Conducting polymer films as model biological membranes

Electrochimica Acta 51 (2006) 2173–2181

Conducting polymer films as model biological membranes Electrochemical and ion-exchange properties of poly(pyrrole) films doped with asparagine and glutamine Beata Paczosa-Bator a , Jan Migdalski a , Andrzej Lewenstam a,b,∗ b

a Faculty of Material Science and Ceramics, AGH – University of Science and Technology, PL-30059 Cracow, Poland Center for Process Analytical Chemistry and Sensor Technology (ProSens), c/o Process Chemistry Center of Excellence, ˚ Akademi University, FIN-20500 Turku-Abo, ˚ Abo Finland

Received 7 December 2004; received in revised form 24 March 2005; accepted 30 March 2005 Available online 26 September 2005

Abstract This paper shows the application of conducting polymers (CPs) for constructing model biological membranes in order to study potential formation mechanism. Two amino acids, asparagine and glutamine, were incorporated in the poly(pyrrole) matrix during electrochemical polymerization. The polymer film was characterized by infrared and X-ray photoelectron spectroscopy. The film morphology was studied by atomic force microscopy. The ion-exchange behavior of PPy-Asn and PPy-Gln membranes in dependence on the conditioning solution are characterized using an open circuit potentiometric measurements. Close-to-Nernstian sensitivity was observed for the films under equilibrium. During equilibration provoked by the change in concentration of magnesium and/or calcium ions, the differences in the shape and evolution of the potential response with time were observed. The varying potential–time behavior after a bulk concentration change has been explained by a different participation of the magnesium and calcium ions on the ground diffusion layer model (DLM). © 2005 Elsevier Ltd. All rights reserved. Keywords: Poly(pyrrole); Amino acids; Ion-exchange kinetics; Dynamic discrimination; Potential transients

1. Introduction Several important functions of the nervous system depend on the behavior of N-methyl-d-aspartate (NMDA) receptor channels. These ligand and voltage gated NMDA channels are highly permeable to both monovalent ions (e.g. Na+ , K+ ) and to Ca2+ ions and are blocked by Mg2+ ions at normal resting potential [1,2]. The calcium filter and magnesium blockade appears to be controlled by amino acids residues in the transmembrane regions of the NMDA receptor [3]. Asparagine and glutamine residues with negatively or/and positively charged side chains are involved in the Mg2+ block by forming the

∗

Corresponding author. Fax: +358 2 2154479. E-mail address: [email protected] (A. Lewenstam).

0013-4686/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2005.03.084

active sites in the channel, which bind the magnesium ions [4–8]. In this paper, we report on a novel way to study competitive ion-binding to asparagine and glutamine with magnesium and calcium ions and its influence on the propagation of a membrane potential with time. For this purpose, poly(pyrrole) (PPy) films as model membranes are applied and p-doped with asparagine (Asn) or glutamine (Gln). These big biologically active ions are expected to result in cationic sensitivity, as previously observed for bulky and immobile anions of metallochroms, e.g. calcion, kalces, tiron or sulfosalicylic acid [9–14]. In order to obtain the model membrane, the PPy-Asn and PPy-Gln films were soaked in solutions of magnesium and calcium salts and made magnesium and/or calcium-sensitive. This work reports on the competitive binding magnesium and calcium ions and the resulting local excess or deficiency

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of these ions during equilibration, previously observed for ion-selective membranes and analyzed in terms of the diffusion layer model (DLM) [19,21]. In particular focus is a possibility of obtaining different transitory potentials by changing the Mg2+ and Ca2+ bulk concentrations. The proof of different discriminating mechanism for magnesium and calcium ions in contact with asparagine and glutamine anions may be highly relevant in considering real biological mechanisms where distinction between local and bulk concentration is rarely applied.

2. Experimental 2.1. Chemicals Pyrrole (Merck) was purified by double distillation under argon gas and then stored under argon at low temperature and protected from light. The l-asparagine, l-glutamine and the TES buffer (N-[Tris(hydroxymethyl)methyl]-2aminoethanesulfonic acid) were obtained from Fluka. Doubly distilled and freshly deionized ELGA water (resistivity 18.2 M
cm) was used throughout this work. The other reagents were purchased from J.T. Baker. All chemicals used were of analytical grade. The solutions of a concentration lower then 0.01 M were prepared just before use. 2.2. Apparatus and procedures 2.2.1. Conditions of electropolymerization The electrochemical synthesis of poly(pyrrole) films was performed with an Autolab General Purpose System (AUT20.Fra2-Autolab, Eco Chemie, B.V., The Netherlands) connected to a conventional, three-electrode cell under argon atmosphere. The working electrode was a glassy carbon disk (area 0.07 cm2 ) or conducting glass pieces with an area of about 1 cm2 (ITO, Lohja Electronics, Finland, used for the XPS and AFM experiments), the reference electrode was an Ag/AgCl/saturated KCl electrode, which was connected to the cell via a bridge filled with supporting electrolyte solution. A glassy carbon (GC) rod was used as the auxiliary electrode. Just before each polymerization, the surface of the working electrode was polished with 0.3 ␮m aluminum oxide powder, rinsed with water and ethanol and cleaned in an ultrasonic bath for at least 15 min before use. The ITO sheets were manually cleaned, then immersed in ethanol and placed in an ultrasonic bath for about 10 min before use. The electrodeposition was performed potentiodynamically and potentiostatically on the working electrode in deoxygenated aqueous solution containing 0.1 M pyrrole monomer and 0.1 M Gln or Asn. Prior to potentiostatic deposition, the cyclic voltammograms were recorded in order to establish the potential required for monomer oxidation. Subsequently, a constant potential of +900 mV was used for growing PPy films with a deposition time of 2400–3800 s. The electrodeposition of these films was very slow, and the maximum attained

current was 10 ␮A. The film thickness as estimated by Faraday’s law was about 1–1.5 ␮m. All experiments were performed at room temperature. 2.2.2. XPS, FTIR, AFM measurements and theoretical calculations The elemental analysis of the poly(pyrrole) films by X-ray photoelectron spectroscopy (XPS) was preformed with a Physical Electronics Quantum 2000 XPS-spectrometer equipped with a monochromatized Al X-ray source to assess qualitatively the influence of soaking on the composition of these films. The size of the analyzed area was 100 ␮m in diameter and the analysis depth was about 2–5 nm depending on the investigated element. The Fourier transform infrared (FTIR) spectra were recorded with a Bruker IFS 66/S instrument. The atomic force microscopy (AFM) images were recorded with a NanoScope IIIa microscope (Digital Instruments Inc., Santa Barbara, CA), equipped with the extender electronics module enabling phase imaging in tapping mode. For numerical calculations, Mathcad 2001 Professional by MathSoft Inc., Canada, was used. 2.2.3. Potentiometric measurements The potentiometric measurements were made with a home made 16-channel set-up. The reference electrode was an Ag/AgCl/3 M KCl electrode. The activity coefficients were calculated using an extended Debye–H¨uckel equation. No correction for the liquid-junction potential (Henderson equation) was applied. Before and after soaking, the potentiometric response in chloride solutions of interfering cations (sodium, potassium and magnesium or calcium) was checked. The first calibration of the PPy-amino acid films was always performed without conditioning. One part of the films was conditioned in solutions at neutral pH: 0.1 M magnesium chloride (pH 6.9) or 0.1 M calcium chloride (pH 6.5), respectively. However, despite the long conditioning time, no cationic response was observed. The sensitivity of the PPy-amino acid films to cations was induced for the second part of films soaked in a mixture of 0.1 M calcium chloride and calcium hydroxide (pH of about 10.5–11.5) or in saturated magnesium hydroxide solution with a pH of about 10.5. After 1 week of soaking, close-to-Nernstian cationic response in the range from 10−1 to 10−5 M of Ca2+ or Mg2+ was observed. Between measurements, the PPy-amino acid films were kept in the same solutions as used for soaking and a very good adhesion of the films to the substrate was observed at least during 6 months after deposition.

3. Results and discussion 3.1. Poly(pyrrole) layer growth It was found that thin films composed of PPy-amino acids could be deposited on GC electrodes using potentiodynamic

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or potentiostatic techniques. The electrochemical oxidation of the pyrrole monomer in 0.1 M amino acid and 0.1 M Py solution was started at potential > 700 mV. A typical curve with the increasing current during electrodeposition (potential cycling in the range 300–900 mV) is shown in Fig. 1(a) (curves i). As can also be seen from Fig. 1(a) (curves ii), pure Gln and Asn did not exhibit any electrochemical activity in such potential range. The exemplary curve of potentiostatic deposition is shown in Fig. 1(b). AFM was used to examine both methods of electropolymerization. The AFM analysis showed that the surface of the films obtained by potentiostatic method was smooth and very regular and superior in comparison with the films obtained potentiodynamically. The AFM analysis showed as well that the conditioning process did not affect the polymer surface. This was the reason why all films used in the measurements were prepared potentiostatically. An example of typical topographies and cross-section plots of the PPy-Asn film obtained by AFM is presented in Fig. 2. 3.2. Chemical characterization of polymer films On the FTIR spectra of the PPy-amino acid films, a large absorbance in the NIR region caused by the oxidized state of PPy was observed. The spectra of the poly(pyrrole) films showed a C O stretching-vibration peak at 1651 cm−1 , O H at 1260 cm−1 , O C O near 800 and 725 cm−1 assigned for Gln or Asn. The peak at 1587 cm−1 is assignable to the C C stretching vibration of the PPy conjugation system. Peak areas at 1531 and 1110 cm−1 are indicative of the C C and C N stretching vibrations in the pyrrole ring, respectively. C H vibrations have been identified with bands near 1330 and 1043 cm−1 [15,16]. The freshly deposited and unsoaked PPy-Gln or PPy-Asn films did not respond to calcium, magnesium, sodium and potassium ions. The exemplary potentiometric response of PPy-Gln film in buffered (by TES, pH 7.4) chloride salts of K, Na, Mg and Ca is presented in Fig. 3. In order to induce potentiometric magnesium or calcium sensitivity, the films were conditioned in solution containing Mg or Ca ions, respectively. The chemical composition of the samples was investigated by XPS. Fig. 4(e) shows on overview spectrum of a freshly deposited PPy-Asn sample. The following peaks were observed at the given binding energies: O 1s at 532.8 eV, N 1s at 400 eV and C 1s at 284.8 eV. These data are in excellent agreement with the literature [17]. The peaks in the region of 347.2 eV binding energy, as shown in Fig. 4(b), are identified as Ca 2p3 signal, which was observed only after conditioning in alkaline calcium solution. It should be noted that after conditioning in neutral pH solution, the calcium peak was not found, see Fig. 4(a). Similarly the XPS analysis taken after soaking of the PPy-amino acid films in alkaline magnesium solution proved Mg 2s peak at the 88.8 eV, see Fig. 4(d), but magnesium was not detected in

Fig. 1. (a) Cyclic voltammograms recorded during poly(pyrrole) electrodeposition (anodic then cathodic, scan rate = 20 mV s−1 ); (curve i) 0.1 M Gln and 0.1 M pyrrole solution; (curve ii) pure 0.1 M Gln solution. (b) Potentiostatic deposition (E = +900 mV) from 0.1 M Gln and 0.1 M pyrrole solution.

the polymer structure after soaking in neutral solution, see Fig. 4(c). The pH of the solutions used to achieve an ionic sensitivity play decisive roles in the final potentiometric sensitivity of the polymer film [11,12,18].

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Fig. 3. Potentiometric responses of unsoaked PPy-Gln film in selected chloride salts buffered with TES (pH 7.4): potassium (♦); sodium (); calcium (×); magnesium ().

Fig. 2. The AFM morphology image and cross-section plots of the PPy-Asn film prepared potentiostatically. The size of image is 10 ␮m × 10 ␮m.

Direct soaking in 0.1 M chloride salt of the main ion did not lead to cationic sensitivity. It was found that the process of making cation-sensitive films is effective only by soaking in a mixture of 0.1 M CaCl2 and Ca(OH)2 with a

pH between 10.5 and 11.5 in the case of calcium ion, and in saturated Mg(OH)2 (pH 10.5) in the case of magnesium. The response of the PPy-amino acid electrode was tested in magnesium and calcium chloride solutions. The cationic response with the close-to-Nernstian slope with linear range from 10−1 to 10−5 M was observed after 1 week of soaking. The induced calcium or magnesium sensitivity of the

Fig. 4. XPS spectra for PPy-Asn films: (a) after conditioning in neutral calcium solution (pH 6.5); (b) after conditioning in alkaline calcium solution (pH 10.5–11.5); (c) after conditioning in neutral magnesium solution (pH 6.9); (d) after conditioning in alkaline magnesium solution (pH 10.5); (e) the overview spectrum before conditioning.

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similar response for these ions was also observed for PPy-Gln films. The conducting polymer (CP) films sensitized towards calcium or magnesium became practically insensitive towards sodium or potassium ions. However, both groups of electrodes showed responses to Mg2+ as well as to Ca2+ . This indicates that the stability constant of calcium or magnesium complexes with Asn and Gln are similar and bigger than those for sodium or potassium complexes, as observed before for CP films doped with adenosine 5-triphosphate (ATP) [13] and heparin [14]. The typical potential–time behavior of PPy-Asn or PPy-Gln magnesium and calcium-sensitive films observed after a bulk concentration change of sodium, potassium and calcium (or magnesium, respectively) is shown in Fig. 6. It should be noted that the concentration of primary ions (Ca2+ or Mg2+ ) was constant and equal to 10−4 M. 3.3. Dynamic response of PPy-Asn and PPy-Gln membranes—the theory and experiment It is assumed that for dynamic behavior of the PPyAsn(Gln)-Me (Me is Mg or Ca) electrodes, the models applied before for ion-selective electrodes [19] are valid. In the absence of interfering ions, the transient potential of the CP film after change of the concentration of the main ion can be described by the coupled system of equations [20]: E = const +

RT [Me2+ ]0 ln 2F [Me2+ ]0 I

(1) A

[Me2+ ]0 = [Me2+ ]0 + ([Me2+ ]
  −2DMe t 2+ I −[Me ]0 ) 1 − exp δ2

(2)

I

Fig. 5. The Ca and Mg potentiometric sensitivity of PPy-Asn films: (♦) before soaking; () after 1 month soaking in calcium (a) and magnesium (b) solution with neutral pH; () after 1 month soaking in alkaline calcium (a) and magnesium (b) solution.

PPy-amino acid films was very stable and almost constant during the test period of 6 months (e.g. for PPy-Gln films: 28.9 ± 0.9 mV/pCa and 29.6 ± 1.2 mV/pMg). In Fig. 5(a and b), typical calibrations curves for calcium and magnesium ions in the case of poly(pyrrole) film doped with Asn (PPy-Asn) are shown. The behavior of unsoaked films and the films after 1 month of soaking in neutral or in alkaline solutions containing Mg2+ or Ca2+ ions are presented. A

where [Me2+ ]0 is the initial primary ion concentration at the membrane surface, [Me2+ ]A the bulk concentration of primary ion (after concentration change), DMe the diffusion coefficient of primary ion with aqueous diffusion layer (cm2 s−l ), δ the diffusion layer thickness (cm), t the time (s) and [Me2+ ]0 represents the ion concentration in the membrane phase at the interface ([Me2+ ]0 = [PPy − Asn(Gln) − (Me2+ )]0 ). If the [Me2+ ]0 is constant and the equilibration process reaches total equilibrium, Eq. (1) can be rewritten as: E = const +

RT A ln [Me2+ ] 2F

(3)

When interfering ions Ca2+ or Mg2+ are added to the bulk of solution containing initially the primary ions, both ions participate in competitive binding at the amino acid sites at the membrane (PPy-amino acid-(Mg2+ ) or (Ca2+ )|solution (Ca2+ , Mg2+ ) interface. The potential of the electrode is influenced by the local concentration of ions in the vicinity of the

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Fig. 6. Comparison of the potentiometric response of the PPy-Gln-Ca film (the bold curve) and PPy-Gln-Mg film (the thin curve) caused by sodium, potassium and magnesium (or calcium) concentration increase.

interface, which evolve during equilibration until total equilibrium is attained. The mass transport that takes place during equilibration is described by Ref. [19]: +JMg2+ = −JCa2+

ntot ds = A dt

(4)

KMg2+ ,Ca2+ =

2+

[Mg2+ ]0 [Ca [Mg

2+

]0

]0 [Ca2+ ]0

is the ion-exchange equilib-

rium constant and s is given by: s=

[Ca2+ ]0 [Mg2+ ]0 + [Ca2+ ]0

=

KMg2+ ,Ca2+ [Ca2+ ]0 [Mg2+ ]0 + KMg2+ ,Ca2+ [Ca2+ ]0 (7)

where s is the parameter describing the extent of equilibrium, t the time (s), A the electrode surface area (cm2 ), ntot the number of active sites occupied by Mg and Ca ions (mol) and JMg2+ and JCa2+ are the fluxes of Mg2+ and Ca2+ to the electrode surface. By adopting the concept of the diffusion layer model used before in a case of ion-selective electrode membranes [21–23], the dynamic response of the PPy doped with Asn or Gln proceeding with the ion-exchange process: PPy − Asn(Gln) − (Mg2+ ) + σCa2+ 2+ 2+  PPy − Asn(Gln) − (Mg2+ 1−σ Ca ) + σMg

(5)

where 0 < σ < 1 The electrode potential can be derived by the sum of the boundary potential and diffusion potential in the membrane:

RT E = const + ln 2F

(1 − s)KMg2+ ,Ca2+ + s

×

A

[Mg2+ ] +

DCa2+ A [Ca2+ ] DMg2+

s = seq =



DCa2+ DMg2+ s

KMg2+ ,Ca2+ [Ca2+ ]

A

A

A

[Mg2+ ] + KMg2+ ,Ca2+ [Ca2+ ]

(8)

And finally, the s(t) function can be obtained [24]:   DCa2+ KMg2+ ,Ca2+ − s − KMg2+ ,Ca2+ DMg2+ ×

DCa2+ 2+ A DMg2+ [Ca ]

  s ln 1 − A seq [Mg2+ ]A + KMg2+ ,Ca2+ [Ca2+ ] A

U Ca2+ K 2+ 2+ U Mg2+ Mg ,Ca

KMg2+ ,Ca2+ (1 − s) +



where: [Mg2+ ]0 + [Ca2+ ]0 is equal to the sum of Mg and Ca ion concentrations in the surface of the film [PPy − Asn(Gln) − (Mg2+ )]0 + [PPy − Asn(Gln) − (Ca2+ )]0 . In total equilibrium, the equilibrium value of the apparent coverage factor equals seq

[Mg2+ ] +

A

= ([Mg2+ ]A + KMg2+ ,Ca2+ [Ca2+ ] )Ct

(9)

where the parameter C is defined as (6)

where U Mg2+ and U Ca2+ represent mobilities in the membrane phase, respectively, of magnesium and calcium ions;

C=

DCa2+ A ntot δ

(10)

To get C is the order 102 cm3 mol−1 s−1 (see Fig. 7), characteristic for solid-state ion-selective membrane [24], DCa2+ ∼ 10−5 cm2 s−1 , A ∼ 1 cm2 and δ ∼ 10−2 cm are used.

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and be ascribed to the different hydration energy of these two ions [13,14]. The lower hydration energy of calcium makes the transport of this ion to and into the membrane faster in comparison to magnesium with resulting redistribution of the surface concentration of ions. The in or outflow of slower magnesium ions determines the speed of the ion-exchange process. This is the reason why after bulk calcium concentration change, the local excess magnesium ions appear at the surface of the PPy-Asn(Gln)-Mg electrode (as shown schematically in the Fig. 8a). And vice versa, after a bulk magnesium concentration change, the deficiency of calcium ions in the vicinity of the membrane surface may be predicted and was observed. When the total equilibrium is reached, then s = seq and Eq. (6) can be reduced to a Nikolskii–Eisenman equation, which for Mg-sensitive film has the form: A

RT E = const + ln 2F

Fig. 7. The time-dependent response of magnesium-sensitive electrode calculated from Eqs. (6) and (9) in the solution of mix magnesium and calcium ions. The bold curve represents response after increase Mg2+ activity while the thin curve potential response after increase Ca2+ activity ( KMg2+ ,Ca2+ = 1;

DCa2+ DMg2+

U Ca2+ U Mg2+

= 1.2;

= 1.12; C = 132 cm3 s−1 mol−1 were assumed).

By coupling Eqs. (6) and (9) through parameter “s”, it is possible to get the function of E versus time and thus a dynamic model of transient effects (for more technical details on derivation of the above equations, see [21,23,24]). This model allows the prediction of characteristic potential overshoots or exponential potential changes during equilibration, the former for changes of faster ion concentration in the bulk (in our case Ca2+ ), and the latter for changes of slower ion concentration (in our case Mg2+ is the main ion) as shown in Fig. 7. The model shows as well that after total equilibrium is achieved (time → ∞), the CP|solution is ruled exclusively by the thermodynamic properties of the system, while at the start of equilibration (time → 0), the system is governed by its kinetic properties. It is obvious that the model can be applied for interpretation of dynamic properties of any other CP-based films what will be shown by us soon elsewhere. A general prediction of the model presented is confirmed experimentally. Fig. 6 shows very characteristic potential changes with time after every bulk concentration change. The time-dependent potential profiles observed have totally different shapes. They are either monotonic (in the case of Mg2+ interference for the calcium electrode) or show overshoots (in the case of Ca2+ interference for the magnesium electrode), as predicted formally. According to the model presented above, the reason for these different potential transients can be sought in slower Mg2+ ion transport in comparison to Ca2+

[Mg2+ ] +

U Ca2+ A K 2+ 2+ [Ca2+ ] U Mg2+ Mg ,Ca

[Mg2+ ]0 + [Ca2+ ]0

(11)

The ion-exchange process of PPy-Gln-(Mg) film in the solution of mixed main and interfering (Ca2+ ) ions under steady state is illustrated in Fig. 8b. In order to exclude the hydrogen ion influence on the potential–time dependence, the measurements with magnesium and calcium-sensitive films were performed also in the solutions buffered by TES to pH 7.4. In Fig. 9, the dynamic response of PPy-Asn-Mg film to magnesium and calcium concentration changes is shown. The basic solution contained a mixture of magnesium and calcium chloride salts with constant (10−2 M) concentrations of magnesium ions when the response to calcium ions was tested or constant calcium concentration when the response to magnesium ions was checked. As can be seen, also in these two cases, distinctively different signals were observed: an overshoot response-type for changes in bulk concentration of calcium ions and a monotonic change for magnesium. In case of the unsoaked PPy-amino acid films as well as PPy films doped with ligands which are not able to bind Ca or Mg ions (e.g. sulfosalicylic acid), such potentials transients were not noticed. These fundamental differences associated with equilibration processes in the Mg–Ca system have already been noticed and discussed for magnesium-selective PCV-based electrodes [25] and recently in the case of conducting polymer films doped with adenosine triphosphate [13] and heparin [14]. Here, for the first time, we observed the same behavior pattern with two doping ions: asparagine and glutamine, which are biologically active ligands responsible for the activity of the NMDA receptor channel. We conclude that the potential-dependent local deficiency or excess of magnesium or calcium at the Aln and Gln sites discussed in this paper may affect the speed (i.e. facilitate or retard) of biological processes. This overlooked possibility

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Fig. 8. The ion-exchange processes on PPy-Gln-Mg electrode in the mixed solution of Mg2+ and Ca2+ ions: (a) just after calcium concentration change and (b) under steady state. GC, glassy carbon substrate; PPy-Gln-Mg, magnesium-sensitive poly(pyrrole) film doped with glutamine anions.

Fig. 9. Transitory response of magnesium-sensitive PPy-Asn film caused by magnesium (the bold curve) or calcium (the thin curve) concentration changes. The measurements were performed for solution buffered with TES (pH 7.4).

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may be of fundamental importance in the case of potentialdependent activity of biological channels.

4. Conclusions It is possible to incorporate asparagine and glutamine into a PPy matrix by electrochemical deposition. The PPy-amino acid membranes are able to exhibit ion-exchange properties towards magnesium and calcium ions after soaking in alkaline solution of respective main ions. The PPy-Asn and PPyGln electrodes showed a stable and reproducible response for activity of Ca2+ and Mg2+ with a close-to-Nernstian slope. By using the poly(pyrrole) matrix, it is possible to explore the competitive ion-exchange properties of asparagine and glutamine with magnesium and calcium ions. Transitory behavior of the films was observed during equilibration provoked by a change of bulk magnesium or calcium concentration, which was ascribed to a different rate of magnesium and calcium ion transfer between the bulk of solution and membrane. PPy-amino acid films can be used as new materials to study the effects of the physicochemical properties of ions on potential formation mechanism and the local distribution these ions in the vicinity of the real biological membranes.

Acknowledgements Financial support from the Polish Committee for Scientific Research (KBN) Project 4T08E09825 is gratefully acknowledged. The Marie Curie Foundation is gratefully acknowledged for financial support in the form of a grant to the Process Chemistry Center, Center of Excellence at ˚ Akademi University. The authors are grateful to Carita Abo Kvarnstr¨om for the FTIR measurements.

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