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Colloids and Surfaces A: Physicochem. Eng. Aspects 317 (2008) 394–399

Layer-by-layer assembly of carbon nanotubes and Prussian bluenanoparticles: A potential tool for biosensing devices

Liang Wang, Shaojun Guo, Xiaoge Hu, Shaojun Dong ∗State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry,

Chinese Academy of Sciences, Changchun, Jilin 130022, China

Received 3 October 2007; received in revised form 2 November 2007; accepted 6 November 2007Available online 22 November 2007

bstract

A promising method for assembling carbon nanotubes (CNTs) and poly(diallyldimethylammonium chloride) protected Prussian blue nanopar-icles (P-PB) to form three-dimensional (3D) nanostructured films is proposed. The electrostatic interaction, combined with layer-by-layerelf-assembly (LBL), between negatively charged CNTs and positively charged P-PB is strong enough to drive the formation of the 3D nanos-ructured films. Thus, prepared multilayer films were characterized by ultraviolet–visible–near-infrared spectroscopy (UV–vis–NIR), scanninglectron microscopy (SEM) and cyclic voltammetry (CV). Regular growth of the mutilayer films is monitored by UV–vis–NIR and CV. SEMrovides the morphology of the multilayer films. The 3D multilayer films exhibit good electrocatalytic activity for the reduction of H2O2. This is a

ery general and powerful technique for the assembling 3D nanostructured films containing carbon nanotubes and nanoparticles. This assemblingethod introduces opportunities for the incorporation of various functionalities into nanotube devices, which, in turn, opens up the possibility of

uilding more complex multicomponent nanostructures with potential application, such as biosensing devices. 2007 Elsevier B.V. All rights reserved.

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eywords: Carbon nanotubes; Prussian blue nanoparticles; Layer-by-layer; Ele

. Introduction

Carbon nanotubes (CNTs), a unique class of one-dimensionalunctional structures, are considered as promising buildinglocks for nanoscience and nanotechnology because of theirigh surface area, good mechanical strength and rich electronicroperties [1–6]. To exploit the potential applications in futureanodevices, it is necessary to develop versatile approaches tossemble or integrate CNTs onto solid surfaces, especially, aomogeneous CNTs thin film is required. However, the tech-iques employed often suffer from nonuniform film coverager a lack of control of the film properties or do not easily allowor the generation of complex film architectures with severalifferent components [7]. Strategies for functionalizing CNTs

nd limitations in assembly methods are important barriers forhe pursuit of potential applications of CNTs. Therefore, facilend effective methods for controlled CNTs thin-film fabrication

∗ Corresponding author. Tel.: +86 431 85262101; fax: +86 431 85689711.E-mail address: dongsj@ciac.jl.cn (S. Dong).

saestud

927-7757/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfa.2007.11.019

atalysis

re still needed. There have been a number of methods on thereparation of CNTs thin films, including solution casting [8],lectrophoretic [9], Langmuir–Blodgett deposition [10], adsorp-ion on modified surfaces [11], stretching polymer films loadedith CNTs [12] and self-assembly [13], but the layer-by-layer

LBL) assembly technique is perhaps the most versatile andommon method today to fabricate robust and uniform thin filmf CNTs [14,15]. The introduction of nanoparticles (NPs) tohe CNTs films can generate new nanostructures with excellentunctions in the fields of optics, electronics, and electrocatalysis,hich are very attractive for practical applications [16].Prussian blue (PB) and its analogues are the prototype of

number of polynuclear transition-metal hexacyanometalatesaving an open, zeolite-like structure. Due to its high activity andelectivity toward the reduction of hydrogen peroxide, PB is usu-lly considered as an “artificial enzyme peroxidase” and has beenxtensively used in the construction of electrochemical biosen-

ors [17,18]. Although PB has been intensively investigated inhe form of thin polycrystalline electrodeposited films [19], these of PB nanostructures for the creation of electrochemicalevices is an extremely promising prospect. The nanostructures

hysicochem. Eng. Aspects 317 (2008) 394–399 395

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fidmwmla(Buehler, Lake Bluff, IL), followed by sonicating in water andethanol. Then, the Au electrode was electrochemically cleanedby potential cycling between 1.5 and −0.2 V (versus Ag/AgCl)in 0.5 M H2SO4 until the cyclic voltammogram characteristic

L. Wang et al. / Colloids and Surfaces A: P

ive rise to porous, high-surface area electrodes, where the localicroenvironment can be controlled by the crosslinking ele-ents and may lead to specific and selective interactions with

ubstrates [20,21]. Various methodologies have been used forhe tailoring of PB nanoparticles on electrode surfaces, in whichlectrostatic assembly is a very general and powerful techniqueor the assembling three-dimensional PB nanoparticles.

Herein a promising method for assembling CNTs andoly(diallyldimethylammonium chloride) protected Prussianlue nanoparticles (P-PB) to form three-dimensional (3D)anostructured films is proposed. The electrostatic interaction,ombined with LBL, between negatively charged CNTs and pos-tively charged P-PB is strong enough to drive the formationf the 3D nanostructured films. The prepared 3D nanostruc-ured films containing P-PB show good electrocatalytic activityn the reduction of H2O2. This assembling method introducespportunities for the incorporation of various functionalitiesnto nanotube devices, which, in turn, opens up the possibil-ty of building more complex multicomponent nanostructuresith potential application, such as biosensing devices.

. Experimental

.1. Reagents

Multi-walled carbon nanotubes (MWNTs) were purchasedrom Nanoport Co., Ltd. (Shenzhen, China). CystamineCYST), poly(diallyldimethylammonium chloride) (PDDA)200,000–350,000 Mw) and poly(sodium styrenesulfonate)PSS) (70,000 Mw) were purchased from Aldrich and used aseceived. K3[Fe(CN)6], FeCl2·4H2O and other chemicals weref analytical grade and were purchased from Beijing Chemicaleagent Company (Beijing, China). All solutions were preparedith deionized water treated in a Millipore water purification

ystem (Millipore Corp.).

.2. Synthesis of PB nanoparticles protected by PDDA

PDDA protected Prussian blue nanoparticles were pre-ared according to a previously published procedure [22].riefly, 4 mL of a 0.025 mol L−1 K3[Fe(CN)6] solution was

lowly added to 16 mL of a 6.25 mmol L−1 FeCl2·4H2O and2.5 mol L−1 PDDA solution under vigorous stirring at roomemperature. The resulting dark blue colloidal solution wassed as a P-PB stock solution. The average diameter of P-PBas about 4 nm, which was measured by transmission elec-

ron microscopy (TEM) (Fig. 1A). Residual PDDA polymeras removed by high-speed centrifugation and the complex was

insed with water for at least three times. The collected complexas redispersed in 5 mL water with mild sonicating to producestable solution of the complex, which was sonicated for 5 min

mmediately before preparing the films.

.3. Preparation of {P-PB/CNTs}n multilayer films

MWNTs were purified using a well-established method14,23], and were characterized by SEM (Fig. 2). Thus, puri-

ig. 1. (A) TEM image of P-PB. (B) UV–vis–NIR absorption spectra of P-PBolloidal solution.

ed MWNTs were rich in negative charges and can be wellispersed in water. Ordinary LBL assembly of CNTs and P-PBultilayer films was carried out as follows. Au disk electrodeith a diameter of 2 mm was used as the substrate to grow theultilayer films. Prior to preparation procedure of the multi-

ayer films, the electrode was polished with diamond pastesnd an alumina slurry down to 0.05 �m on a polishing cloth

Fig. 2. SEM image of purified MWNTs.

396 L. Wang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 317 (2008) 394–399

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Scheme 1. Schematic representation of the assem

or a clean Au electrode was obtained. The cleaned gold elec-rodes were immersed in an aqueous solution of 1 mM CYST forh to form CYST monolayer on gold electrode and introduceositive charges on the surface [24]. The resulting electrode wasashed with copious amount of water and dried with nitrogen

tream. The modified gold electrodes with a positively chargedurface were treated by a PSS aqueous solution (3 mg/mL) andPDDA aqueous solution (3 mg/mL) for 20 min, respectively,

efore LBL assembly. The multilayer films were grown on theu electrodes by alternately dipping the positively charged elec-

rodes into the negatively charged CNTs solution (0.2 mg/mL)nd positively charged P-PB solution for 20 min, respectively.uring each assembling interval, the electrodes were first care-

ully rinsed with distilled water after each dipping step to removehe excess of assembly materials, and then dried with nitrogenas. This sequence was repeated to obtain the desired num-ers of bilayer designated as {P-PB/CNTs}n multilayer films.he “bottom-up” assembling process is schematically shown incheme 1.

.4. Characterization of the {P-PB/CNTs}n multilayerlms

UV–vis–NIR absorption spectra were recorded using a Cary00 Scan UV–vis–NIR spectrometer (Varian Co.) on a quartzlide. The quartz slide was first cleaned with a piranha solutiona 1:3 mixture of 30% H2O2 and concentrated H2SO4) and thenhoroughly rinsed with water. (Caution: piranha solution reactsiolently with almost any organic material and should be han-led with extreme care!) After the treatment, quartz slides wereich in negative charges. The cleaned pieces were kept in abso-ute methanol and were rinsed with water just before use. Theleaned quartz slides were treated by a PDDA aqueous solu-ion (3 mg/mL) for 20 min to obtain a positively charged surfaceefore LBL assembly. The CNTs and P-PB films were alterna-ively assembled on the quartz slide, until the desired multilayersere obtained.The morphology of the formed {P-PB/CNTs}n multilayer

lms was characterized with a PHILIPS XL-30 ESEM at anccelerating voltage of 20 kV on an indium tin oxide (ITO) plate.

efore assembled, the ITO plate was cleaned by sequentially

onication in acetone, 10% KOH and distilled water, each for0 min. The procedure to assemble {P-PB/CNTs}n multilayerlms on ITO plate was the same as that on quartz slide. TEM

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process of the {P-PB/CNTs}n multilayer films.

easurements were made on a JEOL 2000 transmission electronicroscope operated at an accelerating voltage of 200 kV.Electrochemical experiments were performed with a CHI

32 electrochemical analyzer (CH Instruments, Chenhua Co.,hanghai, China). A conventional three-electrode cell was used,

ncluding a Ag/AgCl (saturated KCl) electrode as reference elec-rode, a platinum wire counter electrode and a modified goldorking electrode.

. Results and discussion

.1. UV–vis–NIR absorption spectroscopy characterizationf the {P-PB/CNTs}n multilayer films

The growth of the multilayer films prepared by the LBLethod on a quartz slide was followed by UV–vis–NIR absorp-

ion spectroscopy by recording successive spectra after theeposition of each P-PB/CNTs bilayer. As shown in Fig. 1B,he as-prepared P-PB have a characteristic of the adsorptiont 710 nm, corresponding to the mixed-valence charge-transferbsorbance of the polymeric [Fe(II)–C–N–Fe(III)]. This result isn accordance with the literature [20]. CNTs also have a charac-eristic of the adsorption at about 260 nm [14]. The characteristicf the adsorptions of CNTs and P-PB are usually to be used toollow the uniform growth of multilayer films.

As a result of the absorption intensity being proportional tohe concentration of the deposition material with characteristicbsorption peaks in the UV–vis–NIR region, the growth processf the multilayer films can be monitored by UV–vis–NIR spec-roscopy. Fig. 3 shows the UV–vis–NIR absorption spectra ofhe {P-PB/CNTs}n multilayer films with different bilayer num-er, and P-PB existed in the outmost layer. Spectra of PDDAere featureless in the wavelength range employed. Whereas,

he CNTs multilayer films show a remarkable absorption at60 nm, characteristic of the adsorption of the assembled CNTss described previously [14,25]. Simultaneously, the P-PB mul-ilayer films also show an increasing absorption at 710 nm,hich is characteristic of the adsorption of the assembled P-PBultilayer films as described previously [21,26–28]. The clear

ncrease in the absorbance at 260 nm for CNTs and 710 nm for

-PB versus the bilayer number absorbance with the assem-ling step is indicative of the film deposition on substrate andhe linearity between the absorbance with the bilayer numberf the {P-PB/CNTs}n (inset in Fig. 3) further suggests that the

L. Wang et al. / Colloids and Surfaces A: Physico

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ig. 3. UV–vis–NIR absorbance for {P-PB/CNTs}n layer-by-layer assembledn a quartz slide (P-PB as the outmost layer). The inset shows plots of thebsorbance at 260 nm for CNTs and 710 nm for P-PB vs. the bilayer number (n).

rowth of the {P-PB/CNTs}n multilayer films in each assem-ly step is uniform, which was further confirmed with the SEMemonstrated as below. The weak absorption at 710 nm for P-

B, compared with the intensity of the absorption at 260 nmor CNTs, is due to the amount of assembled P-PB was relativeess, which leads to weakly current intensity and is characterizedsing cyclic voltammetry as demonstrated following.

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ig. 4. SEM images of {P-PB/CNTs}n multilayer films assembled onto ITO plate (Pcale bar: 1 �M.

chem. Eng. Aspects 317 (2008) 394–399 397

.2. SEM characterization of the {P-PB/CNTs}n multilayerlms

SEM is a useful tool for providing the morphology ofultilayer films. The assembly and morphology of the {P-B/CNTs}n multilayer films were also characterized by SEM.ig. 4 displays typical SEM images from one to five bilayer ofP-PB/CNTs}n films assembled onto ITO plates. These SEMrofiles demonstrate that an obvious increase in carbon nan-tubes coverage was observed with increasing number of filmssembly procedures, and thus, one may expect to obtain aomogeneous, porous, and 3D {P-PB/CNTs}n multilayer filmsith a large surface area provided the assembly procedure is

epeated many times. The average diameter of P-PB is aboutnm, which is too small to be obviously observed in the scale ofig. 4. But cyclic voltammetry, as demonstrated below, testified

he existing of the P-PB, in which the electrochemical responsef the corresponding P-PB was distinct.

.3. Electrochemical characterization of theP-PB/CNTs}n multilayer films

Cyclic voltammetry has been proven to be a powerful toolor the electrochemical characterization of the layer-by-layerssembly of electroactive species [26] and has been used inhe present work to characterize the {P-PB/CNTs}n multilayer

-PB as the outmost layer). (a) n = 1, (b) n = 2, (c) n = 3, (d) n = 4 and (e) n = 5.

398 L. Wang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 317 (2008) 394–399

Fig. 5. (A) Cyclic voltammograms of {P-PB/CNTs} multilayer films withnrb

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lms. The electrochemistry of the {P-PB/CNTs}n multilayerlms show a pair of redox waves; therefore, a cyclic voltam-etric study of the {P-PB/CNTs}n multilayer films modified

lectrode was performed during the assembly process. Fig. 5Ahows the cyclic voltammograms of the {P-PB/CNTs}n multi-ayer films modified electrode in phosphate buffer solution ofH 7.4, containing 0.1 M KCl, at a scan rate of 50 mV s−1. Inhe potential range from −0.05 to 0.45 V, there is a pair of redoxeaks centered at ca. 0.2 V. From the formal potential and thencreasing current along with the growing bilayer, it was con-rmed that the redox peaks are attributed to PB, corresponding

o Prussian blue to Prussian white (PW) conversion as describedreviously [26–28]. This suggested that the electrochemicalroperties of PB are not changed in the multilayer films.

Furthermore, the redox peak currents increased with thessembly of PB nanoparticles, which indicated that the {P-B/CNTs}n multilayer films were formed. The plot of the

educed current to the bilayer numbers is shown in Fig. 5B.

nearly linear increase of current to bilayer numbers wasbserved. It further confirmed that similar amount of P-PB wasbsorbed for each assembly, which was consistent with the t

ig. 6. Cyclic voltammograms of {P-PB/CNTs}5 multilayer films modifiedlectrode in phosphate buffer solution of pH 7.4, containing 0.1 M KCl, withouta) and with 50 mmol L−1 H2O2 (b). Scan rate = 50 mV s−1.

btained results based on above absorption measurements ofhe LBL multilayers. The current intensity of the voltammetriceaks increases linearly with the amount of bilayer, putting invidence that P-PB are electrically connected to each other dueo the well-known interpenetration phenomenon among layershat allows charge transfer through an electron-hopping pro-ess [20]. The electron-hopping process of PB and the stabilitynable the {P-PB/CNTs}n multilayer films to be a potential toolor biosensing devices.

.4. Potential biosensing application of the {P-PB/CNTs}n

ultilayer films

Electrocatalysis towards H2O2 reduction was also studiedo extent the potential application of the {P-PB/CNTs}n multi-ayer films. As demonstrated previously, Prussian blue can belectrochemically reduced to Prussian white, which is capablef catalysing the reduction of H2O2 at low potentials. Fig. 6hows the cyclic voltammograms of the {P-PB/CNTs}n multi-ayer films modified electrode in an unstirred 0.1 M phosphateuffer containing 0.1 M KCl without (Fig. 6a) and with (Fig. 6b)0 mM H2O2. In the presence of 50 mM H2O2, the reduction cur-ent obviously increased and the oxidation current intensivelyecreased. It can be described as follows. In the negative poten-ial range, P-PB film can reduce to its reduction state PW, ashown in Eq. (1):

Fe(III)[Fe(II)(CN)6](PB)

+ e− + K+ → K2Fe(II)[Fe(II)(CN)6](PW)

(1)

PW has the catalytic activity for the reduction of H2O2, ashown in Eq. (2):

2K2Fe(II)[Fe(II)(CN)6] + H2O2

→ 2KFe(III)[Fe(II)(CN)6] + 2OH− + 2K+ (2)

Therefore, PB acts as an electron-transfer mediator betweenhe electrode and H2O2 [21]. Besides that, the synergistic effect

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L. Wang et al. / Colloids and Surfaces A: P

f PB and CNTs has also contribution to the electrochemicaleduction of H2O2 [17]. It is confirmed that the {P-PB/CNTs}n

ultilayer films could be used for determination of H2O2 orabrication of biosensors. Due to the less assembled amount of-PB, the {P-PB/CNTs}5 multilayer films did not show highlyatalytic activity toward the reduction of H2O2 (Fig. 6). But,y simply choosing different cycles in the LBL assembly pro-ess, the film thickness and the amount of assembled P-PB wereeadily adjusted, while tuning the electrocatalytic activity toward

2O2 reduction. This advantage make the assembling techniquef the {P-PB/CNTs}n multilayer films could be a potential andromising tool for biosensing devices.

. Conclusion

A promising method based on electrostatic layer-by-layerechnique for assembling negatively charged carbon nanotubesnd positively charged PDDA protected PB nanoparticles is pro-osed. Regular growth of the mutilayer films is characterizedy UV–vis–NIR, SEM and CV. The {P-PB/CNTs}n multilayerlms exhibit good electrocatalytic activity for the reduction of2O2. Furthermore, it is could be expected that the electrocat-

lytic activity of the films could be further tailored by simplyhoosing different cycles in the LBL process. Of central inter-st here is to fabricate and characterize the {P-PB/CNTs}n

ultilayer films. The further applications of such assemblingechnique in the development of biosensors are currently underay.

cknowledgement

This research was supported by National Natural Scienceoundation of China (nos. 20575064 and 20675076).

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