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Evaluation of enzymatic formation of polyaniline nanoparticles
Polymer 115 (2017) 211e216
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Evaluation of enzymatic formation of polyaniline nanoparticles Natalija German a, Anton Popov b, Almira Ramanaviciene b, Arunas Ramanavicius c, * a
Department of Immunology, State Research Institute Center for Innovative Medicine, Santariskiu 5, LT-08406, Vilnius, Lithuania NanoTechnas e Centre of Nanoscience and Nanotechnology, Department of Analytical an Environmental Chemistry, Faculty of Chemistry and Geosciences, Vilnius University, Naugarduko 24, LT-03225, Vilnius, Lithuania c Department of Physical Chemistry, Faculty of Chemistry and Geosciences, Vilnius University, Naugarduko 24, LT-03225, Vilnius, Lithuania b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 January 2017 Received in revised form 22 February 2017 Accepted 14 March 2017 Available online 18 March 2017
Polyaniline (PANI) nanoparticles were synthesized by enzymatic polymerization of aniline monomers using glucose oxidase (GOx). The influence of aniline and GOx concentrations as well as the duration of the polymerization on the formation of PANI nanoparticles was investigated. The UV/Vis spectra of polymerization solution showed absorption peaks at 325 and 434 nm, which are characteristic for PANI emeraldine base. The increase of absorption at 434 nm was exploited for the monitoring of PANI formation. The most optimal duration for the formation of PANI nanoparticles was in the frame of 100 e115 h. Extension of the polymerization time does not increased the efficiency of here proposed polymerization procedure. The highest rate of PANI nanoparticle formation was observed in solution containing 0.50 mol L1 of aniline and 0.75 mg mL1 of GOx. Hydrodynamic diameter of synthesized PANI nanoparticles was evaluated using dynamic light scattering technique. Hydrodynamic radius of formed GOx/PANI-nanoparticles increased from 15.8 nm up to 142 nm by prolongation of polymerization process. Synthesized PANI nanoparticles were characterized by cyclic voltammetry and two oxidation peaks at þ0.51 and þ 0.68 V were observed in cyclic voltammograms. These peaks are attributed to emeraldine form of PANI. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Glucose oxidase Polyaniline nanoparticles Spectrometry Polymerization Nanocomposite
1. Introduction Polyaniline (PANI) is one of mostly studied organic conducting polymers that have been developed over the past 30 years [1e3]. PANI is not toxic and relatively stable. Even in aggressive chemical environments polyaniline is characterized by high thermal stability, low manufacturing cost and loss of conductivity only at relatively high temperature [1,4e6]. The electrochemically conductive (103 S cm1) polyaniline could be synthesized by oxidative polymerization [1,7]. Mostly PANI is synthesized by electrochemical [8e10] or chemical [11e14] oxidation of aniline monomers, and it is characterized by pronounced electron donor properties [4]. Formed PANI could be doped by counterions. Usually aniline polymerization reaction is stoichiometric in the number of electrons accepted from aniline monomer [1] and during the process the dopant anion mostly is incorporated into the structure of formed PANI [7]. Electrochemical polymerization is faster at lower pH values [2]. Chemical polymerization/oligomerization of aniline is typically
* Corresponding author. E-mail address: [email protected] (A. Ramanavicius). http://dx.doi.org/10.1016/j.polymer.2017.03.028 0032-3861/© 2017 Elsevier Ltd. All rights reserved.
initiated by relatively strong chemical oxidants such as H2O2, (NH4)2S2O8, which are able to oxidize dissolved aniline what is leading to the formation of cation radicals of aniline [1,12,15,16]. These cation radicals further react with other monomers or oligomers, ensuring formation prolonged polymeric chains [1]. During such synthesis formed PANI mostly is conductive and is characterized by poor solubility in water [4,17]. But in order to avoid this disadvantage a new strategies, which allow to synthesize water soluble PANI, were developed recently [18e22]. The enzymatic polymerization of PANI can be carried out in aqueous solutions [16,18,19]. Protonated and non-protonated secondary amino groups, which are present in aniline monomer, have different reactivity [23], therefore efficiency of enzymatic polymerization of aniline is strongly depended on the pH of polymerization bulk solution [16,18,22,24]. The conducting polyaniline is produced at lower pH (4.0e4.5). More branched and not conducting form of polyaniline is produced at pH 6.0 or at higher values of pH [18]. Depending on the pH applied during synthesis formed PANI could be doped by negatively or positively charged dopants including enzymes, which are used for initiation of polymerization [10]. Additional advantages of enzymatic polymerization are: (i) possible
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control of the reaction kinetics, (ii) simple, one-step procedure based process [18]. The formation of polyaniline-based films [8,9,11,25], colloidal nanoparticles [19], nanocomposites materials [10,15,26,27] and water-soluble PANI-based materials [18,20,24,28] were reported. The polymerization conditions and composition of initial bulk solution are important factors, which affect the size and morphology of formed nanoparticles and other PANI-based nanostructures [1,7,27]. Chains of polyaniline have ordered repeating structure consisting of phenyl rings and secondary amino groups, which are forming a zigzag lying in one plane, and p-electron clouds overlap with other PANI chains above and below this plane [4]. Depending of the ratio amine/imine nitrogen atoms synthesized electroactive polyaniline might be in various oxidation states. PANI exists in three well-defined stable forms: (i) fully reduced (all nitrogen atoms are in amine form) leucoemeraldine form, (ii) 50% oxidized (amine/imine forms ratio is ~0.5) emeraldine form and (iii) fully oxidized (all nitrogen atoms are imine form) pernigraniline form [1,3]. Depending on oxidation state and pH each of these forms can exist in the form of its base or in the form of its protonated (doped) salt, which have different colours (pale yellow-green-blue-violet), stabilities and electric-conductivities [6]. Leucoemeraldine and electronically conducting emeraldine both can be synthesized by standard chemical or electrochemical oxidation [1,4]. Upon further oxidation of leucoemeraldine and emeraldine a fully oxidized pernigraniline can be formed [1]. Leucoemeraldine, pernigraniline or emeraldine forms may be easily turn one to the other one by the redox processes [29]. The emeraldine form is characterized by high conductivity of 100e101 S cm1 [4]. The formation of aggregates between growing chains of polyaniline could lead to incorporation of some ions within formed aggregates [4]. Polyaniline has attracted a great deal of research interest because electrical, electrochemical and optical properties together with its chemical tunability and easy derivatization together with great promise for applications in rechargeable batteries, light-emitting diodes, electrochromic display devices, a corrosion inhibitors [1] and detecting material for sensors [8,11,30,31]. Due to high stability and unique complex of properties, polyaniline could contribute to the development of portable, sensitive biosensors. PANI-based various microfabricated electrochemical biosensors, such as enzymatic-, DNA or immuno-sensors offer many advantages and new possibilities for the determination of biologically important compounds [7,17,32e34]. Polyaniline nanoparticles are used as an electrocatalysts and immobilization matrixes for biomolecules [1,8,11,30], because it has good environmental stability and biocompatibility [9]. It was demonstrated that in the presence of polyaniline mediatorless redox coupling between the electrode and biomolecular components could be achieved [10,11]. Glucose biosensor based on composite consisting of gold nanoparticles and conductive polyaniline nanofibers was developed, where polyaniline film was exploited not only as excellent matrix for enzyme immobilization, but as perm-selective membrane, which was blocking diffusion of interfering species [15]. In the construction of electrochemical biosensors [30] polyaniline on the electrode leads to a smoother, slightly ordered conductive nanostructured film with good mechanical properties [25,30]. In this research GOx/polyaniline nanoparticles were synthesized by polymerization/oligomerization of aniline monomers using hydrogen peroxide, which was formed during glucose oxidase (GOx) catalyzed enzymatic reaction. During this process GOx was encapsulated within formed PANI nanoparticles. One of the aims of presented research was to select the optimal conditions for enzymatic formation of PANI nanoparticles.
2. Materials and methods 2.1. Materials Glucose oxidase (EC 1.1.3.4, type VII, from Aspergillus niger, 208 units mg1 protein) and D-(þ)-glucose were purchased from Fluka (Buchs, Switzerland) and Carl Roth GmbH&Co (Karlsruhe, Germany), respectively. Before investigations glucose solution was allowed to stay overnight for the formation of equilibrium between a and b optical isomers. All other chemicals used in the present study were either analytically pure or of highest quality. All solutions were prepared using deionized water purified with water purification system Millipore S.A. (Molsheim, France). The solution of sodium acetate (SA) buffer (0.05 mol L1 CH3COONa$3H2O) with 0.1 mol L1 KCl was prepared by mixing of sodium acetate trihydrate and potassium chloride, which were obtained from Reanal (Budapest, Hungary) and Lachema (Neratovice, Czech Republic). Ethanol (C2H5OH) was purchased from Vilniaus degtine (Lithuania). Alumina powder (Al2O3, grain diameter 0.3 mm, Type N) was purchased from Electron Microscopy Sciences (Hatfield, USA). Aniline and hydrochloric acid (HCl) were purchased from Merck KGaA (Darmstadt, Germany). Aniline was filtered before measurements through 5 cm column filled by Al2O3 powder to remove coloured components. All solutions were stored between measurements at þ4 C. 2.2. The separation procedure of polyaniline nanoparticles Polyaniline nanoparticles were prepared at room temperature in darkness in the solution of 0.05 mol L1 SA buffer, pH 6.0, 0.05 mol L1 of glucose, 0.50 mol L1 of aniline and 0.75 mg mL1 of GOx. PANI nanoparticles after 21 h lasting synthesis at 20 ± 2 C were separated from the synthesis solution by centrifugation (5 min, 16.1103 g). PANI nanoparticles were washed with (i) C2H5OH; (ii) 0.05 mol L1 SA buffer, pH 6.0; (iii) 0.001 mol L1 HCl or (iiii) 1.0 mol L1 HCl solution. Washing procedure was repeated 3 times and PANI nanoparticles were collected by centrifugation. Then separated and washed PANI nanoparticles were resuspended in SA buffer and used for further investigations. 2.3. The optimization of PANI nanoparticle formation The efficiency of PANI nanoparticles formation depends on the initial aniline and GOx concentrations in polymerization solution. The selection of optimal aniline concentration was performed by changing the aniline concentration from 0.10 to 0.90 mol L1 in the polymerization solution consisting of 0.05 mol L1 SA buffer, pH 6.0, 0.05 mol L1 of glucose and 0.50 mg mL1 of GOx during 21 h period. The optimization of GOx concentration was performed by changing GOx concentration from 0.125 to 0.75 mg mL1 in the polymerization solution with 0.50 mol L1 of aniline. The optimal duration of polymerization was studied changing the polymerization time from 1 to 331 h in 0.05 mol L1 SA buffer, pH 6.0, 0.05 mol L1 of glucose, 0.50 mol L1 of aniline and 0.75 mg mL1 of GOx. The separation and washing of PANI nanoparticles were carried out in the same way as it was described in the previous section. All experiments were performed at 20 ± 2 C. The influence of aniline and GOx concentrations, as well as polymerization duration was evaluated by UV/Vis spectroscopy. 2.4. UV/Vis spectroscopy based monitoring of polyaniline particle formation The absorbance of solutions was investigated by UV/Vis spectrometer Lambda 25 (Shelton, USA) in 300e700 nm wavelength
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range and it was used for the monitoring of PANI nanoparticle formation in the polymerization solution. The UV/Vis spectrograms were monitored in plastic disposable cuvettes of 1 cm optical path length. Between measurements polymerization solutions were stored at 20 ± 2 C. Spectrometric measurements were evaluated and presented using SigmaPlot software. The main steps of enzymatic polymerization and PANI nanoparticle formation are illustrated in graphical abstract and Fig. 1. 2.5. The evaluation of polyaniline nanoparticles formation by dynamic light scattering Enzymatic polymerization of aniline was performed in the solution consisting of 0.50 mol L1 of aniline and 0.75 mg mL1 of GOx for a defined period of time (from 21 to 163 h) at 20 ± 2 C. After synthesis PANI nanoparticles were washed with 0.05 mol L1 SA buffer, pH 6.0, and collected by centrifugation (5 min, 16.1103 g). Washing procedure was performed three times. Size of formed PANI nanoparticles was evaluated by Zetasizer Nano ZS from Malvern (Herrenberg, Germany) equipped with a 633 nm He-Ne laser and operating at 173 angle using dynamic light scattering (DLS). The obtained data was analysed with Dispersion Technology Software version 6.01 from Malvern. DLS investigations were evaluated and presented using SigmaPlot software. 2.6. Investigation of electrode modified with PANI nanoparticles by cycling voltammetry Enzymatic polymerization of aniline (duration of synthesis 112 h) and washing procedure of PANI nanoparticles were performed in the same way as for DLS measurements. Cyclic voltammograms were registered using a computerized potentiostat PGSTAT 30/Autolab from EcoChemie (Utrecht, Netherlands) with GPES 4.9 software in cyclic voltammetry mode at scan rate of 0.10 V s1. A conventional three-electrode system comprising of modified graphite rod as a working electrode, platinum wire as a counter electrode and Ag/AgCl1 (3 mol L KCl) (Metrohm (Herisau, Switzerland)) as a reference electrode was used in cyclic voltammetry based measurements. Graphite rod was cut and polished on fine emery paper. After this PANI nanoparticles were deposited on the surface of electrode surface. Then modified electrode was covered with a polycarbonate membrane with a pore size of 3 mm, which was received from Merck Millipore (Carrigtwohill, Ireland), in order to avoid the detachment of PANI nanoparticles from the surface of electrode. Cyclic voltammogram was performed in
Fig. 1. Schematic representation of polyaniline formation initiated by hydrogen peroxide formed during enzymatic reaction.
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1 mol L1 HCl electrolyte solution at 20 ± 2 C. The electrode potential was swept from 0.20 to þ1.2 V vs Ag/AgCl1 (3 mol L KCl). 3. Results and discussion Water soluble PANI can be prepared using a template guided enzymatic approach [1]. In the presence of glucose and dissolved oxygen the glucose oxidase generates hydrogen peroxide, which oxidizes the polymer chain and gluconolactone that is hydrolyzed to gluconic acid [16,22]. The process of enzymatic polymerization/ oligomerization is based on a mechanism where monomer of aniline is attacked by a radical cation, leading directly to the aniline reaction with the oxidized oligomer [1,22]. In the presented work, H2O2 was produced during catalytic reaction of glucose oxidase creating conditions for the aniline polymerization. According Liu et al. results the optimal pH for the catalytic activity (the oxidation of polyaniline in the complex begins) has been observed at pH about 6.0 [18], when aniline oligomers with different structures are formed in aqueous solution [12,23]. Such oligomers contain ortoand para-coupled units with cyclic phenazine fragments [12]. The synthesis of polyaniline depends on the reaction conditions (solution pH, the type of oxidant, the nature of the acid protonating the aniline and the concentration of reactants). The selected washing solution, the procedure of the separation and the purification of synthesized PANI nanoparticles from the reaction components have got significant influence on further investigations [35]. In order to select suitable washing solution synthesized PANI nanoparticles were centrifuged and washed with (i) C2H5OH; (ii) 0.05 mol L1 SA buffer, pH 6.0; (iii) 0.001 mol L1 HCl or (iiii) 1.0 mol L1 HCl. Washing/centrifugation procedure was repeated 3 times. The degree of successful separation and purification of PANI nanoparticles was evaluated by UV/Vis spectroscopy. Spectrograms of resuspended PANI nanoparticles and washing solution were compared. Any absorbance peaks were not observed using 0.05 mol L1 SA buffer, pH 6.0, and these results confirmed that the selected washing solution and purification procedure of PANI nanoparticles are appropriate. It should be mentioned that investigated C2H5OH, 0.001 and 1.0 mol L1 HCl solutions dissolved polymer in the first stage of the purification. Synthesized, washed and resuspended in SA buffer, pH 6.0, PANI nanoparticles were used in our further investigation. Successful enzymatic polymerization/oligomerization of polymers depends on such factors like the concentration of monomer, glucose oxidase, the duration of polymerization [18,22]. In the present paper the formation of polyaniline nanoparticles was investigated by UV/Vis spectroscopy. It was demonstrated in previous studies that higher oxidant concentration has influence on the higher yield of polymer [27]. In order to improve PANI formation the influence of aniline concentration on the absorbance was evaluated. To achieve best conditions, aniline monomer concentration in 0.05 mol L1 SA buffer, pH 6.0, with 0.05 mol L1 glucose, 0.50 mg mL1 GOx was changed from 0.10 to 0.90 mol L1. Fig. 2A illustrates that registered absorbance of polymerization solution using 0.05 mmol L1 of glucose increased by increasing concentration of aniline and it was highest at 0.5 mol L1 of aniline. The absorbance of synthesis solution containing 0.50 mol L1 of aniline increased 21.5 times if compared with the absorbance registered in the presence 0.10 mol L1 of aniline. The most significant amount of aniline monomers is oxidized by oxidant during the early stage of the polymerization and oligomers of high molecular weight are formed [22]. The polymerization is faster at higher (up to 0.5 mol L1) aniline concentration [18,27], but further increasing of aniline concentration negatively influenced the polyaniline nanoparticles formation. Thus, 0.5 mol L1 of aniline was selected as the most optimal concentration of
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Fig. 2. The effect of aniline (A) and glucose oxidase (B) concentration in the polymerization solution on the registered absorbance in the presence 0.05 mol L1 of glucose. (Polymerization solution: A e 0.05 mol L1 SA buffer, pH 6.0, with 0.05 mol L1 glucose, 0.50 mg mL1 glucose oxidase; B e 0.05 mol L1 SA buffer, pH 6.0, with 0.05 mol L1 glucose, 0.50 mol L1 aniline; polymerization time 21 h. The absorbance was measured in 0.05 mol L1 SA buffer, pH 6.0.)
monomers. In the next set of experiments spectrometric measurements were performed in order to determine the effect of GOx concentration on the rate of PANI formation. Experiment was performed at 0.50 mol L1 of aniline and GOx concentration varying from 0.125 to 0.75 mg mL1. The influence of GOx concentration on the registered absorbance is presented in Fig. 2B. The registered absorbance significantly increased by increasing GOx concentration in the polymerization solution up to 0.75 mg mL1. Absorbance of PANI nanoparticles, which were prepared using 0.75 mg mL1 of GOx, increased by 6.33 times if compared with the absorbance obtained by PANI nanoparticles prepared using 0.125 mg mL1. Thus, the 0.75 mg mL1 of GOx was selected as optimal concentration for PANI nanoparticles formation. The similar effect of aniline and GOx concentration on PANI formation has been reported by other authors who have investigated enzymatic polymerization of aniline [22]. Polymerization time is one of the most important factors for the synthesis of polymer. Thus, the influence of polymerization time on the absorbance during enzymatic polymerization/oligomerization of aniline was evaluated. Enzymatic polymerization of PANI nanoparticles was performed from 1 to 331 h. Initially polymerization solutions were colourless and any absorption peaks were present in the visible part of UV/Vis spectrogram (Fig. 3A). After some time of polymerization solutions turned to yellowish colour. In the early
Fig. 3. Spectrograms of enzymatic polymerization/oligomerization of aniline (A) and the influence of polymerization time on the absorbance at 434 nm (B) in the presence of 0.05 mol L1 of glucose. (Polymerization solution: 0.05 mol L1 SA buffer, pH 6.0, with 0.05 mol L1 of glucose, 0.50 mol L1 of aniline and 0.75 mg mL1 of glucose oxidase. The absorbance was measured in 0.05 mol L1 SA buffer, pH 6.0.)
stage of aniline oxidative polymerization intermediate form of PANI was formed [3,12,13,22,29] and two absorbance peaks at l ¼ 325 nm and l ¼ 434 nm were registered. Sapurina and Stejskal proposed that at initial stage of aniline polymerization the most probable structure is cyclic aniline dimer (5,10-dihydrophenazine) with the absorbance at 380 nm [12]. During the oxidation of 5,10dihydrophenazine phenazylium radical cation was formed, which absorbs at l ¼ 420 nm [12]. The increase of absorbance peak at 434 nm after longer period of synthesis is characteristic for PANI nanoparticles formation. It means that the starting units of the chain of PANI nanoparticles were formed. It was in an agreement with higher concentration of hydrogen peroxide produced during GOx catalyzed enzymatic reaction [16,21,22] initiated formation of multiple PANI branched structure during polaron band transition [20,24,28,29]. Another peak of the absorbance at 325 nm is due to p-p* transition of the benzenoid ring and is attributed to formation of linear polyaniline structure [5,9,28,29]. These peaks indicate that conducting form of enzymatically synthesized polyaniline is similar to obtained by chemical and electrochemical methods [25,28] and are associated with the stabilization of the composite in the emeraldine form [5], which has got a lower oxidation potential than protonated monomer and is oxidized preferentially [23]. Fig. 3B shows that the registered absorbance at 434 nm increased increasing the time of polymerization and it was highest after 331 h of PANI nanoparticles synthesis. The increase of polymerization rate could be explained by the decrease of pH and the increase of hydrogen peroxide concentration [22]. Absorbance registered after 331 h of enzymatic synthesis of PANI increased by 62.4 times in a comparison to the absorbance registered after 1 h. The maximal rate of PANI nanoparticles formation was achieved after 331 h of enzymatic polymerization/oligomerization. However 331 h of polymerization is inconvenient and too long, so it is recommended to perform polymerization up to 115 h. Although the absorbance after 115 h of enzymatic synthesis was 1.61 time lower in comparison with absorbance registered after almost 3 times shorter polymerization, which was lasting for 331 h. Initial aniline conversion into polymer rate (V), which corresponds to tga in Fig. 3B can be calculated using estimated/approximated optical absorbance (approximately 1.25 (a.u.)) of PANI nanoparticle solution formed if 100% of aniline present in initial 0.50 mol L1 aniline monomer solution would be converted into PANI (Fig. 3B). Then initial aniline polymerization rate (V) is equal:
V¼ ðtgaÞ¼ ð0:950:50Þ=ð1:25100Þ¼ 0:0038 mol L1 h1
Fig. 4. The distribution of PANI nanoparticles size after certain period of enzymatic polymerization/oligomerization determined using DLS (A) and the dependence of PANI nanoparticles size on the time of synthesis (B). (Polymerization solution: 0.05 mol L1 SA buffer, pH 6.0, with 0.05 mol L1 of glucose, 0.50 mol L1 of aniline and 0.75 mg mL1 of glucose oxidase. DLS signal was measured in 0.05 mol L1 SA buffer, pH 6.0. A: 1,2,3 and 4 curves e 21, 45, 115 and 163 h of polymerization.)
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The size of PANI nanoparticles formed after certain period of time was evaluated using DLS method. Results presented in Fig. 4 illustrate that the size of polymeric nanoparticles increased by increasing time of the polymerization. PANI nanoparticles were of 15.8 nm after 21 h of enzymatic synthesis, while size of nanoparticles increased up to 142 nm after 163 h of the polymerization. It could be predicted that after 163 h of enzymatic polymerization the aggregates of PANI nanoparticles were formed. It is in agreement with observations of other authors, as larger nanoparticles were obtained at higher pH value [13]. As it is present in Fig. 4A the formation of smaller PANI nanoparticles (115 nm) allows to use shorter (115 h) polymerization time. In order to confirm the electroactive nature of the PANI obtained by GOx catalyzed polymerization, cyclic voltammograms were performed.During enzymatic reaction in the presence of glucose the GOx generates hydrogen peroxide and gluconolactone that is hydrolyzed to gluconic acid. Fig. 5 illustrates the cyclic voltammogram of electrode modified with enzymatically synthesized PANI nanoparticles in the range between 0.20 and þ 1.2 V vs Ag/ AgCl1 (3 mol L KCl). The cyclic voltammogram of polyaniline showed two oxidation waves with peaks at þ0.51 and þ 0.68 V, which are similar to that obtained in acidic medium in previously reported papers [11,18,21]. The first peak at þ0.51 V is not distinctly resolved, but it is reversible. The peak is slightly shifted towards lower potential in comparison to that reported for PANI nanoparticles, which were synthesized enzymatically by glucose oxidase at pH 6.0 [21] or by Horseradish peroxidase at pH 4.1 [36]. It can be related to the formation of branched or cross-linked structures of the polyaniline backbone [21]. It corresponds to the transformation of leucoemeraldine to emeraldine salt [11]. The second PANI oxidation peak, which is observed at þ0.68 V, is well defined and reversible too. The observed peak could be explained as a radical cationic polaron state and a cationic bipolaron state, and it is associated with the formation of p-benzoquinone and hydroquinone as side products [11]. The enzymatic polymerization/oligomerization of aniline is strongly depended on pH, and this pH-dependence is related to the differences of peaks in cyclic voltammograms of PANI, which was formed in aqueous solution of pH 4.0e4.3, reported by other authors [16,24,36]. Recently PANI based nano and micro scale particles have
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attracted much attention through their broad range of applications [15]. The formation, separation and purification of PANI nanoparticles make these particles particularly suitable for the attachment of proteins and other biologically active materials. Nanocomposite structure of polyaniline with gold [15], Fe3O4 [26] nanoparticles could be use as an excellent matrix for electrocatalysis and enzyme immobilization in biosensors and microfabrications because of their novel properties [32]. 4. Conclusions PANI nanoparticles were synthesized by enzymatic polymerization/oligomerization of aniline monomers during enzymatic reaction of glucose oxidase. The concentration of aniline, GOx and the polymerization time showed a significant influence on PANI nanoparticle formation. It was determined that for the efficient formation of PANI nanoparticles is observed in solution containing 0.50 mol L1 of aniline and 0.75 mg mL1 of glucose oxidase. DLSbased measurements showed that size of PANI nanoparticles increased by increasing duration of polymerization. Optimal duration of enzymatic PANI formation was 115 h. Enzymatically synthesized polyaniline was characterized by two oxidation waves with peaks at þ0.51 and þ 0.68 V. Formed PANI nanoparticles were pure from surfactants and interfering species what increases the applicability of synthesized polymeric nanoparticles for various biomedical applications. Many authors synthesized PANI layers and other structures at pH 4.0 or lower, but if enzyme would be incorporated simultaneously during PANI synthesis at such conditions, then formed composite would be not suitable for biosensors due to degradation/ denaturation of enzyme at low pH. The enzyme e glucose oxidase eis also sensitive to extreme pH. In present paper GOx/PANI nanoparticles were formed at pH 6.0 therefore the glucose oxidase remained active. For this reason such GOx/PANI nanoparticles are potentially suitable for the development of biosensor. Another key issue is related to glucose concentration in the samples: (i) the blood-glucose concentration in human being, which is not afflicted by diabetes, in serums is usually in the interval from 4.1 to 5.9 mmol L1; (ii) the range of glucose for people with diabetes is even more wider and it is 2e30 mmol L1 [37]. The Michaelis constant (KM) of GOx for glucose is relatively low therefore this enzyme in native form can be applied for determination of glucose in relatively low concentration range. But in present our manuscript reported ‘wrapping of GOx within PANI layer’ significantly increases the diffusional limitation for glucose and the KM of GOx for glucose. Therefore, glucose determination limit with such entrapped enzyme based bioanalytical can be significantly extended. Acknowledgements The research was supported by Lithuanian Research Council project number SEN e 21/2015. References
Fig. 5. The cyclic voltammogram of electrode modified with enzymatically synthesized polyaniline nanoparticles. (Cyclic voltammogram was registered in 1 mol L1 HCl, v ¼ 0.10 V s1).
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Polymer journal homepage: www.elsevier.com/locate/polymer
Evaluation of enzymatic formation of polyaniline nanoparticles Natalija German a, Anton Popov b, Almira Ramanaviciene b, Arunas Ramanavicius c, * a
Department of Immunology, State Research Institute Center for Innovative Medicine, Santariskiu 5, LT-08406, Vilnius, Lithuania NanoTechnas e Centre of Nanoscience and Nanotechnology, Department of Analytical an Environmental Chemistry, Faculty of Chemistry and Geosciences, Vilnius University, Naugarduko 24, LT-03225, Vilnius, Lithuania c Department of Physical Chemistry, Faculty of Chemistry and Geosciences, Vilnius University, Naugarduko 24, LT-03225, Vilnius, Lithuania b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 January 2017 Received in revised form 22 February 2017 Accepted 14 March 2017 Available online 18 March 2017
Polyaniline (PANI) nanoparticles were synthesized by enzymatic polymerization of aniline monomers using glucose oxidase (GOx). The influence of aniline and GOx concentrations as well as the duration of the polymerization on the formation of PANI nanoparticles was investigated. The UV/Vis spectra of polymerization solution showed absorption peaks at 325 and 434 nm, which are characteristic for PANI emeraldine base. The increase of absorption at 434 nm was exploited for the monitoring of PANI formation. The most optimal duration for the formation of PANI nanoparticles was in the frame of 100 e115 h. Extension of the polymerization time does not increased the efficiency of here proposed polymerization procedure. The highest rate of PANI nanoparticle formation was observed in solution containing 0.50 mol L1 of aniline and 0.75 mg mL1 of GOx. Hydrodynamic diameter of synthesized PANI nanoparticles was evaluated using dynamic light scattering technique. Hydrodynamic radius of formed GOx/PANI-nanoparticles increased from 15.8 nm up to 142 nm by prolongation of polymerization process. Synthesized PANI nanoparticles were characterized by cyclic voltammetry and two oxidation peaks at þ0.51 and þ 0.68 V were observed in cyclic voltammograms. These peaks are attributed to emeraldine form of PANI. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Glucose oxidase Polyaniline nanoparticles Spectrometry Polymerization Nanocomposite
1. Introduction Polyaniline (PANI) is one of mostly studied organic conducting polymers that have been developed over the past 30 years [1e3]. PANI is not toxic and relatively stable. Even in aggressive chemical environments polyaniline is characterized by high thermal stability, low manufacturing cost and loss of conductivity only at relatively high temperature [1,4e6]. The electrochemically conductive (103 S cm1) polyaniline could be synthesized by oxidative polymerization [1,7]. Mostly PANI is synthesized by electrochemical [8e10] or chemical [11e14] oxidation of aniline monomers, and it is characterized by pronounced electron donor properties [4]. Formed PANI could be doped by counterions. Usually aniline polymerization reaction is stoichiometric in the number of electrons accepted from aniline monomer [1] and during the process the dopant anion mostly is incorporated into the structure of formed PANI [7]. Electrochemical polymerization is faster at lower pH values [2]. Chemical polymerization/oligomerization of aniline is typically
* Corresponding author. E-mail address: [email protected] (A. Ramanavicius). http://dx.doi.org/10.1016/j.polymer.2017.03.028 0032-3861/© 2017 Elsevier Ltd. All rights reserved.
initiated by relatively strong chemical oxidants such as H2O2, (NH4)2S2O8, which are able to oxidize dissolved aniline what is leading to the formation of cation radicals of aniline [1,12,15,16]. These cation radicals further react with other monomers or oligomers, ensuring formation prolonged polymeric chains [1]. During such synthesis formed PANI mostly is conductive and is characterized by poor solubility in water [4,17]. But in order to avoid this disadvantage a new strategies, which allow to synthesize water soluble PANI, were developed recently [18e22]. The enzymatic polymerization of PANI can be carried out in aqueous solutions [16,18,19]. Protonated and non-protonated secondary amino groups, which are present in aniline monomer, have different reactivity [23], therefore efficiency of enzymatic polymerization of aniline is strongly depended on the pH of polymerization bulk solution [16,18,22,24]. The conducting polyaniline is produced at lower pH (4.0e4.5). More branched and not conducting form of polyaniline is produced at pH 6.0 or at higher values of pH [18]. Depending on the pH applied during synthesis formed PANI could be doped by negatively or positively charged dopants including enzymes, which are used for initiation of polymerization [10]. Additional advantages of enzymatic polymerization are: (i) possible
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control of the reaction kinetics, (ii) simple, one-step procedure based process [18]. The formation of polyaniline-based films [8,9,11,25], colloidal nanoparticles [19], nanocomposites materials [10,15,26,27] and water-soluble PANI-based materials [18,20,24,28] were reported. The polymerization conditions and composition of initial bulk solution are important factors, which affect the size and morphology of formed nanoparticles and other PANI-based nanostructures [1,7,27]. Chains of polyaniline have ordered repeating structure consisting of phenyl rings and secondary amino groups, which are forming a zigzag lying in one plane, and p-electron clouds overlap with other PANI chains above and below this plane [4]. Depending of the ratio amine/imine nitrogen atoms synthesized electroactive polyaniline might be in various oxidation states. PANI exists in three well-defined stable forms: (i) fully reduced (all nitrogen atoms are in amine form) leucoemeraldine form, (ii) 50% oxidized (amine/imine forms ratio is ~0.5) emeraldine form and (iii) fully oxidized (all nitrogen atoms are imine form) pernigraniline form [1,3]. Depending on oxidation state and pH each of these forms can exist in the form of its base or in the form of its protonated (doped) salt, which have different colours (pale yellow-green-blue-violet), stabilities and electric-conductivities [6]. Leucoemeraldine and electronically conducting emeraldine both can be synthesized by standard chemical or electrochemical oxidation [1,4]. Upon further oxidation of leucoemeraldine and emeraldine a fully oxidized pernigraniline can be formed [1]. Leucoemeraldine, pernigraniline or emeraldine forms may be easily turn one to the other one by the redox processes [29]. The emeraldine form is characterized by high conductivity of 100e101 S cm1 [4]. The formation of aggregates between growing chains of polyaniline could lead to incorporation of some ions within formed aggregates [4]. Polyaniline has attracted a great deal of research interest because electrical, electrochemical and optical properties together with its chemical tunability and easy derivatization together with great promise for applications in rechargeable batteries, light-emitting diodes, electrochromic display devices, a corrosion inhibitors [1] and detecting material for sensors [8,11,30,31]. Due to high stability and unique complex of properties, polyaniline could contribute to the development of portable, sensitive biosensors. PANI-based various microfabricated electrochemical biosensors, such as enzymatic-, DNA or immuno-sensors offer many advantages and new possibilities for the determination of biologically important compounds [7,17,32e34]. Polyaniline nanoparticles are used as an electrocatalysts and immobilization matrixes for biomolecules [1,8,11,30], because it has good environmental stability and biocompatibility [9]. It was demonstrated that in the presence of polyaniline mediatorless redox coupling between the electrode and biomolecular components could be achieved [10,11]. Glucose biosensor based on composite consisting of gold nanoparticles and conductive polyaniline nanofibers was developed, where polyaniline film was exploited not only as excellent matrix for enzyme immobilization, but as perm-selective membrane, which was blocking diffusion of interfering species [15]. In the construction of electrochemical biosensors [30] polyaniline on the electrode leads to a smoother, slightly ordered conductive nanostructured film with good mechanical properties [25,30]. In this research GOx/polyaniline nanoparticles were synthesized by polymerization/oligomerization of aniline monomers using hydrogen peroxide, which was formed during glucose oxidase (GOx) catalyzed enzymatic reaction. During this process GOx was encapsulated within formed PANI nanoparticles. One of the aims of presented research was to select the optimal conditions for enzymatic formation of PANI nanoparticles.
2. Materials and methods 2.1. Materials Glucose oxidase (EC 1.1.3.4, type VII, from Aspergillus niger, 208 units mg1 protein) and D-(þ)-glucose were purchased from Fluka (Buchs, Switzerland) and Carl Roth GmbH&Co (Karlsruhe, Germany), respectively. Before investigations glucose solution was allowed to stay overnight for the formation of equilibrium between a and b optical isomers. All other chemicals used in the present study were either analytically pure or of highest quality. All solutions were prepared using deionized water purified with water purification system Millipore S.A. (Molsheim, France). The solution of sodium acetate (SA) buffer (0.05 mol L1 CH3COONa$3H2O) with 0.1 mol L1 KCl was prepared by mixing of sodium acetate trihydrate and potassium chloride, which were obtained from Reanal (Budapest, Hungary) and Lachema (Neratovice, Czech Republic). Ethanol (C2H5OH) was purchased from Vilniaus degtine (Lithuania). Alumina powder (Al2O3, grain diameter 0.3 mm, Type N) was purchased from Electron Microscopy Sciences (Hatfield, USA). Aniline and hydrochloric acid (HCl) were purchased from Merck KGaA (Darmstadt, Germany). Aniline was filtered before measurements through 5 cm column filled by Al2O3 powder to remove coloured components. All solutions were stored between measurements at þ4 C. 2.2. The separation procedure of polyaniline nanoparticles Polyaniline nanoparticles were prepared at room temperature in darkness in the solution of 0.05 mol L1 SA buffer, pH 6.0, 0.05 mol L1 of glucose, 0.50 mol L1 of aniline and 0.75 mg mL1 of GOx. PANI nanoparticles after 21 h lasting synthesis at 20 ± 2 C were separated from the synthesis solution by centrifugation (5 min, 16.1103 g). PANI nanoparticles were washed with (i) C2H5OH; (ii) 0.05 mol L1 SA buffer, pH 6.0; (iii) 0.001 mol L1 HCl or (iiii) 1.0 mol L1 HCl solution. Washing procedure was repeated 3 times and PANI nanoparticles were collected by centrifugation. Then separated and washed PANI nanoparticles were resuspended in SA buffer and used for further investigations. 2.3. The optimization of PANI nanoparticle formation The efficiency of PANI nanoparticles formation depends on the initial aniline and GOx concentrations in polymerization solution. The selection of optimal aniline concentration was performed by changing the aniline concentration from 0.10 to 0.90 mol L1 in the polymerization solution consisting of 0.05 mol L1 SA buffer, pH 6.0, 0.05 mol L1 of glucose and 0.50 mg mL1 of GOx during 21 h period. The optimization of GOx concentration was performed by changing GOx concentration from 0.125 to 0.75 mg mL1 in the polymerization solution with 0.50 mol L1 of aniline. The optimal duration of polymerization was studied changing the polymerization time from 1 to 331 h in 0.05 mol L1 SA buffer, pH 6.0, 0.05 mol L1 of glucose, 0.50 mol L1 of aniline and 0.75 mg mL1 of GOx. The separation and washing of PANI nanoparticles were carried out in the same way as it was described in the previous section. All experiments were performed at 20 ± 2 C. The influence of aniline and GOx concentrations, as well as polymerization duration was evaluated by UV/Vis spectroscopy. 2.4. UV/Vis spectroscopy based monitoring of polyaniline particle formation The absorbance of solutions was investigated by UV/Vis spectrometer Lambda 25 (Shelton, USA) in 300e700 nm wavelength
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range and it was used for the monitoring of PANI nanoparticle formation in the polymerization solution. The UV/Vis spectrograms were monitored in plastic disposable cuvettes of 1 cm optical path length. Between measurements polymerization solutions were stored at 20 ± 2 C. Spectrometric measurements were evaluated and presented using SigmaPlot software. The main steps of enzymatic polymerization and PANI nanoparticle formation are illustrated in graphical abstract and Fig. 1. 2.5. The evaluation of polyaniline nanoparticles formation by dynamic light scattering Enzymatic polymerization of aniline was performed in the solution consisting of 0.50 mol L1 of aniline and 0.75 mg mL1 of GOx for a defined period of time (from 21 to 163 h) at 20 ± 2 C. After synthesis PANI nanoparticles were washed with 0.05 mol L1 SA buffer, pH 6.0, and collected by centrifugation (5 min, 16.1103 g). Washing procedure was performed three times. Size of formed PANI nanoparticles was evaluated by Zetasizer Nano ZS from Malvern (Herrenberg, Germany) equipped with a 633 nm He-Ne laser and operating at 173 angle using dynamic light scattering (DLS). The obtained data was analysed with Dispersion Technology Software version 6.01 from Malvern. DLS investigations were evaluated and presented using SigmaPlot software. 2.6. Investigation of electrode modified with PANI nanoparticles by cycling voltammetry Enzymatic polymerization of aniline (duration of synthesis 112 h) and washing procedure of PANI nanoparticles were performed in the same way as for DLS measurements. Cyclic voltammograms were registered using a computerized potentiostat PGSTAT 30/Autolab from EcoChemie (Utrecht, Netherlands) with GPES 4.9 software in cyclic voltammetry mode at scan rate of 0.10 V s1. A conventional three-electrode system comprising of modified graphite rod as a working electrode, platinum wire as a counter electrode and Ag/AgCl1 (3 mol L KCl) (Metrohm (Herisau, Switzerland)) as a reference electrode was used in cyclic voltammetry based measurements. Graphite rod was cut and polished on fine emery paper. After this PANI nanoparticles were deposited on the surface of electrode surface. Then modified electrode was covered with a polycarbonate membrane with a pore size of 3 mm, which was received from Merck Millipore (Carrigtwohill, Ireland), in order to avoid the detachment of PANI nanoparticles from the surface of electrode. Cyclic voltammogram was performed in
Fig. 1. Schematic representation of polyaniline formation initiated by hydrogen peroxide formed during enzymatic reaction.
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1 mol L1 HCl electrolyte solution at 20 ± 2 C. The electrode potential was swept from 0.20 to þ1.2 V vs Ag/AgCl1 (3 mol L KCl). 3. Results and discussion Water soluble PANI can be prepared using a template guided enzymatic approach [1]. In the presence of glucose and dissolved oxygen the glucose oxidase generates hydrogen peroxide, which oxidizes the polymer chain and gluconolactone that is hydrolyzed to gluconic acid [16,22]. The process of enzymatic polymerization/ oligomerization is based on a mechanism where monomer of aniline is attacked by a radical cation, leading directly to the aniline reaction with the oxidized oligomer [1,22]. In the presented work, H2O2 was produced during catalytic reaction of glucose oxidase creating conditions for the aniline polymerization. According Liu et al. results the optimal pH for the catalytic activity (the oxidation of polyaniline in the complex begins) has been observed at pH about 6.0 [18], when aniline oligomers with different structures are formed in aqueous solution [12,23]. Such oligomers contain ortoand para-coupled units with cyclic phenazine fragments [12]. The synthesis of polyaniline depends on the reaction conditions (solution pH, the type of oxidant, the nature of the acid protonating the aniline and the concentration of reactants). The selected washing solution, the procedure of the separation and the purification of synthesized PANI nanoparticles from the reaction components have got significant influence on further investigations [35]. In order to select suitable washing solution synthesized PANI nanoparticles were centrifuged and washed with (i) C2H5OH; (ii) 0.05 mol L1 SA buffer, pH 6.0; (iii) 0.001 mol L1 HCl or (iiii) 1.0 mol L1 HCl. Washing/centrifugation procedure was repeated 3 times. The degree of successful separation and purification of PANI nanoparticles was evaluated by UV/Vis spectroscopy. Spectrograms of resuspended PANI nanoparticles and washing solution were compared. Any absorbance peaks were not observed using 0.05 mol L1 SA buffer, pH 6.0, and these results confirmed that the selected washing solution and purification procedure of PANI nanoparticles are appropriate. It should be mentioned that investigated C2H5OH, 0.001 and 1.0 mol L1 HCl solutions dissolved polymer in the first stage of the purification. Synthesized, washed and resuspended in SA buffer, pH 6.0, PANI nanoparticles were used in our further investigation. Successful enzymatic polymerization/oligomerization of polymers depends on such factors like the concentration of monomer, glucose oxidase, the duration of polymerization [18,22]. In the present paper the formation of polyaniline nanoparticles was investigated by UV/Vis spectroscopy. It was demonstrated in previous studies that higher oxidant concentration has influence on the higher yield of polymer [27]. In order to improve PANI formation the influence of aniline concentration on the absorbance was evaluated. To achieve best conditions, aniline monomer concentration in 0.05 mol L1 SA buffer, pH 6.0, with 0.05 mol L1 glucose, 0.50 mg mL1 GOx was changed from 0.10 to 0.90 mol L1. Fig. 2A illustrates that registered absorbance of polymerization solution using 0.05 mmol L1 of glucose increased by increasing concentration of aniline and it was highest at 0.5 mol L1 of aniline. The absorbance of synthesis solution containing 0.50 mol L1 of aniline increased 21.5 times if compared with the absorbance registered in the presence 0.10 mol L1 of aniline. The most significant amount of aniline monomers is oxidized by oxidant during the early stage of the polymerization and oligomers of high molecular weight are formed [22]. The polymerization is faster at higher (up to 0.5 mol L1) aniline concentration [18,27], but further increasing of aniline concentration negatively influenced the polyaniline nanoparticles formation. Thus, 0.5 mol L1 of aniline was selected as the most optimal concentration of
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Fig. 2. The effect of aniline (A) and glucose oxidase (B) concentration in the polymerization solution on the registered absorbance in the presence 0.05 mol L1 of glucose. (Polymerization solution: A e 0.05 mol L1 SA buffer, pH 6.0, with 0.05 mol L1 glucose, 0.50 mg mL1 glucose oxidase; B e 0.05 mol L1 SA buffer, pH 6.0, with 0.05 mol L1 glucose, 0.50 mol L1 aniline; polymerization time 21 h. The absorbance was measured in 0.05 mol L1 SA buffer, pH 6.0.)
monomers. In the next set of experiments spectrometric measurements were performed in order to determine the effect of GOx concentration on the rate of PANI formation. Experiment was performed at 0.50 mol L1 of aniline and GOx concentration varying from 0.125 to 0.75 mg mL1. The influence of GOx concentration on the registered absorbance is presented in Fig. 2B. The registered absorbance significantly increased by increasing GOx concentration in the polymerization solution up to 0.75 mg mL1. Absorbance of PANI nanoparticles, which were prepared using 0.75 mg mL1 of GOx, increased by 6.33 times if compared with the absorbance obtained by PANI nanoparticles prepared using 0.125 mg mL1. Thus, the 0.75 mg mL1 of GOx was selected as optimal concentration for PANI nanoparticles formation. The similar effect of aniline and GOx concentration on PANI formation has been reported by other authors who have investigated enzymatic polymerization of aniline [22]. Polymerization time is one of the most important factors for the synthesis of polymer. Thus, the influence of polymerization time on the absorbance during enzymatic polymerization/oligomerization of aniline was evaluated. Enzymatic polymerization of PANI nanoparticles was performed from 1 to 331 h. Initially polymerization solutions were colourless and any absorption peaks were present in the visible part of UV/Vis spectrogram (Fig. 3A). After some time of polymerization solutions turned to yellowish colour. In the early
Fig. 3. Spectrograms of enzymatic polymerization/oligomerization of aniline (A) and the influence of polymerization time on the absorbance at 434 nm (B) in the presence of 0.05 mol L1 of glucose. (Polymerization solution: 0.05 mol L1 SA buffer, pH 6.0, with 0.05 mol L1 of glucose, 0.50 mol L1 of aniline and 0.75 mg mL1 of glucose oxidase. The absorbance was measured in 0.05 mol L1 SA buffer, pH 6.0.)
stage of aniline oxidative polymerization intermediate form of PANI was formed [3,12,13,22,29] and two absorbance peaks at l ¼ 325 nm and l ¼ 434 nm were registered. Sapurina and Stejskal proposed that at initial stage of aniline polymerization the most probable structure is cyclic aniline dimer (5,10-dihydrophenazine) with the absorbance at 380 nm [12]. During the oxidation of 5,10dihydrophenazine phenazylium radical cation was formed, which absorbs at l ¼ 420 nm [12]. The increase of absorbance peak at 434 nm after longer period of synthesis is characteristic for PANI nanoparticles formation. It means that the starting units of the chain of PANI nanoparticles were formed. It was in an agreement with higher concentration of hydrogen peroxide produced during GOx catalyzed enzymatic reaction [16,21,22] initiated formation of multiple PANI branched structure during polaron band transition [20,24,28,29]. Another peak of the absorbance at 325 nm is due to p-p* transition of the benzenoid ring and is attributed to formation of linear polyaniline structure [5,9,28,29]. These peaks indicate that conducting form of enzymatically synthesized polyaniline is similar to obtained by chemical and electrochemical methods [25,28] and are associated with the stabilization of the composite in the emeraldine form [5], which has got a lower oxidation potential than protonated monomer and is oxidized preferentially [23]. Fig. 3B shows that the registered absorbance at 434 nm increased increasing the time of polymerization and it was highest after 331 h of PANI nanoparticles synthesis. The increase of polymerization rate could be explained by the decrease of pH and the increase of hydrogen peroxide concentration [22]. Absorbance registered after 331 h of enzymatic synthesis of PANI increased by 62.4 times in a comparison to the absorbance registered after 1 h. The maximal rate of PANI nanoparticles formation was achieved after 331 h of enzymatic polymerization/oligomerization. However 331 h of polymerization is inconvenient and too long, so it is recommended to perform polymerization up to 115 h. Although the absorbance after 115 h of enzymatic synthesis was 1.61 time lower in comparison with absorbance registered after almost 3 times shorter polymerization, which was lasting for 331 h. Initial aniline conversion into polymer rate (V), which corresponds to tga in Fig. 3B can be calculated using estimated/approximated optical absorbance (approximately 1.25 (a.u.)) of PANI nanoparticle solution formed if 100% of aniline present in initial 0.50 mol L1 aniline monomer solution would be converted into PANI (Fig. 3B). Then initial aniline polymerization rate (V) is equal:
V¼ ðtgaÞ¼ ð0:950:50Þ=ð1:25100Þ¼ 0:0038 mol L1 h1
Fig. 4. The distribution of PANI nanoparticles size after certain period of enzymatic polymerization/oligomerization determined using DLS (A) and the dependence of PANI nanoparticles size on the time of synthesis (B). (Polymerization solution: 0.05 mol L1 SA buffer, pH 6.0, with 0.05 mol L1 of glucose, 0.50 mol L1 of aniline and 0.75 mg mL1 of glucose oxidase. DLS signal was measured in 0.05 mol L1 SA buffer, pH 6.0. A: 1,2,3 and 4 curves e 21, 45, 115 and 163 h of polymerization.)
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The size of PANI nanoparticles formed after certain period of time was evaluated using DLS method. Results presented in Fig. 4 illustrate that the size of polymeric nanoparticles increased by increasing time of the polymerization. PANI nanoparticles were of 15.8 nm after 21 h of enzymatic synthesis, while size of nanoparticles increased up to 142 nm after 163 h of the polymerization. It could be predicted that after 163 h of enzymatic polymerization the aggregates of PANI nanoparticles were formed. It is in agreement with observations of other authors, as larger nanoparticles were obtained at higher pH value [13]. As it is present in Fig. 4A the formation of smaller PANI nanoparticles (115 nm) allows to use shorter (115 h) polymerization time. In order to confirm the electroactive nature of the PANI obtained by GOx catalyzed polymerization, cyclic voltammograms were performed.During enzymatic reaction in the presence of glucose the GOx generates hydrogen peroxide and gluconolactone that is hydrolyzed to gluconic acid. Fig. 5 illustrates the cyclic voltammogram of electrode modified with enzymatically synthesized PANI nanoparticles in the range between 0.20 and þ 1.2 V vs Ag/ AgCl1 (3 mol L KCl). The cyclic voltammogram of polyaniline showed two oxidation waves with peaks at þ0.51 and þ 0.68 V, which are similar to that obtained in acidic medium in previously reported papers [11,18,21]. The first peak at þ0.51 V is not distinctly resolved, but it is reversible. The peak is slightly shifted towards lower potential in comparison to that reported for PANI nanoparticles, which were synthesized enzymatically by glucose oxidase at pH 6.0 [21] or by Horseradish peroxidase at pH 4.1 [36]. It can be related to the formation of branched or cross-linked structures of the polyaniline backbone [21]. It corresponds to the transformation of leucoemeraldine to emeraldine salt [11]. The second PANI oxidation peak, which is observed at þ0.68 V, is well defined and reversible too. The observed peak could be explained as a radical cationic polaron state and a cationic bipolaron state, and it is associated with the formation of p-benzoquinone and hydroquinone as side products [11]. The enzymatic polymerization/oligomerization of aniline is strongly depended on pH, and this pH-dependence is related to the differences of peaks in cyclic voltammograms of PANI, which was formed in aqueous solution of pH 4.0e4.3, reported by other authors [16,24,36]. Recently PANI based nano and micro scale particles have
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attracted much attention through their broad range of applications [15]. The formation, separation and purification of PANI nanoparticles make these particles particularly suitable for the attachment of proteins and other biologically active materials. Nanocomposite structure of polyaniline with gold [15], Fe3O4 [26] nanoparticles could be use as an excellent matrix for electrocatalysis and enzyme immobilization in biosensors and microfabrications because of their novel properties [32]. 4. Conclusions PANI nanoparticles were synthesized by enzymatic polymerization/oligomerization of aniline monomers during enzymatic reaction of glucose oxidase. The concentration of aniline, GOx and the polymerization time showed a significant influence on PANI nanoparticle formation. It was determined that for the efficient formation of PANI nanoparticles is observed in solution containing 0.50 mol L1 of aniline and 0.75 mg mL1 of glucose oxidase. DLSbased measurements showed that size of PANI nanoparticles increased by increasing duration of polymerization. Optimal duration of enzymatic PANI formation was 115 h. Enzymatically synthesized polyaniline was characterized by two oxidation waves with peaks at þ0.51 and þ 0.68 V. Formed PANI nanoparticles were pure from surfactants and interfering species what increases the applicability of synthesized polymeric nanoparticles for various biomedical applications. Many authors synthesized PANI layers and other structures at pH 4.0 or lower, but if enzyme would be incorporated simultaneously during PANI synthesis at such conditions, then formed composite would be not suitable for biosensors due to degradation/ denaturation of enzyme at low pH. The enzyme e glucose oxidase eis also sensitive to extreme pH. In present paper GOx/PANI nanoparticles were formed at pH 6.0 therefore the glucose oxidase remained active. For this reason such GOx/PANI nanoparticles are potentially suitable for the development of biosensor. Another key issue is related to glucose concentration in the samples: (i) the blood-glucose concentration in human being, which is not afflicted by diabetes, in serums is usually in the interval from 4.1 to 5.9 mmol L1; (ii) the range of glucose for people with diabetes is even more wider and it is 2e30 mmol L1 [37]. The Michaelis constant (KM) of GOx for glucose is relatively low therefore this enzyme in native form can be applied for determination of glucose in relatively low concentration range. But in present our manuscript reported ‘wrapping of GOx within PANI layer’ significantly increases the diffusional limitation for glucose and the KM of GOx for glucose. Therefore, glucose determination limit with such entrapped enzyme based bioanalytical can be significantly extended. Acknowledgements The research was supported by Lithuanian Research Council project number SEN e 21/2015. References
Fig. 5. The cyclic voltammogram of electrode modified with enzymatically synthesized polyaniline nanoparticles. (Cyclic voltammogram was registered in 1 mol L1 HCl, v ¼ 0.10 V s1).
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