Novel ultrathin polymer films as biomimetic interfaces [Elektronische Ressource] / Florian Rehfeldt
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Novel ultrathin polymer films as biomimetic interfaces [Elektronische Ressource] / Florian Rehfeldt

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Technische Universit¨at Munc¨ henPhysik-DepartmentLehrstuhl fur Biophysik E22¨Novel Ultrathin Polymer Films as Biomimetic InterfacesFlorian RehfeldtVollstandiger¨ Abdruck der von der Fakultat¨ fur¨ Physikder Technischen Universitat¨ Munc¨ hen zur Erlangung des akademischen Grades einesDoktors der Naturwissenschaften (Dr. rer. nat.)genehmigten Dissertation.Vorsitzender: Univ.-Prof. Dr. H. FriedrichPrufer der Dissertation: 1. Univ.-Prof. Dr. E. Sackmann, em.¨2. Univ.-Prof. Dr. J. O. Radler,¨Ludwig-Maximilians-Universitat¨ Munc¨ henDie Dissertation wurde am 1.12.2004 bei der Technischen Universitat Munchen eingereicht¨ ¨und durch die Fakultat¨ fur¨ Physik am 17.12.2004 angenommen.Meinen ElternBernhild und Dr. Klaus Rehfeldtund meiner OmaBerta ZimprichTable of ContentsTable of Contents vSummary 1Introduction 31 Materials and Methods 91.1 DiblockCopolymer............................ 91.2 Chemicals................................. 101.3 Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111.3.1 Cleaning.............................. 11.3.2 Hydrophobization......................... 11.4 LipidBilayerPreparation ........................ 121.5 LangmuirFilmBalance. 131.5.1 Principles of the Langmuir Film Balance . . . . . . . . . . . . 131.5.2 Pressure-Area (π− A)Isotherms................ 141.5.3 In-Situ Subphase Titration . . . . . . . . . . . . . . . . . . . . 141.5.4 Langmuir-SchaeferTransfer................... 141.

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Publié le 01 janvier 2004
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Technische Universit¨at Munc¨ hen
Physik-Department
Lehrstuhl fur Biophysik E22¨
Novel Ultrathin Polymer Films as Biomimetic Interfaces
Florian Rehfeldt
Vollstandiger¨ Abdruck der von der Fakult¨at fur¨ Physik
der Technischen Universitat¨ Munc¨ hen zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften (Dr. rer. nat.)
genehmigten Dissertation.
Vorsitzender: Univ.-Prof. Dr. H. Friedrich
Prufer der Dissertation: 1. Univ.-Prof. Dr. E. Sackmann, em.¨
2. Univ.-Prof. Dr. J. O. R¨adler,
Ludwig-Maximilians-Universitat¨ Munc¨ hen
Die Dissertation wurde am 1.12.2004 bei der Technischen Universitat Munchen eingereicht¨ ¨
und durch die Fakultat¨ fur¨ Physik am 17.12.2004 angenommen.Meinen Eltern
Bernhild und Dr. Klaus Rehfeldt
und meiner Oma
Berta ZimprichTable of Contents
Table of Contents v
Summary 1
Introduction 3
1 Materials and Methods 9
1.1 DiblockCopolymer ............................ 9
1.2 Chemicals ................................. 10
1.3 Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1.3.1 Cleaning . ............................. 11
1.3.2 Hydrophobization......................... 11
1.4 LipidBilayerPreparation ........................ 12
1.5 LangmuirFilmBalance. 13
1.5.1 Principles of the Langmuir Film Balance . . . . . . . . . . . . 13
1.5.2 Pressure-Area (π− A)Isotherms ................ 14
1.5.3 In-Situ Subphase Titration . . . . . . . . . . . . . . . . . . . . 14
1.5.4 Langmuir-SchaeferTransfer ................... 14
1.6 ContactAngleMeasurement ....................... 15
1.7 FluorescenceMicroscopy ......................... 15
1.8 FlowChamberforNeutronReflectometry ............... 15
1.9 Beamlines ................................. 16
1.9.1 X-RayReflectometer 16
1.9.2 NeutronReflectometer ...................... 17
1.9.3 NeutronDiffractometer 17
1.9.4 SAXS/WAXSBeamline . .................... 18
2 Reflectometry Techniques 21
2.1 Ellipsometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.1.1 Theory of Ellipsometry . . . . . . . . . . . . . . . . . . . . . . 21
2.1.2 Ellipsometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.1.3 Imaging Ellipsometer . . . . . . . . . . . . . . . . . . . . . . . 23
2.2 X-RayandNeutronReflectometry.................... 24
2.2.1 Reflectionatasurface . ..................... 24
2.2.2 Reflectionfromlayeredsample.................. 28
v3 Theoretical Concepts - Polymers and Surface Forces 31
3.1 SurfaceandInterfaceForces ....................... 31
3.1.1 Hard-CoreRepulsion 31
3.1.2 vanderWaalsForce 32
3.1.3 HydrationForce. ......................... 32
3.1.4 ElectrostaticForce . 33
3.1.5 Surface Free Energy and Contact Angle . . . . . . . . . . . . . 34
3.2 PolymerandPolyelectrolyteTheory................... 35
3.2.1 FlexiblePolymers . ........................ 35
3.2.2 Copolymers . ........................... 36
3.2.3 Polyelectrolytes . 36
4 Diblock Copolymer at Air/Water & Air/Solid Interface 39
4.1 LangmuirIsotherms............................ 39
4.2 Diblock Copolymer at Solid/Air Interface . . . . . . . . . . . . . . . . 45
4.2.1 Influence of Hydrophobization on LS Transfer . . . . . . . . . 45
4.2.2 ContactAngleMeasurements . ................. 45
4.2.3 X-Ray Reflectivity and Ellipsometry Measurements . . . . . . 46
5 Diblock Copolymer at the Solid/Liquid Interface 51
5.1 pHDependentChangesinAqueousSolution .............. 51
5.2 ProteinAdsorption ............................ 55
5.2.1 Ex-situExperiments . ...................... 55
5.2.2 In-situExperiments........................ 56
5.3 LipidBilayeronDiblockCopolymer................... 57
6 Conclusions 63
7 Outlook 65
A Appendix 67
A.1 PhaseBehaviorofGlycolipids . ..................... 69
A.1.1 MorphologyinBulkDispersions ................. 69
A.1.2 Swelling of Lamellar Stacks of Glycolipids . . . . . . . . . . . 70
A.1.3 Phase Diagram of Gentiobiose Lipid . . . . . . . . . . . . . . 71
A.2 Ultrathin Cellulose Films on Silicon Substrates . . . . . . . . . . . . . 75
A.3 Native Cell Membranes on Patterned Polymer Support . . . . . . . . 83
A.4 Lipids . .................................. 88
A.5 X-RayReflectivityScript. ........................ 89
A.6 NeutronReflectivityScripts ....................... 90
A.7 Abbreviations . .............................. 91
A.8 Symbols 92
Bibliography 93
viSummary
The general aim of this study was the design of ultrathin polymer coatings (with
thicknesses ranging from several nanometers to hundreds of nanometers) which can
serve as interfaces between solid surfaces (e.g. silicon) and living matter and allow
the control of the interfacial forces between these two different states of matter. The
fabrication of such biocompatible interlayers is a key step towards the control of cell
proliferation and immobilization of cells or proteins in their native state onto planar
substrates which are fundamental problems in tissue engineering.
In this study, a new type of diblock copolymer (DMAEMA-b-MMA) was chosen as
the ”tunable” polymer interlayer. It consists of a hydrophobic MMA block and a
weakly cationic DMAEMA block, whose degree of ionization can be adjusted in a
subtle way by pH titration (pK = 7.3). With a sufficiently high hydrophobic fractiona
(50 % MMA, DB 50), DB 50 forms an insoluble monolayer at the air/water interface.
This monolayer can be transferred onto a hydrophobic substrate with the Langmuir-
Schaefer technique, which allows the precise control of the lateral polymer density.
In Chapter 4, the chemical switching of the chain conformation of DB 50 was studied
at the air/water interface. The Langmuir isotherms measured at different pH condi-
tions indicated that the adsorption and desorption of the DMAEMA chains to the
interface can be manipulated by charging and de-charging. Careful optimization of
the preparation protocols based on contact angle measurements, ellipsometry, and
x-ray reflectivity resulted in reproducible, stable, and homogeneous films of DB 50 on
hydrophobized substrates.
Consequently, in Chapter 5, the chemical switching of these DB 50 films at the
solid/liquid interface could be studied quantitatively by neutron reflectivity mea-
surements in D O. The global shape of the reflectivity curves exhibited a distinct2
difference between pH = 8.5 and 5.5, indicating a clear change in thickness, scattering
length density (corresponding to the degree of hydration), and roughness of the DB 50
layer. The reflectivity data were perfectly reproducible after several pH cycles, which2
verified the stability and reversibility of chemical switching.
To study the interaction between DB 50 films and biofunctional molecules, two series
of experiments were performed which are described in Chapter 5.2 and 5.3: one
series addressed the problem of nonspecific physisorption of water soluble proteins
(bovine serum albumin, BSA), while the second dealt with the spreading of model
cell membranes. Although the difference in protein adsorption between pH = 8.5 and
5.5 could hardly be distinguished, for the first time a clear switching of the separation
distance between a model cell membrane and the DB 50 film was observed.
The results demonstrate that the system established in this study has great potential
as a soft, compatible interlayer between hard solids and soft biological material. It is
expected that the fine-tuning of generic interactions between cell membrane models
and planar substrates opens new possibilities for scientific and practical applications.
The new type of biomimetic interface established in this work provides an ideal plat-
form for future studies of proteins in their native state, which is an important issue
in the field of biosensors.Introduction
The understanding of physical interactions at biological interfaces is a challenging
task for interdisciplinary scientific research. In nature, interactions between cells and
tissues are mediated by complex interplays of short-range and long-range forces (e.g.
van der Waals (dispersion) forces, electrostatic forces, steric (entropic) forces, and hy-
dration forces [1, 2]) across hydrated layers of biopolymers, such as the extracellular
matrix (ECM)[3] and the glycocalix [4]. They are necessary to keep a certain distance
between neighboring cells as well as to create hydrating pathways for material trans-
port. However, if one considers even a ”single” plasma membrane that consists of
peripheral and integral proteins, cell surface glycocalix, and cytoskeleton (see Figure
1), the molecular constructs therein are already too complex to be directly recon-
stituted into one model. Therefore, the design of biomimetic molecular assemblies
with a reduced number of components is necessary to create simple physical mod-
els. Here, deposition of artificial extracellular matrix and model cell membranes onto
planar substrates is a straightforward and powerful strategy to apply various surface
sensitive techniques for the quantitative study of their s

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