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Lin Zhang, Jienan Chen, Wenji Yu, Qingfeng Zhao, Jin Liu, "Antimicrobial Nanocomposites Prepared from Montmorillonite/Ag+/Quaternary Ammonium Nitrate", Journal of Nanomaterials, vol. 2018, Article ID 6190251, 7 pages, 2018. https://doi.org/10.1155/2018/6190251
Antimicrobial Nanocomposites Prepared from Montmorillonite/Ag+/Quaternary Ammonium Nitrate
Nanocomposites of Ag with organic montmorillonite (Ag-OMMT), Ag with montmorillonite (Ag-MMT), and organic montmorillonite (OMMT) were successfully prepared via a one-step solution-intercalated method. Sodium MMT, silver nitrate, and dimethyl octadecyl hydroxy ethyl ammonium nitrate were used as precursors. X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy, and energy dispersive spectroscopy analyses confirmed that the MMT layers were intercalated, and Ag+ was partly reduced to silver nanoparticles with diameters within 10–20 nm in Ag-OMMT. The decomposition temperature of the organic cations in OMMT and Ag-OMMT increased to 220°C, as revealed by differential scanning calorimetry-thermogravimetric analysis. The antimicrobial activity of the nanocomposites was tested by measuring the minimum inhibitory concentration (MIC) and killing rate. The MICs of Ag-OMMT against Staphylococcus aureus, Escherichia coli, and Candida albicans were 0.313, 2.5, and 0.625 mg/mL, respectively. Because of the presence of quaternary ammonium nitrate, Ag-OMMT has a better MIC against Gram-positive bacteria compared to Gram-negative bacteria and fungi. OMMT did not show antimicrobial activity against Escherichia coli and Candida albicans. In 2 h, 0.0125 mg/mL Ag-OMMT could kill 100% of S. aureus, E. coli, and C. albicans in solution, and Ag-MMT could kill 99.995% of S. aureus, 90.15% of E. coli, and 93.68% of C. albicans. These antimicrobial functional nanocomposites have the potential for application in the area of surface decoration films.
Montmorillonite (MMT), an all-purpose clay, is widely used in a range of applications because of its high cation exchange capacity, swelling capacity, high surface areas, and strong adsorption and absorption capacities [1–3]. In recent years, the synthesis and application of MMT-based antibacterial materials have attracted great interest due to global concerns regarding public health. Some researchers have reported modified MMT materials with antibacterial activity. For example, silver, copper, and zinc ions have been immobilized on MMT [4–9], and cetylpyridinium, cetyltrimethylammonium [10–12], tetradecyltrimethylammonium , chitosan [14, 15], and chlorhexidine acetate  have been intercalated into the MMT layers. In addition, pharmacology studies have revealed that MMT can adsorb to bacteria such as Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and immobilized cell toxins [17, 18].
Meanwhile, polymers used in several industries such as food processing, biomedical devices, and filtering are required to have antiseptic ability to minimize the transmission of bacterial infections . The dispersibility and compatibility of antimicrobials with polymers is one of the key factors for the preparation of antimicrobial polymers. To improve the compatibility between the antimicrobial and polymer, surface modification of the antimicrobial is required. Furthermore, a lot of researches [20–23] shown that nanoparticles, such as clay and graphene nanoplatelets which was incorporated in antimicrobial polymer nanocomposites, allowed for the tuning of the release of antimicrobial agents, especially reducing the burst release effect, without hindering the antimicrobial activity of the obtained materials.
The aim of this work was to prepare organic antiseptic MMT with good compatibility and dispersibility for use as a nanoadditive in polymers. For this purpose, MMT was modified with Ag+ and quaternary ammonium nitrate via a one-step solution-intercalation technique. The structures of different antimicrobial organic MMTs were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy dispersive spectroscopy (EDS) techniques, and the thermal stability was confirmed by differential scanning calorimetry-thermogravimetric (DSC-TG) analysis. The antimicrobial activity of the nanocomposites was evaluated by examining the minimum inhibitory concentration (MIC) and killing rate.
Sodium MMT used in this study was supplied by Zhejiang Fenghong Clay Chemicals Co., Ltd (China). The cation exchange capacity (CEC) of MMT was 90 meq (100 g)−1. Silver nitrate (AgNO3) with a purity of 99.8% was provided by Hunan Hipure Chemical Reagent Factory (China). Dimethyl octadecyl hydroxy ethyl ammonium nitrate (DOHEAN) at 50% (w/w) in butanol was provided by Jiangsu Hai’an Petrochemical Plant (China). Other reagents used in this study were of analytical grade.
2.2. Synthesis of Antimicrobial Organoclays
10 g sodium MMT was dispersed in 200 mL deionized water and stirred at 80°C. AgNO3 (equimolar with the CEC) was dissolved in deionized water and then slowly dropped into the MMT sol, which was then kept at 80°C for 1 h with stirring. An equimolar quantity of DOHEAN was added to the Ag+ and MMT sol and kept at 80°C for 2 h with stirring. The intercalated montmorillonite (Ag-OMMT) was repeatedly washed with deionized water to remove residual AgNO3 and DOHEAN. This composite was then dried at 100°C for 24 h and then ground to a size less than 300 mesh.
The preparation of Ag-MMT and OMMT were consistent with the above methods but absented the process of addition AgNO3 and DOHEAN, respectively.
XRD measurements were performed using a D/Max 2550 diffractometer (Rigaku Electrical Co., Ltd.) with a Cu target and radiation ( = 0.154 nm). TG and DSC curves were recorded at 20–800°C with a heating rate of 10°C/min under N2 (Netzsch STA 449 C). FTIR spectra were collected from KBr pressed disks on a Nicolet 380 spectrophotometer. SEM images were recorded with a JEOL JSM-6380LV microscope, and TEM and EDS characterizations were performed on a Tecnai G2 20 FEI AEM.
2.4. Evaluation of Antimicrobial Activity
Gram-positive bacteria Staphylococcus aureus, Gram-negative bacteria Escherichia coli, and fungi Candida albicans were provided by the China Center of Industrial Culture Collection (CICC at Beijing).
2.4.1. Minimum Inhibitory Concentration 
MIC tests were performed in MHA for the bacteria and fungi. A serial twofold dilution of Ag-OMMT was added to an equal volume of medium to obtain a concentration of 5000 μg/mL, which was serially diluted by double technique to achieve solutions of 2500–9.77 μg/mL. Control dishes containing equal volumes of distilled water were also prepared. After cooling and drying, the plates were inoculated with 2 μL of 107 CFU/mL strain solutions and incubated aerobically at 27°C for 16–20 h for bacteria or 72–96 h for fungi. Growth control samples of each tested strain were also included. The MIC was defined as the lowest concentration required to inhibit bacterial growth, that is, the concentration at which <5 microorganism colonies were visible.
2.4.2. Antimicrobial Killing Rate 
The microorganism suspension was diluted using 0.9% (w/v) sterile saline water to 104 CFU/mL. 1 mL of cell suspension was added to 95 mL of 0.05, 0.025, and 0.0125 mg/mL nanocomposite (Ag-MMT and Ag-OMMT) solutions that had been autoclaved at 121°C for 20 min. Nanoscale SiO2 was used as a control. The samples were removed after 2 h shake cultivation. 50 μL aliquots were spread on nutrient agar plates, which were incubated at 37°C for 24 h, and the numbers of colonies were counted for each solution. The percent reductions in plate colony counts were calculated by comparing the experiment plates to the control. All presented data were averaged from at least 3 parallel experiments, where the discrepancies among them were <5%.
3. Results and Discussion
3.1. Structure and Morphology
The XRD patterns of unmodified MMT, Ag-MMT, OMMT, and Ag-OMMTs (modified with different amounts of Ag+) are presented in Figure 1. Table 1 shows the -spacing of 001 for MMT as calculated by Bragg’s equation . Ag-MMT features a larger basal spacing (1.381 nm) than MMT (1.258 nm), indicating that Ag+ was exchanged in the silicate layers. Both OMMT and Ag-OMMT feature wide basal spacings with high values between 1.96 nm and 2.03 nm, indicating that DOHEAN has been intercalated into the MMT layers.
As shown in Figure 1, minimal metallic silver is present in all the Ag-OMMTs (at 2θ = 38°) but is absent in Ag-MMT. This is also noted in Figure 2, which shows the XRD patterns of Ag-MMT and Ag-OMMTs having different amounts of Ag+ after calcination at 750°C for 2 h. In the presence of easily oxidizable organic cations, Ag+ as an oxidant was partly reduced to metallic silver during the preparation process; after high temperature calcination, the reaction of Ag+ and DOHEAN was complete. However, in the absence of organic cation, Ag+ could not easily be deoxidized, even after calcination at 750°C. This has also been demonstrated by EDS (Figure 3).
(a) TEM of Ag-MMT
(b) TEM and EDS of Ag-OMMT
Figure 3 shows the EDS pattern of Ag-OMMT and the morphologies of Ag-MMT and Ag-OMMT. The modified MMTs appear as sandwich-like crystals in the TEM images. Numerous black spots are homogeneously dispersed in the MMT crystals, as shown in Figure 3(b), which are metallic silver nanoparticles as demonstrated by EDS and XRD analyses. These silver nanoparticles are smaller in Ag-MMT (particle diameter within 2–5 nm, Figure 3(a)) than in Ag-OMMT (particle diameter within 10–20 nm, Figure 3(a)) because Ag+ is more easily deoxidized in the presence of organic cations; this is in agreement with the XRD results (Figures 1 and 2). Figure 4 presents SEM micrographs showing the morphology change of MMT before and after modification. Unmodified MMT (Figure 4(a)) has a compact and flat surface, while, after modification, the MMT surface becomes crinkled and rough with wide interspacing (Figure 4(b)), which is desirable for use as a nanoadditive.
DSC-TG curves of DOHEAN, MMT, Ag-MMT, OMMT, and Ag-OMMT are shown in Figure 5. The TG curve of each sample shows an endothermal peak below 100°C with a corresponding weight-loss due to the removal of water. The DSC curves of MMT and Ag-MMT both feature a second endothermal peak at 679.2°C and 674°C, respectively, corresponding to the loss of hydrated water of the interlayer cations and the structural hydroxyls .
The sharp exothermal peak on the DSC curve of DOHEAN represents the evaporation or decomposition of DOHEAN. However, there are two exothermal peaks on the DSC curves of OMMT and Ag-OMMT. The low-temperature exothermal peak corresponds to the evaporation or decomposition of DOHEAN on the silicate plate surfaces, and the other peak represents the evaporation or decomposition of DOHEAN between the silicate plates. The TG curves of OMMT and Ag-OMMT reveal that the evaporation or decomposition of DOHEAN occurs at approximately 220°C, which is higher than that of pure DOHEAN (160°C). This indicates that the organic cation has intercalated into the MMT layers, similar to the initial state, and that the silicate platelets have the ability to protect organic molecules from decomposition. . A similar behavior was reported by Scaffaro et al.  during the preparation of poly(ethylene-co-vinyl acetate) films with two commercial formulations of nisin.
Figure 6 shows the FTIR spectra of MMT, DOHEAN, Ag-MMT, OMMT, and Ag-OMMT. Compared to MMT, OMMT has additional absorption peaks appearing at 2921, 2850, and 1384 cm−1. The peaks at 2921 and 2850 cm−1 arise from –CH2– and –CH3 stretching vibrations, while the one at 1384 cm−1 belongs to C–H symmetric deformation vibrations . This further reveals that DOHEAN has intercalated into the MMT layers. The peak at 3100–3700 cm−1 represents O–H stretching vibrations  and the peak at 1638 cm−1 belongs to H–O–H bending vibrations . This band, which is related to the (H–O–H) bending vibration of water adsorbed on MMT, shifted from 1631 cm−1 in MMT to 1638 cm−1 for Ag-OMMT. Meanwhile, the intensity of this band decreased, reflecting that the amount of water adsorbed on MMT decreased after modification of the MMT with organic cations.
3.4. Antimicrobial Activity Assay
As shown in Table 2, Ag-MMT and Ag-OMMT have obvious antimicrobial activity against a wide variety of microorganisms, including Gram-positive bacteria, Gram-negative bacteria, and fungi. They have the same MIC for E. coli and C. albicans, which are 2.5 and 0.625 mg/mL, respectively. In addition, Ag-OMMT has a higher MIC (0.313 mg/mL) for S. aureus than Ag-MMT (1.25 mg/mL). Strong antimicrobial activity was also observed, as outlined in Table 3. At a concentration of 0.0125 mg/mL, Ag-OMMT can kill 100% of the S. aureus, E. coli, and C. albicans population in 2 h, and Ag-MMT can kill 99.995% of the S. aureus, 90.15% of E. coli, and 93.68% of C. albicans in 2 h.
OMMT can inhibit the growth of S. aureus; however, its ability to inhibit the growth of E. coli and C. albicans is less pronounced. This phenomenon is due to the different cell structures of these microbes. S. aureus, a Gram-positive bacterium, consists of a thick peptidoglycan layer and a cytoplasmic membrane. Its peptidoglycan layer is extensively crosslinked in three dimensions to form a solid mesh. Despite its thickness, the peptidoglycan layer of Gram-positive bacteria is not a barrier to the diffusion of foreign molecules. Gram-negative bacteria, however, have a small layer of peptidoglycan and an outer membrane made of a toxic liposaccharide layer. Because of this structure, Gram-negative bacteria are unusually permeable to foreign molecules. Therefore, Gram-negative bacteria are generally less susceptible to antibiotics and antibacterial agents than Gram-positive bacteria .
Novel antimicrobial nanocomposites featuring sodium MMT, Ag+, and dimethyl octadecyl hydroxy ethyl ammonium nitrate were synthesized via a one-step solution-intercalated method. XRD, DSC-TG, FTIR, SEM, TEM, and EDS characterization indicated that Ag+ and DOHEAN were intercalated into the MMT layers. Ag formed both metallic species and Ag+ in the clay layer, while DOHEAN was chemically bonded with the MMT layers. The thermal stability of DOHEAN was improved by the protection from the MMT layers. The nanocomposite surface became crinkled and rough after modification, making it suitable for combining with polymers. Further, the nanocomposites showed a wide range of highly efficient antimicrobial activity. The results of this study may be used as a foundation for the future development of new types of nanocomposites of antimicrobial polymers in many industries, such as in wood adhesives, plastics, paints, and rubbers.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
This work was supported by the National Special Program for International Science and Technology Cooperation (no. 2015DFA01120), the Hunan Province Major Program of Science and Technology (2017NK1010), the Key Projects in the National Science & Technology Pillar Program during the Eleventh Five-year Plan Period of China (2006BAD07A07-08), and the Hunan Province Natural Science Foundation (2015JJ5007).
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