Improved Sensing in Physiological Buffers by Controlling the

Aug 17, 2010 - Xiaoquan Lu , Yan Li , Xia Zhang , Jie Du , Xibin Zhou , Zhonghua Xue ... Zhihua Wang , Hui Li , Jing Chen , Zhonghua Xue , Bowan Wu ...
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14786

J. Phys. Chem. C 2010, 114, 14786–14793

Improved Sensing in Physiological Buffers by Controlling the Nanostructure of Prussian Blue Films Jie Du, Yanfeng Wang, Xibin Zhou, Zhonghua Xue, Xiuhui Liu, Kun Sun, and Xiaoquan Lu* Key Laboratory of Bioelectrochemistry & EnVironmental Analysis of Gansu ProVince, College of Chemistry & Chemical Engineering, Northwest Normal UniVersity, Lanzhou 730070, P. R. China ReceiVed: May 25, 2010; ReVised Manuscript ReceiVed: August 11, 2010

The electrochemical properties of a Prussian blue (PB) electrode were improved by introducing cetyltrimethylammonium bromide (CTAB) and Au nanoparticles (AuNPs) into PB films. The novel hybrid films (PB/ CTAB/AuNPs) were fabricated by electrodepositing PB and AuNPs in the presence of CTAB. The electrochemical behavior of the hybrid film in some supporting electrolyte (cations for the K+, Na+, or K+/ Na+) was investigated in detail, and well-defined and reversible voltammetric responses were obtained in Na+-based electrolytes. The catalytic activity of the PB/CTAB/AuNPs electrode toward hydrogen peroxide (H2O2) reduction at a neutral pH was also investigated, and the results indicated that the electrochemical reduction of H2O2 in the presence of physiological levels of Na+ was superior to that of a PB-modified electrode. Moreover, the PB/CTAB/AuNPs electrode exhibited good performance, a low detection limit (0.1 µM), and high stability at a wide range of concentrations (0.882-195 µM). To determine the performance of PB nanocomposite electrodes in Na+-based phosphate buffers, an amperometric biosensor with a PB/CTAB/ AuNPs electrocatalyst was developed. To fabricate this sensor, the enzyme was immobilized in sol-gel and was electrodeposited onto a PB nanocomposite film. The results indicated that the biosensor can be used at a wide range of concentrations (20-400 µM) and possesses a low detection limit (7 µM) for glucose. These characteristics demonstrate that PB nanocomposite film can be used as an electron mediator for biosensors in potassium-free phosphate buffers. 1. Introduction Prussian blue (PB) is a material that is often used in the electrocatalysis of hydrogen peroxide (H2O2) due to its high rate of peroxide reduction at low operating potentials.1 PB films are easily prepared on the surface of various electrodes; thus, electrodes modified with PB have been used in applications such as electrocatalysis,2,3 electroanalysis,4,5 electrochromism,6,7 and batteries.8,9 Moreover, modified electrodes are suitable for the rapid and accurate determination of H2O2, and many H2O2 sensors based on PB films have been reported. Because hydrogen peroxide is the main product of reactions catalyzed by oxidases, substrate detection can be accomplished by monitoring the formation of hydrogen peroxide. Thus, biosensors based on enzymatic and electrochemical reactions of PBmodified electrodes have been developed.10-12 To achieve superior analytical performance, biosensors with PB-modified electrodes have been developed with lactate oxidase,13,14 cholesterol oxidase,15 glutamate oxidase,1,16 lysine oxidase,17 oxalate oxidase,18 and glucose oxidase.19-22 PB-modified biosensors possess several advantages, including operational stability and low cost. Moreover, by operating at a lower working potential, these biosensors can eliminate interferences from reducing species such as ascorbic acid, uric acid, and acetaminophen. Despite their interesting characteristics, several shortcomings are associated with the use of PB-modified electrodes. For instance, under neutral or alkaline conditions, PB electrodes are unstable over long periods of time because the films decompose in the presence of hydroxide ions. Furthermore, specific cations must be present in the supporting solution of PB-based * Corresponding author: Tel +86-931-7971276; Fax +86-931-7971323; e-mail [email protected].

biosensors. Hydrated cations such as K+, NH4+, Rb+, and Cs+ can enter into the channel of PB lattice (channel diameters of about 0.32 nm and PB lattice with a cubic unit cell of 1.019 nm) and promote electrochemical reactions.17 Alternatively, other hydrated cations such as Na+, Ca2+, and Mg2+ do not fit inside the lattice and block the operation of PB sensors. However, cations such as Na+ or Ca2+ are essential in clinical or physiological measurements and enzyme-based biosensors. Because of the limitations of PB as an electron mediator, the practical use of PB-based biosensors has been restricted.17,23 To expand practical applications, the detection limits and stability of PB films in a variety of supporting solutions must be improved. To overcome the aforementioned problems, the surface of PB electrodes has been modified with composite materials and PB analogues.24 For instance, a hybrid thin film of PB was deposited on an electrode in the presence of a cationic surfactant (cetyltrimethylammonium bromide, CTAB).25,26 Compared to an ordinary PB-modified electrode, the hybrid PB/CTAB electrode displayed enhanced stability and excellent peak currents. Furthermore, the lattice channels in PB/CTAB films were wider than those in PB-modified films, allowing the film to interact with hydrated Na+.27 However, PB/CTAB films did not provide sharp current responses in the presence of Na+ because the channel was not wide enough to allow hydrated Na+ to enter into the matrix.28 Thus, to employ PB-based electrodes in physiological buffers and supporting electrolytes with various cations, further modifications of PB/CTAB composite films are necessary. Another method of improving the applicability of PB films to sensing devices is to integrate PB with nanomaterials or to assemble PB films on the surface of

10.1021/jp104796c  2010 American Chemical Society Published on Web 08/17/2010

Nanostructure of Prussian Blue Films nanomaterials such as carbon nanotubes,11 magnetite,29 polystyrene spheres,30 CdS,31 Au,32,33 or Pt.34 The introduction of nanostructured hybrid materials increases the surface to volume ratio and the surface activity, which leads to a wider pH range, improved electrochemical stability, and excellent electrochemical response. Recently, a direct and facile electrodeposition method for the integration of Au nanoparticles and PB was reported.35,36 Specifically, a Au-Prussian blue (Au/PB) nanocomposite film was deposited on an electrode through a onestep electrochemical deposition process. The integration of Au nanoparticles may have a cooperative effect, which improves the sensing properties of PB. Because of the aforementioned advantages of PB composite films, the fabrication of PB nanocomposite materials is highly desirable. Thus, the main goal of this work was to synthesize a hybrid PB film with improved detection limits and enhanced stability in a variety of supporting solutions. Herein, we present a novel triple-component nanocomposite composed of PB, CTAB, and Au nanoparticles (abbreviated as PB/CTAB/AuNPs). The proposed hybrid material exhibited high electrocatalytic activity toward H2O2 in a supporting solution of Na+ and is a promising electrocatalyst for high-performance oxidase biosensors. Enzyme immobilization is a fundamental step in the fabrication of high-performance oxidase biosensors. A variety of different protocols have been used to immobilize enzymes, including physical adsorption,37-39 covalent attachment,40 and entrapment or encapsulation within a polymer membrane or inorganic matrix.23,41,42 Moreover, materials based on sol-gels are particularly attractive because they can be prepared under ambient conditions and exhibit tunable porosity, high thermal stability, chemical inertness, and negligible swelling in aqueous solution. A variety of technologies have been employed to produce sol-gel films on electrodes, including dipping or spincoating, vapor-deposition, and electrochemical20 methods. Dipping or spin-coating methods are simple and fast; however, this method cannot release the inner stress of the materials, which leads to low porosity and low adhesion to electrode surfaces. Moreover, the thickness of the film cannot be controlled with dipping or spin-coating techniques. Recently, electrochemical methods have been proposed to prepare the porous structure of enzyme encapsulated silica sol-gels.43-45 In these methods, a negative potential is applied to the electrode surface to generate hydroxyl ions, which act as a catalyst for the polycondensation of hydrolyzed alkoxysilane precursors. This method leads to direct incorporation and encapsulation of biomolecules in the course of the deposition process, which results in a more uniform distribution of enzyme and the immobilization of larger amounts of biomolecules. Thus, electrochemical methods are particularly suitable for biosensor fabrication. In this work, novel PB/CTAB/AuNPs nanocomposite films were fabricated by a one-set electrodeposition process, where the surfactant was assembled on the electrode surface. The nanocomposite films were characterized by cyclic voltammetry (CV) and scanning electron microscopy (SEM), and the process of nanocomposite formation was elucidated. The electrochemical behavior of PB/CTAB/AuNPs films was studied in the presence of different alkali metal cations and at various pHs. The results of CV revealed that PB/CTAB/AuNPs electrodes display two pairs of well-defined redox peaks in a supporting electrolyte containing K+ and Na+, and the electrodes retained their electrochemical activity in a Na+-based electrolyte. In Na+-based phosphate buffer solutions (PBS), the PB/CTAB/AuNPs-mediated electrode displayed excellent performance in H2O2 detection, including high sensitivity, a wide linear calibration range,

J. Phys. Chem. C, Vol. 114, No. 35, 2010 14787 and good stability. To fabricate amperometric glucose biosensors, oxidase was embedded in a sol-gel layer and was deposited on the surface of the modified electrode using an electrodeposition technique. The sensor was proven to be successful for the determination of glucose and displayed high sensitivity and a wide linear calibration range in the presence of physiological levels of Na+. 2. Experimental Section 2.1. Chemicals and Apparatus. Glucose oxidase (GOD, from Aspergillus sp., 100 U/mg) was purchased from Toyobo Co. (Osaka, Japan). All other chemicals used in this study were analytical grade or higher and were used as received without further purification. Aqueous solutions of H2O2 were freshly prepared prior to use, and glucose solutions were stored overnight at 4 °C. Phosphate buffer solutions (PBS) with various pHs were made from Na2HPO4 and NaH2PO4. All solutions were prepared using doubly distilled water. A CHI900 electrochemical workstation (CH Instrument Co.) with a conventional three-electrode cell was used to deposit films and to perform voltammetric and amperometric experiments. A glassy carbon electrode (3 mm in diameter, purchased from CH Instruments Inc.) and a Pt ultramicroelectrode (25 µm in diameter) were used as the working electrode, and a platinum wire was applied as the counter electrode. An Ag/AgCl electrode was used as the reference electrode, and all potentials were reported with respect to the reference electrode. Amperometric measurements were conducted in stirred solutions. Prior to amperometric experiments, the potential of each electrode was held at the operating potential, allowing the background current to decay to a steady state. The response current of the electrode was considered to be the difference between the steady-state current and the background current. All experiments and measurements were conducted at room temperature (20 ( 1 °C). 2.2. Preparation of the Hybrid Films. Prior to surface modification, the electrode was polished with alumina slurry (0.30 and 0.05 µm), rinsed thoroughly with redistilled water, and successively ultrasonicated in ethanol and redistilled water for ∼10 min. The pretreated electrode was cast from a growing solution containing 1.0 mM K3Fe(CN)6, 1.0 mM HAuCl4, and 0.4 mM CTAB, and a solution of 0.1 M KCl was used as the supporting electrolyte. Electrodeposition of PB/CTAB/AuNPs films was achieved by conducting 40 cycles of CV between the potential of 0.0 and 1.0 V and a scan rate of 0.05 V/s. Comparative experiments with electrodeposited PB-modified electrodes were accomplished by cycling the potential between -0.2 and 1.0 V at 0.1 V/s in a solution containing 1 mM FeCl3 and 1 mM K3Fe(CN)6. After deposition, the modified electrode was rinsed with doubly distilled water and heated at 110 °C for 1.5 h. 2.3. Preparation of GOD/Sol-Gel Films. Silica sol-gels were prepared by mixing 1.0 mL of tetraethyl orthosilicate (TEOS) and 8.0 mL of PBS (50 mM, pH ) 4.8) for 12 h with a magnetic stirrer until a clear solution was obtained. To ensure a homogeneous mixture, 2.0 mM of CTAB was added under stirring. The deposition solution consisted of 10 mg/mL of GOD, a 50 mM PBS (pH ) 5.8) solution, and silica sol, where equal volumes of the electrolyte and silica were employed. The PB/ CTAB/AuNPs-modified electrode was immersed in the deposition solution, and a constant cathodic current of -20 mA was applied for 300 s. Finally, the electrodeposited electrode was removed from the cell, washed thoroughly with PBS solution (pH ) 5.8), and dried prior to storage at 4 °C.

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Du et al. deposition of Au/PB nanocomposite films by a one-step procedure. In this process, AuCl4- was reduced and deposited on the electrode surface due to the strong interaction between Au particles and CN-. Moreover, ferricyanide (Fe3+/Fe2+) ions were adsorbed on the surface of the Au particles, resulting in the formation of a PB layer. The results of this study indicated that the following equation expresses the mechanism of PB formation:

Figure 1. Electrochemical deposition process used to fabricate PB hybrid films from a solution of 1 mM HAuCl4, 1 mM K3Fe(CN)6, and 0.4 mM CTAB: (a) fabrication on a GC electrode; (b) fabrication on UME in the presence of CTAB; (c) fabrication on UME without CTAB. (scan rates: 50 mV/s).

3. Results and Discussion 3.1. Electrodeposition of PB/CTAB/AuNPs. Many groups have reported the electrodeposition of PB composite films on a wide range of substrates (Pt, Au, glassy carbon) using different multistep or one-step procedures.26,33,35 We improved the oneset deposition method by conducting CV in a solution of 1.0 mM K3Fe(CN)6, 1.0 mM HAuCl4, 0.4 mM CTAB, and 0.1 M KCl. Figures 1a,b show the electrochemical process used to form a PB/CTAB/AuNPs layer on glassy carbon electrode (GC) and Pt ultramicroelectrodes (UME), respectively. The PB/CTAB/ AuNPs layer was deposited by repetitively sweeping the potential from 0.0 to 1.0 V (versus Ag/AgCl) at a scan rate of 0.05 V/s. The cycle voltammograms (CVs) presented a pair of sharp current peaks at ∼0.18 V, and the peak currents of cathodic and anodic waves increased periodically as the scanning time increased. The cathodic peaks centered at 0.17 V corresponded to the reduction of PB to Prussian white, whereas the anodic peaks centered at 0.19 V corresponded to the oxidation of Prussian white to PB, indicating that PB was electrodeposited onto the electrode surface in a stepwise manner. During the first several scans, a reduction peak was observed at 0.4 V, which indicated that Au(0) was formed on the electrode surface. Recently, Yu and Kumar et al.35,36 reported the

AuCl- + 3e f Au(0) + Cl-

(1)

[Fe(CN)6]3- + 6H+ + e f Fe2+ + 6H(CN)

(2)

Fe2+ + [Fe(NC)6]3- f [Fe2+Fe3+(NC)6]-

(3)

To further understand the role of surfactant in the formation of PB composites, electrodeposition experiments were also conducted on Pt-UME, with and without CTAB. Figures 1b,c show the deposition process in the presence and absence of CTAB, respectively. The results indicated that the typical increase in peak current was not observed when CTAB was not present in the solution (Figure 1c). This behavior is inconsistent with the results obtained with a large electrode (similar to Figure 1a, results not shown), which indicated that PB was not formed on the surface of UME. Alternatively, with CTAB, a pair of well-defined peaks at ∼0.18 V was observed (Figure 1b), which strongly suggested the formation of PB. In addition, the CV current under two different types of conditions increased with prolonged cycling, which indicated that Au was deposited and the superficial area of UME increased. Because of the above results, we speculated that the deposition of PB on UME is difficult in the absence of CTAB because Fe3+/ Fe2+ cannot adsorb to UME. Although UME has rather fast mass transport rates46,47 and Au particles are deposited on the surface of the electrode, ferrocyanide (eq 2) quickly diffuses into the bulk solution, and the electrode cannot adsorb Fe3+/ Fe2+; thus, the deposition of PB does not occur. Alternatively, CTAB can be assembled on the Au particles and assist Fe3+/ Fe2+ absorption. The presence of CTAB in solution during film formation can also assist PB deposition on the electrode surface,27,48 which suggests that CTAB becomes adsorbed to hydrophilic ionic groups on the surface of the electrode through its polar head groups.48,49 In the formation of PB/CTAB/AuNPs films, we propose that CTAB adsorbs on Au nanoparticles (eq 1) and self-assembles on the surface of the electrode. The resultant orientation of CTAB allows the molecule to bind to Fe3+/Fe2+, resulting in PB formation (eq 3). The deposition process was repeated by cycling the potential to obtain a dense triple-component nanocomposite (PB/CTAB/AuNPs) multilayer, as described in Scheme 1. 3.2. Morphological Characteristics of PB/CTAB/AuNPs. Figure 2 shows the scanning electron micrographs (SEM) of the surface of PB/CTAB/AuNPs and PB/AuNPs electrodes (prepared according to a literature procedure35). The SEM images revealed that the surface of PB/AuNPs displayed a high degree of roughness. Moreover, the results indicated that the diameter of the spherically shaped composite was between 50 and 200 nm (Figure 2b). Film imperfections in the form of holes and scratches were also observed, and the nanoparticles were primarily composed of PB and Au. In comparison, when the gold surface was coated with a PB/CTAB/AuNPs composite film (Figure 2a), the composition of PB/CTAB/AuNPs films

Nanostructure of Prussian Blue Films

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SCHEME 1: Schematic Illustration of the Formation Process of PB/CTAB/AuNPsa

a Conditions: (1) Au(0) was formed on the electrode surface; (2) CTAB was assembled on the fresh Au particles; (3) Fe3+/Fe2+ was bounded to CTAB; (4) resulting in PB formation and CTAB are attaching to both the AuNPs side and the PB side; (5) cycles (1)-(4), forming dense multilayer.

was relatively smooth and compact, and spherically shaped composite nanoparticles were barely visible, indicating that PB/ CTAB/AuNPs was densely populated on the electrode surface. 3.3. Electrochemistry of PB/CTAB/AuNPs. In the presence of K+ as the supporting electrolyte, PB displayed typical CVs due to the transport characteristics of alkali metal cations. Therefore, CV is a convenient and efficient tool for the characterization of the electrochemical behavior of PB and was used to characterize the PB/CTAB/AuNPs film in 0.1 M KCl. As can be seen in the Supporting Information, Figure S1, one set of redox peaks at 0.165 and 0.195 V were observed. The appearance of this set of peaks is similar to the electrochemical behavior exhibited by other PB analogues and corresponds to the reduction of PB to Prussian white. Thus, these peaks can be used as an indicator of the quality of PB layers.50 For scan rates up to 0.04 V/s, the peak-to-peak separation potential was 0.03 V, which was lower than the separation of other deposition methods, indicating that the electron transfer process at the surface of the electrode is relatively rapid.35,36 The Supporting Information, Figure S1, depicts the CVs of the PB/CTAB/ AuNPs-modified electrode at scan rates between 0.02 and 0.30 V/s. As shown in Figure 3, the peak current was linearly dependent on the square root of scan rate, which suggests that the electron transfer process of PB is reversible and diffusioncontrolled. The anodic-to-cathodic peak current and peak charge ratios approached unity at scan rates from 0.02 to 0.30 V/s, and the peak separation was less than 0.0593 V at all scan rates, indicating that PB/CTAB/AuNPs films displayed ideal Nernst behavior. These results suggested that the electrochemical activity of PB was maintained in the composite films. In the electrochemical process of modified films, ions enter or exit PB, and the film maintains electroneutrality. Several authors have suggested that ions have a significant effect on the electrochemical behavior of the films.27,51,52 To further examine the electrochemical performance of PB/CTAB/AuNPs films, CV was conducted with K+, Na+, and K+/Na+ as the supporting electrolyte. As shown in Figure 4a, a pair of redox peaks was observed in the presence of pure NaCl solution (Figure 4a, blue line). However, the peaks were not as sharp as those obtained with pure KCl solution (Figure 4a, black line); the peak at ∼0.05 V corresponded to the cathodic peak, whereas

the anodic peak was observed at 0.10 V. In a Na+/K+ solution (Figure 4a, red line), the CVs of PB/CTAB/AuNPs displayed two pairs of well-defined redox peaks at 0.02 V (peak 2) and 0.10 V (peak 1). A PB-modified electrode (see section 2.2 for the synthesis of PB-modified electrodes) was studied by CV under the same conditions as the PB/CTAB/AuNPs electrode. With PB as the working electrode (Figure 4b, black line), only one pair of peaks was observed at 0.05-0.15 V, and the peak profile was significantly different from the profile of the PB/ CTAB/AuNPs electrode. These results indicated that the hybridization of PB facilitated Na+ transport. We speculated that the width of the lattice channels in PB films increased due to the presence of CTAB and Au nanoparticles during film formation, which enabled the entry of Na+ and improved ion transport. Several studies27,48,53 on the transport characteristics of PB and PB analogues with ordered zeolite cagelike structures have been conducted, and the results clearly indicated that the size of the lattice channel of PB is ∼0.32 nm. Thus, K+ ions (hydrated radius ) 0.125 nm) can enter into the channels of the PB film, whereas the introduction of Na+, Li+, and NH4+ ions (Na+ hydrated radius is 0.183 nm) is limited. Furthermore, the PB lattice becomes damaged by the introduction of cations with large hydrated ions. Recently, Vittal et al.16 reported that PB films modified with CTAB retain their electrochemical performance after being subjected to CV in a pure solution of NaCl. Presumably, the size of the lattice channels in PB films increased upon interaction with CTAB during film formation, facilitating rapid ion transport. However, in the presence of Na+, PB/CTAB did not provide sharp peaks, suggesting that the increase in the size of the lattice channels in PB films was marginal. In this study, CV was conducted with PB/CTAB/ AuNPs in different supporting electrolytes, and the results support the hypothesis that the introduction of surfactants and nanoparticles increases the size of the lattice channels in PB films, enhancing ion transport. In composite films, Au nanoparticles display a cooperative effect, supplying excellent environmental conditions32,33 and providing a large surface area, which improves the adsorption of CTAB and increases the size of the lattice channel. As a result, Na+ ions can enter into PB composite films. The CVs of PB/CTAB/AuNPs displayed a set

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Figure 3. Relationship between peak current and square root of scan rate (scan rates: 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, and 300 mV/s).

Fe43+[Fe2+(CN)6]3 + 4e + 4Na+ f Na4Fe42+[Fe3+(CN)6]3

(5)

Fe43+[Fe2+(CN)6]3 + 4e + 2Na+ + 2K+ f Na4K2Fe42+[Fe3+(CN)6]3 (6)

Figure 2. SEM images of the hybrid films prepared from solutions (a) with CTAB and (b) without CTAB. The scale bar in figure is 500 nm.

of peaks in pure NaCl and two pairs of peaks in NaCl/KCl. On the basis of these results, the permeability of K+ and Na+ into PB films was determined. Figure 4c shows the CVs of the modified electrode in solutions containing various concentrations of K+ (from 0.03 to 0.09 M) and a constant concentration of Na+ (0.06 M). The results indicated that the formal potential increased with an increase in the activity of K+. At higher concentrations of K+, the two peak currents almost completely overlapped, indicating that the permeability of K+ and Na+ in the lattice channels of PB was identical. On the basis of these results, we speculated that Na+ participates in the electrochemical process of PB hybrid films in a manner that is similar to other PB analogues.23 We suggest that the following equations depict the role of Na+ in the redox reaction of PB films:

Fe43+[Fe2+(CN)6]3 + 4e + 4K+ f K4Fe42+[Fe3+(CN)6]3 (4)

The proposed mechanism is only one of several possibilities; however, to our knowledge, no other attempts to explain the results of modified PB films have been made. Nevertheless, the results obtained in this study revealed that the use of CTAB and Au nanoparticles in the preparation of PB films is an excellent method of improving film performance. Because of the enhanced electrochemical behavior, these films can be considered in electrocatalysis and electroanalytical applications. Furthermore, the films may be able to provide the current responses that are necessary for monitoring electrocatalytic reactions of H2O2 in the presence of physiological levels of Na+. To determine the electrocatalytic activity of modified PB films toward H2O2 in pure Na+ supporting electrolytes, CV was conducted in alkaline solutions and in the presence of H2O2. 3.4. Stabilization of PB/CTAB/AuNPs in Neutral and Alkaline Solution. The instability of PB is the main drawback to the use of PB-modified electrodes. Experiments to evaluate the stability of PB are often based on CV, where the decrease in the peak currents of PB is observed after several cycles. To evaluate the stability of PB/CTAB/AuNPs films, the currents obtained from PB- and PB/CTAB/AuNPs-modified electrodes were recorded after 100 cycles at various pHs. The charge retaining capacity of the film was quantified in terms of the percent residual activity, which was calculated from the ratio of the peak current of the first cycle to the peak current at the end of the cycle, after continuous cycling of the electrode potential between -0.2 and 0.5 V at a scan rate of 0.1 V/s in alkaline electrolyte solution. As shown in Figure 5, when the pH was between 6.33 and 7.24, the current associated with the PB/AuNPs-modified electrode decreased by 8.1-22.4%, whereas the current obtained from the PB/CTAB/AuNPs-modified electrode decreased by only 4.6-9.8% after 100 cycles. These results indicated that the presence of surfactant and nanoparticles improved the electrochemical stability of PB films. The results indicated that PB/CTAB/AuNPs-modified electrodes were more stable than the PB system electrodes. Similar

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Figure 5. Relationship between the peak currents of PB/CTAB/AuNPs films (black line) and PB/AuNPs films (red line) and the pH.

Figure 4. Voltammograms of (a) PB/CTAB/AuNPs in 0.07 M NaCl and 0.03 M KCl (red line), 0.02 M KCl (black line), and 0.08 M NaCl (blue line); (b) PB/CTAB/AuNPs (red line) and PB (black line) modified electrode in 0.07 M NaCl and 0.03 M KCl; (c) PB/CTAB/AuNPs in 0.06 M NaCl and 0.03 M (black line), 0.05 M (red line), 0.07 M (blue line), and 0.09 M (green line) KCl. Scan rate: 20 mV/s.

to the hypotheses presented in previous papers,48,54 we speculated that the stability of PB/CTAB/AuNPs films was due to the method of deposition. Specifically, the deposition of Au nanoparticles during film formation increased the surface area, which provided a high loading capacity for the deposition of PB particles and further stabilized the film. Moreover, the synthesis of PB in the presence of surfactant resulted in an

electrostatic attraction between PB and CTAB, allowing the formation of an ion complex based on Fe2+[Fe3+(CN)6]3- and the cationic headgroup of CTAB, which stabilized the hybrid film in neutral or weakly alkaline solution. Thus, in subsequent experiments, a buffer with a pH of 6.8 was selected. 3.5. Electrocatalytic Activity of PB/CTAB/AuNPs Films toward H2O2. As described in section 3.3, a satisfactory current response was observed for PB/CTAB/AuNPs electrodes in a NaCl solution. Thus, the electrocatalytic activity of PB/CTAB/ AuNPs toward H2O2 was determined at physiological levels of Na+. To this end, linear sweep voltammetry (LSV) was conducted in PBS (sodium-based, 20 mM, pH ) 6.67), and the results are presented in the Supporting Information (Figure S2). When the hydrogen peroxide concentration increases, in the 0.15 to -0.2 V range, the response current clearly increased and the current peak at about 0.02 V, which demonstrates that the modified electrode possessed good catalytic properties for the reduction of H2O2. The catalytic peak was likely derived from the reduced state of PB (Prussian white), which can be oxidized by H2O2. The working mechanism of the reaction is similar to that of pure PB, which is described in the literature.55 However, in modified PB films, PB can be subsequently reduced to Prussian white via Na+-mediated electron exchange on the surface of the electrode (eq 5). This result indicated that CTAB/ PB/AuNPs films act as an effective catalyst for the reduction of H2O2 in a supporting electrolyte without K+. Moreover, the results suggested that CTAB/PB/AuNPs electrodes can be operated in the presence of physiological levels of Na+. As shown in Figure 6, successive injections of H2O2 were made into an electrochemical cell containing 0.02 M Na+-PBS (pH ) 6.67), and the current of the PB/CTAB/AuNPs-modified electrode was monitored over time. To avoid signal interference from electroactive substances, the working potential was maintained at -0.05 V according to the current response of H2O2. The results indicated that the current response of the sensor increased as the concentration of H2O2 increased, reaching 95% of the steady-state current in less than 5 s, indicating that the response of the sensor is rapid. The resulting calibration curve was linear between 8.82 × 10-7 and 1.95 × 10-4 M, and a correlation coefficient of 0.9977 was obtained (see the Supporting Information, Figure S3), indicating that the sensor displays good electrocatalytic activity toward H2O2. The detection limit of the sensor was estimated to be 1.0 × 10-7 M, and the corresponding signal/noise ratio of the method was equal to 3.

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Figure 6. Amperometric response to successive additions of 0.882 µM H2O2 in 20 mM Na+-PBS buffer (pH ) 6.67) at a working potential of -0.05 V.

Figure 7. EIS data obtained from a solution of 1 mM K3Fe(CN)6 and 0.1 M KCl: (a) PB/CTAB/AuNPs; (b) SiO2/ PB/CTAB/AuNPs; (c) SiO2/GOD/PB/CTAB/AuNPs.

3.6. Amperometric Response of the Biosensor to Glucose. To produce a glucose biosensor, a sol-gel film of GOD was immobilized onto PB/CTAB/AuNPs electrodes (see section 2.3 for complete details on the synthesis of the glucose biosensor). Moreover, electrochemical impedance spectroscopy (EIS) was conducted to observe the formation of the films. Figure 7 illustrates typical Nyquist plots obtained from PB/CTAB/AuNPs electrodes, SiO2/ PB/CTAB/AuNPs electrodes, and SiO2/GOD/ PB/CTAB/AuNPs electrodes in a solution of 0.1 M KCl and 1.0 mM [Fe(CN)6]3-/4-. The Rct of the SiO2/PB/CTAB/AuNPs electrode was greater than that of the PB/CTAB/AuNPs electrode (the Rct was close to zero), suggesting that SiO2 sol-gel reduced the rate of electron transfer to and from the redox probe due to electrostatic repulsion between the negatively charged SiO2 sol-gel and [Fe(CN)6]3-/4-. Compared to PB/CTAB/AuNPs electrodes, the observed increase in the Rct of SiO2/PB/CTAB/AuNPs electrodes provided experimental evidence that a layer of sol-gel formed on the electrode surface. Moreover, when GOD was entrapped in the silica sol-gel, an increase in the Rct was observed. As shown in plot c of Figure 7, the large volume of GOD molecules decreased the rate of electron transfer from the

Figure 8. Calibration curve of a SiO2/GOD/PB/CTAB/AuNPs electrode as a function of the glucose concentration in 20 mM Na+-PBS buffer (pH ) 6.67) at a working potential of -0.05 V.

surface of the electrode to [Fe(CN)6]3-/4-, indicating that the adsorption of GOD into SiO2/PB/CTAB/AuNPs was successful. Glucose was added successively into the electrochemical cell, and the chronoamperometric response of the biosensor was recorded (shown in the Supporting Information, Figure S4). When 20 µM glucose was added into the buffer solution, the current rose sharply until a steady-state current was attained. The response time (the time at which 90% of the maximum response was observed) was less than 25 s, indicating that the electron transfer process was rapid and immobilized GOD could catalyze the oxidation of glucose, producing hydrogen peroxide. Figure 8 shows the dependence of the response current on the concentration of glucose. The amperometric current of the biosensor increased linearly with an increase in glucose concentration (20-400 µM), and a correlation coefficient of 0.9988 was obtained. The observed linear relationship suggested that PB-modified films are applicable to glucose sensing devices that operate at low concentrations of Na+-PBS. To increase the number of potential applications of PB nanocomposites, PBbased films could be further modified.

Nanostructure of Prussian Blue Films 4. Conclusions In this study, a new triple-component nanocomposite composed of PB, CTAB, and Au nanoparticles (abbreviated as PB/ CTAB/AuNPs) was fabricated by direct electrodeposition from aqueous solution. CV experiments were conducted under alkaline conditions, and the results indicated that the PB in the hybrid film was more stable than pure PB. Moreover, the performance of PB/CTAB/AuNPs films was evaluated in an electrolyte solution containing sodium cations, and the hybrid films displayed distinct electrochemical behavior. The improved performance of PB/CTAB/AuNPs electrodes was attributed to an increase in the size of the lattice channels, which was due to the introduction of CTAB and Au nanoparticles during film formation. The improved PB hybrid films displayed satisfactory electrocatalytic behavior toward H2O2 in a Na+-based phosphate buffer. Additionally, GOD was embedded in the sol-gel layer, and biosensors based on PB/CTAB/AuNPs modified electrodes were fabricated. Upon immobilization of GOD into PB/CTAB/ AuNPs films, an increase in the response was observed; thus, the fabrication of other PB-based biosensors that require cations such as Na+ for catalytic activity may be achieved. Acknowledgment. This work was supported by the National Natural Science Foundation of China (No. 20927004, 20775060, 20875077) and the Natural Science Foundation of Gansu Province (096RJZA122) and the Key Laboratory of Ploymer Materials of Gansu Province. Supporting Information Available: CVs, LSVs, calibration curve, and typical amperometric response of modified electrode mentioned in the text. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Karyakin, A. A.; Karyakina, E. E.; Gorton, L. Anal. Chem. 2000, 72 (7), 1720–1723. (2) Dong, S. J.; Che, G. L. J. Electroanal. Chem. 1991, 315 (1-2), 191–199. (3) Pandey, P. C.; Singh, B. Biosens. Bioelectron. 2008, 24 (4), 848– 54. (4) Vittal, R.; Gomathi, H.; Rao, G. P. Bull. Electrochem. 1999, 15 (11), 462–465. (5) Karyakin, A. A.; Puganova, E. A.; Budashov, I. A.; Kurochkin, I. N.; Karyakina, E. E.; Levchenko, V. A.; Matveyenko, V. N.; Varfolomeyev, S. D. Anal. Chem. 2004, 76 (2), 474–478. (6) Kaneko, M.; Teratani, S.; Harashima, K. J. Electroanal. Chem. 1992, 325 (1-2), 325–332. (7) Chen, L. C.; Huang, Y. H.; Ho, K. C. J. Solid State Electrochem. 2002, 7 (1), 6–10. (8) Eftekhari, A. J. Power Sources 2004, 126 (1-2), 221–228. (9) Chen, L. C.; Huang, Y. H.; Tseng, K. S.; Ho, K. C. J. New Mater. Electrochem. Syst. 2002, 5 (3), 203–212. (10) Xian, Y.; Hu, Y.; Liu, F.; Xian, Y.; Feng, L.; Jin, L. Biosens. Bioelectron. 2007, 22 (12), 2827–2833. (11) Lin, Y. Q.; Liu, K.; Yu, P.; Xiang, L.; Li, X. C.; Mao, L. Q. Anal. Chem. 2007, 79 (24), 9577–9583. (12) Ulasova, E. A.; Micheli, L.; Vasii, L.; Moscone, D.; Palleschi, G.; Vdovichev, S. V.; Zorin, A. V.; Krutovertsev, S. A.; Karyakina, E. E.; Karyakin, A. A. Electroanalysis 2003, 15 (5-6), 447–451. (13) Curulli, A.; Valentini, F.; Orlanduci, S.; Terranova, M. L.; Palleschi, G. Biosens. Bioelectron. 2004, 20 (6), 1223–1232. (14) Curulli, A. Sens. Lett. 2008, 6 (5), 682–689. (15) Vidal, J. C.; Espuelas, J.; Garcia-Ruiz, E.; Castillo, J. R. Talanta 2004, 64 (3), 655–664. (16) Ricci, F.; Amine, A.; Moscone, D.; Palleschi, G. Biosens. Bioelectron. 2007, 22 (6), 854–862.

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