INTRODUCTION

It is known that oxidoreductases catalyze the oxidative polymerization of various aromatic compounds [1–3]. Lignin is the natural product of the polymerization of these compounds. Synthetic compounds such as aniline, thiophene, pyrrole, and their derivatives can also be used as substrates for oxidoreductases; electrically conducting polymers (ECPs) form as a result of their polymerization [4–9]. Since these polymers perform poorly, they are usually used in composites.

Carbon nanotubes (CNTs) have unique mechanical and electrical properties. This carbon nanomaterial is promising for the production of ECP-based composites. CNTs can be considered “solid” matrices for monomer polymerization. The local environment of the initial substrate (the pH, matrix and monomer charges, and concentration effects), as well as the ability to form an ordered monomer arrangement on the matrices, followed by its enzymatic polymerization, is a feature of matrix polymerization, which makes it possible to direct the enzymatic reaction in the desired direction.

ECP/CNT composites can be used in various devices, for example, supercapacitors and bio- or chemosensors, to protect surfaces from electromagnetic radiation, in bioelectronics, etc. [10–13]. The traditional method for the preparation of ECP/CNT composites involves chemical polymerization of a monomer with large amounts of an oxidizing agent in the presence of acidic dopants, which ensures the electrical conductivity of polymers. Dopants are electron donors or electron acceptors that, in the course of interaction with the main polymer chain, lead to the formation of charges on it (resulting in polymer electrical conductivity). Dopants act as charge-compensating anions of a positively charged polymer chain. Strong low molecular weight acids and polymeric sulfonic acids are used as dopants.

The chemical polymerization of monomers is a kinetically uncontrolled process [14], and the polymer forms not only on the surface of carbon nanotubes but also in the bulk of the solution. An alternative to the chemical method is enzymatic synthesis involving oxidoreductases, in particular, laccases; this process occurs under mild conditions in a kinetically controlled mode [15, 16].

It is known that electrically conductive polyaniline (PANI) is in the dedoping state at pH > 3.0 in the absence of a matrix; that is, the polymer is electrochemically inactive and nonconducting [17].

The purpose of the work was to obtain a PANI/multiwalled carbon nanotube (MWCNT) composite without any acidic dopants using the laccase from basidiomycete Trametes hirsuta as a catalyst and aniline dimer (N-phenyl-p-phenylenediamine) preadsorbed on the surface of carboxylated MWCNTs as an enhancer of the enzyme [18].

MATERIALS AND METHODS

In the work, we used aniline (AN) (LabTekh, Russia) purified by vacuum distillation and N-phenyl-p-phenylenediamine (AD) (Sigma-Aldrich, United States) without additional purification. A Graflex carbon foil (Unikhimtekh, Russia) was used as a current collector to obtain a composite electrode. Taunit-M multiwalled carbon nanotubes (Nanotekh Tsentr, Russia) were pretreated with concentrated nitric acid at 70°C and then washed with deionized water to a neutral pH (they are designated as fMWCNTs). Deionized water purified with a Simplicity device (Millipore, United States) was used in all experiments.

Laccase with a specific activity of 140 U/mL was obtained from the basidiomycete Trametes hirsuta culture fluid according to [19]. The amount of enzyme catalyzing the oxidation of 1 μM of diammonium 2,2'‑azino-bis(3-ethylbenzothiazoline-6-sulfonate) per 1 min at a pH of 4.5 was taken as the unit of activity.

AD was preadsorbed on the fMWCNT surface, and the excess unbound to the surface of the carbon nanomaterial was washed off with deionized water.

A PANI/fMWCNT composite was obtained by in situ AN polymerization in the absence of dopants and components of buffer mixtures on the surface of fMWCNTs, 10 mg of which was dispersed with preadsorbed AD in deionized water (10 mL) in an ultrasonic bath (FinnSonic, Finland). AN was then added in AN/fMWCNT weight ratios of 2 : 1, 5 : 1, and 10 : 1 to the dispersion; the reaction was initiated by the addition of laccase. The specific enzyme activity in the reaction mixture was ~0.6 U/mL. The polymerization was carried out under aerobic conditions at room temperature for 24 h with constant stirring. The PANI/fMWCNT precipitate was then separated via centrifugation, washed with deionized water, dried at 70°C to a constant weight, and used in further studies. For comparison, a composite based on starting (noncarboxylated) MWCNTs with an AN/MWCNT weight ratio of 2 : 1 was synthesized under similar conditions.

Electrochemical measurements were performed via cyclic voltammetry in a three-electrode mode with a CV-50W bioanalytical analyzer (BAS, United States). An Ag/AgCl electrode (BAS, United States) was used as a reference electrode; a platinum sheet was used as a counter electrode. The working electrode was a graphite foil with a deposited composite; for its manufacture, a known volume of alcohol dispersion of the composite was applied to the surface of carbon foil without a binder and dried.

The morphology of the samples was studied by transmission electron microscopy (TEM) with a Supra 40VP scanning electron microscope (Carl Zeiss Microscopy, Germany). Fourier transformed infrared (FTIR) spectroscopy was performed according to the standard procedure with KBr tablets on a Frontier FT-IR/FIR spectrometer (PerkinElmer, United States).

RESULTS AND DISCUSSION

Carboxylation of the MWCNT Surface

When MWCNTs are treated with oxidizing acids, the π–π bond conjugation system of the outer graphite surface of nanotubes is broken, resulting in the formation of hydrophilic carboxyl groups on the MWCNT surface [20–22]; this leads to hydrophilization of the carbon nanomaterial surface and, as a consequence, to enhancement of its compatibility with polar solvents. The treatment of MWCNTs with concentrated nitric acid made it possible to obtain stable fMWCNT dispersions in water. Research on starting and carboxylated fMWCNTs by the FTIR method showed that both spectra (Fig. 1, 1 and 2) contain absorption bands corresponding to stretching vibrations of the aromatic ring (1580 cm–1) and deformation vibrations of the C–H bond (1070 and 820–900 cm–1). After treatment with nitric acid, the carboxyl groups with characteristic bond vibrations in the range of 1750 (C=O) and 1180 cm–1 (C–O) [20, 22] formed on the fMWCNT surface.

Fig. 1.
figure 1

FTIR spectra of (1) starting MWCNTs, (2) carboxylated fMWCNTs, and (3) PANI/fMWCNT composite synthesized at an initial AN/fMWCNT weight ratio of 2 : 1.

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Physicochemical Characteristics of the Enzymatically Synthesized Composite

The structure, morphology, electrical conductivity, and electrochemical stability of the repeating PANI units were determined in the obtained PANI/fMWCNT composite.

The FTIR spectrum (Fig. 1, 3) of the PANI/ fMWCNT composite contains absorption bands at 1564 and 1516 cm–1 that correspond to vibrations of the quinoid diimine and phenylenediamine PANI units in the emeraldine oxidation state [23]. The absorption bands in the region of 1280–1350 cm–1 belonged to vibrations of the C–N bond in the secondary aromatic amines; in the region of 1200–1225 and 650–900 cm–1, they correspond to the deformation vibrations of the C–H bond in 1,4-substituted aromatic structures [24, 25].

The morphology of fMWCNTs and enzymatically synthesized composites was studied by transmission electron microscopy. Figure 2 shows TEM images of (1) AD-modified fMWCNTs and (2) PANI/fMWCNT composite synthesized with an initial AN/fMWCNTs weight ratio = 2 : 1. Figure 2 shows that a sufficiently uniform polymer layer formed as a result of the laccase-catalyzed aniline polymerization on the fMWCNT surface. Besides, PANI/fMWCNT composite had no polymeric phase not bound to the surface of carbon nanomaterial.

Fig. 2.
figure 2

TEM images of (1) fMWCNTs with adsorbed AD and (2) PANI/fMWCNT composite.

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It is known that PANI synthesized without dopants at a neutral or slightly acidic pH of the reaction medium has a branched structure and does not possess electrical conductivity and electrochemical activity [24, 26]. To clarify the effect of charged groups on the “solid” matrix surface on the electrochemical activity of a PANI as part of the PANI/fMWCNT composite, the synthesis was carried out in deionized water.

The PANI/fMWCNT composites obtained at different weight ratios of monomer and fMWCNTs showed electrochemical activity (Fig. 3, 1–3). Cathodic and anodic current peaks corresponding to the PANI redox transformation in the composites are observed in cyclic voltammograms. It should be noted that the specific electrochemical capacitance of the composites decreased significantly with an increasing AN/MWCNT weight ratio. This can be explained by the fact that only PANI bound to the surface of carboxylated fMWCNTs had electrochemical activity; the “free” polymer formed in the bulk of the reaction medium was non-conductive that was a “ballast” that reduced the specific capacity of the composites. At the same time, the PANI in the composite synthesized with the starting MWCNTs also did not possess electrochemical activity (Fig. 3, 4).

Fig. 3.
figure 3

Cyclic voltammograms of PANI/fMWCNT composites synthesized at AN/fMWCNT weight ratios of (1) 2 : 1, (2) 5 : 1, and (3) 10 : 1; and (4) PANI/MWCNT composite. The working electrolyte was 1 M H2SO4, and the potential scan rate was 50 mV/s.

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Thus, the influence of the “solid” matrix (fMWCNTs) on the electroactivity of the polymer in the composite was determined by the presence of charged carboxyl groups on the carbon nanomaterial surface.

We also studied the effect of pH on the electrochemical activity of PANI in a composite synthesized with the initial AN/fMWCNT weight ratio of 2 : 1. It is known that the interaction between the dopant and the PANI chain is violated at pH > 3.0 and the polymer passed into a dedoped state when low molecular weight acids are used as dopants [17]. This PANI form is electrochemically inactive and nonconductive. It was shown by cyclic voltammetry that PANI as a part of the PANI/fMWCNT composite showed electrochemical activity even at a pH of 7.0 (Fig. 4).

Fig. 4.
figure 4

Cyclic voltammograms of a PANI/fMWCNT composite recorded at pH values of (1) 2.8, (2) 5.0, and (3) 7.0. The working electrolyte was 0.1 M citrate-phosphate buffer, and the potential scan rate was 50 mV/s.

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Figure 4 shows that the irreversibility of the oxidation/reduction reaction of PANI in the composite increased with increasing solution pH. Nevertheless, the polymer remained electrochemically active at a neutral pH of the solution. Apparently, a stable PANI/fMWCNT complex formed as a result of the multipoint electrostatic interaction of the positively charged main chain of PANI with the carboxyl groups of the matrix; it was not destroyed when the pH of the solution increased to neutral values.

CONCLUSIONS

Thus, the PANI/fMWCNT composite obtained in situ by enzymatic polymerization of aniline on the surface of carboxylated fMWCNTs had electrochemical activity and electrical conductivity; that is, the fMWCNT surface carboxyl groups acted as a dopant of the main positively charged PANI chain. The composite exhibited electrochemical activity at a pH of 7.0, that is not typical of PANI doped with low molecular weight acids.