Post on 19-Aug-2019
Surface Modification by Plasma Polymerization
and Application of Plasma Polymers as
Biomaterials
Dissertation
Zur Erlangung des Grades
“Doktor der Naturwissenschaften’’
am Fachbereich Chemie und Pharmazie
der Johannes Gutenberg-Universität Mainz
vorgelegt
Zhihong Zhang
Geboren in HeNan, China
Mainz December 2003
DeKan: Prof. Dr. R. Zentel
1. Berichterstatter: Prof. Dr. W. Knoll
2. Berichterstatter: Prof. Dr. A. Janshoff
To my parents and daughter
Abstract
The work described in this thesis concerns the plasma polymers used for
immobilization and adsorption of biomolecules. In particular, thin polymeric films
bearing ether, anhydride, and amine functionalities were synthesis by plasma
polymerization of these group containing monomers, i.e. di-(ethylene glycol) vinyl ether
(EO2), maleic anhydride (MA), and allylamine (AA). Additionally, the characterization
of these films, their surface properties, and solution behavior were investigated in detail.
Self-assembled monolayer (SAM) of octadecanethiol coated Au substrates were
functionalized with different groups by plasma polymerization. The adsorption of the
proteins fibrinogen, bovine serum, and immunoglobulin to these surfaces could be
measured in situ by SPR spectroscopy. The results showed that protein adsorption was
affected significantly by the different surface functional groups, and the polymerization
conditions, as well as the thickness. Among three plasma polymer films, the affinity of
proteins on PEO2 and PPAA is higher than that on PMA. The DNA immobilization
behavior was found to be affected by the amine density and the buffer solution
conditions. The data appear to suggest that the DNA oligonucleotides are able to
penetrate into the low DC polymer network, thus reacting with functional groups deep
within the polymer matrix, which does not seem to be possible with the highly cross-
linked, high Peq films. A strong attraction between the longer chain DNA and the plasma
polymer film was investigated by the pull-force curve using AFM. Also MM0, MM1 and
MM2 are easily distinguished. Since PPAA is three-dimensional network in solution,
DNA hybridization and dissociation is different from the two dimensional matrix.
I
Contents
Chapter 1-------------------------------------------------------------- 1
General Introduction ---------------------------------------------------------------------1
1.1 Plasma, Plasma Polymerization, and Plasma-Surface Modification of
Biomaterials: a Brief Introduction and Historical Background.................... 1
1.2 Concept of this Thesis........................................................................................ 3
References.................................................................................................................. 4
Chapter 2-------------------------------------------------------------- 7
Polymer Surface Modification and Plasma Polymer Treatment -----------7
2.1 Methods of Polymer Surface Modification....................................................... 7
2.1.1 Ion beam modification ------------------------------------------------------------7
2.1.2 Corona discharge------------------------------------------------------------------7
2.1.3 Other surface treatments ---------------------------------------------------------8
2.2 Plasma Surface Treatment................................................................................. 8
2.2.1 The fourth state of materials -----------------------------------------------------8
2.2.2 Main sources of gas discharge plasmas ----------------------------------------9
2.2.2.1 Direct current glow discharges -------------------------------------------9
2.2.2.2 Capacitively coupled radio-frequency discharges----------------------9
2.2.2.3 Pulsed glow discharges -------------------------------------------------- 10
2.2.2.4 Low-pressure, high-density plasmas ----------------------------------- 10
2.2.2.5 Other gas discharge plasmas-------------------------------------------- 11
2.2.3 Applications of gas discharge plasmas --------------------------------------- 12
2.2.3.1 Deposition of thin films -------------------------------------------------- 12
2.2.3.2 Etching --------------------------------------------------------------------- 14
2.2.3.3 Surface activation and functionalization of polymers---------------- 14
2.2.3.4 Plasma polymerization --------------------------------------------------- 15
References................................................................................................................ 17
II
Chapter 3------------------------------------------------------------ 25
Experimental Section ------------------------------------------------------------------ 25
3.1 Plasma Film Deposition.................................................................................... 25
3.1.1 Plasma reactor------------------------------------------------------------------- 25
3.1.2 Plasma polymerization conditions -------------------------------------------- 28
3.2 Surface Analytical Techniques ........................................................................ 28
3.2.1 Contact angle goniometry ------------------------------------------------------ 28
3.2.2 Atomic force microscopy (AFM) ---------------------------------------------- 29
3.3 Thin Film Characterization by Optical Techniques...................................... 31
3.3.1 Waveguide mode spectroscopy (WaMS)-------------------------------------- 31
3.3.2 Surface plasmon resonance spectroscopy (SPS) and surface plasmon
resonance fluorescence spectroscopy (SPFS) -------------------------------------- 33
3.4 Sample Preparation of Solution Behavior Measurements ............................ 38
3.4.1 Phosphate buffer (PB) solution preparation --------------------------------- 38
3.4.2 Self-assembled monolayer (SAM) --------------------------------------------- 38
3.4.3 Plasma polymerization---------------------------------------------------------- 39
3.4.4 SPR measurements -------------------------------------------------------------- 39
3.5 Procedures for Protein Adsorption Measurements ....................................... 39
3.5.1 Plasma polymerization---------------------------------------------------------- 39
3.5.2 Protein solution preparation --------------------------------------------------- 40
3.5.3 SPR measurements -------------------------------------------------------------- 40
3.6 Procedures for DNA Immobilization and Hybridization on PPAA............. 40
3.6.1 Plasma polymerization---------------------------------------------------------- 40
3.6.2 DNA samples --------------------------------------------------------------------- 41
3.6.3 Experimental procedure for DNA hybridization----------------------------- 42
3.6.4 SPFS measurements------------------------------------------------------------- 43
References................................................................................................................ 43
Chapter 4------------------------------------------------------------ 47
III
Properties of Plasma Polymerized Films in Air ------------------------------- 47
4.1 Introduction....................................................................................................... 47
4.2 Plasma Polymerized Di- (ethylene glycol) Vinylether (PEO2) ..................... 48
4.2.1 Spectroscopic characterization ------------------------------------------------ 48
4.2.2 Surface wettability--------------------------------------------------------------- 50
4.2.3 Homogenity and morphology -------------------------------------------------- 51
4.3 Plasma Polymerized Allylamine (PPAA)........................................................ 52
4.3.1 Spectroscopic characterization ------------------------------------------------ 52
4.3.2 Surface wettability--------------------------------------------------------------- 53
4.4 Plasma Polymerized Maleic Anhydride (PMA) ............................................. 54
4.4.1 Spectroscopic characterization ------------------------------------------------ 54
4.4.2 Surface wettability--------------------------------------------------------------- 55
4.4.3 Homogenity and morphology -------------------------------------------------- 56
4.5 Summary............................................................................................................ 56
References................................................................................................................ 57
Chapter 5------------------------------------------------------------ 61
Solution Behavior of Plasma Polymerized Films----------------------------- 61
5.1 Introduction....................................................................................................... 61
5.2 Solution Behavior of Plasma Polymers Investigated by SPR ....................... 62
5.2.1 Solution behavior of PEO2 and PPAA deposited under high input power
-------------------------------------------------------------------------------------------- 62
5.2.2 Solution behavior of PMA deposited under high input power ------------- 63
5.2.3 Different pH solution behavior of PPAA deposited under low input power
-------------------------------------------------------------------------------------------- 64
5.3 Solution Behavior of Plasma Polymers Investigated by WaMS................... 66
5.3.1 Plasma polymerized di-(ethylene glycol) vinyl ether (PEO2) ------------- 66
5.3.1.1 d and n alteration of cw, 90 W plasma PEO2 in PBS -------------- 66
5.3.1.2 d and n alteration of high DC 5/50, 90 W PEO2 in PBS ----------- 67
5.3 .1.3 d and n alteration of low DC 5/100, 90 W plasma PEO2 in PBS- 68
5.3.2 Plasma polymerized allylamine (PPAA)-------------------------------------- 69
IV
5.3.2.1 d and n alteration of cw, 5 W and 90 W PPAA in PBS -------------- 69
5.3.2.2 d and n alteration of 10/50 PPAA in PBS ----------------------------- 70
5.3.3 The model of the solution behavior of plasma polymers ------------------- 71
5.3.4 Plasma polymerized maleic anhydride (PMA) ------------------------------ 73
References................................................................................................................ 74
Chapter 6------------------------------------------------------------ 77
Protein Adsorption to Plasma Functionalized Surfaces ------------------- 77
6.1 Introduction....................................................................................................... 77
6.2 Protein Adsorption on PEO2 ........................................................................... 78
6.2.1 Influence of plasma duty cycles------------------------------------------------ 78
6.2.2 Influence of the plasma polymer thickness ----------------------------------- 83
6.3 Proteins Adsorption on PPAA ......................................................................... 85
6.3.1 Influence of plasma duty cycles------------------------------------------------ 85
6.3.2 Influence of the plasma polymer thickness ----------------------------------- 86
6.4 Proteins Adsorption on PMA........................................................................... 87
6.4.1 Influence of plasma duty cycles------------------------------------------------ 87
6.4.2 Effect of the plasma polymer thickness --------------------------------------- 89
6.6 Conclusions........................................................................................................ 91
References................................................................................................................ 91
Chapter 7------------------------------------------------------------ 95
DNA Immobilization and Hybridization on
Plasma Polymerized Allylamine---------------------------------------------------- 95
7.1 Introduction....................................................................................................... 95
7.2 Backgrounds of DNA immobilization and hybridization.............................. 97
7.3 Factors that Affect DNA Immobilization on PPAA Films ............................ 98
7.3.1 Plasma polymer films ----------------------------------------------------------- 98
7.3.1.1 Typical DNA immobilization procedure on PPAA film -------------- 98
7.3.1.2 Plasma equivalent power employed in plasma polymerization----- 98
V
7.3.1.3 Plasma polymer thickness ----------------------------------------------100
7.3.2 pH value of buffer solution ----------------------------------------------------101
7.3.3 Probe DNA sequences length and concentration---------------------------101
7.4 DNA Immobilization on PPAA Films Investigated by AFM...................... 103
7.5 DNA Hybridization on PPAA........................................................................ 104
7.5.1 Non-specific adsorption between target DNA and PPAA films-----------104
7.5.2 UV treatment and blocking of excess amino groups -----------------------106
7.5.3 DNA hybridization--------------------------------------------------------------108
7.6 Conclusions...................................................................................................... 116
References.............................................................................................................. 116
Summary ---------------------------------------------------------- 119
List of Publications------------------------------------------ 121
Nomenclature -------------------------------------------------- 123
Acknowledgements ----------------------------------------- 125
Curriculum Vitae---------------------------------------------- 127
1
Chapter 1
General Introduction
1.1 Plasma, Plasma Polymerization, and Plasma-Surface Modification of
Biomaterials: a Brief Introduction and Historical Background
The term “plasma polymerization” is widely used to denote the process of forming
high molecular weight products in electrical discharges.1 Over the past 40 years, plasma
has become a very useful method for surface modification and deposition of various
materials.1 An ionized medium consisting of electrons, ions, neutrals and photons, is
called a plasma. According to this definition, the term plasma covers a wide range of
phenomena.2 There are equilibrium (thermal) and non-equilibrium (non-thermal)
plasmas. Low-pressure plasmas as discussed here are of the non-equilibrium type. The
electron energy distribution is close to a thermal distribution of several 10000 K while the
energy distribution of ions an neutrals corresponds to a thermal distribution of about 300
K. Elementary processes with high activation energies of several electron volts are
possible in low-pressure plasmas without elevated gas (ion, neutral) temperatures. At the
same time, there is almost no thermal load for sensitive polymeric materials during low-
pressure plasma treatment.3
Plasma polymerization was first observed in 1874,4 developed at the end of the
1950’s and beginning of the 1960’s in connection with the development of electronics,
where plasma polymer films were investigated systematically.5-6 In those years, plasma
polymer polymerization obtained the rapid advancement in Japan, Germany, and U. K.7-8
Although the plasma chemical technology for producing thin polymer films has entered
widespread use in industry and a large amount of information on the growth kinetics and
properties of polymer films has been accumulated, the mechanism for their formation has
not yet been clarified, mainly due to the extremely complexity of plasma chemical
process, the variety of reactions, end products, intermediate products, the inadequate
development of experimental methods for studying such systems, and the small amount
of research that has been done both on the properties in air, of low-temperature non-
equilibrium plasma molecular gases. The properties of plasma polymer films, their
2
practical applications, and the possible mechanism for their formation are well discussed
reflected in a number of reviews and books.
The advantages of plasma polymer films include excellent coating adhesion on
almost all substrates,9-10 chemical,11 mechanical 12 and thermal stability,13 and high barrier
effects.14 Furthermore, while the chemical structure of a thin surface layer can be changed
significantly, the bulk properties of the substrate remains unchanged.15 An extremely
wide range of surface modifications can be realized with different low-pressure plasmas.
Hence, plasma surface modification has been employed in many technological fields,
such as lubrication surfaces, bio-absorbable polymer, biocompatibility enhancement,
bone internal fixation devices, diagnostic biosensors.16-22 Recently, the application of
plasma polymer as biomaterials has attracted the interest of many researchers.24-26 The
field of biomedical applications needs polymers which, beside satisfying the physical
requirements of their application, show the so-called "biocompatibility" with the
biological environment in which they are employed. Since biocompatibility involves
reactions of the interface of the device and the biological environment, surface
modification techniques can be of a great help to solve this problem, avoiding costly
changes of materials. Among those techniques of surface modification the "cold" plasma
deserves an important place thanks to its characteristics.27-28 For example, in many
application fields, low molecule weight glycols have been utilized as feed gases in the
radio frequency (rf) plasma, where they give rise to PEO-like surface with non-fouling
properties. 29-30 Non-fouling properties have been tested successfully for blood proteins
(fibrinogen and albumin), for antibodies, cells and bacteria.31-32 It is known that heparin
and heparin-like molecules, collagen, albumin and other molecules of biological origin
confer anti-thrombosis properties on polymer surfaces where they are immobilized.30,33-36
The most frequently grafted groups are -NH2, -OH and –COOH, by means of RF glow
discharges fed with non-depositing gases such as NH3, O2, H2O, etc. Such treatments are
known to increase the usually low wettability of conventional polymers, and are utilized
also to improve adhesion and the growth of cells on polymers.37-40
3
1.2 Concept of this Thesis
The research described in this thesis involves the plasma polymerization of three
monomers, i.e., di-(ethylene glycol) vinyl ether (EO2), allylamine (AA), and maleic
anhydride (MA). The work focuses on the study of the plasma polymers used as
biomaterials, such as for protein nonfouling, cell nonfouling, and DNA immobilization
and hybridization. In the present work, the aim is to understand the interaction
mechanism between biomolecules (proteins and DNAs) and plasma polymers. This
interaction is affected by the properties of plasma polymer films and solution behavior,
which should be investigated in detail.
Chapter 2 describes the methods commonly employed techniques for polymer
surface modification. A brief introduction of plasma surface modification, plasma
polymerization, and the application also are discussed.
The plasma reactor and characterization methods used in the analysis of plasma
polymers discussed in this thesis are given in Chapter 3. Surface analytical techniques
such as contact angle goniometry, and Atomic Force Microscopy (AFM) are introduced.
Three optical techniques, Waveguide Mode Spectroscopy (WaMS), Surface Plasmon
Resonance spectroscopy (SPR) and Surface Plasmon resonance Fluorescence
Spectroscopy (SPFS), are also explained. Furthermore, the sample preparation methods
during the work will be described.
Synthesis and characterization of plasma polymers of EO2, AA, and MA in air are
discussed in Chapter 4. A major part this section focuses on the chemical properties,
wettability and morphology.
Since the application of plasma polymers as biomaterials has to be investigated in
solvent environment, it was of major importance to understand the change in properties
of these plasma polymers in solution. Properties such as the chemical structure variation,
swelling behavior, the refractive index, and morphology were studied in solution and are
discussed in Chapter 5.
Chapter 6 focused on the adsorption of the proteins (fibrinogen, Bovine Serum
Albumin (BSA) and Immunoblobulin (IgG)) on plasma polymerized di-(ethylene glycol)
mono vinyl ether (PEO2), allylamine (PPAA), and maleic anhydride (PMA) as a function
4
of the polymer films structure. SPR was employed to provide the real time studies of
protein attachment on these three plasma polymerized films.
DNA immobilization and hybridization on PPAA films are presented in Chapter 7.
The optimum PPAA film for DNA immobilization and hybridization, i.e., cw (continuous
wave) and low input power (5 W) PPAA film, was observed after a series of experiments.
The factors, which affect the DNA immobilization on PPAA films, include polymer
properties, DNA length and concentration, and solution properties. To remove the non-
specific adsorption, succinic anhydride was employed to block extra amino groups after
DNA immobilization. Furthermore, UV light was used to enhance the interaction
between DNA molecules and the plasma polymer chains. Finally, the complementary
DNA strands hybridization on PPAA films was presented. The affinity constant of
hybridization can be obtained by Langumir isotherm model.
References
1. H. Yasuda, Plasma Polymerization, Academic Press Inc.: Orlando, FL, 1985.
2. M. Liston, L. Martinu, R. Wertheimer, Plasma surface modification of polymers
for improved adhesion: a critical review, J. Adhe. Sci. Technol., 7, 1993, 1091.
3. M.A. Lieberman, A.J. Lichtenberg, Principles of Plasma Discharges and
Materials processing, Wiley, New York, 1994.
4. A. Thenard, C.R. Hebd, Seances, Acad. Sci.,78, 1874, 219.
5. M. Stuart, Nature (London), 199, 1963, 59.
6. A. Bradley, J.P. Hammes, J. Electrochem. Soc., 110, 1963, 15.
7. H. Yasuda, Amer. Chem. Soc. Polym. Prep., 19, 1978, 19.
8. F.F. Shin, Surf. Coat. Techn., 82, 1996, 1.
9. B.Feddes, J.G.C. Wolke, A.M. Vredenberg, J.A. Jansen, Biomaterials, 25, 2004,
633.
10. R.V. Dabhade, D. hananjay, S.Bodas, S.A. Gangal, Sensors Actu. B, Corrected
Proof, Available online 30 October 2003.
11. P. Hamerli, Th. Weigel, Th. Groth, D. Paul, Biomaterials, 24, 2003,3989.
12. B D. Beake, G.J. Leggett, M.R. Alexander, Polymer, 42, 2001, 2647.
5
13. J. Zhang, X.f. Feng, H.k. Xie, Y.C. Shi, T.S. Pu, Y. Guo, Thin Solid Films, 435,
2003, 108.
14. C.E. Moffitt, C.M. Reddy, Q.S. Yu, D.M. Wieliczka, H.K. Yasuda, Appl. Surf.
Sci., 161, 2000, 481.
15. M. Strobel, C.S. Lyons, K.L. Mittal, Plasma surface modification of polymers:
Relevances to adhesion, Utrecht, The Netherlands, 1994. 3.
16. C.-M. Chan, T. M. Ko, H. Hiraoka, Surf. Sci . Rep., 24, 1996, 4.
17. A. Harsch, J. Calderon, R.B. Timmons, G.W. Gross, J. Neurosci. Meth., 98, 2000,
135.
18. A. Harsch, J. Calderon, R.B. Timmons, G.W. Gross, J. Biomed Mater Res, 42,
1998, 597.
19. A.T.A. Jenkins, J. Hu, Y.Z. Wang, S. Schiller, R. Foerch, W. Knoll, Langmuir,
16, 2000, 6381.
20. S. Schiller, J. Hu, A.T.A. Jenkins, R.B. Timmons, F.S.Sanchez-Estrada, W.
Knoll, R. Förch, Chem. Mater., 14, 2002, 235.
21. Z. Zhang, B. Menges, R.B. Timmons, W. Knoll, R. Förch, Langmuir, 19, 2003,
4765.
22. M. Malsten, D. Muller, B. Lassen, J. Coll. Interf. Sci.,193, 1997,88.
23. A. Harsch, J. Calderon, R.B. Timmons, G.W. Gross, J. Neurosci. Meth., 98, 2000,
135.
24. X.B. Duan, R.S. Lewis, Biomaterials, 23, 2002, 1197.
25. W. Breemhaar, E. Brinkman, D.J. Ellens, T. Beugeling, A. Banties, Biomaterials,
5, 1984, 269.
26. F.T. Hambrecht, Biomaterials, 3, 1982, 187.
27. F.D. Egitto, L.J. Matienzo, Plasma modification of polymer surfaces for adhesion
improvement, IBM J. Res. Devolop., 38, 1994, 423.
28. C.-M. Chan, T.M. Ko, H. Hiraoka, Surf. Sci. Rep., 1996, 24.
29. G.P. Lopez, B.D. Ratner, C.D. Tidwell, C.L. Haycox, R.J. Rapoza, T.A. Horbett,
J. Biomed. Res.,26, 1992, 415.
30. M.S. Sheu, A.S. Hoffmann, J.G.A. Terlingen, J. Feijen, Clin. Mat., 13, 1993, 41.
31. E.W. Merril, E.W. Saltzmann, Asaio. J, 6 ,1983, 60.
6
32. J.D. Andrade, S. Nagaoka, S. Cooper, T. Okano, S.W. Kim, Asaio. J., 10, 1987,
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33. M.J. Danilich, K. Kottke-Marchant, J.M. Anderson, R.E. Marchant, J. Biomat.
Sci. Polym. Ed., 3, 1992, 195.
34. J.G.A. Terlingen, L.M. Brenneisen, H.T.J. Super, A.P. Pijpers, A.S. Hoffman, J.
Feijen, J. Biomat. Sci. Polym. Ed., 4, 1993, 165.
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36. I. Kang, O.H. Kwon, Y.M. Lee, Y.K. Sung, biomaterials, 17, 1993, 655.
37. T.R. Gegenbach, X. Xie, R.C. Chatelier, H.J. Griesser, J. Adh. Sci. Technol., 8,
1994, 305.
38. B.D. Ratner, A. Chikoti, G.P. Lopez, in: R. d’Agostino (Ed.), Plasma Deposition,
Treatment and Etching of Polymers, Plasma materials Interaction, Academic
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40. S.I. Ertel, A. Chilkoti, Y.M. Sung, Biomaterials, 17, 1996, 841.
7
Chapter 2
Polymer Surface Modification and Plasma Polymer Treatment
2.1 Methods of Polymer Surface Modification
Polymer surface modification is an elegant method for generating functional
polymer surfaces combined with the desirable attributes of bulk polymers.1 Modification
techniques include plasma polymerization,2 plasma spray coating, ion implantation and
ion-beam-assisted deposition, flame,3 corona treatment,4-5 photons, electron beams, X-
rays, and γ-rays.6 Key properties imparted by these technologies include wettability,
adhesion, lubricity, chemical affinity and biocompatibility. Surface modification plays a
very important role in many industrial end-use applications: electronics, packing,
industrial, automotive and aerospace, storage, and medical devices.7-8
2.1.1 Ion beam modification
Ion beam sources have found increasing applications in the past two decades.
Initially developed for space propulsion, their value for surface cleaning, etching, and
thin film deposition was quickly realized. There are several ion beam-processing
techniques that maybe used for surface modification, each having their own relative
advantages and disadvantages. The techniques include ion implantation, ion-beam-
assisted deposition (IBAD), ion heat texturing (IBT), and ion beam polishing and
sharpening technologies. Ion implantation in particular offers a number of advantages
over other techniques because it is known to facilitate both chemical and structural
modification of the near surface volume.9
2.1.2 Corona discharge
Surface treatment by corona discharge is a process to make active groups or the
surfaces of molding and to increase their adhesive force by the collision of electrons
against the surface of the mold. The electrons are generated by the application of high-
tension and high-frequency voltage between two electrodes in air. This process has been
used in the areas of printing and painting as the pretreatment, particular for the surface
8
modification of polyolefins (PE, PP, etc.), polyacetal and fluoroplastics. The effect of
treatment by corona discharge depends on the voltage applied, the distance between the
two electrodes, the kind of plastics, the shape of moldings, and the time of treatment.
When corona discharge occurs in air, the wettability and the reactivity of the surface of
moldings increase, due to the formation of functional groups with high reactivity and
polarity by the effect of ozone and ultraviolet light, which are secondarily generated, in
addition to the collision of electrons.10-11
2.1.3 Other surface treatments
Besides the methods mentioned in last two paragraphs, there are still many other
methods. 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. 12 Photon irradiation should be mentioned, which includes modification
by ultraviolet (UV) and infrared (IR) lasers to treat very small and localized areas.13
Ultraviolet irradiation, typically of wavelength between 250 and 400 nm, produces
photons that can result in activation of polymer surfaces. The adhesion and surface
structuring of polypropylene and poly(ethylene terephthalate) was shown to be improved
by UV treatment.14-15
2.2 Plasma Surface Treatment
2.2.1 The fourth state of materials
Plasma is ionized gases. Hence, they consist of positive and negative ions,
electrons, as well as free radicals. The ionization degree can vary from 100% (fully
ionized gases) to very low values (partially ionized gases). The plasma state is often
referred to as the fourth state of matter. Much of the visible matter in the universe is in
the plasma state. Stars, as well as visible interstellar matter, are in the plasma state.
Besides the astro-plasmas, which are omnipresent in the universe, there are two main
groups of laboratory plasma, i.e., the high-temperature of fusion plasmas, and the so-
called low-temperature plasma or gas discharges. In general, a subdivision can be made
between plasmas which are in the thermal equilibrium and those which are not in the
thermal equilibrium. Thermal equilibrium implies that the temperature of all species
9
(electrons, ions, neutral species) is the same. High temperature is required to form these
equilibrium plasmas, typically ranging from 4000 K to 20 000 K. This is true for stars, as
well as for fusion plasmas. On the other hand, interstellar plasma matter is typically not
in thermal equilibrium.16
2.2.2 Main sources of gas discharge plasmas
2.2.2.1 Direct current glow discharges
When a potential difference is applied between two electrodes, the gases (e.g.
argon) will break down into electrons and positive ions. The latter can cause secondary
electron emission at the cathode. The emitted electrons gives rise to collision in the
plasma, e.g. excitation (which is often followed by de-excitation with emission of visible
light radiation; hence explaining the name of the “glow” discharge) and ionization (which
creates new electrons and ions, and therefore makes the glow discharge a self-sustaining
plasma). Another important process in the glow discharge is the phenomenon of
sputtering, which occurs at sufficiently high voltages. When the ions and fast atoms from
the plasma bombard the cathode, they not only release secondary electrons, but also
atoms of the cathode material, which is called sputtering. A direct discharge can operate
over a wide range of discharge conditions. The pressure can vary from below 1 Pa to
atomospheric pressure. The voltage is mostly in the range between 300 and 1500 V. The
current is generally in the mA range. The discharge can operate in a rare gas or in a
reactive gas, as well as in a mixture of these gases.17-20
2.2.2.2 Capacitively coupled radio-frequency discharges
To sustain a capacitively coupled radio-frequency glow discharge, the electrodes
have to be conducting. When one or both of the electrodes are non-conductive, the
electrodes will be charged up due to the accumulation of positive or negative charges,
and the glow discharge will extinguish. This problem can be overcome by using an
alternating voltage between the two electrodes, so that each electrode will act alternately
as the cathode and anode, and the charge accumulated during one half-cycle will be at
least partially neutralized by the opposite charge accumulated during the next half-cycle.
In practice, many rf glow discharge processes operate at 13.56 MHz, because this is a
10
frequency allotted by international communications authorities at which one can radiate a
certain amount of energy without interfering with communications.21-22
For plasma processing applications, capacitively coupled rf discharges, also called “rf-
diodes”, consist, in the simplest case, of a vacuum chamber containing two planar
electrodes separated by a distance of several cm.
2.2.2.3 Pulsed glow discharges
Besides applying a rf voltage to a glow discharge, the voltage can also be applied in
the form of discrete pulses, typically with lengths in the order of milli- to microseconds.
Because a pulsed discharge can operate at much higher peak voltages and peak current
for the same average power as in a direct current glow discharge, higher instantaneous
sputtering, ionization and excitation can be expected, and hence better efficiencies (e.g.
better sensitivities for analytical spectrochemistry). More recent work has focused mainly
on microsecond discharges, where even higher peak voltage and currents, and hence
better sensitivities, can be obtained. Here, the definition of duty cycle (DC) should be
mentioned, i.e. the ratio of “pulsed-on time” compared to “total pulse time”.23-25
Another advantage of pulsed direct current glow discharges compared to rf
technology is the simpler method of up-scaling due to reduced impedance matching
network and electromagnetic interference problems, and the lower price of power
supplies for large reactors. As far as basic plasma processes are concerned, a pulsed glow
discharge is very similar to a direct current glow discharge, i.e. it can be considered as a
short direct current glow discharge, followed by a generally longer afterglow, in which
the discharge burns out before the next pulse starts.26-27
2.2.2.4 Low-pressure, high-density plasmas
In recent years, a number of low-pressure, high-density plasma discharge have been
developed, mainly as alternatives to capacitively rf discharges and their magnetically
enhanced variants, for etching and deposition applications. Indeed, one of the
disadvantages of rf-diodes is that voltage and current cannot be varied independently of
each other, except when applying different frequencies, which is not always practical.
Thus, for a reasonable ion flux, sheath voltages at the driven electrode must be high,
leading to undesirable damage. To overcome these problems, the mean ion bombarding
11
energy should be controllable independently biasing this electrode with a second rf
source.
The new generation of low-pressure, high-density plasma sources are characterized
by low-pressure (typically 0.1-10 Pa) and by higher plasma density, and consequently by
higher ion fluxes than capacitively current rf discharges of similar pressures. In addition,
the rf or microwave power is coupled to the plasma cross a dielectric window, rather than
by direct connection to an electrode in the plasma, as for an rf diode. To control the ion
energy, the electrode on which the wafer is placed can be independently driven by a
capacitively coupled rf source. Thus, independent control of the ion/radical fluxes and the
ion-bombarding energy is possible.28-30
2.2.2.5 Other gas discharge plasmas
Besides the methods of the glow discharges mentioned above, an atomspheric
pressure glow discharges, can operate over a wide pressure regime. The typical pressure
range is approximately 100 Pa (even atomospheric pressure), but it leads easily to gas and
cathode heating and arcing. Many researchers studied the mechanism and applications of
the atomspheric pressure glow discharges recently.31-32 The main advantage of
atomspheric pressure glow discharges is the absence of vacuum conditions, which greatly
reduces the cost and complexity of the glow discharge operation.33-34 Dielectric barrier
discharges, historically called “silent discharges”,35-36 operate at approximately
atmospheric pressure (typically 0.1-1atm). In 1857, this type of discharge was used to
generate the ozone from air or oxygen. 37Today, these silent discharge ozonizers are
effective tools and a large number of ozone installation are being used worldwide for
water treatment.38 Furthermore, magnetron discharges are applied with a permanent
magnet behind the cathode, in such a way that the magnetic field lines start and return at
the magnet.39-40 Discharges produced by the interaction of an electron beam with a
gaseous medium, called electron beam produced plasmas, are applied in the large area
plasma processing applications.41-42 Finally, all plasmas that are created by the injection
of microwave power, i.e., electromagnetic radiation in the frequency range of 300 MHz
to 10 MHz, can in principle be called microwave induced plasmas.43 The expanding
plasma jet is based on a pressure gradient, inducing bulk transport of the plasma species
12
by supersonic expansion. The major advantage if that the plasma creation region and the
application region are separated, giving rise to remote plasma treatment.44-45 The term
“dusty plasma” does not actually indicate a type of discharges, i.e., the presence of little
dust, particles or clusters, in the plasma. This is typical illustration that the chemical
composition is an important ingredient for the plasma composition.46-47
2.2.3 Applications of gas discharge plasmas
One application field of plasmas is in analytical spectrochemistry.48 The microwave
induces plasma and the glow discharge (in direct current or pulsed mode) are the most
well-known analytical plasmas, but magnetron discharges, direct current plasma jets and
surface wave discharge have also been used for analytical purposes. Plasmas find well-
established use in industrial applications (e.g., for surface modification, lasers, lighting,
etc.), as well as life sciences, related environmental issues and biomedical application.
For example, fluorescence lamps are electroded low-pressure non-local thermal
equilibrium lamps, which operate in the positive column of direct current glow
discharges, in a mixture of a rare gas with mercury, the latter is present both in liquid and
gaseous form.49 Recently, two alternative display technologies, which are based on small
gas discharges: microdischareges, have emerged that offer the possibility of large,
lightweight, flat TV monitors.50-51 Atomic lasers52 ozone generation,53 environmental
application,54-55 biomedical applications56-58 and particle sources are also developed on
base of the gas discharges.59
In the following, surface modification will be described in details. Surface
modification by gas discharge plasmas plays a crucial role in the microelectronics
industry. For the microfabrication of an integrated circuit, one-third of the hundreds of
fabrication steps are typically plasma based.60 Plasma processing is generally used for
film deposition61-64 etching,65-66 and may also be used for resist development and
removal.67 Another important application field of plasma surface modification is in
materials technology.68-69
2.2.3.1 Deposition of thin films
Plasma deposition process can be subdivided in two groups: sputter-deposition and
plasma-enhanced chemical vapor deposition (PE-VCD). Sputter-deposition comprises
13
physical sputtering and reactive sputtering.70 In physical sputtering, ions and atoms from
the plasma bombard the target, and release atoms or molecules of the target material. In
reactive sputtering, use is made of a molecular gas. Besides the positive ions from the
plasma that sputtering bombard the target, the dissociation products from the reactive gas
will also react with the target. Consequently, the film deposited on the substrate will be a
combination of sputtered target material and the reactive gas. When the sputtered atoms
arrive at the substrate they can be temporarily adsorbed, they can, however, also migrate
through the surface or become re-evaporated. When a second atom arrives at the
substrate, it can form a doublet with the first atom, which is more stable than a single
atom, and has more chance to remain ‘stuck’. New atoms arrive at the substrate and can
form triplets, etc. This initial stage is called ‘nucleation’. Little atomic islands are formed
that coalesce together, until a continuous film is formed.
Plasma-enhanced chemical vapor deposition takes place in a reactive gas.71-72 By
chemical reactions in the plasma, different kind of ions and radicals are formed which
diffuse toward the substrate and are deposited by chemical surface reactions. The major
advantage compare to simple chemical vapor deposition is that plasma-enhanced
chemical vapor deposition can operate at much lower temperature. The two most well-
known applications of PECVD are the deposition of amorphous hydrogenated silicon73-75
and carbon76-77 layers. Amorphous hydrogenated silicon layers are often used for the
fabrication of solar cells.78 An advantage of amorphous silicon deposited by PE-CVD
over crystalline produced by a conventional technique is the low cost of production.
Amorphous hydrogenated carbon layers, also called ‘diamond-like carbon layers’ (DLC)
are commonly used as protective hard coatings on various kinds of industrial components
(metals, glass, ceramics and plastics). They have some very interesting characteristics,
eg., a high hardness (in the range 1000–3000 kg.mm2), extremely low friction
coefficients (between 0.01 and 0.28), a good adhesion on most materials, chemical
inertness against most solvents and acids, thermal stability (up to above 300 ?C), and
interesting optical properties (i.e. transparent in the VIS and IR ranges).
14
2.2.3.2 Etching
Plasma etching is essentially used to remove material from a surface.79 It can be
conducted with a variety of discharge sources, such as direct current glow discharges,
capacitively current rf discharge, and so on. The three important parameters for etching
are etched rate uniformity, anisotropy and selectivity.80-81 There are basically for different
low-pressure plasma etched mechanisms, i.e., sputter etching, chemical etching, ion-
enhanced energy etching and ion-enhanced inhibitor etching. The degree of anisotropy
and selectivity depend on the etch mechanism used. An example of ion-enhanced
inhibitor etching is the anisotropic etching of aluminum trenches with CCl4/Cl2 or
CHCl3/Cl2 discharges. Both Cl and Cl2 can efficiently chemically etch aluminum, but it
results in an isotropic pattern. By adding carbon to the mixture, a protective carbon–
chlorine polymer film is formed at the surface. Ion bombardment removes the film at the
bottom of the trench, enabling etching at the bottom. Because the protective film is also
formed at the walls of the trench, whereas the ions bombard only the bottom, steep walls
can be formed. Possible problems of ion-enhanced inhibitor etching are, however, the
contamination of the surface, and the removal of the protective film after the etching
step.82
2.2.3.3 Surface activation and functionalization of polymers
When a plasma is brought into contact with polymers, this will induce chemical and
physical modifications of the surface, eg., producing more reactive sites, or changes in
cross-linking or molecular weight.83 In this way, materials with desired properties can be
obtained, such as wettability, adhesion, barrier protection, material selectivity and even
biocompatibility.84-85 Plasma surface treatments allow the modification of the surface
characteristics of polymers to obtain improved binding, without affecting the bulk
properties. Surface activation of polymers is carried out by exposure to a non-polymer-
forming plasma, such as O2, N2, NH3 and the inert gases.83,86 Additionally, polymer
surface can also be functionalized by plasma induced grafting, which is a combination of
plasma activation and conventional chemistry.82
15
2.2.3.4 Plasma polymerization
Besides the surface activation of polymers, thin polymer films can also be deposited
by so-called plasma polymerization, which is essentially a plasma enhanced chemical
vapor deposition process. It refers to the deposition of polymer films due to the excitation
of an organic monomer gas and subsequent deposition and polymerization of the excited
species on the surface of a substrate. Polymers formed by plasma polymerization are, in
most cases, highly branched and highly cross-linking. Plasma polymerization is
characterized by several features: 82,83
1. Plasma polymers are not characterized by repeating units.
2. The properties of the plasma polymer are not only determined by the monomer being
used, but also by the plasma parameters.
3. The monomer used for plasma polymerization does not have to contain a functional
group, such as a double bond.
(1) Fundamental aspects of plasma polymerization
The ionization of a molecule by collision with an accelerated electron is essential
process for creating plasma of a monomer (with or without carrier gas). The ionization of
molecule is first elementary step of plasma polymerization and is far more complex than
the ionization of an atom. Conventional polymerization is highly dependent on the
structure of the monomer. However, in plasma polymerization, monomers and any
organic compound without a polymerizable structure such as a double bond can
polymerize. Plasma polymerization takes place through several reaction steps. 86 In the
initiation stage, free radicals and atoms are produced by collisions of electrons and ions
with monomer molecules, or by dissociation of monomers adsorbed on the surface of the
sample. Secondarily, i.e., propagation of the reaction is the actual formation of the
polymeric chain. This can take place both in the gas phase and on the substrate film.
Finally, termination can also take place in the gas phase or at the polymer surface, by
similar processes as in the propagation step, but ending either with the final product or
with a closed polymer chain.
(2) Pulsed plasma polymerization
16
Besides the traditional continuous wave (cw) plasma polymerization, the pulsed
plasma polymerization was also very important in the present work. In this approach, the
plasma “on” period produces a burst of reactive species, which are then permitted to
undergo radical decay processes during the plasma “off” relaxation periods. The success
of this process relies on the fact that a more ordered and selective chemistry occurs
during plasma off times, relative to that occurring during the highly energetic plasma
“on” periods. The pulsed plasma is a very simple and unique method to control the film
chemistry. It allows the polymerization of monomers containing labile groups such as
amines, ethers and anhydrides, to name but a few. In fact, dramatic progressive changes
have been observed with sequential changes in the duty cycles, i.e., ratio of plasma on to
plasma off times, employed in previously work from our laboratory. With respect to
retention of monomer functionalities in plasma generated coating, it has been
increasingly apparent that an important feature of the pulsed plasma approach is that
affords an opportunity to generate plasma films under exceptionally low total power input
conditions. This simply reflects the fact that the power is turned off during major portions
of the polymerization process. The average power Peq employed under pulsed conditions
(e.g., relative to a cw run operating at the same peak power) is calculated from the
equation:
Peq = ton / (ton + toff) ? Peak Power
Where ton and toff are the plasma on and off times. The pulsed plasma approach permits
surface modification to be accomplished at Peq values, which are significantly lower than
those attainable under typical cw conditions. In fact, the pulsed plasma technique permits
extension of the examination of the composite the power, the monomer flow rate, and the
molecular weight of the monomer variation on film compositions.
(3) Advantages
The main advantage of plasma polymerization is that it can occur at moderate
temperature compared to conventional chemical reaction. Plasma-based techniques offer
the following advantages with regard to biomaterials engineering. i.e., The benefits of
plasma processing arise from the good understanding of plasma physics and chemistry
learned in other fields such as microelectronics, for example, plasma homogeneity and
effects of non-uniform plasma on the substrate surface.87
17
ii. Plasma engineering is usually reliable, reproducible, relatively inexpensive, and
applicable to different sample geometries as well as different materials such as metals,
polymers, ceramics, and composite.88-90 Plasma processes can be monitored quite
accurately using in situ plasma diagnostic devices.
iii. Plasma treatment can result in changes of a variety of surface characteristics, for
example, chemical, tribological, electrical, optical, biological, and mechanical. Proper
applications yield dense and pinhole free coatings with excellent interfacial bonds due to
the graded nature of the interface.91
vi. Plasma processing can provide sterile surfaces and can be scaled up to industrial
production relatively easily. On the contrary, the flexibility of non-plasma techniques for
different substrate materials is smaller.92
v. Plasma techniques are compatible with masking techniques to enable surface
patterning, 93-94 a process that is commonly used in the microelectronics industry.
(4) Application for biomedical devices
Application of plasma polymerized films are associated with biomedical uses (eg.,
immobilized enzymes,95-96 organelles97 and cells,98 sterilization99 and pasteurization,100
the textile industry,101 electronics (e.g., amorphous semiconductors,102) electrics
(insulators,103 thin film dielectrics,104) optical applications,105-106 chemical processing
(reverse osmosis membrane,107 permselective membrane108 and surface modification
(adhesive improvement,109-110 protective coating111-113). For example, Plasma
polymerization may offer a new alternative in biosensor interface design. The advantage
is that an extremely thin (<1mm) film with good adherence can be produced.
Furthermore, the film is pinhole free and both mechanically and chemically stable, and it
allows a large amount of biological materials to be loaded onto the surface.114The
manufacturing of integrated transducer arrays is now possible by means of plasma
technology. The techniques have actually been used to increase the dynamic range and
sensitivity of urea sensors.115
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24
25
Chapter 3
Experimental Section
Plasma film deposition, characterization methods used in the analysis of plasma
polymers, and the experimental procedures are discussed in this Chapter. Especially, the
plasma reactor is described in detail as the main set-up during the sample preparation,
as well as the definitions of plasma equivalent power and duty cycle are mentioned. To
investigate the basic chemical properties of plasma polymers, surface analytical
techniques such as Contact Angle Goniometry and Atomic Force Microscopy (AFM) are
introduced. Furthermore, Waveguide mode spectroscopy (WaMS) and Surface Plasmon
Resonance Spectroscopy (SPR) were employed in order to understand the kinetic solution
behavior. Surface Plasmon Resonance Fluorescence Spectroscopy (SPFS) are introduced
to investigate the interaction between the biomaterial molumoles and the surface of the
plasma polymers. Finally, the experimental procedures are discussed in detail.
3.1 Plasma Film Deposition
3.1.1 Plasma reactor
A 30 cm long and 10 cm wide cylindrical Pyrex glass reactor was applied for all
plasma depositions described in this thesis. Radio frequency (rf) power of the reactor was
supplied by two external concentric metal rings, with a spacing of 15 cm. Samples were
placed on top of the sample holder located at the center of the reactor chamber. Figure 3.1
shows the plasma reactor and its associated electronics. Non-polymerizable gases such as
O2, N2 and Ar could be introduced from the side arms at the reactor inlet, and their flow
rate could be controlled by MKS gas flow meters (type 159C). The flow rate of the liquid
monomer introduced for the side arms at the reactor inlet was controlled by a Kobold
floating ball flowmeter. A MKS baratron (type 122) was connected to one of the inlets, to
monitor the reactor pressure. A butterfly valve at the other reactor end was employed
with the pressure transducer, to control the process pressure. A central multi-gas
controller (MKS type 647B) controlled the flow meters, the baratron and the butterfly
valve. A pulse generator (Tektronic PG 501) controlled the pulsing of rf signal, which
26
was amplified by a RF amplifier (ENI model A300), and passed via a bi-directional
coupler (BIRD 4266), analogue wattmeter (BIRD 4410A) and matching network to the
electrodes that consists of two external concentric metal rings with a spacing of 10 cm.
An oscilloscope calibrated against the watt under cw conditions was used to adjust the
matching network and minimize reflected power during the plasma operation. All
experiments were carried out at 13.56 MHz.
Table 3.1 The substrates used in the present work
Substrates
Size of substrate
Analytical techniques
Double or single polished Si 10×20 mm2
10×10 mm2
Contact angle and plasma
polymer films thickness
measurements
Double polished Si,
BK7 glasses with 80 nm Au
10×20 mm2 FTIR
Double or single polished Si,
BK7 glasses with 50 nm Au and a
monolayer of SAM
10×10 mm2 AFM
TiO2 / SiOx film (thickness 168 nm,
refractive index 1.82)
on a quartz substrate
10×20 mm2 WaMS
LaSFN9 glasses
with 50 nm gold
20×40 mm2 SPR/SPRF
All monomers, i.e., di-(ethylene glycol) vinyl ether (EO2) and allylamine (AA),
and maleic anhydride (MA)were purchased from Sigma. Prior to use liquid monomers
(EO2 and AA) were outgassed by mulitiple freeze-thaw cycles using liquid N2. In order
to outgas, the inlet was opened for 3 min before plasma polymerizing powder MA.
Monomer gases fed though the system pass two traps, cooled with liquid nitrogen to
collect the excess reactant before going into the pump (Leybold Trivac, D16BCS/PFPE).
27
Substrates employed in the present work are summarized in Table 3.1. All silicon wafers
were cleaned in a mixture gas of argon/oxygen (9/1) plasma for 10 min. All Au/glass
substrates were used immediately after Au evaporated. The waveguide chips were
cleaned in a mixture gas of argon/oxygen (9/1) plasma for 5 min only since they can be
damaged by long time plasma clean.
Prior to plasma polymerization, the system was evacuated to a background pressure
of approximately 0.001 mbar followed by a 5 to 10 min continuous wave (cw) or pulsed
Ar or O2 discharge at a pressure 0.1 mbar. After this cleaning step, the Ar or O2 was
stopped and the system was again evacuated to background pressure. Reactant monomer
was then introduced into the reactor chamber. The polymerization reaction was initiated
after obtaining the monomer flow rate and pressure. Following each experiment the
substrates were removed and chamber was cleaned using acetone. Subsequently, the
chamber was resealed and evacuated and subjected to O2 plasma to remove any
remaining polymer deposits from the inside wall of the plasma reactor.
Figure 3.1 Schematic of the plasma reactor and the electrical components.
Sample holder
Electrodes
Matching Network
Bidirectional Coupler
Monomers
Faraday Cage
Baratron Multigas Controller
Pressure Transducer
Butterfly Valve
Vacuum Pump
RF Amplifier
RF Generator
Pulse Generator Oscillocope
Gas Flow Controller
Standard Gases Ar, N2, O2
Wattmeter
28
Both the continuous wave (cw) and the pulsed mode can employed using this
plasma system. The equivalent power under pulsed operation can be expressed as:
Peq = Ppeak ? ton / (ton+ toff )
where the ratio ton / (ton+ toff) is referred to as the duty cycle (DC).
3.1.2 Plasma polymerization conditions
Di-(ethylene glycol) mono vinyl ether (PEO2) was plasma polymerized at a
monomer pressure of 0.1 mbar, a flow rate of 0.9 sccm (standard temperature and
pressure, STP) and at adsorbed rf power from 90-100 W. Plasma polymerization were
carried out at cw and pulsed discharges, which the duty cycles ranged from 5/25 to 5/115,
including 5/25, 5/45, and 5/115.
Allylamine was polymerized under a constant flow rate of 6.3 sccm and monomer
pressure of 0.1 mbar held constant via a Kobold floating ball flowmeter. The adsorbed rf
input power ranging from 5 W to 100 W was employed, together with cw and the pulsed
duty cycles, which was from 10/50 to 10/200. To investigated the effect of process
pressure on the solution behavior of PPAA films, 0.06mbar was used under 10/50.
0.1mbar process pressure and a flow rate of approximately 1 sccm were used in the
plasma polymerization of maleic anhydride. The rf input power 100 W and cw and the
pulsed duty cycles ranging from 5/45 to 5/100 were used too.
3.2 Surface Analytical Techniques
3.2.1 Contact angle goniometry
The contact angle of liquids on solids are widely used to predict wetting and
adhesion properties of these solids by calculating their solid-vapor surface tension.1-4 The
contact angle is defined as the angle between a solid surface and tangent of the liquid-
vapor interface of a liquid drop.5 The hydrophobicity/hydrophilicity of a solid surface is
usually expressed in terms of wettability that can be quantified by contact angle
measurements.
Contact angle measurement is a simple and convenient method to determine the surface
wettability. Contact angles are not only influenced by the interfacial tensions but also by
other phenomena, such as roughness, chemical heterogeneity, sorption layers, molecular
29
orientation, swelling, and partial solution of the polymer or low-molecular constituents in
the polymeric material.6 There are two kinds of techniques to measure contact angle, i.e.,
Goniomery and Tensiometry (Wilhelmy plate technique)) .7 The former technique can
be described as the static measurement or sessile drop method. While the latter technique
is a dynamic measurement or Drop Shape Analysis 10 (DSA1). The second method was
used in the present work.8
3.2.2 Atomic force microscopy (AFM)
Αtomic force microscopy has been employed for 17 years. The first description of
AFM was published in 1986 by Binnig, Quate and Gerber. 9-11 During the last years,
AFM has been used increasingly to investigate microbial surfaces at high resolution. The
technique can provide three-dimensional images of the surface ultrastructure with
molecular resolution, in real time, under physiological conditions, and with minimal
sample preparation. AFM imaging is performed by sensing the force between a very
gjdlsk
Figure 3.2 General principle of AFM.
sharp tip and the sample surface, (Fig 3.2).12 An AFM image is generated by recording
the force change as the probe (or sample) is scanned in the x and y direction. The sample
is mounted on a piezoelectric scanner, which ensures three-dimensional positioning with
high resolution. The force is monitored by attaching the tip to a pliable cantilever and can
Scanner
Cantilever
Laser
Sample
Photodiode
30
be measured the bending or “deflection” of the cantilever. The larger cantilever
deflection, the higher the force that will be experienced by the probe.12 A laser beam is
focused on the free end of the cantilever, and the position of the reflected beam is
detected by a position-sensitive detector (photodiode). AFM cantilevers and probes are
typically made of silicon or silicon nitride by microfabrication techniques.
AFM can be used to imaging modes and force measurements. A number of AFM
imaging modes are possible. The most widely employed imaging mode is the contact
mode, in which sample topography can be measured in different ways. In the constant-
height mode, one simply records the cantilever deflection while the sample is scanned
horizontally. To prevent sample damage, minimizing large deflections, thus holding the
applied force to small values, is necessary. This is achieved in the constant-deflection
mode, in which the sample height is adjusted to keep the deflection of the cantilever
constant by using a feedback loop. The feedback output is used to display a true “height
image”. The height image provides quantitative height measurements, allowing accurate
measurement of surface roughness, the height of the surface features, or the thickness of
biological layers. The deflection image does not reflect true height variation, but since the
frequency response is much higher, it is more sensitive to fine surface details than the
height signal. In the experiments described in Chapter 4, AFM was employed to measure
the surface roughness of plasma polymers.
Measuring the force acting between the AFM tip and the sample, by means of
force-distance curves, is important in defining the imaging force and thus in optimizing
the image resolution. AFM force measurement can be used to probe the sample’s
physical properties. Force-distance curves are recorded by monitoring, at a given x-y
location, the cantilever deflection as a function of the vertical displacement of the
piezoelectric scanner. A raw curve is a plot of the photodiode voltage versus the scanner
position. By using appropriate corrections, this can be converted into a force-versus-
separation distance curve. The different parts of a force-distance curve can provide a
wealth of information. At large probe-sample separation distances, the force experienced
by the probe is zero. As the tip approaches the surface, the cantilever may bend upwards
due to repulsive forces until it jumps into constant when the gradient of attractive forces
exceeds the spring constant plus the gradient of repulsive force. According to the
31
equation of Fpull-off = K ? ∆Z, the approach portion of the force-distance curve can be
used to measure forces, including van der Waals and electrostatic of the sample. When
the probe is retracted from the surface, the curve often shows a hysteresis referred to as
the adhesion “pull-off” force, which can be used to estimate the surface energy of solids
or the binding forces between complementary molecules.12, 13
3.3 Thin Film Characterization by Optical Techniques
3.3.1 Waveguide mode spectroscopy (WaMS)
An optical waveguide structure is a high refractive index medium in a lower
refractive index environment.14-16 The most simple device possible is a planar waveguide
consisting of a high index waveguide layer on top of a low index substrate in a low index
cover medium such as air or water. If the layer thickness is enough high, guided optical
modes exist because of total internal reflection at the high/low index interfaces. An
exponentially decaying so-called evanescent field of the low index medium is deduced by
solving Maxwell’s equations. A thin adlayer can interact with the guide modes resulting
in a modification of their wave vector due to the non-zero field distribution in the
surrounding medium. This can be measured and employed to monitor optical properties
of a thin film or adlayer deposited on the top of surface. When a thin plasma polymer
film was deposited on the top of the waveguide chip, (Figure 3.3) thus resulted in the
change in the refractive index of the surrounding medium. This in turn induces changes
in the effective refractive indices Neff,TE0 and Neff,TM0 of the guided modes. The effective
refractive indices provide the phase velocity of the guided mode and depend on a
polarization (TM0 and TE0) and the mode number. Shifts in the effective refractive index
(∆Neff i,j) can be determined by measuring the changes in the coupling angle α using
equation (3-1).
Neff i,j = nC sin α + Iλ/Λ ( 3-1)
where i,j are the mode numbers, nC is the refractive index of the cover medium, λ is the
wavelength of the laser used, I is the diffraction order, and Λ the grating constant. From
the effective refractive index shifts both the thickness d and the refractive index n of a
plasma polymer layer on top of the waveguide can be calculated using the dispersion
relationship for a four layer planar waveguide. A more detailed description can be found
32
elsewhere.17The d and n of the TiO2 / SiOx waveguide were determined before the plasma
deposition by measurement against air.
Figure 3.3 Electric or magnetic field distribution for a zeroth mode in a planar multiplayer
waveguide.
Figure 3.4 Schematic diagram of WaMS set-up.
The experiment presented in Chapter 5 was carried out using a home built
spectrometer (Figure 3.4). A curette was used to enable swelling measurements in
solvents. A grating coupler with periodicity Λ was used to couple a polarized HeNe laser
Polarization optics
Laser
Chopper
Lock-in Personal computer
Step-motor
Waveguide chip
θ
Goniometer
Detector
Cuvett
Tube
Substrate
Waveguide
Cover medium
α
Λ
Field distribution TE0 or TM0 mode
Plasma polymer
33
beam (λ = 632.8 nm) from the external cover medium into the waveguide. The
wavevector of the waveguide modes (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 fixed on both ends of the waveguide.
3.3.2 Surface plasmon resonance spectroscopy (SPS) and surface plasmon resonance
fluorescence spectroscopy (SPFS)
The surface interactions of biomaterials are fundamental to determine critical
issues, such as host response and biocompatibility. Indeed, many challenges to
biomaterial design can be directly related to the control of surface interactions. Many
surface analytical techniques have been applied to the study of biomaterials, but few of
these are able to monitor dynamic interactions within a fluid environment, that may be
tailored to model likely conditions encountered in vivo. Scanning probe microscopy
(SPM)19-19 attenuated total reflectance infrared spectroscopy (ATRIR)20 and spectral
ellipsometry21 are three widely used techniques that can offer the possibility to study
dynamic events at a range of surfaces. However, SPR is rapidly gaining recognition and
application as a powerful tool for biomaterial characterization. The use of SPR to probe
surface interactions is advantageous, since it is able to rapidly monitor any dynamic
process, such as adsorption or degradation, to a wide range of biomedically relevant
interfaces in real time without the need to label the adsorbate and without the need for
complex sample preparation. It also can rapidly obtain information on the rate and extent
of adsorption, enabling the determination of dielectric properties, the
association/dissociation kinetics and the affinity constants of specific ligand-ligate
interactions.22 For not too small chemical molecules binding from an aqueous solution to
the interface a sufficient signal can be observed. With a lower limit for a reliable signal
detection corresponding to an effective layer of about 0.1-0.2 nm a sufficient signal-to-
noise can be generated allowing for a detailed kinetic analysis and determination of
binding affinity.23 If very small analytes of low molecular mass are to be detected, only
very thin effective layers are generated, too low to be detected.24 Hence, a sensitivity
limitation is generated. As consequence, to enhance the signal of the interfacial binding
34
events, using (fluorescence) label techniques in connection with surface plasmon
spectroscopy were considered.
Figure 3.5 The Kretchmann configuration for SPR. Resonance of a surface plasmon is excited at
the metal/air interface when the angle of incidence of light is such that the
evanescent component of its wave vector (Kev) is equal to the wave vector of the
propagating surface plasmon (Ksp).
A surface plasmon is a longitudinal charge density propagating wave along the
interface of two media, where one is a metal and the other a dielectric.25 The choice of
metal used is critical, since the metal must exhibit free electron behavior as described by
the free electron model.26 Suitable metals include silver, gold, copper and aluminium of
which silver and gold are more commonly used. Silver is used as it provides a sharp SPR
resonance peak and gold due to its stability.27 Two different experimental systems for the
excitation of surface plasmons were developed by Otto28 and Kretchmann.29 However, it
is the attenuated total reflectance (ATR) configuration developed by Kretchmann that is
widely used within the designs of most SPR instruments. The Kretchmann configuration
(Figure 3.5) relies on the phenomenon of total internal reflection. This occurs when light
traveling through an optically dense medium (e.g. glass) reaches an interface between this
medium and a medium of a lower optical density (e.g. air), and is reflected back into the
dense medium. Although the incident light is totally internally reflected, a component of
this light, the evanescent wave or field, penetrates the interface into the less dense
medium to a distance of one wavelength.30 Total internal reflection fluorescence (TIRF)
relies on the phenomenon of the evanescent field to excite molecules near a glass/liquid
35
interface. In SPR a monochromatic, p-polarized light source is used and the interface
between the two optically dense media is coated with a thin metal film (of thickness less
than one wavelength of light). The wave vector of the evanescent field is given by
where wo is the frequency of incident light, g' the refractive index of the dense medium
(glass), h the angle of incidence of the light and c the speed of light in a vacuum. The
wave vector of a surface plasmon (Ksp) can be approximated to
where εm is the dielectric constant of the metal film and ηs is the refractive index of the
dielectric medium.28 The evanescent wave of the incoming light is able to couple with the
free oscillating electrons (plasmons) in the metal film at a specific angle of incidence
corresponding to when Ksp = Kev, and thus the surface plasmon is resonantly excited.
This causes energy from the incident light to be lost to the metal film resulting in a
reduction in the intensity of reflected light which can be detected by a two-dimensional
array of photodiodes or charge coupled detectors (CCD). Examination of Eq. (3-3) shows
that Ksp is dependent on the refractive index of the water/air medium above the metal
film. Therefore, if the refractive index immediately above the metal surface changes, by
the adsorption of a protein layer, a change in the angle of incidence required to excite a
surface plasmon will occur. By monitoring the angle at which resonance occurs (the SPR
angle) during an adsorption process with respect to time, an SPR adsorption profile can
be obtained.
A quantitative description of all observed phenomena is possible within a
theoretical treatment that goes back to Fresnel and models the optical response of a
layered architecture by solving Maxwell’s equations. Each layer is described by its
complex dielectric function with a real and an imaginary part (for absorbing materials)
and the layer thicknesses. If one monitors the reflectivity, R, i.e. the reflected light
intensity Ir, scaled to the incoming intensity, as a function of the angle of incidence θ
(relative to the normal of the interface) the well-known behavior of the total internal
(3-3)
(3-2)
36
reflection can be observed: Below a certain critical angle θc which is given by Snellius’
law, i.e. by the refractive indices of the solid and the liquid, respectively, most of the light
is transmitted and hence the reflectivity is very low. As one approaches θc, however, the
reflectivity steeply increases and reaches unity above θc, shown in Figure 3.6, which is a
typical SPR scan of bare gold and a monolayer self-assembled layer.
Figure 3.6 Typical reflectivity scans of a) a bare substrate of LaSFN9 glass with a 47 nm thick Au
coating on top, measured in PBS buffer and b) 3 nm thick aldlayer of
octadecanethiol.
In order to increase the sensitivity and specificity of the binding assays, particularly
in the case of antibody binding, advantage is taken from an extension of SPS: Surface
Plasmon Resonance Fluorescence Spectroscopy (SPFS),29 applied simultaneously with
SPR. The electromagnetic field intensity at the gold surface is strongly related to the
reflectivity of the system. As the reflectivity monitored at different incident angles
reaches a minimum corresponding to the excitation of the surface plasmons, the surface
field intensity is maximized, yielding a mirror image of the plasmon reflectivity curve.
This intensity enhancement can be employed to increase the fluorescence emission of the
surface bound dye molecules (e. g. Cy5) excited by the amplified electromagnetic field.
Both SPS and SPFS measurements were carried out using the experimental set-up
shown below, (see Figure 3.7) which is based on the configuration introduced by
Kretschmann and Rather. The resulting high electromagnetic field excites the dye
molecules near the metal surface. A thin metal layer or plasma polymerized adlayer (e. g.
50 55 60 65 70
0
20
40
60
80
100
b)a)
Ref
lect
ivity
/%
Incident angle/degree
37
Au or Ag) is evaporated on the glass slide, and placed on the base of a prism. A HeNe
laser operating at λ= 632.8 nm passes a chopper (EG/G), (used also as the reference for
the amplifier), two polarizers (Berliner Glas) for intensity and polarization control, before
being reflected from the base of a 90 high index (nprism = 3.4069 @ λ=632.8nm) glass
prism mounted on a θ - 2θ goniometer arrangement. A lens collects the reflected light on
the photo diode, the output signal of which is fed into the lock-in amplifier (EG/G 5110).
Monitoring the reflected intensity as a function of the angle of incidence θ, gives the
normal angular reflectivity scans.30 The excitation frequency of the dye molecules is
matched by using a laser of the appropriate wavelength (HeNe laser). The emitted
fluorescence blocked by an interference filter (LOT 670nm, 10nm FWHM) selects a band
of the fluorescence light that is finally measured by a photomultiplier in a photon
counting mode mounted at the backside of the sample. A counter (HP 3553) is employed
to digitize the photomultiplier signal output. The experimental setup is driven by a PC
allowing the real time and simultaneous detection of reflectivity and fluorescence.
Figure 3.7 Schematic of the SPRF set-up that allows for the simultaneous recording of angular or
kinetic scans of surface plasmon spectroscopy and monitoring the fluorescence
originating from chromophores excited by the surface plasmon wave at the base of
the metal-coated coupling prism in this Kretschmann.
laser 632.8 nm
polarizers
photodiode
flow cell filter
2θ
goniometer
lock-in amplifier
PC
θ
chopper
Laser-shutter
attenuator lens
PMT
prism
shutter controller
motor- steering
photon- counter
38
If one monitors the reflectivity at a fixed angle of incidence, then any change of the
interfacial architecture, e.g. induced by adsorption, desorption, and the change of buffer,
can be quantified by evaluating the resulting change of the reflected intensity in this
kinetic mode of operation.
In the present work, SPR was employed in monitoring the adsorption behavior of
proteins and DNA (see Chapter 5, 6, and 7). As while SPFS setup was used to measure
the hybridization and dissociation behavior (see Chapter 7).
3.4 Sample Preparation of Solution Behavior Measurements
3.4.1 Phosphate buffer (PB) solution preparation
(A) 0.2 mol/L NaH2PO4 (27 g in 1L H2O) and (B) 0.2 mol/L Na2HPO4 (53.65 g
Na2HPO4·7H2O, or 71.7g Na2HPO4·12H2O in 1L H2O were used to prepare different PB
solution. They were mixed according to the following recipes (Table 3.2) depending on
the pH required and plus 100 mL H2O diluted to 200 mL solution.
3.4.2 Self-assembled monolayer (SAM)
For studies in aqueous solution an adhesion layer is required to ensure adhesion of
the plasma polymer on the gold SPR substrates. The self-assembled monolayers were
prepared from a solution of 10 nM octadecanethiol dicholoromethane. The substrates
were immersed in the solution for 1 hour. The samples were removed from the solution
and rinsed with dichloromethane and ethanol after self-assembly.
Table 3.2 The recipes of PB solution
Volume A/mL Volume B/mL pH
100 0 4.5
93.5 6.5 5.7
39 61 7
0 100 9
39
3.4.3 Plasma polymerization
The plasma polymerization conditions used in this experiment were summarized in
Table 3.3. Plasma polymer films were always prepared just prior to SPR and WaMS
measurements.
Table3.3. The duty cycles of three plasma polymers employed in protein adsorption.
EO2 cw, 100 W, Peq100 W 5/45, 100 W, Peq 11 W 5/100, 100 W, Peq 5 W 5/115, 100 W, Peq 4.3W
Allylamine cw, 100 W, Peq100 W
90 W, Peq90 W
5 W, Peq 5 W
10/50, 100 W, Peq20 W
90 W, Peq 18 W
5 W, Peq 1 W
10/100, 100 W, Peq10 W
10/200, 100 W, Peq5 W
90 W, Peq 4.5 W
20 W, Peq1 W
MA cw,100 W, Peq100 W
90 W, Peq90 W
5/45, 100 W, Peq11 W
5/100, 100 W, Peq5 W
90 W, Peq 4.5 W
3.4.4 SPR measurements
Before each kinetic measurement, a surface plasma reflectivity scan of the plasma
modified SPR substrate in PBS was recorded, after which the angle is set on the left side
of the reflectivity minimum, in the range where the resonance decreases linearly.
3.5 Procedures for Protein Adsorption Measurements
3.5.1 Plasma polymerization
The plasma polymerization conditions were summarized in Table 3.4. Polymer film
thickness ranged between 10 and 90 nm. Plasma polymer films were always prepared just
prior to the SPR protein studies. Before each protein adsorption experiment, the plasma
polymers were extracted in PBS until they were stable.
Table3.4. The duty cycles of three plasma polymers employed in protein adsorption. PEO2 cw,100 W,Peq100 W 5/45,100 W,Peq11 W 5/100,100W,Peq5 W 5/115,100W, Peq4.3W
PPAA cw,100 W,Peq100 W 10/50,100 W,Peq20 W 10/100,100W,Peq10 W 10/200,100W,Peq5 W
PMA cw,100 W,Peq100W 5/45,100 W,Peq11 W 5/100,100 W,Peq5 W
40
3.5.2 Protein solution preparation
The protein solutions were prepared immediately before use by dispersing
commercially available BSA, fibrinogen and IgG in phosphate buffered saline (PBS, pH
7.4) at a concentration of 1 % by weight. All proteins were purchased from Sigma.
3.5.3 SPR measurements
A surface plasma reflectivity scan of the plasma deposited on the Au/LaSFN9
substrate is recorded in PBS, afterwards the angle was set on the left side of the
reflectivity minimum. Then the kinetic measurement of plasma polymers in PBS was
carried out. Once the polymer was stable, the flow cell was rinsed with PBS buffer
solution, a second reflectivity scan was then recorded, afterwards the protein was injected
into the flow cell and time resolved kinetic measurements of protein adsorption were
carried out. Once the optical properties of the interface were stable (i.e. no more protein
was adsorbing), the cell was rinsed three times with 1% sodium dodecyl sulfate (SDS)
solution in order to remove elutable (denatured) protein. The last reflectivity scan was
recorded, and the layer thickness of the adsorbed protein was calculated from the SPR
angle shift according to the Fresnel theory (a refractive index of n = 1.5 for all proteins
was used to fitting of the curves, as well refractive indices of plasma polymers discussed
in Chapter 5). However, in the case of the experiment of proteins adsorption on plasma
polyallylamine, no SDS was employed due to the interaction between SDS and PPAA
surface.
3.6 Procedures for DNA Immobilization and Hybridization on PPAA
3.6.1 Plasma polymerization
The input powers (Ppeak) used in the continuous wave plasma deposition
experiments were 5 W and 100 W. During the pulsed plasma polymerization
experiments, duty cycles of 10/50 and 10/200 were used. The plasma process pressure
used was 0.1 mbar and 0.06 mbar for some 10/50 PPAA films. Deposition times ranged
between 30 seconds and 15 minutes. Because of the higher deposition rate and high
41
affinity constant for DNA, cw, 5 w and 7 min PPAA was applied in DNA hybridization.
Each polymer film before probe adsorption was immersed in corresponding buffer
solution over 10 hours, so that the whole polymer network can swell completely.
Figure 3.9 The schematic of the sample multilayers architecture.
3.6.2 DNA samples
(1) Probe sequences
P4 25 mer 5’- GGA ATG TGC CAT ACC GAA TCC GTG T –3’
P2 30 mer 5’-TTT TTT TTT TTT TTT TGT ACA TCA CAA CTA-3’, 5’ Biotin
P6 60 mer 5’-GTC TAT ACA TTC CTG AGA TTC TGG GAA AGG TGC TCA AAG
ATG TAC TGA GAG GAG GGG TAA-3’
(2) Target sequences
Mismatch one (MM1) T1 15 mer Cy5-TAG TTG TGA CGT ACA-3’
Mismatch zero (MM0) T2 15 mer Cy5-TAG TTG TGA TGT ACA-3’
Mismatch two (MM2) T3 15 mer Cy5-TAG TTG TCA CGT ACA-3’
Completely mismatch T15 15 mer
spacer
Prism
47 nm Au
PPAA
Quartz slide
3 nm SAM
UV light
Tygon tubing
Sample
Peristatic pumpLaSFN
42
The DNA probe samples used were purchased from MWG BIOTEC AG,
Ebersberg, Germany and stored at – 4 °C until use. The schematic of the custom flow cell
and the position of UV light is shown in Figure 3.9. It consists of a thin polydimethyl
siloxane (PDMS) or steel spacer (300 µm, with a 5 mm × 7 mm ellipse hole) and a quartz
cover slide (Herasil glass) through which two holes were machined and two steel needles
were glued used as inlet and outlet, respectively. The flow cell was attached, via Tygon
tubing with an inner diameter of 0.76 mm, to a peristaltic pump (Ismatec) and the sample
tube to form a closed circulation loop. Buffer and sample solutions were exchanged
manually with little trouble by air bubbles. Once the exchange was completed, the whole
loop was closed and completely sealed allowing for a long interaction time (>24 h). The
loop volume is approximately 300 µm, with a minimum sample consumption of
approximately 400-600 µm to ensure the desired analyte working concentration.
3.6.3 Experimental procedure for DNA hybridization
(1) Circulate 100 nM DNA probe into the flow cell, when reaching the equilibrium,
rinse with PBS.
(2) Irradiate samples with UV light (254 nm/2 W or 366 nm/8 W) situated at the back
of the sample at 4 cm distance.
(3) Circulate 0.1 M NaBorate (Na2B4O7.10H2O), pH 8 for 2 min (to obtain the weak
basic environment which is favorable for the hydrolysis of Succinic anhydride
(SA)).
(4) Dissolve 1.428 g SA in 45 ml of in 1-methel-2-pyrrolidinone, to this, add 5 ml 0.6
M NaBorate, and stir until dissolved.
(5) Circulate this blocking solution for 30 min.
(6) Rinse samples with 0.1 M NaBorate for 2 min, MilliQ for 4 min, and buffer
solution for 10 min.
(7) Circulate 100 nM target DNA to do DNA hybridization.
It should be noted that SA solution was freshly prepared for each experiment, since
the anhydride also reacts with water. In all experiments, a stock solution of
oligonucleotide with the concentration of 100 nM was applied. The Langmuir adsorption
isotherm was measured in a concentration range from C0=1 to 1000 nM.
43
3.6.4 SPFS measurements
All the experiments were performed at room temperature (approximately 21 ?C).
All samples were mounted into the spectrometer and the film equilibration in PBS
solution under a dynamic flow was monitored (1 mL/min). Typically, the injected
volume of sample solution was 1 mL. A neutral filter (attenuator) is used occasionally to
attenuate the fluorescence in cases where it shows a too strong intensity to keep the PMT
running in the linear range (<1-2 million counts/second). It attenuates the intensity by a
factor of 7 as it was obtained from a calibration experiment. All fluorescence data in this
thesis are scaled to the situation with an attenuator in front of the PMT. Routinely, an
angular scan was taken in running buffer before starting the kinetic mode. The angle of
incidence, θ was then fixed at the angle, at which the reflectivity is approximately 40%.
At this angle, the reflectivity changes are approximately linear with the angle shift of the
resonance minimum. Kinetic curves record both, reflectivity and fluorescence signal, as a
function of time. Once the film had stabilized, the previously prepared DNA solution was
passed through the cell. Once no more changes were observed in the % reflected light,
DNA binding was assumed to be complete and the wet cell was rinsed with four cell
volumes of PBS solution to remove excess DNA molecules.
References
1. Ε.D. Meerwall, C.W. Frank, S.N. Semerak, J.L. Koenig, Adv. Poly Sci., Springer-
Verlag Berlin Heidelberg New York Tokyo, 1984, 87.
2. X.M. Wang, Ζ. Gershman, Α.Β. Kharitonov, Ε. Katz, Ι. Willner, Langmuir, 19, 2003,
5413.
3. J.M. Uilk, A.E. Mera, R.B. Fox, K.J. Wynne, Macromolecules, 36, 2003, 3689-3694.
4. H. Kamusewitz, W. Possart, Appl. Phy. A, 76, 2003, 899.
5. W.A. Zisman, Contact Angle-Wettability and Adhesion, R. F. Gould (Ed), Am. Chem.
Soc., Washington, D. C., 1964.
6. P. Atkins, J. de Paula, Physical Chemistry, 7th edition, Oxford University Press, UK,
2001, 154.
7. M.E. Schrader, G. loeb, Modern Approach to Wettability, Plenum Press, N.Y. 1992.
44
8. DAS1 ,user manual,V010924,Krüss Gmbh,Hamburg, 2001
9. G. Binning, H. Rohrer, Helvetica Physica Acta 55, 1982, 726.
10. G. Binning, H. Rohrer, Ch. Gerber, E. Weibel, 1982, Phys. Rev. Lett., 49, 1982, 57.
11. M.J. Miles, in Characterization of Solid Polymers; New Techniques and
Developments Spells S. J. (Ed.), Chapman &Hall, London, UK, 1994.
12. F. Yves, Dufreˆne, J. Bacteriolohy, 184, 2002, 5205.
13. F.J. Giessibl, Rev. Mod. Phys., 75, 2003, 949.
14. S. Busse, V. Scheumann, B. Menges, S. Nittler, Biosensors Bioelectron., 17, 2002,
704.
15. V. Jacobsen, B. Menges, R. Förch, S. Mittler, W. Knoll, Thin Solid Films, 409, 2002,
185.
16. V. Jacobsen, B. Menges, A. Scheller, R. Förch, S. Mittler, W. Knoll, Surf. Coat.
Techn., 142-144, 2001, 1105.
17. K. Tiefenthaler, W. Lukosz, J. Opt. Soc Am. B, 6, 1989, 209.
18. W. Knoll, Ann. Rev. Phys. Chem., 49, 1998, 565.
19. K.M. Shakesheff, M.C. Davies, C.J. Roberts, S.J.B. Tendler, P.M. Williams, The role
of scanning probe microscopy in drug delivery research. Crit Rev Ther Drug Carrier
Syst, 13, 1996, 185.
20. M.C. Davies, C.J. Robert, S.J.B. Tendler, P.M. William, The surface analysis of
polymeric biomaterials. In: Braybrook J, editor. Biocompatibility: assessment of
materials and devices for medical applications. Chichester: Wiley, 1997, 65.
21. B.D. Ratner, Characterization of biomedical surfaces. Cardiovasc Pathol, 2, 1993,
87S-.
22. C. Striebel, A. Brecht, G. Ganglitz, Biosens Bioelectron., 9, 1994, 139.
23. 19. J. Davies, Nanobiology, 3, 1994, 5.
24. L.S. Jung, K.E. Nelson, C.T. Campbell, P.S. Stayton, S.S. Yee, Y. Perez-Luna, G.P.
Lopez, Sens. Actuators B, 54, 1999, 137.
25. J. Spinke, M. Liley, H.J. Guder, L. Angermaier, W. Knoll, Langmuir, 9, 1993, 1821.
26. I. Lundstrom, Biosens Bioelectron., 7, 1994, 725.
27. A.D. Boardman, editor. Electromagnetic surface modes. Chichester: Wiley, 1982.
28. H.E. De Bruijn, R.P.H. Kooyman, J. Greve, Opt., 31, 1992, 440.
45
29. A. Otto, Z Phys., 216, 1968, 398.
30. E. Kretchmann, Z Phys., 241, 1971, 313.
31. L.G. Fagerstam, A. Frostell-Karlsson, R. Karlsson, B. Persson, I. Ronnberg, J.
Chromatogr, 597, 1992, 397.
32. K. Matsubara, S. Kawata, S. Minami, Appl. Spectrosc., 42, 1988, 1375.
33. F. Yu, D. Yao, W. Knoll, Anal. Chem., 75, 2003, 2610.
34. L.A. Luck, M.J. Moravan, J.E. Garland, B.S. Sondi, D. Roy, Biosensors Bioelectron.,
19, 2003, 249.
46
47
Chapter 4
Properties of Plasma Polymerized Films in Air
4.1 Introduction
In recent years, utilization of poly(ethylene glycol) (PEG) for biomedical and
biotechnical applications has grown increasingly important. PEG modified surfaces are
usually effective in protein rejection, non-immunogenicity, and non-antigenicity in an
aqueous environment.1-2 In addition, this polymer is nontoxic and does not harm active
proteins or cells, although it interacts with cell membranes. Using an RF glow discharge
process, Lopez et al.3 demonstrated that the plasma-induced polymerization of
tetraethylene glycol dimethylether (tetraglyme) generates surfaces having high short-term
resistance to the adsorption of biomolecules. Subsequently, Beyer et al.4 showed that the
plasma polymerization of triethylene glycol monoallyl ether (EO3), particularly when the
plasma was operated in a pulsed mode, provided surfaces which are highly efficient with
respect to prevention of protein adsorption. More recently, Johnson et al.5-6 demonstrated
that the gas phase continuous wave plasma deposition of cyclic ethers can also lead to
surfaces with effective non-fouling properties. Wu et al. investigated the protein
adsorption properties of these plasma synthesized one (ethylene oxide vinyl ether) and
two (diethylene oxide vinyl ether) ethylene oxide using 125I-labeled albumin and
fibrinogen, and observed surprisingly effective, non-fouling surfaces were observed with
films synthesized from the monomer containing two ethylene oxide units.7
For many applications, amine rich surfaces are of particular interest, because they
are known to influence protein adsorption and cell adsorption, and provide sites for the
covalent immobilization of graft polymers and biomolecules.8-12 Amino groups are
usually incorporated on the surface using ammonia or organic amines in the plasma
medium. Treatment of various materials with ammonia plasma is the subject of many
papers.13-25 The density of amine groups incorporated their amount depended very
strongly on the plasma apparatus and plasma parameters. Retention of the amine group
can be increased when pulsed or remote plasma is used.17, 26,27 Surface amine groups were
tested to promote cell growth (adhesion for cell culture)24,25,27 to change adsorption of
48
proteins,28 to bond biomolecules chemically19-22,29-32 and to improve membrane
performance,15,17 and adhesion to other materials.23 For example, cell attachment and
viability on plasma polymerized allylamine layers were found to be more intensive than
on the control polyester. The proteins can adsorb on a “bioinert” metal by modifying with
plasma polymerized allylamine, even on the very low NH2 group surfaces.
Maleic anhydride is of particular interest for the synthesis of functional organic thin
films because of the double bond and the reactive anhydride group. Recently plasma
polymerized maleic anhydride films were used as supports for a lipid bilayer.33-36 It is
widely used as an organic reagent and can participate in a variety of chemical reactions
occurring at the double bond by photochemical reactions,37-38 conventional
polymerization,39-40 copolymerization,41-43 and graft polymerization44-45 or at the
anhydride group as, for example, discussed by Kalguthar et al.46 and Evenson et al.47-48
Plasma assisted polymerization of maleic anhydride is, however, difficult, since even at
relatively low input energies the anhydride group is lost and the deposited films contain
mostly dissociation products rather than the desired anhydride group. This could be
overcome by pulsed plasma method.
In all applications of plasma polymers, their surface properties (surface wettablity,
morphology or roughness) and chemical structures are of great importance. The chemical
structure of plasma polymers deposited at different plasma conditions can be observed
using FTIR. Consequently, how plasma conditions affect the chemical structure of
plasma films can be investigated. Contact angle measurements are used to determine if a
plasma film is hydrophilic or hydrophobic. AFM was employed to investigate the surface
morphology of the plasma polymers prepared at different plasma conditions.
4.2 Plasma Polymerized Di- (ethylene glycol) Vinylether (PEO2)
4.2.1 Spectroscopic characterization
Compositional changes in the molecular structure of PEO2 with variations in
plasma deposition conditions were clearly evident in the FT-IR of these polymers. FTIR
spectral analysis of the EO2 plasma polymers deposited under two different plasma
conditions are shown in Figure 4.1. The unreacted EO2 monomer shows the expected
peaks for C=C double bonds (1600 cm-1), the C-H component (2900-3000 cm-1) and –OH
C H
49
groups (3400 cm-1). After plasma polymerization, the C=C double bond absorption band
could not be observed, indicating that the EO2 monomer polymerized. On the other hand,
the plasma films reveal an absorption band at 1716 cm-1 that is not present in the starting
Figure 4.1 FTIR spectra of PEO2 films deposited under (5/25, Peq 22 W) and (5/100, Peq 5.5 W)
compared with neat monomer.
monomer. This band is attributed to carbonyl groups also formed during the plasma
process. High duty cycle (5/25, Peq 22 W) plasma deposited films showed the presence of
carboxyl groups at 1700 cm-1 and only a low relative intensity – OH peak, whereas low
DC (5/100, Peq5.5 W) PEO2 films possess a much lower relative intensity carbonyl peak
with an -OH peak of much higher relative intensity. As shown by these spectra, there are
gradual decreases in the intensity of the C-O and O-H stretching vibrations and increases
in the intensity of the C=O stretching vibration, with increasing the plasma duty cycle.
Also, the wavenumber for the O-H stretching maximum shifts to progressively higher
wavenumbers with decreased plasma duty cycle during film formation. In the present
case, the relative importance of these C-O groups are dramatically reduced as the plasma
duty cycle employed during polymerization was reduced. The same result, which was in
agreement with XPS analysis, was observed by Y.L. Wu and other researchers. An
increase in film C-O/C-C ratio occurred with decreasing the input power at constant duty
cycle or decreasing the duty cycle at constant input power.4-7
3500 3000 2500 2000 1500 1000
C-Hx
5/25, Peq 22 W
5/100, Peq 5.5 W
EO2 monomer
C-O-CC=O
C=C
O-HA
dsor
ptio
n
wavenumber(cm-1)
50
4.2.2 Surface wettability
The advancing and receding contact angle (θa and θr) of PEO2 films, which were
deposited at input power 100w and ton 5 ms (see Figure 4.2), decrease with increasing
plasma off time (toff). According to Young’s Eq., the contact angle can be used to
determine the surface tension of the substrate. As the contact angle rises, the surface free
energy of the interface between the polymer film and the saturated vapor decreases.
Hence, the surface free energy of PEO2 films decreases with increasing the plasma input
power and duty cycle. The hysteresis between the advancing and receding angle, Figure
4.2, could be due to high surface roughness, chemical heterogeneity, surface-
configuration, or adsorption and desorption process. However, in this case, the surface
roughness as measured by AFM is below 0.1 µm (see section 4.1.3), the chemical
structure is heterogeneous (one monomer polymerized), and there is no adsorption and
desorption of other molecules on PEO2 films. These effects mentioned above (the surface
roughness, chemical heterogeneity, and adsorption or desorption) could be negligible.
Hence from the molecule point of view, the most important factor is the surface-
configuration change. The kinds of atoms and groups that exist at the interface determine
the surface properties. Consequently, when a water drop is contact with the polymer
surface, the surface configuration should change following the change of
sgdgdsjgggskldjgl
Figure 4.2 Water contact angle as a function of the plasma offtime for the plasma EO2.
surrounding medium from air to water. The main driving force for the rotation of the
molecules at the surface is the strong interaction between the water and the hydrophilic
20 40 60 80 100 12020
30
40
50
60 θa θr
EO2, 100 W, ontime = 5 ms
Wat
er c
onta
ct a
ngle
/°
off time/ms
RMS=0.097 nm
(a)
groups, which is determined by the bility of the molecules to rotate and hydrophilic
group extent. Higher molecules rotat
higher discrepancy between advancin
for C-O groups on the higher Peq P
polymer-water to the bulk of the poly
higher cross-linking degree, indicatin
4.2.3 Homogenity and morphology
The homogeneity and morphol
the plasma conditions employed dur
Figure 4.3 (a) and (b) show the A
thickness. Compared with 12 nm cw,
Figure 4.3 AFM images of (a) cw, 100 W
100 W, 2 min, dpolymer 50
wafer.
50 nm cw, 100 W film possesses a
surfaces roughness of 5/45 and 5/115
summarized in Table 4.1 together wi
films deposited at 5/45 increases from
to 0.104 nm. As while, for 5/115, 10
a
51
(b)
RMS=0.267 nm
ion ability and hydrophilic group extent induce the
g and receding contact angle. It takes a long time
EO2 surface to rotate back from the interface of
mer when measuring the receding angle due to the
g the higher discrepancy between θa and θr.
ogy of plasma polymers are mainly determined by
ing the deposition process and the films thickness.
FM images of (cw, 100 W) films with different
100 W film (the average roughness Ra = 0.054 nm),
, 30 sec, dpolymer 12 nm, Ra = 0.054 nm And (b) cw,
nm, Ra = 0.133 nm PEO2 films deposited on silicon
higher surface roughness (Ra = 0.133 nm). The
PEO2 films have been investigated too, which are
th the cw conditions. When the thickness of PEO2
20 nm to 50 nm, the Ra increases from 0.034 nm
0 W, 8 min, dpolymer 45 nm PEO2 film, the Ra is
52
about 0.054 nm. If one compares these three plasma polymers, the Ra decreases with
decreasing the duty cycles. The decreased surface roughness under the low duty cycle
could be explained by the absence of high-energy surface bombardment during the
majority of the film formation process. Additionally, the weak film radiation and low
substrate temperature during deposition under low duty cycle also lead to the decreased
surface roughness.
Table 4.1 The Ra and RMS of PEO2 films deposited at cw, 5/45, and 5/115 (100 W)
PEO2 films Thickness/nm Ra/nm RMS/nm
12 0.054 0.097 cw, 100 W
50 0.133 0.267
20 0.034 0.112 5/50, 100 W
50 0.104 0.269
5/115, 100 W 45 0.054 0.095
4.3 Plasma Polymerized Allylamine (PPAA)
4.3.1 Spectroscopic characterization
Figure 4.4 shows the FTIR spectroscopy of PPAA films deposited under different
Peq. The broad band at 3360 cm-1 indicates the presence of amine groups. The intensity of
this peak steadily increases relative to the intensity of the multiple C-H adsorption peaks
at 2960 cm-1, 2930 cm-1, 2875 cm-1 with decreasing input power. The higher density of
the amine peak suggests that under low energy plasma polymerization the amine
functionality is retained to a greater extent than in the high energy process, which is
supported by the increase of the water contact angles with increasing input power (see
Figure 4.5). It should be noted that the bands at 2185 cm-1 are associated with the
presence of nitrile groups or alkyne groups in PPAA films. This suggests that the primary
amino groups have a high tendency to be transformed into nitriles groups. Furthermore,
this tendency increases with increasing the Peq (input power increase or duty cycles
53
decrease) used in the deposition processes. Due to the incorporation of oxygen in the
PPAA films upon exposure to air, the spontaneous aging process can result in the
reaction of residual free radicals in the plasma film with ambient oxygen. The peak
around 1625 cm-1 partly originates from the C=O stretching of amides, but assessment is
hinder due to possible contributions of amines, alkenes and imines.
Figure 4.4 FTIR spectra of PAA films deposited at (a) different DCs and (b) cw, different input
power.
4.3.2 Surface wettability
Figure 4.5 (a) shows the change in water contact angle with decreasing DCs. The θa
and θr of PPAA film deposited at (cw, 5 W) are lower than those of film deposited at
(cw, 50 W). However, under the same input power, θa and θr of PPAA films deposited
decrease with decreasing DCs. From FTIR spectroa of PPAA films, the amino group
density increases with decreasing Peq. Thus, the wettability of the polymer films
increases, and θa and θr decreases. A high hysteresis between the advancing angle and
receding angle was observed on PPAA films, which may be explained by the
rearrangement of functional groups at the surface. The surface of a polymer tends to
restructure and reorient when brought in contact with a different environment, due to the
thermodynamic drive to minimize interfacial tension. In air the polar amino groups tend
to move away from the hydrophobic environment while hydrocarbon parts are attracted
to the interface, inducing relatively high advancing contact angle. However, in wetting
4000 3000 2000
(a)
1625 cm-1
2185 cm-1
2875 cm-1
2930 cm-1
2960 cm-1
3360 cm-1
10/190, 100 w, Peq
2.5W
10/40, 50 w, Peq1W
10/40, 100 w, Peq 20W
Abso
rptio
n
3500 3000 2500 2000 1500 1000
(b)
cw,100w
cw,50w
cw,5w
Wavenumbers, cm -1
54
environment, the amino groups and other polar groups move to the liquid-solid interface
to minimize their energy. The difficulty of amino groups recovery the original
orientation from water-polymer interface to air-polymer interface becomes stronger, due
to the higher cross-linking degree of PPAA films deposited at higher Peq, indicating a
higher hysteresis of contact angle. However, when Peq is over 10 W, the hysteresis of θa
and θr does not increase any more, which may be due to the highly linked structure of the
PPAA films.
Figure 4.5 (a) Water contact angle as a function of DCs and the input power for PPAA films
and (b) the discrepancy of the θa and θr as a function of Peq.
4.4 Plasma Polymerized Maleic Anhydride (PMA)
4.4.1 Spectroscopic characterization
Chemical structure analysis using FTIR showed the plasma deposited films to be
very different in their chemical structure. The FTIR spectrum (Figure 4.6) of PMA
deposited under cw, 50 W shows the carbonyl and ester bonds at wavenumbers of 1630
and 1730 cm-1. The low DC 5/100 films appeared to contain a mixture of the anhydride
groups (1780 and 1860 cm-1) and carbonyl groups (1630 and 1730 cm-1), mainly
anhydride group. Of this former component was consistently reduced and the anhydride
structure became more prominent. Thus, the polymerization reactions were seen to
become more selective leading to improved retention of the anhydride structure.
20
30
40
50
60Allylamine
10/200,50 W10/50,50 Wcw, 50 Wcw, 5 W
θa θr
Wat
er c
onta
ct a
ngle
(°)
55
Figure 4.6 FTIR spectra of PMA films deposited at different DCs.
4.4.2 Surface wettability
Figure 4.7 shows the contact angle of plasma polymaleic anhydride films deposited
under different DCs. The θa and θr increase with decreasing the DCs. Since PMA films
are not stable due to hydrolysis reaction of the maleic anhydride leading to acid groups
and a swelling of the polymeric network as the solvent molecules penetrate into the film,
the measurements of contact angle were done immediately after polymer deposition. The
higher density anhydride can hydrolyze to more COO- when in contact with water. These
COO- groups repel each other due to the same negative charges when in water, which
maybe results in higher contact angle. Around 30 degrees discrepancy of θa and θr are
observed in PMA films, and is independent in the Peq. Since PMA films hydrolyze in
water, this high discrepancy may be due to the chemical structure change.
Figure 4.7 Water contact angle as a function of DCs and the input power for PMA films.
20
30
40
50
60
Duty cycles/ ms/ms
θa θr
Maleic anhydride,100w
5/1005/45cw
wat
er c
onta
ct a
ngle
(°)
3500 3000 2500 2000 1500
maleic anhydride
1630 cm-1
1730 cm-1
1780 cm-1
1860 cm-1
cw,50 W
5/100,50 WA
dsor
ptio
n
Wavenumber(cm-1)
56
RMS=0.888 nm RMS=0.641 nm
(a) (b)
4.4.3 Homogenity and morphology
Figure 4.8 shows the AFM morphology of PMA deposited under cw and 5/100. The
film surface roughness of the cw PMA is higher than that deposited under low DC 5/100,
since the high plasma equivalent power can etch the polymer surface, and the roughness
becomes higher. Compared with the PEO2 surface, the surface of PMA appears to be
rougher, which may be due to the difference of their monomers molecules.
Figure 4.8 AFM images of (a) cw, 90 W, dpolymer 50 nm, Ra = 0.855 nm and (b) 5/100, 90 W,
dpolymer 48 nm, Ra = 0.541 nm PMA films deposited on silicon wafer.
4.5 Summary
The chemical structure, contact angle and the hysteresis between the advancing
angle and receding angle, and surface roughness of PEO2, PPAA, and PMA films were
investigated in this chapter. The surface chemical properties of plasma polymers are
highly dependent on the chemical nature of the monomers used for the plasma
polymerization and the plasma conditions. According to the FTIR spectra of these three
plasma polymers, the functional group density (C-O, NH2, and anhydride) increases with
decreasing the equivalent power (i.e. decreasing the plasma input power and duty cycle),
The contact angle of PEO2 and PPAA films increases with increasing Peq due to
decreased C-O/C-C ratio and amino group density respectively. In contrast, the contact
angle of PMA films decreases with decreasing Peq because of the hydrolysis of the
anhydride groups. Higher hysteresis between advancing angle and receding angle of
plasma polymers was observed, suggesting the surface-configuration change of the
57
plasma polymers. Among three plasma polymers, PPAA and PMA films possess high
hystersis effect, whereas the hysteresis of contact angle of PEO2 films is weak. The
surface roughness of plasma polymers was seen to be affected by the thickness of plasma
polymers and the plasma conditions used. The RMS and Ra increase with increasing Peq
and the thickness of plasma polymers.
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1988, 285.
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Polym. Sci.: Polym. Chem. Ed., 33, 1995, 829.
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39. U.S. Sahu, Polym. Commun , 24, 1983, 61.
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60
61
Chapter 5
Solution Behavior of Plasma Polymerized Films
5.1 Introduction
Thin functionalized plasma polymer films are receiving increased attention as
supports for biological molecules. However, these applications have to be investigated in
solvent environment.1-2 As such, their behavior in aqueous media must be understood to
ensure successful implementation and exploitation of their unique properties. Polymer
dissolution has been of interest for some time and some general behaviors have been
characterized and understood throughout the years. In general, for conventional polymers
the dissolution into a solvent involves two transport processes, namely solvent diffusion
and chain disentanglement. One of the earliest contributors to the study of polymer
dissolution was Ueberreiter3 who outlined the surface layer formation process. First, the
solvent begins its aggression by pushing the swollen polymer substance into the solvent,
and, as time progresses, a more dilute upper layer is pushed in the direction of the solvent
stream. Further penetration of the solvent into the solid polymer increases the swollen
surface layer until, at the end of the swelling time, a quasistationary state is reached
where the transport of the macromolecules from the surface into the solution prevents a
further increase of the layer. Many factors, e.g., molecular weight, polymer thickness,
polymer structure, composition, conformation, different solvent, and environmental
parameters, and processing conditions, can influence the solution behavior of polymers.
However, for plasma polymers it is more difficult to understand the solution
behavior due to their complicated molecular structure and their reaction mechanism. A
multiplayer model for plasma films was described by Snyder et al.4-6 This method is
successfully applied when the dispersion relationship or the chemical composition of the
film is known. The n increases with decreasing pressure and increasing the input power.
The pressure and power dependence of the refractive index is caused not only by a
changed mass density but also by changes of the structure and chemical composition of
the plasma polymer. At the same time, they observed that a notable n gradient arises from
62
the time-dependent process, producing changes of film structure and chemical
composition over the film thickness.7 Hence, on these bases, the plasma dissolution
behavior could be understood and described in detail.
Up to now, three distinct phenomena could be observed when plasma polymers are
submersed in an aqueous phosphate buffer solution (PBS), i.e., swelling, observed as an
increase in the thickness; the dissolution of non-covalently bonded low molecular weight
material; and hydrolysis reactions for some plasma polymers. The extent of each of these
is dependant on the monomers used and plasma conditions employed. A significant
retention of monomer functionality can be achieved by careful choice of the process
conditions, which is influenced significantly by the duty cycle and the equivalent power.8-
13 At the same time, variations in the process parameters induce differences in the cross-
link density within the polymer bulk. When in solution, plasma polymer swelling is
determined by the cross-link density and the chemical nature of the films. The present
study describes a SPR study comparing the swelling behavior of PEO2, PPAA, and PMA
deposited at low DC and high DC. Furthermore, to investigate the exact change in the
thickness and refractive index of plasma polymers in buffer solution WaMS was
employed in the present work.
5.2 Solution Behavior of Plasma Polymers Investigated by SPR
5.2.1 Solution behavior of PEO2 and PPAA deposited under high input power
Figure 5.1 shows typical SPR kinetic curves for the solution behavior of (a) PEO2
and (b) PPAA films when submersed in PBS buffer. Changes in optical thickness (nd) are
shown as changes in the reflectivity measured. Over a period of up to 15 h, high DC
PPAA films and cw PEO2 films show only small changes in ∆nd, suggesting a
comparatively rigid polymeric network with a relatively high cross-link density. Plasma
films deposited at low DC, however, show a substantial decrease in the reflectivity. The
decrease in nd can be understood if one considers that the polymer interaction with water
involves not only swelling, which induces in thickness increase, but also a releasing of
the network due to increased mobility of the polymer chains and due to the dissolution of
material that is not covalently bonded to the substrate. If this dissociation of the polymer
network is large enough, it can lead to a decrease in the refractive index of the film,
63
which in turn contributes to the observed decrease in optical thickness. This would imply
that the relative changes observed in ∆nd could be correlated to the freedom of movement
of the chains and thus the cross-link density of the polymer network. These results
suggest that the low DC plasma polymers in this work consist of highly mobile chains, a
significant proportion of which may be lost to aqueous surroundings. However, those
polymers are that covalently bound to the surface remain after equilibration.
Figure 5.1 SPR kinetics of the solution of (a) PEO2 films deposited at (cw and 5/115, 100 W)
and (b) PPAA films deposited at (cw, 10/50, and 10/200, 100 W).
5.2.2 Solution behavior of PMA deposited under high input power
Compared with PEO2 and PPAA, PMA films behave in a different way in PBS
buffer solution to the other polymers studied. The data in Figure 5.2 (a) appear to be in
agreement with a mechanism in which the cw plasma polymer films lose a high quantity
of soluble material, which is indicated as a decrease in the optical thickness. In contrast,
the low DC (Figure 5.2 (b)) PMA appears to swell as if no unbonded or short chain
material were present, showing an increase in reflectivity with time. Previous work has
shown that with increasing density of anhydride functional group the PMA films show
polyelectrolyte character in aqueous solution.8 As such, the swelling of PMA films is far
more complicated than that for the other polymers studied and a more precise
interpretation can be observed by the results of WaMS, by which the variation of n and d
in aqueous solution with time were investigated (see next section). Figure 5.2 (a) and (b)
also show a dependence of the reflectivity change on the plasma deposition time and the
polymer film thickness before swelling, such that ∆nd for thin films is less than that those
0 10000 20000 30000 40000 50000
20
25
30
35
40
(a) PEO2
5/115,100 w, dpolymer 28.3 nm
cw,100 w, dpolymer 29.2 nm
R%
Time/sec
0 2500 5000 7500 10000 12500 15000
25
30
35
40
(b) PPAA
10/200,100 W, dpolymer 43 nm
10/50,100 W, dpolymer 39 nm
cw,100 W, dpolymer 42 nm
R%
Time/secs
64
of thicker films. Similar observations have been made for the low DC films of the other
polymers, which could be resulted from the irregular molecule structure for thicker
polymer. This phenomena also was observed from conventional polymer dissolution
behavior, i.e., thicker polymer films dissolve much faster than the thinner films in
solvent.10-11
Figure 5.2 SPR kinetics of the solution of PMA films deposited at (a) cw, 100W and (b) 5/100,
100 W.
5.2.3 Different pH solution behavior of PPAA deposited under low input power
In comparison to the low DC (10/200), cw 5W plasma polymer films showed a 30-
40% decrease in the reflectivity with exposure to aqueous buffer (Figure 5.3). This
appears to agree with a polymer network of low cross-link density, consisting of shorter
chain molecules and a comparatively high freedom of movement within the network.
This mobility of the network can lead to a decrease in the refractive index as the polymer
matrix swells, as unbonded low molecular weight material dissolved into solution and as
the network is diluted by solvent molecules. This decrease in n is believed to be the main
reason for the observed decrease in optical thickness. The observed data further suggests
differences between the low DC (10/200) and the cw, 5W plasma polymer films, which
may be related back to subtle differences in the cross link density or the amount of
unbonded material within the network between cw and pulsed plasma deposited films of
comparable Peq. However, the relative significance of these differences is difficult to
assess without absolute measurements of n and film d.
Three different pH (pH 5.7, 7.4, and 9) buffer solutions were employed. At the
same time, thin and thick polymers were investigated. Figure 5.4 summarizes the SPR
-5000 0 5000 10000 15000 20000 25000 30000 3500030
31
32
33
34
35
36
37
38
39
40
41
120 sec,dpolymer>80nm
30 sec, dpolymer 28.1 nm
(a) PMA, cw, 100 W
30 sec, dpolymer 14.2 nm
R%
Time/sec
0 10000 20000 30000 40000
40
42
44
46
48
50
52
1 min,dpolymer=17.7nm
1 min15s,dpolymer=59.3nm
2.5 min,dpolymer>100nm
(b) PMA, 5/100, 100 W
R%
Time/sec
65
kinetic scans taken from the PPAA swelling behaviour in PB buffers described in section
3.5. PPAA films, which deposited at either high DC (cw) or intermediate DC (10/50)
under low input power, were not stable in all kinds of solutions. The reflectivity change
of PPAA films in lower pH solutions is lower than that in higher pH solution. It appears
that the short chain polymers or unpolymerized monomer dissociation behavior of PPAA
films predominates so that the optical thickness changes significantly in higher pH buffer.
Furthermore, the reflectivity variation of thicker polymer is higher than that of the thinner
polymer in the same buffer solution.
Figure 5.3 SPR data on the swelling behaviour in PBS solution of different low DC / low Peq
PPAA films.
Figure 5.4 SPR kinetic scans taken from PPAA swelling behaviour in PB buffer of (a) cw, 5 W
PPAA films and (b) 10/50, 5 W PPAA films.
0 4 8 12 16 20
0.25
0.30
0.35
0.40
cw 5W, dpolymer dry16nm
10/50, Peq1W, dpolymer dry13 nm
10/200, Peq2.5 W, dpolymer dry18 nm
Ref
lect
ivity
Time / hour
0 2 4 6 8 10
22
24
26
28
30
32
34
36
38
40
42 (b)
pH = 9, dpolymer 29 nm
pH = 9, dpolymer 14 nm
pH = 7, dpolymer 14 nm
10/50, 5 w
Ref
lect
ivity
%
Time/ hour-2 0 2 4 6 8 10 12 14 16
18
20
22
24
26
28
30
32
34
36
38
40
42 (a)
pH = 9, dpolymer 21 nm
pH = 7.4, dpolymer
17 nm
CW,5 W
pH = 5.7, dpolymer
19 nm
Ref
lect
ivity
%
Time/ hour
66
0 50 100 150 200-20
0
20
40
60
cw, 90 W
Thick polymer thickness Thin polymer thickness
Time/ min
Thic
knes
s/ n
m
1.4
1.6
1.8
2.0
Thick polymer refractive index Thin polymer refractive index
Refractive index
5.3 Solution Behavior of Plasma Polymers Investigated by WaMS
5.3.1 Plasma polymerized di-(ethylene glycol) vinyl ether (PEO2)
5.3.1.1 d and n alteration of cw, 90 W plasma PEO2 in PBS
Figure 5.5 shows d and n variation of cw, 90 W PEO2 for 31 nm and 68 nm
polymer films in. The points at 0 min are referred to as the thickness and refractive index
of dry PEO2 films. When immersed in PBS, for cw 90 W films, i.e., 68 nm and 31 nm,
approximately 28% was lost within 10 min. The refractive indices increase from 1.429 to
1.526 and 1.406 to 1.444 for thicker and thinner films, respectively (Table 5.1). It
suggests that some short chain polymer or the uppermost layers released from the
polymer matrix, in turn inducing d decreased and n increased. Also, the refractive index
gs Table 5.1 d and n of cw, 90 W PEO2 films against air and in PBS for 10 min cw, 90 W PEO2 Against air In PBS for 10 min
d/nm 68 52 Thicker
polymer n 1.406 1.444
d/nm 31 22 Thinner
polymer n 1.429 1.526
Figure 5.5 Change in n and d of cw, 90 W thick (d = 68 nm, n =1.406 in air) and thin (d = 31 nm,
n = 1.429 in air) PEO2 films with extended exposure to aq. buffer at pH 7.4.
67
of thick film is lower than that of thinner film. It hints that multilayers model determines
the layered structure of the plasma polymer films, which will be discussed in the
following section. The same n and d variation trend of both thicker and thinner polymer
films in PBS were obtained with time. As shown in Figure 5.5, it is clear that cw, 90 W
PEO2 films are stable in PBS buffer at least for 4 hours.
5.3.1.2 d and n alteration of high DC 5/50, 90 W PEO2 in PBS
Compared with the cw, 90 W plasma PEO2, the 5/50, 90w PEO2 films (Figure 5.6)
were less stable in PBS buffer within short time. d and n continually changed over 4
hours. For the thicker polymer (dpolymer 80 nm), it took over 6 hours to become stable.
However, for the thinner polymer (dpolymer 24 nm), the stability was reached in 4 hours.
Approximately 30% polymer thickness decreased for both polymer films within 4 hours
in PBS. The refractive indices of the polymer films jumped from 1.418 (against air) to
1.439 and 1.408 (against air) to 1.448 for 80 nm and 24 nm films, respectively, (Table
5.2). Afterwards, they increased gradually within 4 hours. The crosslinking degree
increases with increasing the plasma equivalent power.15-16 As shown in section 4.1.1, the
ether group density increases with decreasing the duty cycle, and the polymer films
seems to be less crosslinked or branched. Compared with cw plasma polymer films, short
chain polymer molecules are believed to predominated in the 5/50, 90 W plasma polymer
insides due to the low Peq, which induce a lower n. On the other hand, against air, the
refractive indices of the 5/50, 90 W plasma polymers are 1.418 and 1.408 for 80 nm and
24 nm polymers. This slight difference indicates that n of 5/50 plasma polymers is
independent in the polymer d. However, n against PBS of the thicker polymer is a little
gkh
Table 5.2 d and n of 5/50, 90 W PEO2 films against air and in PBS for 10 min 5/50, 90 W PEO2 Against air In PBS for 5 min
d/nm 80 78 Thicker
polymer n 1.418 1.439
d/nm 24 23 Thinner
polymer n 1.408 1.448
68
Figure 5.6 Change in d and n of 5/50, 90 W thick (d = 80 nm, n = 1.418 in air) and thin (d = 24
nm, n = 1.408 in air) PEO2 films with extended exposure to aq. buffer at pH 7.4.
bit lower than that of thinner one, hinting the multilayers structure of the plasma
polymers could be existed in plasma polymer film bulk.
5.3 .1.3 d and n alteration of low DC 5/100, 90 W plasma PEO2 in PBS
For the plasma polymers deposited at 5/100 and 90w (Figure 5.7), the thicker
polymer, the lower n against air. Around 14% decrease of 77 nm film and 55 nm film
exist within 10 min after the polymer films were immersed in PBS. After two hours, the
total decrease of 77 nm and 55 nm polymer films is around 40%. On the other hand, there
are approximately 3% increase in n of 77 nm and 55 nm films within 10 min after
polymer immersing in PBS, shown in Table 5.3. Compared with the cw and 5/50 plasma
Table 5.3 d and n of 5/100, 90 W PEO2 films against air and in PBS for 10 min 5/100, 90 W PEO2 Against air In PBS for 5 min
d/nm 77 58 Thicker
polymer n 1.374 1.409
d/nm 55 48 Thinner
polymer n 1.39 1.437
0 100 200 300 400
0
50
100
150
200
Thick polymer thickness Thin polymer thickness
5/50, 90 W
Time/ min
Thic
knes
s/ n
m
1.2
1.3
1.4
1.5
Thick polymer refractive index Thin polymer refractive index
Refractive index
69
Figure 5.7 Change in n and d of 5/100, 90 W thick (d = 77 nm, n = 1.374 in air) and thin (d = 35
nm, n = 1.39 in air) PEO2 films with extended exposure to aq. buffer at pH 7.4.
polymers, for the low duty cycle 5/100 the short chain or non-polymerized molecules
occupy a significant percent. Hence, the soluble part of the deposit is probably higher and
loss of material in solution is higher. Consequently, the polymer d decrease is higher than
that of 5/50 plasma films.
5.3.2 Plasma polymerized allylamine (PPAA)
5.3.2.1 d and n alteration of cw, 5 W and 90 W PPAA in PBS
Figure 5.8 shows the change in d and n of cw PPAA films deposited at 5 W and 90
W. The effect of the input power on the optical properties of PPAA was significantly. It
was observed that n increased with the increasing of the input power from 5 W to 90 W,
which indicates that the density of PPAA films deposited at 90 W is much higher.
Furthermore, d of the cw, 5 W PPAA decreases approximately 67/%, and then kept
stable. On the other hand, n decreased from 1.419 to 1.372 after the film was immersed in
PBS buffer for 5 min, and then increased to 1.476 after 7 hours. It seems that PPAA film
deposited at cw, 5 W swept quickly when submersed in PBS solution, and at the same
time some short chain or un-polymerized molecules released. However, this competition
between two factors is very complicated, leading to n and d decrease together. In
0 30 60 90 12020
40
60
80
100
120
Thick polymer thickness Thin polymer thickness
Time/ min
Thic
knes
s/ n
m
1.20
1.25
1.30
1.35
1.40
1.45
Thick polymer refractive index Thin polymer refractive index
5/100, 90 W
Refractive index
70
contrast, when the PPAA film deposited at 90 W immersed in PBS for 20 min, n
decreased and d increased, which can be explained by this significantly swelling of
polymer films. Increased power will result in an increased density of energetic electrons
and in an increased bombardment of the electrode by energetic ions. Hence, the dense or
more crosslinked and branched polymer films can be deposited. 13 Higher cross-linking
degree and less short chain or un-polymerized molecules of cw, 90 W PPAA film induce
the polymer stability in PBS solution for 10 hours.
Figure 5.8 Change in n and d of cw, 5 w, 4 min (d =36 nm, n = 1.474 in air) and cw, 90 w, 15 sec
(d = 27 nm, n = 1.593 in air) PPAA films with extended exposure to
aq. buffer at pH 7.4.
5.3.2.2 d and n alteration of 10/50 PPAA in PBS
The variation in n and d for 10/50 plasma polymer films deposited under different
input power (5 W and 90 W) are shown in Figure 5.9. For polymer films of the similar
thickness deposited at 10/50, n against air of the 5 W film is lower than that of 90 W
film. However, n of 5 W film becomes higher than that of 90 W film in PBS solution,
which may be due to the experiment error. These two films express the similar behavior
in PBS solution, i.e., they swept at the very beginning and kept stable in solution above 7
hours. Furthermore, it seems that the swelling dominated the polymer behavior in
solution compared with the releasing of polymer molecules from the polymer, resulting
in the density of polymer films decrease and n decrease. Whereas, for the film deposited
at 5 W n increased with time, since the shorter chain molecules were lost, the remaining
0 200 400 60010
20
30
40
cw, 90 W thickness cw, 5 W thickness
Time/ min
Thic
knes
s/ n
m
1.40
1.45
1.50
1.55
1.60
1.65
cw, 90 W refractive index cw, 5 W refractive index R
efractive index
71
0 150 300 450-20
-10
0
10
20
30
40
50
10/50, 5 W, d 10/50, 90 W, d
Time/ min
Thic
knes
s/ n
m
1.50
1.55
1.60
1.65
10/50, 5 W, n 10/50, 90 W, n
Refractive index
polymer multilayer become dense. In contrast, n of film deposited at 90 W decreased
with time, which resulted from the polymer swelling behavior.
Figure 5.9 Change in d and n of 10/50, 5 W (d = 29 nm, n = 1.583) and 10/50, 90 W (d = 34 nm,
n = 1.594) PPAA films with extended exposure to aq. buffer at pH 7.4.
5.3.3 The model of the solution behavior of plasma polymers
On these bases, a multilayers structure model of the plasma polymers can be
presented. Also the changed process of plasma polymers in PBS solution is assumed to
explain the plasma polymer solution behavior, which is shown in Figure 5.10. When high
cross-linked plasma polymers (such as high DC and input power plasma polymers) are
submersed aqueous solution, the polymer network changes only little (Figure 5.10 (a)).
However, in case of the low cross-linked plasma polymers (Such as low DC and input
power plasma polymers) (Figure 5.10 (b)), the non-bonded materials can be released
from the polymer matrix. At the same time the matrix swells in aqueous solution, as
shown in Figure 5.10 (b’) and (b’’). 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 n, as data in last section that the thicker
polymer possesses lower n against air suggests. Hence, when plasma polymer was
immersed in aqueous solution, the uppermost polymer layers could be washed away,
which would result in n of the remaining film to increase and d to decrease (b’). At the
same time, however plasma polymers always swell in aqueous solution, which could
induce d increase and n decrease (b’’). The two factors of the uppermost layers releasing
72
and the plasma polymer swelling determine the overall changes of n and d in aqueous
solution. If the former one dominates in the solution process, n of the remaining plasma
polymer films should be higher than before, vice versa. If the latter one predominates, n
of the remaining plasma polymers should be lower than before. For example, for PEO2
films deposited at cw, 5/50 and 5/100 under high input power 90 W n of films against air
is lower than those of films even in PBS for 5 min. It would be due to the uppermost
layers releasing from the polymer matrix surfaces. In contrast, for PPAA films the
polymer films readily swell in solution, n against air is higher than those in PBS solution.
The n of PEO2 against air is lower than that in PBS buffer, indicating that the uppermost
layers were washed away and the polymer films did not swell any more.
Figure 5.10 Schematic of the polymer in air and in PBS buffer
water
substrate
(a)
High Peq plasma polymers High cross-linking density
High Peq plasma polymers In aqueous solution
(a’)
Short chain polymers releasing predominates in aqueous solution
(b’)
Polymers swelling predominates in aqueous solution
(b’’)
(b)
Low Peq plasma polymers Low cross-linking density
Aqueous solution
5.3.4 Plasma pol merized maleic anhydride (PMA)
PMA film
cw, 90 W PMA
W PMA film ex
explained. Withi
suddenly from 20
Figure 5.11 Chang
nm an
to aq.
As descr
PMA film is hig
lower. The anhy
structure change
weak due to th
behavior mainly
can be explained
5/100, 90 W PM
polymer structur
the hydrolysis r
0
20
30
40
50
60
70
Thic
knes
s/ n
m
(a) cw, 90 W
y
73
s deposited under different DCs, were investigated (Figure 5.11). For
film d decreased slightly in PBS and n increased. But low DC 5/100, 90
hibited a different behavior in PBS, which can at present not be fully
n 30 min in PBS, the film d and n did not changed. Then, the film swept
nm to 70 nm, and n decreased from 1.488 to 1.379.
e in refractive index and thickness of (a) cw, 90 w, 1min 15 sec, and dpolymer 37
d (b) 5/100, 90 w, 1.5 min and dpolymer 21 nm PMA films with extended exposure
buffer at pH 7.4
ibed in section 4.3.1, the carbonyl and ester bond density in cw, 90 W
her than that of 5/100 PMA, whereas the anhydride group density is
dride bonds hydrolyze in aqueous solution, which induce the chemical
s and in turn alter n. The hydrolysis reaction of cw, 90 W PMA was very
e low anhydride group extent. Consequently, the polymer solution
was d decrease and n increase with the time in aqueous solution, which
by uppermost layers loosing and the polymer swelling. But in case of
A, d increased and n decreased, which resulted from changes in the
e and the interaction between water molecules and the PMA films during
eaction. Additionally, as the hydrolyzing proceeds, the COO- group
100 200
Time/ min
d
1.36
1.40
1.44
1.48
1.52
dpolymer dry 38 nmnpolymer dry 1.469 R
efractive index
n
0 20 40 60 80 100
20
30
40
50
60
70
dpolymer dry 28 nmnpolymer dry 1.496
(b) 5/100,90 W
Time/ min
Thic
knes
s/ n
m d
1.35
1.40
1.45
1.50
Refractive index
n
74
increases, the polymer chain start to repel each other and they have to stretch out of the
surface in order to avoid each other and thus decrease the polymer-polymer repulsion.
5.4 Summary
Surface plasmon resonance spectroscopy (SPR) and waveguide mode spectroscopy
(WaMS) were employed to investigate the solution behavior of plasma polymers in PBS
aqueous solution. Only rough results can be observed from SPR experiment. The exact
variation of d and n of plasma polymers can only be obtained by WaMS. Generally, three
distinct phenomena can be observed when plasma polymers are subjected to an aqueous
phosphate buffer solution, i.e., polymer swelling, the dissolution of non-covalently
bonded low molecular weight material, and the hydrolysis reactions for certain polymers.
For PEO2, PPAA, and PMA films deposited at cw the polymer swelling and the
dissolution play an important role in the solution behavior. If polymer swelling
predominates, d of the plasma polymers decreases and n increases in solution. In contrast,
if the short chain or un-polymerized releasing dominates, d increases and n decreases.
However, for PMA films deposited at low duty cycle, the hydrolysis reaction of
anhydride groups determines the main solution behavior of the plasma polymers.
Additionally, the plasma polymers deposited at cw show a higher stability in solution,
which is due to a highly crosslinked or branched chemical structure. The plasma
polymers deposited at low duty cycles show a variable behavior. Their thickness and
refractive index change with time in solution.
References
1. J.G. Calderon, A. Harsch, G.W. Gross, R.B. Timmons, J. Biomed. Mater. Rea.,
42, 1998, 597.
2. H. Schonherr, M.T. Van Os, R. Foerch, R.B. Timmons, W. Knoll, G.J. Vancso,
Chem. Mater., 12, 2000, 3689.
3. K. Ueberreiter, The solution process. In: J. Crank, G.S. Park, editors. Diffusion in
polymers. New York, NY: Academic Press, 1968, 219.
4. P.G. Snyder, M.C. Rost, G.H. Bu-Abbud, J..A. Woollam, J. Appl. Phy., 60, 1986
3293.
75
5. G.H. Bu-Abbud, N.M. Bashara , J.A. Woollam, Thin Solid Films, 138, 1986, 27.
6. V. Jacobsen, B. Menges, R. Forch, S. Mittler, W. Knoll, Thin Solid Films, 409,
2002, 185.
7. R. Rocotzki, M. Arzt, F. Blaschta, E. Kreyßig, H.U. Poll, Thin Solid Films, 234,
1993, 463.
8. A.T.A. Jenkins, J. Hu, Y.-Z. Wang, S. Schiller, R. Foerch, W. Knoll, Langmuir,
16, 2000, 6381.
9. A.K. Walker, H.B. Qiu, Y.L. Wu, R.B. Timmons, G.R. Kinsel, Anal. Biochem.,
271, 1999, 123.
10. Y. Wu, R.B. Timmons, J.S. Jen, F.E. Molock, Coll. Surf. B, 18, 2000, 235.
11. M. van Os, PhD Thesis, University of Twente, The Netherlands, 1999, 57.
12. R. Rocotzki, M. Arzt, F. Blaschta, E. Kreyßig, H.U. Poll, Thin Solid Films, 234,
1993, 463.
13. J. A. Woollam, P.G. Snyder, M.C. Rost, Thin Solid Films, 166, 1988, 317.
14. Ö. Pekcan, S. Ugˇur, Y. Yilmaz, Polymer, 38, 1997, 2183.
15. Ö. Pekcan, S. Ugˇur, Polymer, 43, 2002, 43, 1937.
76
77
Chapter 6
Protein Adsorption to Plasma Functionalized Surfaces
6.1 Introduction
Protein adsorption at interfaces is important for many applications, such as
stabilizers in food emulsions and foams and in biotechnology.1-3 Adsorption of proteins
on the solid surfaces has been elucidated in terms of the adsorbed amount and
conformational changes upon adsorption. The amount of protein adsorbed at room
temperature is in the order of several milligrams per square meters, varying with the kind
of protein, type of surface, and adsorption conditions. Proteins change their conformation
more or less during the process of adsorption onto the surfaces4 such that there are two
types of configurations for the adsorbed protein layer, i.e., ends-on type and side-on type.
Proteins adsorption include three steps: Firstly, an initial brief period of reversible
adsorption (adsorption and desorption simultaneously); Secondly, the adsorbed protein
undergoes a slow conformational change depending on adsorption time when proteins are
essentially irreversibly adsorbed; Finally, desorption of denaturated material.5 Surfaces
which are hydrophilic and contain groups that are hydrogen donors are overall
electrically neutral and are known to resist the adsorption of proteins. Hence, those
surfaces could be applied as protein nonfouling materials.6-7
How to prepare the matrix that the proteins adsorb on or do not adsorb on is a key
step. Since plasma polymers can be prepared with different functional groups and
different group density, it should be easy to control the chemical structure of plasma
polymers. For example, molecules containing ethylene oxide (EO) units attached to
surfaces exhibit a well documented tendency to reduce the adsorption of proteins.8
Methods employed to attach EO containing molecules to surfaces have included simple
physical adsorption processes9-11 PEO grafting to surfaces,12-15 radiation and chemical
cross-linking methods,16 self-assembled mono-layer techniques17 and plasma
polymerization methods.18-23 Lopez et al. first demonstrated that plasma polymers of
tetraethylene glycol dimethylether lead to surfaces with a high resistance to proteins.20
78
Beyer et al. showed that the pulsed plasma assisted polymerization of triethylene glycol
monoallylether provided highly efficient non-fouling surfaces.21-22Recently, using radio
labeled albumin and fibrinogen, Wu et al. showed that a minimum of two ethylene oxide
units in the monomer molecule are necessary to obtain outstanding non-fouling properties
under low duty cycle pulsed plasma conditions.22-24
In the present work, SPR was used to investigate the proteins adsorption. Films
generated under cw conditions retain their inherent thickness and refractive index even
after prolonged immersions. However, films deposited at low plasma duty cycles exhibit
a marked swelling, accompanied by a decrease in refractive index, after immersion in
aqueous solution. This contrast in film properties is interpreted in terms of the formation
of significantly less cross-linked polymers under low duty cycle conditions leading to
facile penetration of the network structure by water molecules upon immersion in
solution. Films deposited under cw or higher duty cycle conditions exhibited relatively
strong affinity for protein adsorption, along with an unexpected increased protein affinity
with increasing film thickness.
6.2 Protein Adsorption on PEO2
6.2.1 Influence of plasma duty cycles
Protein adsorption was studied for EO2 plasma polymer films approximately 30
nm thick. Figure 6.1 shows kinetic curves obtained for fibrinogen, BSA and IgG on
different EO2 plasma polymer films. The thickness shown were calculated using the
refractive indices previously determined by waveguide mode spectroscopic investigations
in PBS buffer, which provided values of n of 1.58, 1.45 and 1.4 for cw, 5/45 and 5/115,
respectively. For all samples studied, protein physisorption reached equilibrium within a
few minutes after injecting the protein into the cell. The systems were allowed to stabilize
over periods of several hours, but showed no further significant changes. After this
stabilization period, the samples were rinsed with excess PBS which, in the cases of the
highly cross linked, high Peq films always led to a loss of some unbound protein from the
surface. As can be seen for all three proteins studied, no protein adsorption could be
observed by the SPR measurements on the low DC plasma polymer films suggesting
excellent antifouling properties.
79
Figure 6.1 Kinetics of (a) fibrinogen, (b) BSA and (c) IgG binding on EO2 plasma polymers
deposited at cw and DC 5/45 (Peq 11 W) and 5/115 (Peq 4.3 W).
0.0 0.5 1.0 1.5 2.0
0
5
10
15
20 prote in in jection
bufferrinse
bufferrinse
5/115, dpolym er 31 nm
cw , dpolym er33nm
thic
knes
s IgG/n
m
T im e/hour
0.0 0.5 1.0 1.5
0
2
4
6
8
10
12
protein injection
bufferrinse
bufferrinse
5/115, dpolymer30 nm
5/45, dpolymer31 nm,
cw, dpolymer27 nm,
thic
knes
s BSA/n
m
Time/hour
0.0 0.5 1.0 1.5 2.0 2.5
0
5
10
15
20
25
30protein injection
bufferrinse
bufferrinse
cw, dpolymer33 nm
5/45, dpolymer28 nm
5/115, dpolymer28 nm
thic
knes
s Fibr
inog
en/n
m
Time/hour
(a)
(b)
(c)
80
Figure 6.2 Effect of duty cycle on the absorption of fibrinogen, BSA and IgG. At constant plasma
polymer film thickness of d = 30 ? 2 nm and constant input power of 100 W.
As shown in Figures 6.1 and 6.2, the use of low DCs during the polymer synthesis
results in a strongly reduced affinity for protein adsorption. In the present work, excellent
anti-fouling properties were obtained for DC of 5/100 and 5/115 (Peq< 5 W). Yet XPS C
(1s) spectral data when compared to prior studies of this monomer,25-26 which was
discussed in previous paper,27 indicate that the maximum possible density of the ether
functional groups has not been reached in the present study. This is an interesting
observation in that it suggests that it is not essential to maximize the functional group
density in order to achieve effective anti-fouling properties. Since some of the work
presented here and elsewhere has shown that low DC plasma polymer films are often
subject to swelling and loss of low molecular oligomers when immersed in buffer, a
favorable compromise can probably be made between optimization film stability and
functional group density when designing anti-fouling coatings. Finally, the present
suggestion of formation of strongly H-bonded water networks in low DC films in
solution as the basis for their non-fouling properties is in accord with recent studies of
surfaces which are resistant to protein adsorption.28 Additionally, contact angles of the
0 5 10 15 20
SAM
SAM
cw
cw
5/45
5/45
5/115
5/115
cw
5/45
5/115
Dut
y cy
cles
/ ms/
ms
Protein thickness/ nm
Initial adsorption Retention
Fibrinogen
BSA
IgG
81
PEO2 films increases with increasing the DC (section 4.1.2). Consequently, the
hydrophobicity increases. Similar to the role played in maintaining structure of protein,
the role of surfaces hydrophobic interaction is expected to play a major role in the protein
adsorption, even more important than the role of the electrostatic interaction between the
protein and the surfaces.5
It takes long time for protein to adsorb to a surface and to reach the true
equilibrium. Other processes, i.e., conformational change or change in lateral
interactions, may run concurrently with the adsorption process. In the present work, only
the amount of the proteins was measured by SPR, not the conformational changes.
According to the size of the proteins and the adsorbed amount, the conformation of
proteins after adsorption on the plasma polymer surfaces could be assumed out.
Figure 6.3 An assumed model for adsorbed (a) fibrinogen, (b) BSA, and (c) IgG molecules based
on low and high surface coverage.
The structure of albumin has been previously characterized by X-ray
crystallography,29 showing it to be a globular protein with dimensions of approximately
8 nm by 4 nm and a molecular weight of 68,460 Daltons. IgG has a molecular weight of
156,000 Daltons and is a Y shaped molecule of height approximately 12 nm containing
8-10 nm 35-40 nm (a)
4 nm 8 nm
(b)
10-12 nm 8-10 nm
(c)
High surface coverage Low surface coverage
82
two identical antigen binding fibrinogen arms of dimensions 6.5 nm by 3.5 nm and Fc
region of dimensions 5 nm by 4 nm. Fibrinogen (340,000 Da) has been imaged using
electron microscopy30 and atomic force microscopy (AFM),31 showing it to have a triad
structure of length 45 nm with central domain of approximately the size of albumin and
distal domains dilobed and elongated to approximately 10 nm by 6 nm. As anticipated,
albumin, with the smallest molecular weight and dimensions, results in the thinnest
monolayer attached to the polymer surface (approximately 4 nm). It seems that only
monolayer of IgG could adsorb on PEO2 (cw, 33 nm, Figure 6.1 (c)) with the thickness
of approximately 10 nm. However, it is clear that two or three layers fibrinogen adsorb
on PEO2 (cw, 33 nm, Figure 6.1 (a)). Once the buildup of the primary layer of the
irreversibly adsorbed molecules is accomplished, protein molecules continue to assemble
in secondary and subsequent layers, which stack above the primary monolayer. It seems
that this trend is more pronounced in the case of fibrinogen. Due to the voluminosity and
flexibility of fibrinogen molecules may interact more reversibly with air phase than
albumin.32 Furthermore, this observation may be explained by considering the molecular
dimensions of these proteins and their orientations at an interface. Since fibrinogen has a
rod-like structure of width approximately 10 nm, which may lie planar at the polymer
interface, thus, it is easy for the second layer of fibrinogen to lie down on the first layer,
as shown in Figure 6.3 Whereas IgG has a relatively globular Y shaped structure of
height of 12 nm, hence, it is difficult for the second layer to adsorb continuously.
Because of the different molecular dimensions of the protein and their potential
conformation at the polymer/solution interface, there is a different thickness of the
adsorbed proteins in order of increasing value: fibrinogen > IgG > BSA.32 This appears to
be agreement with the result shown in Figure 6.2.
In general, protein molecules are assumed to unfold to a greater extent on the
hydrophobic surfaces, mainly due to the entropy driven strong hydrophobic interaction
operating at these interfaces. When protein molecules diffuse from the bulk to the thin
PEO2 films deposited at high DCs, they attach and make a structure re-conformation of
after adsorption. If the polymer is very thin, perhaps they cannot cover the whole polymer
surface. Consequently, proteins density on surface is low. However, when proteins
adsorb on thick PEO2 films deposited at high DCs, the primary layer of irreversibly
83
adsorbed molecules is accomplished fairly rapidly. Afterwards, protein molecules
continue to assemble in secondary and subsequent layers above the primary monolayer.
This trend seems to be more significant in case of fibrinogen. But in case of BSA, protein
molecules prefer to adsorb on PEO2 films as end-on configuration. It is difficult for IgG
to aggregate on the polymer surface, due to the global structure. Additionally, in that at
low protein surface coverage, the rate of adsorption is diffusion-controlled, while at
higher surface coverage, the protein molecules is able to create space in the existing film,
penetrate it and rearrange, and process becomes according to Graham and Phillips rate
determining.33
6.2.2 Influence of the plasma polymer thickness
While there is a clear dependence of the protein adsorption affinity on the plasma
polymerization condition employed during the film formation, for cw and high DC
generated films the extent of adsorption is also dependent on the plasma polymer
thickness, as shown in Figure 6.4. As described in Chapter 5, though plasma polymers
are pinhole free structure, they could swell or loose short chain molecules when
immersed in aqueous solution. Consequently, the plasma polymer films could change
into porous structure. For thicker plasma polymer films the swelling degree in solution is
higher than that of thinner polymers films (section 5.2.1). Hence it is possible for proteins
molecules to penetrate into the polymer bulk. On the other hand, the configurations of the
proteins adsorbed on plasma polymer films depend on the protein coverage on polymer
surfaces. In general, thicker cw polymer could lead to more protein molecules adsorb on,
which may be due to configuration change from end-on to side-on with increasing the
protein coverage.
Due to the limitation of SPR, very thick polymer films (more than 50 nm) were
not investigated in the present work. In case of PEO2 films, the electrostatic interaction
does not play important role, since PEO2 films do not hydrolyze in pH 7.4 PBS buffer
aqueous solution. From this viewpoint, the hydrophobicity of the PEO2 films determines
the protein adsorption mechanism.
84
Figure 6.4 Effect of the film thickness on the adsorption of (a) Fibrinogen, (b) BSA and (c) IgG
on EO2 plasma polymers deposited at cw and DC 5/45 (Peq 11 W) and 5/115 (Peq
4.3 W) respectively.
0 10 20 30 40 50 60 70 80
0
10
20
30(a)
cw, npolymer=1.58 5/45, npolymer=1.45 5/115, npolymer=1.4
d Fibr
inog
en/n
m
dpolymer/nm
5 10 15 20 25 30 35 40 45 50
0
2
4
6
(b)
5/115, n=1.4 cw, n=1.58d B
SA/n
m
dpolymer/nm
10 20 30 40 50 60
0
2
4
6
8
10
12
(c)
5/115, n=1.4 cw, n=1.58
d IgG/n
m
dpolymer/nm
85
6.3 Proteins Adsorption on PPAA
6.3.1 Influence of plasma duty cycles
Figure 6.6 shows kinetic curves obtained from fibrinogen and BSA on different
DC PPAA films. The optical thicknesses shown were calculated using the refractive
indices previously determined by WaMS in PBS buffer, which provided values of n of
1.52, 1.52, 1.48, and 1.46 for cw, 10/50, 10/90, and 10/200 respectively. After the protein
adsorption reached equilibrium, the flow cell was rinsed with pure PBS buffer. As can be
seen for fibrinogen and BSA studied, no BSA adsorption could be observed by the SPR
measurement on the cw plasma polymer film suggesting anti-fouling property. However,
5 nm fibrinogen adsorbed on the cw plasma polymer film. As shown in Figure 6.5 and
6.6, in the case of PPAA, for similar thickness polymer, the protein thickness adsorbed on
plasma polymer films increases with decreasing the duty cycle. This trend is opposite to
that of obtained for the proteins adsorption on PEO2. It has been discussed in chapter 4
that the amino group density increases with decreasing the duty cycles. It seems that the
higher the amino group density of the plasma polymer films, the more proteins are
adsorbed.
Figure 6.5 Kinetic of (a) fibrinogen and (b) BSA binding on allylamine plasma polymers
deposited at cw and DC 10/50 (Peq 20 W), 10/100 (Peq 10 W), and 10/200 (Peq 5 W),
100 W.
In pH 7.4 PBS buffer aqueous solution, the PPAA films possess the positive
charge NH3+. Since the IEP of fibrinogen is 5.5, that of BSA is 4.4, fibrinogen and BSA
should be negative charged. Hence, the negative charged proteins are strongly adsorbed
0 1 2 3 4 5
0
3
6
9
12
15
(b)
10/200,dpolymer20 nm
10/100,dpolymer28 nm
10/50,dpolymer19.4 nm
cw,dpolymer20.7 nm
Buffer rinse
Buffer rinse Buffer rinse
Buffer rinse
Protein injection
Thic
knes
s BSA/n
m
time/hour0.0 0.5 1.0 1.5 2.0 2.5
-5
0
5
10
15
20
25
30
35
(a)
10/50,dpolymer17 nm
Buffer rinse
10/200,dpolymer
19 nm
10/100,dpolymer20 nm
cw,dpolymer
21 nm
Buffer rinse
Buffer rinse
Buffer rinse
Protein injection
Thic
knes
s Fibr
inog
en/n
m
time/hour
86
by electrostatic interaction. This electrostatic interaction plays an important role in
protein adsorption. The higher amino density of PPAA films deposited at low DCs leads
to stronger electrostatic interaction between proteins and PPAA surface than that of
PPAA deposited at high DC does. As discussed in the last section, the protein
configuration depends on the protein coverage on the polymer surface. Approximately 30
nm fibrinogen and 12 nm BSA were seen to adsorb on 10/200 PPAA films (see Figure
6.6), indicating that fibrinogen could assemble into two or three layers and BSA
molecules aggregate into double layers on 10/200 PPAA films. Consequently, end-on
configuration of adsorbed proteins on low DC PPAA films could be induced. As well as
side-on configuration on high DC PPAA films.
Figure 6.6 Effect of duty cycle on the absorption of fibrinogen and BSA at constant PPAA film
thickness of d = 20 ? 5 nm and constant input power of 100 W.
6.3.2 Influence of the plasma polymer thickness
There appears to be the same obvious dependence of the protein adsorption affinity
on the plasma polymerization condition employed during the film formation. For 10/50
and 10/100 prepared polymer films the extent of adsorption is also dependent on the
plasma polymer thickness, as shown in Figure 6.7. It is clear that there is linear relation
between the polymer thickness and fibrinogen thickness. The reasons could be explained
by proteins penetration into the thick polymer. Furthermore, as the polymer thickness
0 5 10 15 20 25 30 35
Initial adsorption Retention
10/200
10/200
10/100
10/100
10/50
10/50
cw
cw
Dut
y cy
cles
/ ms/
ms
Protein thickness/ nm
Fibrinogen
BSA
87
increases, the surface roughness becomes higher, which described in Chapter 7.5, which
promotes protein molecules to attach to the surface.
Figure 6.7 Effect of the film thickness on the adsorption of fibrinogen on PPAAdeposited at DC
10/50 (Peq 20 W) and 10/100 (Peq 10 W).
6.4 Proteins Adsorption on PMA
6.4.1 Influence of plasma duty cycles
Figure 6.8 shows the kinetic of proteins adsorption on PMA films. Compared with
PPAA films, it is easier for proteins to dissociate from PMA surfaces after rinsing with
PBS, indicating that the interaction between proteins and PMA surfaces is less than that
of PPAA films. For similar plasma polymers the protein preferred to adsorb on the films
deposited at low DCs (Figure 6.9). The thickness of fibrinogen and BSA increase with
decreasing the plasma DC used. It seems that only monolayer fibrinogen can adsorb on
PMA. Also it is no significantly difference among fibrinogen adsorption on PMA
deposited at cw, 5/45, and 5/100, 100 W. On the other hand, no BSA adsorbed on PMA
deposited at high DC (cw, 100 W). Only 4 nm BSA on polymer films prepared at
intermediate DC (5/45, 100 W), and monolayer 7 nm BSA on films deposited at low DC
(5/100, 100 W). As described in Chapter 4.4, cw (100 W) plasma deposited films
consistent predominantly of carbonyl and esters groups. Since the films containing the
carbonyl and esters groups are protein anti-fouling, no protein can attach to this kind of
10 11 12 13 14 15 16 17 180
2
4
6
8
10
12
14
16
18
2010/50, 100 W
Thic
knes
s Fibr
inog
en/n
m
ThicknessPAA/nm
Initinal adsorption Retention
(a)
0 20 40 60 80 100 1200
10
20
30
40
10/100, 100 W
Thic
knes
s Fibr
inog
en/n
mThicknessPAA/nm
Initial adsorption Retention
(b)
88
polymer films. However, the anhydride groups of PMA deposited at low DC will
hydrolyze into COO- in aqueous solution. In consequence, it is difficult for negative
charged fibrinogen and BSA to adsorb onto the films due to the charge repulsion. The
hydrophobicity and the charge repulsion compete with each other, and thus determine the
protein adsorption behavior.
Figure 6.8 Kinetic of (a) fibrinogen and (b) BSA binding on PMA deposited at cw and DC 5/45
(Peq 11 W), 5/100 (Peq 5 W), 100 W.
Figure 6.9 Effect of duty cycle on the absorption of fibrinogen at constant plasma polymer film
thickness of 25 nm and BSA at constant plasma polymer film thickness of 55 nm
(constant input power of 100 W).
0,0 0,6 1,2 1,8 2,4 3,0
0
2
4
6
8
5/100,dpolymer20 nm
5/45,dpolymer30 nm
cw,dpolymer33.2 nm
Buffer rinse
Buffer rinse
Buffer rinse
Protein injection
Thi
ckne
ssfib
rinog
en/n
m
Time/hour
(a)
0,0 0,5 1,0 1,5 2,0 2,5 3,0
0
2
4
6
8
10
Buffer rinse
Buffer rinse
Buffer rinseProtein injection
cw,dpoymer60 nm
5/45,dpoymer54 nm
5/100,dpoymer57 nm
Thi
ckne
ssB
SA/n
m
Time/hour
(b)
0 2 4 6 8 10
5/100
5/100
5/45
5/45
cw
cw
D
uty
cycl
es/ m
s/m
s
Protein thickness/ nm
Initial adsorption Retention
Fibrinogen
BSA
Figure 6.10 Effect of the film thickness on the adsorption of Fibrinogen on PMA deposited at cw
(Peq 100 W), 5/45 (Peq 11 W), and 5/100 (Peq 5 W).
6.4.2 Effect of the plasma polymer thickness
Also, There is a dependence of the protein thickness on PMA thickness. Figure
6.10 shows fibrinogen thickness decreases with increasing cw PMA thickness. In
contrast, for 5/45 and 5/100 PMA films thickness are over 50 nm and fibrinogen
thickness does not increase further. In the case of cw PMA films, as the thickness
increases, the protein non-fouling groups (carbonyl and ester) increase, hence no more
protein could adsorb. In case of 5/45 and 5/100 PMA films, when the polymer thickness
increases, the anhydride group density increases, in consequent, COO- negative charges
rises, and the repulsive interaction becomes more strong. When the polymer thickness is
more than a critical value (50 nm), the protein average thickness does not increase, since
it is up to saturation adsorption.
Figure 6.11 show fibrinogen and BSA adsorption on three plasma polymers. On
the same thick and plas
these surfaces is quite d
show the same trend of
decreases with the DC o
with the DC of PPAA a
20 40 60 80
4
6
8
10
12
5/45,100 W,Maleic anhydride 5/100,100 W,Maleic anhydride cw,100 W,Maleic anhydride
d fib
rinog
en/ n
m
d PMA/ nm
s
89
ma conditions polymer films, the affinity of fibrinogen towards
ifferent from the behavior observed for BSA. All polymer films
protein adsorption. Both fibrinogen and BSA average thickness
f PEO2 decreasing, as while, protein average thickness increases
nd PMA decreasing.
90
Figure 6.11 The schematic of (a) BSA and (b) Fibrinogen adsorption on PEO2, PPAA and PMA
deposited under cw, intermediate DC (5/45, 10/50 and 5/45) and low DC (5/115,
10/200 and 5/115), 100 W.
If one compares the three plasma polymer films in Figure 6.11 (a) and (b), the
affinity of proteins towards PPAA is highest. PEO2 followed, and plasma polymaleic
anhydride is lowest. The typically functional groups are ether (C-O-C, OH-), amino
(NH2), and anhydride bond/carbonyl in PEO2, PPAA, and PMA respectively. The
adhesion forces of proteins on self-assembled mono-layer surface of alkanethiolates with
different functional groups, which have been measured by an atomic force microscope,
was reported by S. Kidoaki and T. Matsuda.34-35 The relative strength of thermodynamic
adhesion of the proteins to the SAM surface was found with statistical signification to be
0 3 6 9 12
Maleic anhydride5/100
5/45
cw
Allylamine10/200
10/50
cw
EO2
5/115
5/45
cw
d BSA/ nm
Initial adsorption Retention
0 5 10 15 20 25 30 35
Maleic anhydride5/100
5/45
cw
Allylamine10/200
10/50
cw
EO25/115
5/45
cw
d fibrinogen/ nm
(a)
(b)
91
in the following orders: For BSA, CH3- >> (OH-, NH2) > COOH-SAM surface; for
fibrinogen, CH3->> OH > NH2 > COOH. Therefore, among three plasma polymer films,
the affinity of proteins on PEO2 and PPAA is higher than that on PMA. Comparing with
PEO2, the electrostatic interaction between PPAA surface and fibrinogen or BSA
molecules plays a more important role than hydrophobicity, leading to a stronger
adsorption.
6.6 Conclusions
The adsorption of proteins on plasma polymer surface was investigated using
SPR. The results showed that protein adsorption was affected significantly by the
different surface functional groups, and the polymerization conditions, as well as the
thickness. Among three plasma polymer films, the affinity of proteins on PEO2 and
PPAA is higher than that on PMA. For low DC PEO2 films, the low degree of cross-
linking, as well as the high retention of ether content or higher hydrophobicity resulted in
the production of biologically non-fouling surface. However, for PPAA, the higher amino
density of PPAA deposited at low DC results in the higher protein affinity, due to the
strong electrostatic interaction. For PMA, the higher hydrophobicity of the polymer films
deposited at low DCs is favorable for protein adsorption. The reason, which the
dependence of the protein thickness on plasma polymers, could be explained by
considering the penetration of proteins into the polymer bulk and the configuration
change of adsorbed protein molecules. The protein molecules adsorb on the thin polymer
as side-on type, but end-on type on thick polymer surface. Whereas in case of adsorption
of fibrinogen on PMA, it seems that very thick polymer films are favorable to protein
adsorption, due to the repulsive interaction.
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14. D.W. Grainger, C. Nojiri, T. Okano, S.W. Kim, J. Biomed. Mater. Res., 22, 1988,
231.
15. D.W. Grainger, K.Knutsen, S.W., Kim, J. Feijin, J. Biomed. Mater. Res., 24,
1990, 403.
16. M.S. Sheu, A.S. Hoffman, B.D. Ratner, J. Feijen, J.M. Harris, in “Plasma Surface
Modification of Polymers: Relevance to Adhesion”, 197-208, M. Strobel, C.S.
Lyons, K.L. Mittal (eds) VSP, The Netherlands, 1994.
17. K.L. Prime, G.M. Whitesides, J. Am Chem. Soc., 115, 1993, 714.
18. J.M. Grunkemeier, W.B. Tsai, M.R. Alexander, D.G. Castner, T.A. Horbett, J.
Biomat. Mat. Res., 51, 2000, 669.
19. P. Favia, R. DAgostino, F. Palumbo, J. Phys. IV, 7, 1997, 199.
20. G.P. Lopez, B.D. Ratner, C.D. Tidwell, L.L. Haycox, R.J. Rapoza, T.A. Horbett,
J. Biomol. Med. Res., 26, 1992, 415.
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21. D. Beyer, W. Knoll, H. Ringsdorf, J.H. Wang, R.B. Timmons, P. Sluka, J.
Biomed. Mater. Res., 36, 1997, 181.
22. D. Beyer, PhD Thesis, Universität Mainz, Germany, 1996.
23. Y.J. Wu, R.B. Timmons J.S. Jen, F.E. Molock, Coll. Surf. B, 18, 2000, 235.
24. Y. J. Wu, PhD Thesis, University of Texas at Arlington, Arlington, USA, 2000.
25. V. Jacobsen, B. Menges, R. Förch, S. Mittler, W. Knoll, Thin Solid Films,409,
2002, 185.
26. V. Jacobsen, B. Menges, A. Scheller, R. Förch, S. Mittler, W. Knoll, Surf. Coat.
Tech., 142-144, 2001, 1105.
27. Z. Zhang, B. Menges, R.B. Timmons, W. Knoll, R. Förch, Langmuir, 19, 2003,
4765.
28. R.L.C. Wang, H.J. Kreuger, M. Grunze, J. Phys. Chem. B, 101, 1997, 9767.
29. M. Marquart, J. Deisenhofer, R. Huber, J. Molec. Biol., 141, 1980, 369.
30. R.F. Doolittle, Ann. Rev. Biochem., 53, 1984, 195.
31. R. Wigren, H. Elurng, R. Erlandsson, S. Welin, I. Lundstrom, FEBS, 280, 1991,
225.
32. A. Baszkin, M.M. Boissonnade, Proteins at Interfaces ACS Sym. Ser., 602, 1995,
209.
33. D.E. Graham, M.C. Phillips, J. Coll. Interf. Sci., 70, 1979, 403.
34. R.J. Green, J. Davies, M.C. Davies, C.J. Roberts, S.J.B. Tendler, Biomaterials,
18, 1997, 405
35. S. Kidoaki, T. Matsuda, Langmuir, 15, 1999, 7639.
94
95
Chapter 7
DNA Immobilization and Hybridization on
Plasma Polymerized Allylamine
7.1 Introduction
The term biosensor describes devices, which are used to monitor living systems or
incorporating biotic elements. The usual aim of a biosensor is to produce discrete or
continuous digital electronic signal, which are proportional to a single analyte or a related
group of analytes.1 Biosensors are finding use in increasingly broader ranges of
application, which includes clinical diagnosis, biomedicine, farm, garden, veterinary
analysis, process control, microbiology, and military applications.2-5
The above clarification allows us to clearly identify Professor Leland C Clark Jnr.
as the father of the biosensor concept. In 1956, Clark published his paper on the oxygen
electrode.6 Guilbault and Montalvo7 were the first to detail a potentiometric enzyme
electrode. The use of thermal transducers for biosensors was proposed in 1974.9 The
paper reported by Divis10 marked the beginning of a major research effort in Japan and
elsewhere into biotechnological and environmental applications of biosensors. Lubbers
and Opitz11 coined the term optode in 1975 to describe a fiber-optic sensor with
immobilized indicator to measure carbon dioxide or oxygen. In 1976, Clemens et.al.12
incorporated an electrochemical glucose biosensor in a bedside artificial pancreas and
this was later marketed.
DNA-based biosensors have received attention over the past years. Recent advances
in molecular genetics have prompted many attempts to develop novel biosensors and
microarray-based devices for the analysis of specific gene sequences and for the study of
nucleic acid-ligand interactions.13,14 A genosensor, or DNA-based biosensor, normally
employs immobilized DNA probes as the recognition element and measures specific
binding processes such as the formation of DNA-DNA and DNA-RNA hybrids and the
interaction between proteins or ligand molecules with DNA at the sensor surface. Most
applications of genosensor have been based on the combination of a sensing component,
96
such as an electrochemical,15,16 fiber optical,17 evanescent wave,18 or acoustic wave
device,19 with an attached single-stranded DNA probe for the detection of a particular
gene sequence by DNA hybridization. Some studies have also used an immobilized
double-stranded DNA as the template for the investigation of protein and drug binding.20
There are many different matrixes to which probe DNA can attach on, such as,
streptavidin21, monoclonal antibodies,22 and cationic lipid membranes.23 For such
systems, the hybridization orientation can easily be controlled. DNA hybridization on the
two-dimensional matrix has been extensively studied and showed broadly commercial
applications as bio-sensors.24,25 Compared with two-dimensional matrix for DNA
immobilization, three-dimensional matrix is higher sensitive. Brett et al. reported that
three-dimensional surfaces have a greater number of potential immobilization sites, hence
they posses higher sensitivity. The net result is that more solution phase probe can be
captured per spot on the three dimensional surface, giving a higher specific signal. On the
other hand, they observed that the extent of hybridization and the initial velocities were
directly dependent on the length of the immobilized species.26 Different analytical
methods have been employed in DNA sensor development. For example, SPFS has been
used for studying the DNA association and dissociation behavior,21,27 AFM has been
applied in assaying the extent of DNA condensation,23, 24 QCM28-30(Quartz Crystal
Microscope) as well as electro catalysis are used to detect the hybridization
behaviors.31,32
Since polymers possess a three-dimensional structure in buffer solution and offer
a lot of advantages for sensor technologies: they are relatively low cost materials, their
fabrication technique are quite simple, they can be deposited on various types of
substrates and the wide choice of their molecular structure and the possibility to build in
side-chains and charged or neutral particles.33 Polylysine has a wide range of application
for DNA immobilization and hybridization, due to the cationic polyamino acid on it.34-35
In the present work, plasma polyallylamine (PPAA) was used as the three-dimensional
matrix for DNA immobilization and hybridization, because of the simplification of the
preparation and the same functionalized group to polylysine.
97
7.2 Backgrounds of DNA immobilization and hybridization
The DNA backbone is a polymer with an alternating sugar-phosphate sequence.
The deoxyribose sugars are joined at both the 3'-hydroxyl and 5'-hydroxyl groups to
phosphate groups in ester links, also known as "phosphodiester" bonds. The single chain
of nucleic acid consists of deoxyribose (a pentose = sugar with 5 carbons), phosphoric
acid, and organic (nitrogenous) bases (Purines - Adenine and Guanine, or Pyrimidines -
Cytosine and Thymine). Each polynucleotide is a linear polymer in which the monomers
(deoxynucleotides), are linked together by means of phosphodiester bridges, or bonds.
These bonds link the 3' carbon in the ribose of one deoxynucleotide to the 5' carbon in the
ribose of the adjacent deoxynucleotide.36
The amino group on the PPAA chains can hydrolyze into NH3+ in buffer solution.
On the other hand the phosphoric acid on DNA strands can hydrolyze into PO4-. Hence
DNA chain can electrostatically adsorb to PPAA films in buffer solution. Thus the
density of R-NH3+ determines the amount of DNA probe that can attach on the polymer
chain. Each PO4- group can be attached onto the polymer chain if it meets a NH3
+ group.
Figure 7.1 Schematic representation of the blocking procedure of succinic anhydride on PPAA.
The key step in DNA hybridization is to block the extra amino and remove the
unspecific adsorption between target DNA and PPAA. The methods include blocking
unrelated amino groups by binding them to a high concentration of BSA,37 or utilizing
succinic anhydride (SA) to chemically react with free amino groups (See Figure 7.1),
thereby preventing ionic binding of DNA target to PPAA films38 in weak basic
circumstance. In the present work, SA was used to block extra amino groups. Compared
with the normal two-dimensional matrix,39 faster dissociation behaviors of double helix
trends was observed after DNA hybridization. In order to increase the degree of
O
O
O
+ R-NH2
HO
O
O
NH-R
98
hybridization, UV light was employed to treat the PPAA/probe surface so that the probe
DNA could covalently link with PPAA films.
7.3 Factors that Affect DNA Immobilization on PPAA Films
7.3.1 Plasma polymer films
7.3.1.1 Typical DNA immobilization procedure on PPAA film
Figure 7.2 summarizes the surface plasmon spectroscopic data demonstrating the
build-up of the multilayered architecture at the sample interface composed of a thiol
SAM, a cw, 5 W PPAA, and the layer of P4 immobilized on PPAA film. Figure 7.2 (a)
gives the angular scans, Figure 7.2 (b) shows the kinetic data taken during the adsorption
process of P4 to PPAA film. With respect to the bare Au surface in contact to the PBS
buffer, the formation of the thiol monolayer and PPAA film can be clearly seen as a
change in the reflectivity when measured as a fixed angle of incidence leading to an
easily measurable shift in the angular position of the resonance (Figure 72 (a)). The final
effective thickness calculated from this angular shift, assuming a refractive index of the
thiol layer of n = 1.50 amounts to d = 3 nm and a measured refractive index (see section
5.4.1) of the PPAA monolayer of n = 1.465 amounts to d = 33 nm. For the probe DNA
layer the final thickness calculated, assuming n = 1.375 amounts to d = 50 nm. All
simulated thicknesses of PPAA film and probe DNA mentioned in next section are
calculated using this method and the same refractive indices.
7.3.1.2 Plasma equivalent power employed in plasma polymerization
After equilibration of the polymer films, the kinetics of DNA probe attachment was
studied using a probe concentration of 100 nM. DNA adsorption was found to be more
efficient for low DC films than for high DC (high Peq) films as seen in the kinetic scan in
Figure 7.3 (a). The thickness of the DNA layer measured for the low DC film, however,
showed a 6-fold improvement and a typical DNA thickness for an 18 nm thick plasma
polymer film was approximately 17 nm. A comparable cw, 5 W plasma polymer film
showed very similar DNA adsorption with a total of approximately 20 nm DNA
adsorbed, but with a somewhat slower kinetic. Figure 7.3 (b) shows the dependence of
the probe DNA thickness on the plasma input power, in which PPAA films deposited at
99
10/200. The optical thickness of the adsorbed probe DNA decreases with increasing the
plasma input power. As discussed in section 4.4.1, when increasing the plasma Peq, the
amino density of the PPAA films decreases. Consequently, the NH3+ density of the
PPAA film deposited under high input power is lower when submersed in aqueous
solution. Hence, the ability of DNA molecules adsorption on PPAA films deposited at
high input power is weak.
Figure 7.2 (a) Angular reflectivity scans and (b) kinetic scans taken during the
build-up of the complex multilayered interfacial architecture (100 nM 25 mer DNA probe
immobilization on PPAA film).
Figure 7.3 SPR kinetic measurements of DNA binding on (a) 10/200 (d ? 18 nm), 5 W cw (d ? 13
nm), and 10/50 (d ? 10 nm) and (b) 10/200, 20 W, 50 W, 100 W PPAA films, Cp4 100
nM, PBS buffer at pH 7.4.
0 2 4 6 50 60 70
0
5
10
15
20 DNAinjection
10/50, Peq20 W, dpoly10 nm
10/200, Peq2.5W, dpoly18 nm
cw 5W, dpoly13nm
DN
A th
ickn
ess/
nm
Time / hour
0 1 2 3 4
0
5
10
15
20
100 W, dpolymer=13.5 nm, dP4=9 nm
50 W, dpolymer=13 nm, dP4=10 nm
20 W, dpolymer=19 nm, dP4=21 nm
10/200
DN
A th
ickn
ess/
nm
Time/ hour
(a) (b)
0 2 4 6 8 10 12
40
45
50
55
60
Rinsing with PBS
100 nM P4 Injection
(b)
Ref
lect
ivity
/%
Time/ hour50 55 60 65 70
0
10
20
30
40
50
60
70
80
90
(a) 47 nm Au 47 nm Au + 3 nm SAM 47 nm Au + 3 nm SAM + 33 nm PPAA 47 nm Au + 3 nm SAM + 33 nm PPAA + P4
Ref
lect
ivity
/ %
Incident angle/ degree
100
7.3.1.3 Plasma polymer thickness
DNA adsorption was found to be dependent not only on the DC and the probe
concentration, but also on the polymer film thickness, Figure 7.4. For polymers of d < 10
nm (after swelling) the DNA thickness measured on the low and high DC plasma
polymers were similar and within a few nm. However, with increasing polymer
thickness, the low DC films indicated a much higher affinity for DNA probe attachment.
For films with d > 20 nm the measured DNA thickness was approximately 4-5 times
greater for the low DC, low cross linked films than for the highly cross linked films, high
DC films. A similar trend could be observed for the 5 W cw films studied, which even
showed some improvement over the low DC films. The total amount of DNA able to
adsorb on a low DC plasma film (10/200) at a probe concentration of 100 nM appeared to
reach a maximum for thickness > 60 nm, showing an optical thickness increase of
approximately 45 nm. Further increases in polymer thickness led to no further significant
increases in adsorbed DNA (25mer). The adsorption of DNA could be studied by the
SPR technique only up to a polymer thickness of approximately 50 nm due to the limited
sampling depth of the evanescent wave.
Figure 7.4 Dependence of the DNA optical thickness on the plasma polymer thickness for DNA
adsorption on high and low duty cycle plasma films, 25 mer DNA (P4), Cp4 100 nM,
pH 7.4
When the polymer film is very thin, DNA molecules attach onto the surface of the
PPAA films. However, as the polymer thickness increases the chains start to repel each
other. The DNA molecules can penetrate into the polymer bulk to attach with the NH3+
0 50 100 1500
10
20
30
40
50
5W cw
cw 100W
10/50, Peq20W
10/200, Peq2.5W
prob
e th
ickn
ess
/ nm
polymer thickness / nm
101
groups. However, beyond a certain thickness, the DNA molecules could not continuously
adsorb on PPAA films, since the same charges repulsion among DNA molecules, as well
as the steric interaction of the DNA molecules continuous adsorption becomes strong.
7.3.2 pH value of buffer solution
The SPR kinetic measurements of 25 mer (100 nM) DNA adsorption on cw (5 W)
PPAA films (d ? 20 nm) in pH 9, 7, and 4.5 (100 nM) PB buffer solution is shown in
Figure 7.5 (a). The DNA optical thickness linearly decreases with the pH value of the
buffer solution increasing (Figure 7.5 (b)). When plasma polymerized allylamine is
subjected to buffers at different pH protonation of the amine group at low pH will lead to
a highly positively charged film. This increased positive charge leads to improved
binding of the negatively charged DNA to the polymer film. Keeping all other conditions
constant and only changing the buffer pH, the thickness of DNA probe measured on low
DC plasma polymers increased significantly. In contrast, the pH effect was significantly
less on the highly cross-linked, high DC films even though the general trend was same.
Figure 7.5 (a) SPR kinetic measurements of DNA binding on cw, 5 W (d ? 20 nm) in different pH
Value buffer solution and (b) dependence of the DNA thickness on the pH value of
buffer solution.
7.3.3 Probe DNA sequences length and concentration
DNA concentration affects the adsorption behaviour significantly. Figure 7.6 shows
it took longer time for DNA probes to reach the adsorption equilibrium at low
concentration. In this case, the whole adsorption procedure is controlled by diffusion, it
0 5 10 15-10
0
10
20
30
40
50
pH 4.5, dP4 46 nm
pH 7, dP4
30 nm
cw, 5 W, CP4100 nM, dpolymer20+/-3 nm
pH 9, dP413 nm
The
optic
al th
ickn
ess P
4/nm
Time/hour4.5 6.0 7.5 9.0
10
20
30
40
50
DN
A th
ickn
ess
/ nm
pH value
(a) (b)
102
needs longer for DNA molecules to diffuse near PPAA films surface and adsorb on it.
However, for high DNA concentration solution DNA molecules can touch the polymer
surface directly and adsorb. On the other hand, for the same PPAA films, DNA thickness
increases with increasing the probe concentration from 0.1 nM to 100 nM.
When increasing the DNA strand length from a 25mer to a 60 mer, the DNA
thickness measured decreased by approximately a factor 2 (Figure 7.7), indicating a
relationship between number of reactive sites available on the plasma polymer surface
and the number of phosphate bases on the DNA strand able to interact with those sites. It
can be explained by difficulty for longer chain DNA molecules to immobilize on PPAA
film. For shorter chain DNA molecules it is easy to penetrate into the PPAA films bulk.
S.dgh
Figure 7.6 SPR kinetic measurements of 25 mer probe DNA binding on cw, 5 W PPAA films
(dpolymer 40 nm ? 3 nm), pH 7.4.
Figure 7.7 25 mer and 60 mer probe DNA binding on cw, 5 W PPAA films (dpolymer 40 nm ? 3
nm), pH 7.4.
0 20 40 60 80 100
10
20
30
40
cw 5 W, 60 mer Probe
cw 5 W, 25 mer Probe
Pro
be th
ickn
ess
/ nm
Probe concentration / nM
-2 0 2 4 6 8 10 12 26 28 30-10
0
10
20
30
40
Cprobe4=0.01 nM
Cprobe4=0.1 nMCprobe4=1 nM
Cprobe4=10 nM
Cprobe4=100 nM25 mer DNA, P4
The
optic
al th
ickn
ess P
4/nm
Time/hour
103
In contrast, since more PO4- groups exist on longer sequence DNA, it leads to the
repulsion between the neighbour DNA chains. As well as the steric interaction becomes
stronger. All of these factors can result in the difficulty of longer chain DNA adsorbing
onto PPAA films.
7.4 DNA Immobilization on PPAA Films Investigated by AFM
Compared with the morphology image of the plasma polymer in air, those of the
PPAA films in PBS and after DNA adsorption changed significantly. These images of
cw, 5 W and 4 min PPAA (dpolymer 25 nm), are shown in Figure 7.8, in which Figure 7.8
(a) is the image of PPAA in air, with the average roughness 0.85 nm, Figure 7.8 (b)
shows the image of the polymer swelled in PBS buffer for 6 hours, with the average
roughness 1.26 nm, and Figure 7.8 (c) shows the image of the sample after DNA (60 mer,
1 nM) adsorption, with the average roughness 1.47 nm. It is clear that the average
roughness of the sample increases in air, in PBS, and after DNA adsorption. Compared
with cw (4 min) PPAA film, cw(11 min) film in air is rougher with surface rms
roughness 1.4 nm (Figure 7.9). After swept in PBS solution for 6 hours, surface rms
roughness increased to 1.55 nm. Afterwards, 200 mer double strands DNA adsorbed on
it, the surface rms roughness rose to 1.73 nm.
Figure 7.8 AFM images of cw, 5 W, 4 min PPAA on Au/SAM substrate (dpolymer 25 nm) (a) in air
(Ra 0.85 nm) and (b) in PBS buffer for 6 hours (Ra 1.26 nm), and (c) after 60 mer, 1
nM DNA adsorption for two hours (Ra 1.47 nm).
(a) (b) (c)
2×2 µm
2×2 µm
2×2 µm
104
Figure 7.9 AFM images of cw, 5 W, 11 min PPAA on Au/SAM substrate (dpolymer 70 nm) (a) in air
(surface rms roughness 1.4 nm ), (b) in PBS buffer for 6 hours (surface rms
roughness 1.55 nm), and (c) after 200 mer, 0.01 nM double strands DNA adsorption
(surface rms roughness 1.73 nm).
7.5 DNA Hybridization on PPAA
The DNA hybridization has been measuring SPFS the present work. Since there is
strong nonspecific adsorption between the target DNA and the plasma polyallylamine
films, the hybridization is not very efficient. Hence, SA was employed to block the extra
amino groups after probe DNA immobilization, and the UV light was applied to make the
covalent link between the DNA chain and the polymer. The affinity constant can be
obtained from the simple Langmuir model of the hybridization and dissociation behavior
and the Langmuir adsorption isotherm experiment.
7.5.1 Non-specific adsorption between target DNA and PPAA films
To make sure that if there is non-specific adsorption between target DNA and
PPAA film, the hybridization of the totally mismatched target with probe was done,
which is shown in Figure 7.10. Figure 7.10 (a) summarizes the angular scans of the
PPAA monolayer, probe DNA adsorption without SA treatment, and the mismatch
labeled target DNA adsorption. A significant shift in the angle of incident indicates the
probe DNA adsorbing on PPAA film. After mismatch target hybridized with probe DNA,
the angle of incident did not shift, besides the reflectivity of the deep angle rose a little,
perhaps due to the surface change of the whole system surface after Cy5’ labeled target
400nm
(a) (b) (c)
105
45 50 55 60 65
0
20
40
60
80Mismatch target 15
Fluorescence background
34 nm Polymer PPAA+25 nm DNA P4 PPAA+P4+T15
Incident angle/degree
Ref
lect
ivity
/%
0
1x104
2x104
3x104
4x104
Fluorescence intensity/cps
injection. Figure 7.10 (b) shows the fluorescence kinetic scan of T15 hybridization on
PPAA/P2. The fluorescence intensity increased to maximum 1.1 × 105 cps. After rinsing
with pure PBS buffer, it decreased to 3×104 cps, which is much more higher than the
fluorescence background 3×103 cps. Consequently, it is clearly that the non-specific
adsorption between target DNA and probe DNA exists. How to remove the non-specific
adsorption will be discussed in next section.
Figure 7.10 (a) Angular reflectivity and fluorescence scans and (b) kinetic scan taken during the
built-up of the complex multilayers interfacial architecture of PPAA/P4/T15 without
SA treatment.
0 2000 4000 6000 800039.5
39.6
39.7
39.8
39.9
40.0
40.1
40.2
Time/sec
Ref
lect
ivity
/%
Flectivity
0.0
2.0x104
4.0x104
6.0x104
8.0x104
1.0x105
1.2x105
Fluorescence intensity/cps
Fluorescence intensity(b)
(a)
106
7.5.2 UV treatment and blocking of excess amino groups
In order to enhance interaction or partly change the electrostatic interaction into the
covalent bond between the probe DNA and PPAA film, UV light was employed in the
present work before blocking extra amino on PPAA films. No shift of incident angle was
observed from the reflectivity scan curves taken before and after UV treatment, Figure
7.11. As well as the reflectivity kept constant (only decreased 0.6%).
Figure 7.11 (a) Angular reflectivity scans and (b) the SPR kinetic measurements of UV
treatment. UV 254 nm, 2 W, the power density 10.19 w/cm2 and 15 min through
quartz and solution.
Figure 7.12 summarizes angular reflectivity scans and the kinetic scans of SA
treatment under UV treatment procedure. The reflectivity increased as 0.1 M NaBorate
solution was injected and circulated for 2 min to pretreat the sample surface. Afterwards,
0 5 10 15 20 25
40,3
40,4
40,5
40,6
40,7
40,8
40,9
41,0
Remove UV light
254nm UV light
2w
15mins
Ref
lect
ivity
/%
Time/min
(b)
50 55 60 65 700
20
40
60
80
100 PPAA 34 nm PPAA 34 nm + P2 24 nm PPAA 34 nm + P2 24 nm after UV treatment
Ref
lect
ivity
/ %
Incident angle/ degree
(a)
107
1000 2000 3000 4000 5000
20
30
40
50
60
70
80
90
PBS
Millin Q H2O
0.1 M NaBorate
Succinic anhydride
0.1 M NaBoratePBS
UV, 366 nm,8 W,7 min UV, 254 nm,2 W,15 min
R%
Time/sec
SA was introduced into the system for 20 min at least, the reflectivity jumped over 80%,
which is beyond the detection of the plasmon resonance. Finally, the whole system was
rinsed with 0.1 M NaBorate, Milli Q water and PBS buffer, the reflectivity decreased
gradually. Comparing the reflectivity before and after SA treatment, the reflectivity
decreased, which suggests that the thickness of the whole film decreased. It may be
because the probe DNA single strand diffused out of the polymer matrix and were
replaced by SA molecules or some polymer chain were lost together with DNA.
Figure 7.13 summarizes the fluorescence kinetic measured for association and
dissociation processes, for samples that were treated by UV (254 nm, and 2 W) and UV
(366 nm, 8 W) lights for different times. Comparing these dissociation behaviours of
samples treated by 254 nm UV light, the dissociation of the sample, which was treated
for 15 min, shows slightly slower process. Furthermore, for samples treated by 366 nm
UV light, the 5 or 7 min treatment time was suitable, even though the dissociation
processes were slower. Thus, in the present work, the 254 nm, 2 W UV light was
employed in DNA hybridization experiments.
Figure 7.12 SPR kinetic curve of SA treatment after no UV, (366 nm, 8 W and 7 min) UV and
(254 nm, 2 W and 15 min) UV though quartz and PBS solution, and (254 nm, 2 W
and 30 sec) UV though 1 mm thick PBS solution.
108
Figure 7.13 Kinetic fluorescence scans taken for hybridization (association) and dissociation
processes between P6 and T6, which were treated by (a) UV, 254 nm 2 W and (b)
UV, 366 nm 8 W light for different length of time.
7.5.3 DNA hybridization
Figure 7.14 summaries angular reflectivity and fluorescence scans. No remarkable
shift in the incident angle of SPR scan curves during the whole process was observed
before and after T3, T1, and T2 hybridized with P2. Although from T3 (MM2)
fluorescence scan curve, no fluorescence intensity increase was noted after rinsing with
pure PBS buffer, the maximum fluorescence intensity increased from background 4200
cps to 9600 cps after T1 (MM1). Afterwards, 3×105 cps maximum intensity for
fluorescence was obtained after T2 (MM0) hybridization with P2. The hybridization steps
of T3, T1, and T2 with P2 are described in next section.
Figure 7.15 shows the hybridization procedure, for MM2, MM1, and MM0. After
UV and SA treatment, T3 with two base mismatches was added to P2. The fluorescence
intensity jumped from 3000 to 6000 cps within 2 min, then kept stable. Switching back to
pure buffer resulted in a step-wise intensity decrease to the initial fluorescence
background. Afterwards MM1 T1 was circulated into the flow cell, the fluorescence
intensity increased to 2×104 cps with a considerably reduced binding rate constant.
However, if the complement solution was replaced by pure buffer, the fluorescence
intensity gradually decreased to background within two hours. Finally, upon the addition
of the T2 (MM0) solution, the fluorescence intensity very rapidly and reached 4×105 cps
0 5000 10000 15000 20000 25000 30000 35000 40000
0.0
0.2
0.4
0.6
0.8
1.0
1.2
15mins 6mins 30mins 18mins
uv,254nm,10.19w/cm2
Nor
mal
izat
ion
Time/sec0 10000 20000 30000
0.0
0.2
0.4
0.6
0.8
1.0 10mins 5mins 7mins 3mins
UV,366 nm,8 W
Nor
mal
izai
on
Time/sec
(a) (b)
109
Figure7.14 Angular reflectivity and fluorescence scans of an experiment of target DNA
hybridization on the interfacial architecture of cw, 5 W PPAA and P2, with P2 as
the probe oligonucleotide and T1 as the corresponding target strand (MM1), T2 as
MM0, and T3 as MM2.
and kept stable. Rinsing with pure PBS buffer could decrease the intensity quickly within
the first 10 min (from 3.75×105 to 3.5×105 cps), and then gradually slowly reduced
with time. MM2 association and dissociation could not be described by Langmuir model
since T3 could not hybridize with P2. But for MM1 and MM0 both kinetic curves could
be described by simple Langmuir model: the oligonucleotides in solution were in
equilibrium with the ones bounds to the sites at the interface, represented by the catcher
probe. Hence, the two reaction rate constants, Kon (adsorption or hybridisation rate
constant) and Koff (dissociation rate constant) can describe the whole process.40 Since the
probe DNA attached to the three-dimensional matrix, the DNA hybridization and
dissociation behaviour differ from the normal two-dimensional matrix. The DNA probe
can regularly immobilized on the surface of the two-dimensional matrix, hence it is ready
45 50 55 60 65 70
0.0
5.0x104
1.0x105
1.5x105
2.0x105
2.5x105
3.0x105MM0 (T2 = 50 nm)
50 55 60 65 70
2,0x103
4,0x103
6,0x103
8,0x103
1,0x104 MM1 (T1 = 100 nM)
MM2 (T3 = 100 nM)
BlackgroundFl
uore
scen
ce in
tens
ity/ c
ps
Incident angle/ degree
Fluo
resc
ence
inte
nsity
/ cps
Indicent angle/ degree
6 2 6 3 6 4 6 5 6 6 6 7T h e t a [ ° ]
0
2
4
6
8
1 0
1 2
1 4
1 6
1 8
R [
%]
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
1 2 0 0
1 4 0 0
1 6 0 0
1 8 0 0
2 0 0 0
2 2 0 0
Cou
nts
S c a n - M e s s u n g
110
Figure 7.15 Kinetic fluorescence scans taken for hybridization (association) and dissociation
process between the (P2) surface and (T3), MM2: (a), (T1), MM1: (b), and (T2),
MM0: (c) target complements from solution.
0 2000 4000 6000 8000 10000 120000.0
5.0x103
1.0x104
1.5x104
2.0x104
2.5x104 (b) MM1
Kon=3.3*103M-1s-1
koff=3.9*10-4s-1
KA=8.46*106M-1
Fluo
resc
ence
inte
nsity
/cps
Time/sec
-2000 0 2000 4000 6000 8000 10000 12000 14000 16000 18000-5.0x104
0.0
5.0x104
1.0x105
1.5x105
2.0x105
2.5x105
3.0x105
3.5x105
4.0x105
(c) MM0
Kon=4.6*104M-1s-1
koff=4*10-5s-1
KA=1.15*109M-1
fluor
esce
nce
inte
nsity
/cps
Time/sec
0 200 400 600
3x103
4x103
5x103
6x103
(a) MM2
Fluo
resc
ence
inte
nsity
/cps
Time/sec
111
for target DNA to hybridize. However, for three-dimensional matrix the DNAs
penetration into the bulk of polymer results in unstable DNA helix strands. If we compare
the rate constant given in Table 1 for the hybridization of the target sequences T1-T2
with the P2, we can see the expected behavior, i.e. with increasing mismatch, the
association rate constant Kon decreases, but the dissociation rate Koff increases. The
affinity constant KA drops from 1.15×109 for the MM0 by about two orders of
magnitude if one single base (out of 15) is replaced in the complement strand resulting in
a single mismatch. Compared with KA (5.3 ×109 M-1) of DNA hybridization on two-
dimensional matrix, KA in this work is lower, due to the fast dissociation. All constants
are summarized in Table 7.1. Furthermore, see Figure 7.14, the reflectivity of the kinetic
scans kept stable, indicating that the film optical thickness did not change so much, since
the binding leads to a thickness increase too low to be detected by SPR alone. Also, a
significant difference of the maximum fluorescence intensity can be obtained from MM2,
MM1, and MM0. (see Figure 7.16).
Figure 7.16 The SPR and fluorescence kinetic profiles take for hybridization (association) and
dissociation processes between and the P2 surface oliogonucleotide and T3 (MM2),
T1 (MM1) and T2 (MM0).
0 5000 10000 15000 20000 25000 300000
20
40
60
80
100
IFlmax 20000 CPS
IFlmax 6000 CPS
time/sec
Ref
lect
ivity
/%
0
1x105
2x105
3x105
4x105
IFlmax 3.9×105 CPS
Rinsing with PBS
MM0=50nM
MM2=100nM
MM1=100nM
Fluorescence intensity/cps
112
Table7.1 kinetic data, i.e. adsorption (hybridization) rate constant Kon, dissociation rate constant
Koff, affinity constant KA=Kon/Koff for the reaction of the probe oligonucleotide
immobilized on the PPA films MM0 MM1
Kon/M-1s-1 4.6×104 3.3×103
Koff/s-1 4×10-5 3.9×10-4
KA/M-1 1.15×109 8.46×106
If PPAA film is assumed to be a three-dimensional network in buffer solution,
probe DNA can penetrate into the polymer matrix. Probe DNA probably adsorbs along
the polymer chain orientation. As the target DNA tries to approach the probe DNA, the
target DNA also has to penetrate into the polymer, and rotates around the probe DNA to
form the more favourable double helix DNA structure. If there are too many functional
amino groups on PPAA, the probe DNA, has no many sites that it is probably tightly
bound to the polymer and thus has a lower freedom of movement. In this situation, when
target DNA comes in, it is difficult to hybridise with the probe DNA. Hence there should
be optimum density of amino group on PPA films, which can produce the highest
hybridizaltion degree. In MM2 case, it is difficult for T3 to grasp the probe DNA quickly,
and hybridise with it partially, so that the fluorescence intensity didn’t rise so much. But
in MM1 case, as shown by the reflectively high fluorescence intensity, most of T1 went
into the matrix and hybridised with probe DNA. Although this interaction is quite weak,
most double strands DNA dissociated just within two hours when rinsed with pure buffer.
Further support for KA comes from the measurement of the Langmuir isotherm,
which is presented in Figure 7.17 and Figure 7.18. By a step-wise increase of the solution
concentration of (T2) and (T1) new equilibrium coverage are reached equivalent to new
stable fluorescence intensities. As predicted by the equation for the adsorption
(hybridization),21 the time needed to reach the new equilibrium coverage gets shorter as
one increases the solution concentration c0. If one plots these equilibrium intensities as a
function of c0 one obtains the well-known Langmuir adsorption isotherm. A plot of c0/Ifl
as a function of c0 yields a straight line from which the affinity constant KA (KA =
Kon/Koff) can be deduced. This plot together with a linear regression is shown in Figure
7.17 (c) and Figure 7.18 (c), giving KA = 1×108 M-1 for MM0 and KA = 6.67 ×106 M-1
113
0 5000 10000 15000 20000 25000 30000 350000,0
0,2
0,4
0,6
0,8
1,0
(a)200nM
50nM100nM
20nM
10nM
1nM
Time/secR
efle
ctiv
ity
0
1x105
2x105
3x105
4x105
5x105
6x105
Fluorescence intensity/cps
0 50 100 150 2000
1x105
2x105
3x105
4x105
5x105
6x105
(b)
Rmax5.67×105 cps
Kd9.98×10-9 M
KA1×108 M-1
Flu
ores
cenc
e in
tens
ity/c
ps
Concentration/nM
0 50 100 150 200
0,0
5,0x10-5
1,0x10-4
1,5x10-4
2,0x10-4
2,5x10-4
3,0x10-4
3,5x10-4
4,0x10-4 (c)
Con
cent
ratio
n/F
luor
esce
nce
inte
nsity
/nM
.cps
-1
Concentration/nM
Figure 7.17 Langmuir adsorption isotherm experiment of (T2) binding to P2. (a):The
fluorescence intensity increases and reaches an equilibrium level as one
increases the T2 concentration c0 in the flow cell. The higher the
concentration, the faster the fluorescence intensity increasing. (b): Plotting
the equilibrium fluorescence intensities Ifl as a function of the solution
concentration c0 gives the known Langmuir behaviour. The full curve is a
corresponding fit with KA=1×108 M-1. (c) Linearism representation of the
data given in (b), i.e. c0/Ifl versus c0 with a linear regression that yields KA .
114
0 10000 20000 30000 400000,0
0,2
0,4
0,6
0,8
1,0
2nM5nM20nM
50nM
100nM200nM
400nM
1000nM
Time/sec
Ref
lect
ivei
ty
0,0
5,0x104
1,0x105
1,5x105
2,0x105
2,5x105(a)
Fluorescence intensity/cps
0 200 400 600 800 10000,000
0,001
0,002
0,003
0,004
0,005 (c)
Con
cent
ratio
n/F
luor
esce
nce
inte
nsity
/nM
.cps
-1
Concentration/nM
0 200 400 600 800 1000
0,0
5,0x104
1,0x105
1,5x105
2,0x105
2,5x105
Rmax2.4×104 cps
Kd1.5×10-7 M
KA6.67×106 M-1
(b)F
luor
esce
nce
inte
nsity
/cps
concentration/nM
Figure 7.18 Langmuir adsorption isotherm experiment of (T1) binding to P2. (a):The
fluorescence intensity increases and reaches an equilibrium level as one
increases the T1 concentration c0 in the flow cell. The higher the
concentration, the faster the fluorescence intensity increasing. (b): Plotting
the equilibrium fluorescence intensities Ifl as a function of the solution
concentration c0 gives the known Langmuir behaviour. The full curve is a
corresponding fit with KA=8.46×106 M-1 (c) Linearied representation of the
data given in (b), i.e. c0/Ifl versus c0 with a linear regression that yields KA.
115
Figure 7.19 Schematic representation of plasma polymer swelling and DNA binding to low and
high DC plasma polymer films.
for MM1. The MM0 DNA hybridization and dissociation can be described by a simple
Langmuir model (KA=1.15×109 M-1) and Langmuir adsorption isotherm (KA = 1×108 M-
1), although there is difference between them. However, for MM1 DNA hybridization KA
obtained from the simple Langmuir model (KA = 8.46×106 M-1) and Langmuir adsorption
isotherm (KA = 6.67×106 M-1) are similar.
Figure 7.19 shows a possible schematic of the plasma polymer swelling and DNA
binding on low and high duty cycles PPAA films. On unswollen PPAA films, DNA
molecules can only lie on the polymer surface, while, on swollen PPAA films, DNA
functional group
DNA probe strand
substrate
DNA target strand Plasma polymer in theswollen state
low DC films are fully swollen
aq. buffer
on low DC films functional groups throughout the films are able to react
DNA solution
(b)
Plasma polymer in the unswollen state
high DC films show little or no swelling
on high DC films only the surface functional groups can react
aq. buffer
DNA solution
(a)
116
molecules could penetrate into the matrix, hence the association and dissociation
behavior differs somewhat from a two-dimensional matrix.
7.6 Conclusions
DNA probe immobilization was found to be optimal for low duty cycle films with
a relatively high density of –NH2 groups, low contact angles and a low cross link density.
When in solution three-dimensional characteristics of low DC films seem to enhance
DNA probe adsorption. Probe thickness observed on these films were up to 7 times
greater than on the highly cross linked films and on poly-L-lysine. The data appear to
suggest that the DNA oligonucleotides are able to penetrate into the low DC polymer
network, thus reacting with functional groups deep within the polymer matrix, which
does not seem to be possible with the highly cross-linked, high Peq films. Low pH value
seems to favor DNA probe attachment. MM0, MM1 and MM2 hybridization reaction are
easily distinguished. Because PPAA is three-dimensional network in solution, DNA
hybridization and dissociation is different from the two dimensional matrix, and has the
higher sensitivity, which is expressed by the high fluorescence intensity of DNA
hybridisation.
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119
Summary
The work described in this thesis concerns the plasma polymers used for
immobilization and adsorption of biomolecules. In particular, thin polymeric films
bearing ether, anhydride, and amine functionalities were synthesis by plasma
polymerization of these group containing monomers, i.e. di-(ethylene glycol) vinyl ether
(EO2), maleic anhydride (MA), and allylamine (AA). Additionally, the characterization
of these films, their surface properties, and solution behavior were investigated in detail.
Finally, these functional groups introduced by plasma polymerization were employed for
proteins adsorption, and plasma polymerized allylamine was used as the three-
dimensional DNA immobilized and hybridized matrix.
Chapter 2 presents some topics that are relevant to this thesis. Also an overview of
the most important surface treatment and surface modification techniques is described.
The fundamental aspects of plasma and plasma polymerization are covered too.
Chapter 3 serves as a brief introduction to plasma reactor and the most important
techniques that were used to synthesis and characterize the plasma polymers discussed in
this thesis. Furthermore, experimental was described in detail.
The characterization of plasma polymers, i.e. PEO2, PMA, and PPAA, is reported
in Chapter 4. The influence of plasma parameters such as peak power and rf duty cycle
on the film composition was investigated using three monomers. It was found that the
functional group density (C-O, NH2, and anhydride) increases with decreasing the
equivalent power (i.e. decreasing the plasma input power and duty cycle), since it is more
difficult for functional groups to be broken at lower equivalent power.
The solution behavior of three plasma polymers was studied in Chapter 5 using
surface plasmon resonance (SPR) and waveguide mode spectroscopy (WaMS). The result
of SPR measurements on plasma polymers shows the films contained low molecular
weight material that could be observed to be related to the duty cycle and input power
employed during polymerization. For PPAA films the solution behavior in different
solution conditions was investigated in detail. WaMS proved to be a power tool for the
characterization for plasma polymerized fills. By allowing a simultaneous study of film
120
thickness and refractive index against air and solution, this method provided insights in
the swelling behavior of plasma polymerized coating. It was observed that three distinct
phenomena occur when plasma polymers are subjected to an aqueous phosphate buffer
solution, i.e., polymer swelling, the dissolution of non-covalently bonded low molecular
weight material, and the hydrolysis reactions for plasma maleic anhydride. The plasma
polymers deposited at cw show the higher stability in solution, which is due to the high
crosslinked or branched chemical structure. However, the plasma polymers deposited at
low duty cycles show a variable behavior. Their thickness and refractive index change
with time in solution.
The proteins adsorption on three plasma polymerized films deposited at different
plasma conditions was described in Chapter 6. Self-assembled monolayer (SAM) of
octadecanethiol coated Au substrates were functionalized with different groups by plasma
polymerization. The adsorption of the proteins fibrinogen, bovine serum, and
immunoglobulin to these surfaces could be measured in situ by SPR spectroscopy. The
results showed that protein adsorption was affected significantly by the different surface
functional groups, and the polymerization conditions, as well as the thickness. Among
three plasma polymer films, the affinity of proteins on PEO2 and PPAA is higher than
that on PMA.
Chapter 7 presents DNA immobilization and hybridization on PPAA films. The
DNA immobilization behavior was found to be affected by the amine density and the
buffer solution conditions. The data appear to suggest that the DNA oligonucleotides are
able to penetrate into the low DC polymer network, thus reacting with functional groups
deep within the polymer matrix, which does not seem to be possible with the highly
cross-linked, high Peq films. A strong attraction between the longer chain DNA and the
plasma polymer film was investigated by the pull-force curve using AFM. Also MM0,
MM1 and MM2 are easily distinguished. Since PPAA is three-dimensional network in
solution, DNA hybridization and dissociation is different from the two dimensional
matrix.
121
List of Publications Published
Z. Zhang, B. Menges, R. B. Timmons, W. Knoll, and R. Förch, Langmuir 19, 4765-4770 (2003). “Surface Plasmon Resonance Studies of Protein Binding on Plasma Polymerized Di(ethylene glycol) Monovinyl Ether Films”. Z. Zhang, Q. Chen, W. Knoll, R. Förch, Surface and Coating Technology, 174-175 , 2003,588-590. “Effect of aqueous solution on functional plasma polymerised films”. Zhihong Zhang, Qiang Chen, Wolfgang Knoll, Renate Foerch, Rob Holcomb, and Daniel
Roitman. Macromolecles; 2003, 36, 7689-7694. “Plasma Polymer Film Structure and DNA Probe Immobilization”. Shan Zou, Zhihong Zhang, Renate Förch, Wolfgang Knoll, Holger Schönherr, and G.
Julius Vancso Langmuir, 2003, 19, 8618-8621. “Tunable Complex Stability in Surface Molecular Recognition Mediated by Self-
Complementary Quadruple Hydrogen Bonds”.
To be submitted Z. Zhang, W. Knoll and R. Förch “DNA hybridisation on continuous wave plasma polyallylamine”. Z. Zhang, B. Menges, R. Förch ,W. Knoll “Plasma polymer swelling behavior in buffer solution detected by waveguide mode spectroscopy” Z. Zhang, R. Förch, W. Knoll
“Proteins adsorption on plasma polymers”.
122
Nomenclature
Techniques AFM atomic force microscopy
ATRIR attenuated total reflectance infrared spectroscopy
CCD charge coupled detectors
FTIR Fourier Transform Infrared Spectroscopy
IBAD ion-beam-assisted deposition
INT ion heat texturing
PE-VCD sputter-deposition and plasma-enhanced chemical vapor deposition
QCM Quartz Crystal Microscope
SPM Scanning probe microscopy
SPR surface plasmon resonance spectroscopy
SPFS surface plasmon resonance fluorescence spectroscopy
TIRF total internal reflection fluorescence
WaMS waveguide mode spectroscopy
UV ultraviolet
XPS X-ray Photoelectron Spectroscopy
Polymers
AA allylamine
BSA Bovine Serum Albumin
DLC diamond-like carbon layers
EO ethylene oxide
EO2 di-(ethylene glycol) vinyl ether
EO3 triethylene glycol monoallyl ether
IgG Immunoblobulin
NaBorate Na2B4O7.10H2O
PB Phosphate buffer
PBS phosphate buffer solution
123
124
PEO2 plasma polymerized di-(ethylene glycol) mono vinyl ether
PMA plasma polymerized maleic anhydride
PPAA plasma polymerized allylamine
SA Succinic anhydride
SDS sodium dodecyl sulfate Physical quantities or constants cw continuous wave
d thickness
DC duty cycle
KA affinity constant
Kon, adsorption (hybridization) rate constant
Koff, dissociation rate constant
MA maleic anhydride
MM1 mismatch one
MM2 mismatch two
MM0 mismatch zero
n refractive index
PEG poly(ethylene glycol)
Peq equivalent power
Ppeak input powers
PMT photomultiplier
P probe
Ra average roughness
rf radio frequency
RMS mean square roughness
SAM Self-assembled monolayer
T target
toff plasma off time
ton plasma on time
θa advancing contact angle
θr receding contact angle
125
Acknowledgements
When I think in retrospective why I came to Mainz and why things went so well, it
is obvious that things are linked more or less directly to my supervisor Professor
Wolfgang Knoll. I would like to express my heartfelt gratitude to him for his support,
guidance, encouragement and patience throughout the course of this work and my
graduate studies.
And also, I would like to thank Prof. Dr. Andreas Janshoff is so interested in my
work and give me a lot promising suggestions on my thesis.
Dr. Renate Förch, my assistant promoter, was always there, and solved the
difficulties that I met during the work. Thank you forever for your kind considers and
introducing the plasma world to me.
Additional thanks are given to Bernhard Menges for discussion and direction of
WaMS experiments. He is so patient during the work, and I really appreciate him to give
me much help.
More thanks will give to my kind colleagues.
Finally, I will thank my family, especially my parents. They bring up my very
young daughter during my study in Germany.
126
127
Curriculum Vitae
Zhihong Zhang was born on June, 1st 1975 in Henan (China). Since June, 10th 1998
she was married to Zhehui Wang. Her daughter Yunshan Wang was born Nov., 22th
1999 (China). Unfortunately, they divorced in 2003.
After attending between 1981 to 1986 Dachen Primary School she studied
between 1986 to 1992 in Luyi Middle School. Then she went to ZhenZhou University
and studied polymer materials. She obtained the bachelor degree in 1996.
Afterwards she passed the matriculation of master student in the 43th Insitute of
China Aerospace Company in 1996. After 2.5 years she got the engineering master
degree on Polymer Compound Material. In Mar, 1999, she worked in this institute for
one year.
In Dec, 2000, she received a chance from Prof. Knoll to study in Germany as PhD
student. After that, new plasma polymerisation world was opened to her.