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pubs.acs.org/JAFC
State Key Laboratory of Applied Organic 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,
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
which has been widely used as blanching and preservative
colorimetry to detect nucleotides,32 H2O2,3335 glucose,3335
agents in a large variety of foodstus (dried fruit, vermicelli,
melamine,36 and so on. To the best of our knowledge, however,
preserves, etc.) to improve the appearance of food and prevent
the colorimetric detection of sulte in food using the enzyme-
bacterial growth.1,2 However, the ingestion of foods containing like property of nanoparticles has not been explored up to now,
large amounts of sulte can cause asthmatic attacks and allergic which might be attributed to the complication of food samples
reactions in sensitive individuals.3,4 Owing to the potential matrices and the diculties in constructing an eective
toxicity, the sulte in foods is strictly limited in many nanoparticles-based enzyme mimic sensing platform to detect
countries.5 Hence, sulte determination in food products is micro amounts of food additive.
essential for the purposes of public health, food assurance, and Herein, we have developed a TMBO2Co3O4 NPs
quality control. In recent years, numerous methods have been colorimetric system for detection of sulte in foods, based on
developed for determining sulte in foods, including titrimetry, the property of oxidase-like activity of Co3O4 NPs. In this
ow injection analysis, anodic stripping voltammetry, ion method, the existence of sulte can cause a change in color of
chromatography, chemiluminescence, gas chromatography, the reaction system, and the color change can be visually
uorometry, high-performance liquid chromatography, and observed directly. We also investigated the mechanism of
colorimetry.68 oxidase-like activity of Co3O4 NPs by XPS, contrast experi-
Colorimetric biosensing has become more attractive because 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)24H2O 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 eld Tetramethylbenzidine (TMB) and 2,2-azinobis(3-ethylbenzothiazo-
analysis.911 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 (2528 wt %) was purchased
the rst 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 sulte, 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 sulde nanomaterials,1417 selenide
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 D/Max-2400 X-ray diractometer with Cu K radiation ( =
Guangfu Chemical Reagent Factory (Tianjin, China). A sulte 0.154056). The chemical state of the coating surface was analyzed
solution was prepared daily by dissolving anhydrous sodium sulte using X-ray photoelectron spectroscopy (XPS, PerkinElmer PHI-5702
in water and was standardized by iodimetric titration when needed. All multifunctional photoelectron spectrometer). Element determination
other chemicals were of analytical reagent grade and used without was measured by a 4300 DV inductively coupled plasmaatomic
further purication. Deionized water was used throughout the emission spectrometer (ICP-AES). Zeta potential measurements of
experiment. Co3O4 NPs were performed with a Malvern Nano-ZS apparatus. ESR
Apparatus and Characterization. A TU-1901 double-beam spectra were acquired using a JES-FA200 electron spin resonance
UVvis spectrophotometer (Beijing Purkine General Instrument Co. spectrometer. Electrochemical experiments were performed on a CHI
Ltd., China) was used to record absorption spectra and measure 832 electrochemical workstation (Shanghai Chenhua Instrument Co.,
absorbance. A FEI Tecnai G2 F30 microscope (TEM) was used for China) and a conventional three-electrode system. The conventional
measuring the morphologies and size of Co3O4 NPs. The X-ray three-electrode system contained a Pt wire counter electrode, a
diraction (XRD) pattern of Co3O4 NPs was determined on a Rigaku saturated calomel electrode as reference electrode, and an as-prepared
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 bond strength of Co3O4 and the larger surface energy of
oxygen in the solution played a role in the TMBO2Co3O4 nanomaterials caused by a large surface to volume ratio.39 In
NPs system, two sets of experiments were conducted: the rst addition, the O 1s peak at 533.0 eV in the XPS spectrum shown
one was carried out in the presence of oxygen; the other was in Figure 2c indicated that there was chemisorbed oxygen on
performed under high-purity nitrogen and the solution was the surface of Co3O4 NPs.40 It is indicated that both the
degassed before the reaction, as in the photograph shown in physically absorbed and chemisorbed oxygen on the surface of
Figure 3a. It was found that they exhibited the same color. The Co3O4 NPs played a key role in the TMBO2Co3O4 NPs
absorbance of the rst one was 0.747; the other was 0.770 at system. In order to further conrm the above conclusion,
652 nm. The absorbance of the latter was higher than the oxidation experiments of TMB, TMB-SO32, and TMB-SO42
former; one possible reason was that nitrogen played a role in catalyzed by Co3O4 NPs were carried out. The results are
mixing and speeded up the reaction. This indicated that the shown in Figure 3c. TMB-SO32 system presented a lower
oxygen in the solution had no eect on the reaction, which was absorbance than the other two systems. Since the volume of
dierent from the behavior of some nanoparticles-based oxidase SO32 is smaller than TMB, and Co3O4 NPs have a positive
mimics reported.8,38 For further study, the Co3O4 NPs without charge at pH 4.0 (Supporting Information), SO32 is more
degassing and Co3O4 NPs degassed by three freezepump easily adsorbed on the surface of Co3O4 NPs. Then SO32
thaw cycles that catalyze the oxidation of TMB were consumed oxygen absorbed on the surface of the Co3O4 NPs
implemented in the degassed solutions. They produced and interfered with the TMBO2Co3O4 NPs system. This
dierent colors, as in the photograph shown in Figure 3b, supported the above result that the oxygen absorbed on the
and the degassed Co3O4 NPs also catalyzed the oxidation of surface of the Co3O4 NPs oxidized TMB in this system.
TMB to produce the typical color reaction. It is suggested that The oxidation of TMB is an electron transfer process.41 The
the oxygen on the surface of Co3O4 NPs was due not only to electrocatalytic behavior of a Co3O4 NP modied glassy carbon
physical adsorption. Chemisorbed oxygen probably existed on electrode (GCE) toward the electrochemical oxidation of TMB
the surface of Co3O4 NPs because of the low oxygen CoO was studied using cyclic voltammetry (CV), in order to
D dx.doi.org/10.1021/jf500950p | J. Agric. Food Chem. XXXX, XXX, XXXXXX
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.
It is suggested that TMB was not oxidized by O2 and OH graphs for the determination of sulte were constructed under
radicals in the TMBO2Co3O4 NPs system, which was the optimum conditions described above. The calibration data
dierent from the behavior of CoFe2O4 NPs.8 Therefore, the of A versus sulte concentration are shown in Figure 7. The
nature of oxidase-like activity of the Co3O4 NPs does not results indicated that the concentration of detectible sulte
originate from the O2 and OH radicals generated. could be 0.2 106 M with a linear range from 0.2 106 to
Taken together, although the detailed mechanism remains 1.6 105 M. The limit of detection (LOD) for sulte was 5.3
unclear, these observations demonstrated that Co3O4 NPs 108 M. (LOD = KS0/S, where K is a numerical factor chosen
accelerated the electron transfer from the peroxidase substrate according to the condence level desired, S0 is the standard
to oxygen absorbed on the surface of the Co3O4 NPs. The deviation (SD) of the blank measurements (n = 10, K = 3), and
possible mechanism of oxidase-like activity of Co3O4 NPs is S is the slope of the calibration curve.44)
presented in Scheme 1; that is, TMB was absorbed on the Eect of Foreign Substances. The eect of foreign
surface of the Co3O4 NPs and donated electrons to the Co3O4 substances was tested by analyzing the mixed solution and
NPs, resulting in an increase in electron density and mobility in standard solution of sulte (12 M). The molar ratio of other
the Co3O4 NPs, thus accelerating the electron transfer to ion and sulte was 20 in the mixed solution. The relative A
oxygen absorbed on the surface of the Co3O4 NPs. After the values ((Atotal Asulfite)/Asulfite) are presented in Figure 8.
completion of the electron transfer, the Co3O4 NPs reverted to As can be seen, Fe and Cu were the main interferences for
their original state. sulte determination, since trace amounts of heavy metal ions
Steady-State Kinetic Assay of Co3O4 NPs. Since Co3O4 in the water are believed to act as the catalyst in the oxidation
NPs exhibited oxidase-like activity, the kinetic parameters of of sulte by oxygen.45 EDTA was selected as a chelate reagent
only TMB/ABTS were determined by changing the concen- for the present study to eliminate the interferences derived
tration of TMB/ABTS. A typical MichaelisMenten curve from Fe, Cu, and other coexisting transient metals in the
(Figure 5a,b) was obtained with TMB/ABTS by monitoring samples.
the absorbance change at 652/417 nm for 4 min (Figure Detection of Sulte in Real Samples. As an illustration
S8a,b). The MichaelisMenten constant (Km) and maximum of analytical application, the proposed method was used to
initial velocity (Vmax) were obtained using a LineweaverBurk determine sulte in real samples under the optimal
plot. The values of Km and Vmax for the Co3O4 NPs with TMB experimental conditions. The results are listed in Table 1.
were 0.051 mM and 3.30 108 M s1. The values of Km and The recoveries of the three samples ranged from 93.8% to
Vmax for the Co3O4 NPs with ABTS were 0.037 mM and 3.20 100.5%. Hence, the proposed method can be considered a
108 M s1. The Km value of Co3O4 NPs with ABTS was useful tool for the quantication of sultes in real samples.
smaller than that with TMB. It is suggested that Co3O4 NPs In summary, Co3O4 NPs were rst found to possess oxidase-
have higher anity toward ABTS than TMB, because ABTS like activity and could catalyze the oxidation of TMB and ABTS
has a higher anity toward a positively charged nanoparticle to produce the typical color reaction in the absence of
surface and TMB has a stronger anity toward a negatively exogenously added H2O2. It was found that the oxygen
charged nanoparticle surface.43 absorbed on the surface of the Co3O4 NPs oxidizes the
Optimization of Experimental Conditions. The reaction peroxidase substrate, rather than the oxygen dissolved in the
conditions were optimized to establish the optimum analytical solution. The oxidase-like activity of Co3O4 NPs was not from
conditions. The pH value not only decides the charge of the the generated O2 or OH radical, probably originating from
surface of Co3O4 NPs (Supporting Information), which aects their ability to transfer an electron between the peroxidase
the interaction between Co3O4 NPs and sulte, but also aects substrate and oxygen absorbed on the surface of the Co3O4
the catalytic activity of Co3O4 NPs. The catalytic activity of NPs. Moreover, as the existence of sulte inhibited TMBO2
Co3O4 NPs is also dependent on the incubation temperature. Co3O4 NPs system and caused a change in color of the reaction
In addition, the concentrations of TMB and Co3O4 NPs system, a new colorimetric method for detecting sulte was
determine the color level of the reaction system. Therefore, developed. The colorimetric method showed a good response
they were investigated in this study as inuencing factors. toward sulte detection with a linear range from 0.2 106 to
Figure 6a indicates that A, where A = A(blank,652nm) 1.6 105 M and good recoveries ranging from 93.8% to
A(sulfite,652nm), increased and then decreased with increasing pH 100.5% in three real samples using Co3O4 NPs as an oxidase
and had a maximum at 4.0. The reaction temperature- mimic. The detection platform showed great potential
dependent response curve is shown in Figure 6b. It showed applications in reducing substrates. In addition, Co3O4 NPs
that A of the reaction system had a peak value at 40 C. can be expected to provide potential applications for chemical
Therefore, 4.0 and 40 C were taken as the optimal pH and sensing, biochemical analysis, electrochemical biosensing, and
temperature. The concentrations of TMB and Co3O4 NPs even clinical chemistry.
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.
ASSOCIATED CONTENT
Analytical Performance for Sulte Determination. As * Supporting Information
S
an oxidase mimic, Co3O4 NPs can catalyze TMB to form This material is available free of charge via the Internet at
colored product in the absence of H2O2. The sulte inhibits the http://pubs.acs.org.
TMBO2Co3O4 NPs system and causes a change in color of
the reaction system, which suggested that sulte can be AUTHOR INFORMATION
indirectly detected. Since sulte is easily adsorbed on the
surface of Co3O4 NPs and consumes absorbed oxygen more Corresponding Author
easily than dissolved oxygen, the sulte that is consumed by *E-mail: chenxg@lzu.edu.cn (X.-G. Chen). Tel: 86-931-
dissolved oxygen is neglected in the reaction. The calibration 8912763 Fax: 86-931-8912582.
F dx.doi.org/10.1021/jf500950p | J. Agric. Food Chem. XXXX, XXX, XXXXXX
Journal of Agricultural and Food Chemistry Article
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