Ligand exchange of QDs

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Table of contents

Chapitre 1 Quantum Dot-DNA Conjugates – An Overview
1.1 Quantum Dots as fluorescent probes
1.1.1 Advantages of QDs over organic fluorophores
1.1.2 Electronic properties
1.1.3 Optical properties
1.1.4 Organic synthesis of core-shell heterostructures
1.2 Methods to disperse QDs in aqueous media
1.2.1 Encapsulation
1.2.2 Ligand exchange
1.3 Strategies to conjugate DNA to QDs dispersed in aqueous media
1.3.1 Properties of DNA
1.3.2 Conjugation of DNA to shells of QD
1.3.3 Conjugation of DNA to ligands on QD
1.4 Types of covalent conjugation reactions
1.5 Purification of conjugates of QD-DNA
1.6 Applications of QD-DNA conjugates
1.7 Conclusions
Chapitre 2 A Novel Method to Conjugate DNA to Quantum Dots
2.1 Dispersion of Quantum Dots in aqueous media
2.1.1QDs in organic solvent
2.1.2 Dispersion of QDs in aqueous buffers
2.1.3 Ligand exchange of QDs
2.2 Conjugation of amine labelled DNA on thiols of QD
2.3 Validation of QD-DNA conjugates
2.3.1 Qualitative approaches
2.3.2 Assessing non-specific interactions of DNA with QD
2.3.3 Quantification of number of DNA conjugated on QD
2.4 Parameters that affect the yield of conjugation of DNA to QDs
2.4.1 Role of the polymer coat
2.4.2 Effect of reducing agent
2.4.3 Effect of pH of medium
2.4.4 Effect of salt concentration
2.4.5 Effect of DNA length
2.5 Experiments on stability of QD-DNA conjugates
2.5.1 Quantum yield measurements on QD-DNA conjugates
2.5.2 Solution stability of QD-DNA conjugates
2.6 Applicability of the coupling strategy
2.6.1 On QDs emitting at different colors
2.6.2 Conjugation of DNA to other nanoparticles
2.6.3 On different lengths of DNA
2.6.4 Conjugation of proteins using similar strategy
2.7 Conclusions
Chapitre 3 Quantum Dot-DNA Conjugates for Controlled Assembly of Transferrin 
3.1 Introduction
3.1.1 Quantum dots as bioimaging agents
3.1.2 Methods to conjugate proteins to QD
3.1.3 Desirable properties of QD-protein conjugate for bioimaging
3.1.4 Transferrin as a model protein system
3.1.5 A novel method to conjugate Transferrin on QDs
3.2 A novel method to functionalize QDs with proteins: synthesis and characterization .
3.2.1 Synthesis of QD-DNA-Tf
3.2.2 Biochemical characterization of QD-DNA-Tf
3.3 Biological properties of QD-DNA-Tf
3.3.1 Receptor-mediated endocytosis of QD-DNA-Tf
3.3.2 Kinetics of endocytosis of QD-DNA-Tf
3.3.3 Steady-state localization of QD-DNA-Tf
3.3.4 Endocytosis of QD-DNA-Tf does not affect the uptake of subsequent Tf-TfR
3.3.5 QD-DNA-Tf recycle out of cells over time
3.3.6 Photostability of QD-DNA-Tf in endosomes
3.3.7 Recycling of conjugates of QD-Tf is affected by several factors
3.3.8 Recycling of QD-DNA-Tf – insights from long duration live imaging
3.4 Conclusions
Chapitre 4 Systematic Evaluation of Quantum Dot Surface Chemistries for Biological Applications
4.1 Introduction
4.2 Quantum dots and the surface chemistries of interest.
4.2.1 Photophysical characterization of QDs in organic solvent
4.2.2 Types of polymers, brief characterization and method of ligand exchange
4.3 Probing biological characteristics of the QDs in-vitro
4.3.1 Electrophoretic mobility of QDs upon incubation with serum
4.3.2 Hard corona and electrophoretic mobility
4.3.3 QY of QDs upon incubation with serum
4.3.4 Quantification of the protein content.
4.4 Probing biological characteristics of these QDs in-cellulo
4.4.1 Dependence on concentration.
4.4.2 Dependence on time
4.5 Conclusion
Chapitre 5 Materials and Methods
5.1 Characterization techniques
5.1.1 Absorption
5.1.2 Fluorescence
5.1.3 Measurement of Quantum yield
5.1.4 Epifluorescence microscope
5.1.4 Spinning disk confocal microscope
5.1.5 Transmission electron microscopy (TEM)
5.1.6 Ultracentrifugation
5.1.7 Size Exclusion Chromatography
5.1.8 Gel Electrophoresis
5.1.9 Dynamic Light Scattering
5.1.10 Zeta Potential
5.1.11 Affinity beads assays
5.2. Chemical methods
5.2.1 Materials
5.2.2 Nomenclature of the amphiphilic polymers
5.2.3 Synthesis of monomer DTMAm
5.2.4 Synthesis of (20-80)n-Zw copolymer
5.2.5 Synthesis of (20-80)20-PEG copolymer
5.2.6 Ligand exchange of QDs (MPA protocol)
5.3 Conjugation methods
5.3.1 Bioconjugation of QDs with dyes or small molecules
5.3.2 Conjugation of DNA to QDs
5.3.3 Conjugation of proteins to QDs
5.3.4 Conjugation of proteins to thiolated DNA
5.4 Biology experiments
5.4.1 Cell maintenance
5.4.2 Plating of cells for experiments
5.4.3 Uptake experiments
5.4.4 Stripping of surface ligands
5.4.5 Fixation of cells
5.4.6 Kinetics experiments
5.4.7 Dual-labelling experiments
5.4.8 Colocalization experiment
5.4.9 Immunofluorescence experiment
5.5 Data Analysis
5.5.1 Analysis of data from epifluorescent microscope
5.5.2 Background subtraction and marking of cell boundary
5.5.3 Quantification of fluorescence intensity
5.5.4 Colocalization of dual colored images
5.5.5 Analysis of data from spinning disc confocal microscope
5.5.6 Particle tracking from live imaging experiments
References
Conclusions
Appendices
Appendix 1: Different QDs used in this study
Appendix 2: Compositions of different buffers used in this thesis
Appendix 3: Sequences of DNA used in this thesis
Appendix 4: Fluorophores and related filters
Appendix 5: Optical set up of the microscope