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Recyclable silver–magnetite nanocomposite for antibacterial application
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Recyclable silver–magnetite nanocomposite for antibacterial application Pawinee Theamdee a, Boonjira Rutnakornpituk a,b, Uthai Wichai a,b, Maliwan Nakkuntod c, Metha Rutnakornpituk a,b,* a b c
Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Naresuan University, Phitsanulok 65000, Thailand Center of Excellence in Biomaterials, Faculty of Science, Naresuan University, Phitsanulok 65000, Thailand Department of Biology, Faculty of Science, Naresuan University, Phitsanulok 65000, Thailand
A R T I C L E I N F O
Article history: Received 26 November 2014 Received in revised form 16 February 2015 Accepted 7 March 2015 Available online xxx Keywords: Surface modification Nanocomposite Magnetite Silver Antimicrobial
A B S T R A C T
Surface modification of magnetite nanoparticle (MNP) with cysteine for efficient conjugation with silver nanoparticle (AgNP) is herein presented. This novel nanocomposite was prepared via a specific binding interaction between thiol groups of cysteine coated on the MNP and the surface of AgNP. Transmission electron microscopy indicated the formation of the nanocomposite with 18–24 nm silver nanoparticles and 8–12 nm magnetite nanoparticles. Energy dispersive spectrometry verified the presence of both Ag and Fe in the nanocomposite. After repetitive adsorption–desorption-washing process, the nanocomposite retained higher than 90% antibacterial efficiency against Escherichia coli K12 for at least five recycling cycles. ß 2015 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Introduction Magnetite nanoparticle (MNP) has been long used in many industrial applications such as magnetic recording media and sensors. Recently, the applications of MNP have been more diversified in biomedicine such as targeted drug delivery, contrast agents for magnetic resonance imaging (MRI) [1–3], specific targeting and imaging of cancer cells [4], hyperthermia treatment of tumors [5], enzyme and protein immobilization [6,7] and RNA and DNA purification [8]. MNP for such biomedical applications should be well dispersible in its media, chemically stable, biocompatible and possesses active functions on its surface [9]. MNP with these properties can be enhanced by modifying its surface with long chain polymers to provide steric repulsion mechanism [10] or charge molecules to provide electrostatic repulsion mechanism [11] to the particles. Conjugation of MNP surface with bioentities such as amino acids [12,13], peptides [14], and antibodies [15] was an efficient method to obtain the particle with active functions for advanced and specific applications.
* Corresponding author at: Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Naresuan University, Phitsanulok 65000, Thailand. Tel.: +66 55 96 3464; fax: +66 55 96 3401. E-mail address: [email protected] (M. Rutnakornpituk).
Synthesis of nanocomposite containing MNP and silver nanoparticle (AgNP) has been extensively studied by many groups [16–18]. AgNP has drawn a lot of attention particularly as an antibacterial agent because of its stability, durability and good antibacterial activity [19–24]. Therefore, nanocomposite of MNP and AgNP combined the functions of both components, magnetic responsiveness from MNP and antibacterial activity from AgNP. Many previous works reported on preparation of binary Ag– magnetite nanocomposite for antibacterial applications [25–28]. However, aggregation of these nanocomposite due to magnetic and electrostatic attractive forces was a major limitation in the synthesis process and particularly when applying it in practice [29,30]. To overcome these problems, coating the nanocomposite with polymer matrix provided good stabilization and good dispersibility in a media. Nonetheless, this method might limit application potential of the nanocomposite from a restriction of its possible transport due to a large size of the polymer matrix [31], and also reduced its magnetic responsiveness and antibacterial effectiveness. In this work, surface modification of MNP with cysteine to obtain specific binding interaction with AgNP is presented. Cysteine, a water-soluble and sulfur-containing amino acid [32,33], was chosen for coating MNP surface in this work because the thiol groups in cysteine molecules can provide strong binding interactions with AgNP [34–37]. In addition, the existence of the
http://dx.doi.org/10.1016/j.jiec.2015.03.018 1226-086X/ß 2015 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
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NH2 and COOH groups in cysteine molecules will enable the cysteine-coated MNP to have good biocompatibility and good dispersibility in aqueous media. Unlike cysteine, the other amino acid containing a thiol group such as methionine is classified as nonpolar and hydrophobic because it has a straight side chain that possesses an S-methyl thioether. In the present work, AgNP, known to have extraordinary inhibitory and bactericidal properties, was conjugated to MNP surface using cysteine as a linker to form magnetic AgNP-containing nanocomposite. It was hypothesized that silver ions interacted with sulfhydryl groups, or thiol groups (–SH), on the membranes of bacteria, causing disruption of their permeability and thereby leading to microbial cell death [38–40]. The novelty of this work is that this is the first report on coating MNP surface with cysteine and its use to effectively bind AgNP on the surface. In addition, taking advantages of MNP, the nanocomposite was efficiently used as a recyclable antibacterial agent. In this research, amino-coated MNP reacted with glycidyl methacrylate as a linker to obtain the MNP coated with methacrylate functional groups. The MNP surface was then further modified via a Michael addition reaction between the methacrylate and cysteine to form cysteine-coated MNP (Fig. 1). The functional groups of the MNP were studied via Fourier transform infrared spectroscopy (FTIR) and the hydrodynamic size and zeta potential values were characterized via photocorrelation spectroscopy (PCS). AgNP was separately synthesized via a chemical reduction method and subsequently conjugated with cysteine-coated MNP. Transmission electron microscopy (TEM) and energy dispersive spectrometry (EDS) techniques were used to verify the successful conjugation of the MNP with AgNP. In addition, the magnetic properties and composition of the nanocomposite were investigated using vibrating sample magnetometry (VSM) and thermogravimetric analysis (TGA), respectively. Antibacterial activity and recyclable efficiency of Ag–magnetite nanocomposite were also discussed.
Experimental Materials Unless otherwise stated, all reagents were used as received; iron(III) chloride anhydrous (FeCl3) (Carlo Erba), iron(II) chloride tetrahydrate (FeCl24H2O) (Carlo Erba), ammonium hydroxide (28–30%, J.T. Baker), oleic acid (Fluka), 3-aminopropyl triethoxysilane (99%, Acros), glycidyl methacrylate (97%, Sigma), L-cysteine (99%, Acros), sodium borohydride (Fisher Scientific) and silver nitrate (99.8%, Merck). Triethylamine (97%, Carto Erba) and toluene were stirred under CaH2 and distilled prior to use. Syntheses Synthesis of MNP and methacrylate-coated MNP (MA-coated MNP) A FeCl3 solution (1.66 g in 20 ml deionized water) and a FeCl24H2O solution (1.00 g in 20 ml deionized water) were mixed together with stirring, followed by adding 25% NH4OH (20 ml). After 30 min stirring, the mixture was centrifuged for 20 min to precipitate large aggregate and the aqueous supernatant was discarded. An oleic acid solution in hexane (2 ml in 20 ml hexane, 10%v/v) was then added into the MNP dispersion with stirring. The dispersion was concentrated by evaporating hexane to obtain concentrated MNP in hexane. The MNP was then re-suspended in toluene in the presence of triethylamine (1.2 ml, 0.0086 mol), followed by adding 3-aminopropyl triethoxysilane (1.2 ml, 0.0068 mol). After stirring at room temperature for 24 h, the dispersion was precipitated in ethanol and washed with toluene to obtain amino-coated MNP. To prepare MA-coated MNP, the aminocoated MNP was stirred in toluene for 15 min, following by adding triethylamine (1.39 ml, 0.0099 mol) and glycidyl methacrylate (1.33 ml, 0.01 mol). After stirring at 60 8C overnight under nitrogen atmosphere, MA-coated MNP was retrieved by a magnet, repeatedly washed with ethanol and toluene, and dried under reduced pressure.
Fig. 1. Schematic diagram for the synthesis of Ag–magnetite nanocomposite.
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Synthesis of cysteine-coated MNP MA-coated MNP dispersion (0.1 g of MA-coated MNP in 20 ml water) was added to the 50 ml round-bottom flask containing a cysteine solution (0.1 g, 0.0083 mol in 10 ml pH 12 phosphate buffer solution). After stirring at 60 8C for 18 h, the particles were then precipitated in water. This procedure was repeated several times to completely remove unreacted cysteine from the cysteinecoated MNP. The particles were finally dried under reduced pressure. Synthesis of silver nanoparticles (AgNP) A large excess of sodium borohydride is needed to reduce the ionic silver and to stabilizer AgNP. A 2 ml volume of 1.0 mM silver nitrate was added dropwise (about 1 drop/s) to 30 ml of a 2.0 mM sodium borohydride solution that was chilled in an ice-bath with stirring. The solution turned light yellow upon addition of 2 ml of a silver nitrate solution, indicating the formation of AgNP. Synthesis of Ag–magnetite nanocomposite by immobilization of cysteine-coated MNP on AgNP surface The as-prepared AgNP dispersion (30 ml water) was added to a cysteine-coated MNP dispersion (1 mg of the MNP in 30 ml water) with stirring for 18 h. The particles were precipitated using an external magnet and re-dispersed in fresh water. This procedure was repeated several times to remove ungrafted AgNP. The particles were finally dried under reduced pressure. Study in the recyclable efficiency of Ag–magnetite nanocomposite in antibacterial applications Antimicrobacterial testing experiments were conducted using a modified ASTM standard (an E2149-01 standard test method for determining the antimicrobial activity of immobilized antimicrobial agents under dynamic contact condition). A colony of Escherichia coli K12 grown on a Luria agar (LB-agar) plate was used to inoculate 50 ml of Luria broth in a sterile conical flask. The culture was incubated at 37 8C with shaking at 300 rpm for 18–20 h. The cells were diluted with 0.85% normal saline to the desired concentrations. Ag–magnetite nanocomposite (10 mg) was shaken in 1 ml of a bacterial suspension containing E. coli K12 for 60 min at 37 8C. The mixture was placed in a magnetic separation stand at room temperature for 10 min. The nanocomposite was attracted onto the wall of the tube using a permanent magnet. The supernatant was decanted, diluted and placed on LB-agar plates. After overnight incubation, the number of viable cells was determined in colony forming units (CFUs). Antibacterial efficiency of Ag– magnetite nanocomposite was shown by the percentage of bacteria killed by the nanocomposite and it was calculated based on Eq. (1). Antibacterial efficiency ¼
F control Ncontrol F sample Nsample F control Ncontrol 100
(1)
where Ncontrol, Nsample are the number of the colonies on the L-agar plates of the control and the sample, respectively, and Fcontrol, Fsample are the dilution factor of the control and the sample, respectively. Characterization of MNP, AgNP and Ag–magnetite nanocomposite FTIR was performed on a Perkin–Elmer Model 1600 Series FTIR Spectrophotometer. The solid samples were mixed with KBr to form pellets. Particles size and size distribution were observed on TEM. TEM images were taken using a Philips Tecnai 12 operated at 120 kV equipped with Gatan model 782 CCD camera. The particles
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were re-suspended in water with sonication before deposition on a TEM grid. PCS was performed on NanoZS4700 nanoseries Malvern instrument at 25 8C. Water, used as a dispersing media, was filtered using nylon syringe filters with a pore size of 0.2 mm before used. The dispersions were sonicated for 15 min before each measurement without filtration. TGA was performed on SDTA 851 MettlerToledo at the temperature ranging between 25 and 600 8C at a heating rate of 20 8C/min under oxygen atmosphere. AgNP was analyzed using Specord S100 UV–Visible spectrophotometer (Analytikjena AG) coupled with a photo diode array detector. Magnetic properties of the particles were measured at room temperature using a Standard 7403 Series, VSM. Magnetic moment was investigated in a range of 10,000 G of applied magnetic fields using 30 min sweep time. Fe and Ag concentrations were analyzed by flame atomic absorption spectroscopy (AAS) (Varian model SpectraAA200) and calculated from sample responses relative to those of standards and blanks. EDS was performed on LEO32 energydispersive X-ray microanalyser. Results and discussion The primary aim in this work was to synthesize a novel nanocomposite of AgNP and MNP for use in antibacterial applications. AgNP is known to have extraordinary inhibitory and bactericidal properties, while MNP can facilitate a separation process due to its magnetically guidable properties, which is greatly useful for recycling purposes. Therefore, this novel nanocomposite possesses advantageous properties of both AgNP and MNP. In this work, MNP surface was designed to have thiol functional groups (–SH), known to have specific interaction with surface of AgNP [40]. Cysteine, a kind of amino acid, was selected for surface modification of MNP because of the existence of –SH groups. In addition, the presence of –COO on the cysteine-coated MNP might be necessary for nanoparticle stabilization due to electrostatic repulsion mechanism. In the first step, MNP was surface modified with 3-aminopropyl triethoxysilane via a ligand exchange and a silanization reaction to obtain amino-coated MNP (Fig. 1). The amino groups on MNP surface were readily reacted with epoxide rings of glycidyl methacrylate to form methacrylate-coated MNP (MA-coated MNP). Glycidyl methacrylate served as a linker to facilitate the accessibility of –NH2 of cysteine to react with MA-coated MNP via a Michael addition reaction. It should be noted that this reaction was performed in a basic condition (pH 12), so that –NH2 of cysteine was in a deprotonated form (pKa of –NH2 of cysteine is 10.28) [41] to serve as a nucleophile to react with methacrylate functional groups coated on MNP surface. Bare MNP and MNPs modified with amino groups, MA and cysteine were characterized using FTIR (Fig. 2). FTIR spectrum of bare MNP shows a distinctive signal of Fe–O bond at 589 cm1 of MNP core (spectrum A, Fig. 2). In the spectrum of amino-coated MNP (spectrum B, Fig. 2), the characteristic signals of N–H stretching (3401 cm1), Si–O stretching cm1 (1115–1001 cm1) and Fe–O bond of MNP core (586 cm1) were observed. The amino groups on MNP surface reacted with epoxide rings of glycidyl methacrylate to form MA-coated MNP as indicated by the presence of carbonyl group signal (1716 cm1) and C5 5C stretching (1606 cm1) (spectrum C, Fig. 2). After grafting the MNP with cysteine, the thiol-derived band (S–H) was observed at 2092 cm1, indicating the formation of cysteine-coated MNP (spectrum D, Fig. 2). It should be noted that amino-coated MNP and MA-coated MNP were not well dispersible in water when compared to those of cysteine-coated MNP. It was rationalized that negative charges of carboxylate ions of cysteine coated on the MNP led to electrostatic repulsion toward neighboring particles and thus prevented
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Fig. 2. FTIR spectra of (A) bare MNP, (B) amino-coated MNP, (C) MA-coated MNP and (D) cysteine-coated MNP.
massive flocculation of the particles. This assumption was supported by the formation of slight particle aggregation in neutral aqueous media and even more in acidic condition, indicating pH-responsive properties of cysteine-coated MNP (Fig. 3). Hydrodynamic size (Dh) and zeta potential values of cysteinecoated MNP were determined as a function of dispersion pH via PCS (Fig. 4). Dh of the particles increased when decreasing the dispersion pH (Fig. 4A). The larger size of the particles in acidic pH dispersion signified that there was some degree of nano-scale agglomeration. Therefore, the smaller size of the particles in basic
Fig. 3. The appearance of cysteine-coated MNP dispersed in acidic, neutral and basic aqueous dispersions. The concentrations of these cysteine-coated MNP in water were 0.05 mg/ml. The arrows indicate the particle aggregation on the bottom of acidic dispersions.
pH dispersion implied the improved dispersibility of the particles in water. Dh was in the range of 80–500 nm in basic pH dispersion, while those in acidic condition was in the range of 500–1400 nm. This was in good agreement with the observation of the particle dispersibility in water shown in Fig. 3. In addition, zeta potential values of the particles in the dispersions with various pHs also supported the aforementioned discussion that cysteine coated on MNP was in a deprotonated form (COO) in neutral and basic pH values and in protonated COOH in acidic pH dispersion (Fig. 4). According to the plot in Fig. 4B, the isoelectric point (PI) of cysteine-coated MNP was 5.8. To prepare Ag–magnetite nanocomposite, AgNP dispersion in water was continuously stirred with cysteine-coated MNP dispersion for 18 h under ambient condition. After removing ungrafted AgNP from the dispersion, the composition of the nanocomposite was analyzed via AAS to determine the ratio of Ag to Fe, corresponding to the ratio of AgNP to the MNP in the nanocomposite. Various feed ratios of AgNP to the MNP were performed to investigate highest percent adsorption efficiency of these two components (Table S1 in the supporting information). It was found that 20 wt% AgNP in feed resulted in about 11% adsorption efficiency. Increasing the ratio of AgNP to the MNP in feed seemed to increase the concentration of AgNP in the composite but significantly lowered percent adsorption efficiency in the reaction. Therefore, 20 wt% AgNP in feed would be used for the synthesis of Ag–magnetite nanocomposites in the following experiments.
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Fig. 4. (A) Hydrodynamic size (Dh) and (B) zeta potentials of cysteine-coated MNP as a function of solution pH.
Fig. 5. TEM images of (A) AgNP (the inset shows a UV–Vis absorption spectrum of AgNP at 410 nm), (B) cysteine-coated MNP and (C) Ag–magnetite nanocomposite. The arrows indicate AgNP conjugating with cysteine-coated MNP.
The TEM images of AgNP, cysteine-coated MNP and Ag– magnetite nanocomposite are shown in Fig. 5. Size of AgNP ranges between 18 nm and 24 nm in diameter and UV–Visible absorption spectrum shows the lmax at 410 nm, which is the characteristic signal of AgNP (Fig. 5A) [42]. TEM of cysteine-coated MNP shows the particles with size of 8–12 nm in diameter with some nanoaggregation (Fig. 5B). This nano-scale aggregation was typical for the MNP synthesized via a co-precipitation method [43,44]. It should be noted that although there was some nano-aggregation observed in TEM, these particles were well dispersible in water (neutral to basic pH dispersions) without noticeable macroscopic aggregation. In Fig. 5C, TEM shows the formation of the nanocomposite between the MNP and AgNP. This nanocomposite retained good dispersibility in water with excellent responsive properties to a permanent magnet. The chemical compositions of cysteine-coated MNP and Ag– magnetite nanocomposite were investigated using EDS technique (Fig. 6). The signals of Fe from MNP core, thiol (S) from cysteine and Si from APS coating (the first layer on the particles) were observed in cysteine-coated MNP (Fig. 6A). In Ag–magnetite nanocomposite, a distinctive signal of Ag was observed in addition to Fe, S and Si signals, indicating the presence of Ag in the nanocomposite (Fig. 6B). TGA studies were carried out to determine the mass loss of the organic components in the surface-modified MNP in each step and Ag–magnetite nanocomposite (Fig. 7A). Bare MNP showed no sign of weight loss due to the absence of organic components in the particles. After each step of the reaction, percent weight loss of the surface-modified MNPs continuously decreased, indicating more organic contents in the complexes. The percent weight loss slightly increased when the MNP was conjugated with AgNP to form Ag– magnetite nanocomposite. This was attributed to the presence of
Fig. 6. EDS spectra showing chemical compositions of (A) cysteine-coated MNP and (B) Ag–magnetite nanocomposite.
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Fig. 7. (A) TGA thermograms and (B) M–H curves of (a) bare MNP, (b) amino-coated MNP, (c) MA-coated MNP, (d) cysteine-coated MNP and (e) Ag–magnetite nanocomposite.
inorganic Ag in addition to Fe from MNP core, resulting in an increase in total inorganic content in the complexes. It should be mentioned that the percentage of organic components in cysteine-coated MNP was rather low (23 wt%), indicating high percentage of magnetite core in the complex (77 wt% iron oxide) (thermogram d in Fig. 7A). This high percent magnetite when compared to polymer-coated MNPs in other systems (20–40 wt% iron oxide) [45,46] was attributed to the existence of thin layers of cysteine on the MNP surface. This might be important that when conjugated with AgNP to form Ag– magnetite nanocomposite, magnetic responsiveness of the nanocomposite should be high enough for facilitating magnetic separation and multiple recycling processes. M–H curves of the particles in each step of the reaction are shown in Fig. 7B. They showed superparamagnetic behavior as indicated by the absence of remanence and coercivity when the external applied magnetic field was removed. The decrease of saturation magnetization (Ms) from 70 emu/g of bare MNP to 48 emu/g of Ag–magnetite nanocomposite was attributed to the presence of the organic components grafted on their surface. The Ms value of the nanocomposite (48 emu/g) was rather high when compared with Ms values of polymer-coated MNP in other systems
(10–30 emu/g) [45–47]. This was attributed to the high magnetite content in its structure due to a lack of polymer coating and this was in good agreement with TGA results. To investigate antibacterial activity and recyclable efficiency of Ag–magnetite nanocomposite and cysteine-coated MNP, bare MNP was used as a control sample. After shaking the particles in bacterial suspension containing E. coli K12 at 37 8C for 50 min, they were then separated from the dispersion using a permanent magnet. The supernatant was diluted and placed on LB-agar plates. After overnight incubation, the number of viable cells was counted in colony forming units (CFUs), which is a direct measurement of bacterial viability. The removed particles were reused by resuspending in an E. coli K12 suspension, and the procedure described above was performed repeatedly (Fig. 8). It should be noted that the particles were not washed with any solvent during recycling process. Fig. 9 shows recyclable efficiency of Ag–magnetite nanocomposite when compared to those of cysteine-coated MNP (without AgNP). Antibacterial efficiency of the nanocomposite remained higher than 90% after five recycling process, whereas cysteinecoated MNP did not or insignificantly show antibacterial efficiency. After repeatedly used for eight cycles, antibacterial efficiency of the
Fig. 8. Recycling processes of Ag–magnetite nanocomposite for antibacterial application.
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Fig. 9. Antibacterial efficiency of Ag–magnetite nanocomposite (&) and cysteine-coated MNP (&) after eight recycling processes.
nanocomposite slightly dropped but still higher than 69%. This excellent antibacterial efficiency was attributed to the presence of AgNP in the nanocomposite. The number of survived E. coli K12 in the experiments of the nanocomposite was provided in Table S2 in the supporting information.
Conclusions This work presented a surface modification of MNP with cysteine for a specific binding interaction with AgNP to obtain Ag– magnetite nanocomposite for antibacterial application. After coating MNP with cysteine, the particles exhibited pH-responsive properties and good dispersibility in water due to the presence of – COO groups on its surface, leading to electrostatic repulsive force and thus stabilizing the particles in water. After conjugation of cysteine-coated MNP with AgNP, the nanocomposite retained its good dispersibility in water with good magnetic responsiveness. Taking advantage of a fast response to an applied magnetic field, the nanocomposite can be easily removed from water for multiple cycles of antibacterial tests without a washing step. The antibacterial efficiency against E. coli K12 of the nanocomposite remained higher than 90% (1011 E. coli/10 mg nanoparticles) after five recycling processes. Acknowledgments The authors thank the Thailand Research Fund (TRF) (DBG5580002) for financial funding. Thailand Toray Science Foundation (TTSF) is also gratefully acknowledged for the support. PT thanks the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Commission on Higher Education, Ministry of Education for the scholarship. The authors thank Dr. Rerngwit Boonyom from the Department of Medical Technology, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok, Thailand, for providing E. coli K12.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jiec.2015.03.018.
References [1] C. Yu, B. YunPeng, T. Bao, L. ZhaoLong, Chin. Sci. Bull. 54 (2009) 1190. [2] A. Singh, F. Dilnawaz, S. Mewar, U. Sharma, N.R. Jagannathan, S.K. Sahoo, ACS Appl. Mater. Interface 3 (2011) 842. [3] A. Manikandan, J.J. Vijaya, J.A. Mary, L.J. Kennedy, A. Dines, J. Ind. Eng. Chem. 20 (2014) 2077. [4] B.F. Pan, F. Gao, H.C. Gu, J. Colloid Interface Sci. 284 (2005) 1. [5] A. Ito, F. Matsuoka, H. Honda, T. Kobavbyashi, Cancer Gene Ther. 10 (2003) 918. [6] K.M. Ponvel, D.G. Lee, E.J. Woo, I.S. Ahn, C.H. Lee, Koren J. Chem. Eng. 26 (2009) 127. [7] R. Khatiri, A. Reyhani, S.Z. Mortazavi, M. Hossainalipour, J. Ind. Eng. Chem. 19 (2013) 1642. [8] K. Kang, J. Choi, J.H. Nam, S.C. Lee, K.J. Kim, S.W. Lee, J.H. Chang, J. Phys. Chem. B 113 (2009) 536. [9] M.E. Park, J.H. Chang, Mater. Sci. Eng., C 27 (2007) 1232. [10] D.K. Kim, M. Mikhaylova, Y. Zhang, M. Muhammed, Chem. Mater. 15 (2003) 1617. [11] N.A. Frey, S. Peng, K. Cheng, S. Sun, Chem. Soc. Rev. 38 (2009) 2532. [12] S.L. Tie, Y.Q. Lin, H.C. Lee, Y.S. Bae, C.H. Lee, Colloids Surf., A 273 (2006) 75. [13] N. Ballav, H.J. Choi, S.B. Mishra, A. Maity, J. Ind. Eng. Chem. 20 (2014) 4085. [14] Z.C. Xing, M.J. Park, S.J. Han, I.K. Kang, M.J. Choi, B.H. Lee, Y. Chang, Macromol. Res. 19 (2011) 897. [15] E.S. Krystofiak, V.Z. Matson, D.A. Steeber, J.A. Oliver, J. Nanomater. (2012) 1. [16] E.I. Silva, J. Rivas, L.M. Leo´n Isidro, M.A. Lo´pez-Quintela, J. Non-Cryst. Solids 353 (2007) 829. [17] B. Lee, S. Koo, J. Ind. Eng. Chem. 17 (2011) 762. [18] B. Lee, S. Koo, J. Ind. Eng. Chem. 18 (2012) 1191. [19] S.D. Solomon, M. Bahadory, A.V. Jeyarajasingam, S.A. Rutkowsky, C. Boritz, J. Chem. Educ. 84 (2007) 322. [20] R. Nithya, R. Ragunathan, Dig. J. Nanomater. Biostruct. 4 (2009) 623. [21] H. Jiang, S. Manolache, A.C. Lee Wong, F.S. Denes, J. Appl. Polym. Sci. 93 (2004) 1411. [22] C. Krishnaraj, E.G. Jagan, S. Rajasekar, P. Selvakumar, P. Kalaichelvan, N. Mohan, Colloids Surf., B 76 (2010) 50. [23] L. Zhang, Y.H. Dou, H.C. Gu, J. Colloid Interface Sci. 297 (2006) 660. [24] C. Lee, K.Y. Nam, D.Y. Kim, H.J. Kim, W.H. Doh, H.K. Choi, M.J. Jung, J. Ind. Eng. Chem. 20 (2014) 1276. [25] M. Mahmoudi, V. Serpooshan, ACS Nano 6 (2012) 2656. [26] X. Zhang, H. Niu, J. Yan, Y. Cai, Colloids Surf., A 375 (2011) 186. [27] T. Mosaiab, C.J. Jeong, G.J. Shin, K.H. Choi, S.K. Lee, I. Lee, I. In, S.Y. Park, Mater. Sci. Eng., C 33 (2013) 3786. [28] M. Sureshkumar, D.Y. Siswanto, C.K. Lee, J. Mater. Chem. 20 (2010) 6948. [29] J.L. Mietta, M.M. Ruiz, P.S. Antonel, O.E. Perez, A. Butera, G. Jorge, R.M. Negri, Langmuir 28 (2012) 6985. [30] S. Mishra, N.G. Shimpi, T. Sen, J. Polym. Res. 49 (2013) 1. [31] R. Prucek, J. Tucek, M. Kilianova´, A. Pana´cek, L. Kvı´tek, J. Filip, M. Kola´r, K. Toma´nkova´, R. Zboril, Biomaterials 32 (2011) 4704. [32] K. Hayashi, K. Ono, H. Suzuki, M. Sawada, M. Moriya, W. Sakamoto, T. Yogo, Chem. Mater. 22 (2010) 3768. [33] H. Cohen, A. Gedanken, Z. Zhong, J. Phys. Chem. C 112 (2008) 15429. [34] D. Lee, R.E. Cohen, M.F. Rubner, Langmuir 21 (2005) 9651. [35] X. Yang, A.P. Gondikas, S.M. Marinakos, M. Auffan, J. Liu, H.H. Kim, J.N. Meyer, Environ. Sci. Technol. 46 (2012) 1119.
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[36] C. Levard, E.M. Hotze, G.V. Lowry, G.E. Brown, Environ. Sci. Technol. 46 (2012) 6900. [37] C.L. Arnaout, C.K. Gunsch, Environ. Sci. Technol. 46 (2012) 5387. [38] C.M. Jones, E.M.V. Hoek, J. Nanopart. Res. 12 (2010) 1531. [39] S.R. Goswami, T. Sahareen, M. Singh, S. Kumar, J. Ind. Eng. Chem. (2014). [40] W. Gan, B. Xu, H.L. Dai, Angew. Chem. Int. Ed. 50 (2011) 6622. [41] A. Mocanu, I. Cernica, T. Gheorghe, L.D. Bobos, O. Horovitz, M.T. Cotisel, Colloids Surf., A 338 (2009) 93. [42] S. Pal, Y.K. Tak, J.M. Song, Appl. Envion. Microbiol. 73 (2007) 1712.
[43] R.A. Frimpong, J. Dou, M. Pechan, J.Z. Hilt, J. Magn. Magn. Mater. 322 (2010) 326. [44] M. Shabanian, M. Khoobi, F. Hemati, H.A. Khonakdar, S.S. ebrahimi, U. Wagenknecht, A. Shafiee, J. Ind. Eng. Chem. (2014). [45] B. Rutnakornpituk, U. Wichai, T. Vilaivan, M. Rutnakornpituk, J. Nanopart. Res. 13 (2011) 6847. [46] F.X. Hu, K.G. Neoh, E.T. Kang, Biomaterials 27 (2006) 5725. [47] P. Theamdee, B. Rutnakornpituk, U. Wichai, M. Rutnakornpituk, J. Nanopart. Res. 16 (2014) 1.
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Recyclable silver–magnetite nanocomposite for antibacterial application Pawinee Theamdee a, Boonjira Rutnakornpituk a,b, Uthai Wichai a,b, Maliwan Nakkuntod c, Metha Rutnakornpituk a,b,* a b c
Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Naresuan University, Phitsanulok 65000, Thailand Center of Excellence in Biomaterials, Faculty of Science, Naresuan University, Phitsanulok 65000, Thailand Department of Biology, Faculty of Science, Naresuan University, Phitsanulok 65000, Thailand
A R T I C L E I N F O
Article history: Received 26 November 2014 Received in revised form 16 February 2015 Accepted 7 March 2015 Available online xxx Keywords: Surface modification Nanocomposite Magnetite Silver Antimicrobial
A B S T R A C T
Surface modification of magnetite nanoparticle (MNP) with cysteine for efficient conjugation with silver nanoparticle (AgNP) is herein presented. This novel nanocomposite was prepared via a specific binding interaction between thiol groups of cysteine coated on the MNP and the surface of AgNP. Transmission electron microscopy indicated the formation of the nanocomposite with 18–24 nm silver nanoparticles and 8–12 nm magnetite nanoparticles. Energy dispersive spectrometry verified the presence of both Ag and Fe in the nanocomposite. After repetitive adsorption–desorption-washing process, the nanocomposite retained higher than 90% antibacterial efficiency against Escherichia coli K12 for at least five recycling cycles. ß 2015 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Introduction Magnetite nanoparticle (MNP) has been long used in many industrial applications such as magnetic recording media and sensors. Recently, the applications of MNP have been more diversified in biomedicine such as targeted drug delivery, contrast agents for magnetic resonance imaging (MRI) [1–3], specific targeting and imaging of cancer cells [4], hyperthermia treatment of tumors [5], enzyme and protein immobilization [6,7] and RNA and DNA purification [8]. MNP for such biomedical applications should be well dispersible in its media, chemically stable, biocompatible and possesses active functions on its surface [9]. MNP with these properties can be enhanced by modifying its surface with long chain polymers to provide steric repulsion mechanism [10] or charge molecules to provide electrostatic repulsion mechanism [11] to the particles. Conjugation of MNP surface with bioentities such as amino acids [12,13], peptides [14], and antibodies [15] was an efficient method to obtain the particle with active functions for advanced and specific applications.
* Corresponding author at: Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Naresuan University, Phitsanulok 65000, Thailand. Tel.: +66 55 96 3464; fax: +66 55 96 3401. E-mail address: [email protected] (M. Rutnakornpituk).
Synthesis of nanocomposite containing MNP and silver nanoparticle (AgNP) has been extensively studied by many groups [16–18]. AgNP has drawn a lot of attention particularly as an antibacterial agent because of its stability, durability and good antibacterial activity [19–24]. Therefore, nanocomposite of MNP and AgNP combined the functions of both components, magnetic responsiveness from MNP and antibacterial activity from AgNP. Many previous works reported on preparation of binary Ag– magnetite nanocomposite for antibacterial applications [25–28]. However, aggregation of these nanocomposite due to magnetic and electrostatic attractive forces was a major limitation in the synthesis process and particularly when applying it in practice [29,30]. To overcome these problems, coating the nanocomposite with polymer matrix provided good stabilization and good dispersibility in a media. Nonetheless, this method might limit application potential of the nanocomposite from a restriction of its possible transport due to a large size of the polymer matrix [31], and also reduced its magnetic responsiveness and antibacterial effectiveness. In this work, surface modification of MNP with cysteine to obtain specific binding interaction with AgNP is presented. Cysteine, a water-soluble and sulfur-containing amino acid [32,33], was chosen for coating MNP surface in this work because the thiol groups in cysteine molecules can provide strong binding interactions with AgNP [34–37]. In addition, the existence of the
http://dx.doi.org/10.1016/j.jiec.2015.03.018 1226-086X/ß 2015 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
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NH2 and COOH groups in cysteine molecules will enable the cysteine-coated MNP to have good biocompatibility and good dispersibility in aqueous media. Unlike cysteine, the other amino acid containing a thiol group such as methionine is classified as nonpolar and hydrophobic because it has a straight side chain that possesses an S-methyl thioether. In the present work, AgNP, known to have extraordinary inhibitory and bactericidal properties, was conjugated to MNP surface using cysteine as a linker to form magnetic AgNP-containing nanocomposite. It was hypothesized that silver ions interacted with sulfhydryl groups, or thiol groups (–SH), on the membranes of bacteria, causing disruption of their permeability and thereby leading to microbial cell death [38–40]. The novelty of this work is that this is the first report on coating MNP surface with cysteine and its use to effectively bind AgNP on the surface. In addition, taking advantages of MNP, the nanocomposite was efficiently used as a recyclable antibacterial agent. In this research, amino-coated MNP reacted with glycidyl methacrylate as a linker to obtain the MNP coated with methacrylate functional groups. The MNP surface was then further modified via a Michael addition reaction between the methacrylate and cysteine to form cysteine-coated MNP (Fig. 1). The functional groups of the MNP were studied via Fourier transform infrared spectroscopy (FTIR) and the hydrodynamic size and zeta potential values were characterized via photocorrelation spectroscopy (PCS). AgNP was separately synthesized via a chemical reduction method and subsequently conjugated with cysteine-coated MNP. Transmission electron microscopy (TEM) and energy dispersive spectrometry (EDS) techniques were used to verify the successful conjugation of the MNP with AgNP. In addition, the magnetic properties and composition of the nanocomposite were investigated using vibrating sample magnetometry (VSM) and thermogravimetric analysis (TGA), respectively. Antibacterial activity and recyclable efficiency of Ag–magnetite nanocomposite were also discussed.
Experimental Materials Unless otherwise stated, all reagents were used as received; iron(III) chloride anhydrous (FeCl3) (Carlo Erba), iron(II) chloride tetrahydrate (FeCl24H2O) (Carlo Erba), ammonium hydroxide (28–30%, J.T. Baker), oleic acid (Fluka), 3-aminopropyl triethoxysilane (99%, Acros), glycidyl methacrylate (97%, Sigma), L-cysteine (99%, Acros), sodium borohydride (Fisher Scientific) and silver nitrate (99.8%, Merck). Triethylamine (97%, Carto Erba) and toluene were stirred under CaH2 and distilled prior to use. Syntheses Synthesis of MNP and methacrylate-coated MNP (MA-coated MNP) A FeCl3 solution (1.66 g in 20 ml deionized water) and a FeCl24H2O solution (1.00 g in 20 ml deionized water) were mixed together with stirring, followed by adding 25% NH4OH (20 ml). After 30 min stirring, the mixture was centrifuged for 20 min to precipitate large aggregate and the aqueous supernatant was discarded. An oleic acid solution in hexane (2 ml in 20 ml hexane, 10%v/v) was then added into the MNP dispersion with stirring. The dispersion was concentrated by evaporating hexane to obtain concentrated MNP in hexane. The MNP was then re-suspended in toluene in the presence of triethylamine (1.2 ml, 0.0086 mol), followed by adding 3-aminopropyl triethoxysilane (1.2 ml, 0.0068 mol). After stirring at room temperature for 24 h, the dispersion was precipitated in ethanol and washed with toluene to obtain amino-coated MNP. To prepare MA-coated MNP, the aminocoated MNP was stirred in toluene for 15 min, following by adding triethylamine (1.39 ml, 0.0099 mol) and glycidyl methacrylate (1.33 ml, 0.01 mol). After stirring at 60 8C overnight under nitrogen atmosphere, MA-coated MNP was retrieved by a magnet, repeatedly washed with ethanol and toluene, and dried under reduced pressure.
Fig. 1. Schematic diagram for the synthesis of Ag–magnetite nanocomposite.
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Synthesis of cysteine-coated MNP MA-coated MNP dispersion (0.1 g of MA-coated MNP in 20 ml water) was added to the 50 ml round-bottom flask containing a cysteine solution (0.1 g, 0.0083 mol in 10 ml pH 12 phosphate buffer solution). After stirring at 60 8C for 18 h, the particles were then precipitated in water. This procedure was repeated several times to completely remove unreacted cysteine from the cysteinecoated MNP. The particles were finally dried under reduced pressure. Synthesis of silver nanoparticles (AgNP) A large excess of sodium borohydride is needed to reduce the ionic silver and to stabilizer AgNP. A 2 ml volume of 1.0 mM silver nitrate was added dropwise (about 1 drop/s) to 30 ml of a 2.0 mM sodium borohydride solution that was chilled in an ice-bath with stirring. The solution turned light yellow upon addition of 2 ml of a silver nitrate solution, indicating the formation of AgNP. Synthesis of Ag–magnetite nanocomposite by immobilization of cysteine-coated MNP on AgNP surface The as-prepared AgNP dispersion (30 ml water) was added to a cysteine-coated MNP dispersion (1 mg of the MNP in 30 ml water) with stirring for 18 h. The particles were precipitated using an external magnet and re-dispersed in fresh water. This procedure was repeated several times to remove ungrafted AgNP. The particles were finally dried under reduced pressure. Study in the recyclable efficiency of Ag–magnetite nanocomposite in antibacterial applications Antimicrobacterial testing experiments were conducted using a modified ASTM standard (an E2149-01 standard test method for determining the antimicrobial activity of immobilized antimicrobial agents under dynamic contact condition). A colony of Escherichia coli K12 grown on a Luria agar (LB-agar) plate was used to inoculate 50 ml of Luria broth in a sterile conical flask. The culture was incubated at 37 8C with shaking at 300 rpm for 18–20 h. The cells were diluted with 0.85% normal saline to the desired concentrations. Ag–magnetite nanocomposite (10 mg) was shaken in 1 ml of a bacterial suspension containing E. coli K12 for 60 min at 37 8C. The mixture was placed in a magnetic separation stand at room temperature for 10 min. The nanocomposite was attracted onto the wall of the tube using a permanent magnet. The supernatant was decanted, diluted and placed on LB-agar plates. After overnight incubation, the number of viable cells was determined in colony forming units (CFUs). Antibacterial efficiency of Ag– magnetite nanocomposite was shown by the percentage of bacteria killed by the nanocomposite and it was calculated based on Eq. (1). Antibacterial efficiency ¼
F control Ncontrol F sample Nsample F control Ncontrol 100
(1)
where Ncontrol, Nsample are the number of the colonies on the L-agar plates of the control and the sample, respectively, and Fcontrol, Fsample are the dilution factor of the control and the sample, respectively. Characterization of MNP, AgNP and Ag–magnetite nanocomposite FTIR was performed on a Perkin–Elmer Model 1600 Series FTIR Spectrophotometer. The solid samples were mixed with KBr to form pellets. Particles size and size distribution were observed on TEM. TEM images were taken using a Philips Tecnai 12 operated at 120 kV equipped with Gatan model 782 CCD camera. The particles
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were re-suspended in water with sonication before deposition on a TEM grid. PCS was performed on NanoZS4700 nanoseries Malvern instrument at 25 8C. Water, used as a dispersing media, was filtered using nylon syringe filters with a pore size of 0.2 mm before used. The dispersions were sonicated for 15 min before each measurement without filtration. TGA was performed on SDTA 851 MettlerToledo at the temperature ranging between 25 and 600 8C at a heating rate of 20 8C/min under oxygen atmosphere. AgNP was analyzed using Specord S100 UV–Visible spectrophotometer (Analytikjena AG) coupled with a photo diode array detector. Magnetic properties of the particles were measured at room temperature using a Standard 7403 Series, VSM. Magnetic moment was investigated in a range of 10,000 G of applied magnetic fields using 30 min sweep time. Fe and Ag concentrations were analyzed by flame atomic absorption spectroscopy (AAS) (Varian model SpectraAA200) and calculated from sample responses relative to those of standards and blanks. EDS was performed on LEO32 energydispersive X-ray microanalyser. Results and discussion The primary aim in this work was to synthesize a novel nanocomposite of AgNP and MNP for use in antibacterial applications. AgNP is known to have extraordinary inhibitory and bactericidal properties, while MNP can facilitate a separation process due to its magnetically guidable properties, which is greatly useful for recycling purposes. Therefore, this novel nanocomposite possesses advantageous properties of both AgNP and MNP. In this work, MNP surface was designed to have thiol functional groups (–SH), known to have specific interaction with surface of AgNP [40]. Cysteine, a kind of amino acid, was selected for surface modification of MNP because of the existence of –SH groups. In addition, the presence of –COO on the cysteine-coated MNP might be necessary for nanoparticle stabilization due to electrostatic repulsion mechanism. In the first step, MNP was surface modified with 3-aminopropyl triethoxysilane via a ligand exchange and a silanization reaction to obtain amino-coated MNP (Fig. 1). The amino groups on MNP surface were readily reacted with epoxide rings of glycidyl methacrylate to form methacrylate-coated MNP (MA-coated MNP). Glycidyl methacrylate served as a linker to facilitate the accessibility of –NH2 of cysteine to react with MA-coated MNP via a Michael addition reaction. It should be noted that this reaction was performed in a basic condition (pH 12), so that –NH2 of cysteine was in a deprotonated form (pKa of –NH2 of cysteine is 10.28) [41] to serve as a nucleophile to react with methacrylate functional groups coated on MNP surface. Bare MNP and MNPs modified with amino groups, MA and cysteine were characterized using FTIR (Fig. 2). FTIR spectrum of bare MNP shows a distinctive signal of Fe–O bond at 589 cm1 of MNP core (spectrum A, Fig. 2). In the spectrum of amino-coated MNP (spectrum B, Fig. 2), the characteristic signals of N–H stretching (3401 cm1), Si–O stretching cm1 (1115–1001 cm1) and Fe–O bond of MNP core (586 cm1) were observed. The amino groups on MNP surface reacted with epoxide rings of glycidyl methacrylate to form MA-coated MNP as indicated by the presence of carbonyl group signal (1716 cm1) and C5 5C stretching (1606 cm1) (spectrum C, Fig. 2). After grafting the MNP with cysteine, the thiol-derived band (S–H) was observed at 2092 cm1, indicating the formation of cysteine-coated MNP (spectrum D, Fig. 2). It should be noted that amino-coated MNP and MA-coated MNP were not well dispersible in water when compared to those of cysteine-coated MNP. It was rationalized that negative charges of carboxylate ions of cysteine coated on the MNP led to electrostatic repulsion toward neighboring particles and thus prevented
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Fig. 2. FTIR spectra of (A) bare MNP, (B) amino-coated MNP, (C) MA-coated MNP and (D) cysteine-coated MNP.
massive flocculation of the particles. This assumption was supported by the formation of slight particle aggregation in neutral aqueous media and even more in acidic condition, indicating pH-responsive properties of cysteine-coated MNP (Fig. 3). Hydrodynamic size (Dh) and zeta potential values of cysteinecoated MNP were determined as a function of dispersion pH via PCS (Fig. 4). Dh of the particles increased when decreasing the dispersion pH (Fig. 4A). The larger size of the particles in acidic pH dispersion signified that there was some degree of nano-scale agglomeration. Therefore, the smaller size of the particles in basic
Fig. 3. The appearance of cysteine-coated MNP dispersed in acidic, neutral and basic aqueous dispersions. The concentrations of these cysteine-coated MNP in water were 0.05 mg/ml. The arrows indicate the particle aggregation on the bottom of acidic dispersions.
pH dispersion implied the improved dispersibility of the particles in water. Dh was in the range of 80–500 nm in basic pH dispersion, while those in acidic condition was in the range of 500–1400 nm. This was in good agreement with the observation of the particle dispersibility in water shown in Fig. 3. In addition, zeta potential values of the particles in the dispersions with various pHs also supported the aforementioned discussion that cysteine coated on MNP was in a deprotonated form (COO) in neutral and basic pH values and in protonated COOH in acidic pH dispersion (Fig. 4). According to the plot in Fig. 4B, the isoelectric point (PI) of cysteine-coated MNP was 5.8. To prepare Ag–magnetite nanocomposite, AgNP dispersion in water was continuously stirred with cysteine-coated MNP dispersion for 18 h under ambient condition. After removing ungrafted AgNP from the dispersion, the composition of the nanocomposite was analyzed via AAS to determine the ratio of Ag to Fe, corresponding to the ratio of AgNP to the MNP in the nanocomposite. Various feed ratios of AgNP to the MNP were performed to investigate highest percent adsorption efficiency of these two components (Table S1 in the supporting information). It was found that 20 wt% AgNP in feed resulted in about 11% adsorption efficiency. Increasing the ratio of AgNP to the MNP in feed seemed to increase the concentration of AgNP in the composite but significantly lowered percent adsorption efficiency in the reaction. Therefore, 20 wt% AgNP in feed would be used for the synthesis of Ag–magnetite nanocomposites in the following experiments.
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Fig. 4. (A) Hydrodynamic size (Dh) and (B) zeta potentials of cysteine-coated MNP as a function of solution pH.
Fig. 5. TEM images of (A) AgNP (the inset shows a UV–Vis absorption spectrum of AgNP at 410 nm), (B) cysteine-coated MNP and (C) Ag–magnetite nanocomposite. The arrows indicate AgNP conjugating with cysteine-coated MNP.
The TEM images of AgNP, cysteine-coated MNP and Ag– magnetite nanocomposite are shown in Fig. 5. Size of AgNP ranges between 18 nm and 24 nm in diameter and UV–Visible absorption spectrum shows the lmax at 410 nm, which is the characteristic signal of AgNP (Fig. 5A) [42]. TEM of cysteine-coated MNP shows the particles with size of 8–12 nm in diameter with some nanoaggregation (Fig. 5B). This nano-scale aggregation was typical for the MNP synthesized via a co-precipitation method [43,44]. It should be noted that although there was some nano-aggregation observed in TEM, these particles were well dispersible in water (neutral to basic pH dispersions) without noticeable macroscopic aggregation. In Fig. 5C, TEM shows the formation of the nanocomposite between the MNP and AgNP. This nanocomposite retained good dispersibility in water with excellent responsive properties to a permanent magnet. The chemical compositions of cysteine-coated MNP and Ag– magnetite nanocomposite were investigated using EDS technique (Fig. 6). The signals of Fe from MNP core, thiol (S) from cysteine and Si from APS coating (the first layer on the particles) were observed in cysteine-coated MNP (Fig. 6A). In Ag–magnetite nanocomposite, a distinctive signal of Ag was observed in addition to Fe, S and Si signals, indicating the presence of Ag in the nanocomposite (Fig. 6B). TGA studies were carried out to determine the mass loss of the organic components in the surface-modified MNP in each step and Ag–magnetite nanocomposite (Fig. 7A). Bare MNP showed no sign of weight loss due to the absence of organic components in the particles. After each step of the reaction, percent weight loss of the surface-modified MNPs continuously decreased, indicating more organic contents in the complexes. The percent weight loss slightly increased when the MNP was conjugated with AgNP to form Ag– magnetite nanocomposite. This was attributed to the presence of
Fig. 6. EDS spectra showing chemical compositions of (A) cysteine-coated MNP and (B) Ag–magnetite nanocomposite.
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Fig. 7. (A) TGA thermograms and (B) M–H curves of (a) bare MNP, (b) amino-coated MNP, (c) MA-coated MNP, (d) cysteine-coated MNP and (e) Ag–magnetite nanocomposite.
inorganic Ag in addition to Fe from MNP core, resulting in an increase in total inorganic content in the complexes. It should be mentioned that the percentage of organic components in cysteine-coated MNP was rather low (23 wt%), indicating high percentage of magnetite core in the complex (77 wt% iron oxide) (thermogram d in Fig. 7A). This high percent magnetite when compared to polymer-coated MNPs in other systems (20–40 wt% iron oxide) [45,46] was attributed to the existence of thin layers of cysteine on the MNP surface. This might be important that when conjugated with AgNP to form Ag– magnetite nanocomposite, magnetic responsiveness of the nanocomposite should be high enough for facilitating magnetic separation and multiple recycling processes. M–H curves of the particles in each step of the reaction are shown in Fig. 7B. They showed superparamagnetic behavior as indicated by the absence of remanence and coercivity when the external applied magnetic field was removed. The decrease of saturation magnetization (Ms) from 70 emu/g of bare MNP to 48 emu/g of Ag–magnetite nanocomposite was attributed to the presence of the organic components grafted on their surface. The Ms value of the nanocomposite (48 emu/g) was rather high when compared with Ms values of polymer-coated MNP in other systems
(10–30 emu/g) [45–47]. This was attributed to the high magnetite content in its structure due to a lack of polymer coating and this was in good agreement with TGA results. To investigate antibacterial activity and recyclable efficiency of Ag–magnetite nanocomposite and cysteine-coated MNP, bare MNP was used as a control sample. After shaking the particles in bacterial suspension containing E. coli K12 at 37 8C for 50 min, they were then separated from the dispersion using a permanent magnet. The supernatant was diluted and placed on LB-agar plates. After overnight incubation, the number of viable cells was counted in colony forming units (CFUs), which is a direct measurement of bacterial viability. The removed particles were reused by resuspending in an E. coli K12 suspension, and the procedure described above was performed repeatedly (Fig. 8). It should be noted that the particles were not washed with any solvent during recycling process. Fig. 9 shows recyclable efficiency of Ag–magnetite nanocomposite when compared to those of cysteine-coated MNP (without AgNP). Antibacterial efficiency of the nanocomposite remained higher than 90% after five recycling process, whereas cysteinecoated MNP did not or insignificantly show antibacterial efficiency. After repeatedly used for eight cycles, antibacterial efficiency of the
Fig. 8. Recycling processes of Ag–magnetite nanocomposite for antibacterial application.
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Fig. 9. Antibacterial efficiency of Ag–magnetite nanocomposite (&) and cysteine-coated MNP (&) after eight recycling processes.
nanocomposite slightly dropped but still higher than 69%. This excellent antibacterial efficiency was attributed to the presence of AgNP in the nanocomposite. The number of survived E. coli K12 in the experiments of the nanocomposite was provided in Table S2 in the supporting information.
Conclusions This work presented a surface modification of MNP with cysteine for a specific binding interaction with AgNP to obtain Ag– magnetite nanocomposite for antibacterial application. After coating MNP with cysteine, the particles exhibited pH-responsive properties and good dispersibility in water due to the presence of – COO groups on its surface, leading to electrostatic repulsive force and thus stabilizing the particles in water. After conjugation of cysteine-coated MNP with AgNP, the nanocomposite retained its good dispersibility in water with good magnetic responsiveness. Taking advantage of a fast response to an applied magnetic field, the nanocomposite can be easily removed from water for multiple cycles of antibacterial tests without a washing step. The antibacterial efficiency against E. coli K12 of the nanocomposite remained higher than 90% (1011 E. coli/10 mg nanoparticles) after five recycling processes. Acknowledgments The authors thank the Thailand Research Fund (TRF) (DBG5580002) for financial funding. Thailand Toray Science Foundation (TTSF) is also gratefully acknowledged for the support. PT thanks the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Commission on Higher Education, Ministry of Education for the scholarship. The authors thank Dr. Rerngwit Boonyom from the Department of Medical Technology, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok, Thailand, for providing E. coli K12.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jiec.2015.03.018.
References [1] C. Yu, B. YunPeng, T. Bao, L. ZhaoLong, Chin. Sci. Bull. 54 (2009) 1190. [2] A. Singh, F. Dilnawaz, S. Mewar, U. Sharma, N.R. Jagannathan, S.K. Sahoo, ACS Appl. Mater. Interface 3 (2011) 842. [3] A. Manikandan, J.J. Vijaya, J.A. Mary, L.J. Kennedy, A. Dines, J. Ind. Eng. Chem. 20 (2014) 2077. [4] B.F. Pan, F. Gao, H.C. Gu, J. Colloid Interface Sci. 284 (2005) 1. [5] A. Ito, F. Matsuoka, H. Honda, T. Kobavbyashi, Cancer Gene Ther. 10 (2003) 918. [6] K.M. Ponvel, D.G. Lee, E.J. Woo, I.S. Ahn, C.H. Lee, Koren J. Chem. Eng. 26 (2009) 127. [7] R. Khatiri, A. Reyhani, S.Z. Mortazavi, M. Hossainalipour, J. Ind. Eng. Chem. 19 (2013) 1642. [8] K. Kang, J. Choi, J.H. Nam, S.C. Lee, K.J. Kim, S.W. Lee, J.H. Chang, J. Phys. Chem. B 113 (2009) 536. [9] M.E. Park, J.H. Chang, Mater. Sci. Eng., C 27 (2007) 1232. [10] D.K. Kim, M. Mikhaylova, Y. Zhang, M. Muhammed, Chem. Mater. 15 (2003) 1617. [11] N.A. Frey, S. Peng, K. Cheng, S. Sun, Chem. Soc. Rev. 38 (2009) 2532. [12] S.L. Tie, Y.Q. Lin, H.C. Lee, Y.S. Bae, C.H. Lee, Colloids Surf., A 273 (2006) 75. [13] N. Ballav, H.J. Choi, S.B. Mishra, A. Maity, J. Ind. Eng. Chem. 20 (2014) 4085. [14] Z.C. Xing, M.J. Park, S.J. Han, I.K. Kang, M.J. Choi, B.H. Lee, Y. Chang, Macromol. Res. 19 (2011) 897. [15] E.S. Krystofiak, V.Z. Matson, D.A. Steeber, J.A. Oliver, J. Nanomater. (2012) 1. [16] E.I. Silva, J. Rivas, L.M. Leo´n Isidro, M.A. Lo´pez-Quintela, J. Non-Cryst. Solids 353 (2007) 829. [17] B. Lee, S. Koo, J. Ind. Eng. Chem. 17 (2011) 762. [18] B. Lee, S. Koo, J. Ind. Eng. Chem. 18 (2012) 1191. [19] S.D. Solomon, M. Bahadory, A.V. Jeyarajasingam, S.A. Rutkowsky, C. Boritz, J. Chem. Educ. 84 (2007) 322. [20] R. Nithya, R. Ragunathan, Dig. J. Nanomater. Biostruct. 4 (2009) 623. [21] H. Jiang, S. Manolache, A.C. Lee Wong, F.S. Denes, J. Appl. Polym. Sci. 93 (2004) 1411. [22] C. Krishnaraj, E.G. Jagan, S. Rajasekar, P. Selvakumar, P. Kalaichelvan, N. Mohan, Colloids Surf., B 76 (2010) 50. [23] L. Zhang, Y.H. Dou, H.C. Gu, J. Colloid Interface Sci. 297 (2006) 660. [24] C. Lee, K.Y. Nam, D.Y. Kim, H.J. Kim, W.H. Doh, H.K. Choi, M.J. Jung, J. Ind. Eng. Chem. 20 (2014) 1276. [25] M. Mahmoudi, V. Serpooshan, ACS Nano 6 (2012) 2656. [26] X. Zhang, H. Niu, J. Yan, Y. Cai, Colloids Surf., A 375 (2011) 186. [27] T. Mosaiab, C.J. Jeong, G.J. Shin, K.H. Choi, S.K. Lee, I. Lee, I. In, S.Y. Park, Mater. Sci. Eng., C 33 (2013) 3786. [28] M. Sureshkumar, D.Y. Siswanto, C.K. Lee, J. Mater. Chem. 20 (2010) 6948. [29] J.L. Mietta, M.M. Ruiz, P.S. Antonel, O.E. Perez, A. Butera, G. Jorge, R.M. Negri, Langmuir 28 (2012) 6985. [30] S. Mishra, N.G. Shimpi, T. Sen, J. Polym. Res. 49 (2013) 1. [31] R. Prucek, J. Tucek, M. Kilianova´, A. Pana´cek, L. Kvı´tek, J. Filip, M. Kola´r, K. Toma´nkova´, R. Zboril, Biomaterials 32 (2011) 4704. [32] K. Hayashi, K. Ono, H. Suzuki, M. Sawada, M. Moriya, W. Sakamoto, T. Yogo, Chem. Mater. 22 (2010) 3768. [33] H. Cohen, A. Gedanken, Z. Zhong, J. Phys. Chem. C 112 (2008) 15429. [34] D. Lee, R.E. Cohen, M.F. Rubner, Langmuir 21 (2005) 9651. [35] X. Yang, A.P. Gondikas, S.M. Marinakos, M. Auffan, J. Liu, H.H. Kim, J.N. Meyer, Environ. Sci. Technol. 46 (2012) 1119.
Please cite this article in press as: P. Theamdee, et al., J. Ind. Eng. Chem. (2015), http://dx.doi.org/10.1016/j.jiec.2015.03.018
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[36] C. Levard, E.M. Hotze, G.V. Lowry, G.E. Brown, Environ. Sci. Technol. 46 (2012) 6900. [37] C.L. Arnaout, C.K. Gunsch, Environ. Sci. Technol. 46 (2012) 5387. [38] C.M. Jones, E.M.V. Hoek, J. Nanopart. Res. 12 (2010) 1531. [39] S.R. Goswami, T. Sahareen, M. Singh, S. Kumar, J. Ind. Eng. Chem. (2014). [40] W. Gan, B. Xu, H.L. Dai, Angew. Chem. Int. Ed. 50 (2011) 6622. [41] A. Mocanu, I. Cernica, T. Gheorghe, L.D. Bobos, O. Horovitz, M.T. Cotisel, Colloids Surf., A 338 (2009) 93. [42] S. Pal, Y.K. Tak, J.M. Song, Appl. Envion. Microbiol. 73 (2007) 1712.
[43] R.A. Frimpong, J. Dou, M. Pechan, J.Z. Hilt, J. Magn. Magn. Mater. 322 (2010) 326. [44] M. Shabanian, M. Khoobi, F. Hemati, H.A. Khonakdar, S.S. ebrahimi, U. Wagenknecht, A. Shafiee, J. Ind. Eng. Chem. (2014). [45] B. Rutnakornpituk, U. Wichai, T. Vilaivan, M. Rutnakornpituk, J. Nanopart. Res. 13 (2011) 6847. [46] F.X. Hu, K.G. Neoh, E.T. Kang, Biomaterials 27 (2006) 5725. [47] P. Theamdee, B. Rutnakornpituk, U. Wichai, M. Rutnakornpituk, J. Nanopart. Res. 16 (2014) 1.
Please cite this article in press as: P. Theamdee, et al., J. Ind. Eng. Chem. (2015), http://dx.doi.org/10.1016/j.jiec.2015.03.018