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Purification of conjugates of QD-DNA
The search for ideal method of purification of functionalized QDs from the excess reactants is as old as the hunt for best strategy of conjugation. Almost no biological or chemical application of these specific conjugates can be carried out without purification of excess (uncoupled) DNA. There are three broad methods of separation discussed herein.
Electrophoresis. Electrophoretic migration of QDs depends on both the type of QD structure and the type of polymer coating. Ligand such as MPA, DHLA and polymeric impart negative charge on QDs, that facilitate migration towards positive terminals. 38,76Conjugation of DNA on QDs results in further decrease of the zeta potential of the surface of QDs without massively altering the molecular weight. This results into faster migration towards the positive terminal. This property has been utilized to separate QDs conjugated with DNA from the unconjugated ones.69,81 This is one of the most simple and routinely used methods. QDs conjugated to DNA have even been purified by the extent of labelling stoichiometry. 57. However, in order to extract conjugated QDs from the gel, tedious extraction processes need to be carried out. The relevant bands of interest should be first excised out of the agarose gel followed by either melting of the gel or prolonged incubation of the gel fragments in relevant buffers. QDs are then re-collected back by centrifugation and concentration. Passing QD-DNA conjugates from several of these methods often result into poor yields and issues with long-term stability.
Dispersion of QDs in aqueous buffers
Organic synthesis of QDs renders the nanocrystals coated with ligands incompatible with biological solvents and buffers. In order to conjugate any biomolecule to QDs, the nanocrystals need to be first dispersed in water. There are several methods to transfer QDs from organic media to aqueous media. These methods have been discussed in great detail in Chapter 1. Briefly, two of the most popular methods to disperse QDs in water include coating them with amphiphilic small molecule ligands (such as Mercaptopropionic acid, MPA) or with higher molecular weight polymers. Small molecule ligands such as MPA have a terminal thiol and a carboxylic acid group. The terminal thiols can attached to the surface of QDs and subsequently displace the original ligands. Additional conjugation reactions can be carried on the carboxylic acid groups. However, small molecule coated QDs have issues with long-term stability. The thiols anchoring to QD shells are prone to photo-oxidation and can desorb in dilute solutions. This exposes hydrophobic patches on the surface of QDs that eventually lead to aggregation. In order to circumvent these issues, multidentate polymeric ligands are used, those bind to the surface of QDs by several appendages and also display several polar groups to impart water solubility.
Previous work from our group have carefully developed and characterized several polymers with exceptional properties. Of particular interest is the thiotic acid-sulfobetaine containing zwitterionic polymer detailed by Giovanelli et al.38 Dots coated with this polymer are soluble in water, retain high QY, compact dimensions and can be easily biofunctionalised. These dots are also stable in wide range of pH, salinity and higher dilution. The design of this polymer also had terminal carboxylic acid functional groups, on which specific bioconjugation reactions were carried out. In the work described in this chapter, the terminal functional group was not utilized for conjugation. In fact systematic investigation was carried out to test conjugation on the thiols of the polymer. Thiols on the polymers anchor to the surface of QDs by the thiolate anion, and keep the polymer in place. This strategy is the first report demonstrating that several of these thiols can be actually used for bioconjugation as well.
Synthesis of polymer. For this work, (20-80)20-Zw was used. Briefly, the polymer synthesis was carried out by co-polymerisation of a monomer 1 (20%) and of 3-[3 methacrylamidopropyl(dimethyl)ammonio]propane-1-sulfonate (SPP, 80%) in presence of (<5%) MPA for chain termination. Monomer 1 was synthesised by peptidylic coupling of thiotic acid and N-(3-aminopropyl) methacrylamide exactly as described in the previous work. Detailed protocol for synthesis of this polymer is described in Section 5.1.3 (Chapter 5). The monomer conversion was assessed by NMR and the polymer was obtained as an off-white solid.
Ligand exchange of QDs
Dispersion of QDs in water was carried out by classical two step ligand exchange procedure described previously.38 The detailed protocol is given in Section 5.1.3 (Chapter 5). Briefly, QDs in organic solvent were first exchanged with small amphiphilic ligand Mercaptopropionic acid (MPA) by overnight incubation at 60°C. The following day, excess of MPA was removed by repeated centrifugation and QDs were dispersed in DMF. Addition of a base in excess (potassium tert-butoxide) deprotonates the MPA (on the surface of QDs), making them insoluble in DMF. The DMF is removed by centrifugation and the QDs could be dispersed in basic (pH>7) buffers. In the next step, QDs coated with MPA were exchanged with the zwitterionic polymer. The polymer was first reduced to convert disulfide to thiols, and then left to facilitate dynamic interactions with the surface of the QDs, that eventually displace the MPA and homogenously coat the surface of QDs.
In this work all QDs used were coated with the (20-80)20-Zw polymer and emitted at 610 nm, unless specified otherwise. Characterization of QDs with (20-80)20-Zw polymer. After the above mentioned ligand exchange procedure, QDs were soluble in aqueous medium such as 0.2 M NaHCO3 (pH 8.3), 1X PBS (pH 7.4) and 20mM NaCl. For long-term stability buffers such as 0.2M NaHCO3 and 20 mM NaCl were observed to preserve the quantum yield (QY) of QDs better that PBS.
Conjugation of amine labelled DNA on thiols of QD
From one of the very first reports of QDs in biological applications, Dubertret et al. demonstrated that specific ligands on QDs can be used to conjugate DNA to these nanocrystals18,91. Since then, there have been a range of strategies employed to conjugate DNA to QDs. Several of these are discussed in detail in Chapter 1. For example, amine functionalized DNA (DNA-NH2) has been coupled to carboxyl group on a polymer-coated QDs by reaction with 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). 72,108 Another method to generate QD-DNA conjugates involves ligand exchange of QDs first with mercaptopropionic acid (MPA) followed by displacement of MPA by thiol-functionalized DNA (DNA-SH) directly,43,45,108 or mediated by a linker.110Alternately, QDs have been functionalized with streptavidin first, followed by addition of a biotinylated DNA.103,113 Nevertheless, each strategy has its own limitations. EDC-NHS coupling suffers from low conjugation efficiencies due to hydrolysis of NHS esters. Ligand exchange with thiol-labelled DNA is limited because the surface bound thiols can be easily oxidized by light or oxygen. Finally, the biotin-streptavidin strategy increases the size of QDs substantially thereby limiting its applicability. Also, conjugation of DNA on the QD surface is limited by the number of streptavidin available on the QD. Along with these limitations specific to chemical reactions, purification of QD-DNA conjugates is often tricky. Most conventional method is gel purification and centrifugation, which are both labor intensive and tend to decrease yields.93,114,115
Assessing non-specific interactions of DNA with QD
As discussed in the previous chapter, specific charged ligands may cause. To further support the above qualitative tests, specific experiments to ascertain non-specific adsorption of biomolecules to QDs were done. The non-specific interaction of DNA with the polymer coat on QDs was assessed by two tests. First was based on SABs binding assays. Solutions of (i) uncoupled QD mixed with DNA-Bt (ii) QD-DNA with non-complementary DNA-Bt were incubated with SAB and visualized under fluorescent microscope. The beads remained non- fluorescent, thereby confirming the absence of non-specific interaction between DNA and QDs (Figure 2-7, A). Several other non-specific absorption controls are also discussed in chapter.
Another approach used dual mode of detection on SEC. Samples of (i) QD mixed with Cy5 labelled DNA and (ii) QD-DNA mixed with Cy5 labelled non-complementary DNA were loaded on the SEC and monitored using absorbance at two wavelengths – 350 nm (black, for QD) and 630 (grey, for Cy5). Then the presence of Cy5 signal during the elution of QD was estimated. No Cy5 fluorescence was detected corresponding to the QD and QD-DNA, validating that there is minimal non-specific adsorption of DNA-Cy5 on QDs (Figure 2-7, B).
Parameters that affect the yield of conjugation of DNA to QDs
In the previous section, several methods to validate the presence of DNA on QDs after conjugation were discussed. Reaction with dual labelled DNA with 5’primary amine and 3’ Cy5 on QD showed that in these conditions, approximately 2-3 DNA molecules could be conjugated to QDs. The ability to conjugate biomolecules on the surface of QDs could depend on variables such as total number of functional groups accessible for conjugation, pH of the reaction media, dimensions of the biomolecule (mostly relevant for proteins), total charge on the biomolecule etc. It was then interesting to see whether this conjugation yield could be improved by further optimizing several of these conditions.
Role of the polymer coat
As discussed in the introduction chapter, conjugation of DNA is very different from conjugation of proteins to QDs (and other NPs). One of the causative factors for such differences is the total number of functional groups available for conjugation. Reactive groups such as NH2 and COOH are abundant on proteins, owing to amino acids such as lysine, arginine, aspartic and glutamic acid. Contrarily, the functional groups on DNA are synthetically incorporated. Hence by design, oligonucleotides are monofunctionalized. Therefore, there is only one reactive group per DNA (unless synthesized otherwise). In the reaction discussed in this chapter, the second reactant is thiol of the polymer on the QDs. By intrinsic design, the polymer synthesis comprises of 20% monomer derived from ɑ-Lipoic acid and 80% zwitterions with average chain length of approximately 20 monomers. This lends into statistically 2-4 dithiol groups per polymer chain. Some of these thiols from the lipoic acid will anchor the QD surface and keep the polymer attached to the QDs. Others remain as disulfides, which can be conjugated to biomolecules. Additionally, the dynamic nature of thiol-metal interactions may have some transient reactive thiols that also facilitate covalent conjugation of biomolecules on the polymer. It was then interesting to investigate whether the QDs could be made more reactive, by incorporation of additional thiols. With that motivation (50-50)10-Zw was synthetized as described in Section 5.1.3 (Chapter 5). As such, polymer should possess approximately 5 (or more) monomer units derived from ɑ-Lipoic acid instead of 2-4 for the (20-80)20-Zw. Further increase in the mole fraction of the thiotic acid derived monomer (and concomitant reduction in mole fraction of sulfobetaine) reduced the solubility of polymers in aqueous buffers and therefore could not be used (data not shown).
Effect of DNA length
Since DNA is a polyanion, it was then interesting to assess the variation in conjugation efficiency with respect to the length of DNA. The experiments for coupling different length of DNA on QD were carried out at NaCl concentration of 1400mM. It was found that despite screening of charges by addition of high salt, the coupling is both size and charge limited. The total number of DNA/QD for single stranded DNA was 12±4 for 15 bases and 6±2 for 45 bases length (Table 2-6). Similar total charge limited conjugation was also seen in reaction of single vs double stranded DNA labelled with Cy5. Upon conjugation ds DNA of 15bp length (same as before), the total number of DNA conjugated was 4±1.3 per QD.
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
