Materials Science and Engineering C 35 (2014) 115–121
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Incredible antibacterial activity of noble metal functionalized magnetic core–zeolitic shell nanostructures M. Padervand a, S. Janatrostami b, A. Kiani Karanji a, M.R. Gholami a,⁎ a b
Department of Chemistry, Sharif University of Technology, Azadi Ave., Tehran, Iran Department of Water Engineering, Tehran University, Karaj, Iran
a r t i c l e
i n f o
Article history: Received 20 April 2013 Received in revised form 4 September 2013 Accepted 24 October 2013 Available online 5 November 2013 Keywords: Magnetic core–zeolitic shell nanostructure Antibacterial activity Nickel ferrite Mordenite E. coli
a b s t r a c t Functionalized magnetic core–zeolitic shell nanostructures were prepared by hydrothermal and coprecipitation methods. The products were characterized by Vibrating Sample Magnetometer (VSM), X-ray powder diffraction (XRD), Fourier Transform Infrared (FTIR) spectra, nitrogen adsorption–desorption isotherms, and Transmission Electron Microscopy (TEM). The growth of mordenite nanoparticles on the surface of silica coated nickel ferrite nanoparticles in the presence of organic templates was also confirmed. Antibacterial activity of the prepared nanostructures was investigated by the inactivation of Escherichia coli as a gram negative bacterium. A new mechanism was proposed for inactivation of E. coli over the prepared samples. In addition, the Minimum Inhibitory Concentration (MIC) and reuse ability were studied. TEM images of the destroyed cell wall after the treatment time were performed to illustrate the inactivation mechanism. According to the experimental results, the core–shell nanostructures which were modified by organic agents and then functionalized with noble metal nanoparticles were the most active. The interaction of the noble metals with the organic components on the surface of nanostructures was studied theoretically and the obtained results were used to interpret the experimental results. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The inactivation of microorganisms is an ongoing issue in the food industry, medical field, and environment. Several inactivation methods, such as pasteurization, chlorination, ozonation, and ultraviolet and ultrasound irradiation, have been proposed and utilized over the past years. In addition, infection control continues to be a growing concern in medical and healthcare facilities. According to the U.S. National Centers for Disease Control and Prevention (CDC), the risk of acquiring a serious infection while being treated in a hospital has increased over 35% in the past two decades. A study found that nearly 2 million American patients contract hospital-acquired infections per year. Of these patients, nearly 90,000 die as a result [1]. The need for infection control guidelines and prevention methods is essential for all the healthcare facilities. Another scenario that shares this common concern is the occurrence of implant-associated infections. Therefore, various types of modified surfaces, which are often coated or integrated in antibiotics or metal ions, have been proposed to disrupt the colonization of bacteria [2,3]. Even though the biocompatibility of most of these antimicrobial
⁎ Corresponding author at: Azadi Ave. P.O. Box 11365-9516, Tehran, Iran. Tel.: +98 2166165314; fax: +98 2166029165. E-mail address: [email protected] (M.R. Gholami). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.10.027
surfaces remains unknown, research activities on new bactericidal materials or devices never cease. Zeolites have a large applicability for decontamination, purification of urban and industrial residual waters, protection of waste disposal areas, purification of industrial gases, etc. Many researchers have studied the usage of natural and synthetic zeolites for removal of heavy metal ions in aqueous mediums [4–6]. Properties such as high surface area, individual micro-pores, variety of channels and high resistance make zeolites very useful for industrial applications and academic researches [6,7]. The combination of zeolitic materials with the magnetic or/and active functional groups to form core–shell structured composites is undoubtedly of special interest in diagnostic analysis [8], bioseparation [9], and controlled drug release [10–12], due to their unique magnetic responsivity, low cytotoxicity, good biocompatibility and mesoporous properties. Over the past decade, the preparation of multifunctional microspheres containing magnetite core with a mesoporous shell has been reported [13–20]. The uniform-sized magnetic particles were normally prepared via a high temperature decomposition method [17], or hydrothermal process [15]. The formation of core–shell nanocomposites is conventionally followed by an encapsulation procedure, where the magnetite core is encapsulated by a silica layer using a sol–gel technique [21]. The aim of the present study was to investigate the antibacterial activity of prepared noble metal functionalized magnetic core–zeolitic shell nanostructures. The effect of initial cell concentration, composite
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dosage and reuse capacity was investigated. In addition, the interaction of used noble metal with the nanostructure's surface was studied theoretically and a new mechanism was proposed for bacterium inactivation over the prepared samples. 2. Experimental 2.1. Materials and instruments Chemicals include NaOH, tetramethylammonium hydroxide (TMA OH) (10% w/w), ethylene glycol (EG), cyclohexane, cyclohexanol, C14H22O(C2H4O)n (Triton X-100), 3-glycidoxypropyltrimethoxysilane (GPTS), and glutamic acid (GLU) and the various salts of polyvalent metals were used as required. Tetraethyl orthosilicate (TEOS) was used as silica source. All chemicals were purchased from Merck Chemical Co. (Germany) and were used without any further purification. The XRD patterns of the prepared samples were recorded on a Bruker D8 Advance X-ray diffractometer with CuKα irradiation (λ = 0.15406 nm). The FTIR spectra were recorded using an NB series spectrometer. The specific surface area of the nanostructures was calculated from the N2 adsorption–desorption isotherms at 77 K, using Belsorp apparatus (Japan). The average particle size and morphology of the samples were examined by TEM analysis. Magnetic studies were carried out on a “Meghnatis Daghigh Kavir Co.” at 300 K.
water and dried at 90 °C for 10 h. Finally, the solid powder was calcined at 500 °C for 5 h and denoted MOR/S/NF. 2.2.4. Surface functionalization with GPTS–GLU MOR/S/NF samples were modified with GPTS and GLU according to the earlier work with some modifications [24]. GLU was dissolved in double distilled water, and the obtained suspension was adjusted to pH 11 with NaOH 10 M. The suspension was transferred into a flask bottle placed in the ice-bath, and GPTS was slowly added while being stirred. The mixed solution was heated to 65 °C for 2 h by stirring. After adjusting the pH to 6 with concentrated HCl, an aqueous suspension of MOR/S/NF was added and stirred for 12 h. The resultant product was separated with the help of a permanent magnet, washed thoroughly with distilled water, dried at 80 °C for 6 h and denoted GPTS–GLU@ MOR/S/NF. 2.2.5. Au and Ag loading on the surface 0.5 g of prepared solid in the earlier section was added to an aqueous solution of H2AuCl4·4H2O and AgNO3, respectively. The suspensions were sonicated for 15 min and then stirred at room temperature for 2 h. After completion of the reaction, the products were separated by an external magnet, were washed repeatedly with double distilled water and then dried at 80 °C overnight. The products were denoted as X/GPTS–GLU@MOR/S/NF (X = Au or Ag). The total formation process of noble metal modified nanostructures is shown in Scheme 1.
2.2. Nanostructure preparation 2.2.1. Synthesis of nickel ferrite (NF) nanoparticles The nanostructures were prepared according to our earlier work with some modifications [22]. NF nanoparticles were prepared via hydrothermal method in a 200 mL stainless steel autoclave with a Teflon liner under autogenous pressure. A transparent solution containing 2.9 g Ni(NO3)2 and 5.4 g FeCl3 in 50 mL of water was prepared and was added to NaOH 2 M solution dropwise under magnetic stirring. Then, a mixture including EG and TMAOH was added to the above suspension dropwise. After stirring for 2 h, the resultant mixture was immediately transferred to the autoclave and kept at 200 °C for 8 h. The resultant solid products were collected by filtration, repeatedly washed with double distilled water, and then dried at 80 °C for 6 h. 2.2.2. Silica coating Silica-coated NF (S/NF) nanoparticles were prepared by water-in-oil microemulsion approach [23] with some modifications. Cyclohexane (120 mL), cyclohexanol (30 mL) and Triton X-100 (30 mL) were placed in a round bottom flask and the solution was stirred. Once the mixture was homogeneous, an aqueous suspension of NF nanoparticles (5 mL) was added to the above solution. The suspension was continuously stirred for 1 h. Ammonium hydroxide (4 mL) and TEOS (4 mL) were then added. After the mixture was stirred at room temperature for 24 h, the reaction solution was decanted with the aid of a magnet. S/NF nanoparticles were redispersed several times in a mixture of double distilled water and ethanol, separated magnetically and dried at 80 °C for 12 h. 2.2.3. Growth of zeolitic layer on the S/NF surface Mordenite nanoparticles were grown on the surface of primary synthesized sample (S/NF) by hydrothermal treatment in the presence of organic templates. In a typical procedure, the specific amount of Al(NO3)3·9H2O was dissolved in NaOH 6 M solution. TMAOH and EG were added to the above solution under stirring. TEOS was added dropwise to obtain a gel when magnetically stirred for 3 h. The resultant mixture was immediately placed in a 200 mL autoclave which was then maintained in a preheated oven at autogenous pressure and static conditions. After the completion of the period of synthesis (24 h and 180 °C), the product was filtered and washed with double distilled
2.2.6. Antibacterial tests All the antibacterial experiments were carried out by preparing the specified colony-forming units (CFU mL−1) of bacteria cell concentration. The reaction mixture in Petri dish, which included a certain amount of bacteria suspension (30 mL) and composites (20 mg), was stirred with a mechanic stirrer to prevent the precipitation of the solid. In certain time intervals (30 min), 2 mL of the reaction mixture was diluted with 0.9% saline. Then 1 mL of diluted sample was incubated at 37 °C for 24 h on soybean casein digest agar, and the remaining colonies were counted. To observe the alteration of the cell wall, TEM analysis was applied. A quantity of 107 CFU mL−1 cells was mixed with 20 mg solid. After the treatment time (120 min), the cell suspension was collected and centrifuged down to a pellet. The bacteria pellets were prefixed in 2.5% glutaraldehyde at 4 °C for 12 h. After washing two times with
Scheme 1. The formation process of noble metal functionalized nanostructures.
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0.1 M phosphate buffer (PBS) (pH 7.2), the specimens were colored by mixing with 2% Na2H5[P(W2O7)6] aqueous solution at a volume ratio of 1:1 for 2 h. Then, the mixing suspension was dropped onto the copper grids with holey carbon films. The grids were used as received and examined using a Philips CM200 TEM. 3. Results and discussion 3.1. Characterization of prepared nanostructures 3.1.1. VSM analysis Fig. 1 shows the magnetic properties of the prepared samples. Magnetic measurements show that NF, S/NF, MOR/S/NF and X/GPTS– GLU/MOR/S/NF have magnetic saturation values of 48, 36.1, 15 and 11.9 emu/g, respectively. It's obvious that the growth of zeolitic layer on the surface reduces the magnetic saturation to less than half. It should also be noted that the multiple modified NF cores still show good magnetization, indicating their suitability for using and separation. The magnified hysteresis loops in Fig. 1 confirm the superparamagnetic feature for all the samples. Moreover, the multifunctional core–shell nanostructures with homogenous dispersion exhibit the re-disperse properties and a suitable response to the external magnetic field due to their high magnetization. 3.1.2. XRD analysis Fig. 2 indicates the XRD patterns of the products. The characteristic peaks of NF nanoparticles and MOR-type zeolite were seen in the samples. The strong peaks in 2θ = 30°, 35.5°, 43°, 57.2° and 63° are attributed to NF structures. The zeolitic phases were identified by comparing the sharp diffraction peaks with the data reported in the International Zeolite Association (IZA). The characteristic peaks of mordenite are well recognizable by their green colors. X/GPTS–GLU/ MOR/S/NF and MOR/S/NF have nearly the same XRD patterns and no excess crystalline phase appears after the surface modification.
Fig. 2. X-ray diffraction (XRD) patterns of (a) NF, (b) MOR/S/NF, and (c) X/GPTS–GLU@ MOR/S/NF.
Some peaks between 700 and 850 cm−1 and between 1000 and 1150 cm−1 are assigned to symmetric and antisymmetric T\O\T stretching vibrations. In contrast, the stretching vibrational mode for
3.1.3. FTIR analysis The FTIR spectra of prepared samples are shown in Fig. 3. From Fig. 3, the strong band in higher than 3400 cm−1 (which is attributed to OH vibrations) and a characteristic peak in 1630 cm−1 (which is attributed to the presence of water molecules) are observable. The absorption band related to Si\O\Si (in 1092 cm−1) also appears in the spectrum of S/NF. All bands observed in the ranges of 920–1250, 650–720, and 420–500 cm−1 in the MOR/S/NF spectrum correspond to the internal tetrahedral: asymmetrical stretch, symmetrical stretch and T\O bonds (where T = Si or Al in the zeolitic structure), respectively.
60
Magnetization (emu/g)
40 20 0 NF S/NF MOR/S/NF X/GPTS-GLU@MOR/S/NF
-20 -40 -60 -10000
-5000
0
5000
10000
Applied Field (Oe) Fig. 1. Magnetization curves of prepared samples.
Fig. 3. FTIR spectra of (a) NF, (b) S/NF, (c) MOR/S/NF, and (d) X/GPTS–GLU@MOR/S/NF.
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−1 AlO− [25]. The absorption band 4 is located in the range 650–900 cm related to X\O (X = Fe, Mn, Co, Ni) (579 cm−1) is also present in all spectra.
3.1.4. BET and BJH analysis The results for Brunauer–Emmett–Teller (BET) analysis are shown in Fig. 4 and Table 1. Fig. 4 presents a typical hysteresis loop obtained from N2 adsorption–desorption procedure for MOR/S/NF and shows that the sample has good porosity. The results revealed that the growth of zeolitic layer on the outer surface of the magnetic core increases the specific surface area and affects the structure porosity. From BET analysis results, by deposition of GPTS–GLU agents on the surface, the basic pore structure of MOR/S/NF sample has not changed. However, BET surface area and total pore volume decrease from 215.7 m2 g−1 and 0.16 cm3 g−1 to 201.58 m2 g−1 and 0.15 cm3 g−1, respectively. Pore size distributions were determined by BJH (Barrett–Joyner–Halenda) method. BJH analysis can be employed to determine the pore area and the specific pore volume using adsorption and desorption techniques. This technique characterizes the pore size distribution independent of the external area due to the particle size of the sample. A typical BJH plot for MOR/S/NF is shown in Fig. 5. Fig. 5 shows that the pore diameter ranges from 1 to 100 nm and the most pore diameters are concentrated between 2 and 50 nm indicating the present mesopores. 3.1.5. TEM analysis TEM photographs demonstrate the morphological and structural features of the samples as shown in Fig. 6. It is clear that MOR/S/NF nanostructures have well spherical structure and high monodispersity in size. The average sizes were about 15–30 nm and 80–100 nm for NF nanoparticles and the spherical MOR/S/NF nanostructures, respectively. From Fig. 6, one can conclude that the growth of zeolitic shell onto the S/NF nanoparticles improved the products' shape and monodispersity. 3.2. Antibacterial activity of prepared nanostructures The results for bacterial inactivation over different nanostructures are shown in Fig. 7. According to the results, GPTS–GLU@MOR/S/NF showed a light antibacterial activity while MOR/S/NF didn't show any activity. This can be attributed to the presence of hydrophilic functional groups on the surface of GPTS–GLU@MOR/S/NF, which enable it to stick to the cell wall. Both Ag and Au contained GPTS–GLU@MOR/S/NF, and MOR/S/NF nanostructures have shown high antibacterial activity.
Fig. 4. A typical N2 adsorption/desorption plot (sample: MOR/S/NF).
Table 1 The results for BET analysis. Sample
BET surface area (m2 g−1)
Total pore volume (cm3 g−1)
NiFe2O4 Z@SiO2@NiFe2O4 GPTS–GLU@Z@SiO2@NiFe2O4
34.66 215.70 201.58
0.29 0.16 0.15
However, the inactivation rate and mechanistic influence of the nanostructures are different. The antibacterial activity of X/MOR/S/NF (X = Ag or Au) samples are attributed to the releasing of Au and Ag ions into the reaction medium (which has been exchanged with the ions existed in the zeolitic channels of porous nanostructure). The interaction between the noble metal ions and the cell wall components (such as cysteine), or inducing a disturbance in the process of DNA replication of microorganism, makes the microorganism inactivate [26]. Fig. 8 shows the TEM images of a destroyed cell, after the treatment time (120 min). It can be inferred from the TEM images that Au and Ag contained GPTS–GLU@MOR/S/NF nanostructures which act through a different mechanism. It can be observed that the adhesion of functionalized nanostructures onto the outer layer of the cell membrane may be a strong reason for microorganism inactivation. The outer membrane of the Escherichia coli cell wall is composed of a two-layer lipid with a thickness of 7 nm. This two-layer lipid consists of lipoproteins, lipopolysaccharides (LPS), phospholipids, and proteins. As shown in Fig. 1S, lipopolysaccharides are located in the outer membrane of the cell wall and mainly contain a specific lipid (lipid A). We suggest that the hydrogen bonding between the GPTS–GLU component on the surface of nanostructures and the head of lipid A in the outer membrane of the cell wall occurred when GPTS–GLU/MOR/S/NF nanostructures approach the microorganism. Forming these bonds makes some channels between the nanostructures and the cell wall, from where Au and Ag ions can be transferred to the microorganism (Fig. 2S). After the adhesion of noble metal contained nanostructures to the outer layer of cell wall of microorganism, some different processes might occur that can interrupt the microorganism viability. The cell wall of microorganisms has the high anionic charge density which enables them to interact strongly with the metal ions dissolved in the environment to accumulate the large quantities of bonded metal [27,28]. However, the transport mechanisms of potentially toxic metals and their various organic derivatives through the microorganism are poorly understood. The outer membrane of the gram-negative cell wall contains magnesium, and sometimes calcium, as integral components [24]. These metals
Fig. 5. A typical BJH plot (sample: MOR/S/NF).
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Fig. 7. The results for antibacterial activity over prepared nanostructures (sample amount: 20 mg, cell concentration: 107 CFU mL−1).
membrane. Enzymes that are not released have been assumed to exist within the cytoplasm, and are termed cell-bound enzymes [30]. Also, adhering of Ag and Au contained nanostructures to the microorganism's cell wall can block the pores where the microorganism nourishes via them. The presence of hydrophilic groups on the surface of nanostructures is an important factor in occurrence of this phenomenon.
3.3. MIC measurements The results for MIC measurements over the prepared nanostructures are represented in Fig. 9 a and b. To do these experiments, various dosages of nanostructures (166–664 ppm) were added to the medium. Fig. 9 shows that increasing the concentration of the nanostructures improves the antibacterial activity. It can also be found from Fig. 9a and b that the MIC quantities are 333 ppm, 468 ppm, 333 ppm and 468 ppm for Au/GPTS–GLU/MOR/S/NF, Au/MOR/S/NF, Ag/GPTS–GLU/MOR/S/NF and Ag/MOR/S/NF, respectively. As the concentration of each composite was optimized to MIC, the great numbers of the cells were killed and only a few of the colonies were observed on the surfaces of nutrient agar plates. The results indicated that the prepared nanostructures have an excellent antibacterial performance against the gram negative bacteria. According to the results, under the same concentrations, X/ MOR/S/NF nanostructures have less MIC compared to the X/GPTS– GLU/MOR/S/NF nanostructures, as expected, because the latter cases inactivate the bacteria via two effective procedures; the inactivation of microorganism over the former cases attributed to releasing of noble metal ions from the zeolitic pores, only. Fig. 6. TEM images of (a) and (b) magnetic nanoparticles and (c) core–shell nanostructures.
3.4. Reuse ability contribute to the outer membrane, and if they are extracted or replaced by exogenous counterions, the loss of the protein or lipopolysaccharide may result. Therefore, it was important to monitor the metal-binding experiments for the lost cell components, which could have produced artificially low metal uptake values [28]. The penetration of noble metal ions to the cell structure can activate some groups of degradative enzymes (such as 5-Nucleotidase) which are released normally by osmotic shock [29]. This process partially disrupts the cell wall of gram-negative bacteria such as E. coli, without causing loss of viability. As a result of osmotic shock damage, some specific enzymes are released from the microorganism, while the majority of bacterial enzymes are not. Enzymes released by osmotic shock have been assigned to a discrete cellular location, the periplasm, which is assumed to be located between the cytoplasmic membrane and the outer
The results for studying the reuse ability of prepared nanostructures are represented in Fig. 10a and b. According to the results, after four times of using, Au/GPTS–GLU/MOR/S/NF shows a better antibacterial activity than Ag/GPTS–GLU/MOR/S/NF. The other samples (Au/MOR/S/NF and Ag/MOR/S/NF) where the antibacterial activities are arisen from releasing the ions existed in the zeolitic pores, the quantity of reuse ability is less than the former cases. This is expected, due to the fact that the amount of available ions decreases after using for several times. In addition, X/GPTS–GLU/MOR/S/NF nanostructures contain X+ ions bonded to the surface via an electrostatic interaction which makes them more effective antibacterial agents. To find this parameter for each sample, 20 mg solid was transferred to a vessel containing 30 mL of E. coli (1 × 107 CFU mL−1) suspension. After each test, the solid was recovered by a 1.4 T magnet, dried at 80 °C and it was then used again.
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Fig. 9. MIC measurements for: (a) X/MOR/S/NF nanostructures (red color: Ag/MOR/S/NF, blue color: Au/MOR/S/NF) and (b) X/GPTS–GLU@MOR/S/NF nanostructures (red color: Ag/GPTS–GLU@MOR/S/NF, blue color: Au/GPTS–GLU@MOR/S/NF).
they are surrounded effectively on the surface by two carboxylate groups, rather than Ag+ ions. In addition, it was observed that by optimizing the configurations Au+ ions were located nearer to oxygen atoms of carboxylate groups. According to the theoretical results, one can conclude that reuse ability of Au/GPTS–GLU/MOR/S/NF must be more suitable than Ag/GPTS– GLU/MOR/S/NF, which was confirmed by the experimental results.
4. Conclusions
Fig. 8. TEM images of E. coli cell wall after the treatment time (120 min).
3.5. Theoretical study of the interaction of noble metal ions with GPTS–GLU component on the surface To compare the antibacterial capability of Ag/GPTS–GLU/MOR/S/NF and Au/GPTS–GLU/MOR/S/NF nanostructures, we studied theoretically the interaction of noble metal ions with the functional group GPTS– GLU, which is located on the surface of prepared nanostructures. This is schematically indicated in Fig. 3S. Calculations were performed by Gaussian code in B3LYP basis set. The results for this study are shown in Table 2. From Table 2, one can conclude that the interaction of Au+ ions with GPTS–GLU is stronger than Ag+ ions. The interaction energies for Ag/GPTS–GLU and Au/GPTS–GLU are −11.0532 and −12.5825 eV, respectively which shows that the former case is more stable bond than the latter case. Au+ ions are more massive than Ag+ ions. Thus,
Magnetic core–zeolitic shell nanostructures were prepared by hydrothermal and coprecipitation methods. Analysis of the products confirmed the growth of zeolitic layer on the surface of S/NF cores. The results of VSM analysis showed that the core–shell nanostructures have good magnetization which reduces with the layer deposition on the surface. Antibacterial activity of prepared samples was investigated by inactivation of E. coli as a gram negative bacterium. The results showed that X/GPTS–GLU@MOR/S/NF nanostructures had better antibacterial activity than the noble metal contained MOR/S/NF samples. Their antibacterial activities were attributed to the bond formation between GPTS–GLU component on the surface and the head of lipid A, which is located in the outer membrane of the cell wall. Reuse ability and MIC measurements also confirmed the higher activity of X/GPTS– GLU@MOR/S/NF nanostructures. The interaction of noble metal ions with GPTS–GLU, as a surface component, was studied theoretically and according to the obtained results, it was concluded that Au+ ions stick to the surface more effectively than Ag+ ions. Thus, it was expected that Au/GPTS–GLU@MOR/S/NF has a better reuse ability compared to Ag/GPTS–GLU@MOR/S/NF.
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Fig. 10. Reuse ability measurements for: (a) X/MOR/S/NF nanostructures (red color: Ag/ MOR/S/NF, blue color: Au/MOR/S/NF) and (b) X/GPTS–GLU@MOR/S/NF nanostructures (red color: Ag/GPTS–GLU@MOR/S/NF, blue color: Au/GPTS–GLU@MOR/S/NF).
Table 2 The theoretical results for studying the interaction of noble metals with GPTS–GLU. GPTS–GLU_X
X = Ag
X = Au
Thermal energy (eV) Enthalpy of interaction (eV) Distance of X-O12 Distance of X-O14
−18941.41 −11.0532 2.17 2.13
−18665.86 –12.5825 2.09 2.07
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2013.10.027. References [1] W.R. Jarvis, Selected aspects of the socioeconomic impact of nosocomial infections: morbidity, mortality, cost, and prevention, Infect. Control Hosp. Epidemiol. 17 (1996) 552–557. [2] A. Gallardo-Moreno, M. Pacha-Olivenza, M.C. Fernandez-Calderonb, C. Perez-Giraldo, J. Bruque, M.L. Gonzalez-Martin, Bactericidal behavior Ti6Al4V surfaces after exposure UV-C light, Biomaterials 31 (2010) 5159–5168. [3] W. Pinggui, A.I. James, K.S. Jian, Mechanism of Escherichia coli inactivation on palladium-modified nitrogen-doped titanium dioxide, Biomaterials 31 (2010) 7526–7533.
121
2+ [4] A. Langella, M. Pansini, P. Cappelletti, B. Gennaro, M. Gennaro, C. Colella, NH+ , 4 , Cu Zn2+, Cd2+ and Pb2+ exchange for Na+ in a sedimentary clinoptilolite, North Sardinia, Italy, Microporous Mesoporous Mater. 37 (2000) 337–343. [5] A. Cincotti, N. Lai, R. Orru, G. Cao, Sardinian natural clinoptilolite for heavy metals and ammonium removal: experimental and modeling, Chem. Eng. J. 84 (2001) 275–282. [6] V. Badillo-Almaraz, P. Trocellier, I. Davila-Rangel, Adsorption of aqueous Zn(II) species on synthetic zeolites, Nucl. Inst. Methods Phys. Res. 210 (2003) 424–428. [7] J. Peric, M. Trgo, N. Vukojevic-Medvidovic, Removal of zinc, copper and lead by natural zeolite — a comparison of adsorption isotherms, Water Res. 38 (2004) 1893–1899. [8] L. Levy, Y. Sahoo, K.S. Kim, E.J. Bergey, P.N. Prasad, Nanochemistry: synthesis and characterization of multifunctional nanoclinics for biological applications, Chem. Mater. 14 (2002) 3715–3721. [9] Y. Li, B. Yan, C.H. Deng, W.J. Yu, X.Q. Xu, P.Y. Yang, Efficient on-chip proteolysis system based on functionalized magnetic silica microspheres, Proteomics 7 (2007) 2330–2339. [10] C.B. Murray, D.J. Norris, M.G. Bawendi, Synthesis and characterization of nearly monodisperse CdE (E ¼ sulfur, selenium, tellurium) semiconductor nanocrystallites, J. Am. Chem. Soc. 115 (1993) 8706–8715. [11] P.P. Yang, Z.W. Quan, L.L. Lu, S.S. Huang, J. Lin, Bioactive, luminescent and mesoporous europium-doped hydroxyapatite as a drug carrier, Biomaterials 29 (2008) 4341–4347. [12] M. Arruebo, M. Galan, N. Navascues, C. Tellez, C. Marquina, M.R. Ibarra, Development of magnetic nanostructured silica-based materials as potential vectors for drug-delivery applications, Chem. Mater. 18 (2006) 1911–1919. [13] S. Giri, B.G. Trewyn, M.P. Stellmaker, V.S.Y. Lin, Stimuli-responsive controlled release delivery system based on mesoporous silica nanorods capped with magnetic nanoparticles, Angew. Chem. Int. Ed. 44 (2005) 5038–5044. [14] W.R. Zhao, J.L. Gu, L.X. Zhang, H.R. Chen, J.L. Shi, Fabrication of uniform magnetic nanocomposite spheres with a magnetic core/mesoporous silica shell structure, J. Am. Chem. Soc. 127 (2005) 8916–8917. [15] Y.H. Deng, D.W. Qi, C.H. Deng, X.M. Zhang, D.Y. Zhao, Superparamagnetic high magnetization micropheres with a Fe3O4@SiO2 core and perpendicularly aligned mesoporous SiO2 shell for removal of microcystins, J. Am. Chem. Soc. 130 (2008) 28–29. [16] J. Guo, W.L. Yang, C.C. Wang, J. He, J.Y. Chen, Poly (N-isopropylacrylamide)-coated luminescent/magnetic silica microspheres: preparation, characterization, and biomedical applications, Chem. Mater. 18 (2006) 5554–5562. [17] J. Kim, J.E. Lee, J. Lee, J.H. Yu, B.C. Kim, K. An, Magnetic fluorescent delivery vehicle using uniform mesoporous silica spheres embedded with monodisperse magnetic and semiconductor nanocrystals, J. Am. Chem. Soc. 128 (2006) 688–689. [18] X. Zhang, H. Wang, Z. Su, Fabrication of Au@Ag core–shell nanoparticles using polyelectrolyte multilayers as nanoreactors, Langmuir 28 (2012) 15705–15712. [19] B. Chudasama, A.K. Vala, N. Andhariya, R.V. Upadhyay, R.V. Mehta, Enhanced antibacterial activity of bifunctional Fe3O4-Ag core–shell nanostructures, Nano Res. 2 (2012) 955–965. [20] A. Dong, J. Huang, S. Lan, T. Wang, L. Xiao, W. Wang, T. Zhao, X. Zheng, F. Liu, G. Gao, Y. Chen, Synthesis of N-halamine-functionalized silica–polymer core–shell nanoparticles and their enhanced antibacterial activity, Nanotechnology 22 (2011) 295602–295611. [21] Y. Piaoping, Q. Zewei, H. Zhiyao, L. Chunxia, K. Xiaojiao, C. Ziyong, L. Jun, A magnetic, luminescent and mesoporous core–shell structured composite material as drug carrier, Biomaterials 30 (2009) 4786–4795. [22] M. Padervand, M.R. Gholami, Removal of toxic heavy metal ions from waste water by functionalized magnetic core–zeolitic shell nanocomposites as adsorbents, Environ. Sci. Pollut. Res. 20 (2013) 3900–3909. [23] L. Chang-Xiang, L. Qiang, G. Can-Cheng, Synthesis and catalytic abilities of silica-coated Fe3O4 nanoparticle bonded metalloporphyrins with different saturation magnetization, Catal. Lett. 138 (2010) 96–103. [24] M. Zhiya, G. Yueping, L. Huizhou, Superparamagnetic silica nanoparticles with immobilized metal affinity ligands for protein adsorption, J. Magn. Magn. Mater. 301 (2006) 469–477. [25] P. Sharma, P. Rajaram, R. Tomar, Synthesis and morphological studies of nanocrystalline MOR type zeolite material, J. Colloid Interface Sci. 325 (2008) 547–557. [26] M. Padervand, M.R. Elahifard, R.V. Meidanshahi, S. Ghasemi, S. Haghighi, M.R. Gholami, Investigation of the antibacterial and photocatalytic properties of the zeolitic nanosized AgBr/TiO2 composites, Mater. Sci. Semicond. Process. 15 (2012) 73–79. [27] T.J. Beveridge, Ultrastructure, chemistry and function of the bacterial wall, Int. Rev. Cytol. 72 (1981) 229–317. [28] T.J. Beveridge, S.F. Koval, Binding of metals to cell envelopes of Escherichia coli K-12, Appl. Environ. Microbiol. 42 (1981) 325–335. [29] H.C. Neu, L.A. Heppel, On the surface localization of enzymes in E. coli, Biochem. Biophys. Res. Commun. 17 (1964) 215–219. [30] D.F. Broad, J.T. Smith, Escherichia coli 5-nucleotidase: purification, properties and its release by osmotic shock, J. Gen. Microbiol. 123 (1981) 241–247.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Incredible antibacterial activity of noble metal functionalized magnetic core–zeolitic shell nanostructures M. Padervand a, S. Janatrostami b, A. Kiani Karanji a, M.R. Gholami a,⁎ a b
Department of Chemistry, Sharif University of Technology, Azadi Ave., Tehran, Iran Department of Water Engineering, Tehran University, Karaj, Iran
a r t i c l e
i n f o
Article history: Received 20 April 2013 Received in revised form 4 September 2013 Accepted 24 October 2013 Available online 5 November 2013 Keywords: Magnetic core–zeolitic shell nanostructure Antibacterial activity Nickel ferrite Mordenite E. coli
a b s t r a c t Functionalized magnetic core–zeolitic shell nanostructures were prepared by hydrothermal and coprecipitation methods. The products were characterized by Vibrating Sample Magnetometer (VSM), X-ray powder diffraction (XRD), Fourier Transform Infrared (FTIR) spectra, nitrogen adsorption–desorption isotherms, and Transmission Electron Microscopy (TEM). The growth of mordenite nanoparticles on the surface of silica coated nickel ferrite nanoparticles in the presence of organic templates was also confirmed. Antibacterial activity of the prepared nanostructures was investigated by the inactivation of Escherichia coli as a gram negative bacterium. A new mechanism was proposed for inactivation of E. coli over the prepared samples. In addition, the Minimum Inhibitory Concentration (MIC) and reuse ability were studied. TEM images of the destroyed cell wall after the treatment time were performed to illustrate the inactivation mechanism. According to the experimental results, the core–shell nanostructures which were modified by organic agents and then functionalized with noble metal nanoparticles were the most active. The interaction of the noble metals with the organic components on the surface of nanostructures was studied theoretically and the obtained results were used to interpret the experimental results. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The inactivation of microorganisms is an ongoing issue in the food industry, medical field, and environment. Several inactivation methods, such as pasteurization, chlorination, ozonation, and ultraviolet and ultrasound irradiation, have been proposed and utilized over the past years. In addition, infection control continues to be a growing concern in medical and healthcare facilities. According to the U.S. National Centers for Disease Control and Prevention (CDC), the risk of acquiring a serious infection while being treated in a hospital has increased over 35% in the past two decades. A study found that nearly 2 million American patients contract hospital-acquired infections per year. Of these patients, nearly 90,000 die as a result [1]. The need for infection control guidelines and prevention methods is essential for all the healthcare facilities. Another scenario that shares this common concern is the occurrence of implant-associated infections. Therefore, various types of modified surfaces, which are often coated or integrated in antibiotics or metal ions, have been proposed to disrupt the colonization of bacteria [2,3]. Even though the biocompatibility of most of these antimicrobial
⁎ Corresponding author at: Azadi Ave. P.O. Box 11365-9516, Tehran, Iran. Tel.: +98 2166165314; fax: +98 2166029165. E-mail address: [email protected] (M.R. Gholami). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.10.027
surfaces remains unknown, research activities on new bactericidal materials or devices never cease. Zeolites have a large applicability for decontamination, purification of urban and industrial residual waters, protection of waste disposal areas, purification of industrial gases, etc. Many researchers have studied the usage of natural and synthetic zeolites for removal of heavy metal ions in aqueous mediums [4–6]. Properties such as high surface area, individual micro-pores, variety of channels and high resistance make zeolites very useful for industrial applications and academic researches [6,7]. The combination of zeolitic materials with the magnetic or/and active functional groups to form core–shell structured composites is undoubtedly of special interest in diagnostic analysis [8], bioseparation [9], and controlled drug release [10–12], due to their unique magnetic responsivity, low cytotoxicity, good biocompatibility and mesoporous properties. Over the past decade, the preparation of multifunctional microspheres containing magnetite core with a mesoporous shell has been reported [13–20]. The uniform-sized magnetic particles were normally prepared via a high temperature decomposition method [17], or hydrothermal process [15]. The formation of core–shell nanocomposites is conventionally followed by an encapsulation procedure, where the magnetite core is encapsulated by a silica layer using a sol–gel technique [21]. The aim of the present study was to investigate the antibacterial activity of prepared noble metal functionalized magnetic core–zeolitic shell nanostructures. The effect of initial cell concentration, composite
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dosage and reuse capacity was investigated. In addition, the interaction of used noble metal with the nanostructure's surface was studied theoretically and a new mechanism was proposed for bacterium inactivation over the prepared samples. 2. Experimental 2.1. Materials and instruments Chemicals include NaOH, tetramethylammonium hydroxide (TMA OH) (10% w/w), ethylene glycol (EG), cyclohexane, cyclohexanol, C14H22O(C2H4O)n (Triton X-100), 3-glycidoxypropyltrimethoxysilane (GPTS), and glutamic acid (GLU) and the various salts of polyvalent metals were used as required. Tetraethyl orthosilicate (TEOS) was used as silica source. All chemicals were purchased from Merck Chemical Co. (Germany) and were used without any further purification. The XRD patterns of the prepared samples were recorded on a Bruker D8 Advance X-ray diffractometer with CuKα irradiation (λ = 0.15406 nm). The FTIR spectra were recorded using an NB series spectrometer. The specific surface area of the nanostructures was calculated from the N2 adsorption–desorption isotherms at 77 K, using Belsorp apparatus (Japan). The average particle size and morphology of the samples were examined by TEM analysis. Magnetic studies were carried out on a “Meghnatis Daghigh Kavir Co.” at 300 K.
water and dried at 90 °C for 10 h. Finally, the solid powder was calcined at 500 °C for 5 h and denoted MOR/S/NF. 2.2.4. Surface functionalization with GPTS–GLU MOR/S/NF samples were modified with GPTS and GLU according to the earlier work with some modifications [24]. GLU was dissolved in double distilled water, and the obtained suspension was adjusted to pH 11 with NaOH 10 M. The suspension was transferred into a flask bottle placed in the ice-bath, and GPTS was slowly added while being stirred. The mixed solution was heated to 65 °C for 2 h by stirring. After adjusting the pH to 6 with concentrated HCl, an aqueous suspension of MOR/S/NF was added and stirred for 12 h. The resultant product was separated with the help of a permanent magnet, washed thoroughly with distilled water, dried at 80 °C for 6 h and denoted GPTS–GLU@ MOR/S/NF. 2.2.5. Au and Ag loading on the surface 0.5 g of prepared solid in the earlier section was added to an aqueous solution of H2AuCl4·4H2O and AgNO3, respectively. The suspensions were sonicated for 15 min and then stirred at room temperature for 2 h. After completion of the reaction, the products were separated by an external magnet, were washed repeatedly with double distilled water and then dried at 80 °C overnight. The products were denoted as X/GPTS–GLU@MOR/S/NF (X = Au or Ag). The total formation process of noble metal modified nanostructures is shown in Scheme 1.
2.2. Nanostructure preparation 2.2.1. Synthesis of nickel ferrite (NF) nanoparticles The nanostructures were prepared according to our earlier work with some modifications [22]. NF nanoparticles were prepared via hydrothermal method in a 200 mL stainless steel autoclave with a Teflon liner under autogenous pressure. A transparent solution containing 2.9 g Ni(NO3)2 and 5.4 g FeCl3 in 50 mL of water was prepared and was added to NaOH 2 M solution dropwise under magnetic stirring. Then, a mixture including EG and TMAOH was added to the above suspension dropwise. After stirring for 2 h, the resultant mixture was immediately transferred to the autoclave and kept at 200 °C for 8 h. The resultant solid products were collected by filtration, repeatedly washed with double distilled water, and then dried at 80 °C for 6 h. 2.2.2. Silica coating Silica-coated NF (S/NF) nanoparticles were prepared by water-in-oil microemulsion approach [23] with some modifications. Cyclohexane (120 mL), cyclohexanol (30 mL) and Triton X-100 (30 mL) were placed in a round bottom flask and the solution was stirred. Once the mixture was homogeneous, an aqueous suspension of NF nanoparticles (5 mL) was added to the above solution. The suspension was continuously stirred for 1 h. Ammonium hydroxide (4 mL) and TEOS (4 mL) were then added. After the mixture was stirred at room temperature for 24 h, the reaction solution was decanted with the aid of a magnet. S/NF nanoparticles were redispersed several times in a mixture of double distilled water and ethanol, separated magnetically and dried at 80 °C for 12 h. 2.2.3. Growth of zeolitic layer on the S/NF surface Mordenite nanoparticles were grown on the surface of primary synthesized sample (S/NF) by hydrothermal treatment in the presence of organic templates. In a typical procedure, the specific amount of Al(NO3)3·9H2O was dissolved in NaOH 6 M solution. TMAOH and EG were added to the above solution under stirring. TEOS was added dropwise to obtain a gel when magnetically stirred for 3 h. The resultant mixture was immediately placed in a 200 mL autoclave which was then maintained in a preheated oven at autogenous pressure and static conditions. After the completion of the period of synthesis (24 h and 180 °C), the product was filtered and washed with double distilled
2.2.6. Antibacterial tests All the antibacterial experiments were carried out by preparing the specified colony-forming units (CFU mL−1) of bacteria cell concentration. The reaction mixture in Petri dish, which included a certain amount of bacteria suspension (30 mL) and composites (20 mg), was stirred with a mechanic stirrer to prevent the precipitation of the solid. In certain time intervals (30 min), 2 mL of the reaction mixture was diluted with 0.9% saline. Then 1 mL of diluted sample was incubated at 37 °C for 24 h on soybean casein digest agar, and the remaining colonies were counted. To observe the alteration of the cell wall, TEM analysis was applied. A quantity of 107 CFU mL−1 cells was mixed with 20 mg solid. After the treatment time (120 min), the cell suspension was collected and centrifuged down to a pellet. The bacteria pellets were prefixed in 2.5% glutaraldehyde at 4 °C for 12 h. After washing two times with
Scheme 1. The formation process of noble metal functionalized nanostructures.
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0.1 M phosphate buffer (PBS) (pH 7.2), the specimens were colored by mixing with 2% Na2H5[P(W2O7)6] aqueous solution at a volume ratio of 1:1 for 2 h. Then, the mixing suspension was dropped onto the copper grids with holey carbon films. The grids were used as received and examined using a Philips CM200 TEM. 3. Results and discussion 3.1. Characterization of prepared nanostructures 3.1.1. VSM analysis Fig. 1 shows the magnetic properties of the prepared samples. Magnetic measurements show that NF, S/NF, MOR/S/NF and X/GPTS– GLU/MOR/S/NF have magnetic saturation values of 48, 36.1, 15 and 11.9 emu/g, respectively. It's obvious that the growth of zeolitic layer on the surface reduces the magnetic saturation to less than half. It should also be noted that the multiple modified NF cores still show good magnetization, indicating their suitability for using and separation. The magnified hysteresis loops in Fig. 1 confirm the superparamagnetic feature for all the samples. Moreover, the multifunctional core–shell nanostructures with homogenous dispersion exhibit the re-disperse properties and a suitable response to the external magnetic field due to their high magnetization. 3.1.2. XRD analysis Fig. 2 indicates the XRD patterns of the products. The characteristic peaks of NF nanoparticles and MOR-type zeolite were seen in the samples. The strong peaks in 2θ = 30°, 35.5°, 43°, 57.2° and 63° are attributed to NF structures. The zeolitic phases were identified by comparing the sharp diffraction peaks with the data reported in the International Zeolite Association (IZA). The characteristic peaks of mordenite are well recognizable by their green colors. X/GPTS–GLU/ MOR/S/NF and MOR/S/NF have nearly the same XRD patterns and no excess crystalline phase appears after the surface modification.
Fig. 2. X-ray diffraction (XRD) patterns of (a) NF, (b) MOR/S/NF, and (c) X/GPTS–GLU@ MOR/S/NF.
Some peaks between 700 and 850 cm−1 and between 1000 and 1150 cm−1 are assigned to symmetric and antisymmetric T\O\T stretching vibrations. In contrast, the stretching vibrational mode for
3.1.3. FTIR analysis The FTIR spectra of prepared samples are shown in Fig. 3. From Fig. 3, the strong band in higher than 3400 cm−1 (which is attributed to OH vibrations) and a characteristic peak in 1630 cm−1 (which is attributed to the presence of water molecules) are observable. The absorption band related to Si\O\Si (in 1092 cm−1) also appears in the spectrum of S/NF. All bands observed in the ranges of 920–1250, 650–720, and 420–500 cm−1 in the MOR/S/NF spectrum correspond to the internal tetrahedral: asymmetrical stretch, symmetrical stretch and T\O bonds (where T = Si or Al in the zeolitic structure), respectively.
60
Magnetization (emu/g)
40 20 0 NF S/NF MOR/S/NF X/GPTS-GLU@MOR/S/NF
-20 -40 -60 -10000
-5000
0
5000
10000
Applied Field (Oe) Fig. 1. Magnetization curves of prepared samples.
Fig. 3. FTIR spectra of (a) NF, (b) S/NF, (c) MOR/S/NF, and (d) X/GPTS–GLU@MOR/S/NF.
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−1 AlO− [25]. The absorption band 4 is located in the range 650–900 cm related to X\O (X = Fe, Mn, Co, Ni) (579 cm−1) is also present in all spectra.
3.1.4. BET and BJH analysis The results for Brunauer–Emmett–Teller (BET) analysis are shown in Fig. 4 and Table 1. Fig. 4 presents a typical hysteresis loop obtained from N2 adsorption–desorption procedure for MOR/S/NF and shows that the sample has good porosity. The results revealed that the growth of zeolitic layer on the outer surface of the magnetic core increases the specific surface area and affects the structure porosity. From BET analysis results, by deposition of GPTS–GLU agents on the surface, the basic pore structure of MOR/S/NF sample has not changed. However, BET surface area and total pore volume decrease from 215.7 m2 g−1 and 0.16 cm3 g−1 to 201.58 m2 g−1 and 0.15 cm3 g−1, respectively. Pore size distributions were determined by BJH (Barrett–Joyner–Halenda) method. BJH analysis can be employed to determine the pore area and the specific pore volume using adsorption and desorption techniques. This technique characterizes the pore size distribution independent of the external area due to the particle size of the sample. A typical BJH plot for MOR/S/NF is shown in Fig. 5. Fig. 5 shows that the pore diameter ranges from 1 to 100 nm and the most pore diameters are concentrated between 2 and 50 nm indicating the present mesopores. 3.1.5. TEM analysis TEM photographs demonstrate the morphological and structural features of the samples as shown in Fig. 6. It is clear that MOR/S/NF nanostructures have well spherical structure and high monodispersity in size. The average sizes were about 15–30 nm and 80–100 nm for NF nanoparticles and the spherical MOR/S/NF nanostructures, respectively. From Fig. 6, one can conclude that the growth of zeolitic shell onto the S/NF nanoparticles improved the products' shape and monodispersity. 3.2. Antibacterial activity of prepared nanostructures The results for bacterial inactivation over different nanostructures are shown in Fig. 7. According to the results, GPTS–GLU@MOR/S/NF showed a light antibacterial activity while MOR/S/NF didn't show any activity. This can be attributed to the presence of hydrophilic functional groups on the surface of GPTS–GLU@MOR/S/NF, which enable it to stick to the cell wall. Both Ag and Au contained GPTS–GLU@MOR/S/NF, and MOR/S/NF nanostructures have shown high antibacterial activity.
Fig. 4. A typical N2 adsorption/desorption plot (sample: MOR/S/NF).
Table 1 The results for BET analysis. Sample
BET surface area (m2 g−1)
Total pore volume (cm3 g−1)
NiFe2O4 Z@SiO2@NiFe2O4 GPTS–GLU@Z@SiO2@NiFe2O4
34.66 215.70 201.58
0.29 0.16 0.15
However, the inactivation rate and mechanistic influence of the nanostructures are different. The antibacterial activity of X/MOR/S/NF (X = Ag or Au) samples are attributed to the releasing of Au and Ag ions into the reaction medium (which has been exchanged with the ions existed in the zeolitic channels of porous nanostructure). The interaction between the noble metal ions and the cell wall components (such as cysteine), or inducing a disturbance in the process of DNA replication of microorganism, makes the microorganism inactivate [26]. Fig. 8 shows the TEM images of a destroyed cell, after the treatment time (120 min). It can be inferred from the TEM images that Au and Ag contained GPTS–GLU@MOR/S/NF nanostructures which act through a different mechanism. It can be observed that the adhesion of functionalized nanostructures onto the outer layer of the cell membrane may be a strong reason for microorganism inactivation. The outer membrane of the Escherichia coli cell wall is composed of a two-layer lipid with a thickness of 7 nm. This two-layer lipid consists of lipoproteins, lipopolysaccharides (LPS), phospholipids, and proteins. As shown in Fig. 1S, lipopolysaccharides are located in the outer membrane of the cell wall and mainly contain a specific lipid (lipid A). We suggest that the hydrogen bonding between the GPTS–GLU component on the surface of nanostructures and the head of lipid A in the outer membrane of the cell wall occurred when GPTS–GLU/MOR/S/NF nanostructures approach the microorganism. Forming these bonds makes some channels between the nanostructures and the cell wall, from where Au and Ag ions can be transferred to the microorganism (Fig. 2S). After the adhesion of noble metal contained nanostructures to the outer layer of cell wall of microorganism, some different processes might occur that can interrupt the microorganism viability. The cell wall of microorganisms has the high anionic charge density which enables them to interact strongly with the metal ions dissolved in the environment to accumulate the large quantities of bonded metal [27,28]. However, the transport mechanisms of potentially toxic metals and their various organic derivatives through the microorganism are poorly understood. The outer membrane of the gram-negative cell wall contains magnesium, and sometimes calcium, as integral components [24]. These metals
Fig. 5. A typical BJH plot (sample: MOR/S/NF).
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Fig. 7. The results for antibacterial activity over prepared nanostructures (sample amount: 20 mg, cell concentration: 107 CFU mL−1).
membrane. Enzymes that are not released have been assumed to exist within the cytoplasm, and are termed cell-bound enzymes [30]. Also, adhering of Ag and Au contained nanostructures to the microorganism's cell wall can block the pores where the microorganism nourishes via them. The presence of hydrophilic groups on the surface of nanostructures is an important factor in occurrence of this phenomenon.
3.3. MIC measurements The results for MIC measurements over the prepared nanostructures are represented in Fig. 9 a and b. To do these experiments, various dosages of nanostructures (166–664 ppm) were added to the medium. Fig. 9 shows that increasing the concentration of the nanostructures improves the antibacterial activity. It can also be found from Fig. 9a and b that the MIC quantities are 333 ppm, 468 ppm, 333 ppm and 468 ppm for Au/GPTS–GLU/MOR/S/NF, Au/MOR/S/NF, Ag/GPTS–GLU/MOR/S/NF and Ag/MOR/S/NF, respectively. As the concentration of each composite was optimized to MIC, the great numbers of the cells were killed and only a few of the colonies were observed on the surfaces of nutrient agar plates. The results indicated that the prepared nanostructures have an excellent antibacterial performance against the gram negative bacteria. According to the results, under the same concentrations, X/ MOR/S/NF nanostructures have less MIC compared to the X/GPTS– GLU/MOR/S/NF nanostructures, as expected, because the latter cases inactivate the bacteria via two effective procedures; the inactivation of microorganism over the former cases attributed to releasing of noble metal ions from the zeolitic pores, only. Fig. 6. TEM images of (a) and (b) magnetic nanoparticles and (c) core–shell nanostructures.
3.4. Reuse ability contribute to the outer membrane, and if they are extracted or replaced by exogenous counterions, the loss of the protein or lipopolysaccharide may result. Therefore, it was important to monitor the metal-binding experiments for the lost cell components, which could have produced artificially low metal uptake values [28]. The penetration of noble metal ions to the cell structure can activate some groups of degradative enzymes (such as 5-Nucleotidase) which are released normally by osmotic shock [29]. This process partially disrupts the cell wall of gram-negative bacteria such as E. coli, without causing loss of viability. As a result of osmotic shock damage, some specific enzymes are released from the microorganism, while the majority of bacterial enzymes are not. Enzymes released by osmotic shock have been assigned to a discrete cellular location, the periplasm, which is assumed to be located between the cytoplasmic membrane and the outer
The results for studying the reuse ability of prepared nanostructures are represented in Fig. 10a and b. According to the results, after four times of using, Au/GPTS–GLU/MOR/S/NF shows a better antibacterial activity than Ag/GPTS–GLU/MOR/S/NF. The other samples (Au/MOR/S/NF and Ag/MOR/S/NF) where the antibacterial activities are arisen from releasing the ions existed in the zeolitic pores, the quantity of reuse ability is less than the former cases. This is expected, due to the fact that the amount of available ions decreases after using for several times. In addition, X/GPTS–GLU/MOR/S/NF nanostructures contain X+ ions bonded to the surface via an electrostatic interaction which makes them more effective antibacterial agents. To find this parameter for each sample, 20 mg solid was transferred to a vessel containing 30 mL of E. coli (1 × 107 CFU mL−1) suspension. After each test, the solid was recovered by a 1.4 T magnet, dried at 80 °C and it was then used again.
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Fig. 9. MIC measurements for: (a) X/MOR/S/NF nanostructures (red color: Ag/MOR/S/NF, blue color: Au/MOR/S/NF) and (b) X/GPTS–GLU@MOR/S/NF nanostructures (red color: Ag/GPTS–GLU@MOR/S/NF, blue color: Au/GPTS–GLU@MOR/S/NF).
they are surrounded effectively on the surface by two carboxylate groups, rather than Ag+ ions. In addition, it was observed that by optimizing the configurations Au+ ions were located nearer to oxygen atoms of carboxylate groups. According to the theoretical results, one can conclude that reuse ability of Au/GPTS–GLU/MOR/S/NF must be more suitable than Ag/GPTS– GLU/MOR/S/NF, which was confirmed by the experimental results.
4. Conclusions
Fig. 8. TEM images of E. coli cell wall after the treatment time (120 min).
3.5. Theoretical study of the interaction of noble metal ions with GPTS–GLU component on the surface To compare the antibacterial capability of Ag/GPTS–GLU/MOR/S/NF and Au/GPTS–GLU/MOR/S/NF nanostructures, we studied theoretically the interaction of noble metal ions with the functional group GPTS– GLU, which is located on the surface of prepared nanostructures. This is schematically indicated in Fig. 3S. Calculations were performed by Gaussian code in B3LYP basis set. The results for this study are shown in Table 2. From Table 2, one can conclude that the interaction of Au+ ions with GPTS–GLU is stronger than Ag+ ions. The interaction energies for Ag/GPTS–GLU and Au/GPTS–GLU are −11.0532 and −12.5825 eV, respectively which shows that the former case is more stable bond than the latter case. Au+ ions are more massive than Ag+ ions. Thus,
Magnetic core–zeolitic shell nanostructures were prepared by hydrothermal and coprecipitation methods. Analysis of the products confirmed the growth of zeolitic layer on the surface of S/NF cores. The results of VSM analysis showed that the core–shell nanostructures have good magnetization which reduces with the layer deposition on the surface. Antibacterial activity of prepared samples was investigated by inactivation of E. coli as a gram negative bacterium. The results showed that X/GPTS–GLU@MOR/S/NF nanostructures had better antibacterial activity than the noble metal contained MOR/S/NF samples. Their antibacterial activities were attributed to the bond formation between GPTS–GLU component on the surface and the head of lipid A, which is located in the outer membrane of the cell wall. Reuse ability and MIC measurements also confirmed the higher activity of X/GPTS– GLU@MOR/S/NF nanostructures. The interaction of noble metal ions with GPTS–GLU, as a surface component, was studied theoretically and according to the obtained results, it was concluded that Au+ ions stick to the surface more effectively than Ag+ ions. Thus, it was expected that Au/GPTS–GLU@MOR/S/NF has a better reuse ability compared to Ag/GPTS–GLU@MOR/S/NF.
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Fig. 10. Reuse ability measurements for: (a) X/MOR/S/NF nanostructures (red color: Ag/ MOR/S/NF, blue color: Au/MOR/S/NF) and (b) X/GPTS–GLU@MOR/S/NF nanostructures (red color: Ag/GPTS–GLU@MOR/S/NF, blue color: Au/GPTS–GLU@MOR/S/NF).
Table 2 The theoretical results for studying the interaction of noble metals with GPTS–GLU. GPTS–GLU_X
X = Ag
X = Au
Thermal energy (eV) Enthalpy of interaction (eV) Distance of X-O12 Distance of X-O14
−18941.41 −11.0532 2.17 2.13
−18665.86 –12.5825 2.09 2.07
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2013.10.027. References [1] W.R. Jarvis, Selected aspects of the socioeconomic impact of nosocomial infections: morbidity, mortality, cost, and prevention, Infect. Control Hosp. Epidemiol. 17 (1996) 552–557. [2] A. Gallardo-Moreno, M. Pacha-Olivenza, M.C. Fernandez-Calderonb, C. Perez-Giraldo, J. Bruque, M.L. Gonzalez-Martin, Bactericidal behavior Ti6Al4V surfaces after exposure UV-C light, Biomaterials 31 (2010) 5159–5168. [3] W. Pinggui, A.I. James, K.S. Jian, Mechanism of Escherichia coli inactivation on palladium-modified nitrogen-doped titanium dioxide, Biomaterials 31 (2010) 7526–7533.
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2+ [4] A. Langella, M. Pansini, P. Cappelletti, B. Gennaro, M. Gennaro, C. Colella, NH+ , 4 , Cu Zn2+, Cd2+ and Pb2+ exchange for Na+ in a sedimentary clinoptilolite, North Sardinia, Italy, Microporous Mesoporous Mater. 37 (2000) 337–343. [5] A. Cincotti, N. Lai, R. Orru, G. Cao, Sardinian natural clinoptilolite for heavy metals and ammonium removal: experimental and modeling, Chem. Eng. J. 84 (2001) 275–282. [6] V. Badillo-Almaraz, P. Trocellier, I. Davila-Rangel, Adsorption of aqueous Zn(II) species on synthetic zeolites, Nucl. Inst. Methods Phys. Res. 210 (2003) 424–428. [7] J. Peric, M. Trgo, N. Vukojevic-Medvidovic, Removal of zinc, copper and lead by natural zeolite — a comparison of adsorption isotherms, Water Res. 38 (2004) 1893–1899. [8] L. Levy, Y. Sahoo, K.S. Kim, E.J. Bergey, P.N. Prasad, Nanochemistry: synthesis and characterization of multifunctional nanoclinics for biological applications, Chem. Mater. 14 (2002) 3715–3721. [9] Y. Li, B. Yan, C.H. Deng, W.J. Yu, X.Q. Xu, P.Y. Yang, Efficient on-chip proteolysis system based on functionalized magnetic silica microspheres, Proteomics 7 (2007) 2330–2339. [10] C.B. Murray, D.J. Norris, M.G. Bawendi, Synthesis and characterization of nearly monodisperse CdE (E ¼ sulfur, selenium, tellurium) semiconductor nanocrystallites, J. Am. Chem. Soc. 115 (1993) 8706–8715. [11] P.P. Yang, Z.W. Quan, L.L. Lu, S.S. Huang, J. Lin, Bioactive, luminescent and mesoporous europium-doped hydroxyapatite as a drug carrier, Biomaterials 29 (2008) 4341–4347. [12] M. Arruebo, M. Galan, N. Navascues, C. Tellez, C. Marquina, M.R. Ibarra, Development of magnetic nanostructured silica-based materials as potential vectors for drug-delivery applications, Chem. Mater. 18 (2006) 1911–1919. [13] S. Giri, B.G. Trewyn, M.P. Stellmaker, V.S.Y. Lin, Stimuli-responsive controlled release delivery system based on mesoporous silica nanorods capped with magnetic nanoparticles, Angew. Chem. Int. Ed. 44 (2005) 5038–5044. [14] W.R. Zhao, J.L. Gu, L.X. Zhang, H.R. Chen, J.L. Shi, Fabrication of uniform magnetic nanocomposite spheres with a magnetic core/mesoporous silica shell structure, J. Am. Chem. Soc. 127 (2005) 8916–8917. [15] Y.H. Deng, D.W. Qi, C.H. Deng, X.M. Zhang, D.Y. Zhao, Superparamagnetic high magnetization micropheres with a Fe3O4@SiO2 core and perpendicularly aligned mesoporous SiO2 shell for removal of microcystins, J. Am. Chem. Soc. 130 (2008) 28–29. [16] J. Guo, W.L. Yang, C.C. Wang, J. He, J.Y. Chen, Poly (N-isopropylacrylamide)-coated luminescent/magnetic silica microspheres: preparation, characterization, and biomedical applications, Chem. Mater. 18 (2006) 5554–5562. [17] J. Kim, J.E. Lee, J. Lee, J.H. Yu, B.C. Kim, K. An, Magnetic fluorescent delivery vehicle using uniform mesoporous silica spheres embedded with monodisperse magnetic and semiconductor nanocrystals, J. Am. Chem. Soc. 128 (2006) 688–689. [18] X. Zhang, H. Wang, Z. Su, Fabrication of Au@Ag core–shell nanoparticles using polyelectrolyte multilayers as nanoreactors, Langmuir 28 (2012) 15705–15712. [19] B. Chudasama, A.K. Vala, N. Andhariya, R.V. Upadhyay, R.V. Mehta, Enhanced antibacterial activity of bifunctional Fe3O4-Ag core–shell nanostructures, Nano Res. 2 (2012) 955–965. [20] A. Dong, J. Huang, S. Lan, T. Wang, L. Xiao, W. Wang, T. Zhao, X. Zheng, F. Liu, G. Gao, Y. Chen, Synthesis of N-halamine-functionalized silica–polymer core–shell nanoparticles and their enhanced antibacterial activity, Nanotechnology 22 (2011) 295602–295611. [21] Y. Piaoping, Q. Zewei, H. Zhiyao, L. Chunxia, K. Xiaojiao, C. Ziyong, L. Jun, A magnetic, luminescent and mesoporous core–shell structured composite material as drug carrier, Biomaterials 30 (2009) 4786–4795. [22] M. Padervand, M.R. Gholami, Removal of toxic heavy metal ions from waste water by functionalized magnetic core–zeolitic shell nanocomposites as adsorbents, Environ. Sci. Pollut. Res. 20 (2013) 3900–3909. [23] L. Chang-Xiang, L. Qiang, G. Can-Cheng, Synthesis and catalytic abilities of silica-coated Fe3O4 nanoparticle bonded metalloporphyrins with different saturation magnetization, Catal. Lett. 138 (2010) 96–103. [24] M. Zhiya, G. Yueping, L. Huizhou, Superparamagnetic silica nanoparticles with immobilized metal affinity ligands for protein adsorption, J. Magn. Magn. Mater. 301 (2006) 469–477. [25] P. Sharma, P. Rajaram, R. Tomar, Synthesis and morphological studies of nanocrystalline MOR type zeolite material, J. Colloid Interface Sci. 325 (2008) 547–557. [26] M. Padervand, M.R. Elahifard, R.V. Meidanshahi, S. Ghasemi, S. Haghighi, M.R. Gholami, Investigation of the antibacterial and photocatalytic properties of the zeolitic nanosized AgBr/TiO2 composites, Mater. Sci. Semicond. Process. 15 (2012) 73–79. [27] T.J. Beveridge, Ultrastructure, chemistry and function of the bacterial wall, Int. Rev. Cytol. 72 (1981) 229–317. [28] T.J. Beveridge, S.F. Koval, Binding of metals to cell envelopes of Escherichia coli K-12, Appl. Environ. Microbiol. 42 (1981) 325–335. [29] H.C. Neu, L.A. Heppel, On the surface localization of enzymes in E. coli, Biochem. Biophys. Res. Commun. 17 (1964) 215–219. [30] D.F. Broad, J.T. Smith, Escherichia coli 5-nucleotidase: purification, properties and its release by osmotic shock, J. Gen. Microbiol. 123 (1981) 241–247.
