ENCAPSULATED DYES FOR BIO-IMAGING AND BIO-LABELING APPLICATIONS
ATIQAH AHMAD
UNIVERSITI SAINS MALAYSIA
2018
by
ATIQAH AHMAD
Thesis submitted in fulfilment of the requirements for the degree of
Master of Science
April 2018
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First and foremost, I would like to express my sincere gratitude to Universiti Sains Malaysia, especially to the School of Materials and Mineral Resources Engineering for letting me fulfil my dream of being a student here. My deepest gratitude and thankfulness to my supervisor, Assoc. Prof. Dr. Khairunisak Abdul Razak for her supervision, guidance and advice throughout the research, as well as giving me constant encouragement to finish my research. Her passion and her knowledge in science inspires and motivates me as a student. Special thanks also go to my co-supervisors; Assoc. Prof. Dr. Zainovia Lockman and not to forget to Dr.
Daruliza Kernain Mohd Azman for their insightful ideas and suggestions throughout this research work. All of them have guided me in a scientific way to conduct a research with their profound knowledge and research experience.
I would also like to extend my deepest gratitudeto all technicians of School of Materials and Mineral Resources Engineering and INFORMM’s staffs especially Mrs.
Dyana, my sincere thank you for their help and support throughout the research. Not to forget to all my friends, especially Ms. Zulfa Aiza, Ms. Amirah, Ms. Syafinaz, Mrs.
Hashimah, Mrs Haslinda and Mr. Lukman for always listening and giving me words of encouragement. Special thanks to my parents and family for always giving me moral support and their du’a throughout the research period.
Last but not least, thank you to Universiti Sains Malaysia for financial support through RU grant 1001/Pbahan/870028 and Graduate Research Assistant Scheme (GRA). Also, not to forget to MyBrain15 program which gave me MyMaster scholarship to further my study.
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TABLE OF CONTENTS
Page
ACKNOWLEDGEMENT ii
TABLE OF CONTENTS iii
LIST OF TABLES viii
LIST OF FIGURES x
LIST OF ABBREVIATIONS xvi
LIST OF SYMBOLS xx
ABSTRAK xxi
ABTRACT xxii
CHAPTER ONE: INTRODUCTION
1.1 Research background 1
1.2 Problem Statement 3
1.3 Objectives 5
1.4 Thesis scope 6
CHAPTER TWO: LITERATURE REVIEW
2.1 Introduction 7
2.2 Fluorophores 8
2.3 Type of fluorophores 10
2.3.1 Organic fluorophore 10
2.3.2 Inorganic fluorophore 12
2.3.2 (a) Quantum Dots 12
2.3.2 (b) Dye doped nanoparticles 16
2.3.2(b)(i) Dye-doped polymer nanoparticles 16 2.3.2(b)(ii) Silica nanoparticles (SiNPs)
incorporated with organic fluorescence dyes
18
2.4 Silica nanoparticles (SiNPs) 22
2.4.1 Structure of silica 24
2.4.2 Size of silica 27
iv
2.5 Method preparing SiNPs incorporated with fluorescence dye 28
2.5.1 Stöber method 28
2.5.2 Microemulsion method 29
2.5.2 (a) Water-in-oil microemulsion 31
2.5.2 (b) Oil-in-water microemulsion 33
2.5.2 (c) Micelle formation 34
2.6 Effect of synthesis parameters on the formation of SiNPs 36
2.6.1 Effect of silica precursor 36
2.6.2 Effect of surfactant 38
2.6.3 Effect of co-solvent 40
2.7 Biological applications of SiNPs incorporated with organic fluorescence dye
43
2.7.1 SiNPs in immunoassay 43
2.7.2 SiNPs for gene delivery 45
2.7.3 SiNPs for bio-imaging and bio-labelling 46
2.7.3 (a) Fluorescence properties 47
2.7.3 (b) Photodegradation 49
2.7.3 (c) Stability of colloidal SiNPs 53
2.7.3 (d) Dye release 55
2.7.3 (e) Cytotoxicity 56
2.8 Summary 60
CHAPTER THREE: METHODOLOGY
3.1 Introduction 61
3.2 Chemical and materials 63
v
3.3 Synthesis of SiNPs 65
3.3.1 Synthesis of SiNPs 65
3.3.2 Synthesis of SiNPs encapsulated fluorescence dyes 68 3.3.3 Preparation of SiNPs mixture with excipient addition 70
3.3.4 Encapsulation efficiency 71
3.4 Sample characterization 71
3.4.1 Phase identification 71
3.4.2 Determination of dye concentration 72
3.4.3 Hydrodynamic particle size and zeta potential determination 75
3.4.4 Morphology observation 75
3.5 Stability study of SiNPs encapsulated fluorescence dye 76
3.5.1 Photostability study 76
3.5.2 Stability in biological medium 76
3.5.3 Dye release study in different pH environment 77
3.6 In-vitro toxicity analysis 78
3.6.1 Preparation of the cells for the testing 78 3.6.2 Preparation of confluence in 96 well microplate 78 3.6.3 Deposition of the nanoparticles product on the cell layer 78
3.6.4 MTT assay 79
3.6.5 Elisa microplate spectrophotometer analysis 80
3.7 Cell fluorescent imaging 80
CHAPTER FOUR: RESULT AND DISCUSSION
4.1 Introduction 81
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4.2 Synthesis of SiNPs 82
4.3 Synthesis of SiNPs encapsulated fluorescein dye (SiFluo) 88
4.3.1 Encapsulation efficiency 95
4.3.2 SiFluo surface modification 97
4.3.3 The effect on photostability of SiFluo 99
4.4 Synthesis of SiNPs encapsulated 1,1%-dioctadecyl 3,3,3%,3%- tetramethylindocarbocyanine perchlorate dye (SiDiI)
104
4.4.1 Encapsulation efficiency 112
4.4.1 SiDiI surface modification 113
4.4.2 The effect on photostability of SiDil 115
4.5 Stability of SiNPs encapsulated DiI dye in biological media 118
4.5.1 Stability in salt solution 119
4.5.2 Stability in mouse serum 123
4.5.3 Dye release profile of SiDiI 128
4.6 In-vitro toxicity analysis 132
4.7 Cellular morphology 134
4.8 Cell fluorescent imaging 137
4.9 Summary 139
CHAPTER 5: CONCLUSIONS AND SUGGESTIONS FOR FUTURE WORK
5.1 Conclusions 142
5.2 Suggestions for future work 143
REFERENCES 144
APPENDICES
vii
Appendix A: Preparation of Fluo dye stock solution Appendix B: Preparation of DiI dye stock solution
Appendix C: Preparation of different concentration of dye solution for calibration curve
Appendix D: Preparation of 5 mL of 20 % of D-glucose Appendix E: Preparation of NaCl stock solution
Appendix F: Preparation of Mouse serum stock solution Appendix G: Dilution of 1.0 M PBS
LIST OF PUBLICATIONS
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LIST OF TABLES
Page Table 2.1 The properties of organic fluorophores used in fluorescence sensing
technologies (Demchenko, 2008)
11
Table 2.2 Overview of silica materials and relevant properties 22 Table 2.3 In vitro studies on silica nanoparticles toxicity 58 Table 3.1 List of chemicals and materials for synthesis of SiNPs encapsulated
fluorescence dye
64
Table 4.1 Hydrodynamic size, polydispersity index (PDI) and TEM size of SiNPs synthesised using different volume of 2-butanol (2, 4 and 6 ml)
87
Table 4.2 Hydrodynamic size, polydispersity index (PDI) and TEM size of SiFluo synthesised using different volume of 2-butanol (2, 4 and 6 ml)
92
Table 4.3 Absorbance value and concentration of SiFluo after synthesis, after dialysis and after re-concentration synthesised using different volume of 2-butanol
94
Table 4.4 The concentration of initial Fluo dye used for SiFluo formation, concentration of supernatant of SiFluo and the entrapment
efficiency of SiFluo synthesised using different volume of 2-butanol 96
Table 4.5 Absorbance value, concentration and filtration efficacy of SiFluo with excipient addition and after filtration with excipient addition synthesised using different volume of 2-butanol
98
Table 4.6 Decolorization efficiency (%) of bare Fluo dye and SiFluo in size 27.7, 41.3 and 46.7 nm after exposure under Halogen lamp for 60 minutes
103
Table 4.7 Hydrodynamic size, polydispersity index (PDI) and TEM size of SiDiI synthesised using different volume of 2-butanol (2, 4 and 6 ml)
108
Table 4.8 Comparison of average particle sizes between SiNPs, SiFluo and SiDiI synthesised synthesised using different volume of 2-butanol
108
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Table 4.9 Absorbance value and concentration of SiDiI after synthesis, after dialysis and after re-concentration synthesised using different volume of 2-butanol
110
Table 4.10 The concentration of initial DiI dye used for SiDiI formation, concentration of supernatant of SiDiI and the entrapment efficiency of SiDiI synthesised using different volume of 2-butanol
112
Table 4.11 Absorbance value, concentration and filtration efficacy of SiDiI with excipient addition and after filtration with excipient addition synthesised using different volume of 2-butanol
114
Table 4.12 Decolourisation efficiency of bare DiI and 30.4, 40.0 and 53.4 nm SiDiI upon irradiation with 200 W Halogen lamp for 60 minutes
117
Table 4.13 Stability results of 30.4 nm SiDiI upon incubation in NaCl medium at t = 0 hour (before incubation), incubated t = 24 hours, after incubation followed by filtration using 0.22 μm membrane filter
121
Table 4.14 Stability results of 40.0 nm SiDiI upon incubation in NaCl medium at t = 0 h (before incubation), incubated t = 24 hours, after
incubation followed by filtration using 0.22 μm membrane filter
121
Table 4.15 Stability results of 53.4 nm SiDiI upon incubation in NaCl medium at t = 0 h (before incubation), incubated t = 24 hours, after
incubation followed by filtration using 0.22 μm membrane filter
122
Table 4.16 Absorbance value, concentration and filtration efficacy of 30.4 nm SiDil in different volume of mouse serum (a) 0, (b) 5 %, (c) 10 % and 25 %
125
Table 4.17 Absorbance value, concentration and filtration efficacy of 40.0 nm SiDil in different volume of mouse serum (a) 0, (b) 5 %, (c) 10 % and 25 %
126
Table 4.18 Absorbance value, concentration and filtration efficacy of SiDil size 53.4 nm in different volume of mouse serum (a) 0, (b) 5 %, (c) 10
% and 25 %
126
Table 4.19 IC50 value of SiDiI size 30.4, 40.0 and 50.4 nm after treated in MCF-7
134
Table 4.20 Summary of results of major findings 141
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LIST OF FIGURES
Page
Figure 2.1 Monomolecular decay pathways (Juris, 2012) 8
Figure 2.2 Jablonski diagram (Juris, 2012) 9
Figure 2.3 (a) Emission colours from small to large CdSe QDs excited by a near-ultraviolet lamp. (b) Photoluminescence spectra of the Cd (Zhang et al., 2012)
14
Figure 2.4 (A-F) represent UV-Vis spectra of HIPPNPs (A-C) and freely dissolved ICG in PBS (D-F) treated with 4 or 37˚C in the presence and absence of light illumination for 48 h (Lee and Lai, 2016)
18
Figure 2.5 Fluorescence spectra of a) R6G dye encapsulated in silica particles and b) R6G dye in aqueous solution (Sokolov et al., 2007)
20
Figure 2.6 Giant structure extending on all 3 dimensions (Nandanwar et al., 2013).
23
Figure 2.7 Mesophase structure of a) MCM-41, b) MCM-48 and MCM-50 (Anderson et al., 2008)
24
Figure 2.8 Effect of oxygen on the fluorescence quantum yield of Ru(bpy)32+; (I) free Ru(bpy)32+ molecules solution in the presence of oxygen, (II) a deoxygenated of free Ru(bpy)32+
molecules solution, (III) a nanomatrix in the presence of oxygen and (IV) a deoxygenated nanomatrix (Liang et al., 2013)
26
Figure 2.9 Hypothetical phase regions of microemulsion systems (Malik et al., 2012)
31
Figure 2.10 A typical structure of water-in-oil microemulsion (Lawrence and Rees, 2000)
32
Figure 2.11 A typical structure of (a) oil-in-water microemulsion and (b) micelle (Lawrence and Rees, 2000)
33
Figure 2.12 Different shape of micelle (Cicuta, 2011) 35
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Figure 2.13 SEM images of silica nanosphere synthesized with (a) TMOS, (b) TEOS, (c) TPOS and (d) TBOS (Yun et al., 2005).
38
Figure 2.14 Different classes of surfactants (Som et al., 2012) 39
Figure 2.15 FESEM images of silica nanoparticles produced in different amount of ethanol (a) 5 ml, (b) 10 ml, (c) 20 ml and (d) 30 ml (Shaharuddin et al.,2015)
41
Figure 2.16 SEM images of silica nanosphere prepared with (a) MeOH, (b) EtOH, (c) PrOH (Yun et al., 2005)
42
Figure 2.17 The detection of Vibrio cholera O1 by the developed dot fluorescence immunoassay strip. The strips were dipped in samples containing VCO1 in ranges (A) 0 cfu/mL (control), (B) 4.3 × 101 cfu/mL, (C) 4.3 × 102 cfu/mL, (D) 4.3 × 103 cfu/mL, (E) 4.3 ×104 cfu/mL, (F) 4.3 × 105 cfu/mL, (G) 4.3 × 106 cfu/mL, (H) 4.3 ×107 cfu/mL, and (I) 4.3 ×108 cfu/mL (Thepwiwatjit et al., 2014).
44
Figure 2.18 UV-vis spectra of RBITC and RBITC fluorescent silica nanoparticles (FSNPs) in ethanol at 25 ºC (dos Santos Neves, 2014)
48
Figure 2.19 Influence of light exposure on ICG degradation in aqueous solution (Saxena et al., 2003)
51
Figure 2.20 Photobleaching behavior of nanoparticle intermediates (blue - free TRITC dye; green - (TRITC dye were covalently
conjugated to Si precursor and condensed); red - (denser Si network around the TRITC core material) and black - fluorescein (Ow et al., 2005)
52
Figure 2.21 Confocal micrographs of continued irradiation of cells leading to photobleaching (a) FITC loaded HUVEC cells and (b) 30 nm fluorescent silica loaded cells (Veeranarayanan et al., 2012)
53
Figure 2.22 (a) Particles size and zeta potential, (b) particles size distribution of colloidal silica suspension at different NaCl concentrations (Orts-Gil et al., 2011)
55
Figure 2.23 Release profile of GLP-1 from SPN-GLP-1 at different pH conditions (pH 1.0 and pH 7.4) at 37 °C (Qu et al., 2012)
56
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Figure 3.1 Summary of experimental procedure for this research 62
Figure 3.2 A schematic of experimental setup 66
Figure 3.3 General flowchart of synthesis process of SiNPs 67 Figure 3.4 General flowchart of synthesis process of SiNPs encapsulated
fluorescence dye
69
Figure 3.5 General flowchart of synthesis process of SiNPs encapsulated fluorescence dye
70
Figure 3.6 Graph of UV-Vis absorbance spectra of Fluo dye in different concentration
73
Figure 3.7 Calibration curve of Fluo dye 73
Figure 3.8 Graph of UV-Vis absorbance spectra of Dil dye in different concentration
74
Figure 3.9 Calibration curve of Dil dye 74
Figure 4.1 Schematic of SiNPs formation using micelle entrapment method
83
Figure 4.2 Hydrodynamic size distribution of silica nanoparticles
synthesised using different volume of 2-butanol (2, 4 and 6 ml) 84
Figure 4.3 TEM images of SiNPs synthesised using different volume of 2- butanol: (a) 2 ml, (c) 4 ml and (e) 6 ml and histograms of particle size distribution of SiNPs: (b)2 ml, (d) 4 ml and (f) 6 ml
86
Figure 4.4 XRD patterns of SiNPs synthesised using different volume of 2-butanol: (a) 2 ml, (b) 4 ml and (c) 6 ml
88
Figure 4.5 Hydrodynamic size distribution of SiFluo synthesised using different volume of 2-butanol (2, 4 and 6 ml)
89
Figure 4.6 TEM images and histograms of particle size distribution of SiFluo synthesised using different volume of 2-butanol: (a) 2 ml, (b) 4 ml and (c) 6 ml
91
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Figure 4.7 XRD patterns of Fluo dye 93
Figure 4.8 Absorbance spectra of SiFluo after synthesis, after dialysis and after re-concentration synthesised using different volume of 2- butanol: (a) 2 ml, (b) 4 ml and (c) 6 ml
94
Figure 4.9 Absorbance spectra of fluorescein dye and SiFluo synthesised using different volume of 2-butanol in water at 25°C with a similar final concentration approximately 0.13 mM
95
Figure 4.10 Absorbance spectra of SiFluo with excipient addition and after filtration with excipient addition synthesised with different volume of 2-butanol: (a) 2 ml, (b) 4 ml and (c) 6 ml
98
Figure 4.11 UV-Vis spectra of (a) bare fluorescein dye and SiFluo
synthesised using different volume of 2-butanol: (b) 2 ml, (c) 4 ml and (d) 6 ml in every 10 minutes of irradiation under
halogen lamp
100
Figure 4.12 Decolorization efficiency (%) of SiFluo and bare Fluo dye after exposure to halogen lamp
102
Figure 4.13 Absorbance spectra of SiFluo after synthesis and one month after synthesis (a) 27.7 nm, (b) 41.3 nm and (c) 46.7 nm
104
Figure 4.14 Hydrodynamic size distribution of SiDiI synthesised using different volume of 2-butanol (2, 4 and 6 ml)
105
Figure 4.15 TEM images and histograms of particle size distribution of SiDil synthesised using different volume of 2-butanol: (a) 2 ml, (b) 4 ml and (c) 6 ml
107
Figure 4.16 XRD pattern of DiI dye, blank SiNPs and SiDiI 109 Figure 4.17 UV-Vis spectra of SiDiI after synthesis, after dialysis and after
re-concentration synthesised using different volume of 2- butanol; (a) 2 ml, (b) 4 ml and (c) 6 ml
110
Figure 4.18 Absorbance spectra of 1.5 μM DiI dye molecules and SiDiI synthesised using different volume of 2-butanol in aqueous solutions
111
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Figure 4.19 Absorbance spectra of SiDiI with excipient addition and after filtration with excipient addition synthesised using different volume of 2-butanol: (a) 2 ml, (b) 4 ml and (c) 6 ml
114
Figure 4.20 Absorbance spectra of (a) bare DiL and SiDiI size (b) 30.4 nm, (c) 40.0 nm and (d) 53.4 nm upon irradiation of Halogen lamp for 60 minutes
116
Figure 4.21 Decolourisation efficiency of bare DiI and 30.4, 40.0 and 53.4 nm SiDiI upon irradiation with 200 W Halogen lamp for 60 minutes
117
Figure 4.22 Absorbance of SiDil size (a) 30.4 nm, (b) 40.0 nm and (c) 53.4 nm against concentration of NaCl medium 0 M, 0.1 M, 0.5 M and 1.0 M
120
Figure 4.23 Stability efficacy of SiDiI size 30.4, 40.0 and 53.4 nm in different concentration of NaCl solution
123
Figure 4.24 Absorbance of SiDil size (a) 30.4 nm, (b) 40.0 nm and (c) 53.4 nm against concentration of mouse serum 5,10 and 25 %
124 Figure 4.25 Stability efficacy of SiDiI size 30.4, 40.0 and 53.4 nm in
different concentration of mouse serum
128
Figure 4.26 Absorbance spectra of release medium in pH 1.4 for (a) SiDiI 30.4 nm, (b) SiDiI 40.0 nm and (c) SiDiI 53.4 nm
130
Figure 4.27 Absorbance spectra of release medium in pH 6.8 for (a) SiDiI 30.4 nm, (b) SiDiI 40.0 nm and (c) SiDiI 53.4 nm
130
Figure 4.28 Absorbance spectra of release medium in pH 7.4 for (a) SiDiI 30.4 nm, (b) SiDiI 40.0 nm and (c) SiDiI 53.4 nm
131
Figure 4.29 Cell viability for bare DiI dye and SiDiI size 30.4, 40.0 and 53.4 nm on MCF-7 cell line treated in 24 hours
134
Figure 4.30 Morphological observation of (a) control of MCF-7 cell and MCF7 cells treated with different size of SiDiI (b) 30.4 nm, (c) 40.0 nm and (d) 53.4 nm under phase-contrast inverted
microscope. Arrows indicate (A) membrane blebbing and (B) cell shrinkage as evidence of apoptosis
136
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Figure 4.31 Fluorescent images of MCF-7 after treated with (a) DiI dye and SiDiI size (b) 30.4 nm, (c) 40.0 nm and (d) 53.4 nm in 1 hour
138
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LIST OF ABBREVIATIONS
A549 Human epithelial cells
Ag Silver
APTS 3-aminopropyltriethoxysilane
Au Gold
CBMN Cytokinesis Block Micronucleus Assay CCC Critical coagulation concentration
Cd2+ Cadmium ion
CdSe Cadmium selenide
cfu Colony forming unit
DCFH Dichloro-dihydro-fluorescein diacetate assay
DI De-ionized water
DiI 1,1%-dioctadecyl 3,3,3%,3%
tetramethylindocarbocyanine perchlorate dye DLS Dynamic light scattering
DLVO Derjaguin, Landau, Verwey, and Overbeek theory
DNA Deoxyribonucleic acid
EtOH Ethanol
FITC Fluorescein isothiocyanate dye
Fluo Fluorescein dye
FMSNP Fluorescent mesoporous silica nanoparticles FRET Förster resonance energy transfer
FSNPs Fluorescence silica nanoparticles GLP-1 Glucagon-like peptide-1
GSH Reduced glutathione level assay
H+ Hydrogen ion
HCl Hydrochloric acid
HEL-30 Mouse keratinocytes
HEp-2 Human epithelial type 2 cells
HER2 Human epidermal growth factor receptor 2
xvii
HIPPNPs Anti-HER2 ICG-encapsulated polyethylene glycol- coated poly(lactic-co-glycolic acid) nano-particles HPRT Hypoxanthine-guanine phosphoribosyltransferase HUVEC Human umbilical vein endothelial
ICG Indocyanine green dye
LDH Lactate dehydrogenase assays
M – Radicals of molecules
M* Photoexcitation of molecules
MCM-41 Silica mesophase structure in hexagonal phase MCM-48 Silica mesophase structure in cubic phase MCM-50 Silica mesophase structure in lamellar phase
MDA Malondialdehyde assay
MeOH Methanol
MET-5A Human mesothelial cell
MonoMac 6 Macrophage
MSN Mesoporous silica nanoparticles
MTT Methyl tetrazolium
N2a Mouse neural crest-derived cell line
Na+ Sodium ion
NaCl Sodium chloride
NaOH Sodium hydroxide
NH3 Ammonia
Ni Nitrogen
NIR Near infrared
NPs Nanoparticles
O/W Oil-in-water
O2 Oxygen
ORMOSIL Organically modified silica O–Si–O Silicon dioxide bonding
Pb2+ Lead (II) ion
PbS Lead sulfide
PBS Phosphate buffer solution/saline
PDI Polydispersity index
xviii
PEG Polyethylene glycol
PLGA Poly(lactic-co-glycolic acid)
PrOH Isopropanol
QDs Quantum dots
R6G Rhodamine 6G
RBITC Rhodamine B isothiocyanate dye
RBS Rhodaminde B
RLE-6TN Rat Lung Epithelial-6-T-antigen Negative ROS Reactive oxygen species
RPMI 2650 Human nasal septum quasidiploid tumour Ru(bpy)32+ Tris(bipyridine)ruthenium(II)
S* Singlet excited state
S0 Electrons from the ground state S1 Excited singlet state 1
S2 Excited singlet state 2
Si Silica
SiDiI Silica nanoparticles encapsulated 1,1%-dioctadecyl 3,3,3%,3% tetramethylindocarbocyanine perchlorate dye
SiFluo Silica nanoparticles encapsulated fluorescein dye SiNPs Silica nanoparticles
SiO2 Silicon dioxide, silica
SPN-GLP-1 silica nanoparticles encapsulated glucagon-like peptide-1
SRB Sulforhodamine B assay
T* Triplet excited state
T1 Lowest vibrational level
TEM Transmission electron microscope TEOS Tetraethyl orthosilicate
THP-1 Macrophage
TMR Tetramethyl rhodamine
TNF-a, IL-6, IL-8 Cytokine expression
TRITC Tetramethylrhodamine isothiocyanate
xix
TSTMP Trisodium trimetaphosphate Tween 80 Polysorbate 80
UV-Vis UV visible spectrophotometer UV-Vis UV-Vis spectrophotometer
VCO1 Vibrio cholera O1
VTMS Vinyltrimethoxysilane
W/O Water-in-oil
WIL2-NS Human B-cell Lymphoblastoid cell Wo Water-to-surfactant molar ratio
XRD X-Ray diffraction
ZnSe Zinc selenide
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LIST OF SYMBOLS
% Percentage
± Plus minus
° Degree
°C Degree Celsius
µg Microgram
µl Microliter
µm Micrometer
cfu Colony-forming unit
d Diameter
ɛ Molar absorbance
g Gram
h Hour
kDa Kilodaltons
l Liter
M Molarity
min Minute
ml Mililiter
mm Milimeter
nm Nanometer
ns Nanosecond
r° Fundamental emission anisotropy
rpm Revolutions per minute
wt Weight
α Two-photonic cross-section
θ Theta
λ Wavelength
τF Excited-state lifetime
φ Fluorescence quantum yield
xxi
SINTESIS SILIKA NANOKOLOID TERKANDUNG PEWARNA UNTUK APLIKASI BIOPENGIMEJAN DAN BIOPELABELAN
ABSTRAK
Dua jenis pewarna pendarflor iaitu 1, 1%-dioctadecyl 3, 3, 3%, 3%- tetramethylindocarbocyanine perchlorate (DiI) dan fluorescein (Fluo) telah dikapsulkan ke dalam nanopartikel amorfus silika dengan mengunakan kaedah pembentukan misel. 2-butanol sebagai pelarut bersama digunakan bagi memudahkan reaksi hidrolisis semasa proses sintesis telah divariasikan untuk mengetahui kesan keatas saiz partikel. Sampel telah dianalisa untuk menentukan purata saiz partikel, morfologi partikel, ciri-ciri spektrum peresapan dan intensiti kependarfloran. Partikel bersaiz 26.2 hingga 53.4nm, berbentuk sfera dengan taburan partikel yang sekata telah dihasilkan dengan menggunakan 2, 4 dan 6 ml isipadu pelarut bersama. Kestabilan warna antara silika berkapsul pewarna Fluo, (SiFluo) dan silika berkapsul pewarna DiI (SiDiI) telah dijalankan. SiDiI mempunyai kestabilan warna yang tinggi berbanding SiFluo. Berikutan itu, SiDiI bersaiz 30.4, 40.0 and 53.4 nm telah diuji untuk menilai kestabilan partikel di dalam media biologi yang berbeza (larutan NaCl dan serum tikus), potensi sitotoksisiti dan paparan pengimejan pendarflor terhadap sel hidup manusia (human breast adenocarcinoma, MCF-7). SiDiI bersaiz 53.4 nm mempunyai kestabilan warna yang tinggi dengan hanya 11 % peratus pemudaran dan mempunyai kestabilan partikel yang bagus di dalam kedua-dua media biologi. Selain itu, SiDiI bersaiz 53.4 nm mempunyai kadar sitotoksisiti yang rendah terhadap sel MCF-7. Sel MCF-7 yang dirawat oleh SiDiI bersaiz 53.4 nm menunjukan kadar kecerahan dan intensiti kependarfloran yang tinggi diperolehi daripada paparan pengimejan pendarflor.
xxii
SYNTHESIS OF SILICA NANOCOLLOIDS ENCAPSULATED DYES FOR BIO-IMAGING AND BIO-LABELING APPLICATIONS
ABSTRACT
Two different types of fluorescent dyes which are 1,1%-dioctadecyl 3,3,3%,3%-tetramethylindocarbocyanine perchlorate (DiI) and fluorescein (Fluo) were encapsulated inside the amorphous silica nanoparticles (SiNPs) using micelle entrapment method. 2-butanol which acted as a co-solvent to facilitate hydrolysis reaction during the synthesis process was varied in order to investigate the effect on the particle size. The synthesised samples were analysed and characterised to determine the hydrodynamic size of particles, average particle size and particles morphology, absorbance properties, fluorescence properties and crystallinity of the samples. Spherical and monodispersed nanoparticles (NPs) with different sizes ranging from 26.2 to 53.4nm, were obtained when synthesised using 2, 4 and 6 ml volume of co-solvent, 2-butanol respectively. The photostability effect between SiNPs encapsulated with Fluo dye (SiFluo) and SiNPs encapsulated with DiI dye (SiDiI) were conducted. SiDiI showed good photostability effect compared to SiFluo. Therefore, selected particle sizes of SiDiI 30.4, 40.0 and 53.4 nm were further analysed to study the particles stability in different biological medium (NaCl and mouse serum), cytotoxicity and fluorescent cell imaging in living cells, human breast adenocarcinoma (MCF-7). SiDiI with size 53.4 nm showed good photostability with only 11 % of decolourisation and obtained good particles stability in both NaCl solution and mouse serum. The cytotoxicity study showed that cytotoxicity of SiDiI is size dependent whereby SiDiI with size 53.4 nm was less toxic. Furthermore, bright and high contrast fluorescent cell images of MCF-7 treated with SiDiI with size 53.4 nm was observed.
1
CHAPTER ONE
INTRODUCTION
1.1 Research background
Nanomaterials are defined as the set of particles which have a very small scale which is less than 100 nm in at least one dimension. Nanomaterials exhibit unique properties in optical, magnetic, electrical or other properties. In the past few decades, nanomaterials were introduced to industries and currently in continuous research due to the potential for great impact which can improve the quality of a product in electrical, medical and other field. Recently, numerous nanomaterials have been developed and employed as the most promising candidates for bio-analysis applications especially in optical bio-imaging and bio-labelling (Yan et al., 2007).
Optical bio-imaging and bio-labelling are researches involving different experimental technique and materials that are being utilized to obtain optical contrast in biological specimens. Optical imaging method is expected to have a substantial impact on the prevention and treatment of diseases especially in cancer treatment. Fast and easy diagnosis process of optical imaging method enables researchers to visualize complete organ to complex biological process by multidimensional and multi parameter data (Deshmukh et al., 2016). Optical bio-imaging and bio-labelling have attracted great attention in biomedical research due to their distinguished advantages in terms of the availability of biocompatible, high resolution and good sensitivity of imaging agents or biomarker (Arunkumar et al., 2005).
In optical bio-imaging and bio-labelling, the imaging agents or biomarker should have high water-solubility, biocompatibility, good chemical stability and
