Article

pubs.acs.org/JAFC

Colorimetric Detection of Sulte in Foods by a TMBO2Co3O4

Nanoparticles Detection System
Wenjie Qin,, Li Su, Chen Yang,, Yanhua Ma,, Haijuan Zhang,, and Xingguo Chen*,,

State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, China

Department of Chemistry, Lanzhou University, Lanzhou 730000, China

School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang 453007, China
*
S Supporting Information

ABSTRACT: In this paper, we rst discovered that Co3O4 nanoparticles (NPs) possess intrinsic oxidase-like activity and can
catalytically oxidize peroxidase substrates, such as 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt
(ABTS) and 3,3,5,5-tetramethylbenzidine (TMB), to form colored products, in the absence of exogenously added H2O2. The
presence of sulte inhibited the TMBO2Co3O4 NPs reaction system and caused a change in color of the reaction system. On
the basis of this phenomenon, a colormetric approach to detect sulte was established with a good linear relationship ranging
from 0.2 106 to 1.6 105 M and a detection limit of 5.3 108 M. The method was used to detect sulte in foods. Good
recoveries ranging from 93.8% to 100.5% were obtained. Furthermore, the mechanism was studied and results showed that the
oxidase-like activity of the Co3O4 NPs was not from OH or O2 radical generated. It may probably originate from their ability
to transfer an electron between the peroxidase substrate and oxygen absorbed on the surface of the Co3O4 NPs.
KEYWORDS: Co3O4 nanoparticles, oxidase mimic, colorimetry, sulte, mechanism of oxidase-like activity

INTRODUCTION
Sulte is one of the oldest and most ubiquitous food additives,
which has been widely used as blanching and preservative
agents in a large variety of foodstus (dried fruit, vermicelli,
preserves, etc.) to improve the appearance of food and prevent
bacterial growth.1,2 However, the ingestion of foods containing
large amounts of sulte can cause asthmatic attacks and allergic
reactions in sensitive individuals.3,4 Owing to the potential
toxicity, the sulte in foods is strictly limited in many
countries.5 Hence, sulte determination in food products is
essential for the purposes of public health, food assurance, and
quality control. In recent years, numerous methods have been
developed for determining sulte in foods, including titrimetry,
ow injection analysis, anodic stripping voltammetry, ion
chromatography, chemiluminescence, gas chromatography,
uorometry, high-performance liquid chromatography, and
colorimetry.68
Colorimetric biosensing has become more attractive because
nanomaterials,18,19 metal nanomaterials,13,20 carbon nanoma-
terials,13,20 nanocomposites,2128 and other nanomaterials.2931
These enzyme-like nanomaterials could be employed in
colorimetry to detect nucleotides,32 H2O2,3335 glucose,3335
melamine,36 and so on. To the best of our knowledge, however,
the colorimetric detection of sulte in food using the enzyme-
like property of nanoparticles has not been explored up to now,
which might be attributed to the complication of food samples
matrices and the diculties in constructing an eective
nanoparticles-based enzyme mimic sensing platform to detect
micro amounts of food additive.
Herein, we have developed a TMBO2Co3O4 NPs
colorimetric system for detection of sulte in foods, based on
the property of oxidase-like activity of Co3O4 NPs. In this
method, the existence of sulte can cause a change in color of
the reaction system, and the color change can be visually
observed directly. We also investigated the mechanism of
oxidase-like activity of Co3O4 NPs by XPS, contrast experi-
ments, and cyclic voltammetry.

of its low cost, simplicity, and practicality. However, the key
challenge for colorimetric sensors is transforming the detection
events into color changes. Since color changes can be read by
the naked eye, a colorimetric sensor does not require expensive
or sophisticated instrumentation and can be applied to eld
analysis.911 To this end, enzyme-like nanomaterials-based
sensors have emerged as an important colorimetric tool. Since
the rst report of Fe3O4 magnetic nanoparticles (MNPs)
possessing intrinsic peroxidase-like activity,12 nanoparticles-
based enzyme mimics have received great attention due to their
merits relative to native enzymes (e.g., low cost, high stability,
and tunability in catalytic activity). More and more nano-
particles have been found as enzymatic mimics, such as metal
oxide nanomaterials,13 sulde nanomaterials,1417 selenide
EXPERIMENTAL PROCEDURES
Materials. Co(CH3COO)24H2O were obtained from Tianjin
Guangfu Chemical Reagent Factory (Tianjin, China). 3,3,5,5-
Tetramethylbenzidine (TMB) and 2,2-azinobis(3-ethylbenzothiazo-
line-6-sulfonic acid) diammonium salt (ABTS) were purchased from
Sigma-Aldrich. Ammonium hydroxide (2528 wt %) was purchased
from Baiyin Liangyou Chemical Reagent Factory (Baiyin, China).
Sodium acetate, glacial acetic acid, anhydrous sodium sulte, sodium
Received: February 23, 2014
Revised: May 28, 2014
Accepted: June 2, 2014

XXXX American Chemical Society A dx.doi.org/10.1021/jf500950p | J. Agric. Food Chem. XXXX, XXX, XXXXXX
Journal of Agricultural and Food Chemistry Article

Figure 1. Images of the oxidation color reaction of TMB and ABTS by Co3O4 NPs (a). UV/vis spectra of TMB solution in 0.2 M acetic acidacetate
buer (pH 4.0) at 40 C for 10 min. The TMB and Co3O4 NPs concentrations were 0.25 mM and 40 g mL1, respectively (b). UV/vis spectra of
an ABTS solution in 0.2 M acetic acidacetate buer (pH 4.0) at 40 C for 10 min. The ABTS and Co3O4 NPs concentrations were 0.25 mM and
40 g mL1, respectively (c).

Figure 2. Survey XP spectra of Co3O4 NPs before and after reaction (a and d). Deconvoluted high-resolution Co 2p3/2 XP spectra of Co3O4 NPs
before and after reaction (b and e). Deconvoluted high-resolution O 1s XP spectra of Co3O4 NPs before and after reaction (c and f).

dihydrogen phosphate, and other regents were obtained from Tianjin
Guangfu Chemical Reagent Factory (Tianjin, China). A sulte
solution was prepared daily by dissolving anhydrous sodium sulte
in water and was standardized by iodimetric titration when needed. All
other chemicals were of analytical reagent grade and used without
further purication. Deionized water was used throughout the
experiment.
Apparatus and Characterization. A TU-1901 double-beam
UVvis spectrophotometer (Beijing Purkine General Instrument Co.
Ltd., China) was used to record absorption spectra and measure
absorbance. A FEI Tecnai G2 F30 microscope (TEM) was used for
measuring the morphologies and size of Co3O4 NPs. The X-ray
diraction (XRD) pattern of Co3O4 NPs was determined on a Rigaku
D/Max-2400 X-ray diractometer with Cu K radiation ( =
0.154056). The chemical state of the coating surface was analyzed
using X-ray photoelectron spectroscopy (XPS, PerkinElmer PHI-5702
multifunctional photoelectron spectrometer). Element determination
was measured by a 4300 DV inductively coupled plasmaatomic
emission spectrometer (ICP-AES). Zeta potential measurements of
Co3O4 NPs were performed with a Malvern Nano-ZS apparatus. ESR
spectra were acquired using a JES-FA200 electron spin resonance
spectrometer. Electrochemical experiments were performed on a CHI
832 electrochemical workstation (Shanghai Chenhua Instrument Co.,
China) and a conventional three-electrode system. The conventional
three-electrode system contained a Pt wire counter electrode, a
saturated calomel electrode as reference electrode, and an as-prepared

B dx.doi.org/10.1021/jf500950p | J. Agric. Food Chem. XXXX, XXX, XXXXXX

Journal of Agricultural and Food Chemistry
working electrode. The specic surface area was measured using a
multipoint BrunauerEmmettTeller technique (ASAP 2020, Micro-
meritics, USA).
Preparation of Co3O4 Nanoparticles. Co3O4 NPs were prepared
using a hydrothermal method as described in the literature.37 Briey,
0.5 g of Co(CH3COO)24H2O was dissolved in 25 mL of ethanol, and
then 2.5 mL of ammonia solution was added under vigorous stirring.
After stirring for 10 min, the obtained suspension was transferred into
a 50 mL autoclave. Then the autoclave was sealed and kept at 150 C
for 3 h followed by cooling to room temperature naturally. Finally, the
product was separated via centrifugation and alternately washed
thoroughly several times with deionized water and ethanol until the
pH of supernatant was about 7.0, then dried at 60 C for 4 h.
Kinetic Assay of Oxidase-like Co3O4 NPs. Kinetic experiments
were carried out at 40 C using 120 L of 1.0 mg mL1 Co3O4 NPs in
2.8 mL of acetic acidacetate buer solution (0.2 M, pH 4.0) with 80
L of TMB/ABTS as substrate, unless otherwise stated. Kinetic
measurements were carried out in time course mode by monitoring
the absorbance change at 652/417 nm for 4 min using a TU-1901
UVvis spectrophotometer. The MichaelisMenten constant was
calculated using a LineweaverBurk plot:12
1/v = K m/Vmax(1/[S] + 1/K m)
where is the initial velocity, Vmax represents the maximal reaction
velocity, and [S] is the substrate concentration. Km is the Michaelis
Menten constant, which is an indicator of enzyme anity for its
substrate.
Preparation of Samples. Dried lily bulbs, vermicelli, and haw roll
were purchased randomly from a local market in Lanzhou, China.
Samples were minced by a mixer. An accurately weighed minced
sample was transferred to a beaker, and 40 mL of EDTA (1.0 104
M) was added.5 After shaking and ultrasonication for 15 min, the
mixture was ltered with a 0.45 m syringe membrane lter. The
ltrate was diluted to 100 mL with EDTA (1.0 104 M). All samples
were stored in a refrigerator at 4 C before analysis.
Detection of Sulte. The amount of sulte in samples was
determined according to the following steps: (a) 80.0 L of sample
solution, 150.0 L of 5 mM TMB, and 120.0 L of Co3O4 NPs
solution (1.0 mg mL1) were added into 2.65 mL of a 0.2 M acetic
acidacetate buer solution (pH 4.0) and then thoroughly mixed to
make it uniform; (b) the as-mixed solution was maintained in a 40 C
water bath for 10 min to hold the reaction and then kept in an ice
water bath for 10 min to terminate the reaction. The solution was then
used to perform the adsorption spectroscopy measurement at 652 nm
wavelength. For the control experiments, 80.0 L of deionized water
was used instead of sample solution.
RESULTS AND DISCUSSION
Oxidase-like Activity of Co3O4 NPs. The oxidase-like
activity of Co3O4 NPs was evaluated in the catalysis of typical
substrates TMB and ABTS in the absence of H2O2. As can be
seen from Figure 1a, Co3O4 NPs catalyzed the oxidation of
TMB and ABTS to produce the typical colored products.
Figure 3. Photographs of TMB solutions in the absence and presence
of an O2 oxidation reaction catalyzed by Co3O4 NPs (a). Photographs
of a TMB solution oxidation reaction catalyzed by Co3O4 NPs without
degassing and Co3O4 NPs degassed by three freezepumpthaw
cycles (b). Photographs of the oxidation color reaction of TMB, TMB-
SO32, and TMB-SO42 catalyzed by Co3O4 NPs, respectively (c).
C dx.doi.org/10.1021/jf500950p | J. Agric. Food Chem. XXXX, XXX, XXXXXX
Article
Figure 4. Cyclic voltammograms of bare GCE (a) and the Co3O4
NPs-modied GCE (b) in a 0.2 M acetic acidacetate buer (pH 4.0)
+ 0.25 mM TMB solution.
Similar to other peroxidase/oxidase mimic reactions, the
maximum absorbance peaks of the oxidation products of
TMB and ABTS were located at 652 and 417 nm,
respectively.12,33 Nevertheless, there was no absorbance at
652 or 417 nm in the absence of Co3O4 NPs in the system
(Figure 1b and c). The results suggested that Co3O4 NPs
exhibit intrinsic oxidase-like activity. The presence of mixed
valence states of Co2+ and Co3+ ions in Co3O4 NPs is
considered the key in their catalysis. As an oxidase mimic, the
states of Co in the Co3O4 NPs did not change before or after
the reaction.
In order to further conrm the property of oxidase mimic of
the Co3O4 NPs, XPS was performed to nd the oxidation state
of Co in the Co3O4 NPs before and after the reaction. Figure 2e
shows the deconvoluted XPS spectra of Co 2p3/2 of Co3O4 NPs
after the reaction. The relative ratio of Co2+ and Co3+ was
almost unchanged in Co3O4 NPs before and after the reaction.
The high-resolution XPS spectra O 1s of unused and used
Co3O4 NPs are presented in Figure 2c and f, respectively. The
relative ratio of OCo2+ and OCo3+ peaks in O 1s XPS
spectra was similar to Co 2p3/2 XPS results (Table S1). It is
suggested that the states of Co in the Co3O4 NPs did not
change before or after the reaction. These results conrmed
that Co3O4 NPs exhibited intrinsic oxidase-like activity.
It is important to conrm the possibility that the observed
activity was not caused by cobalt ions leaching from Co3O4 NPs
in acidic solution. To test this, Co3O4 NPs were incubated in
the reaction buer (pH 4.0) for 2 h and then removed from the
solution by centrifugation. As shown in Figure S5, comparing
the activity of the leaching solution with that of Co3O4 NPs
under the same conditions, the leaching solution had no
activity, showing that the observed oxidase-like activity was due
to intact Co3O4 NPs. Figure S6 shows the amount of cobalt
ions leaked from Co3O4 NPs in the buer solutions (pH 3.0
7.0) for 2 h, which was implemented by ICP-OES. The ICP
results exhibited that there were few cobalt ions in the leaching
solution. They probably came from residual Co3O4 NPs after
centrifugation.
Possible Mechanism of Oxidase-like Activity of Co3O4
NPs. As an oxidase mimic, Co3O4 NPs catalyze an oxidation
reduction reaction involving oxygen (O2) as the electron
acceptor. However, in the reaction system of TMBO2Co3O4
NPs, oxygen is present in the aqueous solution and also exists
on the surface of Co3O4 NPs due to the high specic surface
Journal of Agricultural and Food Chemistry
Scheme 1. Possible Mechanism for the TMBO2Co3O4 NPs System
Figure 5. MichaelisMenten curve of Co3O4 NPs with TMB (a). MichaelisMenten curve of Co3O4 NPs with ABTS (b). Details are included in
the Experimental Procedures.
area of nanomaterials. In order to determine whether the
oxygen in the solution played a role in the TMBO2Co3O4
NPs system, two sets of experiments were conducted: the rst
one was carried out in the presence of oxygen; the other was
performed under high-purity nitrogen and the solution was
degassed before the reaction, as in the photograph shown in
Figure 3a. It was found that they exhibited the same color. The
absorbance of the rst one was 0.747; the other was 0.770 at
652 nm. The absorbance of the latter was higher than the
former; one possible reason was that nitrogen played a role in
mixing and speeded up the reaction. This indicated that the
oxygen in the solution had no eect on the reaction, which was
dierent from the behavior of some nanoparticles-based oxidase
mimics reported.8,38 For further study, the Co3O4 NPs without
degassing and Co3O4 NPs degassed by three freezepump
thaw cycles that catalyze the oxidation of TMB were
implemented in the degassed solutions. They produced
dierent colors, as in the photograph shown in Figure 3b,
and the degassed Co3O4 NPs also catalyzed the oxidation of
TMB to produce the typical color reaction. It is suggested that
the oxygen on the surface of Co3O4 NPs was due not only to
physical adsorption. Chemisorbed oxygen probably existed on
the surface of Co3O4 NPs because of the low oxygen CoO
D dx.doi.org/10.1021/jf500950p | J. Agric. Food Chem. XXXX, XXX, XXXXXX
Article
bond strength of Co3O4 and the larger surface energy of
nanomaterials caused by a large surface to volume ratio.39 In
addition, the O 1s peak at 533.0 eV in the XPS spectrum shown
in Figure 2c indicated that there was chemisorbed oxygen on
the surface of Co3O4 NPs.40 It is indicated that both the
physically absorbed and chemisorbed oxygen on the surface of
Co3O4 NPs played a key role in the TMBO2Co3O4 NPs
system. In order to further conrm the above conclusion,
oxidation experiments of TMB, TMB-SO32, and TMB-SO42
catalyzed by Co3O4 NPs were carried out. The results are
shown in Figure 3c. TMB-SO32 system presented a lower
absorbance than the other two systems. Since the volume of
SO32 is smaller than TMB, and Co3O4 NPs have a positive
charge at pH 4.0 (Supporting Information), SO32 is more
easily adsorbed on the surface of Co3O4 NPs. Then SO32
consumed oxygen absorbed on the surface of the Co3O4 NPs
and interfered with the TMBO2Co3O4 NPs system. This
supported the above result that the oxygen absorbed on the
surface of the Co3O4 NPs oxidized TMB in this system.
The oxidation of TMB is an electron transfer process.41 The
electrocatalytic behavior of a Co3O4 NP modied glassy carbon
electrode (GCE) toward the electrochemical oxidation of TMB
was studied using cyclic voltammetry (CV), in order to
Journal of Agricultural and Food Chemistry Article

Figure 6. A pHA curve (a), a temperatureA curve (b), a TMB concentrationA curve (c), and a Co3O4 NPs concentrationA curve (d)
for 6 M sulte detection, where A = A(blank,652nm) A(sulfite,652nm), in 0.2 M acetic acidacetate buer solutions incubated for 10 min with the as-
prepared Co3O4 NPs as oxidase mimics.

Figure 8. A response of TMB solutions to blank and dierent

common ions in the presence of sulte (12 M). The other ions added
Figure 7. Doseresponse curve for dierent concentrations of sulte are 240 M. Relative A: (Atotal Asulfite)/Asulfite.
standard solutions under the optimum conditions. Inset: Linear
calibration plot for sulte detection. Table 1. Results for the Determination of the Sulte in
Three Kinds of Foods
investigate the mechanism of oxidase-like catalytic activity of
Co3O4 NPs. The CVs of TMB at a bare GCE and the Co3O4 initial added average RSD
NPs-modied GCE are shown in Figure 4. Two pairs of redox amount (g recovery (n = 3)
sample (mg g1) mL1) recovery (%) (%) (%)
peaks of TMB were found under the bare GCE, and increased
current was obviously observed under the Co3O4 NPs-modied dried lily 1.7 0.5 96.399.4 98.9 0.6
bulbs 1.0 94.499.5 96.5 2.8
GCE. It is suggested that Co3O4 NPs exhibited an electro-
catalytic activity to oxidate TMB, which indicated that the 1.5 98.4100.6 100.3 2.6
Co3O4 NPs had the ability to accelerate the electron transfer.42 vermicelli 0.7 0.5 90.595.0 93.1 2.5
In order to further demonstrate that the oxidase-like activity 1.0 91.696.2 93.8 2.5
of the Co3O4 NPs was not from O2 and OH, an EPR spin 1.5 96.897.4 97.1 1.5
trapping experiment was carried out under the optimized haw roll 2.9 0.5 95.299.4 96.3 1.1
experimental conditions. The results revealed that no DMPO- 1.0 99.1101.6 100.5 3.3
O2 or DMPO-OH spin adducts were detected (Figure S7). 1.5 97.899.9 98.9 1.1

E dx.doi.org/10.1021/jf500950p | J. Agric. Food Chem. XXXX, XXX, XXXXXX

Journal of Agricultural and Food Chemistry
It is suggested that TMB was not oxidized by O2 and OH
radicals in the TMBO2Co3O4 NPs system, which was
dierent from the behavior of CoFe2O4 NPs.8 Therefore, the
nature of oxidase-like activity of the Co3O4 NPs does not
originate from the O2 and OH radicals generated.
Taken together, although the detailed mechanism remains
unclear, these observations demonstrated that Co3O4 NPs
accelerated the electron transfer from the peroxidase substrate
to oxygen absorbed on the surface of the Co3O4 NPs. The
possible mechanism of oxidase-like activity of Co3O4 NPs is
presented in Scheme 1; that is, TMB was absorbed on the
surface of the Co3O4 NPs and donated electrons to the Co3O4
NPs, resulting in an increase in electron density and mobility in
the Co3O4 NPs, thus accelerating the electron transfer to
oxygen absorbed on the surface of the Co3O4 NPs. After the
completion of the electron transfer, the Co3O4 NPs reverted to
their original state.
Steady-State Kinetic Assay of Co3O4 NPs. Since Co3O4
NPs exhibited oxidase-like activity, the kinetic parameters of
only TMB/ABTS were determined by changing the concen-
tration of TMB/ABTS. A typical MichaelisMenten curve
(Figure 5a,b) was obtained with TMB/ABTS by monitoring
the absorbance change at 652/417 nm for 4 min (Figure
S8a,b). The MichaelisMenten constant (Km) and maximum
initial velocity (Vmax) were obtained using a LineweaverBurk
plot. The values of Km and Vmax for the Co3O4 NPs with TMB
were 0.051 mM and 3.30 108 M s1. The values of Km and
Vmax for the Co3O4 NPs with ABTS were 0.037 mM and 3.20
108 M s1. The Km value of Co3O4 NPs with ABTS was
smaller than that with TMB. It is suggested that Co3O4 NPs
have higher anity toward ABTS than TMB, because ABTS
has a higher anity toward a positively charged nanoparticle
surface and TMB has a stronger anity toward a negatively
charged nanoparticle surface.43
Optimization of Experimental Conditions. The reaction
conditions were optimized to establish the optimum analytical
conditions. The pH value not only decides the charge of the
surface of Co3O4 NPs (Supporting Information), which aects
the interaction between Co3O4 NPs and sulte, but also aects
the catalytic activity of Co3O4 NPs. The catalytic activity of
Co3O4 NPs is also dependent on the incubation temperature.
In addition, the concentrations of TMB and Co3O4 NPs
determine the color level of the reaction system. Therefore,
they were investigated in this study as inuencing factors.
Figure 6a indicates that A, where A = A(blank,652nm)
A(sulfite,652nm), increased and then decreased with increasing pH
and had a maximum at 4.0. The reaction temperature-
dependent response curve is shown in Figure 6b. It showed
that A of the reaction system had a peak value at 40 C.
Therefore, 4.0 and 40 C were taken as the optimal pH and
temperature. The concentrations of TMB and Co3O4 NPs
response curves are shown in Figure 6c and d; the optimal
concentrations of TMB and Co3O4 NPs were 0.25 mM and 40
g mL1, respectively.
Analytical Performance for Sulte Determination. As
an oxidase mimic, Co3O4 NPs can catalyze TMB to form
colored product in the absence of H2O2. The sulte inhibits the
TMBO2Co3O4 NPs system and causes a change in color of
the reaction system, which suggested that sulte can be
indirectly detected. Since sulte is easily adsorbed on the
surface of Co3O4 NPs and consumes absorbed oxygen more
easily than dissolved oxygen, the sulte that is consumed by
dissolved oxygen is neglected in the reaction. The calibration
F dx.doi.org/10.1021/jf500950p | J. Agric. Food Chem. XXXX, XXX, XXXXXX
Article
graphs for the determination of sulte were constructed under
the optimum conditions described above. The calibration data
of A versus sulte concentration are shown in Figure 7. The
results indicated that the concentration of detectible sulte
could be 0.2 106 M with a linear range from 0.2 106 to
1.6 105 M. The limit of detection (LOD) for sulte was 5.3
108 M. (LOD = KS0/S, where K is a numerical factor chosen
according to the condence level desired, S0 is the standard
deviation (SD) of the blank measurements (n = 10, K = 3), and
S is the slope of the calibration curve.44)
Eect of Foreign Substances. The eect of foreign
substances was tested by analyzing the mixed solution and
standard solution of sulte (12 M). The molar ratio of other
ion and sulte was 20 in the mixed solution. The relative A
values ((Atotal Asulfite)/Asulfite) are presented in Figure 8.
As can be seen, Fe and Cu were the main interferences for
sulte determination, since trace amounts of heavy metal ions
in the water are believed to act as the catalyst in the oxidation
of sulte by oxygen.45 EDTA was selected as a chelate reagent
for the present study to eliminate the interferences derived
from Fe, Cu, and other coexisting transient metals in the
samples.
Detection of Sulte in Real Samples. As an illustration
of analytical application, the proposed method was used to
determine sulte in real samples under the optimal
experimental conditions. The results are listed in Table 1.
The recoveries of the three samples ranged from 93.8% to
100.5%. Hence, the proposed method can be considered a
useful tool for the quantication of sultes in real samples.
In summary, Co3O4 NPs were rst found to possess oxidase-
like activity and could catalyze the oxidation of TMB and ABTS
to produce the typical color reaction in the absence of
exogenously added H2O2. It was found that the oxygen
absorbed on the surface of the Co3O4 NPs oxidizes the
peroxidase substrate, rather than the oxygen dissolved in the
solution. The oxidase-like activity of Co3O4 NPs was not from
the generated O2 or OH radical, probably originating from
their ability to transfer an electron between the peroxidase
substrate and oxygen absorbed on the surface of the Co3O4
NPs. Moreover, as the existence of sulte inhibited TMBO2
Co3O4 NPs system and caused a change in color of the reaction
system, a new colorimetric method for detecting sulte was
developed. The colorimetric method showed a good response
toward sulte detection with a linear range from 0.2 106 to
1.6 105 M and good recoveries ranging from 93.8% to
100.5% in three real samples using Co3O4 NPs as an oxidase
mimic. The detection platform showed great potential
applications in reducing substrates. In addition, Co3O4 NPs
can be expected to provide potential applications for chemical
sensing, biochemical analysis, electrochemical biosensing, and
even clinical chemistry.
ASSOCIATED CONTENT
* Supporting Information
S
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: chenxg@lzu.edu.cn (X.-G. Chen). Tel: 86-931-
8912763 Fax: 86-931-8912582.
Journal of Agricultural and Food Chemistry
Funding
The authors are grateful for nancial support from the National
Natural Science Foundation of China (No. 21375053) and the
Specialized Research Fund for the Doctoral Program of Higher
Education (No. 20130211110039).
Notes
The authors declare no competing nancial interest.
REFERENCES
(1) Bush, R. K. Sulfites in foods: uses, analytical methods, residues,
fate, exposure assessment, metabolism, toxicity, and hypersensitivity.
Adv. Food Res. 1986, 30, 176.
(2) Sapers, G. Browning of foods: control by sulfites, antioxidants,
and other means. Food Technol. (Chicago, IL, U.S.) 1993, 47, 7584.
(3) Adams, J. Food additive-additive interactions involving sulphur
dioxide and ascorbic and nitrous acids: a review. Food Chem. 1997, 59,
401409.
(4) Vally, H.; Carr, A.; El-Saleh, J.; Thompson, P. Wine-induced
asthma: a placebo-controlled assessment of its pathogenesis. J. Allergy
Clin. Immunol. 1999, 103, 4146.
(5) Filik, H.; C etintas, G. Determination of sulfite in water and dried
fruit samples by dispersive liquidliquid microextraction combined
with UVvis fiber optic linear array spectrophotometry. Food Anal.
Methods 2012, 5, 13621367.
(6) Du, J.; Shao, Q.; Yin, S.; Jiang, L.; Ma, J.; Chen, X. Colorimetric
chemodosimeter based on diazonium-gold-nanoparticle complexes for
sulfite ion detection in solution. Small 2012, 8, 34123416.
(7) Pundir, C. S.; Rawal, R. Determination of sulfite with emphasis
on biosensing methods: a review. Anal. Bioanal. Chem. 2013, 405,
30493062.
(8) Zhang, X.; He, S.; Chen, Z.; Huang, Y. CoFe2O4 nanoparticles as
oxidase mimic-mediated chemiluminescence of aqueous luminol for
sulfite in white wines. J. Agric. Food Chem. 2013, 61, 840847.
(9) Stewart, M. E.; Anderton, C. R.; Thompson, L. B.; Maria, J.;
Gray, S. K.; Rogers, J. A.; Nuzzo, R. G. Nanostructured plasmonic
sensors. Chem. Rev. 2008, 108, 494521.
(10) Liu, J.; Cao, Z.; Lu, Y. Functional nucleic acid sensors. Chem.
Rev. 2009, 109, 19481998.
(11) Song, S.; Qin, Y.; He, Y.; Huang, Q.; Fan, C.; Chen, H.-Y.
Functional nanoprobes for ultrasensitive detection of biomolecules.
Chem. Soc. Rev. 2010, 39, 42344243.
(12) Gao, L.; Zhuang, J.; Nie, L.; Zhang, J.; Zhang, Y.; Gu, N.; Wang,
T.; Feng, J.; Yang, D.; Perrett, S. Intrinsic peroxidase-like activity of
ferromagnetic nanoparticles. Nat. Nanotechnol. 2007, 2, 577583.
(13) Wei, H.; Wang, E. Nanomaterials with enzyme-like character-
istics (nanozymes): next-generation artificial enzymes. Chem. Soc. Rev.
2013, 42, 60606093.
(14) Dai, Z.; Liu, S.; Bao, J.; Ju, H. Nanostructured FeS as a mimic
peroxidase for biocatalysis and biosensing. Chem.Eur. J. 2009, 15,
43214326.
(15) He, W.; Jia, H.; Li, X.; Lei, Y.; Li, J.; Zhao, H.; Mi, L.; Zhang, L.;
Zheng, Z. Understanding the formation of CuS concave super-
structures with peroxidase-like activity. Nanoscale 2012, 4, 35013506.
(16) Maji, S. K.; Dutta, A. K.; Dutta, S.; Srivastava, D. N.; Paul, P.;
Mondal, A.; Adhikary, B. Single-source precursor approach for the
preparation of CdS nanoparticles and their photocatalytic and intrinsic
peroxidase like activity. Appl. Catal. B: Environ. 2012, 126, 265274.
(17) Maji, S. K.; Dutta, A. K.; Srivastava, D. N.; Paul, P.; Mondal, A.;
Adhikary, B. Peroxidase-like behavior, amperometric biosensing of
hydrogen peroxide and photocatalytic activity by cadmium sulfide
nanoparticles. J. Mol. Catal. A: Chem. 2012, 358, 19.
(18) Dutta, A. K.; Maji, S. K.; Mondal, A.; Karmakar, B.; Biswas, P.;
Adhikary, B. Iron selenide thin film: peroxidase-like behavior, glucose
detection and amperometric sensing of hydrogen peroxide. Sens.
Actuators, B 2012, 173, 724731.
(19) Dutta, A. K.; Maji, S. K.; Srivastava, D. N.; Mondal, A.; Biswas,
P.; Paul, P.; Adhikary, B. Synthesis of FeS and FeSe nanoparticles from
a single source precursor: a study of their photocatalytic activity,
G dx.doi.org/10.1021/jf500950p | J. Agric. Food Chem. XXXX, XXX, XXXXXX
Article
peroxidase-like behavior, and electrochemical sensing of H2O2. ACS
Appl. Mater. Interfaces 2012, 4, 19191927.
(20) Xie, J.; Zhang, X.; Wang, H.; Zheng, H.; Huang, Y. Analytical
and environmental applications of nanoparticles as enzyme mimetics.
TrAC Trends Anal. Chem. 2012, 39, 114129.
(21) Novoselov, K. S.; Geim, A. K.; Morozov, S.; Jiang, D.; Zhang, Y.;
Dubonos, S.; Grigorieva, I.; Firsov, A. Electric field effect in atomically
thin carbon films. Science 2004, 306, 666669.
(22) Chen, H.; Li, Y.; Zhang, F.; Zhang, G.; Fan, X. Graphene
supported Au-Pd bimetallic nanoparticles with core-shell structures
and superior peroxidase-like activities. J. Mater. Chem. 2011, 21,
1765817661.
(23) Kim, M. I.; Shim, J.; Li, T.; Lee, J.; Park, H. G. Fabrication of
nanoporous nanocomposites entrapping Fe3O4 magnetic nanoparticles
and oxidases for colorimetric biosensing. Chem.Eur. J. 2011, 17,
1070010707.
(24) Ye, Y.; Kong, T.; Yu, X.; Wu, Y.; Zhang, K.; Wang, X. Enhanced
nonenzymatic hydrogen peroxide sensing with reduced graphene
oxide/ferroferric oxide nanocomposites. Talanta 2012, 89, 417421.
(25) Hao, J.; Zhang, Z.; Yang, W.; Lu, B.; Ke, X.; Zhang, B.; Tang, J.
In situ controllable growth of CoFe2O4 ferrite nanocubes on graphene
for colorimetric detection of hydrogen peroxide. J. Mater. Chem. A
2013, 1, 43524357.
(26) Jiang, Y.; Wang, W.; Li, X.; Wang, X.; Zhou, J.; Mu, X. Enzyme-
mimetic catalyst-modified nanoporous SiO2cellulose hybrid compo-
sites with high specific surface area for rapid H2O2 detection. ACS
Appl. Mater. Interfaces 2013, 5, 19131916.
(27) Lin, Y.; Li, Z.; Chen, Z.; Ren, J.; Qu, X. Mesoporous silica-
encapsulated gold nanoparticles as artificial enzymes for self-activated
cascade catalysis. Biomaterials 2013, 34, 26002610.
(28) Lin, Y.; Zhao, A.; Tao, Y.; Ren, J.; Qu, X. Ionic liquid as an
efficient modulator on artificial enzyme system: toward the realization
of high-temperature catalytic reactions. J. Am. Chem. Soc. 2013, 135,
42074210.
(29) Wang, J.; Han, D.; Wang, X.; Qi, B.; Zhao, M. Polyoxometalates
as peroxidase mimetics and their applications in H2O2 and glucose
detection. Biosens. Bioelectron. 2012, 36, 1821.
(30) Wang, W.; Jiang, X.; Chen, K. CePO4: Tb, Gd hollow
nanospheres as peroxidase mimic and magneticfluorescent imaging
agent. Chem. Commun. 2012, 48, 68396841.
(31) Wang, W.; Jiang, X.; Chen, K. Iron phosphate microflowers as
peroxidase mimic and superoxide dismutase mimic for biocatalysis and
biosensing. Chem. Commun. 2012, 48, 72897291.
(32) Song, Y.; Wang, X.; Zhao, C.; Qu, K.; Ren, J.; Qu, X. Label-free
colorimetric detection of single nucleotide polymorphism by using
single-walled carbon nanotube intrinsic peroxidase-like activity.
Chem.Eur. J. 2010, 16, 36173621.
(33) Wei, H.; Wang, E. Fe3O4 magnetic nanoparticles as peroxidase
mimetics and their applications in H2O2 and glucose detection. Anal.
Chem. 2008, 80, 22502254.
(34) Song, Y.; Qu, K.; Zhao, C.; Ren, J.; Qu, X. Graphene oxide:
intrinsic peroxidase catalytic activity and its application to glucose
detection. Adv. Mater. 2010, 22, 22062210.
(35) Su, L.; Feng, J.; Zhou, X.; Ren, C.; Li, H.; Chen, X. Colorimetric
detection of urine glucose based znfe2o4 magnetic nanoparticles. Anal.
Chem. 2012, 84, 57535758.
(36) Ding, N.; Yan, N.; Ren, C.; Chen, X. Colorimetric
determination of melamine in dairy products by Fe3O4 magnetic
nanoparticlesH2O2ABTS detection system. Anal. Chem. 2010, 82,
58975899.
(37) Dong, Y.; He, K.; Yin, L.; Zhang, A. A facile route to controlled
synthesis of Co3O4 nanoparticles and their environmental catalytic
properties. Nanotechnology 2007, 18, 435602.
(38) Zhao, J.; Xie, Y.; Yuan, W.; Li, D.; Liu, S.; Zheng, B.; Hou, W. A
hierarchical CoFe LDH rope-like nanostructure: facile preparation
from hexagonal lyotropic liquid crystals and intrinsic oxidase-like
catalytic activity. J. Mater. Chem. B 2013, 1, 12631269.
Journal of Agricultural and Food Chemistry Article

(39) Tang, X.; Li, J.; Hao, J. Synthesis and characterization of spinel
Co3O4 octahedra enclosed by the {111} facets. Mater. Res. Bull. 2008,
43, 29122918.
(40) Yan, Q.; Li, X.; Zhao, Q.; Chen, G. Shape-controlled fabrication
of the porous Co3O4 nanoflower clusters for efficient catalytic
oxidation of gaseous toluene. J. Hazard. Mater. 2012, 209, 385391.
(41) Josephy, P. D.; Mason, R. P.; Eling, T. Cooxidation of the
clinical reagent 3,5,3,5-tetramethylbenzidine by prostaglandin
synthase. Cancer Res. 1982, 42, 25672570.
(42) Mu, J.; Wang, Y.; Zhao, M.; Zhang, L. Intrinsic peroxidase-like
activity and catalase-like activity of Co3O4 nanoparticles. Chem.
Commun. 2012, 48, 25402542.
(43) Yu, F.; Huang, Y.; Cole, A. J.; Yang, V. C. The artificial
peroxidase activity of magnetic iron oxide nanoparticles and its
application to glucose detection. Biomaterials 2009, 30, 47164722.
(44) Chen, J.; Chen, H.; Zhou, C.; Xu, J.; Yuan, F.; Wang, L. An
efficient upconversion luminescence energy transfer system for
determination of trace amounts of nitrite based on NaYF4Yb 3+ Er
3+
as donor. Anal. Chim. Acta 2012, 713, 111114.
(45) Humphrey, R. E.; Ward, M. H.; Hinze, W. Spectrophotometric
determination of sulfite with 4, 4-dithio-dipyridine and 5,5-dithiobis
(2-nitrobenzoic acid). Anal. Chem. 1970, 42, 698702.

H dx.doi.org/10.1021/jf500950p | J. Agric. Food Chem. XXXX, XXX, XXXXXX