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THE EFFECT OF ANATASE ON RUTILE TIO2
NANOFLOWERS TOWARDS ITS PHOTO-
CATALYTIC ACTIVITY ON CANCER CELLS
NOOR SAKINAH BINTI KHALID
UNIVERSITI TUN HUSSEIN ONN MALAYSIA
THE EFFECT OF ANATASE ON RUTILE TIO2 NANOFLOWERS TOWARDS
ITS PHOTO-CATALYTIC ACTIVITY ON CANCER CELLS
NOOR SAKINAH KHALID
A thesis submitted in
fulfillment of the requirement for the award of the
Degree of Master in Electrical Engineering
Faculty of Electrical and Electronic Engineering
Universiti Tun Hussein Onn Malaysia
2016
iii
This thesis was dedicated to my parents,
parents in-law, my husband; Mohd Ridzuan Abdullah
and my son Ahmad Haziq Mohd Ridzuan
iv
ACKNOWLEDGEMENT
Firstly, I would like to express my sincerest appreciation to my supervisor, Dr. Mohd
Khairul Ahmad, for his continuous support, encouragement and patience throughout
the entire process. To my co-supervisors, En Wan Suhaimizan Wan Zaki and Dr.
Soon Chin Fhong, I wish to express my deepest gratitude for their guidance
throughout my research.
Special thanks are given to Microeletronics & Nanotechnology - Shamsuddin
Research Centre (MiNT-SRC), Universiti Tun Hussein Onn Malaysia (UTHM) for
allowing me to use FESEM, XRD, sputter coater, UV-vis and providing me with
technical support whenever needed. I would also like to acknowledge all MiNT-SRC
technicians, my friends, Mrs. Faezahana, Ms. Isrihetty and final year students for
being there for me through the hard times and the sweet moments leading up to the
submission of this thesis.
Finally, I wish to express my deepest appreciation to Universiti Tun Hussein
Onn Malaysia for the opportunity to further my studies and for giving me a place to
learn and expand my knowledge.
.
v
ABSTRACT
Titanium dioxide (TiO2) rutile nanoflowers were fabricated using acidic
hydrothermal method. TiO2 nanoflowers were successfully grown on fluorine doped
tin oxide (FTO) substrates. The hydrothermal reaction time and amount of titanium
butoxide (TBOT) precursors were optimized to be 10 hours and 2 ml, respectively.
Fabrication of TiO2 using spray pyrolysis technique produced TiO2 nanoparticles.
The surface morphology of the TiO2 nanoflowers and nanoparticles were studied
using Field Emission Scanning Electron Microscope (FESEM) to detect the changes
in surface morphology as a result of varied parameters. The crystalline phases of
TiO2 samples were investigated using X-ray Diffractions (XRD). From the XRD
analysis, TiO2 nanoflowers produced using the hydrothermal method were of rutile
phase while TiO2 nanoparticles produced using the spray pyrolysis technique were of
anatase phase. Thus, when the two fabrication processes were mixed, anatase-rutile
phased TiO2 particles were produced. Photo-degradation of TiO2 nanoflowers were
analyzed using methyl orange dye. UV-Vis-NIR spectrophotometer was used to
observe the absorbance of methyl orange before and after UV light exposure to the
TiO2 samples. The degradation rates of methyl orange by rutile, anatase-rutile and
anatase were 4.35%, 88.8% and 98%, respectively. Thereon, cervical cancer cells
were exposed to the TiO2 samples to check the biocompatibility of the fabricated
TiO2. The cervical cancer cells showed biocompatibility towards the fabricated TiO2.
Lastly, photo-catalytic activity of the fabricated TiO2 was studied on cancer cells.
The fabricated TiO2 showed little effect to the cancer cells as no change in cell size
were observed after UV exposure due to the low electron excitation on TiO2 surface.
Thus, the photo-catalysis reaction did not occur.
vi
ABSTRAK
Titanium dioxide (TiO2) nanobunga rutil dihasilkan menggunakan kaedah
hidroterma berasid. TiO2 nanobunga telah berjaya dihasilkan di atas substrat florin
didopkan atas timah oksida (FTO). Masa reaksi hidroterma dan jumlah titanium
butoxide (TBOT) yang optimum adalah pada 10 jam dan 2 ml. Fabrikasi TiO2 yang
menggunakan proses deposit semburan telah menghasilkan TiO2 nanopartikel dan
dikaji menggunakan Pancaran Medan Mikroskop Imbasan Elektron (FESEM) untuk
mengesan perubahan struktur pada permukaan TiO2 nanobunga dan nanopartikel
dalam pelbagai parameter. Fasa hablur bagi sampel TiO2 telah diperiksa
menggunakan serakan sinar-X (XRD). Analisis daripada XRD menunjukkan TiO2
dari kaedah hidroterma menghasikan hablur rutil, manakala TiO2 dari teknik deposit
semburan menghasilkan hablur anatas. Maka, percampuran diantara dua proses
tersebut menghasilkan fasa partikel TiO2 hablur rutil-anatas. Sifat cahaya-
kemerosotan oleh TiO2 nanobunga dianalisis menggunakan pewarna oren. UV-Vis
NIR spektrofotometer telah digunakan untuk memerhatikan kadar serapan cahaya
sebelum dan selepas pendedahan sinar UV kepada sampel TiO2. Kadar serapan bagi
pewarna oren apabila menggunakan rutil, rutil-anatas dan anatas ialah masing-
masing 4.35%, 88.8% dan 98%. Kemudian, sel-sel kanser serviks telah dibiakkan di
atas sampel TiO2 untuk memeriksa bio-keserasian terhadap sampel TiO2 dan ia
menunjukkan bio-keserasian terhadap fabrikasi TiO2. Akhirnya, aktiviti cahaya-
kemerosotan telah dikaji terhadap sel kanser. Fabrikasi TiO2 menunjukkan kesan
yang kurang kepada sel kanser dimana tiada perubahan diperhatikan terhadap saiz sel
kanser selepas pancaran sinar UV disebabkan oleh kekurangan electron yang keluar
ke permukaan TiO2. Maka, tindak balas foto kemusnahan tidak berlaku.
vii
TABLE OF CONTENTS
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENT iv
ABSTRACT v
ABSTRAK vi
TABLE OF CONTENTS vii
LIST OF TABLES x
LIST OF FIGURES xi
LIST OF SYMBOLS AND ABBREVIATIONS xv
LIST OF APPENDICES xvi
LIST OF PUBLICATIONS xvii
LIST OF AWARDS xix
CHAPTER 1 INTRODUCTION 1
1.1 Background of study 1
1.2 Problem statements 2
1.3 Objectives of the study 3
1.4 Research scope 4
1.5 Research contribution 4
viii
CHAPTER 2 LITERATURE REVIEW 5
2.1 Titanium dioxide nanostructures 5
2.2 Different techniques for the preparation of
nanostructured TiO2 thin film 7
2.2.1 Sol-gel method 7
2.2.2 Spray pyrolysis deposition technique 8
2.2.3 Sputtering 8
2.2.4 Hydrothermal method 9
2.3 Photo-catalytic properties of TiO2 11
2.4 Biocompatibility of TiO2 13
2.5 Photo-toxicity of TiO2 13
2.6 Working principles of devices used for
characterization 14
2.6.1 Field emission scanning electron microscope
(FESEM) 14
2.6.2 X-ray diffraction (XRD) 14
2.6.3 UV-Vis NIR Spectrophotometer 15
2.6.4 Flourescence microscope 16
2.7 Cancer cells 17
2.8 Applications of titanium dioxide 19
CHAPTER 3 METHODOLOGY 20
3.1 Flow chart 20
3.2 Variation of experimental parameters 21
3.3 Fabrication of TiO2 nanoflowers by hydrothermal
method 22
3.3.1 Characterization of the surface morphology
and stuctural phase of TiO2 nanoflowers 23
3.4 Fabrication of TiO2 nanoparticles by spray pyrolysis
method on top of TiO2 nanoflowers 24
3.5 Photo-degradation of TiO2 using MO 25
3.6 HeLa cells culture 26
3.6.1 Counting cells 28
3.6.2 Cells seeding onto TiO2 nanoflowers 31
ix
3.6.3 Cells fixation and dehydration 31
3.4.4 Cells staining 31
3.7 UV exposure to HeLa cells on TiO2 nanoflowers 32
CHAPTER 4 RESULTS AND DISCUSSION 33
4.1 Characterization of TiO2 nanostructures 33
4.1.1 Surface morphology and structural analysis 33
4.1.1.1 Effect of hydrothermal reaction time
and amount of precursor 34
4.1.1.2 Effect of CTAB on TiO2
nanostructures 44
4.1.1.3 TiO2 nanoparticles sprayed on top
of TiO2 nanoflowers 47
4.1.2 Interaction of HeLa cells on TiO2
nanostructures 50
4.2 Photo-catalyst study of TiO2 nanostructures 52
4.3 Cells culture 55
4.3.1 Cells counting using heacytometer 56
4.3.2 Staining of HeLa cells on TiO2 nanostructures 58
4.4 UV exposure to HeLa cells on TiO2 nanostructures 59
CHAPTER 5 CONCLUSIONS AND FUTURE WORKS 62
5.1 Conclusions 62
5.2 Future works 64
REFERENCES 65
APPENDIX 73
VITAE 77
x
LIST OF TABLES
2.1 Bulk properties of TiO2 crystal phases 7
3.1 Variation of synthesis parameters 22
4.1 Measurements of TiO2 nanostructures grown at varied
hydrothermal reaction time and using varied amount of
precursor 43
4.2 Total number of cells 57
xi
LIST OF FIGURES
2.1 Reaction boundaries of phase transition in TiO2 [21] 6
2.2 Crystal structures of (a) anatase, (b) rutile and
(c) brookite [21] 6
2.3 Schematic diagram of spray deposition setup [42] 8
2.4 Diagram of plasma reactor [45] 9
2.5 Schematic diagram of TiO2 nanostructured using
hydrothermal method by Wu (left) and
Hong (right) [29,30] 10
2.6 Schematic diagram of TiO2 photo-catalyst 11
2.7 Schematic diagram of Bragg’s Law 15
2.8 The illustration of flourescence microscope 17
2.9 Cells proliferation from stem cells 18
3.1 Step by step process 21
3.2 (a) Hydrochloric acid, (b) deionized water,
(c) titanium butoxide used as precursor,
(d) FTO glass used as substrates and
(e) autoclave stainless steel lined with Teflon where
process hydrothermal process takes place 23
3.3 FESEM JOEL JSM-7600F and XRD PANalytical
XPert Powder used to characterized samples 24
3.4 (a) Methyl orange and (b) UV lamp for the photo-
degradation of MO in the presence of
TiO2 nanostructures 25
3.5 Experimental set up for photo-catalyst study where
inside black box contains methyl orange with TiO2
nanostructure 26
xii
3.6 HeLa cervical Adenocarcinoma Human (CCL-2)
purchased from ATCC 26
3.7 Incubator to keep the cells in ambient conditions 27
3.8 Safety cabinet used to subculture cells in sterilized
condition 27
3.9 (a) Trypsin EDTA used to detach cells from culture flask,
(b) DMEM supply nutrients to cells, (c) HBSS is
buffer solution to wash out the dead cells, (d) culture
flask used to culture the cells and (e) surrological
pipette to transfer any media 28
3.10 Phased contrast microscope Nikon Eclopse TS 100 28
3.11 (a) Centrifuge PLC series used to separate the cells at
the bottom of culture flask in orderto collect all cells
and (b) precipitation of cells after centrifugation process 29
3.12 The counter and hemacytometer kit used to count cells
and method for counting the cells on the grids 30
3.13 Flourescence microscope Olympus BX53 32
3.14 Schematic diagram on the method for UV exposure
done on HeLa cells grown on TiO2 nanoflowers 32
4.1 Top view of TiO2 structures from FESEM imaging
of 3 ml TBOT for (a) 2, (b) 5, (c) 10 and
(d) 24 hours, respectively 34
4.2 The diameter and cross-section of TiO2 grown for 3
days 35
4.3 Cross-sectional view of TiO2 structures from FESEM
imaging of 3 ml TBOT for (a) 2, (b) 5, (c) 10 and
(d) 24 hours respectively 36
4.4 XRD patterns for TiO2 sample prepared from 3 ml
TBOT for 2, 5, 10 and 24 hours, respectively 37
4.5 Top view of TiO2 structures from FESEM imaging
of sample prepared from 2 ml TBOT for (a) 2,
(b) 5, (c) 10 and (d) 24 hours, respectively 38
xiii
4.6 Cross-sectional view of TiO2 structures from FESEM
imaging of sample prepared from 2 ml TBOT for (a) 2,
(b) 5, (c) 10 and (d) 24 hours, respectively 39
4.7 XRD patterns for TiO2 sample prepared from 2 ml
TBOT for 2, 5, 10 and 24 hours, respectively 40
4.8 Top view of TiO2 structures from FESEM imaging of
sample prepared from 1 ml TBOT for (a) 2, (b) 5,
(c) 10 and (d) 24 hours, respectively 41
4.9 XRD patterns for TiO2 sample prepared from 1 ml
TBOT at 2, 5, 10 and 24 hours, reaction
time respectively 42
4.10 Schematic diagram of the proposed growth mechanism
of TiO2 nanostructures via hydrothermal method 43
4.11 Defined top view (a-1, 2, 3, 4) and defined
cross-sectional view (b-1, 2, 3, 4) of TiO2 structures
fabricated using CTAB for 24, 10, 5 and 2 hours,
respectively 45
4.12 XRD patterns of TiO2 nanostructures sample prepared
at 2, 5, 10 and 24 hours reaction time by using CTAB as
surfactant 46
4.13 Growth of TiO2 nanoparticles on top of TiO2
nanoflowers via spray pyrolysis technique 47
4.14 TiO2 nanoparticles fabrication via spray pyrolysis
technique 48
4.15 XRD pattern of mixture of TiO2 nanoflowers and
nanoparticles and TiO2 nanoparticles 49
4.16 HeLa cells grown on TiO2 nanostructures fabricated at
5, 10 and 24 hours hydrothermal reaction time after
fixation and dehydration process 51
4.17 Absorbance of MO after the exposure to rutile TiO2
nanostructures for 60 minutes 53
4.18 Absorbance of MO after the exposure to rutile-anatase
and anatase TiO2 nanostructures for 60 minutes 54
xiv
4.19 Percentage degradation of MO after the exposure to
different structural phases of TiO2 nanostructures 55
4.20 HeLa cells morphology at (a) 0, (b) 5, (c) 20 and
(d) 44 hours observed under phased-contrast microscope
with 10 x magnification on 25 cm3 culture flask 56
4.21 HeLa cells on hemacytometer grid under phased-contrast
microscope at 10x magnification 57
4.22 HeLa cells with DAPI staining on (a) without TiO2 and
(b) on TiO2 nanostructures under 20x magnification 58
4.23 HeLa cells were grown on rutile TiO2 nanoflowers after
(a)10, (b) 20, (c) 30 and (d) 60 minutes UV exposure 59
4.24 HeLa cells were grown on (a) rutile TiO2,
(b) rutile-anatase TiO2 and (c) anatase TiO2 after 60
minutes UV exposure 60
xv
LIST OF SYMBOLS AND ABBREVIATIONS
TiO2 - Titanium dioxide
TBOT - Titanium butoxide
HCl - Hydrochloric acid
CTAB - Cetyl trimethyl-ammonium bromide
FTO - Fluorine doped tin oxide
MO - Methyl orange
UV - Ultra violet
NCR - National Cancer Registry
FESEM - Field emission scanning electron microscope
XRD - Dispersion X-ray
DSSC - Dye sensitized solar cells
VB - Valence band
CB - Conduction band
DMEM - Dulbecco’s Modified Eagle’s Media
HBSS - Hanks’s Balance Salt Solution
FCS - Fetal calf serum
DAPI - 4’, 6-diamidino-2-phenylindo
xvi
LIST OF APPENDICES
APPENDIX TITLE PAGE
A Table A.1: Gantt Chart of Research Activities 73
B Figure B.1: Datasheet of HeLa cells and their protocols
for subculture and handling 76
xvii
LIST OF PUBLICATIONS
Journal / Proceedings:
(a) Noor Sakinah Khalid, Wan Suhaimizan Wan Zaki, Mohd Khairul Ahmad.
“Study of The Effect of Fluorine Doped Tin Oxide (FTO) Substrate on The
Growth of Titanium Dioxide Nanorods via Hydrothermal Method”,
Proceeding of Malaysia-Japan International Conference on Nanoscience,
Nanotechnology and Nanoengineering 2014 (NANO SciTech 2014 & IC-
NET 2014), UiTM Shah Alam, Selangor, 28 February-3 March 2014,
Advanced Materials Research Volume 1109, pp. 11-14, 2015, Trans Tech
Publications, Switzerland.
(b) Noor Sakinah Khalid, Wan Suhaimizan Wan Zaki, Mohd Khairul Ahmad.
“Growth of Rutile Phased Titanium Dioxide (TiO2) Nanoflowers for HeLa
Cells Treatment”, Proceeding of 5th International Conference on
Biomedical Engineering (BME5 2014), Vietnam, 16‑18 June 2014, IFMBE
Proceedings Volume 46, pp. 243-246, 2015. © Springer International
Publishing Switzerland.
(c) Noor Sakinah Khalid, Siti Harwani Ishak, Mohd Khairul Ahmad. “Effect of
Annealing Time of TiO2 Thin Film Deposited by Spray Pyrolysis
Deposition Method for Dye-Sensitized Solar Cells”, Proceeding of
International Integrated Engineering Summit (IIES 2014), UTHM, Batu
Pahat, Johor, 1-4 December 2014, Advanced Materials Research Volume
773-773, pp. 647-651, 2015, Trans Tech Publications, Switzerland.
(d) Noor Sakinah Khalid, Soo Ren How, Mohd Khairul Ahmad. “Effect of
Deposition Temperature on Surface Morphology and Electrical Properties
of Fluorine Doped Tin Oxide (FTO) Thin Film by Spray Pyrolysis
xviii
Technique”, Proceeding of International Integrated Engineering Summit
(IIES 2014), UTHM, Batu Pahat, Johor, 1-4 December 2014, Advanced
Materials Research Volume 773-773, pp. 652-656, 2015, Trans Tech
Publications, Switzerland.
(e) Noor Sakinah Khalid, Fatin Izyani Mohd Fazli, Noor Kamalia Abd Hamed,
Muhammad Luqman Mohd Napi, Chin Fhong Soon, Mohd Khairul Ahmad.
“Biocompatibility of TiO2 Nanorods and Nanoparticles on Hela Cells.”
Proceeding of Conference on Nano-& Biosource Technology 2015 (NBT
2015), UKM Bangi, Selangor, 28-29 February 2015 (pending publication in
Sains Malaysiana).
(f) Noor Kamalia Abd Hamed , Nafarizal Nayan, Mohd Khairul Ahmad, Noor
Sakinah Khalid, Fatin Izyani Mohd Fazli, , Muhammad Luqman Mohd
Napi. “Influence of Hydrochloric Acid Volume on Titanium Dioxide (TiO2)
Thin Film by Using Hydrothermal Method.” Proceeding of Conference on
Nano-& Biosource Technology 2015 (NBT 2015), UKM Bangi, Selangor,
28-29 February 2015 (pending publication in Sains Malaysiana).
(g) Fatin Izyani Mohd Fazli, Nafarizal Nayan, Mohd Khairul Ahmad, Noor
Sakinah Khalid, Noor Kamalia Abd Hamed, Muhammad Luqman Mohd
Napi. “Effect of Annealing Temperature on TiO2 Thin Film Prepared by
Spray Pyrolysis Deposition Method.” Proceeding of Conference on Nano-&
Biosource Technology 2015 (NBT 2015), UKM Bangi, Selangor, 28-29
February 2015 (pending publication Sains Malaysiana).
(h) Noor Sakinah Khalid, Indah Fitriani Hamid, Noor Kamalia Abd Hamed,
Fatin Izyani Mohd Fazli, Soon Chin Fhong, Mohd Khairul Ahmad.
“Application of TiO2 Nanostructure Using Hydrothermal Method For
Waste Water Treatment.” Proceeding of International Conference on
Electrical and Electronic Engineering 2015 (IC3E2015), Equatorial Hotel,
Melaka, 10-11 August 2015 (pending publication in ARPN Journal of
Engineering and Applied Sciences with Scopus indexed).
xix
LIST OF AWARDS
(i) Bronze Medal in Invention & Innovation Awards 2014, Malaysian
Technology Expo 2014
Mohd Khairul Ahmad, Nafarizal Nayan, Murakami Kenji, Noor Sakinah
Khalid. “Non-Hazardous Solar Cell: Nanostructured Dye-Sensitized Solar
Cells.”
(ii) Bronze Medal in Research and Innovation Festival 2013 (R&I Fest
UTHM)
Noor Sakinah Khalid, Wan Suhaimizan Wan Zaki, Mohd Khairul Ahmad.
“Simple Fabrication of Titanium Dioxide Nanoflowers at Low Temperature.”
(iii) Bronze Medal in Research and Innovation Festival 2014 (R&I Fest
UTHM)
Noor Sakinah Khalid, Soon Chin Fhong, Mohd Khairul Ahmad. “Treatment
on Cancer Cells by Using TiO2 Nanorods/nanoflowers.”
1CHAPTER 1
INTRODUCTION
1.1 Background of study
Photo-catalytic reaction involves oxidation and reduction of a chemical when
exposed to light. Numerous studies were conducted on photo-catalysis over the past
decades. In 1938, a first photo-catalysis study relating to the photo-bleaching of dyes
with ultraviolet (UV) excitation of TiO2 was reported by Goodeve and Kitchener [1].
Later in 1956, Mashio and Kato [2] published their work on the oxidation of different
organic solvent by UV radiation. While in 1972, Fujishima and Honda [3] reported
the success of photo-catalytic splitting of water into hydrogen and oxygen. A few
years later, in 1977, Frank and Bard [4] reported their development of a photo-
catalyst for environmental pollutant treatment.
The first finding on photo-catalytic reaction on living organism was reported
in 1985 by Matsunaga et al., where it was reported that photo-activated TiO2 is
effective at killing bacteria [5]. In 1995, Fujishima et al. discovered that TiO2 attains
superhydrophilic characteristic after UV irradiation [6]. Following the discovery, in
1997, a hydrophilic surface for self-cleaning application was developed using UV
irradiated TiO2 by Wang and his colleagues [7].
Nowadays, there is a high demand for metal oxide semiconductors as photo-
catalyst. Titanium dioxide is the most stable metal oxide semiconductor in
atmospheric conditions. Extensive research has been conducted on the photocatalytic
ability of anatase TiO2. TiO2 photocatalysis finds application in self-cleaning
products [8], dye-sensitized solar cells (DSSC) [3-4], anti-microbial products [5-6],
water treatment and anti-cancer drugs [7-9]. Photocatalytic ability of TiO2 is
influenced by its structure, shape and crystallite phase [10-12]. Anatase TiO2 is the
more popular TiO2 phase for photo-catalysis applications. However, the disadvantage
2
of this phase is that it has an inconvenient shape for removal after a photo-catalysis
reaction. In this study, rutile TiO2 was grown on an active substrate which is fluorine
doped tin oxide (FTO) substrate. Statically grown rutile TiO2 can easily be removed
after photo-catalytic activity. Moreover, anatase TiO2 is metastable and tends to
transform into rutile phase at higher temperatures. However, mixing the two phases
could have a synergistic effect on its photo-catalytic activity. Thus, the growth of
anatase TiO2 on rutile TiO2 nanostructures was studied and optimized to achieve the
objectives that will be explained later.
The cancer cells that were used in this study were HeLa cervical cancer cells.
HeLa cell line was derived from cervical cancer cells taken from Henrietta Lacks, an
African-American woman who died from cervical cancer in 1951. HeLa cervical
cancer cells were chosen as the subject for TiO2 photo-catalysis study due to the
urgency for cervical cancer treatment. This is in reference to a statistic by the
National Cancer Registry (NCR) which reported cervical cancer as the third most
common cancers in female in 2007 [19].
1.2 Problem statements
Several studies have been reported on the use of anatase TiO2 structures, namely
TiO2 nanotubes and TiO2 nanoparticles, for photo-catalytic killing of cancer cells [7-
8, 16]. Cancer cell membranes are composed of phospholipid bilayer, which is a thin
outer layer that separates the cell from its environment. During a photo-catalysis
process, a lot of free radicals hydroxyl groups (OH·) are produced. A free radical
hydroxyl group contains a single unpaired electron which makes it extremely
reactive with proteins, lipids and carbohydrates. Free radical hydroxyl groups
produced during the photo-catalysis process then attacks the membrane of cancer
cells, causing rupture and degradation of cell membrane properties.
The drawback of anatase nanotubes and nanoparticles in previous studies is
the difficulty in removing TiO2 particles from the photo-catalytic reaction system and
the need for an additional catalyst removal step. Furthermore, anatase TiO2 structure
is unstable and may transform to rutile TiO2 at higher temperatures. Rutile TiO2 have
a lower band gap energy and a higher electron mobility in comparison to anatase
such that more free radical hydroxyl groups are generated with rutile TiO2 as catalyst
3
than anatase TiO2 [21]. Higher concentration of free radical hydroxyl groups could
enhance photo-catalytic activity. Thus in this study, photo-catalyst comprising a
mixture of anatase and rutile TiO2 was proposed as it could possibly increase photo-
catalytic activity towards the cancer cells.
TiO2 nanoflowers have a larger surface area in comparison to TiO2
nanoparticles. This is because the petals of the nanoflowers have a larger surface area
to volume ratio in comparison to spherical TiO2 nanoparticles. The TiO2 structure
with a larger surface area will generate more free radical hydroxyl groups. Thus, the
growth of TiO2 in nanoflower structure is favorable as it would increase photo-
catalytic activity of TiO2 towards cancer cells.
1.3 Objectives of the study
Below are the objectives of this research:
(i) To fabricate TiO2 anatase nanoparticles on top of rutile nanoflowers using
hydrothermal and spray pyrolysis method.
(ii) To study the effect of photo-degradation of methyl orange (MO) using TiO2
nanostructure.
(iii) To investigate the biocompatibility of HeLa cells grown on TiO2
nanostructures.
(iv) To apply photo-catalytic activity on HeLa cells using TiO2 nanostructures
after UV radiation.
1.4 Research scope
Hydrothermal method was chosen as the fabrication method for rutile TiO2 because
it is simple and may be performed in a closed system. Spray pyrolysis method was
chosen as the fabrication method for anatase TiO2 as spray method is a simple and
easy method to obtain anatase TiO2 nanoparticles where it involves spraying a
precursor solution on rutile TiO2 substrate.
TiO2 nanostructures were characterized using Field Emission Scanning
Electron Microscope (FESEM) to observe the morphology of TiO2 on its surface and
at its cross-section. It was to determine the size of TiO2 nanostructures that had been
4
fabricated. X-ray Dispersion (XRD) was used to confirm the crystallite phase of the
TiO2 nanostructures after fabrication.
Moreover, to study the photo-degradation property of TiO2 on MO ultra
violet visible near infra-red (UV-Vis-NIR) spectrophotometer was used.
Additionally, a study on the biocompatibility of TiO2 nanostructures was performed
by adding the TiO2 nanostructures to HeLa cells. Furthermore, photo-catalytic effect
of the TiO2 nanostructures on HeLa cells was studied through UV light excitation on
HeLa cells exposed to photo-excited TiO2 nanostructures.
1.5 Research contribution
This study proposed the growth of anatase on rutile TiO2 nanostructure to enhance
photo-catalytic activity. It has been discovered that the rutile TiO2 nanoflowers
grown on FTO substrate provided the base for anatase growth as the rutile TiO2
nanoflowers consist of active sites. After a photo-catalytic reaction has finished, the
TiO2 photo-catalyst may be easily removed from the reaction chamber. This
application could use cervix as a target area.
As mentioned in section 1.2, rutile TiO2 has a higher electron mobility than
anatase. This property will help to accelerate electrons movements to the anatase
TiO2 grown on top of it and increase electron excitation at the contact surface
between cells and TiO2.
2CHAPTER 2
LITERATURE REVIEW
In this chapter, titanium dioxide nanostructures and various methods used to prepare
TiO2 nanostructured thin film were discussed. The photo-catalytic process that
involves TiO2 was described. Then, this chapter also discussed about
biocompatibility and photo-toxicity of TiO2 towards living cells. Moreover, the
working principles of FESEM, XRD, UV-VIS NIR and fluorescence microscope
were explained. Then, what were cancer cells about were reviewed. Studies on the
effect of TiO2 on cancer cells by previous research works were reviewed for
reference purposes. Lastly, the possible applications of TiO2 were described.
2.1 Titanium dioxide nanostructures
The performance of titanium dioxide or titania as metal oxide semiconductor has
been widely studied due to its special photo-catalytic properties [20-22]. TiO2 has
three different forms of crystallite structure, namely, anatase, rutile and brookite [3-4,
23]. Anatase and brookite are metastable and can be transformed to rutile phase
when annealed at high temperatures [21, 24-26]. However, the fabrication of pure
brookite without the presence of anatase or rutile phases is difficult to achieve, thus
brookite is rarely studied. Rutile, on the other hand, is the most stable form of TiO2.
Conventionally, temperatures of around 700 °C and above are used to produce pure
rutile TiO2 structure [23, 25].
It has been reported that the transition onset temperature needs to be around
600 °C to transform anatase to rutile as shown in Figure 2.1 [22].
6
Figure 2.1: Reaction boundaries of phase transition in TiO2 [22].
The crystal structure of anatase consists of 4 edges sharing while rutile
consists of 2 edges sharing, as shown in Figure 2.2. This is as anatase and rutile have
4 and 2 atoms per unit cell, respectively [22]. Both anatase and rutile structures are
tetragonal. The band gap energy for TiO2 is large, around 3.0 eV, depending on its
form. The specific values are 3.26 and 3.05 eV for anatase and rutile, respectively
[21, 25, 27]. The difference is attributed to the arrangement order of its structure, as
shown in Figure 2.2 [22].
Figure 2.2: Crystal structures of (a) anatase, (b) rutile and (c) brookite [22].
As shown in Table 2.1, TiO2 has a high refractive index which results in high
reflectivity from its surface. This property allows TiO2 to absorb more light, thus,
making it an effective photo-catalyst.
7
Table 2.1: Bulk properties of TiO2 crystal phases [28].
Crystal structure System Band gap (eV) Density (kg/m3) Refractive index
ng np
Rutile Tetragonal 3.05 4240 2.946 2.650
Anatase Tetragonal 3.26 3830 2.568 2.658
Brookite Rhombohedral - 4170 2.809 2.677
A lot of research works have been done on the nanostructures of TiO2 in a
variety of area. TiO2 nanostructures include nanoparticles, nanorods, nanobelts,
nanowires and nanoflowers [3, 25, 28-30]. TiO2 nanoparticles are usually made by
sol-gel method and spray pyrolysis deposition technique [31-32]. Normally,
hydrothermal method is utilized to produce nanorods, nanowires, nanobelts and
nanoflowers due to the ability to control pressure and temperature of the reaction in
order to control the shape and size of the resultant TiO2
2.2 Different techniques for preparation of nanostructured TiO2 thin film
In this section, the preparation of nanostructured TiO2 thin film is discussed. There
are a number of methods available for fabricating nanostructures and particles such
as sol-gel method assisted with spin coating, spray pyrolysis deposition technique,
DC or magnetron sputtering and hydrothermal method.
2.2.1 Sol-gel method
Sol-gel process is defined as the process for transiting liquid “sol” into “gel” phase.
A sol is formed after a series of hydrolysis and condensation processes of precursors.
After that, the sol particles are condensed to form a gel. The gel is applied to heat
treatment and drying, until it transforms into dense particles. Sol-gel process
generally uses hydrolysis, condensation and solvent reactions [35]. Puangrat et al.
reported that sol-gel preparation produces nanostructured TiO2 of anatase phase only
and the thin film was coarse [36]. Moreover, in a paper reported by Venkatachalam
et al., it was reported that they were able to fabricate nanostructured anatase TiO2
after calcination at 300 °C. The obtained nanostructured anatase TiO2 transformed to
rutile phase when annealed at 800 °C [37].
8
2.2.2 Spray pyrolysis deposition technique
In spray deposition process, the solution is atomized by at appropriate pressure so
that it deposits are formed on the substrates in tiny droplets. Careful selection of the
solution is needed so that only undesired product will be evaporated at the
temperature of deposition [42-43]. The characteristics of the produced thin film
depend on spray rate, deposition temperature, droplet size and distance of spray
nozzle to substrates [39]. Figure 2.3 shows a schematic diagram of a typical spray
deposition setup [38].
Figure 2.3: Schematic diagram of spray deposition setup [38].
Ranga et al. reported TiO2 nanoparticles fabricated by spray pyrolysis
method. They produced TiO2 nanostructures that has high porosity and with anatase
crystallite structure [40]. Besides, there was study done on modeling spray pyrolysis
for gas sensors. It means that spray deposition is better for sensing application.
2.2.3 Sputtering
The mechanism of sputtering involves bombarding plasma onto a certain target on
the substrates. Ching-Hua et al. published a paper on the principle of sputter systems.
Cations from argon (Ar+) are discharged by plasma at high temperatures. This is
followed by control using an electric field to bombard the target. The particles from
the target metal were then deposited on the substrate surface. To produce TiO2 thin
9
film, pure TiO2 target (99.99 %) was used [18]. Moreover, Kaczmarek et al. also
reported their work on the fabrication of TiO2 nanoparticles using modified
magnetron sputtering. Their work yielded anatase and rutile by using hot target and
high energy, respectively [41]. Another finding by Valencia et al. reported the use of
RF magnetron sputtering to produce anatase and rutile by inductively coupled radio
frequency plasma of calibrated argon/oxygen mixtures without post-annealing [42].
Figure 2.4 shows the plasma reactor used in a sputtering system.
Figure 2.4: Diagram of plasma reactor [42].
2.2.4 Hydrothermal method
The definition of hydrothermal is a process in which a solution is reacted in a closed
system under controlled temperature and pressure with water as the solvent [43]. The
internal pressure is usually determined by the amount of solution and temperature.
By using hydrothermal, many structures could be formed such as nanowires,
nanotubes, nanorods, needle-like structures, nanoflowers, and snowflake-like
structures [3, 28-30, 46-48]. Moreover, hydrothermal method is the best method to
prepare TiO2 nanostructures on conductive substrates due to the presence of active
sites on the substrate’s surface. Thus, nucleation of crystallite TiO2 nanostuctures
may take place [28-30].
There are a few parameters that could influence the properties of TiO2 such as
structure, crystallite phase, pH of solution, and size of particles. TiO2 anatase are
10
produced when the solution is a weak acid or an alkaline solvent [34, 39]. However
when the solution is highly acidic rutile TiO2 particles are produced, as reported in
previous studies [3, 11, 20, 25, 49].
The general chemical reaction occurring in the hydrolysis reaction of Ti
precursor is shown below:-
𝑇𝑖(𝑂𝑅)4 + 𝐻2𝑂 → 𝑇𝑖𝑂2 + 𝑅𝑂𝐻 (2.1)
After hydrolysis process has occurred, the pressure and temperature in the
hydrothermal reaction chamber influences the shape of the TiO2 precipitated onto the
substrate to be crystallite nanostructures. A report by Wu et al. discusses the
formation process of anatase TiO2 on FTO glass. The formation starts with
nucleation of TiO2 on the active sites. They then grew into nanorods with time [31].
Whereas, a study conducted by Hong et al. discussed the fabrication of rutile TiO2
nanorods [32]. Figure 2.5 shows the schematic diagram based on reports by Wu et al.
and Hong et al..
Figure 2.5: Schematic diagram of TiO2 nanostructures using hydrothermal method by
Wu (left) and Hong (right) [29, 30].
As stated in the previous paragraph, acid solution and alkaline solution
produces rutile and anatase TiO2, respectively. A study by Zhiqiao et al. found that
hydrothermal synthesis using strong hydrochloric acid (HCl) produced rutile
nanorods due to the high number of hydroxyl group (OH) ligands thus producing two
edge-shared bonding rutile structure [17]. Whereas, Chin et al. used alkaline sodium
hydroxide (NaOH) and obtained anatase nanotubes where the NaOH breaks the Ti-
O-Ti bond to form four sharing edge bond of TiO2 [49]. So, for this study, the author
had used HCl as a chelating agent using hydrothermal method to produce rutile TiO2
nanostructures.
11
2.3 Photo-catalytic properties of TiO2
The photo excited state of a semiconductor is generally unstable and can be easily
broken down. However, TiO2 is stable when it is photo-excited. Thus, TiO2 can be a
good photo-catalyst [50]. In the previous studies, there were a few reported
applications of TiO2 as a photo-catalyst. It was used in water treatment in which it
could kill the microorganisms and bacteria in contaminated water [5, 36, 51-52],
water splitting to produce hydrogen gas [11, 27], and for cancer cell treatments [7-8,
16]. These are interesting areas to be explored.
Semiconductor with photo-catalytic unique property is characterized by its
valence band (VB) and conductor band (CB). The area between VB and CB is called
a band gap where there are no electrons in that state. However, electrons can jump
from VB to CB when the semiconductor absorbs photons with energy greater than or
equal to its band gap energy, Eg [53]. As shown in Figure 2.6, the photo-catalytic
reaction of TiO2 starts upon exposure to light. The exposure generated hole-electron
pairs. The electrons at VB are excited to CB when the light energy is the same or
greater than the band gap energy. Thus, the processes of oxidation and reduction
occurs when the photo-generated electron reaches the reaction sites [53, 54]. The
electrons in the VB of TiO2 could be excited with light having a wavelength in the
range of UV light (λ<400 nm) which corresponds to TiO2‘s band gap i.e. 3.2 and 3.0
eV for rutile and anatase, respectively [27, 51, 55-57].
Figure 2.6: Schematic diagram of TiO2 photo-catalyst (reproduced figure).
12
The mechanisms of the photo-catalytic reaction may be simplified in the
chemical reactions below [7, 8]. When UV light excites TiO2 particle, it produces
hole+ at the VB and excites one electron to the CB as in equation (2.2) to (2.5).
𝑇𝑖𝑂2 + ℎ𝑣 (𝑈𝑉 𝑙𝑖𝑔ℎ𝑡) → ℎ+ + �̅� (2.2)
𝑇𝑖𝑂2− + 𝑂2 + 𝐻+ → 𝑇𝑖𝑂2 + 𝐻𝑂2
− (2.3)
𝑇𝑖𝑂2− + 𝐻2 𝑂2 + 𝐻+ → 𝑇𝑖𝑂2 + 𝐻2𝑂 + 𝑂𝐻 ∗ (2.4)
�̅� + 𝑂2 → 𝑂2− + 𝐻2𝑂 → 𝑂𝐻 ∗ (2.5)
At the CB, the hole reacts with oxygen and hydrogen ions to produce free radical
hydroxyls group. While at the VB, the electron reacts with oxygen and produces
superoxide anions which later reacts with water molecules to produce free radicals
hydroxyl groups. The free radical hydroxyl groups produced are reactive to organic
substances [7-8, 16, 58].
As reported in previous studies, micro-organisms in waste water consist of
organic substances as well as single cells. What makes them organic is that the
membrane of the cells is made from phospholipid bilayer. When generated free
radical hydroxyl groups comes into close contact with the microorganisms, the cell
wall is the first site attacked [57]. Thereon, the free radical hydroxyl groups will
eventually kill microorganisms, bacteria and cancer cells. Those organisms have a
phospholipid bilayer made from organic compounds. The membrane of cancer cells
may also be attacked by free radical hydroxyl groups released from photo-catalyzed
TiO2 particles. The photo-catalytic mechanism is illustrated using a schematic
diagram shown in Figure 2.6.
Basically, UV light consist of three main regions which are UVA, UVB and
UVC with specific wavelengths range of 315 to 400 nm, 280 to 314 nm and 279 to
100 nm, respectively. UVA is not harmful to our body but too much exposure can
cause aging of skin and eventually can cause cell mutation. On the other hand,
exposure to UVB in long term can cause burning and skin reddening which can
stimulate melanocytes cells to produce pigment in skin cells [59]. However UVC is
very harmful to the cells as it can kill the cells by direct exposure. UVC is widely
used to kill germicides and to sterilize safety cabinets. Generally, in our atmosphere
UVB and UVC are filtered by the ozone layer and only UVA may reach the ground.
However, UVA is less harmful to our skin since we cover ourselves with clothes [67,
68].
13
In this study, UVA was used to activate photo-catalytic activity of TiO2
nanostructures against cells. The energy from UVA is enough to activate the
electrons at the VB of the TiO2 in order for the electrons to be excited to CB for
photo-catalysis to occur since the band gap energy of TiO2 was in range of 3.0 to 3.2
eV and it only absorbs UV light [52]. Methyl orange was used as an indicator to
observe the changes to the main absorption peak which is at 465 nm [11, 50].
2.4 Biocompatibility of TiO2
Biocompatibility is one of the important issue that needs to be addressed when
applying inorganic materials into the human body. The material needs to be non-
toxic, non-carcinogenic, non-thrombogenic, non-immunogenic, non-irritant and so
on [61]. Several research work has been reported on the biocompatibility of TiO2.
Francisco et al. stated that TiO2 thin film was appropriate to culture neuron cells
[62]. Furthermore, a study reported that TiO2 nanoparticles have a stable hydrophilic
biocompatibility to human dermal fibroblast cells [63]. In addition, S. Sangeetha et
al. reported that TiO2 are biocompatible to human blood and may be used in implants
[64]. Moreover, Hangzhou et al. reported antibacterial ability and biocompatibility of
TiO2 nanotubes as implants [65]. Thus, we can conclude that TiO2 is biocompatible
to living cells due to its inert property.
2.5 Photo-toxicity of TiO2
In terms of photo-toxicity, TiO2 could be toxic due to the production of reactive
oxygen species (ROS) by the photo-catalytic process mentioned above. This is as
ROS are very reactive to organic substances. Jun-Jie et al. found TiO2 to be photo-
toxic to human skin keratinocytes when irradiated with UVA at 5.0 J/cm2 [66].
Additionally, Alexandra et al. reported that TiO2 became toxic to cells due to the
presence of ROS upon irradiation of UVA [67]. It can be concluded that TiO2 is not
harmful to living cells unless irradiated with UV light at which they will go through
photo-catalytic process on the surface of cell wall membrane. Therefore, in this study
TiO2 is to be applied to cancer cells and the selected area will be exposed to UV light
in order to generate ROS and kill the cells.
14
2.6 Working principles of devices used for characterization
In this section, the theory and concept of the devices used for characterization such
as FESEM, XRD, UV-VIS NIR Spectrophotometer and fluorescence microscope is
discussed for better understanding on how these devices work to obtain the data
needed.
2.6.1 Field emission scanning electron microscope (FESEM)
The principal of electron microscope is almost the same as conventional microscope
but differs in its energy source. Electron microscope uses electrons as an energy
source while basic microscope utilizes visible light as the energy source. Visible light
is limited to its wavelength range while accelerated electrons have a very short
wavelength. Thus, it is possible for electron microscopes to observe morphology in
micro or nano-scale [68].
FESEM is a type of electron microscope capable for analyzing a surface by
scanning it with high energy beams of electrons in parallel scan patterns. It requires
high vacuum condition so that the electrons could be emitted directly to the sample.
Firstly, electrons are emitted from a tungsten filament. Then they are made to exit the
electron gun where they are then focused and confined into a thin focus. The
monochromatic beam uses metal aperture and magnetic lenses. Lastly, a detector
detects the electrons from the sample and collects the signals to produce an image of
sample [72-73]. The surface morphology of TiO2 nanostructures that produced in this
study were characterized using FESEM JEOL JSM-7600F.
2.6.2 X-ray diffraction (XRD)
The mechanism of XRD applies electromagnetic radiation (x-ray) which has smaller
a wavelength and a higher energy in comparison to visible light. When the x-ray
beam hits an atom, the electrons around the atom start to vibrate with the same
incidence as the incoming beam. This result in a destructive interference and hence
the combined waves were out of phase. No residual energy is left out of the solid
sample. But, the atoms in a crystal are arranged in a regular pattern and there are
15
some small constructive interference. The remaining waves were in phase. Thus, a
diffracted beam could be described as a beam composed of a large number of
scattered rays commonly reinforcing one another [74-75].
Figure 2.7: Schematic diagram of Bragg's Law [71].
The principles of XRD is defined from Bragg’s Law where the beam
reflected from the lower surface travels farther than the one reflected in upper
surface. When the path difference is equal to some integral multiple of the
wavelength, constructive interference occurs. The condition for a constructive
interference is given by Bragg’s law in equation 2.6. A schematic diagram of Bragg’s
Law is shown in Figure 2.7. The angle between the incident beam and the lattice
planes is called θ. The wavelength of the beam is defined by the symbol λ while n is
the order of the lattice. On the other hand, d value is defined as the spacing between
lattice [71].
𝑑 =𝑛𝜆
2𝑠𝑖𝑛𝜃 (2.6)
Thus, XRD can be used to determin crystal structures and the intensity of
crystallinity for the TiO2 nanoflowers sample.
2.6.3 UV-Vis NIR Spectrophotometer
This device was used to measure the absorbance or reflection of a sample. This
spectrometer is capable of measuring wavelength in the ultra violet (300 – 400 nm),
visible (400 – 765 nm) and near-infra red (765 – 3200 nm) regions. The energy
absorbed or emitted by a photon while transitioning from one energy level to another
16
is provided by the equation 2.7 where h is the Plank’s constant and v is the frequency
of the photon.
𝑒 = ℎ𝑣 (2.7)
𝑐 = 𝑣𝜆 (2.8)
𝑇ℎ𝑢𝑠, 𝐸 = ℎ𝑐/𝜆 (2.9)
In essence, the shorter the wavelength, the greater the energy of the photon
and vice versa. When white light is shine on a substance, the light may be totally
reflected, making the substance appear white or the light may be absorbed, making
the substance appear black. Thus, it gives the relationship between light absorption
and colour. For methyl orange dye, the range of absorbed wavelength is at 480 nm to
490 nm [72]. As degradation might occur in methyl orange substance, the intensity of
the absorption may be reduced as a result of the photo-catalytic activity of TiO2
samples.
2.6.4 Fluorescence microscope
Unlike conventional microscopes which uses visible light, this microscope uses
florescence as the reflection source. Sample under observation is illuminated with a
light of certain wavelength which are then absorbed by fluorophores, causing them to
emit light of longer wavelength [73].
The sample is dyed so that it may absorb and emit certain light. The light
source used is either mercury lamp or xenon lamp. For example in living cells, the
sample are immersed with 4’,6-diamidino-2-phenylindo (DAPI) staining that can
only be absorb by the nucleus of cells. Then, when the sample is emitted with a
specific light, the nucleus emits blue color, an indication that the cell is alive.
The concept of this microscope is illustrated in the Figure 2.8. It shows the
components from the light source to where images are captured.
17
Figure 2.8: The illustration of flouroscence microscope by NIKON [73].
2.7 Cancer cells
Cancer is an abnormal growth of cells occurring when the cells split aggressively.
According to the NCR, ten leading cancers among the population of Malaysia in
2007 were breast, colorectal, lung, nasopharynx, cervix, lymphoma, leukemia, ovary,
stomach and liver cancer [19]. Cervical cancer was the third most common cause of
cancer death, after colorectal and breast cancer and was the most common cancer in
women, accounting for 8.4 % of death in women in 2007 [19].
One of the characteristic of cancer cells is the aggressiveness in proliferation
of cells. It means the cells divide themselves vigorously as shown in figure 2.9.
When abnormal cells spread at a certain area, they become cancerous, slowly
destroying normal cells. Moreover, cancer cells have stronger resistance in
comparison to normal cells. Cancer treatment usually involves high doses of drugs
and chemical substances in order to overcome the cancer cells [74].
18
Figure 2.9: Cells proliferation from stem cells [74].
There are many types of cancer cells and it depends on where it develops. For
instant, lung cancer occurs in the lungs and colon cancer occurs in the colon.
Cervical cancer originates from cancerous cells lining the cervix. The normal cervix
cells first develop pre-cancerous changes that will eventually turn them into
cancerous cells. Then, the cells start to develop aggressively in the cervix [75].
Cervical cancer cell line, known as HeLa cells, were taken from an African
American woman called Henrietta Lacks at 1951. After she died her cervical cancer
cells were sub-cultured for research purposed [76].
In this study, the HeLa cells were chosen due to their availability at the
market and their high resistance in comparison to normal cells. For development into
a medical device, TiO2 nanostructures may be inserted at the end of a fiber optic
where the fiber optic may be used in a probe to kill cancer cells in cervix area by
using photo-catalytic effect.
19
2.8 Application of titanium dioxide
In daily uses, TiO2 finds application in paints and sunscreens as white pigments [33-
34]. TiO2 particles are used to increase scattering of visible light. TiO2 applications
have expanded to important technological areas such as dye-synthesized solar cells
(DSSC) [3, 35], environmental areas such as water treatment [5, 36], and medical
areas such as self-sterilizing coatings [79]. Furthermore, TiO2 particles are also used
in gas sensors, corrosion protective coatings, optical coatings and transistors [80].
Moreover, Chutima et al. reported possible TiO2 nanofibers application in a new
area, for anti-microbial and self-cleaning [8]. For gas sensor applications, researchers
found that TiO2 has defined structure suitable for highly sensitive and optoelectronic
sensors [24]. The most promising application of TiO2 reviewed by Markowska et al.
showed the potential of TiO2 to be used in antibacterial, antifungal, antiviral and
anticancer fields [47]. This study is aimed at improving the functionality of TiO2 in
cancer cell treatment.
3CHAPTER 3
METHODOLOGY
Chapter 3 describes and justifies the fabrication methods used for TiO2 nanoflowers
and TiO2 nanoparticles, namely, hydrothermal and spray pyrolysis method, and the
physical characterization methods used, namely, FESEM and XRD analysis. Next,
the photo-catalytic activities of fabricated TiO2 samples were determined using MO
under 20 W UV light exposure. This was followed by photo-catalytic study of HeLa
cells grown on TiO2 samples.
3.1 Flow chart
The overall experimental work was summarized in a flow chart shown in Figure 3.1.
The experimental work was conducted in three main stages, namely, the fabrication
stage, the characterization stage and the verification stage.
21
Figure 3.1: Step by step process.
3.2 Variation of experimental parameters
The parameters used in TiO2 nanoflowers fabrication were hydrothermal reaction
time and the amount of titanium butoxide (TBOT). The hydrothermal reaction time
were varied from 2 to 24 hours while the amount of TBOT were varied from 1 to 3
ml. The parameters were chosen based from the findings of previous studies. For the
photo-catalytic activity study using MO, the parameter used was different TiO2
nanostructures obtained from the varied fabrication methods. For the photo-catalytic
study of all the different TiO2 nanostructures on HeLa cells, the UV light exposure
time to the cells was varied from 10 to 60 minutes. The parameters are summarized
in Table 3.1.
Study the TiO2 nanostructures and their properties
Fabricate TiO2 nanostructures using hydrothermal method
Photo-catalytic study of TiO2 on HeLa cells
Grow HeLa cells on TiO2 nanostructures
Characterized morphological and structural properties of TiO2
Fabrication of TiO2 nanoparticles using spray pyrolysis
method on top of TiO2 nanoflowers (for improvement)
Photo-degrade methyl orange by TiO2 photo-excitation
Culture HeLa cells in laboratory
22
Table 3.1: Variation of experimental parameters
1. Fabrication TiO2
nanostructures
Amount of
TBOT (ml) 1 2 3
Hydrothermal
time (hours) 2 5 10 24 2 5 10 24 2 5 10 24
2. Photo-catalytic
MO
Different TiO2
samples TiO2 rutile TiO2 rutile-anatase TiO2 anatase
3. Photo-catalytic
on HeLa
UV time
exposure
(minutes)
20 30 60
Different TiO2
samples TiO2 rutile TiO2 rutile-anatase TiO2 anatase
3.3 Fabrication of TiO2 nanoflowers by hydrothermal method
The chemicals used to fabricate TiO2 nanoflowers were used as purchased.
Hydrochloric acid (HCl) was used as a chelating agent. TBOT was used as a
precursor material for TiO2 nanoflowers while deionized water was used in the
hydrolysis reaction. Figure 3.2 (a,b,c) shows the chemicals used in the hydrothermal
method. The FTO glass substrates used as templates for growing TiO2 nanoflowers
are shown in Figure 3.2 (d). The FTO glass substrates have a resistivity of about 7
Ω/sq. The substrates were cut into 1 cm by width and 1.5 cm by length. A mixture of
deionized water, acetone and ethanol at a ratio of 1:1:1 was used to clean the
substrates. The substrates were submerged in the mixture and exposed to
ultrasonication in an ultrasonic bath for 10 minutes. Thereon, the substrates were
dried in ambient air. The volume of HCl, TBOT and deionized water were varied to
optimize the structure of the TiO2 nanoflowers.
Firstly, 80 ml of HCl was mixed with 80 ml of deionized water on a hot plate
stirrer at 35°C and a stirring speed of 350 rpm. Then 1 ml of TBOT was dropped
wisely until the solution became transparent. The cleaned FTO substrates were
placed inside Teflon-lined stainless steel autoclave, shown in Figure 3.2 (e). This
process is known the hydrothermal method, a process where a precursor solution is
subjected to heat treatment in a tight chamber. Then, the autoclave was placed inside
10
23
a convection oven and heated at 150 °C for 2, 5, 10 and 24 hours to observe TiO2
structural changes with increasing hydrothermal reaction time.
After the hydrothermal reaction has ended, the autoclave was cooled down to
room temperature. The substrates were taken out and rinsed with deionized water.
Then, the rinsed substrates were dried in an oven at 100 °C for 20 minutes. The
experiment was repeated using 2 and 3 ml of TBOT to monitor changes to the TiO2
structure with an increased amount of TBOT precursor.
Figure 3.2: (a) Hydrochloric acid, (b) deionized water, (c) titanium butoxide as
precursor of TiO2 nanoflowers, (d) FTO glass substrate used as growth template, and
(e) Teflon lined stainless steel autoclave where hydrothermal reaction takes place
3.3.1.1 Characterization of surface morphology and structural phase of TiO2
nanoflowers
The TiO2 samples obtained from hydrothermal method went through surface
morphological analysis using FESEM JEOL JSM-7600F. The crystallinity of the
sample was tested using XRD PANalytical X-Pert Powder with Cu β Kα radiation at
40 kV. The characterization machines used in the experiment are shown in Figure
3.3.
(a) (b) (c) (d) (e)
24
Figure 3.3: FESEM JOEL JSM-7600F and XRD PANalytical XPert Powder used to
characterize the samples.
An important thing to remember when using FESEM is that samples have to
be in dry state. This is because the condition inside the sample chamber has to be in
vacuum to allow the electrons to bombard the sample without any interruptions and
generate clear images. Firstly, the sample was placed onto a sample stage and
clamped. Then, the air inside the sample chamber was removed until the chamber
was fully in vacuum. After that, electrons were bombarded onto the sample and the
current was adjusted to obtain clear images.
For characterization using XRD, the sample was placed on a stage holder.
The beam angle was adjusted to 5°. Then, x-ray was excited onto the sample. Peaks
corresponding to different elements were displayed in a graph showing X-ray
intensity plotted against 2θ. It can be interpreted that the higher the intensity, the
higher the concentration of that element is present in the sample.
3.4 Fabrication of TiO2 nanoparticles by spray pyrolysis method on top of
TiO2 nanoflowers
TiO2 solution was prepared by mixing TiO2 P25 powder with acetic acid in a mortar.
The mixture was grinded until thoroughly combined. Then, TKC-303 was added into
the mortar and the solution was mixed well before it was transferred into a lightproof
bottle. Ethanol and Triton X-100 were added into the solution. The mixture was then
ultrasonicated for 30 minutes. TiO2 nanoflowers grown on FTO substrates fabricated
in the previous experimental step (fabricated using 2 ml of TBOT and 10 hours
hydrothermal reaction time) were used and were aligned on a hotplate with the
65
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