222 M. Delvaux et al. / Analytica Chimica Acta 525 (2004) 221–230catalysed the reduction of hydrogen peroxide, and the elec- both bare Au nanotubes and HRP modiﬁed Au nanotubestrode is promising for the fabrication of simple and low cost have been evaluated with and without a redox mediator. Theenzyme sensors. Horseradish peroxidase (HRP), a heme con- stability, repeatability and selectivity of the nanotubes-basedtaining glycoproteine, is the most thoroughly studied and sensors have also been investigated.used enzyme in the development of enzyme-based H2 O2 am-perometric sensors. Many approaches can be used for the immobilization of 2. Experimental sectionHRP on electrodes such as physical adsorption onto the sur-face of the electrode (e.g. pretreated carbon or graphite elec- 2.1. Reagents and solutionstrodes, Pt) [26–28], coverage with a polymer (conducting ornon-conducting) into which peroxidase molecules are phys- All materials and reagents were used as commer-ically or chemically attached and entrapment in bulk modi- cially received. Horseradish peroxidase (HRP, EC 126.96.36.199,ﬁed composite electrodes, where HRP are distributed in the Type VI), 3-mercaptopropionic acid (MPA, >99%), 2-mixture (graphite oil paste, synthetic lipid ﬁlms, redox hy- mercaptoethylamine (MPE, cysteamine, >98%) were pur-drogel [29–31]). Another approach has been developed by chased from Sigma–Aldrich. Hydroquinone (H2 Q, 99%), 4-Willner et al. [32,33] using self-assembled monolayers as an- acetaminodophenol (AC, 96%), uric acid (UA, >99%) andchor layer for the immobilization of biomolecules. Currently, l(+)-ascorbic acid (AA, 96%) were received from Acros.the well-characterized SAMs on metal electrodes are widely Ethanol (HPLC grade) and hydrogen peroxide (min. 27%)used for the immobilization, orientation and organization of were from Merck.biomolecules at interface. SAMs offer the analytical advan- Glutaraldehyde (GA, 25% in water, Sigma), 1-ethyl-3-tages of reducing the non-faradic backgrounds currents by (3-dimethylaminopropyl)carbodiimide hydrochloride (EDC,preventing close approach of solvents and ions to the elec- Acros), 2-(N-(morpholino)ethanesulfonic acid (MES, Acros)trode surface, provide a high degree of reproducibility and al- and N-hydroxysuccinimide (NHS, Acros) were used for co-low the immobilization, of a variety of biomolecules through valent coupling of enzymes. The phosphate buffer (PB) con-different functional head groups close to the electrode surface tained 0.1 M KH2 PO4 and 0.1 M K2 HPO4 .with a high degree of reproducibility. The accurate concentration of H2 O2 solution was deter- Gold electrodes have been increasingly used in designing mined by titration with potassium permanganate solution.electrochemical biosensors. However, enzyme immobiliza- Pure water, obtained by means of Millipore MilliQ water pu-tion on ﬂat gold surfaces often suffers from low amounts riﬁcation set, was used throughout.of biomolecules and poor electrical contact to the transducer.Currently, most efforts are directed to two new directions: (1) 2.2. Electrochemical devices preparationproduction of composite electrodes made of gold nanoclus-ters and immobilized enzymes, which exploit the enhanced 2.2.1. Construction of gold nanotubes arrayscatalytic activity of the gold nanoparticles [34–40], and (2) Track-etched polycarbonate (PC) membranes with a porethe three-dimensional structuration of gold electrodes with density of 1 × 108 pores/cm2 , a pore diameter of 460 nmnanometer-sized dimensions for biosensor applications (e.g. and a thickness of 20 m realized by the procedure devel-microporous gold electrodes , nanopatterning of porous oped in our lab  were used as templates. Electroless goldgold ﬁlms  or gold nanoelectrode ensembles [43,44]). was deposited on the pore walls and both faces of the mem-Very recently, we reported on the fabrication of ensembles of brane according to the procedure described earlier . Thegold nanotubes, aligned parallel to each other and presenting temperature of the deposition solution was 4 ◦ C. After de-uniform size and shape by electroless plating of gold into the position of gold for 17 h, the pore radius of the membranepores of nanoporous polycarbonate track-etched membranes was reduced to 320 nm. The gold-coated membrane (PC/Au)(nano-PTM) . These nano-PTM are realized by an im- was immersed in 25% nitric acid for 12 h to clean the surfaceproved procedure developed in our lab . By this template and remove any residual Ag, rinsed thoroughly with watermethod, it is possible to prepare cylindrical metallic nan- and dried in air. In order to avoid creeping of the solution be-otubes with an outer diameter ranging from 15 to 1000 nm tween the Au nanotubes and the PC pore walls, the samplesand an inner diameter that can be varied at will by modify- were heated above the glass-transition temperature (150 ◦ C)ing the gold deposition time. Arrays of gold nanotubes were of the PC for 15 min.functionalized through the immobilization of GOx enzymesusing SAMs as anchor layer. The resulting biosensor showed 2.2.2. Construction of HRP modiﬁed gold nanotubesa remarkable sensitivity to glucose in batch and in ﬂow in- arraysjection amperometric measurements [47,48]. In this paper, The gold substrates were ﬁrst freed from contaminants bywe report on the unique properties of gold tubes nanoelec- UV/ozone treatments and then immersed in ethanol to removetrodes ensembles for the electrochemical detection of H2 O2 . any gold oxide deposit before monolayer assembly. The func-HRP was immobilized within gold nanotubes through func- tionalized thiols assemblies were spontaneously adsorbed bytionalized alkylthiol SAMs. The analytical performances of immersing the membrane containing gold nanotubes into
M. Delvaux et al. / Analytica Chimica Acta 525 (2004) 221–230 223dilute (2 × 10−3 M) MPE or MPA solutions of absolute 100/206) spectrometer from Fisons, operating at a pressureethanol. in the low 10−8 Torr range, equipped with an aluminum an- After deposition overnight to ensure a complete coverage, ode and a quartz monochromator. Spectra were recorded at athe substrates were thoroughly rinsed with absolute ethanol takeoff angle of 35◦ (angle between the plane of the sampleand dried under a stream of nitrogen. surface and the entrance of the lens of the analyser). Curve The MPE-modiﬁed electrodes were activated for 2 h in ﬁtting has been done using a Gaussian–Lorentzian (85–15%)a solution of GA (commercial solution diluted 100 times linear combination and a linear background.in 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-modiﬁed 3. Results and discussionnanoelectrodes are further noted PC/Au/MPE/GA/HRP. The terminal carboxylic acid groups of MPA-gold sur- 3.1. Fabrication and characterization of goldfaces were modiﬁed by dipping in a MES buffer solution nanoelectrodes arrays(pH = 3.5) containing 2 mM EDC and 5 mM NHS for 2 h. Af-ter being rinsed with buffer, the MPA–NHS ester monolayers Electroless plating of gold was carried out within the poreswere allowed to react for 2 h with a solution of PB containing of polycarbonate nano-PTM with a pore size of 460 nm, a1 mg/ml of the enzyme. The resulting enzyme-modiﬁed na- pore density of 1 × 108 pores cm−2 and a thickness of 20 m,noelectrodes are further noted PC/Au/MPA/EDC,NHS/HRP. following the procedure described elsewhere . FEG-SEM image (Fig. 1), recorded after dissolution of the PC membrane2.3. Instrumentation and measuring procedures in CH2 Cl2 , clearly shows that nanotubes running the entire thickness of the membrane are so obtained. Indeed gold de- Amperometric experiments were performed using an position proceeds from the activated pore walls rather thanEG&G Princeton Research 273A potentiostat/galvanostat. a base layer of metal. As a result, there is no competitionElectrochemical experiments were carried out in a conven- between elongating the tubules and closure of the tubules totional one-compartment cell with a three-electrode conﬁgu- form solid ﬁbers. Increasing the plating time leads to a thick-ration holding 20 ml of the supporting electrolyte (phosphate ening of the tubules walls . In this work, a depositionbuffer, PB or PB + 0.1 mM hydroquinone, H2 Q). The three- time of 17 h at 4 ◦ C was employed leading to the formationelectrode system consisted of gold nanotubes arrays placed of open nanoscopic channels with a thickness of 70 nm andbetween an Au disk and an O-ring with a surface area of a length of 20 m. This deposition time is a good compro-0.071 cm2 as working electrode, an Ag/AgCl reference elec- mise between the need to have a void space inside the tubestrode and a Pt foil as counter electrode. large enough to allow the immobilization of a huge amount For comparison, a conventional Au foil electrode from of biomolecules and the need to have a mechanically sta-Goodfellow was also used as working electrode. We further ble system, robust enough to be used as an electrochemicalcall this conventional electrode “macroelectrode”. Gold ﬁlms biosensor. A too thin gold layer cannot efﬁciently protect thewere cleaned by cycling potentials continuously between PC membrane against degradation by the reagents used in the−0.2 and 1.4 V versus Ag/AgCl in 0.1 M sulfuric acid at a different steps of functionalization.scan rate of 100 mV/s until reproducible scans were recorded. For amperometric experiments at controlled potential, thebackground current was allowed to decay to a steady valuebefore aliquots of stock hydrogen peroxide solution wereadded. All experiments were carried out at room tempera-ture. The solutions were purged with puriﬁed nitrogen for atleast 20 min to remove oxygen prior to each series of mea-surements. A magnetic stirrer and a stirring bar provided con-vective transport for the electrochemical experiments. Characterization of the morphology of gold nanotubes wascarried out by scanning electron microscopy (SEM) using ahigh resolution FEG digital scanning microscope 982 geminifrom LEO, operating at 1 kV. After Au electroless deposition,the templating membranes were dissolved by dipping themfor 2 min in dichloromethane. The free Au nanotubes werethen recovered from the solution by ﬁltration through a silvermembrane, copiously rinsed with dichloromethane to removeany residual PC and then analysed using FEG-SEM. The surface chemical composition was determined at each Fig. 1. FE-SEM image of an array of Au nanotubes, recorded after dissolu-step of functionalization by XPS using a SSIX probe (SSX tion 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 responses of PC/Au/MPE/GA/HRP, bare PC/Au and bare Au(Fig. 2) in order to compare its morphology and roughness ﬁlm (Goodfellow) to the injections of 5 × 10−4 M H2 O2 werewith the surface of electroless gold deposited on a PC mem- measured at different potentials in the range +0.3 to −0.8 Vbrane. Images in this ﬁgure are depicted at the same magniﬁ- versus Ag/AgCl in PB and PB + H2 Q (0.1 mM) solutionscation (50 000×) in order to emphasize the important differ- (Fig. 3a and b, respectively). Each point in both graphs isence in roughness between the two types of Au electrodes. the mean response of three consecutive additions of H2 O2 . AThe roughness of the electroless gold surface is much higher continuous increase of the reduction current up to −0.8 V wasthan that of evaporated gold foil. Indeed, gold nanotubes are observed for the three studied systems. However, signiﬁcantmade of discrete gold particles with an average size of 30 nm. differences were observed between these three types of elec-Considering their high surface to volume ratio, these gold trodes. Indeed, Fig. 3 clearly shows that gold nanoparticlesnanoparticles are interesting materials for the catalytic ac-tion towards the electrooxidation and the electroreduction ofhydrogen peroxide. Hou et al.  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 wasachieved via carbodiimide activation (EDC + NHS) of thecarboxylic acids to a reactive intermediate that can undergonucleophilic attack by amine moieties on free lysine chains ofthe enzyme. At the end of the functionalization pathways, thepresence of HRP enzyme immobilized on both types of sur-faces was clearly conﬁrmed by XPS. Indeed, modiﬁcationsin the C1s region and the increase of N1s and O1s peak areaswith the concomitant reduction of the Au4f and S2p peaks arein agreement with the ﬁxation of amino acids side chains ofa polypeptide backbone.3.2. Inﬂuence of gold surface on the electrochemical Fig. 3. Hydrodynamic voltammograms obtained using (᭹) bare Au, ( ) PC/Au/MPE/GA/HRP and ( ) PC/Au as working electrode for the analysisbehavior of hydrogen peroxide of 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 M The hydrodynamic voltammograms for H2 O2 detection phosphate buffer (pH = 7.2) with addition of 1.0 × 10−4 M hydroquinoneat various gold electrodes were investigated. The steady-state in the medium.
M. Delvaux et al. / Analytica Chimica Acta 525 (2004) 221–230 225deposited PC membrane (PC/Au) have a remarkable electro- in PB and H2 Q solutions. These high faradic-to-capacitivecatalytic action towards the reduction of H2 O2 throughout all current ratios should give better properties, e.g. a lower limitthe investigated range of potentials both in phosphate buffer of detection for the resulting biosensor.and in hydroquinone solutions. Nano-PC/Au also catalysesthe hydrogen peroxide oxidation (data not shown), although 3.3. Electrode response characteristicsthis effect is less pronounced than in the case of H2 O2 re-duction. On the contrary, with or without any mediator in 3.3.1. Calibration of the sensorsthe solution, the response to hydrogen peroxide at bare Au In order to conﬁrm the catalytic action of the electro-macroelectrode was very low. less gold nanoparticles deposited on PC membrane, the am- The sensitivity of the enzyme-modiﬁed nanoelectrodes perometric response to injections of H2 O2 on bare PC/Au,(PC/Au/MPE/GA/HRP) is between the sensitivity of bare Au PC/Au/MPE/GA/HRP and bare Au macroelectrode weremacroelectrode and that of nano-PC/Au. This lower sensitiv- compared at several ﬁxed potentials (+0.1, 0, −0.1, −0.2,ity of the HRP modiﬁed Au nanotubes compared to bare Au and −0.5 V versus Ag/AgCl) in both PB and H2 Q solutions.nanotubes arises from the coverage of the gold nanoparti- In all cases, when an aliquot of H2 O2 was added into thecles by HRP molecules that considerably reduce the electro- solution, the reductive current rose steeply to reach a sta-less Au active electrode area. The advantages to functional- ble value. However, the response time and the amplitude ofize the gold electroless surface by ﬁxation of HRP is however the current change were drastically different on each typedemonstrated in Fig. 4a and b, where the faradic-to-capacitive of electrode. PC/Au and PC/Au/MPE/GA/HRP systems ex-ratios for the different electrodes in a potential range from hibited a rapid and sensitive response to changes of H2 O2+0.3 to −0.8 V are reported. These graphs show that thanks concentration. These sensors reached 95% of the steady-to a low background current, the enzyme-modiﬁed nano- state current in less than 11 s for PC/Au and in only 8 selectrode (PC/Au/MPE/GA/HRP) gives the highest ratios for PC/Au/MPE/GA/HRP, indicating the excellent electro-throughout nearly all the investigated potential range, both catalytic behavior of the gold nanotubes electrode. The very fast response time of the enzyme-modiﬁed nanoelectrode may also be attributed to the favorable nano-environment of the nanotubes system that could contribute to stabilize the bi- ological activity of the HRP to a large extent. At the bare Au macroelectrode, a much longer response time of about 35 s was obtained and very much lower sensitivity was observed. All sensors (PC/Au/MPE/GA/HRP, bare PC/Au and bare Au macroelectrode) were tested by performing calibration curves for H2 O2 under the same experimental conditions. All the calibration curves were obtained at each potential and for each sensor by the measurement of at least 10 ad- ditions of H2 O2 . Fig. 5 depicts typical calibration curves for one representative electrode, the PC/Au/MPE/GA/HRP sensor, in the presence of hydroquinone by applying sev- eral potential values. The response increases with the ap- plied potential and is linear within the whole investigatedFig. 4. Faradic-to-capacitive current ratios 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 × Fig. 5. Hydrogen peroxide calibration plots obtained at detection poten-10−1 M phosphate buffer (pH = 7.2); (b) electrolyte solution: 1.0 × 10−1 M tial going from 0.1 to −0.5 V vs. Ag/AgCl using PC/Au/MPE/GA/HRP asphosphate buffer (pH = 7.2) with addition of 1.0 × 10−4 M hydroquinone working electrode in a 1.0 × 10−1 M deaerated phosphate buffer solutionin the medium. containing 1.0 × 10−4 M hydroquinone.
226 M. Delvaux et al. / Analytica Chimica Acta 525 (2004) 221–230concentration range (5 × 10−5 to 3 × 10−3 M). Some cali-bration curves were also recorded for the different sensorson wider H2 O2 concentration ranges (1 × 10−5 to 15 ×10−3 M), in both PB solutions and in PB+H2 Q 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 + H2 Q, 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 H2 O2 injection were obtained at the goldmacroelectrode. The PC/Au/MPE/GA/HRP biosensor provides a linearcurrent response to hydrogen peroxide in H2 Q over a con- Fig. 6. Sensitivity of the amperometric response to hydrogen peroxide ascentration range of 1 × 10−5 to 6 × 10−3 M with a a function of the applied potential for the (b, c) PC/Au/MPE/GA/HRP, (a)sensitivity of 9.5 A mM−1 . In PB solution, the dynamic PC/Au/MPA/EDC,NHS/HRP, (d, e) PC/Au and the (f, g) Au macroelectrode sensor. Electrolyte solution 1.0 × 10−1 M deaerated phosphate buffer withrange is extended up to 10 mM but the sensitivity decreases and without 1.0 × 10−4 M hydroquinone.to a value of 1.5 A mM−1 . A signal saturation was ob-served at H2 O2 concentration higher than 6 × 10−3 M. Thiscan be attributed to the saturation of the enzyme-substrate  brought new information about the control of the sur-or enzyme-mediator kinetic or may be due to the forma- face state of a polycrystalline gold. The need for operating attion, at high [H2 O2 ], of the enzymatically inactive form high overvoltage induces the application of potential valuesof peroxidases denoted compound III (oxidation state + 6). at which the interference of other electroactive substances in app real samples is maximized. In the detection potential rangeThe apparent Michaelis–Menten constant (Km ) of HRP atPC/Au/MPE/GA/HRP was calculated to be 7.6 mM accord- of −0.1 to −0.5 V, the highest sensitivities are obtained withing to the Lineweaver–Burk equation. This value is slightly the bare PC/Au system, independently on the presence or ab-higher than 3.69 mM reported for the nano-Au particle/m- sence of the H2 Q mediator in the solution. The high surfacephenylenediamine polymer ﬁlm system  and 2.3 mM area of gold nanotubes compared to the macroscopic goldfor the HRP/Au colloid self-assembled monolayer electrode foil, conjugated to the high roughness of the gold electroless app surface lead to an obvious increase of the efﬁcient area of the. The higher Km value of HRP at our nanotubes-basedsystem could reﬂect diffusional limitations of the substrate electrode surface. Bare PC/Au nanoelectrode can thus be use-within the nanotubes. ful as a probe for a direct electrochemical determination of The other type of enzyme-modiﬁed nanoelectrodes H2 O2 . Nevertheless, it is important to point out that the best(PC/Au/MPA/EDC,NHS/HRP) showed similar results with limits of detection were achieved with the enzyme-modiﬁeda sensitivity of 11.3 A mM−1 at −0.1 V in H2 Q solution. nanoelectrodes, because of higher faradic-to-capacitive cur-As stated previously, the need for a mediator to enhance the rent ratios (Fig. 4). Indeed, the best limits of detection are 4 ×sensor sensitivity is no longer required when using the un- 10−6 and 8 × 10−5 M, based on signal-to-noise of three, formodiﬁed PC/Au electrode. the PC/Au/MPE/GA/HRP and PC/Au systems, respectively, The sensitivity values, calculated by the slope of each while only 10−3 M was reached with the Au macroelectrodecalibration curve in the linear range, of the different sen- device.sors at several applied potentials and for different solutioncompositions (PB or PB + H2 Q) are shown in Fig. 6. Bare 3.3.2. Mechanism of the electron transfer on HRPAu macroelectrode presents the poorest response to H2 O2 . modiﬁed electrodesThe response of such probe is at least ﬁve times lower than As already mentioned, the determination of H2 O2 wasthat of the H2 O2 sensor based on bare PC/Au throughout performed either in the presence of a charge transfer mediator,the investigated potential range. The limit of detection for hydroquinone, or in its absence. For bare PC/Au electrode andthis system is only 8 × 10−4 M at an applied potential of bare Au macroelectrode, the presence of the mediator has a−0.2 V in hydroquinone solution. This poor response on the rather weak effect on the detection of H2 O2 . On the othergold macroelectrode is not surprising, because of the large hand, the enzyme-modiﬁed nanoelectrodes show a largelyoverpotentials associated to both oxidation and reduction of enhanced sensitivity when adding the hydroquinone mediatorthis analyte on Au surfaces. The electrochemical detection in the medium. The catalytic process of HRP immobilizedof hydrogen peroxide on gold has often been neglected in onto gold surfaces can be expressed as follow :the literature because of lack of proper surface control andmechanistic understanding. Only Kauffmann and coworkers HRP(Fe3+ ) + H2 O2 → compound I + H2 O (1)
M. Delvaux et al. / Analytica Chimica Acta 525 (2004) 221–230 227compound I + H2 Q → compound II + BQ (2) tween the active center of HRP and the electrode and/or from the direct reduction of H2 O2 at the remaining gold surface.compound II + H2 Q → HRP(Fe 3+ ) + BQ + H2 O (3) According to the high faradic-to-capacitive current ratio obtained for the PC/Au/MPE/GA/HRP sensor in phosphatenet reaction : BQ + 2e− + 2H+ → H2 Q buffer (Fig. 4a), it is possible that a certain amount of HRPwhere compound I (oxidation state + 5) and II (oxidation participates in direct electron transfer with the electrolessstate + 4) represent the enzyme intermediates in the reaction gold surface. In this case, the enzyme immobilized on theand H2 Q and BQ represent hydroquinone and benzoquinone, electrode is oxidized by hydrogen peroxide according to reac-respectively. In the ﬁrst two electrons transfer step, H2 O2 is tion (1) and then subsequently reduced by electrons providedreduced to water and the HRP is oxidized to compound I. by an electrode as described in reaction (4):Compound I is then reduced in a one electron step to form compound I + 2e− + 2H+ → HRP(Fe3+ ) + H2 O (4)compound II, and then compound II is reduced to the orig-inal form of HRP by the redox mediator (H2 Q). The BQ is This direct electron transfer, which is usually less efﬁcientsubsequently reduced to H2 Q by a rapid reaction involving than mediated electron transfer can also be present in thethe consumption of two electrons from the electrode. The enzyme electrode used in hydroquinone solution. Indeed,resulting cathodic currents are therefore correlated with the Lindgren et al.  have demonstrated that both direct andconcentration of both hydrogen peroxide and mediator in the mediated electron transfers can simultaneously occur in ansolution. The dependence of the amperometric determination enzyme-modiﬁed electrode. Not all the HRP molecules wereof H2 O2 on the concentration of the mediator was investi- equally involved in these two processes. In their work, thegated. Fig. 7 shows that the response of the enzyme elec- high percentage (40–50%) of the HRP able to participatetrode to 5 × 10−4 M H2 O2 at an applied potential of −0.1 V in direct electron transfer has been ascribed to phenolic andincreases sharply as the concentration of hydroquinone in- quinoidal groups present in carbon-based electrodes, whichcreases and then leveled off at 1 × 10−4 M H2 Q. At low me- mimicked the structure of the electron donor substrates of per-diator concentration, the current response is limited by the oxidases. In our system, the use of SAM allowing the immobi-enzyme-mediator kinetic. When the mediator concentration lization of enzymes in close contact with the active surface ofis high, the current response becomes limited by the enzyme- the electrode, as well as the use of gold nanotubes that createsubstrate kinetics. a conﬁned environment surrounding the enzyme molecules, In our enzyme-modiﬁed nanoelectrodes, it is difﬁcult could facilitate the direct electron transfer between the activeto distinguish between the electrocatalytic action of gold center of the biomolecule and the gold surface.nanoparticles and a direct electron transfer between the hemegroup of HRP and the gold nanoparticles on the reduction 3.3.3. Repeatability and reproducibility of the HRPof H2 O2 . Indeed, from previous results , we know that enzyme electrodethe use of short alkyl chain thiols on gold electroless sur- The repeatability of the PC/Au/MPE/GA/HRP system wasface does not lead to a complete coverage of the surface. A tested both in H2 Q (0.1 mM) and in PB solution at −0.1 V.part of the underlying electrode, which was estimated at 24% The relative standard deviations determined for 10 successivefrom cyclic voltammetry measurements, is still available af- injections of a 2.5 × 10−4 M H2 O2 sample was 8 and 7% inter functionalization for the direct electrochemical reduction H2 Q and PB, respectively. At the same applied potential, theof H2 O2 . The signal-response for the enzyme electrode in PB calculated RSD for the PC/Au system was slightly higher, butsolution can thus come from the direct electron transfer be- always below 15%. The electrode-to-electrode reproducibil- ity was estimated from the response to 2.5 × 10−4 M H2 O2 at nine different biosensors in PB and PB + H2 Q solutions and at several ﬁxed potentials (0, −0.2, and −0.5 V). The relative standard deviations are: 14, 9 and 11% at a detection potential of 0, −0.2 and −0.5 V in PB + 0.1 mM H2 Q and 15 and 10% at a potential of −0.2 and −0.5 V in PB. These results show that good reproducibility and repeatability are reached with the enzyme-modiﬁed nanoelectrodes (PC/Au/MPE/GA/HRP system). 3.3.4. Storage stability of the HRP enzyme electrode The storage stability was investigated by measuring every week over a 5 months period, the response to 2.5 × 10−4 MFig. 7. Variation of the amperometric response of PC/Au/MPE/GA/HRP H2 O2 of a PC/Au/MPE/GA/HRP biosensor at −0.1 V. HRPelectrode for the analysis of 5.0 × 10−4 M hydrogen peroxide as a functionof the hydroquinone concentration in phosphate buffer. Applied potential: has been found to be very stable in solution  so the HRP−0.1 V. biosensor was stored in PB at 4 ◦ C between each measure-
228 M. Delvaux et al. / Analytica Chimica Acta 525 (2004) 221–230 1.0 × 10−3 /2.0 × 10−4 1.2 × 10−3 PB + H2 Q 0.4 2.3 × 10−3 /2.0 × 10−4 at leastFig. 8. Selectivity of the PC/Au/MPE/GA/HRP sensor as a function of the 2.3 × 10−3applied potential; comparison of the amperometric current obtained for (a)5.0 × 10−4 M ascorbic acid, (b) 5.0 × 10−4 M H2 O2 and (c) a + b + 5.0× 10−4 M uric acid + 5.0 × 10−4 M acetaminophen (blend). Electrolyte PB Aumedium: 1.0 × 10−1 M phosphate buffer with addition of 1.0 × 10−4 M 1hydroquinone. 1.0 × 10−4 /5.0 × 10−3 at least Analytical characteristics for the PC/Au/MPE/GA/HRP, the PC/Au and Au sensors at −0.1 V vs. Ag/AgCl in PB and in PB + 1.0 × 10−4 M H2 Qment. 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 quite 1.3 × 10−4 PB + H2 Qefﬁcient for retaining the activity of HRP. This could be dueto the strong interaction between the enzyme and the gold 14nanoparticles within the nanotubes. 3.0 × 10−4 /5.0 × 10−3 at least3.3.5. Interference study Despite the low applied potential, many compounds mayinterfere 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 oxidases 3.0 × 10−4that produce H2 O2 and the HRP to measure for instance, glu- PC/Aucose in different types of samples, we have investigated, in the 10.5 PBpotential range +0.4 to −0.8 V the interference of uric acid(UA), ascorbic acid (AA) and acetaminophen (AC), which are 1.0 × 10−5 /6.0 × 10−3common 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 the 9.2 × 10−6 PB + H2 Qcurrent response obtained for each interfering substances inPB in the presence or not of H2 Q. Fig. 8 shows the current 9.5response recorded at the PC/Au/MPE/GA/HRP sensor PB +H2 Q solution to the injection of 5.0 × 10−4 M AA (a), 5.0 PC/Au/MPE/GA/HRP 4 × 10−5 /1.0 × 10−4× 10−4 M H2 O2 (b) and a blend of 5.0 × 10−4 M AA, 5.0× 10−4 M H2 O2 , 5.0 × 10−4 M UA and 5.0 × 10−4 M AC(c). It clearly appears that, in the potential range from 0 to 2.0 × 10−5−0.8 V, these substances do not signiﬁcantly interfere withthe electrode response to H2 O2 . Only ascorbic acid, which 1.5 PBhas a low oxidation potential, causes the electrode to de-viate by more than 74% of its original value in the anodic (A mM−1 )branch (at potential > +0.1 V). The bare PC/Au electrode Detection limit (M) Linear range (M)shows the same behavior than PC/Au/MPE/GA/HRP systemthroughout the whole investigated potential range, except that Electrolyte Sensitivityuric acid produces an anodic signal at 0 and −0.1 V in both Table 1electrolyte solutions. This results in a low negative interfer-ence, less than 18% (decrease in the cathodic response) in
M. Delvaux et al. / Analytica Chimica Acta 525 (2004) 221–230 229the cathodic mode of the sensor operation. On the bare Au Acknowledgementsmacroelectrode, the effect of interfering compounds is morepronounced. Ascorbic acid is oxidizable on the electrode SD-C thanks the Belgian National Fund for Scientiﬁc Re-over a larger potential range (from +0.4 to −0.1 V) while search (FNRS) for her research associate position. MD isUA and AC give anodic and cathodic currents, respectively, ` a fellow of “Fonds pour la formation a la recherche dansfrom −0.1 to −0.6 V. The presence of these compounds in l’industrie et dans l’agriculture” (FRIA, Belgium). The “Ser-the sample can produce a large deviation in the current re- vices F´ d´ raux des Affaires Scientiﬁques, Techniques et Cul- e esponse (e.g. 78% decrease signal at −0.3 V in phosphate turelles” are thanked for their ﬁnancial support in the framebuffer). of the “Pole d’Attraction Interuniversitaire” (IAP network P5/03).4. 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