine dinucleotide and
pyrroloquinoline quinone dependent glucose dehydrogenase, fructose dehydrogenase, cellobiose dehydrogenase and hydrogenases
n
Corresponding author. Tel.: +49 3375508456; fax: +49 3375508458.
E-mail address: ﬂ[email protected] (F. Lisdat).
http://dx.doi.org/10.1016/j.bios.2014.05.027
0956-5663/& 2014 Elsevier B.V. All rights reserved.
are suited for this purpose (Osman et al., 2011; Barton, 2010;
Lojou, 2011).
An efﬁcient electrical communication between the enzyme and
the electrode can be achieved via direct electron transfer (DET).
It allows a current ﬂow at potentials near the E1 of the redox center
of the bound enzyme and avoids side reactions. Alternatively the
addition of shuttle molecules results in a mediated electron transfer
(MET) that may enhance the maximum rate of enzyme–electrode
electron transfer, compared to DET. Here the enzymes do not need
to contact the electrode surface directly (Barton, 2010), but the
redox potential of the mediator inﬂuences the cell potential and
problems with leakage can occur in the case of soluble mediator or
problems in enzyme accessibility for the substrate in the case of
polymer-bound mediators.
The approach to develop a membrane-less EBFC requires a
complete insensitivity toward oxygen during the anodic reaction
of the fuel since oxygen is mostly used as electron acceptor on the
cathode. Hence, the choice of a suitable enzyme and strategies
for an effective competition with oxygen are important. For this
purpose, (PQQ)GDH is an interesting enzyme because it can be
produced in a recombinant way; it has a high catalytic activity at
physiological pH and is oxygen insensitive (Durand et al., 2010).
Several studies report different strategies for functional (PQQ)
632
V. Scherbahn et al. / Biosensors and Bioelectronics 61 (2014) 631–638
GDH–electrode contacts. Often this communication occurs via
mediators e.g. ferrocene derivatives (Razumiene and Meškys,
2000), PQQ (Tanne et al., 2010), cytochrome c (Wettstein et al.,
2012) or osmium containing polymers (Laurinavicius et al., 2002).
DET between the enzyme and the electrode surface has also been
achieved by using self-assembling monolayers on gold with a
covalent enzyme attachment (Sang et al., 2012), by carbon black
modiﬁed carbon paste (Razumiene et al., 2006), aniline derivatives
modiﬁed carbon nanotubes (Schubart et al., 2012), activated
buckypaper with covalently attached (PQQ)GDH (Halamkova et
al., 2012), attaching the enzyme on a carbon cryogel electrode
(Flexer et al., 2011) or titan oxycarbide nanostructures (Sarauli et
al., 2012).
In terms of biocathode, BOD is a favorable enzyme for oxygen
reduction. It has the advantage of being stable at neutral pH, possesses
a high activity and its reduction process starts at potentials 0.5 V vs.
Ag/AgCl (Göbel and Lisdat, 2008). DET between BOD and electrodes
has been achieved at modiﬁed carbon- (Suraniti et al., 2012; Nogala et
al., 2010), gold- (Christenson et al., 2006; Ramírez et al., 2008) and
CNT-electrodes (Ivnitski et al., 2008). Oxygen supply during the reduction reaction has been recognized as a limiting factor for the power
output, thus the development of gas-diffusion cathodes based on
carbon-black modiﬁed carbon toray paper has been shown to avoid
this problem (Gupta et al., 2011; Miyake et al., 2013).
Despite the constant improvement of the EBFCs, their power
output and lifetime are still not optimal for direct and long-time
applications. Nevertheless, ﬁrst implantable EBFCs have been reported
(Halamkova et al., 2012; Cinquin et al., 2010). Besides the choice of the
biocatalysts, suitable electrode materials with a high surface area are
important for improving the EBFC performance. Due to their excellent
electrocatalytic activity and an immense surface area several types
of CNT-architectures have been used for developing bioelectrodes such
as immobilized CNTs on carbon (Ivnitski et al., 2008; Kim and Parkey,
2009) or gold surfaces (Schubert et al., 2009), and vertically aligned
CNTs (Javier et al., 2008; Liu et al., 2009) or buckypaper
(Ahmadalinezhad et al., 2011). In order to increase the bio-compatibility of the rather hydrophobic CNT-surface different pretreatments
can be applied – sonication- (Schubert et al., 2009), acid- (Khabazian
and Sanjabi, 2011), plasma- (Lee and Chang, 2009), electrochemical or
chemical pretreatment (Musameh et al., 2005). Because of their dual
advantages of productive binding of enzymes in an active form and
allowing the electron transport towards the electrode different conducting and biocompatible polymers such as modiﬁed polyaniline- (Li
et al., 2013), polythiophene- (Liu et al., 2010; Hsieh et al., 2009) and
polypyrrole-derivatives (Li et al., 2012) can be combined with CNTs.
The aim of the present study is to develop glucose/O2 EBFCs based
on two different CNT-architectures – (1) buckypaper (BP) and
(2) vertically aligned carbon nanotubes (vaCNT). In order to promote
the direct contact between the enzyme and the electrode surface both
the anode and the cathode have been separately evaluated. At the
anodic side (PQQ)GDH is immobilized at polymer modiﬁed CNTelectrodes. At the cathodic side BOD has been used as the biocatalyst.
The performance of the developed EBFCs has been characterized in
artiﬁcial and physiological solutions (human serum).
gift. The enzyme is recombinantly expressed in Escherichia coli. Poly(3aminobenzoic acid-co-2-methoxyaniline-5-sulfonic acid) – PABM
SA – has been synthesized by chemical oxidative polymerization
according to the procedure described herein (Sarauli et al., 2013).
N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), D-glucose and bilirubon oxidase from Myrothecium
verrucaria (BOD) are purchased from Sigma-Aldrich Chemie GmbH
(Germany). 2-(N-morpholino)ethansulfonic acid (MES) is from ApliChem GmbH (Germany). Calcium chloride (CaCl2) and citric acid are
received from Carl Roth GmbHþ Co. KG (Germany). For all aqueous
solutions 18 MΩ deionized water (Eschborn, Germany) is used.
The vertically aligned vaCNT@Si-electrodes (vaCNT@Si) have
been prepared by a water assisted chemical vapor deposition
(CVD) method (Joshi et al., 2010; Joshi and Schneider 2012). A
silicon substrate (Sb doped, 〈100〉, 0.01–0.02 Ω/cm, from Silicon
Materials), size 5 mm 15 mm is covered with a mesh containing
cavities of 370 mm 370 mm in size. Deposition of 11.6 nm Al and
1.4 nm Fe by e-beam evaporation is followed and the mask is
removed. CVD synthesis has been started by heating the substrate
to 850 1C in a gas mixture of argon and hydrogen (40% hydrogen)
in a quartz tube with inner diameter 85 mm. The patterned
structure of vaCNTs is obtained by introducing a ﬂow of ethine
(200 sccm) as carbon source for 15 min. Microscopic and spectroscopic investigations are performed using SEM (XL 30 FEG,
Philips), TEM (CM20, Philips) and Raman (LabRamHR8000, Horiba) – see Fig. 1.
2. Experimental
2.2.2. Cathodes
The BP- and vaCNT@Si-electrodes have been incubated with a PQQ
solution (1 mM or 2.73 mM) in 100 mM citrate phosphate buffer (CiP),
pH 7 for 1 h. Then the electrodes have been washed three times with
the same buffer. For BOD adsorption on the surface the electrodes are
placed in enzyme solution (10 mM) for 1 h. In order to ﬁx the enzyme
covalently, the PQQ-modiﬁed electrodes have been placed in EDC/
NHS-solution (100 mM/25 mM) in 100 mM CiP for 15–20 min and
washed three times with the same buffer before enzyme incubation.
The BOD/PQQ-electrodes are stored in 100 mM CiP, pH 7, 4 1C.
2.1. Materials
Buckypaper (BP) has been obtained from Buckeye Composites
(USA). Human serum samples containing 3–4 mM glucose has been
received from LIMETEC Biotechnologies GmbH, Germany as a kind
gift. Pyrroloquinoline quinine (PQQ) is purchased from Wako Pure
Chemical Industries. Soluble GDH (Acinetobacter calcoaceticus) is
provided as an apo-enzyme by Roche Diagnostics GmbH as a kind
2.2. Enzyme electrode preparation
(PQQ)GDH is reconstituted by dissolving 2 mg/ml (20 mM) of
apoGDH in 5 mM MES þ1 mM CaCl2, pH 6.5. Next PQQ has been
added with a molar ratio of 1 (PQQ/apoGDH). The solution has
been incubated for 3 h at room temperature in the dark. The
resulting enzyme solution is stored at 4 1C before use. 1 mg BOD
has been dissolved in 1 ml citrate phosphate buffer (100 mM CiP,
pH 7) and stored as 30 ml aliquots at 20 1C before use (concentration amounts to 20 mM).
For the electrode preparation the BP material has been cut into
rectangular pieces. The approximate surface of the electrode
immobilized with enzyme is 0.05–0.11 cm2.
2.2.1. Anodes
The BP- and vaCNT@Si-electrodes have been ﬁrst incubated
with PABMSA solution of different concentrations (0.1, 1, 2, 3 or
5 mg/ml) in 5 mM MES þ1 mM CaCl2, pH 6.5 for one hour. After
this the electrodes are washed three times with the same buffer.
For (PQQ)GDH adsorption the electrodes have been placed in
enzyme solution for 1 h. In order to ﬁx the enzyme covalently,
the PABMSA-modiﬁed electrodes are placed in an EDC/NHS-solution (100 mM/25 mM) in 5 mM MES þ1 mM CaCl2, pH 6.5 for 15–
20 min and after that washed 3 with the same buffer before
enzyme incubation. The (PQQ)GDH/PABMSA-electrodes are stored
in 5 mM MES þ1 mM CaCl2, pH 6.5 at 4 1C.
V. Scherbahn et al. / Biosensors and Bioelectronics 61 (2014) 631–638
2.3. Electrochemical measurements
The voltammetric experiments are performed using the potentiostat PGSTAT 12 (Metrohm-Autolab, Netherlands). A three electrode 1 ml home-made chemical cell is used with a Pt wire as
counter electrode, an Ag/AgCl (1 M KCl) electrode as reference
electrode and the modiﬁed BP- and vaCNT@Si-electrodes as working electrodes. For studying the anodes buffer solutions (5 mM
MES þ1 mM CaCl2 pH 6.5 and 100 mM CiP þ1 mM CaCl2 pH 7) in
absence and in presence of glucose have been used as electrolyte.
For cathode characterization 100 mM CiP buffer, pH 7, (Ar- and airsaturated) has been used. For all bioelectrocatalytic measurements
a scan rate of 10 mV/s is applied.
The biofuel cells are characterized by performing galvanodynamic
measurements using the potentiostat Reference 600 (Gamry Instruments, USA). Since no difference in power output could be seen at
scan rates 2 nA/s and 3 nA/s, the scan rate of 3 nA/s has been applied
resulting in a test period of about 2 h for each measurement. For the
serum measurements 1 ml serum has been placed in a home-made
electrochemical cell and then the prepared electrodes have been
inserted and characterized accordingly.
3. Results and discussion
In the present work we design protein electrodes based on DET for
the application in biofuel cells by using two types of CNT-architectures
and (PQQ)GDH as glucose converting enzyme and BOD as O2-reducing
enzyme. We use here (a) buckypaper and (b) vertically aligned CNT
(Fig. 1) as interface for the immobilization of redox enzymes both for
anodes and cathodes. The vaCNT arrays are grown by a water assisted
chemical vapor process which ensures a high quality of mostly double
walled CNTs. Their structural and spectroscopic characterization gives
633
a main diameter of 6–10 nm containing only a minor amount of
multiwalled CNTs with more graphitic shells. The ID/IG ratio is 0.7 as
obtained by Raman spectroscopy (see Fig. 1).
In order to improve the biocompatibility of the CNT-structure two
types of organic interlayers have been introduced – (1) an aniline-type
polymer (PABMSA, Scheme 1a) for the anodes and (2) a 3-ring
aromatic compound pyrroloquinoline quinone (PQQ, Scheme 1b) for
the cathodes. Furthermore, parameters such as glucose sensitivity, the
inﬂuence of the polymer concentration and different buffer systems
have been investigated in order to elucidate suitable conditions for a
maximal output of enzymatic biofuel cell (EBFC) operation. Finally, the
stability of the EBFCs has been investigated in buffer and in
human serum.
3.1. Buckypaper-based anode
In order to couple PQQ-GDH to the electrode we have used
buckypaper (BP) – a material of high surface area and composed of
multiwalled carbon nanotubes. In addition it has been shown to
interact productively with several redox enzymes (Ahmadalinezhad et
al., 2011; Hussein et al., 2011). Thus, we ﬁrst tried to adsorb (PQQ)GDH
onto an untreated BP-electrode. In the presence of glucose a small
catalytic current can be detected by linear sweep voltammetry (LSV).
At 0.1 V vs. Ag/AgCl a current density of 3–4 mA/cm2 (10 mM glucose)
is obtained. These measurements show that a direct interaction of the
enzyme with the CNT-material is feasible, but efﬁciency of bioelectrocatalysis is very low.
In order to improve the surface properties of the BP for immobilization and DET an aniline-based polymer ﬁlm (PABMSA, see
Scheme 1a) has been adsorbed on the electrode before enzyme
ﬁxation. This idea is based on previous studies showing that
Fig. 1. (a) Photographical image of two vaCNT/Si chips (size 5 mm 1.5 mm); (b) scanning electron micrograph (SEM) of the block-patterned CNT structures;
(c) transmission electron micrograph shows double-walled CNTs with a diameter of 6–10 nm.; (d) Raman spectra of the block-patterned carbon nanotubes.
634
V. Scherbahn et al. / Biosensors and Bioelectronics 61 (2014) 631–638
can be detected (current densities of 0.75 mA/cm2 in presence of
10 mM glucose). The higher ionic strength and higher buffer capacity
of the CiP buffer seem to be beneﬁcial for an efﬁcient glucose
conversion at the (PQQ)GDH/PABMSA/BP-electrode. It can be stated
that mainly the high ionic strength is responsible for the higher
current output since application of a 50 mM MES buffer also results in
higher current values compared to the measurements in 5 mM MES
buffer. Obviously the enzyme electrode interaction is improved and
the system can follow the catalytic ability of the enzyme at higher
glucose concentrations probably also due to the higher buffer capacity
leading to less ﬂuctuation in local pH near the electrode.
Besides the ability to increase active amount of the enzyme on the
electrode, the PABMSA ﬁlm bears carboxylic acid groups within its
structure. In order to increase the stability (PQQ)GDH can be bound
covalently by the EDC/NHS chemistry on the polymer ﬁlm. By means of
LSV a rather similar behavior was found with slightly lower maximal
current densities. This approach is also used in the EBFC measurements.
3.2. vaCNT-based anode
Scheme 1. Chemical structure of (a) poly(3-aminobenzoic acid-co-2-methoxyaniline-5-sulfonic acid) – PABMSA and (b) pyrroloquinoline quinone – PQQ.
sulfonated polyaniline ﬁlms can improve the interaction with (PQQ)
GDH (Göbel et al., 2011).
LSV and cyclic voltammetry (CV) are performed in order to study
whether it is possible to achieve enhanced catalysis in presence of
glucose by adsorbed (PQQ)GDH on the PBMSA-modiﬁed BP-electrode.
In Fig. 2a cyclic voltammograms of PABMSA/BP-electrodes without
and with adsorbed (PQQ)GDH in absence and in presence of glucose
are depicted. It is evident that no glucose conversion occurs at
PABMSA/BP-electrodes. When (PQQ)GDH is adsorbed on the PABM
SA/BP-electrodes an oxidation current in the presence of glucose starts
from about 0.1 V vs. Ag/AgCl and thus indicates efﬁcient sugar
conversion. Since no additional mediator is added a DET from the
sugar reduced enzyme to the polymer-modiﬁed electrode at the
enzymes redox potential can be concluded. The polymer modiﬁcation
of the CNT surface obviously increases the amount of productively
immobilized enzyme on the electrode.
Furthermore, current densities about 400 mA/cm2 at 0.1 V vs. Ag/
AgCl can be achieved which are reasonably high (for the calculation
the geometrical area in contact with the solution has been used).
Thus, it is evident that the PABMSA-ﬁlm improves the biocompatibility and allows a much higher enzyme activity after its immobilization in comparison with unmodiﬁed BP-electrodes.
In a next step we investigate whether different PABMSA
concentrations during the BP modiﬁcation will inﬂuence the
bioelectrocatalysis of the immobilized enzyme in the presence of
substrate. Fig. 2b depicts current densities at 0.1 V vs. Ag/AgCl
achieved at (PQQ)GDH/PABMSA/BP-electrodes prepared with different PABMSA-concentrations in dependence on different glucose
concentrations. Obviously the polymer concentration inﬂuences
the efﬁciency of the glucose conversion and applying 5 mg/ml of
PABMSA for the CNT modiﬁcation maximal current can be
obtained for a given glucose concentration.
It is also studied how the buffer composition inﬂuences the current
output. Ca2 þ -ions are a signiﬁcant parameter for the stabilization of
the enzyme and thus are always added to the buffer. Two buffer
systems with different buffer capacities have been tested with respect
to the performance of the anode – 100 mM citrate-phosphate (CiP)
and 5 mM 2-(N-morpholino)ethanesulfonic acid buffer (MES). Fig. 2c
illustrates the response in presence of different glucose concentrations
in MES and CiP buffer solutions at 0.1 V vs. Ag/AgCl. It is evident that
using CiP higher current densities at higher glucose concentrations
Previous studies reporting about vertically aligned CNTs
(vaCNT) as suitable electrode surfaces for electrochemical applications (Javier et al., 2008; Liu et al., 2009) give the background for
our studies. vaCNTs have been prepared in an array format with
individual spots on n-doped silicon (see Section 2.1). Analogously
to experiments with BP, we ﬁrst adsorb the (PQQ)GDH onto
untreated vaCNT-electrodes. In the presence of 10 mM glucose
current densities in the range of 5 mA/cm2 can be detected at 0.1 V
vs. Ag/AgCl by means of LSV with such an electrode (5 mM MES
pH 6.5, 1 mM CaCl2). These experiments show that the enzyme
interacts directly with the vaCNTs, but the current output is
insufﬁcient for an application in an EBFC.
In order to establish an efﬁcient anode system based on vaCNTs
the surface is modiﬁed in a way, which has been found optimal
during the development of the BP-based anode, namely a PABMSA
concentration of 5 mg/ml for electrode modiﬁcation. Thus,
vaCNT@Si-electrodes have been incubated in the copolymer solution and then (PQQ)GDH has been immobilized by adsorption. Subsequently the bioelectrocatalytic behavior is analyzed.
Fig. 2d depicts CVs of a (PQQ)GDH/PABMSA/vaCNT@Si-electrode in
absence and in presence of glucose using the MES buffer system. It
is evident that a catalytic current in the range of about 590 mA/cm2
can be obtained. This value is about 1.5 times higher in comparison
to the BP-electrode at similar potentials. Communication between
the enzyme and the electrode occurs here in an analogous way
via DET.
Evaluating the glucose sensitivity using CiP buffer by means of LSV
a deﬁned dependence on the enzyme substrate concentration and
higher current values can be found. The results are presented in Fig.
2e. Maximum current densities in the range of about 1.370.18 mA/
cm2 (at 0.1 V vs. Ag/AgCl, n¼3) can be generated in presence of 6 mM
glucose (the geometrical area is used for the calculation as sum of the
individual CNT-squares).
3.3. Buckypaper-based cathode
In order to construct BOD-cathodes three approaches have been
followed – (1) adsorption of BOD on untreated buckypaper (BP),
(2) adsorption of PQQ on BP before BOD adsorption and (3) adsorption
of PQQ on BP before covalent ﬁxation of BOD via EDC/NHS-chemistry.
The idea of applying PQQ as CNT modiﬁer is based on earlier
observations that PQQ can work as promoter for BOD bioelectrocatalysis (Göbel and Lisdat, 2008). The PQQ modiﬁcation of the electrode
has been performed with two different PQQ concentrations – 1 mM
and 2.73 mM. The results of the bioelectrocatalytic reduction of O2 are
depicted in Fig. 3a. Reduction currents can be detected in air-saturated
V. Scherbahn et al. / Biosensors and Bioelectronics 61 (2014) 631–638
635
Fig. 2. Cyclic voltammograms of (a) buckypaper/PABMSA-electrodes: (1) 0 mM glucose, (2) 10 mM glucose, (3) with adsorbed (PQQ)GDH and 0 mM glucose, and (4) with
adsorbed (PQQ)GDH and 10 mM glucose. (b) Voltammetric current responses of (PQQ)GDH/PABMSA/buckypaper-electrodes in presence of different glucose concentrations
at 0.1 V vs. Ag/AgCl, 1 M KCl. Current density changes in dependence on different PABMSA-concentrations used for preparation of the electrodes: (1) 0,2 mg/ml; (2) 1 mg/ml;
(3) 2 mg/ml; (4) 3 mg/ml; (5) 5 mg/ml. Buffer conditions: 5 mM MES þ1 mM CaCl2 pH 6.5, scan rate 10 mV/s. (c) Current density changes in dependence on different buffer
conditions during the measurement (using a PABMSA-concentration of 5 mg/ml for preparation of the (PQQ)GDH electrodes, n¼ 3): (1) 5 mM MESþ 1 mM CaCl2 pH 6.5 and
(2) 100 mM CiP þ CaCl2 pH 7, scan rate 10 mV/s. (d) Cyclic voltammograms of (PQQ)GDH/PABMSA/vaCNT/Si-electrodes: (1) 0 mM glucose, (2) 10 mM glucose, PABMSAconcentration during preparation 5 mg/ml, 5 mM MESþ 1 mM CaCl2 pH 6.5. Scan rate 10 mV/s. (e) Dependence of the catalytic current of (PQQ)GDH/PABMSA/vaCNT
electrodes on the glucose concentration. Buffer conditions: 100 mM CiPþ 1 mM CaCl2 pH 6.5. Scan rate 10 mV/s.
100 mM CiP buffer (quiescent solution, pH 7) by means of LSV starting
at around þ 0.5 V vs. Ag/AgCl which agrees with the behavior of
immobilized BOD on other carbon surfaces (Brocato et al., 2012; Göbel
and Lisdat, 2008; Schubert et al., 2009; Ivnitski et al., 2008) indicating
that the T1 center of the enzyme is in contact with the electrode. The
approach of using PQQ as interface and a covalent attachment of the
BOD shows the highest catalytic currents – about 1 mA/cm2 at 0.1 V
vs. Ag/AgCl in an unstirred solution in comparison to BOD adsorbed
on PQQ-modiﬁed or untreated BP (evaluation of the current change
with respect to the result in Ar-saturated buffer). PQQ works here not
as a mediator since it is not reduced when the electrode starts to
transfer electrons via BOD towards oxygen (at þ0.5 V). It enhances
the productive enzyme–electrode interaction and thus serves as a
promoter here.
Moreover, applying BP as electrode surface a rather weak
diffusion limitation can be observed in the voltammetric curves.
This phenomenon may be explained by the ﬁlter-like structure
and the hydrophobicity of the buckypaper, which allows O2 not
only to diffuse from the solution but also from air along the
electrode down to the BOD-modiﬁed side.
3.4. vaCNT-based cathode
Our second cathode system based on BOD has been established by applying vaCNT@Si-electrodes modiﬁed with PQQ as
interface for the covalent ﬁxation of the enzyme. Fig. 3b depicts
the current behavior of a BOD/PQQ/vaCNT@Si-electrode in
Ar- and in air-saturated solution by means of LSV. It is obvious
that bioelectrocatalytic oxygen reduction takes place and starts
at potential of about þ0.5 V vs. Ag/AgCl. The shape of the
catalytic current indicates a high catalytic activity of the
immobilized enzyme and a diffusion limitation of the electrode
process. This phenomenon can be explained by the measurement setup – during the measurements the spots with the
vaCNTs are completely covered by buffer, thus oxygen can only
be provided from solution. However, the higher reduction
current found in the steepest part of the curve (in comparison
to BP electrode) indicates that the modiﬁed vaCNTs host a
signiﬁcant access amount of active BOD. The steady-state
catalytic current at þ0.1 V vs. Ag/AgCl shows current densities
of about 550 mA/cm2 .
636
V. Scherbahn et al. / Biosensors and Bioelectronics 61 (2014) 631–638
Fig. 3. Linear sweep voltammogram of (a). (1) BOD/buckypaper-electrode, BOD adsorbed, Ar-saturated buffer; (2) BOD/buckypaper, BOD adsorbed, air-saturated buffer; (3)
BOD/PQQ/buckypaper, [PQQ conc. for adsorption]¼ 1 mM, BOD adsorbed, air-saturated buffer; (4) BOD/PQQ/buckypaper, [PQQ conc. for adsorption]¼1 mM, BOD covalently
attached, air-saturated buffer; (5) BOD/PQQ/buckypaper, [PQQ conc. for adsorption]¼ 2.73 mM, BOD covalently attached, air-saturated buffer and (b) of BOD/PQQ/vaCNT/Sielectrode ([PQQ conc. during adsorption]¼2.73 mM) in (1) Ar-saturated buffer and (2) in air-saturated buffer. Buffer conditions: 100 mM CiP pH 7, quiescent solution. Scan
rate 10 mV/s.
Fig. 4. Performance curves of the established biofuel cell based on (a) buckypaper (BP) with a (PQQ)GDH/PABMSA/BP-anode and a BOD/PQQ/BP-cathode obtained from
galvanodynamic measurements at a scan rate of 3 nA/s – cell potential and power density in dependence on current density and (b) fuel cell based on vertically aligned CNTs
with a (PQQ)GDH/PABMSA/vaCNT/Si-anode and a BOD/PQQ/vaCNT/Si-cathode obtained from galvanodynamic measurements at a scan rate of 3 nA/s – cell potential and
power density in dependence on current density. Measuring conditions: quiescent, air-saturated 100 mM CiP þ 1 mM CaCl2, pH 7, 10 mM glucose.
3.5. (PQQ)GDH/BOD – biofuel cell based on buckypaper
In the current study we have shown that BP can be successfully
applied to construct efﬁcient enzyme electrodes. Consequently, an
EBFC based on BP has been assembled applying following preparation conditions: (PQQ)GDH attached on PABMSA/BP as anode
and BOD covalently bound to a PQQ/BP-electrode as cathode. One
of the most important parameter for a fuel cell is the power output
which has been studied under optimal conditions (100 mM CiP pH
7, 10 mM glucose, air saturated) by galvanodynamic sweep measurements. Such an experiment lasts for about 2 h providing real
data of the performance under load. The behavior of this EBFC is
depicted in Fig. 4a. Here the power density and the cell voltage in
dependence on the current density are given.
The power density curve shows a maximum of about 100 mW/cm2
at a rather high cell potential of 490 mV and a current density of
203 mA/cm2. Furthermore, the reproducibility of the electrode preparation can be shown by testing 3 biofuel cells which achieve in
average 104714 mW/cm2 (n¼3). The developed system exhibits an
almost two times higher power density than the EBFCs from our
previous studies also applying BOD and (PQQ)GDH as biocatalysts but
based on different CNT materials and modiﬁcations (Tanne et al.,
2010; Schubart et al., 2012).
After three successive applications of each fuel cell (for about
3 2 h each) a power density decrease up to 30% has been found,
whereas after four days a decrease even down to 13% can be
observed. This behavior indicates a limitation caused probably by
the instability of the adsorbed (PQQ)GDH at the anode. In order to
improve the behavior the enzyme is covalently attached via EDC/
NHS on the PABMSA/BP-electrode (PABMSA contains carboxylic
groups, Scheme 1a). Using these electrodes in a fuel cell set up
about 65% (70 714 mW/cm2, n ¼3) of the original power density
(10775 mW/cm2, n ¼3) can be retained after three successive
measurements of each fuel cell. Comparing this result to the fuel
cells with adsorbed (PQQ)GDH it can be concluded that the
covalent coupling of the enzyme on the PABMSA/BP-electrode
positively inﬂuences the stability of the BP-fuel cells.
3.6. (PQQ)GDH/BOD – biofuel cell based on vaCNT
Our second EBFC system based on vaCNT@Si-electrodes has been
constructed applying the same preparation conditions as the BP-EBFC
– (PQQ)GDH/PABMSA/vaCNT@Si-electrodes as anode and BOD/PQQ/
vaCNT@Si-electrode as cathode. In order to provide improved stability
both BOD and PQQ-GDH are covalently attached on the modiﬁed
electrodes. The performance of the vaCNT@Si-based EBFC is shown in
V. Scherbahn et al. / Biosensors and Bioelectronics 61 (2014) 631–638
Fig. 4b. The power density curve achieves its maximum of about
130 mW/cm2 at a cell potential of about 560 mV. It has to be
mentioned here that the cell voltage is decreasing rather slowly by
increasing the current ﬂow through the system. In addition higher
current densities can be obtained with this electrode combination.
This supports the idea that a high enzymatic activity can be achieved
within the modiﬁed CNT architecture. Further arguments in this
direction can be collected analyzing the stability of the EBFC (see
below).
Compared to our previous studies, the power performance can
be clearly enhanced (23 mW/cm2 (Tanne et al., 2010), 65 mW/cm2
(Schubart et al., 2012)). Moreover, the achieved power density of
the developed fuel cells exceeds the results of reported fuel cells
using GOD/Lac and GOD/BOD (43 mW/cm2 (Rengaraj et al., 2011)),
FDH/BOD on CNT and KetjenBlack electrodes (50 mW/cm2 (Filip et
al., 2013)) or CDH and BOD on nanoporous gold (40 mA/cm2
(Wang et al., 2012)) and reaches the same range with 131 mW/
cm2 using NAD-dependent GDH and Lac immobilized on SWCNT
(Karaśkiewicz et al., 2012). However, the power density is lower
compared to EBFCs with FDH/BOD using Au-NP and carbon paper
achieving 0.66 mW/cm2 or a GOD/Lac-fuel cell based on enzyme–
CNT-composite discs with 1.3 mW/cm2 (Zebda et al., 2011).
The performance of the EBFC has also been analyzed by
repeated measurements (about 2 h each). After 3 successive measurements of the same EBFCs they show only a small decrease
(18%) of the original maximum power density (122 78 mW/cm2,
n ¼3) at 540 750 mV and a rather constant value of current
density of about 230 mA/cm2 (n ¼3). The OCP is about 690 mV.
This can be seen as a ﬁrst hint for an optimized enzyme environment within the vertically aligned CNT architecture and provides
the basis to study the performance within the period of several
days. The key parameters of these experiments are shown in
Table 1a. As one can see there are some ﬂuctuations in the
performance which increase with the storage time of the electrodes (overnight in the refrigerator). Even after 3 days more than
110 mW/cm2 as maximum power density can be retained. This is
clearly a progress compared to the immobilization of (PQQ)GDH
on top of disordered MWCNTs where already after the ﬁrst day a
signiﬁcant loss of activity has been found (Tanne et al., 2010).
Furthermore, a stable cell potential can be provided within several
days of application.
Another aspect of this work relates to the application of the EBFC
in real biological ﬂuids in order to evaluate the performance under
more realistic conditions. Because the vaCNT-based EBFCs exhibit
better cell parameters they are used for these studies in human serum
samples. The results including power density and the cell potential
are presented in Table 1b. A maximum power density of 4177 mW/
637
cm2 (n¼ 3) is achieved at a cell potential of 390770 mV (n¼ 3). These
values are lower than biofuel cells operating in glucose containing
buffer (Fig. 4b, Table 1a). The rather complex matrix inﬂuences
obviously the enzyme–electrode contact since both the maximum
current density and the potential at maximum power are smaller. But,
this performance is found to be rather reproducible using three
similarly prepared fuel cells. Another reason for the diminished
performance might be the lower glucose concentration in serum.
Repeated experiments in serum with added glucose clearly show that
glucose sensitivity is maintained. Taking a medium glucose level of 34 mM, the addition of 5 mM glucose results in a rather small increase
in power (Table 1b). The experiments demonstrate that the glucose
concentration in serum is only a minor point with respect to the
reduced performance found here in comparison to an artiﬁcial buffer
solution.
Nevertheless, even in a biological ﬂuid reasonable power
densities can be achieved compared to previous studies in pure
buffer (Tanne et al., 2010; Schubart et al., 2012; Coman et al., 2010).
Compared to other studies with EBFCs being investigated in
human sera, no signiﬁcant improvement concerning the power
output can be obtained, but a rather good stability has been found
for repeated measurements (no loss of maximum power within
3 repeated measurements).
4. Summary
In the present study we have developed membraneless and
mediatorless, glucose EBFCs based on (PQQ)GDH and BOD as
biocatalysts and two different CNT architectures – buckypaper
(BP) and vertical aligned CNTs (vaCNT).
The separate evaluation of the anodes and cathodes shows a
higher suitability of vaCNT as electrode interface for the enzymes
compared to BP. For both systems a polyaniline based interlayer
has been introduced for (PQQ)GDH coupling and a PQQ ﬁlm for
BOD ﬁxation, thus allowing efﬁcient electrochemical communication between the biocatalyst and the CNT surface. Maximal current
densities of about 1.3 70.18 mA/cm2 (at 0.1 V vs. Ag/AgCl) for the
(PQQ)GDH/PABMSA/vaCNT@Si-anode and about 550 mA/cm2 (at
0.1 V vs. Ag/AgCl) for the BOD/PQQ/vaCNT@Si-cathode can be
achieved. For buckypaper as electrode material these values are
0.75 mA/cm2 for the anode ((PQQ)GDH/PAPMSA/BP) and 1 mA/
cm2 for the cathode (BOD/PQQ/BP).
Applying the developed electrodes in a fuel cell maximum power
density of about 10775 mW/cm2 is detected for the bucky-paper
based system in (10 mM glucose). Covalent coupling of the enzyme to
the polymer layer improves the stability of the system.
Table 1
Key parameters obtained from vaCNT-based enzymatic biofuel cells based on a (PQQ)GDH/PABMSA/vaCNT-anode and a BOD/PQQ/vaCNT-cathode (a) within three days
(EBFCs (n¼ 3), 10 mM glucose in 100 mM CiP þ1 mM CaCl2, pH 7 quiescent, air-saturated solution, galvanodynamic measurements at a scan rate of 3 nA/s) and (b) in human
serum samples (EBFCs (n ¼3), successive addition of 5 mM and 10 mM glucose, air-saturated, quiescent solution, galvanodynamic measurements with a scan rate 3 nA/s).
(a)
Day
1
2
3
Max. power density [mW/cm2]
Potential at power maximum [mV]
Open circuit potential (OCP) [mV]
Current density at power maximum [mA/cm2]
1227 8
540 7 50
7077 68
228 7 5
142 748
519 735
707 738
273 774
1137 26
4477 21
690 7 14
254 7 71
Human serum
Human serum þ5 mM glucose
Human serumþ 10 mM glucose
417 7
3917 70
660 7 43
50 75
418 720
650 724
517 5
3917 23
620 7 20
(b)
Solution
2
Max. power density [mW/cm ]
Potential at power maximum [mV]
Open circuit potential (OCP) [mV]
638
V. Scherbahn et al. / Biosensors and Bioelectronics 61 (2014) 631–638
The second biofuel cell is based on vaCNTs applying the same
enzymes and modiﬁcation steps. Here a power density of
12278 mW/cm2 (at 540 750 mV) in presence of 10 mM glucose
can be generated, which is slightly higher than the BP based cell.
Even on the third day of application of the cell a power density of
more than 110 mW/cm2 is retained. This EBFC has also been
applied in human serum samples. The results show a decreased
power density of about 41 77 mW/cm2. The diminished glucose
serum concentration is only to a minor extent responsible for the
lower performance in serum compared to buffer solutions.
Acknowledgments
The ﬁnancial support by the Bundesministerium für Bildung
und Forschung Germany is kindly acknowledged (Project
03IS2201I). The authors want to thank Roche Diagnostics (Penzberg, Germany) and mainly Dr. Meier and Dr. von der Eltz for
cooperation on supplying us with the GDH enzyme. We also thank
the Karl und Marie Schaak-Stiftung, Frankfurt/Main, for the generous ﬁnancial support.
References
Ahmadalinezhad, A., Wu, G., Chen, A., 2011. Biosens. Bioelectron. 30 (1), 287–293.
Barton S. C., Enzyme catalysis in biological fuel cells. In Handbook of Fuel Cells –
Fundamentals, Technology and Applications. Edited by Wolf Vielstich, Harumi
Yokokawa, Hubert A. Gasteiger. Volume 5: Advances in Electocatalysis, Materials, Diagnostics and Durability (pp. 112-131), 2010, John Wiley & Sons, Ltd.
Brocato, S., Lau, C., Atanassov, P., 2012. Electrochim. Acta 61, 44–49.
Christenson, A., Shleev, S., Mano, N., Heller, A., Gorton, L., 2006. Biochim. Biophys.
Acta 1757 (12), 1634–1641.
Cinquin, P., Gondran, C., Giroud, F., Mazabrard, S., Pellissier, A., Porcu, P., Cosnier, S.,
2010. PLoS One 5 (5), 1–7.
Coman, V., Ludwig, R., Harreither, W., Haltrich, D., Gorton, L., Ruzgas, T., Shleev, S.,
2010. Fuel Cells 10 (1), 9–16.
Durand, F., Stines-Chaumeil, C., Flexer, V., André, I., Mano, N., 2010. Biochem.
Biophys. Res. Commun. 402 (4), 750–754.
Falk, M., Blum, Z., Shleev, S., 2012. Electrochim. Acta 82 (0), 191–202.
Filip, J., Šefčovičová, J., Gemeiner, P., Tkac, J., 2013. Electrochim. Acta 87 (0),
366–374.
Flexer, V., Durand, F., Tsujimura, S., Mano, N., 2011. Anal. Chem. 83, 5721–5727.
Göbel, G., Lisdat, F., 2008. Electrochem. Commun. 10 (11), 1691–1694.
Göbel, G., Schubart, I.W., Scherbahn, V., Lisdat, F., 2011. Electrochem. Commun. 13
(11), 1240–1243.
Gupta, G., Lau, C., Rajendran, V., Colon, F., Branch, B., Ivnitski, D., Atanassov, P., 2011.
Electrochem. Commun. 13 (3), 247–249.
Halamkova, L., Bocharova, V., Szczupak, A., Alfonta, L., Katz, E., 2012. J. Am. Chem.
Soc. 134, 8–11.
Hsieh, G., Li, F.M., Beecher, P., Nathan, A., Wu, Y., Ong, S., Milne, W.I., 2009. J. Appl.
Phys. 106, 123706.
Hussein, L., Urban, G., Kruger, M., 2011. Phys. Chem. Chem. Phys. 13 (13),
5831–5839.
Ivnitski, D., Artyushkova, K., Atanassov, P., 2008. Bioelectrochemistry 74 (1),
101–110.
Javier, F., García-Céspedes, J., Muñoz, F.X., Bertrán, E., 2008. Electrochem. Commun.
10, 1242–1245.
Joshi, R.K., Schneider, J.J., 2012. Chem. Soc. Rev. 41 (15), 5285–5312.
Joshi., R., Yilmazoglu, O., Schneider, J.J., Pavlidis, D., 2010. J. Mater. Chem. 20,
1717–1721.
Karaśkiewicz, M., Nazaruk, E., Żelechowska, K., Biernat, J.F., Rogalski, J., Bilewicz, R.,
2012. Electrochem. Commun. 20, 124–127.
Khabazian, S., Sanjabi, S., 2011. Appl. Surf. Sci. 257 (13), 5850–5856.
Kim, J., Parkey, J., 2009. J. Solid State Electrochem. 13, 1043–1050.
Laurinavicius, V., Razumiene, J., Kurtinaitiene, B., Lapenaite, I., Bachmatova, I.,
Marcinkeviciene, L., Ramanavicius, A., 2002. Bioelectrochemistry 55, 29–32.
Lee, S., Chang, Y.-P., 2009. J. Mater. Sci. 20, 851–857.
Li, D., Wen, Y., He, H., Xu, J., Liu, M., Yue, R., 2012. J. Appl. Polym. Sci. 126, 882–893.
Li, X., Du, Z., Zhang, C., Li, H., Zou, W., 2013. Polym. Adv. Technol. 24, 151–156.
Liu, C., Huang, C., Wu, S., Han, J., 2010. Polym. Int. 59, 517–522.
Liu, X., Baronian, K.H.R., Downard, A.J., 2009. Carbon 47 (2), 500–506.
Lojou, E., 2011. Electrochim. Acta 56 (28), 10385–10397.
Miyake, T., Haneda, K., Yoshino, S., Nishizawa, M., 2013. Biosens. Bioelectron. 40 (1),
45–49.
Musameh, M., Lawrence, N.S., Wang, J., 2005. Electrochem. Commun. 7, 14–18.
Neto, S., Forti, J., Andrade, A. De., 2010. Electrocatalysis 1 (1), 87–94.
Nogala, W., Celebanska, A., Szot, K., Wittstock, G., Opallo, M., 2010. Electrochim.
Acta 55 (20), 5719–5724.
Osman, M.H., Shah, A.A., Walsh, F.C., 2011. Biosens. Bioelectron. 26 (7), 3087–3102.
Ramírez, P., Mano, N., Andreu, R., Ruzgas, T., Heller, A., Gorton, L., Shleev, S., 2008.
Biochim. Biophys. Acta 1777 (10), 1364–1369.
Razumiene, J., Meškys, R., 2000. Electrochem. Commun. 2, 307–311.
Razumiene, J., Vilkanauskyte, A., Gureviciene, V., Barkauskas, J., Meskys, R.,
Laurinavicius, V., 2006. Electrochim. Acta 51 (24), 5150–5156.
Rengaraj, S., Kavanagh, P., Leech, D., 2011. Biosens. Bioelectron. 30 (1), 294–299.
Sang, Y.K., Park, J., Lee, D., Kim, H., 2012. J. Appl. Electrochem. 42 (6), 383–390.
Sarauli, D., Riedel, M., Wettstein, C., Hahn, R., Stiba, K., Wollenberger, U., Lisdat, F.,
2012. J. Mater. Chem. 22 (11), 4615–4618.
Sarauli, D., Xu, C., Dietzel, B., Schulz, B., Lisdat, F., 2013. Acta Biomater. 9 (9),
8290–8298.
Schubart, I.W., Göbel, G., Lisdat, F., 2012. Electrochim. Acta 82, 224–232.
Schubert, K., Goebel, G., Lisdat, F., 2009. Electrochim. Acta 54, 3033–3038.
Suraniti, E., Abintou, M., Durand, F., Mano, N., 2012. Bioelectrochemistry 88, 65–69.
Tanne, C., Göbel, G., Lisdat, F., 2010. Biosens. Bioelectron. 26 (2), 530–535.
Wang, X., Falk, M., Ortiz, R., Matsumura, H., Bobacka, J., Ludwig, R., Shleev, S., 2012.
Biosens. Bioelectron. 31 (1), 219–225.
Wettstein, C., Mö
hwald, H., Lisdat, F., 2012. Bioelectrochemistry 88, 97–102.
Zebda, A., Gondran, C., Goff, A., Le, Cinquin, P., 2011. Nat. Commun. 2, 370–376.

Download Report