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: e.csoregi@biotek.lu.se (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

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