Capacitive Composites Made of Conducting Polymer and Lysozyme

Article pubs.acs.org/JPCC

Capacitive Composites Made of Conducting Polymer and Lysozyme: Toward the Biocondenser Daniel E. López-Pérez,† David Aradilla,†,‡ Luis J. del Valle,† and Carlos Alemán*,†,‡ †

Department of Chemical Engineering, E. T. S. I. B., Technical University of Catalonia, Diagonal 647, 08028 Barcelona, Spain Center for Research in Nano-Engineering, Technical University of Catalonia, Campus Sud, Edifici C’, C/Pasqual i Vila s/n, Barcelona E-08028, Spain

‡

ABSTRACT: Conducting polymer/protein composites, P(EDOT-LZ), of poly(3,4-ethylenedioxythiophene) (PEDOT) and lysozyme, a protein with bactericidal activity, have been prepared by in situ anodic polymerization. The stability of the composite obtained from a polymerization medium consisting of an acetonitrile:water solution (1:4 v/v) with a 1:1 monomer:lysozyme mass ratio is better than that of the individual conducting polymer, while the electrical conductivity and capacitive behavior of the two systems are very similar. Incorporation of the enzyme produces drastic changes in the morphology of PEDOT, resulting in the formation of very homogeneous aggregates with cotton flake morphology that has been shown to be hollow. Multilayered systems formed by alternating layers of PEDOT and P(EDOTLZ) have been examined, even though results indicated that they do not show any practical advantage with respect to the individual P(EDOT-LZ) films. Adhesion assay using four eukaryotic cellular lines have evidenced that the behavior of P(EDOT-LZ) as a supportive matrix is still better than that of PEDOT. The bactericidal activity and the favorable response toward eukaryotic cells combined with its electrochemical properties suggest that P(EDOT-LZ) is a potential candidate for the fabrication of bioelectrochemical capacitors for in vivo implants. In order to examine this possibility, the electrochemical and capacitive properties of P(EDOT-LZ) coated with eukaryotic cellular monolayers have been investigated. Electrochemical impedance spectra reflect important differences in the response of P(EDOT-LZ) and PEDOT surfaces, which are independent of the cell line, when these materials act as supportive cellular matrices.

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INTRODUCTION

Conducting polymers (CPs) provide new opportunities for the preparation of electroactive biocomposite materials for biotechnological applications. For example, CPs have been combined with different biomacromolecules (e.g., proteins, viruses, and polysaccharides) to produce platforms for tissue engineering,5−7 biosensors,8,9 membranes for desalting and extraction of biomolecules,10 scaffolds for nerve regeneration,11 and so on. In a very recent work, we reported a preliminary study about the properties and potential applications of composites made of CP and lysozyme.12 More specifically, poly(3,4-ethylenedioxythiophene), hereafter abbreviated PEDOT, which is one of the most important CPs due to its high conductivity (up to 500 S/ cm), good thermal and chemical stability, fast doping− dedoping processes, and excellent biocompatibility,13−19 was used to prepare composites using the following two strategies: (1) adsorption of enzyme on the surface of PEDOT films, the resulting composite being denoted PEDOT/LZ; and (2) in situ polymerization considering a medium containing both the lysozyme and the 3,4-ethylenedioxythiophene (EDOT) mono-

Chicken egg white lysozyme (also known as muramidase) exhibits antimicrobial activity against both Gram-negative and, especially, Gram-positive bacteria.1 This cationic enzyme (129 amino acids) is nonspecific and, in addition to egg white, it is also abundant in a number of human secretions such as tears, saliva, and mucus. Lysozyme has a two-domain structure. In the first domain, the polypeptide chain is folded forming β-sheets, while the second domain is dominated by the α-helix structure. The presence of four S−S bridges in the lysozyme molecule provides its high pH and thermal stability. Polar and nonpolar patches of amino acids are nonuniformly distributed along the surface of the molecule. The conformation of lysozyme is highly stable in the pH range from 4 to 11. Lysozyme produces damage on the bacterial cell walls by catalyzing the hydrolysis of the glycosidic bonds between N-acetylmuranic acid and Nacetylglucosamine residues in peptidoglycan. This mechanism of action results in a drastic bactericidal effect, which is used to impart protection against bacterial infections.2,3 Moreover, the lytic activity on the cell wall of a broad spectrum of microorganisms led to the of use lysozyme not only in medicine but also in alimentary, as a preservative of many food products (i.e., wine and cheese).4 © XXXX American Chemical Society

Received: December 14, 2012 Revised: February 14, 2013

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dx.doi.org/10.1021/jp3123117 | J. Phys. Chem. C XXXX, XXX, XXX−XXX

The Journal of Physical Chemistry C

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mer, the material obtained from such approach being denoted P(EDOT-LZ). Results indicated that adsorption of the enzyme at the surface of the polymer produces biphasic systems able to retain the properties of PEDOT, but they are not able to protect against bactericidal growth.12 In contrast, the addition of the enzyme to the polymerization medium resulted in a homogeneous composite with excellent bactericidal activity. Unfortunately, the electrochemical properties of P(EDOT-LZ) were relatively poor with respect to those of PEDOT,12 even though it should be mentioned that the characterization of such composite was very scarce. In this work we improve the electrical and electrochemical properties of P(EDOT-LZ) by optimizing the experimental conditions used in the anodic polymerization process. More specifically, the variables re-examined in this investigation are the oxidation potential, the polymerization time, and the EDOT:LZ ratio in the generation medium, which were 1.10 V, 600 s, and 1:7, respectively, in our previous work.12 The resulting material has been characterized at the morphological, electrical, and electrochemical levels. On the other hand, we recently used the electrochemical layer-by-layer technique to fabricate films formed by alternated layers of two different CPs through their electrodeposition.20−23 As these multilayered systems showed an excellent ability to store charge and a very high electrochemical stability, in this work we have prepared and characterized multilayered films formed by PEDOT and P(EDOT-LZ) layers. After this, the response of electroactive P(EDOT-LZ) toward different eukaryotic cellular lines has been investigated to examine the biocompatibility of the composite. The previously proved bactericidal properties of the composite (i.e., the new polymerization conditions are less aggressive than those previously used12 and, therefore, bactericidal activity is expected to be retained) combined with its high capacitive properties and remarkable biocompatibility suggest that P(EDOT-LZ) is a potential candidate for the fabrication of biocondensers. In order to focus on such application, the electrochemical and capacitive properties of P(EDOT-LZ) coated with cell monolayers have been investigated in a physiological medium and compared with those of PEDOT.

P(EDOT-LZ) were carried out under a constant potential of 1.15 V and using a polymerization time of 300 s. This potential, which is intermediate between the potentials optimized for acetonitrile (1.40 V)19 and water (1.10 V)24,25 generation media, has been found by cyclic voltammetry (CV) to be optimum for the acetonitrile:water (1:4 v/v) environment (see below). The layer-by-layer (LbL) electrodeposition procedure, which was detailed in previous studies,20,21 was used to prepare multilayered films made of alternating layers of PEDOT and P(EDOT-LZ). The potential and experimental conditions used to prepare the different layers of multilayered films were identical to those used for individual films. To prevent interferences during the electrochemical assays, the working and counter electrodes were cleaned with acetone before each trial. The reference electrode was an Ag|AgCl electrode containing a KCl saturated aqueous solution (offset potential versus the standard hydrogen electrode, E0 = 0.222 V at 25 °C), which was connected to the working compartment through a salt bridge containing the electrolyte solution. Thickness. The thickness of the PEDOT and P(EDOT-LZ) films was determined using electrochemical techniques. Specifically, the thickness was obtained by determining the current productivity through the mass-charge ratio and, subsequently, the mass of polymer deposited in the electrode. Details about this procedure were reported in previous works. 20,24 For selected cases, the thickness was also determined by measuring the cross-section of the films by scanning electron microscopy (SEM). Electrochemical and Electrical Properties. The electroactivity, which refers to the charge storage ability, and the electrochemical stability (electrostability) were determined by CV using an acetonitrile:water solution (1:4 v/v) with 0.1 M LiClO4. The initial and final potentials were −0.50 V, while the reversal potential was 1.00 V. Specifically, the electrostability was evaluated by determining the loss of electroactivity with the number of consecutive oxidation−reduction cycles (LEA, in %):

METHODS Materials. EDOT monomer and acetonitrile (analytical reagent grade) were purchased from Aldrich and used as received without further purification. Anhydrous LiClO4 (analytical reagent grade, Aldrich) was stored in an oven at 80 °C before use in electrochemical trials. Lysozyme (chicken egg white) was obtained from Sigma and used as received. Synthesis. PEDOT and P(EDOT-LZ) films were prepared potentiostatically on stainless steel electrodes through an Autolab PGSTAT302N equipped with the ECD module to measure very low current densities (100 mA to 100 pA). The polymerizations were performed using a standard threeelectrode system. Steel AISI 316 sheets of 4 and 2 cm2, for electrochemical and biological assays, respectively, were employed as working and counter electrodes. The generation medium used for the preparation of PEDOT films consisted of a 10 mM EDOT solution (0.071 g) in a mixture of acetonitrile and ultrapure Milli-Q water (pH = 7.0) solution (1:4 v/v) containing 0.1 M LiClO4 as the supporting electrolyte at room temperature. P(EDOT-LZ) films were obtained in the same generation medium but considering different EDOT:LZ ratios (see next section). Polymerizations of both PEDOT and

where ΔQ is the difference between the voltammetric charges (in C) of the second and the last oxidation−reduction cycle, and QII is the voltammetric charge corresponding to the second cycle. A scan rate of 100 mV·s−1 was employed in all cases. The specific capacitance (SC) of the active materials in the electrode was calculated as

LEA =

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SC =

ΔQ · 100 Q II

Q ΔV ·A

(1)

(2)

where Q is voltammetric charge, which is determined by integrating either the oxidative or the reductive parts of the cyclic voltammogram curve, ΔV is the potential window, and A is the area of polymer film on the surface of the working electrode. The LEA and SC were also determined using biological samples (i.e., PEDOT and 1:1 P(EDOT-LZ) films coated with living cells). These measures were carried out using a 0.1 M phosphate buffer saline solution (PBS; pH= 7.4) and considering a total number of eight oxidation−reduction cycles. Electrochemical impedance spectroscopy (EIS) diagrams were taken at open circuit (OCP) over the frequency range of 10 kHz to 10 mHz with a potential amplitude of 0.05 V using B



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washing in an alcohol battery (30°, 50°, 70°, 90°, 95° and 100°) at 4 °C for 30 min per wash. Finally, samples were airdried, and sputter-coated with carbon before SEM observation.

an AUTOLAB-302N potentiostat/galvanostat. These electrochemical assays were carried out in both a 0.1 M LiClO4 acetonitrile:water (1:4 v/v) solution and a 0.1 M PBS solution. The electrical conductivity (σ; in S·cm−1) of PEDOT and P(EDOT-LZ) films was determined by the four-probes procedure. Morphological Characterization. SEM micrographs were carried out using a Focused Ion Beam Zeiss Neon 40 scanning electron microscope operating at 3 kV and equipped with an energy dispersive X-ray (EDX) spectroscopy system. Dried samples were mounted on a double-sided adhesive carbon disc and sputter-coated with a thin layer of carbon to prevent sample charging problems. Tapping-mode atomic force microscopy (AFM) measurements were carried out with a Molecular Imaging PicoSPM using a NanoScope IIIa controlled in ambient conditions. The tapping mode AFM was operated at constant deflection and the row scanning frequency was set to 1 Hz and the physical tip− sample motion speed was 10 μm s−1. The scan window size was 5 × 5 μm2, with a total of 65536 topographic data points being computed in each image. The RMS roughness (r) was determined using the statistical application of the Nanoscope software, which calculates the average considering all the values recorded in the topographic image with exception of the maximum and the minimum. AFM measurements were performed on various parts of the films, which produced reproducible images similar to those displayed in this work. Cellular Adhesion. HEp-2 (human epidermoid cancer cell line), LLC-MK2 (rhesus monkey kidney epithelial cell line), MDCK (Madin-Darby canine kidney epithelial cell line) and Vero (African green monkey kidney epithelial cell line) cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin/ streptomycin, and 2 mM L-glutamine at 37 °C in a humidified atmosphere of 5% CO2 in air. The cultured media were changed every 2 days. For subculture, cell monolayers were rinsed with PBS and detached by incubating them with 0.25% trysin/EDTA for 5 min at 37 °C. Cells concentrations were determined by counting at the Newbauer camera using 4% trypan blue as dye vital. The detached cells were cultured following the conditions for the adhesion assays. PEDOT and P(EDOT-LZ) films deposited onto steel AISI 316 sheets of 1 cm2 were placed in plates of 24 wells and sterilized using UV irradiation for 15 min in a laminar flux cabinet. An aliquot of 50 uL containing 5 × 104 (cellular adhesion assays) or 2 × 105 cells (bioelectrochemical assays) was deposited on the substrate of each well. The plate was incubated under culture conditions for 60 min to promote the cell attachment to the film surface. Finally, 1 mL of the culture medium was added to each well. Controls of adhesion were simultaneously performed by culturing cells on the surface of the tissue culture polystyrene (TCPS) plates. Cell adhesion was evaluated after 24 h of culture, respectively, using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, which determines the cell viability.26 The viability results were normalized to TCPS control as relative percentages. Results were derived from the average of four replicates (n = 4) for each independent experiment. ANOVA and Turkey tests were performed to determine the statistical significance, which was considered at a confidence level of 95% (p < 0.05). Before the carbon coating for examination by SEM, samples covered with cells were fixed in a 2.5% glutaraldehyde PBS solution overnight at 4 °C. Then, they were dehydrated by

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RESULTS AND DISCUSSION Preparation of P(EDOT-LZ) and Electrochemical Characterization. P(EDOT-LZ) films were prepared by Table 1. Thickness of PEDOT (1:0) and P(EDOT-LZ) Films Obtained Using Different EDOT:LZ Ratios EDOT:LZ ratio

S (gravimetric; μm)

1:0

1:1

1:2

1:3

1:4

1:7

0.87

0.73

0.95

0.89

0.85

0.88

Figure 1. (a) Control voltammograms of PEDOT and P(EDOT-LZ) films in acetonitrile:water (1:4 v/v) with 0.1 M LiClO4. (b) Loss of electroactivity (LEA) against n, where n refers to the concentration of LZ in the generation medium (1:n EDOT:LZ ratio) used to prepare PEDOT (n = 0) and P(EDOT-LZ) films. (c) Variation of the specific capacitance (SC) against the number of consecutive oxidation− reduction cycles for PEDOT (circles) and 1:1 P(EDOT-LZ) (diamonds) in acetonitrile:water (1:4 v/v) with 0.1 M LiClO4.

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Figure 2. FTIR spectra in the range 1850−800 cm−1 of free lysozyme and 1:1 P(EDOT-LZ) after 50 consecutive oxidation−reduction cycles. Amides I and II are indicated by arrows.

Figure 3. (a) EIS spectra (Nyquist impedance plots) over a frequency of 100 kHz to 10 mHz of PEDOT and 1:1 P(EDOT-LZ) in acetonitrile:water (1:4 v/v) with 0.1 M LiClO4. (b) Equivalent circuit used to simulate the experimental spectra displayed in panel a.

anodic polymerization under a constant potential of 1.15 V in acetonitrile:water (1:4 v/v) using a polymerization time of 300 s. The concentration of EDOT monomer and supporting electrolyte (LiClO4) in the generation medium was 10 mM and 0.1 M, respectively, while the content of enzyme was optimized to achieve the maximum electrochemical stability. Thus, composites with different EDOT:LZ mass ratios (1:1, 1:2, 1:3, 1:4, and 1:7) in the generation media were prepared, their electrochemical stability after 50 consecutive oxidation− reduction cycles being subsequently evaluated. Table 1 lists the thickness of PEDOT and P(EDOT-LZ) films, which was determined by electrochemical measurements.

Figure 4. SEM micrographs of PEDOT, (a) surface and (b) crosssection, and 1:1 P(EDOT-LZ) (c) films prepared in acetonitrile:water (1:4 v/v) under a constant potential of 1.15 V and using a polymerization time of 300 s. Scale bar for high- and low-resolution images = 500 nm and 2 μm, respectively.

As it can be seen, the thickness of all films is close to ∼0.8−0.9 μm, independently of the EDOT:LZ ratio in the generation medium. Figure 1a compares the control voltammograms recorded for PEDOT and P(EDOT-LZ) films in acetonitrile:water solution (1:4 v/v) with 0.1 M LiClO4. The electro-

Table 2. Fitting Parameters Used to Simulate the EIS Data Obtained for PEDOT and 1:1 P(EDOT-LZ) in Acetonitrile:Water (1:4 v/v) with 0.1 M LiClO4 (See Figure 3) system

RS (Ω·cm2)

CPEDL (μF·cm−2·sn−1)

n

RCT (Ω·cm2)

W (Ω·cm−2)

CPS (mF·cm−2·sn−1)

PEDOT P(EDOT-LZ)

28.12 27.21

44.9 66.3

0.85 0.77

476.32 437.86

38.83 60.25

10.11 7.51

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Figure 7. Control voltammograms after (a) 2 and (b) 50 consecutive oxidation−reduction systems for individual PEDOT, PEDOT/P(EDOT-LZ)/PEDOT, and P(EDOT-LZ)/PEDOT/P(EDOT-LZ) films in acetonitrile:water (1:4 v/v). Figure 5. High-resolution SEM micrographs of the (a) section and the (b) interior of a cutted 1:1 P(EDOT-LZ). Scale bar = 100 nm.

Figure 8. Cellular adhesion on PEDOT and P(EDOT-LZ) using Hep2, LLC-MK2, MDCK, and Vero eukaryotic cell lines. The relative viability was established in relation to the TCPS control. p < 0.05 vs TCPS.

activity, which increases with the similarity between the anodic and cathodic areas of the first control voltammogram, is higher for PEDOT than for P(EDOT-LZ). The reduction, which has been calculated with respect to PEDOT, provoked by the enzyme is of 30%, 36%, 36%, 33% and 41% for the composite obtained from 1:1, 1:2, 1:3, 1:4 and 1:7 EDOT:LZ ratios, respectively. Figure 1b represents the variation of loss of electroactivity (LEA; eq 1) after 50 consecutive oxidation−reduction cycles against the concentration of LZ in the medium used to produce the P(EDOT-LZ) films. The electroactivity of PEDOT decreases 37% after 50 cycles, with P(EDOT-LZ) films derived from 1:2, 1:3 and 1:4 EDOT:LZ ratios showing similar reductions (i.e., 38−39%). However, the electrochemical

Figure 6. Height AFM images of (a) PEDOT and (b) 1:1 P(EDOTLZ) prepared in acetonitrile:water (1:4 v/v) under a constant potential of 1.15 V and using a polymerization time of 300 s.

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Figure 9. SEM micrograps of (a,c) LLC-MK2 and (b,d) Hep-2 cells cultured on the surface of (a,b) PEDOT and (c,d) P(EDOT-LZ). Cells were established onto PEDOT and P(EDOT-LZ) surfaces in confluent monolayers, their spreading being through fillopodia (arrows). The substrate surfaces (domains without cells) are marked with asterisks (*), while cell monolayers are indicated with letters (m). Scale bar = (a) 20 μm; (b) 100 μm (left), 50 μm (right, top), and 10 μm (right, bottom); (c) 20 and 10 μm (inset); and (d) 20 μm (left) and 10 μm (right).

Figure 10. Control voltammograms of (a) PEDOT and (b) P(EDOT-LZ) films uncovered and coated with Hep-2, LLC-MK2, MDCK, and Vero cells in PBS. Voltammograms after eight consecutive oxidation−reduction cycles of (c) PEDOT and (d) P(EDOT-LZ) films uncovered and coated with cells in PBS.

stability of P(EDOT-LZ) films obtained using 1:1 and 1:7 ratios is higher and lower, respectively, than that of PEDOT.

Specifically, the electroactivity of the composite obtained using the 1:1 ratio decreases by 19% only. According to a criterion F



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along 1000 consecutive oxidation−reduction cycles. As it was expected, results evidenced that the protecting role played by the enzyme in the composite disappears after such huge amount of cycles (i.e., LEA = 65% and 71% for 1:1 PEDOT and P(EDOT-LZ, respectively). Figure 1c represents the variation of the SC against the number of cycles. As it can been, the SC of 1:1 PEDOT and P(EDOT-LZ) decreases from 77 and 99 F/g, respectively, to 21 and 34 F/g after 1000 oxidation−reduction cycles. It is worth noting that, in spite of this reduction, the SC remains within the same order of magnitude after 1000 cycles. Moreover, assays based on degradation through oxidation−reduction processes induced by CV are considerably more aggressive than other type of electrochemical assays, as, for example, galvanostatic charge− discharge cycles. On the other hand, it should be mentioned that the initial SC could be preserved during more cycles by incorporating inorganic materials to the P(EDOT-LZ) matrix. Thus, for example excellent ultracapacitors with SC ranging from 198 to 375 F/g have been fabricated using nanocomposites of PEDOT and inorganic materials (e.g., MoO3, carbon nanotubes, MnO2, and NiFe2O4).27−30 Nevertheless, these inorganic materials would probably negatively affect the biological response of P(EDOT-LZ) (see below).

Table 3. Specific Capacitance (F/g) Determined by CV for Uncovered and Cell-Coated Films of PEDOT and 1:1 P(EDOT-LZ) in PBS coated matrix

uncovered

Hep-2

LLC-MK2

MDCK

Vero

PEDOT P(EDOT-LZ)

95 79

95 66

101 53

95 57

84 57

based on the electrochemical stability, P(EDOT-LZ) films obtained using the optimum 1:1 EDOT:LZ ratio have been used for subsequent analyses and application studies. Figure 2 shows the FTIR spectrum of 1:1 P(EDOT-LZ) in the 1700− 1400 cm−1 range after 50 cycles, the spectrum of the free enzyme also being included for comparison. As it can be seen, the amide I and II bands of lysozyme, which are detected at 1636 and 1517 cm−1, respectively, remain in the spectrum of P(EDOT-LZ), indicating that the enzyme is completely stable in the composite. These results complement our previous work12 in which the FTIR spectra of free lysozyme, PEDOT and P(EDOT-LZ) as prepared were extensively discussed. Additional analyses were carried out for PEDOT and 1:1 P(EDOT-LZ) by determining the variation of the LEA and SC

Figure 11. SEM micrograps of Hep-2 cells cultured on the surface of (a−c) PEDOT and (d−f) P(EDOT-LZ) after electrochemical assays. The arrows indicate the existence of fillopodia (in b), cellular vacuolization (in e) and the loss of fillopodia (in f). The substrate surfaces (domains without cells) are marked with asterisks (*), while cell monolayers are indicated with letters (m). Scale bar = (a) 20 μm; (b) 10 μm; (c) 50 μm; (d) 20 μm; (e) 10 μm; and (f) 10 μm. G



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P(EDOT-dextrin). The intimate contact between the enzyme and the CP molecules should result not only in the inhibition of the electrical conductivity but also in the promotion of very homogeneous composites (see next subsection). EIS was further employed to monitor the electrochemical behavior of 1:1 P(EDOT-LZ), which has been compared with that of PEDOT. Typical Nyquist diagrams are displayed in Figure 3a. The shape of the two impedance curves, which show two well-characterized regions, is very similar to those reported for PEDOT prepared by potentiostatic polymerization at 1.40 V using an acetonitrile solution containing 10 mM EDOT and 0.1 M LiClO4.22 A capacitive semicircle related with the polymer-electrolyte interface is observed at high frequencies, whereas a nearly vertical line is found in the low frequency region. The latter is due to the faradaic pseudocapacitance of the films, as was previously described for PEDOT.31,32 According to the shape of these spectra, at higher frequencies the process is charge-transfer controlled, while at lower frequencies the diffusion of charges in PEDOT and P(EDOT-LZ) films determines the impedance response. The intercept of the semicircle with the real axis (Z′) at high frequencies is the measure of the ohmic resistance (RS) between the working and the reference electrodes. The RS is 28 and 27 Ω·cm−2 for the PEDOT and 1:1 P(EDOT-LZ) films, respectively, these values being fully consistent with the electrical conductivities measured using the four-probes procedure. The RS values found in this work are at least 1 order of magnitude higher than those reported for PEDOT prepared using a constant potential of 1.40 V and an acetonitrile solution.22 This feature suggests that the experimental conditions used in this work result in less porous structures, as will be displayed in the next subsection. Thus, the lower porosity makes difficult the access and escape of dopant anions into the polymeric matrix, which is consistent with its higher resistance. The origin of the semicircle at the higherfrequency range, which is given by its diameters along the real axis Z′, is due to the ionic charge-transfer resistance at the filmelectrolyte interface (RCT) in parallel with the double layer capacitance (CDL). The RCT determined for PEDOT and P(EDOT-LZ) is 476 and 438 Ω·cm−2. These relatively high values should be also related with a loss of porosity with respect to that obtained in other conditions, which reduces the kinetics of the electron transfer through redox processes. EIS diagrams were analyzed using the equivalent circuit (EC) displayed in Figure 3b, which was previously proposed for CP films.32,33 The proposed EC is given by RS[CPEDL(RCTW)]CPS, where CPEDL is the double layer capacitance, W is the Warburg element in serial connection with RCT, and CPS is the faradic pseudocapacitance at lower frequencies. The double layer capacitance was replaced by a constant phase element (CPE)

Figure 12. EIS spectra (Nyquist impedance plots) over a frequency of 100 kHz to 10 mHz of uncovered and coated (a) PEDOT and (c) 1:1 P(EDOT-LZ) films in PBS. Films were coated with Hep-2, LLC-MK2, MDCK, and Vero cells. The EC used to fit the plots of all PEDOT films and uncovered 1:1 P(EDOT-LZ) is displayed in panel b, while panel d shows the EC used for the simulation of the coated 1:1 P(EDOT-LZ) films.

On the other hand, the electrical conductivity of PEDOT and all P(EDOT-LZ) films was ∼10−3 S·cm−1, independently of the EDOT:LZ ratio in the generation medium, indicating that the enzyme does not act in detriment of this property. Nevertheless, these values are several orders of magnitude smaller than that measured for biocomposites combining PEDOT with linear and cyclic dextrins (i.e., 1 and 3 S·cm−1, respectively).6 These P(EDOT-dextrin) composites were prepared using a constant potential of 1.10 V, the generation medium consisting of an ultrapure Milli-Q water solution containing 10 mM EDOT and 0.1 M LiClO4.6 These experimental conditions are very similar to those used in this work for P(EDOT-LZ), suggesting that the reduction of the electrical conductivity is due to the contact between the CP and the less electrically active biomolecule, which is greater in P(EDOT-LZ) than in

Table 4. Fitting Parameters Used to Simulate the EIS Data Obtained for Uncovered PEDOT and 1:1 P(EDOT-LZ), and PEDOT Coated with Hep-2, LLC-MK2, MDCK and Vero Cells in PBSa

a

system

RS (Ω·cm2)

CPEC (μF·cm−2·sn−1)

nC

RC (Ω·cm2)

CPEIL (mF·cm−2·sn−1)

nIL

RIL (Ω·cm2)

uncovered PEDOT PEDOT/Hep-2 PEDOT/LLC-MK2 PEDOT/MDCK PEDOT/Vero uncovered P(EDOT-LZ)

6.51 6.35 5.96 7.00 8.52 5.46

274 163 152 171 115 384

0.82 0.78 0.78 0.76 0.80 0.67

69.56 49.70 72.53 71.26 89.50 44.42

13.5 79.7 15.1 12.5 12.1 11.7

0.89 0.90 0.90 0.86 0.90 0.83

3.43 2.60 3.11 3.52 2.64 5.40

× × × × × ×

103 103 103 103 103 103

The EC used for the simulation is depicted in Figure 12b. H



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Table 5. Fitting Parameters Used to Simulate the EIS Data Obtained for 1:1 P(EDOT-LZ Coated with Hep-2, LLC-MK2, MDCK, and Vero Cells in PBSa

a

system

RS (Ω·cm2)

CPEC (μF·cm−2·sn−1)

nC

RC (Ω·cm2)

CPEIL (mF·cm−2·sn−1)

nIL

PEDOT/Hep-2 PEDOT/LLC-MK2 PEDOT/MDCK PEDOT/Vero

5.97 6.18 7.80 7.80

891 212 137 250

0.62 0.73 0.76 0.73

155.76 139.71 176.75 148.33

72.1 48.0 65.2 83.6

0.66 0.50 0.61 0.66

The EC used for the simulation is depicted in Figure 12d.

appear at the surface, interrupting the homogeneous structure of the film. The thickness of the PEDOT film, which is displayed in Figure 4b, was estimated to be close to ∼1 μm, in good agreement with the electrochemical estimation. Figure 4c evidence the remarkable influence of LZ in the morphology of PEDOT. Thus, the compact surface found for this CP transforms into a very porous morphology when such enzyme is added to the polymerization medium. The surface of 1:1 P(EDOT-LZ) looks like a superposition of compact aggregates with a cotton flake morphology, each of these elements showing a very smooth and homogeneous surface. The SEM micrographs displayed in Figure 4c clearly indicate that P(EDOT-LZ) is a homogeneous composite in which the CP and enzyme form a single phase. Unfortunately, the mechanical integrity of films with this particular morphology was not good enough to allow the estimation of the thickness by SEM. Figure 4c suggests that the electroactivity reduction provoked by the incorporation of the enzyme (Figure 1a) may be related with both its homogeneous dispersion into the polymeric matrix (i.e., the contact between the CP molecules, which are more active than the enzyme ones, is lower in P(EDOT-LZ) than in PEDOT) and the compact structure of the aggregates at the surfaces, which makes the movement of ions across the polymeric matrix difficult. The internal structure of the 1:1 P(EDOT-LZ) composite was investigated after cutting the aggregates with cotton flake morphology using focused ion beam (FIB) microscopy and subsequent examination of both the section and the interior by SEM. High-resolution SEM micrographs, which are displayed in Figure 5, reveal the existence of a hollow cavity (Figure 5a). Moreover, SEM micrographs of the interior of such cavities (Figure 5b) corroborated the homogeneity of the composite. Thus, the morphologies found at the surface and the hollow interior of the aggregate are identical, proving the intimate contact between the CP and the enzyme. AFM images of PEDOT and 1:1 P(EDOT-LZ), which are included in Figure 6, are consistent with SEM micrographs, reflecting significant differences between the two materials. The surface of PEDOT (Figure 6a) consists on dense distributions of sharp peaks forming leveled clusters, which are separated by deep ravines (i.e., channels). This heterogeneous distribution results in a roughness of r = 181 ± 36 nm. The AFM image of 1:1 P(EDOT-LZ) shows compact blocks of aggregated molecules that grow over plateau regions (Figure 6b). The latter should be associated to the holes formed by the crowding of the aggregates, which are clearly observed in the SEM micrograph (Figure 4c). The roughness of the 1:1 P(EDOTLZ) films (r = 411 ± 83 nm) is significantly higher than that of PEDOT, which is consistent with the morphological characteristics displayed by SEM. Morphological and electrochemical studies suggest that the mechanism of lysozyme immobilization into the PEDOT matrix is essentially due to electrostatic interactions. Thus, the

that describes a nonideal capacitor when the phase angle is different from −90°. The CPE impedance was expressed as ZCPE = [Q (j ·ω)n ]−1

(3)

The CPE represents a capacitor and a resistor for n = 1 and n = 0, respectively, while it is associated to a diffusion process when n = 0.5. The CPE impedance is attributed to the distributed surface reactivity, surface heterogeneity, and roughness of the current and potential distribution, which are, in turn, related with the electrode geometry and the electrode porosity.34 The Warburg impedance was included taking into account the diffusion phenomena.35 The proposed EC was selected considering the minimum number of circuit elements, which can be associated with physical phenomena that are probably taking place at the electrode surface. The quality of the fitting with experimental data was evaluated using the error percentage to each circuit element, with errors smaller than 5% being obtained in all cases. Table 2 shows the simulated values derived from the fitting of the EIS plots represented in Figure 3a to the EC displayed in Figure 3b. The CPE impedance is higher for 1:1 P(EDOT-LZ) than for PEDOT, indicating that the effective surface area for interfacial charge transport is highest for the former than for the latter. Moreover, it should be remarked that for both the PEDOT and 1:1 P(EDOT-LZ) systems, the imaginary part of the impedance Z″ at low frequencies is almost perpendicular to the real part Z′. This feature proves that the two materials show a very similar and good capacitive behavior, as is evidenced by the CPS pseudocapacitance associated with the practically vertical line at the low frequency region of the EIS spectra. This feature is consistent with the specific capacitances (SC; eq 2) determined by CV, which were 95 and 79 F/g for PEDOT and 1:1 P(EDOT-LZ), respectively. Surface Morphology. Figure 4 shows SEM micrographs of the typical morphologies found at the surface of PEDOT and 1:1 P(EDOT-LZ) films deposited on steel and produced using the experimental conditions described above. Both low and high magnification micrographs of PEDOT (Figure 4a) reveal a surface morphology that is significantly different than those reported when this CP is produced in acetonitrile under a constant potential of 1.40 V.6,12,36 Thus, PEDOT generated using the latter conditions shows a porous and spongy morphology in which irregular clusters of molecular aggregates connected by thin elements with a fiber-like shape are uniformly distributed in this surface, this appearance being independent of the thickness of the film (i.e., it was reported for polymerization times of 30,36 100,6 and 30012 s). In contrast, PEDOT obtained at a lower potential in a mixture of water and acetonitrile results in a compact structure formed by a dense aggregation of small shavings with sharp edges (Figure 4a). This produces not only an irregular morphology but also the disappearance of the many well-defined porous observed in acetonitrile. In contrast, a few thin but relatively large cracks I



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logical and bioelectrochemical studies were performed using individual P(EDOT-LZ) films. Cellular Adhesion on P(EDOT-LZ). The response of 1:1 P(EDOT-LZ) toward different eukaryotic cellular lines was compared with that of PEDOT. Cellular adhesion assays were performed considering Hep-2, LLC-MK2, MDCK, and Vero cells, which were selected because of their adherent growth and epithelial-like characteristics. Quantitative results of cellular adhesion assays are displayed in Figure 8, TCPS (or culture plate) being used as control substrate. As it can be seen, the number of adhered cells by area is higher onto PEDOT and P(EDOT-LZ) than onto the controls for the four cellular lines. PEDOT supports a large number of cells adhered to its surface as compared to the control. However, the behavior of P(EDOT-LZ) as supportive matrix for the adhesion of the four cellular lines is still better than that of PEDOT, especially for Hep-2 and LLC-MK2 lines, which should be attributed to specific benefits introduced by the enzyme. SEM micrographs displayed in Figure 9 show the characteristics of cells cultured onto the surface of PEDOT and P(EDOT-LZ) films. In general, there is a significant spreading of cells for the formation of cellular monolayers on the surface of the substrates. The connection sites between the cells and the surface of the films have been marked with arrows, with details about the stress fibers formed by the cells to move along the substrate also being displayed. The latter correspond to filaments for local adhesion, which are known as fillopodia (Figure 9b,d). The overall results presented in this section and the previous ones represent an important complement to those reported in our previous study.12 Thus, P(EDOT-LZ) is not only a composite with bactericidal activity, as was demonstrated previously,12 but also a material compatible with eukaryotic cells that shows both electroactive and capacitive properties similar to those of PEDOT. This feature is particularly relevant for the development of new biotechnological and biomedical applications. In the next section we describe the capabilities exhibited by P(EDOT-LZ) when it is used as a capacitor colonized by cells, mimicking a bioelectrochemical device for implants into animal or human bodies. Within this context, the exhibited antibacterical activity of P(EDOT-LZ)12 would be extremely useful to fight against bactericidal infections. Accordingly, we have investigated the electrochemical and capacitive properties of P(EDOT-LZ) coated with eukaryotic cellular monolayers. Toward a Bioelectrochemical Supercapacitor. The cyclic voltammograms of PEDOT and 1:1 P(EDOT-LZ) coated with Hep-2, LLC-MK2, MDCK, and Vero cells in PBS solution are compared in Figure 10, with voltammograms recorded for uncovered electroactive films being included for the sake of completeness. Comparison with the cyclic voltammograms recorded in acetonitrile:water (1:4 v/v) (Figure 1a) indicates that the electroactivity of the uncovered films is 17% higher in PBS. Moreover, the electroactivity in PBS is slightly higher (∼ 5%) for P(EDOT-LZ) than for PEDOT. However, adhesion of cells on surface of P(EDOT-LZ) produces a reduction of ∼25% in the electroactivity of the composite. This behavior is in opposition with that observed for PEDOT, with uncovered and coated films showing very similar electroactivities independently of the cellular line. On the other hand, Figure 10c,d compares the voltammogram recorded after eight consecutive oxidation−reduction cycles in PBS for PEDOT and 1:1 P(EDOT-LZ) films,

effective charge of lysozyme molecules in solution is expected to be crucial for interpreting the adsorption and deposition phenomena related with this protein. However, although the single phase morphology found for P(EDOT-LZ) suggests that the protein accompanies the counterions during the polymerization process, complete understanding of the mechanism of protein adsorption is not an easy task. Thus, the lysozyme binding is expected to depend on many parameters, such as the substrate topology, the charge and hydrophobicity of the matrix, and the pH and ionic strength of the environment. Within this context, recent FTIR results suggested the possible formation of hydrogen bonding interactions between the protein and the oxygen atoms of PEDOT chains, which was consistent with a reduction in the protein structural integrity.12 Multilayered Films. In recent studies we found that heterogeneous composites that alternate layers of two different CPs show better electrochemical properties and higher ability to store charge than the corresponding individual polymers.20−23 In order to investigate the performance of 1:1 P(EDOT-LZ)-containing multilayered systems with respect to individual PEDOT, PEDOT/P(EDOT-LZ)/PEDOT, and P(EDOT-LZ)/PEDOT/P(EDOT-LZ) three-layered films were prepared using the LbL electrodeposition technique. The two systems were prepared considering a polymerization time of 100 s per layer (i.e., the total polymerization time was 100 s × 3 layers = 300 s), the thickness of the resulting threelayered films (i.e., 0.9−1.0 μ) being very similar to those of individual PEDOT and 1:1 P(EDOT-LZ) films obtained using a polymerization time of 300 s (Table 1). Figure 7a compares the control voltammograms of PEDOT/P(EDOT-LZ)/ PEDOT and P(EDOT-LZ)/PEDOT/P(EDOT-LZ) with that of individual PEDOT. As it can be seen, the electroactivity of the former and latter three-layered systems is higher (∼19%) and lower (∼13%), respectively, than that of PEDOT. By similitude with previous studies on other PEDOT-containing multilayered systems,22−24 the improvement in the ability to store charge of PEDOT/P(EDOT-LZ)/PEDOT film should be attributed to the favorable interactions created in PEDOT layers by the interfaces with 1:1 P(EDOT-LZ). Instead, in P(EDOT-LZ)/PEDOT/P(EDOT-LZ) the coupling between the layers is not favorable enough to compensate the drawback associated to the homogeneous distribution of the enzyme, which is much less active than PEDOT from an electrochemical point of view, in the most external layer. Figure 7b displays the cyclic voltammograms of the two three-layered systems and PEDOT recorded after 50 consecutive oxidation−reduction cycles. As it can be seen, electrochemical degradation results in a loss electroactivity, even though this effect is higher in individual PEDOT than in the three-layered systems. Thus, the LEA determined for PEDOT, PEDOT/P(EDOT-LZ)/PEDOT, and P(EDOTLZ)/PEDOT/P(EDOT-LZ) is 33%, 24% and 23%, respectively. Although the P(EDOT-LZ)/PEDOT/P(EDOT-LZ) is the most electrostable multilayered system, it does not offer any advantage in terms of electroactivity with respect to the individual P(EDOT-LZ) films. Regarding PEDOT/P(EDOTLZ)/PEDOT, the enzyme is trapped in the intermediate layer and, therefore, a very scarce (if any) bactericidal activity should be expected from this heterogeneous composite. The enzyme is far from the surface and separated from it by the interface between layers. Accordingly, despite of the excellent electrochemical properties of PEDOT/P(EDOT-LZ)/PEDOT, bioJ



Article

Nyquist diagrams were fitted to different ECs. Specifically, the EIS plot of uncovered P(EDOT-LZ) was fit to the EC displayed in Figure 12b, which was also used for PEDOT. The simulated values, which have been included in Table 4, indicate a behavior in terms of resistance and capacitance very similar to that of uncovered and coated PEDOT films (Figure 12a). The EIS spectra of coated P(EDOT-LZ) was fitted to the EC displayed in Figure 12d, which is given by RS[CPEC(RC·CPEIL)], the resulting simulated values being listed in Table 5. As it can be seen, cellular monolayers provoke the existence of diffusion processes associated to the capacitors. Moreover, the resistance of the coatings increases by 1 order of magnitude with respect to the uncovered composite. These effects are induced by a decrease in the porosity, as is reflected by the reduction of the slope in long tail at the low frequency region. Thus, cells adhered to P(EDOT-LZ) form relatively compact interfaces affecting the penetration of ions from the PBS solution, which results in an increase of the resistance. This result, which is practically independent of the cellular line, is fully consistent with the fact that P(EDOT-LZ) behaves better than PEDOT as a supportive cellular matrix.

respectively, uncovered and coated with cells. As it can be seen, there is a significant reduction in the electroactivity with respect to the control voltammograms displayed in Figure 10a,b. The electrostability of coated PEDOT films (LEA = 16%, 11% 12%, 12% for Hep-2, LLC-MK2, MDCK, and Vero, respectively) is very similar to that of the uncovered one (LEA = 10%). Results displayed in Figure 10a,c allowed us to conclude that the electrochemical properties of this CP remain practically unaltered when films are coated by cell monolayers. This feature is corroborated by the specific capacitances, which are practically identical for uncovered and coated PEDOT films (Table 3). In contrast, the electrochemical stability of uncovered 1:1 P(EDOT-LZ) (LEA = 28%) is lower than those of composite films coated with cell monolayers (LEA = 18%, 21% 18%, 19% for Hep-2, LLC-MK2, MDCK, and Vero, respectively. These results should be related with the fact that the cellular viability was higher for P(EDOT-LZ) than for PEDOT (Figure 8), adhered cells promoting the exchange of ions in oxidation−reduction processes.18,37−39 Despite of this, the specific capacitances of coated P(EDOT-LZ) films were very similar to those of uncovered P(EDOT-LZ) and PEDOT, as is reflected in Table 3. Figure 11 displays SEM micrographs of Hep-2 cells cultured onto the surface of PEDOT and P(EDOT-LZ) films after eight consecutive oxidation−reduction cycles. The cellular monolayer grown on PEDOT surface retains the integrity after CV assays (Figures 11a and 10c). This is evidenced by the fillopodia displayed in Figure 11b, which shows the adhesion of the cells to the PEDOT substrate. In opposition, cells cultured onto P(EDOT-LZ) collapse after electrochemical assays (Figure 11d). This is reflected by the cytoplasm vacuolization (Figure 11e), which gives place to the appearance of a net of holes due to a cellular contraction, and the loss of fillopodia (Figure 11f). Figure 12 displays the EIS spectra recorded in PBS for PEDOT and 1:1 P(EDOT-LZ) coated with cells, which are compared with those obtained for the uncovered materials. The impedance diagrams of uncovered and all coated PEDOT samples (Figure 12a) show two time constants. The one at high frequency was attributed to the coating layer properties, while that at low frequencies was related to interfacial phenomena. The latter is affected by the presence of pores and defects on the coating layer, which allows the solution to reach the surface of the metallic substrate. The proposed EC is given by RS(RC·CPEC)(CPEIL·RIL) (Figure 12b), where CPEC and RC represent the capacitance and resistance of the coating (i.e., the uncovered film or the film covered with cells), respectively, and CPEIL and RIL correspond to the capacitance and resistance of the substrate/coating interface. Table 4 shows the simulated values derived from the fitting of the EIS plots represented in Figure 12a to the EC displayed in Figure 12b. The capacitive properties of uncovered PEDOT (CPEIL = 13.5 mF·cm−2 and n = 0.89) are similar or, in some cases, worse than those of PEDOT coated with cells (e.g., PEDOT coated with LLC-MK cells: CPEIL = 79.7 mF·cm−2 and n = 0.90) in PBS. Moreover, cultured cells do not affect appreciably the resistance of PEDOT. The RIL values, which are essentially associated with charge transfer processes, are considerably higher than the resistances of the coating and the electrolyte. In contrast, the impedance curves obtained in PBS for uncovered and coated P(EDOT-LZ) films (Figure 12c) evidence that the behavior of the composite is significantly affected by cellular monolayers. This is reflected by the fact that

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CONCLUSIONS

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AUTHOR INFORMATION

The experimental conditions used to prepare P(EDOT-LZ) has been optimized. The composite produced in acetonitrile:water (1:4 v/v) with 0.1 M LiClO4 using a potential of 1.15 V and a 1:1 EDOT:LZ mass ratio has been found to present excellent properties. Specifically, electrochemical assays in acetonitrile:water (1:4 v/v) with 0.1 M LiClO4 indicate that such composite shows higher electrochemical stability than PEDOT, both the electrical conductivity and specific capacitance being similar for the two materials. Moreover, experiments in PBS, which mimics a physiological environment, reveals higher electroactivity for 1:1 P(EDOT-LZ) than for PEDOT. On the other hand, the enzyme produces important morphological changes in PEDOT. Thus, P(EDOT-LZ) consists of a random superposition of hollow aggregates, in which the enzyme and the polymeric matrix organize form a single phase, with a cotton flake morphology. Adhesion assays and EIS measures using four different cellular lines have proved the biocompatibility and good behavior as cellular matrix of the composite. These results have shown to be promising on the potential of P(EDOT-LZ) to act as a bioelectrochemical supercapacitor useful for in vivo implants. Thus, the advantage of these biocomposite resides on the combination of the biocompatibility, electroactivity, and capacitive properties, which are in part intrinsic to the CP, with the bactericidal activity provided by the enzyme. Electrochemical and capacitive results obtained for P(EDOT-LZ) films coated with living cells represent a first step toward the development of new bioinspired organic devices for energy storage and their integration into biological systems.

Corresponding Author

*E-mail: [email protected]; Tel: +34 93 4010883. Notes

The authors declare no competing financial interest. K



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ACKNOWLEDGMENTS This work has been supported by MICINN and FEDER funds (MAT2012-34498) and by the DIUE of the Generalitat de Catalunya (contract number 2009SGR925). Support for the research of C.A. was received through the prize “ICREA Academia” for excellence in research funded by the Generalitat de Catalunya. D.L.-P. and D.A. are thanked for the financial support through FPI-UPC grants.

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Capacitive Composites Made of Conducting Polymer and Lysozyme

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