Nanomolar detection of 4-aminophenol using amperometric sensor based on a novel phthalocyanine

Article abstract of DOI:10.1016/j.electacta.2019.06.097

Novel cobalt (II) tetra-β-[N(2-(1,3-benzothiazole))carboxamide] phthalocyanine (CoTBTCAPc) has been synthesized for the first time. The dark green colored complex was characterized by different physicochemical and analytical techniques. Cyclic voltammetric studies revealed that the synthesized complex is redox-active nature. The CoTBTCAPc modified glassy carbon electrode (GCE/CoTBTCAPc) showed a stagnant charge transfer behavior for the redox probe whereas, composite hybrid of CoTBTCAPc with carbon nanoparticles (CNP) coated on GCE (GCE/CNP-CoTBTCAPc) showed facile and better charge transfer compared to the bare GCE. The GCE/CoTBTCAPc and GCE/CNP-CoTBTCAPc fabricated electrodes exhibited a decrease in over potential with the increase in oxidation peak current. Electrochemical oxidation of 4-aminophenol (4-AP) at nanomolar concentration was obtained in phosphate buffer pH 7.0 electrolytic media. The modified electrodes showed linear electrochemical response in the concentration range of 40–800 nM and the detection limit (LOD) was found as 13 and 11 nM (S/N = 3) for GCE/CoTBTCAPc and GCE/CNP-CoTBTCAPc electrodes respectively. Differential pulse voltammetric (DPV) technique showed linear response for the 4-AP oxidation in the concentration range of 100–1000 nM with sensitivity of 0.0328 and 0.4179 μA nM^?1 cm^?2 for the phthalocyanine and CNP-phthalocyanine modified electrodes respectively. In contrast, the amperometric sensor displayed linear response in the concentration range of 100–900 nM with sensitivity values of 0.4008 and 0.8887 μA nM^?1 cm^?2 and LOD of 40 and 30 nM for GCE/CoTBTCAPc and GCE/CNP-CoTBTCAPc electrodes respectively. The GCE/CNP-CoTBTCAPc showed selectivity for 4-AP even in the presence of interfering organic and inorganic species. This stable and simple sensor offers an opportunity for the electrochemical sensing of 4-aminophenol in real sample analysis like 4-AP in paracetamol tablets.

Full text of DOI:10.1016/j.electacta.2019.06.097

Electrochimica Acta 318 (2019) 342e353
Contents lists available at ScienceDirect
Electrochimica Acta
journal homepage: www.elsevier.com/locate/electacta
Nanomolar detection of 4-aminophenol using amperometric sensor
based on a novel phthalocyanine
Manjunatha Nemakal, Shambhulinga Aralekallu, Imadadulla Mohammed, Malathesh Pari,
K.R Venugopala Reddy, Lokesh Koodlur Sannegowda*
Department of Chemistry, Vijayanagara Sri Krishnadevaraya University, Ballari, 583105, Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel cobalt (II) tetra-b-[N(2-(1,3-benzothiazole))carboxamide] phthalocyanine (CoTBTCAPc) has been
Received 8 April 2019
Received in revised form
13 June 2019
Accepted 16 June 2019
Available online 17 June 2019
synthesized for the first time. The dark green colored complex was characterized by different physico-
chemical and analytical techniques. Cyclic voltammetric studies revealed that the synthesized complex is
redox-active nature. The CoTBTCAPc modified glassy carbon electrode (GCE/CoTBTCAPc) showed a
stagnant charge transfer behavior for the redox probe whereas, composite hybrid of CoTBTCAPc with
carbon nanoparticles (CNP) coated on GCE (GCE/CNP-CoTBTCAPc) showed facile and better charge
transfer compared to the bare GCE. The GCE/CoTBTCAPc and GCE/CNP-CoTBTCAPc fabricated electrodes
exhibited a decrease in over potential with the increase in oxidation peak current. Electrochemical
oxidation of 4-aminophenol (4-AP) at nanomolar concentration was obtained in phosphate buffer pH 7.0
electrolytic media. The modified electrodes showed linear electrochemical response in the concentration
range of 40e800 nM and the detection limit (LOD) was found as 13 and 11 nM (S/N ¼ 3) for GCE/
CoTBTCAPc and GCE/CNP-CoTBTCAPc electrodes respectively. Differential pulse voltammetric (DPV)
technique showed linear response for the 4-AP oxidation in the concentration range of 100e1000 nM
Keywords:
Benzothiazole phthalocyanine
4-Aminophenol
Carbon nanoparticles
Cyclic voltammetry
Amperometry
with sensitivity of 0.0328 and 0.4179
modified electrodes respectively. In contrast, the amperometric sensor displayed linear response in
the concentration range of 100e900 nM with sensitivity values of 0.4008 and 0.8887
A nMꢀ1 cmꢀ2 and
m
A nMꢀ1 cmꢀ2 for the phthalocyanine and CNP-phthalocyanine
m
LOD of 40 and 30 nM for GCE/CoTBTCAPc and GCE/CNP-CoTBTCAPc electrodes respectively. The GCE/
CNP-CoTBTCAPc showed selectivity for 4-AP even in the presence of interfering organic and inorganic
species. This stable and simple sensor offers an opportunity for the electrochemical sensing of 4-
aminophenol in real sample analysis like 4-AP in paracetamol tablets.
© 2019 Elsevier Ltd. All rights reserved.
1. Introduction
pesticides [3,4]. Besides, it is used as a biomarker to check the
health of workers exposed to aniline i.e. an absorbed aniline dose is
4-Aminophenol or p-aminophenol (4-AP) is a phenolic com-
pound and nonopiate derivative. 4-AP is a significant impurity
present in the paracetamol containing pharmaceuticals. In addi-
tion, the decomposition of paracetamol leads to the formation of 4-
AP [1]. Aminophenol is a colorless crystalline solid, easily oxidized
by air and light and gives deep brown tint coloration. 4-AP with a
commercial name Rodinal is used as reducing agent in photo-
graphic black-and-white film developer [2]. It is also used in the
production of sulfur and azo dye components used for textiles, hair,
fur, feathers, wood stains, resins, pharmaceuticals, explosives,
extensively oxidized to p-aminophenol, which is excreted as glu-
curonic and sulfate-conjugates in urine [5]. But, 4-AP is harmful to
humans because it increases the body temperature for an extended
period. Further, 4-AP is a nephrotoxic, teratogenic in nature and
shows mutagenic effects and DNA cleavage in living organisms
[6,7]. The allowed limit of p-aminophenol in paracetamol is 0.005%
w/w (5 ppm), according to the United States Pharmacopeia (USP),
European Pharmacopeia (EP) and Chinese pharmacopeia [8e10].
Hence, the detection and monitoring of phenolic compound in the
clinical, pharmaceutical and industrial process are essential for the
health safety, protection, and remediation purpose [11].
Various methods have been adopted for the sensing and
detection of 4-AP namely gas/liquid chromatography [12], surface-
* Corresponding author.
E-mail address: kslokesh@vskub.ac.in (L.K. Sannegowda).
https://doi.org/10.1016/j.electacta.2019.06.097
0013-4686/© 2019 Elsevier Ltd. All rights reserved.

M. Nemakal et al. / Electrochimica Acta 318 (2019) 342e353
343
enhanced Raman scattering [13], fluorimetry [14], chem-
C9H4O5),
tetrabutylammonium
perchlorate
(TBAP:
iluminescence [15], capillary electrophoresis [16], and micellar
electrokinetic chromatography [17] etc. However, all these instru-
mentation techniques are expensive, time-consuming laboratory
process, difficult detection procedure and need well-equipped
skilled persons. The reproducibility, selectivity and sensitivity pa-
rameters of these methods are low and make them unsuitable for
routine detection of 4-AP. Hence, electroanalytical techniques play
an important role and are advantageous over above methods due to
the biocompatibility, reproducibility, high stability, better reliability
and easy to operate at ambient temperature regarding current and
potential peak response features [18e20]. The 4-AP molecule
contains oxidizable groups and hence, electrochemical methods are
suitable for the detection at lower concentrations by using chem-
ically modified electrodes (CMEs). Different materials such as
graphene-poly(aniline) composite [21] and the droplet-based
microfluidic sensor materials [22] as well as immobilised bis-8-
hydroxyquinoline [23] etc. have been used for the sensing of 4-
AP. The polymeric materials such as poly(aniline), poly(-
((C4H9)4N(ClO4))), dimethylformamide (DMF:(CH3)2NC(O)H) were
purchased from Sigma Aldrich, India. Urea (CO(NH2)2), ammonium
chloride (NH4Cl), potassium thiocyanate (KSCN), aniline (C6H5NH2),
glacial acetic acid (CH3COOH), potassium carbonate (K2CO3), dicy-
clohexacarbodiimide (DCC: C13H22N2), ammonia (NH3), sodium
hydroxide (NaOH), sodium chloride (NaCl), bromine (Br2), nitro-
benzene (C6H5NO2) and 4-aminophenol (C6H4(NH2)OH) were
purchased from Merck, India. All the chemicals procured for the
study were used as received without further purification. Carbon
nanoparticles (CNPs) (CAS #: 7440-44-0) were purchased from the
American element. The CNPs were having an average particle size
of 10e45 nm with specific surface area (SSA) in the range
30e50 m2/g. Double distilled water was used in the preparation of
solutions for electrochemical studies. The electrochemical analysis
was carried out in 0.1 M aqueous phosphate buffer solution (PBS)
obtained by mixing NaH2PO4 and Na2HPO4 and adjusting the pH to
7 by either adding 0.1 M aqueous NaOH/H3PO4.
terthiophene), bio-active poly(
L
-arginine) or other electro-
2.2. Synthesis of amide linked benzothiazolephthalocyanine
complex
conductive/non-conductive polymer and enzymatic methods
have been used as the positive electroactive surfaces [24,25]. Also,
many of the researchers have used carbon active materials namely,
reduced graphene oxide (rGO), multiwalled carbon nanotubes
(MWCNTs), carbon nanoparticles (CNP) at the modified electrode
for the electrochemical sensing and detection of the 4-AP analyte
[21,22,26,27]. These electroactive carbon materials provide very
large surface area, high electrical conductivity, good mechanical
strength and induce the response for 4-AP [28].
Further, the non-enzymatic, non-precious but electroactive ma-
terials like N4 macrocyclic complexes have been used for electro-
catalytic sensing of 4-AP [29e36]. N4-macrocyclic complexes like
phthalocyanine and porphyrin molecules are embedded with a rich
redox behavior originating from the tetradentate ligand as well as
metal ion [31]. The intrinsic properties or characteristics of the
phthalocyanines can be tailored/designed by having a different metal
atom in the cavity or substituents at the peripheral benzene ring.
Benzothiazole is a fused moiety consisting of -S- and -N- hetero
atoms at 1, 3-positions. It is a light-emitting component of luciferin
and plays a vital role in the medicinal field in the preparation of
drugs (riluzole and pramipexole) [37]. This moiety is also used in
dyes (thioflavin), accelerators (rubber vulcanization) and for
nonlinear optical (NLO) applications [38]. The incorporation of
benzothiazole through amide bond at the periphery leads to an
increase in the conjugation as well as ring extension of MPcs. The
amide bond is regarded as a building block of proteins and is ex-
pected to provide biocompatibility and sensitivity in the detection
of biologically important molecules and pollutants. The incorpo-
ration of carbon nanoparticles (CNP) in phthalocyanine molecule
facilitates faster charge transfer and better electrochemical
response with increased sensitivity and LOD.
2.2.1. Synthesis of 2-amino benzothiazole (i)
It was prepared by adopting the modified synthetic procedure
reported in the literature [39,40]. The reaction between aniline
(2.58 g, 15 mmol) and potassium thiocyanate (5.8 g, 60 mmol) was
carried out in 100 mL RB flask containing glacial acetic acid (20 mL)
at 5 ꢁC. The reaction mixture was stirred for 30 min and then
bromine (0.75 mL, 15 mmol) in acetic acid (5 mL) was added drop-
wise with stirring. The temperature was maintained between 0 and
5 ꢁC during the reaction and the solution was allowed overnight at
room temperature under stirring. After the completion of the re-
action, the crude product was poured into ice-cold water to pre-
cipitate the compound and was neutralized with ammonia (50%).
The precipitated compound was filtered through Buckner funnel,
thoroughly washed with cold water, dried and recrystallized with
ethanol to obtain compound i.
Yield: 91%, Melting point: 267 ꢁC (literature: 270 ꢁC). Anal. for 2-
amino benzothiazole, Mol. Wt.: 150.13. C7H6N2S: Cal. (%) C, 56.00;
H, 3.99; N, 18.66; S, 21.73. Found (%): C, 55.85; H, 3.82; N, 18.89, S,
21.98. FTIR absorption bands (KBr pellet, cmꢀ1): 3450, 3360, 3055,
1643, 1530, 1445, 1308, 1283, 1106, 741, 719, 685. 1H-NMR
(400 MHz, DMSO-d6):
d
6.98 (dd, J ¼ 10.80, Hz, 1H), 7.02 (d,
J ¼ 1.20 Hz, 1H), 7.45 (s, 1H), 7.64 (d, J ¼ 9.60 Hz, 1H). Mass:
[MþH2O]ꢀ1 (167.2).
2.2.2. Synthesis of cobalt (II) tetra-[b-(carboxylic acid)]
phthalocyanine (CoTCAPc) (ii)
The compound ii, CoTCAPc was prepared by using the modified
protocol reported in the literature [41]. In brief, a mixture of cobalt
chloride (2.0 g, 8.40 mmol), trimellitic anhydride (6.45 g,
33.62 mmol), urea (4.03 g, 67.24 mmol), ammonium chloride
(2.69 g, 50.43 mmol) and catalytic amount of ammonium molyb-
date (0.51 g) were grounded well in the ratio of 1:4:8:3:0.05
respectively and taken in RB flask containing nitrobenzene (30 mL).
The reaction mixture was heated slowly to 140 ꢁC with continuous
stirring and further heated to 185 ꢁC and the reaction mixture was
held at this temperature with constant stirring for 4 h. The color of
the reaction mixture gradually deepened and finally, the intense
blue solid product was obtained. The solid crude cobalt tetra-
carboxylic acid phthalocyanine (CoTCAPc) was purified by washing
alternatively with 1 M NaOH and 1 M HCl saturated with NaCl.
Finally, the crude compound was treated with hot and cold water to
obtain bluish solid product compound ii, which was dried over
P2O5.
The present work reports the synthesis and characterization of
peripheral amide bridged benzothiazole coupled cobalt phthalo-
cyanine complex. This synthesized Pc complex was immobilised on
GCE and has been used for the detection of 4-AP by cyclic vol-
tammetry and amperometric methods. The composite CNP-
CoTBTCAPc modified electrode is expected to show an increased
electrochemical response, peak current and electrocatalytic activity
towards 4-AP oxidation.
2. Experimental section
2.1. Materials
Cobalt chloride (CoCl2.6H2O), trimellitic anhydride (TMA:

344
M. Nemakal et al. / Electrochimica Acta 318 (2019) 342e353
Yield: 88%. Anal. for CoTCAPc, Mol Wt. 746.93. C36H16N8O8Co:
solution in 0.5 mL isopropanol (IPA) and sonicating for 15 min to
obtain a homogeneous suspension. The dispersed ink sample of
10 mL was drop casted on a cleaned GCE and the modified electrode
was represented as, GCE/CoTBTCAPc. Similarly, the composite
hybrid electrode was prepared by grinding 1 mg carbon nano-
particle with 4 mg of CoTBTCAPc. These composite mixtures were
Cal. (%) C, 58.00; H, 2.10; N,15.00; O, 17.02; Co, 7.88. Found (%): C,
58.43; H, 1.95; N, 15.37, Co, 7.98. UV-VIS (DMSO, lmax, nm (log ℇ)):
333 (4.126), 604 (4.375), 666 (4.482). FTIR absorption bands (KBr
pellet, cmꢀ1): 3414, 2923, 2851, 1683, 1615, 1518, 1397, 1332, 1252,
1155, 1098, 1058, 953, 913 and 743. 1H-NMR (400 MHz, DMSO-d6):
d
11.82 (br s, CO2H, 1H), 9.04 (d, J ¼ 4.00 Hz, 1H), 8.60e8.61 (m, 1H),
8.43e8.44 (m, 2H), 8.07e8.25 (m, 1H). Mass: Mꢀ1 (746); Mꢀ2 (745);
ꢀ3 (744); Mꢀ4 (743).
dispersed in 0.5 mL isopropyl alcohol (IPA) containing 20
mL 5% wt
Nafion by sonication. Then 10 L of ink was drop casted on the GCE
m
M
and the modified electrode was represented as, GCE/CNP-
CoTBTCAPc. The drop casted electrodes were dried in an inert at-
mosphere and kept in a desiccator for later use. These two fabri-
cated electrodes were used for cyclic voltammetry, impedance
characterization and subsequent electrochemical application.
2.2.3. Synthesis of cobalt (II) tetra-[b-(N-(1,3-benzothiazole-2-yl)-
carboxamide]phthalocyanine (CoTBTCAPc) (iii)
Finely ground mixture of CoTCAPc (ii) (0.5 g, 0.66 mmol), 2-
amino benzothiazole (0.4 g, 2.67 mmol) and potassium carbonate
(0.37 g, 2.67 mmol) was charged into a RB flask. Then dry DMF
(20 mL) and coupling reagent dicyclohexylcarbodiimide (DCC)
(0.58 g, 2.81 mmol) were added to RB flask. The reaction mixture
was stirred for 24 h at room temperature and it was noticed that the
color of the solution deepened with time. The solid crude product
was washed several times with DMF (to remove any DCC inter-
mediate by-products) and extracted using a centrifuge. The pre-
cipitate was repeatedly washed with water and then 1:1 hexane
and acetone mixture (2 ꢂ 20 mL) and finally dried the pure com-
pound iii under vacuum to obtain the greenish blue-colored
product.
2.5. Analytical solution preparation
The analytical solution of 1 mM 4-AP analyte was prepared and
then the solution was diluted to 0.04 mM by successive dilution
method. Different volumes of 0.04 mM 4-AP was injected into the
electrochemical cell containing 10 mL of phosphate buffer elec-
trolyte and the 4-AP sensing was performed by recording the cyclic
voltammograms at a scan rate of 50 mV sꢀ1 in the potential
range ꢀ0.4 to þ0.5 V.
Yield: 83%. Anal. for CoTBTCAPc, Mol Wt. 1275.19.
2.6. Collection of water samples for real sample analysis
C
64H32N16O4S4Co: Cal. (%) C, 60.23; H, 2.50; N, 17.56; O, 5.01; S,
10.19; Co, 4.62. Found (%): C, 59.84; H, 2.46; N, 17.32; S, 9.87; Co,
4.59. UV-VIS (DMSO, lmax, nm (log ℇ)): 347 (4.105), 609 (4.356), 682
(4.482). FTIR absorption bands (KBr pellet, cmꢀ1): 3333, 2930, 2857,
2127, 1628, 1572, 1317, 1438, 1244, 1089, 1049, 895, 636 and 536.
Mass: [Mþ4H2O]ꢀ2 (1345.7329).
The developed method and the fabricated sensor was applied
for the analysis of water collected from the Tungabhadra River,
Ballari, Karnataka, India. Also, rainwater, tap water and drinking
water samples were collected for the analysis. These samples were
filtered, labeled and stored in the refrigerator by covering with
aluminum foil to avoid the exposure to light, heat, and air. The
collected samples were used for the detection of 4-aminophenol
without further pre-treatment.
2.3. Instrumentation
The Vario EL III CHNS analyzer was used for elemental analysis
and the gravimetric procedure was used for the determination of
Co [42]. UV-VIS spectra were recorded using an ocean optics in-
strument with 0.1 mM complex in DMSO in the region of
300e800 nm. The FTIR spectra as KBr pellet of the samples were
recorded using PerkinElmer Spectrum-Two spectrophotometer in
the range 4000-450 cmꢀ1. The X-ray diffraction (XRD) patterns
2.7. Preparation of paracetamol tablet sample solution
The two tablets of commercial paracetamol (Paracetamol
Tablets-IP 500 mg, Glaxo Smith Kline Pharmaceuticals Limited
containing 0.5 g of paracetamol), were crushed well and extracted
with 30 mL of anhydrous ethanol using Soxhlet extractor. The
cooled the solution, centrifuged for 10 min and filtered. The filtrate
was collected in a 50 mL volumetric flask and diluted with water
and used for the real sample analysis of paracetamol.
were profiled in the 2
on a Bruker D8 Advance X-ray diffraction machine using Cu-K
q
range of 5e60ꢁ with a scan speed of 2ꢁ/min
a
radiation (0.1542 nm). TGA data were obtained using the STA-6000
instrumenꢀt1in the temperature range 25e600 ꢁC at a heating rate of
10 ꢁC min and flow rate of air at 30 mL minꢀ1. Varian INOVA
400 MHz NMR instrument was used for recording 1H-NMR spectra.
Agilent technologies 6550 instrument was used for obtaining the
mass spectra of the synthesized compounds. CHI6005E Electro-
chemical Analyzer with a three-electrode system consisting of the
silver electrode (Ag/AgCl) as reference, glassy carbon electrode
(GCE) as working, and a platinum wire as the counter electrode was
used for the electrochemical characterization and sensing
applications.
3. Results and discussion
The synthesis of CoTBTCAPc with amide bond linkage and
benzothiazole at the periphery of the benzene ring has been ach-
ieved as shown in Scheme 1. The amide bridged phthalocyanine
macrocyclic complex was prepared according to the synthetic
protocol previously published from our group [43,44]. The syn-
thesized macrocycles were characterized and the structure was
elucidated by using different physical-analytical as well as elec-
trochemical techniques. These compounds are amorphous, deep
blue, planar and highly stable macrocycles. They are soluble in
sulfuric acid (28 N) and aprotic solvents like dimethyl sulphoxide,
dimethyl formamide, pyridine, dimethyl acetamide, and chloro
naphthalene. Elemental analysis data agreed fairly well with the
calculated values which indicate that the complexes synthesized
are pure. The practical yields obtained were also good. The physi-
cochemical characterization of the complexes has been presented
in the Supplementary Information.
2.4. Electrode fabrication
The GCE was fine-polished with 0.3 and 0.05 mm alumina slurry
on a micro cloth pad and then rinsed with double distilled water.
Then electrochemical cleaning was done by cycling in the potential
range ꢀ1.2 to þ1.2 V for 10 cycles in 0.1 M H2SO4 at 50 mV sꢀ1
.
Subsequently, the electrode was rinsed with double distilled water,
and dried by purging N2. The sample ink was prepared by
dispersing 5 mg of CoTBTCAPc complex and 20 mL 5% wt of Nafion

M. Nemakal et al. / Electrochimica Acta 318 (2019) 342e353
345
Scheme 1. Synthetic route for (i) 2-amino bezothiazole, (ii) CoTCAPc and (iii) CoTBTCAPc.
3.1. Electrochemical study
moeity in the benzothiazole unit of phthalocyanine ring to the
central metal atom. In particular, the redox peak separation in-
volves a larger potential difference which may be due to the
thickness and complex nature of the modified electrode [47].
3.1.1. Study of the redox behavior of the new phthalocyanine
derivatives
The cyclic voltammograms were recorded for 0.1 mM CoTCAPc
and CoTBTCAPc in 10 mL DMSO solvent containing 0.1 M TBAP as
supporting electrolyte at 50 mV sꢀ1 in a nitrogen atmosphere and
the cyclic voltammograms obtained are as shown in Fig. 1a. TBAP
improves the conductivity of the solvent, and does not undergo
either oxidation or reduction on electrochemical cycling. The cyclic
voltammograms of CoTCAPc and CoTBTCAPc exhibited two redox
set of peaks; the first redox pair of peaks were observed at ꢀ0.28
and ꢀ0.44 V for CoTCAPc and ꢀ0.30 and ꢀ0.42 V for CoTBTCAPc
which can be ascribed to the (Pcꢀ2/Pcꢀ1) redox couple. Second
redox pair peaks were observed at 0.33 and 0.59 V for CoTCAPc and
3.1.2. Characterization of the Pc modified electrodes in DMSO
The cyclic voltammograms have been recorded for the modified
electrodes in 10 mL DMSO with 0.1 M TBAP as supporting electro-
lyte (Fig. 1b). The redox peaks observed for the modified electrodes
are in agreement with the peaks observed for the CoTBTCAPc
complex in solution. The redox peak for the Pc ligand (Pcꢀ2/Pcꢀ1
)
showed
D
E values of ꢀ0.1733 and ꢀ0.2719 V and the second redox
peak corresponding to Co metal (Coþ2/Coþ1) exhibited
D
E values of
0.2851 and 0.3672 V, respectively for the GCE/CoTBTCAPc and GCE/
CNP-CoTBTCAPc.
0.34 and 0.56 V for CoTBTCAPc which may be responsible for Coþ2
/
Coþ1 redox couple. The difference between the oxidation and
3.1.3. Charge transfer behavior
reduction peak potential ð
D
Ep ¼ Ecp ꢀ EapÞ, is ꢀ0.17 and 0.26 for
The charge transfer behavior of the modified electrodes were
investigated using [Fe(CN)6]ꢀ3/ꢀ4 redox probe. The charge transfer
behavior provides insights into the possible electron transfer
mediation of the analyte at the GCE/CoTBTCAPc and GCE/CNP-
CoTBTCAPc electrode. Fig. 2 shows the cyclic voltammograms for
the electron transfer movement of the [Fe(CN)6]ꢀ3/ꢀ4 redox probe
on the bare and modified electrode surface. The bare electrode
showed a highly resolved and well-defined reversible redox peak
for the redox probe indicating facile electron transfer between the
electrode and electrolyte. The electron transfer rate was slower at
CoTCAPc and ꢀ0.12 and 0.23 V for CoTBTCAPc complexes respec-
tively for
DEpI and DEpII vs Ag/AgCl. The observed redox peaks of
CoTBTCAPc are irreversible and may involve one-electron transfer
during the redox process as the peak separation is more than 59 mV
[45,46]. Further, both the macromolecules CoTCAPc and CoTBTCAPc
have a symmetrical skeleton shape, and Co as the central metal.
But, they differ in their structure with respect to the terminal
groups, i.e. eCOOH and 2-amino benzothiazole substituents. The
benzothiazole substituent in peripheral CoTBTCAPc influences the
p
electron density in the phthalocyanine ring and exhibits excellent
the CoTBTCAPc modified electrode compared to bare GCE. The
DE
electron transfer properties compared to the carboxylic acid-
substituted phthalocyanine. The reduction and oxidation property
of CoTBTCAPc results from the electron transfer from thiazole
value increased for the redox probe on the CoTBTCAPc modified
electrode compared to a bare electrode which may be due to the
blocking behavior of the film for the electron transfer. However, the
Fig. 1. Cyclic voltammograms for (a) (i) bare GCE and 0.1 mM complexes of (ii) CoTCAPc (iii) CoTBTCAPc in DMSO containing 0.1 M TBAP. (b) CVs for (i) bare, (ii) GCE/CoTBTCAPc and
(iii) GCE/CNP-CoTBTCAPc in DMSO with 0.1 M TBAP at a scan rate of 50 mV sꢀ1
.

346
M. Nemakal et al. / Electrochimica Acta 318 (2019) 342e353
Fig. 3. Nyquist plot with the fitted plots for (i) bare, (ii) GCE/CoTBTCAPc and (iii) GCE/
CNP -CoTBTCAPc in the 0.1 M PBS (pH 7.0) in N2 atmosphere and the inset above shows
the magnified image of the low-frequency region and below inset shows the equiva-
lent circuit used for fitting the data.
Fig. 2. CVs for 0.1 mM potassium ferrocyanide solution on (i) bare GCE, (ii) GCE/
CoTBTCAPc and (iii) GCE/CNP-CoTBTCAPc at a scan rate of 50 mV sꢀ1 in N2 atmosphere.
presence of CNP with phthalocyanine enhanced the faster move-
ment of electron between pinhole surface of the GC and the
ferrocyanide system as the composite hybrid possess a larger sur-
face area and high conductivity due to the mixing of surface-active
CNP species. This infers that the CNP-Pc composite hybrid modified
electrode exhibited increased electron transfer kinetics for the
redox probe compared to the bare and Pc modified GCE. The dif-
ference in the oxidation and reduction peak potentials
interfacial electron charge transfer resistance (Rct) at the GCE.
When the composite hybrid material was deposited, the Rct value
decreased owing to the increased conductivity and surface area of
composite material. Rct reflects the restricted diffusion through the
deposited catalyst and relates to the accessibility of the electrode or
the electrode permeability. The Rct value is comparatively less for
GCE/CNP-CoTBTCAPc compared to GCE/CoTBTCAPc.
ðD
Ep ¼ Ecp ꢀEapÞ for the redox probe at the modified electrodes and
the bare electrode increased in the following trend:
3.1.5. Voltammetric detection of 4-aminophenol
0.0636 < 0.0721 < 0.0873 V respectively for GCE/CNP-CoTBTCAPc,
The modified GCE/CoTBTCAPc and GCE/CNP-CoTBTCAPc elec-
trodes were used for the detection and sensing of 4-amino phenol
(4-AP) in 10 mL pH 7.0 PBS under deaerated atmosphere. Fig. 4a
shows the characteristic cyclic voltammetric curves of the modified
electrodes GCE/CoTBTCAPc and GCE/CNP-CoTBTCAPc in pH 7.0
phosphate buffer solution (PBS) with and without 50 nM 4-AP at a
scan rate of 50 mV sꢀ1 in nitrogen inert atmosphere. The inset of
Fig. 4a shows the CVs for bare and CoTBTCAPc modified GCE in PBS.
The bare GCE did not show any characteristic peak in the
measured potential range of ꢀ0.4 to þ0.6 V in PBS. The 4-AP analyte
containing PBS exhibited a redox peak on bare GCE as well as
modified electrodes for the oxidation of 4-AP. The anodic peak
shifted towards positive potential and the cathodic peak shifted
towards less positive potential for the redox behavior of 4-AP on
the modified electrodes compared to bare GCE. Also, the peak
currents were higher for modified electrodes compared to the bare
GCE. The oxidation peak was observed at 0.02 V and a reduction
peak at 0.0 V for the redox behavior of 4-AP on the modified
electrodes. The oxidation of 4-AP may be catalyzed by the redox
peak of the Pc macrocycle at 0.2 V. The cyclic voltammograms for 4-
AP at bare glassy carbon electrode as well as corresponding metal-
free phthalocyanine modified electrode showed oxidation peak for
4-AP at 0.05 V with minimal peak current. Whereas, the CoTBTCAPc
modified electrode exhibited a peak at 0.02 V with an enhanced
peak current for the electrochemical oxidation of 4-AP. The metal
atom of CoTBTCAPc i.e. Co shows a redox peak at 0.2 and 0.45 V
which is not observed for the modified metal-free phthalocyanine
electrode and the Co peak at 0.2 V may be responsible for the
electrocatalytic oxidation of 4-AP. Even the literature supports the
same analogy [29,31].
GCE/CoTBTCAPc, and GCE. Also, it is worth noting that the
DEp
value for Fe2þ/Fe3þ redox system at the modified electrodes ideally
varied between 60 and 70 mV which indicates the highly reversible
one-electron transfer system [48]. The small variation in the
DEp
value infers that the modified electrode works by transferring the
charge in the presence of Fe2þ/Fe3þ. The quick, and faster charge
transfer behavior exhibited by GCE/CNP-CoTBTCAPc electrode, in-
forms that the CNP supported Pc film acts as a mediator to facilitate
the fast transfer of an electron between the surface pinhole of GC
and [Fe(CN)6]ꢀ3/ꢀ4 system in solution.
3.1.4. Electrochemical impedance spectroscopy (EIS)
The electrochemical impedance spectra (EIS) obtained for the
modified electrodes are shown in Fig. 3. The frequency was varied
over a broad range of 0.01 Hze100 kHz with amplitude of 5 mV. The
in-phase and out-of-phase components of the current response are
fitted to an equivalent circuit that models the electrochemical cell.
The equivalent circuit has the cell model as C1 (QC2 (R1W)) R2. The
mass transport effects are minimal at high frequencies for the
working electrode of a simple three-electrode electrochemical cell
[49]. In the equivalence circuit, R1 represents the resistance of the
electrolyte, R2 is the charge-transfer resistance (Rct), C1 is the
double-layer capacitance, C2 is the capacitance of the modified
electrode, Q is the impedance of a capacitor known as constant
phase element i.e. CPE and W is the mass-transfer impedance
represented by a Warburg circuit element [47]. EIS data obtained
from the fitting of the equivalence circuit for the modified elec-
trodes are tabulated in Table 1. It can be seen from Fig. 3 that the
smaller semicircle was observed for GCE/CNP-CoTBTCAPc
compared to GCE/CoTBTCAPc and bare glassy carbon. A straight-
line characteristic with a bigger semicircle was observed for the
bare GCE at higher frequencies indicating a relatively higher
The peak currents for the 4-AP oxidation were significantly
enhanced on the GCE/CNP-CoTBTCAPc compared to GCE/CoTBT-
CAPc due to the high surface area and active sites in CNP. The CNP

M. Nemakal et al. / Electrochimica Acta 318 (2019) 342e353
347
Table 1
Equivalent circuit parameters obtained by EIS studies.
Electrode
C
1 (F)
C2 (F)
R1
(
U
)
R2
1
926.8
861.6
(
U
)
Q-Yo (F s(aꢀ1)
)
Q-n (F s(aꢀ1)
)
W (U )
s(ꢀ0.5)
GCE
66.99x10e5
292.4x10e5
563.5x10e5
1.002x10e6
3.402 x10e10
7.118x10e10
627.90
3273
2271
100.1x10e6
1.264x10e6
19.06x10e6
0.8
0.8
0.8
118.8
GCE/CoTBTCAPc
GCE/CNP-CoTBTCAPc
4.426x10e5
963.6 x10e5
Fig. 4. CV profiles of (a) without 4-AP (i) GCE, (iii) GCE/CoTBTCAPc and (v) GCE/CNP-CoTBTCAPc in PBS (pH ¼ 7) at scan rate 50 mV sꢀ1 and CV for 50 nM 4-AP on (ii) GCE, (iv) GCE/
CoTBTCAPc and (vi) GCE/CNP-CoTBTCAPc in PBS (pH ¼ 7) at a scan rate 50 mV sꢀ1. Inset: magnified CVs in the absence of 4-AP at (i) GCE and (iii) GCE/CoTBTCAPc; and in the
presence of 50 nM 4-AP at (ii) GCE and (iv) GCE/CoTBTCAPc. CVs on (b) GCE/CoTBTCAPc and (c) GCE/CNP-CoTBTCAPc electrode at different concentrations of 4-AP from 40 to 800 nM
in PBS (pH ¼ 7.0) at a sweep rate of 50 mV sꢀ1. Inset, plot of peak current response vs. concentration of 4-AP for the anodic (black line) and cathodic (dotted line) peak currents.
alone on the electrode surface did not show any appreciable current
for the 4-AP oxidation which indicates the synergic and combina-
tion effect of CNP and Pc responsible for the catalysis of 4-AP
oxidation. The catalytic effect of the modified electrodes for 4-AP
oxidation was used to detect the 4-AP at different concentrations.
Fig. 4 shows the CVs for the oxidation of 4-AP with different con-
centrations using the modified electrodes in PBS. The CVs showed
an increase in both the cathodic and anodic peak currents for the
redox behavior of 4-AP with increasing concentrations of 4-AP. The
plot of peak current response Ipa and Ipc vs. concentration of 4-AP
for the oxidation and reduction at 0.02 and 0.0 V respectively
exhibited linear relationship for both the modified electrodes in the
concentration range 40e800 nM. The straight-line characteristics
for the plot of Ipa vs. concentration and Ipc vs. concentration for
GCE/CoTBTCAPc are represented by:
GCE/CNP-CoTBTCAPc electrode follows:
Ipa
Ipc
(
m
A) ¼ 0.0386 (nM) þ 141.5625 (R2 ¼ 0.9959)
A) ¼ ꢀ0.0250 (nM) - 141.7281 (R2 ¼ 0.9895)
(4)
(5)
(m
The correlation coefficient (R2), value for the straight line plot is
greater than 0.99 which refers to an excellent linear relation and
dependence between the variables. The detection limit (LOD) for 4-
aminophenol at GCE/CoTBTCAPc and GCE/CNP-CoTBTCAPc was
found to be 13 and 11 nM (S/N ¼ 3) respectively with sensitivity
values of 0.00955 (Ipa), 0.0082 (Ipc) and 0.1298 (Ipa) and 0.0955 (Ipc
)
m
A nMꢀ1 cmꢀ2
.
3.1.6. Effect of scan rate
The mass transfer behavior of the 4-AP analyte at the modified
GCE/CoTBTCAPc and GCE/CNP-CoTBTCAPc electrodes was investi-
gated by recording the CVs at different scan rates. Figs. S7a and S7b
(supplementary information) show the CVs for the 500 nM of 4-AP
on GCE/CoTBTCAPc and GCE/CNP-CoTBTCAPc electrodes in PBS at
different scan rates. The CVs showed a linear increase in anodic
(Ipa) and cathodic (Ipc) peak current response with a small shift in
the anodic as well as cathodic peak potential slightly towards
positive and negative potentials respectively on varying the scan
Ipa
Ipc
(
m
A) ¼ 0.0030 (nM) þ 10.5130 (R2 ¼ 0.9885)
A) ¼ ꢀ0.0062 (nM) e 11.0790 (R2 ¼ 0.9832)
(2)
(3)
(
m
The correlation coefficient, R2 value for the straight line rela-
tionship refers to a good linear fit and excellent dependence be-
tween the variables. Similarly, the straight-line characteristics for
the linear plot of Ipa vs. concentration and Ipc vs. concentration for

348
M. Nemakal et al. / Electrochimica Acta 318 (2019) 342e353
rate between 5 and 250 mV sꢀ1 at the GCE/CoTBTCAPc and GCE/
CNP-CoTBTCAPc electrodes. The inset of Figs. S7a and S7b (supple-
mentary information) shows the straight line relationship for the
plot of peak current vs. square root of scan rate which suggests that
the oxidation of 4-AP molecule on the modified electrodes follow
diffusion-controlled mass transfer phenomena. The linear equation
for the plot of peak current vs. square root of the scan rate at GCE/
CoTBTCAPc and GCE/CNP-CoTBTCAPc electrodes is represented in
Table S2.
iminobenzoquinone (iv), is stabilized by extended conjugation and
the overall catalytic pathway involves the loss of two protons and
two electrons. The CoTBTCAPc accepts two electrons during the
oxidation of 4-AP and CoTBTCAPc donates two electrons during the
reduction of 4-AP. The analytic species electrochemically exhibits
quasi-reversible redox peaks. In the presence of light, 4-AP is un-
stable and undergoes degradation with time yielding 1,4-
benzoquinone (v) and ammonium ion as final degraded products.
The diffusion coefficient (Do) for the redox reaction of 4-AP was
calculated by using the Randles-Sevcik equation.
3.1.8. Reproducibility and stability of the modified electrodes
The modified GCE/CoTBTCAPc and GCE/CNP-CoTBTCAPc elec-
trodes were used for the sensing of 4-AP in PBS and then washed
with water, and stored in a desiccator for 7 days to conduct the
stability studies. Then these electrodes were again used for the
detection of freshly prepared 4-AP. The electrodes showed almost
the same peak current and sensitivity as that of the freshly pre-
pared electrodes. The three successive determinations of 4-AP
showed good relative stability of the peak current with RSD 2.32%
and 2.09% for CoTBTCAPc and CNP-CoTBTCAPc electrodes respec-
tively. The five sets of the fabricated electrodes were examined for
reproducibility and showed RSD value of 2.42% and 2.87% for the
peak currents of the two different modified electrodes. The GCE/
CoTBTCAPc and GCE/CNP-CoTBTCAPc modified electrodes can be
employed for the practical application of the nanomolar detection
of 4-amino phenol analytic species.
The stability of the modified electrodes was evaluated by per-
forming 50 repeated potential cycles. The cyclic voltammograms
obtained were reproducible without any significant change in the
background current response. The fiftieth cycle of CV was almost
the same as that of the first cycle with RSD value of less than 4%,
indicating that the modified electrode is highly stable and the cyclic
voltammograms obtained are shown in Fig. S8 (Supplementary
Information).
ip ¼ 0:4463 nFACðnFvDo=RTÞ1=2
(6)
where, n is the number of electrons involved in the overall redox
process of 4-AP (n ¼ 2),
F
is the Faraday constant
(96 485.332C molꢀ1), R is the gas constant (8.314 J Kꢀ1 molꢀ1), T is
the temperature (300 K), v is the scan rate in mV sꢀ1, A is the area of
the electrode surface (0.0701 cm2), C is the bulk concentration of 4-
AP (500 nM). The diffusion coefficient (Do) value was found to be
6.873 x 10ꢀ6 cm2 sꢀ1
.
The diffusion coefficient (Do) value provides an information
about the amount of 4-AP that diffuses across a unit area in 1 s
under the influence of a potential gradient of one unit. Higher value
of the diffusion coefficient indicates the faster diffusion of the an-
alytic species across the electrode. The diffusion coefficient ob-
tained from the present method for 4-AP oxidation was compared
with the values reported in the literature for other electrodes and
the
u-mercaptopropionic acid self-assembled monolayer modified
electrode on a gold electrode (MPA/SAM/Au) exhibited Do of
4.55 ꢂ 10ꢀ6 cm2 sꢀ1 [50], whereas nicotinamide adenine dinucleo-
tide and ferrocenyl-tethered dendrimer layer on gold electrode
(NADH/Au and Fc-D/Au) immunosensor showed Do of 6.47 x
10ꢀ6 cm2 sꢀ1 for 4-AP [51]. Our electrode showed a higher Do value
for 4-AP compared to other electrodes in literature which indicates
that the diffusion process of 4-AP is facilitated at the modified
electrodes. The electrons involved in the overall 4-AP redox reac-
tion are found to be two-electrons and the proposed redox mech-
anism for 4-AP at the modified electrodes can be represented as
shown in Scheme 2.
3.1.9. DPV method for the detection of 4-aminophenol
The differential pulse voltammetric (DPV) technique is highly
sensitive towards the catalytic current response for small changes
in the analyte concentration. Hence, the determination of 4-AP was
performed using the DPV method at GCE/CoTBTCAPc and GCE/CNP-
CoTBTCAPc with various concentrations of the 4-AP analytic species
in phosphate buffer solution of pH 7 (Fig. 5). The peak current
increased with the increase of 4-AP analyte concentrations from 50
to 1000 nM. The inset in Fig. 5 shows the linear plot of peak current
vs. 4-AP concentration. The linear plots for 4-AP oxidation at the
modified electrodes followed the regression equation for the
straight-line characteristics as follows for GCE/CoTBTCAPc:
3.1.7. Proposed mechanism for the 4-aminophenol (4-AP) oxidation
The electrocatalytic oxidation of 4-AP on GCE/CoTBTCAPc and
GCE/CNP-CoTBTCAPc electrodes may follow the reaction mecha-
nism pathway [21,26,29,52] as shown in Scheme 2. The analytic
species (i) i.e. 4-aminophenol has two activating groups; one is a
hydroxyl group and another is the amine group. Hence, the mole-
cule undergoes heteroleptic type of oxidation process. Firstly, 4-AP
forms p-aminophenolate (apH-) by the loss of one proton. The p-
aminophenolate (apH-) (ii) reversibly forms the p-iminosemi-
quinonate (isq-) (iii), by the loss of one proton and one-electron.
Further one-electron oxidation of (iii) leads to the formation of p-
iminobenzoquinone (ibq) (iv), which is reversible in nature. The p-
Ipc
(m
A) ¼ e 0.0023 (nM) e 7.5743 (R2 ¼ 0.9935)
(7)
And for the GCE/CNP-CoTBTCAPc:
Ipc
(m
A) ¼ e 0.0293 (nM) e 323.1145 (R2 ¼ 0.9879)
(8)
The detection limit (S/N ¼ 3) was found to be 15 and 10 nM and
the sensitivity was 0.0328 and 0.4179
m
A nMꢀ1 cmꢀ2 for the
detection of 4-AP at the phthalocyanine and composite hybrid
modified electrodes respectively.
3.1.10. Amperometric detection of 4-aminophenol
Fig. 6a illustrates the amperometric i-t response recorded for the
oxidation of 4-AP in neutral media. The i-t response curve gives an
indication of the concentration gradient of 4-AP at the vicinity of
the electrode due to the injection of 4-AP at regular interval of time
which leads to the expansion of the diffusion layer and the deple-
tion of the analyte at the electrode resulting in the step-wise
Scheme 2. The mechanism for the redox behavior of 4-AP.

M. Nemakal et al. / Electrochimica Acta 318 (2019) 342e353
349
Fig. 5. DPVs of 50, 100, 200, 300, 400, 600, 800 and 1000 nM of 4-AP at (a) GCE/CoTBTCAPc and (b) GCE/CNP-CoTBTCAPc in PBS (pH 7.0). Inset, the plot of peak current vs. 4-AP
concentration.
Fig. 6. Amperometric iet curve for (a) (i) GCE/CoTBTCAPc and (ii) GCE/CNP-CoTBTCAPc with 4-AP (100-900 nM) in 10 mL PBS (pH ¼ 7). (b) The plot of amperometric current
response vs. concentration of 4-AP on (i) CoTBTCAPc and (ii) CNP-CoTBTCAPc.
increment of the current response. The amperogram was recorded
for 4-AP with a successive addition of 100 nM at a time interval of
50 s in the concentration range 100e900 nM in10 mL PBS with
continuous stirring at 0.02 V. Fig. 6a shows the amperograms for
bare, GCE/CoTBTCAPc and GCE/CNP-CoTBTCAPc modified elec-
trodes respectively. The current response got stabilized within
3e4 s after the addition of the 4-AP which is an important
parameter for a potential sensor. The continuous stirring of the
electrolyte and complex nature of the CNP-Pc hybrid electrode
leads to noise in the amperogram. Fig. 6b shows the plot of
amperometric current response vs. concentration of 4-AP for the
modified electrodes which showed linear response in the concen-
tration range of 100e900 nM 4-AP and the straight line equation
for GCE/CoTBTCAPc electrode is given by:
reproducible values with a short error bar. This means the data are
clumped around the mean (lesser value of SD) i.e. less spread and
more reliable.
It is evident from amperometric plot that the carbon nano-
particles in the composite hybrid enhance the catalytic activity of
the 4-AP. This is due to the enhancement in the surface area by CNP
for the phthalocyanine accumulation on the GCE surface. This
composite hybrid also promotes the electron transfer between the
pinhole surface of GCE and the electrolytic medium. The modified
electrode has LOD of 30 and 20 nM with sensitivity values 0.4008
and 0.8887 m
A nMꢀ1 cmꢀ2 respectively for GCE/CoTBTCAPc and
GCE/CNP-CoTBTCAPc.
3.2. Sensor application and interference studies at GCE/CNP-
CoTBTCAPc electrode
Ip
(m
A) ¼ 0.0281 (nM) þ 11.4575 (R2 ¼ 0.9950)
(9)
The 4-AP contains alcoholic group as well as the amine group,
and the phenolic and amine-group-containing impurities or co-
existing molecules may cause interference during the determina-
tion of 4-AP. Hence, the interference studies with dopamine which
contains both amine and alcoholic groups and other molecules
which contains an alcoholic group like resorcinol, hydroquinone,
glucose, ascorbic acid, phenol, and chlorophenol has been carried
out. Besides, the interference studies of common metal salts which
exist in our body or industrial effluents have also been carried out.
The GCE/CNP-CoTBTCAPc was used for the sensing of 4-AP
amperometrically in the presence of various interfering bio-
molecules namely organic and inorganic species. The i-t response
And the straight line characteristic for GCE/CNP-CoTBTCAPc
electrode is represented by:
Ip
(m
A) ¼ 0.0623 (nM) þ 64.2275 (R2 ¼ 0.9980)
(10)
The error bar for the modified electrodes towards the detection
of 4-AP by DPV and amperometry are shown in Figs. 5 and 6,
respectively. The error bar in the Figs. 5 and 6 indicates that at
lower concentration, singling of the response is more by having
large disperse from the mean (larger value of SD). As the concen-
tration increases, the deviation reduces and likely to obtain

350
M. Nemakal et al. / Electrochimica Acta 318 (2019) 342e353
curve was recorded at a potential of 0.02 V under stirring condition.
As observed in Fig. 7, fabricated electrode showed the response only
for the addition of 400 nM 4-AP and no appreciable amperometric
or improper storage of paracetamol leads to the formation of 4-AP.
As already mentioned in the introduction part, the consumption of
4- AP leads to various health issues and disorders in the body. The
extracted paracetamol in PBS exhibited a well-defined peak at
0.3730 V in DPV in agreement with the literature values in PBS
[21,29]. But no peak was observed for 4-AP in the detectable range
of the modified electrode in PBS for 500 mg of paracetamol tablet
extract which indicates the absence or smaller amount of 4-AP in
the tablet extract than the detectable range of the sensor. Hence, 4-
AP of different quantities were spiked into the paracetamol extract
in PBS by the standard addition method. The DPV method showed a
clear and distinguishable peak for 4-AP at 0.02 V and peak for
paracetamol at 0.37 V. The peaks observed for 4-AP and paraceta-
mol are well resolved without any overlap and the peak-to-peak
separation in the DPV for 4-AP and paracetamol is 0.35 V, which
is large enough for their simultaneous determination in pharma-
ceutical samples. Fig. S9 a (Supplementary Information), shows the
DPV for 4-aminophenol in the absence of paracetamol (i) and in the
presence of paracetamol (ii). The DPV results obtained by the
spiking of different amounts of 4-AP to paracetamol in PBS showed
recovery values of 98.5%e99.5% with RSD values less than 2.3%. It
was found that the matrix of the paracetamol tablet did not have
any interference or effect during the determination of 4-AP at the
modified electrode as seen from the recovery values of 4-AP and
the results obtained for the paracetamol tablet are summarized in
Table 2. The results from Fig. S9 a (Supplementary Information) and
Table 2 indicates that the modified electrode is an appropriate and
suitable electrode for the simultaneous detection of 4-AP and
paracetamol without any interference from each other. As per the
allowed limit, the concentration of 4-AP in paracetamol is 5 ppm or
response was observed for other interfering molecules like
glucose, hydroquinone, resorcinol, ascorbic acid, dopamine, phenol,
and chlorophenol even at higher concentration of 40 M in PBS (pH
7.0) at 0.02 V. Further, even the presence of 100 fold excess con-
centration of inorganic salts i.e. 40 M KCl, NaNO2 and MgSO4 did
D-
m
m
not show any significant current response in the amperogram. But,
the addition of 400 nM 4-AP to the PBS containing 100 fold excess
of interfering molecules showed an increase in the current
response in the amperogram. Hence, GCE/CNP-CoTBTCAPc elec-
trode shows sensitive, and selective response for the detection and
sensing of 4-AP even in the presence of various bio-molecules and
inorganic co-existing interfering salt species.
3.3. Determination of 4-aminophenol in water samples and
paracetamol sample
The DPV method was employed for the quantitative determi-
nation of 4-aminophenol in real samples. The fabricated electrode
was used for determining 4-AP in a water sample as well as a
commercial sample (paracetamol tablets) to understand the prac-
tical applicability, feasibility, and performance of the developed
electrode in real sample analysis. The water samples were collected
from river water (Tungabhadra River at Ballari, Karnataka); tap
water and potable water. 1 mL of water sample was added to the
experimental electrochemical cell containing 10 mL of PBS (pH 7.0)
and no change in peak current was observed for different water
samples. This indicates that the collected water samples from river,
tap and rain did not contain any 4-AP in the measurable range of
the fabricated sensor. Hence, standard 250 nM 4-AP analyte was
spiked to 1 mL of the water sample by the standard addition
method. The recovery results obtained for 4-AP on GCE/CNP-
CoTBTCAPc are listed in Table 2. The designed sensor and method
provided satisfactory recovery results lying in between 98.6% and
101.6% indicating that the other components present in the sample
did not interfere in the determination of 4-AP at the modified
electrode. The RSD values were less than 3% for the spiked samples
on the modified electrode. The developed sensor showed accuracy,
practical feasibility for the efficient determination of 4-AP in water
samples at nanomolar concentration.
45
the 4-AP in the concentration range of 0.1e0.9
electrode indicating that the paracetamol tablet contains either no
4-AP or less 4-AP than 0.1 M which is much lesser than the
mM. But the paracetamol tablet did not show any response for
m
M at the modified
m
allowed limit. The standard deviation (SD) measures the average
distance between the values of the data set from the mean. The SD
values shown in Table 2, varies between < 2. The data obtained for
the 4-AP analysis in real sample at the modified electrode has low
SD. This means that the data points are tend to be very closely
spread out around the mean i.e. the values are in good appreciable
limit. To understand the interaction of phthalocyanine with 4-AP,
UVeVis spectral characterization was carried out and the spectra
are shown in Fig. S9 b (Supplementary Information). The peaks in the
UV-Vis spectrum of Pc exhibited bathochromic shift which may be
due to the strong interaction between the Pc and 4-AP.
Further, the developed sensor was used for the detection of 4-AP
in paracetamol tablet using the DPV method. In general, the para-
cetamol formulation should not contain 4-AP. But the degradation
Fig. 7. I-t response for (a) 4-AP (400, 800 and 1200 nM) in presence of 40
aminophenol, D-Glu: D-glucose, HQ: hydroquinone, RS: resorcinol, KCl, NaNO2, and MgSO4. (b) Interference of 4-AP (4-aminophenol), AA: ascorbic acid, DA: dopamine, PH: phenol
m
M of different interfering species like biomolecules and inorganic salts in 10 mL of PBS (pH ¼ 7). 4-AP: 4-
and CPH: chlorophenol.

M. Nemakal et al. / Electrochimica Acta 318 (2019) 342e353
351
Table 2
Determination of 4-aminophenol in different water samples and paracetamol tablet on the GCE/CNP-CoTBTCAPc electrode.
Analysis of water samples
Water Samples
Detected (nM)
Spiked (nM)
Foundx/nM ( SD)
RSD (%)
Recovery (%)
Drinking water
Tap water
d
d
d
d
250
250
250
250
247 ( 0.9)
248 ( 1.2)
254 ( 1.9)
248 ( 1.5)
2.31
2.78
3.45
3.23
98.6
99.3
101.6
99.3
River water
Rain water
§Average of five determinations standard deviation.
Analysis of paracetamol tablets
Tablet Samples
Paracetamol-A
Paracetamol-B
Paracetamol-C
Detected (nM)
Spiked (nM)
250
500
Founda/nM( SD)
247 ( 1.8)
498 ( 1.3)
RSD (%)
1.93
Recovery (%)
98.6
d
d
d
2.14
99.3
750
746 ( 0.9)
1.86
99.4
a
Average of five determinations standard deviation.
Table 3
compared with other voltammetric methods in the literature for 4-
AP determination and the results are summarized in Table 4. On
comparing the values for linear range, transduction (methods),
LOD, sensitivity, and the analytical parameters, it could be noticed
that LOD of 4-AP from the proposed method is lower or comparable
with those results based on the complex and costlier modified
electrodes.
Comparison of the present voltammetric and amperometric method with other
methods in the literature.
Methods
Detection limit
Ref.
HPLC Method
Surface enhanced Raman scattering
Fluorimetry
Chemiluminescence
Capillary electrophoresis
Micellar electrokinetic chromatography
Optical reflectance
1.0 ng/mL
0.9 mg/mL
12
13
14
15
16
17
23
3.3
m
g/100 mL
0.19 ng/mL
1.4 ꢂ 10ꢀ6 mol/L
2 ꢂ 10ꢀ6 g/mL
4. Conclusion
0.183
m
M/L
Cyclic voltammetry
Amperometry
13 &11 nM
30 & 25 nM
Present work
Present work
CoTBTCAPc with the amide bridge has been synthesized and
characterized by analytical and electro-analytical techniques. The
composite hybrid of phthalocyanine molecule with CNP was
immobilised on the glassy carbon electrode and used for the
detection of 4-aminophenol at nanomolar concentration. The CNP-
Pc composite hybrid electrode showed better electron transfer
compared to bare GCE and Pc alone modified electrode. CNP pre-
sent in the composite hybrid electrode significantly provides a
larger surface area for the phthalocyanine and increases the con-
ductivity which results in accelerated charge-transfer kinetics be-
tween the electrode surface and the electrolyte. DPV and
amperometric methods showed the good linear response for the
sensing and detection of 4-AP in the nanomolar concentration
range. The detection of 4-AP was selective and reproducible even in
the presence of various interfering organic molecules and inorganic
species on the GCE/CNP-CoTBTCAPc modified electrode. In addi-
tion, the fabricated electrode can be employed for the sensing of 4-
aminophenol in water samples and in paracetamol sample.
3.4. Comparison of the GCE/CNP-CoTBTCAPc electrode with other
methods
The fabricated GCE/CNP-CoTBTCAPc electrode showed better
DPV and amperometric response and analytical parameters than
the sensors/electrodes reported in the literature. Table 3 presents
the comparison of the analytical data obtained for the fabricated
sensor with the reported sensors in the literature for the sensing of
4-AP. The developed method has improved sensitivity and LOD
values similar to that of HPLC. Hence, the developed method is cost-
effective and simple compared to HPLC and chemiluminescence
techniques and can be applied for the detection of 4-AP at nano-
molar concentration.
The analytical parameter obtained by the proposed method was
Table 4
Comparison of analytical parameters of GCE/CNP-CoTBTCAPc sensor with other modified electrodes for the detection of 4AP.
Sensing layer
Linear Range (
m
M)
Transduction
LOD (
m
M)
Sensitivity (
m
A
m
Mꢀ1 cmꢀ2
)
Ref.
aGrapheneechitosan/GCE
bGRePANI/GCE
0.2e550
0.2e20
20e100
50e500
e
Voltammetry
DPV
0.057
0.065
e
18
21
177.6
604.2
e
cG-PANI/CPE
Voltammetry
e
15.68
1
0.09
0.3
0.02
0.011
0.030
0.030
22
24
26
29
30
This work
dp[NVCzVBSA]/CFME
eAu/Nafion/MWNT-Nafion:Cu/Au
faPcCo/CNT hybrid/GC
gCoPc SiO2/C
e
0.2 - 1.6
0.5e1000
5e230
0.04-0.80
0.1 - 0.9
0.1 - 0.9
Voltammetry
DPV
Amperometric
Voltammetry
DPV
7
0.123
9.5/8.2
417.7
888.7
GCE/CNP-CoTBTCAPc
Amperometric
a
Grapheneechitosan/GCE:Grapheneechitosan composite film.
GRePANI/GCE: Grapheneepolyaniline nanocomposite.
G-PANI/CPE: Graphene-polyaniline modified carbon paste coupled with droplet-based microfluidic.
p[NVCzVBSA]/CFME: Poly[N-vinylcarbazole-co-vinylbenzene sulfonic acid]/carbon fibre microelectrode.
Au/Nafion/MWNT-Nafion:Cu/Au: Multi-Wall Carbon Nanotubes Dispersed in Electrochemically Modified Nafion.
b
c
d
e
f
aPcCo/CNT hybrid/GC: Tetra-b-[3-(dimethylamine)phenoxy] phthalocyanine cobalt(II)/multiwalled carbon nanotube hybrid.
CoPc SiO2/C: Cobalt Phthalocyanine modified SiO2/C.
g

352
M. Nemakal et al. / Electrochimica Acta 318 (2019) 342e353
with poly(4-aminophenol), Macromol. Symp. 245e246 (2006) 236e242.
Conflicts of interest
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India (DST-SERB grant No. SERB/F/9388/2016-17). Authors are
highly thankful to the Department of Science and Technology, New
Delhi, Govt. of India, (DST grant No. SB/FT/CS-162/2013) for
providing fellowship and financial support under the Fast Track
Young Scientist Scheme. VGST K-FIST-L1 (GRD No.555), CSIR-EMR
(No.01/(2915)/17/EMR-II) and DST-FIST (grant SR/FST/CSI-274/
2016) support are also acknowledged.
Appendix A. Supplementary data
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