Analytica Chimica Acta 525 (2004) 221–230

Electrocatalytic H2O2 amperometric detection using goldnanotube electrode ensembles

Marc Delvauxa, Alain Walcariusb, Sophie Demoustier-Champagnea,∗a Unite de Physique et de Chimie des Hauts Polym`eres, Universit´e Catholique de Louvain, Place Croix du Sud, 1,

B-1348 Louvain-la-Neuve, Belgiumb Laboratoire de Chimie Physique et Microbiologie pour l’Environnement, Unit´e Mixte de Recherche UMR 7564, CNRS,

Universite H. Poincare Nancy I, 405, Rue de Vandoeuvre, F-54600 Villers-les Nancy, France

Received 5 March 2004; received in revised form 23 August 2004; accepted 23 August 2004Available online 13 September 2004

Abstract

Arrays of nanoscopic gold tubes were prepared by electroless plating of the metal within the pores of nanoporous polycarbonate track-e n the efficienti ylamine orm trodee saT f HH imit ofd tionr©

K

1

gtap[

vro

d

ctive. Con-ableat re-(Pt,

ion

nec-

cing

ighs bi-elec-hich

0d

tched membranes. A procedure for fabricating an ensemble of enzyme-modified nanoelectrodes has been developed based ommobilization of horseradish peroxidase (HRP) to the gold nanotubes array using self-assembled monolayers (mercaptoeth

ercaptopropionic acid) as anchoring layers. Hydrogen peroxide (H2O2) was determined electrochemically by using gold nanoelecnsembles (NEE) functionalized or not in phosphate buffer solution (PB) with or without a mediator (hydroquinone, H2Q). Bare NEE displayremarkable sensitivity (14�A mM−1 in H2Q at−0.1 V versus Ag/AgCl) compared to a classical gold macroelectrode (0.41�A mM−1).he gold nanoparticles that form the tubular structure act as excellent catalytic surfaces towards the oxidation and the reduction o2O2. TheRP modified NEE presents a slightly lower sensitivity (9.5�A mM−1) than bare NEE. However, this system presents an enhanced letection (up to 4× 10−6 M) and a higher selectivity towards the detection of H2O2 over a wide range of potentials. The lifetime, fabricaeproducibility and measurement repeatability of the HRP enzyme electrode were evaluated with satisfactory results.

2004 Elsevier B.V. All rights reserved.

eywords:Gold nanoelectrodes; Immobilized enzyme; Horseradish peroxidase; Hydrogen peroxide; Amperometric detection

. Introduction

The detection and quantitative determination of hydro-en peroxide is of practical importance in food, pharmaceu-

ical, chemical, biochemical, industrial and environmentalnalyses. Numerous methods have been developed for thisurpose, including spectrometry[1–3], chemiluminescence

4–6], titrimetry [7,8] and electrochemistry[9–13].Direct electrochemical detection of H2O2 has been made

ia its oxidation or reduction at a variety of electrode mate-ials but the detection requires operation at a relatively highverpotential (e.g. 0.7 V versus SCE for Pt), which makes the

∗ Corresponding author. Tel.: +32 10 473560.E-mail addresses:walcariu@lcpe.cnrs-nancy.fr (A. Walcarius),

emoustier@poly.ucl.ac.be (S. Demoustier-Champagne).

electrode sensitive to interferences from many electroaspecies in real samples such as ascorbic and uric acidssequently, a lot of work has focused on developing suitelectrocatalytic surfaces where these reactions proceedduced voltages. The usually small size metal particlesPd, Ru, Rh, Ir, Au, Cu, etc.) allow a preferential oxidator reduction of hydrogen peroxide[14–20]without requiringadditional reagents. In numerous papers[21–25], bimetallicalloys dispersed in a matrix (C paste, sol–gel, etc.) areessary to enhance the electrocatalytic detection of H2O2 andbiosensing of glucose or other analytes without sacrifithe selectivity.

An attractive alternative approach is to exploit the hsubstrate specificity and the high activity of enzymes aological catalyst. Amperometric biosensors based ontronic transfer between immobilized peroxidases, w

003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2004.08.054

222 M. Delvaux et al. / Analytica Chimica Acta 525 (2004) 221–230

catalysed the reduction of hydrogen peroxide, and the elec-trode is promising for the fabrication of simple and low costenzyme sensors. Horseradish peroxidase (HRP), a heme con-taining glycoproteine, is the most thoroughly studied andused enzyme in the development of enzyme-based H2O2 am-perometric sensors.

Many approaches can be used for the immobilization ofHRP on electrodes such as physical adsorption onto the sur-face of the electrode (e.g. pretreated carbon or graphite elec-trodes, Pt)[26–28], coverage with a polymer (conducting ornon-conducting) into which peroxidase molecules are phys-ically or chemically attached and entrapment in bulk modi-fied composite electrodes, where HRP are distributed in themixture (graphite oil paste, synthetic lipid films, redox hy-drogel [29–31]). Another approach has been developed byWillner et al.[32,33]using self-assembled monolayers as an-chor layer for the immobilization of biomolecules. Currently,the well-characterized SAMs on metal electrodes are widelyused for the immobilization, orientation and organization ofbiomolecules at interface. SAMs offer the analytical advan-tages of reducing the non-faradic backgrounds currents bypreventing close approach of solvents and ions to the elec-trode surface, provide a high degree of reproducibility and al-low the immobilization, of a variety of biomolecules throughdifferent functional head groups close to the electrode surfacew

ninge iliza-t ntso ucer.C : (1)p clus-t ncedct withn (e.g.m sgV s ofg ntingu thep ranes( im-pm an-o nma ify-i erefu oweda in-j ,w elec-tH nc-t of

both bare Au nanotubes and HRP modified Au nanotubeshave been evaluated with and without a redox mediator. Thestability, repeatability and selectivity of the nanotubes-basedsensors have also been investigated.

2. Experimental section

2.1. Reagents and solutions

All materials and reagents were used as commer-cially received. Horseradish peroxidase (HRP, EC 1.11.1.7,Type VI), 3-mercaptopropionic acid (MPA, >99%), 2-mercaptoethylamine (MPE, cysteamine, >98%) were pur-chased from Sigma–Aldrich. Hydroquinone (H2Q, 99%), 4-acetaminodophenol (AC, 96%), uric acid (UA, >99%) andl(+)-ascorbic acid (AA, 96%) were received from Acros.Ethanol (HPLC grade) and hydrogen peroxide (min. 27%)were from Merck.

Glutaraldehyde (GA, 25% in water, Sigma), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC,Acros), 2-(N-(morpholino)ethanesulfonic acid (MES, Acros)andN-hydroxysuccinimide (NHS, Acros) were used for co-valent coupling of enzymes. The phosphate buffer (PB) con-tained 0.1 M KH2PO4 and 0.1 M K2HPO4.

r-m tion.P pu-r

2

2pore

d ma el-o goldw em-btp anew /Au)w facea atera be-t plesw 0o

2a

ts byU movea func-t ed byi into

ith a high degree of reproducibility.Gold electrodes have been increasingly used in desig

lectrochemical biosensors. However, enzyme immobion on flat gold surfaces often suffers from low amouf biomolecules and poor electrical contact to the transdurrently, most efforts are directed to two new directionsroduction of composite electrodes made of gold nano

ers and immobilized enzymes, which exploit the enhaatalytic activity of the gold nanoparticles[34–40], and (2)he three-dimensional structuration of gold electrodesanometer-sized dimensions for biosensor applicationsicroporous gold electrodes[41], nanopatterning of porouold films [42] or gold nanoelectrode ensembles[43,44]).ery recently, we reported on the fabrication of ensembleold nanotubes, aligned parallel to each other and preseniform size and shape by electroless plating of gold intoores of nanoporous polycarbonate track-etched membnano-PTM)[45]. These nano-PTM are realized by anroved procedure developed in our lab[46]. By this templateethod, it is possible to prepare cylindrical metallic ntubes with an outer diameter ranging from 15 to 1000nd an inner diameter that can be varied at will by mod

ng the gold deposition time. Arrays of gold nanotubes wunctionalized through the immobilization of GOx enzymessing SAMs as anchor layer. The resulting biosensor shremarkable sensitivity to glucose in batch and in flow

ection amperometric measurements[47,48]. In this papere report on the unique properties of gold tubes nano

rodes ensembles for the electrochemical detection of H2O2.RP was immobilized within gold nanotubes through fu

ionalized alkylthiol SAMs. The analytical performances

The accurate concentration of H2O2 solution was deteined by titration with potassium permanganate soluure water, obtained by means of Millipore MilliQ water

ification set, was used throughout.

.2. Electrochemical devices preparation

.2.1. Construction of gold nanotubes arraysTrack-etched polycarbonate (PC) membranes with a

ensity of 1× 108 pores/cm2, a pore diameter of 460 nnd a thickness of 20�m realized by the procedure devped in our lab[46] were used as templates. Electrolessas deposited on the pore walls and both faces of the mrane according to the procedure described earlier[45]. The

emperature of the deposition solution was 4◦C. After de-osition of gold for 17 h, the pore radius of the membras reduced to 320 nm. The gold-coated membrane (PCas immersed in 25% nitric acid for 12 h to clean the surnd remove any residual Ag, rinsed thoroughly with wnd dried in air. In order to avoid creeping of the solution

ween the Au nanotubes and the PC pore walls, the samere heated above the glass-transition temperature (15◦C)f the PC for 15 min.

.2.2. Construction of HRP modified gold nanotubesrrays

The gold substrates were first freed from contaminanV/ozone treatments and then immersed in ethanol to reny gold oxide deposit before monolayer assembly. The

ionalized thiols assemblies were spontaneously adsorbmmersing the membrane containing gold nanotubes

M. Delvaux et al. / Analytica Chimica Acta 525 (2004) 221–230 223

dilute (2 × 10−3 M) MPE or MPA solutions of absoluteethanol.

After deposition overnight to ensure a complete coverage,the substrates were thoroughly rinsed with absolute ethanoland dried under a stream of nitrogen.

The MPE-modified electrodes were activated for 2 h ina solution of GA (commercial solution diluted 100 timesin phosphate buffer) at room temperature. The surface wasrinsed with buffer and immediately placed in a solution ofHRP (1 mg/ml) in PB for 2 h. The resulting enzyme-modifiednanoelectrodes are further noted PC/Au/MPE/GA/HRP.

The terminal carboxylic acid groups of MPA-gold sur-faces were modified by dipping in a MES buffer solution(pH = 3.5) containing 2 mM EDC and 5 mM NHS for 2 h. Af-ter being rinsed with buffer, the MPA–NHS ester monolayerswere allowed to react for 2 h with a solution of PB containing1 mg/ml of the enzyme. The resulting enzyme-modified na-noelectrodes are further noted PC/Au/MPA/EDC,NHS/HRP.

2.3. Instrumentation and measuring procedures

Amperometric experiments were performed using anEG&G Princeton Research 273A potentiostat/galvanostat.Electrochemical experiments were carried out in a conven-tional one-compartment cell with a three-electrode configu-r ateb -e lacedb a of0 lec-t

omG rtherc lmsw een− t as rded.

, theb valueb erea era-t r atl ea-s con-v

wasc ng ah minif ion,t hemf eret lverm ovea

eachs SX

100/206) spectrometer from Fisons, operating at a pressurein the low 10−8 Torr range, equipped with an aluminum an-ode and a quartz monochromator. Spectra were recorded at atakeoff angle of 35◦ (angle between the plane of the samplesurface and the entrance of the lens of the analyser). Curvefitting has been done using a Gaussian–Lorentzian (85–15%)linear combination and a linear background.

3. Results and discussion

3.1. Fabrication and characterization of goldnanoelectrodes arrays

Electroless plating of gold was carried out within the poresof polycarbonate nano-PTM with a pore size of 460 nm, apore density of 1× 108 pores cm−2 and a thickness of 20�m,following the procedure described elsewhere[45]. FEG-SEMimage (Fig. 1), recorded after dissolution of the PC membranein CH2Cl2, clearly shows that nanotubes running the entirethickness of the membrane are so obtained. Indeed gold de-position proceeds from the activated pore walls rather thana base layer of metal. As a result, there is no competitionbetween elongating the tubules and closure of the tubules toform solid fibers. Increasing the plating time leads to a thick-e nt iono anda ro-m tubesl unto sta-b micalb theP in thed

F solu-t

ation holding 20 ml of the supporting electrolyte (phosphuffer, PB or PB + 0.1 mM hydroquinone, H2Q). The threelectrode system consisted of gold nanotubes arrays petween an Au disk and an O-ring with a surface are.071 cm2 as working electrode, an Ag/AgCl reference e

rode and a Pt foil as counter electrode.For comparison, a conventional Au foil electrode fr

oodfellow was also used as working electrode. We fuall this conventional electrode “macroelectrode”. Gold fiere cleaned by cycling potentials continuously betw0.2 and 1.4 V versus Ag/AgCl in 0.1 M sulfuric acid a

can rate of 100 mV/s until reproducible scans were recoFor amperometric experiments at controlled potential

ackground current was allowed to decay to a steadyefore aliquots of stock hydrogen peroxide solution wdded. All experiments were carried out at room temp

ure. The solutions were purged with purified nitrogen foeast 20 min to remove oxygen prior to each series of murements. A magnetic stirrer and a stirring bar providedective transport for the electrochemical experiments.

Characterization of the morphology of gold nanotubesarried out by scanning electron microscopy (SEM) usiigh resolution FEG digital scanning microscope 982 ge

rom LEO, operating at 1 kV. After Au electroless deposithe templating membranes were dissolved by dipping tor 2 min in dichloromethane. The free Au nanotubes when recovered from the solution by filtration through a siembrane, copiously rinsed with dichloromethane to remny residual PC and then analysed using FEG-SEM.

The surface chemical composition was determined attep of functionalization by XPS using a SSIX probe (S

ning of the tubules walls[45]. In this work, a depositioime of 17 h at 4◦C was employed leading to the formatf open nanoscopic channels with a thickness of 70 nmlength of 20�m. This deposition time is a good compise between the need to have a void space inside the

arge enough to allow the immobilization of a huge amof biomolecules and the need to have a mechanicallyle system, robust enough to be used as an electrocheiosensor. A too thin gold layer cannot efficiently protectC membrane against degradation by the reagents usedifferent steps of functionalization.

ig. 1. FE-SEM image of an array of Au nanotubes, recorded after dision of the PC template membrane.

224 M. Delvaux et al. / Analytica Chimica Acta 525 (2004) 221–230

Fig. 2. FE-SEM images of (a) the surface morphology of Au electroless deposited on nanoporous membranes (b) the surface of the commercial gold foil.

The surface of a commercial gold foil was imaged by SEM(Fig. 2) in order to compare its morphology and roughnesswith the surface of electroless gold deposited on a PC mem-brane. Images in this figure are depicted at the same magnifi-cation (50 000×) in order to emphasize the important differ-ence in roughness between the two types of Au electrodes.The roughness of the electroless gold surface is much higherthan that of evaporated gold foil. Indeed, gold nanotubes aremade of discrete gold particles with an average size of 30 nm.Considering their high surface to volume ratio, these goldnanoparticles are interesting materials for the catalytic ac-tion towards the electrooxidation and the electroreduction ofhydrogen peroxide. Hou et al.[49] demonstrated that suchroughly Au surfaces remained however compatible with theformation of densely packed alkylthiol SAMs. In our previousarticles[47,48], we also showed that glucose oxidase couldbe efficiently fixed on electroless gold surface using SAMs asanchoring layers. In this work, a similar procedure was usedto immobilize HRP within gold nanotube arrays. Two typesof thiol-functionalized monolayers (MPE and MPA) wereemployed as base nanoglue interfaces to link the enzymeto the electrode support. In the system modified with NH2terminated thiol (PC/Au/MPE), the HRP molecules were at-tached by using glutaraldehyde as linking agent. The covalentattachment of HRP to the carboxylic terminated SAM wasa thec ergon s oft , thep sur-f ionsi sw ei s ofa

3b

a -state

responses of PC/Au/MPE/GA/HRP, bare PC/Au and bare Aufilm (Goodfellow) to the injections of 5× 10−4 M H2O2 weremeasured at different potentials in the range +0.3 to−0.8 Vversus Ag/AgCl in PB and PB + H2Q (0.1 mM) solutions(Fig. 3a and b, respectively). Each point in both graphs isthe mean response of three consecutive additions of H2O2. Acontinuous increase of the reduction current up to−0.8 V wasobserved for the three studied systems. However, significantdifferences were observed between these three types of elec-trodes. Indeed,Fig. 3 clearly shows that gold nanoparticles

Fig. 3. Hydrodynamic voltammograms obtained using (�) bare Au, (�)PC/Au/MPE/GA/HRP and (�) PC/Au as working electrode for the analysisof 5.0× 10−4 M hydrogen peroxide samples. (a) Electrolyte solution: 1.0×10−1 M phosphate buffer (pH = 7.2); (b) electrolyte solution: 1.0× 10−1 Mphosphate buffer (pH = 7.2) with addition of 1.0× 10−4 M hydroquinonein the medium.

chieved via carbodiimide activation (EDC + NHS) ofarboxylic acids to a reactive intermediate that can unducleophilic attack by amine moieties on free lysine chain

he enzyme. At the end of the functionalization pathwaysresence of HRP enzyme immobilized on both types of

aces was clearly confirmed by XPS. Indeed, modificatn the C1s region and the increase of N1s and O1s peak areaith the concomitant reduction of the Au4f and S2p peaks ar

n agreement with the fixation of amino acids side chainpolypeptide backbone.

.2. Influence of gold surface on the electrochemicalehavior of hydrogen peroxide

The hydrodynamic voltammograms for H2O2 detectiont various gold electrodes were investigated. The steady

M. Delvaux et al. / Analytica Chimica Acta 525 (2004) 221–230 225

deposited PC membrane (PC/Au) have a remarkable electro-catalytic action towards the reduction of H2O2 throughout allthe investigated range of potentials both in phosphate bufferand in hydroquinone solutions. Nano-PC/Au also catalysesthe hydrogen peroxide oxidation (data not shown), althoughthis effect is less pronounced than in the case of H2O2 re-duction. On the contrary, with or without any mediator inthe solution, the response to hydrogen peroxide at bare Aumacroelectrode was very low.

The sensitivity of the enzyme-modified nanoelectrodes(PC/Au/MPE/GA/HRP) is between the sensitivity of bare Aumacroelectrode and that of nano-PC/Au. This lower sensitiv-ity of the HRP modified Au nanotubes compared to bare Aunanotubes arises from the coverage of the gold nanoparti-cles by HRP molecules that considerably reduce the electro-less Au active electrode area. The advantages to functional-ize the gold electroless surface by fixation of HRP is howeverdemonstrated inFig. 4a and b, where the faradic-to-capacitiveratios for the different electrodes in a potential range from+0.3 to−0.8 V are reported. These graphs show that thanksto a low background current, the enzyme-modified nano-electrode (PC/Au/MPE/GA/HRP) gives the highest ratiosthroughout nearly all the investigated potential range, both

FPo1pi

in PB and H2Q solutions. These high faradic-to-capacitivecurrent ratios should give better properties, e.g. a lower limitof detection for the resulting biosensor.

3.3. Electrode response characteristics

3.3.1. Calibration of the sensorsIn order to confirm the catalytic action of the electro-

less gold nanoparticles deposited on PC membrane, the am-perometric response to injections of H2O2 on bare PC/Au,PC/Au/MPE/GA/HRP and bare Au macroelectrode werecompared at several fixed potentials (+0.1, 0,−0.1, −0.2,and−0.5 V versus Ag/AgCl) in both PB and H2Q solutions.In all cases, when an aliquot of H2O2 was added into thesolution, the reductive current rose steeply to reach a sta-ble value. However, the response time and the amplitude ofthe current change were drastically different on each typeof electrode. PC/Au and PC/Au/MPE/GA/HRP systems ex-hibited a rapid and sensitive response to changes of H2O2concentration. These sensors reached 95% of the steady-state current in less than 11 s for PC/Au and in only 8 sfor PC/Au/MPE/GA/HRP, indicating the excellent electro-catalytic behavior of the gold nanotubes electrode. The veryfast response time of the enzyme-modified nanoelectrodemay also be attributed to the favorable nano-environment oft e bi-o Aum t 35 sw rved.

areA tionc ns.A ntiala 0 ad-d sf RPs sev-e e ap-p ted

ig. 4. Faradic-to-capacitive current ratios obtained using (�) bare Au, (�)C/Au/MPE/GA/HRP and (�) PC/Au as working electrode for the analysisf 5.0× 10−4 M hydrogen peroxide samples. (a) Electrolyte solution: 1.0×0−1 M phosphate buffer (pH = 7.2); (b) electrolyte solution: 1.0× 10−1 Mhosphate buffer (pH = 7.2) with addition of 1.0× 10−4 M hydroquinone

n the medium.

F oten-t asw ionc

he nanotubes system that could contribute to stabilize thlogical activity of the HRP to a large extent. At the bareacroelectrode, a much longer response time of abouas obtained and very much lower sensitivity was obseAll sensors (PC/Au/MPE/GA/HRP, bare PC/Au and b

u macroelectrode) were tested by performing calibraurves for H2O2 under the same experimental conditioll the calibration curves were obtained at each potend for each sensor by the measurement of at least 1itions of H2O2. Fig. 5 depicts typical calibration curve

or one representative electrode, the PC/Au/MPE/GA/Hensor, in the presence of hydroquinone by applyingral potential values. The response increases with thlied potential and is linear within the whole investiga

ig. 5. Hydrogen peroxide calibration plots obtained at detection pial going from 0.1 to−0.5 V vs. Ag/AgCl using PC/Au/MPE/GA/HRPorking electrode in a 1.0× 10−1 M deaerated phosphate buffer solutontaining 1.0× 10−4 M hydroquinone.

226 M. Delvaux et al. / Analytica Chimica Acta 525 (2004) 221–230

concentration range (5× 10−5 to 3× 10−3 M). Some cali-bration curves were also recorded for the different sensorson wider H2O2 concentration ranges (1× 10−5 to 15 ×10−3 M), in both PB solutions and in PB+H2Q medium, ata detection potential of−0.1 V. For a better characteriza-tion of the enzyme-modified nanotube-based bioelectrode,the response of the PC/Au/MPA/EDC,NHS/HRP biosen-sor, in PB + H2Q, was also evaluated. As expected fromprevious results, higher sensitivity for the reduction of hy-drogen peroxide was obtained with all the sensors basedon gold nanoparticles (PC/Au, PC/Au/MPE/GA/HRP andPC/Au/MPA/EDC,NHS/HRP). On the other hand, very lowcurrent responses to H2O2 injection were obtained at the goldmacroelectrode.

The PC/Au/MPE/GA/HRP biosensor provides a linearcurrent response to hydrogen peroxide in H2Q over a con-centration range of 1× 10−5 to 6 × 10−3 M with asensitivity of 9.5�A mM−1. In PB solution, the dynamicrange is extended up to 10 mM but the sensitivity decreasesto a value of 1.5�A mM−1. A signal saturation was ob-served at H2O2 concentration higher than 6× 10−3 M. Thiscan be attributed to the saturation of the enzyme-substrateor enzyme-mediator kinetic or may be due to the forma-tion, at high [H2O2], of the enzymatically inactive formof peroxidases denoted compound III (oxidation state + 6).T app

P ord-i htlyh /pf rode[ seds ratew

des( itha .A e thes un-m

achc en-s tioncAT thant outt fort f− theg largeo on oft ctiono d int l andm kers

Fig. 6. Sensitivity of the amperometric response to hydrogen peroxide asa function of the applied potential for the (b, c) PC/Au/MPE/GA/HRP, (a)PC/Au/MPA/EDC,NHS/HRP, (d, e) PC/Au and the (f, g) Au macroelectrodesensor. Electrolyte solution 1.0× 10−1 M deaerated phosphate buffer withand without 1.0× 10−4 M hydroquinone.

[50] brought new information about the control of the sur-face state of a polycrystalline gold. The need for operating athigh overvoltage induces the application of potential valuesat which the interference of other electroactive substances inreal samples is maximized. In the detection potential rangeof −0.1 to−0.5 V, the highest sensitivities are obtained withthe bare PC/Au system, independently on the presence or ab-sence of the H2Q mediator in the solution. The high surfacearea of gold nanotubes compared to the macroscopic goldfoil, conjugated to the high roughness of the gold electrolesssurface lead to an obvious increase of the efficient area of theelectrode surface. Bare PC/Au nanoelectrode can thus be use-ful as a probe for a direct electrochemical determination ofH2O2. Nevertheless, it is important to point out that the bestlimits of detection were achieved with the enzyme-modifiednanoelectrodes, because of higher faradic-to-capacitive cur-rent ratios (Fig. 4). Indeed, the best limits of detection are 4×10−6 and 8× 10−5 M, based on signal-to-noise of three, forthe PC/Au/MPE/GA/HRP and PC/Au systems, respectively,while only 10−3 M was reached with the Au macroelectrodedevice.

3.3.2. Mechanism of the electron transfer on HRPmodified electrodes

As already mentioned, the determination of H2O2 wasp iator,h e andb has ar rh rgelye iatori zedo

H

he apparent Michaelis–Menten constant (Km ) of HRP atC/Au/MPE/GA/HRP was calculated to be 7.6 mM acc

ng to the Lineweaver–Burk equation. This value is sligigher than 3.69 mM reported for the nano-Au particlem-henylenediamine polymer film system[38] and 2.3 mM

or the HRP/Au colloid self-assembled monolayer elect28]. The higherKapp

m value of HRP at our nanotubes-baystem could reflect diffusional limitations of the substithin the nanotubes.The other type of enzyme-modified nanoelectro

PC/Au/MPA/EDC,NHS/HRP) showed similar results wsensitivity of 11.3�A mM−1 at −0.1 V in H2Q solutions stated previously, the need for a mediator to enhancensor sensitivity is no longer required when using theodified PC/Au electrode.The sensitivity values, calculated by the slope of e

alibration curve in the linear range, of the different sors at several applied potentials and for different soluompositions (PB or PB + H2Q) are shown inFig. 6. Bareu macroelectrode presents the poorest response to H2O2.he response of such probe is at least five times lower

hat of the H2O2 sensor based on bare PC/Au throughhe investigated potential range. The limit of detectionhis system is only 8× 10−4 M at an applied potential o0.2 V in hydroquinone solution. This poor response onold macroelectrode is not surprising, because of theverpotentials associated to both oxidation and reductihis analyte on Au surfaces. The electrochemical detef hydrogen peroxide on gold has often been neglecte

he literature because of lack of proper surface controechanistic understanding. Only Kauffmann and cowor

erformed either in the presence of a charge transfer medydroquinone, or in its absence. For bare PC/Au electrodare Au macroelectrode, the presence of the mediatorather weak effect on the detection of H2O2. On the otheand, the enzyme-modified nanoelectrodes show a lanhanced sensitivity when adding the hydroquinone med

n the medium. The catalytic process of HRP immobilinto gold surfaces can be expressed as follow[51]:

RP(Fe3+) + H2O2 → compound I+ H2O (1)

M. Delvaux et al. / Analytica Chimica Acta 525 (2004) 221–230 227

compound I+ H2Q → compound II+ BQ (2)

compound II+ H2Q → HRP(Fe3+) + BQ + H2O (3)

net reaction : BQ+ 2e− + 2H+ → H2Q

where compound I (oxidation state + 5) and II (oxidationstate + 4) represent the enzyme intermediates in the reactionand H2Q and BQ represent hydroquinone and benzoquinone,respectively. In the first two electrons transfer step, H2O2 isreduced to water and the HRP is oxidized to compound I.Compound I is then reduced in a one electron step to formcompound II, and then compound II is reduced to the orig-inal form of HRP by the redox mediator (H2Q). The BQ issubsequently reduced to H2Q by a rapid reaction involvingthe consumption of two electrons from the electrode. Theresulting cathodic currents are therefore correlated with theconcentration of both hydrogen peroxide and mediator in thesolution. The dependence of the amperometric determinationof H2O2 on the concentration of the mediator was investi-gated.Fig. 7 shows that the response of the enzyme elec-trode to 5× 10−4 M H2O2 at an applied potential of−0.1 Vincreases sharply as the concentration of hydroquinone in-creases and then leveled off at 1× 10−4 M H2Q. At low me-diator concentration, the current response is limited by theenzyme-mediator kinetic. When the mediator concentrationi yme-s

ultt oldn hemeg tiono tt sur-f ce. Ap 24%f af-t tiono n PBs r be-

F RPe ono ntial:−

tween the active center of HRP and the electrode and/or fromthe direct reduction of H2O2 at the remaining gold surface.

According to the high faradic-to-capacitive current ratioobtained for the PC/Au/MPE/GA/HRP sensor in phosphatebuffer (Fig. 4a), it is possible that a certain amount of HRPparticipates in direct electron transfer with the electrolessgold surface. In this case, the enzyme immobilized on theelectrode is oxidized by hydrogen peroxide according to reac-tion (1) and then subsequently reduced by electrons providedby an electrode as described in reaction (4):

compound I+ 2e− + 2H+ → HRP(Fe3+) + H2O (4)

This direct electron transfer, which is usually less efficientthan mediated electron transfer can also be present in theenzyme electrode used in hydroquinone solution. Indeed,Lindgren et al.[52] have demonstrated that both direct andmediated electron transfers can simultaneously occur in anenzyme-modified electrode. Not all the HRP molecules wereequally involved in these two processes. In their work, thehigh percentage (40–50%) of the HRP able to participatein direct electron transfer has been ascribed to phenolic andquinoidal groups present in carbon-based electrodes, whichmimicked the structure of the electron donor substrates of per-oxidases. In our system, the use of SAM allowing the immobi-lization of enzymes in close contact with the active surface oft createa ules,c ctivec

3e

astT ssivei inH l, thec , buta ibil-in das entialoa wt witht RPs

3very

wHhb re-

s high, the current response becomes limited by the enzubstrate kinetics.

In our enzyme-modified nanoelectrodes, it is diffico distinguish between the electrocatalytic action of ganoparticles and a direct electron transfer between theroup of HRP and the gold nanoparticles on the reducf H2O2. Indeed, from previous results[47], we know tha

he use of short alkyl chain thiols on gold electrolessace does not lead to a complete coverage of the surfaart of the underlying electrode, which was estimated at

rom cyclic voltammetry measurements, is still availableer functionalization for the direct electrochemical reducf H2O2. The signal-response for the enzyme electrode iolution can thus come from the direct electron transfe

ig. 7. Variation of the amperometric response of PC/Au/MPE/GA/Hlectrode for the analysis of 5.0× 10−4 M hydrogen peroxide as a functif the hydroquinone concentration in phosphate buffer. Applied pote0.1 V.

he electrode, as well as the use of gold nanotubes thatconfined environment surrounding the enzyme molec

ould facilitate the direct electron transfer between the aenter of the biomolecule and the gold surface.

.3.3. Repeatability and reproducibility of the HRPnzyme electrode

The repeatability of the PC/Au/MPE/GA/HRP system wested both in H2Q (0.1 mM) and in PB solution at−0.1 V.he relative standard deviations determined for 10 succe

njections of a 2.5× 10−4 M H2O2 sample was 8 and 7%2Q and PB, respectively. At the same applied potentiaalculated RSD for the PC/Au system was slightly higherlways below 15%. The electrode-to-electrode reproduc

ty was estimated from the response to 2.5× 10−4 M H2O2 atine different biosensors in PB and PB + H2Q solutions ant several fixed potentials (0,−0.2, and−0.5 V). The relativetandard deviations are: 14, 9 and 11% at a detection potf 0,−0.2 and−0.5 V in PB + 0.1 mM H2Q and 15 and 10%t a potential of−0.2 and−0.5 V in PB. These results sho

hat good reproducibility and repeatability are reachedhe enzyme-modified nanoelectrodes (PC/Au/MPE/GA/Hystem).

.3.4. Storage stability of the HRP enzyme electrodeThe storage stability was investigated by measuring e

eek over a 5 months period, the response to 2.5× 10−4 M2O2 of a PC/Au/MPE/GA/HRP biosensor at−0.1 V. HRPas been found to be very stable in solution[10] so the HRPiosensor was stored in PB at 4◦C between each measu

228 M. Delvaux et al. / Analytica Chimica Acta 525 (2004) 221–230

Fig. 8. Selectivity of the PC/Au/MPE/GA/HRP sensor as a function of theapplied potential; comparison of the amperometric current obtained for (a)5.0 × 10−4 M ascorbic acid, (b) 5.0× 10−4 M H2O2 and (c) a + b + 5.0× 10−4 M uric acid + 5.0× 10−4 M acetaminophen (blend). Electrolytemedium: 1.0× 10−1 M phosphate buffer with addition of 1.0× 10−4 Mhydroquinone.

ment. After five measurements, the electrode response wasdecreased by 18% due to the desorption of enzyme moleculesthat were only physisorbed on the surface. After 5 months ofstorage, the biosensor retained about 70% of its original re-sponse. The PC/Au/MPE/GA/HRP biosensor is thus quiteefficient for retaining the activity of HRP. This could be dueto the strong interaction between the enzyme and the goldnanoparticles within the nanotubes.

3.3.5. Interference studyDespite the low applied potential, many compounds may

interfere at the electrode surface, because HRP has a broadsubstrate activity. As one of our final goal is to fabricate abi-enzymatic nanotubes-based biosensors including oxidasesthat produce H2O2 and the HRP to measure for instance, glu-cose in different types of samples, we have investigated, in thepotential range +0.4 to−0.8 V the interference of uric acid(UA), ascorbic acid (AA) and acetaminophen (AC), which arecommon metabolites in blood samples. The interference wasevaluated for the three systems: PC/Au/MPE/GA/HRP, barePC/Au and bare Au macroelectrode sensors by measuring thecurrent response obtained for each interfering substances inPB in the presence or not of H2Q. Fig. 8 shows the currentresponse recorded at the PC/Au/MPE/GA/HRP sensor PB +H2Q solution to the injection of 5.0× 10−4 M AA (a), 5.0× −4 −4

×( 0 to− witht hh de-v dicb odes stemt t thatue fer-e se) in Ta

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10 M H2O2 (b) and a blend of 5.0× 10 M AA, 5.010−4 M H2O2, 5.0× 10−4 M UA and 5.0× 10−4 M AC

c). It clearly appears that, in the potential range from0.8 V, these substances do not significantly interfere

he electrode response to H2O2. Only ascorbic acid, whicas a low oxidation potential, causes the electrode toiate by more than 74% of its original value in the anoranch (at potential > +0.1 V). The bare PC/Au electrhows the same behavior than PC/Au/MPE/GA/HRP syhroughout the whole investigated potential range, excepric acid produces an anodic signal at 0 and−0.1 V in bothlectrolyte solutions. This results in a low negative internce, less than 18% (decrease in the cathodic respon

M. Delvaux et al. / Analytica Chimica Acta 525 (2004) 221–230 229

the cathodic mode of the sensor operation. On the bare Aumacroelectrode, the effect of interfering compounds is morepronounced. Ascorbic acid is oxidizable on the electrodeover a larger potential range (from +0.4 to−0.1 V) whileUA and AC give anodic and cathodic currents, respectively,from −0.1 to−0.6 V. The presence of these compounds inthe sample can produce a large deviation in the current re-sponse (e.g. 78% decrease signal at−0.3 V in phosphatebuffer).

4. Conclusions

Arrays of gold nanotubes were prepared through the elec-troless deposition of gold within the nanopores of polycar-bonate track-etched membranes. The versatility of the pro-cess, the possibility to bend the nanotubes and to use themin various configurations, as well as the high surface area ofthese new materials make them very attractive for the fabri-cation of biosensors.

Enzyme-modified nanoelectrodes arrays were constructedby the covalent immobilization of HRP on pre-formed self-assembled monolayers onto the gold surface. Ensembles ofbare gold nanotubes as well as ensembles of HRP modifiednanotubes were used as working electrodes for the direct elec-t ticalp t theH Pa e ex-pPp ameg f 10.T lytica ree-m duc-e iv-i g oftu s at-t tiono at-i lim-i lace,a lay-e

theP f thea its ab kesis s wella ad-v directe p ar

Acknowledgements

SD-C thanks the Belgian National Fund for Scientific Re-search (FNRS) for her research associate position. MD isa fellow of “Fonds pour la formationa la recherche dansl’industrie et dans l’agriculture” (FRIA, Belgium). The “Ser-vices Federaux des Affaires Scientifiques, Techniques et Cul-turelles” are thanked for their financial support in the frameof the “Pole d’Attraction Interuniversitaire” (IAP networkP5/03).

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