Home Search Collections Journals About Contact us My IOPscience Synthesis of nanosilver particles by reverse micelle method and study of their bactericidal properties This content has been downloaded from IOPscience Please scroll down to see the full text 2009 J Phys.: Conf Ser 187 012054 (http://iopscience.iop.org/1742-6596/187/1/012054) View the table of contents for this issue, or go to the journal homepage for more Download details: IP Address: 192.154.54.11 This content was downloaded on 28/04/2015 at 13:50 Please note that terms and conditions apply APCTP–ASEAN Workshop on Advanced Materials Science and Nanotechnology (AMSN08) IOP Publishing Journal of Physics: Conference Series 187 (2009) 012054 doi:10.1088/1742-6596/187/1/012054 Synthesis of nanosilver particles by reverse micelle method and study of their bactericidal properties Tran Thi Ngoc Dung1, Ngo Quoc Buu1, Dang Viet Quang1, Huynh Thi Ha2, Le Anh Bang1, Nguyen Hoai Chau1, Nguyen Thi Ly1 and Nguyen Vu Trung3 Institute of Environmental Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay Distr., Hanoi, Vietnam Institute of Materials Science, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet Road, Cau Giay Distr., Hanoi, Vietnam National Institute for Infectious and Tropical Diseases, Ton That Tung, Dong Da Distr., Hanoi, Vietnam E-mail: ttndzung@yahoo.com, buu_nq@yahoo.com Abstract Nanosilver particles have been synthesized by the reverse micelle method, where AgNO3 was used as a silver ions source, NaBH4 and quercetin - as reducing agents, CTAB, SDOSS and AOT- as surfactants, while the stabilizer was Vietnamese chitosan Studying the factors influencing the process of nanosilver particle formation, it was shown that the particle size of the nanosilver products depends on the concentration of the reaction components and their stoichiometric ratio It was also shown that the reaction system using AOT surfactant is capable of producing nanosilver particles with smallest nanoparticles (φav ~ nm) and good particle-size distribution The study on bactericidal activity of the nanosilver products indicated that the disinfecting solution with a nanosilver concentration of ppm was able to inhibit all E.coli and Coliforms, TPC and fungi at 15 ppm, while Vibrio cholerae cells were inactivated completely with 0.5 ppm of nanosilver after 30 minutes exposition Keywords: Reverse micelle, nanosilver, AOT, antibacterial agent Introduction Among inorganic antibacterial agents, silver has been employed most extensively since ancient times to fight infections and control spoilage Catalytic oxidation by metallic silver and reaction with dissolved monovalent silver ion probably contribute to its microbicidal effect [1] Microbes are unlikely to develop resistance against silver, as they against narrow-target antibiotics, because the metal attacks many targets in the organisms, which means that they would have to develop a host of mutations simultaneously to protect themselves [2, 3] Thus, at present silver ion is being widely used for disinfection, especially due to the advances in nanotechnology, which make possible the delivery of ionic silver during disinfection process [3-6] For these reasons, we studied the synthesis of nanosilver for disinfection purposes using reverse micelle systems as one of the simplest methods for nanosilver production and its antibacterial activity Water/carbohydrate reverse microemulsions in the presence of a surfactant are used as a microreactor for synthesizing nanoparticles from different metals such as Au, Ag, Cu, Zn, and Fe In a solvent, different micelle can be formed under different conditions [7-10] as shown in figures and From figure it can be seen that the formation of micelles depends on the geometrical ratio P So, to obtain a reverse micelle emulsion it is necessary to have P >1 [8, 9] c 2009 IOP Publishing Ltd APCTP–ASEAN Workshop on Advanced Materials Science and Nanotechnology (AMSN08) IOP Publishing Journal of Physics: Conference Series 187 (2009) 012054 doi:10.1088/1742-6596/187/1/012054 OIL WATER Normal micelle WATER WATER Double-layer membrane Micellar vesicle OIL Reverse micelle Figure Different forms of micellae in a water/surfactant/carbohydrate system [7, 8] P= Hydrophobic tail Hydrophilic head v a.l ℓ, v – lengh and volume of the hydrophobic tail a – cross-section of the hydrophilic head P < normal micelle P > reverse micelle P ~ double-layer membrane or vesicle Figure Structure of a surfactant and influence of its geometrical parameters on the micellar formation [9] The procedure of nanosilver preparation can be done by using reverse micelle method as follows Micellar solutions are produced by successively mixing silver nitrate water solution and water solution of a reductant with surfactant in a solvent Then the reductant-containing microemulsion is added to the silver nitrate -containing microemulsion while stirring vigorously for two hours The exchange of the solubles (AgNO3 and NaBH4) between the micelles takes place according to the following stages: 1) diffusion process of the micelles resulting in their collision; 2) destruction of certain parts of surfactant layer (CTAB or AOT) around the micelles; 3) diffusion exchange of the solubles in the micelles; 4) formation of new micelles with appearance of nanosilver particle therein [9-11] These stages are illustrated in figure 3, where the third one was approved to be the slowest and, thus, the limiting stage, and considerably depending on the speed of stirring In a triple component system “carbohydrate – water – surfactant” the solubility ratio ω ([H2O]mol / [Surfactant]mol) is a crucial factor for the formation of nano-sized silver particles [9] St.1 St.2 St.3 St.4 Figure Stages of the nanosilver particles formation in reverse micelles [10] Figure illustrates the influence of the solubility ratio on the size of water micelle in a triple system “Hexan – H2O – AOT”, where one can see that diameter of a water micelle increases with increasing of solubility ratio AOT H2O HEXAN Water micelle diameter, nm APCTP–ASEAN Workshop on Advanced Materials Science and Nanotechnology (AMSN08) IOP Publishing Journal of Physics: Conference Series 187 (2009) 012054 doi:10.1088/1742-6596/187/1/012054 Figure Diameter of water micelles depends upon the solubility ratio in system “Hexan- H2O- AOT” [7] ω = [H2O]/[AOT] Although mechanism of inhibitory action of nanosilver on microbes is not fully clear, there is a lot of proof on the bactericidal action of silver [4, 5, 12-14] Nanosilver can provide a control delivery of ionic silver and works in a number of ways to disrupt critical functions in an organism It has a high affinity for negatively charged side groups on biological molecules such as sulfohydryl, carboxyl, phosphate and other charged groups distributed throughout microbial cells This binding reaction alters the molecular structure of the macromolecule, rending it worthless to the cell Silver simultaneously attacks many sites within the cell to inactivate critical physiological functions such as cell-wall synthesis, membrane transport, nucleic acid synthesis and translation, electron transport, which is important in generating energy for the cell Without these functions, the microorganism is inhibited or killed Therefore at present, with more and more bacteria developing resistance to antibiotic drugs, the healthcare researchers began to consider nanosilver as one of the most potent antimicrobial agents Experimental 2.1 Materials Chemicals such as silver nitrate, sodium borohydride, quercetin, chloroform, isooctan, cetyltrimethyl ammonium bromide (CTAB), sodium bis(2-ethylhexyl) sulfosuccinate (AOT) and sodium dioctyl sulfosuccinate (SDOSS) were of high purity (Merk, Aldrich, Canto Chemical) Vietnamese β-chitozan (10% deacetylated) was provided by Institute of Chemistry, VAST E.coli, Coliforms and Vibrio cholerae were isolated from hospital pathogenous waste water, while total aerobic bacteria (TPC) and fungi were isolated from air Nutrient broth Chromocult and PCA in agar medium was used to grow and maintain the bacterial cultures 2.2 Methods Silver nanoparticles were synthesized by reverse micelle method using two representative reverse micelle systems AgNO3/NaBH4/CTAB/Chloroform and AgNO3/quercetin/SDOSS(or AOT)/isooctan In these systems isooctan and chloroform were used as solvent, quercetin and sodium borohydride as reductant, while CTAB, AOT and SDOSS were used as surfactant which met the requirement of the reverse micelle formation (P>1) For experiment, the following solutions were prepared: Water silver nitrate solutions, M and M Water solutions of the reducing agents: NaBH4 (0.1 M, 1.0 M, 1.5 M) and quercetin (0.06 M) dissolved in M NaOH solution (20 mg/ml) 0.1 M CTAB solution in chloroform 0.1 M SDOSS and 0.1 M AOT solutions in isooctan Water solution of β-chitosan 0.5% In the first system, to restrict the nanoparticle aggregation a stabilizing agent should be introduced into solution during the nanoparticle formation Figure illustrated the formation of an anti-agglomeration layer around a nanosilver cluster in a system AgNO3/NaBH4/CTAB/CHCl3 using thiolglycerin as a stabilizing agent [7] APCTP–ASEAN Workshop on Advanced Materials Science and Nanotechnology (AMSN08) IOP Publishing Journal of Physics: Conference Series 187 (2009) 012054 doi:10.1088/1742-6596/187/1/012054 Ag Ag CHLOROFORM Figure Thiolglycerin stabilizes nanosilver particle against aggregation in a reverse micelle system AgNO3/NaBH4/CTAB/CHCl3 As mentioned above, another important factor which controls the parameters of nanosilver particles in reverse micelles is to keep the molar solubility ratio (ω) as small as possible [15] To make nanosilver of better quality, the two reaction systems with different constituents have been studied A procedure for preparation of a nanosilver solution according to the reverse micelle system AgNO3/NaBH4/CTAB/chloroform could be as follows: • • • • 0.5 ml of M silver nitrate water solution was added to 30 ml of 0.1 M CTAB in chloroform 0.4 ml of M NaBH4 water solution was added to 30 ml of 0.1 M CTAB in chloroform The two solutions were vigorously stirred for one hour to form reverse micelle emulsions (RMEs) Then the emulsions were mixed together and ultrasonic stirring was continued for hours, and during this time 0.2 ml of 0.5% chitosan solution was added in order to stabilize the nanoparticles obtained For the reverse micelle system AgNO3/quercetin/AOT(or SDOSS)/isooctan the synthesis could be performed in a similar manner: • • • 0.1 ml of M AgNO3 solution was added to 25 ml of 0.1 M AOT or 0.1 SDOSS in isooctan 0.2 ml of 0.06 M quercetin solution was added to 25 ml of 0.1 M sodium dioctyl sulfosuccinate in isooctan The two solutions were vigorously stirred for two hours to form RME, then mixed together and ultrasonic stirring was continued for 2-3 hours to obtain nanosilver particles For the second reaction system, according to the Russian researchers [12], due to the peculiar reducing and stabilizing properties of quercetin, the use of stabilizers becomes unnecessary The same researchers confirmed that quercetin makes it possible to prepare only one quercetin-containing RME solution, whereas silver nitrate water solution could be poured directly into the quercetin RME This peculiarity allows to considerably decrease the molar solubility ratio, giving rise to the decrease of nanosilver particle size The nanosilver products then underwent antibacterial activity tests To examine the bactericidal effect of nanosilver particles on Escherichia coli, Coliforms, TPC and fungi, bacteria was incubated with nanosilver particles at concentration of 3, 5, 10, 15 and 30 ppm for 30 minutes Then, bacteria were cultured on Chromocult agar plates and colony-forming units were counted Results and discussion Reaction parameters of the nanosilver formation in reverse micelles were presented in table In the first system with borohydride reductant, CTAB in chloroform was used as a surfactant and chitosan – as a stabilizer, while in the second one with quercetin reductant, AOT or SDOSS in isooctan was used instead of CTAB In this system a stabilizer was not required because quercetin possesses stabilizing property to keep silver nanoparticles from oxidizing and agglomeration APCTP–ASEAN Workshop on Advanced Materials Science and Nanotechnology (AMSN08) IOP Publishing Journal of Physics: Conference Series 187 (2009) 012054 doi:10.1088/1742-6596/187/1/012054 Table Experimental parameters and results of preparation of nanosilver using different reverse micelle systems a) AgNO3/ NaBH4/ CTAB/ chloroform/ stabilizer N0 [AgNO3] (M/ml) 1.0/ 0.4 1.0/ 0.3 Rednt Stabilizer (ml) NaBH4 (M/ml) 1.5/ 0.4 1.5/ 0.3 Surfactant CTAB (M) Water [H2O] CHCl3 phase (ml) (ml) [CTAB] [Ag]* (ppm) 0 0.1 80.8 0.8 5.5 531 Chts** 0.2 0.1 81.1 0.8 7.5 400 Remarks and particles features Precipitate after 48 h staying; φav ~ 15 nm φav ~10 nm; good particle size distribution; stable [Ag]* - maximally available nanosilver concentration; **Chts - chitosan, concentration 0.5% b) AgNO3/ quercetin/ SDOSS/ isooctan Rednt [H2O] Stabi lizer (ml) Surfactant (M) Isooctan (ml) Water phase (ml) [DOSS] Ag* (ppm) 0.08 0 SDOSS 0.1 35 0.23 3.62 460 0.2 0 SDOSS 0.1 50 0.35 3.86 320 N [AgNO3] (M/ml) Qr** 0.06M (ml) 3C 1.0/0.15 4F 1.0/0.15 Remarks and particles features Slight precipitation after 24 h staying; less uniform particle size distribution; φav ~ 10 nm, Stable particles, more or less uniform particle size distribution; φav ~10 nm *[Ag] - maximally available nanosilver concentration; ** Qr - quercetin (0.06 M) c) AgNO3/ quercetin/ AOT/ isooctan N0 5K 6I [AgNO3] (M/ml) 3.0/0.06 3.0/0.10 Rednt Qr** 0.06M (ml) 0.3 0.3 Stabi lizer Surfac tant Isooctan Water phase [H2O] (ml) (M) (ml) (ml) [AOT] AOT 0.1M 50 0.36 3.97 0 AOT 0.1M 50 0.40 4.44 Ag* (ppm) Remarks and particles features 380 Stable particles after staying; more uniform particle size distribution; φav ~ 5-7 nm 640 Water silver nitrate solution was poured directly into quercetin RME and then ultrasonically stirred Silver partly precipitated; less uniform particle size distribution; φav ~ 10 nm Experimental data depicted in table 1a confirmed the stabilizing role of chitosan for the reaction system using sodium borohydride as a reductant In experiment N01 without stabilizer a black precipitate appeared in the finished solutions after 48 hours, meanwhile for the experiment using chitosan stabilizer (N02) nanosilver solutions remained transparent after staying For the system AgNO3/NaBH4/ CTAB/chloroform, nanosilver particles of the best quality have been obtained by using chitosan as a stabilizer and solubility ratio ω = 7.5 (exp N02, table 1a) TEM image of this nanosilver product was shown in figure 6, where one can see a rather good particle-size distribution with an average size of 10 nm Tables 1b and 1c represent the experimental data for the system AgNO3/quercetin/AOT (or SDOSS)/isooctan The results show that using quercetin reductant it is possible to obtain nanosilver particles stable without stabilizer, but in condition that the quercetin concentration should be enough, comparable with that of silver nitrate (exp N03,4) In the case of low quercetin concentration, nanoparticles can not be avoided from oxidation (exp N03); consequently a precipitate appeared in the nanosilver reverse micelle emulsion after 24 hours staying TEM image of nanosilver particles prepared following the system AgNO3/quercetin/ SDOSS/isooctan and presented in figure 6b shows small average particle size (1) b a a)AgNO3/NaBH4/CTAB/CHCl3/ chitosan c b)AgNO3/quercetin/SDOSS/ isooctan c) AgNO3/quercetin/AOT/isooctan Figure Nanosilver particles obtained by reverse micelle method in the systems Table Bactericidal activity of the nanosilver produced by using reverse micelle method Exposition time 30 minutes TPC Inhibite (cfu/ml) (%) E.coli Inhibited (cfu/ml) (%) Coliforms Inhibited (cfu/ml) (%) Fungi Inhibited (cfu/ml) (%) Control 3.5 x107 2.2 x104 3.1 x104 2.9 x106 ppm 7.3 x103 99.98 100 100 ppm 6.8 x103 99.98 100 100 ppm x 102 99.99 100 100 x 102 99.97 10 ppm x 102 99.99 100 100 1.1 x102 99.99 15 ppm x 102 99.999 100 100 50 99.99 Nanosilver concentration The results of bactericidal activity tests were illustrated in table The experimental data indicated that E.coli and Coliforms were totally killed in a solution with nanosilver concentration of ppm after 30 minutes of exposition, whereas TPC bacteria and fungi being more resistant were inactivated 99.99% only at 10 ppm of nanosilver Figure illustrates the antimicrobial activity of the reverse micelle nanosilver against TPC bacteria and fungi on the tile and wood surfaces respectively after being exposed 30 minutes to 10 ppm of nanosilver APCTP–ASEAN Workshop on Advanced Materials Science and Nanotechnology (AMSN08) IOP Publishing Journal of Physics: Conference Series 187 (2009) 012054 doi:10.1088/1742-6596/187/1/012054 Contro Control (b) (a) Log10 [cfu/ml] Figure TPC bacteria (a) and fungi (b) were inactivated on the tile and wood surfaces respectively after being exposed 30 minutes to 10 ppm of nanosilver -• 53• -• - - • • • 10 15 20 Nanosilver concentration, ppm - • - 0• Figure Influence of nanosilver concentration on the inactivation rate of Vibrio cholerae Exposition time 30 minutes Reverse micelle nanosilver solutions of different concentration have also been tested against Vibrio cholerae bacterium Figure depicted the inactivation rate of Vibrio cholerae cells depending on the silver concentration The data proved that this bacterium is very sensitive to the destructive action of nanosilver: 0.5 ppm of nanosilver was able to inactivate completely Vibrio cholerae during 30 minutes of exposition Microscopic image on the right shows that almost Vibrio cholerae cells were destroyed in the presence of ppm of nanosilver after 30 minutes of exposition Conclusion Nanosilver particles have been synthesized by reverse micelle method, where AgNO3 was used as a silver ions source, NaBH4 and quercetin - as reducing agents, CTAB, SDOSS and AOT- as surfactants, while for the system using sodium borohydrite as a reducing agent, a stabilizer was Vietnamese chitosan Studying the factors influencing the process of nanosilver particle formation, it was shown that the particle size of the nanosilver products depends on the concentration of the reaction components and their stoichiometric ratio, as well as the way of their introduction into reaction mixture It was also shown that the reaction system using AOT surfactant is capable of producing nanosilver particles with smaller particles (φav ~ nm) and good particle-size distribution The study on bactericidal activity of the nanosilver products indicated that a disinfecting solution with a nanosilver concentration of ppm was able to inhibit all E.coli and Coliforms, TPC and fungi at 10 ppm, while Vibrio cholerae cells were inactivated completely with 0.5 ppm of nanosilver after 30 minutes exposition References [1] James G V 1971 Water treatment (Cleveland OH: CRC Press) p 38 [2] Elechiguerra J L, Burt J L, Morones J R et al 2005 Interaction of nanosilver particles with HIV- J Nanobiotechnol (6) 41 [3] Sucdeb P, Yu K T, Joon M S 2007 Does the antibacterial activity of silver nanoparticles depend on theshape of the nanoparticle Appl & Env Microbiol 73 (6) 1712 APCTP–ASEAN Workshop on Advanced Materials Science and Nanotechnology (AMSN08) IOP Publishing Journal of Physics: Conference Series 187 (2009) 012054 doi:10.1088/1742-6596/187/1/012054 [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] Oka M, Tomioka T, Tomita K et al 1994 Inactivation of enveloped viruses by a silver thiosulfate complex Metal-based druds 511 Sondi I, Salopek-Sondi 2004 Silver nanoparticles as antimicrobial agent: a case study on E coli as a model for gram-negative bacteria J Colloid Interface Sci 275 177 Wiley B, Sun Y, Mayers B and Yxia 2005 Shape controlled synthesis of metal nanoostructures: the case of silver Chem Eur J 11 454 Razumov V F 2003 Nanoparticles and chemical reactions in micellar systems Report on the scientific session of the Section of Chemical Sciences (Department of Chemistry and Material Sciences Russian Academy of Sciences 9-11 April) Robinson B H, Khan-Lodhi A N, Towey T 1989 Microparticle synthesis and characterization in reverse micelles ed Pileni M P (Amsterdam: Elsevier) p 199 Pileni M-P 1989 Structure and reactivity in reverse micelles ed Pileni M P (Amsterdam: Elsevier) Petit C, Lixon P, Pileni M P 1993 In situ synthesis of silver nanocluster in AOT reverse micelles J Phys Chem 97 12974 Ershov B G 1997 Ions of metals in unusual and unstable oxidation states in aqueous solutions: the receipt and properties Successes Chemistry 66 (2) 103 Fung M C, Bowen D L 1996 Silver products for medical indications: risk-benefit assessment Clinical Toxicol 34 119 Yakabe Y, Sano T, Ushiho H, Yasumaga T 1980 Kinetic studies of the interaction between silver ion and deoxyribonucleic acid Chem Lett 373 Gibbins B 2003 The antimicrobial benefits of silver and the relevance of microlattice Technology Ostomy Wound Management 49 (6) Egorova E M, Revina A A 2002 Optical properties and size of nanoparticles of silver in mitselyarnyh solutions Colloid Journal 64 (3) 334 Acknowledgement This research was partially sponsored by the Basic Research Program from Ministry of Science & Technology of Vietnam ... Science and Nanotechnology (AMSN08) IOP Publishing Journal of Physics: Conference Series 187 (2009) 012054 doi:10.1088/1742-6596/187/1/012054 Synthesis of nanosilver particles by reverse micelle method. .. Chromocult and PCA in agar medium was used to grow and maintain the bacterial cultures 2.2 Methods Silver nanoparticles were synthesized by reverse micelle method using two representative reverse micelle. .. Figure Nanosilver particles obtained by reverse micelle method in the systems Table Bactericidal activity of the nanosilver produced by using reverse micelle method Exposition time 30 minutes TPC