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Enzymatically synthesized polyaniline film deposition studied by simultaneous open circuit potential and electrochemical quartz crystal microbalance measurements
Journal of Colloid and Interface Science 369 (2012) 103–110
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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Enzymatically synthesized polyaniline film deposition studied by simultaneous open circuit potential and electrochemical quartz crystal microbalance measurements Norma Carrillo a, Ulises León-Silva a, Tatiana Avalos a, M.E. Nicho a, Sergio Serna a, Felipe Castillon b, Mario Farias b, Rodolfo Cruz-Silva c,⇑ a
Centro de Investigación en Ingeniería y Ciencias Aplicadas, UAEM, Av. Universidad, 1001, Col. Chamilpa, CP 62210 Cuernavaca, Morelos, Mexico Centro de Nanociencias y Nanotecnología de la UNAM, Apdo. Postal 2681 C.P. 22800, Ensenada, B.C., Mexico c Research Center for Exotic Nanocarbon (JST), Shinshu University, 4-17-1 Wakasato, Nagano 380-8553, Japan b
a r t i c l e
i n f o
Article history: Received 11 September 2011 Accepted 6 December 2011 Available online 16 December 2011 Keywords: Enzymatic polymerization Open circuit potential Quartz crystal microbalance Conducting polymers
a b s t r a c t The chemical and enzymatic deposition of polyaniline (PANI) films by in situ polymerization was studied and the resulting films were characterized. The film formation and polymerization processes were simultaneously monitored by the evolution of the open circuit potential and quartz-crystal microbalance measurements. Different substrates, such as Indium-Tin oxide electrodes and gold-coated quartz-crystal electrodes were used as substrates for PANI deposition. Electroactive PANI films were successfully deposited by in situ enzymatic polymerization at low oxidation potential. The electrogravimetric response of the enzymatically deposited PANI film was studied by cyclic voltammetry in monomer-free acidic medium. The morphology of the films was observed by scanning electron microscopy, revealing a granular structure in enzymatically deposited PANI. The PANI films were also characterized by thermogravimetric analysis, electrochemical impedance spectroscopy, and X-ray photoelectron and Fourier-transformed infrared spectroscopy. The simultaneous use of quartz crystal microbalance and open circuit potential is presented as a very useful technique to monitor enzymatic reactions involving oxidoreductases. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Polyaniline (PANI) is a conductive polymer that combines chemical stability, relatively high electrical conductivity and low-cost synthetic route [1]. These characteristics make this polymer one of the most promising for several applications, such as sensors [2], corrosion protection [3], and electronic devices [4]. Many of these applications require the deposition of PANI as films. There are several one-step methods to prepare PANI films by aniline polymerization, such as electrochemical [5], plasma [6] and in situ chemical [7–13] polymerization. Both electrochemical and plasma polymerization methods have major drawbacks that difficult their practical applications. For instance, the electrochemical method is restrained to electrically conducting substrates whereas the plasma polymerization requires a low-vacuum special environment to carry out the reaction. In contrast, in situ chemical polymerization is simple and has been extensively used to prepare PANI films adhered to a wide range of substrates. The main restriction is that the substrate must withstand the low-pH and highly oxidizing environment employed during the polymerization. Consequently, ⇑ Corresponding author. E-mail address: [email protected] (R. Cruz-Silva). 0021-9797/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2011.12.021
substrates that can be oxidized or dissolved in acidic conditions cannot be used during in situ chemical polymerization of aniline. Indeed, the strong oxidizing medium generated during the aniline polymerization makes this polymerization incompatible with delicate molecules, particularly those from biological origin, which can be destroyed by oxidation. Regarding the mechanism of film formation, most researchers agree with the mechanism proposed by Sapurina et al. [10] where the aniline autopolymerization plays a key role. Taking into account this, an aniline polymerization method mechanistically distinct from the chemical polymerization, may offer a way to compare and to study more deeply the formation of PANI films. In the last years, enzymatic oxidation of aniline has been used to prepare electrically conductive PANI [14–22]. This environmentally friendly polymerization method involves the use of a peroxidase enzyme as catalyst for aniline oxidation and hydrogen peroxide as oxidizer. The reaction conditions employed are milder than those employed in chemical synthesis. Another advantage of the enzymatic oxidation method is its high selectivity. Peroxidases reacts only with their substrate, in this case the aniline monomer, reducing the oxidation by-products and resulting in a very selective oxidation process. This has made possible the synthesis of PANI in presence of complex biomolecules, such as nucleic acids [17–19],
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without affecting drastically their structures. PANI films have been prepared with enzymatically synthesized PANI by using different approaches, such as polyelectrolyte immobilization on surfaces [14], enzyme immobilization [15], solution casting [16], and layer-by-layer self-assembly [22]. Nevertheless, the formation of PANI films by in situ enzymatic polymerization has not been studied, being a very easy and straightforward method to prepare PANI films. Among the several techniques that can be employed to monitor the film growth process of polyaniline, quartz crystal microbalance (QCM) stands out due to its high mass sensitivity, but unfortunately, it does not provide additional information about the deposited material. For this reason, QCM has often been combined with ellipsometry [23] and Fourier-transform infrared spectroscopy (FTIR) [24] or UV–Visible spectroscopy [25]. Open circuit potential measurement (OCP) has been used previously to monitor the polymerization stages in aniline chemical polymerization [26,27], giving information about the oxidation potential during the reaction. Very recently, Ramanaviciene et al. used QCM to study the adsorption of glucose oxidase to gold and the further polypyrrole formation [28]. Nevertheless, these OCP and QCM techniques have not been combined to study enzymatic oxidative polymerizations, and in fact, as far as we know there is only one report by Bressel et al. using simultaneous OCP and QCM to study biofilm growth [29]. In this work, we studied the PANI films grown by in situ enzymatic and chemical polymerization. The progress of the reactions was followed by simultaneous open-circuit potential evolution and quartz crystal microbalance measurements whereas the morphology was studied by scanning electron microscopy. Finally, the resulting PANI films were also characterized by cyclic voltagravimetry, electrochemical impedance spectroscopy (EIS), thermogravimetric analysis (TGA), FTIR, and X-ray photoelectron spectroscopy (XPS) techniques. 2. Experimental part 2.1. Materials Aniline was obtained from Aldrich and distilled at reduced pressure before use. Hydrogen peroxide (30 wt%), ammonium persulfate (ACS reagent grade), p-toluenesulfonic acid and ±10camphorsulfonic acid were acquired from Aldrich. Horseradish Peroxidase (HRP, 560 U/mg) was a product from Biochimica. Indium Tin-oxide electrodes (resistivity 8–12 X/sq) were purchased to Delta Technologies. All other reagents were of analytical grade and used as received. Gold coated AT-cut quartz crystals (5 MHz resonance frequency) were acquired from Stanford research systems. 2.2. Methods Aniline enzymatic polymerization was carried out at room temperature using a pH 4.0 potassium phthalate buffer solution. In a typical reaction, aniline was added to 20 mL of pH 4.0 buffer to reach a 50 mM concentration. In order to study the effect of doping agents, an equimolar amount relative to aniline of camphorsulfonic acid or toluenesulfonic acid was added to the reaction mixture Then, 2.0 mg of horseradish peroxidase were added. Immediately after peroxidase dissolution, hydrogen peroxide (3.0 wt%) was added drop wise until a 1:2 M ratio between hydrogen peroxide and aniline was reached. After 1 h of reaction under gentle stirring, the polyaniline-coated glass slides or ITO electrodes were rinsed with either water or ammonium hydroxide and dried at room temperature in a desiccator. PANI films were prepared also by in situ chemical polymerization of aniline, by mixing two solutions, the first one containing 0.4 mL of aniline (dissolved in 30 mL HCl
1.0 N) and the second one containing 1.37 g of ammonium persulfate (dissolved in 80 mL HCl 1.0 N). The films were deposited over a corning glass slide carefully cleaned with mild soap, acetone and water. 2.3. Characterization Electrochemical measurements were done in a BAS 100 EW potentiostat/galvanostat using a three-electrode cell, containing a Ag/AgCl electrode as reference electrode. During cyclic voltammetry, a platinum wire was used as counter electrode and either Indium-Tin oxide or gold electrodes coated with PANI by in situ enzymatic polymerization were used as working electrodes. A gold-coated quartz crystal was used as working electrode and substrate for PANI deposition during simultaneous open circuit potential and gravimetric measurements. Voltagravimetric studies were carried out using the same set-up, but instead of the polymerization medium, a 0.2 N HCl monomer-free electrolyte solution was used with a 20 mV/s scan rate. Additional cyclic voltagravimetry experiments were carried under the same conditions but using PANI films deposited onto ITO coated conductive glass as working electrode. Gravimetric deposition of the film on the gold coated surface was followed by using a quartz crystal microbalance QCM200 from Stanford Research Systems. The frequency and electrical resistance of the quartz crystal were recorded at 1.0 Hz. Sauerbrey equation [30] was used to transform the frequency shift into the gravimetric response using the physical constants provided by the quartz crystal manufacturer. Electrochemical impedance spectroscopy was carried out using a PGSTAT100/FRA2 potentiostat. The oscillation amplitude was set to 10 mV and the electrode was set to its open circuit potential, 0.2 N hydrochloric acid was used as electrolyte and the PANI films deposited onto ITO were used as working electrode. Thermogravimetric analyses were done in a TA Instruments Q500 equipment, using a 40 mL/min nitrogen flow and a 10 °C/min heating rate. SEM images of the polyaniline coated corning glass-slides were obtained on a Topcon 510 scanning electron microscope. X-ray photoelectron spectroscopy analysis was carried out on a modified laser ablation system, Riber LDM32. The X-ray Al Ka line at 1486.6 eV was used as X-ray source. The binding energies were calibrated with reference to Cu 2p3/2 at 932.67 eV and Ag 3d5/2 at 368.26 eV, respectively. The base pressure in the analysis chamber was approximately 10 10 Torr. Survey spectra were obtained by acquiring data every 1.0 eV with an energy resolution of 3.0 eV whereas high resolution spectra were collected by acquiring data every 0.2 eV with an energy resolution of 0.8 eV. Background was subtracted using the Tougaard method. Charging effect was corrected by shifting the binding energies considering the C 1s signal at 284.6 eV. Non-linear fit, using Gaussian curves was performed maintaining the Full-Width at Half-Maximum constant for all components in a particular spectrum. FTIR measurements of the PANI films were done in a Nicolet Magna 500 equipment using KBr pellets containing PANI scratched from the glass substrates. 3. Results and discussion 3.1. In situ polymerization and film formation We applied for the first time simultaneous OCP and QCM measurements to monitor the chemical and enzymatic polymerization of aniline, in order to analyse the oxidation stage by OCP and the film deposition by microgravimetric measurements at the same time. This was achieved by connecting the gold-coated quartzcrystal electrode from the QCM simultaneously to a potentiostat (Fig. 1). This set-up was also used during the cyclic voltagravimet-
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Fig. 1. Set-up for simultaneously monitoring in situ PANI polymerization and film deposition. (A) 3.0 M NaCl solution, (B) saline bridge, (C) reaction mixture, (D) goldcoated quartz crystal, (E) constant temperature bath, (W) working electrode, (C) counter electrode (Pt wire) (R) reference electrode (Ag/AgCl).
ric study of the enzymatically deposited PANI films in monomerfree electrolyte. The evolution of the OCP during chemical and enzymatic polymerization, along with the gravimetric response and the change in resistance of the quartz crystal versus time is plotted in Fig. 2. Wei et al. [26] divided the OCP change during the chemical polymerization into three stages, namely the induction, polymerization, and reduction stages. During the induction period of the chemically synthesized PANI, the reaction media gradually changed its color
Fig. 2. Simultaneous open circuit potential and quartz crystal microbalance measurements during the chemical (solid line) and enzymatic (dotted line) polymerization of aniline. The (a) resistance and (b) frequency shift of the quartz crystal, as well as it (c) open circuit potential (vs Ag/AgCl) plotted versus time is shown. In (c) vertical arrows indicate the peroxide addition during enzymatic polymerization.
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from transparent to slight blue. Fig. 2c shows that this stage started immediately after the oxidizer addition, (at t = 5 min), and took approximately 6 min. In this stage, the oxidation potential drastically increased after the ammonium persulfate addition from 0.25 V to 0.60 V, and then within a minute decreased to 0.55 V followed by a steady increment until it reached 0.75 V (at t = 11 min). In the second stage the polymerization took place, so the reaction media became dark green and the OCP was 0.75 V, due to the presence of persulfate ions. In Fig. 2b, it is shown the mass change of the electrode, which results from PANI film deposition. By comparing Fig. 2a and b we can conclude that during this stage the PANI films grew continuously during seven minutes. When the oxidizer was depleted, the reduction period is evidenced by a constant decrease of the OCP from 0.75 V to 0.5 V. In this stage, the pernigraniline was reduced to emeraldine [7]. On the other hand, aniline enzymatic polymerization revealed a different mechanism. In this case, after the first addition of hydrogen peroxide, the oxidation started immediately, as evidenced by the rapid change of color, from transparent to dark black. Horseradish peroxidase can quickly inactivate in presence of excess hydrogen peroxide, making the gradual addition the only way to add a stoichiometric amount of hydrogen peroxide to the reaction. However, Fig. 2c shows that after adding hydrogen peroxide, instead of increasing, there is a slight decrease in the OCP of the working electrode, in spite of the higher OCP of the peroxide solution. We believe that there is no increase in the OCP because the hydrogen peroxide is rapidly consumed by the enzyme. This is further supported by a control experiment that shows that after the first addition of hydrogen peroxide, the OCP reaches a steady value of 0.5 V in absence of the enzyme. As the polymerization proceeds, the enzyme activity decreases and after the third addition there is a moderate increment in the OCP (ca. 0.15 V) caused by the presence of the oxidizer in the solution. Nevertheless, in the enzymatic reaction the increment of the OCP is lower compared with that shown in the chemical reaction (ca. 0.5 V), most likely because hydrogen peroxide is added gradually allowing its consumption, and has a lower potential (1.8 V vs NHE) than ammonium persulfate (2.1 V vs NHE). This electrochemical study demonstrate the lower oxidation potential of the reaction media during the enzymatic oxidation of aniline, which is an interesting feature that enables its use of this polymerization mechanism even in the presence of complex biological molecules such as nucleic acids [17–19]. QCM was used to analyze the kinetics of PANI thin film deposition during enzymatic polymerization and the results are shown in Fig. 2b. Interestingly, the plot shows that even though the enzymatic oxidation of aniline does not show an induction period before oxidation, the deposition of the film did show an induction period of similar length to that shown during chemical polymerization. Induction period has been observed by QCM previously and is ascribed to the autocatalytic nature of the film formation [31]. In the enzymatic deposition, during the first six minutes there was no polymer deposition on the electrode, however, after the PANI starts to attach to the electrode, the frequency of the crystal decreased due to the mass gain and the OCP increased simultaneously. The induction period in the enzymatic polymerization should be the result of adsorption instead of autopolymerization, because the enzymatic polymerization is not autocatalytic. Simultaneous observation of the electrical resistance (Fig. 2a) and the frequency shift (Fig. 2b) of the quartz crystal electrode showed that there was no drastic change in the damping ability of the PANI film, since both curves show a similar trend. The amount of PANI deposited by enzymatic polymerization (12 lg/cm2) was slightly lower than the amount deposited by chemical polymerization (15 lg/ cm2), and very similar to other chemically synthesized films in the literature [32,33].
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3.2. Polyaniline film morphology The Fig. 3 shows the SEM images of the films formed under different reaction conditions. Fig. 3a and b is the PANI films obtained by enzymatic polymerization using either TSA or CSA as doping agents, respectively. In both cases the morphology of the films is granular, with some colloidal particles attached on their surfaces. Interestingly, some colloids in the sample prepared using CSA show a hollow structure, most likely induced by the camphorsulfonic, which has been previously used as template to prepare PANI nanotubes [31]. The average thickness measured by profilometry of PANI films prepared using TSA and CSA as doping agents, are 163 nm and 176 nm respectively (Supporting info., Fig. S1a and Fig. S1b, respectively). Films prepared by enzymatic polymerization are less mechanically stable, as evidenced by the crater-like
marks left by debonded colloids, and the morphology suggests that there is no brush ordering (Fig. 3a, black circles). For comparison purposes, Fig. 3c shows a chemically synthesized PANi film. We can see that the surface is rough and some PANI colloids are strongly attached to the film. Profilometry reveals that chemically synthesized PANI films are more uniform in thickness (Figure S1c) due to a morphology that results from a combination of aniline adsorption autocatalytic polymerization, and surface promoted polymerization with brush-like ordering [8,10,31]. In Fig. 4 we propose a different mechanism of film formation for the enzymatic polymerization pathway. Briefly, before the hydrogen peroxide addition some enzyme and aniline cations are adsorbed on the surface (Fig. 4a). After adding the hydrogen peroxide, polymerization starts in the solution, and it is highly likely that the PANI film grows from aggregation of particles formed in solution and later attached rather loosely to the surface by electrostatic adsorption and weak Van der Waal forces (Fig. 4b). Indeed, the formation of PANI colloidal particles has been reported by enzymatic polymerization even in absence of stabilizers [21]. This is a different behaviour compared to chemical polymerization, where the polymerization on the surface of the substrate occurs before the polymerization in solution [12]. Even though enzymatic polymerization of aniline is not autocatalytic, the adsorption and further polymerization of free radicals should contribute to bond these particles with each other and to the surface (Fig. 4c), although the mechanism results in films loosely attached to the surface. Direct oxidation of aniline with H2O2 is possible due to the high oxidation potential of hydrogen peroxide, but under acidic conditions and room temperature, the reaction rate is very slow. Under these conditions the low concentration of oxidized monomer favours the reaction with the solvent and dissolved oxygen, and consequently there is no significant polymer formation. Indeed, it has been reported that without a catalytic amount of metal salt, such as FeCl2, the bulk synthesis of PANI using H2O2 usually does not proceed or results in a PANI with very low yield and a high amount of structural defect [34,35]. One exception is when the H2O2 is produced in situ in low concentrations by an enzymatic method in near neutral media [21,36]. In our experiments, the non-enzymatic formation of PANI by H2O2 was ruled out by control experiments carried out without peroxidase. 3.3. Electrogravimetric behaviour of enzymatically synthesized PANI films
Fig. 3. Scanning electron microscopy of PANI films prepared by enzymatic polymerization using (a) toluenesulfonic acid as doping agent, and (b) camphorsulfonic acid as doping agent. Compare with (c) PANI films prepared chemical polymerization using hydrochloric acid as doping agent.
After the enzymatic deposition of the film the electrochemical response was analysed by cyclic voltagravimetry (Fig. 5). The enzymatically synthesized PANI film in presence of TSA showed two broad oxidation peaks at 300 mV and 500 mV during the anodic scan (Fig. 5a), indicating the transitions between different oxidation states of PANI, specifically, from leucoemeraldine to emeraldine, and from emeraldine to pernigraniline [13]. The respective reductions are shown during the cathodic scan, indicating that the oxidation is reversible. During the potential scan, the polymers undergo dedoping during the leucoemeraldine stage, and the loss of mass is reflected in the change of frequency (Fig. 5b) that corresponds to a mass of 0.5 lg/cm2. This is ascribed to the loss and uptake of chlorine anions during the doping–redoping process. The mass gain occurs between the emeraldine stage, at a potential between 300 mV and 500 mV, as expected. Sudden changes in the resistance (Rm) are due to changes in the energy dissipation of the quartz crystal [32,33], mainly because of changes in the damping capability of the deposited film, which in turn result from changes in the mechanical properties of the deposited PANI film. Nevertheless, in the enzymatically synthesized films the change in the resistance (Rm) of the quartz crystal is consistent with the mass
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Fig. 4. Schematic representation of the proposed mechanism for film formation during aniline enzymatic polymerization. (a) Reaction mixture before peroxide addition, (b) reaction mixture after peroxide addition, and (c) after polymerization.
Fig. 5. (a) Cyclic voltammetry, (b) electrogravimetry and (c) change in the quartz crystal resistance during potential cycling of enzymatically synthesized polyaniline films. Dotted arrows indicate the direction of potential cycling. 0.2 N hydrochloric acid was used as electrolyte.
changes (Fig. 5c), i.e. both curves have the same shape, indicating that there is no significant change in the elastic properties of the PANI film during the test. The lack of change in elastic properties of the enzymatically synthesized PANI film might result from its granular morphology, which impedes to behave as a continuous coating. Chemically synthesized PANI films showed also two oxidation peaks in the anodic scan (Fig. 5d), the first one at 200 mV, and the second one was interrupted by reversing the scan but started around 800 mV. Similarly to the enzymatically synthesized film, there is a mass gain in the anodic scan during the emeraldine stage, from 200 mV to 800 mV, which is reversible (Fig. 5e). Unlike the enzymatically synthesized PANI, the resistance is not concomitant
with the mass change, and apparently three transitions can be observed, the first one occurs at 200 mV during the anodic scan, and is associated with the stiffening of the film due to leucoemeraldine to emeraldine transition, and a softening of the film when the potential is close to 800 mV due to the emeraldine to pernigraniline transition. Finally, another stiffening of the film is observed around 600 mV in the reverse scan, and coincides with the pernigraniline to emeraldine transition observed in the voltammogram. Another characteristic feature of all runs is an apparent and gradual mass loss from the electrode with the number of cycles. However, this was observed also in the control run in bare electrodes and it is ascribed to slow dissolution of the exposed quartz in the acidic solution.
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3.4. Electrochemical impedance study of the enzymatically deposited PANI films Electrochemical impedance spectroscopy of the PANI films was carried out to study the AC electrochemical response of the enzymatically synthesized PANI films. Fig. 6a shows the electrical circuit equivalents used to fit the impedance data and Fig. 6b shows the EIS data in Nyquist impedance plots for the as obtained PANI-coated ITO electrodes compared with the bare ITO electrode. Whereas the ITO electrode can be fitted to an uniformly accessible non-ideal electrode [37], the PANI coated ITO electrodes displayed a more complex behaviour that can be represented by adding a resistor in parallel with a non-ideal capacitor representing the PANI film. In this model, RX represents the sum of the connections resistance and electrolyte resistance, and Cdl corresponds to the double layer capacitance of the electrode, and in the PANI coated ITO electrodes, RF is the Faradaic resistance and CF is a pseudocapacitance, both due to the electroactive PANI film. Constant phase elements were employed in all cases instead of ideal capacitors to account for the non-homogeneities in the system. Table 1 shows the values of the constants of the electrical models after the fitting. At high frequencies, a compressed semicircle indicates the electron transfer limited process, whereas the diffusion limited process is observed at lower frequency as a linear part of the data. Films enzymatically prepared in presence of CSA or TSA showed film resistances values lower to chemically synthesized films redoped with the same acids [38], which might result from a high permeability to the electrolyte, most likely due to the globular morphology and the pores seen in the SEM images.
Table 1 Values of resistance and capacitance of the electrical circuit elements for the ITO electrode, and the enzymatically synthesized PANI films prepared with toluenesulfonic acid or camphorsulfonic acid as doping agent.
RX Q1
a1 RF Q2
a2
TSA-PANI
CSA-PANI
ITO
51.04 1.55E 04 0.6 31.27 3.24E 04 0.79
33.63 5.00E 04 0.57 24.12 6.18E 04 0.84
75.47 3.88E 06 0.84 – – –
Resistance is in X and capacitances in (F cm2).
are shown in Fig. 7a–c. The sample is compared with chemically synthesized PANI film prepared using hydrochloric acid as doping agent (Fig. 7d–f). The silicon signals from the enzymatic sample (Fig. 7a) indicates that the film is not completely covering the whole surface, most likely due to the mechanical instability of the film and the grain like morphology of the film, as observed in the SEM. For the same reason, the C 1s to O 1s signal ratio for this film is lower than that of the chemically synthesized PANI (Fig. 7d). The C 1s signals for both samples (Fig. 7b and e) show the presence of surface species, such as CAO and OAC@O, which might result from overoxidation of the PANI and are typical of the enzymatically synthesized polymer [16]. On the other hand, the N 1s signals (Fig. 6c and f) show that most of the nitrogen is present in its amine form, with apparent doping levels (N+/N) of about 23% for both samples, and similar overall features.
3.5. Spectroscopic and thermal characterization of PANI films The XPS spectra of the PANI film prepared by enzymatic polymerization using toluenesulfonic as doping agent on a glass slide
Fig. 6. (a) The electrical circuit equivalents used to fit the experimental electrical impedance spectra. (b) Nyquist complex plots of the PANI films prepared in presence of toluenesulfonic and camphorsulfonic acid, compared with the response of the bare ITO electrode.
3.6. Thermogravimetric and Fourier transformed infrared analysis of the enzymatically synthesized PANI films The thermal stability of the enzymatically synthesized PANI has been shown to be dependent of the chemical structure, with more ordered structures resulting in higher thermal stability. Fig. 8 shows the thermogravimetric curve for the samples studied in this work. A great difference between the enzymatically and chemically synthesized PANI films was observed. The enzymatically synthesized samples of PANI had similar thermal stability regardless of the acid used during synthesis, but they were considerable less stable than the PANI film chemically synthesized. The difference is attributed to chain defects present in the polymer backbone [16]. The reason is the relatively high pH employed during the synthesis as well as the absence of templates to direct the structure of the polymer. The partially crosslinked structure model was supported by the FTIR analysis of the sample shown in Fig. 9. The main signals are due to PANI, such as the CAC stretching signals at 1588 cm 1 in the quinoid and at 1498 cm 1 in benzenoid units. The CAN@ and CANA stretching signals appears at 1373 cm 1 and 1292 cm 1, respectively. The in-plane (1180 cm 1) and out of plane (830 cm 1) bending of aromatic hydrogens are also observed [20]. On the other hand, a strong carbonyl stretching signal at 1692 cm 1 evidences the presence of phthalic acid either co-doping the polymer. Another signals due to this compound are the aromatic CAC stretching at 1551 cm 1, the out of plane due to ortho substituted aromatic rings at 700 cm 1. This later signal is very common in branched and ortho coupled PANI, and is evidence of the presence of chain defect on the polymer backbone. In addition, the several small peaks and shoulders indicate many kinds of couplings in the PANI structure [16] as opposed to the regular structure obtained by chemical polymerization.
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Fig. 7. (a) Wide scan spectrum, (b) C 1s, and (c) N 1s core-level spectra of polyaniline film deposited by in situ enzymatic polymerization using toluenesulfonic as doping agent. (d) Wide scan spectrum, (e) C 1s, and (f) N 1s core-level spectra of chemically synthesized polyaniline using hydrochloric acid as doping agent.
4. Conclusions
Fig. 8. Thermogravimetric analysis of enzymatically synthesized polyaniline prepared using toluenesulfonic (dashed line) and camphorsulfonic (dotted line) acids as doping agent. Compare with the high thermal stability of the chemically synthesized polyaniline (solid line).
Simultaneous QCM and OCP measurements can be used as a powerful tool to monitor chemical or enzymatic oxidative reactions involving film deposition, such as the aniline polymerization. Both techniques provide complementary information, i.e. while the QCM is sensitive to film deposition; the OCP of the electrode is related to oxidative chemical species present at the interface, even before film deposition. The set up presented here is a new electrochemical method for monitoring enzymatic polymerizations involving oxidoreductases. PANI films prepared by in situ enzymatic polymerization of aniline are very different to those obtained by in situ chemical polymerization of aniline, the structure is irregular, but on the other hand the reaction proceeds under very low oxidation potential. This low oxidation potential allows enzymatic polymerization to be carried out in presence of delicate molecules without affecting them. A mechanism for the enzymatic deposition of PANI was proposed. Acknowledgments Authors want to acknowledge Antonio Diaz (CNYN-UNAM) for his technical assistance in XPS measurements, Hector Barrientos for his help in profilometry, and E. Saucedo for the SEM technical assistance. Reviewers are acknowledged by their valuable suggestions. This research was partially supported by CONACYT through Grants J-50313 and PROMEP NPTC-151. R. Cruz-Silva acknowledges the support from the Exotic Nanocarbon Project, Japan Regional Innovation Strategy Program by the Excelence, JST. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jcis.2011.12.021. References
Fig. 9. Fourier transform infrared spectra of polyaniline films prepared by in situ enzymatic polymerization using (a) camphorsulfonic acid and (b) toluenesulfonic acid.
[1] [2] [3] [4]
A.G. MacDiarmid, Synth. Met. 125 (2001) 11–22. A. Talaie, Polymer 38 (1997) 1145–1150. W.K. Lu, R.L. Elsenbaumer, B. Wessling, Synth. Met. 71 (1995) 2163–2166. D. Chaudhuri, D.D. Sarma, Chem. Comm. 1 (2006) 2681–2683.
110
N. Carrillo et al. / Journal of Colloid and Interface Science 369 (2012) 103–110
[5] S.X. Xiong, P.T. Jia, K.Y. Mya, J. Ma, F. Boey, X.H. Lu, Electrochim. Acta 53 (2008) 3523–3530. [6] J.G. Wang, K.G. Neoh, L.P. Zhao, E.T. Kang, J. Colloid. Interace Sci. 251 (2002) 214–224. [7] J. Stejskal, I. Sapurina, J. Prokes, J. Zemek, Synth. Met. 105 (1999) 195–202. [8] A. Riede, M. Helmstedt, I. Sapurina, J. Stejskal, J Colloid Interface Sci. 248 (2002) 413–418. [9] A. Riede, M. Helmstedt, V. Riede, J. Zemek, J. Stejskal, Langmuir 16 (2000) 6240–6244. [10] I. Sapurina, A. Riede, J. Stejskal, Synth. Met. 123 (2001) 503–507. [11] I. Sapurina, A.Y. Osadchev, B.Z. Volchek, M. Trchova, A. Riede, J. Stejskal, Synth. Met. 129 (2002) 29–37. [12] I. Sedenkova, M. Trchova, N.V. Blinova, J. Stejskal, Thin Solid Films 515 (2006) 1640–1646. [13] A. Malinauskas, Polymer 42 (2001) 3957–3972. [14] S. Trakhtenberg, Y. Hangun-Balkir, J.C. Warner, F.F. Bruno, J. Kumar, R. Nagarajan, L.A. Samuelson, J. Am. Chem. Soc. 127 (2005) 9100–9104. [15] S. Alvarez, S. Manolache, F. Denes, J. Appl. Polym. Sci. 88 (2003) 369–379. [16] R. Cruz-Silva, J. Romero-Garcia, J.L. Angulo-Sanchez, E. Flores-Loyola, M.H. Farias, F.F. Castillon, J.A. Diaz, Polymer 45 (2004) 4711–4717. [17] Y.F. Ma, J.M. Zhang, G.J. Zhang, H.X. He, J. Am. Chem. Soc. 126 (2004) 7097– 7101. [18] Z.Q. Gao, S. Rafea, L.H. Lim, Adv. Mater. 19 (2007) 602–606. [19] P. Nickels, W.U. Dittmer, S. Beyer, J.P. Kotthaus, F.C. Simmel, Nanotechnology 15 (2004) 1524–1529. [20] M. Thiyagarajan, L.A. Samuelson, J. Kumar, A.L. Cholli, J. Am. Chem. Soc. 125 (2003) 11502–11503. [21] A. Kausaite, A. Ramanaviciene, A. Ramanavicius, Polymer 50 (2009) 1846– 1851.
[22] C. Espinosa-Gonzalez, I. Moggio, E. Arias-Marin, J. Romero-Garcia, R. CruzSilva, J. Le Moigne, J. Ortiz-Cisneros, J. Synth. Met. 139 (2003) 155–161. [23] J. Rishpon, A. Redondo, C. Derouin, S. Gottesfeld, J. Electroanal. Chem. 294 (1990) 73–85. [24] Q. Yang, Y. Zhang, H. Li, Y. Zhang, M. Liu, J. Luo, L. Tan, H. Tang, S. Yao, Talanta 81 (2010) 664–672. [25] L. Jiang, Q. Xie, L. Yang, X. Yang, S. Yao, J. Colloid Interface Sci. 274 (2004) 150– 158. [26] Y. Wei, K.S. Hsueh, G.W. Jang, Polymer 35 (1994) 3572–3575. [27] D. Li, R.B. Kaner, J. Am. Chem. Soc. 128 (2006) 968–975. [28] A. Ramanaviciene, A. Kausaite-Minkstimiene, Y. Oztekin, G. Carac, J. Voronovic, N. German, A. Ramanavicius, Microchim. Acta 175 (2011) 79–86. [29] A. Bressel, J.W. Schultze, W. Khan, G.M. Wolfaardt, H.-P. Rohns, R. Irmscher, M.J. Schöningd, Electrochim. Acta 48 (2003) 3363–3372. [30] G. Sauerbrey, Z. Physik 155 (1959) 206–222. [31] J. Stejskal, I. Sapurina, M. Trchová, Prog. Polym. Sci. 35 (2010) 1420–1481. [32] M.M. Ayad, M.A. Shenashin, Eur. Polym. J. 40 (2004) 197–202. [33] M.A. Shenashen, M.M. Ayad, N. Salahuddin, M.A. Youssif, React. Funct. Polym. 70 (2010) 843–848. [34] M. Bláha, M. Riesová, J. Zedník, A. Anzlovar, M. Zigon, J. Vohlidal, Synth. Met. 161 (2011) 1217–1225. [35] Y. Wang, X. Jing, J. Kong, Synth. Met. 157 (2007) 269–275. [36] A. Kausaite-Minkstimiene, V. Mazeiko, A. Ramanaviciene, A. Ramanavicius, Sens. Actuat. B 158 (2011) 278–285. [37] Mark E. Orazem, Bernard Tribollet, Electrochemical Impedance Spectroscopy, John Wiley & Sons, Hoboken, 2008. [38] Q. Hao, W. Lei, X. Xia, Z. Yan, X. Yang, L. Lu, X. Wang, Electrochim. Acta 55 (2010) 632–640.
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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Enzymatically synthesized polyaniline film deposition studied by simultaneous open circuit potential and electrochemical quartz crystal microbalance measurements Norma Carrillo a, Ulises León-Silva a, Tatiana Avalos a, M.E. Nicho a, Sergio Serna a, Felipe Castillon b, Mario Farias b, Rodolfo Cruz-Silva c,⇑ a
Centro de Investigación en Ingeniería y Ciencias Aplicadas, UAEM, Av. Universidad, 1001, Col. Chamilpa, CP 62210 Cuernavaca, Morelos, Mexico Centro de Nanociencias y Nanotecnología de la UNAM, Apdo. Postal 2681 C.P. 22800, Ensenada, B.C., Mexico c Research Center for Exotic Nanocarbon (JST), Shinshu University, 4-17-1 Wakasato, Nagano 380-8553, Japan b
a r t i c l e
i n f o
Article history: Received 11 September 2011 Accepted 6 December 2011 Available online 16 December 2011 Keywords: Enzymatic polymerization Open circuit potential Quartz crystal microbalance Conducting polymers
a b s t r a c t The chemical and enzymatic deposition of polyaniline (PANI) films by in situ polymerization was studied and the resulting films were characterized. The film formation and polymerization processes were simultaneously monitored by the evolution of the open circuit potential and quartz-crystal microbalance measurements. Different substrates, such as Indium-Tin oxide electrodes and gold-coated quartz-crystal electrodes were used as substrates for PANI deposition. Electroactive PANI films were successfully deposited by in situ enzymatic polymerization at low oxidation potential. The electrogravimetric response of the enzymatically deposited PANI film was studied by cyclic voltammetry in monomer-free acidic medium. The morphology of the films was observed by scanning electron microscopy, revealing a granular structure in enzymatically deposited PANI. The PANI films were also characterized by thermogravimetric analysis, electrochemical impedance spectroscopy, and X-ray photoelectron and Fourier-transformed infrared spectroscopy. The simultaneous use of quartz crystal microbalance and open circuit potential is presented as a very useful technique to monitor enzymatic reactions involving oxidoreductases. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Polyaniline (PANI) is a conductive polymer that combines chemical stability, relatively high electrical conductivity and low-cost synthetic route [1]. These characteristics make this polymer one of the most promising for several applications, such as sensors [2], corrosion protection [3], and electronic devices [4]. Many of these applications require the deposition of PANI as films. There are several one-step methods to prepare PANI films by aniline polymerization, such as electrochemical [5], plasma [6] and in situ chemical [7–13] polymerization. Both electrochemical and plasma polymerization methods have major drawbacks that difficult their practical applications. For instance, the electrochemical method is restrained to electrically conducting substrates whereas the plasma polymerization requires a low-vacuum special environment to carry out the reaction. In contrast, in situ chemical polymerization is simple and has been extensively used to prepare PANI films adhered to a wide range of substrates. The main restriction is that the substrate must withstand the low-pH and highly oxidizing environment employed during the polymerization. Consequently, ⇑ Corresponding author. E-mail address: [email protected] (R. Cruz-Silva). 0021-9797/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2011.12.021
substrates that can be oxidized or dissolved in acidic conditions cannot be used during in situ chemical polymerization of aniline. Indeed, the strong oxidizing medium generated during the aniline polymerization makes this polymerization incompatible with delicate molecules, particularly those from biological origin, which can be destroyed by oxidation. Regarding the mechanism of film formation, most researchers agree with the mechanism proposed by Sapurina et al. [10] where the aniline autopolymerization plays a key role. Taking into account this, an aniline polymerization method mechanistically distinct from the chemical polymerization, may offer a way to compare and to study more deeply the formation of PANI films. In the last years, enzymatic oxidation of aniline has been used to prepare electrically conductive PANI [14–22]. This environmentally friendly polymerization method involves the use of a peroxidase enzyme as catalyst for aniline oxidation and hydrogen peroxide as oxidizer. The reaction conditions employed are milder than those employed in chemical synthesis. Another advantage of the enzymatic oxidation method is its high selectivity. Peroxidases reacts only with their substrate, in this case the aniline monomer, reducing the oxidation by-products and resulting in a very selective oxidation process. This has made possible the synthesis of PANI in presence of complex biomolecules, such as nucleic acids [17–19],
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without affecting drastically their structures. PANI films have been prepared with enzymatically synthesized PANI by using different approaches, such as polyelectrolyte immobilization on surfaces [14], enzyme immobilization [15], solution casting [16], and layer-by-layer self-assembly [22]. Nevertheless, the formation of PANI films by in situ enzymatic polymerization has not been studied, being a very easy and straightforward method to prepare PANI films. Among the several techniques that can be employed to monitor the film growth process of polyaniline, quartz crystal microbalance (QCM) stands out due to its high mass sensitivity, but unfortunately, it does not provide additional information about the deposited material. For this reason, QCM has often been combined with ellipsometry [23] and Fourier-transform infrared spectroscopy (FTIR) [24] or UV–Visible spectroscopy [25]. Open circuit potential measurement (OCP) has been used previously to monitor the polymerization stages in aniline chemical polymerization [26,27], giving information about the oxidation potential during the reaction. Very recently, Ramanaviciene et al. used QCM to study the adsorption of glucose oxidase to gold and the further polypyrrole formation [28]. Nevertheless, these OCP and QCM techniques have not been combined to study enzymatic oxidative polymerizations, and in fact, as far as we know there is only one report by Bressel et al. using simultaneous OCP and QCM to study biofilm growth [29]. In this work, we studied the PANI films grown by in situ enzymatic and chemical polymerization. The progress of the reactions was followed by simultaneous open-circuit potential evolution and quartz crystal microbalance measurements whereas the morphology was studied by scanning electron microscopy. Finally, the resulting PANI films were also characterized by cyclic voltagravimetry, electrochemical impedance spectroscopy (EIS), thermogravimetric analysis (TGA), FTIR, and X-ray photoelectron spectroscopy (XPS) techniques. 2. Experimental part 2.1. Materials Aniline was obtained from Aldrich and distilled at reduced pressure before use. Hydrogen peroxide (30 wt%), ammonium persulfate (ACS reagent grade), p-toluenesulfonic acid and ±10camphorsulfonic acid were acquired from Aldrich. Horseradish Peroxidase (HRP, 560 U/mg) was a product from Biochimica. Indium Tin-oxide electrodes (resistivity 8–12 X/sq) were purchased to Delta Technologies. All other reagents were of analytical grade and used as received. Gold coated AT-cut quartz crystals (5 MHz resonance frequency) were acquired from Stanford research systems. 2.2. Methods Aniline enzymatic polymerization was carried out at room temperature using a pH 4.0 potassium phthalate buffer solution. In a typical reaction, aniline was added to 20 mL of pH 4.0 buffer to reach a 50 mM concentration. In order to study the effect of doping agents, an equimolar amount relative to aniline of camphorsulfonic acid or toluenesulfonic acid was added to the reaction mixture Then, 2.0 mg of horseradish peroxidase were added. Immediately after peroxidase dissolution, hydrogen peroxide (3.0 wt%) was added drop wise until a 1:2 M ratio between hydrogen peroxide and aniline was reached. After 1 h of reaction under gentle stirring, the polyaniline-coated glass slides or ITO electrodes were rinsed with either water or ammonium hydroxide and dried at room temperature in a desiccator. PANI films were prepared also by in situ chemical polymerization of aniline, by mixing two solutions, the first one containing 0.4 mL of aniline (dissolved in 30 mL HCl
1.0 N) and the second one containing 1.37 g of ammonium persulfate (dissolved in 80 mL HCl 1.0 N). The films were deposited over a corning glass slide carefully cleaned with mild soap, acetone and water. 2.3. Characterization Electrochemical measurements were done in a BAS 100 EW potentiostat/galvanostat using a three-electrode cell, containing a Ag/AgCl electrode as reference electrode. During cyclic voltammetry, a platinum wire was used as counter electrode and either Indium-Tin oxide or gold electrodes coated with PANI by in situ enzymatic polymerization were used as working electrodes. A gold-coated quartz crystal was used as working electrode and substrate for PANI deposition during simultaneous open circuit potential and gravimetric measurements. Voltagravimetric studies were carried out using the same set-up, but instead of the polymerization medium, a 0.2 N HCl monomer-free electrolyte solution was used with a 20 mV/s scan rate. Additional cyclic voltagravimetry experiments were carried under the same conditions but using PANI films deposited onto ITO coated conductive glass as working electrode. Gravimetric deposition of the film on the gold coated surface was followed by using a quartz crystal microbalance QCM200 from Stanford Research Systems. The frequency and electrical resistance of the quartz crystal were recorded at 1.0 Hz. Sauerbrey equation [30] was used to transform the frequency shift into the gravimetric response using the physical constants provided by the quartz crystal manufacturer. Electrochemical impedance spectroscopy was carried out using a PGSTAT100/FRA2 potentiostat. The oscillation amplitude was set to 10 mV and the electrode was set to its open circuit potential, 0.2 N hydrochloric acid was used as electrolyte and the PANI films deposited onto ITO were used as working electrode. Thermogravimetric analyses were done in a TA Instruments Q500 equipment, using a 40 mL/min nitrogen flow and a 10 °C/min heating rate. SEM images of the polyaniline coated corning glass-slides were obtained on a Topcon 510 scanning electron microscope. X-ray photoelectron spectroscopy analysis was carried out on a modified laser ablation system, Riber LDM32. The X-ray Al Ka line at 1486.6 eV was used as X-ray source. The binding energies were calibrated with reference to Cu 2p3/2 at 932.67 eV and Ag 3d5/2 at 368.26 eV, respectively. The base pressure in the analysis chamber was approximately 10 10 Torr. Survey spectra were obtained by acquiring data every 1.0 eV with an energy resolution of 3.0 eV whereas high resolution spectra were collected by acquiring data every 0.2 eV with an energy resolution of 0.8 eV. Background was subtracted using the Tougaard method. Charging effect was corrected by shifting the binding energies considering the C 1s signal at 284.6 eV. Non-linear fit, using Gaussian curves was performed maintaining the Full-Width at Half-Maximum constant for all components in a particular spectrum. FTIR measurements of the PANI films were done in a Nicolet Magna 500 equipment using KBr pellets containing PANI scratched from the glass substrates. 3. Results and discussion 3.1. In situ polymerization and film formation We applied for the first time simultaneous OCP and QCM measurements to monitor the chemical and enzymatic polymerization of aniline, in order to analyse the oxidation stage by OCP and the film deposition by microgravimetric measurements at the same time. This was achieved by connecting the gold-coated quartzcrystal electrode from the QCM simultaneously to a potentiostat (Fig. 1). This set-up was also used during the cyclic voltagravimet-
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Fig. 1. Set-up for simultaneously monitoring in situ PANI polymerization and film deposition. (A) 3.0 M NaCl solution, (B) saline bridge, (C) reaction mixture, (D) goldcoated quartz crystal, (E) constant temperature bath, (W) working electrode, (C) counter electrode (Pt wire) (R) reference electrode (Ag/AgCl).
ric study of the enzymatically deposited PANI films in monomerfree electrolyte. The evolution of the OCP during chemical and enzymatic polymerization, along with the gravimetric response and the change in resistance of the quartz crystal versus time is plotted in Fig. 2. Wei et al. [26] divided the OCP change during the chemical polymerization into three stages, namely the induction, polymerization, and reduction stages. During the induction period of the chemically synthesized PANI, the reaction media gradually changed its color
Fig. 2. Simultaneous open circuit potential and quartz crystal microbalance measurements during the chemical (solid line) and enzymatic (dotted line) polymerization of aniline. The (a) resistance and (b) frequency shift of the quartz crystal, as well as it (c) open circuit potential (vs Ag/AgCl) plotted versus time is shown. In (c) vertical arrows indicate the peroxide addition during enzymatic polymerization.
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from transparent to slight blue. Fig. 2c shows that this stage started immediately after the oxidizer addition, (at t = 5 min), and took approximately 6 min. In this stage, the oxidation potential drastically increased after the ammonium persulfate addition from 0.25 V to 0.60 V, and then within a minute decreased to 0.55 V followed by a steady increment until it reached 0.75 V (at t = 11 min). In the second stage the polymerization took place, so the reaction media became dark green and the OCP was 0.75 V, due to the presence of persulfate ions. In Fig. 2b, it is shown the mass change of the electrode, which results from PANI film deposition. By comparing Fig. 2a and b we can conclude that during this stage the PANI films grew continuously during seven minutes. When the oxidizer was depleted, the reduction period is evidenced by a constant decrease of the OCP from 0.75 V to 0.5 V. In this stage, the pernigraniline was reduced to emeraldine [7]. On the other hand, aniline enzymatic polymerization revealed a different mechanism. In this case, after the first addition of hydrogen peroxide, the oxidation started immediately, as evidenced by the rapid change of color, from transparent to dark black. Horseradish peroxidase can quickly inactivate in presence of excess hydrogen peroxide, making the gradual addition the only way to add a stoichiometric amount of hydrogen peroxide to the reaction. However, Fig. 2c shows that after adding hydrogen peroxide, instead of increasing, there is a slight decrease in the OCP of the working electrode, in spite of the higher OCP of the peroxide solution. We believe that there is no increase in the OCP because the hydrogen peroxide is rapidly consumed by the enzyme. This is further supported by a control experiment that shows that after the first addition of hydrogen peroxide, the OCP reaches a steady value of 0.5 V in absence of the enzyme. As the polymerization proceeds, the enzyme activity decreases and after the third addition there is a moderate increment in the OCP (ca. 0.15 V) caused by the presence of the oxidizer in the solution. Nevertheless, in the enzymatic reaction the increment of the OCP is lower compared with that shown in the chemical reaction (ca. 0.5 V), most likely because hydrogen peroxide is added gradually allowing its consumption, and has a lower potential (1.8 V vs NHE) than ammonium persulfate (2.1 V vs NHE). This electrochemical study demonstrate the lower oxidation potential of the reaction media during the enzymatic oxidation of aniline, which is an interesting feature that enables its use of this polymerization mechanism even in the presence of complex biological molecules such as nucleic acids [17–19]. QCM was used to analyze the kinetics of PANI thin film deposition during enzymatic polymerization and the results are shown in Fig. 2b. Interestingly, the plot shows that even though the enzymatic oxidation of aniline does not show an induction period before oxidation, the deposition of the film did show an induction period of similar length to that shown during chemical polymerization. Induction period has been observed by QCM previously and is ascribed to the autocatalytic nature of the film formation [31]. In the enzymatic deposition, during the first six minutes there was no polymer deposition on the electrode, however, after the PANI starts to attach to the electrode, the frequency of the crystal decreased due to the mass gain and the OCP increased simultaneously. The induction period in the enzymatic polymerization should be the result of adsorption instead of autopolymerization, because the enzymatic polymerization is not autocatalytic. Simultaneous observation of the electrical resistance (Fig. 2a) and the frequency shift (Fig. 2b) of the quartz crystal electrode showed that there was no drastic change in the damping ability of the PANI film, since both curves show a similar trend. The amount of PANI deposited by enzymatic polymerization (12 lg/cm2) was slightly lower than the amount deposited by chemical polymerization (15 lg/ cm2), and very similar to other chemically synthesized films in the literature [32,33].
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3.2. Polyaniline film morphology The Fig. 3 shows the SEM images of the films formed under different reaction conditions. Fig. 3a and b is the PANI films obtained by enzymatic polymerization using either TSA or CSA as doping agents, respectively. In both cases the morphology of the films is granular, with some colloidal particles attached on their surfaces. Interestingly, some colloids in the sample prepared using CSA show a hollow structure, most likely induced by the camphorsulfonic, which has been previously used as template to prepare PANI nanotubes [31]. The average thickness measured by profilometry of PANI films prepared using TSA and CSA as doping agents, are 163 nm and 176 nm respectively (Supporting info., Fig. S1a and Fig. S1b, respectively). Films prepared by enzymatic polymerization are less mechanically stable, as evidenced by the crater-like
marks left by debonded colloids, and the morphology suggests that there is no brush ordering (Fig. 3a, black circles). For comparison purposes, Fig. 3c shows a chemically synthesized PANi film. We can see that the surface is rough and some PANI colloids are strongly attached to the film. Profilometry reveals that chemically synthesized PANI films are more uniform in thickness (Figure S1c) due to a morphology that results from a combination of aniline adsorption autocatalytic polymerization, and surface promoted polymerization with brush-like ordering [8,10,31]. In Fig. 4 we propose a different mechanism of film formation for the enzymatic polymerization pathway. Briefly, before the hydrogen peroxide addition some enzyme and aniline cations are adsorbed on the surface (Fig. 4a). After adding the hydrogen peroxide, polymerization starts in the solution, and it is highly likely that the PANI film grows from aggregation of particles formed in solution and later attached rather loosely to the surface by electrostatic adsorption and weak Van der Waal forces (Fig. 4b). Indeed, the formation of PANI colloidal particles has been reported by enzymatic polymerization even in absence of stabilizers [21]. This is a different behaviour compared to chemical polymerization, where the polymerization on the surface of the substrate occurs before the polymerization in solution [12]. Even though enzymatic polymerization of aniline is not autocatalytic, the adsorption and further polymerization of free radicals should contribute to bond these particles with each other and to the surface (Fig. 4c), although the mechanism results in films loosely attached to the surface. Direct oxidation of aniline with H2O2 is possible due to the high oxidation potential of hydrogen peroxide, but under acidic conditions and room temperature, the reaction rate is very slow. Under these conditions the low concentration of oxidized monomer favours the reaction with the solvent and dissolved oxygen, and consequently there is no significant polymer formation. Indeed, it has been reported that without a catalytic amount of metal salt, such as FeCl2, the bulk synthesis of PANI using H2O2 usually does not proceed or results in a PANI with very low yield and a high amount of structural defect [34,35]. One exception is when the H2O2 is produced in situ in low concentrations by an enzymatic method in near neutral media [21,36]. In our experiments, the non-enzymatic formation of PANI by H2O2 was ruled out by control experiments carried out without peroxidase. 3.3. Electrogravimetric behaviour of enzymatically synthesized PANI films
Fig. 3. Scanning electron microscopy of PANI films prepared by enzymatic polymerization using (a) toluenesulfonic acid as doping agent, and (b) camphorsulfonic acid as doping agent. Compare with (c) PANI films prepared chemical polymerization using hydrochloric acid as doping agent.
After the enzymatic deposition of the film the electrochemical response was analysed by cyclic voltagravimetry (Fig. 5). The enzymatically synthesized PANI film in presence of TSA showed two broad oxidation peaks at 300 mV and 500 mV during the anodic scan (Fig. 5a), indicating the transitions between different oxidation states of PANI, specifically, from leucoemeraldine to emeraldine, and from emeraldine to pernigraniline [13]. The respective reductions are shown during the cathodic scan, indicating that the oxidation is reversible. During the potential scan, the polymers undergo dedoping during the leucoemeraldine stage, and the loss of mass is reflected in the change of frequency (Fig. 5b) that corresponds to a mass of 0.5 lg/cm2. This is ascribed to the loss and uptake of chlorine anions during the doping–redoping process. The mass gain occurs between the emeraldine stage, at a potential between 300 mV and 500 mV, as expected. Sudden changes in the resistance (Rm) are due to changes in the energy dissipation of the quartz crystal [32,33], mainly because of changes in the damping capability of the deposited film, which in turn result from changes in the mechanical properties of the deposited PANI film. Nevertheless, in the enzymatically synthesized films the change in the resistance (Rm) of the quartz crystal is consistent with the mass
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Fig. 4. Schematic representation of the proposed mechanism for film formation during aniline enzymatic polymerization. (a) Reaction mixture before peroxide addition, (b) reaction mixture after peroxide addition, and (c) after polymerization.
Fig. 5. (a) Cyclic voltammetry, (b) electrogravimetry and (c) change in the quartz crystal resistance during potential cycling of enzymatically synthesized polyaniline films. Dotted arrows indicate the direction of potential cycling. 0.2 N hydrochloric acid was used as electrolyte.
changes (Fig. 5c), i.e. both curves have the same shape, indicating that there is no significant change in the elastic properties of the PANI film during the test. The lack of change in elastic properties of the enzymatically synthesized PANI film might result from its granular morphology, which impedes to behave as a continuous coating. Chemically synthesized PANI films showed also two oxidation peaks in the anodic scan (Fig. 5d), the first one at 200 mV, and the second one was interrupted by reversing the scan but started around 800 mV. Similarly to the enzymatically synthesized film, there is a mass gain in the anodic scan during the emeraldine stage, from 200 mV to 800 mV, which is reversible (Fig. 5e). Unlike the enzymatically synthesized PANI, the resistance is not concomitant
with the mass change, and apparently three transitions can be observed, the first one occurs at 200 mV during the anodic scan, and is associated with the stiffening of the film due to leucoemeraldine to emeraldine transition, and a softening of the film when the potential is close to 800 mV due to the emeraldine to pernigraniline transition. Finally, another stiffening of the film is observed around 600 mV in the reverse scan, and coincides with the pernigraniline to emeraldine transition observed in the voltammogram. Another characteristic feature of all runs is an apparent and gradual mass loss from the electrode with the number of cycles. However, this was observed also in the control run in bare electrodes and it is ascribed to slow dissolution of the exposed quartz in the acidic solution.
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3.4. Electrochemical impedance study of the enzymatically deposited PANI films Electrochemical impedance spectroscopy of the PANI films was carried out to study the AC electrochemical response of the enzymatically synthesized PANI films. Fig. 6a shows the electrical circuit equivalents used to fit the impedance data and Fig. 6b shows the EIS data in Nyquist impedance plots for the as obtained PANI-coated ITO electrodes compared with the bare ITO electrode. Whereas the ITO electrode can be fitted to an uniformly accessible non-ideal electrode [37], the PANI coated ITO electrodes displayed a more complex behaviour that can be represented by adding a resistor in parallel with a non-ideal capacitor representing the PANI film. In this model, RX represents the sum of the connections resistance and electrolyte resistance, and Cdl corresponds to the double layer capacitance of the electrode, and in the PANI coated ITO electrodes, RF is the Faradaic resistance and CF is a pseudocapacitance, both due to the electroactive PANI film. Constant phase elements were employed in all cases instead of ideal capacitors to account for the non-homogeneities in the system. Table 1 shows the values of the constants of the electrical models after the fitting. At high frequencies, a compressed semicircle indicates the electron transfer limited process, whereas the diffusion limited process is observed at lower frequency as a linear part of the data. Films enzymatically prepared in presence of CSA or TSA showed film resistances values lower to chemically synthesized films redoped with the same acids [38], which might result from a high permeability to the electrolyte, most likely due to the globular morphology and the pores seen in the SEM images.
Table 1 Values of resistance and capacitance of the electrical circuit elements for the ITO electrode, and the enzymatically synthesized PANI films prepared with toluenesulfonic acid or camphorsulfonic acid as doping agent.
RX Q1
a1 RF Q2
a2
TSA-PANI
CSA-PANI
ITO
51.04 1.55E 04 0.6 31.27 3.24E 04 0.79
33.63 5.00E 04 0.57 24.12 6.18E 04 0.84
75.47 3.88E 06 0.84 – – –
Resistance is in X and capacitances in (F cm2).
are shown in Fig. 7a–c. The sample is compared with chemically synthesized PANI film prepared using hydrochloric acid as doping agent (Fig. 7d–f). The silicon signals from the enzymatic sample (Fig. 7a) indicates that the film is not completely covering the whole surface, most likely due to the mechanical instability of the film and the grain like morphology of the film, as observed in the SEM. For the same reason, the C 1s to O 1s signal ratio for this film is lower than that of the chemically synthesized PANI (Fig. 7d). The C 1s signals for both samples (Fig. 7b and e) show the presence of surface species, such as CAO and OAC@O, which might result from overoxidation of the PANI and are typical of the enzymatically synthesized polymer [16]. On the other hand, the N 1s signals (Fig. 6c and f) show that most of the nitrogen is present in its amine form, with apparent doping levels (N+/N) of about 23% for both samples, and similar overall features.
3.5. Spectroscopic and thermal characterization of PANI films The XPS spectra of the PANI film prepared by enzymatic polymerization using toluenesulfonic as doping agent on a glass slide
Fig. 6. (a) The electrical circuit equivalents used to fit the experimental electrical impedance spectra. (b) Nyquist complex plots of the PANI films prepared in presence of toluenesulfonic and camphorsulfonic acid, compared with the response of the bare ITO electrode.
3.6. Thermogravimetric and Fourier transformed infrared analysis of the enzymatically synthesized PANI films The thermal stability of the enzymatically synthesized PANI has been shown to be dependent of the chemical structure, with more ordered structures resulting in higher thermal stability. Fig. 8 shows the thermogravimetric curve for the samples studied in this work. A great difference between the enzymatically and chemically synthesized PANI films was observed. The enzymatically synthesized samples of PANI had similar thermal stability regardless of the acid used during synthesis, but they were considerable less stable than the PANI film chemically synthesized. The difference is attributed to chain defects present in the polymer backbone [16]. The reason is the relatively high pH employed during the synthesis as well as the absence of templates to direct the structure of the polymer. The partially crosslinked structure model was supported by the FTIR analysis of the sample shown in Fig. 9. The main signals are due to PANI, such as the CAC stretching signals at 1588 cm 1 in the quinoid and at 1498 cm 1 in benzenoid units. The CAN@ and CANA stretching signals appears at 1373 cm 1 and 1292 cm 1, respectively. The in-plane (1180 cm 1) and out of plane (830 cm 1) bending of aromatic hydrogens are also observed [20]. On the other hand, a strong carbonyl stretching signal at 1692 cm 1 evidences the presence of phthalic acid either co-doping the polymer. Another signals due to this compound are the aromatic CAC stretching at 1551 cm 1, the out of plane due to ortho substituted aromatic rings at 700 cm 1. This later signal is very common in branched and ortho coupled PANI, and is evidence of the presence of chain defect on the polymer backbone. In addition, the several small peaks and shoulders indicate many kinds of couplings in the PANI structure [16] as opposed to the regular structure obtained by chemical polymerization.
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Fig. 7. (a) Wide scan spectrum, (b) C 1s, and (c) N 1s core-level spectra of polyaniline film deposited by in situ enzymatic polymerization using toluenesulfonic as doping agent. (d) Wide scan spectrum, (e) C 1s, and (f) N 1s core-level spectra of chemically synthesized polyaniline using hydrochloric acid as doping agent.
4. Conclusions
Fig. 8. Thermogravimetric analysis of enzymatically synthesized polyaniline prepared using toluenesulfonic (dashed line) and camphorsulfonic (dotted line) acids as doping agent. Compare with the high thermal stability of the chemically synthesized polyaniline (solid line).
Simultaneous QCM and OCP measurements can be used as a powerful tool to monitor chemical or enzymatic oxidative reactions involving film deposition, such as the aniline polymerization. Both techniques provide complementary information, i.e. while the QCM is sensitive to film deposition; the OCP of the electrode is related to oxidative chemical species present at the interface, even before film deposition. The set up presented here is a new electrochemical method for monitoring enzymatic polymerizations involving oxidoreductases. PANI films prepared by in situ enzymatic polymerization of aniline are very different to those obtained by in situ chemical polymerization of aniline, the structure is irregular, but on the other hand the reaction proceeds under very low oxidation potential. This low oxidation potential allows enzymatic polymerization to be carried out in presence of delicate molecules without affecting them. A mechanism for the enzymatic deposition of PANI was proposed. Acknowledgments Authors want to acknowledge Antonio Diaz (CNYN-UNAM) for his technical assistance in XPS measurements, Hector Barrientos for his help in profilometry, and E. Saucedo for the SEM technical assistance. Reviewers are acknowledged by their valuable suggestions. This research was partially supported by CONACYT through Grants J-50313 and PROMEP NPTC-151. R. Cruz-Silva acknowledges the support from the Exotic Nanocarbon Project, Japan Regional Innovation Strategy Program by the Excelence, JST. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jcis.2011.12.021. References
Fig. 9. Fourier transform infrared spectra of polyaniline films prepared by in situ enzymatic polymerization using (a) camphorsulfonic acid and (b) toluenesulfonic acid.
[1] [2] [3] [4]
A.G. MacDiarmid, Synth. Met. 125 (2001) 11–22. A. Talaie, Polymer 38 (1997) 1145–1150. W.K. Lu, R.L. Elsenbaumer, B. Wessling, Synth. Met. 71 (1995) 2163–2166. D. Chaudhuri, D.D. Sarma, Chem. Comm. 1 (2006) 2681–2683.
110
N. Carrillo et al. / Journal of Colloid and Interface Science 369 (2012) 103–110
[5] S.X. Xiong, P.T. Jia, K.Y. Mya, J. Ma, F. Boey, X.H. Lu, Electrochim. Acta 53 (2008) 3523–3530. [6] J.G. Wang, K.G. Neoh, L.P. Zhao, E.T. Kang, J. Colloid. Interace Sci. 251 (2002) 214–224. [7] J. Stejskal, I. Sapurina, J. Prokes, J. Zemek, Synth. Met. 105 (1999) 195–202. [8] A. Riede, M. Helmstedt, I. Sapurina, J. Stejskal, J Colloid Interface Sci. 248 (2002) 413–418. [9] A. Riede, M. Helmstedt, V. Riede, J. Zemek, J. Stejskal, Langmuir 16 (2000) 6240–6244. [10] I. Sapurina, A. Riede, J. Stejskal, Synth. Met. 123 (2001) 503–507. [11] I. Sapurina, A.Y. Osadchev, B.Z. Volchek, M. Trchova, A. Riede, J. Stejskal, Synth. Met. 129 (2002) 29–37. [12] I. Sedenkova, M. Trchova, N.V. Blinova, J. Stejskal, Thin Solid Films 515 (2006) 1640–1646. [13] A. Malinauskas, Polymer 42 (2001) 3957–3972. [14] S. Trakhtenberg, Y. Hangun-Balkir, J.C. Warner, F.F. Bruno, J. Kumar, R. Nagarajan, L.A. Samuelson, J. Am. Chem. Soc. 127 (2005) 9100–9104. [15] S. Alvarez, S. Manolache, F. Denes, J. Appl. Polym. Sci. 88 (2003) 369–379. [16] R. Cruz-Silva, J. Romero-Garcia, J.L. Angulo-Sanchez, E. Flores-Loyola, M.H. Farias, F.F. Castillon, J.A. Diaz, Polymer 45 (2004) 4711–4717. [17] Y.F. Ma, J.M. Zhang, G.J. Zhang, H.X. He, J. Am. Chem. Soc. 126 (2004) 7097– 7101. [18] Z.Q. Gao, S. Rafea, L.H. Lim, Adv. Mater. 19 (2007) 602–606. [19] P. Nickels, W.U. Dittmer, S. Beyer, J.P. Kotthaus, F.C. Simmel, Nanotechnology 15 (2004) 1524–1529. [20] M. Thiyagarajan, L.A. Samuelson, J. Kumar, A.L. Cholli, J. Am. Chem. Soc. 125 (2003) 11502–11503. [21] A. Kausaite, A. Ramanaviciene, A. Ramanavicius, Polymer 50 (2009) 1846– 1851.
[22] C. Espinosa-Gonzalez, I. Moggio, E. Arias-Marin, J. Romero-Garcia, R. CruzSilva, J. Le Moigne, J. Ortiz-Cisneros, J. Synth. Met. 139 (2003) 155–161. [23] J. Rishpon, A. Redondo, C. Derouin, S. Gottesfeld, J. Electroanal. Chem. 294 (1990) 73–85. [24] Q. Yang, Y. Zhang, H. Li, Y. Zhang, M. Liu, J. Luo, L. Tan, H. Tang, S. Yao, Talanta 81 (2010) 664–672. [25] L. Jiang, Q. Xie, L. Yang, X. Yang, S. Yao, J. Colloid Interface Sci. 274 (2004) 150– 158. [26] Y. Wei, K.S. Hsueh, G.W. Jang, Polymer 35 (1994) 3572–3575. [27] D. Li, R.B. Kaner, J. Am. Chem. Soc. 128 (2006) 968–975. [28] A. Ramanaviciene, A. Kausaite-Minkstimiene, Y. Oztekin, G. Carac, J. Voronovic, N. German, A. Ramanavicius, Microchim. Acta 175 (2011) 79–86. [29] A. Bressel, J.W. Schultze, W. Khan, G.M. Wolfaardt, H.-P. Rohns, R. Irmscher, M.J. Schöningd, Electrochim. Acta 48 (2003) 3363–3372. [30] G. Sauerbrey, Z. Physik 155 (1959) 206–222. [31] J. Stejskal, I. Sapurina, M. Trchová, Prog. Polym. Sci. 35 (2010) 1420–1481. [32] M.M. Ayad, M.A. Shenashin, Eur. Polym. J. 40 (2004) 197–202. [33] M.A. Shenashen, M.M. Ayad, N. Salahuddin, M.A. Youssif, React. Funct. Polym. 70 (2010) 843–848. [34] M. Bláha, M. Riesová, J. Zedník, A. Anzlovar, M. Zigon, J. Vohlidal, Synth. Met. 161 (2011) 1217–1225. [35] Y. Wang, X. Jing, J. Kong, Synth. Met. 157 (2007) 269–275. [36] A. Kausaite-Minkstimiene, V. Mazeiko, A. Ramanaviciene, A. Ramanavicius, Sens. Actuat. B 158 (2011) 278–285. [37] Mark E. Orazem, Bernard Tribollet, Electrochemical Impedance Spectroscopy, John Wiley & Sons, Hoboken, 2008. [38] Q. Hao, W. Lei, X. Xia, Z. Yan, X. Yang, L. Lu, X. Wang, Electrochim. Acta 55 (2010) 632–640.