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myoglobin biofilm for direct electrochemistry
Biosensors and Bioelectronics 25 (2010) 1447–1453
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Facile preparation of magnetic core–shell Fe3 O4 @Au nanoparticle/myoglobin biofilm for direct electrochemistry Jian-Ding Qiu a,∗ , Hua-Ping Peng a , Ru-Ping Liang a , Xing-Hua Xia b,∗ a b
Department of Chemistry, Nanchang University, Nanchang 330031, China Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China
a r t i c l e
i n f o
Article history: Received 27 July 2009 Received in revised form 8 October 2009 Accepted 28 October 2009 Available online 6 November 2009 Keywords: Fe3 O4 @Au nanoparticles Myoglobin Direct electrochemistry Magnetic glassy carbon electrode Biosensor
a b s t r a c t In this work, the magnetic core–shell Fe3 O4 @Au nanoparticles attached to the surface of a magnetic glassy carbon electrode (MGCE) were applied to the immobilization/adsorption of myoglobin (Mb) for fabricating Mb/Fe3 O4 @Au biofilm. The morphology, structure, and electrochemistry of the nanocomposite were characterized by transmission electron microscope, UV–vis spectroscopy, electrochemical impedance spectroscopy, and cyclic voltammetry, respectively. The resultant Fe3 O4 @Au NPs not only have the magnetism of Fe3 O4 NPs that make them easily manipulated by an external magnetic field, but also have the good conductivity and excellent biocompatibility of Au layer which can maintain the bioactivity and facilitate the direct electrochemistry of Mb in the biofilm. The modified electrode based on this Mb/Fe3 O4 @Au biofilm displayed good electrocatalytic activity to the reduction of H2 O2 with a linear range from 1.28 to 283 M. The proposed method simplified the immobilization methodology of proteins and showed potential application for fabricating novel biosensors and bioelectronic devices. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Since the end of 1970s, the direct electron transfer (DET) has attracted considerable attention in life science and analytical chemistry, because the DET between redox proteins and electrodes cannot only be used to study enzyme-catalyzed reactions in biological systems, but also to establish a foundation for fabricating mediator-free biosensors, biomedical devices, and enzymatic bioreactors (Zhang et al., 2002). Nevertheless, the establishment of DET between redox proteins and conventional electrodes is still challenging because of the inaccessibility of the redox centers which are usually buried in large three-dimensional protein shells and the occurrence of denaturation adsorption of proteins on and resulting passivation of the electrode surfaces (Shi et al., 2007). Many efforts have been made to facilitate the electron transfer of the heme proteins by modifying the electrodes with various nanostructured materials, including inorganic non-metallic materials (Cui et al., 2009; Liu et al., 2007), nano metal oxides (Xiang et al., 2009; Zhou et al., 2005; Milsom et al., 2008), and macroporous gold films (Araci et al., 2008; Song et al., 2007). Among these materials, due to their high electrical conductivity, strong adsorptive ability, good mechanical strength, relatively large surface and excellent biocompatibility, Au nanoparticles (NPs) have been extensively
∗ Corresponding authors. Tel.: +86 791 3969518. E-mail addresses: [email protected], [email protected] (J.-D. Qiu), [email protected] (X.-H. Xia). 0956-5663/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2009.10.043
used as building blocks for the immobilization of biomolecules (Zhang et al., 2009; Willner et al., 2007; Jia et al., 2002). For example, many methods including electrostatic absorption, solvent casting, polyelectrolyte deposition, self-assembly, covalent binding, sol–gel, and layer by layer have been developed to fabricate functional interfaces containing Au NPs and biomolecules (Pingarron et al., 2008). On the other hand, magnetic nanoparticles have exhibited their advantages for their novel properties. The magnetic NPs can be controllably separated from bulk systems by means of an external magnetic field. This property enables us to immobilize enzymes on substrate surfaces (Dyal et al., 2003) and thus construct magnetically controllable bio-electrochemical systems (Willner and Katz, 2003; Katz and Willner, 2005, 2002; Hirsch et al., 2000). Among the magnetic materials, iron oxide nanoparticles have been extensively studied. However, the reactivity of iron oxide nanoparticles increases with the decrease of particle size, i.e., ion oxide NPs particles with relatively small sizes may undergo rapid degradation upon directly exposing to certain environments (Salgueirino-Maceira et al., 2006). To avoid such problems, magnetic core–shell nanoparticles with Fe3 O4 as the core and metal or metal oxide such as gold (Xu et al., 2007; Cho et al., 2005), silica (Qiu et al., 2007; Deng et al., 2008), titania (Chen and Chen, 2005), and alumina (Chen et al., 2007) as the shell have been extensively proposed. Among them, Au as the shell has been considered as one of the best candidates for building novel magnetic core–Au shell NPs, which exhibits well intrinsic properties of the magnetic core and Au shell. Due to the good biocompatibility, the magnetic
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Scheme 1. Schematic illustration of the procedure for preparation of Mb/Fe3 O4 @Au film-modified MGCE.
core–Au shell NPs have been proven to be a simple, cheap and effective way to construct bio-electrochemical interfaces for biocatalysis and biosensors (Riskin et al., 2007; Qiu et al., 2009). In this paper, a simple approach to the immobilization of proteins using Fe3 O4 @Au NPs as the building block was developed, and the DET between the immobilized redox proteins and electrode was studied. The Fe3 O4 @Au NPs was initially deposited on the electrode surface by applying a constant magnetic field, and then horse heart myoglobin (Mb) was immobilized on the Fe3 O4 @Au NPs surfaces via the interaction between Au coatings and the cysteine or NH4 + lysine functional groups of the Mb (Scheme 1). The interaction between Mb and the Fe3 O4 @Au NPs was investigated using UV–vis spectroscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. This protein immobilization method was facile, neither the complex pretreated procedure nor the special agents for enzyme immobilizing were required. The immobilized Mb well retained its biological activity and displayed an excellent response to the reduction of hydrogen peroxide.
ing paste, and a nummular magnet (3 mm in diameter and 2 mm in thickness, 0.2 T at the surface) was embedded in the tube with 2 mm apart from the electrode surface. Then, a glassy carbon (3 mm in diameter and 2 mm in depth) was inserted in the tube to support the magnet and the edge of the glassy carbon was sealed by adhesives. The prepared MGCE was carefully polished with 1.0, 0.3 and 0.05 m alumina slurry successively and then was cleaned ultrasonically in water for a few minutes. After the MGCE was immersed in 0.2 mL as-prepared Fe3 O4 @Au NPs suspension (11.0 mg mL−1 ) at room temperature, Fe3 O4 @Au NPs were firmly attached to the surface due to the magnetic force. After washing with water, the obtained Fe3 O4 @Au/MGCE was dipped into Mb solution (3 mg mL−1 at pH 6.98) at 4 ◦ C for protein immobilization. The resulted Mb/Fe3 O4 @Au/MGCE was washed thoroughly with water and stored at 4 ◦ C when not in use. 2.3. Characterization
Horse heart myoglobin (Mb, MW 17,800) and (3-aminopropyl) triethoxysilane (APTES) were purchased from Sigma. All other chemicals, such as hydrogen peroxide (30%), potassium ferricyanide, and HAuCl4 ·4H2 O were of analytical grade and used as received. A permanent magnet was purchased from As One Ltd. (Osaka, Japan). Phosphate buffer solution (0.1 M) at pH 6.98 was used as the supporting electrolyte for electrochemical experiments. All solutions were prepared with doubly distilled water.
Electrochemical experiments were performed on an Autolab PGSTAT30 electrochemical workstation (Eco Chemie). A threeelectrode system consisting of an Ag/AgCl reference electrode, a platinum wire auxiliary electrode, and the modified MGCE as the working electrode was used. The electrolyte solution was purged with N2 for at least 20 min to remove dissolved O2 and kept under N2 atmosphere during measurements. Electrochemical impedance spectroscopy (EIS) measurements were recorded at an open potential of 210 mV within the frequency range of 10−2 to 105 Hz. The amplitude of the applied sine wave potential was 5 mV. UV absorption spectra were acquired with a UV-2450 spectrophotometer (Shimadzu). The size of Fe3 O4 NPs and Fe3 O4 @Au NPs were characterized by a transmission electron microscope (TEM, Hitach-600).
2.2. Preparation of Mb/Fe3 O4 @Au/MGCE
3. Results and discussion
Fe3 O4 NPs were prepared using the Massart’s method (Massart, 1981). Core–shell Fe3 O4 @Au NPs were prepared via sonolysis of a mixture of HAuCl4 and APTES-coated Fe3 O4 NPs with further drop-addition of sodium citrate (Wu et al., 2007). The magnetic glassy carbon electrode (MGCE) was prepared according to the Wang’s method with little modification (Wang and Kawde, 2002a). Briefly, paraffin oil (1.2 mL) was mixed with graphite powder (10 mg) thoroughly to get a homogeneous paste. A copper wire was inserted into a Teflon tube (3 mm in diameter and 50 mm in depth) which was initially filled with a portion of the result-
3.1. Assembly of Mb/Fe3 O4 @Au/MGCE
2. Materials and methods 2.1. Materials
Scheme 1 shows schematically the procedure for fabricating the Mb/Fe3 O4 @Au film-modified MGCE. Core–shell Fe3 O4 @Au NPs were prepared by growing Au layers onto the Fe3 O4 NPs Surfaces. TEM images showed that both the Fe3 O4 and Fe3 O4 @Au NPs were of well spherical structure and high monodispersity in size. The average diameter were about 8–10 nm (Fig. 1A) and 25–30 nm for the purified Fe3 O4 NPs and the formed core–shell Fe3 O4 @Au NPs (Fig. 1B), respectively. Upon deposition of gold
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Fig. 1. TEM images of pure Fe3 O4 NPs (A) and core–shell Fe3 O4 @Au NPs (B). Photographic images of the Fe3 O4 @Au NPs in absence (C) and presence (D) of external magnetic field.
shell to the Fe3 O4 NPs, the diameter of the particles increased by ∼18 nm, demonstrated that the Au shell was ∼9-nm thick. The core–shell Fe3 O4 @Au NPs were then attached to the MGCE surface with the help of magnetic force (Scheme 1c). Although it has been reported that the thickness of the Au shell could be adjusted by varying the experimental parameters (Wang et al., 2005a), the proper thickness should be controlled to keep the high enough magnetic-field intensity and stability of the magnetic NPs which ensures the good magnetic response of the resultant core–shell Fe3 O4 @Au NPs under the external magnetic field. In the present case, the core–shell Fe3 O4 @Au NPs could be easily captured onto the MGCE surface with the presence of an external magnet. As shown in Fig. 1C, the Fe3 O4 @Au NPs suspension was homogeneous and dark purple. Once an external magnetic field was applied, the Fe3 O4 @Au NPs were attracted quickly toward the magnet, leaving the bulk solution clear and transparent (Fig. 1D). These results demonstrated the excellent magnetic separation ability of the core–shell Fe3 O4 @Au magnetic NPs enables them to be easily deposited on the MGCE surface by the magnetic force. When the electrode was immersed in a solution containing Mb, the adsorption of Mb on the Fe3 O4 @Au film electrode surface occurred via the interactions between the cysteine or NH4 + -lysine functional groups of the Mb and the gold shell of the NPs (Scheme 1d). The interconnected three-dimensional Fe3 O4 @Au film structure provided a biocompatible microenvironment to preserve the biological and electrochemical activities of the immobilized Mb. The
charged amino acid groups around the heme cleft of Mb molecule make the cleft side of Mb face to the negatively charged Au surface. This favorable orientation of Mb would greatly shorten the distance between the electroactive heme group of Mb and Au NPs, and thus, facilitated electron communication can occur (Scheme 1f). 3.2. UV–vis spectroscopy The Soret absorption band of the heme proteins may provide information on conformational integrity of the proteins (George and Hanania, 1953). The UV–vis spectrum (Fig. 2a) showed that the absorbance intensity decreased monotonically with the increase of light wavelength in the range 300–800 nm, which is in agreement with the result in literature (Tang et al., 2006). When Au coating were deposited around the Fe3 O4 NPs, a new absorption band centered at 547 nm was remarkably observed (Fig. 2b), which indicated the formation of (Fe3 O4 )core –Aushell NPs. As is well known, the Soret band of Mb is sensitive to its microenvironment, substructure, and oxidation state (Rusling and Nassar, 1993). Therefore, upon immobilization of Mb on the Fe3 O4 @Au NPs, the characteristic Soret band of the immobilized Mb appeared at 408 nm (Fig. 2c), which is exactly the same as that of Mb in neutral pH solution (Fig. 2d) (Zhang et al., 2006). The result suggested that the interaction between Fe3 O4 @Au NPs and Mb molecule did not change the natural secondary structure of Mb.
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Fig. 2. UV–vis spectra of the Fe3 O4 NPs (a), Fe3 O4 @Au NPs (b), Mb/Fe3 O4 @Au (c), and native Mb (d).
3.3. Electrochemical impedance spectroscopy Electrochemical impedance spectroscopy (EIS) was employed to monitor the Mb/Fe3 O4 @Au film-modified electrode with different adsorption time of Mb using a 5 mM Fe(CN)6 3−/4− solution as the electrochemical probe (Fig. 3). For comparison, the EIS of a Fe3 O4 @Au/MGCE is also shown. The EIS (Fig. 3A, curve a) of a Fe3 O4 @Au/MGCE exhibited almost a straight line that is characteristics of a diffusional limiting step of the electrochemical process. When the Fe3 O4 @Au/MGCE was immersed in Mb solution for different adsorption time, the electron-transfer resistance (Ret ) increased dramatically as indicated by the enlarged semicircle domains in Fig. 3A (curves b–h). This change indicated that Mb molecules could be immobilized on the electrode surface, inhibiting the electron communication of the electrochemical probe. The diameter of the semicircle is equal to the electrontransfer resistance (Ret ) which shows the electron-transfer kinetics of the redox probe (Katz and Willner, 2003), could be estimated by using the Randles equivalent circuit as inset in Fig. 3A (Randles, 1947). Fig. 3B shows the plot of Ret against adsorption time. It is clear that the Ret increased quickly with the adsorption time (<90 min) and then reached a relatively stable value with longer adsorption times (>90 min). A saturated adsorption of Mb was reached after 90 min. At saturation adsorption of Mb, the Ret was estimated to be 2203 (Fig. 3A, curve g). These results demonstrated that Mb could be immobilized on the Fe3 O4 @Au/MGCE. 3.4. Direct electrochemistry of Mb/Fe3 O4 @Au/MGCE Fig. 4A shows the typical cyclic voltammograms (CVs) for Mb/MGCE, Fe3 O4 @Au/MGCE and Mb/Fe3 O4 @Au/MGCE in 0.1 M PBS (pH 6.98) in the potential range of −0.75 to 0.15 V at a scan rate of 100 mV s−1 . As shown in Fig. 4A, the Mb-modified MGCE did not show discernible redox peaks (curve a), indicating that electron transfer of Mb was usually limited due to the inaccessibility of the heme group, unfavorable orientation and/or the denaturization of Mb molecules at the bare electrode surface. For the Fe3 O4 @Au/MGCE, the CV showed only an increased background curve (curve b) as compared to the bare electrode due to the three-dimensional matrix of the Fe3 O4 @Au layer. When Mb molecules were immobilized on the Fe3 O4 @Au/MGCE surface, however, a couple of stable and well-defined redox peaks centered at −310 mV appeared clearly (curve c). Obviously, the presence of Fe3 O4 @Au NPs accelerated the electron transfer of Mb due to the shortened distance among Fe3 O4 @Au NPs and the favorable
Fig. 3. (A) Electrochemical impedance spectra of the Fe3 O4 @Au/MGCE (a) and Mb/Fe3 O4 @Au/MGCE with Mb adsorption time of 1 (b), 3 (c), 10 (d), 40 (e), 60 (f), 90 (g), 150 (h) min in 5.0 mM K3 Fe(CN)6 /K4 Fe(CN)6 (1:1) containing 0.1 M KCl. Inset: Equivalent circuit used to model impedance data in the presence of redox couples. (B) Plot of the electron-transfer resistance against Mb adsorption time. Rs , Zw , Ret and Cdl represent the solution resistance, the Warburg diffusion resistance, the electron-transfer resistance and the double layer capacitance, respectively.
orientation of Mb (Riskin et al., 2007). Compared with {Au/Mb}n film developed by layer-by-layer assembly method (Zhang et al., 2006), {TiO2 /protein}n films constructed by vapor-surface sol–gel technique (Lu et al., 2006), and Mb/DL-homocysteine/Au film prepared by self-assembled method (Zhang and Li, 2001), the present method not only greatly simplified the immobilizing process, but also retained the bioactivity of the immobilized Mb to the utmost extent due to the excellent biocompatibility of the Fe3 O4 @Au NPs and the independent of the special agents for protein immobilizing. The influence of scan rate on the response of the Mb/Fe3 O4 @Au/MGCE in PBS was also investigated. As shown in Fig. 4B, with increasing the scan rate from 0.05 to 1.0 V s−1 , both the cathodic and anodic peak currents increased and their potentials did not show considerable shift, indicating a diffusionless, surface-controlled electrode process of the DET (Yoon et al., 2000; Dai et al., 2004a). According to the Laviron equation (1) (Laviron, 1979), Ip =
n2 F 2 Av nFQ v = 4RT 4RT
(1)
where (mol cm−2 ) is the surface amount of the adsorbed Mb on electrode surface, A is the electrode area (cm2 ), v is the scan rate, Ip is the peak current, n is the number of electron transferred,
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the Fe3 O4 @Au/MGCE can be obtained by Eq. (2) (Laviron, 1979), log Ks = ˛ log(1 − ˛) + (1 − ˛) log ˛ − log
nFEp RT − ˛(1 − ˛) nF v 2.3RT (2)
where ˛ is the charge-transfer coefficient, Ep is the peak potential separation, v is the scan rate, n is the number of electron transferred, and F is the Faraday’s constant. Our experimental results showed that the scan rate in the range of 50–1000 mV s−1 did not affect the Ks value. Taking the charge-transfer coefficient ˛ of 0.5, at a scan rate of 300 mV s−1 , the Ks of the adsorbed Mb on the Fe3 O4 @Au/MGCE was estimated to be 3.2 s−1 . The Ks value was much larger than the one obtained for Mb immobilized on DL-homocysteine self-assembled gold electrode (0.93 s−1 , Zhang and Li, 2001), Mb immobilized in silk fibroin film (1.34 s−1 , Wu et al., 2006), and Mb entrapped in agarose hydrogel film in room-temperature ionic liquid (1.2 s−1 , Wang et al., 2005b). The accelerated electron communication of Mb in the present case could be due to the function of electronic wire of Fe3 O4 @Au NPs and the favorable orientation of Mb. It is well known that the direct electrochemistry of Mb is a one-electron coupled with a one-proton reaction. Therefore, the solution pH should influence the electrochemical behavior of the immobilized Mb on Fe3 O4 @Au/MGCE. Fig. 4C shows the CVs of the Mb/Fe3 O4 @Au/MGCE in the PBS solutions with different pH value. As shown in Fig. 4C, the CVs with stable and well-defined peaks were observed in the pH range of 4.49–9.18. Increase of the solution pH caused a negative shift of both cathodic and anodic peak potentials due to the proton transfer from MbFe(III) to the reduced MbFe(II) (MbFe(III) + H+ + e− MbFe(II)). In addition, it was found that the formal potential of the Mb/Fe3 O4 @Au/MGCE decreased linearly with the pH in the range of 4.49–9.18 with a slop of −51.8 mV/pH (r = 0.998) (inset in Fig. 4C). This value was close to the expected one of −57.8 mV/pH for a single proton-transfer coupled to a reversible single electron transfer at 291 K (Nassar et al., 1997; Chen et al., 1999; Shire et al., 1974). 3.5. Electrocatalytic activity of the Mb/Fe3 O4 @Au/MGCE
Fig. 4. (A) Cyclic voltammograms of the Mb/MGCE (a), Fe3 O4 @Au/MGCE (b), and Mb/Fe3 O4 @Au/MGCE (c) in a 0.1 M pH 6.98 PBS at a scan rate of 100 mV s−1 . (B) Cyclic voltammograms of the Mb/Fe3 O4 @Au/MGCE in PBS (pH 6.98) at scan rate of 50, 100, 200, 300, 400, 500, 600, 800, and 1000 mV s−1 (from internal to external). Inset: Plots of the peak currents vs. scan rate. (C) Cyclic voltammograms of the Mb/Fe3 O4 @Au/MGCE in PBS with pH values of 4.49 (a), 5.29 (b), 5.91 (c), 6.47 (d), 6.98 (e), 8.04 (f), and 9.18 (g). Inset: Plot of formal potential vs. pH.
Q is the charge involved in the reaction, and F is the Faraday’s constant. The average surface amount ( ) of the adsorbed Mb on the Fe3 O4 @Au/MGCE was estimated to be 9.18 × 10−10 mol cm−2 , which was much larger than that reported for the {Au/Mb}n films (Zhang et al., 2006) and {PAMAM-Au/Mb}n films (Zhang and Hu, 2007a). This result demonstrated that the biofilm could considerably increase the functional density of Mb. Since nEp < 200 mV (Ep : peak potential), the direct electron-transfer rate constant (Ks ) of the adsorbed Mb on
Immobilized Mb on an electrode surface with retained conformation normally exhibits electrocatalytic activity for oxygen, H2 O2 , trichloroacetic acid (TCA), and NO2 − (Dai et al., 2004b; He et al., 2002). By using H2 O2 as model, the electrocatalytic properties of the Mb/Fe3 O4 @Au/MGCE were studied. Inset A of Fig. 5 shows the bioelectrocatalytic activity of the Mb/Fe3 O4 @Au/MGCE toward the reduction of H2 O2 at −0.35 V. A pair of reversible CV peaks appeared in the absence of H2 O2 (curve a). Upon addition of H2 O2 into the 0.1 M pH 6.98 PBS, the reduction peak current of immobilized Mb increased dramatically and oxidation peak current decreased. The reduction peak current increased with H2 O2 concentration (curves b–d), displaying an obvious electrocatalytic behavior of Mb to the reduction of H2 O2 (Lu et al., 2007). These results indicated that the immobilized Mb on Fe3 O4 @Au/MGCE retains its electrocatalytic activity to the reduction of H2 O2 . The performance of the Mb/Fe3 O4 @Au/MGCE was also studied by the amperometric current–time method. Fig. 5 illustrates a typical current–time curve of Mb/Fe3 O4 @Au/MGCE at −0.35 V upon successive addition of H2 O2 . When H2 O2 was added to the stirring PBS, the reductive current increased steeply to a stable value. The time to reach 95% of the maximum current was ca. 5 s, which is much faster than that of 10 s reported in the Mb/ZrO2 /chitosan film electrode (Zhao et al., 2005), 9 s in the Mb/ZrO2 -grafted collagen/DMSO/GE (Zong et al., 2007), and 10 s in the Mb/HMS/GCE (Dai et al., 2004b). Such a short response time can be mainly attributed to the fast diffusion process of hydrogen peroxide in the
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3.7. Reproducibility and stability of the biosensor
Fig. 5. Amperometric responses of the Mb/Fe3 O4 @Au/MGCE at −0.35 V to successive addition of H2 O2 in a stirred 0.1 M PBS (pH 6.98). Inset: (A) Cyclic voltammograms of the Mb/Fe3 O4 @Au/MGCE in 0.1 M pH 6.98 PBS containing 0 (a), 15 (b), 50 (c), and 100 M (d) H2 O2 at 100 mV s−1 . (B) Plot of chronoamperometric current vs. H2 O2 concentration.
To prove the precision and practicability of the proposed method, the reproducibility and storage stability of the biosensor were also examined. The relative standard deviation of the modified electrode response to 0.1 mM H2 O2 was 3.0% for ten successive measurements. The relative standard deviation for detection of 0.1 mM H2 O2 with five sensors prepared under the same conditions was 4.3%. The modified electrode was stored in pH 6.98 PBS at 4 ◦ C and measured at intervals of 1 week. The biosensor retained 96% of its original sensitivity after 1 week, and remained about 89% of its original current after 4 weeks. The excellent reproducibility and stability of the biosensor are due to the good biocompatibility and stability of the Mb/Fe3 O4 @Au biofilm. On the one hand, the biocompatibility of Fe3 O4 @Au NPs provides an interconnected three-dimensional matrix to deposit Mb and maintain the native structure of the immobilized proteins. On the other hand, the good affinity of Fe3 O4 @Au NPs to Mb improves the electron transfer and prevents leakage of Mb molecules from the Mb/Fe3 O4 @Au biofilm structure efficiently. 4. Conclusions
three-dimensional matrix and high electronic conductivity of the Fe3 O4 @Au NPs film. The modified electrode displayed increasing amperometric responses to H2 O2 with a good linear ranging from 1.28 to 283 M with a detection limit of 0.4 M (S/N = 3) (inset B of Fig. 5). When the H2 O2 concentration was higher than 283 M, the electrochemical response deviated from linearity and reached a plateau at much higher concentration of hydrogen peroxide, showing the characteristics of the Michaelis–Menten kinetic mechanism. app The apparent Michaelis–Menten constant (Km ) can be obtained from the Lineweaver–Burk equation (Shu and Wilson, 1976; Kamin and Wilson, 1980): app
1 K 1 1 = m · + Iss Imax C Imax
(3)
Here, Iss is the steady state current after the addition of substrate, C is the bulk concentration of the substrate, and Imax is the maximum app current measured under saturated substrate conditions. The Km value in this work was found to be 0.145 mM, which is much lower than those ever reported values (Dai et al., 2004b; Zhang and Zhen, app 2008; Zhang et al., 2007b; Liu and Ju, 2003). The smaller Km value indicates that the Mb immobilized in the Mb/Fe3 O4 @Au biofilm possesses higher enzymatic activity due to the higher affinity of the Mb to H2 O2 (Shi et al., 2007). 3.6. Interference study In real samples, the coexisting electroactive species might affect the biosensors response. In order to assess the anti-interference capability of the present sensor, the electrochemical responses of some common interfering substances were studied. No significant interference was observed upon addition of CO3 2− , ClO3 − , SO4 2− , Cl− , Br− , I− , glycin, l-cystine, l-tyrosine, choline, acetylcholine, catechol, nitrite, uric acid and ascorbic acid at concentration 10 times higher than H2 O2 at 50.0 M. However, Fe3+ might be a main interference to Mb for the electrocatalytic reduction of H2 O2 . When the concentration of Fe3+ increased to ten times higher than H2 O2 , a minor interference was observed. This possibly resulted from the interaction between Fe3+ and H2 O2 similar to that reported previously (Wang et al., 2002b). This interference could be excluded by adding EDTA in the sample solutions.
In this study, we have successfully developed a novel and simple myoglobin immobilization method by using biocompatible Fe3 O4 @Au magnetic composite material as the building block. The interconnected three-dimensional Fe3 O4 @Au magnetic nanocomposites film possessed superior conductivity, high stability, large surface area, and a good microenvironment for the immobilization of proteins. The immobilized Mb with retained biological activity could directly shuttle electron with the electrode. Compared to other immobilization procedures, the present method shows advantageous such as much larger adsorption capacity, free orientation possibility of the proteins, short immobilization time, and no need for an electron-transfer mediator or specific reagent. The designed Mb/Fe3 O4 @Au/MGCE exhibited good performances for electrocatalytic reduction of H2 O2 , such as high sensitivity, low detection limit, short response time, long-time stability and reproducibility. Acknowledgment This work was supported by grants from the National Natural Science Foundation of China (20605010, 20865003, 20805023), the Jiangxi Province Natural Science Foundation (0620039, 2007JZH2644), and the Opening Foundation of State Key Laboratory of Chem/Biosensing and Chemometrics of Hunan University (2006022, 2007012). References Araci, Z.O., Runge, A.F., Doherty, W.J., Saavedra, S.S., 2008. J. Am. Chem. Soc. 130, 1572–1573. Chen, C.T., Chen, W.Y., Tsai, P.J., Chien, K.Y., Yu, J.S., Chen, Y.C., 2007. J. Proteome Res. 6, 316–325. Chen, C.T., Chen, Y.C., 2005. Anal. Chem. 77, 5912–5919. Chen, X., Hu, N., Zeng, Y., Rusling, J.F., Yang, J., 1999. Langmuir 15, 7022–7030. Cho, S.J., Idrobo, J.C., Olamit, J., Liu, K., Browning, N.D., Kauzlarich, S.M., 2005. Chem. Mater. 17, 3181–3186. Cui, H.F., Ye, J.S., Zhang, W.D., Sheu, F.S., 2009. Biosens. Bioelectron. 24, 1723–1729. Dai, Z.H., Liu, S.Q., Ju, H.X., Chen, H.Y., 2004a. Biosens. Bioelectron. 19, 861–867. Dai, Z.H., Xu, X.X., Ju, H.X., 2004b. Anal. Biochem. 332, 23–31. Deng, Y., Qi, D., Deng, C., Zhang, X., Zhao, D., 2008. J. Am. Chem. Soc. 130, 28–29. Dyal, A., Loos, K., Noto, M., Chang, S.W., Spagnoli, C., Shafi, K.V.P.M., Ulman, A., Cowman, M., Gross, R.A., 2003. J. Am. Chem. Soc. 125, 1684–1685. George, P., Hanania, G.I.H., 1953. Biochem. J. 55, 236–243. He, P.L., Hu, N.F., Zhou, G., 2002. Biomacromolecules 3, 139–146. Hirsch, R., Katz, E., Willner, I., 2000. J. Am. Chem. Soc. 122, 12053–12054. Jia, J.B., Wang, B.Q., Wu, A.G., Cheng, G.J., Li, Z., Dong, S.J., 2002. Anal. Chem. 74, 2217–2223.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Facile preparation of magnetic core–shell Fe3 O4 @Au nanoparticle/myoglobin biofilm for direct electrochemistry Jian-Ding Qiu a,∗ , Hua-Ping Peng a , Ru-Ping Liang a , Xing-Hua Xia b,∗ a b
Department of Chemistry, Nanchang University, Nanchang 330031, China Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China
a r t i c l e
i n f o
Article history: Received 27 July 2009 Received in revised form 8 October 2009 Accepted 28 October 2009 Available online 6 November 2009 Keywords: Fe3 O4 @Au nanoparticles Myoglobin Direct electrochemistry Magnetic glassy carbon electrode Biosensor
a b s t r a c t In this work, the magnetic core–shell Fe3 O4 @Au nanoparticles attached to the surface of a magnetic glassy carbon electrode (MGCE) were applied to the immobilization/adsorption of myoglobin (Mb) for fabricating Mb/Fe3 O4 @Au biofilm. The morphology, structure, and electrochemistry of the nanocomposite were characterized by transmission electron microscope, UV–vis spectroscopy, electrochemical impedance spectroscopy, and cyclic voltammetry, respectively. The resultant Fe3 O4 @Au NPs not only have the magnetism of Fe3 O4 NPs that make them easily manipulated by an external magnetic field, but also have the good conductivity and excellent biocompatibility of Au layer which can maintain the bioactivity and facilitate the direct electrochemistry of Mb in the biofilm. The modified electrode based on this Mb/Fe3 O4 @Au biofilm displayed good electrocatalytic activity to the reduction of H2 O2 with a linear range from 1.28 to 283 M. The proposed method simplified the immobilization methodology of proteins and showed potential application for fabricating novel biosensors and bioelectronic devices. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Since the end of 1970s, the direct electron transfer (DET) has attracted considerable attention in life science and analytical chemistry, because the DET between redox proteins and electrodes cannot only be used to study enzyme-catalyzed reactions in biological systems, but also to establish a foundation for fabricating mediator-free biosensors, biomedical devices, and enzymatic bioreactors (Zhang et al., 2002). Nevertheless, the establishment of DET between redox proteins and conventional electrodes is still challenging because of the inaccessibility of the redox centers which are usually buried in large three-dimensional protein shells and the occurrence of denaturation adsorption of proteins on and resulting passivation of the electrode surfaces (Shi et al., 2007). Many efforts have been made to facilitate the electron transfer of the heme proteins by modifying the electrodes with various nanostructured materials, including inorganic non-metallic materials (Cui et al., 2009; Liu et al., 2007), nano metal oxides (Xiang et al., 2009; Zhou et al., 2005; Milsom et al., 2008), and macroporous gold films (Araci et al., 2008; Song et al., 2007). Among these materials, due to their high electrical conductivity, strong adsorptive ability, good mechanical strength, relatively large surface and excellent biocompatibility, Au nanoparticles (NPs) have been extensively
∗ Corresponding authors. Tel.: +86 791 3969518. E-mail addresses: [email protected], [email protected] (J.-D. Qiu), [email protected] (X.-H. Xia). 0956-5663/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2009.10.043
used as building blocks for the immobilization of biomolecules (Zhang et al., 2009; Willner et al., 2007; Jia et al., 2002). For example, many methods including electrostatic absorption, solvent casting, polyelectrolyte deposition, self-assembly, covalent binding, sol–gel, and layer by layer have been developed to fabricate functional interfaces containing Au NPs and biomolecules (Pingarron et al., 2008). On the other hand, magnetic nanoparticles have exhibited their advantages for their novel properties. The magnetic NPs can be controllably separated from bulk systems by means of an external magnetic field. This property enables us to immobilize enzymes on substrate surfaces (Dyal et al., 2003) and thus construct magnetically controllable bio-electrochemical systems (Willner and Katz, 2003; Katz and Willner, 2005, 2002; Hirsch et al., 2000). Among the magnetic materials, iron oxide nanoparticles have been extensively studied. However, the reactivity of iron oxide nanoparticles increases with the decrease of particle size, i.e., ion oxide NPs particles with relatively small sizes may undergo rapid degradation upon directly exposing to certain environments (Salgueirino-Maceira et al., 2006). To avoid such problems, magnetic core–shell nanoparticles with Fe3 O4 as the core and metal or metal oxide such as gold (Xu et al., 2007; Cho et al., 2005), silica (Qiu et al., 2007; Deng et al., 2008), titania (Chen and Chen, 2005), and alumina (Chen et al., 2007) as the shell have been extensively proposed. Among them, Au as the shell has been considered as one of the best candidates for building novel magnetic core–Au shell NPs, which exhibits well intrinsic properties of the magnetic core and Au shell. Due to the good biocompatibility, the magnetic
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Scheme 1. Schematic illustration of the procedure for preparation of Mb/Fe3 O4 @Au film-modified MGCE.
core–Au shell NPs have been proven to be a simple, cheap and effective way to construct bio-electrochemical interfaces for biocatalysis and biosensors (Riskin et al., 2007; Qiu et al., 2009). In this paper, a simple approach to the immobilization of proteins using Fe3 O4 @Au NPs as the building block was developed, and the DET between the immobilized redox proteins and electrode was studied. The Fe3 O4 @Au NPs was initially deposited on the electrode surface by applying a constant magnetic field, and then horse heart myoglobin (Mb) was immobilized on the Fe3 O4 @Au NPs surfaces via the interaction between Au coatings and the cysteine or NH4 + lysine functional groups of the Mb (Scheme 1). The interaction between Mb and the Fe3 O4 @Au NPs was investigated using UV–vis spectroscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. This protein immobilization method was facile, neither the complex pretreated procedure nor the special agents for enzyme immobilizing were required. The immobilized Mb well retained its biological activity and displayed an excellent response to the reduction of hydrogen peroxide.
ing paste, and a nummular magnet (3 mm in diameter and 2 mm in thickness, 0.2 T at the surface) was embedded in the tube with 2 mm apart from the electrode surface. Then, a glassy carbon (3 mm in diameter and 2 mm in depth) was inserted in the tube to support the magnet and the edge of the glassy carbon was sealed by adhesives. The prepared MGCE was carefully polished with 1.0, 0.3 and 0.05 m alumina slurry successively and then was cleaned ultrasonically in water for a few minutes. After the MGCE was immersed in 0.2 mL as-prepared Fe3 O4 @Au NPs suspension (11.0 mg mL−1 ) at room temperature, Fe3 O4 @Au NPs were firmly attached to the surface due to the magnetic force. After washing with water, the obtained Fe3 O4 @Au/MGCE was dipped into Mb solution (3 mg mL−1 at pH 6.98) at 4 ◦ C for protein immobilization. The resulted Mb/Fe3 O4 @Au/MGCE was washed thoroughly with water and stored at 4 ◦ C when not in use. 2.3. Characterization
Horse heart myoglobin (Mb, MW 17,800) and (3-aminopropyl) triethoxysilane (APTES) were purchased from Sigma. All other chemicals, such as hydrogen peroxide (30%), potassium ferricyanide, and HAuCl4 ·4H2 O were of analytical grade and used as received. A permanent magnet was purchased from As One Ltd. (Osaka, Japan). Phosphate buffer solution (0.1 M) at pH 6.98 was used as the supporting electrolyte for electrochemical experiments. All solutions were prepared with doubly distilled water.
Electrochemical experiments were performed on an Autolab PGSTAT30 electrochemical workstation (Eco Chemie). A threeelectrode system consisting of an Ag/AgCl reference electrode, a platinum wire auxiliary electrode, and the modified MGCE as the working electrode was used. The electrolyte solution was purged with N2 for at least 20 min to remove dissolved O2 and kept under N2 atmosphere during measurements. Electrochemical impedance spectroscopy (EIS) measurements were recorded at an open potential of 210 mV within the frequency range of 10−2 to 105 Hz. The amplitude of the applied sine wave potential was 5 mV. UV absorption spectra were acquired with a UV-2450 spectrophotometer (Shimadzu). The size of Fe3 O4 NPs and Fe3 O4 @Au NPs were characterized by a transmission electron microscope (TEM, Hitach-600).
2.2. Preparation of Mb/Fe3 O4 @Au/MGCE
3. Results and discussion
Fe3 O4 NPs were prepared using the Massart’s method (Massart, 1981). Core–shell Fe3 O4 @Au NPs were prepared via sonolysis of a mixture of HAuCl4 and APTES-coated Fe3 O4 NPs with further drop-addition of sodium citrate (Wu et al., 2007). The magnetic glassy carbon electrode (MGCE) was prepared according to the Wang’s method with little modification (Wang and Kawde, 2002a). Briefly, paraffin oil (1.2 mL) was mixed with graphite powder (10 mg) thoroughly to get a homogeneous paste. A copper wire was inserted into a Teflon tube (3 mm in diameter and 50 mm in depth) which was initially filled with a portion of the result-
3.1. Assembly of Mb/Fe3 O4 @Au/MGCE
2. Materials and methods 2.1. Materials
Scheme 1 shows schematically the procedure for fabricating the Mb/Fe3 O4 @Au film-modified MGCE. Core–shell Fe3 O4 @Au NPs were prepared by growing Au layers onto the Fe3 O4 NPs Surfaces. TEM images showed that both the Fe3 O4 and Fe3 O4 @Au NPs were of well spherical structure and high monodispersity in size. The average diameter were about 8–10 nm (Fig. 1A) and 25–30 nm for the purified Fe3 O4 NPs and the formed core–shell Fe3 O4 @Au NPs (Fig. 1B), respectively. Upon deposition of gold
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Fig. 1. TEM images of pure Fe3 O4 NPs (A) and core–shell Fe3 O4 @Au NPs (B). Photographic images of the Fe3 O4 @Au NPs in absence (C) and presence (D) of external magnetic field.
shell to the Fe3 O4 NPs, the diameter of the particles increased by ∼18 nm, demonstrated that the Au shell was ∼9-nm thick. The core–shell Fe3 O4 @Au NPs were then attached to the MGCE surface with the help of magnetic force (Scheme 1c). Although it has been reported that the thickness of the Au shell could be adjusted by varying the experimental parameters (Wang et al., 2005a), the proper thickness should be controlled to keep the high enough magnetic-field intensity and stability of the magnetic NPs which ensures the good magnetic response of the resultant core–shell Fe3 O4 @Au NPs under the external magnetic field. In the present case, the core–shell Fe3 O4 @Au NPs could be easily captured onto the MGCE surface with the presence of an external magnet. As shown in Fig. 1C, the Fe3 O4 @Au NPs suspension was homogeneous and dark purple. Once an external magnetic field was applied, the Fe3 O4 @Au NPs were attracted quickly toward the magnet, leaving the bulk solution clear and transparent (Fig. 1D). These results demonstrated the excellent magnetic separation ability of the core–shell Fe3 O4 @Au magnetic NPs enables them to be easily deposited on the MGCE surface by the magnetic force. When the electrode was immersed in a solution containing Mb, the adsorption of Mb on the Fe3 O4 @Au film electrode surface occurred via the interactions between the cysteine or NH4 + -lysine functional groups of the Mb and the gold shell of the NPs (Scheme 1d). The interconnected three-dimensional Fe3 O4 @Au film structure provided a biocompatible microenvironment to preserve the biological and electrochemical activities of the immobilized Mb. The
charged amino acid groups around the heme cleft of Mb molecule make the cleft side of Mb face to the negatively charged Au surface. This favorable orientation of Mb would greatly shorten the distance between the electroactive heme group of Mb and Au NPs, and thus, facilitated electron communication can occur (Scheme 1f). 3.2. UV–vis spectroscopy The Soret absorption band of the heme proteins may provide information on conformational integrity of the proteins (George and Hanania, 1953). The UV–vis spectrum (Fig. 2a) showed that the absorbance intensity decreased monotonically with the increase of light wavelength in the range 300–800 nm, which is in agreement with the result in literature (Tang et al., 2006). When Au coating were deposited around the Fe3 O4 NPs, a new absorption band centered at 547 nm was remarkably observed (Fig. 2b), which indicated the formation of (Fe3 O4 )core –Aushell NPs. As is well known, the Soret band of Mb is sensitive to its microenvironment, substructure, and oxidation state (Rusling and Nassar, 1993). Therefore, upon immobilization of Mb on the Fe3 O4 @Au NPs, the characteristic Soret band of the immobilized Mb appeared at 408 nm (Fig. 2c), which is exactly the same as that of Mb in neutral pH solution (Fig. 2d) (Zhang et al., 2006). The result suggested that the interaction between Fe3 O4 @Au NPs and Mb molecule did not change the natural secondary structure of Mb.
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Fig. 2. UV–vis spectra of the Fe3 O4 NPs (a), Fe3 O4 @Au NPs (b), Mb/Fe3 O4 @Au (c), and native Mb (d).
3.3. Electrochemical impedance spectroscopy Electrochemical impedance spectroscopy (EIS) was employed to monitor the Mb/Fe3 O4 @Au film-modified electrode with different adsorption time of Mb using a 5 mM Fe(CN)6 3−/4− solution as the electrochemical probe (Fig. 3). For comparison, the EIS of a Fe3 O4 @Au/MGCE is also shown. The EIS (Fig. 3A, curve a) of a Fe3 O4 @Au/MGCE exhibited almost a straight line that is characteristics of a diffusional limiting step of the electrochemical process. When the Fe3 O4 @Au/MGCE was immersed in Mb solution for different adsorption time, the electron-transfer resistance (Ret ) increased dramatically as indicated by the enlarged semicircle domains in Fig. 3A (curves b–h). This change indicated that Mb molecules could be immobilized on the electrode surface, inhibiting the electron communication of the electrochemical probe. The diameter of the semicircle is equal to the electrontransfer resistance (Ret ) which shows the electron-transfer kinetics of the redox probe (Katz and Willner, 2003), could be estimated by using the Randles equivalent circuit as inset in Fig. 3A (Randles, 1947). Fig. 3B shows the plot of Ret against adsorption time. It is clear that the Ret increased quickly with the adsorption time (<90 min) and then reached a relatively stable value with longer adsorption times (>90 min). A saturated adsorption of Mb was reached after 90 min. At saturation adsorption of Mb, the Ret was estimated to be 2203 (Fig. 3A, curve g). These results demonstrated that Mb could be immobilized on the Fe3 O4 @Au/MGCE. 3.4. Direct electrochemistry of Mb/Fe3 O4 @Au/MGCE Fig. 4A shows the typical cyclic voltammograms (CVs) for Mb/MGCE, Fe3 O4 @Au/MGCE and Mb/Fe3 O4 @Au/MGCE in 0.1 M PBS (pH 6.98) in the potential range of −0.75 to 0.15 V at a scan rate of 100 mV s−1 . As shown in Fig. 4A, the Mb-modified MGCE did not show discernible redox peaks (curve a), indicating that electron transfer of Mb was usually limited due to the inaccessibility of the heme group, unfavorable orientation and/or the denaturization of Mb molecules at the bare electrode surface. For the Fe3 O4 @Au/MGCE, the CV showed only an increased background curve (curve b) as compared to the bare electrode due to the three-dimensional matrix of the Fe3 O4 @Au layer. When Mb molecules were immobilized on the Fe3 O4 @Au/MGCE surface, however, a couple of stable and well-defined redox peaks centered at −310 mV appeared clearly (curve c). Obviously, the presence of Fe3 O4 @Au NPs accelerated the electron transfer of Mb due to the shortened distance among Fe3 O4 @Au NPs and the favorable
Fig. 3. (A) Electrochemical impedance spectra of the Fe3 O4 @Au/MGCE (a) and Mb/Fe3 O4 @Au/MGCE with Mb adsorption time of 1 (b), 3 (c), 10 (d), 40 (e), 60 (f), 90 (g), 150 (h) min in 5.0 mM K3 Fe(CN)6 /K4 Fe(CN)6 (1:1) containing 0.1 M KCl. Inset: Equivalent circuit used to model impedance data in the presence of redox couples. (B) Plot of the electron-transfer resistance against Mb adsorption time. Rs , Zw , Ret and Cdl represent the solution resistance, the Warburg diffusion resistance, the electron-transfer resistance and the double layer capacitance, respectively.
orientation of Mb (Riskin et al., 2007). Compared with {Au/Mb}n film developed by layer-by-layer assembly method (Zhang et al., 2006), {TiO2 /protein}n films constructed by vapor-surface sol–gel technique (Lu et al., 2006), and Mb/DL-homocysteine/Au film prepared by self-assembled method (Zhang and Li, 2001), the present method not only greatly simplified the immobilizing process, but also retained the bioactivity of the immobilized Mb to the utmost extent due to the excellent biocompatibility of the Fe3 O4 @Au NPs and the independent of the special agents for protein immobilizing. The influence of scan rate on the response of the Mb/Fe3 O4 @Au/MGCE in PBS was also investigated. As shown in Fig. 4B, with increasing the scan rate from 0.05 to 1.0 V s−1 , both the cathodic and anodic peak currents increased and their potentials did not show considerable shift, indicating a diffusionless, surface-controlled electrode process of the DET (Yoon et al., 2000; Dai et al., 2004a). According to the Laviron equation (1) (Laviron, 1979), Ip =
n2 F 2 Av nFQ v = 4RT 4RT
(1)
where (mol cm−2 ) is the surface amount of the adsorbed Mb on electrode surface, A is the electrode area (cm2 ), v is the scan rate, Ip is the peak current, n is the number of electron transferred,
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the Fe3 O4 @Au/MGCE can be obtained by Eq. (2) (Laviron, 1979), log Ks = ˛ log(1 − ˛) + (1 − ˛) log ˛ − log
nFEp RT − ˛(1 − ˛) nF v 2.3RT (2)
where ˛ is the charge-transfer coefficient, Ep is the peak potential separation, v is the scan rate, n is the number of electron transferred, and F is the Faraday’s constant. Our experimental results showed that the scan rate in the range of 50–1000 mV s−1 did not affect the Ks value. Taking the charge-transfer coefficient ˛ of 0.5, at a scan rate of 300 mV s−1 , the Ks of the adsorbed Mb on the Fe3 O4 @Au/MGCE was estimated to be 3.2 s−1 . The Ks value was much larger than the one obtained for Mb immobilized on DL-homocysteine self-assembled gold electrode (0.93 s−1 , Zhang and Li, 2001), Mb immobilized in silk fibroin film (1.34 s−1 , Wu et al., 2006), and Mb entrapped in agarose hydrogel film in room-temperature ionic liquid (1.2 s−1 , Wang et al., 2005b). The accelerated electron communication of Mb in the present case could be due to the function of electronic wire of Fe3 O4 @Au NPs and the favorable orientation of Mb. It is well known that the direct electrochemistry of Mb is a one-electron coupled with a one-proton reaction. Therefore, the solution pH should influence the electrochemical behavior of the immobilized Mb on Fe3 O4 @Au/MGCE. Fig. 4C shows the CVs of the Mb/Fe3 O4 @Au/MGCE in the PBS solutions with different pH value. As shown in Fig. 4C, the CVs with stable and well-defined peaks were observed in the pH range of 4.49–9.18. Increase of the solution pH caused a negative shift of both cathodic and anodic peak potentials due to the proton transfer from MbFe(III) to the reduced MbFe(II) (MbFe(III) + H+ + e− MbFe(II)). In addition, it was found that the formal potential of the Mb/Fe3 O4 @Au/MGCE decreased linearly with the pH in the range of 4.49–9.18 with a slop of −51.8 mV/pH (r = 0.998) (inset in Fig. 4C). This value was close to the expected one of −57.8 mV/pH for a single proton-transfer coupled to a reversible single electron transfer at 291 K (Nassar et al., 1997; Chen et al., 1999; Shire et al., 1974). 3.5. Electrocatalytic activity of the Mb/Fe3 O4 @Au/MGCE
Fig. 4. (A) Cyclic voltammograms of the Mb/MGCE (a), Fe3 O4 @Au/MGCE (b), and Mb/Fe3 O4 @Au/MGCE (c) in a 0.1 M pH 6.98 PBS at a scan rate of 100 mV s−1 . (B) Cyclic voltammograms of the Mb/Fe3 O4 @Au/MGCE in PBS (pH 6.98) at scan rate of 50, 100, 200, 300, 400, 500, 600, 800, and 1000 mV s−1 (from internal to external). Inset: Plots of the peak currents vs. scan rate. (C) Cyclic voltammograms of the Mb/Fe3 O4 @Au/MGCE in PBS with pH values of 4.49 (a), 5.29 (b), 5.91 (c), 6.47 (d), 6.98 (e), 8.04 (f), and 9.18 (g). Inset: Plot of formal potential vs. pH.
Q is the charge involved in the reaction, and F is the Faraday’s constant. The average surface amount ( ) of the adsorbed Mb on the Fe3 O4 @Au/MGCE was estimated to be 9.18 × 10−10 mol cm−2 , which was much larger than that reported for the {Au/Mb}n films (Zhang et al., 2006) and {PAMAM-Au/Mb}n films (Zhang and Hu, 2007a). This result demonstrated that the biofilm could considerably increase the functional density of Mb. Since nEp < 200 mV (Ep : peak potential), the direct electron-transfer rate constant (Ks ) of the adsorbed Mb on
Immobilized Mb on an electrode surface with retained conformation normally exhibits electrocatalytic activity for oxygen, H2 O2 , trichloroacetic acid (TCA), and NO2 − (Dai et al., 2004b; He et al., 2002). By using H2 O2 as model, the electrocatalytic properties of the Mb/Fe3 O4 @Au/MGCE were studied. Inset A of Fig. 5 shows the bioelectrocatalytic activity of the Mb/Fe3 O4 @Au/MGCE toward the reduction of H2 O2 at −0.35 V. A pair of reversible CV peaks appeared in the absence of H2 O2 (curve a). Upon addition of H2 O2 into the 0.1 M pH 6.98 PBS, the reduction peak current of immobilized Mb increased dramatically and oxidation peak current decreased. The reduction peak current increased with H2 O2 concentration (curves b–d), displaying an obvious electrocatalytic behavior of Mb to the reduction of H2 O2 (Lu et al., 2007). These results indicated that the immobilized Mb on Fe3 O4 @Au/MGCE retains its electrocatalytic activity to the reduction of H2 O2 . The performance of the Mb/Fe3 O4 @Au/MGCE was also studied by the amperometric current–time method. Fig. 5 illustrates a typical current–time curve of Mb/Fe3 O4 @Au/MGCE at −0.35 V upon successive addition of H2 O2 . When H2 O2 was added to the stirring PBS, the reductive current increased steeply to a stable value. The time to reach 95% of the maximum current was ca. 5 s, which is much faster than that of 10 s reported in the Mb/ZrO2 /chitosan film electrode (Zhao et al., 2005), 9 s in the Mb/ZrO2 -grafted collagen/DMSO/GE (Zong et al., 2007), and 10 s in the Mb/HMS/GCE (Dai et al., 2004b). Such a short response time can be mainly attributed to the fast diffusion process of hydrogen peroxide in the
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3.7. Reproducibility and stability of the biosensor
Fig. 5. Amperometric responses of the Mb/Fe3 O4 @Au/MGCE at −0.35 V to successive addition of H2 O2 in a stirred 0.1 M PBS (pH 6.98). Inset: (A) Cyclic voltammograms of the Mb/Fe3 O4 @Au/MGCE in 0.1 M pH 6.98 PBS containing 0 (a), 15 (b), 50 (c), and 100 M (d) H2 O2 at 100 mV s−1 . (B) Plot of chronoamperometric current vs. H2 O2 concentration.
To prove the precision and practicability of the proposed method, the reproducibility and storage stability of the biosensor were also examined. The relative standard deviation of the modified electrode response to 0.1 mM H2 O2 was 3.0% for ten successive measurements. The relative standard deviation for detection of 0.1 mM H2 O2 with five sensors prepared under the same conditions was 4.3%. The modified electrode was stored in pH 6.98 PBS at 4 ◦ C and measured at intervals of 1 week. The biosensor retained 96% of its original sensitivity after 1 week, and remained about 89% of its original current after 4 weeks. The excellent reproducibility and stability of the biosensor are due to the good biocompatibility and stability of the Mb/Fe3 O4 @Au biofilm. On the one hand, the biocompatibility of Fe3 O4 @Au NPs provides an interconnected three-dimensional matrix to deposit Mb and maintain the native structure of the immobilized proteins. On the other hand, the good affinity of Fe3 O4 @Au NPs to Mb improves the electron transfer and prevents leakage of Mb molecules from the Mb/Fe3 O4 @Au biofilm structure efficiently. 4. Conclusions
three-dimensional matrix and high electronic conductivity of the Fe3 O4 @Au NPs film. The modified electrode displayed increasing amperometric responses to H2 O2 with a good linear ranging from 1.28 to 283 M with a detection limit of 0.4 M (S/N = 3) (inset B of Fig. 5). When the H2 O2 concentration was higher than 283 M, the electrochemical response deviated from linearity and reached a plateau at much higher concentration of hydrogen peroxide, showing the characteristics of the Michaelis–Menten kinetic mechanism. app The apparent Michaelis–Menten constant (Km ) can be obtained from the Lineweaver–Burk equation (Shu and Wilson, 1976; Kamin and Wilson, 1980): app
1 K 1 1 = m · + Iss Imax C Imax
(3)
Here, Iss is the steady state current after the addition of substrate, C is the bulk concentration of the substrate, and Imax is the maximum app current measured under saturated substrate conditions. The Km value in this work was found to be 0.145 mM, which is much lower than those ever reported values (Dai et al., 2004b; Zhang and Zhen, app 2008; Zhang et al., 2007b; Liu and Ju, 2003). The smaller Km value indicates that the Mb immobilized in the Mb/Fe3 O4 @Au biofilm possesses higher enzymatic activity due to the higher affinity of the Mb to H2 O2 (Shi et al., 2007). 3.6. Interference study In real samples, the coexisting electroactive species might affect the biosensors response. In order to assess the anti-interference capability of the present sensor, the electrochemical responses of some common interfering substances were studied. No significant interference was observed upon addition of CO3 2− , ClO3 − , SO4 2− , Cl− , Br− , I− , glycin, l-cystine, l-tyrosine, choline, acetylcholine, catechol, nitrite, uric acid and ascorbic acid at concentration 10 times higher than H2 O2 at 50.0 M. However, Fe3+ might be a main interference to Mb for the electrocatalytic reduction of H2 O2 . When the concentration of Fe3+ increased to ten times higher than H2 O2 , a minor interference was observed. This possibly resulted from the interaction between Fe3+ and H2 O2 similar to that reported previously (Wang et al., 2002b). This interference could be excluded by adding EDTA in the sample solutions.
In this study, we have successfully developed a novel and simple myoglobin immobilization method by using biocompatible Fe3 O4 @Au magnetic composite material as the building block. The interconnected three-dimensional Fe3 O4 @Au magnetic nanocomposites film possessed superior conductivity, high stability, large surface area, and a good microenvironment for the immobilization of proteins. The immobilized Mb with retained biological activity could directly shuttle electron with the electrode. Compared to other immobilization procedures, the present method shows advantageous such as much larger adsorption capacity, free orientation possibility of the proteins, short immobilization time, and no need for an electron-transfer mediator or specific reagent. The designed Mb/Fe3 O4 @Au/MGCE exhibited good performances for electrocatalytic reduction of H2 O2 , such as high sensitivity, low detection limit, short response time, long-time stability and reproducibility. Acknowledgment This work was supported by grants from the National Natural Science Foundation of China (20605010, 20865003, 20805023), the Jiangxi Province Natural Science Foundation (0620039, 2007JZH2644), and the Opening Foundation of State Key Laboratory of Chem/Biosensing and Chemometrics of Hunan University (2006022, 2007012). References Araci, Z.O., Runge, A.F., Doherty, W.J., Saavedra, S.S., 2008. J. Am. Chem. Soc. 130, 1572–1573. Chen, C.T., Chen, W.Y., Tsai, P.J., Chien, K.Y., Yu, J.S., Chen, Y.C., 2007. J. Proteome Res. 6, 316–325. Chen, C.T., Chen, Y.C., 2005. Anal. Chem. 77, 5912–5919. Chen, X., Hu, N., Zeng, Y., Rusling, J.F., Yang, J., 1999. Langmuir 15, 7022–7030. Cho, S.J., Idrobo, J.C., Olamit, J., Liu, K., Browning, N.D., Kauzlarich, S.M., 2005. Chem. Mater. 17, 3181–3186. Cui, H.F., Ye, J.S., Zhang, W.D., Sheu, F.S., 2009. Biosens. Bioelectron. 24, 1723–1729. Dai, Z.H., Liu, S.Q., Ju, H.X., Chen, H.Y., 2004a. Biosens. Bioelectron. 19, 861–867. Dai, Z.H., Xu, X.X., Ju, H.X., 2004b. Anal. Biochem. 332, 23–31. Deng, Y., Qi, D., Deng, C., Zhang, X., Zhao, D., 2008. J. Am. Chem. Soc. 130, 28–29. Dyal, A., Loos, K., Noto, M., Chang, S.W., Spagnoli, C., Shafi, K.V.P.M., Ulman, A., Cowman, M., Gross, R.A., 2003. J. Am. Chem. Soc. 125, 1684–1685. George, P., Hanania, G.I.H., 1953. Biochem. J. 55, 236–243. He, P.L., Hu, N.F., Zhou, G., 2002. Biomacromolecules 3, 139–146. Hirsch, R., Katz, E., Willner, I., 2000. J. Am. Chem. Soc. 122, 12053–12054. Jia, J.B., Wang, B.Q., Wu, A.G., Cheng, G.J., Li, Z., Dong, S.J., 2002. Anal. Chem. 74, 2217–2223.
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