Ind ian Journal of Chemistry Vol. 44A, May 2005. pp. 950-955

Functionalization of carbon nanotubes for direct electrochemistry of horseradish peroxidase

Wei Zheng l, Qingfen Lil , Yiming Yan 2, Lei Su2 & Lanqun Ma02.*

[School of M echanical and Electrical Engineering. Harbin Engineering Uni versity. Harbin 15000 I , PR China lCenter for M olecular Sc ience. Institute of Chemistry. The Chinese Academy of Sciences, Beijing 100080. PR China

Email: Iqmao@iccas.ac.cn

Received 9 November 2004

New strategies for functi onali za ti on of carbon nanotubes (CNTs) for facilitating direct electron transfer of horseradi sh perox idase (HRP) are reported. The CNTs are covalently functionali zed with hyd roxy l and carboxy l groups and noncova lentl y functiona li zed wi th block copolymer (Pluronic PI 23) . The functionali zed CNTs are found to possess all improved solubility in aqueous solution and may havc all enhanced biocompatibility w ith biomacromolecules such as proteins and enzymes. M oreover. all the functionali zed CNTs have been demonstrate for the first time to be capable of facilitating the di rec t electron transfer of HRP. ill which the Iloncova lelltly Pl 23- functi onali zed CNTs are showll 10 be advantageous over those cova lentl y fUll cti onali zed w ith - OH and -COOH groups. The catal yti c activities of I ~ RP adsorbed onlo the PI 23- functi onali zed CNTs towards the reductions of dioxygen (0]) and hydrogen perox ide (H20 l ) are also discussed.

II'C Code: Int. CI.7 B82 B: C25B

The preparation and basic studies on carbon nanotubes (CNTs) are being carried out because of their unique elec tronic, structural and mechanical properti es 1.2. These attracti ve properties of the CNTs enable them to be particularly useful for practica l app li cat ions such as for the development of nanodev ices, quantum wires, ultrahigh-strength engineering fibers and sensors3

.4. Recent studies show that the CNTs possess distinct electrochemical properties from other carbon-based materials, e.g., glassy carbon, graphite and diamond. The nanotubes have open ends and sidewa lls that may respect i vely hehave like edged and basal plane graphite elec trochemicall y, and possess beller electrocataly tic acti vity and higher surface area compared with other kinds of carbon-based material s5

.7

. The untque propert ies of CNTs make them parti cularly use ful for vari ous electrochemi cal investi gation s, e.g. , elec trocatalys is. direc t elec trochemistry of protein:, and fabricati on of elec trochemica l sensors and b· X- 15 0 .. . CNT tosensors . ur recent tn vesttgat tons on electrochemi stry are primaril y focused on the devc lopmen t of CNT-based bioe lec tron ic nallocleviccs such as biosensors and hiofuel ce ll s, mainly by virtlle

"

I . . ,. h C T 16- I Q o t le altraclt ve propert tes 0 t 12 S .

combinat ion of the str iking electrochemical properti es or the CNTs wi th the spec ific enzy lllc-

substrate reacti vity of the biomacromol ecu les (e.g. , proteins and enzymes) could y ield funct ional nanohybrid systems. This may pave an effecti ve way for the development of new bioel ec tronic nanodev ices, which is largely limited by : (i) the CNTs tend to aggregate in most so lvents, rendering difficulties in manipulation of the C Ts for pract ica l applications and their integrati on w ith biomacromolecu les20

; ( i i) the electronic communication between the proteins and the CNTs, which is a critical step for developing redox protein­based bioelec tronic nanodeviees, is rather slow at the pristine CNTs2t -D ; and, ( iii ) The strong interact ions between the proteins and the CNTs substantiall y distort the protei ns, leadi ng to the loss of thei r biocatalytie acttVltI es toward subs tra t es2~. Using horseradi sh peroxidase (HRP) and single- walled carbon nanotubes (S WNTs), we have tri ed to address these limitations by rati onall y funct ionali zing the CNTs through covalent and noneovalent ways so as to fac ilitate direc t elec tron ic commun icati on between the protei ns and the CNT s. These approaches may offer a straightforward way LO bioelec tron ic nanodev ices such as elec trochem ica l biosensors dnd enzy me-based biofu el ce ll s and wou ld be ve ry usefu l for investigating prote in elec tron trans fer at n<tno­interfaces.

ZHENG ('/ (fl. : FUNCTIONALIZATION OF CARBO NANOTUBES 95 1

Materials and Methods CNTs (d ia.30-40 nm. purity>95%. length 0.5-40

ILm) were purchased from Shenzhen Nanotech Port Co., Ltd (Shenzhen. Ch ina). Prior to use, the CNTs were purified by reflux ing the as-received CNTs in 2.6 M HNO, for 36 h. Covalent sidewall func ti ona li zation of the CNTs wi th hydroxyl groups was performed as already reponed25 Briefly, the CNTs ( 10 III g) and potassium hydroxide (200 mg) were weighted into a stain less steel capsule containing a milling ball. Then. the capsule was vigorously shaken for '2 h in air at room temperature. The reaction mixture was disso lved in 10 mL of distilled water and repeated ly precipitated into methanol to ensure complete removal of potassium hydrox ide residue. The obtained CNTs functionalized with multiple hydroxyl groups (CNT-O Hs) were highly hyd rophilic and cou ld be readily dispersed in distilled water and ethanol. The CNTs sidewall functionali zed with carboxy l groups (CNT-COOHs) were received as a gi ft from Dr. Zhengzhong Yang at Institute of Chemistry , CAS. Structures of the CNT-OHs, CNT­COOHs and Pluronic P 123 are shown in Scheme I .

Horseradi sh peroxidase (HRP. m.WI. 42100) and 1-ethyl-3-(3-di methy lami nopropy l) carbod i imide hydrochloride (EDC) were purchased from Sigma and used without further purification. Pluronic PI23 (E02oP07oE02Ih m.wl. 5800) was purchased from Beijing Chemical Co (Beijing, Ch ina). Other chemica ls were of analytical grade or higher and used as received. Aq ueous solutions were prepared with doubly disti li ed water.

Glassy carbon (GC, 3 mm-diameter) elec trodes purchased from Bioanalytical Systems lnc. (BAS, West Lafayette, IN) were first polished with emery paper (# 2000), 0.3 and 0.05 11m alumina slurry on a woolen cloth , and then cleaned under bath sonica ti on for 10 min . and finally rinsed thoroughly with distilled water.

CNT-OHs or CNT-COOHs ( 1.0 mg)were dispersed into I mL distilled water and the mixture was sonica ted to give a homogeneous and black dispersion, which was stable for at leas t two weeks. The dispersion (4 ILL) was dip-coated onto the GC electrodes and the electrodes (denoted as CNT­OH/GC and CNT-COO H/GC) were air-dried. HRP was adsorbed onto the CNT-OH/GC electrodes by immersing the as-prepared CNT -O H/GC electrodes into 0.10 M phosphate buffer (PH 7.0) containing 20 mg/mL HRP for 8 h. After dry ing in air, the

electrodes (HRP/CNT-O H/GC) were coated with 10 ilL of 0.5% Nafion so lution to improve the electrode stabi lity. For the preparation of HRP/CNT-COOH/GC electrodes, a mixture consisting of 1.0 mg/mL CNT­COOHs, 10 mg/mL HRP and 8 mM EDC was first prepared. A n aliquot (4 ILL) of the resulting mixture was then coated on GC elec trodes. The electrodes were finally air-dried and rinsed with distill ed water.

For the preparation of HRP/CNT-P I 23/GC electrodes, the CNTs were dispersed into OAO mM aqueous so lution of P 123 and the mixture was sonicated to give a homogenous black dispersion contai ning I mg/mL of the CNT:~ . The dispersion was stable fo r about one month . An aliquot (4 ~lL) of the dispersion was dip-coated onto GC electrodes and the electrodes (CNT- PI 23/GC) were rin sed with distilled water and air-dried. HRP was immobilized on the CNT -P1 23/GC electrodes by inlnlersing the electrodes into 20 mg/mL HRP so lution containing OAO mM PI 23 for 8 h. The electrodes (HRP/C T­PI23/GC) were rinsed w ith distilled water prior to use. For compari son, the pristine CNTs without any f unctionali zati on were dispersed into DMF and 4 pL of the dispersion was coated on GC electrode. The elec trodes (CNT/GC) were air-dried, rin sed with distilled water, and immersed into 20 mg/mL HRP solution for 8 h. The resulting electrodes (HRP/CNT/GC) were rinsed w ith distilled water to remove non-adsorbed H RP before electrochemical measurements.

Electrochemical measurements were carried out with a computer-controlled CHI660A Electrochemical Analyzer (CHI, Austin , USA) in a conventional and two-compal1ment cell. The modified GC electrodes were used as working electrode and a platinum spi ral wire as counter electrode. All potentials were referred to a KCI-saturNed Agi AgCI electrode. Phosphate buffer (0. 10 M. pH 7.0) was used as supp0l1ing electrolyte. The electrolyte was deoxygenated by purging pure nitrogen into the solution for more than 30 min. and nitrogen gas was kept flowing over the solution during the electrochemical measurements. All electrochemical measurements were pelformed at room temperature.

Results and Discussion Solubility and biocompatibility of the functionalized CNTs

The use of carbon nanotubes to develop new bioelectronic nanodevices such as electrochemical biosensors and enzyme-based biofuel cells requ ires the CNTs to have a good solubility for convenient manipulation . ft is known that the CNTs easi ly

952 INDIAN.I CHEM. SEC A. MA Y 2005

aggregate and have poor so lubility in most solvents, which makes it difficult to manipulate them for various appli cations26. Homogeneously dispersed and well­separated single nanotubes are required for the development of bioelectronic nanodevices since most of the well dispersed functional nanotubes are electrochemica lly accessible to analytc and can be thus used for biosensing units, y ielding a high signal output. We found that the strategies demonstrated here lo r the functionali zati on of the CNTs essentiall y so lubili zed the C Ts in aqucous solution and the rcsulting aqueous dispersions were quite stable.

On the other hand, the strong interactions between the CNTs and biomacromolecules (enzymes and proteins) esscntially distort the biomacromolecules and thereby decrease their biocatalytic acti v it/·2~ . Thus, the biocoillpatibility of the CNTs w ith the enzymes and proteins used for specilic recognition toward substrate constitutes another key point to bioelectronic nanodevices. To meet such a requirement, we have tried to chemically functionalize the C Ts through both covalent and noncovalent ways. In the covalent way. we intentionally introduce oxygenated groups sLlch as hydroxy l and carboxyl groups on the sidewall of the C Ts since the sidewall functionali zation of covalently bound moieti es. (-OH and -COOH groups in this case). to the CNT framework inev itably destroys the inherent electronic structure of the nanotubes. i.e .. change the .Ii carbons into Spl hybridization, while maintaining the tubular structure of the nanotubes25 Moreover, such covalent sidewall functionali zation of the nanotubes with - OH and -COOH groups substantially improves their hydrophilic property. These changes are believed to weaken the interactions (both electronic and hydrophobic) between the CNTs and the proteins and thus may well retain the biocatalytic activity of the proteins although it is difli cult to determine the activity of the proteins adsorbed onto the functionalized CNTs in the present stage.

Al though the covalent functionali zation of the CNTs endows the CNTs with better solubilization and possibly enhanced biocompatibility, a noncovalent strategy is still highly desired since such a strategy maintains the attracti ve electron ic and structural properties of the C TS27. As a typical example, we used one of the block copolymers (i.e. , Pluronic PI23. structure shown in Scheme I ) to noncovalently functionali ze the CNTs. The copolymer Pluronic PI 23 consists of two hydrophilic polymeric chains of E020 and one hydrophobic chain of P07() . The hydrophobic P070 chain

essentially interacts with the CNTs through hydrophobic interaction, leading to the adsorption of P 123 onto the CNTs. The hydrophilicity of the E02() double chai ns substantiall y solubilizes the CNTs in aqueous solu tion. Indeed, our experimental results show that the CNTs are well so lubili zed into PI 23 solution; the ~ uspension of the CNTs into PI23 aqueous so lution essentiall y gives a homogenous black dispersion that is stab le for at least one month. Furthermore, the P 123 is very biocoJl1patible which is believed to retain the biocatalyti c ac ti vity 0(' the proteins adsorbed. The improved solubility and biocompatibility of the CNTs fu ncti onali zed through covalcnt and noncovalent ways may facilitate the manipulation of the CNTs for practica l applications, especially ('or development or bioelectron ic dev ices such as biosensors.

Electrochemistry of H I{P at the covalently functiunalizcd CNTs

We used HRP as an example to demonstratc the possible applications of the ('unctionalized CNTs in faci I i tati on of direct elect ron tran sfer of hCJl1e­containing proteins. Figure. I depicts typi ca l cyc li c voltammograms (CYs) obtained at HRP/CNT-OH/GC (A) and HRP/CNT-COOH/GC (B) clectrodes. For compari son, the CYs obta ined at CNT-O H/GC and CNT-COOH/GC electrodes (w ithout HRP) were also displuyed (In set A and B). As shown, the CYs obtained w ith the HRP/CNT-OH/GC electrode exhibi t a pair of wave at -0.20 Y with a large peak-to-peak separation of co. 200 mY (at 100 mYs·l

) (A), 'vvh ich could be ascribed to the irreversible redox process of HRP at the CNT-OHs. Similarl y. the irrevers ible direct elec tron transfer of HRP was al so observed at the CNT-COOHs (8); a pair o f redox wave was observed at -0 .27 Y w ith a peak separat ion of ca. 150

R c

(n =20.01=70) I)

Structures of pristine CNT (A). CNTs functionalized covalent ly with -DH (B) and -COOH (C) groups and P1 23 (D)

Scheme I

ZHENG el 01.: FUNCTIONALIZATION OF CARBON ANOTUBES 953

mY (at 100 mYs"\ The CNT-COOH/GC electrode showed a cathod ic peak at -0.90 Y and a pair of wave at 0.0 Y, of which the wave at 0.0 Y was attributed to redox process of the oxygenated groups at the CNT­COOHs. The disappearance of these peaks upon the immobili zation of HRP is indicat ive of cross- linking of HRP at the CNTs.

A lthough the strategies for covalent functiona li zat ion of the CNTs with oxygenated groups, i .e ., - OH and - COO H groups. essentiall y made i t possib le to realize the direct electron transfer of HRP, the faci litated redox process of HRP was yet

5 A

o

- 5

- 10

5 B

o

-5

-\

-0.5

o ~ -sV

-o.s 0 E / Vvs.Ag/AgCI

o E l Y vs. Ag/ AgCl

-0.5

-1 ·0.5 0 0.5 E / Vvs.Ag/AgCI

o 0.5

ElY vs. Ag/ AgCl

Fig. I - Cyc lic vo l lalll lllog ral11 s in N2-sa turated 0. 10 M phosphate buffer [(pH 7.0: scan rate 100 I11YS"I: A: HRP/CNT­OH/GC: and. B: HRP/CNT-COO H/GC e lectrodes. (lnsel : CYs with CNT-OH /GC and CNT-COO H/GC e lec trodes in Nr sa turated phosphate)l.

relatively irreversible and ill-defined. Such a sluggish electron transfer process could be largely improved at the CNTs noncovalently functionalizecl with P 123 as demonstrated below.

Electrochemistry and electrocatalysis of 1"\1{ I' at P123-functionalized CNTs

Figure 2 depicts typi ca l CY s obtai ned at HRP/CNT-Pl 23/GC (so lid line) and HRP/CNT/GC (dotted line) and CNT/GC (dashed line) electrodes. A pair of well-defined redox peaks is observed at a formal potential of -0 .25 Y wi th a peak-to-peak separat ion of ca. 65 mY and a near unity of the ratio or cathodic- to-anod ic peak current (at 500 mYs"\ characteri stic of revers ible elec trode process of heme Fe1l1/Fe11 in HRP~S.29 . This observation indi ca tes that HRP adsorbed onto the CNTs noncovalentl y funct ional ized w ith P 123 effici ently real izes its di rect electrochemistry. In compari son. the HRP/CNT/GC electrode exhibits no redox peaks wh ich could be ascribed to the redox process of HRP, suggesting that it is difficult to realize the direct electron transfer or HRP at the pristine CNTs withoLit any functionalization , which co incides with previ oLi s reports22

.23

. Although , the mechanism for the observed facilitation of the electron transfer of HRP at the functi onali zed CNTs (i .e., CNT-P 123. CNT-OH and CNT -COOH) sti II requi res more experi mental ev idences, it is very likely that the proper ori entation and/or conformation of the enzymes onto the

-0.5 o

E l Y vs. Agi AgCl

Fig. 2 - CYs obta ined at HRP/CNT-PI 2J/GC (so lid line) , HRP/CNT/GC (dOlled line) and CNT/GC (dashed linc) e lec trodes in 0. 10 M Nrsaturated phosphate buffer. IScan rate 500 IllV s· l l.

954 INDIAN J CHEM. SEC A. MA Y 2005

functionalized nanotube surface, which are believed to be favorable for inteli'acial electron transfer of the proteins' O, constitutes one of the consequences for the above res ults.

Figure 3 displays the CVs of the HRP/CNT­P l23/GC electrode in phosphate buffer at different potential sweep rates. As shown , the peak currents are linear with potenti al scan rate in a range from 100 to 900 mVs· 1 (in set, using cathodic current as an exampl e), while the potential does not change clearly , demonstrati ng that the electron transfer process of HRP adsorbed onto the CNT-P123 is a fast and surface-confined process .

We next investigated the electrocatalytic activities of HRP confined onto the CNT-P123/GC electrodes . Figure 4 shows typical CVs of HRP/CNT-PI23/GC electrode in phosphate buffer. The presence of H20 2

in solution remarkabl y increases the cathodic peak current , and decreases the reversed ox idation peak current (solid line) as compared with the peak currents in its absence (dotted line). This observation , along with the large positive shift of the potential for H20 2 reducti on at the HRP/CNT-PI23/GC electrode compared with that at the CNT-PI23/GC electrode (inset) , indicate that the HRP/CNT -P l23/GC electrode possesses a good catalytic activity toward H20 2 reduction . A similar catalytic activity has also

o 1

-1 0

-0.5 o E I V vs. AgJAgCI

Fig. 3 - CVs obtained at HRP/CNT -PI23/GC electrode in 0.10 M Nrsaturated phosphate buffer. [Scan rate ( from inner to outer): 100, 200, 300, 400. 500, 600. 700, 800, and 900 mVs·l

; Inset shows a plot of cathod ic peak current against potential scan rate).

o

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1 - 20

-

- 40

. ", .. , ,I" ....

,,"

... .... ...

- 0 .5

.... ..... '

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E I V vs. Ag/ AgCl

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Fig. 4 - CVs obtai ned at HRP/CNT-PI23/GC electrode in 0.10 M N2-salUrated phosp ate buffer in the absence (dOlled line) and presence (solid line) of 10 pM H20 2. [Inset shows CVs obtained at CNT-P I 23/GC electrode in 0.1 0 M N2-saturated phosphate buffer in the absence (dOlled line) and presence (so lid line) of 10 ~lM H20 1: scan rate. 500 mVs· I

] .

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- 50

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orr········ ·· ····················· · (~~ ... ~ ..... ........ .

-20 <: ;?; -10'---_-:-::-_ _ ------'

-0 .5 0 E I VYs.AgI~1

o E I V vs. Ag/ Agel

Fig. 5 - CVs obtai ned at HRP/CNT-P I23!GC electrode in 0. 10 M phosphate buffer saturated with N2 (dOlled line) and ai r (solid line). [Inset shows CVs obtained at CNT-P I23/GC electrode in 0.10 M phospha t.e buffer saturated with N2 (dotted line) and air (solid line); scan rate, 500 mVs·I

].

ZHENG et al.: FUNCTIONALIZATION OF CARBON NANOTUBES 955

been observed for O2 reduction as shown in Fig. 5. As shown, the O~ reduction occurs at the CNT-P I 23/GC electrode at -0.70 V under the present experimental conditions (inset in Fig. 5). Very similar to the case of H20~ , the introduction of O2 in solution clearly increases the cathodic peak current, and decreases the reversed oxidation peak current (solid line) at the HRP/CNT-PI23/GC electrode compared with the peaks in its ab ence (dotted line) . Such an excellent

• catalytic activity of HRP coupled with its fast and direct electron transfer property at the CNT -P 123 is responsible for the large positive shift of the potential (ca. 400 mV) for O2 reduction at the HRP/CNT­PI23/GC elec trode compared with that at the CNT­P I 23/GC electrode. The observed catalytic acti vi ties of HRP adsorbed onto the functionalized CNTs may be use ful for the development of new bioelectronic nanodev ices, e.g., electrochemical biosensors and enzyme-based biofuel cell s.

Conclusions Three strategies have been utilized here for both

covalent and noncovalent functionalizing of the CNTs to facilitate direc t electronic communicati on between the proteins (i.e., HRP in thi s work) and the CNTs. Functionali zation of the CNTs with hydroxyl and carboxy l groups and block copolymer (i.e" P123) essentially increases the solubility of the CNTs and facilitates the manipulation of the CNTs and may enhance the biocompatibility of the CNTs with biomacromolecules such as proteins and enzymes. These properti es are believed to be very useful for the development of bioelec tronic nanodev ices such as electrochemical biosensors and enzy me-based biofuel cells. M oreover. the functionali zed CNTs have been found to be capable of facilitating the direct electron transfer of HRP. The noncovalently P123-functionalized CNTs have been found to be advantageous over those covalently functionalized with - OH and - COOH groups. The combination of the direct electron transfer property of HRP with its biocatalytic ac tivity substantially enables electrochemical reductions of dioxygen and hydrogen peroxide to occur with a low overpotential.

Acknowledgement Financial support from National Natural Science

Foundation of China (Grant Nos. 20375043 and 20435030) and Chinese Academy of Sciences (Grant No. KJCX2-SW-H06) is sincerely acknowledged. The

authors thank Prof. Zhi-Xin Guo for helpful discussions.

References I Baughman R H. Zakhidov A A & de Hcer W A. Sci('I/('('. :'97

(2002) 787. :2 Sun Y P. Fu K F. Lin Y & Huang W J. Ace Chelll Res. ])

(2002) 1096. ] Haddon R C. Ace Chelll Res. 35 (2002) 997. 4 Katz E & Willner L Chelll Ph V.I' Chelll. 5 (2004) IOX4. 5 Moore R R. Banks C E & Compton R G. 1\ 1/01 Chell/. 76

(2004) 2677. 6 Li J. Casse ll A, Delzeit L. Han J & Meyyappan M . .J 1)/1\'.1'

Chelll B. 106 (2002) 9299. 7 Poh W C. Loh K P. Zhang W D, Tripanh y S, Ye J & Sheu F

S. Lal/glllllir. 20 (2004) 5484. 8 Zhao Q. Gan Z & Zhuang Q, Elect/'{}w/{/lys i.l. 14 (200:')

1690. 9 Davis J J. Co les R J. Allen H & Hill O . .J E/ectw(///(// Ch(,l//.

440 ( 1997) 279. 10 Sheri gara B S, Kutner W & D'Souza F. EleClwul/{//vsi.l'. 15

(2003) 75]. II Rubi anes M D & Ri vas GA. E/('ct J'(}chelll COIl/lillI/I . 5 (2003 )

689. 12 Hrapovic S, Liu Y L. Male K B & Luong J H T. I\c(' ChI'lli

Res. 76 (2004) 1083. I ] Deo R P & Wang J, EleClmcilelll COIIIIIIIII/. 6 (2004) 284. 14 Zhao G C. Zhang L. Wei X W & Yang Z S. Elel'lmchelll

COIIIIIII/II , 5 (2003) 825. 15 Yang P. Zhao Q. Gu Z & Zhuang Q. Eleetmwlah·si.l'. 16

(2004) 97 . 16 Zhang M, Gong K. Zhan g H & Mao L. Biosel/s Bio('/('('/rl/Il.

20 (2005 ) 1270. 17 GO llg K. Zhang M. Yan Y. Su L. Mao L. Xiong S & Chen Y.

AI/a / Chelll. 76(2004) 6500. 18 Zhang M, Yan Y, Gong K. Mao L. Guo Z & Chen Y.

LUllg III II iI', 20 (20U4) 878 1. 19 Gong K. Dong Y. Xiong S. Chen Y & Mao L, iJiosel/s

Bioel eClr(lII . 20 (2004) 253. 20 Bianco A & Prato M, Adv Mater, 15 (2003) 1765. 2 1 Wang J. Li M. Shi Z. Li N & Gu Z, A llul Chem. 74 (2002)

199]. 22 Yu X, ChatlOpadhyay D. Galeska I, Papadilllitrakopoul os F

& Rusling J F, Electroch elll COllI/JIll//. 5 (200] ) 40S. 23 Goodin g J J, Wibowo R, Liu J. Yang W. Losic D. Orbons S.

Mearns F J. Shapter J G & Hibben D B . .J Alii Chelll Soc. 125 (2003) 9006.

24 Xu J. Zhu J. Wu Q. Hu Z & Chen H. Chil/.J CI,ell/. 2 1 (20m) 1088.

25 Pan H, Liu L, Guo Z, Dai L, Zha ng F, Zhu D. Czerw R & Carro ll D L. Nal/o Letl. 3 (2003) 29.

26 Ri chard C. Balavo ine F. Schultz P. Ebbesen T W & Mi oskowski C. Sciellce . 300 (2003) 775 .

27 Chcn J. Lill H. Weimer W A, Hall s M D. Waldec k D H & Walker G c. .J A lii ChI'lli Soc, 124 (2003) 9034.

28 Nassar A F. Zhang Z. Hu N. Rusling J F & KUlllos inski T F. .J Phvs Chelll B, 101 ( 1997) 2224.

29 Zhang Z, Chollchane S. Magli ozzo R S & Ru sling J F. AI/al Chelll , 74 (2002) 163.

30 Jellken L J C, Biochilll Biophys Acta, 1604 (2003) 67.