Biosensors and Bioelectronics ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Immobilization of superoxide dismutase on Pt–Pd/MWCNTs hybrid modified electrode surface for superoxide anion detection Xiang Zhu a, Xiangheng Niu a, Hongli Zhao a,n, Jie Tang a, Minbo Lan a,b,nn a b
Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, PR China State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 14 May 2014 Received in revised form 3 July 2014 Accepted 3 July 2014
Monitoring of reactive oxygen species like superoxide anion (O2∙ ) turns to be of increasing significance considering their potential damages to organism. In the present work, we fabricated a novel O2∙ electrochemical sensor through immobilizing superoxide dismutase (SOD) onto a Pt–Pd/MWCNTs hybrid modified electrode surface. The Pt–Pd/MWCNTs hybrid was synthesized via a facile one-step alcoholreduction process, and well characterized by transmission electron microscopy, X-ray photoelectron spectroscopy and X-ray diffraction. The immobilization of SOD was accomplished using a simple dropcasting method, and the performance of the assembled enzyme-based sensor for O2∙ detection was systematically investigated by several electrochemcial techniques. Thanks to the specific biocatalysis of SOD towards O2∙ and the Pt–Pd/MWCNTs – promoted fast electron transfer at the fabricated interface, the developed biosensor exhibits a fast, selective and linear amperometric response upon O2∙ in the concentration scope of 40–1550 μM (R2 ¼ 0.9941), with a sensitivity of 0.601 mA cm 2 mM 1 and a detection limit of 0.71 μM (S/N¼ 3). In addition, the favorable biocompatibility of this electrode interface endows the prepared biosensor with excellent long-term stability (a sensitivity loss of only 3% over a period of 30 days). It is promising that the proposed sensor will be utilized as an effective tool to quantitatively monitor the dynamic changes of O2∙ in biological systems. & 2014 Elsevier B.V. All rights reserved.
Keywords: Pt–Pd/MWCNTs hybrid Superoxide dismutase Enzyme-based electrochemcial sensor Superoxide anion detection High sensitivity and stability
1. Introduction Various evidences suggest that reactive oxygen species (ROS) play a vital role in atherosclerosis, hypertension, diabetes, inflammation and chronic nitrate tolerance as well as in postischemic myocardium (Dröge, 2002; Valko et al., 2007). Among ROS, superoxide anion (O2∙ ) is of critical importance for its harmful interaction with biological molecules and its function as a message in signaling pathways (Valko et al., 2007). Up to date, there have been a number of approaches developed to measure O2∙ qualitatively and quantitatively, mainly including electron spin resonance (Stolze et al., 2000; Wang et al., 2013b; Zhang et al., 2000), spectrophotometry (Manjare et al., 2014; Yang et al., 2006; Zhao et al., 2005), chemiluminescence (Lu et al., 2006; Skatchkov et al., 1999), chromatography (Dikalov and Harrison, 2014 ; Fink et al., 2004; Zhao et al., 2005) and electrochemical measurement (Kim et al., 2012 ; Liu et al., 2009; Wang et al., 2012). In comparison with n
Corresponding author. Tel.: þ 86 21 64253574; fax: þ86 21 64252947. Corresponding author at: Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, PR China. Tel.: þ 86 21 64253574; fax: þ86 21 64252947. E-mail addresses: [email protected] (H. Zhao), [email protected] (M. Lan). nn
other methods, electroanalysis is well known as a simple and efficient tool due to its merits of low-cost instrumentation, fast operation and favorable performance, and has drawn considerable attention and interest in recent years. The widely studied enzymebased sensors were fabricated by immobilizing copper–zinc superoxide dismutase (Cu–Zn SOD) onto the electrode surface (Kim et al., 2012). However, the electron transfer between SOD and electrode is very difficult because of the deeply buried redox centers of SOD and its unfavorable orientations (Wang et al., 2012). Besides, the instability of enzymes results in great difficulties for the assembly, storage and use of enzyme-involved electrochemical biosensors. In order to promote the electron transfer and prolong the life of enzyme electrodes, many immobilization methods have been developed, such as directly binding to a support, encapsulation and cross-linking (Hanefeld et al., 2009; Mateo et al., 2007; Sheldon, 2007). Among these methods, employing nanomaterials as carriers/hosts to immobilize enzyme is a promising way (Sheldon, 2007; Shi et al., 2011b). Nanomaterials including conducting polymers, composite matrixes, metallic/polymeric nanoparticles and nanoclays are ideal carriers for the immobilization of enzymes for their large surface area, good biocompatibility and excellent stability (Pusch et al., 2013). Recently, biosensors based on SOD for O2∙ detection
http://dx.doi.org/10.1016/j.bios.2014.07.004 0956-5663/& 2014 Elsevier B.V. All rights reserved.
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encounter with a low sensitivity and narrow linear range due to the lack of a friendly interface for immobilization of SOD (Wang et al., 2012). As common materials for modifying electrodes, Pt, Pd and carbon nanotubes (CNTs) are usually used to fabricate enzyme sensors for H2O2, glucose and other biomolecular detection because they can effectively improve the analytical performance of the modified sensor due to the faster electron transfer (Kang et al., 2007; Niu et al., 2012; Santhosh et al., 2011). However, to the best of our knowledge, there are no reports employing these materials as a biocompatible interface to immobilize SOD for O2∙ detection. We suspect whether it is possible to utilize Pt, Pd and CNTs to construct an interface for immobilizing SOD and promote analytical properties. CNTs are fascinating materials and commonly utilized to fabricate electrochemical sensing interfaces. CNTs can not only enhance the electrochemical reactivity of biomolecules but also promote the electron-transfer reaction of proteins whose redox centers are embedded deeply within glycoprotein shells (Shan et al., 2009; Wang, 2005, 2009). Although CNTs are a good matrix for the immobilization of enzymes, their catalytic activities remain to be promoted. To solve this problem, metallic nanoparticles have been introduced to form various hybrids with CNTs to improve the catalytic activities of CNTs (McLamore et al., 2011; Shi et al., 2011a, 2012). In recent years, metallic nanoparticles (NPs), especially bimetallic NPs, have been investigated for their importance in catalysis due to the bifunctional or synergistic effects. This combination of two metallic elements in a single particle results in a material with interesting properties, including chemical selectivity and catalytic activity superior to those of monometallic NPs (Fu et al., 2013; Huang et al., 2013; Pusch et al., 2013). Among these bimetallic NPs, Pt–Pd NPs are fascinating materials, and can combine with CNTs to fabricate electrochemical sensing interfaces. On the one hand, the synergistic effect of Pt and Pd has great catalytic activities to enhance the performance of modified electrodes for H2O2 detection (Janyasupab et al., 2013; Niu et al., 2012); on the other hand, both Pt–Pd nanoparticles and CNTs have the properties of good biocompatibility, excellent stability and large surface area, providing an ideal interface for the immobilization of enzymes (Kim et al. 2006; Siriviriyanun et al., 2013). Driven by the increasing demands of monitoring O2∙ in biological systems with ultra-sensitive, well selective and rapid response, we here recommend a captivating electrochemical sensing interface composed of Pt–Pd NPs and CNTs for the immobilization of SOD, and use the assembled sensor to qualify and quantify O2∙ . The fabricated interface shows three advantages: (1) the Pt–Pd/CNTs hybrid is synthesized using a facile onepot method; (2) the interface can facilitate the electron transfer between SOD and electrode due to the favorable electro-conductivity of Pt–Pd NPs and CNTs; and (3) the good biocompatibility of the prepared hybrid increases the stability of SOD, and thus prolongs the usage time of the sensor. It is found that the Pt–Pd/ CNTs modified enzyme electrode offers attractive sensitivity and excellent selectivity for O2∙ detection.
Industrial Inc. Ethylene glycol and trisodium citrate were provided by Shanghai Lingfeng Chemical Reagent Co. D-fructose (Fru), ascorbic acid (AA), dopamine (DA), glucose (Glu), uric acid (UA), 4-acetamidophenol (AP) and superoxide dismutase from bovine erythrocytes were commercially provided by Sigma-Aldrich. Ultrapure water (18.2 MΩ cm, Laboratory Water Purification Systems) was used in all experiments. All other chemicals were of analytical grade and utilized as received. A field emission scanning electron microscope (FESEM, S-4800, Hitachi) was utilized to observe the surface morphology of Pt–Pd/ MWCNTs with/without the immobilization of SOD. A transmission electron microscope (TEM, JEM-1400, JEOL) was also used to capture the morphology of Pt–Pd/MWCNTs. An X-ray photoelectron spectrometer (XPS, ESCALAB 250Xi, Thermo Fisher) was employed to detect the surface composition of the as-prepared Pt–Pd/MWCNTs hybrid. Powder X-ray diffraction (XRD) measurements were carried out in a D/MAX2550 diffractometer (Rigaku International Co.) using Cu Kα (λ ¼0.15408 nm) radiation. Ultraviolet–visible spectroscope (UV–Vis, EVOLUTION 220, Thermo Scientific) was used to detect the concentration of O2∙ obtained from the KO2 stock solution. All electrochemical experiments were performed at room temperature on a CHI660D workstation equipped with a conventional three-electrode system consisting of a screen-printed working electrode, a platinum wire counter electrode, and an Ag/AgCl reference electrode. 2.2. Synthesis of the Pt–Pd/MWCNTs hybrid Before use, the purchased MWCNTs were acid-treated by refluxing in a mixture of concentrated HNO3 and H2SO4 (v/v, 1:3) for 8 h at 80 °C. The Pt–Pd/MWCNTs hybrid was prepared by a facile one-step alcohol-reduction process. In brief, 10 mg MWCNTs were dispersed into the solution of 20 mL ethylene glycol and 20 mL deionized water. Then, 300 mg trisodium citrate, 50 μmol H2PtCl6 and 50 μmol Na2PdCl4 were added to the mixture above. After sonication for 30 min, the pH was adjusted to 10 with NaOH. The mixture solution was deoxygenated by nitrogen for 30 min and reacted for 8 h. Finally, the hybrid was washed with water and dried for characterization and measurement. 2.3. Fabrication of the modified electrode Screen-printed gold film electrodes as the substrate were home-prepared according to our previous work (Niu et al., 2011), and the diameter of the working electrode is 3 mm. As for the Pt– Pd/MWCNTs modified SOD-based sensor (SOD/Pt–Pd/MWCNTs/ SPGE), 2 mg Pt–Pd/MWCNTs were added into 1 mL ultrapure water and sonicated for 2 h to form a well-dispersed mixture. Afterwards, 5 μL of the mixture, different volumes of SOD (3000 U/mL) and 2 μL of 0.05 wt% Nafion were drop-casted onto the electrode surface, and dried at room temperature before use. Besides, the Pt–Pd/MWCNTs sensor was prepared without immobilizing SOD. 2.4. Generation of superoxide anion
2. Experimental 2.1. Chemicals and apparatus Multi-walled carbon nanotubes (MWCNTs) were obtained from Shenzhen Nanotech Port Co., with a mean diameter of 15 nm and a tube length of 1–2 μm. H2PtCl6 6H2O and Na2PdCl4 were purchased from Sinopharm Chemical Reagent Co. and used directly without further purification. Anhydrous dimethyl sulfoxide, 18crown-6 and potassium dioxide were obtained from Aladdin
A stock solution of KO2 was prepared by adding KO2 to anhydrous dimethyl sulfoxide (containing 18-crown-6). After sonicating the solution for 2 min, KO2 dissociated and produced O2∙ . The solubility of KO2 in DMSO can be increased by addition of 18-crown-6, which complexes K þ (Hyland and Auclair, 1981). According to the molar absorptivity of O2∙ in DMSO (2006 M 1 cm 1 at 271 nm), the concentration of O2∙ could be estimated by a UV–Vis spectroscope (Hyland and Auclair, 1981; Thandavan et al., 2013).
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2.5. Electrochemical measurements The behaviors of the fabricated electrodes were studied using the cyclic voltammetric, chronoamperometric and electrochemical impedance techniques. All chronoamperometric measurements were implemented by successively injecting the analyte into 5 mL 0.05 M PBS (pH 7.4, containing 0.1 M KCl) under a constant stirring.
3. Results and discussion 3.1. Synthesis and characterization of the Pt–Pd/MWCNTs hybrid The Pt–Pd/MWCNTs hybrid was synthesized by a facile onestep alcohol-reduction process. Ethylene glycol was used as the solvent and the reducing agent; trisodium citrate was utilized as a complexing agent and stabilizer that prevented alloys from aggregation; and MWCNTs were employed as the support for Pt–Pd alloys reduction in situ (Wang et al., 2008). In this work, the TEM technique was used to confirm the formation of the Pt–Pd/MWCNTs hybrid firstly. Fig. 1(A) shows the morphology of the as-prepared hybrid. It is found that Pt–Pd nanoparticles are uniformly deposited on the surface of MWCNTs without obvious aggregation. The size distribution is evaluated statistically by measuring the diameter of Pt–Pd nanoparticles in
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the selected area. As shown in Fig. 1(B), the Pt–Pd nanoparticles have a mean size of 5.2 nm, with a narrow size distribution. The well-defined size distribution is mainly due to the use of trisodium citrate as the complexing agent and the stabilizer and MWCNTs as an idea carrier for alloys. The XPS technique was used to further confirm the surface composition of the Pt–Pd/MWCNTs hybrid. As depicted in Fig. 1(C), the peaks located at 69.5, 72.8, 183.2 and 291.2 eV are assigned to the binding energy of Pt4f7/2, Pt4f5/2, Pd3d5/2 and Pd3d3/2, respectively (Fig. S1), revealing the coexistance of Pt(0) and Pd(0) (Kadirgan et al., 2009; Li et al., 2014 ). The calculated atom ratio of Pt and Pd is approximately 1:1, which is in accordance with the XPS analysis from the table of elemental component. Meanwhile, the peaks at 283.1 eV and 531.1 eV are attributed to the binding energy of C1s and O1s, corresponding to the sp2 C–C peak from MWCNTs and the oxygen functionalities originating from the functional groups (–OH, –COOH) of acid treated-MWCNTs. Fig. 1(D) represents the typical XRD patterns of the as-prepared hybrid. MWCNTs show a broad diffraction peak observed at 26°. This peak corresponds to the (002) plane of the graphitic carbon, indicating the presence of MWCNTs in the as-prepared hybrid (Kannan et al., 2013). Due to that Pt and Pd have almost the same crystal structures, the difference of the XRD peak positions of Pt and Pd is not distinct. The representative diffraction peaks at 40°, 46°, 67° and 82° are attributed to the (111), (200), (220) and (311) planes of Pt–Pd alloys with the face-centered cubic (FCC) structure.
Fig. 1. TEM image (A), size distribution (B), XPS pattern (C) and XRD pattern (D) of Pt–Pd/MWCNTs hybrid.
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Fig. 2. FESEM images of (A) Pt–Pd/MWCNTs/SPGE and (B) SOD/Pt–Pd/MWCNTs/SPGE, (C) The EIS curves for the bare SPGE, Pt–Pd/MWCNTs/SPGE and SOD/Pt–Pd/MWCNTs/ SPGE in 100 mM KCl solution containing 5 mM Fe(CN)63 /4 with the frequency ranging from 100 kHz to 0.01 Hz.
To note that these diffraction peaks are slightly shifted to higher 2θ values in comparison with the commercial Pt black (JCPDS 040601) (Li et al., 2014), indicating the formation of Pt–Pd alloys, and this result agrees with the XPS result. According to the value of the full-width at half-maximum (FWHM) of the (111) diffraction peaks, the mean crystallite size of Pt–Pd particles is calculated to be 5.4 nm by the Scherrer equation (D ¼0.9λ/β cos θ, where D is the particle size, λ is the radiation wavelength, β is the FWHM, and θ is the Bragg diffraction angle). The calculated average size is closely matched with that obtained from the TEM image (Kannan et al., 2013). 3.2. Electrochemical properties of the SOD/Pt–Pd/MWCNTs/SPGE sensor FESEM was used to characterize the morphology of electrodes during the step-wise modification. As shown in Fig. 2(A), the synthesized hybrid is uniformly distributed on the surface SPGE. After SOD is immobilized, the hybrid looks rough and incompact with (Fig. 2(B)) some agglomerates, (Wang et al., 2013a) indicating the existence of SOD, and the successful fabrication of enzyme electrode.
Electrochemical impedance spectroscopy (EIS) is carried out to identify the change of the charge transfer resistance and/or capacitance on the electrode surface, which can further reflect the step-wise modification process. The EIS curves of the bare SPGE, Pt–Pd/MWCNTs/SPGE and SOD/Pt–Pd/MWCNTs/SPGE are shown in Fig. 2(C). The charge transfer resistance (Rct) of the bare SPGE is 17,526 Ω. After the modification of Pt–Pd/MWCNTs on the SPGE substrate, the Rct decreases dramatically to 286 Ω, which demonstrates that the presence of the Pt–Pd alloy and MWCNTs can greatly enhance the conductivity of the electrode and promotes the electron transfer between the electrolyte and the electrode interface. When SOD is immobilized on the electrode, the Rct shows a slight increase to 669 Ω, indicating that the immobilization of SOD will cause an insulating effect on the electrode and increases the charge transfer resistance (Emregul et al., 2013). These results are in complete accordance with the typical CVs of the bare SPGE, Pt–Pd/MWCNTs/SPGE and SOD/Pt– Pd/MWCNTs/SPGE in 0.05 M PBS (0.1 M KCl) containing 5 mM Fe(CN)6 3 − /4 − at a scan rate of 50 mV s 1 in the potential range of 0.2 V to 0.6 V (shown in Fig. S2). We further checked the electrochemistry behavior of SOD/Pt– Pd/MWCNTs/SPGE in 0.05 M PBS (0.1 M KCl) containing 5 mM Fe(CN)6 3 − /4 − at different scan rates of 10–200 mV s 1.
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Fig. 3. Cyclic voltammograms of the SOD/Pt–Pd/MWCNTs/SPGE sensor in 0.05 M PBS (pH 7.4, containing 0.1 M KCl) with the absence and presence of different amounts of KO2 (different concentrations of O2d ) at scan rate of 50 mV s 1.
As presented in Fig. S3(A), with increasing scan rate, both the peak-to-peak separation (ΔEp) and the ΔIp increase along with the increasing scan rate. Besides, the oxidation peak shifts towards more positive potential and the reduction peak shifts towards more negative potential. It is further estimated that the ΔIp is linearly proportional to the square root of scan rate (Fig. S3(B)), suggesting the diffusion confined process. 3.3. Amperometric detection of O2∙ The electrochemical properties of modified electrodes towards the detection of O2∙ was evaluated by the CV technique. As displayed in Fig. 3, when different volumes of KO2 are added, both the oxidation and reduction currents of the sensor gradually increases, indicating that the SOD/Pt–Pd/MWCNTs/SPGE sensor has excellent response towards O2∙ . The enhanced reduction and oxidation currents can be explained by the following reaction mechanism (Di et al., 2004):
SOD(Cu(II)) + e− → SOD(Cu(I)) H+
SOD(Cu(I)) + O2∙− → SOD(Cu(II)) + H2 O2 SOD(Cu(I)) − e− → SOD(Cu(II))
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7.4. The results indicate that SOD retains the highest enzymatic activity and natural structure on the fabricated interface when the pH value is around the physical condition. Therefore, pH 7.4 is determined to be the optimum pH for further experiments. The loading of SOD was further optimized by immobilization different amounts of SOD (1–5 μL) on the modified electrode. When the amount of enzyme is lower than 3 μL, the sensor exhibits a very low sensitivity due to the scarce loading of enzyme molecules. When the amount is higher than 3 μL, the current gradually decreases, due to that SOD can act as a resistance material. As a result, the optimum amount of enzyme used for immobilization onto the electrode surface is 3 μL (displayed in Fig. S5). Then, the proper potential applied at the working electrode is studied to achieve the lowest detection limit. Fig. S6 displays the typical chronoamperometric responses of the proposed sensor for O2∙ detection at different applied potentials. It is obvious that the proposed sensor provides the maximum response at 0.1 V, which fits well with the DPV curves (as shown in Fig S7 in Supplementary information). Therefore, an applied potential of 0.1 V vs. Ag/AgCl is selected as the optimized potential in the following measurements. Finally, some other factors of the amperometric response to O2∙ , such as oxygen deficiency, the presence of chlorides ions and 18-crown-6, are also studied. As depicted in Fig. S8(A and B), it is observed that the response of the electrode in air saturated solution is lower than that of the Ar saturated electrolyte. Oxygen is thus a potential interfering compound to detect the superoxide radicals for the present system and has to be taken into account. Prior to analysis, the solution should bubble Ar gas for 10 min to eliminate the dissolved oxygen. In addition, the electrode offers equal current response in the presence/absence of 0.1 M KCl in the analyte solution, indicating the excellent poison resistance/antiinterference behavior of the sensor due to the formation of Pt–Pd alloys reduction the possibility of catalyst poisoning against chlorides ions. 18-crown-6 is used as co-solvent for preparation of KO2 stock solution (Cao et al., 2013). We have also studied the effect of the using 18-crown-6 (as shown in Fig. S9). The results demonstrate this species produces negligible response and does not interfere with the detection of O2∙ . Under the above optimized conditions, typical chronoamperometric responses of the prepared sensor for successive additions of O2∙ into constantly stirred 0.05 M PBS (pH 7.4, containing 0.1 M KCl) at the applied potential of 0.1 V are recorded. As shown in Fig. 4, a gradual increase of current is observed when adding the KO2 stock solution, indicating that SOD can catalyze O2∙ to H2O2, and then the Pt–Pd/MWCNTs hybrid has the
SOD(Cu(II)) + O2∙− → SOD(Cu(I)) + O2 SOD can efficiently catalyze the dismutation of O2∙ to O2 and H2O2 via a redox cycle of the copper complex moiety (Cu(I/II)) couple in Cu–Zn SOD. Two O2∙ ions are stoichiometrically converted to one O2 molecule and one H2O2 molecule with consumption of two H þ ions during the dismutation (Di et al., 2004). Then, the oxidation and reduction of H2O2 is effectively electrocatalyzed by Pt–Pd/MWCNTs, leading to the increase of both oxidation (0.6 V) and reduction current (0 V), which is consistent with the literature (Bo et al., 2011; Chen et al., 2012; Kang et al., 2007). Prior to amperometric analysis of O2∙ , experimental parameters possibly affecting the analytical performance of the fabricated sensor are optimized. The effect of pH, ranging from 6.8 to 8.6, on the electrochemical behavior of the SOD/Pt–Pd/MWCNTs sensor was firstly measured by the CV technique in 0.05 M PBS containing 5 mM Fe(CN)63 − /4 − and 0.1 M KCl. As shown in Fig. S4, the reduction current gradually increases from pH 6.8 to 7.4, and the highest magnitude of the current response is obtained at pH
Fig. 4. Amperometric response of the SOD/Pt–Pd/MWCNTs/SPGE for the successive addition of different concentration of O2d into constantly stirred 0.05 M PBS, pH 7.4 (containing 0.1 M KCl). Applied potential: 0.1 V.
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Table 1 Comparison of the analytical performance of various electrodes for O2d sensing. Electrode
Applied potential (V)
Sensitivity (μA cm 2 mM 1)
LOD (μM)
Linear range (μM)
Ref.
SOD/SA SOD/nano-Fe3O4/Au electrode SOD-CNT-PPy-Pt electrode PMMA/PANI-Au-nano/SOD-ESCFM SOD-CS-gold nanoparticles-ITO SOD/GNP/Cys/SG/Au SOD/Pt–Pd/MWCNTs/SPEG
0 0.5 – 0.3 0 0.15 0.1
0.782 – – 42.5 – – 601
0.23 0.2 0.1 0.3 0.2 0.2 0.71
0.4–229.9 0.2–1.4 0.1–750 0.5–2.4 0.08–0.64 0.05–0.40 40–1550
Wang et al. (2012) Thandavan et al. (2013) Rajesh et al. (2010) Santhosh et al. (2011) Wang et al. (2009) Di et al. (2007) This work
SOD: superoxide dismutase; SA: sodium alginate; PPy: polypyrrole; PMMA: poly(methyl-methacrylate); PANI: polyaniline; ESCFM: electrospun nanofibers; CS, Cys: cysteine; and GNP: gold nanoparticle; SG: sol–gel.
effective electron-transfer ability towards the reduction of enzymatically produced H2O2. The incremental current reaches a steady state within 5 s after addition of O2∙ . The inset in Fig. 4 shows that the amperometric responses are linear with the O2∙ concentration in the range of 40–1550 μM (R2 ¼0.9941), with a sensitivity of 0.601 mA cm 2 mM 1. The limit of detection is further calculated to be as low as 0.71 μM (S/N ¼3). In comparison with other sensors based on SOD for O2∙ detection and the sensor without nanomaterials (as shown in Fig. S10), our proposed sensor provides higher sensitivity and wider linear range (as list in Table 1). app ) is an indication The apparent Michaelis–Menten constant (Km of the enzyme–substrate kinetics which is generally used to app can be evaluate the biological activity of enzyme. The Km calculated from the electrochemical version of the Linweaver– Burk equation (Kamin and Wilson, 1980):
K app 1 1 1 = + m × iss imax imax c where iss is the steady-state current, imax is the maximum current, Kmapp is the apparent Michaelis–Menten constant, C is the concenapp tration of O2∙ (Turkmen et al., 2014). The Km value for the Pt–Pd/ MWCNTs sensor is 3.44 μM, indicating that SOD immobilized on the Pt–Pd/MWCNTs has a high enzymatic activity and affinity to O2∙ . Several potential interfering substances were used to evaluate the sensitivity of the fabricated SOD/Pt–Pd/MWCNTs/SPGE. The typical chronoamperometric responses upon successive addition of 0.08 mM O2∙ , 0.5 mM Glu, Fru, AA, UA, AP and DA into 0.05 M PBS (pH 7.4, containing 0.1 M KCl) are recorded. As depicted in Fig. 5(A), it is found that these species produce negligible response for the amperometric detection of O2∙ , indicating that
the proposed electrode can be employed to selectively detect O2∙ . This attractive specificity is mainly ascribed to the immobilization of SOD, which has a high selectivity towards O2∙ ; relatively low potential being used ( 0.1 V), where these species cannot be electrochemically oxidized, and the outermost Nafion layer as an effectively pre-selective barrier against anionic interferences. The reproducibility of the SOD/Pt–Pd/MWCNTs/SPGE sensor was estimated according to the responses provided by eight newly-prepared electrodes for 0.08 mM O2∙ . The results reveal that the recommended sensor exhibits a satisfied reproducibility with a RSD of 2.8%. The long-term stability is also an important factor to evaluate the performance of sensors. The amperometric responses of 0.08 mM O2∙ were collected every three days to check the long-term stability, and the electrode was stored at 4 °C when not in use. As depicted in Fig. 5(B), the sensitivity remains 97% of its original value after a month, suggesting that the fabricated electrode has a considerable stability for O2∙ detection. Two main factors lead to the good reproducibility and stability: on the one hand, good biocompatibility and excellent stability of the hybrid provide the enzyme with a friendly interface for immobilization and thus prolong the usage time of the enzyme electrode; on the other hand, the Nafion layer grasps the Pt–Pd/MWCNTs hybrid modified on the electrode surface well against leakage when testing. 3.4. Real sample analysis The test for O2∙ detection in Dulbecco's modified Eagle's medium (DMEM) cell culture medium containing fetal bovine serum (FBS, 10%) and penicillin/streptomycin (1%). This solution was used to mimic the environment of cell culture and the test in this sophisticated system was meaningful to carry out real sample
Fig. 5. (A) Effect of the potential interferents in O2d biosensor. The arrows show each moment at successive addition of 0.08 mM O2d , 0.5 mM Glu, 0.5 mM Fru, 0.5 mM AA and 0.5 mM UA, 0.5 mM AP and 0.5 mM DA. (B) Stability in sensitivity of the fabricated sensor in a period of one month.
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analysis by the proposed sensor. The real sample analysis was conducted on DMEM cell culture medium containing 10% FBS, and penicillin/streptomycin (1%) using a calibration curve method to determine the recoveries of various concentration of O2∙ . The sample was prepared by spiking different concentration of O2∙ into the mixture of 4 mL PBS and 1 mL DMEM containing 10% FBS and 1% penicillin/streptomycin, and the final concentration of O2∙ in sample was 0 (as control group), 40, 60, and 80 μM, respectively. The recovery is 102.4%, 98.5% and 97.4%, and the relative standard deviation is 1.5%, 3.6% and 2.9%. The results (as shown in Table S1) indicated that the methodology employed in the present work might be applied for sensitive and selective detection of O2∙ in real samples.
4. Conclusions As a conclusion, in this work we immobilized SOD onto the Pt– Pd/MWCNTs-constructed electrochemical interface, and fabricated a new enzyme-based O2∙ electrochemical sensor. Owning to the good biocompatibility and excellent stability of the interface, which provides SOD with a friendly immobilization environment, the proposed sensor exhibited high selectivity and sensitivity for O2∙ detection.
Acknowledgments This research was financially supported by National Natural Science Foundation of China (NSFC, No. 21305044), the Fundamental Research Funds for the Central Universities (No. 222201314026) and Science and Technology Commission of Shanghai Municipality (STCSM, No. 13510710900).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at: http://dx.doi.org/10.1016/j.bios.2014.07.004.
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Immobilization of superoxide dismutase on Pt–Pd/MWCNTs hybrid modified electrode surface for superoxide anion detection Xiang Zhu a, Xiangheng Niu a, Hongli Zhao a,n, Jie Tang a, Minbo Lan a,b,nn a b
Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, PR China State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 14 May 2014 Received in revised form 3 July 2014 Accepted 3 July 2014
Monitoring of reactive oxygen species like superoxide anion (O2∙ ) turns to be of increasing significance considering their potential damages to organism. In the present work, we fabricated a novel O2∙ electrochemical sensor through immobilizing superoxide dismutase (SOD) onto a Pt–Pd/MWCNTs hybrid modified electrode surface. The Pt–Pd/MWCNTs hybrid was synthesized via a facile one-step alcoholreduction process, and well characterized by transmission electron microscopy, X-ray photoelectron spectroscopy and X-ray diffraction. The immobilization of SOD was accomplished using a simple dropcasting method, and the performance of the assembled enzyme-based sensor for O2∙ detection was systematically investigated by several electrochemcial techniques. Thanks to the specific biocatalysis of SOD towards O2∙ and the Pt–Pd/MWCNTs – promoted fast electron transfer at the fabricated interface, the developed biosensor exhibits a fast, selective and linear amperometric response upon O2∙ in the concentration scope of 40–1550 μM (R2 ¼ 0.9941), with a sensitivity of 0.601 mA cm 2 mM 1 and a detection limit of 0.71 μM (S/N¼ 3). In addition, the favorable biocompatibility of this electrode interface endows the prepared biosensor with excellent long-term stability (a sensitivity loss of only 3% over a period of 30 days). It is promising that the proposed sensor will be utilized as an effective tool to quantitatively monitor the dynamic changes of O2∙ in biological systems. & 2014 Elsevier B.V. All rights reserved.
Keywords: Pt–Pd/MWCNTs hybrid Superoxide dismutase Enzyme-based electrochemcial sensor Superoxide anion detection High sensitivity and stability
1. Introduction Various evidences suggest that reactive oxygen species (ROS) play a vital role in atherosclerosis, hypertension, diabetes, inflammation and chronic nitrate tolerance as well as in postischemic myocardium (Dröge, 2002; Valko et al., 2007). Among ROS, superoxide anion (O2∙ ) is of critical importance for its harmful interaction with biological molecules and its function as a message in signaling pathways (Valko et al., 2007). Up to date, there have been a number of approaches developed to measure O2∙ qualitatively and quantitatively, mainly including electron spin resonance (Stolze et al., 2000; Wang et al., 2013b; Zhang et al., 2000), spectrophotometry (Manjare et al., 2014; Yang et al., 2006; Zhao et al., 2005), chemiluminescence (Lu et al., 2006; Skatchkov et al., 1999), chromatography (Dikalov and Harrison, 2014 ; Fink et al., 2004; Zhao et al., 2005) and electrochemical measurement (Kim et al., 2012 ; Liu et al., 2009; Wang et al., 2012). In comparison with n
Corresponding author. Tel.: þ 86 21 64253574; fax: þ86 21 64252947. Corresponding author at: Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, PR China. Tel.: þ 86 21 64253574; fax: þ86 21 64252947. E-mail addresses: [email protected] (H. Zhao), [email protected] (M. Lan). nn
other methods, electroanalysis is well known as a simple and efficient tool due to its merits of low-cost instrumentation, fast operation and favorable performance, and has drawn considerable attention and interest in recent years. The widely studied enzymebased sensors were fabricated by immobilizing copper–zinc superoxide dismutase (Cu–Zn SOD) onto the electrode surface (Kim et al., 2012). However, the electron transfer between SOD and electrode is very difficult because of the deeply buried redox centers of SOD and its unfavorable orientations (Wang et al., 2012). Besides, the instability of enzymes results in great difficulties for the assembly, storage and use of enzyme-involved electrochemical biosensors. In order to promote the electron transfer and prolong the life of enzyme electrodes, many immobilization methods have been developed, such as directly binding to a support, encapsulation and cross-linking (Hanefeld et al., 2009; Mateo et al., 2007; Sheldon, 2007). Among these methods, employing nanomaterials as carriers/hosts to immobilize enzyme is a promising way (Sheldon, 2007; Shi et al., 2011b). Nanomaterials including conducting polymers, composite matrixes, metallic/polymeric nanoparticles and nanoclays are ideal carriers for the immobilization of enzymes for their large surface area, good biocompatibility and excellent stability (Pusch et al., 2013). Recently, biosensors based on SOD for O2∙ detection
http://dx.doi.org/10.1016/j.bios.2014.07.004 0956-5663/& 2014 Elsevier B.V. All rights reserved.
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encounter with a low sensitivity and narrow linear range due to the lack of a friendly interface for immobilization of SOD (Wang et al., 2012). As common materials for modifying electrodes, Pt, Pd and carbon nanotubes (CNTs) are usually used to fabricate enzyme sensors for H2O2, glucose and other biomolecular detection because they can effectively improve the analytical performance of the modified sensor due to the faster electron transfer (Kang et al., 2007; Niu et al., 2012; Santhosh et al., 2011). However, to the best of our knowledge, there are no reports employing these materials as a biocompatible interface to immobilize SOD for O2∙ detection. We suspect whether it is possible to utilize Pt, Pd and CNTs to construct an interface for immobilizing SOD and promote analytical properties. CNTs are fascinating materials and commonly utilized to fabricate electrochemical sensing interfaces. CNTs can not only enhance the electrochemical reactivity of biomolecules but also promote the electron-transfer reaction of proteins whose redox centers are embedded deeply within glycoprotein shells (Shan et al., 2009; Wang, 2005, 2009). Although CNTs are a good matrix for the immobilization of enzymes, their catalytic activities remain to be promoted. To solve this problem, metallic nanoparticles have been introduced to form various hybrids with CNTs to improve the catalytic activities of CNTs (McLamore et al., 2011; Shi et al., 2011a, 2012). In recent years, metallic nanoparticles (NPs), especially bimetallic NPs, have been investigated for their importance in catalysis due to the bifunctional or synergistic effects. This combination of two metallic elements in a single particle results in a material with interesting properties, including chemical selectivity and catalytic activity superior to those of monometallic NPs (Fu et al., 2013; Huang et al., 2013; Pusch et al., 2013). Among these bimetallic NPs, Pt–Pd NPs are fascinating materials, and can combine with CNTs to fabricate electrochemical sensing interfaces. On the one hand, the synergistic effect of Pt and Pd has great catalytic activities to enhance the performance of modified electrodes for H2O2 detection (Janyasupab et al., 2013; Niu et al., 2012); on the other hand, both Pt–Pd nanoparticles and CNTs have the properties of good biocompatibility, excellent stability and large surface area, providing an ideal interface for the immobilization of enzymes (Kim et al. 2006; Siriviriyanun et al., 2013). Driven by the increasing demands of monitoring O2∙ in biological systems with ultra-sensitive, well selective and rapid response, we here recommend a captivating electrochemical sensing interface composed of Pt–Pd NPs and CNTs for the immobilization of SOD, and use the assembled sensor to qualify and quantify O2∙ . The fabricated interface shows three advantages: (1) the Pt–Pd/CNTs hybrid is synthesized using a facile onepot method; (2) the interface can facilitate the electron transfer between SOD and electrode due to the favorable electro-conductivity of Pt–Pd NPs and CNTs; and (3) the good biocompatibility of the prepared hybrid increases the stability of SOD, and thus prolongs the usage time of the sensor. It is found that the Pt–Pd/ CNTs modified enzyme electrode offers attractive sensitivity and excellent selectivity for O2∙ detection.
Industrial Inc. Ethylene glycol and trisodium citrate were provided by Shanghai Lingfeng Chemical Reagent Co. D-fructose (Fru), ascorbic acid (AA), dopamine (DA), glucose (Glu), uric acid (UA), 4-acetamidophenol (AP) and superoxide dismutase from bovine erythrocytes were commercially provided by Sigma-Aldrich. Ultrapure water (18.2 MΩ cm, Laboratory Water Purification Systems) was used in all experiments. All other chemicals were of analytical grade and utilized as received. A field emission scanning electron microscope (FESEM, S-4800, Hitachi) was utilized to observe the surface morphology of Pt–Pd/ MWCNTs with/without the immobilization of SOD. A transmission electron microscope (TEM, JEM-1400, JEOL) was also used to capture the morphology of Pt–Pd/MWCNTs. An X-ray photoelectron spectrometer (XPS, ESCALAB 250Xi, Thermo Fisher) was employed to detect the surface composition of the as-prepared Pt–Pd/MWCNTs hybrid. Powder X-ray diffraction (XRD) measurements were carried out in a D/MAX2550 diffractometer (Rigaku International Co.) using Cu Kα (λ ¼0.15408 nm) radiation. Ultraviolet–visible spectroscope (UV–Vis, EVOLUTION 220, Thermo Scientific) was used to detect the concentration of O2∙ obtained from the KO2 stock solution. All electrochemical experiments were performed at room temperature on a CHI660D workstation equipped with a conventional three-electrode system consisting of a screen-printed working electrode, a platinum wire counter electrode, and an Ag/AgCl reference electrode. 2.2. Synthesis of the Pt–Pd/MWCNTs hybrid Before use, the purchased MWCNTs were acid-treated by refluxing in a mixture of concentrated HNO3 and H2SO4 (v/v, 1:3) for 8 h at 80 °C. The Pt–Pd/MWCNTs hybrid was prepared by a facile one-step alcohol-reduction process. In brief, 10 mg MWCNTs were dispersed into the solution of 20 mL ethylene glycol and 20 mL deionized water. Then, 300 mg trisodium citrate, 50 μmol H2PtCl6 and 50 μmol Na2PdCl4 were added to the mixture above. After sonication for 30 min, the pH was adjusted to 10 with NaOH. The mixture solution was deoxygenated by nitrogen for 30 min and reacted for 8 h. Finally, the hybrid was washed with water and dried for characterization and measurement. 2.3. Fabrication of the modified electrode Screen-printed gold film electrodes as the substrate were home-prepared according to our previous work (Niu et al., 2011), and the diameter of the working electrode is 3 mm. As for the Pt– Pd/MWCNTs modified SOD-based sensor (SOD/Pt–Pd/MWCNTs/ SPGE), 2 mg Pt–Pd/MWCNTs were added into 1 mL ultrapure water and sonicated for 2 h to form a well-dispersed mixture. Afterwards, 5 μL of the mixture, different volumes of SOD (3000 U/mL) and 2 μL of 0.05 wt% Nafion were drop-casted onto the electrode surface, and dried at room temperature before use. Besides, the Pt–Pd/MWCNTs sensor was prepared without immobilizing SOD. 2.4. Generation of superoxide anion
2. Experimental 2.1. Chemicals and apparatus Multi-walled carbon nanotubes (MWCNTs) were obtained from Shenzhen Nanotech Port Co., with a mean diameter of 15 nm and a tube length of 1–2 μm. H2PtCl6 6H2O and Na2PdCl4 were purchased from Sinopharm Chemical Reagent Co. and used directly without further purification. Anhydrous dimethyl sulfoxide, 18crown-6 and potassium dioxide were obtained from Aladdin
A stock solution of KO2 was prepared by adding KO2 to anhydrous dimethyl sulfoxide (containing 18-crown-6). After sonicating the solution for 2 min, KO2 dissociated and produced O2∙ . The solubility of KO2 in DMSO can be increased by addition of 18-crown-6, which complexes K þ (Hyland and Auclair, 1981). According to the molar absorptivity of O2∙ in DMSO (2006 M 1 cm 1 at 271 nm), the concentration of O2∙ could be estimated by a UV–Vis spectroscope (Hyland and Auclair, 1981; Thandavan et al., 2013).
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2.5. Electrochemical measurements The behaviors of the fabricated electrodes were studied using the cyclic voltammetric, chronoamperometric and electrochemical impedance techniques. All chronoamperometric measurements were implemented by successively injecting the analyte into 5 mL 0.05 M PBS (pH 7.4, containing 0.1 M KCl) under a constant stirring.
3. Results and discussion 3.1. Synthesis and characterization of the Pt–Pd/MWCNTs hybrid The Pt–Pd/MWCNTs hybrid was synthesized by a facile onestep alcohol-reduction process. Ethylene glycol was used as the solvent and the reducing agent; trisodium citrate was utilized as a complexing agent and stabilizer that prevented alloys from aggregation; and MWCNTs were employed as the support for Pt–Pd alloys reduction in situ (Wang et al., 2008). In this work, the TEM technique was used to confirm the formation of the Pt–Pd/MWCNTs hybrid firstly. Fig. 1(A) shows the morphology of the as-prepared hybrid. It is found that Pt–Pd nanoparticles are uniformly deposited on the surface of MWCNTs without obvious aggregation. The size distribution is evaluated statistically by measuring the diameter of Pt–Pd nanoparticles in
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the selected area. As shown in Fig. 1(B), the Pt–Pd nanoparticles have a mean size of 5.2 nm, with a narrow size distribution. The well-defined size distribution is mainly due to the use of trisodium citrate as the complexing agent and the stabilizer and MWCNTs as an idea carrier for alloys. The XPS technique was used to further confirm the surface composition of the Pt–Pd/MWCNTs hybrid. As depicted in Fig. 1(C), the peaks located at 69.5, 72.8, 183.2 and 291.2 eV are assigned to the binding energy of Pt4f7/2, Pt4f5/2, Pd3d5/2 and Pd3d3/2, respectively (Fig. S1), revealing the coexistance of Pt(0) and Pd(0) (Kadirgan et al., 2009; Li et al., 2014 ). The calculated atom ratio of Pt and Pd is approximately 1:1, which is in accordance with the XPS analysis from the table of elemental component. Meanwhile, the peaks at 283.1 eV and 531.1 eV are attributed to the binding energy of C1s and O1s, corresponding to the sp2 C–C peak from MWCNTs and the oxygen functionalities originating from the functional groups (–OH, –COOH) of acid treated-MWCNTs. Fig. 1(D) represents the typical XRD patterns of the as-prepared hybrid. MWCNTs show a broad diffraction peak observed at 26°. This peak corresponds to the (002) plane of the graphitic carbon, indicating the presence of MWCNTs in the as-prepared hybrid (Kannan et al., 2013). Due to that Pt and Pd have almost the same crystal structures, the difference of the XRD peak positions of Pt and Pd is not distinct. The representative diffraction peaks at 40°, 46°, 67° and 82° are attributed to the (111), (200), (220) and (311) planes of Pt–Pd alloys with the face-centered cubic (FCC) structure.
Fig. 1. TEM image (A), size distribution (B), XPS pattern (C) and XRD pattern (D) of Pt–Pd/MWCNTs hybrid.
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Fig. 2. FESEM images of (A) Pt–Pd/MWCNTs/SPGE and (B) SOD/Pt–Pd/MWCNTs/SPGE, (C) The EIS curves for the bare SPGE, Pt–Pd/MWCNTs/SPGE and SOD/Pt–Pd/MWCNTs/ SPGE in 100 mM KCl solution containing 5 mM Fe(CN)63 /4 with the frequency ranging from 100 kHz to 0.01 Hz.
To note that these diffraction peaks are slightly shifted to higher 2θ values in comparison with the commercial Pt black (JCPDS 040601) (Li et al., 2014), indicating the formation of Pt–Pd alloys, and this result agrees with the XPS result. According to the value of the full-width at half-maximum (FWHM) of the (111) diffraction peaks, the mean crystallite size of Pt–Pd particles is calculated to be 5.4 nm by the Scherrer equation (D ¼0.9λ/β cos θ, where D is the particle size, λ is the radiation wavelength, β is the FWHM, and θ is the Bragg diffraction angle). The calculated average size is closely matched with that obtained from the TEM image (Kannan et al., 2013). 3.2. Electrochemical properties of the SOD/Pt–Pd/MWCNTs/SPGE sensor FESEM was used to characterize the morphology of electrodes during the step-wise modification. As shown in Fig. 2(A), the synthesized hybrid is uniformly distributed on the surface SPGE. After SOD is immobilized, the hybrid looks rough and incompact with (Fig. 2(B)) some agglomerates, (Wang et al., 2013a) indicating the existence of SOD, and the successful fabrication of enzyme electrode.
Electrochemical impedance spectroscopy (EIS) is carried out to identify the change of the charge transfer resistance and/or capacitance on the electrode surface, which can further reflect the step-wise modification process. The EIS curves of the bare SPGE, Pt–Pd/MWCNTs/SPGE and SOD/Pt–Pd/MWCNTs/SPGE are shown in Fig. 2(C). The charge transfer resistance (Rct) of the bare SPGE is 17,526 Ω. After the modification of Pt–Pd/MWCNTs on the SPGE substrate, the Rct decreases dramatically to 286 Ω, which demonstrates that the presence of the Pt–Pd alloy and MWCNTs can greatly enhance the conductivity of the electrode and promotes the electron transfer between the electrolyte and the electrode interface. When SOD is immobilized on the electrode, the Rct shows a slight increase to 669 Ω, indicating that the immobilization of SOD will cause an insulating effect on the electrode and increases the charge transfer resistance (Emregul et al., 2013). These results are in complete accordance with the typical CVs of the bare SPGE, Pt–Pd/MWCNTs/SPGE and SOD/Pt– Pd/MWCNTs/SPGE in 0.05 M PBS (0.1 M KCl) containing 5 mM Fe(CN)6 3 − /4 − at a scan rate of 50 mV s 1 in the potential range of 0.2 V to 0.6 V (shown in Fig. S2). We further checked the electrochemistry behavior of SOD/Pt– Pd/MWCNTs/SPGE in 0.05 M PBS (0.1 M KCl) containing 5 mM Fe(CN)6 3 − /4 − at different scan rates of 10–200 mV s 1.
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Fig. 3. Cyclic voltammograms of the SOD/Pt–Pd/MWCNTs/SPGE sensor in 0.05 M PBS (pH 7.4, containing 0.1 M KCl) with the absence and presence of different amounts of KO2 (different concentrations of O2d ) at scan rate of 50 mV s 1.
As presented in Fig. S3(A), with increasing scan rate, both the peak-to-peak separation (ΔEp) and the ΔIp increase along with the increasing scan rate. Besides, the oxidation peak shifts towards more positive potential and the reduction peak shifts towards more negative potential. It is further estimated that the ΔIp is linearly proportional to the square root of scan rate (Fig. S3(B)), suggesting the diffusion confined process. 3.3. Amperometric detection of O2∙ The electrochemical properties of modified electrodes towards the detection of O2∙ was evaluated by the CV technique. As displayed in Fig. 3, when different volumes of KO2 are added, both the oxidation and reduction currents of the sensor gradually increases, indicating that the SOD/Pt–Pd/MWCNTs/SPGE sensor has excellent response towards O2∙ . The enhanced reduction and oxidation currents can be explained by the following reaction mechanism (Di et al., 2004):
SOD(Cu(II)) + e− → SOD(Cu(I)) H+
SOD(Cu(I)) + O2∙− → SOD(Cu(II)) + H2 O2 SOD(Cu(I)) − e− → SOD(Cu(II))
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7.4. The results indicate that SOD retains the highest enzymatic activity and natural structure on the fabricated interface when the pH value is around the physical condition. Therefore, pH 7.4 is determined to be the optimum pH for further experiments. The loading of SOD was further optimized by immobilization different amounts of SOD (1–5 μL) on the modified electrode. When the amount of enzyme is lower than 3 μL, the sensor exhibits a very low sensitivity due to the scarce loading of enzyme molecules. When the amount is higher than 3 μL, the current gradually decreases, due to that SOD can act as a resistance material. As a result, the optimum amount of enzyme used for immobilization onto the electrode surface is 3 μL (displayed in Fig. S5). Then, the proper potential applied at the working electrode is studied to achieve the lowest detection limit. Fig. S6 displays the typical chronoamperometric responses of the proposed sensor for O2∙ detection at different applied potentials. It is obvious that the proposed sensor provides the maximum response at 0.1 V, which fits well with the DPV curves (as shown in Fig S7 in Supplementary information). Therefore, an applied potential of 0.1 V vs. Ag/AgCl is selected as the optimized potential in the following measurements. Finally, some other factors of the amperometric response to O2∙ , such as oxygen deficiency, the presence of chlorides ions and 18-crown-6, are also studied. As depicted in Fig. S8(A and B), it is observed that the response of the electrode in air saturated solution is lower than that of the Ar saturated electrolyte. Oxygen is thus a potential interfering compound to detect the superoxide radicals for the present system and has to be taken into account. Prior to analysis, the solution should bubble Ar gas for 10 min to eliminate the dissolved oxygen. In addition, the electrode offers equal current response in the presence/absence of 0.1 M KCl in the analyte solution, indicating the excellent poison resistance/antiinterference behavior of the sensor due to the formation of Pt–Pd alloys reduction the possibility of catalyst poisoning against chlorides ions. 18-crown-6 is used as co-solvent for preparation of KO2 stock solution (Cao et al., 2013). We have also studied the effect of the using 18-crown-6 (as shown in Fig. S9). The results demonstrate this species produces negligible response and does not interfere with the detection of O2∙ . Under the above optimized conditions, typical chronoamperometric responses of the prepared sensor for successive additions of O2∙ into constantly stirred 0.05 M PBS (pH 7.4, containing 0.1 M KCl) at the applied potential of 0.1 V are recorded. As shown in Fig. 4, a gradual increase of current is observed when adding the KO2 stock solution, indicating that SOD can catalyze O2∙ to H2O2, and then the Pt–Pd/MWCNTs hybrid has the
SOD(Cu(II)) + O2∙− → SOD(Cu(I)) + O2 SOD can efficiently catalyze the dismutation of O2∙ to O2 and H2O2 via a redox cycle of the copper complex moiety (Cu(I/II)) couple in Cu–Zn SOD. Two O2∙ ions are stoichiometrically converted to one O2 molecule and one H2O2 molecule with consumption of two H þ ions during the dismutation (Di et al., 2004). Then, the oxidation and reduction of H2O2 is effectively electrocatalyzed by Pt–Pd/MWCNTs, leading to the increase of both oxidation (0.6 V) and reduction current (0 V), which is consistent with the literature (Bo et al., 2011; Chen et al., 2012; Kang et al., 2007). Prior to amperometric analysis of O2∙ , experimental parameters possibly affecting the analytical performance of the fabricated sensor are optimized. The effect of pH, ranging from 6.8 to 8.6, on the electrochemical behavior of the SOD/Pt–Pd/MWCNTs sensor was firstly measured by the CV technique in 0.05 M PBS containing 5 mM Fe(CN)63 − /4 − and 0.1 M KCl. As shown in Fig. S4, the reduction current gradually increases from pH 6.8 to 7.4, and the highest magnitude of the current response is obtained at pH
Fig. 4. Amperometric response of the SOD/Pt–Pd/MWCNTs/SPGE for the successive addition of different concentration of O2d into constantly stirred 0.05 M PBS, pH 7.4 (containing 0.1 M KCl). Applied potential: 0.1 V.
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Table 1 Comparison of the analytical performance of various electrodes for O2d sensing. Electrode
Applied potential (V)
Sensitivity (μA cm 2 mM 1)
LOD (μM)
Linear range (μM)
Ref.
SOD/SA SOD/nano-Fe3O4/Au electrode SOD-CNT-PPy-Pt electrode PMMA/PANI-Au-nano/SOD-ESCFM SOD-CS-gold nanoparticles-ITO SOD/GNP/Cys/SG/Au SOD/Pt–Pd/MWCNTs/SPEG
0 0.5 – 0.3 0 0.15 0.1
0.782 – – 42.5 – – 601
0.23 0.2 0.1 0.3 0.2 0.2 0.71
0.4–229.9 0.2–1.4 0.1–750 0.5–2.4 0.08–0.64 0.05–0.40 40–1550
Wang et al. (2012) Thandavan et al. (2013) Rajesh et al. (2010) Santhosh et al. (2011) Wang et al. (2009) Di et al. (2007) This work
SOD: superoxide dismutase; SA: sodium alginate; PPy: polypyrrole; PMMA: poly(methyl-methacrylate); PANI: polyaniline; ESCFM: electrospun nanofibers; CS, Cys: cysteine; and GNP: gold nanoparticle; SG: sol–gel.
effective electron-transfer ability towards the reduction of enzymatically produced H2O2. The incremental current reaches a steady state within 5 s after addition of O2∙ . The inset in Fig. 4 shows that the amperometric responses are linear with the O2∙ concentration in the range of 40–1550 μM (R2 ¼0.9941), with a sensitivity of 0.601 mA cm 2 mM 1. The limit of detection is further calculated to be as low as 0.71 μM (S/N ¼3). In comparison with other sensors based on SOD for O2∙ detection and the sensor without nanomaterials (as shown in Fig. S10), our proposed sensor provides higher sensitivity and wider linear range (as list in Table 1). app ) is an indication The apparent Michaelis–Menten constant (Km of the enzyme–substrate kinetics which is generally used to app can be evaluate the biological activity of enzyme. The Km calculated from the electrochemical version of the Linweaver– Burk equation (Kamin and Wilson, 1980):
K app 1 1 1 = + m × iss imax imax c where iss is the steady-state current, imax is the maximum current, Kmapp is the apparent Michaelis–Menten constant, C is the concenapp tration of O2∙ (Turkmen et al., 2014). The Km value for the Pt–Pd/ MWCNTs sensor is 3.44 μM, indicating that SOD immobilized on the Pt–Pd/MWCNTs has a high enzymatic activity and affinity to O2∙ . Several potential interfering substances were used to evaluate the sensitivity of the fabricated SOD/Pt–Pd/MWCNTs/SPGE. The typical chronoamperometric responses upon successive addition of 0.08 mM O2∙ , 0.5 mM Glu, Fru, AA, UA, AP and DA into 0.05 M PBS (pH 7.4, containing 0.1 M KCl) are recorded. As depicted in Fig. 5(A), it is found that these species produce negligible response for the amperometric detection of O2∙ , indicating that
the proposed electrode can be employed to selectively detect O2∙ . This attractive specificity is mainly ascribed to the immobilization of SOD, which has a high selectivity towards O2∙ ; relatively low potential being used ( 0.1 V), where these species cannot be electrochemically oxidized, and the outermost Nafion layer as an effectively pre-selective barrier against anionic interferences. The reproducibility of the SOD/Pt–Pd/MWCNTs/SPGE sensor was estimated according to the responses provided by eight newly-prepared electrodes for 0.08 mM O2∙ . The results reveal that the recommended sensor exhibits a satisfied reproducibility with a RSD of 2.8%. The long-term stability is also an important factor to evaluate the performance of sensors. The amperometric responses of 0.08 mM O2∙ were collected every three days to check the long-term stability, and the electrode was stored at 4 °C when not in use. As depicted in Fig. 5(B), the sensitivity remains 97% of its original value after a month, suggesting that the fabricated electrode has a considerable stability for O2∙ detection. Two main factors lead to the good reproducibility and stability: on the one hand, good biocompatibility and excellent stability of the hybrid provide the enzyme with a friendly interface for immobilization and thus prolong the usage time of the enzyme electrode; on the other hand, the Nafion layer grasps the Pt–Pd/MWCNTs hybrid modified on the electrode surface well against leakage when testing. 3.4. Real sample analysis The test for O2∙ detection in Dulbecco's modified Eagle's medium (DMEM) cell culture medium containing fetal bovine serum (FBS, 10%) and penicillin/streptomycin (1%). This solution was used to mimic the environment of cell culture and the test in this sophisticated system was meaningful to carry out real sample
Fig. 5. (A) Effect of the potential interferents in O2d biosensor. The arrows show each moment at successive addition of 0.08 mM O2d , 0.5 mM Glu, 0.5 mM Fru, 0.5 mM AA and 0.5 mM UA, 0.5 mM AP and 0.5 mM DA. (B) Stability in sensitivity of the fabricated sensor in a period of one month.
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analysis by the proposed sensor. The real sample analysis was conducted on DMEM cell culture medium containing 10% FBS, and penicillin/streptomycin (1%) using a calibration curve method to determine the recoveries of various concentration of O2∙ . The sample was prepared by spiking different concentration of O2∙ into the mixture of 4 mL PBS and 1 mL DMEM containing 10% FBS and 1% penicillin/streptomycin, and the final concentration of O2∙ in sample was 0 (as control group), 40, 60, and 80 μM, respectively. The recovery is 102.4%, 98.5% and 97.4%, and the relative standard deviation is 1.5%, 3.6% and 2.9%. The results (as shown in Table S1) indicated that the methodology employed in the present work might be applied for sensitive and selective detection of O2∙ in real samples.
4. Conclusions As a conclusion, in this work we immobilized SOD onto the Pt– Pd/MWCNTs-constructed electrochemical interface, and fabricated a new enzyme-based O2∙ electrochemical sensor. Owning to the good biocompatibility and excellent stability of the interface, which provides SOD with a friendly immobilization environment, the proposed sensor exhibited high selectivity and sensitivity for O2∙ detection.
Acknowledgments This research was financially supported by National Natural Science Foundation of China (NSFC, No. 21305044), the Fundamental Research Funds for the Central Universities (No. 222201314026) and Science and Technology Commission of Shanghai Municipality (STCSM, No. 13510710900).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at: http://dx.doi.org/10.1016/j.bios.2014.07.004.
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