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

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Materials Science and Engineering C

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

⁎ Corresponding author. Tel.: +91 3712 267009; fax: +E-mail address: karakniranjan@gmail.com (N. Karak).

http://dx.doi.org/10.1016/j.msec.2015.06.0230928-4931/© 2015 Published by Elsevier B.V.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 28 September 2014Received in revised form 24 February 2015Accepted 10 June 2015Available online xxxx

Keywords:Hyperbranched epoxy nanocompositeBiocide immobilizationCarbon dot reduced Cu2O-OMMT nanohybridMechanicalAntimicrobial activityOptical property

The present work demonstrated a transparent thermosetting nanocomposite with antimicrobial andphotoluminescence attributes. The nanocomposites are fabricated by incorporation of different wt.% (1, 2 and3) of a biocide immobilized OMMT-carbon dot reduced Cu2O nanohybrid (MITH-NH) in the hyperbranchedepoxy 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 nanohybridis prepared by reduction of cupric acetate using carbon dot as the reducing agent in the presence of OMMT at70 °C. The significant improvements in tensile strength (~2 fold), elongation at break (3 fold), toughness (4fold) and initial thermal degradation temperature (30 °C) of the pristine hyperbranched epoxy system areachieved by incorporation of 3 wt.% of MITH-NH in it. The nanocomposites exhibit strong antimicrobial activityagainst Staphylococcus aureus, Bacillus subtilis, Klebsiella pneumoniae and Pseudomonas aeruginosa bacteria andCandida albicans, a fungus. The nanocomposite also shows significant activity against biofilm formation com-pared to the pristine thermoset. Further, the nanocomposite films emit different colors on exposure of differentwavelengths of UV light. The properties of these nanocomposites are also compared with the same nanohybridwithout OMMT.

© 2015 Published by Elsevier B.V.

1. Introduction

In recent years, antimicrobial materials are gaining strong impe-tus for prevention of infection, water purification, marine coating,etc. In this milieu, inorganic nanoparticles and organic biocides areconsidered as effective antimicrobial agents [1–4]. However, directuse of thermo-labile organic biocide as an antimicrobial agent maycause risk to human health and environmental hazards [2]. Althoughcopper based inorganic nanomaterials possess strong inhibitory/kill-ing effect to different microorganisms [5–8], but they also sufferfrom some practical drawbacks. First of all, metallic Cu nanoparticlesare highly toxic to animals too [2]. Further, formation and stabiliza-tion of Cu nanoparticles are also challenging issues. On contrary,Cu2O nanoparticles are nontoxic to animals, easily available and rel-atively cheap. Further, they act as p-type semiconductor with a bandgap 2.2 eV, which may be explored to generate active free radicals tokill 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 oralgae. In addition, controlled reduction of Cu2+ to produce Cu2O

91 3712 267006.

nanoparticles is difficult, as most of the cases mixture of Cu andCuO nanoparticles are also formed [13,14]. In this vein, carbon dotsmay reduce metal salts in a controlled manner and one pot such re-duction are gaining importance in recent times [15,16]. Carbon dotis now a topic of current interest in nanomaterial research becauseof its simple synthetic protocol and multifaceted applications[17–20]. The aqueous solubility, functionalizability, resistance tophotobleaching, toxicity and biocompatibility are attested it as a su-perior material over other nanomaterials [21,22]. Moreover, it hastunable emissions from near-infrared to blue wavelength and it ex-hibits unique up-conversion photoluminescence property [18].Thus carbon dot can generate electron/hole (e−/h+) pairs by the ex-citation of semiconductor metal oxide nanoparticles like Cu2O andTiO2. [18,23]. These electron/hole pairs react with H2O and O2 to pro-duce active oxygen radicals like •OH, •O2

− which may also help in kill-ing the microorganisms. However, direct nanoparticles cannot beused as antimicrobial agents for long duration as they lose their sta-bility and functions due to agglomeration. In this milieu, fabricationof nanocomposite of the metal nanoparticles with polymer or immo-bilization of the organic biocides into such polymer nanocompositesis an advanced technique for destruction of microorganisms by slowrelease of the active agents [24,25]. This technique reduces the toxiceffect 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 highthermostability, highmechanical strength, excellent chemical resistanceand high adhesive strength of epoxy thermoset suggested it as thematrix of choice [26,27]. Further, compared to the conventional linearepoxy resin, hyperbranched one enjoys superiority with respect to itslower viscosity, higher solubility, higher functionality and reactivity aswell as easier processing [28–30]. Again, stable andhomogenous disper-sions of metal nanoparticles are achieved by using polymer supportedorgano-modified clay templated system. The resultant nanocompositealso offers excellent mechanical performance to the pristine polymers[31,32]. This organo-modified clay has layer structure with high aspectratio, which allows efficient load tolerance to the polymer matricesand thus provides high strength and stiffness to the nanocomposites[33–35]. Further, clay is also inherently nontoxic and has the capacityto absorb inorganic and organic molecules [2]. Thus unison of inorganicnanoparticle and organic biocidemay result in an advanced antimicrobi-al agent with enhanced activity to address a spectrum of microorgan-isms of different characters [1,2]. In the present report, therefore, abiocide immobilized OMMT-carbon dot reduced Cu2O nanohybrid/hyperbranched epoxynanocompositewas investigated as a high perfor-mance advanced antimicrobial material with photoluminescenceattribute.

2. Experimental

2.1. Materials

Hyperbranched epoxy resin used in this study was prepared frombisphenol-A, triethanol amine and epichlorohydrin by a polyconden-sation reaction as reported earlier [28]. Epoxy equivalent, degree ofbranching and viscosity of the prepared hyperbranched epoxy were358 g/eq., 0.79 and 19 Pa s (at 25 °C) respectively. Carbon dot wasprepared using earlier method by direct heating of banana (Musaacuminate) juice at 150 °C for 4 h [21]. Octadecyl amine modifiedmontmorillonite 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 ob-tained from Ciba Giegy, Mumbai, India and used as hardener for epoxy.Ethanol (EtOH) and tetrahydrofuran (THF) (Merck, India) were usedafter 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 re-duction of Cu(OAC)2 solution using carbon dot in the presence ofOMMT. In a typical process, 0.5 g Cu(OAC)2 was dissolved in 25 mLEtOH by stirring for 15 min in a two necked round bottom flask. Anamount of 20mL aqueous solution of NH3 (30%) was added to the solu-tion and stirred for another 15 min at room temperature to form a cop-per ammonium complex. 1.0 g OMMT was dispersed in 50 mL EtOH bystirring for 30 min in a 100 mL reagent bottle and it was added dropwise to the above copper ammonium complex under constant stirringat room temperature. After 1 h, the round bottom flask was equippedwith a water condenser and the temperature was raised to 70 °C.Then a 25 mL ethanolic carbon dot solution (0.5 g) was added dropwise to the mixture and stirred for 6 h. The whole process was also re-peated under the same conditions in the absence of OMMT to obtaincarbon dot reduced Cu2O system, which was used for comparison pur-pose. In both the cases after completion of the reaction the solutionwas cooled down naturally and separation of the nanohybrid particleswas done by ultracentrifugation at 5000 rpm for 10 min. Thenanohybrid particles were washed with EtOH for 2–3 times and finallydispersed in 20mL THF by ultrasonication for 5min. The nanohybrids in

the presence and absence of OMMT were coded as ECDCONC andECDCO respectively.

2.3. Immobilization of antibiotic on nanohybrid

2-Methyl-4-isothiazolin-3-one hydrochloride (MITH) was immo-bilized on ECDCONC by combined effect of mechanical shearing and ul-trasonic forces at room temperature. In a typical process, 0.5 g ECDCONCwas dispersed in 25 mL THF under constant stirring for 30 min in a60 mL glass bottle. An amount of 20 wt.% of MITH (0.1 g) with respectto ECDCONC nanohybrid was added to it and stirred continuously for2 h at room temperature followed by ultrasonication for 10 min. MITHimmobilized nanohybrid was coded as MITH-NH.

2.4. Preparation of nanocomposites

The hyperbranched epoxy nanocomposites with different amountsof MITH-NH (1, 2 and 3 wt.% MITH-NH containing biocide: 0.2, 0.4and 0.6 wt.% with respect to hyperbranched epoxy) were prepared bysolution technique using our earlier method [20,35]. The requisiteamount of the THF dispersed nanohybrid particles were added to thehyperbranched epoxy resin and stirred magnetically for 2 h at roomtemperature followed by ultrasonication for 10 min. A 50 wt.% ofpoly(amido-amine) was mixed homogeneously with it and coated onmild glass plates and steel plates. Before curing the plates were keptunder vacuum at room temperature for 24 h to remove all the volatiles.Finally the plateswere cured at 100 °C for 1 h followed by post-curing at130 °C for 30 min. The nanocomposites were coded as MITH-NH1,MITH-NH2 andMITH-NH3 for 1, 2 and 3 wt.% of MITH-NH respectively.The pristine epoxy thermoset was coded as MITH-NH0. One nanocom-posite with 1 wt.% of carbon dot reduced Cu2O nanohybrid withoutOMMT was also prepared by same procedure for comparison purposeand coded as ECDCO1. The hyperbranched epoxy nanocomposite with1, 2 and 3 wt.% ECDCONC nanohybrid are only used in antimicrobialstudy for comparison with biocide immobilized nanocomposites andare coded as ECDCONC1, ECDCONC2 and ECDCONC3 respectively.

2.5. Antibacterial study of the nanocomposites

Antimicrobial tests were done by well diffusion method as reportedin literatures [32,36]. Two gram positive: Staphylococcus aureus (MTCC3160) and Bacillus subtilis (MTCC 121) and two gram negative: Klebsiellapneumoniae (MTCC 618) and Pseudomonas aeruginosa (MTCC 1688) bac-terial stains and Candida albicans (MTCC 3017) as a fungal strain wereused for antimicrobial assay. An amount of 200 μL of a log phase cultureof the test microbes was seeded on the surface of theMuller Hinton agar(potato dextrose agar for fungal study) on Petri dishes. Thenanocompos-ites were dispersed in sterilized DMSO by ultrasonication for 10min and100 μL of each put into the individualwells (diameter 6mm). In onewell,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. Thezone of inhibition diameters was measured using a transparent rulerafter 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 cultureswere taken in conical flasks. An amount of 200 μL of the samples wereadded to the corresponding conical flasks and incubated for 24 h at37 °C (for bacteria) and for 48 h at 28 °C (for fungus). One conical flaskwithout sample was taken as the control for each microbe. The growthof the microbes was measured by checking optical density (OD) at620 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 biofilmassay using representative MITH-NH0 and MITH-NH2 nanocomposites[37,38]. In the present study, slightmodificationwas done, where direct

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thermoset films (size 1 cm2) were used instead of microtiter plates. Thefilms were incubated for a total time of 72 h in potato dextrose broth(PDB) medium. After every 24 h, the used media was decanted fromthe plate and a freshmediawas added to the plate. The filmswere gent-ly washed thrice with phosphate buffer saline (PBS, pH 7.4) to removethe planktonic bacteria. Then the filmswere resuspended and homoge-nized in PBS by vortexing for 5 min and the cells were serially dilutedand plated onto PDB agar. Finally, colony-forming units (CFU) wereenumerated after 48 h of incubation at 28 °C.

2.7. Characterization

FTIR spectra were recorded on a Nicolet FTIR spectrometer (Im-pact-410) using KBr pellet. The wide angle X-ray diffraction patternsof ECDCONC, ECDCO, MITH-NH and MITH-NH2 were recorded by X-ray diffractometer, Miniflex (Rigaku Corporation) using CuΚα radiation(0.154 nm). Morphology of the nanohybrid and the nanocompositewas studied by high resolution transmission electron microscope,HRTEM (JEOL, JEMCXII, Transmission Electron Microscope operatingvoltage at 200 kV). Tensile strength of nanocomposite films (size:60 × 10 × 0.3 mm3) was measured by Universal Testing Machine(UTM WDW10) with a 500 N load cell at a crosshead speed of10 mm/min using the standard test ASTM D822. Scratch hardnesstest (ASTM G171) was done using scratch hardness tester (Sheen In-strument) 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 carriedout by ASTM D522 method using a mandrel with a diameter1–100 mm. All the tests for the measurement of mechanical proper-ties were repeated for five times and average values were taken.Thermal stability of the thermosets was measured by thermogravi-metric analysis (PerkinElmer TG4000) with nitrogen flow rate of30 mL/min and the heating rate of 10 °C/min from 30 to 700 °C. TheUV–visible absorption spectra were recorded using a UV spectropho-tometer, Hitachi (U2001, Tokyo, Japan) for calculation of percent oflight transmittance. The visual transparency of nanocomposite filmswas checked by printed words covered with the thin thermosettingfilms (thickness 0.5 mm). The optical color emission photos of the

Scheme 1. Formation of E

nanocomposite films were recorded in a UV fluorescence inspectioncabinet (Labotec Solutions, Mumbai).

3. Results and discussion

3.1. Formation and characterization of nanohybrid

The OMMT-carbon dot reduced Cu2O nanohybrid particles wereprepared in a single pot by reduction of Cu(OAc)2 solution by carbondot in the presence of OMMT. Here, carbon dot act as a reducing aswell as a capping agent. Carbon dots contain a large number ofpolar functional groups like, hydroxyl, carbonyl, acid, and epoxy, ontheir surface, as revealed in our earlier study [21]. The peripheral hy-droxyl and aldehyde groups of carbon dots help to reduce Cu2+ intoCu+. The hydroxyl groups reduce Cu2+ by polyphenolic mechanismand the aldehyde groups reduce Cu2+ by the Benedict test reactionas shown in Scheme 1. OMMT acts as a stabilizer for carbon dot aswell as Cu2O nanoparticles by absorbing them on its surface and in-side the platelets (Scheme 1). From TEM images of ECDCONC(Fig. 1a and b), it can be seen that the small tiny particles of carbondot and Cu2O are attached on the surface as well as inside the plate-lets of OMMT. However, ECDCO particles started to coalesce asshown in Fig. 1d. Carbon dots contain large numbers of polar func-tional groups on their surface and their intermolecular attractionscause to coalesce. Again, this nanomaterial possesses large surfacearea 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 stabilizedby employing steric or electrostatic stabilizer. As ECDCO does nothave any such stabilizer so the particles are aggregated. However,in the presence of OMMT, these groups help to interact with thepolar groups of the OMMT platelets by different polar–polar interac-tions and thus nanoparticles are stabilized on the surface of OMMT.The formation of Cu2O was confirmed from the XRD patterns asshown in Fig. 2. In ECDCO, the crystallographic spacing d111, d200,d220 and d311 of Cu2O were found at 2θ (°) = 36.4, 42.3, 61.4 and73.5 respectively, which are comparable with the other reports ofCu2O as well as JCPDF #78-2076 data [39,40]. The crystallized struc-ture of Cu2O was also confirmed from the selected area electron dif-fraction (SAED) pattern (Fig. 1c) in TEM study of the nanohybrid,

CDCONC nanohybrid.

Fig. 1. TEM images of ECDCONC at (a) 100 nmand (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.

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where bright crystalline spots were found. Carbon dots possessamorphous to poor crystalline structure as indicated in one of theSAED patterns (Fig. 1c), which is also supported by our earlierstudy [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 spac-ing of Cu2O and d002 spacing of carbon dots, respectively as shown inFig. 1c [23]. In the XRD pattern (Fig. 2) of ECDCONC, the same crystal-lographic spacing like ECDCO was found along with d001 and d002

peaks at 2θ (°) = 7.08 and 19.82 respectively for OMMT crystal.However, in this case the intensity of Cu2O peaks were very lowdue to the absorption of Cu2O nanoparticles into the OMMT plateletsas well as masking effect by OMMT as amount is higher than Cu2O.Again after immobilization of MITH the d001 crystallographic peakof OMMT was shifted from 2θ (°) = 7.08 to 5.45. Thus the interlayerspacing of OMMT increases from 1.2 to 1.6 nm after immobilizationof MITH. This increase of interlayer spacing is due to the combiningeffect of mechanical shearing and ultrasonication forces which helpin dispersion of OMMT and interactions of its layers with MITH bythe presence of polar groups in both. The ultrasonication and me-chanical shearing forces also help to interact MITH with the surfaceof carbon dots and Cu2O nanoparticles. The polar functional groupsof carbon dots and quantum size of both carbon dot and Cu2O nano-particles strengthen the interactions with MITH molecules. MITH isimmobilized under ambient condition. Even though biocide is storedat low temperature but on immobilization, its storage stability is en-hanced. The biocide is adsorbed immediately on the surface of thenanohybrid because of large surface area and thereby gaining thestability with retaining activity. Literature supports similar observa-tion 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 3wt.% of MITH-NHwere prepared by solution techniquewith thehelp of combined effect of mechanical shearing and ultrasonic forces.Mechanical shearing helps to mix the matrix and nanohybrid homoge-neously, whereas ultrasonication generates small cavities in the liquidmedium by mechanical vibrations with high frequency. These cavitiesrapidly create microscopic shock waves. This cavitation is extremelypowerful when all the imploding cavities are combined. Such cavitiesare formed and collapsed within microseconds, thereby releasing tre-mendous energy within the liquid medium. This energy is utilized todisperse the nanohybrid and OMMT in hyperbranched epoxy matrix.Thus, the use of ultrasonication force prevents the agglomeration ofthe nanohybrid by strong interactions of the OMMT layers, carbon dotand Cu2O nanoparticles with hyperbranched epoxy matrix during thepreparation of nanocomposites. These forces therefore, help theOMMT platelets to interact with hyperbranched epoxy and therebywell dispersed platelets into the matrix were obtained. As a result,d001 peak in XRD analysis (2θ = 5.45°) was completely vanished afterformation of nanocomposite as found in Fig. 2. The direct visualizationof well dispersed OMMT platelets as well as carbon dot reduced Cu2Onanohybrid particles were found in TEM images (Fig. 3) of nanocom-posite. Thewell dispersed and disordered arrangement of clay layers in-side the hyperbranched epoxy matrix was found in Fig. 3a and c. Thedispersion of the OMMT platelets and the carbon dot reduced Cu2Onanohybrid particles are shown in Fig. 3b, where the carbon dot re-duced Cu2O nanohybrid particles are attached with the OMMT surface

Fig. 3. TEM images of MITH-NH2 at different resolutions and positions: (a) at 0.2 μm, (b) 100

and well-separated from each other without aggregation. This is dueto the strong physico-chemical interactions between the polar function-al groups of matrix, nanohybrid particles (mainly OMMT platelets andcarbon dots) and MITH. In Fig. 3d the intercalation behavior of OMMTplatelets by the matrix was found. The interlayer spacing between theOMMT platelets was found to be ~1.3 nm as shown in Fig. 3d. This is be-cause of the hyperbranched epoxy chains intercalate the clay galleriesby the strong interactions with the OMMT platelets assisted byimmobilized MITH.

The percentage of swelling values of the cured nanocomposites andthe pristine thermoset are given in Table 1. The swelling values of thenanocomposites decrease with the increase of amount of nanohybridloading and thus the extent of crosslinking is increased. This is due tothe fact that the nanohybrid particles act both as physical crosslinkerand chemical crosslinker with the hyperbranched epoxy as well aspoly(amido-amine) hardener [20,35]. Here, polar functional groups ofcarbon dots can chemically react with hyperbranched epoxy andpoly(amido-amine) hardener as shown in earlier study [20]. OMMTcan also act as chemical crosslinker and assisted by polar groups ofimmobilized MITH.

3.3. Mechanical properties

The values of mechanical properties for the nanocomposites and thepristine thermoset are given in Table 1. Tensile strength, elongation atbreak and toughness values of the pristine hyperbranched epoxy ther-moset sharply increased after formation of nanocomposites withMITH-NH. The tensile strength of pristine thermoset increased from

nm, (c) 100 nm at different positions and (d) OMMT layer spacing at 20 nm resolution.

Table 1Performance 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 (%) 24 22 22 21 23Tensile strength (MPa) 40 ± 1.0 53.4 ± 1.4 63.5 ± 1.2 72.5 ± 1.5 45 ± 0.8Elongation at break (%) 18.5 ± 1.0 27 ± 0.8 43.5 ± 0.5 54.6 ± 1.4 40 ± 0.4Toughnessa (MPa) 540 748 1553 2317 1370Scratch hardnessb (kg) 9.0 ± 0.5 N10.0 N10.0 N10.0 N10.0Impact resistancec (cm) N100 N100 N100 N100 N100Bending diameterd (mm) b1 b1 b1 b1 b1Initial degradation temperature (°C) 267 285 288 296 278

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

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40 to 72.5 MPa after formation of MITH-NH3. From the stress–strainprofiles (Fig. 4), it is found that elongation at break and toughness(area under stress–strain curve) of pristine thermoset were dramatical-ly improved after formation of nanocomposites with MITH-NH. A 3 foldincrement in elongation at break and 4 fold increment in toughness ofthe pristine system were observed for MITH-NH3. The improvementin tensile strength may be due to chemical crosslinking of peripheralpolar functional groups of aromatic carbonized carbon dot and polargroups of immobilized MITH-OMMT with hyperbranched epoxy andpoly(amido-amine) matrices [20]. The strong physical interactionsamong partially exfoliated OMMT platelets, quantum size carbon dotsand Cu2O nanoparticles with the polymer matrix also increase the ten-sile strength [20,35]. Dramatic enhancement in elongation at break andtoughness is due to the physical crosslinking of the nanoparticles withhyperbranched epoxy and plasticizing effect of poly(amido-amine).The mobility of partially exfoliated OMMT platelets assisted by theimmobilized MITH as well as spherical carbon dots and Cu2O nanopar-ticles inside the clay galleries also provides a mode of energy dissipa-tion. The different flexible moieties like aliphatic hydrocarbon andether of hyperbranched epoxy and poly(amido-amine) (as observedin Fig. S1) increase both strain and toughness of the material by plasti-cizing effect [20]. However, in case of ECDCO1 only 12.5% improvementin tensile strength was occurred though improvements in elongation atbreak and toughness were more than 2 fold. The improvements inscratch hardness and impact resistance could not be measured as thevalues for the nanocomposites have reached the highest limit of the in-struments for scratch hardness (10 kg) and impact resistance (100 cm)as given in Table 1. The nanocomposites also exhibited the lowest limit

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

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

3.4. Thermal stability

The initial thermal degradation (5% weight loss) temperatures ofthe pristine thermoset and its nanocomposites are given in Table 1and TGA curves are shown in Fig. 5. From the results it was foundthat the initial degradation temperature of hyperbranched epoxythermoset increased up to 30 °C after formation of nanocompositewith 3 wt.% MITH-NH nanohybrid. The initial degradation temperatureincreased with the increase in the amount of nanohybrid loading. Thisimprovement in thermal stability is due to the strong interactions ofthe quantum sizes carbon dots and Cu2O nanoparticles withhyperbranched epoxy and poly(amido-amine) matrices, as statedabove. The quantum sizes of carbon dots and Cu2O nanoparticles pro-vide large surface area for such strong interactions. Moreover, the pres-ence of aromatic carbonized structure and peripheral polar functionalgroups of carbon dots which provide strong physico-chemical interac-tions with the matrix also enhances the thermal stability of nanocom-posites. Another reason for increase in thermal stability of pristinethermoset after formation of nanocomposites is due to the intercalationof OMMT clay galleries by hyperbranched epoxy and poly(amido-amine) chains which restricted the segmental motion of the polymerchains by different physico-chemical interactions. In addition to theabove, the improvement in initial decomposition temperature of thenanocomposite by incorporation of OMMT is due to the fact that clayis a heat insulator and acts as mass transport barrier to the volatile

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

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products generated during decomposition by providing long paths forthem to travel [38]. However, in case of ECDCO1 only 10 °C improve-ment in initial degradation temperature was noticed. The pristine ther-moset as well as nanocomposites were degraded mainly by two stagepatterns in TGA curves (Fig. 5), where first step (~300 °C) is related tothe degradation of the aliphatic moieties of the matrix and the secondstage (~400 °C) is due to degradation of aromaticmoieties of thematrix.In case of ECDCO1 slight weight loss was observed after 100 °C. This isdue to moisture absorption by the polar functional groups of carbondots as a weight loss of about 2% was observed on heating at 105 °Cfor 3 h without any other change of this nanocomposite. Whereas, incase of nanocomposites with MITH-NH such type of weight loss wasnot found as the carbon dots aswell as Cu2O nanoparticleswere embed-ded inside the organophilic OMMT layers.

3.5. Antimicrobial activity

Antibacterial test of nanocomposites was done by well diffusionmethod. Hyperbranched epoxy/ECDCONC nanocomposites showedsignificant antimicrobial activity towards different Gram positive andGram negative bacteria as shown in Fig. 6(a–c) However, they showedpoor antifungal activity against C. albicans as shown in Fig. 6d. OnlyCu2O showed less antimicrobial activity as it is less toxic than Cu orCuO nanoparticles. Again, formation of nanohybrid with carbon dotsas well OMMT and fabrication of nanocomposites, reduces the toxicitytowards fungal strain. Whereas MITH immobilized nanocomposites ex-hibited excellent antifungal and antibacterial activity. The bacterialgrowth curves for MITH-NH1, MITH-NH2, and MITH-NH3 againstGram positive and Gram negative bacteria are shown in Fig. 7. From

Fig. 6. Antimicrobial activity of hyperbranched epoxy nanocomposites with ECDCONC nanohyband (d) Candida albicans fungus.

the curves it is found that the bacterial growths for all the tested bac-teria were completely inhibited by the nanocomposites at high doseof MITH-NH. In Fig. 7d the increase of P. aeruginosa bacterial growthfor MITH-NH1 after 10 h is due to the presence of insufficient amountof biocide which unable to kill bacterial strain completely. The zonesof inhibition against different bacterial strains are given in the Sup-plementary information as Fig. S2. The antifungal activity of thenanocomposites against C. albicans is given in Fig. 8. From these fig-ures, it is seen that the nanocomposites formed clear inhibitionzones against the tested microbes. The diameter of the zone in-creases with an increase in the amount of MITH-NH in the nanocom-posite and MITH-NH3 shows even larger zone of inhibition than thecontrol. The fungal growth curves of the nanocomposites are shownin Fig. 8b. Where, it is seen that MITH-NH3 completely inhibits thefungal growth. The strong antimicrobial activity of the nanocompos-ites is due to the combined effect of inorganic Cu2O nanoparticlesand MITH biocide. In case of Cu2O, the release of copper ion fromnanocomposites strongly interacts with cell surface of the bacteria.The quantum size of Cu2O provides large surface area which makesthe interaction stronger. The released copper ion of nanocompositesbind with the DNAmolecules and lead to disorder structure betweenthe nucleic acids and also disrupts the biochemical process inside thecell of bacteria [7]. In addition to that, carbon dots absorb visible lightas well as near infrared light during the test and emits shorter wave-length of light (300–500 nm) which again excites Cu2O nanoparticlesand forms electron/hole (e−/h+) pairs as reported in literatures[18,23]. These electron/hole pairs react with H2O and O2 to produceactive oxygen radicals like •OH and •O2

− which may also participatein killing process of microbes by damaging cell membrane via lipid

rid against (a) Bacillus subtilis, (b) Klebsiella pneumoniae (c) Staphylococcus aureus bacteria

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.

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peroxidation [42,43]. On the other hand, MITH has also been used asa powerful biocide since long time for controlling the growth of mi-croorganisms [44]. However, MITH is highly toxic to environment,mainly marine environment. In this case immobilization of MITHon the nanoparticles and formation of polymer nanocomposite mayreduce its toxicity to the environment. After immobilization intothe nanocomposites it is slowly released from the nanoparticle sur-face (as shown in Fig. S3) and inhibits the growth of the tested mi-crobes along with the Cu2O nanoparticles.

Fig. 8. Antifungal activity of MITH-NH1, MITH-NH2 and MITH-NH3 aga

3.6. Biofilm formation study

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

inst Candida albicans, (a) zone of inhibition and (b) growth curves.

Fig. 9.Number of Candida albicans adherence on the surface ofMITH-NH0 andMITH-NH2.

82 B. De et al. / Materials Science and Engineering C 56 (2015) 74–83

Fig. 9. Thus it is confirmed thatMITH-NH2 inhibitedmore fungal adher-ence on the surface of the film as compared to MITH-NH0 which is dueto the presence of MITH as well as Cu2O nanoparticles on the surface ofMITH-NH2 film. The inhibition of biofilm formation by the nanocom-positefilm is due to the combined effect of inorganic Cu2O nanoparticlesand MITH biocide. They killed the adherence fungus from the surface ofthe film by samemechanism as stated earlier in the antimicrobial activ-ity subsection. C. albicans biofilm formation proceeds in an organizedfashion through the early, intermediate, and maturation phases of de-velopment like many other microorganisms. Development of biofilmis closely associated with the generation of matrix which is commonlyknown as extracellular polymeric substances (EPS). It can induce thebiofilm formation on any biotic or abiotic surface. For the transfer ofthe bacteria from the film, other methods like sonication are also usedbut reports suggested that vortexing without sonication increased theyield of adherent bacteria to a considerable extent [45].

3.7. Optical properties

The optical properties like, transparency andUV luminescence of thenanocomposites are illustrated in Fig. 10. From Fig. 10a it is seen that thepercentage of transmittance of the pristine thermoset is decreased from90 to 40 at low visible wavelength (500 nm). However, the percent oftransmittance is not much effected (changes from 90 to 80) at high vis-ible wavelengths (700–800 nm). From the inset picture of Fig. 10a, it isalso found that the transparency and the color of the nanocomposite

Fig. 10. (a) Transparency and (b) optical color emissionof thenanocomposites. (For interpretatithis article.)

films (thickness 0.5 mm) also changes with the increase in amount ofMITH-NH. The transparency of the pristine film is not much affectedafter formation of MITH-NH1. However, at high amount of MITH-NH(2 and 3 wt.%) the transparency decreases. This is due to the fact thatcarbon dots as well as Cu2O nanoparticles can absorb visible light(mainly lower wavelength ~500 nm) [20,21,23]. The luminescence ofthe 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 waschanged into bluish green by illumination with 254 nm UV light andto dark blue at 365 nm UV light. The green and blue color emissions innanocomposites are due to the corresponding band gaps of quantumsize carbon dots [20]. The change of color with the change of wave-length of UV light is due to the presence of different sizes of carbondots in the nanocomposites. The nanoparticles with small and largesizes get excited in the short and long UV region, respectively [20,21].

4. Conclusions

Thus in this study we demonstrated a toughened antimicrobialtransparent hyperbranched epoxy nanocomposite with added interest-ing luminescence property by the incorporation of biocide immobilizedOMMT-carbon dot reduced Cu2O nanohybrid. The nanocompositeshowed excellent antimicrobial activity against both Gram positiveand Gram negative bacteria as well as against a fungus at 3 wt.%nanohybrid loading. The nanocomposite is fabricated by a simple proto-col at room temperature using one pot prepared above nanohybrid. Im-mobilization of organic biocide on OMMT-carbon dot reduced Cu2Onanohybrid resulted advanced antimicrobialmaterial against testedmi-crobes. Thus this work contributes a light in the field of advanced anti-microbial functional thermosetting material.

Acknowledgments

The authors express their gratitude to the NRB for financial assis-tance 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.

on of the references to color in thisfigure legend, the reader is referred to theweb versionof

83B. De et al. / Materials Science and Engineering C 56 (2015) 74–83

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