Biosensors Based on Graphene Modified Screen-Printed Electrodes for The
Detection of Catecholamines
Received for publication, March 25, 2014
Accepted, June 14, 2014
I. M. APETREIa, C. V. POPA (UNGUREANU) b, C. APETREIc,*, D. TUTUNARUa
a
Department of Pharmaceutical Sciences, Faculty of Medicine and Pharmacy, “Dunarea de
Jos” University of Galati
b
Faculty of Food Science and Engineering, “Dunarea de Jos” University of Galati
c
Department of Chemistry, Physics and Environment, Faculty of Sciences and Environment,
“Dunarea de Jos” University of Galati
*Address correspondence to: “Dunarea de Jos” University of Galati, Faculty of Sciences and
Environment, 47 Domneasca Street, 800201, Galati, Romania; E-mail: apetreic@ugal.ro
Abstract
This work reports tyrosinase based electrochemical biosensors using graphene modified
screen-printed carbon based electrodes for the determination of catecholamines. The enzyme has been
immobilized onto the graphene modified carbon working electrodes by cross-linking with
glutaraldehyde. The detection has been performed by measuring the cathodic current due to the
reduction of the corresponding quinone a low potential, 0.025 V for dopamine and -0.025 V for
epinephrine, respectively. The experimental conditions for the tyrosinase immobilization and the main
variables that can influence the cathodic current have been optimized. Under optimum conditions, the
electrochemical biosensors have been characterized. A linear response range from 0.2 up to 25 M of
dopamine and from 1 to 27.5 M of epinephrine was obtained. The detection limits are in the range
2.42×10-7 - 6.56×10-7 M for developed biosensors. The biosensors construction was highly
reproducible. Finally, the developed biosensors have been applied to the determination of dopamine
and epinephrine content in pharmaceutical formulation samples.
Keywords: biosensor, screen-printed electrode, tyrosinase, graphene, catecholamine
1.
Introduction
Catecholamines (dopamine, epinephrine and norepinephrine) play a major role in the
nervous system as central and peripheral neurotransmitters [1]. Catecholamines are biological
amines released mainly from the adrenal glands in response to stress, acting as
neurotransmitters. Therefore, they are maintaining normal physical activity of the body
including blood pressure, heart rate and the reactions of the sympathetic nervous system [2].
The identification and quantification of catecholamines in biological fluids is of great
importance in medical diagnostics, principally for patients suffering from Parkinson's disease
and stress. Consequently, there is a necessity for their quantitative determination in
pharmaceutical products and biological fluids for detecting any endocrine disorders or any
other physiological and pathological abnormality in the body [3].
Various techniques have been implemented for the determination of catecholamines
such as high performance liquid chromatography [4], capillary electrophoresis [5],
chemiluminescence [6] and fluorescence [7]. Usually, these methods are relative complicated
as they need complex derivatization procedures or combination of various detection methods.
Furthermore, some of these methods do not have enough sensitivity and selectivity in the
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corresponding determinations. Contrary, electrochemical detection methods have shown
several advantages such as high sensitivity, low cost, not time consuming and simple surface
modification of electrodes. As a consequence of these advantages, several electrochemical
methods are available for the determination of dopamine (DA) [8-10] and epinephrine (EP)
[11-13].
Biosensors, which combine biological recognition through enzyme specificity with
construction simplicity, have been reported as a good and cheap alternative for the traditional
ones [14]. The use of disposable transducers, such as screen-printed electrodes (SPEs), boosts
the intrinsic characteristics of biosensors.
Screen-printing technology, which offers design flexibility, process automatization
and good reproducibility in the transducers fabrication, had been shown as a method for mass
production of biosensors at low cost [15].
In the case of biogenic amines, however, some results have been described in the
literature. Lange and Wittmann [16], as well as Chemnitius and Bilitewski [17] have
described amino oxidase-sensors based on platinum screen printed electrodes, using a
glutaraldehyde - bovine serum albumin cross-linking immobilization procedure, for the
determination of histamine, putrescine and tyramine. Alonso-Lomillo et al. report
monoamine oxidase/horseradish peroxidase and diamine oxidase/horseradish peroxidase
based biosensors using screen-printed carbon electrodes for the determination of several
biogenic amines [18]. Apetrei et al. report the development of disposable biosensors based on
carbonaceous screen-printed electrodes and diamine oxidase [19].
In this work, biosensors based on graphene modified screen-printed carbon electrodes
and containing tyrosinase as biocatalyst were prepared and their capability to detect
dopamine and epinephrine was evaluated. For this purpose, dopamine and epinephrine
detection was carried out in buffered aqueous solutions. The amperometric characteristics
including kinetics, calibration curves and limits of detection in the detection of dopamine and
epinephrine were investigated. The potential of biosensors to detect and quantify dopamine
and epinephrine in pharmaceutical formulations was studied.
2.
Materials and Methods
2.1. Chemical and solutions All reagents were of high purity and used without further
purification. Dopamine and epinephrine were purchased from Sigma-Aldrich. Tyrosinase
(EC 1.14.18.1, from mushroom) was purchased from Sigma. A 5 mg×mL-1 solution of
tyrosinase in buffer phosphate solution (0.01 M, pH 7.0) was used for the enzyme
immobilization. The phosphate buffer solutions were prepared from phosphoric acid,
potassium monobasic and dibasic phosphate salts from Aldrich. Ultrapure water (18 MΩ×cm,
Millipore Milli-Q) was used for preparation of all aqueous solutions.
2.2. Apparatus Electrochemical measurements were performed on a Biologic Science
Instruments SP 150 potentiostat/galvanostat using the EC-Lab Express software. An UV-Vis
spectrophotometer from Labomed Inc. connected to a PC (software UVWin) was used for
spectrophotometric experiments. An Elmasonic S10H ultrasonic bath was used for dissolving
and homogenization of solutions. For pH measurements an Inolab pH 7310 was used. For
stirring of solutions in amperometric measurements a magnetic stirrer (Velp, Italy) was used.
2.3. Electrodes and electrochemical cell Screen-printed carbon electrodes (4 mm
diameter, S=12.56 mm2) purchased from Dropsens, www.dropsens.com, model DRP110GPH (screen-printed carbon electrodes modified with graphene) were used for biosensor
construction. A three-electrode configuration was used. The reference and the counter
electrode integrated in the device were used (counter electrode - carbon, reference electrode -
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silver). Cyclic voltammograms were registered from -0.5 to +0.5V (the scan started at 0V) at
a scan rate of 0.05 V×s-1 (except otherwise indicated).
2.4. Fabrication of biosensors Ty was immobilized over graphene-carbon thick film by
casting technique followed by cross-linking with glutaraldehyde. 50 L of 0.01 M phosphate
buffer (pH 7.0) containing 5 mg×mL-1 of Ty was added onto 12.56 mm2 area of graphenecarbon thick film. After drying, the Ty-graphene-carbon films were exposed to a 2.5% (v/v)
glutaraldehyde solution (in PBS 0.01 M of pH 7.0) for 20 min at room temperature. The
enzyme-immobilized film was dried at 10°C and rinsed with phosphate buffer solution to
remove any unbound enzyme from the electrode surface. Finally, the biosensors were further
dried at 10°C and stored at 4°C [30].
To study the repeatability and stability of the biosensor based on tyrosinase immobilized on
graphene screen-printed carbon electrode, amperometric measurements were carried out in a
10−5 M dopamine solution in 0.01 M PBS at pH 7.0, using the same biosensor device.
2.5. Pharmacopoeia method The spectrophotometric methods established in the X
Romanian Pharmacopoeia (1993) [29] were used to check the accuracy of the results
obtained with the proposed biosensors.
3.
Results and Discussions
In this study, a nanomaterial based on carbon, graphene (GPH), was used as immobilization
matrix for tyrosinase (Ty). The screen-printed carbon electrodes modified with GPH are
designed for the development of (bio)sensors with an enhanced electrochemical active area
and enhanced electronic transfer properties.
3.1. Cyclic voltammetry Thermal The cyclic voltammograms of biosensor in 100 M
dopamine solution and 100 M epinephrine (in 0.01 M phosphate buffer solution, pH 7.0),
respectively, were shown in Figure 1.
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Figure 1. Cyclic voltammogram of biosensor in a) 100 M dopamine solution (0.01 M PBS, pH
7.0). b) 100 M epinephrine solution (0.01 M PBS, pH 7.0). Scan rate 0.050 V×s-1
As can be seen, in the cyclic voltammetry of DA (a catecholamine), one oxidation peak and
one reduction peak are observed (Figure 1a). The oxidation peak is due to the oxidation of
catecholamine to o-quinone, which is reversible in nature:
Dopamine + Tyrosinase (O2) → o-Dopaquinone + H2O
The o-dopaquinone is electrochemically active is electrochemically reduced to biosensor
surface:
o-Dopaquinone + 2H+ + 2e- → Dopamine
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EP follows the same reaction mechanism. EP gets oxidized to epinephrinequinone, which is
electrochemically reduced to biosensor surface.
In DA solution (supporting electrolyte PBS 0.01 M, pH 7.0), the CV gave two well defined
peaks locating, one cathodic at 0.025 V, and one anodic at 0.30 V. In the presence of EP (100
M) in PBS (Figure 1b), the CV curves gave two well defined peaks locating, one cathodic at
-0.025 V, and one anodic at +0.40 V. These results are in good agreement with results
published using poly(indole-5-carboxylic acid)/tyrosinase electrode or carbon
black/tyrosinase biosensor [20,21].
These results demonstrate that the tyrosinase enzyme retains its bioactivity to a large extent
when immobilized on GPH-C thick film. Tyrosinase immobilized in GPH efficiently
catalyzes the oxidation of DA or EP. The sharp and intense oxidation and reduction peaks
reveal a fast electron transfer at tyrosinase immobilized GPH-C electrode [22].
Additionally, the effect of potential scan rate on the peak current of the two catecholamines
was studied. From Figure 2, it can also be seen that the oxidation peak shifts to a more
positive value and the reduction peak for both molecules shifts to the more negative values
with increasing scan rates along with a concurrent increase in current.
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Figure 2. (a) Cyclic voltammograms of biosensor in 100 M DA (pH 7.0) registered with
different scan rates. (b) Cyclic voltammograms of biosensor in 100 M EP (pH 7.0) registered
with different scan rates.
The cyclic voltammetric results point out the cathodic peak currents (Ip) for both
catecholamines varied linearly with the scan rate () ranging from 0.050 V×s-1 to 1.000 V×s-1
(Figure 2) which implies that the reduction of DA and EP is kinetically controlled on
Ty/GPH-C/SPE.
3.2. Optimization of experimental parameters The pH of the supporting electrolyte
showed a significant effect on the electrochemical behavior of DA (100 M) and EP (100
M) at the surface of the biosensor. Cyclic voltammetry (CV) was carried out to characterize
the influence of pH of the buffer solution (in the range of 4.0-9.0). The influence of the pH on
the oxidation peaks currents of DA and EP was investigated employing phosphate buffer
solutions. It was observed that as the pH of the medium was progressively augmented, the
potential kept on shifting towards less positive values. The involvement of proton in the
reaction is suggested. Over the pH range of 4.0-9.0, the peak potential (Ep) for DA and EP is
found to be a linear function of pH.
From the plot of Ep vs. pH, slopes of 0.051, and 0.0534 V/pH unit were obtained for DA
(Figure 3a) and EP (Figure 3b), respectively. These slopes reveal that an equal number of
protons and electrons are involved in the reduction reactions of DA and EP. Additionally, it
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was observed that the peak current for both DA and EP was maximum at pH 7.0. Thus, these
optimized pH values were employed for further studies.
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Figure 3. a) A plot of peak current (ip) vs. pH and peak potential (Ep) vs. pH for 100 M DA at
Ty/GPH-C/SPE employing CV; b) A plot of peak current (ip) vs. pH and peak potential (Ep) vs. pH
for 100 M EP at Ty/GPH-C/SPE employing CV.
The applied potential has a key influence over the biosensor response, because the applied
potential contributes to the sensitivity and selectivity of the biosensor. The potential
dependence on the biosensor response is shown in Figure 4 using 100 M DA in 0.01 M
phosphate buffer (pH 7.0) and 100 M EP in 0.01 M phosphate buffer (pH 7.0), respectively.
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Figure 4. Current-potential dependence in (a) 100 M DA and (b) 100 M EP (supporting
electrolyte 0.01 M PBS, pH 7.0, constant stirring).
The maximum of the signal is obtained at +0.025 V. In the case of EP The maximum of the
signal is obtained at -0.025 V. Therefore, +0.025 V was used as the applied potential for DA
detection, and -0.025 V for EP detection.
3.3. Amperometric response of biosensor Figure 5 (a) illustrates a typical amperometric
response for the Ty/GPH-C/SPE biosensor at +0.025 V after the addition of successive
aliquots of DA to the 0.01 M PBS (pH 7.0) under constant stirring. As presented in the
Figure, a well-defined reduction current proportional to the concentration of catecholamine is
observed, which results from the electrochemical reduction of o-dopaquinone species
enzymatically formed.
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Figure 5. (a) Amperometric response of Ty/GPH-C/SPE biosensor to DA (in 0.01 M PBS
solution, pH 7.0), with levels increasing in 40 M increments; Applied potential +0.025V (b)
Amperometric response of Ty/GPH-C/SPE biosensor to EP (in 0.01 M PBS solution, pH 7.0),
with levels increasing in 40 M increments; Applied potential -0.025V.
The Ty/GPH-C/SPE biosensor achieves 95% of steady-state current in less than 5 s. The
response rate is much faster than that of 10 s reported in a Ty-conducting polymer film based
biosensor [23] and comparable with that reported for a Ty-graphite paste based biosensor
[24]. Such fast response is attributed to a fast electron transfer between the enzymaticallyproduced dopaquinone and the biosensor.
Figure 6 (a) showed the relationship between the response current of the biosensor and the
DA concentration in PBS (pH 7.0) at +0.025 V (calibration curve).
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Figure 6. (a) Calibration curve between the cathodic current and the concentration of DA in 0.01
M PBS solution (pH 7.0) of Ty/GPH-C/SPE biosensor; (b) Calibration curve between the
cathodic current and the concentration of EP in 0.01 M PBS solution (pH 7.0) of Ty/GPH-C/SPE
biosensor.
It can be seen from Figure 6 (a) that the response current is linear with DA concentration in
the range from 0.2 up to 25 M with sensitivity of 0.64A/M for the cathodic process. The
corresponding detection limit was calculated according to the 3sb/m criterion. In this equation
m is the slope of the calibration graph. sb is the standard deviation (n = 5) of the voltammetric
signals of the substrate at the concentration level corresponding to the lowest concentration of
the calibration plot. The detection limit calculated was 2.42×10-7 M. The detection limit is in
the range of the detection limits published for biosensors based on tyrosinase [25].
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From the above results, the Hill coefficient (h) can be calculated by representing the
log[I/(Imax-I)] vs. log [DA] (the logarithm of the substrate concentration). A Hill coefficient of
1.04 was calculated for the enzymatic process (R2=0.9915). The finding that the h parameter,
calculated from the corresponding Hill’s plot, was close to unity, demonstrated that the
kinetics of the enzymatic reaction fitted into a Michaelis-Menten type kinetics [26].
The enzymatic kinetic parameters were obtained using the linearization of Lineweaver-Burk
expressed by equation (1) [27]:
K app
1
1
 M
(1) 
I I max I max S 
where [S] is the concentration of the substrate, I is the cathodic current at 0.025V, KMapp is
the apparent Michaelis-Menten constant for the enzymatic reaction and Imax is the steady-state
current. Using the Lineweaver-Burk equation and representing 1/I vs. 1/[DA] the apparent
Michaelis-Menten constant was calculate from the slope and the Imax from the intercept.
KMapp of 18.75 M and an Imax of 24.84Awere obtained for DA. KMapp is lower than the
values usually reported for tyrosinase biosensors (in the range of mM) using other
immobilization techniques [28]. This proves the benefits of the GPH in construction of
biosensor with high quality performances.
In the case of epinephrine similar results were obtained as was summarized in Table 1. The
graphical results are presented in the Figures 5 (b) and 6 (b).
Table 1. Response characteristics of the Ty/GPH-C/SPE biosensor to catecholamines
Compound
h
LOD (M)
Linear range (M)
KMapp (M)
Imax (A)
-7
Dopamine
1.04
0.2 - 25
2.42×10
18.75
24.84
Epinephrine
0.96
1 - 27.5
6.56×10-7
15.56
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12.34
3.4. Repeatability and stability of the biosensor To study the repeatability and stability
of the biosensor based on tyrosinase immobilized on graphene screen-printed carbon
electrode, amperometric measurements were carried out in a 10−5 M dopamine solution in
0.01 M PBS at pH 7.0 using the same biosensor device. Among replicate measurements, the
biosensor was tacked off from the electrolyte solution containing dopamine and rinsed with
ultrapure water and 0.01 M PBS (pH 7.0). The relative standard deviation (%RSD) of 10
measurements was 4.1%. Therefore, several measurements can be carried out without a
significant variation of the biosensor response, lower than 5%.
The stability of the biosensor was studied by monitoring the amperometric response at
regular intervals of 24 hours for 3 days. Between measurements, the biosensor was stored in a
refrigerator at 4°C in 0.01 M PBS at pH 7.0. The amperometric responses of the biosensor
decrease constantly. After 3 days of storage, the biosensor maintains 67% of its initial current
response. Therefore, the biosensor can be used as a disposable single use device.
3.5. Application of the biosensor to determination of DA and EP in pharmaceutical
formulas The UV spectrophotometric methods indicated in the X Romanian Pharmacopoeia
suggest the measurement of dopamine at 280 nm, in 1 g×L-1 Na2SO3, and the measurement of
epinephrine at 279 nm, in presence of 10-2 M HCl [29].
Calibration curves were constructed using pure dopamine, and epinephrine, respectively.
These were used for quantification of dopamine and epinephrine, respectively, in different of
pharmaceutical formulations.
Table 2 shows the results obtained for the analyses of pharmaceutical formulations using the
UV spectrophotometric procedure and biosensor, respectively (at +0.025 V for DA and 0.025 V for EP).
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Table 2. Determination of DA and EP in pharmaceutical formulations by UV spectrophotometry and biosensor
No.
Compound
Sample
a
b
c
1.
Dopamine
Dopamine chlorhydrate (S.C. Zentiva S.A., Romania)
50
501.2
491.6
2.
Dopamine
Dopamin (Admeda Arzneimittel GmbH, Germany)
20
200.7
19 0.8
3.
Epinephrine
Adrenalina (S.C. Terapia S.A., Romania)
1
1.00.05 1.10.07
4.
Epinephrine
Anapen® (Lincoln Medical Limited, UK)
1
1.00.06 0.90.08
a - Amount of catecholamine in the sample (mg).
b - Amount of catecholamine obtained by the proposed method (mg) RSD (n=5).
c - Amount of catecholamine obtained by the spectrophotometric method (mg)  RSD (n=5).
RSD - Relative standard deviation
4. Conclusions
A novel biosensor has been developed for the determination of catecholamines
employing tyrosinase immobilized on graphene screen-printed carbon electrodes. The
advantages of this biosensor comparing with traditional analytical methods are sensitivity,
fabrication simplicity and short time for analysis. The proposed biosensor is very sensitive
having sub-micromolar detection limits for dopamine and epinephrine. The biosensor has
been employed for the determination of dopamine and epinephrine in pharmaceutical
formulations and the results are satisfactory, which suggests that biosensor can act as a
promising biosensor for the determination of dopamine and epinephrine. The concentration
results have been in a good agreement with the Pharmacopoeia method.
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Acknowledgments
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This work was supported by a grant of the Romanian National Authority for Scientific
Research, CNCS - UEFISCDI, project number PN-II-ID-PCE-2011-3-0255.
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