Oxidase enzyme immobilisation through electropolymerised films toassemble biosensors for batch and flow injection analysis

Mihaela Badea a,*, Antonella Curulli b, Giuseppe Palleschi a

a Dipartimento di Scienze e Tecnologie Chimiche, Universita di Roma ‘Tor Vergata’, Via della Ricerea Scientifica, 00133 Rome, Italyb CNR Istituto per lo Studio di Materiali Nanostrutturati Sezione Roma 2, Rome, Italy

Received 24 June 2002; received in revised form 26 November 2002; accepted 2 December 2002

Abstract

Glucose oxidase, lactate oxidase, L-aminoacid oxidase and alcohol oxidase were immobilised on new films based on 2,6-

dihydroxynaphthalene (2,6-DHN) copolymerised with 2-(4-aminophenyl)-ethylamine (AP-EA) onto the Pt electrodes. The

electropolymerisation was performed by cyclic voltammetry. Different scan rates and scan potential ranges were investigated and

selected according to the monomers used. These sensors were tested for hydrogen peroxide, ascorbic acid and acetaminophen by

cyclic voltammetry and amperometry. The amperometric studies were carried out in batch as well as in a flow injection analysis

(FIA) system. Analytical parameters such as reproducibility, interference rejection, response time, buffer, storage and operational

time of the sensors have been studied. These films were also characterised by X-ray photoelectron spectroscopy (XPS). Different

strategies for enzyme immobilisation were performed and discussed: enzyme entrapment in the film during the electropolymerisation

and covalent attachment of the enzyme to the film via a carbodiimide (1-ethtl-3-(3-dimethylaminopropyl)carbodiimide, EDC) or

glutaraldehyde. Different parameters were considered in order to optimise the immobilisation procedures. Results provide a guide to

design high sensitive, stable and interference-free biosensors. In addition, studies were performed using these probes in an original

FIA based on solenoidal valves. Sensor stability, life time and dynamic range were also optimised in these conditions.

# 2003 Elsevier Science B.V. All rights reserved.

Keywords: Electropolymerisation; 2-(4-Aminophenyl)-ethylamine; 2,6-Dihydroxynaphtalene; Oxidases; Flow injection analysis

1. Introduction

Electrochemically deposited polymers have shown

considerable interest among the scientific community

for the preparation of enzyme membrane and their use

for biosensor assembling and application. It has been

reported that an accurate control over the charge passed

during the polymer formation allows a control over the

thickness of the deposited enzyme layers (Emr and

Yacynych, 1995). So, enzyme entrapment within elec-

tropolymerised films has recently been confirmed as a

versatile and powerful immobilisation technique for the

construction of enzyme electrode probes.

Since the pioneering works of Zambonin (Malitesta et

al., 1990) and Yacynych (Sasso et al., 1990), a particular

attention in the last decade was focused on electro-

synthesised polymers for biosensor designing (Garjonyte

and Malinauskas, 1999; Vidal et al., 1999; Palmisano et

al., 2000).

In the field of biosensors, the ‘electrochemical im-

mobilisation’ technique was introduced as an alternative

way for enzyme entrapment in a polymer matrix

(Malitesta et al., 1990; Sasso et al., 1990). This very

simple technique involves the electrosynthesis of the film

on the electrode surface, starting from a solution

containing the monomer and the enzyme. This techni-

que is one-step, fast and only electrochemical. However,

a drawback of this approach is the enzyme leaching

from the film.

On the other hand, covalent attachment of the

enzyme onto the polymer surface provides a simple

* Corresponding author. Tel.: �/39-06-7259-4423; fax: �/39-06-

7259-4328.

E-mail address: mihaela.badela@uniroma2.it (M. Badea).

Biosensors and Bioelectronics 18 (2003) 689�/698

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)00036-8

method to assemble reproducible enzyme electrodes.

Different coupling reagents can be used, the most used

being the glutaraldehyde (Madaras and Buck, 1996 and

references cited therein; Curulli et al., 2001) and 1-ethtl-

3-(3-dimethylaminopropyl)carbodiimide (EDC) with N -

hydroxy-succinimide (NHS) (Situmorang et al., 1998).

The immobilisation of the enzyme onto the surface of

the polymer did, however, increase the biosensor

response time.

The non-conduction electropolymerised films can be

successfully used for biosensor design because of their

perm-selectivity for hydrogen peroxide over interferents

as ascorbic acid and acetaminophen (Christie and

Vadgama, 1993). The non-conducting film acts as

selective barrier for the rejection of the electroactive

interferents on the basis of the charge and size exclusion

(Warriner et al., 1996).

Recently, an electrochemically depositable non-con-

ducting polymer that has received attention was poly-

tyramine (4-hydroxyphenethylamine) (Situmorang et

al., 1998, 2002), because of the presence of free amino-

groups on the polymer backbone, which are ideal for the

covalent attachment of enzymes. On the other hand, the

limitation of polytyramine was determined by the

polymerisation conditions (0.3 M NaOH in methanol

solution), which were incompatible with activity main-

taining of the enzymes.

An interesting approach for the electropolymerised

film based biosensors is the immobilisation of the

enzyme throughout the polymer layer, which is reported

to improve dramatically the sensitivity and the dynamic

range of the biosensor (Situmorang et al., 1999).

The purpose of this work is to demonstrate the

versatility of the new non-conducting films proposed

by us, not only as a cut-off membrane to reject the

electrochemical interferences but also as an immobilisa-

tion matrix for oxidase based biosensors. For the first

time, in our knowledge, the electropolymerisation of the

new monomer 2-(4-aminophenyl)-ethylamine (AP-EA),

which has a similar structure with tyramine, was

reported.

Moreover, a copolymer between AP-EA and 2,6-

dihydroxynaphthalene 2,6-dihydroxynaphthalene (2,6-

DHN), which improved the stability and the interfer-

ence rejection properties of the poly(AP-EA), was

electrosynthesised. The poly(substituted naphthalenes)

resulted in a better probe stability and rejection of the

interferences (Badea et al., 2001).

High probe performances were attained using this

copolymer and the glucose oxidase (GOD) as a

model enzyme. The ability of such copolymer to provide

a general system to assemble biosensors was demon-

strated by assembling enzyme electrode probes based on

L-amino acid oxidase, lactate oxidase and alcohol

oxidase.

2. Experimental

2.1. Reagent and solutions

The following monomers: 2,6-DHN and AP-EA werereceived from Aldrich (Steinheim, Germany).

Glucose oxidase type VII from Aspergillus niger 200

U/mg (Sigma Chemical Co, St. Louis, MO), alcohol

oxidase from Pichia pastoris 464 U/ml (Fluka, Buchs,

Switzerland), L-aminoacid oxidase from Crotalus ada-

manteus 0.5 U/mg (Fluka), lactate oxidase from Ped-

ioccoccus sp. 40 U/mg (Sigma) were used as received.

Ascorbic acid, acetaminophen, L-(�/)-lactic acid(lithium salt) were received from Sigma and hydrogen

peroxide 30% from J.T. Baker (Deventer, Netherlands).

D-(�/)-glucose and absolute ethanol were purchased

from Carlo Erba (Milan, Italy) and L-leucine from

Merck (Darmstadt, Germany).

Glutaraldehyde grade I: 25% aqueous solution

(Sigma), EDC (Sigma), NHS (Aldrich) and albumin

from bovine serum (BSA) (Fluka) were used forcovalent coupling of enzymes.

Phosphate buffers (0.1 M) were prepared with bidis-

tilled-deionized water using sodium dihydrogen phos-

phate dihydrate (Fluka). For pH adjustments, pellets of

sodium hydroxide were used.

Diluted solutions were prepared just before use.

Glucose stock solutions were allowed to mutarotate

overnight at room temperature before use.

2.2. Apparatus

An AMEL polarographic system Model 433A

(AMEL, Milan, Italy) was used for voltammetric

studies.

Amperometric measurements in batch system were

carried out with an AMEL detector model 559. Currentswere recorded with a LKB 2210 recorder (Delft, Hol-

land). For batch measurements platinum (Pt) working

electrodes (2 or 3 mm) (Model 492) from AMEL, an

auxiliary Pt electrode and an Ag/AgCl (3 M KCl)

reference electrode (BAS, Bioanalytical System West

Lafayette, IN, USA) were used.

For the FIA system a Metrohm wall-jet cell (Model

656) (Metrohm, Herisau, Switzerland) was assembledwith the modified Pt electrodes, used for batch analysis.

A peristaltic pump Minipuls 3 (Gilson, France) and an

injection system based on solenoidal valves (Bio-Chem

Valve Inc, NJ, USA) were used. The injected volume

was 50 ml. For FIA determinations we used a measure-

ment system and a software by S. Kalinowsy (Olsztyn,

Poland). The PTFE connection tubes (i.d.�/0.5 mm)

were from Supelco (Bellofonte, CA, USA).X-ray photoelectron spectroscopy (XPS) was per-

formed with a VG Scientific ESCALAB MKII, using

a sorgent Al K (1486.6 eV).

M. Badea et al. / Biosensors and Bioelectronics 18 (2003) 689�/698690

2.3. Electrode cleaning

The platinum electrode surface was polished with 1,

0.3 and 0.05 mm alumina slurry (Al2O3, Buehler,Evanston, IL, USA), washed with distilled water

followed by sonication for 10 min. Electrodes were

pre-treated by potential cycling in 0.5 M H2SO4 from �/

0.2 to�/1.2 V (vs. SCE) at a scan rate of 20 mV/s, until

no changes were observed in the cyclic voltammograms

(Curulli et al., 2001).

2.4. Electropolymerisation

2,6-DHN (0.5�/1 mM) and AP-EA (10�/100 mM)

were electropolymerised on the Pt electrode by cycling

voltammetry. Both the monomers were dissolved in 0.1

M phosphate buffer solution, pH 7.4 and the resultingsolutions were deareated for 15 min with argon. The

potential was continuously cycled from 0 to �/1200 mV

for 2,6-DHN, from �/300 to �/1300 mV for AP-EA and

from �/150 to 1300 mV for the copolymerisation of 2,6-

DHN with AP-EA. The potential was continuously

cycled until a minimum value of current, which

remained constant after further cycling, was observed.

2.5. Biosensor assembling

Different strategies were used to immobilise the

enzyme, for GOD biosensor assembling.

2.5.1. (a) Via EDC/NHS after copolymer formation

The copolymer modified electrode was dipped in a

solution containing 200 U/ml GOD, 0.015 M EDC and

0.03 M NHS in 0.1 M phosphate buffer, pH 6.3 andstirred for 1.5 h.

2.5.2. (b) Via EDC/NHS with an initial preactivation of

the film (three steps)

1) The copolymer modified electrode was dipped in asolution containing 0.015 M EDC and 0.03 M NHS

in 0.1 M phosphate buffer, pH 6.3 and stirred for 1

h.

2) The preactivated film was washed with distilled

water.

3) Finally, the electrode was immersed in a solution

countering 400 U/ml GOD and stirred for 1 h.

2.5.3. (c) Via glutaraldehyde and BSA after polymer or

copolymer formation

Successive additions of 2 ml solution of 1000U/ml

GOD, 1% BSA and 0.04% glutarlaldehyde in 0.1 M

phosphate buffer, pH 7.4, were quickly deposited on theworking modified electrodes, using a microsyringe. The

electrodes were allowed to cross-link in air, at room

temperature.

2.5.4. (d) Entrapment of GOD during the

electropolymerisation

The electropolymerisation was carried out in the

presence of monomer/monomers (0.5�/10 mM) and 500U/ml GOD in 0.1 M phosphate, pH 7.4 (potential range:

100�/1150 mV; scan rate: 10 mV/s; 10�/20 cycles).

2.5.5. (e) Entrapment of GOD during the

electropolymerisation followed by the cross-linking via

EDC and NHS

The modified electrode, prepared as in (d), was

immersed in a solution of 0.015 M EDC and 0.03 MNHS in 0.1 M phosphate buffer, pH 6.3 and stirred for

1.5 h.

2.5.6. (f) Immobilisation of the GOD throughout the

polymer layer during the copolymer formation in the

presence of glutaraldehyde

The electropolymerisation was carried out in the

presence of the monomers (0.5�/10 mM), 500 U/ml

GOD and 0.01% glutaraldehyde in 0.1 M phosphate,pH 7.4 (potential range: 100�/1150 mV; scan rate: 10

mV/s; 10�/20 cycles).

For the other oxidase based biosensors, we used

procedure (f), the activity of the LOD, AOD and L-

AAOD being 40, 10 and, respectively, 1.8 U/ml. The

glutaraldehyde concentration was 0.01% for LOD

biosensor assembling and, respectively, 0.005% for

AOD and L-AAOD biosensor.

2.6. Electrochemical measurements

The polymer films were characterised by cyclic

voltammetry (range �/400 to �/1000 mV, scan rate 20

mV/s) and amperometry (E�/�/650 mV vs. Ag/AgCl) in

presence of hydrogen peroxide, ascorbic acid and

acetaminophen.In order to evaluate the biosensor performance, the

hydrogen peroxide produced by the enzyme reaction

was oxidised at the electrode by applying a constant

potential of �/650 mV versus Ag/AgCl.

Before each addition of the substrate, the current was

allowed to reach a constant baseline in phosphate buffer

(0.1 M, pH 7.4). Additions of the analyte were made

from a stock solution under stirring. The current wasrecorded continuously and further additions were made

once the current reached a stable steady state.

3. Results and discussion

3.1. Electropolymerisation and characterisation of the

polymeric films

Cyclic voltammograms for electrodeposition of 2,6-

DHN and for AP-AE are presented in Figs. 1 and 2. The

M. Badea et al. / Biosensors and Bioelectronics 18 (2003) 689�/698 691

decrease of the oxidation current after the first cycle

indicates the passivation of the electrode surface by the

films. The polymers formed were transparent and

strongly adherent on the surface of the platinum

electrode.

The presence of the free amino-groups on the

poly(AP-EA) surface permits the covalent attachment

of the enzymes via peptide bond formation. However,

during stability studies, poly(AP-EA) showed a poor

stability in time. In order to improve this stability and to

maintain the presence of the amino groups in the

polymer structure, a possible copolymerisation of the

AP-EA with 2,6-DHN was studied. Fig. 3a. shows the

cyclic voltammogram registered for the copolymer

poly(2,6-DHN�/AP-EA) formation. It can be observed

that for the first cycle two oxidation peaks are present,

which clearly indicate the copolymer synthesis. Different

ratios of 2,6-DHN/AP-EA were studied (data not

shown) and the best results in terms of stability and

interference rejection were obtained using a mixture 0.5

mM 2,6-DHN and 10 mM AP-EA.

The permeability of the new polymeric films to

hydrogen peroxide, ascorbic acid and acetaminophen

was tested. The permeability (P ) was defined as follows:

P�Ifilm

Ibare

� 100

where, Ifilm refers to the current measured at theelectrode covered with the film and Ibare is the current

measured at the naked electrode. Ascorbic acid and

acetaminophen were used as model molecules to test the

interference rejection of the films.

For each polymer, the preparation conditions were

optimised by study of the scan range, the scan rate and

the number of cycles. Fig. 4 shows the effect of scan rate

on the film rejection of the interferent, ascorbic acid. Itcan be seen that poly(2,6-DHN) and the copolymer

obtained at low scan rates (5�/10 mV/s) are characterised

by a very good rejection of ascorbic acid, while poly(AP-

Fig. 1. Cyclic voltammogram for electropolymerisation of 1 mM 2,6-

DHN at a scan rate of 10 mV/s in phosphate buffer 0.1 M, pH 7.4.

Fig. 2. Cyclic voltammogram for electropolymerisation of 100 mM

AP-EA at a scan rate of 50 mV/s in phosphate buffer 0.1 M, pH 7.4.

Fig. 3. (a) Cyclic voltammogram for copolymerisation of 0.5 mM 2,6-

DHN with 10 mM AP-EA at a scan rate of 10 mV/s in phosphate

buffer 0.1 M, pH 7.4. (b) Cyclic voltammogram for GOD biosensor

preparation using procedure (f).

M. Badea et al. / Biosensors and Bioelectronics 18 (2003) 689�/698692

EA) with the lowest permeability for the intereferent

was obtained at higher scan rate (50 mV/s).

Table 1 reports the permeability for hydrogen perox-

ide, ascorbic acid and acetaminophen for the films

obtained in optimised conditions. It is also reported

the permselectivity of the films for hydrogen peroxide

towards the interferents (ascorbic acid and acetamino-

phen). The permselectivity was calculated as the ratio

Phydrogen peroxide/Pinterferent.

It can be observed that the new synthesised films are

characterised by a high permeability for hydrogen

peroxide and by a very good rejection of the interferents.

This permeability of these films is due mainly to the size

exclusion properties of the polymeric films, which

permits the diffusion of the hydrogen peroxide (a small

and uncharged molecule) to the electroactive surface of

the electrode.

The ferro�/ferricyanide redox couple has been used to

check for pinholes and gross defects of the non-

conducting films (Long et al., 2001). All the films

prepared in our study suppressed the reaction of the

ferro�/ferricyanide couple at 0.25 V versus Ag/AgCl.

These results showed the presence of a continuous,

pinhole free non-conducting film on the electrodes

surface, which prevented the oxidation of ferro�/ferri-cyanide couple. The poly(2,6-DHN) and the copolymer

showed no electrochemistry of the ferro�/ferricynide

system after 3 months storage (in phosphate buffer pH

7.4, stored in the refrigerator), which indicates the great

improvement of the polymer stability when compared

with the poly(AP-EA) which started to deteriorate after

1 week storage.

The polymeric films were characterised using the XPStechnique (Table 2). From these results we can assume

that, during the polymerisation of AP-EA a link is

formed through the aromatic amino groups, while the

aminoethyl group remains available for covalent attach-

ment of the enzyme. In the case of the copolymer,

though the number of the free aminoethyl groups is

lower, the possibility of the enzyme coupling still occurs.

For biosensors assembling we choose to work withthe copolymer because its characteristics are a compro-

mise among presence of the free amino groups, the

stability in time, and the interference rejection.

3.2. Biosensors characterisation

In our studies GOD was used as a model enzyme for

the evaluation and optimisation of the new copolymer

(2,6-DHN�/AP-AE) as an immobilisation matrix.

3.2.1. GOD biosensor

Table 3 reports the analytical characteristics of the

GOD sensors relating to different immobilisation pro-

cedures. The interference rejection was evaluated mea-suring the polymer permeability to 1 mM ascorbic acid.

After all procedures for GOD immobilisation were

performed, we observed that the sensitivity and the

linear range for glucose determination and the perme-

ability for ascorbic acid were different. When EDC and

NHS were used in absence of the enzyme in the same

mixture (procedure (b) and (e), entries 2, 5 and 9) a poor

rejection of the ascorbic acid was observed, but thiseffect did not appear in procedure (a) (entry 1). We

suppose that the electropolymerised film was affected by

EDC and NHS, which induced the formation of pin-

Fig. 4. Influence of the scan rate used in the film preparation on the

permeability to 1 mM ascorbic. Conditions for film preparation: (I)

poly(2,6-DHN) c2,6-DHN�/1 mM; range: 0�/1200 mV; 20 cycles (II)

poly(AP-EA) cAP-EA�/100 mM; range: 300�/1300 mV; 20 cycles (III)

poly(2,6-DHN�/AP-EA) c2,6-DHN�/0.5 mM; cAP-EA�/10 mM; range:

150�/1300 mV; ten cycles.

Table 1

Permeability of electropolymerised films to 1mM hydrogen peroxide, 1 mM ascorbic acid and 1 mM acetaminophen (E�/�/650 mV)

Film Phydrogen peroxide (%) Pascorbic acid (%) Pacetaminophen (%) Phydrogen peroxide/Pascorbic acid Phydrogen peroxide/Pacetaminophen

Poly(2,6-DHN) 89.39/1.32 1.259/0.70 2.109/0.38 71.44 42.52

Poly(AP-EA) 95.69/1.44 0.629/0.45 3.579/0.56 154.19 26.77

Poly(2,6-DHN�/AP-EA) 93.39/1.76 0.799/0.40 2.319/0.61 118.10 40.39

Conditions for films preparation: (IV) poly(2,6-DHN) c2,6-DHN�/1 mM; scan rate 5 mV/s; range: 0�/1200 mV; 20 cycles; (V) poly(AP-EA) cAP-

EA�/100 mM; scan rate 50 mV/s; range: 300�/1300 mV; 20 cycles; (VI) poly(2,6-DHN�/AP-EA) c2,6-DHN�/0.5 mM; cAP-EA�/10 mM; scan rate 10

mV/s; range: 150�/1300 mV; ten cycles.

M. Badea et al. / Biosensors and Bioelectronics 18 (2003) 689�/698 693

holes. To clarify this point, we checked the rejection of

ascorbic acid and acetaminophen immediately after the

copolymer modified electrode was prepared, and then

after it was left immersed for 1.5 h in a solution

containing EDC and NHS. Indeed, the permeability of

the membrane versus interferences increased drastically

after the treatment with the coupling reagents. The good

results, obtained in the procedure (a), probably are due

to the fact that in this case the enzyme is acting as a

protective agent for the membrane against EDC and

NHS.

In the case of covalent attachment of the GOD on the

polymer surface via glutaraldehyde and BSA (procedure

(c), entries 3 and 7), the sensitivity for glucose determi-

nation was lower than that via EDC and NHS, also the

interference rejection was not satisfactory.

For the biosensor with GOD entrapped in the

membrane, during the electropolymerisation (procedure

(d), entries 4 and 8), even the rejection of the ascorbic

acid was the highest, but the response to glucose was

very low.

The best results were obtained for the GOD immo-

bilised throughout the polymer layer during the copo-

lymer formation in the presence of glutaraldehyde

(procedure (f), entry 6). Fig. 3b shows the cyclic

voltammogram for the GOD biosensor preparation

using procedure (f). In this figure a decrease of the

oxidation peak after the first cycle can be observed. This

demonstrates the formation of the non-conducting film.

This procedure was then used for further studies.

Fig. 5 shows the influence of the glutaraldehyde

concentration in the polymerisation media on the

retained enzymatic activity. When the electropolymer-

isation was performed immediately after mixing the

monomers with GOD and glutaraldehyde, the response

for glucose was 30% lower than that obtained when the

electropolymerisation was started after 30 min from the

reagents mixing. An explanation of this could be that,

before the electropolymerisation step, the AP-EA is

covalently coupled to the enzyme via glutarladehyde.

In the case of biosensors prepared using 0.02%

glutaraldehyde, after 10 days it was observed a drastic

decrease of the response for glucose and an increase of

the permeability for ascorbic acid and acetaminophen.

The influence of the number of cycles performedduring the electropolymerisation on the response of

GOD/copolymer/Pt electrode was checked for five, ten,

15 and 20 cycles. If more than ten cycles were

performed, the GOD biosensors showed a very good

rejection for ascorbic and acetaminophen, but a low

response for glucose because of the reduced access of the

substrate (glucose) to the catalytic centre of the enzyme.

For five cycles of electropolymerisation, the GODsensors presented a high permeability for interferences.

Further, all the oxidase/copolymer/Pt electrodes were

prepared performing ten cycles with a scan rate of 10

mV/s (see Section 3.1).

Fig. 6 shows GOD biosensor calibration graphs

obtained during days.

The very good stability during time of this new

biosensor is shown also in Fig. 7. Stability of theGOD/copolymer/Pt electrode was checked for glucose,

ascorbic acid and acetaminophen over a period of 60

days. Between measurements the biosensor was kept in

phosphate buffer (0.1 M, pH 7.4) at 4 8C.

Stability studies were also performed for the GOD

biosensors storing them dry or in phosphate buffer, at

the room temperature or at 4 8C. The best stability for

GOD/copolymer/Pt electrode was obtained, whenstored in phosphate buffer (0.1 M, pH 7.4) at 4 8C.

The amperometric response of GOD/copolymer/Pt

electrode in batch analysis indicated satisfactory dy-

namic properties of this sensor. The response time was

25 s calculated as the time necessary for reaching 90% of

the maximum signal.

A suitable test of this probe was its application in flow

analysis conditions. These studies were performed in aone-line flow injection analysis (FIA)system, where

glucose solution was injected into a stream of phosphate

buffer (0.1 M, pH 6.5). The injection of the sample was

performed using a solenoidal valve and selecting the

time necessary for injecting the volume of 50 ml sample.

The flow rate was 0.30 ml/min.

The resulting flow-injection peaks were well shaped,

also we obtained a linear response for glucose within therange 0.1�/10 mM. A comparison with the glucose

determinations in batch showed an extension of the

dynamic range and, as expected, a decrease of the

sensitivity (448.5 nA/mM). Fig. 8 shows a FIA registra-

Table 2

XPS results for poly(2,6-DHN), poly(AP-EA) and copolymer (2,6-DHN-AP-EA)

Atom Type Binding energy (eV) Poly(2,6-DHN) (% atom) Poly(AP-EA) (% atom) Copolymer (2,6-DHN with AP-EA) (% atom)

C Aromatic 285.0 58.1 56.0 58.7

C�/OH 286.4 15.2 15.5 15.6

N NH2�/Ph 399.0 �/ 1.8 �/

H2N�/C 400.2 �/ 4.5 2.8

O 533.6 21.4 17.9 18.7

Impurities SiO2 102.3 5.3 4.4 4.2

M. Badea et al. / Biosensors and Bioelectronics 18 (2003) 689�/698694

Table 3

Analytical characteristics for the GOD sensors obtained used different immobilisation strategies

Number of

crt.

Sensor Diameter

(mm)

Immobilisation proce-

dure

Detection limit

(mM)

Linear range

(mM)

Sensitivity (mA/

mM)

RSD

(%)

Pascorbic acid

(%)

Response time

(s)

Stability in time

(days)

1 GOD/copolymer/

Pt

2 a 0.05 0.1�/2 0.324 3.43 1.22 25 15

2 GOD/copolymer/

Pt

2 b 0.1 0.2�/2 0.386 4.12 14.11 20 8

3 GOD/copolymer/

Pt

2 C 0.1 0.2�/2.5 0.204 3.90 6.45 25 10

4 GOD/copolymer/

Pt

3 D 0.1 0.1�/1.0 0.088 3.25 2.05 25 15

5 GOD/copolymer/

Pt

3 E 0.05 0.1�/2.0 0.218 2.87 12.85 20 7

6 GOD/copolymer/

Pt

2 F 0.01 0.05�/4.0 1.133 1.88 1.64 25 60

7 GOD/poly(AP-

EA)/Pt

3 C 0.05 0.1�/2.5 0.253 3.26 5.22 25 5

8 GOD/poly(AP-

EA)/Pt

3 D 0.1 0.1�/1.4 0.076 2.92 1.98 25 12

9 GOD/poly(AP-

EA)/Pt

3 E 0.05 0.1�/2.0 0.195 2.23 18.68 25 8

E�/�/650 mV vs. Ag/AgCl; in 0.1 M phosphate buffer, pH 7.4.

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tion obtained for glucose determination using the GOD

biosensor. The RSD for 15 consecutive injections of 5

mM glucose was 1.88%, which indicates a good repro-

ducibility of the GOD sensor in flow conditions. The

detection limit was estimated to be 0.07 mM glucose for

S /N�/3.

The stability of the GOD sensor in operational

conditions was evaluated by successive injections of 2

mM glucose. After 10 h, only 4.5% of the enzymatic

activity was lost.

3.3. Applications of the copolymer (2,6-DHN�/AP-EA)

to other oxidase based biosensors

The versatility of the new copolymer (2,6-DHN�/AP-

EA) as an immobilisation matrix and its rejection of

interferents was also checked assembling enzyme elec-

trodes for ethanol, lactate and L-leucine by immobilisa-

tion of the respective oxidase enzymes as for GOD.

The performances of the four different enzyme

electrodes are presented in Table 4. Relative to pub-

lished data for similar enzyme electrodes for glucose, L-

lactate, ethanol and L-leucine, the copolymer (2,6-

DHN�/AP-EA) enzyme electrodes gave similar perfor-

mances (Situmorang et al., 1999; Palmisano et al., 1997).

These results demonstrate the versatility of the copoly-

Fig. 5. Influence of the glutaraldehyde concentration in the polymer-

isation media on the GOD biosensor response to glucose (cglucose�/2

mM).

Fig. 6. Calibration plots for GOD biosensor made in different days

from the preparation. Conditions for biosensor preparation: c2,6-

DHN�/0.5 mM; cAP-EA�/10 mM; GOD activity�/500 U/ml;

cglutaraldehyde�/0.01%; scan range: 100�/1150 mV; scan rate: 10 mV/s;

ten cycles.

Fig. 7. Stability of the GOD biosensor for repeated measurements of 2

mM glucose, 1 mM ascorbic acid and 1 mM acetaminophen.

Fig. 8. FIA registration for glucose determination using the GOD

biosensor.

M. Badea et al. / Biosensors and Bioelectronics 18 (2003) 689�/698696

mer (2,6-DHN�/AP-EA) to obtain enzyme biosensors

based on oxidases that produce hydrogen peroxide.

The preparation is simple and sufficiently fast (less

than 1 h, including the deaereation step). The character-istic of this new procedure for biosensor fabrication is

the fact that the enzyme is, at the same time, entrapped

and covalently attached on the polymeric film during

the electropolymerisation step. Also the interference

rejection and the good stability showed by the polymer

films are maintained during the biosensor assembling

and use.

The main advantage of the new co-polymer poly(2,6-DHN�/AP-EA) is the very good resistance and the high

stability in flow conditions, which make this method

robust and reliable.

3.4. Real sample analysis

The GOD biosensor was used for glucose determina-

tion in some commercial products like fruit juices and

lemon tea. Recovery studies were carried out using the

GOD biosensor inserted in the flow analysis system.The samples were diluted for 100 times with phos-

phate buffer 0.1 M, pH 6.5 and were analysed without

any another treatment. Results reported in Table 5

indicate a good recovery of glucose when working with

this kind of samples.

4. Conclusions

Enzyme electrodes have been assembled using the

copolymer (2,6-DHN�/AP-EA), and GOD as a model

enzyme. The GOD biosensor based on enzyme immo-

bilisation within the copolymer exhibits good perfor-

mances, a rapid response (25 s), a low detection limit (10

mM glucose). The GOD biosensor was reproducible,

stable and showed good rejection versus common

intereferences like ascorbic acid and acetaminophen.Features of the copolymer (2,6-DHN�/AP-EA) to

provide a general single step method to prepare oxidase

electrode probes are reported.

L-aminoacid oxidase, alcohol oxidase and lactate

oxidase based probes have been assembled with the

same preparation procedure used for GOD.

Acknowledgements

The authors thank the European Community

(MCFA-2000-000725) and the CNR Target Project

MSTA II for the financial support.Tab

le4

Per

form

an

ces

of

dif

fere

nt

cop

oly

mer

(2,6

-DH

N� /

AP

-EA

)b

ase

db

iose

nso

rs

Bio

sen

sor

An

aly

teE

nzy

me

(U/m

l)G

luta

rlad

ehyd

e(%

)D

etec

tio

nli

mit

(mM

)L

inea

rra

nge

(mM

)S

ensi

tivit

y(n

A/m

M)

Pasc

orb

icacid

(%)

Res

po

nse

tim

e(s

)

GO

D/c

op

oly

mer

/Pt

Glu

cose

50

00

.01

0.0

10

.05� /4

.01

13

3.4

39

/0.0

11

.64

25

LO

D/c

op

oly

mer

/Pt

L-(�

/)-la

ctic

aci

d4

00

.01

0.0

10

.01�/0

.30

91

5.9

49

/29

.02

1.9

82

5

AO

D/c

op

oly

mer

/Pt

Eth

an

ol

10

0.0

05

0.1

00

.25� /5

.08

8.6

49

/3.6

41

.85

25

L-A

AO

D/c

op

oly

mer

/Pt

Leu

cin

e1

.80

.00

50

.08

0.1

0�/0

.70

68

.589

/0.7

11

.76

25

M. Badea et al. / Biosensors and Bioelectronics 18 (2003) 689�/698 697

References

Badea, M., Amine, A., Palleschi, G., Moscone, D., Volpe, G., Curulli,

A., 2001. New electrochemical sensors for detection of nitrites and

nitrates. J. Electroanal. Chem. 509, 66�/72.

Christie, I.M., Vadgama, P., 1993. Modification of electrode surfaces

with oxidised phenols to confer selectivity to amperometric

biosensor for glucose determination. Anal. Chim. Acta 274, 191�/

199.

Curulli, A., Dragulescu, S., Cremisini, C., Palleschi, G., 2001.

Bienzyme amperometric probes for choline and choline esters

assembled with nonconducting electrosynthesized polymers. Elec-

troanalysis 13, 236�/241.

Emr, A., Yacynych, A.M., 1995. Use of polymer films in amperometric

biosensors. Electroanalysis 7, 10�/15.

Garjonyte, R., Malinauskas, A., 1999. Amperometric glucose biosen-

sor based on glucose oxidase immobilized in poly(o -phenylendia-

mine) layer. Sensors Actuators B 56, 85�/92.

Long, D.D., Marx, K.A., Zhou, T., 2001. Amperometric hydrogen

peroxide sensor electrodes coated with electropolymerized tyrosine

derivative and phenolic films. J. Electroanal. Chem. 501, 107�/113.

Madaras, M.B., Buck, R.P., 1996. Miniaturised biosensor employing

electropolymerized permselective films and their use for cretine

assay in human serum. Anal. Chem. 68, 3832�/3839.

Malitesta, C., Palmisano, F., Torsi, L., Zambonin, P.G., 1990. Glucose

fast-response amperometric sensor based on glucose oxidase

immobilised in an electropolimerised poly(o -phenylenediamine)

film. Anal. Chem. 62, 2735�/2740.

Palmisano, F., De Benedetto, G.E., Zambonin, P.G., 1997. Lactate

miniaturised biosensor based on an lectrosunthesuzed bilayer film

with covalently immobilized enzyme. Analyst 122, 365�/369.

Palmisano, F., Rizzi, R., Centonze, D., Zambonin, P.G., 2000.

Simultaneous monitoring of glucose and lactate by an interference

and cross-talk free dual electrode amperometric biosensor based on

electropolymerized thin film. Biosens. Bioelectron. 15, 531�/539.

Sasso, S.V., Pierce, R.J., Walla, R., Yacynych, 1990. Electropoly-

merised 1.2-diaminobenzene as a means to prevent interferences

and fouling and to stabilize immobilised enzyme in electrochemical

biosensors. Anal. Chem. 62, 1111�/1117.

Situmorang, M., Gooding, J.J., Hibbert, D.B., Barnett, 1998. Electro-

deposited polytyramine as an immobilisation matrix for enzyme

biosensors. Biosens. Bioelectron. 13, 953�/962.

Situmorang, M., Gooding, J.J., Hibbert, D.B., 1999. Immobilisation of

enzyme throughout a polytyramine matrix: a versatile procedure

for fabricating biosensors. Anal. Chim. Acta 394, 211�/223.

Situmorang, M., Gooding, J.J., Hibbert, D.B., Barnett, D., 2002. The

development of a pyruvate biosensors using electrodeposited

polytyramine. Electroanalysis 14, 17�/21.

Vidal, J.-C., Garcia, E., Mendez, S., Yarnoz, P., Castillo, J.R., 1999.

Three approaches to the development of selective bilayer ampero-

metric biosensors for glucose by in situ electropolymerization.

Analyst 124, 319�/324.

Warriner, K., Higson, S., Christie, I., Ashworth, D., Vadgama, P.,

1996. Electrochemical characteristics of two model electropoly-

merised films for enzyme electrodes. Biosens. Bioelectron. 11 (6/7),

615�/623.

Table 5

Glucose determination in real samples using the GOD biosensor in flow system

Sample Found (mM) Added (mM) Expected after addition (mM) Found after addition (mM) Recovery (%)

Orange juice 132 50 182 168 92.3

Grapefruit juice 147 50 197 186 94.4

Lemon tea 123 50 173 169 97.6

M. Badea et al. / Biosensors and Bioelectronics 18 (2003) 689�/698698