Charles University in Prague. Faculty of Mathematics and Physics DOCTORAL THESIS. Artemenko Anna

Transcription

1 Charles University in Prague Faculty of Mathematics and Physics DOCTORAL THESIS Artemenko Anna XPS analysis of plasma polymers and nanocomposite films without breaking vacuum Department of Macromolecular Physics Supervisor of the doctoral thesis: prof. RNDr. Hynek Biederman, DrSc. Study programme: Physics Specialization: F4 Biophysics, chemical and macromolecular physics Prague 2013

2 Acknowledgements This work would not appear on a paper without the help of many colleagues and friends from the Charles University. That s why I would like to acknowledge the generous support of all these people. First of all I am very indebted to my supervisor Prof. RNDr. H. Biederman DrSc and co-supervisor Doc. D. Slavinska CSc for their continuous interest, advices and help in accomplishing the experiments and writing the thesis. For inestimable help and support in current discussion of results during the course of this work I would like to thank to Ondrej Kylian PhD. Also I would like to mention a great help in writing this thesis and in neat remarks made by Associate professor Ing. Andrey Shukurov PhD. I am very thankful to MUDr Lucie Bacakova MD, PhD Marta Vandrovcova and their colleagues for the help in implementation of biological tests; to Ivan Khalahan for SEM measurements; to Jan Somvarsky PhD for theoretical support in simulations. At last I am very obliged to thank to fellow members of Prof. Biederman s group: Jan Hanus PhD, Oleksandr Polonskyi PhD, Jaroslav Kousal PhD, Jindrich Matousek, Martin Drabik PhD, Pavel Solar, Ivan Gordeev PhD, Dmitry Arzhakov, Anton Serov, Juraj Cechvala, Martin Petr and Marcela Buryova for a help and support. Special thanks to my family and friends (Vitalina Kyselyova, Olga Orel, Ivan Orel and others) for their support and faith in me.

3 This work was financially supported by: SVV KAN GAUK

4 I declare that I carried out this doctoral thesis independently, and only with the cited sources, literature and other professional sources. I understand that my work relates to the rights and obligations under the Act No. 121/2000 Coll., the Copyright Act, as amended, in particular the fact that the Charles University in Prague has the right to conclude a license agreement on the use of this work as a school work pursuant to Section 60 paragraph 1 of the Copyright Act. In Prague date..... signature

5 Název práce: XPS analýza plazmových polymerů a nanokompozitních vrstev bez přerušení vakua. Autor: Artemenko Anna Katedra/Ústav: Katedra makromolekalární fyziky/univerzita Karlova v Praze Vedoucí doktorské práce: prof. RNDr. Hynek Biederman, DrSc., Katedra makromolekulární fyziky Abstrakt: Plazmové polymery a nanokompozity kov/ plazmový polymer byly široce používány pro různé biomedicínské účely. Pro bioaplikace přirozeně požadovány vlastnosti jakými jsou vysoká smáčivost, stabilita materiálů na vzduchu, ve vodném prostředí odolnost vůči různým sterilizačním procesům, adhese buněk. Tato práce se věnuje zejména zkoumání chemického složení připravených vrstev pomocí XPS analýzy. Plazmový polymer na bázi nylonu, PEO-podobné vrstvy, fluorouhlikové vrstvy a nanokompozitní vrstvy Au/PEO-podobné, Ag/C:H a Al/C:H nanokompozity byly vybrány jako zkoumané materiály. Kromě toho byly výsledky měření XPS použity pro počítačovou simulaci pro výpočet faktoru plnění nanokompozitů kov/plazmový polymer. Byla dosažena dobrá shoda s experimentem. Klíčová slova: plazmový polymer, nanokompozity, XPS analýza, bioaplikace, simulace. Title: XPS analysis of plasma polymers and nanocomposite films without breaking vacuum. Author: Artemenko Anna Department/Institute: Department of Macromolecular Physics/Charles University in Prague Supervisor of the doctoral thesis: prof. RNDr. Hynek Biederman, DrSc., Department of Macromolecular Physics Abstract: Plasma polymers and metal/ plasma polymer nanocomposites have been widely used for various biomedical proposes. Naturally, surface properties of the coatings such as high wettability, stability on the open air and in aqueous media, resistance towards different sterilization processes and cells adhesion are required for bioapplications. This thesis is mainly dedicated to the investigation of chemical composition of deposited coatings using XPS analysis. Nylon-like plasma polymer, PEO-like coatings, fluorocarbon plasma polymer (PTFE) films and Au/PEO-like, Ag/C:H, Al/C:H nanocomposites were chosen as the subject material. In addition, results of XPS measurements were used for the computer simulation for calculation of filling factor of metal/ plasma polymer nanocomposites. These results were in a good agreement with experimental data. Keywords: plasma polymer, nanocomposite, XPS analysis, bioapplication, simulation.

6 Content 1. INTRODUCTION PLASMA POLYMERIZATION AND PLASMA POLYMERS PROPERTIES AND APPLICATIONS OF THIN POLYMERIC FILMS Poly(ethylene oxide)-like plasma polymer coatings (PEO) Polytetrafluoroethylene plasma polymer films (PTFE) Properties and applications of amine containing plasma polymers NYLON-SPUTTERED NANOPARTICLES DEPOSITED BY MEANS OF A GAS AGGREGATION PARTICLE SOURCE BASED ON THE RADIO FREQUENCY HOLLOW CATHODE MAGNETRON DEPOSITION OF METAL/ PLASMA POLYMER NANOCOMPOSITES, THEIR PROPERTIES AND APPLICATIONS EXPERIMENTAL DEPOSITION METHODS Equipment and technology for deposition of plasma polymers a) Amine containing plasma polymers b) PEO-like coatings c) Fluorocarbon plasma polymer films Preparation of metal/ plasma polymer nanocomposites a) Au/PEO-like plasma polymer nanocomposites b) Ag clusters/ch x plasma polymer nanocomposites c) Al clusters/ch x plasma polymer Fabrication of Nylon-sputtered particles DIAGNOSTIC INSTRUMENTS X-ray photoelectron spectroscopy (XPS) and chemical derivatization a) Chemical derivatization Fourier Transform Infra-Red spectroscopy (FTIR) Atomic Force Microscopy (AFM) Thickness measurements (spectroscopical ellipsometry)... 48

7 Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) Measurements of wettability Quartz Crystal Microbalance (QCM) BIOLOGICAL TESTS MATERIALS RESULTS AND DISCUSSION INVESTIGATION OF PROPERTIES OF AMINO-RICH PLASMA POLYMERS FILMS RELATED TO THE POSSIBLE BIOMEDICAL APPLICATIONS Influence of working gas mixture on properties of amine containing coatings Influence of storage time on properties of amino-rich plasma polymers and their stability in aqueous media BASIC CHARACTERIZATION OF NYLON-SPUTTERED NANOPARTICLES PREPARED IN A GAS AGGREGATION PARTICLE SOURCE BASED ON THE HOLLOW CATHODE MAGNETRON RESISTANCE OF PEO-LIKE COATINGS, FLUOROCARBON AND AMINO-RICH FILMS TOWARDS VARIOUS STERILIZATION TECHNIQUES STUDY OF THE GROWTH OF FLUOROCARBON FILMS ON THE SUBSTRATE AGING OF AL CLUSTERS/ C:H PLASMA POLYMER NANOCOMPOSITES COMPUTER SIMULATION OF FILLING FACTOR OF METAL/ PLASMA POLYMER NANOCOMPOSITES USING XPS MEASUREMENTS Model Theoretical basis for the computer simulation for calculation of filling factor of metal/ plasma polymer nanocomposites using XPS measurements Comparison with the experiment CONCLUSION BIBLIOGRAPHY LIST OF ABBREVIATIONS AUTHOR S CONTRIBUTION LIST OF PUBLICATIONS

8 Aims of doctoral thesis Increased attention was paid to the surface analysis of various materials for the last 50 years. Nowadays, different applications of polymeric products (nanocomposite materials, semiconductors, covering biocompatible prosthesis and implants with plasma polymer films, etc.) require accurate chemical characterization of the surfaces. A lot of techniques were used for this propose. According to the literature, X-ray photoelectron spectroscopy (XPS) is one of the most powerful methods for the characterization of the surface properties of a wide variety of materials. XPS is a quantitative non-destructive spectroscopic technique that measures the elemental composition, chemical state and electronic state of the elements that exist within a material. In this thesis the main emphasis is on using XPS analysis for plasma polymers and nanocomposites in order to characterize them after various modifications for biomedical applications. The aims were as follows: Characterize deposited amine containing plasma polymers in order to determine their structure, morphology and surface energy using XPS, atomic force microscopy (AFM) and water contact angle (WCA) measurements. Characterize modified amino-rich film surfaces their structure, chemical composition, morphology and surface energy by means of derivatization process, AFM, XPS, fourier transform infra-red spectroscopy (FTIR) and WCA analysis. Study biological response of the amino-rich, poly(ethylene oxide) PEO-like and fluorocarbon plasma polymer films in terms of adsorption and adhesion of proteins and cells; adhesion and growth of human osteoblast-like cells; resistance towards various sterilization techniques. Study of the aging of nanocomposite metal/plasma polymer films using aluminium as a metal and C:H plasma polymer as a matrix by XPS measurements. Study of deposited nanocomposite (Au/PEO-like, Ag/CH x, Al/CH x ) films by XPS method for the calculation of filling factor using MatLab2011 calculation program.

9 1. Introduction 1.1. Plasma polymerization and plasma polymers Plasma is often called a fourth state of matter. Moreover, plasma is known as a collection of free charged particles moving in random directions that is, on the average, electrically neutral. The free electric charges (electrons and ions) make plasma electrically conductive, internally interactive, and strongly responsive to electromagnetic fields. Plasma is also called as ionized gas. It could be divided into two kinds with respect to temperature: high temperature (or hot ) plasma and low temperature (or cold ) plasma. The term hot plasma is commonly used for plasmas which are fully ionized. As for low temperature plasma, it has to be pointed out, that this kind of plasma could be ignited at different working gas pressure: low pressure (in range of few to a hundred millitorr) and high pressure (up to atmospheric pressure) discharges [1]. These types of plasma are applied in different glow discharges: direct current (DC) or alternating current (AC) discharges (frequency of voltage up to 1 khz); RF discharge (frequency of voltage 100 khz - 30 MHz, typically MHz is used); microwave (MW) discharge (frequency of voltage is 100 MHz or more into the microwave region). Using molecular organic gases (e.g. hydrocarbons, fluorocarbons, organo silicon compounds) as working gas in low pressure discharges with the dissociation of the precursor molecules results in a reactive plasma species (neutral atoms, ions and molecule fragments) which produce new stable gas compounds (plasma chemical gas conversion) and leads to the deposition of thin organic films (plasma polymerization) on discharge electrodes, walls and immersed substrates [2, 3]. The polymerization reaction initiated by plasma is called as plasma polymerization, and is a useful technique for creation of new surfaces with different chemical functionalities. It started to be investigated by scientists in the second half of 19 th century because of many potential applications. It could be used not only in electrical but also, optical field, various surface modification especially for biomedical proposes [3, 4]. 1

10 Plasma polymerization can take a variety of forms, depending on the chemical nature of the monomers or molecular fragments deposited on the surface. In addition, some researchers suggested that it is more accurate to use the term PECVD (plasma enhanced chemical vapor deposition) of organic compounds instead plasma polymerization [5, 6]. Deposition of polymeric films using PECVD processes have been modeled in details by Yasuda in [7] using a mechanistic (flow) phenomenological approach. The geometry of the deposition system, reactivity of starting compound, its inlet flow rate, working gas pressure, the power delivered to the discharge, and etc. are taken into account. Deposition systems (reactors) could be divided into 3 groups: internal electrode reactors (frequently used bell-jar-type reactor with internal parallel plate metal electrodes); external electrode reactors (frequently used tubular-type reactors with external ring electrodes or an external coil); electrodeless microwave or high frequency reactors [3, 4]. However, Shen in [8] pointed out that from these models could be seen, at least qualitatively, the interplay between power, flow rate and pressure for a deposition process using a certain starting compound. From the other side, discharge power, working gas pressure and flow rate are not independent parameters for determination of the optimum conditions for polymerization process. Various conditions are needed for different monomers even to start a glow discharge [4]. Chemical reactions that occur under plasma conditions are generally very complex and nonspecific in nature. Glow discharge polymerization of organic compounds seems to proceed by the free radical mechanism and the extent of ionization is small. Recombination of these radicals results in formation of high molecular weight compounds called polymers. The free radicals are trapped in these films which continue to react and change the polymer network over time. Since radicals are formed by fragmentation of monomer, some elements and groups may be absent in resulting polymer [9]. Very often organic gases or vapours (which are not used as monomers in conventional polymerization) are used for a plasma polymerization process. However, even if a 2

11 traditional monomer is used the resulting polymer film from the plasma polymerization process is very different from the corresponding conventional polymeric film [3]. In spite of the use of the word polymer, the plasma polymer means a new class of material that has only a little in common with the conventional polymer that has regularly repeating units (Figure 1). In the case of the plasma polymer, the chains are short and in addition they are randomly branched and randomly terminated with a high degree of crosslinking (see Figure 2). Figure 1. Conventional high-density polyethylene (adapted from [4]) As usual a lot of free radicals are trapped within the network. These radicals cannot recombine rapidly. Therefore when the plasma polymer is exposed to the open atmosphere they starts to react the air (generally, with the oxygen and water vapor). Aging processes which are observed in plasma polymers are caused often by free radicals. Plasma polymers possess a rather disordered structure and this depends on the intensity and energy of the species bombarding the growing film [3, 4]. The overall scheme of plasma polymerization based on Poll s et al. [10] work was proposed by Yasuda and Hsu [11] and is shown in Figure 3 that involves the competitive ablation and polymerization mechanism (CAP). Monomer gas passesing through the plasma zone is turned into the complex mixture of intact monomer, excited or ionized fragments as well as gaseous products that do not participate in plasma polymerization. Yasuda suggests that plasma polymerization can be explained by a bicyclic step-growth mechanism. Figure 4 is a schematic representation of this mechanism, where M identifies any neutral species, M represents the monofunctional activated species, particularly free radicals, capable of covalent bond formation. Bifunctional activated species are shown by M. 3

12 Figure 2. Hypothetical structure of a hydrocarbon plasma polymer (adapted from [4]) Figure 3. The plasma polymerization scheme (adapted from [12]) 4

13 Figure 4. The bicyclic step-growth mechanism of plasma polymerization (adapted from [12]) There are two major ways of step growth, which involve the polymerization via mono- (Cycle I) and bifunctional (Cycle II) radicals. Reactions 1 and 4 are the first step with chain propagating through the repeated addition of radicals to the unactivated species. The latter must contain a chemical structure allowing the addition of M. Reaction 2 represents the termination through radical recombination. Reaction 3 is a recombination of mono- and biradicals, which yields another monofunctional radical. Reaction 5 is a recombination of two bifunctional radicals. The bicycle mechanism proposed by Yasuda describes reality in most cases when reaction step (1-5) take place on the surface while the excited species (free radicals) are excited in the plasma volume. Nevertheless, the same plasma polymerization process is occurring for the different monomers and influences the deposition process in rather different ways [3, 4]. 5

14 Film growth mechanism occurring through the reaction of radicals with polymer sites activated by charged particle bombardment (Activated Growth Model) was suggested by d Agostino [4] in the case of fluorocarbon coatings. This model was proposed as the most complete because it explains the role of precursor species from the fragmentation of the monomer in gas phase, of ion bombardment and temperature of the substrate during the growth of the coatings. Homogeneous and heterogeneous reactions in plasma polymer processes of teflon-like coatings have been rationalized by d Agostino and, as a result, simplified Activated Growth Model (AGM) scheme is shown in the following reactions (adapted from [4]): fragmentation of the monomer in the plasma monomer n CF x (1 x 3) (1) ion-activation of the coating (substrate) I + (low energy) + (coating) n (coating) n * (2) growth of the coating CF x CF x (adsorbed) (3.1) CF x (adsorbed) + (coating) n * (coating) n+1 (3.2) The production of the CF x precursors from the monomer is schematically described by reaction (1). The ion-activation step (2) depends highly on the energy of the ions bombarding the growing film (or the substrate, in the early deposition stages), thus on the related external parameters (pressure, power, bias, geometry). The ions create surface defective sites (e.g., dangling bonds) that act as preferential adsorption sites for the precursor CF x radicals from the plasma. Reaction (2) significantly influences the deposition rate, the composition and the cross-linking of the coating. The adsorptiondesorption equilibrium of the CF x radicals on the substrate (film) and the following reaction of polymerization with the surface active sites originated by the ionbombardment are described by reaction (3.1) and (3.2), respectively. As an overall effect of the increased substrate temperature, a reduced deposition rate is usually recorded and a reduced F/C ratio (increased cross-linking) in the deposited coating [4]. 6

15 Various kinds of coatings can be deposited by plasma polymerization using the proper monomers or monomer/inorganic gas mixtures. According to the used type of monomer several types of plasma polymers are distinguished: 1. Hydrocarbon plasma polymers. 2. Fluoro(chloro)carbon plasma polymers. 3. Nitrogen-containing plasma polymers. 4. Oxygen-containing plasma polymers. 5. Composite. A lot of attention was paid to the investigation of properties and possible application of plasma polymers mentioned above. Nowadays advantages of surface, bulk, optical, barrier, electrical and biomedical properties of plasma polymer coatings are used for different purposes in various spheres of human life Properties and applications of thin polymeric films Poly(ethylene oxide)-like plasma polymer coatings (PEO) Poly(ethylene oxide) (PEO) belongs to the large family of hydrogels with remarkable properties, which have been widely investigated for the use in biomedical field as protein resistant material. Plasma polymers based on PEO chemistry have retained interest among the polymeric films community for about the last twenty years. One of the most important and interesting features of PEO is its biologically non-fouling behavior, i. e. ability to resist protein and cell adhesion, which directly depends on chemical composition and structure of this polymer, because of the presence of repeating ethylene oxide monomer units. These groups are responsable for the structuring of water in aqueous environment by non-covalent interaction, where hydrogen bonding held together polymeric chains and water molecules. Hence, the coatings underwent considerable volume changes in contact with water, i.e. they behaved as hydrogels [13]. What is more, the non-fouling properties of PEO are tuned not only by the presence and relative orientation of the functional groups with water molecules but also due to a steric repulsive interaction of the macromolecules with proteins. Flexibility of PEO chains, which are acting as macromolecular springs and repulse the approaching proteins, is 7

16 known as the other crucial factor affecting the non-fouling properties [13, 14]. Generally, concentration of the C-O-C groups of 70 % and higher was found to be sufficient for achievement of the non-fouling behavior of the PEO plasma polymers [15]. It is known, that cells, bacteria, biological tissues, proteins and other biological molecules generally behave differently after their reaction with the surface of various materials [16]. The main strategic importance for a growing number of biomaterials applications in vitro and in vivo is the orientation of cell which is responsible for optimization of material/tissue interactions. There is no full understanding of the interactions process of cells with material surfaces until now. Though, it is clear, that possibility to tailor chemical composition, surface energy, morphology and other surface properties could result in the healing of living tissues around the host material. This approach has opened many research paths, which were connected with investigation of various plasma polymers and PEO-like coatings often play a paramount role [16]. Sardella et al. confirmed in [17] that among several approaches shown in literature to obtain surfaces resistant towards protein adsorption and cell adhesion, PEOlike coatings are the most effective. Moreover, PEO-like film can greatly reduce platelet adhesion, platelet activation and fibrinogen adsorption, but also show a strong cell repulsive behavior [18]. These properties put it into the list of potential candidates for development of biofouling resistant surfaces, which are used as coatings of blood-contacting biomedical devices liking retrievable vena cava filters, surface-based diagnostic devices or in vivo sensors. Thin PEO-like plasma polymer coatings have been successfully prepared by low pressure plasma polymerization processes [19]. Recently, the atmospheric pressure discharge is also used for obtaining these films [20]. In addition, various deposition methods with plasma sources and reactors (from micro-discharges to large scale reactors) are commonly used for preparation of PEO-like coatings, for example, such as: - radiofrequency (RF, MHz) parallel plate reactors for low pressure regimes [19]; - dielectric barrier discharges (DBD) [21]; 8

17 - evaporation (thermal decomposition/degradation) of PEO under vacuum with combination with a glow discharge [13]; - atmospheric pressure plasma liquid deposition [20]. Short review of the studying and practical use of PEO films by different scientific groups is given below. PEO coatings were successfully grafted to the silicone rubber by Hong Chen [22]. Normally silicones do not have appropriate surface functional groups that could be used to passivating polymers such as PEO. As a consequence, several approaches have been developed to introduce organic functionalities on silicone surfaces, for example: - using of UV irradiation to create radicals [23]; - oxidation by an O 2 -based plasma to give alcohols and more highly oxidized species [24]. Plasma deposited PEO-like coatings were also investigated as surface modifications [13, 18]. However, application of PEO for surface modification of medical devices is rather complicated. The reason for this is good solubility of coatings in aqueous solutions, which leads to loss of non-fouling properties of the surface over extended periods of time due to releasing of the macromolecules into a biological medium. Therefore, the one possible way to improve the stability of PEO treated surfaces, which are immersed into aqueous environment, is to tune the covalent attachment of the PEO macromolecules to the surface and crosslinking between them. Moreover, several research groups suggested and demonstrated that this can be achieved by the application of plasma polymerization both in continuous wave (CW) and pulsed mode. Applying of plasma polymerization as a technique of thin film deposition, allowed precise adjustment of the chemical properties, as well as crosslinking density within the PEO-like films. Thermal decomposition of PEO-like coatings under vacuum was used by Choukourov et al. [13] in combination with a glow discharge in order to deposit PEO-like plasma polymer films. Resultant coatings were stable in water, because of application of a glow discharge inorder to enhance the crosslinking. Deposited films possess a chemical composition very close to that of original PEO. Furthermore, presence of significant fragmentation of the emitted species under the influence of plasma made a strong impact on physical and chemical properties 9

18 of prepared films. Such coatings behaved as non-fouling and did not adsorb blood proteins (albumin, immunoglobulin and fibrinogen). In addition, reduced thrombogenicity of the plasma polymers expressed in ineffective adhesion of fibrin network was detected. Plasma deposited PEO-like films were investigated as surface modification by Yang et al. in [18]. Mixture of gas composed of tetraethylene-glycol-dimethyl-ether (tetraglyme) vapor and oxygen was used as precursor in this work. High content of ether groups was observed for the films fabricated with the plasma polymerization under high ratio of oxygen/tetraglyme. Such kind of PEO-like films was stable in phosphate buffer solution. Low platelet adhesion, fibrinogen adhesion, platelet activation, endothelial cell (EC) adhesion and proliferation on this plasma deposited PEO-like coatings were revealed by use of tests for in vitro hemocompatibility and EC adhesion. These properties made a potential candidate for the applications in antifouling surfaces of blood-contacting biomedical devices from PEO-like films. Recently, wide use of microstructured surfaces in cell culture experiments facilitates to understand the fundamentals of cell-material interactions by a spatial control of cell adhesion and spreading. Numerous studies have demonstrated that cell behaviours are tightly correlated by substrate chemistry and topography. For this reason great amount of techniques have been investigated to obtain (in a simple and cheap way) reproducible patterned substrates in [17]. Furthermore, such substrates should allow possibility of drive cell adhesion, growth and physiology in many applications: biomaterials, prostheses, tissue/cell engineering, biosensors, microfluidics and biochips, regenerative medicine [25]. Moreover, plasma-deposited non-fouling PEO-like films became very popular and reliable for many different plasma-aided patterning protocols [15, 17, 25]. The main aim of their application is to transfer micro- and nano-metric domains with different properties (e.g., protein adhesive and non-fouling; biomolecule functionalized and nonfouling, etc.) at the surface of platforms, devices, biosensors and biochips intended to function in water media. Sardella et al. [17] described the way for the production of micropatterned substrates by a spatial micro arrangement of chemically different domains, prepared by plasma deposition. Patterns with non-fouling zones of PEO-like 10

19 coatings were alternated with cell-adhesive tracks, namely plasma deposited Acrylic Acid (pdaa) films. It was revealed by that such patterns could be used for chemical and topographical limiting of cell-adhesion; they also supported migration of cells inside the produced pattern. Cells migration inside the tracks of pattern demonstrated possibility to utilize such kind of patterned substrates for tissue engineering or in the development of medical implants. In addition, it was shown in [17] that prepared PEO-like films were able to limit protein and cell adhesion in order to defined micrometric areas of biomedical devices. It was achieved by using of proper physical masking techniques to plasma deposit micropatterned PEO-like coatings were cell-adhesive and non-fouling micrometric domains were alternated on the same substrate. Bretagnol et al. [26] described the method for production of micro-patterned surfaces based on plasma modification of PEO-like coatings using a combination of plasma and photolithographic processes. First, a non-adhesive layer of PEO-like film was obtained by pulsed plasma polymerization of Diethylene Glycol Dimethyl Ether. Second, pattern generation with the help of photolithography was combined with the fabrication of bio-adhesive areas by activation of the first layer by a second plasma treatment. This activation was made using post-discharge plasma of an argon-hydrogen mixture, which allows controlling the concentration of ether bonds of the coatings without introducing new reactive chemical moieties (e.g. hydroxyl or carboxyl) at the surface. As a result, protein and cell attachment experiments confirm that micropatterned surfaces obtained by this combination of plasma processes allow the spatial control of protein adsorption and cell attachment Polytetrafluoroethylene plasma polymer films (PTFE) Fluorocarbon polymer films could be truly named as one of the most studied materials in the field of thin film deposition since the end of the sixties until nowadays [27]. 11

20 PTFE coatings are widely used for various applications because of their numerous specific properties (which are obtained by enhancing of the properties of a conventional bulk polytetrafluoroethylene (PTFE, (CF2)n)): - excellent thermal and chemical stability [28]; - unsurpassed electrical properties including low dielectric loss, low dielectric constant and high dielectric strength [28, 29]; - low dynamic friction coefficient [30, 31]; - biocompatibility [32, 33, 34, 35, 36]; - low surface energy enabling water and oil repellency with water contact angles reaching over 160 (super-hydrophobic surface) [37, 38] and oil contact angles of about 70 [39]. Commonly, the wetting property of a film surface depends on two factors: the surface chemistry of the material and the surface roughness. Hence, the wetting property of a surface can be tuned by controlling these two parameters of the coatings. In last decades, the fabrication of superhydrophobic surfaces with combination effect of high surface roughness and low surface energy had attracted a great deal of attention because such surfaces have unique properties including self-cleaning and antiadhesion [40]. Apparently, successful industrial application of PTFE-like coatings requires both chemical composition of the prepared films resemble with PTFE and tuned morphology of their surface. What is more, nowadays, the great interest has been focused on fluorocarbon polymer films because of utilization of their properties in biomedical field [27]. As a consequence, different methods of fabrication of PTFE-like coatings were suggested by many scientific groups [41, 42, 43, 44, 45, 46, 47, 48, 49]: Deposition methods for preparing of PTFE-like films Template method [41] Extension [42] Electrospraying [43, 44] Physical vapor deposition [45, 46, 47] Irradiation methods [48, 49] 12

21 Numerous scientists have used such methods for fabrication and utilization of PTFE-like coatings. Super-hydrophobic and slippery fluorocarbon plasma polymer films were prepared by using plasma polymerization from gaseous precursors C 2 F 4 by d'agostino and co-workers [50, 51, 52]. Resulted coatings had appropriate chemical composition and roughness in order to achieve the super-hydrophobicity. From the other side, the deposition rate was rather low. Furthermore, several scientific groups investigated deposition of PTFE-like films using plasma polymerization of various perfluorocarbon gases and liquids [53, 54]. It is known from the literature, that cells can (in vitro and in vivo) react differently with chemical [55] and topographical stimuli [56]. Many studies have demonstrated that control and manipulation of cell-substrate and cell-cell interactions could be made by the presence of topographical micro/nanofeatures on the surface. It was investigated by several scientists that nanostructured PTFE-like coatings can be plasma deposited by modulated (MW, i.e., the discharge is pulsed on/off at defined time intervals) PECVD and afterglow (AG) conditions. Thus, Favia and his scientific group have showed in several publications that cell lines (osteoblasts, fibroblasts, condrocytes and others) were grown on surfaces prepared by depositing a rough (MW or AG) plasma polymerized fluorocarbon layer. These films were characterized by different topographical features (ribbons, bumps, petal-like structures) followed by the deposition of a conformal CW layer to obtain surfaces with pre-determined chemical composition and tunable roughness [16]. Stelmashuk et al. [57] have made attempts to prepare such films by RF magnetron sputtering of PTFE target in argon at the target-substrate distance of 4 cm. It was considered by authors that the surface roughness is important for the targeted superhydrophobic effect. Whereas, the roughness did not increase gradually with the pressure, its significant increase was observed after the sudden jump to the highest pressure of 70 Pa. Moreover, Drabik et al. [38] reinvestigated the influence of the substrate-target distance on the roughness and chemical composition of the film surfaces. These authors demonstrated that for the distance of 25 cm between target and substrate with the 13

22 combination of the pressures above 40 Pa allowed to deposit coatings with sufficiently high surface roughness and chemical composition resembling the structure of conventional PTFE. Testing of the prepared films confirmed their super-hydrophobic character. RF magnetron sputtering of PTFE using balanced magnetron and different deposition parameters were also utilized by [38] for fabrication of fluorocarbon plasma polymer films with different chemical compositions and surface roughness. It was find out, that the chemical structure of the coatings could be changed from highly disordered and rather crosslinked fluorocarbon plasma polymer (C/F = 1.5) to more PTFEresembling film (C/F = 1.9) when the target-substrate distance is increased from 14 cm to more than 20 cm and Ar pressure was higher than 40 Pa. It was shown that sufficient roughness for superhydrophobicity and slippery character of the films were provided by the certain granular objects developed in the coatings. Moreover, such properties of the films were stable even after 12 months of storage in the open air. Kylian et al. [58] demonstrated possible way for the deposition of thin, flexible and optically transparent fluorocarbon plasma polymer films with super-hydrophobic character by means of RF magnetron sputtering of PTFE target. Actually, superhydrophobicity of the prepared coatings was achieved by both the high surface roughness of the deposited films and their F/C ratio. It was obtained using higher pressures and longer distances from the sputtered target to substrate. These deposition conditions permitted the CF 2 radicals to have more time to diffuse to the surface to facilitate nucleation and, eventually, formation of the rough surface. Furthermore, mass spectroscopy revealed that longer fluorocarbon molecules also can reach the substrate at long target-substrate distance, which promotes the formation of fluorocarbon plasma polymer coatings with F/C ratio close to 2. Gleason et al. [59, 60] used hexafluoropropylene oxide (HFPO) as a monomer in pulsed RF glow discharges for the deposition of extremely flexible, highly dielectric, very thick (10 micron) fluorocarbon coatings with high retention of the monomer structure on very thin (< 100 micron radius) metal wires. Such finding made possible to use fluoropolymer insulation for electrodes, probes, Micro Electro Mechanic Systems (MEMS) and other very small parts in various biomedical devices. 14

23 Conformal fluoropolymer coatings have been utilized by [55, 61] for the development of smooth surfaces imprinted with protein-recognition nanocavities due to a very elegant sequence of processes. The production sequence of such surfaces includes next steps: 1) protein adsorption on a mica sheet; 2) coating with a polysaccharide solution; 3) drying; 4) CW PECVD of conformal fluoropolymer layer; 5) removal of the mica sheet and wash-off of the template protein. A polysaccharide surface with the molecular imprints was left, because by means of hydrogen bonds it could selectively recognize the template proteins (albumin, immunoglobulin G, etc). Roughness of micro-structured fluorocarbon coatings of ribbon- and petalstructure were induced on polystyrene substrates by means of plasma etching by [62]. The conformal surface chemistry of coatings was provided with a plasma polymerized fluorocarbon layer. Previously mentioned experiments demonstrated that plasma polymerization of micro/nano-structured PTFE-like films can be utilized for independent modulating of surface morphology and surface chemistry of substrates for various purposes, for example: - investigating of adhesion and growth of defined cell types; - for driving, in specific applications, their proliferation and integration with biological tissues. Also, it was found [16] that the spreading of the cells, increasing of the density of focal sites and of stress fibers on the membrane of the cells depends on the presence of fluorinated nanometric roughness. Two plasma deposited fluocorarbon surfaces of identical CFx chemical composition (F/C = 1.62) and different roughness were used for seeding of MG63 osteoblast cells. One of the samples was smooth, the other one was previously plasma coated with a ribbon-like fluorocarbon coating of defined nanometric roughness. It was clearly seen that cells spread better on the rough surface. 15

24 From the other side, Lee et al. [40] presented a facile and straightforward method for controlling the morphology of PTFE films and fabricating superhydrophobic surfaces using electron irradiation. The irradiation of an adjusted electron fluence 2, cm -2 allowed to obtain PTFE coatings with a rough surface structure with micrometer-sized pores and a superhydrophobic property. Consequently, the irradiated PTFE films exhibit superhydrophobic property with a water contact angle (WCA) greater than 150 at fluences ranging from to cm -2 because of high surface roughness. Furthermore, other low-surface energy materials including various fluoropolymers might be used for the fabricating of superhydrophobic surfaces applying such method. The possible way to improve the performance of the fluorocarbon plasma polymers in various applications is to introduce particular functional groups onto the surface. Numerous studies were dedicated to the problem of improving the surface properties of PTFE-like films. Some scientists approaches are chemical etching with sodium naphthalene, electron and ion beams irradiation, UV-lasers and plasma modification [63]. Among these methods, plasma treatments (plasma polymerization and plasma induced grafting polymerization) are rather attractive for their high efficiency Properties and applications of amine containing plasma polymers Recently, thin functionalized plasma polymer films are receiving increased attention as supports for biological molecules. A great number of investigations were dedicated to the problem of enriching surface of the coatings with -NH 2 functional groups. Such so-called tailored surfaces are essential for various biomedical applications because of active functionalities dispersed over the surface which serve as bonding agents for specific molecules [64]. The bonding mechanism is caused by the presence of primary amines (-NH 2 ) and their associated positive charges that are attractive (in aqueous solutions at physiological ph values) for the negatively charged biomolecules (proteins, DNA) and living cells. Additionally, -NH 2 functional groups are used in biochemistry for covalent coupling of proteins in aqueous environments because they are chemically reactive [65]. 16

25 Surfaces with amino functional groups (for example, derived of ammonia, see Figure 5) are widely used in biotechnology but, unfortunately, a lack of information about stability, aging, chemical composition and its dependence on the deposition parameters influence even more their use. This is why huge part of this work is dedicated to the investigation of amino-rich plasma polymers and their properties related to biomedical purposes. It is known for a long time, that surfaces of synthetic polymers are chemically quite inert. It is necessary to suitably vary their surface chemical structures in order to control their interactions with biological systems. This is often done with the help of electrical discharges, via two possible routes to modify macromolecules in the nearsurface region: Ammonia NH 3 Primary NH 3 amine R-NH 2 Secondary amine R NH R Tertiary amine R R N R Figure 5. Classification of amino functional groups derived of ammonia So called grafting (adding new chemical groups via substitutive radical reactions); Plasma polymerization (adding a suitable organic coating onto the surface) [65]. According to the literature, amine containing coatings were prepared by various plasma based methods employing either the plasma polymerization or copolymerization from gaseous precursors performed both at low and atmospheric pressures [64, 65, 66]. Allylamine was used as the N-containing monomers for the plasma polymerization by [66] and plasma copolymerization of hydrocarbon monomers (C 2 H 4, CH 4, C 2 H 2, etc.) in 17

26 mixtures with N-containing gases (N 2 or NH 3 ) [67] were used for the fabrication of N- rich surfaces. Deposition of amine containing films was also made by means of RF magnetron sputtering of Nylon targets in Ar/N 2 and N 2 /H 2 atmospheres [68, 69]. Application of amine containing plasma polymers in biomedical field require specific physico-chemical properties of the surface like chemical functionality (high density of amino groups), hydrophilicity, roughness, surface charge, etc., which should be developed to a certain extent before the interaction with a given biological environment. However, besides all mentioned above there are numerous chemical and physical properties of deposited amino-rich coatings important for various applications. The main utilizations of amine containing plasma polymers are summarized in Figure 6. Application of amine containing plasma polymers Membrane modification Biomolecule immobilization Treatment of different materials Food-packaging Cell growth and adhesion QCM sensors SPR sensors Enzyme electrodes Figure 6. Application of amine containing plasma polymers The applications of microfiltration membranes spread from biotechnology and medical analysis to chemical engineering. Recently, technological development required to diversify the industrial waste-water composition and to solve problem of their environmental purification problem. For this purpose the membrane process was 18

27 proposed as alternative one. Various kinds of membrane materials were tested in order to confirm the necessary requirement of thermally stable and chemically inert. However, the low surface energy of polymers and inevitably tends to fouling during operation were not avoided. Plasma polymerization was found to be very effective in creating thin films on membrane surface with good adhesion, containing specific hydrophilic functional groups useful for improving the flow. At last, it was investigated that amine containing plasma polymers increase significantly the performance of membranes [70], could be utilized for waste-water purification [71, 72], desalination [73] and ultra-pure water preparation [74]. Variety of objects with complicated geometry was successfully treated by glow discharge of amine containing monomers. Oye at el. [75] performed plasma chemical amine functionalization of porous polystyrene beads using allylamine glow discharge. Received result of operation can be utilized for combinatorial chemistry, solid-state organic synthesis, polymer supported catalysis and ion-exchange resins. Evidence of effectivity of amine containing plasma polymers in removing of heavy metal ions from aqueous media (in adsorption of Cu (II) and Pb (II) ions from water solutions) was investigated by [76]. Treatment of polyhydroxyethylmetacrylate (PHEMA) microspheres by ethylenediamine (EDA) and hexamethylenediamine (HMDA) plasma was used for these. Great amount of amino containing plasma polymers are also widely used in biomedical field. Biomedical devices are required to have three major issues: - biocompatibility (the surface of the film should not produce any toxic or allergic reactions after contact with biological system); - resistance to different sterilization techniques (the coating should not have significant changes in structure and properties after applied sterilization); - mechanical stability (the device should be flexible and resist the mechanical loads). The biocompatibility of polymeric materials is usually verified be the seeding of endothelial cells onto the surface. The dependence of cell-surface interactions on surface chemistry is clear. Whereas, the most of the used polymers have good mechanical properties, unfortunately, they are hydrophobic and, generally, are not suitable for 19

28 significant cell attachment. Plasma polymerization is known as nontoxic method for modification of surface properties of the coatings. Moreover, by tuning deposition process and introducing various specific functional groups on the surface, it is possible to enhance or inhibit the cell attachment. Meanwhile, using of some polymeric materials as multilayer in food-packaging industry have expand their ability for biotechnological applications and further investigations in these field. France et al. [77] have find out, that increasing of nitrogen content of the surfaces prepared by plasma co-polymerization of allylamine and octa-1, 7-diene result on improvement of attachment of human keratinocytes. Intereaction between natural or synthetic surfaces and keratinocytes is important in wound care and healing. Endothelialization of Silastic polymers were significantly improved after their treatment with allylamine and/or acrylamide plasma [78]. Stable neuronal cell adhesion was achieved [79] as a result of treatment of the hydrophobic polysiloxane resin with allylamine pulsed plasma. Applying of several autoclaving cycles confirmed stability of the samples. Even after three autoclaving cycles the surfaces were still usable as cell growing substrates. However, there is still a lot of vagueness in mechanisms of the cell-surface interaction. Commonly, it is used to think, that immobilization of proteins on the surface is favor for the cell attachment and growth. Various researches dealt with the immobilization of biomolecules (e.g. coupling of proteins [80], DNA [81]) or facilitation of cell colonization [82] on amine-containing plasma polymers. Investigation of the fibrinogen adsorption on plasma functionalized surfaces was made by Tang et al. [83]. Amino-rich surfaces, prepared by plasma polymerization of allylamine on PET disks, shown the high level of fibrinogen attachment. Subsequently, such samples with immobilized fibrinogen were implanted into peritoneal cavity of mice and positive reaction was observed. Organosilicon polymers and PTFE were coated with n-heptylamine plasma polymer for the covalent attachment of polysacharides [84]. Various biomedical aims are pursued using wide range of compositions and structures of polysacharides. A microwave excited, pulsed, low-pressure plasma was used for coating of polished titanium with an amino group containing plasma polymer (Ti PPA) [84]. 20

29 Human osteoblasts were cultured on the surface of Ti PPA. The final results demonstrate that functionalization of positively charged amino-groups on Ti is sufficiently enough to significantly improve initial steps of the cellular contact to the material surface. Amine containing plasma polymers also serve as a base for development of various types of sensors. Classification of sensor categories is given below: 1. Sensors on quartz crystal microbalance system (QCM). Basically, main function of QCM is based on the change fixation of the oscillation resonant frequency of the crystal with adsorption of material on its surface. Generally, after the deposition of plasma polymer with specific functional groups (amines) on the surface of QCM, the spacer molecules (e.g. glutharaldehyde) are attached to the amine groups. As a result, prepared surface with the big mass is active to react with various biomolecules and measurable frequency shift are achieved. The detection of carboxylic acid vapors was made by application of n- buthylamine plasma deposited QCM sensor [85], which possesses good reproducibility, rapid response time and high stability. The development the QCM immunosensor of antibodies was reported by Nakanishi et al. [86]. First, ethylenediamine (EDA) was plasma polymerized on the quartz crystal surface. Then binding of various antibodies with the film are followed after the activation by glutaraldehyde. The resulted sensors have higher sensitivity and low noise, in comparison with sensors fabricated by the conventional immobilization methods. 2. Surface plasmon resonance (SPR) based on sensors. A phenomenon occurring when light is reflected off thin metal films is called surface plasmon resonance. Detailed description of the working principle of this type of optical sensor is given in [87]. Various antigen-antibodies interactions were monitoring by SPR sensors developed on the base of EDA plasma polymers [88]. In addition, Sasaki at el. [89] used such sensors for the fast estimation of insecticide (etofenprox) concentration in rice. 3. Enzyme electrode sensors. Enzyme electrodes are devices combining a recognition layer consisting of an enzyme and an electrochemical transducer. Their application is connected with the food and biomedical industries. Amino-rich plasma polymer were used as a base for 21

30 preparing of the recognition layer, on account of ability of primary amine groups to serve as linking agents for other species [90]. The resulted layer on the surface of cellulose acetate membranes after treatment with EDA plasma was activated by glutaraldehyde then the model enzyme (glucose oxidase) was immobilized. The functionalized membrane was afterwords placed on the top of electrode cell (usually made of Pt working electrode and Ag/AgCl reference electrode). After reaction between the immobilized enzyme and the target molecules eliminated hydrogen peroxide was detected amperometrically. Detection of alcohol concentration in beers was made by polycarbonate membrane after the EDA plasma treatment and with immobilized alcohol oxidase [91]. However, estimation of the amine concentration in plasma polymers have become rather significant because of biomedical applications for which high amount of primary amines is the most important components, since they react specifically with target molecules. A lot of techniques to evaluate the content (in most cases relative) of amino groups in plasma polymers were tested. Coexistence of amino groups with a manifold of other nitrogen-containing species with similar chemical shifts makes impossible direct identification and quantification of amine concentration at aminated surfaces by X-ray photoelectron spectroscopy (XPS). Chemical derivatization by mean of XPS (CD XPS) ussually solves this problem. Generally, amino groups are reactively tagged with molecules which contain elements (for example fluorine) not originally present on the surface of the samples. Different derivatization reagents for primary amino groups are often utilized during applying CD XPS to plasma deposited aminated surfaces (Table 1) [92]. Different chemical derivatization methods were reported: 1. Favia et al. [93] have made derivatization experiments as gas phase reactions at 50 o C in a small vacuum chamber (cca. 250 ml). The chamber was separated from the pumping (down to a pressure ca.10 mbar) by membrane pump line and connected to a reservoir with the liquid derivatization reagent (TFBA or PFB). Establishing of derivatization reagent vapor pressure was kept in the chamber during derivatization. When derivatization was finished the chamber was 22

31 pumped again for 30 min via the membrane pumping line after the separation of reservoir with derivatization reagent from it. 2. The chemical derivatization was also conducted in the gas phase by Choukourov [64]. On the contrary form [93] the preliminary experiments were done in order to establish the time necessary for the derivatization processes to be entirely completed. For this reason, the samples were treated for 30 min with TFBA. Afterward XPS analysis was applied for determination of the fluorine and carbon concentrations. Reference Derivatization reagents Chemical configuration [64] 4-trifluoromethylbenzaldehyde (TFBA) [93] Pentafluorobenzadehyde (PFB) Table 1. Derivatization reagents for amine groups frequently used for CD XPS 3. The surface concentrations of primary amines were determined by the method of Truica-Marasescu et al. [65] using the selective derivatization reaction of TFBA vapor with amino groups on the sample surface. The derivatization reactions were carried out in a small glass enclosure, where 2 mm diameter glass beads were placed. A tiny amount (~200 µl) of TFBA liquid was dripped onto ~1 cm deep layer of glass beads. Undesirable direct contact between the sample surface and the TFBA liquid was avoided by the putting the coatings on microscope glass slide on the layer of glass beads. The glass enclosure was then placed in an oven at 45 o C for ~ 2 hours at atmospheric pressure. 23

32 Various authors have tried diversity of deposition conditions in order to prepare amino-rich thin films and used different techniques for the calculation of amine concentration. According to the [92], the searching for the optimal derivatization reaction for amines at plasma processed surfaces is not finished yet. As a consequence, there are significant differences in estimation of amine content in plasma polymers. Detailed description of the estimation method of amino content of coatings in this thesis is given in Diagnostic instruments (section 2.2.1) Nylon-sputtered nanoparticles deposited by means of a gas aggregation particle source based on the radio frequency hollow cathode magnetron A growing interest in production of surfaces with scalable nanoroughness using nanoscale sized particles is driven mainly by their possible biomedical applications. [94]. First reports about formation of micrometre- and sub-micrometre-sized particles of plasma polymers refer to the 70s of the last century by Kobayashi et al [95, 96]. Since then, a great attention was paid to the investigation of the mechanisms of particle formation as well as to the evaluation of changes in thier properties. Though, only little interest was devoted to the possibility of technological use of plasma-polymerized particles. According to last data from literature, nowadays controlled deposition of these particles gives a large potential for their using in various applications. For example, fabrication of super-hydrophobic coatings based on films of plasma-polymerized fluorocarbon nanoparticles [97, 98] or biomedical applications, where plasmapolymerized nanoparticles are used for tuning the nanoroughness of surfaces for enhancement of their bioadhesive properties [99]. Great interest is paid to the different methods of fabrication of polymeric surfaces with polymerized particles that allow the tuning of their hydrophilic/hydrophobic character. Generally, so prepared coatings can be used in food packaging or biomedical applications, where such surfaces are utilized for instance as assays for selective protein or cell adhesion [94]. 24

33 Different plasma-based methods have been developed that enabled deposition of plasma-polymerized nanoparticles of various sizes using RF plasmas (e.g. [97, 98, 100]) or pulsed direct current (dc) magnetron plasma sources [101, 102]. This work presents a novel approach to produce Nylon-sputtered nanoparticles by means of gas aggregation source (GAS) based on the radio frequency hollow cathode magnetron that involves a low-temperature plasma in the process of production of nanoparticles. More detailed description of GAS experimental technique is given in next section. According to the literature, in spite of the fact that hollow cathodes were discovered already in 1916, they represent relatively new and unexploited sources of high density reactive plasmas. Nevertheless, it was shown by [103] that by machining hollow structures into the sputtered target can sustain hollow cathode discharges and (or) inject the sputtering gas directly inside the magnetron discharge. The current-voltage characteristics of a magnetron source can be efficiently controled by changing of the size and number of holes, allowing an extended range of operation source. A simple cylindrical radio frequency hollow cathode has been used in this work. Detailed description of its main work principles are given in [104]. There is insufficient amount of information in literature about fabrication of Nylon particles by means of GAS based on the radio frequency hollow cathode magnetron. Therefore, basic characterisation of Nylon-sputtered nanoparticles from the point of view of their chemistry, morphology and wettability was made. Aggregation chamber pressure and input power were varied, which resulted in deposition of particles with different properties including size and chemistry. The possibility to prepare nanocomposites - nylon-sputtered nanoparticles covered with conventionally RF magnetron sputtered Nylon was tested. In addition, the possibility to tune wettability and amine character of overlaid coatings by controlling Nylon particles roughness was investigated. 25

34 1.4. Deposition of metal/ plasma polymer nanocomposites, their properties and applications The first reports of incorporation metal inclusions into a plasma polymer (composites) appeared in the 1970s [105]. In the literature can be found several methods of metal/ plasma polymer nanocomposite film deposition. Those methods are mainly based od sputtering or thermal evaporation of a metal with simoultaneous plasma polymerization process [3, 106, 107]. A short historical overview of basic deposition techniques is given in the following (adapted from [106]). 1. Plasma polymerization of metal organic compounds was first published in 1973 by Tkachuk et al. 2. Simultaneous plasma polymerization and sputtering of a metal using an RF discharge was introduced by E. Kay et al. in DC magnetron sputtering and simultaneous plasma polymerizationwas applied in 1979 by G. L. Harding and S. Craig. 4. RF sputtering from the composite target or targets of a polymer and a metal appeared in 1983 in a paper by H. Biederman and Holland. 5. Metal cluster beam deposition and simultaneous plasma polymerization that was utilized first by Lamber et al. in Simultaneous evaporation of a metal and a polymer was introduced by N. Boonthanom and M. White in Nanocomposite coatings have been studied for several decades mainly because of their unique chemical, physical or bioresponsive properties. Nowadays, the main emphasis is on metal (metal oxide)/ plasma polymer nanocomposites prepared by simultaneous plasma polymerization and metal co-sputtering or evaporation. Simple explanation of successfull developement of such nanocomposites is their possible application for instance as barrier, protective or decorative layers, for the modification of wetting properties of coated materials or for control of interaction of various biological systems with surfaces [108]. 26

35 Numerous investigations of metal/ plasma polymer nanocomposites by various scientists and their studies have established some basic concepts for the films property explanations [4]. Structure, composition and morphology of metal/plasma polymer composite films can be schematically viewed as shown in Figure 7. The amount of a metal is usually described by volume fraction ratio so called filling factor ( f ) defined as [4] The determination of f can be made by means of a quartz crystal microbalance in order to determine the mass (weight) of the composite film. The volume of the film and therefore, its density can be obtained by measuring (using an independent method) the thickness of the film deposited on the known area. In this case, the filling factor is given by: where ρ with the indices of comp, met and p are densities of composite, metal and plasma polymer, respectively. [5] Plasma polymer Metal Figure 7. Schematic structure of metal/plasma polymer composite film (adapted from [4]) 27

36 The size of the metal inclusions depends on the combination of plasma polymer and the metal reactivity and its surface diffusion properties, which are assessed by the melting point. Also, the temperature of the substrate during deposition and changing of the ratio of the monomer/argon working gas mixture have an influence on the inclusions [4]. The main reason for the increase of interest for the nanocomposites lies in their properties. The electrical properties of a metal/ plasma polymer composite film usually depend on metal contents and its arrangement on a nanostructural level. Metals (Au, Ag, Pt, Ni, Mo etc.) are dispersed in the matrix in the form of inclusions with the size from 1 nm to 100 nm. Figure 8 shows, on the example for Ag/C:H, that the DC electrical conductivity of composites strongly dependents on the filling factor, where it is presented as the dependence of the sheet resistance (resistance per square) R on the metal filling factor (volume fraction ratio). Figure 8. Sheet resistance as a function of filling factor for Ag/C:H (adapted from [4]) There are 3 regimes of electrical conduction expressed as the sheet resistance as a function of the metal filling factor: 1. Dielectric regime ( f < 0.2) 28

37 Resistance is very high because the metal inclusions are separated by the dielectricplasma polymer and the dominant conductivity mechanism is then electric charge transport by the electrons and holes tunneling from one metal inclusion to the other. 2. Percolation threshold ( f = ) The inclusions start to touch, a very steep decrease of the resistance is observed. 3. Metallic regime ( f > 0.4) The filling factor is so high that the plasma polymer inclusions are in a metallic continuum [4, 106]. Optical properties of nanocomposites are also very interesting due to so called anomalous optical absorption which takes place because of the optical resonance caused by the collective resonant oscillations of the conduction electrons in metal inclusions. This phenomenon is shown as transmission and reflection in a range of visible and near infrared light for Au/ polytriflurochlorethylene (C 2 F 3 Cl) plasma polymer nanocomposite on Figure 9 [109]. The color of the composite film on glass in the transmitted light changes from pink ( f = 0.01) to red ( f = 0.06), to violet ( f = 0.24) to blue ( f = 0.37). Obviously, the kind of metal and plasma polymer matrix, filling factor, the size and shape of the metal inclusions and their distribution in the plasma polymer matrix influence the position, size and width of the (anomalous) absorption maximum. All these facts led to the first steps in application of nanocomposite metal/ plasma polymers coatings because of their different colors. Biederman [110] proposed to use large area optical filters based on Au/fluorocarbon plasma polymer films. Nanocomposite film composed of Au-Ag alloy particles embedded in a Teflon AF (DuPont) matrix were prepared by simultaneous vapor phase co-evaporation from the three independent evaporation sources [111]. Subsequently, it has been shown, that for the changing of the ratio of Au/Ag from 0.74 to 5.39 nanocomposites can exhibit absorption maximum in the visible range of light in an arbitrary position ranging from 450 to 554 nm. Ability to prepare coating with tailored (arbitrary) color is possible due to this important finding. Useful mechanical properties as decreasing of hardness were found [112] for a- C:H films after co-deposited with Au or Ag (co-evaporated and incorporated into the a- 29

38 C:H grown on the RF excitation electrode possessing DC bias below V). Also the same effect was found for other metals and ion beam techniques [113]. A great interest was devoted to deposition of graded and multilayered system such as Ti/TiC:H/TiC/a-C:H on steel substrates with success for tribological application [114]. This concept was later followed by other authors. Figure 9. Spectral dependence of the optical transmission T (a) and reflection R (b) for gold/halocarbon plasma polymer films, numbers at the curves show filling factor values (adapted from [108]) Investigation of magnetic properties of sputtered Fe-Ni-Co cluster embedded in a hydrophobic protective fluoropolymer matrix (co-sputtered PTFE) was made by Greve [115]. Presence of such properties led to the proposal of development of a toroidal microinductor with minimization of stray fields due to a closed magnetic core. The quality factor was ~ 12 at 1 GHz and the resonance frequency was at about 5 GHz. The research dedicated to the investigation of magnetic properties of nanocomposite films of plasma polymerized propane with cobalt inclusions was made by [116]. 30

39 Recently, the metal nanoparticles/ plasma polymer nanocomposites have gained intense interest in their investigation due to numerous useful properties. While free metal particles offer the possibility to study single species unperturbed by the surrounding medium, practical applications often require nanoparticle distributed in gaseous, liquid or solid environments [117]. Therefore, a greate attention was attracted to the the embedding of metal nanoparticles into matrices with regards to chemical reactions and possible changes of their physical properties. In general, any material may be chosen for the embedding matrix. Though, using plasma polymers matrices offers several significant advantages. Cluster interface with surrounding functionalized polymeric medium may give to plasma polymer (with diverse chemical composition) the rise of new properties which are not known from conventional matter. Moreover, it is possible in a wide range easily to change physical properties of plasma polymers that allow tuning volume diffusion/segregation of the clusters [118]. For instance, PEO was chosen as non-toxic and biologically non-fouling polymeric matrix for the fabrication of metal/ plasma polymer nanocomposites with different metal nanoparticles by some scientists. According to the literature, several research groups have focused on plasma deposition of nanocomposite metal/ PEO-like plasma polymers [119, 120] but there not many published results. Silver sputtering in combination with plasma polymerization of ether-bearing low molecular weight precursors was used by mentioned scientists. The main motivation was to combine the anti-bacterial properties of silver with non-fouling properties of PEO. As a result, the obtained films had only several percent of silver and rather poor retention of the nonfouling properties of the PEO structure was observed. Choukourov et al. [13] suggested using vacuum evaporation of conventional PEO in combination with magnetron sputtering of gold. This process allows deposition of nanocomposite films with very high retention of the PEO character. Special attention was paid in this work to the interaction of coatings with water, because of the possible application of such films in biomedicine, i.e. in contact with aqueous biological solutions. Consequently, gold/plasma polymer nanocomposites could be interesting 31

40 candidates for use in biomedicine as biologically non-fouling materials and as a media with controlled release of gold nanoparticles. Meanwhile, utilization of nanocluster sources had allowed further development in the field of production of nanocomposite materials. Gas phase deposition of clusters became very popular within the last two decades and also various cluster sources have been designed by different research groups [121, 122]. Generally, Gas Aggregation (condensation) nanoclusters Sources (GAS) are based on metal evaporation into a rather high pressure of Ar or He and creating of clusters by a homogeneous nucleation (balanced condensation and evaporation of molecules (atoms) to and from clusters). These processes occurred in an aggregation chamber enclosed by an orifice. The expanding gas carries the clusters through an orifice into the low pressure deposition chamber (typically ultra-high vacuum (UHV) one). Totally new possibilities of nanoclusters production were found out after the replacement of evaporator with a planar magnetron [123]. It was observed, that the influence of plasma from the planar magnetron enables charging (negative or positive) of a considerable amount of clusters. Such fact facilitates electromagnetic manipulation with clusters and allows their mass filtration or acceleration in the direction to the substrate [121]. More detailed description of GAS designs can be found in reviews [124, 125]. Unfortunattly, very low intensity of the nanoclusters beam is principal drawback of a mass filtered ion cluster beams generated in UHV systems. This impact on cluster film deposition rates and make them inappropriate in most of the applications. Therefore, scientific group from Department of Macromolecular Physics from Faculty of Mathematics and Physics of Charles University in Prague has developed a simple and compact GAS without a mass separation (see Figure 10). Such source can be used in a high vacuum only. In this case the beams of all produced nanoclusters (charged and neutral of all sizes), even with certain mass distribution, are used to form cluster coatings with reasonable deposition rates [121]. Such cluster sources were utilized for the incorporation of various metal clusters into the growing matrix of a hydrocarbon plasma polymer. The production of such nanocomposite materials is interesting from the technological point of view since it 32

41 could be used in a wide range of applications (for example, deposition of optical coatings, sensor development or for the production of antimicrobial surfaces). Figure 10. Schematics of experimental setup used for deposition of nanocomposite films (adapted from [121]) For instance Polonskyi et al. [126] and Hanus et al. [125] have made an embedding of Ag clusters into C:H plasma polymer because of the prospect to use their antibacterial effect in biomedical applications. However, interesting optical properties of Ag/C:H plasma polymer nanocomposite were investigated by [121] because of strong absorption maximum due to particle plasmon resonance effect. It was found that the absorption maximum can be shifted with increasing filling factor to higher wavelength for Ag embedded into C:H. The enlarged size of Ag inclusions distribution could cause this shift of some extent overall increase of the absorption in the red part of the part of the spectra. Nowadays, many applications in air or under humidity require fabrication of desirable coatings that better withstand oxidation or corrosion. An ideal class of materials for these purposes are oxides due to their resistance towards high temperatures and other useful properties made them attractive for using in various applications (for 33

42 utilization in microelectronics, food industry, optical applications and for biomedicine) [127]. The mechanical, optical and electrical properties are very important as well. According to the [128], significant amount of research was dedicated to the applying of metal oxides such as Al 2 O 3 as dielectric coatings and as an effective gas diffusion barrier for food packaging. Recently, increased interest was paid to nanocomposites consisting of Al and Al oxide nanoparticles embedded into the different polymer matrix due to their novel functional properties. Al/Al x O y nanoclusters were prepared by Polonskyi et al. [128] by means of simple gas aggregation cluster source based on the magnetron sputtering. Nanoclusters were embedded into the C:H plasma polymer matrix in order to solve the problem of low adhesion to the substrate. It was found out the the surface of nanocomposits deposited by co-deposition of Al clusters and C:H polymer matrix is very rough. In addition, at least 30% of aluminium was present in form of metallic clusters. Applications of these nanocomposites are planned as barrier and protective films either for organic electronics or in a simpler case for food packaging. 34

43 2. Experimental 2.1. Deposition methods Equipment and technology for deposition of plasma polymers a) Amine containing plasma polymers The thin plasma polymer films were deposited by RF magnetron sputtering of Nylon 6,6 (Goodfellow) target (81 mm diameter, 3 mm thickness) in different argon, nitrogen and hydrogen working gas mixtures using a cylindrical plasma reactor depicted in Figure 11. Such equipment was used in numerous papers [68, 69, 129, 130, 131]. Figure 11. Experimental set-up (adapted from [69]) Cylindrical processing chamber (volume of 50 l) was equipped for in-situ characterization (FTIR, XPS) of thin films of plasma polymers and had also several ports for plasma diagnostics. The gas inlet system with needle valves connected to argon and nitrogen gas containers and hydrogen generator (HG 2200, Claind) was used for the introduction of the working gas (total gas flow of 5 sccm) through the tubes into the reactor. The processing chamber was pumped to the base pressure of Pa by means of a rotary and a oil diffusion pumps. 35

44 The plasma was sustained using water-cooled RF planar magnetron, operated at a frequency of MHz, at an applied power of 40 W and at a pressure of 2 Pa. The distance between substrates and the magnetron was ~ 50 mm. Generally, after each deposition the working gas mixture flow was shut off and the samples were left in the chamber under vacuum for 30 minutes in order to avoid an oxidation after the extraction of the samples from the chamber which will cause decrease of the concentration of the radicals entrapped in the films. b) PEO-like coatings The deposition of PEO-like plasma polymers was made by plasma assisted vapor deposition in an experimental set-up presented in Figure 12 and described in details in articles [132]. Figure 12. Experimental arrangement for plasma-assisted thermal vapour deposition of PEO-like films: Q - quartz crystal microbalance; C - crucible; T - thermocouple; M - magnetron (adapted from [132]) 36

45 Deposition chamber was pumped with rotary and diffusion pumps to a base pressure of 10-3 Pa. A copper crucible designed for the evaporation of PEO (received from Sigma-Aldrich, M=2500) was electrically heated up to a temperature of C. The crucible was placed coaxially with and above the RF magnetron equipped with the graphite target (Goodfelow). The magnetron was connected to the RF generator (13.56 MHz, Dressler Ceasar). The substrates ( cm one side polished Si wafers or glass slides) were located 10 cm from the crucible. Argon was used as a working gas (with the pressure of 1 Pa and flow rate of 5 sccm). Such deposition conditions allowed to fabricate films with high retention of the PEO character and with good stability in aqueous environment. c) Fluorocarbon plasma polymer films The deposition of PTFE-like films was performed in a deposition chamber schematically depicted in Figure 13 and described in more details in the studies [38, 131]. Figure 13. Scheme of experimental setup for deposition of plasma sputtered PTFE (S - substrate, M magnetron) (adapted from [38]) 37

46 Deposition equipment consisted of a cylindrical processing chamber (volume of 50 l) pumped by means of a rotary and a diffusion pump (base pressure Pa). The plasma was sustained using RF planar magnetron cooled by water, operated at applied power of 100 W and frequency of MHz. PTFE (Goodfellow) target with the thickness of 3 mm and diameter of 80 mm was used. The substrates ( cm one side polished Si wafers or glass slides) were located 170 mm from the magnetron. Loadlock system was used for the sample loading into the deposition chamber. Argon at pressure of 2 Pa and a total gas flow of 12 sccm was utilized for the operation of RF discharge Preparation of metal/ plasma polymer nanocomposites a) Au/PEO-like plasma polymer nanocomposites The experimental setup was the same as described in previously in b). The crucible was located coaxially with and above a water-cooled planar RF magnetron equipped with a gold target (Au 99.9 %, 51 mm diameter, 2 mm thickness, Safina, a. s.). The substrates ( cm one side polished Si wafers or glass slides) were placed at a distance of 10 cm above the crucible. The intensity of magnetic field of 0.2 T above the erosion track was ensured by using in magnetron neodymium magnets. In such configuration, the concentration of gold in the deposited films was adjusted both by the evaporation rate of PEO controlled by the temperature of the crucible and by the power delivered to the magnetron from an RF generator (13.56 MHz, Dressler Cesar). b) Ag clusters/ch x plasma polymer nanocomposites Deposition of nanocomposite films composed of silver clusters with hydrocarbon plasma plymer was made by simultaneous plasma polymerization using a mixture of Ar/n-hexane and metal cluster beam deposition. Scheme of a simple compact cluster gas aggregation source utilized for the production of Ag clusters is presented on Figure 14 38

47 [121]. It consisted of cylindrical aluminum aggregation chamber: 100 mm inner diameter and 3 mm long nozzle of variable diameter (from 1.5 mm to 3 mm). The aggregation length (distance between target surface and nozzle) was 90 mm. Water was used for cooling the aggregation chamber. DC planar magnetron (80 mm in diameter) in side aggregation chamber was equipped with silver target (3 mm thick). Ag was sputtered with dc current up to 250 ma. Argon was used as a working gas (its pressure in the aggregation chamber with 1.5 mm orifice was 50 Pa, the flow rate was in the range 2-5 sccm). Figure 14. Schematic drawing of gas aggregation cluster source. 1 - vacuum chamber wall, 2 - aggregation chamber, 3 - output nozzle, 4 - cooling of cluster source, 5 - gas inlet, 6 - magnetron, 7 - Ag target, 8 - power supply, 9 - plasma, 10 - cluster beam (adapted from [121]) Ag nanoclusters and plasma polymer matrix were deposited from the two independent sources as schematically shown previously in Figure 10 [121]. The deposition chamber was pumped up with the rotary and diffusion pumps. RF unbalanced magnetron (13.56 MHz) with graphite target was placed on the top of the chamber and operated at a constant power of 20 W (it served as a source for plasma polymerization process). The graphite target was selected for these experiments due to its low sputter yield. However, during plasma polymerization process the target covers with carbonaceous film (C:H hard plasma polymer) that limits further possible 39

48 effects connected with the release of the target material. A cluster source (from Figure 14) was placed vertically to the magnetron as shown above on Figure 10. The pressure in the deposition chamber was tuned to 1 Pa (Ar/n-hexane ratio was 5:2). A rotating substrate holder (with cm one side polished Si wafers, glass slides or glass slides covered with gold) was placed between cluster source and the magnetron with the frequency of rotation of approximately 5 Hz. The distances of the sample holder from both cluster source nozzle and magnetron were 5 cm. c) Al clusters/ch x plasma polymer Preparation of these nanocomposites was made by experimental setup shown on Figure 10 [121] by covering metal clusters with the plasma polymer using mixture of Ar/n-hexane. The pressure in the chamber was tuned to 0.5 Pa with Ar/n-hexane ratio 1:5. RF magnetron (13.56 MHz) with graphite target was mounted on the top of the chamber and used for the plasma polymerization by applying constant power of 100 W. Al clusters were prepared using the same cylindrical aluminum aggregation chamber as for the deposition of Ag clusters (described in details above). DC planar magnetron (80 mm in diameter) with aluminium target (2 mm thick) was placed in side aggregation chamber. Al was sputtered with dc current up to 0,2-0,5 ma. The pressure of working gas (Ar) in the aggregation chamber with 1.5 mm orifice was 30 Pa, the flow rate was 1 sccm. Glass slides, glass slides covered with gold and one side polished Si wafers ( cm) were used as substrates for deposition. The distance from the orifice to substrate holder was 30 cm and from the RF magnetron to the substrate was 20 cm. The sample holder was turned in to 90 degrees to each clusters source and RF magnetron during the deposition of nanocomposites. 40

49 Fabrication of Nylon-sputtered particles Experimental arrangement for the preparation of Nylon-sputtered nanoparticles by means of GAS based on the radio frequency hollow cathode magnetron is schematically described on Figure 15. Deposition equipment consisted of a cylindrical processing chamber (volume of 100 l) pumped by means of a rotary and a diffusion pump (base pressure Pa). The pressure in the processing chamber was settled from 1 to 5 Pa. Cylindrical aluminum gas aggregation chamber (60 mm inner diameter and 3 mm long nozzle with 2,5 mm diameter) was used for the preparation of Nylon nanoparticles. The distance between target surface and nozzle was 5 mm. Water was used for cooling the aggregation chamber. The pressure in GAS chamber was in the range from 15 to 150 Pa. Pure argon was used as working gas for the fabrication of Nylon-sputtered nanoparticles. Figure 15. Schematic drawing of the experimental setup for deposition of Nylonsputtered nanoparticles. Ar argon, RF radiofrequency, C cooling, M magnetron, T nylon target, N output nozzle, G gauge, W window, S sample, P pump 41

50 Internal view of water-cooled hollow cathode magnetron with Nylon target (3mm diameter with the thickness 5 mm) operated with RF power up to 100 W and frequency of MHz is shown on Figure 16. Figure 16. Picture of the internal view of the hollow cathode with Nylon target and Nylon on the sides of the gas aggregation source chamber The thin plasma polymer films used for covering of Nylon nanoparticles were deposited by RF planar magnetron sputtering of Nylon 6,6 (Goodfellow) target (81 mm diameter, 3 mm thickness) in pure argon. This water-cooled magnetron was placed on the top of the chamber and operated at applied power 60 W and frequency of MHz. Substrate holder (with cm one side polished Si wafers, glass slides or glass slides covered with gold) was placed between nanoparticles source and RF planar magnetron. Sample holder was possible to rotate in to 90 degrees to each nanoparticle source and RF magnetron during the deposition of nanocomposites. The distance from sample holder to nanoparticle source nozzle was 18 cm and 10 cm to RF magnetron. 42

51 2.2. Diagnostic instruments This section contents the main information of the diagnostics instruments for the in-situ measurements installed permanently at the reactor chamber for the deposition of amino-rich films and other several techniques used throughout the experimental work which were used ex-situ X-ray photoelectron spectroscopy (XPS) and chemical derivatization X-ray photoelectron spectroscopy is a widely used technique for surface analysis of thin films, which require ultra-high vacuum conditions (pressure in the analysis chamber vacuum ranges from 10-8 to mbar). Generally, XPS instrument consists of such basic elements as: X-ray source, electron energy analyser and detector system (see Figure 17). XPS data are usually obtained using X-ray gun as X-ray source in which an electron beam of several kev kinetic energy strikes an anode (usually cooled by water), providing characteristic radiation. The sample is exposed to photon beam which induces process of photoelectric ionization. By absorbing photons, atoms release electrons to regain their original energy state. The released electrons (photoelectrons) gain the energy from photons and escape from the sample surface with kinetic energy given by the energy of incoming photons, binding energy and work function. As a result, first, electronic analyser measure kinetic energy of electrons emitted from solid surface in order to determine the binding energies of electrons in a substance and then electron detector count the number of electrons [133]. In other words, photoelectrons that actually escaped into the vacuum are collected, energy resolved, slightly retarded and counted, which results in a spectrum of electron intensity as a function of the measured kinetic energy. Since the energy of an X-ray with particular wavelength is known, the electron binding energy of each of the emitted electrons can be determined using an equation that is based on the work of Ernest Rutherford (1914): 43

52 [6], where E B is the binding energy of the electron, hv is the energy of the X-ray photons being used, E kin is the kinetic energy of the electron as measured by the instrument and φ is the work function of the spectrometer (not the material). 2 hv Figure 17. Schematic experimental arrangement for photoelectron spectroscopy (XPS) of the sample surface: 1 - sample, 2 X-ray source, 3 electron detector, 4 electron analyser (adapted from [133]) Generally, obtained XPS spectrum is a plot of the number of detected electrons versus the binding energy of the detected electrons. Each element produces a characteristic set of XPS peaks at characteristic binding energy values that directly identify each element that exists on the surface of the material being analyzed. These characteristic peaks correspond to the electron orbitals within the atoms, e.g., 1s, 2s, 2p, 3s, etc. The number of detected electrons in each of the characteristic peaks is directly related to the amount of element within the area (volume) irradiated [133]. The main advantages and disadvantages of X-ray photoelectron spectroscopy as diagnostic method are gathered in Table 2. 44

53 1. X-ray photoelectron spectroscopy Advantages Disadvantages The energy resolution is limited by the Quantitative non-destructive primary radiation, rather than by the spectroscopic technique. properties of the energy analyzer. 2. Allows measuring the elemental composition, chemical state and electronic state of the chemical elements (except, H and He). During bombardment by photons sample can significantly heat up. Table 2. The main advantages and disadvantages of XPS Detailed description of the XPS technique used in this work is given bellow. The XPS measurements were made using the ultrahigh vacuum (UHV) system (base pressure about Pa). The system consists of a multi-channel hemispherical electrostatic analyser (Phoibos 150, Specs) and a dual (Al/Mg) anode X-ray source. The Al Kα x-ray source ( ev, Specs) was used with the incidence angle of 45 o to the surface plane. The analyser was operated in the retarding-field mode. Survey spectra were acquired once at 40 ev pass energy with step 0.05 ev and dwell time 0.1 s. High resolution spectra were measured at 10 ev pass energy with step 0.5 ev, dwell time 0.1 s and 10 repetitions. In the case of RF sputtered nylon, all the binding energies were referenced to the C 1s carbon peak at ev to compensate the effect of surface charging. In contrast, PEO-like plasma polymers prepared by plasma-assisted vacuum evaporation of PEO have been recently shown to consist predominantly of the C-O-C groups and therefore the XPS spectra were charge corrected for ethers at ev. Finally, in the case of plasma sputtered PTFE films, the XPS spectra were referenced to peak at 292 ev, which corresponds to CF 2 functional group. XPS peak positions were determined with an 45

54 accuracy of 0.1 ev. Curve fitting was performed with CasaXPS software using linear baseline and Gaussian line shapes of variable widths. a) Chemical derivatization XPS equipment was used for determinaton of fluorine concentration after the chemical derivatization in this work. As was mentioned above in Introduction (see section 1.2.3), chemical derivatization can be utilized to determine primary and secondary amine concentration in plasma polymers. In this work primary amine surface concentration was estimated by performing the derivatization reactions in gas phase in accordance with [62]. The reaction was carried out in a small plastic container, which was put into the derivatization oven. A slight amount of TFBA liquid was dropped onto a 1 cm deep layer of 2 mm diameter glass beads placed on the bottom of the box. TFBA had been used in a gas phase to tag primary amines with fluorine atoms like it is shown on Figure 18. Figure 18. Derivatization reaction for primary amines (NH 2 ) with TFBA Then the coatings with the plasma polymer on Si wafers were placed on the layer of glass beads, in order to avoid direct contact between the coated sample surface and the TFBA liquid. The plastic container was left in an oven at 45 o C for 2 hour. Fluorine concentration had been determined thereafter by XPS. According to the literature, in the case of relatively low nitrogen content, the most common way to evaluate primary amine concentration after derivatization is to use the ratio [NH 2 ]/[C] which gives a better idea about the real [NH 2 ] concentration [134]. Consequently, the primary amine [NH 2 ] concentration was derived as follows: 46

55 [NH 2 ] = [7] where [F] and [C] are the fluorine and carbon concentrations, respectively, determined by XPS after the reaction with TFBA. [F]/3 then indicates the absolute amount of primary amines, 8[F]/3 corresponds to the amount of carbon atoms added by TFBA per each NH 2 group and so the term. [C] 8[F]/3 is the original carbon concentration on the surface [63, 134] Fourier Transform Infra-Red spectroscopy (FTIR) The FTIR measurements were performed by FTIR spectrometer (Equinox 55, Bruker) allowing in-situ and ex-situ determination of the chemical composition of the deposited coatings. For the FTIR measurements gold coated glass slides were used as substrate material. The structure of the films was investigated in a spectral range from 400 to 4000 cm -1. For each sample 250 scans with the resolution of 4 cm -1 were acquired Atomic Force Microscopy (AFM) The surface images of deposited thin polimeric coatings were recorded using Quesant Q-Scope 350 atomic force microscope operated in the semicontact mode. NSC- 16 silicon cantilevers (Schaefer Technologie, GmbH) were used for the measurements. The films were deposited on silicon wafers for analyses. The average RMS roughness was calculated from 10 μm x 10 μm to 1 μm x 1 μm scans and resolution points). The standard deviation of measured values of RMS roughness was in less than 0.2 nm. The nanocomposite deposits were characterized using an atomic force microscope (AFM) (Dimension 3100, Veeco Instruments, Inc.). 47

56 Thickness measurements (spectroscopical ellipsometry) The thickness of the coatings was determined by means of spectral ellipsometry (SE) using a variable angle spectroscopic ellipsometer (Woolam M-2000DI) in the wavelength range of λ= nm at an angle of incidence AOI=55-75 in air and at room temperature. In order to obtain the thickness of the coatings, recorded SE spectra were fitted with multilayer model (Si/SiO2/plasma polymer) using the CompleteEASE (Woolam) analysis software Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) The topography of the deposited samples was determined by scanning electron microscopy (SEM) employing Mira II microscope (Tescan) using 10 kv operational voltages. Structure of the nanocomposites was studied by transmission electron microscopy (TEM, Jeol FX 2000) performed on the films deposited on carbon foil supported with copper mesh grids (S160, Agar Scientific Ltd). The TEM images were taken with , and magnification. ImageJ software ( was used for the SEM and TEM image analysis Measurements of wettability Static water contact angles (WCA) of the plasma polymer films deposited on glass substrates were measured by a sessile droplet method at three different spots (about 2 mm in diameter) on the sample surface at a room temperature (18-20 C). In order to fully characterize the wettability of the samples, advancing and receding contact angles were determined in a dynamic mode following the procedure described in the work of Di Mundo et al. [135]. 48

57 Quartz Crystal Microbalance (QCM) The dissolving of deposited films in phosphate buffer saline (PBS) was performed by measuring a Quartz Crystal Microbalance (Maxtek, Inc.) resonance frequency shift, which is directly related to the mass on the crystal surface. In this work, the crystals pre-deposited with a plasma polymer were submerged into a beaker with 100 ml of a PBS solution and the temporal evolution of QCM frequency was measured. The deposition rate was also monitored by QCM placed in-plane with the substrates and analysed by using Lutron, FC-2700TCXO, Frequency counter (MHz) Biological tests Samples were seeded with human osteoblast-like MG 63 cells (European Collection of Cell Cultures, Salisbury, UK), suspended in Dulbecco s modified Eagle s Minimum Essential Medium (DMEM; Sigma, U.S.A., Cat. N D5648) with 10% fetal bovine serum (FBS; Sebak GmbH, Aidenbach, Germany) and gentamicin (40 µg/ml, LEK, Ljubljana, Slovenia). Each sample was well contained with cells (i.e., approximately cells/cm 2 ) and 3 ml of the medium. The cells were cultured for 1 day at 37 C in a humidified air atmosphere containing 5% CO 2. For each experimental group three samples were used. One day after the seeding the samples were rinsed with phosphate-buffered saline (PBS; Sigma, USA), fixed with 70% frozen ethanol (room temperature, 20 minutes) and stained with a combination of two fluorescence dyes, i.e. cell membrane dye Texas Red C2-maleimide (excitation maximum 595 nm, emission maximum 615 nm; Molecular Probes, Invitrogen, USA, Cat. No. T6008; 20 ng/ml of PBS) and nuclear dye Hoechst #33342 (excitation max. 346nm, emission max. 460nm; Sigma, USA; 5μg/ml of PBS) for 2 hours at room temperature. The number of cells on the surface was evaluated on microphotographs taken under an IX-50 microscope, equipped with a DP 70 digital camera (both from Olympus, Japan, obj. 20x). Cell adhesion was estimated by the number of initially adhered cells and cell adhesion area. 49

58 2.4. Materials Silicon polished wafers (n-type (100), On Semiconductor Czech Republic, a.s.) were used as substrates for XPS, AFM and ellipsometry and also for derivatization. Microscopic glass slides (Marienfeld, Germany, mm) were used as substrates for biological tests. These slides were covered with gold for FTIR measurements. Gold-mirror covered microscopic glass substrates (1 1 cm) were used for FTIR- RAS analysis. The Cr/Au polished QCM crystals (5 MHz, Maxtek, Inc.) pre-mounted in a crystal-holder (CHC-100, Maxtek) were used for analysis of adsorption of proteins. Human MG 63 osteoblast-like cells (European Collection of Cell Cultures, Salisbury, UK) and Balb/3T3 immortalized mouse fibroblasts were used for in vitro study of cell adhesion on the amino-rich, PTFE and PEO-like plasma polymers. TFBA was purchased from Aldrich for making the derivatization. 50

59 Thickness of the film [nm] Deposition rate [nm/min] 3. Results and discussion 3.1. Investigation of properties of amino-rich plasma polymers films related to the possible biomedical applications Influence of working gas mixture on properties of amine containing coatings The feasibility of RF magnetron sputtering of Nylon target for the fabrication of the amino-rich films suitable for biomedical applications was investigated in terms of finding the relationship between deposition parameters and the properties of resulting coatings. According to the [69], the fastest film growth can be achieved in Ar/N 2 (50 : 50) discharge, followed by the discharge sustained in a N 2 /H 2 (50 : 50) mixture and pure Ar plasma. Thus, the characterization of the coatings deposited using these working gas mixtures was performed [130]. First, the influences of the working gas mixture have been studied (Figure 19) Ar Ar/N 2 50:50 N 2 /H 2 50: Time [s] Ar N 2 /H 2 Ar/N Figure 19. Deposition rate determined by QCM (right) and ellipsometry (left) as a function of working gas mixture (2 Pa, 40 W) 51

60 The presence of hydrogen in the discharge mixture results in a decrease in the deposition rate, whereas nitrogen causes its increase. The highly reactive hydrogen atoms in the case of H 2 containing mixtures caused significant contribution in etching of the growing films, whereas the enhancement of the amount of sputtered molecular fragments connected with nitrogen was observed for N 2 containing mixtures. The last effect can be explained by the ability of N atoms to effectively passivate dangling bonds created on the target surface by energetic ions. This leads to a lower cross-linking of the target surface exposed to plasma, which, consequently, facilitates the release of shorter molecular fragments by subsequent impact of ions. The presence of higher fluxes of sputtered fragments on the substrate surface then caused increasing of the deposition rate. The chemical composition of the films is also strongly linked to the used working gas mixture. Figure 20 shows results of XPS measurements: the elemental composition of the fabricated coatings (without inclusion of hydrogen in them) is almost the same when Ar/N 2 and N 2 /H 2 discharge mixtures were used (oxygen atom concentration is in both cases less than 4 %, C/N ratio is 1.40 or 1.39, respectively), in contrast to films prepared by Nylon 6,6 sputtering in pure argon that were found to have chemical composition similar to the Nylon 6,6 (C/N ratio close to 5 and O atom concentration 13.8 %). Additionally, results of chemical derivatization revealed that highest [NH 2 ]/C ratio was observed when N 2 /H 2 mixture was used, followed by Ar/N 2 and pure argon (see Table 3). Nevertheless, the high resolution XPS scans showed distinct differences in the chemical structure of the films with similar elemental composition deposited in both nitrogen containing working gas mixtures. This fact was proved by the measurements of the high resolution C 1s peaks (Figure 21), which were deconvoluted in accordance with [65]. When pure Ar was used as the working gas, the most dominant peak in the C 1s spectrum is the one at ev, which corresponds to C-C and C-H bonds. Using of Ar/N 2 and N 2 /H 2 mixtures lead either to enhancement of the peak at ev belonging to various amine C-N and C=N, C N, C-O, C C groups. The contribution from hydroxyls and ethers can be neglected and the second component could be assigned predominantly to nitriles, imines and amines because of the relatively low density (less 52

61 Elemental composition [%] Nylon 6,6 Ar Ar/N 2 50:50 N 2 /H 2 50: C N O Figure 20. Elemental composition of amine containing films deposited in diverse working gas mixtures (2 Pa, 40 W) Ar Ar/N 2 N 2 /H 2 NH 2 /C NH 2 /N Table 3. Amino-selectivity NH 2 /N and amino efficiency NH 2 /C (2 Pa, 40 W) then 4 %) of oxygen in the films. The minor component at ev is attributed to the C=O and N-C=O, N-C-O species and is present in equal amounts in all the spectra. XPS measurements are not capable to distinguish from spectra the primary, secondary and tertiary amines. However, the high density of primary amino groups in such coatings was determined by the derivatization technique. AFM scans of the deposited coatings showed that different working gas mixture composition had no influence on the films morphologies. Fabricated films were rather smooth in all cases (root mean square roughness (RMS) less than 10 nm). 53

62 Intensity [a.u.] Intensity [a.u.] Intensity [a.u.] C-O, C-N, C=N, C N, C C C=O, N-C=O, N-C-O C-C, C-H N 2 /H 2 1: C-O, C-N, C=N, C N, C C C=O, N-C=O, N-C-O C-C, C-H Ar/N 2 1: C-O, C-N, C=N, C N, C C C=O, N-C=O, N-C-O C-C, C-H Ar Binding energy [ev] Figure 21. The C1s spectra of samples prepared in pure Ar (top), Ar/N 2 1:1 (middle) and pure N 2 /H 2 (bottom) (2 Pa, 40 W, measured in-situ) Meanwhile, the wettability of the coatings is governed by the used working gas mixture. Water contact angle measurements (Figure 22) revealed that samples prepared with pure argon pronounced hydrophobic character then films deposited with N 2 /H 2 gas mixture. The samples deposited using different working gas mixtures have also different capability to adsorb proteins. This has been proved by experiments using QCM (Figure 23). 54

63 f [Hz] WCA 51 WCA 47 WCA 28 Figure 22. Picture of WCA measurements of the sample prepared in Ar (left), Ar/N 2 1:1, (middle) and N 2 /H 2 1:1, (right) (2 Pa, 40 W) Ar (NH 2 /C= 2.4 %) Ar/N 2 50:50 (NH 2 /C= 5 %) N 2 /H 2 50:50 (NH 2 /C= 18 %) Time [min] Figure 23. Protein adsorption as measured by QCM on Au coated quartz crystals (2 Pa, 40 W, BSA in PBS) (adapted from [69]) As can be seen in Figure 23, the rate of albumin adsorption corresponds to the NH 2 density determined by XPS measurements, i.e. the fastest process was observed for samples prepared in N 2 /H 2 mixture, followed by Ar/N 2, Ar discharges. Presented part of the investigation aimed at the influence of various working gas mixture on the properties of nitrogen-rich thin films prepared by means of RF magnetron sputtering of Nylon target. First, it was demonstrated that the chemical composition of the deposited coatings can be tuned by changing of working gas mixture. Second, it was found out that chemical composition of the films is strongly linked with used working gas mixture: the highest amino efficiency is found for samples prepared in N 2 /H 2 1:1 mixture. 55

64 Influence of storage time on properties of amino-rich plasma polymers and their stability in aqueous media. Amine containing coatings are widely used in biomedical applications, since their high surface density of -NH 2 functional groups promote adsorption of diverse biomolecules or facilitate cell growth. According to the literature, the coatings gradually lose their amino rich character, which is ascribed mainly to the oxidation of primary amino groups and formation of more stable amides. Although, the recent studies indicated that in some cases loosening of primary amino groups does not have an influence on the ability of such films to promote cells adhesion and proliferation [136]. Nevertheless, the temporal stability of the amino-rich films prepared by RF magnetron sputtering of Nylon target using various working gas mixtures (Ar, Ar/N 2 1:1, N 2 /H 2 1:1) was investigated as important property for their applications. Comparison of in-situ and ex-situ XPS measurements of C 1s peaks of deposited coatings demonstrated in Figure 24 showed that all samples readily oxidize after their exposure to air. In a case for Ar/N 2 and N 2 /H 2 discharge mixtures, increasing in oxygen fraction in the coatings occurs almost solely at the expense of N 2. Nitrogen fraction was found to rapidly decrease after the exposure of the samples to open air ([N]/[C] ratio decreases by more than 30%). This can be explained by the fact that the loss of one nitrogen atom is accompanied with incorporation of one oxygen atom. Gengenbach et al. [137] made a suggestion, that the hydrolysis by atmospheric humidity can affect such behavior. Changes in the chemical structure of the samples exposed to air can be clearly witnessed by the deconvolution of the high resolution XPS scans of C 1s peak (see Figure 24) made with accordance to [131]. The C 1s spectral deconvolution revealed the presence of hydrocarbon peak (C-C, C-H) at ev, amine groups (C-N) that might be primary, secondary, or tertiary at ev, C-O/C=N/C-N at ev, and carbonyl (C=O) and/or amide (O=C-NH) at ev. First of all, it can be seen that the exposure of samples prepared using pure argon, Ar/N 2, N 2 /H 2 working gas mixture to open air causes an increase of N-C=O and C=O bonds. 56

65 Intensity [a.u.] Intensity [a.u.] Intensity [a.u.] Ar C 4 C 3 C 2 C 1 In-situ C 4 C 3 C 2 C 1 1 day on air Binding energy [ev] Binding energy [ev] Ar/N2 (1:1) C 4 C 3 C 2 C 1 In-situ C 4 C 3 C 2 C 1 1 day on air Binding energy [ev] Binding energy [ev] N2/H2 (1:1) C 4 C 3 C 2 C 1 In-situ C 4 C 3 C 2 C 1 1 day on air Binding energy [ev] Binding energy [ev] Figure 24. High resolution XPS scans of C1 s peak of samples measured in-situ (left) and after 1 day on air (right). Assignment of components of C 1s peak is C1) C-C and C-H C2) C-N C3) C=N, C N and C-O and C4) C=O and N-C=O 57

66 absorbance [a.u.] In contrast to the films deposited in pure Ar, the films prepared in Ar/N 2, N 2 /H 2 mixtures showed signifiant increase of corresponding C4 component of C 1s peak (more than 40%) after being exposed to the open air. FTIR measurements shown in Figure 25 also confirmed significant increase of C=O stretching vibrations after the exposure of coatings to the open air. Such changes are consistent with hydrolysis of imines that give rise to the C=O functionalities as well as with formation of amides by the oxidation of primary amines. Furthermore, results of XPS measurement revealed differences in ageing of the samples prepared in various working gas mixtures. Study of the C 1s peaks of coatings fabricated in all three discharge mixtures demonstrated that dramatic influence of the residence time on the open air on the chemical composition was observed in the case of Ar/N 2 and N 2 /H 2 gas mixtures. 0,040 C=O C=N C=C insitu exsitu 0,035 0,030 0,025 NH OH CH 2 CH 3 C N C C CH x N 2 /H 2 1:1 0,020 0,015 Ar/N 2 1:1 0,010 0,005 Ar 0, wavenumber [cm -1 ] Figure 25. The FTIR spectra of samples prepared in pure Ar (bottom), Ar/N 2 1:1 (middle) and N 2 /H 2 1:1 (top) measured before and after their exposure to air (2 Pa, 40 W) 58

67 Decrease of both C2 and C3 components of C 1s peak (see Figure 26), which can be attributed according to [130] mainly to amines and imines, was observed for samples prepared in Ar/N 2 mixture, the C3 component is relatively stable in the case of N 2 /H 2 mixture. In addition, high resolution XPS scans of C 1s peak revealed increase of C=O and N-C=O bonds with time on air. C-N and C=N bonds are reduced as compared to C- C and C-H ones. Results of chemical derivatization (see Figure 27) also confirm expected decreasing of amino efficiency [NH 2 /100C] the films prepared Ar/N 2 and N 2 /H 2 gas mixtures with ageing on the open air. It was found, that coatings exposed to the air for 24 hours after deposited lost almost a half of the amine content. However, the nature of biomedical applications requires also sufficient stability of the deposited coatings in aqueous environments. Ar/N 2 C4 C3C2 C1 C3 C2 C4 C1 N 2 /H 2 In situ In situ 1 hour on air 1 hour on air 24 hours on air 24 hours on air 72 hours on air 72 hours on air Binding energy [ev] Binding energy [ev] Figure 26. The C 1s spectra of samples prepared in Ar/N 2 1:1 (left) and N 2 /H 2 1:1 (right) measured before and after their exposure to air (2 Pa, 40 W) C1) C-H, C-C; C2) C-N, C3) C=N, C N, C-O; C4) N-C=O, C=O 59

68 NH 2 /[100C] The thickness of films submersed into liquids reflects two effects: swelling and loss of material that is dissolved in liquids. The procedure introduced by Abbas et al. [138] was employed in order to estimate the influence of these two effects. Primarily, the initial thickness of the samples T 0 was measured immediately after the deposition by means of spectroscopical ellipsometry. Afterwards, the samples were soaked for 24 hours in de-ionized water and then dried by a flush of air. The thickness T 1 of the films was measured immediatly after the drying step. Subsequently, the thickness T 2 of the films was measured after heating of the samples in an oven with 100 C for 30 min in order to evaporate water absorbed in the plasma polymer network. Moreover, it is important to mention that no considerable change of the thickness of reference samples that were not soaked into water was observed after 2 hours in an oven. The thickness of the swelling T swell of the coatings can be then determined as T swell =T 1 T 2, whereas the loss of the thickness T loss is given by relation T loss =T 0 T N 2 /H 2 Ar/N Storage time [houres] Figure 27. Amino efficiency of the samples as a function of storage time (2 Pa, 40 W) Thus, spectroscopical ellipsometry was used for the thickness measurements of the films in dependence on their residence time in de-ionized water as mention above. Decrease of the thickness of coatings as well as their swelling was observed for all tested samples immersed in water (see Figure 28). 60

69 Thickness [nm] Thickness [nm] Left diagram on Figure 28 shows measurements of the swelling. It was found that values of T swell were around 10% of the initial thickness of the coatings deposited using all three discharge mixtures. However, it was observed that changes it the rates at which the coatings dissolve in water strongly depepend on the working gas mixture employed for the thin films deposition. Namely, the coatings prepared in Ar or Ar/N 2 mixture dissolve rather slow (reduction of the thickness was less than 9%) in contrast to the samples fabricated using N 2 /H 2,which dissolve readily already after being 1 day in water (reduction of the thickness was 35%). The same behaviuor was demonstrated by the samples immersed into PBS. However, the results are affected by the presence of salt residuals that have to be rinsed first in this case. Therefore, QCM measurements were applied in addition to the ellipsometry in order to measure the evolution of mass of films presented on the surface directly in PBS. Such experiments showed stability of the coatings fabricated using Ar and Ar/N 2 in contrast to the films deposited in N 2 /H 2 mixtures that readily dissolved in PBS (see Figure 29) T 0 as deposited T 1 after 1 day in H 2 O T 2 after 1 day in H 2 O and heated swelling dissolving Ar Ar/N 2 N 2 /H 2 0 Ar Ar/N 2 N 2 /H 2 Ar Ar/N N2/H2 Figure 28. Thicknesses of films prepared in pure argon, Ar/N 2 1:1 and N 2 /H 2 1:1 mixtures after the deposition, after immersion into water for 24 hours and after additional heating (left diagram) together with estimated thicknesschanges caused by swelling and dissolving (right diagram) 61

70 f [Hz] QCM measurements were made in two steps: first, the crystals pre-deposited with a plasma polymer were submerged into a beaker with 100 ml of a PBS solution; second, the temporal evolution of QCM frequency was measured. The higher solubility of the films prepared in N 2 /H 2 mixture can not be explained just by the presence of higher amino content in comparison with the coatings fabricated in Ar/N 2 mixture. Great amount of authors have already reported in their studies (e.g. [139]) that the higher portion of polar -NH 2 groups in plasma polymers is connected with presence of higher amount of low molecular weight fractions (so called oligomers) in the coatings that are highly soluble in water. Additionally, as can be seen from Figure 30, the comparison of dissolving the coatings deposited using Ar/N 2 and N 2 /H 2 working gas mixtures in various liquids (ethanol, de-ionized water and PBS) was measured by ellipsometry. It was found that films prepared using Ar/N 2 are rather stable in all three tested liquids then coatings fabricated in the other working gas mixtures. Samples deposited in N 2 /H 2 mixture rapidly dissolved in water and PBS. However, no effect was observed after their soaking into ethanol Ar Ar/N 2 1:1 N 2 /H 2 1: Time [min] Figure 29. Temporal evolution of frequency shift of samples immersed into PBS (2 Pa, 40 W) 62

71 Thickness [nm] Thickness [nm] AFM measurements on Figure 31 demonstrated different trends in the evolution of the morphology of films which were observed after the immersion of samples into the distilled water. Whereas water caused only negligible changes of RMS roughness of the samples prepared in Ar/N 2 mixture (from 0.2 nm to 0.4 nm), considerably higher alterations of the morphology were observed on samples deposited in N 2 /H 2 mixture, for which randomly distributed mounds on the surface were observed (see Figure 32). These mounds can be soluble part of the films that remained on the surface after drying. Moreover, even in the later case the surfaces remained rather smooth with the values of RMS roughness around 1 nm. Furthermore, no formation of porous microstructures reported previously by Vasilev et al. [140] was observed. Immersion of the samples fabricated in Ar/N 2 and N 2 /H 2 mixtures into various liquids for 1 day also confirm slight changes in roughness in contrast to dry one (see Figure 32). However, AFM measurements revealed that 1 day immersion in ethanol has no effect on morphologies of both films. In PBS and water considerably higher increase of RMS roughness was observed for coatings prepared in N 2 /H 2 mixture Ar/N 2 Before and After immersion to liquids N 2 /H EthanolWater PBS EthanolWater PBS 0 Figure 30. Thickness of the coatings before and after 24 hours immersion in liquids as measured by ellipsometry (2 Pa, 40 W) 63

72 So it can be concluted that study of the influence of the storage time on the amino-rich coatings prepared by RF magnetron sputtering of Nylon target using various working gas mixtures (Ar, Ar/N 2 1:1, N 2 /H 2 1:1) revealed that these films readily oxidize in contact with air and exhibit fast ageing and loss of their amino-rich character. Furthermore, these films are easily dissolved both on water or PBS and thus their use in biomedical application is limited. However, coatings prepared in N 2 /H 2 were founded to dissolve in liquids much more readily as compared to samples deposited in Ar and Ar/N 2 gas mixtures which did not reach high values of [NH 2 ]/C ratio, but seem to be more stable in liquids and this makes them more suitable for the real technological application. a) b) c) d) Figure 31. AFM scans (5 5 µm): a) typical as deposited nitrogen-rich film, b) film after 24 hours in H 2 O (Ar, 2 Pa, 40 W), c) film after 24 hours in H 2 O (Ar/N 2, 2 Pa, 40 W), d) film after 24 hours in H 2 O (N 2 /H 2 2 Pa, 40 W) 64

73 RMS rougness [nm] Moreover, distilled water or PBS did not strongly influence topography and thickness of the films prepared in Ar/N 2 mixture. This distinguished such method of samples deposition from sputtering in N 2 /H 2 mixture, which results in the deposition of coatings that readily dissolve both in water and PBS Ar/N 2 N 2 /H As dep 1 Day in H 2 O 1 Day in PBS - rinsed 1 Day in ethanol Figure 32. RMS roughness of the films before and after 24 hours immersion in liquids as measured by AFM (2 Pa, 40 W) 65

74 3.2. Basic characterization of Nylon-sputtered nanoparticles prepared in a gas aggregation particle source based on the hollow cathode magnetron The first step in the characterization of Nylon-sputtered paricles was dedicated to investigation of deposited tracks on the glass substrate (see Figure 33). It was found out that using 2.5 mm orifice and certain deposition parameters (100 W, Ar pressure in GAS = 50 Pa, deposition time = 5 min) it is possible to achieve visible, relatively thick and homogenious covering of the substrate with Nylon particles on the distance of 18 cm from orifice. 6 cm 9 cm 12 cm 15 cm 18 cm 21 cm Figure 33. Dependence of the deposit tracks of Nylon nanoparticles on the distance from the glass substrate to 2.5 nm orifice (100 W, pressure in GAS = 50 Pa, deposition time = 5 min) Change of aerodynamic focusing of Nylon-sputtered nanoparticles prepared at 60 W was investigated in dependence on the different pressure in working chamber and gas aggregation source. As can be seen from Figure 34 increasing the pressure in deposition chamber (for various values of Ar pressure in GAS), probably, creats gas vortexes which prevent intensive deposition of particles on the glass substrate. Also, these vortexes significantly affect the form of the deposit track on the substrate. The spot of deposit track become much more wide and blurred with the increasing of pressure in the deposition chamber. The next part of the study of the properties of Nylon particles was focused on their chemistry in dependence on the pressure of pure argon in aggregation chamber. Results of XPS analysis shown in Figure 35 confirmed that various input power and 66

75 pressure in GAS have almost no influence on chemical composition of deposited Nylon nanoparticles. Figure 34. Aerodynamic focusing of Nylon nanoparticles in dependence on the pressure in working chamber and GAS (60 W, deposition time = 5 min, distance to the substrate = 18 cm) Fabrication of nanoparticles involves such an important aspect as possibility to control their size by a proper choice of operational conditions (for example, power, pressure in GAS and working chamber). Figure 36 presents SEM images as an example of Nylon nanoparticles produced in this work. It can be seen from these images that the particles have a cauliflower-like shape, commonly observed for particles produced in dusty plasmas [141]. The size distribution and SEM images of deposited Nylon-sputtered nanoparticles demonstrated that increase of input power caused dramatic changes in the mean size and amount of the nanoparticles (see Figure 37 and Table 4). 67

76 Concentration of chemical bond, % Concentration of chemical bond, % Pa C-C,C-H C-N C=N,C-=N N-C=O 20 W 60 W 100 W Pa C-C,C-H C-N C=N,C-=N N-C=O 20 W 60 W 100 W Figure 35. Concentration of chemical bonds in Nylon particles (measured by XPS) prepared with different RF powers and pressures in the aggregation chamber (orifice = 2.5 mm, distance to the substrate = 18 cm) 5 µm 1 µm 500 nm Figure 36. SEM images of the Nylon nanoparticles with different magnifications prepared under 50 Pa, 60 W, orifice = 2.5 mm, deposition time = 15 s 68

77 Count Count Count 20 W W Diameter [nm] 60 W W Diameter [nm] 100 W W Figure 37. SEM scans (2 µm) (left side) and sizes distribution (right side) of Nylon particles prepared with different input powers (50 Pa, orifice = 2,5 mm, distance to the substrate = 18 cm) 69 Diameter [nm]

78 Input power, W Average diameter of Nylon particles, nm 20 ~ ~ ~ 45 Table 4. Dependence of the average diameter of Nylon nanoparticles on input power (50 Pa, orifice = 2,5 mm, distance to the substrate = 18 cm) The increase of input power from 20 to 100 W caused decrease of the average diameter of Nylon particles from ~ 65 to ~ 45 nm. The slowing down of the nanoparticle growth can be explained by the increasing of particle temperature. Maurer H. et al [142] have shown that the temperature of the particles fabricated in low-temperature plasmas may reach high values and exceed the gas temperature of the plasma by several hundred kelvins. As a consequence, these may occurs thermal cleavage of bonds with release of small fragments from the surface of the polymeric particles. Some scientists have even reported that higher temperatures delay the appearance of particles in the plasma volume [143]. Therefore, an integral energy influx density (in which electron/ion recombinations at the particle surface play the dominant role) strongly influences the growth of the particles [142]. The intensification of the recombination processes on the particle surface is caused by the increasing electron density that increases with rise of discharge power. In this case, a larger recombination energy released to the nanoparticles hinders their growth at a higher discharge power. The influence of RF power on the formation of Nylon-sputtered particles film tracks on the glass substrate is shown in Figure 38. Increase of the pressure in the gas aggregation source resulted in decrease of the size of clusters, but increased their amount on the substrate as can be seen from Figure 39. Such behaviour is similar to the growth of nanoparticles in PEVCD systems, and can be explained in a three-step process described below [141]. At first, as the result of complex plasmochemical reactions stable nanosized particles are created. These 70

79 particles (if created in sufficient number) readily coagulate and form particles with diameters in the range of tens of nanometres. However, this step is accompanied by a steep decrease in the number of nanoparticles, which also lowers the probability of their collisions and thus their further growth is on a longer time scale driven mainly by sticking of gasphase particles (radicals) to big nanoparticles [144]. As for our case, the particles produced in GAS are dragged by a carrier gas (Ar) towards the exit orifice of the aggregation chamber and thus the above-mentioned time evolution may be translated to the position in the aggregation chamber. Taking into account that the first two phases occur in the vicinity of the sputtered Nylon target, the main process that determines the final size of the particles is the sticking of gas-phase species to nanoclusters travelling through the aggregation chamber. Apparently, increasing the pressure of carrier gas in GAS chamber in this moment causes decreasing the possibility of gas-phase species sticking to particles (which are transferred through the orifice much faster) and this influences the deposition rate of nanoparticles. 20 W 40 W 60 W 100 W Figure 38. Deposited tracks of Nylon nanoparticles on glass substrates as they depend on input RF power (50 Pa, deposition time = 5 min, distance to the substraten = 18 cm) 71

80 15 Pa 50 Pa 150 Pa Figure 39. SEM images (2 µm) of Nylon nanoparticles produced with different pressures in GAS chamber (orifice = 2,5 mm, distance to the substrate = 18 cm) As the next step, dependence of sample roughness on mean particle size was studied. Nylon nanoparticles were covered with thin Nylon-sputtered plasma polymer (thickness 15 nm) because of weak adhesion of particles to the substrate. AFM measurements (see Figure 40) releaved that the size of the particles had a strong influence on the roughness of the coatings. In addition, the possibility to prepare nanocomposites of Nylon-sputtered nanoparticles covered with conventionally RF magnetron sputtered Nylon was tested. According to the AFM and WCA measurements presented in Figure 41, strong hydrophilic character was observed for nanocomposites with rather high values of RMS roughness. Increase of roughness of nanosomposites using operation condition for fabrication of different amount of Nylon particles with various diameters caused changes in water contact angle of the samples. It can be summarized that, gas aggregation particle source based on the hollow cathode magnetron was successfully used for production of Nylon-sputtered nanoparticles. Results of characterization of fabricated nanoparticles in dependence on deposition conditions showed that alteration of input power and pressure in the aggregation chamber have strong influence on the chemistry of Nylon nanoparticles as 72

81 well as on average size and deposition rate of the particles. In addition, size of the particles had a dramatic influence on the roughness of the films. First samples of Nylon nanoparticles/nylon plasma polymer nanocomposites were fabricated and characterized. WCA measurements revealed that nanocomposites with rather high roughness had strong hydrophilic character. Furthermore, WCA of nanocomposites can be tuned by using different amount of Nylon particles with various diameters. RMS ~ 110 nm RMS ~ 114 nm a) b) RMS ~ 77 nm RMS ~ 42 nm c) d) Figure 40. AFM scans (5x5 µm) of Nylon particles covered with 15 nm of Nylon-sputtered plasma polymer and prepared with: a) 15 Pa, b) 40 Pa, c) 75 Pa, d) 150 Pa (orifice = 2,5 mm, distance to the substrate = 18 cm) 73

82 RMS ~ 1nm WCA = 30 a) RMS ~ 35nm WCA = 10 b) RMS ~ 56nm WCA = 8 c) RMS ~ 50nm WCA = 6 d) Figure 41. AFM scans (5x5 µm) and pictures of WCA of : a) Nylon plasma polymer (50 nm), b) Nylon particles (55 nm), c) Nylon particles (40 nm), d) Nylon particles (45 nm) (Nylon particles were deposited for 30 s and covered with Nylon thin film (50 nm)) 74

83 3.3. Resistance of PEO-like coatings, fluorocarbon and amino-rich films towards various sterilization techniques The main objective of investigations related to biomedical use of plasma polymers was a fabrication of plasma polymers having desired bioresponsive properties, high stability in time as well as stability in aqueous environments. Nevertheless, due to the nature of biomedical applications, the coatings should also withstand a sterilization process. Therefore, three kinds of plasma polymers mentioned above were subjected to three different sterilization procedures commonly employed in biomedical praxis i.e. dry heat, autoclave and UV radiation treatment. The physical, chemical and bioresponsive properties of such treated samples were determined by means of different techniques [137]. First, the variation of the thickness of the plasma polymers depending on the used sterilization method was studied. The thicknesses comparison of plasma polymerized PEO films before and after their sterilization by UV radiation, dry heat performed at 160 o C and autoclaving (120 o C) is presented in Figure 42 a). It can be clearly seen, that only UV radiation did not change the thickness of the coating. Both autoclave and dry heat caused reduction of the film thickness, which suggests thermal instability of ppeo films. Regarding the films of pptfe, no significant variations of their thickness were observed after their sterilization by UV radiation, dry heat or autoclave as can be seen in Figure 42 b). Finally, in the case of plasma sputtered nylon, only autoclaving which is combination of moist environment with elevated temperature caused a significant decrease of the films thickness. In this case almost a half of the deposited coating was removed (see Figure 42 c)). 75

84 Thickness [nm] Thickness [nm] Thickness [nm] UV radiation UV radiation UV radiation Plasma polymerized PEO as deposited after sterilization a) Autoclave Plasma sputtered PTFE as deposited after sterilization Autoclave Autoclave Figure 42. Comparison of thicknesses of plasma polymers before and after their Dry heat Plasma sputtered Nylon as deposited after sterilization Dry heat Dry heat sterilization: a) plasma polymerized PEO b) plasma sputtered PTFE b) c) and c) plasma sputtered Nylon 76

85 It was shown that different plasma polymers possess different resistance towards various sterilization techniques in terms of their thickness. Such information is important for the application of ultrathin films (i.e. films having thickness of several nanometers), which are often used for various biosensors including those based on surface plasmon resonance. The thickness reduction in this case can cause exposure of underlyining substrate, that subsequently interact with biological samples and can give false signal. On the contrary to the thickness, AFM measurements revealed that the roughness of the deposited films was not markedly modified by any of the used sterilization techniques. All measured coatings remain very smooth (RMS roughness is below 1 nm), with only few spikes on the surface induced by the sterilization process. The XPS analysis of the chemical composition of non-sterilized and sterilized coatings was made. Moreover, the wettability of the films, which is highly surface sensitive, was also measured. Following results were achieved. As was already shown ppeo films before sterilization are composed of carbon and oxygen with O/C ratio 0.60 (see Table 5). O [%] C [%] O/C C-C, C-H % C-O-C % C=O % O-C=O % As deposited UV radiation Dry heat Autoclave Table 5. The XPS analysis of the non-sterilized and sterilized ppeo films High resolution XPS spectra of as-deposited ppeo demonstrated in Figure 43 revealed that C 1s peak can be decomposed into three peaks: C-C/C-H bonds at binding energy ev, C-O-C bonds at ev and C=O bonds at ev. It can be seen that the amount of the C-O-C bonds (77 %) prevails over of the C=O bonds (12 %) and the C- C/C-H bonds (11 %). 77

86 Intensity [a.u.] Intensity [a.u.] Intensity [a.u.] Intensity [a.u.] a) C-O WCA = 36 o C=O C-C C-H b) C-O WCA = 37 o O-C=O C=O C-C C-H c) WCA = 46 o C-O C=O O-C=O C-C C-H d) C-O WCA = 49.6 o O-C=O C=O C-C C-H Binding energy [ev] Figure 43. High resolution XPS scans of C 1s peak of ppeo films (left) and water droplets on ppeo films (right): a) non-sterilized sample b) sample sterilized by UV radiation c) sample treated by dry heat and d) autoclaved sample 78

87 The contribution of these three peaks to the C 1s peak remained almost unaffected when the samples were exposed to UV radiation. WCA measurements also confirmed similarity between the non-sterilized and UV irradiated samples by giving 36 o and 37 o for both. Good retention of PEO-like character was also observed for autoclaved samples. This can be explained by the fact that ethers do not hydrolyze during autoclaving. Slight decrease of the C-O-C bonds to 72 % was detected, which was accompanied by the increase of C-C/C-H bonds (14 %). Significant increase of WCA to the value of 46 o was caused by higher fraction of hydrophobic C-C/C-H bonds. Although, autoclaving made slight changes in chemical composition, the loss of mass for ppeo coatings was significant after this sterilization technique. Since hydrolisis can be excluded from consideration, another mechanism should account for the observed loss of the material. It has been previously shown by Choukourov et al. in [13] that ppeo films prepared by plasma-asisted evaporation are heterogeneous systems in which a cross-linked network co-exists with a mixture of oligomers with very broad molecular mass distribution. The oligomers are held within the network by physical entanglements but not linked chemically to it. Hence, after the contact with water, such macromoleculs leave plasma polymer by diffusion and can be detected in liquid phase, for example, by Nuclear magnetic resonance (NMR). Generally, the ppeo film, prepared at indentical conditions as those used in this work, lost about 12 % of its thickness by out-diffusion of the unbounded species away from the film. Nevertheless, in our case, the coating was autoclaved, i. e. brought in contact with water vapours. It can be assumed that the elevated temperature is responsible for intensification of the macromolecular dynamics and unbounded oligomers, especially of low molar mass, get higher probability to disentangle and to escape from the plasma polymer. The most pronounced variations of chemical structure of ppeo coatings were observed after dry heat treatment at which high temperature was used. The C-O-C content drops down to 55 %, which is accompanied by an increase of C-C/C-H bonds (21 %) and formation of O-C=O bonds (15 %). In this case significant change in the O/C ratio was observed. Oxygen content increased on expense of carbon and the O/C raised to the value 0.73 (see Table 5). Formation of O-C=O bonds after dry heating is in 79

88 agreement with the work made by Han et al. [145], who elucidated esterification of PEO by the random chain scission mechanism during thermal degradation in air. In agreement with these changes of the chemical composition, an increase of WCA to the value close to 50 o was observed. In addition, the film lost about 70 % of its thickness during the sterilization (see Figure 42). The XPS analysis showed that the pptfe coatings were composed mainly of F and C atoms. Insignificant incorporation of oxygen was also detected (see Table 6). For the pptfe coatings, the C 1s XPS spectra were referenced to the peak at 292 ev, which corresponded to the CF 2 functional groups. Other components were positioned at ev (C-CF), at 290 ev (C-F) and at ev (CF 3 ). The additional component at ev (C-C) was added where relevant. F [%] C [%] O [%] F/C % C-C % C-CF % CF% CF 2 % CF 3 % As deposited UV radiation Dry heat Autoclave Table 6. The XPS analysis of the non-sterilized and sterilized pptfe films It was find out, that the F/C ratio of non-sterilized films derived from the high resolution spectra is After the sterilization process the F/C ratio remained the same for the UV-treated and dry heated samples, but in a case of autoclaved samples the F/C value was changed to Such changes in F/C ratio are further evidenced by the highresolution scan and by the WCA measurements showed on Figure 44. It can be seen, that the C 1s spectra of non-sterilized, UV-treated and dry heated samples are indentical and their WCA value almost the same is around 110. However, the C 1s of the autoclaved sample differs from the others by enchanced contribution from the C-C/C-H bonds, which occurs on the expense of F-containing bonds (C-CF, C-F, CF 2 and CF 3 ). These changes resulted in significant decrease of WCA down to 83 o typical for hydrocarbon polymers (80-90 o, PE). 80

89 According to the literature, conventional PTFE is very stable thermally and chemically. Beyler et al. [146] investigated, that its thermal degradation starts at temperatures exceeding 477 o C. From the other side, other fluorocarbon polymers, especially those containing tertiary carbon atoms in their structure, are less stable and degrade at temperatures below 297 o C [147]. The pptfe plasma polymers are much more branched than classical polymers and contain considerable amounts of tertiary carbon atoms (represented in the highresolution XPS by the CF groups). Although, it can be expected that the pptfe coatings have worse thermal stability than conventional PTFE, the temperature of 160 o C used for the dry heat treatment is neither sufficient to induce significant changes in chemical composition of the pptfe coatings nor to result in their weight loss. As can be seen form Figure 44, only autoclaving have caused dramatic chemical changes of the samples. It can be notice, that both the decrease of the F/C ratio and the enhancement of the C-C bonds evidence that the pptfe films become deficient with fluorine. It was considered that the release of the fluorine-bearing species is compensated from the outside, because there was no thickness lost of the film after the autoclaving. The reason for this could be oxygen contribution in to coatings during sterilization: for the as-deposited film oxygen concentration was about 1 %, but after autoclaving it increased to 5 %. Unfortunately, there is no exact information about the mechanism of such substitution as well as type of the fluorine-bearing species released. Also the data on thermal degradation of fluorocarbon polymers under purely water vapour atmosphere are scarce in the literature, yet. As a consequence, only certain speculations can be drawn. According to literature [148, 149], the main product of thermal degradation of conventional PTFE under anaerobic environment (vacuum, dry nitrogen) is tetrafluoroethylene (TFE), while oxidative pyrolysis leads to production of COF 2 as the principal evolved gas with smaller amounts of TFE, CO, CO 2, and the other species. Baker et al. [148] found in their study of the thermal degradation of commercial fluoropolymers in 50 % humid air a significant formation of hydrogen fluoride (HF). The controversial release of HF by perfluorinated (and therefore dehydrogenated) polymers was explained by the reaction of oxygen with polymer radical which leads to 81

90 the emission of COF 2 which further hydrolyzed by reaction: COF 2 + H 2 O CO 2 + 2HF (8) Autoclaving was performed, as mentioned above, at much lower temperature, with lack of oxygen and at excess of water vapours. So, it was the action of water molecules themselves that lead to the pptfe coatings partially losing fluorine and acquiring oxygen. Probably, in this process the fluorine-bearing species should be sufficiently small to be volatile. The fact that sample did not loss its weight leads to a conclusion that the release of low molecular mass fluorocarbons including TFE is unlikely during the autoclaving. The lack of oxygen should also render the release of COF 2 impossible. Thus, HF could be chosen as an appropriate candidate as an escaping species. Moreover, in this situation, hydrogen fluoride is formed rather as a result of a direct attack of H 2 O on the fluorocarbon chain and not as a product of hydrolysis of COF 2. Since the CF bond weakens with less fluorine atoms attached to carbon [137], tertiary carbon atoms aremost likely to take part in hydrolysis as they are weakest and therefore most vulnerable to such attack. The assumption of cleavage of the F atoms from the tertiary carbon atoms is supported by the decrease of the CF and C-CF bonding environments in the C 1s XPS (see Figure 44, Table 6). Finally, films of plasma sputtered nylon (psn) were investigated. It was found that they composed of O, N and C (see Table 7). Deconvolution of the C 1s peak showed consists that it could be compoused of three components: C-C/C-H bonds at ev (charging reference), collective contribution from the C-N, the C=N and the C N groups at ev and C=O, N-C=O at ev. As can be seen from Figure 45, UV sterilization did not cause any changes in the elemental content and produced the minimal changes in chemical bonding. The concentration of various CN bonds slightly decreased and the concentration of the C- C/C-H bonds insignificantly increased. Also the WCA value did not change and stayed around 47 o. More pronounced changes were, however, observed for samples both autoclaved and subjected to dry heat treatment. 82

91 Intensity [a.u.] Intensity [a.u.] Intensity [a.u.] Intensity [a.u.] a) CF3 CF2 CF C-CF WCA = 110 o C-C C-H b) CF2 CF C-CF CF3 WCA = o C-C C-H c) CF3 CF2 WCA = 83 o CF C-CF C-C C-H d) CF3 CF2 CF C-CF C-C C-H WCA = 133 o Binding energy [ev] Figure 44. High resolution XPS scans of C1 s peak of pptfe films (left) and water droplets on pptfe films (right): a) non-sterilized sample b) sample sterilized by UV radiation c) sample treated by dry heat and d) autoclaved sample 83

92 CN, C=O, N- O [%] C [%] N[%] O/N N/C C-C, C-H % % C=O, % As deposited UV radiation Dry heat Autoclave Table 7. The XPS analysis of the non-sterilized and sterilized psn film Dry heated films became more carbonized with the C-C/C-H groups reaching 44% as compared to 34% in the as-deposited samples. The total concentration of various CNgroups remained almost at the initial level (39% against 41% in the as-deposited samples) but the concentration of the carbonyl- and amide-based species decreased significantly from 25% to 16%. Although nylon sputtered films have little in common with nylon itself, certain correlation in their thermal behavior can be noticed. According to the [150], conventional nylons including nylon 6,6 do not decompose thermally below 342 o C. The temperatures above 342 o C caused cleavage of the weakest C-N and CO- CH 2 bonds and, as a result, volatile species are emitted. CO 2 and H 2 O are the main gaseous products of thermal degradation of nylon. At even higher temperature Beyler et al. [146] suggested, that the release of hydrogen cyanide (HCN). In this work, significantly lower temperature of 160 o C was applied. On the other hand, detection of slight loss of mass (see Figure 42) implies that small amount of volatile products was released as a result of thermal degradation. Moreover, as in the case of conventional nylons, the reduction of the C=O/N-C=O XPS peaks indicates, that carbon dioxide may be considered as the main escaping species. Evidently, oxidation of the psn coatings by oxygen from air does not play a significant role in dry heat treatment as it should proceed to formation of the carbonyl-based species (via peroxy- and hydroperoxy radicals) [151] and thus lead to increase of oxygen content which was not detected by XPS. However, autoclaving, performed at lower temperature than dry heat sterilization, produced the most pronounced changes in the chemical composition of the psn coatings. The decrease of the N content and simultaneous increase of oxygen was 84

93 detected by XPS. Such changes had caused an increase of O/N ratio approximately by 40% as compared to O/N ratios observed for as-deposited, UV-treated and dry heated samples. Oxidation can also be ruled out here as autoclaving is performed in water vapors without presence of air. Consequently, reactions of certain functional groups with water, i. e. hydrolysis, should be considered. Significant loss of mass observed for the psn films (see Figure 42) indicated that hydrolysis should be accompanied by the release of volatile species. It was find out from XPS analysis of the C 1s that the hydrocarbon content increases similar to the dry heat, the concentration of the C=O/N- C=O species remains constant and the concentration of the CN species decreases from initial 41 % to 31 %. It should be pointed out that the CN component of the C 1s peak is contributed by the the C-N (primary, secondary and tertiary amines), the C=N (imines) and the C N (nitriles) groups. Nevertheless, only imines are the most prone to hydrolysis by reaction: -C=N- + H 2 O -C=O +NH 3 (9) This reaction was proposed by Gengenbach [137] as a possible route of aging of aminecontaining plasma polymers in air. Such plasma polymers degraded slowly with the loss of nitrogen over extended periods of time. In this work, the mechanism of the imine hydrolysis is accelerated due to higher concentration of water and elevated temperature. According to the [151], amines and nitriles can hardly take part in hydrolysis with elimination of ammonia. Amines get partially protonated upon reaction with water and this is the final step of their hydrolysis which cannot explain the observed transformation of the XPS spectrum. Nitriles undergo hydrolysis with formation of amides and with further elimination of ammonium ions or ammonia. However, the rate of the reaction is extremely low and normally acidic or basic catalysts should be used which is not the case here. Thus, it is likely hydrolysis of imines that is predominantly responsible for the loss of nitrogen content and decrease of the CN species in the psn films during autoclaving. 85

94 Intensity [a.u.] Intensity [a.u.] Intensity [a.u.] Intensity [a.u.] a) 7000 C-N C=N C N C-C, C-H WCA = 48 o N-C=O C=O N-C=O C=O C N b) N-C=O C=O C-N C=N C-C, C-H WCA = 47 o C N c) N-C=O C=O C-N C=N C-C, C-H WCA = 45 o C-N C 288 N d) N-C=O C=O C=N C-C, C-H WCA = 80 o Binding energy [ev] Figure 45. High resolution XPS scans of C1 s peak of psn films (left) and water droplets on psn films (right): a) non-sterilized sample b) sample sterilized by UV radiation c) sample treated by dry heat and d) autoclaved sample 86

95 It should be mentioned, that hydrolysis of imines influence formation of the carbonyl-based species and also it should lead to the increase of the C=O/N-C=O component of the C 1s XPS peak. Although, it remains at the same level as in nonsterilized coatings. It was suggested, that the mechanism of thermal degradation described above for the dry heat treatment is also active at some extent in the case of autoclaving. The hydrolysis of imines supplies carbonyls, which is counterbalanced by the loss of carbonyls due to release of CO 2. Wettability measurements revealed that mentioned above chemical changes are accompanied by dramatic change of WCA values for dry heated samples. As can be seen from Figure 45, for non-sterilized, UV-treated and autoclaved samples the WCA was measured of approximately 47 o whereas for dry heated films it reached value of 80 o. This strongly correlates with lower amount of the C=O/N-C=O groups after dry heat treatment as compared to other samples. Therefore its seems that carbonyl-based species, even though being in minority, govern polarity of the surface and have strong influence on the wettability of the psn films. Futhermore, the final step of this part of the work was dedicated to the evaluation of bioadhesive properties of ppeo, pptfe and psn coatings before and after different sterilization processes. First, it has been found that the poor MG63 cell adhesion to the non-sterilized pptfe samples remained also for the UV irradiated samples and for the samples sterilized by dry heat (see Figure 46 and Figure 47). This fact is in a good aggrement with the minimal changes of the chemical composition as described above. However, significant increase of the number of adhering cells was observed for the autoclaved samples, which was even higher as compared to the polystyrene dish used as a control. Such behavior could be explained by the dramatic changes of the chemical composition of the autoclaved samples described previously (the decrease of the F/C ratio resulted in lowering of hydrophobic character of the surface). According to the literature [151], more beneficial effects on cell colonization and cell phenotypic maturation is observed for the materials with higher surface hydrophilia. This phenomenon can be explained by the fact that the surface hydrophilia, i.e. with specific amino acid sequences, serving as ligands for cell adhesion receptors, ex- posed to these receptors [60, 61]. 87

96 Not sterilized UV sterilization Autoclave Dry heat PS Figure 46. Human osteoblast-like MG63 cells in 1-day-old cultures on pptfe A) without sterilization or subjected to B) UV radiation treatment, C) autoclave treatment, D) dry heat treatment cells/cm * p<0.05 relative to not sterilzed sample * * * Figure 47. Number of adhering MG63 cells 1 day after seeding on pptfe films sterilized by different methods. For comparison the number of cells adhering on polystyrene dish (PS) is also given 88

97 This phenomenon can be explained by the fact that the surface hydrophilia caused the adsorption of cell adhesion-mediating (ECM), i.e. with specific amino acid sequences, serving as ligands for cell adhesion receptors [15]. Regarding plasma sputtered nylon, the WCA values did not correlate with the number of adhering cells. As can be seen Figure 48 and Figure 49, the as-deposited, UVtreated and autoclaved samples had the similar wettability but different numbers of attached cells. However, the correlation can be found with the total amount of the C-N, C=N and C N species. The non-sterilized and dry heated samples have almost the same (highest) concentration of the CN groups and both accumulate the largest amount of cells. Note also significantly different wettability of the two surfaces (46 against 80 ) which almost does not manifest in the number of cells attached. The number of cells and the concentration of CN are lower for the UV-treated sample. Although, the wettability of the autoclaved samples is comparable to the as-deposited ones, they contain the lowest amount of the CN groups and are least favorable for the cell adhesion. Figure 48. Human osteoblast-like MG63 cells in 1-day-old cultures on psn A) without sterilization or subjected to B) UV radiation treatment, C) autoclave treatment, D) dry heat treatment 89

98 Not sterilized UV sterilization Autoclave Dry heat cells/cm 2 PS * p<0.05 relative to not sterilzed sample * * * Figure 49. Number of adhering MG63 cells 1 day after seeding on psn films sterilized by different methods. For comparison also the number of cells adhering on polystyrene dish (PS) is given Also for the ppeo samples, the cell attachment on their surfaces also depend on by sterilization methods (seen in Figure 50 and Figure 51). It is known from [13] that the MG63 cells do not adhere to the non-sterilized films, which supports our previous reports about the non-fouling properties of such plasma polymers. The UV treatment does not influence the ability of the films to withstand the accumulation of cells either. However, dry heated and autoclaved samples lost their non-fouling character and the number of cells adhering to them approached the value observed for the polystyrene dish. These results are consistent with the change of the chemical composition of the films: enhancement of the cell adhesion is caused by the loss of the ether bonds. The critical concentration of 65-70% of the C-O-C bonds at which the non-fouling properties of ppeo coatings disappear were established previously by [13, 15]. In this work, significant accumulation of cells was showed on the autoclaved sample with 72 % of the C-O-C groups. From the other side, the dry heated samples with the lowest concentration (55%) of ethers are more cell repellent than autoclaved samples. Such 90

99 effect cannot be explained by the chemical changes and is, probably, related with other properties of the films (elasticity, flexibility of the chain segments etc). It can be concluded that the effect of various sterilization techniques on the properties of distinctly different kinds plasma polymers is of high importance with respect to their possible use in biomedical applications. First of all, it was demonstrated that sterilization methods may have strong impact on the thickness of plasma polymers: - dry heat sterilization reduced the thickness of ppeo films; - autoclaving caused decrease of the thickness of psn coatings; - the thickness of pptfe films was not changed by any of the tested sterilization methods. Figure 50. Human osteoblast-like MG63 cells in 1-day-old cultures on ppeo A) without sterilization or subjected to B) UV radiation treatment, C) autoclave treatment, D) dry heat treatment 91

100 Not sterilized UV sterilization Autoclave Dry heat PS The changes in the chemical composition of the plasma polymers induced by the sterilization depend strongly on a sterilized plasma polymer and the sterilization method. The psn and pptfe coatings were most sensitive to autoclaving due to hydrolysis whereas the ppeo films exhibited the strongest chemical changes after the dry heat treatment due to thermal degradation/oxidation. Because of that, there is no universal sterilization method that assures preservation of the properties of all kinds of plasma polymers and thus resistance of each plasma polymer towards sterilization methods has to be tested separately. cells/cm * p<0.05 relative to not sterilzed sample * * * Figure 51. Number of adhering MG63 cells 1 day after seeding on ppeo films sterilized by different methods. For comparison also the number of cells adhering on polystyrene dish (PS) is given 92

101 3.4. Study of the growth of fluorocarbon films on the substrate It is well known, that different deposition methods can be utilized for deposition of conformal ultra thin films (several nm) as well as relatively thick films (several mm thick). However, plasma deposition techniques are highly system dependent and influenced by process parameters [152]. Nevertheless, the fundamental mechanisms that explain how functional groups in the plasma bond with the substrate atoms, begin to nucleate and eventually grow into complete films are not enough experimantally proved. Consequently, study of the growth of these films is needed in order to obtain an estimation of the minimum possible thickness of the coating that will allow effective surface modification. One of the objects of this part of the work was to characterize growth of plasma polymer coatings. Also, the influence of the substrate on the chemical composition of the plasma polymer coatings was studied. This was accomplished by depositing thin plasma polymer films as a function of thickness on flat and rough surfaces and analyzing changes in chemical composition, morphology and water contact angle value. It has been already mentioned in the Introduction, that fluorocarbon films are well known for making the surface inert and hydrophobic due to the presence of -CF 2 /- CF 3 functional groups. The WCA value of rough fluorocarbon coated surfaces is around indicating super-hydrophobic surfaces. The pptfe surface has a water contact angle very close to these values. Therefore pptfe films were selected as an object for the investigation. First of all, it was found, that the pptfe coating with the thickness over 100 nm consist of the F and C atoms (see Figure 52). Other elements such as nitrogen, oxygen, detected by XPS had less than 1% of concentration in such film. Futher XPS measurements demonstrated that the chemical composition of pptfe coatings with sufficient thickness (i.e. thickness higher than the sensing depth of XPS) does not depend on the used substrate. XPS spectra of this plasma polymer were measured not only on Si wafers, but also on polypropylene (PP) foils and Ti substrates. 93

102 Intensity [a.u.] 800 F 1s F KLL 200 C 1s Binding energy [ev] Figure 52. The XPS spectrum of pptfe (100 W, 5 Pa) The same deposition conditions were preserved for all depositions, however, there were no significant differences between them as shown on Figure 53 and Figure 54. In addition, the influence of the roughness of the underlying surface on chemical composition of pptfe coatings was investigated. The RMS roughness of the substrate was changed by covering various amount of the C:H nanoparticles (deposited at 20 W and 160 Pa) with Ti film (thickness ~ 100 nm). It was found out that chemical composition of pptfe films does not depend on the underlying surface roughness, as shown in Figure 53 and Table 8. Deposition time of C:H nanoparticles, t [min] CF 3 [%] CF 2 [%] CF [%] C-CF [%] C-C [%] F [%] C [%] ,28 F/C Table 8. Dependence of the percentage of carbon bonds and elements F and C in pptfe film deposited on Ti substrate on the deposition time (t) of C:H nanoparticles 94

103 Intensity [a.u.] Intensity [a.u.] 4 -CF 3 -CF 2 -CF -C-CF a) 2 C-C b) Binding energy [ev] Figure 53. The C 1s peaks of pptfe coatings on: a) substrate with C:H nanoparticles (deposited for 8 min) covered with Ti film (~ 100 nm), b) Ti substrate Both spectra are almost identical, although, the RMS roughness of the sample a) was over 100 nm. This result is particularly important with regard to the possibility of using nanoparticles for the preparation of nanostructured pptfe coatings. As it was shown above, the chemical composition of the deposited coatings does not depend on the substrate material. However, this statement is valid only for sufficiently thick films, where the substrate does not play an important role. Study of the initial growth phase pptfe coatings (i.e. the study of chemical composition of pptfe films depending on the deposited thickness) was investigated in detail for the case of deposition on PP foils (see Figure 54). As shown in Table 9, with increase of pptfe film thickness leads to gradual decrease of C-C and C-H bonds in the spectrum - from the value of 83 % for pure PP foils to 4 % for pptfe film with a thickness of 20 nm. It is known, that XPS method can reach to the depth of ~ 10 nm of the continuous film without presence of the signal from the substrate. Hence, the presence of 4 % of 95

104 C-C and C-H bonds in the XPS spectra confirms that grown pptfe coating is not continuous. Thickness of pptfe films [nm] CF 3 [%] CF 2 [%] CF [%] C-O,C- CF [%] C-C, C- H [%] F [%] C [%] O [%] , , , ,26 F/C Table 9. The percentage of carbon bonds and elements F, C and O in pptfe film on PP foils in dependence on thickness Furthermore, it was found that the ratio between different CFx functional groups is changing with the film thickness. In the initial stages of growth pptfe coating has large amount of the CF groups (or C-CF, the peak of these bonds in the XPS spectrum is superimposed with the peak C-O) in contrast with the CF 2 or CF 3. In the coating with the thickness of 2 nm, the ratio of CF group to the CF 3 group was equal to 3.25; in the film with the thickness of 20 nm this ratio was only Apparently, the CF bonds are dominant in the coatings with low fluorine content and only with further growth of the film CF 2 and CF 3 bond appears in it. Another important parameter of the deposited pptfe coatings is their roughness, which can significantly change the interaction of deposited film with the environment (such as their wettability). Figure 55 shows that the pptfe film deposited on Si wafer with the thickness of approximately 10 nm is rather smooth (RMS roughness is less than 0.3 nm). The growth of the pptfe coating continuously as solid film without formation of islands on the substrate (which is also a possible growth mechanism of thin films) was confirmed by the measurements that were performed on various underlying films polymerized PEO (polyethylene oxide) and C:H plasma polymer. These coatings have significantly different wettability (C:H film has a contact angle of approximately 90 and 36 ppeo). AFM scans on Figure 56 shows pptfe coatings with the thickness of 2 96

105 Intensity [a.u.] Intensity [a.u.] Intensity [a.u.] Intensity [a.u.] Intensity [a.u.] nm CF3 -CF2 -CF C-O C-CF C-C C-H nm nm nm PPp foil Binding energy [ev] Figure 54. The C 1s peaks of pptfe films with different thicknesses deposited on PP foils: 20 nm, 10 nm, 5 nm, 2 nm, PP foil without pptfe 97

106 Figure 55. AFM scan of pptfe films (thickness 10 nm) deposited on Si wafers (5 Pa, 100 W, 30 s deposition time) (adapted from [153]) a) b) Figure 56. AFM scans of pptfe films (thickness 2 nm) on underlayer a) ppeo b) C:H plasma polymer (adapted from [153]) 98

107 nm deposited over ppeo and C:H films. It can be seen that even such a thin thickness the film is continuous and is able to change the contact angle of the coating to about 110 (contact angle for classic pptfe) and it does not depend on the substrate (Si wafer or underlying films of plasma polymer - ppeo and C:H). As it was already mentioned, WCA on the pptfe coatings deposited on Si wafer was around 110 (see Figure 57). Figure 57. Picture of the water drop on the pptfe film (thickness 10 nm) (adapted from [153]) It is known that significant changes in the chemical composition or morphology can cause the change of the contact angle of fabricated coatings. Therefore, WCA was also measured on the pptfe films deposited on the PP foils. On the contrary to the coatings deposited on the Si substrate, water contact angle in this case reached 120 degrees. The reason is, probably, higher value of the PP foils surface roughness than in the case of films deposited on the polished Si substrate. This part of thesis was focused on the study of growth stages of the hydrophobic thin films of pptfe plasma polymer fabricated by RF magentron sputtering of PTFE target and also influence of the substrate on their properties. As a consequence, it was revealed that the chemical composition of the coatings does not depend on the material or substrate roughness. Moreover, pptfe films with thickness of 2 nm deposited on different substrates were found to be rather smooth, and they had caused significant changes on the initial contact angle of the substrate to the value between according to the initial roughness of the substrate. 99

108 3.5. Aging of Al clusters/ C:H plasma polymer nanocomposites Nowadays nanocomposite materials are used in many current and emerging technologies. Great variety of different deposition methods were used to prepare such kind of nanocomposites which will be resistant to oxidation and corrosion. In particular, numerous researches were dedicated to the investigation of Al clusters which are widely used in technical and biological fields. Therefore, aging process of Al cluster/ C:H plasma polymers nanocomposites with general characterization of their properties was studied in this part of thesis. The main emphasis was put on nanocomposites consisting of metal and metal oxide nanoparticles covered with polymer. The polymer film is used to solve the problem of low cluster adhesion to the substrate. First, the general characterization of Al clusters prepared with various DC currents (0.2 and 0.4 A) deposited on the Si wafers and covered with thin layer of hydrocarbon plasma polymer (about 3 nm) using experimental equipment described previously in the Experimental. SEM scans (see Figure 58) and size distributions in Figure 59 revealed that the value of DC current had signicant influence both on the size of fabricated Al clusters and their amount in the nanocomposite. Probably, much higher value of DC current caused more intensive sputtering of Al atoms and therefore nucleation into clusters inside the GAS. 0.2 A 0.4 A Figure 58. SEM pictures (1µm 1µm) of Al/ C:H composites containing Al clusters prepared with different currents 100

109 Counts Counts A Diameter of the coverd clusters [nm] A Diameter of the covered clusters [nm] Figure 59. The corresponding size histogram of Al/ C:H composites containing Al clusters prepared with different current Different size of underlying Al clusters also influenced the morphology of the resulted nanocomposite. As can be seen from AFM scans (Figure 60), nanocomposites with lower amount of large Al clusters had higher value of RMS roughness (about 40 nm) in comparison with the other one with numerous smaller nanoparticles for which the RMS roughness was about 30 nm. a) b) Figure 60. AMS scans (5µm 5µm) of Al/ C:H composites containing Al clusters with different sizes: RMS a) ~ 40 nm, b) ~ 30 nm 101

110 Concentration of O, % Important parameter for possible application of Al/ C:H composites is the stability of their chemistry. Thus, it was suggested to study not only the aging process of nanocomposites, but also aging of clusters and C:H plasma polymer individually. The detailed investigation of the effect of time on the open air on the chemical properties was made for the nanocomposites with clusters fabricated with 0.4 DC current. The XPS analysis of C:H plasma polymer, Al clusters and Al/ C:H nanocomposites was made in situ and after defined time on the open air. As can be seen from Figure 61, dramatic increase of the oxygen content after 24 hours on the open air in all three cases was observed C:H plasma polymer Al clusters Al cluster/ C:H plasma polymer nanocomposites in situ 1 day on the air 7 days on the air Figure 61. Changing of oxygen concentration in C:H plasma polymer, Al clusters and Al/ C:H nanocomposites with time on the open air measured by XPS Moreover, it was found that Al clusters oxidize more rapidly then those covered even with 3 nm of plasma polymer. Apparently, thin C:H coating not only enhance the adhesion of the Al clusters to the substrate but also reduce their oxidation. The deconvolution of the high resolution Al 2p3 peak into two components (Al metal at 72.7 ev and Al 2 O 3 at 75.1 ev) revealed that there is no significant difference 102

111 Intensity [a.u.] Intensity [a.u.] Intensity [a.u.] Intensity [a.u.] Intensity [a.u.] Intensity [a.u.] between aging of the Al clusters and aluminium covered by C:H plasma polymer (see Figure 62 and Table 10) day on air Al metal Al 2 O day on air Al metal Al 2 O day on air Al metal Al 2 O day on air 2500 Al metal Al 2 O in situ Al metal Al 2 O in situ Al metal Al 2 O Binding energy [ev] Binding energy [ev] Figure 62. The Al 2p3 spectra of Al clusters (left) and Al/ C:H nanocomposites (right) with time on the open air It can be concluded that covering of Al clusters with thin layer (over 3 nm) of C:H plasma polymer significantly prevent their rapid oxidation as a ratio Al/Al 2 O 3 to 103

112 0.48 in 7 days on the open air when pure aluminium clusters oxidize to 0.33 during the same time period. In situ [Al/Al 2 O 3 ] 1 day on air [Al/Al 2 O 3 ] 7 day on air [Al/Al 2 O 3 ] Al clusters Al clusters/ C:H plasma polymers nanocomposit Table 10. The Al/Al 2 O 3 ratio in the C 1s peak for Al clusters and Al/ C:H nanocomposites 104

113 3.6. Computer simulation of filling factor of metal/ plasma polymer nanocomposites using XPS measurements Model Theoretical basis for the computer simulation for calculation of filling factor of metal/ plasma polymer nanocomposites using XPS measurements X-ray photoelectron spectroscopy is a widely used technique for surface analysis of thin films. The relative elemental composition of the coating can be directly measured from the XPS signal. It is hard to obtain the bulk properties of the film using XPS measurements because the signal comes from the depth of the first nanometers of the coating. Such complications in chemical analysis are present in the case of nanocomposites, where the "matrix" often overcoats the top layers of the "filler" and the elemental composition of the films obtained by XPS underestimates the amount of filler. If the underestimation of the amount of filler material could be quantified, it should be possible to correct the detected XPS elemental composition of nanocomposite in order to obtain a real "bulk" composition (total composition throught the full depth of the film). Thus, this part of thesis was focused on the computer simulation of the XPS data of the metal/ plasma polymer nanocomposite for the possibility to estimate of filling factor. This investigation was made in steps described below. The core of the problem of XPS analysis of nanonocomposites is linked with the dependence of the intensity of detected signal on the depth (path length of the electron to the surface). As was mentioned previously, when photons strike a surface of the sample, mainly photoelectron emission is observed. Then electrons, extracted from atoms, before reaching sample surface, travel on a certain distance accross matter. This distance depends on several parameters. Two of them are of great influence: the initial electron energy and the nature of the medium interacting with this electron. It is known, that in XPS analysis the electron kinetic energy characterizes the chemical state of the atom. Therefore, in order to obtain an accurate quantitative analysis of a surface whose composition in inhomogeneous with depth, one needs to know the depth of origin of the 105

114 measured electrons. Moreover, it becomes interesting to know the distance which an electron can traverse while keeping its kinetic energy. What value of the signal intensity of the material can be obtained if the signal weakens with path length of the electron to the surface? The distance covered by an electron between two inelastic shocks is called inelastic mean free path (IMFP) and denoted as λ. It was suggested in the 1980s, that the appropriate length scale to substitute into the quantification analysis was not the IMFP but the "attenuation length" (AL). Powell et al. [154] defined the AL as a value resulting from overlayer-film experiments on the basis of a model in which elastic electron scattering is assumed to be insignificant. Therefore, we have used the AL values in the simulation of the intensity signal of the material. Such intensity simulation was based on the Beer-Lambert law [154] which says that the intensity decays exponentially with distance traveled through the sample:, [10] where I 0 is the intensity of the incoming beam, h - Planck's constant, λ - attenuation length. It has been found that carbon predominates in the chemical composition of PEO-like plasma polymers [132] and C:H plasma polymers [121]. Therefore, approximate values of attenuation length (for metal λ is equal to 1,5 nm, for carbon (polymer) λ = 3,5 nm) were used for the simulation in the case of Ag/C ratio for the Ag/ C:H nanococmposite. This approach was also approptiate for the Au/C ratio for the Au/ PEO-like nanococmposite. As the next step, the simulation model structure of metal/ plasma polymer nanocomposite shown on the Figure 63 was taken into consideration. Structure of such 3D model was generated as follows: - all the metal particles in nanocomposite are spherical; - fixed amount of particles (20000) was placed in the layer of minimal thickness of 15 nm or 3 times the diameter of the particles; 106

115 - the area of the simulated nanocomposite layer was adapted according to the desired filling factor; - metal particles were randomly scattered in the plasma polymer matrix. 1 2 Figure 63. 3D model structure of metal/ plasma polymer nanocomposite: 1 - metal particle, 2 - plama polymer matrix Then, dependence of the apparent XPS composition of the coating on the real bulk filling factor and size of the filler nanoparticles were investigated by computer simulation of the matrix and of the filler in the form of nanospheres by MathLab2011 ( programming language. The algorithm of the simulation of the process of measurement is based on "probing" of the above described generated nanocomposite structure. The material was studied along vertical lines in an evenly spaced grid. Along each line an intersections with surfaces of the nanoparticles are calculated. From top to down the integral signal of each part of each material is calculated. The attenuation of the signal between the intersections takes into account the attenuation of the materials between previous intersections as is schematically shown in Figure 64. Calculations of simulated ratio of signal of plasma polymer matrix and metal particles were repeated for many times in order to get statistics and for various values of filling factor of nanocomposites. 107

116 Depth [nm] Finally, the results of the simulations were compared with experimental data obtained using various methods (QCM, SE) for the model of metal/ plasma polymer nanocomposite. 1 2 Intensity [a.u.] Figure D probing model for the metal/ plasma polymer nanocomposites: 1 - metal particle, 2 - plasma polymer Comparison with the experiment Two different coatings were chosen for the investigation: Au/PEO-like and Ag/C:H nanocomposites. Experimental filling factor of nanococmposites was estimated by making QCM and SE measurements and using them in equation [4] for calculation. In addition, XPS measurements of these nanocomposites were made and atomic concentrations of Au and Ag in composites from XPS spetrum were obtained. Afterwards, computer simulation of XPS results were made for Au/PEO-like and Ag/C:H nanocomposites and compared with real one. Au/PEO-like nanocomposites with different size of Au particles and their filling factors (f) which were used in simulations (see Figure 65). Table 11 represents experimentally obtained Au filling factors in Au/PEO-like nanocomposites from XPS, QCM ans SE measurements. Various Ag/C:H nanocomposites were used for the simulation as well. Different Ag particle sizes and their filling factors were taken to the account (see Figure 66). 108

117 Relative Frequency [%] Relative Frequency [%] Relative Frequency [%] a) b) c) a) 38,5% Au 25 b) 16,7% Au 20 c) 5,0% Au Particle diameter [nm] Particle diameter [nm] Particle diameter [nm] Figure 65. Au/PEO-like nanocomposites with different size of Au particles and their filling factors. Top panel - TEM images (4µm 4µm) with different f: a) 38,5 % Au, b) 16,7 % Au, c) 5,0 % Au. Bottom panel - Au particles size distribution with different f: a) 38,5 % Au, b) 16,7 % Au, c) 5,0 % Au Au filling factors in Au/PEO nanocomposites, % QCM XPS SE Table 11. Experimentally obtained Au filling factors in different Au/PEO-like nanocomposites measured by QCM, XPS and SE 109

118 Relative Frequency, % Relative Frequency, % Relative Frequency, % 0.18% 0.25% 0.33% a) b) c) a) 2,9 % Ag 30 b) 3,6 % Ag 10 c) 11,9%Ag Particle diameter [nm] Particle diameter [nm] Particle diameter [nm] Figure 66. Ag/C:H nanocomposites with different size of Ag particles and their filling factors. Top panel - TEM images (4µm 4µm) with different f: a) 0,5 % Ag, b) 2,9 % Ag, c) 3,6 % Ag. Bottom panel - Ag particles size distribution with different f: a) 0,5 % Au, b) 2,9 % Au, c) 3,6 % Au Experimentally obtained Ag filling factor in nanococmposites was measured by XPS and QCM (see Table 12). Ag filling factor in Ag/C:H nanocomposites, % QCM XPS Table 12. Experimentally obtained Ag filling factor in different Ag/C:H nanocomposites measured by QCM and XPS 110