Eco-friendly method for synthesis of zeolitic imidazolate framework 8 decorated graphene oxide for antibacterial activity enhancement

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Eco-friendly method for synthesis of zeolitic imidazolate framework 8 decorated graphene oxide for antibacterial activity enhancement Nazerah Ahmad a,b , Nik Abdul Hadi Md Nordin c , Juhana Jaafar a , Nik Ahmad Nizam Nik Malek e , Ahmad Fauzi Ismail a,∗ , Muhammad Nabil Fikri Yahya d , Siti Aishah Mohd Hanim e , Mohd Sohaimi Abdullah a a

Advanced Membrane Technology Research Centre, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia Malaysian Institute of Chemical Engineering Technology-Universiti Kuala Lumpur, Lot 1988, Bandar Vendor, Taboh Naning, 78000 Alor Gajah, Melaka, Malaysia c Chemical Engineering Department, Universiti Teknologi PETRONAS, 32610 Seri Iskandar, Perak, Malaysia d Centre for Student Innovation and Technology Entrepreneurship, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia e Department of Biosciences, Faculty of Science, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia b

a r t i c l e

i n f o

Article history: Received 14 January 2019 Received in revised form 11 March 2019 Accepted 13 April 2019 Available online xxx Keywords: Graphene oxide Metal–organic framework Hybrid nanocomposite Zeolitic imidazolate framework Antibacterial agent

a b s t r a c t We report a rapid method for synthesis of zeolitic imidazolate framework 8 (ZIF-8)-decorated graphene oxide (GO) composites (ZGO) with good antibacterial properties. The ZGO composites were synthesized at room temperature with low GO to metal salt ratios. The samples were characterized by X-ray diffraction, transmission electron microscopy, Fourier transform infrared spectroscopy, thermal gravimetric analysis, and surface area analysis. The characterization results show that ZIF-8 with a size of approximately 120 nm is successfully decorated on the surface of GO sheets with the host ZIF-8 framework maintained in the synthesized composite, but there is a significant reduction in the Brunauer–Emmett–Teller surface area. The antibacterial activities of the samples against Escherichia coli ATCC 11229 and Staphylococcus aureus ATCC 6538 as model strains of gram-negative and -positive bacteria, respectively, were determined by disc diffusion and minimum inhibitory concentration (MIC) tests. ZGO-1.0 (1 wt% of ratio of GO to metal salt) shows the highest antibacterial activity with MIC values required to inhibit bacterial growth of E. coli and S. aureus of 5 times lower than those of pristine ZIF-8. Different antibacterial mechanisms are proposed based on field-emission scanning electron microscope images of the two bacteria after contact with the synthesized composite. Overall, owing to the simple synthesis, good stability, low chemical usage, and excellent antibacterial activity of the ZGO composites, they show great potential for application in the field of microbial contamination control. © 2019 Chinese Society of Particuology and Institute of Process Engineering, Chinese Academy of Sciences. Published by Elsevier B.V. All rights reserved.

Introduction Metal–organic frameworks (MOFs) have recently shown great potential for various applications, and they have emerged as important materials for catalytic application (Ke, Wang, & Zhu, 2015), gas storage (Ma & Zhou, 2010), adsorption (Petit, Levasseur, Mendoza, & Bandosz, 2012), and gas separation (Rodenas et al., 2015). MOFs are crystalline compounds consisting of metal ions and secondary building units known as organic ligands. The interesting characteristics of MOFs, such as their high micropore volume, large pore

∗ Corresponding author. E-mail address: [email protected] (A.F. Ismail).

sizes, high phase crystallinity, and high metal content, provide valuable active sites for the key features of this new emerging class of porous materials (Butova, Soldatov, Guda, Lomachenko, & Lamberti, 2016). The large surface area of MOFs has advantages over other porous materials like activated carbon and zeolite, resulting in more contact with the target species and thus increasing the effectiveness of the particles. The antibacterial properties of MOFs have recently received attention and been reported in several studies (Alavijeh, Beheshti, Akhbari, & Morsali, 2018; Berchel et al., 2011). The presence of inorganic and organic components in the MOF structure makes them promising antibacterial agents because it provides a platform to generate high antibacterial activity (Horcajada et al., 2008). More importantly, their threedimensional structure can act as a reservoir for metal ions, stabilize

https://doi.org/10.1016/j.partic.2019.04.007 1674-2001/© 2019 Chinese Society of Particuology and Institute of Process Engineering, Chinese Academy of Sciences. Published by Elsevier B.V. All rights reserved.

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release of the metal ions, and result in long-term antibacterial activity. These properties of MOFs mean that they show promise as antibacterial agents in water treatment and biomedical applications (Prince et al., 2014; Quirós et al., 2015). Another material that has recently been extensively investigated is graphene oxide (GO). GO has a layered structure with multiple oxygen-containing groups, such as carboxyl groups on the edges and epoxide and hydroxyl groups on the basal planes (Li, Jiang, Zhao, & Zhang, 2015). These functional groups provide sites for chemical reactions that lead to GO modification. Owing to its unique structure, GO has been extensively used in preparation of composite materials with promising adsorptive properties (Petit & Bandosz, 2015) and good antibacterial properties, which are prerequisites for biomedical applications (Li et al., 2015). The antibacterial properties of GO originate from the physical and chemical interactions between GO and bacterial cell membranes (Szunerits & Boukherroub, 2016). Several studies have demonstrated the strong antimicrobial properties of GO against a wide variety of microorganisms, including gram-positive and -negative bacterial pathogens, phytopathogens, and biofilmforming microorganisms (Bao et al., 2014; Perreault, De Faria, Nejati, & Elimelech, 2015; Rojas-Andrade et al., 2017). Combining MOFs with GO is a promising technique for improving the performance in various applications, such as lithium ion batteries (Bao et al., 2014; Yang, Tang, & Wu, 2015), gas adsorption (Bian et al., 2014; Lin, Ge, Liu, Rudolph, & Zhu, 2015), supercapacitors (Srimuk, Luanwuthi, Krittayavathananon, & Sawangphruk, 2015), and biosensors (Song et al., 2015). The drawback of synthesizing MOF/GO composites by common methods, like the solvothermal process, is that it requires time and energy because of dissolution in an organic solvent at elevated temperature for 2–24 h. For example, Kumar, Jayaramulu, Maji, and Rao (2013) successfully synthesized a MOF/GO composite using methanolic solution at room temperature. However, because their method requires a relatively expensive solvent (methanol), using an aqueous system at room temperature would reduce the synthesis cost and improve the process for wider application. Modification of MOFs for enhanced CO2 separation properties has been intensively investigated (Nordin et al., 2014; Wang et al., 2016), but their potential as antibacterial agents has not been widely investigated. Recently, Wang et al. (2016) reported that the synergistic effect of ZIF-8 and GO in an aqueous system results in effective antibacterial activity. However, they did not investigate the effect of the GO content on the overall antibacterial activity. Nordin et al. (2014) recently proposed an eco-friendly method for synthesis of ZIF-8 to reduce the use of toxic chemicals and improve the reaction rate and product yield. In this study, we used a similar method to functionalize ZIF-8 using GO and the effect of the GO to metal salt ratio on the antibacterial activity was investigated. A variety of characterization techniques were used to investigate the structural, morphological, and antibacterial properties. Mechanisms of the antibacterial activity of the nanocomposite are proposed. The findings of this research are expected to promote research on development of MOF/GO composites.

Synthesis of the ZIF-8/GO composites The ZIF-8/GO (ZGO) composites were prepared by modifying the ZIF-8 synthesis procedure described in our previous study (Nordin et al., 2014). The average size of GO used in this study was 758.3 nm with a C/O ratio of 2.74. In brief, a metal salt solution was prepared by dissolving Zn(NO3 )2 ·6H2 O (2 g, 6.72 mmol/L) in deionized water (12 g). For the ligand solution, 2-MeIM (3.312 g, 40.34 mmol/L) was dissolved in deionized water (48.56 g) and then TEA (3 mL) was added. A predetermined amount of GO sheets based on the mass of the Zn salt (see Table 1) was added to the ligand solution and sonicated for 30 min to break the GO sheets. The actual amount of GO in the composite was calculated based on elemental analysis (energy dispersive X-ray (EDX) analysis). The ligand suspension was stirred for 2 h to allow better dispersion of GO. Zn salt solution was gently added to the ligand suspension, resulting in an almost instantaneously cloudy solution. The solution was vigorously stirred for 30 min, followed by centrifugation of the reaction product. The obtained ZGO product was washed several times with deionized water to remove excess reactants and then dried in an oven at 60 ◦ C for 12 h. The collected powder was ground into fine particles before drying in an oven at 100 ◦ C for a minimum of 12 h to evacuate the guest molecules from the composite pores. The pure ZIF-8 phase was prepared by a similar procedure but without the GO sheet dispersion steps. Characterization X-ray diffraction (XRD) analysis was performed to determine the crystallinities of pristine ZIF-8 and the ZGO composites using a diffractometer (D5000, Siemens, Germany) with CuK␣ radiation ( = 1.54 Å at room temperature). The functional groups of the ZGO composites were determined by Fourier transform infrared (FTIR) spectroscopy using a Nicolet IS5-IR spectrometer (Thermo Scientific, USA). The FTIR spectra were recorded in the wavelength range 400–4000 cm−1 using pressed KBr pellets. The specific surface areas of pristine ZIF-8 and the ZGO composites were determined with a ASAP 2010 gas adsorption analyzer (Micromeritics, USA) at −196 ◦ C after degassing for 24 h at 150 ◦ C under vacuum at 1 × 10−3 Torr. The Brunauer–Emmett–Teller (BET) surface area was calculated by the BET equation based on the obtained adsorption isotherm (Kobayashi, Hiroishi, Tokunoh, & Saegusa, 1987; Zhao et al., 2018). The total pore volume and micropore volume were measured based on the amount of nitrogen adsorbed at relative pressure P/P◦ = 0.99 by converting the adsorbed amount to the volume of the liquid adsorbate and by a t-plot, respectively. To observe the microstructures of the ZGO composites, transmission electron microscope microscopy (TEM; JSM-6701 F, JEOL, Japan) was performed at 200 kV. For this analysis, the samples were prepared by dispersing the ZGO sample powder in methanol and then dropping the suspension on a carbon-coated copper grid. Thermogravimetric analysis (TGA) was performed to determine the thermal stabilities of the composites. The samples were heated from 50 to 900 ◦ C at a heating rate of 10 ◦ C/min under a nitrogen atmosphere with a nitrogen flow rate of 20 mL/min.

Experimental

Antibacterial activity

Materials

The antibacterial activities of the synthesized materials against gram-negative Escherichia coli (E. coli) ATCC 11229 and grampositive Staphylococcus Aureus (S. Aureus) ATCC 6538 were determined by disc diffusion tests (DDTs). These bacterial species were selected because they are considered to be a great threat to human health and can possibly cause multiple human diseases, such as skin, bone, joint, and respiratory infections (Chase-Topping et al., 2012; Kobayashi et al., 1987). Before the test, 0.1 g of the pow-

Zinc nitrate hexahydrate (Zn(NO3 )2 ·6H2 O), 2-methylimidazole (2-MeIM), triethylamine (TEA), and GO sheets were purchased from Sigma Aldrich, St Louis,USA. Mueller–Hinton agar (MHA) and Luria–Bertani (LB) broth were purchased from Merck Chemicals, Darmstadt, Germany. All of the chemicals were used without further purification.

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Table 1 Amounts of GO used to prepare the ZGO composites. Amount of GO loading (g)

Actual amount of GO(g)-extracted from EDX

Ratio of GO to Zn(NO3 )2 (%)

Sample name

0.00 0.01 0.02 0.06 0.10

0.0000 0.0098 0.0194 0.0580 0.0967

0 0.5 1.0 3.0 5.0

ZIF-8 ZGO-0.5 ZGO-1.0 ZGO-3.0 ZGO-5.0

Fig. 1. XRD patterns of the synthesized ZIF-8 and ZGO samples. (b) High magnification of the (211) peaks for the ZGO-5.0 and ZIF-8 samples.

der sample was pressed into a round disc with a diameter of 1.20 cm by an E-Z Press (International Crystal Laboratories). The bacterial suspension was prepared by inoculating a single colony of the bacteria in sterile saline at a solution concentration of 9 mg/mL, and its turbidity was adjusted based on the turbidity of 0.5 McFarland standard (1.5 × 108 CFU/mL). The bacterial suspension was then uniformly swabbed onto MHA plates using sterile cotton swabs. The previously prepared sample discs were then placed on the surface of the MHA agar plates. The plates were incubated overnight at 37 ◦ C and then the clear inhibition zones formed around the discs were measured and images of the plates were taken. Minimum inhibitory concentration (MIC) tests of pristine ZIF-8 and the ZGO composites against E. coli and S. aureus were performed to determine the lowest concentrations of the synthesized samples that inhibit bacterial growth. The bacterial suspension was first prepared by adding a single colony of the bacteria into LB broth before incubating overnight at 200 rpm and 37 ◦ C. Fresh LB medium (100 mL) was then inoculated with the bacterial culture (1 mL) and incubated in an incubator shaker at 100 rpm until the optical density reached 0.6 at 550nm . Finally, the bacterial cells were harvested by centrifugation at 1643 xg(relative centrifugal force) for 15 min, washed twice with distilled water, and finally suspended in distilled water (100 mL). A predetermined amount of ZIF-8 or the ZGO composite sample was then added into the bacterial suspension, in which the concentration of the sample in the bacterial suspension varied from 0.5 to 10.0 g/L. The sample mixture was shaken in an incubator shaker at 100 rpm for 30 min and then 10 ␮L of the bacterial suspension was dropped onto a MHA plate using the dropped plate method (Nik Malek, Williams, Dhanabal, Bhall, & Ibrahim, 2014). The plates were incubated overnight at 37 ◦ C and then the MIC values of pristine ZIF-8 and the ZGO composites were determined. Results and discussion Characterization of the ZGO composites XRD was performed to identify the phase changes of ZIF-8 after incorporation of GO, and the XRD patterns are shown in Fig. 1(a).

The strong peaks at 2 = 7.30◦ , 10.35◦ , 12.70◦ , 14.80◦ , 16.40◦ , and 18.00◦ , which correspond to the (110), (200), (211), (220), (310), and (222) Miller indices, respectively, confirm the polycrystalline structure of ZIF-8, which is in good agreement with a previous experimental study (Gross, Sherman, & Vajo, 2012). The similar XRD patterns for the prepared ZGO composites with different GO contents reveal formation of highly crystalline ZGO composites without any significant changes of the ZIF-8 structure. The small shoulder peak at 2 = 11.04◦ for 1 and 3 wt% GO content (of the metal salt weight) corresponds to the (101) plane of GO, which is because of decoration of ZIF-8 with a thin layer of GO. However, for lower (0.5 wt%) and higher GO content (5 wt%), the (101) peak is not observed. It is presumed that low GO content provides limited active sites to interact with ZIF-8 and only a small portion of the ZGO composite forms. Therefore, most of the X-ray reflection of ZGO originates from the ZIF-8 planes and there are no peaks of GO. For high GO content (5 wt%), the absence of the (101) signal can be attributed to agglomeration of the GO layers, and GO may be present at a specific point that cannot be detected by XRD. Moreover, the unit cell parameters were determined for the two samples ZIF-8 and ZGO-5.0 using the (211) peak (Fig. 1(b)). ◦ originates from decoration of ZIF-8 with GO, ˜ The peak shift of 0.06 which causes a decrease in the lattice parameter (see the insert table in Fig. 1(b)). TEM images of the morphologies and microstructures of the synthesized nanocomposites with different GO contents are shown in Fig. 2(a). For comparison, TEM images of ZIF-8 and GO are also included. The presence of GO and ZIF-8 in the synthesized composites was confirmed by field-emission scanning electron microscopy (FESEM)–EDX elemental mapping (Fig. 2(b)). Pristine ZIF-8 has a rhombic dodecahedron shape within nanoscale size. For low GO content (0.5 wt%), ZIF-8 in deposited on the GO surfaces and the shape remains rhombic dodecahedron, similar to that of pristine ZIF-8. This may result from growth of ZIF-8 nanocrystals starting from the edge of the GO sheets (Zhou, Zhang, Ji, Yuan, & Shen, 2016). For a moderate GO ratio, the shape of the ZIF-8 particles slightly changes and a few irregular rhombic dodecahedra are observed in the micrograph. This is because of the affinity of the oxygen-containing functional groups of GO, especially epoxy or

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Fig. 2. (a) TEM images of the morphologies of synthesized ZIF-8, commercial GO, and the ZGO composites with different GO contents with respect to ZIF-8. (b) FESEM-EDX maps of the ZGO-1.0 sample.

hydroxyl groups from the basal plane, for Zn2+ ions, which provides more nucleation sites for ZIF-8 crystal growth, as shown in Fig. 3(a). There are also higher percentages of C and O for ZGO-1.0, indicating that GO exists in the synthesized composite based on FESEM-EDX elemental mapping, as shown in Fig. 2(b). As the GO content increases, the morphology of ZGO is similar to ZGO with low GO content. This is in agreement with XRD analysis, where a peak at 2 = 11.04◦ is present for GO contents of 1 and 3 wt%. This is probably because of exfoliation of the GO lay-

ers, thereby causing notable changes in the XRD peaks without significant changes in the morphology, as shown in Fig. 3(b). However, when the GO content further increases to 5 wt%, noticeable aggregation is observed for ZGO-5.0. This is probably caused by the tendency of GO sheets to restack and agglomerate, as shown schematically in Fig. 3(c). It is clear that the GO content has a significant effect on the morphology and structure of the synthesized composite. This result is in good agreement with the XRD observations.

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Fig. 3. Schematics of (a) the concept of ZGO prepared in this study, (b) exfoliation of the GO layers by ZIF-8 formed at the interlayers of the GO sheets, and (c) restacking of the GO layers at high GO content.

Table 2 Surface textural properties of synthesized ZIF-8, commercial GO, and ZGO composites with different GO contents with respect to ZIF-8. Sample

GO content (g)

BET surface area (m2 /g)

ZIF-8 ZGO-0.5 ZGO-1.0 ZGO-3.0 ZGO-5.0 Pure GO

– 0.01 0.02 0.06 0.10

715.54 675.31 772.68 606.74 632.98 253.06

The textural parameters, such as the surface area and total pore volume, of pristine ZIF-8 and the ZGO composites were determined by N2 adsorption analysis, and the results are summarized in Table 2. Pristine ZIF-8 has a BET surface area of 715.54 m2 /g. After incorporation of GO, the surface area decreases for all the composites with the exception of ZGO-1.0. This low BET surface area could be related to incomplete solvent removal owing to insufficient activation (100 ◦ C for 12 h) (Kaye, Dailly, Yaghi, & Long, 2007). Incomplete solvent removal means that guest molecules remain in the pores and/or dense by-product because the framework is sensitive to moisture and/or oxygen (Pan, Liu, Zeng, Zhao, & Lai, 2011). The increase of the surface area for ZGO-1.0 can be explained by generation of new pores at the interface between the GO layers and the ZIF-8 structure. Similar findings have been reported by Petit and Bandosz (2015). However, for higher GO content, there is a significant decrease in the BET surface areas for ZGO-3.0 and ZGO-5.0. The decrease in the surface area can be attributed to the large amounts of GO in the composites, which tends to lead to distortion of the ZIF-8 structure (Kumar et al., 2013). This result is in agreement with the results reported by Petit and Bandosz (2011), who modified the mesopores of the MIL-100 (Fe) MOF using GO. The FTIR spectra of pristine ZIF-8 and the ZGO composites are shown in Fig. 4(a). The distinctive features of GO in the FTIR spectra are the major stretching vibrations of O H (3460 cm−1 ), C O

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(1622 cm−1 ), C C (1167 cm−1 ), and C O and C O C (1051 cm−1 ) (Lai et al., 2016). Most of the FTIR spectra of the ZGO composites are consistent with ZIF-8 and are related to the vibrations of the methylimidazole unit. The peaks at 3135 and 2929 cm−1 originate from the stretching modes of the aromatic and aliphatic C H bonds of methylimidazole. The peaks in the range 1350–1500 cm−1 are attributed to complete ring stretching and the peak at 1584 cm−1 is attributed to C N stretching (Hu, Kazemian, Rohani, Huang, & Song, 2011). Additionally, the peaks between 1200 and 1300 cm−1 are in-plane bending of the ring, whereas those below 800 cm−1 are attributed to out-of-plane bending. The peak between 980 and 1010 cm−1 corresponds to combination of the C O bonds of GO and in-plane bending of the ZIF-8 ring, as shown in Fig. 4(b). Increasing the amount of GO leads to the structure converting from staircaselike to a multilayer configuration, which reduces the active surface area of GO and decreases the C O stretching vibration. This phenomenon decreases the peak intensity, as shown in Fig. 4(b). For GO, the absorption band at about 3430 cm−1 can be ascribed to the −OH groups, and the corresponding small peak for ZIF-8 is moisture from the synthesis process. The absence of this signal for the other samples is because of an insufficient amount of GO and formation of O–metal bonds on the surfaces of the GO sheets. TGA analysis was performed to investigate the thermal stabilities of the ZGO composites. The TGA curves of GO, ZIF-8, and the ZGO composites with different GO contents are shown in Fig. 5. There is slight weight loss in the temperature range 30–150 ◦ C for all of the samples, which is mainly because of vaporization of physically adsorbed water on the surfaces of the nanocomposites (Martin-Betancor et al., 2017; Wang et al., 2016). There is also a clear weight loss step of over 80% for pristine ZIF-8 and the ZGO composites in the temperature range 220–800 ◦ C. This is because of decomposition of the organic linker molecules, leading to ZIF-8 collapse (Nguyen, Le, Truong, & Phan, 2012), and decomposition of GO in the ZGO hybrid nanosheets. The same trend was observed in a previous study (Wang et al., 2016). There is gradual weight loss from 220 to 800 ◦ C, of which approximately 20% is attributed to decomposition of GO.

Antibacterial activity The antibacterial activities of the ZGO nanocomposites with various GO contents against the two representative bacteria E. coli ATCC 11229 and S. aureus ATCC 6538 were determined by DDTs and MIC tests. Images of the inhibition zones and the inhibition zone diameters of pristine ZIF-8 and the ZGO composites with different GO contents are shown in Fig. 6. Pristine ZIF-8 shows antibacterial activity against both bacteria. The antibacterial effect of ZIF-8 might be because of the existence of Zn in the structure of ZIF8 (Martin-Betancor et al., 2017). With the presence of GO in the structure, the ZGO samples show significant inhibition abilities for both bacteria. The enhanced antibacterial effect can be explained based on the synergistic effect of Zn in the ZIF-8 structure and the sharp edges of the GO nanosheets when in direct contact with ˜ bacterial cells (Maillard, 2002; Munoz-Bonilla & Fernández-García, 2012; Sirelkhatim et al., 2015). Among the synthesized samples, the ZGO-1.0 sample has the largest inhibition zone diameters for both bacteria. This is because of two main reasons: (1) its staircase-like structure with a larger number of sharp edges of GO, which damage the cell walls and increase the death rate compared with the other samples, and (2) the larger surface area of ZGO-1.0 (see the BET results in Table 2). The larger surface area provides more available active sites and increases the bactericidal activity, because the composite material strongly adheres to the bacterial cells. The inhibition zone diameter decreases when the GO content is higher than 1 wt%. This weak antibacterial performance is related to restack-

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Fig. 4. (a) FTIR spectra of pristine ZIF-8 and the ZGO composites with different GO contents. (b) High magnification of the dashed region in (a) (C O bond).

Fig. 5. TGA curves of ZIF-8, GO, and the ZGO composites.

ing of the GO nanosheets (Fig. 3(c)). This decreases the number of Zn2+ ions on the surface, which prevents surface contact with the bacterial membrane and decreases the killing rate. Both antibacterial assays show that the antibacterial effect of the ZGO composite is more pronounced against S. aureus (gram-positive bacteria) than E. coli (gram-negative bacteria). This is possibly because of the simpler cell membrane structure of gram-positive bacteria compared with gram-negative bacteria (Sirelkhatim et al., 2015). Even though gram-positive bacteria have a thicker peptidoglycan layer, the loosely packed structure of this layer may not assist in protection against biocide (antibacte˜ rial) attack (Munoz-Bonilla & Fernández-García, 2012). In addition, the absence of a phospholipid bilayer structure in gram-positive bacteria exposes their cytoplasmic membrane, so they are easily damaged (Maillard, 2002). Because the DDT results only represent the magnitude of the susceptibility to the bacteria, MIC tests were performed to further investigate the antibacterial activities of the ZGO composites, and the results are summarized in Table 3. A lower MIC value corresponds to higher antibacterial effectiveness. The concentration of ZGO-1.0 required to inhibit bacterial growth is much lower than those of the other samples, and only 2.0 g/L is required to completely kill E. coli and S. aureus. Similar to the DDTs, ZGO with higher GO content shows lower antibacterial effectiveness, and thus a significantly higher concentration is required to inhibit bacterial growth. The results obtained from the MIC tests confirm the

Fig. 6. Inhibition zone diameters of the prepared samples against E. coli and S. aureus and images of the halo zones.

trend observed in the DDTs. Modification of ZIF-8 with a moderate amount of GO results in an excellent synergistic effect, which results in high antibacterial effectiveness for the tested bacteria. Both the DDTs and MIC tests reveal that the antibacterial activity of ZGO-5.0 is similar to that of pristine ZIF-8. This is related to distortion of the MOF crystal when ZIF-8 is decorated with a high content of GO (Hafizovic et al., 2007), which reduces the surface area of the composite (Table 2). The smaller surface area of the composite provides fewer available active sites for bactericidal activity, because it reduces the surface contact between bacterial cells and the composite. Therefore, our hypothesis of restacking of GO at high GO content (Fig. 3(c)) is confirmed.

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Fig. 7. Morphologies of the bacteria before and after exposure to the ZGO nanocomposite: (A) E. coli and (B) S. aureus.

Table 3 MIC values of the synthesized composites against E. coli and S. aureus in distilled water. Sample

ZIF-8 ZGO-0.5 ZGO-1.0 ZGO-3.0 ZGO-5.0

MIC values (g/L) E. coli

S. aureus

>10.0 7.0 2.0 7.0 >10.0

>10.0 5.0 2.0 5.0 >10.0

Mechanisms of the bactericidal effects of the ZGO composites Based on the results of the antibacterial assays, the ZGO composites exhibit strong antibacterial activity against E. coli and S. aureus. However, the exact mechanism of the bactericidal effect of the ZGO composites is still unknown. A number of mechanisms are proposed to understand the bactericidal action of the ZGO composites. It is suggested that release of Zn metal ions (Zn2+ ) from ZIF-8 and direct interaction of ZGO with the bacterial cell wall are responsible for the bactericidal action of the ZGO composites. It is essential to determine the mechanism of the bactericidal effect of ZGO based on the morphology changes of the bacteria via FESEM images. Images of the bacteria before and after exposure to the ZGO1.0 composite are shown in Fig. 7. Based on the changes of the morphologies of both bacteria, the difference in the susceptibility between E. coli and S. aureus can be attributed to the different interaction mechanisms of the composite sample with the bacterial cells (Azam et al., 2012). It is proposed that the synergistic effect of ZIF8 and GO could contribute to the antibacterial activity of ZGO. The capability of ZIF-8 as an antibacterial agent results from the presence of Zn metal ions in its structure (Wyszogrodzka, Marszałek, ˙ nski, ´ Gil, & Dorozy 2016). In addition, according to studies by Ji, Sun, and Qu (2016) and Akhavan and Ghaderi (2010), the sharp edges of GO are a crucial element of the action of GO in causing physical damage to the bacterial membranes. The characteristic cell shape of untreated gram-negative E. coli can be represented as a rod with an intact normal morpholog-

ical structure (Fig. 7(A)) (Furchtgott, Wingreen, & Huang, 2011). However, after being exposed to the ZGO composite for 24 h, significant morphological changes are observed in the E. coli cells. Most of the bacteria change from rod-shaped to a globular shape with substantial damage to the membrane integrity (Fig. 7(A1)). It is proposed that the Zn2+ ions released from ZIF-8 are the main contributors to the substantial damage of the E. coli cells. A similar mechanism has been suggested by Li, Liu et al. (2008); Li, Mahendra et al., 2008, who investigated the toxicity of ZnO towards E. coli. In brief, the Zn2+ ions damage the outer cell membrane structure and penetrate the intracellular content, which leads to an osmotic imbalance and causes bacterial death. Normal S. aureus cells are shown in Fig. 7(B). After being treated with the ZGO composite, the ZGO composite adheres to the bacterial cell membrane, but there are no significant changes in the S. aureus morphology (Fig. 7(B)). Another possible mechanism of the antibacterial activity of ZGO is direct interaction of the ZGO composite with the cell membrane. As mentioned by Esmailzadeh, Sangpour, Shahraz, Hejazi, and Khaksar (2016), positively charge ions like Zn tend to attach to the surface of gram-positive bacteria because their surface has a negative charge. In this study, the tight contact between the GO sheets and bacteria cells may enable the Zn ions of the composites to tightly adhere to the S. aureus cell membrane, thereby inducing rupture of the bacterial cell. Heinlaan, Ivask, Blinova, Dubourguier, and Kahru (2008) reported that binding of ZnO nanoparticles on the surface of bacteria by electrostatic forces directly damages the cell wall, which eventually causes bacterial death. It is likely that the ZGO composites affect the cell walls and membranes of both types of bacteria, destroying the cell membrane or wall integrity (Wang et al., 2014).

Conclusions ZGO composites with low GO to metal salt ratios have been successfully synthesized at room temperature. FTIR and XRD characterization confirm the composites and crystal structure of ZIF-8. The morphology of the composite determined by TEM indicates the presence of ZIF-8 decorated on the GO nanosheets. All of the samples show outstanding antibacterial activity against E. coli and S. aureus. ZGO-1.0 shows the best antibacterial activity with a

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