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Bienzyme biosensors for glucose, ethanol and putrescine built onoxidase and sweet potato peroxidase
Jaime Castillo a, Szilveszter Gaspar a, Ivan Sakharov b, Elisabeth Csoregi a,*a Department of Biotechnology, Lund University, P.O. Box 124, SE 221 00 Lund, Sweden
b Department of Chemical Enzymology, Chemical Faculty, Moscow State University, Moscow 119899, Russia
Received 28 May 2002; received in revised form 14 October 2002; accepted 22 November 2002
Abstract
Amperometric biosensors for glucose, ethanol, and biogenic amines (putrescine) were constructed using oxidase/peroxidase
bienzyme systems. The H2O2 produced by the oxidase in reaction with its substrate is converted into a measurable signal via a novel
peroxidase purified from sweet potato peels. All developed biosensors are based on redox hydrogels formed of oxidases (glucose
oxidase, alcohol oxidase, or amine oxidase) and the newly purified sweet potato peroxidase (SPP) cross-linked to a redox polymer.
The developed electrodes were characterized (sensitivity, stability, and performances in organic medium) and compared with
similarly built ones using the ‘classical’ horseradish peroxidase (HRP). The SPP-based electrodes displayed higher sensitivity and
better detection limit for putrescine than those using HRP and were also shown to retain their activity in organic phase much better
than the HPR based ones. The importance of attractive or repulsive electrostatic interactions between the peroxidases and oxidases
(determined by their isoelectric points) were found to play an important role in the sensitivity of the obtained sensors.
# 2003 Elsevier Science B.V. All rights reserved.
Keywords: Amperometric biosensor; Bienzyme electrode; Redox hydrogel; Sweet potato peroxidase
1. Introduction
The amperometric, enzyme-based, biosensors are
bearing great potential to be used in different fields
such as environment monitoring (Cowell et al., 2001;
Dennison and Turner, 1995; Karube and Nomura, 2000;
Karube et al., 1998; Marco and Barcelo, 2000; Rekha et
al., 2000), food and beverage quality analysis (Csoregi et
al., 2001), process monitoring (Brooks et al., 1991; Pons,
1993) or biomedicine (Wang, 1999). They are simple,
sensitive, and fast responding analytical tools con-
structed by immobilization of enzymes on the electro-
de’s surface. The major problem that appears in the
operation of these devices is the electron transfer
between the enzyme’s active center and the electrode.
The first developed, oxidase-based, amperometric bio-
sensors usually involved oxidation of H2O2 resulting
from the enzyme’s reaction with its natural cofactor, O2.
In this type of biosensors high operational potential
must be applied on the interface that leads to high
background currents and to the non-selectivity of the
electrodes. Since this drawback became very important
in real sample measurements, many solutions to avoid
high applied potentials have been developed, leading to
the second and third generation of the amperometric
enzyme-based biosensors (Cass, 1990).
Addressing the interference elimination problem
Kulys et al. designed the first oxidase/peroxidase
bienzyme electrode with a film containing horseradish
peroxidase (HRP), and glucose oxidase (GOx) that is
detecting glucose by measuring the produced amount of
H2O2 through the peroxidase (Kulys et al., 1981). Using
oxidase/peroxidase bienzyme systems the detection
principle switches from an electrochemical oxidation to
a reduction process that is happening at much lower
potentials, and therefore, is improving considerably the
selectivity of the device.
Peroxidases are among the very few enzymes able to
directly transfer electrons to an electrode at relatively
low applied potentials (Csoregi et al., 1993b; Gorton et
* Corresponding author. Tel.: �/46-46-222-4274; fax: �/46-46-222-
4713.
E-mail address: [email protected] (E. Csoregi).
Biosensors and Bioelectronics 18 (2003) 705�/714
www.elsevier.com/locate/bios
0956-5663/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0956-5663(03)00011-3
al., 1992, 1999) but in order to improve sensitivity,
mediated electron transfer involving multitude of redox
mediators was also experimented. One of the most
promising approaches, involving an electron transferthrough immobilized Os complexes, is ‘wiring’ the
peroxidase by using redox polymers (Gregg and Heller,
1990; Ohara et al., 1994, 1993). This design offers a
hydrophilic environment where enzymes can maintain
their native properties and activity. In the same time, a
redox hydrogel is not only an immobilization method
but also an electron transfer solution. Redox centers
dispersed in the hydrogel assure the transfer of theelectrons to/from the enzyme’s redox center from/to the
electrode by the so-called ‘electron hopping’ phenom-
enon (Katakis and Heller, 1997).
Nowadays this ingenuous combination of two en-
zymes is widely used to develop biosensors with
improved characteristics. A wide spectrum of oxidases
was coupled with peroxidase electrodes for the detection
of the appropriate substrates: amine oxidase (Niculescuet al., 2000), alcohol oxidase (Boguslavsky et al., 1995),
glucose oxidase (Tian and Zhu, 2002), diamine oxidase
(Tombelli and Mascini, 1998), choline oxidase (Bogus-
lavsky et al., 1995), putrescine oxidase (Yang and
Rechnitz, 1995), glutamate oxidase (Collins et al.,
2001), oxalate oxidase (Perez et al., 2001), galactose
oxidase (Tkac et al., 2000), xanthine oxidase (Mao and
Yamamoto, 2000), and lysine oxidase (Saurina et al.,1999).
Peroxidase from horseradish is usually used in
bienzyme biosensors. However, there are at least two
major drawbacks displayed by this peroxidase: (1) it has
a very broad specificity for reducing substrates (Dun-
ford, 1991) which in turn produces reduced selectivity of
the biosensor and (2) even though HRP exhibits a good
stability at room temperature, its stability is unsatisfac-tory at more elevated temperatures (Kenausis et al.,
1997; Vreeke et al., 1995). Therefore, an extensive
investigation of peroxidases of different origin has
been carried out involving screening for peroxidases
with improved properties (Csoregi et al., 1993a; Gorton
et al., 1999; Lindgren et al., 1997; Munteanu et al., 1998;
Sakharov et al., 1999, 2000). In a recent work of our
group the peroxidase purified from sweet potato peelsand immobilized in redox hydrogels was resulting in
H2O2 biosensors characterized by high sensitivities and
low detection limits. Our work proved that sweet potato
peroxidase (SPP) could be a promising alternative to the
commonly used HRP when used in bienzyme biosensor
design (Gaspar et al., 2000).
The objective of the present work is to assess the
performance of SPP in bienzyme electrode designs.Three different oxidases were selected for biosensor
development, namely: glucose-, amine-, and alcohol
oxidase. The choice of these oxidases was motivated
by the medical and biotechnological importance of their
substrate, and also by the properties of the different
enzymes. The targeted analytes were selected covering
interest from many different fields. The determination of
glucose in biological fluids is extremely important forthe diagnosis and management of diabetes (Wang et al.,
2000). Biogenic amines are toxic substances, which
causes disease in man and animal (Shalaby, 1996). In
the present work, we target especially putrescine because
its level also reflects tumor growth rates (Harik and
Sutton, 1979), and is the cause of hypotension, brady-
cardia, lockjaw, paresis of extremities, and potentiates
the toxicity of other amines (Shalaby, 1996). Thedetermination of the alcohol levels in clinical chemistry
and in alcoholic drinks is also of great importance.
Ethanol is one of the aliphatic alcohols, which are most
detected and quantified.
2. Experimental
2.1. Chemicals
HRP (EC 1.11.1.7, Cat No. P6782, specific activity of
1100 U mg�1 solid), alcohol oxidase (from Hansenula
sp.) (AlcOx; EC 1.1.3.13, Cat No. A0438, specific
activity of 11 U mg�1 solid), glucose oxidase (from
Aspergillus niger ) (GOx; EC 1.1.3.4, Cat No. G2133,
specific activity of 1 81 000 U g�1 solid) were purchased
from Sigma�/Aldrich (Tyreso, Sweden) and used asreceived. Amine oxidase from grass pea (AO; EC
1.4.3.6) isolated and purified according to a previously
published protocol (Sebela et al., 1998), was kindly
provided by Dr I. Frebort (Palacky University, Olo-
mouc, Czech Republic). SPP was purified from sweet
potato peels as described under Section 2.2. Polyethylen
Glycol 10000 (Cat. No. 30,902-8) was purchased from
Sigma, Phenyl-Sepharose 6 Fast Flow (low sub, Cat.No. 17-0965-05) was purchased from Amersham Phar-
macia Biotech AB (Uppsala, Sweden), DEAE-Toyo-
pearl (Cat. No. 007473) was from Toyo Soda MFG Co.
Ltd. (Tokyo, Japan), while NaCl (Cat. No. 106404),
K2HPO4 (Cat. No. 105202), (NH4)2SO4 (Cat. No.
101217), C4H11NO3 (Cat. No. 108382) were from Merck
(Darmstadt, Germany). The Lower Molecular Weight
calibration kit for SDS electrophoresis (Cat. No. 17-0446-01) was from Amersham Pharmacia Biotech
(Buckinghamshire, England). Poly(ethylene glycol)
(400) diglycidyl ether (PEGDGE, Cat. no. 08210) was
from Polysciences, (Warrington, PA, USA). Poly(viny-
limidazole) complexed with Os(4,4? dimethylbipyridi-
ne)2Cl (PVI7-dme-Os) was synthesized according to a
previously published protocol (Ohara et al., 1994). The
subscript 7 indicates that every 7th vinylimidazole unit iscomplexed to an osmium center. Borate�/HCl buffer
solution was prepared using Na2B4O7 �/ 10H2O pur-
chased from Sigma�/Aldrich and HCl (Cat. No.
J. Castillo et al. / Biosensors and Bioelectronics 18 (2003) 705�/714706
113386) from Merck. Phosphate buffer (PB) solution
was prepared using Na2HPO4 �/ 2H2O (Cat. No. 106580)
and NaH2PO4 �/ H2O (Cat. No. 106346) all Merck.
Hydrogen peroxide solutions were prepared daily from35% H2O2 solution (Cat. No. 20246-0010) from Acros
Organics (Geel, Belgium). Eastman Kodak (EK) AQ
55D 28% was obtained from Eastman Chemical Pro-
ducts (Kingsport, TN, USA). Ethanol (99.5%) was from
Kemetyl AB (Haninge, Sweden), putrescine dihy-
drochloride (Cat. No. 100450) and D-(�/)-glucose
(Cat. No. 152527) were from ICN Biochemicals Inc.
(Aurora, OH, USA). All solutions were prepared usingtridistilled water produced in a Milli-Q system (Milli-
pore, Bedford, MA) if not otherwise stated.
2.2. Methods
2.2.1. Sweet potato peroxidase purification
Details of the purification process has been described
elsewhere (Castillo et al., 2002). Briefly, the purification
involved steps as follows: peels of sweet potato were
milled and incubated with constant agitation in 10 mM
Borate�/HCl buffer pH 9.0 also containing 0.5 mM
NaCl. The tissue debris was removed by filtration and
centrifugation. After this, solid PEG and solid K2HPO4
were dissolved in the supernatant up to 14% (w/v) and
8.5% (w/v), respectively. Two phases were formed and
the lower clear phase was applied directly to a Phenyl-
Sheparose column and finally was eluted by decreasing
the (NH4)2SO4 concentration. The fractions having
peroxidase activity were collected and dialyzed against
distilled water and Tris�/HCl buffer pH 8.3. After
dialysis, the extract was applied to a DEAE-Toyopearlcolumn. The peroxidase was eluted with a linear NaCl
gradient. The optimized purification resulted in a 116-
fold purification producing nearly homogeneus enzyme.
The peroxidase migrated in SDS-electrophoresis is a
single band corresponding to the molecular weight of 35
kDa.
2.2.2. Electrode preparation
Graphite electrodes (i.d. 0.305 cm, type RW 001,
Ringsdorff Werke, Bonn, Germany) were wet polished
on emery paper (Tufbak Durite type P1200, Allar,Sterling Heights, MI, USA) and thoroughly washed
with distilled water, before modification. Next, pre-
mixed hydrogels were made using different volumes of
peroxidase stock solutions, (0.23 mg ml�1 HRP or
SPP), oxidase stock solutions (20 mg ml�1 GOx, AlcOx,
or AO), PVI7-dme-Os (3.3 mg ml�1) and PEGDGE (2.5
mg ml�1, freshly prepared and used within 15 min). A 5
ml hydrogel droplet was placed on the electrode surfaceusing a pipette and cured for 20 h at room temperature.
The final hydrogel film composition consisted of 57% of
oxidase, 14% of peroxidase, 23% of redox polymer and
6% of cross-linker (weight percentages). Two types of
sensing layers were developed:
1) The first hydrogel contained both the oxidase and
the peroxidase and was prepared exactly as de-
scribed above.
2) The second sensing layer consisted of a hydrogellayer containing only the peroxidase separated by
an EK film from a layer containing cross-linked
oxidase. Accordingly, first a hydrogel containing
only the peroxidase was deposited on the electrode
surface and cured for 20 h. Then an EK film was
placed over the peroxidase containing hydrogel by
dipping the electrode into 0.56% v/v polymer
solution and curing it for 90 min. Finally a layerof oxidase cross-linked with PEGDGE was pipetted
on top of the EK film making sure that the amount
of oxidase is the same as in the first type of sensing
layer. Prior to use, all modified electrodes were
thoroughly washed with deionized water. All pre-
sented results are the mean of at least three
identically prepared electrodes, if not otherwise
mentioned.
2.3. Instrumentation
Amperometric measurements were done in a home
built flow-through wall-jet cell (Appelqvist et al., 1985)
inserted in a single line flow injection (FI) system. The
experimental set-up also contained a peristaltic pump
(type U4-MIDI, Alitea AB, Stockholm, Sweden) and a
100 ml manual injector (Vici AG, Valco Europe,
Schenkon, Switzerland). The output signal was recorded
on a strip chart recorder (Model BD 111, Kipp andZonen, Delft, The Netherlands). All FI experiments
were done using a low current potentiostat (Zata-
Elektronik, Hoor, Sweden). Operational stability ex-
periments were performed using the same system but
with the appropriate substrate passed continuously
through the cell at a flow rate of 0.5 ml min�1 (i.e.
the injector was excluded). Electrodes modified with
hydrogels containing only peroxidase were tested in abatch system using a beaker instead of the flow-through
cell (in order to avoid working with organic solvents in a
flow system). A deaerated 0.1 M PB solution at pH 7.0
or acetonitrile containing 5% of the same buffer was
used as supporting electrolyte and all experiments were
carried out at room temperature.
3. Results and discussion
SPP has been previously found to successfully replaceHRP when constructing biosensors for monitoring of
hydrogen peroxide (Gaspar et al., 2000). The SPP based
electrodes displayed lower detection limits and higher
J. Castillo et al. / Biosensors and Bioelectronics 18 (2003) 705�/714 707
sensitivities than the ones using HRP. These preliminary
results motivated further work considering the use of
SPP-based electrodes in organic phase and for the
construction of bi-enzyme based electrodes.
3.1. Electrodes in organic phase
There are applications were analytes must be detected
in non-aqueous phases like dioxane, chlorobenzene,
alcohols and chloroform. The use of enzyme based
sensors in non-aqueous phases is problematic since
organic solvents distort the essential water layer around
the enzyme. A more hydrophilic solvent requires higherwater content in the solution in order to maintain the
essential water molecules around the enzyme, and thus,
its right conformation and activity. The detrimental
effect of the organic solvents on enzymes is primarily
due to interactions with the essential enzyme-bound
layer of water rather than with the enzyme itself (Carrea
and Riva, 2000). Since it has been previously shown that
there are structural differences between plant peroxi-dases (Munteanu et al., 2000), the properties of the SPP
and HRP electrodes for hydrogen peroxide detection
were studied in both acetonitrile and aqueous solutions.
The electrode design was based on the enzymes inte-
grated into redox hydrogels using beside the perox-
idases, a cross-linker and a redox polymer as described
under Section 2.2. The two different electrodes displayed
an almost linear current response to H2O2 in the studiedconcentration range in both aqueous and non-aqueous
phase (see Fig. 1). As seen, the HRP based electrode
displayed twice higher sensitivity in aqueous phase
Fig. 1. Calibration curves for hydrogen peroxide, obtained with biosensors based on SPP and HRP in 0.1 M PB pH 7.0 (inset) and in acetonitrile
with in 0.1 M TBAP and 5% of 0.1 M PB pH 7.0. Experiment conditions: batch system, �/50 mV vs. Ag/AgCl, 0.1 M KCl.
Table 1
Characteristics of the obtained biosensors
Sensing film composition Km (mM) Imax (nA) Sensitivity (nA mM�1) Conversion efficiency (%) Detection Limit (mM)
SPP�/GOX�/PVI-Os 1.319/0.02 2358.19/1.3 1.809/0.03 15.229/0.7 1.329/0.2
HRP�/GOX�/PVI-Os 2.149/0.01 6004.39/5.3 2.819/0.03 7.89/0.3 1.149/0.5
SPP�/PVI-Os/EK/GOX �/ �/ 1.049/0.03 1.459/0.2 1.99/0.1
HRP�/PVI-Os/EK/GOX 4284.89/0.8 5362.59/1.3 1.029/0.03 10.49/0.5 0.89/0.1
SPP�/AO�/PVI-Os 0.859/0.01 5326.69/0.98 6.39/0.2 22.19/6.1 0.39/0.1
HRP�/AO�/PVI-Os 0.449/0.01 1562.79/0.92 3.69/0.1 42.89/8.5 0.59/0.1
SPP�/PVI-Os/EK/AO 1373.59/0.2 2222.89/1.5 16.29/0.3 29.59/1.1 0.49/0.2
HRP�/PVI-Os/EK/AO 517.239/0.1 4074.79/0.03 7.889/0.2 31.579/1.3 0.59/0.2
SPP�/AlcOx�/PVI-Os 2.389/0.3 249.059/2.6 0.109/0.01 0.629/0.02 0.239/0.02
HRP�/AlcOx�/PVI-Os 4.719/0.4 813.959/3.2 0.179/0.02 0.249/0.01 0.39/0.01
SPP�/PVI-Os/EK/AlcOx 839.319/0.04 29.0039/0.41 0.039/0.001 0.149/0.01 0.69/0.01
HRP�/PVI-Os/EK/AlcOx 5188.79/0.36 118.399/1.1 0.029/0.002 0.069/0.02 39/0.02
J. Castillo et al. / Biosensors and Bioelectronics 18 (2003) 705�/714708
(113.3 vs. 47.6 nA mM�1) than the SPP based ones (see
inset of Fig. 1). This result is in contradiction with
previously obtained ones (Gaspar et al., 2000), a fact
attributed to the two different SPP enzymes used in the
experiments; the former results were obtained with sweet
potato from Colombia while present ones with sweet
potato purchased in Sweden.
However, comparing the calibration curves of the two
biosensors in acetonitrile, it is obvious that the SPP-
based ones display a better resistance to non-aqueous
mediums as shown by the relatively higher sensitivity of
these electrodes compared with the HRP-based ones
(35.4 vs. 31.8 nA mM�1). Changing the PB to acetoni-
trile produced a 70% decrease in sensitivity for the
sensors based on HRP and only a 33% decrease in
sensitivity for those based on SPP.
3.2. Bi-enzyme electrodes
The sensing chemistry of the developed biosensors for
glucose, ethanol, and putrescine, is based on SPPcoupled with the appropriate oxidase integrated into
the same type of redox hydrogel, as mentioned above for
the hydrogen peroxide electrodes. Table 1 and Fig. 2A�/
Fig. 2. Calibration curves for glucose (a), putrescine (b), and ethanol (c) obtained with biosensors based on SPP and HRP. Experimental conditions:
FI analysis, 0.1 M PB pH 7.0 as carrier, flow rate 0.5 ml min�1, applied potential of �/50 mV vs. Ag/AgCl, 0.1 M KCl.
J. Castillo et al. / Biosensors and Bioelectronics 18 (2003) 705�/714 709
C summarizes the results obtained with the biosensors
based on SPP or HRP for glucose, ethanol and
putrescine.
Considering the detection limits (defined as three
times the signal to noise ratio), the biosensors for
putrescine and ethanol based on SPP displayed better
detection limits than the ones based on HRP, while the
detection limit of the GOX electrode was not signifi-
cantly different for the two used peroxidases.
Higher substrate affinity has been observed for SPP-
GOX and SPP-AlcOX systems than for the HRP-based
ones (see corresponding lower Michaelis constants, Km).
However, the HRP using electrodes displayed higher
sensitivity in these cases (obtained as the ratio between
the maximum current and the apparent Michaelis
constant). In the case of the AO-peroxidase system,
the situation is the opposite, lower Km was obtained for
the HRP, but the sensitivity of the bienzyme electrode
was higher for the SPP-AO system.
The results contained in Table 1 clearly demonstrates
that in a sensor design both the nature of the oxidase
and peroxidase play an important role and for certain
substrates the SPP might be a better counterpart of the
oxidase than HRP. Attractive electrostatic interactions
leading to enzyme�/enzyme complexes seem to have an
important role in this aspect by reducing the distance the
produced hydrogen peroxide has to diffuse until the
active site of the peroxidase and resulting in sensors
which display higher sensitivities. Considering the iso-
electric point of the used enzymes (4.2 for GOX (Pazur
and Kleppe, 1964), 7.2 for AO (Sebela et al., 1998), 6.2
for AlcOx (Van Der Klei et al., 1990), 3.5 for SPP
(Castillo et al., 2002) and 7.2 for HRP (Maehly, 1955)),
it can be noticed that, at the working pH of 7.0,
attractive electrostatic interactions between SPP and
oxidase occur only in the case of AO. These interactions
seem to play an important role in the efficiency of a
hydrogel since the most sensitive biosensor for putres-
cine is obtained using SPP as the counterpart of AO.
Accordingly, since HRP is able to attractively interact
with the other two studied oxidases, the biosensors for
glucose and ethanol have higher sensitivities using HRP.
The conversion efficiency (defined as the ratio of the
current signal given by injecting similar concentrations
of substrate and H2O2) could be considered another
measure of the communication between the oxidase and
peroxidase. The obtained results showed indeed, that the
AO-peroxidase couple displayed higher conversion effi-
ciency as the other two bienzyme systems (GOX and
AlcOX) in both cases (SPP and HRP). However, while
GOX and AlcOX showed higher conversion efficiencies
for the SPP based system, the opposite result was
observed for AO situation, which seems contradictory
with the obtained values for sensor sensitivities. One
possible explanation can be given if one considers this
time the electrostatic interactions between the enzymes
and the polycationic polymer. Even though the amount
of the two peroxidases in the different hydrogels is
theoretically equal, based on the attractive interactions
between SPP and the polymer, we can suppose that the
real peroxidase concentration in the hydrogel is always
higher when using SPP than when using HRP (since
some of the components can be washed away). A higher
peroxidase content of the hydrogel when using SPP,
combined with the high substrate affinities observed for
the AO-peroxidase systems (i.e. with a more pronounced
Fig. 2 (Continued)
J. Castillo et al. / Biosensors and Bioelectronics 18 (2003) 705�/714710
dependency of the signal on the amount of the enzyme)
and the repulsive interaction between polymer and AO
could be an explanation of the low conversion efficiency
but still high sensitivity of the SPP-AO sensors (high
sensitivity based on the attractive interaction between
SPP and AO).
Coupled enzyme biosensors have usually a small
drawback, which is very often not considered in pub-
lications. Namely, the involved electron transfer med-
iator could act first as oxidizing substrate for the oxidase
and then as reducing substrate for the peroxidase. In this
way, the mediator is not regenerated at the interface
Fig. 3. Operational stability of biosensors for glucose based on SPP and HRP for glucose (a), putrescine (b), and ethanol (c). Experimental
conditions: 0.1 M PB pH 7.0 as carrier with 0.5 mM glucose (a), 100 mm putrescine (b) or 2 mM ethanol (c), flow rate of 0.5 ml min�1 applied
potential of �/50 mV vs. Ag/AgCl, 0.1 M KCl.
J. Castillo et al. / Biosensors and Bioelectronics 18 (2003) 705�/714 711
(resulting in the useful signal) but rather in the bulk of
the hydrogel, i.e. the system ‘short-circuits’ itself (Ke-
nausis et al., 1997; Vijayakumar et al., 1996). A common
way of testing the biosensors for this effect is to
immobilize the oxidase separated from the peroxidase
by using an inert polymer film. In our case significant
‘short-circuit’ was observed just in case of the biosensors
designed for putrescine where using an EK film resulted
in sensors with increased sensitivities besides an ex-
tended diffusion limited linear range. For glucose and
ethanol the EK acted only as a diffusion-limiting barrier
producing extended linear ranges but lower sensitivities
(see Table 1).
Certain practical applications (especially continuous
monitoring) need biosensors with a stable signal over
time periods ranging from hours to days or even weeks.
One of the key factors to obtain a stable biosensor is the
lifetime of the enzymes used in the sensor design.
Therefore, operational stability tests were performed
for the biosensors developed for glucose, ethanol and
putrescine by passing through the measuring cell a
carrier solution containing the substrate for 400 min at
room temperature (see Fig. 3A�/C). In two of the
studied cases the SPP coupled enzyme electrodes showed
a better stability. The current signal after 400 min was
77.5 and 71% of its initial value when measuring glucose
(500 mM) with sensors based on SPP and HRP,
respectively. The putrescine biosensors based on SPP
and HRP retained 30 and 35%, respectively, of their
initial response to 100 mM putrescine. Different behavior
of the two peroxidases was observed only in the case of
ethanol biosensors where the HRP-based biosensor
retained 60% of its initial current signal to 2 mMethanol while SPP-based biosensor only 37.5%.
4. Conclusions
Amperometric biosensor for glucose, ethanol and
putrescine were developed using the appropriate oxi-
dases coupled with either SPP or HRP. The SPP wasfound to be a better alternative than HRP in the case of
putrescine sensor, giving higher sensitivity and lower
detection limit. This result might be true even for other
enzymes (oppositely charged to SPP), considering the
observed importance of the isoelectric point of the used
enzymes. A better resistance of the SPP-based hydrogen
peroxide electrodes in non-aqueous phase was also
proved. Accordingly, SPP is a promising alternative tothe commercially available HRP, also indicating the
importance of purifying and using enzymes from
different sources as a way of improving existing
biosensor designs.
Acknowledgements
The authors thank the Swedish Institute (JC), and theINTAS Project Number 00-0751, for financial support
and Dr Ivo Frebort for the kind gift of the amine
oxidase.
Fig. 3 (Continued)
J. Castillo et al. / Biosensors and Bioelectronics 18 (2003) 705�/714712
References
Appelqvist, R., Marko-Varga, G., Gorton, L., Torstensson, A.,
Johansson, G., 1985. Enzymatic determination of glucose in a
flow system by catalytic oxidation of the nicotinamide coenzyme at
a modified electrode. Anal. Chim. Acta 169, 237�/247.
Boguslavsky, L., Kalash, H., Xu, Z., Beckles, D., Geng, L., Skotheim,
T., Laurinavicius, V., Lee, H.S., 1995. Thin film bienzyme
amperometric biosensors based on polymeric redox mediators
with electrostatic bipolar protecting layer. Anal. Chim. Acta 311
(1), 15�/21.
Brooks, S.L., Higgins, I.J., Newman, J.D., Turner, A.P.F., 1991.
Biosensors for process control. Enzyme Microb. Technol. 13 (12),
946�/955.
Carrea, G., Riva, S., 2000. Properties and synthetic applications of
enzymes in organic solvents. Angew Chem. Int. Edit. 39 (13),
2226�/2254.
Cass, A.E.G. (Ed.), 1990. Biosensors: A Practical Approach. IRL Press
at Oxford University Press, Oxford.
Castillo, J.L., Alpeeva, I.S., Chubar, T.A., Galaev, I.Y., Csoregi, E.,
Sakharov, I.Y., 2002. Purification and substrate specificity of
peroxidase from sweet potato tubers. Plant Sci. 163 (5), 1011�/1019.
Collins, A., Mikeladze, E., Bengtsson, M., Kokaia, M., Laurell, T.,
Csoregi, E., 2001. Interference elimination in glutamate monitoring
with chip integrate enzyme microreactors. Electroanalysis 13 (6),
425�/431.
Cowell, D.C., Abass, A.K., Dowman, A.A., Hart, J.P., Pemberton,
R.M., Young, S.J., 2001. Screen-printed disposable biosensors for
environmental pollution monitoring. Environ. Sci. Res. 56, 157�/
174.
Csoregi, E., Gorton, L., Marko-Varga, G., 1993a. Carbon fibres as
electrode materials for the construction of peroxidase-modified
amperometric biosensors. Anal. Chim. Acta 273 (1�/2), 59�/70.
Csoregi, E., Jonsson-Pettersson, G., Gorton, L., 1993b. Mediatorless
electrocatalytic reduction of hydrogen peroxide at graphite electro-
des chemically modified with peroxidases. J. Biotech. 30 (3), 315�/
337.
Csoregi, E., Gaspar, S., Niculescu, M., Mattiasson, B., Schuhmann,
W., 2001. Amperometric enzyme-based biosensors for application
in food and beverage industry. In: De Cuyper, M., Bulte, J.W.M.
(Eds.), Physics and Chemistry Basis of Biotechnology. Kluwer
Academic Publisher, London, pp. 105�/129.
Dennison, M.J., Turner, A.P.F., 1995. Biosensors for environmental
monitoring. Biotechnol. Adv. 13 (1), 1�/12.
Dunford, H.B., 1991. Horseradish peroxidase: structure and kinetic
properties. In: Everse, J., Everse, K.E., Grisham, M.B. (Eds.),
Peroxidases in Chemistry and Biology. CRC, Boca Raton, pp. 1�/
24.
Gaspar, S., Popescu, I.C., Gazaryan, I.G., Gerardo Bautista, A.,
Sakharov, I.Y., Mattiasson, B., Csoregi, E., 2000. Biosensors based
on novel plant peroxidases: a comparative study. Electrochim.
Acta 46 (2�/3), 255�/264.
Gorton, L., Jonsson-Pettersson, G., Csoregi, E., Johansson, K.,
Dominguez, E., Marko-Varga, G., 1992. Amperometric biosensors
based on apparent direct electron transfer between electrodes and
immobilised peroxidases. Analyst 117 (8), 1235�/1241.
Gorton, L., Lindgren, A., Larsson, T., Munteanu, F., Ruzgas, T.,
Gazaryan, L., 1999. Direct electron transfer between heme-contain-
ing enzymes and electrodes as basis for third generation biosensors.
Anal. Chim. Acta 400 (1�/3), 91�/108.
Gregg, B.A., Heller, A., 1990. Cross-linked redox gels containing
glucose oxidase for amperometric biosensor applications. Anal.
Chem. 62 (3), 258�/263.
Harik, S.I., Sutton, C.H., 1979. Putrescine as a biochemical marker of
malignant brain tumors. Cancer Res. 39 (12), 5010�/5015.
Karube, I., Nomura, Y., 2000. Enzyme sensors for environmental
analysis. J. Mol. Cat. B: Enzym. 10 (1�/3), 177�/181.
Karube, I., Yano, K., Sasaki, S., Nomura, Y., Ikebukuro, K., 1998.
Biosensors for environmental monitoring. Ann. New York Acad.
Sci. 864, 23�/36.
Katakis, I., Heller, A., 1997. Electron transfer via redox hydrogels
between electrodes and enzymes. In: Scheller, F.W., Schubert, F.,
Fedrowitz, J. (Eds.), Frontiers in Biosensorics Fundamental
Aspects. Birkhauser Verlag, Basel, pp. 229�/241.
Kenausis, G., Chen, Q., Heller, A., 1997. Electrochemical glucose and
lactate sensors based on ‘‘Wired’’ thermostable Soybean Perox-
idase operating continuously and stably at 37 8C. Anal. Chem. 69
(6), 1054�/1060.
Kulys, J., Pesliakiene, M., Samalius, A., 1981. The development of
bienzyme glucose electrodes. Bioelectrochem. Bioenerg. 8, 81�/88.
Lindgren, A., Emneus, J., Ruzgas, T., Gorton, L., Marko-Varga, G.,
1997. Amperometric detection of phenols using peroxidase-mod-
ified graphite electrodes. Anal. Chim. Acta 347 (1�/2), 51�/62.
Maehly, A.C., 1955. Plant peroxidase. In: Colowick, S.P., Kaplan,
N.O. (Eds.), Methods in Enzymology, Vol. II, Academic Press,
Inc., New York, pp. 807.
Mao, L., Yamamoto, K., 2000. Amperometric on-line sensor for
continuos measurement of hypoxanthine based on osmium-poly-
vinilpyridine gel polymer and xhantine oxidase bienzyme modified
glassy carbon electrode. Anal. Chim. Acta 415, 143�/150.
Marco, M.P., Barcelo, D., 2000. Fundamentals and applications of
biosensors for environmental analysis. Tech. Instrumen. Anal.
Chem. 21, 1075�/1105.
Munteanu, F., Lindgren, A., Emneus, J., Gorton, L., Ruzgas, T.,
Csoregi, E., Ciucu, A., Van Huystee, R.B., Gazaryan, G.,
Lagrimini, L., 1998. Bioelectrochemical monitoring of phenols
and aromatic amines in flow injection using novel plant perox-
idases. Anal. Chem. 70 (13), 2596�/2600.
Munteanu, F.D., Gorton, L., Lindgren, A., Ruzgas, T., Emneus, J.,
Csoregi, E., Gazaryan, I.G., Ouporov, I.V., Mareeva, E.A.,
Lagrimini, L.M., 2000. Direct and mediated electron transfer
catalyzed by anionic tobacco peroxidase: effect of calcium ions.
Appl. Biochem. Biotechnol. 88 (1�/3), 321�/333.
Niculescu, M., Nistor, C., Frebort, I., Pec, P., Mattiason, B., Csoregi,
E., 2000. Redox hydrogel-based amperometric bienzyme electrodes
for fish freshness monitoring. Anal. Chem. 72 (7), 1591�/1597.
Ohara, T.J., Vreeke, M.S., Battaglini, F., Heller, A., 1993. Bienzyme
sensors based on ‘electrically wired’ peroxidase. Electroanalysis 5
(9�/10), 825�/831.
Ohara, T.J., Rajagopalan, R., Heller, A., 1994. Wired enzyme
electrodes for amperometric determination of glucose or lactate
in the presence of interfering substances. Anal. Chem. 66 (15),
2451�/2457.
Pazur, J.H., Kleppe, K., 1964. The oxidation of glucose and related
compounds by glucose oxidase from Aspergillus niger . Biochem-
istry 3, 578�/583.
Perez, E.F., De Oliveira Neto, G., Kubota, L.T., 2001. Bi-enzymatic
amperometric biosensor for oxalate. Sens. Actuators B: Chem. 72
(1), 80�/85.
Pons, M.N., 1993. Biosensors for fermentation control. Curr. Opin.
Biotech. 4 (2), 183�/187.
Rekha, K., Thakur, M.S., Karanth, N.G., 2000. Biosensors for the
detection of organophosphorous pesticides. Crit. Rev. Biotechnol.
20 (3), 213�/235.
Sakharov, I.Y., Bautista, G., Sakharova, I., Rojas, A., Pletyuschkina,
O.Y., 1999. Peroxidase in tropical plants. Rev. Colombiana Quım.
1999 (28), 97�/106.
Sakharov, I.Y., Castillo, L.J., Areza, J.C., Galaev, I.Y., 2000.
Purification and stability of peroxidase of African oil palm Elaies
guineensis . Bioseparations 9 (3), 125�/132.
Saurina, J., Hernandez-Cassou, S., Alegret, S., Fabregas, E., 1999.
Amperometric determination of lysine using a lysine oxidase
J. Castillo et al. / Biosensors and Bioelectronics 18 (2003) 705�/714 713
biosensor based on rigid-conducting composites. Biosens. Bioelec-
tron. 14 (2), 211�/220.
Sebela, M., Luhova, L., Febort, I., Faulhammer, H.G., Heinz, G.,
Hirota, S., Zajoncova, L., Stuzka, V., Pec, P., 1998. Analysis of the
active sites of copper/topa quinone-containing amine oxidases from
Lathyrus odoratus and L. sativus seedlings. Phytochem. Anal. 9 (5),
211�/222.
Shalaby, A.R., 1996. Significance of biogenic amines to food safety
and human health. Food Res. Int. 29 (7), 675�/690.
Tian, F., Zhu, G., 2002. Bienzymatic amperometric biosensor for
glucose based on polypyrrole/ceramic carbon as electrode material.
Anal. Chim. Acta 451, 251�/258.
Tkac, J., Sturdık, E., Gemeiner, P., 2000. Novel glucose non-
interference biosensor for lactose detection based on galactose
oxidase-peroxidase with and without co-immobilised b-galactosi-
dase. Analyst 125 (7), 1285�/1289.
Tombelli, S., Mascini, M., 1998. Electrochemical biosensors for
biogenic amines: a comparison between different approaches.
Anal. Chim. Acta 358 (3), 277�/284.
Van Der Klei, I.J., Bystrykh, L.V., Harder, V., 1990. Alcohol oxidase
from Hansenula polymorpha CBS 4732. Methods Enzymol. 188,
420�/427.
Vijayakumar, A.R., Csoregi, E., Heller, A., Gorton, L., 1996. Alcohol
biosensors based on coupled oxidase-peroxidase systems. Anal.
Chim. Acta 327 (3), 223�/234.
Vreeke, M.S., Yong, K.T., Heller, A., 1995. Thermostable Hydrogen
Peroxide sensor based on ‘Wiring’ of Soybean Peroxidase. Anal.
Chem. 67 (23), 4247�/4247.
Wang, J., 1999. Amperometric biosensors for clinical and therapeutic
drug monitoring: a review. J. Pharm. Biomed. Anal. 19 (1-2), 47�/
53.
Wang, J., Chen, L., Hocevar, S.B., Ogorevc, B., 2000. One-step
electropolymeric co-immobilization of glucose oxidase and heparin
for amperometric biosensing of glucose. Analyst 125 (8), 1431�/
1434.
Yang, X., Rechnitz, G.A., 1995. Dual enzyme amperometric biosensor
for putrescine with interference suppression. Electroanalysis 7 (2),
105�/108.
J. Castillo et al. / Biosensors and Bioelectronics 18 (2003) 705�/714714