Increment of Density of Au Nanoparticles Deposited in Situ within

May 8, 2009 - Poly(allylamine hydrochloride) (PAH) and SiO2 nanoparticles were assembled into {PAH/SiO2}n layer-by-layer (LbL) films on the surface of...
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J. Phys. Chem. C 2009, 113, 9831–9837

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Increment of Density of Au Nanoparticles Deposited in Situ within Layer-by-Layer Films and Its Enhancement on the Electrochemistry of Ferrocenecarboxylic Acid and Bioelectrocatalysis Xihong Guo and Naifei Hu* Department of Chemistry, Beijing Normal UniVersity, Beijing, 100875, P. R. China ReceiVed: March 30, 2009; ReVised Manuscript ReceiVed: April 15, 2009

Poly(allylamine hydrochloride) (PAH) and SiO2 nanoparticles were assembled into {PAH/SiO2}n layer-bylayer (LbL) films on the surface of pyrolytic graphite (PG) electrodes. The films were then placed in HAuCl4 solutions to load Au(III) ions into the films, followed by chemical reduction of Au(III) into Au(0) nanoparticles (AuNPs) by immersion of the films in NaBH4 solutions. This procedure of in situ deposition of AuNPs could be repeated for several cycles to enhance the density of AuNPs in the films. The presence of AuNPs in the films with high density improved the electrochemical responses of ferrocenecarboxylic acid (Fc(COOH)) greatly and then enhanced the electrocatalytic oxidation of glucose by glucose oxidase (GOD) with Fc(COOH) as a mediator. Various techniques, such as quartz crystal microbalance (QCM), scanning electron microscopy (SEM), energy dispersive X-ray (EDX) analysis, and different electrochemical approaches, were used to characterize the films. The mechanism of enhancement of AuNPs in the films on Fc(COOH) electrochemistry was explored and discussed in detail. Introduction The development of biological devices is of great importance in environmental, medical, electronic, and other applications. Bioelectrocatalysis based on direct or mediated electrochemistry of various enzymes can be used to detect and determine the corresponding substrates, thus establishing the foundation of electrochemical biosensing. In the progress, the improvement of electrochemical performances of biosensors has continued to be the main challenge. One of the important strategies for this purpose is to develop a new type of biosensor that combines enzymes or other biomacromolecules with nanomaterials.1,2 Among different nanomaterials, gold nanoparticles (AuNPs) have attracted increasing interest in the construction of biosensors because of unique properties such as high biocompatibility, distinctive size-related behavior, good conductivity, and high catalytic activity.2-5 For example, AuNPs have been used as electron relays to enhance electron exchange between redox enzymes immobilized on the surface of electrodes and underlying electrodes.6-8 Recently, Willner and co-workers reported that biocatalytic enlargement of AuNPs immobilized on enzyme glucose oxidase (GOD) electrodes could greatly enhance the ferrocene-mediated bioelectrocatalytic oxidation of glucose.9 To apply AuNPs in biosensors, it is highly desired to effectively immobilize or deposit AuNPs on the surface of electrodes.10 Among various strategies in the deposition of AuNPs, layer-by-layer (LbL) assembly, originally developed by Decher and co-workers for fabricating polyelectrolyte multilayers,11,12 has demonstrated remarkable advantages over other film-forming approaches in the precise control of film composition and thickness at a molecular or nanometer level with extreme simplicity and high versatility. Different types of LbL films containing AuNPs have been used as the foundation of electrochemical biosensors.13-19 For example, oppositely charged presynthesized AuNPs and myoglobin (Mb) were assembled * Corresponding author. E-mail: [email protected]., tel: (+86) 105880-5498, fax: (+86) 10-5880-2075.

into LbL films on the electrode surface, and the direct electrochemistry of Mb in the films was greatly enhanced by AuNPs.18 Recently, a novel approach to prepare noble metal nanoparticles in situ within LbL films has been developed.20 This method essentially includes three steps: (1) the regular LbL films of polyelectrolytes and/or nanoparticles are assembled on the surface of solid substrates; (2) the films are immersed in the solution of noble metal ions to load or incorporate the ions into the films; (3) the noble metal ions in the films are chemically reduced in situ by reductants, finally forming the noble metal nanoparticle-loaded LbL films. Numerous metal nanoparticleloaded LbL films have been fabricated by this approach and have found their applications in various fields.21-27 This in situ synthetic methodology using preassembled LbL films as nanoreactors demonstrates some unique advantages over the conventional LbL films assembled directly by the presynthesized nanoparticles, including better control of film structure and nanoparticle size, more flexibility in a variety of chemical approaches in the postreduction, and higher density of nanoparticles in the films by repeating the deposition cycles. For example, in the work of Rubner’s group, the size and density of Ag nanoparticles (AgNPs) in polyelectrolyte multilayers were well controlled by the number of deposition cycles and assembly pH.26 The amine-rich polyelectrolyte LbL films27 and titania precursor composite LbL films containing “free” imine groups24 as the nanoreactors for in situ synthesis of AuNPs were also reported. However, to the best of our knowledge, no work on applying AuNP-loaded LbL films in electrochemistry and bioelectrocatalysis has been described up to now. In the present work, the above strategy was used to assemble AuNP-loaded LbL films on the surface of electrodes, and the electrochemistry of ferrocenecarboxylic acid (Fc(COOH)) at the film electrodes and the corresponding bioelectrocatalysis were investigated. First, oppositely charged poly(allylamine hydrochloride) (PAH) and SiO2 nanoparticles were assembled into {PAH/SiO2}n LbL films on electrodes. The films were then placed in HAuCl4 solutions to load Au(III) ions into the films,

10.1021/jp902862g CCC: $40.75  2009 American Chemical Society Published on Web 05/08/2009

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followed by immersion of the films in NaBH4 solutions to chemically reduce Au(III) into AuNPs in the films. The Au(III) loading and reduction procedure could be repeated for several cycles to enhance the density of AuNPs in the films. In this work, PAH was chosen mainly because the protonated amine groups of PAH carried positive charges and might become the binding sites for anionic Au(III) complex ions.27 SiO2 nanoparticles were used in the work mainly because of their good biocompatibility.28-30 The high rigidity of SiO2 nanoparticles was also helpful to form porous LbL films,8 which was important not only for Au(III) ion loading but also for electron exchange of Fc(COOH) with underlying electrodes. Herein, Fc(COOH) acted as both electroactive probe and electrocatalytic mediator. Ferrocene and its derivatives are well-known electron mediators in electrocatalytic oxidation of glucose in the presence of GOD.31-33 While the {PAH/SiO2}n films as a barrier limited the cyclic voltammetric (CV) response of Fc(COOH) at the electrodes, the presence of AuNPs in the films could greatly increase the CV peaks of Fc(COOH) accompanied by the decrease of peak separation (∆Ep ) Epa - Epc). The enhancement of CV response of Fc(COOH) by AuNPs in the films was also used to improve the bioelectrocatalysis of glucose by GOD. Various techniques, such as quartz crystal microbalance (QCM), scanning electron microscopy (SEM), energy dispersive X-ray (EDX) analysis, and different electrochemical approaches were used to characterize the films. The mechanism of enhancement of AuNPs in the films on Fc(COOH) electrochemistry was also explored and discussed in detail. A better understanding of the function of AuNPs in this model system would be helpful to design and develop a novel type of LbL film containing highly densified AuNPs, which may greatly facilitate the direct or mediated electron transfer of enzymes and improve the performance of electrochemical biosensors. Experimental Section 1. Chemicals. Glucose oxidase (GOD, EC 1.1.3.4, type VII, from Aspergillus niger, 192 000 units g-1), poly(allylamine hydrochloride) (PAH, MW ∼ 70 000), HAuCl4 · 3H2O, 3-mercapto-1-propanesulfonate (MPS), and ferrocenecarboxylic acid (Fc(COOH)) were purchased from Sigma-Aldrich. Hexaammineruthenium(III) chloride (Ru(NH3)6Cl3) was obtained from Alfa Aesar. K3Fe(CN)6 and K4Fe(CN)6 were obtained from Beijing Chemical Plant. SiO2 nanoparticles (15 ( 5 nm) were from Zhoushan Nanoparticle Technology. Glucose was from Beijing Yili Chemicals. A glucose stock solution was prepared in pH 7.0 phosphate buffers and was allowed to mutarotate for 24 h at room temperature before use. All other chemicals were of reagent grade. Solutions were prepared with water purified twice by ion exchange and subsequent distillation. Buffers were 0.05 M sodium acetate (pH 5.0), potassium dihydrogen phosphate (pH 7.0), or boric acid (pH 9.0) solutions, all containing 0.1 M NaCl. The pH of the buffers was adjusted to the desired value by dilute HCl or NaOH. 2. Film Assembly. Basal plane pyrolytic graphite (PG, Advanced Ceramics, geometric area 0.16 cm2) disk electrodes were used for electrochemical studies. Prior to assembly, the PG electrodes were abraded with 320-grit metallographic sandpaper, ultrasonicated in water for 30 s, and then dried in air. The electrodes were then alternately immersed in PAH solution (1 mg mL-1 at pH 9.0) and an aqueous suspension of SiO2 nanoparticles (3 mg mL-1 at pH 9.0) for 20 min with intermediate water washing, forming {PAH/SiO2}n LbL films on the PG surface. The films were then immersed in 10 mM HAuCl4 solutions at pH 7.0 for about 1 h to allow the Au(III) ions to diffuse into the films. After being washed with water

Guo and Hu and dried in air, the Au(III)-loaded films were placed in freshly prepared 0.05 M NaBH4 solutions at pH 7.0 for 30 min to transform the Au(III) ions loaded in the films into Au(0) nanoparticles. To increase the amount or the surface concentrations of AuNPs in the films, the Au(III) loading and reduction procedure described above could be repeated for several cycles, and the resultant films were designated as {PAH/SiO2}n-xAu, where n is the number of bilayers of LbL films and x is the number of Au(III) loading and reduction cycles. The {PAH/ SiO2}n films fabricated on PG disks and the corresponding {PAH/SiO2}n-xAu films were also used as samples for scanning electron microscopy (SEM) measurements. For the quartz crystal microbalance (QCM) study, the clean QCM gold electrodes were first immersed in MPS solutions for 24 h to chemisorb MPS as a precursor layer, making the electrode surface become negatively charged. The subsequent assembly of {PAH/SiO2}n films on the surface of Au/MPS was the same as on the PG electrodes. 3. Apparatus and Procedures. A CHI 621B or CHI 660A electrochemical workstation (CH Instruments) was used for electrochemical measurements. A three-electrode cell was used with a saturated calomel electrode (SCE) as the reference, a platinum wire as the counter, and the PG disk with films as the working electrode. QCM measurements were performed with a CHI 420 electrochemical analyzer (CH Instruments). The quartz crystal resonator (AT-cut, fundamental resonance frequency 8 MHz) covered by thin gold films on both sides (geometric area 0.196 cm2 per one side) was used. After each adsorption step, the QCM gold electrodes were washed with water and dried under a nitrogen stream, and the frequency change was then measured in air by QCM. On the basis of the Sauerbrey equation,34 the relationship between QCM frequency shift (∆F, Hz) and micromass change (∆M, g) for each adsorption step can be expressed as ∆F ) -7.40 × 108∆M by taking into account the properties of quartz resonators used in this work. Thus, 1 Hz of frequency decrease corresponds to 1.35 ng of mass increase. If assuming that the adsorption layer was densely packed on the surface without any imperfection, the QCM data can also be used to estimate the nominal thickness (d, cm) of each adsorption layer according to the equation of d ) -(3.4 × 10-9)∆F/F,35 where F is the density of the layer material (g cm-3). For SiO2 nanoparticles, the density is about 2.2 ( 0.1 g cm-3,36 while for polymers such as PAH, the density was assumed to be 1.2 ( 0.1 g cm-3.37 The SEM images of films and the spectra of energy dispersive X-ray (EDX) were obtained using a scanning electron microscope (Hitachi, S-4800) equipped with an energy dispersive X-ray analyzer (Horiba, EMAX-350). The test regions for EDX measurements were randomly selected on the film surface for the elemental analysis. All experiments were performed at an ambient temperature of 20 ( 2 °C. Results 1. Fabrication of {PAH/SiO2}10-xAu Films. With a pI at about 238 or 3,39 SiO2 nanoparticles are negatively charged at pH 9.0, while PAH carries partial positive charges at pH 9.0 because its solution pKa is about 8.5.40-42 Thus, the assembly driving force of {PAH/SiO2}n LbL films should mainly be electrostatic interaction between oppositely charged SiO2 and PAH. The assembly of {PAH/SiO2}n films on the Au/MPS surface was monitored and confirmed by QCM (Figure 1). The frequency decrease (-∆F) showed a roughly linear relationship with adsorption step, indicating that the {PAH/SiO2}n multilayer films are successfully assembled and the assembly is in a regular

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Figure 1. QCM frequency shift (∆F) with adsorption step for {PAH/ SiO2}n films on Au/MPS surface: (b) PAH and (O) SiO2 adsorption steps.

TABLE 1: QCM Results for Adsorption Layers of {PAH/ SiO2}n Films on the Au/MPS Surface layer

frequency decrease (-∆F), Hz

amounts adsorbed (∆m), ng

nominal thickness (d), nM

PAH SiO2

165 ( 95 855 ( 85

223 1154

4.7 13.2

fashion with nearly equal adsorption amount for each PAH/ SiO2 bilayer. The quantitative QCM results are also listed in Table 1. For the layer of SiO2 nanoparticles, the nominal thickness was about 13.2 nm, very close to the average diameter of SiO2 nanoparticles (15 ( 5 nm) provided by the manufacturer, suggesting that SiO2 nanoparticles are probably arranged in an approximate monolayer mode. CVs of the electroactive probe Fc(COOH) in solution were also used to confirm the LbL assembly of {PAH/SiO2}n films on PG electrodes. Fc(COOH) showed a well-defined and nearly reversible CV peak pair at about 0.3 V vs SCE, characteristic of a Fc/Fc+ redox couple.43 Both oxidation and reduction peak currents of the probe decreased with the number of bilayers (n) after the {PAH/SiO2}n films were assembled on PG electrodes, accompanied by the increase of peak separation (∆Ep) (Figure 2). This result indicates the successful assembly of {PAH/SiO2}n films because the films formed on the PG electrodes act as a barrier and hinder the probe from approaching the electrode surface and transferring electrons. The {PAH/SiO2}10 films on PG electrodes were immersed in HAuCl4 solution for about 1 h to allow the Au(III) ions to diffuse into the films. The Au(III)-loaded films were then placed in NaBH4 solutions for 30 min to reduce the Au(III) into AuNPs. This Au(III) loading and reduction procedure was repeated for several cycles to increase the amount or density of AuNPs in the films. The formation of AuNPs in the films by this way was confirmed by SEM and EDX. Compared with {PAH/ SiO2}10 films containing no AuNPs, additional small bright particles with a size of less than 20 nm were observed on the

Figure 2. CVs of 0.2 mM Fc(COOH) in pH 7.0 buffers at 0.05 V s-1 for {PAH/SiO2}n films assembled on PG electrodes with a different number of bilayers (n): (a) 0, (b) 3, (c) 7, and (d) 10.

surface of {PAH/SiO2}10-xAu films in SEM pictures (Figure 3), which should be attributed to the formation of AuNPs. Moreover, the amount of bright nanoparticles increased obviously when the Au deposition cycles (x) increased from 3 to 8. To further support the speculation that the AuNPs are formed in the {PAH/SiO2}10-xAu films, EDX was used to characterize the different films. For {PAH/SiO2}10 films containing no AuNPs, only one peak characteristic of Si at 1.74 keV44,45 was observed in the region from 1.3 to 2.7 keV (Figure 4, curve a). For {PAH/SiO2}10-3Au and {PAH/SiO2}10-8Au films, however, in addition to the Si peak, another peak characteristic of Au at 2.2 keV46,47 was observed, and the peak area for {PAH/SiO2}108Au films was obviously larger than that for {PAH/SiO2}103Au films (curves b and c). By EDX analysis combined with SEM, it is very clear that the bright nanoparticles observed on the surface of {PAH/SiO2}10-xAu films are AuNPs, which are formed in situ by chemical reduction of Au(III) ions previously loaded into the films. The density of AuNPs in the films can thus be controlled by adjusting the Au(III) loading and reduction cycles. In addition, the porosity or roughness of the films increased when AuNPs were deposited (Figure 3). 2. Enhancement of AuNPs in {PAH/SiO2}10-xAu Films on CV Responses of Fc(COOH). Fc(COOH) showed a welldefined CV peak pair at bare PG electrodes with ∆Ep ) 0.063 V (Figure 5, curve a). When {PAH/SiO2}10 films were assembled on PG electrodes, the CV peak currents of Fc(COOH) decreased, accompanied by the increase of ∆Ep value (curve b). However, at {PAH/SiO2}10-3Au film electrodes, Fc(COOH) demonstrated much higher CV response at a similar potential (curve c), indicating that the incorporation of AuNPs greatly improves the CV response of Fc(COOH). A similar improvement of CV responses of Ru(NH3)63+ at {PAH/SiO2}10-3Au film electrodes was also observed (Supporting Information Figure S1), indicating the generality of enhancement of AuNPs in the multilayer films on electron transfer of small electroactive probes in solution. For {PAH/SiO2}10-xAu films, the CV response of Fc(COOH) in solution was greatly influenced by the number of Au deposition cycles (x) (Figure 6). The oxidation peak current of Fc(COOH) (Ipa) increased linearly with the deposition cycle at least when x e 8. Moreover, the peak separation (∆Ep) showed a general decreasing trend with x accompanied with some small fluctuation (Figure 6B), indicating that the AuNPs deposited in the films can improve the reversibility of CV behavior of Fc(COOH) to some extent, and the higher density of AuNPs in the films will lead to the better reversibility. The number of bilayers (n) of {PAH/SiO2}n-xAu films also demonstrated the remarkable influence on CV response of Fc(COOH) in solution, and the oxidation peak currents (Ipa) of Fc(COOH) for different n and x values are summarized in Figure 7. For {PAH/SiO2}n films containing no AuNPs (x ) 0), the Ipa decreased with the increase of n (curve a), indicating that the assembly of multilayer films may block or hinder the electron transfer of Fc(COOH) with electrodes to some extent. However, for {PAH/SiO2}n-xAu films with x g 1, the Ipa always increased with n (curves b-d), suggesting that the thicker {PAH/SiO2}n films with a larger n value can accommodate larger amounts of AuNPs and then improve the electron transfer of Fc(COOH) to a greater degree. Considering that the {PAH/SiO2}10-xAu films with n ) 10 showed quite a large CV response for Fc(COOH) and the assembly took relatively limited time, 10 bilayer films were usually used in this work. The great improvement of CV response of Fc(COOH) by {PAH/SiO2}n-xAu films could be used to detect Fc(COOH) at

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Figure 3. SEM top views of (A) {PAH/SiO2}10, (B) {PAH/SiO2}10-3Au, and (C) {PAH/SiO2}10-8Au films fabricated on PG disks.

Figure 4. EDX spectra of (a) {PAH/SiO2}10, (b) {PAH/SiO2}10-3Au, and (c) {PAH/SiO2}10-8Au films.

Figure 5. CVs of 0.2 mM Fc(COOH) in pH 7.0 buffers at 0.05 V s-1 for (a) PG electrode, (b) {PAH/SiO2}10, and (c) {PAH/SiO2}10-3Au films.

very low concentration. For example, when bare PG electrodes were placed in 1 µM Fc(COOH) solution, no CV peak of Fc(COOH) could be detected (Supporting Information Figure S2, curve a) because the concentration of Fc(COOH) was too low. However, for {PAH/SiO2}10-xAu films, an obvious CV peak pair of Fc(COOH) was observed in the same 1 µM Fc(COOH) solution, and as expected, the CV peak currents of

Fc(COOH) for {PAH/SiO2}10-8Au films were larger than those for {PAH/SiO2}10-3Au films (curves b and c). 3. Bioelectrocatalytic Oxidation of Glucose by GOD Using Fc(COOH) as a Mediator at {PAH/SiO2}10-xAu Film Electrodes. The enhancement of AuNPs in {PAH/SiO2}10-xAu films on CV response of Fc(COOH) in solution could be used to improve bioelectrocatalytic oxidation of glucose by GOD. It is well-known that Fc(COOH) is a good mediator in electrocatalytic oxidation of glucose by GOD,31-33 and the Fc(COOH)GOD-glucose system was used in the present work as a model to investigate the feasibility and prospect of using {PAH/SiO2}nxAu films in bioelectrocatalysis. For example, when {PAH/ SiO2}10-1Au film electrodes were placed in pH 7.0 buffers containing Fc(COOH), glucose, and GOD, a large increase in CV oxidation peak at about 0.3 V for Fc(COOH) was observed, accompanied by the decrease or even disappearance of the reduction peak (Figure 8). The oxidation peak increased with the concentration of glucose in solution. All these are characteristic of electrochemical catalysis, suggesting that the glucose can be catalytically oxidized by GOD mediated by Fc(COOH) at {PAH/SiO2}10-1Au film electrodes. The related reactions can be expressed by the following equations:43,48,49

GOD(FAD) + glucose f GOD(FADH2) + gluconolactone GOD(FADH2) + 2Fc(COOH)ox f GOD(FAD) + 2Fc(COOH)red Fc(COOH)red - e- S Fc(COOH)ox at electrode where GOD(FAD) and GOD(FADH2) represent oxidized and reduced forms of glucose oxidase, respectively. The effect of AuNPs in {PAH/SiO2}10-xAu films on CV catalytic response of glucose in the presence of GOD and

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Figure 6. (A) CVs of 0.2 mM Fc(COOH) in pH 7.0 buffers at 0.05 V s-1 for {PAH/SiO2}10-xAu films on the PG electrode with a different number of Au(III) loading and reduction cycles (x). (B) Dependence of CV oxidation peak currents (Ipa) and CV peak separation (∆Ep) of Fc(COOH) on x for {PAH/SiO2}10-xAu films.

the Ipa was obviously larger than that for {PAH/SiO2}10/PAH/ GOD films containing no AuNPs (curve c), suggesting that the AuNPs incorporated in the LbL films can also enhance the detection sensitivity of glucose when the enzyme GOD is immobilized in the films. Discussion

Figure 7. Dependence of CV oxidation peak current (Ipa) of 0.2 mM Fc(COOH) in pH 7.0 buffers at 0.05 V s-1 on the number of bilayers (n) for {PAH/SiO2}n-xAu films with different number of Au deposition cycles (x): (a) 0, (b) 1, (c) 2, and (d) 3. Data were the average of at least three parallel experiments.

Figure 8. CVs at 0.005 V s-1 in pH 7.0 buffers containing 0.2 mM Fc(COOH) and 1 mg mL-1 GOD for {PAH/SiO2}10-1Au films in the presence of (a) 0, (b) 1, (c) 5, (d) 20, and (e) 200 mM glucose.

Fc(COOH) was also investigated by changing the number of Au(III) loading and reduction cycles (x) under the same conditions (Figure 9). The CV oxidation peak (Ipa) demonstrated an increasing trend with x value, suggesting that more amounts of AuNPs deposited in the films will lead to larger quantities of Fc(COOH) oxidized and resulting in the increase of catalytic oxidation peaks of glucose. The enhancement of AuNPs deposited in the films on CV response of Fc(COOH) could be further used to improve the bioelectrocatalysis of glucose with GOD incorporated in the films. The immobilization of enzymes on the electrode surface is of great importance because it provides a foundation for fabricating electrochemical biosensors.50-52 For example, the {PAH/SiO2}10-3Au films could be further immersed in PAH (1 mg mL-1 at pH 9.0) and then GOD solution (3 mg mL-1 at pH 5.0) for 20 min to sequentially adsorb PAH and GOD on the surface, forming {PAH/SiO2}10-3Au/PAH/GOD films. When the GOD film electrode was placed in pH 7.0 buffers containing Fc(COOH) and glucose, the CV catalytic oxidation of glucose was observed (Supporting Information Figure S3, curve d), and

For {PAH/SiO2}n LbL films assembled at pH 9.0, some amine groups of PAH are “free” and not pair-linked with SiO2 nanoparticles, and these unpaired amine groups may become positively charged in Au(III) loading solutions at pH 7.0 and provide the binding sites for the negatively charged AuCl4ions.27 Furthermore, upon reduction of Au(III) in the films into AuNPs by NaBH4, these protonated amine groups of PAH can be regenerated and act as the binding sites again for AuCl4- in the next loading cycle. Therefore, the in situ synthesis of AuNPs within {PAH/SiO2}n LbL films can be repeatedly cycled and used to increase the density of AuNPs in the films. The {PAH/SiO2}n films can obstruct the Fc(COOH) probe from reaching the electrode surface to some extent because of the barrier effect and lead to the decrease of CV peak currents of the probe and increase of ∆Ep value with the number of bilayers (n) (Figure 2). However, the quite large CV response can still be observed even with n ) 10, indicating that the films have considerable permeability for the probe and the electrode surface is not completely covered by the polyelectrolytes and nanoparticles. The incorporation of AuNPs in the films demonstrates a great improvement in CV response of Fc(COOH) in comparison with not only {PAH/SiO2}10 films but also bare PG (Figure 5), and the response is further improved with the increase of Au(III) loading and reduction cycles (x) (Figure 6). Possible explanation for this improvement is discussed as follows. Conductive AuNPs can act as “electron relays” between electroactive species and underlying electrodes, which are well recognized.53-56 In the present system, AuNPs deposited in {PAH/SiO2}n films may take two different ways to act as the conductive relays. On the one hand, because the PG surface is not completely covered by the films, some AuNPs may be deposited directly on the uncovered surface of PG electrodes. The electron transfer of Fc(COOH) may take place on these conductive AuNPs. On the other hand, some fraction of the PG surface was covered by the PAH layer, and a certain amount of AuNPs may be deposited on the surface of the PAH layer. Because the PAH layer is quite thin (Table 1), the AuNPs deposited on its surface may tunnel electrons across the layer with underlying PG electrodes. The tunneling effect of AuNPs on enhancing electron transfer has been observed for various monolayer or multilayer modified electrodes.57,58 In the present work, the tunneling effect of AuNPs may lead to the transition

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Figure 9. (A) CVs at 0.005 V s-1 in pH 7.0 buffers containing 0.2 mM Fc(COOH), 200 mM glucose, and 1 mg mL-1 GOD for {PAH/SiO2}10-xAu films with different x values: (a) 0, (b) 1, (c) 3, (d) 5, and (e) 8. (B) Influence of the number of Au(III) loading and reduction cycles (x) of {PAH/SiO2}10-xAu films on CV catalytic oxidation peak currents (Ipa).

of some section of electrode surface covered by the PAH layer from electrochemical inactive to active, resulting in the improvement of CV response of Fc(COOH). The ∆Ep value of CV usually reflects the reversibility of an electrochemical system and can be used to estimate the corresponding heterogeneous electron transfer rate constant.59 After deposition of AuNPs in the {PAH/SiO2}10 films, the ∆Ep value of Fc(COOH) becomes smaller and shows a general decreasing trend with increasing the x value for {PAH/SiO2}10-xAu films (Figure 6), suggesting that incorporation of AuNPs into {PAH/SiO2}10 films greatly improves the reversibility of Fc(COOH) electrochemistry and enhances the electron transfer rate of the probe. Moreover, the deposition of AuNPs may also make the {PAH/SiO2}n films become more porous (Figure 3), and the improvement of film permeability is beneficial to the electron exchange of Fc(COOH) with PG electrodes. However, the observation that the CV peak currents of Fc(COOH) at {PAH/SiO2}10-xAu film electrodes are much higher than those at bare PG electrodes (Figures 5 and 6) cannot be well explained just by the tunneling effect. According to the literature,58 while the increase of AuNPs density on the surface of SAM-modified Au electrodes will result in the increase in number of the tunneling channels and then the increase of CV response of the electroactive probe, the maximum peak currents usually cannot exceed those at bare Au electrodes.54,58 Therefore, two possible reasons should be responsible for the observed CV behavior in the present work: (1) Fc(COOH) is adsorbed on and/or loaded into the {PAH/SiO2}10-xAu films, and the higher surface concentration of the probe in the films leads to its better CV response than that for bare PG electrodes; (2) deposition of AuNPs in the films results in the increase of effective electrode area and leads to a CV response of Fc(COOH) larger even than that at bare PG electrodes. These two explanations are discussed in the following sections, respectively. First, the influence of scan rate on CV peak currents of Fc(COOH) at {PAH/SiO2}10-3Au film electrodes was investigated and compared with that at bare PG electrodes. Both oxidation and reduction peak currents of Fc(COOH) increased linearly with the square root of scan rates in the range of 0.01 - 0.25 V s-1 for both {PAH/SiO2}10-3Au film and bare PG electrodes (Supporting Information Figure S4), suggesting that the electrode reaction of Fc(COOH) at {PAH/SiO2}10-3Au film electrodes is also a diffusion-controlled process, just like that at bare PG electrodes. Thus, the improvement of CV response of the probe in {PAH/SiO2}10-3Au films does not originate from those Fc(COOH) molecules adsorbed on and/or loaded into the films. This speculation was further supported by an experiment, in which {PAH/SiO2}10-3Au film electrodes were first placed in Fc(COOH) solution for CV scans and then transferred into blank buffers containing no Fc(COOH) for CV

Figure 10. Schematic diagram of how deposition of AuNPs in {PAH/ SiO2}n-xAu films would increase the effective electrode area.

test again. The CV response of Fc(COOH) in blank buffers was much smaller than that in Fc(COOH) solution, and continuously decreased with CV scans until to complete disappearance after five cycles (Supporting Information Figure S5). These results imply that Fc(COOH) is unable to be adsorbed on and/or incorporated into the films stably, and the improvement of CV response of Fc(COOH) for {PAH/SiO2}10-xAu films cannot be explained by accumulation of the probe within the film phase. Therefore, the remarkable increase in CV peak currents of Fc(COOH) for {PAH/SiO2}10-xAu films in comparison with bare PG electrodes is most probably attributed to the increase of effective electrode area after deposition of AuNPs in the films, as also observed in other AuNPs systems.55,60 Some of the AuNPs formed in {PAH/SiO2}10 films may be directly attached to the bare PG electrode surface, and some may be located on the surface of PAH monolayer adsorbed on the PG surface. In both cases, the AuNPs would become “active” for the Fc(COOH) probe through either the direct contact of AuNPs with PG or the tunneling effect of AuNPs. With an increase of deposition cycles, some subsequently deposited AuNPs may be attached tightly on the surface of these “active” AuNPs, and because of the conductivity of AuNPs, those followers also become “active” and act as the “electron relays” for the probe, thus resulting in the increase of effective electrode area and then greater CV response of Fc(COOH). More deposition cycles would lead to more amounts of “active” AuNPs in the films and result in higher effective electrode area. Figure 10 shows the schematic diagram of how the multiple depositions of AuNPs in the films would increase the effective electrode area. To further support this speculation, CVs of Fe(CN)63- were performed at various scan rates for different electrodes to estimate the effective electrode area. Herein we chose Fe(CN)63instead of Fc(COOH) as the electroactive probe mainly because the diffusion coefficient of Fe(CN)63- is known61,62 (D ) 7.6 × 10-6 cm2 s-1). Moreover, the independent determination of electrode area with Fe(CN)63- is more convincing. For all three studied electrodes including bare PG, {PAH/SiO2}10-3Au, and {PAH/SiO2}10-8Au films, the reduction peak current (Ipc) demonstrated good linear relationship with the square root of

Increment of Density of Au Nanoparticles the scan rate (V1/2) (Supporting Information Figure S6), indicating that the electrode process of the probe for all three types of electrodes is diffusion-controlled. From the slope of straight line of Ipc vs V1/2, the effective electrode area of different electrodes could be estimated59 and showed the sequence of {PAH/SiO2}108Au (0.34 cm2) > {PAH/SiO2}10-3Au (0.24 cm2) > bare PG (0.11 cm2). These results indicate again that the deposition of AuNPs in the {PAH/SiO2}10 films greatly increases the effective electrode area and thus leads to the remarkable increase of CV response of Fc(COOH). Conclusion After Au(III) complex ions in solution are incorporated into {PAH/SiO2}n LbL films, the chemical reduction of Au(III) will lead to the in situ formation of AuNPs in the films. This Au(III) loading and reduction procedure can be repeated for numerous cycles to enhance the density of AuNPs in the films, and the increase of AuNPs amount in the films greatly improves the electrochemistry of Fc(COOH) and the corresponding bioelectrocatalysis of glucose in the presence of GOD. While other AuNP-modified electrodes can also enhance electron transfer of electroactive probes with underlying electrodes, the present system demonstrates a prominent advantage in the increase of AuNP density in the films by repeating the deposition cycles. The improvement of CV response of Fc(COOH) is attributed to not only the tunneling effect of AuNPs formed on the surface of the PAH layer directly adsorbed on PG surface but also the increase of effective electrode area after deposition of AuNPs in the films. This model AuNP-loaded LbL film will open a new way to improve electrochemical performance of biosensors. Better understanding of the function of AuNPs in electron transfer will also be beneficial to the design and construction of this new type of electrochemical biosensors. Acknowledgment. Financial support from the National Natural Science Foundation of China (NSFC 20775009 and 20475008) is acknowledged. Supporting Information Available: Six figures showing the improvement of CV responses of Ru(NH3)63+ by AuNPs in {PAH/SiO2}10-3Au films, CVs of Fc(COOH) with low concentration at different electrodes, CVs of bioelectrocatalytic oxidation of glucose mediated by Fc(COOH) at {PAH/SiO2}10/PAH/ GOD and {PAH/SiO2}10-3Au/PAH/GOD films, dependence of CV peak currents on the square root of scan rates for Fc(COOH) and Fe(CN)63- at different electrodes, and CVs of Fc(COOH) at {PAH/SiO2}n-3Au film electrodes in Fc(COOH) and blank solutions. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Willner, I. Science 2002, 98, 2407. (2) Daniel, M.-C.; Astruc, D. Chem. ReV. 2004, 104, 293. (3) Shipway, A. N.; Lahav, M.; Willner, I. AdV. Mater. 2000, 12, 993. (4) Liu, S.; Leech, D.; Ju, H. Anal. Lett. 2003, 36, 1. (5) Guo, S.; Wang, E. Anal. Chim. Acta 2007, 598, 181. (6) Xiao, Y.; Patolsky, F.; Katz, E.; Hainfeld, J. F.; Willner, I. Science 2003, 299, 1877. (7) Jia, J.; Wang, B.; Wu, A.; Cheng, G.; Li, Z.; Dong, S. Anal. Chem. 2002, 74, 2217. (8) Guo, X.; Zheng, D.; Hu, N. J. Phys. Chem. B 2008, 112, 15513. (9) Yan, Y.-M.; Tel-Vered, R.; Yehezkeli, O.; Cheglakov, Z.; Willner, I. AdV. Mater. 2008, 20, 2365. (10) He, J.; Kunitake, I. In Nanocrystals Forming Mesoscopic Structures; Pileni, M. P., Ed.; Wiley-VCH: Weinheim, 2005; p 91. (11) Decher, G. Science 1997, 277, 1232.

J. Phys. Chem. C, Vol. 113, No. 22, 2009 9837 (12) Decher, G.; Hong, J. D.; Schmitt, J. Thin Solid Films 1992, 210/ 211, 831. (13) Yang, W.; Wang, J.; Zhao, S.; Sun, Y.; Sun, C. Electrochem. Commun. 2006, 8, 665. (14) Zhang, S.; Yang, W.; Niu, Y.; Li, Y.; Zhang, M.; Sun, C. Anal. Bioanal. Chem. 2006, 384, 736. (15) Chen, S.; Yuan, R.; Chai, Y.; Yin, B.; Xu, Y. Electroanalysis 2008, 19, 2141. (16) Liu, Y.; Geng, T.; Gao, J. Microchim. Acta 2008, 161, 241. (17) Yu, A.; Liang, Z.; Cho, J.; Caruso, F. Nano Lett. 2003, 9, 1203. (18) Zhang, H.; Lu, H.; Hu, N. J. Phys. Chem. B 2006, 110, 2171. (19) Zhang, H.; Hu, N. J. Phys. Chem. B 2007, 111, 10583. (20) Shi, X.; Shen, M.; Mo¨hwald, H. Prog. Polym. Sci. 2004, 29, 987. (21) Joly, S.; Kane, R.; Radzilowski, L.; Wang, T.; Wu, A.; Cohen, R. E.; Rubner, M. F. Langmuir 2000, 16, 1354. (22) Wang, T. C.; Chen, B.; Rubner, M. F.; Cohen, R. E. Langmuir 2001, 17, 6610. (23) Wang, T. C.; Rubner, M. F.; Cohen, R. E. Chem. Mater. 2003, 15, 299. (24) Miyazaki, Y.; Shiratori, S. Thin Solid Films 2006, 499, 29. (25) Logar, M.; Janeˇar, B.; Suvorov, D.; Kostanjsˇek, R. Nanotechnology 2007, 18, 325601. (26) Wang, T. C.; Rubner, M. F.; Cohen, R. E. Langmuir 2002, 18, 3370. (27) Chia, K.-K.; Cohen, R. E.; Rubner, M. F. Chem. Mater. 2008, 20, 6756. (28) He, P.; Hu, N.; Rusling, J. F. Langmuir 2004, 20, 722. (29) Guo, X.; Zhang, H.; Hu, N. Nanotechnology 2008, 19, 055709. (30) Liu, H.; Hu, N. Electroanalysis 2007, 19, 884. (31) Badia, A.; Carlini, R.; English, A. M. J. Am. Chem. Soc. 1993, 115, 7053. (32) Koide, S.; Yokoyama, K. J. Electroanal. Chem. 1999, 468, 193. (33) Cass, A. E. G.; Davis, G.; Francis, G. D.; Hill, H. A. O.; Aston, W. J.; Higgins, J. I.; Plotkin, E. V.; Scott, L. D. L.; Turner, A. P. F. Anal. Chem. 1984, 56, 667. (34) Sauerbrey, G. Z. Phys. 1959, 155, 206. (35) Lu, H.; Hu, N. J. Phys. Chem. B 2007, 111, 1984. (36) Lvov, Y.; Ariga, K.; Onda, M.; Ichinose, I.; Kunitake, T. Langmuir 1997, 13, 6195. (37) Brandrup, J.; Immergut, E. H. Polymer Handbook; Wiley: New York, 1975. (38) Franks, G. V. J. Colloid Interface Sci. 2002, 249, 44. (39) Fisher, M. L.; Colic, M.; Rao, M. P.; Lange, F. F. J. Am. Ceram. Soc. 2001, 84, 713. (40) Burke, S. E.; Barrett, C. J. Langmuir 2003, 19, 3297. (41) Park, M. K.; Deng, S.; Advincula, R. C. J. Am. Chem. Soc. 2004, 126, 13723. (42) Burke, S. E.; Barrett, C. J. Macromolecules 2004, 37, 5375. (43) Murthy, A. S. N.; Sharma, J. Anal. Chim. Acta 1998, 363, 215. (44) Wu, Y.; Fan, R.; Yang, P. Nano Lett. 2002, 2, 83. (45) Tshabalala, M. A.; Sung, L. P. J. Coat. Technol. Res. 2007, 4, 483. (46) Qian, L.; Yang, X. J. Phys. Chem. B 2006, 110, 16672. (47) Wang, B.; Chen, K.; Jiang, S.; Reincke, F.; Tong, W.; Wang, D.; Gao, C. Biomacromolecules 2006, 7, 1203. (48) Liao, C. W.; Chou, J. C.; Sun, T. P.; Hsiung, S. K.; Hsieh, J. H. Sens. Actuators, B 2007, 123, 720. (49) Blonder, R.; Katz, E.; Willner, I.; Wray, V.; Bu¨ckmann, A. F. J. Am. Chem. Soc. 1997, 119, 11747. (50) Scouten, W. H.; Luong, J. H. T.; Brown, R. S. Trends. Biotechnol. 1995, 13, 178. (51) Lojou, E´.; Bianco, P. Electroanalysis 2004, 16, 13. (52) Wei, X.; Cruz, J.; Gorski, W. Anal. Chem. 2002, 74, 5039. (53) Brown, K. R.; Fox, A. P.; Natan, M. J. J. Am. Chem. Soc. 1996, 118, 1154. (54) Lu, M.; Li, X. H.; Yu, B. Z.; Li, H. L. J. Colloid Interface Sci. 2002, 248, 376. (55) Cheng, W.; Dong, S.; Wang, E. Langmuir 2002, 18, 9947. (56) Zhang, J.; Oyama, M. Anal. Chim. Acta 2005, 540, 299. (57) Bharathi, S.; Nogami, M.; Ikeda, S. Langmuir 2001, 17, 1. (58) Diao, P.; Guo, M.; Zhang, Q. J. Phys. Chem. C 2008, 112, 7036. (59) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamental and Applications, 2nd ed.; Wiley: New York, 2001. (60) Musick, M. D.; Pena, D. J.; Botsko, S. L.; McEvoy, T. M.; Richardson, J. N.; Natan, M. J. Langmuir 1999, 15, 844. (61) Yun, Y.; Dong, Z.; Shanov, V. N.; Doepke, A.; Heineman, W. R.; Halsall, H. B.; Bhattacharya, A.; Wong, D. K. Y.; Schulz, M. Sens. Actuators, B 2008, 133, 208. (62) Schro¨der, U.; Wadhawan, J. D.; Compton, R. G.; Marken, F.; Suarez, P. A. Z.; Consorti, C. S.; de Souza, R. F.; Dupont, J. New J. Chem. 2000, 24, 1009.

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