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Electrochimica Acta 138 (2014) 62–68

Contents lists available at ScienceDirect

Electrochimica Acta

j our na l ho me pa g e: www.elsev ier .com/ locate /e lec tac ta

lectrosynthesis and characterisation of poly(folic acid) films

aimonda Celiesiute, Tautvydas Venckus, Sarunas Vaitekonis, Rasa Pauliukaite ∗,1

epartment of Nanoengineering, Center for Physical Sciences and Technology, Savanoriu Ave. 231, LT-02300, Vilnius, Lithuania

r t i c l e i n f o

rticle history:eceived 7 April 2014eceived in revised form 18 June 2014ccepted 18 June 2014vailable online 25 June 2014

eywords:

a b s t r a c t

Electropolymerisation of folic acid (FA) was performed in different media in order to optimise the bestconditions for the formation of a stable poly(folic acid) (PFA) film. Different substrates were also usedfor PFA deposition. The electrochemical stability of the PFA film was insufficient; therefore, graphene-chitosan (G-Chit) composite was deposited on the top of PFA film in order to prevent it from washing outof the electrode surface. A possible electropolymerisation mechanism was proposed.

The obtained films were characterised microscopically and electrochemically applying atomic force

olic acidlectropolymerisationlectrochemical impedance spectroscopyraphene composite

microscopy (AFM) and electrochemical methods such as cyclic voltammetry (CV), square wave voltam-metry (SWV) and electrochemical impedance spectroscopy (EIS) in different media. The best film wasobtained when PFA was electrosynthesised from 0.1 mol L−1 KCl/HCl solution, pH 2.0, covered with G-Chitcomposite film. The best electrochemical behaviour of such electrode was in the same buffer solution.This electrode will be further applied to (bio)sensor construction.

© 2014 Elsevier Ltd. All rights reserved.

. Introduction

Folic acid (FA) or vitamin B9, an IUPAC name–(2S)-2-[[4-(2-amino-4-oxo-1H-pteridin-6-yl)methylamino]benzoyl]amino]entanedioic acid, is a B group vitamin essential for DNA synthesisnd the metabolism of homocysteine. FA deficiency is associatedith anaemia, unfavourable pregnancy outcomes, cardiovascularisease, and possibly neoplasms of the colon [1]. For these reasons,A is intensively determined in food [2–4], biofluids [1,5], etc.oreover, conjugated foliates are used as the targeting drug

elivery carriers and disease therapies [6–9] as well as for theiagnosis of some diseases [1,10,11].

Fig. 1 shows the chemical structure of folic acid. As seen, thisompound is electroactive as most of the B group vitamins; there-ore, this peculiarity allows employing it in electrochemistry forifferent purposes as well as detecting it electrochemically. Elec-ropolymerisation of folic acid was performed in order to create aensitive and a selective dopamine sensor [12]. However, this workaid little attention to the electrochemical behaviour of poly(folic

cid) (PFA) itself as well as the polymerisation mechanism wasiscussed briefly.

∗ Corresponding author. Tel.: +370 5 2644886; fax: +370 5 260 2317.E-mail address: pauliukaite@ftmc.lt (R. Pauliukaite).

1 ISE member

ttp://dx.doi.org/10.1016/j.electacta.2014.06.103013-4686/© 2014 Elsevier Ltd. All rights reserved.

In order to attract FA to the electrode surface, a polymer oranother compound is often deposited on the surface. Chitosan(Chit) is one of such polymers which attracts folic acid due toan interaction of amino groups from chitosan and carboxy-groupsfrom FA; stabilise it and fixes to the surface [13]. Chitosan or �-(1-4)-linked D-glucosamine is a biologically friendly polymer obtainedfrom chitin and it is applied in many fields due to its propertiesallowing fabricate membranes, thin films, three-dimensional struc-tures, graphene and carbon nanotube immobilisation [14–17]. Insome cases, FA by itself can be that particular compound attractingother compounds, for example dopamine [12].

Electrodes are also often modified with materials enhancing anelectrochemical signal, for example, electrode for FA determina-tion was modified with ordered mesoporous carbon [18] carbonnanotubes [19–21], metal nanoparticles [20], DNA [22], polypyr-role [23] and imprinted polymers [24,25]. Lately, graphene was alsoused for such purposes because it showed electrocatalytical activ-ity towards various compounds [26] especially in electrochemicalbiosensing [27–31].

In this work the electropolymerisation of folic acid is presented.FA was polymerised from different media on glassy carbon elec-trodes and on HOPG for morphology studies. The electrochemicalbehaviour of PFA was studied electrochemically employing voltam-

metric techniques and electrochemical impedance spectroscopy.Finally, the mechanism of PFA formation was proposed. The poly-merisation conditions were optimised according to the stability ofthe PFA film.

R. Celiesiute et al. / Electrochimic

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Fig. 1. Chemical structure of folic acid.

. Experimental

.1. Chemicals

Folic acid, KCl, chitosan from shrimp shells, H2SO4, HNO3,nd CH3COOH were obtained from Sigma Aldrich (Germany).aH2PO4·H2O, Na2HPO4, NaOH and HCl were from ROTH GmbH

Germany). Graphene flakes with the thickness of 8 nm were pur-hased from Graphene Supermarket (USA). All reagents used weref analytical grade and used as received, except graphene, whichas chemically functionalised as described below. All solutionsere prepared with nanopure MilliQ-water (resistivity of 18.2 M�

m) directly taken from Synergy 185 unit equipped with a UV lampMillipore, USA).

Aqueous chitosan solution of 1% was prepared as describedlsewhere [32]. Chitosan was dissolved in aqueous 1% CH3COOHolution with magnetic stirring for approximately 1 h, pH wasdjusted to pH 5.0 with 20% NaOH solution. The solution was fil-ered using glass fibre filter (70 g m−2) (ROTH GmbH, Germany).

Supporting electrolytes used for electrochemical experimentsere 0.1 mol L−1 KCl/HCl, pH from 1.0 to 3.0, and 0.1 mol L−1 sodiumhosphate buffer saline (PBS) with 0.15 mol L−1 NaCl solutions, pHrom 4.0 to 8.0.

.2. Apparatus

Atomic force microscopy (AFM) images were recorded withhe atomic force microscope (CP-II, Veeco Instruments Inc., USA).he imaging of the surface was performed in the contact moden air under normal laboratory conditions (20 ◦C, relative humid-ty 30-60%) using Bruker MLCT probes. The highly orientedyrolytic graphite (HOPG) 10 × 10 mm (NT-MTD, Ireland) surfaceas renewed by cleaving the surface with the adhesive Scotch taperior to AFM investigation.

All electrochemical measurements were carried out with theotentiostat/galvanostat CompactStat (Ivium Technologies, Theetherlands) assembled with the three-electrode system. Theorking electrode was either bare or modified glassy carbon elec-

rode (diameter of 3.0 mm) or HOPG, a Pt wire served as theounter electrode and an Ag/AgCl (sat. KCl) electrode was aseference. All potentials in this paper are given versus this refer-nce. Electrochemical characterisation was carried out using cyclicoltammetry (CV), square wave voltammetry (SWV) and electro-hemical impedance spectroscopy (EIS). The SWV parameters weres follows: the start potential -1.0 V, the end potential 1.0 V, theotential step 5 mV, the frequency 10 Hz and the amplitude 10 mV.IS investigation was performed at a constant applied potential,hosen at the peak position (see Results and Discussions Section),n a frequency range from 100 kHz to 0.1 Hz, with the potentialerturbation of 10 mV.

.3. Electrosynthesis of poly(folic acid)

Prior to modification, the surface of the glassy carbon electrodeas carefully polished to a mirror-like plane with 1.0, 0.3 and 0.05

a Acta 138 (2014) 62–68 63

micron aluminium oxide slurry successively and rinsed with theMiliQ water followed by the sonication in water and ethanol for1 min after each polishing step. After the mechanical polishing,the electrode was polished electrochemically in 0.1 mol L−1 KClby scanning the applied potential repeatedly between -1.0 V and+1.0 V vs. Ag/AgCl at a scan rate of 100 mV s−1 for at least 10 cyclesuntil constant voltammograms were obtained. Then the electrodewas successively rinsed with the MiliQ water and transferred to theelectropolymerisation solution containing buffer solution, either0.1 mol L−1 PBS, pH 5.0, or 0.1 mol L−1 KCl/HCl, pH 2.0, or 1.0 molL−1 HCl, with 0.1 mmol L−1 folic acid. PFA was obtained by therepeated scanning of the applied potential between -1.0 and +1.5 Vat the potential scan rate of 50 mV s−1 for at least 10 cycles. Thescan number was optimised according to film stability during theelectrochemical characterisation.

When PFA was electrosynthesised on HOPG, the electrode sur-face was cleaved with the adhesive tape and immediately a dropletof 150 �L of the same solutions containing FA was dropped onthe surface. Pt-wire counter electrode and micro Ag/AgCl referenceelectrode were inserted into the droplet and PFA was synthesisedapplying the same procedure as in the case of GCE.

PFA was also covered with graphene-chitosan (G-Chit) compos-ite film. Graphene was cast on the electrode surface as describedelsewhere [32]. Chemically functionalised graphene was obtainedas indicated previously [32]. 50 mg of graphene was sonicated for20 h in a mixture of 5 mol L−1 H2SO4 and 5 mol L−1 HNO3, volumeratio 3:1, at +40 ◦C; then the mixture was filtrated and washed withthe MiliQ water until pH of the washing water was neutral. Afterthat, such functionalised graphene was dried in an oven at 80 ◦C for24 h. Finally, this freshly functionalised graphene was added to the1% acetate solution (pH 5) and sonicated for 2 h, until a homoge-neous dispersion was reached. Next, chitosan solution was addedin order to get 1 �g mL−1 graphene dispersion in aqueous 0.5%chitosan solution. The dispersion was shacked with the Vortex-Genie® 2 (Germany) and then sonicated for 5 min to reach the finalhomogeneous dispersion. In order to prepare the G-Chit/PFA/GCE,the aliquot of 3 �L graphene dispersion in aqueous 0.5% chitosansolution was dropped onto PFA/GCE surface immediately after son-ication and left to dry in air.

PFA was also synthesised on the graphene-chitosan (G-Chit)modified GCE. Graphene was cast on the electrode surface asdescribed above, the only difference–the suspension was droppedonto the pre-treated GCE surface immediately after sonication andleft to dry in air. PFA was synthesised in the same way as indicatedabove.

3. Results and discussion

3.1. Electrosynthesis of poly(folic acid)

First PFA was synthesised by the electropolymerisation of0.1 mmol L−1 folic acid from 0.1 mol L−1 PBS solution, pH 5.0, or0.1 mol L−1 KCl/HCl, pH 2.0, either on the GCE or on the HOPGsurface by the repeated potential cycling. HOPG was introducedbecause it was more suitable for microscopic investigations. Thesesolutions were chosen due to the fact that polymerisation of simi-lar heterocyclic compounds such as phenazines usually is catalysedand polymers are stabilised by small anions such as Cl-, NO3-, SO4

2-,etc. [33,34]. The pH values were chosen according to dissociationconstants pK’ for N(1) and N(10), which were 2.35 and 0.20, respec-tively at the ionic strength of 1.0 mol L−1 [35]. However, at the ionic

strength of 0.3 mol L−1, pKa1 (carboxylic group) was 3.23; pKa2 (N5)was 5.13, and pKa3 (N3) was 8.39 [36]. The best counterion for folicacid was found to be Cl− ion. The potential window and the scan ratewere optimised for each deposition solution. Since the most stable

64 R. Celiesiute et al. / Electrochimica Acta 138 (2014) 62–68

Fig. 2. (A) CVs of electropolymerisation of folic acid from 0.1 mmol L−1 FA in0(a

Po

patsTbtsfsgupa

.1 mol L−1 KCl/HCl, pH 2.0 on GCE. Potential scan rate 50 mV s−1. Scheme of PFAB) radical formation and (C) possible structure of FA tetramer; R = 2‘-(4‘-(methylmino)benzoamide pentanedioic acid.

FA film was obtained from 0.1 mol L−1 KCl/HCl, pH 2.0 solution,nly this film was presented in this work.

The PFA formation from 0.1 mol L−1 KCl/HCl, pH 2.0, on GCE isresented in Fig. 2A. Fig. 2B presents the plausible radical formationt 0.875 V (Fig. 2A, peak IV) which initiated the polymerisation reac-ion. The radicals either react with pre-treated carbon at the GCEurface [12,37] or with other FA molecules forming the oligomers.he polymerisation of FA was going via amino group binding to car-on in the aromatic ring as reported for phenasine [33,34] ratherhan “ring-to-ring” coupling. A possible mechanism of polymeri-ation via nitrogen radical formation from a secondary amine byorming–N–C–linkage on one hand, and, on the other hand, the pos-ible formation of amide groups from primary amine and carboxy

roup was reported [12]. The first mechanism was more crediblender these conditions. Since the oligomers formed, an exampleresented in Fig. 2 C, changed the length of the molecule as wells its energy, the polymer oxidation was observed at different

Fig. 3. AFM image of PFA deposited from 0.1 mmol L−1 FA in 0.1 mol L−1 KCl/HCl,pH 2.0; scanned area 1 × 1 �m.

potentials (Fig. 2A peaks I and II) than that of monomer (peaks I’,II’, and III). Other deposition solutions lead to low film stability,therefore, they will be discussed just shortly.

Stability of such PFA film was insufficient, i.e. the polymer filmdegraded electrochemically (see below), therefore, the GCE sur-face was modified with the graphene-chitosan composite film (notshown) expecting that amino groups from chitosan attract FA to thesurface [13]. Since FA was protonated, the electrochemical reactionbetween chitosan and FA was not observed in this case.

3.2. Characterisation of electrosynthesised poly(folic acid)

3.2.1. Atomic force microscopyAFM images of the PFA electropolymerised on HOPG from

0.1 mol L−1 KCl/HCl, pH 2.0 were taken in order to study the mor-phology of the PFA film. The typical morphology of the film ispresented in Fig. 3. A uniform film composed from the separatenanoparticles was formed on the electrode surface as seen fromFig. 3. The PFA film deposited from this solution was soft and vis-cous, i.e. had ability to stick to the AFM tip.

3.2.2. Electrochemical characterisation3.2.2.1. Voltammetric characterisation. Since the peak current den-sity of the PFA in CV mode was rather small, it was difficult toestimate its changes under different conditions, therefore, a squarewave voltammetry has been applied for the characterisation of thepolymer film.

First, buffer solutions with different pH were applied to evaluatethe peak potential (Ep) dependence on pH, and afterwards, differ-ent scan rate/frequency was used to determine the limiting stepof the electrochemical process. Finally, the surface concentrationand stability of the film was calculated from electrochemical dataobtained from square wave voltammograms (SWVs). Typical SWVsof PFA, deposited at pH 2.0, are presented in Fig. 4A in the bufferedsolutions of pH 2.0, 4.0 and 5.0. As seen, three oxidation peakswere observed in strongly acidic medium: a well-defined peak IIat -0.320 V, peak III as a small wave at -0.020 V, and the oxidationwave as the peak IV was observed at 0.845 V indicating radical for-mation what means that polymerisation might go further. The peakI was at more negative potential, out of this potential range, i.e. at-0.885 V (not shown). At pH 4.0, the peak I shifted to less nega-tive potentials and it was already observed in this potential range,while with further increase in solution pH the peak I disappearedcompletely, i.e. it again shifted to more negative potentials showingthat the oxidation of the protonated form as well as fully deproto-nated form was more complex because PFA was in a reduced form

(Fig. 4A, dashed line). The peaks II and III shifted to less negativeand/or more positive potentials from pH 2.0 to 5.0. Moreover, thepeak III also increased in height. The peak IV decreased and dis-appeared with increase in pH, moreover, it shifted to less positive

R. Celiesiute et al. / Electrochimica Acta 138 (2014) 62–68 65

Fig. 4. SWVs after background subtraction (A) in different buffers: 0.1 mol L−1

KCl/HCl pH 2.0 (solid curve), 0.1 mol L−1 PBS pH 4.0 (dashed curve) and 0.1 molL2K

pd

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pdgiGPPpe

3Eic

Fig. 5. (A) Ep (empty symbols) and jp (filled symbols) dependence on pH calculatedfrom SWVs for peak I (circles) and peak II (squares) from Fig. 5A. All other con-

−1 PBS pH 5.0 (dash-dot-dash curve), at PFA deposited from 0.1 mol L−1 KCl/HCl pH.0, containing 0.1 mmol L−1 FA (B) at differently modified electrodes in 0.1 mol L−1

Cl/HCl solution pH 2.0. SWV parameters are presented in Experimental.

otentials. This fact indicates that further polymerisation of fullyeprotonated FA is not possible.

According to the peak width at a half height of the peak, peakV was a one-electron process, i.e. the width of a peak at the halfeight was ∼90 mV [38] and peaks I, II and III were two-electronlectrochemical reactions.

Modification of the GCE surface with G-Chit layer prior to FAolymerisation caused much higher peak currents of PFA (Fig. 4B)ue to increase in the electroactive surface area in the presence ofraphene, which eased PFA oxidation. However, only small changesn the peak current were obtained when PFA/GCE was covered with-Chit layer, except peak I, which was less negative than that atFA/GCE. Although, the best electrochemical properties were atFA/G-Chit/GCE, but its electrochemical stability of this film wasoor in opposite to G-Chit/PFA/GCE which was the most promisinglectrode composition for further possible applications.

.2.2.2. Dependence on pH. The dependence of the peak potential,p, on pH was studied in order to determine number of protons tak-ng part in the electrochemical reactions. The influence of pH wasonducted in interval from 1.0 to 8.0, while 0.1 mol L−1 KCl/HCl

ditions like in Fig. 4A. (B) Possible scheme of electrochemical FA transformations.Roman numbers indicate peaks in CV or SWV. R = 2‘-(4‘-(methyl amino)benzoamidepentanedioic acid or methyl group.

electrolyte was for 1.0 to 3.0 and 0.1 mol L−1 PBS solutions forpH from 4.0 to 8.0 with the step of 1.0. The results demonstratedthe shift of the oxidation peaks to more negative or less positivepotentials (depending on the peak position) with the increase in pH(Fig. 5A) and the peak shift was linear over whole pH range studied.The slope of the shift of the peak I was -61 mV/pH, this value is closeto the theoretical value of 59 mV from the Nernst equation, there-fore, it confirmed that the proton number was equal to the electronnumber in the electrochemical reactions occurring at the electrodesurface. This peak was visible just up to pH 6.0, hence, no electro-chemical reaction occurred at this potential in neutral and alkalinemedia. Peak II was clear over whole pH range and its slope was-68 mV, again showing proton: electron ratio 1:1. Unfortunately,

other peaks existed in a narrow pH range and their analysis wascomplicated as well as dependence of Ep on pH was non-linear.

The dependence of the peak height on pH was also plotted anddisplayed in Fig. 5A with the filled symbols. The peak I significantly

66 R. Celiesiute et al. / Electrochimica Acta 138 (2014) 62–68

Table 1Surface coverage and short term stability of differently modified electrodes withpoly(folic acid) deposited from 0.1 mol L−1 KCl/HCl solution, pH 2.0.

Electrode Medium* Surface coverage,pmol cm−2

Relative peakheight after 10SWV scans%

PFA/GCE KCl/HCl, pH 2.0 21.7 94.0PBS, pH 4.0 1.29 115PBS, pH 5.0 2.49 65.1

PFA/G-Chit/GCE KCl/HCl, pH 2.0 267 91.3PBS, pH 4.0 4.77 47.5PBS, pH 5.0 0** 0**

G-Chit/PFA/GCE KCl/HCl, pH 2.0 15.7 94.6PBS, pH 4.0 123 105PBS, pH 5.0 11.2 190

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Fig. 6. EIS complex plane spectra at different electrodes at -0.32 V. Inset presents

* Concentration of all buffers was 0.1 mol L−1

** No peaks found

ecreased with pH and, finally, it disappeared at pH 7.0, while theeak II had no clear dependence on pH but it had a slight minimumt pH 5.0. This shows that the peak I defined the strongly proton-ependent process.

According to these data, PFA exists at the surface in a reducedorm and the oxidation mechanism is proposed in the scheme ofig. 5B; for simplicity, the electrochemical transformations of theonomer are given. Depending on pH, FA exists in two different

orms [39] which can be oxidised or reduced (peaks I-III). The peakV was found at low pH but it disappeared at pH 5.0 and higher.his leads to the conclusion that the peak IV was from amide groupxidation in glutamic part of FA. There are data in literature that inome cases FA lost 2‘-(4‘-(methyl amino)benzoamide pentanedioccid part of the molecule and R in the peaks II and III was CH3 [39].n this case, the peak IV (Fig. 4) indicated that entire FA molecule

as polymerised and no cleavage in the polymer was observed, thisas confirmed changing electrolyte from pH 5.0 to pH 2.0 and peak

V appeared again showing that amide group from glutamic part ofhe molecule still existed.

.2.2.3. Electrochemical stability of PFA. SWVs were registered atifferent scan rates and the peak current density was plotted versusither the scan rate or the square root of the scan rate in ordero estimate the limiting step of the electrochemical process. Thedsorption was found to be the rate determining step in all bufferolutions at PFA.

Surface coverage was calculated from the charge and electroderea according to the equation:

= nFA� (1)

r

= /nFA, (2)

here � is the surface concentration in mol cm−2, n is the num-er of electrons, F is the Faraday constant, A is the surface area inm2, and Q the charge in C. The results are presented in Table 1.s seen, the surface concentration depended on the medium while

he largest surface concentration of PFA was found in strongly acidicedium. This was related to the stability of PFA in acidic media. The

ighest surface coverage with PFA was obtained at G-Chit/PFA/GCEecause chitosan on the top of PFA stabilised the conducting poly-er film and protected it from degradation and fouling.An electrochemical stability of the film was evaluated by the

epeated sweep of potential in square wave mode: the changesn the current density of the peak I were determined during 10epeated potential scans. As seen from Table 1, the stability of theolymer film was related to the surface concentration. However,

equivalent circuit. Supporting electrolyte 0.1 mol L−1 PBS, pH 4.0. Numbers indicatefrequency at the points indicated with arrows.

the uncovered films were washed out or almost washed after 20 ormore repeated cycles.

3.2.2.4. Electrochemical impedance spectroscopy. EIS was per-formed at all electrodes covered with PFA at the position of thepeak I in order to identify processes taking place at the electrodesurface. EIS at bare GCE was also recorded for comparison withthe modified electrodes. The complex plane spectra are shown inFig. 6A. As seen, the deposition of PFA increased impedance valuescomparing to bare GCE.

The spectra were analysed by fitting them to equivalent cir-cuit models consisting of two interfaces: solution/PFA-film andPFA-film/GCE. The equivalent circuit model consisted of the cellresistance, R�, in series with the parallel couple of the chargetransfer resistance, Rct, and the constant phase element (CPE) as anon-ideal double layer capacitance, Cdl, in series with the next par-allel couple of the film resistance, Rf, and CPE as a non-ideal filmcapacitance, Cf. In the case of GCE electrode, only one R-CPE couplewas used in the model because the film consisted of one interfacesolution/GCE; while one more couple for film resistance and filmcapacitance, Rf2-CPEf2, was added for the electrodes modified withthe graphene-chitosan composite film, for the interface PFA/G-Chit(Fig. 6A inset). The data are presented in Table 2. The errors of thefitting were less than 10%.

R�, as usually, depended on solution and the electrode compo-sition, but it was independent on the applied potential and variedfrom 5 to 8 � cm2. Interestingly, the correlation of R� was obtainedwith the proton concentration, i.e. the cell resistance decreasedwith increase in solution pH in the studied pH range from 2.0 to5.0.

The charge transfer resistance also depended on the pH valueand on the applied potential. Both these dependences were relatedto the changes at the interface during electrochemical reaction. Rct

also depended on the electrode composition; it increased after theapplication of G-Chit layer on the top of PFA. The lowest R was at

ct

bare GCE while each additional film increased charge transfer resis-tance. However, the lowest film resistance was at PFA/G-Chit/GCEor G-Chit/PFA/GCE and it decreased from hundreds of k� cm2 to

R. Celiesiute et al. / Electrochimic

Table 2Some EIS parameters obtained at PFA modified electrodes in different pH at differentpotentials. Data calculated from EIS spectra presented in Fig. 6.

Electrode pH Ep, V Rct, k� cm2 Cdl, �F cm−2 ˛1

PFA/GCE 2.0 −0.315 10.8 58.5 0.955−0.060 77.9 59.4 0.9990.670 258 24.7 0.999

4.0 −0.220 54.7 57.7 0.9990.020 317 70.0 0.89860.680 610 19.6 0.9811

5.0 −0.260 33.8 84.9 0.9630.670 285 20.9 0.974

G-Chit/PFA/GCE 2.0 −0.300 0.798 204 0.8970.000 7.43 97.0 0.8590.800 9.82 63.0 0.879

4.0 −0.450 0.756 89.4 0.9850.675 4.12 88.8 0.883

5.0 −0.505 0.186 87.5 0.995−0.190 0.217 170 0.9990.805 1.105 166 0.999

PFA/G-Chit/GCE 2.0 −0.320 2.03 61.7 0.9530.215 144 76.1 0.9990.740 332 25.8 0.999

4.0 0.060 308 102 0.8500.610 299 20.8 0.935

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undreds � cm2, since at this interface the electrochemical processas the fastest and surface changes were most notable.

Generally, the double layer and the film capacitances increasedith the decrease in the charge transfer and the film resistances. As

xpected the highest capacitances were obtained when electrodesere modified with G-Chit film, since graphene increased the effec-

ive surface area and increased the conductivity of the modifiedlectrode [38]. As stated above, capacitance was modelled as CPE,here

PE = −1/(Ciw)˛, (3)

he capacitance C describes the charge separation at the doubleayer interface and the exponent is due to the heterogeneity of theurface [40]. The flattest surface was obtained when PFA depositedn the top of the electrode according to values (Table 2), sincehe situation when = 1 means perfectly flat and uniform surface40]. Further deposition of the G-Chit film made the surface lessomogeneous.

. Conclusions

PFA was electropolymerised from folic acid in buffer solutionsrom pH 2.0 to 5.0 on GCE. Cl− ions were found to be the best cata-ysts for the electropolymerisation of FA. A poor electrochemicaltability of the PFA films was significantly increased by cover-ng the PFA film with the graphene-chitosan composite layer. Theest electropolymerisation conditions were optimised accordingo electrochemical behaviour of obtained PFA: the optimal filmas obtained at G-Chit/PFA/GCE electrode when the PFA film waseposited from 0.1 mol L−1 KCl/HCl, pH 2.0. This film had the longestperational stability.

Electropolymerisation of FA from KCl/HCl, pH 2.0 led to nano-tructured morphology when deposited from KCl/HCl solution pH.0.

Possible polymerisation and electrochemical redox mecha-isms are proposed from obtained data. The best electrode,-Chit/PFA/GCE, deposited from 0.1 mol L−1 KCl/HCl, pH 2.0 wille further applied in sensor construction.

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a Acta 138 (2014) 62–68 67

Acknowledgements

This research is funded by the European Social Fund under theGlobal Grant measure, Project No. VP1-3.1-SMM-07-K-01-124.

References

[1] S. Vinker, E. Krantman, M. Shani, S. Nakar, Low Clinical Utility of Folate Determi-nations in Primary Care Setting, Amer. J. Managed Care 19 (2013) e100–e105.

[2] H.E. Indyk, An Optical Biosensor Assay for the Determination of Folate in Milkand Nutritional Dairy Products, Intern. Dairy J. 21 (2011) 783–789.

[3] S.H. Zhao, P.P. Zhang, X.H. Liang, D.L. Hua, T.S. Ma, G.L. Pei, A New PotassiumTetrabromoaurate (III)-Luminol Chemiluminescence System for the Determi-nation of Folic Acid in Milk Powder, J.Food Sci. 77 (2012) C102–C106.

[4] K. Zhao, L. Zhao, Y. He, Y. Cai, D. Tian, S. Zang, Y. Zhang, Electrochemical Behav-ior of Folic Acid in Neutral Solution on the Modified Glassy Carbon Electrode:Platinum Nanoparticles Doped Multi-Walled Carbon Nanotubes with Nafion asAdhesive, J. Electroanal. Chem. 677-680 (2012) 105–112.

[5] C. Ferreiro-Vera, F. Priego-Capote, M.D.L. de Castro, An Approach for Quantita-tive Analysis of Vitamins D and B9 and Their Metabolites in Human Biofluidsby On-Line Orthogonal Sample Preparation and Sequential Mass SpectrometryDetection, Analyst 138 (2013) 2146–2155.

[6] P. Huang, C. Xu, J. Lin, C. Wang, X.S. Wang, C.L. Zhang, X.J. Zhou, S.W. Guo, D.X.Cui, Folic Acid-conjugated Graphene Oxide loaded with Photosensitizers forTargeting Photodynamic Therapy, Theranostics 1 (2011) 240–250.

[7] D. Depan, J. Shah, R.D.K. Misra, Controlled Release of Drug From Folate-Decorated and Graphene Mediated Drug delivery system: Synthesis, LoadingEfficiency, and Drug Release Response, Mater. Sci. Eng. C 31 (2011) 1305–1312.

[8] L. Yu, M.Y. Cao, H.W. Zhang, H. Xiong, Y.C. Jin, Y. Lu, H.Y. Jia, D.S. Luo, L.Q. Wang,H.L. Jiang, Synthesis of Well-Defined Chitosan-Based Tricomponent Copoly-mers for Constructing Multifunctional Delivery Systems, J. Polym. Sci. A 50(2012) 4936–4946.

[9] X.H. Wang, X.K. Sun, G.H. Jiang, R.J. Wang, R.B. Hu, X.G. Xi, Y. Zhou, S. Wang, T.Wang, Synthesis of biomimetic hyperbranched zwitterionic polymers as tar-geting drug delivery carriers, J. Appl. Polym. Sci. 128 (2013) 3289–3294.

10] A.J. Shen, D.L., Li, X.J., Cai, C.Y., Dong, H.Q., Dong, H.Y., Wen, G.H., Dai, P.J., Wang,Y.Y. Li, Multifunctional nanocomposite based on graphene oxide for in vitrohepatocarcinoma diagnosis and treatment, J. Biomed. Mater. Res. A 100A (2012)2499-2506.

11] J.J. Castillo, W.E. Svendsen, N. Rozlosnik, P. Escobar, F. Martineza, J. Castillo-Leon, Detection of cancer cells using a peptide nanotube-folic acid modifiedgraphene electrode, Analyst 138 (2013) 1026–1031.

12] M. He, X. Zheng, A highly sensitive and selective method for dopamine detectionbased on poly(folic acid) film modified electrode, J. Mol. Liq. 173 (2012) 29–34.

13] Z. Wanga, C. Zhou, J. Xia, B. Via, Y. Xia, F. Zhang, Y. Li, L. Xia, Fabrication andcharacterization of a triple functionalization of graphene oxide with Fe3O4,folic acid and doxorubicin as dual-targeted drug nanocarrier, Coll. Surf. B 106(2013) 60–65.

14] M.Z. Elsabee, E.S. Abdou, Chitosan based edible films and coatings: A review,Mater. Sci. Eng. C 33 (2013) 1819–1841.

15] K. Pal, B. Behera, S. Roy, S.S. Ray, G. Thakur, Chitosan based delivery systems ona length scale: Nano to macro, Soft Mater. 11 (2013) 125–142.

16] K. Bustos-Ramírez, A.L. Martínez-Hernández, G. Martínez-Barrera, M. de Icaza,V.M. Castano, C. Velasco-Santos, Covalently Bonded Chitosan on GrapheneOxide via Redox Reaction, Materials 6 (2013) 911–926.

17] R. Pauliukaite, M.E. Ghica, O. Fatibello-Filho, C.M.A. Brett, Comparative study ofdifferent cross-linking agents for the immobilization of functionalized carbonnanotubes within a chitosan film supported on a graphite-epoxy compositeelectrode, Anal. Chem. 81 (2009) 5364–5372.

18] H. Yang, B. Lu, B. Qi, L. Guo, Voltammetric sensor based on ordered mesoporouscarbon for folic acid determination, J. Electroanal. Chem. 660 (2011) 2–7.

19] B. Unnikrishnan, Y.L. Yang, S.M. Chen, Amperometric determination of folicacid at multi-walled carbon nanotube-polyvinyl sulfonic acid composite filmmodified glassy carbon electrode, Int. J. Electrochem. Sci. 6 (2011) 3224–3237.

20] K. Zhao, L. Zhao, Y. He, Y. Cai, D. Tian, S. Zang, Y. Zhang, Electrochemical behav-ior of folic acid in neutral solution on the modified glassy carbon electrode:Platinum nanoparticles doped multi-walled carbon nanotubes with Nafion asadhesive, J. Electroanal. Chem. 677-680 (2012) 105–112.

21] H. Beitollahi, M.A. Taher, F. Mirrahimi, R. Hosseinzadeh, Electrochemical sensorfor selective determination of N-acetylcysteine in the presence of folic acidusing a modified carbon nanotube paste electrode, Mater. Sci. Eng. C 33 (2013)1078–1084.

22] L. Mirmoghtadaie, A.A. Ensafi, M. Kadivar, P. Norouzi, Highly selective elec-trochemical biosensor for the determination of folic acid based on DNAmodified-pencil graphite electrode using response surface methodology,Mater. Sci. Eng. C 33 (2013) 1753–1758.

23] M.R. Majidi, H. Dastangoo, M. Hasannejad, J. Malakouti, Voltammetric deter-mination of folic acid with a overoxidized polypyrrole film modified sol-gelcarbon ceramic electrode, Int. J. Pol. Anal. Charact. 16 (2011) 486–495.

24] B.B. Prasad, R. Madhuri, M.P. Tiwari, P.S. Sharma, Imprinted polymer–carbonconsolidated composite fiber sensor for substrate-selective electrochemicalsensing of folic acid, Biosens. Bioelectron. 25 (2010) 2140–2148.

25] B.B. Prasad, R. Madhuri, M.P. Tiwari, P.S. Sharma, Electrochemical sen-sor for folic acid based on a hyperbranched molecularly imprinted

6 chimic

[

[

[

[

[[

[

[

[

[

[

[

[

[

8 R. Celiesiute et al. / Electro

polymer-immobilized sol–gel-modified pencil graphite electrode, Sens. Actuat.B 146 (2010) 321–330.

26] D.A.C. Brownson, D.K. Kampouris, C.E. Banks, Graphene electrochemistry: Fun-damental concepts through to prominent applications, Chem. Soc. Rev. 41(2012) 6944–6976.

27] Y. Shao, J. Wang, H. Wu, J. Liu, I.A. Aksay, Y. Lin, Graphene based electrochemicalsensors and biosensors: A review, Electroanalysis 22 (2010) 1027–1036.

28] T. Gan, S. Hu, Electrochemical sensors based on graphene materials, Microchim.Acta 175 (2011) 1–19.

29] K.R. Ratinac, W. Yang, J.J. Gooding, P. Thordarson, F. Braet, Graphene andrelated materials in electrochemical sensing, Electroanalysis 23 (2011) 803–826.

30] M. Pumera, Graphene in biosensing, Mater. Today 14 (2011) 308–315.31] B. Pérez-López, A. Merkoc i, Carbon nanotubes and graphene in analytical sci-

ences, Microchim. Acta 179 (2012) 1–16.32] R. Celiesiute, G. Grinciene, S. Vaitekonis, T. Venckus, T. Rakickas, R. Pauliukaite,

Application of carbon electrodes modified with graphene and chitosan to elec-trochemical sensing of ascorbate, Chemija 24 (2013) 296–306.

33] R. Pauliukaite, C.M.A. Brett, Poly(neutral red): Electrosynthesis, character-ization, and application as a redox mediator, Electroanalysis 20 (2008)1275–1285.

[

a Acta 138 (2014) 62–68

34] R. Pauliukaite, M.E. Ghica, M.M. Barsan, C.M.A. Brett, Phenazines andpolyphenazines in electrochemical sensors and biosensors, Anal. Lett. 43 (2010)1588–1608.

35] M. Poe, Acidic dissociation constants of folk acid, dihydrofolic acid, andmethotrexate, J, Biol. Chem. 252 (1977) 3724.

36] J. Ghasemi, S. Ghobadi, B. Abbasi, M. Kubista, Spectrophotometric determina-tion of acidity constants of group B vitamins in different ionic strengths at25 ± 0.1 ◦C, J. Korean Chem. Soc. 49 (2005) 269.

37] L. Zhang, X. Lin, Covalent modification of glassy carbon electrode with glutamicacid for simultaneous determination of uric acid and ascorbic acid, Analyst 126(2001) 367–370.

38] S. Komorsky-Lovric, M. Lovric, Theory of square-wave voltammetry of twoelectron reduction with the intermediate that is stabilized by complexation,Electrochim. Acta 69 (2012) 60–64.

39] H. Lund, Electrolysis of heterocyclic compounds, in: H. Lund, O. Hammerich(Eds.), Organic Electrochemistry, 4th ed., Marcel Dekker, Inc, New York, 2001,

pp. 682–724.

40] R. Celiesiute, R. Trusovas, G. Raciukaitis, G. Niaura, V. Svedas, Z. Ruzele, R.Pauliukaite, Influence of the laser irradiation on the electrochemical and spec-troscopic peculiarities of graphene-chitosan composite film, Electrochim. Acta132 (2014) 265–276.