New sensing platforms based on nanoscopic devices for probing protein-membrane interactions [Elektronische Ressource] / vorgelegt von Cristina-Luminita Baciu

Sujets

• Naturwissenschaften

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2009
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 15 Mo

Extrait

New sensing platforms
based on nanoscopic devices
for probing protein-membrane interactions
Dissertation zur Erlangung des Grades
“Doktor der Naturwissenschaften”
am Fachbereich
Chemie, Pharmazie und Geowissenschaften
der Johannnes Gutenberg-Universität Mainz
vorgelegt von
Cristina-Luminita Baciu
geboren in Ploie ti /Rumänien
Mainz, 2009
?Dekan:
1. Berichterstatter:
2. Berichterstatter:
Tag der mündlichen Prüfung:Contents
Introduction..................................................................................................................... 7
Chap.1 Basic concepts .................................................................................................... 9
1.1 Biological membranes ............................................................................................ 9
1.2 Model lipid bilayer systems .................................................................................. 11
1.3 Adsorption of proteins to lipid membranes .......................................................... 12
1.4 Analysis of equilibrium coverage fluctuations for quantification of reversible
adsorption of proteins: a theoretical approach ...................................................... 13
1.5 When small is different: Localized Surface Plasmon Resonance (LSPR) ................. 15
1.5.1 Absorption and scattering by small ellipsoidal particles ................................. 16
1.5.2 Nanohole plasmons in optically thin films ...................................................... 18
1.5.3 Beyond the quasi-static approximation .......................................................... 18
1.5.4 Shape dependence of the plasmonic particle resonance ................................ 19
Chap.2 Instrumentation ................................................................................................ 23
2.1 Atomic Force Microscopy (AFM) ........................................................................... 23
2.1.1 Basics ............................................................................................................. 23
2.1.2 The cantilevers and the piezo scanners 24
2.1.3 The PID controller .......................................................................................... 24
2.1.4 Imaging modes .............................................................................................. 25
2.2 Ellipsometry.......................................................................................................... 26
2.2.1 Basic principles .............................................................................................. 26
2.2.2 Nulling ellipsometry ....................................................................................... 28
2.2.3 Optical models for determination of ellipsometric thickness.......................... 29
2.2.4 Imaging ellipsometry ..................................................................................... 30
2.3 Epifluorescence and Confocal Microscopy ............................................................ 31
2.3.1 Fluorescence .................................................................................................. 31
2.3.2 Epifluorescence microscopy .......................................................................... 32
2.3.3 Confocal laser scanning microscopy (CLSM) .................................................. 33
2.4 Dark field microscopy (DFM)................................................................................. 34
2.5 Single particle spectroscopy.................................................................................. 36
Chap.3 Materials and experimental procedures ........................................................... 37
3.1 Materials .............................................................................................................. 37
3.1.1 Lipids ............................................................................................................. 37
3.1.2 Proteins ......................................................................................................... 41
3.1.3 Gold nanorods ............................................................................................... 42
3.2 Preparation of solid substrates for supported lipid membranes ........................... 44
3.2.1 Hydrophilization of the glass substrates and silica wafers ............................. 44
3.2.2 Functionalization with EDSPA ........................................................................ 443.3 Formation of large unilamelar lipid vesicles (LUVs) .............................................. 45
3.4 Formation of giant unilamellar vesicles (GUVs) ..................................................... 46
3.5 Microstructuring lipid membranes on solid supports ............................................ 46
3.6 Fluorescence recovery after photobleaching (FRAP) ............................................. 48
3.6.1 Experimental.................................................................................................. 48
3.6.2 Data analysis .................................................................................................. 48
3.7 Monte Carlo simulations ....................................................................................... 49
Chap.4 Lipid bilayers on solid supports ........................................................................ 51
4.1 Formation of lipid bilayer patches on silicon dioxide substrates............................ 51
4.2 Micro-domains and nano-domains in multicomponent lipid bilayers .................... 55
4.3 Conclusions .......................................................................................................... 59
Chap.5 Reversible and irreversible adsorption of proteins ........................................... 61
5.1 Calcium-induced and annexin A1-induced clustering of acidic phospholipids in
ternary lipid membranes ....................................................................................... 63
5.2 Ellipsometric characterization of reversible adsorption of prothrombin on mixed
anionic lipid bilayers .............................................................................................. 65
5.3 Conclusions 70
Chap.6 Fast imaging with conventional AFM for detecting surface coverage
fluctuations: pitfalls and drawbacks .................................................................. 71
6.1 Thermal drift and small scan areas........................................................................ 72
6.2 Image resolution and long-time imaging stability and repeatability ...................... 74
6.3 How fast? How long ? – a matter of the chosen system ........................................ 76
6.4 Conclusions .......................................................................................................... 78
Chap.7 Nanostructuring Gold Surfaces ......................................................................... 79
7.1 Nanosphere lithography ....................................................................................... 80
7.2 Regular arrays of triangle shaped gold nanoparticles ............................................ 81
7.3 Random arrays of nanoholes in thin gold layers .................................................... 85
7.4 Conclusions 89
Chap.8 Lipid membrane coated gold nanorods as sensing elements............................ 91
8.1 Self-assembling of gold nanorods on glass substrates ........................................... 92
8.1.1 Immobilization of gold nanoparticles on amine-functionalized glass .............. 92
8.1.1.1 Influence of CTAB on the particles’ adsorption efficiency ........................ 93
8.1.1.2 Incubation time ....................................................................................... 93
8.1.2 Self-assembling of gold nanorods on hydrophilic glass surface ....................... 95
8.2 Lipid bilayer formation on glass substrates with gold nanorods ............................ 96
8.2.1 FRAP analysis ................................................................................................. 98
8.2.2 Imaging with AFM ........................................................................................ 100
8.3 Sensing the lipid membrane with single particle spectroscopy............................ 102
8.4 Configurational limitations of membrane-nanoparticle interaction .................... 105
8.5 Conclusions ........................................................................................................ 109Chap.9 Protein-membrane interaction probed by single plasmonic nanoparticles .... 111
9.1 Sensing the adsorption of streptavidin to biotinylated lipid membranes ............ 112
9.2 Detection sensitivity ........................................................................................... 114
9.3 Potential extension of the nanosensor applicability ............................................ 115
9.4 Conclusions ........................................................................................................ 117
Chap.10 Phase imaging with AFM reveals increased activity of PLA on highly curved2
membranes ................................................................................................... 119
10.1 Structure and function of phospholipase PLA .................................................. 1202
10.2 Characterization of PLA activity with ellips