Synthesis and Investigation of the Properties of Water Soluble Quantum Dots for Bioapplications

A Thesis by Fatemeh Mir Najafi Zadeh Submitted in Partial Fulfilment of the Recruitment for the Degree of Doctor of Philosophy in Chemistry

Supervisor: A/Prof. John A.Stride Co-supervisor: A/Prof. Marcus Cole

School of Chemistry Faculty of Science August 2014

The University of New South Wales Thesis/ Dissertation Sheet Surname or family name: Mir Najafi Zadeh First name: Fatemeh Other name/s: Abbreviation for degree as given in the University calendar: PhD School: Chemistry Faculty: Science Title: Synthesis and Investigation of the Properties of Water Soluble Quantum Dots for Bioapplications

Abstract Semiconductor nanocrystals or quantum dots (QDs) have received a great deal of attention over the last decade due to their unique optical and physical properties, classifying them as potential tools for biological and medical applications. However, there are some serious restrictions to the bioapplications of QDs such as water solubility, toxicity and photostability in biological environments for both in-vivo and in-vitro studies. In this thesis, studies have focused on the preparation of highly luminescent, water soluble and photostable QDs of low toxicity that can then be potentially used in a biological context. First, CdSe nanoparticles were synthesized in an aqueous route in order to investigate the parameters affecting formation of nanoparticles. Then, water soluble CdSe(S) and ZnSe(S) QDs were synthesized. These CdSe(S) QDs were also coated with ZnO and Fe2O3 to produce CdSe(S)/ZnO and CdSe(S)/Fe2O3 core/shell QDs. The cytotoxicities of as-prepared CdSe(S), ZnSe (S) and CdSe(S)/ZnO QDs were studied in the presence of two cell lines: HCT-116 cell line as cancer cells and WS1 cell line as normal cells. Finally, CdSe(S) QDs were linked to Donkey-anti mouse (H+L) (IgG) antibody Cy3 fluorophore to prepare a CdSe(S)-antibody conjugated compound and the photostability of CdSe(S) QDs both after linking to antibody and in the presence of HCT-116 cells was investigated. The obtained QDs exhibited high crystallinity, water solubility, low toxicity and photostability, demonstrating that highly crystalline nanoparticles can be formed by sufficient control of the experimental parameters. It was determined that coating the CdSe(S) QDs with ZnO led to a reduced cytotoxicity of the CdSe(S) QDs. It was found that ZnSe(S) QDs have no cytotoxicity toward both HCT-116 and WS1 cell lines across all concentrations studied. Finally, the CdSe(S) QDs were found to be photostable both in a CdSe(S)-antibody conjugated compound and in the presence of HCT-116 cancer cells.

Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International. …………………………… ………………………...…… …………………….. Signature Witness Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research.

FOR OFFICE USE ONLY Date of completion of requirements for Award:

Certificate of Originality

I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, nor material which to a substantial extent has been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgment is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project’s design and conception or in style, presentation and linguistic expression is acknowledged.

Signed……………………………………

Date………………………………………

I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.

I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International.

I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.

Signed ......

Date ......

Authenticity Statement

I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.

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Acknowledgments

All praise and thanks goes to God who has created a unique order in everything, from the minuscule particles to magnificent universes.

The years spent for PhD training was an excellent opportunity for me to be familiar with the fascinating world of nanoparticles, along with contacting with many people who assisted me to accomplish this research. I would like to express my gratitude to all of them.

First and foremost, it is my absolute pleasure to thank my supervisor A/Prof. John Stride for teaching me many useful lessons and providing me with unlimited guidance and support to reach goals of my research. He has been so patient, encouraging, supportive and helpful in all of stages of this thesis and I am grateful to have worked with him.

I would like to extend my special thanks to Dr. Deborah Ramesy and A/Prof. Shelly McAlpine for their kind collaboration about providing the cells and performing cytotoxicity assays.

I would like to thank my co-supervisor, A/Prof. Marcus Cole and postgraduate coordinators of School of Chemistry, A/Prof. Jonathan Morris and Prof. Martina Stenzel for their kind cooperation, Prof. Brynn Hibbert for organizing Electronic Lab Notebook (ELN), Prof. Barbara Messerle and Prof. Scott Kable the heads of School of Chemistry for their great management and providing facilities.

I am grateful to all the staff of School of Chemistry for assisting me, especially Ms. Lucy Stride, Mr. Ken McGuffin, Ms. Jodee Anning, Ms. Anne Ayres, Mr. Rick Chan, Mr. Steve Yonnoulatos, Mr. Ray Arnold, Dr. Tobby Jackson, Ms. Peta Di Bella, Dr. Route Devakaram, Mr. Rama Anning, Mr. Grant Platt and Dr. Doug Lawes.

I am thankful to the staff of Mark Wainwright Analytical Centre of University of New South Wales for training me to use facilities: Dr. Yu Wang (XRD), Ms. Katie Levick and Dr. Aaron Dodd (TEM), Dr. Anne Rich (CD spectroscopy), Ms. Katerina Bendowa and Dr. Renee Wan (confocal microscopy).

I would like to thank Dr. Fan Wang and Dr. Peter Reece for their kind assistance with photoluminescence spectroscopy and Dr. Bin Gong for performing XPS spectroscopy, Dr. Quadir Zakaria for his good comments about SAED, A/Prof. Grainne Moran for her useful guidance about CD spectroscopy, Mr. Stephan Parker for providing antibody, Mr. John Ronaldo, Mr. Teddy Chang and Dr. Michael Whittaker for their help about DLS and A/Prof. Ritha Khanna for her good advice.

I gratefully acknowledge Australian Nanotechnology and Nano Science Network (ANN), Australian X-ray Analytical Association (AXAA) and Australian Microscopy Microanalysis Society (ACMM) for encouraging me with awarding several student bursaries to present my work, especially Ms.Liz Micallef, Dr.Vanessa Peterson, Prof. Chennuputi Jagadish, Prof. Ajayan Vinu, Prof. Jin Zou and Prof. Graeme Auchterlonie.

I acknowledge all of my colleagues for their kind contribution: Fehmida, Mohammad, Arif, Tom, Trung, Maggi, Sanghun, Rob, Daniel, Roman, Mehandra, Boon, Madhuka, Rebecca, Eric and Jess. I would like to thank Mr.Abbas Barfidokht, Mr. Xiayn (Jet) Cheng, Mr. Adrian Plummer and Dr. Simone Ciampi for their good advice.

I would also like to thank all of my former trainers and supervisors in University of Tehran and the University of Bu-Alisina of Hammadan.

I am extremely thankful to Mrs. Anna Lumberou and her family who are the best friends for me and my family members. I am also grateful to all of my friends: Hamideh, Mitra, Roya, Maryam, Rima, Elham, Lida, Parisa and Leila for their kindness and support.

Finally, I would like to express my utmost appreciation to my mother for her endless love, affections, inspirations and dedications and also my brothers Dr. Ali and Dr. Reza Mir Najafi Zadeh for their kindness, encouragements, guidance and support. I would not be motivated enough to complete this research without their help and I would like to dedicate this thesis to them.

Fatemeh Mir Najafi Zadeh

August 2014

To: My Lovely Mother

And

My Kind Brothers

Foreword

The chapters of this thesis is based upon studies of synthesis and properties of water soluble, photostable and nontoxic QDs for applying in biological context, as summarized in the poem below :

“A Scientific poem about this research”

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Abstract

Semiconductor nanocrystals or quantum dots (QDs) have received a great deal of attention over the last decade due to their unique optical and physical properties, classifying them as potential tools for biological and medical applications. However, there are some serious restrictions to the bioapplications of QDs such as water solubility, toxicity and photostability in biological environments for both in-vivo and in- vitro studies. In this thesis, studies have focused on the preparation of highly luminescent, water soluble and photostable QDs of low toxicity that can then be potentially used in a biological context. First, CdSe nanoparticles were synthesized in an aqueous route in order to investigate the parameters affecting formation of nanoparticles. Then, water soluble CdSe(S) and ZnSe(S) QDs were synthesized. These

CdSe(S) QDs were also coated with ZnO and Fe2O3 to produce CdSe(S)/ZnO and

CdSe(S)/Fe2O3 core/shell QDs. The cytotoxicity of as-prepared CdSe(S), ZnSe (S) and CdSe(S)/ZnO QDs was studied in the presence of two cell lines: HCT-116 cell line as cancer cells and WS1 cell line as normal cells. Finally, CdSe(S) QDs were linked to Donkey-anti mouse (H+L) (IgG) antibody Cy3 fluorophore to prepare a CdSe(S)- antibody conjugated compound and the photostability of CdSe(S) QDs both after linking to antibody and in the presence of HCT-116 cells was investigated. The obtained QDs exhibited high crystallinity, water solubility, low toxicity and photostability, demonstrating that highly crystalline nanoparticles can be formed by sufficient control of the experimental parameters. It was determined that coating the CdSe(S) QDs with ZnO led to a reduced cytotoxicity of the CdSe(S) QDs. It was found that ZnSe(S) QDs have no cytotoxicity toward both HCT-116 and WS1 cell lines across all concentrations studied. Finally, the CdSe(S) QDs were found to be photostable both in a CdSe(S)-antibody conjugated compound and in the presence of HCT-116 cancer cells.

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Table of Contents

Title Page Thesis Dissertation Sheet ...... i Certificate of Originality ...... ii Copyright Statement ...... iii Authenticity Statement ...... iii Acknowledgments ...... iv Foreword ...... vii Abstract ...... viii Table of Contents ...... ix List of Figures ...... xvi List of Tables...... xx List of Abbreviations...... xxi

Chapter 1: Introduction ...... 1 1.1 Quantum Confinement Effect ...... 2 1.2 Electronic Structure of QDs ...... 3 1.3 Optical Properties of QDs ...... 5 1.4 Synthesis of QDs ...... 8 1.4.1 Physical Methods ...... 8 1.4.2 Gas Phase Syntheses ...... 8 1.4.3 Liquid Phase Synthesis ...... 8 1.4.3.1 Organic Route ...... 9 1.4.3.2 Aqueous Route ...... 11 1.5 Core/shell QDs ...... 13 1.6 Applications of QDs ...... 15 1.6.1 Industrial Applications ...... 15 1.6.2 Biological Applications ...... 17 1.6.2.1 QDs in Compare with Organic Fluorophores ...... 17 1.6.2.2 Bioimaging Applications of QDs ...... 19 1.6.2.3 QDs in Biosensors ...... 23 1.6.2.4 QDs in Drug Delivery and Therapies ...... 24

Table of Contents

Title Page 1.6.3 Limitations of Bioapplications of QDs ...... 25 1.7 Project Aims ...... 27 References ...... 29

Chapter 2: Experimental Procedures ...... 39 2.1 Materials ...... 39 2.2 Chemical Synthesis ...... 40 2.2.1 Synthesis of CdSe-thiol Capped Nanoparticles ...... 41 2.2.2 Synthesis of Water Soluble QDs ...... 44 2.2.2.1 Synthesis of NaHSe ...... 44 2.2.2.2 Synthesis of CdSe(S) QDs ...... 45 2.2.2.3 Synthesis of ZnSe(S) QDs ...... 45 2.2.3 Synthesis of Core/shell QDs ...... 46 2.2.3.1 Formation of CdSe(S)/ZnO QDs ...... 46

2.2.3.2 Formation of CdSe(S)/Fe2O3 QDs ...... 47 2.2.4 Separation of QDs from Aqueous Phase ...... 47 2.2.5 Cytotoxicity Assays ...... 47 2.2.5.1 Preparation of Aqueous Solutions of QDs ...... 48 2.2.5.2 Cell Culture ...... 48 2.2.5.3 Sample Preparation ...... 48 2.2.5.4 Determination the Viability of the Cells...... 49 2.2.6 Preparation of a QD-antibody Conjugated Compound ...... 51 2.2.6.1 Modification of CdSe(S) QDs ...... 51 2.2.6.2 Formation of QD-antibody Compound ...... 52 2.2.7 Photostability of CdSe(S) QDs in Presence of HCT-116 cells ...... 52 2.2.7.1 Sample Preparation ...... 52 2.2.7.2 Microscopy Techniques ...... 53 2.2.8 The Investigation of Photostability of CdSe(S) QDs in Cell Media ...... 53 2.2.8.1 Preparation of Cell Media Solution ...... 54 2.2.8.2 Spectral Properties of CdSe(S) QDs in Cell Media ...... 54 2.3 Characterization Techniques ...... 54 2.3.1 Powder X-ray Diffraction ...... 54

Table of Contents

Title Page 2.3.2 High Resolution Transmission Microscopy ...... 56 2.3.3 Optical Spectroscopy ...... 57 2.3.4 Fourier Transform Infra-red Spectroscopy ...... 57 2.3.5 Circular Dichroism Spectroscopy ...... 58 2.3.6 Elemental Analysis ...... 58 2.3.7 Dynamic Light Spectroscopy ...... 58 References ...... 59

Chapter 3: Synthesis of CdSe Nanoparticles (NPs) ...... 61 3.1 Introduction ...... 61 3.1.1 Nanoparticles Formation……………… ...... ……….61 3.1.2 Nanoparticle Structure…………………… ...... ……....63 3.1.3 Synthetic Methods…………………… ...... …………...65 3.2 Experimental Procedure ...... 67 3.2.1 Synthesis……………………… ...... ………………………….….67 3.2.2 Characterization…………………… ...... ……………...67 3.3 Results and Discussion ...... 67 3.3.1 Formation Mechanism……………… ...... ………….67 3.3.2 Formation of Cubic CdSe NPs under Different Conditions ...... 69 3.3.2.1 Formation of CdSe NPs in Acidic pH………… ...... ……………..69 3.3.2.2 Formation of CdSe NPs in Basic pH………………………… ...... …74 3.3.2.3 Formation of CdSe NPs in Presence of L-cysteine……………… ...... 78 3.3.2.4 Formation of CdSe NPs in Presence of Ethanol……………… ...... ….82 3.3.3 Formation of Hexagonal CdSe NPs…………………… ...... ….…84 3.3.4 Particle Size of As-prepared NPs……………………… ...... …87 3.4 Conclusion ...... 89 References ...... 90

Chapter 4: Synthesis of Water Soluble QDs...... 94 4.1 Introduction…………………………………………………………… ...... 94 4.1.1 Ligand Exchange Method……………………………………… ...... …...94 4.1.2 Silanization…………………………………………………… ...... …..96 xi

Table of Contents

Title Page 4.1.3 Surface Coating of QDs in Amphiphilic Polymers ...... 97 4.1.4 Direct Synthesis of QDs in Water ...... 97 4.2 Experimental Procedure ...... 99 4.2.1 Chemical Synthesis ...... 99 4.2.2 Characterization ...... 99 4.3 Results and Discussion………………………… ...... …………………..99 4.3.1 Synthesis of CdSe(S) QDs ...... 99 4.3.2 Synthesis of ZnSe(S) QDs ...... 112 4.4 Conclusion ...... 118 References ...... 118

Chapter 5: Synthesis of Water Soluble Core/shell QDs ...... 123 5.1 Introduction ...... 123 5.2 Experimental Procedure ...... 125 5.2.1 Chemical Synthesis ...... 125 5.2.2 Characterization ...... 126 5.3 Result and Discussion ...... 126 5.3.1 Synthesis of CdSe(S)/ZnO QDs ...... 126

5.3.2 Synthesis of CdSe(S)/Fe2O3 QDs ...... 134 5.4 Conclusion ...... 140 References ...... 141

Chapter 6: The Investigation of Cytotoxicity of QDs ...... 144 6.1 Introduction ...... 144 6.1.1 Production of Reactive Oxygen Species ...... 144 6.1.2 Immunotoxicity ...... 145 6.1.3 Effective Parameters on Cytotoxicity of QDs ...... 145 6.1.4 Short Term and Long Term Toxicity ...... 147 6.1.5 Control the Toxicity of QDs ...... 147 6.1.6 Toxicity Assays of QDs ...... 148 6.2 Experimental Procedure ...... 149

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Table of Contents

Title Page 6.3 Results and Discussion ...... 149 6.3.1 Cytotoxicity of QDs toward Cancer Cells ...... 150 6.3.1.1. Cytotoxicity of CdSe(S) QDs ...... 150 6.3.1.2 Cytotoxicity of Precursor Solutions toward HCT Cell Line ...... 151 6.3.1.3 Cytotoxicity of CdSe(S) QDs in Compare with Hsp-90 Inhibitor ...... 153 6.3.1.4 Cytotoxicity of Dialyzed CdSe(S) QDs ...... 153 6.3.1.5 Cytotoxicity of CdSe(S)/ZnO QDs ...... 154 6.3.1.6 Cytotoxicity of ZnSe(S) QDs ...... 155 6.3.2 Cytotoxicity of QDs toward Normal Cells ...... 156 6.3.2.1 Cytotoxicity of CdSe(S) QDs ...... 156 6.3.2.2 Cytotoxicity of CdSe(S)/ZnO QDs ...... 157 6.3.2.3 Cytotoxicity of ZnSe(S) QDs ...... 158 6.4 Conclusion ...... 160 References ...... 160

Chapter 7: The Investigation of Photostability of QDs in Biological Context ...... 164 7.1 Introduction ...... 165 7.2 Experimental Procedure ...... 165 7.2.1 Preparation of a QD-antibody Conjugated Compound ...... 165 7.2.1.1 Preparation ...... 165 7.2.1.2 Characterization ...... 165 7.2.2 Photostability of CdSe(S) QDs in Presence of HCT-116 Cells ...... 165 7.2.3 The Investigation of Photostability of CdSe(S) QDs in Cell Media ...... 165 7.3 Results and Discussion ...... 166 7.3.1 Optical Properties of the QD-antibody Conjugated Compound ...... 166 7.3.1.1 Conjugation Method ...... 167 7.3.1.2 Modification of QDs ...... 170 7.3.1.3 The Properties of As-prepared QD-antibody Conjugated Compound ...... 172 7.3.1.4 Comparison of Cy3 with QDs ...... 173 7.3.2 Photostability of CdSe(S) QDs in Presence of Cancer Cells ...... 174

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Table of Contents

Title Page 7.3.3 The Investigation of Photostability of CdSe(S) QDs in Cell Media ...... 176 7.4 Conclusion ...... 177 References ...... 177

Chapter 8: Conclusions& Future Works ...... 179 8.1 Conclusions ...... 179 8.2 Future Works ...... 180 References ...... 181

Appendix ...... -1- 1. Calculation of Bohr Radius ...... -1- 2. La Mer Theory ...... -1- 3. Calculation of the Number of MPA Molecules ...... -2- 4. Calculation of the Particle Size of CdSe NPs using PXRD Spectra ...... -4- 5. Estimation the Particle Size of CdSe NPs & CdSe(S) QDs from HRTEM Images .. -5- 6. Calculation of the Band Gap Energy of CdSe NPs ...... -6- 7. UV Spectrum of As-synthesized CdSe NPs in Acidic pH ...... -7- 8. Electronic Transitions for ZnSe Nanocrystals ...... -8- 9. PXRD Patterns of Cubic CdSe NPs under Different Conditions ...... -8- 10. Calculation of the Particle Size of QDs ...... -9- 10.1 Particle Size of CdSe(S) QDs ...... -9- 10.2 Particle Size of ZnSe(S) QDs ...... -9- 11. The Correspondence of Planes in PXRD and SAED Patterns ...... -10- 12. Atomic Percentage of Elements in CdSe(S) QDs ...... -11- 13. Calculation of the Particle Size of Core/shell QDs using PXRD ...... -11- 13.1 Particle Size of CdSe(S)/ZnO QDs ...... -11-

13.2 Particle Size of CdSe(S)/ Fe2O3 QDs ...... -12-

14. XPS Standard Spectrum of α-Fe2O3...... -12-

15. XPS Spectra of CdSe(S) /Fe2O3 Core/shell QDs ...... -13- 16. Optical Properties of Cy3 ...... -14- 17. Photostability of As-synthesized CdSe(S) QDs ...... -14- References ...... -15- xiv

Table of Contents

Title Page List of Publications ...... I

List of Figures

Chapter 1 Figure 1. 1. A CdSe QD ...... 1 Figure 1.2. Electron confinement effect in QDs ...... 3 Figure 1. 3. Electronic structure of a CdSe QD compared to a bulk semiconductor ...... 4 Figure 1.4. Size-tunable optical properties of CdSe QDs ...... 6 Figure 1.5. The size-dependent unique optical properties of CdSe/ZnS QDs ...... 7 Figure 1.6. Schematic of organometalic synthesis of QDs ...... 10 Figure 1.7. Synthesis of CdTe-thiol capped QDs in an aqueous route ...... 12 Figure 1.8. The structure of CdSe/ZnS core/shell QDs ...... 14 Figure 1.9. Improving efficiency in solar cells using PbSe QDs ...... 16 Figure 1.10. Labelling of surface and intracellular targets with QDs Probes ...... 20 Figure 1.11. In vivo targeting of hepartocarcinoma with QD-AFP-Ab probes ...... 21 Figure 1.12. Fluorescence micrograph of a mixture of CdSe/ZnS QDs ...... 22 Figure 1.13. Schematic diagram of the QDs-based FRET maltose sensor ...... 24 Figure 1.14. The steps of formation of this thesis ...... 28

Chapter 2 Figure 2.1. Schematic of formation of core/shell QDs ...... 46 Figure 2.2. Image of a 96-well plate ...... 49 Figure 2.3 Structures of WST-8 and WST-8 formazan ...... 50 Figure 2.4 Principle of the cell viability detection with cell counting kit-8 ...... 50 Figure 2.5 Schematic of EDC coupling ...... 51 Figure 2.6. Schematic of a PXRD diffractometer ...... 55

Chapter 3 Figure 3.1. The steps of CdSe nanoparticles formation ...... 62 Figure 3.2. The crystal structures in WZ and ZB ...... 64 Figure 3.3. PXRD patterns of ZB and WZ of CdSe NPs ...... 65 Figure 3.4. The structures of 3-mercaptopropionic acid and L-cysteine ...... 68 Figure 3.5. The structure of MPA-dithio complex ...... 68

xvi

List of Figures

Figure 3.6. PXRD of CdSe NPs in acidic pH ...... 70 Figure 3.7. TEM images of CdSe NPs in acidic pH ...... 71 Figure 3.8. UV-visible spectrum of CdSe NPs in acidic pH ...... 72 Figure 3.9. FT-IR spectra of MPA and CdSe-MPA capped NPs...... 73 Figure 3. 10. PXRD of CdSe NPs in basic pH ...... 75 Figure 3.11. TEM images of CdSe NPs in basic pH ...... 76 Figure 3.12. UV-visible spectra of CdSe NPs in basic pH ...... 77 Figure. 3.13. PXRD of CdSe NPs in presence of L-cysteine and MPA ...... 79 Figure 3.14. TEM images of CdSe NPs in presence of both L-cysteine and MPA ...... 80 Figure 3.15. UV-visible spectrum of CdSe NPs in presence of L-cysteine ...... 81 Figue 3.16. PXRD of CdSe NPs in presence of both ethanol and water ...... 82 Figure 3.17.TEM images of CdSe NPs in presence of both ethanol and water ...... 83 Figure 3.18. UV-visible spectrum of CdSe NPs in presence of ethanol ...... 84 Figure 3.19. PXRD pattern of hexagonal CdSe NPs ...... 85 Figure 3.20. TEM images of hexagonal CdSe NPs ...... 86 Figure 3.21. UV-visible of hexagonal NPs ...... 87 Figure 3.22. Correlation of particle size and Eg in as-synthesized NPs ...... 89

Chapter 4 Figure 4.1. Ligand exchange method ...... 95 Figure 4.2. CdSe/ZnS QDs with DHLA ...... 96 Figure 4.3. The synthesized QDs under UV light ...... 98 Figure 4.4. HRTEM of spherical CdSe(S) QDs ...... 101 Figure 4.5. Vertical atomic planes in CdSe(S) QDs ...... 101 Figure 4.6. PXRD from amorphous CdSe-MPA yellow complex and CdSe QDs ...... 102 Figure 4. 7. PXRD of CdSe QDs in different reaction times ...... 103 Figure 4.8. SAED pattern of CdSe(S) QDs ...... 104 Figure 4.9. Optical spectra of CdSe(S) QDs after 1h ...... 104 Figure 4.10. FT-IR spectra of MPA and CdSe(S) QDs ...... 106 Figure 4.11. XPS spectra of CdSe(S) QDs ...... 107 Figure 4.12 Size distribution histogram of CdSe(S) QDs ...... 108 Figure 4.13. PL spectra CdSe(S) QDs prepared with conventional heating up ...... 109 Figure 4.14. PL spectra of CdSe(S) QDs in different hydrothermal reaction times ..... 110 xvii

List of Figures

Figure 4.15. PL spectra of CdSe(S) QDs in different molar ratios of Cd to MPA ...... 111 Figure 4.16 PXRD pattern of ZnSe(S) QDs ...... 113 Figure 4.17. Optical spectra of ZnSe(S) QDs ...... 114 Figure 4. 18. HRTEM images of ZnSe(S) QDs ...... 115 Figure 4.19. XPS spectra of ZnSe(S) QDs ...... 116 Figure 4.20. FT-IR spectra of ZnSe(S) QDs ...... 117

Chapter 5 Figure 5.1. Different types of core/shell QDs ...... 124 Figure 5.2. PXRD of CdSe(S)/ZnO core/shell QDs ...... 127 Figure 5.3. TEM image of CdSe(S)/ZnO core/shell QDs ...... 128 Figure 5.4 Optical spectra of CdSe(S)/ZnO core/shell QDs ...... 129 Figure 5.5. XPS spectrum of CdSe(S) QDs ...... 130 Figure 5.6. XPS surveys of CdSe(S) QDs and CdSe(S)/ZnO core/shell QDs ...... 130 Figure 5.7. XPS spectra of CdSe(S)/ZnO core/shell QDs ...... 131 Figure 5.8. EDS spectrum of CdSe(S)/ZnO core/shell QDs ...... 132 Figure 5.9. PL spectra of CdSe(S) QDs & CdSe(S)/ZnO core/shell QDs ...... 133

Figure 5.10. PXRD of CdSe(S)/Fe2O3 core/shell QDs ...... 134

Figure 5.11. SAED pattern of CdSe(S)/Fe2O3 core/shell QDs ...... 135

Figure 5.12. HRTEM image of spherical CdSe(S)/Fe2O3 core/shell QDs ...... 136

Figure 5.13. HRTEM images of CdSe(S)/Fe2O3 core/shell QDs ...... 137

Figure 5.14. Optical spectra of CdSe(S)/Fe2O3 core/shell QDs ...... 138

Figure 5.15. XPS spectrum of CdSe(S)/Fe2O3 core/shell QDs ...... 139

Figure 5.16. XPS survey of CdSe(S)/Fe2O3 core/shell QDs ...... 140

Chapter 6 Figure 6.1. Interaction of ROS with cells ...... 145 Figure 6.2. Nanoparticles toxicity in major organs ...... 146 Figure 6.3. Cytotoxicity of CdSe(S) QDs toward HCT-116 cell line ...... 150 Figure 6.4. Cytotoxicity of Cd-MPA and Cd-Se-MPA toward HCT-116 cell line ..... 152 Figure 6.5. Cytotoxicity of dialyzed CdSe(S) QDs...... 153 Figure 6.6. Cytotoxicity of CdSe(S)/ZnO QDs toward HCT-116 cell line ...... 154 Figure 6.7. The effect of free cadmium ion on the liver cells ...... 155 xviii

List of Figures

Figure 6.8. Cytotoxicity of ZnSe(S) QDs toward HCT-116 cells ...... 156 Figure 6.9. Cytotoxicity of CdSe(S) QDs toward WS-1 cells ...... 157 Figure 6.10. Cytotoxicity of CdSe(S)/ZnO QDs toward WS-1 cells ...... 158 Figure 6.11 Cytotoxicity of ZnSe(S) QDs toward WS-1 cells ...... 159

Chapter 7 Figure 7.1. Schematic of applying secondary antibodies to detect antigens ...... 167 Figure 7.2. The steps of formation of CdSe(S)-antibody conjugated compound ...... 169 Figure 7.3. XPS spectrum of the modified CdSe(S) QDs ...... 170 Figure 7.4. PL spectra of CdSe(S) QDs and the modified CdSe(S) QDs ...... 171 Figure 7.5. UV-visible spectra of CdSe(S) QDs and the modified CdSe(S) QDs ...... 171 Figure 7.6. Optical spectra of as- prepared CdSe(S) QD-antibody compound ...... 172 Figure 7.7. CD spectra of as- prepared CdSe(S) QD-antibody compound ...... 173 Figure 7.8. Confocal image of as-prepared CdSe(S) QDs ...... 174 Figure 7.9. Confocal image of HCT-116 fixed cells-CdSe(S) QDs ...... 175 Figure 7.10. Confocal image of HCT-116 live cells-CdSe(S) QDs ...... 175 Figure 7.11. PL spectra of CdSe(S) QDs in cell media ...... 176

Appendix Figure 1. La Mer plot ...... -2- Figure 2. Schematic of complete coating of a CdSe nanoparticle with MPA...... -4- Figure 3. UV-visible spectrum of CdSe nanoparticles in acidic pH after reflux ...... -7- Figure 4. Molecular energy levels for a ZnSe nanoparticle ...... -8- Figure 5. PXRD of cubic CdSe nanoparticles ...... -9- Figure. 6. PXRD and SAED pattern of as-synthesized CdSe(S) QDs ...... -10-

Figure 7. XPS standard spectrum of Fe in α-Fe2O3 ...... -12-

Figure 8. XPS spectra of CdSe(S)/ Fe2O3 core/shell QDs ...... -13- Figure 9. Photostability of CdSe(S) QDs over the time ...... -14-

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List of Tables

Chapter 2 Table 2.1. list of the required materials...... 39 Table 2.2. Preparation of cubic CdSe NPs in different conditions ...... 43

Chapter 3 Table 3.1. A comparison between formation of CdSe NPs in presence of L-cysteine and without L-cysteine ...... 81 Table 3.2. Calculation of the particle size of as-synthesized CdSe NPs ...... 88

Chapter 4 Table 4.1. The effect of molar ratio of Cd to MPA on optical properties of CdSe(S) QDs ...... 110

Chapter 6 Table 6.1. A comparison of cytotoxicity and stability of as-synthesized CdSe(S) QDs & CdSe-TOPO capped QDs ...... 151 Table 6.2. Summary of Cytotoxicity of QDs ...... 160

Appendix Table 1. Elemental analysis of CdSe(S) QDs………………………………………...-11- Table 2. Optical properties of CY3 ...... -14-

List of Abbreviations

Ab ...... Antibody AFP ...... Alpha Feto Protein CdSe ...... Cadmium Selenide CdS ...... Cadmium Sulfide CdTe ...... Cadmium Telluride CVD ...... Chemical Vapour Deposition CD ...... Circular Dichroism CB ...... Conductive Band DLS ...... Dynamic Light Scattering DHLA ...... Dihydrolipoic Acid EG ...... Energy Gap EPR ...... Enhanced Retention Permeability EDS ...... Energy Dispersive X-ray Spectroscopy FT-IR ...... Fourier Transform Infra-red FFT ...... Fast Fourier Transform FRET ...... Forster Resonance Energy Transfer HgTe ...... Mercury Telluride HRTEM ...... High Resolution Transmission Electron Microscopy HOMO ...... Highest Occupied Molecular Orbital HSP ...... Heat Shock Protein InP ...... Indium Phosphide LUMO ...... Lowest Unoccupied Molecular Orbital MBP ...... Microphage Bone Protein MPA ...... Mercapto Propionic Acid MPTS ...... Mercapto Propyl Trimethoxy Silane NaHSe ...... Sodium Hydrogen Selenide NPs ...... Nanoparticles OLEDs ...... Organic Light Emitting Diodes PbSe ...... Lead Selenide PL ...... Photoluminescence

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List of Abbreviations

PXRD ...... Powder X-ray Diffraction QDs ...... Quantum Dots ROS ...... Reactive Oxygen Species SAED ...... Selected Area Electron Diffraction TEOS ...... Tetra Ethyl Ortho Silicate TGA ...... Thio Glycolic Acid

TiO2 ...... Titanium Oxide TOPO ...... Tri Octyl Phosphine Oxide TOP ...... Tri Octyl Phosphine SK ...... Stranski Krastanov VLS ...... Vapour Liquid Solid VB ...... Valance Band WZ...... Wurtzite XPS ...... X-ray Photon Spectroscopy ZB ...... Zincblende ZnSe ...... Zinc Selenide ZnS ...... Zinc Sulfide

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Chapter1

Introduction

Semiconductor nanoparticles, or quantum dots (QDs), were introduced to the world of nanotechnology in the early 1980s by two independent researchers; Alexi Ekimov who prepared CuCl microcrystals in a glass matrix [1, 2] and Louis Brus who observed size effects in the excited electronic properties of CdS nanocrystals in colloidal solutions [3].

A QD is a semiconductor nonocrystal typically consisting of elements in groups II-VI or III-V or IV-VI of the periodic table, with only 1000 to 100000 atoms and as such lie in the size range of 1-10 nm [4, 5]. Figure 1.1 shows a CdSe QD [6].

Figure 1. 1. A CdSe QD: the blue represents cadmium, the yellow represents selenium and the red represents a cloud of electrons in their excited state [6].

At this length-scale, surface to volume ratios of materials become large and their electronic energy states become discrete, resulting in electrons exhibiting quantum confinement [7, 8].

Chapter 1 Introduction

1. 1 Quantum Confinement Effect

A bulk semiconductor has a filled valance band (VB) separated from a largely empty conduction band (CB) by an energy gap (Eg). When a semiconductor is excited with light in the near infrared (IR) or UV-visible spectrum, the photonic energy is greater than Eg and so electrons are excited across the band gap from the VB to the CB, leaving a positively charged hole behind. This hole behaves as a particle with its own charge and mass in the solid. Electrostatic Columbic interactions then result in bound states between the generated hole and the excited electron, known as excitations. The average distance between the electron in the CB and the hole that it leaves behind in the VB is called Bohr excitation radius, which is calculated using Bohr model (Appendix 1) [7-9].

In QDs, the diameter of the nanocrystal is smaller than the size of the Bohr excitation radius for the material and rather like the energy levels of an atom, the bound state energy levels are quantized according to Pauli’s exclusion principle. The confinement of the electrons in a relatively small volume, physically a quantum box, leads to the phenomenon known as quantum confinement [10]. One outcome of quantum confinement in QDs is that the band gap increases in energy with decreasing particle size; this is analogous to the quantum mechanical particle in a box model in which the energy states of the trapped particle decrease in magnitude as the size of the box increases [11].

Figure 1.2 shows quantum confinement effect in QDs compared with a bulk semiconductor [12].

Chapter 1 Introduction

(a) (b) (c)

Figure 1.2. Electron confinement effect in QDs: discrete energy states in QDs (a, b), continuous energy levels in bulk semiconductors (c). Diagram also shows that

Eg decreases when particle size increases [12].

1. 2 Electronic Structure of QDs

QDs have a very specific electronic structure. In bulk semiconductors, the atomic orbitals overlap to form continuous energy levels in both the VB and CB. Electrons then occupy all of the states up to the edge of the VB, whilst the electronic states in the CB are almost empty and only occupied by thermal excitation across Eg. In semiconductor QDs, the energy states are quantized due to quantum confinement effects and can be labelled with atom-like nominations, such as 1S, 1P and 1D. This electronic structure is more similar to that of atoms than bulk semiconductors and as such, QDs have also been referred to as “artificial atoms” [13, 14].

Chapter 1 Introduction

Indeed, the electronic structure of QDs can be considered to be intermediate between that of bulk semiconductors and discrete atoms or molecules; in bulk semiconductors, the electrons lie in bands, whilst in molecules electrons exist in bonds or molecular orbitals. In QDs, the electronic structure is an intermediate regime between bands and bonds, whilst the band gap energy can be considered to be analogous to the HOMO- LUMO energy of molecules [7]. Figure 1.3 gives a schematic overview of the electronic structure of QDs.

Figure 1. 3. Electronic structure of a CdSe QD compared to a bulk semiconductor and its analogous molecule [7, 13].

These electronic states and their corresponding energy levels can be interpreted using a spherical quantum well model. In this model, wave functions corresponding to the S, P, D,…,etc. states originate from the associated Schrödinger equations, and the energy of electronic states can be calculated as:

ℏ2푛2 퐸푛,푙 = 2 2 (Equation 1.1) 8 휋 푚푒,ℎ 푑

Where n and l are quantum numbers, ℏ is Planck’s constant, d is the diameter of the nanoparticle and m is the mass of an electron or hole. In QDs, when an excited electron vacates the VB, transferring to the CB, the electronic levels are more separated in

Chapter 1 Introduction

energy than the hole levels because the electron mass is much smaller than effective mass of the hole [15].

There are many possible electronic transitions between various energy levels in the QDs, however, it is not possible to observe all of them because the wave functions have different quantum numbers and some of them are orthogonal and not accessible [15]. However, some of the electronic transitions are observable as fluorescence, classifying QDs as one of the most remarkable of optical materials.

1. 3 Optical Properties of QDs

QDs have unique optical properties that include narrow emission and wide absorption spectra, large extinction coefficients, high fluorescence quantum yields and photostability [16]. When a photon has an excitation energy exceeding the semiconductor band gap, the QD may absorb the photon, creating an excited electron in the CB, resulting in a wide-band absorption, usually in the UV-visible region. The excited electron can relax to its ground state by the emission of another photon with energy equal to the band gap, resulting in a narrow and symmetric emission known as photoluminescence (PL) [11, 17].

The optical properties of QDs are size dependent due to the effects of quantum confinement. As the particle size decreases, the energy levels become more discrete and both absorption and emission occur at shorter wavelengths as the energy between the HOMO and LUMO in large QDs is less than the same electronic transition in smaller QDs [18, 19].

This quantum confinement effect can be physically observed as a red shift in the emission and absorption spectra for QDs as the diameters grow larger (Figure 1.4).

Chapter 1 Introduction

(A)

(B)

Figure 1.4. Size-tunable optical properties of CdSe QDs with sizes ranging from 2.2 to 7.3 nm: fluorescence emission (A) and absorption spectra (B) [20].

Chapter 1 Introduction

Therefore, as the difference in energy between the discrete ground and excited states

(Eg) increases with decreasing particle size, the colour of emitted light can be fine-tuned by adjusting the size of the QDs [21]. The size-dependent optical properties of QDs can be observed when exposed to UV light because the dots fluoresce with a colour determined by the dot size (Figure 1.5) [18, 22].

Figure 1.5. The size-dependent unique optical properties of CdSe/ZnS QDs [18, 22].

Chapter 1 Introduction

1.4 Synthesis of QDs

There are different approaches to the synthesis of QDs such as physical methods, gas phase syntheses and liquid-phase colloidal synthesis of QDs.

1.4.1 Physical Methods

Semiconductor nanoparticles can be produced by breaking down bulk semiconductors using attrition or milling to produce nanoparticles. The advantage of this method is the production of a large quantity of nanoparticles, however it suffers from a relatively large heterogeneity of the products. Moreover, the structure of the obtained nanoparticles can be damaged and the particles might contain significant levels of impurities due to the milling medium [23, 24].

1.4.2 Gas Phase Syntheses

QDs can be synthesized using gas phase syntheses including the vapour-liquid–solid (VLS), chemical vapour deposition (CVD) and thermal evaporation methods. These syntheses are based upon Stranski-Krastanov (SK) growth and utilize epitaxial growth of thin–films on a crystal surface to form three-dimensional nanomaterials, including semiconductor nanocrystals. SK growth occurs when there is a large lattice mismatch between the substrate and the growth layer, but with same lattice structure. These methods are mainly used to produce QDs in industrial equipment such as transistors, detectors and electronic devices. The limitations of this method are a lack of fabrication and controlled deposition of individual dots [25-29].

1.4.3 The Liquid Phase Colloidal Synthesis

The colloidal method of synthesis is a chemical approach that yields uniform nanocrystals of controlled particle size, simply by varying the reaction conditions. In colloidal synthesis, precursors are allowed to react together in solution to produce the QD materials as colloids. This method has two general synthetic routes: those occurring in organic or aqueous media [30, 31].

Chapter 1 Introduction

1.4.3.1 Organic Route

1.4.3.1.1 Organometalic Synthesis

QDs can be obtained after mixing the precursors in organic coordinating solvents at high temperatures (T = 200-300 ºC) under an inert atmosphere. The selection of appropriate coordinating solvents is an important parameter to obtain high quality QDs due to the aggregation of QDs in inappropriate solvents. The organic solvent can also act as a surfactant and the QDs are synthesized after careful control of experimental parameters such as precursor injection rate, temperature and concentration. This approach was first established by Bawendi and co-workers to synthesise high quality CdE (E = S, Se, Te) QDs using tri-n-octyl phosphine (TOP) and tri-n-octyl phosphine oxide (TOPO) as solvents. Then, the method was developed further by other researchers to produce a wide range of QDs including ZnS, CdS, ZnSe and InP nanocrystals [18, 31-35].

QD seed nuclei form after rapid injection of organometallic precursors to the coordinating organic solvents at high temperature and the growth of nanoparticles occurs after aging the reaction at lower temperatures. Therefore, the average size and size distribution can be adjusted by controlling the growth steps over different temperatures and reaction times [36, 37].

The advantages of this method are the high crystallinity and monodispersity of the obtained nanoparticles. Indeed, as-synthesized nanocrystals can be dispersed in organic solvents and exhibit quantum confinement [24]. Figure 1.6 shows a schematic of this method.

Chapter 1 Introduction

(b) (a)

Coordinating organic solvent

stabilizer at 150-350 ºC

Figure 1.6. Schematic of organometalic synthesis (a) & formation of homogenous nanocrystals (b) [29].

However, the organometallic pathway has some inherent disadvantages, such as difficulties in controlling the particle size due to rapid growth rates of nanocrystals. In addition, the obtained nanoparticles are often toxic and insoluble in water as major limitations in their potential usage in bioapplications [38, 39]. QDs obtained by this approach require additional steps to render them water soluble, as described in Chapter 4, Sections 4.1.1 to 4.1.3.

1.4.3.1.2 Solvothermal Method

Solvothermal synthesis is a commonly-used method to produce inorganic nanoparticles including QDs. This method involves the use of organic solvents at higher temperatures than the nominal boiling points under increased pressure; it often results in an increase in reactivity of reactants and an improvement of nanoparticle growth. Reaction parameters including pH, concentration, temperature and time influence the particle size and size distribution [23, 40, 41]. This method has been successfully used to produce various nanocrystals such as CuInS2, CdS, SnO2, CeO2, Ag2 Se and CdSe QDs [42-48].

However, the obtained nanoparticles are not soluble in water and precise control of the particle size is difficult, similar to the organometallic method. 10

Chapter 1 Introduction

1.4.3.2 Aqueous Route

Aqueous synthesis of QDs is an alternative approach to organic synthesis. The method is based upon using hydrophilic stabilizing thiols or phosphates as capping agents to produce QDs in water as a nontoxic and inexpensive solvent. This method has been widely reported and is now a common approach to produce a wide range of nanoparticles including CdSe, CdS, CdTe , HgTe, CdTe/HgTe and ZnSe QDs [49-57].

The first aqueous synthesis of QDs was reported in 1993 by Rajh and co-workers when they synthesized CdTe nanoparticles using hexametaphosphate as a hydrophilic capping agent and water as the solvent [56]. The method was improved upon by Rogach in 1996 by replacing 2-mercaptoethanol and thioglycerol as thiol capping agents and to produce CdTe QDs [57].

In the aqueous synthetic method, the separation of nucleation and growth steps is similar to that of the organometallic method first established by Bawendi. Initially nucleic QD particles form upon reaction between the capping agent and reactants, which then grow through thermal treatment of the reaction system [58, 59]. For example, formation of CdTe-thiol capped QDs occurs in two steps: the first step involves introducing H2Te gas to an aqueous solution of Cd(ClO4) and a thiol capping agent under an inert gas to form a CdTe QDs precursor solution and the second step is the particle growth through refluxing the initial precursor solution, as shown in Figure 1.7 [59].

Chapter 1 Introduction

Figure 1.7. Synthesis of CdTe-thiol capped QDs in an aqueous route: formation of CdTe precursor solution (I) & the growth of QDs after heating the aqueous precursor solution (II) [59].

The aqueous approach can be achieved using hydrothermal and microwave assisted method.

1.4.3.2.1 Hydrothermal Method

Similar to the solvothermal method, this synthetic route is based upon taking advantage of the high reactivity of the initial QD precursor solution at high temperature and pressure. The aqueous QD precursor solution forms after mixing the reactants in the presence of hydrophobic capping agents and growth occurs upon hydrothermal treatment. Hydrothermal synthesis produces monodisperse particles and is usually carried out below the supercritical point of water [23, 60, 61].

This approach, has been extensively reported to synthesise both bulk particles and nanomaterials such as metal oxides, metal sulfides, magnetic nanoparticles and a wide range of QDs including InP, CdSe , CdS and CdTe QDs [61-67].

The advantages of this method are the enhancement of the reaction kinetics, production of pure and homogenous materials whilst experimental parameters influence particle

Chapter 1 Introduction

size and the phase formation of products. Therefore, finding optimal reaction conditions restricts formation of high quality nanoparticles [40, 68].

1.4.3.2.2 Microwave Assisted Method

Microwave dielectric heating has been adopted as a new technique in various fields such as plasma chemistry, analytical chemistry, organic reactions, chemical catalysis and also the synthesis of high quality QDs [69, 73-78]. This method of synthesis has some advantages such as the production of uniform nanoparticles, the enhancement of reaction selectivity and increased reaction kinetics. Thermal gradient effects are effectively reduced due to the volumetric heating of microwaves, which is favourable to realizing homogenous heating and providing more control of the size distribution [70- 72]. As the polarity of solvents and precursors are different and the polar materials absorb microwave energy preferentially, the growth process can be achieved by careful selection of the reaction system such as precursors, capping agent, time, power and temperature. Microwave heating has been used to produce high quality nanocrystals such as CdS, ZnSe, CdSe, ZnS and CdSe/ZnS QDs via an aqueous route [73-77].

In all aqueous synthetic methods of QDs, using water instead of organic solvents is the main advantage of aqueous synthetic route compared to organic routes, whilst the obtained nanoparticles had poor crystallinity and weak optical properties when compared with the QDs synthesized in organic solvents [78].

1.5 Core/shell QDs

The cores of core/shell QDs have a high surface area to volume ratio with unsaturated bonds or dangling bonds existing on their surface. These atoms are under coordinated, making them more active than those in the bulk materials. Therefore, core QDs can be coated by appropriate materials and form core/shell QDs [79-81]. Core/shell QDs exhibit higher quantum yield and more stability than core QDs because the shell growth confines the excitation to the core and protects the core against oxidation and degradation [82-84].

Epitaxial shell growth processes, necessitate the selection of suitable shell materials to allow the formation of core/shell QDs with improved optical properties. For example, 13

Chapter 1 Introduction

ZnS is a suitable shell material for over coating of CdSe core QDs because the small lattice mismatch between ZnS and CdSe prevents a change of crystal structure, minimizes surface-defects and leads to increased quantum yields in CdSe/ZnS core/shell QDs. [85, 86].

Control of the thickness of the shell over the nanoparticle cores is another important parameter in core/shell QDs because a thick shell induces surface defects that result in reduced photoluminescence [87].

Figure 1.8 shows the structure of a CdSe/ZnS core/shell QD [88].

Figure 1.8. The structure of CdSe/ZnS core/shell QDs [88].

Core/shell QDs are mostly synthesized in two steps: first, the synthesis of the core QDs and then overcoating of these QDs through shell growth reactions [89]. The epitaxial shell growth process can occur in organic or aqueous synthetic methods [90-100].

The organic shell growth is based upon addition of organic shell precursor to a solution of colloidal QDs using organic solvents to produce core/shell QDs such as CdSe/CdS, CdSe/ZnS, CdS/ZnS and CdTeSe/CdZnS QDs [90-94]. For instance, Bawendi and co- workers reported formation of CdSe/ZnS QDs after injection of Diethylzinc (ZnEt2) and 14

Chapter 1 Introduction

hexamethyldisilathiane ((TMS)2S) as zinc and sulfur precursors in a solution of tricyclophosphine oxide (TOPO) ,tricyclophosphine (TOP) and CdSe QDs at high temperature. The overcoated QDs showed an improvement in quantum yield of 50 % at room temperature [90]. However, the core/shell QDs synthesized in organic synthetic route are soluble only in nonpolar solvents such as hexane and they are not soluble in water.

In addition to producing core/shell QDs via organic routes, core/shell QDs can be formed via aqueous synthetic methods. The process of shell growth in an aqueous route occurs by adjusting experimental parameters such as temperature, pH and reaction time in the presence of QDs through heating up approach, hydrothermal process or microwave assisted method to produce core/shell QDs [95-101].

The obtained core/shell QDs are water soluble but similar to the synthesis of core QDs in aqueous media, poor crystallinity and weak optical properties of the nanoparticles are disadvantageous of shell growth processes in the aqueous route, due to the fact that high quality core/shell QDs can be obtained by the overcoating of highly crystalline core QDs.

1.6 Applications of QDs

1.6.1 Industrial Applications

The unique optical properties of QDs promotes their usage in a wide range of photonic applications in various optoelectronic devices such as solar cells, photodetectors, photorefractives, organic light emitting diodes (OLEDs) and field-effect transistors [102-110].

The size dependent optical properties of QDs enable them to be used in various optoelectronic devices. The tunable optical properties of QDs in light-sensitive layers of optical equipment enable the formation of electroluminescence devices. Therefore, QDs can be potentially used in flat panel TV screens, digital cameras and mobile phones. As QDs emit in different observable colours, they provide a wider range of colours with higher intensity, more brightness, and lower contrast than traditional LEDs and LCDs.

Chapter 1 Introduction

These optical properties also promote usage of QDs in QD-displays as a new technology [102-104, 111, 112].

QDs can be incorporated in organic polymers for use in optical devices such as solar cells. Utilization of conjugated polymers provides greater light efficiency in devices than using available traditional semiconductors. QDs can be used to limit optical losses, thereby enhancing the overall efficiency [106-108]. For example, QDs can be utilized in solar cells as sensitizers to improve the photo-efficiency of solar cells by maximizing photo-induced charge separation and electron transfer processes [109]. As Figure 1.9 shows, QDs can be used in solar cells to absorb light in the visible spectrum and inject excited electrons into large band gap semiconductors such as TiO2 and SnO2, resulting in the enhancement of light conversion processes in photovoltaic cells [109, 113].

Figure 1.9. Improving efficiency in solar cells using PbSe QDs.

The advantages of utilizing QDs in optical devices include ease of fabrication, low cost and flexible substrate capability [110].

Chapter 1 Introduction

Another example of industrial applications of QDs is the development of high-speed optical devices for telecommunication applications. In such devices, the replacement of traditional semiconductors with QDs allows the use of basic quantum mechanical effects to control microscopic material properties relevant to device applications, resulting in enhanced power telecom devices such as high speed semiconductor amplifiers and high power QD laser devices [114, 115].

1.6.2 Biological Applications

The first bioapplications of QDs was reported by two independent research groups in 1998 when Alivisators and his group used CdSe/CdS QDs as fluorescent probes for labelling 3T3 mouse fibroblast cells and at around the same time, Nie and co-workers reported utilizing CdSe/ZnS QDs as detection labels by coupling of QDs with biomolecules [116, 117]. Since these works, QDs have been widely used as a new powerful class of tools for biological and medical applications in bioimaging, biosensing, biodetection, cell tracking, biomolecule labelling, drug delivery and therapy in both research and diagnostic environments [118-125].

1.6.2.1 QDs in Compare with Organic Fluorophores

Organic fluorophores or chromophores are commonly used in fluorescence techniques to detect or label biological components in biodetection or bioimaging for the analysis of biological samples. These are typically small organic dyes or metal-ligand complexes that can re-emit light upon light excitation [126]. In comparison to organic dyes, QDs have distinct optical properties that are not typical for organic fluorophores, including higher photostability, larger absorption cross-section and longer decay lifetimes than organic fluorophores [127,128]. Additionally, as previously described, QDs have size- dependent optical properties, narrow emission and wide absorption spectra as opposed to organic dyes. Moreover, QDs can undergo the phenomenon known as Förster resonance energy transfer (FRET), which is widely used in the development of biosensors and detection assays [122, 129].

I) Photostability: QDs are more stable than organic chromophores against photo bleaching because the cycles of excitation and fluorescence emission can be repeated in

Chapter 1 Introduction

QDs for hours, with high levels of brightness and minimum photobleaching, as opposed to organic dyes which bleach after a few minutes on exposure to light. For example, QDs are more stable than Alexa 488 which is reported as one of the most stable organic dyes. The high stability of QDs allows them to be used in long-time monitoring of labelled biomolecules, which is important in many cell labelling studies [22, 4, 128].

II) Absorption cross-section: QDs have large absorption cross-sections that make them at least 10 times brighter than organic fluorescent dyes. For example, QDs are 20 times brighter than rhodamin 6G, a common organic dye [117].

III) Decay lifetime: QDs have a longer decay lifetime at room temperature (30 to 100 ns) compared to the decay lifetime of organic dyes, which is less than 5 ns. This long decay lifetime prevents a reduced signal to noise ratio, which is always observed by utilizing organic dyes due to having fast florescence that causes an autofluorescence background from many naturally occurring species. In contrast, QDs re-emit light slower than autofluorescence background due to having longer decay lifetime, enhancing the intensity of the fluorescence signal [128, 130, 131].

IV) Excitation spectrum: traditional organic dyes have a narrow excitation spectrum and can be only excited by light of specific wavelengths, whilst QDs can be excited by light of a wide range of wavelengths due to their inherently broad excitation spectra. Therefore, multicolour QDs can be excited by a single wavelength of light, making them ideal for bioimaging [22, 132].

V) Emission spectrum: organic fluorophores have broad emission spectra and can overlap to a large extent, leading to a limitation of the number of fluorescence probes that can be used to tag different biomolecules. In contrast, QDs have narrow and symmetric emission that can be controlled by particle size, surface chemistry and size distribution that make them suitable for use in bioimaging [22, 133-135]. In addition, the narrow emission spectra of QDs enables their usage in barcoding of specific analytes including DNA and miRNA barcode analysis, another advantage of using QDs compared to organic dyes [136].

Chapter 1 Introduction

1.6.2.2 Bioimaging Applications of QDs

QDs have been widely used as fluorescence probes in bioimaging in both in vivo and in vitro including cellular labelling [119, 120, 123], detection of primary tumours [137, 121], detection of biomolecules [22, 133, 135] and tracking the drugs in animal cells [125].

1.6.2.2.1 Cellular Labelling

Cellular labelling is an important method to investigate phenotypes of healthy cells as well as detection of molecular signature of diseases for both research and diagnostic purposes. In cell labelling processes, organic fluorophores have been widely used either as staining agents for highlighting specific probes or for labelling biomarkers in fluorescence microscope imaging. However, the applications of organic dyes is limited in labelling techniques due to the fact that organic dyes undergo a rapid bleaching and can be only excited at specific wavelengths. In contrast, QDs exhibit unique photophysical properties and can be used in cell labelling as powerful fluorescence probes [119, 120, 123, 138, 139].

For instance, the applications of QDs as stains for either intracellular organelles or cell lines have been extensively reported such as nuclear antigens and microtubules [120], mitochondria [140], F-actin in fibroblast cells [119], labelling Human embryonic kidney cell line 293T [141], labelling Hella cells [135] or labelling biomarkers [142]. Figure 1.10 shows labelling of cellular components using QDs as fluorescence probes [138].

Chapter 1 Introduction

Figure 1.10. Labelling of surface and intracellular targets with QDs Probes: membrane-Her 2 receptors are detected with primary antibodies and QD-labelled secondary IgG (A, green), nuclear antigens (B, red), microtubules (C, red), nuclei are counterstained with Hoeshst 33 342 (blue in A, C). Both labelling routes can be applied simultaneously for a two-colour staining (D) [138].

1.6.2.2.2 Targeting Tumours

Application of QDs for cancer detection was reported by Akerman and his colleagues in 2002 as the first demonstration of in vivo imaging of tumour targeting in mice [143]. Since this work, QDs have been extensively used to target cancer cells such as targeting prostate tumour [144], detection of cancer cells sentinel lymph node [145] and targeting melanoma cancer cells [146].

In tumour targeting methods, QDs are utilized as immunoflourescent probes and a high sensitivity to cancer cells QD is required. This can be accomplished with linking of QDs with specific biomolecules such as antibodies or proteins to form QD-conjugated compounds. The QD-conjugated compounds have affinity for biomarkers in cancer cells and it makes QDs to target tumours [147, 150].

For example, alpha-feto-protein (AFP) is an important biomarker for detection of the hepatocarcinoma cell line HCCLM6, a common type of liver cancer [148]. For in-vivo tumour targeting and imaging of hepatoma cancerous lump, a specific immunofluorescence probe can be synthesized by linking QDs to AFP monocolonal antibody (Ab) for specific recognition of AFP biomarker. The QD-Ab compound can be injected to the mouse and used as QD-AFP-Ab probe to detect the cancer cells as shown in Figure 1.11 [149, 150].

Chapter 1 Introduction

Figure 1.11. In vivo targeting and imaging of hepartocarcinoma with QD-AFP-Ab probes: the spectra analysis of tumour site (red arrow) and the normal tissue adjacent to normal site (green arrow) (A) & whole body imaging (B). The diagram shows that the fluorescence spectra of tumour site was in the same with QD-AFP- Ab probes but the characteristic peak of QDs was not observed in normal site [150].

1.6.2.2.3 Detection of Biomolecules

QDs can be utilized in multiplexed imaging due to the potential for a combination of multiple colours and intensities of different multicoloured QDs, making them suitable as photostable fluorescence probes [22]. QDs can be used to monitor the long-term interactions of multiply-labelled biological molecules in cells and be applied in a wide range of biological assays such as separation of cell, proteins and small biomolecules. [128].

Chapter 1 Introduction

Besides, multicolour QDs can be used to encode genes. It has been reported that QDs in 5-6 colours with 6 intensity levels can be used to detect approximately 10,000 to 40,000 different recognisable codes [128, 22]. For example, biobeads are bioreactive molecules that can absorb small biological molecules and multicolour QDs can be applied for separation of different beads in a mixture of microbead- biomolecules compounds (Figure 1.12) [22].

Figure 1.12. Fluorescence micrograph of a mixture of CdSe/ZnS QDs-tagged biomolecules emitting single colours at 484, 508, 547, 575, 611 nm [22].

As Figure 1.12 shows, QDs emit single colour signals at different wavelength whilst this type of simultaneous excitation is not possible with fluorescent microphores containing organic dyes [22]. In contrast, QDs have narrow and symmetric emission that can be controlled by particle size, surface chemistry and size distribution that makes them to utilize for multiplexed bioimaging to encode genes, proteins and small biomolecules [22, 128].

Chapter 1 Introduction

1.6.2.3 QDs in Biosensors

QDs can undergo the phenomenon known as Förster resonance energy transfer (FRET), which is one of the most popular used processes in the development of biosensors and detection assays [122]. This mechanism is based upon the transfer of fluorescence energy from a donor particle to an acceptor compound where the distance between the donor and acceptor is smaller than a critical radius, known as the Förster radius [151]. FRET has been extensively used in biosensing systems to detect biomolecule such as proteins, nucleic acids and small molecules [152, 153].

QDs can be used as energy donors in FRET mechanisms because the photoemission properties of QDs allow efficient energy transfer with a number of traditional organic dyes which are energy acceptors [122]. Besides, QDs have narrower and more symmetric emission spectra than conventional dyes, making it possible to distinguish the emission of donors from the emission spectra of acceptors [154]. Therefore, QDs can be used in biosensors based upon the photochemically-induced fluorescence or FRET [122].

For example, CdSe QDs can be used in a maltose sensor [155]. As shown in Figure 1.13, a maltose-binding protein (MBP)-QD bioconjugated compound acts as energy donor and a cyclodextrin (CD) conjugated with an organic dye acts as energy acceptor. In the absence of maltose, cyclodextrin-dye complex bounds to MBP and transferring energy from QDs to dye leads to quenching flouresence of QDs whilst in the presence of maltose, maltose bounds to MPB, QDs have fluorescence and maltose can successfully be detected [155].

Chapter 1 Introduction

Figure 1.13. Schematic diagram of the QDs-based FRET maltose sensor: quenching fluorescence of QDs in absence of maltose (a) & recovery of fluorescence in QDs in presence of maltose (b), MBP is maltose binding protein and CD is cyclodextrin-dye complex [155].

1.6.2.4 QDs in Drug Delivery and Therapies

QDs can be used in drug delivery as a beneficial tool for drug loading, targeting and drug labelling based upon the ability of QDs to target cells and their unique photophysical properties. These applications can be monitored using ultrasensitive imaging [156, 157].

The major advantages of using QDs in drug delivery systems is to prevent systematic toxicity which is a common problem in delivering chemotherapeutic agents to cancer cells because QDs have potential ability for both targeting the cancer cells and delivering drugs to tumours [157].

Chapter 1 Introduction

In the process of utilizing QDs for drug delivery, QD probes can target and accumulate in tumours with two mechanisms including enhanced retention and permeability (EPR) effect which is similar to the function of anticancer agents and also with recognition of cancer cell surface biomarkers [158]. As chemotherapeutic drugs can be linked to QDs, they will recognize and bind to cancer cells without affecting healthy cells. Therefore, using QDs in drug delivery offer a new strategy to overcome systematic side effects [159].

1.6. 3 Limitations of Bioapplications of QDs

In spite of the ability of QDs for utilizing in biological context, there are some serious restrictions for bioapplications of QDs as listed below.

I) Solubilisation: one of the most critical requirements for introducing QDs to biological media is water solubility. As previously described in section 1.4.3, most high quality QDs are synthesized by organic routes, but they are insoluble in water due to their hydrophobic surface chemistry, requiring additional steps to render them water soluble [160-162]. Moreover, the process of water solubilisation leads to a quenching of the photoluminescence of QDs [78]. In contrast, the synthesized QDs via an aqueous route are soluble in water whilst they suffer of low crystallinity and poor optical properties [78].

II) Cytotoxicity: QDs are toxic materials due to the composition of the core, surface coating, size and surface charge. The core of many QDs may be formed from cadmium, tellurium and selenium, which are known as toxic compounds. Therefore, QDs have been classified as poisonous materials for both human and animal cells, as discussed in Chapter 6 [163-165].

III) Photostability of QDs in biological media: the interaction of QDs with cellular components, cell media and other biological or chemical compounds in biological context leads to the alteration of nanocrystal structure that can influence optical properties of QDs in the biological environment [166,167]. Besides, degradation of QDs induces free radical formation and causes cell necrosis in live cells [168]. Additionally, spectral fluctuation and heterogeneity in the emission profiles of QDs including on and off luminescence intermittency, or blinking, of QDs are other challenges that limit 25

Chapter 1 Introduction

applications of QDs in bio imaging [169-171]. Blinking can occur when the QD rapidly alternates between emissive and non-emissive states, leading to an alteration of the emission spectra and a quenching of fluorescent properties of QDs in a biological media [157]. Therefore, photostability of QDs is an essential requirement to introduce QDs for bioapplications.

IV) Biocompatibility: QDs are not able to bind with biomolecules due to the absence of any biocompatibility. However, the high surface to volume ratio of QDs enables them to be functionalized with a number of surface reactive groups, including amines, carboxylic acids and imides for specific targeting actions. After functionalization, the obtained QDs are biocompatible and they are able to bind with biomolecules such as cells, antibodies, proteins, DNA and albumin [172]. However, functionalization of QDs is a challenging process because the optical properties of QDs can be changed either after functionalization or targeting biomolecules [173].

V) Delivery of QDs to the cells: the delivery of QDs to cytoplasm and organelles including nuclei and mitochondria is a challenging area in biological research because in spite of the potential for the labelling of intracellular organelles with QDs, the majority of QDs are unable to penetrate subcellular organelles and more modification in their size and surface properties are required to apply them in intracellular assays [174- 176].

VI) Size limitation: the size of QDs is an important parameter in establishing appropriate binding to the cell membrane and QDs with a large hydrodynamic size cannot be adhered to the cells [177]. However, reducing the crystallite size can diminish the emissive brightness of QDs because smaller QDs are not as efficiently excited as larger QDs [178, 179]. Consequently, QDs should have an appropriate dynamic diameter for imaging applications.

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1. 7 Project Aims

The aims of this thesis are: the synthesis of water soluble, highly fluorescent, photostable and nontoxic QDs in an aqueous route and the investigation of the toxicity and optical properties of QDs in biological context.

To achieve these goals:

 CdSe nanoparticles were synthesized in water as a nontoxic solvent and the effects of experimental parameters such as pH, the capping agent and solvent on the formation and particle size of CdSe nanoparticles were investigated (Chapter 3).  Water Soluble CdSe(S) and ZnSe(S) QDs were synthesized using a hydrothermal approach (Chapter 4).  CdSe(S) QDs were coated with zinc oxide and ferric oxide to synthesis water

soluble CdSe(S)/ZnO and CdSe(S)/Fe2O3 core/shell QDs (Chapter 5).  The cytotoxicity of CdSe(S), ZnSe(S) and CdSe(S)/ZnO QDs were investigated in the presence of human colorectal carcinoma cells as cancer cells (HCT-116) and human skin fibroblast cell line (WS-1) as normal cells (Chapter 6).  A CdSe(S) QD-antibody conjugated compound was prepared and the photostability of the CdSe(S) QDs after linking to antibodies was investigated. In addition, the photostability of the CdSe(S) QDs in the presence of HCT-116 cells was studied using confocal microscopy (Chapter 7).

The steps of formation of this thesis have been illustrated in Figure 1.14.

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Figure 1.14. The steps of formation of this thesis.

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[160]. Zhao, Y.; Li, Y.; Song, Y.; Jiang, W.; Wu, Z.; Wang, Y. A.; Sun, J.; Wang, J. J. Colloid Interface Sci. 2009, 339, 336. [161]. Gerion, D.; Pinaud, F.; Williams, S. C.; Parak, W. J.; Zanchet, D.; Weiss, S.; Alivisatos, A. P. J. Phys. Chem. B 2001, 105, 8861. [162]. Yu, W. W.; Chang, E.; Falkner, J. C.; Zhang, J.; Al-Somali, A. M.; Sayes, C. M.; Johns, J.; Drezek, R.; Colvin, V. L. J. Am. Chem. Soc. 2007, 129, 2871. [163]. Nel, A.; Xia, T.; Maedler, L.; Li, N. Science 2006, 311, 622. [164]. Chen, N.; He, Y.; Su, Y.; Li, X.; Huang, Q.; Wang, H.; Zhang, X.; Tai, R.; Fan, C. Biomaterials 2012, 33, 1238. [165]. Sadaf, A.; Zeshan, B.; Wang, Z.; Zhang, R.; Xu, S.; Wang, C.; Cui, Y. J. Nanosci. Nanotechnol. 2012, 12, 8287. [166]. Galeone, A.; Vecchio, G.; Malvindi, M. A.; Brunetti, V.; Cingolani, R.; Pompa, P. P. Nanoscale 2012, 4, 6401. [167]. Morelli, E.; Cioni, P.; Posarelli, M.; Gabellieri, E. Aquat Toxicol 2012, 122-123, 153. [168]. Garnett, M. C.; Kallinteri, P. Occup Med (Lond) 2006, 56, 307. [169]. Mandal, A.; Nakayama, J.; Tamai, N.; Biju, V.; Isikawa, M. J Phys Chem B 2007, 111, 12765. [170]. Zhang, L.; Niu, W.; Yang, H.; Pan, M. Shengwu Yixue Gongchengxue Zazhi 2011, 28, 636. [171]. Zou, H.; Cao, Y.; Xu, Y.; Gan, L. Huaxue Tongbao 2012, 75, 209. [172]. Alivisatos, A. P.; Gu, W.; Larabell, C. Annu. Rev. Biomed. Eng. 2005, 7, 55. [173]. Oh, E.; Hong, M.-Y.; Lee, D.; Nam, S.-H.; Yoon, H. C.; Kim, H.-S. J. Am. Chem. Soc. 2005, 127, 3270. [174]. Chan, W. C.; Nie, S. Science 1998, 281, 2016. [175]. Lidke, D. S.; Nagy, P.; Heintzmann, R.; Arndt-Jovin, D. J.; Post, J. N.; Grecco, H. E.; Jares-Erijman, E. A.; Jovin, T. M. Nat. Biotechnol. 2004, 22, 198. [176]. Bharali, D. J.; Lucey, D. W.; Jayakumar, H.; Pudavar, H. E.; Prasad, P. N. J Am Chem Soc 2005, 127, 11364. [177]. Rejman, J.; Oberle, V.; Zuhorn, I. S.; Hoekstra, D. Biochem. J. 2004, 377, 159.

Chapter 1 Introduction

[178]. Kostic, R.; Stojanovic, D. Optoelectron. Adv. Mater., Rapid Commun. 2012, 6, 121. [179]. Hoshino, A.; Fujioka, K.; Suga, M.; Sasaki, Y. F.; Ohta, T.; Yasuhara, M.; Dohi, T.; Suzuki, K.; Yamamoto, K. Proc. SPIE-Int. Soc. Opt. Eng. 2005, 5705, 263.

Chapter 2 Experimental Procedures

2.1 Materials

All materials were of high purity and used as received, as listed in Table. 2.1.

Table 2.1. List of the required materials

Materials Chemical Supplier Grade Formula / Abbreviation Sodium sulfite Na2SO3 Fluka > 98% Selenium (powder) Se Ajax > 97% Sodium hydroxide NaOH Sigma-Aldrish Pellets (98%)

Ethanol C2H5OH Fluka > 99% 3-mercaptopropionic acid MPA Fluka > 99% L-cysteine Sigma-Aldrish > 97% Cadmium chloride CdCl2.2.5 H2O Fluka Hemi pentahydrate, > 99%

Sodium brohydrate NaBH4 Sigma-Aldrish Pellets > 98%

Oleic acid Ajax > 98% Human colorectal HCT-116 ATCC(Manassas, carcinoma cells Virginia, USA) Human skin fibroblast WS-1 ATCC (Manassas, cells Virginia, USA) N-hydroxysuccinimide NHS Sigma-Aldish > 99% N-(3- EDC Sigma-Aldrish > 99% dimethylaminopropyl)- N′-ethylcarbomide hydrochloride Sodium chloride NaCl Ajax > 99% Potassium chloride KCl Ajax > 99% Potassium bromide KBr Sigma Aldrich > 99% Disodium hydrogen Na2HPO4 Ajax > 99% phosphate Potassium dihydrogen KH2PO4 Ajax > 99% phosphate

Chapter 2 Experimental Procedures

Dimethyl sulfoxide DMSO Sigma-Aldrish > 98% 17-allyamino-17- 17-AAG demethoxy- (Hsp90 inhibitor) Sigma-Aldrish 100 nM, 98% geldanamycine Paraformaldehyde Sigma-Aldrish > 95% Dulbecco’s modified DMEM Invitrogen High glucose eagle medium C glucose= 4500 mg/lit Fetal bovine serum FBS Sigma-Aldrish Penicillin Invitrogen Concentratio n = 100X Streptomycin Invitrogen Concentratio n = 100X Nonessential amino acids Sigma-Aldrish Concentratio n = 100X L-glutamine Invitrogen Concentratio n =100 X Zinc chloride ZnCl2 Ajax > 99% Iron (III) chloride FeCl3. 2.5H2O Ajax Hemi pentahydrate > 98% Bisbenzimide Hoechst 33342 Invitrogen > 99% Donkey anti mouse IgG Jackson Immuno Concentratio (H+L) CY-3 fluorophore Research n =0.7 mg/ml Laboratories Slide-A-Lyzer dialysis Thermo Scientific 10k MWCO cassette Millipore filtration Millipore Membrane funnel grade= 0.1 µm

Additionally, phosphate buffered saline (PBS) was made up as follow; 136 mM NaCl,

2.6 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH = 7.4. Ultrapure water (18 MΩ) was collected from a MilliporeTM water purification system and used in all experiments.

2.2 Chemical Synthesis

Aqueous synthetic method was selected as an environmentally friendly approach to synthesis of CdSe nanoparticles (NPs) and various water soluble QDs including

CdSe(S), ZnSe(S), CdSe(S)/ZnO and CdSe (S)/Fe2O3 QDs.

Chapter 2 Experimental Procedures

2.2.1 Synthesis of CdSe-thiol Capped NPs

The general synthetic process for obtaining all thiol capped-nanocrystals is to dissolve a metal salt in the presence of the stabilizing thiol, followed by injection of the chalcogen source under an inert gas. A widely used approach reported in several works was used.

This approach is based upon using Na2SeSO3 and cadmium salts as the precursors and 3-mercaptopropionic acid (MPA) or L-cysteine as the capping agent to prepare CdSe nanoparticles via an aqueous route [1-6]. Zhang used these reactants in basic pH and prepared CdSe nanoparticles after refluxing the reaction system for 10 hours [1]. Chen used the same reactants and same synthetic method to prepare CdSe nanoparticles with an increased reflux time of up to 64 hours [2], Gupta obtained CdSe nanoparticles using the same approach and synthesized CdSe nanoparticles in different sizes [3], Wageh used this method to synthesise CdSe nanoparticles and to investigate the thermal degradation of the obtained particles [4]. Meanwhile, Liu and Sivasankar replaced MPA with L-cysteine in forming CdSe nanoparticles [5, 6].

In this work, this common synthetic method was used to prepare CdSe nanoparticles in different conditions including acidic and basic pH, in the presence of L-cysteine and ethanol.

All of these works are similar in appearance; however, the thermal conditions, experimental parameters and the aims of the experiments distinguish them. In the current work, Na2SeSO3, CdCl2 and MPA were used, similar to previous reports [1-4], but the preparation of CdSe NPs in an acidic pH instead of a basic pH, the synthesis of nanoparticles in presence of two thiol capping agents at the same time, the preparation of CdSe in the presence of ethanol and the formation of hexagonal CdSe NPs in the presence of L-cysteine in acidic pH are new aspects of this work.

In following experiments, sodium selenosulfite (Na2SeSO3) was used as the selenide precursor, MPA (3-mercaptopropionic acid) and L-cysteine were used as the capping agents, ultrapure water and ethanol were used as solvents, CdCl2.2.5H2O was the source of cadmium and 1M NaOH was employed to adjust the pH.

Chapter 2 Experimental Procedures

I)-Synthesis of Sodium Selenosulfite (Na2SeSO3)

Na2SeSO3 was synthesized according to the literature methods [7]. First, Na2SeSO3 was obtained by refluxing a solution of 70 ml of ultrapure water, 0.79 g (10 mmol) of Se

(powder) and 2.52 g (20 mmol) of Na2SO3. During reflux, Se and Na2SO3 react gradually to form a colourless transparent solution of Na2SeSO3 due to reduction of Se

(powder) with Na2SO3. The excess amount of Na2SO3 protects Na2SeSO3 from oxidation [7]. The reflux was continued for 3 hours and then the obtained solution was filtered to remove any unreacted selenium using a filtration funnel.

Finally according to the literature [8], a simple test was performed to confirm the formation of sodium selenosulfite, as follow; 0.2 g NaOH and 8 ml oleic acid dissolved in a mixture of 15 ml of C2H5OH and 10 ml ultrapure water to form a clear solution.

Then, 5 ml of as-prepared Na2SeSO3 was added to this solution with stirring. The observation of colour change of the solution from colourless to red indicated the presence of Na2SeSO3.

II)-Preparation of Cubic CdSe NPs in Different Conditions

A Cd precursor solution (100 ml, 3.6 mmol or 1.2 mmol) was prepared by dissolving

CdCl2.2.5H2O in ultrapure water. MPA or L-cysteine was added to the solution with adjusting the molar ratio for Cd: capping agent in each experiment and the pH was adjusted using 1M NaOH. The reaction was heated to 80 ºC. Then, heating was interrupted and CdSe nanoparticles were formed upon addition of the fresh Na2SeSO3 (70 ml, 4 mmol) to the Cd precursor solution at temperature 60 ºC in different conditions, as described in Table.2.2. The temperature of the reaction after adding

Na2SeSO3 was 60 ºC, the system was cooled in room temperature and the CdSe NPs were formed as cloudy solutions.

In order to investigate the effect of heating of reaction on the formation of NPs, the obtained nanoparticles in both acidic and basic pH (Table 2.1, Experiments 1, 2); were put under reflux at T= 100 ºC for 6 hours.

Chapter 2 Experimental Procedures

Table 2.2 Preparation of cubic CdSe NPs in different conditions

Experiment Condition of Molar Capping Solvent pH Colour preparation ratio of agent of Cd: Se (mmol) Particles (mmol) 1 Acidic pH 3.6: 4 MPA(52 Water 3.5-4.5 Orange mmol) 2 Basic pH 3.6:4 MPA (52 Water 9.5- Brown mmol) 10.5 3 Basic pH and 3.6:4 MPA ( 52 Water 9.5- Brown in presence of mmol) 80% and 10.5 ethanol ethanol 20% 4 In presence of 1.2:4 5.6 mmol Water 3.5-4.5 Brown MPA + 3 L-cysteine and mmol L- MPA Cysteine 5 Control of the 1.2:4 5.6 mmol Water 3.5- 4.5 Orange experiment 4 MPA

III)-Preparation of Hexagonal CdSe NPs

A Cd precursor solution (100 ml, 1.2 mmol) was prepared by dissolving CdCl2.2.5H2O in ultrapure water, then, 0.78 g (4.4 mmol) L-cysteine was added to the solution followed by adding fresh Na2SeSO3 (70 ml, 4 mmol) to the Cd precursor solution at temperature 80 ºC and pH was adjusted to 4. Then, the reaction was refluxed for 4.5 hours at temperature 100 ºC. Then, the system was cooled in room temperature and CdSe NPs were obtained as a cloudy brown solution.

Chapter 2 Experimental Procedures

IV)-Separation of CdSe NPs from Solutions

As-synthesized CdSe NPs were obtained in cloudy solutions with partial solubility in the water. A part of each sample was stored in room temperature in water and another part, was filtered using a filtration funnel. The obtained precipitate was dried in room temperature for characterization.

2.2.2 Synthesis of Water Soluble QDs

An aqueous hydrothermal method was employed to synthesise water soluble CdSe(S) and ZnSe(S) QDs. This approach is based on the properties of the reactants including their solubility and reactivity in the water at high pressure and temperature, as described in Chapter 1, Section 1.4.3.2.1. The particle size distribution and phase formation of the particles can be influenced with reaction parameters such as temperature, pressure, reactant concentration and pH [9-12]. By using a modified literature method, the experimental parameters such as the molar ratio of precursors to capping agent and pH were optimized to produce high quality CdSe(S) QDs [12]. Then, the hydrothermal method was used to synthesis ZnSe(S) QDs because until now, formation of ZnSe(S) QDs has been reported using microwave assisted method [13] and reflux in the presence of hydrazine as a capping ligand [14, 15], but the formation of ZnSe(S) QDs in a hydrothermal method has not been reported previously.

2.2.2.1 Synthesis of Sodium Hydrogen Selenide (NaHSe )

A fresh aqueous NaHSe (1 mmol in 20 ml) was synthesized according to the literature methods [16, 17]. Briefly, selenium powder (0.079 g, 1 mmol) and NaBH4 (0.076 g, 2 mmol) were reacted in the presence of water (4 ml) with N2-bubbling through the solution at T = 4 ºC, according to reaction 2.1.

4NaBH4 + 2Se (powder) + 7 H2O NaHSe + Na2B4O7 +14 H2

(Reaction. 2.1)

After 3 hours, the black selenium powder was no longer visible, indicating the formation of NaHSe as a white transparent solution. This was transferred to 20 ml of

N2-saturated ultrapure water and stored under N2 for use in experiments as source of selenide to prepare CdSe(S) and ZnSe(S) QDs. 44

Chapter 2 Experimental Procedures

2.2.2.2 Synthesis of CdSe(S) QDs

A Cd-MPA precursor solution (65 mM) was prepared by dissolving 1.48 g (6.5 mmol)

CdCl2 in 100 ml ultrapure water and adding 1.1 ml (13 mmol) MPA to the solution, the molar ratio of Cd: MPA was 1:2 and the pH was adjusted to 12.5 using 1M NaOH. The Cd-MPA precursor solution was then put under nitrogen for 10 minutes and NaHSe (1 mmol, 20 ml), was injected to the Cd-MPA solution to form a CdSe-MPA complex as a clear yellow liquid. The reaction solution was transferred to a Teflon-lined autoclave and placed in a conventional oven for 1 hour, under hydrothermal treatment at T = 150 ºC. Finally, water soluble CdSe-MPA capped QDs were formed as clear aqueous orange solutions. The obtained QDs were orange in colour in contrast to that previous reported [12]. The molar ratio of Cd: MPA: Se was 6.5:13:1. The sample was labelled as Sample1 and fully characterized.

In order to investigate different parameters on the formation of CdSe(S) QDs, the reaction was repeated as follows:

I)-The experiment was performed in a range of reaction times including 0.5, 4, 20 and 73 hours.

II)-The experiment was repeated with refluxing of the CdSe-MPA complex solution for 3 hours to prepare CdSe(S) QDs in conventional condition.

III)-Two other samples were prepared under the same experimental conditions, but in different molar ratios of Cd to MPA including: Sample 2 (Cd: MPA = 8: 13) and Sample 3 (Cd: MPA = 6.5: 8). These samples were synthesized to be compared with Sample 1 (Cd: MPA = 6.5:13).

2.2.2.3 Synthesis of ZnSe(S) QDs

An aqueous Zn precursor solution was prepared by dissolving 1.09 g (8 mmol) anhydrous ZnCl2 in 100 ml ultra-pure water and adding 2.5 ml MPA (~29 mmol) to the solution. A colourless solution of ZnSe-MPA complex was obtained in a clear solution after the addition 20 ml of fresh NaHSe (0.5 mmol of Se in 20 ml) to the Zn precursor solution, under a nitrogen atmosphere. The pH was adjusted with 1M NaOH to 7.3, with

Chapter 2 Experimental Procedures

the Zn:Se:MPA molar ratio of 16:1:58. The solution was transferred to a Teflon-lined autoclave and placed in a conventional oven under hydrothermal treatment at T = 150 ºC for 165 minutes to form ZnSe(S) water soluble QDs as a transparent colourless solution. The obtained sample was labelled as Sample 4.

2.2.3 Synthesis of Core/shell QDs

CdSe(S) QDs were prepared, as described in section 2.2.2.2 and coated with ZnO according to previous literature report [12] based upon the control of hydrolysis of zinc salts in basic pH. Besides, in a similar approach, coating of CdSe(S) QDs with Fe2O3 was performed to produce CdSe(S)/Fe2O3 QDs that has not previously reported.

Figure 2.1 shows a schematic of the coating of CdSe(S) QD cores with ZnO or Fe2O3 shells.

Figure 2.1. Schematic of formation of CdSe(S)/ZnO and CdSe(S)/ Fe2O3 core/shell QDs.

2.2.3.1 Formation of CdSe(S)/ZnO Core/shell QDs

A)-According to a previous literature method [12], an aqueous solution of as-prepared water soluble CdSe(S) QDs (Sample 1, Section 2.2.2.2) was diluted using ultrapure water. The concentration of CdSe(S) QDs was 1.7 mg in 5 ml and the pH was adjusted to 13 using 1M NaOH. This solution was refluxed for 2 hours. Then, an aqueous

Chapter 2 Experimental Procedures

solution of Zn(OAC)2.2 H2O (18 ml, 0.01M) was prepared and injected to the solution of CdSe(S) QDs at T = 100 ºC. The molar ratio of Zn: Cd was 6:1 in the final solution. Reflux was continued for another hour and CdSe(S)/ZnO core/shell QDs were obtained and labelled as Sample 5.

B)-The experiment was repeated using Sample 3 (Section 2.2.2.2, III), as the core QDs and CdSe(S)/ZnO QDs were obtained under the same experimental conditions. The as- prepared sample was labelled as Sample 6.

2.2.3.2 Formation of CdSe(S)/Fe2O3 Core/shell QDs

First, an aqueous solution of water dispersed CdSe(S) QDs (Sample 1, Section 2.2.2.2) was diluted using ultrapure water. The concentration of CdSe(S) QDs was 1.7 mg in 5 ml and the pH was adjusted to 13 using 1M NaOH. The solution was refluxed for 2 hours. Then, an aqueous solution of FeCl3.2.5H2O (18 ml, 0.01M) was injected into the solution of CdSe(S) QDs at T = 100 ºC. The injection speed of ferric chloride was 0.001 ml/minute and the molar ratio of Cd to Fe was 3:2 in final solution. After continuing reflux for another hour, CdSe(S)/Fe2O3 QDs were formed and labelled as sample 7.

2.2.4 Separation of Water Soluble QDs from Aqueous Solutions

All as-synthesized QDs (Sections 2.2.2 & 2.2.3) were obtained in an aqueous phase, whilst solid phase QDs were required for XRD, XPS and IR characterisation. Ethanol and acetone were tested as solvents with which to induce precipitation of QDs from aqueous solutions and it was found that acetone is the most effective solvent for obtaining QDs in the solid phase. Therefore, all QDs were precipitated with acetone, washed with water, filtered with a Millipore filtration funnel (membrane grade = 0.1 µm) and used for characterization.

2.2.5 Cytotoxicity Assays

The cytotoxicity of the obtained CdSe(S) QDs (Sample 3, Section 2.2.2.2, III), ZnSe(S) QDs (Sample 4, Section 2.2.2.3), and CdSe(S)/ZnO QDs (Sample 6, Section 2.2.3.1, B)

Chapter 2 Experimental Procedures

were determined in presence of two cell lines: human colorectal carcinoma cells (HCT- 116) and human fibroblast skin cells (WS1) in following steps.

2.2.5.1 Preparation of Aqueous QDs

A total of fifteen different solutions of QDs were prepared with diluting the aqueous solutions of CdSe(S), ZnSe(S) and CdSe(S)/ZnO QDs with ultra-pure water to achieve different concentrations of 25, 50, 100, 250, 500 (µg/ml) for all QDs solutions. Two aqueous solutions containing a Cd-Se-MPA complex and Cd-MPA were prepared as described in Section 2.2.2.2 and then diluted with ultra-pure water to provide ten solutions with different concentrations of 25, 50, 100, 250, 500 (µg/ml) for both precursor solutions. Each sample was used in cytotoxicity assays without further purification. However, a Slide-A-Lyzer dialysis cassette (10 kMWCO) was used as an effort to remove excess amounts of cadmium and capping agent from the aqueous solution of CdSe(S) QDs and the cytotoxicity of CdSe(S) QDs in the presence of HCT- 116 cell line was investigated at concentrations 25, 50, 100, 250 and 500 (µg/ml).

2.2.5.2 Cell Culture

Cell cultures of human colorectal carcinoma cells (HCT-116) and human skin fibroblast cell line (WS-1) were obtained according to the standard protocol [18]. As cells are usually received frozen in the culture medium with 5-10% dimethyl sulfoxide (DMSO) in the vapour of liquid nitrogen at 77 K, they were first defrosted at 37 ºC and the culture medium was discarded by using a centrifuge at 1500 rpm for 5 minutes. Then, the HCT-116 and WS-1 cells were separately resuspended in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, (1%) nonessential amino acid and L-glutamine (2 mM) as growth medium and transferred in two 75 cm2 flasks. Finally, the cells were grown in a humidified incubator at 37 ºC with 5% CO2 and as cells reached confluence; they were immediately used for cytotoxicity tests in separate experiments.

2.2.5.3 Sample Preparation

HCT-116 and WS-1 cells were separately seeded in two 96-well plates (3000 cells/well) and allowed to adhere to the dish for 24 hours in a humidified incubator. To control,

Chapter 2 Experimental Procedures

both cell media alone (3000 cell/well) and cell media in presence of Hsp90 inhibitor 17- AAG (100 nM = 0.06 µg/ml) were seeded and used.

Then, 10 µl of each aqueous solution of QDs (described in section 2.2.5.1) was added to the plates, as shown in Figure 2.2.

Figure 2.2. Image of a 96-well plate [19].

As shown in Figure 2.2, 96-well plates were used to prepare the cell-QD samples at different concentrations (25, 50, 100, 250, 500 µg/ml): HCT-116- CdSe(S) QDs (A1-

A5), HCT-116-ZnSe(S) QDs (D1-D5), HCT-116- CdSe(S)/ZnO QDs (G1-A5), cell media

(D10), Hsp90 inhibitor 17- AAG –HCT-116 (H10). In the other plate, WS1-QDs were similarly seeded.

Finally, the plates were put under incubation in the presence of QDs for 72 hours at 37

ºC with 5% CO2. The thus-prepared samples were used to determine proliferation of the cells.

2.2.5.4 Determination of Cell Viability

Viability of the cells was performed using a Cell Counting Kit-8 assay to determine the proliferation of both HCT-116 and WS-1 cells in the presence of different types of water soluble QDs. This instrument allows standard assays by utilizing Dojindo’s highly water soluble tetrazolium salt (WST-8). As shown in Figures 2.3 and 2.4, WST-8 reduces by dehydrogenases in the cells to produce formazan dye, which is soluble in

Chapter 2 Experimental Procedures

tissue culture medium cells. Therefore, the amount of generated formazan dye and hence the absorption at λ=450 nm, can be equated to the number of living cells [20, 21].

Figure 2.3 Structures of WST-8 and WST-8 formazan [22].

Figure 2.4 Principle of the cell viability detection with cell counting kit-8 [22].

According to the manufacturer’s instructions [22], 1 µl of CCK-8 solution was added to each well of the plate and the plate was incubated up to 4 hours. Then the absorbance of each well of the plate was measured at 450 nm, using a Chromate 4300 micro plate reader. Each measurement was repeated in triplicate to eliminate errors and the results were plotted using Graph Pad prism 5 and Excel software.

Chapter 2 Experimental Procedures

The lethal concentration that kills 50% of cells (LC50) was estimated for CdSe(S) QDs as 105 (µg/ml) for CdSe (S) QDs in the presence of HCT-116 cell lines.

2.2.6 Preparation of a QD-antibody Conjugated Compound

As discussed in Chapter 1, QDs have the ability to bind with biomolecules, including proteins, DNA and antibodies, producing compounds that have properties of both components, so-called inorganic-biological hybrids [23]. Following this assumption, a conjugated compound of CdSe(S)-donkey anti mouse IgG CY3 fluorophore was prepared after modification of CdSe(S) QDs via EDC coupling.

2.2.6.1 Modification of CdSe(S) QDs

EDC coupling is a standard method to synthesis bioconjugated compounds via linking of carboxylic acid capped nanoparticles to biomolecules including antibodies. The first step of EDC coupling is the reaction of carboxyl-QDs with EDC to form an unstable intermediate compound which is stabilised by reacting with NHS to produce an amine- reactive ester in a second step. This amine-reactive ester then reacts with amine groups in antibody surface molecules to form a QDs-antibody bioconjugated compound [24- 26]. Figure 2.5 shows the steps of EDC coupling.

Figure 2.5 Schematic of EDC coupling [27].

Chapter 2 Experimental Procedures

First, water soluble CdSe(S) QDs (Sample 3, section 2.2.2.2, III) were synthesized. Then, a solution of as-prepared CdSe(S) QDs (1 ml, 1 mg/ml), EDC (0.1 ml, 5 mM), NHS (0.1 ml, 1 mM) in 0.2 ml PBS was prepared and incubated in room temperature for 30 minutes to form a solution of amine-modified QDs. Additionally, the modification process was repeated using different molar ratios of NHS to EDC, including 1:1, 1:2 and 1:5 in order to find the optimum molar ratio of NHS: EDC in the modification of CdSe(S) QDs.

2.2.6.2 Formation of QD-antibody Compound

After modification of QDs, donkey anti mouse IgG antibody (1 µl, 0.7 mg/ml) was added to as-prepared solution of the modified CdSe(S) QDs ( 0.2 ml, 100 µg/ml) , incubated in room temperature for 2.5 hours with light protection and an antibody- CdSe(S) conjugated compound was obtained. The compound was then stored at 4 ºC.

The incubation step was repeated using different volume ratios of antibody to the modified CdSe(S) QDs such as 0.1 µl of antibody to 0.4 ml, 1 ml and 2 ml of the modified QDs. Besides, the incubation process using 1 µl antibody to 0.2 ml of the modified QDS was repeated at 4 ºC in order to compare with incubation at room temperature.

2.2.7 The Investigation of Photostability of CdSe(S) QDs in Presence of HCT-116 Cells

The photostability of CdSe(S) QDs was investigated in the presence of HCT-116 cell line using confocal microscopy. First, the as-synthesized CdSe(S) water soluble QDs (Sample 3, Section 2.2.2.2, III), were incubated with HCT-116 cell line to prepare two kinds of samples: fixed HCT 116 cells-CdSe(S)QDs and live HCT 116 cells-QDs samples. Then, the images of QDs in the presence of cells were recorded using confocal microscopy. The details of these steps described below.

2.2.7.1 Sample Preparation

(A)-Fixed HCT 116 cells-CdSe(S) QDs sample: cells were cultured according to section 2.2.5.2 and were seeded in 35 mm fluorodish cell culture dishes at a density of 52

Chapter 2 Experimental Procedures

20000 cell/dish for 24 hours. The cells were then treated with an aqueous solution of

CdSe(S) QDs (250 µg/ml) and incubated at 37 ºC with 5% CO2 for 24 hours. After incubation, the cells were fixed with 4% paraformaldehyde in PBS for 1 hour. Finally, they were washed with PBS and stained with 5 µg/ml bisbenzimide (Hoechst 33342) for 20 minutes in room temperature.

(B)–Live HCT 116 cells-QDs samples: cells were cultured according to the protocols outlined in section 2.2.5.2 in similarity with fixed cells and were seeded in a 24-well glass bottom plate at a density of 20000 cells/dish for 24 hours. Then, cells were treated with an aqueous solution of CdSe(S) QDs (100 µg/ml) and incubated at 37 ºC with 5%

CO2 for 24 hours.

As-prepared samples were used for confocal microscopy studies.

2.2.7.2 Microscopy Techniques

The investigation into the stability of QDs in the presence of HCT 116 cells was performed using Leica TCS SP5 CW STED and Zias LSM 780 confocal microscopes. The sample of fixed HCT 116 cells-CdSe(S) QDs was dropped on a flourodish polycarbonate and images were recorded with the Leica TCS SP5 CW STED confocal microscope. Images of live HCT 116 cells incubated with CdSe(S) QDs solution for 24 hours were recorded with the Zias LSM 780 confocal microscope. Cells were stained by adding a Hoechst solution (5 µg /ml) for 10 minutes before recording images due to the cell permeability of Hoechst and its DNA interaction.

2.2.8 The Investigation of Photostability of CdSe(S) QDs in Cell Media

As described in Chapter 1, Section 1.6.3, cell media may influence the photostability of QDs [28]. In order to investigate the spectral properties and stability of QDs in cell media, the emission profiles of the CdSe(S) in both water and cell media solution were recorded using confocal microscopy. The cell media was prepared as described below.

Chapter 2 Experimental Procedures

2.2.8.1 Preparation of Cell Media Solution

First, 25 ml of heat-inactivated fetal bovine serum (FBS), 25 ml of nonessential amino acids and 2.5 ml of L-glutamine (200 mM) were added into a 250 ml filtration unit. Then, Dulbecco’s modified eagle medium (DMEM) was added up to a total volume of 250 ml. Finally, the media was filtered and stored at 4 ºC.

2.2.8.2 The Investigation of Spectral Properties of QDs in Cell Media

A solution of as prepared CdSe(S) QDs (Sample 3, Section 2.2.2.2, III), were diluted to 100 µg/ml. Then, 1 µl of the diluted solution dropped on a flourodish polycarbonate, labelled as Sample A and the emission profile of QDs in aqueous solution was recorded using Zias LSM 780 confocal microscope. Finally, 0.1 µl of as-prepared cell media (Section 2.2.8.1) was added to Sample A and the Zias LSM 780 confocal microscope was used to record the emission profile of QDs in cell media.

2.3 Characterization Techniques

2.3.1 Powder X- ray Diffraction

Powder X- ray diffraction (PXRD) is a powerful technique to identify phase information of the nanocrystals due to the fact that X-rays diffract from crystalline lattices according to Bragg’s Law. Since a unique crystalline phase of materials has a characteristic diffraction pattern, the obtained diffraction pattern of materials recorded by a powder X-ray diffractometer, can be used to characterize the phase information of compounds using a database of diffraction patterns [29].

A typical PXRD diffractometer consists of an X-ray source, a sample stage, a detector and a computer control system, as shown in Figure 2.6. When the X-rays scatter from the sample, they are detected, generating the diffraction pattern compiled in the computer system [30]. The computer system processes the information and crystal structure is determined using standard diffraction data.

Chapter 2 Experimental Procedures

Figure 2.6. Schematic of a PXRD diffractometer [31].

Any impurities can be determined by identifying and quantifying the diffraction patterns. Moreover, the particle size of nanocrystalline materials can be calculated using the Scherrer equation [30], as shown in Equation 2.1.

B λ d = (Equation. 2.1) β cosθ

The Scherrer equation relates the particle size to the peak width; B is the measured FWHM in radians, θ is the Bragg angle of the peak, λ is the X-ray wavelength (1.5418 Å),  is a dimensionless shape factor = 0.8-1~ 0.94 and d is the crystallite size.

All X-ray PXRD patterns were recorded on a Philips X’pert Multipurpose X-ray diffraction system (MPD system), using a Cu Kα source (λ = 0.154056 nm). Samples were first precipitated from aqueous solution using acetone and then washed with water. They were dried at room temperature and ground into a fine powder using a mortar and pestle. Then, they were transferred to sample holders having a diameter of 15 mm and a depth of 1 mm; data were collected over the 2θ range of 5º to 90º using a continuous 55

Chapter 2 Experimental Procedures

scan with an angular step size of 0.0263º. The obtained data were analyzed using High Score Plus software.

2.3.2 High Resolution Transmission Electron Microscopy

The direct imaging of the samples using high-resolution transmission electron microscopy (HRTEM) is a powerful tool to study the properties of nanomaterials. This imaging technique, allows one to directly observe particles, along with some structural details of the nanocrystals at high resolution. Additionally, HRTEM can be used to estimate the particle size and the spacing of atomic planes in nanocrystalline structures by direct observation. This is complementary to scattering techniques (e.g. PXRD), but can also be used to record selected area diffraction patterns (SAED) of nanocrystals and energy dispersive X-ray spectroscopy (EDS). This wide range of information is necessary for characterization of nanocrystals.

In order to investigate the properties of the obtained nanomaterials, described in Sections 2.2.1 to 2.2.3, a Philips CM 200 instrument was used for recording TEM micrographs, obtaining SAED patterns and performing EDS analysis.

Prior to imaging, samples were sonicated in water for several minutes and then dropped onto a carbon grid and dried in air. TEM images were then recorded by adjusting the instrument resolution. Image-j software (http://imagej.en.softonic.com) was used to obtain fast Fourier transforms (FFT) of regions within the HRTEM images and also to determine the d-spacing of atomic planes.

SAED images were recorded at 300 keV. Samples deposited from aqueous nanoparticle solutions onto amorphous carbon grids and dried at room temperature.

Additionally, the EDS analysis was performed. A sapphire Si(Li) EDAX detector with 30 mm active area was employed to acquire and analysis the chemical spectra from the TEM images. First, the TEM images of samples were recorded and then the EDS spectra were obtained from the selected TEM image.

Chapter 2 Experimental Procedures

2.3.3 Optical Spectroscopy

UV-vis absorption spectra of as-prepared samples, described in Sections 2.2.1 to 2.2.3 were obtained with a Varian Cary UV spectrometer. First, aqueous solutions of samples were used without precipitation or re-dissolving, the samples were dropped in quartz cuvette and diluted with adding ultrapure water. The concentration of samples was 0.1 mg/ml. Then, the excitation spectra of the samples were recorded in wavelength range of 200 to 800 nm.

Photoluminescence spectroscopy was conducted on a custom-built microscope coupled to an Acton 2300 spectrometer using an excitation laser at 514.5 nm to measure photoluminescence properties of CdSe(S) QDs, CdSe(S)/ZnO and CdSe(S)/Fe2O3 core/shell QDs. The photoluminescence spectra of ZnSe(S) QDs were recorded using with J/B SPEX 270M spectrometer using a power max PMT detector and excitation laser at 350 nm. Additionally, a Carry Eclipsed Fluorescence instrument was used to record fluorescence spectra of CdSe(S) QD-antibody conjugated compound and its relevant CdSe(S) QDs sample using an excitation wavelength at 350 nm. In similarity with UV-visible spectroscopy, fluorescence spectra of the samples were recorded using aqueous samples.

The images of QDs under UV-light were taken using a Spectroline UV equipment. The aqueous samples of QDs were put under UV exposure and the images of QDs under UV-light, were recorded using a digital camera. The excitation wavelength of the equipment was 365 nm.

2.3.4 Fourier Transform Infra-red Spectroscopy

Fourier transform infra-red (FT-IR) measurements were conducted with Thermonicolet Avatar 370 FT-IR spectrometers. First, aqueous solution of nanoparticles, described in Section 2.2.1 and 2.2.2; were precipitated and then solid samples were mixed with potassium chloride (KBr) as an IR-inactive compound and compressed as solid tablets consistent of KBr and compounds. Then, KBr-compound tablets were transferred to sample holders and FT-IR spectra of compounds were recorded using the instrument in wavenumber of 500 to 4000 cm -1.

Chapter 2 Experimental Procedures

2.3.5 Circular Dichroism Spectroscopy

Circular dichroism spectroscopy (CD) is a useful technique for confirmation of the structure of macromolecules such as proteins and antibodies after interaction with other molecules. CD spectroscopy of a CdSe(S) QD-antibody conjugated compound, described in Section 2.2.6 was performed on a Chrascan TM-plus spectrometer using a dual polarising momochromator equipped with temperature control and simultaneous UV acquisition. The light source was a 150W air-cooled Xe arc lamp and the wavelength range was 170 nm to 1150 nm with a single detector. First, both samples of aqueous solution of QD-antibody conjugated compound and antibody were diluted with FBS and dropped in quartz cuvette (0.5 mm in length). Then, CD spectra were recorded at 4ºC due to the fact that the antibody structure is not stable at higher temperatures.

2.3.6 Elemental Analysis

X-ray photoelectron spectroscopy (XPS) can provide elemental analysis of materials including QDs and particularly core/shell QDs. XPS spectroscopy of the obtained QDs, described in Sections 2.2.2 and 2.2.3; was performed on a Escalab 250i XL spectrometer using a monochromated Al Kα X-ray source (hν = 1486.6 eV) operated at 10 kV and 12 mA. The pressure in the measurement chamber was typically ~1 × 10-9 Torr. Samples were either screwed down or attached to aluminum holders to use in the instrument. The Varian Eclipse software was used for data analyzing.

2.3.7 Dynamic Light Scattering

Dynamic light scattering (DLS) is used to characterize nanoparticles dissolved in a liquid. The average hydrodynamic diameters of the nanoparticles can be determined using DLS and can be compared with the obtained particle size using either calculations or other methods. DLS studies of CdSe(S) QDs (Sample 1, Section 2.2.2.2), were conducted using a Malvern Instruments Zetasizer Nano ZS instrument equipped with a 4 mV He-Ne laser operating at λ = 633 nm with a high quantum efficiency avalanche photodiode detector and an ALV/LSE-5003 multiple tau digital correlator electronics system [32]. A sample of as-prepared CdSe(S) QDs was used as an aqueous solution directly after synthesis, without precipitation or redissolving and was filtered through 58

Chapter 2 Experimental Procedures

Minisart RC25 syringe filters to removing any unwanted dust particles that can influence the particle size. The filtered solution was transported to quartz cuvettes (1 cm length) and put into the sample chamber of the DLS instrument. The size distribution histograms were obtained to determine the average hydrodynamic diameters of as- prepared QDs and the obtained data were analysed using Malvern software.

References

[1]. Zhang, S.; Yu, J.; Li, X.; Tian, W. Nanotechnology 2004, 15, 1108. [2]. Chen, X.; Hutchison, J. L.; Dobson, P. J.; Wakefield, G. J. Mater. Sci.2009, 44, 285. [3]. Gupta, P.; Ramrakhiani, M. Open Nanosci. J. 2009, 3, 15. [4]. Wageh, S.; Higazy, A. A.; Hassouna, A. S. J. Mater. Sci.: Mater. Electron. 2013, 24, 3049. [5]. Sivasankar, K.; Padmavathy, N. Micro Nano Lett. 2011, 6, 144. [6]. Liu, P.; Wang, Q.; Li, X. J. Phys. Chem. C 2009, 113, 7670. [7]. Gao, Y.; Zhang, Q.; Gao, Q.; Tian, Y.; Zhou, W.; Zheng, L.; Zhang, S. Mater. Chem. Phys. 2009, 115, 724. [8]. Liu, L.; Peng, Q.; Li, Y. Nano Res. 2008, 1, 403. [9]. Zhang, H.; Wang, L.; Xiong, H.; Hu, L.; Yang, B.; Li, W. Adv. Mater. 2003, 15, 1712. [10]. Yang, W.-h.; Li, W.-w.; Dou, H.-j.; Sun, K. Mater. Lett. 2008, 62, 2564. [11]. Wang, J.; Han, H. J. Colloid Interface Sci. 2010, 351, 83. [12]. Aldeek, F.; Mustin, C.; Balan, L.; Medjahdi, G.; Roques-Carmes, T.; Arnoux, P.; Schneider, R. Eur. J. Inorg. Chem. 2011, 794. [13]. Qian, H.; Qui, X.; Li, L.; Ren, J. J. Phys. Chem. B 2006, 110, 9034. [14]. Saikia, K.; Deb, P.; Kalita, E. Physica Scripta 2013, 87, 065802. [15]. Senthilkumar, K.; Kalaivani, T.; Kanagesan, S.; Balasubramanian, V. J. Mater. Sci.: Mater. Electron. 2012, 23, 2048. [16]. Klayman, D. L.; Griffin, T. S. J. Amer. Chem. Soc. 1973, 95, 197. [17]. Filgueiras, d. A.-F. P.; Gouveia, d. S. A.; Alves, d. M. S.; Botelho, J. R.; Barbosa- Filho, J. M.; Miller, J.; Lira, B. F. ARKIVOC 2004, 22. [18]. Phelan, M. C. In Current Protocols in Cell Biology; John Wiley & Sons, Inc.: 2007, Chapter 1, p 1.1.1- 1.1.18.

Chapter 2 Experimental Procedures

[19]. Image from http://www.4ti.co.uk. [20]. Ishiyama, M.; Miyazono, Y.; Sasamoto, K.; Ohkura, Y.; Ueno, K. Talanta 1997, 44, 1299. [21]. Tominaga, H.; Ishiyama, M.; Ohseto, F.; Sasamoto, K.; Hamamoto, T.; Suzuki, K.; Watanabe, M. Anal. Commun. 1999, 36, 47. [22]. Technical manual for Cell Counting kit-8, General information cell proliferation assay and cytotoxicity assay, Dojindo Molecular Technologies, Inc. [23]. Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Nat. Mater 2005, 4, 435. [24]. Song, F.; Chan, W. C. W. Nanotechnology 2011, 22, 494006/1. [25]. Wang, S.; Mamedova, N.; Kotov, N. A.; Chen, W.; Studer, J. Nano Lett. 2002, 2, 817. [26]. Shan, Y.; Wang, L.; Shi, Y.; Zhang, H.; Li, H.; Liu, H.; Yang, B.; Li, T.; Fang, X.; Li, W. Talanta 2008, 75, 1008. [27]. www.piercenet.com. [28]. Kulvietis, V.; Streckyte, G.; Rotomskis, R. Lith. J. Phys. 2011, 51, 163. [29]. Ermrich, M. XRD for Analyst, PANalytical, 2011, Almelo, The Netherlands, 2011, p 8-12. [30]. Cullity, B.D.; Stock, S.R.; Elements of X-Ray Diffraction, 3rd Ed., Prentice-Hall Inc., 2001, p 167-171. [31]. Image from http://lipidlibrary.aocs.org. [32]. ALV-5000 Reference Manual for Software Version 5.0, ALV-laser, Langen, 1993, p 7.

Chapter 3

Synthesis of CdSe nanoparticles (NPs)

3.1 Introduction

The synthesis of CdSe nanoparticles and the investigation of effective control by virtue of reaction parameters governing the formation of nanostructured materials with dimensions in the range of 1 to 20 nm, have attracted a great attention in nano-science due to the potential industrial and biological applications of these materials [1-3]. There are various reports in the literature for the synthesis of semiconductor nanoparticles including CdS, CdSe and CdTe via different synthetic methods to produce highly crystalline nanoparticles and among all nano-scale materials, CdSe nanoparticles have been the focus of much interest due to their unique optical properties. For instance, CdSe nanparticles can exhibit the effects of quantum confinement when sized below 10 nm and CdSe quantum dots are one of the most popular demonstrations of nanocrystals [4- 9].

3.1.1 Nanoparticles Formation

The solution phase formation of CdSe nanoparticles consists of nucleation and growth processes according to La Mer Theory [10] (Appendix 2), as shown in Figure 3.1[11].

Chapter 3 Synthesis of CdSe NPs

Figure 3.1. The steps of CdSe nanoparticles formation [11].

This reaction is not quite as simple as shown schematically and a detailed understanding of the mechanism of nanoparticle formation is required in order to produce uniform and highly crystalline CdSe nanoparticles. Here, the use of the word uniform refers to the particles having the same characteristics in composition, phase, size and shape. The production of uniform particles is very important due to the potential for sample inconsistency with respect to the chemical and physical properties displayed, often referred to as sample inhomogeneity [12, 13, 14].

I) Nucleation step: the nucleation stage involves the presence of many seed nuclei of CdSe nanoparticles that form upon mixing the precursors in the presence of a suitable capping agent. Whilst this initial step is important in the formation nanoparticles, it is mostly too fast to be completely understood [14].

However, it has been classified into three categories, which can be generalized for the formation of all nanoparticles, including CdSe:

A) Primary homogeneous nucleation: nucleation occurs when molecular solutes are completely dissolved in a liquid in the absence of any solid interface. This is an ideal nucleation step to produce homogeneous nanoparticles [14, 15].

B) Primary heterogeneous nucleation: nucleation induced by any solid surfaces that are present; this is not an ideal nucleation step due to the potential of introducing inhomogeneity [14, 16].

Chapter 3 Synthesis of CdSe NPs

C) Secondary nucleation: nucleation is the result of growing particles that form on the walls of the reaction container or stirrer, secondary nucleation acts to inhibit the formation of homogeneous particles [14, 17] and is therefore undesirable in the process of nanoparticle formation.

II) Growth step: after the formation of homogeneous seed nuclei, solute molecules, atoms or ions must be transported from the bulk solution to the particle surface. In this mass transport step, solute molecules are adsorbed onto the particle surface and nanoparticle growth takes place when solute molecules are integrated into the initial seed nuclei [14].

Nanoparticle growth depends on the synthetic method and occurs over an appropriate reaction time and temperature [18, 19].

The nucleation and growth mechanisms are correlated and both strongly depend on various parameters that include pH, molar ratio of precursors, appropriate capping agents, solvents, reaction time, temperature and synthetic procedure [14, 20].

The mechanism of nanoparticle formation is a complicated process due to the formation of unstable intermediate compounds during the nucleation stage. In addition, experimental parameters such as reaction time, temperature and pH can influence particle size, morphology or crystal phase formation during nanoparticle growth [17, 21].

3.1.2 Nanoparticle Structure

CdSe nanoparticles can form in two crystalline phases, namely cubic zincblende (ZB) and hexagonal wurtzite (WZ). The ZB structure has cubic unit cells in which the atoms stack in an ABCABC… pattern. In contrast, the WZ crystal structure is in a hexagonal system and the atoms in unit cells are stacked in ABAB…pattern, as shown in Figure 3.2 [22-24].

Chapter 3 Synthesis of CdSe NPs

Figure 3.2. The crystal structures in WZ (a) and ZB (b). The blue and red balls represent cadmium and selenium atoms, respectively. (c) shows ABAB…stacking in WZ and (d) illustrates ABCABC …stacking in ZB [24].

ZB and WZ crystal phases both have tetrahedral coordination about the Cd and Se ions and the difference between their associated total energy is very small, of the order of few tens of meV/atom. However, they can be easily distinguished as they have different X-ray diffraction patterns [24, 25].

The ZB crystalline phase is characterized in the PXRD with the presence of the characteristic (111), (220) and (311) reflections, referring to reflections at 2θ = 25º, 42º and 49.7º (λ = 0.154056 nm), whilst the WZ crystal structure has a broad peak at 2θ = 25º due to the overlap of the (100), (002) and (101) reflections. This is much broader than the two other observed peaks in 2θ = 42º and 49.5º that assigned to the reflections (110)

Chapter 3 Synthesis of CdSe NPs

and (112). These observed peak positions are similar to the peak positions of ZB, but WZ has two peaks at 2θ = 35.1º and 45.8º related to reflections (102) and (103) which are unique characteristics of the WZ phase and cannot be observed in ZB (Figure 3.3) [25-27].

Figure 3.3. PXRD patterns of ZB and WZ CdSe NPs [25].

The stability of WZ and ZB CdSe nanoparticles is different. ZB is the most stable phase for CdSe nanoparticles at low temperature, such as at temperatures less than 120°C [24].

However, the formation of WZ or ZB crystal phases depends on the synthetic method and the temperature [26].

3.1.3 Synthetic Methods

There are various synthetic methods based upon colloidal chemistry used to control the crystallization process to produce highly crystalline nanoparticles including CdSe nanoparticles; these include thermal decomposition methods, hydrothermal and solvothermal methods and heating. Each of these synthetic approaches has been

Chapter 3 Synthesis of CdSe NPs

proposed as a simple synthetic method of producing nanoparticles and can be utilized with the use of both organic and aqueous solvents [28-36].

A)-Thermal decomposition pathway: this method is an organic route based upon thermal decomposition reactions of organometallic compounds and metal surfactant complexes. It occurs in hot surfactant solutions. For instance, organometallic synthesis (described in Chapter 1, Section 1.4.3.1.1), is a typical example of this method [28-31].

B)-Solvothermal and hydrothermal synthesis: as discussed in Chapter 1, Sections 1.4.3.1.2 & 1.4.3.2.1, these approaches have been proposed based upon taking advantage of the high reactivity of metal salts and complexes at high temperature, leading to the formation of homogeneous and uniform particles in both aqueous and organic route [32-34].

C)-Heating up approach: in this synthetic method, the growth of nanoparticles occurs with thermal treatment of the reaction system, usually under reflux, to produce nanoparticles [35, 36].

These methods mainly involve either high temperatures or complex processes that remain challenging to find convenient conditions of nanoparticle formation [37].

In addition, the synthesis of nanoparticles using organic reactants leads to the formation of potentially toxic nanoparticles due to the presence of residual solvent molecules, which is a significant restriction in bio-applications of nanoparticles [38].

The aims of this chapter are the synthesis of CdSe nanoparticles under less harsh conditions, such as low temperatures and the use of water as a nontoxic solvent. The effects of different parameters including pH, capping agent and solvent on formation of CdSe nanoparticles were also investigated.

Chapter 3 Synthesis of CdSe NPs

3.2 Experimental Procedure

3.2.1 Synthesis

An aqueous colloidal synthetic procedure was used to form of CdSe NPs under different experimental conditions at relatively low temperature (T = 60 ºC); the resultant thiol- stabilized CdSe nanocrystals formed, as described in details in Chapter 2, Section 2.2.1.

3.2.2 Characterization

The X-ray Powder diffraction (PXRD) spectra were taken on a Philips X’pert Multipurpose X-ray Diffraction System (MPD system), high resolution transmission electron microscopy (HRTEM) was performed by a Philips CM 200 instrument and Image-j software was used to obtain Fourier fast transform (FFT) patterns and to analyse the HRTEM images. UV-vis absorption spectra were obtained with a Varian Cary UV spectrometer. The Fourier transform infra-red (FT-IR) spectroscopy was performed on Thermonicolet Avatar 370 FT-IR spectrometers. The details of the characterization techniques have been detailed in Chapter 2, Section 2.3.

3.3 Results and Discussion

3.3.1 Formation Mechanism

Thiol-capped CdSe nanoparticles can be formed in the presence of thiol capping agents, including MPA and L-cysteine.

Figure 3.4 shows the structure of these stabilizing thiols.

Chapter 3 Synthesis of CdSe NPs

(a) (b) Figure 3.4. The structures of 3-mercaptopropionic acid (a) and L-cysteine (b).

The use of capping agent limits particle growth or Ostwald ripening and promotes nucleation of thiol-capped CdSe nanoparticles through two steps, including the formation of a cadmium-thiol complex as the first step and nucleation of thiol-capped CdSe nanoparticles, to which the addition of selenide is the second step.

Step1: Formation of Cadmium-thiol complex

Thiol is a weak acid and upon de-protonation forms the thiolate anion (RS-) according to Equation 3.1:

RSH RS- + H+ (Equation 3.1)

This thiolate ion reacts with cadmium in two consecutive steps according to Equation 3.2 & 3.3, forming the dithio cadmium complex as shown in Figure 3.5 [39].

Cd 2+ + RS- (Cd-SR)+ (Equation 3.2)

+ - (Cd-SR) + RS Cd (SR) 2 (Equation 3.3)

Figure 3.5. The structure of MPA-dithio complex [39].

Chapter 3 Synthesis of CdSe NPs

It was observed that during the synthesis, the solubility of the dithio complex depended on pH due to the formation of a turbid white solution in the pH range 5.5 – 8, whilst in pH less than 5.5 or higher than 8, a clear solution was formed.

Step 2: Nucleation of thiol-capped CdSe NPs

After introducing selenide ions to thiol capped cadmium ions, CdSe-thiol capped particles can be formed according to Equation 3.4 [40]:

2+ 2- Cd(T) + Se CdSe (T) + H2O (T = Thiol capping agent) (Equation 3.4)

CdSe-capped nanoparticles tend to incorporate other Cd-thiol complexes and form larger particles [40].

It was determined that there was complete coating of the obtained CdSe nanoparticles with the capping agent, evidenced with the estimation of the numbers of molecules of capping agent on the surface of each CdSe nanoparticles. The calculation also showed that each molecule of capping agent takes up ~3.5 Å2 in surface area of each CdSe particle, as described in Appendix 3.

3.3.2 Synthesis of Cubic CdSe NPs under Different Conditions

3.3.2.1 Formation of CdSe NPs in Acidic pH

Up until now, the preparation of thiol-capped CdSe nanoparticles has been reported under basic pH due to the assumption that at high pH, carboxylate anions form by the hydrolysis of carboxyl groups in the thiol capping agent, rendering it hydrophilic [40- 45]. In addition to this, sodium selenosulfate is stable in alkaline solution because at room temperature the dismutation reaction occurs only in acidic or neutral solution, as described in Equation 3.5 [46]:

Na2SeSO3 Na2SO3 + Se (Equation 3.5)

However, according to the mechanism of formation of CdSe NPs and the high reactivity of the thiolate anion (RS-) toward cadmium ions, acidic pH promotes the formation of a

Chapter 3 Synthesis of CdSe NPs

dithiol cadmium complex [39]. Therefore, CdSe NPs were found to form even at acidic pH.

The formation of CdSe NPs showed that Na2SeSO3 is stable in acidic solution when the selenide ions were introduced to the cadmium-MPA complex.

The PXRD pattern showed that the obtained nanoparticles were in the cubic zinc blend crystalline phase, as shown in Figure 3.6. The Particle size was calculated as 2.97 nm with Scherrer equation (Appendix 4).

111

220

311 Intensity (a.u) Intensity

20 25 30 35 40 45 50 55 60 2 Theta (degree)

Figure 3.6. PXRD of as-prepared CdSe nanoparticles in acidic pH.

Three distinct diffraction peaks at 2θ values of 26.6º, 44.2º and 51.6º, corresponding to the lattice planes (111), (220) and (311) were found to match to a standard CdSe cubic pattern [47], indicating that the as-prepared CdSe NPs are highly crystalline nanoparticles.

TEM images confirmed the formation of highly crystalline particles in agreement with PXRD results, evidenced with the direct observation of lattice planes and symmetric FFT pattern. The particle size with TEM images was estimated to be about 3 nm (Appendix 5).

Chapter 3 Synthesis of CdSe NPs

The obtained particle size using Scherrer equation was in agreement with the estimated particle size from TEM images, as described in Table 3.2, Section 3.3.

The approximate d-spacing was found to be 3.2 Å, which matches the d-spacing of the (111) planes in cubic ZB, (Figure 3.7).

20 nm 5 nm

(a) (b)

d111 =0.32 nm

5 nm

Figure 3.7. TEM images of as-synthesized CdSe nanoparticles in acidic pH (a, b, c) & FFT of selected area (d).

The UV-visible absorption spectrum indicated that there is a strong absorption onset at 320 nm with a small excitonic transition at 260 nm. The onset was determined by drawing a straight line on the linear part of the graph and finding its intersection with the x axis, as shown in Figure 3.8.

Chapter 3 Synthesis of CdSe NPs

260n

onset Intensity (a.u) Intensity

220 320 420 520 620 720

Wavelength (nm)

Figure 3.8. UV-visible spectrum of as-synthesized CdSe nanoparticles in acidic pH. The intersection of two drawn lines shows the onset.

From the onset of the absorption edge, the value of the band gap energy was calculated to be 3.88 eV, a blue shift of 2.18 eV from that of standard bulk CdSe, which is 1.7 eV, indicating the formation of small particles (Appendix 6).

Besides, the UV absorption spectrum was obtained using reflux for 6 hours and showed that there is no change in the absorption of nanoparticles after heating the sample (Appendix 7).

FT-IR spectroscopy confirmed that the formation of CdSe nanoparticles in acidic pH depends on the formation of thiolate anions because according to Figure 3.9, MPA has absorbance bands at 1708, 1251 and 2574 cm-1 corresponding to C=O, C-O, S-H stretching bands and the sharp absorption at 1427 cm-1 assigned to the O-H bending band. The infrared spectrum of CdSe-MPA capped nanoparticles showed that the sharp and strong peak at 2574 cm-1, corresponding to the S-H stretching band in the mercapto group disappeared in the infrared spectrum of CdSe-MPA capped nanoparticles, indicating the formation of a new S-H bond in Cd-thiolate complexes and selenide ions on the nanoparticle surface, in accord with the formation mechanism as described in Section 3.3.1.

Chapter 3 Synthesis of CdSe NPs

Figure 3.9. FT-IR spectra of MPA (a) & CdSe-MPA capped nanoparticles (b).

In addition, the IR spectrum of the obtained nanoparticles showed two sharp peaks at 1697 cm-1 and 1422 cm-1, assigned as the C=O stretching and O-H bending, respectively. The existence of these two absorbance bands indicates that the carboxylic acid functional group in MPA is maintained, with no change in the structure of CdSe- MPA capped particles owing to the fact that the carboxylate ion cannot be formed in acidic pH and so the formation mechanism depends on the formation of thiolate ions, as discussed in Section 3.3.1.

Chapter 3 Synthesis of CdSe NPs

This observation is in accord with literature precedents in that the FT-IR spectra of the obtained MPA-capped nanoparticles in basic pH. The IR spectrum of analogous CdSe NPs in basic pH shows two absorbance bands at 1567 cm-1 and 1397 cm-1, which correspond to the asymmetric and symmetric vibrations of the COO-, respectively. The existence of both of these peaks indicates that the carboxylate ion has been formed in basic pH due to the de-protonation of the carboxylic acid group of MPA in alkaline solutions [48]. These peaks did not appear the in IR spectrum of the as-synthesized CdSe nanoparticles, further evidence that the formation of nanoparticles in acidic pH occurs based upon formation of thiolate ions.

It was found that unlike free MPA molecules, the absorbance at 1251 cm-1 assigned to the C-O stretch, was not observed in the MPA capped nanoparticles; consistent with the formation of covalent bonds between MPA and CdSe nanoparticles that restricts the C- O stretch of MPA on the surface of CdSe-MPA capped nanoparticles.

3.3.2.2 Formation of CdSe NPs in Basic pH

Although the formation of highly crystalline CdSe nanoparticles has been extensively reported in basic pH during high temperature reflux of reactants [41-44] the work performed here showed that highly crystalline CdSe nanoparticles can also be obtained at relatively low temperatures such as T = 60 °C.

PXRD showed that the CdSe NPs are in the cubic zinc blend crystalline phase, similar to those particles obtained in acidic pH, Figure 3.10. The particle size estimated using Scherre equation was found to be about 3.24 nm, as detailed in Appendix 4.

Chapter 3 Synthesis of CdSe NPs

111

220

311 Intensity (a.u) Intensity

20 25 30 35 40 45 50 55 60

2 Theta (degree)

Figure 3. 10. PXRD of the obtained CdSe NPs in basic pH.

HRTEM images further demonstrated the presence of crystalline nanoparticles, with a d-spacing determination of the observed lattice fringes of 3.34 Å, consistent with the (111) lattice plane of zinc blend. The FFT pattern of the as-synthesized nanoparticles was obtained as a symmetric pattern, further indicating that the nanoparticles are highly crystalline (Figure 3.11). Meanwhile, the particle size was estimated with TEM images to be around 3nm, as detailed in Appendix 5.

Chapter 3 Synthesis of CdSe NPs

5 nm 2 nm

(b) (a)

d111 =0.334 nm

5 nm

(d) (c )

Figure 3.11. TEM images of as-synthesized CdSe nanoparticles in basic pH (a, b, c) & FFT pattern of selected area (d).

UV absorption spectra showed that the particles have a broad absorption edge around 600 nm. Meanwhile, a weak shoulder was observed around 330 nm corresponding to first excitonic transition, the absorption onset was observed at 625 nm and the energy gap was estimated as 1.98 eV, a blue shift of 0.28 eV from bulk CdSe (Figure. 3.12, a). The calculated energy gap was in agreement with the particle size, which is further evidence of quantum confinement effects, as discussed in Section 3.3.3 and illustrated in Figure 3.22.

Chapter 3 Synthesis of CdSe NPs

The obtained nanoparticles were heated up using reflux for 6 hours at 100 ºC and the UV-visible spectrum of the nanoparticles showed that the nanoparticles have two well- defined and sharp excitonic shoulders at 280 nm and 330 nm (Figure.3.12, b).

625 nm

Figure 3.12. UV-visible spectra of as-synthesized CdSe nanoparticles in basic pH without reflux (a) & after 6 hours reflux (b).

The sharp distinct shoulder at 330 nm is attributed to the first electronic transition (1S- 1S) that occurs in CdSe nanoparticles as a pseudo HOMO-LUMO electronic transition [40]. The second excitonic shoulder at 280 nm corresponds to a higher spin-orbit component of the (1S-1S) transition. Such electronic transitions have been previously seen in ZnSe, CdSe and CdS nanoparticles, as shown in Appendix 8 in ZnSe NPs [49, 50].

Indeed, UV-absorption spectra revealed that the nanoparticles obtained after reflux are smaller than nanoparticles prepared at 60 ºC, by virtue of the band gap value for the

Chapter 3 Synthesis of CdSe NPs

nanoparticles being 3.55 eV after 6 hours of reflux, a 1.85 eV blue shift compared with bulk CdSe, indicative of a decrease in the particle size on heating the nanoparticles.

The observed excitonic transitions in as-prepared nanoparticles in basic solution indicate that heating up the nanoparticles during reflux promoted the mono-dispersity of CdSe NPs in basic pH.

3.3.2.3 Formation of CdSe NPs in Presence of L-cysteine

L-cysteine is a popular capping ligand that has a low toxicity, being classified as a semi- essential amino acid. This capping agent has two sulfur atoms in its structure that can promote the formation of sulfur bridges when acting as a capping molecule, in addition to the capacity of L-cysteine to participate in biochemical reactions [51, 52].

There are several reports in the literature that have proposed the preparation of nanoparticles including CdS, ZnSe and CdSe using L-cysteine as a capping agent [45, 53-56] but until now, no report has been found for obtaining nanoparticles with L- cysteine in the presence of MPA at the same time. We used these two thiol capping agents in an optimized molar ratio to investigate the effect of the presence of L-cysteine on formation of CdSe nanoparticles.

PXRD revealed that the obtained nanoparticles using both L-cysteine and MPA were formed in the cubic phase, indicating that the presence of L-cysteine does not influence the phase of CdSe nanoparticles, as would be expected for the action of a capping agent (Figure 3.13).

Chapter 3 Synthesis of CdSe NPs

111

220

311 Intensity (a.u) Intensity

20 25 30 35 40 45 50 55 60 2 Theta (degree)

Figure. 3.13. PXRD of the obtained CdSe NPs in presence of L-cysteine and MPA as capping agent.

HRTEM images of the as-obtained CdSe NPs showed that they are highly crystalline nanoparticles due to the observation of atomic planes within the particles and the observation a symmetric pattern in the FFT images of the nanoparticles. The d-spacing of the observed lattice planes was estimated as 3.39 Å, consistent with the (111) plane of ZB in standard cubic structure (Figure 3.14).

Chapter 3 Synthesis of CdSe NPs

2 nm 2 nm

(a) (b)

d111= 0.339 nm

2nm

Figure 3.14. TEM images of as-synthesized CdSe NPs in presence of both L- cysteine and MPA (a, b, c) & FFT pattern of selected area (d).

UV showed that there is an excitonic shoulder at 270 nm with an onset of absorption at 570 nm (Figure 3.15). The band gap energy value was calculated as 2.17 eV, a 0.48 eV blue shift compared to bulk CdSe (Appendix 6).

Chapter 3 Synthesis of CdSe NPs

270 nm Intensity (a.u) Intensity

200 300 400 500 600 700 800 Wavelength (nm)

Figure 3.15. UV-visible spectrum of as-synthesized CdSe nanoparticles in presence of L-cysteine.

Indeed, it was found that the presence of L-cysteine promotes the formation of CdSe nanoparticles, even when using MPA at low concentration, because in the absence of L- cysteine CdSe NPs were not formed, as described in Table 1.3.

Table 3.1. A comparison between formation of CdSe NPs in presence of L-cysteine and without L-cysteine

Name of CdCl2 Na2SeSO3 Capping pH product sample (mmol) (mmol) agent 1 1.2 4 5.6 (mmol) acidic Cadmium and MPA without selenium l-cysteine oxides 2 1.2 4 5.6 (mmol) acidic Cubic CdSe MPA+(3mmol) NPs l-cysteine

Chapter 3 Synthesis of CdSe NPs

3.3.2.4 Preparation of CdSe NPs in presence of Ethanol

Although pure water has been frequently used as a solvent when producing highly crystalline nanoparticles, CdSe nanoparticles were prepared in the presence of ethanol in order to investigate the influence of ethanol on the formation of the nanoparticles.

PXRD revealed once again that cubic phase nanoparticles were formed, indicating that the presence of ethanol in the aqueous solution did not influence the crystalline phase of the nanoparticles (Figure 3.16).

111

220

311 Intensity (a.u) Intensity

20 25 30 35 40 45 50 55 60 2 Theta (degree)

Figue 3.16. PXRD of the obtained CdSe NPs in presence of both ethanol and water as solvent.

HRTEM revealed that highly crystalline nanoparticles were obtained, as evidenced with direct observation of lattice planes; the d-spacing for these nanoparticles was found to be around 3.5 Å, assignable to the 111 lattice planes of the standard cubic structure, as shown in Figure 3.17.

Chapter 3 Synthesis of CdSe NPs

Nanoparticle in Nanoparticle in area B area A

5 nm 2 nm (a) (b)

d111 = 0.35 nm

2 nm

d111 = 0.357 nm

2 nm

(e) (f)

Figure 3.17.TEM images of as-synthesized CdSe NPs in presence of both ethanol and water: nanoparticles in area A, B (a, b, c, e) and their corresponding FFT (d, f).

Chapter 3 Synthesis of CdSe NPs

UV absorption, illustrated in Figure 3.18, showed that there is an excitonic transition at 260 nm which is similar to UV spectrum of the obtained CdSe NPs in basic pH after 6 hours reflux (Figure 3.12, b) whilst the onset of absorption was observed at 650 nm, similar to the nanoparticles synthesized without the use of ethanol in basic pH (Figures 3.18 and 3.12, a).

The energy band gap value was estimated to be 1.91 eV and the blue shift was 0.21 eV, similar to that of the nanoparticles synthesized in the absence of ethanol (section

3.3.2.2), which was found to be 0.28 eV (Appendix 6).

260 nm Intensity (a.u) Intensity

200 300 400 500 600 700 800 Wavelength (nm)

Figure 3.18. UV-visible spectrum of as-synthesized CdSe nanoparticles in presence of ethanol.

3.3.3 Formation of Hexagonal CdSe NPs

Preparation of hexagonal-phase CdSe nanoparticles has been reported in several works [26, 57, 58]. Whilst the formation of cubic-phase CdSe NPs has been reported at room temperature using L-cysteine under basic pH [45, 53], in this work hexagonal-phase CdSe NPs were formed using L-cysteine in acidic pH after 4.5 hours reflux at 100 ºC.

The results of PXRD confirmed formation of hexagonal phase (Figure 3.19).

Chapter 3 Synthesis of CdSe NPs

CdSeSO4.H2O (100, 002 ,101)

110

)

102 103 (200, 112, 201) Intensity (a.u Intensity

10 20 30 40 50 60

2 Theta (degree)

Figure 3.19. PXRD pattern of as-synthesized hexagonal CdSe Nanoparticles.

Five distinct peaks are attributed to reflections (100, 002, 101), (102), (110), (103), (200, 112, 201) that are consistent with the hexagonal phase. The peak at 2θ = 18º is assigned to the presence of CdSeSO4.H2O, which was not observed in the PXRD patterns of the cubic-phase CdSe nanoparticles, described in section 3.3.2.1 to 3.3.2.4 (The relevant XRD patterns of cubic CdSe NPs at 2θ = 10º to 60º, have been shown in Appendix 9).

HRTEM images showed that they are highly crystalline nanoparticles because of the observation of lattice having a d-spacing of 2.25 Å, corresponding to the (110) plane of hexagonal CdSe. Besides, FFT patterns of the obtained nanoparticles are symmetric, indicating the formation of highly crystalline nanoparticles (Figure 3.20).

Chapter 3 Synthesis of CdSe NPs

10 nm 2 nm (a) (b)

d110 = 0.225 nm

2 nm

Figure 3.20. TEM images of as-prepared hexagonal CdSe nanoparticles (a, b, c) & FFT of selected area (d).

The UV-visible absorption spectrum showed that the particles have a broad absorption edge around 610 nm. It was also observed that there is an excitonic transition at 348 nm next to the absorption onset, which is around 370 nm (Figure 3.21).

Chapter 3 Synthesis of CdSe NPs

Wavelength (nm)

Figure 3.21. UV-visible of as-prepared hexagonal NPs.

The band gap energy was calculated as 3.36 eV, a 1.66 eV blue shift from that of bulk CdSe (Appendix 6).

3.3.4 Particle size of As-prepared NPs

The particle size of the as-obtained nanoparticles was calculated using two complementary techniques. Firstly, the Scherrer equation (Chapter 2, Equation 2. 1), which is based upon the observed FWHM in PXRD patterns of nanoparticles was used [59], augmented with the estimation of particle size by direct measurement from the HRTEM images. However, the calculation of particle size with TEM is an approximation because of the size of water soluble QDs are difficult to determine by TEM due to their small dimensions and being aggregated rapidly during drying on a carbon grid in time of recording images.

Chapter 3 Synthesis of CdSe NPs

The results showed that the calculated particle size using PXRD were in agreement with the estimated particle size from TEM images as described in Table 3.2. The particle size range was found to be between 3 to 3.4 nm, approximately.

The details of calculations have been mentioned in Appendix 4 & 5.

Table 3.2 Calculation of particle size of as-synthesized CdSe NPs

Condition of XRD (nm) TEM (nm) Eg (eV) Preparation Acidic pH 2.97 3 3.88 Basic pH 3.24 3.4 1.98 In presence of 3.31 3 2.17 both MPA and l- cysteine In presence of 3.43 3.2 1.91 both water and ethanol Hexagonal CdSe 2.9 3.4 3.36 nanoparticles

It was found that pH influences the optical properties of the obtained nanoparticles. As Table 3.2 shows, changing pH from acidic to basic led to decreasing the particle size (∆ = 0.4 nm) and increasing Eg (∆= 2 eV). This significant change of Eg indicates that pH influences optical properties of CdSe NPs with changing absorption onset in UV-visible patterns of as-prepared nanoparticles (Sections 3.3.2.1 & 3.3.2.2).

It was determined that the presence of ethanol has no effect on optical properties of CdSe NPs. According to Table 3.2, the particle size of as-prepared nanoparticles in the presence of ethanol (Section 3.3.2.4) is about 0.2 nm larger than the obtained nanoparticles in absence of ethanol (Section 3.3.2.2), whilst the Eg increases about 0.07 eV, which is not a significant change. This data indicates that the presence of ethanol has no effect on both optical properties and particle size of CdSe NPs.

Chapter 3 Synthesis of CdSe NPs

Moreover, as shown in Figure 3.22 for the as-prepared cubic NPs, the correlation between the size and Eg was found to be as a linear graph. This indicates that the as- synthesized NPs exhibited the quantum confinement effect in that the increasing the particle size led to decrease in Eg.

4.5 y = -3.6302x + 14.168 4 R² = 0.833 3.5

2.5

2 Eg(eV) 1.5 1 0.5 0 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 Particle size (nm)

Figure 3.22. Correlation of particle size and Eg in as-synthesized NPs. Figure 3.22. Correlation of particle size and Eg in as-synthesized NPs (the particle size estimated from PXRD patterns, as described in Table 3.2)

3.4 Conclusion

In summary, highly crystalline cubic CdSe nanoparticles were synthesized in an aqueous pathway, under various conditions. Hexagonal-phase CdSe nanoparticles were also obtained.

In addition, the investigation of effective experimental parameters in the formation of nanoparticles allowed the following conclusions to be reached: a) Despite the general assumptions in the preparation of MPA-capped CdSe nanoparticles in basic pH, nanoparticles can be prepared in acidic pH under a different mechanism based upon controlling the concentration of thiolate ions. This is completely different to the mechanism of formation of these nanoparticles in basic pH in which the carboxylic acid functional group of MPA undergoes de-

Chapter 3 Synthesis of CdSe NPs

protonation. It was found that although the nanoparticles were formed in a different mechanism, the final formation phase of nanoparticles was not influenced. b) The formation of CdSe nanoparticles using two thiol capping agents, MPA and L-cysteine, led to the formation of highly crystalline nanoparticles that were not obtained in the absence of L-cysteine under the same experimental conditions.

C) The presence of ethanol induces the formation of highly crystalline nanoparticles, in accord with the use of pure water as the solvent.

D) It was determined that the particle size of as-synthesized NPs were in accord with their relevant estimated band gap due to quantum confinement effect.

References

[1]. Alivisatos, P. Nat. Biotechnol. 2004, 22, 47. [2]. Coe, S.; Woo, W.-K.; Bawendi, M.; Bulovic, V. Nature 2002, 420, 800. [3]. Huang, M. H.; Mao, S.; Feick, H.; Yan, H.; Wu, Y.; Kind, H.; Weber, E.; Russo, R.; Yang, P. Science 2001, 292, 1897. [4]. Wu, D.; Kordesch, M. E.; Van, P. P. G. Chem. Mater. 2005, 17, 6436. [5]. Yang, Y. A.; Wu, H.; Williams, K. R.; Cao, Y. C. Angew. Chem., Int. Ed. 2005, 44, 6712. [6]. Rodriguez-Fragoso, P.; Gonzalez, d. l. C. G.; Tomas, S. A.; Zelaya-Angel, O. Appl. Surf. Sci. 2010, 257, 581. [7]. Kalasad, M. N.; Rabinal, M. K.; Mulimani, B. G. Langmuir 2009, 25, 12729. [8]. Chen, C.-C.; Yet, C.-P.; Wang, H.-N.; Chao, C.-Y. Langmuir 1999, 15, 6845. [9]. Zhong, P.; Yu, Y.; Wu, J.; Lai, Y.; Chen, B.; Long, Z.; Liang, C. Talanta 2006, 70, 902. [10]. La Mer, V. K.; Dinegar, R. H. J. Am. Chem. Soc. 1950, 72, 4847. [11]. Park, J.; Joo, J.; Kwon, S. G.; Jang, Y.; Hyeon, T. Angew. Chem., Int. Ed. 2007, 46, 4630. [12]. Leubner, I. H. Curr. Opin. Colloid Interface Sci. 2000, 5, 151. [13]. Rao, C. N. R.; Ramakrishna, M. H. S. S.; Voggu, R.; Govindaraj, A. Dalton Trans. 2012, 41, 5089.

Chapter 3 Synthesis of CdSe NPs

[14]. Salas, G.; Costo, R.; Morales, M. d. P. In Frontiers of Nanoscience; Jesus, M. d. l. F., Grazu, V., Eds.; Elsevier: 2012; Vol. Volume 4, p 35. [15]. La, M. V. K.; Dinegar, R. H. J. Am. Chem. Soc. 1950, 72, 4847. [16]. Sugimoto, T. Adv. Colloid Interface Sci. 1987, 28, 65. [17]. Dirksen, J. A.; Ring, T. A. Chem. Eng. Sci. 1991, 46, 2389. [18]. Nann, T.; Riegler, J. Chem. - Eur. J. 2002, 8, 4791. [19]. Williams, J. V.; Adams, C. N.; Kotov, N. A.; Savage, P. E. Ind. Eng. Chem. Res. 2007, 46, 4358. [20]. Li, L.; Qian, H.; Fang, N.; Ren, J. J. Lumin. 2005, 116, 59. [21]. Vairavamurthy, M. A.; Goldenberg, W. S.; Ouyang, S.; Khalid, S. Mar. Chem. 2000, 70, 181. [22]. Yeh, C. Y.; Lu, Z. W.; Froyen, S.; Zunger, A. Phys. Rev. B: Condens. Matter 1992, 46, 10086. [23]. Lim, S. J.; Chon, B.; Joo, T.; Shin, S. K. J. Phys. Chem. C 2008, 112, 1744. [24]. Datta, S.; Saha-Dasgupta, T.; Sarma, D. D. arXiv.org, e-Print Arch., Condens. Matter 2011, 1. [25]. Erwin, S. C.; Zu, L.; Haftel, M. I.; Efros, A. L.; Kennedy, T. A.; Norris, D. J. Nature .2005, 436, 91. [26]. Soni, U.; Arora, V.; Sapra, S. Cryst Eng Comm 2013, 15, 5458. [27]. The International Centre for Diffraction Data (ICDD), 1978, JCPDS files, No.88- 2346 & No.772307. [28]. Wang, X.; Zhuang, J.; Peng, Q.; Li, Y. Nature 2005, 437, 121. [29]. Murray, C. B.; Norris, D. J.; Bawendi, M. G. J. Am. Chem. Soc. 1993, 115, 8706. [30]. Hudry, D.; Apostolidis, C.; Walter, O.; Gouder, T.; Courtois, E.; Kuebel, C.; Meyer, D. Chem. - Eur. J. 2012, 18, 8283. [31]. Tian, Q.; Tao, K.; Li, W.; Sun, K. J. Phys. Chem. C 2011, 115, 22886. [32]. Williams, J. V.; Adams, C. N.; Kotov, N. A.; Savage, P. E. Ind. Eng. Chem. Res. 2007, 46, 4358. [33]. Bu, H.-B.; Kikunaga, H.; Shimura, K.; Takahasi, K.; Taniguchi, T.; Kim, D. Phys Chem Chem Phys 2013, 15, 2903. [34]. Cao, Y.; Hu, P.; Jia, D. Appl. Surf. Sci. 2013, 265, 771.

Chapter 3 Synthesis of CdSe NPs

[35]. Xu, Z.-C.; Shen, C.-M.; Yang, T.-Z.; Zhang, H.-R.; Li, H.-L.; Li, J.-Q.; Gao, H.-J. Chem. Phys. Lett. 2005, 415, 34. [36]. Wang, K.; Liu, J.; Yang, X.; Zhang, P.; Hunan University, Peop. Rep. China . 2008, p 11pp. [37]. Oluwafemi, O. S. Colloids Surf B Biointerfaces 2009, 73, 382. [38]. Hardman, R. Environ Health Perspect 2006, 114. [39]. Vairavamurthy, M. A.; Goldenberg, W. S.; Ouyang, S.; Khalid, S. Mar. Chem. 2000, 70, 181. [40]. Liu, P.; Wang, Q.; Li, X. J. Phys. Chem. C 2009, 113, 7670. [41]. Zhang, S.; Yu, J.; Li, X.; Tian, W. Nanotechnology 2004, 15, 1108. [42]. Chen, X.; Hutchison, J. L.; Dobson, P. J.; Wakefield, G. J. Mater. Sci. 2009, 44, 285. [43]. Gupta, P.; Ramrakhiani, M. Open Nanosci. J. 2009, 3, 15. [44]. Wageh, S.; Higazy, A. A.; Hassouna, A. S. J. Mater. Sci.: Mater. Electron. 2013, 24, 3049. [45]. Sivasankar, K.; Padmavathy, N. Micro Nano Lett. 2011, 6, 144. [46]. Liu, L.; Peng, Q.; Li, Y. Nano Research 2008, 1, 403. [27]. The International Centre for Diffraction Data (ICDD), 1978, JCPDs files, No. 19- 0191. [48]. Saikia, K.; Deb, P.; Kalita, E. Physica Scripta 2013, 87, 065802. [49]. Bawendi, M. C.; Steigerwald, M. L.; Brus, L. E. Annu. Rev. Phys. Chem. 1990, 41, 477. [50]. Chestnoy, N.; Hull, R.; Brus, L. E. J. Chem. Phys. 1986, 85, 2237. [51]. Ayyar, R. R. Z. Kristallogr. 1967, 126, 227. [52]. Roux, M. V.; Foces-Foces, C.; Notario, R.; Ribeiro, d. S. M. A. V.; Ribeiro, d. S. M. d. D. M. C.; Santos, A. F. L. O. M.; Juaristi, E. J. Phys. Chem. B 2010, 114, 10530. [53]. Feng, B.; Teng, F.; Tang, A.-W.; Wang, Y.; Hou, Y.-B.; Wang, Y.-S. J. Nanosci. Nanotechnol. 2008, 8, 1178. [54]. Qu, J.; Zhu, Z.; Bai, X.; Qu, J. Nanosci. Nanotechnol. Lett. 2013, 5, 1051. [55]. Ozturk, S. S.; Selcuk, F.; Acar, H. Y. J. Nanosci. Nanotechnol. 2010, 10, 2479.

Chapter 3 Synthesis of CdSe NPs

[56]. Xia, W.; Zhang, X.; Zhou, L.; Zhou, W.; Ou, Y.; Liang, L. ECS Solid State Lett. 2013, 2, R41. [57]. Kumar, P.; Singh, K. Curr. Nanosci. 2010, 6, 89. [58]. Ramalingam, G.; Madhavan, J. Arch. Appl. Sci. Res. 2011, 3, 217. [59]. Patterson, A. L. Phys. Rev. 1939, 56, 978.

Chapter 4

Synthesis of Water Soluble Quantum Dots (QDs)

4.1 Introduction

As discussed in Chapter 1, Section 1.6.2, quantum dots are potentially useful imaging tools in a range of biological and medical applications. They are very good candidate materials for medical imaging, assisting as markers, in the detection of cancer and in the active delivery of chemotherapy drugs to cancer cells [1-5]. In order to make use of QDs in biological systems, they should be both water soluble and bio-compatible [6]. There are many different methods for the synthesis of water soluble QDs, including ligand exchange methods, silanization, surface coating of QDs with amphiphilic polymers and the direct synthesis of water soluble QDs via aqueous routes.

4.1.1 Ligand Exchange Method

Ligand exchange methods involve replacing organic hydrophobic ligands on the surface of QDs with hydrophilic and bio-functional ligands. The QDs synthesized in organic reactions tend to have hydrophobic ligands such as trioctylphosphine oxide (TOPO), trioctylphosphine (TOP), oleic acid and others at the surface [7-12]. These hydrophilic ligands bind to metal atoms on the QD surface but can be replaced by stronger hydrophobic ligands with a higher binding affinity in so-called ligand exchange reactions [13-15]. They have been widely used to produce water soluble QDs by transferring QDs from organic solvents to aqueous solvents. For example, mercapto- alkyl carboxyl ligands are monodentate thiols and the mercapto group in their structure has a high binding affinity for the surface atoms of the QDs, rendering the QDs water soluble [16-18].

Chapter 4 Synthesis of Water Soluble QDs

Figure 4.1 shows the synthesis of water soluble TOPO-capped CdSe QDs via replacement of TOPO with 3-mercaptopropionic acid (MPA) [19].

Ligand Exchange

QDs in organic solvents QDs in aqueous solvents

Figure 4.1. Ligand exchange method in synthesis of water soluble CdSe QDs [19].

The other advantage of this method is providing further functionality for coupling of QDs with biomolecules [20]. For example, CdSe/ZnS-TOPO capped QDs after surface functionalization with dihydrolipoic acid (DHLA) through a ligand exchange reaction have homogenous atomic orientations to bind to microphage bone protein (MBP), as shown in Figure 4.2 [21].

Besides, the functional group in the structure of DHLA can be activated through EDC coupling reactions to form bioconjugated compounds consisting of QDs and biomolecules [22].

Chapter 4 Synthesis of Water Soluble QDs

MBP

Figure 4.2. CdSe/ZnS QDs (blue) modified with DHLA (red) with homogenous orientation to bind with MBP [21].

4.1.2 Silazination

The silanization, or silica coating, method is modification of hydrophobic QDs with a layer of hydrophilic silica to form water soluble and stable QDs [23].

The formation of a silica shell around the QDs by the replacement of hydrophobic domains of QDs with organosilicone molecules incorporating thiols, amines or carboxylic acids into the silica shell, provides surface functionality, stability and water solubility for QDs [24].

Modification of the silica shells occurs in a second step to provide QD biocompatibility [25,26]. For example, TOPO-coated CdSe/ZnS QDs have been mixed with mercaptopropyltrimethoxysilane (MPTS); the mercapto group was found to bind to the ZnS shell, substituting the TOPO. The silanol groups form a siloxane shell on the QDs surface, thus encapsulated silica QDs can be functionalized with different functional groups to attach to biomolecules [25]. Sillanization can occur in sol-gels, micellanization and microemulsion methods [27-31].

The sol gel process can be viewed as a combination of ligand exchange and silica coating methods in which organic ligands on the surface of the QDs are replaced with water soluble ligands and silica shell forms after adding tetraethylorthosilicate (TEOS) or silanes with functional groups including amines, thiols or carboxylic acids. These can

Chapter 4 Synthesis of Water Soluble QDs

partition the QDs from organic solvents to protic solvents, thus providing further functionalization for QDs to apply in biological environments [14, 31, 32].

The micellanization method is a silica coating procedure based upon formation of hydrophilic micelles from siloxane surfactants. The hydrophobic core of micelles stabilizes and encapsulates hydrophobic QDs and creates silica shell on QDs surface while the hydrophilic tail of the surfactant enables QDs to transfer from organic to aqueous phase [27].

The microemulsion method is essentially the hydrolysis of a silica precursor on the surface of the hydrophobic QDs in a water-oil microemulsion. The products are single QDs encapsulated in silica spheres which can be used for bio applications [29, 33].

4.1.3 Surface Coating of QDs in Amphiphilic Polymers

Coating of QDs with amphiphilic polymers is another method of forming water soluble QDs. This method is based upon the formation of a uniform polymeric coating around the QDs and involves interactions between the hydrophobic parts of the ligands on the QDs surface and hydrophobic parts of the polymer, meanwhile the hydrophilic part of the polymer provides water solubility [34, 35].

4.1.4 Direct Synthesis of QDs in Water

Unlike organic-approaches, water soluble QDs can be synthesized in the aqueous phase without additional processes for rendering the QDs water soluble. This method is based upon using hydrophilic stabilizing thiols or phosphates as capping agents, resulting in QDs coated with hydrophilic ligands with functional groups, to which the biomolecules can be attached after further modifications [36-40]. Several common capping agents used in synthesising QDs in water are: 3-mercaptopropionic acid (MPA), L-cysteine, thioglycolic acid (TGA), 2-mercaptoethylamine, 2-dimethylamino ethanethiol and 2- mercaptorthanol [40, 41].

As discussed in Chapter 1, Section 1.4.3.2, until now, a wide range of QDs such as CdS, ZnSe, CdSe, CdSeZn and CdTe have been synthesized through aqueous direct synthesis with various stabilizers and precursors, using different thermal conditions [42-50]. Both 97

Chapter 4 Synthesis of Water Soluble QDs

hydrothermal or microwave assisted methods have been used to prepare water soluble QDs using water as a solvent, making them more suited to bio applications [51-54]. However, there are some restrictions to this, such as low stability, poor photo properties, decreased quantum yield and limited reproducibility [55-59].

This chapter details the synthesis and characterization of water soluble CdSe(S) QDs using a modified literature method [53], resulting in QDs having high quality optical properties. In addition, ZnSe(S) QDs were obtained in a water-based hydrothermal method aimed at reducing the toxicity of the final QDs by eliminating the use of both cadmium and viscous organic solvents. Figure 4.3 shows the obtained QDs under UV light [Sections 4.3.1, 4.3.2 and 5.3.1].

Figure 4.3. The synthesized QDs glowing under UV light (λ excitation = 365 nm): ZnSe(S) QDs (blue), CdSe(S)/ZnO QDs (green) and CdSe(S) QDs (yellow, orange and red ).

Chapter 4 Synthesis of Water Soluble QDs

4.2 Experimental Procedure

4.2.1 Chemical Synthesis

Water soluble CdSe(S) QDs (Samples 1 to 3) and ZnSe(S) QDs (Sample 4), were synthesized via an aqueous hydrothermal method, as described in Chapter 2, Sections 2.2.2.2 & 2.2.2.3.

4.2.2 Characterization

The X-ray Powder diffraction (PXRD) patterns were obtained on a Philips X’pert Multipurpose X-ray Diffraction System (MPD system), TEM micrographs and SAED images were recorded by a Philips CM200 instrument and Image-j software was used to obtain Fourier fast transform (FFT) patterns. UV-vis absorption spectra were obtained with a Varian Cary UV spectrometer. Photoluminescence spectroscopy was conducted on a custom-built microscope coupled to an Acton 2300 spectrometer using an excitation laser at 514.5 nm for CdSe(S)QDs and with a J/B SPEX 270M spectrometer using a power max PMT detector and excitation laser at 350 nm for ZnSe(S)QDs. Fourier transform infra-red (FT-IR) measurements were performed on Thermonicolet Avatar 370 FT-IR spectrometers. X-Ray photoelectron spectroscopy (XPS) was performed on an Escalab 250i XL spectrometer. Dynamic light scattering studies were conducted using a Malvern Instruments Zetasizer Nano ZS instrument. The details of characterization techniques have been detailed in Chapter 2, section 2.3.

4.3 Results and Discussion

4.3.1 Synthesis of CdSe(S) QDs

Direct aqueous synthesis of CdSe nanocrystals employing thiols as the stabilizer has been widely used [60-67] in addition to the aqueous synthesis of CdTe, CdS and ZnSe QDs [68-71]. There have been several reports in the literature that use a common experimental method; this focuses on the initial formation of a complex compound between inorganic precursors of cadmium and the thiols that react to form CdSe QDs

Chapter 4 Synthesis of Water Soluble QDs

upon addition of a selenide precursor in the appropriate thermal conditions including heating under long time reflux, hydrothermal or microwave assisted method, leading to formation of QDs [46, 53, 57, 66-69, 72]. However, the crystal quality, stability and optical properties of the QDs obtained using such direct aqueous methods was found to depend on physical and chemical parameters such as the molar ratio of precursors to capping agent, pH, reaction time and temperature.

In this work, the synthetic method was based on hydrothermal reactions, described in the literature [51, 53, 60, 61] and as such has some similarity with previous work by Aldeek and co-workers. However, after repeating experiments in different molar ratios, the molar ratio of cadmium to the capping agent was selected as 1:2 instead of 1:1 as used previously [53]. This simple modification led to the formation of highly crystalline CdSe(S) QDs that suffered less from aggregation and had a narrow emission; this is a distinct improvement over the previous report [53], highlighting that the experimental parameters directly influence the crystallinity and optical properties of the resultant CdSe(S) QDs.

As described in Chapter 2, Section 2.2.2.2, sample 1 was selected to be fully characterized and the following results were obtained.

HRTEM images revealed that the CdSe(S) QDs were spherical (Figure 4.4) and crystalline; the direct observation of atomic planes of d-spacing equal to 3.51 Å matches that of (111) planes in cubic ZB CdSe (Figure 4.5, a & b). The particle size was estimated to be 3.4 nm from TEM images, as detailed in Appendix 5, B. Besides, the FFT of as-synthesized QDs was obtained as a highly symmetric pattern, further evidence for formation of highly crystalline QDs (Figure 4.5, d).

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Chapter 4 Synthesis of Water Soluble QDs

5 nm

Figure 4.4. HRTEM of spherical CdSe(S) QDs.

5 nm 2 nm

(a) (b)

5 nm

(c) (d) Figure 4.5. Vertical atomic planes in as-synthesized CdSe(S) QDs (a, b) & A single particle and its corresponding FFT (c, d).

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PXRD results were in accord with the TEM images, showing that the CdSe(S) QDs were highly crystalline spherical nanoparticles of cubic symmetry, in accord with the standard pattern of cubic CdSe [73, 41].

The high crystallinity of the QDs is contrasted by the amorphous nature of the CdSe- MPA yellow complex prior to hydrothermal reaction; whilst 1h hydrothermal treatment was found to yield highly crystalline cubic QDs (Figure 4.6, a & b).

111

220

311 Intensity (a.u) Intensity

a 20 30 40 50 60 70 80 2 Theta (degree)

Figure 4.6. PXRD from amorphous CdSe-MPA yellow complex without hydrothermal treatment (a) & CdSe(S) QDs after 1 hour hydrothermal treatment (b).

By performing a series of reactions (described in Chapter 2, Section 2.2.2.2, I), it was determined that reaction time was not a factor in determining the crystalline phase of the CdSe (S) QDs, evidenced by the formation of cubic QDs after 4h, 20h and 73h of hydrothermal reaction time, as shown in Figure 4.7.

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Chapter 4 Synthesis of Water Soluble QDs

Intensity (a.u) Intensity b

20 30 40 50 60 70 80

2 Theta (degree)

Figure 4. 7. PXRD of CdSe(S) QDs in different reaction times under hydrothermal conditions: 4h (a), 20h (b) and 73h (c).

The average of crystallite size of the QDs after 1h hydrothermal treatment was calculated as 3.56 nm using the Scherrer equation [74, Appendix 4. 1]. The obtained particle size was in agreement with the estimated particle size from TEM images which was estimated as 3.4 nm.

The selected area electron diffraction pattern (SAED) appeared as sharp rings, indicating that the mean particle size of the QDs was much smaller than the electron spot size, which is in accord with the particle size calculated from PXRD. The distance between planes that could be derived from the SAED patterns agreed with PXRD data and both diffraction patterns indicated the existence of 111, 220 and 311 planes in the structure of cubic CdSe QDs (Appendix 11). Figure 4.8 shows the typical SAED image of the obtained CdSe(S) QDs.

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Chapter 4 Synthesis of Water Soluble QDs

Figure 4.8. SAED pattern of as-prepared CdSe(S) QDs.

Optical spectroscopy revealed that the obtained nanoparticles had a broad absorption excitation starting at 550 nm and a narrow emission spectrum at 580 nm, corresponding to their orange-red colouration, as shown in Figure 4.9.

Figure 4.9. Optical spectra of CdSe(S) QDs after 1h hydrothermal reaction time:

(a) UV/Vis absorption and (b) photoluminescence spectrum (λ excitation = 514 nm).

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Chapter 4 Synthesis of Water Soluble QDs

From UV-spectra, the particle size of CdSe QDs can be obtained using the Yu formula, as described in Equation 4.1.

D = (1.6122 ⨯ 10-9 ) λ4 – (2.6575 ⨯ 10-6 ) λ3 + (1.6242 ⨯ 10-3 ) λ2 – (0.4277) λ + (41.57)

(Equation 4.1)

Where D is the size of the nanocrystals (nm) and λ is the wavelength of the first excitonic absorption peak of the corresponding sample.This formula is an empirical equation and can be used only for highly luminescent nanoparticles in similarity with Yu’s experimental products [75].

The average particle size of as-prepared QDs obtained as 3.04 nm (λ = 550 nm) according to the formula of Yu, in agreement with the calculated particle size either in TEM images or using the Scherrer equation.

FT-IR spectroscopy identified the presence of MPA absorbance bands at 1708, 1251 and 2574 cm-1, corresponding to the C=O, C-O and S-H stretching bands, along with the sharp absorption at 1427 cm-1 assigned to the OH bending. These peaks all disappeared in the infrared spectrum of CdSe–MPA capped QDs, indicating the ionization of carboxylic functional groups in basic pH and formation of new covalent bonds between sulfur in the structure of MPA and the surface of CdSe QDs.

In addition, two sharp and strong peaks at 1558 and 1402 cm-1, corresponding to the symmetric and asymmetric COO- stretches, appeared in the FT-IR spectrum of CdSe MPA-capped QDs, indicating that the carboxylic acid functional group of MPA had ionized in basic pH and MPA was bound to Cd through the thiol group. The observed peak at 1641 cm-1 in CdSe-MPA capped QDs is assigned to the -OH stretching bond of water (Figure 4.10).

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Figure 4.10. FT-IR spectra of MPA (a) and CdSe(S) QDs (b).

The presence of a signature of MPA in the FT-IR spectrum of the QDs is in accord with the appearance of peaks characteristic of S2p at 161.8 eV and three peaks for 3C1S at 284.5, 286.5, and 288.5 eV in XPS data; thereby confirming the presence of sulfur and three atoms of carbon from 3-mercaptopropionic acid (MPA) in the structure of QDs (Figure 4.11, a & b).

XPS data also show peaks typical of Cd3d at 404 eV and Se3d at 52 eV, confirming that cadmium and selenium are the main elements in the formation of the obtained QDs (Figure 4. 11, c and d). Meanwhile, the XPS data showed the atomic percentage of elements in the obtained QDs, as described in Table 1, Appendix 12.

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Chapter 4 Synthesis of Water Soluble QDs

Binding energy (eV) Binding energy (eV) (b) (a)

Binding energy (eV) Binding energy (eV)

Figure 4.11. XPS spectra of CdSe(S) QDs binding energy of (a) S2p, (b) C1s , (c)

Cd3d and (d) Se3d.

It is also in agreement with previous reports in the literature where MPA releases sulfur at high temperature [57, 58], resulting in sulfur from the 3-mercaptopropionic acid (MPA) participating the growth of CdSe(S) particles.

The particle size distribution was determined using DLS and the obtained size distribution histograms showed that the QDs are well dispersed in water. The average size of MPA-capped CdSe(S) QDs in aqueous solution is about 7.16 nm with narrow size distribution. Figure 4.12 shows a typical histogram of as-prepared QDs.

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Chapter 4 Synthesis of Water Soluble QDs

Figure 4.12 Size distribution histogram of CdSe(S) QDs.

The hydrodynamic diameter is about two times larger than the estimated size with HRTEM, UV and PXRD due to the fact that the hydrodynamic diameter contains organic molecules on the surface of QDs, originating from capping agent (MPA), whereas HRTEM, PXRD and UV reflect the particle size of the inorganic core of the nanoparticles [76].

Photoluminescence spectra were recorded for QDs obtained under different experimental conditions (Figures 4.13 to 4.15), from which it appears as though some reaction parameters influence the eventual photoluminescence of the QDs:

A)- Thermal Conditions: the photoluminescence spectrum of as-prepared CdSe(S) QDs using reflux (Chapter 2, Section 2.2.2.2, II), indicated that the thermal conditions strongly influence the optical properties of the QD such that simple refluxing of the CdSe–MPA complex for 3 hours yielded QDs in which the photoluminescence was significantly diminished. As shown in Figure 4.13, as-prepared CdSe(S) QDs using reflux (Sample 2) had a weak photoluminescence when compared with CdSe(S) QDs after 1 h hydrothermal treatment (Sample 1, Chapter 2, Section 2.2.2.2).

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Chapter 4 Synthesis of Water Soluble QDs

Figure 4.13. Photoluminescence spectra CdSe(S) QDs prepared with conventional

heating up (red) and after 1hour hydrothermal treatment (black) (λ excitation = 514 nm).

B)-Effect of reaction time: the photoluminescence spectra of as-prepared QDs after different reaction times (described in Chapter 2, Section 2.2.2.2, I), indicated that there is no photoluminescence from the CdSe-MPA complex prior to hydrothermal reaction. The shortest hydrothermal reaction time studied was 30 mins, after such time, photoluminescence consisting of a narrow emission profile was observed; however the emissive intensity was observed to diminish with reaction times longer than 1h, suggesting that the step is essential to form QDs but prolonged heating may result in sample aggregation. At increased hydrothermal reaction times of 4h, 20h and 73h, quenching of the QD photoluminescence was observed (Figure 4.14).

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Chapter 4 Synthesis of Water Soluble QDs

Figure 4.14. Photoluminescence spectra of CdSe(S) QDs prepared in different hydrothermal reaction times: (a) CdSe-MPA complex without hydrothermal treatment and (b), (c), (d), (e), (f) show spectra of CdSe(S) QDs prepared after 73,

20 , 4 , 1 and 0.5 hour hydrothermal treatment (λ excitation = 514 nm).

C)-Effect of Changing molar ratio of cadmium to capping agent: the photoluminescence spectra of as-synthesized CdSe(S) QDs ( Samples 1 to 3, described in Chapter 2, Section 2.2.2.2 ), revealed that changing the molar ratio of cadmium to capping agent (MPA), led to a shift in the emission profile of the CdSe(S) QDs, as summarized in Table 4.1.

Table 4.1. The effect of molar ratio of Cd to MPA on the optical properties of

CdSe(S) QDs (λ excitation = 350 nm).

Sample Molar ratio of λ emission Colour under UV

Cd: MPA (mmol) (nm) light 1 6.5: 13 580 Red 2 8: 13 572 Orange 3 6.5: 8 560 Yellow

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Chapter 4 Synthesis of Water Soluble QDs

This change in emission wavelength of QDs was observed as a change of the colour of as-synthesized QDs under UV light, as illustrated in Figure 4.15.

As-synthesized CdSe(S) QDs under UV light

Figure 4.15. Photoluminescence spectra of the synthesized CdSe(S) QDs in

different molar ratios of Cd to MPA (λ excitation = 350 nm): (a) Sample 1, (b) Sample

2, (c) Sample 3 and (inset) as-prepared CdSe(S) QDs under UV light (λ excitation = 365 nm): from left to right: yellow (Sample 3), (orange) Sample 2 and red (Sample 1).

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Chapter 4 Synthesis of Water Soluble QDs

4.3.2 Synthesis of Water Soluble ZnSe(S) QDs

The synthesis of semiconducting zinc selenide nanocrystals has attracted remarkable attention over the past decade due to their potential applications in blue lasers, light emitting diodes, photo detectors and full colour displays, along with its considerable attraction as heavy-metal free nanoparticles having a wide band gap energy (2.7 eV) at room temperature, enabling it to be used as organic passivation shells for a wide range of core/shell semiconductor nanoparticles [57, 77-81].

Up until now, there have been several reports in the literature that use a common experimental method; this focuses on the initial formation of a complex compound between inorganic precursors of zinc and the thiols that react to form ZnSe QDs upon addition of a selenide precursor in the appropriate thermal conditions including heating under long time reflux or microwave assisted method, leading to aqueous formation of

QDs. Ren [82] reported preparation of ZnSe(S) QDs from ZnCl2 and NaHSe in presence of MPA as capping agent using reflux at 95ºC in high pH, Shavel [44] reported formation of ZnSe(S) QDs using ZnClO4 and Al2Se3 after refluxing at 100ºC, Kalita [83] reported formation of ZnSe(S) QDs in presence of both MPA and hydrazine after refluxing the reaction system for 24 hours, Senthilikumar [45] prepared ZnSe QDs in presence of hydrazine and ethylene glycole in the absence of thiol capping agents using reflux and Chang reported formation of ZnSe(S) QDs in presence of thiol capping agents after refluxing at 160ºC in oil bath for 9 hours [84]. Meanwhile, Qian [57] and Xin [85] utilized microwave assistant method to prepare ZnSe(S) QDs. The hydrothermal approach has also been used in various reports including synthesis of ZnSe hollow nanospheres in presence of hydrazine [77], formation of ZnSe/ZnS QDs in the presence of N-acetyl–l-cysteine [86] and production of ZnSe nanoparticles via a reaction between zinc and selenium powder in the absence of capping agent [87] but preparation of ZnSe(S) QDs via hydrothermal route in the presence of 3- mercaptopropionic acid (MPA) has not been reported previously.

The current experimental approach in the synthesis of ZnSe(S) QDs is based upon taking advantage of hydrothermal reactions to make highly luminescent QDs and focuses on easily-controlled parameters such as reaction time, reactant molar ratios and pH.

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PXRD data confirmed that the obtained ZnSe(S) QDs, were cubic in structure (Figure 4.16) and the particle size calculated with Scherrer equation was 2.59 nm (Appendix 10.2).

111

220

311 Intensity ( a.u) ( Intensity

20 30 40 50 60 70 80

2 Theta (degree)

Figure 4.16. PXRD pattern of ZnSe(S) QDs.

Optical spectroscopy data revealed that the as-synthesized ZnSe(S) QDs have a wide excitation absorption band below 380 nm and a narrow emission band at 420 nm, in accord with blue nanoparticles and in agreement with the calculated particle size of 2.59 nm obtained from PXRD patterns (Appendix 10.2). The obtained QDs had blue emission under UV light, as shown in Figure 4.17. Meanwhile, both the estimated particle size and the observed blue shift (420 nm) were in accord with quantum confinement effect because when the particle size decreased around 2.6 nm, the emission peak was blue shifted when compared with bulk ZnSe, as previously reported in several works [83, 86, 88, 89].

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Chapter 4 Synthesis of Water Soluble QDs

Wavelength (nm)

Figure 4.17. Optical spectra of ZnSe(S) QDs: (a) UV/Vis absorption, (b)

photoluminescence spectrum (λ excitation = 350 nm) and (inset) ZnSe(S) QDs under

UV light (λ excitation = 365 nm).

HRTEM images of the as-prepared ZnSe(S) QDs showed spherical nanoparticles with clearly visible atomic planes, indicating high crystallinity of the nanoparticles. The d- spacing was determined to be 3.53 Å, approximately equal to the d-spacing of the (111) lattice plane of cubic ZnSe (Figure 4.18).

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5 nm

(a)

2 nm (c)

(b)

Figure 4. 18. HRTEM images of ZnSe(S) QDs (a), (b) & vertical planes (c).

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Chapter 4 Synthesis of Water Soluble QDs

XPS data revealed the presence of zinc and selenium as the main elements in the structure of the QDs due to the characteristic peaks of Zn2p at 1021.5 eV and Se3d at 54.5 eV. However, sulfur from the mercaptan group in MPA was detected at 163.5 eV and three distinct carbons at 285, 286.5 and 288.5eV, corresponding to the binding energies of C-S, C=O and C-H, indicating that ZnSe(S) QDs have MPA in their structure (Figure 4.19, a-d) similar to CdSe(S) QDs (Figure 4.11, a-d).

Figure 4.19. XPS spectra of ZnSe(S) QDs binding energy of (a) Zn3p,(b) Se3d , (c)

S2p and (d) C1s.

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Chapter 4 Synthesis of Water Soluble QDs

FT-IR spectroscopy confirmed the binding of MPA in the ZnSe(S) QDs, in agreement with the XPS results, suggesting an analogous mechanism for formation both CdSe(S) and ZnSe(S) QDs. As Figure 4.20 shows, the absence of absorbance bands at 1708, 1251 and 2574 cm-1, corresponding to C=O, C-O and S-H stretching bands, in addition to the sharp absorption at 1427 cm-1 assigned to OH bending, indicates new covalent bonds between sulfur in the structure of MPA and the surface of ZnSe –MPA capped QDs. Meanwhile, two sharp and strong peaks in 1560 cm-1 and 1406 cm-1 corresponding to symmetric and asymmetric COO-, appeared in the FT-IR spectrum of ZnSe-MPA capped QDs, indicating that the ionization of carboxylic functional group of MPA involved with QDs in basic pH in agreement with IR spectroscopy data of CdSe-MPA capped QDs (Figure 4.20). Meanwhile, the obtained FT-IR spectrum was in agreement with the result of IR spectroscopy of the obtained ZnSe QDs with refluxing the reaction system, as previously reported [90].

-1 Wavenumbers (cm )

Figure 4.20. FT-IR spectra of ZnSe(S) QDs.

Consequently, XPS and IR spectroscopic data for CdSe(S) and ZnSe(S) QDs suggests a similar behaviour for both types of nanocrystals under an aqueous hydrothermal condition.

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4.4 Conclusion

In this work, literature procedures of synthesizing CdSe(S) QDs in aqueous media were modified, resulting water soluble and stable QDs having highly luminescent properties. It was clearly shown that increasing the molar ratio of cadmium to the capping agent promotes the formation of highly crystalline CdSe(S) QDs [91].

UV-blue emitting ZnSe(S) QDs were also synthesized using hydrothermal methods and aqueous reactions. It was determined that the obtained QDs are highly crystalline, stable nanoparticles with highly luminescent properties. As such, hydrothermal conditions have been shown to lead to QDs displaying both high optical quality and water solubility.

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[76]. Wang, J.; Han, H. J. Colloid Interface Sci. 2010, 351, 83. [77]. Jiang, C.; Zhang, W.; Zou, G.; Yu, W.; Qian, Y. Nanotechnology 2005, 16, 551. [78]. Quinlan, F. T.; Kuther, J.; Tremel, W.; Knoll, W.; Risbud, S.; Stroeve, P. Langmuir 2000, 16, 4049. [79]. Zheng, Y.; Yang, Z.; Ying, J. Y. Adv. Mater. 2007, 19, 1475. [80]. Pradhan, N.; Battaglia, D. M.; Liu, Y.; Peng, X. Nano Lett. 2007, 7, 312. [81]. Dong, B.; Cao, L.; Su, G.; Liu, W. Chem. Commun. 2010, 46, 7331. [82]. Zan, F.; Ren, J. Luminescence 2010, 25, 378. [83]. Saikia, K.; Deb, P.; Kalita, E. Physica Scripta 2013, 87, 065802. [84]. Lan, G.-Y.; Lin, Y.-W.; Huang, Y.-F.; Chang, H.-T. J. Mater. Chem. 2007, 17, 2661. [85]. Bailon-Ruiz, S.; Perales-Perez, O.; Su, Y.-f.; Xin, Y. Curr. Nanosci. 2013, 9, 117. [86]. Zhao, D.; Li, J.-T.; Gao, F.; Zhang, C.-l.; He, Z.-k. RSC Adv. 2014, 4, 47005. [87]. Wang, M.; Hao, H.; Xi, Z.; Gong, H.; Yao, X. Ferroelectrics 2007, 356, 220. [88]. Shu, C.; Huang, B.; Chen, X.; Wang, Y.; Li, X.; Ding, L.; Zhong, W. Spectrochim. Acta, Part A 2013, 104, 143. [89]. Peng, L. L.; Wang, Y. H.; Li, C. Y. J. Nanosci. Nanotechnol. 2010, 10, 2113. [90]. Wang, C.; Gao, X.; Ma, Q.; Su, X. J. Mater. Chem. 2009, 19, 7016. [91]. Mirnajafi, F.; Wang, F.; Reece, P.; Stride, J. A. Nanotech. 2012, 1, 441.

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Chapter 5

Synthesis of Water Soluble Core/shell Quantum Dots (QDs)

5.1 Introduction

Core/shell QDs incorporate the novel properties of QDs such as a high photostability and fluorescence. They are potentially advantageous due to having lower toxicity than uncoated QDs. Core/shell QDs are the product of additional engineering of the nanocrystal structures, passivating the surface of QDs by coating them with suitable materials, resulting in core/shell QDs [1-5]. Such coatings may promote their optical and physical properties due to the fact that the shell may protect the QD core from oxidation, degradation and photoionization, leading to greater photo stability [6-8]. Moreover, by controlling the epitaxial growth of the shells, core/shell nanocrystals with various shell thicknesses can be made [9, 10]. As described in Chapter 1, Section 1.5, core/shell QDs can be formed either via organic or aqueous synthetic routes [11-15].

Surface coating of QDs with other semiconductors is a common approach for the synthesis of new types of nanoparticles, referred to heterogeneous core/shell QDs [16]. This strategy has been widely used for the synthesis of a variety of core /shell QDs such as CdSe/ZnS, CdSe/CdS, CdSe/ZnSe, InAs/CdSe, CdS/HgS, CdS/CdSe, CdTe/CdSe and CdS/ZnSe QDs [17-24]. The coating processes predominantly occur in organic reaction media, requiring additional steps to render the products water soluble, as mentioned in Chapter 4, Sections 4.1.1 to 4.1.3.

Core/shell QDs can be categorized according to the band gap and energy levels of their components in three groups: type I, reverse type I and type II [18].

(a)- Type I: in this type of nanoparticle, either the conduction or the valance bands of the core align within the band gap of the shell, such that both the electrons and holes are localized in the core. For example in CdSe/CdS type-I core/shell QDs, the band gap of 123

Chapter 5 Synthesis of Water Soluble Core/shell QDs

the CdSe core is 1.74 eV and the band gap of the CdS shell is 2.42 eV. Therefore, both the holes and electrons are confined to the CdSe core [18, 19]. CdSe/ZnS, and InAs/CdSe core/shell QDs are other examples for type-I core/shell nanocrystal systems [17, 20].

(b)- Inverse type I: in inverse type-I materials the band gap of the core is wider than the band gap of the shell and both the conduction and valence bands of the shell are therefore localized within the band gap of core. Consequently, the holes and electrons are confined in the shell [25]. Examples include CdS/HgS, CdS/CdSe and ZnSe/CdSe core/shell QDs [21, 22, 23].

(c)- Type II: in type-II systems both the valence and conduction band edges of the core are lower or higher than those in the shell and both the hole and the electron are confined to the core [19, 26, 27]. CdSe/ZnSe, CdTe/CdSe and CdS/ZnSe core/shell QDs are some examples of type-II core/shell QDs [19, 24].

Figure 5.1 shows different types of core/shell QDs [18].

Figure 5.1. Different types of core/shell QDs: (A) type I with localization of both electron and hole in the core, (B) type II with localization of electrons in the shell, (C) type II with localization of the hole in the shell and (D) inverse type I with localization of both electron and hole in the shell [18]. 124

Chapter 5 Synthesis of Water Soluble Core/shell QDs

Core/shell QDs can also be formed in aqueous media, often consisting of the overcoating of QD cores with metal sulfides, metal oxides or other semiconductors as the shell. The overgrowth of the shell occurs in an analogous manner to the initial QD growth reactions using the appropriate precursors, adjusting the experimental parameters and utilizing simple heating reactions, hydrothermal method and microwave assisted approach [15, 28-31].

In all of the QD coating methods, the selection of a shell with an appropriate band gap is very important to form the desired type of QD. Meanwhile, when metal oxides or sulfides are used as the shell material, the resultant shells are amorphous which prohibits perturbation of the crystalline structure of the cores [15, 31, 32, 33].

Clearly the properties of core/shell nanocrystals depend on the individual properties of both the cores and shells; high quality core/shell QDs are naturally obtained by coating highly crystalline and photo stable QDs cores with appropriate shell materials [34]. Photostable core/shell QDs can be used for bio imaging, in photovoltaic devices and photo emitting diodes [35- 37].

This chapter contains studies focused on the over-coating of the CdSe(S) QDs with zinc [31] and ferric oxide shells. By controlling the reaction parameters in the hydrolysis of the metal precursors in the presence of QDs in aqueous conditions, type-II

CdSe(S)/Fe2O3 core/shell QDs were found to form. The effect of having either ferric or zinc oxide as the shell around the CdSe(S) QD cores was compared with reference to a previous report of type-I CdSe(S)/ZnO core/shell QDs [31].

5.2 Experimental Procedure

5.2.1 Chemical Synthesis

Water soluble CdSe(S) QDs were synthesized (Sample 1, Chapter 2, Section 2.2.2.2) were coated with ZnO and Fe2O3 in two separated syntheses, thus forming core/shell QDs (Samples 5 and 7, Chapter 2, Section 2.2.3).

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Besides, another sample of CdSe(S) QDs (Sample 3, Chapter 2, Section 2.2.2.2-III) was utilized to form CdSe(S)/ZnO core/shell QDs (Sample 6, Chapter 2, Section 2.2.3.1-B).

The details of the experiments have been detailed in Chapter 2, Section 2.2.3.

5.2.1.3 Characterization

UV-vis absorption spectra were measured with a Varian Cary UV spectrometer, whilst photoluminescence spectra were measured with on a custom-built microscope coupled to an Acton 2300 spectrometer using an excitation laser at 514.5 nm. X-ray powder diffraction patterns (PXRD) were obtained on a X'pert PRO Multi-purpose X-ray diffraction System (MPD system). X-ray photoelectron spectroscopy (XPS) was performed using an Escalab 250Xi spectrometer and transmission electron micrographs (TEM) and energy dispersive X-ray spectra (EDS) were recorded by a Philips CM 200 instrument. The details of characterization mentioned in Chapter 2, section 2.2.3.

5.3 Results and Discussion

5.3.1 Synthesis of CdSe(S)/ZnO QDs

The growth of a zinc oxide shell onto CdSe(S) QDs by controlling the hydrolysis of metal precursors under basic pH has been previously reported in the literature [31]. However, the experimental parameters at the time of the coating process, or during the synthesis the CdSe(S) QDs core, influence the properties of the resultant core/shell QDs.

As mentioned in Chapter 2, Section 2.2.2.2, CdSe(S) QDs were synthesized using optimized experimental parameters (Samples 1 and 3). The obtained QDs, were then coated with a ZnO shell by the hydrolysis of Zn(OAC)2 in basic pH to produce CdSe(S)/ZnO QDs as Samples 5 and 6. Sample 5 was selected to be characterized and the following results were obtained.

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PXRD results showed that the resultant CdSe(S)/ZnO maintained the cubic nanocrystal CdSe(S) structure as the cores after coating with the inorganic zinc oxide shell (Figure 5.2).

111

220

311 Intensity (a.u) Intensity

20 30 40 50 60 70 80 90

2 Theta (degree)

Figure 5.2. PXRD of CdSe(S)/ZnO core/shell QDs.

HRTEM images revealed that the obtained nanoparticles were spherical in nature with clearly visible atomic planes, confirming the retention of crystalline CdSe(S) cores even after the zinc oxide coating. The d-spacing measured for the atomic planes in the HRTEM images was 3.5 Å in accord with the (111) lattice spacing in ZB CdSe, which is 3.58 Å (Figure 5.3).

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Chapter 5 Synthesis of Water Soluble Core/shell QDs

Figure 5.3. TEM image of CdSe(S)/ZnO core/shell QDs and (inset) TEM of a single particle with its corresponding atomic planes.

Optical spectroscopy showed that the synthesized CdSe(S)/ZnO core/shell QDs have a broad absorption excitation starting at 550 nm, similar to that of the CdSe(S) cores, with a narrow emission spectrum at 572 nm corresponding to orange nanoparticles (Figure 5.4).

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Chapter 5 Synthesis of Water Soluble Core/shell QDs

Intensity (a.u) Intensity

b a 300 400 500 600 700 800

Wavelength (nm)

Figure 5.4. Optical spectra of CdSe(S)/ZnO core/shell QDs: (a) UV/Vis absorption

and (b) photoluminescence spectrum (λ excitation = 514 nm).

The emission wavelength of the QDs was observed to shift from 580 nm (λemission

CdSe(S) QDs) to 572 nm (λemission CdSe(S)/ZnO QDs), potentially indicating a decrease in the size of the QD core during the heating process to yield the zinc oxide shell. Indeed, the average CdSe particle size of the cores in the CdSe(S)/ZnO QD sample was estimated to be 3 nm using the Scherrer equation and PXRD data (Appendix 13.1); this should be compared to the size obtained for the uncoated core, which was 3.56 nm (Appendix 10.1). The decrease in size of the core in the CdSe(S)/ZnO QDs is strong evidence for effective quantum exciton confinement; with a decrease in the particle size, the effective band gap increases and the emitted photons have higher energy or shorter wavelength [38].

XPS data confirmed the existence of both Zn and O in the core/shell CdSe(S)/ZnO QDs, with the observation of a new peak related to Zn 2P at 1022 eV, indicating the formation of a ZnO shell around the CdSe(S) cores; this is consistent with the standard X-ray photo electron spectrum of zinc oxide [39] and is in accord with previous report [31] (Figures. 5.5 & 5.6).

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Chapter 5 Synthesis of Water Soluble Core/shell QDs

Zn2P

Counts/s

1015 1020 1025 1030 Binding energy (ev)

Figure 5.5 XPS spectrum of CdSe(S) QDs: binding energy of Zn2P.

(b)

(a)

Figure 5.6. XPS surveys of CdSe(S) QDs (a) and CdSe(S)/ZnO core/shell QDs (b) and insets show that binding energy of Zn 2P was observed at 1022 eV in and CdSe(S)/ZnO QDs while it was not observed in CdSe(S) QDs.

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Chapter 5 Synthesis of Water Soluble Core/shell QDs

Besides, the existence of the peaks assigning to selenium, cadmium, sulfur and three distinct carbons, indicated that the shell growth does not influence the structure of CdSe(S) QDs as the core (Figure 5. 7).

Figure 5.7. XPS spectra of CdSe(S)/ZnO core/shell QDs binding energy of (a) Cd3d

, (b) Se3d , (c) S2p and (d) C1S.

In conjunction with the XPS data, EDS spectrum of CdSe(S)/ZnO QDs confirmed the existence of zinc, cadmium, selenium and sulfur as the major elements present (Figure 5.8).

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Chapter 5 Synthesis of Water Soluble Core/shell QDs

Figure 5.8. EDS spectrum of CdSe(S)/ZnO core/shell QDs.

The observed peaks at 8.03 eV and 8.9 eV are characteristic of copper and the peak at 0.27 eV is characteristic of carbon; both are produced by the grid. The peak at 0.52 eV is an artefact of the measurement.

The coating process was also performed using another sample of CdSe(S) QDs (Sample

3, λemission = 560 nm), obtaining CdSe(S)/ZnO QDs (Sample 6, described in Chapter 2, Section 2.2.3.1, B) and resulting in a small change of emission wavelength. However, the colour of the core/shell QDs was markedly different under a UV lamp. These ZnO- coated and non-coated CdSe(S) QD samples were found to have λemission = 550 nm and 560 nm, corresponding to the green and yellow coloured samples, respectively (Figure 5.9). Given that the wavelength shift is relatively modest (10 nm), a greater influence on the observed colour of the coated and non-coated QDs is the shape of the emission peak. The full widths of half maximum (FWHM) were measured as 47 nm and 62 nm for CdSe(S)/ZnO QDs and CdSe(S) QDs, respectively. The decrease in the FWHM of CdSe(S)/ZnO QDs indicates that the range of particle sizes had decreased post coating, giving a narrower emission band than the parent CdSe(S) QDs. This may be due to the formation of homogenous nanoparticles through heating as described in Chapter 3,

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Chapter 5 Synthesis of Water Soluble Core/shell QDs

Section 3.3.2.2. The observation of broader emission peaks as a result of a larger distribution in the size of the nanocrystalline QDs is in accord with the literature reports [38].

Figure 5.9. Photoluminescence spectra of uncoated CdSe(S) QDs (a) &

CdSe(S)/ZnO core/shell QDs (b) (λ excitation = 350 nm) and (inset) the images of QDs

under UV light (λ excitation = 365 nm) : CdSe(S) QDs (yellow) and CdSe(S)/ZnO QDs (green).

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Chapter 5 Synthesis of Water Soluble Core/shell QDs

5.3.2 Synthesis of CdSe(S)/Fe2O3 QDs

Ferric oxide is a semiconductor material with a narrow band gap (Eg bulk = 2.2 eV), 0.46 eV larger than the band gap of CdSe (Eg bulk =1.74 eV) [40]. In addition, the formation of ferric oxide can be controlled in aqueous phase reactions and as such it is potentially a convenient shell with which to synthesis type-II CdSe(S)/Fe2O3 core/shell QDs in completely aqueous method. Up until now, synthesis of type II- core/shell QDs has been widely reported using both organic and aqueous approach [19, 41-43]. Lui synthesized type-II CdTe/CdSe via a hydrothermal method [41]. Bawendi prepared both CdTe/CdSe and CdSe/ZnTe QDs using a colloidal organic pathway [42]. Jia synthesized CdSe/ZnSe in an aqueous route [19], and Toprak reported the synthesis of type II -CdS/CdSe in an organic pathway [43]. Here, type II, CdSe(S)/Fe2O3 core/shell QDs were synthesized via an aqueous route.

The results of PXRD showed that the obtained CdSe(S)/Fe2O3 nanoparticles were composed of cubic CdSe cores (Figure 5.10), with an average particle size of 3 nm according to the Scherrer equation (Appendix 13.2).

111

11 220

311 Intensity ( a.u ) a.u ( Intensity

20 30 40 50 60 70 80

2 Theta (degree)

Figure 5.10. PXRD of CdSe(S)/Fe2O3 core/shell QDs.

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Chapter 5 Synthesis of Water Soluble Core/shell QDs

The selected area electron diffraction pattern (SAED) of the CdSe(S)/Fe2O3 QDs revealed that the distance between the planes derived from the SAED patterns are in agreement with the PXRD data, indicating that the core remained highly crystalline (Figure 5.11).

311 220

111

Figure 5.11. SAED pattern of CdSe(S)/Fe2O3 core/shell QDs.

HRTEM images of the obtained CdSe(S)/Fe2O3 QDs showed that they are spherical nanoparticles with visible atomic planes in their structure (Figure 5.12).

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Chapter 5 Synthesis of Water Soluble Core/shell QDs

5nm

Figure 5.12. HRTEM image of spherical CdSe(S)/Fe2O3 core/shell QDs.

The d-spacing of the as-synthesized CdSe/Fe2O3 QDs was determined to be 3.12 Å, similar to the inter-planar spacing of the of the (220) plane in cubic CdSe which is 3.10 Å. The symmetric FFT pattern also confirmed the formation of highly crystalline nanoparticles, as shown in Figure 5.13.

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Chapter 5 Synthesis of Water Soluble Core/shell QDs

2 nm

(a)

(c)

(b)

Figure 5.13. HRTEM images of CdSe(S)/Fe2O3 core/shell QDs (a) A single nanoparticle and its corresponding FFT pattern (b), (c).

Optical spectroscopy revealed that the Fe2O3 coated CdSe(S) QDs, have a wide excitation absorption band starting at 570 nm and a narrow emission band at 595 nm, consistent with the observation of them being orange-red in colour (Figure 5.14).

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Chapter 5 Synthesis of Water Soluble Core/shell QDs

Intensity (a.u) Intensity

b a

300 350 400 450 500 550 600 650

Wavelength (nm)

Figure 5.14. Optical spectra of CdSe(S)/Fe2O3 core/shell QDs: (a) UV/Vis

absorption and (b) photoluminescence spectrum (λ excitation = 514 nm).

The emission wavelength of CdSe(S)/Fe2O3 QDs shifted from 580 nm as uncoated

CdSe(S) QDs to 595 nm after Fe2O3 shell growth. The particle sizes of the CdSe cores, claculated from PXRD data, were 3 nm when coated and 3.56 nm uncoated, similar to that observed in the CdSe(S)/ZnO core/shell QDs (Appendix 11.1 & 9.1). This indicates that the observed red shift in the emission wavelength of core/shell QDs can be related to the relative band gaps in Fe2O3 (Egbulk = 2.2 eV) and CdSe (Egbulk = 1.74 eV). This difference is much less than the difference of energy gap values in the core CdSe QDs and ZnO, which is 1.56 eV. The relative band gaps in the respective bulk materials, result in the CdSe(S)/Fe2O3 being classified as type II-core/shell QDs, whilst CdSe(S)/ZnO QDs can be categorized in type I ( Section 5.1, Figure 5.1). The decrease in size of CdSe(S) QD cores during heating is rendered insignificant in the strongly interacting type-II system, whereas the type-I shell is more passive. Therefore, the optical properties of core/shell QDs have been shown to strongly depend on the properties of the shell materials.

XPS data confirmed the presence of Fe2O3 on the surface of the CdSe(S) QDs with the 3+ 3+ observation of peaks at 711.5 eV and 726 eV that correspond to Fe 2P3/2 and Fe 2P1/2 (Figure 5.15). 138

Chapter 5 Synthesis of Water Soluble Core/shell QDs

Fe 2P3/2

Fe 2p1/2

Counts/s

711.5 726

705 715 725 735 Binding energy (ev)

Figure 5.15. XPS spectrum of CdSe(S)/Fe2O3 core/shell QDs: binding energy of

Fe2P3/2 & Fe2P1/2.

3+ The characteristic peak of Fe 2P3/2 for standard α-Fe2O3 was at 710.9 eV in a standard

XPS spectrum [39, Appendix 14] and the observed slight shift in the CdSe/Fe2O3 nanoparticles (~0.6 eV), can be assigned to the interaction of CdSe(S)-MPA capped

QDs as the core with Fe2O3 as the shell during over coating process.

In addition, XPS spectra indicate the formation of ferric oxide as the product of hydrolysis of ferric chloride in basic pH, evidenced with the disappearance of the peaks - - characteristic of Cl 2p1/2 at 201.4 eV and Cl 2p3/2 at 199.3 eV in XPS survey spectra (Figure 5.16).

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Chapter 5 Synthesis of Water Soluble Core/shell QDs

Figure 5.16. XPS survey of CdSe(S)/Fe2O3 core/shell QDs and inset shows disappearing of the peaks corresponding to chloride to indicate completing the

hydrolysis of FeCl3.

XPS results also showed that the components in the structure of as prepared

CdSe(S)/Fe2O3 core/shell QDs are cadmium, selenium, sulfur and carbon, similar to that observed with CdSe(S)/ZnO QDs (Appendix 15).

5.4 Conclusion

A shell growth method using water as the solvent was employed to overcoat CdSe(S) QDs with amorphous layers of both zinc oxide and ferric oxide to produce

CdSe(S)/ZnO and CdSe(S)/Fe2O3 core/shell QDs. Whilst literature reports of type I-

CdSe(S)/ZnO QDs exist [31], the preparation of type II-CdSe(S)/Fe2O3 core/shell QDs are reported here for the first time. It was observed that the crystalline structure of the CdSe(S) core was unchanged after coating with either of zinc or ferric oxide shells. However, it was found that surface coating of CdSe(S) QDs with either ferric oxide or zinc oxide led to the decrease in the particle size of the CdSe cores. Besides, it was determined that the properties of the shell materials, including Eg of the bulk material, influence the optical properties of the resultant core/shell QDs.

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Chapter 5 Synthesis of Water Soluble Core/shell QDs

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Chapter 5 Synthesis of Water Soluble Core/shell QDs

[20]. Xie, R.; Peng, X. Angew. Chem., Int. Ed. 2008, 47, 7677. [21]. Mews, A.; Eychmueller, A.; Giersig, M.; Schooss, D.; Weller, H. J. Phys. Chem. 1994, 98, 934. [22]. Tian, Y.; Newton, T.; Kotov, N. A.; Guldi, D. M.; Fendler, J. H. J. Phys. Chem. 1996, 100, 8927. [23]. Hu, D.; Zhang, P.; Gong, P.; Lian, S.; Lu, Y.; Gao, D.; Cai, L. Nanoscale 2011, 3, 4724. [24]. Ning, Z.; Yuan, C.; Tian, H.; Fu, Y.; Li, L.; Sun, L.; Aagren, H. J. Mater. Chem. 2012, 22, 6032. [25]. Korsunska, N. E.; Dybiec, M.; Zhukov, L.; Ostapenko, S.; Zhukov, T. Semicond. Sci. Technol. 2005, 20, 876. [26]. Ivanov, S. A.; Piryatinski, A.; Nanda, J.; Tretiak, S.; Zavadil, K. R.; Wallace, W. O.; Werder, D.; Klimov, V. I. J. Am. Chem. Soc. 2007, 129, 11708. [27]. Balet, L. P.; Ivanov, S. A.; Piryatinski, A.; Achermann, M.; Klimov, V. I. Nano Lett. 2004, 4, 1485. [28]. Wang, J.; Han, H. J. Colloid Interface Sci. 2010, 351, 83. [29]. Law, W.-C.; Yong, K.-T.; Roy, I.; Ding, H.; Hu, R.; Zhao, W.; Prasad, P. N. Small 2009, 5, 1302. [30]. Fu, T.; Qin, H.-Y.; Hu, H.-J.; Hong, Z.; He, S. J. Nanosci. Nanotechnol. 2010, 10, 1741. [31]. Aldeek, F.; Mustin, C.; Balan, L.; Medjahdi, G.; Roques-Carmes, T.; Arnoux, P.; Schneider, R. Eur. J. Inorg. Chem. 2011, 794. [32]. Jia, G.; Hao, B.; Lu, X.; Yao, J. Int. J. Electrochem. Sci. 2013, 8, 8167. [33]. Xie, H.-Y.; Liang, J.-G.; Liu, Y.; Zhang, Z.-L.; Pang, D.-W.; He, Z.-K.; Lu, Z.-X.; Huang, W.-H. J Nanosci Nanotechnol 2005, 5, 880. [34]. Kong, J.; Qiu, H.; Yu, M.; Zhang, B. Huaxue Xuebao 2012, 70, 789. [35]. Blanco-Canosa, J. B.; Medintz, I. L.; Farrell, D.; Mattoussi, H.; Dawson, P. E. J. Am. Chem. Soc. 2010, 132, 10027. [36]. Kostic, R.; Stojanovic, D. Optoelectron. Adv. Mater., Rapid Commun. 2012, 6, 121. [37]. Li, Y.; Rizzo, A.; Mazzeo, M.; Carbone, L.; Manna, L.; Cingolani, R.; Gigli, G. J. Appl. Phys. 2005, 97, 113501/1. [38]. Gupta, P.; Ramrakhiani, M. Open Nanosci. J. 2009, 3, 15.

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143

Chapter 6 The Investigation of Cytotoxicity of Quantum Dots (QDs)

6.1. Introduction

Quantum dots can be considered as potentially being toxic materials due to both their nanoscale size and the presence of heavy metals in their composition [1, 2]. As nanomaterials, QDs have a greater toxicity than their bulk counterparts simply because the small size of the QDs may enable them to penetrate much deeper into cells than the equivalent bulk chemical material [3].

The interaction of QDs with mitochondria and cell nuclei leads to an alteration or disruption of cell function, inhibition of cell proliferation and a decrease cell viability due to the production of reactive oxygen species (ROS), ultimately resulting in cell death and may also promote chronic diseases and gene mutation or induced immunotoxicity [4- 7].

6.1.1 Production of Reactive Oxygen Species (ROS)

The generation of reactive free radicals, ROS, as a result of cell exposure to nanomaterials is a significant cause of nanoparticle cytotoxicity [7]. ROS are induced as a phagocytic cell response to foreign materials, or in the presence of transition metals, which incidentally are often incorporated into nanoparticle structures [7, 8]. Normally, ROS such as superoxide are produced during cellular metabolism processes and are maintained in equilibrium in the body with antioxidant enzymes and glutathione. Increased rates of ROS production leads to damage of biological components through the oxidation of lipids, proteins and DNA [4, 6, 7]. Indeed, high levels of ROS disturbs mitochondria and perturbs electronic transfer leading to induced cell-cycle arrest, mutagenesis and apoptosis, as shown schematically in Figure 6.1[9].

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Chapter 6 The Investigation of Cytotoxicity of QDs

Figure 6.1. Interaction of ROS with cells [9].

6.1.2 Immunotoxicity

Nanoparticles, including QDs, can stimulate the immune system and induce immunotoxicity or inflammation due to adverse effects on the function of the immune system. Foreign substances may lead to an alteration of the immune function and ultimately cause infectious diseases or cancer [10, 11].

6.1.3 Effective Parameters on Cytotoxicity of QDs

The toxicity of QDs depends on various physicochemical and environmental parameters including size, composition, degradation and dosage of QDs in biological environments [12].

I)-Size: particle size is an important factor in the distribution or elimination of nanomaterials in the body because the particle size influences the reactivity of particles and affects cellular responses during cellular uptake processes [ 8, 13, 18]. For example, the small size of QDs can lead to them being internalized in the cells in a rapid process due to the increased surface reactivity of nanoparticles that affects the kinetics of intercellular processes [14].

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Moreover, decreasing the particle size leads to an increase in the ratio of the reactive surface area relative to volume that makes nanoparticles even more active, resulting in an accumulation of nanocrystals in the tissues and long term toxicity in organs [15], particularly organs with a high blood flow such as spleen, kidneys, liver, lungs and the blood circulation system (Figure 6.2) [16].

Figure 6.2. Nanoparticles toxicity in major organs [16].

In addition, smaller QDs may be more aggregated in the biological environment, leading to precipitation on the cell surface and thereby altering cell functions [17].

II)-Composition: chemical components incorporated into the nanoparticles define the reactivity, toxicity and also chemical interactions between the cells and the nanoparticles [18]. Many QDs have functional groups on their surface to increase their biocompatibility, which can also lead to specific interactions with biological components and potentially alter their biological function [19]. As QDs often contain transition metals or other toxic compounds in their structures, they can induce ROS and other poisonous components that influence environment and human health [18, 20].

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III) Degradation: degradation of QDs in biological media is a complex issue for in- vivo applications of QDs because both non-degradable and degradable QDs have been classified as toxic materials [16]. Degradable QDs can accumulate in organs and cause detrimental effects to the cells. They can also induce free radical formation and release toxins, resulting in unpredicted toxicity due to the unexpected toxic degradation products [21].

On the contrary, stable and non-degradable QDs are less toxic than degradable QDs in in-vitro studies primarily due to the prohibition of free radical formation and the surface oxidation that is restricted in forming stable QDs [22].

IV) Dosage: the dose of QDs is an important parameter in determining toxicity. The correlation of dosage and toxicity of QDs have been widely reported [4, 12, 17, 23-26] and show that the determination of an appropriate dose of QDs depends on factors such as the type of QDs, cell type, exposure time and the interaction of cell/QDs.

6.1.4 Short term and Long Term Toxicity

Some QDs have been assessed to be non-toxic materials in both in-vivo and in-vitro toxicology studies due to the absence of any evidence of poisonous effects to the cells after several hours to several weeks [27]. However, long-term toxicity of QDs has been clearly perceived because they can accumulate in organs and influence intracellular processes, leading to organ damage and chronic illnesses [28].

6.1.5 Control the Toxicity of QDs

In spite of the toxicity of QDs, researchers have tried to control and minimize the hazardous effects of QDs by following several pathways:

A) Further engineering in QDs structure: as the toxicity of QDs critically depends on free heavy metal ion formation and the oxidation of components involved in the structure of QDs, researchers have introduced core/shell QDs as a new class of QDs to control the toxic effects of QDs [29, 30]. It is anticipated that core/shell QDs exhibit lower toxicity than uncoated QDs because the presence of the shell around the core QDs protects the core against oxidation, preventing the possible formation of free heavy 147

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metal ions particularly when the core contains transition metals such as cadmium [31, 32].

B) Development of modified synthetic approaches: the synthetic method has an important role in the toxicity of QDs. According to literature reports, QDs synthesized in an organic route are more toxic than those synthesized in an aqueous pathway. For example various organic solvents such as trioctylphosphine oxide or hydrazine have been used to synthesize QDs; however despite the formation of highly luminescent QDs, they exhibit greater toxicity than those synthesized in purely aqueous routes [33, 34].

C) Size control: as the size of nanoparticles is an important factor in the toxicity of QDs (section 6.1.3), identification of optimally sized QDs can be considered as a significant parameter in controlling the toxicity of QDs. For example, small CdTe QDs, with a particle size (2.2±0.1 nm), were assessed as being more toxic than larger QDs of the same material, (5.2±0.1 nm), toward rat pheochromocytoma cells at equal concentrations [4, 35, 36].

D) Synthesis of heavy metal-free QDs: it is well understood that toxicity of QDs is related to the presence of transition metals in their structures, releasing free metal ions in solution which is one of the most important factors in QD toxicity [32, 37]. Potentially this may be overcome by simply avoiding such elements and heavy metal- free QDs including, silicon QDs, zinc sulfide and zinc selenide QDs, have been widely synthesized as an effort to limit free radical formation and minimize toxicity [38- 41].

6.1.6 Toxicity Assays of QDs

There are various methods to determine the toxicity of QDs in both in-vivo and in-vitro studies. These methods involve introducing QDs to microorganisms, using animal models to determine in-vivo QD toxicity and treatments of various cell types with different QDs to investigate the in-vitro toxicity of nanocrystals in mammalian cell lines [42- 46].

The present chapter details the determination of cytotoxicity of three groups of QDs synthesised in this thesis; water soluble CdSe(S), ZnSe(S) QDs and CdSe(S)/ZnO 148

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core/shell QDs in the presence of human colorectal carcinoma cells (HCT-116) and human skin fibroblast cell line (WS1). The cytotoxicities of the precursor solutions during the synthesis of CdSe(S) QDs were determined in the presence of the HCT-116 cell line, whilst the toxicity of CdSe(S) QDs was compared with the heat-shock protein (Hsp90) inhibitor, 17-AAG.

6.2 Experimental Procedure

Water soluble CdSe(S) (Sample 3, Chapter 2, Section 2.2.2.2-III), ZnSe(S) (Sample 4, Chapter 2, Section 2.2.2.3) and CdSe(S)/ZnO QDs (Sample 6, Chapter 2, Section 2.2.3.1-B), were synthesized and used for cytotoxicity assays in presence of two cell lines including human colorectal carcinoma cells (HCT-116) and human skin fibroblast cells (WS1). Initially, aqueous solutions of QDs in different concentrations were prepared and cell cultures of both HCT-116 and WS1 cells were performed separately. The cell lines were then separately treated with QDs and incubated. Finally, the viability of the cells after incubation with QDs was determined using a Cell Counting Kit-8 assay. The details of these steps have been detailed in Chapter 2, Section 2.2.5.

6.3 Results and Discussion

HCT-116 and WS1 cell lines were chosen in order to investigate the toxicity of these QDs in the presence of both cancer cells and normal cells. In addition to providing general assays of cytotoxicity of QDs, the selection of HCT-116 and WS1 cell lines is based upon following reasons:

(i)- Similar to other types of cancer cells, HCT-116 cells have irregular DNA pattern that makes them more sensitive than healthy cells against cell death originating from free heavy metal ions including cadmium whilst skin normal cells (WS1) are known as one of the most resistant cells against penetration of free metal ions when compare with other types of human cells. Therefore, these two cell lines were selected to investigate effect of cell resistance in toxicity of QDs.

(ii)- Fifty percent of cancer cells including HCT-116 cells need to use heat-shock protein (Hsp90) to survive [53]. The viability of these types of cancer cells decrease in 149

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the presence of the heat-shock protein (Hsp90) inhibitor, 17-AAG, which is a common anti-tumour compound. Therefore, HCT-116 cells were selected to compare viability of the cancer cells either in presence of Hsp90 inhibitor, 17-AAG or after treatment of the cells with QDs. This comparison provides an initial effort to use QDs as an alternative to anti-tumour drugs.

6.3.1 Cytotoxicity of QDs toward Cancer Cells

HCT-116 cells are cancer cells that have a DNA mutation with the capacity to form tumours when injected into mice and are also fatal in the human body due to metastasis [47, 48]. Cytotoxicity assays of the synthesized QDs (Chapters 4 & 5) toward these cancer cells were performed.

6.3.1.1 Cytotoxicity of CdSe(S) QDs

The cytotoxicity of CdSe(S) QDs was assessed based upon LC50 = 105 µg/ml as shown in Figure 6.3.

Figure 6.3. The results of cytotoxicity assays of CdSe(S) QDs toward HCT-116 cell line. Error bars indicate standard error of the mean.

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The results indicate that CdSe(S) QDs are nontoxic at concentrations 25, 50 and 100 µg/ml, with 100%, 91% and 52% cell proliferation (assuming 50% mortality to equate to being toxic), whilst they were identified as being toxic at concentrations 250 and 500 µg/ml, indicating that the cytotoxicity of CdSe(S) QDs is dose-dependent.

Comparisons between the cytotoxicity of CdSe(S) QDs synthesized in aqueous media to those synthesized in an organic approach [49] showed that aqueous QDs exhibit less toxicity and greater stability, as summarized in Table.6.1. This comparison is based upon disregarding the effects of cell type in the cytotoxicity assays because no other reports on the cytotoxicity of CdSe QDs synthesized by organic routes against HCT-116 cell line are available.

Table 6.1. The comparison of cytotoxicity and stability as-synthesized CdSe(S) QDs and CdSe-TOPO capped QDs .

QDs- Synthesis Cell viability Cell viability Cytotoxicity Stability type method (%) (%) in the air (C=62.5µg/ml) (C = 250µg/ml) CdSe(S) Aqueous 78 29 Noncytotoxic Stable QDs method at C < 105 (Chapter 4) µg/ml CdSe Organic 21 5 Cytotoxic at Unstable QDs approach [49] all concentrations

6.3.1.2 Cytotoxicity of Precursor Solutions toward HCT Cell Line

The viability of HCT-116 cells was measured after treatment of the cells with Cd-MPA and the Cd-Se-MPA yellow complex precursor solutions (Figure 6.4).

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Figure 6.4. The results of cytotoxicity assays of Cd-MPA and Cd-Se-MPA precursor solutions toward HCT-116 cell line. Error bars indicate standard error of the mean.

The results indicated that both precursor solutions have no significant toxicity at concentrations 25 and 50 µg/ml but they are cytotoxic at concentration 500 µg/ml, similar to that of CdSe(S) QDs.

The Cd-MPA solution was found to be more toxic than the solution of the Cd-Se-MPA complex due to being toxic at concentrations of 100 and 250µg/ml, whilst the complex was nontoxic at these same concentrations. In addition, after treatment with the Cd- MPA precursor solution, the cells had a lower viability (67%) at 50 µg/ml in comparison with that of the Cd-Se-MPA precursor, which was measured as 96%. This indicates that formation of the Cd-Se-MPA complex during chemical synthesis of CdSe(S) QDs (Chapter 2. Section 2.2.2.2) leads to a decrease in the toxicity relative to the Cd-MPA solution, presumably due to the decreased number of free cadmium ions as the source of the cytotoxicity in cell incubation.

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6.3.1.3 Cytotoxicity of CdSe(S) QDs in Compare with Function of Hsp90 inhibitor 17-AAG

Hsp-90 inhibitor 17-AAG is an anti-tumour drug that inhibits the activity of Hsp-90 heat shock protein, which is a necessary protein for the growth of several kinds of cancer cells [50]. This anti-cancer drug has been widely used in cancer treatment and its function is based on destroying cancer cells by inhibiting the activity of Hsp-90 [51]. The results of the cytotoxicity studies of CdSe(S) QDs in the presence of HCT-116 cell line showed that CdSe(S) QDs inhibit cell viability by 64% at a concentration of 500 µg/ml, similar to the cell proliferation profile of Hsp-90 with 17-AAG at concentration 0.06 µg/ml, which is 72%, as shown in Figure 6.3, Section 6.3.1.1. It indicated that the cytotoxicity of CdSe(S) QDs is much less than that of Hsp90 inhibitor 17-AAG.

6.3.1.4 Cytotoxicity of Dialyzed CdSe(S) QDs

A dialysis cassette was used in an attempt to remove excess amounts of either capping agent or cadmium ions; the results showed that the dialyzed CdSe(S) QDs were in fact more toxic than those of CdSe(S) QDs without using dialysis, as shown in Figure 6.5.

Figure 6.5. The results of cytotoxicity assays of dialyzed CdSe(S) QDs. Error bars indicate standard error of the mean.

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The obtained data were in accord with the literature [52] because QDs were unstable after removing excess amount of cadmium and capping agent, leading to increased free cadmium ions as a source of toxicity.

6.3.1.5 Cytotoxicity of CdSe(S)/ZnO QDs

The cytotoxicity of CdSe(S)/ZnO core/shell QDs toward HCT-116 cancer cells was assessed, with the results showing that CdSe(S)/ZnO QDs are nontoxic at all concentrations (Figure 6.6).

Figure 6.6. The results of cytotoxicity assays of CdSe(S)/ZnO core/shell QDs toward HCT-116 cell line. Error bars indicate standard error of the mean.

These data indicate that passivation of the CdSe(S) QD cores with a zinc oxide shell promotes cell viability, as evidenced with an increased cell proliferation, from 52% for CdSe(S) QDs at a concentration of 100 µg/ml, to 69% after coating with zinc oxide as shown in Figure 6.6. Indeed, CdSe(S)/ZnO core/shell nanocrystals had no cytotoxicity at concentrations 250 and 500 µg/ml in contrast to CdSe(S) QDs, confirming that core/shell QDs are potentially less toxic than uncoated QDs. The rationale for this behaviour is the protection that the shell affords to the core against oxidation and the

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prohibition of free cadmium ion formation that can lead to cell death, as described in Figure 6.7 [49].

Figure 6.7. The effect of free cadmium ion on the liver cells [49].

6.3.1.6 Cytotoxicity of ZnSe(S) QDs

Heavy metal-free nanoparticles including ZnSe(S) QDs, are expected to have lower toxicity than heavy-metal QDs. However, the cytotoxicity of these nanoparticles should be investigated due to their nanoscale properties and the possible toxicity of their components including capping agents. For example both zinc and selenium are less toxic than other elements, but the presence of 3-mercaptopropionic acid in the structure of the ZnSe(S) QDs may yield some level of toxicity. Toxicology data showed that ZnSe(S) QDs are nontoxic over all concentrations, with 100% cell viability of the HCT- 116 cell line after incubation with the as–synthesized ZnSe(S) nanocrystals (Figure 6.8).

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Figure 6.8. The results of cytotoxicity assays of ZnSe(S) QDs toward HCT-116 cell line. Error bars indicate standard error of the mean.

This indicates that the replacement of cadmium with less-toxic zinc leads to nontoxic nanoparticles, even toward cancer cells that have mutated DNA and that can be easily killed after treatment with nanocrystals. These data are in accord with previous studies to support this idea that cadmium is the primary source of cytotoxicity in heavy metal QDs [49].

6.3.2 Cytotoxicity of QDs toward Normal Cells

A human skin fibroblast cell line (WS1) was used to determine cytotoxicity of the as- synthesized QDs toward normal cells. The viability of WS1 cell line was measured after treatment of the cells with the QDs and cytotoxicity of QDs was assessed in different concentrations.

6.3.2.1 Cytotoxicity of CdSe(S) QDs

The results of cytotoxicity assays of CdSe(S) QDs toward normal cells showed that they are nontoxic at all concentrations (Figure 6.9).

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Figure 6.9 The results of cytotoxicity assays of CdSe(S) QDs toward WS1 cell line. Error bars indicate standard error of the mean.

As described in Figures 6.3 and 6.9, when compared to the HCT-116 cell line, CdSe(S) QDs do not have toxic effects in the presence of normal cells, indicating that cancer cells are more likely to die in the presence of QDs.

6.3.2.2 Cytotoxicity of CdSe(S)/ZnO QDs

The results of cytotoxicity assessment indicated that CdSe(S)/ZnO core/shell QDs are nontoxic in the presence of normal cells at all concentrations (Figure 6.10).

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Figure 6.10. The results of cytotoxicity assays of CdSe(S)/ZnO core/shell QDs toward WS1 cell line. Error bars indicate standard error of the mean.

In comparison to the cell proliferation of the HCT-116 cell line after treatment with CdSe(S)/ZnO core/shell QD that is 69% at a concentration of 100 µg/ml, the cell viability of WS1 was determined to be 100% at the same concentration, indicatinge that the CdSe(S)/ZnO QDs exhibit no toxicity toward normal cells.

6.3.2.3 Cytotoxicity of ZnSe(S) QDs

Cell proliferation of the WS1 cell line in the presence of ZnSe(S) QDs was determined to be 100% at all concentrations, similar to the cell viability of HCT-116 cell line after treatment with ZnSe(S) QDs (Figure 6.11).

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Figure 6.11 The results of cytotoxicity assays of ZnSe(S) QDs toward WS-1 cell line. Error bars indicate standard error of the mean.

It was observed that the viability of normal cells toward all of the obtained QDs was significantly higher than cancer cells. This is because normal cells have a regular DNA pattern and they have more persistence against free ions originating from cadmium or other possible toxic compounds which can otherwise potentially be produced during treatment of the cells with QDs nanoparticles.

The summary of the toxicology assays toward both HCT-116 and WS1 cell lines are displayed in Table. 6.2. These results is based upon the assumption that 50% mortality equates to being toxic.

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Table 6.2 Summary of cytotoxicity of QDs

QDs-type Toxicity at concentrations (25-500 µg/ml) CdSe(S) QDs Nontoxic toward HCT-116 cells at concentrations < 105 µg/ml, nontoxic toward WS1 cells at all concentrations.

CdSe(S)/ZnO Nontoxic toward both HCT-116 and WS1 cell lines at all concentrations. ZnSe(S) QDs Nontoxic toward both HCT-116 and WS1 cell lines with 100% cell viability at all concentrations.

6.4 Conclusion

In this work, it was clearly shown that the cytotoxicity of QDs can be controlled with the synthesis of stable QDs via an aqueous route. It was determined that the cytotoxicity of CdSe(S) QDs decreases after passivation with zinc oxide shells. Moreover, replacing zinc to cadmium led to QDs displaying no toxicity at all concentrations toward both HCT-116 and WS1 cell lines.

References

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[28]. Mejias, R.; Gutierrez, L.; Salas, G.; Perez-Yague, S.; Zotes, T. M.; Lazaro, F. J.; Morales, M. P.; Barber, D. F. J. Controlled Release 2013, 171, 225. [29]. Kirchner, C.; Liedl, T.; Kudera, S.; Pellegrino, T.; Javier, A. M.; Gaub, H. E.; Stoelzle, S.; Fertig, N.; Parak, W. J. Nano Lett. 2005, 5, 331. [30]. Yang, F.; Yang, P. J. Cluster Sci. 2013, 24, 643. [31]. Liu, Y.; Wang, P.; Wang, Y.; Zhu, Z.; Lao, F.; Liu, X.; Cong, W.; Chen, C.; Gao, Y.; Liu, Y. Small 2013, 9, 2440. [32]. Rzigalinski, B. A.; Strobl, J. S. Toxicol. Appl. Pharmacol. 2009, 238, 280. [33]. Wang, Q.; Zhou, X.; Fang, T.; Liu, P.; Li, X.; Min, X. Powder Technol. 2013, 247, 81. [34]. Li, Y.; Jing, L.; Qiao, R.; Gao, M. Chem. Commun. 2011, 47, 9293. [35]. Bruneau, A.; Fortier, M.; Gagne, F.; Gagnon, C.; Turcotte, P.; Tayabali, A.; Davis, T. L.; Auffret, M.; Fournier, M. Environ. Sci.: Processes Impacts 2013, 15, 596. [36]. Xu, Z.-Q.; Lai, L.; Li, D.-W.; Li, R.; Xiang, C.; Jiang, F.-L.; Sun, S.-F.; Liu, Y. Mol. Biol. Rep. 2013, 40, 1009. [37]. Clift, M. J. D.; Varet, J.; Hankin, S. M.; Brownlee, B.; Davidson, A. M.; Brandenberger, C.; Rothen-Rutishauser, B.; Brown, D. M.; Stone, V. Nanotoxicology 2011, 5, 664. [38]. Moret, S.; Becue, A.; Champod, C. Forensic Sci. Int. 2013, 224, 101. [39]. Chinnathambi, S.; Chen, S.; Ganesan, S.; Hanagata, N. Adv Healthc Mater 2014, 3, 10. [40]. Lee, S.; Cho, W.-J. Proc. SPIE-Int. Soc. Opt. Eng. 2004, 5510, 26. [41]. Senthilkumar, K.; Kalaivani, T.; Kanagesan, S.; Balasubramanian, V. J. Mater. Sci.: Mater. Electron. 2012, 23, 2048. [42]. Wang, L.; Zheng, H.; Long, Y.; Gao, M.; Hao, J.; Du, J.; Mao, X.; Zhou, D. J. Hazard. Mater. 2010, 177, 1134. [43]. Hauck, T. S.; Anderson, R. E.; Fischer, H. C.; Newbigging, S.; Chan, W. C. W. Small 2010, 6, 138. [44]. Ballou, B.; Lagerholm, B. C.; Ernst, L. A.; Bruchez, M. P.; Waggoner, A. S. Bioconjugate Chem. 2004, 15, 79. [45]. Yan, M.; Zhang, Y.; Xu, K.; Fu, T.; Qin, H.; Zheng, X. Toxicology 2011, 282, 94. [46]. Brunetti, V.; Chibli, H.; Fiammengo, R.; Galeone, A.; Malvindi, M. A.; Vecchio, G.; Cingolani, R.; Nadeau, J. L.; Pompa, P. P. Nanoscale 2013, 5, 307.

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Chapter 7

The Investigation of Photostability of Quantum Dots in Biological Context

7.1 Introduction

As described in Chapter 1, Section 1.6.2, nanocrystalline semiconductors or quantum dots (QDs) have narrow and symmetrical emission spectra, high quantum yields and a size-dependent wavelength tunability that have promoted their use in bio imaging and bio diagnostic applications [1-4].

However, the photo stability of QDs is an important issue when applying them in the biological context as degradation of the QDs may alter the optical properties of the QDs in biological environments [5-10]. In addition, degradation of QDs can induce free radical formation and cause cell necrosis in live cells [5, 6]. Moreover, spectral modulation of QD emission profiles including blinking are other challenges that limit bio-applications; these may arise due to ionization of the QDs on emission, leading to a changes in the emission spectrum and fluorescence quenching [7, 8].

Consequently, potential alteration of the optical properties of QDs in both biological media and bioconjugated molecules needs to be considered. In this work, we prepared a CdSe(S)QD-antibody conjugated compound, in order to investigate the photostability of the QDs after linking to biomolecules. In addition, the stability and optical properties of as-synthesized CdSe(S) QDs in the presence of both colon cancer cells and cell media were also studied.

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7.2 Experimental Procedure 7.2.1 Preparation and Characterization of a QDs-antibody Conjugated Compound

7.2.1.1 Preparation

Water soluble CdSe(S) QDs (Sample 3, Chapter 2, Section 2.2.2.2-III) were synthesized and then surface-modified using EDC and NHS and linked to donkey anti mouse IgG CY3 fluorophore antibody via EDC coupling reactions, as detailed in Chapter 2, Section 2.2.6.

7.2.1.2 Characterization

UV-vis absorption spectra were measured with a Varian Cary UV spectrometer. Photoluminescence spectra were measured with on a Carry Eclipsed Fluorescence instrument using an excitation wavelength at 350 nm. X-ray photoelectron spectroscopy (XPS) was performed using an Escalab 250Xi spectrometer. Circular dichroism (CD) spectroscopy was performed on a ChrascanTM-plus spectrometer. The details of characterization described in Chapter 2, Section 2.3.

7.2.2 The Investigation of Photostability of CdSe(S) QDs in Presence of HCT-116 Cell Line

Water soluble CdSe(S) QDs (Sample 3, Chapter 2, Section 2.2.2.2-III), were synthesized. These were then separated into two samples containing HCT 116 cells: fixed HCT-116 cells and live HCT-116 cells as detailed in Chapter 2, Section 2.2.7.1. The images of the QDs in the presence of the HCT-116 cell line were recorded using confocal microscopy technique as described in Chapter 2, Section 2.2.7.2.

7.2.3 The Investigation of Photostability of Watersoluble QDs in Cell Media

First, an aqueous solution of CdSe(S) QDs (Sample 3, Section 2.2.2.2-III) diluted to 100 µg/ml using ultrapure water. Then, the cell media were prepared and added to CdSe(S) 165

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QDs. Finally, the emission profiles of the QDs in both the cell media and solution were recorded using Zias LSM780 confocal microscope, as detailed in Chapter 2, Section 2.2.8.

7.3 Results and Discussion

7.3.1 Optical Properties of the CdSe(S) QD-antibody Conjugated Compound

QDs can be surface-modified using a number of reactive chemical groups, including amines, carboxylic acids and esters. The modified QDs can be conjugated to various biomolecules including proteins, DNA and antibodies to produce inorganic-biological hybrids [9]. These conjugated compounds may have use in bioimaging or biodiagnostic applications. For instance, a secondary antibody conjugated fluorophore can be used as a biological label, because it has affinity to attach with its relevant primary antibody. These primary antibodies are mostly immunoglobulin G (IgG) or immunoglobulin M (IgM), meanwhile the primary antibody has an affinity to bind with specific targets such as biomarkers, antigens, proteins, hydrocarbons or other small biomolecules. The detection of these specific targets is crucial as biomarkers for cancer, HIV, diabetes, Parkinson’s and Alzheimer’s disease. Secondary antibodies can be used for indirect detection of these specific targets and a QD-antibody conjugated compound can potentially have the properties of both the antibody and QDs. An advantage of this approach is the better optical properties that QDs have compared to traditional fluorophores such as cyanine 3 (CY3) or cyanine 5 (CY5), that may also be conjugated to secondary antibodies, Figure 7.1 [10, 11].

Figure 7.1 shows schematic of using secondary antibodies as cell label [12].

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Figure 7.1. Schematic of applying secondary antibodies to detect antigens: primary antibody has affinity to bind with Antigen B (red) and secondary antibody has affinity to bind with primary antibody to join in indirectly detection of antigen B. Primary antibody does not have affinity to bind with Antigen A (blue) [12].

Following these assumptions, a QD-antibody conjugated compound was prepared from as synthesized CdSe(S) water soluble QDs and Donkey-anti mouse (H+L) (IgG) antibody having the Cy3 fluorophore attached; this is a secondary antibody developed in the donkey and has an affinity to bind with mouse IgG proteins. It has been specifically designed for the detection of multiple primary antibodies and the presence of the Cyanine 3 (Cy3) as a conjugated fluorophore (λexcitation = 550 nm, λemission = 570 nm) within its structure, allowed us to compare the optical properties of the QDs

(λexcitation = 540 nm, λemission = 560 nm) with that of Cy3.

7.3.1.1 Conjugation Method

The inorganic–biological hybrids can be obtained by different methods of conjugation that can be classified in three categories including direct conjugation, non-covalent self- assembly and EDC coupling.

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The direct method involves binding of biomolecules to QDs using thiolated peptides or polyhistdine (HIS) residues [13]. Another method is non–covalent self-assembly and is based upon using engineered proteins that have been positively charged and can be linked to negatively charged QDs [14]. The EDC coupling method is a common method for particles with carboxylic acid function group [15-19]. This approach makes use of 1- ethyl-3-(3-dimethylaminopropyl carbamide (EDC) condensation reactions with thiol- carboxilic acid capped QDs to form modified QDs that can be linked to biomolecules via binding to amine groups involved with biomolecules [19].

We aimed to make use of EDC coupling to link CdSe(S) QDs to antibodies in a two steps process, including the modification of QDs via EDC coupling and formation of antibody-QD conjugated compound.

As shown in Figure 7.2, first, carboxyl-QDs were treated with EDC (as catalyst) to form compound A, an intermediate known as QD-O-acylisourea. This then was reacted with NHS (as a stabilizer) to form compound (B), a QD-amine-reactive sulfo NHS ester. Compound (B) then reacts with terminal amine groups on the antibody structure to obtain a QD-antibody conjugated compound (Compound C).

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Compound + (A)

EDC

+ Compound (B) Compound (A)

NHS

Compound (C) +

Antibody

Compound

(B)

Compound (C) or QD-Antibody Bioconjugated Compound

Figure 7.2. The steps of formation of CdSe(S)-antibody conjugated compound.

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7.3.1.2 Modification of QDs

In accord with the reactions illustrated in Figure 7.2, the synthesized CdSe(S) QDs have carboxylic functional groups on their surface (described in Chapter 4, Section 4.3.1), they were modified using EDC and NHS in order to conjugate with antibody via EDC coupling.

The results of XPS spectroscopy confirmed the presence of nitrogen courtesy of the peak at 399.9 eV, consistent with an amine bond in the modified QDs; in addition to C, Cd, Se and S, indicating that the QDs were successfully modified with EDC and NHS (Figure 7.3).

Figure 7.3. XPS spectrum of CdSe (S) – modified QDs and (inset) energy binding of N1S.

The optical properties of the modified QDs were then determined to verify whether the emission profile of the QDs was altered post modification. Spectroscopic results showed that the obtained modified nanoparticles have an emission spectrum that peaks at 562 nm, which cannot be considered as a significant shift when compared to the non- modified CdSe(S) QDs (λemission = 560 nm) as shown in Figure 7.4.

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Figure 7.4. Photoluminescence spectrum of CdSe(S) QDs (a) in compare with the

modified CdSe(S) QDs (b) (λ excitation = 350 nm). The excitation wavelength for both the modified and unmodified QDs were determined to be 540 nm, showing that modification of the QDs did not influence their excitation spectrum (Figure 7.5).

Figure 7.5. UV-visible spectrum of CdSe(S) QDs (a) in compare with the modified CdSe(S) QDs (b).

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The molar ratio of NHS to EDC QDs was found to be an important parameter when forming modified QDs having good photoluminescent properties; different molar ratios including 1:1, 1:2 and 1:5 were explored in preparing the modified QDs and it was observed that the best QD photoluminescence was obtained with the molar ratio of NHS: EDC of 1:5, with other molar ratios not having photoluminescence.

7.3.1.3 The Properties of the as-prepared QD-antibody Conjugated Compound

Photoluminescence spectra of as-prepared antibody-conjugated compound showed that the QDs have an emission spectrum at 562 nm, indicating that they retain their photoactive after conjugation with antibodies. The excitation wavelength of the QD- antibody conjugated compound was observed at 540 nm, no change when compared with both the modified and unmodified QDs (Figure 7.6).

(b) (a)

Figure 7.6. Optical spectra of as-prepared CdSe(S) QD-antibody conjugated

compound: (a) Photoluminescence spectrum (λ excitation = 350 nm) & (b) UV-visible absorption.

CD was used to confirm of formation of antibody-conjugated compound and showed that CD spectrum of antibody-conjugated compound has not been changed in comparison with the CD spectrum of the antibody (Figure 7.7)

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Chapter 7 The Investigation of Photostability of QDs

Figure 7.7. CD spectra of antibody (a) & as-prepared CdSe(S) QD-antibody conjugated compound (b).

It was experimentally found that the photoluminescent properties of the QD-antibody conjugated compound was not quenched when the ratio of antibody to modified QDs was adjusted as (1 µl, 0.7mg/ml) to (0.2 ml, 100 µg/ml), whilst outside of this range, the compound did not have photoluminescence.

In addition, it was determined that temperature is an important parameter in the formation of the antibody-conjugated material; the compound was not obtained at a temperature of 4 ºC, but did form after incubation of the QDs and antibodies at room temperature.

7.3.1.4 Comparison of the Organic Dye CY3 with QDs

The QD-antibody conjugated compound exhibited a strong emission at 562 nm (Figure 7.6, a) which is related to the emission profile of CdSe(S) QDs and it cannot be assigned to the emission of CY3 which is at 570 nm (Table 1, Appendix 14). This indicates that QDs have significant photoluminescence and supports this idea that QDs are potentially better fluorescent probes compared to most fluorescent dyes such as CY3. 173

Chapter 7 The Investigation of Photostability of QDs

7.3.2 Photostability of QDs in Presence of Cancer Cells

The photostability of QDs in the presence of the HCT-116 cell line was investigated to determine any effects of interaction with the components of cell media or oxidation of QDs, leading to quenching of the fluorescent properties of QDs as described in section 7.1. Images of the as-synthesized CdSe(S) QDs in aqueous solution showed that the nanoparticles had luminescent properties under confocal microscope to indicate the photo stability of QDs and having potential ability for imaging applications (Figure 7.8).

Figure 7.8. Confocal image of as-prepared CdSe(S) QDs: QDs can be observed as green illuminated small dots.

The obtained images of both HCT-116 fixed and live cells after treatment with CdSe(S) QDs showed that highly luminescent CdSe(S) QDs were present in samples of both the fixed and live cells, with no determinable loss of brightness, indicating that the QDs were stable during cell incubation, as illustrated in Figure 7.9 & 7.10.

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Chapter 7 The Investigation of Photostability of QDs

Figure 7.9. Confocal image of HCT-116 fixed cells-CdSe(S) QDs: cells (red) & CdSe(S) QDs (green).

Figure 7.10. Confocal image of HCT-116 live cells-CdSe(S) QDs: Cells (blue) & CdSe(S) QDs (green).

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Chapter 7 The Investigation of Photostability of QDs

It was found that all live cells were stable after treatment with QDs at a concentration of 100 µg/ml with no cell necrosis, confirming previous data (Chapter 6) and indicating no sign of cytotoxicity at concentrations less than 105 µg/ml. These data confirm the potential capacity of the as-synthesized QDs for biological imaging due to high photo stability, water solubility and low toxicity.

7.3.3 The Investigation of Photostability of QDs in Cell Media

The emission profile of as prepared CdSe(S) QDs in cell media was recorded because it is important to investigate the interaction of QDs with the components of cell media to understand the effects of media cells on the emission spectra. For instance, it has reported in the literature that water soluble CdTe-MPA capped QDs are stable only in water and the cell growth media alters their emission profile [20]. The recorded emission profiles of as-synthesized CdSe(S) QDs in both cell media and water were recorded using a confocal microscope and the obtained spectra indicated that CdSe(S) QDs have a maximum in the emission spectrum at 548 nm and there is no change or shift in the photoluminescence spectrum of QDs in cell media when compared to the emission profile of QDs in water, as shown in Figure 7.11.

Figure 7.11. Photoluminescence spectra of CdSe(S) QDs in water (a) and in cell media (b). The excitation wavelength of the equipment was adjusted at 405 nm.

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Chapter 7 The Investigation of Photostability of QDs

This suggests that the as-synthesized QDs have photostability in biological context on the contrary with CdTe QDs as reported in the literature [22]. In addition, as- synthesized CdSe(S) QDs were stable in water over one year as shown in Figure 9, Appendix 17. 7.4 Conclusion

In this work a new CdSe(S) QD-antibody conjugated compound with the potential to be used in bio-imaging was successfully synthesized and characterized. In addition, the photostability of CdSe(S) QDs in the presence of colon cancer cells (HCT-116 cells) was also investigated, determining that the as-obtained QDs were stable in biological media.

References

[1]. Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Science 2005, 307, 538. [2]. Wu, X.; Liu, H.; Liu, J.; Haley, K. N.; Treadway, J. A.; Larson, J. P.; Ge, N.; Peale, F.; Bruchez, M. P. Nat. Biotechnol. 2003, 21, 41. [3]. Jaiswal, J. K.; Mattoussi, H.; Mauro, J. M.; Simon, S. M. Nat. Biotechnol. 2003, 21, 47. [4]. Alivisatos, P. Nat. Biotechnol. 2004, 22, 47. [5]. Galeone, A.; Vecchio, G.; Malvindi, M. A.; Brunetti, V.; Cingolani, R.; Pompa, P. P. Nanoscale 2012, 4, 6401. [6]. Morelli, E.; Cioni, P.; Posarelli, M.; Gabellieri, E. Aquat Toxicol 2012, 122-123, 153. [7]. Mandal, A.; Nakayama, J.; Tamai, N.; Biju, V.; Isikawa, M. J Phys Chem B 2007, 111, 12765. [8]. Hoshino, A.; Fujioka, K.; Suga, M.; Sasaki, Y. F.; Ohta, T.; Yasuhara, M.; Dohi, T.; Suzuki, K.; Yamamoto, K. Proc. SPIE-Int. Soc. Opt. Eng. 2005, 5705, 263. [9]. Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Nat. Mater 2005, 4, 435. [10]. Strohl, W. R. Curr. Drug Discovery Technol. 2014, 11, 3. [11]. Beck, A.; Wurch, T.; Bailly, C.; Corvaia, N. Nat. Rev. Immunol. 2010, 10, 345

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[12]. Image from www.wikipedia.com.org. [13]. Lao, U. L.; Kim, J.-Y.; Mulchandani, A.; Chen, W.; American Chemical Society 2006, p BIOT. [14]. Jin, T.; Tiwari, D. K.; Tanaka, S.-I.; Inouye, Y.; Yoshizawa, K.; Watanabe, T. M. Mol Biosyst 2010, 6, 2325. [15]. Song, F.; Chan, W. C. W. Nanotechnology 2011, 22, 494006/1. [16]. Hua, X.-F.; Liu, T.-C.; Cao, Y.-C.; Liu, B.; Wang, H.-Q.; Wang, J.-H.; Huang, Z.- L.; Zhao, Y.-D. Anal. Bioanal. Chem. 2006, 386,1665. [17]. Shan, Y.; Wang, L.; Shi, Y.; Zhang, H.; Li, H.; Liu, H.; Yang, B.; Li, T.; Fang, X.; Li, W. Talanta 2008, 75, 1008. [18]. Zhang, H.; Sun, P.; Liu, C.; Gao, H.; Xu, L.; Fang, J.; Wang, M.; Liu, J.; Xu, S. Luminescence 2011, 26, 86. [19]. Sperling, R. A.; Parak, W. J. Philos. Trans. R. Soc., A 2010, 368, 1333. [20]. Kulvietis, V.; Streckyte, G.; Rotomskis, R. Lith. J. Phys. 2011, 51, 163.

178

Chapter 8 Conclusions & Future Works

8.1 Conclusions

All chemical synthesises presented in this thesis are based upon utilizing aqueous routes to prepare either CdSe nanoparticles or QDs. Although aqueous synthesis has previously been proposed as an alternative to organic routes, the synthesis of highly crystalline, stable and highly luminescent nanoparticles has remained challenging due to difficulties in reproducibility and the low quality of products that are strongly dependent on experimental parameters. Therefore, in this work, much focus was made on finding optimal experimental parameters for the formation of nanoparticles in aqueous media and the investigation of the properties of the obtained products including particle size, crystallinity and optical properties of both CdSe nanoparticles and QDs.

In Chapter 3 a new mechanism for the formation of cubic CdSe nanoparticles in acidic pH was proposed; whilst previous reports were based upon formation of CdSe nanoparticles in basic pH. Meanwhile, CdSe nanoparticles were prepared under different experimental parameters, allowing us to investigate the effect of experimental conditions on the properties of as-prepared nanoparticles. Moreover, highly crystalline hexagonal CdSe nanoparticles were obtained in acidic pH.

In Chapter 4, the knowledge of CdSe nanoparticle formation from Chapter 3 was employed to synthesise CdSe(S) QDs; this work was based upon a modification of a previous literature method reported by Aldeek and co-workers [1] and it was found that by optimizing the molar ratio of cadmium precursor to capping agent from 1:1 to 1:2, CdSe(S) QDs were obtained with highly crystallinity, good stability and narrow emission profile. Then, the blue-emitting highly luminescent ZnSe(S) QDs were prepared by replacing the cadmium precursor with a zinc precursor and finding optimal experimental parameters including proper pH, appropriate reaction time and suitable molar ratio of Zn: Capping agent: Se.

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Chapter 8 Conclusions & Future Works

Chapter 5 expanded on previous work, demonstrated by Aldeek [1] to prepare

CdSe(S)/ZnO QDs whilst coating of CdSe(S) core QDs with Fe2O3 led to formation of type II- CdSe(S)/Fe2O3 core/shell QDs as a new core/shell nanostructure.

Chapter 6 presented the results of cytotoxicity studies of three kinds of QDs including CdSe(S) QDs as representative of heavy-metal QDs, CdSe(S)/ZnO QDs as a core/shell type QDs and ZnSe(S) QDs as an example for free heavy metal QDs. The results showed that cytotoxicity of CdSe(S) QDs can be reduced using coating of the core QDs with the shells. Additionally, heavy metal-free QDs such as ZnSe(S) QDs exhibited nontoxicity in all cytotoxicity assays.

In Chapter 7, a CdSe(S) QD-antibody conjugated compound was synthesised, indicating that QDs can be bound to biomolecules including antibodies. Moreover, this was achieved without significant change in the structure of the antibody or the emission profile of the QDs. Besides this, the results of photostability using confocal microscopy showed that the as-synthesized CdSe(S) QDs are photostable in the presence of HCT- 116 cancer cells and the emission profile of QDs was unaffected by incubation with the cells. Moreover, the CdSe(S) QDs exhibited photostability even in as-prepared antibody conjugated compound, indicating that the QDs are much brighter than other potential flouresence probes such as fluorophore Cy3.

8.2 Future Works

Although the manner in which QDs act towards cells is complex and the toxicology of nanomaterials including QDs is a relatively young field, under the conditions of this study the degree of cytotoxicity of the CdSe(S) QDs is much less than the cytotoxicity of recognised anti-tumour agents such as 17-AAG. As the cytotoxicity of QDs in biological media is most likely the result of QD degradation yielding free ions, the kinetics of this have not studied here and may be the scope of further work.

Given that QDs may have only limited toxicity, further studies focussed on delivering QDs to DNA and cell nuclei could be envisaged, providing intra-cellular imaging of assays.

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References

[1]. Aldeek, F.; Mustin, C.; Balan, L.; Medjahdi, G.; Roques-Carmes, T.; Arnoux, P.; Schneider, R. Eur. J. Inorg. Chem. 2011, 794.

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Appendix

1. Calculation of Bohr Radius

휀 ɦ2 r = 2 (Bohr Formula) 휋 푚 푟 푒

In this equation, r is the radius of the sphere defined by the three-dimensional separation of the electron-hole pair, 휀 is the dielectric constant of the semiconductor, ɦ is Planck’s constant, 푚 푟 is the reduced mass of the electron-hole pair and 푒 is the charge of electron [1].

2. La Mer Theory

In 1950, La Mer and co-workers proposed a new mechanism to describe formation of nanoparticles in solution based upon two separated processes: nucleation and crystal growth [2]. This mechanism was utilized by La Mer to study formation of sulfur sols from sodium thiosulfate that occurs in two steps: the first to form free sulfur from sodium thiosulfate and the second to form sulfur sols [2]. La Mer mechanism was then adopted as the first proposed mechanism that describes formation of nanoparticles in two separated steps [3].

According to the La Mer theory, the nucleation and growth can be divided in three stages [4]:

(I)- The concentration of solute increases with time.

(II)- Nucleation occurred. The concentration of solute is high enough to overcome energy barrier for nucleation, resulting in formation of stable nuclei.

(III)- First, the concentration of solute decreases until the number of nuclei formed per unit reaches to zero, indicating that nucleation step is completed. Then, the system undergoes growth stages [4].

These stages have been shown in Figure 1.

- 1 -

Appendix

Figure 1. La Mer Plot: three stages of formation of nanoparticles: increasing concentration of solute (I), nucleation (II), growth (III) [3].

3. Calculation of the Number of MPA Molecules on the Surface of CdSe Nanoparticles

CdSe NPs were synthesized in acidic pH by reacting 3.6 mmol of cadmium chloride

(containing 2.5 molecules of H2O) (Mw = 228.32 g/mol) and 4 mmol selenium (Mw = 78.96 g/mol).

Cd (3.6 mmol) + Se (4 mmol) + MPA CdSe nanoparticles

Therefore, the maximum mass yield of CdSe is 0.82 g, however a typical yield was 1.86 g.

This difference in mass () is related to the presence of MPA (Mw = 106.14 g/mol) in the final NPs.

W1 = 0.82 g , W2 =1.86 g, ∆ = 1.04 g = 9.79 mmol of MPA - 2 -

Appendix

9.79/3.6 = 2.71 ~ 3; so the no. of moles of MPA = 3 × no. of moles of CdSe

If we consider a particle of size ~3 nm, then r = 1.5 nm

Surface area of a CdSe particle = 4πr2 = 2.82 × 10-17m2

Volume of a CdSe particle = 4/3 πr3 = 1.41× 10-26 m3

The radius of Cd2+ = 1.09 Å = 1.09× 10-10 m and the radius of Se2- = 1.98 Å = 1.98 × 10- 10m

Therefore the volume of Cd2+ = 5.42 × 10-30 m3 and Se2- = 3.25 ×10-29 m3

The sum of the volumes: Cd2+ + Se2- = 3.79 ×10-29 m3

CdSe is a close-packed structure with a 74% filling so the packed volume per ion pair is:

3.79 ×10-29 m3 / 0.74 = 5.13 ×10-29 m3

As each CdSe nanoparticle (approximate size = 3 nm) is 1.41 ×10-26 m3 and each nanoparticle consists of a number of ion pairs, the number of ion pairs per particle CdSe = 1.41 ×10-26 m3 / 5.13 ×10-29 m3 = 275

But we know that:

The number of MPA molecules per particle is 3 × the number of ion pairs = 3 × 275 = 825

Therefore, the number of MPA molecules on the surface of each CdSe nanoparticle is 825.

Also, we know that the surface area of a CdSe particle = 2.82 ×10-17 m2

Therefore, each MPA molecule takes up (2.82 ×10-17 m2 / 825) = 3.4 ×10-20 m2 ~ 3.5 Å2 .

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Appendix

Figure 2.Schematic of complete coating of a CdSe nanoparticle with MPA.

4. Calculation of the Particle Size of CdSe NPs using PXRD Spectra

B λ d = (Scherrer equation) β cosθ

β = FWHM in radian

θ = the Bragg angle of the peak

λ = the X-ray wavelength (1.5418 Å)

= dimensionless shape factor = 0.8-1~ 0.94 d= the crystallite size

The particle size for as-prepared CdSe nanoparticles in different conditions has been calculated as above:

A) Acidic pH (Figure 3.6) β = 3 degree = 0.0523 radian Cos θ = Cos 21.5º = 0.93 d = 0.94 (0.154 nm) / 0.0523 (0.93) = 2.97 nm

B) Basic pH ((Figure 3.10) β = 2.8 degree = 0.048 radian Cos θ = Cos 22.5º = 0.93 d = 0.94 (0.154 nm) / 0.048 (0.93) = 3.24 nm - 4 -

Appendix

C) In presence of both MPA and L-cysteine (Figure 3.13) β = 2.7 degree = 0.047 radian Cos θ = Cos 22.5º = 0.93 d = 0.94 (0.154 nm) / 0.047 (0.93) = 3.31 nm

D) In presence of both water and ethanol (Figure 3.16) β = 2.6 degree = 0.045 radian Cos θ = Cos 21.5º = 0.93 d = 0.94 (0.154 nm) / 0.045 (0.93) = 3.43 nm

E) Hexagonal nanoparticles (Figure 3.19) β = 3 degree = 0.0523 radian Cos θ = Cos 17.5º = 0.9537 d = 0.94 (0.154 nm) / 0.0523(0.9537) = 2.9 nm

5. Estimation the Particle size of As-prepared CdSe NPs & CdSe(S) QDs from HRTEM Images

All the obtained particle size estimated from TEM images, as detailed below. The values were rounded based upon resolution of TEM Philips Cm 200 which is 0.2.

A- CdSe NPs

I) Acidic pH

Figure 3.7, a: 0.85 cm = 5 nm, daverage of particles = 0.5 cm = 2.94 nm (the rounded value = 3 nm).

II) Besic pH

Figure 3.11, b: 0.45 cm = 2 nm, daverage of particles = 0.75 cm = 3.33 nm (the rounded value = 3.4 nm).

III) In presence of L-cysteine

Figure 3.14, a : 0.4 cm = 2 nm, daverage of particles = 0.6 cm = 3 nm

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Appendix

IV) In presence of ethanol

Figure 3.17, a : 0.4 cm = 2 nm, for particles in area A : d average = 1 cm = 2.5 nm, for particles in area B : daverage = 0.75 cm = 3.75 nm so daverage for particles in area A and B = 3.12 nm (the rounded value = 3.4 nm).

V) Hexagonal nanoparticles

Figure 3.20 : 0.5 cm = 2 nm, daverage = 0.85 cm = 3.4 nm. B- CdSe(S) QDs

Figure 4.4: 0 .9 cm= 5 nm, daverage = 0.9 cm = 3.33 nm ( the rounded value = 3.4 nm).

6. Calculation of the Band Gap Energy (Eg) of CdSe NPs

According to the literature [5, 6], the energy gap can be obtained using Equation 1:

ℎ 푐 Eg = (Equation 1) 휆

Where h is Planck’s constant = 6.626 × 10-34 Joules sec, c = 3× 108 m/s, λ = cut off wavelength (absorption onset) and 1 eV = 1.6 × 10-19 J

I) As- prepared nanoparticles in acidic pH

Band gap value of CdSe NPs prepared in acidic pH = 6.626 × 10-34 × 3 × 108 / 320 ×

10-9 = 0.078 × 10-17 Joules = 3.88 eV

∆ E = Enanoparticle – Ebulk = 3.88– 1.7 = 2.18 eV ( acidic pH)

Eg for other nanoparticles was estimated using similar calculation as below:

II) As-prepared nanoparticles in basic pH:

A- without reflux: λ = 625 nm, Eg = 1.98 eV, ∆ E =1.98 -1.7 = 0.28 eV

B- After 6 hours reflux: λ = 350 nm, Eg = 3.55 eV, ∆ E = 3.55- 1.7 = 1.85 eV

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Appendix

III) As-prepared nanoparticles in presence of L-cysteine: λ= 570 nm, Eg = 2.17 eV,

∆E = 2.17 - 1.7 = 0.48 eV.

IV) As-prepared nanoparticles in presence of ethanol: λ = 650 nm, Eg = 1.91 eV, ∆E = 1.91- 1.7 = 0.21 eV.

V) Hexagonal CdSe nanoparticle: λ = 370 nm, Eg = 3.36 eV, ∆E = 3.36 - 1.7 = 1.66 eV.

7. UV Spectrum of As-synthesized CdSe NPs in Acidic pH after 6 hours reflux

250 nm Intensity (a.u) Intensity

200 300 400 500 600 700 800 Wavelegth (nm)

Figure 3. UV-visible spectrum of as-prepared CdSe nanoparticles in acidic pH after 6 hours reflux.

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Appendix

8. Electronic Transitions for ZnSe Nanocrystals

According to molecular orbital theory, for a small crystallite, all molecular orbitals are characterized by quantum numbers in partial analogy with that of the hydrogen atom. The calculated molecular orbitals (MOs) for a 45 Å diameter ZnSe crystallite appear in Figure 2 and show that the lowest state is N = 1 and L = 0 which is called 1S state and electronic transition occurs as 1S-1S transition, as shown in Figure 4 [7, 8].

Figure 4. Molecular energy level for a ZnSe crystallite [5].

9. PXRD Patterns of Cubic CdSe NPs under Different Conditions (10° ≤ 2 ≤ 60°)

Low angle PXRD spectra of cubic CdSe nanoparticles in different conditions are shown in Figures 4, (a) to (d) and indicate that the obtained cubic nanoparticles does not show the formation of extra phases or other compounds on the contrary with hexagonal CdSe nanoparticles (described in section 2.3.3).

- 8 -

Appendix

(b) (a (d) Intensity (a.u) Intensity (c) 10 20 30 40 50 60 2 Theta (degree)

Figure 5. PXRD low angle of as-prepared cubic CdSe nanoparticles: in acidic pH (a), in basic pH (c), in presence of L-cysteine (C) and in presence of ethanol (d).

10. Calculation of the Particle Size of QDs

10.1 Particle size of CdSe(S) QDs

B λ d = (Sherrer equation) β cosθ β = 2.5 degree = 0.044 radian cos θ = cos 22.5º = 0.94 0.94 (0.154) d = 0.044 ( 0.924) d = 3.56 nm

10.2 Particle size of ZnSe(S) QDs β = 3.5 degree = 0.061 radian cosθ = cos24 = 0.913 0.94 (0.154) d = 0.061 ( 0.913) d = 2.59 nm

- 9 -

Appendix

11. The correspondence of planes in PXRD and SAED patterns

111

220

311 Intensity (a.u) Intensity

20 30 40 50 60 70

2 Theta (degree))

Figure 6. PXRD and SAED pattern of as-synthesized CdSe (S) QDs.

As it has been shown in Figure 1, there are 3 peak positions in PXRD pattern: 2θ1 = X =

X (2θ1 ) 26 X 26, 2θ2 = Y = 44 and 2θ3 =Z = 52. The position ratios are: = = = 0.59, = Y (2θ2) 44 Z

(2θ1 ) 26 Y (2θ2 ) 44 = = 0.5 and = = = 0.84 and in SAED pattern there are 4 rings: (1), (2θ3) 52 Z (2θ3) 52 (2), (3), (4). The diameter ratios for these rings are:

(1) 6 (1) (6) (1) 6 (2) 7.6 (3) 9 = =0.79, = = 0.66 and = =0.5, = = 0.63, = =0.75 (2) 7.6 (3) (9) (4) 12 (4) 12 (4) 12

With comparison of diameter ratios in SAED to position ratios in PXRD, it can be X figured that diameter ratio in ring 1 to ring 4 is equal to ratio position of and both are Z equal to 0.5. Therefore, the ring 1 and ring 4 in SAED pattern correspondence to planes 111 and 311 in PXRD. It was also determined that ring 3 in SAED pattern correspondence to plane 220 in XRD pattern because one of rings 2 or 3 correspondence X to plane 220 and ratio position of = 0.59 has further agreement with diameter ratio of Y ring1 to ring 3 which is equal to 0.66 rather than diameter ratio of ring 1 to ring 2

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Appendix

Y which is 0.79. Meanwhile in a similar comparison, position ratio of = 0.84 is more Z comparable with diameter ratio of ring 3 to ring 4 which is 0.75 in compare with diameter ratio of ring 2 to ring 4 which is 0.63.

Consequently, it can be approximately determined that ring 1, 3, 4 in SAED correspondence to planes 111, 220 and 311, respectively. The other rings with similar calculation, belonged to other planes which they cannot be observed in XRD pattern for example ring 2 correspondence to plane 200 and rings 5, 6, 7, 8 corresponding to planes 222, 400, 331 + 420 and 422.

12. Atomic Percentage of Elements in CdSe(S) QDs

XPS Analysis can be used as a quantitative method to determine elemental composition. The atomic percentage of as-prepared QDs (Chapter 4, Section 4.3.1 have been summarized in Table 1.

Table 1: Elemental analysis of CdSe(S) QDs

Sample Element Atomic % CdSe(S) QDs Carbon 34.02 Cadmium 10.85 Sulfur 10.35 Selenium 0.57

13. Calculation of the Particle Size of Core/shell QDs using PXRD

13.1 CdSe(S)/ZnO QDs

B λ d = (Scherrer equation) β cosθ

β = 3 degree = 0.0523 radian cos θ = cos 22º = 0.927 d = 0.94 (0.154 nm)/0.0523 (0.927) =2.985 nm ~ 3 nm

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Appendix

13.2 Particle size of CdSe(S)/ Fe2O3 QDs β = 3 degree = 0.0523 radian cosθ = cos 21.5º = 0.93 d = 0.94 (0.154 nm)/0.0523(0.93) = 2.99nm ~ 3 nm

14. XPS Standard Spectrum of α-Fe2O3

Figure 7. XPS standard spectrum of Fe in α-Fe2O3: peak positions at 710.9 ev and 3+ 3+ 726 ev are assigned to Fe 2P3/2 and Fe 2P1/2 , respectively [9].

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Appendix

15. XPS Spectra of CdSe(S) /Fe2O3 Core/shell QDs

Figure 8. XPS spectra of CdSe(S)/ Fe2O3 core/shell QDs binding energy

of (a) Cd3d , (b) Se3d , (c) S2p and (d) C1S.

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Appendix

16. Optical Properties of Cy3

Approximate peak wavelengths of excitation and emission of Cy3 has been mentioned in Table 2 [10].

Table 2. Optical properties of CY3

Fluorophore Excitation peak (nm) Emission peak(nm) CY3 550 nm 570 nm

17. Photostability of As-synthesized CdSe(S) QDs

The obtained CdSe(S) QDs were stable in water over one year. As described in Figure 9, the emission wavelength of QDs was determined as 550 nm after 1 hour whilst emission wavelength of QDs was 562 nm either after 4 months or 14 months which is not a significant shift, indicating that QDs have photostability over the time.

Figure 9. Photostability of as-synthesized CdSe(S) QDs over the time: after 1 hour (a), 4 months (b) and 14 months (c).

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Appendix

References

[1]. Murphy, C. J.; Coffer, J. L. Appl. Spectrosc. 2002, 56, 16A. [2]. La Mer, V. K.; Dinegar, R. H. J. Am. Chem. Soc. 1950, 72, 4847. [3]. Thanh, N. T. K.; MacLean, N.; Mahiddine, S. Chem. Rev.2014, 114, 7610. [4]. Park, J.; Joo, J.; Kwon, S. G.; Jang, Y.; Hyeon, T. Angew. Chem., Int. Ed. 2007, 46, 4630. [5]. Sivasankar, K.; Padmavathy, N. Micro Nano Lett. 2011, 6, 144. [6]. Li, J. H.; Ren, C. L.; Liu, X.; Hu, Z. D.; Xue, D. S. Mater. Sci. Eng., A 2007, A458, 319. [7]. Bawendi, M. C.; Steigerwald, M. L.; Brus, L. E. Annu. Rev. Phys. Chem. 1990, 41, 477. [8]. Chestnoy, N.; Hull, R.; Brus, L. E. J. Chem. Phys. 1986, 85, 2237. [9]. Moulder, F. J.; Stickle, F. W.; Sobol. E. P.; Bomben, D. K. Hand book of X-ray photoelectron spectroscopy. Second Edition., Perkin Elements., 1992, p 81. [10]. http://www.jacksonimmuno.com/technical/f-cy3-5.asp.

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List of Publications

The following manuscripts are under preparation for submitting in peer review journals:

 F. Mirnajafi and J.A. Stride. “The Investigation of Effective Parameters on the Formation of CdSe Nanoparticles in Aqueous Reactions”.  F. Mirnajafi, D. M. Ramsey, Sh. R. McAlpine, F. Wang, P. Reece and J. A. Stride. “Highly Luminescent Blue-emitting and Nontoxic ZnSe(S) QDs “.  F. Mirnajafi and J.A. Stride. “Synthesis of type-II CdSe(S)/Fe2O3 Core/shell QDs “.  F. Mirnajafi and J.A. Stride. “The Investigation of Photostability of CdSe(S) QDs”.  F. Mirnajafi, D. M. Ramsey, Sh. R. McAlpine, F. Wang, P. Reece and J. A. Stride. “Investigation of Cytoxicity of Water Soluble Quantum Dots in the Presence of Colon Cancer Cells”.  F. Mirnajafi, D. M. Ramsey, Sh. R. McAlpine, F. Wang, P. Reece and J. A. Stride. “Review of Synthesis of Water Soluble QDs in an Aqueous Route”.

Published Papers

Some parts of work have been published in conference Proceedings as below:

 F. Mirnajafi, F. Wang, P. Reece and J.A..Stride, "Highly Luminescence Quantum Dots: New Tools for Biological Applications". NIST-Nanotecl 2012, volume 1, 441.  F. Mirnajafi and J.A. Stride “The Investigation of Optical Properties of Water Soluble Quantum Dots in a Quantum Dot-Antibody Conjugated Compound “, Accepted for publication in IEEE 2014.

Presentations/Posters

 F. Mirnajafi and J. A. Stride. “ Biocompatible Quantum Dots: Next Generation of Nanoparticles ”, Australian X-ray Analytical Association International Conference, Sydney, Australia, February 2011 (oral presentation).

List of Publications

 F. Mirnajafi, F. Wang, P. Reece and J. A. Stride. “ Highly Luminescent Quantum Dots: New Tools for Biological Applications “, International Conference of Nanotech 2012, Santa Clara, San Francisco, California, U.S.A, June 2012 (poster presentation).

 F. Mirnajafi, D. M. Ramsey, Sh. R. McAlpine, F. Wang, P. Reece and J. A. Stride. “The Investigation of Cytoxicity of Water Soluble Quantum Dots in Presence of Colon Cancer Cells”, International Conference of on Emerging Advanced Nanomaterials (ICEAN 2012), Brisbane, Australia, October 2012

(oral presentation) *.  F. Mirnajafi and J.A. Stride “The Investigation of the Properties of Water Soluble Quantum Dots in Biological Context”, International Conference of Nanotechnology and Nanoscience (ICONN 2014), Adelaide, Australia, February 2014 (oral presentation) *.

 *: It was also presented as a poster in poster day competition, Faculty of Science, University of New South Wales and selected as the best poster in the field of Nanotechnology, Sydney, Australia, August 2013.