3ZnO/TiO2 Coupled Oxides LLDPE Nanocomposite: Effect of Various Weight Percent of Sol-gel Synthesized Catalyst on Structural and Bacteriostatic Activity Against S. Aureus and E. Coli

In this study, coupled oxide 3ZnO:1TiO2 (3ZT) synthesized by sol-gel method with different weight percentage were incorporated in linear lowdensity polyethylene (LLDPE) for antimicrobial application. The nanoparticles were homogeneously distributed across the surface due to the casting method adopted in this work. The formation of heterojunction of 3ZT and crystalline nature of the phases and high quantity of surface •OH resulted in LLDPE nanocomposites with higher adsorption water molecules property, thus causing a substantial improvement in photocatalytic reaction. The antimicrobial results indicate LLDPE incorporated with coupled oxide 3ZT was active in inactivating Gram-positive; Staphylococcus aureus (S. aureus) and Gram-negative; Escherichia coli (E. coli). S. aureus was found to be more susceptible in killing as compared to E. coli, under visible light. The best inactivation up to 100% was able to achieve for both bacteria in LLDPE with 10wt.% under visible light due to more Zn ion and •OH release at a specific incubation time. Abbreviations: LLDPE: Linear Low-Density Polyethylene; ZAD: Zinc Acetate Dehydrate; Ag: Silver; Cu: Copper; TiO2: Titanium Dioxide; ZnO: Zinc Oxide; GRAS: Generally Recognized As Safe; TTIP: Titanium Isopropoxide; DSC: Differential Scanning Calorimetry; Tm: Melting Temperature; FTIR: Fourier Transform Infrared Spectrometer; HRTEM: High-resolution Transmission Electron Microscopy; MB: Methylene Blue; BQ: Benzoquinone; AMDI: Advanced Medical and Dental Institute; LB: Luria-Bertani; CFU: Colony Forming Unit; DSC: Differential Scanning Calorimetry; ACN: Acetonitrile; NCLLS: National Committee for Clinical Laboratory Standards; MOE: Ministry of Education; TRGS: Transdisciplinary Research Grant Scheme; USM: Universiti Sains Malaysia Biomedical Journal of Scientific & Technical Research Volume 8Issue 4: 2018 Cite this article: Saharudin K A, Sreekantan S, Basiron N, Mydin R B S, Harun N H et al. 3ZnO/TiO2 Coupled Oxides LLDPE Nanocomposite: Effect of Various Weight Percent of Sol-gel Synthesized Catalyst on Structural and Bacteriostatic Activity Against S. Aureus and E. Coli. BJSTR MS.ID.001681. DOI: 10.26717/ BJSTR.2018.08.001681. 6604 verified its ability to reduce E. coli contamination on food surface. TiO2 nanoparticles was reported to kill viruses including hepatitis B virus [14] and herpes simplex virus [15]. Recently, Ramesh and co-workers [16] reported the potential application of TiO2 photocatalyst in the food sector. The U.S. FDA has approved the use of TiO2 in human food, drugs, cosmetics and food contact materials [17]. Zinc oxide (ZnO) is frequently considered as an alternative to TiO2 for photocatalytic applications [18]. Currently, it is listed as one of five zinc compounds that is generally recognized as safe (GRAS) by the U.S. FDA [17]. Wang et al. [19] reported ZnO nanoparticles exhibit strong antibacterial property over a broad range of microorganism. Azam and co-workers [20] found that ZnO has the highest bactericidal activity against both Gram-negative (E. coli and P. aeruginosa) and Gram-positive (S. aureus and B. subtilis) bacteria compared to CuO and Fe2O3 nanoparticles. Nevertheless, the potential of single phase TiO2 and ZnO to achieve complete inactivation is hindered due to high recombination of electron and holes. Besides, the incorporation of these particles in polymer matrix further reduce the water uptake, ion and ROS migration thus reduce their inactivation capacity. Attempt has been made to combined TiO2 and ZnO particles to improve its individual antimicrobial property. These particles would gallop the properties of both of the nanoparticles simultaneously while retaining their individual characteristics. There have been multiple reports of these coupled oxides which perform dually well when combined than in their individual states [21-23]. However, most of the literatures concerning only on coupled oxides nanoparticles and not polymer nanocomposites. In our previous work, bacteriostatic activity of LLDPE nanocomposite embedded with sol–gel synthesized TiO2/ZnO coupled oxides with various ratios at 5wt.% has been reported [24]. However, the effect of various percentage of best performed catalyst 3ZT was not addressed in the reported work. Besides total inactivation of S. aureus was not achieved. Therefore, in this work structural aspects, metal ion and ROS release of LLDPE nanocomposites with various percentage (1 ,3, 7, 10 wt.%) 3ZT and prolonged incubation time for complete inactivation of Gram-positive; Staphylococcus aureus (S. aureus) and Gram-negative; Escherichia coli (E. coli) is performed. The structural and functional relationship and the resultant outcomes of this work have the potential benefits to design antimicrobial polymer nanocomposites that function under visible light to reduce disease transfer via biomedical devices.


Introduction
Microbial contamination poses a major threat to human health.
Many diseases spread due to the bacterial infections, which cause significant economic and personal losses [1]. Hence, antimicrobial modification of surfaces to prevent growth of detrimental microorganism is highly desired. In biomedical devices such as catheters, prosthetics and implants, surface microbial invasion can result in serious infection and device failure [2]. Surface-centered infections also implicated in food spoilage, spread of foodborne disease and biofouling of materials [3]. Hence, there is significant interest in the development of antimicrobial materials and surfaces for applications in the health and biomedical device industry, food industry and personal hygiene industry. Incorporation of antimicrobial agents directly into polymers [4][5][6] have gained priority in research [7] due to unique properties such as strong antibacterial activity at low concentrations [8], stable in extreme conditions [9], non-toxic [10], and some of them even contain mineral elements essential to the human body [9]. Various metal and metal oxides such as silver (Ag), gold (Au), copper (Cu), titanium dioxide (TiO 2 ), and zinc oxide (ZnO) has been incorporated in polymeric materials [11,12].
Among them TiO 2 has been one of the most versatile antimicrobial agents due to its excellent photocatalytic antimicrobial activity over a broad spectrum of microorganism. The antimicrobial properties of TiO 2 are attributed to the high redox potential of ROS generated by the photo-excitation. Chawengkijwanich and Hayata, [13]  6604 verified its ability to reduce E. coli contamination on food surface.
TiO 2 nanoparticles was reported to kill viruses including hepatitis B virus [14] and herpes simplex virus [15]. Recently, Ramesh and co-workers [16] reported the potential application of TiO 2 photocatalyst in the food sector. The U.S. FDA has approved the use of TiO 2 in human food, drugs, cosmetics and food contact materials [17]. Zinc oxide (ZnO) is frequently considered as an alternative to TiO 2 for photocatalytic applications [18]. Currently, it is listed as one of five zinc compounds that is generally recognized as safe (GRAS) by the U.S. FDA [17]. Wang et al. [19] reported ZnO nanoparticles exhibit strong antibacterial property over a broad range of microorganism. Azam and co-workers [20] found that ZnO has the highest bactericidal activity against both Gram-negative (E.

Preparation of 3ZT Coupled Oxide
In this study, sol-gel process was carried out to synthesis the 3ZT

Preparation of LLDPE Nanocomposite
The LLDPE nanocomposites denoted as LLDPE/3ZT/1, LLDPE/3ZT/5, LLDPE/3ZT/7 and LLDPE/3ZT/10 were prepared from the mixture containing 1 g of LLDPE matrix and 1, 5, 7 and 10 wt% of 3ZT coupled oxide. Each mixture was prepared separately. Finally, the mixture solution was poured into 90 mm diameter petri dish and oven dried at 80 °C for 18 h. Then, the mixture was allowed to continuous stir for 1 min to allow homogeneous mixing.
The solution mixture was then poured into a 90 mm diameter petri dish and allowed to oven dry for 18 h [25,26]. The procedure was repeated for the rest of wt.% of 3ZT coupled oxide.

Characterization
The melting temperature (T m ) and the enthalpy of fusion (∆H f ) were determined using differential scanning calorimetry (DSC,   Later, the colony forming unit (CFU) of each plate were determined.

Bacterial Strains, Media and Materials
Colonies were counted and compared to those on control plates to measure changes in the cell growth inhibition. The bare LLDPE nanocomposite and bacterial inoculum only served as control.
The percentage of bacteria reduction (R %) was calculated using the following equation: where R is antibacterial rate (%), B are the average number of cell colony of blank sample (CFU/sample) and A are the average number of colony of treated sample (CFU/sample) at specified contact time.  The FTIR spectra of 3ZT coupled oxide, bare LLDPE and LLDPE nanocomposites with 1-10 wt.% 3ZT coupled oxide are is shown in Figure 1. This results also provide insights on the reactive candidates that are liable for microbe inactivation. Acetonitrile (ACN), methanol (MeOH) and benzoquinone (BQ) were used as scavengers for hydroxyl radicals, photo-generated holes and superoxide anion radicals, respectively. As seen in Figure 5, a positive correlation was found between MB degradation and wt% of 3ZT coupled oxide.

Results and Discussion
With the addition of the scavengers, the photo-degradation of MB 6607 decelerated drastically with wt% of catalyst, which means more hydroxyl radicals, photogenerated holes and superoxide anion radicals are produced and active during the degradation process.
Upon the introduction of ACN, the photodegradation efficiency of LLDPE with 1wt% to 10wt% 3ZT are decelerated drastically by 32% to 75%, respectively (Table 2).      during incubation period will cause cell membrane destructions [33]. Therefore, the Zn ion released from LLDPE composites is analyzed by measuring the dynamic zinc ion by ICP method and displayed in Figure 6.     Apart from that, a superior bacteriostatic effect was found in LLDPE nanocomposite with high percentage of 3ZT (7 and 10wt%) and the reason is due to the relatively low crystallinity and high polarity induced by the hydrophilic characteristic of 3ZT coupled oxides in LLDPE nanocomposite. Such characteristic expedite water uptake through LLDPE matrix, hence promotes high photocatalytic activity to release reactive species as well as Zn ion migration.
From the above observation, the mechanism for the S. aureus and E. coli inactivation of LLDPE nanocomposites with different weight percentage of 3ZT is illustrated in Figure 8.