Chemical Physics Letters 716 (2019) 177–185

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Chemical Physics Letters

journal homepage: www.elsevier.com/locate/cplett

Research paper

Synthesis and characterization of novel nanocomposite (MWCNTs/CC-SH/ T

Au) and its use as a modifier for construction of a sensitive sensor for
determination of low concentration of levothyroxine in real samples
⁎
Shahram Lotfi , Hojat Veisi
Department of Chemistry, Payame Noor University (PNU), 19395-4697 Tehran, Iran

H I GH L IG H T S

• AThenewelectrochemical
nanocomposite, (MWCNTs/CC-SH/Au) was synthesized and used as a sensor in T4 detection.
• The designed sensor behavior of the T4 at physiological pH (7.2) was investigated at the surface of MWCNTs/CC-SH/Au/GCE.
• was successfully used for determination of nanomolar level of T4 in real samples.

A R T I C LE I N FO A B S T R A C T

Keywords: A new nanocomposite based on gold nanoparticles supported on mercaptoethanol bonded to melamine modified
Levothyroxine multi-walled carbon nanotubes (MWCNTs/CC-SH/AuNPs) was synthesized, characterized and used as sensitive
Electrochemical sensor sensor for the determination of levothyroxine (T4) using glassy carbon electrode. In order to characterize the
Gold nanoparticles nanocomposite, different methods including field emission scanning electronic microscopy (FE-SEM), Fourier
Multi-walled carbon nanotubes
transform infrared (FTIR), transmission electron microscopy (TEM), energy dispersion X-ray spectroscopy (EDX)
Differential pulse voltammetry
and Raman spectroscopy were used. The differential pulse voltammetry data indicated that the acquired re-
sponse was linearly reliant on the T4 concentration within 10–120 nM, with a detection limit of 2.84 nM.

1. Introduction hyperthyroidism and hypothyroidism [8].

Levothyroxine (T4), 3, 5, 3′, 5′-tetraiodothyronin, is a key biological
hormone of iodoamino acid derivative of thyronine which is created in
the thyroid gland. The T4 not only has a crucial role in variety of
functions including carbohydrate metabolism, oxygen consumption,
maturation of sexual organs, protein synthesis and fetal neuro-devel-
opment, but also affects the regular growth as well as the development
of the body [1–4]. Therefore, T4 is the most often prescribed drug for
hypothyroidism replacement therapy. Following the replacement of
therapy post-operatively, serum thyroid stimulating hormone (TSH)
levels are measured, and levothyroxine dosages are titrated until to the
point that a normal level of TSH serum is achieved. It has been common
practice to start patients on a 1.6 µg/kg (kg: actual body weight) dose of
levothyroxine following total thyroidectomy, a standard dose for
thyroid hormone replacement in patients with primary hypothyroidism
[5–7]. Owing to the biological and pharmacological importance of T4,
its determination has practical significance for the diagnosis of
So, different methods have been reported for determination of T4
including chemiluminescence [9,10], gas chromatography mass spec-
troscopy [11,12], capillary electrophoresis [13,14], time-resolved
fluorescence [15], immunoassay methods [16–21], high performance
liquid chromatography (HPLC) [8,22–24] and electrochemical methods
[1,4,25–30]. Among various methods of T4 determination, im-
munoassay and high performance liquid chromatography remain the
most popular. The fact is that most of non-electrochemical techniques,
despite their being sensitive and accurate, however, they are time
consuming, expensive instrumentation, and require sample prepara-
tions. Electroanalytical methods involve nomerous advantages such as
simplicity, sensitivity, versatility, inexpensive instrumentation, high
and wide linearity and quick alternative for determining the pharma-
ceutical compounds compared with other methods [31].
Modified electrodes were developed to enhance the electrode re-
sponse in electroanalysis. There is currently an intense interest in uti-
lizing nanomaterials for the fabrication of electrochemical sensors and a

⁎
Corresponding author.
E-mail address: sh.lotfei@gmail.com (S. Lotfi).

https://doi.org/10.1016/j.cplett.2018.12.029
Received 16 September 2018; Received in revised form 23 November 2018; Accepted 12 December 2018
Available online 27 December 2018
0009-2614/ © 2018 Elsevier B.V. All rights reserved.
S. Lotfi, H. Veisi
wide range of bioscience applications [32]. Nanomaterials were ex-
tensively developed as a result of their unique properties. Nanoparticles
can act as conduction centers enabling the transfer of electrons and
deliver great catalytic surface areas. Various nanomaterials were uti-
lized in modifying the electrode surfaces such as carbon nanotube
[30,33], gold [34], polymer films [35], graphene [36], Ag [37], Fe3O4
nanoparticle [38], palladium [39] etc, as a result of the response signal
improvement, increased sensitivity and better reproducibility.
Among the wide range of electrode modifiers, carbon nanotubes
(CNTs) are entirely considered as metallic conductors because of their
unique properties. They enjoy such useful characteristics as ordered
structure with high aspect ratio, high electrical conductivity, good
chemical stability, relative chemical inertness in most electrolyte so-
lutions, etc. It also has a wide operation potential window and high
surface area besides being extensively applied for the improvement of
chemically modified electrodes [37,40]. Metallic nanoparticles such as
gold, platinum and palladium have also attracted much more attention,
possess important properties for electroanalysis and permit amplified
electrochemical detection, including roughening of the conductive
sensing interface, large surface-to-volume ratio, improved conductivity
at nanoscale dimensions and their catalytic properties. Gold nano-
particles (AuNPs) have been utilized increasingly in many electro-
analytical systems to improve analytical selectivity and sensitivity due
to their excellent biological compatibility, high conductivity and good
catalytic properties [35,41]. Therefore, MWCNT/AuNPs are of great
interest among the nanocomposites and composites.
Ibrahim et al. [42] prepared f-MWCNT decorated with AuNPs using
glassy carbon paste (GCP) as a cross linker (AuNPs/f-MWCNT/GCPE)
for determination of cyproterone acetate (CPA) drug. This nano-
composite material exhibited an enhanced peak current compared to
the bare GCPE and displayed non-interference against the physiological
common interferents. Shahrokhian et al. [43] electrodeposited a thin
film of Pd–Au nanoparticles decorated with MWCNT on glassy carbon
electrode. This nanostructured film, having been modified on electrode
effectively, exhibited enhanced properties for detecting ceftazidime
with a linear dynamic range of 0.05–50 μM and detection limit of 1 nM.
Afkhami et al. [44] examined AuNPs/MWCNTs nanoparticles on glassy
carbon electrode to measure diclofenac sodium. They showed good
repeatability and reproducibility with relative standard deviation of
2.52 and 3.4% respectively. Mpanza et al. [45] immobilized MWCNTs
decorated with AuNPs on glassy carbon electrode. This electrode ex-
hibited an excellent electrocatalytical activity towards the determina-
tion of capsaicin and silymarin. Gholivand et al. [46] immobilized
MWCNs/poly(methylene blue)/AuNPs on the surface of graphite elec-
trode to determine the amount of nevirapine (NVP). The designed
sensor exhibited catalytic activity toward NVP oxidation. It also in-
creased the response current and lowers the oxidation overpotential.
In the present study, we fallowed two steps. First, we synthesized a
new nanocomposite including multi walled carbon nanotubes; mela-
mine; mercaptoethanol and gold nanoparticles (MWCNTs/CC-SH/Au).
The organized nanocomposite was described by FT-IR, FE-SEM, TEM
and EDX. Then, this nanocomposite was utilized as a sensitive modifier
for the construction of novel electrochemical sensor modified glassy
carbon electrode (GCE) with the purpose of determining of T4. The
sensor is achieved in a simple way by the pouring of MWCNTs/CC-SH/
Au nanocomposite immobilized onto the surface of a GCE. The ex-
perimental results indicated that MWCNTs/CC-SH/Au hybrid exhibited
significant ability to increment the electroactive surface area and the
electron-transfer between the analyte and the electrode. Sensitivity and
selectivity of MWCNTs/CC-SH/Au/GCE were investigated using vol-
tammetry techniques besides its successful application for T4 mon-
itoring in pharmaceutical and serum samples. The designed sensor in-
dicated a great improvement in voltammetric response. This new sensor
showed reproducible behavior and high level stability during the ex-
periments. So, it makes it predominantly appropriate for the analytical
determination of T4 in the real samples without any serious side
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Chemical Physics Letters 716 (2019) 177–185
interferences.
2. Experimental
2.1. Chemicals
Levothyroxine as a pharmaceutical grade material (purity > 99.7%)
was purchased from the Aburaihan Pharmaceutical Company Tehran,
Iran. The pure MWCNTs with no functional groups were bought from
Research Institute of Petroleum Industry (RIPI) Tehran, Iran. The
pharmaceutical sample, levothyroxine tablet (0.1 mg) Iran Hormone
pharmaceutical Company, Iran was obtained from local drug stores.
HCl, H3PO4, H2SO4, HNO3, NaH (80%), ethylenediamine (EDA), acet-
onitrile, HAuCl4, diisopropylethylamine (DIPEA), cyanuric chloride
(CC), N, N-dicyclohexylcarbodiimide (DCC), Tetrahydrofuran (THF)
and phosphate salts were obtained from Merck, Fluka or Sigma-Aldrich.
A stock solution of T4 substance was prepared daily in 0.02 M ethanolic
Hcl solution and then the solution was stored in a refrigerator at 4 °C
darkness. Phosphate buffered solutions (PBS) at various pH values also
were prepared by H3PO4, KH2PO4 and K2HPO4.
2.2. Apparatus
A potentiostat/golvanostat (Autolab PGSTAT 12) was utilized for
record of cyclic and differential pulse voltammograms (DPVs). The
electrochemical cell consisted of a glassy carbon electrode (unmodified
or modified with MWCNTs/CC-SH/Au nanocomposite), as working
electrode, saturated Ag/AgCl and platinum wire were used as reference
and auxiliary electrode. A Metrohm pH-meter (model 827 pH Lab) was
applied for pH measurements. Nanocomposite were characterized using
Fourier transform infrared (FT-IR) spectroscopy (Shimadzu, IR Prestige
21), scanning electron microscopy (SEM) (Holland Philips XL30), and
X-ray powder diffraction (XRD) (Holland Philips PW-1840).
2.3. Synthesis of MWCNTs/CC-SH/Au nanocomposite
2.3.1. Preparation of amino-MWCNTs
First MWCNTs were added to 50 mL HCl (10%). Next, following
sonication for 15 min, the solution was stirred for 24 h to obtain pur-
ification of MWCNTs through removing the metal ions and other im-
purities adsorbed on it. Then the purified MWCNTs were precipitated
and separated utilizing 9000 rpm centrifuging. After that, 1000 mg of
purified MWCNTs was suspended with vigorous stirring in 400 mL of
concentrated HNO3 and refluxed for 12 h. The product (MWCNTs-
COOH) was separated by 9000 rpm centrifuging, rinsed with deionized
water to make it neutral and then dried at 80 °C for 12 h under a va-
cuum. In the next stage, 1000 mg of MWCNTs-COOH was added to
50 mL of EDA. The DCC was added to the solution after heating and
stirring for 5 min and refluxed for 48 h. Then the obtained MWCNTs-
EDA was separated by a centrifuge, rinsed with ethanol and dried in an
oven for 8 h at 80 °C.
2.3.2. Preparation of MWCNTs/CC-SH
Initially, 100 mL of anhydrous THF, 0.5 g of MWCNTs-EDA and
0.2 mL of DIPEA were added to a 250 mL of round-bottom flask. Then,
0.23 g (1.5 mmol) of cyanoric chloride (CC) was added in 0 °C. After 3 h
of stirring, the solution was decanted and washed with 2 × 25 of fresh
THF and then decanted. 100 mL of acetonitrile and 0.5 mL of DIPEA
were then added to the residue. 0.31 g of 2-mercaptoethanol (4 mmol)
was added to the mixture, stirred for 3 h in room temperature and re-
fluxed for 12 h. The solid products were filtered, washed with deionized
water and acetone sequentially and dried at 50 °C for 12 h, following
completion of the reaction. The clung 2-mercaptoethanol on triazine
functionalized MWCNTs (MWCNTs/CC-SH) was obtained through this
simple procedure.
S. Lotfi, H. Veisi Chemical Physics Letters 716 (2019) 177–185

OH HO
O O
O OH
HNO3 EDA, DCC
HO O
Reflux Reflux

Cl N Cl Cl N Cl

N N N N

H2N NH2 HN NH
Cl
HN NH HN NH
O O N N O O
H2N NH2
O HN O HN
Cl N Cl
N O N
H H O
DIPEA, THF, 0 C

HO
SH
CH3CN,DIPEA
Reflux
SH HS SH HS SH HS SH HS

Au Au
O N O O N O O N O O N O

N N N N N N N N

HN NH HN
Au NH

HN NH Au HN NH
Au
O O O Au O
O HN O HN
N O N Au Au O
H HAuCl4 H
NaBH4

Scheme 1. Stepwise preparation process of MWCNTs/CC-SH/Au nanocomposite.

2.3.3. Preparation of the MWCNTs/CC-SH/Au
The MWCNTs/CC-SH (500 mg) was dispersed in H2O (100 mL) in an
ultrasonic bath for 30 min. Next, the solution of HAuCl4 (0.012 mmol,
4.8 mg) was added. Then, aqueous solution of NaBH4 (0.3 mmol,
11.3 mg in 1 mL water) was added slowly and the mixture was stirred
for 24 h at room temperature. Finally, the solid was separated with
centrifuge, rinsed with water (2 × 10 mL) and ethanol (2 × 10 mL) and
dried in an oven at 60 °C. Scheme 1, represents the synthetic trend of
MWCNTs/CC-SH/Au.
2.4. Preparation of modified electrode (MWCNTs/CC-SH/Au/GCE)
To support MWCNTs/CC-SH/Au nanocomposite powder on glassy
carbon electrode, following polishing the glassy carbon electrode with
emery paper, it was polished with an alumina slurry, then rinsed several
times with double distilled water. Next the polished electrode was so-
nicated with water and ethanol and dried at room temperature. After
cleaning and drying, nanocomposite suspension was formed by sus-
pending 1 mg of the MWCNTs/CC-SH/Au nanocomposite in 1 mL of
Dimethylformamide (DMF) solvent under sonication for 30 min. Then,
6 μL of the homogeneously dispersed DMF– nanocomposite solution
was cast onto the surface of the cleaned GCE to make the MWCNTs/CC-
SH/Au/GCE. The fabricated electrode was dried in ambient tempera-
ture and rinsed with distilled water.
However, the best response for the current was achieved in the case
of 6 μL MWCNTs/CC-SH/Au utilization. Therefore, 6 μL of MWCNTs
were utilized for the electrode modification.
2.5. Preparation of human serum
The blood serum samples were achieved based on the procedure
that was described in details in earlier work [38].
2.6. Preparation of pharmaceutical samples
Ten tablets of levothyroxine (0.1 mg) were finely pulverized in a
mortar with pestle. An accurately-weighed portion of the powders,
equivalent to 1 × 10−5 M of T4, was placed into a 50 mL beaker and
extracted with 10 mL of 0.02 M HCl in ethanol. Following sonicating
the content of beaker for 10 min and filtering the resulting solution, the
clear filtrate was collected in 25 mL volumetric flask and stored in the
fridge for further uses.
2.7. Analytical procedure
Electrochemical measurements on MWCNTs/CC-SH/Au/GCE were
performed in voltammetric cell with 10 mL phosphate buffer solution
pH 7.2. DPVs were recorded with an amplitude of 50 mV, pulse width of
50 s and scan rate of 100 mV s−1. Then, under optimal conditions, and
with successive addition of appropriate concentration of T4 to the
voltammetric cell, their voltammograms were recorded and the oxida-
tion peak net current was used for calibration curve.

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S. Lotfi, H. Veisi Chemical Physics Letters 716 (2019) 177–185

Fig. 1. (I) FTIR spectra of (a) MWCNTs, (b) MWCNTs-COOH, (c) MWCNTs/CC-SH, and (d) MWCNTs/CC-SH/Au. (II) Raman spectra of the (a) MWCNTs, and (b)
MWCNTs/CC-SH.

The modified electrode was washed with double distilled water
following each experiment.
3. Results and discussion
3.1. Characterization of the MWCNTs/CC-SH/Au nanocomposite
Fourier transform infrared (FTIR) spectroscopy was used to confirm
the exterior structure of the materials. The FTIR spectra collected are
seen in Fig. 1 part I for (a) MWCNTs, (b) MWCNTs-COOH, (c)
MWCNTs/CC-SH, and (d) MWCNTs/CC-SH/Au. As it can be seen in the
curve b rather than a, the appearance absorption bands at 1705 cm−1
was credited to the CO widening of the carboxylic acid groups on the
MWCNTs during oxidation. The absorption band in curve c at
1664 cm−1 is allocated to the carbonyl stretching of the amide groups
(eCONHe), while the bands at around 2950 cm−1 were ascribed to the
bending vibration of CH2. The bands at approximately 3400 cm−1 were
attributed to the NH stretching vibrations, which reinforces the

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S. Lotfi, H. Veisi
successful bonding of the EDA to carboxyl groups. Additionally, in
curve c, the appearance peak at ≈1550–1650 cm−1 and ≈1200 cm−1
can be ascribed to the C]N and CeN groups in the triazine ring re-
spectively [47]. The band within the spectral region of 1221 cm−1 can
be attributed to the side-chain in plane CeNeC and NeCeN bending of
triazine ring [48]. The new weak peak at ≈2688 cm−1 in curve c may
be attributed to the SeH bonded vibration. These outcomes confirmed
that the mercapto groups were bonded to triazine rings on the exterior
of MWCNTs. IR scanning revealed paralleled patterns in the MWCNTs/
CC-SH and MWCNTs/CC-SH/Au, which showed that the structure of
CNTs was preserved through the deposition process of Au NPs. The
vanishing of SeH and NeH frequency at curve d demonstrated the
efficacious construction of a metal-ligand bond on the exterior of al-
tered nanotubes.
Another method for assessment of functional reaction is Raman
spectroscopy. Fig. 1, part II shows the Raman spectra of the samples.
Two main peaks in the Raman spectra appeared in the samples at 1332
and 1594 cm−1 which are known as D and G bonds respectively. D band
is related to disordered carbon atoms of MWCNTs corresponding to sp3
hybridized and G band shows the sp2-hybridized of carbon atoms in the
graphene sheets. Area ratio of the D to G bands (ID/IG) can be used to
assess the amount of defects in nano particles structure. ID/IG ratio was
increased for MWCNTs/CC-SH (ID/IG = 1.41) which approves the
successful conversion of MWCNTs to MWCNTs/CC-SH. In the absence
of amorphous carbon, the increase of ID is related to increase of carbon
containing sp3 hybridized and implies to successful functional reaction.
The morphology of the MWCNTs/CC-SH/Au was characterized by
field emission scanning electron microscopy (FE-SEM) in Fig. 2A. It is
observed that in the image 2A, the surface of CNTs was uniformly
coated thus clearly indicative of CC-SH units attached to MWCNTs.
Fig. 2. (A) FE-SEM image of MWCNTs/CC-SH/Au, (B) TEM images of MWCNTs/CC-SH/Au and (C) Energy-dispersive X-ray spectroscopy (EDS) of the MWCNTs/CC-
SH/Au.
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Chemical Physics Letters 716 (2019) 177–185
Also, the decorated Au nanoparticles on the MWCNTs/CC-SH surface
were distinguishable. To observe the morphology and distribution of Au
NPs supported on mercaptoethanol bonded to melamine modified
multi-walled carbon nanotubes, transmission electron microscopy
(TEM) investigations were performed. The presence of Au NPs de-
posited on f-CNTs was clearly distinguishable as dark spots in Fig. 2B.
From Fig. 2B, we can observe that Au was dispersed on the surface of
modified MWCNTs. The findings showed that thio groups play a key
role in enhancing the dispersibility of Au NPs. In Fig. 2C, energy dis-
persion X-ray spectroscopy (EDX) was reported. The characteristic peak
allocated to gold in EDX spectrum as well as the effective formation of
Au species were detected. Other prevailing elements exposed by the
EDX analysis was comprised of carbon, sulfur, and nitrogen which
proved that the mercapto-malamine groups existed on the surface.
The wavelength-dispersive X-ray spectroscopy (WDX)-coupled
quantified FE-SEM plotting of the sample was also examined (Fig. 3).
The WDX can provide qualitative data regarding the dispersal of var-
ious chemical elements in the catalytic matrix. Observing the compo-
sitional maps C, N, S, and Au, the presence of Au NPs with proper
dispersion is obviously noticeable in the composite.
3.2. Electrochemical behavior of T4 at MWCNTs/CC-SH/Au/GCE
In the nonexistence and existence of T4, the electrochemical beha-
viors of the bear GCE and MWCNTs/CC-SH/Au nanocomposite film
which coated GCE were studied in 0.2 mol/L PBS at physiological pH
(7.2) using cyclic voltammetry. Fig. 4A depicts the cyclic voltammo-
grams of bear GCE (curve a) and MWCNTs/CC-SH/Au/GCE (curve c) in
the blank solution and also the cyclic voltammetric responses for the
electrochemical oxidation of 1 µM T4 at bare GCE (curve b), MWCNTs/
S. Lotfi, H. Veisi Chemical Physics Letters 716 (2019) 177–185

Fig. 3. FESEM image of MWCNTs/CC-SH/Au and elemental maps of C, N, S, and Au atoms.

CC-SH/GCE (curve d) and MWCNTs/CC-SH/Au/GCE (curve e) at a scan
rate of 100 mV s−1. In the absence of T4, no peaks were seen for bear
GCE (curve a), but at MWCNTs/CC-SH/Au/GCE (curve c) a couple of
redox peaks (A1 and C1) were observed at 0.18 and 0.07 V in blank
solution. This is the result of the electrochemical responses of the CNTs
at the surface of GCE [30,33,49]. A well-defined oxidation peak (A2)
was observed at a potential value of 0.54 V vs. Ag/AgCl subsequent
1 µM of T4, which is associated with the electro-oxidation of the
phennolic hydroxyl group on the thyroxine molecule. No cathodic peak
appeared in the reverse scan, suggesting that the oxidation of T4 at the
surface of MWCNTs/CC-SH/Au/GCE is irreversible. The same behavior
had been reported for electrooxidation of T4 in previous work
[1,28,33].
In order to gain a further insight into the proposal that peaks A1 and
C1 are responsible for electrochemical responses of the CNTs, the
electrochemical responses of T4 were studied with different con-
centrations of T4 at MWCNTs/CC-SH/Au/GCE using cyclic voltam-
metry technique. Fig. 4B shows cyclic voltammograms of different
concentration of T4. As shown, during the variation of concentration of
T4, the peak current of A1 and C1 remained almost stable but the height
of peak A2 increased. This data confirmed the redox peaks A1 and C1 at
0.18 and 0.07 V at MWCNTs/CC-SH/Au/GCE, are due to electro-
chemical responses of the CNTs at surface of GCE.
The recorded cyclic voltammograms (Fig. 4A) of three different
electrodes were compared for sensitivity and catalytic activity of the
modifier films. As it can be seen in Fig. 4A, a relatively weak peak was
obtained on the bare GCE (curve b). MWCNTs/CC-SH/Au nano-
composite film which coated GCE (Fig. 4A curve e) showed a higher
current density compared to MWCNTs/CC-SH modified electrode
(Fig. 4A curve d) due to the high surface area, conductivity and cata-
lytic effect of Au nanoparticles. Thus, the electro-oxidation of T4 was
investigated at the resulted electrode (MWCNTs/CC-SH/Au/GCE).
Subsequently, the effect of pH on the cyclic voltammetry response of
A2 to the electro -oxidation of a 1 μM T4 solution in PBS at MWCNTs/
CC-SH/Au/GCE surface was investigated over the pH range of 1–10.
Fig. 5A depicts that oxidation peak current and the peak potential of A2
are dependent on the pH medium. As the pH increased, the oxidation
peak potential (Ep) of T4 shifted toward negative value potentials. This
result implies the contribution of protons in the oxidation process. A
potential–pH diagram is constructed for T4 on MWCNTs/CC-SH/Au/
GCE by plotting the Ep values as a function of pH (Fig. 5B). The Ep–pH
diagram comprises two linear segments with different equations and
slopes around pH 7.4.
In pHs < 7.4

Fig. 4. (A) Cyclic voltammograms of different electrodes in PBS with pH 7.2 at scan rate 100 mV s−1; the bare GCE (a, b), MWCNTs/CC-SH/Au/GCE (c, e) and
MWCNTs/CC-SH/GCE (d). Electrodes were exposed for to a PBS (a and c) and a 1 µM T4 solution (b, d and e). Inset: a part of curves a – e in different scale and (B)
Cyclic voltammograms of T4 in PBS (pH 7.2, C = 0.2 M) at modified electrode at various concentrations. Concentrations from a to e are: 0.5, 0.6, 0.8 and 1.0 µM
respectively. Scan rate: 100 mV s−1.

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S. Lotfi, H. Veisi Chemical Physics Letters 716 (2019) 177–185

Fig. 5. (A) Cyclic voltammograms of PBS containing 1 µM T4 with different pH at the surface of MWCNTs/CC-SH/Au/GCE. (B) Plot of the anodic peak potential (A2)
vs. pH of the solution.

52mV respectively, which, in itself indicates the sensitivity of the re-

E pA = 0.8643 − 0.0521pH or slope = −
pH
In pHs > 7.4
20mV
E pA = 0.63 − 0.02pH or slope = −
pH
Based on the above-mentioned slope, suggesting that, in the pH
range of 1–7.4, the number of protons involved in the oxidation of T4 is
equal to that of electrons transferred and the reaction occurring is a
two-electron, two-proton process. When the pH is higher than pH 7.4,
the hydroxyl group in T4 molecule begins to dissociate and thereby
hardly becomes oxidized in basic solution. Similar results for electro-
chemical oxidation process of T4, had been reported previously
[14,27,28]. However, the maximum current was achieved at pH 7, but
considering the importance of physiological medium, pH 7.2 was
chosen for determination of T4.
3.3. Analytical application
To outline calibration curves, the achieved optimal conditions and
parameters on the response of the MWCNTs/CC-SH/Au/GCE sensor
were utilized. The DPVs for different concentrations of T4 from 10 to
120 nM in PBS at pH 7.2 were found under the selected optimum
conditions at MWCNTs/CC-SH/Au/GCE, as shown in Fig. 6A. The plot
of oxidation peak current (IpA2) against concentration of T4 (Fig. 6B)
shows a linear dynamic range of 10 to 120 nM with a regression
equation of Ip (µA) = 0.017 CT4 (nM) + 0.787, (R2 = 0.989). The limits
of detection (LOD) and quantification (LOQ) were signified as
LOD = KSb/m, where k = 3 for LOD and 10 for LOQ, Sb the standard
deviation of the peaks current of the blank (n = 10) and m the slope of
the calibration curve, which were found to be 2.84 and 9.5 nM,
commended method. Furthermore, the analytical characteristics of the
current sensor were compared to other electrochemical techniques,
which are published for specifying the amount of T4, the results of
which are presented in Table 1. The comparative data indicated su-
periority of the fabricated sensor over some earlier published electro-
chemical techniques. This can be associated with the immobilization of
MWCNTs/CC-SH/Au nanocomposite at the surface of GCE with large
surface area, good conductivity and good electrocatalytic effect.
3.4. Stability, repeatability and reproducibility of the MWCNTs/CC-SH/
Au/GCE
To study the stability of the proposed sensor for the determination
of T4, DPVs of 0.1 μM of T4 solutions were recorded over a period of
30 days. The response current of the sensor was 97.6% of its original
value after this period, indicating a good stability of the fabricated
sensor in the determination of T4. To study the repeatability, five de-
terminations of 0.1 µM T4 were performed utilizing the MWCNTs/CC-
SH/Au/GCE. The relative standard deviation (RSD) value was obtained
as 2.8%, indicating that the MWCNTs/CC-SH/Au/GCE has outstanding
repeatability. Furthermore, the reproducibility of the modified elec-
trode was assessed by preparing five individual electrodes from the
same batch of modifier (6 µL), at the same procedure independently,
which were constructed in five consecutive days. These electrodes were
used for the determination of a 0.1 μM T4 solution, under the optimized
conditions. The relative standard deviation for the responses between
electrodes was obtained less than 3.5%.

Fig. 6. (A) DPVs obtained for various concentrations of T4. (B) Calibration curve of T4. From down to up correspond 10 to 120 nM.

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Table 1
Comparison of the analytical parameters of the proposed electrode with the various electrochemical methods for the determination of T4.
Electrode Method pH Detection limit (nM) Linear range (µM) Real samples Reference

a b
CPE LSV 4 6.5 0.2–9 Tablet [50]
EPPG electrodec CV 1 3 0.01–10 Serum [4]
CPE CV 13 80 0.2–9 Aqueous solution [30]
Hg(Ag)FEd DPAdSVe 2.3 18 0.025–4 Tablet [26]
SPCEf DPV 7.2 3 0.005–0.035 Tablet [1]
MWNTs/GCE DPV 1 6.4 0.02–0.5 Human serum [35]
SWNT/GCE LSV 1 20 0.1–7 Aqueous solution [32]
CPE CV 1 2500 25–100 Tablet [29]
MWCNTs/CC-SH/Au/GCE DPV 7.2 2.84 0.01–0.12 Tablet human serum This work

a
Carbon Paste Electrode.
b
Linear sweep voltammetry.
c
Edge plane pyrolytic graphite.
d
Mercury film silver based electrode.
e
Differential pulse adsorptive stripping voltammetry.
f
Screen-printed carbon electrode.

Table 2
Determination results of T4 in real samples.
sample DPV method HPLC method

a
Added (nM) Founded (nM) Relative recovery% RSD% Added (nM) Founded (nM)b Relative recovery% RSD%

Tablet 0.00 19.32 – 3.2 0.00 19.43 – 2.3

40 39.46 98.2 2.1 40 39.71 99.3 1.9
50 50.21 100.4 2.9 50 50.16 100.3 2.5
Human serum 20 19.72 98.6 2.7
40 40.24 100.6 2.1
60 60.18 100.3 2.4

a
Average of six replicate measurements (rounded).
b
Average of three replicate measurements (rounded).

3.5. Interference study 4. Conclusions

The selectivity of the designed sensor were studied under the op-
timum conditions by affecting some organic and ions substances, which
may interfere with the detection of T4. By adding different ions and
biological substances to PBS (pH 7.2) in the presence of 0.1 μM T4,
possible interference was evaluated and the peak heights obtained were
measured. The results indicated that in the presence of the common
ions such as Zn2+, Mg2+, Na+, K+, Cu2+, Fe2+,Cl− did not interfere
with T4 determination. Moreover, examination interference of biolo-
gical samples such as citric acid, ascorbic acid, uric acid, glucose, gly-
cine, l-cysteine, Vitamin B6, and Vitamin C with adding 100-fold excess
concentration of interference for the detection of T4 was testified and it
was found that these substances had no effective interference for the
determination of T4 (current change ± 5%), by DPV method. These
results indicate that this sensor has good selectivity and is suitable to-
wards the measurement of T4 in pharmaceutical and biological sam-
ples.
3.6. Real sample analysis
To prove the applicability of the recommended sensor it has been
applied for determination of T4 in tablet and human blood serum
samples using a standard addition method. Table 2 represents the ob-
tained results. In order to test the precision of the recommended elec-
trochemical procedure, the results obtained by MWCNTs/CC-SH/Au/
GCE were compared with those by HPLC technique in Table 2. Ac-
cording to Table 2, the recovery values using voltammetric method
were from 98.2 to 100.6% and the RSD of the proposed method was less
than 3.2%, which implies to be comparable with those obtained by
HPLC. This means that the determination of T4 using the MWCNTs/CC-
SH/Au/GCE is very effective and can be successfully employed to the
measurement of T4 in real samples with various matrices.
A nanocomposite based on gold nanoparticles supported on the
mercaptoethanol which was bonded to melamine modified multi-
walled carbon nanotubes (MWCNTs/CC-SH/Au) was synthesized,
characterized and utilized as a sensitive sensor for simple and fast de-
termination of T4 using glassy carbon electrode. Being easy to prepare,
long-term stability and suitable reproducibility as well as being cheap
are some important advantages of fabricated sensor. This is a very in-
teresting amplification of anodic peak current density observed in this
electrochemical process, compared to unmodified electrode, which can
lead to an increased sensitivity of the analytical method. It should be
stated that the enhancement of the anodic peak current of T4, following
the modification of GCE with MWCNTs/CC-SH/Au nanocomposite in-
dicates the role of modifier on facilitating the electron transfer kinetics,
increasing surface area and electro-catalytic activity. The analytical
procedure was appropriate for T4 analysis with excellent electro-
analytical performance, having satisfactory linearity, sensitivity, re-
peatability and low detection limit. The proposed modified electrode
was effectively utilized for determining T4 in tablet and human blood
serum samples, which was validated with HPLC technique.
Acknowledgments
This study was conducted under financial support of Payame Nour
University (Grant No. 7/49103).
References
[1] A. Das, M.V. Sangaranarayanan, Electroanalytical sensor based on unmodified
screen-printed carbon electrode for the determination of levo-thyroxine,
Electroanalysis 27 (2015) 360–367.
[2] P.A. Insel, L.L. Brunton, J.S. Lazo, K.L. Parker, Goodman & Gilman’s, The

184
S. Lotfi, H. Veisi
Pharmacological Basis of Therapeutics, United States of America, 2006.
[3] E. Mutschler, G. Gesslinger, H.K. Kroemer, P. Ruth, M. Schafer-Korting,
Farmakologia i toksykologia, Med Pharm, Wroclaw, Poland, 2010.
[4] M. Khafaji, S. Shahrokhian, M. Ghalkhania, Electrochemistry of levo-thyroxin on
edge-plane pyrolytic graphite electrode: application to sensitive analytical de-
terminations, Electroanalysis 23 (8) (2011) 1875–1880.
[5] L.H. Fish, H.L. Schwartz, J. Cavanaugh, et al., Replacement dose, metabolism, and
bioavailability of levothyroxine in the treatment of hypothyroidism, N Eng. J. Med.
316 (1987) 764–770.
[6] S.J. Mandel, G.A. Brent, P.R. Larsen, Levothyroxine therapy in patients with thyroid
disease, Ann. Intern. Med. 119 (1993) 492–502.
[7] M.D. Judy Jin, T. Matthew, M.D. Allemang, R. Christopher, M.D. McHenry,
Levothyroxine replacement dosage determination after thyroidectomy, Am. J.
Surgery 205 (2013) 360–364.
[8] H.G. Gika, V.F. Samanidou, I.N. Papadoyannis, Development of a validated HPLC
method for the determination of iodotyrosines and iodothyronines in pharmaceu-
ticals and biological samples using solid phase extraction, J. Chromatogr. B 814
(2005) 163–172.
[9] J. Cao, H. Wang, Y. Liu, Determination of L-thyroxine in pharmaceutical prepara-
tions by flow injection analysis with chemiluminescence detection based on the
enhancement of the luminol–KMnO4 reaction in a micellar medium, Spectrochim.
Acta Part A: Mol. Biomol. Spectroscopy 140 (2015) 162–165.
[10] E. Gok, S. Ates, Determination of thyroxine hormone by luminol chemilumines-
cence, Anal. Chim. Acta 505 (2004) 125–127.
[11] L.M. Thienpont, V.I. De Brabandere, O. Stoeckl, Development of a new method for
the determination of thyroxine in serum based on isotope dilution gas chromato-
graphy mass spectrometry, Biol. Mass Spectrom. 23 (1994) 475–482.
[12] S.J. Soldin, N. Soukhova, N. Janicic, J. Jonklaas, O.P. Soldin, The measurement of
free thyroxine by isotope dilution tandem mass spectrometry, Clin. Chim. Acta 358
(2005) 113–118.
[13] D. Schmalzing, L.B. Koutny, T.A. Taylor, W. Nashabeh, M. Fuchs, Immunoassay for
thyroxine (T4) in serum using capillary electrophoresis and micromachined devices,
J. Chromatogr. B, Biomed. Sci. Appl. 697 (1997) 175–180.
[14] H. Zhihui, J. Wenrui, Capillary electrophoretic enzyme immunoassay with elec-
trochemical detection for thyroxine, Anal. Biochem. 313 (2003) 34–40.
[15] E. Taimela, V. Kairisto, P. Koskinen, A. Leino, K. Irjala, Reference intervals for
serum thyrotropin, free thyroxine and free triiodothyronine in healthy adults in
Finland, measured by an immunoautomate based on time-resolved fluorescence
(AutoDELFIA), Eur. J. Clin. Chem. Clin. Biochem. 35 (1997) 889–890.
[16] F. Tagliaro, M. Camilot, R. Valentini, F. Mengarda, F. Antoniazzi, L. Tato,
Determination of thyroxine in the hair of newborns by radioimmunoassay with
high-performance liquid chromatographic confirmation, J. Chromatogr. B 716
(1998) 77–82.
[17] H. Silvaieh, R. Wintersteiger, M.G. Schmid, O. Hofstetter, V. Schurig, G. Gbitz,
Enantioselective sequential-injection chemiluminescence immunoassays for 3,3′,5
triiodothyronine (T3) and thyroxine (T4), Anal. Chim. Acta 463 (2002) 5–14.
[18] C.D. Karapitta, A. Xenakis, A. Papadimitriou, T.G. Sotiroudis, A new homogeneous
enzyme immunoassay for thyroxine using glycogen phosphorylase b-thyroxine
conjugates, Clin. Chim. Acta 308 (2001) 99–106.
[19] M.R. Oates, W. Clarke, A. Zimlich, D.S. Hage, Optimization and development of a
high-performance liquid chromatography-based one-site immunometric assay with
chemiluminescence detection, Anal. Chim. Acta 470 (2002) 37–50.
[20] S. Georgiou, I. Christofidis, Radioimmunoassay of free thyroxine (T4) using 125I-
labeled T4-IgG complex with very large molecular weight, Clin. Chim. Acta 244
(1996) 209–220.
[21] M.I. Becker, J.E. Aguayo, A. Jamett, F. Juica, A. Yudelevich, A. Foradori,
A.E.D. Ioannes, An alternative ELISA for T4 determination based on idiotype anti-
idiotype interaction and a latex method for anti-idiotype monoclonal antibody se-
lection, J. Immunol. Methods 192 (1996) 73–85.
[22] S. Yong, Y. Chen, T.K. Lee, H.K. Lee, Determination of total thyroxine in human
serum by hollow fiber liquid-phase microextraction and liquid chromato-
graphy–tandem mass spectrometry, Talanta 126 (2014) 163–169.
[23] J.W. Collier, R.B. Shahb, A.R. Bryant, M.J. Habib, M.A. Khanb, P.J. Faustino,
Development and application of a validated HPLC method for the analysis of dis-
solution samples of levothyroxine sodium drug products, J. Pharma. Biomed. Anal.
54 (2011) 433–438.
[24] J.F. Brower, D.Y. Toler, J.C. Reepmeyer, Determination of sodium levothyroxine in
bulk, tablet, and injection formulations by high-performance liquid chromato-
graphy, J. Pharma. Scien. 73 (1984) 1315–1317.
[25] A. Alimadadi, F. Faridbod, B. Larijani, R. Heshmat, B. Akbari-Adergani, Analysis of
levothyroxine in pharmaceutical formulation by a novel levothyroxine potentio-
metric membrane sensor, Anal. Bioanal. Electrochem. 6 (2014) 355–366.
[26] J. Smajdor, R. Piech, M. Rumin, B. Paczosa-Bator, Z. Smajdor, High sensitive vol-
tammetric levothyroxine sodium determination on renewable mercury film silver
based electrode, J. Electrochem. Society 163 (2016) 605–609.
[27] S. Chitravathi, B.E. Kumara Swamy, Umesh Chandra, G.P. Mamatha, B.S. Sherigara,
185
Chemical Physics Letters 716 (2019) 177–185
Electrocatalytic oxidation of sodium levothyroxine with phenyl hydrazine as a
mediator at carbon paste electrode: A cyclic voltammetric study, J. Electroanal.
Chem. 645 (2010) 10–15.
[28] Q. He, X. Dang, C. Hu, S. Hu, The effect of cetyltrimethyl ammonium bromide on
the electrochemical determination of thyroxine, Colloids Surf. B: Biointerfaces 35
(2004) 93–98.
[29] S. Chitravathi, B.E. Kumaraswamy, E. Niranjana, Umesh Chandra, G.P. Mamatha,
B.S. Sherigara, Electrochemical studies of sodium levothyroxine at surfactant
modified carbon paste electrode, Int. J. Electrochem. Sci. 4 (2009) 223–237.
[30] F. Wang, J. Fei, S. Hu, The influence of cetyltrimethyl ammonium bromide on
electrochemical properties of thyroxine reduction at carbon nanotubes modified
electrode, Colloids Surf. B: Biointerfaces 39 (2004) 95–101.
[31] A. Afkhami, A. Bahiraei, T. Madrakian, Application of nickel zinc ferrite/graphene
nanocomposite as a modifier for fabrication of a sensitive electrochemical sensor for
determination of omeprazole in real samples, J. Colloid Interface Sci. 495
(2017) 1–8.
[32] M.H. Mashhadizadeh, E. Afshar, Electrochemical investigation of clozapine at TiO2
nanoparticles modified carbon paste electrode and simultaneous adsorptive vol-
tammetric determination of two antipsychotic drugs, Electrochim. Acta 87 (2013)
816–823.
[33] K. Wu, X. Ji, J. Fei, S. Hu, The fabrication of a carbon nanotube film on a glassy
carbon electrode and its application to determining thyroxine, Nanotechnology 15
(2004) 287–291.
[34] E. Haghshenas, T. Madrakian, A. Afkhami, A novel electrochemical sensor based on
magneto Au nanoparticles/carbon paste electrode for voltammetric determination
of acetaminophen in real samples, Mater. Sci. Eng., C 57 (2015) 205–214.
[35] Ya-Ping Hsiao, Wan-Yu Su, Jin-Ru Cheng, Shu-Hua Cheng, Electrochemical de-
termination of cysteine based on conducting polymers/gold nanoparticles hybrid
nanocomposites, Electrochim. Acta 56 (2011) 6887–6895.
[36] R. Jain, Dhanjai, Nanographene based sensor for antiarrhythmic agent quinidine
insolubilized system, Colloids Surf. B Biointerfaces 105 (2013) 278–283.
[37] M. Baghayeri, A. Amiri, S. Farhadi, Development of non-enzymatic glucose sensor
based on efficient loading Ag nanoparticles on functionalized carbon nanotubes,
Sens. Actuators, B 225 (2016) 354–362.
[38] E. Tammari, A. Nezhadali, Sh. Lotfi, H. Veisi, Fabrication of an electrochemical
sensor based on magneticnanocomposite Fe3O4/_-alanine/Pd modified glassy
carbon electrodefor determination of nanomolar level of clozapine in biological
modeland pharmaceutical samples, Sens. Actuators, B 241 (2017) 879–886.
[39] M. Baghayeri, H. Veisi, Fabrication of a facile electrochemical biosensor for hy-
drogenperoxide using efficient catalysis of hemoglobin on the porous Pd@Fe3O4-
MWCNTnanocomposite, Biosens. Bioelectron 74 (2015) 190–198.
[40] Ke-Jing Huang, Xue Liu, Wan-Zhen Xie, Hong-Xia Yuan, Electrochemical behavior
and voltammetric determination of norfloxacin at glassy carbon electrode modified
with multi walled carbon nanotubes/Nafion, Colloids Surf. B: Biointerfaces 64
(2008) 269–274.
[41] E. Katz, I. Willner, J. Wang, Electroanalytical and bioelectroanalytical systems
based on metal and semiconductor nanoparticles, Electroanalysis 16 (2004) 19–44.
[42] M. Ibrahim, H. Ibrahim, N.R. Almandil, A.N. Kawde, Gold nanoparticles/f-MWCNT
nanocomposites modified glassy carbon paste electrode as a novel voltammetric
sensor for the determination of cyproterone acetate in pharmaceutical and human
body fluids, Sens. Actuators, B. l 274 (2018) 123–132.
[43] S. Shahrokhian, R. Salimian, Sh. Rastgar, Pd–Au nanoparticle decorated carbon
nanotube as a sensing layer on the surface of glassy carbon electrode for electro-
chemical determination of ceftazidime, Mater. Sci. Eng., C 34 (2014) 318–325.
[44] A. Afkhami, A. Bahiraei, T. Madrakian, Gold nanoparticles/multi-walled carbon
nanotubes modified glassy carbon electrode as a sensitive voltammetric sensor for
the determination of diclofenac sodium, Mater. Sci. Eng., C 59 (2016) 168–176.
[45] T. Mpanza, M.I. Sabela, S.S. Mathenjwa, S. Kanchi, K. Bisetty, Electrochemical
determination of capsaicin and silymarin using a glassy carbon electrode modified
by gold nanoparticle decorated multiwalled carbon nanotubes, Anal. Lett. 47
(2014) 2813–2828.
[46] M.B. Gholivand, E. Ahmadi, M. Haseli, A novel voltammetric sensor for nevirapine,
1 based on modified graphite electrode by MWCNs/poly(methylene blue)/gold
nanoparticle, Anal. Biochem. 527 (2017) 4–12.
[47] K. Mori, Y. Sasaki, S. Sai, S. Kaneda, H. Hirahara, Y. Oishi, Electrochemical poly-
merization of 2-(dioctylamino)-1, 3, 5-triazine-4, 6-dithiol on iron plates, Langmuir
11 (1995) 1431–1434.
[48] P.J. Larkin, M.P. Makowski, N.B. Colthup, L.A. Flood, Vibrational analysis of some
important group frequencies of melamine derivatives containing methoxymethyl,
and carbamate substituents: mechanical coupling of substituent vibrations with
triazine ring modes, Vib. Spec. 17 (1998) 53–72.
[49] P.J. Britto, K.S.V. Santhanam, V. Alonso, A. Rubio, P.M. Ajayan, Improved charge
transfer at carbon nanotube electrodes, Adv. Mater. 11 (1999) 154–157.
[50] C. Hu, Q. He, Q. Li, Sh Hu, Enhanced reduction and determination of trace thyr-
oxine at carbon paste electrod in the presence of trace cetyltrimethyl ammonium
bromide, Anal. Sci. 20 (2004) 1049–1054.