You are on page 1of 9

Full Paper

Electrochemically Prepared Polypyrrole-2-Carboxylic Acid Films:
Synthesis Protocols and Studies on Biosensors
M. Foschini,a A. Marletta,a R. C. Faria,b D. Leonard,c F. Bessueille,c N. Jaffrezic-Renault,c D. GonÅalves*d
a Instituto de Fsica, Universidade Federal de Uberlndia, Av. Jo¼o Naves de vila, Uberlndia, MG, postcode 593, 38400-902, Brazil
b Departamento de Qumica, Universidade Federal de S¼o Carlos, S¼o Carlos, SP, postcode 676,13565-905, Brazil
c Institut des Sciences Analytiques (ISA), UMR 5280, Universit Lyon 1-CNRS-ENS de Lyon, Universit de Lyon, 5 rue de la Doua,

69 100 Villeurbanne, France.
d Instituto de Fsica de S¼o Carlos, Universidade de S¼o Paulo, Av. Trabalhador S¼o-carlense 400, S¼o Carlos, SP, postcode 369,

13560-970, Brazil
*e-mail: gdebora@ifsc.usp.br

Received: October 17, 2012
Accepted: December 4, 2012
Published online: February 22, 2013

Abstract
The process for obtaining polypyrrole-2-carboxylic acid (PPY-2-COOH) films in acetonitrile was investigated using
cyclic voltammetry, electrochemical quartz crystal microgravimetry (EQCM), and infrared spectroscopy (FTIR).
Different potential ranges were applied during cyclic voltammetry experiments with the aim of obtaining films with-
out and with the presence of controlled amounts of water added in acetonitrile. The FTIR spectra of the films have
evidenced that cations and anions from the electrolyte solution were incorporated into the PPY-2-COOH structure,
with a preferential adsorption of cations. After chemically immobilizing polyphenoloxidase (tyrosinase, PPO), PPY-
2-COOH/PPO films were build for amperometric detection of catechol, establishing a linear limit of concentrations
ranging from 5.0  10 4 to 2.5  10 2 mol L 1.

Keywords: Biosensors, Functionalized polypyrrole, Conducting polymers.

DOI: 10.1002/elan.201200574

1 Introduction ly described its use in biosensors [9–11]. In particular,
studies focused on the electrochemical preparation of
Polypyrrole (PPY) films have been frequently prepared PPY-2-COOH films are far away from entirely detailed.
by electrochemical synthesis methods aiming towards ap- Here, we studied the protocols for the electrosynthesis of
plication in displays, batteries, and sensors [1]. When PPY-2-COOH films in acetonitrile (ACN) without and
used as an active layer in enzymatic sensors, PPY allows with traces of water. We have chosen ACN because this
one to combine stability and sensibility in different envi- solvent has been frequently used in the electropolymeri-
ronments [2, 3]. However, biosensors based on the use of zation medium of pyrrole, thiophene, and common deriv-
PPY have not been competitively employed yet because atives; furthermore, only a small amount of water in
they usually exhibit relatively slow response times and ACN can have a strong influence on the kinetics of for-
short lifetimes mainly related to problems of stability mation and properties of conjugated polymers such as
and, consequently, degradation of the PPY matrix [4]. poly(1,4-phenylene ether) and poly(p-phenylene) [12, 13].
Biosensors designed by immobilizing glucose oxidase in The use of dried ACN in the electrosynthesis medium of
PPY matrices have shown better performances after parent PPY yields a partly conjugated and poorly conduc-
using ferrocene as redox mediator [5]. Faster and repro- tive polymer structure mainly due to the formation of
ducible electroanalytical responses can be also obtained a passive layer on the electrode surface [14]. However,
in composite films made by combining PPY and hydro- previous studies have indicated that small amounts of
gels in a biocompatible microenvironment for the entrap- water intentionally added to ACN yield PPY films with
ment of oxidases [6]. Therefore, these results give us improved properties such as better adhesion and higher
a strong indication that novel polymeric matrices appoint conductivity [15].
as alternatives at achieving efficient and selective biosen- Here, PPY-2-COOH films were characterized by cyclic
sors, especially when functionalized PPYs can be used in voltammetry and Fourier transform infrared spectroscopy
order to covalently immobilize bio-receptors [7, 8]. (FTIR), and also, using an electrochemical quartz crystal
Among the PPY derivatives, polypyrrole-2-carboxylic microbalance (EQCM) in order to understand the overall
acid (PPY-2-COOH) is not completely unknown, but to deposition process under successive doping-undoping cy-
the best of our knowledge, only a few authors have brief- cling steps. The films were also deposited onto Au micro-

Electroanalysis 2013, 25, No. 3, 741 – 749  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 741

Amperometric system was connected to a thermal bath. nolic compounds under flow injection conditions. (2. the water.Full Paper M. (PFP) (Aldrich.1 mol L 1 HCl solution (Figure 1 d). Acetonitrile (ACN) with 32 ppm of water (la. No.0. PPO films were obtained at different concentrations of All electrochemical measurements made by using the catechol.1 mol L 1AgNO3 and AgCl in nase). PPY-2-COOH films in an ethanol solution containing before and after immersing the biosensor in a solution 0. beled value) was dried under molecular sieves (4 ) it allowed one to use these biosensors for detecting phe- treated at 300–400 8C for 4 h. After obtaining the Au tracks. 741 – 749 . mainly in soil and electrode (Au). Flow injection analysis (FIA) were performed under tiostat/galvanostat with M270 software.1 mol L 1 1-ethyl-3(3-dimethylaminopropyl)carbodii- containing phenols at certain concentrations. the electrode studies. This activa- tion procedure has been employed for cross-linking car- Pyrrole-2-carboxylic acid (PY-2-COOH) ( 97 %) and boxylate groups with amines of enzymes to form peptidic tetramethylammonium tetrafluoroborate (TMABF4) ( bonds [16]. the reflection mode with a smart specular reflector acces- trode.5) containing PPO (7360 U mL 1). leaving the Au tracks totally clean tion are shown in Figures 2 a and 2 b. 18]. and then. pH 6.0 to 1. corded with a Nicolet Nexus 470 spectrophotometer in trochemical Teflon cell. electrodes in order to build amperometric transductors deposited Ag layers.0 cm2) as the counter electrode. this protocol was conducted in order to 97 %) (Sigma-Aldrich) were used after drying under prepare stable polymer films that can be used in vacuum.wiley-vch. tivated PPY-2-COOH films was obtained by cross-linking after immersing these films in phosphate buffer (Synth. 99 + %) for 25 min at pH 7. biosensors can be constructed by immobilizing PPO on The activation of the carboxylic acid groups of PY-2- a polymer matrix.95 V. electrode with a radius of 500 mm. Au-QCM as the working elec. (PY-2-COOH) was initially studied over a wide potential strates. low-cost. and the cyclic voltammograms were ob- 742 www. comparing how the enzymat.5–7. As a result. The role of PPO in the biosensor is to catalyze the Teflon plate was fixed facing on the Au surface. tial of the Ag/AgCl microelectrode in 0. which were previously prepared by lithography range. 25.05 mol L 1 phos- terminated biotin on PET modified with -COOH groups phate buffer at pH 6. Phenolic compounds have been widely found as ag. which have been used for immobilizing amine. extracted from 2 Experimental mushrooms (Fluka) (2260 U mg 1) for 20 min. and then.60 V. The printing ink was dissolved in ace. In this case. water. one compartment electrochemical cell injection valve filling with 200 mL of 0. and a Pt plate sory for the films deposited on Au electrodes. pared in an aqueous 0. including the Au working of PPY-2-COOH with cross-linker 1-ethyl-3-(3-dimethyla. Ag/AgCl as the reference electrode.de  2013 Wiley-VCH Verlag GmbH & Co. Here. bilization of polyphenoloxidase (PPO) (tyrosinase) on ac- able means of analysis of pollutants. ACN.05 mol L 1 phosphate buffer at pH 6. All electrochemical experi.05 mol L 1 phos- with a Pt plate as the counter electrode and Ag/AgCl as phate buffer containing catechol (Mallinckrodt) at vari- the reference electrode.5 was measured as 87  9 mV vs. the films was also obtained by using a Stanford QCM200 Fourier transformed infrared (FTIR) spectra were re- microbalance (EQCM) with a three-compartment elec.0.  97 %) and pentafluorophenol the use of novel polymeric biosensors seems to be a prom. 0. After these initial (Figure 1 c). cell with a total volume of 100 mL built with two rubber tants. Au microelectrodes built on polyamide substrates and prepared by lithography using a laser printing. which were electrochemically pre- prior to immobilizing polyphenoloxidase (PPO) (tyrosi. 3.95 V to 1. in a solution containing PY-2- with a laser printed mask (Figure 1 a). and dispos. The Au layers COOH and tetramethylammonium tetrafluoroborate were evaporated onto the substrate surfaces and then. The poten- nol (PFP). The enzyme was covalently bonded to the polymer 0. an elec- matrix after activating the carboxylate groups (-COOH) trochemical cell was obtained. in this case. which was intentionally added to the solvent. Ag/ [16]. The whole nols to intermediate quinones [17. Weinheim Electroanalysis 2013. Au counter electrode. The results obtained under this experimental condi- tone. Foschini et al. which al- oxidation of monophenols to o-diphenols. When inside the electrochemical cell. from 0. The working electro- des used here were 5 MHz AT-cut quartz crystals coated with polished Au films on both sides (Au-QCM). which was chosen here due to its importance as biosensor arrays were performed with a flow injection a model compound for biosensing environmental pollu. The immo- ising technology to address portable.electroanalysis. lowed both inlet and outlet solution fluxes. rinsed. The amperometric responses of the PPY-2-COOH/ AgCl. The electropolymerization of pyrrole-2-carboxilic acid The Au microelectrodes were built on polyamide sub. mide (EDAC) (Aldrich. and 3 Results and Discussion also. ments were carried out with an EG&G PAR 283 poten. and Ag/AgCl reference electrode (Figure 1 e). when measured by a current signal. o-rings between two Teflon plates separated by a micro- ricultural and industrial residues. the final (positive) potential was reduced from area was delimited by using epoxy resin and previously 1. The electrochemical response of ous concentrations. on a standard optimal conditions of pH and temperature with the loop three-electrode. (TMABF4) in acetonitrile (ACN) containing 1 % (v/v) heated at 240 8C during 10 min in order to improve adhe. and o-diphe. COOH was performed by immersing freshly obtained ic activity may vary. sion (Figure 1 b). minopropyl) carbodiimide (EDAC) and pentafluorophe. KGaA.

and continuously oxidized during the overall process of film formation. 10 cycles. where it can be noticed 1. been discussed in detail for monomers such as thiophenes.30 V (see v) water. Electroanalysis 2013. After the first cycles. the current density Au electrode in a medium containing PY-2-COOH over decreases rapidly with time. however. Manufacturing methods for electrode miniaturization: a) ink-mask deposition. b) metallization and heat treatment for im- proving adherence of the metal film. now. 1.60 V. cannot be related to an effect of dissolution of the a crossover current can be seen in the curves of Fig. uniform film were visibly deposited on the the Au-QCM electrode in a solution containing PY-2- electrode surface. and for the first time investigated seen in Figure 2 b (see arrow). No. metric curves become more defined. Then. 3. this narrow potential range. COOH in ACN containing a specific amount of water pyrrole. about 6 min after starting the poten. (1 % v/v). final potential initially chosen (1. and only arrow) due to the oxidation of PY-2-COOH. one infers that the electrode surface be. this narrower potential range was reapplied. Weinheim www. and this effect can be better a wide potential range.Electrochemically Prepared Polypyrrole-2-Carboxylic Acid Films Fig. i. During the first five cycles. as it can be seen in ization taking place up to reach 1. Since a poorly defined redox response COOH in ACN with a known amount of water (1. and e) final array structure. KGaA. The appearance of a crossover current in Fig- higher values anodic currents were achieved at potentials ure 2 a can be related to a high amount of oxidized spe- close to 1.0 % v/ (typical of nonconductive polymer films) can be noticed v). by applying a final potential of 1.70 V. however. this effect has havior of bare electrodes in a solution containing PY-2. experiments was the same. After 10 cycles. and derivatives [19. Figure 2 c shows an anodic peak at 1.0 % (v/ Figure 2 d. d) deposition of the reference electrode. Au overlayers in ACN containing water. tained again for a cleaned Au electrode in a solution pre. PY.de 743 . cies that was generated at the electrode/surface after 2-COOH. but. reaching a sufficiently high final potential. here.45 V due to the oxidation of the monomer.wiley-vch. but not for PY-2-COOH. but in The results described up to now have indicated that the a solution containing ACN without adding water (Figur.95 V. Under these experimental conditions.60– are shown in Figures 2 a and 2 b. 1. phene in ACN [20]. which is after a couple of cycles. Over in Figure 2 b.e. ble oligomers). evidencing an increase of the electrode area due before during the electropolymerization of 3-methylthio- to a coverage with nuclei formed at positive potentials. with the current creases with successive scanning. it was unnecessarily high because the oxidation The results from Figure 2 can be compared. and up to reach a relatively high value of final Furthermore. the current density still de- comes blocked during the first cycles. this typical voltammetric response of the potential. and they of PY-2-COOH had already been achieved at 1. The number of cycles achieved in all these to promote the polymer growth on the electrode surface. no colored products were visibly formed in solution (solu- pared exactly as the previous one (Figures 2 c and 2 d). 20].95 V in ACN/1. Figures 2 c and 2 d show the voltammetric responses of tial scanning. 25. the voltam- density decreasing with the number of cycles for polymer. as it was verified ure 2 a. 741 – 749  2013 Wiley-VCH Verlag GmbH & Co. These curves exhibit the electrochemical be.95 V) was high enough es 2 e and 2 f).electroanalysis. a colored film was obtained. c) ink-mask removal.

and shown in Fig.74 mg C 1. Asymmetric stretching of reach five complete cycles. b) with water (1 % v/v) at a final potential (Ef) of 1.90 mg C 1 calculated for ure 2 e. These be suitable and high enough to promote the growth of values indicated that ions from electrolyte are incorporat- a film on the electrode surface. becomes greater with the number of cycles as an in. as a result of an oxida- during the polymerization of PY-2-COOH at a final po. the molar cm 2. Figures 2 e and 2 f show the vol. it can be observed how the mass gain varies up to the electrolyte (TMABF4). The use of an electrochemical quartz crystal microba. mass was 0. potential curves for Au-QCM electrodes were included in this figure for comparative purposes.1 mol L 1 TMABF4 : a. the mass gain.87 mg C 1. 1. which is in accord- trode surface. 741 – 749 . there is no significant variation of the PPY-2-COOH films on Au-QCM electrodes within current density (or mass gain). where the mass loss is still small.06 mg. v = 50 mV s 1.10 V to 1. and they ure 2 e. This film exhibited a stable.60 V. direction. it can be noticed that the mass gain (Dm). Fig. tion process.60 V. without intentionally adding process. 2. in accordance with a theoretical dicative that the final potential chosen. seemed to value of 0.Full Paper M.wiley-vch. negative. 25. with the current density pensate the charges created during the oxidation-reduc- increasing gradually with the number of cycles (see Fig. noticed. The molar mass obtained in the anodic ure 2 f) due to a continuous growth of a film on the elec. as it was also obtained in FTIR studies.de  2013 Wiley-VCH Verlag GmbH & Co. In the cathodic. as a result of a reduction tential of 1.05 mol L 1 PY-2-COOH and 0. and e. From the EQCM curves. ance with a theoretical value of 0.electroanalysis. can be related to the balance of incoming and water in ACN. d) with water (1 % v/v) at Ef = 1. respectively. each cycle. in a mono. when a stable response was CH2 groups. Weinheim Electroanalysis 2013. about tammetric responses of the working electrode. Au-QCM.6 V. electroactive response can be assigned. in mg BF4– ions. Figure 4 a shows the FTIR spectrum of a PPY-2-COOH mer-free solution. c. In this case. tion processes. ed into the film. Foschini et al. Cyclic voltammograms for Au electrodes in acetonitrile (ACN) containing 0.60 V. metric stretching of CH3 groups from (CH3)4N + ions of ure 3 b. now.77 mg C 1 calculated for TMA + ions. After increasing the number of cycles (more lance (EQCM) allowed us to investigate the deposition of than 4–5 cycles). range of potentials was 0. In Fig. In this case. 0. in particular at the end of a narrow potential range. No. Strong and tained according to the procedures described in Fig. it is evident that the curves in outcoming fluxes of ions into the film in order to com- dry ACN became more defined. and the mass loss. Figure 3 a shows the voltammetric response. of a PPY-2-COOH film that was ob. film prepared in ACN without added water. and asymmetric and symmetric deformation 744 www.0 V. to the asymmetric and sym- after successive cycling from 0. sharp signals can be seen at 2963 and 2877 cm 1.95 V. 3. KGaA. f) without adding water at Ef = 1. Mass variation vs. but.

an increase in intensity at 1572 cm 1 is veri- COOH structure with 1-ethyl-3-(3-dimethylaminopropyl) fied for the PPY-2-COOH/PPO film. When Figures 4 a and 4 c are compared (see after activating the carboxylate groups from the PPY-2. lapping of most typical signals from amides I and II Electroanalysis 2013. Mean.de 745 . and b) mass variation vs potential curves for PPY-2-COOH films electrodeposited on Au-QCM electrodes in 0. KGaA. and then attributed from pyrrole structure. a medium containing water [23. 3. Weinheim www. where it can be noticed a strong signal at 1727 cm 1. A weak and broad peak at 3313 cm 1 can be also seen in Figure 4 b due to a shifted stretching vibration of the secondary amine while the signal at about 1700 cm 1. v = 50 mV s 1. can be noticed at 2937. (c) PPY-2-COOH/PPO film.1 mol L 1 TMABF4 in dried ACN up to reach five complete cycles. 25. (a) without and (b) with the presence of water (1 %) in Similar signal has been observed in the FTIR spectra of TMABF4 . also from the electrolyte. FTIR spectra of PPY-2-COOH films prepared in ACN be attributed to a superoxidized PPY-2-COOH structure. Figure 4 b shows the FTIR spectrum of a PPY-2- COOH film prepared in ACN with traces of water. 1474 and 1383 cm 1.electroanalysis. indicating an over- carbodiimide (EDC) and pentafluorphenol (PFP). (d) PPY-2-COOH/PPO superoxidized PPY obtained at high positive potentials in and PPY-2-COOH films in a narrow wavenumber range. to a superoxidized PPY-2-COOH structure.22]. becomes less trum of a PPY-2-COOH film with immobilized polyphe. 1064 and 1035 cm 1 can be attributed to the presence of BF4– anions incorporated into the film during the polymerization process. Weak and broad signal centered at around 3430 cm 1 can be assigned to the N H stretching vibration mode of secondary amines. vibrations of CH3 groups. which have been typically seen in the spectra of parent PPY [21. 3. a) Cyclic voltammetric response. a well-defined noloxidase (PPO) (tyrosinase) (PPY-2-COOH/PPO film). 24]. signal can be distinguish with high intensity at 1572 cm 1 This film was prepared in ACN without adding water and in Figure 4 c. Figure 4 c shows the FTIR spec.wiley-vch. Figure 4 d). The weak signal observed at 1703 cm 1 in Fig- ure 4 a can be attributed to the presence of C=O groups from the carboxylic acid of the PPY-2-COOH structure.Electrochemically Prepared Polypyrrole-2-Carboxylic Acid Films Fig. 741 – 749  2013 Wiley-VCH Verlag GmbH & Co. Strong and sharp signals at 1095. which can Fig. 4. No. evident for the PPY-2-COOH/PPO film.

When PPO is grafted into the PPY-2-COOH film a flow injection analysis system. in this case. which is a relatively high value when it is compared to the optimum pH of free PPO (5. were ob- tained in a blank solution.0 [31]) in a PPY matrix. 28]. no visible elec- tron transfer process (redox process) is evident in curves a1 and b1.wiley-vch. As discussed before. No. Foschini et al. 3. ranging from 6. the curves were di- vided into positive and negative branches of the cyclic voltammograns in terms of current scales. When PPY-2-COOH is used as a matrix for the immobilization of PPO.10 V. Electrochemical responses of: a) PPY-2-COOH b) PPY.06–0. Ag/QRE. into the buffer solution. allows one to achieve a stability condition under a wide 2-COOH/PPO films between 0. 0. 746 www. where o-quinones are enzymatically varying from 6. The maximum amperometric response for this biosensor was found to be close to 9. Response obtained by chronoamperometry of a PPY-2- o-quinones generated from catechol.05 mol L 1 phosphate buffer at pH 6.5 and 0. in phosphate buffer (Figure 6 a). In this case. [25. Multiple bands centered at 3284 cm 1 can be related to stretching vibrations of the N-H groups characteristics of peptide linkage [27. KGaA. 5. COOH/PPO biosensor at 0. 0.0–8. 741 – 749 . PPY-2-COOH Fig.1 mol L 1 catechol in phosphate buffer solutions at (a) pHs range of potentials. The voltammetric responses of the PPY-2-COOH films.5 to 11 at 20 8C and (b) different temperatures in formed. In the absence of catechol. PPO. Figure 6. Therefore. 6. Weinheim Electroanalysis 2013. a reduction peak becomes to be noticed at 0.5 [29. A maximum signal-to-background current can be also seen at 0. 26] from the peptide bonds of the enzyme. curves a2 and b2 exhibit well de- fined peaks due to the oxidation of catechol in the pres- ence of dissolved oxygen.07 V with a higher cathodic current. 0. Figures 5 a and 5 b. The effect of pH on the amperometric response of the PPY-2-COOH/PPO film was examined by applying a fixed potential. Thus. v = range of pHs. 25.07– 0. these results suggest a covalent attachment of the enzyme that can be achieved by activation of the polymer with EDC and PFP. in phosphate buffer at 20 8C.5. structure.4. one infers that the use of PPY-2-COOH in biosensors is advantageous because sev- eral enzymes may lose their catalytic activities at extreme low and high values of pHs.1 mol L 1. and 0.30]) and immobilized PPO (8.electroanalysis. Figure 5 shows the voltammetric curves of the PPY-2-COOH and PPY-2-COOH/PPO films before and after adding catechol at fixed concentration.10 V was chosen here as the optimum work- ing potential for further amperometric measurements. PPO.5 to 11. which show only low background currents.1 V vs. PPY-2-COOH/PPO films were also obtained on Au mi- croelectrodes and used as amperometric biosensors. After adding catechol. Ag/QRE at 20 8C in the detection of catechol can be studied within a cathodic 0.0–9. In order to better analyze the voltammetric responses of the films. 50 mV s 1. a specific value of potential can be chosen as the opti- mum working potential.Full Paper M. and a concomitant reduction of Fig. Catechol at 10 3 mol L 1 was injected into the FIA system at different values of pHs.10 V. before and after immobilizing the enzyme. This type of analysis allows one to define a potential range where the catalytic effect from the immobilized enzyme is more evident.1 mol L 1. 0.5 mV vs. it raised the optimum pH value as related to this different environment used for the immobilization of PPO.de  2013 Wiley-VCH Verlag GmbH & Co.0. 1) without and 2) with adding catechol at fixed con- centration.

60 8C (Figure 6 b). The current for this film increased ex.4. the enzyme when immobilized may be protected in phosphate buffer containing catechol at wide range of at high concentrations of OH–. Response obtained by chronoamperometry of a PPY-2-COOH/PPO biosensor at 0. tained the maximum rate for enzymatic reaction.wiley-vch. Higher values of optimum pH have been also found in here to be ranging from 38 8C to 47 8C. tion of the signal occurs. Vmax = chol (10 2 mol L 1) ranging the temperature from 5 8C to 6.0  10 1 mol L 1) at pH 9.0.0  10 1 mol L 1 in a flow injection analysis system.5  10 2 mol L 1 (Figure 7). matrix. 30]. After that. justifying their stability at concentrations (5. exhibiting stable. been seen for free PPO a this temperature [29. and the highest sensitivity Another parameter obtained here was the optimum for an optimal signal-to-noise ratio in the nonlinear temperature for PPO entrapped within the PPY-2-COOH region was 7. similar results.10 V phase.1 V vs. KGaA. 3. A linearity was obtained since for thin films an optimal pH value would be close by ranging the concentration of catechol from 5. that is in the bulk phase. The optimum temperatures were assumed value may be optimized after employing cationic media- Electroanalysis 2013. This value of optimum pH. the amperometric responses of a PPY-2-COOH/ Lineweaver Burk plot (Inset of Figure 7). Km = 3.85 mmol min 1 cm 2 and Michaelis Menten constant.0  10 6 to 1.de 747 . This effect yields a current and a controlled temperature of 40 8C. 25.4 higher values of pHs [32. two flux injections were per- should be reduced after decreasing remaining carboxylic formed for each concentration of catechol. 33]. as a suggestion.0  10 4 to 7. this beyond 50 8C. where we ob- PPO film were obtained at a fixed concentration of cate. thickness will have an effect on the pH value. to that of the corresponding free enzyme (44 mmol L 1) comes to decreases. unstable values of current [34]. the enzyme-catalyzed reaction. For obtaining this The kinetic parameters were also estimated from the value. Weinheim www.5  10 6 mol L 1. titioning of protons [32]. which was chosen as 40 8C. Ag/QRE under an optimal working temperature of 40 8C in phosphate buffer solution and catechol at concentrations ranging from 5. When this value of Km is compared ponentially with temperature up to reach 47 8C. with the current cross-linked by covalent bonds. 9.electroanalysis. Once the pH at the immobilized Figure 7 shows the amperometric response of a PPY-2- PPO/solution interface is lower than that of the bulk COOH/PPO film under flow injection by applying 0. but.4 than that at double-check the concentration value in which the satura- pH 6. it ing that here enzyme and matrix are not completely can be expected a fully saturated signal. No. 7. but here the value biosensors based on the use of PPY matrices [7]. to 2. constant signals due to a limited rate for In this case. it can be verified that Km for the PPY-2-COOH/ were obtained due to a loss of the enzymatic activity PPO film is ten times smaller. Insert: Linearweaver Burk plot.0  10 6 mol L 1 to 1. 741 – 749  2013 Wiley-VCH Verlag GmbH & Co. and yielded acid ends from the PPY-2-COOH structure.Electrochemically Prepared Polypyrrole-2-Carboxylic Acid Films Fig. thus evidenc. of 40 8C was chosen since minor losses in activity have usual high values of pHs have been explained by the par. when be.5 (free enzyme). In order to response that is 15 times higher at pH 9. At higher concentrations of catechol.78 mmol L 1. and un.

748 www. Centonze. 3. H. Chilkoti.de  2013 Wiley-VCH Verlag GmbH & Co. within the PPY-2-COOH matrix. C. 2000. Kvarnstrçm. 2002. Mater. Ouyang. Synth. for films synthe. ski. J. 117. Electroanal. Xu. 25. 2000. 2002. G. Krysinski. G. U. values of detection limit and linear regime of quantifica. C. E. Narinesingh. As related to the stabili. Ghosh. 2003. istry. 207. S.4 [35] Phosphate-dopedpolypyrrolefilm Catechol 0. G.wiley-vch. Inzelt. J. Rodrguez-L pez. X. Curulli. ty of PPY-2-COOH/PPO as a biosensor. 55. Shmulevich. [7] A. Mange- ney. [8] S. F. Electrochim. P. Zhong. Palle- schi. Toppare. 1731. J. Bowmaker. R. Rodrguez-L pez. Bio- ble biosensor mainly because the presence of unstable Au electron. F. 3109. The value of Km obtained here [1] G. J. ing that its preferential use can be as a disposable. 10. Adv. O’Sullivan. Chem. and proceeded with the immobilization of [14] M. 2527. 63. L. F. 5031. mobilized in different matrices (Table 1) as an indicative [2] B. 2001. 1293. Soc. J. 103. Besides. Dairy Sci. J. Kinetic constante. Dai. D. 17. Brahim. Y. R. I. Biochim. J. Struct. Results from FTIR spectroscopy have indicated Garca-C novas. Met. Y.2 [39] 4-Methylcatechol 2. Foschini et al. Fenoll. 95. 38. Biosens. J. Ivaska. Leipzig. [23] Y. D. H. 401. predominantly formed.08 [36] Cobalt (II) phthalocyanine modified cellulose-graphite composite electrode Catechol 1. A. 29. Yang. J. Met. Amine. 14. Liu. Heinze. Chem. Tudela. Majumdar. 2779. Mol. 1986. C. Li. Rogal- cooperation project between France and Brazil (COFE. 38. which characterized a relatively short lifetime. Int. Pallini. CUB Project 507/05). 8451. N. for different PPO biosensors. A. W. 159.6 [37] Eggshell membrane activated by glutaraldehyde Dopamine 0. Cosnier. 139. 332. 729. Langmuir 2001. tracks deposited on polyamide substrates. 35. J. Bioelectron. used in the [10] M. [25] C. Electrochem. Mazur. which is out References of the scope of this work. de Torresi. Chem. 2010. surface. S. Struct. 1245. J. Li. M. 1010. No. 741 – 749 . Chem. sis 1998. measurements were obtained under two consecutive days. Y. [19] A. R. Hiner. G. Fiorito. J. Krywko-Cendrowska. Biosens. Y. also. 4 Conclusions [9] A. [21] S. Garca-Molina. Biosensor Analyte Km (mmol L 1) Reference Aminopropyl-controlled pore-glass Catechol 2. Bousalem. 344 – 348. 140. tion for catechol suggest that the PPY-2-COOH/PPO [24] S. N. preserve functionali. Bioanal. Adhikari. S. Benabderrahmane. Reedijk. Zambonin. A. Acta mild conditions (at low oxidation potentials in a mild sol. Curulli. Y. PPY-2-COOH/PPO film showed to be similar to those re- 1988. G. Chem. 2005. J. Scharifker. 1999. zation of pyrrole. quartz crystal microbalance (EQCM) allowed us to con- 1597. an overoxidized structure was 147. 2001. Tudela. Cooney. 1727. 45. 13. Irene. 206. Yoneyama. A. Cogan. A. from the electrolyte (TMABF4). Guiseppi-Elie. Q. [12] P. The value of Km obtained for the [20] D. Zhou. 699. 366. [27] C. vent). Doblhofer. A. S. Anal. Arslan. A. Polymer J. M. The [22] C. We have successfully synthesized a new Challa. The use of an electrochemical [17] M. Mac- romolecules 2005. G. Kuwabata. Table 1. Palmisano. Palleschi. Acta 2002. 644. 1971. obtain electroactive films and. P. Yang. Weinheim Electroanalysis 2013. [3] S. Garca-C novas. Soc. Polymer 1997. Biosens. Kiralp. GonÅalves.67 [38] Agarose guargum Dopamine 0. Seakins. B. K. firm the deposition of PPY-2-COOH on the electrode [18] L. Downard. 15. ACN.38 PPY-2-COOH/PPO Catechol 3. 37. Gamez. reproducible [4] D. Biotechnology 2006. Simons. A. 2008. Linker. Steensma. 17. J. Synth. Wu. COOH) showed to be extremely important in order to [11] F. Shi. Ito. J. Mol. 53. Chehimi. Cell Biol. 1998. N.electroanalysis. Xu. I. agencies FAPESP. Synth. [6] S. 139. Braz. sized with traces of water. J. Y. Mater. Teruel. 1990. polyphenoloxidase (PPO). X. 413. Acknowledgements [26] Y. Bioelectron. Xie. Electroanal. C. 1998. Electroanaly- electropolymerization of pyrrole-2-carboxilic acid (PY-2. Driessen. films can be used as biosensors for monitoring of phenols. ported for PPO immobilized in different matrices. under [13] M. J. and defin- 12. R. Met. Ya Dai. and CAPES from a bilateral [28] M. Shi. 5. A. D. C. Km. Phys. Progress in Polymer Science of strong affinity for cathecol when PPO is immobilized 2004. Biol. J. Sang. [29] Z. Electrochim. Liu. PeÇalver. 2003. Pletcher. F.Full Paper M. 2004. Wagner. S. that was covalently [15] J. 12. J. P.87 This work tors during the preparation steps of the film. 586. The authors acknowledge the support from the Brazilian 644. J. A. 135. 2009. Biophys. polymer. [5] P. flexi. Chem. a enzyme. M. G. The absence of water in the solvent. amperometric biosensors. P. linked to the polymer matrix in order to obtain stable [16] Z. Q. Fermn. pyrrole-2-carboxilic acid (PPY-2-COOH). Int. J. C. B 1999. Eur. Etzion. Yagci. 163. Acta 2000. Guilbault. Y. 1996. Biochem. Springer. G. S. 443. Kelly. Qian. L. Xie. R. KGaA. J. that the PPY-2-COOH films had incorporated the salt 34. 1691. Conducting Polymers: A New Era in Electrochem- is comparable to those for biosensors based on PPO im. 87. CNPq.

212. Karve. Toppare. D Souza. A. A. Funct. [37] Y. D. 72. Yildiz. Paranjpe. Kiralp. L. S. D Souza. 33. 63. Apetrei. chim. Karve. S. Inamdar. Melo. React. S. M. Kiralp. Anal. Messina. Electro- Polymers 2005. Yagci. Electroanalysis 2013. G. 25. S. [35] A. 40. [33] S. Chim. Kiralp. 72. Haram. 155. Ferreira. Tanimoto de Albuquerque. Anal. F. Yagci. B. Toppare. Macromolecules [38] S. 2003. 741 – 749  2013 Wiley-VCH Verlag GmbH & Co. Girelli. E. Yagci. Y. 37.Electrochemically Prepared Polypyrrole-2-Carboxylic Acid Films [30] P. 40. Dorantes-lvarez. L. Acta 2008. Tembe. Polymers 2005. de Saja. B. J. KGaA. [34] G. 82. 349. Anal. S. J. 210. Toppare. M. 2006. 155. Chim. S. Biol. M. Padhye. S. L. Int. Karve. Palma-Orozco. 612. Biochem. 63. Weinheim www. 580. 2003. M. Sampedro. A. Acta 2007. 8919. Phytochemistry 2011. Tembe.electroanalysis. B. Indian J. Chim. Y. J. Kubal. L. J. Acta Biophys. H. [39] S. 271. [31] H. L. No. React. 3. 596. S. Yildiz. F. [36] C. Biochem. S. 56. Mattei. M. N jera. 2006.de 749 . Y. [32] H. S. Anal. Rodrguez-Mndez. Ortiz-Moreno.wiley-vch. Acta 2011. L. F. B. Funct.