Aggregation Study of Ag-TiO<sub>2</sub> Composites

doi:10.4236/msa.2011.212229

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Materials Sciences and Applicatio ns, 2011, 2, 1719-1723

doi:10.4236/msa.2011.212229 Published Online December 2011 (http://www.SciRP.org/journal/msa)

1719

Aggregation Study of Ag-TiO2 Composites

María Eugenia Noriega-Treviño1,2*, Claudia Cristina Quintero-González1,

José Elpidio Morales-Sánchez1,2,3, Jesús María Guajardo-Pacheco1,2,3,

Martha Eugenia Compeán-Jasso1, Facundo Ruiz4

1Science Faculty, Universidad Autónoma de San Luis Potosí, San Luis Potosí, México; 2Mathematics and Physics Department,

Universidad Autónoma de San Luis Potosí, San Luis Potosí, México; 3Materials Advanced Research Center, Chihuahua, México;

4Materials Advanced Research Center, Apodaca, México.

E-mail: *[email protected]

Received October 21st, 2011; revised November 30th, 2011; accepted December 7th, 2011.

ABSTRACT

Most of the toxicity data presented in the literature are obtained in relatively simple media, like distilled water. The

literature reported that nanoparticles agglomerate immediately upon being added to cell culture media and if the ag-

glomerates are used directly in antimicrobial studies, the interpretation of the toxicity results tends to be complicated.

Six different molar ratios Ag-TiO 2 composites were synthesized by a reduction method using two different commercial

TiO2 particles as base ma terials and were used to find the agg regate size in distilled water and Mueller-Hinton Broth,

and to obtain the minimum inhibitory concentrations (MIC) against E. coli and E. faecalis. To evaluate the evolution of

the Ag-TiO2 particle size (z-average) three dilutions of each of the synthesized composites 100 µg/ml, 250 µg/mL and

500 µg/ml were realized in deionized water an d Mueller Hinton broth. It was found that Ag-TiO2 composites increased

in size after being diluted in Mueller-Hinton Broth, but once they grew in size, they remained constant for 24 minutes,

and after this time, did not affect the MIC for the microorganisms invo lved.

Keywords: Ag-TiO2, Composites, Nanoparticles, Particles, Aggregate Size, MIC

1. Introduction

The most common method of producing silver nanopar-

ticles is a chemical reduction of silver salt dissolved in

water with a reducing compound [1,2]. Silver nanomate-

rials exhibit broad-spectrum biocidal activity toward

bacteria, fungi, viruses and algae [3].

Several factors have been reported to influence silver

nanoparticle toxicity like particle size, shape, pH, ionic

strength and the presence of divalent cations and mac-

romolecules [4-8]. The stability of silver nanoparticles

also influences toxicity since the formation of aggregates

tends to decrease biocidal activity [9,10]. Most of the

toxicity data presented in the literature are obtained in

relatively simple media like distilled water.

It was found that TiO2, when irradiated with UV radia-

tion, acted as an antimicrobial [11]. It had been reported

that photocatalytic and antimicrobial properties of TiO2

can be improved by growing particles of a noble metal

like Ag, Au or Cu over its surface or inside a matrix

[12-14]. Composites of silver coatings over titanium di-

oxide nanoparticles are used in products to produce anti-

bacterial activity [15].

The literature reported that nanoparticles agglomerate

immediately upon being added to cell culture media and

if the agglomerates are used directly in antimicrobial

studies, the interpretation of the toxicity results tends to

be complicated. Agglomerates of nanoparticles have been

shown to exert lower antibacterial effects as compared to

well dispersed nanoparticles [16]. It was reported that the

presence of proteins within the nanoparticle solution can

stabilize the silver nanoparticles against aggregation [16-

19].

It was the purpose of this study to evaluate the aggre-

gate size of Ag-TiO2 composites in deionized water and

Mueller Hinton Broth, and analyze the bactericidal activ-

ity of the composites using two bacterial strains.

2. Materials and Methods

2.1. Materials

Two commercial TiO2 particles were used as a base ma-

terials, Degussa P25 and DuPont™ Ti-Pure R-902,

AgNO3 (Sigma Aldrich, ACS reagent) was used as a

precursor, NaBH4 (Sigma Aldrich, ACS reagent) were

used as a reducing agent and NH4OH (30% w/w aqueous

solution, Sigma Aldrich, ACS reagent), were used to ad-

just de pH.

Aggregation Study of Ag-TiO Composites

1720 2

2.2. Synthesis of the Composites

Silver nanoparticles were synthesized over the surface of

two different commercial TiO2 particles. The composites

with three molar ratios were prepared following a method

reported by Nino-Martinez et al. and they demostrated that

the nature of de nanoparticles is elemental silver [12].

All preparations started as follows: 0.2 g of TiO2 par-

ticles were dispersed in 100 mL of deionized water by

ultrasound for five minutes. Afterwards the 1:10, 1:25

and 1:50 molar ratios Ag-TiO2 composites were obtained

by addition of 0.0425 g, 0.0169 g, 0.00845 g of AgNO3

respectively. The solution was magnetically stirred for 30

min at pH 7, then predetermined amount of NaBH4, pre-

viously dissolved in deionized water, was added. The pH

of the reaction was adjusted to 10 by adding NH4OH, and

magnetically stirred for another 30 min.

2.3. Characterization

The composites obtained were characterized by using

Dynamic Light Scattering in a Malvern Zetasizer Nano

Zs. Transmission electron microscopy (TEM) analysis

were performed on a JEOL JEM-1230 at an accelerating

voltage of 100 kV.

2.4. Bacterial Strains

Two bacteria were evaluated, Escherichia coli (ATCC

25922) Gram-negative and Enterococcus faecalis, (ATCC

29212) Gram-positive.

2.5. Antibacterial Test

The applied antibacterial test was the standard microdilu-

tion method (NCCLS-CLSI N7 A7 Vol. 26 No. 2, 1996),

which determines the minimum inhibitory concentration

(MIC, as the minimum concentration of tested substance

that inhibited the growth of the bacterial strain). The

MIC was determined on 96-well microdilution plates.

Microorganisms (105 CFU/mL) were exposed to serial

dilutions of Ag-TiO2 particles with Mueller-Hinton Broth

(Fluka), and the endpoints were determined when no

turbidity in the well was observed after 24 hours of in-

cubation at 37˚C. All assays were carried out in triplicate

and the Ag-TiO2 composites were used in the form in

which they had been prepared.

3. Results and Discussion

3.1. Characterization

Six different samples were synthesized with Ag-TiO2

molar ratios 1:10, 1:25 and 1:50, three with TiO2 P25,

and three with TiO2 R-902 as base materials.

TEM images shows that TiO2 P-25 had a size between

10 - 70 nm, TiO2 DuPont R902 had a size 200 - 300 nm.

The Ag nanoparticles deposited on TiO2 P25 surface had

a size between 5 to 40 nm, and Ag nanoparticles depos-

ited in TiO2 R902 surface had a size between 5 to 50 nm.

Figures 1(a) and (b) shows the TEM images for 1:10

Ag-TiO2 R902 and 1:50 Ag-TiO2 R902 composites. Fig-

ures 1(c) and (d) shows the TEM images for 1:10

Ag-TiO2 P25 and 1:50 Ag-TiO2 P25 composites.

In DLS analyses, Ag-TiO2 composites present an over-

all particle diameter (z-average) 377.5, 288, 282, 254.6,

252.4 and 251.5 nm, for samples Ag-TiO2 R902 1:10,

Ag-TiO2 R902 1:25 Ag-TiO2 R902 1:50, Ag-TiO2 P25

1:10, Ag-TiO2 P25 1:25, Ag-TiO2 P25 1:50 respectively.

The polydispersity index (PDI) was below 0.3 in all

cases.

( )

( ) ( )

Figure 1. TEM images of Ag-TiO2 composites (a) 1:10 Ag-TiO2 R902; (b) 1:50 Ag-TiO2 R902; (c) 1:10 Ag-TiO2 P25; (d) 1:50

Ag-TiO2 P25.

Aggregation Study of Ag-TiO Composites1721

The zeta potential of the same samples were –58.8 ±

6.92, –47.8 ± 6.03, –46.78 ± 5.9, –37.7.8 ± 4.8, –49.8 ±

5.89 and –58.8 ± 6.92. Figures 2(a) and (b) show the DLS

results for 1:10 Ag-TiO2 R902 and 1:50 Ag-TiO2 R902

composites. Figures 2(c) and (d) show the DLS results for

1:10 Ag-TiO2 P25 and 1:50 Ag-TiO2 P25 composites.

Acording to the DVLO Theory, the stability of particle

dispersions depends on the balance between attractive

and repulsive forces between the particles. With electro-

static stabilization, the zeta potential of the particles pro-

vides a repulsive force. In practice if the zeta potential of

the particles is higher than 30 mV or lower than −30 mV

the dispersion is stable [19].

To evaluate the evolution of the Ag-TiO2 particle size

(z-average) there were realized three dilutions of each of

the six synthesized composites 100 µg/ml, 250 µg/mL

and 500 µg/ml in deionized water and Mueller Hinton

broth. It is interesting to note that the particles size re-

mained stable in deionized water for about 24 minutes,

235 - 290 nm for Ag-TiO2 R902 1:10 (Figure 3(a)), 240 -

290 for Ag-TiO2 R902 1:25, 257 - 285 nm for Ag-TiO2

R902 1:50, 170 - 190 nm for Ag-TiO2 P25 1:10, 160 -

200 nm for Ag-TiO2 P25 1:25 and 190 - 210 nm f

Ag-TiO2 P25 1:50. In contrast the particles became larger

in Mueller Hinton Broth but once they grew in size, they

remained constant for about 24 minutes, 280 - 360 nm

for Ag-TiO2 R 9021:10 (Figure 3(b)), 260 - 360 nm for

Ag-TiO2 R 902 1:25, 260 - 350 nm for Ag-TiO2 R 902

1:50,190 - 215 nm for Ag-TiO2 P25 1:10, 180 - 220 nm

for Ag-TiO2 P25 1:25 and 210 - 240 nm for TiO2 P25

1:50. The size of de Ag-TiO2 composites was influenced

by the TiO2 base materials in both media.

Steric stabilization is used for nanoparticle dispersion

stabilization, where a stabilizer is added to the dispersion

and it is adsorbed onto the particle surface, preventing

them from coming close to one another [16-19]. It is be-

lieved that nanoparticles are covered by proteins imme-

diately upon contact with a cell culture media and

physiological environment, resulting in a protein ar-

rangement also referred to as protein corona on the parti-

cle surface [16] and this protein corona is exchanging

with other nearby proteins [20]. Mueller-Hinton Broth is

a complex system containing a lot of different proteins.

Therefore, it is possible that Ag-TiO2 composite adsorb

proteins from Mueller-Hinton Broth, as indicated by an

increase in the particle size (z-average), which could

make them become more stable. The identification of the

protein corona composition was not the focus of this work.

3.2. Antibacterial Results

Minimum inhibitory concentration values were obtained

(Table 1). The six different samples have antibacterial

activity. The TiO2 particles present no antibacterial activ-

ity. The test was performed on dark and it is reported that

TiO2 particles in dark condition present no antibacterial

activity [12,15] which is consistent with our results. The

six composites show antibacterial activity without light.

We evaluated the antibacterial activity after 24 minutes

of exposure of the Ag-TiO2 composites in Mueller-Hin-

ton Broth, and this did not affect the MIC for the micro-

organisms involved. According to Lynch and coworkers

[20] if the protein corona exchanges with other proteins

in the medium faster than the time it takes for the particle

to attach to the bacteria surface, then the particle-bacteria

interactions will not be greatly affected by the presence

of the corona. Therefore the MIC of the composite would

not be affected. We found the best results in Ag-TiO2

1:10 composite.

Figure 2. DLS results (a) 1:10 Ag-TiO2 R902; (b) 1:50 Ag-TiO2 R902; (c) 1:10 Ag-TiO2 P25; (d) 1:50 Ag-TiO2 P25.

Aggregation Study of Ag-TiO Composites

1722 2

(a) (b)

Figure 3. Temporal evolution of the Ag-TiO2 DuPont 1:10 composite size (z-average): (a) deionized water; (b) Mueller Hinton

Broth.

Table 1. Minimu n inhibitory concentr ation of Ag-TiO2 nano-

particles against E. coli and E. faecalis.

Material MIC

(µg/mL)

E. coli

(Gram-negative)

E. faecalis

(Gram-positive)

Ag-TiO2 1:10

(P25)

500 ± 0

500 ± 0a

500 ± 0

500 ± 0a

Ag-TiO2 1:10

(R902)

500 ± 0

500 ± 0a

375 ± 144.3

375 ± 144.3a

Ag-TiO2 1:25

(P25)

500 ± 0

500 ± 0a

1000 ± 0

1000 ± 0a

Ag-TiO2 1:25

(R902)

500 ± 0

500 ± 0a

1000 ± 0

1000 ± 0a

Ag-TiO2 1:50

(P25)

1000 ± 0

1000 ± 0a

1000 ± 0

1000 ± 0a

Ag-TiO2 1:50

(R902)

1000± 0

1000 ± 0a

1000 ± 0

1000 ± 0a

TiO2 (P25)

TiO2 (R902)

>2000b

>2000b >2000b

>2000b

4. Conclusions

Six different molar ratios Ag-TiO2 composites were pre-

pared and characterized. We found that Ag-TiO2 com-

posites increased in size with respect to deionized water

when be diluted in Muller-Hinton Broth, but once they

grew in size, they remained constant for 24 minutes, and

did not affect the MIC for the microorganisms involved.

The Ag-TiO2 1:10 composites showed promising results

as an antibacterial agent against E. faecalis Gram-posi-

tive and E. coli. Gram-negative bacteria.

5. Acknowledgements

M. E. Noriega-Treviño, J. E. Morales-Sánchez and J. M.

Guajardo-Pacheco would like to thank CONACYT for

grant of scholarships.

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