Development of bioconjugated quantum dots for cancer imaging and detection

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DEVELOPMENT OF BIOCONJUGATED QUANTUM DOTS FOR CANCER IMAGING

AND DETECTION

GURPAL SINGH

Centre for Biomedical Engineering

INDIAN INSTITUTE OF TECHNOLOGY DELHI NEW DELHI-16, INDIA

JULY, 2012

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DEVELOPMENT OF BIOCONJUGATED QUANTUM DOTS FOR CANCER IMAGING

AND DETECTION

GURPAL SINGH

Centre for Biomedical Engineering

Submitted

In fulfillment of the requirement of the degree of

Doctor of Philosophy

to the

INDIAN INSTITUTE OF TECHNOLOGY DELHI

July, 2012

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DEDICATED TO

ALMIGHTY

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CERTIFICATE

This is to certify that the thesis entitled "Development of Bioconjugated Quantum Dots for Cancer Imaging and Detection" submitted by Mr. Gurpal Singh to the Indian Institute of Technology, Delhi for the award of degree of Doctor of Philosophy, in Biomedical Engineering is a record of bonafide research work carried out by him. Mr.

Gurpal Singh has worked under our guidance and supervision and has fulfilled the requirement for the submission of this thesis.

The results contained in this thesis are original and have not been submitted in partial or full, to any other university or institute for the award of any degree or diploma.

(Dr. Harpal Singh) (Dr. Sameer Sapra)

Professor Assistant Professor

Centre for Biomedical Engineering Department of Chemistry

Indian Institute of Technology, Delhi Indian Institute of Technology, Delhi Hauz Khas, New Delhi-110016 Hauz Khas, New Delhi-110016

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ACKNOWLEDGEMENT

I wish to place on record my deep sense of gratitude and feeling of reverence to my thesis supervisor Prof Harpal Singh, Centre for Biomedical Engineering, Indian Institute of Technology, Delhi for his guidance, constant inspiration, invaluable suggestions, broad vision and constructive criticism during the course of research work It is more than mere formality that I express my heartfelt gratitude for his understanding and generosity bestowed on me without which this thesis would have been an uphill task.

I am equally grateful to my co-supervisor Dr. Sameer Sapra, Assistant Professor, Department of Chemistry, Indian Institute of Technology, Delhi for his generosity, constant support, invaluable discussion and encouragement throughout and for introducing me to the field of quantum dots. His sincerity, punctuality and disciplined work attitude groomed my research aptitude. I am also thankful to research scholars in his laboratory for constant help. My thanks are also due to previous and present heads, Prof Sneh Anand and Prof. Alok R. Ray, Centre for Biomedical Engineering, for their timely help in all academic pursuits.

I am grateful to Dr. Amit Tyagi, Sc. C, INMAS, DRDO for his help during animal toxicity studies at INMAS.

I sincerely thank Dr. Veena Koul and Dr. Nivedita Gohil, Centre for Biomedical Engineering for their concern to wards my work

I feel elated in acknowledging the precious help, suggestions and support by Mr Manoj Kumar, Dr. Lomas Tomar, Dr. Neelam. H. Zaidi, Dr. Shalini, Dr. Vinay and Dr. Sruti Chatopadhyay, who helped me to accomplish this arduous work.

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It's my pleasant duty to convey my affectionate thanks to Mr. Manoj Kumar, Dr. Loma Tomar, Mr. Sita Ram, Dr. Sapna, Mrs. Dikshi Gupta, Mr. Sumeet Kapoor, Mr. Vivek Bansal, Ms. Alka Khanna, Ms. Swati Jain, Mr. Bikesh Kumar, Mr. Arun, Mr. Shantanu, Mr. Alex, Mr. Manu Dalela, Mr. Rajkumar Sinha, Mr. Udit, Mr. Vikas Arora, Mr.

Ankoor, Ms. Suchi, Ms. Sushma and all other friends and colleagues for their mutual help and support during my research work.

My heartfelt thanks are due to all the staff members of the Centre for Biomedical Engineering, especially, Mr. Rajesh Prasher, Mr. Loknath and Mr. Nirmal for their help during the research work.

I am thankful to Indian Institute of Technology, Delhi for providing me financial aid and necessary facilities to carry out the research work

Finally, I would like to express my gratitude and deepest regards for my parents, my parents in law and my family without whose affection, constant encouragement and blessings, I could never have reached this stage of academic pursuit. In the end, I would like to extend my affectionate thanks to my wife, and my daughter, Princess for being there, without their encouragement and support I would not have reached this pinnacle in life.

(Gurpal Singh)

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Abstract

Current diagnostic methods are not very sensitive and accurate for early cancer detection.

Fluorescent organic dyes and polymer encapsulated fluorescent dyes have limited use in early cancer detection due to photobleaching and photodeterioration of organic dyes.

Semiconductor nanocrystals quantum dots (QDs) with high fluorescence intensity and resistant to photobleaching have been developed by various research groups. Quantum dots, despite their many advantages, have drawbacks of hydrophobicity and incompatibility with the aqueous biological environment and are also toxic to the living system. Therefore, there is an utmost need to synthesize water dispersible QDs with strong photoluminescence as well as good colloidal and stability and reduced toxicity for medical diagnostics especially in the area of cancer detection. Present thesis is devoted towards the development of hydrophilic bioconjugated quantum dots for cancer imaging and detection.

Cadmium telluride quantum dots (CdTe QDs) were synthesized by direct aqueous synthesis using thiol stabilizers such as thioglycolic acid (TGA), 3-mercaptopropionic acid (MPA) and mercaptosuccinic acid (MSA) at 100 °C at pH 12.0. Modified CdTe QDs were characterized by fluorescence and absorption spectroscopy, ATR-FTIR, TEM, AFM and XRD. It was observed that TGA and MPA capped CdTe QDs have the tendency to aggregate on changing pH from 12.0 to 7.4 over a period of time, while MSA capped CdTe QDs remain dispersed for a prolonged period of time at pH 7.4 due to high pKa value. Therefore, MSA capped CdTe quantum dots were selected for conjugation with folic acid using EDCINHS technique for targeting human cancer cells expressing folate receptor. The MCF-7 cells treated with folic acid (FA) conjugated and non-conjugated

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CdTe-MSA quantum dots at different time intervals, showed that both FA conjugated and non-conjugated CdTe-MSA QDs were distributed randomly throughout the cells. During the study, it was also observed that the cells treated with CdTe-MSA quantum dots damaged the morphology of cells and resulted in 90% cellular death, confirming the high toxicity of synthesized CdTe-MSA quantum dots. Therefore, MSA capped CdTe QDs despite showing high photoluminescence are not suitable candidates for cell targeting and imaging studies.

Quantum dots doped silica nanoparticles were synthesized by nanoemulsion technique using tetraethylorthosilicate (TEOS). 3-aminopropyltriethoxysilane (APTES) and trihydroxysilylpropylmethylphosphonate (THPMP) were then added to give surface functionality and colloidal stability to QDs doped silica nanoparticles in aqueous solution. QDSNPs were characterized by fluorescence and absorption spectroscopy, ATR-FTIR and TEM. The bioconjugation of QDSNPs with different concentration of rabbit anti-goat (RAG IgG) antibodies (37.8, 19.1 µl, 1 mg/ml in PBS, pH 7.4) was done using glutaraldehyde chemistry. Fluorescence intensity of nonconjugated QDSNPs shows prominent emission whereas antibody conjugated QDSNPs exhibited 50-60% diminished intensity of emission peak at concentration of 19.1 jig antibodies and 60-70% dimished intensity of emission peak at concentration of 37.8 µg antibodies. RAG IgG conjugated and non-conjugated QDSNPs were further subjected to ELISA studies. Enzyme (HRP) labeled goat anti-rabbit immunoglobulins (GAR IgG-HRP) were incubated with rabbit anti-goat immunoglobulins (RAG IgG) conjugated and non-conjugated QDSNPs. Finally, the developed yellow color was quantified using a microtitre plate reader at 450 nm. The successful bioconjugation of antibody conjugated QDSNPs was confirmed by absorbance

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values of 2.5, whereas non-conjugated QDSNPs shows the negligible absorbance. FA was conjugated to quantum dots (CdSe/CdS/ZnS) doped silica nanoparticles using glutaraldehyde chemistry for specific targeting and imaging of MCF-7 cancer cell.

Maximum photoluminescence intensity for non-folated QDSNPs was observed at 603 rim at excitation of 400 rim. Decrease in the intensity of photoluminescence and minor shift in peak position towards shorter wavelength was observed for folated QDSNPs. Folated and non-folated QDSNPs were characterized by fluorescence and absorption spectroscopy, ATR-FTIR and AFM. Internalization studies were carried out to confirm the uptake of folated QDSNPs by MCF-7 cells which is much higher as compared to non- folated QDSNPs. XTT assay showed > 90 % viability of the cells at QDSNPs concentration from 10 to 80 µg. In-vivo toxicity was performed in mice (Balb/C) model that showed QDSNPs were non-toxic to the mice. Inspite of low toxicity folated QDSNPs are not suitable for cancer detection due to low quantum yield.

In another approach ligand exchange hydrophilic CdSe/CdS/ZnS-MPA QDs were also evaluated for imaging and detection of cancer. FA conjugated CdSe/CdS/ZnS-MPA QDs showed high photoluminescence properties. Internalization studies were carried out to confirm the uptake of folated QDs by MCF-7 cells which is much higher as compared to non-folated QDSNPs. XTT studies showed > 80 % viability of the cells and in-vivo toxicity performed in mice (Balb/C) model also showed that folated QDs were non-toxic to the mice.

Glass matrix was selected for the development of fluoroimmuno-based detection because of its non-autofluorescence properties which allowed the imaging and detection of fluorescent sandwich immunocomplex formed on the matrix. Glass matrices were treated

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with piranha solution to generate surface hydroxyl groups and react with 3-aminopropyltriethoxysilane (APTES) to obtained amine modified glass matrices.

Amine modified glass matrices were characterized using contact angle measurements, ATR-FTIR and ELISA. Anti-HER2 antibodies were immobilized on amine modified glass matrices using glutaraldehyde chemistry. A sandwich immunocomplex formed between the anti-HER2 IgG immobilized glass matrices and anti-HER2 IgG conjugated QDs after the incubation of glass matrices with cancer cells. Intense red fluorescence was observed under confocal laser scanning microscope confirming the attachment of anti-HER2 conjugated QDs onto the cancer cells. Developed detection method enabled the detection of cancer cells upto a minimum level of 103 cells. Anti-HER2 IgG QDs were also evaluated with actual human cancer tissue expressing HER2 receptor. Intense red fluorescence of anti-HER2 conjugated QDs attached to human breast cancer tissue was confirmed by confocal laser scanning microscopy.

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Topic Page No.

Chapter-I Introduction and literature survey 1

1.1 Introduction 3

1.2 Cancer and its causes 5

1.2.1 Pathophysiology of cancer 5

1.2.2 Common causes of cancer 7

1.3 Current diagnostic methods 10

1.3.1 Physical examination 10

1.3.2 Cancer marker detection 10

1.3.3 Imaging techniques 12

1.3.3.1 Computerized tomography 12

1.3.3.2 Positron emission tomography 12

1.3.3.3 Magnetic resonance imaging 12

1.3.3.4 Ultra sound imaging 13

1.3.4 Pathological techniques 13

1.3.4.1 Biopsy 13

1.3.4.2 Histochemistry 13

1.3.4.3 Immunohistochemistry 13

1.4 Fluorescent dyes for cancer detection 14

1.4.1 Mechanism of photobleaching 15

1.5 Advantages of inorganic QDs over organic fluorophores 17

1.6 Quantum dots (QDs) 18

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1.6.1 Quantum confinement effect 20

1.6.2 Synthesis of QDs 21

1.6.3 Photoluminescence 22

1.6.4 Functionalization of QDs: solubilization and bioconjugation 24

1.7 Quantum dots in bioimaging 24

1.8 Application of QDs in bioimaging 25

1.8.1 QDs for multiplexing imaging 26

1.8.2 QDs for live cell imaging and tracking of membrane receptors 27

1.8.3 In vivo animal imaging 27

1.8.4 In vivo tumor imaging 28

1.8.5 In vivo vascular imaging 30

1.9 Rationale and objective of the work 31

References 36

Chapter II Synthesis, characterization and biological evaluation of cadmium telluride (CdTe) based quantum dots

2.1 Introduction 49

2.2 Experimental 51

2.2.1 Materials 51

2.2.2 Direct aqueous synthesis of TGA, MPA and MSA capped CdTe QDs 51 2.2.3 Bioconjugation of CdTe-MSA QDs with folic acid 53 2.2.4 Characterization of folic acid conjugated quantum dots 53 2.2.4.1 Stability of thiolated CdTe QDs at different pH 53 2.2.4.2 Determination of concentration of CdTe QDs 53

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2.2.4.3 Fluorescence and absorption spectroscopy 54 2.2.4.4 Effect of concentration on emission intensity of CdTe-MSA QDs 54 2.2.4.5 Transmission electron microscopy (TEM) 55 2.2.4.6 X-ray diffraction pattern of CdTe-MSA QDs 55 2.2.4.7 Dynamic light scattering and zeta potential measurement 56 2.2.4.8 Fluorescence and absorption spectroscopy of folic acid conjugated CdTe-

MSA QDs 56

2.2.4.9 Measurement of quantum yield 56

2.2.4.10 Fourier transforms infrared spectroscopy (FTIR) 57

2.2.4.11 Atomic force microscopy 57

2.2.5 In vitro cell culture studies 57

2.2.5.1 Nuclear (DAPI) staining 58

2.2.5.2 Cellular uptake studies of FA conjugated CdTe-MSA QDs 58

2.2.5.3 Cell viability assay 58

2.3 Results and discussion 59

2.3.1 Synthesis of CdTe QDs using thiol stabilizer 59 2.3.2 Bioconjugation of CdTe-MSA QDs with folic acid 60 2.3.3 Characterization of folic acid conjugated QDs 61 2.3.3.1 Determination of concentration of CdTe QDs 61 2.3.3.2 Absorption and fluorescence spectroscopy 62 2.3.3.3 Effect of concentration on emission intensity of CdTe-MSA QDs 63 2.3.3.4 Transmission electron microscopy (TEM) 64 2.3.3.5 X-ray diffraction pattern of CdTe-MSA QDs 65

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2.3.3.6 Dynamic light scattering (DLS) measurements 66 2.3.3.7 Fluorescence and absorption spectroscopy of folic acid conjugated and non- conjugated CdTe-MSA QDs

2.3.3.8 Measurement of quantum yield

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2.3.3.9 Surface charge (zeta potential) measurements 69 2.3.3.10 Fourier transform infrared spectroscopy (FTIR) 70

2.3.3.11 Atomic force microscopy (AFM) 72

2.3.4 Cellular uptake studies 75

2.3.5 Cell viability assay 77

References 78

Chapter III Synthesis, characterization and biological evaluation of quantum dot doped silica nanoparticles

3.1 Introduction

Part A Synthesis of bioconjugated QDs (CdSeICdS/ZnS) doped silica nanoparticles and their characterization

3.2 Experimental 86

3.2.1 Materials 86

3.2.2 Synthesis of CdSe core QDs 87

3.2.3 Synthesis of core/shell/shell CdSe/CdS/ZnS QDs (SILAR) 88 3.2.4 Synthesis of QDs (CdSe/CdS/ZnS)doped silica nanoparticles 88 3.2.5 Bioconjugation of QDs (CdSe/CdS/ZnS) doped silica nanoparticles with rabbit

anti-goat (RAG IgG) antibodies 89

3.2.6 Characterization of QDs doped silica nanoparticles 90

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3.2.6.1 Fluorescence and absorption spectroscopy 90 3.2.6.2 Confocal laser scanning microscopy

3.2.6.3 Fourier transform infrared spectroscopy (FTIR) 90 3.2.6.4 Transmission electron microscopy (TEM) 91 3.2.7 Fluorescence detection of Bioconjugated QDSNPs 91 3.2.8 Confirmation of Bioconjugated QDSNPs by ELISA 91

3.3.1 Synthesis of CdSe/CdS/ZnS QDs 92

3.3.2 Synthesis of QDs (CdSe/CdS/ZnS) doped silica nanoparticles 93 3.3.3 Characterization of QDs doped silica nanoparticles 95 3.3.3.1 Absorption and fluorescence spectroscopy 95 3.3.3.2 Confocal laser scanning microscopy

3.3.3.3 Fourier transform infrared spectroscopy (FTIR) 97 3.3.3.4 Transmission electron microscopy (TEM) 98 3.3.4 Fluorescence detection of bioconjugated QDSNPs 99 3.3.5 Confirmation of bioconjugated QDSNPs by ELISA 100

Part B Cancer cell targeting using folic acid conjugated QDs (CdSe/CdS/ZnS) doped silica nanoparticles and its characterization

3.4 Experimental 102

3.4.1 Materials 102

3.4.2 Bioconjugation of NH2 functionalized QDSNPs with folic acid 102 3.4.3 Characterization of folic acid conjugated QDSNPs 103 3.4.3.1 Determination of concentration of CdSe core QDs 103

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3.4.3.2 Fluorescence and absorption spectroscopy of folic acid conjugated

QDSNPs 104

3.4.3.4 Dynamic light scattering (DLS) and zeta potential measurements 104 3.4.3.5 Fourier transform infrared spectroscopy (FTIR) 105

3.4.4.1 Cellular uptake studies of FA conjugated QDSNPs 106

3.4.5 In-Vivo toxicity assessment of folated QDSNPs (CdSe/CdS/ZnS) in

Balb/C mice 107

3.4.5.1 Animal studies 107

3.4.5.2 Hematological studies 107

3.4.5.3 Biochemistry panel assay 108

3.4.5.4 Histopathological studies 108

3.5.1 Bioconjugation of QD (CdSe/CdS/ZnS) doped silica nanoparticles 108 3.5.2 Characterization of folic acid conjugated QDSNPs 109 3.5.2.1 Determination of concentration of CdSe core QDs 109 3.5.2.2 Fluorescence and absorption spectroscopy 110

3.5.2.4 Dynamic light scattering (DLS) measurements 112 3.5.2.5 Surface charge (Zeta potential) measurements 112

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3.5.2.6 Fourier transform infrared spectroscopy (FTIR) 113

3.5.2.8 Cellular uptake studies 115

3.5.3 In vivo toxicity assessment of QDSNPs in Balb/C mice 119

3.5.3.2 Hematology studies 120

3.5.3.4 Microscopic examination 125

References 131

Chapter IV Synthessis, characterization and biological evaluation of cadmium selenide (CdSe) based quantum dots

4.1 Introduction 137

Part A Synthesis and characterization of CdSe/ZnS and CdSe/CdS/ZnS QDs

4.2.1 Materials 139

4.2.2 Synthesis of CdSe/ZnS QDs (SILAR) 139

4.2.3 Synthesis of CdSe/CdS/ZnS QDs (SILAR) 140

4.2.4 Ligand exchange of CdSe based QDs 140

4.2.5 Bioconjugation of QDs with rabbit anti-goat (RAG IgG) antibodies 141

4.2.6 Characterization of QDs 141

4.2.6.1 Fluorescence and absorption spectroscopy 141 4.2.6.2 Confocal laser scanning microscopy (CLSM) 141

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4.2.6.3 Fourier transform infrared spectroscopy (FTIR) 142 4.2.6.4 Transmission electron microscopy (TEM) 142 4.2.7 Fluorescence detection of bioconjugated QDs 142 4.2.8 Confirmation of bioconjugation of QDs by ELISA 142

4.3.1 Synthesis of CdSe based core/shell QDs 143 4.3.2 Ligand exchange of CdSe based core/shell QDs 144 4.3.3 Bioconjugation of CdSe based core/shell QDs with RAG IgG antibodies 144

4.3.4 Characterization of QDs 145

4.3.4.1 Absorption and fluorescence spectroscopy 145 4.3.4.2 Confocal laser scanning microscopy 146 4.3.4.3 Fourier transform infrared spectroscopy (FTIR) 147

4.3.4.4 Transmission electron microscopy 150

4.3.5 Fluorescence detection of bioconjugated QDs 150 4.3.6 Confirmation of bioconjugation of QDs by ELISA 153 Part B Cancer cell targeting using folic acid conjugated CdSe/CdS/ZnS-MPA QDs

4.4.1 Materials 155

4.4.2 Bioconjugation of CdSe/CdS/ZnS-MPA QDs with folic acid 155 4.4.3 Characterization of folic acid conjugated QDs 156 4.4.3.1 X-ray diffraction (XRD) pattern of CdSe/CdS/ZnS QDs 156 4.4.3.2 Effect of concentration on emission intensity of CdSe/CdS/ZnS-MPA

QDs 156

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4.4.3.3 Fluorescence and absorption spectroscopy of QDs 156

4.4.3.5 Fourier transform infrared spectroscopy (FTIR) 157 4.4.3.6 Dynamic light scattering (DLS) and zeta potential measurements 158

4.4.4.1 Cellular uptake studies of FA conjugated QDs 158 4.4.4.2 Cellular uptake studies of FA conjugated QDs by TEM 159 4.4.4.3 Quantification of cellular uptake of QDs by fluorescence 159 4.4.4.3.1 Time dependent cellular uptake of QDs 160

4.4.5 In vivo toxicity assessment of folated CdSe/CdS/ZnS-MPA QDs

in Balb/C mice 161

4.5.1 Bioconjugation of CdSe/CdS/ZnS-MPA QDs with folic acid 162 4.5.2 Characterization of folic acid conjugated QDs 163 4.5.2.1 X-ray diffraction (XRD) pattern of hydrophobic QDs (CdSe/CdS/ZnS) 163 4.5.2.2 Effect of concentration on emission intensity of CdSe/CdS/ZnS-MPA

QDs 164

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4.5.2.3 Fluorescence and absorption spectroscopy of FA conjugated QDs 165

4.5.2.5 Dynamic light scattering (DLS) measurements 167 4.5.2.6 Surface charge (zeta potential) measurements 168 4.5.2.7 Fourier transform infrared spectroscopy 169

4.5.3.1 Cellular uptake studies 172

4.5.3.2 Cellular uptake studies of FA conjugated QDs by TEM 174 4.5.3.3 Quantification of cellular uptake of QDs by fluorescence 175

4.5.4 In vivo toxicity assessment of folated QDs in Balb/C mice 179

Part C Selective capturing and detection of cancer cell on glass matrix using anti- HER2 antibodies conjugated quantum dots

4.6.1 Materials 190

4.6.2 Amine functionalization of glass matrices 190 4.6.3 Characterization of amine functionalized glass matrices 191

4.6.3.1 Contact angle measurements 191

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4.6.3.2 FTIR characterization of glass matrices 191 4.6.4 Bioconjugation of NH2 functionalized glass matrices with RAG IgG 191 4.6.5 Bioconjugation of NH2 functionalized glass matrices with

anti-HER2 antibodies 192

4.6.6 Bioconjugation of CdSe/CdS/ZnS-MPA QDs with anti-HER2 antibodies 192 4.6.7 Confirmation of bioconjugation of RAG IgG antibodies with glass matrices by

sandwich ELISA 192

4.6.8 Fluorescence properties of anti-HER2 conjugated CdSe/CdS/ZnS-MPA

QDs 193

4.6.9 Detection of cancer cells using anti-HER2 conjugated glass matrices

and QDs 193

4.6.10 Immunofluorescence detection of HER2 positive human breast cancer tissue using anti-HER2 IgG conjugated CdSe/CdS/ZnS-MPA QDs 193

4.7.1 Amine functionalization of glass matrices 194 4.7.2 Characterization of amine functionalized glass matrices 194

4.7.2.1 Contact angle measurements 194

4.7.2.2 FTIR characterization of glass matrices 195 4.7.3 Bioconjugation of NH2 functionalized glass matrices with anti-HER2

antibodies 196

4.7.4 Bioconjugation of CdSe/CdS/ZnS-MPA QDs with anti-HER2 antibodies 198 4.7.5 Confirmation of bioconjugation of RAG IgG antibodies with glass matrices by

sandwich ELISA 199

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4.7.6 Fluorescence properties of anti-HER2 conjugated CdSe/CdS/ZnS-MPA

QDs 200

4.7.7 Detection of cancer cell using anti-HER2 conjugated glass matrices and

QDs 201

4.7.8 Immunofluorescence detection of HER2 positive human breast cancer tissue using anti-HER2 conjugated CdSe/CdS/ZnS-MPA QDs 203

References 205

Chapter V — Summary and scope for future work

5.1 Summary 209

5.2 Scope for future work 216

Development of bioconjugated quantum dots for cancer imaging and detection

DEVELOPMENT OF BIOCONJUGATED QUANTUM DOTS FOR CANCER IMAGING

AND DETECTION

GURPAL SINGH

Centre for Biomedical Engineering

INDIAN INSTITUTE OF TECHNOLOGY DELHI NEW DELHI-16, INDIA

JULY, 2012

DEVELOPMENT OF BIOCONJUGATED QUANTUM DOTS FOR CANCER IMAGING

AND DETECTION

GURPAL SINGH

Centre for Biomedical Engineering

Submitted

In fulfillment of the requirement of the degree of

Doctor of Philosophy

to the

INDIAN INSTITUTE OF TECHNOLOGY DELHI

July, 2012

DEDICATED TO

ALMIGHTY

CERTIFICATE

ACKNOWLEDGEMENT

Abstract

Contents

Topic Page No.

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