Surface Modification by Plasma Polymerization and ... · Surface Modification by Plasma...

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


3.5.2 Protein solution preparation --------------------------------------------------- 40

3.5.3 SPR measurements -------------------------------------------------------------- 40

3.6 Procedures for DNA Immobilization and Hybridization on PPAA............. 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



4.4 Plasma Polymerized Maleic Anhydride (PMA) ............................................. 54



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


6.3.2 Influence of the plasma polymer thickness ----------------------------------- 86

6.4 Proteins Adsorption on PMA........................................................................... 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,

75.

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.

35. J. anilich, D. Gervasio, R.E. Marchant, Ann. Biomed. Eng., 21, 1993, 655.

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

Press, San Diego, 1990.

39. W.R. Gombotz, A.S. Hoffmann, CRC Crit. Rev. Biocompatility, 4, 1987, 1.

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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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


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


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.

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)


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.


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)


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.


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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37. H.J.F. Angus, D. Bryce-Smith, Proc. Chem. Soc., 1959, 326.

38. E. Grovenstein, D.V. Rao, W. J.Jtaylor, J. Am. Chem. Soc., 83, 1961, 1705.

59

39. U.S. Sahu, Polym. Commun , 24, 1983, 61.

40. M.S. Kellou, G. Jenner, Eur. Polym. J., 12, 1976, 883.

41. B. Schneider, J. Polym. Sci. B, 10, 1972, 245.

42. T.A. Duplessis, A. Lustigand, D. Greyling, J. Macromol. Sci.Chem., 11, 1977,

1015.

43. K. Fujimori, W.S. Schiller, I.E. Craven, Macromol. Chem., 192, 1991, 959.

44. B.D. Roover, M. Sclavons, V. Carlier, J. Devaux, R. Legras, A.J. Momtaz, Polym.

Sci.: Polym. Chem. Ed., 33, 1995, 829.

45. R.M. Ho, A.C. Su, C.H. Wu, S. Chen, Polymer, 34, 1993, 3254.

46. M.S. Kalguthar, B.C. Crews, C. Brenda, L.J. Marnett, J. Med. Chem., 39, 1996,

1692.

47. S.A. Evenson, J.P.S. Badyal, J. Phy. Chem., 102, 1998, 5500.

48. S.A. Evenson, C.A. Fail, J.P.S. Badyal, Chem. Mater., 12, 2001, 3038.

49. S. Schiller, J. Hu, A.T.A. Jenkins, R.B. Timmons, F.S. Sanchez-Estrada, W.

Knoll, R. Förch, Chem. Mater., 14, 2002, 235.

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



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 = 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


5/50, 90 W

Time/ min

Thic

knes

s/ n

m

1.2

1.3

1.4

1.5


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


Time/ min

Thic

knes

s/ n

m

1.20

1.25

1.30

1.35

1.40

1.45


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.

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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.

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


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


10/50,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


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


10/200

10/200

10/100

10/100

10/50

10/50

cw

cw

Dut

y cy

cles

/ ms/

ms


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


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


(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



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


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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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.

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(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

/ %


(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


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


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


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)


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”.

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.

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.

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