Original article

Textile Research Journal

84(1) 40–51

ZnO-modified hybrid polymers as an ! The Author(s) 2014

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antibacterial finish for textiles DOI: 10.1177/0040517513485623
trj.sagepub.com

Asmaa Farouk1,3, Shaaban Moussa4, Mathias Ulbricht1,2,

Eckhard Schollmeyer1 and Torsten Textor1

Abstract
The antibacterial activity of ZnO is reported by several authors. We present the preparation and application of inor-
ganic–organic hybrid polymers modified/filled with ZnO nanoparticles of varying particle sizes. Inorganic–organic hybrid
polymers employed here are based on 3-glycidyloxypropyltrimethoxysilane (GPTMS). ZnO is prepared by hydrolysis of
zinc acetate in different solvents (methanol, ethanol or 2-propanol) using lithium hydroxide (LiOH  H2O). The hybrid
materials prepared are applied to cotton (100%) and cotton/polyester (65/35%) fabrics. The antibacterial performance of
these sol-gel derived hybrid materials is exemplarily investigated against Gram-negative bacterium Escherichia coli and
Gram–positive Micrococcus luteus. Effects of particle size and concentration for the antibacterial performance are
examined. Literature discusses various (active) species and processes responsible for the antibacterial action of ZnO.
Therefore, particular attention is paid to investigate active species available in the described systems as well as to observe
possible interaction between the nanoparticles and bacteria; the first results are presented.

Keywords
Zinc oxide nanoparticles, antibacterial sol-gel coating, hybrid polymers

The growth of bacteria on textile material during use The inorganic materials are discussed as advanta-
or storage harmfully affects the textile or in case of geous for several reasons. In many cases the mode of
clothes the person wearing the garment. By employing action is less specific which allows reducing a wide
durable antimicrobial finishing of textiles using broad- range of germs and prevents the development of resist-
spectrum biocides, on can limit at least the unpleasant ances at least to a certain degree. Some argue that the
and sometimes dangerous growth. Biocides or bac- increase of resistances is excluded for the antibacterial
teriostatic agents employed can be divided into two activity of silver.4 The durability of metals or metal
types, organic or inorganic ones respectively. Organic oxides is high compared with various organic alterna-
biocides include organic acids or tertiary alkyl amines; tives and the harmful potential is often lower with metals
a widely used bacteriostatic agent is triclosan, a poly- or metal oxides.3 Some of these metal oxides such as
chloro phenoxy phenol.1,2 Although such compounds ZnO and TiO2 have been used in the production of
are widely used there are certain drawbacks as they
are often less stable at higher temperatures and show a
limited durability. Furthermore different organic bio-
1
cides or bacteriostatic agents bear an allergic potential Deutsches Textilforschungszentrum Nord-West gGmbH (DTNW),
Germany and CENIDE - Center for Nanointegration Duisburg-Essen,
that should not be disregarded. Specifically, the
Germany
organic compounds employed are discussed as being 2
Technical Chemistry II, University of Duisburg-Essen, Germany
responsible for the spreading of resistant germs. Some 3
National Research Center, Textile Research Division, Cairo, Egypt
4
organic biocides are pollutants and/or enrich in the Genetic Engineering and Biotechnology Research Institute, Minofya
environment.1 Inorganic biocides are metals such as University, Egypt
silver or copper and oxidic materials, e.g. ZnO,
Corresponding author:
CuO, MgO or TiO23. Silver is actually a widely Torsten Textor, Deutsches Textilforschungszentrum Nord-West gGmbH
employed antibacterial material not only used in tex- (DTNW), Adlerstr. 1 47798 Krefeld, Germany.
tile finishing.3 Email: textor@dtnw.de
Farouk et al. 41

cosmetics or personal care products, e.g. sun-creams.4 The study presented here is a part of an ongoing
Such materials are of interest for many further applica- research project aimed at the development of a stable
tions. As an example, ZnO, a semiconductor with a wide and highly durable multifunctional product for finish-
band-gap (3.36 eV), has achieved increased attention ing textiles. We recently reported on the use of such
concerning possible electronic applications due to its inorganic–organic hybrid polymers filled with ZnO
unique optical, electrical and chemical properties.5 nanoparticles first focusing on a use as an ultraviolet
Antibacterial properties of the inorganic materials protection finish for textile.21 The investigations shown
are often explained by the release of ions interfering here prove that ZnO-containing hybrid materials are
with bacteria6 which has been reported in detail for suitable to develop antibacterial textiles with excellent
silver ions.4 In the case of titanium dioxide, the biocidic activities. This work describes the synthesis, character-
effect can be explained by the so-called photo-catalytic ization and application of appropriate ZnO nanoparti-
effect. The photo-catalytic effect is very complex; one of cles. Furthermore, it specifies the investigation of the
the main reasons for its antibacterial activity is in the antibacterial activity as well as the experiments helping
fact that water or hydrogen ions respectively are to understand the mechanisms responsible for the
reduced on the oxide’s surface producing hydroxyl rad- antibacterial action of our ZnO-based materials.
icals.7 When these very reactive species interfere with
organic material as well as with microorganisms they
cause decomposition. Since these radicals are able to Experimental section
decompose nearly all organic molecules the develop-
Materials
ment of bacterial resistances is highly improbable.
The release of the active ions as well as the photo-cat- The coating experiments were carried out using plain-
alytic effect is strongly dependent on the specific surface weaved fabrics made of scoured and bleached cotton
area of the materials used. The specific surface area is (100%), with a mass per unit area of 250 g/m2, and
well known and is connected to the particle size of the blended fabrics made of polyester/cotton (65/35%),
materials; therefore, nanoparticles are of an enormous with a mass per unit area of 162 g/m2. Both materials
interest in this field. When the primary particle is smal- were standard test materials supplied by WFK,
ler, the specific surface area is higher.8 Germany. Prior to application, the fabrics were purified
There are many studies investigating the antibacterial by washing with warm water (40 C) and methanol. For
effect of ZnO nanoparticles. Sawai et al.,9–12, for exam- the washing, 1 g/l of non-ionic detergent (MarlipalÕ O
ple, reported that ZnO particles show antibacterial 80/13) was added. After washing, the fabric was rinsed
activity against both Gram-positive and Gram-negative with warm water and dried, acclimatized for 24 h (22 C,
bacteria. ZnO particles are known to be active even 65% rel. humidity) and weighted before using.
against spores that are high temperature and high pres-
sure resistant. ZnO treatment influences the activity of
the particles; high-temperature treatment of the oxide
Chemicals
lowers the particle’s antibacterial activity.9–12 Zinc acetate dihydrate (ZnAc), lithium hydroxide
Yamamoto et al.13 reported that the surface area and monohydrate (LiOHH2O, 98%), methanol, ethanol
the concentration play an important role in the antibac- and 2-propanol were obtained from Merck. 3-
terial activity, while the particle shape and crystalline Glycidyloxypropyltrimethoxysilane (GPTMS, 98%)
structure have lower impact. Yamamoto et al.13 also was obtained from ABCR. Further chemicals used
reported that the antibacterial activity increased as the were fluorescein isothiocyanate (FITC, Aldrich, 90%),
particle size decreased (which necessarily leads to an catalase from bovin liver, (activity  2000units/mg from
increased surface area)3. Adams et al.,14 Brayner Sigma-Aldrich, Germany) and 2,2-diphenyl-1-pikrylhy-
et al.15 and Jeng et al.16 reported that ZnO behaves in drazyl (DPPH, Aldrich, 98%). For catalyzing the
a different way towards microorganisms than towards cross-linking reaction of the epoxy group of the
other metal oxides such as SiO2, MgO, TiO2 and GPTMS 1-methylimidazole (97%, Fluka) was used.
CaO.15–17 All observations reported by the researchers All chemicals were used without further purification.
were explained by a number of mechanisms. These Lysogeny broth (LB broth) was obtained from Sigma-
mechanisms include the formation of active oxygen spe- Aldrich, Germany.
cies in the presence of the corresponding nanoparti-
cles12,18,19 which damages the membrane cell wall
Microorganisms
because of the binding of the particles on the bacteria
surface due to the electrostatic forces19,20 or the Escherichia coli DSMZ498 and Micrococcus luteus
penetration of particles or ions through the cell ATCC-No. 9341 were incubated overnight at 37 C in
membrane.18 a nutrient broth (LB broth medium: 20 g LB broth/liter
42 Textile Research Journal 84(1)

water). The cultures obtained were diluted with auto-

claved nutrient broth to obtain cell suspensions which
Preparation of the hybrid polymer
were adjusted to a final working concentration. Before the finishing of the textiles, the zinc oxide dis-
persion and the GPTMS-sol were mixed in different
ratios and finally 1-methylimidazol (0.5 ml/10 ml
Preparation of zinc oxide nanoparticles
GPTMS) was added as a catalyst for the cross-linking
The preparation procedure employed has been described reaction of the epoxy group of the GPTMS. The label-
in previous work21 and is a variation of a preparation ing used in the text gives the amount of ZnO in relation
described by Spanhel et al.22 A two-neck round bottom to GPTMS, therefore 10% ZnO means 1 g ZnO per
distillation flask was used to suspend 2.8 g of 10 g GPTMS.
(ZnAc2H2O) in 100 ml of the particular solvent (metha-
nol, ethanol or 2-propanol) by reflux heating for 3 h.
Characterization of the ZnO nanoparticles
0.75 g of lithium hydroxide was dissolved in 100 ml of
solvent at room temperature by magnetic stirring. The The size of the ZnO nanoparticles was measured by
ZnAc suspension was cooled to 0 C before the lithium dynamic light scattering (DLS), using a Zetasizer,
hydroxide solution was added by drops under vigorous Nano-S, produced by Malvern. For all samples high
stirring. The mixture was treated in an ultrasonic bath polydisperity index (PDI) values in the range of 0.4–
(SONOREX TK 52H) at room temperature for about 0.5 were calculated indicating broad dispersions.
2 h. The resulting sol theoretically contains 0.675 wt.% Samples were only analyzed and used for further inves-
ZnO. Higher amounts of ZnAc and lithium hydroxides tigations if the size quality report of the calculation
were used (using constant amounts of solvent) to pre- showed no errors.
pare colloidal solutions of higher concentration.
Coating process
Preparation of zinc oxide nanoparticles labeled
The hybrid polymer sol was applied to the fabrics by a
with FITC pad–cure–method. The coating was carried out by a
ZnO prepared as described above had been centrifuged padding process with a laboratory padder equipped
and washed, before 0.1 g were resuspended in 100 ml with roles with a standard rubber hardness of 70
2-propanol. 20 mg of FITC were added to this solution Shore (Mathis, Switzerland). The nip-pressure was
before it had been stirred for 1 h. Subsequently 100 ml of adjusted to a value guaranteeing a wet pick up of
the resulting ZnO/FITC solution was added to 50 ml of 100%; the role speed was 4 m/min. After padding, the
an aqueous solution containing E. coli cultures (108 samples were dried in a labcoater (Mathis, Switzerland)
colony forming units [CFU]/ml) which were grown over- at 130 C for 30 min before they were washed to remove
night at 37 C in a nutrient broth medium (LB broth residual by-products.
medium). A control solution of the same concentration
was prepared in parallel. FTIC is expected to link chem- Inductively coupled plasma–optical emission
ically to ZnO and is therefore used to label the nanopar-
spectrometry
ticles. The dye FTIC shows a distinct green fluorescence
which allows a visualization of the labeled nanoparticles The amount of ZnO or zinc respectively was investi-
in the fluorescence microscope. Furthermore, the attach- gated by inductively coupled plasma–optical emission
ment of the labeled particles to the bacteria can be visua- spectrometry (ICP–OES). Coated cotton samples were
lized. The approach is employed to prove an occurring treated with concentrated nitric acid (65%) before the
of the attachment of the ZnO nanoparticles to the bac- decomposition of the material was carried out at
teria surface. If it occurs, a decomposition of the bacteria 180 C in a microwave digester (MarsXpress, CEM,
cells by time can also be visualized. The stained bacteria Kamp-Lintfort, Germany). The resulting solutions
were observed using a microscope (KEYENCE all-in- were completely transferred in volumetric flasks and
one-Type Fluorescence Microscope, BZ-Analyzer diluted to a defined volume with distilled water. ICP–
8100E, Japan). OES measurements were carried out with a Varian
720-ES.
Preparation of the GPTMS-sols
Scanning electron microscopy
10 ml of GPTMS were dissolved in 100 ml of solvent
before hydrolyzation using 0.01 M hydrochloric acid. For qualitative assessment, the samples were studied by
The resulting sol was stirred for at least 3 h to form scanning electron microscopy (SEM). The micrographs
the basis sol. in Figure 1 were prepared using a Topcon-Microscope
Farouk et al. 43

inoculated and placed in an incubator for 24 h at

37 C. The pure agar medium will be overgrown by
the bacteria, if antimicrobial substances diffuse into
the agar medium growth will be prevented which can
be observed by a halo (zone of inhibition). The diam-
eter of the halo can be used as a measure of the anti-
bacterial activity.
Modified AATCC test method 100-2004: the pur-
pose of the AATCC test method is to ‘‘provide a quan-
titative procedure for the evaluation of the degree of
antibacterial activity’’ of finishes on textile materials.12
The method is based on an inoculating test and was
modified for the experiments presented; the procedure
valid here is as follows. Textile samples (diameter 6 cm)
are placed in a flask sealed with aluminum foil
before sterilization. In the next step, 3 ml of inoculum
(1-2*106 CFU/ml) are added. Samples are taken imme-
diately after the addition and after 24 h. Samples were
taken by the addition of 100 ml 0.9% NaCl before
shaking vigorously for 1 minute, followed by extracting
1.5 ml of the solution, and applying 500 ml to agar
plates in logarithmic dilutions 0, 1, 2 (except the
blanks after 24 hours, which were plated with up to
six logarithmic dilutions since stronger growth was
expected). The plates were incubated at 37 C for 48
hours, after which they were removed from the incuba-
tor and any formed colonies were counted. After that
the number of bacteria was counted and the reduction
of bacteria, R, was calculated as follows:9
Figure 1. SEM micrographs of a blank cotton fabric (upper
micrograph) and a cotton fabric treated with a GPTMS-sol BP
R% ¼  100
modified with 10 wt.% ZnO particles (size 30–60 nm) (lower B
micrograph).
R ¼ reduction in percentage
(ATB-55) those in Figure 6 using a Hitachi S-3400 B ¼ CFU/test sample in blank
Type II (Tokyo, Japan). P ¼ CFU/test sample for treated samples

Tetrazolium/formazan test (TTC): in the presence of

Antimicrobial testing bacteria, TTC is reduced to red formazan.23 The red
The antimicrobial activity of the coated fabrics was formazan obtained therefore indicates the activity and
tested against the Gram-negative bacterium E. coli viability of the cells. Therefore, the TTC test method is
and the Gram–positive M. luteus using the following considered as a fast method for evaluating the antibac-
qualitative as well as quantitative tests: terial activity of fabrics. To complete this test, circular
swatches of test fabrics of a diameter 3.8  0.1 cm were
. zone of inhibition test (disc diffusion test); cut into a small size. The number of swatches to be used
. American Association of Textile Chemists and was six. Both, the test and control swatches, were ster-
Colorists (AATCC) Test Method 100-2004; ilized at 110 C before incubation. All swatches were
. tetrazolium/formazan test (TTC). placed in a 40 ml nutrient broth medium containing
10 ml an E. coli solution (108 CFU\ml) as an inoculum
The zone of inhibition method (disc diffusion test) has volume. All flasks were incubated while shaking at
been employed as a method to evaluate the antibacter- 37 C/200 rpm for 3 h; then, 1 ml from each flask con-
ial activity of textiles. The test is carried out according taining the fabrics to be tested was added to the ster-
to EN ISO 20645:2004. Sterilized agar plates are pre- ilized test tubes containing 100 ml TTC solutions (0.5%
pared before a textile sample to be investigated is w/v). All tubes were incubated at 37 C for another
placed onto the surface. Afterwards the agar plate is 20 min. The resulting formazan was centrifuged at
44 Textile Research Journal 84(1)

4000 rpm for 3 min followed by decantation of the Table 1. Particles size of ZnO nanoparticles pre-
supernatants. The pellets obtained were dissolved in pared in different solvents measured by DLS
ethanol and centrifuged again. The red formazan solu-
Particle size, Average
tion obtained at the end which indicated the activity Solvent DLS [nm] PDI
and viability of the cells was measured by a photometer
at 480 nm. isopropanol 30–60 0.399
ethanol 250–300 0.395
High-performance anion exchange methanol 600–650 0.460
chromatography–pulsed amperometric
detection measurements fiber sample shows a very smooth, less structured sur-
The generation of H2O2 from ZnO were detected using face. Big agglomerates are not observed indicating a
a high-performance anion exchange chromatography– homogeneous distribution of ZnO in the coating layer.
pulsed amperometric detection (HPAEC–PAD) system
from DIONEX (Idstein, Germany). The system was Evaluation of antibacterial activity for
equipped with a Carbo Pac PA 1 column and 0.5 M
NaOH acted as eluent. Calibration was carried out in
ZnO-treated fabrics
a range from 0.2 down to 0.0012 g H2O2/l. The calibra- As mentioned before, fabrics are excellent media for the
tion curve is the average area of three repeated meas- growth of microorganisms when the basic requirements
urements. For measuring the amount of H2O2 formed such as nutrients, moisture, oxygen and appropriate
in ZnO dispersions, different amounts (0.5, 0.8 and temperature are available. Natural fibers like cotton
1 g/l) of ZnO particles (30 nm) were suspended in are more susceptible to microbial attack than synthetic
water before the resulting dispersions were injected. fibers. Antibacterial activity of cotton and cotton/poly-
ester blended fabrics coated with hybrid polymers
containing ZnO nanoparticles were evaluated to
Laundering assess the effectiveness of ZnO nanoparticle coating.
A single laundering was performed in a laboratory Antibacterial activity is evaluated qualitatively and
washing machine (Linitest, ATLAS in accordance to quantitatively.
DIN EN ISO 105-C06 using a non-ionic surfactant Results for cellulosic cotton fabrics are shown as
(MarlipalÕ O 80/13), 40 C). All textile samples were exemplarily for the evaluation of the antibacterial activ-
washed once before the different investigations were ity of textiles finished with hybrid polymer coatings
carried out. prepared with ZnO-sols. The hybrid polymers were
either prepared with ZnO-sols of various concentra-
tions of particles of a given size, or with widely differing
Results and discussion particle sizes of a given concentration. Antibacterial
The synthesis of ZnO nanoparticles basically described activity was tested against E. coli and M. luteus on
by Spanhel et al.22 leads to the formation of colloidal agar plates by evaluating the resulting zone of inhib-
ZnO particles with a comparably homogenous distribu- ition (area of halo). The results of the zone of inhibition
tion and an average particle size as summarized in test for a sample coated with a hybrid polymer loaded
Table 1. The results presented here show typical particle with different amounts of ZnO particles of a given par-
sizes observed for the concentration resulting from the ticle size are shown in Figure 2 (left photograph). The
receipt presented in the experimental part. Further vari- test is carried out against M. luteus and it can be
ations in the particle size were achieved by increasing observed that the blank sample (1) and the one
the overall concentration of the ZnO-sols. coated with the hybrid polymer without ZnO (5)
After preparation of ZnO nanoparticles, particles yields no antibacterial effect. Samples modified with
dispersed in the mentioned solvents were mixed with ZnO yield a distinct activity which increases with the
a freshly prepared GPTMS-sol. The resulting sol was increasing amount of ZnO. We investigated the amount
applied to textile materials by a padding process. After of zinc oxide deposited on corresponding samples by
drying and curing all samples were laundered before ICP-OES supplementary. The values measured for zinc
further experiments were carried out. To investigate are ranging from 8 g/kg fabric (for sample ‘‘10% ZnO’’)
the homogeneity of the coating and to exclude the to 28 g/kg fabric (for sample ‘‘50% ZnO’’). The values
occurrence of undesired agglomeration of the ZnO increase linearly (R2 ¼ 0.99642) increasing the amount
nanoparticles SEM investigations were carried out. of ZnO particles added to the GPTMS-sol. Figure 2
The corresponding SEM micrographs are shown in (right photograph) shows the results for the hybrid
Figure 1. Compared to the blank sample, the treated polymer coatings of the same amount of ZnO with
Farouk et al. 45

Figure 2. Disc diffusion tests with M. luteus (left) and E. coli (right). Samples in the left dish were coated with the hybrid polymer
loaded with ZnO particles of a given size distribution (30–157 nm) and various concentrations. (1) untreated, (2) 10% ZnO, (3) 30%
ZnO, (4) 50% ZnO, (5) treated with GPTMS-sol only. Samples in the right dish were coated with the hybrid polymer loaded with ZnO
particles of a given concentration (10% ZnO) and various size distributions, (1) untreated, (2) 600–650 nm, (3) 250–300 nm,
(4) 30–60 nm. (The relative share of ZnO is always calculated as wt.% in relation to GPTMS in the sol.)

varying particle sizes. As can be clearly seen, when the ZnO (10%, 30–60 nm) leads to a 98.8% reduction of
particle size is decreased, the antibacterial activity E. coli after 5 h and a 97.3% reduction of M. luteus.
increases. These tests are carried out with E. coli. The same measurements were carried out to evaluate
In general, from Figure 2 it can be concluded that the antibacterial activity of cotton/polyester blended
the untreated fabrics as well as the fabrics treated with fabrics coated with the hybrid polymer modified with
only GPTMS had no antibacterial activity. An antibac- a constant mass of ZnO but with a varying particle size.
terial activity can be observed when fabrics are treated Measurements were performed against E. coli and
with the hybrid polymer modified with ZnO nanopar- M. luteus. As shown in Figure 4, treatment of the bac-
ticles. Activity of treated fabrics increases with increas- teria for 5 h with the fabric coated with the hybrid poly-
ing nanoparticle concentration as well as with mer loaded with ZnO particles of the smaller particle
decreasing particle size. Both variations mean an size (30–60 nm) leads to a complete reduction of the
increase of the active surface area of ZnO and therefore E. coli and M. luteus populations, while fabrics
an increased biocidal effect. coated with hybrid polymers loaded with bigger ZnO
For a given set of experiments, either by increasing particles (600–650 nm) achieve a 93% and 97% reduc-
the amount of particles of a given size or by decreasing tion of the E. coli and M. luteus populations, respect-
the particle size at a given mass of particles the active of ively. The results for ZnO particles in the range of
ZnO surfaces increase which seems to be the reason for 245 nm (PDI ¼ 0.456) are in between.
an increased activity. A further quantitative investigation of the antibac-
In further experiments, antibacterial activity of the terial activity of fabrics coated with the hybrid polymer
coated fabrics was determined for E. coli and M. luteus modified with different amounts of the active ZnO par-
according to the AATCC 100-2004 standard method. ticles – with a given particles size in the range of
Following a slightly modified AATCC Test Method 30–60 nm– has been carried out using the TTC test.
100-2004 (see the experimental section)12 the antimicro- Details about this method were described in the experi-
bial activity of the treated fabrics was evaluated in sev- mental part. This test serves as indicating the system for
eral test series. Qualitative results for the antibacterial the determination of the viability of microorganisms.
activity of cellulosic fabrics coated with the ZnO- The absorbance of formazan that is formed in the pres-
modified hybrid polymers are presented in Figures 3 ence of vital microorganisms is directly proportional to
and 4. As expected, no reduction of the bacteria the amount of living bacteria. Corresponding data
E. coli and M. luteus was detected for the untreated shown are in good agreement with the results shown
fabrics. On the contrary, there was a decrease in the before. We observe a strong reduction of the vital cells
number of bacteria counted after 5 h incubation (com- due to the presence of ZnO-modified textiles. Raising
pared with 0 h contact time). As shown in Figure 3, a the relative amount of ZnO in the hybrid poly-
cotton sample coated with a GPTMS-sol loaded with mer increases the resulting antibacterial activity.
46 Textile Research Journal 84(1)

Figure 3. Reduction rate of E. coli and M. luteus versus time for cotton fabrics treated with a GPTMS-sol modified with ZnO
nanoparticles (10% ZnO).

Figure 4. Reduction rate of E. coli and M. luteus after 5 h in the presence of cotton/PET blended fabric (65/35%) treated with the
GPTMS-based hybrid polymer loaded with 10% ZnO nanoparticles of different particle sizes.
Farouk et al. 47

Figure 5. Upper row: comparison of microscopic investigation (visible light and fluorescence) of bacteria broth with and without
FITC. Proving that a staining of the bacteria itself with FITC cannot be observed, neither in visible nor in fluorescence mode. Second
and third row: cell imaging (fluorescence light) with FITC-modified ZnO nanoparticles. Pictures show the solution after up to 4 h after
adding the ZnO to the bacteria broth.

A concentration of 10 wt% ZnO in the coating yields a the number of bacteria. The reduction in the number
reduction of more than 75% within 3 h; an increase of the fluorescing ZnO-covered bacteria observed on the
this concentration to 50 wt% improves this value to images indicates that the bacteria are more or less com-
approximately 90%. pletely decomposed. To verify this observation samples
To follow the reduction of bacteria in the presence of were taken from the same bacteria solution after 4 h
ZnO, visually particles were labeled with the fluores- and bred on agar – no colonies were grown proving
cence dyestuff FITC. After combining labeled ZnO par- the absence of vital bacteria.
ticles (30–60 nm) and an E. coli solution, the particles Further investigations aiming on the visualization of
cover the bacteria surface (as described by Wang the bacteria – ZnO nanoparticle interactions were car-
et al.24). This allows a visualization of the particle-cov- ried out by SEM analyses. SEM analyses were per-
ered bacteria in the fluorescence microscope as can be formed to investigate morphological changes of
seen in Figure 5 (photograph labeled 0 h). Further M. luteus in the presence of ZnO particles (30–60 nm).
microscopic images taken after repeated sample We mixed 50 ml of the M. luteus culture with autoclaved
taking over four hours prove the drastic reduction of LB broth medium and ZnO nanoparticles dispersion.
48 Textile Research Journal 84(1)

Figure 6. SEM micrographs of (a) blank M. luteus culture, (b) M. luteus culture treated with ZnO nanoparticles (30–60 nm) after 1 h,
(c) M. luteus culture treated with ZnO after 2 h, (d) M. luteus culture treated with ZnO after 3 h.

Cultures were grown at 37 C under agitation. Sample pH of 9.0.25 Since the pH of all solutions employed here
of these solutions were taken, placed on a support, and is 7.0, a positive charging of the particle’s surface is
dried in a vacuum before the investigation by SEM. expected while the surface of the bacteria is expected
Figure 6(a) shows cultures of bacteria not treated to be negative. The observations described are in good
with ZnO. The cell radius is in the range of 840 nm agreement with the observations shown above, e.g. in
and therefore much larger than the ZnO particles Figure 6(b). Certainly this does not explain the mech-
employed for the following samples. Comparing this anisms that are responsible for the antibacterial activity
micrograph with the one showing bacteria that were in of the ZnO-modified hybrid polymer coatings that were
contact with the nanoparticles one observes a coverage applied to the textiles. The particles immobilized on the
of the bacteria and a distinct lumping (compare to textile surface will not be available for covering the
Figure 6(b)). By the time, a reduction of the number of bacteria. The halos in the tests described before prove
bacteria can be observed as shown in Figure 6(c) and (d), that there must be a mechanism that does not need a
supporting the results of the fluorescence microscopy. direct contact between ZnO particles and the bacteria.
In the following paragraph, experiments are described
Investigation of the effects responsible for the that were carried out to investigate possible explan-
ations for the antibacterial properties. It should be
antibacterial action
noted that antibacterial activity of ZnO in our work
The results presented above show that if bacteria and has been studied in a normal laboratory environment
zinc oxide particles are available in an aqueous medium and without ultraviolet (UV) light, which might be
there will be coverage of the bacteria with the oxide important since ZnO might show a photo-catalytic
particles; obviously a lumping occurs to a certain activity.
degree and the number of intact bacteria is drastically To exclude the possibility that zinc ions are released
reduced within hours. Electrostatic interaction between into the aqueous ZnO suspension were responsible for
the surface charge of ZnO particles and the membrane the antibacterial activity, control experiments were car-
charge surface of bacteria could contribute to the anti- ried out by dissolving a zinc salt (zinc acetate, 0.045 M)
bacterial behavior of the ZnO dispersions. It has been in water. Tests were carried out for antibacterial activ-
reported that the point of zero charge for ZnO is at a ity against E. coli and it was found that under the
Farouk et al. 49

conditions used in this study, zinc (II) ions are not proving that H2O2 is involved in the antibacterial activ-
responsible for the strong antibacterial activity ity, a possible release of H2O2 from ZnO was tested
observed for the ZnO nanoparticle systems. using HPAEC–PAD. The corresponding results are
Shantikumar et al.8 gives hints, that hydrogen per- presented in Table 2. It has to be noticed that the
oxide is generated in aqueous ZnO dispersion. For values are below the lower limit of the calibration
range (and near the lower edge of the accuracy of the
equipment). Therefore the measurements were repeated
Table 2. Amount of H2O2 generated from three times with comparable results. This is why the
ZnO nanoparticles (30–60 nm) authors state that the measurements give at least a
qualitative proof for the presence of hydrogen perox-
Conc. ZnO
nanoparticle [g/l] Conc. H2O2[mg/l] ide. So, chemical reactions between the cell envelope
components and chemical species such as hydrogen
0.5 0.15 peroxide, generated due to the presence of ZnO par-
0.8 0.29 ticles, could contribute to the antibacterial behavior
1.0 0.38 of the ZnO-sol.
To support that H2O2 is in fact involved in the anti-
bacterial activity, antibacterial activity of ZnO disper-
Table 3. Effect of catalase for the antibacterial sion in presence of catalase was tested. Catalase is an
activity of ZnO dispersions enzyme that decomposes hydrogen peroxide to water
and oxygen. We found that the antibacterial activity
Bacterium ZnO ZnO /Catalase
decreased when catalase is present in the solution (cp
E. coli +  Table 3) which is a strong hint supporting the explan-
M. luteus +  ation that hydrogen peroxide is at least jointly respon-
sible for the antibacterial properties of ZnO.
(+): distinct antibacterial activity is observed
(): no antibacterial activity observed
Another suggested mechanism responsible for the
antibacterial activity of ZnO nanoparticles is the

Figure 7. Relative absorption of aqueous ZnO dispersions containing a given mass of ZnO with varying particle sizes. Initial
dye concentration was 20 mg DPPH per 100 ml dispersion. Changes occurring in ambient light have been observed for up to
90 min by measuring absorbance. Absorbance was measured at the maximum of aqueous DPPH solutions, which is at a wavelength
of 514 nm.
50 Textile Research Journal 84(1)

generation of oxy- or hydroxyl radicals. If avail- followed by a lumping which obviously also contributes
able, these radicals will be able to be involved in the to the observed decomposition of the bacteria.
decomposition of the cell membrane, for example. For The results are very promising since the antibacterial
proving the existence of radicals 2,2-diphenyl-1-pikryl- activity of the textile material is comparably high;
hydrazyl (DPPH) a colored and stable free radical has ongoing work aims at a combination with other anti-
been employed. DPPH is used as scavenger for radicals bacterial additives (e.g. biopolymers) to further
(e.g. hydroxyl radical, superoxide anion radical and improve the performance.
many more). As a result of a combination process
with other radicals the violet color of DPPH converts Funding
to colorless. In this experiment 20 mg of DPPH was The authors wish to convey a very special thanks to the
added to 100 ml ZnO nanoparticle dispersions of a Egyptian Missions Department for giving Asmaa Farouk
given concentration (1 g/l) but with different particle the opportunity and the financial support to pursue a PhD
sizes. The mixtures were initially stirred for 10 min degree in Germany. Further financial support by the
under the dark condition before they were stored for Ministerium für Innovation, Wissenschaft, Forschung und
a certain time at the bench (daylight). The UV-visible Technologie des Landes Nordrhein-Westfalen (Department
spectra shown in Figure 7 were recorded immediately. of Innovation, Science, Research and Technology of
the state of Nordrhein-Westfalen) is also gratefully
The spectra clearly show a discoloration proving the
acknowledged.
presence of radicals. For a given dispersion, the discol-
oration of DPPH proceeds with time as can be seen in
Acknowledgment
Figure 7. The results furthermore show that the amount
of radicals available in the solution is also dependent on We thank Prof Dr Nickisch-Hartfiel, HS-Niederrhein, for her
kind support in carrying out the antibacterial tests.
the particle size. When the particles at a given mass
concentration are smaller, the discoloration of the
References
DPPH is stronger.
1. Sawai J. Quantitative evaluation of antibacterial activities
of metallic oxide powders (ZnO, MgO and CaO) by con-
ductimetric assay. Microbiol Meth 2003; 54: 177–182.
Conclusion 2. Schweizer HP. Triclosan: a widely used biocide and its
link to antibiotics. FEMS Microbiol Lett 2001; 202: 1–7.
Nano sized zinc oxide particles were successfully 3. Fu G, Vary PS and Lin CT. Anatase TiO2
synthesized, applied and added to inorganic–organic Nanocomposites for Antimicrobial Coatings. Phys
hybrid polymer sols. The resulting hybrid materials Chem B 2005; 109: 8889–8898.
can be applied to textile materials by a simple pad– 4. Li Y, Leung PL and Yao QW. Song and Newton E.
dry–cure method – here shown as exemplarily for Antimicrobial effect of surgical masks coated with nano-
cotton (100%) and polyester/cotton (65/35%) fabrics. particles. Hosp Infect 2006; 62: 58–63.
The resulting textile materials exhibit antibacterial 5. Baxter JB and Aydil ES. Nanowire-based dye-sensitized
activity. The antibacterial performance of fabrics solar cells. Appl Phys Lett 2005; 86: 53–114.
coated with different amounts of active ZnO particles 6. Wiegand A, Buchalla W and Attin T. Review on fluoride-
releasing restorative materials—Fluoride release and
or different particle sizes was investigated respectively.
uptake characteristics, antibacterial activity and influence
The antibacterial activity of textiles treated with ZnO
on caries formation. Dent Mater 2007; 23: 3.
nanoparticles increases when the amount of nanoparti- 7. Zhang L, Jiang Y, ding Y, Daskalakis N, Jeuken L,
cles deposited is increased and it increases with decreas- Povey M, O’Neill AJ, York DW. Mechanistic
ing particle size. The enhanced bioactivity of smaller Investigation into antibacterial behaviour of suspensions
particles is attributed to the higher surface area to of ZnO nanoparticles against E. Coli, J Nanopart Res
volume ratio; up to 98.8% of E. coli and 97.3% of 2010; 12: 1625–1636.
M. luteus were killed within 5 h by the best samples 8. Shantikumar N and Abhilash S. Role of size scale of ZnO
prepared for this report. nanoparticles and microparticles on toxicity toward bac-
Further experiments were carried out to explain the teria and osteoblast cancer cells. Mater Sci 2008; 5: 3548.
mechanism responsible for the antibacterial action. It 9. Sawai J, Igarashi H, Hashimoto A, et al. Evaluation of
growth inhibitory effect of ceramics powder slurry on
was proven that the activity is not due to a release of
bacteria by conductance method. Chem Engr Japan
zinc ions. Results showed that hydrogen peroxide and/
1995; 28: 288–293.
or radical species are developed by the ZnO-modified 10. Sawai J, Saito I, Kanou F, et al. Mutagenicity test of
hybrid polymer and may contribute to the antibacterial ceramic powder which have growth inhibitorory effect
activity. SEM analyses show that direct interaction on bacteria. Chem Engr Japan 1995; 28: 352–354.
between ZnO particles and the membrane surface of 11. Sawai J, Igarashi H, Hashimoto A, et al. Effect of particle
bacteria may lead to extensive coverage of the bacteria size and heating temperature of ceramic powders on
Farouk et al. 51

antibacterial activity of their slurries. Chem Engr Japan 19. Zhang L, Jiang Y, Ding Y, et al. Antibacterial behaviour
1996; 29: 251–256. of suspensions of ZnO nanoparticles (Nanofluids).
12. Sawai J, Kawada E, Kanou F, et al. Detection of Nanopart Res 2007; 9: 479–489.
active oxygen generated from ceramic powders having 20. Zhang L, Jiang Y, Ding Y, et al. Mechanistic investiga-
antibacterial activity. Chem Engr Japan 1996; 29: tion into antibacterial behavior of suspensions of ZnO
627–633. nanoparticles against E. coli. Nanopart Res 2010; 12:
13. Yamamoto O, Hotta M, Sawai J, et al. Influence of 1625–1636.
powder characteristic of ZnO on antibacterial activ- 21. Farouk A, Textor T, Schollmeyer E, et al. Sol-gel derived
ity—effect of specific surface area. Ceram Soc Japan inorganic–organic hybrid polymers filled with ZnO nano-
1998; 106: 1007–1011. particles as ultraviolet protection finish for textiles.
14. Adams LK, Lyon DY and Alvarez J. Comparative eco- AUTEX Res 2009; 9: 114–120.
toxicity of nanoscale TiO2, SiO2, and ZnO water suspen- 22. Spanhel L and Anderson MA. Semiconductor clusters in
sions. Water Res 2006; 40: 3527–3532. the sol-gel process: quantized aggregation, gelation,
15. Brayner R, Ferrarilliou R, Brivois N, et al. Toxicological and crystal growth in concentrated zinc oxide colloids.
impact studies based on Escherichia coli bacteria in ultra- Am Chem Soc 1991; 113: 2826.
fine ZnO nanoparticles colloidal medium. Nano Lett 23. Wan-Chun C, Ming-Hwa C and Tse-Min L. 2,3,5-
2006; 6: 866–870. Triphenyltetrazolium reduction in the viability assay of
16. Jeng HA and Swanson J. Toxicity of metal oxide nano- Ulva fasciata (Chlorophyta) in response to salinity stress.
particles in mammalian cells. Environ Sci Health 2006; 41: Bot Bull Acad Sin 1999; 40: 207–212.
2699–2711. 24. Hua Wang, Wingett D, Engelhard MH, et al. Fluorescent
17. Makhluf S, Dror R, Nitzan Y, et al. Microwave-assisted dye encapsulated ZnO particles with cell-specific toxicity
synthesis of nanocrystalline MgO and its use as a bacter- for potential use in biomedical applications. Mater Sci:
iocide. Adv Funct Mater 2005; 15: 1708–1715. Mater Med 2009; 20: 11–22.
18. Stoimenov PK, Klinger RL, Marchin GL, et al. Metal 25. Parks GA. The isoelectric points of solid oxides, solid
oxide nanoparticles as bactericidal agents. Langmuir hydroxides, and aqueous hydroxo complex system.
2002; 18: 6679–6686. Chem Rev 1965; 65: 177–198.