Design of a biosensor based on 1-(4-nitrophenyl)-2,5-di(2-thienyl)-1H pyrrole

Article abstract of DOI:10.1016/j.molcatb.2009.06.002

Immobilization of polyphenol oxidase (tyrosinase, E.C. 1.14.18.1) was achieved on a copolymer of 1-(4-nitrophenyl)-2,5-di(2-thienyl)-1H-pyrrole [SNS(NO₂)] with pyrrole ([SNS(NO₂)]/PPy) via electrochemical polymerization. Two different substrates; catechol and l-tyrosine were used for the characterization of biosensor. The kinetic parameters of the biosensor, maximum reaction rate of the enzyme (V_max) and Michaelis-Menten constant (K_m) were determined for two different substrates. V_max was found as 0.02 μmol/min electrode for both substrates. K_m values were determined as 250 and 2 mM for catechol and l-tyrosine respectively. Calibration curves for enzyme activity versus substrate concentration were plotted between 0.05 and 0.5 M catechol and between 0.8 and 2.5 mM l-tyrosine. Optimum temperature and pH, operational and storage stabilities of immobilized enzyme were examined.

Full text of DOI:10.1016/j.molcatb.2009.06.002

Journal of Molecular Catalysis B: Enzymatic 64 (2010) 195–199
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Design of a biosensor based on 1-(4-nitrophenyl)-2,5-di(2-thienyl)-1H pyrrole
Sevinc Tuncagil, Serhat Varis, Levent Toppare∗
Department of Chemistry, Middle East Technical University, 06531 Ankara, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 10 June 2009
Immobilization of polyphenol oxidase (tyrosinase, E.C. 1.14.18.1) was achieved on a copolymer of 1-(4-
nitrophenyl)-2,5-di(2-thienyl)-1H-pyrrole [SNS(NO2)] with pyrrole ([SNS(NO2)]/PPy) via electrochemical
polymerization. Two different substrates; catechol and l-tyrosine were used for the characterization
Keywords:
of biosensor. The kinetic parameters of the biosensor, maximum reaction rate of the enzyme (Vmax
)
Conducting polymers
Electrochemical biosensors
Enzyme immobilization
Catechol
l-Tyrosine
Tyrosinase
and Michaelis–Menten constant (Km) were determined for two different substrates. Vmax was found as
0.02 ␮mol/min electrode for both substrates. Km values were determined as 250 and 2 mM for catechol
and l-tyrosine respectively. Calibration curves for enzyme activity versus substrate concentration were
plotted between 0.05 and 0.5 M catechol and between 0.8 and 2.5 mM l-tyrosine. Optimum temperature
and pH, operational and storage stabilities of immobilized enzyme were examined.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
biomolecules in organic polymers during their electrogeneration
on an electrode surface. The polymer formation is carried out by
Conducting polymers (CPs) are intelligent materials containing
␲-electron backbone responsible for their electrical conductivity,
low energy optical transitions, low ionization potential and high
electron affinity. This extended ␲-conjugated system of the con-
ducting polymers have single and double bonds alternating along
the polymer chain. Those polymers, called as ‘synthetic metals’,
possess the electrical, electronic, magnetic and optical properties
of a metal while retaining the ease of processing associated with a
common polymer [1,2]. Many applications of conducting polymers
including biosensing devices have been reviewed [3–5]. CPs have
been studied extensively in the recent decades for various applica-
tions as light emitting diodes [6], batteries [7], solar cells [8], optical
displays [9], enzyme immobilization matrices [10], electrochromic
devices [11], chemical sensors and arrays [12].
Since the first demonstration of an enzyme as an electrode
in a biosensor, the development of such electrochemical devices
has made considerable progress [13,14]. Enzymes have been
widely applied on biosensor construction as bioreceptor which is
the main part of a biosensor. Since enzymes are very expensive
biological catalysts, immobilization procedure is effective because
immobilized enzyme can be recovered from the solution and
can be repetitively used. The immobilization of biomolecules in
electropolymerized films is gaining importance since the elec-
trochemical formation of polymer layers of controlled thickness
constitutes a reproducible procedure for biosensor fabrication
[15]. The electrochemical method involves the entrapment of
controlled potential electrolysis of an aqueous solution containing
monomers and biomolecules [16].
Tyrosinase is a tetrameric enzyme with a molecular size of
128 kD. The enzyme has two distinct substrate binding sites one
with a high affinity for aromatic compounds including pheno-
lic substrates, the other for metal-binding agents and oxygen.
The latter site presumably involves enzyme copper. It is a cop-
per containing oxidoreductase type enzyme with an EC number of
1.14.18.1. It catalyzes two different reactions: (Schemes 1 and 2) the
orthohydroxylation of monophenols (cresolase activity), and the
oxidoreduction of orthodiphenols to orthoquinones (catecholase
activity) [17,18]. Tyrosinase is commonly found in yeast, mush-
room, apples, and potatoes and responsible for enzymatic browning
of some fruits and vegetables during handling and storage hence,
affecting the taste and nutrition value of them [19]. In plants,
sponges and many invertebrates, they are important components
of wound healing and the primary immune [20,21]. In previous
works tyrosinase was immobilized on different conducting poly-
mer matrices [22–24].
We here report a novel biosensor based on immobilization of
tyrosinase on a copolymer of 1-(4-nitrophenyl)-2,5-di(2-thienyl)-
1H-pyrrole [SNS(NO2)] with pyrrole. Two different substrates; cate-
chol and l-tyrosine were used for the characterization of biosensor.
2. Experimental
2.1. Reagents
∗
Tyrosinase (o-diphenol; oxidoreductase; EC 1.14.18.1) obtained
from mushroom as lyophilized powder, l-tyrosine, l-ascorbic acid,
Corresponding author. Tel.: +90 312 2103251; fax: +90 312 2103200.
E-mail address: toppare@metu.edu.tr (L. Toppare).
1381-1177/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2009.06.002

196
S. Tuncagil et al. / Journal of Molecular Catalysis B: Enzymatic 64 (2010) 195–199
Scheme 1. Reaction mechanism of PPO when the substrate is catechol.
hydrochloric acid, sodium hydroxide, sodium molybdate, and
sodium nitrite were purchased from Sigma and used as received
without further purification. Sodium dodecylsulfate (SDS) was sup-
plied from Merck. Pyrrole (Py) was purchased from Aldrich and
used without further purification. NaH2PO4·H2O (sodium phos-
phate monobasic) and NaHPO4·7H2O (sodium phosphate dibasic)
were purchased from Fisher Scientific Company. Sodium dodecyl-
sulfate (SDS) was supplied from Merck. For spectrometric activity
determination, 3-methyl-2-benzothiazolinone hydrazone (MBTH),
acetone and sulfuric acid were purchased from Sigma. Catechol was
obtained from Sigma. For the preparation of citrate buffer, citric acid
and sodium hydroxide were used as received.
Scheme 3. Synthesis of [SNS(NO2)].
matography (GPC) and the monomer was characterized in a
previous study [25]. The monomer [SNS(NO2)] was synthesized
from 1,4-di(2-thienyl)-1,4-butanedione and 4-nitroaniline in the
presence of p-toluene-sulphonic acid (PTSA). 1,4-Di(2-thienyl)-1,4-
butanedione was obtained through the following pathway: To a
suspension of AlCl3 in CH2Cl2, a solution of 3-methylthiophene
succinyl chloride in CH2Cl2 was added drop wise. The mixture
was stirred at room temperature for 4 h, then poured into ice
and concentrated HCl mixture. The dark green organic phase was
washed with concentrated NaHCO3 and brine, and then dried
over MgSO4. After evaporation of the solvent a blue green solid
remained. Filtration and washing with ethanol yielded 1,4-bis(2-
thienyl)butane-1,4-dione. A round-bottomed flask equipped with
an argon inlet and magnetic stirrer was charged with 1,4-di(2-
thienyl)-1,4-butanedione, 4-nitroaniline, PTSA and toluene. The
2.2. Instrumentation
Potentioscan Wenking POS-73 potentiostat, Shimadzu UV-1601
spectrophotometer, Memmert D-91126 model water bath and JEOL
JSM-6400 model scanning electron microscope (SEM) were used.
2.3. Synthesis of monomer
The structures of both the monomer and the soluble poly-
mer were elucidated by Nuclear Magnetic Resonance (1H and
13C-NMR) and Fourier Transform Infrared (FTIR). The average
molecular weight has been determined by gel permeation chro-
Scheme 2. Reaction mechanism of PPO when the substrate is l-tyrosine.

S. Tuncagil et al. / Journal of Molecular Catalysis B: Enzymatic 64 (2010) 195–199
197
Scheme 4. Schematic representation of enzyme immobilization procedure.
mixture was stirred and refluxed (110 ◦C) for 24 h under argon.
[SNS(NO2)] was obtained as pale brown powder after evaporation
of the toluene, followed by flash column chromatography [12,26].
Schematic representation of the synthesis was shown in Scheme 3.
spectrochemical analysis at 460 nm exactly after 1 h. Enzyme
electrodes were kept in phosphate buffer at 4 ◦C when not in use
and daily prepared electrodes were used in all experimental steps.
All experiments were done in constant temperature water bath
while shaking.
2.4. Immobilization of tyrosinase
2.6. Optimum pH and temperature experiments
Electrochemical polymerizations were achieved by constant
potential electrolysis carried out in a three electrode cell con-
sisted of a Pt working electrode coated with [SNS(NO2)], a counter
electrode, and a Ag wire as the pseudo reference electrode. For
immobilization of tyrosinase in [SNS(NO2)]/PPy biosensor, a solu-
tion of 0.1 mg/mL PPO, 2 mg/mL supporting electrolyte (SDS),
0.005 M pyrrole and 10 mL buffer were put in a typical three elec-
trode cell. Immobilization was carried out at constant potential of
1.0 V for 20 min at room temperature. Tyrosinase was immobilized
by entrapment during electropolymerization. Simple representa-
tion for immobilization is shown in Scheme 4.
The effect of pH was determined by changing reaction medium
pH between 6.0 and 7.5 at a constant substrate concentration
for both matrices. The effect of temperature was determined by
changing the reaction medium temperature between 10 and 80 ◦C
at a constant substrate concentration for both catechol and l-
tyrosine. For all experiments the enzyme activity determination
was performed as described above, and the relative enzyme activ-
ity was calculated by assigning the maximum value of activity as
100%.
2.7. Storage and operational stability experiments
2.5. Enzymatic assay
The activity of immobilized enzyme on [SNS(NO2)]/PPy biosen-
sor after storage in citrate buffer at 4 ◦C were measured for 60 days
with the experimental conditions given above.
Operational stability of the immobilized enzyme was studied by
repetitive use of the same [SNS(NO2)]/PPy electrode for 50 succes-
sive measurements in the same day. For all experiments, relative
enzyme activity was calculated by assigning the maximum value of
activity as 100%.
For catechol, immobilized enzyme activity determination was
performed by Besthorn’s hydrazone method [27]. In this method 3-
methyl-2-benzothiozolinone (MBTH) interacts with the quinines
produced by the enzyme to yield red products instead of brown
colored pigments in the absence of color reagent [28]. Different
concentrations of catechol solutions were prepared in citrate buffer
(pH 6.5) and were put in water bath at 25 ◦C. 1.0 mL MBTH (0.3%)
in ethanol was added to produce a red complex with the product.
Enzyme electrode was immersed into the solution. After a spe-
cific reaction time 1 mL sample was drawn to achieve enzymatic
assay.
3. Results and discussions
For the determination of the enzymatic activity of
[SNS(NO2)]/PPy biosensor towards catechol, 1.0 mL sulfuric acid
solution (5%, v/v) was added to stop the enzymatic reaction and
1.0 mL acetone was added to dissolve the colored complex. After
mixing, absorbances were determined at 495 nm. For l-tyrosine,
different concentrations of l-tyrosine and l-ascorbic acid solutions
were prepared in phosphate buffer (pH 7). Electrodes were put
into test tubes containing substrate solutions. After a specific
reaction time 1 mL sample was drawn to achieve enzymatic assay.
1 mL HCl (2 M) was added to stop the reaction. 1 mL NaOH (2 M)
was added to prevent bubbling and 1 mL 15% NaMoO4 and NaNO2
were added to have the yellow complex in specific reaction time
intervals (5, 10 and 15 min). Since the formation of l-dopa complex
is time dependent, l-dopa concentrations were determined by
3.1. Kinetic parameters for soluble and immobilized enzyme
When a biocatalyst is immobilized, maximum velocity (Vmax
)
and Michaelis–Menten constant (Km) can undergo variations with
respect to corresponding parameters of the soluble enzyme. These
changes are results of several factors; such as protein confor-
mational changes induced by the support, steric hindrances and
diffusional effects. It is very important to know these variations
since they reveal the relation between the substrate and the
enzyme.
To determine kinetic parameters for [SNS(NO2)]/PPy, enzymatic
assay was applied for different concentrations of catechol and l-
tyrosine solutions as mentioned in Section 2.5. Vmax and Km values
were determined from Lineweaver–Burk plot at 25 ◦C pH 6.5 for cat-
echol and pH 7 for l-tyrosine. In previous works kinetic parameters
of soluble enzyme for both substrates were calculated [29,30]. The
kinetic parameters of the free and immobilized enzymes are pre-
sented in Table 1. Vmax of the immobilized enzyme was decreased
fourfold upon immobilization for catechol whereas there was a five-
fold decrease for l-tyrosine. Km value for the catechol was higher
than the soluble enzyme resulted from whenever the enzyme sub-
strate comes together they gives products.
Table 1
Properties of the soluble PPO and immobilized PPO on [SNS(NO2)]/PPy.
Substrate
Vmax (␮mol/min electrode)
Km (mM)
Soluble PPO
SNSNO2/PPy/PPO
Soluble PPO
Catechol
Catechol
l-Tyrosine
l-Tyrosine
0.073
0.02
0.1
4
250
4
SNSNO2/PPy/PPO
0.02
2

198
S. Tuncagil et al. / Journal of Molecular Catalysis B: Enzymatic 64 (2010) 195–199
Fig. 1. Effect of pH on [SNS(NO2)]/PPy biosensor.
Fig. 3. Storage stabilities of [SNS(NO2)]/PPy biosensor.
3.2. Optimum pH
As shown in Fig. 1 the maximum activity was found at pH 6.5
for catechol. In previous works, the maximum activity was found at
pH 5 for the soluble enzyme [29]. For l-tyrosine, both soluble (free)
enzyme [30] and [SNS(NO2)]/PPy biosensor have maximum activity
at pH 7. For catechol at pH 6.5–7.5 [SNS(NO2)]/PPy biosensor shows
90% activity, whereas for l-tyrosine maximum activity is achieved
at pH 7. [SNS(NO2)]/PPy biosensor can be used in wide pH range for
catechol.
3.3. Optimum temperature
The effect of temperature on relative enzyme activity was
examined between 10 and 80 ◦C as illustrated in Fig. 2. For both
substrates, [SNS(NO2)]/PPy biosensor has maximum activity at
60 ◦C. Biosensor shows higher activity towards catechol compare to
l-tyrosine at lower temperatures. For the soluble enzyme, the maxi-
mum enzyme activities were seen at 30 and 40 ◦C for l-tyrosine and
catechol respectively [29,30]. Enzyme stability toward temperature
wasachieveduponimmobilization. Immobilizedenzymepossesses
a better heat resistance than free enzyme since immobilization pro-
cedure protects the enzyme active conformation from damage by
heat exchange.
Fig. 4. Operational stabilities of [SNS(NO2)]/PPy biosensor.
iments. At the end of the 20th experiment for catechol, biosensor
has retained 85% of its original activity. This is found to be 40% for
l-tyrosine. The half-life value was determined as 14 assays for l-
tyrosine. The operational stabilities of the enzyme electrodes were
given in Fig. 4.
3.5. Analytical characteristics
The analytical characteristics of the [SNS(NO2)]/PPy biosensor
was examined using catechol in citrate buffer pH 6.5 and l-tyrosine
in potassium phosphate buffer (pH 7) at 25 ◦C. Calibration curves
were plotted for enzyme activities versus substrate concentrations.
A linear calibration graph was obtained for enzyme activity versus
substrate concentration between 0.05 and 0.5 M. A linear relation
was defined by the equation of y = 0.0388x + 0.001 (R2 = 0.9387)
for catechol. For l-tyrosine a calibration graph was obtained
for enzyme activity versus substrate concentration between 0.08
and 2.5 mM. A linear relation was defined by the equation of
y = 2.4904x + 0.0039 (R2 = 0.9944) for l-tyrosine. At higher concen-
trations enzyme activity remained constant reaching to saturation
for both systems.
3.4. Storage and operational stabilities
For the storage stability measurements of the [SNS(NO2)]/PPy
biosensor, the activity of the enzyme electrode was checked for
catechol and l-tyrosine every 5 days for 60 days (Fig. 3). At the end
of the 35th day the biosensor activity was lost for catechol and the
same happened at the end of the 60th day for l-tyrosine. The half-
lives were calculated as 25 and 13 days for l-tyrosine and catechol
respectively. Operational stability of the [SNS(NO2)]/PPy biosensor
for both substrates were also determined with 20 repetitive exper-
4. Conclusion
In this study, immobilization of polyphenol oxidase was
carried out successfully on a copolymer of 1-(4-nitrophenyl)-
2,5-di(2-thienyl)-1H-pyrrole with pyrrole via electrochemical
polymerization. The[SNS(NO2)]/PPybiosensorwastestedandchar-
acterized for two substrates: catechol and l-tyrosine. The kinetic
parameters, of the biosensor were determined for the substrates.
Analytical characterization was achieved by calibration curves.
Optimization of the [SNS(NO2)]/PPy biosensor was achieved by
determining the optimum pH, temperature values. Storage and
operational stabilities were also examined.
Fig. 2. Effect of temperature on [SNS(NO2)]/PPy biosensor.

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199
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