A study of magnetic, antibacterial and antifungal behaviour of a novel gold anchor of polyaniline/itaconic acid/Fe₃O₄ hybrid nanocomposite: Synthesis and characterization

2.1 Synthesis of PANI

2 mL (0.02 mol) of aniline monomer was dissolved in 50 mL of 0.1 M HCl with stirring till complete dispersion and homogeneity of solution. Anhydrous FeCl₃ (3.5 g) dissolved in 50 mL of distilled water was added dropwise to aniline solution with constant stirring for 5 h. The resulting intermediate PANI was washed several times by double distilled water followed by methanol. The final product was dried in a vacuum oven at 50 °C for 24 h (Ayad et al., 2012).

2.2 Synthesis of PANI–IA–Fe₃O ₄ nanocomposite

Initially, 2.70 g of IA (0.02 mol) was dissolved into 50 mL of double distilled water and stirred for 30 min then 2 mL (0.02 mol) of aniline monomer also added. The resulting solution was left constant under stirring for 1 h to obtained homogenize compound. In an ice bath, 6.5 g (0.04 mol) of anhydrous FeCl₃ was added to the mixture and left under mechanical stirring overnight. The sodium hydroxide solution was added slowly to the mixture and maintain the temperature at 10 °C. The resulting product was collected and washed several times with double distilled water and ethanol. The obtained product was dried in an oven at 50 °C for 24 h.

2.3 Preparation of piper betel leaf extract

The piper betel leaves were collected and washed several times with double distilled water. 30 g of piper betel leaves were cut in to a small pieces and put it into a conical flask containing 300 mL double distilled water. The whole content was boiled for about 15 min and filtered through Whatmann filter paper. The obtained extract of leaves was stored at refrigerator at 4 °C till further use of the compound (Punuri et al., 2012).

2.4 Synthesis of gold nanoparticles

A single-step reduction was used to synthesis of AuNPs (Zhan et al., 2011). Briefly, 40 mL of HAuCl₄ (1 mM) was prepared and 20 mL of leaf extract was then added to the solution with constant stirring for 2 h. The formation of AuNPs could be indicated by the change of solution color to be a typical dark-red solution. The feed composition of gold anchor Au@PANI-IA-Fe₃O₄ magnetic nanocomposites shown in Table 2.

2.5 Synthesis of Au nanoparticles @PANI– IA–Fe₃O ₄ nanocomposite

The prepared PANI–IA–Fe₃O₄ nanocomposite was added to 50 mL of 0.5 M NH₄OH for complete deprotonation and then washed using double distilled water. 0.5 g of the previously deprotonated PANI–IA–Fe₃O₄ nanocomposite was added to 40 mL of AuNPs solution under constant mechanical stirring for 4 h. The obtained product was washed with double distilled water with ethanol several times. The collected final product was dried at room temperature for 3 days for further characterization. The schematic representation of the development of Au@ PANI–IA–Fe₃O₄ nanocomposite has shown in Scheme 1.

Close

Scheme 1

Shows the schematic representation of Au@PANI–IA–Fe₃O₄ nanocomposites.

Export to PPT

3.1 Antibacterial activity

The antibacterial activity of magnetic nanocomposites was evaluated against Staphylococcus aureus (gram +ve) and Escherichia coli (gram −ve) pathogens spread on Mueller-Hinton agar (MHA) plates. A well of diameter 6 mm was made using a sterile cork borer and loaded with the required concentration sample over the agar. The test plates were incubated for 24 h at 37 °C. The zone of inhibition (mm in diameter) activity were observed against the test pathogens, Ciproflaxin (20 μg) was used as a positive control.

3.2 Antifungal activity

To estimate the impacts of antibiotic unloaded Au@PANI–IA–Fe₃O₄ nanocomposites on Aspergillus niger and Candida albicans growth were analysed by well diffusion method. Petri plates were also been used to prepare 20 mL of sterile MHA (Hi- media, Mumbai). The test culture was washed on the top of solidified media and then allowed to dry for 10 min. Wells were prepared in the media using a well borer. The different concentrations of the samples (50, 100, 250 and 500 μg per well) were loaded on the wells. Ciproflaxin (20 μg/well) used as a positive control. These plates were incubated at 37 °C for 24 h in closed condition. The zone of inhibition was noticed in millimeters (mm).

4.1 Fourier transform infrared spectroscopy (FT-IR) studies

FT-IR spectra tool has been utilized to finding the chemical structure of PANI, IA, PANI–IA–Fe₃O₄ nanocomposite of before and after anchoring the AuNPs. Fig. 1 has shown the comparison of FT-IR spectra of PANI (Fig. 1a) and IA (Fig. 1b) through the FT-IR spectra of PANI–IA–Fe₃O₄ (Fig. 1c) and Au@PANI–IA–Fe₃O₄ (Fig. 1d). The formation of PANI (Fig. 1a) has been confirmed by the characteristics peaks at 1553 cm⁻¹ and 1447 cm⁻¹ corresponds to C⚌C stretching of quinonoid and benzenoid respectively (Sampreeth et al., 2018). The sharp peaks at 1315 cm⁻¹ and 1221 cm⁻¹ was assigned to the C—N stretching vibration in (Q-B-Q) unit and vibration of the secondary aromatic amine. The sharp peaks were observed at 1095 cm⁻¹ and 850 cm⁻¹ could be assigned to in plane C—H stretching vibration and out of plane C—H bending stretching vibrations respectively (Ayad et al., 2017).

Close

Fig. 1

FTIR spectra of (a) PANI, (b) IA, (c) PANI–IA–Fe₃O₄ nanocomposite and (d) Au@PANI–IA–Fe₃O₄ magnetic nanocomposite.

Export to PPT

A broad peak has been notified at 3011 cm⁻¹, which is due to O—H stretching vibrations of IA. The (Fig. 1b) sharp peaks have been observed at around 1686, 1431 and 1213 cm⁻¹, indicating the stretching vibrations such as C⚌O (carboxylic acid), C—O—H in-plane and C—O, respectively. Fig. 1c showed an overlapped peak at 3163 cm⁻¹ and 2630 cm⁻¹ which is due to the interaction between the PANI and IA. It leads to form PANI–IA–Fe₃O₄. A new peak has been noticed at 1639 cm⁻¹ corresponds to PANI–IA–Fe₃O₄ nanocomposite which is confirms the disappearance peaks. The peak at 1686 cm⁻¹ was also observed in the interaction between PANI and IA Fig. 1c (Zeghioud et al., 2015).

Except for the two peaks at 1300 cm⁻¹ and 825 cm⁻¹, other characteristic absorption peaks in the infrared spectrum of the IA doped polyaniline have shown redshift with a broader shape undoped one. The other stretching vibrations such as 1576 cm⁻¹, 1396 cm⁻¹ and 1161 cm⁻¹ of quinone and benzene rings of C-N stretch in a secondary aromatic amine has been observed. The redshift might be due to the delocalization of positive charge from the imine nitrogen atom to the benzene ring and decrease the electronic interaction intensity. The new absorption peak has been observed at 1238 cm⁻¹ which is indicates the doped polyaniline in the nanocomposite (Wang et al., 2014).

The two peaks at 580 cm⁻¹ and 464 cm⁻¹ has shown the association of pristine magnetite. In Fig. 1c has shown the new appearance of peaks at 553 cm⁻¹ and 445 cm⁻¹ which well support the presence of magnetite (vibrational Fe—O bond). The shifting of the magnetite peaks from the parent peak exposed to the interaction of magnetite with PANI and IA. A small shift of NH and OH stretching vibration has been noticed in the position at 1639 cm⁻¹ and 3263 cm⁻¹ (Fig. 1c) to 1608 cm⁻¹ and 3246 cm⁻¹ (Fig. 1d) which is due to gold metal reduction and its deposition over PANI–IA–Fe₃O₄ nanocomposite. Hence, this FT-IR gold anchor PANI–IA magnetic nanocomposites has been well supporting the structure of Au@ PANI–IA–Fe₃O₄ nanocomposites. Therefore, FT-IR suggest good interaction between IA and PANI with the presence of magnetite and gold nanoparticles on the PANI–IA–Fe₃O₄ nanocomposite (Peymanfar et al., 2019; Ayad et al., 2017).

4.2 Thermal analysis

Thermal properties of the developed magnetic nanocomposites were examined by the TGA thermogram method. The magnetic nanocomposites such as PANI–IA–Fe₃O₄ and Au@PANI–IA–Fe₃O₄ has shown in Fig. 2. The different stages of thermal degradation of these materials could be observed as a multistep process. The three-stage weight loss behavior has been noticed due to the evaporation of the humidity or expulsion of water, residual solvents and small molecule form PANI at 40 °C to 150 °C and 40 °C to 325 °C could be attributed to the loss of bounded water moieties and dopant. The samples (a) and (b) an amount of weight loss due to the evolution of water are 21.94% and 30.60% composites cleavages respectively. The second stage of weight loss observed from 150 °C to 545 °C and 325 °C to 500 °C is typical for the degradation of IA. The samples (a) and (b) an amount of weight loss owing to the evolution of the dopant molecules are 10.86% and 27.14% composites, respectively. The third stage of weight loss has been examined between 545 °C and 780 °C and 500 °C to 780 °C which is due to the decomposition of PANI polymer matrix. The complete decomposition of the composite occurred at 780 °C. The samples (a) and (b) an amount of weight loss due to the evolution of water are 28.25% and 16.34% composites, respectively. The total weight loss is about 61.05% and 74.07%, thus the residue of Fe₃O₄ is about 38.95% and 25.93%. The above percentage was close to the presence of Fe content. The addition of itaconic acid which enhances the thermal stability of magnetic nanocomposite such as PANI–IA–Fe₃O₄ and Au@PANI–IA–Fe₃O₄ (Mu et al., 2017; Minisy et al., 2018). Differential analysis (DTA) curve for PANI–IA–Fe₃O₄ (Fig. 2a) has been observed an endothermic peak at 50 °C to 120 °C and an exothermic peak at 320 °C to 390 °C. The endothermic peak for removal of moisture and water molecule and exothermic for crosslinking of a polymeric matrix with metal and sharp endothermic peak 700 °C to 720 °C has also observed. Similarly, the DTA curve for Au@PANI–IA–Fe₃O₄ (Fig. 2b) has been noticed an endothermic peak at 70 °C to 120 °C due to the traces amount of water molecules. The exothermic peak at 390 °C to 420 °C has been observed and it is due to the crosslinking of a polymer and strong binding of AuNPs. A small peak at 690 °C has also been observed.

Close

Fig. 2

TGA/DTA curves of (a) PANI–IA–Fe₃O₄ and (b) Au@PANI–IA–Fe₃O₄ magnetic nanocomposite.

Export to PPT

4.3 Solubility analysis

The solubility test was performed in PANI, PANI–IA–Fe₃O₄ and Au@PANI–IA–Fe₃O₄ nanocomposite using different organic solvents such as ethyl alcohol, chloroform, acetic acid, DMF, DMSO, DEG, THF, THF/water, NMP/water, hot water, and distilled water. The FT-IR spectra were used for qualitative recording before and after their submerging of organic solvents for 5 min. By comparing these FT-IR spectra for each polymer. The intensities of the FTIR peaks of poly(aniline), PANI–IA–Fe₃O₄ and Au@PANI–IA–Fe₃O₄ nanocomposites were observed. The results indicate that the 50% solubility was obtained in organic solvents for polyaniline, whereas 100% solubility was noticed in PANI–IA–Fe₃O₄. PANI based synthetic nanocomposite were soluble in THF/water and NMP/water (Zeghioud et al., 2015; Butoi et al., 2017). Hence, the prepared Au@PANI–IA–Fe₃O₄ were not diminished in any solvents that exposed the stability of the compound. The obtained results are shown in Table 2.

4.4 X-ray powder diffraction (XRD)

The crystallinity has been investigated through the XRD pattern. Fig. 3 shows the XRD spectra of PANI, PANI–IA–Fe₃O₄ and Au@PANI–IA–Fe₃O₄ nanocomposite. The main reflections correspond to a pseudo orthorhombic phase of PANI (Mirmohseni et al., 2012). The slight sharps peaks have been observed at 2θ of 15.36°, 20.65°, 25.22°, and 35.73° which representing the crystal planes of [0 1 1], [1 0 0], [1 1 0]and [2 0 0] of PANI, respectively Fig. 3a (Yin et al., 2019). The peak centered at 2θ of 25.22° could be attributed to periodicity perpendicular to the polymer chain of PANI (Hasik et al., 2001). Whereas, pure PANI, a broad amorphous peak has been observed at 2θ of 20.65° might due to the attribution of the conjugated polymer chain (Belaabed et al., 2012; Zhang et al., 2018). The diffractogram of PANI–IA–Fe₃O₄ magnetic nanocomposite has shown two peaks at 14.26° and 33.21° with a characteristic distance of 6.23 Å and 2.69 Å, respectively (Fig. 3b). The formation of PANI–IA–matrix was also supported by the comparison of PANI and PANI–IA (Zeghioud et al., 2015). The Fig. 3b, shows peaks at 2θ of 21.45°, 32.98°, 42.88°, 53.89°, 64.35° and 84.19° could be indexed as [h k l] to [2 2 0], [3 1 1], [4 0 0], [4 2 2], [4 4 0] and [5 1 1] planes of face-centered cubic (fcc) of Fe₃O₄ observed in PANI-IA-Fe₃O₄ nanocomposite (Mu et al., 2017; Minisy et al., 2018). The above results Au@PANI–IA–Fe₃O₄ nanocomposite after the formation of gold nanoparticles over its surface was performed well. The four main diffraction lines were observed at 2θ of 38.47, 44.71, 64.35 and 77.96 that can be indexed with (h k l) crystallographic planes to [1 1 1], [2 0 0], [2 2 0] and [3 1 1] with related to FCC gold crystals. The formation of AuNPs on the PANI–IA–Fe₃O₄ magnetic nanocomposite of XRD was also well supported to FT-IR results (Punuri et al., 2012; Ayad et al., 2017).

Close

Fig. 3

XRD patterns of the prepared (a) PANI, (b) PANI–IA–Fe₃O₄, and (c) Au@ PANI–IA–Fe₃O₄ magnetic nanocomposites.

Export to PPT

4.5 SEM–EDX studies of Au@PANI–IA–Fe₃O₄ nanocomposite

The surface morphology and the particle sizes of Au@PANI–IA–Fe₃O₄ size distribution of the magnetic nanocomposite were identified using SEM/EDX. Fig. 4a show the typical SEM image of Au@PANI–IA–Fe₃O₄ magnetic nanocomposite. The SEM images reveal the nanostructure of the synthesized nanocomposite and the triangle and bar shape of the Fe₃O₄–Au nanostructures (without aggregation) on PANI – IA matrix with a particle size of about 20 μm (Ayad et al., 2017; Duc Quan et al., 2019).

Close

Fig. 4

SEM micrographs of (a) Au@PANI–IA–Fe₃O₄ and EDX images of (b) Au@PANI–IA–Fe₃O₄ magnetic nanocomposite.

Export to PPT

The energy dispersive X-ray analysis of gold nanoparticles in the PANI–IA–Fe₃O₄ nanocomposite has shown in Fig. 4b. The Au@PANI–IA–Fe₃O₄ magnetic nanocomposite has shown an optical absorption peak at ∼2.0 keV, which is a typical absorption of metallic gold nanoparticles (Duc Quan et al., 2019; Elavazhagan et al., 2011). The obtained result reveals the elemental composition and confirmed the presence of C, O, Fe, and Au in Au@PANI–IA–Fe₃O₄ nanocomposite. The embedded HAuCl₄ was successfully reduced into AuNPs and it has supported the Au signals. The gold nanoparticles peak was obtained at 2 Kev with 0.29% gold, 46.73% carbon, 10.485% iron, and 42.495% oxygen (Fig. 4b). There are no impurity peaks has been investigated which is support the purity of gold nanoparticles (Chitra et al., 2018). The distribution of iron and Au in Au@PANI–IA–Fe₃O₄ magnetic nanocomposite was demonstrated via elemental mapping technique (Fig. 4b). The obtained results were indicated that the Fe₃O₄ and AuNPs over PANI-IA matrix.

4.6 Transmission electron microscopy

The TEM analysis was used to identify the structure of PANI–IA–Fe₃O₄ and Au@PANI–IA–Fe₃O₄ magnetic nanocomposite. A uniform distribution of gold nanoparticles within the nanocomposite matrix were observed in different TEM images. The TEM images of PANI–IA–Fe₃O₄ and Au@PANI–IA–Fe₃O₄ nanocomposite (Fig. 5a & b) have shown the dispersion of Fe₃O₄ and AuNPs through PANI–IA matrix with low aggregation at high magnification resolution 20 nm. A uniform distribution of Fe₃O₄–Au nanostructures demonstrated with low aggregation to some extent was examined. The TEM image of Au@PANI–IA–Fe₃O₄ nanocomposite has been observed in Fig. 5b. The crystalline AuNPs as the lattice fringes spacing is 50 nm, which is consistent with the crystal structure of Fe₃O₄ formation. The typical HRTEM image were 20 nm reveal the crystal structure of Au nanoparticles within the polymer matrix in Fig. 5c. Hence, Au@PANI–IA–Fe₃O₄ magnetic nanocomposites with small size could reveled larger surfaces (20 nm and 50 nm) and a more competent active center, which is a favorite of bacterial activity (Min et al., 2007). Chitra et al, Similar to our observation, the indole-3-acetic acid-based nanocomposite systems. The selected area via electron diffraction (SAED) of Au@PANI–IA–Fe₃O₄ nanocomposite and the arrays with bright circles supported the crystallinity of these nanoparticles has showed Fig. 5d. (Chitra et al., 2018). The magnetic, biological and thermal behavior of Au@PANI–IA–Fe₃O₄ might be due to the presence of AuNPs crystal in different sizes and shapes to account for the polymer matrix.

Close

Fig. 5

TEM images of PANI–IA–Fe₃O₄ magnetic nanocomposite (a), Au@PANI–IA–Fe₃O₄ magnetic nanocomposite (b), HRTEM of Au@PANI–IA–Fe₃O₄ magnetic nanocomposite (c), and SAED of Au@PANI–IA–Fe₃O₄ magnetic nanocomposite (d).

Export to PPT

4.7 Magnetic properties (VSM)

Magnetization measurements of PANI–IA–Fe₃O₄ and Au@PANI–IA–Fe₃O₄ magnetic nanocomposites were done by vibrating sample magnetometer (VSM) at room temperature. Fig. 6. Shows the images of magnetization of nanocomposites para, dia, and super paramagnetic materials, when the applied magnetic field is removed, they should exhibit no coercivity and remanence. Paramagnetic materials have a linear relationship between magnetization (M) and applied filed (H) with a positive slope. The suggesting paramagnetic character with a saturation magnetization of PANI–IA–Fe₃O₄ and Au@PANI–IA–Fe₃O₄ values were found to be 0.925 emu/g and 1.140 emu/g. The net magnetization of the PANI based nanocomposite has been reduced due to a further reduction in the magnetization. The prepared PANI based complex exhibits paramagnetic character, which indicates it could act as a potent MRI positive contrast agent (Thenmozhi et al., 2015; Azizian et al., 2012). The obtained paramagnetic character is due to the interactions of ferrite and polymeric matrix with the PANI-IA prepared ferrite. Donescu et al, also been examined the one without similar PANI-based ferrite complex (Donescu et al., 2017). This behavior of the M−H curves is a well-known characteristic of materials with paramagnetic properties.

Close

Fig. 6

Magnetization curves of PANI–IA–Fe₃O₄ and Au@PANI–IA–Fe₃O₄ magnetic nanocomposite.

Export to PPT

4.8 Antibacterial activity of Au@PANI–IA–Fe₃O₄ nanocomposite

The in-vitro antibacterial activity of Au@PANI–IA–Fe₃O₄ nanocomposite tested against Staphylococcus aureus (gram +ve) and Escherichia coli (gram −ve) pathogens Mueller-Hinton agar (MHA) plates test as shown in Fig. 7. The synthesized AuNPS embedded Au@PANI–IA–Fe₃O₄ magnetic nanocomposite has been exhibited good antibacterial activity against two pathogens compared to Ciproflaxin (Fig. 7a-b). This could be related to a more antibacterial feature of AuNPs, largely due to the inert chemical nature of gold present within the nanocomposite (Zhou et al., 2012). The Ciproflaxin was chosen as a broad-spectrum antibiotic standard. The zone of inhibition of itaconic acid based nanocomposite against pathogens was determined with varying concentrations of 50 μg, 100 μg, 250 μg and 500 μg (Chitra et al., 2018; Sakthivel et al., 2018). The zone of inhibition of Au@PANI–IA–Fe₃O₄ has been exhibited anti-bacterial efficacy towards S. aureus (gram +ve) was 10 mm, 11 mm, 12 mm and 14 mm for 50 μg, 100 μg, 250 μg, 500 μg respectively. The obtained results did not support the antibacterial zone of inhibition against pathogens and it mainly depends on the concentration of IA. The synthesized magnetic nanocomposite has shown better sensitivity against S.aureus (gram +ve) bacterial strains. Whereas, Au@PANI–IA–Fe₃O₄ magnetic nanocomposite did not exhibit a significant antibacterial effect observed against E. coli (gram −ve) bacteria. Sakthivel et al and Kenawy et al, also mentioned the positively charged antibacterial polymers tendency to bind with (gram −ve) bacteria and there by splitting of negatively charged bacterial membranes (Sakthivel et al., 2018; Kenawy et al., 2007). Hence, the prepared Au@PANI–IA–Fe₃O₄ based nanocomposite has been enhanced after the incorporation of AuNPs with anionic charged polymers against +ve pathogen (S.aureus) (Pulat et al., 2008). The Au@PANI–IA–Fe₃O₄ magnetic nanocomposite has been revealed a better antibacterial property. This could be attributed to the combined antibacterial effect of Au and IA in Au@PANI–IA–Fe₃O₄ magnetic nanocomposite.

Close

Fig. 7

Antibacterial effect of Au@PANI–IA–Fe₃O₄ magnetic nanocomposite.

Export to PPT

4.9 Antifungal activity of Au@PANI–IA–Fe₃O₄ nanocomposite

In recent years anti-fungal activity based polymeric material has been focused in different biomedical applications. The most common species of Aspergillus nigeris has been derived from the genus Aspergillus. In general, some fruits and vegetables have been affected on the surface of fruits which causes black mould disease. Whereas, ear infections and serious lung disease have been observed in humans. The Candida albicans originated from the Candida genus has also produced various disease viz., candidiasis, fungemia, morbidity, etc. Fig. 8. has shown the antifungal activity of gold anchor PANI–IA–Fe₃O₄ nanocomposites with a different zone. Here, two pathogens such as Aspergillus niger and Candida albicans were utilized to observe the antifungal activity. The Ciproflaxin (20 μg/well) was preferred as a positive control. The anti-fungal zone of inhibition has been examined, with respect to different Au@PANI–IA–Fe₃O₄ nanocomposite concentrations viz., 50 μg, 100 μg, 250 μg, and 500 μg. The antifungal activity of gold anchor PANI–IA–Fe₃O₄ nanocomposites of C. albicans was found to 1 mm, 2 mm, 3 mm, & 7 mm respectively. The achieved results have clearly shown that the gold anchor PANI–IA–Fe₃O₄ nanocomposite as a better anti-fungal activity. This might be due to the increasing stoichiometric amount and hydrophilic nature of IA in the polymeric nanocomposite (Tomic et al., 2009; Zhang et al., 2013). However, no such kind of the microbial growth was observed against A. Niger. Sakthivel et al, has also been studied using Itaconic acid based pH-sensitive hydrogels systems (Sakthivel et al., 2018). Hence, the results imply gold anchor PANI based magnetic nanocomposite has a significant inhibition activity against C. albicans.

Close

Fig. 8

Antifungal activity of Au@PANI–IA–Fe₃O₄ magnetic nanocomposite.

Export to PPT