Cerium doped magnetite nanoparticles for highly sensitive detection of metronidazole via chemiluminescence assay

Article abstract of DOI:10.1016/j.saa.2020.118272

Cerium doped magnetite nanoparticle (CDM) was synthesized via a co-precipitation method and used as the co-reactant of luminol-K₃Fe(CN)₆ chemiluminescent system. The physical-chemical features of CDM were studied by XPS, XRD, HRTEM, FESEM, VSM, BET, and FTIR analyses. This simple and highly sensitive nanoprobe enabled the determination of minor concentrations of metronidazole (MNZ). Owing to the quenching efficacy of MNZ in the studied chemiluminescence system, a linear range of 3.47 × 10^?6-9.37 × 10^?5 mol/L was obtained with a limit of detection of 3.91 × 10^?7 mol/L. This biosensor was used for MNZ detection in human serum samples, which was highly efficient. The outcomes of this study give credit to the proposed biosensor to be applied for detection of MNZ in biological samples.

Full text of DOI:10.1016/j.saa.2020.118272

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 234 (2020) 118272
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Cerium doped magnetite nanoparticles for highly sensitive detection of
metronidazole via chemiluminescence assay
Yasin Orooji a,b, Mahsa Haddad Irani-nezhad c, Ramin Hassandoost c, Alireza Khataee c,d,e,
⁎
,
Shima Rahim Pouran f, Sang Woo Joo g,
⁎
a
College of Materials Science and Engineering, Nanjing Forestry University, No. 159, Longpan Road, Nanjing, 210037, Jiangsu, People's Republic of China
b
c
d
e
f
Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, Nanjing Forestry University, Nanjing 210037, China
Research Laboratory of Advanced Water and Wastewater Treatment Processes, Department of Applied Chemistry, Faculty of Chemistry, University of Tabriz, 51666-16471 Tabriz, Iran
Department of Environmental Engineering, Gebze Technical University, 41400 Gebze, Turkey
Institute of Research and Development, Duy Tan University, Da Nang 550000, Viet Nam
Department of Biology, Faculty of Science, University of Mohaghegh Ardabili, P.O. Box 179, Ardabil, Iran
School of Mechanical Engineering, Yeungnam University, Gyeongsan 712-749, South Korea
g
a r t i c l e i n f o
a b s t r a c t
Article history:
Cerium doped magnetite nanoparticle (CDM) was synthesized via a co-precipitation method and used as the co-
reactant of luminol-K3Fe(CN)6 chemiluminescent system. The physical-chemical features of CDM were studied
by XPS, XRD, HRTEM, FESEM, VSM, BET, and FTIR analyses. This simple and highly sensitive nanoprobe enabled
the determination of minor concentrations of metronidazole (MNZ). Owing to the quenching efficacy of MNZ
in the studied chemiluminescence system, a linear range of 3.47 × 10−6-9.37 × 10−5 mol/L was obtained with
a limit of detection of 3.91 × 10−7 mol/L. This biosensor was used for MNZ detection in human serum samples,
which was highly efficient. The outcomes of this study give credit to the proposed biosensor to be applied for de-
tection of MNZ in biological samples.
Received 28 January 2020
Received in revised form 14 March 2020
Accepted 15 March 2020
Available online 17 March 2020
Keywords:
Antibiotics detection
Chemiluminescence
Human plasma
Magnetic sensor
Metronidazole
© 2020 Elsevier B.V. All rights reserved.
1. Introduction
Nowadays, inspiring applications of sensors propel scientists to inte-
grate them with nanotechnology. Given that the intrinsic CL illumina-
Currently, detecting the signals of the targeted analytes such as anti-
biotics, toxins, proteins, glucose, hormones, and DNA is facilitated by
biosensors. These devices, with their unique features such as the early
diagnosis of diseases by detecting analytes and biomarkers from
human blood, urine, and saliva, are at the forefront of attention. Detect-
ing the signals of different nature such as capacitance, electric potential,
impedance, current, and/or light intensity is the basis of biosensors op-
eration [1]. Chemiluminescence (CL), a method that requires no light
source, is used to detect the target analyte based on the produced pho-
tons during some specific chemical reactions. This property makes CL
more applicable than the widely used fluorescence (FL) technique
[2,3]. The other advantages of CL over FL are being appropriate in
small-sized systems and for miniaturization, facile instrumentation
and operation, and a low limit of detection with a wide linear range
for quantifying various analytes [4].
tion of luminol and its low quantum yield can be enhanced by various
compounds such as nanoparticles, metal oxides, metal ions, and en-
zymes [5]. Hence, one of the appealing research interests of scientists
is to modify nanomaterials in order to advance some particular
physical-chemical properties of them [6]. It has been demonstrated
that the surface defects, chemical composition, crystal structure, size
and surface properties are of critical factors of engineered
nanomaterials for sensing applications [7]. Rare earth metal doped
nanomaterials have shown exceptional chemical, physical, and me-
chanical properties, which are imperative for analytical approaches [8].
Magnetite (Fe3O4, MAG) has been frequently used in several applica-
tions such as catalysis, sensor, nanofluid, and electronics, owing to its
unique properties including low toxicity, narrow bandgap, easy modifi-
cation, and superparamagnetism [9,10]. Both chemical and physical
properties of MAG nanoparticles (NPs), such as morphology, particle
size, magnetic behavior, and surface functionalization have great effects
on the enhancement of detection sensitivity [11,12]. Zhang et al. [13]
fabricated an innovative and intrinsically selective MAG-based
chemosensor for simultaneous detection of 2,4-dichlorophenoxyacetic
acid, ethoprophos, profenofos, and dylox. In their study, the effects of
⁎
Corresponding authors.
E-mail addresses: alirezakhataee@duytan.edu.vn, a_khataee@tabrizu.ac.ir (A. Khataee)
, swjoo@yu.ac.kr (S.W. Joo).
https://doi.org/10.1016/j.saa.2020.118272
1386-1425/© 2020 Elsevier B.V. All rights reserved.

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Y. Orooji et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 234 (2020) 118272
the surface modification of MAG NPs were assessed on the adjustable CL
switching. In another work, Shen et al. [14] reported the usage of the
zinc ferrite (ZnFe2O4) in electro-chemiluminescence immune-sensor
for detection of carcinoembryonic antigen. For this purpose, ZnFe2O4@
hydrochloric acid (HCl, 37%), zinc nitrate (Zn(NO3)2, 98%), urea
(CO(NH2)2, ≥98%), sodium acetate (NaCH3COO, ≥99%), acetone (OC
(CH3)2, ≥99.5%), calcium chloride (CaCl2, ≥97%), sodium sulfate
(Na2SO4, ≥97%), sodium carbonate (Na2CO3, ≥99%), pure ethanol
(C2H5OH, 95%), luminol (C8H7N3O2, 97%), and potassium ferricya-
nide (K3Fe(CN)6, 99%) were acquired from Merck (Germany). Also,
valine (C5H11NO2, ≥98%), starch ((C6H10O5)n -(H2O), ≥97%), alanine
(C3H7NO2, ≥98%), glucose (C6H12O6, ≥99.5%), and lactose
(C12H22O11, ≥99%) were purchased from Aladdin (China). All of the
chemicals used in this work were of analytical grade without further
purification and used as obtained.
Au and CdTe-graphene combined with anti-bodies to form
a
sandwich-like structure and enhanced the electrochemiluminescence
efficiency. Recent studies reported the fabrication of a number of probes
using modified MAG chemiluminescent sensors such as silanized mag-
netic graphene, oxide-molecularly imprinted polymer [15] or β–
cyclodextrins-CoFe2O4 magnetic NPs [16]. However, either their linear
range was low or the detection limit was high. These limitations moti-
vated us to modify MAG in an effort to enhance its properties for
sensor-based applications.
2.2. Apparatus
One method to increase MAG catalytic activity is to be doped with
rare earth metals such as Terbium [17], Europium [18], and Lanthanide
[19]. In this method, several factors such as the particle size, amount of
the incorporation, and valence states of metals have influential effects
on the catalytic activity of MAG NPs [20]. Moreover, the metal cation's
distribution through the octahedral and tetrahedral sites of the MAG
crystal lattice plays a big role in its catalytic activity. This is because
the octahedral sites of MAG are more catalytically active due to their ex-
position on the surface of MAG spinel crystallites [21]. Notably, the sub-
stitution or incorporation of rare earth metals changes the surface
properties of MAG such as specific surface area and surface functional
groups (e.g. hydroxyl groups) to some extent. From the earth rare
metals, cerium has been studied by many researchers because of its
Ce3+/ Ce4+ redox property [22]. The Ce3+/Ce4+ redox pair have
shown considerable synergistic effects on the catalytic activity of Fe-
bearing composites. Basically, doping of Ce ions with MAG enhances
the electron transfer process among the Ce3+/Ce4+ and Fe2+/Fe3+
redox pairs and elevates the catalytic activity of MAG via the
oxidation-based reactions [23].
The chemical structure and morphology of the synthesized nano-
particles were determined by the X-ray photoelectron spectrometer
(XPS, Thermo Scientific, K-Alpha, UK), X-ray diffraction (XRD,
PW1730, Philips, the Netherlands, under 0.15406 nm of Cu-Kα radi-
ation at current and accelerating voltage of 40 mA, 45 kV), Cs-
corrected high-resolution transmission electron microscopy
(HRTEM) images 200 kV (JEM-2200FS, JEOL, Japan, working at
200 kV), field emission scanning electron microscopy (FESEM) mi-
crographs (Tescan Mira3 microscope, Czech Republic), vibrating
sample magnetometer (VSM, Lakeshore Cryotronics, 7400 Series,
USA), Brunauer-Emmett-Teller (BET, Belsorp Mini II, Japan, deter-
mined by nitrogen adsorption/desorption isotherms at 77 K), and
Fourier transform infrared spectrometer (FTIR, Bruker Tensor 27,
Germany). Moreover, for the chromatographic analysis, an HPLC sys-
tem (Smartline 1000 Knauer, Germany) equipped with a C18 column
and UV detector was used. To study the mechanism of the action of
CDM nanoparticles, UV–Vis absorption spectra (S2000 spectropho-
tometer, WPA Lightwave, England) were recorded.
Metronidazole (MNZ) tablets have been used as an antibiotic for the
treatment of giardiasis, trichomoniasis, dental infection, amoebiasis,
and acute ulcerative gingivitis [24]. Though the explicit toxicity of met-
ronidazole has still remained an issue, some of the side effects such as
ataxia, peripheral neuropathy, and seizures have been identified after
MNZ accumulation in human body and wildlife. On the other hand,
due to its high solubility in water and non-biodegradability, the precise
detection and removal of MNZ is technically challenging [25].
In this study, a novel and sensitive CL system were developed using
cerium doped magnetite nanoparticles (CDM). It was inspected that the
Fe2.8Ce0.2O4 NPs had the highest enhancement effects than those of
Fe3O4, CeO2, and ceria mechanically doped magnetite (CMDM) nano-
particles on the luminol-K3Fe(CN)6 CL system. The CL intensity of
luminol, which was reacted with a weak oxidant, K3Fe(CN)6, in a contin-
uous flow injection was examined when undergone a catalytic reaction
by CDM nanoparticles. The fabricated sensor was used to determine
MNZ in human plasma and spiked water samples. Moreover, the effect
of the MNZ analogues was investigated on the designed CL system. Be-
sides, the possible mechanism of the proposed CL system was
investigated.
2.3. Synthesis of nanoparticles
As reported earlier [26], for the synthesis of Fe3O4, MAG, Argon
bubbling was used to deoxygenate the aqueous solution. Then, in a
three-neck flask, an acidic solution containing 0.9 mol/L of iron (II)
chloride tetrahydrate was dissolved in 1.2 mol/L of hydrochloric
acid to prepare 0.90 mol/L of Fe2+. After that, 1 mL of hydrazine
was added and pH was adjusted under 1.0 to prevent oxidation of
Fe2+ and hydroxide precipitation. In the next step, the solution was
boiled up to 100 °C and a basic solution containing sodium nitrate
(0.90 mol/L) and sodium hydroxide (4.0 mol/L) with equal volumes
was added slowly into the Fe2+ acidic solution. Then, while the me-
chanical stirring at 500 rpm for 2 h, the reaction was adjusted at 90 °C
with Argon flux passing through. After 2 h, when the reaction com-
pleted, the solution was cooled to room temperature. The synthe-
sized MAG was reaped by an external magnet and 10 min
centrifugation at 3500 rpm and washed for 4–5 times with ethanol
and boiling DI water. Then, MAG was collected via an external mag-
net and dried in a vacuum oven for 24 h at 100 °C. Finally, the MAG
was finely powdered and filtered through a 200-mesh screen.
Production of CDM, Fe2.8Ce0.2O4, was exactly similar to the MAG syn-
thesis, with one difference: the acidic solution was produced by the
0.90mol/L total concentration of the cerium and iron cations prepared
from 2.60 g of Ce(NO3)3·6H2O and 16.85 g of FeCl2·4H2O dissolved in
1.2mol/L of HCl. The schematic of CDM NPs synthesis is shown in
Scheme 1. The synthesis of ceria and CMDM NPs are provided in the
Supplementary data (S1 and S2).
2. Experiments
2.1. Reagents
Metronidazole (C6H9N3O3, 99%) was purchased from Jalinous
pharmaceutical company (Iran). Lead (II) nitrate (Pb(NO3)2,
≥99.0%), copper(II) chloride (CuCl2, 97%), potassium bromide (KBr,
≥99%), cerium nitrate hexahydrate (Ce(NO3)3.6H2O, 99%), cobalt
(II) chloride (CoCl2, ≥98%), sodium hydroxide (NaOH, ≥98%), magne-
sium chloride (MgCl2, ≥98%), iron (II) chloride tetrahydrate
(FeCl2.4H2O, 99.99%), Nickel(II) sulfate (NiSO4, ≥98%), iron(II) sul-
fate heptahydrate (FeSO4.7H2O, ≥99%), sodium nitrate (NaNO3,
≥99%), hydrazine (N2H4, 65%), trisodium phosphate (Na3PO4, 96%),
2.4. Preparation of samples
2.4.1. Preparation of the stock and working solutions
The weight of MNZ commercial tablets (five pieces) was measured
and then grounded in a mortar. Next, a weight equal to 10 mg of MNZ

Y. Orooji et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 234 (2020) 118272
3
Scheme 1. The schematics of synthesis of CDM NPs.
was scaled and dissolved in double-distilled water (DDW). Whatman
no. 42 filter paper was used to filter the resultant solution of which
was diluted with DDW to prepare 5.84 × 10−4 mol/L MNZ stock solu-
tion. The serial dilution of the as-prepared MNZ stock solution gave
rise to the working solutions.
was injected into the carrier and a premixing was accomplished with
an oxidant current in a Y-shaped glassy connecter, and finally, the
blend was transferred into the flow cell. The CL emission peak resulted
from the chemical reactions was steadily monitored in a computer con-
nected to the luminometer (FB12 luminometer, Bad Wildbad,
Germany).
2.4.2. Preparation of the calibration standards and quality control samples
One mL of each pre-known MNZ working solution was added to
drug-free human plasma (10 mL) to prepare eight MNZ calibration
standards other than zero, varying from 3.47 × 10−6 mol/L to
9.73 × 10−5 mol/L. A similar method was used to prepare the quality
control samples (QC) at three concentration ranges including low, me-
dium and high concentrations of MNZ (6.92 × 10−6, 3.39 × 10−5 and
8.20 × 10−5 mol/L).
2.6. Preparation of real samples
The real samples were prepared via modified methods. No pretreat-
ment step was carried out for the tap water before analyzing. To prepare
river water samples, the freshly sampled water was filtered through
0.45 μm polyamide membrane filter to eliminate the suspended solids
and then stored in a refrigerator at 4 °C under dark condition. The sam-
ples were kept and used no longer than one week. Before analyses, dif-
ferent volumes of the standard solution of MNZ (5.84 × 10−4 mol/L)
were mixed with 30 mL of water samples and diluted with DDW up
to 50 mL.
For measuring MNZ in human plasma, it was procured from an
Iranian Blood Transfusion Center (Tabriz, Iran). The only pre-
treatment step employed for human serum samples was
deproteinization using nitric acid without any extraction course.
The spiked samples were prepared by spiking pre-known values of
MNZ into the serum (1.0 mL), following the addition of nitric acid
(5.0 mL) into each sample and centrifugation for 15 min at
3000 rpm. The protein-free supernatant (2.5 mL) was diluted with
DDW up to 50 mL.
2.5. MNZ determination procedure
The schematic of the flow injection CL (FI-CL) system, employed for
the detection of MNZ using luminol-K3Fe(CN)6 reaction catalyzed by
CDM is displayed in Scheme 2. The setup featured
polytetrafluoroethylene (PTFE) tubes (inner diameter of 1.0 mm), peri-
staltic pump, six-port valve, mixing tube, Y-shaped glassy connecter,
luminometer detector, and a computer. Luminol solution (10−3 mol/
L), alone or together with CDM (10−4 mol/L) and metronidazole (via
line c), NaOH solution (1.5 mol/L, via line d), DI water (as carrier, via
line a), and K3Fe(CN)6 solution (0.05 mol/L, as oxidant via line b) was
pumped into the FI-CL system. The c and d solutions were combined
by a blender tube (PTFE), and then, 100 μL of the resulted solution
Scheme 2. The schematics of CL set-up.

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3. Results and discussion
the HRXPS spectra of O 1s, Ce 3d, and Fe 2p that were used to identify
the valance states of iron, cerium, and oxygen elements in the CDM
sample. The O 1 s spectrum can be deconvoluted to three characteristic
peaks at 529.32 eV (lattice O binding of Fe3O4), and the peaks at
530.03 eV and 531.25 eV energies, which are attributed to the lattice
binding of oxygen with Ce ions and exterior hydroxyl radicals, respec-
tively [30]. In the Ce 3d spectrum, the deconvoluted peaks at the binding
energies of 903.77 and 885.27 eV are attributed to the Ce3+ 3d3/2 and
Ce3+ 3d5/2, respectively. The peaks located at the binding energies of
916.65, 907.09, 900.99, 898.45, 888.74, and 882.11 eV are corresponding
to Ce+4 3d3/2 and Ce+4 3d5/2 [31]. The Fe 2p1/2 and Fe 2p3/2 peaks were
also detected at about 723.9 eV and 710.44 eV. The Fe 2p3/2 can be de-
convoluted to two peaks at 712.35 and 710.25 eV with a shakeup satel-
lite, which confirms the presence of both Fe3+ and Fe2+ in the CDM
sample [32].
The specific surface area and porosity of the samples were mea-
sured using the N2 adsorption-desorption and calculated by BET
and BJH models. The results showed that the cerium doping had a
positive effect on the specific surface area of Fe3O4 NPs. Furthermore,
as,BET of bare MAG and ceria increased from 42.113 m2/g to
51.272 m2/g and about 50.034 m2/g, respectively. On the contrary,
the as,BET of mechanically doped ceria- MAG was reduced to
18.353 m2/g. The detailed data on the BET results are provided in Ta-
ble S1. As shown in Fig. 3a, the hysteresis loops of all the nanoparti-
cles were H3 type, indicating the multi-layer structure of the NPs,
with loose interactions between the adsorbent and adsorbate [33].
In addition, the BJH plots of MAG, CDM, and CDMD are also provided
in Fig. S4, and the N2 adsorption-desorption and the BJH plots of bare
ceria is given in Fig. S5.
3.1. Characterization of nanoparticles
From the FESEM images depicted in Fig. 1a–c, the uniform spherical
shape of MAG NPs with an average diameter of 10–16 nm can be ob-
served. Fig. 1 (b and c) shows that the cerium doping of magnetite did
not have a significant effect on the shapes and sizes of CDM and
CMDM NPs [27]. For more information, FESEM images of the so-
synthesized ceria NPs are displayed in Fig. S1. The HRTEM images
(Fig. 1d,e) of nanoparticles indicated that both CDM and CMDM NPs
were in the octahedral shape, which is regarded as the signature struc-
ture of MAG NPs. The d-spacing fringes of CDM NPs (Fig. 1d) were fig-
ured out to be 0.251 nm and 0.293 nm, which can be attributed to the
(311) and (220) planes of MAG. Besides, for CMDM NPs (Fig. 1e). the
characteristic lattice fringes of 0.249, 0.268, 0.292, and 0.313 nm were
well-matched with the planes of MAG (311), ceria (200), MAG (220),
and ceria (111), respectively [28].
The purity of the phase structure and crystallinity of the prepared
MAG, CDM, and CMDM were studied by XRD patterns. As displayed in
Fig. 1f, all the samples were quite consistent with the pattern of pure
MAG (JCPDS File No.19–629) [29]. However, because of the low
amounts of Ce and ceria in CDM and CMDM samples, the pure MAG pat-
tern prevailed in the XRD patterns of CDM and CMDM. Furthermore, the
XRD pattern of ceria is in good agreement with the cubic fluorite lattice
structure of pure ceria (JCPDS File No.34-0394) as depicted in Fig. S2.
The chemical composition of the near-surface elements and their
relative valance states were investigated by XPS analysis. The XPS sur-
vey scan of CDM sample is shown in Fig. S3. Moreover, Fig. 2, shows
Fig. 1. The FESEM micrographs of (a) MAG NPs, (b) CDM NPs, and (c) CMDM NPs. The HRTEM images of (d) CDM NPs, (e) CMDM NPs, and (f) The XRD pattern of so-synthesized samples.

Y. Orooji et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 234 (2020) 118272
5
Fig. 2. The HRXPS of CDM samples for O 1 s, Ce 3d, and Fe 2p.
The FTIR analysis was conducted to study the functional groups of
3.2. Effect of nanoparticles
nanoparticles and the possible structural impurities. As shown in
Fig. 3b, the characteristic peaks of Fe\\O bond appeared at about 650
to 680 1/cm in all the samples [34]. The small shift in the CDM and
CMDM samples confirmed the successful doping of cerium with magne-
tite. In the FTR spectra of CDM and CMDM, the characteristic peaks ap-
peared at 494, 542, 649, and 1383 1/cm could be attributed to the
stretching vibration of the Ce\\O bond [30]. The absorption peaks de-
tected at 3425 and 1634 1/cm corresponds to the adsorbed water mol-
ecules. The FTIR spectrum of bare ceria can be seen in Fig. S6.
The magnetic behavior of the samples was studied using vibrating
sample magnetometer. The hysteresis loops of “S” shape and small
squareness ratios proved the superparamagnetic nature of the synthe-
sized NPs (Fig. 3c, Table S2.). Since the saturation magnetization value
of 38.19 emu/g for CDM sample was comparable to that of the bare
MAG (Ms of 54.58 emu/g), it can be concluded that the doping of cerium
doesn't have a great effect on the magnetic behavior of MAG [35].
The CL reaction of luminol-ferricyanide was utilized to study the ef-
fect of the prepared NPs. The CL reaction mechanism of the luminol-
ferricyanide system has been initially investigated by Shevlin and
Neufeld [36]. It was established that the relaxation of the excited state
⁎
of 3-aminophthalate (3-aminophthalate ) at 425 nm lead to the CL
⁎
emission. 3-aminophthalate as a product of luminol oxidation is pro-
duced as a result of the reaction of luminol radical and diazaquine
with reactive oxygen species (ROS such as •OH, O2•− or the other radical
derivatives) [37].
The kinetic profiles (Fig. 4a) and UV–vis absorption spectra (Fig. 4b)
were studied to figure out the mechanism of the target CL system. In the
absence of the NPs, the CL emission of luminol-K3Fe(CN)6 was relatively
weak in the alkaline medium (Fig. 4a). However, the CL intensity of the
system was enhanced about 1.5 times in the presence of CDM, wherein
the enhancement effect of CDM was greater than those of MAG, CeO2,
Fig. 3. (a) The BET isotherms of so-synthesized samples, (b) FTIR spectra of so-synthesized samples, and (c) The hysteresis loops of the so-synthesized samples.

6
Y. Orooji et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 234 (2020) 118272
Fig. 4. (a) Kinetic profiles for different CL systems. (b) UV–Vis absorption spectra of luminol- K3Fe(CN)6-CDM in alkaline media; The concentrations of luminol, K3Fe(CN)6, NaOH and
different catalysts (MAG, CeO2, CDM, and CMDM) were 10−3, 0.05, 1.5, and 9.52 × 10−5 mol/L, respectively.
and CMDM. Because, as mentioned in the characterization section, the
reduction in the CMDM surface area could reduce its enhancing effect.
Since the application of CDM could give rise to the generation of reactive
oxygen species such as •OH, O2•−, the boosting effect of CDM on CL in-
tensity can be attributed to the formation of O2•− [38]. Under this con-
dition, the oxidation reaction of luminol was facilitated by O2•− thus
enhancing the CL emission (Scheme 3). In contrast, the application of
Fe3O4 and CeO2 had lower effects on CL intensity, thus revealing the
interactional contribution of these compartments for the enhanced in-
fluence of CDM.
As illustrated in Fig. 4b, ferricyanide has 3 absorption peaks at about
260, 310, and 425 nm (curve a). The maximum absorption peak of MNZ
was located at the wavelength of about 315 nm (curve g) and CDM gave
rise to a linear absorption peak (curve f). Considering the diminished in-
tensity of the absorption peak of luminol-ferricyanide system (curve
b) in the presence of CDM (curve d), it can be concluded that CDM
Scheme 3. Schematic illustration of the proposed CL assay for MNZ detection.

Y. Orooji et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 234 (2020) 118272
7
Fig. 5. (a) Kinetic profiles for luminol- K3Fe(CN)6-CDM CL system after injection of different concentrations of MNZ solution [luminol = 10−3 mol/L, NaOH = 1.5 mol/L, K3Fe(CN)6
=
0.05 mol/L, and CDM = 9.52 × 10−5 mol/L]. (b) Calibration graph for the determination of MNZ, the inset shows the linear part of the calibration graph.
Fig. 6. (a) CL response of luminol- K3Fe(CN)6-CDM system towards MNZ and its analogues; The concentrations of luminol, K3Fe(CN)6, NaOH and CDM were 10−3, 0.05, 1.5, and
9.52 × 10−5 mol/L, respectively. (b) Catalytic activity of prepared CDM in certain times after its synthesis.
has had an interaction with the reactants or intermediates in the sys-
tem. The inhibitory effect of MNZ was also studied on the proposed CL
system. The absorbance of luminol-ferricyanide-CDM-MNZ (curve
c) system was not equal to those of the two individual systems (curves
d and g). This indicates that MNZ could be involved and oxidized in the
luminol-ferricyanide-MNZ (curve e) reaction system and resulted in
new products. Therefore, the quenching role of MNZ could be derived
from a reducing group (e.g. OH). As reported previously [37,39], the
Table 1
Comparison of the purposed method with some methods reported in literature for determination of MNZ.
Method
Catalyst
Matrix
LODs (mg/L)
Linear range (mg/L)
Ref.
Fluorescence
Fluorescence
Spectrophotometric
Spectrophotometric
SERSc
Fluorescent membrane
Human serum
Human serum
0.360
0.026
0.080
83.100
10.000
0.250
9.927
0.040
2.500
0.067
1.23–35.48
0.034–2.567
10.0–60.0
5.0–25.0
10.0–50.0
0.85–71.0
0.85–275.56
0.06–10.0
50.0–6030.0
0.59–16.66
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
GQDs-embedded SMIPa
Vanillin and PDABb
–
Silver nanorod array
Nanostructure thin film on the gold electrode
Ni/Fe-LDH
–
–
Tablet formulations
Pharmaceutical dosage forms and human urine
Environmental Samples
Pharmaceutical and human urine
Tablets
Human serum
Tablets and ovules
Human serum
Electrochemical
Electrochemical
HPLC
GC-FIDd
CL
[50]
This work
Fi2.8Ce0.2O4
a
Graphene quantum dots embedded silica molecularly imprinted polymer.
P-dimethyl amino benzaldehyde.
Surface Enhanced Raman Spectroscopy.
b
c
d
Gas Chromatography and Flame Ionization Detector.

8
Y. Orooji et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 234 (2020) 118272
Table 2
Table 4
Interference of different species on the determination of 3 × 10−5 mol/L MNZ (in optimum
condition).
Results for the determination of MNZ in spiked environmental water samples.
Sample
Added
(mol/L)
Found (CL method)
(mol/L)
Recovery
(%)
Coexisting
substance
Tolerance limit (interference
to analyte ratio)
Water
Tap water
0
–
–
Cu2+ and Cl−
1000
500
200
100
50
1.75 × 10−6
3.50 × 10−6
5.84 × 10−6
1.75 × 10−5
1.63 × 10−6
3.56 × 10−6
5.66 × 10−6
1.72 × 10−5
93.14
101.71
96.91
98.28
Na+, Co2+, Mg2+, CH3COO−, NO23−, and SO42−
Alanine, glucose, lactose, Fe2+, and Zn2+
Starch, valine, Ca2+, Ni2+, Pb2+, and CO23−
K+, Br−, and PO34−
River
water
0.9991) (Fig. 5b). The limit of detection (LOD) was also
3.91 × 10−7 mol/L.
functional groups of MNZ could react by ROS and prevent their produc-
tion, leading to the decreased CL intensity. Consequently, the MNZ mol-
ecules could cover CDM surface and prevent its catalytic action in the
studied CL system.
Furthermore, the CL sensor was tested for the detection of some
other drugs including tinidazole (TNZ) and mebendazole (MBZ)
and the results are shown in Fig. 6a. The studies revealed that the
compounds with a chemical structure similar to MNZ could also
quench the CL intensity owing to the existence of the same func-
tional group on them. This outcome indicated the sensitivity of the
suggested CL tool for the detection of the same group of drugs, in par-
ticular MNZ. The analytical properties of different approaches were
compared to those of the investigated CL system in this study
(Table 1). This comparison shows that the fabricated CL method in
this study provides a relatively good sensitivity and it can be used
for detection of MNZ and its analogues owing to its reliability and
promptness.
3.3. Optimization of CL reaction parameters
In order to access the sensitivity of the system for MNZ determina-
tion, the CL reaction conditions were optimized. The effects of the influ-
ential parameters were investigated in reference to the ΔI value, where
ΔI = I0-Is, I0 is the CL signal in the absence of MNZ, and Is is the CL signal
in the presence of MNZ. The ΔI value revealed the decreased efficacy of
MNZ on the emitted CL intensity of luminol-K3Fe(CN)6-CDM system.
The efficacy of some key factors is shown in Fig. S7.
Fig. S7(a and b) shows that the most sensitive CL system was
achieved using 10−3 mol/L luminol and 1.5 mol/L NaOH. These low
quantities could be limited to the kinetic of CL reaction.
The effect of the concentration of ferricyanide solution on ΔI of
the CL system was also investigated (Fig. S7c). The optimum concen-
tration of ferricyanide was 0.05 mol/L, which could be attributed to
the absorption of the emitted radiation by a strong colored oxidant
[40]. The quantity of the cerium used for the synthesis of CDM
could also play an important role in its catalytic yield. Accordingly,
several NPs were synthesized with different quantities of cerium
and applied to the CL system (Fig. S8). The maximum CL intensity
was obtained by the CDM sample containing 0.2 of cerium (Fe3-
xCexO4, x = 0.2). The lower amounts of Ce gave rise to less active
CDM samples due to the insignificant active sites. on the other
hand, the higher amounts of Ce had negative effect on CDM activity
because of the minimized porosity of the synthesized samples.
Fig. S7 d shows that the highest CL emission was obtained at
9.52 × 10−5 mol/L of CDM. Notably, once the concentration of
nanomaterial increases, strong interactions take place [41]. On this
basis, the catalytic efficiency of CDM NPs was decreased in the CL
system. Besides, the absorption of the emitted radiation by CDM
NPs at higher concentrations could reduce the catalytic performance
of the NPs.
The precision of the studied system was also investigated by mea-
suring 10−5 and 3 × 10−5 mol/L of MNZ solution, for 5 times. The rela-
tive standard deviation (%) was 1.94% and 3.33%, respectively.
Moreover, the CL solution offered good stability during the study
(Fig. 6b).
3.5. Selectivity
The selectivity of the developed sensor was investigated by measur-
ing a constant concentration of MNZ (3 × 10−5 mol/L) in the presence of
various ionic species and intervening compounds with different con-
centrations. An interfering effect of b5% on the signal of MNZ was pre-
sumed as the tolerance limit (Table 2). Most of the studied
compounds demonstrated insignificant effects on the MNZ determina-
tion results. Besides, for further elimination of the intervening com-
pound efficacies, a dilution ratio of 1:500 of the original samples was
very helpful.
3.6. Analysis of real samples
The applicability of the proposed system for real samples was
assessed through MNZ determination in human serum and spiked
water samples. The studied samples were supplied and examined
based on the procedures outlined in the experimental subdivision. The
obtained results were then compared with corresponding values
3.4. Analytical figures of merit
Table 5
Results for the determination of MNZ in pharmaceutical formulation.
The CDM (Fe2.8Ce0.2O4) supported CL system was used for the deter-
mination of MNZ. The CL intensity was decreased when the high magni-
tudes of MNZ was used (Fig. 5a). Moreover, a linear relationship was
Nominal
(mg/vial)
Official method
(mg/vial)
Proposed CL method
(mg/vial)
t-Statistica
1.79
achieved for the concentration range of 3.47
×
10−6 to
10
10.17
10.23
9.37 × 10−5 mol/L of MNZ (ΔI = (9E+10) (CMNZ) + 263,584, R2
=
a
t-Critical = 4.30 for n = 6 and P ≥ .05.
Table 3
Results for the determination of MNZ in human plasma.
QC sample
Added concentration of drug in plasma (mol/L)
Found concentration of drug in plasma (mol/L)
Mean recovery (%)
CV (%)a
Accuracy
nb
Low
Medium
High
6.92 × 10−6
3.39 × 10−5
8.20 × 10−5
6.94 × 10−6
3.34 × 10−5
8.15 × 10−5
100.36
98.57
99.43
2.57
5.19
2.44
98.11
94.82
105.68
6
6
6
a
Coefficient of variation.
Number of determinations.
b

Y. Orooji et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 234 (2020) 118272
9
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4. Conclusion
We have reported a CL assay based on the catalytic activity of cerium
doped magnetite in the luminol-K3Fe(CN)6 CL system with the
quenching effect of MNZ, which could effectively detect MNZ. Excellent
selectivity, high sensitivity, and simplicity of this approach were the
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range of 3.47 × 10−6-9.37 × 10−5 mol/L, which has not been reported
so far. More importantly, this sensor was applicable to detect MNZ in in-
tricate mediums such as plasma. Hence, the application of this sensor in
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Declaration of competing interest
We would like to confirm that there is no known conflict of interest
associated with this publication.
Acknowledgment
The authors are thankful to the University of Tabriz for the support
provided. This work is funded by Nanjing Forestry University [Grant
nos. 163020139, 163040109 and 163020236] and the grant no. NRF-
2019R1A5A8080290 of the National Research Foundation of Korea.
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