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Novel colorimetric sensor based on peroxidase-like

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Cite this: DOI: 10.1039/c8ay01975d

activity of chitosan-stabilized Au/Pt nanoclusters
for trace lead†
Zahra Dehghani,a Morteza Hosseini, *ab Javad Mohammadnejadc
and Mohammad Reza Ganjalicd

Received 9th September 2018
Accepted 25th December 2018
DOI: 10.1039/c8ay01975d
rsc.li/methods
In this study we propose a new colorimetric sensor for trace lead ions in milk samples. Au/Pt nanoclusters
were synthesized using chitosan as the stabilizer at room temperature. Chitosan-protected Au/Pt
nanoclusters exhibit strong peroxidase-like activity, and could catalyze the oxidation of 3,3,5,5-
tetramethylbenzidine (TMB) in the vicinity of hydrogen peroxide (H2O2) and create a visual blue color.
The detection mechanism is based on the Pb2+ ions-induced aggregation of chitosan–Au/Pt NCs that
causes changes in size and impairs the enzymatic performance of Au/Pt NCs. Experiments indicated that
a step-by-step increase in concentration of Pb2+ ions gradually decreased peroxidase activity and this
reduction was linear. Based on the declared procedure, a colorimetric detection for Pb2+ was obtained
with a detection limit (LOD) of 16 nM and a linear range from 25 nM to 1 mM. The method was
considerably selective toward Pb2+ over other metal ions. Low price and simplicity support the
application of sensing Pb2+ in milk samples.

priced sophisticated instruments, time-consuming and labor-

1 Introduction
Sensitive and quantitative monitoring of lead (Pb2+) has been
given enormous consideration in order to alleviate the under-
lying problems of this environmentally toxic pollutant.1 As
indicated, lead ions (Pb2+) bring health threats to the kidneys,
liver and blood.2 Since it damages the nervous system and
creates brain and blood disorders, it is considered a neurotoxin
with greater toxicity than other heavy materials.3 Due to the
widespread applications of lead in industrial sectors and its
release into the environment, humans' exposure to polluted
natural resources has been intensied considerably. Over the
last two decades, numerous conventional analytical techniques
including atomic absorption spectrometry (AAS), atomic emis-
sion spectrometry, inductively coupled plasma mass spec-
trometry (ICP-MS),4 ICP-AES,5 MS and HPLC, have been
employed for Pb2+ monitoring. Despite the efficiency of these
techniques, their applications in real-time monitoring and
diagnostics have been markedly limited by the need for high-
a
Department of Life Science Engineering, Faculty of New Sciences & Technologies,
University of Tehran, Tehran, Iran. E-mail: smhosseini@khayam.ut.ac.ir
b
Department of Pharmaceutical Biomaterials, Medical Biomaterials Research Center,
Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
c
Center of Excellence in Electrochemistry, School of Chemistry, College of Science,
University of Tehran, Tehran, Iran
d
Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular
Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8ay01975d
intensive operations, complicated pretreatment processes and
lack of portable and online applications.6 In this regard, it is
necessary to improve such portable, cost-effective, rapid means
with long-term stability for Pb2+ detection. Furthermore, the
need for procient operators and multifaceted analytic systems
should be excluded.
In light of breakthroughs in nanotechnology, ultrasensitive
detection tools have been provided for environmental moni-
toring. Numerous sensors and biosensors have been reported in
terms of environment protection via detection processes.7–12
Noticeably sensitive and exceptionally selective nanosensors for
the detection of Pb2+ ions have been obtained using efficient
measurements such as uorescent, colorimetric,13 electro-
chemical,7 electro chemiluminescence13,14 and surface plasmon
resonance (SPR) techniques.15 Liu et al. reported luminescent
sensors based on lanthanide metal–organic frameworks (MOFs)
for detecting heavy metal ions consisting of Pb2+ ions, organic
molecules and biomaterials,16 but uorescent measurement
requires high price tools, and also some uorescent sensors
based on quantum dots and lanthanide metals are highly toxic,
which can be a hindrance for the widespread application of
these sensors.17,18 Pelossof et al. designed an electrochemical
and SPR sensor based on gold nanoparticles and an Au elec-
trode, and although this method has a high LOD, its several step
functionalization takes time, is tedious15 and also being an
electrochemical method produces high background signals that
are difficult to analyse.7 In another study, Tan and coworkers
reported a SERS nano sensor for lead ion detection using Au

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Analytical Methods
nanoparticles,19 but the costly fabrication of SERS sensors, and
the requirement for expensive instruments as well as expert
operators are underlying issues limiting their usage for the
speciation of poisonous metal ions. Nevertheless, among
various detection methods, colorimetric detection approaches
based on metal nanoparticles have drawn wide research inter-
ests thanks to their bigger absorption coefficients and suitable
size- and shape-dependent optical properties.20–23 Colorimetric
biosensors demonstrate sensitive and selective capabilities and
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also require only simple instrumentation.24 Since gold nano-
materials have exceptional size- and shape-dependent optical
properties, high extinction coefficients and light scattering
features, optical probes based on them have been researched
signicantly in the analytical processing of environmental and
biological samples owing to their simplicity, sensitivity and
selectivity.18 In Wey's research, a label-free colorimetric sensor
for Pb2+ monitoring was developed by using gold nanoparticles
and DNAzyme with a tunable dynamic response toward Pb2+
and a detection limit as low as 5 pM Pb2+. SsDNA can protect
gold nanoparticles (Au NPs) against aggregation using the
electrostatic stabilization of Au NPs, but it is released by DNA-
zyme in the presence of Pb2+. The absence of Pb2+ leads to
unstable purple-blue Au NP aggregates owing to an un-cleaved
complex.25,26 In another colorimetric sensor built by Chen
et al., G-quartet single-strand DNA was used as a stabilizer of
gold nanoparticles against salt, but in the presence Pb2+ ions,
the gold nanoparticles aggregate and their red color changes to
purple; the LOD for this sensor is estimated at 5 mM.27
Furthermore, in previous work, some peptides such as gluta-
thione were applied as capping agents to protect the gold
nanoparticles. The mechanism of Pb2+ sensing was based on
the interaction of lead ions with glutathione, which induces
gold nanoparticle aggregation, changing the color of the solu-
tion and creating a shi in the UV absorption peak.28 With
regard to former work, there are some limitations in working
with gold nanoparticles: for example low stability and difficult
regulation of the concentration of NaCl, and also the high price
of protecting agents such as DNA and peptides. Thus, it is felt
that there is a necessity to introduce another colorimetric
method. With regard to the literature over the last decade, the
peroxidase-like activity of nanoparticles could be used in
a colorimetric study.29–35 So far DNA–Ag/Pt nanoclusters have
been used for the fabrication of biosensors for L-cysteine36 and
for a thrombin colorimetric assay37 and ions.38 Bimetallic Au/Pt
nanozymes with peroxidase-like activity were synthesized using
C-rich DNA as the nucleation template and were used for the
colorimetric assay of bio thiols.39 But limitations of this system
were the time taken to order DNA and the cost of DNA synthesis
being high. Also the conditions for keeping the nucleotides are
sensitive and require a refrigerator or icebox. By searching
scientic reports about nanoclusters with catalytic activity, we
found a chitosan-stabilized nanocluster with high catalytic
activity that is used for the oxidation of various alcohols.40
Chitosan has a low price and high stability and keeping it in
optimum condition is easy.
In this work, we synthesized chitosan–Au/Pt NCs and the
peroxidase-like activity of this system was tested in the presence
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of metal ions. Pb2+ ions have a selective inhibitory effect on the
peroxidase activity of chitosan–Au/Pt NCs. Thus Au/Pt NCs were
proposed as a novel tool for the colorimetric detection of Pb2+
for the rst time (Scheme 1).
2 Experimental section
2.1 Materials
All chemicals and solvents were used as received without
further purication. Hydrogen chloroauric acid tetrahydrate
(HAuCl4$3H2O), sodium borohydride (NaBH4), acetic acid and
other metal salts were obtained from the Merck Millipore
company. K2PtCl6, 3,30 ,5,50 -tetramethylbenzidine (TMB) and
hydrogen peroxide solution (H2O2) were obtained from Sigma-
Aldrich. Chitosan, poly(D-glucosamine), with a medium molec-
ular weight of 75–85% deacetylated was purchased from Sigma-
Aldrich. Milli-Q grade distilled water was utilized for the prep-
aration of all solutions in the experiments.
2.2 Apparatus
UV-vis spectra were measured by a JASCO V-670 spectropho-
tometer at 24  C. The size-measurements of the DNA–Au NCs
were conducted using an EM10C, 80 kV transmission electron
microscope (TEM), Zeiss, Germany, as well as through dynamic
light scattering (DLS) evaluations.
The high-resolution TEM images of chitosan-stabilized
metal NCs were recorded with a Zeiss, EM10C, 80 kV TEM,
Germany. The hydrodynamic size of the NCs was measured by
DLS (Malvern Zetasizer Nano ZS). FT-IR spectra of chitosan and
also chitosan–Au NCs, chitosan–Au/Pt NCs and chitosan–Au/Pt
NCs interacted with Pb2+ ions were achieved with a Perkin
Elmer device. All the spectra were considered from 450 to
3986 cm1. EDX spectra of chitosan–Au/Pt NCs were attained by
TESCAN MIRA II (Czech).
2.3 Preparation of chitosan-stabilized Au/Pt NCs
All glasswork used in the experiments was washed in a bath of
freshly prepared Aqua Regia (HCl:HNO3), and dipped thor-
oughly in ethanol and water prior to use. Chitosan-stabilized
Au/Pt NCs were synthesized based on the Murugadoss and
Sakurai method.40 Briey 0.15 g of chitosan powder was
Scheme 1
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dissolved in 50 mL of 0.18% aqueous acetic acid. Then 0.4 mM
of HAuCl4 and 0.1 mM of H2PtCl6 were added and the mixture
was agitated at 1600 rpm for 30 min at room temperature. The
molar ratio of metal ions used in these experiments was 4 : 1
(Au/Pt). Aer stirring for 30 minutes, 2.5 mL (0.1 M) of NaBH4
aqueous solution was quickly added at 6  C under vigorous
stirring. The hydrosol chitosan–Au/Pt NCs solution was kept in
a refrigerator at 4  C.
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2.4 Assessment of peroxidase-like activity for chitosan–Au/Pt
NCs
For the evaluation of the peroxidase-like activity of chitosan–Au/
Pt NCs, 60 mL of NCs was transferred into a 200 mL mini tube
and 1 mM of TMB and H2O2 were added to the tube. Aer a few
seconds a blue color was visually observed. This response was
also attained in the absence of H2O2.
2.5 Optimization of TMB and H2O2 concentration
A minimum level of TMB and H2O2 (100 mM) was applied as the
lowest concentration and a range of 100 to 1000 mM of TMB and
H2O2 in two separate experiments was evaluated. In the rst
examination, different concentrations of TMB (100, 200, 400,
600, 800 and 1000 mM) in 6 vials containing 60 mL of chitosan–
Au/Pt NCs were incubated at room temperature while in all the
vials the H2O2 level was 1000 mM. In this trial the best TMB
concentration was selected and utilized in the next test to
determine the optimum H2O2 concentration. To dene the best
H2O2 concentration various concentrations (100, 200, 400, 600,
800, 1000 mM) were tested.
2.6 Optimization of temperature and pH
To identify the optimum conditions for the chitosan–Au/Pt NCs
peroxidase properties, the interaction of chitosan–Au/Pt NCs
with TMB at several pH values and temperatures was
performed.
2.7 Colorimetric detection of ions
In order to evaluate the effects of various ions on the peroxidase-
like activity of chitosan–Au/Pt NCs, 1 mM of some ions (Na+,
Cu2+, Co2+, Hg2+, Sn2+, Mn2+, Zn2+, Ni2+, Cd2+, Mg2+, Cr3+, Pb2+,
Br, I) in a volume of 60 mL of nanoclusters was incubated at
room temperature for 10 min. In the next step 4 mL of TMB (10
mM) and 4 mL of H2O2 (10 mM) were imported into each vial
and the UV absorption spectra were recorded by a Perkin Elmer
spectrophotometer in the range 250 to 800 nm.
2.8 Colorimetric detection of Pb2+
In a typical test, 60 mL of as-prepared chitosan–Au/Pt NCs
solution (pH 4.0) was mixed with a certain amount of Pb2+. The
mixture was incubated at room temperature for 10 min. Then 4
mL of TMB (10 mM) and 4 mL of H2O2 (10 mM) were added. The
UV absorption spectra were obtained by a Perkin Elmer spec-
trophotometer in the range 250 to 800 nm.
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3 Results and discussion
3.1 Characterization of chitosan–Au/Pt NCs
Chitosan-protected gold/platinum nanoclusters (chitosan–Au/
Pt NCs) with blue-emitting uorescence were produced by
a simple one-pot method in aqueous solution. The optical
properties of chitosan–Au/Pt NCs were dened by UV-vis
absorption spectroscopy and uorescence spectroscopy. The
UV-vis absorption of the chitosan–Au/Pt NCs and chitosan are
exhibited in Fig. 1a. It was revealed that chitosan–Au NCs dis-
played a new absorption peak near 530 nm compared to chi-
tosan. Showing the creation of Au NCs, this peak was generated
due to the surface plasmon resonance (SPR) of gold nano-
clusters,40 and in chitosan–Au/Pt NCs with the deposition of Pt
ions on the Au nanoclusters, the absorption intensity at 530 nm
was decreased. The spectrum inserted in Fig. 1a shows that the
created chitosan–Au NCs solution was pink and in Fig. 1b, the
chitosan–Au/Pt NCs solution was light orange in color under
visible light, while the chitosan solution was colorless (Fig. 1c).
The TEM images of the chitosan–Au/Pt NCs are shown in
Fig. 2. It was conrmed that the distribution of Au/Pt NCs is
suitable and the particles are about 1 to 2 nm in diameter.
Assessment of TEM photos for chitosan-stabilized Au/Pt NCs in
previous work by Murugadoss and Sakurai also revealed an NC
size of about 2.4 nm.40 In addition, these results are in accor-
dance with the size of bimetallic Ag/Pt NCs in other work where
they were stabilized by DNA and the size of the nanoclusters in
TEM image was about 2.11 nm.37 DLS analysis was performed to
detect the hydrodynamic diameters and size distribution of the
Au/Pt NCs (Fig. 4) and the results showed that the size of Au/Pt
NCs was about 1 nm (range from 3 to 8 nm). EDX spectra
(Fig. S1†) conrmed that chitosan–Au/Pt NCs contain Au and Pt
atoms, showing the construction of Au/Pt NCs.
3.2 Peroxidase-like activity for chitosan–Au/Pt NCs
Peroxidase-like activity has been mentioned for Pt nanoclusters
stabilized by DNA that catalyze TMB oxidation in the presence
of H2O2. In other work by Gao et al., Au@Pt nanoparticles as
substitutes for HRP enzyme, in the presence of H2O2, take an
electron from TMB and produce a visual blue color in aqueous
solution.41 But the catalytic activity of chitosan–Au/Pt NCs in the
Fig. 1 UV-visible absorption spectra of chitosan, chitosan–Au NCs
and chitosan–Au/Pt NCs.
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Fig. 2 TEM image (a) and size distribution (from DLS) analysis (b) of
chitosan–Au/Pt NCs.
oxidation of TMB has not been studied in the literature. In this
work, initially, 60 mL of chitosan–Au/Pt NCs, 0.8 mM of TMB and
1 mM of H2O2 were examined to create a blue color and the UV-
vis spectrum showed a strong absorption peak at 650 nm
(Fig. 3a). In another test, even in the absence of H2O2 and just
with the interaction of TMB and chitosan–Au/Pt NCs, a visual
blue color was observed but in comparison with the previous
test it was weaker (Fig. 3b), while TMB and H2O2 in the absence
of chitosan–Au/Pt NCs did not generate any peak in the UV
spectrum. This occurrence illustrates the intrinsic peroxidase-
like activity of chitosan–Au/Pt NCs (Fig. 3b).
Fig. 3 Evaluation of peroxidase-like activity of chitosan Au/Pt NCs.
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In the following step, the concentrations of H2O2 and TMB
were optimized, and the results are given in Fig. S2.† Based on
the obtained results, 800 mM of TMB and 1 mM of H2O2 showed
the best results with an absorption peak of 1 at 650 nm. These
quantities were preferred for the following experiments. These
obtained quantities in this study were lower than those in other
work (0.921 mM of TMB and 7.63 mol of H2O2).30 In the case of
Ag/Pt NCs the blue color was observed in the presence of 20 mL
of H2O2 (1.0 M) and 40 mL of TMB (3.0 mM).
The effect of pH on biosensor performance was also inves-
tigated by testing solutions with different pH values (ranging
from 4 to 8) (Fig. S3†). It was found that at a pH of 6, the
nanoclusters exhibit the best color change aer the addition of
TMB and H2O2. By increasing the pH, H2O2 could decompose
into H2O and O2.
Also, the catalytic property of chitosan–Au/Pt NCs was
improved by increasing the temperature up to 45  C. However,
at temperatures higher than 35  C, due to decomposition of
H2O2, a lower performance was obtained, as shown in Fig. S3.†
As can be seen in the gure, the highest DA was at 30  C.
Therefore, we used this temperature in further experiments.
3.3 Selectivity of chitosan–Au/Pt NCs peroxidase-like activity
for lead ions
In this step, the effect of different cations, including Cd2+, Zn2+,
Ca2+, Mn2+, Mg2+, Co2+, Ni2+, Cu2+, Al3+, Sn2+, Hg2+, Na+ and Pb2+
on the peroxidase-like activity of chitosan–Au/Pt NCs was tested.
Fig. 4 (a) Selectivity of chitosan–Au/Pt NCs toward lead ions and (b)
photograph of color change of reaction system in the presence of
cations and anions.
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An experiment was carried out to explore the selectivity of the
above-mentioned nanostructures for ion detection. As shown in
Fig. 4, UV-visible absorption spectra and associated photographs,
chitosan–Au/Pt NCs in the presence of 1 mM of anions and
cations, have no apparent inuence on the UV-vis absorption
peak or visual blue color, while the negative effect of the Pb2+ ions
on the color and the decrease in the absorption peak in the UV
spectra are obvious. In fact, Pb2+ ions have an inhibitory effect on
chitosan–Au/Pt NCs peroxidase-like activity and this outcome
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indicated that this structure has specicity for Pb2+ ions.
3.4 Colorimetric detection of Pb2+ ions by chitosan–Au/Pt
NCs peroxidase-like activity
Different concentrations of Pb2+ ions were utilized to determine
the limit of detection of the chitosan–Au/Pt NCs. Fig. 5 shows
Fig. 5 (a) Dose–response curve for lead detection using the combi-
nation of chitosan–Au/Pt NCs and TMB (4 diluted samples). (b) Linear
calibration plot for lead (25–1000 nM). (c) Color variation in the
presence of different concentrations of lead. The error bars represent
the standard deviation of three measurements.
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the results of detection sensitivity attained visually aer addi-
tion of TMB and H2O2. As shown in Fig. 5, the aggregation with
increasing amounts of Pb2+ can specically decrease the UV
absorbance at 650 nm. The decrease in absorbance at 650 nm is
linearly proportional to the concentrations of Pb2+. All the
positive samples were found to have the characteristic absor-
bance peak at 650 nm with a gradual decrease in absorbance
relative to the concentration of Pb2+ present in the test sample.
Furthermore, the absorption intensity dropped linearly in
proportion to the concentrations of Pb2+ ions in the range from
25 nM to 1 mM (Fig. 5c), with a calibration function of A ¼ 6.0
 104C + 0.809 (R2 ¼ 0.96). The detection limit was calculated
to be 16 nM. As observed in Table 1, various nanoparticles with
and without a capping agent were used for colorimetric detec-
tion of lead ions. The results show that the limit of detection
and ease of our method are comparable with previous works.
3.5 Mechanism of colorimetric detection of Pb2+
The efficiency of the novel sensor was assessed by detecting Pb2+
ions via the aggregation process. The TEM and DLS results of
chitosan-stabilized Au/Pt NCs 40 min aer the addition of Pb2+
ions can be observed in Fig. 6. The size increase might have
happened as a result of the interaction of Pb2+ ions with the
amine and OH group of chitosan and a decrease in the capping
role of chitosan in protecting the Au/Pt nanocluster, causing
a change in the structure of the nanocluster. Interaction of Pb2+
ions with the NH2 and OH groups of chitosan were described in
a former study.45 As is shown in Fig. S4,† for chitosan, the
characteristic bands were observed at 3050–30 650 cm1 (–OH
and –NH2 stretching), 2929 and 2869 cm1 (–CH stretching),
1584 cm1 (–NH amide band) and 1085 cm1 (–C–O stretch-
ing).45 With the addition Pb2+ these bands are shied to longer
wave numbers at 1065 cm1, 1404 cm1 and 3430 cm1 which
are assigned to the formation of intramolecular interactions.
Mao et al. in 2011, used L-glutathione as a gold nanoparticle
stabilizer and the effect of Pb2+ ions on the COOH and NH2 of L-
glutathione triggered gold nanoparticle aggregation and
changed the red color of the solution to purple.28 Fu et al. in
2012 also reported on glutathione-stabilized Au nanoparticles
decorated on a graphene sheet for the detection of Pb2+ ions.
The addition of Pb2+ caused the leaching of Au nanoparticles
stabilized by glutathione, releasing them from the graphene
sheet. This process occurred due to the interaction of lead ions
Table 1 Comparison of different colorimetric systems for the detec-
tion of lead ions
Nanostructure Functional or capping agent LOD References
Gold nanoparticle DNAzyme 5 pM 26
Gold nanoparticle G-quartet 5 mM 27
Gold nanoparticle L-Glutathione 0.1 mM 28
Gold nanoparticle DNAzyme molecular beacon 20 pM 42
Gold nanoparticle Valine 30.5 mM 43
Gold nanoparticle Maleic acid 0.5 mM 44
Au/Pt nanocluster Chitosan 16 nM Our work
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Fig. 6 (a) TEM image of aggregated chitosan–Au/Pt NCs after addition
of lead ions and (b) size distribution of chitosan–Au/Pt NCs after
addition of lead ions.
with glutathione that in the end ultimately separated the gold
nanoparticles and turned on the uorescence.46
3.6 Real sample analysis
To consider the applicability of the suggested system, chitosan–
Au/Pt NCs were utilized in monitoring Pb2+ in milk samples. In
the rst step, 1 mL of milk was centrifuged three time at
14 000 rpm for 10 min, and the transparent phase was trans-
ferred to a clean tube. Then, this uid was ltrated with a 0.45
mM syringe lter, and was diluted 20 times with water. Next,
several concentrations of Pb2+ ions (from 100 to 1000 nM) were
prepared in milk samples. Finally, the peroxidase-like activity of
chitosan–Au/Pt NCs was measured in the presence of lead ions.
Fig. 7 shows the colorimetric sensor response with different
Pb2+ concentrations in real samples. To validate the accuracy of
this method, the results were compared with those obtained by
inductively coupled plasma optical emission spectrometry (ICP-
OES) (Table S2†). A percentage recovery of spiked Pb2+ in the
range of 102–106% was obtained, indicating the acceptable
accuracy of the proposed method for the detection of Pb2+ in
real samples. Thus, the proposed colorimetric sensor has high
potential for the selective and quantitative detection of Pb2+
ions at trace levels in real samples.
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Fig. 7 Absorption spectra and visual detection of colorimetric sensor
with different concentrations of Pb2+ in milk samples.
4 Conclusions
In this paper, for the rst time, we used the peroxidase-like
activity of chitosan–Au/Pt NCs for the colorimetric assay of
Pb2+. The presented sensing method based on chitosan–Au/Pt
NCs revealed a response based on the interaction of Pb2+ ions
with chitosan functional groups and also Au/Pt NCs and the
induced aggregation of nanoclusters. This phenomenon
decreases the catalytic surface of the nanoclusters that
sequentially creates defects in the peroxidase-like activity.
Compared with the previous method, this suggested sensor is
easy, low-price and does not need costly apparatus.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
This study was sponsored by the Research Council of University
of Tehran (28645/01/02) and authors gratefully appreciated for
commercial support.
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