179 Pages
English

Characterization of structure and dynamics of submicrometric systems via fluorescence correlation spectroscopy [Elektronische Ressource] / vorgelegt von Riccardo Raccis

-

Gain access to the library to view online
Learn more

Description

Characterization of Structure and Dynamics of Submicrometric Systems via Fluorescence Correlation Spectroscopy Dissertation zur Erlangung des Grades Doktor der Naturwissenschaften im Fachbereich Physik, Mathematik und Informatik der Johannes Gutenberg-Universität Mainz vorgelegt von Riccardo Raccis geboren in Florenz, Italien Mainz, im Dezember 2010 1 2 3 4 “There are more things in heaven and earth, Horatio, Than are dreamt of in your philosophy.” - William Shakespeare, Hamlet 5 6 To Eva, and Mariona. I miss you girls 7 8 Summary Summary..............................................................................................................................9 1. List of abbreviations .................................................................................................11 2. Abstract.....................................................................................................................13 2.1. English version ..................................................................................................13 2.2. German version .................................................................................................14 3. Introduction and motivation .....................................................................................17 4. FCS: general theory and practical application.........................

Subjects

Informations

Published by
Published 01 January 2010
Reads 5
Language English
Document size 7 MB



Characterization of Structure and Dynamics
of Submicrometric Systems
via Fluorescence Correlation Spectroscopy



Dissertation



zur Erlangung des Grades

Doktor der Naturwissenschaften
im
Fachbereich Physik, Mathematik und Informatik
der Johannes Gutenberg-Universität Mainz


vorgelegt von
Riccardo Raccis
geboren in Florenz, Italien

Mainz, im Dezember 2010


1





2







3 4





“There are more things in heaven and earth, Horatio,
Than are dreamt of in your philosophy.”
- William Shakespeare, Hamlet
5 6





To Eva, and Mariona.
I miss you girls




7 8 Summary



Summary..............................................................................................................................9
1. List of abbreviations .................................................................................................11
2. Abstract.....................................................................................................................13
2.1. English version ..................................................................................................13
2.2. German version .................................................................................................14
3. Introduction and motivation .....................................................................................17
4. FCS: general theory and practical application..........................................................21
4.1. Theory................................................................................................................21
4.2. Practical application ..........................................................................................29
5. Experimental ensemble ............................................................................................33
5.1. FCS setup...........................................................................................................33
5.2. Chambers...........................................................................................................34
5.3. Materials employed ...........................................................................................35
5.3.1. Gel constituents .........................................................................................35
5.3.1.1. PNIPAAm..........................................................................................35
5.3.1.2. BP-silane............................................................................................36
5.3.2. Solvents .....................................................................................................36
5.3.2.1. Water .................................................................................................36
5.3.2.2. Ethanol...............................................................................................36
5.3.2.3. Water-based buffers...........................................................................37
5.3.2.3.1. PBS .................................................................................................37
5.3.2.3.2. HEPES............................................................................................37
5.3.2.3.3. Acetate............................................................................................37
5.3.3. Fluorescent probes.....................................................................................38
5.3.3.1. Molecular dyes ..................................................................................38
5.3.3.1.1. Rh6G...............................................................................................38
5.3.3.1.2. Cy5 .................................................................................................38
5.3.3.1.3. Alexa 647........................................................................................38
5.3.3.2. Green Fluorescent Protein .................................................................39
5.3.3.3. Quantum dots.....................................................................................40
5.3.4. Gel functionalization material ...................................................................42
5.3.4.1. GFP antibodies ..................................................................................42
5.3.4.2. TFPS ..................................................................................................43
5.3.4.3. EDC ...................................................................................................43
5.3.4.4. Ethanolamine.....................................................................................43
5.3.5. Inverse opals..............................................................................................43
6. Experiment 1: PNIPAAm-Rhodamine interactions in a water solution..................47
6.1. Motivation .........................................................................................................47
6.2. Experimental......................................................................................................48
6.3. Diffusion and possible Models..........................................................................52
9 6.4. Data analysis and theory development..............................................................61
6.5. Conclusions .......................................................................................................96
7. Experiment 2: Size-dependent diffusion in inverse opals ........................................97
7.1. Motivation .........................................................................................................97
7.2. Experimental .....................................................................................................98
7.3. Blinking effect and FCS theory re-engineering ..............................................100
7.3.1. Data simulation........................................................................................106
7.3.2. Diffusion in iOpals and experimental-simulation comparison................111
7.4. Conclusions .....................................................................................................115
8. Experiment 3: PNIPAAm hydrogel nanostructure and dynamics for biosensing
implementation 117
8.1. Motivation .......................................................................................................117
8.2. Sample preparation..........................................................................................119
8.2.2. Gel spincoating and drying......................................................................120
8.2.3. Gel crosslinking and gel-substrate grafting.............................................121
8.2.4. Pre-FCS gel characterization and rinsing................................................122
8.2.5. Gel annealing...........................................................................................122
8.2.6. Gel functionalization ...............................................................................123
8.3. Experimental ...................................................................................................123
8.3.1. Non-annealed gels ...................................................................................126
8.3.1.1. Variable: crosslink density ..............................................................126
8.3.1.2. Variable: temperature ......................................................................133
8.3.1.3. Variable: buffer ...............................................................................141
8.3.2. Annealed gels ..........................................................................................147
8.3.2.1. Variable: protein diffusion and probe size dependence ..................151
8.3.2.2. Variable: antibody functionalization...............................................156
8.4. Conclusions .....................................................................................................165
9. Concluding remarks and outlook ...........................................................................167
10. References ..............................................................................................................171
11. Curriculum Vitae....................................................................................................177


10