COLÓIDES TERMICAMENTE SENSÍVEIS
SÍNTESE, ADSORÇÃO DE OLIGONUCLEÓTIDOS E CARACTERIZAÇÃO FOTOFÍSICAResumoNanopartículas de núcleo-coroa, com um núcleo vítreo de poli(metilmetacrilato) (PMMA) e uma coroa termossensível de poli(N-isopropilacrilamida) (PNIPAM) foram sintetizadas por polimerização em emulsão envolvendo duas etapas. Estas partículas têm várias aplicações, entre as quais devemos salientar a sua utilidade como suportes em testes de diagnóstico, testes imunológicos e em processos de separação. A adsorção de oligonucleótidos carregados negativamente compostos por 25 unidades (ou meros) de timina marcados com rodamina X (ROX) na coroa carregada positivamente obedece à isotérmica de Hill. Usando as propriedades solvatocrómicas da rodamina X, a polaridade da coroa termossensível pode ser obtida abaixo e acima da temperatura de transição do volume de fase (TTVF). Utilizando a técnica de anisotropia de fluorescência estacionária e resolvida no tempo, a dinâmica dos oligonucleótidos marcados com ROX e adsorvidos na coroa pode ser seguida e correlacionada com a variação do volume da coroa durante a transição de fase. Transferência não radiativa de energia de Förster (FRET) entre doadores (oligonucleótidos marcados com ROX) e aceitantes (oligonucleótidos marcados com malaquite verde) foi utilizada para obter informação sobre a distribuição e a conformação dos oligonucleótidos adsorvidos abaixo e acima da TTVF. Finalmente, uma série de copolímeros de bloco anfifílicos, com um bloco hidrofóbico curto de poli(N-decilacrilamida) e blocos hidrofílicos com vários comprimentos de poli(N,N-dietilacrilamida) foram sintetizados por polimerização radicalar controlada (polimerização RAFT). Os copolímeros de bloco e os seus agregados micelares formados em água, por dissolução dos polímeros foram caracterizados por dispersão de luz e fluorescência.
Palavras - ChaveNanopartículas de polímero.
Nanopartículas de núcleo-coroa
Termossensível
Oligonucleótidos
Dispersão de luz
Polímeros
Polimerização
Fluorescência
Colóides
Micelas
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THERMALLY RESPONSIVE COLLOIDS
SYNTHESIS, ADSORPTION OF OLIGONUCLEOTIDES AND PHOTOPHYSICAL CHARACTERIZATIONAbstractCore-shell polymer nanoparticles, with a glassy core of poly(methylmethacrylate) (PMMA) and a thermally responsive shell of poly(isopropylacrylamide) (PNIPAM) were synthesized by a two-stage emulsion polymerization. These particles have several applications, among those they can be used as supports in diagnosis, immunoassay tests and separation processes. The adsorption of negatively charged oligonucleotides composed of 25 mers of thymine (dT25-ROX) labeled with rhodamine X (ROX) in the positively charged thermosensitive shell follows a cooperative Hill isotherm. By using the solvatochromism of the ROX, the polarity of the shell was determined below and above the Volume Phase Transition Temperature (TVPT). Using steady-state and time-resolved anisotropy, the dynamics of the ROX labeled-oligonucleotides adsorbed on the shell was followed and correlated with the variation of volume of the shell during the phase transition. Förster resonance energy transfer (FRET) between a donor (oligonucleotides labeled with ROX) and an acceptor (oligonucleotides labeled with malachite green) was used to obtain information about the distribution and the conformation of the oligonucleotides within the shell below and above the TVPT. Finally, a series of amphiphilic block copolymer, with a similar short poly(N-decylacrylamide) hydrophobic block and hydrophilic blocks of poly(N,N-diethylacrylamide) of several lenghts, were synthesized by controlled/living radical polymerization (RAFT). The block copolymers and their micelle-like aggregates in water were characterized by light-scattering and fluorescence. Key WordsPolymer nanoparticles
Core-shell nanoparticles
Thermally responsive
Oligonucleotides
Light scattering
Polymers
Polymerization
Fluorescence
Colloids
Micelles------------------------------------------------------------
Index - Table of ContentsDedicatory
Abstract
Resumo
Keywords
Palavras chave
Acknowledgements / Agradecimentos
Index
Objectives and Organization of the Thesis ................... 1Chapter 1 – General Introduction ............................ 71.1 Colloids ....................................................... 10
1.1.1 Colloidal Stability........................................... 12
1.1.1.1 Surface Charge and Stability ............................... 12
1.1.1.2 Electrokinetics and Zeta-potential ......................... 16
1.1.1.3 van der Waals Forces ....................................... 18
1.1.1.4 Derjaguin-Landau-Verwey-Overbeek Theory (DLVO Theory) ............................................................ 20
1.1.1.5 Effect of Polymers. Steric Stabilization ................... 22
1.2 Polymers ....................................................... 24
1.2.1 Flory-Huggins Theory ......................................... 26
1.2.2 Phase Transitions and Miscibility ............................ 29
1.2.3 Stimuli-Responsive Polymers .................................. 34
1.2.3.1 Thermally Responsive Polymers .............................. 34
1.2.3.2 pH Responsive Polymers ..................................... 35
1.2.4 Nucleic Acids and Oligonucleotides ........................... 36
1.3 Polymerization Techniques ...................................... 38
1.3.1 Radical Polymerization ....................................... 39
1.3.2 Controlled/Living Radical Polymerizations .................... 41
1.3.3 Emulsion Polymerization ...................................... 46
1.4 Association Colloids ........................................... 50
1.4.1 Self-Assembly of Small Amphiphiles ........................... 51
1.4.2 Self-Assembly of Block Copolymers ............................ 57
1.5 References ..................................................... 61
Chapter 2 – Experimental Methods and Techniques ............ 712.1 Optical Spectroscopic Methods .................................. 72
2.1.1 Ultraviolet and Visible Absorption Spectroscopy .............. 72
2.1.1.1 The Beer-Lambert Law and the Absorption Spectrum ........... 73
2.1.1.2 Spectrophotometers and Experimental Considerations ......... 75
2.1.2 Fluorescence Spectroscopy .................................... 77
2.1.2.1 Emission Spectrum .......................................... 79
2.1.2.2 Excitation Spectrum ........................................ 81
2.1.2.3 Spectrofluorometers and Experimental Conditions ............ 82
2.1.2.4 Excited State Kinetics and Quantum Yield ................... 83
2.1.2.4.1 Quantum yield ............................................ 85
2.1.2.5 Time-Resolved Fluorescence ................................. 86
2.1.2.5.1 Single Photon Timing (SPT) Instrumentation .............. 86
2.1.2.5.2 Fluorescence Decay Analysis .............................. 90
2.1.3 Light Scattering ............................................. 93
2.1.3.1 Static Light Scattering .................................... 96
2.1.3.2 Brownian Motion and Stokes-Einstein Equation .......... 103
2.1.3.3 Dynamic Light Scattering .................................. 107
2.1.3.3.1 Time-dependent Correlation Function of Light Scattering Intensity ......................................................... 109
2.1.3.3.2 Time-dependent Correlation Function of the Scattered Electric Field .......................................................... 112
2.1.3.3.3 Signal Processing - Correlator ....................... 117
2.1.3.3.4 Polydisperse Samples ................................. 119
2.1.3.3.5 Analysis Methods ..................................... 120
2.1.3.3.5.1 Cumulants Method ................................... 121
2.1.3.3.5.2 Regularization or Inversion Methods (CONTIN) ....................................................... 122
2.1.3.3.5.3 Overlay Histogram Methods with Exponential Sampling(EXPSAM) ....................................................... 123
2.1.3.4 Preparation of Samples ................................. 123
2.1.3.5 Light Scattering Spectrometer Components ............... 125
2.1.4 Nuclear Magnetic Resonance Spectroscopy (NMR) ............ 125
2.1.4.1 Pulsed or Fourier-Transform NMR experiment: FT-NMR .. 126
2.1.4.2 Experimental Conditions and Applications ............... 128
2.2 Chromatographic Methods .................................... 128
2.2.1 Size Exclusion Chromatography (SEC) ...................... 129
2.2.1.1 Experimental Conditions and Intrumentation ............. 132
2.3 Mass Spectrometry .......................................... 132
2.3.1 Mass Spectrometer ........................................ 132
2.3.2 Matrix Assisted Laser Desorption/Ionisation Time-of-Flight (MALDI-TOF) Mass Spectrometry ........................ 134
2.3.2.1 Ionisation ............................................. 134
2.3.2.2 The Time-Of-Flight (TOF) Analyser ...................... 136
2.3.3 Experimental Conditions and Instrumentation .............. 138
2.4 References ................................................. 138
Chapter 3 - A Fluorescence Anisotropy Standard Dye: Rhodamine 101 ............................................................... 1433.1 Introduction ............................................... 144
3.2 Rhodamines ................................................. 146
3.3 Fluorescence Anisotropy .................................... 152
3.3.1 Steady-State Anisotropy .................................. 156
3.3.2 Time-Resolved Fluorescence Anisotropy .................... 157
3.3.2.1 Analysis of Time-Resolved Anisotropy Decays ............ 157
3.3.2.2 Construction of Anisotropy Decay Methods ............... 159
3.3.2.2.1 Method 1: Construction of anisotropy decay without deconvolution .................................................. 160
3.3.2.2.2 Method 2: Deconvolution of I//(t) and I(t) using an arbitrary function to create r(t) ........................................ 161
3.3.2.2.3 Method 3: Global fitting knowing empirically the equations of I//(t) and I(t) in terms of and ............................ 162
3.3.2.3 Steady-State Fluorescence Anisotropy from Time-Resolved Anisotropy ......................................... 162
3.4 Limiting Anisotropy of Rhodamine 101 ........... 163
3.4.1 Anisotropy of Rh101 in a High Viscosity Solvent: Glycerol ........................................... 163
3.4.2 Anisotropy of Rh101 in Acidified Ethylene Glycol.......... 167
3.4.3 Anisotropy of Rhodamine 101 in Rigid Medium: Films of PMMA .. 170
3.5 Rotational Time and Hydrodynamic Volume of Rhodamine 101 in Aqueous Solution ........................................... 173
3.6 Conclusions ................................................ 174
3.7 References ................................................. 175
Chapter 4 - Adsorption of Oligonucleotides on PMMA/PNIPAM Core-Shell Latexes: Polarity of the PNIPAM Shell .......................... 1774.1 Introduction ............................................... 178
4.2 Synthesis and Characterization of PMMA/PNIPAM Core-Shell Latexes .. 180
4.3 Adsorption of Oligonucleotides (dT25-ROX) onto PMMA/PNIPAM Core-Shell Latexes ................................................ 184
4.4 Photophysical Properties of dT25-ROX Oligonucleotides in Solution and Adsorbed on PMMA/PNIPAM Core-Shell Latexes ...................... 187
4.5 Effect of Temperature on Shell Polarity of PMMA/PNIPAM Core-Shell Latexes ................................................ 193
4.5.1 Polarity Scales .................................. 193
4.5.2 Shell Polarity Probed by Fluorescence ............ 200
4.6 Conclusions ........................................ 202
4.7 References ......................................... 203
Chapter 5 - Dynamics of Oligonucleotides in Solution and Adsorbed on Thermosensitive Core-Shell Latex Particles ............................ 2075.1 Introduction ........................................... 208
5.2 Fluorescence Anisotropy in Restrict Environments ....... 209
5.3 Dynamics of dT25-ROX Oligonucleotides in Solution ...... 212
5.3.1 The “two step model” ................................. 216
5.4 Dynamics of Adsorbed dT25-ROX Oligonucleotides on Thermosensitive Core-Shell Latex Particles ............................................ 220
5.4.1 Dynamical Models ..................................... 224
5.4.1.1 Heterogeneous Population Model ..................... 224
5.4.1.2 “Wobbling-in-two-cones” Model ...................... 226
5.4.2 Application of the Dynamical models to dT25-ROX Oligonucleotides Adsorbed on Latex Particles ................................ 226
5.5 Conclusions ............................................ 230
5.6 References ............................................. 231
Chapter 6 - Distribution of Adsorbed Oligonucleotides on the Shell of Thermo Responsive Polymer Nanoparticles ........................... 2356.1 Introduction ........................................... 236
6.2 PMMA/PNIPAM Core-Shell Latex Particles ................. 239
6.3 Adsorption of Oligonucleotides ......................... 241
6.4 Distribution Model for Adsorbed ODNs on the PNIPAM Shell ... 243
6.5 Förster Resonance Energy Transfer (FRET) ............... 245
6.5.1 Förster Radius for the Donor/Acceptor Pair: dT25-ROX / dT25-MG ......................................................... 246
6.6 Förster Resonance Energy Transfer in Thermo-Responsive Particles .................................................. 248
6.7 Analysis of the Distribution of ODNs Adsorbed on the Nanoparticles .............................................. 248
6.8 Conclusions ............................................ 254
6.9 References ............................................. 255
Chapter 7- Synthesis of Amphiphilic Block Copolymers Using a New Fluorescent RAFT Agent ................................................. 2597.1 Introduction ........................................... 260
7.2 RAFT Polymerization .................................... 263
7.3 Synthesis of the Phenanthrene-Labeled RAFT Agent ....... 271
7.3.1 Experimental Procedure ............................... 273
7.4 Synthesis of N-Decylacrylamide (DcA) ................... 274
7.4.1 Experimental Procedure ............................... 274
7.5 RAFT Polymerization of N-decylacrylamide Mediated by Phenanthrene-Labeled RAFT Agent ............................................... 275
7.5.1 Experimental Procedure ............................... 279
7.6 Characterization of Phenanthrene End-labeled Poly(N-decylacrylamide) ........................................... 280
7.6.1 Characterization by 1H NMR ........................... 281
7.6.2 Characterization by UV/Vis Absorption Spectroscopy ... 281
7.6.3 Characterization by MALDI-TOF MS ..................... 284
7.7 Synthesis of Amphiphilic Block Copolymers of Poly(N-decylacrylamide-b-N,N-diethylacrylamide) Using Sequential RAFT Polymerization ... 288
7.7.1 Experimental Procedure ............................... 290
7.8 Characterization of Poly(DcA-b-DEA) Amphiphilic Block Copolymers ................................................. 291
7.9 Conclusion ............................................. 295
7.10 References ............................................ 295
Chapter 8 - Characterization of Micellar Aggregates of Poly(DcA–b–DEA) Amphiphilic Copolymers in Water ............................ 3018.1 Introduction ........................................... 302
8.2 Spectroscopic Methods for the Determination of Critical Micelle Concentrations ............................................. 305
8.3 7-Aminocoumarins ....................................... 308
8.4 Determination of Critical Micelle Concentrations of Surfactants Using Coumarin 153 ................................................. 316
8.5 Self-Assembly of Poly(DcA-b-DEA) Amphiphilic Block Copolymers in Water ............................................... 321
8.5.1 Micellization of Poly(DcA-b-DEA) Amphiphilic Copolymers in Water ... 323
8.5.2 Micellar Aggregates of Poly(DcA-b-DEA) Copolymers in Water ... 324
8.5.3 Elimination of the End-dithioester Group by Aminolysis ... 332
8.5.4 Critical Aggregation Concentrations of Poly(DcA-b-DEA) Copolymers in Water ............................................... 334
8.6 Conclusion ...................................... 339
8.7 References ...................................... 340
Conclusions and Future Perspectives ................ 347Publicações Relacionadas:Accurate determination of the limiting anisotropy of rhodamine 101. Implications for its use as a fluorescence polarization standardPrazeres, T. J. V.; Fedorov, A.; Barbosa, S. P; Martinho, J. M. G.; Berberan-Santos, M. N.
J. Phys. Chem. A 2008, 112, 5034–5039.Adsorption of Oligonucleotides on PMMA/PNIPAM Core-Shell Latexes: Polarity of the PNIPAM Shell Probed by Fluorescence Prazeres, T. J. V.; Santos, A. M.; Martinho, J. M. G.; Elaissari, A.; Pichot, C.
Langmuir 2004, 20, 6834-6840Dynamics of Oligonucleotides Adsorbed on Thermosensitive Core-Shell Latex Particles Prazeres, T. J. V.; Fedorov, A.; Martinho, J. M. G.
J. Phys. Chem. B 2004, 108, 9032-9041.Control of Oligonucleotide Distribution on the Shell of Thermo-Responsive Polymer NanoparticlesPrazeres, T. J. V.; Farinha, J. P. S.; Martinho, J. M. G.
J. Phys. Chem. C 2008, 112, 16331-16339.Thermo-responsiveness of poly(N,N-diethylacrylamide) polymers at the air–water interface: The effect of a hydrophobic blockGonçalves da Silva, A. M.P.S.; Lopes, S. I.C.; Brogueira, P.; Prazeres, T. J. V.; Beija, M.; Martinho, J. M. G.
J. Colloid Interface Sci. 2008, 327, 129-137.Understanding Fluorescence Quenching in Polymers Obtained by RAFTRelógio, P.; Charreyre, M.-T.; Prazeres, T. J. V.; Martinho, J. M. G.; Farinha, J. P. S.
Macromolecules 2007, 40, 4680-4690.
