Biocide immobilized OMMT-carbon dot reduced Cu2O nanohybrid/ hyperbranched epoxy nanocomposites: Mechanical, thermal, antimicrobial and optical properties

Materials Science and Engineering C 56 (2015) 74–83

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Biocide immobilized OMMT-carbon dot reduced Cu2O nanohybrid/ hyperbranched epoxy nanocomposites: Mechanical, thermal, antimicrobial and optical properties Bibekananda De a, Kuldeep Gupta b, Manabendra Mandal b, Niranjan Karak a,⁎ a b

Advanced Polymer and Nanomaterial Laboratory, Center for Polymer Science and Technology, Department of Chemical Sciences, Tezpur University, Napaam 784028, Assam, India Department of Molecular Biology and Biotechnology, Tezpur University, Napaam 784028, Assam, India

a r t i c l e

i n f o

Article history: Received 28 September 2014 Received in revised form 24 February 2015 Accepted 10 June 2015 Available online xxxx Keywords: Hyperbranched epoxy nanocomposite Biocide immobilization Carbon dot reduced Cu2O-OMMT nanohybrid Mechanical Antimicrobial activity Optical property

a b s t r a c t The present work demonstrated a transparent thermosetting nanocomposite with antimicrobial and photoluminescence attributes. The nanocomposites are fabricated by incorporation of different wt.% (1, 2 and 3) of a biocide immobilized OMMT-carbon dot reduced Cu2O nanohybrid (MITH-NH) in the hyperbranched epoxy matrix. MITH-NH is obtained by immobilization of 2-methyl-4-isothiazolin-3-one hydrochloride (MITH) at room temperature using sonication on OMMT-carbon dot reduced Cu2O nanohybid. The nanohybrid is prepared by reduction of cupric acetate using carbon dot as the reducing agent in the presence of OMMT at 70 °C. The significant improvements in tensile strength (~ 2 fold), elongation at break (3 fold), toughness (4 fold) and initial thermal degradation temperature (30 °C) of the pristine hyperbranched epoxy system are achieved by incorporation of 3 wt.% of MITH-NH in it. The nanocomposites exhibit strong antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumoniae and Pseudomonas aeruginosa bacteria and Candida albicans, a fungus. The nanocomposite also shows significant activity against biofilm formation compared to the pristine thermoset. Further, the nanocomposite films emit different colors on exposure of different wavelengths of UV light. The properties of these nanocomposites are also compared with the same nanohybrid without OMMT. © 2015 Published by Elsevier B.V.

1. Introduction In recent years, antimicrobial materials are gaining strong impetus for prevention of infection, water purification, marine coating, etc. In this milieu, inorganic nanoparticles and organic biocides are considered as effective antimicrobial agents [1–4]. However, direct use of thermo-labile organic biocide as an antimicrobial agent may cause risk to human health and environmental hazards [2]. Although copper based inorganic nanomaterials possess strong inhibitory/killing effect to different microorganisms [5–8], but they also suffer from some practical drawbacks. First of all, metallic Cu nanoparticles are highly toxic to animals too [2]. Further, formation and stabilization of Cu nanoparticles are also challenging issues. On contrary, Cu2O nanoparticles are nontoxic to animals, easily available and relatively cheap. Further, they act as p-type semiconductor with a band gap 2.2 eV, which may be explored to generate active free radicals to kill the microorganisms under suitable conditions [9,10]. However, Cu2O nanoparticles are used occasionally as antibacterial agent [11, 12], because of their low activity, particularly against fungus or algae. In addition, controlled reduction of Cu2 + to produce Cu 2 O ⁎ Corresponding author. Tel.: +91 3712 267009; fax: +91 3712 267006. E-mail address: [email protected] (N. Karak).

http://dx.doi.org/10.1016/j.msec.2015.06.023 0928-4931/© 2015 Published by Elsevier B.V.

nanoparticles is difficult, as most of the cases mixture of Cu and CuO nanoparticles are also formed [13,14]. In this vein, carbon dots may reduce metal salts in a controlled manner and one pot such reduction are gaining importance in recent times [15,16]. Carbon dot is now a topic of current interest in nanomaterial research because of its simple synthetic protocol and multifaceted applications [17–20]. The aqueous solubility, functionalizability, resistance to photobleaching, toxicity and biocompatibility are attested it as a superior material over other nanomaterials [21,22]. Moreover, it has tunable emissions from near-infrared to blue wavelength and it exhibits unique up-conversion photoluminescence property [18]. Thus carbon dot can generate electron/hole (e−/h+) pairs by the excitation of semiconductor metal oxide nanoparticles like Cu2O and TiO2. [18,23]. These electron/hole pairs react with H2O and O2 to produce active oxygen radicals like •OH, •O− 2 which may also help in killing the microorganisms. However, direct nanoparticles cannot be used as antimicrobial agents for long duration as they lose their stability and functions due to agglomeration. In this milieu, fabrication of nanocomposite of the metal nanoparticles with polymer or immobilization of the organic biocides into such polymer nanocomposites is an advanced technique for destruction of microorganisms by slow release of the active agents [24,25]. This technique reduces the toxic effect of the active agents to the environment as well as provides

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stability to the same with long durability. Moreover, attributes like high thermostability, high mechanical strength, excellent chemical resistance and high adhesive strength of epoxy thermoset suggested it as the matrix of choice [26,27]. Further, compared to the conventional linear epoxy resin, hyperbranched one enjoys superiority with respect to its lower viscosity, higher solubility, higher functionality and reactivity as well as easier processing [28–30]. Again, stable and homogenous dispersions of metal nanoparticles are achieved by using polymer supported organo-modified clay templated system. The resultant nanocomposite also offers excellent mechanical performance to the pristine polymers [31,32]. This organo-modified clay has layer structure with high aspect ratio, which allows efficient load tolerance to the polymer matrices and thus provides high strength and stiffness to the nanocomposites [33–35]. Further, clay is also inherently nontoxic and has the capacity to absorb inorganic and organic molecules [2]. Thus unison of inorganic nanoparticle and organic biocide may result in an advanced antimicrobial agent with enhanced activity to address a spectrum of microorganisms of different characters [1,2]. In the present report, therefore, a biocide immobilized OMMT-carbon dot reduced Cu2O nanohybrid/ hyperbranched epoxy nanocomposite was investigated as a high performance advanced antimicrobial material with photoluminescence attribute. 2. Experimental 2.1. Materials Hyperbranched epoxy resin used in this study was prepared from bisphenol-A, triethanol amine and epichlorohydrin by a polycondensation reaction as reported earlier [28]. Epoxy equivalent, degree of branching and viscosity of the prepared hyperbranched epoxy were 358 g/eq., 0.79 and 19 Pa s (at 25 °C) respectively. Carbon dot was prepared using earlier method by direct heating of banana (Musa acuminate) juice at 150 °C for 4 h [21]. Octadecyl amine modified montmorillonite nanoclay (OMMT) was purchased from Sigma-Aldrich, Germany and used after vacuum drying. Cupric acetate [Cu(OAc)2] monohydrate was purchased from Rankem, India and used as received. Poly(amido-amine) hardener (HY840, amine value 5–7 eq./kg) was obtained from Ciba Giegy, Mumbai, India and used as hardener for epoxy. Ethanol (EtOH) and tetrahydrofuran (THF) (Merck, India) were used after distillation. 2-Methyl-4-isothiazolin-3-one hydrochloride (MITH) biocide was purchased from Sigma-Aldrich, Germany and used as such. All other chemicals used in this study, were of reagent grade. 2.2. Preparation of OMMT-carbon dot reduced Cu2O nanohybrid OMMT-carbon dot reduced Cu2O nanohybrid was prepared by reduction of Cu(OAC)2 solution using carbon dot in the presence of OMMT. In a typical process, 0.5 g Cu(OAC)2 was dissolved in 25 mL EtOH by stirring for 15 min in a two necked round bottom flask. An amount of 20 mL aqueous solution of NH3 (30%) was added to the solution and stirred for another 15 min at room temperature to form a copper ammonium complex. 1.0 g OMMT was dispersed in 50 mL EtOH by stirring for 30 min in a 100 mL reagent bottle and it was added drop wise to the above copper ammonium complex under constant stirring at room temperature. After 1 h, the round bottom flask was equipped with a water condenser and the temperature was raised to 70 °C. Then a 25 mL ethanolic carbon dot solution (0.5 g) was added drop wise to the mixture and stirred for 6 h. The whole process was also repeated under the same conditions in the absence of OMMT to obtain carbon dot reduced Cu2O system, which was used for comparison purpose. In both the cases after completion of the reaction the solution was cooled down naturally and separation of the nanohybrid particles was done by ultracentrifugation at 5000 rpm for 10 min. The nanohybrid particles were washed with EtOH for 2–3 times and finally dispersed in 20 mL THF by ultrasonication for 5 min. The nanohybrids in

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the presence and absence of OMMT were coded as ECDCONC and ECDCO respectively. 2.3. Immobilization of antibiotic on nanohybrid 2-Methyl-4-isothiazolin-3-one hydrochloride (MITH) was immobilized on ECDCONC by combined effect of mechanical shearing and ultrasonic forces at room temperature. In a typical process, 0.5 g ECDCONC was dispersed in 25 mL THF under constant stirring for 30 min in a 60 mL glass bottle. An amount of 20 wt.% of MITH (0.1 g) with respect to ECDCONC nanohybrid was added to it and stirred continuously for 2 h at room temperature followed by ultrasonication for 10 min. MITH immobilized nanohybrid was coded as MITH-NH. 2.4. Preparation of nanocomposites The hyperbranched epoxy nanocomposites with different amounts of MITH-NH (1, 2 and 3 wt.% MITH-NH containing biocide: 0.2, 0.4 and 0.6 wt.% with respect to hyperbranched epoxy) were prepared by solution technique using our earlier method [20,35]. The requisite amount of the THF dispersed nanohybrid particles were added to the hyperbranched epoxy resin and stirred magnetically for 2 h at room temperature followed by ultrasonication for 10 min. A 50 wt.% of poly(amido-amine) was mixed homogeneously with it and coated on mild glass plates and steel plates. Before curing the plates were kept under vacuum at room temperature for 24 h to remove all the volatiles. Finally the plates were cured at 100 °C for 1 h followed by post-curing at 130 °C for 30 min. The nanocomposites were coded as MITH-NH1, MITH-NH2 and MITH-NH3 for 1, 2 and 3 wt.% of MITH-NH respectively. The pristine epoxy thermoset was coded as MITH-NH0. One nanocomposite with 1 wt.% of carbon dot reduced Cu2O nanohybrid without OMMT was also prepared by same procedure for comparison purpose and coded as ECDCO1. The hyperbranched epoxy nanocomposite with 1, 2 and 3 wt.% ECDCONC nanohybrid are only used in antimicrobial study for comparison with biocide immobilized nanocomposites and are coded as ECDCONC1, ECDCONC2 and ECDCONC3 respectively. 2.5. Antibacterial study of the nanocomposites Antimicrobial tests were done by well diffusion method as reported in literatures [32,36]. Two gram positive: Staphylococcus aureus (MTCC 3160) and Bacillus subtilis (MTCC 121) and two gram negative: Klebsiella pneumoniae (MTCC 618) and Pseudomonas aeruginosa (MTCC 1688) bacterial stains and Candida albicans (MTCC 3017) as a fungal strain were used for antimicrobial assay. An amount of 200 μL of a log phase culture of the test microbes was seeded on the surface of the Muller Hinton agar (potato dextrose agar for fungal study) on Petri dishes. The nanocomposites were dispersed in sterilized DMSO by ultrasonication for 10 min and 100 μL of each put into the individual wells (diameter 6 mm). In one well, 100 μL DMSO was taken as the blank and in another 100 μL (25 mg/mL) gentamicin (nystatin for fungal) was used as the positive control. The zone of inhibition diameters was measured using a transparent ruler after incubating for 24 h at 37 °C (for bacteria) and for 48 h at 28 °C (for fungus). For growth curve analysis of the microbes, the cultures were taken in conical flasks. An amount of 200 μL of the samples were added to the corresponding conical flasks and incubated for 24 h at 37 °C (for bacteria) and for 48 h at 28 °C (for fungus). One conical flask without sample was taken as the control for each microbe. The growth of the microbes was measured by checking optical density (OD) at 620 nm after every 2 h and the OD was taken up to 14 h. 2.6. Biofilm formation study Biofilm formation was studied by means of microtiter plate biofilm assay using representative MITH-NH0 and MITH-NH2 nanocomposites [37,38]. In the present study, slight modification was done, where direct

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thermoset films (size 1 cm2) were used instead of microtiter plates. The films were incubated for a total time of 72 h in potato dextrose broth (PDB) medium. After every 24 h, the used media was decanted from the plate and a fresh media was added to the plate. The films were gently washed thrice with phosphate buffer saline (PBS, pH 7.4) to remove the planktonic bacteria. Then the films were resuspended and homogenized in PBS by vortexing for 5 min and the cells were serially diluted and plated onto PDB agar. Finally, colony-forming units (CFU) were enumerated after 48 h of incubation at 28 °C. 2.7. Characterization FTIR spectra were recorded on a Nicolet FTIR spectrometer (Impact-410) using KBr pellet. The wide angle X-ray diffraction patterns of ECDCONC, ECDCO, MITH-NH and MITH-NH2 were recorded by Xray diffractometer, Miniflex (Rigaku Corporation) using CuΚα radiation (0.154 nm). Morphology of the nanohybrid and the nanocomposite was studied by high resolution transmission electron microscope, HRTEM (JEOL, JEMCXII, Transmission Electron Microscope operating voltage at 200 kV). Tensile strength of nanocomposite films (size: 60 × 10 × 0.3 mm 3 ) was measured by Universal Testing Machine (UTM WDW10) with a 500 N load cell at a crosshead speed of 10 mm/min using the standard test ASTM D822. Scratch hardness test (ASTM G171) was done using scratch hardness tester (Sheen Instrument) on the surface of glass coated thermoset films (size: 75 × 25 × 0.3 mm3 ). Impact test was carried out by impact tester (S. C. Dey) as per the standard falling ball method (ASTM D1709) using the mild steel plate coated nanocomposite films (size: 150 × 50 × 0.3 mm3). Bending test of thermoset films was carried out by ASTM D522 method using a mandrel with a diameter 1–100 mm. All the tests for the measurement of mechanical properties were repeated for five times and average values were taken. Thermal stability of the thermosets was measured by thermogravimetric analysis (PerkinElmer TG4000) with nitrogen flow rate of 30 mL/min and the heating rate of 10 °C/min from 30 to 700 °C. The UV–visible absorption spectra were recorded using a UV spectrophotometer, Hitachi (U2001, Tokyo, Japan) for calculation of percent of light transmittance. The visual transparency of nanocomposite films was checked by printed words covered with the thin thermosetting films (thickness 0.5 mm). The optical color emission photos of the

nanocomposite films were recorded in a UV fluorescence inspection cabinet (Labotec Solutions, Mumbai). 3. Results and discussion 3.1. Formation and characterization of nanohybrid The OMMT-carbon dot reduced Cu2O nanohybrid particles were prepared in a single pot by reduction of Cu(OAc)2 solution by carbon dot in the presence of OMMT. Here, carbon dot act as a reducing as well as a capping agent. Carbon dots contain a large number of polar functional groups like, hydroxyl, carbonyl, acid, and epoxy, on their surface, as revealed in our earlier study [21]. The peripheral hydroxyl and aldehyde groups of carbon dots help to reduce Cu2 + into Cu+. The hydroxyl groups reduce Cu2 + by polyphenolic mechanism and the aldehyde groups reduce Cu2 + by the Benedict test reaction as shown in Scheme 1. OMMT acts as a stabilizer for carbon dot as well as Cu2O nanoparticles by absorbing them on its surface and inside the platelets (Scheme 1). From TEM images of ECDCONC (Fig. 1a and b), it can be seen that the small tiny particles of carbon dot and Cu2O are attached on the surface as well as inside the platelets of OMMT. However, ECDCO particles started to coalesce as shown in Fig. 1d. Carbon dots contain large numbers of polar functional groups on their surface and their intermolecular attractions cause to coalesce. Again, this nanomaterial possesses large surface area due to its quantum size as evidence from TEM image (Fig. 1d) with extremely large numbers of surface atom compared to interior. Thus to avoid the agglomeration the nanoparticles must be stabilized by employing steric or electrostatic stabilizer. As ECDCO does not have any such stabilizer so the particles are aggregated. However, in the presence of OMMT, these groups help to interact with the polar groups of the OMMT platelets by different polar–polar interactions and thus nanoparticles are stabilized on the surface of OMMT. The formation of Cu2 O was confirmed from the XRD patterns as shown in Fig. 2. In ECDCO, the crystallographic spacing d 111 , d 200 , d220 and d311 of Cu 2O were found at 2θ (°) = 36.4, 42.3, 61.4 and 73.5 respectively, which are comparable with the other reports of Cu2O as well as JCPDF #78-2076 data [39,40]. The crystallized structure of Cu2O was also confirmed from the selected area electron diffraction (SAED) pattern (Fig. 1c) in TEM study of the nanohybrid,

Scheme 1. Formation of ECDCONC nanohybrid.

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Fig. 1. TEM images of ECDCONC at (a) 100 nm and (b) 20 nm resolutions, (c) SAED patterns of Cu2O, carbon dot and their interlayer spacings obtained from TEM images of ECDCONC; and (d) The resolution will be 20 nm.

where bright crystalline spots were found. Carbon dots possess amorphous to poor crystalline structure as indicated in one of the SAED patterns (Fig. 1c), which is also supported by our earlier study [21]. Again, from the TEM images, two types of lattice spacing

Fig. 2. XRD patterns of ECDCO, ECDCONC, MITH-NH and MITH-NH2.

of 0.27 and 0.36 nm were found, which correspond to the d111 spacing of Cu2O and d002 spacing of carbon dots, respectively as shown in Fig. 1c [23]. In the XRD pattern (Fig. 2) of ECDCONC, the same crystallographic spacing like ECDCO was found along with d001 and d 002 peaks at 2θ (°) = 7.08 and 19.82 respectively for OMMT crystal. However, in this case the intensity of Cu 2 O peaks were very low due to the absorption of Cu2O nanoparticles into the OMMT platelets as well as masking effect by OMMT as amount is higher than Cu2O. Again after immobilization of MITH the d001 crystallographic peak of OMMT was shifted from 2θ (°) = 7.08 to 5.45. Thus the interlayer spacing of OMMT increases from 1.2 to 1.6 nm after immobilization of MITH. This increase of interlayer spacing is due to the combining effect of mechanical shearing and ultrasonication forces which help in dispersion of OMMT and interactions of its layers with MITH by the presence of polar groups in both. The ultrasonication and mechanical shearing forces also help to interact MITH with the surface of carbon dots and Cu2O nanoparticles. The polar functional groups of carbon dots and quantum size of both carbon dot and Cu2O nanoparticles strengthen the interactions with MITH molecules. MITH is immobilized under ambient condition. Even though biocide is stored at low temperature but on immobilization, its storage stability is enhanced. The biocide is adsorbed immediately on the surface of the nanohybrid because of large surface area and thereby gaining the stability with retaining activity. Literature supports similar observation for nanomaterial immobilized enzyme [41].

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3.2. Preparation and characterization of nanocomposites Hyperbranched epoxy nanocomposites with 1 wt.% of ECDCO and 1, 2 and 3 wt.% of MITH-NH were prepared by solution technique with the help of combined effect of mechanical shearing and ultrasonic forces. Mechanical shearing helps to mix the matrix and nanohybrid homogeneously, whereas ultrasonication generates small cavities in the liquid medium by mechanical vibrations with high frequency. These cavities rapidly create microscopic shock waves. This cavitation is extremely powerful when all the imploding cavities are combined. Such cavities are formed and collapsed within microseconds, thereby releasing tremendous energy within the liquid medium. This energy is utilized to disperse the nanohybrid and OMMT in hyperbranched epoxy matrix. Thus, the use of ultrasonication force prevents the agglomeration of the nanohybrid by strong interactions of the OMMT layers, carbon dot and Cu2O nanoparticles with hyperbranched epoxy matrix during the preparation of nanocomposites. These forces therefore, help the OMMT platelets to interact with hyperbranched epoxy and thereby well dispersed platelets into the matrix were obtained. As a result, d001 peak in XRD analysis (2θ = 5.45°) was completely vanished after formation of nanocomposite as found in Fig. 2. The direct visualization of well dispersed OMMT platelets as well as carbon dot reduced Cu2O nanohybrid particles were found in TEM images (Fig. 3) of nanocomposite. The well dispersed and disordered arrangement of clay layers inside the hyperbranched epoxy matrix was found in Fig. 3a and c. The dispersion of the OMMT platelets and the carbon dot reduced Cu2O nanohybrid particles are shown in Fig. 3b, where the carbon dot reduced Cu2O nanohybrid particles are attached with the OMMT surface

and well-separated from each other without aggregation. This is due to the strong physico-chemical interactions between the polar functional groups of matrix, nanohybrid particles (mainly OMMT platelets and carbon dots) and MITH. In Fig. 3d the intercalation behavior of OMMT platelets by the matrix was found. The interlayer spacing between the OMMT platelets was found to be ~1.3 nm as shown in Fig. 3d. This is because of the hyperbranched epoxy chains intercalate the clay galleries by the strong interactions with the OMMT platelets assisted by immobilized MITH. The percentage of swelling values of the cured nanocomposites and the pristine thermoset are given in Table 1. The swelling values of the nanocomposites decrease with the increase of amount of nanohybrid loading and thus the extent of crosslinking is increased. This is due to the fact that the nanohybrid particles act both as physical crosslinker and chemical crosslinker with the hyperbranched epoxy as well as poly(amido-amine) hardener [20,35]. Here, polar functional groups of carbon dots can chemically react with hyperbranched epoxy and poly(amido-amine) hardener as shown in earlier study [20]. OMMT can also act as chemical crosslinker and assisted by polar groups of immobilized MITH. 3.3. Mechanical properties The values of mechanical properties for the nanocomposites and the pristine thermoset are given in Table 1. Tensile strength, elongation at break and toughness values of the pristine hyperbranched epoxy thermoset sharply increased after formation of nanocomposites with MITH-NH. The tensile strength of pristine thermoset increased from

Fig. 3. TEM images of MITH-NH2 at different resolutions and positions: (a) at 0.2 μm, (b) 100 nm, (c) 100 nm at different positions and (d) OMMT layer spacing at 20 nm resolution.

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Table 1 Performance of pristine hyperbranched epoxy and its nanocomposites with MITH-NH and ECDCO nanohybrids. Parameters

MITH-NH0

MITH-NH1

MITH-NH2

MITH-NH3

ECDCO1

Swelling value (%) Tensile strength (MPa) Elongation at break (%) Toughnessa (MPa) Scratch hardnessb (kg) Impact resistancec (cm) Bending diameterd (mm) Initial degradation temperature (°C)

24 40 ± 1.0 18.5 ± 1.0 540 9.0 ± 0.5 N100 b1 267

22 53.4 ± 1.4 27 ± 0.8 748 N10.0 N100 b1 285

22 63.5 ± 1.2 43.5 ± 0.5 1553 N10.0 N100 b1 288

21 72.5 ± 1.5 54.6 ± 1.4 2317 N10.0 N100 b1 296

23 45 ± 0.8 40 ± 0.4 1370 N10.0 N100 b1 278

a b c d

Calculated by integrating the area under stress–strain curves. Instrument limit of the scratch hardness was 10.0 kg (highest). Instrument limit of the impact strength was 100 cm (highest). Instrument limit of the mandrel diameter was 1 mm (lowest).

of the instruments for flexibility evaluation (bending diameter of mandrel is 1 mm) as they possessed high elongation at break.

40 to 72.5 MPa after formation of MITH-NH3. From the stress–strain profiles (Fig. 4), it is found that elongation at break and toughness (area under stress–strain curve) of pristine thermoset were dramatically improved after formation of nanocomposites with MITH-NH. A 3 fold increment in elongation at break and 4 fold increment in toughness of the pristine system were observed for MITH-NH3. The improvement in tensile strength may be due to chemical crosslinking of peripheral polar functional groups of aromatic carbonized carbon dot and polar groups of immobilized MITH-OMMT with hyperbranched epoxy and poly(amido-amine) matrices [20]. The strong physical interactions among partially exfoliated OMMT platelets, quantum size carbon dots and Cu2O nanoparticles with the polymer matrix also increase the tensile strength [20,35]. Dramatic enhancement in elongation at break and toughness is due to the physical crosslinking of the nanoparticles with hyperbranched epoxy and plasticizing effect of poly(amido-amine). The mobility of partially exfoliated OMMT platelets assisted by the immobilized MITH as well as spherical carbon dots and Cu2O nanoparticles inside the clay galleries also provides a mode of energy dissipation. The different flexible moieties like aliphatic hydrocarbon and ether of hyperbranched epoxy and poly(amido-amine) (as observed in Fig. S1) increase both strain and toughness of the material by plasticizing effect [20]. However, in case of ECDCO1 only 12.5% improvement in tensile strength was occurred though improvements in elongation at break and toughness were more than 2 fold. The improvements in scratch hardness and impact resistance could not be measured as the values for the nanocomposites have reached the highest limit of the instruments for scratch hardness (10 kg) and impact resistance (100 cm) as given in Table 1. The nanocomposites also exhibited the lowest limit

The initial thermal degradation (5% weight loss) temperatures of the pristine thermoset and its nanocomposites are given in Table 1 and TGA curves are shown in Fig. 5. From the results it was found that the initial degradation temperature of hyperbranched epoxy thermoset increased up to 30 °C after formation of nanocomposite with 3 wt.% MITH-NH nanohybrid. The initial degradation temperature increased with the increase in the amount of nanohybrid loading. This improvement in thermal stability is due to the strong interactions of the quantum sizes carbon dots and Cu2O nanoparticles with hyperbranched epoxy and poly(amido-amine) matrices, as stated above. The quantum sizes of carbon dots and Cu2O nanoparticles provide large surface area for such strong interactions. Moreover, the presence of aromatic carbonized structure and peripheral polar functional groups of carbon dots which provide strong physico-chemical interactions with the matrix also enhances the thermal stability of nanocomposites. Another reason for increase in thermal stability of pristine thermoset after formation of nanocomposites is due to the intercalation of OMMT clay galleries by hyperbranched epoxy and poly(amidoamine) chains which restricted the segmental motion of the polymer chains by different physico-chemical interactions. In addition to the above, the improvement in initial decomposition temperature of the nanocomposite by incorporation of OMMT is due to the fact that clay is a heat insulator and acts as mass transport barrier to the volatile

Fig. 4. Stress–strain profiles of pristine hyperbranched epoxy thermoset and its nanocomposites with MITH-NH nanohybrid.

Fig. 5. TGA thermograms for pristine hyperbranched epoxy and its nanocomposites with MITH-NH and ECDCO nanohybrids.

3.4. Thermal stability

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products generated during decomposition by providing long paths for them to travel [38]. However, in case of ECDCO1 only 10 °C improvement in initial degradation temperature was noticed. The pristine thermoset as well as nanocomposites were degraded mainly by two stage patterns in TGA curves (Fig. 5), where first step (~300 °C) is related to the degradation of the aliphatic moieties of the matrix and the second stage (~400 °C) is due to degradation of aromatic moieties of the matrix. In case of ECDCO1 slight weight loss was observed after 100 °C. This is due to moisture absorption by the polar functional groups of carbon dots as a weight loss of about 2% was observed on heating at 105 °C for 3 h without any other change of this nanocomposite. Whereas, in case of nanocomposites with MITH-NH such type of weight loss was not found as the carbon dots as well as Cu2O nanoparticles were embedded inside the organophilic OMMT layers. 3.5. Antimicrobial activity Antibacterial test of nanocomposites was done by well diffusion method. Hyperbranched epoxy/ECDCONC nanocomposites showed significant antimicrobial activity towards different Gram positive and Gram negative bacteria as shown in Fig. 6(a–c) However, they showed poor antifungal activity against C. albicans as shown in Fig. 6d. Only Cu2O showed less antimicrobial activity as it is less toxic than Cu or CuO nanoparticles. Again, formation of nanohybrid with carbon dots as well OMMT and fabrication of nanocomposites, reduces the toxicity towards fungal strain. Whereas MITH immobilized nanocomposites exhibited excellent antifungal and antibacterial activity. The bacterial growth curves for MITH-NH1, MITH-NH2, and MITH-NH3 against Gram positive and Gram negative bacteria are shown in Fig. 7. From

the curves it is found that the bacterial growths for all the tested bacteria were completely inhibited by the nanocomposites at high dose of MITH-NH. In Fig. 7d the increase of P. aeruginosa bacterial growth for MITH-NH1 after 10 h is due to the presence of insufficient amount of biocide which unable to kill bacterial strain completely. The zones of inhibition against different bacterial strains are given in the Supplementary information as Fig. S2. The antifungal activity of the nanocomposites against C. albicans is given in Fig. 8. From these figures, it is seen that the nanocomposites formed clear inhibition zones against the tested microbes. The diameter of the zone increases with an increase in the amount of MITH-NH in the nanocomposite and MITH-NH3 shows even larger zone of inhibition than the control. The fungal growth curves of the nanocomposites are shown in Fig. 8b. Where, it is seen that MITH-NH3 completely inhibits the fungal growth. The strong antimicrobial activity of the nanocomposites is due to the combined effect of inorganic Cu2O nanoparticles and MITH biocide. In case of Cu2 O, the release of copper ion from nanocomposites strongly interacts with cell surface of the bacteria. The quantum size of Cu2O provides large surface area which makes the interaction stronger. The released copper ion of nanocomposites bind with the DNA molecules and lead to disorder structure between the nucleic acids and also disrupts the biochemical process inside the cell of bacteria [7]. In addition to that, carbon dots absorb visible light as well as near infrared light during the test and emits shorter wavelength of light (300–500 nm) which again excites Cu2O nanoparticles and forms electron/hole (e −/h+) pairs as reported in literatures [18,23]. These electron/hole pairs react with H2O and O2 to produce active oxygen radicals like •OH and •O− 2 which may also participate in killing process of microbes by damaging cell membrane via lipid

Fig. 6. Antimicrobial activity of hyperbranched epoxy nanocomposites with ECDCONC nanohybrid against (a) Bacillus subtilis, (b) Klebsiella pneumoniae (c) Staphylococcus aureus bacteria and (d) Candida albicans fungus.

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Fig. 7. Bacterial growth curves of hyperbranched epoxy nanocomposites with MITH-NH nanohybrid against (a) Staphylococcus aureus, (b) Bacillus subtilis, (c) Klebsiella pneumoniae and (d) Pseudomonas aeruginosa bacteria.

peroxidation [42,43]. On the other hand, MITH has also been used as a powerful biocide since long time for controlling the growth of microorganisms [44]. However, MITH is highly toxic to environment, mainly marine environment. In this case immobilization of MITH on the nanoparticles and formation of polymer nanocomposite may reduce its toxicity to the environment. After immobilization into the nanocomposites it is slowly released from the nanoparticle surface (as shown in Fig. S3) and inhibits the growth of the tested microbes along with the Cu2O nanoparticles.

3.6. Biofilm formation study Biofilm formation study was done only for MITH-NH0 and MITHNH2 against C. albicans fungal strain. The whole antimicrobial study of the nanocomposites was done in dispersed state in DMSO. However the application of the nanocomposites is more important in solid film. So the biofilm formation study was done on the nanocomposite film surface. From the study it was found that greater number of fungus adhered on MITH-NH0 film compared to MITH-NH2 film as shown in

Fig. 8. Antifungal activity of MITH-NH1, MITH-NH2 and MITH-NH3 against Candida albicans, (a) zone of inhibition and (b) growth curves.

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Fig. 9. Number of Candida albicans adherence on the surface of MITH-NH0 and MITH-NH2.

Fig. 9. Thus it is confirmed that MITH-NH2 inhibited more fungal adherence on the surface of the film as compared to MITH-NH0 which is due to the presence of MITH as well as Cu2O nanoparticles on the surface of MITH-NH2 film. The inhibition of biofilm formation by the nanocomposite film is due to the combined effect of inorganic Cu2O nanoparticles and MITH biocide. They killed the adherence fungus from the surface of the film by same mechanism as stated earlier in the antimicrobial activity subsection. C. albicans biofilm formation proceeds in an organized fashion through the early, intermediate, and maturation phases of development like many other microorganisms. Development of biofilm is closely associated with the generation of matrix which is commonly known as extracellular polymeric substances (EPS). It can induce the biofilm formation on any biotic or abiotic surface. For the transfer of the bacteria from the film, other methods like sonication are also used but reports suggested that vortexing without sonication increased the yield of adherent bacteria to a considerable extent [45].

films (thickness 0.5 mm) also changes with the increase in amount of MITH-NH. The transparency of the pristine film is not much affected after formation of MITH-NH1. However, at high amount of MITH-NH (2 and 3 wt.%) the transparency decreases. This is due to the fact that carbon dots as well as Cu2O nanoparticles can absorb visible light (mainly lower wavelength ~ 500 nm) [20,21,23]. The luminescence of the hyperbranched epoxy nanocomposites with 1 wt.% carbon dot (ECD1), 1 wt.% ECDCO and 2 wt.% MITH-NH in the visible, short UV (254 nm) and long UV (365 nm) regions are shown in the Fig. 10b. The brown color of the nanocomposites in the visible range was changed into bluish green by illumination with 254 nm UV light and to dark blue at 365 nm UV light. The green and blue color emissions in nanocomposites are due to the corresponding band gaps of quantum size carbon dots [20]. The change of color with the change of wavelength of UV light is due to the presence of different sizes of carbon dots in the nanocomposites. The nanoparticles with small and large sizes get excited in the short and long UV region, respectively [20,21]. 4. Conclusions Thus in this study we demonstrated a toughened antimicrobial transparent hyperbranched epoxy nanocomposite with added interesting luminescence property by the incorporation of biocide immobilized OMMT-carbon dot reduced Cu2O nanohybrid. The nanocomposite showed excellent antimicrobial activity against both Gram positive and Gram negative bacteria as well as against a fungus at 3 wt.% nanohybrid loading. The nanocomposite is fabricated by a simple protocol at room temperature using one pot prepared above nanohybrid. Immobilization of organic biocide on OMMT-carbon dot reduced Cu2O nanohybrid resulted advanced antimicrobial material against tested microbes. Thus this work contributes a light in the field of advanced antimicrobial functional thermosetting material. Acknowledgments

3.7. Optical properties The optical properties like, transparency and UV luminescence of the nanocomposites are illustrated in Fig. 10. From Fig. 10a it is seen that the percentage of transmittance of the pristine thermoset is decreased from 90 to 40 at low visible wavelength (500 nm). However, the percent of transmittance is not much effected (changes from 90 to 80) at high visible wavelengths (700–800 nm). From the inset picture of Fig. 10a, it is also found that the transparency and the color of the nanocomposite

The authors express their gratitude to the NRB for financial assistance through the grant no. DNRD/05/4003/NRB/251 dated 29.02.12. SAIF of NEHU, Shillong is gratefully acknowledged for the TEM imaging. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2015.06.023.

Fig. 10. (a) Transparency and (b) optical color emission of the nanocomposites. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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References [1] A. Rai, A. Prabhune, C.C. Perry, Antibiotic mediated synthesis of gold nanoparticles with potent antimicrobial activity and their application in antimicrobial coatings, J. Mater. Chem. 20 (2010) 6789–6798. [2] Y. Wu, N. Zhou, W. Li, H. Gu, Y. Fan, J. Yuan, Long-term and controlled release of chlorhexidine–copper(II) from organically modified montmorillonite (OMMT) nanocomposites, Mater. Sci. Eng. C 33 (2013) 752–757. [3] V.K. Sharma, R.A. Yngard, Y. Lin, Silver nanoparticles: green synthesis and their antimicrobial activities, Adv. Colloid Interface Sci. 145 (2009) 83–96. [4] M. Stewart, W.H. Miles, C. Depree, Antifouling activity of synthetic γhydroxybutenolides, Int. Biodeter. Biodegr. 88 (2014) 176–184. [5] M.S. Usman, M.E.E. Zowalaty, K. Shameli, N. Zainuddin, M. Salama, N.A. Ibrahim, Synthesis, characterization, and antimicrobial properties of copper nanoparticles, Int. J. Nanomedicine 8 (2013) 4467–4479. [6] G. Ren, D. Hu, E.W.C. Cheng, M.A. Vargas-Reus, P. Reip, R.P. Allaker, Characterisation of copper oxide nanoparticles for antimicrobial applications, Int. J. Antimicrob. Agents 33 (2009) 587–590. [7] B. Bagchi, S. Kar, S.K. Dey, S. Bhandary, D. Roy, T.K. Mukhopadhyay, S. Dasa, P. Nandy, In situ synthesis and antibacterial activity of copper nanoparticle loaded natural montmorillonite clay based on contact inhibition and ion release, Colloids Surf., B 108 (2013) 358–365. [8] L.A. Tamayo, P.A. Zapata, N.D. Vejar, M.I. Azocar, M.A. Gulppi, X. Zhou, G.E. Thompson, F.M. Rabagliati, M.A. Paez, Release of silver and copper nanoparticles from polyethylene nanocomposites and their penetration into Listeria monocytogenes, Mater. Sci. Eng. C 40 (2014) 24–31. [9] M. Deo, D. Shinde, A. Yengantiwar, J. Jog, B. Hannoyer, X. Sauvage, M. Moreb, S. Ogale, Cu2O/ZnO hetero-nanobrush: hierarchical assembly, field emission and photocatalytic properties, J. Mater. Chem. 22 (2012) 17055–17062. [10] K. Tu, Q. Wang, A. Lu, L. Zhang, Portable visible-light photocatalysts constructed from Cu2O nanoparticles and graphene oxide in cellulose matrix, J. Phys. Chem. C 118 (2014) 7202–7210. [11] J. Ren, W. Wang, S. Sun, L. Zhang, L. Wang, J. Chang, Crystallography facet-dependent antibacterial activity: the case of Cu2O, Ind. Eng. Chem. Res. 50 (2011) 10366–10369. [12] Y.J. Lee, S. Kim, S.H. Park, H. Park, Y.D. Huh, Morphology-dependent antibacterial activities of Cu2O, Mater. Lett. 65 (2011) 818–820. [13] Y. Abboud, T. Saffaj, A. Chagraoui, A.E. Bouari, K. Brouzi, O. Tanane, B. Ihssane, Biosynthesis, characterization and antimicrobial activity of copper oxide nanoparticles (CONPs) produced using brown alga extract (Bifurcaria bifurcata), Appl. Nanosci. 4 (2014) 571–576. [14] M. Yin, C.K. Wu, Y. Lou, C. Burda, J.T. Koberstein, Y. Zhu, S. O'Brien, Copper oxide nanocrystals, J. Am. Chem. Soc. 127 (2005) 9506–9511. [15] D. Dey, T. Bhattacharya, B. Majumdar, S. Mandani, B. Sharma, T.K. Sarma, Carbon dot reduced palladium nanoparticles as active catalysts for carbon–carbon bond formation, Dalton Trans. 42 (2013) 13821–13825. [16] H. Choi, S.J. Ko, Y. Choi, P. Joo, T. Kim, B.R. Lee, J.W. Jung, H.J. Choi, M. Cha, J.R. Jeong, I.W. Hwang, M.H. Song, B.S. Kim, J.Y. Kim, Versatile surface plasmon resonance of carbon-dot-supported silver nanoparticles in polymer optoelectronic devices, Nat. Photonics 7 (2013) 732–738. [17] S.N. Baker, G.A. Baker, Luminescent carbon nanodots: emergent nanolights, Angew. Chem. Int. Ed. 49 (2010) 6726–6744. [18] H. Li, Z. Kang, Y. Liu, S.T. Lee, Carbon nanodots: synthesis, properties and applications, J. Mater. Chem. 22 (2012) 24230–24253. [19] M.D. Purkayastha, A.K. Manhar, V.K. Das, A. Borah, M. Mandal, A.J. Thakur, C.L. Mahanta, Antioxidative, hemocompatible, fluorescent carbon nanodots from an “end-of-pipe” agricultural waste: exploring its new horizon in the food-packaging domain, J. Agric. Food Chem. 62 (2014) 4509–4520. [20] B. De, B. Voit, N. Karak, Transparent luminescent hyperbranched epoxy/carbon oxide dot nanocomposites with outstanding toughness and ductility, ACS Appl. Mater. Interfaces 5 (2013) 10027–10034. [21] B. De, N. Karak, A green and facile approach for the synthesis of water soluble fluorescent carbon dots from banana juice, RSC Adv. 3 (2013) 8286–8290. [22] E.J. Goh, K.S. Kim, Y.R. Kim, H.S. Jung, S. Beack, W.H. Kong, G. Scarcelli, S.H. Yun, S.K. Hahn, Bioimaging of hyaluronic acid derivatives using nanosized carbon dots, Biomacromolecules 13 (2012) 2554–2561.

83

[23] H. Li, R. Liu, Y. Liu, H. Huang, H. Yu, H. Ming, S. Lian, S.T. Lee, Z. Kang, Carbon quantum dots/Cu2O composites with protruding nanostructures and their highly efficient (near) infrared photocatalytic behavior, J. Mater. Chem. 22 (2012) 17470–17475. [24] H. Palza, S. Gutierrez, K. Delgado, O. Salazar, V. Fuenzalida, J.I. Avila, G. Figueroa, R. Quijada, Toward tailor-made biocide materials based on poly(propylene)/copper nanoparticles, Macromol. Rapid Commun. 31 (2010) 563–567. [25] S. Barua, N. Dutta, S. Karmakar, P. Chattopadhyay, L. Aidew, A.K. Buragohain, N. Karak, Biocompatible high performance hyperbranched epoxy/clay nanocomposite as an implantable material, Biomed. Mater. 9 (2014) 025006 (14 pp.). [26] N. Karak, Fundamentals of Polymers: Raw Materials to Applications, PHI Learning Pvt. Ltd., New Delhi, 2009. [27] H. Lee, K. Neville, Handbook of Epoxy Resins, McGraw-Hill, New York, 1967. [28] B. De, N. Karak, Novel high performance tough hyperbranched epoxy by an A2 + B3 polycondensation reaction, J. Mater. Chem. A 1 (2013) 348–353. [29] B. De, K. Gupta, M. Mandal, N. Karak, Biodegradable hyperbranched epoxy from castor oil-based hyperbranched polyester polyol, ACS Sustainable Chem. Eng. 2 (2014) 445–453. [30] D. Zhang, D. Jia, Toughness and strength improvement of diglycidyl ether of bisphenol-A by low viscosity liquid hyperbranched epoxy resin, J. Appl. Polym. Sci. 101 (2006) 2504–2511. [31] G. Das, R.D. Kalita, P. Gogoi, A.K. Buragohain, N. Karak, Antibacterial activities of copper nanoparticle-decorated organically modified montmorillonite/epoxy nanocomposites, Appl. Clay Sci. 90 (2014) 18–26. [32] B. Roy, P. Bharali, B.K. Konwar, N. Karak, Silver-embedded modified hyperbranched epoxy/clay nanocomposites as antibacterial materials, Bioresour. Technol. 127 (2013) 175–180. [33] K. Wang, L. Chen, J. Wu, M.L. Toh, C. He, A.F. Yee, Epoxy nanocomposites with highly exfoliated clay: mechanical properties and fracture mechanisms, Macromolecules 38 (2005) 788–800. [34] J.H. Park, S.C. Jana, Mechanism of exfoliation of nanoclay particles in epoxy–clay nanocomposites, Macromolecules 36 (2003) 2758–2768. [35] B. De, N. Karak, Tough hyperbranched epoxy/poly(amido-amine) modified bentonite thermosetting nanocomposites, J. Appl. Polym. Sci. 131 (2014) 40327 (8 p.). [36] P. Radhika, B.S. Sastry, B.M. Harica, Antibacterial screening of Andrographis paniculata (Acanthaceae) root extracts, Res. J. Biotechnol. 3 (2008) 62–63. [37] D. Djordjevic, M. Wiedmann, L.A. McLandsborough, Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation, Appl. Environ. Microbiol. 68 (2002) 2950–2958. [38] B. Roy, P. Bharali, B.K. Konwar, N. Karak, Modified hyperbranched epoxy/clay nanocomposites: a study on thermal, antimicrobial and biodegradation properties, Int. J. Mater. Res. 105 (2014) 296–307. [39] S.B. Kalidindi, U. Sanyal, B.R. Jagirdar, Nanostructured Cu and Cu@Cu2O core shell catalysts for hydrogen generation from ammonia-borane, Phys. Chem. Chem. Phys. 10 (2008) 5870–5874. [40] P. He, X. Shen, H. Gao, Size-controlled preparation of Cu2O octahedron nanocrystals and studies on their optical absorption, J. Colloid Interface Sci. 284 (2005) 510–515. [41] R. Konwarh, D. Kalita, C. Mahanta, M. Mandal, N. Karak, Magnetically recyclable, antimicrobial, and catalytically enhanced polymer-assisted “green” nanosystemimmobilized Aspergillus niger amyloglucosidase, Appl. Microbiol. Biotechnol. 87 (2010) 1983–1992. [42] H. Bodaghi, Y. Mostofi, A. Oromiehie, Z. Zamani, B. Ghanbarzadeh, C. Costa, A. Conte, M.A.D. Nobile, Evaluation of the photocatalytic antimicrobial effects of a TiO2 nanocomposite food packaging film by in vitro and in vivo tests, LWT-Food Sci. Technol. 50 (2013) 702–706. [43] Z. Lu, C.M. Li, H. Bao, Y. Qiao, Y. Toh, X. Yang, Mechanism of antimicrobial activity of CdTe quantum dots, Langmuir 24 (2008) 5445–5452. [44] T.M. LaMarre, C.H. Martin, M.T. Wilharm, Synergistic biocide of 2(thiocyanomethylthio)benzothiazole with a mixture of 5-chloro-2-methyl-4isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one, US patent 4595691, 1986. [45] H. Kobayashi, M. Oethinger, M.J. Tuohy, G.W. Procop, T.W. Bauer, Improved detection of biofilm-formative bacteria by vortexing and sonication, Clin. Orthop. Relat. Res. 467 (2009) 1360–1364.