Synthesis of nanosilver particles by reverse micelle method and study of their bactericidal properties

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

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Synthesis of nanosilver particles by reverse micelle method and study of their bactericidal properties

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2009 J Phys.: Conf Ser 187 012054

(http://iopscience.iop.org/1742-6596/187/1/012054)

Synthesis of nanosilver particles by reverse micelle method and study of their bactericidal properties

Tran Thi Ngoc Dung 1 , Ngo Quoc Buu 1 , Dang Viet Quang 1 , Huynh Thi Ha 2 , Le Anh Bang 1 , Nguyen Hoai Chau 1 , Nguyen Thi Ly 1 and Nguyen Vu Trung 3

1 Institute of Environmental Technology, Vietnam Academy of Science and Technology,

18 Hoang Quoc Viet Road, Cau Giay Distr., Hanoi, Vietnam

2 Institute of Materials Science, Vietnam Academy of Science and Technology,

18 Hoang Quoc Viet Road, Cau Giay Distr., Hanoi, Vietnam

3 National Institute for Infectious and Tropical Diseases,

1 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 ~ 5 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 3 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

1 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 do 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 1 and 2 From figure

2 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]

Figure 1 Different forms of micellae in a water/surfactant/carbohydrate system [7, 8]

Figure 2 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]

Figure 3 Stages of the nanosilver particles formation in reverse micelles [10]

Figure 4 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

OIL

Normal micelle Reverse micelle Double-layer membrane Micellar vesicle

l

a

v

ℓ, v – lengh and volume of the hydrophobic tail

a – cross-section of the hydrophilic head

P < 1 normal micelle

P > 1 reverse micelle

P ~ 1 double-layer membrane or vesicle Hydrophobic tail Hydrophilic head

St.2

St.1

2

Figure 4 Diameter of water micelles depends upon the solubility ratio in system “Hexan- H2O- AOT” [7]

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

2 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:

1 Water silver nitrate solutions, 1 M and 3 M

2 Water solutions of the reducing agents: NaBH4 (0.1 M, 1.0 M, 1.5 M) and quercetin (0.06 M) dissolved in 1 M NaOH solution (20 mg/ml)

3 0.1 M CTAB solution in chloroform

4 0.1 M SDOSS and 0.1 M AOT solutions in isooctan

5 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 5 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]

ω = [H2O]/[AOT]

H 2 O

HEXAN

Figure 5. 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 1 M silver nitrate water solution was added to 30 ml of 0.1 M CTAB in chloroform

• 0.4 ml of 1 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 2 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 3 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

3 Results and discussion

Reaction parameters of the nanosilver formation in reverse micelles were presented in table 1 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

CHLOROFORM

4

a) AgNO3/ NaBH4/ CTAB/ chloroform/ stabilizer

N 0 [AgNO 3 ]

(M/ml)

Rednt

Stabilizer (ml)

Surfactant Water

phase (ml)

[H 2 O] [Ag]*

(ppm)

Remarks and particles features

NaBH 4

(M/ml) CTAB (M) CHCl(ml) 3 [CTAB]

1 1.0/ 0.4 1.5/ 0.4 0 0 0.1 80.8 0.8 5.5 531 Precipitate after 48 h staying; φav ~

15 nm

2 1.0/ 0.3 1.5/ 0.3 Chts** 0.2 0.1 81.1 0.8 7.5 400 φav ~10 nm; good particle size

distribution; stable

[Ag]* - maximally available nanosilver concentration; **Chts - chitosan, concentration 0.5%

b) AgNO3/ quercetin/ SDOSS/ isooctan

N 0 [AgNO 3 ]

(M/ml)

Rednt

Stabi lizer (ml)

Surfact-ant (M)

Iso-octan (ml)

Water phase (ml)

[H 2 O]

Ag*

(ppm) particles features Remarks and

Qr**

0.06M

3C 1.0/0.15 0.08 0 0 SDOSS 0.1 35 0.23 3.62 460 less uniform particle size distribution; Slight precipitation after 24 h staying;

φav ~ 10 nm, 4F 1.0/0.15 0.2 0 0 SDOSS 0.1 50 0.35 3.86 320 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

N 0 [AgNO 3 ]

(M/ml)

Rednt Stabi lizer (ml)

Surfac tant (M)

Iso-octan (ml)

Water phase (ml)

[H 2 O]

Ag*

(ppm) particles features Remarks and

Qr**

0.06M

5K 3.0/0.06 0.3 0 0 AOT

0.1M 50 0.36 3.97 380

Stable particles after staying; more uniform particle size distribution; φav ~ 5-7 nm

6I 3.0/0.10 0.3 0 0 AOT

0.1M 50 0.40 4.44 640

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(<10 nm), but not uniform (exp N03) In exp

N04 quercetin concentration was considerably increased, comparable with that of silver nitrate, resulting

in stable and more uniform nanoparticles

Nanosilver particles produced according to the system AgNO3/quercetin/AOT/isooctan presented in table 1c demonstrate the higher quality of the particles with an average particle diameter 5 nm (figure 1c)

in comparison with a system, where reducing agent was SDOSS (table 1b) It is due to the fact that, although both AOT and SDOSS have the same molecular weight (444Da), in SDOSS molecule there are two linear octyl chains, but in AOT there are two branched 2-ethylhexyl groups which better fit the reverse micelle formation requirement (P>1)

a)AgNO3/NaBH4/CTAB/CHCl3/

chitosan b)AgNO3/quercetin/SDOSS/ isooctan c) AgNO3/quercetin/AOT/isooctan

Figure 6 Nanosilver particles obtained by reverse micelle method in the systems

Table 2. Bactericidal activity of the nanosilver produced by using reverse micelle method Exposition time 30 minutes

Nanosilver

concentration

(cfu/ml) Inhibite (%) (cfu/ml) Inhibited (%) (cfu/ml) Inhibited (%) (cfu/ml) Inhibited (%)

Control 3.5 x107 2.2 x104 3.1 x104 2.9 x106

7 ppm 7 x 102 99.99 0 100 0 100 8 x 102 99.97

10 ppm 6 x 102 99.99 0 100 0 100 1.1 x102 99.99

15 ppm 4 x 102 99.999 0 100 0 100 50 99.99

The results of bactericidal activity tests were illustrated in table 2 The experimental data indicated that

E.coli and Coliforms were totally killed in a solution with nanosilver concentration of 3 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 7 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

6

Figure 7 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

Figure 8 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 8 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 5 ppm of nanosilver after 30 minutes of exposition

4 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 ~ 5 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 3 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

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Acknowledgement

This research was partially sponsored by the Basic Research Program from Ministry of Science & Technology of Vietnam

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