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Synthesis of biocidal polymers containing metal NPs using an electron beam
Radiation Physics and Chemistry 81 (2012) 975–977
Contents lists available at SciVerse ScienceDirect
Radiation Physics and Chemistry journal homepage: www.elsevier.com/locate/radphyschem
Synthesis of biocidal polymers containing metal NPs using an electron beam Kwonyong Choi a, Seong-Eun Kim b, Hee-Yeon Kim a, Jeyong Yoon a, Jong-Chan Lee a,n a b
Department of Chemical and Biological Engineering, Seoul National University, Gwanak-Ro, Gwanak-Gu, Seoul 151-742, Republic of Korea Department of Polymer Science and Engineering, Inha University, Icheon, Republic of Korea
a r t i c l e i n f o
abstract
Article history: Received 24 June 2011 Accepted 27 December 2011 Available online 10 January 2012
Metal containing antibacterial polymers were prepared by the polymerization of methylmethacrylate and methacrylic acid with copper or zinc. When the thin film of the polymers coated on a glass was irradiated with an electron beam, nanoparticles were obtained. It was found that these polymers exhibited a potent antibacterial activity against the Gram-negative bacteria, Escherichia coli. The metal containing polymers showed a 99.999% (5.0 logs) reduction in E. coli at a contact time of 12 h. In addition, polymers had a good antifouling effect against marine organisms. & 2012 Elsevier Ltd. All rights reserved.
Keywords: E-beam Antibacterial Antifouling Nanoparticle
1. Introduction Copper is considered as one of the most important biocidal agents besides organotin compounds. The biocidal property of copper has been widely used in coating technologies, such as the design of materials for biomedical devices, hospital equipment, household materials, and antifouling paint (Omae, 2003). Copper metals, copper alloys, and organic copper compounds are used principally for biocides because these materials are able to form Cu ions, which have a biocidal activity. Recently, Cu nanoparticle/ polymer composites have been reported, since they have stability in a polymer matrix and good biocidal activity due to their small size and large surface area (Anyaogu et al., 2008). The aims of this study were to produce copper nanomaterials dispersed in methacrylic polymers prepared by the reduction of copper containing methacrylic polymers and to evaluate its biocidal activities. The process included the synthesis of copper containing methacrylic polymers, the preparation of copper nanomaterials from methacrylic polymers, and the preliminary electron beam process for the preparation of the copper nanoparticles.
2. Experimental 2.1. Materials Commercially available reagents including cuprous chloride, methylmetacrylate (MMA), methacrylic acid (MAA), and azobis n
Corresponding author. E-mail addresses: [email protected] (K. Choi), [email protected] (J.-C. Lee). 0969-806X/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2011.12.045
(isobutyronitrile) (AIBN) were used without any further purification. THF was distilled under nitrogen from sodium and benzophenone. 2.2. Preparation of copper containing methacrylic polymers Copper containing methacrylic polymers were prepared by free radical polymerization of MMA, MAA using AIBN as the initiator and THF as the solvent. The reaction mixture was refluxed for 17 h under a dry nitrogen atmosphere and poured into n-hexane. The precipitate was further purified by several washes using n-hexane, and then dried in a vacuum oven. 2.3. Electron beam reduction Electron beam irradiation was done by a linear electron accelerator at the Korea Atomic Energy Research Institute (KAERI, Daejeon, Korea). Electron beams of absorbed irradiation at a dosage range of 14–96 kGy, at acceleration voltages from 0.3 to 2 MV, and at beam currents from 0.06 to 0.24 mA can be produced and irradiated in the air at room temperature without any vacuum system present. In addition, the pulse width was 30 ps and the scanning range was 7 cm 45 cm. The absorbed irradiation dose was measured by GEX B3WINDOSETM radiochromic film dosimeters (GEX Corporation, Centennial, CO, USA) attached to the surfaces at the top and bottom of the sample holder. 2.4. Biocidal activity Escherichia coli ATCC8739 was selected as model strain for Gram-negative bacteria. E. coli was inoculated in 40 mL of nutrient broth (Difco Co., Michigan) and Luria–Bertani
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K. Choi et al. / Radiation Physics and Chemistry 81 (2012) 975–977
(Difco Co., Michigan) medium, and cultured overnight at 37 1C. Then, the bacterial cells were centrifuged and washed three times with 150 mM phosphate-buffered saline solution (PBS, pH 7.2) to remove any remaining growth medium. The cells were diluted to 0.5 106 colony-forming units (CFU)/mL with PBS and 0.1 mL of the bacterial suspension was exposed to the copper complex polymer. The exposed bacterial suspensions were covered with sterile polypropylene film to prevent vaporization, and kept in a thermo-hygrostat at 25 1C. After exposure time had elapsed, the bacterial cells were collected with 0.9 mL of PBS. The viable cells were ascertained by the plate count method. 2.5. Antifouling ability Slides were placed in individual compartments of ‘quadriperm’ polystyrene culture dishes (Fisher) and 10 mL of a diatom suspension was added. Six replicates were used for each treatment. The cells were allowed to settle for 1 h in an illuminated incubator at 188 1C before the slides were rinsed gently in F/2 medium to remove any unattached cells. Slides were incubated for a further 1 h. Three replicate slides were fixed in 2.5% glutaraldehyde in sea water, desalted by first washing with a 50:50 mix of sea water/distilled water, followed by distilled water and dried before counting. Cells were counted in 30 fields of view with each at 0.075 mm2 to provide cell settlement data from each of the three replicate slides. The remaining three replicates were used to evaluate the strength of the diatom attachment as detailed below. The cells were enumerated as above and the mean number of cells remaining attached to the surface after exposure to a turbulent flow in a water channel or by pressure from a water jet was compared to the mean number adhered to the control slides. Data were expressed as the percentage removal; 95% confidence limits were calculated from arcsinetransformed data.
3. Results and discussion Copper containing methacrylate polymers were prepared by copolymerization using a radical initiator, AIBN, because these polymers are soluble in organic solvents such as CHCl3 and THF if the content of copper in the copolymer is not very high. The polymerization solutions of MMA/MAA/CuCl had a haze at the beginning of the reactions, while they become transparent during the polymerization. The copper containing methacrylic polymers had a very high solubility in MeOH, even a 10 wt% polymer
solution could be prepared, while they were less soluble in a less polar solvent, such as CHCl3 and THF. IR and 1H NMR results clearly indicate that all the monomers were removed through the purification process. IR: n 2996 cm 1 (O–CH3 stretch methacrylate), 2951 cm 1 (CH2 asym. stretch of polymer chain), 2882 cm 1 (CH3 stretch of a-methyl), 2843 cm 1 (CH2 sym. stretch of polymer chain), 1731 cm 1 (CO2 stretch of methacrylate),1642 cm 1, 1481 cm 1 (CH2 bending of polymer chain), 1445 cm 1, 1433 cm 1 (O–CH3 deformation of methacrylate), 1385 cm 1 (CH3 bending of amethyl). 1H NMR (CDCl3) d: 3.60 (s, OCH3), 1.95 (d, CH2), 1.89 (m, CH2), 1.81 (s, CH2), 1.65 (m, CH2), 1.44 (m, CH2), 1.21 (m, CH3), 1.02 (s, CH3), 0.84 (s, CH3). It has been known that carboxylic acid and carboxylate compounds can be coordinated on metallic surfaces to stabilize metallic nanomaterials and methacrylic polymers that have a carboxylate group and can stabilize copper nanonetworks (Dhas et al., 2008; Lisiecki and Pileni, 1995). The copper ions in the copper containing polymers were reduced into metallic copper materials using an electron beam because metal nanoparticles have good antibacterial properties and antifouling ability due to their broad surface area. Generally, an electron beam is used for the preparation of metallic nanomaterials from metallic complexes. For example, gold nanoparticles with various sizes were prepared from gold (I)alkanthiolate complexes through electron beam irradiation in TEM (Kim et al., 2005). Similarly, when thin film of copper containing methacrylic polymers coated on glass were irradiated with an electron beam, copper nanoparticles were obtained. For example, copper nanoparticles with an average diameter of about 6.470.9 nm and 4.470.7 nm were obtained through electron beam irradiation of copolymers from MMA/MAA at a ratio of 9/1 and copolymers from MMA/MAA at a ratio of 7/3 (Fig. 1). The biocidal activity of the copper nanoparticle/polymer composite was tested against Gram-negative bacteria (E. coli). The copolymers were prepared from MMA/MAA (ratio 9:1) film without E-beam irradiation and copper NPs/polymer composites were prepared by E-beam irradiation and were used for the test. Copper NPs/polymer composite film had a strong biocidal activity against the Gram-negative bacteria, E. coli (Fig. 2). However, the copolymer film without E-beam irradiation had no biocidal activity. This means that the copper ions in the copper containing polymer film are not released to the bacterial suspension because the copper ions in polymer were stably bound. The antifouling effect of the Cu–polymer was evaluated with the marine microorganism, Amphora coffeaformis (Holland et al., 2004).
Fig. 1. TEM image of copper particles prepared by the electron beam reduction process. (a) MMA/ MAA = 9/1, (b) MMA/ MAA = 7/3.
K. Choi et al. / Radiation Physics and Chemistry 81 (2012) 975–977
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in adhesion occurred on the surface of the copper NPs/polymer composites.
4. Conclusion Copper containing polymer was synthesized with MMA and MAA by free radical polymerization. This copper containing polymer was soluble in organic solvent and easy to make thin uniform films on glass substrates. Copper ions in the copper containing polymers were reduced to metallic copper nanomaterials using the E-beam, which formed nanonetworks. This composite film had a strong biocidal activity against Gramnegative bacteria. From the results of the antifouling test, it was confirmed that marine organism had a hard time attaching to and had a reduced mobility on the composite films.
Acknowledgment Fig. 2. Biocidal activity of Cu nanoparticle/polymer composite film against Gramnegative bacteria (E. coli).
This research was supported by Maritime Affairs (MLTM) of Korean government and a part of the project titled ‘‘Development of Environmental Friendly Antifouling System for Marine Vessels’’ funded by the Ministry of Land, Transport and Maritime Affairs, Republic of Korea. References
Fig. 3. Antifouling property of Cu NPs/polymer composites against Amphora coffeaformis.
The adhesion and mobility of the microorganism on the surface of copper NPs/polymer composites declined after 12 h compared with to the control. As shown in Fig. 3, 76.5% reduction
Anyaogu, K.C., Fedorov, A.V., Neckers, D.C., 2008. Langmuir 24, 4340–4346. Dhas, N.A., Raj, C.P., Gedanken, A., 2008. Chem. Mater. 10, 1446–1452. Holland, R., Dugdale, T.M., Wetherbee, R., Brennan, A.B., Finlay, J.A., Callow, J.A., Callow, M.E., 2004. Biofouling 20, 323–329. Kim, J.U., Cha, S.H., Shin, K., Jho, J.Y., Lee, J.C., 2005. J. Am. Chem. Soc. 127, 9962–9963. Lisiecki, I., Pileni, M.P., 1995. J. Phys. Chem. 99, 5077–5082. Omae, I., 2003. Chem. Rev. 103, 3431–3448.
Contents lists available at SciVerse ScienceDirect
Radiation Physics and Chemistry journal homepage: www.elsevier.com/locate/radphyschem
Synthesis of biocidal polymers containing metal NPs using an electron beam Kwonyong Choi a, Seong-Eun Kim b, Hee-Yeon Kim a, Jeyong Yoon a, Jong-Chan Lee a,n a b
Department of Chemical and Biological Engineering, Seoul National University, Gwanak-Ro, Gwanak-Gu, Seoul 151-742, Republic of Korea Department of Polymer Science and Engineering, Inha University, Icheon, Republic of Korea
a r t i c l e i n f o
abstract
Article history: Received 24 June 2011 Accepted 27 December 2011 Available online 10 January 2012
Metal containing antibacterial polymers were prepared by the polymerization of methylmethacrylate and methacrylic acid with copper or zinc. When the thin film of the polymers coated on a glass was irradiated with an electron beam, nanoparticles were obtained. It was found that these polymers exhibited a potent antibacterial activity against the Gram-negative bacteria, Escherichia coli. The metal containing polymers showed a 99.999% (5.0 logs) reduction in E. coli at a contact time of 12 h. In addition, polymers had a good antifouling effect against marine organisms. & 2012 Elsevier Ltd. All rights reserved.
Keywords: E-beam Antibacterial Antifouling Nanoparticle
1. Introduction Copper is considered as one of the most important biocidal agents besides organotin compounds. The biocidal property of copper has been widely used in coating technologies, such as the design of materials for biomedical devices, hospital equipment, household materials, and antifouling paint (Omae, 2003). Copper metals, copper alloys, and organic copper compounds are used principally for biocides because these materials are able to form Cu ions, which have a biocidal activity. Recently, Cu nanoparticle/ polymer composites have been reported, since they have stability in a polymer matrix and good biocidal activity due to their small size and large surface area (Anyaogu et al., 2008). The aims of this study were to produce copper nanomaterials dispersed in methacrylic polymers prepared by the reduction of copper containing methacrylic polymers and to evaluate its biocidal activities. The process included the synthesis of copper containing methacrylic polymers, the preparation of copper nanomaterials from methacrylic polymers, and the preliminary electron beam process for the preparation of the copper nanoparticles.
2. Experimental 2.1. Materials Commercially available reagents including cuprous chloride, methylmetacrylate (MMA), methacrylic acid (MAA), and azobis n
Corresponding author. E-mail addresses: [email protected] (K. Choi), [email protected] (J.-C. Lee). 0969-806X/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2011.12.045
(isobutyronitrile) (AIBN) were used without any further purification. THF was distilled under nitrogen from sodium and benzophenone. 2.2. Preparation of copper containing methacrylic polymers Copper containing methacrylic polymers were prepared by free radical polymerization of MMA, MAA using AIBN as the initiator and THF as the solvent. The reaction mixture was refluxed for 17 h under a dry nitrogen atmosphere and poured into n-hexane. The precipitate was further purified by several washes using n-hexane, and then dried in a vacuum oven. 2.3. Electron beam reduction Electron beam irradiation was done by a linear electron accelerator at the Korea Atomic Energy Research Institute (KAERI, Daejeon, Korea). Electron beams of absorbed irradiation at a dosage range of 14–96 kGy, at acceleration voltages from 0.3 to 2 MV, and at beam currents from 0.06 to 0.24 mA can be produced and irradiated in the air at room temperature without any vacuum system present. In addition, the pulse width was 30 ps and the scanning range was 7 cm 45 cm. The absorbed irradiation dose was measured by GEX B3WINDOSETM radiochromic film dosimeters (GEX Corporation, Centennial, CO, USA) attached to the surfaces at the top and bottom of the sample holder. 2.4. Biocidal activity Escherichia coli ATCC8739 was selected as model strain for Gram-negative bacteria. E. coli was inoculated in 40 mL of nutrient broth (Difco Co., Michigan) and Luria–Bertani
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K. Choi et al. / Radiation Physics and Chemistry 81 (2012) 975–977
(Difco Co., Michigan) medium, and cultured overnight at 37 1C. Then, the bacterial cells were centrifuged and washed three times with 150 mM phosphate-buffered saline solution (PBS, pH 7.2) to remove any remaining growth medium. The cells were diluted to 0.5 106 colony-forming units (CFU)/mL with PBS and 0.1 mL of the bacterial suspension was exposed to the copper complex polymer. The exposed bacterial suspensions were covered with sterile polypropylene film to prevent vaporization, and kept in a thermo-hygrostat at 25 1C. After exposure time had elapsed, the bacterial cells were collected with 0.9 mL of PBS. The viable cells were ascertained by the plate count method. 2.5. Antifouling ability Slides were placed in individual compartments of ‘quadriperm’ polystyrene culture dishes (Fisher) and 10 mL of a diatom suspension was added. Six replicates were used for each treatment. The cells were allowed to settle for 1 h in an illuminated incubator at 188 1C before the slides were rinsed gently in F/2 medium to remove any unattached cells. Slides were incubated for a further 1 h. Three replicate slides were fixed in 2.5% glutaraldehyde in sea water, desalted by first washing with a 50:50 mix of sea water/distilled water, followed by distilled water and dried before counting. Cells were counted in 30 fields of view with each at 0.075 mm2 to provide cell settlement data from each of the three replicate slides. The remaining three replicates were used to evaluate the strength of the diatom attachment as detailed below. The cells were enumerated as above and the mean number of cells remaining attached to the surface after exposure to a turbulent flow in a water channel or by pressure from a water jet was compared to the mean number adhered to the control slides. Data were expressed as the percentage removal; 95% confidence limits were calculated from arcsinetransformed data.
3. Results and discussion Copper containing methacrylate polymers were prepared by copolymerization using a radical initiator, AIBN, because these polymers are soluble in organic solvents such as CHCl3 and THF if the content of copper in the copolymer is not very high. The polymerization solutions of MMA/MAA/CuCl had a haze at the beginning of the reactions, while they become transparent during the polymerization. The copper containing methacrylic polymers had a very high solubility in MeOH, even a 10 wt% polymer
solution could be prepared, while they were less soluble in a less polar solvent, such as CHCl3 and THF. IR and 1H NMR results clearly indicate that all the monomers were removed through the purification process. IR: n 2996 cm 1 (O–CH3 stretch methacrylate), 2951 cm 1 (CH2 asym. stretch of polymer chain), 2882 cm 1 (CH3 stretch of a-methyl), 2843 cm 1 (CH2 sym. stretch of polymer chain), 1731 cm 1 (CO2 stretch of methacrylate),1642 cm 1, 1481 cm 1 (CH2 bending of polymer chain), 1445 cm 1, 1433 cm 1 (O–CH3 deformation of methacrylate), 1385 cm 1 (CH3 bending of amethyl). 1H NMR (CDCl3) d: 3.60 (s, OCH3), 1.95 (d, CH2), 1.89 (m, CH2), 1.81 (s, CH2), 1.65 (m, CH2), 1.44 (m, CH2), 1.21 (m, CH3), 1.02 (s, CH3), 0.84 (s, CH3). It has been known that carboxylic acid and carboxylate compounds can be coordinated on metallic surfaces to stabilize metallic nanomaterials and methacrylic polymers that have a carboxylate group and can stabilize copper nanonetworks (Dhas et al., 2008; Lisiecki and Pileni, 1995). The copper ions in the copper containing polymers were reduced into metallic copper materials using an electron beam because metal nanoparticles have good antibacterial properties and antifouling ability due to their broad surface area. Generally, an electron beam is used for the preparation of metallic nanomaterials from metallic complexes. For example, gold nanoparticles with various sizes were prepared from gold (I)alkanthiolate complexes through electron beam irradiation in TEM (Kim et al., 2005). Similarly, when thin film of copper containing methacrylic polymers coated on glass were irradiated with an electron beam, copper nanoparticles were obtained. For example, copper nanoparticles with an average diameter of about 6.470.9 nm and 4.470.7 nm were obtained through electron beam irradiation of copolymers from MMA/MAA at a ratio of 9/1 and copolymers from MMA/MAA at a ratio of 7/3 (Fig. 1). The biocidal activity of the copper nanoparticle/polymer composite was tested against Gram-negative bacteria (E. coli). The copolymers were prepared from MMA/MAA (ratio 9:1) film without E-beam irradiation and copper NPs/polymer composites were prepared by E-beam irradiation and were used for the test. Copper NPs/polymer composite film had a strong biocidal activity against the Gram-negative bacteria, E. coli (Fig. 2). However, the copolymer film without E-beam irradiation had no biocidal activity. This means that the copper ions in the copper containing polymer film are not released to the bacterial suspension because the copper ions in polymer were stably bound. The antifouling effect of the Cu–polymer was evaluated with the marine microorganism, Amphora coffeaformis (Holland et al., 2004).
Fig. 1. TEM image of copper particles prepared by the electron beam reduction process. (a) MMA/ MAA = 9/1, (b) MMA/ MAA = 7/3.
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in adhesion occurred on the surface of the copper NPs/polymer composites.
4. Conclusion Copper containing polymer was synthesized with MMA and MAA by free radical polymerization. This copper containing polymer was soluble in organic solvent and easy to make thin uniform films on glass substrates. Copper ions in the copper containing polymers were reduced to metallic copper nanomaterials using the E-beam, which formed nanonetworks. This composite film had a strong biocidal activity against Gramnegative bacteria. From the results of the antifouling test, it was confirmed that marine organism had a hard time attaching to and had a reduced mobility on the composite films.
Acknowledgment Fig. 2. Biocidal activity of Cu nanoparticle/polymer composite film against Gramnegative bacteria (E. coli).
This research was supported by Maritime Affairs (MLTM) of Korean government and a part of the project titled ‘‘Development of Environmental Friendly Antifouling System for Marine Vessels’’ funded by the Ministry of Land, Transport and Maritime Affairs, Republic of Korea. References
Fig. 3. Antifouling property of Cu NPs/polymer composites against Amphora coffeaformis.
The adhesion and mobility of the microorganism on the surface of copper NPs/polymer composites declined after 12 h compared with to the control. As shown in Fig. 3, 76.5% reduction
Anyaogu, K.C., Fedorov, A.V., Neckers, D.C., 2008. Langmuir 24, 4340–4346. Dhas, N.A., Raj, C.P., Gedanken, A., 2008. Chem. Mater. 10, 1446–1452. Holland, R., Dugdale, T.M., Wetherbee, R., Brennan, A.B., Finlay, J.A., Callow, J.A., Callow, M.E., 2004. Biofouling 20, 323–329. Kim, J.U., Cha, S.H., Shin, K., Jho, J.Y., Lee, J.C., 2005. J. Am. Chem. Soc. 127, 9962–9963. Lisiecki, I., Pileni, M.P., 1995. J. Phys. Chem. 99, 5077–5082. Omae, I., 2003. Chem. Rev. 103, 3431–3448.