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Electrochimica Acta 55 (2010) 4247–4255

Contents lists available at ScienceDirect

Electrochimica Acta

journa l homepage: www.e lsev ier .com/ locate /e lec tac ta

AM-modified microdisc electrode arrays (MDEAs) with functionalizedarbon nanotubes

nthony Guiseppi-Eliea,b,c,∗, Abdur Rub Abdur Rahmana, Nikhil K. Shuklaa

Center for Bioelectronics, Biosensors and Biochips (C3B), College of Engineering and Science, Clemson University, 100 Technology Drive, Anderson, SC 29625, USADepartment of Chemical and Biomolecular Engineering, Clemson University, Clemson, SC 29634, USADepartment of Bioengineering, Clemson University, Clemson, SC 29634, USA

r t i c l e i n f o

rticle history:eceived 19 August 2008eceived in revised form 12 December 2008ccepted 22 December 2008vailable online 30 December 2008

eywords:icrodisc electrode array

arbon nanotubes

a b s t r a c t

The construction of a novel voltammetric transducer employing mixed acid-chopped, oxidativelymodified, single-walled carbon nanotubes (HOOC–SWNT–COOH) covalently coupled to alkane thiol self-assembled monolayers (SAMs) that were deposited onto gold microdisc electrode arrays (MDEA-Au)(1296 microdiscs, 100 �m diameter each) has been demonstrated. The chemisorption of cysteamine (CA)and 11-amino-1-undecanethiol (11-AUT) SAMs onto the MDEAs resulted in MDEA-Au|CA and MDEA-Au|11-AUT, which when conjugated with the HOOC–SWNT–COOH produced MDEA-Au|CA|SWNT andMDEA-Au|11-AUT|SWNT electrodes. Electrodes were characterized by multiple scan rate cyclic voltam-metry in the presence of ferrocene monocarboxylic acid and by AFM and compared. As expected, CA

oltammetryonolayers

ransducers

modification of MDEA-Au produced a disordered film that did not alter the effective surface area of theMDEA while 11-AUT modification resulted in passivation and a 50% reduction in effective surface area. Theconjugation of the SWNT to the CA-modified and the 11-AUT-modified MDEA-Au surface returned well-resolved voltammograms with highest peak currents (Ipa = 5.54×10−4 A, Ipc = 2.34×10−4 A at 100 mV/s).The effective area of MDEA-Au|CA|SWNT was increased by 200% while that of MDEA-Au|11-AUT|SWNTwas increased by 100%. AFM showed SWNTs to be vertically oriented on the CA-modified surface but to

and b

be horizontally arranged

. Introduction

Carbon nanotubes (CNTs), with the unique properties of highhemical stability, high surface area, high tensile strength, excel-ent stiffness and high electrical conductivity [1] are now beingxtensively investigated for the construction of supramolecularano-bio-assemblies and nanobiosensors [2–8]. CNTs are expectedo be widely used in many fields including nanoelectronic devices,s electron field emission sources, and potentially, as hydrogentorage materials [9]. The electronic properties of CNTs, includ-ng their support of electron transfer and redox mediation, makehem suitable for the preparation of electrode materials and forse in chemical and biological sensors [10,11]. Immediately after

heir discovery and observed remarkable properties [12,13], CNTsave attracted interest and excitement across a broad spectrum ofcience and technologies including engineering, materials, chem-stry, biology and medicine [14]. The reports from Baughman et

∗ Corresponding author at: Center for Bioelectronics, Biosensors and BiochipsC3B), College of Engineering and Science, Clemson University, 100 Technology Drive,nderson, SC 29625, USA. Tel.: +1 864 656 1712; fax: +1 864 656 1713.

E-mail address: guiseppi@clemson.edu (A. Guiseppi-Elie).

013-4686/$ – see front matter © 2009 Published by Elsevier Ltd.oi:10.1016/j.electacta.2008.12.043

undled on the 11-AUT surface.© 2009 Published by Elsevier Ltd.

al., regarding their impressive mechanical and electronic proper-ties, suggest many potential applications, ranging from their use aselectrochemical actuators to conductive, high strength compositematerials [15,16]. The use of CNTs as biosensor electrode materialshas been demonstrated [17,18] and their application [19,20], sincethe first report of Guiseppi-Elie et al., as direct biosensors [11] hasgained significant momentum [21–24].

The quest for novel nanodevices and the growing interest innanobiotechnology has produced an explosion of work in thestudies of carbon nanotubes. The physicochemical properties andstructure of un-functionalized nanotubes make them suitable foruse in many potential technological applications. However, func-tionalization of CNTs results in multiple options for their refinementand use. Covalent functionalization changes the chemical proper-ties of nanotubes dramatically, and has been successfully appliedin the covalent binding of the redox proteins [25,26]. The oxidativefunctionalization of nanotubes allows for the efficient immobiliza-tion of glucose oxidase allowing the CNTs to simultaneously act as

an immobilization matrix, as a redox mediator or as an electronicshuttle.

We have previously reported on the combination of CNT withGOx and demonstrated the oxygen-free, direct bioelectrochemistryof flavin adenine dinucleotide (FAD), the redox active co-factor, on

4 chimica Acta 55 (2010) 4247–4255

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248 A. Guiseppi-Elie et al. / Electro

NT beds cast onto glassy carbon macro electrodes [11]. This sys-em offered the opportunity for research on the electron transferhemistry of these and other biological redox molecules such asseudoazurin [27]. In this paper, we report the strategic chemi-al functionalization of SWNTs and the covalent immobilization ofunctionalized SWNTs to alkane thiol-modified, microlithograph-cally fabricated microdisc electrode arrays (MDEA, gold). The

icrodisc Electrode Arrays (MDEAs) are inert, array microelec-rodes formed from a microlithographically patterned passivatingayer of insulating silicon nitride on magnetron sputter-depositedold. MDEAs offer improved signal-to-noise, enhanced dynamicange and greater signal compared to planar electrodes of similarrea; the result of hemispherical diffusion to individual discs of therray. This electrode format thus offers significant advantages in theevelopment of chemical and biological sensors [23]. The resultingovel supramolecular assembly has been demonstrated to serve aswide dynamic range chemical transducer. In this report, MDEA-

00Au (1296 discs, 100 �m in diameter) comprising an hexagonallose packed array with uniform center-to-center disc spacing thatas four times the disc radius (d/r = 4) and of total electrode area of.1 cm2, were alkane thiol-modified and functionalized by covalent

mmobilization of acid-chopped SWNTs.Here is examined the effect of the covalently tethered SWNT

ayer on the dynamic electrochemical behavior of a modellectroactive specie, ferrocene monocarboxylic acid, at the SWNT-unctionalized microdisc array electrodes. Aspects of electronransfer and mass transport in the presence and absence of theWNTs are examined using Multiple Scan Rate Cyclic VoltammetryMSRCV). In addition to CV experiments, finite element simulationsFEM) were performed to augment the experimental study of the

DEA electrochemical performance and AFM was used to examinehe nanostructure of the supra-molecular surfaces.

. Experimental

.1. Materials

Gold microdisc electrode arrays (MDEA-100Au) with individ-al disc diameters of 100 �m and total active area of 0.1 cm2 wereurchased from ABTECH Scientific, Inc. (Richmond, VA). The MDEA-00Au possessed 1296 microdiscs, each 100 �m in diameter andositioned 200 �m on centers in a hexagonal close-packed arrange-ent. The single-walled carbon nanotubes (SWNT) used in this

tudy were commercially supplied (Nanotubes @ Rice) as an aque-us suspension stabilized with laurel sulfate. Cysteamine (CA) wasurchased from Sigma (St Louis, MO). Aminoundecane thiol (11-UT) was obtained from Dojindo Laboratories (Kumamoto, Japan).ll other chemicals were of analytical grade and were used without

urther purification. The water used was Milli-Q quality (18 M�).

.2. Methods

.2.1. Electrode preparationThe gold MDEAs were cleaned according to the standard oper-

ting procedures A and E provided by ABTECH Scientific, Inc.28,29]. Briefly, this involved sequentially immersing the elec-rodes in boiling solutions of trichloroethylene and acetone formin each followed by ultrasonicating in isopropyl alcohol (3 min),ashing profusely with DI water, exposure to UV generated ozone

UV-CleanTM; Model 135500, Boekel Industries, Inc., Feasterville,

ennsylvania, USA) for 15 min followed by DI water washing andven drying. The cleaned electrode surfaces were then function-lized by overnight immersion in 2 mM ethanolic solutions of CAr 11-AUT at RT. This treatment resulted in the formation of self-ssembled monolayers of the alkane thiols on the gold electrode

Fig. 1. Schematic illustration of the final multilayered carbon nanotube-SAM conju-gate structure showing the microdisc electrode array (MDEA) substrate, the alkanethiol SAM and the amide conjugated acid-chopped SWNT.

surface and the presentation of terminal amine functionalities tothe electrode-solution interface.

2.2.2. CNT modificationThe SWNT used in this work were fabricated by the pulsed-laser

vaporization method. This method produced hexagonally packedbundles of carbon nanotubes with diameters between 12 and 14 Å,intertube separation within a bundle of ∼17 Å and an average bun-dle diameter of ∼100 Å. The process of purification and shorteningof the tubes involved sonication in a 3:1 ratio of H2SO4:HNO3 solu-tion followed by cycles of washing and cross-flow filtration withDI water [30,31]. This treatment produced SWNTs that were ter-minated at both ends with –COOH groups, and the generationof the acid functionalities on the SWNT was confirmed by FTIRtransmission spectroscopy. The activated SWNTs (2 mg/mL) wereallowed to stand for 4 h in 5 mL of freshly prepared solution of25 mM 1-ethyl-3-(3-diethylaminopropyl) carbodiimide (EDC) and25 mM N-hydroxysulfosuccinimide (NHS) prepared in HEPES buffer(0.01 M, pH 7.4). A small volume (3 �L) of this solution containingthe activated SWNTs was transferred to the alkane thiol-treatedsurface of an MDEA and allowed to chemically conjugate with thepreviously treated electrode surface overnight at room tempera-ture and to yield the configuration MDEA-100Au|SAM|SWNT-COOHwhere the SAM may be CA or 11-AUT.

Fig. 1 schematically illustrates the structure of the finalsupramolecular assembly on the MDEA. In addition to these twosystems, an MDEA-100Au electrode that was not functionalizedby alkane thiol, MDEA-100Au|SWNT–COOH, served as the control,while an unmodified MDEA without any alkane thiol modification(MDEA-100Au) served as the blank in all analyses.

2.2.3. Characterization of devicesThe electrochemical characterization of the various supramolec-

ular assemblies, controls and blanks was performed at 20 ◦C±1 ◦Cusing a computer controlled Electrochemical Analyzer model BAS100B (Bioanalytical Systems, Inc., Lafayette, IN). Multiple scan ratecyclic voltammetry (MSRCV) within 1.0 mM FcCOOH prepared in0.1 M Tris/0.1 M KCl buffer solution (pH 7.2) was performed in astandard three-electrode electrochemical cell by sweeping overthe range of 0 to +0.80 V at scan rates that ranged from 10 to100 mV/s. Each of the six fabricated electrodes was made the work-ing electrode in turn. A miniature Ag/AgCl, 3 M Cl− electrode (RE

803, ABTECH Scientific, Inc., Richmond, VA) and a large surfacearea platinum gauze (3 in.×2 in.) electrode (Alfa Aesar, USA) wereused as a reference and counter electrodes, respectively. AtomicForce Microscopy (AFM) was performed on a Veeco AFM instrument(USA) with a CP-II scanner in a tapping mode. The image analysis

A. Guiseppi-Elie et al. / Electrochimica Acta 55 (2010) 4247–4255 4249

crolith

wS

2

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k

k

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wor[

Fig. 2. Wafer traveler for the mi

as performed using WSxM 4 software from Nanotec Electronica.L. (Spain).

.2.4. Simulation of MDEA MSRCV responsesFinite Element modeling of the MDEA electrodes was performed

sing the method described by Lavacchi et al. [32] and based onhe model developed by Davies and Compton [33]. The simulatedeaction is a simple, single electron transfer of the type:

+ e− k←→R (1)

The initial and bulk concentrations of species O are 1 mM andhe reduced species R is assumed to initially absent in the bulkolution. The concentration–overpotential relationship was giveny the Butler–Volmer equation:

f = k0 exp[−˛F(E − Estd)

RT

](2)

b = k0 exp[

(1− ˛)F(E − Estd)RT

](3)

here k0 is the standard electron transfer rate constant, taken as.08×10−2 m/s, for FcCOOH. The value of ˛ is assumed to be 0.5.std is fixed at 0 V for the purpose of this simulation. The value ofis the gas constant (8.31 J/K), T is the temperature (298 K) and F

s the Faraday’s constant (=96,487 C/mol). The mass transport of Ond R species is often governed by diffusion, convection and migra-ion according to the Nernst–Planck equation. By considering annstirred solution containing an excess supporting electrolyte, theffects of convection and migration can be neglected. Thus, theass transport mechanism of these species is reduced to primarilydiffusion-controlled process,

∂ci = Di∇2ci (4)

∂t

here ci = concentration of the ith specie, Di = diffusion coefficientf the ith specie. The diffusion coefficient of both the oxidizing andeducing specie was taken as 5.73e−10 m2/s for FcCOOH as reported34]. All boundaries were assigned insulation/symmetry boundary

ographic fabrication of MDEAs.

condition (BC) except for boundary 2, which was assigned a flux BCwith flux given by the following relationship:

M = Cox kf − Cred kb (5)

Optimal mesh density was determined by refining the meshuntil no further changes in output occurred upon mesh refinement.Geometric singularities were particularly meshed at a higher den-sity than other areas. Simulations were performed for all the scanrates corresponding to the experimental scans.

3. Results and discussion

3.1. Unmodified ultra-microelectrodes and microdisc electrodearrays

The microdisc electrode array is a single electrode, here fabri-cated from 100 nm of magnetron sputter-deposited gold on top of10 nm adhesion promoting titanium/tungsten (Ti/W) alloy, whichis presented as a hexagonal close-packed array of discrete, disc-likeopenings formed through an insulating silicon nitride (Si3N4) layer.The discs or openings are formed by fluoro plasma etching of theSi3N4 through a photolithographically patterned hard-baked pho-toresist. Fig. 2 summarizes the several additive and subtractive stepsin the microlithographic fabrication of microdisc arrays [35]. TheMDEA-100Au electrochemical transducer comprises 1296 discrete100 �m diameter discs (r = 50 �m) arranged in an HCP configura-tion with a center-to-center distance between discs d of 200 �m(d/r = 4). The MDEA electrode geometry has the potential to transi-tion from semi-infinite linear diffusion to hemispherical diffusionof electroactive species to the electrified interface during redoxreactions. For reversible redox reactions this transition is a func-tion of the diameter of the opening, the center-to-center spacingbetween discs, and the voltammetric scan rate. MSRCV was first

performed on an isolated 100 �m diameter disc and its electro-chemical characteristics were compared with that of an isolated10 �m disc. Isolated disc electrodes have an independent diffu-sion zone as opposed to potentially overlapping diffusion zones inthe case of microdisc electrode arrays. The knowledge of diffusion

4250 A. Guiseppi-Elie et al. / Electrochimica Acta 55 (2010) 4247–4255

F and ub 00 mV

cts

dCaadiidtrstwmCaitstus

1mmis

where n is the number of electrons involved in the redox process,F is Faraday’s constant (96,485), D is the diffusion coefficient, [Cred]is the molar concentration of the reduced form of the electroactiveanalyte (15 mM) and ro is the radius of the working ultra-microdiscelectrode (5 �m). Applying this equation yields a diffusion coeffi-

ig. 3. Multiple scan rate cyclic voltammetry (MSRCV) of micro- (A and B = 100 �m)uffered 0.1 M KCl solution (pH 7.2) over the range of 0 to +0.80 V at scan rates 10–1

haracteristics at individual 100 �m disc is germane to the interpre-ation of the voltammetric responses of a collection of such discs,uch as in the case of MDEA-100 transducer.

Fig. 3 presents the experimentally obtained MSRCV of 100 �miameter and a 10 �m diameter isolated disc electrodes (Fig. 3A and, respectively) and their associated COMSOL simulations (Fig. 3Bnd D, respectively). The 100 �m disc exhibited peak current char-cteristic which was indicative of analyte transport in the lineariffusion regime. This is due to the fact that the diameter of the disc

s much larger than the diffusion length of the electroactive speciesn the time duration of the potential scan. The 10 �m diameterisc on the other hand displays steady state current characteris-ics and shows a plateau current that was independent of scanate. The efficiency of the ultra-microelectrode was clearly demon-trated by the observation that at 100 mV/s the current density athe 100 �m diameter microdisc electrode was 6.4×10−1 pA/�m2

hereas at 100 mV/s the current density at the 10 �m diametericrodisc electrode was 1.6 pA/�m2; a 10-fold advantage. Thus, the

Vs of un-modified and CNT derivatized MDEA-100Au electrodesre expected to always exhibit peak current characteristics, evenf the discs are placed sufficiently far away from each other so aso avoid diffusion zone overlap. COMSOL simulation was used toimulate these responses and those results are plotted alongsidehe actual data. For the reversible one-electron transfer reactionsed in these experiments, these voltammograms can be accuratelyimulated using the formalism described earlier.

The scan rate dependence of the anodic peak currents of the

00 �m diameter microdisc electrode and the 10 �m diametericrodisc electrode are presented in Fig. 4. The 100 �m diametericrodisc electrode showed the familiar Randles–Sevcik character-

stics while the 10 �m diameter ultra-microdisc electrode revealedteady state currents that were independent of the square root of

ltra-micro- (C and D = 10 �m) electrodes in 15 mM FcCOOH prepared in phosphate-/s. A and C are experimental data and B and D are simulations.

scan rate. The steady state current of the 10 �m diameter ultra-microdisc electrode is given by Refs. [33,36].

iss = 4nFD[Cred]ro (6)

Fig. 4. Anodic peak current, Ipa, as a function of the square root of the voltammetricscan rate for the 100 �m micro- and 10 �m diameter ultra-microelectrodes in 15 mMFcCOOH prepared in phosphate-buffered 0.1 M KCl solution (pH 7.2) for scan ratesof 10–100 mV/s at RT.

A. Guiseppi-Elie et al. / Electrochimica Acta 55 (2010) 4247–4255 4251

F 100 �S in 15r

cloirs

i

wi[tefevetd

umKapi1oatacafteigitc(ewtkdf

ig. 5. Multiple scan rate cyclic voltammetry (MSRCV) of (A) unmodified MDEA-AuWNT modified MDEA-100 electrode, experimentally obtained. CVs were obtainedange of 0 to +0.80 V at scan rates of 10–100 mV/s at RT.

ient of 4.21×10−10 m2/s, which compares quite favorably with theiterature value of 5.73×10−10 m2/s for FcCOOH [34]. The responsesf the 100 �m diameter microdisc electrode may be taken as aris-ng from a reversible redox reaction operating in the linear diffusionegime and so the diffusion coefficient may be obtained from thelope of the Randles–Sevcik:

pa = 2.686× 105n3/2[Cred]D1/2appt�

1/2A (7)

here the other parameters in the equation have their usual mean-ng, n is the number of electrons involved in the redox process,Cred] is the molar concentration of the oxidized form of the elec-roactive analyte (15 mM) and A is active contact area of the workinglectrode (0.1 cm2). The value of D = 1.59×10−10 m2/s was obtainedrom the use of this approach and the 100 �m diameter microdisclectrode. This value compares poorly with literature values and thealue obtained using the steady state current of the ultra-microdisclectrode, suggesting that under these test conditions, this elec-rode is not operating in the purely linear regime or the radialiffusion regime.

Multiple scan rate cyclic voltammetry (MSRCV) using thenmodified MDEA-100Au electrode was performed in ferroceneonocarboxylic acid solution (15 mM in 0.1 M phosphate-buffered

Cl) and resulted in voltammograms shown in Fig. 5A. The peak sep-ration between the anodic peak potential (Epa) and the cathodiceak potential (Epc) values (�Ep) were noticeably increased upon

ncreasing the scan rate from 10 mV/s (�Ep = 137 mV) up to00 mV/s (�Ep = 166 mV) indicating increasing non-idealities in thene electron transfer process. The formal potential, E◦, which is theverage of the cathodic and anodic peak potentials, showed littleo no change as the scan rate was increased from 10 to 100 mV/snd averaged around ∼317 mV vs. Ag/AgCl. The ratio of anodic andathodic peak currents, Ipa/Ipc, for this system was found to averageround 1.6, suggesting that the oxidation of the ferrocene moiety isast under these conditions. The deviation from unity is also indica-ive of the contribution of kinetic or other complications to thelectrode process. The cathodic peak current (Ipc) was observed toncrease with the square root of the potential scan rate (�1/2), sug-esting that the analyte diffusion pattern at the electrode-solutionnterface is dominated by linear diffusion to the electrode surfacehat comprised the 100 �m discs. The apparent diffusion coeffi-ient, Dappt, was determined using the familiar Randles–Sevcik Eq.7) and the total geometric area of the electrode, 0.1 cm2. The appar-nt diffusion coefficient was estimated to be 1.74×10−10 m2 s−1,

hich is different than values obtained at single microdisc elec-

rodes above. The heterogeneous electron transfer rate constant,s, was determined using a technique developed by Laviron foriffusion-controlled electrode reactions [37]. Developed primarilyor the determination of rate constants influenced by adsorption,

m experimentally obtained, (B) unmodified MDEA-Au 100 �m simulated, and (C)mM FcCOOH prepared in phosphate-buffered 0.1 M KCl solution (pH 7.2) over the

the ks values can be calculated at each scan rate using the equation:

log ks=˛ log(1− ˛)+ (1− ˛) log ˛−log(

RT

nF�

)− ˛(1− ˛)

nF �Ep

2.3RT(8)

where the parameters have their usual meanings and values and� is the scan rate. The value of ˛ was determined from the slopeof the Epc vs. log � plot, which is equal to 2.53RT/˛nF. The averageks calculated using this method was found to be 5.17×10−3 s−1.Fig. 5B shows the MSRV simulation of the unmodified elec-trode in ferrocene monocarboxylic acid solution (15 mM in 0.1 Mphosphate-buffered KCl). Here the diffusion coefficient is parame-tized at the experimentally determined value of 1.74×10−10 m2 s−1.

It is possible to simply adsorb, via the casting method, a bed ofacid-chopped SWNTs directly onto the un-modified MDEA and toevaluate its contribution to the dynamic electrochemical behav-ior of the MDEA-100 by performing MSRCV in 15 mM in 0.1 Mphosphate-buffered KCl. The resulting voltammograms over thepotential range of 0 to 800 mV at MDEA-Au|SWNT are shown inFig. 5C. These voltammograms are to be directly compared withthose obtained at unmodified gold MDEAs shown in Fig. 5A andthe simulations shown in Fig. 5B. Like at the unmodified MDEA-100Au (blank), well-defined cyclic voltammograms were obtainedat the various scan rates. Also, the values of Epa and Epc for theMDEA-Au|SWNT system were observed to be identical to those atthe unmodified MDEA-Au electrode at corresponding scan rates.This is quantitatively reflected in a comparison of the E◦ and �Ep

parameters of both systems. The average E◦ and �Ep values of315 and 158 mV, respectively, exactly match the average E◦ valueof 317 mV and average �Ep values of 158 mV at unmodified gold,indicating that while the oxidation and reduction reactions wererendered lass facile at the SWNT-modified electrodes, the over-all fundamental mechanism of the ferrocene–ferrocenium reactionremained unchanged [38]. Just like at the unmodified MDEA-Au, theIpa/Ipc ratio for the Au|SWNT system was greater than unity. How-ever, the effect of the presence of the nanotubes can be clearly seenby the increase in the magnitude of both peak anodic and cathodiccurrents at all scan rates investigated compared to the MDEA-Auelectrode without SWNT. For example, at 100 mV/s, the Ipa and Ipc

were increased by almost twofold in the presence of SWNT.

3.2. SAM-modified microdisc electrode arrays

MDEAs were surface modified with alkane thiol SAMs as a fore-runner to their subsequent derivatization with SWNTs. These thenwere similarly characterized by MSRCV in 15 mM ferrocene mono-carboxylic acid solution. Fig. 6A and B shows the multiple scanrate cyclic voltammograms of FcCOOH measured over the poten-

4252 A. Guiseppi-Elie et al. / Electrochimica Acta 55 (2010) 4247–4255

F MDEAe olutio

tmmatasbwasfii3feMwfsce

3

s

FA(

ig. 6. Multiple scan rate cyclic voltammetry (MSRCV) of (A) Cysteamine modifiedlectrode (MDEA-Au|11-AUT). CVs were obtained in phosphate-buffered 0.1 M KCl s

ial range of 0–800 mV at CA-modified (MDEA-Au|CA) and 11-AUTodified (MDEA-Au|11-AUT) MDEA’s, respectively. There was aarked difference in the CVs of ferrocene monocarboxylic acid

t the CA-modified and 11-AUT-modified 100 �m MDEA-Au elec-rodes. At CA-modified MDEAs there were clearly defined anodicnd cathodic peaks while at 11-AUT-modified MDEAs, only a steadytate anodic current was discernable as the system approached theehavior of an ultra-microelectrode with a steady state current thatas independent of scan rate. The average peak separation between

nodic and cathodic potential values (�Ep) for the MDEA-Au|CAystem (198 mV) was now larger than that observed for the unmodi-ed MDEA-Au (159 mV). The formal potential, E◦, however, was sim-

lar to that for the unmodified MDEA-Au with an average value of22 mV. The ratio of the anodic and cathodic peak currents, Ipa/Ipc,or the CA-modified MDEA-Au|CA was found to be 1.3. The het-rogeneous electron transfer rate constant, ks, for the CA-modifiedDEA system was determined to be 6.41×10−3 s−1, slightly largerhen compared to the value obtained at the unmodified MDEA sur-

ace. Oxidation profiles obtained with the use of MDEA-Au|11-AUTystem in ferrocene monocarboxylatic acid solution were not dis-ernable; consequently, typical electrochemical parameters for thelectroactive specie at this surface could not be obtained.

.3. SWNT deraviatized microdisc electrode arrays

MDEAs that were surface modified with alkane thiol SAMs wereubsequently derivatized via conjugation with acid-functionalized

ig. 7. Multiple scan rate cyclic voltammetry (MSRCV) of: (A) Unmodified MDEA-Au 100 �u 100 �m electrode conjugated to acid-chopped SWNT (MDEA-Au|CA|SWNT), and (C)MDEA-Au|11-AUT|SWNT). CVs were obtained in phosphate-buffered 0.1 M KCl solution (

-Au 100 mm electrode (MDEA-Au|CA) and (B) 11-AUT modified MDEA-Au 100 mmn (pH 7.2) over the range of 0 to +0.80 V at scan rates of 10–100 mV/s at RT.

SWNTs using hetero-bifunctional cross-linking with carbodiimide.These electrodes were then similarly characterized by MSRCVin phosphate-buffered 15 mM ferrocene monocarboxylic solu-tion. The results of multiple scan rate cyclic voltammetry forSWNT-derivatized, CA-modified MDEA (MDEA-Au|CA|SWNT) areshown in Fig. 7B. The CVs show well-defined voltammogramswith peak potentials for oxidation and reduction now occurringat more positive and more negative (less positive) potentials,respectively, compared to both the unmodified MDEA-Au andthe MDEA-Au|SWNT (Fig. 7A) systems. This translated into alarger peak potential separation, �Ep, of 172 mV compared to theMDEA-Au and MDEA-Au|SWNT systems which both had iden-tical �Ep values of 158 mV. The average E◦ value of 317 mVwas, however, exactly the same as that obtained at unmodi-fied MDEA-Au (317 mV) and similar to E◦ at MDEA-Au|SWNT(315 mV), indicating similar discharge mechanisms at all three-electrode surfaces and that covalent immobilization and possiblepreferential orientation of the SWNTs did not alter the funda-mental electrochemistry of the ferrocene–ferrocenium reaction.Most noticeable, however, is the very sharp increase in thepeak currents observed (10−4 A) compared to the unmodifiedMDEA-Au and MDEA-Au|CA systems (10−5 A). The peak cur-

rents exhibited by the MDEA-Au|CA|SWNT system were also ca.two-fold larger than corresponding peak currents at the SWNT-adsorbed MDEA without any SAM alkane thiol. The Ipa/Ipc ratiofor MDEA-Au|CA|SWNT was ca. 1.3 at all scan rates investi-gated.

m electrode with adsorbed acid-chopped SWNT, (B) Cysteamine modified MDEA-11-AUT modified MDEA-Au 100 �m electrode conjugated to acid-chopped SWNTpH 7.2) over the range of 0 to +0.80 V at scan rates of 10–100 mV/s at RT.

A. Guiseppi-Elie et al. / Electrochimica Acta 55 (2010) 4247–4255 4253

Fig. 8. Plots of the anodic peak current, Ipc, as a function of the square root ofthe voltammetric scan rate for all three supra-molecular electrode configurationscontaining SWNTs and controls; (a) un-modified MDEA-Au 100 �m electrode, (b)SWNT modified MDEA-Au 100 �m electrode, (c) Cysteamine modified MDEA-Au100 �m electrode conjugated to acid-chopped SWNT (MDEA-Au|CA|SWNT), (d)1(r

tirAa�sidbtoP

pepi(sbspt

ftasgDpafif1tt

Table 1Summary of the electrochemical parameters for each of the six electrode systemsinvestigated in aqueous ferrocene monocarboxylic acid (15 mM, PBS 7.2 Buffer at20 ◦C).

Electrode design E◦ �Ep (mV) Ipa/Ipc Aapp (cm2)* ks (S−1)

MDEA-Au 317 158 1.6 0.10 5.17E−03MDEA-Au|CA 322 198 1.3 0.10 6.41E−03MDEA-Au|11AUT NA NA NA 0.05 NAMDEA-Au|SWNT 315 158 1.3 0.21 1.21E−02MDEA-Au|CA|SWNT 317 172 1.3 0.29 3.17E−03

ified electrodes, respectively, there are larger features sizes (up to70 nm) compared to the AFM images of MDEA-Au|CA and MDEA-Au|11-AUT electrodes that showed feature sizes up to 12 nm. Thesefeature sizes confirm the successful immobilization via conjugation

Table 2Tapping mode AFM parameters for each of the electrode systems investigated.

Electrode Grain size/featureheights (nm)

RMS roughness(nm)

Average roughness(nm)

1-AUT modified MDEA-Au 100 �m electrode conjugated to acid-chopped SWNTMDEA-Au|11-AUT|SWNT), and (e) Simulation of the MDEA-Au 100 �m electrodeesponse.

For the SWNT-derivatized, 11-AUT-modified MDEA gold elec-rodes, referred to as MDEA-Au|11-AUT|SWNT, the results of MSRCVn 15 mM ferrocene monocarboxylate at various scan rates (Fig. 7C)evealed response profiles that resemble those of the MDEA-u|SWNT (Fig. 7A), i.e., without the SAM layer. In fact, similarverage electrochemical parameters (E◦ = 316 mV and 315 mV andEp = 156 mV and 158 mV, respectively) were obtained for both

ystems, suggesting a similar mechanism of discharge even uponntroduction of the 11-AUT SAM. Compared to the same SWNT-erivatized system containing CA, the observed peak currents foroth oxidation and reduction cycles were decreased. This reduc-ion in peak currents is consistent with the electrode passivationbserved at 11-AUT–modified gold MDEAs (MDEA-Au|11-AUT).eak current ratio, Ipa/Ipc, at all scan rates were of the order of 1.4.

For all three-electrode systems containing SWNT, the anodiceak current (Ipa) corresponding to ferrocene oxidation at eachlectrode surface increased linearly with the square root of theotential scan rate (�1/2) (Fig. 8). As was observed with the unmod-

fied electrode (MDEA-Au: line “a” of Fig. 8), the anodic peak currentIpa) also increased linearly with the square root of the potentialcan rate (�1/2) but with a slope that was lower than that predictedy the idealized Randles–Sevcik equation, which was simulated andhown as line “e”. All three-electrode systems containing SWNTossess slopes (lines “b”, “c” and “d” of Fig. 8) that are greater thanhat predicted by the idealized Randles–Sevcik equation.

Changes in the slopes of Fig. 8 may be interpreted as arisingrom changes in the apparent diffusion coefficient of the electroac-ive specie. However, these changes are more logically argued asrising from changes in the effective surface area, Aeff, of the nano-tructured MDEAs. The effect of the short-chain alkane thiol on theold MDEA surface did not alter the apparent diffusion coefficient,appt, (1.74×10−10 m2 s−1) for ferrocene monocarboxylic acid com-ared to the unmodified MDEA surface (also 1.74×10−10 m2 s−1),ccordingly, its effective area was the same as that of the unmodi-ed MDEA-Au, 0.10 cm2. However, there was a 3.5-fold decrease in

errocene Dappt to 4.96×10−11 m2 s−1 when the C-11 alkane thiol,1-AUT, was deposited on the gold MDEA surface. This is equivalento an effective area of 0.050 cm2; a 50% reduction in area relative tohe MDEA-Au and MDEA|CA.

MDEA-Au|11-AUT|SWNT 316 156 1.4 0.21 8.71E−03

* The apparent area, Aapp, was calculated on the basis of the apparent diffusioncoefficient obtained for the MDEA-Au electrode.

Comparison of the effective areas of all three SWNT-containingsystems resulted in values of 0.21 cm2, a 100% increase, for bothMDEA-Au|SWNT and MDEA-Au|11-AUT|SWNT and 0.29 cm2, a200% increase, for CA-modified SWNT MDEA (MDEA-Au|CA|SWNT).Clearly, the combined effect of the C-2 chain SAM in associationwith the SWNT resulted in an electrified interface that promotedthe enhanced flux or discharge of the electroactive solution-bornespecie. On the other hand, the use of the C-11 chain SAM, 11-AUT,did not enhance the effective area relative to the unmodified MDEAsurface with SWNT. The average ks calculated using the techniqueof Laviron gave values of 1.21×10−2 cm s−1, 3.17×10−3 cm s−1 and8.71×10−3 cm s−1 for MDEA-Au|SWNT, MDEA-Au|CA|SWNT andMDEA-Au|11-AUT|SWNT systems, respectively. Contrary to resultsobtained for the effective areas, the unmodified MDEA surface con-taining SWNT exhibited the highest ks value, followed by 11-AUTthen by CA. The electrochemical parameters for each of the sixelectrode systems investigated in ferrocene monocarboxylate aresummarized in Table 1.

3.4. AFM of modified and derivatized microdisc electrode arrays

Atomic force microscopy was used to determine the presence,distribution and organization of conjugated SWNTs to the twoalkane thiol-modified MDEAs. Fig. 9 shows tapping mode AFMimages of the several supra-molecular electrode assemblies of thiswork. Fig. 9A shows an un-modified MDEA-Au 100 �m electrode,which served as the control for the cysteamine modified MDEA-Au100 �m electrode (Fig. 9B) and the a 11-AUT modified MDEA-Au100 �m electrode (Fig. 9E). The CA modified and 11-AUT modifiedMDEA-Au 100 �m in turn served as controls for the conjugated,acid-chopped SWNT electrodes, MDEA-Au|CA|SWNT (Fig. 9C) andMDEA-Au|11-AUT|SWNT (Fig. 9F). Finally, Fig. 9D shows the edgestep of the transition between the Au and Si3N4 layers of a microdiscelectrode array. Images were evaluated and compared for grain size,RMS roughness and average roughness and these are presented inTable 2.

Clearly, by visual inspection of Fig. 9C and F, representing AFMimages of MDEA-Au|CA|SWNT and MDEA-Au|11-AUT|SWNT mod-

Unmodified Au 5.1 ± 1.7 1.63 6.5Au|CA 2.8 ± 0.8 0.97 9.7Au|CA|SWNT 24 ± 4.9 6.4 15.2Au|11-AUT 4.8 ± 2.9 1.8 8.9Au|11-AUT|SWNT 25.5 ± 6.7 9.5 28.7

4254 A. Guiseppi-Elie et al. / Electrochimica Acta 55 (2010) 4247–4255

F ode, (M T), (da u 100

ot(tmumvSCdauSmMlea

vaCAatthgolA5

4

u

ig. 9. Tapping mode AFM images of: (a) an un-modified MDEA-Au 100 mm electrDEA-Au 100 mm electrode conjugated to acid-chopped SWNT (MDEA-Au|CA|SWN11-AUT modified MDEA-Au 100 mm electrode, and (f) a 11-AUT modified MDEA-A

f the SWNT to the CA and 11-AUT SAM-modified MDEAs. When thewo electrodes MDEA-Au|CA|SWNT and MDEA-Au|11-AUT|SWNTFig. 9C and F) were directly compared, the conjugation of SWNTso the two different alkane thiol-modified electrodes appeared dra-

atically different. SWNT of short lengths (<70 nm) were arrangedpright, being vertically aligned assemblies, on the MDEA-Au|CAodified electrodes as single tubes or aggregates. Similar obser-

ations have been reported by Huang et al. [39], where similarWNT assemblies were created on planar gold electrodes using aA SAM. Cysteamine, being a short length, loosely organized andisordered alkane thiol SAM, is able to anchor the SWNT via theircid functionalized ends. However, the SWNTs are forced into anpright arrangement by the lateral forces of attraction amongstWNTs which dominate over the interaction with the amine ter-inated CA SAM. The feature heights and RMS roughness of theDEA-Au|CA|SWNT were about 24.0±4.9 and 6.4, which were

arger compared to both unmodified MDEA-Au and MDEA-Au|CAlectrode, which had feature heights and RMS roughness of 5.1±1.7nd 2.8±0.8, respectively.

In case of MDEA-Au|11-AUT|SWNT electrodes, the SWNTs ofarying lengths and degrees of aggregation are seem to be lyings aggregates on the 11-AUT SAM bed with a possibility of a fewNT bundles being partly buried within the 11-AUT SAM bed. 11-UT, being a longer, better organized, although still disorderedlkane thiol SAM (evidenced the electrochemistry), is similarly ableo anchor the SWNT via their acid functionalized ends. However,he SWNTs are forced into a lateral arrangement by the favorableydrophobic interactions between the SWNTs and the methyleneroups of the 11-AUT SAM. The feature heights and RMS roughnessf the MDEA-Au|11-AUT|SWNT were 25.5±6.7 and 9.5, which werearger compared to both unmodified MDEA-Au and MDEA-Au|11-UT electrodes, which had feature heights and RMS roughness of.1±1.7 and 4.8±2.9, respectively.

. Conclusions

Amine terminated alkane thiol SAMs may be convenientlysed to immobilize SWNTs to gold microdisc electrode arrays.

b) a cysteamine modified MDEA-Au 100 mm electrode, (c) a cysteamine modified) the edge step of the transition between the Au and Si3N4 layers of a microdisc (e)mm electrode conjugated to acid-chopped SWNT (MDEA-Au|11-AUT|SWNT).

Short chain, disordered SAMs such as cysteamine were shown byexperiment and simulation to not appreciably block the gold elec-trode towards electrochemical activity of the common mediatormolecule, ferrocene monocarboxylic acid. However, cysteamine didallow spontaneous, vertically oriented immobilization of SWNTsthat enhanced the effective electrode area and so increased MDEAvoltammetric responses to FcCOOH. This was in sharp contrast tothe longer chain alkane thiol SAM, 11-AUT, which effectively pas-sivated the gold electrode, reducing the electrochemical activityof FcCOOH. When 11-AUT modified MDEAs were conjugated withSWNTs, the CNTs were found to lie on and within the SAM layer anddid not contribute appreciably to the enhanced area of the electrodebut nonetheless enhanced electrochemical activity of the otherwisepartially passivated gold electrode. Unique chemical and biologi-cal sensing devices can thus be generated by bringing together thesurface assembly and molecular recognition properties of enzymesand the electronic properties of carbon nanotubes. The microdiscelectrode array is seen as a convenient means to bridge the micro-to nano-length scales in the design of electrochemical biosensors[35].

Acknowledgments

This work was supported by the consortium of the Center forBioelectronics, Biosensors and Biochips (C3B). NKS of MahatamaGandhi P.G. College, Gorakhpur, 273001, U.P., India, acknowledgesthe Department of Biotechnology, Ministry of Science and Tech-nology, Government of India for the award (BT/IN/BTOA/24/04) ofthe Biotechnology Overseas Associateship. The authors thank Dr.Ashwin K. Rao for assistance with AFM images.

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