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Rheological and antimicrobial properties of epoxy-based hybrid nanocoatings
Journal Pre-proof Rheological and antimicrobial properties of epoxy-based hybrid nanocoatings M.R. Islam, M. Parimalam, M.G. Sumdani, A. Taher, F. Asyadi, T.W. Yenn
PII:
S0142-9418(19)31043-8
DOI:
https://doi.org/10.1016/j.polymertesting.2019.106202
Reference:
POTE 106202
To appear in:
Polymer Testing
Received Date: 24 June 2019 Revised Date:
9 October 2019
Accepted Date: 2 November 2019
Please cite this article as: M.R. Islam, M. Parimalam, M.G. Sumdani, A. Taher, F. Asyadi, T.W. Yenn, Rheological and antimicrobial properties of epoxy-based hybrid nanocoatings, Polymer Testing (2019), doi: https://doi.org/10.1016/j.polymertesting.2019.106202. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
RHEOLOGICAL AND ANTIMICROBIAL PROPERTIES OF EPOXYBASED HYBRID NANOCOATINGS M. R. Islam, M. Parimalam, M. G. Sumdani, A. Taher, F. Asyadi and T. W. Yenn Section of Chemical Engineering Technology, Malaysian Institute of Chemical and Bioengineering Technology, University of Kualalumpur, Alor Gajah, 78000, Melaka, Malaysia. *e-mail: [email protected]; [email protected]
ABSTRACT The modification of nanocomposite coatings with fillers having unique characteristics in the polymeric matrix is a promising strategy to enhance the durability as well as to prevent the growth of microorganisms that decrease the stability of the materials. This study was conducted to evaluate the rheological and antimicrobial behavior of epoxy-based nanocomposite coatings filled with nanosilica, titanium oxide (TiO2) and zinc oxide (ZnO) against Staphylococcus aureus and Escherichia coli. A rheometer was used for characterizing the rheological properties of the various fillers embedded epoxy nanocomposite coatings. All of the composites inhibited the growth of Staphylococcus aureus and Escherichia coli on modified Kirby Bauer antimicrobial testing, only when they are in contact with samples. Upon quantitative analysis, bioactive constituent dependent antimicrobial activity was observed which increased with the exposure of contact times. The epoxy/silica/TiO2/ZnO (ESTZ) coating showed the highest bacterial reduction of more than 95% for 4h of treatment. The bioactivity was decreased for the case of epoxy/silica/ZnO (ESZ) or epoxy/silica/TiO2 (EST). The combined effect of the nanosilica, TiO2, and ZnO shows the highest performance in terms of stress, viscosity and torque compared to the individual effect of these three fillers onto the epoxy. Results showed that the shear stress of ESZ, EST, epoxy/silica (ES), and ESTZ coating was increased by 4.4%, 7.7%, 32.2%, and 42%, respectively, compared to the neat epoxy (NE) coating. The torque versus strain curve also showed that the torque of ESTZ composites was the highest (0.52 mN.m) compare to NE (0.36 mN.m), ESZ (0.38 mN.m), EST (0.40 mN.m), and ES (0.45 mN.m). The studies indicate that the epoxy-based thermoset
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nanocomposite coatings can be utilized as bactericidal surfaces for the industrial and medical purpose to reduce microbial growth. Keywords: epoxy; nanocomposite coatings; hybrid; antimicrobial activities; rheological properties.
INTRODUCTION The epoxy resin is one of the most important and widely-used polymers. They exhibit some superior properties such as high strength, excellent adhesion to various substrates, low shrinkage, effective electrical insulation, ease of cure, chemical and solvent resistance, and low toxicity due to a three-dimensional cross-linked structure [1-2]. This cross-linked structure may be a reason for their fragile and brittle characteristics [3]. However, epoxy provides low impact strength, low fracture resistance, low thermal stability, and poor hydrophobicity due to the brittleness [4-5]. As a consequence, they exhibit premature failure when load is applied [6]. These problems can be solved by modifying epoxy resin with various additives or fillers. Different attempts have been taken to improve the strength, adhesiveness, and thermal stability of polymers using various types of reinforcing agents, such as glass fibers, graphene, silica, zinc oxide, iron, copper, cobalt, titanium oxide, etc. [7-10]. The properties of the final products depend on the specific function of the fillers. The physico-chemical properties of the fillers and their size are important for the applications of the fillers into the matrix [11]. The inorganic fillers are usually used with the epoxy matrix because they can modify the epoxy matrix without deteriorating the properties. The inorganic fillers improve the chemical resistance, mechanical, thermal, and electrical properties of the epoxy [12]. The silica-reinforced epoxy composites have become attractive for their physical and chemical stability, good moisture resistance, and high thermal expansion coefficient [13]. The epoxy exhibits a significant change in the properties at small amount of loading of silica. The 2
addition of silica to the epoxy can improve the viscosity, modulus of elasticity and relaxation time. The interaction between the epoxy molecules and the layers of silicates forms network structures which provides a high stress yields and sometimes a very slow stress relaxation. The rheological properties of the layered silicates-based composites are influenced by the degree of exfoliation of the layered silicates. The greater degree of exfoliation increases the friction force between the layers of silicates and the epoxy resin which results a higher viscosity of the composites [14]. For example, fume-silica improved the shear stress and the viscosity of the epoxy by 50% [15]. Chen et al. produced epoxy-based hybrid composites using silica nanoparticles and silver nanowires and investigated rheological properties of the composites [16]. It was found that the viscosity of the composites was higher than neat epoxy at low shear rate, and the viscosity decreased with the increase of shear rate. The toughness and the fracture energy of the hybrid composites was also improved. Mejía et al. have shown that epoxy coatings exhibit anti-bacterial activity when silica is added with the epoxy [17]. Recently, ZnO has become a leading filler in the polymer composite areas due to unique mechanical, electrical, thermal, and optical properties [18]. In addition, ZnO has antimicrobial and bactericide activity and it can absorb radiation in a wide range. Matei et al. used ZnO to improve the antimicrobial properties of the composites [19]. Results showed that the antimicrobial performance of the polymer composites increases for introducing ZnO fillers into the polymers. ZnO is a multifunctional inorganic material and can be used as a semiconductor. Moreover, ZnO exhibits piezoelectric and pyroelectric properties [20]. Due to these properties, ZnO is used in the sensor, ceramics, aerospace engineering, biomedical and structural materials. Moreover, ZnO can improve various properties of the polymer based composites for its high surface area. The interfacial adhesion becomes strong between the ZnO and polymers for its high surface area, and the dispersion of ZnO becomes stable into the composites. 3
Titanium dioxide (TiO2) is a common filler used in polymer composites. It is the whitest filler among the available commercial fillers. It possesses a high degree of fastness to light and vulcanization. TiO2 particles have a great impact on the polymer matrix composites. TiO2 particles have improved mechanical properties, low cost, high surface area, high effective pigment, high refractive index, and high chemical inertness [21]. TiO2 contains hydroxyl groups on the surface which makes a strong interaction between the fillers and the matrix resulting a good dispersion of TiO2 into the matrix. For these properties, TiO2 particles have been used in various industries including heat-resistant coatings, corrosion-resistant coatings, paints, paper, plastics, anti-microbial agent, water purification and food additives [22]. The rheological and volume shrinkage properties were investigated at different amount of the loading of TiO2 in the epoxy [23]. Result showed that TiO2 nanoparticles improved the viscosity and reduced the volume shrinkage behavior of the epoxy-based composites. Santhosh et al. utilized TiO2 with epoxy to produce anti-biofilm coating [24]. Result showed that the Ag+ ion doped TiO2 performed anti-biofilm activity in the surface of the epoxy coating. Besides the reveal of unique physical and chemical properties, the modifications of surfaces of the composite with antimicrobial agents are to prevent the growth of microorganisms. The diverse chemical and mechanical characteristics of the filler coated surfaces do not readily encourage sufficient microbial growth due to the lack of suitable enzymes for biodegradation [25]. However, a group of heterotrophic bacteria and fungi produce some exo-enzymes which are able to degrade polymeric materials [26]. As a result, the surfaces are subjected to some microbial attacks over the times that gradually lead to their growth and biofilm formation [27]. Furthermore, these happen when environmental conditions such as pH, temperature, redox potential and humidity favorable to microorganisms, then they adhere to the polymeric coating surfaces and start growing since 4
they already have gained the ability to produce material induced enzymes[25]. Microbial invasions on coating surfaces can be minimized by focusing on potential antimicrobial compounds having potent efficacy but relatively safe to the environment with low toxicity as expected attributes while designing the composition of the fillers. In this study, we considered surface modification approaches using polymeric matrix directly incorporated with various combinations of antibacterial agents such as ZnO and TiO2. These compounds were chosen since they have the ability to prevent the growth of microorganisms on the surfaces which have been revealed in many studies [28-30]. So the filler coatings possessing significant rheological properties coupled with stable and resistant from microbial attack are expected to be more efficient upon applications in the industrial fields including food as well as health and biomedical devices. In this study, a combined effect of the fillers, such as silica, ZnO and TiO2 nanoparticles on the rheological and antimicrobial performance of the epoxybased nanocomposite coating was evaluated. The antimicrobial activities were performed using Staphylococcus aureus and Escherichia coli. As the produced nanocomposite coating is a multi-filler system, therefore, it is really necessary to study its rheological properties for the assessment of the application. The performance of the antimicrobial activities was also important to investigate as different types of prominent anti-microbial fillers were used together. EXPERIMENTAL Materials The type of epoxy resin used in this research is EPIKOTETM Resin 240 manufactured and supply by Momentive Speciality Chemicals B.V. from Singapore. The epoxy equivalent is 185 g/equivalent and epoxy group content is 5400 mmol/kg. Silica nanoparticles, zinc oxide (ZnO), titanium dioxide (TiO2), Tween 80 and Acetic acid were purchased from
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Sigma-Aldrich, USA. Nutrient agar and Mueller Hinton agar media were procured from Merck, Germany.
Preparation of the nanocomposite coatings Five 250 ml of beakers were used to prepare different types of epoxy nanocomposite coatings. An analytical balance was used to measure 30 g of neat epoxy. After that, 6 g nanosilica 4.5 g of ZnO 1.5 g of TiO2 6 g silica, 1.5 g TiO2 and 4.5 g ZnO were added into the epoxy and stirred using a glass rod for few minutes. The mixture was then stirred with a magnetic stirrer for 2 h at 250 rpm. The samples were dried in the drying oven for 5 h at 75 °C. Microorganisms and culture standardization Antimicrobial activity of the composites was carried out against Gram-positive and Gram-negative bacteria included Staphylococcus aureus and Escherichia coli, respectively. The microorganisms were received from Bioengineering Laboratory, Universiti Kuala Lumpur, Malaysian Institute of Chemical and Bioengineering Technology, Melaka, Malaysia. A fresh culture of the representative bacteria was grown on nutrient agar medium (Merck, Germany) at 37°C for 18 h was used at the experimental sections. Bacterial suspensions were standardized in 0.85% sterile NaCl equivalent to 0.5 McFarland standard of BaSO4 turbidity prepared from 0.5 mL of 1.175% BaCl2 consolidated to 99.5 ml of 1% H2SO4. Rheological measurements The rheological measurements were carried out using a Rheometer (Model: MCR 302) to characterize shear flow properties through the shear stress and shear viscosity. The 6
tests were performed with a wide range of shear rate ranging from 10 to 1500 S-1at a temperature 60 °C. The capillary die of rheometer was 30 mm in length, 2 mm in diameter, and 180° entry angle with 16:1 aspect ratio (L/D). The samples were preheated in a barrel under pressure around 3-5 MPa for 5 minutes for achieving a compact mass. The excess sample was automatically purged until no bubbles were found. A set of shear rate was controlled via a microprocessor during the tests. Antimicrobial Activity: Qualitative Analysis Qualitative antibacterial activity of the composites materials was conducted following the modified Kirby Bauer method [31]. The composite samples and Whatman filter paper for as controls were cut into approximately 10×8 mm dimensions based on the availability of materials. Prior to use, the materials were subjected to surface disinfection by dipping in 70% ethanol (v/v) for 5 minutes then rinsed with sterile distilled water to remove the disinfectant. The samples were transferred to a sterile petri dish and excess water was removed by air drying. In addition, the repetition of microbial contamination was minimized by carefully handling the samples under laminar air flow cabinet. The Mueller Hinton agar (Merck, Germany) was used as the growth medium for testing antimicrobial activity against the test bacteria, S. aureus, and E. coli. Each organism was inoculated over the dried surface of agar plates through evenly repeated streaking by a sterile cotton swab dipped in test organisms. The disinfected samples were placed onto the agar plates inoculated with the test bacteria and gently pressed down with sterile forceps to ensure contact with the agar surface. A volume of 20 µL of 70% ethanol and sterile distilled water was applied on sterile Whatman filter paper as the positive and negative control, successively. After 24 h of incubation at 37ºC, the samples and controls were removed and the plates were re-incubated for another 24 h at the same growth conditions to observe the recovery of growth inhibition.
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Antimicrobial Activity: Quantitative Analysis Antimicrobial activity of the composite samples was performed adopting the previously executed method for polymeric surface materials with minor modifications [3233]. The representative microbes, S. aureus, and E. coli were tested for quantitative analysis. Same procedures were followed for disinfection and preparation of samples for inoculation. Bacterial suspensions were prepared by comparing with the 0.5 McFarland standard. The initial concentration of the test microbes was also determined by solid plate counting of suitable dilutions on nutrient agar medium. An aliquot of 20 µL of bacterial suspension standardized in nutrient broth medium was applied on the upper surface of each sample to be tested and spread with the head of a tip to cover maximum surface contact. The plates containing the inoculated samples were incubated at 37ºC for 2h and 4h as sample contact time. Then samples were separately transferred in test tubes containing 10 pieces of sterile glass beads with 5 ml of 0.85% sterile NaCl and 1% of Tween 80 (Sigma-Aldrich, USA). Each tested sample was thoroughly scrubbed by vortex mixing for 5 min then serially diluted by ten folds to reduce microbial concentration. From the dilutions, an aliquot of 100 µL was spread on nutrient agar medium. The plates were incubated at 37ºC for 24h and the number of surviving microorganisms was counted as the colony-forming unit (CFU). RESULTS AND DISCUSSION Shear stress versus shear rate Shear stress versus shear rate curves are plotted in Figure 1. The effect of shear stress on the shear rate is shown in figure 1 for epoxy and different fillers reinforced epoxy coatings. All the samples exhibited straight lines of the flow curves. Moreover, different curves for the different composites indicates that the composites possess various kinds of flow characteristics. ESTZ exhibited the highest shear stress over the shear rate. The ESZ
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composites exhibited the lowest shear stress compared to EST, ES and ESTZ. The higher resistance of flow of the epoxy resin leads to a higher shear stress of the epoxy over shear rate [34]. From the curves of the composites, it is found that reinforcements of the fillers into the epoxy increase the flow resistance of the composites. The strong interfacial interaction between fillers and epoxy leads uniform shear stress transfer from matrix to fillers. The fillers, TiO2, have good interaction with epoxy resin which increase the shear stress of the epoxy/TiO2 composites (EST) compared to the epoxy [35]. For the high surface area of the silica, a strong physical interaction is formed between silica and epoxy which significantly improves the stress of the epoxy/silica composites (ES) compared to the neat epoxy. High adhesion of zinc oxide to the epoxy matrix forms a strong physical bond between zinc oxide and epoxy which reduces the free volume [36]. Thus, zinc oxide provides a higher ratio of shear stress to shear rate of the epoxy/zinc oxide composites (ESZ). Viscosity vs shear rate In Figure 2, the viscosity of the composites is plotted against shear rate. Figure 2 shows that the viscosity of the epoxy increases for the addition of the zinc oxide, titanium oxide and silica fillers. It is also found that the viscosity of the composites slightly decreases with the shear rate first and then remains constant over the shear rate. Figure 3 represents the temperature dependence of the viscosity of the composites. It can be seen in Figure 3 that the viscosity of the composites slightly decreases with the increase of the temperature. The viscosity of the EC, ESZ, EST and ES composites was observed to fall drastically where the viscosity of the ESTZ composites was slowly decreased. The use of three types of fillers in ESTZ composites may provide good resistance against shear rate with temperature. For the pseudo-plastic behavior of polymer, the viscosity of the epoxy and epoxy-based composites decreases with the increase of the shear rate and temperature. The viscosity of the of the epoxy decreases for the applied rotating force of the cylinder because the molecular chain 9
structure of the polymer undergoes rearrangements towards parallel to the rotation of the cylinder surface. Thus, the cylinder rotation decreases the resistance force of the epoxy polymer. At high shear rate, the rotation speed is high which makes rapid rearrangement of the epoxy polymer chain structure. Consequently, the epoxy molecular chains move in the same direction that reduces the viscosity of the epoxy polymer [37]. It can be seen that the viscosity of the silica reinforced epoxy composites is improved compared to the neat epoxy and is decreased for the increase of the shear rate. This is because of the strong interaction between the epoxy groups and silica. The strong bonding between silica and epoxy prevents the movement of the epoxy molecular chain resulting the high viscosity of the ES composites under shearing stress [38]. The addition of TiO2 enhances the hydrophilic characteristics of the epoxy and increases the number of hydrogen bonds between the TiO2 and epoxy molecules. For the hydrogen bonds, the mobility of the epoxy molecular chain decreases resulting the improvement of the viscosity of the EST composites [39]. The addition of ZnO also increases the viscosity of the epoxy. The epoxide group of the epoxy has affinity to form hydrogen bond with the ZnO fillers [40]. However, ZnO has a strong tendency to form agglomeration among them which leads a poor dispersion of the ZiO into the epoxy. For the poor dispersion, the viscosity of the ESZ composites slightly increases compared to the neat epoxy. Torque vs shear rate Figure 4 shows the relation between the torque and the strain of the epoxy-based coatings. The torque versus strain curves are plotted in Figure 3. It can be seen in Figure 4 that the torque of the filler modified epoxy coating is higher than the unmodified epoxy coating. Figure 4 shows that the torque increases at a high rate at the beginning with the increase of shear strain. At higher strain, the torque increases mostly linearly for all composites. The torque of the ESTZ composites is found higher than other composites in this 10
experiment. The neat epoxy exhibits lower torque against shear strain than the filler reinforced epoxy composites. The improvement of the torque is related to the viscosity of the epoxy liquid [41]. The reinforcement of the filler particles makes hindrance to the flow of melted epoxy resin. The embedded filler particles with epoxy resin increase the friction forces between the rotating plate and the filler particles and also between the fillers and matrix. The strong entanglement between the fillers and epoxy matrix obstructs the flow of the epoxy resin. The entanglement between the silica and epoxy is stronger for the high surface area of the silica and the strong interfacial adhesion between silica and epoxy matrix [42]. The addition of TiO2 slightly improves the viscosity as well as torque of the epoxy for the poor dispersion. The agglomeration of the TiO2 particles entraps the epoxy molecule between the TiO2 particles which prevents the formation of the cross-linked network structure of the epoxy [43]. For this reason, the torque of the EST coating is increased slightly compared to the neat epoxy. The ZnO particles have good interfacial interaction with the epoxy which reduces the flow ability of the epoxy resin resulting epoxy more viscous. As a result, the torque of the ZiO reinforced epoxy increases compared with the neat epoxy. The fillers, silica, titanium and zinc oxide, also have tendency to form agglomeration when added with epoxy resin. The presence of the agglomeration into the fluid also make obstacles to flow the resin [44]. FTIR Spectra Figure 5 shows the FTIR spectrum of the composites which provides a clear overview about the structural properties of the composites [7]. The main functional groups of the composites were identified in Figure 5. The peak was observed at around 825 cm-1 for the stretching vibration of epoxy group in the composites [45]. There was a peak at around 1607 cm-1 for the Zn-O bond in the ESZ and ESTZ composites [46]. The stretching vibration was found near about 1177 cm-1 wave length for the Si-O-Si bonds for the presence of silica in the 11
composites [47]. The effect of loading of TiO2 on the epoxy was observed at the 1418 cm-1 for the vibration due to the Ti-O-Ti bonds. The functional group of Ti-O-Si showed a peak at 917 cm-1 in the composites [48]. Qualitative Analysis of Antimicrobial Activity The antimicrobial activity of the samples was performed following basic Kirby Bauer technique which is most commonly used for testing bioactivity of compounds. Focusing on the physical condition of samples, the necessary modification was installed by directly placing the samples on agar plates seeded with the test bacteria. A commonly practiced disinfectant, 70% ethanol was applied to remove surface microorganisms which might be retained from raw materials or sample processing. Before inoculation on the testing media, samples were washed in the sterile distilled water to dispel the trace of disinfectant. In this study, S. aureus and E. coli were selected from two main groups of microorganisms including Gram-positive and Gram-negative bacteria respectively. This consideration was based on the reality that they are commonly associated surface infections that have been studied well and they can interpret most of the cases of infection causing surface contamination [49]. In the qualitative test, all of the samples arrayed on the surface of bacteria inoculated plates showed inhibition of microbial growth only beneath the samples. Figure 6 depicts the antimicrobial activity of the composites against the test bacteria. After 24h of incubation, bacterial inhibitory activity was found only under the contact area of samples. No clear zone was observed around or away from the composites against S. aureus and E. coli. This means the samples restricted the growth of the test bacteria only when they were in direct contact with the samples. There might be a reason that the bioactive ingredients used in the composite as filler did not diffuse on the surrounding media from the solid polymeric matrix. In this stage, no microbial growth was observed under the filter papers used as positive and
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negative controls. A possible explanation might be the restriction of bacterial growth due to unfavorable physical conditions resulting from the unavailability of free space, lacking oxygen and pressure of weight exerted by the paper itself [50]. Hence, the plates were reincubated for another 24h at 37ºC after removing the filter papers and the samples to verify the restriction of microbial growth. In contrast to samples, the controls recovered the microbial growth on the following day being escaped from the adverse physical conditions. So, the sustaining of clear zones with equal consistency after re-incubation indicates that the composite samples had the ability to resist the growth of S. aureus and E. coli. The findings indicate that some of the fillers incorporated in the polymeric matrix inhibited the growth of the target bacteria [51-52]. Quantitative Analysis of Antimicrobial Activity Quantitative bioactivity activity of the composites was investigated to assess microbial growth reduction over different time intervals. On spread plate counting, initial concentration of S. aureus and E. coli cells were found at 3×107and 4.7×107 CFU/mL, successively. The inoculated bacterial suspensions prepared with nutrient broth medium were spread to cover the maximum upper surface of the composites. The bacterial growth was facilitated under standard conditions and the survivals were determined at 2 h and 4 h intervals. After elapsing the incubation period, samples were vortexed with glass bead and Tween 80 to remove the microbes attached on the treating surface. Suitable serial dilutions were prepared for counting surviving cells as colony forming unit on nutrient agar medium by spread plate technique. Figure 7 shows the survival of S. aureus and E. coli upon exposure with the composites. The pattern of bacterial survival was almost similar for the samples other than ESTZ. In general, the rate of bacterial reduction was increased over the times and the lowest
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number of bacterial survival was observed at 4 h of contact time. This means the composites have the ability to kill bacteria upon long time exposure with the slow releasing of antibacterial compounds. The probable reason is there might be a delay of releasing of bioactive ions from the integrated polymeric matrix of the composites. So the tendency was that longer contact times between the samples and microorganisms cause more ion release that leading to the reduction of bacterial survival [53]. Nevertheless, the effect of releasing bioactive ion over the period of time was not analyzed in this study. In addition, it was also noticed that the inhibitory activity was dependent on the bioactive constituents of the composites. Figure 8 demonstrates the effect of the fillers on the reduction of bacterial growth at two different time intervals. The highest antimicrobial activity was noticed for the composite, ESTZ consisting of both ZnO and TiO2 against the two bacteria with the reduction values more than 95% except S. aureus at 2h. On the other hand, the number of bacterial killing was decreased with those composed of either ZnO (ESZ) or TiO2 (EST) followed by the presence of neither component (ES). These findings demonstrate that the fillers for instances ZnO and TiO2 might play the key roles in the killing of the microorganisms. Many investigators reported that ZnO and TiO2 have broad-spectrum antimicrobial activity against wide ranges of microorganisms together with Gram-positive and Gram-negative bacteria as well as fungal and bacterial spores [54-55]. The basic mechanism of bacterial inhibition by TiO2 and ZnO are almost same. Being irradiated under aerobic conditions, both of the oxides involved to the generation of the highly reactive oxygen species including hydrogen peroxide, superoxide and hydroxyl radicals [55]. Although these radicals and molecules are short lived but they are very reactive to most kinds of vital cellular ingredients including cell wall constituents, membrane proteins, genetic materials (DNA) and enzymes [57]. These usually result to inactivation of the respective cellular components by oxidation which leads to the microbial killing under the exposure of 14
metal oxides (Figure 9). Furthermore, S. aureus was noticed to be more susceptible to the composites than E. coli at the highest contact time. The disparity of sensitivity of the bacteria might be due to differences of their cell wall materials in which the later contains an extra outer membrane composed of lipopolysaccharides (LPS) maintaining selective permeability of the toxic molecules [58]. Although, the degree of sensitivity of TiO2 between Grampositive and Gram-negative bacteria was found controversial with Foster et al. where they described the antimicrobial activity of photocatalytic titanium oxide [59]. CONCLUSION The dispersion of silica together with the bioactive fillers included TiO2 and ZnO into the epoxy coating enhances the rheological as well as antimicrobial properties. As conforms to the bioactivity, the composite, ESTZ composed of silica, zinc oxide, and titanium oxide exhibited the highest rheological performance in terms of stress, viscosity, and torque. Although, the rheological properties increased with the composites ESZ, EST, and ES but antimicrobial activities decreased accordingly. Furthermore, the antimicrobial analyses suggest that the composites have a significant inhibitory activity to the test bacteria and microbial reduction increases with the length of exposure time and presence of bioactive constituents. Finally, it can be assessed that the amalgamation of bioactive fillers coupling with silica and epoxy makes a significantly better composite in terms of rheological property and antimicrobial activity which may potentially be applicable to food and biomedical devices.
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FIGURES 0.55 0.50 0.45
Torque (mN.m)
0.40 0.35 0.30 0.25 0.20
ESTZ ES EST ESZ
0.15 0.10 0.05
EC
0.00 2*10
9
4*10
9
6*10
9
9
8*10
Strain (%) Figure 1. Relationship between the torque and strain of the nanocoatings.
22
23
ESTZ
Viscosity [a.u.]
ES EST
ESZ EC
200
400
600
800
1000
1200
1400
Shear rate [1/s] Figure 2. Relationship between the viscoties and shear rate of the nanocoatings.
24
Figure 3: Viscosity versus temperature curves of the composites.
25
140 EC ESZ EST ES ESTZ
Shear Stress (Pa)
120 100 80 60 40 20 0 0
200
400
600
800
1000
1200
1400
Shear Rate (1/s) Figure 4. Relationship between the shear stress and shear rate of the nanocoatings.
26
27
Figure 5: FTIR analysis of the composites [7].
28
i
ii
iii
Figure 6. Antimicrobial activity of the composites against E. coli on nutrient agar medium (iiii). Plate (ii) shows inhibition of microbial growth underlying the contact of samples and controls upon 24h of incubation. After 48h, the controls recovered bacterial growth (iii) whereas the samples areas’ were still clear as an indication of their bioactivity.
29
Number of Survivors (CFU/mL)
108
ES
EST
ESZ
ESTZ
107
106 105
104 0H
2H
4H
Contact Time (h)
Number of Survivors (CFU/mL)
(a) 108
ES
EST
ESZ
ESTZ
107
106
105 104 0H
2H
4H
Contact Time (h) (b) Figure 7. Survival profile of (a) S. aureus and (b) E. coli upon contact with the composites. Antimicrobial activity depends on the length of contact time and the bioactive constituent of the polymeric matrix of composites.
30
% of Bacterial Growth Reduction
100
90
ES EST
80 ESZ
70 ESTZ
60 SA (2 h)
SA (4 h)
EC (2 h)
EC (4 h)
Antimicrobial Activity of Samples at Different times
Figure 8. Effect of composite fillers on the reduction of bacterial growth at different time intervals.
31
Figure 9: Microbial killing mechanism of TiO2 and ZnO. (a) Generation of highly reactive superoxide and hydroxyl radicals, and (b) degradation of vital cellular constituents leading to microbial death.
32
Table 1: Formulation table of Epoxy-based coatings filled with various fillers. Sample Epoxy (g) Silica (g) Titanium oxide (g) Zinc oxide (g) EC
30
-
-
-
ES
30
6.0
-
-
EST
30
-
1.5
-
ESZ
30
-
-
4.5
ESTZ
30
6.0
1.5
4.5
33
Highlights 1. The rheological and antimicrobial behavior of epoxy-based nanocoatings filled with nanosilica, titanium oxide (TiO2) and zinc oxide (ZnO) were evaluated. 2. Modified Kirby Bauer antimicrobial testing was performed using Staphylococcus aureus and Escherichia coli 3. The shear stress of the nanocoating increased by the maximum of 42%. 4. The highest bacterial reduction (95%) was found for hybrid nanocoating composed of nanosilica, ZnO and TiO2. 5. The bioactivity was decreased significantly.
Dear Editor, I do not have any conflict regarding this manuscript. Thank you. Kind regards, Dr. Remanul.
PII:
S0142-9418(19)31043-8
DOI:
https://doi.org/10.1016/j.polymertesting.2019.106202
Reference:
POTE 106202
To appear in:
Polymer Testing
Received Date: 24 June 2019 Revised Date:
9 October 2019
Accepted Date: 2 November 2019
Please cite this article as: M.R. Islam, M. Parimalam, M.G. Sumdani, A. Taher, F. Asyadi, T.W. Yenn, Rheological and antimicrobial properties of epoxy-based hybrid nanocoatings, Polymer Testing (2019), doi: https://doi.org/10.1016/j.polymertesting.2019.106202. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
RHEOLOGICAL AND ANTIMICROBIAL PROPERTIES OF EPOXYBASED HYBRID NANOCOATINGS M. R. Islam, M. Parimalam, M. G. Sumdani, A. Taher, F. Asyadi and T. W. Yenn Section of Chemical Engineering Technology, Malaysian Institute of Chemical and Bioengineering Technology, University of Kualalumpur, Alor Gajah, 78000, Melaka, Malaysia. *e-mail: [email protected]; [email protected]
ABSTRACT The modification of nanocomposite coatings with fillers having unique characteristics in the polymeric matrix is a promising strategy to enhance the durability as well as to prevent the growth of microorganisms that decrease the stability of the materials. This study was conducted to evaluate the rheological and antimicrobial behavior of epoxy-based nanocomposite coatings filled with nanosilica, titanium oxide (TiO2) and zinc oxide (ZnO) against Staphylococcus aureus and Escherichia coli. A rheometer was used for characterizing the rheological properties of the various fillers embedded epoxy nanocomposite coatings. All of the composites inhibited the growth of Staphylococcus aureus and Escherichia coli on modified Kirby Bauer antimicrobial testing, only when they are in contact with samples. Upon quantitative analysis, bioactive constituent dependent antimicrobial activity was observed which increased with the exposure of contact times. The epoxy/silica/TiO2/ZnO (ESTZ) coating showed the highest bacterial reduction of more than 95% for 4h of treatment. The bioactivity was decreased for the case of epoxy/silica/ZnO (ESZ) or epoxy/silica/TiO2 (EST). The combined effect of the nanosilica, TiO2, and ZnO shows the highest performance in terms of stress, viscosity and torque compared to the individual effect of these three fillers onto the epoxy. Results showed that the shear stress of ESZ, EST, epoxy/silica (ES), and ESTZ coating was increased by 4.4%, 7.7%, 32.2%, and 42%, respectively, compared to the neat epoxy (NE) coating. The torque versus strain curve also showed that the torque of ESTZ composites was the highest (0.52 mN.m) compare to NE (0.36 mN.m), ESZ (0.38 mN.m), EST (0.40 mN.m), and ES (0.45 mN.m). The studies indicate that the epoxy-based thermoset
1
nanocomposite coatings can be utilized as bactericidal surfaces for the industrial and medical purpose to reduce microbial growth. Keywords: epoxy; nanocomposite coatings; hybrid; antimicrobial activities; rheological properties.
INTRODUCTION The epoxy resin is one of the most important and widely-used polymers. They exhibit some superior properties such as high strength, excellent adhesion to various substrates, low shrinkage, effective electrical insulation, ease of cure, chemical and solvent resistance, and low toxicity due to a three-dimensional cross-linked structure [1-2]. This cross-linked structure may be a reason for their fragile and brittle characteristics [3]. However, epoxy provides low impact strength, low fracture resistance, low thermal stability, and poor hydrophobicity due to the brittleness [4-5]. As a consequence, they exhibit premature failure when load is applied [6]. These problems can be solved by modifying epoxy resin with various additives or fillers. Different attempts have been taken to improve the strength, adhesiveness, and thermal stability of polymers using various types of reinforcing agents, such as glass fibers, graphene, silica, zinc oxide, iron, copper, cobalt, titanium oxide, etc. [7-10]. The properties of the final products depend on the specific function of the fillers. The physico-chemical properties of the fillers and their size are important for the applications of the fillers into the matrix [11]. The inorganic fillers are usually used with the epoxy matrix because they can modify the epoxy matrix without deteriorating the properties. The inorganic fillers improve the chemical resistance, mechanical, thermal, and electrical properties of the epoxy [12]. The silica-reinforced epoxy composites have become attractive for their physical and chemical stability, good moisture resistance, and high thermal expansion coefficient [13]. The epoxy exhibits a significant change in the properties at small amount of loading of silica. The 2
addition of silica to the epoxy can improve the viscosity, modulus of elasticity and relaxation time. The interaction between the epoxy molecules and the layers of silicates forms network structures which provides a high stress yields and sometimes a very slow stress relaxation. The rheological properties of the layered silicates-based composites are influenced by the degree of exfoliation of the layered silicates. The greater degree of exfoliation increases the friction force between the layers of silicates and the epoxy resin which results a higher viscosity of the composites [14]. For example, fume-silica improved the shear stress and the viscosity of the epoxy by 50% [15]. Chen et al. produced epoxy-based hybrid composites using silica nanoparticles and silver nanowires and investigated rheological properties of the composites [16]. It was found that the viscosity of the composites was higher than neat epoxy at low shear rate, and the viscosity decreased with the increase of shear rate. The toughness and the fracture energy of the hybrid composites was also improved. Mejía et al. have shown that epoxy coatings exhibit anti-bacterial activity when silica is added with the epoxy [17]. Recently, ZnO has become a leading filler in the polymer composite areas due to unique mechanical, electrical, thermal, and optical properties [18]. In addition, ZnO has antimicrobial and bactericide activity and it can absorb radiation in a wide range. Matei et al. used ZnO to improve the antimicrobial properties of the composites [19]. Results showed that the antimicrobial performance of the polymer composites increases for introducing ZnO fillers into the polymers. ZnO is a multifunctional inorganic material and can be used as a semiconductor. Moreover, ZnO exhibits piezoelectric and pyroelectric properties [20]. Due to these properties, ZnO is used in the sensor, ceramics, aerospace engineering, biomedical and structural materials. Moreover, ZnO can improve various properties of the polymer based composites for its high surface area. The interfacial adhesion becomes strong between the ZnO and polymers for its high surface area, and the dispersion of ZnO becomes stable into the composites. 3
Titanium dioxide (TiO2) is a common filler used in polymer composites. It is the whitest filler among the available commercial fillers. It possesses a high degree of fastness to light and vulcanization. TiO2 particles have a great impact on the polymer matrix composites. TiO2 particles have improved mechanical properties, low cost, high surface area, high effective pigment, high refractive index, and high chemical inertness [21]. TiO2 contains hydroxyl groups on the surface which makes a strong interaction between the fillers and the matrix resulting a good dispersion of TiO2 into the matrix. For these properties, TiO2 particles have been used in various industries including heat-resistant coatings, corrosion-resistant coatings, paints, paper, plastics, anti-microbial agent, water purification and food additives [22]. The rheological and volume shrinkage properties were investigated at different amount of the loading of TiO2 in the epoxy [23]. Result showed that TiO2 nanoparticles improved the viscosity and reduced the volume shrinkage behavior of the epoxy-based composites. Santhosh et al. utilized TiO2 with epoxy to produce anti-biofilm coating [24]. Result showed that the Ag+ ion doped TiO2 performed anti-biofilm activity in the surface of the epoxy coating. Besides the reveal of unique physical and chemical properties, the modifications of surfaces of the composite with antimicrobial agents are to prevent the growth of microorganisms. The diverse chemical and mechanical characteristics of the filler coated surfaces do not readily encourage sufficient microbial growth due to the lack of suitable enzymes for biodegradation [25]. However, a group of heterotrophic bacteria and fungi produce some exo-enzymes which are able to degrade polymeric materials [26]. As a result, the surfaces are subjected to some microbial attacks over the times that gradually lead to their growth and biofilm formation [27]. Furthermore, these happen when environmental conditions such as pH, temperature, redox potential and humidity favorable to microorganisms, then they adhere to the polymeric coating surfaces and start growing since 4
they already have gained the ability to produce material induced enzymes[25]. Microbial invasions on coating surfaces can be minimized by focusing on potential antimicrobial compounds having potent efficacy but relatively safe to the environment with low toxicity as expected attributes while designing the composition of the fillers. In this study, we considered surface modification approaches using polymeric matrix directly incorporated with various combinations of antibacterial agents such as ZnO and TiO2. These compounds were chosen since they have the ability to prevent the growth of microorganisms on the surfaces which have been revealed in many studies [28-30]. So the filler coatings possessing significant rheological properties coupled with stable and resistant from microbial attack are expected to be more efficient upon applications in the industrial fields including food as well as health and biomedical devices. In this study, a combined effect of the fillers, such as silica, ZnO and TiO2 nanoparticles on the rheological and antimicrobial performance of the epoxybased nanocomposite coating was evaluated. The antimicrobial activities were performed using Staphylococcus aureus and Escherichia coli. As the produced nanocomposite coating is a multi-filler system, therefore, it is really necessary to study its rheological properties for the assessment of the application. The performance of the antimicrobial activities was also important to investigate as different types of prominent anti-microbial fillers were used together. EXPERIMENTAL Materials The type of epoxy resin used in this research is EPIKOTETM Resin 240 manufactured and supply by Momentive Speciality Chemicals B.V. from Singapore. The epoxy equivalent is 185 g/equivalent and epoxy group content is 5400 mmol/kg. Silica nanoparticles, zinc oxide (ZnO), titanium dioxide (TiO2), Tween 80 and Acetic acid were purchased from
5
Sigma-Aldrich, USA. Nutrient agar and Mueller Hinton agar media were procured from Merck, Germany.
Preparation of the nanocomposite coatings Five 250 ml of beakers were used to prepare different types of epoxy nanocomposite coatings. An analytical balance was used to measure 30 g of neat epoxy. After that, 6 g nanosilica 4.5 g of ZnO 1.5 g of TiO2 6 g silica, 1.5 g TiO2 and 4.5 g ZnO were added into the epoxy and stirred using a glass rod for few minutes. The mixture was then stirred with a magnetic stirrer for 2 h at 250 rpm. The samples were dried in the drying oven for 5 h at 75 °C. Microorganisms and culture standardization Antimicrobial activity of the composites was carried out against Gram-positive and Gram-negative bacteria included Staphylococcus aureus and Escherichia coli, respectively. The microorganisms were received from Bioengineering Laboratory, Universiti Kuala Lumpur, Malaysian Institute of Chemical and Bioengineering Technology, Melaka, Malaysia. A fresh culture of the representative bacteria was grown on nutrient agar medium (Merck, Germany) at 37°C for 18 h was used at the experimental sections. Bacterial suspensions were standardized in 0.85% sterile NaCl equivalent to 0.5 McFarland standard of BaSO4 turbidity prepared from 0.5 mL of 1.175% BaCl2 consolidated to 99.5 ml of 1% H2SO4. Rheological measurements The rheological measurements were carried out using a Rheometer (Model: MCR 302) to characterize shear flow properties through the shear stress and shear viscosity. The 6
tests were performed with a wide range of shear rate ranging from 10 to 1500 S-1at a temperature 60 °C. The capillary die of rheometer was 30 mm in length, 2 mm in diameter, and 180° entry angle with 16:1 aspect ratio (L/D). The samples were preheated in a barrel under pressure around 3-5 MPa for 5 minutes for achieving a compact mass. The excess sample was automatically purged until no bubbles were found. A set of shear rate was controlled via a microprocessor during the tests. Antimicrobial Activity: Qualitative Analysis Qualitative antibacterial activity of the composites materials was conducted following the modified Kirby Bauer method [31]. The composite samples and Whatman filter paper for as controls were cut into approximately 10×8 mm dimensions based on the availability of materials. Prior to use, the materials were subjected to surface disinfection by dipping in 70% ethanol (v/v) for 5 minutes then rinsed with sterile distilled water to remove the disinfectant. The samples were transferred to a sterile petri dish and excess water was removed by air drying. In addition, the repetition of microbial contamination was minimized by carefully handling the samples under laminar air flow cabinet. The Mueller Hinton agar (Merck, Germany) was used as the growth medium for testing antimicrobial activity against the test bacteria, S. aureus, and E. coli. Each organism was inoculated over the dried surface of agar plates through evenly repeated streaking by a sterile cotton swab dipped in test organisms. The disinfected samples were placed onto the agar plates inoculated with the test bacteria and gently pressed down with sterile forceps to ensure contact with the agar surface. A volume of 20 µL of 70% ethanol and sterile distilled water was applied on sterile Whatman filter paper as the positive and negative control, successively. After 24 h of incubation at 37ºC, the samples and controls were removed and the plates were re-incubated for another 24 h at the same growth conditions to observe the recovery of growth inhibition.
7
Antimicrobial Activity: Quantitative Analysis Antimicrobial activity of the composite samples was performed adopting the previously executed method for polymeric surface materials with minor modifications [3233]. The representative microbes, S. aureus, and E. coli were tested for quantitative analysis. Same procedures were followed for disinfection and preparation of samples for inoculation. Bacterial suspensions were prepared by comparing with the 0.5 McFarland standard. The initial concentration of the test microbes was also determined by solid plate counting of suitable dilutions on nutrient agar medium. An aliquot of 20 µL of bacterial suspension standardized in nutrient broth medium was applied on the upper surface of each sample to be tested and spread with the head of a tip to cover maximum surface contact. The plates containing the inoculated samples were incubated at 37ºC for 2h and 4h as sample contact time. Then samples were separately transferred in test tubes containing 10 pieces of sterile glass beads with 5 ml of 0.85% sterile NaCl and 1% of Tween 80 (Sigma-Aldrich, USA). Each tested sample was thoroughly scrubbed by vortex mixing for 5 min then serially diluted by ten folds to reduce microbial concentration. From the dilutions, an aliquot of 100 µL was spread on nutrient agar medium. The plates were incubated at 37ºC for 24h and the number of surviving microorganisms was counted as the colony-forming unit (CFU). RESULTS AND DISCUSSION Shear stress versus shear rate Shear stress versus shear rate curves are plotted in Figure 1. The effect of shear stress on the shear rate is shown in figure 1 for epoxy and different fillers reinforced epoxy coatings. All the samples exhibited straight lines of the flow curves. Moreover, different curves for the different composites indicates that the composites possess various kinds of flow characteristics. ESTZ exhibited the highest shear stress over the shear rate. The ESZ
8
composites exhibited the lowest shear stress compared to EST, ES and ESTZ. The higher resistance of flow of the epoxy resin leads to a higher shear stress of the epoxy over shear rate [34]. From the curves of the composites, it is found that reinforcements of the fillers into the epoxy increase the flow resistance of the composites. The strong interfacial interaction between fillers and epoxy leads uniform shear stress transfer from matrix to fillers. The fillers, TiO2, have good interaction with epoxy resin which increase the shear stress of the epoxy/TiO2 composites (EST) compared to the epoxy [35]. For the high surface area of the silica, a strong physical interaction is formed between silica and epoxy which significantly improves the stress of the epoxy/silica composites (ES) compared to the neat epoxy. High adhesion of zinc oxide to the epoxy matrix forms a strong physical bond between zinc oxide and epoxy which reduces the free volume [36]. Thus, zinc oxide provides a higher ratio of shear stress to shear rate of the epoxy/zinc oxide composites (ESZ). Viscosity vs shear rate In Figure 2, the viscosity of the composites is plotted against shear rate. Figure 2 shows that the viscosity of the epoxy increases for the addition of the zinc oxide, titanium oxide and silica fillers. It is also found that the viscosity of the composites slightly decreases with the shear rate first and then remains constant over the shear rate. Figure 3 represents the temperature dependence of the viscosity of the composites. It can be seen in Figure 3 that the viscosity of the composites slightly decreases with the increase of the temperature. The viscosity of the EC, ESZ, EST and ES composites was observed to fall drastically where the viscosity of the ESTZ composites was slowly decreased. The use of three types of fillers in ESTZ composites may provide good resistance against shear rate with temperature. For the pseudo-plastic behavior of polymer, the viscosity of the epoxy and epoxy-based composites decreases with the increase of the shear rate and temperature. The viscosity of the of the epoxy decreases for the applied rotating force of the cylinder because the molecular chain 9
structure of the polymer undergoes rearrangements towards parallel to the rotation of the cylinder surface. Thus, the cylinder rotation decreases the resistance force of the epoxy polymer. At high shear rate, the rotation speed is high which makes rapid rearrangement of the epoxy polymer chain structure. Consequently, the epoxy molecular chains move in the same direction that reduces the viscosity of the epoxy polymer [37]. It can be seen that the viscosity of the silica reinforced epoxy composites is improved compared to the neat epoxy and is decreased for the increase of the shear rate. This is because of the strong interaction between the epoxy groups and silica. The strong bonding between silica and epoxy prevents the movement of the epoxy molecular chain resulting the high viscosity of the ES composites under shearing stress [38]. The addition of TiO2 enhances the hydrophilic characteristics of the epoxy and increases the number of hydrogen bonds between the TiO2 and epoxy molecules. For the hydrogen bonds, the mobility of the epoxy molecular chain decreases resulting the improvement of the viscosity of the EST composites [39]. The addition of ZnO also increases the viscosity of the epoxy. The epoxide group of the epoxy has affinity to form hydrogen bond with the ZnO fillers [40]. However, ZnO has a strong tendency to form agglomeration among them which leads a poor dispersion of the ZiO into the epoxy. For the poor dispersion, the viscosity of the ESZ composites slightly increases compared to the neat epoxy. Torque vs shear rate Figure 4 shows the relation between the torque and the strain of the epoxy-based coatings. The torque versus strain curves are plotted in Figure 3. It can be seen in Figure 4 that the torque of the filler modified epoxy coating is higher than the unmodified epoxy coating. Figure 4 shows that the torque increases at a high rate at the beginning with the increase of shear strain. At higher strain, the torque increases mostly linearly for all composites. The torque of the ESTZ composites is found higher than other composites in this 10
experiment. The neat epoxy exhibits lower torque against shear strain than the filler reinforced epoxy composites. The improvement of the torque is related to the viscosity of the epoxy liquid [41]. The reinforcement of the filler particles makes hindrance to the flow of melted epoxy resin. The embedded filler particles with epoxy resin increase the friction forces between the rotating plate and the filler particles and also between the fillers and matrix. The strong entanglement between the fillers and epoxy matrix obstructs the flow of the epoxy resin. The entanglement between the silica and epoxy is stronger for the high surface area of the silica and the strong interfacial adhesion between silica and epoxy matrix [42]. The addition of TiO2 slightly improves the viscosity as well as torque of the epoxy for the poor dispersion. The agglomeration of the TiO2 particles entraps the epoxy molecule between the TiO2 particles which prevents the formation of the cross-linked network structure of the epoxy [43]. For this reason, the torque of the EST coating is increased slightly compared to the neat epoxy. The ZnO particles have good interfacial interaction with the epoxy which reduces the flow ability of the epoxy resin resulting epoxy more viscous. As a result, the torque of the ZiO reinforced epoxy increases compared with the neat epoxy. The fillers, silica, titanium and zinc oxide, also have tendency to form agglomeration when added with epoxy resin. The presence of the agglomeration into the fluid also make obstacles to flow the resin [44]. FTIR Spectra Figure 5 shows the FTIR spectrum of the composites which provides a clear overview about the structural properties of the composites [7]. The main functional groups of the composites were identified in Figure 5. The peak was observed at around 825 cm-1 for the stretching vibration of epoxy group in the composites [45]. There was a peak at around 1607 cm-1 for the Zn-O bond in the ESZ and ESTZ composites [46]. The stretching vibration was found near about 1177 cm-1 wave length for the Si-O-Si bonds for the presence of silica in the 11
composites [47]. The effect of loading of TiO2 on the epoxy was observed at the 1418 cm-1 for the vibration due to the Ti-O-Ti bonds. The functional group of Ti-O-Si showed a peak at 917 cm-1 in the composites [48]. Qualitative Analysis of Antimicrobial Activity The antimicrobial activity of the samples was performed following basic Kirby Bauer technique which is most commonly used for testing bioactivity of compounds. Focusing on the physical condition of samples, the necessary modification was installed by directly placing the samples on agar plates seeded with the test bacteria. A commonly practiced disinfectant, 70% ethanol was applied to remove surface microorganisms which might be retained from raw materials or sample processing. Before inoculation on the testing media, samples were washed in the sterile distilled water to dispel the trace of disinfectant. In this study, S. aureus and E. coli were selected from two main groups of microorganisms including Gram-positive and Gram-negative bacteria respectively. This consideration was based on the reality that they are commonly associated surface infections that have been studied well and they can interpret most of the cases of infection causing surface contamination [49]. In the qualitative test, all of the samples arrayed on the surface of bacteria inoculated plates showed inhibition of microbial growth only beneath the samples. Figure 6 depicts the antimicrobial activity of the composites against the test bacteria. After 24h of incubation, bacterial inhibitory activity was found only under the contact area of samples. No clear zone was observed around or away from the composites against S. aureus and E. coli. This means the samples restricted the growth of the test bacteria only when they were in direct contact with the samples. There might be a reason that the bioactive ingredients used in the composite as filler did not diffuse on the surrounding media from the solid polymeric matrix. In this stage, no microbial growth was observed under the filter papers used as positive and
12
negative controls. A possible explanation might be the restriction of bacterial growth due to unfavorable physical conditions resulting from the unavailability of free space, lacking oxygen and pressure of weight exerted by the paper itself [50]. Hence, the plates were reincubated for another 24h at 37ºC after removing the filter papers and the samples to verify the restriction of microbial growth. In contrast to samples, the controls recovered the microbial growth on the following day being escaped from the adverse physical conditions. So, the sustaining of clear zones with equal consistency after re-incubation indicates that the composite samples had the ability to resist the growth of S. aureus and E. coli. The findings indicate that some of the fillers incorporated in the polymeric matrix inhibited the growth of the target bacteria [51-52]. Quantitative Analysis of Antimicrobial Activity Quantitative bioactivity activity of the composites was investigated to assess microbial growth reduction over different time intervals. On spread plate counting, initial concentration of S. aureus and E. coli cells were found at 3×107and 4.7×107 CFU/mL, successively. The inoculated bacterial suspensions prepared with nutrient broth medium were spread to cover the maximum upper surface of the composites. The bacterial growth was facilitated under standard conditions and the survivals were determined at 2 h and 4 h intervals. After elapsing the incubation period, samples were vortexed with glass bead and Tween 80 to remove the microbes attached on the treating surface. Suitable serial dilutions were prepared for counting surviving cells as colony forming unit on nutrient agar medium by spread plate technique. Figure 7 shows the survival of S. aureus and E. coli upon exposure with the composites. The pattern of bacterial survival was almost similar for the samples other than ESTZ. In general, the rate of bacterial reduction was increased over the times and the lowest
13
number of bacterial survival was observed at 4 h of contact time. This means the composites have the ability to kill bacteria upon long time exposure with the slow releasing of antibacterial compounds. The probable reason is there might be a delay of releasing of bioactive ions from the integrated polymeric matrix of the composites. So the tendency was that longer contact times between the samples and microorganisms cause more ion release that leading to the reduction of bacterial survival [53]. Nevertheless, the effect of releasing bioactive ion over the period of time was not analyzed in this study. In addition, it was also noticed that the inhibitory activity was dependent on the bioactive constituents of the composites. Figure 8 demonstrates the effect of the fillers on the reduction of bacterial growth at two different time intervals. The highest antimicrobial activity was noticed for the composite, ESTZ consisting of both ZnO and TiO2 against the two bacteria with the reduction values more than 95% except S. aureus at 2h. On the other hand, the number of bacterial killing was decreased with those composed of either ZnO (ESZ) or TiO2 (EST) followed by the presence of neither component (ES). These findings demonstrate that the fillers for instances ZnO and TiO2 might play the key roles in the killing of the microorganisms. Many investigators reported that ZnO and TiO2 have broad-spectrum antimicrobial activity against wide ranges of microorganisms together with Gram-positive and Gram-negative bacteria as well as fungal and bacterial spores [54-55]. The basic mechanism of bacterial inhibition by TiO2 and ZnO are almost same. Being irradiated under aerobic conditions, both of the oxides involved to the generation of the highly reactive oxygen species including hydrogen peroxide, superoxide and hydroxyl radicals [55]. Although these radicals and molecules are short lived but they are very reactive to most kinds of vital cellular ingredients including cell wall constituents, membrane proteins, genetic materials (DNA) and enzymes [57]. These usually result to inactivation of the respective cellular components by oxidation which leads to the microbial killing under the exposure of 14
metal oxides (Figure 9). Furthermore, S. aureus was noticed to be more susceptible to the composites than E. coli at the highest contact time. The disparity of sensitivity of the bacteria might be due to differences of their cell wall materials in which the later contains an extra outer membrane composed of lipopolysaccharides (LPS) maintaining selective permeability of the toxic molecules [58]. Although, the degree of sensitivity of TiO2 between Grampositive and Gram-negative bacteria was found controversial with Foster et al. where they described the antimicrobial activity of photocatalytic titanium oxide [59]. CONCLUSION The dispersion of silica together with the bioactive fillers included TiO2 and ZnO into the epoxy coating enhances the rheological as well as antimicrobial properties. As conforms to the bioactivity, the composite, ESTZ composed of silica, zinc oxide, and titanium oxide exhibited the highest rheological performance in terms of stress, viscosity, and torque. Although, the rheological properties increased with the composites ESZ, EST, and ES but antimicrobial activities decreased accordingly. Furthermore, the antimicrobial analyses suggest that the composites have a significant inhibitory activity to the test bacteria and microbial reduction increases with the length of exposure time and presence of bioactive constituents. Finally, it can be assessed that the amalgamation of bioactive fillers coupling with silica and epoxy makes a significantly better composite in terms of rheological property and antimicrobial activity which may potentially be applicable to food and biomedical devices.
15
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FIGURES 0.55 0.50 0.45
Torque (mN.m)
0.40 0.35 0.30 0.25 0.20
ESTZ ES EST ESZ
0.15 0.10 0.05
EC
0.00 2*10
9
4*10
9
6*10
9
9
8*10
Strain (%) Figure 1. Relationship between the torque and strain of the nanocoatings.
22
23
ESTZ
Viscosity [a.u.]
ES EST
ESZ EC
200
400
600
800
1000
1200
1400
Shear rate [1/s] Figure 2. Relationship between the viscoties and shear rate of the nanocoatings.
24
Figure 3: Viscosity versus temperature curves of the composites.
25
140 EC ESZ EST ES ESTZ
Shear Stress (Pa)
120 100 80 60 40 20 0 0
200
400
600
800
1000
1200
1400
Shear Rate (1/s) Figure 4. Relationship between the shear stress and shear rate of the nanocoatings.
26
27
Figure 5: FTIR analysis of the composites [7].
28
i
ii
iii
Figure 6. Antimicrobial activity of the composites against E. coli on nutrient agar medium (iiii). Plate (ii) shows inhibition of microbial growth underlying the contact of samples and controls upon 24h of incubation. After 48h, the controls recovered bacterial growth (iii) whereas the samples areas’ were still clear as an indication of their bioactivity.
29
Number of Survivors (CFU/mL)
108
ES
EST
ESZ
ESTZ
107
106 105
104 0H
2H
4H
Contact Time (h)
Number of Survivors (CFU/mL)
(a) 108
ES
EST
ESZ
ESTZ
107
106
105 104 0H
2H
4H
Contact Time (h) (b) Figure 7. Survival profile of (a) S. aureus and (b) E. coli upon contact with the composites. Antimicrobial activity depends on the length of contact time and the bioactive constituent of the polymeric matrix of composites.
30
% of Bacterial Growth Reduction
100
90
ES EST
80 ESZ
70 ESTZ
60 SA (2 h)
SA (4 h)
EC (2 h)
EC (4 h)
Antimicrobial Activity of Samples at Different times
Figure 8. Effect of composite fillers on the reduction of bacterial growth at different time intervals.
31
Figure 9: Microbial killing mechanism of TiO2 and ZnO. (a) Generation of highly reactive superoxide and hydroxyl radicals, and (b) degradation of vital cellular constituents leading to microbial death.
32
Table 1: Formulation table of Epoxy-based coatings filled with various fillers. Sample Epoxy (g) Silica (g) Titanium oxide (g) Zinc oxide (g) EC
30
-
-
-
ES
30
6.0
-
-
EST
30
-
1.5
-
ESZ
30
-
-
4.5
ESTZ
30
6.0
1.5
4.5
33
Highlights 1. The rheological and antimicrobial behavior of epoxy-based nanocoatings filled with nanosilica, titanium oxide (TiO2) and zinc oxide (ZnO) were evaluated. 2. Modified Kirby Bauer antimicrobial testing was performed using Staphylococcus aureus and Escherichia coli 3. The shear stress of the nanocoating increased by the maximum of 42%. 4. The highest bacterial reduction (95%) was found for hybrid nanocoating composed of nanosilica, ZnO and TiO2. 5. The bioactivity was decreased significantly.
Dear Editor, I do not have any conflict regarding this manuscript. Thank you. Kind regards, Dr. Remanul.