Surface Science 599 (2005) 69–75

www.elsevier.com/locate/susc

Surface functionalization of cellulose fibers with

titanium dioxide nanoparticles and their combined
bactericidal activities
Walid A. Daoud *, John H. Xin, Yi-He Zhang
Nanotechnology Centre, ITC, The Hong Kong Polytechnic University, Hung Hom, Hong Kong

Received 3 June 2005; accepted for publication 23 September 2005

Available online 8 November 2005

Abstract

A well-adherent surface of titanium oxide nanoparticles was produced on cellulose fibers at low temperature from an
aqueous titania sol that was obtained via hydrolysis and condensation reactions of titanium isopropoxide in water.
SEM investigations of the formed titania films revealed a semi-spherical particle morphology with grain size about
10 nm in diameter. The coated substrates showed substantial bactericidal properties under UV radiation, ambient fluo-
rescent white light and dark conditions. The possible mechanisms for the antibacterial activity are discussed. The sta-
bility of the titania coatings was investigated by comparing the UV transmission profiles of coated fibers before and
after repeated washing.

2005 Elsevier B.V. All rights reserved.

Keywords: Titanium oxide; Nanostructures; Photochemistry; Scanning electron microscopy

1. Introduction on environmental surfaces makes infection trans-

mission a critical issue, and studies have shown
Extensive efforts have been made to control that some infectious bacteria can survive on the
emerging disease infections in the past several dec- surface of various polymeric and textile materials
ades [1]. Nevertheless, the spreading of antibiotic for more than 90 days [3]. Antimicrobial surfaces
resistant pathogens is still a growing concern glob- not only provide protection against infectious dis-
ally [2]. The ability of microorganisms to survive eases but also against odor, staining, deterioration,
and allergies [4]. As a preventive measure, anti-
*
Corresponding author. Tel.: +852 2766 6415/9500 9607; fax:
microbial surfaces should be necessary features
+852 2773 1432. of daily used materials, especially in some high-
E-mail address: [email protected] (W.A. Daoud). risk environments, such as medical and related

0039-6028/$ - see front matter
2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.susc.2005.09.038
70 W.A. Daoud et al. / Surface Science 599 (2005) 69–75

health-care and hygienic applications. Therefore, Recently, we have succeeded in forming nano-
new strategies are required for conferring common structured photocatalytic titania from an alcohol-
materials, such as glass, plastics and textiles with based sol at low temperatures [23,24]. However,
bactericidal properties. Due to the poor heat resis- for practical applications, the use of water as a
tance of plastics, wood and textiles, low tempera- medium may offer several economical and ecolo-
ture strategies are essential. The application of gical advantages over alcohols.
several antibacterial surfaces to polymeric and tex- In this contribution, we describe a novel
tile materials has been discussed [5–10]. method to form an antibacterial titania surface
In recent years, great interests in the bacterici- on organic cellulose fibers from an aqueous TiO2
dal activity of TiO2 photocatalyst have been grow- sol at low temperatures. The antibacterial activity
ing [11,12]. Several techniques such as sol–gel [13], was performed under various conditions, and a
spray pyrolysis [14], chemical vapor deposition [15] high level of bactericidal effect of titania coated
can be used to prepare titanium dioxide thin films. cellulose substrates was observed. Although it is
Among these preparation techniques, the relatively generally accepted that TiO2 is bactericidally
simple sol–gel method is the most widely used. inactive in absence of light, we found that the cellu-
However, the disadvantage of all these techniques lose-TiO2 composite possesses a bactericidal prop-
is that a high temperature process is required to erty in presence and absence of light with different
produce highly photocatalytic thin films. The for- rates of cell destruction. Titania coating on a low
mation of photocatalytic titania films at low tem- heat resistance material such as cellulose is a poten-
peratures is required for any practical application tial process in functionalizing thermosensitive com-
of titania in thermosensitive materials. In addition, mon materials with self-cleaning and antibacterial
the destructive effect of strong acids, used in the properties for the decomposition of organic dirt,
sol–gel process to keep aqueous sols in the pep- environmental pollutants and harmful micro-
tized state, on such substrates at elevated tempera- organisms.
ture hinders such applications. A third obstacle is
the need for UV radiation as titania is reported
to perform as a photocatalyst in their antibacterial 2. Experimental
application [11,12]. Several attempts have been
made to sensitize titanium dioxide for the much 2.1. Chemicals
longer visible range [16–18]. In presence of light,
TiO2 is excited with energy equal to (or higher All chemicals were used as received without fur-
than) its band gap (3.2 eV) and electrons transfer ther purification. Titanium isopropoxide (97%)
from the valence band (O 2p) to the conduction was purchased from Aldrich. Nitric acid (68%)
band (Ti 3d) occurs. As a result, electron/hole was supplied by Riedel de-Häen. Water was deion-
pairs are formed in the conduction/valence band ized and distilled in glass apparatus. Staphylococ-
region. In the presence of oxygen and/or H2O cus aureus was grown in our laboratory.
superoxide (O2) and/or hydroxyl (OH) radi-
cals are formed. These radicals attack adsorbed 2.2. Sol preparation and surface coating
organic species on the surface of TiO2 and decom-
pose them. Although, the radical mechanism of the The aqueous sol was prepared at room temper-
bactericidal activity of TiO2 in presence of UV ature by mixing titanium tetraisopropoxide (10 g)
light has been extensively studied [19,20], dark with acidic water (200 ml) containing nitric acid
catalysis of titanium dioxide is reported to result (2 ml). The mixture was vigorously stirred for
in dehydrogenation and dehydration of organic 18 h prior to coating. The substrates were cleaned
substances at elevated temperatures [21]. The by a nonionic detergent to remove surface impuri-
destruction of some oral microorganisms in pres- ties before coating. The cleaning process was per-
ence of TiO2 powder was also discussed and found formed at 80 C for 30 min. The cleaned cellulose
to be equally achieved in light and dark [22]. fibers were dried before being dipped in the nano-
 W.A. Daoud et al. / Surface Science 599 (2005) 69–75 71

sol for 1 min and then pressed using an automatic nutrient broth and an appropriate volume was
press at a nip pressure of 2.75 kg/cm2. The pressed extracted into sterile phosphate buffer solution
substrates were air dried for 24 h, rinsed with (PBS) (containing 0.9 ml of 0.5 M KH2PO4
sodium carbonate solution (1%) and water, dried in 900 ml ddH2O) in order to obtain a final con-
at 80 C for 10 min in a preheated oven to drive centration of 1.5–3 · 105 cell forming colonies
off ethanol and finally annealed at 100 C in a pre- (CFU) per ml. Two pieces of each test specimen
heated oven for 30 min. This was followed by a (pristine cellulose fibers and fibers coated with
hydrothermal treatment at 97 C to induce anatase TiO2) with a fixed surface area of 42 cm2 (cut into
crystallization and densification as previously re- small pieces of about 0.5 cm2) were transferred
ported [25,26]. into 250 ml capped Erlenmeyer flasks. The flasks
were autoclaved before the addition of 50 ml of
2.3. Washing sterile PBS. The flasks were capped, placed in a
wrist-action shaker (Sheldon Manufacturing Inc
Washing was performed using a laundering SI4-2) and shaken at 250 rpm at 37 C. At the pre-
machine (Atlas Launder-Ometer, Atlas electric determined time, 1 ml of solution from each flask
devices company, Chicago, USA) at 49 C in a was transferred to a test tube containing 4 ml of
1.2 L stainless steel lever lock canister. The sub- ddH2O and plated out in agar plates (containing
strates were leached in 200 ml of 0.15% aqueous 10 g/l beef extract, 10 g/l peptone, 5 g/l NaCl and
solution of sodium lauryl sulfate (SLS) and in 15 g/l agar powder) in duplicate. A drop of
presence of 50 steel balls for 45 min. The sub- 0.1 ml of the diluted sample was then spread onto
strates were then rinsed intensively with water solid growth agar plates. After incubation of the
and dried at room temperature prior to further plates at 37 C for 24 h, the number of viable cells
investigations. (colonies) was counted manually and the results
after multiplication with the dilution factor were
2.4. Surface characterization expressed as mean colony forming units (CFU)
per ml after averaging the duplicate counts. The
SEM images were obtained with a JEOL JSM- UV irradiation was performed using a UV lamp
6335F field emission scanning electron microscopy (Philips TL05 8 W) with a maximum intensity
(FESEM) operated at 3 kV of accelerating voltage wavelength (km) at 365 nm.
and equipped with energy dispersive X-ray facility.
The UV transmission of the fibers was compared 2.6. Assessment of static antibacterial activities
before and after washing to determine the level
of interfacial adhesion between the fibers and Four swatches of each test specimens (pristine
TiO2 coating using a Varian Cary 300 UV cellulose fibers, and fibers coated with TiO2) with
spectrophotometer. a diameter of 5 cm were stacked up and in separate
150 ml wide-mouth jars. 1 ml of inoculum was
2.5. Assessment of dynamic antibacterial activities added to each jar. The tops were immediately
screwed tightly to prevent evaporation. For, ‘‘0’’
The dynamic antibacterial activity assessment contact time sample, 100 ml of sterile phosphate
was carried out according to a modified procedure buffer were added immediately after inoculation
of the shake flask method (ASTM E2149-01). and the jars were shaken for one minute. 1 ml of
S. aureus, a gram-positive type of bacteria, was the resulting solution was transferred from each
cultivated in a nutrient broth (containing 10 g/l jar to a test tube containing 4 ml of ddH2O and pla-
beef extract, 10 g/l peptone, 5 g/l NaCl) by shaking ted out in duplicate as explained before. For ‘‘5 h’’
for 18 h at 37 C. All glassware and test samples contact sample, the jars were incubated for 5 h in a
were sterilized in an autoclave at 120 C for 37 C incubator before the plate count technique
20 min or with UV irradiation before experiments. was performed. All the Petri dishes were incubated
The formed inoculum was further diluted with for 24 h in a 37 C incubator. After incubation, the
72 W.A. Daoud et al. / Surface Science 599 (2005) 69–75

number of viable cells (colonies) was counted man-

ually and the results after multiplication with the
dilution factor were expressed as mean colony
forming units (CFU) per ml after averaging the
duplicate counts.

3. Results and discussion

3.1. Formation of titania coating

It was noticed that even though a low concen-

tration of 0.15 mol/L of nitric acid was used in
the preparation of the aqueous sol, coated sub-
strates lost their mechanical strength upon anneal-
ing at 100 C. To overcome this problem, the
titania coatings were first air-died for 24 h and
then neutralized by immersion in a 1 weight%
sodium carbonate solution prior to annealing.
Substrates so produced retained their mechanical
properties as evidenced by a comparison study of
their tensile strength before and after coating. It
was found that while annealed substrates without
a preceding neutralization step were easily torn,
neutralized substrates had a tensile strength of Fig. 1. High resolution field emission SEM images of (a)
643.5 kg m/s2, compared to 666.2 kg m/s2 for pris- pristine cellulose fiber and (b) titania surface coated on a
tine cellulose. This drop in strength might be due cellulose fiber.
to the stiffness of the inorganic coating layer or
perhaps due to the drop of the degree of polymer-
ization that may have been caused by nitric acid nia particles may have been removed as sodium
during the dipping step. Nevertheless, this 3% of titanate. Nevertheless, Fig. 1b shows an even coat-
tensile loss is relatively insignificant compared to ing of titania grains. This suggests that these parti-
that of substrates annealed without a preceding cles have formed strong bonding with cellulose
neutralization. and hence were not removed during neutralization
with sodium carbonate solution and that only
3.2. Microstructural observations loosely deposited particles (in excess) may have
been leached out. The chemical composition of
Fig. 1a shows a high magnification FESEM the film coating was studied by EDX (see Supple-
micrograph of a pristine cellulose fiber. The obser- mentary data). For coated cellulose fibers, tita-
vation of the titania coating shows that the surface nium and oxygen were the only two detected
texture appears to have dense and low porosity elements in addition to gold due to the gold coat-
characteristics and consists of near spherical grains ing layer.
of about 10 nm in diameter as shown in Fig. 1b.
The particles appears to be uniform in size, how- 3.3. Antibacterial assessments
ever, Fig. 1b also shows the formation of surface
aggregates with grains greater than 10 nm in dia- The antibacterial activity has been quantita-
meter. It is anticipated that during washing with tively measured by means of two types of antibac-
sodium carbonate solution, it is likely that the tita- terial assessments. The first antibacterial effect of
 W.A. Daoud et al. / Surface Science 599 (2005) 69–75 73

the functionalized cellulose fibers was investigated 32 lW/cm2, the cell count decreased after 1 h,
by a comparison of the number of viable cells in but with much faster rate of cell destruction so that
the suspension in contact with different substrates a 100% reduction was achieved after 3 h only of
as a function of contact time. contact time. It is very convenient to account the
From Fig. 2, it can be seen that with pristine antibacterial activity in presence of UV and white
cellulose irradiated with a UV intensity of light that also contains a very small fraction of UV
32 lW/cm2, the number of viable cell count (0.47 lW/cm2) to a photocatalytic destruction of
increased in the suspension by over 40% after the bacterial cells [27]. Thus, the difference in cell
5 h. This behavior of substantial cell growth on destruction rate is due to the difference in the
pristine cellulose was consistent regardless of the amount of given energy intensity. However, the
conditions of the experiment, i.e., in presence or unexpected bactericidal property of the coated
absence of light. This may be attributed to the fact substrates under dark conditions was difficult to
that cellulosic materials are in fact good media for account for and forced us to repeat the test. The
bacterial growth [3]. antibacterial assessment was indeed repeated four
For cellulose fibers coated with TiO2 in absence times and the results were close with an error mar-
of light, the cell count increased by 4% after 1 h, gin of ±1%. The average of which is quoted in this
i.e., the growth was significantly constrained com- manuscript.
pared to a 39% increase after 1 h with pristine In a control study carried out under illumina-
fibers. However, unlike pristine cellulose, the cell tion with the same UV source, in which neither
count started dropping from the second hour of pristine nor coated substrates were present, a total
contact onwards until a 79% reduction was reduction of 81% was observed after 5 h. The
observed after 5 h. For coated fibers and in pres- reduction trend closely resembled that of coated
ence of white light, the cell count decreased by substrates under dark conditions. This resem-
19% after 1 h and continued to drop reaching a blance can be explained by the fact that UV radi-
reduction level of 93% after 5 h. Likewise, with ation has no direct bactericidal effect on the
coated fibers irradiated with UV intensity of viability of the cells, hence the cell reduction
occurred naturally as it did in absence of a protec-
tive biofilm in the case of titania coated substrates
under dark conditions, where the bacterial cells
18
were as vulnerable as in the sample with no sub-
16 strates. Biofilms provide a protective structure
Viable Cell Count (kCFU)

14 for bacterial growth rendering them less suscepti-

12 ble than their free counterparts [28–30]. This, in
turn, would suggest that the 40% increase in
10
CFU under UV radiation after 5 h for pristine
8 substrates is attributable to the formation of bio-
6 films on the fibers surface.
4 In a second assessment that does not involve agi-
tation the fibers were made to absorb the inoculum
2
and kept still in dark for 5 h of incubation time as
0 explained before. Results from this assessment, as
0 1 2 3 4 5
shown in Fig. 3, revealed a 69% reduction, which
Contact Time (h)
is slightly less that that obtained from the dynamic
Fig. 2. Viable Staphylococcus aureus cell count of as a function method after identical contact time. This may be
of time while in contact with different substrates: Pristine due to the fact that shaking enhances the contact
cellulosic fibers (h), coated fibers under dark conditions (m), no
substrates under UV radiation (r), coated fibers under white
and hence enhances the reduction rate. Thus, these
light radiation (d), and coated fibers under UV radiation (j). results confirm our observations from the dynamic
The cell count was determined by the surface-spread method. assessment method.
74 W.A. Daoud et al. / Surface Science 599 (2005) 69–75

Fig. 3. Viable cells recovered after 5 h of static contact with pristine cellulose (left plate) and coated cellulose (right plate) under dark
conditions.

Our interpretation is that TiO2 may simply pro- 14

vide no sustenance for bacteria, whereas cellulose, (a)
12
being a hospitable medium, offers good pores for
their growth and maintains good respiration for 10
Transmission%

the host. In this context, the TiO2 surface may

have prevented the formation of a protective bio- 8
film of adsorbed bacteria rather than actively kill- 6
ing them via free radical formation. (c)
4
3.4. Stability of the titania coating 2
(b)
Although, it can be observed from Fig. 1a that 0
the titania films have withstood washing with 280 300 320 340 360 380
Wavelength (nm)
sodium carbonate solution and the hydrothermal
treatment suggesting a direct chemical bonding Fig. 4. UV transmission spectra of (a) pristine cellulose, (b)
with cellulose, we have decided to examine the sta- coated cellulose, and (c) coated cellulose after 20 washings.
bility of the titania coating by comparing the UV
transmission profile of coated substrates before
and after repeated washing. Fig. 4 shows the UV attributable to the formation of covalent bonding
transmission profiles before and after 20 washings. at their interface as a result of dehydration reac-
It can be seen from Fig. 4 that the transmission of tions between the cellulosic hydroxyl groups and
the coated substrates was almost cut off in the the hydroxyl groups of titania.
UVB region (280–315 nm) and up to 356 nm in
the UVA region due to the UV absorption of
TiO2. In the spectral region above 356 nm, the 4. Conclusions
transmission increased gradually. Although the
transmission after 20 washings was slightly higher We have successfully formed a well-adherent
in this region, it remained much lower than that of bactericidal surface on organic cellulose fibers by
pristine cellulose fibers. These results suggest a a simple deposition method at low temperatures.
high level of adhesion and confirm our conclusions It was found that TiO2-coated cellulose fibers
from the FESEM observations. This might be possess substantial antibacterial properties. These
 W.A. Daoud et al. / Surface Science 599 (2005) 69–75 75

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