SOME RESULTS IN MANUFACTURING OF NANOSILVER AND

INVESTIGATION OF ITS APPLICATION FOR DISINFECTION

Nguyen Hoai Chau*, Le Anh Bang, Ngo Quoc Buu, Tran Thi Ngoc Dung

Huynh Thi Ha, Dang Viet Quang

Institute of Environmental Technology, VAST

Abstract. Nanosilver particles have been manufactured by using aqueous solution method, where

AgNO3 is used as a silver ion source and NaBH4 as a reducing agent, while Vietnamese β-chitozan is used as a stabilizator. Studying the factors affecting the process of nanosilver particle formation showed that the particle size of the nanosilver products depends on the concentration of the reaction components and their stoichiometric ratio, as well as the dropping speed of the reducing agent solution. The optimum conditions allowing to obtain nanosilver with an average particle size of

20nm have been set: [AgNO3] ≤ 1000ppm, [β-chitozan] = 250-300ppm, [NaBH4] = 200ppm, mole ratio [NaBH4]/[AgNO3] = 1/4 and dropping rate of borohydride solution = 10-12 drops/min. It was found that the shelf life of the nanosilver colloids produced is at least 7 months. The study on disinfection capacity of the nanosilver product indicated that the disinfecting solution with a nanosilver concentration of 10ppm is able to completely inhibit E.coli and coliforms.

I. INTRODUCTION

Among inorganic antibacterial agents, silver has been employed most extensively since

ancient times as tableware such as bowls, chopsticks, spoons etc. to fight infections and control

spoilage. The antibacterial and antiviral actions of silver, silver ions, silver compounds have

been thoroughly investigated [1,2,3,4]. It was known that silver can absolutely suppress almost

all single cell pathogens, but practically do not harm human organisms [3].

As a wound disinfectant silver ions require a special delivery system in order to provide

their control release into the treating place in the body. Different forms of silver consist of those

that is either made up of atomic silver with no ions, thereby being inert, or of compounds such

as silver nitrate, silver oxide, silver hydroxide, silver chloride etc. These substances contain

silver ions but the bonds they share with the other constituents are either too weak to provide

any stable effective delivery in the body or are too strong to release the ions (as the case with

atomic silver, silver oxide and silver chloride), or release them instantaneously which will bind

with other components and become inert (as the case of silver nitrate). Among these forms only

nanosilver particles are able to overcome these challenges. Many research results report that in a

nano state silver is more effective against microbes than other forms and can kill as many as 650

kinds of bacteria and other microbes. Silver nanoparticles are thought to inhibit bacterial

enzymes, interfere with electron transport and bind to DNA [5]. Microbes are unlikely to

develop resistance against silver, as they do against conventional and narrow-target antibiotics,

because the metal attacks a broad spectrum of targets in the bacterial organisms, which means

that they would have to develop a host of mutations simultaneously to protect themselves.

Due to its unique bactericidal properties nanosilver is now widely used against

microorganisms in wounds and burns, although the synthesis of nanosilver particles is

confronted with a number of difficulties, especially those related to the control of surface

reactivity and agglomeration. An effective control of average particle size and size distribution,

as well as control of particle-particle interaction for obtaining a stable dispersion of

nanoparticles, still continue to be the most difficult challenge for researchers working on the

synthesis of coloidal metal dispersions.

Manufacturing of nanosilver particles at relatively low temperature is possible by using

soft-chemistry methods, which present many advantages over traditional physical methods and

high-temperature procedures and offer the possibility to control nanoparticle parameters. This report presents some results in manufacturing of nanosilver using aqueous bulk-

solution method and investigation of its application as a disinfectant agent.

II. EXPERIMENTAL

II.1. MATERIALS

Chemical reagents of p.a. purity such as silver nitrate, sodium borohydride, sodium

hydroxide and acetic acid were taken from Merk and Sigma companies and used as received,

while β-chitozan was provided by Institute of Chemistry, VAST. Stock solutions of 20mM

AgNO3, 15mM NaBH4 and β-chitozan were prepared using bi-distilled water.

II.2. METHODS

For manufacturing nanosilver the aqueous solution method [6] was used. The reaction of

nanosilver formation was taking place in a homogeneous solution of AgNO3 with β-chitozan as

a stabilizator and permanent stirring at ambient temperature, while reducing agent was being

added successively by drops. Nanoparticle characteristics of the nanosilver solutions obtained

were studied by UV spectroscopy and scanning and transmission electron microscopy methods.

The antibacterial activity of the nanosilver products have been studied by two routes: (i)

dissolving bacterial suspension directly in an aqueous solution with a given nanosilver

concentration followed by pouring agar solution into the aliquate of the bacterial suspension in a

petri dish for bacterial counting ; (ii) spraying the solution onto the surface of different materials

such as wood, fabric, ceramic tile, plastics etc., then after an appropriate exposition samples

were taken from these surfaces by using tampon for bacterial counting. Total aerobic bacteria,

E.coli, coliforms and fungi have been chosen for the experiment. The bacteria were isolated

from a waste water source and kept in a nutrient broth Chromocult or PCA medium for growing,

while fungi were taken from air.

III. RESULTS AND DISCUSSION

10 11 12 13 14 15 16 17 18 19

0 10 20 30

Average particle size, nm

Drops speed of NaBH4, drops/min

15

16

17

18

19

20

21

0 200 400 600

Average particle size, nm

Chitozan concentration, ppm

12

14

16

18

20

22

24

0.10 0.20 0.30 0.40 0.50 0.60

Average particle size, nm

Mole ratio [NaBH4]/[AgNO3]

Fig.1. Dependence of the

nanosilver particle size on the

speed of the reductant adding

to reaction mixture.

Fig.2. Influence of the

chitozan concentration on

nanosilver particle size.

Fig.3. Influence of the mole

ratio [NaBH4]/[AgNO3] on

nanosilver particle size.

Studying the influence of different factors on

the nanosilver formation it was shown that

nanoparticle size depends upon the concentration and

the ratio of the reacting components as well as the

dropping speed of reducing agent. The experimental

data displayed in Fig. 1,2,3 showed that the smallest

particle size resulted when dropping speed of the

sodium borohydride solution into the reaction

mixture was 10 - 12 drops/min, chitozan

concentration 200 - 300 ppm and mole ratio

[NaBH4]/[AgNO3] = 1 : 4. From these pictures one

can see also that average silver particle size strongly

depends on the borohydride to silver nitrate mole

ratio as well as the dropping rate of the reductant.

Fig. 4 depicts a plasmon resonance absorption

spectrum of a nanosilver colloid produced by

aqueous solution method with the following reaction parameters: [AgNO3] = 750 ppm; [NaBH4]

: [AgNO3] = 1 : 4; [Chitozan] = 250 ppm and borohydride dropping rate = 10 drops/min. The

fact that plasmon resonance absorption maximum was found at a wavelength around 420 nm

confirmed the nano nature of the manufactured silver particles.

Fig. 5 and 6 illustrate the dependence of the nanosilver average particle size upon the

reductant dropping rate and the mole ratio [NaBH4]/[AgNO3]. The SEM and TEM images of

some nanosilver samples obtained under different experimental conditions show that the

formation of nanosilver under unfavourable conditions may result in much more large particle

size as can be seen in fig.5. Figure 6 presents a TEM image of a nanosilver sample prepared by

the aqueous solution method with optimum reaction parameters allowing to obtain an average

particle size of 20 nm.

The shelf live of a nanosilver colloid with a concentration of 750 ppm produced by

aqueous solution method has been estimated to be at least 7 months. Fig.7 illustrates the TEM

images of the nanosilver particles manufactured June 05-2007 and electron-microscopically

investigated just after and 7 months after preparation. The images show almost no difference in

particle size of the two analyzed samples.

Fig. 5. SEM image of a nanosilver sample

prepared by aqueous solution method under

following reaction parameters: [AgNO3] =

1000 ppm; [Chitozan] = 250ppm; [NaBH4]

= 200 ppm; [NaBH4] : [AgNO3] = 1 : 2 ;

reductant dropping rate = 3 drops/min.

Fig. 6. TEM image of a nanosilver sample

prepared by aqueous solution method under

the following reaction parameters: [AgNO3] = 750 ppm; [Chitozan] = 250

ppm; [NaBH4] : [AgNO3] = 1 : 4; reductant

dropping rate = 10-12 drops/min.

Fig. 4. Surface plasmon

absorption spectrum of a

nanosilver solution produced by

aqueous solution method

Fig. 8 demonstrates one of the anti-

microbial mechanisms of nanosilver against

different microorganisms. It can be seen that

the cell wall of fungus Candida albican was

destroyed by nanosilver particles resulting

in inactivation of the fungal cells.

Bactericidal effectiveness of the

manufactured nanosilver in compare with

that of Korean NANOGIST product against

E.coli was shown in Table 1. The data show

that the manufactured nanosilver solution of

10 ppm concentration inactivated

completely E. coli 106

cfu/ml as did

NANOGIST silver from Korea.

Table 1. Disinfection efficiency of the manufactured nanosilver solution against E. coli in

compare with that of Korean NANOGIST product

Nanosilver source

Nanosilver

concentration

(ppm)

Exposition

(min)

E. coli density (cfu/ml)

Dilution order

100 10

1 10

2 10

3

Control 0 30 >> >> >> 1090

Manufactured

nanosilver

10 30 0 0 0 0

50 30 0 0 0 0

Nanosilver

NANOGIST, Korea

10 30 0 2 37 0

50 30 0 0 0 0

Control 0 60 >> >> >> 1126

Manufactured

nanosilver

10 60 0 0 0 0

50 60 0 0 0 0

Nanosilver

NANOGIST, Korea

10 60 0 0 0 0

50 60 0 0 0 0

The results of surface disinfection of different materials against total aerobic bacteria

(TPC) and fungi by spraying nanosilver solution of different concentration presented in tables 2

Fig.8. Destructive interaction of nanosilver particles with the cell wall of fungus Candida

albican. The nanosilver solution produced by

aqueous solution method with a concentration of 100ppm. Microscope OLYMPUS BX-51.

Fig. 7. TEM images of a nanosilver sample manufactured by aqueous solution method June 05-2007 and analyzed on EM June 08-2007 (a) and January 06-2008 (b).

a) b)

and 3 indicated that nanosilver solution of 7 ppm concentration is able to inactivate more than

98% of bacteria and fungi.

Table 2. Result of surface disinfection of different materials against total aerobic bacteria by

spraying the manufactured nanosilver solution, cfu/cm2.

Material Nanosilver concentration (ppm) and inactivation (%)

Control 3 (%) 5 (%) 7 (%) 10 (%)

Lavatory ceramics 3000 150 95

Ceramic tile 2500 30 98,8 30 98,8 15 99,4

Table wood 100 12 88,0 7 93,0

Door wood 2500 6 99,7

Interior plastics 120 45 62,5 11 90,8

Waste bin plastics 460 12 97,4

Table 3. Result of surface disinfection of different materials against fungi by spraying the

manufactured nanosilver solution, cfu/cm2.

Material Nanosilver concentration (ppm) and inactivation (%)

Control 3 (%) 5 (%) 7 (%) 10 (%)

Lavatory ceramics 120 35 70,8 11 90,8 5 95,8 3 97,5

Door wood 150 13 91,3 7 95,3 2 98,7 1 99,3

Waste bin ceramics 160 23 85,6 3 98,1 2 98,7

Fig.9. Inactivation of total aerobic

bacteria (TPC) by spraying the nanosilver

solution onto a ceramic tile. [Ag] = 7

ppm; exposition 30min.

Fig.10. Inactivation of TPC bacteria by

spraying the nanosilver solution onto a

plastic surface. [Ag] = 7 ppm; exposition 30min.

Control Control

Fig.11. Inactivation of fungi by spraying the

manufactured nanosilver solution onto a

wood surface. [Ag] = 10 ppm; exposition

30min.

Fig.12. Inactivation of E. coli by a piece

of cotton fabric impregnated with a

manufactured nanosilver solution of 750

ppm concentration.

Control

Control

Fig. 9,10,11 and 12 illustrate the inactivation of bacteria and fungi (taken from air) by

spraying nanosilver solutions onto the surface of different materials as well as impregnating

them by a concentrated nanosilver solution. The result showed that the manufactured nanosilver

solutions of rather low concentration ( ≤ 10 ppm) are capable to inactivate effectively bacteria

and fungi.

IV. CONCLUSION

Nanosilver particles have been manufactured by the aqueous solution method using

AgNO3 as a silver ion source and NaBH4 as a reducing agent, while β-chitozan as a stabilizator

to protect them from oxidization and agglomeration. Studying the factors which affect the

process of nanosilver particle formation showed that the particle size of the nanosilver product

depends on the concentration of each reaction component, their stoichiometric ratio, as well as

the dropping speed of the reducing agent solution.

The optimum conditions allowing to obtain nanosilver with an average particle size ≤

20nm have been set: [AgNO3] = 750 - 1000 ppm, [β-chitozan] = 250-300 ppm, [NaBH4] = 200

ppm, mole ratio [NaBH4]/[AgNO3] = 1/4 and dropping rate of the borohydride solution = 10-12

drops/min.

Study on the bactericidal efficiency of the manufactured nanosilver products indicated that

with a nanosilver concentration of 10ppm they are able to inactivate effectively bacteria and fungi.

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(2003) 4278 5. R. B. Thurman and C. P. Gerba, CRC Crit. Rev. Environ. Contam. 18 (1989) 295

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Corresponding author address:

Nguyen Hoai Chau

18 Hoang Quoc Viet Str., Cau Giay,Hanoi

Tel.: 84-4-7569134; Fax: 84-4-7911203

E-mail: chauhoainguyen@Yahoo.com