Myoglobin immobilized on LaF3 doped CeO2 and ionic liquid composite film for nitrite biosensor

Sensors and Actuators B 173 (2012) 704–709

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Myoglobin immobilized on LaF3 doped CeO2 and ionic liquid composite film for nitrite biosensor Sheying Dong a,∗ , Nan Li a , Tinglin Huang a , Hongsheng Tang b , Jianbin Zheng b a b

College of Sciences, Xi an University of Architecture and Technology, Xi’an 710055, China Institute of Analytical Science/Shaanxi Provincial Key Laboratory of Electroanalytical Chemistry, Northwest University, Xi’an 710069, China

a r t i c l e

i n f o

Article history: Received 31 May 2012 Received in revised form 15 July 2012 Accepted 18 July 2012 Available online 27 July 2012 Keywords: LaF3 -DP-CeO2 Ionic liquid Myoglobin Nitrite Biosensor

a b s t r a c t This study described a simple and reliable method for the electrochemical determination of nitrite based on the immobilization of myoglobin (Mb) on LaF3 doped CeO2 (LaF3 -DP-CeO2 ) and ionic liquid (IL) composite film. Meantime, ultraviolet visible spectra (UV–vis), Fourier transform infrared spectra (FT-IR) and circular dichroism spectra (CD) were utilized to characterize the composite film. The results demonstrated that Mb in the composite membrane retained its secondary structure similar to the native state. Furthermore, the LaF3 -DP-CeO2 provided a biocompatible microenvironment for protein immobilization and a suitable electron transfer distance between Mb and electrode surface. Amperometric responses showed that the biosensor exhibited a fast response time (within 5 s), good stability and a broad linear range of nitrite (NO2 − ) concentration from 5 ␮M to 4650 ␮M with a detection limit of 2 ␮M (S/N = 3). The low value of Michaelis–Menten constant KM (2.19 mM) indicated a high affinity of Mb to NO2 − . The attractive features of LaF3 -DP-CeO2 could provide potential applications in sensor and biosensor design. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Nitrite (NO2 − ) exists widely in the environment, beverages and food products, which is an important precursor in the formation of N-nitrosamines, has been shown as potent carcinogens in human bodies [1,2]. Current methods in literatures for measuring NO2 − include spectrophotometry [3], chromatography [4], capillary electrophoresis [5] and electrochemistry [6]. Especially, the sensors based on the electrochemical methods are favorable with the advantages of high sensitivity, relatively good selectivity and fast response [7]. Recently, the biosensors of NO2 − have been constructed based on immobilized protein or enzyme in nanomaterials due to the quantum size effect and surface effect [8,9]. Meanwhile, their large surface areas provide abundant active sites to immobilize protein or enzyme and a suitable microenvironment to retain their bioactivity. Besides these unique structure, CeO2 has attracted much attention due to its properties such as non-toxicity, biocompatibility, electrocatalysis, high chemical stability and oxygen storage capacity, which has been widely used to detect hydrogen dioxide or glucose with the complex methods such as pulsed laser deposition (PLD) and electrophoretic method (EPD) [10–12]. In particular, the development of the fabrication of metal or LaF3 doped CeO2 with well controllable microstructure has generated considerable interest because of their potential utilization in fuel cell,

solid electrolyte and catalyst [13–15]. When the LaF3 doped CeO2 , anionic and/or cationic exchange between metal oxide and metal fluoride lattices took place, leading to formation of O− ions, anion vacancies and partial reduced Ce4+ centers. Most importantly, these factors should be responsible for the improvement of conductivity of CeO2 nanomaterial to some extent. Nevertheless, to our knowledge, there was no relative application in the LaF3 doped rare earth oxides materials realizing the direct electrochemistry of proteins reported in literatures. In our work, LaF3 -DP-CeO2 nanomaterial was introduced to realize the direct electron transfer of Mb at carbon paste electrode with a very simple casting procedure. At the same, 1-butyl-3methylimidazolium tetrafluoroborate ionic liquid ([BMIm]BF4 , IL) was chosen to disperse LaF3 -DP-CeO2 to promote the electron transfer of redox proteins. The structure of Mb entrapped in LaF3 DP-CeO2 /IL composite film was evaluated by UV–vis, FT-IR and circular dichroism (CD) spectroscopy, while the electrochemical behavior of Mb was investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). In addition, the catalytic ability of the modified electrode to NO2 − was also studied, and a simple as well as novel NO2 − biosensor with high selectivity based on LaF3 -DP-CeO2 /IL was fabricated. 2. Materials and methods 2.1. Reagents

∗ Corresponding author. Tel.: +86 29 82201203; fax: +86 29 82205332. E-mail address: [email protected] (S. Dong). 0925-4005/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2012.07.073

Bovine myoglobin (Mb, MW. 17,800) was purchased from Sigma Chemical Co.. [BMIm]BF4 IL was purchased from Hangzhou Chemer

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Chemical Limited Company. High purity graphite powder was obtained from China National Medicine Corporation. K3 Fe(CN)6 , K4 Fe(CN)6 , KCl, K2 HPO4 , KH2 PO4 , H3 PO4 , NaOH, NaNO2 and liquid paraffin were obtained from Xi’an Chemical Reagent Corporation. All the chemicals were of analytical reagent grade and used without further purification. 0.1 M phosphate buffer solutions (PBS) with different pH values were prepared by mixing the stock standard solutions of K2 HPO4 and KH2 PO4 and adjusting the pH with 0.1 M H3 PO4 and 0.1 M NaOH. All solutions were made up with twice-distilled water. 2.2. Apparatus All electrochemical measurements were performed with CHI660B electrochemical workstation (Shanghai Chenhua Co., China). A conventional three-electrode system was employed with a modified carbon paste electrode (CPE) as the working electrode, a saturated calomel electrode (SCE) and a platinum wire electrode were served as the reference electrode and auxiliary electrode, respectively. UV–vis spectra were recorded on a Nicolet Evolution 300 spectrophotometer in 0.1 M PBS, the CD spectra were recorded on a Applied Photophysics Limited and the FT-IR spectra (KBr) were recorded on an IR-spectrometer FTIR-8400S Shimadzu at room temperature. The surface morphology of LaF3 -DP-CeO2 nanomaterial were observed through scanning electron microscopy (SEM) on JEOL JSM-5800 at an accelerating voltage of 15 kV. 2.3. Preparation of the modified electrodes LaF3 doped CeO2 (LaF3 -DP-CeO2 ) was prepared as follows: the appropriate amounts of Ce2 (CO3 )3 (90%) and La2 (CO3 )3 (90%) were dissolved in distilled water to obtain a slurry. After adjusting the pH value to 7 with ammonia water, the fluorinated solutions were precipitated by addition of silicofluoric acid (5–7%) to the slurry under continuous stirring at room temperature. The above products were ground for 2 h in the QML mix ball-grind machine and then sprayed drying. Immediately following, the product of intermediates was dried in oven for 3.5 h at 950 ◦ C. Finally, the LaF3 -DP-CeO2 was obtained by grinding for 1–3 h and filtrating, followed by washing with twice-distilled water. Carbon paste electrode (CPE) was fabricated as follows: 0.6 g of liquid paraffin and 3.4 g of graphite powder were hand-mixed to produce a homogenous paste. Then the prepared carbon paste was firmly packed into a PVC tube (3 mm internal diameter) and a copper wire (1.5 mm external diameter) was introduced into the other end for electrical contact. The surface of CPE was carefully smoothed on a weighing paper before used. The Mb modified electrode was prepared according to the following procedure: Firstly, 5 ␮L Mb solution (5 mg/mL) was casted onto the surface of a freshly polished CPE and the modified electrode was dried at 4 ◦ C in a refrigerator to obtain Mb-CPE. Then 5 mg LaF3 -DP-CeO2 and 10 ␮L [BMIm]BF4 were dispersed into 1 mL 0.1 M pH 7.0 PBS under sonication. Finally, 10 ␮L above suspension was casted onto Mb-CPE to get Mb/LaF3 -DP-CeO2 /IL-CPE, which was evaporated at 4 ◦ C in a refrigerator to form a stable film and rinsed with doubly distilled water for twice or thrice to remove the unimmobilized Mb molecules. When not in use, the enzyme electrode was stored in 0.1 M pH 7.0 PBS at 4 ◦ C in a refrigerator. 2.4. Electrochemical measurements CV measurements were done in an undivided electrochemical Teflon cell at 25 ◦ C. EIS measurements were carried out in 0.5 mM K3 Fe(CN)6 /K4 Fe(CN)6 (1:1) containing 0.1 M KCl, at applied perturbation amplitude of 0.005 V and frequency ranging from 105 to 10−2 Hz. Amperometric experiments were performed in a

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constant stirred cell with the successive addition of NaNO2 solution into 20.0 mL PBS supporting electrolyte at 25 ◦ C, while the electrode potential was set at 0.8 V vs. RE(SCE). Experimental solutions were deoxygenated by purging them with highly pure nitrogen for 30 min and maintained under nitrogen atmosphere during measurements. 3. Results and discussion 3.1. Spectroscopic characterization of modified electrodes The shape and position of the Soret absorption band of Mb could provide structural information on the possible denaturation of heme protein, especially the conformational change around the heme groups, so the UV–vis absorption spectrum of modified films were shown in Fig. 1(A). It could be seen that the Soret band of Mb/LaF3 -DP-CeO2 /IL (408.8 nm) which had subtracted the matrix of LaF3 -DP-CeO2 /IL as a background was similar to that of Mb in 0.1 M pH 7.0 PBS (408.5 nm), indicating that Mb retained its native structure in the presence of LaF3 -DP-CeO2 and [BMIm]BF4 . Meanwhile, the secondary structure of Mb could be investigated by FT-IR. As well known, the characteristic infrared adsorption bands of amide I (1700–1600 cm−1 ) and amide II (1600–1500 cm−1 ) can provide detailed information on the secondary structure of polypeptide chain [16]. As shown in Fig. 1(B), the amide I and amide II bands of Mb/LaF3 -DP-CeO2 /IL were located at 1645 and 1568 cm−1 respectively, which were slightly shifted compared to those of native Mb (1652 and 1545 cm−1 ). The results proved that Mb was not denatured in Mb/LaF3 -DP-CeO2 /IL film which had subtracted the matrix of LaF3 -DP-CeO2 /IL as a background. Furthermore, circular dichoism (CD) method was used to analysis the change of components about ␣-helix, ␤-fold, ␤-turn and random coil in the secondary structure of protein [17]. Fig. 1(C) was the CD spectra of Mb and Mb/LaF3 -DP-CeO2 /IL. It was apparently observed that CD spectrum of Mb exhibited characteristic features of the ␣helix structure of protein with a positive absorption band at 192 nm as well as negative absorption bands at 209 and 222 nm. When Mb was added into the LaF3 -DP-CeO2 /IL mixture solution, the position and intensity of absorption band did not alter obviously and superposed almost. This result indicated that the content of ␣-helix with a dominant position did not change and Mb was not denatured. In other words, the UV–vis, FT-IR and CD spectra results yielded to the conclusion that the native structure of Mb had retained because of the convenient microenvironment provided by the excellent biocompatible LaF3 -DP-CeO2 /IL composite. This can be explained by the fact that the dissolution of LaF3 in CeO2 and the exchange of anionic and/or cationic would lead to the formation of the active sites and a partial reduced state of Ce4+ center [18]. 3.2. Electrochemical impedance spectroscopy The electrochemical impedance spectroscopy (EIS) was applied to monitor the whole procedure in preparing the modified electrodes, which could provide useful information between each step and often be used for probing the change of surface-modified electrode [19]. EIS results of different electrodes were described in Fig. 2. For LaF3 -DP-CeO2 /IL-CPE, there was almost no resistance owing to excellent conductivity of LaF3 -DP-CeO2 /IL matrix compared with the others. After Mb was immobilized on the LaF3 -DP-CeO2 /IL matrix, the resistance (Ret ) of the modified electrode increased obviously because the dielectric behavior of Mb molecules would result in a higher barrier for interfacial electron transfer. But the Ret of Mb/LaF3 -DP-CeO2 /IL-CPE was smaller than that of IL-CPE, which indicated that the synergistic action of LaF3 DP-CeO2 and IL made the electron transfer easier.

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Fig. 2. Electrochemical impedence spectra of CPE, IL-CPE, LaF3 -DP-CeO2 /IL-CPE and Mb/LaF3 -DP-CeO2 /IL-CPE. Supporting solution: 0.5 mM K3 Fe(CN)6 /K4 Fe(CN)6 (1:1) containing 0.1 M KCl.

process of the heme Fe(III)/Fe(II) couples in Mb. From the comparison of electrochemical behavior of Mb at different modified electrodes, it was clearly seen that LaF3 -DP-CeO2 played an important role in facilitating the direct electron transfer between Mb and underlying electrode by the synergetic effect with [BMIm]BF4 . The dispersion of “inert” fluorides on the CeO2 surface in the ionic liquid will be also beneficial for the isolation of the surface active centers, and thus be favorable for the improvement of direct electrochemistry of Mb. The reason is that the LaF3 doped CeO2 not only increases the conductivity of CeO2 nanomaterial but also makes the structure of CeO2 more stable [18,21]. In addition, the similar snowflake nanoparticles of LaF3 -DP-CeO2 (inset in Fig. 3) had high specific surface area so that Mb molecules were more accessible to be penetrated inside the LaF3 -DP-CeO2 microspheres. Thus, more protein molecules were aggregated onto the surface of the nanoparticles. Following that, the influence of solution pH on the direct electrochemistry of Mb at Mb/LaF3 -DP-CeO2 /IL-CPE was examined by CV, as shown in Fig. 4. With the increasing of solution pH from 3.0 to 9.0, the negative shifts of both oxidation and reduction peak potentials were observed. The plot of formal potential vs. pH showed a slope of −22.82 mV pH−1 (inset in Fig. 4), which was smaller than the theoretically expected value of −59 mV pH−1 for a proton-coupled

Fig. 1. (A) UV–vis spectra of Mb and Mb/LaF3 -DP-CeO2 /IL in 0.1 M pH 7.0 PBS solution. (B) FT-IR spectra of Mb and Mb/LaF3 -DP-CeO2 /IL film. (C) CD spectrum of Mb and Mb/LaF3 -DP-CeO2 /IL in 0.1 M pH 7.0 PBS solution. Solution concentration: 3 × 10−6 M Mb and Mb/LaF3 -DP-CeO2 /IL in 0.1 M pH 7.0 PBS.

3.3. Direct electrochemistry of Mb In order to research the direct electron transfer of Mb in the different modified electrodes, CVs of Mb-CPE, Mb/IL-CPE and Mb/LaF3 -DP-CeO2 /IL-CPE in 0.1 M pH 7.0 PBS were examined over a potential range from −0.7 V to 0.2 V at the scan rate of 0.1 V s−1 . As shown in Fig. 3, the Mb-CPE gave a weak and irreversible reduction peak (curve (a)). At Mb/IL-CPE (curve (b)), a couple of unstable redox peaks were observed because the molecular film formed by water-miscible imidazolium-based ionic liquid, which possessed the capability to facilitate direct electron transfer [20]. In contrast, when Mb was immobilized in the composite film of LaF3 DP-CeO2 /IL (curve (c)), a pair of well-defined and quasi-reversible redox peaks with Ep,a = −0.250 V and Ep,c = −0.329 V (vs. SCE) in PBS  with the formal potential E0 = −0.290 V, indicating the electrode

Fig. 3. Cyclic voltammograms of (a) Mb-CPE, (b) Mb/IL-CPE and (c) Mb/LaF3 -DPCeO2 /IL-CPE. Supporting solution: 0.1 M pH 7.0 PBS, scan rate: 0.1 V s−1 . Inset shows the SEM image of the LaF3 -DP-CeO2 .

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Fig. 4. Cyclic voltammograms of Mb/LaF3 -DP-CeO2 /IL-CPE in 0.1 M pH 7.0 PBS with  pH values at 0.1 V s−1 . Inset shows the linear relationship of E0 vs. solution pH.

single electron transfer process [22]. This might be attributed to the influence of the protonation states of the ligands coordinated to the Mb iron and the amino acids around the Mb, or to the protonation of the water molecule coordinated to the central iron. In general, all changes in the peak potentials and currents with solution pH were reversible in the pH range from 3.0 to 9.0, which was the same CVs could be obtained if the electrode was transferred from a solution with a different pH values to its original solution. But when pH > 9.0 or pH < 3.0, the structure of Mb was destroyed, leading to the denaturalization of the protein. 3.4. Kinetics parameters of Mb/LaF3 -DP-CeO2 /IL-CPE The effect of scan rate on electrochemistry of the immobilized Mb was shown in Fig. 5. With an increasing scan rate from 0.02 to 1.00 V s−1 , the anodic and cathodic peak currents increased linearly ip,a (␮A) = −105.6v − 4.209, R = 0.9971; ip,c (␮A) = 102.6v + 14.00, R = 0.9973), indicating a surface-controlled process [23]. When the scan rate was greater than 0.5 V s−1 , the peak currents (ip ) were proportional to the square root of scan rate (v1/2 ). These phenomena, which were also observed in other heme protein electrode modified

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Fig. 6. Amperometric current–time response curves of Mb/LaF3 -DP-CeO2 /IL-CPE upon successive addition of NaNO2 into pH 7.0 PBS solution. Applied potential: 0.8 V. Inset: calibration curve of steady-state currents vs. NaNO2 concentration at Mb/LaF3 -DP-CeO2 /IL-CPE.

by ionic liquid, were consistent with heme protein immobilized in surfactant films [24]. For a surface-controlled process, the surface coverage ( *) was estimated from integration of reduction peaks of the cyclic voltammograms according to the Faraday’s law: Q = nAF *, here Q was the charge involved in the reaction, n was the number of electron transferred, F was Faraday’s constant, and A was the effective surface area of electrode. The surface concentration ( *) of Mb was calculated as 2.07 × 10−9 M cm−2 for Mb/LaF3 -DP-CeO2 /IL-CPE, which was far greater than 1.36 × 10−10 M cm−2 at Mb/ZrO2 /MWCNT [25] and 9.5 × 10−11 M cm−2 at Mb-QDs-MCFs/GC [26]. It was possible that the LaF3 -DP-CeO2 /IL nanocomposite provided larger surface areas available for protein binding and increased the loading of Mb. On the other hand, the anodic peak potentials slightly shifted to the positive direction and the cathodic peak potentials slightly shifted to the negative direction at higher scan rate, which resulted in an increase of the peak separation between anodic and cathodic peak. The peak separation at higher scan rate could be used to estimate the heterogeneous electron transfer rate constant (ks ). According to the method of Laviron [27], the average value of ks was estimated to be 1.01 s−1 , which was larger than those of SnO2 0.53 s−1 [28] and CeO2 0.68 s−1 [29], suggesting a reasonably fast electron transfer between the immobilized Mb and the electrode ascribed to the presence of LaF3 -DP-CeO2 /IL nanocomposite. 3.5. Biocatalytic determination of nitrite at the Mb/LaF3 -DP-CeO2 /IL-CPE

Fig. 5. Cyclic voltammograms of Mb/LaF3 -DP-CeO2 /IL-CPE in 0.1 M pH 7.0 PBS with scan rates from 0.02 to 1.00 V s−1 . Insets show the plots of cathodic and anodic peak currents vs. (a) scan rates and (b) square root of scan rates.

Fig. 6 displayed the amperometric responses of the Mb/LaF3 -DPCeO2 /IL-CPE upon successive addition of NaNO2 to 0.1 M pH 7.0 PBS at an applied potential of 0.8 V under stirring. The biosensor exhibited a very rapid and sensitive response to the changes of NO2 − concentration, the steady-state current being reached in 5 s. Under these conditions, a calibration graph was constructed for nitrite (inset in Fig. 6), with a linear range between 5 ␮M and 4650 ␮M (R = 0.9989). The limit of detection, determined as 3 times the standard deviation of the single for buffer (blank), was 2.0 ␮M of nitrite. In addition, the apparent Michaelis–Menten constant (KM ) value of Mb/LaF3 -DP-CeO2 /IL-CPE for NO2 − was calculated to be 2.19 mM, indicating a higher affinity of Mb/LaF3 -DP-CeO2 /IL composite film modified electrode to NO2 − . Compared with other NO2 − biosensors listed in Table 1, the biosensor based on Mb/LaF3 -DP-CeO2 /ILCPE had wider linear range, lower detection limit and smaller KM .

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Table 1 Performances of different NO2 − biosensors. Biosensor

Detection range(␮M)

Detection limit(␮M)

KM (mM)

Reference

Cytc/1-Cys/P3MT/MWCNT/GCE Hb-ZnO-Nafion/GC Nafion-BMIMPF6 /Mb/CILE Nafion/Mb/MWCNTs/CILE Mb/LaF3 -DP-CeO2 /IL-CPE

10–100 10–2700 100–8400 8.0–196 5.0–4650

0.5 4.0 50 6.0 2.0

– – 1.46 11.1 2.19

[30] [31] [32] [33] This work

Table 2 Test for recovery of NO2 − with the Mb/LaF3 -DP-CeO2 /IL-CPE. Samples of pickle juice

Content c/(×10−5 M)

Added c/(×10−5 M)

Total after addition c/(×10−5 M)

Recovery (%)

1 2 3

10.3 9.9 10.7

12.5 12.5 12.5

22.9 21.8 22.5

100.3 97.3 97.0

The prominent electrocatalytic ability of Mb/LaF3 -DP-CeO2 /IL-CPE might be attributed that the LaF3 -DP-CeO2 /IL composite provided more sites for protein binding and a short diffusion distance for NO2 − to access the immobilized Mb. 3.6. Interference study Possible interference for the detection of nitrite on Mb/LaF3 -DPCeO2 /IL-CPE was investigated by addition of various species into 0.1 M pH 7.0 PBS containing 0.1 mM NO2 − . The interference study showed that 40 mM of NaCl and KCl, 20 mM of NaNO3 , 5 mM of CuSO4 , NiSO4 , uric acid as well as 1 mM ascorbic acid did not interfere with the detection of 0.1 mM of NO2 − . These results should make the biosensor an ideal analytical tool for sensitive detection of NO2 − in the environment. 3.7. Real sample analysis To evaluate the ability of the sensor for routine analysis, the sensor was applied to determination of NO2 − in pickle juice samples. The 0.3 mL real sample of pickle juice was mixed with 20 mL 0.1 M pH 7.0 PBS for determination NO2 − with the proposed procedure. Recovery studies were completed on samples by adding NO2 − standards solution. The obtained results were displayed in Table 2. 3.8. Stability and reproducibility of the biosensor The stability and reproducibility of Mb/LaF3 -DP-CeO2 /IL-CPE was examined by CV. The biosensor could retain about 95% of its initial response after storage at 4 ◦ C in 0.1 M pH 7.0 PBS for 2 weeks. After a month, the sensor retained 87% of its initial response to NaNO2 . Thus, LaF3 -DP-CeO2 /IL composite film was very efficient for retaining the bioactivity of immobilized Mb and preventing it from leaking out of the sensor. The relative standard deviations (RSD) of catalytic peak currents for six successive determinations of 100 ␮M NaNO2 were 4.2%. The fabrication reproducibility of six electrodes made independently, showed an acceptable reproducibility with the RSD of 3.9% in PBS for the current determination of 100 ␮M NaNO2 . These results obtained suggested that the proposal biosensor displayed good stability and reproducibility, which could be satisfactorily used for NO2 − determination. 4. Conclusions The developed NO2 − biosensor based on the direct electron transfer of myoglobin immobilized on LaF3 -DP-CeO2 and ionic liquid composite film exhibited excellent electrocatalytic activity, electron transfer ability and stability owing to the advantages of both LaF3 -DP-CeO2 and ionic liquid. The simple LaF3 -DP-CeO2 /IL

composite film provided an efficient strategy and a new promising platform for the development of biosensors. Moreover, the proposed i–t method was applied to the determination of NO2 − in practical samples and satisfactory results were obtained. Thus, this study was expected to provide important insight into the application of LaF3 -DP-CeO2 in electroanalysis. Acknowledgments The authors appreciate the support from the National Natural Science Foundation of China (No. 50830303), Program for Changjiang Scholars and Innovative Research Team in University (Grant No. IRT0853), the Program of International S & T Cooperation (No. 2010 DFA 94550) and the Research Achievements Foundation of Xi an University of Architecture and Technology (No. ZC1004). References [1] W. Lijinsky, S.S. Epstein, Nitrosamines as environmental carcinogens, Nature 225 (1970) 21–23. [2] S.S. Mirvish, Role of N-nitroso compounds (NOC) and N-nitrosation in etiology of gastric, esophageal, nasopharyngeal and bladder cancer and contribution to cancer of known exposures to NOC, Cancer Letters 93 (1995) 17–48. [3] V. Kuznetsov, S. Zemyatova, Flow-injection spectrophotometry of nitrites based on the diazotization reactions of azine dyes, Journal of Analytical Chemistry 62 (2007) 637–644. [4] P. Niedzielski, I. Kurzyca, J. Siepak, A new tool for inorganic nitrogen speciation study: simultaneously determination of ammonium ion, nitrite and nitrate by ion chromatography with post-column ammonium derivatization by Nessler reagent and diode-array detection in rain water samples, Analytica Chimica Acta 577 (2006) 220–224. [5] O. Nadzhafova, M. Etienne, A. Walcarius, Direct electrochemistry of hemoglobin and glucose oxidase in electrodeposited sol–gel silica thin films on glassy carbon, Electrochemistry Communications 9 (2007) 1189–1195. [6] B. Sljukic, R. Compton, Manganese dioxide graphite composite electrodes formed via a low temperature method: detection of hydrogen peroxide, ascorbic acid and nitrite, Electroanalysis 19 (2007) 1275–1280. [7] C.A. Caro, F. Bedioui, J.H. Zagal, Electrocatalytic oxidation of nitrite on a vitreous carbon electrode modified with cobalt phthalocyanine, Electrochimica Acta 47 (2002) 1489–1494. [8] H. Zhang, H.Y. Lu, N.F. Hu, Fabrication of electroactive layer-by-layer films of myoglobin with gold nanoparticles of different sizes, Journal of Physical Chemistry B 110 (2006) 2171–2179. [9] D. Shan, S.X. Wang, H.G. Xue, S. Cosnier, Direct electrochemistry and electrocatalysis of hemoglobin entrapped in composite matrix based on chitosan and CaCO3 nanoparticles, Electrochemistry Communications 9 (2007) 529–534. [10] A.A. Ansari, P.R. Solanki, B.D. Malhotra, Hydrogen peroxide sensor based on horseradish peroxidase immobilized nanostructured cerium oxide film, Journal of Biotechnology 142 (2009) 179–184. [11] S. Saha, S.K. Arya, S.P. Singh, K. Sreenivas, B.D. Malhotra, V. Gupta, Nanoporous cerium oxide thin film for glucose biosensor, Biosensors and Bioelectronics 24 (2009) 2040–2045. [12] D. Patil, N.Q. Dung, H. Jung, S.Y. Ahn, D.M. Jang, D. Kim, Enzymatic glucose biosensor based on CeO2 nanorods synthesized by non-isothermal precipitation, Biosensors and Bioelectronics 31 (2012) 176–181. [13] Z. Khakpour, A.A. Youzbashi, A. Maghsoudipour, K. Ahmadi, Synthesis of nanosized gadolinium doped ceria solid soution by high energy ball milling, Powder Technology 214 (2011) 117–121.

S. Dong et al. / Sensors and Actuators B 173 (2012) 704–709 [14] F.Y. Wang, S.Y. Chen, S.F. Cheng, et al., Gd3+ and Sm3+ co-doped ceria based electrolytes for intermediate temperature solid oxide fuel cells, Electrochemistry Communications 6 (2004) 743–746. [15] S. Sharma, Z.P. Hu, P. Zhang, E.W. McFarland, H. Metiu, et al., CO2 methanation on Ru-doped ceria, Journal of Catalysis 278 (2011) 297–309. [16] J.K. Kauppinen, D.J. Moffat, H.H. Mantsch, D.G. Cameron, Fourier selfdeconvolution: a method for resolving intrinsically overlapped bands, Applied Spectroscopy 35 (1981) 271–276. [17] M.K. Haron, J.J. Thomas, C.P. Nicholas, How to study proteins by circular dichroism? Biochimica et Biophysica Acta 1751 (2005) 119–139. [18] J.Z. Luo, H.L. Wan, Oxidative dehydrogenation of ethane over LaF3 –CeO2 catalysts, Applied Catalysis A: General 158 (1997) 137–144. [19] H.Y. Gu, A.M. Yu, H.Y. Chen, Direct electron transfer and characterization of hemoglobin immobilized on a Au colloid cysteamine-modified gold electrode, Journal of Electroanalytical Chemistry 516 (2001) 119–126. [20] A. Safavi, F. Fariami, Hydrogen peroxide biosensor based on a myoglobin/hydrophilic room temperature ionic liquid film, Analytical Biochemistry 402 (2010) 20–25. [21] Y.S. Han, C.Z. Wang, Y.C. Zhai, Y.W. Tian, Property and application of LaF3 or doped LaF3 solid-electrolyte, Chinese Journal of Rare Metals 27 (2003) 596–600. [22] H. Sun, N.F. Hu, H.Y. Ma, Direct electrochemistry of hemoglobin in polyacrylamide hydrogel films on pyrolytic graphite electrodes, Electroanalysis 12 (2000) 1064–1070. [23] J.F. Rusling, A.E.F. Nassar, Enhanced electron-transfer for myoglobin in surfactant films on electrodes, Journal of the American Chemical Society 115 (1993) 11891–11897. [24] X.Q. Li, R.J. Zhao, Y. Wang, X.Y. Sun, W. Sun, C.Z. Zhao, K. Jiao, An electrochemical biosensor based on nafion-ionic liquid and a myoglobin-modified carbon paste electrode, Electrochimica Acta 55 (2010) 2173–2178. [25] R.P. Liang, M.Q. Deng, S.G. Cui, H. Chen, J.D. Qiu, Direct electrochemistry and electrocatalysis of myoglobin immobilized on zirconia/multi-walled carbon nanotube nanocomposite, Materials Research Bulletin 45 (2010) 1855–1860. [26] Q. Zhang, L. Zhang, B. Liu, X.B. Lu, J.H. Li, Assembly of quantum dots–mesoporous silicate hybrid material for protein immobilization and direct electrochemistry, Biosensors and Bioelectronics 23 (2007) 695–700. [27] E. Laviron, General expression of the linear potential sweep voltammogram in the case of diffusionless electrochemical systems, Journal of Electroanalytical Chemistry 101 (1979) 19–28. [28] E. Topoglidis, Y. Astuti, F. Duriaux, M. Grätzel, J.R. Durrant, Direct electrochemistry and nitric oxide interaction of heme proteins adsorbed on nanocrystalline tin oxide electrodes, Langmuir 19 (2003) 6894–6900.

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[29] Y. Li, Y.F. Gao, Y.C. Liu, Y. Zhou, J.R. Liu, Direct electrochemistry of hemoglobin immobilized on electrode modified by ceria, Acta Chimica Sinica 68 (2010) 1161–1166. ˜ ˜ J.M. Pingarrón, A biosensor based on [30] M. Eguílaz, L. Agüí, P. Yánez-Sede no, cytochrome c immobilization on a poly-3-methylthiophene/multi-walled carbon nanotubes hybrid-modified electrode. Application to the electrochemical determination of nitrite, Journal of Electroanalytical Chemistry 644 (2010) 30–35. [31] X.B. Lu, H.J. Zhang, Y.W. Ni, Q. Zhang, J.P. Chen, Porous nanosheet-based ZnO microspheres for the construction of direct electrochemical biosensors, Biosensors and Bioelectronics 24 (2008) 93–98. [32] W. Sun, X.Q. Li, K. Jiao, Direct electrochemistry of myoglobin in a nafion-ionic liquid composite film modified carbon ionic liquid electrode, Electroanalysis 21 (2009) 959–964. [33] W. Sun, X.Q. Li, P. Qin, K. Jiao, Electrodeposition of Co nanoparticles on the carbon ionic liquid electrode as a platform for myoglobin electrochemical biosensor, Journal of Physical Chemistry C 113 (2009) 11294–11300.

Biographies Sheying Dong is a Professor in College of Sciences at Xi’an University of Architecture and Technology. She obtained his Ph. D. in 2003 at the Department of Chemistry of Northwest University, China. Her main research fields include electroanalytical chemistry, bioelectrochemical sensor, ionic liquid electrochemistry and environmental science. Nan Li is studying for master in the in College of Sciences at Xi an University of Architecture and Technology. She received her BS at Xi an University of Architecture and Technology in 2010. Tinglin Huang is a Professor at Xi an University of Architecture and Technology. His main research fields include bioelectrochemical sensor and environmental science. Hongsheng Tang is a teacher of Institute of Analytical Science/Shaanxi Provincial Key Laboratory of Electroanalytical Chemistry, Northwest University. Jianbin Zheng is a scientific expert Institute of Analytical Science/Shaanxi Provincial Key Laboratory of Electroanalytical Chemistry, Northwest University. His research interests include electroanalytical chemistry, bioelectrochemical sensor, ionic liquid electrochemistry, HPLC electrochemistry and chemometrics.