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Colloids and Surfaces A: Physicochem. Eng. Aspects 355 (2010) 167–171

Contents lists available at ScienceDirect

Colloids and Surfaces A: Physicochemical andEngineering Aspects

journa l homepage: www.e lsev ier .com/ locate /co lsur fa

reparation and antibacterial activity of silver-doped organic–inorganic hybridoatings on glass substrates

ung Min Leea, Bum Suk Leeb, Tae Gang Byunc, Ki Chang Songa,∗

Department of Chemical and Biochemical Engineering, Konyang University, 26 Naedong, Nonsan, Chungnam 320-711, Republic of KoreaKorea Institute of Energy Research, 71-2 Jangdong, Yuseong-gu, Daejeon 305-343, Republic of KoreaDepartment of Food Science and Biotechnology, Konyang University, 26 Naedong, Nonsan, Chungnam 320-711, Republic of Korea

r t i c l e i n f o

rticle history:eceived 29 September 2009eceived in revised form 1 December 2009ccepted 3 December 2009

a b s t r a c t

Silver-doped organic–inorganic hybrid coating solutions were prepared by incorporating AgNO3 intoorganic–inorganic hybrid sols derived from methacryloxypropyl trimethoxysilane (MEMO) and vinyl-triethoxysilane (VTES) using the sol–gel method. They were applied on the soda-lime slide glass, andcrack-free and transparent coatings were obtained by a low temperature heat treatment at 150 ◦C for 3 h.

vailable online 5 December 2009

eywords:ol–geloating

The formation of Ag colloids in the coating films was confirmed by UV–vis spectroscopy and scanningelectron microscopy. The size control of Ag colloids in the coating films was possible by adjusting initialAgNO3 concentrations in the coating solutions. The antibacterial activity of the coating films was testedagainst Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria by film contact

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ntibacterial activityilverlass

method. The coating film

. Introduction

Silver or silver ions have been known to have strong inhibitorynd bactericidal effects [1]. Silver-doped glass and ceramics arexpected to be candidates for antibacterial materials, since theyhow high chemical durability and antibacterial activity [2]. In addi-ion to the antibacterial properties, silver has also been used fortaining glasses yellow since the medieval age. The coloring effects due to a surface plasmon resonance of the conductive electronsn the nano-sized silver metal particles [3]. Moreover, coating filmsontaining silver nanoparticles have also attracted much attentionn the last two decades, because of their potential application inonlinear optics [4].

Many methods, sol–gel method [5], high temperature glassusion [6], and ion implantation [7] have been developed for theynthesis of silver-doped coating films on glass or ceramic sub-trates. Among the methods, sol–gel method was widely studied,ue to its advantages such as high purity, homogeneity of coatings,nd low processing temperatures [5]. The sol–gel method offers anique opportunity to incorporate silver components into a pure

r organically modified silica matrix.

There have been a few attempts for the preparation of silver-oped pure silica coatings on glass substrates by dissolving AgNO3

n a sol made from tetraethoxysilane [2,8]. These silver-doped pure

∗ Corresponding author. Tel.: +82 41 730 5681; fax: +82 41 730 5762.E-mail address: songkc@konyang.ac.kr (K.C. Song).

927-7757/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfa.2009.12.010

ibited an excellent antibacterial activity against both S. aureus and E. coli.© 2009 Elsevier B.V. All rights reserved.

silica coatings on glass substrates showed high antibacterial activ-ity, but required a heat treatment at high temperatures of 600 ◦Cin air, which led to a migration of Ag+ ions away from the surfaceof the coatings and into the bulk, reducing the amount of avail-able surface silver [8]. As the initial release rate of antibacterialagent during the early stages is high, which is important in prevent-ing bacterial attachment, a reduction in the availability of surfacesilver may be undesirable [9]. Thus the use of low temperature pro-cessed silver-doped coatings on glass substrates may be proposedas antibacterial coatings. Organic–inorganic hybrid coating solu-tions are effective in preparing relatively thick silver-doped coatingfilms at low temperatures [10].

In this study, we report the preparation of silver-dopedorganic–inorganic hybrid coatings on glass substrates at a relativelylow temperature of 150 ◦C by sol–gel method. The formation of Agcolloids in the coating films was confirmed by the UV–vis spec-troscopy, and scanning electron microscopy. The effect of initialAgNO3 concentrations in the coating solutions on the size of Ag col-loids was also investigated. In addition, the antibacterial activity ofthe silver-doped hybrid films was studied against Gram-positiveStaphylococcus aureus and Gram-negative Escherichia coli.

2. Experimental

2.1. Preparation of coating films

Silver-doped hybrid coating solutions were prepared bymethacryloxypropyl trimethoxysilane (MEMO, 98%, Aldrich

1 hysicochem. Eng. Aspects 355 (2010) 167–171

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hemicals), vinyltriethoxysilane (VTES, 98%, Aldrich Chemicals),ilver nitrate (99.5%, Junsei Chemicals), water, nitric acid (50%,amchun Chemicals), and ethanol (99%, Samchun Chemicals). Sev-ral AgNO3 solutions with different concentrations (0.01–0.5 M)ere prepared by dissolving the required amounts of AgNO3 in

.2 ml distilled water. To these solutions, MEMO (0.07 mole), VTES0.03 mole), ethanol (0.2 mole), and nitric acid (0.001 mole) weredded, and stirred at room temperature for 3 h.

Coatings were prepared from the above solutions by dip-coatingechnique on the soda-lime glass microscope slides, and then fol-owed curing at 150 ◦C for 3 h in dry oven. Prior to coating, the glassubstrates were first cleaned with neutral detergent, followed byinsing with distilled water.

.2. Characterization

The optical absorption spectra of the films were measured inhe range of 300–800 nm using UV–vis spectroscopy (UV-2450,himadzu, Japan). The Ag colloid formation was investigated byigh resolution scanning electron microscopy (HR-SEM, MIRA IIMH, TESCAN). An energy dispersive spectroscopy (EDS, BRUKER)ttached to the HR-SEM was used to study the surface chemicalomposition of the coating films.

.3. Antibacterial test

The antibacterial activity of the silver-doped and un-dopedoatings, against Gram-positive and Gram-negative strains, wasetermined using a modified version of the Japanese standard (JIS2801) by film contact method. Two different bacteria, Gram-

Fig. 2. SEM images of the surface morphology of the coating films prepared at differ

Fig. 1. UV–vis absorption spectra of the coating films prepared with different initialAgNO3 concentrations.

positive S. aureus (ATCC 6538P) and Gram-negative E. coil (ATCC8739), were used for testing the antibacterial activity of coatingfilms. Stock cultures of the bacteria were grown on plate countagar (PCA—3 w/v% trypton, 1 w/v% glucose, 2.5 w/v% yeast, 9 w/v%agar, Oxoid). The microorganisms were grown overnight in nutri-

ent rich broth to give a bacterial concentration of approximately108 CFU/ml. These were diluted one in a hundred with maximumrecovery diluent (MRD—1 w/v% peptone, 8.5 w/v% NaCl, Oxoid)to give a working culture of approximately 106 CFU/ml. Doped

ent initial AgNO3 concentrations. (a) 0.01 M, (b) 0.1 M, (c) 0.2 M, and (d) 0.5 M.

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agent for metal ions. In this study, it is also thought that the organicgroups of used organically modified silanes (MEMO, VTES) act asthe reducing agent for silver ions, resulting in the reduction ofsilver ions to silver colloids by thermal treatment in air. Severalresearchers showed that heating temperature in the formation of

S.M. Lee et al. / Colloids and Surfaces A: P

nd un-doped coatings (25 cm2) were inoculated with 400 �l ofhe working bacteria cultures, and incubated at 37 ◦C overnight.he coatings were then agitated with MRD (20 ml) in steriletomacher® 400 polybags. To determine the number of colonies,he MRD was serially diluted tenfold, and the resulting dilutionslated (100 �l) onto plate count agar (PCA) for overnight incubationt 37 ◦C.

. Results and discussion

Silver-doped organic–inorganic hybrid coating solutionsere prepared by dissolving different amounts of AgNO3 into

rganic–inorganic hybrid sols derived from MEMO and VTES. Theyere applied on the soda-lime slide glass, and crack-free and

ransparent hybrid coating films were obtained by a low tempera-ure heat treatment at 150 ◦C. Fig. 1 shows the optical absorptionpectra of the hybrid coating films prepared with different initialgNO3 concentrations. Thermal curing at 150 ◦C induced thevolution of a peak at around 410 nm due to the surface plasmonesonance (SPR) of Ag colloids [4]. The coating films, preparedt relatively low concentrations of AgNO3 below 0.1 M, wereolorless, and showed no absorption peaks in the visible region.owever, the films, prepared with high concentrations of AgNO3

0.1, 0.2, 0.4, 0.5 M), turned to yellow, followed by brown in colorith increasing AgNO3 concentrations, and showed absorptioneaks near 410 nm due to the SPR of Ag colloids. These results

ndicate that Ag colloids are formed in the hybrid films afterhermal curing at 150 ◦C. Also, the increase in optical density oflms with increasing AgNO3 concentrations suggests an increasingrecipitation of Ag colloids in the films.

Fig. 2 shows the SEM images of the films prepared with differ-nt initial AgNO3 concentrations in the coating solutions. It is foundhat surface morphology of the films is greatly affected by the initialgNO3 concentrations in the coating solutions. The film preparedith a low AgNO3 concentration of 0.01 M showed no Ag colloids.owever, the films, prepared with initial AgNO3 concentrations of.1, 0.2 and 0.5 M, exhibited Ag colloids with mean diameters of 30,

0 and 50 nm, respectively, which increased with increasing initialgNO3 concentrations. This means that a reduction of ionic silver

o colloidal silver happened, and the control of mean size of col-oidal Ag was possible by adjusting initial AgNO3 concentrationsn the precursor solutions. For this reason, it can be explained that

ig. 3. SEM image of the cross-sectional morphology of the coating film preparedt initial AgNO3 concentration of 0.1 M.

chem. Eng. Aspects 355 (2010) 167–171 169

Ag clusters, formed by reduction of the silver ions in the precur-sor solutions, aggregate to form bigger colloids through thermaltreatment at 150 ◦C in air [11]. With increasing the initial AgNO3concentrations in the precursor solutions, the Ag loading amountson the coating films increase, thus the number of Ag clusters formedalso increases, resulting in the increase of mean diameter of Ag col-loids through more aggregation between Ag clusters [2]. Mennig etal. [12] reported that in sol–gel derived SiO2 coatings on soda-limeglass, the formation and growth of Au-, Pd-, Ag colloids was con-trolled by using silanes with functional amino, thio or thiocyantogroups, and the organically modified silanes acted as a reducing

Fig. 4. SEM image of (a) the surface morphology of the coating film prepared atinitial AgNO3 concentration of 0.1 M, (b) EDS spectrum taken on the spot labeled as1 and, (c) EDS spectrum taken on that labeled as 2 in (a).

170 S.M. Lee et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 355 (2010) 167–171

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Fig. 5. Antibacterial activity of silver-doped coatings against S.

g colloids by a thermal treatment is an important factor for theilver particle size [2,3]. However, in this study, we report that theize control of Ag colloids in the coating films is also possible bydjusting initial AgNO3 concentrations in the coating solutions at aow heating temperature of 150 ◦C.

A cross-sectional morphology of coating film, prepared with annitial AgNO3 concentration of 0.1 M, is shown in Fig. 3. The filmhickness is about 3.8 �m. The figure shows that the adhesion ofhe coating to the substrate is quite good.

Fig. 4a indicates that the coating film, prepared with an initialgNO3 concentration of 0.1 M, has two kinds of surface morpholo-ies, labeled as 1 and 2. Fig. 4b and c shows the EDS spectra takenrom the spots 1 and 2 on the surface in Fig. 4a, respectively. EDSnalysis not only confirms the existence of silver in coating films,nd but also reveals quantitatively the content of silver. The sur-ace composition analysis by EDS shows that the Ag atomic wt% ofhe spots outside and inside Ag colloids is 1.85 and 3.12, respec-

ively. This means that the Ag content of the spot inside Ag colloidss slightly higher than that outside Ag colloids.

Figs. 5 and 6 show the antibacterial effect of silver-doped coat-ng films, prepared with an initial AgNO3 concentration of 0.1 M,

Fig. 6. Antibacterial activity of silver-doped coatings against E. coli

s after 24 h. (a) Un-doped coating and (b) silver-doped coating.

against S. aureus and E. coli, respectively. There is a big differencein antibacterial effect between silver-doped and un-doped coat-ings. These figures show that the silver ions, released from thecoating films, have strong bactericidal effect against both S. aureusand E. coli bacteria. The antibacterial effect of silver-doped and un-doped coating films, against S. aureus and E. coli, are summarizedin Table 1. After 24 h incubation, the percent reductions of bac-teria were 99.99847% and 99.99995% against S. aureus and E. coli,respectively. This result shows slightly greater bacterial activity ofsilver-doped coating films against E. coli compared to S. aureus. Ithas been known that S. aureus has a thicker cell wall comparedto E. coli [13]. It seems that this results in slightly greater bacte-rial activity of E. coli compared to S. aureus in our experimentalcondition.

It has been reported that ionic silver strongly interacts withthiol groups of vital enzymes, and inactivates them [14]. Feng etal. [15] also investigated the inhibition mechanism of silver ions

on microorganism, and concluded that the silver ions affect theDNA replication ability, which in turn induces the inactivationof bacterial proteins. Marini et al. [10] prepared the silver-dopedorganic–inorganic hybrid coatings on polymer substrates by a

after 24 h. (a) Un-doped coating and (b) silver-doped coating.

S.M. Lee et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 355 (2010) 167–171 171

Table 1Antibacterial test results against S. aureus and E. coli.

Bacteria Case At beginning (cells/ml) After 24 h (cells/ml) Reduction of bacteria (%)

S. aureus Blank 2.9 × 105 5.9 × 105 –Coating film 2.9 × 105 <10 99.99847

E. coli Blank 1.5 × 105 1.9 × 107 –Coating film 1.5 × 105 <10 99.99995

Table 2Comparison of antibacterial performance against S. aureus and E. coli of Ag-doped coating films prepared with different methods.

Researchers Preparation method Reduction of S. aureus (%) Reduction of E. coli (%) Reference

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Jeon et al. Silver-doped silica coatings on glass substratesMarini et al. Silver-doped organic–inorganic hybrid coatings on polymer suLoher et al. Silver-doped calcium phosphate coatings on polymer substratLee et al. Silver-doped organic–inorganic hybrid coatings on glass subst

ol–gel method. They found that the antibacterial activity of coatinglms is diffusion-controlled, and the diffusion of silver ions occurshrough the organic phase. Recently, Loher et al. [16] made silver-oped tricalcium phosphate (Ag-TCP) nanoparticles by flame-sprayynthesis, and demonstrated the rapid self-sterilization of poly-er substrates containing Ag-TCP nanoparticles using human

athogens. They stated that the enhanced activity of silver onalcium phosphate can be linked to a microorganism’s uptake ofutrition minerals, triggering the timely release of silver towardsrowing microorganism. In our study, it seems that the antibacte-ial activity is related to the release of silver ions from the coatinglms. Compared to the microorganism-triggered silver release iniodegradable Ag-TCP carriers of Loher et al., in our coating, silver

on is released from inert glass substrates, and the silver ion releases controlled by water diffusion in the surface pores of the coatinglms [11].

Table 2 shows the comparison of antibacterial performance,gainst S. aureus and E. coli, between our Ag-doped coating films andhose prepared by the above methods. The comparison betweeng-doped coatings reveals a slightly improved performance of ouroating against S. aureus and E. coli compared to other meth-ds.

Jeon et al. [2] showed that the silver-doped silica coating films onlass substrates, made at a high temperature of 600 ◦C, had an excel-ent antibacterial performance against S. aureus and E. coli bacteria.n this article, we report that the silver-doped organic–inorganicybrid coating films on glass substrates, made at a relatively

ow temperature of 150 ◦C by the sol–gel method, also show atrong antibacterial activity against S. aureus and E. coli bacte-ia.

. Conclusions

Sol–gel method was successfully used for synthesizing silver-oped organic–inorganic hybrid coating solutions. They werepplied on the slide glass by dip-coating, and crack-free andransparent coatings were obtained by a low temperature heatreatment at 150 ◦C. The formation of Ag colloids in the coatinglms was confirmed by UV–vis spectroscopy and scanning electron

icroscopy. The control of mean size of Ag colloids in the coating

lms was possible by adjusting initial AgNO3 concentrations in theoating solutions. The excellent antibacterial activity of the coat-ng films, against Gram-positive S. aureus and Gram-negative E. coliacteria, was demonstrated.

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– 99.998 [16]99.99847 99.99995 This study

Acknowledgments

This work was supported by the Energy-Resources TechnologyR&D task implemented by the Ministry of Knowledge Economy inKorea.

References

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[2] H.J. Jeon, S.C. Yi, S.G. Oh, Preparation and antibacterial effects of Ag-SiO2 thinfilms by sol–gel method, Biomaterials 24 (2003) 4921–4928.

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