Poly (fast sulphone black F) modified pencil graphite electrode sensor for serotonin

Abstract

Development of sensitive and rapid biosensor for the investigation of serotonin has great significance because it is a key neurotransmitter and its unusual concentrations associated with serious mental disorders. In this study, an electrochemically modified serotonin-sensing electrode was fabricated by simple electropolymerisation of fast sulphone black F on pencil graphite electrode (PGE) using cyclic voltammetric technique. This modified pencil graphite electrode was applied for selective determination of serotonin (5-HT) and shows increased current responses of 5-HT in 0.2 M PBS of pH 7.4. The various analytical parameters such as effect of scan rate, concentration of 5-HT and solution pH were investigated. The diffusion controlled electrode process was observed for 5-HT and detection limit was found to be 1.7 μM. Interference study of 5-HT was analysed in presence of dopamine (DA) by cyclic voltammetry (CV) and differential pulse voltammetry (DPV).

Full text

Poly (fast sulphone black F) modified pencil graphite electrode sensor
for serotonin
Rukaya Banu, B.E. Kumara Swamy
*
, S. Deepa
Department of P.G. Studies and Research in Industrial Chemistry, Jnana Sahyadri, Kuvempu University, Shankaraghatta, 577 451, Shivamogga, Karnataka, India
ARTICLE INFO
Keywords:
Serotonin
Fast sulphone black F
Cyclic voltammetry
Differential pulse voltammetry
Pencil graphite electrode
ABSTRACT
Development of sensitive and rapid biosensor for the investigation of serotonin has great significance because it is
a key neurotransmitter and its unusual concentrations associated with serious mental disorders. In this study, an
electrochemically modified serotonin-sensing electrode was fabricated by simple electropolymerisation of fast
sulphone black F on pencil graphite electrode (PGE) using cyclic voltammetric technique. This modified pencil
graphite electrode was applied for selective determination of serotonin (5-HT) and shows increased current re-
sponses of 5-HT in 0.2 M PBS of pH 7.4. The various analytical parameters such as effect of scan rate, concen-
tration of 5-HT and solution pH were investigated. The diffusion controlled electrode process was observed for 5-
HT and detection limit was found to be 1.7
μ
M. Interference study of 5-HT was analysed in presence of dopamine
(DA) by cyclic voltammetry (CV) and differential pulse voltammetry (DPV).
1. Introduction
Pencil leads often referred as pencil graphite electrodes (PGEs)
gained more prominence in recent days as a working electrode material
for various electrochemical applications. PGEs act as a crucial substi-
tution for other carbon electrodes due to their affordable price and thin
dimensions. Also shows great potential in designing a disposable bio-
sensing electrode materials [1,2]. A fine particles of graphite is used to
produce a pencil leads, which is composed of graphite powder mixing
with clay or mica and a high polymeric binders are sometimes added
[3]. PGEs are simple and easy to use because of their good adsorption
properties, high conductivity, good mechanical strength and easy
methods of modifications [4–6]. The renewal of electrode surfaces is
simple and faster in case of PGEs among various solid electrodes
involving common polishing and cleaning techniques and has larger
surface area. Therefore, it is able to detect the analyte in its lower
concentration [7–9].
Serotonin and Dopamine are important neurotransmitters belong to
catecholamine family and serves as chemical carriers for transporting
information between the nerve cells [10]. Both serotonin and dopamine
are responsible for various phenomenon occurs in the living organisms.
Serotonin, Which is chemically known as 5-hydroxytryptamine(5-HT)
(Scheme 1) derived from
α
amino acid tryptophan and widely distrib-
uted inside and outside of brain tissues. 5-HT together with other
neurotransmitter plays a significant role in regulating and controlling the
several biological and physiological functions like sleep disturbances,
memory, wound healing, appetite, thermoregulation, behaviour and
drug dependency [11–14]. Many life functions are depends on the con-
centration of 5-HT level in blood. The normal range of 5-HT levels in
blood is 101–283 ng/mL. Any unbalance in the concentration of 5-HT
leads to numerous health issues such as low concentration level causes
anxiety, chronic pain, blood clotting. While extremely high concentration
leads to potentially fatal effects like serotonin syndrome, carcinoid syn-
drome, liver regeneration and autism [15–17].
3,4-dihydroxyphenylethylamine commonly known as dopamine (DA)
(Scheme 2) is an inhibitory neurotransmitter. It exhibits significant
contribution in the proper functioning of hormonal, renal and cardio-
vascular system [18,33]. The normal level of DA in blood plasma is in the
range of 0.04–450 nM [19]. Lower concentration of DA than the normal
level causes severe neurological diseases such as Parkinson's disease,
* Corresponding author.
E-mail address: bek@kuvempu.ac.in (B.E.K. Swamy).
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https://doi.org/10.1016/j.sintl.2020.100044
Received 6 July 2020; Received in revised form 18 September 2020; Accepted 18 September 2020
2666-3511/©2020 The Authors. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-
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Sensors International 1 (2020) 100044

Huntington's chorea, schizophrenia, drug addiction and HIV infection
whereas an excess level can leads to hallucinations, mania and euophia
[20–22].
Analysis of 5-HT is more complicated as a result of interference of
other neurotransmitters present in the biological fluids. In this regard,
several conventional analytical methods were employed for the detection
of 5-HT such as, capillary electrophoresis, luminescence [23] and HPLC
provides a certain results. However, these methods are time consuming,
complex, expensive equipments and required sample management. The
use of electrochemical methods are more advantageous than other
techniques because of its experimental simplicity, good selectivity, usage
of small quantity of sample, rapid monitoring and inexpensiveness
[24–28]. The polymeric film coated electrodes have excellent stability,
more active sites and strong adherence nature to the surface of the
electrode [29–33]. Due to this properties, the electropolymer film
modified electrodes attracted more in the field of sensor. The main
purpose of our study was to fabricate a stable working electrode by
electropolymerising fast sulphone black F (Scheme 3), a metallochromic
dye on the surface of bare pencil graphite electrode to achieve the
determination of 5-HT in the presence of DA in biological pH.
2. Experimental section
2.1. Chemicals and stock solutions
The pencil-lead rods (HB 0.7 mm) were purchased from bookstore.
Serotonin (5-HT), dopamine (DA) and fast sulphone black F (FSBF) was
purchased from Himedia (Bangalore, India). Stock solution of 25 10
4
M
Serotonin, 25 10
3
M fast sulphone black F and 0.1 M NaOH were pre-
pared by dissolving in doubly distilled water and 25 10
4
M dopamine
solution was prepared in 0.1 M perchloric acid. Phosphate buffer solution
(PBS) of different pH was obtained by mixing an appropriate proportions
of 0.2 M sodium dihydrogen phosphate (NaH
2
PO
4
) and 0.2 M disodium
hydrogen phosphate (Na
2
HPO
4
). All the chemicals were of analytical grade
quality and used without any additional treatment.
2.2. Apparatus and procedure
The electrochemical investigations were carried out in analytical in-
strument of Model CHI-660c Potentiostat (CH Instrument-660 electro-
chemical workstation). All the experiments were performed in standard
three-electrode one component glass cell. The cell containing a bare pen-
cil graphite electrode (BPGE) and poly (fast sulphone black F) modified
pencil graphite electrode (poly ((FSBF)MPGE) as a working electrode.
Additionally, a platinum wire as auxiliary electrode anda saturated calomel
electrode as a reference electrode. All the redox potentials of electroanalysis
of 5-HT were referenced to SCE at an optimum temperature.
2.3. Fabrication of poly(FSBF) modified pencil graphite electrode
The poly (FSBF)MPGE was prepared by electropolymerisation of 1
mM fast sulphone black F on the surface of BPGE in the presence of 0.1 M
NaOH as supporting electrolyte in electrochemical cell. With the cyclic
scanning of potential from 0.0 V to 1.4 V for 15 multiple cycles at the
sweep rate of 50 mV s
-1
. The uniform film of poly (FSBF) formed on
surface of BPGE. Then the electrode was carefully rinsed with double
distilled water to remove unreacted FSBF from the surface and this
electrode was used for the determination of 5-HT.
3. Result and discussion
3.1. Electropolymerisation of FSBF on BPGE
The electrochemical polymerization process of fast sulphone black F
onto the PGE surface was achieved between the potential window 0.0 V
and þ1.4 V at the scan rate of 50 mV s
-1
through cyclic voltammetry for 15
multiple cycles. During the process of multiple scanning, the voltammo-
gram gradually decreased as the cyclic time increases. This result shows
that the film of poly (FSBF) was coated on the surface of BPGE [34–36].
The extent of thickness of the polymer film formed on the BPGE will also
affect the electrocatalytic response of the electrode and thickness of the
film can be controlled by varying the number of cycles on the BPGE (from 5
to 25 multiple cycles). Fifteen cycles shows the maximum anodic peak
current as shown in Fig. 1A. Therefore, 15 cycles were considered as most
Scheme 1. Structure of Serotonin.
Scheme 2. Structure of Dopamine.
Scheme 3. Structure of Fast Sulphone Black F.
R. Banu et al. Sensors International 1 (2020) 100044
2

favorable for the electro-polymerization of FSBF on BPGE. The electro-
catalytic response of 5-HT increased at initially up to 15 cycles. After that,
the peak currents of 5-HT was decreased as shown in Fig. 1B. It was due to
the increase in thickness of the polymeric film that would prevent the
electron transfer process. The corresponding electrocatalytic performance
towards oxidation of 5-HT in PBS of pH 7.4 was investigated. An appro-
priate estimated amount of incorporated FSBF polymeric film on BPGE
surface was calculated by using equation (1) [37].
Ip ¼n
2
F
2
AΓ
υ
/4RT (1)
Where, Ip is the peak current, Γ(M/cm
2
) is the surface coverage concen-
tration, A and
υ
are the area and scan rate of the working electrode
respectively, n is the number of exchanged electrons, R, F and Τare
physical constants with their normal meanings. The surface coverage
concentration(Γ) of FSBF on BPGE was calculated to be 0.0274 10
10
M/
cm
2
.
3.2. Characterization of fabricated poly(FSBF)MPGE
The electrocatalytic behaviour of poly (FSBF)MPGE was examined by
using 1 mM potassium ferrocyanide ([K
4
Fe(CN)
6
]) in presence of sup-
porting electrolyte (1 M KCl) with sweep rate of 50 mV s
-1
by CV tech-
niques. Fig. 2 represents the cyclic voltammogram for 1 mM potassium
ferrocyanide recorded at BPGE (dashed line) shows lower redox peak
current signals and large difference between the redox peak potentials
(ΔEp). While at poly (FSBF)MPGE the significant enhancement in the
redox peak current was observed. This results indicates that the modified
electrode shows better surface properties and electrocatalytic properties
for potassium ferrocyanide system. The total active surface area of
modified electrode can be computed by Randles–Sevick's equation (2)
[38–40].
I
p
¼(2.69 10
5
)n
3/2
AD
1/2
C
0
ν
1/2
(2)
Where, Ip is the peak current, A is the active surface area of electrode
(cm
2
), n is the number of exchanged electrons, C
0
is the concentration of
Fig. 1. (A) Electropolymerisation of 1 mM FSBF on surface of BPGE recorded in
presence of 0.1 M NaOH at 15 multiple cycles with scan rate of 50 mVs
1
.(B)
Graph of anodic peak current (Ipa) versus number of polymerization cycles.
Fig. 2. Cyclic voltammogram of BPGE(dashed line) and poly (FSBF)MPGE
(solid line) for 1 mM potassium ferrocyanide in presence of 1 M KCl at scan rate
of 50 mV s
-1
.
Fig. 3. Cyclic voltammogram of 10
μ
M 5-HT in 0.2 M phosphate buffer solution
of pH 7.4 at BPGE (solid line) and poly (FSBF) film coated PGE (dashed line)
with scan rate of 50 mV s
-1
.
R. Banu et al. Sensors International 1 (2020) 100044
3

electroactive species (mol cm
3
) in solution, D is the diffusion co-
efficient (cm
2
s
1
) and
ν
is the scan rate (Vs
1
). The electro active sur-
face area calculated for BPGE and poly (FSBF)MPGE was found to be
0.021 cm
2
and 0.052 cm
2
respectively.
3.3. Electrocatalytic behaviour of 5-HT at bare and poly (FSBF) modified
PGE
Fig. 3 shows the cyclic voltammogram recorded for the electro-
chemical response of 10
μ
M 5-HT at BPGE (solid line) and poly (FSBF)
MPGE (dashed line) in 0.2 M PBS of pH 7.4 with the scan rate of
50 mVs
1
. It is observed that voltammogram obtained at BPGE shows
poor voltammetric signal and low current signal. However, in the iden-
tical condition, the poly (FSBF)MPGE exhibited well defined oxidation
peak with a higher magnitude of oxidation current than that of bare PGE.
Which is ascribed to the larger surface area of the poly (FSBF) film. This
results demonstrated that the poly (FSBF) modified electrode possessed a
strong electrocatalytic action for the oxidation of 5-HT. When potential
initially swept from þ0.1 V to þ0.6 V, cathodic peak could not be found,
indicating that the electrochemical oxidation of 5-HT at modified PGE
was an irreversible process. The electro oxidation of 5-HT at poly (FSBF)
MPGE was represented in Scheme 4.
3.4. Influence of scan rate on peak current of 5-HT
The effect of potential scan rate on the electrocatalytic current
response of 5-HT was illustrated for different scan rates. Fig. 4A shows
the voltammograms obtained for 10
μ
M 5-HT in 0.2 M PBS at pH 7.4 with
different scan rate from 50 to 500 mVs
1
at poly (FSBF)MPGE. The
oxidation peak current (Ipa) of 5-HT increases linearly with the increase
in potential scan. To study the type of electrode process, anodic peak
current (Ipa) of 5-HT was plotted with different scan rate(
ν
) for poly
(FSBF)MPGE as shown in Fig. 4B. The plotted graph shows excellent
linearity between anodic peak currents and scan rates. The correspond-
ing linear regression equation is Ipa(
μ
A) ¼0.7251 (log
υ
)þ4.6037 with
correlation co-efficient (r
2
) 0.9798. This result suggested that diffusion
controlled electrode process occurs at poly (FSBF) modified pencil
graphite electrode [41].
3.5. Effect of solution pH on 5-HT at poly (FSBF) MPGE
The influence of solution pH on the redox response of modified
electrode towards the determination of 5-HT was examined. Cyclic vol-
tammogram of 10
μ
M 5-HT in 0.2 M PBS at poly (FSBF)MPGE with
different solution pH values from 5.8 to 7.8 are shown in Fig. 5A. The
peak potentials of 5-HT were shifted towards less positive side with
Scheme 4. The electrochemical oxidation of 5-HT at poly(FSBF)MPGE.
Fig. 4. (A) Cyclic voltammograms of 10
μ
M 5-HT at poly (FSBF)MPGE with
different scan rate (a–j:50-500 mVs
1
) using 0.2 M phosphate buffer solution at
pH 7.4. (B) Graph of log Ipa versus log
υ
.
R. Banu et al. Sensors International 1 (2020) 100044
4

increasing in the pH of the electrolyte. The oxidation peak potential of 5-
HT moved from 400 mV to 290 mV with respect to pH from 5.8 to 7.8.
The pH 7.4 was selected for all subsequent electrochemical analysis of 5-
HT because pH 7.4 is a biological pH. A linear relationship was estab-
lished by plotting oxidation peak potential (Epa) with pH of the solution
as illustrated in Fig. 5B. From the plot, the slope value was found to be
53 mV/pH. This was near to the theoretical Nernstian value of 59 mV/
pH. This result corresponds to the electrochemical oxidation of 5-HT was
two-electron and two-proton transfer reaction process.
3.6. Effect of concentration of 5-HT at poly (FSBF) MPGE
The poly (FSBF)MPGE was employed towards the electrocatalytic
oxidation of 5-HT by varying it's concentration in the range of
(10–50
μ
M) in 0.2 M PBS of pH 7.4 with the scan rate of 50 mVs
1
using
cyclic voltammetric (CV) method as shown in Fig. 6A and Fig. 6B. From
the graph it confirms that the oxidation peak current increases with in-
crease in concentration of 5-HT. The calibration plot of Ipa versus con-
centration of 5-HT gives good linearity which was given by equation
Ipa(
μ
A) ¼0.1435(C
0
μ
M/L) þ1.9078 and correlation coefficient of (r
2
)
0.9867. The detection limit (LOD) and quantification limit (LOQ) for 5-
HT at poly (FSBF)MPGE were calculated through the ensuing formulas
[42,43].
LOD ¼3 S/M
LOQ ¼10 S/M
Where ‘S’is the standard deviation of oxidation peak currents and ‘M’is
the slope of the calibration plot. The LOD of 5-HT was found to be 1.7
μ
M
and LOQ was found to be 5.8
μ
M for 5-HT. The comparison of detection
limit of 5-HT at poly (FSBF)MPGE with other reported modified elec-
trodes is provided in Table 1.
3.7. Simultaneous determination of 5-HT and DA at poly(FSBF)MPGE
The prime objective of the present investigation is to apply the fast
sulphone black F modified electrode for the selective and sensitive
determination of 5-HT in presence of DA. Both 5-HT and DA are co-exist
in biological fluid. The simultaneous detection of these molecules in a
binary mixture was difficult at most solid electrodes because of similar
oxidation potentials of the molecules. The cyclic voltammetric response
Fig. 5. (A) Cyclic voltammograms of 10
μ
M 5-HT at different pH (5.8–7.8) in
0.2 M PBS of pH 7.4 with the scan rate of 50 mVs
1
at poly (FSBF) MPGE. (B)
The plot of anodic peak potential versus different pH.
Fig. 6. (A) Cyclic voltammogram of variation of concentration of 5-HT
(10–50
μ
M) at poly (FSBF) MPGE in 0.2 M PBS of pH 7.4 with the scan rate of
50 mVs
1
.(B) Graph of the anodic peak current (Ipa) versus the concentration
of 5-HT (
μ
M).
R. Banu et al. Sensors International 1 (2020) 100044
5

of 10
μ
M 5-HT and 10
μ
M DA in 0.2 M phosphate buffer solution of pH
7.4 at the BPGE (dashed line) and the poly (FSBF) MPGE (Solid line) with
the sweep rate of 50 mV s
-1
is displayed in Fig. 6. Voltammetric signals of
5-HT and DA shows less sensitive and broad anodic peaks at BPGE, so the
peak potentials for 5-HT and DA are not well separated at unmodified
electrode. On the other hand, in case of Poly (FSBF) MPGE the problem of
overlapped peak was resolved into two well-defined peaks of 5-HT and
DA with different peak potentials located at 325 mV and 142 mV
respectively. The corresponding peak to peak separation of 5-HT to DA
was 183 mV. This result was large enough to determine 5-HT and DA
individually and simultaneously (see Fig. 7).
3.8. Interference study
Differential pulse voltammetry (DPV) was used to examine the anti-
interference ability of the poly (FSBF)MPGE due to better resolution
and high current sensitivity of the technique. Fig.8 shows the voltam-
mogram recorded by DPV for homogeneous mixture of 10
μ
M 5-HT and
10
μ
M DA in 0.2 M PBS of pH 7.4 at the sweep rate of 50 mV s
-1
gives well
separated voltammograms corresponding to their oxidation at poly
(FSBF)MPGE. The oxidation potentials of 5-HT and DA was situated at
243 mV and 95 mV respectively. The difference between the peak po-
tential of 5-HT to the peak potential of DA was 148 mV. This suggested
that the fabricated modified electrode exhibits good tendency for the
determination of 5-HT in presence of DA .
3.9. Real sample analysis
To validate the practical utility of prepared electrode, the poly (FSBF)
MPGE was used for the detection of 5-HT in real sample. The human
sample obtained from hospital was diluted with 0.2 M PBS of pH 7.4 and
spiked with 5-HT. The analysis results were given in Table 2. This result
implied that the sensing activity of modified electrode was reliable for 5-
HT detection in real sample.
4. Conclusion
In this work, Poly (FSBF)MPGE was developed by electro-
polymerisation of FSBF on the surface of PGE and successfully employed
for the determination of 5-HT and DA. The modified electrode shows
better electrocatalytic properties with higher sensitivity and selectivity.
The poly (FSBF)MPGE resolves the problem of broad oxidation peaks of
5-HT and DA and gives well separated oxidation peaks for the binary
mixture of 5-HT and DA. This result indicates the anti-interference ability
of the modified electrode. The proposed electrochemical sensor was
simple, cost effective and can be used in the detection of other bioactive
molecules.
Conflict of interest
There is no conflict of Interest.
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