Development of Enzyme Biosensor Based On Ph-Sensitive Field-Effect Transistors For Detection of Phenolic Compounds

Bioelectrochemistry 55 (2002) 79 81
www.elsevier.com/locate/bioelechem
Development of enzyme biosensor based on pH-sensitive field-effect

transistors for detection of phenolic compounds
S.V. Dzyadevych a,b,*, T. Mai Anh b,c, A.P. Soldatkin a,d, N. Duc Chien c, N. Jaffrezic-Renault d,
J.-M. Chovelon b
a
b
Institute of Molecular Biology and Genetics, National Academy of Science of Ukraine, 150 Zabolotnogo St., Kiev 03143, Ukraine
LACE, UMR/CNRS 5634, University of Claude Bernard Lyon 1, 43, bld du 11 Novembre 1918, 69622 Villeurbanne Cedex, France
c
International Training Institute for Materials Science, ITIMS Building, Dai hoc Bach Khoa, 1 Dai Co Viet, Hanoi, Viet Nam
d
IFoS, UMR/CNRS 5621, Ecole Centrale de Lyon, BP 163, 69131 Ecully Cedex, France
Received 1 June 2001; received in revised form 4 September 2001; accepted 26 September 2001
Abstract
This article describes a biosensor based on pH-sensitive field-effect transistors (pH-FETs) as transducer, and immobilised enzyme
tyrosinase as biorecognition element, which was used for the determination of phenolic compounds in water solutions. The biologically
active membrane was formed by cross-linking of tyrosinase with bovine serum albumin (BSA) in saturated glutaraldehyde (GA) vapours on
the sensitive transducer surface. The main analytical characteristics were studied under different conditions as well as the possibility to
optimise these working parameters. Different factors such as the pH of immobilisation, the enzyme loading, the time of exposition to
glutaraldehyde vapours were investigated in regards to the influence on sensitivity, limit of detection, dynamic range, and operational and
storage stability. D 2002 Elsevier Science B.V. All rights reserved.
Keywords: Biosensor; pH-FET; Tyrosinase; 4-Chlorophenol
1. Introduction
Chlorophenols are a major group of chemicals, which are
widely used in the manufacture of various industrial products such as pesticides, disinfectants, antioxidants, dyes, etc.
[1]. Organophosphorous and chlorinated phenoxyacids also
yield chloro and nitrophenols as major degradation products
[2].
The conventional methods for phenols determination are
chromatography and spectrometry [3]. However, these
techniques do not easily allow continuous on-site monitoring, are expensive, time-consuming, need skilled operators,
and sometimes require preconcentration steps. The application of biosensors is favourable due to some generally
claimed advantages such as the selectivity, the relatively
low cost of realisation, sometimes the good storage stability,
the potential for miniaturisation and the easy automation,
*
Corresponding author. Institute of Molecular Biology and Genetics,

National Academy of Science of Ukraine, 150 Zabolotnogo St., Kiev
03143, Ukraine. Tel.: +380-44-266-07-49; fax: +380-44-266-07-59.
E-mail addresses: dzyad@yahoo.com (S.V. Dzyadevych),
chien@itims.edu.vn (N. Duc Chien), Nicole.Jaffrezic@ec-lyon.fr
(N. Jaffrezic-Renault), chovelon@univ-lyon1.fr (J.-M. Chovelon).
and for the construction of simple portable device for fast

screening purposes and in-field/on-site monitoring. In particular, several biosensors based on tyrosinase were elaborated for the determination of phenols. They include the
various kinds of electrochemical detection, such as the
amperometric of dioxygen consumption [4], the direct
reduction of generated o-quinone [5] and the mediated
reduction of o-quinone by hexacyanoferrate(II) [6]. This
article describes the biosensor based on pH-sensitive fieldeffect transistors (pH-FETs) as transducer, and immobilised
enzyme tyrosinase as biorecognition element, which was
used for the determination of phenolic compounds in water
solutions.
2. Experimental
Tyrosinase (EC 1.14.18.1, 6680 U/mg) from mushroom,
bovine serum albumin (BSA) and aqueous solutions (25%
w/v) of glutaraldehyde (GA) were purchased from Sigma.
All other chemicals were of analytical grade.
The biologically active membrane was formed by crosslinking of tyrosinase with BSA in saturated GA vapour on
1567-5394/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
PII: S 1 5 6 7 - 5 3 9 4 ( 0 1 ) 0 0 1 6 5 - 7
80
S.V. Dzyadevych et al. / Bioelectrochemistry 55 (2002) 7981
the sensitive transducer surface [7]. The 10% solutions of

tyrosinase and BSA were prepared in 20 mM phosphate
buffer. Prior to the deposition on a sensitive area of a
transducer, these solutions were mixed, and glycerol was
added to the final concentration of 10%. As a differential
experimental set-up was used, one drop of the enzymecontaining mixture was deposited on the working ISFET,
while on the reference ISFET, only a mixture containing
10% BSA and 10% glycerol was deposited. Then the sensor
chip was placed in a saturated glutaraldehyde vapour for a
certain time called immobilisation time. After exposure in
GA, the membranes were dried at room temperature for 15
min.
The potentiometric sensor chip contains two identical
pH-FETs, the design and operation mode are given elsewhere [8]. Measurements were conducted in daylight at
room temperature (25 C) in a glass cell (5-ml volume)
filled with phosphate buffer. The biosensors were immersed
in a vigorously stirred sample solution. After the initial drift
of the output signal, 4-chlorophenol was added to the vessel.
The differential output signal between the measuring and
reference pH-FET was registered using laboratory ISFETmeter from the Institute of Microtechnology (Neuchatel,
Switzerland) and the steady-state response of the biosensor
was plotted as a function of 4-chlorophenol concentration.
3. Results and discussion

Tyrosinase (phenol oxidase) contains copper, which
forms part of the prosthetic group in its active site, and acts
as a built-in carrier undergoing reversible oxidation and
reduction of the enzyme. The conversion of phenols by
tyrosinase proceeds in two consecutive steps involving
molecular oxygen. The first step is referred to as the
Fig. 1. A typical response curve of tyrosinase biosensor for 4 mM

4-chlorophenol. Measurements were conducted in 5 mM phosphate buffer,
pH 6.0.
Fig. 2. Calibration curves for 4-chlorophenol determination for pH of

membrane immobilisation mixture of 6.0 (1) and 7.4 (2). Measurements
were conducted in 5 mM phosphate buffer, pH 6.0. Time of exposition to
glutaraldehyde vapours30 min.
enzymes hydroxylase activity (also cresolase activity)

where phenol is hydroxylated by the aid of molecular
oxygen to produce catechol (o-hydroquinone). In the second
step, known as enzymes catecholase activity, catechol is
oxidized to an o-quinone, and simultaneously, tyrosinase is
oxidized by oxygen to its original form with the production
of water. The potentiometric detection of phenols can be
used, since the subsequent local decreasing of concentration
of protons inside the membrane results in a change of the
measuring FET gate voltage.
A typical dependence of the potentiometric tyrosinase
biosensor response on the time after 4-chlorophenol additions to a vessel is shown in Fig. 1. After the biosensor
reached a stable baseline in blank phosphate buffer solution,
injection of 4-chlorophenol stock solutions into blank solution caused significant sensor response, which resulted
from subsequent local decreasing of concentration of protons inside the membrane from the enzymatic oxidation of
phenol. As it can be seen, the biosensor steady-state
response time, i.e. time necessary to reach 90% of the
steady-state amplitude was about 5 min.
The main analytical characteristics of tyrosinase biosensor based on pH-sensitive field-effect transistors for the
detection of phenolic compounds were studied under different conditions such as the pH of sample solution and
immobilisation mixture, the enzyme loading in immobilisation mixture, time of exposition to glutaraldehyde vapours.
An optimum for the sensor response was observed at
solution pH 6.0. This result is in good agreement with
results obtained for tyrosinase by other authors [9,10]. The
pH in the immobilisation mixture may also influence the
analytical characteristics of enzyme biosensors. Fig. 2
presents the calibration curves for 4-chlorophenol determinations for different pH of membrane immobilisation mixture. Here, it is clearly seen that the sensitivity of biosensor
S.V. Dzyadevych et al. / Bioelectrochemistry 55 (2002) 7981
is higher in case of immobilisation at pH 6.0, i.e. the pH

range where the tyrosinase activity is optimal. This pH of
immobilisation mixture was selected for all further experiments.
The effect of the enzyme loading in immobilisation
mixture on tyrosinase biosensor response to 4-chlorophenol
was investigated. Different ratios tyrosinase/(BSA + tyrosinase) were tested to optimise the amount of loaded tyrosinase with the sensor response. It was shown that the best
ratio for enzyme loading was about 0.4 with general
containing of protein 10%. For this ratio, the best combination of operational stability and response value was
obtained.
Concerning time of incubation in glutaraldehyde vapours, the best result was obtained for 30 min. For longer
incubation time, a dramatic decrease of response values was
occurred. This phenomenon can be connected with the
formation of a great number of covalent bonds between
the glutaraldehyde and the enzyme molecules, which block
enzyme active centres.
The response of the tyrosinase biosensor based on pHsensitive field-effect transistor is reproducible; the relative
standard deviation of intra-sensor responses was about 3%
for each concentration. The relative standard deviation of
inter-sensor responses was about 7%.
These biosensors could be used also for the detection of
diuron-type herbicides (atrazines, simazine, diuron, isoproturon, etc.), because these substances can inhibit the activity
of tyrosinase [11].
Acknowledgements
This work was supported by French CNRS, Department
of Chemistry (to SVD) and Department of International
Relation (to TMA). SVD and TMA are grateful quatrie`me
81
etage de Bat. J. Raulin de Universite Claude Bernard Lyon

1 for their friendly climate and assistance in the work.
References
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of phenolic compounds, Anal. Chim. Acta 387 (1999) 309 326.
[3] G. Marko-Varga, Phenols and substituted derivatives in water samples, in: D. Barselo (Ed.), Environmental Analysis: Techniques, Applications and Quality Assurance, Elsevier, Amsterdam, 1993, pp.
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Bioelectrochemistry 55 (2002) 79 81

Development of enzyme biosensor based on pH-sensitive field-effect

Corresponding author. Institute of Molecular Biology and Genetics,

and for the construction of simple portable device for fast

S.V. Dzyadevych et al. / Bioelectrochemistry 55 (2002) 7981

the sensitive transducer surface [7]. The 10% solutions of

3. Results and discussion

Fig. 1. A typical response curve of tyrosinase biosensor for 4 mM

Fig. 2. Calibration curves for 4-chlorophenol determination for pH of

enzymes hydroxylase activity (also cresolase activity)

S.V. Dzyadevych et al. / Bioelectrochemistry 55 (2002) 7981

is higher in case of immobilisation at pH 6.0, i.e. the pH

etage de Bat. J. Raulin de Universite Claude Bernard Lyon

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