In this study, the nanocomposites of POPD and MMT were successfully synthesized via intercalation and in-situ polymerization of o-phenylenediamine within MMT using sonochemical technique by taking the former to be as filler in one case and as matrix in the other. The results confirmed the formation of NC which is published in our previously paper (Riaz et al., 2016).
TEM micrograph of the different ratios of POPD:MMT and MMT:POPD (i.e. 1:0.25, 1:0.50, 1:1) exhibited particle size in the range of 20–90 nm and showed the spherical chain like structure (Fig. 1). POPD has shown the formation of core and MMT was found to encapsulate the dense POPD particles like a shell. The POPD:MMT-1:1 and MMT:POPD-1:1 NC showed clustering and aggregation of the spherical nanoparticles of 50–90 nm range (Fig. 1d and 1g). The results obtained were in compliance with as described in our previously published paper (Riaz et al., 2016).
4.4 Broth microdilution Assay
The effects of different ratios of MMT/POPD and POPD/MMT and standard antibiotic drug (ampicillin) on bacterial strains were determined by measuring postincubation absorbance readings at 600 nm and %MGI was calculated. The results were compared with standard ampicillin as shown in Fig. 2. NC had exhibited various degree of growth inhibition against tested bacterial strains such as E. coli, S. aureus, B. subtilis, P. aeruginosa etc. Among the all bacterial strains tested, B. subtilis was the most sensitive and S.aureus was the least sensitive strains towards the NC. When MMT: POPD − 1:0.25, MMT: POPD − 1:0.5 and MMT: POPD − 1:1 were tested, they showed the highest resistance against B. subtilis i.e. %MGI were 31.609 ± 0.349, 86.565 ± 0.440 and 77.733 ± 0.749 respectively whereas they showed least % inhibition against S. aureus which were 18.235 ± 1.014, 47.819 ± 0.793 and 38.403 ± 0.943 respectively (Fig. 2).
When bacterial cells were incubated with POPD: MMT – 1:0.25, POPD: MMT – 1:0.5, and POPD: MMT – 1:1, they showed the highest susceptibility towards B. subtilis and hence high mean growth inhibition was observed i.e. %MGI were 38.866 ± 0.974, 79.591 ± 0.429 and 77.971 ± 1.026 respectively whereas S. aureus showed least sensitivity towards these POPD/MMT NC. The % mean growth inhibition against S. aureus was observed approximately 15.524 ± 0.427, 61.878 ± 0.347 and 37.616 ± 0.896 respectively. The results were quite comparable with standard ampicillin which showed highest % inhibition against B. subtilis (%MGI-78.798 ± 0.366) and least against S. aureus (%MGI 56.236 ± 0.364) (Fig. 3).
S. aureus was the only strain which was less susceptible to the antibacterial effect of the all NC and ampicillin as compared to the other strains. NC exhibited different degree of growth percentage inhibition among tested bacterial strains which were presented in the form of EC50 values .
Both the assays confirmed that NC is capable to inhibit the growth of bacterial strains in a range of 15.625–2000 µg ml− 1. The results showed that B. subtilis is the highest sensitive strains among all NC and exhibited the highest % inhibition i.e. sensitivity towards all the ratios of NC that’s why MIC was performed for this strain only in presence of all NC. However, the results also proved that NC was bacteriostatic to B. subtilis at its MIC80 concentration where its 80% growth was inhibited and the clear solution was obtained. Inhibitory concentration of B. subtilis was evaluated by determining MIC80 as depicted in Figs. 4 and 5.
The antibacterial activity of NC showed that, B. subtilis displayed susceptibility to all NC ranging from 15–2000 µg ml− 1. This method is based on a broth micro dilution method in 96-well microtitre plates. The MIC (MIC80) of MMT/POPD and POPD/MMT against the B. subtilis was shown below. No matter whichever the NC were tested, all of them exhibited good antibacterial activity and a broad spectrum of activity.
Determination of zone of inhibition by agar well-diffusion method
NC which was synthesized from MMT and POPD has shown good zone of inhibitions which were provided in and Fig. 6, 7, 8. The antibacterial activity was conducted against the pathogenic bacterial strains such as E. coli, S. aureus, P. aeruginosa and B. subtilis. The well-diffusion method also showed the highest antimicrobial activity against B. subtilis followed by P. aeruginosa, E. coli, and S. aureus. The stock concentration used for different ratios of MMT:POPD and POPD:MMT were 2 mg ml− 1 and approximately 40 µl of the NC were diffused into each well.
NC had showed various degree of zone of inhibition against tested bacterial strains and among them, B. subtilis showed the highest inhibition zone whereas S. aureus possess the minimum zone towards the NC. When MMT: POPD − 1:0.25, MMT: POPD − 1:0.5 and MMT: POPD − 1:1 were diffused into the wells, they showed the maximum zone of inhibition in B. subtilis i.e. 30, 35 and 32 mm respectively whereas they showed minimum inhibition zone in S. aureus which were 21, 24 and 23 mm respectively (Fig. 6).
When ~ 40 µl of POPD: MMT – 1:0.25, POPD: MMT – 1:0.5, and POPD: MMT – 1:1 was diffused into each well of agar plate of separate bacterial strains, they showed the highest susceptibility towards B. subtilis and hence maximum zone of inhibition was observed i.e. 20, 25 and 24 mm respectively whereas S. aureus showed minimum zone of inhibition on agar plate towards these POPD/MMT NC. The observed inhibition zone in S. aureus was approximately 16, 20 and 17 mm respectively. The results were quite comparable with standard ampicillin which showed maximum zone of inhibition in B. subtilis (34 mm) and minimum zone of inhibition in S. aureus (30 mm) (Figs. 7 and 8). The positive control where the LB broth was used, there were no zone of inhibition produced suggesting that the NC are antibacterial agent as they have cleared the bacterial growth zone on agar plate.
The synthesized NC was energetically involved in the antibacterial activity against B. subtilis, P. aeruginosa, E. coli and S. aureus. B. subtilis had the maximum zone of inhibition among all NC tested and S. aureus had the minimum zone of inhibition because of the maximum resistant capacity of the bacterial isolates and having lowest growth inhibition with all NC .
The organic-inorganic hybrid nanocomposites generation using clay minerals was aided by their properties like large surface area, ultra-fine particle size, and intercalation property. Although, in construction of these nanocomposites comprising controlled properties the interaction between matrix and filler serves as a substantial feature. Hence, for a better understanding of the role played by play as well as conducting polymer as a matrix and filler, in this research paper nanocomposites of poly(o-phenylenediamine) (POPD) and MMT have been synthesized using sonochemical intercalation methods via two methods – by keeping the former as a filler in one case and as matrix in other. To navigate the optimum loading of clay and conducting polymer for the construction of nanocomposites with property of self-assembled morphology so that they could be employed in optoelectronic devices, the spectral, fluorescence, and morphological properties were investigated (Pontes et al. 2013; Hosseini 2011; Wang et al. 2010; Anuar et al. 2004; Chiu et al. 2014; Riaz et al. 2016). The antimicrobial property of such nanocomposites makes it a versatile material in various bacterial mediated disorders. In this regard, Rhim et al. (Rhim et al. 2006) reported that the nanocomposite films containing certain organically modified nanoclay offers strong antimicrobial function against both Gram-positive and Gram negative bacteria. They postulated that the quaternary ammonium groups present in the organically modified clays are responsible for the antimicrobial function of nanocomposite films (Rhim et al. 2009).
Some of the chemical antimicrobial agents are irritant and toxic, while plants are easily available, safe, and nontoxic in most cases, but do not have antimicrobial potential as effective as other chemical agents. Therefore, there is vital need and much interest in finding ways to formulate new types of safe and cost-effective biocidal materials. This study helped us to use these NC as safe and strong antimicrobial material. It may be due to the strong adsorption and immobilization capacity of modified layered silicates (Guo et al. 2005; Hu et al. 2005). It is a well-established fact that at pH 7.4, a net negative charge is exhibited by the parent layered silicate (Nzengung et al. 1996). Bacteria possessing negative charge will not be adsorbed notably onto the clay under these conditions. Nevertheless, after modification of POPD, nanocomposites were synthesized with several degrees of hydrophobicity. The diameters exhibited by inhibition zone fell between 10 and 14 mm. Furthermore, Gram-positive bacteria displayed a greater effect than the Gram-negative bacteria. The basic difference is the presence of an outer membrane in Gram-negative whereas absent in Gram-positive bacteria. Dissimilar barricades serves as the explanation of variation in their sensitivity as their capability to avoid the entrance of microbicides varies. Antimicrobial agents kill bacteria through various means depending on the type of bacteria. Most antiseptics and disinfectants kill bacteria immediately on contact by causing the bacterial cell to burst, or by depleting the nutrients preventing bacterial reproduction such as bacterial conjugation. Antimicrobial polymers might kill bacteria by adsorbing onto the bacterial cell wall. Most bacterial surfaces are negatively charged; therefore, the adsorption of polymeric cations has proved to be more effective than adsorption of polymeric anions. The antimicrobial agent must then diffuse through the bacterial cell wall and adsorb onto the cytoplasmic membrane. This might lead to the disruption of the cytoplasmic membrane and subsequent leakage of cytoplasmic constituents leads to the death of the cell (Nonaka et al. 2003; Uemura et al. 1999).