AOT vesicles as templates for the horseradish peroxidase-triggered polymerization of aniline

Abstract

In dilute aqueous solution at pH = 4.3 in the presence of 0.1 M sodium dihydrogen phosphate, AOT (bis-(2-ethylhexyl)sulfosuccinate) was found to form vesicles. The average diameter of the vesicles was adjusted to about 70 nm by polycarbonate membrane extrusion. The vesicles were applied as chemical structure-controlling templates for the horseradish peroxidase/H₂O₂-triggered polymerization of aniline to yield the green emeraldine salt form of polyaniline. The enzyme-containing vesicular reaction system was optimized with respect to obtaining a reaction product with high absorbance in the NIR region of the spectrum which is known to be a characteristic property of the conductive emeraldine salt form of polyaniline. The reaction system was analyzed by cryo transmission electron microscopy, ¹H NMR, UV/VIS/NIR, circular dichroism and fluorescence measurements. The peroxidase was found to be bound to the vesicles leading to an initiation of the reaction preferentially on the vesicles surface and not in the bulk aqueous solution. Before the reaction was started by H₂O₂ addition, the anilinium cations were found to only weakly interact with the surface of the vesicles. After polymerization, a stable suspension containing vesicles which were coated with polyaniline was obtained. The reaction product was isolated and analyzed by FTIR measurements. With respect to the vesicle system used previously, SDBS/decanoic acid (1 : 1) (Z. Guo, H. Rüegger, R. Kissner, T. Ishikawa, M. Willeke and P. Walde, Langmuir, 2009, 25, 11390–11405), the AOT system has several advantages for further explorations of this type of in situ formation of conductive vesicle-based polymer capsules.

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1. Introdution

The oxidative polymerization of aniline can lead to a number of different polymeric products which may not only differ in the degree of oxidation and protonation but also in the level of branching, on the nature of the chemical bond between the aniline monomers and on the morphology of the polymerization products obtained.¹ With respect to the polymerization conditions, the chemical structure of the polyaniline (PANI) obtained, its morphology and physico-chemical properties depend on many factors. These include the type of oxidant used, the concentrations of aniline and oxidant and their molar ratio, the temperature and acidity.² If H₂O₂ and the enzyme horseradish peroxidase (HRP) are used as oxidants, the green electrically conductive emeraldine salt form of PANI can easily be obtained at room temperature under mild, environmentally friendly conditions in slightly acidic aqueous medium (pH = 4.3).³ One of the key requirements is, however, that an appropriate chemical structure-controlling template⁴ is added, as originally demonstrated by Samuelson and coworkers.⁵

The template can be a negatively charged polymer (particularly poly(styrene sulfonate)),^5–7 negatively charged micelles,^6,8 negatively charged vesicles,⁹ or even negatively charged inorganic particles.¹⁰ It seems that the template plays several roles. The template may concentrate and pre-orient the positively charged anilinium ions, possibly via N–H⋯O–S hydrogen bonds, so that the polymerization reaction is regioselective in the sense that the monomers are predominantly added at the para-position with respect to the amino group of aniline. This regioselective coupling determines the amount of linear para-directed units formed as compared to ortho-directed units. Other roles of the template are as efficient counter ion (dopant), most likely again via N–H⋯O–S hydrogen bonds,¹¹ and as complexing agent for the polymerization products to increase the dispersability of the otherwise insoluble PANI.

If the reaction conditions are not optimized, the enzymatic polymerization product obtained is not the desired emeraldine salt (Scheme 1), as can be determined conveniently by UV/VIS/NIR spectroscopy.^12,13 Absorption bands in the NIR region of the spectrum (at λ > 750 nm) are typical for the conductive emeraldine salt form of PANI,^12–17 while deprotonation (to form the emeraldine base) or overoxidation (to obtain the pernigraniline form) leads to a blue shift with absorption maxima at ∼650 nm (for the emeraldine base)¹⁸ and ∼750 nm (pernigraniline salt).⁹ In the case of the emeraldine salt form of PANI (Scheme 1), the absorption maximum in the NIR region seems to strongly depend on the chain conformation which influences the conjugation length.¹⁹ “Compact coils” lead to a localized absorption near 750 nm,^19,20 while “expanded coils” show broad absorption bands with maximum absorption centered at around 1000 nm or at even higher wavelengths.²² The fully reduced form of PANI (leucoemeraldine) has absorption maxima below 400 nm.^10,12 Furthermore, branched ortho-directed PANI has strong absorption around 550 nm with negligible absorption in the region of 1000 nm.¹³ Based on this strong chemical structure and conformation dependence of the VIS/NIR spectrum of PANI, absorption measurements are often used as convenient and simple tools for optimizing aniline polymerization conditions. Unfortunately, the low solubility of the emeraldine salt form of PANI makes the determination of the molar mass by size exclusion chromatography and determination of the chemical structure by solution NMR difficult. Furthermore, the (paramagnetic) unpaired electrons expected to be present in the polaron state of the conductive emeraldine salt form of PANI (Scheme 1) hinder NMR measurements even if oligomeric products would be soluble. Often, infrared absorption measurements are used for a confirmation of the presence of the expected structural units in the PANI synthesized (for example benzenoid units show a characteristic absorption at about 1500 cm⁻¹ and quinoid units at about 1590 cm⁻¹ in the emeraldine base form),^13,23 although the assignment of all peaks is not always possible unequivocally.²⁴

Scheme 1 Emeraldine salt form of polyaniline with its bipolaron and polaron states.^1f

Recently, we have studied the HRP/H₂O₂-catalyzed polymerization of aniline in the presence of SDBS/decanoic acid (1 : 1, mole ratio) vesicles,⁹ and we found that this type of vesicles is a promising surfactant template since the enzyme was more stable as compared to the enzyme stability in the presence of corresponding micellar templates that were used previously.^6,8a Furthermore, we proposed that the localization of the enzyme on the surface of the vesicular templates may be essential for obtaining preferentially the para-linked, linear emeraldine salt form of PANI at pH = 4.3 from the very beginning of the reaction.⁹ In addition, we carried out NOESY NMR measurements of the vesicle system before polymerization since we were interested in gaining information about the possible association of aniline with the vesicle template before the reaction was initiated. Drawbacks of using SDBS, however, were (i) the presence of an aromatic ring in this surfactant which gave broad aromatic signals that overlapped with the signals of the aromatic aniline protons and (ii) the fact that commercial SDBS is actually a mixture of surfactants.⁹ Furthermore, below a temperature of about 10 °C, SDBS/decanoic acid (1 : 1) vesicle precipitation was observed since the phase transition temperature of this surfactant mixture was found to be at 9 °C.⁹ This was another clear drawback which hindered carrying out HRP-catalyzed polymerization reactions in the presence of the vesicles at low temperature. Template-assisted polymerization reactions at low temperature may be advantageous since they may lead to more uniform polymerization products due to a decreased aniline mobility on the vesicle surface as compared to 25 °C, i.e. a more ordered aniline pre-orientation before polymerization is started.

In this work we have used vesicles from AOT, sodium bis-(2-ethylhexyl)sulfosuccinate (Scheme 2), which do not have the mentioned drawbacks of the SDBS/decanoic acid (1 : 1) system, i.e. (i) no aromatic ring in the chemical structure, (ii) no chemical heterogeneity in the surfactant sample apart from the presence of different stereoisomers, and (iii) no precipitation of the vesicles at a temperature between 5 and 10 °C. This latter property allowed studying the HRP/H₂O₂-catalyzed polymerization reaction under various experimental conditions also below 10 °C.

Scheme 2 Chemical structure of AOT (sodium bis-(2-ethylhexyl)sulfosuccinate).

To the best of our knowledge, this is only the second time that vesicles are used as chemical structure-controlling templates⁴ for directing an enzymatic polymerization reaction. We hope that the promising data presented in this work stimulate further investigations by others on related systems. With respect to our previous study with SDBS/decanoic acid (1 : 1) vesicles,⁹ the current investigation has shown that the AOT vesicle system is clearly superior, as outlined in the following.

2. Materials and methods

2.1. Materials

Horseradish peroxidase (HRP, type II, RZ = 2.0), sodium bis(2-ethylhexyl) sulfosuccinate (AOT, 99.0%), 2-(p-toluidino)-6-naphthalenesulfonic acid potassium salt (TNS) and sodium phosphate (NaH₂PO₄, ≥99%) were purchased from Sigma. Based on the absorption at 403 nm, the HRP content in the solid product was found to be 54 wt%, taking into account the molar absorbance of HRP, ε₄₀₃ = 1.02 × 10⁵ M⁻¹ cm⁻¹.²⁵ All HRP concentrations given are in amount of solid product per volume, except for the circular dichroism (CD) measurements (see below). Hydrogen peroxide (30%) was from Merck and aniline (≥99.5%) from Fluka. Horseradish peroxidase isoenzyme C (HRPC, grade I-C, RZ ≥ 3.0) for CD measurements was purchased from Toyobo Enzymes. The HRPC concentration was determined by using the molar absorbance given above.

2.2. Preparation of AOT vesicles

AOT vesicles were prepared either by a combined bath/tip sonication procedure or by the freezing–thawing extrusion method with a pH = 4.3 solution containing 0.1 M H₂PO₄⁻, as described previously.⁹ In both cases, a thin film of AOT was first formed by dissolving 0.178 g (0.4 mmol) AOT in 5 mL chloroform in a 50 mL round-bottom flask, followed by chloroform removal with a rotary evaporator under reduced pressure. The film was dried overnight under high vacuum. In the case of sonication, bath sonication (with a Bandelin Sonorex RK 100 H) was carried out for 4 min at room temperature, followed by tip sonication (with a Branson Sonifier 250) for another 4 min at room temperature. In the case of the extrusion method,²⁶ final extrusions were through 100 nm Nucleopore polycarbonate membranes.

2.3. Polymerization of aniline by HRP/H₂O₂ in the presence of AOT vesicles

The polymerization of aniline initiated by HRP/H₂O₂ in the presence of AOT vesicles was carried out at pH = 4.3 (0.1 M H₂PO₄⁻) at room temperature. A volume of 6 mL AOT suspension (20 mM AOT) and 4 mL 40 mM aniline (prepared in 0.1 M H₂PO₄⁻ solution, pH = 4.3) was added into a 200 mL flask containing a magnetic stirrer bar, together with 108.8 mL 0.1 M H₂PO₄⁻ solution (pH = 4.3). To this mixture, 0.6 mL of a solution containing 5 mg mL⁻¹ HRP in water was then added. The reaction was finally started by quick addition of 0.6 mL 0.2 M H₂O₂ (prepared in H₂PO₄⁻ solution, pH = 4.3). The total reaction volume was 120 mL and the total reaction time 5 h. [AOT] = 1 mM, [aniline] = 1.33 mM, [HRP] = 25 µg mL⁻¹, and [H₂O₂] = 1 mM. For reactions carried out in quartz cuvettes inside a spectrophotometer, the volumes were reduced accordingly.

2.4. UV/VIS/NIR measurements

Absorption measurements in the ultraviolet (UV), visible (VIS) and near infrared (NIR) region of the spectrum were recorded with a Perkin Elmer Lambda 20 or a JASCO V-670 instrument at 25 °C, using quartz cuvettes with a path length of either 0.1 cm or 0.5 cm (from Hellma). UV/VIS measurements of small volume samples (below 0.1 mL) were also measured with a NanoDrop ND1000 instrument from Thermo Scientific.

2.5. Circular dichroism (CD) measurements

Circular dichroism (CD) measurements were performed on a JASCO J-715 spectropolarimeter at 25 °C by using HRPC at a concentration of 12 µM. For the CD spectrum recorded in the Soret band region of the spectrum, quartz cuvettes with a path length of 0.5 cm (from Hellma) were used. For the far UV region, the quartz cuvettes had a path length of 0.1 cm. The AOT concentration was 1 mM. The spectra were recorded with a wavelength step of 1 nm and a time constant of 1 s. The reported mean residue ellipticity [θ] is defined as follows: [θ] = θ/(10cln); θ measured ellipticity (in mdeg); c molar concentration of HRPC (in M); l path length of the cuvette (in cm); n: number of amino acids per HRPC (n = 308).²⁷

2.6. Fluorescence measurements

Fluorescence spectra of TNS as fluorescent membrane probe were recorded on a SPEX Fluorolog 2 instrument from Jobin Yvon (UK) using 1 cm quartz cells, as described previously.⁹ The excitation and emission slits were set to 0.5 mm and 1 mm, respectively. TNS was dissolved in methanol at a concentration of 3 mM and a small volume of this solution was added to preformed AOT vesicles prepared at pH = 4.3 (0.1 M H₂PO₄⁻). The vesicle suspension was stored for 30 min at room temperature to ensure complete TNS incorporation into the vesicle membrane. A solution of aniline or HRP was added to the vesicle suspension, followed by storage in the dark for 1 h at room temperature, before the fluorescence measurements were started. The concentration of TNS was 1 µM in the measurements with aniline and 3.2 µM in the measurements with HRP, [AOT] = 1 mM.

2.7. NMR measurements

For the NMR measurements, a phosphate solution in deuterated water was prepared by first dissolving sodium phosphate (Na₃PO₄) in D₂O, followed by adjustment of the pH to 4.3 (pH meter reading, corresponding to pD = 3.9)⁹ by using 1 M DCl and 1 M NaOD solution. The NMR measurements were carried out on a Bruker Avance 700 MHz instrument. For NOESY measurements, the aniline and AOT concentrations were 8 mM each. For the ¹H NMR measurements of the reaction system, the concentrations of AOT and aniline were 1 mM and 1.33 mM respectively; [HRP] = 25 µg mL⁻¹ and [H₂O₂] = 1 mM.

2.8. Cryo-TEM measurements

The cryo transmission electron microscopy (Cryo-TEM) analysis of the AOT vesicles was performed by using vesicle suspensions prepared by the freezing–thawing extrusion method. Details of the sample preparation and of the electron microscopy analysis are described elsewhere.^9,28 For the analysis of the AOT vesicles in 0.1 M H₂PO₄⁻ (pH = 4.3, no reaction), the AOT concentration was 6 mM. For the analysis of the reaction mixture, the following conditions were used: [AOT] = 3 mM, [aniline] = 4 mM, [HRP] = 75 µg mL⁻¹, [H₂O₂] = 3 mM, T = 7 °C, and reaction time: 12 h. Before analysis by cryo-TEM, the vesicle suspension was concentrated by using a Millipore ultrafiltration unit with a molar mass cut-off of 10 000 D and a Hermle Z 320K table centrifuge (30 min, 2000 rpm, 25 °C). The suspension which did not pass through the ultrafiltration membrane (retentate) was analyzed.

2.9. Determination of the amount of remaining aniline in the reaction system

To analyze the amount of aniline which was not converted into PANI during the polymerization reaction, the reaction was carried out with a volume of 20 mL. During the reaction, 0.5 mL portions were withdrawn at different reaction times and transferred into a Millipore ultrafiltration unit with a molar mass cut-off of 10 000 D. After centrifugation with a Hermle Z 320K table centrifuge (5 min, 3000 rpm, 25 °C), the absorption spectrum of the filtrate containing aniline was measured with a NanoDrop ND1000 spectrometer. Analysis of the system before initiating the reaction was taken as reference.

2.10. ATR-FTIR measurements

Attenuated total reflectance-Fourier transformed infrared (ATR-FTIR) spectra of PANI were recorded with an Alpha instrument from Bruker. Around 6 mg sample was used. PANI was isolated by first adding acetone to the reaction system to precipitate the formed PANI. The precipitate was collected by centrifugation using a Hermle Z 320K table centrifuge, followed by washing of the precipitate first with acetone/water (1 : 1) and then with pure acetone. The sample was then dried in vacuum overnight.

3. Results and discussion

3.1. AOT vesicles

AOT is a widely used anionic amphiphile with two short branched hydrophobic tails (Scheme 2). The most popular application of AOT is as surfactant for the formation of reverse micelles in organic solvents like isooctane.²⁹ AOT is actually thesurfactant for reverse micelle formation due to its molecular geometry which is in a first approximation like a truncated inverted cone, prone to form inverted structures in an apolar organic solvent. In dilute aqueous solution, AOT may aggregate into micelles or vesicles, depending on the experimental conditions. In the present work, we took advantage of this latter fact that AOT vesicle formation indeed can occur at low AOT concentration. For a better understanding of the AOT vesicle system which we later used as chemical structure-controlling template for the polymerization reaction, literature data and our own observations on the aggregation behavior of AOT in dilute aqueous solution are summarized in this section.

In dilute aqueous solutions prepared with deionized water—without added salt—AOT forms micelles above the critical concentration for micelle formation (cmc). The recently determined cmc value for AOT in deionized water at 25 °C varies between 0.63 mM³⁰ and 2.4–2.7 mM.³¹ In 1987, Ghosh and Miller reported that dispersing AOT in an aqueous solution at concentrations below 1 wt% (corresponding to 23 mM AOT) at 30 °C led to the formation of micelles (isotropic L₁ phase), which were found to coexist with a lamellar liquid crystalline phase (dispersed L_α phase, i.e.vesicles) if salt in the form of NaCl was added.³² Regions of coexistence of L₁ and L_α phases appeared if the AOT concentration was above about 0.25 wt% (= 5.6 mM AOT) and if the aqueous solution contained NaCl at a concentration between about 0.1 wt% (= 17 mM) and about 1.4 wt% (= 239 mM).^30,32 The driving force for the formation of vesicles in the presence of salt is the screening of the electrostatic repulsion between adjacent sulfonate head groups leading to a transformation of AOT micelles into AOT vesicles.³³ Originally, this transformation was found to occur at the reported L₁ + L_α phase boundary, at about 5.6 mM for 17–239 mM NaCl.^30,32 This seems to be incorrect since vesicle formation was also observed in the region of the phase diagram which was originally assigned to the pure L₁ phase.³⁰ This means that the originally reported cmc values in the presence of NaCl probably represent values for the critical concentration for the formation of vesicles (cvc).²⁰ At 4.7 mM NaCl, the cvc for AOT was found to be 7.8 mM at 30 °C.³⁴ At NaCl concentrations above 25 mM, the cvc values were reported to be below 1 mM AOT, with a tendency to decrease with increasing NaCl concentration (up to 170 mM).³⁰ All these literature data clearly showed that AOT vesicle formation in dilute aqueous solution occurs if the aqueous solution contains added NaCl.

In the present work, we did not use NaCl but we dispersed AOT in 0.1 M phosphate solution (pH = 4.3), which was found to be optimal for the HRP-catalyzed polymerization of aniline,^6–9 and we wondered whether AOT vesicle formation also occurs under these conditions. Since this was the case, the vesicles were used later on as templates for the polymerization reaction. The AOT vesicle formation was studied as follows. In a first set of experiments, the transmission (expressed as absorbance at 400 nm (arbitrarily chosen)) was measured as a function of AOT concentration (Fig. 1). A decrease in light transmission at 400 nm was taken as the indication of the presence of vesicles (large aggregates) which scatter visible light. In control measurements, solutions at different AOT concentrations in deionized water and in methanol were also analyzed in the same way. The results for 0.1 M phosphate solution (curve a), for deionized water (curve b) and for methanol (curve c) are shown in Fig. 1. Since the hydrophilic head group and the hydrophobic tails of AOT molecules have a good solubility in methanol, it is expected that no large aggregates form in methanol solution, which is consistent with the measurements (no turbidity up to at least 6 mM AOT (curve c in Fig. 1). In deionized water, the solutions also did not scatter visible light (curve b in Fig. 1), indicating again that no large aggregates formed up to 6 mM AOT. Therefore, there was no indication of vesicle formation in deionized water in the concentration range investigated, in agreement with literature.^30–33 Whether micelles formed in deionized water below 6 mM AOT was not of interest and also could not be determined by these simple measurements since the micelles would be too small to scatter visible light. The determined aggregation number of AOT micelles is 29, see Grillo et al.³³ In 0.1 M phosphate solution (pH = 4.3), the samples were turbid above about 0.4 mM AOT (curve a in Fig. 1), indicative of the formation of large aggregates (vesicles). The presence of vesicles was confirmed by cryo-TEM measurements (Fig. 2). For this electron microscopy analysis, the sample was homogenized with the freezing–thawing extrusion method,^9,26 see Section 2.2. Even if the few small dark dots present in the images—most likely arising from a contamination by small ice crystallites—would be AOT micelles, they would amount to less than 5% of the AOT present as vesicles (as judged from samples containing 6 mM AOT). There was no indication of the presence of cylindrical micelles or flat bilayered sheets.

Fig. 1 Determination of the critical concentration for vesicle formation, cvc, of AOT by measuring the turbidity of AOT solutions and suspensions as a function of AOT concentration. The optical density was measured at λ = 400 nm (OD₄₀₀), path length = 1 cm. (a, filled square) AOT in 0.1 M H₂PO₄⁻ (pH = 4.3); (b, open triangle) AOT in water; and (c, open circle) AOT in methanol.

Fig. 2 Cryo-TEM images of AOT vesicles, prepared by the freezing–thawing extrusion method (see Materials and methods) in 0.1 M H₂PO₄⁻, pH = 4.3. [AOT] = 20 mM (a) and 6.0 mM (b). Length of the bar: 100 nm.

The AOT vesicles obtained by extrusion were rather homogeneous with respect to size and most of the vesicles were unilamellar (Fig. 2). The average size of the vesicles determined by dynamic light scattering (DLS) measurements was 72 nm immediately after the preparation with an increase in size upon storage at room temperature up to 108 nm after 7 days (Fig. 3). Such vesicle size increase upon storage was also observed for SDBS/decanoic acid (1 : 1) vesicles,⁹ indicating (i) that the average size to which the vesicles were forced by extrusion was not the thermodynamically most stable size and (ii) that the activation energy for vesicle fusion was low enough so that significant vesicle fusion occurred at room temperature within a few days of storage.

Fig. 3 Dynamic light scattering measurements of AOT vesicles, prepared by the freezing–thawing extrusion method (see Materials and methods) and stored at room temperature. [AOT] = 1 mM, 0.1 M H₂PO₄⁻, pH = 4.3.

3.2. Optimization of the reaction system

3.2.1. VIS/NIR absorption measurements as analytical tool for reaction optimization. The reaction system was optimized for obtaining mainly the emeraldine salt form of PANI. For this, the polymerization reaction was analyzed by measuring the VIS/NIR absorption spectrum at reaction equilibrium and by comparing the absorbance at 1000 nm (A₁₀₀₀) with the absorbance at 550 nm (A₅₅₀). A high ratio of A_{1000 nm}/A₅₅₀ was taken as signature for the presence of mainly the linear conductive emeraldine salt form of PANI (Scheme 1), since the absorbance at 1000 nm is characteristic for this type of polyaniline and the absorbance at 550 nm is characteristic for (unwanted) branched chains.^{7–9,12,13,17} Theoretical calculations showed that the location of the absorption maximum in the NIR and VIS region of the spectrum depends on the chemical structure of the possible subunits present in the emeraldine salt form of PANI.^15,16 Furthermore, the precise position of the absorption maximum of the emeraldine salt in the NIR region of the spectrum is expected to strongly depend on the conformation of the polymer chain,¹⁹ which in turn depends on the interaction of the PANI chain with the dopant or with other counter ions present; and it depends on the interaction of the PANI chain with other PANI chains in close proximity. Absorbance in the NIR region seems to arise from the presence of delocalized polarons (Scheme 1).^1f

Like in the HRP/H₂O₂ systems studied previously with SDBS/decanoic acid (1 : 1) vesicles as templates,⁹ the absorption maximum of the emeraldine salt form of PANI under optimal reaction conditions in the presence of AOT vesicles was found to be at about 1000 nm. This agrees with the observations made with sulfonated polystyrene as template⁷ or with micellar templates formed from surfactants which have a sulfonate head group (SDBS or sodium dodecyldiphenyloxidedisulfonate).⁸

The AOT vesicle reaction system was optimized by varying the AOT concentration, the sodium dihydrogen phosphate concentration and the temperature.

3.2.2. Variation of AOT concentration. The influence of the AOT concentration on the HRP/H₂O₂-triggered polymerization of aniline was investigated by performing the polymerization at various AOT concentrations from 0.3 mM to 15 mM at fixed HRP, aniline and H₂O₂ concentrations. The initial conditions for the polymerization at T ≈ 25 °C were pH = 4.3 (0.1 M H₂PO₄⁻) with [aniline] = 1.33 mM, [HRP] = 25 µg mL⁻¹ and [H₂O₂] = 1 mM. The reaction was characterized by measuring the VIS/NIR spectrum 1 hour after the start of the reaction, after there were no obvious changes in the absorption spectrum anymore. The ratio of absorbance at 1000 nm to absorbance at 550 nm was calculated (see Section 3.2.1) and is plotted as a function of AOT concentration in Fig. 4.

Fig. 4 HRP-catalyzed polymerization of aniline in the presence of AOT vesicle templates. Variation of the ratio A_{1000 nm}/A_{550 nm} as a function of AOT concentration at pH = 4.3 (0.1 M H₂PO₄⁻), [aniline] = 1.33 mM, [HRP] = 25 µg mL⁻¹, [H₂O₂] = 1 mM, t = 1 h, T ≈ 25 °C.

As the AOT concentration increased from 0.3 mM to 15 mM, A_{1000 nm}/A_{550 nm} first increased and reached a maximum value at 1 mM AOT, then decreased. This observation can be explained as follows: at pH = 4.3, about 67% of the aniline molecules are protonated and have a positive charge which may efficiently complex with the negatively charged AOT vesicles prior to the reaction (pK_a (anilinium) = 4.6). If the AOT concentration is low (as compared to the aniline concentration), most of the aniline molecules are expected to freely move in the solution before and during the polymerization, leading to the formation of more branched structures since there is no direct control of the polymer chain elongation by the vesicle template. At too high AOT concentration, the local concentration of aniline on the vesicle surface decreases which may result in the formation of increased amounts of short polymer chains. This would explain the decrease in A_{1000 nm}/A_{550 nm} at high AOT concentration. In any case, the optimum AOT concentration at [aniline] = 1.33 mM was found to be 1 mM (1 mM H₂O₂, 0.1 M H₂PO₄⁻, pH = 4.3), see Fig. 4.

3.2.3. Variation of sodium dihydrogen phosphate concentration. At pH = 4.3, most of the phosphate is present as dihydrogen phosphate, H₂PO₄⁻. The effect of varying the concentration of the added NaH₂PO₄ on the HRP-triggered polymerization of aniline in the presence of AOT vesicles was studied by starting the reaction at pH = 4.3. The AOT, aniline and HRP concentrations were always 1 mM, 1.33 mM and 25 µg mL⁻¹, respectively. The polymerization was initiated by the addition of H₂O₂ (1 mM initial concentration in the reaction system). The reaction system was analyzed by recording the VIS/NIR spectrum 24 h after the start of the reaction. The absorbance at 1000 nm is plotted as a function of the added NaH₂PO₄ concentration in Fig. 5. Upon increasing the NaH₂PO₄ concentration from 25 mM to 240 mM, the absorbance at 1000 nm first increased until 120 mM then decreased. When the concentration was 320 mM, which is not shown in Fig. 5, precipitation was observed already 30 min after the start of the reaction.

Fig. 5 Effect of added NaH₂PO₄ concentration on the HRP-catalyzed polymerization of aniline in the presence of AOT vesicles. The absorbance at 1000 nm of the reaction solution was measured 24 h after the start of the reaction. [AOT] = 1 mM; [aniline] = 1.33 mM; [HRP] = 25 µg mL⁻¹; [H₂O₂] = 1 mM; path length = 0.1 cm; T ≈ 25 °C.

These observations can be understood on the basis of the following consideration. Sodium ions compete with anilinium ions and H₃O⁺ and with (protonated) oligoaniline chains for binding to the vesicle surface. It is therefore likely that the amount of bound anilinium ions at constant pH is decreased at high NaH₂PO₄ concentration. As a consequence, the AOT vesicles will lose their template effect. Furthermore, since protons are released from the amino groups during polymerization,^1d,35 there may be a significant decrease of the pH value if the buffering capacity of the phosphate solution at low phosphate concentration is insufficient. If the pH value drops to a too low value, the HRP may become less active, and the relative amount of unprotonated aniline molecules which can be oxidized by HRP decreases at low pH. Although the observed effect of NaH₂POP₄ concentration requires further investigations, it seems that a concentration of about 0.1 M NaH₂PO₄ is optimal.

3.2.4. Variation of reaction temperature. The HRP-catalyzed polymerization of aniline was performed at two different temperatures, at 7 °C and at room temperature (T ≈ 25 °C). Otherwise the reaction conditions were the same: [AOT] = 1 mM, [aniline] = 1.33 mM, [HRP] = 25 µg mL⁻¹, [H₂O₂] = 1 mM and pH = 4.3 (0.1 M H₂PO₄⁻). The VIS/NIR spectra of the two reaction solutions were recorded 24 h after the start of the reaction, i.e. after reaching equilibrium, see Fig. 6.

Fig. 6 HRP-catalyzed synthesis of PANI in the presence of AOT vesicles. VIS/NIR spectra of PANI obtained after 24 h at two different temperatures: (a, solid line) at T = 7 °C and (b, dashed line) at T = 25 °C. [AOT] = 1 mM, [aniline] = 1.33 mM, [HRP] = 25 µg mL⁻¹, [H₂O₂] = 1 mM, t = 24 h pH = 4.3 (0.1 M H₂PO₄⁻), and path length = 0.1 cm.

The absorption bands at about 1000 nm and about 420 nm were more intense when the reaction was carried out at 7 °C, as compared to 25 °C. Absorption in both regions of the spectrum can be attributed to delocalized electrons of the radical cations (separated and delocalized polarons) that are characteristic for the conductive emeraldine salt form of PANI.^{1f,19–21,36} The higher band intensities around 1000 nm and 420 nm of PANI obtained at 7 °C as compared to room temperature may indicate that the PANI obtained at the lower reaction temperature contained more structural units with extended conjugation, possibly longer chains, if compared to the PANI obtained at room temperature. This latter interpretation would be consistent with reports on the temperature dependency of the formation of PANI by purely chemical, non-enzymatic methods.³⁷ Although in that case, the experimental conditions were very different from the conditions used in our system, a lower reaction temperature led to the formation of longer polymer chains.³⁷ The possibility that the chains obtained at 7 °C had a more extended coil-like conformation¹⁹ as compared to the chains obtained at 25 °C would be consistent with the observed decreased absorbance at ∼300 nm (π–π* transition).¹⁹ A decrease in band intensity at ∼300 nm—with an increase in the NIR region of the spectrum (above ∼800 nm)—was observed if compact coils of the emeraldine salt form of PANI were converted into extended coils by changing the solvent.¹⁹

The higher absorbance at ∼420 nm and ∼1000 nm observed at 7 °C was not related to a higher reaction yield at 7 °C, as compared to 25 °C, see Fig. 7. Independent of whether the polymerization reaction was carried out at 7 °C or 25 °C, the remaining aniline in the reaction system quickly dropped to about 40% within the first hour and to 30% after 24 h, and then stayed constant for at least another day. This reveals that the HRP-catalyzed polymerization of aniline under the experimental conditions used is a fast process and the consumption of aniline in the presence of AOT vesicles mainly occurred during the first hour.

Fig. 7 Change in the percentage of remaining aniline in the reaction systems during the reaction carried out at two different reaction temperatures, at T = 7 °C (filled circle) and at 25 °C (open square). The reaction conditions were: [AOT] = 1 mM, [aniline] = 1.33 mM, [HRP] = 25 µg mL⁻¹, [H₂O₂] = 1 mM, in pH = 4.3 (0.1 M H₂PO₄⁻).

3.3. Interaction of HRP with AOT vesicles

3.3.1. Use of TNS as vesicle membrane probe. The interaction of HRP with AOT vesicles was elucidated by using the fluorescent vesicle membrane probe TNS (2-(p-toluidino)-6-naphtalenesulfonic acid potassium salt). TNS is known to bind to vesicle membranes⁹ where it senses changes in the polarity of the environment (change in fluorescence intensity). Fig. 8 shows the variation of the TNS fluorescence intensity with increasing HRP concentration. There was a steady increase in fluorescence intensity until the HRP concentration reached ∼6 µg mL⁻¹. Afterwards, the fluorescence intensity remained almost constant. This observation can be taken as an indication of a strong binding of HRP to the AOT vesicle surface. If all HRP molecules were bound to the outer vesicle surface at 25 µg HRP per mL, the condition used for the polymerization reaction, then each vesicle would contain ∼30 HRP molecules.³⁸

Fig. 8 Effect of HRP on the fluorescence of TNS in the presence of AOT vesicles (1 mM AOT) at pH = 4.3 (0.1 M H₂PO₄⁻). [TNS] = 3.2 µM, λ_ex = 320 nm, λ_em = 422 nm, T = 25 °C. The relative fluorescence intensity of TNS is plotted as a function of HRP concentration.

3.3.2. CD measurements. As shown in Fig. 9, at room temperature, the CD spectra of HRP in the dihydrogen phosphate solution (pH = 4.3, without any vesicles) and in the presence of AOT vesicles overlap in the far UV region, which shows that the presence of AOT surfactants did not unfold the secondary, α-helical structure present in HRP (Fig. 9a). Similarly, in the Soret band region of the spectrum of HRP, the presence of AOT vesicles had no measurable effect (Fig. 9b). This indicates that the heme group embedded in the protein matrix of HRP remained largely intact without being “washed out” by AOT surfactants. This is different from the case of SDBS/decanoic acid (1 : 1) vesicles,⁹ where the presence of vesicles led to a measurable decrease of the ellipticity of HRP in the Soret band region.⁹ Based on the CD analysis it can be concluded that the AOT vesicles (1 mM) did not affect the structure of HRP at pH = 4.3 significantly, despite there being evidence that the enzyme interacts with the vesicles (Fig. 8). Enzyme binding to the vesicles appears to occur away from the active site, without altering the heme group environment.

Fig. 9 Effect of AOT vesicles on the CD spectrum of horseradish peroxidase C (12 µM = 480 µg mL⁻¹) in (a) the far UV region and (b) the Soret band region of the spectrum. [AOT] = 1 mM, 0.1 M H₂PO₄⁻, pH = 4.3, T = 25 °C. (a) Path length = 0.1 cm and (b) path length = 0.5 cm. Solid line: in the presence of AOT vesicles and dashed line: in phosphate solution (no vesicles).

3.3.3. HRP stability measurements. To investigate the stability of HRP in the presence of AOT vesicles, HRP at a concentration of 25 µg mL⁻¹ was pre-incubated in dihydrogen phosphate solution (pH = 4.3) in the presence of AOT vesicles (1 mM AOT) and 1.33 mM aniline at room temperature (T ≈ 25 °C). After incubation for a certain period of time, 1 mM H₂O₂ was added to initiate the reaction. The reaction system was analyzed by recording the VIS/NIR spectrum 1 h after H₂O₂ addition. The absorbance at 1000 nm was taken as a measure of the activity of HRP, i.e. of the incubated enzyme's ability to trigger aniline polymerization. The results are shown in Fig. 10. The data are normalized by the absorbance measured at 1000 nm after 1 h without any enzyme pre-incubation. Results obtained from similar experiments in dihydrogen phosphate solution (pH = 4.3, no vesicles) and in dihydrogen phosphate solution in the presence of SDBS/decanoic acid (1 : 1) vesicles ([SDBS] = [decanoic acid] = 0.665 mM) as templates are also shown in Fig. 10.

Fig. 10 Stability of HRP in different reaction systems prepared at pH = 4.3 (0.1 M H₂PO₄⁻) in the absence of vesicles, in the presence of AOT vesicles and in the presence of SDBS/decanoic acid (1 : 1) vesicles. HRP (25 µg mL⁻¹), aniline (1.33 mM) and the vesicles were incubated for a certain period of time, followed by the addition of H₂O₂ (1.0 mM in the reaction mixture) to start the reaction. After 1 h, the VIS/NIR spectrum was recorded and the value of absorbance at λ = 1000 nm was taken as a measure for the activity of the enzyme, plotted as a function of time (path length = 0.1 cm). The values given are relative values, normalized by the absorbance obtained without incubation. Filled bar: in the presence of AOT vesicles ([AOT] = 1.0 mM); dashed bar: in the absence of vesicles; and empty bar: in the presence of SDBS/decanoic acid (1 : 1) vesicles ([SDBS] = [decanoic acid] = 0.665 mM).

After 3 h pre-incubation, HRP showed similar activity in phosphate solution and in the presence of AOT vesicles or in the presence of SDBS/decanoic acid (1 : 1) vesicles. In all three cases, the activity was almost the same as without pre-incubation (set to 100% in Fig. 10). If the HRP was pre-incubated for 20 h at 25 °C, the enzyme in the presence of AOT vesicles was still fully active like without incubation or after incubation in phosphate solution without vesicles. In contrast, the activity of HRP in the presence of SDBS/decanoic acid (1 : 1) vesicles decreased to about 70%. Based on these results it is evident that HRP remains more stable in the presence of AOT vesicles as compared to the SDBS/decanoic acid (1 : 1) vesicles used previously.⁹ In this respect, the AOT vesicle system is a better template system than the SDBS/decanoic acid (1 : 1) system, which in turn was already shown to be better than SDBS micelles.⁹

3.4. Interaction of aniline with AOT vesicles

3.4.1. Use of TNS as vesicle membrane probe. Fig. 11 shows the variation of the fluorescence intensity of TNS in the presence of AOT vesicles as a function of aniline concentration. With increasing aniline concentration up to 2.5 mM (1 mM AOT), the fluorescence intensity of TNS did not change significantly, similarly to what was found for SDBS/decanoic acid (1 : 1) vesicles.⁹ This fully supports the view that aniline molecules do not have strong interactions with AOT vesicles under the conditions used, which is in clear contrast to the behavior of HRP (Fig. 8). Aniline probably just loosely associates on the vesicle surface. It may, however, also be that possible aniline–AOT interactions are not sensed by the vesicle embedded membrane probe TNS. Therefore, NMR measurements were also carried out, see below.

Fig. 11 Effect of aniline on the fluorescence of TNS in the presence of AOT vesicles (1 mM AOT) at pH = 4.3 (0.1 M H₂PO₄⁻). [TNS] = 1 µM, λ_ex = 320 nm, λ_em = 422 nm, T = 25 °C. The relative fluorescence intensity of TNS is plotted as a function of aniline concentration.

3.4.2. ¹H–¹H NOESY NMR measurements. The interaction of aniline with the vesicles was studied with ¹H–¹H NOESY NMR spectroscopy. The experiments were carried out in deuterated phosphate solution which was made from Na₃PO₄ with the addition of DCl in D₂O. The AOT and aniline concentrations were 8 mM. The NOESY NMR spectrum of the aniline and AOT vesicle mixture at pD = 3.9 is shown in Fig. 12 in which strong NOEs originating from intramolecular interactions between the protons in aniline (δ = 7–8 ppm) and from intramolecular interactions between the protons of the aliphatic chains of AOT (δ = 0.5–4 ppm) can clearly be seen. There were also intermolecular cross-peaks between aniline and AOT, indicating intermolecular interactions between aniline and AOT vesicles. However, these interactions were relatively weak, as judged from the peak intensities, indicating that aniline monomers do not strongly interact with the AOT chains but are most likely only loosely associated on the surface of the vesicles. Although the AOT and aniline concentrations were higher in the NMR measurements (8 mM each), as compared to the measurements with TNS (1 mM AOT, up to 2.5 mM aniline), the fluorescence measurements reported in Section 3.4.1 are consistent with the interpretation of the NMR data. There are only weak interactions between aniline and the vesicles.

Fig. 12 700.13 MHz ¹H–¹H NOESY spectrum of a mixture of aniline and AOT vesicles in deuterated phosphate solution. [AOT] = [aniline] = 8 mM, pD = 3.9, T = 25 °C. Strong intramolecular NOE cross-peaks are indicated with the solid squares. These NOEs originate from proton interactions in aniline and from proton interactions in AOT, respectively. Weak intermolecular NOE crosspeaks are found between resonances of aniline and the aliphatic chains of AOT, as indicated with the dashed square.

3.5. Analysis of the reaction system during polymerization

3.5.1. VIS/NIR measurements. The kinetics of the HRP/H₂O₂-catalyzed polymerization of aniline (1.33 mM) in the presence of AOT vesicles (1 mM AOT) was analyzed by recording the VIS/NIR spectrum during the polymerization at 25 °C and at 7 °C. The HRP concentration was 25 µg mL⁻¹ and the initial H₂O₂ concentration 1 mM. At both temperatures, the same qualitative observations were made. The spectrum measured two minutes after the start of the reaction had an intense absorption band at ∼750 nm. The intensity of this band decreased with time and two new maxima centered around 400 nm and 1000 nm appeared, the latter being indicative of the formation of more and more delocalized polarons in the emeraldine salt form of PANI.^1f The spectral changes observed during the reaction at 7 °C are shown in Fig. 13. At 25 °C, the situation was similar. The only difference was that the band intensities in the spectra recorded at 7 °C were higher as compared to 25 °C, and the time changes in the spectrum were slower at 7 °C than at 25 °C, see also Fig. 6. Similarly to the results obtained with the SDBS/decanoic acid (1 : 1) vesicle system, the VIS/NIR data may indicate that during the early phase of the reaction the reaction products obtained were overoxidized (peak at 750 nm). The typical spectrum of the emeraldine salt form with its characteristic absorption in the NIR region of the spectrum and at 400 nm only developed in the second phase of the reaction, when the intensity of the peak at 750 nm continuously decreased. Our interpretation about the formation of overoxidized PANI at the early stage of the enzymatic polymerization reaction would be in agreement with reports on the purely chemical, non-enzymatic polymerization of aniline with (NH₄)₂S₂O₈ in 1.0 M HCl.³⁹ In that case, the fully oxidized pernigraniline form of PANI was found to form first before further reaction leads to the formation of the half oxidized emeraldine form.³⁹ There are, however, two other points to consider. First, a similar transition with a decrease of the band intensity at 750 nm and an increase in the band intensity in the NIR region of the spectrum (at λ > 800 nm) was observed when coiled emeraldine salt conformations transformed into expanded coil-like conformations,¹⁹ apparently without any externally triggered chemical reactions occurring. Second, Fig. 7 shows that after 1 h of reaction the concentration of non-reacted aniline remained almost constant; the monomer integration into the polymer chains apparently was already almost completed. Experiments are now in progress with the aim of hopefully clarifying the situation at the early stage of the reaction.

Fig. 13 Time dependent changes of the VIS/NIR absorption spectrum during the HRP-catalyzed polymerization of aniline in the presence of AOT vesicles carried out at T = 7 °C. [AOT] = 1 mM; [aniline] = 1.33 mM; [HRP] = 25 µg mL⁻¹; [H₂O₂] = 1 mM; pH = 4.3 (0.1 M H₂PO₄⁻), path length: 0.2 cm. (a) Absorption spectrum of the reaction system as a function of reaction time; the first spectrum was recorded 10 min after the start of the reaction (after the addition of H₂O₂), the following spectra were recorded in intervals of 20 min; the arrows indicate direction of the changes of the intensities with time. (b) Changes of the absorbance at λ = 1000 nm, 750 nm and 400 nm with reaction time.

3.5.2. ¹H NMR measurements. The HRP/H₂O₂-catalyzed polymerization of aniline in the presence of AOT vesicles was further studied at 7 °C by using ¹H NMR spectroscopy and [AOT] = 1 mM; [aniline] = 1.33 mM; [HRP] = 25 µg mL⁻¹ and [H₂O₂] = 1 mM.

Sections of the ¹H NMR spectrum of the reaction system recorded at different times after the start of the reaction are presented in Fig. 14, together with a reference spectrum of the reaction solution before the start of the reaction, i.e. before the addition of H₂O₂ (Fig. 14). Three obvious time-dependent changes could be identified in comparison with the reference spectrum: (i) the signal intensity of the aromatic protons at 7.0–7.3 ppm showed a significant decrease one hour after the start of the reaction; (ii) a clear peak shift to higher frequency of the meta-, para- and ortho-protons of aniline could be observed. After one hour, the signal intensity and the position of the peaks corresponding to the meta-, para- and ortho-protons remained constant; and (iii) the NMR signals attributed to the AOT molecules (at δ ≈ 1 ppm) and the aniline resonances at 7.0–7.3 ppm became broader. No signals originating from reaction products could be detected, despite the fact that the integral of the aniline protons between 7.0 and 7.3 ppm after 4.5 h of reaction dropped to ∼45% of the integral measured at the beginning of the reaction. This means that ∼55% of the initial aniline reacted. This is comparable with the independently determined amount of unreacted aniline left at reaction equilibrium, see Fig. 7.

Fig. 14 HRP-catalyzed polymerization of aniline in the presence of AOT vesicles. ¹H NMR spectrum of the reaction solution recorded at different time. (a) Between 7.00 and 7.35 ppm and (b) between 0.00 and 2.00 ppm. From bottom to top: in the absence of H₂O₂ (no reaction); 1 h after the addition of H₂O₂ to start the reaction; 2 h after the start of the reaction; 4.5 h after the start of the reaction. [AOT] = 1 mM; [aniline] = 1.33 mM; [H₂O₂] = 1 mM, pD = 3.9 (0.1 M D₂PO₄⁻).

The absence of NMR signals arising from the reaction product(s) is in agreement with the formation of delocalized unpaired (paramagnetic) electrons, which lead to broad signals that are not measurable. Such delocalized unpaired electrons are expected to be present in the emeraldine salt form of PANI. Therefore, this interpretation of the NMR measurements would be in agreement with the interpretation of the VIS/NIR spectrum in the sense that most of the reaction products obtained had the expected chemical structure of the emeraldine salt form of PANI (Scheme 1). An alternative explanation for the absence of NMR signals from the polymeric product(s) is the polymer binding to the vesicles which leads to a slowing down of the molecular motions and consequently to a broadening of the signals. If the signals become too broad they are not detectable.

The absence of ¹H NMR signals for the reaction products is in agreement with literature data from a ¹H NMR study of the chemical synthesis of PANI with Na₂S₂O₈.⁴⁰ In that case, the reaction products were also not visible in a ¹H NMR spectrum,⁴⁰ independent of whether the products precipitated—absence of signals from insoluble polymers—or whether they were kept as stable dispersion with the help of SDBS.⁴⁰

The broadening of the AOT ¹H NMR signals at ∼1 ppm and of the aniline resonances at 7.0–7.3 ppm is probably due to intermolecular paramagnetic dipolar relaxation caused by the presence of unpaired electrons. The peak shift of the aromatic protons of aniline in the spectrum recorded 1 h, 2 h and 4.5 h after H₂O₂ addition (Fig. 14) may indicate that the remaining, unreacted, aniline molecules are no more so close to the vesicle surface. This possibility is based on the fact that the chemical shifts of the aniline protons in aqueous solution at pH = 4.3 (in the absence of vesicles) are similar to the chemical shifts of aniline measured after 1 h, 2 h, or 4.5 h of reaction (Fig. 14). This would mean that the unreacted aniline monomers are displaced from the vesicle surface by the PANI formed which itself binds to the vesicles. Further experiments are certainly needed to hopefully clarify this point.

3.5.3. Cryo-TEM measurements. The morphology of the vesicles was analyzed after aniline polymerization at 7 °C, see Fig. 15. The reaction was carried out with the following initial conditions: [AOT] = 3 mM, [aniline] = 4 mM, [HRP] = 75 µg mL⁻¹, [H₂O₂] = 3 mM and pH = 4.3 (0.1 M H₂PO₄⁻). The reaction time was 15 h and the formation of the emeraldine salt form of PANI was confirmed by recording the VIS/NIR absorption spectrum (A₁₀₀₀/A₅₅₀ = 3.1). Fig. 15 clearly shows that the reaction system still contained vesicles, although they were more polydisperse in size than pure extruded AOT vesicles (Fig. 2). Furthermore, some of the vesicles were not perfectly spherical. The vesicle membranes in Fig. 15 generally are darker and thicker than the vesicle membranes in Fig. 2. This indicates that the PANI formed remained bound on the vesicle surface. The vesicle surface appears uniform; no individual particles are distinguishable in terms of thickness. It seems that the vesicles were relatively regularly coated by the PANI obtained.

Fig. 15 Cryo-TEM images of the AOT vesicular reaction system after reaching reaction equilibrium. The polymerization reaction was carried out at T = 7 °C under the following conditions: [AOT] = 3 mM, [aniline] = 4 mM, [HRP] = 75 µg mL⁻¹, [H₂O₂] = 3 mM, pH = 4.3 (0.1 M H₂PO₄⁻). The reaction time was 15 h. The vesicles were prepared by the freezing–thawing extrusion method (see Materials and methods). Length of the bar in both images, a and b, is 100 nm.

3.6. Characterization of the PANI product obtained by FTIR spectroscopy

The reaction product obtained from aniline with HRP/H₂O₂ in the presence of AOT vesicles at 7 °C was isolated from the reaction mixture (see Section 2.10) and then analyzed by ATR-FTIR spectroscopy (Fig. 16). The individual bands in the FTIR spectrum can be assigned as follows. The two distinct peaks at 1595 cm⁻¹ and 1501 cm⁻¹ are characteristic bands of the vibrational modes of the quinoid units (C–C stretching at 1595 cm⁻¹) and of the benzenoid units (C–C stretching at 1501 cm⁻¹) of the emeraldine form of PANI^{1d,f,9,23a,41} The absorption band at 1595 cm⁻¹ supports the presence of aniline units in the PANI obtained that are connected via a head-to-tail monomer coupling during polymerization.^1d,42 The strong absorptions at 1306 cm⁻¹ and 1160 cm⁻¹ are caused by the C–N stretching of aromatic amines and by the C–H in plane deformation vibration, respectively.^1d,f,9,41,42 The C–H out-of-plane bending mode located at 819 cm⁻¹, which is characteristic for para-disubstituted aromatic rings,^1d,f,9 is clearly observed. The band at 1248 cm⁻¹ can be assigned to the C–N⁺˙ stretching in the polaron form of the emeraldine salt of PANI.⁴¹ Based on the literature,^1d,41 the stretching vibration of –NH⁺= in the bipolaron structure of the emeraldine salt form of PANI should be at 1146 cm⁻¹ which is not clearly evident in the spectrum shown in Fig. 16. The band—if present—may be localized below the peak with maximum absorbance at 1160 cm⁻¹. The weak bands located at 695 cm⁻¹ and 749 cm⁻¹ can be assigned to C–H out-of-plane deformation vibrations of monosubstituted aromatic rings.^1f The presence of a band at ∼690 cm⁻¹ has also been taken as an indication of ortho-disubstituted aromatic rings within the PANI chains.^1d,9,43 Furthermore, the band at 749 cm⁻¹ may also indicate the presence of ortho-coupled units.⁴⁴ The small peak at ∼860 cm⁻¹ may indicate the presence of some branched units,^1d,9 occurring through an ortho-coupling of the aniline molecules during polymerization. If the intensities of the peaks which may arise from ortho-coupled aniline units (at 875 cm⁻¹, 795 cm⁻¹ and 695 cm⁻¹) are compared with the intensities of the band characteristic for para-coupled aniline units (819 cm⁻¹), it seems that most of the PANI obtained was linear with para-coupled aniline chains, as expected for the emeraldine form of PANI.

Fig. 16 ATR-FTIR spectrum of PANI synthesized at 7 °C in the presence of AOT vesicles and isolated by using the acetone precipitation method (see Materials and methods). The reaction conditions were: [AOT] = 1 mM, [aniline] = 1.33 mM, [HRP] = 25 µg mL⁻¹, [H₂O₂] = 1 mM, t = 24 h and pH = 4.3 (0.1 M H₂PO₄⁻).

Ortho-coupling of aniline units, particularly occurring in chemical polymerizations at the early stage of the reaction,^2b may lead to the formation of phenazine units. Typical band positions for vibrations of substituted phenazines are expected at 1623 cm⁻¹, 1414 cm⁻¹, 1208 cm⁻¹, 1144 cm⁻¹, 1136 cm⁻¹, and 1108 cm⁻¹.^1f Since there was no clear absorption at these frequencies (see Fig. 16), the presence of large amounts of phenazine units in the PANI obtained can be excluded.

The assignment of the bands observed at 1072 cm⁻¹ and 933 cm⁻¹ is not clear at the moment. Although AOT has a strong band at ∼1050 cm⁻¹ (S=O stretching), the other characteristic band of AOT at ∼1730 cm⁻¹ (C=O stretching) is missing. The FTIR spectrum of the PANI obtained indicates that AOT could be removed from the reaction product quite efficiently, in contrast to the case of the SDBS/decanoic acid (1 : 1) vesicles used previously,⁹ where complete SDBS removal was not possible.⁹ In this respect, there is another advantage of the AOT vesicles over the previously used system.

4. Conclusions

This is the second time that experimental data on the use of vesicles as chemical structure-controlling templates for enzymatic polymerization reactions are presented. The reaction investigated was the HRP/H₂O₂-triggered polymerization of aniline at pH = 4.3 in 0.1 M H₂PO₄⁻ solution and the vesicles used were formed from AOT. It was shown that AOT vesicles have clear advantages if compared with the SDBS/decanoic acid (1 : 1) vesicle system used previously.⁹ First, the enzyme, HRP, is more stable if incubated together with the AOT vesicles as compared to the SDBS/decanoic acid (1 : 1) vesicles. Second, the AOT vesicles are also stable at a temperature below 10 °C which is not the case for the SDBS/decanoic acid (1 :1 ) vesicles.⁹ Since the reaction temperature has an influence on the conjugation length and/or the conformation of the PANI obtained (Fig. 6), the AOT system allows further studies of this point. Although several aspects of the entire reaction need to be elaborated further, the first results obtained indicate that the polymerization of aniline leads to a stable aqueous suspension which is composed of vesicles which are coated with the conductive emeraldine salt form of PANI. Compared to polymers and micelles as templates for the HRP/H₂O₂-catalyzed polymerization of aniline,^5–8 the vesicle system is different since it allows the preparation of hollow polymeric capsules, which is not possible with polymers or micelles as templates. Hollow capsules of conductive polymers have recently attracted the interest of several groups for various potential applications, i.e. for the development of sensor or delivery systems.⁴⁵

Whether these vesicle polymer capsules can be processed, for example to form thin films, remains to be seen. Furthermore, the applicability of vesicles as chemical structure-controlling templates for other enzymatic polymerization reactions needs to be clarified. Based on a more general consideration,⁴ there seems to be a restriction for using chemical structure-controlling templates (polymers, micelles, vesicles, etc.) for those enzymatic reactions in which the growth of the polymer chain does not occur at the active site of the enzyme, similarly to the final steps in the biosynthesis of lignin.⁴ Further investigations toward a better understanding of the reaction mechanism in the vesicle system are currently underway.

Acknowledgements

The authors thank Dr Martin Willeke, Department of Materials, ETH Zürich, for many discussions and for the critical and careful reading of the manuscript. We acknowledge EMEZ (Electron Microscopy Center, ETH Zürich), especially Peter Tittmann and Dr Roger Wepf, for technical support. The financial support by the Swiss National Science Foundation (200021-111696 and 200020-124690) is highly appreciated.

Notes and references

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