Enzymatic Formation of Polyaniline, Polypyrrole, and Polythiophene Nanoparticles with Embedded Glucose Oxidase

nanomaterials

Article Enzymatic Formation of Polyaniline, Polypyrrole, and Polythiophene Nanoparticles with Embedded Glucose Oxidase

Natalija German 1,2, Anton Popov 1 , Almira Ramanaviciene 1 and Arunas Ramanavicius 3,4,*

1 NanoTechnas – Center of Nanotechnology and Materials Science, Faculty of Chemistry and Geosciences, Vilnius University, Naugarduko 24, LT-03225 Vilnius, Lithuania; [email protected] (N.G.); [email protected] (A.P.); [email protected] (A.R.) 2 Department of Immunology, State Research Institute Centre for Innovative Medicine, Santariskiu 5, LT-08406 Vilnius, Lithuania 3 Department of Physical Chemistry, Faculty of Chemistry and Geosciences, Vilnius University, Naugarduko 24, LT-03225 Vilnius, Lithuania 4 Division of Materials Science and Electronics, State Scientiﬁc Research Institute Center for Physical Sciences and Technology, Savanoriu˛ave. 231, LT-02300 Vilnius, Lithuania * Correspondence: [email protected]

Received: 13 May 2019; Accepted: 24 May 2019; Published: 27 May 2019

Abstract: Polyaniline (PANI), polypyrrole (Ppy), and polythiophene (PTh) composite nanoparticles with embedded glucose oxidase (GOx) were formed by enzymatic polymerization of corresponding monomers (aniline, pyrrole, and thiophene). The influence of monomers concentration, the pH of solution, and the ratio of enzyme/substrate on the formation of PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles were spectrophotometrically investigated. The highest formation rate of PANI-, Ppy-, and PTh-based nanoparticles with embedded GOx was observed in the sodium acetate buffer solution, pH 6.0. The increase of optical absorbance at λmax = 440 nm, λmax = 460 nm, and λmax = 450 nm was exploited for the monitoring of PANI/GOx, Ppy/GOx and PTh/GOx formation, respectively. It was determined that the highest polymerization rate of PANI/GOx, Ppy/GOx, and 1 PTh/GOx composite nanoparticles was achieved in solution containing 0.75 mg mL− of GOx and 1 0.05 mol L− of glucose. The influence of the enzymatic polymerization duration on the formation of PANI/GOx and Ppy/GOx composite nanoparticles was spectrophotometrically investigated. The most optimal duration for the enzymatic synthesis of PANI/GOx and Ppy/GOx composite nanoparticles was in the range of 48–96 h. It was determined that the diameter of formed PANI/GOx and Ppy/GOx composite nanoparticles depends on the duration of polymerization using dynamic light scattering technique (DLS), and it was in the range of 41–167 nm and 65–122 nm, when polymerization lasted from 16 to 120 h.

Keywords: glucose oxidase; polyaniline; polypyrrole; polythiophene; nanoparticles; polymerization; spectrophotometry

1. Introduction Conducting polymers (CPs) such as polyaniline (PANI),polypyrrole (Ppy), and polythiothene (PTh) are very attractive due to the number of technological advantages [1–4]. The electrical conductivity of PTh, Ppy, and PANI reaches 10, 1 102, and 1 103 S cm 1, respectively [1,5–8]. Therefore, × × − nanocomposites based on these CPs are especially interesting from the applicability-related point of view [9–13], because these CPs are nontoxic and stable in aggressive chemical environments.

Nanomaterials 2019, 9, 806; doi:10.3390/nano9050806 www.mdpi.com/journal/nanomaterials Nanomaterials 2019, 9, 806 2 of 16

In addition, CPs possess unique electrical, mechanical, and charge transfer properties, which are also very attractive for many practical applications [1,14–16]. Most of the CPs are insoluble in the common organic solvents, which are explained by favourable interactions between π-systems of proximal polymeric chains [7,17]. Such interactions are stronger than between the polymer and the solvent; therefore, CPs are forming nanoparticles and/or larger aggregates [17]. PANI, Ppy, and PTh can be dependently doped by corresponding ions on the pH value of polymerization bulk solution during oxidation or reduction processes [1,5,6,18–20]. Novel strategies for the enzymatic synthesis of PANI [21,22], Ppy [23–26], and PTh [27] were developed during the last decade. In this context, the enzymatic polymerization seems to be very attractive method, which is suitable for the formation of CPs, because it is carried out in neutral aqueous solutions containing redox enzyme [21,22,24,25,27]. PANI is commonly used in the fabrication of electrochemical biosensors, due to controllable conductivity, good processability, controllable degree of protonation, charge transfer capability, good biocompatibility, and relatively good environmental stability [8,20,28]. PANI exists in three well-defined oxidation states: leucoemeraldine, emeraldine, and pernigraniline [1,19]. The oxidation/reduction of PANI depend on the pH value. PANI can also be in the form of bases or protonated salts, which are characterized by different colours (yellow, green, blue, or violet) and conductivities [19]. The amine and imine groups of PANI can be protonated or deprotonated depending on the pH values [19]. 1 Conducting (4–10 S cm− ) PANI is produced during chemical polymerization in the solution of high acidity (pH < 2.5). The morphology of formed PANI composite depends on the pH value [5,8,19]. In the pH range of 2.5 to 4.6, a considerable part of aniline is protonated, but the imine groups in polymeric chains still remain unprotonated. Therefore, oxidative processes are attenuated and the rate of proton release is decreased [6]. PANI presents in the form of neutral molecules and it can be more easily oxidized than anilinium cation, which dominates at higher acidity (pH < 1.0) under the conditions of low acidity (pH 4.6) [5,28]. Oxidized terminal imine groups of PANI replace hydrogen atoms in benzene rings of the neutral aniline molecules, then the polymerization reaction is occurring at pH > 4.6. Furthermore, PANI is not formed in regular polymer structure, but in the form of oligomers of different structures, based on orto- and para-coupled units with cyclic phenazine fragments [6,19]. One of the most widely investigated CPs is Ppy, which is characterized by a relatively stable electrical conductivity and it can be formed by electrochemical synthesis at low oxidation potentials and in solutions of neutral pH [7,18,20,24,26]. Ppy in aqueous solution can be synthesized by various methods and it can be used in the design of biosensors that are based on immobilized enzymes, antibodies, or DNA [26,29–31]. Black oligomers of pyrrole start to form solid particles and then the polymerization is continuing on the surface of such polymeric particles [18]. Some cross-linked Ppy-based structures are formed after a certain time of polymerization reaction [18]. Generated by the oxidation of the pyrrole cation radicals are deprotonated and neutral radicals are formed during chemical polymerization [18]. Conducting polymer PTh has a great potential for the application in the fields of organic electronics, photovoltaic devices, solar cells, gas, and biological sensors [16,32,33]. High environmental stability, electrical conductivity, useful redox, and optical properties characterize PTh [13,15,16,26,33,34]. The molecule of PTh has π-π conjugated sp2 hybridized backbone [32]. Electrochemical [16,35], chemical [16,34], and enzymatic [27] polymerization based methods are used for PTh formation. The polymerization of thiophene is relatively easy and it proceeds via electrochemical polymerization, chemical oxidative polymerization, and Suzuki polycondensation based routes [16,32]. During the polymerization, firstly, the thiophene monomer is oxidized to a cation and was combined with another ionized or electrostatically neutral monomer’s molecule [16]. The dimer is further connected with another monomer by the same route [16]. Oxidized and reduced forms of PTh are red/brown and blue coloured, respectively [34]. The structure of PTh is mostly regular. The formation of soluble PTh is still a challenge [12,13], because PTh is usually formed in insoluble form, even if polymerization is Nanomaterials 2019, 9, 806 3 of 16 performed in organic solutions [32,34–36]. Depending on polymerization conditions [12,32], PTh can be synthesized in form of spherical nanoparticles [12,13,36], thin films [34,35,37], or nanocomposites [34]. Enzymatic polymerization has been proven by PANI formation while using enzymes (glucose oxidase [21], horseradish peroxidase [22,38], and laccase [38], which catalyse the generation of respective aniline free radicals [19]. Peroxidases contain a heme cofactor in their active sites, or alternatively redox-active cysteine or seleno-cysteine residues [38]. The action of peroxidases mostly is based (i) on a two electron transfer step or (ii) on two clearly observable subsequent single electron transfer steps, which enable the reduction of peroxides to water or alcohols [38]. The most optimal substrate is hydrogen peroxide for many peroxidases, which is produced in some biological oxidation reactions [22,38]. The hydrogen peroxide acts as an oxidizer in oxidative-polymerization reactions, which are used for formation of conducting polymers, which is generating cation-radicals that tends to form polymeric structures [18,19,27]. Glucose oxidase (GOx) was used for the enzymatic formation of PANI, Ppy, and PTh composite nanoparticles in this research, with embedded GOx (PANI/GOx, Ppy/GOx, and PTh/GOx), respectively. The effect of pH, the ratio of enzyme/substrate on the formation of these composite nanoparticles and the influence of polymerization duration on the formation of PANI/GOx, Ppy/GOx were investigated by UV/Vis spectrophotometry. The diameter of the PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles was evaluated while using the dynamic light scattering (DLS) technique.

2. Materials and Methods

2.1. Materials

1 Glucose oxidase (EC 1.1.3.4, type VII, from Aspergillus niger, 201 units mg− protein) and D-(+)-glucose were purchased from Fluka (Buchs, Switzerland) and Carl Roth GmbH + Co.KG (Karlsruhe, Germany). Glucose solution was allowed to mutarotate for 24 h before all of the investigations, and the equilibrium between α and β optical isomers was established during this time. All other chemicals that were used in present study were either analytically pure or of highest quality. All of the solutions were prepared using deionized water. The solution of sodium acetate (SA) buffer (0.05 mol L 1 CH COONa 3H O) with 0.1 mol L 1 KCl was prepared by the mixing of sodium acetate − 3 · 2 − trihydrate and potassium chloride, which both were purchased from Reanal (Budapest, Hungary) and Lachema (Neratovice, Czech Republic). Alfa alumina powder (Al2O3, grain diameter 0.3 micron, Type N) was purchased from Electron Microscopy Sciences (Hatfield, MA, USA). Aniline, sodium hydroxide (NaOH), and thiophene were purchased from Merck KGaA (Darmstadt, Germany), pyrrole — from Acros Organics (New Jersey, NJ, USA) and hydrochloric acid (HCl) — from Sigma-Aldrich (Saint Louis, MO, USA). The polymers were filtered before measurements through 5 cm column that was filled by Al2O3 powder to remove the coloured components. All of the solutions were stored between measurements at +4 ◦C.

2.2. The Formation and Separation of PANI/GOx, Ppy/GOx and PTh/GOx Composite Nanoparticles PANI, Ppy, and PTh composite nanoparticles with embedded GOx were synthesized at room temperature (+20 2 ◦C) in darkness while using polymerization bulk solution containing 0.05 1 ± 1 1 mol L− of glucose, 0.75 mg mL− of GOx, and 0.50 mol L− of aniline, pyrrole, or thiophene. The enzymatically formed polymeric nanoparticles were separated from the polymerization bulk solution by the centrifugation (6 min, 16.1 103 g) with IEC CL31R Multispeed centrifuge (ZI Aze × Bellitourne, France). Subsequently, PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles were 1 washed two times with 0.05 mol L− SA buﬀer, pH 6.0, and they were collected by the centrifugation. Separated and washed polymeric nanoparticles with embedded GOx were resuspended in SA buﬀer, pH 6.0, and used for further investigations. All of the experiments were performed at +20 2 C. Figure1 ± ◦ illustrates the main steps of enzymatic polymerization and the formation of polymeric nanoparticles. Nanomaterials 2019, 9, 806 4 of 16

2.3. The Optimization of PANI/GOx, Ppy/GOx and PTh/GOx Composite Nanoparticles Formation Conditions The efficiency of polymeric nanoparticles formation depends on the pH of solution and the duration of enzymatic polymerization. The optimal pH value for the formation of PANI/GOx, Ppy/GOx and PTh/GOx composite nanoparticles was studied in pH range from pH 1.0 to 12. Polymerization 1 1 bulk solution containing 0.05 mol L− of glucose, 0.50 mol L− of polymerizable monomers (aniline, 1 pyrrole, or thiophene), and 0.75 mg mL− of GOx was used for 48 h lasting polymerization. Deionized 1 1 6 4 water, 0.1, 0.01, or 0.001 mol L− HCl solutions, 0.05 mol L− SA buffer, pH 6.0, and 1 10− , 1 10− 1 × × or 0.01 mol L− NaOH solutions were used to prepare solutions in pH range between 1.0 and 12. During 48 h lasting polymerization, several concentrations of glucose (were 0.015, 0.02, 0.03, 0.04, 0.05, 1 1 1 0.06 and 0.1 mol L− ) were tested in the 0.05 mol L− SA buffer, pH 6.0, containing 0.75 mg mL− of 1 GOx and 0.50 mol L− of aniline, pyrrole, or thiophene. The optimal duration of polymerization was 1 determined by varying the duration of polymerization from 5 to 288 h in 0.05 mol L− SA buffer, pH 6.0, 1 1 1 with 0.05 mol L− of glucose, 0.50 mol L− of aniline or pyrrole, and 0.75 mg mL− of GOx. A UV/Vis spectrophotometer was used to perform the investigations.

2.4. The Monitoring of PANI/GOx, Ppy/GOx, and PTh/GOx Formation by UV/Vis Spectrophotometry The UV/Vis spectrophotometer Lambda 25 (Shelton, CT, USA), which operates in 230–1000 nm wavelength range, was used for the registration of optical absorbance spectra of the formed PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles colloidal solution. The measurements were performed at room temperature in plastic disposable cuvettes of 1 cm optical path length. The optical absorbance of PANI/GOx, Ppy/GOx, and PTh/GOx solutions was registered after the polymerization reaction. UV/Vis measurements were evaluated and presented using SigmaPlot software 12.5 (San Jose, CA, USA).

2.5. The Evaluation of PANI/GOx, Ppy/GOx, and PTh/GOx Composite Nanoparticles Formation by DLS The influence of solution’s pH on the diameter and zeta-potential of PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles was studied in pH range from 2.0 to 8.0 at +20 2 C. In this ± ◦ experiment, 0.01 or 0.001 mol L 1 HCl, 0.05 mol L 1 SA buffer, pH 6.0, and 1 10 6 mol L 1 NaOH − − × − − were used to prepare solutions in pH range between 2.0 and 8.0. Polymerization solutions containing 1 1 1 0.05 mol L− of glucose, 0.50 mol L− of aniline, pyrrole, or thiophene, and 0.75 mg mL− of GOx were monitored during 15–120 h lasting polymerization. Polymeric nanoparticles after enzymatic synthesis were separated, washed two times with 0.05 mol L 1 SA buffer, pH 6.0, and collected by the centrifugation (6 min, 16.1 103 g), as it was − × described in chapter 2.2. The diameter and zeta-potential of PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles were evaluated by DLS and microelectrophoresis, while using the Zetasizer Nano ZS from Malvern (Herrenberg, Germany) equipped with a 633 nm He-Ne laser. The obtained data was analysed with Dispersion Technology Software version 6.01 from Malvern. DLS and microelectrophoresis investigations were evaluated and visualized by SigmaPlot software 12.5.

2.6. Imaging of PANI/GOx and Ppy/GOx Composite Nanoparticles by field Emission Scanning Microscope FE-SEM The enzymatic polymerization of the PANI/GOx and Ppy/GOx composite nanoparticles was proceeded in darkness at room temperature within 24 h. The procedure of the formation, separation, washing, and collection of nanoparticles was similarly performed, as it was described in chapter 2.2. To reduce the effect of dissolved salts, colloidal solutions of composite nanoparticles were diluted in 50 µL of deionized water and homogenized using an ultrasound Bandelin Sonorex RK 31 H (Berlin, Germany). Afterwards, 6 µL of nanoparticle solution was placed on the surface of graphite rod and dried, this procedure was repeated eight times, and then the surface was characterized by Hitachi SU-70 field emission scanning microscope equipped by energy dispersive X-ray spectrometry EDS Nanomaterials 2019, 9, 806 5 of 16

Nanomaterials 2019, 9, x FOR PEER REVIEW 5 of 16 and electron backscatter diﬀraction (EBSD) modules from Hitachi (Dublin, Ireland). Turbosputer was appliedand for electron the preparation backscatter diffraction of synthesized (EBSD) CPs module for SEM-baseds from Hitachi imaging. (Dublin, Ireland). Turbosputer was applied for the preparation of synthesized CPs for SEM-based imaging. 3. Results and Discussion 3. Results and Discussion 3.1. The Inﬂuence of Polymerization Solution pH on PANI/GOx, Ppy/GOx and PTh/GOx Composite Nanoparticles3.1. The Influence Formation of Polymerization Solution pH on PANI/GOx, Ppy/GOx and PTh/GOx Composite Nanoparticles Formation The large number of research papers is dealing with the chemical and electrochemical The large number of research papers is dealing with the chemical and electrochemical polymerizationpolymerization of aniline,of aniline, pyrrole, pyrrole, and and thiophene, thiophene, but only only few few researches researches are are dealing dealing with with the the enzymaticenzymatic formation formation of theseof these CPs. CPs. The The enzymaticenzymatic formation formation of of these these CPs CPs can can be bebased based on the on the applicationapplication of four of majorfour major compounds: compounds: corresponding corresponding monomer, monomer, glucose glucose oxidase, oxidase, glucose, glucose, and and oxygen, whichoxygen, is naturally which dissolved is naturally in dissolved used solution in used (Figure solution1). (Figure 1).

FigureFigure 1. The 1. The formation formation of compositeof composite conducting conducting polymerpolymer nanoparticles nanoparticles with with embedded embedded glucose glucose oxidaseoxidase (GOx) (GOx) by enzymatic by enzymatic polymerization polymerization and and the the schematic schematic presentation presentation of of polyanilinepolyaniline (PANI) (A), (A), polypyrrole (Ppy) (B), and polythiothene (PTh) (C) polymerization mechanisms. polypyrrole (Ppy) (B), and polythiothene (PTh) (C) polymerization mechanisms. GOx generates hydrogen peroxide and gluconolactone in the presence of glucose and dissolved GOx generates hydrogen peroxide and gluconolactone in the presence of glucose and dissolved oxygen, which is hydrolyzed to gluconic acid in an aqueous solution. Hydrogen peroxide as a strong oxygen, which is hydrolyzed to gluconic acid in an aqueous solution. Hydrogen peroxide as a strong oxidizer generates radical cation of pyrrole, aniline [18,19,25], and, in such a way, it initiates the oxidizerpolymerization generates radicalreaction cationin acidic of environment pyrrole, aniline that is created [18,19, 25by], gluconic and, in acid such [27]. a These way, free it initiates radical the polymerizationcations undergo reaction coupling in acidic and, environment in this way, oligom that isers created and polymeric by gluconic structures acid [ 27are]. formed These free[18,19]. radical cationsThe undergo diffusional coupling permeability and, in of this formed way, polymeric oligomers stru andctures polymeric is sufficient; structures therefore, are a formeddecent substrate [18,19]. The diffusionaland product permeability mobility of towards/outwards formed polymeric the structures enzyme, iswhich sufficient; is encapsulated therefore, awithin decent the substrate formed and productpolymer mobility layer, towards is retained/outwards [27]. the enzyme, which is encapsulated within the formed polymer layer, is retainedPANI and [27 Ppy]. are very often used for the immobilization of biomolecules [1]. Neutral pH values PANIare required and Ppy for are the very enzymatic often usedsynthesis for theof PANI immobilization- and Ppy-based of biomolecules composites [21,24,39]. [1]. Neutral However, pH values good polymerization efficiency at neutral pHs is difficult to achieve; in addition, PANI and Ppy are required for the enzymatic synthesis of PANI- and Ppy-based composites [21,24,39]. However, usually are better conducting and are more electroactive only in their protonated form, which is good polymerization efficiency at neutral pHs is difficult to achieve; in addition, PANI and Ppy usually formed at low pH [1]. The concentration of the reactant and the velocity of oxidation process both are betterincrease conducting at low values and of are pHmore [36]. At electroactive pH > 4.6, both only aniline in theirand pyrrole protonated monomers form, and which corresponding is formed at low pHpolymer [1]. The chains concentration are not protonated of the reactantand, in such and case, the velocityonly neutral of oxidation nitrogen-containing process both species increase are at low values of pH [36]. At pH > 4.6, both aniline and pyrrole monomers and corresponding polymer chains are not protonated and, in such case, only neutral nitrogen-containing species are involved in PANI or Ppy formation reactions which are characterized by low oxidation potential [6]. Therefore, the Nanomaterials 2019, 9, 806 6 of 16

pH valueNanomaterials of the 2019 polymerization, 9, x FOR PEER REVIEW solution is a very important parameter for the formation of6 of PANI 16 or Ppy [18]. Theinvolved enzymatic in PANI formation or Ppy formation of PANI-, reactions Ppy-, and which PTh-based are characterized composite by low nanoparticles oxidation potential with embedded [6]. glucoseTherefore, oxidase the was pH monitored value of the by polymerization UV/Vis spectrophotometry solution is a very inimportant the pH intervalparameter from for the 1.0 formation to 12. During of PANI or Ppy [18]. the first few minutes, the polymerization bulk solutions containing aniline, pyrrole, or thiophene were The enzymatic formation of PANI-, Ppy-, and PTh-based composite nanoparticles with colourlessembedded and glucose any absorbance oxidase was peaks monitored were by observed UV/Vis spectrophotometry in the visible part in the of spectra.pH interval The from formation 1.0 of polymericto 12. During nanoparticles the first few (PANIminutes,/GOx, the polymerizati Ppy/GOx,on or bulk PTh solutions/GOx, respectively) containing aniline, was observedpyrrole, or after 24 hthiophene of polymerization. were colourless In theand caseany absorbance of the formation peaks were of PANIobserved/GOx in andthe visible Ppy/GOx, part of the spectra. solutions becomeThe coloured formation yellow-brown of polymeric andnanoparticles black-grey, (PANI/GOx, respectively. Ppy/GOx, It was lateror PTh/GOx, observed respectively) that the intensity was of colourobserved and the after value 24 h of of optical polymerization. absorbance In the were case dependent of the formation on the of pHPANI/GOx value ofand the Ppy/GOx, polymerization the solution.solutions Preliminary become coloured spectrophotometric yellow-brown investigations and black-grey, showed respectively. that the It was enzymatic later observed polymerization that the of anilineintensity [21], pyrrole of colour [24 ,and25] andthe value thiophene of optical [12,15 abso] wasrbance followed were bydependent the formation on the ofpH peaks value with of the optical polymerization solution. Preliminary spectrophotometric investigations showed that the enzymatic absorbance maximum (λmax) at 440 nm, 460 nm, and 450 nm, respectively (Figures2A,3A and4A). polymerization of aniline [21], pyrrole [24,25] and thiophene [12,15] was followed by the formation These λmax corresponds to the formation of aniline, pyrrole and thiophene oligomers. The optical of peaks with optical absorbance maximum (λmax) at 440 nm, 460 nm, and 450 nm, respectively (Figures absorbance peaks at λ = 260 nm were registered in all cases; this absorbance can be explained 2A, 3A and 4A). Thesemax λmax corresponds to the formation of aniline, pyrrole and thiophene oligomers. by aThe presence optical of absorbance protein—glucose peaks at λ oxidase—inmax = 260 nm thewere polymerization registered in all solutions,cases; this absorbance because aminocan be acid phenylalanineexplained by optical a presence absorbance of protein peak—glucose is at λoxidasemax =—260in the nm. polymerization solutions, because amino PANIacid phenylalanine/GOx colloidal optical solution absorbance after 48peak h lastingis at λmax enzymatic = 260 nm. polymerization was characterised by one for PANIPANI/GOx characteristic colloidal solution optical after absorbance 48 h lasting peak enzymatic with maximum polymerizationλmax was= 440 characterised nm (Figure by 2A), λ whichone is for in agreementPANI characteristic with another optical investigationsabsorbance peak related with maximum to synthesis max = of 440 PANI nm (Figure [8,11,28 2A),]. Some which of the is in agreement with another investigations related to synthesis of PANI [8,11,28]. Some of the authors authors reported the optical absorbance peak at λmax = 360 nm, which is attributed to emeraldine salt, reported the optical absorbance peak at λmax = 360 nm, which is attributed to emeraldine salt, corresponding to π-π* transition of PANI benzene ring delocalized on nitrogen atoms and related to corresponding to π-π* transition of PANI benzene ring delocalized on nitrogen atoms and related to the formation of cyclic aniline dimer 5,10-dihydrophenazine [8,11,28]. It was reported that optical the formation of cyclic aniline dimer 5,10-dihydrophenazine [8,11,28]. It was reported that optical absorbance peaks at λ =360 nm and λ = 420 nm are able to combine into a flat or distorted absorbance peaks atmax λmax =360 nm and λmaxmax = 420 nm are able to combine into a flat or distorted single singlepeak peak with with a new a new maximum maximum being being formed formed between between these initial these two initial peaks two [28]. peaks The [oxidation28]. The oxidationof 5,10- of 5,10-dihydrophenazinedihydrophenazine generates generates phenazyliu phenazyliumm radical-cation, radical-cation, which which absorbs absorbs at λ atmaxλ =max 440= nm.440 Such nm. Sucha a phenazyliumphenazylium radical-cation radical-cation is usuallyis usually insoluble insoluble inin the most most organic organic solvents solvents and and in aqueous in aqueous medium medium of lowof pHlow value pH value [6]. All[6]. ofAll the of PANIthe PANI/GOx/GOx composite composite nanoparticles nanoparticles that that formed formed in in polymerization polymerization bulk solutionbulk in solution the pH in range the pH from range 5.0 from to 8.0 5.0 were to 8.0 formed were formed in the emeraldinein the emeraldine oxidation oxidation form, form, which which has been confirmedhas been by confirmed the formation by the of formation brown-coloured of brown-coloured precipitate. precipitate.

A B 0.8 pH 3.0 4 , a.u. , a.u.

0.4 2 pH 6.0 Absorbance Absorbance pH 10 0 0.0 0 300 600 900 0369 λ , nm pH FigureFigure 2. ( A2.) ( SpectraA) Spectra of of colloidal colloidal solutions solutions ofof PANIPANI/GOx/GOx composite composite nanopartic nanoparticlesles synthesized synthesized at at variousvarious solution solution pH valuespH values and (Band) the (B inﬂuence) the influence of polymerization of polymerization bulk solutionbulk solution pH on pH the on absorbance the absorbance at λmax = 440 nm. (The composition of polymerization solution: 10.05 mol L−1 of glucose, 1 at λmax = 440 nm. (The composition of polymerization solution: 0.05 mol L− of glucose, 0.50 mol L− of 0.50 mol L−1 of aniline and 0.75 mg mL−1 of glucose oxidase at pH 1.0–10; 48 h lasting polymerization aniline and 0.75 mg mL 1 of glucose oxidase at pH 1.0–10; 48 h lasting polymerization was performed. − −1 was performed. Optical absorbance was registered1 in 0.05 mol L SA buffer, pH 6.0). Optical absorbance was registered in 0.05 mol L− SA buﬀer, pH 6.0).

Figure2B presents the inﬂuence of pH value of polymerization solution on the formation of PANI/GOx composite nanoparticles. It was determined that, by the increase of pH value from 1.0 to Nanomaterials 2019, 9, 806 7 of 16

Nanomaterials 2019, 9, x FOR PEER REVIEW 7 of 16 10, the optical absorbance of formed colloidal PANI/GOx nanoparticle solution at λmax = 440 nm has decreasedFigure 5.52 times2B presents (from the 0.690 influence to 0.125 of a.u.). pH value The highestof polymerization value of opticalsolution absorbance on the formation (0.690 of a.u. at λmaxPANI/GOx= 440 nm) composite was registered nanoparticles. in the solutionIt was determined of pH 1.0 that, after by48 the h increase lasting of polymerization pH value from 1.0 of aniline.to However,10, the the optical pH 1.0absorbance is not suitable of formed for PANIcolloidal/GOx PANI/GOx formation nanoparticle due to the solution inactivation at λmax of = GOx440 nm and has due to the non-enzymaticdecreased 5.52 times polymerization (from 0.690 to at 0.125 this a.u.). extreme The pHhighest value. value In of the optical research absorbance that was (0.690 performed a.u. at by λ anothermax authors,= 440 nm) it was was registered presented in that, the solution by the increaseof pH 1.0 of after pH 48 value, h lasting the polaronpolymerization optical of absorbance aniline. at However, the pH 1.0 is not suitable for PANI/GOx formation due to the inactivation of GOx and due λmax = 420 nm and λmax = 823 nm gradually decreases and a strong optical absorbance is observed to the non-enzymatic polymerization at this extreme pH value. In the research that was performed due to the transition of the quinoid rings, which emerges at λ = 560–600 nm [22]. The most suitable by another authors, it was presented that, by the increase of pH value, the polaron optical absorbance solution for the formation of emeraldine form of PANI/GOx composite nanoparticles was SA buﬀer at λmax = 420 nm and λ max = 823 nm gradually decreases and a strong optical absorbance is observed solution,due to pH the 6.0 transition (absorbance of the valuequinoid of rings, 0.329 which a.u. at emergesλmax = at440 λ nm= 560–600 was observed). nm [22]. The most suitable Thesolution relationship for the formation between of emeraldine the optical form absorbance of PANI/GOx of colloidal composite solution nanoparticles of Ppy was/GOx SA buffer composite nanoparticlessolution, pH and 6.0 the (absorbance pH values value in theof 0.329 range a.u. from at λmax 2.0 = to440 12 nm of was polymerization observed). solution is presented in Figure3The. The relationship optical absorbance between the peak optical at absorbanceλmax = 460 of nm colloidal has been solution observed of Ppy/GOx during composite the formation of Ppynanoparticles/GOx composite and the nanoparticles, pH values in the which range is from in an 2.0 agreement to 12 of polymerization with the results solution that is werepresented obtained in Figure 3. The optical absorbance peak at λmax = 460 nm has been observed during the formation of during the chemical polymerization of pyrrole [7]. Optical absorbance at λmax = 460 nm is attributed to a transitionPpy/GOx composite from valence nanoparticles, bond to the which polaron is in or an bipolaron agreement state with [ 7the]. The results peak that of opticalwere obtained absorbance during the chemical polymerization of pyrrole [7]. Optical absorbance at λmax = 460 nm is attributed with λ = 460 nm was observed at the early stage of pyrrole oxidation, and it was identiﬁed that it tomax a transition from valence bond to the polaron or bipolaron state [7]. The peak of optical absorbance belongs to the intermediate that formed during the oxidative polymerization of pyrrole. Some authors with λmax = 460 nm was observed at the early stage of pyrrole oxidation, and it was identified that it mentionedbelongs the to opticalthe intermediate absorbance that peak formed at λmax during= 360 the nm, oxidative which is polymerization related to π-π* of transition pyrrole. [7Some]. During enzymaticauthors polymerization, mentioned the optical the formedabsorbance Ppy peak/GOx at compositeλmax = 360 nm, nanoparticles which is related were to π of-π intensive* transitionblack colour[7]. when During polymerization enzymatic polymerization, was performed the atform pHed 2.0 Ppy/GOx and pH 3.0;composite and, intensive nanoparticles dark-grey—in were of pH rangeintensive from 5.0 black to 12. colour Black when colour polymerization is the most characteristicwas performed forat pH the 2.0 delocalised and pH 3.0;π-electron and, intensive system of dopeddark-grey—in Ppy [25]. pH range from 5.0 to 12. Black colour is the most characteristic for the delocalised π- Itelectron was determined system of doped that, Ppy by the[25]. increase of pH value of polymerization bulk solution from 3.0 to It was determined that, by the increase of pH value of polymerization bulk solution from 3.0 to 6.0, the absorbance at λmax = 460 nm has increased by 1.60 times (Figure3B). Additionally, the decrease 6.0, the absorbance at λmax = 460 nm has increased by 1.60 times (Figure 3B). Additionally, the decrease of the absorbance was followed by the increase of pH value from 8.0 to 12, and it is in agreement with of the absorbance was followed by the increase of pH value from 8.0 to 12, and it is in agreement with investigations of other researchers [18]. The absorbance at λmax = 460 nm has decreased by 9.17 times investigations of other researchers [18]. The absorbance at λmax = 460 nm has decreased by 9.17 times whenwhen polymerization polymerization was was performed performed in in pHspHs rangingranging from from 8.0 8.0 to to 12 12 (Figure (Figure 3B).3B). Alkaline Alkaline solutions solutions werewere considered considered to beto lessbe less suitable suitable for for Ppy Ppy formation. formation. According According to toinvestigations investigations [18], [ 18the], neutral the neutral radicalradical that formedthat formed in strong in strong bases bases is not is able not toable form to theform dimer the dimer that consists that consists of two of pyrrole two pyrrole molecules. The highestmolecules. value The ofhighest the absorbancevalue of the (0.207absorbance a.u.) (0.207 was registereda.u.) was registered at pH 6.0 at afterpH 6.0 48 after h of 48 enzymatic h of polymerizationenzymatic polymerization in all cases. in all cases.

A B

0.2 2 , a.u. , a.u.

1 pH 3.0 0.1 pH 10 Absorbance Absorbance

0 pH 6.0 0.0

0 300 600 900 04812 λ pH , nm FigureFigure 3. (A 3.) ( TheA) The spectra spectra of of formed formed Ppy Ppy/GOx/GOx compositecomposite nanoparticles nanoparticles colloidal colloidal solution solution at various at various solution pH values and (B) dependence of optical absorbance at λmax = 460 nm on the solution pH. solution pH values and (B) dependence of optical absorbance at λmax = 460 nm on the solution pH. (The composition of polymerization solution: 0.05 mol L−1 of 1glucose, 0.50 mol L−1 of pyrrole,1 and 0.75 (The composition of polymerization solution: 0.05 mol L− of glucose, 0.50 mol L− of pyrrole, and mg mL−1 of glucose oxidase at pH 2.0–12; 48 h lasting polymerization was performed. Optical 0.75 mg mL 1 of glucose oxidase at pH 2.0–12; 48 h lasting polymerization was performed. Optical − −1 absorbance was registered in 0.05 mol L1 SA buffer, pH 6.0). absorbance was registered in 0.05 mol L− SA buﬀer, pH 6.0). Nanomaterials 2019, 9, 806 8 of 16 Nanomaterials 2019, 9, x FOR PEER REVIEW 8 of 16

The main charge carriers in PTh are polarons and bipolarons [15]. Figure 4 presents the optical The main charge carriers in PTh are polarons and bipolarons [15]. Figure4 presents the optical absorbance spectra of formed PTh/GOx composite nanoparticles in the pH interval from 1.0 to 12. absorbance spectra of formed PTh/GOx composite nanoparticles in the pH interval from 1.0 to The UV/Vis absorbance spectra of colloidal solutions of PTh/GOx are characterized by optical 12. The UV/Vis absorbance spectra of colloidal solutions of PTh/GOx are characterized by optical absorbance peak at λmax = 450 nm. Optical absorbance peak (λmax = 450 nm) is attributed to the π-π* absorbance peak at λmax = 450 nm. Optical absorbance peak (λmax = 450 nm) is attributed to the π-π* transition for large conjugated structure [12,34]. Some authors mentioned optical absorbance peak at transition for large conjugated structure [12,34]. Some authors mentioned optical absorbance peak at λmax = 890 nm, which is related to the bipolaron state [15]. The appearance of optical absorbance peak λmax = 890 nm, which is related to the bipolaron state [15]. The appearance of optical absorbance peak at λmax = 450 nm was in agreement with the investigations related to chemical-oxidative formation of: at λmax = 450 nm was in agreement with the investigations related to chemical-oxidative formation (i) PTh-based 60–100 nm nanoparticles in water solution containing copper (II) ions and H2O2 as of: (i) PTh-based 60–100 nm nanoparticles in water solution containing copper (II) ions and H2O2 as oxidizer [12] and (ii) PTh-based 25–45 nm nanoparticles in the presence of FeCl3 as an oxidizer [15]. oxidizer [12] and (ii) PTh-based 25–45 nm nanoparticles in the presence of FeCl3 as an oxidizer [15].

A B 0.16 1.0 , a.u. , a.u. ,

0.5 0.08

pH 3.0 pH 6.0 Absorbance Absorbance 0.0 pH 10 0.00 0 300 600 900 04812 λ , nm pH Figure 4. ((AA)) Spectra Spectra of of PTh/GOx PTh/GOx composite nanoparticles colloidal solutions formed at various λ solution pH values and (B) dependencedependence ofof opticaloptical absorbanceabsorbance atat λmax == 450450 nm nm on on the the solution solution pH. pH. −11 −1 (The composition ofof polymerizationpolymerization solution:solution: 0.050.05 molmol LL− of glucose, 0.50 molmol LL− ofof thiophene, and −11 0.75 mg mL− ofof glucose glucose oxidase oxidase at at pH 1.0–12; 48 h lastinglasting polymerizationpolymerization was performed.performed. Optical −11 absorbance was registered in 0.05 mol L− SASA buffer, buﬀer, pH pH 6.0). 6.0).

As presentedpresented inin Figure Figure4B, 4B, the the absorbance absorbance at λ maxat λ=max450 = nm450 wasnm dramaticallywas dramatically changed changed (4.96 times), (4.96 times),from 0.124 from to 0.124 0.025 to a.u. 0.025 by a.u. the increaseby the increase of pH valueof pH from value 1.0 from to 12. 1.0 The to 12. absorbance The absorbance of 0.023 of a.u. 0.023 at a.u.λmax at= λ450max = nm 450 was nmregistered was registered at pH at 6.0 pH after 6.0 48after h of 48 enzymatic h of enzymatic polymerization. polymerization.

3.2. The The Comparison Comparison of Optical Absorbance of PANI/GOx, PANI/GOx, PpyPpy/GOx/GOx andand PThPTh/GOx/GOx Composite Composite Nanoparticles Colloidal Solution and the Choice of Optimal Enzyme/Substrate Ratio Nanoparticles Colloidal Solution and the Choice of Optimal Enzyme/Substrate Ratio The optical absorbance of PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles colloidal The optical absorbance of PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles solutions at λmax = 440 nm, λmax = 460 nm, and λmax = 450 nm, respectively, was assessed in SA colloidal solutions at λmax = 440 nm, λmax = 460nm, and λmax = 450 nm, respectively, was assessed in SA buﬀer, pH 6.0, after 48 h lasting polymerization and was 0.329, 0.207, and 0.023 a.u., respectively buffer, pH 6.0, after 48 h lasting polymerization and was 0.329, 0.207, and 0.023 a.u., respectively (Figure5A). It was noticed that the optical absorbance of PTh /GOx composite nanoparticles after 48 h (Figure 5A). It was noticed that the optical absorbance of PTh/GOx composite nanoparticles after 48 lasting enzymatic polymerization was 14.3 and 9.00 times lower than that for the PANI/GOx and h lasting enzymatic polymerization was 14.3 and 9.00 times lower than that for the PANI/GOx and Ppy/GOx composite nanoparticles. As it is presented in Figure5A, the absorbance of formed PANI /GOx Ppy/GOx composite nanoparticles. As it is presented in Figure 5A, the absorbance of formed colloidal solution after 48 h of polymerization was 1.59 times higher than that of the Ppy/GOx colloidal PANI/GOx colloidal solution after 48 h of polymerization was 1.59 times higher than that of the solution. The optical control of PTh/GOx composite nanoparticles formation was more complicated in Ppy/GOx colloidal solution. The optical control of PTh/GOx composite nanoparticles formation was comparison to that of PANI/GOx or Ppy/GOx composite nanoparticles formation due to the slower more complicated in comparison to that of PANI/GOx or Ppy/GOx composite nanoparticles polymerization rate. It should be noted that it is quite diﬃcult to prepare the solution of well-dispersed formation due to the slower polymerization rate. It should be noted that it is quite difficult to prepare polythiophene particles, because they tend to aggregate and precipitate [12,34]. Therefore, further the solution of well-dispersed polythiophene particles, because they tend to aggregate and precipitate investigations of a polymerization’s duration were focused on the enzymatic formation of PANI/GOx [12,34]. Therefore, further investigations of a polymerization’s duration were focused on the and Ppy/GOx composite nanoparticles. enzymatic formation of PANI/GOx and Ppy/GOx composite nanoparticles. Nanomaterials 2019, 9, 806x FOR PEER REVIEW 9 of 16

A B 0.4 0.3 3 , a.u. , a.u. , 0.2

0.2 0.1 2 Absorbance Absorbance

1 0.0 0.0 PANI/ Ppy/ PTh/ PANI Ppy 30/20 30/40 30/60 GOx GOx GOx The ratio of GOx/Glucose value Figure 5. The diagram of optical absorbance of PANI-, Ppy-, and PTh-based composite nanoparticles in the the presence presence and and absence absence of of GOx GOx (A (A) and) and the the influence inﬂuence of ofGOx/Glucose GOx/Glucose ratio ratio on onthe theabsorbance absorbance for forPTh/GOx PTh/GOx (1 curve), (1 curve), Ppy/GOx Ppy/GOx (2 (2curve), curve), or or PANI/GOx PANI/GOx (3 (3 curve) curve) composite composite nanoparticles colloidal 1 solutions at pH 6.06.0 ((BB).). ((((AA)) TheThe compositioncomposition ofof polymerizationpolymerization solution,solution, pHpH 6.0:6.0: 0.050.05 molmol LL−−1 of 1 1 glucose, 0.50 mol LL−−1 ofof aniline, pyrrole, or thiophene and 0.75 mg mLmL−−1 ofof glucose oxidase (grey 1 columns); the composition ofof solutionsolution usedused forfor autopolymerization:autopolymerization: 0.050.05 molmol LL−−1 of glucose, 0.50 mol 1 L−1 ofof aniline aniline or or pyrrole pyrrole at at pH pH 1.0 or pH 2.0, respectively (dash columns); ( B) The composition of 1 1 polymerization solution,solution, pHpH 6.0:6.0: 0.500.50 molmol LL−−1 of aniline, pyrrole or thiophene, 0.750.75 mgmg mLmL−−1 of glucose 1 oxidase and 0.015–0.05 molmol LL−−1 of glucose. Polymerization Polymerization lasted lasted for 48 h. Optical absorbance was registered for PANIPANI/GOx/GOx andand PANI,PANI, Ppy Ppy/GOx,/GOx, and and Ppy, Ppy, PTh PTh/GOx/GOx at atλ maxλmax= = 440440 nm,nm, λλmaxmax = 460460 nm, 1 and λmax == 450450 nm nm in in 0.05 0.05 mol mol L L−1− SASA buffer, buﬀer, pH pH 6.0). 6.0).

As it has been mentioned by other authors, the Ppy formation can be performed in the solution without any any chemical chemical oxidant oxidant or or enzyme enzyme [18], [18 ],and and this this process process is named is named autopolymerization. autopolymerization. An 1 1 Anexperiment experiment was was performed performed in solutions in solutions containing containing only only 0.05 0.05 mol mol L−1 L of− glucoseof glucose and and 0.5 0.5 mol mol L− L1 −of ofaniline, aniline, pyrrole, pyrrole, or orthiophene thiophene at atpH pH from from 1.0 1.0 to to 12 12 without without GOx, GOx, to to evaluate evaluate the the influence influence of the autopolymerization onon thethe formationformation of of PANI, PANI, Ppy Ppy and and PTh. PTh. Autopolymerization Autopolymerization was was not not observed observed in thein the pH pH range range from from 1.0 to1.0 12 to for 12 thiophene.for thiophene. The slowThe slow autopolymerization autopolymerization has only has beenonly observedbeen observed at pH 1.0at pH for 1.0 aniline for aniline and at and pH belowat pH 2.0below for pyrrole2.0 for pyrro and itle reachedand it reached 0.030 and 0.030 0.035 and a.u. 0.035 after a.u. 48 after h lasting 48 h polymerization,lasting polymerization, respectively. respectively. This autopolymerization This autopolymerization phenomenon phenomenon was not observed was not atobserved a higher at pH a value.higher ThepH polymerizationvalue. The polymerization rate of aniline rate was of 1.20aniline times was slower 1.20 times in the slower comparison in the of comparison pyrrole at pH of 2.0pyrrole at pH at 1.0pH in 2.0 the at absencepH 1.0 in of the GOx. absence The polymerizationof GOx. The polymerization rate of pyrrole rate at pHof pyrrole 2.0 in the at pH absence 2.0 in andthe inabsence the presence and in the of GOxpresence was of the GOx same. was It the means same. that It means GOx wasthat inactivatedGOx was inactivated at this low at pH. this Welow have pH. performedWe have performed the polymerization the polymerization at pH values at higher pH thanvalues 3.0 higher in order than to avoid 3.0 thein order autopolymerization. to avoid the Theautopolymerization. results of enzymatic The polymerizationresults of enzymatic were inpolyme an agreementrization were with thein an results agreement that were with obtained the results for PANIthat were [21] andobtained Ppy [ 24for]. SignificantPANI [21] diandfferences Ppy [24]. of the Significant absorbance differences were detected of the in theabsorbance absence and were in thedetected presence in the of absence GOx demonstrate and in the apresence significant of impactGOx demonstrate of the enzyme a significant to polymerization impact of reaction the enzyme rate. Theto polymerization most optimal formation reaction raterate. ofThe PANI most/GOx op andtimal Ppy formation/GOx composite rate of nanoparticlesPANI/GOx and was Ppy/GOx observed 1 incomposite 0.05 mol nanoparticles L− SA buffer, was pH observed 6.0, where in GOx 0.05 exhibitsmol L−1 theSA maximalbuffer, pH catalysed 6.0, where reaction GOx rateexhibits and the highestmaximal stability, catalysed according reaction torate our and measurements the highest st [24ability,,39]. according to our measurements [24,39]. The type type of of oxidizing oxidizing agent agent has hassignificant significant influence influence on the onoxidative the oxidative polymerization polymerization reaction reaction [18]. It was determined that the polymerization is faster at higher (0.50 mol L 1) monomer’s [18]. It was determined that the polymerization is faster at higher (0.50 mol·L· −1) monomer’s concentration [22], [22], but further increasing of monomer’smonomer’s concentration negatively influencedinfluenced the formation of polymeric nanoparticles. The The rate rate of of polymerization reaction and the properties of the formed polymer not only dependdepend onon thethe preparationpreparation technique,technique, selectedselected solvent,solvent, polymerizationpolymerization duration, temperature, kind, and concentration of monomer, but also on the enzyme and substrate ratio [[18,21].18,21]. Nanomaterials 2019, 9, 806 10 of 16

The effect of enzyme/substrate ratio on the rate of PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles formation was investigated in the next set of spectrophotometric measurements. The investigations were performed during 48 h in the polymerization solution, pH 6.0, containing 1 1 0.50 mol L− of aniline, pyrrole, or thiophene, 0.75 mg mL− of GOx and glucose concentration varying 1 from 0.015 to 0.05 mol L− . Figure5B presents the influence of the ratio of GOx /Glucose on the registered absorbance. The registered absorbance significantly increased by increasing glucose concentration from 1 1 0.015 mol L− in the polymerization solution up to 0.05 mol L− . The absorbance of PANI/GOx, 1 Ppy/GOx, and PTh/GOx composite nanoparticles, which were prepared using 0.75 mg mL− of GOx 1 and 0.05 mol L− of glucose, increased by 3.39, 3.79, and 10.1 times in a comparison to the absorbance 1 that was obtained for polymeric nanoparticles with embedded GOx using 0.015 mol L− of glucose. 1 By the increase of glucose concentration up to 0.015 mol L− , the absorbance of PANI/GOx, Ppy/GOx, and PTh/GOx decreased 1.25, 1.31, and 3.53 times, if compared with the results that were obtained 1 1 for the absorbance with 0.75 mg mL− of GOx and 0.05 mol L− of glucose. Thus, the ratio of 1 1 GOx/Glucose 0.75 mg mL− /0.05 mol L− was selected as the most optimal for polymeric nanoparticle composites formation.

3.3. The Evaluation of PANI/GOx, Ppy/GOx and PTh/GOx Composite Nanoparticles Formation by DLS and the Morphology of PANI/GOx, Ppy/GOx Nanocomposites Depending on pH, the negatively or positively charged enzymes can be entrapped within the CPs during oxidation/reduction reactions [20]. Enzymatic polymerization strongly depends on the solution’s pH. For instance, the conducting form of linear PANI is synthesized at pH 4.0–4.5, wherein, at pH 6.0 or higher, more branched and insulating form of PANI is formed [11,22]. The diameter of 48 h enzymatically synthesized PANI-, Ppy-, and PTh-based nanocomposites with embedded GOx in solution of pH 2.0–8.0 was evaluated by the DLS technique. The results of PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles diameter and their distribution are presented in Table1 and Figure6, respectively. The diameter of PANI /GOx, Ppy/GOx, and PTh/GOx composites nanoparticles depended on 5h3 pH of polymerization solution and it decreased by the increase of pH. It was determined that, in SA buﬀer, pH 6.0, PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles of 54, 86, and 98 nm diameter were formed, respectively. The DLS results (Table1) represent that formed PANI /GOx composite nanoparticles were 1.59 and 1.81 times smaller if compared with Ppy/GOx and PTh/GOx. Due to the relatively large (535 nm) diameters of PTh/GOx, which were formed at pH 8.0, we decided that such relatively large particle colloidal solution will be not very stable and mostly focused on the investigation of PANI/GOx and Ppy/GOx composite nanoparticles, which are smaller; therefore, it was expected that colloidal solution of these nanoparticles will be more stable.

Table 1. The dependence of PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles diameter on 1 1 the pH of solutions. The composition of polymerization solution: 0.05 mol L− of glucose, 0.75 mg mL− 1 of GOx and 0.50 mol L− of aniline, pyrrole, or thiophene. The particles were formed by 48 h lasting enzymatic polymerization. The diameter of particles was determined by dynamic light scattering (DLS) 1 in 0.05 mol L− SA buﬀer, pH 6.0.

Diameter, nm pH PANI/GOx Ppy/GOx PTh/GOx 2.0 180 94 180 46 169 62 ± ± ± 5.0 56 22 155 61 63 9.4 ± ± ± 6.0 54 6.9 86 18 98 28 ± ± ± 8.0 106 41 308 49 535 106 ± ± ± Nanomaterials 2019, 9, 806 11 of 16 NanomaterialsNanomaterials 20192019,, 99,, xx FORFOR PEERPEER REVIEWREVIEW 1111 ofof 1616

PANI/GOxPANI/GOx 3030 Ppy/GOxPpy/GOx PTh/GOxPTh/GOx , % , , % , 2020 Number Number 1010

00 1010 100 100 1000 1000 DiameterDiameter,, nmnm

FigureFigure 6.6. TheThe distribution distribution ofof PANI/GOx,PANI/GOx, PANI/GOx, Ppy/GOx, PpyPpy/GOx,/GOx, an andandd PTh/GOx PThPTh/GOx/GOx composite composite nanoparticlesnanoparticles diameter.diameter. TheThe particlesparticles werewere formedformed byby 4848 hhh lastinglastinglasting enzymaticenzymaticenzymatic polymerization polymerizationpolymerization at atat pH pHpH 6.0. 6.0.6.0.

SomeSome of ofof the thethe researchers researchersresearchers evaluated evaluatedevaluated the thethe diameter diameterdiameter of synthesized ofof synthesizedsynthesized polymer polymerpolymer nanocomposites nanocompositesnanocomposites by FE-SEM. byby FE-FE- DuringSEM.SEM. DuringDuring the enzymatic thethe enzymaticenzymatic polymerization polymerizationpolymerization of aniline using ofof chitosananilineaniline andusingusing poly( chitosanchitosanN-isopropylacrylamide) andand poly(poly(NN-- asisopropylacrylamide)isopropylacrylamide) steric stabilizers 50 asas nm stericsteric PANI stabilizersstabilizers nanoparticles 5050 nmnm werePANIPANI synthesized nanoparticlesnanoparticles [11 werewere]. In synthesizedsynthesized our study, [11].[11]. PANI InIn/GOx ourour andstudy,study, Ppy PANI/GOxPANI/GOx/GOx composite andand Ppy/GOxPpy/GOx nanoparticles compositecomposite formed nanoparticlesnanoparticles during 24 h formed lastingformed enzymatic duringduring 2424 polymerization hh lastinglasting enzymaticenzymatic were investigatedpolymerizationpolymerization by FE-SEMwerewere investigatedinvestigated (Figure7). byby FE-SEMFE-SEM (Figure(Figure 7).7).

FigureFigure 7.7. FE-SEMFE-SEM imagesimages ofof PANI/GOxPANI/GOx PANI/GOx ( (AA)) and and PpyPpy/GOxPpy/GOx/GOx ((BB)) compositecomposite nanoparticlesnanoparticles after after 24 24 hh −−11 lastinglasting enzymaticenzymatic polymerization.polymerization.polymerization. (The(The polymerizationpolymerizationpolymerization solution:solution:solution: 0.050.050.05 molmolmol LLL− SASA buffer,buffer, buﬀer, pH pH 6.0, 6.0, with with −−11 −−111 −−11 1 0.050.05 molmol LL− ofofof glucose,glucose, glucose, 0.750.75 0.75 mgmg mg mLmL mL− ofofof GOx,GOx, GOx, andand and 0.500.50 0.50 molmol mol LL L− ofof of anilineaniline aniline oror or pyrrole).pyrrole). pyrrole).

SphericalSpherical PANI/GOx PANIPANI/GOx/GOx andand PpyPpy/GOxPpy/GOx/GOx compositecompositecomposite nanoparticlesnananoparticlesnoparticles werewere formedformed duringduring enzymaticenzymatic polymerization.polymerization. ItItIt isisis seen seenseen that thatthat the thethe PANI PANI/GOxPANI/GOx/GOx (Figure (Figure(Figure7A) 7A)7A) nanoparticles nanoparticlesnanoparticles were werewere randomly randomlyrandomly distributed distributeddistributed on theonon thethe surface surfacesurface of graphiteofof graphitegraphite rod rodrod used usedused for forfor investigations. investigations.investigations. The TheThe Ppy Ppy/GOxPpy/GOx/GOx nanoparticles nanoparticlesnanoparticles were werewere densely denselydensely stuckstuck togethertogether (Figure (Figure 7 7B).7B).B). The TheThe diameter diameterdiameter of ofof nanoparticles nanoparticlesnanoparticles dependsdependsdepends ononon thethethe kindkindkind ofofof polymerizedpolymerizedpolymerized monomer.monomer. ItIt waswas determineddetermineddetermined thatthatthat the thethe diameter diameterdiameter of ofof PANI PANI/GOxPANI/GOx/GOx and andand Ppy Ppy/GOxPpy/GOx/GOx composites compositescomposites was waswas in inin the thethe range rangerange of ofof 38–50 38–38– and5050 andand 50–100 50–10050–100 nm, nm,nm, respectively. respectively.respectively.

3.4.3.4. The The InfluenceInfluenceInfluence of ofof Polymerization PolymerizationPolymerization Duration DurationDuration on onon PANI PANI/GOxPANI/GOx/GOx and andand Ppy Ppy/GOxPpy/GOx/GOx Composite CompositeComposite Nanoparticles NanoparticlesNanoparticles Formation FormationFormationOne of most important parameters of polymer‘s formation is the duration of a polymerization. The influenceOneOne ofof mostmost of polymerization importantimportant parametersparameters duration (fromofof polymer‘spolymer‘s 5 to 288 formationformation h) on the formation isis thethe durationduration of PANI ofof/ GOx aa polymerization.polymerization. and Ppy/GOx 1 compositeTheThe influenceinfluence nanoparticles ofof polymerizationpolymerization was investigated. durationduration (from(from The polymerization 55 toto 288288 h)h) onon was thethe performedformationformation inofof 0.05PANI/GOxPANI/GOx mol L− andandSA 1 1 1 buPpy/GOxPpy/GOxffer, pH compositecomposite 6.0, with 0.05nanoparticlesnanoparticles mol L− of waswas glucose, investigated.investigated. 0.50 mol TheThe L− polymerizationpolymerizationof aniline or pyrrole, waswas performedperformed and 0.75 inin mg 0.050.05 mL molmol− ofLL−−11GOx. SASA buffer,buffer, Visible pHpH aggregation 6.0,6.0, withwith 0.050.05 of molmol PANI LL−−/11GOx ofof glucose,glucose, and Ppy 0.500.50/GOx molmol was LL−−11 ofof observed anilineaniline oror after pyrrole,pyrrole, 48 h lasting andand 0.750.75 enzymatic mgmg mLmL−−11 polymerization.ofof GOx.GOx. VisibleVisible aggregationaggregation Monomers of areof PANI/GOxPANI/GOx oxidized during andand Ppy/GOxPpy/GOx the enzymatic waswas observedobserved polymerization afterafter 4848 hh and lastinglasting the amountenzymaticenzymatic of polymerization.polymerization. MonomersMonomers areare oxidizedoxidized duringduring ththee enzymaticenzymatic polymerizationpolymerization andand thethe amountamount ofof Nanomaterials 2019, 9, 806 12 of 16 Nanomaterials 2019, 9, x FOR PEER REVIEW 12 of 16 polymerspolymers is is increasing increasing by by the the prolongation prolongation of of synthesis’s synthesis’s duration. duration. It It should should be be noted noted that that the the enzyme enzyme tendstends to to loss loss catalytic catalytic activity activity due due to to long long lasting lastin polymerizationg polymerization [18 [18].]. The The amount amount and and the the intensity intensity of of absorbance for the obtained PANI/GOx and Ppy/GOx composite nanoparticles at λmax = 440 nm absorbance for the obtained PANI/GOx and Ppy/GOx composite nanoparticles at λmax = 440 nm and and λmax = 460 nm increased by the prolongation of polymerization duration from 5 to 168 h (Figure 8). λmax = 460 nm increased by the prolongation of polymerization duration from 5 to 168 h (Figure8).

0.9 1

, a.u. 0.6

0.3 2 Absorbance

0.0

050100150 Time, h FigureFigure 8. 8.The The influence influence of polymerization of polymerization duration duration on PANI /GOxon PANI/GOx and Ppy/GOx and composite Ppy/GOx nanoparticles composite 1 opticalnanoparticles absorbance. optical (The absorbance. composition (The of composition polymerization of polymerization solution: 0.05 mol solution: L− SA 0.05 bu molffer, pHL−1 SA 6.0, buffer, with 1 1 1 0.05pH mol6.0, Lwith− of 0.05 glucose, mol 0.50L−1 of mol glucose, L− of aniline,0.50 mol or L pyrrole−1 of aniline, and 0.75 or pyrrole mg mL− andof glucose0.75 mg oxidase. mL−1 of Opticalglucose 1 absorbanceoxidase. Optical was registered absorbance in 0.05 was mol registered L− SA in bu ff0.05er, pHmol 6.0, L−1 at SAλmax buffer,= 440 pH nm, 6.0, and at λmax == 440460 nm, nm forand

PANIλmax =/GOx 460 nm and for Ppy PANI/GOx/GOx, respectively). and Ppy/GOx, respectively).

SuchSuch an an e effectffect indicated indicated the the formation formation of of PANI PANI/GOx/GOx and and Ppy Ppy/GOx/GOx composite composite nanoparticles nanoparticles and and it isit in is anin an agreement agreement with with some some previous previous researches researches [18 [18,24].,24]. The The maximal maximal concentration concentration of of PANI PANI/GOx/GOx andand Ppy Ppy/GOx/GOx composite composite nanoparticle nanoparticle formation formation was was achieved achieved after after 168 168 h. h. Optical Optical absorbance absorbance after after 168168 h h at atλ maxλmax == 440440 nmnm andand λλmaxmax = 460 nm has increased by 74.774.7 andand 11.111.1 times,times, respectively,respectively, inina a comparisoncomparison with with the the absorbance absorbance that that was was registered registered after after 5 h 5 (Figure h (Figure8). After8). After 168 168 h lasting h lasting formation formation of Ppyof Ppy/GOx/GOx composite composite nanoparticles nanoparticles the optical the optical absorbance absorbance at λmax at =λ max460 = nm 460 was nm 2.48was times 2.48 times lower lower than thatthan of that PANI of/ PANI/GOxGOx at λmax at= λ440max = nm 440 after nm after the same the same duration duration of polymerization. of polymerization. It was Itdetermined was determined that thethat absorbance the absorbance of PANI of /PANI/GOxGOx and Ppy and/GOx Ppy/GOx composite composite nanoparticles nanoparticles has decreased has decreased during long-lastingduring long- polymerizationlasting polymerization (more than (more 168h). than The 168 limited h). The solubility limited of solubility synthesized of polymerssynthesized and polymers the precipitation and the ofprecipitation particles or of clusters particles of PANIor clusters/GOx andof PANI/GOx Ppy/GOx fromand Ppy/GOx the polymerization from the polymerization solution could explainsolution thiscould phenomenon explain this [ 18phenomenon]. When the [18]. formation When ofthe PANI formation/GOx andof PANI/GOx Ppy/GOx compositesand Ppy/GOx lasted composites longer thanlasted 168 longer h, the than significant 168 h, the part significant of formed part nanoparticles of formed nanoparticles has precipitated. has precipitated. Though, optical Though, absorbance optical atabsorbanceλmax = 440 at nm λmax and = λ440max nm= 460 and nm λmax of PANI= 460/GOx nm andof PANI/GOx Ppy/GOx afterand 48Ppy/GOx h lasting after polymerization 48 h lasting waspolymerization 2.52 and 1.94 was times 2.52 lower and than1.94 times that registered lower than after that 168 registered h lasting after polymerization. 168 h lasting polymerization. 48–96 h lasting polymerization48–96 h lasting seemspolymerization to be more seems efficient to be for more PANI efficient/GOx andfor PANI/GOx Ppy/GOxcomposite and Ppy/GOx nanoparticles composite formation,nanoparticles because formation, smaller because PANI/GOx smaller and PANI/GOx Ppy/GOx will and be Ppy/GOx formed. will be formed. TheThe initial initial aniline aniline and and pyrrole pyrrole conversion conversion into polymerinto polymer reaction reaction rate (V )rate was ( evaluatedV) was evaluated by formula by Vformula= (tgα), V where = (tg tgαα),was where calculated tgα fromwas Figurecalculated8 by thefrom evaluation Figure of8 estimatedby the /approximatedevaluation of absorbanceestimated/approximated of PANI/GOx and absorbance Ppy/GOx of solutions PANI/GOx (1.27 and a.u. Ppy/GOx for PANI solutions/GOx and (1.27 0.446 a.u. a.u. for for PANI/GOx Ppy/GOx) thatand formed0.446 a.u. when for 100%Ppy/GOx) of monomers, that formed which when were 100% present of monomers, in the solution, which will were be converted present in into the polymer.solution, The will initial be converted aniline and into pyrrole polymer. polymerization The initial rateaniline was and determined pyrrole aspolymerization 0.0037 and 0.0106 rate molwas 1 1 Ldetermined− h− , respectively. as 0.0037 It and is seen 0.0106 that mol the L initial−1 h−1, polymerizationrespectively. It is rate seen of that aniline the initial is 2.86 polymerization times slower than rate thatof aniline of pyrrole. is 2.86 times slower than that of pyrrole. TheThe diameterdiameter ofof PANIPANI/GOx/GOx and and Ppy Ppy/GOx/GOx composite composite nanoparticles nanoparticles and and their their aggregates aggregates was was investigatedinvestigated byby DLSDLS andand FigureFigure9 and9 and Table Table2 present 2 present the the results. results. The The diameter diameter of of polymeric polymeric nanoparticlesnanoparticles increased increased by by the the increase increase of polymerization of polymerization duration duration from 16 from to 120 16 h. 41to nm120 PANI h. 41/GOx nm PANI/GOx and 65 nm Ppy/GOx were formed after 16 h of enzymatic polymerization. The diameter Nanomaterials 2019, 9, 806 13 of 16 Nanomaterials 2019, 9, x FOR PEER REVIEW 13 of 16 andof PANI/GOx 65 nm Ppy /andGOx Ppy/GOx were formed increased after 16 up h of to enzymatic 167 and polymerization.122 nm, respectively, The diameter after of120 PANI h of/GOx the andpolymerization. Ppy/GOx increased up to 167 and 122 nm, respectively, after 120 h of the polymerization. The aggregates of PANIPANI/GOx/GOx andand PpyPpy/GOx/GOx compositecomposite nanoparticles were formed from the 120th hourhour ofof enzymaticenzymatic polymer’spolymer’s formation.formation. During During 168–288 168–288 h lasting polymerization, a lot of agglomerates that are characterized by large diameter were formed, which was in an agreement with our previous researches, where 142 nm PANIPANI/GOx/GOx compositecomposite nanoparticlesnanoparticles were formed after 168 h lasting polymerizationpolymerization [ 21[21].]. For For the the enzymatic enzymatic synthesis synthesis of smaller of smaller polymeric polymeric nanoparticles, nanoparticles, 48 h lasting 48 h polymerizationlasting polymerization is the most is the suitable, most suitable, because, because, in such in way, such 72 way, nm PANI72 nm/GOx PANI/GOx (Figure (Figure9B) and 9B) 43 nmand Ppy43 nm/GOx Ppy/GOx composite composite nanoparticles nanoparticles were formed. were Otherformed. authors Other have authors reported have that,reported during that, enzymatic during polymerizationenzymatic polymerization of aniline using of aniline chitosan using and poly(chitosanN-isopropylacrylamide) and poly(N-isopropylacrylamide) as steric stabilizers as steric and horseradishstabilizers and peroxidase horseradish as enzyme,peroxidase 50 nmas enzyme, PANI nanoparticles 50 nm PANI werenanoparticles synthesized were [11 synthesized]. However, [11]. the PANIHowever, matrix the doped PANI by matrix small doped anions by is characterizedsmall anions is by characterized poor stability. by Therefore, poor stability. it is recommended Therefore, it tois doperecommended PANI by largerto dope anions PANI to by solve larger this anions problem to solve [28]. this problem [28].

A B 30 150

, nm 16 h r, % 100 48 h 120 h 10 Numbe

Diameter 50

0 0 10 100 Time, h Diameter, nm

Figure 9. The influence influence of enzymatic polymerization dura durationtion on the diameter of formed PANI/GOx PANI/GOx (dash(dash column)column) and PpyPpy/GOx/GOx (grey column)column) composite nanoparticles (A) and thethe distributiondistribution of PANIPANI/GOx/GOx nanoparticlesnanoparticles diameter afterafter 16, 48, and 120 hh polymerizationpolymerization (B). (The(The compositioncomposition of 1 1 1 polymerization solution:solution: 0.050.05 molmol LL−−1 SA buffer, buffer, pHpH 6.0,6.0, withwith 0.050.05 mol mol L L−−1 ofof glucose,glucose, 0.500.50 molmolL L−−1 of 1 1 aniline oror pyrrole,pyrrole, andand 0.750.75 mgmg mLmL−−1 of glucose oxidase. DLS signal was registeredregistered inin 0.050.05 molmolL L−−1 SA bubuffer,ffer, pHpH 6.0).6.0). Table 2. The characteristics of PANI/GOx and Ppy/GOx composite nanoparticles depending of the polymerizationTable 2. The characteristics duration determined of PANI/GOx by DLS. and (Conditions Ppy/GOx composite like in Figure nanoparticles9, SD—standard depending deviation.). of the polymerization duration determined by DLS. (Conditions like in Figure 9, SD—standard deviation.). PANI/GOx Ppy/GOx Time, h PANI/GOx Ppy/GOx Time, h Diameter, nm SD Diameter, nm SD Diameter, nm SD Diameter, nm SD 16 41 11 65 15 ± ± 16 4841 72 ±1127 4365 20±15 ± ± 48120 16772 ±2728 12243 23±20 ± ± 120 167 ±28 122 ±23 The electrical charge of particles is usually expressed by zeta-potential. The zeta-potential was determinedThe electrical in SA bu chargeffer, pH of 6.0,particles for polymeric is usually nanoparticles expressed by formed zeta-potential. by 48 h lasting The zeta-potential polymerization was in ourdetermined investigations. in SA buffer, The zeta-potentials pH 6.0, for polymeric of PANI/GOx, nanoparticles Ppy/GOx, formed and PTh by/GOx 48 h nanoparticles lasting polymerization were 6.2, − in9.4, our and investigations.8.6 mV, respectively. The zeta-potentials of PANI/GOx, Ppy/GOx, and PTh/GOx nanoparticles were −6.2, −9.4, −and −8.6 mV, respectively.

4. Conclusions PANI/GOx, Ppy/GOx, and PTh/GOx composite nanoparticles were synthesized by the enzymatic polymerization of aniline, pyrrole, and thiophene, respectively. The PANI/GOx, Ppy/GOx, and PTh/GOx nanoparticles were characterized by optical absorbance maximums at λmax = 440 nm, λmax = 460 nm, and λmax = 450 nm, respectively. The formation of PANI/GOx in emeraldine base form was observed. Both factors (i) the pH of polymerization bulk solution and (ii) the duration of polymerization have significant influence on the formation of polymeric nanoparticles. The highest rate and the smallest diameter of formed PANI/GOx, Ppy/GOx, and PTh/GOx were observed in solution of sodium acetate buffer, pH 6.0. The optimal ratio of glucose oxidase/glucose for the formation of PANI/GOx, Ppy/GOx, 1 1 and PTh/GOx composite nanoparticles was determined as 0.75 mg mL− / 0.05 mol L− . Spherical particles of PANI/GOx and Ppy/GOx were formed during enzymatic polymerization. The most optimal duration for the enzymatic synthesis of PANI/GOx and Ppy/GOx composite nanoparticles was in the range of 48–96 h. The diameter of polymeric nanoparticles, which was determined by DLS, was dependent on the duration of polymerization: 41–167 nm PANI/GOx and 65–122 nm Ppy/GOx particles were formed by polymerization lasting from 16 to 120 h, respectively. The formed PANI/GOx and Ppy/GOx composite nanoparticles were pure from surfactants and other interfering species, which increases the applicability of synthesized polymeric composite nanoparticles for possible biomedical applications. Here, the evaluated PANI/GOx and Ppy/GOx composite nanoparticles formation conditions can be applied in further researches. Some advantages of the application of gold nanoparticles in the formation of conducting polymers-based composite materials have been demonstrated [37,39]. Therefore, we are predicting that the extension of PANI/GOx and Ppy/GOx applicability in biosensorics can be achieved by embedment of gold nanoparticles within PANI/GOx and Ppy/GOx composite structure.

Author Contributions: Conceptualization, A.R. (Arunas Ramanavicius) and N.G.; methodology, N.G.; software, N.G.; validation, A.R. (Almira Ramanaviciene), A.R. (Arunas Ramanavicius) and N.G.; formal analysis, N.G.; investigation, N.G. and A.P.; resources, A.R. (Arunas Ramanavicius); data curation, A.R. (Almira Ramanaviciene); writing-original draft preparation, N.G.; writing-review and editing, A.R. (Arunas Ramanavicius); visualization, A.R. (Almira Ramanaviciene); supervision, A.R. (Arunas Ramanavicius); project administration, A.R. (Arunas Ramanavicius); funding acquisition, A.R. (Arunas Ramanavicius). Funding: This research is/was funded by the European Regional Development Fund according to the supported activity ‘Research Projects Implemented by World-class Researcher Groups’ under Measure No. 01.2.2-LMT-K-718. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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