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-

Extensive efforts have been made to control
emerging disease infections in the past several dec-
ades [1]. Nevertheless, the spreading of antibiotic
resistant pathogens is still a growing concern glob-
ally [2]. The ability of microorganisms to survive
*
Corresponding author. Tel.: +852 2766 6415/9500 9607; fax:
+852 2773 1432.
E-mail address: tcdaoud@polyu.edu.hk (W.A. Daoud).
mission a critical issue, and studies have shown
that some infectious bacteria can survive on the
surface of various polymeric and textile materials
for more than 90 days [3]. Antimicrobial surfaces
not only provide protection against infectious dis-
eases but also against odor, staining, deterioration,
and allergies [4]. As a preventive measure, anti-
microbial surfaces should be necessary features
of daily used materials, especially in some high-
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,
new strategies are required for conferring common
materials, such as glass, plastics and textiles with
bactericidal properties. Due to the poor heat resis-
tance of plastics, wood and textiles, low tempera-
ture strategies are essential. The application of
several antibacterial surfaces to polymeric and tex-
tile materials has been discussed [5–10].
In recent years, great interests in the bacterici-
dal activity of TiO2 photocatalyst have been grow-
ing [11,12]. Several techniques such as sol–gel [13],
spray pyrolysis [14], chemical vapor deposition [15]
can be used to prepare titanium dioxide thin films.
Among these preparation techniques, the relatively
simple sol–gel method is the most widely used.
However, the disadvantage of all these techniques
is that a high temperature process is required to
produce highly photocatalytic thin films. The for-
mation of photocatalytic titania films at low tem-
peratures is required for any practical application
of titania in thermosensitive materials. In addition,
the destructive effect of strong acids, used in the
sol–gel process to keep aqueous sols in the pep-
tized state, on such substrates at elevated tempera-
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
application [11,12]. Several attempts have been
made to sensitize titanium dioxide for the much
longer visible range [16–18]. In presence of light,
TiO2 is excited with energy equal to (or higher
than) its band gap (3.2 eV) and electrons transfer
from the valence band (O 2p) to the conduction
band (Ti 3d) occurs. As a result, electron/hole
pairs are formed in the conduction/valence band
region. In the presence of oxygen and/or H2O
superoxide (O2) and/or hydroxyl (OH) radi-
cals are formed. These radicals attack adsorbed
organic species on the surface of TiO2 and decom-
pose them. Although, the radical mechanism of the
bactericidal activity of TiO2 in presence of UV
light has been extensively studied [19,20], dark
catalysis of titanium dioxide is reported to result
in dehydrogenation and dehydration of organic
substances at elevated temperatures [21]. The
destruction of some oral microorganisms in pres-
ence of TiO2 powder was also discussed and found
to be equally achieved in light and dark [22].
Recently, we have succeeded in forming nano-
structured photocatalytic titania from an alcohol-
based sol at low temperatures [23,24]. However,
for practical applications, the use of water as a
medium may offer several economical and ecolo-
gical advantages over alcohols.
In this contribution, we describe a novel
method to form an antibacterial titania surface
on organic cellulose fibers from an aqueous TiO2
sol at low temperatures. The antibacterial activity
was performed under various conditions, and a
high level of bactericidal effect of titania coated
cellulose substrates was observed. Although it is
generally accepted that TiO2 is bactericidally
inactive in absence of light, we found that the cellu-
lose-TiO2 composite possesses a bactericidal prop-
erty in presence and absence of light with different
rates of cell destruction. Titania coating on a low
heat resistance material such as cellulose is a poten-
tial process in functionalizing thermosensitive com-
mon materials with self-cleaning and antibacterial
properties for the decomposition of organic dirt,
environmental pollutants and harmful micro-
organisms.
2. Experimental
2.1. Chemicals
All chemicals were used as received without fur-
ther purification. Titanium isopropoxide (97%)
was purchased from Aldrich. Nitric acid (68%)
was supplied by Riedel de-Häen. Water was deion-
ized and distilled in glass apparatus. Staphylococ-
cus aureus was grown in our laboratory.
2.2. Sol preparation and surface coating
The aqueous sol was prepared at room temper-
ature by mixing titanium tetraisopropoxide (10 g)
with acidic water (200 ml) containing nitric acid
(2 ml). The mixture was vigorously stirred for
18 h prior to coating. The substrates were cleaned
by a nonionic detergent to remove surface impuri-
ties before coating. The cleaning process was per-
formed at 80 C for 30 min. The cleaned cellulose
fibers were dried before being dipped in the nano-
 W.A. Daoud et al. / Surface Science 599 (2005) 69–75
sol for 1 min and then pressed using an automatic
press at a nip pressure of 2.75 kg/cm2. The pressed
substrates were air dried for 24 h, rinsed with
sodium carbonate solution (1%) and water, dried
at 80 C for 10 min in a preheated oven to drive
off ethanol and finally annealed at 100 C in a pre-
heated oven for 30 min. This was followed by a
hydrothermal treatment at 97 C to induce anatase
crystallization and densification as previously re-
ported [25,26].
2.3. Washing
Washing was performed using a laundering
machine (Atlas Launder-Ometer, Atlas electric
devices company, Chicago, USA) at 49 C in a
1.2 L stainless steel lever lock canister. The sub-
strates were leached in 200 ml of 0.15% aqueous
solution of sodium lauryl sulfate (SLS) and in
presence of 50 steel balls for 45 min. The sub-
strates were then rinsed intensively with water
and dried at room temperature prior to further
investigations.
2.4. Surface characterization
SEM images were obtained with a JEOL JSM-
6335F field emission scanning electron microscopy
(FESEM) operated at 3 kV of accelerating voltage
and equipped with energy dispersive X-ray facility.
The UV transmission of the fibers was compared
before and after washing to determine the level
of interfacial adhesion between the fibers and
TiO2 coating using a Varian Cary 300 UV
spectrophotometer.
2.5. Assessment of dynamic antibacterial activities
The dynamic antibacterial activity assessment
was carried out according to a modified procedure
of the shake flask method (ASTM E2149-01).
S. aureus, a gram-positive type of bacteria, was
cultivated in a nutrient broth (containing 10 g/l
beef extract, 10 g/l peptone, 5 g/l NaCl) by shaking
for 18 h at 37 C. All glassware and test samples
were sterilized in an autoclave at 120 C for
20 min or with UV irradiation before experiments.
The formed inoculum was further diluted with
71
nutrient broth and an appropriate volume was
extracted into sterile phosphate buffer solution
(PBS) (containing 0.9 ml of 0.5 M KH2PO4
in 900 ml ddH2O) in order to obtain a final con-
centration of 1.5–3 · 105 cell forming colonies
(CFU) per ml. Two pieces of each test specimen
(pristine cellulose fibers and fibers coated with
TiO2) with a fixed surface area of 42 cm2 (cut into
small pieces of about 0.5 cm2) were transferred
into 250 ml capped Erlenmeyer flasks. The flasks
were autoclaved before the addition of 50 ml of
sterile PBS. The flasks were capped, placed in a
wrist-action shaker (Sheldon Manufacturing Inc
SI4-2) and shaken at 250 rpm at 37 C. At the pre-
determined time, 1 ml of solution from each flask
was transferred to a test tube containing 4 ml of
ddH2O and plated out in agar plates (containing
10 g/l beef extract, 10 g/l peptone, 5 g/l NaCl and
15 g/l agar powder) in duplicate. A drop of
0.1 ml of the diluted sample was then spread onto
solid growth agar plates. After incubation of the
plates at 37 C for 24 h, the number of viable cells
(colonies) was counted manually and the results
after multiplication with the dilution factor were
expressed as mean colony forming units (CFU)
per ml after averaging the duplicate counts. The
UV irradiation was performed using a UV lamp
(Philips TL05 8 W) with a maximum intensity
wavelength (km) at 365 nm.
2.6. Assessment of static antibacterial activities
Four swatches of each test specimens (pristine
cellulose fibers, and fibers coated with TiO2) with
a diameter of 5 cm were stacked up and in separate
150 ml wide-mouth jars. 1 ml of inoculum was
added to each jar. The tops were immediately
screwed tightly to prevent evaporation. For, ‘‘0’’
contact time sample, 100 ml of sterile phosphate
buffer were added immediately after inoculation
and the jars were shaken for one minute. 1 ml of
the resulting solution was transferred from each
jar to a test tube containing 4 ml of ddH2O and pla-
ted out in duplicate as explained before. For ‘‘5 h’’
contact sample, the jars were incubated for 5 h in a
37 C incubator before the plate count technique
was performed. All the Petri dishes were incubated
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
the functionalized cellulose fibers was investigated
by a comparison of the number of viable cells in
the suspension in contact with different substrates
as a function of contact time.
From Fig. 2, it can be seen that with pristine
cellulose irradiated with a UV intensity of
32 lW/cm2, the number of viable cell count
increased in the suspension by over 40% after
5 h. This behavior of substantial cell growth on
pristine cellulose was consistent regardless of the
conditions of the experiment, i.e., in presence or
absence of light. This may be attributed to the fact
that cellulosic materials are in fact good media for
bacterial growth [3].
For cellulose fibers coated with TiO2 in absence
of light, the cell count increased by 4% after 1 h,
i.e., the growth was significantly constrained com-
pared to a 39% increase after 1 h with pristine
fibers. However, unlike pristine cellulose, the cell
count started dropping from the second hour of
contact onwards until a 79% reduction was
observed after 5 h. For coated fibers and in pres-
ence of white light, the cell count decreased by
19% after 1 h and continued to drop reaching a
reduction level of 93% after 5 h. Likewise, with
coated fibers irradiated with UV intensity of
18
16
Viable Cell Count (kCFU)
14
12
10
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-
2
0 explained before. Results from this assessment, as
0 1 2 3 4 5
Contact Time (h)
Fig. 2. Viable Staphylococcus aureus cell count of as a function
of time while in contact with different substrates: Pristine
cellulosic fibers (h), coated fibers under dark conditions (m), no
substrates under UV radiation (r), coated fibers under white
light radiation (d), and coated fibers under UV radiation (j).
The cell count was determined by the surface-spread method.
73
32 lW/cm2, the cell count decreased after 1 h,
but with much faster rate of cell destruction so that
a 100% reduction was achieved after 3 h only of
contact time. It is very convenient to account the
antibacterial activity in presence of UV and white
light that also contains a very small fraction of UV
(0.47 lW/cm2) to a photocatalytic destruction of
the bacterial cells [27]. Thus, the difference in cell
destruction rate is due to the difference in the
amount of given energy intensity. However, the
unexpected bactericidal property of the coated
substrates under dark conditions was difficult to
account for and forced us to repeat the test. The
antibacterial assessment was indeed repeated four
times and the results were close with an error mar-
gin of ±1%. The average of which is quoted in this
manuscript.
In a control study carried out under illumina-
tion with the same UV source, in which neither
pristine nor coated substrates were present, a total
reduction of 81% was observed after 5 h. The
reduction trend closely resembled that of coated
substrates under dark conditions. This resem-
blance can be explained by the fact that UV radi-
ation has no direct bactericidal effect on the
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
were as vulnerable as in the sample with no sub-
strates. Biofilms provide a protective structure
for bacterial growth rendering them less suscepti-
ble than their free counterparts [28–30]. This, in
turn, would suggest that the 40% increase in
CFU under UV radiation after 5 h for pristine
tation the fibers were made to absorb the inoculum
and kept still in dark for 5 h of incubation time as
shown in Fig. 3, revealed a 69% reduction, which
is slightly less that that obtained from the dynamic
method after identical contact time. This may be
due to the fact that shaking enhances the contact
and hence enhances the reduction rate. Thus, these
results confirm our observations from the dynamic
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
properties were found less efficient in dark condi-
tions than in presence of UV radiation. The dark
antibacterial behavior of the formed composite
has been attributed to the non-sustenance nature
of TiO2, in contrast to the hospitable nature of cel-
lulose for bacterial growth. These results suggest
that a titania coating may not only perform as a
photocatalytic bactericidal agent but also as a pro-
tective shield against the formation of biofilms in
dark and light conditions. Thus, titania coating
on a low heat resistance material such as cellulose
may open up new applications of TiO2 as a bacte-
ricidal surface for a wide variety of materials espe-
cially for those with low heat resistance such as
textiles, wood, plastics, papers and biomaterials.
Acknowledgments
This work was supported by the Hong Kong
Polytechnic University and the government of
Hong Kong Special Administrative Region (Grant
No. K14. 56.ZPOD).
Appendix A. Supplementary data
Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.susc.2005.09.038.
References
[1] J. Lederberg, R.E. Shope, S.S. Oaks, Emerging Infections:
Microbial Threats to Health in the United States, National
Academic, Washington, DC, 1992.
[2] J. Lederberg, Science 288 (2000) 287.
[3] A.N. Neely, M.P. Maley, J. Clin. Microbiol. 38 (2000) 724.
[4] Y. Shin, D.I. Yoo, J. Jang, J. Appl. Polym. Sci. 80 (2001)
2495.
[5] Y. Shin, D.I. Yoo, K. Min, J. Appl. Polym. Sci. 74 (1999)
2911.
75
[6] J. Bozja, J. Sherrill, S. Michielsen, I. Stojiljkovic, J. Polym.
Sci. A 41 (2003) 2297.
[7] J. Lin, S. Qiu, K. Lewis, A.M. Klibanov, Biotechnol.
Bioeng. 83 (2003) 168.
[8] M. Tsukada, H. Katoh, D. Wilson, B. Shin, T. Arai, R.
Murakami, G. Freddi, Appl. Polym. Sci. 86 (2002) 1181.
[9] U. Klueh, V. Wagner, S. Kelly, A. Johnson, J.D. Bryers, J.
Biomed. Mater. Res. 53 (2000) 621.
[10] L. Qian, G. Sun, J. Appl. Polym. Sci. 89 (2003) 2418.
[11] P.A. Christensen, T.P. Curtis, T.A. Egerton, S.A.M. Kosa,
J.R. Tinlin, Appl. Catal. B 41 (2003) 371.
[12] K. Sunada, Y. Kikuchi, K. Hashimoto, A. Fujishima,
Environ. Sci. Technol. 32 (1998) 726.
[13] B. OÕRegan, J. Moser, M. Anderson, M. Grätzel, J. Phys.
Chem. 94 (1990) 8720.
[14] C.R. Bickmore, K.F. Waldner, R. Baranwal, T. Hinklin,
D.R. Treadwell, R.M. Laine, J. Eur. Ceram. Soc. 18 (1998)
287.
[15] A. Sandell, M.P. Andersson, M.K.J. Johansson, P.G.
Karlsson, Y. Alfredsson, J. Schnadt, H. Siegbahn, P.
Uvdal, Surf. Sci. 530 (2003) 63.
[16] H. Kisch, L. Zang, C. Lange, W.F. Maier, C. Antonius, D.
Meissner, Angew. Chem. Int. Ed. 37 (1998) 303.
[17] G. Burgeth, H. Kisch, Coord. Chem. Rev. 230 (2002) 41.
[18] S. Sakthivel, H. Kisch, Angew. Chem. Int. Ed. 42 (2003)
4908.
[19] P.C. Maness, S. Smolinski, D.M. Blake, Z. Huang, E.J.
Wolfrum, W.A. Jacoby, Appl. Environ. Microbiol. 65
(1999) 4094.
[20] Z. Huang, P.C. Maness, D.M. Blake, E.J. Wolfrum, S.
Smolinski, W.A. Jacoby, J. Photochem. Photobiol. A 130
(2000) 163.
[21] T. Reztsova, C.H. Chang, J. Koresh, H. Idriss, J. Catal.
185 (1999) 223.
[22] S. Nagame, T. Oku, M. Kambara, K. Konishi, J. Dent.
Res. 68 (1999) 1696.
[23] W.A. Daoud, J.H. Xin, J. Sol–Gel Sci. Technol. 29 (2004)
25.
[24] W.A. Daoud, J.H. Xin, Y.H. Zhang, K.H. Qi, J. Non-
Cryst. Solids 351 (2005) 1486.
[25] W.A. Daoud, J.H. Xin, J. Am. Ceram. Soc. 87 (2004) 953.
[26] W.A. Daoud, J.H. Xin, G.K.H. Pang, J. Am. Ceram. Soc.
88 (2005) 443.
[27] T. Saito, T. Iwase, J. Horis, T. Morioka, J. Photochem.
Photobiol. B 14 (1992) 369.
[28] T. Saito, T. Iwase, J. Horis, T. Morioka, Science 284 (1999)
1318.
[29] M.R. Parsek, P.K. Singh, Annu. Rev. Microbiol. 57 (2003)
677.
[30] M.R. Brown, J. Barker, Trends Microbiol. 7 (1999) 46.