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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