International Journal of Environmental Analytical

Chemistry

ISSN: 0306-7319 (Print) 1029-0397 (Online) Journal homepage: https://www.tandfonline.com/loi/geac20

Dopamine biosensor based on screen-printed

electrode modified with reduced graphene oxide,
polyneutral red and gold nanoparticle

Muhammed Altun, Melike Bilgi Kamaç, Alper Bilgi & Merve Yılmaz

To cite this article: Muhammed Altun, Melike Bilgi Kamaç, Alper Bilgi & Merve Yılmaz (2020)
Dopamine biosensor based on screen-printed electrode modified with reduced graphene oxide,
polyneutral red and gold nanoparticle, International Journal of Environmental Analytical Chemistry,
100:4, 451-467, DOI: 10.1080/03067319.2020.1720669

To link to this article: https://doi.org/10.1080/03067319.2020.1720669

Published online: 05 Feb 2020.

Submit your article to this journal

Article views: 18

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=geac20
INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY
2020, VOL. 100, NO. 4, 451–467
https://doi.org/10.1080/03067319.2020.1720669

ARTICLE

Dopamine biosensor based on screen-printed electrode

modified with reduced graphene oxide, polyneutral red and
gold nanoparticle
Muhammed Altun , Melike Bilgi Kamaç , Alper Bilgi and Merve Yılmaz
Department of Chemistry, Faculty of Science, Cankiri Karatekin University, Cankiri, Turkey

ABSTRACT ARTICLE HISTORY

A new amperometric dopamine sensor has been developed on the Received 22 December 2019
basis of modified screen-printed electrode (SPCE) with reduced Accepted 24 December 2019
graphene oxide (RGO), polyneutral red (PNR) and gold nanoparti- KEYWORDS
cles (AuNP). The electrochemical behaviour of dopamine on SPCE/ Dopamine biosensor;
RGO/PNR/AuNP was investigated. The prepared sensor showed reduced graphene oxide;
a high electrocatalytic effect on the oxidation of dopamine. In the polyneutral red; gold
amperometric determination of DA, optimum working conditions nanoparticles; screen-printed
(optimum pH, optimum working potential, linear working range) electrodes
were determined. The optimum values of these parameters in DA
determination were 7.0 for pH and +0.25 V for working potential.
The results of analytical characterisations, repeatability for 50 µM
DA, sensitivity, the limit of detection, the limit of quantification and
the linear range were found to be 2.14% (n = 10) as relative
standard deviation (RSD.), 13.38 µA mM−1; 0.17 µM; 0.57 and 0.57–-
500 µM, respectively. Operational stability studies have shown that
the initial amperometric response of the sensor to dopamine
decreased by 90.05% on day 60. Storage life studies showed that
the sensitivity of the biosensor decreased by 18.88% at the end of
the 8th week. Amperometric electrochemical analysis of DA using
SPCE/RGO/PNR/AuNP electrode was performed in the presence of
interfering electroactive species AA and UA. The developed sensor
was tested for DA determination in artificial blood serum samples.

1. Introduction
Dopamine (DA) is one of the most important neurotransmitters that play a critical role in
regulating the functions of the renal, hormonal, central nervous and cardiovascular
systems and in emotional control [1–3]. Abnormal DA levels can cause various neurolo-
gical disorders such as Parkinson’s, Huntington’s, Alzheimer’s and Schizophrenia [4–11].
Health problems such as Alzheimer’s and Parkinson’s disease increase with the ageing of
the individuals forming the society [8]. One of the symptoms of dopamine deficiency is
depression. In addition, chronic boredom, lack of satisfaction, low physical energy, chronic
fatigue and lack of interest in body exercise come. In general, the amount of DA in the
brain fluid is between 10−6 and 10−8 M concentrations [12–14]. The plasma and urinary
reference ranges for dopamine are respectively as follows: Ambulatory adults: <20 ng/mL

CONTACT Muhammed Altun maltun80@gmail.com

© 2020 Informa UK Limited, trading as Taylor & Francis Group
452 M. ALTUN ET AL.

(conventional units); <0.13 nmol/L (SI units) [15] and 65–400 mcg/24 hr [16]. Dopamine,
involved in the communication between nerve cells in the brain, plays a role in regulating
reward, motivation, memory, attention and even body movements. Dopamine is also
responsible for the formation of pleasure in the brain. This hormone, also known as
happiness hormone; it is of great importance in the realisation of undeniable operations
in daily life, such as movement, learning and concentration [17]. Some pathological
conditions such as schizophrenia, autism, attention deficit and hyperactivity disorder
have been associated with DA dysfunction [18,19]. The determination of DA plays
a critical role in neurochemical and industrial applications.
Therefore, the precise detection of the DA at the physiological level is important for the
early diagnosis of these neurological diseases [1,11,12,20]. The response signals of DA are
often sensitive to interference from the other metabolites [14]. Electrochemical sensors
are widely used in the determination of dopamine with rapid response and sensitivity.
A wide variety of DA biosensors have been developed with many nanomaterial modifica-
tions [20].
Neutral red (NR; N8, N8, 3-trimethyl-phenazine-2,8,-diamine chloride) is a phenazine
dye that is used for various purposes such as colourant to examine biological materials
such as viruses, pH indicator in biochemical systems, determination of DNA using optical
or electrochemical methods [21]. NR, like other phenazine dyes, can be electrochemically
polymerised to form a conductive polymer. Recently, poly (neutral red) (PNR) has been
a polymer used as both conductive polymer and a redox mediator in sensors and
biosensors [21]. The polymerisation of NR can be controlled both by the applied potential
responsible for the radical formation and by the pH [22–25]. The polymerisation mechan-
ism involves the formation of radicals, dimerisation of the monomer, and tetramerization
and subsequent to polymer formation [26].
The morphologies of the nanomaterials have great importance in improving the
detection performance of the modified electrodes [11,27]. Graphene has been widely
used in the modification of electrochemical sensors due to its large surface area, excellent
conductivity, good chemical stability, unique electronic and mechanical strength proper-
ties [28,29]. Significant synergistic effects were observed for target analytes in graphene-
based electrodes modified with noble metal nanoparticles [11,28–30].
Graphene is a very useful nanomaterial due to its mechanical, optical, thermal and
electrochemical properties [31]. Graphene, graphene oxide (GO) and reduced graphene
oxide (RGO) are indispensable nanomaterials for the production of biosensors due to their
biocompatibility [32]. Graphene-based materials contain functional groups that are essen-
tial for electrode surface functionality. Physically, these nanomaterials exhibit a high
surface area/volume ratio that increases bio-receptor immobilisation in biosensing pro-
cesses [33]. The detection performances of graphene-based electrochemical biosensors
are very high due to their high selectivity, precision and advanced reproducibility [34–36].
Due to the advantages of electrochemical methods, biosensors are widely used for the
diagnosis of neurological diseases [11,37]. The importance of developing modified elec-
trodes with high selectivity and precision to meet clinical demands is becoming more
important [28,38].
In this study, SPCE was modified with RGO, PNR and AuNP to generate amperometric
DA sensor. The generated DA sensor has been successfully used for the detection of DA in
the human serum sample. No studies have been found in which NR was deposited in
 INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 453

SPCE/RGO by electropolymerization and using SPCE/RGO/AuNP electrode for the electro-

oxidation of DA.

2. Experimental
2.1. Reagents
Reduced graphene oxide (RGO), neutral red (NR), colloidal gold nanoparticle solution
(AuNP) (10 nm), dopamine hydrochloride (DA), L-ascorbic acid (AA) and uric acid (UA)
were obtained from Sigma-Aldrich. Potassium dihydrogen phosphate (KH2PO4), potassium
hydrogen phosphate (K2HPO4), potassium chloride (KCl), dimethylformamide (DMF), hydro-
chloric acid (HCl, 37%), sodium hydroxide (NaOH) and potassium nitrate (KNO3) were
purchased from Merck. Water taken from Millipore Milli-Q Direct Q-3 ultrapure water system
was used in experiments. K2HPO4, KH2PO4 and KCl were used in phosphate buffer solution
(PBS) preparations and DA, AA and UA solutions were prepared in these PBSs (in 0.1 M KCl).

2.2. Electrochemical measurements

The electrochemical measurements were conducted using a portable Bipotentiostats/
Galvanostate μStat 400 that was purchased from DropSens (Spain) and controlled by PC
with DropView 800 software. Disposable screen-printed carbon electrodes (SPCEs) were
also supplied from DropSens (Oviedo, Spain, ref. DRP110). These electrodes consist of
three parts, a carbon working electrode (WE), a silver reference electrode (RE) and
a carbon counter electrode (CE). The amperometric responses of SPCE/RGO/PNR/AuNP
sensor towards DA detection were recorded under stirred conditions in 0.05 M PBS (pH
7.0) by applying a constant potential of +0.25 V at the working electrode. A Mettler Toledo
pH meter was used for pH measurements. SEM images of SPCE electrodes were made by
ÇOBİLTUM with JEOL SEM-7100-EDX.

2.3. Preparation of RGO-PNR-AuNP modified SPCEs

A stable and homogeneous suspension was prepared by a 3 h sonicator application to the
mixture of 0.5 mg of RGO and 1 mL of DMF:H2O (1:1). Electrochemical pretreatment was
applied for cleaning and activation of the surfaces of SPCE electrodes by applying
potential for 300 s at +1.8V in 50 mM pH 7.5 phosphate buffer [39,40]. One microlitre of
the dispersed RGO was dropped onto the surface of the working electrode (WE) and
allowed to dry at room temperature. Thus, RGO modified SPCE (SPCE/RGO) was prepared.
The PNR was then electropolymerized by cyclic voltammetry at a scanning rate of 50 mV
s−1 to form a thin PNR film on SPCE/RGO. The potential was converted from −1.1 to +0.9
V for 15 cycles in a solution containing 1 mM NR monomer, 0.025 M pH 5.5 PBS and 0.1
M KNO3. The electrode (SPCE/RGO/PNR) was rinsed in PBS and several more potential
cycles were applied to remove any residual monomer NR. Finally, it was further modified
with AuNPs. Four microlitres of AuNP suspension was deposited on the surface of the
SPCE/RGO/PNR working electrode and then allowed to dry at room temperature. The
developed sensor was tested for the determination of DA in blood serum sufficiently
diluted with PBS (pH = 7.0) at a working potential of +0.25 V.
454 M. ALTUN ET AL.

3. Results and discussion

3.1. Characterisation of SPCE modified RGO, PNR and AuNP
SEM images of the bare SPCE, SPCE/RGO, SPCE/RGO/PNR and SPCE/RGO/PNR/AuNP
electrodes were taken and given in Figure 1(a–d), respectively. Figure 1(a) indicates that
bare SPCE electrode has a smooth surface. In Figure 1(b), RGO’s layered, wrinkled surface,

B
1 µm

1 µm

1 µm 100 nm

1 µm 100 nm

Figure 1. SEM images of bare SPCE (a), SPCE/RGO (b), SPCE/RGO/PNR (c) and SPCE/RGO/PNR/AuNP (d).
 INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 455

randomly distributed thin layers and scattered overlapping structures are observed, and
these images are consistent with the literature [41,42]. These results clearly show that RGO
has been successfully modified to the SPCE surface. Figure 1(c) presents polymer on the
SPCE/RGO/PNR electrode surface, while Figure 1(d) shows an intensive layer on the SPCE/
RGO/PNR/AuNP surface.
To examine the effect of RGO, PNR and AuNP in the formulation of the modified
electrodes, SPCE, SPCE/RGO, SPCE/RGO/PNR and SPCE/RGO/PNR/AuNP electrodes were
characterised by applying cyclic voltammetry between −1.0 and +1.0 V in a solution of
5 mM K3Fe(CN)6/K4Fe(CN)6 in 0.1 M KCl supporting electrolyte using scan rate 50 mV s−1
and obtained voltammograms are given in Figure 2. The values of anodic peak current
(Ipa), cathodic peak currents (Ipc), anodic peak potentials (Epa) and cathodic peak
potentials (Epc) obtained from Figure 2 are given in Table 1. It is understood from
Figure 2 and Table 1 that the Ipa and Ipc increase the difference between the peak
potentials decreases with the presence of RGO in the electrode formulation. RGO
increased the electronic conductivity and the reversibility of the electrochemical reaction.
The introduction of PNR into the SPCE/RGO formulation increased Ipa and Ipc as in RGO.
The coexistence of RGO and PNR in the electrode formulation has created a synergistic
effect, increasing the electronic conductivity and sensitivity considerably. With the addi-
tion of AuNP in SPCE/RGO/PNR formulation, Ipa and Ipc are further increased. It was

bare SPCE
4.0E-04
SPCE/RGO
SPCE/RGO/PNR

2.0E-04 SPCE/RGO/PNR/AuNP
I / µA

0.0E+00

-2.0E-04

-4.0E-04
-1.2 -0.8 -0.4 0.0 0.4 0.8 1.2
E / V vs Ag/AgCl

Figure 2. Cyclic voltammograms of bare SPCE, SPCE/RGO, SPCE/RGO/PNR and SPCE/RGO/PNR/AuNP in

5 mM K3Fe(CN)6/K4Fe(CN)6 in 0.1 M KCl between potentials of −1.0 V to +1.0 V (vs Ag/AgCl) at a scan
rate of 50 mV s−1.

Table 1. Epa, Ipa, Epc and Ipc values obtained from Figure 2.
Electrode formulation Epa/(V) Ipa/(µA) Epc/(V) Ipc/(µA)
Bare SPCE 0.258 135 0.008 −124.8
SPCE/RGO 0.221 169.8 0.078 −170.7
SPCE/RGO/PNR 0.240 182.1 0.054 −190.2
SPCE/RGO/PNR/AuNP 0.230 195.7 0.032 −211.7
456 M. ALTUN ET AL.

observed that Epa shifted negatively, and Epc shifted positively. Although the effect of
AuNP is less than that of RGO and PNR, coexistence of RGO, PNR and AuNP in the
electrode formulation showed a synergistic effect, increasing the electroactive surface
area of the electrode and increasing the conductivity and electrode reversibility.
To compare the electroactive surface area (Aea) of different modified electrodes,
the Aea values of SPCE, SPCE/RGO, SPCE/RGO/PNR and SPCE/RGO/PNR/AuNP electro-
des were calculated from Randles-Sevcik equation. According to Randless–Sevcik
equation,

ip ¼ 2:96x105 n3=2 Aea D1=2 Cv1=2 (1)

where ip is the oxidation peak current (A), n is the number of electrons transferred, Aea is
the electroactive area (cm2), D is the diffusion coefficient of 7.63 × 10−6 cm2 s−1 [43] for
Fe(CN)63-, (cm2 s−1), C is the concentration of the redox species in bulk solution (mol cm−3)
and v is the potential scan rate (V s−1) [44]. The Aea values of SPCE, SPCE/RGO, SPCE/RGO/
PNR and SPCE/RGO/PNR/AuNP electrodes were calculated to be 0.163 cm2, 0.205 cm2,
0.220 cm2 and 0.237 cm2, respectively. The Aea values of the different modified electrodes
are higher than the bare SPCE electrode. As the introduction of RGO, PNR and AuNP,
respectively, into the electrode formulation, the Aea values of these electrodes increased.
As a result, Ipc and Ipc increase with increasing the electroactive surface area of the
different modified electrodes.

3.2. Electrooxidation of DA on SPCE/RGO/PNR/AuNP

The effect of modification of SPCE with RGO, PNR and AuNP on electrooxidation of DA
was studied by cyclic voltammetry in 1 mM DA in 50 mM PBS of pH 7.0 at a scan rate of
50 mV s−1 (Figure 3(a)). Ipa, Epa, Ipc and Epc obtained from the resulting CVs for bare
SPCE, SPCE/RGO SPCE/AuNP, SPCE/PNR, SPCE/RGO/AuNP, SPCE/RGO/PNR and SPCE/
RGO/PNR/AuNP are given in Table 2. When Figure 3(a) and Table 2 are examined, it is
observed that RGO, PNR and AuNP show electrocatalytic effect towards electrooxidation
of DA separately and together. PNR showed the highest peak current effect towards DA
(Ipa: 127.09 μA). The Ipa values of SPCE/PNR (127.09 μA), SPCE/RGO/PNR (122.13 μA) and
SPCE/RGO/PNR/AuNP (114.88 μA) were higher than SPCE/RGO (104.75 μA), SPCE/AuNP
(110.13 μA) and SPCE/RGO/AuNP (89.20 μA). In the literature, it was found that PNR
showed the electrocatalytic effect towards DA [45,46]. Both SPCE/RGO/PNR and SPCE/
RGO/PNR/AuNP electrodes exhibited considerable electrocatalytic effect towards the
electrooxidation of DA. Peak potentials are also close to each other (Epa: 0.190 V for
SPCE/RGO/PNR; Epa: 0.194 V for SPCE/RGO/PNR/AuNP). Although the Ipa value of SPCE/
RGO/PNR (122.13 μA) electrode was higher than SPCE/RGO/PNR/AuNP (114.88 μA),
SPCE/RGO/PNR/AuNP electrode was preferred for the DA sensor. This is because AuNP
increases the operation and storage stability of the developed DA sensor due to the
biocompatibility of its.
In order to test the electrocatalytic activity of SPCE/RGO/PNR/AuNP to the electroox-
idation of DA, cyclic voltammetry was applied in the presence and absence of 1 mM DA in
50 mM pH 7.0 phosphate buffer (in 0.1 M KCl) at a scan rate of 50 mV s−1. In the absence of
DA, only the peaks of PNR in the electrode formulation were observed in the buffer
 INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 457

180
BARE SPCE
SPCE/RGO
SPCE/AuNP a
120 SPCE/PNR
SPCE/RGO/AuNP
SPCE/RGO/PNR
SPCE/RGO/PNR/AuNP
60
I / µA

-60

-120
-0.4 -0.2 0 0.2 0.4 0.6
E / V vs Ag/AgCl

150
PBS b
1 mM DA
100

50
I / µA

-50

-100
-0.4 0 0.4 0.8
E/V vs Ag/AgCl

Figure 3. (a) The CVs of bare SPCE, SCPCE/RGO, SPCE/PNR, SPCE/AuNP, SPCE/RGO/AuNP, SPCE/RGO/
PNR and SPCE/RGO/PNR/AuNP recorded in 1 mM DA (pH = 7.0) between potentials of −0.3 V and +0.6
V (vs Ag/AgCl) at a scan rate of 50 mV s−1. (b) The CVs of SPCE/RGO/PNR/AuNP electrode obtained in
PBS (pH 7.0) in the absence of DA and in the presence of 1 mM DA, between potentials of −0.3 V and
+0.7 V (vs Ag/AgCl) at a scan rate of 50 mV s−1.

solution, but a redox pair was observed in the presence of DA (Figure 3(b)). The Epa and
Epc values of the redox pair observed in the presence of 1 mM DA are +0.158 V and +0.076
V. Ipa and Ipc values are 57.52 µA and 8.43 µA, respectively.
Figure 4 shows cyclic voltammograms of 1 mM DA at different scanning rates (10, 25,
50, 75, 100, 125 mV s−1) on SPCE/RGO/PNR/AuNP. It can be seen from these graphs that
458 M. ALTUN ET AL.

Table 2. Epa, Ipa, Epc and Ipc values for electrooxidation of DA on various electrode
formulations obtained from CVs taken in 1 mM DA in 50 mM PBS of pH 7.0 at a scan
rate of 50 mV s−1 with each electrode.
Electrode formulation Epa/(V) Ipa/(µA) Epc/(V) Ipc/(µA)
Bare SPCE 0.168 31.98 0.008 −19.27
SPCE/RGO 0.152 104.75 0.056 −62.67
SPCE/AuNP 0.166 110.13 0.076 −25.45
SPCE/PNR 0.204 127.09 0.060 −66.45
SPCE/RGO/AuNP 0.162 89.20 0.064 −28.62
SPCE/RGO/PNR 0.190 122.13 0.066 −52.76
SPCE/RGO/PNR/AuNP 0.194 114.88 0.066 −22.87

200

50
I / µA

1.4E-04 Ipa

Ipc
y = 2.71E-04x + 4.08E-05
6.0E-05 R² = 1.00E+00
I/A

-100
-2.0E-05 y = -1.91E-04x - 1.21E-05
R² = 9.99E-01

-1.0E-04
0.05 0.151/2 0.25 0.35
v / V1/2.s-1/2
-250
-0.4 0 0.4 0.8
E / V vs Ag/AgCl

Figure 4. Cyclic voltammograms of SPCE/RGO/PNR/AuNP recorded in 1 mM DA for different scan rates.

Inset: (a) The variation of anodic (■) and cathodic (●) peak currents with square root of scan rate (scan
rates: 10 to 125 mV s−1, pH 7.0). (b) The variation of peak potentials observed in Figure 4 with natural
logarithm of scan rate.

both anodic and cathodic peak currents are linear with the square root of the scanning
speed (inset Figure 4). This situation shows that the electro-oxidation and reduction of DA
on SPCE/RGO/PNR/AuNPs takes place in a diffusion-controlled process. The variation of
peak potentials observed in Figure 4 with natural logarithm of scan rate is given inset
b Figure 4. The values of the electron-transfer coefficient (α) for the electrooxidation of DA
at the surface of SPCE/RGO/PNR/AuNP can be obtained from the following equation [47]:
Ep ¼ ½ðRT Þ=ð2αF Þlnv þ constant (2)
where Ep is the peak potential, v is the scan rate, T is the temperature (293.15 K), R is the
gas constant (8.314 J K−1 mol−1) and F is the Faraday constant (96,485 C mol−1). This
equation is valid for an irreversible diffusion-controlled process. Using the above values
given in parentheses for R, T and F in the slope of Ep vs. lnv plot we obtained α to be 0.241.
 INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 459

The diffusion coefficient of 1 mM DA on SPCE/RGO/PNR/AuNP was determined as 1.37 ×

10−3 cm2 s−1 calculated by using equation 1.

3.3. Amperometric detection of DA on SPCE/RGO/PNR/AuNP

In order to increase the sensitivity of SPCE/RGO/PNR/AuNP in DA analysis, solution pH
and working potential were investigated. To determine the optimum pH of the buffer
used as the supporting electrolyte, acetate and phosphate buffer solutions at different
pHs (range 4–8) were prepared at a concentration of 50 µM. DA solutions were prepared
at a constant concentration of 500 µM using buffer solutions with different pH and
cyclic voltammograms of these solutions are given in Figure 5(a). PNR shows different
redox behaviours at different pH values due to its chemical structure. At pH 7.0, PNR
shows redox behaviour with 2-electron and 2-proton [4]. Optimum pH was determined

150
a pH 4
pH 4,5
pH 5
50
pH 5,5
I / µA

pH 6
pH 6,5
-50 pH 7
pH 7,5
pH 8

-150
-0.4 0 0.4 0.8
E / V vs Ag/AgCl

0.4
b y = -0.0507x + 0.53
0.35 R² = 0.9908

0.3
Epa / V

0.25

0.2

0.15

0.1
3.5 4.5 5.5 6.5 7.5 8.5
pH

Figure 5. (a) Cyclic voltammograms of SPCE/RGO/PNR/AuNP recorded in 500 µM DA for different pHs.
(b) Anodic peak potential-pH graph obtained from cyclic voltammograms at different pHs.
460 M. ALTUN ET AL.

to be 7.0 because the pH had to be 7.0 for PNR to play a role as mediator in the
electrooxidation of DA. Epa and pH change are given in Figure 5(b). Epa is linearly
related to the pH, while the corresponding regression equation is Epa (V) = −0.0507pH +
0.53 (r = 0.9954). The slope (50.7 mV/pH) approaches the theoretical value of 57.6 mV/
pH, implying that the equal number of electrons and protons takes part in the DA
oxidation. The oxidation potential of DA decreases with increasing pH. This result is
consistent with the literature [48–52].
Amperometric determination of DA with SPCE/RGO/PNR/AuNP biosensors in PBS (pH
= 7) was performed at +0.20 V, +0.25 V and +0.30 V potentials. Using the obtained
amperograms, current–concentration graphs were drawn (Figure 6). The sensitivity and
correlation coefficient values obtained from these graphs are given in Table 3. Although
the highest sensitivity was obtained from the equation of the line obtained at +0.3
V potential, the correlation coefficient was the lowest. During amperometric determina-
tion, current stability was highest at +0.2 V and +0.25 V. At +0.30 V, current stability
decreased as the analyte concentration increased. The current stability and correlation
coefficient were considered, and the optimum working potential was determined as
+0.25 V.
Amperometric measurements for DA determination were carried out with SPCE/RGO/
PNR/AuNP electrode under stirring at a fixed potential of +0.25 V in 9 mL PBS (pH = 7). In
Figure 7, a typical amperogram obtained for amperometric determination of DA is given.
The current values obtained from these amperograms and the concentration values of the
DA are plotted in Figure 8. Sensitivity and limit of detection (based on 3σ/sensitivity) were
calculated to be 13.38 μA mM−1 and 0.17 μM, respectively. The repeatability of the SPCE/
RGO/PNR/AuNP sensor was determined at +0.25 V with 50 µM DA solution. The relative

18

15

12
I / µA

0
0 200 400 600 800 1000

C / µM

Figure 6. Current-concentrations plots for amperometric detection of DA with SPCE/RGO/PNR/AuNP in

PBS (pH 7.0) at different working potentials: +0.20 V (▲), +0.25 V (●) and +0.30 V (■).
 INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 461

Table 3. Sensitivities and correlation coefficients obtained from current–con-

centration plots for amperometric detection of DA with SPCE/RGO/PNR/AuNP at
various working potentials.
Working potential/(V) Sensitivity/(µA mM−1) Correlation coefficient (r)
+0.200 10.69 0.9997
+0.250 13.76 0.9998
+0.300 16.40 0.9956

6
I / (µA)

0
0 400 800 1200 1600
t/s

Figure 7. Amperometric current response of SPCE/RGO/PNR/AuNP at +0.25 V upon successive

additions of 50 µM DA in PBS (E = +0.25 V, pH = 7.0).

8.0
y = 0.0134x - 0.0008
7.0 R² = 0.9995

6.0

5.0
I / µA

4.0

3.0

2.0

1.0

0.0
0 100 200 300 400 500 600

C / µM

Figure 8. Calibration plots for DA using SPCE/RGO/PNR/AuNP.

standard deviation (RSD) was 2.14% (for n = 10). The analytical characteristics of the SPCE/
RGO/PNR/AuNP sensor were compared with some recently reported data from the
literature and the results are presented in Table 4.
462 M. ALTUN ET AL.

Table 4. The comparison of the SPCE/RGO/PNR/AuNP with the other recently published amperometric
DA sensors.
Working potential/ Linear range/ Limit of detection/ Sensitivity/
Modified electrode (V) (µA) (µA) (µA mM−1) References
HAu/G/GCE +0.40 0.08–600 0.05 – [53]
GNC/CMG/GCE +0.30 0.1–80 0.028 – [54]
MWCNTs/MoS2-Co3O4 PHs/ +0.34 2150.2–5540 0.013 3.486 [55]
SPCE
N-G/NiTsPc +0.4 0.1–200 0.1 88.9 [56]
GN-PSS-Pt +0.5 0.2–4000 0.04 302.2 [57]
3D graphene–CNT +0.177 2.0–64.0 0.02 470.7 [58]
Graphene/Pt-modified GC +0.3 0.03–8.13 0.03 – [59]
PVP/GR/GCE +0.4 0.0005–1130 0.0002 – [60]
β-CD/GS +0.1 0.9–200 0.005 – [61]
Fe2O3 NPs/GRS/GCE +0.2 0.006–635 0.001 4.066 [62]
SPCE/RGO/PNR/AuNP +0.25 0,57–500 0,17 13,38 This work
HAu/G/GCE: Highly dispersed hollow gold-graphene glassy carbon electrode, GNC: gold nanocage, CMG: chemically
modified graphene oxide, MWCNTs: Multiwall carbon nanotubes, MoS2: Molybdenum disulphide, Co3O4PHs: Cobalt
oxide polyhedrons, SPCE: screen-printed carbon electrode, N-G: nitrogen-doped graphene, NiTsPc: nickel tetrasulfo-
nated phthalocyanine, GN-PSS-Pt: Graphene-poly(styrene sulphonate)-Platinum, 3D graphene–CNT: Three-dimensional
(3D) graphene–carbon nanotube, Graphene/Pt-modified GC: graphene/Platinum modified glassy carbon electrode,
PVP/GR/GCE: Polyvinylpyrrolidone (PVP)/graphene modified glassy carbon electrode, β-CD/GS: b-cyclodextrin and
graphene sheet, Fe2O3 NPs/GRS/GCE: iron oxide nanoparticle-capped graphene sheet modified glassy carbon electrode,
RGO: reduced graphene oxide, PNR: polyneutral red, AuNP: gold nanoparticles.

The operation stability of the SPCE/RGO/PNR/AuNP biosensor was measured at +0.25

V for 60 days in response to 50 µM DA in PBS (pH = 7.0). The relative current values, as
a measure of the response of the sensor to DA over 60 days are compared (Figure 9).
Initially, the amperometric response of the biosensor for DA decreased by 13.99% at the
end of day 2, 69.8% at the end of day 10, 79.8% at the end of day 30 and 90.05% at the end
of day 60. Although the application stability of the SPCE/RGO/PNR/AuNP biosensor
decreased after the first use, it gave an amperometric response to DA up to day 60.
From these results, it can be concluded that for a single-use biosensor, this application
stability is good and that RGO, PNR and AuNP in the biosensor formulation increase the
application stability of the biosensor.
The storage stability of the SPCE/RGO/PNR/AuNP sensor was also examined by ampero-
metric DA analysis with stirring at +0.25 V for 12 weeks. During this period, the prepared
biosensors were kept in a constant humidity environment at +4◦C temperature. The SPCE/
RGO/PNR/AuNP biosensor responded to DA up to 12 weeks. The obtained relative current
values for 12 weeks are given in Figure 10. Compared to the first-day analysis of DA, the
amperometric response of the biosensor decreased by 15.82% at the end of the 1st and
2nd weeks, 17.35% at the end of the 4th week and 18.88% at the end of the 8th week. The
biosensor was found to maintain 26.03% of its sensitivity at the end of the 12th week.
In the electrochemical determination of DA, the effects of interfering electroactive
analytes ascorbic acid (AA) and uric acid (UA) on SPCE/RGO/PNR/AuNP electrode were
investigated by differential pulse voltammetry and cyclic voltammetry. DPVs and CVs of
the SPCE/RGO/PNR/AuNP electrodes in 3 mM DA, AA, UA, PBS and mixture of 3 mM DA,
AA and UA are given in Figure 11(a,b), respectively. According to DPV and CV results,
peaks of AA, UA and DA a little interfere with each other. However, when the peaks
obtained with the mixture of these three species were examined, it was observed that AA
 INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 463

100
100
80
% Relative Current 86.01

71.1
60

40 44.48

30.2
20 23.72
21.1
13.03 9.95
0
1 2 3 5 10 15 30 45 60
t / day

Figure 9. Operational stability of SPCE/RGO/PNR/AuNP sensor. % Relative current vs time plots over 60
days for amperometric detection of DA in PBS (pH 7.0).

100
100
% Relative Current

80
84.18 84.18 83.67 82.65 81.122
73.97
60

40

20

0
t / week

Figure 10. Storage-stability stability of SPCE/RGO/PNR/AuNP sensor. % Relative current vs time plots
over 12 weeks for amperometric detection of DA in PBS (pH 7.0).

and UA interfered in the analysis of DA, Epa values shifted to the left and Ipa values
decreased due to the interaction between the species.

3.4. Analysis of real sample

The developed DA biosensors were used in real sample analysis. The human blood serum
sample purchased from Merck was diluted 1:100 with a pH 7.4 buffer solution. A certain
concentration of DA was added to the blood serum sample and artificial blood serum
samples containing DA were prepared. Electrochemical determination of blood serum
464 M. ALTUN ET AL.

a 3 mM DA
250 3 mM AA
3 mM UA
200 3 mM DA,AA,UA mix
PBS

150
I/µA

100

50

0
-0.5 -0.3 -0.1 0.1 0.3 0.5 0.7 0.9
E / V vs Ag/AgCl

240
b

140
I/µA

40

3 mM DA

-60 3 mM AA
3 mM UA
3 mM DA,AA,UA mix
PBS
-160
-0.5 -0.3 -0.1 0.1 0.3 0.5 0.7 0.9
E / V vs Ag/AgCl

Figure 11. (a) Differential pulse voltammograms of the SPCE/RGO/PNR/AuNP electrode obtained at
a scanning rate of 50 mV s−1 in 3 mM DA, AA, UA, PBS and mixture of 3 mM DA, AA and UA at
a potential range of −0.4 V to +0.8 V. (b) Cyclic voltammograms of the SPCE/RGO/PNR/AuNP electrode
obtained at a scanning rate of 50 mV s−1 in 3 mM DA, AA, UA, PBS and mixture of 3 mM DA, AA and UA
at a potential range of −0.4 V to +0.8 V.

samples containing a certain amount of DA was performed with amperometric measure-

ments with SPCE/RGO/PNR/AuNP biosensors at +0.25 V. The results of real sample analysis
are given in Table 5. The recoveries for 20 µM and 50 µM DA were found to be 101.5% and
102.5%, respectively.

Table 5. Real sample analysis results.

Human blood serum sample Added (µM) Found (µM) (%) Recovery
1 20 20,30 ± 0,18 101,5
2 50 51,25 ± 0,15 102,5
 INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 465

4. Conclusion
In this study, a new amperometric sensor for the detection of DA was developed using
a screen-printed electrode modified with RGO, PNR and AuNP. The synergistic effect of
RGO, PNR and AuNP showed a high electrocatalytic effect on DA oxidation. The ampero-
metric DA sensor exhibited a successful result with a linear range between 0.57 and 500
μM, 0.17 μM detection limit, good reproducibility and storage stability. In addition, the
results obtained for DA analysis in human blood samples showed satisfactory recoveries.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The support of this work by the Scientific Research Projects Coordination Unit of Cankiri Karatekin
University (Project Number: FF061218L07) is acknowledged.

ORCID
Muhammed Altun http://orcid.org/0000-0002-0387-1819
Melike Bilgi Kamaç http://orcid.org/0000-0002-3381-7522
Alper Bilgi http://orcid.org/0000-0003-3381-6578
Merve Yılmaz http://orcid.org/0000-0003-3690-2455

References
[1] Z. Cai, Y. Ye, X. Wan, J. Liu, S. Yang, Y. Xia, G. Li and Q. He, Nanomaterials 9, 835 (2019).
doi:10.3390/nano9060835.
[2] J.W. Dalley and J.P. Roiser, Neuroscience 215, 42 (2012). doi:10.1016/j.
neuroscience.2012.03.065.
[3] Q. He, J. Liu, X. Liu, G. Li, D. Chen, P. Deng and J. Liang, Nanomaterials 8, 194 (2018).
doi:10.3390/nano8040194.
[4] A. Carlsson, Psychol. Med. 7, 583 (1977). doi:10.1017/S003329170000622X.
[5] R.M. Wightman, L.J. May and A.C. Michael, Anal. Chem. 60, 769A (1988). doi:10.1021/
ac00164a001.
[6] M. Zhang, C. Liao, Y. Yao, Z. Liu, F. Gong and F. Yan, Adv. Funct. Mater. 24, 978 (2014).
doi:10.1002/adfm.201302359.
[7] F. Gao, X. Cai, X. Wang, C. Gao, S. Liu, F. Gao and Q. Wang, Sens. Actuators B Chem. 186, 380
(2013). doi:10.1016/j.snb.2013.06.020.
[8] Q. He, J. Liu, J. Liang, X. Liu, W. Li, Z. Liu, Z. Ding and D. Tuo, Cells 7, 24 (2018). doi:10.3390/
cells7040024.
[9] A. Liu, I. Honma and H. Zhou, Biosens. Bioelectron. 21, 809 (2005). doi:10.1016/j.
bios.2005.03.005.
[10] C. Wang, R. Yuan, Y. Chai, Y. Zhang, F. Hu and M. Zhang, Biosens. Bioelectron. 30, 315 (2011).
doi:10.1016/j.bios.2011.08.035.
[11] Q. He, G. Li, X. Liu, J. Liu, P. Deng and D. Chen, Catalysts 8, 323 (2018). doi:10.3390/
catal8080323.
[12] Q. He, J. Liu, X. Liu, G. Li, D. Chen, P. Deng and J. Liang, Electrochim. Acta 296, 683 (2019).
doi:10.1016/j.electacta.2018.11.096.
466 M. ALTUN ET AL.

[13] R. Sivasubramanian and P. Biji, Mater. Sci. Eng. B 210, 10 (2016). doi:10.1016/j.
mseb.2016.04.018.
[14] X. Wan, S. Yang, Z. Cai, Q. He, Y. Ye, Y. Xia, G. Li and J. Liu, Nanomaterials 9, 847 (2019).
doi:10.3390/nano9060847.
[15] D.G. Gardner and D. Shoback, Greenpan’s Basic & Clinical Endocrinology, 9th ed ed. (The
McGraw-Hill Companies, New York, NY, 2011).
[16] K. Pagana, T. Pagana and T. Pagana, Mosby’s Diagnostic & Laboratory Test Reference, 14th ed.
(Elsevier, St. Louis, Mo, 2019).
[17] Ó. Arias-Carrión and E. Pöppel, Acta Neurobiol. Exp. (Wars) 67, 481 (2007).
[18] L. Liu, J. Du, S. Li, B. Yuan, H. Han, M. Jing and N. Xia, Biosens. Bioelectron. 41, 730 (2013).
doi:10.1016/j.bios.2012.09.061.
[19] J.W. Mo and B. Ogorevc, Anal. Chem. 73, 1196 (2001). doi:10.1021/ac0010882.
[20] Q. He, J. Liu, X. Liu, G. Li, P. Deng and J. Liang, Sens. (Switzerland) 18, 199 (2018). doi:10.3390/
s18010199.
[21] R. Pauliukaite and C.M.A. Brett, Electroanalysis 20, 1275 (2008). doi:10.1002/elan.200804217.
[22] A.A. Karyakin, E.E. Karyakina and H.L. Schmidt, Electroanalysis 11, 149 (1999). doi:10.1002/
(SICI)1521-4109(199903)11:3<149::AID-ELAN149>3.0.CO;2-G.
[23] A.A. Karyakin, O.A. Bobrova and E.E. Karyakina, J. Electroanal. Chem. 399, 179 (1995).
doi:10.1016/0022-0728(95)04300-4.
[24] B.P. Nikolski, V.V. Palchevskii, L.A. Polyanskaya and A.G. Rodichev, Dokl. Akad. Nauk SSSR 194,
1334 (1970).
[25] C.S. Halliday and D.B. Matthews, Aust. J. Chem. 36, 507 (1983). doi:10.1071/CH9830507c.
[26] G. Broncová, T.V. Shishkanova, P. Matějka, R. Volf and V. Král, Anal. Chim. Acta 511, 197 (2004).
doi:10.1016/j.aca.2004.01.052.
[27] Q. He, J. Liu, Y. Xia, D. Tuo, P. Deng, Y. Tian, Y. Wu, G. Li and D. Chen, J. Electrochem. Soc. 166,
B805 (2019). doi:10.1149/2.1271910jes.
[28] Q. He, J. Liu, X. Liu, G. Li, P. Deng and J. Liang, Colloids Surf. B Biointerfaces 172, 565 (2018).
doi:10.1016/j.colsurfb.2018.09.005.
[29] Q. He, Y. Wu, Y. Tian, G. Li, J. Liu, P. Deng and D. Chen, Nanomaterials 9, 115 (2019).
doi:10.3390/nano9010115.
[30] G. Li, Y. Xia, Y. Tian, Y. Wu, J. Liu, Q. He and D. Chen, J. Electrochem. Soc. 166, B881 (2019).
doi:10.1149/2.0171912jes.
[31] V. Singh, D. Joung, L. Zhai, S. Das, S.I. Khondaker and S. Seal, Prog. Mater. Sci. 56, 1178 (2011).
doi:10.1016/j.pmatsci.2011.03.003.
[32] S. Taniselass, M.K.M. Arshad and S.C.B. Gopinath, Biosens. Bioelectron. 130, 276 (2019).
doi:10.1016/j.bios.2019.01.047.
[33] W. Dong, Y. Ren, Y. Zhang, Y. Chen, C. Zhang, Z. Bai, R. Ma and Q. Chen, Talanta 165, 604
(2017). doi:10.1016/j.talanta.2017.01.017.
[34] A. Bonanni, A.H. Loo and M. Pumera, TrAC-Trends Anal. Chem. 37, 12 (2012). doi:10.1016/j.
trac.2012.02.011.
[35] J. Zhao, Z. Wang, J.C. White and B. Xing, Environ. Sci. Technol. 48, 9995 (2014). doi:10.1021/
es5022679.
[36] C. Wang, X. Han, P. Xu, X. Zhang, Y. Du, S. Hu, J. Wang and X. Wang, Appl. Phys. Lett. 98, 72906
(2011). doi:10.1063/1.3555436.
[37] G. Li, S. Wang and Y.Y. Duan, Sens. Actuators B Chem. 277, 250 (2018). doi:10.1016/j.
snb.2018.08.155.
[38] J. Ning, Q. He, X. Luo, M. Wang, D. Liu, J. Wang, G. Li and J. Liu, Catalysts 8, 407 (2018).
doi:10.3390/catal8100407.
[39] M. Sahin and E. Ayranci, Electrochim. Acta. 166, 261 (2015). doi:10.1016/j.
electacta.2015.03.030.
[40] M. Bilgi and E. Ayranci, Sens. Actuators B Chem. 237, 849 (2016). doi:10.1016/j.
snb.2016.06.164.
[41] İ. Tiyek, U. Dönmez, B. Yıldırım, M.H. Alma, M.S. Ersoy, Ş. Karataş and M. Yazıcı, Sak. Univ. J. Sci.
20, 349 (2016). doi:10.16984/saufenbilder.29009.
 INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 467

[42] A. Shalaby, D. Nihtianova, P. Markov, A.D. Staneva, R.S. Iordanova and Y.B. Dimitriev, Bulg.
Chem. Commun. 47, 291 (2015).
[43] A.J. Bard and L.R. Faulkner., Fundamentals and Applications, 2nd ed. (Electrochemical
Methods, New York, 2001).
[44] C.M.A. Brett and A.M. Oliveira Brett, Electrochemistry: Principles, Methods and Applications
(Oxford University Press, Oxford, 1993).
[45] Y. Sun, B. Ye, W. Zhang and X. Zhou, Anal. Chim. Acta 363, 75 (1998). doi:10.1016/S0003-
2670(98)00061-0.
[46] U. Yogeswaran and S.M. Chen, Electrochim. Acta 52, 5985 (2007). doi:10.1016/j.
electacta.2007.03.047.
[47] J.A. Harrison and Z.A. Khan, J. Electroanal. Chem 28, 131 (1970). doi:10.1016/S0022-0728(70)
80288-1.
[48] D. Zhang, L. Li, W. Ma, X. Chen and Y. Zhang, Mater. Sci. Eng. C 70, 241 (2017). doi:10.1016/j.
msec.2016.08.078.
[49] L. Yang, D. Liu, J. Huang and T. You, Sens. Actuators B Chem. 193, 166 (2014). doi:10.1016/j.
snb.2013.11.104.
[50] H.S. Han, H. Seol, D.H. Kang, M.S. Ahmed, J.M. You and S. Jeon, Sensors Actuators, B Chem
204, 289 (2014). doi:10.1016/j.snb.2014.07.075.
[51] M.A. Kamyabi, M.A. Shafiee and J. Braz, Chem. Soc. 23, 593 (2012). doi:10.1016/j.
nano.2009.06.003.
[52] W. Sun, M. Yang and K. Jiao, Anal. Bioanal. Chem. 389, 1283 (2007). doi:10.1007/s00216-007-
1518-2.
[53] W. Zhu, T. Chen, X. Ma, H. Ma and S. Chen, Colloids Surf. B Biointerfaces 111, 321 (2013).
doi:10.1016/j.colsurfb.2013.06.026.
[54] S. Daemi, A.A. Ashkarran, A. Bahari and S. Ghasemi, J. Colloid Interface Sci. 494, 290 (2017).
doi:10.1016/j.jcis.2017.01.093.
[55] K. Sakthivel, M. Govindasamy, S.M. Chen, A. Muthumariappan, V. Mani, T.W. Chen and
S. Selvaraj, Int. J. Electrochem. Sci. 12, 7435 (2017). doi:10.20964/2017.08.156.
[56] H. Xu, J. Xiao, L. Yan, L. Zhu and B. Liu, J. Electroanal. Chem. 779, 92 (2016). doi:10.1016/j.
jelechem.2016.04.032.
[57] W.L. Liu, C. Li, L. Tang, Y. Gu and Z.Q. Zhang, Chinese J. Anal. Chem. 41, 714 (2013).
doi:10.1016/S1872-2040(13)60656-8.
[58] X. Dong, Y. Ma, G. Zhu, Y. Huang, J. Wang, M.B. Chan-Park, L. Wang, W. Huang and P. Chen,
J. Mater. Chem. 22, 17044 (2012). doi:10.1039/c2jm33286h.
[59] C.L. Sun, H.H. Lee, J.M. Yang and C.C. Wu, Biosens. Bioelectron. 26, 3450 (2011). doi:10.1016/j.
bios.2011.01.023.
[60] Q. Liu, X. Zhu, Z. Huo, X. He, Y. Liang and M. Xu, Talanta 97, 557 (2012). doi:10.1016/j.
talanta.2012.05.013.
[61] L. Tan, K.G. Zhou, Y.H. Zhang, H.X. Wang, X.D. Wang, Y.F. Guo and H.L. Zhang, Electrochem.
Commun. 12, 557 (2010). doi:10.1016/j.elecom.2010.01.042.
[62] T. Kokulnathan, A.J. Anthuvan, S.M. Chen, V. Chinnuswamy and K. Kadirvelu, Inorg. Chem.
Front. 5, 705 (2018). doi:10.1039/c7qi00716g.