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Transcript of 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
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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.
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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
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B . Serra et al . / Biosensors & Bioelectronics 17 (2002) 217 – 226 220
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.
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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
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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.
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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.
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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.
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B . Serra et al . / Biosensors & Bioelectronics 17 (2002) 217 – 226 225
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
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B . Serra et al . / Biosensors & Bioelectronics 17 (2002) 217 – 226 226
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.
References
Alegret, S., Alonso, J., Bartoli, J., Cespedes, F., Martınez-Fabregas,
E., Del Valle, M., 1996. Amperometric biosensors based on
bulk-modified epoxy graphite biocomposite. Sens. Mater. 8, 147 – 153.
Alonso, L., Parrado, C., Pedrero, M., Aguı, L., Pingarron, J.M., 1999.
Graphite – ethylene/propylene/diene terpolymer composite elec-
trodes. A new electrode material for electrochemical detection.
Electroanalysis 11, 161 – 166.
Byfield, M.P., Abuknestra, R.A., 1994. Biochemical aspects of biosen-
sors. Biosens. Bioelectron. 9, 373 – 399.
Cayuela, G., Pena, N., Reviejo, A.J., Pingarron, J.M., 1998. Develop-
ment of a bienzymic graphite – Teflon composite electrode for the
determination of hypoxanthine in fish. Analyst 123, 371 – 377.
del Cerro, M.A., Cayuela, G., Reviejo, A.J., Pingarron, J.M., Wang,
J., 1997. Graphite-Teflon – Peroxidase composite electrodes. Appli-
cation to the direct determination of glucose in musts and wines.Electroanalysis 9, 1113 – 1119.
Cespedes, F., Martinez-Fabregas, E., Alegret, S., 1996. New materials
for electrochemical sensing. I. Rigid conducting composites. Trends
Anal. Chem. 15, 296 – 304.
Cosnier, S., Lepellec, A., Guidetti, B., Rico-Lattes, I., 1998. Enhance-
ment of biosensor sensitivity in aqueous and organic solvents using
a combination of poly(pyrrole-ammonium) and poly(pyrrole-lacto-
bionamide) films as host matrixes. J. Electroanal. Chem. 449,
165 – 171.
Daigle, F., Leech, D., 1997. Reagentless tyrosinase enzyme electrodes.
Effects of enzyme loading, electrolyte pH, ionic strength, and
temperature. Anal. Chem. 69, 4108 – 4112.
Ducey, M.W. Jr, Meyerhoff, M.E., 1998. Microporous gold electrodes
as combined biosensors/electrochemical detectors in flowing
streams. Electroanalysis 10, 157 – 162.
Eggins, B.R., Hickey, C., Toft, S.A., Zhou, D.M., 1997. Determination
of flavanols in beers with tissue biosensors. Anal. Chim. Acta 347,
281 – 288.
Fresht, A., 1980. Serie de Biologıa Fundamental. Estructura y Mecan-
ismos de los Enzimas, Reverte S.A., Badalona.
Gorton, L., 1995. Carbon paste electrodes modified with enzymes,
tissues, and cells. Electroanalysis 7, 23 – 45.
Hedenmo, M., Narvaez, A., Domınguez, E., Katakis, I.J., 1997.
Improved mediated tyrosinase amperometric enzyme electrodes.
Electroanal. Chem. 425, 1 – 11.
Kalcher, K., Kauffmann, J.M., Wang, J., Svancara, I., Vytras, K.,Neuhold, C., Yang, Z., 1995. Sensors based on carbon paste in
electrochemical analysis: a review with particular emphasis on the
period 1990 – 1993. Electroanalysis 7, 5 – 22.
Kotte, H., Grundig, B., Vorlop, K-D., Strehlitz, B., Stottmeister, U.,
1995. Methylphenazonium-modified enzyme sensor based on poly-
mer thick films for subnanomolar detection of phenols. Anal.
Chem. 67, 65 – 70.
Li, J., Chia, L.S., Goh, N.K., Tan, S.N., 1998. Silica sol – gel immobi-
lized amperometric biosensors for the determination of phenolic
compounds. Anal. Chim. Acta 362, 203 – 211.
Narvaez, A., Guinea, M., Domınguez, E., 1996. Characterization and
optimization of tyrosinase solid graphite electrodes for the detection
of phenolic compounds. Quımica Analıtica 15, 83 – 90.
Nistor, C., Enmeus, J., Gorton, L., Ciucu, A., 1995. Improved stability
and altered selectivity of tyrosinase based graphite electrodes for
detection of phenolic compounds. Anal. Chim. Acta 387, 309 – 326.
Ortiz, G., Gonzalez, C., Reviejo, A.J., Pingarron, J.M., 1997.
Graphite – Poly(tetrafluoroethylene) composite enzyme electrodes
as suitable biosensors in predominantly nonaqueous media. Anal.
Chem. 69, 3521 – 3526.
Pena, N., Romero, M., Manuel de Villena, F.J., Reviejo, A.J.,
Pingarron, J.M., 1999. Reticulated vitreous carbon-based com-
posite enzyme electrodes as suitable biosensors in both aqueous and
predominantly nonaqueous media. Electroanalysis 11, 85 – 92.
Peter, M.G., Wollenberger, U., 1997. In: Scheller, F.W., Schubertn, F.,
Fedrowitz, Y.J. (Eds.), Frontiers in Biosensorics I, Fundamental
Aspects. Birkhauser Verlag, Basel.
Serra, B., Mateo, E., Pedrero, M., Reviejo, A.J., Pingarron, J.M.,
1999a. Graphite – teflon – tyrosinase composite electrodes for the
monitoring of phenolic compounds in predominantly non-aqueous
media. Analusis 27, 592 – 599.
Serra, B., Reviejo, A.J., Parrado, C., Pingarron, J.M., 1999b. Graphite-
Teflon composite bienzyme electrodes for the determination of
L-lactate: Application to food samples. Biosens. Bioelectron. 14,
505 – 513.
Tallman, D.E., Petersen, S.L., 1990. Composite electrodes for electro-
analysis: principles and applications. Electroanalysis 2, 499 – 510.
Wang, J., Fang, L., Lopez, D., 1994. Amperometric biosensors for
phenols based on a tyrosinase – graphite epoxy biocomposite. Ana-
lyst 119, 455 – 458.
Wang, J., Reviejo, A.J., Angnes, L., 1993. Graphite – teflon enzyme
electrodes. Electroanalysis 5, 575 – 579.
Wang, J., Varughese, K., 1990. Polishable and robust biological
electrode surface. Anal. Chem. 62, 318 – 320.