Applied Clay Science 90 (2014) 18–26
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Applied Clay Science journal homepage: www.elsevier.com/locate/clay
Research paper
Antibacterial activities of copper nanoparticle-decorated organically modified montmorillonite/epoxy nanocomposites Gautam Das a,1, Ranjan Dutta Kalita b, Pankaj Gogoi a, Alok K. Buragohain b, Niranjan Karak a,⁎ a b
Advanced Polymer and Nanomaterial Laboratory, Chemical Sciences Department, Tezpur University, India Department of Molecular Biology and Biotechnology, Tezpur University, Napaam 784028, India
a r t i c l e
i n f o
Article history: Received 11 June 2012 Received in revised form 31 December 2013 Accepted 2 January 2014 Available online 1 February 2014 Keywords: Nanostructures Coatings Mechanical properties Biomaterials Clay mineral
a b s t r a c t Organically modified montmorillonite (OMt) was decorated by copper nanoparticles (with average diameters of 10 to 20 nm) at three different mass percentages at room temperature. Such OMt-Cu nanohybrid was incorporated into Mesua ferrea L. seed oil modified epoxy resin (BPSE) to obtain clay mineral polymer nanocomposites (CPN). The nanohybrids and CPN were characterized by SEM, XRD, HRTEM and FTIR and UV–visible spectroscopic techniques. The antimicrobial efficacy of the as-prepared OMt-Cu nanohybrid and OMt-Cu/epoxy nanocomposites was also premeditated and significant antibacterial activity against ubiquitous Gram negative bacteria Klebsiella pneumonia and Gram positive bacteria Staphylococcus aureus was observed. In addition the OMt-Cu/epoxy nanocomposites exhibited enhancement in thermostability over the pristine system by 15 °C and tensile strength and in scratch hardness by 2.5 and 2.1 units respectively along with marginal improvement in the elongation at break value. The study reveals that the OMt-Cu/epoxy nanocomposites have the potentiality to be used as advanced antimicrobial materials. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Nanotechnology has emerged as a rapid growing field with multifaceted application for new materials at the nanoscale level (Atiyeh et al., 2007). The unique combination of different nanostructures creates material properties which have a great potential in optical, electrical, medical, antibacterial etc. applications due to interfacial interactions that can be established between nanoscaled architectures (Drelich et al., 2011; Huang et al., 1997; Liu and Bando, 2003; Magdassi et al., 2010). Metallic nanoparticles for biomedical applications are the most promising as they show good antibacterial properties due to their large specific surface area (SSA) and high specificity (Gong et al., 2007). Copper and its complexes have found their uses as efficient materials for sterilizing liquids, clothing and also antibiotics for centuries (Borkow and Gabbay, 2009; Dutkiewicz and Fallowfield, 1998; Kaali et al., 2011; Pang et al., 2009; Perelshtein et al., 2009). Compared to other metals, copper has the advantage of insignificant sensitivity to human tissues (Hostynek and Maibach, 2004), however microorganisms have been found to show high sensitivities to copper (Michels et al., 2005). Recently, due to the development of some resistant bacterial strains (Kyriacou et al., 2004) the antibacterial activity of nanomaterials, such as silver (Akhavan and Ghaderi, 2009a, 2011, 2009b; Ferraris et al., 2010) and
⁎ Corresponding author. Tel.: +91 3712 267009; fax: +91 3712 267006. E-mail address:
[email protected] (N. Karak). 1 Present address: Department of Environment & Energy Engineering, Gachon University, Gyeonggi-Do 461-701, South Korea. 0169-1317/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.clay.2014.01.002
copper based nanostructures (Akhavan and Ghaderi, 2009c Akhavan et al., 2011; Gao et al., 2009; Kim and Park, 2008; Pachoalino et al., 2008), with their unique size dependent properties has attracted great attention. The use of clay and clay minerals as a support for the synthesis of nanoparticles has been recognized as a promising method. Montmorillonite (Mt) in this regard, has been an apt choice due to its chemical and physical nature. Mt has been used for preparation of a wide range of nanoparticles like gold and silver, among others (Manikandan et al., 2007). This hybrid material is vouched for application in advanced coating materials such as antibacterial coatings, in biomedical devices or in antimicrobial packaging (Cioffi et al., 2005; Sunada et al., 2003). The rationale for the use of copper lies in the strong toxic action this metal exerts against prokaryotes (i.e., all types of bacteria), while it is much less toxic against eukaryotes (i.e., all other organisms). Thus dispersion of copper or copper oxide particles into organic matrixes has been employed as antifouling coatings by the paint industry, mainly for maritime applications. There are reports on the use of copper/polymer nanocomposites as a bioactive coating against bacteria (Anyaogu et al., 2008). However the use of the combined system of OMt decorated copper nanoparticles for such application is not known. This combined system has the advantage of being both a reinforcing filler as well as a bioactive material. Clay with its high aspect ratio provides effective reinforcement of the matrix, further it also serves as a site for the anchorage of the copper nanoparticles. Today, the quest is for nontoxic antimicrobial protection, which can prevent the surface from biofouling without causing environmental pollution and poisoning. A scrutiny of the
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plethora of literature reveals no report of epoxy/OMt-Cu system as antimicrobial system. The aim of the present investigation is the decoration of OMt with copper nanoparticles at three different mass percentages. Further this modified OMt was incorporated in Mesua ferrea L. seed oil modified epoxy resin at three different mass% to form OMt-Cu/epoxy nanocomposites (CPN). The antimicrobial activities of the OMt-Cu as well as of the CPN were studied. Further the hemolytic activity of the CPN was evaluated by the RBC hemolysis protection assay. The performance characteristics including thermal stability of the CPN were also studied to view the applicability of the system as advanced antimicrobial materials.
at room temperature by vigorous stirring for 20 min. A high vacuum was applied for about 15 min to remove any volatile generated during the mixing. The thin film of the mixture was cast on a glass plate and kept for 2 h under ambient conditions. The plates were then heated at 100 °C in a muffle furnace to determine the touch free time (minimum time, when no impression will appear on touching the film) and hard dry time (when no indentation on the film can be made by the nail of the thumb) of the pristine resin and the nanocomposites. The mixtures were also uniformly spread on mild steel plates (150 mm × 50 mm × 1.60 mm), tin plates (150 mm × 50 mm × 0.40 mm) and glass plates (75 mm × 25 mm × 1.75 mm) for impact strength, gloss and scratch resistance tests.
2. Experimental
2.1.5. Antimicrobial study OMt-Cu/BPSE nanocomposites and the nanoparticles N1, N0.50 and N0.25 were investigated for their antimicrobial activity, separately, using the agar well diffusion study. Staphylococcus aureus, Gram positive bacteria and Klebsiella pneumoniae, Gram negative bacteria were taken for the study. The bacteria were grown overnight in Luria broth media and 50 μL of the broth culture was taken and plated in the agar plates. Agar plates for the antimicrobial test were prepared using the Mueller Hinton Agar media for the antibacterial assay, as per the manufacturer's instructions. Wells were punched using a well borer with a diameter of 6 mm. 50 μL of the nanocomposite solutions and also the nanoparticles were poured into the wells. Antibiotic solution containing streptomycin sulfate at a concentration of 5 mg/mL was taken as the positive control. The plates were then incubated overnight at 37 °C. The formation of zones of inhibition around the wells indicated the antimicrobial activity of the tested materials.
2.1. Materials M. ferrea L. (Nahar) seeds (Jamugurihat, Assam) were utilized for the extraction of the oil. Epichlorohydrin, phenyl hydrazine, copper acetate (Merck, Mumbai, India), bis(4-hydroxy phenyl) sulfone (Aldrich, Germany) were used as received. Bisphenol-A (BG & Co., India) was used after purification by recrystallization from toluene. Organomontmorillonite (OMt), (Nanomer I.30E, octadecylamine modified) was purchased from Nanomer PGV, Aldrich, Germany. All other reagents used in the present investigation were of reagent grade. 2.1.1. Preparation of copper nanoparticles The clay dispersion was prepared by dispersing 1 g of OMt in 50 mL DMF. It was then again dispersed by sonication for 20 min. To achieve theoretical copper loading in the clay/copper mass ratio of 1:1, 1:0.5 and 1:0.25 (coded as N1, N0.5 and N0.25, respectively), 3.16 g, 1.58 g and 0.792 g of copper acetate were dissolved in 30 mL, 15 mL and 8 mL respectively. The full copper acetate solutions were then added to the clay dispersion and stirred for 24 h at 60 °C, followed by sonications for 10 min. The copper precursor was then reduced in the presence of OMt by adding phenyl hydrazine in the mole ratio of 1:3 (copper acetate/phenyl hydrazine). The whole solution was stirred at room temperature for 48 h until reddish brown coloration appears. The whole solution was further sonicated for 15 min. The obtained dispersion consisting of copper nanoparticles embedded in OMt was centrifuged and washed repeatedly with acetone. The resulting solid phase was dried at 45–50 °C. 2.1.2. Preparation of diglycidyl ether bisphenol-S epoxy resin M. ferrea L. seed oil based sulfone epoxy resin was prepared by the similar method as reported earlier (Das and Karak, 2010). Briefly, monoglyceride of the oil (obtained by glycerolysis technique), epichlorohydrin, bisphenol-A (BPA) and bisphenol-S (BPS) were reacted together by maintaining the mole ratio of 1:5:2:1 at 110 ± 5 °C for 14 h in a slightly alkaline medium. The resinous product was isolated, washed and purified. The purified and dried product is coded as BPSE. 2.1.3. Fabrication of nanocomposites The dried OMt-Cu systems were added into the BPSE matrix by fixing the percentage of OMt fixed at 3 mass%. The copper nanoparticle concentrations in the matrix varies viz., 3, 1.5 and 0.75 mass% with respect to 1:1, 1:0.5 and 1:0.25 OMt-Cu and were coded as ECN1, ECN0.5 and ECN0.25 respectively. BPSE and OMt-Cu were mixed vigorously for 10 min at room temperature followed by sonication for 15 min using a single probe sonicator (UP200S, Hielscher, Germany) at half cycle and amplitude of 50%. 2.1.4. Curing of the resin and nanocomposites A homogenous mixture of the BPSE and OMt-Cu/BPSE nanocomposites with 50 phr (parts per hundred gram with respect to epoxy resin) of poly(amido amine) hardener was prepared separately in a glass beaker
2.1.6. RBC hemolysis protection assay The hemolytic activity (Nair et al., 2007) of the final OMt-Cu/BPSE nanocomposite and polymer films was investigated to find out if the materials possessed any membrane damaging activity. For this study goat blood was collected in a 50 mL centrifuge tube containing an appropriate anticoagulant. The blood was centrifuged at 2000 rpm, the supernatant discarded and the pellet was washed further using PBS (pH 7.4). Finally the pellets were mixed with PBS at a ratio of 1:50 (v/v). From this 2 mL of blood was collected in 2 mL centrifuge tubes and 10 mg of the CPN and polymer films was added to the tubes and incubated for 2 h. After the incubation period was over the tubes were centrifuged and then 200 μL of the supernatant was collected and taken separately. To this 2.8 mL of PBS was added and then the final solution was measured in a spectrophotometer (Cecil, UK) at 415 nm. Triton X100 and PBS were taken as the positive and negative control respectively. 2.2. Measurements FTIR spectra of resin, OMt-Cu hybrid system and CPN were recorded in FTIR spectroscopy (Impact-410, Nicolet, USA) using KBr pellet. The surface morphology of the samples was studied by a JEOL scanning electron microscope (JSM-6390LV SEM) after platinum coating on the surface. Wide angle X-ray scattering (WAXS) studies were carried out using a powder diffractometer Rigaku X-ray diffractometer (Miniflex, UK) at room temperature (about 25 °C), operated at 30 kV and 15 mA. The scanning rate used was 2.0 min−1 over the range of 2θ = 0–60° for the above study. The distribution of OMt-Cu in the polymer matrix was studied by using a JEOL, JSM-2100CX transmission electron microscope (TEM). UV–visible spectra of the OMt-Cu and OMt-Cu/BPSE nanocomposites were recorded by a UV–visible spectrophotometer, UV-2550 (Shimadzu, Japan) with a 0.001% solution in DMF at room temperature (~27 °C). The scratch hardness test (ASTM D5178/1991) of the cured films was done by using a scratch hardness tester (Sheen instrument Ltd, UK). The
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front impact resistance test was carried out by applying the falling ball method using an impact tester (S.C. Dey Co., Kolkata) with a maximum test height of 100 cm. In this test a mass of 850 g was allowed to fall on the film coated on a mild steel plate from minimum to maximum falling heights. The maximum height is taken as the impact resistance up to which the film is not tearing out. The tensile strength and elongation at break (as per the ASTM D 41251T) were measured with the help of Universal Testing Machine (UTM) of model Zwick Z010 (Germany) by using 10 kN load cell and at 40 mm/ min jaw separation speed. Thermogravimetric (TG) analysis was carried out in a Shimadzu TG 50 thermal analyzer using the nitrogen flow rate of 30 mL/min and at the heat rate of 10 °C/min. 3. Result and discussion 3.1. Formation of OMt-Cu/BPSE nanocomposites The nanoparticles by virtue of their high aspect ratio and surface energy have high tendency to agglomerate. Therefore stabilization of the nanoparticles is a prerequisite for obtaining specific surface chemistry and/or architectures bestow desired surface properties to a given material without significantly altering other desirable properties of the material (Zhu et al., 2002). Polymers play important roles in dictating nanoparticle size, shape, and interparticle spacing, and also in determining the properties of both the interface between the surfaces of the polymer and the nanoparticles and the interface between the nanoparticle and its environment. The OMt was incorporated as a reinforcing nanofiller to compensate for the low mechanical and thermal properties of pristine BPSE by forming CPN. The epoxy/carboxyl/ hydroxyl groups of the BPSE can easily interact with the hydroxyl groups of OMt through H-bonding or other polar–polar interaction to form stable and well dispersed CPN. The use of high shear force and ultrasonication results in the delamination of the OMt layers in the polymer matrix. The Cu2 + ions intercalated in between the layers were reduced using phenyl hydrazine. OMt by virtue of its ionizale hydroxyl sites can preferentially interact with metal cations (Borchardt, 1989). Further, the permanent negative layer charge originating from the isomorphous substitution of Mg(II) or Fe(II) for octahedral Al(III) is the origin of the binding of exchangeable cations to the interlayer sites of OMt. It has a great adsorption capacity, which is attributed to its large specific SSA and high cation exchange capacity (CEC) (Grim, 1998). The OMt surface by virtue of its high surface functionality stabilizes the nanoparticles onto its surface. The reduction performed at room temperature resulted in nanoparticles with uniform particle size distribution, good antibacterial properties and good dispersion stabilities. 3.2. UV–visible spectroscopy The formation of copper nanoparticles in clay mineral system was first observed by UV–visible absorption spectra studies (Fig. 1). Chemical reduction of Cu2+-loaded OMt with excess of phenyl hydrazine results formation of intra OMt-Cu nanohybrids. Evidence for this comes from the change in solution color from blue to reddish brown. The absorbance band in UV spectrum shows a broad band from about 588 nm to 612 nm (Fig. 1i) (Ramos and Hernandez, 1996). The surface-plasmon band may shift depending upon the particle size and shape. The strong surfaceplasmon absorption band observed is due to the formation copper nanoparticles. The broadness of the absorption band (Fig. 1) probably arises from the wide size distribution of copper nanoparticles. The incorporation of the nanohybrids in the BPSE matrix does not affect the surface plasmon resonance significantly as can be observed from the UV–visible spectra. The absorbance band 589–615 broadens in the CPN (Fig. 1ii).
Fig. 1. Plot of (αhν)2 versus hν for N0.25, N0.5 and N1.
The band gaps (Eg) of the OMt-Cu nanohybrids were determined by the absorbance data from the UV–visible spectra following the equation: n ahv ¼ A hv−Eg
ð1Þ
where A is a constant and η is another constant which depends on the nature of the transition. The η for copper based materials is assumed to be 1/2 corresponding to the directly allowed electron transition mechanism (Akhavan et al., 2009c; Thakur and Karak, 2012). Thus, the absorption region where the plot of (αthν)2 versus hν exhibits a linear behavior, the band gap energy is obtained by the extrapolation of the intercept of the fitted line with the hν axis (Fig. 2). The band gaps of N0.25, N0.5 and N1 were found to be 2.83 eV, 2.91 eV and 3.01 eV respectively. Thus the band gap energy increases with the increase in the copper loading. Further the band gap value indicated that Cu was stable and minimum oxide formation resulted. 3.3. XRD analysis The X-ray diffractogram (Fig. 3i–ii) of the powder sample is well agreed with the literature values (Haas et al., 2006). All the reflections at 2θ values of about 43.61°, 50.59° and 74.47° are representing the (911), (200) and (220) Bragg's reflections of cubic structure of copper. Besides these the peaks appearing in the XRD spectrum are 35.62° ― ― (1 11) and 61.81° (1 13) corresponding to copper oxide (CuO), though UV–visible is silent about this. The appearance of the oxide reflection in the diffractogram suggested the presence of CuO and Cu structures intercalated within the clay mineral layers. However the presence of the CuO is negligible as can be seen from the XRD and UV measurements. The reflection for the layered silicate was also observed, however the reflection at 4.15° corresponding to basal reflection (110) shifts to 3.64° indicating an increase in the interlayer space with an increase in the nanoparticle loading, other reflection observed was at 20.1° (002). The observation of low-angle peak of OMt suggests the possible coexistence of staked nanostructures made up of Cu/CuO nanoparticles in the layers. The intensity of the reflection corresponding to the nanoparticles increases with the increase in the loading within the layered silicate. As shown by the sharper and more intense band, the degree of crystallinity increases with copper concentrations. The oxidation of the Cu nanoparticles might be responsible for the presence of reflection for copper oxide nanoparticles. However the structure provides sufficient stability and no structural changes were observed in the spectra taken after two months. This suggested that the OMt layer provides sufficient stability to the copper nanoparticles.
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Fig. 2. UV-visible spectra for (i) OMt-Cu systems and (ii) nanocomposites.
The XRD diffractograms of the CPN with 1, 0.5 and 0.25 mass% of copper loading are shown in Fig. 3ii. Diffraction reflection is observed at 43.8°, 50.57° and 74.68° corresponding to (111), (200) and (220) of Bragg's reflection for cubic structure of copper (ICSD 851362). The basal reflection at 30.7° for (200) plane corresponding to the tetragonal structure whereas 65.58° and 83.45° observed for copper oxide nanoparticles (CuO) corresponds to (022) and (400) of Bragg's reflection for monoclinic structure of CuO. The basal reflection at (110) plane corresponding to the reflection value of 4.15° was seen to be absent in the CPN, which is due to the loss of structural regularity of the layered silicates in the CPN. A broad
reflection was observed around 18.9°–20.13° in the diffractogram of the CPN. This appearance is due to the amorphous nature of the polymer. 3.4. FTIR analysis Fig. 3iii depicts the FTIR spectrum of copper nanoparticles in OMt. Obviously the features of the OMt can also be recognized among the bands of copper nanoparticles in the spectrum. The spectrum of OMt shows the presence of bands (cm− 1) at 2926 for \CH2 stretching of modifying hydrocarbon, 3263 for \N\H stretching, 1619 for \OH
Fig. 3. XRD spectra of (i) OMt-Cu system for (a) N0.25, (b) N0.5 and (c) N1, and (ii) nanocomposites viz. (a) ECN0.25, (b) ECN0.5 and (c) ECN1, (*= Cu, ○= CuO and ●= OMt) and (iii) FTIR spectra of (a) OMt and (b) OMt-Cu, and (iv) FTIR spectra of (a) BPSE, (b) ECN0.25, (c) ECN0.5 and (d) ECN1.
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bending, 1045–980 and 524–429 for oxide bands of metals (Al, Mg, Si, etc.) present in the clay mineral. Multiple broad bands were detected for OMt in the range 3627–3403 cm−1 that are attributed to the \OH stretching frequencies. In the high frequency range a well-defined peak appears at 3627 cm−1, associated to the stretching mode of the \OH group coordinated to Al cations (Damonte et al., 2007). This band gets minimized in the OMt-Cu nanohybrids (Fig. 3iii), indicating the insertion and stabilization of the Cu nanoparticles. The broad \OH stretching band at about 3446 cm−1 due to the Al\OH and Si\OH shifts to 3412 cm−1 on the insertion of Cu nanoparticles. The interaction of the OMt-Cu with the BPSE matrix is shown in Fig. 3iv. The structural features of the CPN and the matrix are not so different although the intensity decreases in all the cases. The \OH stretching band in all the cases exhibits a red shifting with the increase of the OMt-Cu content (Fig. 3iv). The shift (about 4–13 cm−1) primarily occurs due to the H-bonding between the BPSE matrix and the surface \OH group of the OMt. There is a decrease of \C_O stretching vibrations with simultaneous blue shifting in all the CPN. The \C_O group can be involved in numerous interactions viz. with the \NH2 group of the hardener or with epoxy group and results in chemical bond formation or H-bonding. This supports the above observation where the intensity decreases nearly half of the original, thus the crosslinking helps in the stabilization of the nanoparticles. Further the formation of Cu⋯O\C structure cannot be neglected in the CPN, the appearance of a broad band at around 1650–1605 cm−1 is indicative of such interactions. The epoxide stretching vibration gets vanished in the CPN indicating the completion of curing via the ring opening of the epoxy group.
unevenly distributed in size and dispersion across the platelet surface of the OMt (Fig. 6). The dark spheres anchored onto the clay mineral surface have a particle distribution of 10–20 nm in size. The polymer proves to be a strong stabilizer for the nanohybrids, as observed in the TEM images (Fig. 7) the nanohybrids are uniformly distributed in the matrix. It can be observed from Fig. 7 that Cu nanoparticles with an average particle size of less than 20 nm and preponderant distribution were developed and assembled. There is no agglomeration phenomenon observed in the TEM images of this kind of nanoparticles and Cu particles are well separated into single ones. The pendant hydroxyl group of the matrix stabilizes the nanoparticles. The hydroxyl groups act as a kind of confined nanoreactors and/or superficial modifiers of Cu nanoparticles, inhibiting their growth and avoiding aggregation (Raffi et al., 2010). The predominate interaction results in the immobilization of copper nanoparticles. The efficient interaction between the Cu and OMt in the OMt-Cu nanohybrids and between the OMt-Cu and BPSE matrix, helps to reduce aggregation and protects from chemical environment resulting in complete stabilization.
3.5. Morphology
3.6. Thermal analysis
The SEM micrographs of N1 and ECN1 are shown in Fig. 4. It was observed that the treated clay mineral contains heterogeneous particles. The white nanoparticles in the case of N1 were clearly seen to anchor onto the surface of OMt and the well dispersed exhibited no agglomeration. The EDX data (Table 1) further supports the presence of the copper nanoparticles in the clay mineral, wherein 6.16% of Cu in OMt and 0.35% in case of ECN1 were observed (Fig. 5). The results indicate that the room temperature reduction was effective in modifying the surface of the OMt with Cu nanoparticles. The larger interlayer space of OMt provides a greater area of contact for the metal ion to penetrate, however longer time was required for intrinsic adsorption because of the diffusion barrier of the interlayer part. The adsorption of the metal ion on the OMt layer includes electrostatic adsorption, complex formation and reduction upon exposure to phenyl hydrazine. Further upon CPN formation the crosslinked network of the polymer matrix provides an effective stabilization. The OMt with its high SSA extensively interacts with the matrix resulting in the immobilization of OMt. The HRTEM of N1, copper nanoparticles can be spotted as dark spherical nanodots of diameters ranging from 10 to 20 nm, which seem
Fig. 8 shows the thermal profile of OMt, N1 and the CPN. OMt exhibits three degradation steps, the first step corresponds to the loss of externally adsorbed water. The second degradation step at about 290 °C is due to the thermal loss of the organic modifier and the third step corresponds to OMt dehydroxylation at 670 °C. The sharp loss of mass at higher temperature (690 °C) is attributed to desorption of any ligand, followed by the combustion at higher temperature of the residual ligands trapped in the clay mineral matrix. The initial degradation at about 77 °C was not observed in OMt-Cu nanohybrids. The presence of Cu nanoparticles has a significant effect on the high temperature stability of the OMt. The OMt-Cu exhibited a mass residue of 79.6% at 600 °C, whereas for OMt it was about 67%. The fact that there are different combustion temperatures for the residual organic ligands in these types of clay minerals is an indication of different interactions between the copper nanoparticles and the clay mineral surfaces. The thermal stability of the CPN enhances with the increase in the incorporation of the OMt-Cu nanohybrids with respect to the pristine system. However the difference in the peak temperature among the
Table 1 EDX data of OMt-Cu/epoxy nanocomposites. Content of element (%) Samples
C
O
Mg
Al
Si
S
Fe
Cu
N
Mn
N1 ECN1
17.71 75.36
48.97 20.39
0.94 0.10
4.36 0.24
12.47 0.36
– 0.88
0.39 0.27
6.16 0.35
7.77 –
0.15 –
Fig. 4. SEM micrographs for (a) N1 and (b) ECN1.
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Fig. 5. EDX spectra of (a) N1 and (b) ECN1.
CPN was not significant. It is known that the clay mineral plays an effective role in enhancing the thermal stability of the matrix system. The hard aluminum-silicate layer acts as a thermal insulator thereby preventing the volatilization of the underlying matrix. The small size of the copper nanoparticles can enhance the thermal stability of the system by virtue of a barrier. The nanohybrids prevent the escape of volatiles that may generate from the decomposition of the matrix at higher temperature. The mass residue of the CPN was enhanced after the incorporation of the nanoparticles. ECN0.25 showed a mass residue at 650 °C of about 18%, ECN0.5 20.41% and ECN1 24.5%, whereas BPSE showed a mass residue of only 6.9% (Fig. 8). Higher amount of mass residue signifies the non-combustible materials (i.e. char). These char residues form a shield over the polymer and thus reducing the heat and mass transport from or into the matrix, thereby enhancing the thermostability of the system.
The gloss value was found to increase with the copper content as large amount of light is reflected from the smooth surface. The scratch hardness value of the films was found to be increased for the nanocomposites with the increase of amount of copper nanoparticles as compared to the BPSE. The OMt layers along with uniformly disseminated nanosized copper particles proficiently restrict indentation in the CPN. At high copper content, it was observed that the viscosity of the resin during the processing of CPN increases significantly and hence the entrapped air cannot escape from the matrix. The impact resistance of the prepared CPN increases as the amount of copper loading increases. The evenly distributed nanosize copper particles have an imperative role for this improvement by interacting with the polyester segments of the matrix along with the OMt platelets. The presence of high interactions by small copper nanoparticles resulted high rigidity. So, they interact with the polymer chains and restrict their mobility which increases the strength of the CPN films.
3.7. Performance characteristics 3.8. Antibacterial properties The mechanical properties like tensile strength, elongation at break, gloss, impact resistance and scratch hardness of the prepared BPSE/ OMt-Cu nanocomposites were determined and are given in Table 2. The tensile strength of BPSE/OMt-Cu nanocomposites was found to be enhanced to a noticeable degree from 6 to 13.2 MPa with the increase of amount of copper nanoparticles from 0 to 3 mass%. This is attributed to the inclusion of homogeneously dispersed copper nanoparticles into the clay mineral layers that are further delaminated by the polymer chains, thereby strengthening the matrix system. Also maximum surface of the silicate layers is available for strong interaction with the polymer chains and copper nanoparticles after delamination of the aluminosilicate layers which leads to an increase in tensile strength with the copper contents. The increase of crosslinking density as indicated by the swelling results (Table 2) due to the increase of copper loading is also responsible for the enhancement of strength property of the films.
The details of the relative retention of activity (zone of inhibition) of the nanohybrids, BPSE and CPN against Gram positive bacteria (S. aureus) and Gram negative bacteria (K. pneumoniae) are given in Tables 3 and 4. After 24 h of incubation the nanohybrids and CPN exhibited good zones of inhibition against both Gram negative and Gram positive bacteria (Table 4), whereas pristine BPSE did not show any zone of inhibition. The CPN exhibited significant efficacy against bacteria, especially against K. pneumoniae, and also the efficacy increased with the increase of copper content. These results thus confirmed that the CPN with copper have good antimicrobial efficacy against these bacteria. However, the mechanism of bactericidal action of copper nanoparticles is still not well understood (Scheme 1). The copper nanoparticles bind strongly to electron donor groups in biological molecules containing sulfur, oxygen or nitrogen (Esteban-Tejeda et al., 2009; Heinlaan et al., 2008;
Fig. 6. TEM image of N1.
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Fig. 7. TEM image of ECN1.
Weir et al., 2008). This may result in the formation of defects in the bacterial cell wall so that cell contents are lost. It is also reported that the structure of the outer cell membrane responsible for the cell permeability was substantially changed for the Escherichia coli (ATCC 25922), after being in contact with the copper containing stainless steel, showing that cell walls were seriously damaged and a lot of contents in the cells leaked. It is reasonable to state that the binding of the particles to the bacteria depends on the SSA available for such interactions. The smaller particles with a larger surface-to-volume ratio provided a more efficient means of antibacterial activity than larger particles. The clay mineral plays a manifold role in enhancing the interaction by virtue of its ability to interact closely with the bacteria. The binding of clay
mineral to the surface of the bacteria then helps copper nanoparticles for its action. A complex formation with proteins may disturb the metabolism of bacterial cells and their power functions, such as permeability and respiration (Damm et al., 2008). Both effects lead to death of the bacterial cells. Streptomycin which is a very good antibiotic is taken as the positive control in the present case and DMSO was taken as the negative control. Overall comparison of the microbial reduction rates in the present study revealed Gram-negative bacteria to be more susceptible to the antimicrobial effects of copper than Gram positives, presumably due to their thinner murine wall, which may allow more rapid absorption of copper into the cell (Rhim et al., 2006). 3.9. Hemolytic protection assay To investigate the effect of CPN on the mammalian membranes, the hemolysis test was carried out. The CPN exhibit comparable inhibition assay as to the negative control i.e. PBS (Fig. 9). This observation indicates that the presence of clay mineral has a definite role to play in RBC hemolysis prevention. However, compared to Triton X100, the CPN did not exhibit any significant hemoglobin release and showed almost similar results as that of the negative control PBS. This indicates Table 2 Performance characteristics of BPSE and its OMt-Cu nanocomposites.
Fig. 8. TGA thermogram of OMt, N1, BPSE and nanocomposites.
Sample code
BPSE
ECN0.25
ECN0.5
ENC1
Tensile strength (MPa) Elongation at break (%) Impact resistance (cm)a Scratch hardness (kg) Gloss (60°) Swelling value (%)
6 95 100 3.4 62 27
7.2 97 100 3.8 70 25
11.5 105 100 4.6 74 22
14.2 112 100 8 80 20
a
100 cm is the maximum limit of the instrument.
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Table 3 Antimicrobial activity of OMt-Cu nanohybrids. Zone of inhibition (mm) Bacteria
N1
N0.5
N0.25
Streptomycin (positive control) 5 mg/mL
Negative control (DMSO)
Klebsiella pneumoniae Staphylococcus aureus
27 ± 0.51 25 ± 0.32
22 ± 0.21 21 ± 0.25
18 ± 0.35 20 ± 0.30
33 ± 0.54 28 ± 0.27
– –
Table 4 Antimicrobial activity of BPSE/OMt-Cu nanocomposites. Zone of inhibition (mm) Bacteria
BPSE
ECN1
ECN0.5
ECN0.25
Streptomycin (positive control) 5 mg/mL
Negative control (DMSO)
Klebsiella pneumoniae Staphylococcus aureus
0 0
26 ± 0.21 23 ± 0.25
21 ± 0.43 20 ± 0.19
18 ± 0.33 18 ± 0.36
33 ± 0.44 27 ± 0.30
– –
Scheme 1. Plausible representation of antibacterial action of OMt-Cu/BPSE nanocomposites.
that it did not cause any lysis of the erythrocyte membranes. The above observation reveals the antihemolytic behavior of the CPN to the living cells with concomitant prevention of cell damage against any harmful free radicals.
4. Conclusion Copper nanoparticles decorated OMt was successfully prepared at room temperature. The antibacterial activity study showed enhancement of activity after CPN formation. The hemolysis protection assay test reveals the antihemolytic behavior of the CPN to mammalian RBC. The thermal and performance characteristics of the CPN were enhanced after nanocomposite formation. The observations vouch for the use of these as advanced antimicrobial surface coating materials. Acknowledgment The authors express their gratitude to UGC for the financial assistance given for carrying out the work via Sanction letter no. F. 16-1532(SC)/ 2009 [SA-III]. Also the authors express their gratitude to Miss Purnima Chowdhury, Department of English and Foreign Languages, Tezpur University for helping in language correction of the manuscript. References
Fig. 9. Bar diagrams for RBC hemolysis protection assay of BPSE and CPN.
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