Green Synthesis of Gold−Chitosan Nanocomposites for Caffeic AcidSensingGabriella Di Carlo,*,† Antonella Curulli,‡ Roberta G. Toro,† Chiara Bianchini,‡ Tilde De Caro,†

Giuseppina Padeletti,† Daniela Zane,‡ and Gabriel M. Ingo†

†Istituto per lo Studio dei Materiali Nanostrutturati (ISMN) − CNR, Via Salaria Km 29300, 00015, Monterotondo Stazione, Roma,Italy‡Istituto per lo Studio dei Materiali Nanostrutturati (ISMN) − CNR, Via del Castro Laurenziano 7, 00161 Roma, Italy

*S Supporting Information

ABSTRACT: In this work, colloidal gold nanoparticles (AuNPs) stabilized into a chitosan matrix were prepared using a greenroute. The synthesis was carried out by reducing AuIII to Au0 in an aqueous solution of chitosan and different organic acids (i.e.,acetic, malonic, or oxalic acid). We have demonstrated that by varying the nature of the acid it is possible to tune the reductionrate of the gold precursor (HAuCl4) and to modify the morphology of the resulting metal nanoparticles. The use of chitosan, abiocompatible and biodegradable polymer with a large number of amino and hydroxyl functional groups, enables thesimultaneous synthesis and surface modification of AuNPs in one pot. Because of the excellent film-forming capability of thispolymer, AuNPs−chitosan solutions were used to obtain hybrid nanocomposite films that combine highly conductive AuNPswith a large number of organic functional groups. Herein, Au−chitosan nanocomposites are successfully proposed as sensitiveand selective electrochemical sensors for the determination of caffeic acid, an antioxidant that has recently attracted muchattention because of its benefits to human health. A linear response was obtained over a wide range of concentration from 5.00 ×10−8 M to 2.00 × 10−3 M, and the limit of detection (LOD) was estimated to be 2.50 × 10−8 M. Moreover, further analyses havedemonstrated that a high selectivity toward caffeic acid can be achieved without interference from catechin or ascorbic acid(flavonoid and nonphenolic antioxidants, respectively). This novel synthesis approach and the high performances of Au−chitosan hybrid materials in the determination of caffeic acid open up new routes in the design of highly efficient sensors, whichare of great interest for the analysis of complex matrices such as wine, soft drinks, and fruit beverages.

■ INTRODUCTIONIn the last decades, gold nanoparticles (AuNPs) have receivedincreasing amounts of attention because of their uniquephysical and chemical properties, which make them interestingfor applications in many fields such as biotechnology, optics,electronics, and catalysis.1−4 An intriguing feature of thesenanoparticles is the possibility to tune their properties througha molecular-level design by varying the size of the core and bysurface modification with suitable functional molecules. Amongthe various stabilizing agents used to prevent nanoparticlesfrom aggregating, polysaccharides represents an attractivechoice for the preparation of functional materials.5 In particular,an increasing amount of attention has recently been focused onthe synthesis of AuNPs with chitosan, a biocompatible,

biodegradable, and nontoxic polymer composed of β-(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine and obtainedby the partial deacetylation of chitin derived from crustaceanshells.6−11 The great interest in this polymer is derivedespecially from the large number of functional groups andfrom its excellent film-forming capability. Because of thepresence of amino and hydroxyl groups, chitosan canparticipate in both reduction and stabilization processes inthe synthesis of colloidal AuNPs, avoiding the use of toxicreductants such as hydrazine and sodium borohydride.

Received: December 14, 2011Revised: February 1, 2012Published: March 2, 2012

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Moreover, the film-forming capability of chitosan makespossible the production of highly dispersed AuNPs, which areembedded into the polymer matrix. This can significantly slowthe aggregation processes that typically affect the stability ofAuNPs.The design of hybrid films that combine highly conductive

AuNPs with organic functional groups, which are able tointeract specifically with target molecules, is very attractive forthe development of electrochemical sensors. In the presentwork, we focus our attention on the determination of caffeicacid (3,4-dihydroxycinnamic acid), a phenolic compound ofgreat interest, because of its antioxidant action against humanaging processes, its potential pharmacological applications as ananti-inflammatory, anticarcinogenic agent, and its protectiverole against cardiovascular diseases.12−17 Several analyticalmethods including chromatography18−27 and capillary electro-phoresis28 have been employed for the determination ofphenolic acids. However, in the literature just a few studies inwhich electrochemical techniques are used for a quantitativeanalysis of caffeic acid in different matrices are pro-posed.17,29−34 Generally, the linear range of detection is limitedand not applicable to different matrices, making the sensorunsuitable for general purposes.17,21,29−31 A high efficiency hasbeen reported for enzymatic biosensors,33−35 although in thiscase the immobilization of enzymes is often a critical issue.The determination of caffeic acid at low levels and in

different complex matrices, as human fluids or beverages usinga procedure that allows the achievement of higher sensitivityand selectivity and a rapid response time with respect toconventional analytical methodologies, is still an importantchallenge.Herein, we present the first study on the design of chitosan−

AuNP nanocomposites (AuNP−CHIT) with tailored morpho-logical properties as a sensing platform that is able to interactdirectly and specifically with caffeic acid, our target molecule.To prepare these materials, we have used a facile green routethat allows the tuning of the morphological and electrochemicalproperties of chitosan-stabilized AuNPs. The synthesis wascarried out in the presence of different organic acids (i.e., acetic,malonic, and oxalic acid), which, besides enhancing chitosansolubility through the protonation of amino groups, canparticipate to a different extent in gold precursor reduction.To the best of our knowledge, the influence of carboxylic acidon the size and shape of AuNPs stabilized in chitosan matriceshas not been investigated so far.

■ EXPERIMENTAL SECTIONSample Preparation. Synthesis of Chitosan-Stabilized AuNPs.

Medium-molecular-weight chitosan (5800 g mol−1), composedof β-(1-4)-linked D-glucosamine and N-acetyl-D-glucosaminewith a degree of deacetylation of 75−85%, was purchased fromAldrich. In a typical preparation, a suitable amount of chitosan(0.5 wt %) was dissolved in a 0.1 M aqueous solution of aceticacid (AA), malonic acid (MA), or oxalic acid (OA) by stirringthe system overnight.An appropriate amount of HAuCl4 aqueous solution was added to

the chitosan/organic acid solution in order to obtain a 1 mM goldconcentration. After the addition of the gold precursor, the solutionwas heated to 70 °C with reflux under magnetic stirring until the colorturned red. The resulting samples were labeled as AuXX−CHIT,where XX refers to the organic acid that was used in the synthesis. Toevaluate the contribute of organic acid and chitosan to the reductionand stabilization processes, the synthesis of AuNPs was also carried outwithout chitosan using the same experimental conditions.

Gold nanoparticles stabilized with citrate were prepared as areference material by using the classical Turkevich method.36 Anaqueous solution of HAuCl4 (1 mM) was brought to a boil undermagnetic stirring with a reflux condenser, and 1 mL of trisodiumcitrate (38.8 mM) was quickly added. The solution was refluxed understirring for 15 min, and during this time, its color changed from paleyellow to red. The resulting sample was labeled AuCITR.

Synthesis of Chitosan−Organic Acid Matrices. Chitosan matrices,in the presence of different organic acids and without gold precursor,were prepared by heating the solution to 70 °C with reflux undermagnetic stirring using the same conditions of AuNP synthesis. Theresulting samples were used as a reference in order to evaluate the roleof AuNPs in the electrochemical sensor.

Electrochemical Measurements. Polishing Steps. The firststep in bare gold electrode (AMEL Milano, Italy, d = 2 mm)pretreatment was the complete removal of trace adsorbatesfrom previous experiments by reductive desorption in 0.1 MNaOH in deionized water by carrying out cyclic voltammetry(CV) from −0.5 to −1.5 V versus Ag/AgCl at a scan rate of 1 Vs−1 (40 scans).37 This was followed by mechanical polishing ofthe gold electrodes to a mirrorlike finish with an alumina slurry(1, 0.3, and 0.05 μm) for 3 to 4 min on a polishing cloth(Buehler, U.K.). Finally, the electrodes were rinsed withdeionized water and cleaned ultrasonically in deionized waterfor 1 to 2 min to remove residual alumina particles. Thechemical pretreatment procedure consists of dipping the goldelectrodes into hot piranha solution (3:1 conc H2SO4/H2O2,work with caution) for ca. 15 min followed by rinsing withdeionized water and ultrasonication for 1 min. Electrochemicalpolishing was performed by 25 successive CV scans from −0.2to +1.5 V versus Ag/AgCl in a 0.1 M H2SO4 aqueous solutionat 0.1 V s−1. Electrochemical gold oxide stripping was carriedout by running 10 successive CV scans from 0.75 to 0.2 Vversus Ag/AgCl in a 0.1 M H2SO4 aqueous solution at 0.1 V s−1.Chemical reduction of gold oxide by incubation with absoluteEtOH was performed at ambient temperature under nonstirredconditions for 20 min. Capacitance changes were monitored byrunning chronoamperometry by stepping a working potentialfrom 0.00 to 0.05 V.37

Assembly of the AuNP−CHIT-Modified Gold Electrode. After thepolishing steps, Au electrodes were modified by drop casting 1 μL of aAuNP−CHIT solution onto the electrode surface and subsequent airdrying at room temperature. To increase the number of particles at theelectrode, this procedure was repeated three times.

Assembly of the Chitosan-Modified Gold Electrode. Au electrodeswere modified with chitosan−organic acid solutions using the sameprocedure for AuNP−CHIT-modified electrodes. These chitosan-modified electrodes were used as a reference to evaluate the role ofAuNPs.

Procedure. Electrochemical measurements were performed in aconventional two-compartment, three-electrode cell with a workingelectrode (bare or modified gold electrode), an Ag/AgCl electrode as areference, and a Pt electrode as a counter.

The modified electrode was electrochemically characterized in 0.2M KCl using (K3[Fe(CN)6]) as an electrochemical probes at aconcentration of 0.01 M by cyclic voltammetry, and the experimentswere carried out at a scan rate ranging from 20 to 500 mV s−1.

Finally, a AuNP−CHIT-modified electrode was used in phosphatebuffer for the electrochemical investigation of caffeic acid at aconcentration of 0.004 M. Phosphate buffer solutions (pH 7.00, 0.1M) were prepared using sodium dihydrogen phosphate dihydrate(Fluka, Microselect for molecular biology), with adjustment of the pHwith NaOH drops. The buffer solutions were prepared using distilled−deionized water and were kept refrigerated to minimize bacterialgrowth.

The following optimized instrumental parameters were used torecord differential pulse voltammograms (DPV): a potential rangefrom 0.0 to 0.7 V, a step potential of 0.006 V, and a modulationamplitude of 0.025 V.

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The limit of detection (LOD) and the limit of quantification(LOQ) for caffeic acid were obtained by using the equations LOD =3sx/y/b and LOQ = 10sx/y/b, where sy/x and b are the estimatedstandard deviation and the slope of the analytical calibration functionof each substance, respectively, with a 95% (degrees of freedom = 3)confidence level.38,39 The reproducibility of the method was evaluatedby measurements on 10 solutions of 2 × 10−7 M, leading to a relativestandard deviation of 3.9%.The impedance of the samples was measured at room temperature

at open circuit voltage (OCV) with a signal amplitude of 10 mV over afrequency range of 10 kHz−0.1 Hz. For the fitting of the data obtainedby EIS, we used Z-views software (Scribner Associates, Inc.) using the

Randles equivalent circuit40 shown in Scheme 1. This equivalentcircuit is given as a model for describing the electrolyte/electrodeinterphase. It consists of Rs (bulk solution resistance) in series with aparallel combination of Rct (interfacial charge transfer resistance), Zw(diffusion of the analytes in solution), and Cdl (double layercapacitance). In the presence of an analyte, the capacitive and resistivefeatures resulting from reactions under study at the electrode/electrolyte interphase are described by the Cdl and Rct elements.Characterization. UV−vis absorbance spectra of the as-prepared

chitosan-stabilized gold solutions were collected using a double-beamV-660 spectrophotometer (Jasco). The measurements were carried outusing a quartz cell of thickness 1 cm in the wavelength range of 300−900 nm.X-ray diffraction profiles of AuNP−CHIT were recorded with a

Siemens D5000 using Cu Kα radiation (λ = 1.5418 Å). The samplesfor the analysis were prepared by depositing gold−chitosan colloidalsolutions over a glass substrate and subsequent drying at roomtemperature.FE-SEM characterizations were carried out with a high-brilliance

LEO 1530 field emission scanning electron microscope apparatusequipped with an INCA 450 energy-dispersive X-ray spectrometer(EDS) and a four-sector backscattered electron detector (BSD). FE-SEM images were recorded both in SEI and BSD modes at differentacceleration voltages ranging from 3 to 20 kV. The samples for theanalysis were prepared by depositing gold−chitosan solutions onto asilicon substrate and subsequent drying at room temperature. Beforethe analysis, the surfaces were coated with a thin layer of carbon inorder to avoid charging effects. The carbon coating was deposited byusing an Emitech sputter coater K550 unit, a K 250 carbon-coatingattachment, and a carbon cord at a pressure of 1 × 10−2 mbar in orderto produce a carbon film with a constant thickness of about 3.0 nm.

■ RESULTS AND DISCUSSIONColloidal gold nanoparticles were prepared by addingtetrachloroauric acid (HAuCl4) to an aqueous chitosan solutionin the presence of acetic, malonic, or oxalic acid at 70 °C, andthe resulting solutions were labeled as AuAA−CHIT, AuMA−CHIT, and AuOA−CHIT, respectively. The first opticalindication of AuNP formation was given by the color changeof the solution from light yellow to red, and it was monitoredby UV−vis spectroscopy. According to the reaction time inTable 1, we can affirm that the reduction rate of AuIII to Au0

was strongly affected by the nature of the carboxylic acid andfollows the order AuOA−CHIT > AuMA−CHIT > AuAA−CHIT. When oxalic or malonic acid is used to dissolve chitosan,the reduction process becomes an order of magnitude fasterthan using acetic acid, suggesting the direct participation ofthese acids in the reaction. The reduction rate is related to the

reducing power of the acid; however, a pH effect cannot beruled out. By lowering the pH (Supporting Information, TableS1), a higher degree of protonation of chitosan amino groupscould favor the electrostatic interaction between the −NH3

+

groups and AuCl4− ions, promoting the reduction of the gold

precursor by means of chitosan.9

The formation of metallic AuNPs in AuAA−CHIT andAuMA−CHIT solutions was confirmed by the presence of thesurface plasmon resonance (SPR) band at 525 nm in thecorresponding UV−vis spectra (Figure 1). On the basis of the

SPR peak position and intensity,41 we have estimated theaverage diameter of AuNPs, which is 10 nm for both samples(Table 1). Different behavior is observed when AuNPs aresynthesized in chitosan−oxalic acid solution; the UV−visspectrum shows a significant red shift and broadening of theSPR band (Figure 1), which is indicative of larger metalnanoparticles and a certain degree of heterodispersion.To evaluate the electrochemical efficiency of chitosan-

stabilized AuNPs, the colloidal solutions were deposited onthe surface of a bare Au electrode. On the basis of the excellentfilm-forming capability, mechanical strength, and waterpermeability of chitosan, we aimed to obtain a modifiedelectrode surface that consists of a hybrid organic−inorganicfilm containing highly dispersed and accessible AuNPs. Theanalysis of the film cross sections by FE-SEM measurements(Figure 2) shows the existence of a fairly uniform network ofmetal nanoparticles that are dispersed in the chitosan matrices.According to Figure 2a,b, AuAA−CHIT and AuMA−CHITfilms consist of spherical AuNPs with a diameter of about 10−15 nm in agreement with the values estimated from UV−visspectra. The morphology of AuAA−CHIT and AuMA−CHITfilm surfaces (Figure 3a,b) is comparable to the corresponding

Scheme 1. Randles Equivalent Circuit

Table 1. Reduction Time for the Synthesis of AuNPs,Particle Size Estimated from UV−Vis Spectra, and theWavelength of Maximum Absorbance

sample reduction time (min) dAu‑UV (nm) λmax (nm)

AuAA−CHIT 70 10 530AuMA−CHIT 8 10 525AuOA−CHIT 4 nda 607

aNot determined.

Figure 1. UV−vis spectra of chitosan-stabilized AuNP solutions:AuAA−CHIT, AuMA−CHIT, and AuOA−CHIT.

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cross section. It is worth noting that the density of metalnanoparticles in the cross section of AuAA−CHIT is higherwith respect to AuMA−CHIT (Figure 2a,b). On the contrary,larger spherical and polygonal nanocrystals were detected forAuOA−CHIT (Figure 2c). For this sample, the morphology ofthe film surface differs significantly from the cross section, andgold enrichment was observed on the upper layers. The SEMimages (Figures 2c and 3c) suggest the formation of a highlyreactive surface layer in the AuOA−CHIT film.The presence of metal gold nanocrystals in AuNP−CHIT

films was also confirmed by X-ray diffraction (XRD) measure-ments (Supporting Information, Figure S1). In agreement withUV−vis and FE-SEM findings, the average diameter of Aucrystallites, as calculated with the Scherrer equation, corre-sponds to about 10 nm for AuAA−CHIT and AuMA−CHIT,whereas larger crystalline domains with a mean size of 37 nmwere detected for AuOA−CHIT (details in SupportingInformation).Our findings clearly show the important role played by

organic acid in combination with chitosan in the morphology of

AuNPs, which depends on the AuIII reduction rate and thedifferent interaction between chitosan and AuNPs. Theformation of large polygonal gold nanoparticles in AuOA−CHIT can be attributed to haphazard crystal growth mainlybecause of the strong reducing properties of oxalic acid. Theparticipation of oxalic and malonic acids in the reductionprocess was clearly demonstrated because performing thesynthesis without chitosan leads to a quick precipitation ofmetallic gold, whereas the contribution of acetic acid is notsignificant. However, the AuNP morphological propertiescannot be explained only on the basis of carboxylic acidreducing properties. By considering the reduction time (Table1), it was expected that the morphology of AuMA−CHITwould be similar to that of AuOA−CHIT. Nevertheless, FE-SEM, UV−vis, and XRD measurements have shown that thesize and shape of AuNPs in these two samples are significantlydifferent. When malonic acid is used, the average diameter ofthe AuNPs is smaller with respect to that when oxalic acid isused. The different morphological properties of AuNPs inAuMA−CHIT and AuOA−CHIT can be attributed to the

Figure 2. FE-SEM cross-section of AuNP−CHIT films prepared usingcolloidal gold solutions: (a) AuAA−CHIT, (b) AuMA−CHIT, and (c)AuOA−CHIT.

Figure 3. FE-SEM surface images of AuNP−CHIT films preparedusing colloidal gold solutions: (a) AuAA−CHIT, (b) AuMA−CHIT,and (c) AuOA−CHIT.

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combined effect of a different reduction rate and a differentinteraction between AuNPs and chitosan. It has to be pointedout that the number of carboxylic groups, the degree of aciddissociation (Supporting Information, Table S1), and thedensity of negative charge in carboxylate anions couldsignificantly affect the electrostatic interaction between theorganic acids and the amino groups of chitosan. The higher thestrength of the carboxylic acid−chitosan interaction, the lesseffective the stabilizing effect of polysaccharide on goldnanoparticles because of a smaller concentration of freeamino groups. On the basis of these considerations, theeffectiveness of chitosan as a stabilizer of AuNPs should followthe order AuAA−CHIT > AuMA−CHIT > AuOA−CHIT. Asimilar explanation was reported by Huang et al.9 when apolyanion such as tripolyphosphate is added to a chitosan−acetic acid solution. The authors demonstrated that the growthof large polygonal gold nanocrystals was promoted by loweringthe number of amino groups available to absorb on the surfacesof gold nanoparticles.Therefore, we have demonstrated that by using a facile green

route it is possible to modify the morphology of AuNPs andtheir distribution into a chitosan matrix by varying the nature ofthe organic acid in the solution.To evaluate the influence of the synthesis procedure on the

electrochemical properties of AuNP−CHIT films, the efficiencyof modified electrodes was preliminarily investigated by cyclicvoltammetry (CV) using K3[Fe(CN)6] as a redox probe. All ofthe samples lead to an amplification of the electrochemicalresponse with respect to the bare gold reference electrode(Table 2). According to the literature,14,42 the increase in thecurrent signal could be explained by the higher surface area ofnanostructured electrodes.43 The electroactive surfaces of thesemodified Au electrodes was calculated by CV in 10 mMK3[Fe(CN)6] according to the Randles−Sevcik equation44 anda well-known literature protocol45−49 (details in SupportingInformation). All of the AuNP−CHIT-modified electrodesshowed an increase in the peak current intensity with respect tothe bare gold electrode and an amplification of the anodiccurrent higher than 77% (Table 2). In addition, on the basis ofipa/ipc values (ranging from 1.07 and 0.83) and ΔEp (rangingfrom 0.10 to 0.07 V), the redox process can be considered to beat least quasi-reversible.44 More efficient electron transfer hasbeen observed for all of the AuNPs−chitosan-modifiedelectrodes, and we are going to consider a surface where Aunanoparticles are in electrical contact with a gold electrodethrough the chitosan structure.To demonstrate the significant role of chitosan, we have

compared the electrochemical performance of our samples withthat of an electrode modified with citrate-stabilized AuNPs(AuCITR). AuCITR nanoparticles were prepared by theclassical Turkevich method,36 and according to the literature,their mean diameter is 14 nm (Supporting Information, Figure

S2). As shown in Table 2, the current intensity of the AuCITR-modified electrode is even lower than that of a bare goldelectrode, probably because of the absence of an efficientdispersing agent. These findings suggest that chitosan plays akey role, keeping metal nanoparticles highly dispersed andinterconnected through the formation of a collaborative hybridnetwork.The efficiency of AuNP−CHIT for the determination of

caffeic acid was investigated by CV (Figure 4 and Table 3).

According to the literature,29,50,51 this process implies theoxidation of caffeic acid on a modified electrode involving twoelectrons per molecules, which likely leads to the formation ofcaffeic acid o-quinone.16

The oxidation of caffeic acid has been studied at differentsolid electrodes by many authors.16,29 An electrochemical studyvia cyclic voltammetry on a gold electrode has shown that the

Table 2. Electrochemical Activity by Cyclic Voltammetry in 10 mM K3[Fe(CN)6] at a Scan Rate of 0.02 V s−1

sample Epa (V) Epc (V) ipa (μA) ipc (μA) ipa/ipc ΔEp (V) Δipa (%)a area (cm2)

bare Au electrode 0.27 0.19 24.01 −22.38 1.07 0.08AuAA−CHIT 0.28 0.21 46.45 −55.95 0.83 0.07 93.46 4.74 × 10−2

AuMA−CHIT 0.28 0.19 42.56 −46.55 0.91 0.09 77.26 2.45 × 10−2

AuOA−CHIT 0.28 0.21 46.77 −52.78 0.88 0.07 94.79 4.77 × 10−2

AuCITR 0.30 0.20 18.29 −17.38 1.05 0.10 −23.82 1.86 × 10−3

aΔipa was calculated using the equation (ipaM − (ipaB/ipaB)) × 100, where ipaM and ipaB are the anodic current intensities at modified and bare Auelectrodes, respectively.

Figure 4. Cyclic voltammetry in 0.004 M caffeic acid, recorded in 0.1M phosphate buffer (pH 7.0, scan rate 0.02 V s−1, potential range from−0.2 to +0.8 V) at different electrodes: (a) bare Au electrode, (b)AuAA−CHIT-modified Au electrode, (c) AuOA−CHIT-modified Auelectrode, and (d) AuMA−CHIT-modified Au electrode.

Table 3. Electrochemical Activity by Cyclic Voltammetry in0.004 M Caffeic Acid at a Scan Rate of 0.02 V s−1

sample Epa (V) ipa (μA) Δipa (%)a

bare Au electrode 0.33 14.37AuAA−CHIT 0.33 19.76 37.51AuMA−CHIT 0.40 17.27 20.18AuOA−CHIT 0.43 17.62 22.62AuCITR 0.33 16.14 12.32

aΔipa was calculated using the equation (ipaM − (ipaB/ipaB)) × 100,where ipaM and ipaB are the anodic current intensities at modified andbare Au electrodes, respectively.

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first oxidation step involves a number of electrons per moleculeequal to 2 with the formation of caffeic acid o-quinone.16,29 Itshould be mentioned that in caffeic acid a double bond ispresent; however, its conjugation with the aromatic ring makesthe molecule more difficult to reduce. Therefore, the cyclicvoltammogram does not exhibit any voltammetric wave for thereduction of caffeic acid in the useful potential range, and thereduction of the olefinic bond in this compound cannot be usedfor analytical purposes.The cyclic voltammograms in caffeic acid, recorded in the

potential range from −0.2 to +0.8 V versus Ag/AgCl, showed awell-shaped anodic peak (Figure 4).As shown in Figure 4 and Table 3, the anodic peak potential

(Epa) values at AuNP-modified electrodes for caffeic acid do notdiffer significantly from those detected at a bare electrode. Theevident electroanalytical improvement concerns the higheranodic current intensity from the oxidation process.It is well known that although gold is a poor catalyst in bulk

form, nanosized gold exhibits excellent catalytic52 and electro-catalytic activities.53,54 Consequently, AuNPs can significantlyaccelerate the electrooxidation of caffeic acid because of theirhigh activity in oxidation processes.52−54

The most pronounced amplification of the electrochemicalresponse was observed for AuAA−CHIT (Δipa = 37.51%),whereas for AuMA−CHIT and AuOA−CHIT the increases inthe current intensity are comparable (Δipa = 20.18 and 22.62%,respectively).The high performance of the AuAA−CHIT-modified

electrode is probably due to a better interconnection betweenAuNPs. According to FE-SEM analysis (Figure 2), the higherdensity of metal nanoparticles into the chitosan matrix wasobserved in the cross section of a AuAA−CHIT film. Inaddition, a greater number of chitosan free amino groups inAuAA−CHIT, according to the smaller number of carboxylicgroups from organic acid, could also play a crucial role. Indeed,chitosan free amino groups can be responsible for an

electrostatic interaction with the carboxylic group of the caffeicacid. Chitosan could act as a scavenger for our target molecule,which, once close to AuNPs, can easily react with a consequentenhancement of the anodic current. It is worth noting that thepresence of AuNPs is of crucial importance because nosignificant signal amplification with respect to the bare Auelectrode was detected for chitosan-modified electrodeswithout AuNPs (Supporting Information, Figures S3 and S4).For the sake of completeness, the CV in caffeic acid was also

carried out using a AuCITR-modified electrode. As shown inTable 3, the amplification of the current intensity is lesspronounced with respect to the AuNPs−chitosan nano-composites. This suggests that both the high surface area ofnanosized AuNPs and the ability of chitosan to bring theanalyte molecules to the electrode contribute to enhance theelectrochemical performance of the nanocomposite films.The oxidation of caffeic acid was further investigated by

electrochemical impedance spectroscopy (EIS) in the presenceof caffeic acid (Supporting Information, Figure S5). The EIS ofall three modified electrodes versus the bare electrode indicateda comparable behavior with respect to the cyclic voltammetry.In particular, it was observed that the AuAA−CHIT-modifiedAu electrode had the smallest charge-transfer resistance (Rct):11.8 ± 0.16 KΩ versus 28.5 KΩ ± 0.42 KΩ for bare goldelectrodes, 17.7 ± 0.39 KΩ for AuMA−CHIT, and 22.6 ± 0.30KΩ for AuOA−CHIT-modified gold electrodes: this wasconsistent with voltammetric measurements. The smallestcharge-transfer resistance of AuAA−CHIT indicates lessopposition to the passage of an electric current.To investigate the analytical potential of the AuAA−CHIT-

modified electrode as a sensing platform, we have detectedcaffeic acid by means of differential pulse voltammetry (DPV).According to the literature,17−35,55,56 several analytical

methods have been employed for the detection of caffeicacid, from liquid chromatography to the electrochemical ones.By comparing the common analytical parameters (Table 4),

Table 4. Some Analytical Methods for the Determination of Caffeic Acid

detection method linearity range (M) LOD (M) LOQ (M) ref

liquid chromatography (LC) 2.90 × 10−4− 2.00 × 10−3 2. × 10−4 6.11 × 10−4 193.67 × 10−7−3.67 × 10−5 3.44 × 10−8 1.17 × 10−6 56

high performance liquid chromatography (HPLC) 5.55 × 10−5−1.88 × 10−4 3.49 × 10−7 205.55 × 10−8− 1.11 × 10−7 1.67 × 10−9 212.55 × 10−8− 1.72 × 10−6 222.77 × 10−6− 5.55 × 10−5 9.47 × 10−7 238.60 × 10−8− 8.60 × 10−6 1.17 × 10−8 2.83 × 10−8 245.55 × 10−6− 5.55 × 10−4 4.44 × 10−7 1.44 × 10−6 254.88 × 10−8− 2.77 × 10−4 1.17 × 10−8 2.83 × 10−8 263.71 × 10−6− 1.11 × 10−4 4.44 × 10−9 1.49 × 10−6 271.00 × 10−5− 1.00 × 10−4 2.00 × 10−4 18

ultraviolet spectroscopy 4.44 × 10−7− 4.70 × 10−5 1.11 × 10−7 55electrophoresis 5.00 × 10−6− 3.00 × 10−3 2.50 × 10−6 28electrochemical methodssquare wave voltammetry (SWV) 1.00 × 10−8− 5.00 × 10−5 4.00 × 10−9 31

9.00 × 10−6− 4.00 × 10−5 4.00 × 10−6 3.00 × 10−5 32cyclic voltammetry (CV) 6.00 × 10−5− 5.00 × 10−4 3.12 × 10−5 30

1.00 × 10−4− 1.00 × 10−3 29amperometry (tyrosinase biosensor) 3.00 × 10−7− 8.30 × 10−5 2.00 × 10−7 33amperometry (laccase biosensor) 5.00 × 10−7− 1.30 × 10−4 5.24 × 10−7 34amperometry (laccase-MWCNT-chitosan biosensor) 7.35 × 10−7− 1.05 × 10−5 2.75 × 10−7 35differential pulse voltammetry (DPV) 9.65 × 10−7− 1.10 × 10−5 2.90 × 10−7 9.70 × 10−7 17

5.00 × 10−8 −2.00 × 10−3 2.50 × 10−8 4.50 × 10−8 this work

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such as the linearity range, LOD, and LOQ, it was observedthat restricted linearity ranges generally limit the field ofapplication to specific matrices (e.g., only in wine or in plasmaor in a plant material). Most of the analytical methods for thedetermination of caffeic acid (Table 4) show a very limitedlinearity range, generally 1 or 2 orders of magnitude.21,29−35

Concerning the LOD values, in many cases they are notparticularly low and are suitable only for the analysis of fruitjuices or not well-specified food.29−31 In other papers, lowerLOD values were observed,17,21,30,31 starting from 2.90 × 10−7

to 1.67 × 10−9 M, but with restricted linearity ranges that makethe sensor nonapplicable to different matrices.In our case, the DPV technique showed a wide linear

response range with concentration varying from 5.00 × 10−8 to2.00 × 10−3 M (correlation coefficient, r, of 0.999, Figure 5).

The limit of detection (LOD) and the limit of quantification(LOQ) for caffeic acid resulted in 2.50 × 10−8 and 4.50 × 10−8

M, respectively, which were calculated using a number of pointsequal to 5 (n = 5).39 These results demonstrate the analyticalpotential of the proposed sensing platform. Moreover, a highreproducibility of the method was obtained because measure-ments made on 10 solutions of 2.00 × 10−7 M lead to a relativestandard deviation (RSD) of 3.9%, which proves that theconstruction procedure of the sensor was reliable, thus allowingreproducible responses. The RSD values for the differentanalytical methods are in the range from 1%18,23 to 6%,26 andthe value obtained for our work is in the above-mentionedrange.Potential interfering agents for the electrochemical determi-

nation of caffeic acid in food and beverages are flavonoids (suchas catechin) and nonphenolic antioxidants (such as ascorbicacid).57 These species might hinder the interaction between thecaffeic acid molecule and the modified electrodic surface.CVs for the AuAA−CHIT-modified electrode have shown

that the anodic potential and the current intensity in thepresence of catechin (Epa = 0.15 V, ipa = 0.15 μA) differsignificantly from those observed in the presence of caffeic acid

(Epa = 0.33 V, ipa = 19.76 μA). This means that the two anodicpeaks can be easily distinguished without interference.On the contrary, the anodic peak of ascorbic acid can be

considered to be absent at the AuAA−CHIT-modifiedelectrode (Figure 6). To evaluate the selectivity of our sensor

for the determination of caffeic acid in products such as wineand fruit beverages, the calibration of caffeic acid has beencarried out in the presence of ascorbic acid (0.5 mM) by DPV.Under these experimental conditions, the electrochemical signaldue to ascorbic acid is not detectable, so no interference ofascorbic acid has been observed (correlation coefficient, r, of0.998, Figure 6).These findings can be extended to a concentration of

ascorbic acid of up to 1 mM (Supporting Information, FigureS6) because its contribution is still absent. At concentrationshigher than 1 mM (i.e., 2 and 3 mM), the electrochemicalsignal of caffeic acid has to be corrected by subtracting thecurrent intensity for ascorbic acid.On the basis of these results, AuNPs−chitosan-modified

electrodes are of great interest for the determination of caffeicacid in wine,32 isotonic drinks,58 tea drinks,58 and fruitbeverages18,58 without the interference of ascorbic acid becauseits mean concentration is usually not much higher than 1 mM.The procedure can be extended to fruit juice with a highercontent of ascorbic acid by subtracting the contribution of thisacid to the current intensity.Our preliminary results for the determination of caffeic acid

are quite promising and make Au−chitosan-modified electrodesworthy of further investigation. In particular, it is of greatinterest to investigate the electrochemical study of othermolecules, structurally related to caffeic acid, at AuNPs−chitosan-modified gold electrodes and to evaluate the potentialof the proposed sensors for the analysis of complex matrices.

Figure 5. DPVs of different concentrations of caffeic acid (5 × 10−8, 2× 10−7, 5 × 10−7, 1 × 10−6, 2 × 10−4, 5 × 10−4, 1 × 10−3, and 2.0 ×10−3 M) at a AuAA−CHIT-modified Au electrode in 0.1 M phosphatebuffer (pH 7.0, potential range from 0.0 to 0.7 V, step potential 0.006V, and modulation amplitude 0.025 V). The inset shows thecalibration curve.

Figure 6. DPVs at a AuAA−CHIT-modified Au electrode in 0.1 Mphosphate buffer (pH 7.0, potential range from 0.0 to 0.7 V, steppotential 0.006 V, and modulation amplitude 0.025 V): (a) phosphatebuffer, (b) 5 × 10−4 M ascorbic acid, (c) 5.0 × 10−4 M ascorbic acid +1.0 × 10−4 M caffeic acid, (d) 5.0 × 10−4 M ascorbic acid + 3.0 × 10−4

M caffeic acid, (e) 5.0 × 10−4 M ascorbic acid + 5.0 × 10−4 M caffeicacid, (f) 5.0 × 10−4 M ascorbic acid + 7.0 × 10−4 M caffeic acid, (g) 5.0× 10−4 M ascorbic acid + 9.0 × 10−4 M caffeic acid, (h) 5 × 10−4 Mascorbic acid + 1.0 × 10−3 M caffeic acid. The inset shows thecalibration curve.

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dx.doi.org/10.1021/la204924d | Langmuir 2012, 28, 5471−54795477

■ CONCLUSIONS

We propose a green route to produce a collaborative networkof AuNPs that are highly dispersed in a chitosan matrix, whosefunctional groups favor the interaction with caffeic acid. Theelectrode modification with AuNP−CHIT nanocompositefilms allows significant improvements to the electron-transferprocess with respect to the bare Au electrode, to the electrodesmodified with a chitosan−organic acid matrix (withoutAuNPs), and to the one with citrate-stabilized AuNPs. Thesefindings suggest the key role of a collaborative hybrid networkthat is composed of AuNPs and chitosan. The electrochemicalperformance of these nanocomposite films can be varieddepending on the synthesis procedure. In particular, we havedemonstrated that the nature of the organic acid in chitosansolutions has a significant effect on the size and shape of goldnanoparticles because of its involvement in the reduction of thegold precursor and the interaction with chitosan. Consequently,by changing the nature of the acid in Au−chitosan solutions, itis possible to modify the morphological properties of the metalnanoparticles greatly.In this work, these materials are successfully proposed to be

sensitive, selective electrochemical sensors for the determi-nation of caffeic acid, an antioxidant that has recently attractedmuch attention because of its benefits on human health and itspharmacological properties. The very low limit of detection andlinear response over a wide range of caffeic acid concentrationmake these materials suitable for the analysis of differentmatrices. Moreover, catechin (a flavonoid) does not causeinterference, and a high selectivity toward caffeic acid wasachieved in the presence of ascorbic acid (a nonphenolicantioxidant).These preliminary results on AuNP−CHIT hybrid nano-

composites open up new routes in the design of highly efficientsensors, which are attractive and worthy of further inves-tigations for the analysis of complex matrices, such as wine andfruit beverages. In addition, the use of an environmentallyfriendly process that involves a biocompatible polysaccharide tostabilize and functionalize AuNPs makes these materialssuitable for large-scale production.

■ ASSOCIATED CONTENT

*S Supporting InformationFurther details on X-ray diffraction and electrochemicalmeasurements and additional figures and tables as describedin the text. This material is available free of charge via theInternet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*Phone: +39 0690672214. E-mail: gabriella.dicarlo@ismn.cnr.it.

NotesThe authors declare no competing financial interest.

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