Article

pubs.acs.org/JAFC

Colorimetric Detection of Sulfite in Foods by a TMB−O2−Co3O4

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 first 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 sulfite inhibited the TMB−O2−Co3O4 NPs reaction system and caused a change in color of the reaction system. On
the basis of this phenomenon, a colormetric approach to detect sulfite was established with a good linear relationship ranging
from 0.2 × 10−6 to 1.6 × 10−5 M and a detection limit of 5.3 × 10−8 M. The method was used to detect sulfite 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, sulfite, mechanism of oxidase-like activity

■ INTRODUCTION
Sulfite 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 foodstuffs (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 sulfite can cause asthmatic attacks and allergic
reactions in sensitive individuals.3,4 Owing to the potential
toxicity, the sulfite in foods is strictly limited in many
countries.5 Hence, sulfite 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 sulfite in foods, including titrimetry,
flow injection analysis, anodic stripping voltammetry, ion
chromatography, chemiluminescence, gas chromatography,
fluorometry, high-performance liquid chromatography, and
colorimetry.6−8
Colorimetric biosensing has become more attractive because
nanomaterials,18,19 metal nanomaterials,13,20 carbon nanoma-
terials,13,20 nanocomposites,21−28 and other nanomaterials.29−31
These enzyme-like nanomaterials could be employed in
colorimetry to detect nucleotides,32 H2O2,33−35 glucose,33−35
melamine,36 and so on. To the best of our knowledge, however,
the colorimetric detection of sulfite 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 difficulties in constructing an effective
nanoparticles-based enzyme mimic sensing platform to detect
micro amounts of food additive.
Herein, we have developed a TMB−O2−Co3O4 NPs
colorimetric system for detection of sulfite in foods, based on
the property of oxidase-like activity of Co3O4 NPs. In this
method, the existence of sulfite 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 EXPERIMENTAL PROCEDURES
events into color changes. Since color changes can be read by
Materials. Co(CH3COO)2·4H2O were obtained from Tianjin
the naked eye, a colorimetric sensor does not require expensive Guangfu Chemical Reagent Factory (Tianjin, China). 3,3′,5,5′-
or sophisticated instrumentation and can be applied to field Tetramethylbenzidine (TMB) and 2,2′-azinobis(3-ethylbenzothiazo-
analysis.9−11 To this end, enzyme-like nanomaterials-based line-6-sulfonic acid) diammonium salt (ABTS) were purchased from
sensors have emerged as an important colorimetric tool. Since Sigma-Aldrich. Ammonium hydroxide (25−28 wt %) was purchased
the first report of Fe3O4 magnetic nanoparticles (MNPs) from Baiyin Liangyou Chemical Reagent Factory (Baiyin, China).
possessing intrinsic peroxidase-like activity,12 nanoparticles- Sodium acetate, glacial acetic acid, anhydrous sodium sulfite, sodium
based enzyme mimics have received great attention due to their
merits relative to native enzymes (e.g., low cost, high stability, Received: February 23, 2014
and tunability in catalytic activity). More and more nano- Revised: May 28, 2014
particles have been found as enzymatic mimics, such as metal Accepted: June 2, 2014
oxide nanomaterials,13 sulfide nanomaterials,14−17 selenide Published: June 2, 2014

© 2014 American Chemical Society 5827 dx.doi.org/10.1021/jf500950p | J. Agric. Food Chem. 2014, 62, 5827−5834
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 acid−acetate
buffer (pH 4.0) at 40 °C for 10 min. The TMB and Co3O4 NPs concentrations were 0.25 mM and 40 μg mL−1, respectively (b). UV/vis spectra of
an ABTS solution in 0.2 M acetic acid−acetate buffer (pH 4.0) at 40 °C for 10 min. The ABTS and Co3O4 NPs concentrations were 0.25 mM and
40 μg mL−1, 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 sulfite
solution was prepared daily by dissolving anhydrous sodium sulfite
in water and was standardized by iodimetric titration when needed. All
other chemicals were of analytical reagent grade and used without
further purification. Deionized water was used throughout the
experiment.
Apparatus and Characterization. A TU-1901 double-beam
UV−vis 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
diffraction (XRD) pattern of Co3O4 NPs was determined on a Rigaku
D/Max-2400 X-ray diffractometer with Cu Kα radiation (λ =
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 plasma−atomic
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

5828 dx.doi.org/10.1021/jf500950p | J. Agric. Food Chem. 2014, 62, 5827−5834

Journal of Agricultural and Food Chemistry
working electrode. The specific surface area was measured using a
multipoint Brunauer−Emmett−Teller technique (ASAP 2020, Micro-
meritics, USA).
Preparation of Co3O4 Nanoparticles. Co3O4 NPs were prepared
using a hydrothermal method as described in the literature.37 Briefly,
0.5 g of Co(CH3COO)2·4H2O 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 μL of 1.0 mg mL−1 Co3O4 NPs in
2.8 mL of acetic acid−acetate buffer solution (0.2 M, pH 4.0) with 80
μL 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
UV−vis spectrophotometer. The Michaelis−Menten constant was
calculated using a Lineweaver−Burk plot:12
1/v = K m/Vmax(1/[S] + 1/K m)
where ν 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 affinity 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 × 10−4
M) was added.5 After shaking and ultrasonication for 15 min, the
mixture was filtered with a 0.45 μm syringe membrane filter. The
filtrate was diluted to 100 mL with EDTA (1.0 × 10−4 M). All samples
were stored in a refrigerator at 4 °C before analysis.
Detection of Sulfite. The amount of sulfite in samples was
determined according to the following steps: (a) 80.0 μL of sample
solution, 150.0 μL of 5 mM TMB, and 120.0 μL of Co3O4 NPs
solution (1.0 mg mL−1) were added into 2.65 mL of a 0.2 M acetic
acid−acetate buffer 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 μL 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 freeze−pump−thaw
cycles (b). Photographs of the oxidation color reaction of TMB, TMB-
SO32−, and TMB-SO42− catalyzed by Co3O4 NPs, respectively (c).
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Figure 4. Cyclic voltammograms of bare GCE (a) and the Co3O4
NPs-modified GCE (b) in a 0.2 M acetic acid−acetate buffer (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 confirm the property of oxidase mimic of
the Co3O4 NPs, XPS was performed to find 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 O−Co2+ and O−Co3+ 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 confirmed
that Co3O4 NPs exhibited intrinsic oxidase-like activity.
It is important to confirm 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 buffer (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 buffer 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 TMB−O2−Co3O4
NPs, oxygen is present in the aqueous solution and also exists
on the surface of Co3O4 NPs due to the high specific surface
dx.doi.org/10.1021/jf500950p | J. Agric. Food Chem. 2014, 62, 5827−5834
Journal of Agricultural and Food Chemistry
Scheme 1. Possible Mechanism for the TMB−O2−Co3O4 NPs System
Figure 5. Michaelis−Menten curve of Co3O4 NPs with TMB (a). Michaelis−Menten 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 TMB−O2−Co3O4
NPs system, two sets of experiments were conducted: the first
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 first 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 effect on the reaction, which was
different 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 freeze−pump−
thaw cycles that catalyze the oxidation of TMB were
implemented in the degassed solutions. They produced
different 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 Co−O
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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 TMB−O2−Co3O4 NPs
system. In order to further confirm 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 TMB−O2−Co3O4 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 modified glassy carbon
electrode (GCE) toward the electrochemical oxidation of TMB
was studied using cyclic voltammetry (CV), in order to
dx.doi.org/10.1021/jf500950p | J. Agric. Food Chem. 2014, 62, 5827−5834
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Figure 6. A pH−ΔA curve (a), a temperature−ΔA curve (b), a TMB concentration−ΔA curve (c), and a Co3O4 NPs concentration−ΔA curve (d)
for 6 μM sulfite detection, where ΔA = A(blank,652nm) − A(sulfite,652nm), in 0.2 M acetic acid−acetate buffer solutions incubated for 10 min with the as-
prepared Co3O4 NPs as oxidase mimics.

Figure 8. ΔA response of TMB solutions to blank and different

common ions in the presence of sulfite (12 μM). The other ions added
Figure 7. Dose−response curve for different concentrations of sulfite are 240 μM. Relative ΔA: (ΔAtotal − ΔAsulfite)/ΔAsulfite.
standard solutions under the optimum conditions. Inset: Linear
calibration plot for sulfite detection. Table 1. Results for the Determination of the Sulfite 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-modified GCE are shown in Figure 4. Two pairs of redox amount (μg recovery (n = 3)
sample (mg g−1) mL−1) recovery (%) (%) (%)
peaks of TMB were found under the bare GCE, and increased
current was obviously observed under the Co3O4 NPs-modified dried lily 1.7 0.5 96.3−99.4 98.9 0.6
bulbs 1.0 94.4−99.5 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 98.4−100.6 100.3 2.6
Co3O4 NPs had the ability to accelerate the electron transfer.42 vermicelli 0.7 0.5 90.5−95.0 93.1 2.5
In order to further demonstrate that the oxidase-like activity 1.0 91.6−96.2 93.8 2.5
of the Co3O4 NPs was not from O2•− and •OH, an EPR spin 1.5 96.8−97.4 97.1 1.5
trapping experiment was carried out under the optimized haw roll 2.9 0.5 95.2−99.4 96.3 1.1
experimental conditions. The results revealed that no DMPO- 1.0 99.1−101.6 100.5 3.3
O2•− or DMPO-•OH spin adducts were detected (Figure S7). 1.5 97.8−99.9 98.9 1.1

5831 dx.doi.org/10.1021/jf500950p | J. Agric. Food Chem. 2014, 62, 5827−5834

Journal of Agricultural and Food Chemistry
It is suggested that TMB was not oxidized by O2•− and •OH
radicals in the TMB−O2−Co3O4 NPs system, which was
different 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 Michaelis−Menten 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 Michaelis−Menten constant (Km) and maximum
initial velocity (Vmax) were obtained using a Lineweaver−Burk
plot. The values of Km and Vmax for the Co3O4 NPs with TMB
were 0.051 mM and 3.30 × 10−8 M s−1. The values of Km and
Vmax for the Co3O4 NPs with ABTS were 0.037 mM and 3.20 ×
10−8 M s−1. The Km value of Co3O4 NPs with ABTS was
smaller than that with TMB. It is suggested that Co3O4 NPs
have higher affinity toward ABTS than TMB, because ABTS
has a higher affinity toward a positively charged nanoparticle
surface and TMB has a stronger affinity 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 affects
the interaction between Co3O4 NPs and sulfite, but also affects
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 influencing factors.
Figure 6a indicates that ΔA, where ΔA = 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 ΔA 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
μg mL−1, respectively.
Analytical Performance for Sulfite Determination. As
an oxidase mimic, Co3O4 NPs can catalyze TMB to form
colored product in the absence of H2O2. The sulfite inhibits the
TMB−O2−Co3O4 NPs system and causes a change in color of
the reaction system, which suggested that sulfite can be
indirectly detected. Since sulfite is easily adsorbed on the
surface of Co3O4 NPs and consumes absorbed oxygen more
easily than dissolved oxygen, the sulfite that is consumed by
dissolved oxygen is neglected in the reaction. The calibration
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graphs for the determination of sulfite were constructed under
the optimum conditions described above. The calibration data
of ΔA versus sulfite concentration are shown in Figure 7. The
results indicated that the concentration of detectible sulfite
could be 0.2 × 10−6 M with a linear range from 0.2 × 10−6 to
1.6 × 10−5 M. The limit of detection (LOD) for sulfite was 5.3
× 10−8 M. (LOD = KS0/S, where K is a numerical factor chosen
according to the confidence 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)
Effect of Foreign Substances. The effect of foreign
substances was tested by analyzing the mixed solution and
standard solution of sulfite (12 μM). The molar ratio of other
ion and sulfite was 20 in the mixed solution. The relative ΔA
values ((ΔAtotal − ΔAsulfite)/ΔAsulfite) are presented in Figure 8.
As can be seen, Fe and Cu were the main interferences for
sulfite determination, since trace amounts of heavy metal ions
in the water are believed to act as the catalyst in the oxidation
of sulfite 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 Sulfite in Real Samples. As an illustration
of analytical application, the proposed method was used to
determine sulfite 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 quantification of sulfites in real samples.
In summary, Co3O4 NPs were first 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 sulfite inhibited TMB−O2−
Co3O4 NPs system and caused a change in color of the reaction
system, a new colorimetric method for detecting sulfite was
developed. The colorimetric method showed a good response
toward sulfite detection with a linear range from 0.2 × 10−6 to
1.6 × 10−5 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.
dx.doi.org/10.1021/jf500950p | J. Agric. Food Chem. 2014, 62, 5827−5834
Journal of Agricultural and Food Chemistry
Funding
The authors are grateful for financial 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 financial interest.
■
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