Ne

CD

a

ARRAA

KHGSTl

1

teuomemebdsc

mssoet

0d

Sensors and Actuators B 149 (2010) 129–135

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

journa l homepage: www.e lsev ier .com/ locate /snb

anostructures on gold electrodes for the development of an l-tyrosinelectrochemical sensor based on host–guest supramolecular interactions

armen Quintana ∗, Sonia Suárez, Lucas Hernándezpto. Química Analítica y Análisis Instrumental, Facultad de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain

r t i c l e i n f o

rticle history:eceived 22 December 2009eceived in revised form 19 May 2010ccepted 8 June 2010vailable online 17 June 2010

eywords:

a b s t r a c t

Supramolecular host–guest interactions between �-cyclodextrins and l-tyrosine have been employed todevelop a selective electroanalytical method for the determination of this amino acid. For this purpose,self-assembled monolayers of thiolated �-cyclodextrins on a gold electrode have been built up. The ben-efits derived from the immobilization of these macromolecular receptors on the electrode surface areshown by the resolution of a mixture of electroactive amino acid analysed with the proposed modifiedelectrode. In addition to this, all parameters affecting the gold electrode modification and the l-tyrosine

ost–guest interactionsold electrodeelf-assembled monolayershiolated �-cyclodextrins-Tyrosine

determination through differential pulse voltammetry have been optimized. During the electrode modifi-cation step, pentanethiol was employed to fill up the exposed surface between �-cyclodextrin molecules.The current is proportional to the concentration of l-tyrosine from 3.6 × 10−5 M to 2.4 × 10−4 M. The detec-tion limit and the sensitivity were found to be 1.2 × 10−5 M and 16.9 × 10−4 AxM−1 respectively, with Ervalues smaller than 13.0% and a RSD (%) (n = 5) values equal or lower than 5.4 achieving good accuracy andprecision. Moreover, the developed methodology was successfully applied to l-tyrosine determination in

on sa

pharmaceutical formulati

. Introduction

The use of modified electrodes is strategy widely employed byhe electroanalytical researches in order to get a better control oflectrode surface properties. Many advantages are derived from these of modified electrodes respect to the electroanalytical method-logies developed with unmodified ones, i.e. higher sensitivity orore stable devices. However, one of the aims of using modified

lectrodes is to get more selective methodologies. The modifierust be selective or even specific to the target analyte and must

nsure the charge transfer between the electrode and the solutionearing it. Usually, metallic electrodes (Pt, Ag or Au) or graphiteerivatives such as glassy carbon or carbon paste ones, are used asupports [1–3]. On the other hand, plenty of different compoundsan be chosen as modifiers [4,5].

Among all the modification strategies, the chemisorption of theodifiers on metallic surfaces is a spontaneous process leading to

elf-assembled monolayers (SAM’s) highly ordered, compact and

table. The high S–Au affinity makes the chemisorption of thi-lated organic species on gold surfaces, an ideal way to designlectrochemical sensors. Alkanethiols with NH2, CH2OH and COOHerminal functional groups have been usually employed for this

∗ Corresponding author. Tel.: +34 914977626; fax: +34 914974931.E-mail address: carmen.quintana@uam.es (C. Quintana).

925-4005/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.snb.2010.06.011

mples with a RSD (n = 5) of 6.3% in average and an Er (n = 5) minor than 7.0%.© 2010 Elsevier B.V. All rights reserved.

purpose [6]. On the other hand, although non-functionalized alka-nethiols self-assembled monolayers have served as model organicsurfaces in fundamental studies of molecular adsorptions, wet-ting and lubrication [7], some examples of the improvements ofselectivity in the electroanalytical procedures using SAM’s of non-functionalized alkanethiols, have been previously reported [8,9].The use of modifiers with hosts–guest interactions properties inaddition to the easy and reproducible sulphide chemisorptionson gold, leads to successfully modified electrodes which work asspecific electrochemical sensors and allow the development ofhighly selective electroanalytical methods. It is well known thatcyclodextrins (CDs) are natural receptors that have demonstratedtheir molecular recognition properties and their capability asenantiomeric selectors [10]. These compounds are cyclic oligosac-charides consisting of six (�-cyclodextrin), seven (�-cyclodextrin)or eight (�-cyclodextrin) glucopyranose units, that are producedas a result of the action of some enzymes on starch [11]. The �-cyclodextrin (�-CD) cavity size, is the most appropriate to includea wide range of analytes. All CDs present a toroidal shape with theprimary hydroxyl groups of the glucose units expose to the outside[12] and an apolar, well-defined cavity which is responsible of theirhost–guest interactions to form inclusion complexes. Because of

these properties, CDs have aroused great interest in supramolecularchemistry studies and a wide range of industrial or pharmaceuticalapplications have been found [13,14].

By substitution of CD hydroxyl groups by sulphide ones, it is pos-sible to obtain thiolated cyclodextrins. Depending on the number

1 d Actu

occsstosewwwbsgd

tIptmis[diom

ehetcrgtiv(d

2

2

pSlfw

�(dL(gt

2

(

30 C. Quintana et al. / Sensors an

f groups replaced, they are classified as mono- or multithiolatedyclodextrins. These thiolated molecular receptors can be easilyhemisorbed on gold electrodes to develop electrochemical sen-ors. To date, there are numerous papers dealing with fundamentaltudies about the adsorption of thiolated CDs and their deriva-ives on gold surfaces [15,16]. However, no examples of the usef these compounds as modifiers of gold electrodes to developelective electroanalytical methodologies can be found in the lit-rature. It is described that the chemisorption of cyclodextrinsith all their OH replaced by SH groups allows an optimal packingithin the adsorbed layer [17] in contrast those monolayers builtith monothiolated ones [15,16]. In this work, the combination of

oth, self-assembled monolayers and host–guest chemistry is con-idered to prepare self-assembled host–guest nanostructures on aold electrode surface for the electroanalytical l-tyrosine (l-tyr)etermination.

Tyrosine is an amino acid precursor of important neurotransmit-ers such as dopamine [18]. Tyrosine is indispensable for humans.t maintains a positive nitrogen balance [19] and its absence couldroduce albinism, hypochondria, or depression. In contrast, highyrosine concentration in culture medium increases sister chro-

atid exchange [20]. Thus, tyrosine has been of great interestn research studies. Different analytical methodologies involvingpectrophotometric [21], fluorimetric [22] or gas [23] and liquid24] chromatographic techniques, have been proposed for tyrosineetermination. The high sensitivity and low cost of electroanalyt-

cal methods result in an interesting choice for the determinationf those electroactive amino acids, however, less examples of theseethodologies can be found in the literature [25,27–29].As previously mentioned, the aim of this work is to develop an

lectrochemical sensor for l-tyrosine determination based on theost–guest �-cyclodextrins supramolecular interactions. To thisnd, the investigation explored (1) the study in solution of the l-yr–�CD inclusion complex formation and the optimization of thehemical variables related to the differential pulse voltamperomet-ic response on a bare gold electrode, (2) the optimization of theold electrode modification by chemisorption of �-cyclodextrinhiolated in all its hydroxide groups, (3) the optimization of allnstrumental variables related to the complex differential pulseoltamperometric response on a �-CD modified gold electrode and4) the application of the developed methodology to l-tyrosineetermination in a pharmaceutical formulation.

. Experimental

.1. Reagent

Pure standard of l-tyrosine (99%) and �-cyclodextrin (98%) wereurchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA).tock solutions were prepared in ultrapure water at a concentrationevel of 9.0 × 10−3 M and 4.9 × 10−3 M respectively and used forurther dilution in supporting electrolyte. The standard solutionsere kept at 4 ◦C and protected from light.

Heptakis (6-deoxy-6-thio)-�-cyclodextrin, C46H78O28S7 (SH-CD), was produced by Cyclolab (Budapest, Hungary). Pentanethiol

C5SH12), ferrocene, phenylalanine, methionine, l-glutathione andopamine were purchased from Sigma–Aldrich Chemical Co. (St.ouis, MO, USA). Finally, l-cysteine was obtained from MerckDarmstadt, Germany). All reagents used were of analytical reagentrade and ultrapure water was purified with a Millipore MilliQ Sys-em (Waters). All diluted solutions were prepared just prior to use.

.2. Apparatus

A Potentiostat workstation (�Autolab III) from Eco-ChemieUtrecht, The Netherlands), equipped with a conventional three

ators B 149 (2010) 129–135

electrode cell, was employed for all the electrochemical measure-ments. A coiled platinum wire was employed as a counter electrode.All potentials are reported against an Ag/AgCl/KCl 3 M referenceelectrode. A pH-meter Methrom C831 was also used for pH adjust-ment.

2.3. Procedure

SH-�CDs were immobilized on gold electrodes by direct adsorp-tion. Prior to immobilization, gold electrodes were conditionedin 0.1 M H2SO4 solution by successive cyclic voltammetric scansbetween 0.0 V and 1.5 V at 100 mV s−1. For SH-�CDs immobiliza-tion, the conditioned gold electrode was immersed in a thiolated�-cyclodextrin solution during 5 h. The electrode was then takenout from the solution, rinsed with ultrapure water and dried undernitrogen flow. Next, the modified Au electrode was immersed ina 0.75 mM ferrocene/0.75 mM pentanethiol solution for 1 h. Afterrinsing with pure ethanol and ultrapure water, the modified elec-trode was transferred to an electrochemical cell containing 10.0 mLof l-tyrosine at 1 M phosphate buffer pH = 1, and the differentialpulse voltammograms were recorded under the optimal conditionsdeveloped.

3. Results and discussion

3.1. l-Tyrosine complexation by ˇ-cyclodextrin

The spectroscopic study reported by Shanmugam et al. [26]related to the l-tyrosine and �-cyclodextrin interaction, proposesan exergonic and spontaneous process for the formation of a1:1 l-tyr–�CD complex. Based on these results, the possibility ofmonitoring the complex formation through electrochemical mea-surements and applying the results to develop electroanalyticalmethod for l-tyr determination was studied. To this end, a set ofexperiments involving the study of the pH, nature and concen-tration of the supporting electrolyte were carried out. Differentialpulse voltammetry was chosen as measurement technique. As ini-tial instrumental conditions a pulse amplitude of 50 mV, a step of2 mV, a scan rate of 10 mV s−1 with an initial accumulation during5 s at 0.0 V vs. Ag/AgCl/KCl 3 M, were selected. Later on, all theseparameters affecting sensitivity and selectivity of the method wereindividually optimized and fixed at the optimal values.

Differential pulse voltammograms of pure 10−4 M tyrosine solu-tions and with increasing �CD additions were recorded in different0.1 M electrolytes in a wide range of pH varying from 1 to 11. Thepresence of an excess of �CD in solution produces a variation in thel-tyr electrochemical oxidation signal that can be attributed to thel-tyr–�CD complex formation (Fig. 1). From the results depictedin this figure, different effects can be observed: (1) in all cases, anincrement in the peak current and a variation of the peak poten-tial to minor potential values are observed. (2) These variationsare quantitatively higher at pH 1 than at the rest of pH conditionsassayed (i.e. �E 50 mV and �Ip = 3.7 × 10−7 A and �E 40 mV and�Ip = 1.3 × 10−7 A for pHs 1 and 3 respectively). (3) At basic pHvalues, the oxidation signals corresponding to gold and l-tyr, areoverlapped (Fig. 1c, voltammograms a and b respectively). This cir-cumstance makes unfeasible the monitoring of the analyte at basicpH conditions. Therefore, the best selectivity and sensitivity condi-tions for l-tyr oxidation monitoring are obtained at pH = 1. So, thispH was selected for further experiments.

Among the different electrolytes studied, 0.1 M H3PO4 producedthe highest peak current and lowest peak width values. Therefore,the following step was to study the influence of H3PO4 concentra-tion in the analytical signal taking into account that in this case,this variable could also influence the complex formation equilib-

C. Quintana et al. / Sensors and Actuators B 149 (2010) 129–135 131

Fv

reatlse

3

ttaSo

shows that this procedure leads to relative imperfect monolayerswith some “defects” (uncovered gold areas) between CD molecules[15–17]. Therefore, to cover those defects, a second modificationstep was performed. The modified electrode was immersed in

ig. 1. Differential pulse voltammograms of 10−4 M l-tyr solutions at different pH

alues.

ium. As shown in Fig. 2a and b, the increase in the supportinglectrolyte concentration seems to benefit the complex formationnd the electrochemical processes leading to both, an increase inhe peak current recorded and a peak potential displacement toess positive values, like if salt-out effect is produced. Finally, theupporting electrolyte concentration was fixed in 1.0 M for furtherxperiments.

.2. Electrode modification

Following experiments were focused on optimizing the elec-

rode modification step. As it was stated on the procedure section,hiocyclodextrins were chemisorbed on gold electrodes by directdsorption. To this end, the clean electrode is immersed in a 10−3 MH-�CD solution in DMSO/H2O (60/40, v/v) for different periodsf time ranging from 5 min to 15 h. Next, the electrode is gently

Fig. 2. Influence of the H3PO4 pH = 1 concentration on the (A) peak current and (B)peak potential of the l-tyr–�CD complex oxidation.

washed up with ultrapure water and the differential pulse voltam-mograms of 10−4 M l-tyr solutions in 1 M H3PO4 are recorded.

The results obtained from these set of experiments showed adecrease in the peak current as a result of the reduced available goldsurface (see Fig. 3). This Ip variation does not occur for modificationtimes higher than 5 h. This fact could lead us to think that the wholegold surface is covered by the cyclodextrins however the literature

Fig. 3. Influence of the modification time on the peak current.

1 d Actuators B 149 (2010) 129–135

aEartwcvitpCwi

3

tdw

rfepv

mspbiw

tiwrmrtipa

fa(bfcitrtIslaotatqb

−4

32 C. Quintana et al. / Sensors an

0.75 × 10−3 M pentanethiol:0.75 × 10−3 M ferrocene solution intOH/H2O (50:50, v/v) for 1 h. Pentanethiol is an alkanethiol withsimilar length than �-cyclodextrin high. The addition of fer-

ocene at this stage blocks the pentanethiol adsorption throughhe inner cyclodextrin cavities. Finally, the electrode is rinsed upith pure ethanol and ultrapure water before the electrochemi-

al measurements. The absence of ferro/ferri signal in the cyclicoltammograms recorded in supporting electrolyte with the mod-fied electrode demonstrates that any ferrocene remains neither onhe electrode surface nor into the cavities. With the modificationrocedure described, a mixed monolayer is built in which the �-Ds widest portals are oriented to the bulk solution and the onlyay for the analyte to reach the electrode surface is through the

nner cyclodextrins cavities [15–17].

.3. Instrumental variables optimization

With the aim of developing a highly sensitive and selective elec-roanalytical method, all the instrumental variables involved in theifferential pulse response with the �-CDs modified gold electrode,ere individually optimized.

From the variation of the initial potential (Ei) in the 0.0–0.9 Vange, a slight decrease in the peak current recorded was observedor Ei values higher than 0.2 V which corresponds to the high-st analytical signal. On the other hand, no influence on the peakotential value was observed so, 0.2 V was chosen as optimal Eialue.

The analyte l-tyr can be accumulated on the electrode surface byaintaining the Ei up to 10 s before the differential pulse potential

can was carried out. Higher accumulation times leads to constanteak current values as corresponds to an equilibrium situationetween the analyte concentration on the electrode surface and

n the solution. From these results, an accumulation time of 10 sas set for the rest of the experiments.

Regarding the optimal pulse amplitude value, the variation ofhis parameter between 10 mV and 90 mV leads to both, an increasen the peak current up to 60 mV and a moderate increase in the peak

idth from 55 mV to 65 mV. For the experimental measurementsemaining pulses of 60 mV in amplitude were applied as a compro-ise situation between sensitivity and selectivity. Finally, the scan

ate was optimized as a result of the individual study of the step andhe interval time parameters. The peak current diminished while Vbncreased showing some kinetic limitation to the electrochemicalrocess. Therefore, 10 mV s−1 (as a result of applying a step of 5 mVnd an interval time of 0.5 s) was the scan rate chosen as optimal.

If a comparison among the pulse voltamperograms recordedrom solutions of l-tyr and �CD (1:2) with a bare gold electrodend solutions of l-tyr with the SH-�CD modified electrode is doneFig. 4, voltammograms a–c), we can observe a great differenceetween the oxidation response when the inclusion complex isormed either in solution or on the electrode surface. In this latterase, an adequate analyte orientation is necessary to be includedn the �CD cavity before being oxidated. This fact could explainhe Ep displacement to more positive values. The decrease in the Ipecorded can be explained with the minor gold surface available inhe modified electrode (just that present in the modifier cavities).n contrast, with the unmodified electrode both, the wave inten-ity and morphology and the decrease of the peak potential value,eads us to think in a rapid l-tyr adsorption process indicating a littlenalyte accumulation followed by the �CD diffusion from the bulkf the solution to the electrode to form the inclusion complex. All

his process is favoured by a �CD excess in solution. Moreover, thedded selectivity to the electrochemical methodology in additiono the improvement in the signal/noise ratio produced as a conse-uence of the decrease in the background noise, demonstrates theenefits of the proposed methodology.

Fig. 4. Differential pulse voltammograms of (a) 10 M l-tyr on a bare gold electrode,(b) l-tyr + 2�-CD mixture solutions with a bare gold electrode and (c) 10−4 M l-tyrwith a �-CD modified gold electrode. (d) 0.1 M H3PO4, pH = 1. Ei = 0.2 V, Tac = 10 s,a = 60 mV, Vb = 10 mV s−1.

3.4. Relation between peak current and l-tyr concentration.Analytical data

Once the chemical and instrumental variables for l-tyr deter-mination with a SH-�CD modified gold electrode were optimized,the relation between the amino acid concentration and thepeak current (Ip) was studied. A linear increase of the peakintensity with increasing l-tyr concentrations was observed inthe 3.6 × 10−5–2.4 × 10−4 M range according to the equation Ip(A) = 1.93 × 10−8 + 16.9 × 10−4 [l-tyr] (M); r = 0.997. The sensitivityof the proposed method was inferred from a LOD and LOQ calcu-lated values of 1.2 × 10−5 M and 3.3 × 10−5 M respectively whichdoes not improve others previously reported [27–31] as expectedby the lower gold surface available. The RSD (%) and Er (%) (n = 5) val-ues were evaluated at different concentration levels (9.0 × 10−5 M,2.0 × 10−4 M, 3.0 × 10−4 M). A very good reproducibility and accu-racy are observed as it is shown from RSD (%) n = 5 values of 5.4%or minor and Er (%) values of 13.0% or less.

3.5. Interferences study

To evaluate the capability of discrimination of the developedsensor, some experiments related to the influence of other elec-troactive amino acids present in solution were performed. Theamino acids, l-cysteine (l-cys), methionine (met), phenylalanine(phe), the peptide l-glutatione (l-glu) and the neurotransmitterdopamine, were chosen for this purpose.

The most interesting results were obtained from the study withl-cys. As it can be observed in Fig. 5a, the oxidation of a 10−4 M l-cys solution on an unmodified gold electrode shows one wave at apotential of ca. 0.95 V (Fig. 5a, voltammogram b), relatively close tothe 10−4 M l-tyr oxidation wave (Fig. 5a, voltammogram a). Whenthe differential pulse voltammograms are recorded from solutionscontaining a mixture of both amino acids, the bare electrode cannotdistinguish between the analytes showing only one oxidation wavecorresponding to both. However, if the same experiment is carriedout with the modified electrode under the optimized conditionsdescribed above, several effects can be observed as a consequenceof the modification. First, an increase in the l-tyr peak potential

value. Second, a decrease in the Ep corresponding to the l-cys oxida-tion respect to that recorded with a bare electrode. These results canbe rationalized on the base of the molecular structures of both com-pounds. l-tyr presents an aromatic ring to be included in the inner

C. Quintana et al. / Sensors and Actuators B 149 (2010) 129–135 133

Fce

�[taa(amtcgadt

fdhgwaccotttw

ig. 5. Differential pulse voltammograms of 10−4 M l-tyr, 10−4 M l-cys and l-tyr + l-ys mixture solutions with (A) a bare gold electrode and (B) a �-CD modified goldlectrode. 0.1 M H3PO4, pH = 1, Ei = 0.2 V, Tac = 10 s, a = 60 mV, Vb = 10 mV s−1.

CD cavity which benefits the stabilization of the complex formed26]. In addition, when the �CD is immobilized on the electrode,he kinetic of the l-tyr complex formation on the electrode surfaceffects the electrochemical response because a reorientation of thenalyte is required to be included through their aromatic structuresee discussion in page 10). In contrast, the l-cys is an aliphaticmino acid which minor affinity to be bound by �CD but with lowerolecular size than l-tyr and with a SH group in its structure which

ends to be chemisorpted on gold and benefits the global electro-hemical process when working with a gold electrode. Both factsive rise to two different signals allowing the identification of bothnalytes in a mixture (Fig. 5b, voltammogram c). The voltamogramsepicted in Fig. 6, show the benefits of the developed sensor respecto an unmodified electrode.

Finally, the maximum amount of l-cys in solution to inter-ere on the l-tyr determination was evaluated (interference weefine the concentration that leads to an l-tyr intensity variationigher than the 10%). To this aim, differential pulse voltammo-rams of 10−4 M l-tyr solutions with increasing l-cys amountsere recorded. Although the peak current of l-tyr is slightly

ffected by the presence of l-cys, employing the method l-tyran be determined in the presence of l-cys up to 25 fold l-tyr/l-ys concentration ratio. The high Ep values recorded for both

xidation processes (very close to gold oxides formation) andhe difference between both peak potentials is not high enougho ensure the complete interference to avoid without any addi-ional pre-treatment. Nevertheless, the resolution of the mixtureith the proposed method is guaranteed, which is not possi-

Fig. 6. Differential pulse voltammograms of l-tyr and l-cys mixtures with a �-CD modified gold electrode. 0.1 M H3PO4 pH = 1, Ei = 0.2 V, Tac = 10 s, a = 60 mV,Vb = 10 mV s−1.

ble with the unmodified electrode, and even the simultaneousanalysis of both amino acids at the concentration level statedabove.

The oxidation of the amino acid methionine on a bare electrodeunder the experimental conditions occurs at 1.05 V very close tothe formation of gold oxides. That circumstance allows the identi-fication of these two analytes in a mixture when an unmodifiedgold electrode is used. Nevertheless, when the proposed modi-fied electrode is employed, the presence of methionine affects thel-tyr current and causes interference from a 20 fold l-tyr/met con-centration ratio. In addition to this, the proposed method allowsthe l-tyr determination in the presence of 100 fold phenylalanineconcentration respect to l-tyr. On the other hand, the presence ofthe trypeptide l-glutathione affects in a great extent the oxidationsignal of l-tyr at the 1:1 concentration ratio. Although no electro-chemical signal was recorded under the experimental conditions,in a mixture solution of 10−4 M l-tyr and 10−4 M l-glu (either in abare electrode or in a modified one), an increase of the blackgroundcurrent and a displacement of the peak potential corresponding tothe l-tyr oxidation to more positive values up to overlap with thegold oxidation signal, are produced.

Finally, in the case of the neurotransmitter dopamine, the pres-ence of this analyte in solutions containing 10−4 M of l-tyr, doesnot produce any interference on the determination of the aminoacid up to a 10−5 M concentration.

3.6. Analytical application. Determination of l-tyr inpharmaceutical formulations

Once all the experimental variables were optimized, the valid-ity of the proposed sensor was evaluated for the determinationof l-tyrosine in a pharmaceutical formulation (vegetable tabletsfor human consumption, Solgar, 500 mg). The standard additionmethod was employed for quantification. The procedure was asfollows: an accurate amount of about 0.02 g of the sample (throw-ing the wrapper away) was dissolved in 10.0 mL of ultrapure water.Next, a subsample of 0.1 mL was diluted to 25.0 mL with support-ing electrolyte. This latter solution was set in the electrochemicalcell and the corresponding differential pulse voltammograms wererecorded. This procedure was applied to unspiked samples and todifferent 0.1 mL subsamples spiked with increasing l-tyr amountsin the range 1.0 × 10−5–3.0 × 10−4 M. Fig. 7 shows the voltam-

mograms recorded and the good fitting of the experimental datarecorded. All the experiments were done in triplicate and theresults are summarized in Table 1. Recoveries consistently betterthan 87% were obtained at all concentration levels assayed. A goodagreement between the l-tyr reported by the manufacturer and the

134 C. Quintana et al. / Sensors and Actu

Fig. 7. Differential pulse voltammograms of unspiked (a) and spiked samples (b–d)in the range 1.0 × 10−5 M (voltammogram b), 3.0 × 10−4 M l-tyr (voltammogramd) recorded with a �-CD modified gold electrode. 0.1 M H3PO4, pH = 1, Ei = 0.2 V,Tac = 10 s, a = 60 mV, Vb = 10 mV s−1.

Table 1Analytical data for l-tyrosine determination in real samples.

l-Tyr (mg/tablet) l-Tyr found (mg/tablet) Er (%) RSD (n = 3) (%)

lc

4

cleAdtrmteost

am

A

CU

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

500 478 4.4 6.7500 467 6.6 3.4500 465 7.0 8.7

-tyr was found, so, it can be concluded that the proposed methodan be applied with enough accurate and precision.

. Conclusions

An electrochemical sensor based on nanostructurated �-yclodextrins on a gold electrode surface has been developed for-tyrosine determination. The modification procedure proposednsures the inclusion complex formation on the electrode surface.fter the optimization of the experimental variables affecting theifferential pulse response of l-tyrosine with the modified elec-rode, the selectivity of the proposed methodology is shown by theesults recorded by analysis of amino acid mixtures. Although theodification proposed leads to a lower available gold surface with

he subsequent loss of sensitivity respect to the use of a bare goldlectrode, the LOQ achieved is good enough to apply the method-logy to the l-tyr determination in real samples and the addedelectivity showed by the method is quite better to demonstratehe benefits of the proposed method.

In addition, the complete proposed method is enough accuratend reproducible to be successfully applied to the l-tyrosine deter-ination in real samples.

cknowledgements

Authors thank to Universidad Autónoma de Madrid and to theomunidad Autónoma de Madrid for the financial support (CCG08-AM/PPQ-4439).

eferences

[1] N. Oh, J.H. Kim, C.S. Yoon, Self-assembly of silver nanoparticles synthesized byusing a liquid-crystalline phospholipid membrane, Adv. Mater. 20 (18) (2008)3404–3409.

[2] B. Wrzosek, J. Bukowska, Molecular structure of 3-amino-5-mercapto-1,2,4-triazole self-assembled monolayers on Ag and Au surfaces, J. Phys. Chem. C111 (46) (2007) 17397–17403.

[

ators B 149 (2010) 129–135

[3] S. Lee, J. Park, R. Ragan, S. Kim, Z. Lee, D.K. Lim, D.A.A. Ohlberg, R.S. Williams,Self-assembled monolayers on Pt (111): molecular packing structure and straineffects observed by scanning tunneling microscopy, J. Am. Chem. Soc. 128 (17)(2006) 5745–5750.

[4] Y. Wang, Y. Bai, M. Liang, W. Zheng, Differential pulse voltammetric determina-tion of selenocystine and selenomethionine using SAM nanoSe0/Vc/SeCys-filmmodified Au electrode, Electroanalytical 21 (19) (2009) 2145–2152.

[5] T. Kondo, A. Tamura, T. Kawai, Cobalt phthalocyanine-modified boron-dopeddiamond electrode for highly sensitive detection of hydrogen peroxide, J. Elec-trochem. Soc. 156 (11) (2009) 145–150.

[6] N. Wanichacheva, E.R. Soto, C.R. Lambert, W.G. McGimpsey, Surface-basedlithium ion sensor: an electrode derivatized with a self-assembled monolayer,Anal. Chem. 78 (20) (2006) 7132–7137.

[7] G.E. Poirier, M.J. Tarlov, H.E. Rushmeir, Two-dimensional liquid phase and thepx

√3 phase of alkanethiol self-assembled monolayers on Au(1 1 1), Langmuir

10 (10) (1994) 3383–3386.[8] E. Blanco, C.S.H. Domínguez, P. Hernández, J.V. Hernández, C. Quintana, L.

Hernández, Electroanalytical 21 (2009) 495–500.[9] I. Carrillo, M.C. Quintana, A.M. Esteva, L. Hernández, P. Hernández, Electroana-

lytical 20 (2008) 2614–2620.10] S.E. Lucanglioli, V. Tripodi, E. Masrian, S.L. Scioscia, C.N. Carducci, E. Kenndler,

Enantioselective separation of rivastigmine by capillary electrophoresis withcyclodextrines, J. Chromatogr. A 1081 (1) (2005) 31–35.

11] K.A. Connors, The stability of cyclodextrin complexes in solution, Chem. Rev.97 (1997) 1325–1357.

12] G. Nelles, M. Weisser, R. Back, P. Wohlfart, G. Wenz, S. Mittler-Neher, Controlledorientation of cyclodextrin derivatives immobilized on gold surfaces, J. Am.Chem. Soc. 118 (1996) 5039–5046.

13] T. Loftsson, M.E. Brewster, Pharmaceutical applications of cyclodextrins. 1. Drugsolubilization and stabilization, J. Pharm. Sci. 18 (10) (1996) 1017–1025.

14] J. Szejtli, Introduction and general overview of cyclodextrin chemistry, Chem.Rev. 98 (1998) 1743–1753.

15] D. Velic, M. Knapp, G. Köhler, Supramolecular inclusion complexes betweena coumarin dye and �-cyclodextrin, and attachment kinetics of thiolated �-cyclodextrin to gold surface, J. Mol. Struct. 598 (2001) 49–56.

16] D. Velic, G. Köhler, Supramolecular layer: coumarin/thiolated cyclodex-trin/gold, Chem. Phys. Lett. 371 (2003) 483–489.

17] M.T. Rojas, R. Koniger, J.F. Stoddart, A.E. Kaiffer, Supported monolayerscontaining preformed binding sites. Synthesis and interfacial binding prop-erties of a thiolated �-cyclodextrin derivative, J. Am. Chem. Soc. 117 (1995)336–343.

18] B. Fang, H. Liu, G. Wang, Y. Zhou, M. Li, Y. Yu, W. Zhang, The electrochemicalbehaviour and direct determination of tyrosine at a glassy carbon electrodemodified with poy(9-aminoacridine), Ann. Chim. 97 (2007) 1005–1013.

19] G.P. Jin, X.Q. Lin, The electrochemical behaviour and amperometric determi-nation of tyrosine and tryptophan at a glassy carbon electrode modified withbutyrylcholine, Electrochem. Commun. 6 (5) (2004) 454–460.

20] Q. Xu, S.F. Wang, Electrocatalytic oxidation and direct determination of l-tyrosine by square wave voltammetry at multi-wall carbon nanotubes modifiedglassy carbon electrodes, Microchim. Acta 151 (1–2) (2005) 47–52.

21] Y. Azuma, M. Maekawa, Y. Kuwabara, T. Nakajima, K. Taniguchi, T. Kanno, Deter-mination of branched-chain amino acids and tyrosine in serum of patients withvarious hepatic diseases, and its clinical usefulness, Clin. Chem. 35 (7) (1989)1399–1403.

22] F. Wang, K.Z. Wu, Y. Qing, Y.X. Ci, Spectrofluorometric determination of thesubstrates based on the fluorescence formation with the peroxidase-like con-jugates of hemin with proteins, Anal. Lett. 25 (1992) 1469–1478.

23] C.H. Deng, Y.H. Deng, B. Wang, X.H. Yang, Gas chromatography–mass spec-trometry method for determination of phenylalanine and tyrosine in neonatalblood spots, J. Chromatogr. B 780 (2) (2002) 407–413.

24] H. Orhan, N.P.E. Vermeulen, C. Tump, H. Zappey, J.H.N. Meerman, Simultaneousdetermination of tyrosine, phenylalanine and deoxyguanosine oxidation prod-ucts by liquid chromatography–tandem mass spectrometry as non-invasivebiomarkers for oxidative damage, J. Chromatogr. B 799 (2) (2004) 245–254.

25] G.H. Zhao, Y. Qi, Y. Tian, Simultaneous and direct determination of tryptophanand tyrosine at boron-doped diamond electrode, Electroanalytical 18 (8) (2006)830–834.

26] M. Shanmugam, D. Ramesh, V. Nagalakshmi, R. Kavitha, R. Rajamohan, T. Stalin,Host–guest interaction of l-tyrosine with �-cyclodextrin, Spectrochim. ActaPart A 71 (2008) 125–132.

27] J. Okuno, K. Maehashi, K. Matsumoto, K. Kerman, Y. Takamura, E. Tamiya,Single-walled carbón nanotube-array microelectrode chip for electrochemicalanalysis, Electrochem. Commun. 9 (2007) 13–18.

28] X. Yu, Z. Mai, Y. Xiao, X. Zou, Electrochemical behaviour and determination of l-tyrosine at single-walled carbon nanotubes modified glassy carbon electrode,Electroanalytical 20 (2008) 1246–1251.

29] X. Tang, Y. Liu, H. Hou, T. You, Electrochemical determination of l-tryptophan,l-tyrosine and l-cysteine using electrospun carbon nanofibers modified elec-trode, Talanta 80 (2010) 2182–2186.

30] G.P. Jin, X. Peng, Q.Z. Chen, Preparation of novel arrays silver nanoparticles

modified polyrutin coat paraffin-impregnated graphite electrode for tyrosineand tryptophanıs oxidation, Electroanalytical 20 (2008) 907–915.

31] K.J. Huang, D.F. Luo, W.Z. Xie, Y.S. Yu, Sensitive voltammetric determina-tion of tyrosine using multi-walled carbon nanotubes/4-aminobenzeresulfonicacid film-coated glassy carbon electrode, Colloids Surf. B 61 (2008)176–181.

d Actu

B

CA1Iir

S2D

Hernández has a wide recognized career in electroanalysis especially in modifiedelectrodes with applications in different areas. Among others, he got the prize “Pre-mio de Investigación 2003” from the Real Sociedad Espanola de Química (Analytical

C. Quintana et al. / Sensors an

iographies

armen Quintana received her degree in Chemistry in 1991 from the Universidadutónoma de Madrid and her PhD degree in 1998 from the same university. Since993 she is an Assistant Professor at the Department of Analytical Chemistry and

nstrumental Analysis at the Universidad Autónoma de Madrid. Her current research

s focused on the development of electrochemical sensors based on macrocycliceceptors.

onia Suárez received her bachelorıs degree in Industrial Chemical Engineering in009 from the Universidad Autónoma de Madrid and this work makes up her Finalegree Project.

ators B 149 (2010) 129–135 135

Lucas Hernández is, since 1982, Full Professor in the Department of AnalyticalChemistry and Instrumental Analysis at the Universidad Autónoma of Madrid. Hereceived his degree in Chemistry in 1969 from the Universidad de Salamanca andhis PhD degree in 1972 from the Universidad de Sevilla (Facultad de Badajoz). Prof.

Chemistry category) and, in 2008, the CIDETEC prize from the Electrochemical Groupof the Real Sociedad Espanola de Química in recognition of his entire electrochemicalcareer.