compozite electrochimice

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Biosensors & Bioelectronics 17 (2002) 217–226

Composite electrochemical biosensors: a comparison of threedifferent electrode matrices for the construction of amperometrictyrosinase biosensors

B. Serra, S. Jimenez, M.L. Mena, A.J. Reviejo, J.M. Pingarron *

Department of Analytical Chemistry, Faculty of Chemistry, Complutense Uni ersity of Madrid , 28040 Madrid , Spain

Received 7 November 2000; received in revised form 16 July 2001; accepted 7 August 2001

Abstract

A comparison of the behaviour of three different rigid composite matrices for the construction of amperometric tyrosinase

biosensors, which are widely used for the detection of phenolic compounds, is reported. The composite electrode matrices were,

graphite–Teflon; reticulated vitreous carbon (RVC)–epoxy resin; and graphite–ethylene/propylene/diene (EPD) terpolymer. After

optimization of the experimental conditions, different aspects regarding the stability of the three composite tyrosinase electrode

designs were considered and compared. A better reproducibility of the amperometric responses was found with the graphite–EPD

electrodes, whereas a longer useful lifetime was observed for the graphite– Teflon electrodes. The kinetic parameters of the

tyrosinase reaction were calculated for eight different phenolic compounds, as well as their corresponding calibration plots. The

general trend in sensitivity was graphite–EPDgraphite–TeflonRVC–epoxy resin. A correlation between sensitivity and the

catalytic efficiency of the enzyme reaction for each phenolic substrate was found. Furthermore, differences in the sensitivity order

for the phenolic compounds were observed among the three biocomposite electrodes, which suggests that the nature of the

electrode matrix influences the interactions in the tyrosinase catalytic cycle. © 2002 Elsevier Science B.V. All rights reserved.

Keywords: Composite electrode matrices; Tyrosinase biosensors; Phenolic compounds

www.elsevier.com/locate/bios

1. Introduction

Nowadays, the use of composite electrode matrices is

well established as an efficient strategy to design robust

electrochemical biosensors. These matrices are materials

consisting of at least one conductor phase mixed with at

least one insulator phase (Tallman and Petersen, 1990),

and present several advantageous characteristics whichcan be of use in biosensors development. Probably, one

of the most important features is the versatility that can

be achieved with this type of approach. Thus, different

species improving selectivity and/or sensitivity

(biomolecules, cofactors, mediators) can be incorporated

into the bulk of the electrode matrix. In this way,

three-dimensional biocomponents reservoirs can be fab-

ricated whose surface can be easily regenerated by

polishing. In addition, fast responses to the involved

substrates can be expected because of the absence of

supporting membranes on the electrode surface and the

closeness of the biomolecules (and other components) to

the electrode material.

A lot of materials have been used to construct com-

posite electrodes. In general, carbon-based matrices are

ideal conductor phases to be used for the developmentof amperometric sensors (Cespedes et al., 1996), whereas

epoxy resins, silicone, polyurethane, metacrylate, Teflon,

etc. can be employed as insulator materials. The most

popular group of bulk-modified bioelectrodes is carbon-

paste biosensors (Byfield and Abuknestra, 1994; Gorton,

1995; Kalcher et al., 1995), but some problems associated

with a lack of long-term stability, mainly under flowing

conditions, as well as of mechanization ability and

compatibility with nonaqueous media, have led to the use

of rigid composite matrices such as graphite– epoxy

(Wang and Varughese, 1990; Alegret et al., 1996) or

graphite–Teflon (Wang et al., 1993).

* Corresponding author. Tel.: +34-91-394-4315; fax: +34-91-394-

4329.E -mail address: [email protected] (J.M. Pingarron).

0956-5663/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.

PII: S 0 9 5 6 - 5 6 6 3 ( 0 1 ) 0 0 2 6 9 - X

mailto:[email protected]



B . Serra et al . / Biosensors & Bioelectronics 17 (2002) 217 – 226 218

The aim of this work is to compare the behaviour of

three different rigid composite matrices, developed in

our research group, for the construction of amperomet-

ric tyrosinase biosensors, which are widely used for the

detection of phenolic compounds (Hedenmo et al.,

1997; Ducey and Meyerhoff, 1998; Wang et al., 1994;

Kotte et al., 1995; Nistor et al., 1995; Li et al., 1998;

Narvaez et al., 1996; Daigle and Leech, 1997; Eggins et

al., 1997; Cosnier et al., 1998). The composite electrodematrices to be compared were, graphite – Teflon; reticu-

lated vitreous carbon (RVC) – epoxy resin; and

graphite – ethylene/propylene/diene (EPD) terpolymer.

The use of graphite – Teflon pellets for the fabrication

of robust enzyme electrodes has been extensively docu-

mented by our group recently (Serra et al., 1999a,b;

Cayuela et al., 1998; del Cerro et al., 1997; Ortiz et al.,

1997), the bioelectrodes being prepared simply by inclu-

sion of the enzyme(s) into the bulk of the graphite –

Teflon pellet with no need of covalent attachments.

Furthermore, we reported recently the fabrication and

performance, in different aqueous and predominantlynonaqueous media, of a composite RVC amperometric

peroxidase – ferrocene electrode in which the insulator

material used for filling the holes of the electrode

matrix is an epoxy resin (Pena et al., 1999). No covalent

bindings were either needed to fabricate these com-

posite polishable bioelectrodes. Finally, we have re-

cently prepared a new composite material by mixing

graphite and the EPD terpolymer (Alonso et al., 1999),

a rubbery material with a 70 wt.% ethylene and a 4

wt.% 5-methylene-2-norbornene content. This material,

with EPD contents around 2%, can be used as voltam-metric electrode material as well as indicator electrode

under flowing conditions, and exhibits good signal-to-

background ratios for analytes of different hydropho-

bicity, and a very good resistance to fouling with no

need of electrode surface pretreatment. Up to date, no

data on the fabrication of enzyme electrodes by using

this type of composite material are found in the

literature.

The comparison of capabilities of the amperometric

composite tyrosinase biosensors are illustrated in this

work by their response to different phenolic compounds

(phenol, catechol, 4-chloro-3-methylphenol, 4-chloro-2-

methylphenol, 4-chlorophenol, 2,4-dimethylphenol, 2,3-

dimethylphenol, and 3,4-dimethylphenol), most of them

included in the EPA pollutant list, in a phosphate

buffer working solution. The enzyme reaction involves

the catalytic oxidation of these compounds to the corre-

sponding quinones, and the electrochemical reduction

of these quinones was employed to monitor this reac-

tion (Fig. 1).

2. Experimental

2 .1. Apparatus, electrodes and electrochemical cells

Experiments were performed on a Metrohm

(Herisau, Switzerland) 641 VA potentiostat connected

to a Linseis (Serb, Germany) L6512 recorder. The

electrochemical cell was a BAS (W. Lafayette, IN,

USA) Model VC-2 cell with a BAS RE-1 Ag/AgCl/KCl

(3 M) reference electrode and a platinum wire auxiliary

electrode. Other apparatus used were a Metrohm 628-10 rotating electrode connected to a E-510 Metrohm

potentiostat, and a 728 Metrohm magnetic stirrer.

2 .2 . Composite tyrosinase electrodes

Graphite – Teflon – tyrosinase composite electrodes

were fabricated in the form of cylindrical pellets as

described previously (Serra et al., 1999a).

Regarding RVC – epoxy resin – tyrosinase electrodes,

RVC cylinders were firstly chemically pretreated as

described in a former article (Pena et al., 1999). Then,the enzyme (from mushroom, EC 1.14.18.1, activity

3000 U per mg of solid, Sigma, St Louis, MO, USA)

was immobilized by direct adsorption on the RVC by

immersing the pretreated RVC cylinder for 30 min in a

0.05 mol l−1 phosphate buffer solution (pH 6.5) con-

taining 3.0 mg of enzyme (9000 U). Next, the solvent

was evaporated by passing an Ar stream through, and

pores of RVC were filled with the epoxy resin (Araldit),

as described in the above-mentioned article (Pena et al.,

1999).

Graphite – EPD (Aldrich, Milwaukee, WI, USA) – ty-

rosinase composite electrodes were fabricated, also inthe form of cylindrical pellets, as follows. Graphite

(ultra-F purity; Carbon of America, Bay City, MI,

USA), 0.540 g, and tyrosinase, 0.030 g, were accurately

weighed and thoroughly mixed by mechanic stirring for

2 h in a 0.70 ml suspension of a 0.05 mol l−1 phosphate

buffer solution of pH 6.5 at 4 °C. Then, water was

evaporated by passing an argon stream through the

mixture, and 3.0 ml of a 1.0% (w/v) EPD solution in

cyclohexane (Panreac, Barcelona, Spain) were added.

The resulting mixture was thoroughly hand-mixed until

cyclohexane was completely evaporated. Next, the mix-

ture was pressed into pellets (1.3 cm diameter) by

Fig. 1. Schematic diagram displaying the enzyme and electrode

reactions involved in the detection of phenolic compounds at com-posite tyrosinase electrochemical biosensors.




Fig. 2. Current – time recording obtained for successive additions of a stock solution of phenol (1.0 ×10−6 mol l−1) in phosphate buffer (0.05 M)

of pH 6.5 at: (A) RVC – epoxy resin – tyrosinase, (B) graphite – Teflon – tyrosinase and (C) graphite – EPD – tyrosinase electrodes. Applied potential,

−0.15 V.

means of a Carver pellet press (Perkin – Elmer, Nor-

walk, CT, USA) at 10 000 kg cm−2 for 10 min. Several

3.0 mm diameter cylindrical portions of the pellet were

bored, and each portion was press-fitted into a Teflon

holder. Electrical contact was made through a stainless

steel screw.

2 .3 . Reagents and solutions

Other reagents used were phenol (Sigma), catechol

(Sigma), 2,4-dimethylphenol (Aldrich), 2,3-dimethyl-phenol (Aldrich), 3,4-dimethylphenol (Aldrich),

4-chlorophenol (Aldrich), 4-chloro-2-methylphenol

(Aldrich), 4-chloro-3-methylphenol (Aldrich), and meth-

anol (Panreac). All chemicals were of analytical-reagent

grade and the water used was obtained from a Milli-Q

purification system (Millipore, Bedford, NA, USA).

Stock solutions of the phenolic compounds, 0.10 mol

l−1, were prepared in a 0.05 mol l−1 phosphate buffer

of pH 6.5, or in methanol depending on the solubility

of these compounds in water. More dilute standards

were prepared by suitable dilution with the 0.05 moll−1 phosphate buffer solution.

2 .4 . Procedure

A similar experimental procedure was followed with

all the three enzyme composite electrodes. Thus, amper-

ograms in stirred solutions were recorded by immersing

the biosensor, at room temperature, in the electrochem-

ical cell containing 5.0 ml of the phosphate buffer

solution which was mechanically stirred at a constant

rate. Then, the selected potential was applied and the

background current was allowed to stabilize. Next, the

appropriate volume of the stock solution of the corre-

sponding phenolic compound was added with a mi-

cropipete and amperometric measurements were carried

out at the same potential and allowing the steady-state

current to be reached. When the response obtained with

any of the composite tyrosinase electrodes was signifi-

cantly lower than the original response (see below),

regeneration of the electrode surface was performed in

all cases by polishing for 5 s on a 150 grit SiC paper.

After use, graphite – Teflon and RVC – epoxy resin bio-

composite electrodes were stored at 4 °C in a refrigera-tor, whereas graphite – EPD electrodes were stored at

ambient temperature.

Calculation of the bioelectrodes active area was ac-

complished by constructing rotating disc electrodes

with each of the composite matrices, and recording

amperograms after adding 50 l of 0.1 mol l−1 potas-

sium ferricyanide to the electrochemical cell containing

0.1 mol l−1 phosphate buffer (pH 7.0). These ampero-

grams were registered at −0.20 V versus Ag/AgCl for

different rotation rates of the electrodes. The value of

the active area was obtained from the limiting current

vs. the square root of the rotation rate plots.

3. Results and discussion

Fig. 2 shows current-time recordings obtained for

successive 1.0×10−6 mol l−1 phenol additions at the

three composite tyrosinase electrodes. It can be seen

that the graphite-EPD biosensor yielded the most sensi-

tive response (almost 3-fold higher than that at the

graphite – Teflon electrode and around 100-fold higher




than that obtained with the RVC – epoxy resin bioelec-

trode). The response times were comparable at the

three electrodes, although somewhat longer at the

RVC – epoxy resin biosensor. Nevertheless, the steady-

state current was always reached in no more than 3

min. Furthermore, the background current at the

graphite – Teflon and graphite – EPD electrodes was sim-

ilar, whereas this current was much lower with the

RVC – epoxy electrode. As a consequence, the signal-to-noise ratio was comparable in the three cases.

3 .1. Optimization of the composition of the electrode

matrices

Concerning graphite – Teflon electrodes, both the per-

centage of Teflon in the matrix (70%) and the amount

of tyrosinase immobilized (90 000 U), were the same as

those optimized previously by our group (Serra et al.,

1999a).

Regarding graphite – EPD electrodes, composite pel-lets with EPD contents lower than 1% had poor com-

pactness, whereas EPD contents above 7 – 8% showed

poor conductivity (Alonso et al., 1999). Therefore,

composite enzyme electrodes with EPD percentages

between 1 and 7% were compared using 1.0×10−6 mol

l−1 phenol as the substrate. Although the highest

steady-state currents were achieved for EPD contents of

1% (0.900.07 A), the mechanical consistency im-

proved remarkably as the terpolymer percentage in-

creased. Taking into account that not very big

differences in the steady-state current were observed

between 2% (0.750.05 A) and 5% (0.680.02 A)

EPD, this later content was finally chosen to carry out

further studies. Regarding the amount of enzyme en-

trapped into the electrode matrix, it was the same as for

graphite – Teflon electrodes (90 000 U), since higher ty-

rosinase loading (a highest loading of 100 000 U was

tested) did not improve the amperometric responses.

Finally, concerning RVC – epoxy resin – tyrosinase

electrodes, only the enzyme loading in the RVC matrix

was optimized, the remainder variables for the fabrica-

tion of the electrodes being the same as those optimized

previously (Pena et al., 1999). This was done by con-

structing three different biosensors containing tyrosi-

nase loading of 4500, 9000 and 13 500 U, respectively.

Sets of 10 amperometric measurements for 1.0×10−5

mol l−1 phenol at −0.15 V showed a high increase in

the steady-state current when the enzyme content

passed from 4500 (0.020

0.004 A for a significancelevel of 0.05 – 9000 U (0.0400.006 A), but only a

slight increase from 9000 to 13 500 U (0.0450.006

A). Moreover, no significant differences in stability

were found for all of them. Consequently, an amount

of 9000 U tyrosinase was selected for further work.

3 .2 . Optimization of other experimental conditions

The pH of the phosphate buffer working solution

was optimized in the range 5.5 – 8.0. For the three

composite enzyme electrodes, there was not a notice-

able variation of the steady-state current between pH5.5 and 6.5. However, the current decreased rapidly for

higher pHs. Then, a 0.05 mol l−1 phosphate buffer

solution of pH 6.5 was chosen for subsequent work.

Regarding the influence of the applied potential on

the amperometric response, a similar behaviour was

found for the three composite electrodes. A rapid in-

crease of the current occurred for more reducing poten-

tials than 0.10 V up to −0.15 V, following which the

steady-state current remained practically constant. As

an example, Fig. 3 shows the trend observed for the

graphite – EPD biosensor. A potential value of −0.15V was selected because it is the less negative value in

the current plateau, and, therefore, in this way, the

number of potential interferents able to be reduced at

the electrode can be minimized. Moreover, the back-

ground current and the stabilization time of the base-

line also increased as the potential became more

negative.

3 .3 . Stability of the composite tyrosinase biosensors

One of the most claimed advantages of the enzyme

rigid composite electrodes is their stability and robust-ness, which is related in part to the simplicity of the

electrode surface regeneration process, if needed. Dif-

ferent aspects regarding the stability of the three com-

posite tyrosinase electrode designs were considered and

compared.

Firstly, the repeatability of successive amperometric

measurements without regeneration of the electrode

surface was evaluated. Sets of 10 measurements of

1.0×10-5 mol l−1 phenol, for graphite – Teflon and

RVC – epoxy resin electrodes, and of 1.0×10−6 mol

l−1 phenol for the graphite-EPD electrode (the linear

range of the response vs. the concentration plot was

Fig. 3. Influence of the applied electrode potential on steady-state

current obtained from a 1.0×10−6 mol l−1 phenol solution in

phosphate buffer (0.05 M) of pH 6.5 at a composite graphite-EPD – tyrosinase electrode.




Fig. 4. Control charts constructed for: (A) graphite-Te flon-tyrosinase,

(B) graphite – EPD – tyrosinase and (C) RVC – epoxy resin – tyrosinase

composite electrodes. Three measurements of different 1.0×10−5

mol l−1 (A and C) or 1.0×10−6 mol l−1 (B) phenol solutions daily.

E app=−0.15 V.

electrode matrix. Nevertheless, again the R.S.D. was

considerably lower for the graphite – EPD biosensor,

which can be attributed to the differences in the fabri-

cation procedures. Actually, in the case of graphite –

EPD electrodes the insulator material was dissolved

before mixing with graphite and the enzyme, which did

not occur with the other enzyme electrodes. This can

facilitate the obtention of a more homogeneous final

biomaterial.The useful lifetime of one single biosensor was also

compared for the three composite biomaterials. This

was done by performing everyday three measurements

of different 1.0×10−5 mol l−1 phenol (or 1.0×10−6

mol l−1 in the case of the graphite – EPD electrode)

solutions. Control charts were constructed for the three

electrodes (Fig. 4), considering as the central value the

mean steady-state current calculated from the 10 suc-

cessive measurements performed with no regeneration

of the electrode surface mentioned above. In all cases,

when a mean value was out of the lower limit (consid-

ered as the central value minus three times the standarddeviation), the electrode surface was polished and the

signal could then be restored inside the control limits.

For the three electrode designs, it was necessary to

polish the electrode surface at the beginning of each

working day to recover the response. As can be seen in

Fig. 4, the graphite – Teflon tyrosinase biosensor gave

mean values of the amperometric signal inside the

control limits over 30 days. However, the repetitive

regeneration of the electrode surface resulted in a too

thin composite pellet, and, consequently, the electrode

became useless after approximately such a period of time.

The control chart for the graphite – EPD electrode

shows a much shorter lifetime (5 days). This was due to

the need of applying a longer polishing time (around 10

s) to recover the signal, which was attributed to a

deeper fouling of the electrode matrix by the products

of the enzyme reaction.

Regarding the RVC – epoxy resin tyrosinase biosen-

sor, the useful lifetime of a single electrode was deter-

mined, in this case, by a loss of the enzyme activity,

since from approximately 17 days, the amperometric

response could not be recovered by polishing. We thinkthat the enzyme activity is altered by the electrode

matrix, as a consequence of a maturing process of the

epoxy resin with time which could cause the enzyme

deactivation.

In any case, either because the electrode became

useless after a repetitive regeneration of the sensing

surface or due to a loss of the enzyme activity, the

biocomposite electrode needed to be changed when the

amperometric responses could not be restored by pol-

ishing. Therefore, the reproducibility of the analytical

responses obtained with different electrodes constructed

in the same manner for each design, is an important

obtained at lower concentrations for this electrode, as it

will be shown below), yielded relative standard devia-

tion (R.S.D.) values for the steady-state current of 4.0,

5.0, and 2.8%, respectively. Although, the graphite−

EPD biosensor offered a better repeatability, no big

differences were observed for any electrode.

On the other hand, the possibility of obtaining a

fresh electrode surface simply by polishing is one of the

most advantageous properties of the use of composite

bioelectrodes. Thus, the reproducibility of the ampero-

metric response was checked after regeneration of theelectrode surfaces by polishing as described in the Sec-

tion 2. Sets of 10 polishing, and three different mea-

surements after each polishing, were carried out at the

same phenol concentration levels commented above.

R.S.D. values of 4.4, 5.6, and 2.4% were obtained for

the ten steady-state current mean values of each series

with the graphite – Teflon, the RVC – epoxy resin, and

the graphite – EPD electrode, respectively. These results

indicated that all the composite tyrosinase electrodes

yielded reproducible amperometric responses after be-

ing subjected to the regeneration procedure, which

means that the enzyme was uniformly distributed in the




aspect to evaluate the real practical capabilities of the

composite biosensors. In the case of graphite – Teflon

and graphite – EPD biosensors, this reproducibility was

evaluated for four different electrodes, two of them

fabricated from the same main pellet, and the other two

from a different pellet, whereas in the case of the

RVC – epoxy resin design, the reproducibility was evalu-

ated from the measurements obtained with six different

electrodes.

The results obtained are summarized in Table 1. The

R.S.D. for the mean values of all the four electrodes

was 5.0 and 4.3% for graphite – Teflon and graphite – EPD electrodes, respectively, whereas it was 5.6% for

the six RVC – epoxy resin electrodes. These results indi-

cate that the fabrication procedures of the composite

tyrosinase biosensors are reliable in all cases, and allow

the obtention of reproducible electroanalytical re-

sponses with different electrodes constructed in the

same manner.

Finally, it is important to remark that after 5 months

of storage, at 4 °C in a refrigerator, of graphite – Teflonand graphite – EPD composite pellets, no significant loss

of the enzyme activity occurred, and similar (and repro-

ducible) amperometric responses to those shown in

Table 1 were obtained when the corresponding bioelec-

trodes were fabricated from such stored pellets.

As a conclusion of all these studies, it can be said

that a rather good stability and robustness of the three

composite biosensor designs has been demonstrated.

Nevertheless, it should be remarked the better repro-

ducibility of the amperometric responses obtained with

the graphite – EPD electrodes in comparison with the

other two biosensor designs, as well as the longer usefullifetime observed for the graphite – Teflon electrodes.

3 .4 . Kinetics of the enzyme reaction at the composite

electrodes

Under the experimental conditions optimized for

phenol, the kinetic parameters of the tyrosinase reac-

tion were calculated and compared at the three com-

posite matrices for the following compounds: phenol,

catechol, 3,4-dimethylphenol, 4-chloro-3-methylphenol,

4-chlorophenol, 4-chloro-2-methylphenol, 2,4-dimethylphenol and 2,3-dimethylphenol. The kinetics of

the enzyme reaction for these phenolic compounds

fitted in all cases into a Michaelis – Menten type kinetic,

as demonstrated by the calculation of the parameter x

from the Hill’s plots (log((i max/i )−1] vs. the log of the

substrate concentration). As can be seen in Table 2, this

parameter was very close to 1 in all cases, indicating

that the differences between the composite matrices did

not affect the Michaelis – Menten behaviour. Calcula-

tion of the apparent Michaelis – Menten constants (K m,

app.), and the maximum rate of the reaction (V m) was

accomplished from the corresponding Lineweaver – Burk plots. The obtained values are shown in Table 2.

When comparing K m, app. values obtained with each

of the three tyrosinase composite biosensors developed,

it can be observed that, in general, these values are

lower for the graphite – EPD electrode and higher for

the RVC – epoxy resin electrode. Regarding V m, it could

be found that this parameter was higher for the

graphite – EPD – based biosensor and lower for the one

constructed with RVC and epoxy resin. These trends

depend mainly on two factors. On the one hand, they

depend on the amount of immobilized enzyme on theelectrode surface. Taking into account the bioelectrodes

Table 1

Reproducibility of the amperometric responses (n=10) obtained with different electrodes

Pellet R.S.D. (%)Electrode R.S.D. (%)I (A) i (A)

Graphite – Teflon – tyrosinase electrode

1.80.11 1 1.760.04 5.04.8

5.62 1.800.08

2 5.51.830.071

1.970.07 4.82

Graphite – EPD – tyrosinase electrode0.660.011 2.81 0.690.05 4.3

0.680.02 3.22

1 0.690.02 3.82

2 0.730.02 4.1

RVC – epoxy resin – tyrosinase

5.60.0410.0020.0391

0.0402

0.0443

4 0.038

0.0435

0.0406

1.0×10

−5

mol l

−1

phenol (graphite – Teflon and RVC – epoxi resin tyrosinase biosensors), and 1.0×10

– 6

mol l

−1

phenol (graphite – EPD electrodes).E app=−0.15 V.




Table 2

Kinetic parameters of the tyrosinase reaction at composite tyrosinase

electrodes: (1) graphite – Teflon, (2) graphite – EPD and (3) RVC – ep-

oxy resin

ElectrodeCompound x V m, app K m,app

(M×105)(A)

Phenol 0.9991 24.4 7.76

2 0.98 38.4 6.26

0.98 0.983 24.01Catechol 0.98 17.1 4.99

2 0.997 29.9 5.63

0.97 0.963 20.0

3,4- dimethyl- 1 0.99 6.04 0.788

phenol

0.99 10.72 1.04

1.01 0.633 11.0

1 1.0054-chloro-3-methyl- 13.1 3.76

phenol

0.97 2.442 0.089

0.96 0.353 6.80

0.96 12.51 6.904-chlorophenol

2 0.97 14.4 2.53

3 0.98 3.76 97.01 0.97 3.384-chloro-2-methyl- 17.2

phenol

2 0.95 5.79 5.80

1.1 0.083 120

1 1.0032,4-dimethyl- 7.90 53.3

phenol

1.002 8.292 22.9

1.1 0.50 64.03

1 1.01 2.292,3-dimethyl- 435

phenol

0.97 4.51 36.12

1.2 0.113 1122

On the other hand, and assuming that the rate limit-

ing step of the reaction is in all cases the enzymatic

oxidation of the phenolic compound, the K m, app values

will depend on the phenolic compound recycling pro-

cess at the electrodes surface. When the electrode active

area is larger, such a process gives rise to an increase of

the phenolic compound concentration in the diffusion

layer. Then, assuming that the phenolic compound

oxidation process is slower than the correspondingquinones electrode reduction, an enzyme saturation will

be observed for lower concentrations of substrate,

which implies lower K m, app values.

As stated in the Section 2, the bioelectrodes active

area was calculated by recording amperograms after

adding 50 l of a 0.1 mol l−1 potassium ferricyanide to

the electrochemical cell containing 0.1 mol l−1 phos-

phate buffer (pH 7.0). These amperograms were regis-

tered at −0.20 V versus Ag/AgCl for different rotation

rates of the electrodes, and the value of active area was

obtained from the limiting current versus the square

root of the rotation rate plots. These values were:2.05×10−2 cm2, 7.00×10−2 cm2, and 5.96×10-3 cm2,

for the graphite – Teflon, graphite – EPD and RVC – ep-

oxy resin tyrosinase electrodes, respectively.

Fig. 5 shows the plots of the current density (thus

avoiding the active area contribution) vs. the rotation

rate of enzyme electrodes constructed with each of the

composite matrices studied, as well as a comparison

with the theoretical current density (calculated from the

Levich equation) when there is not enzyme recycling. It

can be seen that the current density was 15.5-fold

higher (for a rotation rate of 1500 rpm) at thegraphite – EPD bioelectrode, 12.7-fold higher at the

graphite – Teflon electrode, and only 1.3-fold higher for

the RVC – epoxy resin biosensor than the current den-

sity obtained with no enzyme recycling. These differ-

ences indicate that the phenolic compound enzyme

recycling was dependent on the electrode matrix used.

On the other hand, when comparing the K m, app

values obtained with the same composite electrode for

the tested phenolic compounds, a higher Michelis –

Menten constant is always observed for those com-

pounds with one ortho-position occupied; this means a

lower af finity for the enzyme, and, therefore, lowerresponses than those of phenolic compounds substi-

tuted in para- and meta-positions.

3 .5 . Calibration plots and analytical characteristics

Table 3 summarises the characteristics of the calibra-

tion plots obtained for the phenolic compounds tested

with the three composite tyrosinase electrodes under the

optimised working conditions selected above. The cor-

responding limits of detection, calculated according to

the 3sb/m criterium, where s

b was estimated as the S.D.

(n=10) of the signals from different solutions of the

Fig. 5. Current density vs. the square root of the rotation rate of: ()

graphite – Teflon – tyrosinase, () graphite – EPD – tyrosinase, and ()

RVC – epoxi resin tyrosinase electrodes. () theoretical current den-

sity in the absence of enzyme recycling. Catechol 1.0×10−6 mol l−1.

fabrication procedures, in all of which tyrosinase was

firstly homogeneously adsorbed on the conductor

phase, it can be assumed that a larger electrode active

surface will imply a larger conductor material area,

and, therefore, a higher amount of enzyme at the

electrode surface. This factor will influence mainly V m.




Table 3

Calibration data for the different phenolic compounds obtained at composite tyrosinase electrodes: (1) graphite – Teflon, (2) graphite – EPD and (3)

RVC – epoxy resin

Compound Linear range, (mol l−1)Electrode r Slope, (A mol−1 l) LOD, mol l−1

(0.1 – 25)×10−6 0.997Phenol (2.790.09)x1051 9.9×10−8

2 (0.05 – 6)×10−6 0.998 (6.20.2)x105 2.63×10−8

(0.02 – 4)×10−5 0.99973 (3.43)x103 1.1×10−7

(0.1 – 15)×10−6 0.997Catechol (3.00.1)x1051 1.0×10−7

(0.05 – 8)×10−6

0.9992 (6.60.1)x105

2.8×10−8

3 (0.01 – 5)×10−5 0.999 (4.110.09)x103 9.2×10−8

(0.1 – 6)×10−6 0.9981 (5.20.2)x1053,4-dimethyl phenol 1.0×10−7

(0.2 – 9)×10−7 0.998 (1.20.1)×1062 8.7×10−9

(0.01 – 3)×10−5 0.99973 (4.780.06)×103 3.0×10−8

(0.1 – 20)×10−6 0.9994-chloro-3-methylphenol (2.910.07)×1051 3.1×10−8

(0.2 – 3)×10−7 0.9982 (2.00.4)×106 5.4×10−9

(0.01 – 0.2)×10−5 0.999 (5.90.9)×1033 3.5×10−8

(0.74 – 8)×10−6 0.9981 (2.50.1)×1054-chlorophenol 7.4×10−8

(0.05 – 7)×10−6 0.999 (4.60.1)×1052 2.3×10−8

(0.01 – 8)×10−5 0.9993 (3.940.06)×103 7.1×10−8

14-chloro-2-methylphenol (5 – 10)×10−5 0.998 (65)×103 2.3×10−5

2 (2 – 30)×10−6 0.999 (7.10.4)×104 5.4×10−9

(5 – 10)×10−5 0.9983 (2.10.4)×102 4.2×10−5

12,4-dimethyl Phenol (0.7 – 100)×10−

6 0.998 (1.860.06)×104 7.1×10−

7

(1 – 50)×10−6 0.9992 (3.30.1)×104 6.7×10−7

(0.2 – 30)x10−5 0.997 (5.80.3)×1023 1.3×10−6

(1.0 – 10)x10−4 0.9991 (4.00.8)×1022,3-dimethyl phenol 6.7×10−5

2 (1 – 7)x10−5 0.998 (1.040.08)×104 8.0×10−6

(2 – 40)x10−4 0.999 (7.70.4) 8.1×10−53

substrates at the concentration level corresponding to

the lowest concentration of the calibration plot, are

also given in Table 3. The confidence intervals were

calculated for a significance level of 0.05. As expected,

the general trend in sensitivity, when comparing theelectrode matrices, was graphite – EPDgraphite –

TeflonRVC – epoxy resin. As an example, and to

allow a graphical view of the responses, Fig. 6 com-

pares the calibration plots obtained for phenol, catechol

and 4-chlorophenol with the three composite tyrosinase

biosensors.

As theoretically predicted, the sensitivity of the

biosensors for each phenolic compound is higher as the

corresponding K m, app is lower and the V m value (which

is proportional to the catalytic constant for the conver-

sion of the enzyme-substrate complex into the product

plus the enzyme) is higher. Thus, the ratio V m/K m, app,

the catalytic ef ficiency (Fresht, 1980], gives an a priori

indication of the substrates sensitivity trend. Further-

more, this parameter indicates the enzyme specificity

for the different substrates (Peter and Wollenberger,

1997]. Table 4 collects the V m/K m, app values for each of

the enzyme biosensors developed. By comparing the

slopes of the calibration plots (Table 3) with data of

Table 4, it can be deduced that, actually, there is a

correlation between sensitivity and the catalytic ef fi-

ciency of the enzyme reaction for each phenolic

substrate.

Fig. 6. Calibration graphs obtained for phenol, 4-chlorophenol and

catechol at: () graphite – Teflon – tyrosinase, () graphite – EPD –

tyrosinase, and () RVC – epoxy resin – tyrosinase electrodes. E app=−0.15 V.




Table 4

Values of the catalytic ef ficiency, V m/K m, app, and slopes of the calibration graphs for the different phenolic compounds obtained at composite

tyrosinase electrodes of graphite – Teflon, graphite – EPD and RVC – epoxy resin

Slope, A mol−1 lElectrode V m/(K m, app×105), mol l−1Compound

52×104Graphite – Teflon – tyrosinase 7.673,4-DMPh

30×104Catechol 3.43

4-Cl-3-MPh 29×104 3.48

Phenol 28×104 3.14

25×104

4-ClPh 1.811.9×104 0.152,4-DMPh

0.6×1044-Cl-2-MPh 0.20

2,3-DMPh 0.04×104 0.0053

Graphite – EPD-tyrosinase 4-Cl-3-MPh 200x×04 27.4

120×1043,4-DMPh 10.3

66×104 5.31Catechol

62×104Phenol 6.13

4-ClPh 46×104 5.69

4-Cl-2-MPh 7.1×104 1.00

3.3×1042,4-DMPh 0.36

1.0×104 0.1252,3-DMPh

0.59×104RVC – epoxy resin – tyrosinase 0.0524-Cl-3-MPh

3,4-DMPh 0.48×104 0.0570.41×104 0.048Catechol

0.39×1044-ClPh 0.039

Phenol 0.34×104 0.041

2,4-DMPh 0.058×104 0.0078

0.021×1044-Cl-2-MPh 0.00067

2,3-DMPh 0.00077×104 0.000098

4-Cl-3-MPh, 4-chloro-3-methylphenol; 3,4-DMPh, 3,4-dimethylphenol; 4-ClPh, 4-chlorophenol; 2,4-DMPh, 2,4-dimethylphenol; 4-Cl-2-MPh,

4-chloro-2-methylphenol; 2,3-DMPh, 2,3-dimethyl phenol.

On the other hand, it can be observed that the trend

in sensitivity is not exactly the same for the three

composite tyrosinase electrodes. Nevertheless, a generaltrend can be found for all of them. The three phenolic

compounds with one ortho-position occupied are re-

markably less sensitive than the others (and therefore

they have lower V m/K m, app. values). Moreover, the

phenolic compounds with the positions 3 and 4 of the

aromatic ring occupied show a high slope of their

calibration graphs, probably because these substituents

produce a stabilization of the enzyme-substrate link,

which is corroborated by the low K m, app. values ob-

tained for these compounds with the three composite

electrodes. Catechol is also detected in all cases with ahigh sensitivity as a consequence of having a hydroxyl

group in ortho position which facilitates the conversion

to quinone, whereas phenol and 4-chlorophenol gave

similar sensitive responses since both of them have the

two ortho-positions free.

The existing differences on the phenolic compounds

sensitivity order for each biocomposite electrode sug-

gest the influence of other factors on the analytical

responses, besides those commented above. As we have

already stated, the nature of the electrode matrix influ-

ences the interactions of the tyrosinase catalytic cycle.

Apart from the af finity between the enzyme and the

substrate and the rate of the catalytic conversion, fac-

tors depending directly on the electrode matrix nature,

such as the ability of accumulation of the analyte onthe electrode surface, and the hydrophobicity of this

electrode surface can promote differences in the amper-

ometric responses obtained for the same compounds.

4. Conclusions

From the comparison of the behaviour of the three

tyrosinase composite electrode matrices, it can be con-

cluded that all of them can be used for the monitoring

of the phenolic compounds used as substrates. Thesecomposite matrices are reusable and show a rather

good stability and robustness when compared with

other tyrosinase biosensors designs found in the litera-

ture. Among the three composite biosensors tested, the

graphite – EPD – tyrosinase electrodes show a better re-

producibility of the amperometric measurements both

with and without regeneration of the electrode surface

by polishing. However, the graphite – Teflon electrode

matrix exhibits a longer useful lifetime. The trend in

sensitivity towards the different phenolic compounds

tested is dependent on the nature of the electrode

matrix used, and the limits of detection obtained are, in




general, better than those found with other tyrosinase

electrochemical biosensors designs. Actually, these

composite biosensors can be used in the monitorization

of phenolic compounds in industrial waste waters, in

which the highest concentration level permitted is 2 mg

l−1. The lowest limit of detections are obtained with

the graphite – EPD electrodes.

Acknowledgements

The financial support of Comunidad de Madrid,

Project 07M/0033/99, and EC (Inco-Copernicus), Pro-

ject PL 965022, is gratefully acknowledged.

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