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Electrochimica Acta 55 (2010) 8686–8695

Contents lists available at ScienceDirect

Electrochimica Acta

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

ne step gold (bio)functionalisation based on CS2-amine reaction

nês Almeidaa, António C. Cascalheirab, Ana S. Vianaa,∗,1

Centro de Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa, Ed. C8, Campo Grande, 1749-016 Lisboa, PortugalLumisense, Lda, Campus Faculdade de Ciências da Universidade de Lisboa, Ed. ICAT, Campo Grande, 1749-016 Lisboa, Portugal

r t i c l e i n f o

rticle history:eceived 4 May 2010eceived in revised form 23 July 2010ccepted 27 July 2010vailable online 6 August 2010

eywords:n situ dithiocarbamate formationelf-assembled monolayers

a b s t r a c t

Dithiocarbamates have been regarded as alternative anchor groups to thiols on gold surfaces, and claimedto be formed in situ through the reaction between secondary amines and carbon disulphide. In thispaper, we further exploit this methodology for a convenient one step biomolecule immobilisation ontogold surfaces. First, the reactivity between CS2 and electroactive compounds containing amines, pri-mary (dopamine), secondary (epinephrine), and an amino acid (tryptophan) has been investigated byelectrochemical methods. Cyclic voltammetric characterisation of the modified electrodes confirmedthe immobilisation of all the target compounds, allowing the estimation of their surface concentration.The best result was obtained with epinephrine, a secondary amine, for which a typical quasi-reversible

lectrochemistrynzyme covalent attachmentlucose oxidase

behaviour of surface confined electroactive species could be clearly depicted. Electrochemical reductivedesorption studies enabled to infer on the extent of the reaction and on the relative stability of the gener-ated monolayers. Bio-functionalisation studies have been accomplished through the reaction of CS2 withglucose oxidase in aqueous medium, and the catalytic activity of the immobilised enzyme was evaluatedtowards glucose, by electrochemical methods in the presence of a redox mediator. Scanning tunnellingmicroscopy (STM) and Atomic force microscopy (AFM) were used respectively, to characterize the gold

xidas

electrodes and Glucose O

. Introduction

In the last decades, numerous strategies have been developed inhe preparation of biosensing surfaces, where the biological activ-ty of the biomolecule is not compromised during and upon themmobilisation to the transduction surface [1,2]. The direct elec-ron transfer between redox proteins and electrodes is frequentlyifficult to occur mainly due the three-dimensional structure ofroteins that may hinder the interaction with the transducer orecause protein may undergo denaturation upon adsorption ontohe surface with the subsequent passivation of the electrode [3,4].or the specific case of enzymes, which demonstrate direct electronransfer to the electrode, the orientation, distribution, organisation,nd vicinity to the surface are parameters of highest importance [5].ithin the studied methodologies for the anchoring of enzymes or

ther bio-compounds to the electrode surfaces, such as encapsu-

ation onto sol–gel inorganic matrixes [6], physical electrostatic orovalent adsorption onto polymer films [7] and on flat and nanos-ructured carbon and gold surfaces [8], wired enzymes throughelf-assembled monolayers (SAMs), have been largely investigated

∗ Corresponding author. Tel.: +351 217500000; fax: +351 217500088.E-mail address: anaviana@fc.ul.pt (A.S. Viana).

1 ISE member.

013-4686/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.electacta.2010.07.084

e coverage and distribution on the modified surfaces.© 2010 Elsevier Ltd. All rights reserved.

[9,10]. SAMs can be easily prepared with high reproducibility andare the most elementary form of an organised nanometer-scaleorganic thin-film material [11]. The adsorption of organosulphurcompounds onto gold enjoy the greatest popularity among themodified electrodes by SAMs [12–15], since they rely on the strongaffinity between Au and S, which together with the Van der Waalsinteractions between adjacent tail groups (usually alkyl chains)provide very stable and ordered monolayers. The methods for thecovalent attachment of enzymes to functionalised SAMs bearingappropriate groups for the chemical coupling of the biomolecules,such as amine, carboxylic acid, carbonyl, lactone, etc., have beenthoroughly investigated and described in a review of Willner andKatz [16]. The most reported routes, involve the anchorage ofenzymes via carbodiimide derivatives [17], and glutaraldehyde[18], all of them representing an intermediate step in the prepa-ration of the biosensing interface. With a view of eliminatingthis activation process N-hydroxysuccinimide-ester functionaliseddisulphide has been used to prepare SAMs on gold electrodes,where the exposed N-succinimide group could promptly reactto Laccase [5], decreasing the number of preparation steps to

two (SAM adsorption and further chemical reaction with biocom-pound). Notwithstanding, it has been shown [19] that terminalsuccinimide group on a SAM is unstable and may undergo hydrol-ysis to carboxylic acid, which would require the use of couplingagents to chemical attach any biomolecule.

mica Acta 55 (2010) 8686–8695 8687

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Other studies concerning gold functionalisation involve dithio-arbamate (DTC) monolayers as alternative to alkanethiol-basedAMs [20,21]. DTCs have been commonly used as chelating lig-nds in coordination chemistry [22,23], and the formation ofis(dithiocarbamate)copper(II) complex built on Au (1 1 1) sur-aces, using a layer-by-layer procedure has been reported [24].t was also demonstrated that monolayers formed from lithiumioctadecyldithiocarbamate [21] display similar capacitance, thick-ess, crystallinity and wetability than equivalent alkanethiols,nd that dithiocarbamates–gold linkages are not disrupted whenAMs are exposed to alkanethiols [25]. The major structural aspectistinguishing dithiocarbamates from thiolates is the resonancetructure among nitrogen, carbon and sulphur atoms, due to theree electron pairs on the sulfur and nitrogen atoms, providingifferent binding properties than thiolates, namely higher sul-hur densities with proper overlapping of molecular and metaltates [20,21]. This leads to characteristic physical and chemicalroperties, such as the striking optical and electronic behaviour26,27], and also sensor ability [28] described for DTC derivativeshen interlinking nanoparticles. The pioneer work of Wei and

o-workers [25,29] showed that a variety of secondary amines,ncluding n-alkane, branched-alkane, aromatic and ring substi-uted, condense with carbon disulfide onto gold surfaces at roomemperature, forming dithiocarbamate monolayers.

Very recently [30], electroactive SAMs based on the coadsorp-ion of ferrocene dialkyldithiocarbamates with dithiocarbamatesere prepared on gold electrodes, for the study of the electron

ransfer processes in such in situ formed dithiocarbamate SAMs,onfirming the efficiency of this surface modification methodology.

The purpose of the present paper is to further exploit the reac-ion between CS2 and amines for direct biomolecule immobilisationnto gold surfaces. In this way, the reactivity between CS2 andeveral electroactive compounds containing amines, primary (e.g.opamine), secondary (e.g. hexylmethylamine, and epinephrine),nd amino acids (tryptophan, with primary and secondary amines)ave been primarily investigated by electrochemical methods.opamine (DA) and epinephrine (EPIN) are catecholamines, play-

ng important roles respectively, on the function of the centralervous, renal, hormonal and cardiovascular systems, and as a neu-otransmitter in the mammalian central nervous system, and areommonly detected by electrochemical methods [31–35]. Trypto-han (TRP) is an essential amino acid, with relevance in humanutrition responsible to maintaining a positive nitrogen balance,nd several methods have been developed for its electrochemi-al detection [36,37]. The studies concerning Bio-functionalisationave been attempted through the reaction of CS2 with aminesresent in the well studied glucose oxidase [3,10,38], and the cat-lytic activity of the immobilised enzyme towards glucose, has beenemonstrated by chronoamperometric assays. Scanning tunnellingicroscopy (STM) and Atomic force microscopy (AFM) were used

o characterize the gold electrodes and glucose oxidase coveragend distribution on the surface, respectively. Scheme 1 illustrateshe surface modification approach presented and discussed in thisaper.

. Experimental

.1. Chemicals

Carbon disulfide (Acros Organics), dopamine, epinephrine

nd tryptophan were acquired from Sigma–Aldrich, N-exylmethylamine and sodium hydroxide from Fluka, sodiumiethyldithiocarbamate (DEDTC) from Riedel-de Häen, sulphuriccid (Pancreac) and absolute ethanol (Riedel-de Häen) were allnalytical grade and used without further purification.

Scheme 1. Methodology used for surface attachment of different compounds con-taining amine groups, including glucose oxidase.

Glucose oxidase from Aspergillus niger (GOx, Type X–S,lyophilised powder, 100,000–250,000 units/g, Sigma–Aldrich) wasalso used without further purification procedure, d(+)Glucose fromMerck and ferrocenemonocarboxylic acid were obtained fromAldrich.

Ultra pure water was obtained from a MILI-Q A10 Gradientpurification system (18 M� cm at 25 ◦C) and used to prepare allthe solutions.

2.2. Buffer solution

Phosphate-buffered saline solution (PBS: 8.0 mmol dm−3

Na2PO4; 1.14 mmol dm−3 KH2PO4; 138 mmol dm−3 NaCl and2.7 mmol dm−3 KCl) has been used for the assessment of enzy-matic activity. The pH of the solution was adjusted to 6.8 by addinga diluted solution of HCl.

2.3. Substrates

Gold film (200 nm) on borosilicate glass (pre-layer of chromium,2–4 nm), ArrandeeTM, was used as substrate for monolayerpreparation. The substrates were cleaned in piranha solution(H2SO4:H2O2 = 3:1, v/v) for few minutes, washed with water andethanol, dried under N2 (purity > 99.99997%) flow and flame-annealed in a butane–oxygen flame [39] to produce a flat surfacewith predominant (1 1 1) crystallographic orientation, confirmedby STM. The average roughness factor of the substrates, 1.2, wasestimated by the iodine chemisorption method [40] and is in agree-ment with reported values for similar thin gold electrodes [41,42].

2.4. Electrode modification

For the in situ dithiocarbamate formation gold electrodes havebeen immersed in an ethanolic solution containing 0.1 mol dm−3 ofcarbon disulphide and 0.1 mol dm−3 amine-containing compounds(DA, EPIN and TRP) for 16 h, at room temperature (RT, 22 ◦C). CS2

and DEDTC monolayers were prepared by immersion of the goldsubstrates in 1 mmol dm−3 ethanolic solution of these compoundsfor 16 h at RT. Biofuncionalisation of gold was carried out in an aque-ous solution of 0.1 mol dm−3 of carbon disulphide and 0.1 mg/mLof Glucose oxidase for 16 h at 4 ◦C. For the chronoamperomet-

8688 I. Almeida et al. / Electrochimica Acta 55 (2010) 8686–8695

F , of iml curren

rb

2

S(es

ig. 1. Cyclic voltammograms of 1 mmol dm−3 dopamine on bare gold electrode (a)ine) (b), in 0.1 mol dm−3 H2SO4 at 50 mV s−1. The inset in figure (b) represents the

ic essays, glucose solution was allowed to come to mutarotationefore its employment.

.5. Electrochemistry

Electrochemical measurements were performed using a PAR-TAT 2263 electrochemical work station produced by AMETEKPrinceton Applied Research). A three electrode Teflon cell wasmployed to perform the electrochemical experiments with goldlide as working electrode. A saturated calomel electrode (SCE)

Scheme 2. Representation of the 2e− reversible redox process between hy

mobilised dopamine via CS2 reaction in one step (thick line) and of a CS2 SAM (thint density vs. sweep rate for the oxidation and reduction processes shown in (b).

and a platinum foil served as the reference electrode and thecounter-electrode, respectively. The geometric area of workingelectrode was defined to be a 0.57 cm2. The electrolyte solutions,0.1 mol dm−3 NaOH or 0.1 mol dm−3 H2SO4, were degassed withnitrogen (99.99997%) prior to each experiment.

2.6. Morphological studies

The morphology of modified gold surfaces was characterisedby Atomic force microscopy (AFM) and Scanning tunnelling

droquinone and quinone forms of (a) dopamine and (b) epinephrine.

I. Almeida et al. / Electrochimica Acta 55 (2010) 8686–8695 8689

F (a), anS sentsi

mcmu

3

3a

thtemdi0ihsof

ig. 2. Cyclic voltammograms of 1 mmol dm−3 epinephrine on bare gold electrodeAM (thin line) (b), in 0.1 mol dm−3 H2SO4 at 50 mV s−1. The inset in figure (b) repren (b).

icroscopy (STM). The measurements were performed in ambientonditions in a Nanoscope IIIa Multimode AFM (Digital Instru-ents, Veeco). Etched silicon tips (∼300 KHz) and Pt/Ir tips were

sed for Tapping mode AFM and STM, respectively.

. Results and discussion

.1. Covalent attachment of electroactive compounds containingmines

In order to investigate the reactivity of CS2 with distinctypes of amines in the presence of a gold surface, the electrodesave been exposed to ethanolic solutions of equimolar concen-rations of carbon disulphide and dopamine (primary amine),pinephrine (secondary amine) and tryptophan (containing pri-ary and secondary amines). The cyclic voltammetric response of

opamine in H2SO4 solution on a bare gold electrode is shownn Fig. 1a. The redox process with the anodic current peak at.615 V and the cathodic current peak at 0.455 V (�Ep = 0.160 V)

s assigned to the two electron, two proton conversion betweenydroquinone/quinone forms of dopamine (Scheme 2a). In acidicolutions the redox process is more reversible than the onebserved in phosphate buffer solution pH 7 [5], since the oxidisedorm of dopamine is more stable at low pH, and decomposition

d of immobilised epinephrine via CS2 reaction in one step (thick line) and of a CS2

the current density vs. sweep rate for the oxidation and reduction processes shown

through ring closure is retarded [43]. The cyclic voltammogramshown in Fig. 1b confirms the successful attachment of dopamineto gold via reaction with CS2, in only one step. In contrast withthe electrochemical behaviour of dopamine in solution, the oxi-dation and reduction peaks appear at 0.580 mV and 0.540 mV(�Ep = 0.040 V), respectively. The increase in reversibility of theelectron transfer process confirms molecule adsorption to thesurface, also supported by the linear behaviour exhibited bythe anodic and cathodic peak currents with scan rate (inset ofFig. 1b).

Fig. 2 shows representative cyclic voltammograms ofepinephrine on a bare gold electrode (Fig. 2a) and after sur-face modification (Fig. 2b), as above described. The redox peaksobserved for epinephrine in H2SO4 solution, Eox

p = 0.600 V andEred

p = 0.515 V (�Ep = 0.085 V), are in similarity with those ofdopamine attributed to the 2e−, 2H+ hydroquinine/quinoneconversion of epinephrine molecule, as illustrated in Scheme 2b.Upon reaction with CS2 and in the presence of gold substrateepinephrine becomes immobilised on the surface as clearly

demonstrated by the typical electrochemical behaviour ofcovalently attached electroactive molecules. Upon adsorption,epinephrine oxidation and reduction peaks are nearly symmetric,shifted to 0.580 V and 0.560 V (�Ep = 0.020 V) and the anodic andcathodic current peaks are proportional to the potential sweep

8690 I. Almeida et al. / Electrochimica Acta 55 (2010) 8686–8695

F (a); anS sentsi

rFeacaawtso3e

useptttp(E

ig. 3. Cyclic voltammograms of 1 mmol dm−3 tryptophan on bare gold electrodeAM (thin line) (b), in 0.1 mol dm−3 H2SO4 at 50 mV s−1. The inset in figure (b) repren (b).

ate (inset of Fig. 2b). The electrochemical responses depicted inigs. 1b and 2b strongly indicate that although both dopamine andpinephrine could be adsorbed onto gold, epinephrine, containingsecondary amine is more effectively bounded to the surface,

ompared to the dopamine, which has an available primarymine. The results using this simple approach are therefore ingreement with previous studies involving the reaction of CS2ith distinct amines mainly secondary amines, which indicates

hat the reaction is favoured for secondary amines [29,30]. Theurface coverage values estimated based on the integrationf the anodic peaks and assuming the transfer of 2e− were.6 × 10−11 mol cm−2 and 1.3 × 10−10 mol cm−2 for dopamine andpinephrine, respectively.

The immobilisation via the reaction of amines with CS2 has asltimate purpose the direct attachment of bio-compounds on goldurfaces, and therefore it would be important to check whether anlectroactive amino acid, such as tryptophan, that contains bothrimary and secondary amines would be attached to gold usinghis simple route. Fig. 3 shows the electrochemical response for

ryptophan in a bare gold electrode and after surface modifica-ion. In both cyclic voltammograms it is possible to observe aair of non symmetrical redox peaks, Eox

p = 0.575 V, Eredp = 0.495 V

�Ep = 0.080 V) for the unmodified electrode and Eoxp = 0.520 V,

redp = 0.375 V (�Ep = 0.145 V), with a common feature: the abso-

d of immobilised tryptophan via CS2 reaction in one step (thick line) and of a CS2

the current density vs. sweep rate for the oxidation and reduction processes shown

lute value for the oxidation peak current is much higher that thereduction counterpart. The nature of the redox peaks of trypto-phan can be very complex. It is known that in most surfaces a singleoxidation peak is detected, which is due to an irreversible 2e− reac-tion giving an extremely reactive methylene–imine intermediate[44,45]. This intermediate, can be easily attacked by nucleophilespresent in the reaction solution, such as water with the formationof oxindolalanyl species, which may exhibit some electrochemi-cal reversibility, although the extent of reduction reaction is muchsmaller than the oxidation process [36,46], which supports theobserved behaviour in Fig. 3. In spite of the broad anodic pro-cess observed upon immobilisation, it is possible to conclude thattryptophan has been immobilised onto gold through the reac-tion with CS2, fostering the use of this methodology for furtherattachment of enzymes that contain a large number of tryptophanresidues.

The cyclic voltammograms shown in Fig. 4a correspond tothe electrochemical reductive stripping of a CS2 SAM, a DEDTCSAM (prepared by gold immersion in a sodium diethyldithiocar-

bamate solution) and of a monolayer resulting from the reactionbetween CS2 and hexylmethylamine (HMA) in one step procedure,as described previously. As it is well known from the literature,SAMs bearing sulphur species as anchoring groups (e.g. thiols[11,12], disulphides [47] and thiotic acids [39]), desorb quantita-

I. Almeida et al. / Electrochimica Acta 55 (2010) 8686–8695 8691

Fh0

tiprfaps[talmtsa

Fig. 5. UV–vis spectra of the HMA–DTC formed in situ (appearance of the bands

assumed the transfer of one electron per sulphur atom in simi-

TR

ig. 4. Cyclic voltammograms of gold modified with (a) CS2, DEDTC, and CS2 andexylmethylamine; and (b) CS2 and dopamine, epinephrine and tryptophan, in.1 mol dm−3 NaOH at 20 mV s−1.

ively from gold surface in a strong alkaline solution, giving risen general, to a sharp peak at negative potentials. The shape of thiseak as well as the potential at which the desorption process occurs,eflecting monolayer stability, are strongly dependent on severalactors, namely surface crystallinity, monolayer packing densitynd type of sulphur species [11,12]. The very negative reductionotential observed for CS2 (−0.920 V) regarding the desorption ofhort thiol derivatives, such as mercaptopropanol (Ep = −0.784 V48]) or mercaptopropionic acid SAM (E = −0.800 V [49]), revealshe great stability of the resonant structure among the sulphurnd carbon as the result of a good overlap of the orbitals of theigand and the gold surface. In the case of the pure dithiocarba-

ate SAM reductive desorption, there is a shift of the potentialowards positive direction most probably due to electron delocali-ation between the resonant structure N–C(–S2) which may lead toweaker surface bond [24]. In fact, the desorption of the monolayer

able 1eduction potentials and surface coverage values obtained from the cyclic voltammogram

SAMs CS2 CS2 + DA CS2 + EPIN

Eredp vs SCE/V −0.92 −0.89 −0.90

� (2S)/mol cm−2 4.3 × 10−10 4.6 × 10−10 3.2 × 10−10

at 260 nm and 280 nm) from the reaction between CS2 and HMA and diluted (10-fold from the initial concentrations: 0.25 mmol dm−3 CS2 + 0.50 mmol dm−3 HMA)in ethanol, at 5 min ( ), 15 min ( ) and 30 min ( ) upon thebeginning of the reaction.

prepared by the surface reaction between gold, CS2 and hexyl-methylamine presents two reduction peaks, one more negative at−0.880 V, most probably associated with a fraction of CS2 that wasadsorbed and did not react with the amine, and the other less neg-ative, at −0.800 V due to dithiocarbamate formation. The reductionpotentials are summarised in Table 1.

The reductive desorption of the monolayers formed bydopamine, epinephrine and tryptophan reaction with CS2 as dis-cussed above in this paper, are shown in Fig. 4b. Although thestripping potentials (Table 1) for the three modified electrodesare less negative than the one observed for a pure CS2 SAM, asexpected due to the formation of dithiocarbamate–gold bond, theyare noticeably more negative than the value obtained for DEDTCSAMs. This behaviour suggests in agreement with a previous stud-ies on pure dithiocarbamate SAMs [20] that the aromaticity ofthe adsorbed molecules should be responsible for monolayer sta-bilisation through �-interactions between adjacent immobilisedmolecules. Besides, the presence of broad cathodic peaks can beclearly depicted in Fig. 4b, most probably arising from distinct sul-phur environments within the monolayers which desorb at slightlydifferent potentials. These should correspond to CS2 and to dithio-carbamate with aromatic pending groups that establish bonds withgold with different energies.

The amount of adsorbed sulphur species, evaluated throughthe charge involved in the reductive desorption, was estimatedfor all modified electrodes and are presented in Table 1. It was

larity with the generally accepted for the desorption of thiolates.CS2 and DEDTC monolayers have similar coverages, taking intoaccount the total amount of adsorbed sulphur, in the range of theexpected for a packed alkylthiol

(√3 ×

√3)

/R30◦ overlayer struc-

s presented in Fig. 4.

CS2 + HMA CS2 + TRP DEDTC

−0.80; −0.88 −0.88 −0.741.7 × 10−10; 1.0 × 10−10 5.2 × 10−10 4.0 × 10−10

8692 I. Almeida et al. / Electrochimica Acta 55 (2010) 8686–8695

F SCE) ur h CS2

a

tteiaicrsbgsdDwts

ig. 6. (a) Chronoamperograms (current sampling: 300 ms) obtained at 0.4 V (vs.ocenemonocarboxylic acid in PBS (0.05 mol dm−3, pH 6.8) of (a) modified gold witnodic currents obtained from the chronoamperograms vs. glucose concentration.

ure at Au (1 1 1), 7.6 × 10−10 mol cm−2 [50]. It is worth to notehat lower surface coverages have been obtained for the modifiedlectrodes with secondary amines, HMA and Epinephrine, wheret was observed the more successful reaction between CS2 andmine group, as supported by the electrochemical data shownn Fig. 2, for epinephrine and by the reductive stripping in thease of HMA. Since this latter compound is not electroactive, theeaction has been confirmed by UV–vis spectroscopy of a diluteolution of CS2 and HMA (Fig. 5) that clearly shows the typicalands at 252 nm and 283 nm expected for the dithiocarbamateroup [29]. The reason for the lower coverage obtained for theecondary amines is attributed to the fact that dithiocarbamate

erivatives with epinephrine and HMA are bulkier that CS2 andEDTC (shorter alkylchains) and even adsorbing in simultaneousith a fraction of non-reacted CS2 it is expected some disorganisa-

ion that should lead to a lower amount of sulphur on the electrodeurface.

pon successive addition of glucose (0.25–28 mmol dm−3) in aerated 0.01 mM fer-+ GOx and (b) bare gold electrode (absence of GOx). The figure insets represent the

3.2. Covalent attachment of an enzyme to gold

The great novelty of this work, as mentioned before, is to usethe reaction between CS2 and the amine groups present in thestructure of the enzyme for the direct bio-functionalisation ofsurfaces. To ascertain this possibility, glucose oxidase has been cho-sen for immobilisation since its electrocatalytic properties havebeen thoroughly investigated [3,10,38]. The biological activity ofthe modified surface with GOx was addressed by using a redoxmediator, ferrocenemonocarboxylic acid, in order to enhance theelectrochemical response, since a monolayer or sub-monolayercoverages are expected as a result of the methodology used. The

following equations [38] should be the base for the electrochemicalsignals observed in Fig. 6, due to the immobilised enzyme.

GOx(FAD) + glucose → GOx(FADH2) + gluconolactone (1)

I. Almeida et al. / Electrochimica Acta 55 (2010) 8686–8695 8693

F withf ows tc

G

2

ifoosoauispdtiiabtpcFglib(G

ret

ig. 7. Chronoamperograms (current sampling: 300 ms) of a modified gold electrodeerrocenemonocarboxylic acid in PBS (0.05 mol dm−3, pH 6.8). The figure inset shoncentration (b).

Ox(FADH2) + 2Fc+ → GOx(FAD) + 2Fc + 2H+ (2)

Fc → 2Fc+ +2e− (3)

The currents obtained from the chronoamperograms shownn Fig. 6a recorded at 0.4 V, associated with the oxidation of theerrocene centre to the ferricinium cation due to the enzymaticxidation of glucose to gluconolactone, increase linearly (insetf Fig. 6a), with the successive additions of glucose to the PBSolution, from 0.25 mmol dm−3 (first addition) to 22 mmol dm−3

f glucose, after which value the current reaches its maximumnd tends to a plateau, that should correspond to enzyme sat-ration. In spite of the routine use of ferrocene derivatives, and

n particular the ferrocenemonocarboxylic acid in glucose biosen-ors [38,51], it is clearly visible from Fig. 6b, a control experimenterformed on bare gold without enzyme, that ferrocene oxi-ation current decreases with the first additions of glucose tohe buffer solution. This side effect, which may arise from somenteraction between ferrocenemonocarboxylic acid and glucoses currently under investigation, in order to overcome it and beble to extract accurate kinetic enzymatic data on these type ofiosensing surfaces. This phenomenon should be the reason whyhe observed catalytic current shown in Fig. 6a, exhibiting a linearart and the beginning of a plateau, is not following the typi-al Michaelis–Menten curve. Nevertheless, the contrast betweenig. 6a and b undoubtedly demonstrates that Au/CS2–GOx modifiedold is biologically active towards glucose oxidation, exhibiting aarge linear response, with a slope of 7.9 × 10−5 A (mol dm−3)−1,n a range of concentration suitable for sensing glucose in diabeticlood samples [51]. Assuming that imax is attained at 1.85 �A cm−2

Fig. 6a) the Michaelis–Menten constant (KM) for the immobilised

Ox using CS2 reaction should be approximately 10 mmol dm−3.

It is worth noting that no signal has been observed for the directedox conversion of GOx in deaerated solutions, which could bexplained by the low protein-loading capacitance on flat gold elec-rode.

GOx at 0.4 V (vs. SCE) upon successive addition of glucose in aerated 0.01 mmol dm−3

he anodic currents evolution obtained from the chronoamperograms vs. glucose

In order to validate the data presented for the modified elec-trodes with glucose oxidase and CS2, the enzyme has been adsorbeddirectly on bare gold electrode (in the absence of carbon disulphide)and the electrocatalytic performance towards glucose studied, asillustrated in Fig. 7, in the same conditions as those used forFig. 6a. The current for ferrocene oxidation decreases with succes-sive additions of glucose to the PBS solution, following a similartrend observed for a bare gold electrode in the absence of enzyme(Fig. 6b), as discussed above. It is worth to point out that currents forferrocene oxidation are much lower in the case of Fig. 7, since thephysically adsorbed GOx is blocking the available electrode areafor this electrochemical process. More importantly, the contrastbetween Figs. 6a and 7, strongly indicates that the direct enzymeadsorption onto unmodified gold preclude enzyme activity, whichcould be a result of protein denaturation on the bare surface, asreported before [4,52].

The morphology of bare gold electrode, showing the typicalgold (1 1 1) terraces, separated by 0.24 nm [39], after the flame-annealing process was analysed by STM (Fig. 8a) and a larger areaby AFM (Fig. 8b). AFM was used in the characterisation of themodified gold electrodes with GOx in the presence and absenceof carbon disulphide, in order to obtain qualitative information onthe enzyme coverage and surface distribution, as shown in Fig. 8cand d, respectively. The presence of individual enzyme molecules(ca. 10 nm), as well as small aggregates (ca. 20–25 nm), can bedenoted in Fig. 8c. The small globular features exhibited in Fig. 8dare also attributed to physically adsorbed GOx; however, it can beseen that there is more than one layer of protein although notcovering uniformly all the gold surface. Even though this latterenzyme–electrode did not exhibit activity towards glucose, AFMimaging revealed the presence of enzyme on the gold surface,

supporting that enzyme looses activity when deposited directlyon gold. In fact, AFM images obtained after the chroamperomericstudies still exhibit similar enzyme loading on the gold surface,indicating that the absence of activity of the immobilised enzymeis not due to enzyme leak to the solution.

8694 I. Almeida et al. / Electrochimica Acta 55 (2010) 8686–8695

F y (a) Si x in th

4

aaTtcc(otafbtp

ig. 8. Images of a bare gold electrode after the flame-annealing process obtained bmage); and (b) AFM, z = 4 nm. AFM images of the modified gold electrodes with GO

. Conclusions

Electroactive compounds containing primary and secondarymine groups could be readily immobilised on gold surfaces usingconvenient and simple one-pot reaction with carbon disulphide.he redox behaviour in acidic solutions of the modified elec-rodes with dopamine, epinephrine and tryptophan undoubtedlyonfirmed their immobilisation on gold. The best electrochemi-al behaviour was attained for gold modified with epinephrinebearing a secondary amine) achieving a surface concentrationf epinephrine molecule of 1.3 × 10−10 mol cm−2. This observa-ion is in line with previous studies involving other secondary

mines. The electrochemical reductive desorption studies providedurther evidence for surface modification via in situ dithiocar-amate formation, and the obtained surface coverages indicatedhat relatively packed monolayers, regarding the number of sul-hur molecules can be formed, where the short amine derivatives

TM, z = 2 nm (the inset in figure (a) corresponds to the cross section marked on thee (c) presence z = 30 nm, and (d) absence of carbon disulphide; z = 20 nm.

should be diluted with pure CS2 molecules. It was demon-strated that glucose oxidase could be successfully immobilisedon the electrode, using this simple methodology in aqueoussolutions, displaying biological activity towards glucose, as evi-denced by the chronoamperometric assays. A linear increase ofthe anodic current with the increment of glucose concentration,up to 22 mmol dm−3, could be clearly depicted. In contrast, whenthe glucose oxidase was adsorbed directly on the gold surfacein the absence of CS2, the electrodes were covered with a largeamount of enzyme, as illustrated by AFM imaging, but no cat-alytic activity towards glucose could be detected. Electrochemicaldata revealed the impact that the present methodology may have

in biomolecule surface attachment. Gold nanoparticles are cur-rently being modified using this procedure in order to increasethe amount of electroactive compound and enzyme loading andalso to enhance the electronic properties of the modified elec-trodes.

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cknowledgements

The authors acknowledge Prof. L.M. Abrantes for support andelpful discussions and Fundacão para a Ciência e a Tecnologia

or funding (Project PTDC/QUI/66612/2006 and Program Ciência007).

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