In recent decades, much attention has been paid to hybrid systems and composites based on conducting polyaniline (PANI), which is associated with the prospects of their use in various fields [1–5]. The properties of PANI can be varied over a wide range depending on the synthesis conditions [6, 7]. Usually, PANI is synthesized by chemical or electrochemical oxidation of ANI in a strongly acidic medium [1, 2, 8, 9]. An enzymatic method for obtaining conducting PANI, which meets the requirements of “white” biotechnology, has also been described [1, 10–15]. From an ecological point of view, the enzymatic synthesis of PANI is a good alternative to traditional methods of synthesis.

Laccase is a promising catalyst for the oxidative polymerization of ANI, since atmospheric oxygen is the oxidizing agent in the reaction. Laccase (p-diphenol:oxygen oxidoreductase, EC 1.10.3.2) belongs to copper-containing oxidases and catalyzes the oxidation of various organic compounds, resulting in the formation of radicals that enter into coupling reactions to form oligomeric/polymeric products [16–18].

The electrical and optical properties of PANI can be reversibly controlled by redox reactions or protonation [19, 20]. The oxidative state of PANI can vary from fully oxidized (pernigraniline) to fully reduced (leucoemeraldine). The semi-oxidized state of the polymer backbone is called emeraldine [1, 2]. The conducting form of PANI, the emeraldine salt, can be obtained both by protonation [1, 2, 21] and by doping the polymer chain with various dopants, such as Lewis acids, iodine, and transition metal salts [22–26]. Dopants of polymers are called electron donors or acceptors, which, when interacting with the polymer backbone, lead to the formation of charges on it, which determine the conductivity of the polymer.

It has been shown in a number of works that chemically synthesized PANI has antimicrobial properties [27–32]. The effectiveness of chemically synthesized PANI to inhibit the growth of gram-positive and gram-negative bacteria and fungi was first described in [27]: cotton fabrics coated with conducting PANI inhibited the growth S. aureus 95% E. coli by 85% and the fungus Candida albicans by 92%. The authors attributed the antibacterial effects of PANI to the electrostatic interaction between polymer molecules and bacterial cells, which led to the destruction of cell walls and cell death. Shi et al. [28] showed that polyvinyl alcohol films containing 1–10 wt % PANI completely inhibited bacterial growth of E. coli and S. aureus.

It is known that copper and its alloys are antimicrobial materials [33, 34]. It can be assumed that Cu2+ copper ions act as a polymer backbone dopant; they can enhance the antimicrobial properties of PANI.

The aim of this work was to carry out an template-guided enzymatic synthesis and study the physicochemical and antibacterial properties of polyaniline doped with copper(II) ions.

MATERIALS AND MEETHODS

Citric acid, NaH2PO4, KH2PO4, and NaOH were produced by Riedel-de Haën (Germany), CuSO4⋅5H2O, polystyrenesulfonate (PSS, 30 wt%) Sigma-Aldrich (United States) were used without additional purification. ANI (Sigma-Aldrich, United States) was purified by vacuum distillation.

Laccase was isolated from the culture liquid of the basidiomycete Trametes hirsuta (Wulfen) Pilat (strain T. hirsuta 56) according to the method in [35]. The enzyme was homogeneous according to SDS electrophoresis and had a specific activity of 161 U/mg protein. Enzyme activity was determined spectrophotometrically using 1 mM ABTS solution as a chromogenic substrate (λ = 420 nm; ε = 36 000 M–1 cm–1) in 0.1 M Na-citrate-phosphate buffer, pH 4.5. One unit of activity is defined as the amount of laccase oxidizing 1 µM of ABTS per min at 22°C. The protein concentration measured according to the method [36] was 7.8 mg/mL.

All solutions were prepared using water purified with Milly-Q Simplicity system (Millipore, United States).

Synthesis of PANI/PSS was carried out as follows: 10 µL of ANI (concentration 11 mM) was added to 10 mL of a PSS solution in 0.1 M citrate-phosphate buffer, pH 3.5 (concentration 11 mM per repeating unit of the polymer), and stirred for 1 h. The polymerization of ANI was initiated by adding an enzyme whose specific activity in the reaction mixture was ~1 U/mL. The reaction was carried out at room temperature (21–22°C) with constant stirring at a speed of 400 rpm on an RT-10 magnetic stirrer (IKA®-Werke GmbH & Co, Germany) for 24 h. The reaction mixture was then dialyzed against deionized water in order to remove low-molecular-weight compounds.

Dedoping of the PANI/PSS interpolymer complex was carried out by adding 1 M NaOH to the dialyzed solution to pH 10.0. After 12 h, the solution was dialyzed against water, whose pH was adjusted to a value of 7.8 with a NaOH solution. For redoping the complex with copper ions, 27.5 mg of CuSO4⋅5H2O (molar ratio ANI/CuSO4⋅5H2O = 1 : 1) was stirred at room temperature (21–22°C) on a magnetic stirrer at 400 rpm for 12 h and dialyzed against deionized water.

UV-visible spectra were recorded on a UV1240 mini spectrophotometer (Shimadzu, Japan). Fourier transform infrared spectroscopy with Attenuated Total Reflection sampling (FTIR-ATR) was carried out on an FT/IR-67000 spectrometer (Jasco, Japan). The copper content in the samples was determined by energy dispersive X-ray spectroscopy (EDX) using a JSM-6510LV scanning electron microscope (JEOL Ltd, Japan) equipped with a cX-MAX energy dispersive attachment (Oxford Instruments, United Kingdom). To determine the copper content and study by FTIR-ATR spectroscopy, the samples were dried at 60°C to a constant weight.

For studies on antimicrobial activity, aqueous solutions of the complexes were concentrated by evaporation at 37°C to 5.8 (PANI/PSS) and 5.3 mg/mL (PANI-Cu/PSS). The minimum inhibitory concentrations (MIC) for the synthesized complexes were determined by the standard serial two-fold dilution method in LB nutrient medium (Luria-Bertani, Miller, Sigma, United States). The Gram-positive bacterium Staphylococcus aureus 209P and Gram-negative bacterium Escherichia coli K-12 were used as a test cultures. 200 µL of presterile LB media containing various concentrations of the complexes was added to the wells of a 96-well plate in three replicates for each concentration. Then, 4 µL of cells of test cultures of the stationary growth phase (1 day) were added to each well and incubated at 28°C with stirring on a rotary shaker (150 rpm). After 24 h of incubation, the optical density of each well was measured relative to controls (without inoculum) at 540 nm using an Absorbance Microplate Reader (Azure Biosystems, UK). The growth of microorganisms was assessed by the change in optical density compared to the initial value (immediately after inoculation of the medium). The MIC was defined as the lowest concentration of a compound that inhibited bacterial growth within 24 h. Each culture was tested three times.

RESULTS AND DISCUSSION

One of the key problems with the use of conducting PANI is the poor performance characteristics of the polymer (insolubility, infusibility, and poor mechanical properties), which prevent its wide industrial use. One of the options for solving this problem is the synthesis of PANI using a water-soluble template [1, 2, 37, 38]. In this work, PSS was used as a template.

As a result of the oxidative polymerization of ANI on the PSS catalyzed by laccase, an conducting PANI/PSS interpolyelectrolyte complex was obtained, whose UV-visible spectrum is shown in Fig. 1 (curve 1). The PANI/PSS spectrum contained bands that are characteristic of conducting PANI: a band at 340 nm, corresponding to π–π* electronic transitions in aromatic rings, and two absorption bands (420 and 787 nm), indicating the formation of a polaron in the PANI structure [39 , 40]. When the complex was dedoped these bands disappeared and an absorption band appeared in the region of 510 nm, corresponding to PANI in the nonconductive form of the emeraldine base (Fig. 1, 2). During this transition, the color of the solution changed from green to blue. Since the dedoping/doping process is reversible, after dialysis, bands reappear in the spectrum, which are characteristic of the conducting form of PANI (Fig. 1, 3). However, at pH 7.8, the main absorption band corresponding to the polaron shifted to shorter wavelengths (755 nm). When the polymer was redoped with Cu2+ ions (Fig. 1, 4) this band shifted to 764 nm, which was indirect evidence of the interaction of copper ions with the PANI backbone.

Fig. 1.
figure 1

The UV-visible spectra of the enzymatically synthesized PANI/PSS complex (1, pH 3.5), dedoped complex (2, pH 10.0), complex after dialysis (3, pH 7.8) and copper-redoped PANI-Cu/PSS complex (4).

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The study of PANI‑Cu/PSS by energy-dispersive X-ray spectroscopy (Fig. 2) showed that the copper content in the complex, calculated as an average value over four measurements, was 1.66 ± 0.31 at % or 6.81 ± 1.18 wt %.

Fig. 2.
figure 2

The EDX spectrum of the PANI‑Cu/PSS.

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Figure 3a shows the general formula for PANI. The ratio of phenylenediamine (benzenoid) and quinoiddiimine (quinoid) units in the PANI backbone determines several possible redox states of the polymer: leucoemeraldin (completely reduced state), emeraldine (semioxidized state), and pernigraniline (completely oxidized state). During acid doping (Fig. 3b), protons interact with imine nitrogen atoms, which leads to the formation of a polycation. Positive charges localized on nitrogen atoms increase the total energy of the polymer system, resulting in a redistribution of the electron density and depairing of the lone electron pair of nitrogen atoms. Delocalized electrons appear in the polymer chain and electronic conductivity occurs, which depends on the degree of PANI protonation and on the nature of the doping acid. Thus, the conductivity of PANI is due to the presence of radical cations (polarons) localized on the nitrogen atoms of quinoid diimine rings in the polymer chain. Doping of the PANI chain with transition metal ions refers to oxidative doping. Based on the results of experiments with transition metal salts that can act as an oxidizing agent, Dimitriev [25] proposed the following mechanism for oxidative doping of the PANI emeraldine base. Transition metal ions oxidize the nitrogen atoms of the benzenoid units; the reduced metal ions then coordinate with the imine nitrogen atoms of the quinoid units. The reduced metal cations are then oxidized and the imine groups are reduced, which leads to the formation of the doped conducting form of PANI and the oxidized cation.

Fig. 3.
figure 3

The general formula for PANI (a): y = 1, leucoemeraldine; y = 0.5, emeraldine and y = 0, pernigraniline; schematic representation of interconversions of various redox states of PANI (leucoemeraldin, emeraldine, pernigraniline) upon acid doping (b).

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Figure 4 shows the FTIR-ATR spectra of the PSS (curve 1) and PANI/PSS (curve 2) and PANI‑Cu/PSS (curve 3) complexes. Stripes in the area of 600–1150 cm–1 corresponded to bond vibrations in the PSS molecule, which confirmed the formation of the PANI/PSS complex as a result of the enzymatic reaction. The most significant differences in the spectra of PANI/PSS and PANI-Cu/PSS were observed in the region of 1150–1600 cm–1. Both spectra had a band at 1496 cm–1 related to vibrations of the C–C bond of benzenoid units in the PANI structure [41, 42]. At the same time, the intensity of the absorption band corresponding to vibrations of the C=C bond of the quinoid fragments increased significantly when PANI was doped with copper ions and the position of the band shifted to the side of the lower wavenumber (from 1581 cm–1 in PANI/PSS up to 1567 cm–1 in PANI-Cu/PSS), indicating an increase in the relative amount of quinoid fragments in the PANI chain [24]. There was also a shift in the band corresponding to the vibrations of the ‒NH+=, from 1173 to 1163 cm–1. In addition, additional bands appeared in the PANI-Cu/PSS spectrum, corresponding to vibrations of the C–N bond near the quinoid ring (1324 and 1358 cm–1), and a band related to bond vibrations of the radical cation C‒N•+ (1259 cm–1). When PANI was doped with copper ions, the intensity of the band related to vibrations of the C–N bond of secondary aromatic amines increased in the region of 1307 cm–1. Such changes in the FTIR spectra indicated that the main chain of PANI was doped with copper ions.

Fig. 4.
figure 4

FTIR-ATR spectra of the PSS template (1), PANI/PSS (2) and PANI‑Cu/PSS (3).

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For antimicrobial studies, aqueous solutions of PANI/PSS and PANI-Cu/PSS were concentrated to 5.8 and 5.3 mg/mL, respectively. Studies have shown that the PSS did not have antimicrobial activity; for the PANI/PSS complex, although it exhibited antimicrobial activity against S. aureus and E. coli, its MIC for both organisms is higher than the maximum reached in the studied concentration (2.9 mg/mL). It should be noted that in there are very few data in the literature on the MIC of “pure” PANI, which is apparently due to its poor solubility. It was shown in [29] that the MIC of a suspension in a nutrient medium of chemically synthesized PANI for E. coli and S. aureus was above 10 mg/mL. Shalini et al. [31] showed that the MICs of a suspension of PANI in dimethyl sulfoxide for E. coli and S. aureus were 10  and 2.5 mg/mL, respectively.

The PANI-Cu/PSS complex showed a higher inhibitory activity than PANI/PSS. The MIC of PANI-Cu/PSS vs. S. aureus was 0.66 mg/mL, and E. coli—2.65 mg/mL. Since there are no data in the literature on the MIC values for composites containing PANI doped with copper ions, it does not seem possible to make a direct comparison with our results. However, it can be concluded that the copper ions in the composition of the PANI-Cu/PSS complex enhanced the antimicrobial properties of the polymer and their use as a dopant in the PANI main chain is promising for the development of antimicrobial film coatings.