Surface Modification by Plasma Polymerization: Film Deposition, Tailoring of Surface Properties and Biocompatibility

Menno T. van Os SURFACE MODIFICATION BY PLASMA POLYMERIZATION:

FILM DEPOSITION, TAILORING OF SURFACE PROPERTIES

AND BIOCOMPATIBILITY

PROEFSCHRIFT

ter verkrijging van

de graad van doctor aan de Universiteit Twente,

op gezag van de rector magnificus,

prof. dr. F. A. van Vught,

volgens besluit van het College voor Promoties

in het openbaar te verdedigen

op vrijdag 17 november 2000 te 16.45 uur.

door

Menno Thomas van Os

geboren op 26 september 1970

te Nijmegen Dit proefschrift is goedgekeurd door:

Promotor: Prof. dr. G. J. Vancso Promotor: Prof. dr. W. Knoll Assistant promotor: Dr. R. Förch

ii A man in the corner approached me for a match. I knew right away he was not ordinary. He said, “Are you looking for something easy to catch?” I said, “I got no money.” He said, “That ain’t necessary.”

Isis – Bob Dylan

iii CIP-GEGEVENS KONINKLIJKE BIBLIOTHEEK, DEN HAAG

Os, Menno Thomas van

Surface Modification by Plasma Polymerization: Film Deposition, Tailoring of Surface Properties and Biocompatibility / Menno T. van Os Thesis University of Twente, Enschede, The Netherlands With references – With summary in Dutch ISBN 90 36515130

 2000 M. T. van Os, Enschede, The Netherlands

No part of this book may be reproduced in any form by any means, nor transmitted, nor translated into a machine language without written permission from the author.

Cover illustration: Lightning Over Clovelly – Shark Pt.,  Anthony Roach Press: Print Partners Ipskamp, Enschede, The Netherlands, 2000 All rights reserved iv &217(176

Chapter 1

General Introduction ______1

1.1 Plasma and Plasma Polymerization: a Brief Introduction and Historical Background______1

1.2 Concept of this Thesis______2

1.3 References ______4

Chapter 2

Surface Modification Techniques and Fundamental Aspects of Plasma Polymerization______5

2.1 Polymer Surface Modification Techniques ______5

2.2 Surface Treatments of Polymers ______5 2.2.1 Flame Treatment 5 2.2.2 Corona Discharge 5 2.2.3 Plasma Treatment 6 2.2.4 Other Surface Treatments 7

2.3 Surface Modification by Attachment of Monolayers or Thin Films ______8 2.3.1 Langmuir-Blodgett-Kuhn Technique 8 2.3.2 Polyelectrolyte Multilayer Deposition 9 2.3.3 Block Copolymer Thin Films 9 2.3.4 Radiation Grafting 9

2.4 Plasma Polymerization ______9 2.3.1 The Fourth State of Materials 9 2.4.2 Fundamental Aspects of Plasma Physics 11 2.4.3 Types of Collisions 12

2.5 References ______14

v Chapter 3

Characterization Methods for Plasma Polymers ______17

3.1 Surface Analytical Techniques ______17 3.1.1 Contact Angle Goniometry 17 3.1.2 X-ray Photoelectron Spectroscopy (XPS) 18 3.1.3 Atomic Force Microscopy (AFM) 19

3.2 Thin Film Characterization by Optical Techniques ______21 3.2.1 Waveguide Mode Spectroscopy (WaMS) 21 3.2.2 Surface Plasmon Resonance (SPR) Spectroscopy 24

3.3 References ______25

Chapter 4

Tailoring of Film Properties by Variation of Plasma Deposition Parameters ______29

4.1 Introduction______29

4.2 Plasma Reactor______30

4.3 Influence of Plasma Parameters on Film Chemistry and Deposition Rate 31 4.3.1 Influence of the Monomer Flow 31 4.3.2 Input Power Variation under Continuous Wave (CW) Conditions 33 4.3.3 Pulsed Plasma: Variation of the Plasma-on Time 36 4.3.4 Pulsed Plasma: Variation of the Plasma-off Time 39 4.3.5 Pulsed Plasma: Plasma-on- and off Time Variation at Constant Duty Cycle 42 4.3.6 Influence of the Deposition Time on the Film Structure 43

4.4 Conclusions ______45

4.5 Experimental Section______46 4.5.1 Sample Preparation 46 4.5.2 Plasma Polymerization 46 4.5.3 Film Characterization 47

4.6 References ______47

vi Chapter 5

Film Stability and Surface Aging______51

5.1 Introduction______51

5.2 Effect of Solvent Treatment on Plasma Polymerized Allylamine Films __ 52 5.2.1 Effect of Solvent Treatment on Film Composition 52 5.2.2 Effect of Solvent Treatment on Film Thickness 53 5.2.3 Swelling Behavior of PPAA Monitored by WaMS 55

5.3 Post-plasma Oxidation______57 5.3.1 Effect of Deposition Conditions on Post-plasma Oxidation 57 5.3.2 Influence of Amino Groups on the Aging Process 59 5.3.3 Surface Restructuring and Reorientation of Functional Groups during Aging 60

5.4 Conclusions ______62

5.5 Experimental section ______62 5.5.1 Sample Preparation 62 5.5.2 Plasma Polymerization 63 5.5.3 Characterization 63

5.6 References ______63

Chapter 6

Mapping of Amino Group Distributions in Plasma Polymerized Allylamine Films by Atomic Force Microscopy using Functionalized Probe Tips ______65

6.1 Introduction______65

6.2 Pull-off Force Measurements ______66

6.3 Determination of the Surface pKa of Plasma Polymerized Allylamine Films ______70

6.4 Mapping of functional group distributions ______72

6.5 Conclusions ______74

6.6 Experimental Section______74 6.6.1 Materials 74

vii 6.6.2 Scanning Force Microscopy and Tip Modification 74

6.7 References ______75

Chapter 7

Attachment of Polymer Monolayers to Plasma Polymerized Allylamine Surfaces by Radical Chain Polymerizations Initiated by Azo Compounds and Residual Free Radicals ______77

7.1 Introduction______77 7.1.1 “Grafting-to” Polymerization 77 7.1.2 Grafting-from Polymerization 77 7.1.3 Plasma Treatment and Polymerization 78 7.1.4 Concept 79

7.2 Qualitative Evidence for the Attachment of PMMA and PS Brushes to PPAA Films ______81 7.2.1 FT-IR Spectroscopy 81 7.2.2 Contact Angle Goniometry 82 7.2.3 AFM Imaging 83

7.3 Grafting-from Polymerization of MMA and Styrene Initiated by an Azo Compound and/or Peroxide Groups: Quantitative Evaluation ______84 7.3.1 Increasing the Number of Reactive Sites at PPAA Surfaces through Variation of Plasma Parameters 84 7.3.2 Influence of Plasma Parameters on the Free Radical and Peroxide Concentration 85 7.3.3 Grafting Conditions and Characterization 86 7.3.4 Peroxide Initiated Polymerization 86 7.3.5 Azo Initiation Supported by Peroxide Initiation 87 7.3.6 Influence of the Plasma Deposition Time. 88 7.3.7 Influence of the Peak Power 90 7.3.8 Influence of the Plasma-off Time 91

7.4 Conclusions ______94

7.5 Experimental section ______94 7.5.1 Substrates 94 7.5.2 Synthesis 95 7.5.3 Characterization 96

viii 7.6 References ______97

Chapter 8

Protein Adsorption to Plasma Functionalized Surfaces Studied by Surface Plasmon Resonance Spectroscopy and Atomic Force Microscopy ______99

8.1 Introduction______100 8.1.1 Amine-functionalized Surfaces 100 8.1.2 (Polyethylene Oxide)-functionalized Surfaces 101 8.1.3 Characterization 102

8.2 Results and discussion ______103 8.2.1 Adsorption and Denaturation of Proteins on Test Surfaces 104

8.3 Conclusions ______114

8.4 Experimental Section______114 8.4.1 Substrates 114 8.4.2 Plasma Polymerization and SAM Preparation 115 8.4.3 Contact Angle Goniometry 115 8.4.4 Proteins 115 8.4.5 Surface Plasmon Resonance (SPR) Spectroscopy 116 8.4.6 Tapping Mode Atomic Force Microscopy (TM-AFM) 116

8.5 References ______116

Summary ______121

Samenvatting ______125

List of Publications______129

Dankwoord ______131

ix Curriculum Vitae______135

x General Introduction

&KDS WHU

General Introduction

1.1 Plasma and Plasma Polymerization: a Brief Introduction and Historical Background

Plasma is not an invention of mankind.1 It has been around for much longer than we have. The word plasma is derived from the ancient Greek language, where it meant ‘that what is built’, or ‘that what is formed’. In modern language the term plasma denotes a more or less ionized gas, also referred to as ‘glow discharge’. This so-called state of ionized gas was first described by Crookes in 1879 as ‘a world where matter may exist in a fourth state’. Fifty years later, first in 1928, this fourth state of matter was finally given a name of its own by Irving Langmuir, when he introduced the term ‘plasma’ in his studies of electrified gases in vacuum tubes.2 Perhaps without realizing, every person reading this work has had his or her own encounter with one or another form of plasma. Lightning for instance, an electric discharge in air, can be considered a plasma. Also the stars or the polar light belong to the better known plasma phenomena. In general, when a molecule is subjected to ‘severe’ conditions such as intense heat, the molecule will ionize. The sun, and other stars in our universe, have temperatures ranging from 5000 to 70,000 K or more, and consist entirely of plasma.3 In the laboratory, plasma can be generated by combustion, flames, lasers, controlled nuclear reactions and shocks, but most experimental work, especially in the field of plasma polymerization, is carried out using an electrical discharge.

Plasma polymerization can be defined as ‘the formation of polymeric materials under the influence of plasma.3 Solid deposits from organic compounds formed in a plasma, generated by some kind of electrical discharge, were already described as early as in 1874.4 However, at that time very little was known about polymers, and these deposits were considered to be nothing more than undesirable by-products of phenomena associated with electrical discharge. Systematic investigation of plasma polymerization started only in the 1960’s,5 following the rapid advancement of polymer science in those years.3,6 Only over the past two decades, the advantages of plasma polymerization have been fully recognized. Today, plasma polymerization is accepted as an important process for the formation of entirely new materials, and as a valuable technique to modify the surfaces of polymers or of other materials.

Advantages of plasma polymerization include the fact that pinhole free, conformal thin films can be deposited on most substrates, using a relatively simple one-step coating Chapter 1

procedure.7 Additionally, a wide range of compounds can be chosen as a monomer for plasma polymerization, even saturated hydrocarbons, providing a great diversity of possible surface modifications. These advantages have resulted in the rapid development of plasma technology during the past decades, for applications ranging from adhesion to composite materials, protective coatings, printing, membranes, biomedical applications and so on.

Despite the efforts that have been made however, plasma polymerization remains a very complex process that is not well understood.8 The structure of plasma deposited films is not as well defined as that of conventional polymers, and depends on many different factors. Several groups have reported that the structure, as well as the surface chemistry of plasma polymers, can be influenced by parameters such as the design of the reactor,9 input power,10 monomer flow rate,11 substrate temperature,12 and frequency.13 Initially, most of the studies on plasma polymerization focussed on the production of highly cross-linked films. Reactions were generally carried out under high-energy plasma conditions, leading to a wide variety of ionization and chemical bond dissociation processes.14

More recently, interest has shifted towards the synthesis of films that contain high concentrations of specific functional groups at the surface. Different techniques to improve the control over the film chemistry of plasma films have been studied, most of them based on decreasing the input energy during plasma polymerizations. One approach in particular has been very successful in achieving this enhanced film chemistry controllability. By employing a pulsed radio frequency (rf) plasma instead of the traditional continuous wave (CW) plasma, Timmons et al. reported a high control over the film chemistry by variation of the plasma-on or plasma-off times. For many monomers, it was found that a more selective chemistry occurs during the plasma-off relaxation periods, leading to less cross-linked and more ‘polymer-like’ structures at relatively long off-times.15

1.2 Concept of this Thesis

The research described in this Thesis deals with the surface modification of a large variety of substrates by plasma polymerization. The major part of the work presented is directed towards the functionalization of materials with amino groups, through the pulsed plasma polymerization of allylamine or other amino group containing monomers. Thin films bearing amino groups are of great interest for many applications, because they are known to influence protein adsorption and cell adhesion, and provide sites for the covalent immobilization of graft polymers and biomolecules.

Chapter 2 gives an overview of the most commonly used techniques for surface modification of polymers. This chapter also serves as a general introduction to plasma polymerization, and covers the fundamental aspects of plasma physics that are necessary for a good understanding of this work.

2 General Introduction

In Chapter 3 the characterization methods that have been used for the analysis of the plasma polymers discussed in this thesis are presented. Surface sensitive techniques such as contact angle goniometry, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM) are introduced. The principles of two optical techniques, surface plasmon resonance (SPR) spectroscopy and waveguide mode spectroscopy (WaMS), are also explained, as these have been used frequently in our work to determine the thickness and refractive index of plasma films.

Synthesis and characterization of amine functionalized plasma films are discussed in Chapter 4. A major part of this section focuses on the molecular tailoring of surfaces by variation of plasma parameters such as the input power and the monomer flow, or by changing the pulse characteristics. A description of the plasma reactor is also given.

Plasma polymers are known to be susceptible to oxidation upon storage in air. Restructuring of plasma surfaces or loss of material may also occur, depending on the environment to which they are exposed. Chapter 5 deals with stability aspects of plasma polymerized allylamine (PPAA) films. Material loss and swelling behavior were studied mainly by SPR spectroscopy and WaMS. The effects of aging on these films were investigated by Fourier transform infrared (FT-IR) spectroscopy, water contact angle measurements, and electron spin resonance (ESR) spectroscopy.

Force microscopy with chemically functionalized probe tips was utilized in order to study the lateral distribution of functional groups, and characterize their local environment in pH dependent pull-off force measurements on a sub-50 nm level (Chapter 6). With the obtained results, the ‘surface pKa’ of PPAA films was determined.

In Chapter 7, a novel method for the covalent attachment of polymer monolayers to plasma modified surfaces is presented. The study comprises a three-step surface modification, consisting of a plasma film depositing step to provide the appropriate reactive sites (e.g. amino groups), followed by the immobilization of an ‘azo compound’, which can than be used to initiate the radical chain polymerization of styrene or methyl(methacrylate) from the surface. Residual free radicals present in the plasma films contribute to the ‘grafting-from’ polymerization as well.

In Chapter 8, protein adsorption to a number of plasma modified surfaces is described. The influence of surface functionalities on the adsorption and retention of the studied proteins is discussed. Adsorption of fibrinogen, bovine serum albumin and immunoglobulin G to these surfaces was measured in situ with SPR spectroscopy. After drying, the protein layers were studied by tapping mode atomic force microscopy (TM-AFM).

3 Chapter 1

1.3 References

1 Kickuth, R. Plasmatechnik; Prozessvielfalt + Nachhaltigkeit, Roco Druck GmbH: Wolfenbüttel, 2000. 2 Boenig, H. V. Fundamentals of Plasma Chemistry and Technology, Technomic Publishing Co., Inc.: Lancaster, PA, 1988. 3 Yasuda, H. Plasma Polymerization, Academic Press Inc.: Orlando, FL, 1985. 4 (a) de Wilde, P. Ber. Dtsch. Chem. Ges. 1874, 7, 4658.; (b) Thenard, A.; C. R. Hebd. Seances Acad. Sci. 1874, 78, 219. 5 (a) Goodman, J. J. Polym. Sci. 1960, 44, 551; (b) Stuart, M. Nature (London) 1963, 199, 59; (c) Bradley, A.; Hammes, J. P. J. Electrochem Soc. 1963, 110, 15. 6 d’Agostino, R. Plasma Deposition, Treatment and Etching of Polymer Films, Acadamic Press, Inc.: San Diego, CA, 1990. 7 Rinsch, C. L.; Chen, X.; Panchalingam, V.; Eberhart, R. C.; Wang, J. H.; Timmons, R. B. Langmuir 1996, 12, 2995. 8 Chan, C.-M. Polymer surface modification and characterization, Hanser/Gardner Publications, Inc., Cincinnatti, OH, 1994. 9 Yasuda, H.; Hirotsu, T. J. Polym. Sci. Polym. Chem. Ed. 1978, 16, 313. 10 Yasuda, H.; Hirotsu, T. J. Polym. Sci. Polym. Chem. Ed. 1978, 16, 2587. 11 (a) Yasuda, H.; Hirotsu, T. J. Appl. Polym. 1977, 21, 3167; (b) Yasuda, H.; Hirotsu, T. J. Appl. Polym. 1977, 21, 3139. 12 Ohkubo, J.; Inagaki, N. J. Appl. Polym. 1990, 41, 349. 13 Morita, S.; Bell, A. T.; Shen, M. J. Polym. Sci. Polym. Chem. Ed. 1979, 17, 2775. 14 Wu, Y. Pulsed Plasma Surface Modification to Improve the Biocompatibility of Materials, Ph.D. Thesis, The University of Texas at Arlington, 1998. 15 (a) Panchalingam, V.; Poon, B.; Huo, H.-H.; Savage, C. R.; Timmons, R. B.; Eberhart, R. C. J. Biomater. Sci. Polymer Edn. 1993, 5, 131; (b) Panchalingam, V.; Chen, X.; Savage, C. R.; Timmons, R. B.; Eberhart, R. C. J. Appl. Polym. Sci.: Appl. Polym. Symp. 1994, 54, 123; (c) Hynes, A.M.; Shenton, M.J.; Badyal, J.P.S. Macomolecules 1996, 29, 4220; (d) Chen, X.; Rajeshwar, K.; Timmons, R. B.; Chen, J.-J.; Chyan, M. R. Chem. Mater. 1996, 8, 1067; (e) Rinsch, C. L.; Chen, X.; Panchalingam, V.; Eberhart, R. C.; Wang, J. H.; Timmons, R. B. Langmuir 1996, 12, 2995; (c) Wang, J.-H.; Chen, J.-J.; Timmons, R.B. Chem. Mater. 1996, 8, 2212; (f) Han, L.M.; Rajeshwar, K.; Timmons, R.B. Langmuir 1997, 13, 5941; (g) Calderon, J. G.; Timmons, R. B. Macromolecules 1998, 31, 3216.

4 Surface Modification Techniques and Fundamental Aspects of Plasma Polymerization

&KDS WHU

Surface Modification Techniques and Fundamental

Aspects of Plasma Polymerization

2.1 Polymer Surface Modification Techniques

Many polymers possess good bulk mechanical properties which make them very useful for a number of applications. In fields such as protective coatings, adhesion, and biomaterials however, their surface properties are equally important for their success. Since polymers often do not possess the required surface properties for these, or similar applications, intensive research efforts have been made in recent years to develop surface treatment methods that alter and improve the chemical and physical properties of polymer surfaces. Improving adhesion characteristics, increasing hydrophobicity, introducing special functional groups at a surface, or modifying the surface morphology are examples for the purposes of these surface treatments.1 Some of these surface modification techniques will be discussed briefly in the next two paragraphs. Firstly, some surface treatment methods will be presented, that enable the alteration of chemical and physical properties of polymer surfaces without affecting their bulk properties (§2.2). Secondly, surface modification by attachment of a monolayer or thin polymer film to the surface will be discussed (§2.3).

2.2 Surface Treatments of Polymers

2.2.1 FLAME TREATMENT

Flame treatments have been used commonly in the polymer industry to improve adhesive characteristics of surfaces, or more particularly to enhance ink permanence on polymer surfaces.2 The high flame temperature (1000-2000 °C) and reaction with excited species in the flame, basically leads to an increased oxygen concentration at the treated surface.3

2.2.2 CORONA DISCHARGE

Corona discharges are widely used in surface modification of polymers for printing4 and adhesion.5 A corona discharge (atmospheric pressure plasma) is produced when air is ionized by a high electric field. Often a corona discharge system is used for continuous Chapter 2

treatment of films, installed downstream of an extruder. The general setup is shown in Figure 2.1.2

High voltage

Electrode

Polymer film

Dielectric Grounded covering metal roll

Figure 2.1 A schematic diagram of a corona discharge setup.

When a high voltage is applied across the electrodes a plasma is formed, and a light blue color can be observed in the air gap between the electrodes. The insulating covering of the grounded roll prevents a direct arc between the two electrodes. Similar to a flame treatment, a corona treatment causes surface oxidation of polymers. Electrons, ions, excited species and photons that are present in a discharge react with the polymer surface to form radicals. These radicals react rapidly with atmospheric oxygen.6

Advantages of corona and flame treatments are that these processes can be used in continuous operation, and that the required equipment is very simple and cost effective. The disadvantages arise from the fact that both treatments are carried out in open air, which often makes it difficult to control the uniformity or chemical nature of the modification, due to variations in ambient conditions such as temperature and humidity or contaminations.2

2.2.3 PLASMA TREATMENT

Although the previously mentioned flame treatment and corona discharge are both based on the formation of a plasma, plasma treatment will be referred to in this thesis as the modification of a surface with a non-polymerizable gas such as argon, oxygen, nitrogen or fluorine, in a vacuum system. The most common applications of plasma treatments are surface cleaning or etching,7 to increase the wettability and adhesion of polymers,8 to reduce friction,9 and more recently, to improve cell attachment in tissue culture studies,10 or to alter surface- protein interactions.11 Functional groups and cross-links are introduced at the surface of the polymer by reaction of gas-phase species and surface species.

6 Surface Modification Techniques and Fundamental Aspects of Plasma Polymerization

Advantages of plasma processes (plasma treatment as well as plasma polymerization) include the following: 1. The modification is limited to the top surface layer and does not affect the bulk properties of the polymer. 2. The modification by plasma processes is largely independent on the structure or chemical reactivity of the substrate. 3. A broad range of functional groups can be introduced at the surface, by variation of the gas that is used. 4. In general, the modification is fairly uniform over the whole substrate. 5. The plasma treatment or polymerization is a simple one-step procedure, and is an all-dry process.

Some of the disadvantages inherent to plasma processes are summarized in the following list: 1. A vacuum system is required for plasma treatments. This demand increases the cost of the operation. 2. Due to the complexity of plasma processes, it is not easy to achieve a good control over the chemical composition of the surface after modification. The influence of process parameters such as reactor geometry, input power, and gas flow on the chemical composition of the modified material should be investigated separately to find the optimal treatment conditions for each gas.

2.2.4 OTHER SURFACE TREATMENTS

In industry, large objects are often subjected to wet chemical treatments. Chemical etchants like chromic or sulfuric acid can be used to convert smooth hydrophobic surfaces to rough hydrophilic surfaces by dissolution of amorphous regions and surface oxidation.2,12 When polymer surfaces are prepared for adhesive bonding for instance, the etching of these surfaces is an important step. Ion-beam modification has been used to texturize polymer (especially fluoropolymer) surfaces, to increase adhesion.13 The bombardment of polymer surfaces with ions has been shown to lead to reduction, oxidation, cross-linking, ion implantation, loss of heteroatoms and loss of aromaticy via ring opening, depending on the polymer, ion, ion beam energy and dose.2 Finally, photon irradiation should be mentioned, which includes e.g. modification by ultraviolet (UV) and infrared (IR) lasers to treat very small and localized areas. Figure 2.2 gives an example of the proposed mechanism for the photochemical reactions occurring during successive UV irradiation of poly(vinyl chloride) in chlorine and nitrogen, and argon laser irradiation in air, after which the surface becomes electrically conductive.14

7 Chapter 2

CH 2 CH n Poly(vinyl chloride) Cl

UV irradiation in chlorine λ > 300 nm

Chlorination CH CH n Chlorinated PVC Cl Cl

UV irradiation in nitrogen λ > 250 nm

Dehydrochlorination CH C CH C n Chlorinated polyenes Cl Cl

Ar-laser irradiation in air λ = 488 nm

Carbonization CH CH n + HCl

Figure 2.2 The mechanism of the carbonization of poly(vinyl chloride) by UV and laser irradiation.2

2.3 Surface Modification by Attachment of Monolayers or Thin Films

2.3.1 LANGMUIR-BLODGETT-KUHN TECHNIQUE

The Langmuir-Blodgett-Kuhn (LBK) technique15 can be used to deposit highly oriented, ultrathin films onto planar substrates. In the first step, amphiphilic molecules are spread on an aqueous subphase in a trough. In the second step, the surface area of the molecules is slowly reduced by two barriers, resulting in orientation of the molecules. The hydrophilic headgroups are dissolved in the subphase, while the compressed hydrophobic chains stand out of the solution. In the third step, a substrate is dipped in and out of the solution, while the surface pressure is kept constant by the barriers. At every dip a well defined monolayer is transferred on to the substrate, and highly ordered multilayers can be deposited. Comprehensive reviews on LBK films have been written by Petty,16 and by Roberts.17

8 Surface Modification Techniques and Fundamental Aspects of Plasma Polymerization

2.3.2 POLYELECTROLYTE MULTILAYER DEPOSITION

A fairly simple technique for the deposition of polymer multilayers has been developed recently by Decher et al..18 By dipping a charged substrate alternatingly in two polyelectrolyte solutions with oppositely charged polymers, it is possible to transfer over 100 monolayers of constant thickness to the substrate. By functionalization of polyelectrolytes with various chemical groups, stable thin films with different properties can be prepared.19

2.3.3 BLOCK COPOLYMER THIN FILMS

Another method to prepare thin polymer coatings is by adsorption of block copolymers. Block copolymers are macromolecular architectures, consisting of two or more chemically different, covalently linked polymer chains.20 In general, a substrate is subjected to the block copolymer solution. In principle, the polymer chains in solution are highly mobile, and the thermodynamic driving force minimizes the total free energy of the system by segregation of the component with the lowest surface free energy at the surface.2,21

2.3.4 RADIATION GRAFTING

The chemical and physical properties of polymers are frequently modified by surface grafting methods. This can be performed in a one-step process, when a polymer is irradiated in the presence of a solvent containing a monomer. However, a two-step process is often used to minimize homopolymerization, initiated by free radicals formed during irradiation of the monomer. In this method, the polymer is first irradiated in air, or subjected to a corona discharge, plasma treatment or ozone treatment to introduce peroxide groups at the surface. In a second step, the grafting is mostly initiated thermally in contact with the monomer. Several energy sources can be used in radiation grafting, such as high-energy electrons,22 γ radiation,23 X-rays, and UV light.24

2.4 Plasma Polymerization

2.4.1 THE FOURTH STATE OF MATERIALS

In general, plasma is referred to as a partially ionized gas that contains positively and negatively charged particles.25 The plasma state is more highly activated than the solid, liquid or gas state, and is often called the fourth state of materials. As an example, the transition of ice to aqueous vapor can be considered.26 In ice (the solid state), the thermal motion of the

H2O molecules is restricted, but they still vibrate with a small amplitude around a given mean position. As the temperature of the ice increases, the vibration of the H2O molecules becomes stronger, until at 0 °C they leave the position determined by the free energy, and transition from ice to water occurs. As the temperature of water is raised further, the kinetic energy of

9 Chapter 2

the H2O molecules increases. At 100 °C, the kinetic energy of the molecules becomes larger than their potential energy, and the molecules escape from the liquid state into the gas phase

(vaporization). In the gas state, the H2O molecules move around freely, until they collide with other particles. At temperatures of more than a few thousand degrees Celsius, the kinetic energy of the H2O molecules becomes so high that collisions with other molecules leads to the dissociation of H2O, and ionization of the atoms. This whole transition process is visualized schematically in Figure 2.3.

Plasma

Ionization Kinetic energy Kinetic

Dissociation

Boiling

Melting

Melting Boiling Temperature point point

Figure 2.3 Schematic diagram of the state transition processes.26

A plasma can also be created by other conditions than intense heat, such as an electric glow discharge. A plasma generated in this manner is frequently called a low temperature plasma, and is used in most of the experimental work on plasma polymerization. A glow discharge can be generated by the transfer of power from an electric field to electrons. In a direct current (DC) glow discharge, this can be established by passing a DC electric current through a gas under low pressure. The cathode is bombarded with positive ions, resulting in the generation of secondary electrons. These electrons are accelerated away from the cathode until they gain sufficient energy to ionize the gas molecules or atoms that collide with the electrons.6,25 In alternating current (AC) glow discharges, the mechanism depends on the frequency of the alternation. At low frequencies, the system can be looked upon as a DC glow discharge with alternating polarity. Upon increasing the frequency of the applied voltage, positive ions become immobile, because they can no longer follow the periodic changes in

10 Surface Modification Techniques and Fundamental Aspects of Plasma Polymerization

field polarity, and only respond to time-averaged fields. At frequencies above 500 kHz, the half-cycle is so short that all electrons and ions stay within the interelectrode volume. This reduces the loss of charged particles from the system significantly, and regeneration of electrons and ions occurs within the body of the plasma through collisions of electrons with gas molecules. In rf plasma (13.56 MHz) therefore, no contact between the electrodes and the plasma is required. The plasma can be initiated and sustained by external electrodes, at a much lower voltage than is required for maintaining a DC glow discharges.2

2.4.2 FUNDAMENTAL ASPECTS OF PLASMA PHYSICS

This paragraph deals with some of the basic concepts of plasma physics that are useful for the understanding of plasma polymerization. First of all, the negative particles in a glow discharge plasma are mostly electrons, however negative ions are also formed. When an electric field is applied, the electrons gain energy according to Newton’s law, q a = E (2-1) me where a is the acceleration of the electron, q is its electric charge, me is its mass, and E is the electric field.25 Three different types of collisions can occur between an electron and an atom, depending on the energy K that is transferred to an electron in the atom: 1. K = 0. The electrons in the atom remain in the ground state. The collision is elastic, and causes no change in the structure of the atom.

2. 0 < K < qVi, where Vi is the ionization potential of the atom. An electron in the atom is excited to a higher energy level, but returns to the ground state in a short time, releasing the gained energy again. The collision is inelastic.

3. K > qVi. The atom is ionized by inelastic collision, and becomes positive with charge +q.

Between the energy level of the ground state and that of the ionized state, a number of other energy levels can exist. An electron in an atom that receives energy from a primary electron can jump to a higher energy level, but after a short period of time (of the order of 10-8 s) it falls back to lower energy levels or to the ground state. In this process, the electron's excess energy is released e.g. by emission of a photon. When an electron falls back from energy level Em to En, the frequency ν of this photon is given by:

hν = Em - En (2-2) where h is Planck’s constant.

The temperature of colliding species plays an important role on the collision processes occurring in a glow discharge. The electron temperature Te in a plasma is given by Eq. 2-3,

1 1  e  Eλ  m  2  π  4 =  e  m  Te       (2-3)  k  2 2  me   6 

11 Chapter 2

in which k is the Bolzmann constant, λe the mean free path of electrons, and mm the mass of 26 particles (neutral atoms and molecules). Since the ratio of mm/me is very high, the electron temperature in a low pressure plasma is extremely high (e.g. on the order of 104 K). Because ions have roughly the same mass as the corresponding neutral atoms and molecules, they lose most of their kinetic energy in collisions with molecules. The ion temperature Ti is therefore much lower than Te (in the range of 300-1000 K), and it is only slightly higher than the 25 temperature of molecules Tm (~ 300 K).

2.4.3 TYPES OF COLLISIONS

A number of different collision processes occur in a glow discharge, including ionization, excitation, dissociation and attachment.2 Ionization is the essential step in creating and sustaining a plasma, but is not necessarily the primary step in initiating plasma polymerization.25 The ionization of a helium atom for example can be visualized as follows:

He + e- → He+ + 2e- (ionization)

A positive ion and two electrons are produced in this reaction. The two electrons are accelerated by the electric field, and can produce further ionization. It was mentioned that electrons in the ground state can jump to higher energy levels (excitation), without resulting in ionization:

He + e- → He* + e- (excitation)

After a very short period of time the electron falls to a lower energy level, or returns to the ground state by radiative decay (deexcitation):

He* → He + hν (deexcitation)

Some excited states however have much higher stability than others, and have lifetimes of 1 ms or longer. Such a state is called a metastable state.27 Penning discovered in 1937 that the voltage necessary to initiate a neon glow discharge was reduced dramatically by the addition of only 0.1 % of argon to the gas.28 The reason behind this behavior is that the excitation energy of the metastable state of neon is higher than the ionization energy of argon. The metastable neon atom returns to the ground state upon collision, but transfers the excess energy to the colliding argon atom. The neon gas acts as a catalyst in the gas ionization of argon:

Ne* + Ar → Ne + Ar+ + e- (Penning ionization)

A metastable atom can also be ionized by electron impact directly. Negative ions such as F-, - - - - - Cl , Br , I , O and O2 can also be produced in a glow discharge, if a free electron is captured by a molecule, for example:

12 Surface Modification Techniques and Fundamental Aspects of Plasma Polymerization

- - - - Cl2 + e → Cl + Cl or O2 + e → O2 (formation of negative ions)

Collisions between charged species take place in ionized gas as well, but do not play an important role overall, simply due to the fact that most plasmas are only partially ionized, and contain far more neutral than charged species. An example of such a collision is recombination:

Ar+ + e- → Ar (electron-ion recombination)

O- + Ar+ → O + Ar (ion-ion recombination)

Recombination also occurs at the surface of the reactor. Collisions of electrons with the reactor wall charge the surface (depicted as S) negatively, which then attracts positive ions that neutralize the charge:

S + e- → S- and S- + Ar+ → S + Ar (surface recombination)

In this reaction the energy gained in the ionization process is released, and absorbed by the surface in the form of thermal energy.25

Cycle II

Figure 2.4 Schematic diagram of the bicyclic step-growth mechanism of plasma polymerization.25,29

Although the ionization of monomer molecules is necessary to create and maintain a plasma, the actual polymerization is considered to be a side reaction of the ionization process. According to Yasuda, the polymerization is based on a rapid step-growth process, as is shown in Figure 2.4.25 M can be either the original monomer molecule, or any of the dissociation

13 Chapter 2

products, including atoms. The reactive species are denoted here as monofunctional (M•), or difunctional (•M•) free radicals, but other activated species should also be considered in the reaction mechanism.27 In cycle I the reaction products from monofunctional activated species are repeatedly activated. Cycle II proceeds through the reaction of difunctional radicals with other species.

2.5 References

1 Polymer Surface Modification: Relevance to Adhesion, Mittal, K. L. (Ed.); VSP BV, Utrecht, The Netherlands, 1996. 2 Chan, C.-M. Polymer surface modification and characterization, Hanser/Gardner Publications, Inc., Cincinnatti, OH, 1994. 3 (a) Briggs, D.; Brewis, D. M.; Konieczko, M. B. J. Mater. Sci. 1979, 14, 1344; (b) Garbassi, F.; Ochiello, E.; Polato, F. J. Mater. Sci. 1987, 22, 207; (c) Sutherland, I.; Brewis, M. D.; Heath, R. J.; Sheng, E. Surface Interface Anal. 1991, 17, 507. 4 (a) Stradal, M.; Goring, D. A. I. Polym. Eng. Sci. 1977, 17, 38; (b) Gatenholm, P.; Bonnerup, C.; Wallström, E. J. Adhesion Sci. Technol. 1990, 4, 817. 5 (a) Blythe, A. R.; Briggs, D.; Kendall, C. R.; Rance, D. G.; Zichy, V. J. I. Polymer 1978, 19, 1273; (b) Briggs, D. J. Adhesion 1982, 13, 287; (c) Briggs, D.; Kendall, C. R. Int. J. Adhesion Adhesives 1982, 2, 13. 6 Strobel, M.; Dunatov, C.; Strobel, J. M.; Lyons, C. S.; Perron, S. J.; Morgen, M. C. J. Adhesion Sci. Technol. 1989, 3, 321. 7 Shi, M. K.; Selmani, A.; Martinu, L.; Sacher, E.; Wertheimer, M. R.; Yelon, A. in Polymer Surface Modification: Relevance to Adhesion, Mittal, K. L. (Ed.); VSP BV, Utrecht, The Netherlands, 1996. 8 Hudis, M. in Techniques and Applications of Plasma Chemistry, Hollahan, J. R.; Bell, A. T. (Eds), John Wiley, New York, 1974. 9 Triolo, P. M.; Andrade, J. D. J. Biomed. Mater. Res. 1983, 17, 129. 10 Amstein, C. F.; Hartman, P. A. J. Clin. Microbiol. 1975, 2, 46. 11 Holly, F. J.; Owen, M. J. in Physicochemical Aspects of Polymer Surfaces, Mittal, K. L. (Ed.) Vol. 2; Plenum Press, New York, 1983. 12 Busscher, H. J.; Arends, J. J. Colloid Interface Sci. 1981, 81, 75. 13 Kojima, M.; Satake, H. J. Polym. Sci. Polym. Phys. Ed. 1982, 20, 2153. 14 Decker, C. in Chemical Reactions on Polymers, ACS Symposium Series, Vol. 364; Benham, J. L.; Kinstle, J. F. (Eds), Washington, DC, 1988. 15 (a) Langmuir, I. JACS, 1917, 39, 1848; (b) Blodgett, K. B. JACS, 1935, 57, 1007; (c) Thiele, J. Ph.D. Thesis, Cuvillier Verlag, Göttingen, 1997. 16 Petty, M. C.; Langmuir-Blogett films; Plenum Press: New York, 1990. 17 Roberts, G. G.; Langmuir-Blodgett films; University Press: Cambridge, 1996. 18 (a) Decher, G.; Hong, J. D. Makrom. Chem. Makrom. Symp. 1991, 49, 321; (b) Decher, G.; Hong, J. D. Ber. Bunsen-Ges. Phys. Chem. 1991, 11, 1430. 19 (a) Fou, A. C.; Rubner, M. F. Macromol. 1995, 28, 7115; (b) Knobler, C. M. Science 1991, 249, 249. 20 Lammertink, R. G. H. Ph.D. Thesis University of Twente, Enschede, The Netherlands, 2000, ISBN 90 36514517. 21 (a) Alexander, S. J. Phys. 1977, 38, 977; (b) Clark, D. T.; Peeling, J.; O’Malley, J. M. J. Polym. Sci., Polym. Chem. 1976, 14, 543.

14 Surface Modification Techniques and Fundamental Aspects of Plasma Polymerization

22 Kim, H.-C.; Song, J. H.; Wilkes, G. L.; Smith, S. D.; McGrath, J. E. J. Appl. Polym. Sci. 1989, 38, 1515. 23 (a) Yamamoto, F.; Yamakawa, S.; Kato, Y. J. Polym. Sci. Polym. Chem. Ed. 1978, 16, 1883; (b) Yamamoto, F.; Yamakawa, S.; Kato, Y. J. Polym. Sci. Polym. Chem. Ed. 1978, 16, 1897; 24 (a) Allmer, K.; Hilborn, J.; Larsson, P. H.; Hult, A.; Ranby, B. J. Polym. Sci. Polym. Chem. Ed. 1990, 28, 173; (b) Allmer, K.; Hult, A.; Ranby, B. J. Polym. Sci. Polym. Chem. Ed. 1989, 27, 3405. 25 Yasuda, H. Plasma Polymerization, Academic Press Inc.: Orlando, FL, 1985. 26 Inagaki, N. Plasma Surface Modification and Plasma Polymerization, Technomic Publishing Comapny, Inc., Lancaster, PA, 1996. 27 Biederman, H.; Osada, Y. Plasma Polymerization Processes, Elsevier Science Publishers B.V.: Amsterdam, The Netherlands, 1992. 28 Loeb, L. B. Rev. Mod. Phys. 1940, 12, 87. 29 Yasuda, H.; Wang, C. R. J. Polym. Sci., Polym. Chem. Ed. 1985, 23, 87.

15

Characterization Methods for Plasma Polymers

&KDS WHU

Characterization Methods for Plasma Polymers

The ever-increasing importance of polymeric thin films in every day life and in modern technology has greatly increased the activity in the field of thin film analysis. Plasma films are in general insoluble, and therefore not all the characterization methods commonly used for conventional polymers are suitable to analyze plasma polymers. In this section, the most important techniques that have been used to characterize the plasma polymers in this research will be briefly discussed.

3.1 Surface Analytical Techniques

In many applications of plasma polymers, their surface properties are of great importance. Using one or more of the following surface characterization techniques can give extensive qualitative and quantitative information about the chemical composition, morphology, or the surface energy of the material investigated. In general, the term surface is not well defined, and its definition depends on the sampling depth of the used surface analysis technique.1

3.1.1 CONTACT ANGLE GONIOMETRY

Contact angle measurements give information about the top several Å of a plasma film.2 The contact angle is defined as the angle between a solid surface and the tangent of the liquid-vapor interface of a liquid drop.3 Contact angle measurements with water can be used to determine if a plasma film is hydrophilic (low contact angle) or hydrophobic (high contact angle). In static measurements, the angle θ is measured at a stationary liquid front.1 Figure 3.1-a shows a picture of a sessile drop on a solid surface.

a) Vapour γ b) c) LV Liquid θ θ γ s γ a θ SL SV r

Solid

Figure 3.1 Schematic diagram of the measurement of the a) sessile, b) advancing and c) receding contact angle. Chapter 3

The contact angle depends on the force balance at the three-phase boundary, defined by Young’s equation:

γLV cosθ = γSV - γSL (3-1) where γLV is the surface tension of the liquid in equilibrium with its saturated vapor, γSV the surface tension of the solid in equilibrium with the vapor, and γSL the interfacial tension between the solid and the liquid.1 A simple technique to measure dynamic contact angles is to insert a needle in the drop, and measure the contact angle during the movement of the three- phase boundary while water is added (advancing contact angle θa; Figure 3.1-b) or removed

(receding contact angle θr; Figure 3.1-c). Differences between θa and θr, so-called contact angle hysteresis, are often observed, and may be due to surface roughness,4 surface heterogeneity,5 or surface mobility of functional groups.6 Contact angle measurements provide a simple tool to study changes of the surface composition that polymers undergo upon surface treatment,7 aging,8 or migration of functional groups in certain environments.6 To illustrate the difference between a hydrophilic and a hydrophobic surface, one piece of polyester fabric was treated with a CHF3/Ar plasma, while another piece was treated with an O2 plasma. When a drop of ink was placed on the hydrophobic piece, the contact area between the drop and the polyester was very small, while complete wetting occurred on the hydrophilic surface (Figure 3.2).

Figure 3.2 The difference in water contact angle can be seen clearly for this drop of ink on a piece of polyester fabric. The left piece has been treated with a CHF3/Ar plasma, the right piece with O2 plasma.

3.1.2 X-RAY PHOTOELECTRON SPECTROSCOPY (XPS)

Another widely used technique to characterize plasma polymer surfaces (± 3-10 nm) is XPS, also known as electron spectroscopy for chemical analysis (ESCA).1,9 A sample is irradiated in vacuum with X-rays. The interaction between an X-ray photon and an inner-shell electron causes a complete transfer of the photon energy to the electron, which then has enough energy to leave the atom and escape from the surface (photoelectron).1 The binding energy Eb of the inner-shell electron can be calculated from the difference between the known

X-ray photon energy hv, and the kinetic energy Ek of the photoelectron, which is measured by an electron energy analyzer:

Eb = hv – Ek (3-2)

18 Characterization Methods for Plasma Polymers

Knowing the binding energy of a particular shell of an atom allows identification of the element. An example of a XPS spectrum is shown in Figure 3.3, taken from a plasma polymerized ethylenediamine film. The photoelectron peaks are associated with a particular core level of a particular element.

N1s C1s

O1s

Figure 3.3 XPS survey spectrum of a film plasma polymerized from ethylenediamine at Ppeak = 200 W, ton = 3 ms and toff = 15 ms.

The peaks shown in Figure 3.3 can also be scanned individually. These so-called high- resolution spectra allow one to detect small shifts in the binding energies, and can thus provide information about the chemical environment of the atom.

3.1.3 ATOMIC FORCE MICROSCOPY (AFM)

The first description of the atomic force microscope was published in 1986 by Binnig,10 Quate and Gerber.11 It can produce three-dimensional images of solid surfaces at very high resolution, and unlike scanning tunneling microscopy (STM), it can also image nonconducting samples such as polymers and ceramics.8 When the force microscope is operated in ‘contact mode’, the sample surface is scanned by a sharp tip, which is mounted on a soft spring, also referred to as ‘cantilever’.12 The sample is positioned on a piezoelectric tube ortripod, which controls the scanning motion. Features on the sample surface cause a deflection of the cantilever. This deflection is most commonly measured by an optical beam technique: laser light is focused onto the end of the cantilever, and reflected onto a split photodiode. The feedback signal from the photodiode is used to control the height of the piezoelectric crystal as the sample is scanned. The corresponding height adjustment signal is directly related to the topography of the surface. Interaction between the sample surface and the AFM tip is determined by the interaction between the molecules or atoms on the surface of the sample and the tip. The relevant forces in force microscopy (e.g. electrostatic forces, dipolar actions, van der Waals forces, H-bonding) have been summarized e.g. by Schönherr.12 His work also gives an excellent overview of the most important theories and models (e.g. DLVO theory,13 Hertz model,14 JKR model,15 Cobblestone model of interfacial friction,16 etc.) that are used nowadays for the quantitative evaluation of force measurements.

19 Chapter 3

When AFM is used to obtain topographic information on soft samples such as adsorbed biolayers, the lateral force exerted by the tip can lead to image artefacts due to disrupture of the surface.17 This problem has been solved recently by the development of a new AFM technique, known as tapping mode AFM (TM-AFM). This tapping mode employs a cantilever oscillating with a high amplitude. The vibration is set such that the tip contacts the sample surface once in every vibration period.17a Since the tip only intermittently ‘taps’ the surface, the tip-sample interactions (especially shear forces) are greatly reduced, which ensures minimal disturbance of the adsorbed molecules.18 The use of this technique has enabled researchers to image the molecular packing and aggregation of proteins on surfaces such as silicon,17a methylated silicon, 17b self-assembled monolayers,17c spin-cast polymer films,19 and on mica.20 Some of the plasma films discussed in this Thesis were seen to be rather soft, and contact mode AFM sometimes resulted in disrupture of the surfaces. The AFM images of the plasma surfaces presented in this work were therefore taken in tapping mode, or in one particular case (Figure 4.7) in non-contact mode11. TM-AFM was also used in the research described in Chapter 8, to image proteins adsorbed to plasma polymerized films.

In the experiments described in Chapter 6, AFM was used to measure both attractive and repulsive forces between the AFM tip and the sample surface. It will be shown that pH- dependent force measurements with chemically modified tips21 were able to provide valuable information about the environment of ionizable functional groups. A schematic representation of such a ‘force spectroscopy’ experiment is given in Figure 3.4, which is reproduced from Schönherr.12

Figure 3.4 Schematic diagram of a force spectroscopy experiment. Adapted from Schönherr.12

Starting at 1, the sample is moving up, but is out of contact with the tip. In situation 2, the gradient of the force overcomes the cantilever spring constant, and the tip jumps into contact. The sample continues to move up (3), and causes deflection of the cantilever. If the attractive forces are higher than the repulsive forces, the tip sticks to the surface during retraction (4). In situation 5 finally, the tip snaps off when the spring constant overcomes the

20 Characterization Methods for Plasma Polymers

force gradient. Adhesion between a (chemically modified) tip and a surface can thus be characterized by this so-called "pull-off" force. The corresponding force-distance plot is shown in Figure 3.5 (the tip jumps off in piezo position labeled by 5).

Figure 3.5 Schematic force-distance plot. The numbers correspond to the situations sketched in Figure 3.4. Adapted from Schönherr.12

3.2 Thin Film Characterization by Optical Techniques

A powerful and very promising technique for the characterization of polymeric thin films is based on evanescent wave optics. The use of different types of surface- electromagnetic waves22 has proven to be particularly sensitive for monitoring optical properties of ultrathin layers.23 Surface plasmons24 (or plasmon surface polaritons) and guided optical waves25 are two examples of such surface-bound modes, and form the basis for a wide range of optical techniques developed by Knoll and co-workers,23,26 that use the interaction of this ‘surface light’ with thin films deposited onto such a surface. This paragraph describes two techniques that have been used to investigate the optical properties of plasma polymerized allylamine films in different environments.

3.2.1 WAVEGUIDE MODE SPECTROSCOPY (WAMS)

This method is based on the fact that light can be trapped in very thin films of transparent materials under certain conditions. These thin films are called optical waveguides. When a beam of light impinges with angle θ to the normal of a planar boundary between two dielectrics F and C with refractive indices respectively nF and nC, the refracted beam propagates away from the interface according to Snell’s law, which is given by

nF sinθ = nC sin θC (3-3)

If nF > nC is assumed, and the angle of incidence is increased from 0, a certain angle θ will be reached where sinθ = nC/nF, or sinθC = 1. Since the angle θC of the refracted beam cannot exceed 90°, a further increase in θ results in total internal reflection at the interface.27 If F is

21 Chapter 3

the optical waveguide, bounded by media with lower refractive indices on both sides, then a light ray can be confined in the film, and propagate through the waveguide by total internal reflection alternately at each interface. What makes guided optical waves a particularly valuable diagnostic tool is the fact that they can be excited with both transverse magnetic (TM) and transverse electric (TE) light, i.e. with p- and s-polarized photons. The field distribution in and around the waveguide for both TE and TM modes, can be derived using Maxwell’s equations, assuming that the material is isotropic, linear, nonconducting, charge- free and nonmagnetic. For a waveguide F with a support S for mechanical strength on one side, cover C on the other side (e.g. air or solvent) and a thin adlayer A (e.g. a plasma polymer) between F and C (Figure 3.6), the mode equations can be calculated from the Fresnel reflection coefficients. The complete derivitizations have been described in the literature.27,28 D C A θ F deff d F θ

S

Figure 3.6 Laser light propagating through a waveguide composite by total internal reflection. The waveguide consists of a substrate S, the waveguide film F, an adlayer A and the cover medium C.27

The waveguide modes can be excited by coupling laser light into the waveguide by means of a prism or a diffraction grating. To characterize a thin plasma polymer adlayer, very thin planar waveguides of high refractive index modes are used as a substrate, with which the TM0 and TE0 mode can be measured. This is schematically depicted in Figure 3.7. By binding an adlayer to the surface of the waveguide, the refractive index of the surrounding medium changes in the vicinity of the evanescent field of the waveguide modes. This in turn induces changes in the effective refractive indices Neff,TE0 and Neff,TM0 of the guided modes. The effective refractive index provides the phase velocity ν of the guided mode (ν = c/Neff, where c is the velocity of light in vacuum) and depends on the polarization and the mode number. Changes in the effective refractive index, ∆Neff i,j, can be measured using a grating coupler.28b A laser beam can be coupled into the planar waveguide at an angle of incidence α if the in-coupling condition

Neff i,j = nC sinα + I λ / Λ (3-4) is satisfied. In this equation nC is the refractive index of the covering medium (air or solvent), λ the wavelength of the laser used, I the diffraction order and Λ the grating period. Thus, if a

22 Characterization Methods for Plasma Polymers

plasma polymer is deposited as an adlayer onto the waveguide, then its optical parameters

(thickness da and refractive index na) change the effective refractive indices Neff i,j of the waveguide modes. The effective index shift ∆Neff i,j can be determined from Eq. 3-4 above, by measuring the change ∆α. From the effective index shifts ∆Neff i,j the thickness and refractive index of the plasma polymer adlayer can be calculated using the dispersion relationship for a four layer planar waveguide.28b

nc Cover medium

da na Plasma polymer Λ df nf Waveguide

ns Substrate substrate

α Field distribution TM0 or TE0 mode

Figure 3.7 Electric or magnetic field distribution for a zeroth mode in a planar multilayer waveguide.

Waveguide Waveguide with adlayer 3 TM0' TE0 step 1.0 1.0 TM0 TE0' PC motor 2 lock-in 0.5 0.5 1 Intensity detector 0.0 0.0 0 01234567 α [°] α

laser waveguide polarization chopper chip optics cuvette

goniometer

Figure 3.8 Schematic diagram of the WaMS setup. The angles at which the TE0 and TM0 modes are excited before and after (TE0’ and TM0’) deposition of a plasma polymerized film, are shown as an example.

The experiments that are presented in Chapter 4 and 5 were carried out using a home built spectrometer (Figure 3.8). A grating with periodicity Λ, incorporated into the waveguide

23 Chapter 3

was used to couple a HeNe laser beam (λ = 632.8 nm) from the external cover medium into the waveguide. Neff i,j is determined by scanning the angle of incidence of the incoming laser beam onto the grating, while the in-coupled power is measured by two photo-detectors situated at both ends of the waveguide. The waveguide was designed for optimal sensitivity for the TE0 and TM0 modes. The system was equipped with a cuvette to enable swelling measurements in solvents.

3.2.2 SURFACE PLASMON RESONANCE (SPR) SPECTROSCOPY

Surface plasmons, or plasmon surface polaritons, are oscillations of the quasi-free electron gas in a metal that are coupled to an electromagnetic wave at the surface. They propagate along the interface between a metal and a dielectric medium.26i Their evanescent field peaks at the interface, and decays exponentially into the metal and the dielectric medium.

Experiments were carried out on a home-built SPR setup (Figure 3.9), which is based on the configuration introduced by Kretschmann and Raether.29 A thin metal layer (typically Ag or Au) is evaporated on a glass slide, and placed on the base of a prism. The light from a HeNe laser is linearly polarized, reflected at the base of the prism, and monitored with a detector. If the angle of incidence is scanned, a sharp minimum in the reflected light intensity occurs, when the light is resonantly coupled to the surface plasmon modes. At this angle, the energy and the momentum between the incoming photons and the surface plasmon waves are matched, and the reflectivity goes virtually to zero. Detector

Polarizer θ Laser

Prism Sample Figure 3.9 Schematic diagram of a SPR setup.

As an example, Figure 3.10 shows the reflectivity scans of a gold-coated glass substrate in water, a similar substrate after modification with plasma polymerized allylamine, and a dimyristoylphosphatidylcholine (DMPC) lipid bilayer adsorbed to this surface. Kinetic information can also be obtained from SPR spectroscopy. In these measurements, the angle of incidence is kept constant, on the left side of the initial reflectivity minimum, in the range where the resonance decreases linearly. Then the change in the reflectivity is measured as a function of time, until equilibrium is reached.

24 Characterization Methods for Plasma Polymers

1.0

0.8

0.6

0.4 Reflectivity

a) b) 0.2 c)

0.0 40 50 60 70 80 Incident angle [°]

Figure 3.10 Reflectivity scans of a) a bare substrate of LaSFN9 glass with a 47.4 nm thick Au coating on top, measured in water, b) with a 44.4 nm thick adlayer of plasma polymerized allylamine (P = 100 W CW tdep = 1 min) after 11 h of swelling in water, and c) a 3.7 nm thick adsorbed DMPC lipid bilayer, after rinsing with water. The full lines show the reflection curves calculated from Fresnel equations.

3.3 References

1 Chan, C.-M. Polymer surface modification and characterization, Hanser/Gardner Publications, Inc., Cincinnatti, OH, 1994. 2 Anand, M.; Cohen, R. E.; Baddour, R. F. Polymer 1981, 22, 361. 3 (a) d’Agostino, R. Plasma Deposition, Treatment and Etching of Polymer Films, Acadamic Press, Inc.: San Diego, CA, 1990; (b) Zisman, W. A. Contact Angle-Wettability & Adhesion, Gould, R. F. (Ed.); Am. Chem. Soc., Washington, D.C., 1964. 4 (a) Johnson Jr., R. E.; Dettre, R. E.; Surface Colloid Sci. 1969, 2, 85; (b) Wenzel, R. N. J. Phys. Colloid Chem. 1949, 53, 1466; (c) Oliver, J. F.; Huh, C.; Mason, S. G. Colloids Surfaces 19880, 1, 79. 5 (a) Morra, M.; Occhiello, E.; Garbassi, F. Surface Interface Anal. 1990, 16, 412; (b) Garbassi, F.; Morra, M.; Occhiello, E.; Barino, L.; Scordamaglia, R. Surface Interface Anal. 1989, 14, 585. 6 (a) Yasuda, H.; Sharma, A. K.; Yasuda, T. K. J. Polym. Sci. Polym. Phys. Ed. 1981, 19, 285; (b) Yasuda, T.; Okuno, A. K.; Yoshida, K.; Yasuda, H. J. Polym. Sci. Polym. Phys. Ed. 1988, 26, 1781; (c) Yasuda, T.; Yoshida, K.; Okuno, T.; Yasuda, H. J. Polym. Sci. Polym. Phys. Ed. 1988, 26, 2061; (d) Kennedy, V. O. J. Coating Technol. 1988, 60, 37, (e) Brennar, W. J.; Feast, W. J.; Munro, H. S.; Walker, S. A. Polymer 1991, 32, 1527 (f) van Damme, H. S.; Hogt, A. H.; Feijen, J. J. Colloid Interface Sci. 1986, 114, 167 (g) Owen, M. J.; Gentle, T. M.; Orbeck, T.; Williams, D. E. in Polymer Surface Dynamics, Andrade, J. (Ed.), Plenum Press, New York, 1988. 7 (a) Ho, C.-P.; Yasuda, H. J. Biomed. Mater. Res. 1988, 22, 919; (b) Inagaki, N; Tasaka, S.; Kawai, H.; Kimura, Y. J. Adhesion Sci. Technol. 1990, 4, 99.

25 Chapter 3

8 Gengenbach, T. R.; Vasic, Z. R.; Chatelier, R. C.; Griesser, H. J. J. Polym. Sci.: Part A: Polym. Chem. 1994, 32, 1399; (b) Gengenbach, T. R.; Griesser, H. J. Surf. Interface Anal. 1998, 26, 498; (c) Gengenbach, T. R.; Chatelier, R. C.; Griesser, H. J. Surf. Interface Anal. 1996, 24, 271; (d) Gengenbach, T. R.; Chatelier, R. C.; Griesser, H. J. Surf. Interface Anal. 1996, 24, 611; (e) Gengenbach, T. R.; Griesser, H. J. J. Polym. Sci.: Part A: Polym. Chem. 1999, 37, 2191; (f) Gengenbach, T. R.; Griesser, H. J. J. Polym. Sci.: Part A: Polym. Chem. 1998, 36, 985; (g) Gengenbach, T. R.; Griesser, H. Polymer 1999, 40, 5079; (h) Boenig, H. V. Fundamentals of Plasma Chemistry and Technology, Technomic Publishing Co., Inc.: Lancaster, PA, 1988; (i) Yasuda, H. K. J. Macromol. Sci.-Chem. 1976, A10, 383. 9 XPS was developed by Professor Kai Siegbahn and his collaborators at the University of Uppsala. For his discovery he received the Nobel prize in 1981. See also: a) Siegbahn, K.; Nordling, C.; Fahlman, R.; Hamrin, K.; Hedman, J.; Johansson, T.; Bergmark, T.; Karlsson, S.-E.; Lindgren, I.; Lindberg, B. ESCA: Atomic, Molecular, and Solid State Structure Studied by Means of Electron Spectroscopy, Nova Acta Regiae Sco. Sci. Upsaliensis, Ser. IV, Vol. 20, Almqvist and Wiksells, Stockholm, 1967; (b) Siegbahn, K.; Nordling, C.; Johansson, G.; Hedman, J.; Heden, P. F.; Hamrin, J.; Gelius, U.; Bergmark, T.; Werme, L. O.; Manne, R.; Baer, Y. ESCA Applied to Free Molecules, North-Holland, Amsterdam, 1969. 10 Gerd Binnig and Heinrich Rohrer received the Nobel prize for Physics in 1986 for there invention of the scanning tunneling microscope. See also: (a) Binnig G.; Rohrer, H. Helvetica Physica Acta 1982, 55, 726; (b) Binnig, G.; Rohrer, H.; Gerber, Ch.; Weibel, E. 1982, Phys. Rev. Lett. 1982, 49, 57. 11 Miles, M. J. in Characterization of Solid Polymers; New Techniques and Dvelopments Spells, S. J. (Ed.), Chapman & Hall, London, UK, 1994. 12 Schönherr, H. Ph.D. Thesis University of Twente, Enschede, The Netherlands, 1999, ISBN 90 365 1347 2. 13 (a) Derjaguin, B.; Landau, L. Acta Physiochem. 1941, 14, 633; (b) Verwey, E. G. W.; Overbeck, J. J. G. Theory of the Stability of Lyophobic Colloids; Elsevier, Amsterdam, 1948. 14 (a) Hertz, H. Reine Angew. Math. 1881, 92, 156; (b) Timoshenko, S. P.; Goodier, J. N. Theory of elasticity; McGraw-Hill, New York, 1970. 15 Johnson, K. L.; Kendall, K.; Roberts, A. D. Proc.R. Soc. London A 1971, 324, 301. 16 (a) Gee, M. L.; McGuiggan, P. M.; Israelachvili, J. N.; Honola, A. M. J. Chem Phys. 1990, 93, 1895; (b) Yoshizawa, H.; Chen, Y. L.; Israelachvili, J. N. J. Phys. Chem. 1993, 97, 4128; (c) Yoshizawa, H.; Israelachvili, J. N. J. Phys. Chem. 1993, 97, 11300; (d) Yoshizawa, H. ; Israelachvili, J. N. Thin Solid Films 1994, 246, 71. 17 (a) Ortega-Vinuesa, J. L.; Tengvall, P.; Lundström, I. Thin Solid Films 1998, 324, 257; (b) Ortega-Vinuesa, J. L.; Tengvall, P.; Lundström, I. J. Colloid Interface Sci. 1998, 207, 228; (c) Lin, J. N.; Drake, B.; Lea, A. S.; Hansma, P. K. Langmuir 1990, 6, 509. 18 Baty, A. M.; Leavitt, P. K.; Siedlecki, C. A.; Tyler, B. J.; Suci, P. A.; Marchant, R. E.; Geesey, G. G. Langmuir 1997, 13, 5702. 19 Baty, A. M.; Leavitt, P. K.; Siedlecki, C. A.; Tyler, B. J.; Suci, P. A.; Marchant, R. E.; Geesey, G. G. Langmuir 1997, 13, 5702. 20 Shibata-Seki, T.; Masai, J.; Ogawa, Y.; Sato, K.; Yanagawa, H. Appl. Phys. A 1998, 66, 625. 21 (a) Frisbie, C. D.; Rozsnyai, L. F.; Noy, A.; Wrighton, M. S.; Lieber, C. M. Science 1994, 265, 2071; (b) Noy, A.; Vezenov, D. V.; Lieber, C. M. Annu. Rev. Mater. Sci. 1997, 27, 381.

26 Characterization Methods for Plasma Polymers

22 Burstein, E.; Chen, W. P.; Chen, Y. J.; Hartstein, A. J. Vac. Sci. Technol. 1974, 11, 1004. 23 Aust, E. F.; Ito, S.; Sawodny, M.; Knoll, W. Trends in Polymer Science 1994, 2, 313 24 Raether, H. Springer Tracts in Modern Physics 1988, Springer Verlag, 111. 25 Tien, P. K. Rev. Mod. Phys. 1977, 49, 361. 26 (a) Rothenhaüsler, B.; Knoll, W. Opt. Commun. 1987, 63, 301; (b) Rothenhaüsler, B.; Knoll, W. Nature 1988, 332, 615; (c) Hickel, W.; Kamp, D.; Knoll, W. Nature 1989, 339, 186; (d) Hickel, W.; Knoll, W. J. Appl. Phys. 1990, 67, 3572; (e) Knoll, W. MRS Bull. 1991, 16, 29; (f) Knoll, W. Makromol. Chem. 1991, 192, 2827; (g) Aust, E. F.; Knoll, W. J. Appl. Phys. 1993, 73, 2705; (h) Fernadez, U.; Fischer, T. M.; Knoll, W. Opt. Commun. 1993, 102, 49; (i) Aust, E. F.; Sawodny, M.; Ito, S.; Knoll, W. Scanning 1994, 16, 353; (j) Lawall, R.; Knoll, W. Opt. Commun. 1995, 115, 265 (k) Knoll, W. in Handbook of Optical Properties, Vol. II, Optics of Small Particles, Interfaces and Surfaces, (eds.) Hummel, R. E.; Wißmann, P. 1997, 373; (l) Bock, H.; Christian, S.; Knoll, W.; Vydra, J. Appl. Phys. Lett. 1997, 71, 3643; (m) Knoll, W. Annu. Rev. Phys. Chem. 1998, 49, 569; (n) Kano, H.; Knoll, W. Opt. Commun. 1998, 153, 235. 27 Ramsden, J. J. J. Statistical Phys. 1993, 73, 853. 28 (a) Adams, M.J. An Introduction to Optical Waveguides John Wiley & Sons, 145-155, 1981; (b) Tiefenthaler, K.; Lukosz, W. J. Opt. Soc. Am. B 1989, 6(2), 209; (c) Lee, D.L. Electromagnetic Principles of Integrated Optics John Wiley & Sons, 116-127, 1986. 29 (a) Kretschmann, E.; Raether, H. Z. Naturforsch. 1968, 23, 2135; (b) Kretschmann, E. Opt. Commun. 1972, 6, 185.

27

Tailoring of Film Properties by Variation of Plasma Deposition Parameters

&KDS WHU

Tailoring of Film Properties by Variation of Plasma

∗ Deposition Parameters

4.1 Introduction

The surface modification of chemically inert materials based on gas plasma treatment is an important field of research for applications ranging from protective coatings, biomaterials and electronic devices.1,2 For many applications, amine rich surfaces are of particular interest, because they are known to influence protein adsorption and cell adhesion, and provide sites for the covalent immobilization of graft polymers and biomolecules.3 There are basically two plasma-assisted processes used for the introduction of amino groups onto a surface. In the first process, a surface is exposed to the plasma of a non-polymerizable gas such as pure nitrogen or ammonia,4 leading to surfaces containing amino groups, amides and imines. The second approach is the plasma-assisted deposition of an amino group containing organic monomer.3b,c,5 This technique enables the fabrication of adhesive, pin-hole free films on a large range of substrates in a solvent-free, single-step process.

Many different organic compounds can be used as a monomer for plasma polymerization. By careful selection of the process conditions, it is possible to produce thin films with a great variety in chemical structure and physical properties. The molecules of the precursor are excited in the rf discharge and can undergo polymerization reactions in the gas phase and on the solid substrate. Due to the complex nature of the plasma deposition process however, the functional groups of the monomer are often lost during polymerization. Several reports have placed emphasis on improving the control over the film composition by variation of process parameters, such as substrate location,6 substrate temperature,7 peak power,3c,8 monomer flow or process pressure.9 Other studies have shown good results by using a pulsed plasma instead of the traditional continuous wave (CW) plasma. Timmons and co-workers

∗ Part of the work described in this chapter has been published or will be submitted for publication: van Os, M. T.; Menges, B.; Förch, R.; Vancso, G. J.; Knoll, W. Chem. Mater. 1999, 11, 3252; van Os, M. T.; Menges, B.; Timmons, R. B.; Knoll, W.; Förch, R. ISPC-13 Symp. Proc. 1997, 3, 1298; Menno T. van Os, Renate Förch, Richard B. Timmons, G. Julius Vancso, and Wolfgang Knoll; Plasma Polymerization of Amine Functionalized Monomers; Tailoring of the Film Chemistry and Surface Aging, to be submitted. Chapter 4

reported large progressive changes in the film chemistry with variations of the radio frequency (rf) duty cycle10 for a number of organic precursors.11

In this chapter we compare plasma-polymerized coatings from a number of different monomers containing amine functional groups. The selected monomers vary in amino group content, as well as in chemical structure. The results obtained show that these monomers enable us to fabricate amine functionalized plasma polymers with a broad range of surface properties due to variation of the functional group density at the surface. This work also gives an overview of the various techniques that can be employed to tailor the plasma film chemistry. Emphasis is placed on variation of the rf duty cycle during plasma polymerization as a tool to optimize the surface properties.

4.2 Plasma Reactor

Figure 4.1 provides a schematic diagram of the home-built apparatus employed for all the plasma depositions described in this work.11b,12 The reaction chamber consists of a Pyrex glass cylindrical tube, 30 cm in length and 10 cm in width. Gases fed through the system pass two cold traps, cooled with liquid nitrogen, for collection of excess reactant before reaching the pump (Leybold Trivac, D16BCS / PFPE).

Multigas Baratron Controller

Pressure Electrodes Transducer

Sample Gas Flow Holder Butterfly Controller Valve

To Vacuum Pump Matching Monomers Network O 2 N2 Ar Bidirectional Wattmeter RF Amplifier Faraday Coupler Cage RF Generator Oscillocope Pulse Generator Figure 4.1 Schematic diagram of the plasma reactor and the electrical components.

30 Tailoring of Film Properties by Variation of Plasma Deposition Parameters

Side arms at the reactor inlet allow the introduction of non-polymerizable gases such as oxygen, nitrogen and argon, and the monomer vapors. The flow rate of the O2, N2 and Ar is controlled by MKS gas flow meters (type 1259C), while that of the monomer vapor is controlled by a Kobold floating ball flowmeter. A MKS baratron (type 122) is also connected to one of the inlets, to monitor the reactor pressure. A butterfly valve at the other reactor end is used in conjunction with the pressure transducer, to allow accurate control over the process pressure. The flow meters, the baratron and the butterfly valve are controlled by a central multigas controller (MKS type 647B). A Tektronix PG 501 pulse generator controls the pulsing of the radio frequency signal, obtained from a 20 MHz function generator (Wavetek model 166). The signal is amplified by an ENI 300 W amplifier, and passed via a bi- directional coupler (BIRD 4266), an analogue wattmeter (BIRD 4410A) and a matching network to the electrodes, consisting of two external concentric metal rings with a spacing of 10 cm. An oscilloscope, connected to the bi-directional coupler, was used to tune the rf circuit, by minimizing the reflected power.

The geometry of the two plasma reactors that were used at the Max-Planck-Institut für Polymerforschung is in principle the same as the reactor that was used at the University of Texas at Arlington. However, even the smallest differences in geometry, the presence of very small leaks, or differences in monomer quality can result in relatively large differences in deposition rate or film chemistry, even between films that are polymerized ‘at the same conditions’. Therefore each figure and table in this thesis contains only results from experiments carried out in one and the same reactor.

4.3 Influence of Plasma Parameters on Film Chemistry and Deposition Rate

In most of the experiments described in this Thesis, allylamine was chosen as the monomer for the plasma polymerization. The main motivation behind this choice is the presence of the double bond in this compound. It is believed that the reaction mechanisms occurring during the plasma deposition of an unsaturated monomer like allylamine are more selective in nature than for saturated monomers. A thorough preliminary study was undertaken to investigate the influence of the different process parameters on the resulting film structure and chemistry. This knowledge could then be used to tailor the film characteristics for specific applications of these films.

4.3.1 INFLUENCE OF THE MONOMER FLOW

Water contact angle measurements reveal progressive changes in the hydrophilic character of the pulsed plasma polymerized allylamine films with variations of the monomer flow. This is illustrated in Figure 4.2, where both the deposition rate, and the advancing (θa), sessile (θs) and receding water contact angle (θr) are shown for runs carried out at different allylamine gas flows.

31 Chapter 4

70

7 θ a 60 θ s θ 6 r 50

5 40

4 30

20 3 Water contact angle [°] angle Water contact Deposition rate [nm/min] 10 2

0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Gas flow [sccm]

Figure 4.2 Deposition rate (left) and water contact angles (right) as a function of the monomer flow for 13 the pulsed plasma deposition of allylamine (Ppeak = 200 W, ton = 10 ms, toff = 100 ms).

It can be seen clearly that the deposition rate decreases with increasing monomer flow. This effect has also been observed by others.1,3c The power absorbed by the gas due to the rf field is inversely proportional to the collision frequency of the electrons with the allylamine molecules. At higher gas flows, the electron mean free path decreases, so that less active species can be generated, when all other variables are being kept constant. The decrease in the plasma light intensity with increasing flow at constant power is also an indication that the energy of a plasma is greatly influenced by the gas flow. In similar fashion, the change in the water contact angles with varying gas flow can be explained. At low pressures, a relatively large amount of reactive species is generated in the glow discharge. This leads to a high degree of cross-linking in the plasma polymer, as well as significant fragmentation of the monomer functional groups. Less amines are retained in the film, thus accounting for the more hydrophobic character of those films.

A remarkable feature of the amine functionalized plasma films studied in this work is the high hysteresis between the advancing and receding water contact angle observed on these films. In Figure 4.2 for instance at the lowest gas flow, the difference between θa and θr is almost 55 degrees. The most likely explanation for this behavior is based on the rearrangement of functional groups at the surface. In general, the surface of a polymer tends to restructure and reorient when brought in contact with a different environment, because of the thermodynamic drive to minimize interfacial tension.14 Extremely large contact angle hysteresis has also been found by Yasuda and co-workers on a number of plasma polymer surfaces, and was associated with the reorientation of hydrophilic moieties of polymer molecules at the surface.15 In air the polar amino groups tend to move away from the hydrophobic environment while hydrocarbon segments are attracted to the interface, resulting

32 Tailoring of Film Properties by Variation of Plasma Deposition Parameters

in relatively high advancing water contact angles. Upon wetting however, the amino groups and other polar groups move to the liquid-solid interface to minimize their free energy in the aqueous environment.

200 [nm/min] 70 [mÅ/J] 180 60 160

140 50 120 θ a 40 θ 100 s θ r 80 30

Deposition rate Deposition 60 20

40 Water contact angle[°]

20 10

0 0 0 50 100 150 200 0 50 100 150 200 Power [W]

Figure 4.3 Deposition rate (left) and water contact angles (right) as a function of the rf power for the continuous wave plasma deposition of allylamine.

4.3.2 INPUT POWER VARIATION UNDER CONTINUOUS WAVE (CW) CONDITIONS

In a series of CW runs, the influence of the power on the deposition rate and the hydrophilicity was studied using allylamine as a monomer (Figure 4.3). The deposition rate is expressed as an absolute value (nm/min), as well as relative to the energy input (mÅ/J). The absolute deposition rate was seen to steadily increase with increasing power. However, the relative deposition rate shows an opposite dependence on the power input; at low powers the film growth per unit energy input is higher than at high powers.

Several factors have been previously suggested to explain dynamic differences between the film formation processes occurring at high and low power input conditions.11a,g With respect to the deposition rate, the charging of the substrate plays an important role. At high CW power conditions, the substrate becomes negatively charged. This potential enhances high-energy collisions of positively charged particles in the plasma with the surface. This is believed to lead to ablation processes, which have a negative effect on the deposition rate.2 Another important factor is the generation of short-wavelength photons in the plasma under high power conditions, which are known to induce photodecomposition of plasma films.16 Finally, the substrate temperature may influence the deposition rate during polymerization.11g For obvious reasons, the temperature increases with increasing power, thereby negatively affecting the polymerization rate. Although it is difficult to determine the contribution of each of these factors on the chemistry and selectivity during the film

33 Chapter 4

formation, the overall influence of the power input is clearly demonstrated in Figure 4.4, which shows the FT-IR transmittance spectra of four films polymerized from allylamine at different CW power conditions. A summary of the mode assignments and their wavenumbers of the functional groups that can be expected in this type of films3b,17 is given in Table 4.1.

-1 -1 -1 3360 cm 2930 cm 2185 cm 1625 cm-1 -1 -1 1455 cm 2875 cm 1370 cm-1 -1 2960 cm 1100 cm-1 4W

10W

50W

200W Transmittance [a.u.] Transmittance

4000 3500 3000 2500 2000 1500 1000

Wavenumber [cm-1]

Figure 4.4 FT-IR transmission spectra as a function of the rf power for the continuous wave plasma deposition of allylamine.

The broad band at 3360 cm-1 mainly results from amine groups. With decreasing input power, the intensity of this peak steadily increases relative to the intensity of the multiple absorption peak at 2960, 2930 and 2875 cm-1, which originates from the C-H stretching of aliphatic groups. This suggests that in the low energy plasma polymerization the amine functionality is retained to a greater extent than in the high energy process, which is confirmed by the sharp decrease of the water contact angles with decreasing power in Figure 4.3.

It has been reported previously that during the plasma polymerization of amine functionalized gases, the primary amino groups have a high tendency to be transformed into imine or nitrile groups.5a-c Elimination of nitrogen fragments has also been observed previously during the plasma polymerization of allylamine and related monomers.6a Both this elimination process and the tendency of transformation were seen to increase with increasing power.6a These results are in good agreement with the changes in the relative IR peak intensities observed in this work. The band at 2185 cm-1 can be associated with the presence of nitrile groups (C≡N stretching) or alkyne groups (C≡C stretching) in the film. The intensity

34 Tailoring of Film Properties by Variation of Plasma Deposition Parameters

of this peak progressively increases with increasing plasma power. If we would assume that this peak is mainly due to alkynes, the increase in intensity with increasing power would be accompanied by a relative decrease in the peaks due to the C-H stretching (2960, 2930 and 2875 cm-1) and the C-H deformations (1455 and 1370 cm-1) of aliphatic groups. Since the opposite is the case, we assume that this peak is mainly due to nitrile groups. The fact that at the same time the N-H absorption peak (3360 cm-1) decreases with increasing power, leads us to believe that abstraction of hydrogen atoms from amino groups is the most important reason for the low chemical selectivity achieved during plasma polymerization at high power input conditions.

Table 4.1 Frequencies and IR band assignments of possible functional groups in plasma polymerized films from allylamine. Frequency (cm-1) Assignmentsa Functional group

3360 νs and νas NH2 Primary amine and amide ν NH Secondary amine and amide

2960 νas CH3 Methyl 2930 νas CH2 Methylene

2875 νs CH3 Methyl 2185 C≡C Alkyne ν C≡N Nitrile 1625 C=O Primary and secondary amide

NH2 Primary amine and amide NH Primary and secondary amide C=C Alkene ν C=N Imine

1455 δas CH3 Methyl

δs CH2 Methylene

1370 δs CH3 Methyl 1100 ν C-N Primary amine a ν = stretching; δ = bending; s = symmetric; as = antisymmetric

A well-known complication with amino group containing plasma polymers is the incorporation of oxygen in the films upon exposure to air. This spontaneous aging process is believed to be the result of the reaction of residual free radicals in the plasma film with ambient oxygen, and has been investigated extensively by Griesser and co-workers.5a-c,18 They found this oxidation reaction to predominantly take place at the nitrogen-carrying carbon atoms, resulting in conversion from amines to amides. Although the samples are measured by FT-IR spectroscopy within ten minutes after deposition, the broad absorption band at 3360 cm-1 most probably also contains contributions from amide groups. The peak around 1625 cm- 1 partly originates from the C=O stretching of amides, but assessment is hindered due to possible contributions of amines, alkenes and imines. The changes that these films undergo upon aging will be addressed further in Chapter 5. It should be noted however that the

35 Chapter 4

functional groups listed in Table 4.1 are therefore not complete. Plasma polymerized allylamine films contain many more groups that are not directly associated with the monomer, like carbonyls, carboxyls, imides, hydroperoxides etc. These can be formed by many combinations of monomer fragmentation in the reactor during deposition, in combination with above-mentioned post-plasma oxidation reactions of the films.

4.3.3 PULSED PLASMA: VARIATION OF THE PLASMA-ON TIME

A number of pulsed plasma experiments were carried out with allylamine, in which the plasma-on time ton was varied while all other process variables were kept constant. In order to compare pulsed plasma experiments with runs carried out under CW conditions, the equivalent power

can be used.

= Ppeak ⋅ ton/(ton + toff) (4-1) where Ppeak is the peak power. The ratio ton/(ton + toff) is also referred to as the duty cycle.

120 [nm/min] 70 [mÅ/J]

100 60

50 80 θ 40 a θ 60 s θ 30 r 40

Deposition rate 20 Water contact angle [°] angle contact Water 20 10

0 0 0 50 100 150 200 0 50 100 150 200 Equivalent power [W]

Figure 4.5 Deposition rate (left) and water contact angles (right) as a function of

for the pulsed plasma deposition of allylamine with ton varying from 1 to 100 ms (Ppeak = 200 W, toff = 100 ms).

Figure 4.5 shows the changes in deposition rate and in water contact angles with increasing ton. When we compare the deposition rates per unit energy input of these runs with the deposition rates under CW conditions (Figure 4.3), we observe a very different trend. Under CW conditions, the deposition rate (mÅ/J) decreases with increasing power, whereas the deposition rate under pulsed plasma conditions increases with increasing

. This reflects the significance of the competing mechanisms of ablation and deposition generally known to take place in plasma polymerization processes. During the CW process the ablation reactions appear to play an increasingly important role as the input energy is increased,

36 Tailoring of Film Properties by Variation of Plasma Deposition Parameters

leading to a decrease of the relative deposition rate. In the pulsed experiments, the ablation reactions only take place during the plasma-on times, since during the plasma-off times the reactive species are rapidly quenched from the reactor chamber. For this reason the negative effect of ablation processes on the deposition rate is less pronounced than in the CW runs. As the plasma-on time is increased however, the influence of the quenching during the plasma- off time diminishes, and ablation processes become more dominant again. Eventually, the deposition rate per unit energy input equals that of the CW run (ton → ∞), which is shown in Figure 4.5 as the point connected by the dotted line. The changes in the chemical structure of the films are reflected by the increasing hydrophobicity of the film surface with increasing plasma-on time.

100 ms

50 ms

25 ms

10 ms

1 ms Transmittance [a.u] Transmittance

4000 3500 3000 2500 2000 1500 1000

Wavenumber [cm-1] Figure 4.6 FT-IR transmission spectra as a function of the plasma-on time for the pulsed plasma deposition of allylamine (Ppeak = 200 W, toff = 100 ms).

Figure 4.6 shows the FT-IR spectra of films polymerized at five different plasma-on times. The trend observed is similar to that in Figure 4.4; a progressive increase in the retention of the monomer’s amino group content with decreasing plasma-on time (e.g., decreasing

). However, one major advantage of utilizing a pulsed instead of a CW plasma becomes clear if we look carefully at the runs at the lowest energy inputs (4 W CW and 1/100 ms/ms pulsed). During the CW experiments, the minimum power that was required to maintain a glow discharge in the reactor was 4 W. During the pulsed experiments, a glow discharge can be maintained at equivalent powers much lower than 4 W. Even if the plasma- on time is as low as 1 ms, the applied power during this period is still 200 W. This short on time is sufficient to initiate the glow discharge under these conditions, and it can be seen that the intensity of the absorption band at 3360 cm-1 (amines) in the spectrum of the film polymerized at 1/100 ms/ms (

= 2 W) becomes so high that it overlaps the bands around

37 Chapter 4

2900 cm-1 (aliphatic groups). One could also argue that the increasing broadness of the band around 3360 cm-1 is due to an increasing number of functionalities associated with this band. However, this is very unlikely if the increasing hydrophilicity of the surface with decreasing ton is considered, together with the decreasing intensity of the nitrile band.

a) 0.43 nm b) 0.40 nm

c) 0.35 nm d) 0.25 nm

Figure 4.7 AFM images of pulsed plasma polymerized allylamine (Ppeak = 200 W, toff = 100 ms) with surface rms roughnesses. The rf duty cycle was varied by changing the plasma-on time from a) 100 ms, b) 25 ms, c) 10 ms to d) 1 ms.

The influence of the plasma-on time on the morphology has also been investigated by AFM measurements. The images in Figure 4.7 reveal a decrease in the average surface roughness as the plasma-on time is decreased. The surface root mean square (rms) roughness in these series of films polymerized from allylamine decreased from 0.43 nm at ton = 100 ms, to 0.25 nm at ton = 1 ms. These morphological changes with changing duty cycle have been observed also for other monomers.11g,19 The absence of high-energy surface bombardment during the majority of the film formation process accounts for part of the decreased surface roughness at lower plasma-on times. Additionally, the decreased surface roughness at lower

38 Tailoring of Film Properties by Variation of Plasma Deposition Parameters

duty cycles may also reflect the elimination of film radiation damage and lower substrate temperatures during deposition.

4.3.4 PULSED PLASMA: VARIATION OF THE PLASMA-OFF TIME

In a similar way as discussed in §4.3.3, longer plasma-off times also lead to higher selectivities during the plasma deposition process. Many of the reactive species created during the plasma-on periods disappear during the plasma-off periods, thus allowing the film formation to take place under less energetic conditions at long off times. The deposition rates per unit energy input for four different monomers are plotted in Figure 4.8. Starting at the data points at zero plasma-off time, which represent CW runs at 200 W, the deposition rate increases with increasing plasma-off time (decreasing

) for all of the monomers investigated. A further increase of the off time results in a decrease of the relative deposition rate, after reaching a maximum. Similar behavior has been observed previously for other monomers plasma polymerized under pulsed conditions.11b Complete disappearance of radical species from the reactor gas has been given as a possible explanation, which has a negative influence on the coupling of the subsequent plasma-on pulse to the reactor gas.

180 Cycloheptylamine 160 Allylamine n-Butylamine 140 Methylamine 120

100

80

60

Deposition rate [mÅ/J] rate Deposition 40

20

0

0 50 100 150 200 Equivalent power [W]

Figure 4.8 Deposition rate as a function

with toff varying from 0 ms (CW) to 117 ms (Ppeak = 200 W, ton = 3 ms) for four different monomers.

With respect to the deposition rates of the different monomers, it can be seen as expected, that the unsaturated allylamine and the cyclic cycloheptylamine compound are on average more readily polymerizable than the saturated monomers. The lowest deposition rates were found for methylamine.

Table 4.2 gives an overview of all the amino group containing monomers that were investigated in this work, as well as some XPS data and water contact angles of a selection of films polymerized from these monomers. The monomers were chosen in such a way that the

39 Chapter 4

nitrogen over carbon ratio (N/C) varied over a large range, from 0.14 (n-heptylamine and cycloheptylamine) to 1.00 (ethylenediamine and methylamine).

Table 4.2 Surface atom concentrations, N/C ratios and water contact angles of films polymerized from seven different monomers with amine functionality. All depositions were carried out at a peak power of 200 W.

ton/toff C1s N1s O1s N/C θa θs θr [ms/ms] [%] [%] [%] [°] [°] [°] n-Heptylamine (N/C = 0.14) 3/15 82.3 7.5 10.2 0.09 98 89 21 3/45 76 54 14 Cycloheptylamine (N/C = 0.14) 3/3 767333 3/57 81 74 19 n-Butylamine (N/C = 0.25) CW 72 66 23 3/30 74 68 8 Allylamine (N/C = 0.33) 3/5 71.2 21.9 6.9 0.31 63 59 7 3/45 63.2 29.1 7.7 0.46 28 23 6 1,2-Diaminopropane (N/C = 0.67) CW 74.3 23.6 2.1 0.32 46 16 10 3/45 63.4 34.2 2.5 0.54 13 9 7 Ethylenediamine (N/C = 1.00) CW 19 13 9 3/45 15 10 8 Methylamine (N/C = 1.00) 3/5 60.0 33.7 6.3 0.56 14 10 6 3/30 57.1 34.9 8.0 0.61 11 7 5

The structure of the monomer has a large effect on the types of active species that are formed in the glow discharge, and thus on the structure of the resulting plasma polymer. For instance, when allylamine is used as a monomer, it may be expected that a major part of the polymerization is initiated by allyl radicals that are formed upon excitation in the plasma. Similarly, one would expect a ring opening polymerization to be the dominant process occurring during the plasma deposition of cycloheptylamine.

It is obvious that the success of controlling the film chemistry by variation of the rf duty cycle when a pulsed plasma is utilized, depends largely on the monomer structure. In the case of allylamine plasma polymerization, the plasma-off chemistry can be visualized primarily as polymerization of the allylamine through free radical additions to the carbon- carbon double bond, and thus to a great extent retaining the –NH2 functional groups in the resulting films: H H

H2C CH H2CCH CC

CH2 CH2 H CH2

NH2 NH2 NH2 n

40 Tailoring of Film Properties by Variation of Plasma Deposition Parameters

As has been shown above, the controllability of the film chemistry is indeed remarkable with this monomer, and can be easily achieved by variation of a number of different plasma parameters (§4.3.1-4.3.3).

Despite the excellent film chemistry control achieved with allylamine, we found that the advantage of utilizing the pulsed plasma approach is not nearly as advantageous for some of the other monomers. When saturated materials such as n-heptylamine or n-butylamine were used as monomer, FT-IR spectra revealed only small differences for films polymerized at different power input conditions. Also the range over which the duty cycle could be varied was smaller than with allylamine. Stable n-heptylamine plasma films for instance were obtained at ton/toff = 3/15 ms/ms (Ppeak = 200 W), but increasing the off time to 45 ms results in an oily deposit rather than an adhesive and stable film. The reason for this is the absence of a double bond or other reactive bond in the monomer. The binding energy of a single C-C bond is significantly higher than that of a double C=C bond, so a higher

is required to activate this molecule. Since relatively high power conditions are necessary to obtain a good film quality, fragmentation of the monomer functional amino groups is also more likely to occur, which makes it more difficult to control the retention of these groups in the plasma film.

Another limitation that was encountered for some of the investigated monomers, was the very low deposition rate at high plasma-off times. In the case of the plasma deposition of methylamine, the deposition rate at 3/30 ms/ms (Ppeak = 200 W) was found to be only 1 nm/min. Increasing the off time to 90 ms resulted in a further decrease of the deposition rate to 0.07 nm/min (15 times less than in the case of allylamine under the same conditions).

The molecular tailoring of surfaces when these other monomers are used, is apparently not nearly as successful as with allylamine. Despite this fact, from Table 4.2 it is clear that this whole set of chemicals allows us to tune the hydrophilicity of the surfaces over a very broad range. Provided that the process parameters for each individual monomer are chosen in such a way that stable and adhesive films are produced, the N/C ratio at the surface can be controllably varied from ± 0.1 to ± 0.6. Extremely hydrophilic surfaces could be produced when methylamine was polymerized, on which θa was seen to be as low as 11°. On the other hand, the plasma polymerization of n-heptylamine surfaces resulted in relatively hydrophobic surfaces (with θa as high as 98°), however still bearing amino- and other nitrogen containing groups. This variation in film chemistry is reflected in Figure 4.9 as well, where the FT-IR spectra of the different monomers are plotted. The spectra are normalized with respect to the peak at 1625 cm-1.

It can be seen from this figure that the contribution of aliphatic groups diminishes as the N/C content of the monomer is reduced. Although XPS revealed that the N/C content of the films polymerized from methylamine (a) and 1,2-diaminopropane (b) was typically higher than that of plasma polymerized allylamine at comparable duty cycles, the broadness of the peak around 3360 cm-1 suggests that a major part of the nitrogen containing functional groups

41 Chapter 4

may be present in a form other than amino groups in films polymerized from these monomers with the highest N/C ratio. The peak around 2185 cm-1 that is associated with nitrile groups is also much smaller in the allylamine spectrum, confirming the previous mentioned hypothesis that the unsaturation in the monomer allows for a more orderly polymerization mechanism to occur.

a) b) c) d) e) f) g) Transmittance [a.u.]

4000 3500 3000 2500 2000 1500 1000 Wavenumber [cm-1]

Figure 4.9 FT-IR transmission spectra of pulsed plasma polymerized films from seven different monomers (Ppeak = 200 W, ton = 3 ms, 15 ms ≤ toff ≤ 20 ms): a) methylamine; b) ethylenediamine; c) 1,2- diaminopropane; d) allylamine; e) n-butylamine; f) cycloheptylamine and g) n-heptylamine.

4.3.5 PULSED PLASMA: PLASMA-ON- AND OFF TIME VARIATION AT CONSTANT DUTY CYCLE

In another series of experiments, the plasma-on and off times were varied in such a way that the duty cycle remained constant. Even though the equivalent power input during these runs was constant, remarkable differences were observed in both deposition rate and the surface energy of the films with varying plasma-on and off times (Figure 4.10).

The deposition rate reaches the highest value at ton = 0.1 ms and toff = 1 ms (± 8.0 nm/min). The plasma-on time is apparently long enough to establish a glow discharge, but the plasma-off time is too short in absolute terms to effectively remove all the reactive species from the reactor chamber. This explanation is supported by the appearance of the plasma; the pulsing can not be observed by eye, but the glow discharge looks similar to a low energy CW plasma. At ton = 25 ms and toff = 250 ms, the deposition rate reaches a minimum of 2.9 nm/min. Increasing the plasma-on and off time even further to ton = 50 ms and toff = 500 ms

42 Tailoring of Film Properties by Variation of Plasma Deposition Parameters

results in an increase of the deposition rate again. The plots for the advancing, sessile and receding water contact angles show a similar trend as the deposition rate; the most hydrophilic surface is produced at ton = 10 ms and toff = 100 ms. Apparently, the pulsed plasma deposition process of allylamine is a trade-off between a high deposition rate and a high density of polar groups on the surface of the plasma coating.

9

50 8

7 40

6 30 θ a θ 5 s θ 20 r 4 Deposition rate [nm/min] rate Deposition Water contact angle [°] angle contact Water 10 3

2 0 0 1020304050 0 1020304050 Plasma-on time [ms] Figure 4.10 Deposition rate (left) and water contact angles (right) of pulsed plasma polymerized allylamine as a function of ton. The plasma-on and off times were both varied while the duty cycle was kept constant at 1/11 (Ppeak = 200 W,

= 18 W).

4.3.6 INFLUENCE OF THE DEPOSITION TIME ON THE FILM STRUCTURE

Film properties were studied using WaMS on plasma polymerized allylamine films, 12a deposited at of ton/toff = 10/90 and 10/190 ms/ms. For both of these duty cycles, films were deposited onto waveguide chips for different deposition times. For both duty cycles the film thickness increased linearly with the deposition time (Figure 4.11). At the highest duty cycle

(ton/toff = 10/90, Figure 4.11-a), the refractive index n obtained from measurement of the TE0 and TM0 mode, was seen to increase from 1.48 at a film thickness of 4 nm, to a value of 1.56 at a thickness of 129 nm. At a lower duty cycle (ton/toff = 10/190, Figure 4.11-b) a similar trend was observed: an increase in n from 1.40 for the thinnest film (5 nm), to 1.55 for the thickest film (74 nm). It must be noted that the error bars that are shown in these figures are based on the WaMS experiments only, and do not take into account any errors introduced by the plasma deposition process. In both cases, starting at low deposition times, the refractive index increased rapidly with increasing deposition time, to eventually level off and reach a maximum value.

The measured refractive indices may give indications regarding the degree of cross- linking within the film during the early stages of the deposition mechanism. Low refractive

43 Chapter 4

indices could be taken to indicate a lower density, less intermolecular bonding and a lower cross-link density within the polymer film. The change in refractive index observed with increasing deposition time and film thickness could thus be taken to suggest structural changes occurring within the polymer film as the film grows. It could thus be envisaged that as material is depositing at the solid/gas interface, the underlying bulk material is continuing to undergo considerable structural changes.

a) 140 1.62

Thickness 120 1.60 Refractive index

1.58 Refractive index 100 1.56 80 1.54 60 1.52

Thickness [nm] Thickness 40 1.50

20 1.48

0 1.46 024681012 Deposition time [min]

b) 1.60 80 Thickness 1.58 70 Refractive index 1.56 1.54 60 index Refractive 1.52 50 1.50 1.48 40 1.46 30 1.44 Thickness [nm] Thickness 20 1.42 1.40 10 1.38 0 1.36 0246810121416182022 Deposition time [min]

Figure 4.11 Thickness and the increase of the refractive index ∆n of plasma polymerized allylamine after varying deposition times, a) ton = 10 ms, toff = 90 ms, Ppeak = 200 W, and b) ton = 10 ms, toff = 190 ms, Ppeak = 200 W.

Possible structural changes may be due to intermolecular reactions, initiated by the UV light emitted during the plasma-on times, or due to cross-linking and molecular rearrangement within the network during the plasma-off times. As the molecules rearrange within the bulk, a more densely packed polymer network results which is assumed to be

44 Tailoring of Film Properties by Variation of Plasma Deposition Parameters

reflected in the higher refractive indices measured. At high duty cycles, the equivalent power per cycle is greater than during the low duty cycle deposition, which results in a higher initial refractive index at short deposition times. Such a model could imply that the uppermost layers of a plasma polymer near the solid/gas interface have a lower cross-link density and probably show a lower refractive index. Below this interface layer the underlying bulk material continues to react, to form a more densely packed network with a higher refractive index. As the overall film thickness increases, the influence of the refractive index of the bulk material eventually dominates the total average refractive index measured by WaMS, leading to negligible further index changes at greater thicknesses.

Finally, Figure 4.12 shows the refractive indices of PPAA films polymerized at five different duty cycles, plotted as a function of the equivalent power. The thickness of each of these films was greater than 70 nm by using longer deposition times at lower duty cycles. Variations in refractive index between films deposited at different duty cycles were seen to remain within the experimental error of the data.

1.57

1.56

1.55

1.54

Refractive index Refractive 10 / 90 6.5 min 1.53

10 / 190 10 / 40 10 / 15 10 / 5 20 min 3 min 1.8 min 1 min 1.52 0 20406080100120140 Equivalent power [W] Figure 4.12 Refractive index as a function of the equivalent power for pulsed plasma polymerized allylamine films of similar thickness (d > 70 nm), deposited at 200 W at different duty cycles. The deposition time and duty cycle for each sample are also given.

4.4 Conclusions

Plasma assisted polymerization of allylamine and six other amine-functionalized monomers was successfully established. The different concentrations of nitrogen in the monomers resulted in N/C ratios on the plasma polymer surfaces ranging from 0.1 (n- heptylamine) to 0.6 (methylamine). The surface properties could be controlled by variation of deposition parameters such as monomer flow, peak power, plasma-on time and plasma-off

45 Chapter 4

time. The amino group functionality of the monomer was increasingly retained in the plasma polymer upon decreasing the average power input conditions. The N/C ratio could be controllably varied from 0.09 to 0.61 by variation of the monomer and the deposition conditions, while the advancing water contact angles accordingly decreased from 89 to 11 degrees. The best film composition controllability was achieved using allylamine as a monomer, due to the presence of a double bond in the monomer structure. A large contact angle hysteresis was found on all the surfaces examined in this thesis, most probably resulting from reorientation of the functional groups upon exposure to different environments. WaMS experiments indicated that the refractive index of PPAA was a function of the deposition time. This possibly indicates the presence of a refractive index gradient in the films. The bulk of the material, which has been subjected to the glow discharge for a longer period of time than the surface layer, may have a higher degree of cross-linking and therefore a higher refractive index.

4.5 Experimental Section

4.5.1 SAMPLE PREPARATION

The films were deposited either on 10 x 20 mm2 pieces of silicon or KBr windows for FT-IR analysis, microscope glass slides for water contact angle measurements, 5 x 5 mm2 silicon wafers for XPS or AFM analysis, or on waveguide chips for the WaMS experiments.

A TiO2 / SiOx film (dF ≈ 168 nm, nF ≈ 1.82, Λ = 417 nm) on a glass substrate was used as the waveguide chip in the measurements of the plasma polymerized allylamine against air. The exact refractive index and thickness of the waveguide were determined before each deposition by a measurement against air. All substrates were cleaned in an argon/oxygen (9/1) plasma for 10 minutes before deposition of the allylamine. All other substrates were cleaned in ethanol under ultrasound prior to plasma polymerization.

4.5.2 PLASMA POLYMERIZATION

The n-heptylamine, cycloheptylamine, n-butylamine, allylamine, 1,2-diaminopropane, ethylenediamine and methylamine were obtained from Aldrich Chemical Co. All these chemicals were outgassed by multiple freeze-thaw cycles before use, but not subjected to any further purification. Reactions were carried out in an all-glass cylindrical reactor (Figure 4.1). The 13.56 MHz rf power was delivered to the reactor through two concentric electrodes located around the exterior of the reactor. A detailed description of the system is provided in §4.2. To compare the total consumed power during pulsed experiments with the power conditions during CW experiments, an equivalent power

is used in some cases.20 The substrates were placed on a glass substrate holder in the center of the reaction chamber between the two electrodes. Monomer flow (8 sccm), process pressure (0.2 mbar), and sample position within the reactor were held constant (between the two electrodes) in all experiments,

46 Tailoring of Film Properties by Variation of Plasma Deposition Parameters

unless noted otherwise. The deposition time tdep, plasma-on time ton, plasma-off time toff, and the peak power Ppeak were set individually for each experiment. After each deposition, the substrates were removed from the chamber and the reactor was cleaned with an oxygen plasma for 60 min at 200 W.

4.5.3 FILM CHARACTERIZATION

The film compositions of plasma polymerized films were studied using XPS and FT- IR Spectroscopy. The XPS spectra were obtained using a Perkin-Elmer PHI 5000 series instrument equipped with an X-ray source monochromator. The X-ray source was an Al Kα at 1486.6 eV. A pass energy of 17.90 eV giving a resolution of 0.6 eV with Ag(3d5/2) was used. Spectra were recorded using a 70° take-off angle relative to the sample surface. An electron flood gun was employed to neutralize charge build-up on the nonconductive films examined in this work. FT-IR spectra were recorded using a Bruker Vector 22 spectrometer operated at 4 cm-1 resolution. Film thickness measurements were obtained using a Tencor Alpha Step 200 profilometer.21 The surface energy of the films was evaluated using a Ramé- Hart goniometer.22 To minimize the effects of aging, the samples were analyzed within 10 minutes after removal from the reactor unless noted otherwise. The morphology of selected films was studied using a METRIS-2000 atomic force microscope (AFM), which was operated in the non-contact mode. WaMS experiments were carried out using a home built spectrometer (Figure 3.8). A grating with periodicity Λ, incorporated in the waveguide, was used to couple a HeNe laser beam (wavelength λ = 632.8 nm) from the external cover medium into the waveguide. Neff i,j can be determined by scanning the angle of incidence of the incoming laser beam onto the grating, while the in-coupled power is measured by two photodetectors situated at both ends of the waveguide. The waveguide was designed for optimal sensitivity for the zeroth transversal electric (TE0) and transversal magnetic (TM0) modes.

4.6 References

1 Boenig, H. V. Fundamentals of Plasma Chemistry and Technology, Technomic Publishing Co., Inc.: Lancaster, PA, 1988. 2 d’Agostino, R. Plasma Deposition, Treatment and Etching of Polymer Films, Acadamic Press: Boston, MA, 1990. 3 (a) Jui-Che Lin; Cooper, S.L. J. Appl. Polym. Sci: Appl. Polym. Symp. 1994, 54, 157; (b) Fally, F.; Doneux, C.; Riga, J.; Verbist, J. J. J. Appl. Polym. Sci. 1995, 56, 597; (c) Gombotz, W.R.; Hoffman, A.S. J. Appl. Polym. Sci.: Appl. Polym. Symp. 1988, 42, 285. 4 (a) Foerch, R.; Hunter, D.H. J. Polym. Sci.: Part A: Polym. Chem. Ed. 1992, 30, 279; (b) Foerch, R.; Kill, G.; Walzak, M.J. J. Adhes. Sci. Technol. 1993, 7, 1077; (c) Klemberg- Sapieha, J.E.; Küttel, O.M.; Martinu, L.; Wertheimer, M.R. J. Vac. Sci. Technol. 1991, A9, 2975; (d) Lub, J.; van Vroonhoven, F.C.B.M.; Bruinx, E.; Benninghoven, A. Polymer 1989, 30, 40.

47 Chapter 4

5 (a) Gengenbach, T. R.; Chatelier, R. C.; Griesser, H. J. Surf. Interface Anal. 1996, 24, 271; (b) Gengenbach, T. R.; Chatelier, R. C.; Griesser, H. J. Surf. Interface Anal. 1996, 24, 611; (c) Gengenbach, T. R.; Griesser, H. J. J. Polym. Sci.: Part A: Polym. Chem. 1999, 37, 2191; (d) Walker, A.K.; Wu, Y.; Timmons, R.B.; Kinsel, G.R.; Nelson, K.D. Anal. Chem. 1999, 71, 268. 6 (a) Fally, F.; Virlet, I.; Riga, J.; Verbist, J. J. J. Appl. Polym. Sci., Appl. Polym. Symp. 1994, 54, 41; (b) O’Kane, D. F.; Rice, D. W. J. Macrom. Sci. Chem. 1976, A10, 567. 7 (a) Lopez, G. P.; Ratner, B. D. J. Appl. Polym. Sci., Appl. Polym. Symp. 1990, 46, 493; (b) Lopez, G. P.; Ratner, B. D. Langmuir 1991, 7, 766. 8 (a) Fally, F.; Virlet, I.; Riga, J.; Verbist, J. J. J. Appl. Polym. Sci., Appl. Polym. Symp. 1996, 59, 1569; 9 (a) D’Agostino, R.D.; Favia, P.; Fracassi, F. J. Polym. Sci., Polym. Chem. Ed. 1990, 28, 3387; (b) D’Agostino, R.D.; Cramarossa, F; Illuzi, F. J. Appl. Phys.1987, 61, 2754; (c) Ratner, B.D.; Lopez, G.P. J.Polym. Sci.: Polym. Chem. Ed. 1992, 30, 2415; (d) Shard, A.G.; Munroe, H.S.; Badyal J.P.S. Polym. Comm. 1991, 32, 152. 10 The duty cycle is defined as: plasma-on time / (plasma-on time + plasma-off time) 11 (a) Panchalingam, V.; Poon, B.; Huo, H.-H.; Savage, C. R.; Timmons, R. B.; Eberhart, R. C. J. Biomater. Sci. Polymer Edn. 1993, 5, 131; (b) Panchalingam, V.; Chen, X.; Savage, C. R.; Timmons, R. B.; Eberhart, R. C. J. Appl. Polym. Sci.: Appl. Polym. Symp. 1994, 54, 123; (c) Hynes, A.M.; Shenton, M.J.; Badyal, J.P.S. Macomolecules 1996, 29, 4220; (d) Chen, X.; Rajeshwar, K.; Timmons, R. B.; Chen, J.-J.; Chyan, M. R. Chem. Mater. 1996, 8, 1067; (e) Rinsch, C. L.; Chen, X.; Panchalingam, V.; Eberhart, R. C.; Wang, J. H.; Timmons, R. B. Langmuir 1996, 12, 2995; (c) Wang, J.-H.; Chen, J.-J.; Timmons, R.B. Chem. Mater. 1996, 8, 2212; (f) Han, L.M.; Rajeshwar, K.; Timmons, R.B. Langmuir 1997, 13, 5941; (g) Calderon, J. G.; Timmons, R. B. Macromolecules 1998, 31, 3216. 12 (a) van Os, M. T.; Menges, B.; Förch, R.; Vancso, G. J.; Knoll, W. Chem. Mater. 1999, 11, 3252; (b) van Os, M. T.; Menges, B.; Timmons, R. B.; Knoll, W.; Förch, R. ISPC-13 Symp. Proc. 1997, 3, 1298. 13 The plasma-on- (ton) and off time (toff) are discussed in §4.3.3 14 Chan, C.-M. Polymer surface modification and characterization, Hanser/Gardner Publications, Inc., Cincinnatti, OH, 1994. 15 (a) Yasuda, H.; Sharma, A. K.; Yasuda, T. J. Polym. Sci. Polym. Phys. Ed. 1981, 19, 285; (b) Yasuda, H.; Okuno, T.; Yoshida, K.; Yasuda, H. J. Polym. Sci. Polym. Phys. Ed. 1988, 26, 1781. 16 Holländer, A.; Klemberg-Sapieha, J. E.; Wertheimer, M. R. J. Polym. Sci. A 1995, 33, 2013. 17 (a) Li, Z.-F.; Netravali, A. N. J. Appl. Polym. Sci. 1992, 44, 319; (b) Silverstein, R. M.; Webster, F. X. Spectrometric identification of organic compounds, 6th ed.; John Wiley & Sons: New York, 1998. 18 (a) Griesser, H. J.; Chatelier, R. C.; Gengenbach, T. R.; Johnson, G.; Steele, J. G. J. Biomater. Sci. Polymer Edn. 1995, 5, 531; (b) Griesser, H. J.; Chatelier, R. C. J. Appl. Polym. Sci.: Appl. Polym. Symp. 1990, 46, 361. 19 Wang, J.-H.; Chen, X.; Chen, J.-J.; Calderon, J. G.; Timmons, R. B. Plasmas and Polymers 1997, 2, 241. 20 The equivalent power

can be calculated from the equation

= ton/(ton + toff) ⋅ Ppeak, where ton and toff are the plasma-on and plasma-off times, and Ppeak is the peak power. For example, a pulsed run carried out at 200 W peak power and a pulse period of 3 ms on and 45 ms off corresponds to an equivalent power of 12.5 W [i.e. (3/48) ⋅ 200]. The multiplication factor 3/48 [i.e. ton/(ton + toff)] is referred to as the duty cycle.

48 Tailoring of Film Properties by Variation of Plasma Deposition Parameters

21 A metal tipped pen was employed to scribe a thin scratch on films deposited on silicon wafers, after which the film thickness was calculated as the average of measurements at ten different positions for each sample. 22 The sessile contact angle (θs) was determined by placing a drop of water on the surface and measuring the angle between the plane of the surface and the tangent to the drop at the point of contact with the substrate. The advancing contact angle (θa) was measured by inserting the needle of the syringe into the drop, adding water to the drop, and measuring the maximum angle achieved. The receding contact angle (θr) was measured by removing water from the drop and measuring the minimum contact angle achieved.

49

Film Stability and Surface Aging

&KDS WHU

∗ Film Stability and Surface Aging

5.1 Introduction

It has been known since the early days of research on plasma polymers that they are susceptible to oxidation upon storage in air.1,2 Oxygen-free surface layers can be prepared, but most of these layers quickly take up oxygen when brought in contact with air.3 To investigate the applicability of plasma films, many studies have been devoted to this so-called aging process. Since many plasma deposited coatings have shown promising behavior in biomedical and other applications, the film properties at the time of end use is often considered more important than the film characteristics immediately after deposition. A large contribution to a better understanding of oxidative reaction mechanisms occurring during aging of plasma polymers has been made by Griesser and co-workers. XPS, FT-IR spectroscopy and contact angle goniometry has given a good insight in the long-term compositional changes of a number of plasma deposited coatings. Their work includes detailed oxidation studies of hydrocarbon plasma polymers,4 fluorocarbon films,5 nitrogen-containing plasma coatings6 and other plasma deposited films.7 In general, it is accepted that the aging process is initiated by the reaction of ambient oxygen with residual radicals in plasma polymers. Free radicals have been detected in freshly deposited plasma films by ESR spectroscopy, slowly disappearing upon storage in air.1,2 However, the rates, mechanisms and products of oxidative aging depend on many factors, such as the structure of the film, the functional groups that it contains and the mobility of the surface.5,6,8

Another important factor to consider with respect to possible applications of plasma films is the stability of these films when subsequent exposure to a solvent environment is required. Examples of such wet treatments are sterilization procedures for plasma coatings for cell tissue culture studies,9 and the post plasma grafting of monomers on the surface of plasma films.10 A poor surface stability and swelling of plasma polymerized films after solvent treatment has been observed in our laboratory and by others.11

∗ Part of the work described in this Chapter has been published or will be submitted for publication: van Os, M. T.; Menges, B.; Förch, R.; Vancso, G. J.; Knoll, W. Chem. Mater. 1999, 11, 3252; van Os, M. T.; Menges, B.; Foerch, R.; Knoll, W.; Timmons, R. B.; Vancso. G. J. Mat. Res. Soc. Symp. Proc. 1999, 544, 45; Menno T. van Os, Renate Förch, Richard B. Timmons, G. Julius Vancso, and Wolfgang Knoll; Plasma Polymerization of Amine Functionalized Monomers; Tailoring of the Film Chemistry and Surface Aging, to be submitted. Chapter 5

In this chapter, the aging behavior of plasma polymerized allylamine films will be discussed. Changes at the surface were studied by XPS and water contact angle measurements, while changes in the bulk were studied using FT-IR spectroscopy. The results provide evidence that the wettability of these films change remarkably during storage in air, and that the amount of oxygen incorporation is strongly dependent on the plasma deposition conditions. ESR spectroscopy was used to investigate the decay of free radicals in the films. In an effort to understand the swelling behavior of plasma polymerized allylamine films in solvents, their optical properties were monitored during swelling in ethanol. For this purpose, WaMS was used, which enables an independent determination of the film thickness and the refractive index. The effect of ethanol treatment on the composition and the thickness of these films was also studied by XPS, FT-IR spectroscopy, water contact angle measurements and SPR spectroscopy.

5.2 Effect of Solvent Treatment on Plasma Polymerized Allylamine Films

The major focus in this paragraph was to investigate both the chemical and physical effects of solvents on plasma polymerized allylamine (PPAA) films.

5.2.1 EFFECT OF SOLVENT TREATMENT ON FILM COMPOSITION

Figure 5.1 shows the FT-IR spectra of a pulsed plasma polymerized allylamine film, before and after cold extraction in ethanol. Both spectra show absorption bands suggesting the presence of primary and secondary amines (3340 cm-1), and the multiple absorption band due -1 to the CHx bond stretches (2960, 2935 and 2875 cm ).

Before extraction

Absorbance [a.u.] After extraction

4000 3500 3000 2500 2000 1500 1000 Wave number [cm-1]

Figure 5.1 FT-IR absorbance spectra of plasma polymerized allylamine films deposited at 250 W for 10 minutes and a duty cycle of 10/40 ms/ms, before and after 24 hours of cold extraction in ethanol.

52 Film Stability and Surface Aging

At lower wavenumbers, the peak at 2185 cm-1 indicates the presence of nitrile groups, while the broader bands around 1660 cm-1 can be assigned to the bending modes of primary amine groups, and the stretching of imine groups (see §4.3.2 for more details). There are no major changes in the FT-IR spectral profiles before and after extraction of the plasma polymerized allylamine (PPAA) films in ethanol. Only subtle changes in the absolute peak intensities are observed, due to a decrease in film thickness.

XPS data were also obtained on two PPAA films before and after cold extraction (Table 5.1). The surface atom concentrations indicate only slight changes in the surface composition after extraction in ethanol.

Table 5.1 Surface atom concentrations of plasma polymerized allylamine films, before and after 24 hr extraction in ethanol (Ppeak = 250 W, tdep = 10 min). atomic concentration [%] before extraction after extraction carbon nitrogen oxygen carbon nitrogen oxygen CW 70.6 17.4 12.0 70.9 15.8 13.3 10/40 65.9 21.7 12.5 67.4 21.3 11.3

5.2.2 EFFECT OF SOLVENT TREATMENT ON FILM THICKNESS

In order to investigate the influence of a solvent environment on the stability of the PPAA films, SPR experiments were performed before and after cold extraction of the films in ethanol. A refractive index of 1.58 (determined by WaMS) was used to calculate the thickness from the measured reflectivity minima. After each extraction the films were dried in a vacuum oven at 40° C for 2 hours. Figure 5.2-a shows the decrease in film thickness of various films after extraction in ethanol. The film thickness is plotted as a function of the equivalent power

.

It was observed from these experiments that the films that were deposited at lower duty cycles showed a larger relative decrease in film thickness upon extraction (more than 80 % for the film deposited at 10/240 ms/ms), than the films polymerized at higher duty cycles. However, the absolute thickness decrease appeared to be similar for all the films in Figure 5.2-a, which could indicate that the dissolution of PPAA in ethanol could be a surface process rather than a bulk process.

To study this further, similar experiments were performed on a series of films, of which the deposition times were varied in such way that the film thicknesses were of the same order of magnitude after deposition. The results are shown in Figure 5.2-b, and show that, within the first 24 hours of extraction, the relative decrease in thickness is much higher for the film deposited at 10/240 ms/ms (95 %), than for the CW deposited film (55 %). Since the film

53 Chapter 5

thicknesses before extraction were practically the same, the thickness decrease expressed as an absolute value increased with decreasing duty cycle. This indicates that the dissolution process takes place throughout the whole bulk of the film, and not only at the surface. This can be understood if the effect of the rf duty cycle on the degree of cross-linking is considered. At low energy input, only a relatively small amount of radical species is present in the glow discharge and at the surface of the growing film. Because of this, the number of unselective cross-linking reactions occurring in the film is greatly reduced. At the same time this means that, a plasma film polymerized under such conditions contains more low molecular weight material, which can be dissolved and removed during washing with ethanol.

a) 10/15 2500 Before extraction [Å] 80 After extraction [Å] thicknessFilm decrease [%] Thickness decrease [%]

2000 60

10/40 1500

40

1000

Film thickness [Å] Film thickness 10/120 20 500 10/240

0 0 0 20406080100 Equivalent power [W]

100

CW, 3 min, d0=186 nm

10/40, 23 min, d0=176 nm 10/90, 60 min, d =177 nm 80 0

10/240, 164 min, d0=187 nm

60

40

20 Relative film thickness [%] b) 0 0 1020304050 Extraction time [h]

Figure 5.2 a) Film thickness of PPAA films (Ppeak = 250 W, tdep = 5 min.) polymerized at different duty cycles, before and after 24 hours of cold extraction in ethanol and the relative decrease (right axis); b) Relative film thickness of PPAA (Ppeak = 50 W) polymerized at different duty cycles, as a function of the extraction time in ethanol.

54 Film Stability and Surface Aging

If the same films were extracted in ethanol for another 24 hours, no further significant changes in the thickness were observed for all the films. This supports the assumption that, immediately after deposition, the films contain short molecular chains and probably unreacted monomer, but once these have been washed away during the solvent extraction, a stable film remains, that is not affected by further extraction in ethanol.

5.2.3 SWELLING BEHAVIOR OF PPAA MONITORED BY WAMS

The fact that the films could be stabilized by this post-plasma extraction treatment made it possible to study the swelling behavior of the plasma polymerized allylamine films in ethanol, by following the refractive index and the film thickness using WaMS. Basically, the effective refractive indices (Neff) for both the TE0 and TM0 mode, were determined by measuring the angle of incidence of an external beam onto the grating coupler at which resonant excitation of the respective guided mode occurred (see §3.2.1).

1.94635 a) 1.7922

1.94630 Neff(TE0) N (TM0) eff 1.7920 1.94625

1.7918 1.94620 eff,TE0 N 1.7916 1.94615

1.94610 1.7914

1.94605 1.7912 0 2 4 6 8 10 12 14 16 18 20 Time [h]

1.605 11.0 Thickness Refractive index 1.600 10.5

1.595 Refractive index

10.0 1.590

1.585 9.5 1.580 9.0 1.575 Thickness [nm] Thickness

8.5 1.570

1.565 8.0 b) 1.560 0 2 4 6 8 101214161820 Time [h]

Figure 5.3 a) Effective refractive indices of the TE0 and TM0 mode and b) thickness and refractive index of PPAA during swelling of ppaa in ethanol (Ppeak = 200 W, 10/190 ms/ms, tdep = 5 min) after extraction in ethanol for 24 h and drying.

55 Chapter 5

If an adlayer of PPAA is deposited onto the waveguide, its parameters (thickness and refractive index) also enter into the mode equation (see Eq. 3-3). If the assumption is made that the films are isotropic and uniform, the thickness and refractive index of the plasma film can be calculated from the Neff of the two modes, if the waveguide parameters and the refractive index of the covering medium are known. Figure 5.3-a shows Neff of the TE0 and the TM0 mode respectively, monitored during the swelling of a PPAA film for 19 hours in ethanol. Figure 5.3-b shows the thickness and the refractive index of this film during the swelling process.

1.64 A E B 1.62

F 1.60 C

1.58

Refractive index Refractive D 1.56

0 1000 2000 3000 4000 14 A 13 12 11 D 10 F 9 C 8

Thickness [nm] Thickness B E 7

0 1000 2000 3000 4000 Time [min]

Figure 5.4 a) Refractive index and b) thickness of PPAA (Ppeak = 200 W, 10/190 ms/ms, tdep = 5 min) immediately after deposition (A), after 24 h cold extraction in ethanol and drying in vacuum for 12 h (B), after exposure to ethanol (C), after swelling for 19 h (D), after drying in vacuum for 12 h (E) and after 55 h in air (F).

Figure 5.4 provides the data for the same film before (A) and after (B) extraction measured against air, during swelling measured against ethanol (C and D), and during drying in vacuum (E and F), measured against air again. The values corresponding with the points A to E are summarized in Table 5.2.

Table 5.2 Refractive index and film thickness of PPAA (Ppeak = 200 W, 10/190 ms/ms, tdep = 5 min), before and after extraction, before and after swelling, and after drying.

nd D covering [nm] medium A: after deposition 1.633 13.92 air B: after 24 h extraction in EtOH 1.628 7.61 air C: after submersion in EtOH 1.603 8.22 ethanol D: after 19 h swelling in EtOH 1.564 10.98 ethanol E: after drying 1.626 7.61 air

56 Film Stability and Surface Aging

Firstly, extraction caused an almost negligible change in the refractive index, but a considerable change in thickness (Figure 5.4, points A and B). When the film was then submersed and left in ethanol for a period of 19 hours, a decrease in refractive index of 0.04, accompanied by an increase in thickness of approximately 50 % was observed (points C and D). During swelling, the solvent molecules penetrate the plasma polymer network, and the measured refractive index decreases, due to the lower refractive index of the ethanol. After subsequent drying in vacuum overnight, the ethanol was almost completely removed from the polymer network, and the thickness and refractive index were seen to return to their value before solvent treatment (point E). This is again an indication that once the film is subjected to an extraction in ethanol, it shows good stability and no further dissolution from the bulk occurs.

Monitoring the films against air for another 50 hours resulted in minimal changes in refractive index and negligible variations in thickness, possibly due to contamination or absorption of water from the air.

5.3 Post-plasma Oxidation

A well-known complication that plasma polymers can present is that they usually undergo compositional changes upon storage after deposition. In our work we have observed significant amounts of oxygen incorporation in plasma polymerized allylamine films after aging in air.

5.3.1 EFFECT OF DEPOSITION CONDITIONS ON POST-PLASMA OXIDATION

Table 5.3 shows XPS data for four films deposited from allylamine at different duty cycles, after two weeks of aging in air. It illustrates that the oxygen uptake is dependent on the rf duty cycle employed during the film formation. The maximum amount of oxygen that has been found by XPS on the surface of a plasma film polymerized from allylamine immediately after deposition was 2.8 %. This oxygen can result from residual air in the reactor,12 from the exposure to air during the time it takes to transfer the samples from the reactor to the XPS (± 10 min) or from contamination of the monomer. After two weeks of storage in air, the N/C ratio is, as expected, the highest on the surface of the PPAA film polymerized at the lowest duty cycle. However, the O/C ratio for this film is almost a factor 4 higher compared to the film polymerized under CW conditions. The film polymerized at 5/200 ms/ms is apparently more susceptible to oxidation than the CW film.

57 Chapter 5

Table 5.3 Surface atom concentrations, N/C and O/C ratios of plasma polymerized allylamine after two weeks of aging in air (Ppeak = 40 W).

ton/toff

C1s N1s O1s N/C O/C [ms/ms] [W] [%] [%] [%] CW 40 77.1 18.5 4.4 0.24 0.06 5/5 20 70.9 20.9 8.2 0.29 0.12 5/50 3.6 67.9 19.9 12.2 0.29 0.18 5/200 0.2 63.7 21.4 14.9 0.34 0.23

The influence of the aging process on the wettability was also investigated (Figure 5.5) for allylamine films polymerized at two different rf duty cycles. A sharp increase in the water contact angles is observed during the initial stage of aging for both films. However, the film polymerized at the lowest duty cycle (3/45 ms/ms) shows a higher relative increase than the more crosslinked and hydrophobic film polymerized at a higher duty cycle (3/5 ms/ms). After several days the water contact angles reach a maximum, and further aging leads to a decrease of the water contact angles.

80 a)80 b) Advancing 70 70 Sessile

60 60 Receding

50 50

40 40

30 30

20 20 Water contact angle [°] angle contact Water 10 10

0 0 0 100 200 300 400 500 0 100 200 300 400 500 Hours of aging in air

Figure 5.5 Changes in water contact angles upon storage in air of PPAA films (Ppeak = 200 W, ton = 3 ms) polymerized at (a) toff = 5 ms and (b) toff = 45 ms.

To study the aging of these films further, FT-IR spectra have been taken after various times of storage in air, some of which are plotted in Figure 5.6 as difference spectra. A strong increase and broadening of the band at 3360 cm-1 is observed, due to the increasing degree of association over time. The amide I band (1680 cm-1) and the amide II band (1560 cm-1) appear as separate bands now, showing a conversion of amines to amides during the aging process. The band that appears around 1380 cm-1 after long storage times is assigned to amide III

58 Film Stability and Surface Aging

vibrations. A reduction of the intensity of the band at 2170 cm-1 also becomes apparent after longer times of storage, indicating the loss of unsaturated structures during oxidation.

5.3.2 INFLUENCE OF AMINO GROUPS ON THE AGING PROCESS

It is well known that the oxidation of plasma polymers is initiated by residual free radicals in the plasma polymer, which react with oxygen upon exposure to air. Even though the initial oxidation mechanism might be very similar for many different plasma polymers, the amine functionality in this particular case plays a considerable role in the following reactions. In a particular aging study by Gengenbach et al.,6b ethylenediamine was used as a monomer for plasma polymerization. The shifts of the binding energies of the N(1s) and O(1s) peaks over time, studied by XPS, suggested that the oxidation process predominantly takes place at the carbon atoms that have amino groups attached to them, leading mainly to the formation of amide groups.

as deposited

2 hours 1 day

19 days

85 days Transmittance [a.u]

4000 3500 3000 2500 2000 1500 1000 Wavenumber [cm-1]

Figure 5.6 FT-IR spectrum of a freshly deposited pulsed plasma polymerized allylamine film (Ppeak = 200 W, 3/45 ms/ms) and difference spectra, recorded after various times of storage.

Other work by their group, involving the plasma polymerization of n-heptylamine, revealed a rapid increase in the oxygen content during the first days of aging, followed by a slower oxygen uptake which continued for several years.6a Finally, their work on amine functionalized surfaces includes the aging of 1,3-diaminopropane plasma polymer samples, studied by XPS and FT-IR spectroscopy.6c The freshly deposited plasma polymer consisted of a random hydrocarbon network with a considerable amount of unsaturation and a high concentration of nitrogen-containing functional groups, mainly primary and secondary amines and imines. These groups strongly influenced the oxidation reactions. In contrast to

59 Chapter 5

hydrocarbon-based materials, where hydrogen abstraction and reaction of carbon-centered radicals with in-diffusing oxygen lead to a wide range of oxidative products, both XPS and FT-IR spectroscopy identified a rather narrow range of products in the 1,3-diaminopropane plasma polymers. Mainly amides and similar groups were detected, even after extensive aging for more than 2 years. The dominant reaction after plasma deposition was found to be the conversion of amines to amides. Again, preferential hydrogen abstraction from carbon atoms adjacent to amines was given as the reason for this behavior, since the lone electron of the so formed carbon radical can be stabilized by the lone pair of an amine nitrogen atom. In later stages of the oxidation (after four months) they observed a growing but still minor contribution by functionalities other than amides, possibly imides, carbonyls and hydroxides.

In our work, the N/C ratio on the surface, and thus the number of nitrogen-carrying carbon atoms in the allylamine films polymerized at low duty cycles is significantly higher than in the films deposited at high duty cycles or under CW conditions. Since the oxidative radical reaction preferentially takes place at the amine-carrying carbon atoms, the oxidation rate of the film polymerized at 5/200 ms/ms is much faster than the oxidation of the CW film. This effect is even more pronounced because of the higher fragmentation of amino groups at higher power input conditions. As has been shown in Chapter 4, FT-IR spectra revealed a progressive increase of the nitrile peak intensity with increasing power or duty cycle. If a large part of the nitrogen is incorporated in the plasma polymer in the form of nitrile groups, which are not susceptible to oxidation, this will also negatively influence the overall oxidation process.

5.3.3 SURFACE RESTRUCTURING AND REORIENTATION OF FUNCTIONAL GROUPS DURING AGING

The mobility of the surface layer also plays an important role in the surface aging process. Angle dependent XPS studies previously revealed reorganization of the surface layers of amine functionalized plasma films upon storage in air (see also §4.3.1).6b The hydrophilic amino groups showed a tendency to move away from the surface and the more hydrophobic environment, thus decreasing the N/C ratio at the surface. Again, this effect would be more pronounced for films polymerized at lower input powers, due to the lesser degree of cross-linking of these films, which gives the chains a higher flexibility.

It has been shown in Figure 5.5 that during the first days of aging, the PPAA films became more hydrophobic. After a certain time the water contact angles reached a maximum, followed by a slow decrease over time. This behavior can be understood if the separate processes contributing to the overall aging process are considered. During the initial stage, reorganization and restructuring of the surface is probably the dominating process. The hydrophilic amino groups move away from the surface into the bulk of the film, leading to a more hydrophobic surface layer with a higher density of hydrocarbon segments. At the same time, oxygen is incorporated in the film. The continuous reaction of free radicals with ambient

60 Film Stability and Surface Aging

oxygen results in the formation of polar, oxygen containing groups at the surface, and the surface energy slowly increases again.

Finally, another important factor that should be taken into consideration is the variation in the concentration of free radicals with the duty cycle after deposition. To investigate the changes in the amount of free radicals present in the film during aging, ESR spectra of a PPAA film have been taken immediately after deposition, and after various times of storage in air (Figure 5.7). The spectra reveal a large relative decrease in the radical density during the first two days of aging, followed by a slower rate of oxidation after this period. To our knowledge, nothing is known about the dependence of the free radical density on the rf duty cycle for plasma polymerized allylamine. According to Yasuda,2 the formation of free radicals from monomers containing a triple bond, a double bond or a cyclic structure, proceeds by the cleavage of a covalent bond, resulting in the formation of a diradical. Once these diradicals are formed, polymer formation can continue during the plasma-off times, without complete quenching of free radicals (see also Figure 2.4).

Yasuda reported that the changes of the ESR spin concentrations upon pulsing the plasma are completely dependent on the particular monomer, and the growth mechanism that is followed during deposition. For some unsaturated monomers, the spin concentration in the polymer was lower when they were polymerized under pulsed conditions than under CW conditions (vinyl acetate, methyl acrylate, tetrafluoroethylene, styrene). For other monomers the trapped radicals increased significantly in number when a pulsed plasma was used (ethylene, acetylene, acrylic acid, vinyl fluoride).2

1500

1000

500

0

-500 Intensity

-1000

-1500

3430 3440 3450 3460 3470 3480 3490 3500 3510 3520 Magnetic field [G]

Figure 5.7 ESR spectra of a pulsed plasma polymerized allylamine film (Ppeak = 200 W, 3/15 ms/ms). From high to low intensity: as deposited, after 2 days, after 1 week and after 6 weeks of storage in air.

61 Chapter 5

We have reasons to believe that in the case of allylamine, there is no major change in the free radical density in the polymer when the plasma is pulsed. In other work in this laboratory, the free radicals in these films have been utilized to initiate the subsequent grafting reaction of poly(methyl methacrylate) (PMMA) to the surface, and no dependence of the thickness of the grafted PMMA layer on the duty cycle employed was found.10b This will be further discussed in Chapter 7.

5.4 Conclusions

The work described in this chapter and in §4.3.6 presents waveguide mode spectroscopy as a powerful tool for the characterization of plasma polymerized films. Although allylamine was the only monomer used in this work, the WaMS technique in general can easily be used for the study of other plasma deposited films. By allowing a simultaneous study of film thickness and refractive index against different cover media, this method can give new insights in the structure, aging and swelling behavior of plasma polymerized coatings.

The results discussed in this chapter show, that plasma polymerized allylamine films are partly soluble in ethanol, and that treatment with a suitable solvent leads to the loss of material. Films produced under low energetic conditions (low duty cycles) have shown to result in films with a higher -NH2 content, but are also less stable upon subsequent solvent treatment. After extraction in ethanol for 24 hours and drying, the remaining films were apparently stable, since no further decrease in film thickness was observed after a second extraction. This indicates that immediately after deposition, the films contain short molecular chains or unreacted monomer molecules that are trapped in the network, and which are removed during extraction.

The aging of plasma polymerized allylamine films was found to be dependent on the deposition conditions. The amount of oxygen incorporation in the films after storage in air increased with decreasing duty cycle. This behavior was associated with preferential oxidation of carbon atoms that have amine groups attached to them, leading predominantly to amide groups.

5.5 Experimental section

5.5.1 SAMPLE PREPARATION

The films were deposited on pieces of silicon for FT-IR and XPS analysis, on waveguide chips for the WaMS experiments, or on polished glass slides (BK7, Berliner Glas) covered with 500 Å of silver topped with 300 Å of silicon oxide for SPR spectroscopy. The

62 Film Stability and Surface Aging

WaMS experiments described in this chapter were carried out with a waveguide chip consisting of a Ta2O5 film (dF ≈ 163 nm, nF ≈ 2.22, Λ = 745 nm) on glass (nS = 1.523). This film showed an increase in thickness of only 2 Å after swelling in ethanol for 10 hours, an order of magnitude lower than the TiO2 / SiOx waveguide that was used in Chapter 4. All substrates were cleaned in an Ar/O2 (9/1) plasma for 10 minutes prior to deposition. Silicon substrates were cleaned in ethanol under ultrasound. The samples were placed on a 40 x 40 mm glass holder on top of a small glass beaker half way between the electrodes. After each deposition process the reactor was cleaned for 1 hour using an O2 plasma at 200W.

5.5.2 PLASMA POLYMERIZATION

The allylamine monomer (Aldrich, Germany) was outgassed three times before use, but was not further purified. Reactor variables such as rf frequency (13.56 MHz), monomer flow (8 sccm), process pressure (0.2 mbar), and sample position (between the two electrodes) within the reactor were held constant in all experiments. More details regarding the deposition process can be found in §4.5.2.

5.5.3 CHARACTERIZATION

XPS and FT-IR spectroscopy were used to determine film compositions. FTIR spectra were recorded with a NICOLET 730 FTIR spectrometer. XPS analysis was performed on a Perkin Elmer PHI-500 series spectrometer equipped with an X-ray monochromator (see §4.5.3 for more details. WaMS offered the unique possibility of measuring changes in refractive index and thickness separately, unlike SPS and ellipsometry. The setup is described in detail in §3.2.1. The system was equipped with a cuvette, which made it possible to perform measurements in solvents. The film thicknesses on the SPR substrates (Figure 5.2) were determined using a homebuilt SPR spectrometer (§3.2.2), where p-polarized light from a helium-neon laser (λ = 632.8 nm) is coupled into the silver film on the substrates through a glass prism (BK7, Spindler & Hoyer). The reflectivity curves were fitted according to the Fresnel theory, using a refractive index n = 1.515 for BK7 glass, n = 1.460 for silicon oxide, and the dielectric constants ε’ = -16.000 and ε” = 0.600 for silver.

5.6 References

1 Boenig, H. V. Fundamentals of Plasma Chemistry and Technology, Technomic Publishing Co., Inc.: Lancaster, PA, 1988. 2 Yasuda, H. Plasma Polymerization, Academic Press Inc.: Orlando, FL, 1985. 3 Gerenser, L. J.; J. Adhes. Sci. Tech. 1987, 1, 303 4 Gengenbach, T. R.; Vasic, Z. R.; Chatelier, R. C.; Griesser, H. J. J. Polym. Sci.: Part A: Polym. Chem. 1994, 32, 1399. 5 Gengenbach, T. R.; Griesser, H. J. Surf. Interface Anal. 1998, 26, 498.

63 Chapter 5

6 (a) Gengenbach, T. R.; Chatelier, R. C.; Griesser, H. J. Surf. Interface Anal. 1996, 24, 271; (b) Gengenbach, T. R.; Chatelier, R. C.; Griesser, H. J. Surf. Interface Anal. 1996, 24, 611; (c) Gengenbach, T. R.; Griesser, H. J. J. Polym. Sci.: Part A: Polym. Chem. 1999, 37, 2191. 7 (a) Gengenbach, T. R.; Griesser, H. J. J. Polym. Sci.: Part A: Polym. Chem. 1998, 36, 985; (b) Gengenbach, T. R.; Griesser, H. Polymer 1999, 40, 5079. 8 (a) Occhiello, E.; Morra, M.; Morini, G.; Garbassi, F.; Humphrey, P. J. Appl. Polym. Sci. 1991, 42, 551; (b) Garbassi, F.; Morra, M.; Occhiello, E.; Barino, L.; Scordamaglia, R. Surf. Interface Anal. 1989, 14, 585. 9 (a) Calderon, J. G.; Harsch, A.; Gross, G. W.; Timmons, R. B. J. Biomed. Mater. Res. 1998, 42, 597; (b) Magnee, R.; Girardeaux, C.; Gillon, B.; Pireaux, J. J.; Caudano, R. in Surfaces in Biomaterials ’96, Surfaces in Biomaterials Foundation, Minneapolis, 1996, 80. 10 (a) Gombotz, W. R.; Guanghui, W.; Hoffmann, A. S. J. Appl. Polym. Sci. 1989, 37, 91; (b) van Os, M. T.; Stöhr, T.; Förch, R.; Vancso, G. J.; Knoll, W.; Rühe, J. R. to be submitted. 11 (a) Griesser, H.J.; Chatelier, R.C. J. Appl. Polym. Sci.: Appl. Polym. Symp. 1990, 46, 361. (b) van Os, M. T.; Menges. B.; Foerch, R.; Knoll, W.; Timmons, R. B.; Vancso, G. J. Mat. Res. Soc. Symp. Proc., 1999, 44, 45; (c) van Os, M. T.; Menges, B.; Foerch, R.; Vancso, G. J.; Knoll. W. Chemistry of Materials, 1999, 11, 3252. 12 Before each plasma polymerization the reactor was evacuated to a background pressure of typically 0.005-0.010 mbar before the monomer was introduced into the reactor chamber.

64 Mapping of Amino Group Distributions in PPAA Films by AFM with Functionalized Tips

&KDS WHU

Mapping of Amino Group Distributions in Plasma

Polymerized Allylamine Films by Atomic Force Microscopy

∗ using Functionalized Probe Tips

6.1 Introduction

It has been demonstrated in Chapter 4 and 5 that the composition and the structure of plasma polymerized films can be successfully analyzed by a combination of spectroscopic techniques. These techniques, including FT-IR spectroscopy, XPS, SPR spectroscopy, and WaMS, allow one to identify and analyze the functional groups present in the polymer films, and to determine the thickness and refractive index of the films. This information can be of great value to explain or predict adhesion, friction, or wetting behavior of polymers, and in particular, their biocompatibility.

However, besides the presence and the density of functional groups, the lateral distribution of functional groups in surface-treated or modified polymers also plays an important role in determining the interfacial properties of these materials.1 In biomedical applications for instance, the homogeneous distribution of functional groups in plasma modified or plasma polymerized materials is desirable to obtain biocompatible polymeric coatings.2 The determination of this spatial distribution, and the characterization of their local environment on a sub-100 nm scale remains difficult to impossible with the aforementioned techniques.3,4

In this chapter, the investigations of plasma polymerized allylamine films5 in a pulsed plasma6 are described. Due to the high density of amino functionalities, thin films of poly(allylamine) are potential candidates as substrates for studies involving the growth of cells on surfaces. Atomic force microscopy (AFM) with chemically functionalized tips7,8 was utilized in order to study the lateral distribution of functional groups and to characterize their local environment in pH dependent pull-off force measurements on a sub-50 nm level. The work described combines the strategies of AFM tip modification9 and active external control of tip-sample interactions in nanometer scale AFM adhesion measurements on polymers that

∗ Part of the work described in this Chapter has been published or has been accepted for publication: Schönherr, H.; van Os, M. T.; Hruska, Z.; Kurdi, J.; Förch, R.; Arefi-Khonsari, F.; Knoll, W.; Vancso, G. J. Chem. Comm. 2000, 1303; Holger Schönherr, Menno T. van Os, Renate Förch, Richard B. Timmons, Wolfgang Knoll, G. Julius Vancso Chem. Mater. 2000, accepted. 65 Chapter 6 contain ionizable functional groups.8b,10 In particular, it is shown that pH-dependent force titration measurements allow one to map functional group distributions with a sub-50 nm resolution. Results were obtained on thin films, plasma polymerized from allylamine at different duty cycles. Oxyfluorinated isotactic polypropylene (iPP),11 containing carboxylic acid groups,12 and ammonia plasma modified polypropylene (PP), containing basic (amino) groups, were also investigated for comparison reasons.13

6.2 Pull-off Force Measurements

Allylamine was polymerized in a pulsed glow-discharge plasma reactor as described previously.5,14 The films were characterized by SPR spectroscopy and WaMS, as well as by XPS and FT-IR spectroscopy (see Chapter 4 and 5). The duty cycle15 employed during the pulsed plasma experiments has an effect both on the film thickness and on the content of amino groups. In Table 6.1 the corresponding thickness data are shown.

Table 6.1 Process parameters and thickness data16 of plasma polymerized allylamine films studied (Pressure 0.20 mbar, gas flow 8 sccm, peak power 50 W).

Film ton/toff Equivalent Deposition Film [ms/ms] power [W] time [min] thickness 1 CW 50 30 1180 nm 2 10/40 10 30 160 nm 3 10/90 5 30 60 nm 4 10/240 2 30 24 nm

The content of amino groups depends on the duty cycle employed during deposition. As is evident from the transmission FT-IR spectra shown in Figure 6.1, the carbon to nitrogen ratio decreases with increasing off times. The broad absorbance for the amino groups (ca. 3200-3500 cm-1) increased with increasing off times, while the aliphatic C-H stretching vibrations (ca. 2800-2900 cm-1) and the typical nitrile band at ca. 2200 cm-1 were drastically reduced in absorbance.

The nitrogen / carbon ratio as measured in XPS experiments rises steadily from 0.22 for films prepared at continuous wave to 0.32 for low duty cycles. The latter value is almost identical to the N/C ratio of the monomer (0.33). Thus, the films can be expected to present significant numbers of hydrophilic amino groups at the surface in addition to hydrocarbon segments.

66 Mapping of Amino Group Distributions in PPAA Films by AFM with Functionalized Tips

a)

b)

c)

d) Transmittance [a.u] Transmittance

4000 3500 3000 2500 2000 1500 1000

Wavenumber [cm-1]

Figure 6.1 Transmission FT-IR spectra for plasma polymerized allylamine films as a function of the duty cycle. The films were deposited at a peak power of 50 W. The plasma-on time was kept constant at 10 ms, while the plasma off time was varied from (a) 0 ms (CW), (b) 40 ms, (c) 90 ms to (d) 240 ms.

The interfacial properties, i.e. the exposed amino groups, were characterized by AFM force-distance measurements using carboxylic acid functionalized tips in ethanol. The pull-off forces measured between amino groups and carboxylic acid groups in highly ordered model systems (self-assembled monolayers (SAMs) of amino-terminated disulfides) were shown to be much stronger than the interaction between methylene groups and carboxylic acid groups (Figure 6.2). This qualitative trend can be understood considering dipolar interactions and the strong hydrogen bonding between carboxylic acid and amino groups.

-NH -CH 45 2 75 3 70 40 65 60 35 55 30 50 45 25 40 35 Counts 20 Counts 30 15 25 20 10 15 10 5 5 0 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 Pull-off force [nN] Pull-off force [nN]

Figure 6.2 Histograms of pull-off forces measured with a -COOH tip on -NH2 (left) and -CH3 terminated 17 SAMs (right) in ethanol (curves: Gaussian fits). The average pull-off forces are 0.90 ± 0.22 nN (-NH2) and 0.12 ± 0.08 nN (-CH3).

According to the JKR theory,18 the pull-off force is proportional to the work of adhesion (Eq. 6-1), while the work of adhesion is the sum of the surface free energies of the 67 Chapter 6 functional groups (tip and sample) in contact with the liquid minus the interfacial free energy (Eq. 6-2). For the symmetric cases the work of adhesion is two times the surface free energy.

Fad = - 3/2 π R WST (6-1) where WST is the work of adhesion and R the radius of the tip.

WST = γS + γT - γST (6-2) where γS and γT denote the surface free energies of the sample and the tip (in contact with ethanol), and γST is the interfacial free energy of the two surfaces in contact. As stated in Eq. 6-3, the total surface free energy can be written as sum of Lifshitz-van der Waals and acid- base contributions:

LW AB γ = γs + γs (6-3)

LW where γs denotes the contribution of Lifshitz-van der Waals interactions to the surface free AB energy and γs the acid-base component of the surface free energy.

The acid-base parameters for -CH3 terminated SAMs are zero. If we assume that in a first rough approximation the Lifshitz-van der Waals surface free energies are similar for -

COOH, -NH2 and -CH3 SAMs, it follows that γCOOH > γCH3 and γNH2 > γCH3. Furthermore, the 19 positive interfacial energy between -COOH and -CH3 groups and the negative interfacial 10a,20 energy between -NH2 and -COOH groups reduces the work of adhesion for the former combination as compared to the combination -NH2 and -COOH. Thus, we conclude that the acid-base contributions of the -NH2 and -COOH terminated SAMs are responsible for the observed trends. The trends observed can also be qualitatively explained by the fact that forces involving dipolar interactions and hydrogen bonding (-COOH and -NH2) are stronger than interactions between dipoles and non-polar molecules (-COOH and -CH3) in ethanol.

The average pull-off forces for the PPAA films described above were estimated from the corresponding pull-off force distributions (Figure 6.3). The average data are summarized in Figure 6.4 as a function of the plasma-off time. The trend of increasing average pull-off forces with increasing off times, and its correlation with the increasing N/C ratios as seen by FT-IR spectroscopy and XPS (vide supra) are evident.

It can be concluded at this point that AFM force data obtained with chemically modified tips can be successfully correlated with the amino group content. The homogeneity of the distribution of the amino groups in these very flat films21 was investigated in more detail in aqueous environment.

68 Mapping of Amino Group Distributions in PPAA Films by AFM with Functionalized Tips

10 10 / 240

0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 20 10 / 90 10 0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 20

Counts 10 / 40 10

0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 30 CW 20 10 0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Pull-off force [nN] Figure 6.3 Pull-off force istributions measured with a -COOH tip in ethanol on plasma polymerized allylamine films polymerized at different duty cycles [ms/ms] (curves: Gaussian fits).

0.35

0.30

0.25

0.20

0.15

0.10 Average pull-off force [nN] force Average pull-off 0.05

0.00 0 50 100 150 200 250 Plasma off time [ms]

Figure 6.4 Average pull-off forces measured with -COOH tip in ethanol on allylamine films prepared at different duty cycles.22 The error bars correspond to the standard deviations obtained from the pull-off force distributions shown in Figure 6.3. The dotted line has been added to guide the eye.

69 Chapter 6

6.3 Determination of the Surface pKa of Plasma Polymerized Allylamine Films

By performing pH dependent pull-off force measurements, the contribution of the ionizable functional groups (amino groups) can be studied separately. In aqueous solutions, the sum of the different attractive forces is counteracted by the electrostatic repulsion of protonated amino groups if the pH is close to or lower than the pKa of these groups. Thus, in these systems the balance of attractive forces and repulsive electrostatic forces is measured as ‘pull-off force’.

For the pH dependent SFM experiments, hydroxyl-terminated (-OH) tips were used as these do not show any pH dependence in force measurements, while the interaction between these hydrophilic tips and various surfaces is relatively weak in aqueous environment.10a-c,20 By measuring pull-off forces as a function of pH, a corresponding ‘force titration’ curve and a ‘force pKa’ value can be obtained.23

In the average pH dependent force measurements a pronounced dependence of the magnitude of the average pull-off forces on the pH was observed. In Figure 6.5 a typical force titration curve is shown. Due to the protonation of the amino groups the pull-off forces decrease significantly for pH values below 5.5. However, there was surprisingly no pronounced difference between films prepared at different plasma deposition conditions. The data obtained in experiments with six different films were normalized and plotted in one graph (Figure 6.6). Despite the scatter of the data points one can clearly notice the reduction of the adhesive interactions between pH 6.2 and 5.2.

0.7

0.6 Force

0.5 (a)

0.4 (b) 0.3 (c) 0.2

(d) 0.1 Average pull-off force [nN] force Average pull-off 0.0 0.15 nN 345678 12.5 nm pH Piezo position Figure 6.5 Force titration of plasma polymerized allylamine film (left, the dotted line has been added to guide the eye).24 On the right the corresponding force-distance curves, which display (depending on the pH of the buffered aqueous solution) (a) exclusively repulsive (pH 4.7), (b) and (c) repulsive and attractive (pH 5.3 and pH 6.2, respectively), or (d) exclusively adhesive interactions (pH 6.8).25

70 Mapping of Amino Group Distributions in PPAA Films by AFM with Functionalized Tips

1.5

1.0

0.5 Normalized pull-off force

0.0 345678 pH Figure 6.6 Normalized force titration curves of six different plasma polymerized allylamine films. The data were normalized to the pull-off force measured at pH 8.0.26

Typical thermodynamic pKa values of amines in solution are in the range of 10.5.27 A comparison with the force data presented here indicates that the local environment of the amino groups in the allylamine films is somewhat hydrophobic. Upon lowering the pH the amino groups will resist protonation since the stabilization of the charge is low in the (hydrophobic) environment. This causes a shift of the thermodynamic pKa, and thus also the force pKa measured by SFM, to lower values. Similar shifts have been previously reported for -NH2 terminated SAMs.20a For disordered SAMs of amino-terminated silanes, a force pKa of 3.9 was reported previously in the literature.20a,28 In general, these shifts are in accordance with simulation results.29

Figure 6.7 Force titration data acquired with hydroxy-terminated tips for representative samples of the three model surfaces studied. For PPAA (left) and ammonia plasma modified PP (middle) the average pull-off forces are shown, for the untreated and oxyfluorinated iPP (right) the values were normalized to the values found for pH 3.8 (errors bars correspond to the standard deviation of the mean determined from the pull-off force distribution histograms). The dotted lines have been added to guide the eye.

As it is shown in Figure 6.7, the ammonia plasma treated PP samples,30 which contain basic amino groups, show a similar force titration curve as PPAA. The pull-off forces are negligible at low pH values, while at pH > 5 the adhesion was quite pronounced. It can be noted however that the inflection is not as sharp as for the PPAA specimens. An opposite

71 Chapter 6 trend was observed for the carboxylic acid-containing oxyfluorinated iPP,31 while the untreated iPP (without functional groups) showed no pH dependent pull-off forces.

The reduction of the pull-off forces as a result of repulsive interactions can again be attributed to the presence of protonated amino groups for the PP (low pH), and deprotonated carboxylic acid groups in the iPP (high pH), respectively. The inflection points (force pKa) are closely related to the thermodynamic surface pKa values.32 It is obvious that the force pKa values are shifted relative to the known pKa values (in solution) for carboxylic acids and amino groups (acetic acid: pKa 4.8; ethylamine: pKa 10.7). The force pKa values are shifted to a higher pH for the acidic groups, while for basic groups they are lower.

6.4 Mapping of functional group distributions

In all the allylamine films studied, there seems to be on average little difference in hydrophobicity for different films. This is probably related to the possible restructuring of the thin PPAA films in contact with water (see also Chapter 5). The films consist of a flexible network, which has been previously shown to facilitate rearrangements of the polymer network and the amino groups.33

The laterally resolved measurements of pull-off forces with OH-terminated SFM tips on plasma polymerized allylamine films at pH 5.2 showed inhomogeneities of adhesive interactions on a sub-50 nm scale. In contrast, the interaction between the tips and the polymer surface became almost exclusively repulsive at pH 3.8 (Figure 6.7). In the plasma polymerized allylamine films coupling of topography into the force images can be neglected due to the featureless, flat surface of these films.26 An inhomogeneous coverage of the surface, i.e. areas covered with polymer and areas without film coverage, can also be excluded since the observations were fully reproducible after changing the pH.34

On a number of different films similar observations were reproduced. Analogous to the oxyfluorinated iPP films it can be concluded that the distribution of the functional groups is inhomogeneous. Differences in the local hydrophobicity can be assumed to cause a shift of the force pKa. In general, the more hydrophobic the local environment is, the lower the force pKa. Thus, at pH 5.2, areas with high adhesion correspond to the more hydrophobic regions. The origin of the inhomogeneous distribution of functional groups is not known at present. A possible explanation is the previously observed restructuring of the polymer and the reorientation of the amino groups upon initial aging of the films in air.

72 Mapping of Amino Group Distributions in PPAA Films by AFM with Functionalized Tips

Figure 6.7 Height (left) and pull-off force (right) images of a plasma polymerized allylamine film (Ppeak = 175 W, ton/toff = 10/40 ms/ms) obtained with OH-terminated tips at different pH. In the height image, the color scales ranges from dark (0 nm) to bright (25 nm). In the force images dark color indicates high adhesion (≈ 0 nN, -2.0 nN for pH 3.8 and pH 5.2, respectively), bright color indicates low adhesion (≈ 0 nN and -0.1 nN for pH 3.8 and pH 5.2, respectively).

The data shown confirm earlier results from Schönherr et al. on laterally resolved imaging of functional group distributions on polymers on a sub-50 nm level using atomic force microscopy with chemically modified tips (‘chemical force microscopy’).35 The inhomogeneous forces observed can be related to variations of local ‘pKa’ values and different local hydrophobicity, and thus to inhomogeneous distribution of the polar functional groups introduced by aging of the films or the plasma polymerization process.

73 Chapter 6

6.5 Conclusions

Atomic force microscopy measurements were performed on plasma polymerized allylamine films using chemically modified AFM tips. Pull-off force measurements with carboxylic acid terminated tips were successfully correlated with the amino group content of the films. The average pull-off force measured increased from ± 0.09 nN for films polymerized under CW conditions, to ± 0.30 nN for pulsed PPAA films deposited at ton/toff = 10/240 ms/ms.

The distribution of amino groups in plasma polymerized allylamine films was investigated by AFM using hydroxyl-terminated tips. By measuring pull-off forces as a function of the pH, a corresponding ‘force-titration’ curve was obtained. The adhesive interactions between the –OH terminated tip and the –NH2 groups on the surface greatly decreased between pH 6.2 and 5.2 due to repulsion by the protonated amino groups. This ‘force pKa’ was found to be independent of the duty cycle employed during plasma polymerization. Laterally inhomogeneous pull-off forces were related to the inhomogeneous distribution of the polar groups on the surface.

6.6 Experimental Section

6.6.1 MATERIALS

Details on the preparation, characterization, and swelling behavior of the allylamine films can be found in references 5 and 36, or in Chapter 4 and 5 of this thesis. The films for the AFM studies were extracted in cold ethanol for 24 h prior to examination.

6.6.2 SCANNING FORCE MICROSCOPY AND TIP MODIFICATION

Triangular shaped silicon nitride cantilevers with silicon nitride tips (Digital Instruments (DI), Santa Barbara, CA, USA), which were coated with 2 nm Ti and 75 nm Au in high vacuum (Balzers), were functionalized with 11-mercaptoundecanoic-acid (-COOH) or 11-hydroxyundecanol (-OH) following the procedures described previously.37 The force- distance data were acquired using a NanoScope III multimode SFM (DI). Measurements were performed with 11-mercaptoundecanoic-acid modified tips in ethanol (p.a., Merck) or 11- hydroxyundecanol modified tips in buffered aqueous solutions of constant ionic strength utilizing a liquid cell (DI).25 The force-distance curves were obtained on at least 10 different positions of each sample, and the pull-off forces of the corresponding single events were plotted in a histogram. The values of at least 2 independent tip-sample combinations were averaged. For laterally resolved pull-off force measurements the force microscope was operated in the force volume (FV, DI) mode. Only identical images acquired in subsequent up and down scans were considered.

74 Mapping of Amino Group Distributions in PPAA Films by AFM with Functionalized Tips

6.7 References

1 Ferguson, G. S.; Whitesides, G. M. in Modern Approaches to Wettability; Schrader, M. E.; Loeb, G. I. (Eds.); Plenum, New York, 1992, p. 143, and references therein; Hupfer, B.; Ringsdorf, H. in Surface and Interfacial Aspects of Biomedical Polymers; Andrade, J. D. (Ed.); Vol. 1; Surface Chemistry and Physics; Chapter 4, Plenum, New York, 1985, pp. 77; van Oss, C. J. in Polymers and Interfaces II; Feast, W. J.; Munro, H. S.; Richards, R. W. (Eds.); Chapter 11, Wiley & Sons, New York, 1993, pp. 268-290; Ulman, A. An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self-Assembly; Academic Press, New York, 1991. 2 Gombotz, W. R.; Hoffman, A. S. Critical Reviews in Biocompatibility 1987, 4, 1. 3 Practical Surface Analysis; Briggs, D.; Seah, M. P. (Eds.); Wiley, Chichester, 1992 4 Chan, C.-M. Polymer Surface Modification and Characterization; Hanser Publishers, Munich, 1994, p.100 5 van Os, M. T.; Menges, B.; Förch, R.; Vancso, G. J.; Knoll, W. Chem. Mater. 1999, 11, 3252. 6 Radiofrequency (13.56 MHz). 7 (a) Vezenov, D. V.; Noy, A.; Rozsnyai, L. F.; Lieber, C. M. J. Am. Chem. Soc. 1997, 119, 2006; (b) Noy, A.; Vezenov, D. V.; Lieber, C. M. Annu. Rev. Mater. Sci. 1997, 27, 381; (c) van der Vegte, E. W.; Hadziioannou, G. Langmuir 1997, 13, 4357; (d) van der Vegte, E. W.; Hadziioannou, G. J. Phys. Chem. 1997, 101, 9563. 8 (a) Sinniah, S. K.; Steel, A. B.; Miller, C. J.; Reutt-Robey, J. E. J. Am. Chem. Soc. 1996, 118, 8925; (b) Feldmann, K.; Tervoort, T.; Smith, P.; Spencer, N. D. Langmuir 1998, 14, 372. 9 Frisbie, C. D.; Rozsnyai, L. F.; Noy, A.; Wrighton, M. S.; Lieber, C. M. Science 1994, 265, 2071. 10 (a) Noy, A.; Vezenov, D. V.; Lieber, C. M. Annu. Rev. Mater. Sci. 1997, 27, 381; (b) van der Vegte, E. W.; Hadziioannou, G. J. Phys. Chem. 1997, 101, 9563. (c) Schönherr, H.; Vancso, G. J. J. Polym. Sci. B, Polym. Phys. 1998, 36, 2486. 11 Schönherr, H.; Hruska, Z.; Vancso, G. J. Macromolecules 1998, 31, 3679. 12 Kranz, G.; Lüschen, R.; Gesang, T.; Schlett, V.; Hennemann, O. D.; Stohrer, W. D. Int. J. Adh. Adh. 1994, 14, 243. 13 Shahidzadeh, N.; Arefi-Khonsari, F.; Chehimi, M. M.; Amouroux, J. Surf. Sci. 1996, 352 - 354, 888. 14 van Os, M. T.; Menges, B.; Timmons, R. B.; Knoll, W.; Förch, R. ISPC-13 Symp. Proc. 1997, 3, 1298. 15 The duty cycle is defined as the ratio [plasma-on time/(on time + off time)]. 16 Thicknesses and refractive indices were measured by surface plasmon resonance spectroscopy and waveguide spectroscopy ((a) Aust, E. F.; Sawodny, M.; Ito, S.; Knoll, W. Scanning 1994, 16, 353; (b) Ramsden, J. J. J. Statistical Phys. 1993, 73, 853). 17 Monolayers were prepared by self-assembly of 11-mercaptoundecanoic-acid (-COOH) and bis(12-aminododecyl) disulfide onto Au(111) substrates as described in: Schönherr, H.; Ringsdorf, H. Langmuir 1996, 12, 3891. 18 Johnson, K. L.; Kendall, K.; Roberts, A. D. Proc. R. Soc. London A 1971, 324, 301. 19 van Oss, C. J. in Polymers and Interfaces II; Feast, W. J.; Munro, H. S.; Richards, R. W. (Eds.); Wiley & Sons, New York, 1993, Chapter 11, 267 20 Vezenov, D. V.; Noy, A.; Rozsnyai, L. F.; Lieber, C. M. J. Am. Chem. Soc. 1997, 119, 2006; (b) van der Vegte, E. W.; Hadziioannou, G. Langmuir 1997, 13, 4357. 21 The roughness RA of these films was 1.0 ± 0.2 nm as determined by tapping mode AFM. 22 The increase in pull-off forces with increasing amino group content can be rationalized by taking the hydrogen bonding between carboxylic acid and amino groups into account. The

75 Chapter 6

interfacial free energy for similar combinations of functional groups was shown to be negative and quite large, which results in large pull-off forces.20b 23 The force pKa is related to the thermodynamic pKa. However, the relation is very difficult to analyze quantitatively. The interactions in our experiments are probed on a non- continuum level using a rather ill defined geometry (tip covered with granular gold vs. rough polymer surfaces). A theory which would allow one to relate forces measured with this geometry to surface charge is currently not available. Different methods for the determination of surface pKa values have been discussed in the literature. These include: (a) An, S. W.; Thomas, R. K. Langmuir 1997, 13, 6881 (combined surface tension and neutron reflection measurements); (b) Holmes-Farley, S. R.; Reamey, R. H.; McCarthy, T. J.; Deutch, J.; Whitesides, G. M. Langmuir 1985, 1, 725 (contact angle titration); (c) Smith, C. P.; White, H. S. Langmuir 1993, 9, 1 (electrochemical determination of interfacial capacitance). For the determination of a surface pKa by force microscopy using a well-defined tip geometry (micrometer-sized spherical particles) see: Hu, K.; Bard, A. J. Langmuir 1997, 13, 5114. 24 Ppeak = 175 W, ton/toff = 10/10 ms/ms. 25 The data presented here was acquired at an ionic strength of 1.87 × 10-3. Phosphate and acetate buffers were used. It should be noted that the force titration curves and the corresponding inflection points were found to be independent of the ionic strength (in the range of 2 × 10-1 to 2 × 10-4). 26 The films with the following deposition parameters were investigated: Ppeak = 175 W; ton/toff = 10/10, 10/40 and 10/90 ms/ms. The roughness RA of these films was 1.0 ± 0.2 nm as determined by tapping mode AFM. 27 Typical pKa values measured for amines in solution are: methylamine (10.6), dimethylamine (10.7), ethylamine (10.7), propylamine (10.6) (Risch, K.; Seitz, H. Organische Chemie; Schroedel, Hannover, 1981; p. 115). 28 The extraordinary low pKa which was confirmed by independent contact angle titrations (4.3), was explained by the conformational disorder of the silane SAM. The disorder leads to a exposition of hydrophobic methylene groups. 29 Smart, J. L.; McCammon, J. A. J. Am. Chem. Soc. 1996, 118, 2283. 30 Details on the ammonia plasma treated PP samples can be found in reference 13. 31 Details on the oxyfluorinated iPP samples can be found in reference 11. 32 A strict quantitative evaluation, such as described by Hu and Bard (Hu, K.; Bard, A. J. Langmuir 1997, 13, 5114), is at this point not possible due to uncertainties of the tip shape (granular gold coating) and the unknown effect of the morphology of the polymer films. 33 Gengenbach, T. R.; Vasic, Z. R.; Li, S.; Chatelier, R. C.; Griesser, H. J. Plasmas and Polymers 1997, 2, 91. 34 Further support is the observation of practically pH independent pull-off forces measured on a bare Si wafer used as substrate for polymer deposition. Here the adhesion was homogeneous. 35 (a) Holger Schönherr, Zdenek Hruska, G. Julius Vancso PMSE Prepr. 1999, 81, 838; (b) Holger Schönherr, Zdenek Hruska, G. Julius Vancso Macromolecules 2000, 33, 4532; (c) Schönherr, H. Ph.D. Thesis University of Twente, Enschede, The Netherlands, 1999, ISBN 90 365 1347 2. 36 van Os, M. T.; Menges, B.; Timmons, R. B.; Knoll, W.; Förch, R. ISPC-13 Symp. Proc. 1997, 3, 1298. 37 (a) Schönherr, H.; Hruska, Z.; Vancso, G. J. Macromolecules 1998, 31, 3679; (b) Schönherr, H.; Vancso, G. J. J. Polym. Sci. B, Polym. Phys. 1998, 36, 2486; (c) Schönherr, H.; Vancso, G. J. Polym. Prepr. 1998, 39(2), 1177.

76 Attachment of Polymer Monolayers to PPAA Films by Grafting-from Polymerization

&KDS WHU

Attachment of Polymer Monolayers to Plasma Polymerized

Allylamine Surfaces by Radical Chain Polymerizations

∗ Initiated by Azo Compounds and Residual Free Radicals

7.1 Introduction

The surface modification of solid substrates has become an increasingly important field of research for applications ranging from protective coatings, to the improvement of biocompatibility of materials, the fabrication of electronic devices and the reduction of fouling of ultrafiltration membranes.

7.1.1 “GRAFTING-TO” POLYMERIZATION

An important factor in the preparation of thin polymer films is to have a sufficient long-term stability of the films in different environments. The establishment of a covalent bond between the surface of the substrate and the polymer molecules is one way to improve this stability. Most approaches in this direction use preformed polymers with a functional group, that can react with an appropriate surface site, better known as the ‘grafting-to’ method.1 However, these methods are usually limited to thicknesses of 1-5 nm, due to kinetic hindrance of the surface-attached polymer chains and for thermodynamic reasons.2

7.1.2 GRAFTING-FROM POLYMERIZATION

Recently, a few groups have found a way to overcome this limitation, by starting the polymerization from a self-assembled monolayer of initiator, so that the polymer is formed directly at the surface of the substrate (almost exclusively high surface area silica gels).3 In these ‘grafting-from’ techniques, the polymerization is no longer limited by diffusion, since the small monomer molecules only have to reach the end of the growing chains. Several disadvantages however still limit the applicability of such systems. Firstly, stepwise procedures are generally employed, sometimes requiring up to eight surface modification

∗ Part of the work described in this Chapter will be submitted for publication: M.T. van Os, T. Stöhr, R. Foerch, G.J. Vancso, J. Rühe, and W. Knoll, Attachment of Polymer monolayers to Plasma Modified Surfaces by Radical Chain Polymerizations Initiated by Azo Compounds and Residual Free Radicals, to be submitted.

77 Chapter 7 reactions.2,4 Secondly, due to incomplete conversion in the different reaction steps and side reactions, the amounts of immobilized polymer remain very low, and not very reproducible.2

A system has recently been developed by Prucker and Rühe, in which the initiator molecules are attached to the surface in a one-step reaction, and in which reproducible polymer layers with high graft density can be obtained.5 The initiator in the system described, consists of three basic components: an anchoring group to link the initiator to the surface, the initiator itself, and a cleavable group to degraft the formed macromolecules after polymerization for analytical purposes.5a A drawback in this method is that the anchoring groups of the initiator molecules have to react with appropriate reactive sites at the surface, thus limiting the number of materials that could be used as a substrate for this surface modification method. Most of the common commercial polymers for instance are unreactive.

7.1.3 PLASMA TREATMENT AND POLYMERIZATION

As an alternative for the surface modification of a substrate, plasma treatment has been proposed. A major advantage of this technique is that it is largely independent of the substrate that is used. Non-polymerizable gases such as argon or nitrogen are often used to introduce free radicals at the surface region of a material. When this treated substrate is then subjected to a (vaporized) monomer or immersed in a monomer solution, grafting reactions can proceed through a radical mechanism, illustrated in a simplified way by the following steps:6

• R → R (initiation)

• • R + M → R-M (propagation)

• • R-Mn + M → R-Mn+1 (propagation)

• • R1-Mm + R2Mn → R1-Mm+n-R2 (termination)

It has already been discussed in Chapter 5 that the free radicals present in plasma polymers can undergo changes in the presence of air through the formation of peroxide functional groups.7 These groups can also initiate grafting reactions, for example by the following mechanism:

• ROOH + H2C=CH-R1 → RO-H2C-C H-R1 + OH (peroxide initiation)

It has indeed been shown by several groups that these peroxides can be used for the subsequent graft polymerization of radically polymerizable monomers. Polymeric substrates such as poly(ethylene),8 poly(propylene),9 poly(ethylene terephtalate),10 and poly(urethanes)11 have been modified successfully by this process. To some extent, the grafting yield in such experiments can be controlled by the plasma treatment time or the plasma power.11,12

78 Attachment of Polymer Monolayers to PPAA Films by Grafting-from Polymerization

Many groups have reported on the use of low-temperature plasmas for plasma polymerization or deposition, in which a thin cross-linked layer of polymeric material is deposited on the substrate. In some cases, plasma deposition and plasma treatment have been observed to produce reactive and unstable surfaces. An increasing amount of oxygen may be incorporated in the surface layer over time, even if no oxygen was present in the monomer used.13 Instability of plasma modified surfaces may also be caused by the restructuring and reorientation of chains or polar groups in the surface region, leading to a gradual loss of the desired functional groups at the surface over time (see also §5.3.3).

7.1.4 CONCEPT

The present study examines a three-step surface modification, consisting of a plasma film-depositing step to provide the appropriate reactive sites (e.g. amino groups), followed by reaction of an azo-initiator with the amino groups, and the subsequent grafting-from polymerization. An overview of the concept is given in Figure 7.1.

The ability of the plasma polymerization technique to deposit a thin, strongly adherent and cross-linked film onto almost any desired surface is utilized in the first step. By selecting allylamine as the monomer for the plasma polymerization, reactive amino groups are introduced at the surface of the plasma film.

In the second step, these amino groups are then used for the subsequent immobilization of the radical initiator 4,4’-azobis-(4-cyano-pentanoic acid chloride). The concentration of the amino groups at the surface can be controlled by variation of plasma parameters such as the input power and the rf duty cycle (see reference 14 or Chapter 4). At the same time, some of the free radicals may have been transformed into peroxide groups through reaction with ambient oxygen.

In the third and final step, the azo groups are utilized to start the thermally induced polymerization of methyl methacrylate or styrene, leading to surface-attached poly(methyl methacrylate) (PMMA) or polystyrene (PS) chains, also denoted as polymer “brushes”. In addition to the created reactive amino groups, this study will investigate the influence of the peroxides (or free radicals) present at the surface of the plasma deposited film on the grafting reaction. The dependency of the graft density on the plasma polymerization conditions will be described qualitatively as well as quantitatively.

79 Chapter 7

silicon oxide substrate

deposition of a plasma NH2 polymerized allylamine allylamine network

NH amino groups NH 2 free radicals 2 · ·

ambient oxygen peroxide groups

OH OH O NH2 O NH2

NC Me O Cl N N Cl MeCN NEt , toluene O 3 4,4'-azobis-(4-cyano-pentanoic acid chloride)

N azo groups N N N OH NH OH NH O O

toluene O 60 °C methyl methacrylate

PMMA brushes

NH NH

Figure 7.1 Concept for the synthesis of covalently attached PMMA monolayers to a silicon oxide surface. Amino and peroxide groups are introduced through the plasma polymerization of allylamine and the subsequent oxidation of residual free radicals with ambient oxygen respectively. After immobilization of the azo initiator, both the azo groups and the peroxide groups can in principle start the polymerization of MMA molecules from the PPAA surface.

80 Attachment of Polymer Monolayers to PPAA Films by Grafting-from Polymerization

7.2 Qualitative Evidence for the Attachment of PMMA and PS Brushes to PPAA Films

7.2.1 FT-IR SPECTROSCOPY

Poly(methyl methacrylate) (PMMA) brushes. The attachment of a PMMA layer to a thin film of plasma polymerized allylamine (PPAA) could be detected with FT-IR spectroscopy. Figure 7.2-a shows the spectrum of a PPAA film deposited on a Si wafer. The broad absorption band around 3350 cm-1 indicates the presence of primary and secondary amines. The multiple absorption band around 2900 cm-1 is due to the C-H stretching vibrations. The peak at 2195 cm-1 indicates the presence of nitrile groups, while the bands around 1640 cm-1 are assigned to the bending modes of amines, the stretching of imine groups, and the stretching vibrations of amide groups. A transmission spectrum of pure PMMA is shown in 7.2-c. The C-H stretching vibrations are observed between 2800 and 3000 cm-1. A strong single absorption band due to the stretching of carbonyl groups appears at 1730 cm-1. The bands at 1480, 1440 and 1380 cm-1 are attributed to the C-H deformation vibrations -1 of CH2 and CH3 groups, while the two groups of absorption bands around 1260 and 1170cm originate from C-O stretch modes. These bands characteristic for PMMA can clearly be observed in the spectra of the PMMA films that were grafted onto the PPAA (7.2-b), confirming the formation of a PMMA adlayer on the PPAA film.

c) ν(>C=O) 1271 1242 1192 1150 1730 ν (C-Hx) 2951

b) 1732 1271 1242 1194 1150

2951 1630 3343 2193 Absorbace[a.u.] a) ν ν (-C(=O)-NHx), (>C=N-) 1638 ν (-NHx) ν(-C≡N) 3353 2195

4000 3500 3000 2500 2000 1500 1000 Wavenumber [cm-1]

Figure 7.2 FT-IR transmission spectra of a) a plasma polymerized allylamine film (Ppeak = 250 W, ton = 10 ms, toff = 25 ms, tdep = 5 min) on a silicon wafer, b) with an adlayer of PMMA polymerized for 24 h at 60 °C (MMA/toluene = 1/1), and c) pure PMMA.

Polystyrene (PS) brushes. Qualitative evidence for the successful grafting of PS to a thin film of PPAA by the described method was also obtained by FT-IR spectroscopy (Figure 81 Chapter 7

7.3). The PS bulk spectrum (Figure 7.3-c) shows C-H stretching vibrations around 3000 cm-1, and the absorption bands at 1597, 1489, and 1452 cm-1 can be assigned to the stretching vibrations of the C=C double bond. The FT-IR spectrum of a polystyrene-modified PPAA film is depicted in Figure 7.3-b, and appears as the superposition of the individual spectra.

ν(=CH ) ν(-CH ) ν(C=C) c x x ) 3084 3060 3024 2921 2849 1597 1489 1452

b) 3059 3024 2924 2851 1600 1495 1449 ~1650

~3310

Absorbance [a.u.] Absorbance ν ν (-C(=O)-NHx), (>C=N-) a) ν(-NH ) x ~1640 ~3300

4000 3500 3000 2500 2000 1500 1000 Wavenumber [cm-1]

Figure 7.3 FT-IR transmission spectra of a) plasma polymerized allylamine film (Ppeak = 250 W, ton = 10 ms, toff = 190 ms, tdep = 5 min) on a silicon wafer, b) with an adlayer of PS polymerized for 24 h at 60 °C and c) pure PS.

7.2.2 CONTACT ANGLE GONIOMETRY

Table 7.1 Water contact angles of PPAA (Ppeak = 20 W, ton = 10 ms, toff = 120 ms), PMMA- and PS- modified PPAA, and PMMA and PS spin coated on silicon substrates. Advancing Sessile Receding PPAA 65 62 6 PMMA brush (peroxide initiated) 69 67 58 PMMA brush (azo and peroxide 68 66 58 initiated) PMMA (spin coated) 70 66 58 PS brush (peroxide initiated) 93 87 76 PS (spin coated) 94 88 75

The PPAA films were also investigated by water contact angle measurements, before and after modification with PMMA and PS respectively. The results of those measurements are summarized in Table 7.1. PMMA and PS were also spin coated on to Si wafers for reasons of comparison. The contact angles of the spin coated films were all found to be within a margin of 2° of the contact angles measured on the graft polymers (Table 7.1). The large

82 Attachment of Polymer Monolayers to PPAA Films by Grafting-from Polymerization increase of the receding contact angle on PPAA upon modification clearly indicates the presence of a PMMA or PS layer on top of this film.

7.2.3 AFM IMAGING

a) b)

0 2 µm 0 2 8.0 nm c)

0.0 nm

0 2 µm Figure 7.4 TM-AFM images of a) a silicon wafer (rms surface roughness 0.9 Å), b) modified with plasma polymerized allylamine (ton = 10 ms, toff = 25 ms, Ppeak = 250 W, tdep = 300 s, rms surface roughness = 0.48 nm), and c) after polymerization of methyl methacrylate (rms surface roughness 0.38 Å).

To obtain an impression of the film morphology, tapping mode AFM measurements were performed on the different samples. Figure 7.4-a shows an image taken from a 2 x 2 µm2 large area of a silicon wafer. An image of this substrate after modification with a PPAA layer is shown in Figure 7.4-b. The rms surface roughness over this area of the plasma film is 0.48 nm. It can be seen in Figure 7.4-c that the surface becomes more homogeneous after attachment of a PMMA film (rms surface roughness 0.38 nm). This proves that the

83 Chapter 7 polymerization of MMA on the PPAA film by the concept depicted in Figure 7.1 leads to films with good homogeneity on this scale.

7.3 Grafting-from Polymerization of MMA and Styrene Initiated by an Azo Compound and/or Peroxide Groups: Quantitative Evaluation

7.3.1 INCREASING THE NUMBER OF REACTIVE SITES AT PPAA SURFACES THROUGH VARIATION OF PLASMA PARAMETERS

One of the goals of this study was to investigate the dependence of the thickness of the grafted polymer layers, by varying the plasma parameters during deposition. First of all, the influence of the amino group density at the surface is expected to play an important role in the subsequent grafting reaction. The higher the concentration of the amino groups, the larger the number of reactive sites that are available for the immobilization of the azo compound. It has been described in Chapter 4 that the film chemistry and structure of plasma polymerized allylamine depends largely on polymerization parameters such as the peak power (Ppeak) or the rf duty cycle employed.14a,b

c) ~1650

1456 1381 ~3300 2961 2936 2876

2184

δ ν (-NHx), (C=N-) b - ) ~1640

δ (-CHx) ν(-CH ) ν(-NH ) x 1455 1381 x ν ≡ 2963 2934 2876 (-C N)

~3330 2240 2188

a) ~1630 Absorbance [a.u.] 2965 2938 2876

1458 1381 2240 2191 ~3360

4000 3000 2000 1000 Wavenumber [cm-1]

Figure 7.5 FT-IR transmission spectra of PPAA films, polymerized at different duty cycles. The off time was increased systematically from a) toff = 0 ms (CW), b) toff = 40 ms, to c) toff = 90 ms, while the other parameters were kept constant (Ppeak = 50 W, ton = 10 ms).

A typical example of the variation in film chemistry that can be achieved is given in Figure 7.5, where the FT-IR spectra of PPAA films polymerized at increasing plasma-off times (e.g. decreasing duty cycles) are shown, keeping the other parameters constant. The relative increase of the absorption band around 3300 cm-1 indicates that upon increasing the 84 Attachment of Polymer Monolayers to PPAA Films by Grafting-from Polymerization plasma-off time, the amino group in the monomer is increasingly retained in the plasma polymer. Reduced fragmentation with decreasing duty cycle is also shown by the decreasing intensity of the band around 2200 cm-1, which is due to nitrile groups.

7.3.2 INFLUENCE OF PLASMA PARAMETERS ON THE FREE RADICAL AND PEROXIDE CONCENTRATION

Secondly, the amount of free radicals in the plasma film should be taken into account. As was mentioned in the introduction of this chapter, many plasma polymers contain a certain amount of free radicals, caused by the bombardment of the surface by plasma species, electrons and UV radiation. These trapped free radicals are known to react with oxygen over time, in some cases leading to changes in the characteristics of a plasma polymer, as has been extensively discussed in Chapter 5. Although allylamine itself contains no oxygen, XPS spectra reveal the incorporation of oxygen at the surface of PPAA films after aging in air (Table 5.3). The oxygen content has been previously shown to increase rapidly during the first days of aging, after which the films become less reactive (see §5.3 for more details). The influence of the peroxide group concentration on the graft density of the attached PMMA layer will be described.

b) xO = 12.4 %

xN = 18.1 %

xC = 69.5 %

xN/xC = 0.26

x = 14.0 % a) O(1s) C(1s) O N(1s) xN = 19.9 % Intnesity [a.u.] xC = 66.2 %

xN/xC = 0.30

1000 800 600 400 200 0 Binding energy [eV]

Figure 7.6 X-ray photoelectron spectra of PPAA films (Ppeak = 250 W, ton = 10 ms, toff = 190 ms) before (a) and after (b) cold extraction in ethanol for 24 hours and subsequent drying in vacuum. The spectra were taken approximately two weeks after deposition.

It should be noted that all the PPAA films used in this study were subjected to a 24 h cold extraction in ethanol prior to further use. It has been reported in §5.2 that plasma polymerized allylamine films contain oligomers and other small extractable molecules, that can be removed by treatment with a suitable solvent. It was demonstrated that after this

85 Chapter 7 washing procedure, the films could be reversibly swollen, and no further leaching of extractable material occurs.14a As is shown by XPS, the extraction in ethanol causes only minimal changes in the N/C content of these films (Figure 7.6).

7.3.3 GRAFTING CONDITIONS AND CHARACTERIZATION

All the grafting polymerization reactions described in this chapter were carried out at 60 °C for 24 hours. Prucker and Rühe reported that the amount of poly(styrene) grafted to azo-modified SiO2 samples at this temperature increased steadily during the first 20 h of polymerization, after which the increase of the graft density over polymerization time slowly started to level off.5a SPR spectroscopy was used to quantify the thicknesses of the different polymer layers. The thicknesses of the PPAA films were always determined after the 24 h extraction process and the subsequent drying in vacuum for 2 h. The graft density Γ of the grafted polymer was calculated from the following equation:

Γ = (d ⋅ ρ) / Mn (7-1) where d is the thickness of the polymer layer as measured by SPR spectroscopy, ρ the density of the polymer, and Mn the number average molecular mass. The values of the molecular weights of the attached polymer chains were determined by GPC after degrafting.15

16 7.3.4 PEROXIDE INITIATED POLYMERIZATION

In order to study and understand the grafting of polymer brushes to plasma deposited allylamine films through the immobilized azo initiator, it must first be investigated if, and to what extent the peroxide groups (and free radicals) contribute to the overall polymerization. A number of PPAA films have been prepared at different process conditions, and subjected to a cold extraction in ethanol for 24 hours. These films were not exposed to the azo initiator, but subjected directly to the respective monomers.

The right graph in Figure 7.7 shows a typical example of a grafting-from polymerization of MMA, initiated only by the residual free radicals at the surface of the plasma polymerized allylamine film. It is assumed that there is no direct reaction of amino groups with MMA, or that it can be neglected compared to the reaction of MMA with the free radicals. The shift of the surface plasmon resonance minimum after polymerization corresponds to a PMMA thickness of 19 nm, despite the fact that no azo compound was present to initiate the reaction.

86 Attachment of Polymer Monolayers to PPAA Films by Grafting-from Polymerization

e)

III d)

c)

Reflectivity [a.u.] II Reflectivity [a.u.]

b)

a) I

40 50 60 70 80 90 40 50 60 70 80 90 Angle [°] Angle [°] Figure 7.7 SPR reflectivity scans taken after each step of the PMMA attachment process, with (left), and without (right) the immobilization of 4,4-azobis-(4-cyano-pentanoic acid chloride). The left graph shows the reflectivity scans a) of the unmodified substrate (glass / 50 nm Ag / 30 nm SiOx), b) of a 4.6 nm thick adlayer of PPAA (Ppeak = 20 W, ton = 10 ms, toff = 120 ms), c) after immobilization of the initiator (+ 1.2 nm), d) after polymerization measured with p-polarized light, and e) after polymerization, measured with s-polarized light (+ 92.9 nm). On the right side: the reflectivity scans of the bare SPR substrate (I), a 3.4 nm thick adlayer of PPAA (II), and after deposition of a 18.6 nm thick PMMA layer.

7.3.5 AZO INITIATION SUPPORTED BY PEROXIDE INITIATION

In most of the experiments described here, the grafting-from polymerization was initiated by 4,4’-azobis-(4-cyano-pentanoic acid chloride), which was covalently attached to the amino groups present at the surface of PPAA films prior to polymerization. The thickness of the PPAA film, the immobilized azo initiator layer, and the PMMA or PS brush was determined by taking a reflectivity scan after each step. A typical example of the polymerization of MMA initiated by the azo groups is shown in the left graph of Figure 7.7. The resulting PMMA layer is significantly thicker (93 nm) than in the experiment described above without the use of the azo compound (19 nm), where the polymerization was only initiated by peroxide groups. The plasma films in this example (Figure 7.7) were deposited under exactly the same conditions, therefore it is assumed that they contain roughly the same amount of peroxide groups at the surface, that contribute to the overall polymerization. The fraction fazo of the overall PMMA layer thickness that is due to the azo initiator can be estimated by fazo = (93 - 19) / 93 = 0.80.

Typical GPC traces of the surface grafted polymers after degrafting are depicted in

Figure 7.8. The molar mass Mn of both PS and PMMA were seen to be almost constant over the whole range of plasma parameters investigated (± 5.5⋅105 g⋅mol-1 and ± 2.4⋅106 g⋅mol-1 resp.) The polydispersity D for each polymer is also given in the figure. The molecular mass 87 Chapter 7 of the degrafted polystyrene is remarkably high in comparison with the values reported by Prucker and Rühe in a study on the radical chain polymerization of styrene to high surface area silica gels.17 In this work they used an immobilized layer of an AIBN18-like azo initiator to initiate the grafting-from polymerization. They found by GPC measurements that Mn steadily increased with polymerization time at 60 °C. After 14 h of polymerization, when the 5 -1 molecular mass started to level off, a value of Mn = 1.75⋅10 g⋅mol was obtained.

The reason for the high molecular weight of PS found in our study can probably be found in the planar substrates employed in this work, as opposed to the high surface area silica gels employed in their work. The homolysis of an immobilized initiator molecule generates a second nonattached radical, which can generate a freely growing polymer chain in the solution. It has been demonstrated that the most important pathway for deactivation of the surface-attached chains is the reaction between a grafted and a freely growing polymer chain.5a When planar substrates are used, the total number of surface-attached initiator molecules, and accordingly the number of freely growing chains in solution, is much lower than in the experiments with the high surface area silica gel. Therefore this termination mechanism occurs less frequently on planar substrates, leading to longer surface-attached PS chains (higher Mn).

5 -1 M = 2.4·106 g·mol-1 Mn,PS = 5.5·10 g·mol n,PMMA D = 1.9 DPS = 2.0 PMMA w [a. u.] w [a.

45678 log(M / [g·mol-1]) Figure 7.8 GPC traces of poly(styrene) (left) and poly(methyl methacrylate) (right) isolated after degrafting by transesterification.

7.3.6 INFLUENCE OF THE PLASMA DEPOSITION TIME.

The influence of the plasma deposition time on the PMMA layer thickness was studied, while all the other plasma parameters were kept constant. Firstly it is shown in Figure 7.9-a, that the PPAA thickness after extraction increases almost linearly from ± 4 to 26 nm with increasing deposition time. If the polymerization is carried out without previously

88 Attachment of Polymer Monolayers to PPAA Films by Grafting-from Polymerization reacting the PPAA substrate with the initiator, and is only initiated by peroxide groups present at the surface, the thickness of the additional PMMA brush is fairly constant (between 10 and 20 nm), and seems to be independent of the plasma deposition time (7.9-b, lower curve). Other authors have described a linear relationship between the grafting yield and the peroxide concentration at the surface. For example, Dogué et al. reported a maximum acrylamide grafting yield on polypropylene after 8 s of argon plasma treatment, when the peroxide concentration was at a maximum.9a In the system described here, where the mechanism is film deposition as opposed to surface modification, it seems reasonable to assume that the peroxide concentration at the surface is independent of the plasma deposition time. The concentration of reactive sites would then remain constant with varying deposition time, thus explaining the constant thicknesses of the PMMA films.

85 c)

[%] 75

Azo f 65 Γ 1500 PMMA b) azo and peroxo initiated 6x10-8 [mol·m 1000 peroxo initiated -8 [Å] 4x10 500 -8 PMMA 2x10 d -2 0 0 ] 500 a) untreated 400 [Å] 300 immobilized PPAA 200 d 100 0 0 500 1000 1500 2000 2500 3000 3500 4000 Deposition time [s]

Figure 7.9 (a) Layer thickness of PPAA (Ppeak = 20 W, ton = 10 ms, toff = 120 ms) after extraction, as a function of the deposition time, with and without the azo initiator treatment. (b) Layer thickness and graft density of PMMA, as a function of the deposition time, with and without the azo initiator. (c) Fraction of the overall PMMA layer thickness that is due to the presence of the azo initiator.

When the azo compound was immobilized prior to the polymerization, to support the initiation of the grafting reaction, the PMMA layer thickness greatly increased, especially at short deposition times. For a very thin PPAA film, the PMMA layer thickness was found to be remarkably high, up to almost 140 nm on an 8 nm thick plasma film. However, with increasing PPAA thickness (or deposition time), the PMMA thickness gradually decreases to below 50 nm. Following the assumption mentioned above concerning the constant peroxide concentration, the amino group concentration would also be expected to be independent of the deposition time in a film depositing mechanism, and remain constant as well. A possible explanation for this discrepancy could be sought in the previously reported restructuring and reorientation of amine functionalized plasma films.19 Besides the incorporation of oxygen during aging in air, these surfaces are known to build up a concentration gradient over time, in nitrogen- as well as in oxygen containing groups (see §5.3.3 of this Thesis). After several

89 Chapter 7 days of aging in air, the immediate surface region becomes relatively depleted in N, as the polar nitrogen containing groups tend to bury themselves below the surface, away from the hydrophobic atmosphere. So even if the average amino group density of each film is fairly constant for all the PPAA films in Figure 7.9, the density of the amines at the surface may be lower than the density of these groups in the bulk of the film. At the shortest deposition time in these experiments, the film is less than 10 nm thick, so that even after a possible reorientation of the surface, the polar groups are still fairly close to the surface and therefore more readily accessible by the azo initiator molecules, depending on their penetration depth. As the PMMA thickness increases, it would then become more difficult for the azo initiator molecules (or maybe the MMA molecules) to penetrate the plasma polymer deep enough to reach the amino groups, which could account for the decreasing PMMA thickness with increasing PPAA thickness.

A second and more likely explanation is based on the postulation that the average degree of cross-linking of PPAA films increases with deposition time. A WaMS study has shown that the refractive index of these films increase rapidly with increasing deposition time, to eventually level off and become more or less constant (see §4.3.6).14a This suggested the presence of an index gradient within PPAA films. During the growth of each new ‘surface layer’, the already deposited underlying material is still undergoing structural changes. Further crosslinking of the bulk leads to a more densely packed network, which would be less accessible for the initiator- or MMA molecules. In addition to this effect, the underlying bulk material is also subjected to UV radiation emitted during the plasma-on times, which initiates further intermolecular reactions, resulting in a reduction of amino groups. Both the increasing cross-link density and the decreasing amino group density with increasing deposition time are in accordance with the results shown in Figure 7.9.

7.3.7 INFLUENCE OF THE PEAK POWER

It was shown in §4.3.2 that milder power conditions during plasma polymerization result in an increased retention of amine functionalities in the film. Figure 7.10 shows the results of a series of experiments where the plasma peak power was varied between 25 and 250 W at a constant duty cycle of 10/120 ms/ms and at constant deposition time of 5 min. The highest PMMA layer thickness was found on a PPAA film polymerized at the lowest power (25 W). Increasing the plasma peak power leads to a decrease in thickness of the grafted monolayer. It is not clear from this figure if this trend is primarily a result of the variation in the amino group concentration at the surface. From a chemical point of view one could clearly argue that the film deposited at 25 W contains more amines than the high power films, and therefore more reactive sites for the subsequent grafting reaction.

Caution must be taken regarding this point however, since the thickness of the particular PPAA film is only ± 6 nm, i.e. it is significantly less thick than the other three films. The film could also have a lower cross-link density due to the mild power conditions, making it easy for the initiator- and the MMA molecules to reach the reactive sites upon

90 Attachment of Polymer Monolayers to PPAA Films by Grafting-from Polymerization swelling in toluene. The films polymerized at 100, 175 and 250 W show no significant variation with respect to the thickness of the grafted PMMA layer. The decreasing retention of amino groups with increasing power seems to have no (further) influence on the graft density.

1600 -8

6x10 Γ PMMA

1200 [mol·m 4x10-8 [Å]

PMMA 800 d -8 2x10 -2 ]

400 b) 0 200

150 [Å]

100 PPAA

d untreated 50 immobilized a) 0 0 50 100 150 200 250

Ppeak [W]

Figure 7.10 Layer thickness of PPAA (ton = 10 ms, toff = 120 ms) after extraction, with and without the azo initiator treatment (a), and layer thickness and graft density20 of PMMA (b), as a function of the peak power.

7.3.8 INFLUENCE OF THE PLASMA-OFF TIME

Duty cycle [%] 0 5 10 15 20 25 30 35 40 45 1600 -8

6.0x10 Γ 1400 PMMA

[Å] 1200 4.0x10-8 [mol·m 1000 PMMA

d 800 -8

2.0x10 -2

600 ] b) 400 0.0 2000 immobilized 1500 untreated [Å]

1000 PPAA d 500 a) 0 0 20406080100 Equivalent power [W]

Figure 7.11 Layer thickness of PPAA (Ppeak = 250 W, ton = 10 ms) after extraction, with and without the azo initiator treatment (a), and layer thickness and graft density20 of PMMA (b), as a function of the equivalent power or duty cycle.

91 Chapter 7

Similar results as presented above were obtained when the rf duty cycle was varied at constant peak power (Figure 7.11). By increasing the plasma off-time, the equivalent power during deposition decreases, and both FT-IR spectroscopy and XPS revealed an increased retention of the monomer structure in the plasma coating (see also Chapter 4). Different PPAA films were produced at different plasma-off times and used for immobilization with initiator molecules. The two films polymerized under relatively severe power conditions (toff =

15 ms and toff = 40 ms) show no dependency of dPMMA on the off time. Only at much higher off times (e.g. toff = 190 ms), the thickness of the grafted PMMA increases significantly, up to ~ 130 nm.

In an attempt to study the role of the peroxide groups and free radicals in the grafting- from polymerization of PMMA in more detail, a number of reference experiments have been carried out. More specifically, 16 PPAA films were plasma polymerized under the same conditions, and subjected to several different treatments before further polymerization, to investigate the stability of the peroxide groups present in these films. Figure 7.12 gives an overview of the effect of each pretreatment on the resulting polymer layer thickness. The experimental details corresponding to each of the columns are summarized in Table 7.2. Each experiment was carried out in duplo: the given thickness is the average value of the two measurements.

1000 -8 2 800 3.9·10 mol/m a

600

Thickness [Å] Thickness 400 -8 200 2 3.8·10 mol/m b 0 a b I II III IV V VI

Figure 7.12 Layer thickness of PMMA and PS grafted on allylamine plasma polymerized at Ppeak = 250 W, ton = 10 ms, and toff = 190 ms after different treatments. See Table 7.2 for experimental details.

In column I-a, the extracted PPAA films were subjected to the MMA solution in toluene without further treatment. Column I-b shows the result of the polymerization of styrene on the same surface. Although the thickness of the PMMA layer is more than four times higher than the thickness of the PS layer, the graft density of both polymers on this

92 Attachment of Polymer Monolayers to PPAA Films by Grafting-from Polymerization substrate is almost identical (3.9 ⋅10-8 mol/m2 and 3.8 ⋅10-8 mol/m2 resp.), due to the difference in molecular weight. This indicates that the grafting-from polymerization is initiated successfully by the peroxide groups in the plasma film, and that this process is independent of the choice of the monomer (e.g. styrene or methyl methacrylate).

Table 7.2 Layer thickness of PMMA and PS grafted on allylamine plasma polymerized at Ppeak = 250 W, ton = 10 ms, and toff = 190 ms after different treatments. Treatment Thickness [nm] I Peroxide initiated polymerization of MMA (a) and styrene (b) 88 (a) 21 (b) II After treatment with a solution of high molecular weight PMMA (a) 5 (a) and PS (b) in toluene (100 mg/ml) at 60 °C for 24 h 4 (b) III as in I, but after pretreatment with boiling ethanol at 78 °C for 24 h 47 IV as in I, but after pretreatment with boiling toluene at 111 °C for 24 h 50 V as in I, but after pretreatment with boiling pivalinic acid methylester 38 at 101 °C for 24 h VI as in I, but after pretreatment with a solution of 2,2’- 24 azobis(isobutyronitrile) in toluene (0.1 mol/l) at 70 °C for 24 h

In the experiments corresponding to column II, the plasma films were treated with the respective polymers, dissolved in toluene. Only 5 and 4 nm of polymer was found on the PPAA films after 24 h, for the PMMA and PS respectively. If the process occurring during the experiments described in I (Table 7.2) would be an adsorption process of polymer formed in solution, rather than a grafting reaction from the surface, the thicknesses of column II would be comparable to the values given for I. We can therefore conclude that the process in I is truly a grafting-from polymerization, and not an adsorption process.

In the next series of experiments, it was investigated if the amount of peroxide groups in the PPAA could be decreased or removed, by reaction with other peroxide groups or radicals within the film. The films were pretreated with boiling ethanol (column III), boiling toluene (column IV), and with pivalinic acid methylester (column V). This last treatment was chosen because the structure of this compound is very similar to the structure of methyl methacrylate, as is shown below.

O O O pivalinic acid methylester methyl methacrylate

However, pivalinic acid methylester lacks a double bond to start a polymerization reaction. It is assumed that the swelling behavior of a PPAA film in this compound is similar to the swelling in methyl methacrylate. After these respective pretreatments, the films were subjected to the MMA solution for polymerization again. The results clearly indicate a decrease in the PMMA thickness with 40-50 % after any of these pretreatments. The swelling in these solvents, in combination with the high temperatures, apparently resulted in reactions 93 Chapter 7 between peroxide groups or radicals. These reactions also increase the number of cross-links in the film. Both the decrease in the number of reactive groups and the increased cross-link density may contribute to the lower graft thickness of PMMA after polymerization (Table 7.2). On the other hand, even after these treatments under harsh conditions, the grafted PMMA layers are still very thick (e.g. between 38 and 50 nm). The free radicals or peroxide groups that are present in the plasma polymer can be concluded to be very stable, and not likely to be removed completely by a wet chemical treatment.

Finally, the PPAA films were treated with a solution of 2,2’-azobis(isobutyronitrile) (AIBN) in toluene prior to the polymerization. This molecule is probably the most extensively studied initiator for the radical chain polymerization of styrene.2 Reaction with this ‘radical scavenger’ resulted in a further decrease of the thickness of the subsequently grafted PMMA layer to 24 nm.

7.4 Conclusions

Plasma polymerized allylamine films were used successfully as a substrate for the ‘grafting- from’ polymerization of methyl methacrylate and styrene. The amino groups present at the surface of PPAA films were immobilized with the radical initiator 4,4’-azobis-(4-cyano- pentanoic acid chloride). The azo groups could then be utilized to start the thermally induced polymerization of methyl methacrylate or styrene, leading to surface-attached poly(methyl methacrylate) (PMMA) or polystyrene (PS) ‘brushes’. It was found in this study that the peroxides or free radicals present in the plasma deposited film contributed to the grafting 6 -1 reaction as well. High molar masses were determined for both PMMA (Mn = 2.4⋅10 g⋅mol ) 5 -1 and for PS (Mn = 5.5⋅10 g⋅mol ). PMMA layer thicknesses as high as 130 nm were found on the plasma surfaces after polymerization following the proposed modification concept. Although progressive changes in the thickness of the surface attached brush were observed with varying plasma polymerization conditions, the properties of the PPAA films responsible for the trends observed are not fully clear at present. Both the amino group density and the degree of cross-linking in the films are suspected to play a role in the subsequent grafting reaction mechanism.

7.5 Experimental section

7.5.1 SUBSTRATES

For SPR spectroscopy, polished glass slides (BK7, Berliner Glas) were cut in pieces of 2.0 x 2.5 cm2, and cleaned under ultrasound (Sonorex Super RK 510 H, Bandelin) in Hellmanex (Hellma), ultrapure water (Milli-Q Plus 185, Millipore), and ethanol (Riedel-de Haën). A 500 Å thick silver layer topped with a 300 Å thick siliconoxide layer were thermally evaporated (BA 250 E, Balzers) on the substrates. For FT-IR spectroscopy, contact angle measurements, XPS, and AFM, double-sided polished silicon wafers (Aurel or VSI) of

94 Attachment of Polymer Monolayers to PPAA Films by Grafting-from Polymerization orientation <100> and 0.5 mm thick were used. The thickness of the natural silicon oxide layer was typically 30 Å. The substrates were cleaned in ethanol under ultrasound prior to use.

7.5.2 SYNTHESIS

4,4’-azobis-(4-cyano-pentanoic acid chloride). The chemicals and solvents (4,4’- azobis-(4-cyano-pentanoic acid chloride), phosphorus pentachloride (PCl5), dichloromethane, and n-hexane) were purchased from Aldrich, Fluka, or Riedel-de Haën. 28 g (0.10 mol) of 4,4’-azobis-(4-cyano-pentanoic acid chloride) (M = 280.28 g/mol) was carefully added to a suspension of 104 g (0.50 mol) of PCl5 (M = 208.24 g/mol) in 200 ml of dichloromethane, cooled with an icebath. The reaction mixture was allowed to warm to room temperature and stirred overnight. The excess of PCL5 was filtered off, and the volume of the remaining dichloromethane was reduced with approximately 20 % by evaporation. Some additional

PCL5 separated, and was filtered off again. The filtrate was then added to 300 ml of cold n- hexane, causing the separation of the product. The product was filtered off, washed with cold hexane, and dried in vacuum. The yield of 4,4’-azobis-(4-cyano-pentanoic acid chloride) (M = 1 317.17 g/mol) was 22 g (0.07 mol), or 69 %. H-NMR (CDCl3, δ in ppm): 3.23-2.87 (m, 4H,

=N-C(CH3)(CN)-CH2-CH2-CO-Cl), 2.64-2.37 (m, 4H, =N-C(CH3)(CN)-CH2-CH2-CO-Cl),

1.72, 1.66 (2s, 3+3 H, =N-C(CH3)(CN)-CH2-CH2-CO-Cl). Melting point Tm = 91 °C. Plasma polymerized allylamine (PPAA). Allylamine was purchased from Aldrich, and outgassed by multiple repetitive freeze-thaw cycles using liquid nitrogen. The plasma depositions were carried out in the plasma reactor described in §4.2 at 13.56 MHz (radio frequency). The substrates were placed on a glass substrate holder between the two electrodes. After evacuation to a background pressure of approximately 0.005 mbar, the allylamine was introduced into the reaction chamber. Plasma polymerization was initiated after establishing allylamine flow rate (8 sccm) and the reactor pressure (0.20 mbar). The deposition time tdep, plasma-on time ton, plasma-off time toff, and the peak power Ppeak were set individually for each experiment. After each deposition, the substrates were removed from the chamber and the reactor was cleaned with an oxygen plasma for 60 min at 200 W. The substrates were then extracted continuously in ethanol (Riedel-de Haën) for 24 h, in a water-cooled Soxhlet extractor under atmospheric conditions. A second extraction with ethanol did not result in further decrease of the film thickness (see §5.2.2). The reference experiments as described in §7.3.8, Figure 7.12 and Table 7.2, refer to treatments with boiling ethanol (Riedel-de Haën), boiling toluene (Riedel-de Haën), boiling pivalinic acid methylester (Aldrich), and a solution of AIBN (Fluka) in toluene. Immobilization. All the steps were carried out under argon atmosphere. A solution of 5 g/l (0.016 mol/l) 4,4’-azobis-(4-cyano-pentanoic acid chloride) in dry toluene (Riedel-de Haën) was added to the samples. Additionally, 25 ml/l (0.18 mol/l) triethyleneamine (Riedel- de Haën, M = 101.19 g/mol) was added. The mixture was stirred gently for 2 h. The substrates were then rinsed thoroughly with toluene and ethanol, to remove unreacted 4,4’- azobis-(4-cyano-pentanoic acid chloride) and triethyleneamine. Surface polymerization. Polymerizations were performed in toluene/methyl methacrylate mixtures (1/1 v/v) and in pure styrene at 60 °C. All solutions were carefully degassed through at least three freeze-thaw cycles to remove all oxygen traces. After

95 Chapter 7 polymerization for 24 h, the reactions were stopped by allowing the mixture to cool down to room temperature. Subsequently every sample was extracted in toluene using a Soxhlet apparatus for 16 h. This procedure was found to be mandatory to remove all of the nonbonded homopolymer that is formed in solution.2 A second extraction of PMMA or PS modified PPAA in boiling toluene at 111 °C resulted in no further film thickness decrease. Thick films of PMMA and PS for the reference experiments described in §7.2, Table 7.1, were spin- coated (spin-coater, Headway Research) from a 1 % (w) solution in toluene.

7.5.3 CHARACTERIZATION

FT-IR spectroscopy. The IR spectra of polymer films, spin-coated from toluene on NaCl windows, were taken on a Perkin Elmer FTIR spectrometer Paragon 1000 at a resolution of 2 cm-1. All the other spectra were recorded on a Nicolet 730 FTIR Spectrometer at a resolution of 2 cm-1. The spectra of unmodified silicon wafers were used as background spectra. Typically 300 scans were accumulated. Contact angle goniometry. Contact angles of ultrapure water (Milli-Q Plus 185, Millipore) on the various substrates were determined with a contact angle microscope (Krüss or Olympus). The average value of a minimum of six measurements was reported. XPS. X-ray photoelectron spectra were measured using a Perkin-Elmer PSI-5000 series instrument equipped with an X-ray source monochromator. The X-ray source employed is an AlKα at 1486.8 eV. Spectra recorded were taken using a 70 ° take-off angle relative to the sample surface. An electron flood gun was employed to neutralize charge build-up on the films. SPR spectroscopy. The film thicknesses on SPR-silicon oxide substrates were determined using a homebuilt SPR spectrometer, where s- or p-polarized light from a helium- neon laser (λ = 632.8 nm) is coupled into the silver film on the substrates through a glass prism (BK7, Spindler & Hoyer). The reflectivity curves were fitted according to the Fresnel theory, using a refractive index n = 1.515 for BK7 glass, and the dielectric constants ε’ = - 16.000 and ε” = 0.600 for silver. Additionally, the refractive indices n = 1.460 for silicon oxide, n = 1.580 for PPAA films,14a n = 1.489 for PMMA, and n = 1.519 for PS were used. The thickness of the very thin 4,4’-azobis-(4-cyano-pentanoic acid chloride) layer was fitted using the refractive index of PPAA. 1H-NMR-spectroscopy. A Bruker Avance 250 was used to record the 1H-NMR spectra, at a frequency of 250 MHz. CDCl3 (Cambridge Isotope Laboratories) was used as the solvent. AFM. The atomic force microscopy images were taken on a Digital Instruments NanoScope III in tapping mode. GPC. The molar mass of PS and PMMA were determined by GPC (Polymer Standards Service) using THF (Riedel-de Haën) as the eluent. The equipment was calibrated with poly(styrene)-standards (Polymer Standards Service). A differential refractometer (RI ERC 7512, Erma) was used as the detector.

96 Attachment of Polymer Monolayers to PPAA Films by Grafting-from Polymerization

7.6 References

1 a) Fleer, G. J.; Cohen-Stuart, M. A.; Scheutjens, J. M. H. M.; Cosgrove, T.; Vincent, B. Polymers at Interfaces; Chapman & Hall: London, 1993; (b) Bridger, K.; Vincent B. Eur. Polym. J. 1980, 16, 1017; (c) Ben Ouada, H.; Hommel, H.; Legrand, A. P.; Balard, H.; Papirer, E. J. Colloid Interface Sci. 1988, 122, 441. 2 Prucker, O.; Rühe, J. Langmuir 1998, 14, 6893. 3 (a) Boven, G.; Oosterling, M. L. C. M.; Challa, G; Schouten, A. J. Polymer 1990, 31, 2377; (b) Carlier, E.; Guyot, A.; Revillon A. React. Polym. 1992, 16, 115; (c) Prucker, O.; Rühe, J. J. Mater. Res. Soc. Symp. Proc. 1993, 304, 1675. 4 Fery, N.; Laible, R.; Hamann, K. Angew. Makromol. Chem. 1973, 46, 81. 5 (a) Prucker, O.; Rühe, J. Macromolecules 1998, 31, 592.; (b) Rühe, J.; Nachr. Chem. Tech. Lab. 1994, 42, 1237; (c) Rühe, J.; Habilitation Thesis, University of Bayreuth, Bayreuth, Germany, 1995. 6 Mastihuba, M.; Blecha, J.; Kamenická, V.; Lapcík, L.; Topolský, I. Acta Phys. Slov. 1985, 35, 355. 7 Yasuda, H. Plasma Polymerization, Academic Press Inc.: Orlando, FL, 1985. 8 (a) Suzuki, M.; Kishada, A.; Iwata, H.; Ikada, Y. Macromolecules 1986, 19, 1804; (b) Tretinnikov, O. N.; Ikada, Y. Macromolecules 1997, 30, 1086. 9 (a) Dogué, I. L. J.; Förch, R.; Mermilliod, N. J. Adhesion Sci. Technol. 1995, 9, 1531; (b) Dogué, I. L. J.; Mermilliod, N.; Förch, R. Nuclear Instruments and Methods in Physics Research B 1995, 105, 164. 10 Sugiyama, K.; Kato, K.; Kido, M.; Shiraishi, K.; Ohga, K.; Okada, K.; Matsuo, O. Macromol. Chem. Phys. 1998, 199, 1201. 11 Lee, K. R.; Yu, S. J.; Huang, S. L.; Wang, D. M.; Lai, J. Y. J. Appl. Polym. Sci. 1998, 67, 1789. 12 Suzuki, M.; Kishida, A.; Iwata, H.; Ikada, Y. Macromolecules 1986, 19, 1804. 13 (a) Gengenbach, T. R.; Vasic, Z. R.; Chatelier, R. C.; Griesser, H. J. J. Polym. Sci.: Part A: Polym. Chem. 1994, 32, 1399; (b) Gengenbach, T. R.; Griesser, H. J. Surf. Interface Anal. 1998, 26, 498; (c) Gengenbach, T. R.; Chatelier, R. C.; Griesser, H. J. Surf. Interface Anal. 1996, 24, 271; (d) Gengenbach, T. R.; Chatelier, R. C.; Griesser, H. J. Surf. Interface Anal. 1996, 24, 611; (e) Gengenbach, T. R.; Griesser, H. J. J. Polym. Sci.: Part A: Polym. Chem. 1999, 37, 2191; (f) Gengenbach, T. R.; Griesser, H. J. J. Polym. Sci.: Part A: Polym. Chem. 1998, 36, 985; (g) Gengenbach, T. R.; Griesser, H. Polymer 1999, 40, 5079. 14 (a) van Os, M. T.; Menges, B.; Förch, R.; Vancso, G. J.; Knoll, W. Chem. Mater. 1999, 11, 3252; (b) van Os, M. T.; Menges, B.; Timmons, R. B.; Knoll, W.; Förch, R. ISPC-13 Symp. Proc. 1997, 3, 1298. 15 Details about the degrafting procedure can be found in: Stöhr, T. Ph.D. Thesis Universität Mainz, 2000. 16 It was not possible from the experiments described in this chapter to distinguish between the contributions on the poymerization process of the free radicals on the one hand, and the peroxide groups on the other hand. When the term “peroxide initiated polymerization” is used, this refers to the polymerization initiated by the peroxide groups and the free radicals. 17 Prucker, O.; Rühe, J. Macromolecules 1998, 31, 602. 18 2,2’-azobis(isobutyronitrile). 19 Gengenbach, T. R.; Chatelier, R. C.; Griesser, H. J. Surf. Interface Anal. 1996, 24, 611. 20 The graft density Γ was calculated from Eq. 7-1.

97

Protein Adsorption to Plasma Modified Surfaces studied by SPR Spectroscopy and AFM

&KDS WHU

Protein Adsorption to Plasma Functionalized Surfaces

Studied by Surface Plasmon Resonance Spectroscopy and

∗ Atomic Force Microscopy

Plasma modification provides a powerful tool to tailor the surface properties of materials. Surface characteristics such as wettability, chemistry and morphology are known to influence protein adsorption, and the subsequent attachment and spreading of cells on biomaterials. To improve the understanding of protein-surface interactions we functionalized gold and silicon surfaces with amino or ether groups, using radio frequency plasma polymerization of ethylenediamine, allylamine, cycloheptylamine and di(ethylene glycol)-vinyl ether (EO2V). The functional group density at the surface was controlled by using different monomers or by variation of the input power during the plasma deposition. The adsorption of the proteins fibrinogen, bovine serum albumin and immunoglobulin G to these surfaces was measured in situ with surface plasmon resonance spectroscopy. The tenacity of the protein adsorption on the different substrates was also measured, after removing elutable protein with 1 % sodium dodecyl sulfate (SDS) solution. After drying, the protein layers were studied by tapping mode atomic force microscopy (TM-AFM). The results obtained show that both the protein adsorption to and the retention on the surfaces are affected greatly by the surface functionalities. All the amine functionalized surfaces showed a high affinity toward the proteins, and thin dense layers of adsorbed protein remained on these surfaces, even after rinsing with SDS solution. A large contrast in protein affinity was observed between the EO2V films polymerized at different power input conditions. Dramatic reduction in both initial adsorption and retention of all proteins was observed on these films with decreasing power. The low degree of cross-linking, as well as the high retention of ether content during the polymerization of EO2V under low power input conditions is thought to result in the production of biologically non-fouling surfaces.

∗ Part of the work described in this Chapter will be submitted for publication: Menno T. van Os, A. Toby A. Jenkins, Mária Péter, Renate Förch, Richard B. Timmons, G. Julius Vancso, Wolfgang Knoll, Protein Adsorption to Plasma Functionalized Surfaces Using Surface Plasmon Resonance Spectroscopy and Atomic Force Microscopy, to be submitted. 99 Chapter 8

8.1 Introduction

One of the most promising applications of plasma deposited thin films and plasma treated materials is to be found in the field of developing new biomaterials and to improve the performance of existing materials and devices.1 Biomaterials have been defined as ‘non- viable materials used in a biomedical device intended to interact with biological systems’.2 They include polymers (fibers, rubbers, molded plastics, emulsions, coatings, porous solids), metals, ceramics, reconstituted or specially treated natural tissues, and composites made from various combinations of such materials.3 Plasma polymerized films for biomedical applications offer unique advantages, such as: • Plasma deposition can be used to modify almost any solid substrate, including metals, ceramics, and polymers. Other surface modification techniques are highly dependent on the chemical nature of the substrate.1 • The coating can modify the chemical nature of a surface without affecting the surface topography and without altering the bulk characteristics of the substrate material.4 • The pinhole-free and crosslinked nature of plasma films makes them interesting as permeation barriers and protective films.5

There is a substantial need for biomaterials that can interact with blood and tissue without triggering undesirable responses such as thrombosis,6 complement activation,7 inflammation (typically accompanied by the accumulation of phagocytic cells),7c,8 and device- associated infections.9,10 Surface properties of biomaterials, such as chemical composition, wettability, domain composition, and morphology, can strongly influence biological reactions at foreign interfaces.3,10 It is believed that the first response when a biomaterial is brought in contact with blood or tissue fluids, is the adsorption of biomolecules, usually proteins.3 The adsorption of protein is much faster than the migration of cells to foreign surfaces, and it is generally accepted that this initial adsorbed protein layer dictates subsequent cellular responses at the interface.10,11 Although significant research efforts have been made to improve the biocompatibility of materials by modification of surface and chemical properties of the surface,12 it is still unclear which particular surface properties are critical to the host responses.10 Several groups assume that hydrophobic interactions between the surface and hydrophobic patches on the surface of the protein play an important role in the protein adsorption processes.13 Following the initial adsorption, that is probably weak and reversible, the protein may undergo conformational changes, and unfold to expose more hydrophobic groups,13a and ultimately develop tight bonds with the biomaterial surface.14 Earlier studies have shown that the resistance of adsorbed protein to sodium dodecyl sulfate (SDS) wash is a good indicator of the extent of denaturation of proteins.10,12d,15

8.1.1 AMINE-FUNCTIONALIZED SURFACES

Materials that are functionalized with amine groups offer promising characteristics for a variety of biomedical applications. Ammonia plasma discharges have been used to provide binding sites for immobilization of heparin on different polymer surfaces.16 The wettability of a n-butane plasma polymer substrate has successfully been increased by adding nitrogen to 100 Protein Adsorption to Plasma Modified Surfaces studied by SPR Spectroscopy and AFM the n-butane gas, resulting in an increased fibrinogen adsorption but a slightly lower human serum albumin (HSA) adsorption.17 Plasma coatings from allylamine on poly(ethylene terephtalate) (PET) have been found to both adsorb and denature much more fibrinogen than unmodified PET surfaces.10,15b In a study by Burns et al., silicon substrates were modified by plasma deposition of 1,2-diaminocyclohexane (DACH), acrylic acid (ACA), and hexamethyldisiloxane (HMDSO). Adsorption of the three test proteins HSA, immunoglobulin G (IgG) and fibrinogen was found to be the greatest on the DACH surface, which was attributed to a positive charge of the DACH surface at pH = 7.2, while these proteins are slightly negatively charged at this pH.18

Allylamine deposited films proved their value in immunoassays. By adsorbing a relatively high amount of F(ab’)2 anti-human IgG antibody, these surfaces have been successful in increasing the sensitivity towards the detection of IgG.19 Karube et al. demonstrated that ethylenediamine, plasma polymerized on gold, was also suitable for use in immunosensing, detected by SPR spectroscopy20 or a quartz crystal microbalance.21

Coatings containing amine groups also received much attention as useful substrates in nerve cell tissue culture studies.22 Timmons et al. addressed the advantages of the use of plasma polymerization over other, generally more complex, wet chemical surface maintain processes.23 Utilization of a pulsed plasma resulted in stable allylamine surfaces for neuronal cell adhesion, even after three autoclaving cycles.24 Adsorbed fibrinogen has been implicated by several groups in mediating the adhesion of blood platelets to different polymers.25 Surprising results to this effect were obtained by Horbett et al. on the adsorption of baboon fibrinogen and the adhesion of platelets to a plasma polymerized allylamine film. Plasma preadsorption studies with fibrinogen deficient media suggested that adsorbed fibrinogen is indeed necessary for platelet adhesion. However, platelet adhesion was found to be strongly dependent on the conformation of adsorbed fibrinogen. A strong decrease in the SDS elutability of fibrinogen was observed, when the preadsorbed substrates were stored in a buffer solution for five days before platelet contact, reflected by a diminished platelet adhesion on the more tightly adherent fibrinogen after these five days.26 A study by Timmons et al. showed that there is a strong correlation between the amounts of surface denatured fibrinogen and the total numbers of adherent phagocytes after implantation of variously treated substrates in mice.10 More phagocytes were found on surfaces with high amounts of denatured (i.e. SDS non-elutable) fibrinogen, indicating that these surfaces could trigger stronger inflammatory responses. Ko et al. found that allylamine plasma modification of low- density polyethylene (LDPE) had a negative effect on blood thromboresistance.

8.1.2 (POLYETHYLENE OXIDE)-FUNCTIONALIZED SURFACES

It is well known these days that polyethylene oxide (PEO) containing surfaces can greatly reduce the adsorption of proteins and the adhesion of cells to biomaterials.27 Because of the medical significance of these biological non-fouling coatings, intense research efforts have been directed towards attachment procedures of various PEO molecules to a range of substrates.28

101 Chapter 8

These attachment procedures include the adsorption of PEO homopolymers directly on a substrate,29 or the adsorption of PEO block copolymers to hydrophobic substrates.30 PEO has also been attached to various substrates covalently, through the coupling of (modified) PEO molecules with targeted surfaces,31 grafting to a surface via a backbone polymer,32 or by direct PEO incorporation as block segments in polymeric substrates.33 Crosslinked PEO homopolymer networks have been prepared, by radiation34 or chemical cross-linking.35

The plasma deposition technique has also been proposed as an attractive alternative approach to PEO surface modification. Gombotz et al. introduced amino groups on a PET substrate by exposing it to an allylamine plasma glow discharge, which were then immobilized with PEO using cyanuric chloride chemistry.28 In addition to this indirect approach, the direct PEO modification through plasma deposition has also been reported. Plasma deposition of tetraethylene glycol dimethyl ether resulted in surfaces with high short- term resistance to biomolecular adsorption.36 In a study from Beyer et al., tri(ethylene glycol) monoallyl ether was plasma polymerized under both CW and pulsed conditions. On the films that were deposited using low rf power during the CW runs and low rf duty cycles during the pulsed plasma experiments, dramatic reductions in both initial adsorption and retention of bovine albumin were observed.37 Intensive research on the generation of biologically non- fouling PEO surfaces was performed by Wu.15b Films plasma polymerized from EO2V or 12- crown-4 ether were shown to greatly reduce the fouling characteristics of PET and polyethylene (PE), as was evaluated by adsorption studies of radioactively labeled proteins and in vivo cell adhesion studies using mouse intrapertoneal implantation.

8.1.3 CHARACTERIZATION

In response to the increasing need to understand protein-surface interactions, and the urge to test new biomaterial coatings, many techniques have been developed to probe protein adsorption behavior. The binding affinity between a surface and a protein is most commonly assessed through exposure of the substrate to a solution containing 125I-radiolabeled proteins.10,15b,26,28,37,38 Although this technique is extremely accurate and sensitive, it is also expensive, requires specialized training and equipment, and the efficiency of the reaction and the stability of the product may vary from protein to protein.39 Fluorescence spectroscopy has the advantage that it can be used to study protein adsorption in situ,40 but the fluorescent tags may have unknown effects on the adsorption characteristics. Other methods that have been used include colorimetric techniques,41 MALDI mass spectrometry,39,42 ellipsometry,43 and X- ray reflectivity.44

In this study we have combined the thickness sensitivity of surface plasmon resonance (SPR) spectroscopy with the lateral information obtained from atomic force microscopy (AFM) to explore the extent of protein adsorption to various plasma deposited test surfaces. When AFM is used to obtain topographic information on soft samples such as adsorbed biolayers, the lateral force exerted by the tip can lead to image artefacts due to disrupture of the surface.43b,43c,45 This problem has been solved recently by the development of a new AFM technique, known as tapping mode AFM. This tapping mode employs a cantilever oscillating 102 Protein Adsorption to Plasma Modified Surfaces studied by SPR Spectroscopy and AFM with high amplitude, thereby contacting the surface once every period.43b Since the tip only intermittently “taps” the surface, the tip-sample interactions are greatly reduced, allowing for minimal disturbance of the adsorbed molecules.46 The use of this technique has enabled researchers to image the molecular packing and aggregation of proteins on surfaces such as silicon,43b methylated silicon,43c self-assembled monolayers,44 spin-cast polymer films,46 and on mica.47 SPR spectroscopy has been used extensively to measure protein adsorption in situ with great sensitivity.20,48

To the best of our knowledge, results on AFM imaging of proteins adsorbed on plasma modified substrates has not been published previously. However, in one particular study from Holland et al., proteins have been observed in topographic images, adsorbed to biomaterial surfaces with significant roughnesses (i.e. polydimethylsiloxane, low-density polyethylene, and expanded polytetrafluoroethylene).49 It is well known that plasma films in general are considered to be relatively smooth and pinhole-free. If the differences in morphology between the surface of the plasma polymer and the adsorbed protein layer are distinctive enough, it may be possible to obtain valuable information about the molecular structuring of proteins at plasma modified biomaterials by AFM imaging.

8.2 Results and discussion

In earlier studies, the influence of the monomer and the deposition conditions on the presence of specific functional groups in plasma films used in this work, has been investigated using XPS, FTIR spectroscopy, and water contact angle measurements.15b,50 The wettability of the films polymerized from the monomers containing amine groups increased in the order cycloheptylamine < allylamine < ethylenediamine, as can be seen in Table 8.1. This was confirmed by XPS analysis, which revealed an increasing N/C atom ratio in the same order.

Table 8.1 Monomers, deposition conditions, and water contact angles of the plasma coatings, and water contact angles of the self-assembled monolayer.

Monomer Abbreviation Ppeak Duty cycle θa θr [W] [ms/ms] [°] [°] Ethylenediamine EDA 100 5/95 14 7 Allylamine AA 100 5/95 38 19 Cycloheptylamine CHA 100 5/95 80 33

Di(ethylene glycol)-vinylether EO2Vlow 50 5/95 69 44

Di(ethylene glycol)-vinylether EO2Vhigh 200 10/40 94 53 Octadecanethiol SAM SAM - - 112 104

It has been shown by Wu that decreasing the input power during the pulsed plasma polymerization of EO2V leads to a higher retention of ether linkages in the coating.15b At the lowest power conditions (e.g. 10/300 ms/ms and Ppeak = 10 W), high resolution C1s XPS spectra and FTIR spectra were practically identical to those of conventionally synthesized EO2V polymer. All the plasma films used in this work were subjected to a cold extraction in 103 Chapter 8 ethanol prior to protein adsorption, to remove any unreacted monomer or other low molecular weight material from the film. The self-assembled monolayer of octadecanethiol was chosen for its smoothness, to obtain information about the influence of the relative surface roughness of plasma coatings on the resolution of the AFM images.

8.2.1 ADSORPTION AND DENATURATION OF PROTEINS ON TEST SURFACES

It is generally acknowledged that the protein adsorption process is driven by a combination of factors. Firstly, it has been demonstrated that surface functionality can influence protein-surface interactions.10,12a,d,51 Secondly, partly related to the surface functionality, hydrophobic interactions between the protein and the surface are believed to play an important role in protein adsorption and retention.13a-c,43b,52 Thirdly, electrostatic interactions can contribute to the adsorption process. If ionizable groups are present at the biomaterial surface, the pH and the ionic strength of the protein solution are important parameters to consider.10,17,18,53,54 It is however extremely difficult to determine which factors are most important as driving forces for protein adsorption, if not practically impossible. If one of the former parameters is varied, other surface properties are likely to be affected as well, directly or indirectly. In addition to this, there are many other factors that to some extent affect the adsorption, such as: protein concentration, structural stability of the protein, isoelectric point (IP) of the protein, morphology of the surface, and domain composition.

Figure 8.1 A schematic representation of structure and surface adsorption of a) BSA, b) IgG, and c) fibrinogen, taken from Green et al..55

Figure 8.1 gives a schematic representation of the proteins studied in this work. Albumin is known to be a globular protein with dimensions of approximately 8 nm by 4 nm, and has a molecular weight of 68,460 Da. The molecular weight of IgG is 156,000 Da. It is a

Y shaped molecule, consisting of two identical antigen binding Fab arms, and a Fc region. Fibrinogen has been shown to have a triad structure of length 45 nm, and has a molecular weight of 340,000 Da.55 104 Protein Adsorption to Plasma Modified Surfaces studied by SPR Spectroscopy and AFM

Figure 8.2 shows the adsorption profiles of the proteins on the five plasma coatings and the SAM. It can be seen in Figure 8.1-a,b that the highest amount of BSA and IgG is adsorbed to the EO2Vhigh surface. This observation is in large contrast with the extremely low adsorption to the EO2Vlow film. The reason for the dramatic difference in affinity towards these two films polymerized from the same monomer can be explained by the difference in input power during deposition. It has been shown previously that during the pulsed plasma polymerization of EO2V monomer, both the peak power and the duty cycle have a large influence on the retention of both the hydroxyl and the ether content in the resulting film. Characterization by XPS and FTIR spectroscopy pointed out that at high power input conditions (e.g. EO2Vhigh), the ether as well as the hydroxyl content in the film were greatly reduced as compared to milder polymerization conditions.15b

In addition to the effect on the film chemistry, a lower input power also results in a lower degree of cross-linking within the film. A decreased cross-linking density results in a larger conformational freedom of the polymer chains between the cross-links. Protein adsorption would reduce this conformational freedom, thus making it entropically unfavourable.17

As for the other substrates, no significant variations in BSA adsorption were observed. Although the advancing water contact angles of the surfaces varied from 14° (EDA) to 112° (SAM), the thickness of the BSA layer adsorbed to these surfaces was almost identical (± 20 Å). A similar conclusion can be drawn from Figure 8.2-b about the IgG adsorption, with the exception of the allylamine modified substrate, to which the adsorption is roughly three times lower than to the SAM, EDA, and CHA surface. It may carefully be concluded until this point that hydrophobic interactions do not seem to be the major driving force for the adsorption behavior of these two proteins.

The affinity of fibrinogen towards these surfaces (Figure 8.2-c) is quite different from the behavior observed for BSA and IgG. The thickest layer of fibrinogen is formed on the AA substrate (± 170 Å), whereas the EO2V film is covered with only ± 65 Å of fibrinogen after 1.5 h of incubation. Again, no dependency of the protein adsorption on the surface wettability can be found.

105 Chapter 8

60 a) EO2V 50 high

40

AA 30

SAM 20 Thickness [Å] Thickness CHA 10 EDA

EO2V 0 low

0 1000 2000 3000 4000 5000 Adsorption time [s]

140 b) 120

EO2Vhigh 100

80

60 CHA SAM

Thickness [Å] Thickness 40 EDA

20 AA

0 EO2Vlow

0 1000 2000 3000 4000 5000 Adsorption time [s]

200 c) 180

160 AA 140 Buffer wash 120

100 CHA

80 EDA SAM Thickness [Å] Thickness 60 EO2Vhigh

40

20

0 EO2Vlow

0 1000 2000 3000 4000 5000 Adsorption time [s] Figure 8.2 Adsorption of a) BSA, b) IgG, and c) fibrinogen to the various test surfaces at pH 7.

As far as electrostatic interactions are concerned, the IP values of the proteins, the pH value during the adsorption experiments, and the ionic strength of the solutions are important parameters to consider. Since all the experiments were carried out at adsorption pH 7.0, both the BSA (IP 4.7) and the fibrinogen (IP 5.8) can be expected to be negatively charged, while 106 Protein Adsorption to Plasma Modified Surfaces studied by SPR Spectroscopy and AFM the IgG (IP 6.5-8.0) may be slightly positively charged or neutral.43c In the literature, plasma polymerized films containing amine groups are often referred to as being positively charged at pH 7, due to protonation of amine groups. Indeed, typical thermodynamical pKa values of amines in solution are in the range of 10.5.56 We have previously reported that the actual ‘surface pKa’ of plasma polymerized allylamine films is lower than 7 (e.g. ~ 5.5), indicating that these surfaces would still be neutral at this pH value.57 The reason for this shift is that the local environment of the amino groups in the allylamine films is somewhat hydrophobic. Upon lowering the pH, the amino groups resist protonation since the stabilization of the charge is low in the hydrophobic environment. This causes a shift of the thermodynamic pKa 58 to lower values. Similar shifts have been previously reported for -NH2 terminated SAMs. For disordered SAMs of amino-terminated silanes, a force pKa of 3.9 was reported previously in the literature.58,59 Negative surface charges have also been observed on this type of films at sufficiently high pH values. This phenomenon however is due to oxygen-containing groups at the surface, resulting from the reaction of residual free radicals in the film with atmospheric oxygen.17,60

The results in Figure 8.2 are in accordance with the assumption that the surfaces of the amine-functionalized films carry no net charge at pH 7. If the amino groups would be 2 protonated at the adsorption pH, the surface charge σ0 [µC/cm ] would most probably increase in the order CHA < AA < EDA, based on the assumption that fragmentation of the amines present in the monomer is minimal at the mild power conditions used during polymerization of these films. Albumin is a protein with a high charge density, but no significant differences in adsorption are observed between the three substrates, despite the negative charge of the protein.

In Figure 8.2-b, where the adsorption of IgG is plotted, electrostatic interactions can probably be neglected, due to the (almost) neutral character of surface and protein at the adsorption pH. In this case, the influence of hydrophobic interactions may be more pronounced, based on the high adsorption to EO2Vhigh, CHA, and SAM, the surfaces with the highest contact angles.

As shown in Figure 8.3-(c), the total amount of SDS nonelutable (denatured) fibrinogen is extremely high on EDA, the most hydrophilic surface. This low elutability of fibrinogen has been observed previously on plasma polymerized allylamine surfaces.10,26 Only relatively small amounts of fibrinogen are retained on the more hydrophobic SAM and CHA surface, as compared to the initial adsorption to these surfaces. It is possible that hydrophobic interactions dictate the initial adsorption of fibrinogen to the surface, but that these forces are not strong enough to change the conformation of the protein dramatically. On hydrophilic surfaces, the fibrinogen apparently unfolds almost completely, suggesting the formation of strong bonds with the amino groups at the interface.

107 Chapter 8

700 Initial adsorption a) ]

2 Retention 600

500

400

300

200

Protein adsorption [ng/cm adsorption Protein 100

0 EDA CHA EO2V EO2V SAM high low

1600 b) Initial adsorption 1400 ] Retention 2

1200

1000

800

600

400

200 Protein adsorption [ng/cm

0 EDA CHA EO2V EO2V SAM high low

2400 Initial adsorption c)

] 2200

2 Retention 2000 1800 1600 1400 1200

1000 800 600 400 Protein adsorption [ng/cm adsorption Protein 200 0 EDA CHA EO2V SAM EO2Vhigh low

Figure 8.3 The extent of a) BSA, b) IgG, and c) fibrinogen adsorption and retention (e.g. SDS nonelutable protein) to various test surfaces after ± 1.5 h of incubation.

108 Protein Adsorption to Plasma Modified Surfaces studied by SPR Spectroscopy and AFM

a) b)

0 1 µm 0 1 µm

c) d)

0 1 µm 0 1 µm Figure 8.4 TM-AFM images of a) plasma polymerized allylamine (AA) film on a silicon substrate (rms surface roughness 6.7 Å) and the proteins b) BSA (rms surface roughness 14.3 Å), c) IgG (rms surface roughness 11.3 Å), and d) fibrinogen (rms surface roughness 10.7 Å) adsorbed on AA after 1.5 h of incubation at pH 7, rinsing with SDS, rinsing with water and drying in air. The color scale ranges from dark (0 nm) to bright (10 nm).

Figure 8.4-a shows an AFM image of an AA film, with a rms surface roughness of 6.7 Å. Single molecules of BSA can be recognized in Figure 8.4-b, as well as aggregated molecules. The typical very small feature sizes that are observed in the image of the plasma film can not be seen after BSA adsorption, which could indicate that a complete layer of protein remains on the surface, even after rinsing with SDS. That the rms surface roughness does not increase dramatically after BSA adsorption also points in the direction of a very high surface coverage. The same can be said for the IgG and especially for the fibrinogen, where also the larger topographical features of the plasma polymerized AA film have been ‘smoothened’ by a dense protein layer. 109 Chapter 8

a) b)

0 1 µm 0 1 µm

c) d)

0 1 µm 0 1 µm Figure 8.5 TM-AFM images of a) plasma polymerized cycloheptylamine (CHA) film on a silicon substrate (rms surface roughness 4.9 Å) and the proteins b) BSA (rms surface roughness 5.2 Å), c) IgG (rms surface roughness 5.8 Å), and d) fibrinogen (rms surface roughness 7.0 Å) adsorbed on CHA after 1.5 h of incubation at pH 7, rinsing with SDS, rinsing with water and drying in air. The color scale ranges from dark (0 nm) to bright (10 nm).

The single protein molecules can be seen even more clearly on the CHA substrate (Figure 8.5), where the spots formed by the fibrinogen are slightly larger in size than the BSA and IgG spots. Again, it appears that the whole surface is covered with protein in a homogeneous way. In the topographic image of BSA, shown in Figure 8.5-b, some smearing can be observed, due to lateral forces exerted by the probe tip.

110 Protein Adsorption to Plasma Modified Surfaces studied by SPR Spectroscopy and AFM

a) b)

0 1 µm 0 1

c) d)

0 1 µm 0 1

Figure 8.6 TM-AFM images of a) plasma polymerized di(ethylene glycol) vinyl ether (EO2Vlow) film on a silicon substrate (rms surface roughness 3.1 Å) and the proteins b) BSA (rms surface roughness 3.5 Å), c) IgG (rms surface roughness 4.3 Å), and d) fibrinogen (rms surface roughness 4.7 Å) adsorbed on EO2Vlow after 1.5 h of incubation at pH 7, rinsing with SDS, rinsing with water and drying in air. The color scale ranges from dark (0 nm) to bright (5 nm).

The results of the AFM imaging of the EO2V films after subjection to the various protein solutions are shown in Figure 8.6. Some IgG molecules seem to be present at this surface, which is surprising considering that no shift of the SPR minimum angle was observed in the kinetic studies. The surface concentration of this protein is very low however, and the image hardly differs from the image of the EO2V surface. Both BSA and the fibrinogen are not detected in this figure, confirming the biologically non-fouling character of this film.

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a) b)

0 1 µm 0 1 µm

c) d)

0 1 µm 0 1 µm Figure 8.7 TM-AFM images of a) octadecanethiol SAM on gold (rms surface roughness 5.2 Å) and the proteins b) BSA (rms surface roughness 47.6 Å), c) IgG (rms surface roughness 26.0 Å), and d) fibrinogen (rms surface roughness 7.1 Å) adsorbed on the SAM after 1.5 h of incubation at pH 7, rinsing with buffer and drying in air. The color scale ranges from dark (0 nm) to bright in a) 7 nm, b) 40 nm, c) 40 nm, and d) 10 nm.

Interesting patterns are found on the SAM surface after incubation with BSA solution (Figure 8.7-b). The images in this figure were taken before the washing step with SDS, and presumably show the initially adsorbed protein (after possible restructuring in air). Remarkably similar aggregation patterns as the one showed in Figure 8.7-b have been observed by Sheller et al. after adsorption of human serum albumin (HSA) to a SAM of hexadecyltrichlorosilane.44 It is interesting to note that this pattern disappeared completely after washing with SDS and subsequent rinsing with water and drying in air (Figure 8.8-a). It seems that the SDS treatment not only removes adsorbed albumin from the surface, but also causes changes in the conformation of the protein. Before the SDS wash, the BSA covers

112 Protein Adsorption to Plasma Modified Surfaces studied by SPR Spectroscopy and AFM roughly half of the surface, leaving the rest of the surface uncovered. It is likely that in its original conformation, it is energetically favorable for the protein to form local clusters, rather than to unfold and cover the whole substrate. It is not unlikely that the SDS interacts not only with the protein, but also with the surface of the film, causing the protein to cover the film in a more homogeneous way. Another possibility is that the SDS treatment changes the charge distribution of the protein (e.g. albumin is negatively charged at the experimental conditions with an IP of 4.7, and has a high charge density), thus increasing the influence of electrostatic interactions in the overall adsorption process. Alternatively, subjection of the adsorbed BSA to the SDS could also cause internal restructuring of the protein, thereby exposing more hydrophobic groups to the solution and the surface. This restructuring and desorption is accompanied by a decrease in the rms surface roughness from 47.6 Å to 12.2 Å.

a) b)

0 1 µm 0 1 µm

c)

0 1 µm Figure 8.8 TM-AFM images of the proteins a) BSA (rms surface roughness 12.2 Å), b) IgG (rms surface roughness 20.9 Å), and d) fibrinogen (rms surface roughness 48.7 Å) adsorbed on the SAM after 1.5 h of incubation at pH 7, rinsing with SDS, rinsing with water and drying in air. The color scale ranges from dark (0 nm) to bright in a) 15 nm, b) 30 nm, and c) 60 nm.

113 Chapter 8

The IgG initially forms a dense layer on the SAM, only leaving a small part of the surface uncovered (the black holes in Figure 8.7-c). Possibly a multilayer is formed, since the individual IgG molecules can not be detected in the image. After SDS treatment, these single proteins can be seen very clearly Figure 8.8-b). It appears that initially, one monolayer of IgG is tightly bound to the surface during incubation. Other IgG molecules may be adsorbed loosely on to this monolayer, and are removed by rinsing the sample with SDS. The fibrinogen forms a smooth and dense layer on the SAM surface, with a rms surface roughness of only 7.1 Å. After SDS treatment, significant amounts of protein were desorbed locally from the surface, leaving large holes in the otherwise smooth layer.

8.3 Conclusions

The adsorption of proteins to plasma polymer surfaces was investigated in detail in by surface plasmon resonance spectroscopy and tapping mode atomic force microscopy. Si and Au substrates were successfully functionalized with amino or ether groups by plasma polymerization, or with a self-assembled monolayer (SAM) of octadecanethiol. The results obtained showed that both the initial protein adsorption to and the retention on the surfaces were affected greatly by the different surface functional groups. All the amine-functionalized surfaces exhibited a high affinity toward the proteins, and thin dense layers of adsorbed protein remained on these surfaces, even after rinsing with SDS solution. A large contrast in protein affinity was observed between the ether group containing plasma films polymerized at different power input conditions. Dramatic reduction in both initial adsorption and retention of all the proteins tested was observed on these films upon decreasing the power input. The low degree of cross-linking, as well as the high retention of ether content during the polymerization under mild power input conditions was thought to result in the production of biologically non-fouling surfaces. TM-AFM and SPR spectroscopy form a powerful combination of techniques for the characterization of protein adsorption to plasma modified surfaces. Single protein molecules, as well as formed aggregates could be detected by TM- AFM on most of the substrates.

8.4 Experimental Section

8.4.1 SUBSTRATES

For SPR spectroscopy, polished glass slides (LaSFN9) were cut in pieces of 2.0 x 2.5 cm2, and cleaned under ultrasound (Sonorex Super RK 510 H, Bandelin) in Hellmanex (Hellma), ultrapure water (Milli-Q Plus 185, Millipore), and ethanol (Riedel-de Haën). A 48 nm thick gold layer was thermally evaporated (BA 250 E, Balzers) on the glass slides. Double-sided polished silicon wafers were used as substrates in the contact angle studies and the AFM analysis for all the plasma films. In all the measurements performed on the self- assembled monolayer clean Au(111) was used as the substrate.

114 Protein Adsorption to Plasma Modified Surfaces studied by SPR Spectroscopy and AFM

8.4.2 PLASMA POLYMERIZATION AND SAM PREPARATION

Plasma Polymerization. Ethylenediamine, allylamine, cycloheptylamine, and di(ethylene glycol)-vinyl ether were obtained from Aldrich. Prior to use monomers were outgassed by multiple freeze-thaw using liquid nitrogen. Reactions were carried out in an all- glass cylindrical reactor (Figure 4.1). The 13.56 MHz rf power was delivered to the reactor through two concentric electrodes located around the exterior of the reactor. A detailed description of the system is provided in §4.2. The substrates were placed on a glass substrate holder in the center of the reaction chamber between the two electrodes. The reactant monomer was introduced into the reaction chamber after the system was evacuated to a background pressure of 0.004-0.010 mbar. Plasma polymerization of ethylenediamine was carried out at a monomer pressure of 0.078 mbar, and at an adsorbed rf power of 100 W. The plasma-on time was 5 ms. To obtain good adhesion between the plasma film and the substrate, the plasma-off time was 20 ms during the first 10 min, after which the off time was increased to 95 ms for the final 15 min of deposition (EDA). The monomer pressure of the allylamine was 0.080 mbar, and polymerization was carried out at Ppeak = 100 W, ton = 5 ms, toff = 20 ms (5 min) and 95 ms (15 min) (AA). Cycloheptylamine was polymerized after establishing a monomer pressure of 0.073 mbar, at 5/20 ms/ms for 2 min and at 5/95 ms/ms for 6 min (CHA). The monomer pressure of the di(ethylene glycol)-vinyl ether was 0.018 mbar. Films were polymerized at Ppeak = 100 W at 10/40 ms/ms for 15 min (EO2Vhigh), and at

Ppeak = 50 W at 5/20 ms/ms for 4 min and continued at 5/95 ms/ms for 8 min (EO2Vlow). After each deposition, the substrates were subjected to a cold extraction in ethanol (Riedel-de Haën) for 24 h, and dried for 2 h in a vacuum oven at 40 °C, to remove any loosely bound material from the films. Self-assembled Monolayer (SAM). The self-assembled monolayers were prepared from a solution of 10 mM octadecanethiol in dicholoromethane. The substrates were immersed in the solution for 1 h. After self-assembly the samples were removed from the solution and rinsed with dichloromethane and ethanol.

8.4.3 CONTACT ANGLE GONIOMETRY

Contact angles of ultrapure water (Milli-Q Plus 185, Millipore) on the various substrates were determined with a contact angle microscope (Olympus). The average value of a minimum of six measurements was reported.

8.4.4 PROTEINS

The bovine serum albumin (BSA), fibrinogen (human plasma, 60 % protein, over 90 % clottable) and human immunoglobulin G (IgG) (reagent grade, liquid) were purchased from Sigma. The protein solutions were freshly prepared by dissolution of 1 wt % of protein in phosphate buffered saline (PBS, pH 7.4).

115 Chapter 8

8.4.5 SURFACE PLASMON RESONANCE (SPR) SPECTROSCOPY

The substrates were determined using a homebuilt SPR spectrometer, which has been discussed in detail in §3.2.2. Briefly, the SPR instrument uses a monochromatic laser light source (HeNe, λ = 632.8 nm) which is focussed, through a glass prism (LaSFN9), onto the gold-coated glass slide. Before each kinetic measurement, a surface plasmon reflectivity scan of the plasma modified SPR substrate in PBS is recorded, after which the angle is set on the left side of the reflectivity minimum, in the range where the resonance decreases linearly. Then the buffer is replaced by the buffered protein solution and the change in reflectivity is recorded as a function of time. The adsorption process is halted after it reaches an equilibrium state by flushing the cell with PBS. A second reflectivity scan is then recorded, and the layer thickness of the adsorbed protein is calculated from the SPR angle shift according to the Fresnel theory (a refractive index of n = 1.5 for all proteins was used for fitting of the curves). In order to remove elutable (denatured) protein, the cell was rinsed three times with 1 % sodium dodecyl sulfate (SDS) solution. A final reflectivity scan is recorded after this step, and the amount of protein that is retained on the surface after this SDS wash is taken as a measure for the amount of denatured protein.

8.4.6 TAPPING MODE ATOMIC FORCE MICROSCOPY (TM-AFM)

The AFM measurements were made using a Digital Instruments NanoScope III system. The instrument was operated in tapping mode. The substrates were incubated in the respective protein solution for 1 h. After removing the substrates from the solution, they were rinsed thoroughly with PBS, and in some cases with SDS solution and ultrapure water. The samples were carefully dried with argon and subsequently allowed to dry in air overnight before analysis.

8.5 References

1 d’Agostino, R. Plasma Deposition, Treatment and Etching of Polymer Films, Acadamic Press, Inc.: San Diego, CA, 1990. 2 Williams, D. F. Definitions in Biomedicals. Proceedings of a Consensus Conference of the European Society for Biomaterials, Elsevier, Vol. 4, Chester, England, 1987. 3 Hoffman, A. S. J. Appl. Polym. Sci.: Appl. Polym. Symp. 1988, 42, 251. 4 (a) Biederman, H.; Osada, Y. Plasma Polymerization Processes, Elsevier Science Publishers B.V.: Amsterdam, The Netherlands, 1992; (b) Hoffman, A. S. Advances in Polymer Sci. 1984, 57, 142. 5 (a) Nichols, M. F.; Hahn, A. W.; James, W. J.; Sharma, A. K.; Yasuda, H. K. Biomaterials 1981, 2, 161; (b) Johnson, H. J.; Northup, S. J.; Seagraves, P. A.; Atallah, M.; Garvin, P. J.; Lin, L.; Darby, T. D. J. Biomed. Mater. Res. 1985, 19, 489; (c) Ratner, B. D. in Contemporary Biomaterials: Material and Host Response, Clinical Applications, New Technology and Legal Aspects, Boretos, J. W.; Eden, M. (eds.), Noyes Publications, p. 193, 1984.

116 Protein Adsorption to Plasma Modified Surfaces studied by SPR Spectroscopy and AFM

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117 Chapter 8

23 Calderon, J. G.; Harsch, A.; Gross, G.; Timmons, R. B. J. Biomed. Mater. Res. 1998, 42, 597. 24 Harsch, A.; Calderon, J.; Timmons, R. B.; Gross, G. W. Pulsed Plasma Deposition of Allylamine on Polysiloxane: A Stable Surface for Neuronal Cell Adhesion, to be submitted. 25 (a) Zucker, M. B.; Vroman, L. Proc. Soc. Exp. Biol. Med. 1969, 131, 318; (b) Poot, A.; Beugeling, T.; Cazenave, J. P.; Bantjes, A.; van Aken, W. G. Biomaterials 1988, 9, 126; (c) Adams, G. A.; Feuerstein, I. A. Trans. Am. Artif. Int. Organs 1980, 26, 17; (d) Kim, S. W.; Lee, E. S. J. Polym. Sci.: Polym. Symp. 1976, 66, 429. 26 Chinn, J. A.; Ratner, B. D.; Horbett, T. A. Biomaterials 1992, 13, 322. 27 (a) Harris, J. M.; Herron J. N. in Poly(Ethylene) Glycol Chemistry: Biotechnical and Biomedical Applications, Harris, J. M. (Ed.), pp.1, Plenum Press, New York, 1992; (b) Neff, J. A.; Caldwell, K. D. in Handbook of Biomaterials Evaluation: Scientific, Technical, and Clinical Testing of Implant Materials, von Recum, A. F. (Ed.), pp. 205, Edwards Brothers, Ann Arbor, MI, 1998. 28 Gombotz, W. R.; Guanghui, W.; Horbett, T. A.; Hoffman, A. S. J. Biomed. Mater. Res. 1991, 25, 1547. 29 (a) Wasiewski, W.; Fasco, M. J.; Martin, B. M.; Detwiler, T. C.; Fenton, J. W. Thrombo Res. 1976, 8, 881; (b) George, J. N. Blood 1972, 40, 862. 30 (a) Illum, L.; Jacobsen, L. O.; Muller, R. H.; Mak, E.; Davis, S. S. Biomaterials 1987, 8, 113; (b) Kayes, J. B.; Rawlins, D. A.; Colloid Polym. Sci. 1979, 257, 622; (c) Lee, J. H.; Kopecek, K. J.; Andrade, J. D. J. Biomed. Mater. Res. 1989, 23, 351. 31 (a) Desai, N. P.; Hubbel, J. A. ACS Polym. Mater. Sci. Eng. 1990, 62, 731; (b) Gombotz, W. R.; Guanghui, W.; Hoffman, A. S. J. Appl. Polymer Sci. 1989, 37, 91; (c) Gregonis, D. E.; Buerger, D. E.; van Wagenen, R. A.; Hunter, S. K.; Andrade, J. D. Trans. Soc. Biomat. 1984, 10, 266; (d) Han, D. K.; Jeong, S. Y.; Kim, Y. H. J. Biomed. Mater.: Appl. Biomat. 1989, 23, 87. 32 (a) Nagaoka, S.; Mori, Y.; Takiuchi, H.; Yokoata, K.; Tanzawa, H.; Nishiumi, S. in Polymers as Biomaterials, Shalaby, A. S.; Hoffman, A. S.; Ratner, B. D.; Horbett, T. A. (Eds.), pp. 361, Plenum Press, New York, 1985; (b) Sun, Y.; Hoffman, A. S.; Gombotz, W. R. ACS Polym. Prepr. 1987, 28, 282. 33 (a) Grasel, T. G.; Cooper, S. L. Biomaterials 1987, 7, 315; (b) Grainger, D. W.; Okano, T.; Kim, S. W. J. Colloid Interface Sci. 1989, 132, 161; (c) Brasch, J. L.; Uniyal, S.; Samak, Q. Trans. Amer. Soc. Artif. Int. Organs 1974, 20, 69; (d) Sa Da Costa, V.; Brier- Russel, D.; Salzman, E. W.; Merrill, E. W. J. Colloid Interface Sci. 1981, 80, 445. 34 Merrill, E. A.; Salzman, E. W. ASAIO J. 1983, 6, 60. 35 Graham, N. B.; Nwachuku, N. E.; Walsh, D. J. Polymer 1982, 23, 1345. 36 Lopez, G.; Ratner, B. D.; Tidwell, C. D.; Haycox, C. L.; Rapoza, R. J.; Horbett, T. A. J. Biomed. Mater. Res. 1992, 26, 415. 37 Beyer, D.; Ringsdorf, H.; Knoll, W.; Timmons, R. B. J. Biomed. Mater. Res. 1997, 36, 181. 38 (a) Ratner, B. D.; Caster, D. G.; Horbett, T. A.; Lenk, T. J.; Lewis, K. B.; Rapoza, R. J. Vac. Sci. Technol. A 1990, 8, 2306; (b) van Delden, C. J.; Bezemer, J. M.; Engbers, G. H. M.; Feijen, J. J. Biomater. Sci. Polymer Edn. 1996, 8, 251. 39 Walker, A. K. The Development of MALDI Mass Spectrometry for the Investigation of Surface-Protein Interactions, Ph.D. Thesis, The University of Texas at Arlington, 1999. 40 Kiremitci, M.; Gok, E.; Ates, S. J. Biomater. Sci. Polymer Edn. 1994, 6, 425. 41 (a) Suzawa, T.; Shirahama, H.; Fujimoto, T. J. Colloid Interface Sci. 1982, 86, 144; (b) Shirahama, H.; Suzawa, T. J. Colloid Interface Sci. 1985, 104, 416. 42 Walker, A. K.; Wu, Y.; Timmons, R. B.; Kinsel, G. R.; Nelson, K. D. Anal Chem. 1999, 71, 268.

118 Protein Adsorption to Plasma Modified Surfaces studied by SPR Spectroscopy and AFM

43 (a) Tiberg, F.; Malmsten, M.; Linse, P.; Lindman, B. Langmuir 1991, 7, 2723; (b) Ortega- Vinuesa, J. L.; Tengvall, P.; Lundström, I. Thin Solid Films 1998, 324, 257; (c) Ortega- Vinuesa, J. L.; Tengvall, P.; Lundström, I. J. Colloid Interface Sci. 1998, 207, 228. 44 Sheller, N. B.; petrash, S.; Foster, M. D.; Tsukruk, V. V. Langmuir 1998, 14, 4535. 45 Lin, J. N.; Drake, B.; Lea, A. S.; Hansma, P. K. Langmuir 1990, 6, 509. 46 Baty, A. M.; Leavitt, P. K.; Siedlecki, C. A.; Tyler, B. J.; Suci, P. A.; Marchant, R. E.; Geesey, G. G. Langmuir 1997, 13, 5702. 47 Shibata-Seki, T.; Masai, J.; Ogawa, Y.; Sato, K.; Yanagawa, H. Appl. Phys. A 1998, 66, 625. 48 (a) Mrksich, M.; Sigal, G. B.; Whitesides, G. M. Langmuir 1995, 11, 4383; (b) Lofas, S.; Malmquist, M.; Ronnberg, I.; Stenberg, E.; Liedberg, B.; Lundstrom, I. Sensors Actuators B 1991, 5, 79; (c) McGurk, S. L.; Green, R. J.; Sanders, G. H. W.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Langmuir 1999, 15, 5136; (d) Silin, V. I.; Balchytis, G. A.; Yakovlev, V. A. Opt. Commun. 1993, 97, 19. 49 Holland, N. B.; Marchant, R. E. J. Biomed. Mater. Res. 2000, 51, 307. 50 For synthesis and characterization of amine functionalized films, see Chapter 3 of this thesis. 51 Ekdahl, K. N.; Nilsson, B.; Golander, C. G.; Elwing, H.; Lasen, B.; Nilsson, U. R. J. Colloid Interface Sci. 1993, 158, 121. 52 Spijker, H. T.; Bos, R.; van Oeveren, W.; de Vries, J.; Busscher, H. J. Colloids and Surfaces B: Biointerfaces 1999, 15, 89. 53 Werner, C.; König, U.; Augsburg, A.; Arnhold, C.; Körber, H.; Zimmermann, R.; Jacobasch, H.-J. Colloids and Surfaces A: Physicochem. Eng. Aspects 1999, 159, 519. 54 Burns, N. L.; Holmberg, K.; Brink, C. J. Colloid Interface Sci. 1996, 178, 116. 55 Green, R. J.; Davies, J.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B. Biomaterials 1997, 18, 405. 56 Typical pKa values measured for amines in solution are: methylamine (10.6), dimethylamine (10.7), ethylamine (10.7), propylamine (10.6) (Risch, K.; Seitz, H. Organische Chemie; Schroedel, Hannover, 1981; p. 115). 57 (a) Schönherr, H.; van Os, M. T.; Hruska, Z.; Kurdi, J.; Förch, R.; Arefi-Khonsari, F.; Knoll, W.; Vancso, G. J. Chem. Comm. 2000, 1303; (b) See also Chapter 5 of this thesis for details. 58 Vezenov, D. V.; Noy, A.; Rozsnyai, L. F.; Lieber, C. M. J. Am. Chem. Soc. 1997, 119, 2006. 59 The extraordinary low pKa which was confirmed by independent contact angle titrations (4.3), was explained by the conformational disorder of the silane SAM. The disorder leads to a exposition of hydrophobic methylene groups. 60 (a) Parker, J. L.; Claesson, P. M.; Wang, J.-H.; Yasuda, H. K. Langmuir 1994, 10, 2766; (b) Lassen, B.; Malmsten, M. J. Mater. Sci. Mater. Med. 1994, 5, 662.

119

Summary 6XPPDU\

The work described in this thesis concerns the surface modification of materials by thin film deposition in a plasma reactor. In particular, thin polymeric films bearing amine functionalities were synthesized by plasma polymerization of amino group containing monomers. In addition to the synthesis, attention was directed towards the characterization of these films, and the tailoring of their surface properties on a molecular level. Finally, the amino groups introduced by plasma polymerization were used for the subsequent immobilization of graft polymers and biomolecules.

Chapter 2 presents a number of topics that are relevant to this thesis. An overview of the most important surface treatment and surface modification techniques is given. Some of the fundamental aspects of plasma and plasma polymerization are also covered.

Plasma films are in general insoluble, and therefore not all the characterization methods commonly used for conventional polymers are suitable to analyze plasma polymers. Chapter 3 serves as a brief introduction to the most important techniques that were used to characterize the plasma polymers discussed in this thesis.

The synthesis and characterization of amine functionalized plasma polymers is reported in Chapter 4. The influence of plasma parameters such as monomer flow, peak power, and rf duty cycle on the film composition was investigated using different monomers. By careful selection of monomer and process conditions, it was possible to produce thin films with a large variety in chemical structure and physical properties. In general it was found that the amino group functionality of the monomer was increasingly retained in the plasma polymer with decreasing power input. Waveguide mode spectroscopy (WaMS) measurements suggested the presence of an index gradient within the plasma polymerized allylamine films.

The oxidation behavior of plasma polymerized allylamine was studied in Chapter 5. The loss of material after extraction in ethanol and the swelling behavior of these films in this solvent were also considered. Surface plasmon resonance (SPR) thickness measurements on plasma polymers showed that the films contained low molecular weight material that could be removed by extraction in ethanol. The film thickness decrease upon this solvent treatment was found to be related to the duty cycle employed during polymerization. WaMS proved to be a powerful tool for the characterization of plasma polymerized films. By allowing a simultaneous study of film thickness and refractive index against different cover media, this method provided new insights in the aging and swelling behavior of plasma polymerized coatings. The duty cycle employed during plasma polymerization of allylamine strongly affected the aging mechanism during storage in air. It was found that films polymerized under low power conditions were more susceptible to surface aging than films deposited at high

121 power input. This behavior was explained by the preferential oxidation of carbon atoms adjacent to amines and by the reorientation of the polar amino groups at the surface.

The characterization of plasma polymerized allylamine films using atomic force microscopy (AFM) with chemically functionalized tips is described in Chapter 6. Pull-off force measurements with carboxylic acid functionalized tips in ethanol were found to correlate with the amino group content of these films. The ionization of the amino groups was studied separately by pH-dependent pull-off force measurements with hydroxyl-terminated tips. Adhesive interactions were found to decrease significantly between pH 6.2 and 5.2 upon lowering the pH, due to protonation of the amino groups. The detected ‘force pKa’ of plasma polymerized allylamine films was independent of the power input during deposition. Laterally inhomogeneous pull-off forces were related to the inhomogeneous distribution of the polar groups on the surface.

In Chapter 7, a novel method for the attachment of polymer monolayers to plasma modified surfaces is presented. Plasma modified surfaces containing amino groups were successfully immobilized with the radical initiator 4,4’-azobis-(4-cyano-pentanoic acid chloride). The azo groups were then utilized to start the thermally induced polymerization of methyl methacrylate or styrene, leading to surface-attached poly(methyl methacrylate) (PMMA) or polystyrene (PS) chains. It was found in this study that the peroxides or free radicals present in the plasma deposited film contributed to the grafting reaction as well. The dependency of the graft density on the plasma polymerization conditions is described qualitatively as well as quantitatively. High molar mass values were determined for both 6 -1 5 -1 PMMA (Mn = 2.4⋅10 g⋅mol ) and for PS (Mn = 5.5⋅10 g⋅mol ). PMMA layer thicknesses as high as 130 nm were found on the plasma surfaces after polymerization following the proposed modification concept.

The adsorption of proteins to plasma polymer surfaces was investigated in detail in Chapter 8. Si and Au substrates were functionalized with amino or ether groups by plasma polymerization, or with a self-assembled monolayer (SAM) of octadecanethiol. The functional group density at the surface was controlled by using different monomers, or by variation of the average input power during plasma deposition. The adsorption of the proteins fibrinogen, bovine serum albumin and immunoglobulin G to these test surfaces could be measured in situ by SPR spectroscopy. The tenacity of the protein adsorption on the different substrates was also measured, after removing elutable protein with 1 % sodium dodecyl sulfate (SDS) solution. After drying in air, the protein layers were studied by tapping mode atomic force microscopy (TM-AFM). The results obtained showed that both the initial protein adsorption to and the retention on the surfaces were affected greatly by the surface functionalities. All the amine-functionalized surfaces exhibited a high affinity toward the proteins, and thin dense layers of adsorbed protein remained on these surfaces, even after rinsing with SDS solution. A large contrast in protein affinity was observed between the ether group containing plasma films polymerized at different power input conditions. Dramatic reduction in both initial adsorption and retention of all proteins was observed on these films upon decreasing the power input. The low degree of cross-linking, as well as the high 122 Summary retention of ether content during the polymerization under mild power input conditions was thought to result in the production of biologically non-fouling surfaces.

123

Samenvatting 6DPHQYDWWLQJ

Het werk dat in dit proefschrift wordt beschreven omvat de oppervlakte modificatie van materialen door het aanbrengen van een dunne organische coating in een plasma reactor. Het grootste deel van het onderzoek richt zich op de functionalisering van oppervlakken met aminogroepen door plasma polymerisatie van allylamine en andere monomeren die aminogroepen bevatten. Naast de synthese van deze films wordt aandacht besteed aan de karakterisering ervan, alsmede aan de invloed van een aantal procesvariabelen op de chemische en fysische eigenschappen van de films. Tenslotte wordt beschreven hoe de aan het oppervlak geïntroduceerde aminogroepen gebruikt kunnen worden voor de immobilisatie van andere polymeren en eiwitten. Het concept van dit proefschrift wordt beschreven in Hoofdstuk 1.

Hoofdstuk 2 dient als een inleiding op een aantal onderwerpen die van belang zijn voor een goed begrip van dit promotieonderzoek. Er wordt een overzicht gegeven van de belangrijkste oppervlakte behandelingstechnieken. Ook komen een aantal fundamentele aspecten van plasma en plasma polymerisatie aan de orde.

Omdat plasma polymeren in het algemeen onoplosbaar zijn, zijn niet alle conventionele karakteriseringsmethoden voor polymeren geschikt om plasma coatings te analyseren. Hoofdstuk 3 beschrijft de belangrijkste technieken die gebruikt zijn om de in dit proefschrift beschreven films te karakteriseren.

De synthese en de karakterisering van plasma polymeren die aminogroepen bevatten staat beschreven in Hoofdstuk 4. Het effect van proces parameters als de monomeer flux, het piekvermogen, en de radio frequentie (rf) duty cycle op de samenstelling van de film is bestudeerd met fourier-transform infrarood (FT-IR) spectroscopie, röntgen foto-electron spectroscopie (XPS), randhoekmetingen en atomaire krachtmicroscopie (AFM). Door een zorgvuldige selectie van het monomeer en de procescondities bleek het mogelijk om dunne films te produceren met een grote variatie in chemische en fysische eigenschappen. In het algemeen bleek het aantal aminogroepen in het plasma-polymeer toe te nemen naarmate het gemiddeld vermogen tijdens de polymerisatie afnam. Waveguide mode spectroscopie (WaMS) metingen suggereerden de aanwezigheid van een brekingsindex gradiënt in de plasma-gepolymeriseerde allylamine coatings.

Hoofdstuk 5 beschrijft het oxidatiegedrag van plasma-gepolymeriseerde allylamine films. Uit XPS- en randhoekmetingen bleek dat de rf duty cycle een belangrijke rol speelt in het verouderingsmechanisme van dit soort oppervlakken. Films die bij een laag gemiddeld vermogen werden gepolymeriseerd, waren gevoeliger voor oppervlakte-oxidatie dan films die bij een hoog vermogen werden afgezet. De preferentiële oxidatie van koolstofatomen die zich

125 naast een stikstofatoom bevinden en reoriëntatie van polaire aminogroepen aan het oppervlak worden als mogelijke verklaringen voor dit gedrag gegeven. Ook wordt in dit hoofdstuk het uitzettingsgedrag van deze films in ethanol beschreven. Oppervlakte plasmonen resonantie (SPR) metingen wezen uit dat de plasma-polymeren materiaal bevatten met een lage moleculaire massa, dat verwijderd kan worden door extractie in ethanol. De afname van de dikte van de film bleek eveneens gerelateerd te zijn aan de duty cycle tijdens polymerisatie. WaMS bewees in dit promotieonderzoek zijn waarde in de karakterisering van plasma gepolymeriseerde films. Door tegelijkertijd een studie van de filmdikte en de brekingsindex mogelijk te maken, gaf deze methode nieuwe inzichten in het uitzettingsgedrag van plasma- coatings.

De resultaten van AFM experimenten met chemisch gemodificeerde tips ter karakterisering van plasma-gepolymeriseerde allylamine films worden gepresenteerd in Hoofdstuk 6. Pull-off force metingen in ethanol met tips die gefunctionaliseerd waren met zuurgroepen correleerden met de hoeveelheid aminogroepen in deze films. De ionisatie van de aminogroepen werd apart bestudeerd door pH-afhankelijke pull-off force metingen uit te voeren met tips die van tevoren gemodificeerd waren met alcoholgroepen. Uit deze reeks experimenten werd duidelijk dat de aantrekkingskrachten tussen tip en coating significant afnamen tussen pH 6.2 en 5.2 bij verlaging van de zuurgraad, hetgeen wordt toegeschreven aan protonering van de aminogroepen. De zo waargenomen ‘kracht pKa’ van plasma- gepolymeriseerde allylamine films was onafhankelijk van het vermogen tijdens plasma polymerisatie. Tenslotte wezen de pull-off force metingen op een inhomogene verdeling van de polaire groepen aan het oppervlak.

In Hoofdstuk 7 wordt een nieuwe methode voor de hechting van polymere monolagen (ook wel borstels genoemd) aan plasma-gemodificeerde oppervlakken beschreven. In de eerste stap reageert een radicaal-initiator met aminogroepen die zich aan het oppervlak van plasma- gepolymeriseerde allylamine films bevinden. Vervolgens worden de azogroepen van de geïmmobiliseerde initiator in de tweede stap gebruikt om de thermisch geïnduceerde polymerisatie van methyl methacrylaat of styreen te starten, hetgeen resulteert in covalent aan het oppervlak gebonden poly(methyl methacrylaat) (PMMA) of polystyreen (PS) ketens. Uit deze studie bleek dat de in de plasma-film aanwezige vrije radicalen of peroxiden eveneens een bijdrage leveren aan dit proces. De afhankelijkheid van de keten dichtheid van de plasma polymerisatie condities is zowel kwalitatief als kwantitatief beschreven. Er werden hoge 6 -1 waarden gevonden voor zowel de molaire massa van PMMA (Mn = 2.4⋅10 g⋅mol ), als voor 5 -1 die van PS (Mn = 5.5⋅10 g⋅mol ). PMMA borstels met een dikte tot 130 nm konden met dit voorgestelde mechanisme bereikt worden.

De adsorptie van eiwitten aan plasma polymeren wordt besproken in Hoofdstuk 8. Silicium- en goud-substraten werden gefunctionaliseerd met amino- of ethergroepen door plasma-polymerisatie, of met methylgroepen door modificatie met zelf-organiserende monolagen van octadecaan thiol. De dichtheid van de functionele groepen aan het oppervlak kon worden geregeld door het gebruik van verschillende monomeren, of door variatie van het gemiddeld vermogen tijdens het plasma polymerisatie proces. De adsorptie van fibrinogeen, 126 Samenvatting albumine en immunoglobuline aan deze testoppervlakken kon in situ gevolgd worden met SPR spectroscopie. De retentie van de eiwitten op de verschillende substraten werd ook gemeten, nadat zwak gebonden eiwit verwijderd was met 1 % natrium dodecyl sulfaat (SDS) oplossing. Naast deze kwantitatieve studie naar het adsorptiegedrag van genoemde eiwitten op de geprepareerde oppervlakken, werden de eiwitten ook onderzocht met AFM, na te zijn gedroogd aan lucht. De resultaten lieten zien dat zowel de initiële adsorptie als de retentie van de eiwitten voor een belangrijk deel beïnvloed worden door de functionele groepen aan het oppervlak van het substraat. De met aminogroepen gemodificeerde oppervlakken toonden in het algemeen een hoge affiniteit ten opzichte van de verschillende eiwitten en dunne dichte lagen eiwit bleven op deze oppervlakken achter, zelfs na het spoelen met SDS oplossing. Een groot contrast in affiniteit werd waargenomen tussen de ethergroep-bevattende plasma- polymeren die bij verschillende vermogens geprepareerd waren. Een lager vermogen tijdens de plasma-polymerisatie leidde tot een drastische verlaging van zowel de initiële adsorptie als de retentie van alle eiwitten op deze films. Verondersteld wordt dat de geringe vernettingsgraad, alsmede de hogere retentie van ethergroepen in het plasma-polymeer onder milde polymerisatiecondities, resulteren in de productie van biologisch inerte oppervlakken.

127

List of Publications /LVWRI3XEOLFDWLRQV

Published / in press

Holger Schönherr, Menno T. van Os, Renate Förch, Richard B. Timmons, Wolfgang Knoll, G. Julius Vancso Chem. Mater. 2000, in press. “Functional Groups Distributions in Plasma Polymerized Allylamine Films by Scanning Force Microscopy Using Functionalized Probe Tips”.

Schönherr, H.; van Os, M. T.; Hruska, Z.; Kurdi, J.; Förch, R.; Arefi-Khonsari, F.; Knoll, W.; Vancso, G. J. Chem. Comm. 2000, 1303. “Towards Mapping of Functional Group Distributions in Functional Polymers by AFM Force Titration Measurements”. van Os, M. T.; Menges, B.; Foerch, R.; Vancso, G. J.; Knoll. W. Chem. Mater. 1999, 11, 3252-3257. “Characterization of Plasma Polmerized Allymine using Waveguide Mode Spectroscopy”.

Peter, M.; Lammertink, R. G. H.; Hempenius, M. A.; van Os, M.; Beulen, M.W.J.; Reinhoudt, D.N.; Knoll, W.; Vancso, G. J. Chem. Comm. 1999, 359-360. “Synthesis, Characterization and Thin Film Formation of End-Functionalized Organometallic Polymers”. van Os, M. T.; Menges, B.; Foerch, R.; Timmons, R. B.; Vancso, G. J.; Knoll. W. Mat. Res. Soc. Symp. Proc. 1999, 544, 45-50. “Thin Film Plasma Deposition of Allylamine: Effects of Solvent Treatment”.

Pieter Stroeve, Menno van Os, Renee Kunz, John F. Rabolt Thin Solid Films 1996, 284-285, 200-203. “Stabilization of Water Soluble Surfactants at the Air-Water Interface with Gegenion Complexation”.

To be submitted

M.T. van Os, T. Stöhr, R. Foerch, G.J. Vancso, J. Rühe, and W. Knoll “Attachment of Polymer monolayers to Plasma Modified Surfaces by Radical Chain Polymerizations Initiated by Azo Compounds and Residual Free Radicals”.

129 Menno T. van Os, Renate Förch, Richard B. Timmons, G. Julius Vancso, and Wolfgang Knoll “Plasma Polymerization of Amine Functionalized Monomers; Tailoring of the Film Chemistry and Surface Aging”.

Menno T. van Os, A. Toby A. Jenkins, Mária Péter, Renate Förch, Richard B. Timmons, G. Julius Vancso, Wolfgang Knoll “Protein Adsorption to Plasma Functionalized Surfaces Using Surface Plasmon Resonance Spectroscopy and Atomic Force Microscopy”.

130 Dankwoord 'DQNZRRUG

Met het afronden van dit proefschrift komt voor mij een einde aan een periode van bijna vier jaar onderzoek. Ik bevond mij in de bevoorrechte positie dat ik gedurende de loop van mijn promotietraject verschillende malen van thuisbasis kon veranderen, wat niet alleen de kwaliteit van het onderzoek ten goede is gekomen, maar waardoor deze periode ook voor mij persoonlijk een fantastische ervaring is geworden. Via deze weg wil ik iedereen die hieraan bijgedragen heeft hartelijk bedanken.

It started about four years ago, when my promotor, professor Knoll, offered me a position as a Ph.D. student in his group at the Max-Planck-Institut für Polymerforschung in Mainz. Wolfgang, I can not thank you enough for your enthusiasm and supervision during the time I spent in your group. I have profited a great deal from your international orientation, something that also reflects on the cultural diversity of the group, and I greatly appreciate the opportunities you gave me to visit conferences abroad, and to organize the joint seminars with the University of Twente. The summerschool in Hirschegg and the winterschool in Schloss Ringberg are just two of the highlights I will never forget.

Renate Förch, my assistant promotor, was always there for troubleshooting and the direct supervision. Without her theoretical and practical knowledge, this work would have never been possible. Renate, thank you for sharing so many of your ideas with me, and for introducing me into the world of plasma. It has been great working with you!

After two wonderful years in Mainz, I gladly took up an invitation from professor Timmons to spend eight months in his group at the University of Texas at Arlington. This visit proved to be very fruitful, and the experimental work I was able to do in his lab formed the basis for Chapter 4 and 5 of this thesis. Richard, thank you very much for the many interesting discussions we had, the new insights you gave me in pulsed plasma polymerization, and most of all for your great hospitality. You and your wife (I still dream of her cooking sometimes) made my stay in the Lone Star State a very pleasurable one.

Na deze omzwervingen kwam ik uiteindelijk weer in Enschede terecht, waar mijn promotor, professor Vancso, mij de kans gaf het onderzoek af te ronden en dit proefschrift te schrijven in zijn groep aan de Universiteit Twente. Zijn inbreng blijft echter allerminst beperkt tot deze laatste fase van mijn promotieonderzoek. Julius, ik wil je graag hartelijk bedanken voor de steun en begeleiding die je mij vanaf het begin gegeven hebt. Ondanks mijn fysieke afwezigheid in de eerste jaren heb ik me altijd welkom gevoeld in je groep, en heb ik bijzonder veel profijt gehad van onze tussentijdse discussies, de groepslezingen en de perfecte samenwerking vanuit Mainz.

131 In addition to the aforementioned people, there are several others I would like to acknowledge individually for their valuable scientific contributions to the research described in this thesis. Kim Pham contributed to the basic understanding of pulsed plasma deposition of fluorocarbon films during her visits at the Max-Planck-Institut as a summer student from UC Santa Barbara. I thank Bernard Menges for his valuable contribution to the waveguide mode spectroscopy measurements (Chapter 5), and for the time he spent analyzing the resulting data. The work described in Chapter 7 was done in collaboration with Torsten Stöhr. Although the number of experiments we needed to carry out steadily increased over time, the pressure never got the best of us. Und Torsten, was machen die Schichten? I would like to thank Haibo Qiu for the XPS measurements, especially those carried out after midnight. The discussions with professor Kinsel and Angela Walker resulted in a better insight in the aging behavior of plasma polymerized films. The collaboration with Holger Schönherr proved to be extremely fruitful (Chapter 6). Holger, it was always a pleasure working with you, and you often surprised me with your efficiency and scientific instincts. Another example of the successful “crossing border” collaboration between Mainz and Twente is illustrated in Chapter 8 of this thesis. Toby Jenkins and Mária Péter are responsible for the presented SPR data and AFM images.

I also wish to express my thanks to the people who played an important role in the social aspects of my life over the past few years. Looking back on my time in Mainz, I vaguely recall names such as Kuz, Interbar, and worst of all, Schachtel. But also the famous Karaoke nights, Wolfgang’s birthday parties, Hup Holland Hup in the Kaffee Ecke, the party office (sponsored by Napster), and the Schlager parties made my stay in Mainz an unforgettable experience. Thanks to Torsten, Toby, Kirstin, Martin, Ralf, Thomas, Lisa, Alex, Heike, Stephan, Andreas (imdf) and many other (former) Knollies.

The same can be said for the crew I worked with at UTA. I even managed to improve my Chinese by listening carefully to the lively discussions between Haibo, James and Maggie. Samuel Sanchez Estrada often tortured us with his elevator music, but he makes the best Margarita’s outside of Mexico. I thank all of you for the good time in the lab, not to forget Jay and Vance. After work I always enjoyed the company of Nikola, Bala, Olivera, Brad and Campbell at UTA village. I will never forget the jam sessions, Bob and Bitch, the poodle from hell, and the power of the black widow. And how to take a curb in a wheelchair.

Ook mijn collega’s van de MTP groep wil ik hartelijk bedanken voor de goede tijd in Enschede. De hulpvaardigheid van Geneviève, Gerda, Karin en Clemens heb ik zeer gewaardeerd. En aan alle andere (ex)-MTP’ers: bedankt voor de prettige samenwerking, de leuke borrels en de zoekgeraakte kloten.

Dan blijven er tot slot nog een paar mensen over die ik graag wil bedanken voor hun onmisbare steun vanaf de zijlijn. Dan heb ik het bijvoorbeeld over iedereen die mij door de jaren heen in Mainz heeft opgezocht, een aantal van jullie kwam zelfs regelmatig langs. Zes man in mijn kamertje van 4 x 5 meter tijdens een jaarclubweekend bleek geen enkel probleem te zijn. Anton, Cor, Menno en Bart stonden niet alleen met de regelmaat van de klok in Mainz 132 Dankwoord voor de deur, maar kwamen mij zelfs in Amerika opzoeken. Zal ik zeggen dat het voor herhaling vatbaar is, Anton?

Aan Bert Koning en Sacha Kersten ben ik bijzonder veel dank verschuldigd. Zij waren zo vriendelijk om mij in Enschede een dak boven mijn hoofd te bieden. Naast de gezellige tijd zal zeker ook het gezonde eten mij bij blijven. Hugo Schotman en Bart Smulders hebben zich vanaf het prille begin bereid verklaard als paranimf op te willen treden tijdens de promotieplechtigheid. Mijn dank hiervoor! Niet in het minst omdat Hugo speciaal uit Bangla Desh moet overkomen...

Mijn ouders kan ik niet genoeg bedanken voor alles wat ze voor mij gedaan hebben. Jullie onvoorwaardelijke steun, enthousiasme en interesse voor alles wat ik onderneem is voor mij belangrijker dan ik ooit zal kunnen laten blijken. Ook Bas en Tamar, bedankt voor de gezellige weekends in Overasselt, Mainz en Texas!

En tenslotte, Tineke, jou wil ik bedanken voor je ‘grenzeloze’ flexibiliteit en steun in de afgelopen jaren. Op thuisloze momenten was het voor mij een fijne gedachte dat er ergens in Nederland een plekje was waar ik mij altijd thuis mocht voelen en welkom was.

133

Curriculum Vitae &XUULFXOXP9LWDH

Menno van Os was born in Nijmegen (The Netherlands), on the 26th of September 1970. After visiting elementary school in Overasselt (The Netherlands), he attended the ‘Elshof College’ in Nijmegen (The Netherlands) between 1982 and 1989. He received his ‘VWO’ diploma in June 1989.

He began his undergraduate studies at the University of Twente in Enschede (The Netherlands) in August 1989, where he studied Chemical Technology. During his undergraduate studies he also worked as a visiting scientist at the University of California, Davis (USA), in the group of Prof. dr. P. Stroeve (July 1995 to November 1995). He received his ‘ingenieurs’ diploma in August 1996, after completing a graduation project in the group Industrial Processes and Products, under the supervision of Prof. dr. ir. K. R. Westerterp. His graduation thesis was titled ‘Modeling of the thermal behavior of runaways in a semi-batch reactor for multiple liquid-liquid reaction systems’.

The major part of his Ph.D. Thesis work was carried out at the Max-Planck-Institut für Polymerforschung in Mainz (Germany). He received a stipend from the Max-Planck Gesellschaft, and started his research in February 1997 in the group of Prof. dr. W. Knoll. The research was focussed on the pulsed plasma deposition of thin organic films. Menno van Os left Mainz in April 1999, to continue his Ph.D. research at The University of Texas at Arlington (USA) in the group of Prof. dr. R. B. Timmons. This visit was financially supported by NWO and the University of Texas at Arlington. After he returned from the USA in December 1999, he worked for two more months at the Max-Planck-Institut für Polymerforschung. The final stage of his Ph.D. Thesis work was carried out at the University of Twente in Enschede (The Netherlands), in the group of Prof. dr. G. J. Vancso. He will defend his Ph.D. Thesis on November the 17th in the year 2000 at the University of Twente.

Menno van Os presented his results at several international conferences. In August 1997 he attended the 13th International Symposium on Plasma Chemistry in Beijing (China). He visited the fall meeting of the Materials Research Society in Boston (USA) in November 1998, and in October 2000 he presented part of his work at the 47th International Conference of the American Vacuum Society in Boston.

135