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Pulsed Laser Deposited Metal Oxide Thin Films Mediated Controlled Adsorption of Proteins

Permanent Link: http://ufdc.ufl.edu/UFE0022878/00001

Material Information

Title: Pulsed Laser Deposited Metal Oxide Thin Films Mediated Controlled Adsorption of Proteins
Physical Description: 1 online resource (108 p.)
Language: english
Creator: Kim, Se
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: charge, pld, protein, ta2o5, wettability, zno
Materials Science and Engineering -- Dissertations, Academic -- UF
Genre: Materials Science and Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Protein adsorption experiments were conducted on metal oxide thin films in order to control or reduce adsorption. Several metal oxide thin films (Ta2O5, ZnO, binary Ta-Zn oxide film and nanostrcutured oxide film) were grown on Si substrate using pulsed laser deposition (PLD). The film properties such as cyratllinity, surface roughness, surface charge and wettability were investigated by many techniques in order to obtain the relationship with protein adsorption. Fibrinogen, BSA, and lysozyme were used as model protein in this study. The protein thickness results by ellipsometry showed that protein adsorption on oxide film surface were influenced by surface electrical charge and wettability of films. Especially, binary oxide surface having neutral charge showed the smallest thickness of proteins compared with Ta2O5 and ZnO. Nanostructured oxide film on which two oxide materials are coexisted, showed zero adsorption of lysozyme.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Se Kim.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Singh, Rajiv K.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022878:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022878/00001

Material Information

Title: Pulsed Laser Deposited Metal Oxide Thin Films Mediated Controlled Adsorption of Proteins
Physical Description: 1 online resource (108 p.)
Language: english
Creator: Kim, Se
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: charge, pld, protein, ta2o5, wettability, zno
Materials Science and Engineering -- Dissertations, Academic -- UF
Genre: Materials Science and Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Protein adsorption experiments were conducted on metal oxide thin films in order to control or reduce adsorption. Several metal oxide thin films (Ta2O5, ZnO, binary Ta-Zn oxide film and nanostrcutured oxide film) were grown on Si substrate using pulsed laser deposition (PLD). The film properties such as cyratllinity, surface roughness, surface charge and wettability were investigated by many techniques in order to obtain the relationship with protein adsorption. Fibrinogen, BSA, and lysozyme were used as model protein in this study. The protein thickness results by ellipsometry showed that protein adsorption on oxide film surface were influenced by surface electrical charge and wettability of films. Especially, binary oxide surface having neutral charge showed the smallest thickness of proteins compared with Ta2O5 and ZnO. Nanostructured oxide film on which two oxide materials are coexisted, showed zero adsorption of lysozyme.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Se Kim.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Singh, Rajiv K.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022878:00001


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PULSED LASER DEPOSITED METAL OXIDE THIN FILMS MEDIATED CONTROLLED ADSORPTION OF PROTEINS By SE JIN KIM A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1

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2008 Se Jin Kim 2

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To my wife, my parents, and other family members Their endless support made it possible for me to complete this work. 3

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ACKNOWLEDGMENTS First, I would like to express my heartfelt thanks to my advisor, Dr. Rajiv K. Singh, for his continuous support and endless guidance. I was also honored to meet and work for him. I shall never forget his endless advice and help. I also would like to express my thanks to my other committee members (Dr. Stephen J. Pearton, Dr. Hassan El-Shall, Dr. Valentin Craciun, and Dr. Ben Koopman) for their helpful advice. I would also like to express my special thanks to all of my fellow group members: Myoung Hwan Oh, Tae Kon Kim, Jae Seok Lee, Feng-chi Chang, Prushottam Kumar, Sushant Gupta and Anirrddh Khanna. I thank the staff of MAIC and PERC, and my friends for their help and sincere discussion. Finally, I would like to thank all of my family members, my parents and brothers, for their continuous trust and supports. I owed a lot to my wife (Sun Hyung) for my Ph.D study. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................8 LIST OF FIGURES .........................................................................................................................9 ABSTRACT ...................................................................................................................................11 1 INTRODUCTION..................................................................................................................13 2 LITERATURE REVIEW.......................................................................................................17 2.1 Basic Aspects of Proteins ..............................................................................................17 2.1.1 Synthesis of Protein ..........................................................................................17 2.1.2 Structure of Protein ...........................................................................................18 2.1.3 Stability of Protein ............................................................................................19 2.1.3.1 Protein Folding ...................................................................................19 2.1.3.2 Protein Denaturation ...........................................................................20 2.2 Protein Adsorption at Solid Interface ............................................................................21 2.3 Driving Force of Protein Adsorption at Solid Surface ..................................................22 2.3.1 Conformational Entropy of Proteins .................................................................23 2.3.2 Hydrophobic Interaction ...................................................................................24 2.3.3 Electrostatic Interaction ....................................................................................25 2.3.4 Other Contributions ...........................................................................................26 2.4 Resistance to Protein Adsorption ..................................................................................27 2.4.1 Current Techniques for Protein Resistance .......................................................27 2.4.2 Mechanism of protein resistance .......................................................................29 3 EXPERIMENTAL PROCEDURE.........................................................................................35 3.1 Oxide Thin Film Deposition .........................................................................................35 3.1.1 Oxide Film Growth by PLD ..............................................................................35 3.1.2 Laser Ablation System [50&57] .......................................................................36 3.2 Protein Adsorption Process ...........................................................................................39 3.3 Characterizations ...........................................................................................................39 3.3.1 Characterization of Oxide Thin Films ...............................................................40 3.3.1.1 X-ray Diffraction (XRD) ....................................................................40 3.3.1.2 X-ray Photoelectron Spectroscopy (XPS) ..........................................40 3.3.1.3 Contact Angle Measurement ..............................................................41 3.3.1.4 Zeta Potential () Measurement ..........................................................42 3.3.2 Evaluations of Adsorbed Protein Surface .........................................................42 3.3.2.1 Ellipsometry ........................................................................................43 3.3.1.3 Atomic Force Microscopy (AFM) ......................................................43 5

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4 OXIDE THIN FILMS-MEDIATED CONTROLLED ADSOPTION OF PROTEINS.........49 4.1 Introduction ...................................................................................................................49 4.2 Experimental .................................................................................................................51 4.2.1 Preparation of Oxide Thin Films ......................................................................51 4.2.2 Protein Adsorption Experiment ........................................................................51 4.3 Results and Discussion ..................................................................................................52 4.3.1 Characterization of Oxide Thin Film ................................................................52 4.3.2 Adsorption of Proteins ......................................................................................53 4.4 Summary .......................................................................................................................55 5 INFLUENCE OF THERMALLY ANNEALED OXIDE THIN FILMS ON FIBRINOGEN ADSORPTION..............................................................................................63 5.1 Introduction ...................................................................................................................63 5.2 Experimental .................................................................................................................64 5.2.1 Preparation of Annealed Oxide Thin Films ......................................................64 5.2.2 Fibrinogen Adsorption ......................................................................................65 5.2.3 Surface Sensitive Techniques ...........................................................................65 5.3 Results and Discussion ..................................................................................................65 5.3.1 Annealing Effect on Properties of Oxide Thin Films .......................................65 5.3.2 Annealing Effect on Fibrinogen Adsorption .....................................................68 5.4 Summary .......................................................................................................................69 6 BINARY TA-ZN OXIDE THIN FILM-INDUCED REDUCTION OF PROTEINS ADSORPTION.......................................................................................................................77 6.1 Introduction ...................................................................................................................77 6.2 Experimental .................................................................................................................78 6.2.1 Preparation of Binary Oxide Thin Films ...........................................................78 6.2.2 Protein Adsorption Experiment ........................................................................79 6.3 Results and Discussion ..................................................................................................79 6.3.1 Characterization of Ta-Zn Oxide Thin Films ....................................................79 6.3.2 Adsorption of Proteins on TZ Oxide Film ........................................................81 6.3.3 Comparison of Protein Adsorption ...................................................................82 6.4 Summary .......................................................................................................................82 7 PULSED LASER ABLATED NANOSTRUCTURED OXIDE FILMS-MEDIATED ZERO ADSORPTION OF LYSOZYME...............................................................................91 7.1 Introduction ...................................................................................................................91 7.2 Experimental .................................................................................................................92 7.3 Results and Discussion ..................................................................................................93 7.4 Summary .......................................................................................................................95 8 CONCLUSION.......................................................................................................................99 8.1 Effect of Surface Charge on Protein Adsorption ..........................................................99 6

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8.2 Annealing Effect on Fibrinogen Adsorption .................................................................99 8.3 Effect of Binary Ta-Zn Oxide Films on Protein Adsorption ......................................100 8.4 Influence of Nanostructured Oxide Films on Lysozyme Adsorption .........................100 LIST OF REFERENCES .............................................................................................................101 BIOGRAPHICAL SKETCH .......................................................................................................108 7

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LIST OF TABLES Table page 3-1 Pulsed laser deposition parameters of metal oxide thin films ............................................45 4-1 Surface roughness data of adsorbed-protein samples by AFM .........................................56 5-1 Comparison in properties between as grown and annealed oxide films ............................70 6-1 Summarized properties of three oxide thin films ...............................................................84 8

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LIST OF FIGURES Figure page 1-1 Process of blood coagulation due to protein adsorption ....................................................16 2-1 General formula of amino acid ..........................................................................................30 2-2 Simple schematic of peptide bond .....................................................................................30 2-3 Example of four levels of protein structure .......................................................................31 2-4 Process of folding and denaturation of protein ..................................................................32 2-5 Simplified chronology of protein adsorption to a solid surface in aqueous solution .........32 2-6 Structure of a peptide unit in a polypeptide chain .............................................................33 2-7 Hydrophobic interaction ....................................................................................................33 2-8 Gouy-Stern model of an electrical double layer ................................................................34 2-9 Examples of several techniques for reducing protein adsorption ......................................34 3-1 The UVPLD system with excimer laser and optics setup ..................................................46 3-2 Formation of thin film by laser ablation ............................................................................46 3-3 Laser energy distribution during target ablation ................................................................47 3-4 Contact angle of liquid on horizontal solid surface described by youngs equation .........48 3-5 Schematic diagram of ellipsometry experiment ................................................................48 4-1 Grazing incidence X-ray diffraction pattern for as grown oxide thin films......................57 4-2 SEM images of as grown oxide thin films .........................................................................57 4-3 Images of a deionized water drop on oxide thin films .......................................................58 4-4 Zeta potential curve as a function of pH ............................................................................59 4-5 Thickness vs time graph of adsorbed proteins onto TaO surface in pH 7.4 solution. 2 5 .....60 4-6 Thickness vs time graph of adsorbed proteins onto ZnO surface in pH 7.4 solution .......60 4-7 The 3-D AFM images of protein-attached Ta2O5 surfaces ...............................................61 4-8 The 3-D AFM images of protein-attached ZnO surface ....................................................62 9

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5-1 AFM images of Oxide films before and after air annealing at 800C. ..............................71 5-2 Grazing incidence XRD spectra of two oxide films ..........................................................72 5-3 X-ray photoemission spectroscopy of two oxide films ......................................................73 5-4 Photographs of water contact angle on two oxide film surfaces .......................................74 5-5 Thickness vs time graph of adsorbed fibrinogen onto as grown and annealed TaO surface in pH 7.4 solution 2 5 ..................................................................................................75 5-6 Thickness vs time graph of adsorbed fibrinogen onto as grown and annealed ZnO surface in pH 7.4 solution ..................................................................................................75 5-7 The RMS roughness variation of oxide thin films during fibrinogen adsorption ..............76 6-1 X-ray diffraction patterns of (a) target material and (b) binary Ta-Zn oxide film. ...........85 6-2 The XPS spectra of as grown binary Ta-Zn oxide thin films ............................................86 6-3 The SEM (A) and AFM (B) images of binary TZ oxide films ..........................................87 6-4 Zeta potential vs pH curve (A) and water drop images (B) of TZ oxide films ..................88 6-5 Adsorption density of proteins vs time on Ta-Zn binary oxide film .................................89 6-6 Comparison graph of Fibrinogen thickness onto three oxide films in pH 7.4 solution .....89 6-7 Comparison graph of BSA thickness onto three oxide films in pH 7.4 solution ...............90 6-8 Comparison graph of lysozyme thickness onto three oxide films in pH 7.4 solution .......90 7-1 Structures of nanostructured oxide thin films ....................................................................96 7-2 Contact angle variation of nanostructured oxide films ......................................................96 7-3 The XPS spectra of nanostructured oxide films ................................................................97 7-4 Thickness diagrams of adsorbed lysozyme on nanostructured oxide films .......................98 10

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy PULSED LASER DEPOSITED METAL OXIDE THIN FILMS MEDIATED CONTROLLED ADSORPTION OF PROTEINS By Se Jin Kim December 2008 Chair: Rajiv.K. Singh Major: Materials Science and Engineering Several metal oxide thin films were grown on Si substrate by pulsed laser deposition for controlling adsorption of proteins. No intentional heating of substrate and introduction of oxygen gas during growth were employed. Additionally, fibrinogen, bovine serum albumin (BSA), and lysozyme were used as model protein in this study. The film properties such as cyratllinity, surface roughness, surface electrical charge and chemistry were investigated by many techniques in order to obtain the relationship with protein adsorption. Firstly, as grown Ta 2 O 5 and ZnO thin film were used to study the effects of surface charge on the behaviors of BSA and lysozyme adsorption. The protein thickness results by ellipsometry showed that negatively charged Ta 2 O 5 had a stronger affinity to positively charged lysozyme, while positively charged ZnO had a stronger affinity to negatively charged BSA. The results confirmed electrostatic interaction due to surface charge is one of main factors for determining adsorption of proteins. Furthermore, annealing studies were performed by heat treatment of as grown Ta 2 O 5 and ZnO at 800C in air ambience. Annealed Ta 2 O 5 thin film had almost wetting property (from 10.02 to less than 1~2) and the change of cystallinity (from amorphous to cyrsalline) while 11

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annealed ZnO thin film had a reduced contact angle (from 75.65 to 39.41) and remained to crystalline structure. The fibrinogen thickness on annealed Ta 2 O 5 film was increased compared with as grown sample, while heat treated ZnO film showed much reduction of fibrinogen adsorption. Binary Ta-Zn oxide thin films (TZ) were grown by preparing PLD target composed of 50 wt% Ta 2 O 5 and 50 wt% ZnO. This binary film had IEP pH 7.1 indicating nearly neutral charge in pH 7.4 PBS solution, and hydrophilic property. Ellipsometrical results showed that TZ film had the lowest fibrinogen, BSA and lysozyme thickness after 120 min adsorption compared with Ta 2 O 5 and ZnO. Other samples, bilayer oxide films in which Ta 2 O 5 and ZnO coexist were also employed to study adsorption behaviors. Especially, Ta 2 O 5 -based bilayer films revealed zero adsorption of lysozyme. 12

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CHAPTER 1 INTRODUCTION The biological response to artificial materials is partially dictated by the adsorption of proteins from human body (Figure1-1). Nonspecific adsorption and bio-fouling, which lead to uncontrollable and adverse biological consequences, occur because many proteins adsorb spontaneously and simultaneously onto synthetic foreign materials. Therefore, control over protein adsorption is required for many applications of biomedical and bloodcontacting devices [1-2]. Currently, over 54 million blood contacting biomaterials and devices are used each year in the US. However, since blood coagulation due to nonspecific protein adsorption is one of the greatest challenges, research into blood-compatible and protein-resistant surfaces have been aggressively pursued. The fabrication of protein-resistant surfaces is of considerable interest for a number of applications. Recent applications include materials for use in implanted devices and contact lenses and systems for patterned cell cultures, tissue regeneration, and drug delivery. With the recent intense development of new biomedical applications, there is a great need to develop new biomaterials with novel structures and properties [3]. In particular, the complete avoidance of adsorption of all proteins has been the subject of numerous studies because non-specific adsorption and fouling can lead to false response and degradation of the blood contacting devices over time. Since the early 1990s, many researchers have investigated protein-resistant materials to optimize the protein compatibility of surfaces or develop new and better surface coating techniques suitable for biological purposes [4]. Most of them focused on understanding biocompatible properties of polymeric biomaterials and already reported many hydrophilic polymer-based materials as a suitable protein-resistant surface layer [5-9]. Among them, poly 13

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(ethylene glycol) (PEG) is the most commonly used material because of its exceptional biocompatibility [10]. PEG shows excellent resistance to nonspecific protein binding. However, even though PEG has big advantages, this polymeric material has several limitations [3]: PEG can only be functionalized at the chain ends, and the polymer is not biodegradable. For many biomedical applications, biodegradability and flexibility to incorporate desired functionalities are critical. Furthermore, PEG oxidizes relatively rapidly, especially in the presence of oxygen and transition metal ions, and also has low adsorption to oxide surfaces due to its net neutral charge. Therefore, complex surface modifications are required to functionalize PEG. Furthermore, despite extensive research, the molecular mechanism for PEGs excellent protein-resistant ability remains to be fully understood. Surface modification is another way of providing blood compatibility. Protein adsorption phenomena depend to a large extent on surface characteristics such as electric charge, hydrophobicity, morphology, roughness and chemistry [11]. Proteins easily attach to hydrophobic materials or surfaces with opposite electric charge. Surface morphology and roughness can affect the hydrophobicity. Control of surface properties could help gain understanding of fundamentals of protein adsorption, which could facilitate development of protein-resistant surface. Recently, use of metal oxide thin films has received increased attention for wide ranges of applications.Ta 2 O 5 and ZnO thin films have been extensively investigated for their excellent electrical, magnetic and optical properties [13-15]. Recently, these oxide materials have been used in bio-related applications such as coating of implants, biosensors and biomedicines due to their good biocompatibility and chemical stability [16,17] However, studies of inhibition of protein adsorption by using metal oxide coatings are lacking. 14

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Ta 2 O 5 and ZnO have opposite surface charges at physiological pH, because isoelectric points of these materials are approximately pH 2.8 to 2.9 and pH 8.7 to 9.3, respectively [22]. Ta 2 O 5 is hydrophilic and ZnO is relatively hydrophobic, thus differing in surface wettability [23]. Fabrication of well controlled oxide film surfaces of complementary surface charge and wettability offers a promising method for reducing protein adsorption. In our study, one promising route to achieve well controlled surfaces and to overcome the limitations of polymer-based materials was investigated. This route is pulsed laser deposited metal oxide thin films. Deposition of oxide films is relatively simple and oxide surface is less sensitive to the presence of oxygen than polymeric materials, and functionalization of coating material is not needed. Our study investigated and compared the behaviors of protein adsorption on single oxide film layer such as Ta 2 O 5 and ZnO. Another single layer oxide surface, binary Ta-Zn oxide film is studied for its effect on reducing adsorption of proteins. Furthermore, the adsorption behavior onto nanostructured oxide thin films such as ZnO-based or Ta 2 O 5 -based thin films is also presented. With these oxide surfaces, we can easily control surface charge and wettability by means of using different oxide materials because individual oxide surface has its inherent surface property. 15

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red blood Figure 1-1. Process of blood coagulation due to protein adsorption cell fibrin and protein platelet thrombus adsorption adhesion formation fibrin platelet 16

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CHAPTER 2 LITERATURE REVIEW 2.1 Basic Aspects of Proteins The word protein comes from the Greek word proteious which means of the first rank or importance. Protein is indeed important for life and is involved in every biological process within body [12]. The average human body is approximately 18% protein. Proteins are large complex molecules with molecular weights ranging from 1000 to over 1,000,000 Da. All proteins are made up of varying numbers of amino acids attached to together in a specific sequence and having a specific architecture. The sequence and structure of the amino acids differentiates one protein from another, and gives the protein special physiological and biological properties. In this part, simple basic structure and properties of protein will be covered [12]. 2.1.1 Synthesis of Protein Proteins are organic compounds that contain nitrogen in addition to carbon, hydrogen and oxygen. The subunits of proteins are called amino acids. Amino acids are characterized by the presence of an amino (NH 2 ) group with basic properties and a carboxyl (COOH) group with acidic properties, attached to the same carbon atom, the -carbon. The rest of the molecule varies with the particular amino acid [13]. The structure of an amino acid may be represented by the formula (Figure 2-1). The -carbon is also bonded to hydrogen and to the side chain group, which is represented by the letter R. The R group determines the identity of the particular amino acid. In the formation of protein, amino acids are linked together by the peptide linkage in which amino group of one amino acid is linked to the carboxyl group of another, with the elimination of a molecular of water (Figure 2-2). The resultant chain of amino acids therefore has an amino 17

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group at one end (the N-terminus) and a carboxyl group at the other end (the C-terminus) [14]. These amino acid chains are called peptide or proteins. Proteins fall into the general class of polymers, which are simply linear molecules built up from simple repeating units, the monomers. In the case of proteins, the monomers are the amino acids; 20 different amino acids are used. In one sense, proteins are more complex than most polymers in that 20 different monomers are used in their construction. There is more to a protein than the number of amino acids that makes it up. A protein is a polypeptide chain (a chain of up to 100 amino acids) that has attained a unique, stable, three-dimensional shape (referred to as its native conformation), and is biologically active as a result. 2.1.2 Structure of Protein Proteins are long, unbranched chains of amino acids that fold up into complex shapes because of attractive and repulsive forces between the R groups of different kinds of amino acids in the polypeptide chain. Scientists describe the structure of a protein on four levels of complexity: primary, secondary, tertiary, and quaternary levels (Figure 2-3) [15]. The primary structure of proteins is the sequence of amino acids in the polypeptide chain. This determines protein properties and may be thought of as a complete description of all of the covalent bonding in a polypeptide chain or protein. Secondary structure is the ordered arrangement or conformation of amino acids in localized regions of a protein molecule. This structure occurs when the sequence of amino acids are linked by hydrogen bonds. The two main secondary structures are the alpha helix and the anti-parallel beta-pleated sheet. There are other periodic conformations, but the -helix and -pleated sheet are the most stable. A single polypeptide or protein may contain multiple secondary structures. 18

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The tertiary structure of a protein is the three-dimensional arrangement of the atoms within a single polypeptide chain. The formation of tertiary structure is usually driven by the hydrophobic interaction among the R groups of the different amino acids, but other interactions such as hydrogen bonding, ionic interaction and disulfide bonds can also stabilize the tertiary structure. The tertiary structure encompasses all the noncovalent interactions that are not considered secondary structure and is usually indispensable for the function of the protein. Quaternary structure is used to describe proteins composed of multiple subunits (multiple polypeptide molecules). Most proteins with a molecular weight greater than 50,000 consist of two or more noncovalently-linked monomers. 2.1.3 Stability of Protein The unique native structure is for most proteins a basic requirement for proper functioning. It was demonstrated in many cases that the ability to build up and to keep this native and functional structure in a particular range of temperature (as well as of pH, of pressure, and of salinity) is an intrinsic property of the protein itself which is already determined by the amino acid sequence. Sometimes, native conformation proteins undergo denatured state because of a variety of reagents and conditions. Denaturation occurs because the bonding interactions responsible for the secondary structure (hydrogen bonds to amides) and tertiary structure are disrupted. In tertiary structure there are four types of bonding interactions between "side chains" including hydrogen bonding, salt bridges, disulfide bonds, and non-polar hydrophobic interactions, which may be disrupted. 2.1.3.1 Protein Folding The amino acid sequence of a protein predisposes it towards its native conformation. It will fold spontaneously during or after synthesis. Protein folding is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure [16]. 19

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Each amino acid in the chain can be thought of having certain gross chemical features. These may be hydrophobic, hydrophilic or electrically charged. These interact with each other and their surroundings in the cell to produce a well-defined, three dimensional shape, the folded protein known as the native state. The resulting three-dimensional structure is believed to be determined by the sequence of amino acids and has lowest energy level. Most folded proteins have a hydrophobic core in which side chain packing stabilizes the folded state, and charged or polar side chains on the solvent-exposed surface where they interact with surrounding water molecules. It is generally accepted that minimizing the number of hydrophobic side-chains exposed to water is the principal driving force behind the folding process. In addition to hydrophobic interaction, the process of the protein folding is mainly guided by formation of intramolecular hydrogen bonds, and van der Waals forces, and it is opposed by conformational entropy [17-19]. 2.1.3.2 Protein Denaturation In certain solutions and under some conditions, proteins will not fold into their biochemically functional forms. Denaturation of proteins involves the disruption and possible destruction of both the secondary and tertiary structures. Since denaturation reactions are not strong enough to break the peptide bonds, the primary structure (sequence of amino acids) remains the same after a denaturation process [20-22]. Denaturation disrupts the normal alpha-helix and beta sheets in a protein and uncoils it into a random shape. Protein denaturation has been defined in several ways, for example as a change in solubility or by simultaneous changes in chemical, physical and biological properties under some standard reference set of conditions. Denatured proteins can exhibit a wide range of characteristics, from loss of solubility to communal aggregation. Communal aggregation is the phenomenon of aggregation of the hydrophobic properties to come closer and form the bonding between them, 20

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so as to reduce the total area exposed to water [23-25]. The denaturation process can be achieved by any one of the following methods: increasing temperature, changing pH, using denaturants, inorganic salts, organic solvents, detergents, high pressure and ultrasonic homogenization. However, in many proteins (unlike egg whites), denaturation is reversible in that the proteins can regain their native state when the denaturing influence is removed (Figure 2-4). 2.2 Protein Adsorption at Solid Interface Protein adsorption at interface is involved in a wide variety of phenomena natural and man-made systems. It gives great impact in medical, environmental and biotechnological processes. Protein adsorption can be disadvantageous such as in biofouling of intraand extracorporeal devices [26-27] and of processing equipment. In other cases, proteins are adsorbed on purpose, e.g. in drug targeting and drug delivery systems [28], biosensors [29-30], in vitro immunoassay [31] immobilization of enzymes in biocatalysis [32] and stabilization of dispersions [33]. The existence of compact structure of protein molecules in an aqueous environment requires that the importance of the decreased conformational entropy of the folded state is determined by the net enthalpy and entropy of the various structure including intraand intermolecular interactions. Introduction of a sorbent surface in Figure 2-5, which is more or less hydrophobic and which is usually electrically charged, causes a shift in the delicate balance of the interactions. This may lead to spontaneous adsorption, possibly accompanied by structural rearrangements in the protein molecules [2, 34]. In step (1), protein transports to the energetic boundary layer where the potential at the surface influences the rate of approach of the protein (including diffusion through the stagnant boundary layer), and in step (2), interaction and attachment of the 21

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protein with the surface which may involve perturbations in protein structure. Relaxation of the adsorbed protein to its steady-state conformations step occurs in step (3). In this study, step (2) will be focused, which are primarily controlled by direct forces such as electrostatic and hydrophobic forces between the protein, the sorbent surface, solvent (water) molecules and other ions in the interfacial region. Whatever the mechanism of the process is, protein adsorption under conditions of constant temperature (T) and pressure (P) only occurs spontaneously if the change in the Gibbs energy (G) of the system is negative. According to equation 2-1, this can be realized by a decrease in the enthalpy H and/or an increase in the entropy S. ads G = ads H T ads S .... 2-1 The ads indicates the change in the thermodynamic functions of state resulting from the adsorption process. The irreversible nature of the protein adsorption process eliminates the possibility of direct measurement of the overall driving force for adsorption, ads G, as well as determination of ads S. This leaves ads H and the heat capacity change upon adsorption, ads C p as the only directly measurable thermodynamic parameters describing the irreversible adsorption process. Regretably, such data are severely limited for protein adsorption systems. However, an answer to the question why proteins prefer interfaces may thus be found by considering which interactions contribute to ads G. 2.3 Driving Force of Protein Adsorption at Solid Surface Protein adsorption is the net result of the various interactions between and within the system components, which include the sorbent surface, the protein molecules, the solvent (water) and any other solutes present such as low molecular mass ions. The interactions recognized as 22

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occurring in protein adsorption are mostly noncovalent. Examples of covalent adsorption (as opposed to intentional covalent immobilization) are rare. The origins of these interactions include Van der Waals forces (i.e. dispersion), hydrogen bonding forces, electrostatic forces (including ion pairing) and more entropically based effects such as the hydrophobic interaction (at least under ambient conditions) [35-41]. Clearly, intermolecular interactions between, for example, protein and surface, solvent and surface, or solvent and solvent are important in protein adsorption. In addition, intramolecular forces between atoms and residues within the protein macromolecules may contribute to ads G. Thus, understanding of the driving force for protein adsorption helps us achieve well-made protein-resistant/ adsorptive sample. 2.3.1 Conformational Entropy of Proteins Most globular proteins contain 40% ~ 80% of ordered secondary structure. These structures are predominantly found in the interior of the molecule where they are stabilized by hydrogen bonds between peptide units and also by hydrophobic interaction between amino acid side groups. Hydrogen bonding between peptide units largely restricts the rotational mobility around the bonds in the polypeptide backbone and, consequently, reduces the conformational entropy of the protein (Figure 2-6). Additional losses of conformational entropy result from the freezing of other parts of the polypeptide backbone, and probably some side groups, into densely packed random structures in the interior of the protein molecules. The entropy loss due to the folding into the native state is considered to be a characteristic of a given protein independent of environment and temperature. 23

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Hence, -T confo S becomes proportionally more positive with increasing temperature. As a result, the native structure becomes less stable at high temperature and, the change of conformational entropy can affect the adsorption of proteins. 2.3.2 Hydrophobic Interaction The notion hydrophobic interaction stems from the observation that solubility of non-polar solutes in water is extremely low. When there are two regions containing hydrophobic substances, each of the substances is excluded from the water matrix. Over a period of time, the two areas of hydrophobic substances will encounter one another, combine and form one larger hydrophobic region that is excluded from the water matrix (Figure 2-7). This combined state is more energetically favorable than the one in which the hydrophobic substances were separate. Thus this combined state will persist. For example, when you add some drops of oil to water, the drops combine to form a larger drop. This comes about because water molecules are attracted to each other and are cohesive because they are polar molecules. Oil molecules are non polar and thus have no charged regions on them. This means that they are neither repelled nor attracted to each other. The attractiveness of the water molecules for each other then has the effect of squeezing the oil drops together to form a larger drop. Since it looks like the oil molecules are avoiding the water, this type of interaction is called a hydrophobic interaction. If contacting surface is hydrophobic, dehydration of that surface would promote protein adsorption. It has been estimated that dehydration of hydrophobic surface results in a reduction in the Gibbs energy which is mainly due to entropy increase. Therefore, from the hydrophobicity phenomenon, if the sorbent surface is hydrophilic, hydration of water molecules at sorbent surface will resist the adsorption of proteins. 24

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2.3.3 Electrostatic Interaction The interaction between two charged atoms or molecules is potentially the strongest form of physical interaction to be considered at interfaces and in colloidal systems. For two point charges Q1 and Q2, the free energy of interaction w(r) is given by rezzrQQrw022102144)( .2-2 where 0 is the permittivity of a vacuum or free space, is the relative permittivity or dielectric constant of the medium, and r is the distance between the two charges. The right-hand form of the equation is commonly used, where the value of Q can be readily specified in terms of the sign and valency of each ion, z, and the elementary charge, e (= 1.602 X 10 -19 C). The force of the coulombic interaction, F c is the differential with respect to r of the free energy ()CdwrFdr 20221202144rezzrQQ ....... 2-3 According to Coulombs law, for two charges of the same sign, both w(r) and F will be positive, which means that the interaction will be repulsive; for unlike charges they will be attractive. Both the protein molecules and the sorbent surface are electrically charged in aqueous environment. These charged species are surrounded by counter-ions, which, together with the surface charge form the so-called electrical double layer. Close approach between the protein molecules and the surface implies overlap of the electrical double layers and, hence, redistribution of ions. As shown in Figure 2-8, within the charge-free regions 0 < X < d, potential (x) drops linearly with x. This layer is called Stern layer. For the region X d, Gouy assumed that 25

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counterions to the charged surfaces are exponentially distributed. This led to the well-known diffuse double layer. When the charges on the protein and the surface, respectively, have opposite signs these components would attract each other, at least as long as these charges more or less compensate each other. If one of the components carries a great excess of charge, it would result in a considerable net amount of charge in the contact region between the protein layer and the surface upon adsorption. This non-aqueous, proteinaceous region has a low dielectric permittivity relative to that of bulk water and, therefore, accumulation of net charge in such an environment is energetically very unfavorable. A similar situation would result on adsorption of a charged protein molecule on a sorbent surface that has the same charge sign. Nevertheless, even under such electrostatically adverse conditions, proteins often adsorb spontaneously. 2.3.4 Other Contributions Most peptide unit in a proteins backbone contains a hydrogen donor (>NH) and a strong proton acceptor (>C=O), the basic constituents of a hydrogen bond. For a series of globular proteins, it is found that 88% of all peptide amide groups and 89% of all peptide carbonyl groups are involved in hydrogen bonds and some amino acid side-chains can participate in hydrogen bonds as well. However, their contribution to the total number of internal hydrogen-bonds is typically small. Clearly, hydrogen bonds make important contributions to secondary and tertiary structures of proteins. Van der Waals attractions arise from interactions between fixed or induced dipoles. They are very sensitive to the distance r between atoms, varying as 6 r Since atomic packing densities are unusually large in the interiors of proteins, Van der Waals attractions should favor the folded conformation. However, this interaction hardly influences adsorption of proteins from 26

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an aqueous solution because the Hamaker constant for proteins is only slightly larger than that for water. 2.4 Resistance to Protein Adsorption The ability to resist nonspecific protein adsorption is a major indicator of a materials biological inertness or biocompatibility. Protein repelling surfaces are used, for example, as substrates for cell culture, and as coatings for contact lenses [3, 4]. More recent applications include systems for patterned cell cultures, tissue regeneration, microfluidic systems, drug delivery, and systems for high-throughput screening of proteins or cells [42]. To optimize the protein compatibility of surfaces or to develop new and better surface coatings suitable for suppressing nonspecific protein adsorption, a fundamental understanding of mechanisms of protein resistance is required. 2.4.1 Current Techniques for Protein Resistance Since the early 20 th century, many researchers have investigated a lot of materials including heparin, Polysaccharide, phosphorylcholine (PC), propane sulfone (PS), thiolated dextran, and polyethylene glycol (PEG) for protein resistance. Many of their studies of resistant surfaces have been focused on improving the biocompatibility of materials for biomedical applications; hence, the majority of these works has been performed with bulk polymers or thin polymeric films. Among them, PEG is the most commonly used material due to its exceptional biocompatibility. Even though there are PEGs limitations, it has been used as a surface coating to prevent the adsorption of proteins and cells in buffered aqueous solution [43]. Furthermore, several techniques [44] for reducing protein adsorption are currently tried and used (Figure 2-9). Self assembled monolayers (SAMs) are surfaces consisting of a single layer of molecules on a substrate and have been intensively studied with respect to their interfacial properties. Regarding investigation of biocompatibility, SAMs provide an ordered, predictable 27

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and monotonous surface with defined end-group functionality, chemical specificity and density. A variety of terminal groups was seen to provide protein resistance. Rather than having to use a technique such as CVD or MBE to add molecules to surface, SAMs can be prepared simply by adding a solution of the desired molecule onto the substrate and washing off the excess. Polyelectrolytes are polymers whose repeating units bear an electrolyte group. These groups will dissociate in aqueous solutions, making the polymers charged. Polyelectrolyte multi-layers provide new coating opportunities and are obtained by simply dipping charged solid substrate into polyelectrolyte solution. As dipping occurs in alternating sequence into solutions of opposite charge, multilayer films are grown. These may be applied to complex geometries as represented by tissue engineering scaffolds, catheters or filtration membranes, all of which require low fouling. Such films have been the focus of increasing attention. Biomimetic surfaces are variously attempted to present cell membrane-like contact zones for tissue and blood interaction or a tissue matrix-type of reactive surface for controlled cell deposition and organization. The end requirement may be either to have defined, high levels of cell attachment without adverse toxic or inflammatory changes or to fully avoid protein attachment. Lipid bilayer membrane (BLM) is a zone of a membrane composed of lipid molecules (usually phospholipids) and constitutes the ultimate boundary modifying structure capable variously of controlled protein attachment and complex cellcell interactions. Their inherent fluidity is one factor in their ability to control interfacial reactions. In the case of natural BLM, the presence of orientated and topographically organized arrays of complex membrane proteins confers the further modality of site-specific surface affinity. Background non-specific binding 28

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is largely rejected, and an essential reason for this natural biocompatibility is the presence of phosphorylcholine head groups. Hydrogels with their high water content provide elastomeric materials for both reservoir and barrier function, notably for drug delivery cell encapsulation and soft contact lenses. Their bulk swelling and hydration have dominated reports, but they do, nevertheless, have surface attributes which influence their interfacial biocompatibility. 2.4.2 Mechanism of protein resistance Adsorption involves a number of interactions and steps, but is not mechanistically well-defined. In many cases, a combination of attractive components causes adsorption. In most cases of nonspecific adsorption, proteins adsorb onto surfaces irreversibly by process that do not lend themselves to detailed kinetic treatment. Individual proteins often exhibit remarkable differences in their kinetics of adsorption. The adsorbed proteins can undergo conformational changes that increase interactions (especially hydrophobic interactions) between the proteins and the surfaces [45]. Despite extensive research, the molecular mechanism for protein resistance remains to be fully solved. Many researchers believe that the interaction of the surface with water is a key component of the problem [44]. To generate structure-property correlation and to discover new structures which are resistant to protein attachment, Whitesides and Mrksich groups prepared self-assembled monolayers presenting substrates with various functional groups to test the protein resistance of those substrates. Through their studies, Whitesides and co-workers observed the following common properties among protein resistant substrates: (i) they are hydrophilic, (ii) they include hydrogen-bond acceptors, (iii) they do not include hydrogen-bond donor, (iv) their overall electrical charge is neutral [3, 43]. (ii) and (iii) properties are not generally applicable to inorganic oxide surface. 29

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Figure 2-1. General formula of amino acid H H Figure 2-2. Simple schematic of peptide bond C COOH R NH2 C COOH R NH2 H H C CO R NH2 C COOH R -NH H2O 30

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Figure 2-3. Example of four levels of protein structure 31

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Folded state Denatured state Figure 2-4. Process of folding and denaturation of protein Native-state conformation Transport to interfacial region Attachment to surface Steady-state perturbed structure (3)Further structural Rearrangements surface Figure 2-5. Simplified chronology of protein adsorption to a solid surface in aqueous solution 32

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Figure 2-6. Structure of a peptide unit in a polypeptide chain. The shaded bond is fixed. Two ( and ) of the three (back bone) bonds are free to rotate, unless intramolecular hydrogen bonds are formed; R and R represent amino acid side-groups. Figure 2-7. Hydrophobic interaction C C O C H R N R H H 33

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o Figure 2-8. Gouy-Stern model of an electrical double layer [26]. Figure 2-9. Examples of several techniques for reducing protein adsorption. A) Self-assembled monolayer (SAM), B) Polyelectrolyte multi-layers, C) Biomimetic surface, D) lipid bilayer, and E) hydrogel A B C D E m d 34

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CHAPTER 3 EXPERIMENTAL PROCEDURE Preparation of adsorbed protein layer on metal oxide thin films deposited by pulsed laser ablation method includes the fabrication of Ta 2 O 5 ZnO and binary Ta 2 O 5 -ZnO PLD targets, the deposition of various oxide thin films, and the process of proteins adsorption. Characterization of this work includes the properties of oxide films, thickness measurements of adsorbed protein layer on oxide films and topographical analysis. 3.1 Oxide Thin Film Deposition An extensive variety of film growth techniques with vacuum chamber, such as molecular beam epitaxy (MBE), chemical vapor deposition (MOCVD & PECVD), thermal evaporator and sputter deposition have been used to grow oxide thin films [46-49]. A relatively simple technique, pulsed laser deposition (PLD) which provides the stoichiometry of target to deposited film and a fast response in exploiting new material systems, has been used very successfully, despite its limitations for large-area deposition [50-53]. In the present study, single layer metal oxide films such as Ta 2 O 5 [54] and ZnO [55], and binary Ta 2 O 5 -ZnO (TZ) [56] were grown by pulsed laser deposition method in an ultra-high vacuum compatible chamber utilizing an excimer laser system. 3.1.1 Oxide Film Growth by PLD Figure 3-1 shows a schematic diagram of the PLD system, which was used in the present work. Solid oxide targets were prepared by hydraulic press using 99.99% Ta 2 O 5 and 99.99% ZnO powder respectively and TZ target was composed of 50 wt% Ta 2 O 5 powder and 50 wt% ZnO powder. After pressing, targets were sintered at 1300C for 4hrs in the air atmosphere and cooled in the furnace. 35

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Silicon (001) substrates that cleaned by acetone, methanol, HF (10%) and deionized water, which are loaded into substrate holder inside the main growth chamber. This chamber was a stainless steel vessel which can be reached into 10 -6 Torr within 2 hours with a help of mechanical and turbomolecular pumping system. No intentional substrate heating was employed and substrate was rested on platter during the film growth. Ultra-high-purity O 2 gas flow was used to kept constant vacuum level of 0.3 Torr throughout the film growth. During deposition, target material was rotated and nanosecond KrF excimer (248nm) laser was used with laser energy density of 3.3 J/cm 2 and frequency of 5 Hz. Due to the lack of a loadlock for the system, the chamber was backfilled with nitrogen to bring it to atmosphere each time a sample was unloaded. 3.1.2 Laser Ablation System Pulsed laser deposition (PLD) is a thin film deposition technique where a high power pulsed laser beam is focused inside a vacuum chamber to strike a target of the desired composition. Material is then vaporized from the target and deposited as a thin film on a substrate. This process can occur in ultra high vacuum or in the presence of a background gas, such as oxygen which is commonly used when depositing oxides to full oxygenate the deposited films [50&57]. Many techniques are available for the deposition of thin films onto a substrate, however pulsed laser deposition has several key advantages that make it an ideal small batch research tool. PLD provides near-stoichiometric transfer of molecules from the target to the substrate, rapid testing of many different materials, and offers a wide range of applications, though it does not lend itself well for industrial scale production. In general, the method of pulsed laser deposition is simple. Only few parameters need to be controlled during the process. Targets used in PLD are small compared with other targets used in other sputtering techniques. It is quite easy to produce multi-layer film composed of two or more 36

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materials. Besides, by controlling the number of pulses, a fine control of film thickness can be achieved. Pulsed laser deposition, in contrast to the simplicity of the system set-up, is a very complex physical phenomenon. It not only involves the physical process of laser-material interaction with the impact of high-power pulsed radiation on solid target, but also the formation of plasma plume with high energetic species and transfer of the ablated material through the plasma plume onto the substrate surface. The thin film formation process in PLD generally can be divided into the following four stages (Figure 3-2); (1) Laser radiation interaction with the target, (2) Laser-plasma plume interaction, (3) Plasma expansion, (4) Nucleation and growth of a thin film on the substrate surface. Each stage in PLD is critical to the formation of good quality, stoichiometric, uniform and small surface roughness thin film. The ablation process itself is characterized by an input of energy from the laser to a given target material. Figure 3-3 shows a schematic of the energy distribution at the target surface during ablation. The total energy for the system is represented in equation 3-1. E = E r + E p + E d + E c ..... 3-1 E = incident energy E r = reflected energy E p = plasma plume energy E d = energy of disintegration E c = energy absorbed by the cavity wall There are additional factors related to the laser energy that play an important role in surface response to pulse energy. The incident pulse energy must exceed the ablation threshold energy for the material for ablation to occur. If the pulse energy is less than the ablation threshold, material will not be physically removed from the surface (i.e., E p and E d tend to zero), but that energy will be absorbed by the cavity wall (E c ) as heat, which can be useful for laser annealing 37

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applications. The degree to which the target material is ablated depends on the extent the energy is exceeded. If excessive energy is used, large particulates from the target can be blown off. This is unwanted for high-quality films. A Lambda Physik LPX 305 I KrF excimer laser was used to deposit all the samples in the following experiments. This model laser uses a pulsed mode delivering 25 nanosecond duration square shaped wave pulses at frequencies from 1-50 Hz and output energies from 10-1100 mJ yielding ultimate fluences from 0.1-4 J/cm 2 depending on optics used. The laser is fired or triggered by either an internal computer module near the laser or from a remote external computer placed near the deposition chamber. This laser uses a mixture of Kr, F, and a buffer gas, typically Ne or He. High voltages ranging from 16-23 kV elevate electrons in the Kr and F atoms into excited states causing excited KrF* complexes to form. Upon their decay, 248 nm single wavelength radiation is given off. A series of lenses and an aperture are used to select the highest energy portion of the beam, direct, collimate, and focus the pulse until it impinges the sample. For the PLD system, the laser beam first passed through a 2 cm x 1 cm aperture to select the center portion of the spot from the laser. This eliminates the lower energy diffuse side portions of the spot and gives the initial rectangular spot shape. Once reflected, the incident beam is passed through a collimating lens to keep the radiation from diverging due to scattering as it passes through the ambient air. One more mirror is used to reflect the beam to the chamber. The beam then is passed through a focusing optic with a 30 cm focal length placed just outside of the chamber. The beam enters the chamber through a fused silica window port rated for use with excimer radiation. This window passes 90% of 248 nm radiation. 38

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The laser ablation spot impinging the target had dimensions of 2 x 6 mm, maintaining the shape of the rectangular aperture used at the start of the beam path. Substrate was placed with 6cm distance from the target and with an angle of 45 from the target. 3.2 Protein Adsorption Process Protein adsorption onto different oxide thin film surfaces were conducted with 3 kinds of protein solution. Fibrinogen from bovine plasma, Albumin from bovine serum (BSA) and lysozyme from chicken egg white were purchased from Sigma and used without further purification: fibrinogen, a large blood plasma protein (MW 340kDa, IEP pH 5.5-5.8) that is part of the coagulation cascade of protein and adsorbs strongly to hydrophobic surfaces, BSA (MW 66kDa, IEP pH 4.6-4.7) that makes up to approximately 60% of the total protein in serum, and lysozyme, a small protein (MW 14.3kDa, IEP pH 11.2) that is positively charged under the conditions of this experiment [48,58]. The proteins were dissolved in diluted phosphate buffer saline (PBS) solution. When it (raw PBS) is diluted to a 1X concentration, this product will yield a phosphate buffered saline solution with a phosphate buffer concentration of 0.01M and a sodium chloride concentration of 0.154M and the solution pH will be 7.4. All adsorption experiments were carried out with 0.1mg/ml protein solution at room temperature. This solution was dropped onto oxide surfaces and remained to 10, 30, 60, and 120 min, respectively. After adsorption, samples were cleaned 2 times with deionized water and, PBS solution and then were blown dry with a stream of nitrogen gas. 3.3 Characterizations The information about the properties of metal oxide thin films used as substrates is given in Section 3.3.1. The information about thickness measurements and some evaluations of adsorbed protein is given in Section 3.3.2. 39

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3.3.1 Characterization of Oxide Thin Films Because the surface properties of oxide films are critical factors for determining adsorption behaviors, oxide surface analysis should be preceded to understand the effect of surface characteristics on protein adsorption. In this work, metal oxide thin films grown by PLD will be characterized using a variety of methods. 3.3.1.1 X-ray Diffraction (XRD) X-ray diffraction (XRD) is a non-destructive tool for studying material properties such as crystallinity, strain, and grain size. Also, the phase identification and orientation can be easily analyzed with this technique. The incident X-rays interact with the periodic crystal planes and results in constructive and destructive interference. Only the condition satisfying the Braggs Law gives the constructive interference. n = 2dsin .................................................3-2 where n is the order of diffraction, is the x-ray wavelength, d is the interplanar spacing of crystal planes, and is the incident angle of x-ray. X-ray diffraction analysis has been performed on the samples to examine the crystalline structure of the oxide target materials. In addition to providing a quick measurement of crystalline quality, XRD analysis also yields information on the crystalline lattice structure as well as phase identification. Phillips APD 3720 x-ray diffractometer system using Cu K ( = 1.5405) will be used for quick evaluation of target materials. In addition, grazing incidence-angle XRD (GIXRD) will be carried out using Xpert system since diffraction peaks originating from the underlying oxide layers can not be obtained from conventional XRD method. 3.3.1.2 X-ray Photoelectron Spectroscopy (XPS) X-ray photoelectron spectroscopy (XPS) is an analytical technique used to investigate the chemical state, elemental composition, and electronic state at the sample surface. Monoenergetic 40

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X-rays irradiate a material and core electrons are ejected from the sample surface. These ejected electrons have characteristic binding energy which can be determined by the Rutherford equation. E binding = E photon E kinetic where E photon is the characteristic energy of X-ray photon, E kinetic is the kinetic energy of ejected photoelectron, and is the work function of the spectrometer. Each element can be identified with this specific binding energy of these ejected photoelectrons. The number of detected photoelectrons is related to the element concentration within the irradiated material surface. XPS was used to evaluate compositional analyses and chemical bonding of oxide thin layer in this study. In addition, this method can confirm the phase change between as-grown and heat treated oxide layer, and measure metal atom/ oxygen atom concentration ratio of oxide thin films. 3.3.1.3 Contact Angle Measurement The contact angle is the angle at which a liquid/ vapor interface meets the solid surface. The contact angle is specific for any given system and is determined by the interactions across the three interfaces. Mostly, this concept is illustrated with a small liquid droplet resting on a flat horizontal solid surface. Youngs equation is the basis for a quantitative description of this phenomenon (Figure 3-4). SLSVLV cos where LV is liquid-vapor surface tension, SV is solid-vapor surface tension, SL is solid-liquid surface tension, and is contact angle. Contact angle measurement is widely used for studying surface wettability because it is a simple and sensitive method for evaluating surface chemistry changes. In this study, contact angles were measured by the sessile drop technique using a Rame-Hart 100 goniometer under ambient conditions. Three different regions on the surface of oxide sample were measured using 41

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goniometer. A 2 Ldrop of ultrapure water (> 18Mcm) was deposited to the surface and contact angle value based on drop image was calculated by Image J software. 3.3.1.4 Zeta Potential () Measurement The zeta potential is a very important property of charged solid-liquid interfaces. From a theoretical viewpoint, zeta potential is electrical potential in the interfacial double layer at the location of the slipping plane versus a point in the bulk fluid away from the interface. In other words, zeta potential is the potential difference between the medium solvent and the stationary layer of fluid attached to the dispersed particle. Streaming potential measurements were used to determine the zeta potential data with pH and isoelectric points (IEP) of oxide thin films. The Paar Physica Electro Kinetic Analyzer measures the voltage drop across a flat plate over which the conductive aqueous solvent is forced under pressure. The zeta potential of the surface is calculated based on the flow of the solvent (1mM KCl), the streaming potential, the current, and the conductivity of the solution as the solvent flows across the sample material [59-60]. This potential is very sensitive to the pH of solvent and the pH where the potential is zero is called isoelectric point. 3.3.2 Evaluations of Adsorbed Protein Surface When metal oxide thin films are introduced in protein solution, proteins are adsorbed onto them naturally with time. At this point, the kinetics of adsorption of proteins is frequently measured because it is useful method for evaluating biocompatibility of oxide surface. Furhtermore, the variation of surface topography due to protein adsorption is one of main interest. In this study, several surface sensitive techniques such as ellipsometry and AFM are applied in order to evaluate adsorbed protein-surface. 42

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3.3.2.1 Ellipsometry Ellipsometry is an optical technique for measuring the thickness and optical properties of extremely thin films, or even organic layers. The measurable properties are the refractive index, or how much light is bent, and the level of light absorption, called the absorption coefficient. Ellipsometer works by shining a well-defined source of light on a material and capturing the reflection. Electromagnetic radiation is emitted by a light source and linearly polarized by a polarizer, it can pass an optional compensator, and falls onto the sample. After reflection the radiation passes a compensator (optional) and a second polarizer, which is called analyzer, and falls into the detector. Upon the analysis of the change of polarization of light, which is reflected off a sample, ellipsometry can yield information about layers that are thinner than the wavelenth of the probing light itself, even down to a single atomic layer or less (Figure 3-5). Measurements for thickness of adsorbed protein onto oxide surfaces in this study were performed using a variable angle spectroscopic ellipsometer from J.A. Woollam. This optical technique was operated as a function of both wavelength and angle of incidence light. Measurements were performed from 2000 A to 9000 A at an angle of incidence of 70. To determine thickness of adsorbed protein, cauchy optical model was used [61-63]. The (Root) Mean Squared Error (MSE) is used to quantify the difference between the experimental and predicted data. A smaller MSE implies a better model fit to the data. Since the MSE value for all actual experiments was less than 2, the thickness of protein was thought as reliable data. Results are the average of 4 measurements on respective oxide layer and protein-adsobed layer. 3.3.1.3 Atomic Force Microscopy (AFM) Atomic force microscope (AFM) was used to characterize the surface topography and morphology of the films. AFM operates by measuring an interatomic force between a sharp tip at the end of the cantilever and the sample. The interactomic force contributes to the cantilever 43

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deflection. As the tip scan the sample surface, the cantilever bends by responding to the topography of the sample surface. The position of the laser beam on the detector reflecting the position of the cantilever, shifts with displacement. As a result, the position sensitive photodiode generates topographic data by sensing this cantilever deflection. Measurements for surface roughness evaluation were performed using a VEECO Dimension 3100 Atomic Force Microscope by digital instrument. All imaging was conducted in the tapping mode, with 256 256 data acquisitions as a scan speed of 1 Hz at room temperature in air. Etched silicon tips with integrated cantilevers with a nominal spring constant of 20-100 N/m were used. Various roughness parameters, such as the RMS roughness (R q ) and average roughness (R a ) were used to understand surface topographies of oxide layers and adsorbed protein samples. 44

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Table 3-1. Pulsed laser deposition parameters of metal oxide thin films Oxide target material Ta 2 O 5 ZnO, TZ (50% Ta 2 O 5 + 50% ZnO) Target diameter 2.54 cm Background gas pressure Ultra high purity O 2 0.3 Torr Substrate Si wafer (1cm X 1cm) Substrate temperature Room temperature Laser energy 600 mJ Laser frequency 5 HZ Distance between target and substrate 6 cm Film thickness 80 120 45

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Figure 3-1. The UVPLD system with excimer laser and optics setup Figure 3-2. Formation of thin film by laser ablation Vacuum Chamber UV Transport Window KrF Excimer La se r Oxide Target Substrate Plume Optical Lens To vacuum pump unit substrate target (3) (4) (2) (1) 46

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Figure 3-3. Laser energy distribution during target ablation [12] 47

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Figure 3-4. Contact angle of liquid on horizontal solid surface described by youngs equation Figure 3-5. Schematic diagram of ellipsometry experiment solid LV vapor liquid SV SL Detector Light source Polarizer Analyzer Compensator Compensato Sample 48

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CHAPTER 4 OXIDE THIN FILMS-MEDIATED CONTROLLED ADSOPTION OF PROTEINS 4.1 Introduction It is well known that proteins spontaneously adsorb to foreign solid surfaces. Protein adsorption at solid-liquid interfaces is an important process for blood contacting devices, biomedical applications such as medical implants, biosensors and drug delivery, and non-fouling surfaces [64-67]. When proteins from blood are exposed to solid surfaces, subsequent unnecessary reactions due to protein adsorption are activated. Avoiding adsorption of proteins when biomaterials are introduced into human body has been one of big issues for the success of bio-applications [68-69]. Therefore, the control over protein adsorption at solid interface has been of considerable interest for a number of blood-contacting applications. Various interactions are involved in a protein adsorption process. These complex interactions between proteins and solid surfaces include electrostatic interaction, hydrophobic interaction, van der Waals force, and hydrogen bonding [2, 35, 70-74]. Especially, electrostatic interaction due to electrical surface charge and hydrophobic interaction due to hydration change between proteins and biomaterials are important. Moreover, adsorption to solid surface is determined by surface physical or chemical properties such as electrical charge and wettability, the net charge of proteins and pH of the medium [75-79]. Many methods and materials have been prepared to reduce or control the adsorption of proteins and especially, hydrophilic polymeric-based materials such as poly (ethylene glycol) have been of high attention due to their excellent adsorption resistance. However, even though they showed unsurpassed blood compatible ability, several limitations should be solved; complex 49

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surface modification for strong bonding between solid surface and polymer, oxidation control of polymer, and biodegradability to incorporate desired functionality. Recently, metal oxide materials such as TiO 2 ZnO, Ta 2 O 5 and HfO 2 have been studied and received much attention due to their good blood compatibility, corrosion resistance and mechanical properties for blood-contacting applications. Thin films using these oxide materials are not sensitive to oxidation during adsorption and complex modification process of surfaces is not needed. Furthermore, since these oxide surfaces have positive or negative charge in aqueous solution depending on their natural isoelectric points (IEP) [80-81] and inherent wettability, it is possible to control of protein adsorption by using electrostatic and hydrophobic interaction between proteins and oxide films. In this study, we describe the adsorption characteristics of two proteins. The first protein is bovine serum albumin (BSA), which is the most abundant plasma protein and the second one is lysozyme from chicken egg white that is commercially valuable enzyme. It is important to investigate the effects of different surface properties, i.e., electrical charge and wettability. According to research paper and document, isoelectric point of BSA and lysozyme is pH 4.6~4.7 [82] and pH 11.2 [83], respectively. Therefore, BSA is negatively charged in pH 7.4 solution and lysozyme is positively charged. Here we focus on the influence of surface electrical charge and relationship of different charged oxide surfaces (ZnO and Ta 2 O 5 ) with the adsorption of proteins. Ellipsometry will be used for evaluating and characterizing protein adsorption by measuring the thickness of adsorbed proteins on oxide film surfaces. Because it is non-destructive and quick to perform, it is currently used for characterization of the deposition and growth mode of thin organic films including protein films [84-86]. Atomic force microscopy (AFM) is a powerful 50

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technique used for obtaining the surface topography and roughness on a scale of nanometers before and after protein adsorption [87-88]. 4.2 Experimental 4.2.1. Preparation of Oxide Thin Films The two metal oxide thin films were grown on silicon (001) substrates using pulsed laser deposition system (PLD). The substrates were cleaned prior to growth using acetone, methanol and deionized water for 5 minutes, respectively. Natural oxide layer on silicon surface is removed by hydrofluoric acid (10%) for 60 seconds. Oxide targets prepared by high purity oxide powder 99.99% Ta 2 O 5 and ZnO, respectivelywere used for the ablation experiments using a pulsed KrF laser, which provides a beam with 248 nm wavelength, 600 mJ pulse energy and at a repetition rate of 5 Hz. Both oxide films were deposited to thickness of 80-120 at a room temperature under 3.0 10 -1 Torr oxygen pressure. 4.2.2. Protein Adsorption Experiment Albumin from bovine serum (A1933, MW 66kDa) and lysozyme from chicken egg white (L6876, MW 14.3kDa) were purchased from Sigma and used without further purification. The proteins were dissolved in phosphate buffer saline (PBS) solution (pH 7.4) and all adsorption experiments were carried out with 0.1 mg/ml protein solution at a room temperature. This solution was dropped onto oxide surfaces and remained to 10, 30, 60, and 120 minutes, respectively. After adsorption, samples were cleaned 2 times with deionized water and, PBS solution. Subsequently, they were blown dry with a stream of nitrogen gas. 51

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4.3 Results and Discussion 4.3.1 Characterization of Oxide Thin Film Because the surface properties of oxide films are critical factors for determining adsorption behaviors, surface analysis should be preceded to understand the effect of surface properties on protein adsorption behaviors. Figure 4-1 displays a grazing incidence XRD pattern of two as grown oxide films by PLD. The X-ray pattern obtained from tantalum oxide film shows that there are no distinct peaks and this film is amorphous structure. During deposition at room temperature of substrate, it is not easy for atoms to diffuse and bond between Ta ions and O ions in tantalum oxide material with complex stoichiometry [53, 89]. The strong and sharp diffraction peak in zinc oxide film revealed this film is grown with (002) preferential orientation and relatively well crystallized. Figure 4-2 shows the morphology of two as grown thin films on Si substrates (at 100,000 magnification) obtained by SEM. These images confirmed that tantalum oxide surface is much coarser and agglomerated. Furthermore, we found that the surface of zinc oxide thin film is much smoother than tantalum oxide because the borders of images on ZnO are not clear even though the thicknesses of both films are similar. Wettability of as grown oxide films was also checked by measuring contact angle. The drop images are shown in Figure 4-3. The measured results showed that the contact angle of tantalum oxide surface was 10.1 2 which indicates that this oxide has a hydrophilic property and a high surface energy. However, the angle of zinc oxide surface was 75.7 2 which indicates that the surface has a low surface energy and a relatively high hydrophobicity. The zeta potential versus pH curves of two metal oxide films are plotted in Figure 4-4. In these curves, the isoelectric points (point at which the zeta potential is zero) of the tantalum 52

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oxide and zinc oxide thin films are at approximately pH 2.9 and pH 8.5 respectively. In addition, the plots can be used to predict pH values at which the oxide sample can be stable and the sign of electrical charge of oxide surface at certain pH. When the oxide surface are suspended to a lower pH solution than its isoelectric point, the oxide film is positively charged by adsorbing protons on its surface while the protons are desorbed and oxide is charged negative in a higher pH solution. Therefore, the surface of tantalum oxide film has a negative charge in pH 7.4 solution and zinc oxide has a positive charge at same pH. Comparing properties of two different oxide films, tantalum oxide thin film is amorphous and negatively charged in pH 7.4 solution, and it has higher roughness and hydrophilic property. While zinc oxide film has crystalline structure and relatively hydrophobic property, and surface of it is smoother and positively charged. 4.3.2 Adsorption of Proteins The kinetics graphs of adsorbed proteins on oxide thin films obtained by ellipsometry are shown in Figure 4-5 and 4-6. As can be seen, the kinetics of protein adsorption is similar for both graphs. Initially, the trend of adsorption thickness is steep. However, the amount of protein adsorption on the oxide surface begins to stabilize with time. This trend is much clear in tantalum oxide surface. In Figure 4-5, after 10 minutes of adsorption time, the thickness of BSA and lysozyme was approximately 25.8 3 and 69.2 4 respectively and final protein thickness was 48.7 3 and 78.2 4 respectively. The difference of thickness between BSA and lysozyme on tantalum oxide surface may be due to primarily electrostatic interaction between the protein and the oxide surface. According to Coulombs law, the surface of tantalum oxide film with negative charge can attract positively charged lysozyme more easily. Ellipsometrically obtained thickness on the oxide surfaces can be used to influence of electrical surface charge on protein adsorption behaviors 53

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Figure 4-6 shows the thickness variation of adsorbed proteins on the zinc oxide film. Unlike data of tantalum oxide surface, the thickness trend of the adsorbed protein on zinc oxide was opposite. After 10 and 120 minutes of adsorption time, the thickness of BSA was approximately 69.1 3 and 332.1 4, respectively while lysozyme thickness was 55.6 3 and 133.3 3 respectively. As shown in this graph, the positively charged zinc oxide surface showed a stronger affinity to BSA, but less so with lysozyme, which is a positively charged protein. Again, the electrostatic force is one of the main factors governing the protein adsorption in this system. Even though the surface charge between proteins and oxide surface is critical in these adsorption systems, we also need to consider oxide surface wettability such as the surface energy and the hydrophobicity. For the positively charged lysozyme system, thickness of adsorbed protein in zinc oxide surface is a little higher than the tantalum oxide, even though lysozyme and the zinc oxide surface have the same charge. From this result, it is important to note that the hydrophobic effect is also involved in adsorption process. This is consistent with other observation [74-75] that proteins are attached to a hydrophobic surface more easily than a hydrophilic surface. AFM measurements were carried out to visualize the surface nano-topography and measure roughness of protein-adsorbed surface. Figure 4-7 displays the 3-D images of adsorbed proteins onto tantalum oxide surfaces. These images can show visible differences in morphology and roughness of surfaces with respect to adsorption. As grown sample without adsorption has RMS roughness with 7.86 nm. During laser ablation for tantalum oxide film, larger particles were ejected from the target material and formed a thin film onto the substrate. Thus, the film surface has a relatively high roughness. Interestingly, after 120 minutes of adsorption process, the roughness of obtained image was less than that of no adsorption. The detailed roughness data of 54

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the various surfaces are presented in Table 4-1. The data show that the roughness value for Ta 2 O 5 system was decreased as adsorption time increased, while ZnO system responded in opposite manner. This can be explained by the fact that the starting tantalum oxide film had a relatively high surface roughness and adsorption of proteins would make rougher surface become smoother by filling in grooved areas. Figure 4-8 also contains representative AFM images of adsorbed BSA and lysozyme on the zinc oxide films. The morphology and roughness of smooth zinc oxide surfaces were largely changed after 120 minutes of adsorption process. The increased roughness introduced by the protein adsorption is evident from AFM images in the fig. 4-8. From the roughness data, values were increased with adsorption time. With increased adsorption time, the roughness data of ZnO surface increased from 1.5 nm to 6.34 nm for BSA and 3.75 nm for lysozyme. 4.4 Summary We used metal oxide thin films grown by PLD to investigate adsorption behaviors of proteins. Adsorption kinetics of BSA and lysozyme for tantalum oxide and zinc oxide thin films were studied using ellipsometry and AFM methods. The thickness and roughness data obtained by two techniques showed that the adsorption of proteins is generally increased with time. We also found that surface charges of oxide films play an important role in controlling the amount of adsorbed proteins. Negatively charged tantalum oxide thin films were found to behave as expected when positively charged protein is introduced into an aqueous PBS solution. Less BSA was attached to tantalum oxide surface than lysozyme. However, in the positively charged ZnO system, more BSA was adsorbed onto the zinc oxide surface than lysozyme. Furthermore, when we take the lysozyme adsorption into consideration and compare the thickness data of two samples, surface hydrophobicity may be another key factor for understanding the behavior of protein adsorption. 55

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Table 4-1. Surface roughness data of adsorbed-protein samples by AFM Surface roughness (nm) Tantalum oxide film Zinc oxide film BSA Lysozyme BSA Lysozyme Adsorption time (min) Rq Ra Rq Ra Rq Ra Rq Ra 0 7.86 6.97 7.86 6.97 1.55 1.21 1.55 1.21 10 5.40 4.11 4.64 3.57 2.27 1.85 2.39 1.92 30 5.13 3.87 4.43 3.42 4.88 3.87 2.75 2.16 60 4.33 3.35 3.73 2.73 5.89 4.68 3.22 2.59 120 4.08 3.15 3.60 2.65 6.34 5.06 3.75 3.01 56

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Figure 4-1. Grazing incidence X-ray diffraction pattern for as grown oxide thin films. A) Ta 2 O 5 and B) ZnO A B Figure 4-2. SEM images of as grown oxide thin films. A) Ta 2 O 5 surface and B) ZnO surface 57

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A B Figure 4-3. Images of a deionized water drop on oxide thin films. A) Ta 2 O 5 surface and B) ZnO surface 58

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A B Figure 4-4. Zeta potential curve as a function of pH. A) Ta 2 O 5 thin film and B) ZnO thin film 59

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Figure 4-5. Thickness vs time graph of adsorbed proteins onto Ta 2 O 5 surface in pH 7.4 solution. Figure 4-6. Thickness vs time graph of adsorbed proteins onto ZnO surface in pH 7.4 solution 60

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A B C Figure 4-7. The 3-D AFM images of protein-attached Ta2O5 surfaces. A) as grown Ta 2 O 5 B) 120 min. adsorption of BSA, and C) 120 min. adsorption of lysozyme 61

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A B C Figure 4-8. The 3-D AFM images of protein-attached ZnO surface. A) as grown ZnO, B) 120 min. adsorption of BSA, and C) 120 min. adsorption of lysozyme 62

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CHAPTER 5 INFLUENCE OF THERMALLYU ANNEALED OXIDE THIN FILMS ON FIBRINOGEN ADSORPTION 5.1 Introduction Metal oxide materials have attracted much attention for utility in wide range of industry. Especially, well controlled ZnO and Ta 2 O 5 thin films have been frequently chosen for development of electronic, photonic and optical applications [90-91]. They also have exhibited good mechanical property, chemical stability and biocompatibity [92-95]. These biological properties have led to study the behaviors of protein adsorption on metal oxide films. Extensive investigations for protein adsorption over past decades have shown that this is one of key challenges in development of many biological applications including medical implants and blood contacting devices. The adsorption of proteins occurs naturally when foreign solid materials come into contact with blood, followed by cell attachment [96-100]. Furthermore, the physical and chemical characteristics of substrate surface are detrimental to adsorption behaviors [2, 70-71, 74, 101-102]. Many researchers have tried to control the adsorption of proteins on solid surface because nonspecific adsorption and biofouling can damage implants and devices. For reducing amount of adsorption, considerable studies have been focused to the substrate modification using hydrophilic polymers as poly (ethylene glycol). Even though much progress in resisting adsorption has been made, the interaction between proteins and solid surfaces are still not fully understood because of complexity of adsorption process. The protein used in this study is fibrinogen, which is a major plasma protein. Its molecular weight is 340 kDa and isoelectric point is in the range from pH 5.5~5.8 [103-104]. It plays an important role in blood coagulation and regulation of both haemostasis and thrombosis [105-106]. Furthermore, the fibrinogen adsorbs nonspecifically onto both hydrophobic and hydrophilic 63

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surfaces, following denaturation and reorientation on the solid surfaces [107]. To improve and affect the physical and chemical properties of the oxide thin films, a furnace annealing technique is applied in this experiment. Generally, post-deposition annealing at suitable temperature has been known to be able to reduce the oxygen vacancies and other organic impurities [49]. Additionally, the heat treatment is also an effective method for influencing properties and improving the qualities of oxide thin films. We thermally annealed as grown Ta 2 O 5 and ZnO thin films deposited by PLD and studied the variation of film properties such as phase transition, surface chemistry and roughness. In order to clarify the effects of changes of oxide film characteristics on the fibrinogen adsorption, the relationships between the adsorption and the oxide films before and after annealing will also be reported using the surface sensitive techniques. 5.2 Experimental 5.2.1 Preparation of Annealed Oxide Thin Films The Ta 2 O 5 and ZnO thin films in this work were deposited on silicon (100) substrates using pulsed laser deposition system (PLD). The substrate surface was cleaned in ultrasonic baths using acetone, methanol and deionized water. Prior to the growth, natural oxide layer on the silicon surface is removed by hydrofluoric acid (10%) for 60 seconds. Oxide targets prepared by high purity oxide powder 99.99% Ta 2 O 5 and ZnO, respectivelywere used for the ablation experiments using a pulsed KrF laser with 600 mJ pulse energy with a repetition rate of 5 Hz. Both oxide films were grown to a thickness of 80-120 at a room temperature under 3.0 10 -1 Torr oxygen pressure. To understand the effects of annealed oxide films on protein adsorption, the heat treatment was performed in air atmosphere at 800 C for 30 min to complete the preparation of films after deposition. 64

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5.2.2 Fibrinogen Adsorption Fibrinogen from bovine plasma (A4753, MW 340 kDa) was purchased from Sigma and used without further purification. The protein powder was first dissolved in phosphate buffer saline (PBS) solution (pH 7.4) and all the adsorption experiments were carried out with 0.1 mg/ml protein solution at a room temperature. This solution was dropped onto the oxide film surfaces and remained there for 10, 30, 60, and 120 min. After adsorption, samples were cleaned twice using deionized water and, PBS solution. Subsequently, they were blown dry with a stream of nitrogen gas. Dried samples were then transferred to ellipsometry and AFM to evaluate the behaviors of fibrinogen adsorption. 5.2.3 Surface Sensitive Techniques Ellipsometry is an optical surface sensitive technique for studying the behaviors of a protein adsorption occurring at solid surfaces [62, 108]. In this study, measurements for thickness of the adsorbed fibrinogen were performed using a variable angle spectroscopic ellipsometer from J.A. Woollam. Cauchy optical model (N=A+B/ 2 +C/ 4 ) was used to determine thickness of the adsorbed protein. The surface topography of the oxide films and the protein-adsorbed samples was investigated using AFM method [109-110]. The changes to Topographical and surface roughness after protein adsorption were studied with a Digital Instruments Nanoscope III system. All images were obtained in the tapping mode, with 256 256 data acquisitions as a scan speed of 1 Hz at a room temperature in air. 5.3 Results and Discussion 5.3.1 Annealing Effect on Properties of Oxide Thin Films Figure 5-1 shows the amplitude AFM images of as grown and annealed oxide samples at 800C for 30 min. The scan area is 1 m 1 m, and scale bar of image is 200nm for Ta 2 O 5 and 65

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50nm for ZnO, respectively. Even though both Ta 2 O 5 and ZnO have a similar film thickness with 8~10nm, the film surface for Ta 2 O 5 is much coarser than ZnO. Compared with as grown sample, the film surface of annealed Ta 2 O 5 becomes much smoother (RMS of from 7.86 nm to 6.97 nm) and reveals the dramatic change in film surface; large clusters appear on the annealed surface in the case of Ta 2 O 5 Whereas, annealed ZnO shows relatively larger cluster than as grown surface, and no big change in surface roughness (RMS of from 1.55 nm to 1.51 nm). These AFM images indicate that the surface morphologies of both annealed oxide films had been improved and surface roughness had decreased due to high temperature annealing, which could help metal atom (Ta or Zn) and O atom diffuse on their lattice sites. To investigate the structure of thermally annealed films, an XRD analysis was performed. Figure 5-2 A and B display the XRD spectra of Ta 2 O 5 and ZnO films before and after annealing, respectively. As shown in this figure, as grown Ta 2 O 5 film has a amorphous structure, while annealed film has a polycrystalline structure showing three distinct diffraction peaks with (001), (1 11 0), and (1 11 1). The spectra of as grown and annealed ZnO films showed that these are poly crystalline hexagonal wurtzite structure, with dominant (002) C-axis orientation. Furthermore, two diffraction peaks with small intensity such as (100) and (101) were observed in the spectra of annealed ZnO. The position of the (002) peak is shifted from 34.35 to 34.49. As a result of evaluating the average grain size of ZnO films using the Scherrer formula [111], grain size of annealed sample was increased from 19.65 nm to 24.11 nm. An increase in intensity of (002) peak and a decrease in full width at half maximum (FWHM) value confirm that annealing process improved the quality and crystallinity of ZnO film. 66

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XPS spectra of both oxide films before and after annealing are measured and the results are compared. Figure 5-3 A illustrates the Ta 4f high resolution spectra in two different Ta 2 O 5 films. Even though the binding energy of Ta metal atoms in both films is almost same, the spectrum of annealed surface shows that the chemical state of Ta is the fully oxidized Ta 2 O 5 state [112]. This is confirmed by the fact that the atomic ratio of Ta : O was 1 : 4.7 before annealing and 1 : 6.5 after annealing. Figure 5-3 B shows XPS of Zn LMM. In this spectrum, the shoulder peak became relatively weaker and this observation is believed that ZnO was changed into O-rich after thermal annealing in atmosphere. The atomic ratio of Zn : O was 1: 1.5 before annealing and 1:2 after annealing. The contact angle between deionized water droplet and oxide film surface was observed to be changed significantly with thermal heating. Figure 5-4 shows the representative images of a water drop on as grown and annealed oxide thin films by sessile drop method at room temperature. Clearly, the contact angle of each oxide surface was decreased after annealing. Figure 5-4 A and B show that contact angle on annealed Ta 2 O 5 surface had decreased from 10.1 2 to < 1~2 which is indicative that the hydrophilic as grown surface became almost wettable surface after annealing. The contact angle on the ZnO surface was also changed from 75.6 2 into 39.4 2, indicating that as grown ZnO is relatively hydrophobic and becomes hydrophilic after annealing. Decrease in contact angle is believed to be higher O atom ratio on annealed film surfaces. Thus, characterizations of the oxide thin films showed that annealing process affects physical and chemical properties structure, roughness and wettabilityof the oxide films. The summary of properties of as grown and annealed oxide films are presented in Table 1. 67

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5.3.2 Annealing Effect on Fibrinogen Adsorption It is known that fibrinogen adsorbs easily onto hydrophobic and even hydrophilic surfaces. The kinetics of adsorbed fibrinogen on Ta 2 O 5 thin film studied by ellipsometry is shown in Figure 5-5. Thickness data show that the adsorption of fibrinogen increases with time for both as grown and annealed films, and gradually stabilizes; Over 50% of the total adsorption occurred in the first 10 min, followed by gradual adsorption to 120 min. Surprisingly, the thickness of adsorbed protein on annealed surface was higher by 17 ~ 25 than as grown surface. Even though the annealed Ta 2 O 5 film has cyratalline structure and superhydrophilicity (contact angle < 5), fibrinogen has less attachment to amorphous as grown film. Especially, this trend of fibrinogen adsorption kinetics observed here is reverse of the data that were reported previously. It is generally accepted that adsorption of fibrinogen is more resistant to hydrophilic surface. Choukourov et al. [113] also reported that adsorption on hydrophilic gold is faster and has higher surface coverage than hydrophobic self-aligned monolayer (SAM). They mentioned that large adsorption of fibrinogen on hydrophilic surface is due to surface charge effect. However, comparing surface charge and wettability of annealed Ta 2 O 5 is still negative and hydrophilic, respectively, the phase change from amorphous to crystalline structure may influence adsorption behaviors. The kinetics graph of adsorption in Figure 5-6 shows that the fibrinogen adsorbs more readily on hydrophobic surface. The difference in fibrinogen adsorption between as grown and annealed ZnO film was reached to 185 after 120 min. This is believed to be a decrease in hydrophobicity of oxide surface after annealing. Interestingly, difference in the thickness of fibrinogen between 30 min and 60 min is very small or even decreased for annealed surface because desorption as well as adsorption may be active for this period. 68

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Furthermore, the reason for the thickness of adsorbed fibrinogen on as grown ZnO is much higher than as grown Ta 2 O 5 is due to surface charge and the hydrophobicty; in pH 7.4 PBS solution, Ta 2 O 5 and fibrinogen has negative surface charge, while ZnO is positively charged. Figure 5-7 displays the RMS (root mean square) roughness of the adsorbed fibrinogen on the oxide film surfaces. As shown in this figure, the roughness trend of the adsorbed protein on the Ta 2 O 5 films is opposite to that of the ZnO films. The surface roughness of as grown Ta 2 O 5 film is much higher due to ejection of large particles from PLD target during the film deposition. As the adsorption proceeds, fibrinogen would make rougher surface become smoother by filling in the grooved areas. Therefore, RMS of as grown and annealed Ta 2 O 5 is decreased with the adsorption time. On the contrary, ZnO film has smoother surface and RMS is increased with the adsorption time. In this case, RMS of as grown ZnO is higher than that of annealed ZnO because amount of adsorbed fibrinogen is proportional to the surface roughness. 5.4 Summary Ta 2 O 5 and ZnO thin films with an initial thickness of 8~12 nm were grown by PLD, and subsequently annealed in air ambience at 800C for 30min. The effects of annealing process on fibrinogen adsorption onto oxide films have been studied using spectroscopic ellipsometry and AFM. It was found that the changes of physical and chemical properties of the oxide film due to heat treatment have influenced the behaviors of fibrinogen adsorption. Annealed Ta 2 O 5 film showed higher adsorption of fibrinogen. The adsorption on annealed ZnO film was much decreased compared with as grown ZnO because of surface wettability change. 69

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Table 5-1. Comparison in properties between as grown and annealed oxide films Crystallinity RMS roughness Atomic ratio of metal and oxygen Contact angle as grown amorphous 7.86 nm 1 : 4.7 10.1 Ta 2 O 5 annealed crystalline 6.97 nm 1 : 6.5 < 1~2 as grown crystalline 1.55 nm 1 : 1.5 75.6 ZnO annealed crystalline 1.51 nm 1 : 2 39.4 70

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A B C D Figure 5-1. AFM images of Oxide films before and after air annealing at 800C. A) as grown Ta 2 O 5 B) annealed Ta 2 O 5 C) as grown ZnO, and D) annealed ZnO. Scale bar is for 200 nm for Ta2O5 and 50 nm for ZnO. 71

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A B Figure 5-2. Grazing incidence XRD spectra of two oxide films. A) Ta 2 O 5 surface and B) ZnO surface 72

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A B Figure 5-3. X-ray photoemission spectroscopy of two oxide films.A) Ta 2 O 5 surface and B) ZnO surface. 73

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A B C D Figure 5-4. Photographs of water contact angle on two oxide film surfaces. A) as grown Ta 2 O 5 ,B) annealed Ta 2 O 5 C) as grown ZnO, and D) annealed ZnO. 74

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Figure5-5. Thickness vs time graph of adsorbed fibrinogen onto as grown and annealed Ta 2 O 5 surface in pH 7.4 solution Figure 5-6. Thickness vs time graph of adsorbed fibrinogen onto as grown and annealed ZnO surface in pH 7.4 solution 75

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Figure 5-7. The RMS roughness variation of oxide thin films during fibrinogen adsorption 76

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CHAPTER 6 BINARY TA-ZN OXIDE THIN FILM-INDUCED REDUCTION OF PROTEINS ADSORPTION 6.1 Introduction Minimization of nonspecific protein adsorption and biofouling onto biomaterial surfaces is a key issue for success of many blood contacting applications. Considerable attention for reducing adsorption of proteins has been paid using many materials and techniques. Recently, the most widespread method for obtaining biocompatible surfaces is immobilizing polymeric materials onto substrate [6, 114-117]. Even though this approach showed much improvement for resisting adsorption, several limitations still remain to be solved. In many cases, substrate should be modified for strong bonding with biopolymers. Furthermore, due to delicacy of oxidation and degradability of some polymers, resistance property of surfaces can be changed over time [118-120]. According to observation of several researchers, hydrophilicity and net neutral charge of surfaces are decisive properties for resistance of protein adsorption [3, 43]. Therefore, techniques that control substrate properties such as surface charge, wettability and roughness are important for the development for protein resistant materials. It was observed in previous studies of chapter 4 & 5 that fabrication of metal oxide thin films such as Ta 2 O 5 and ZnO is very useful approach to control protein adsorption and these oxide films can be promising materials for reducing adsorption. Especially, because Ta 2 O 5 and ZnO have an opposite surface charge and hydrophobicity, hydrophilic oxide surface with a neutral charge can be obtained by mixing these oxide materials. Moreover, the deposition of oxide thin film is simple and very useful due to no worry for oxidation and complicated polymer functionalization. 77

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In this paper, in order to acquire well controlled oxide materials, Ta-Zn binary oxide thin film is prepared by pulsed laser deposition (PLD). This deposition method is very versatile technique for depositing multi-component materials with a high melting temperature. The physical and chemical properties of Ta-Zn binary oxide thin film are analyzed and compared with data of Ta 2 O 5 and ZnO [43]. Fibrinogen, bovine serum albumin (BSA) and lysozyme are used as model proteins, and the thickness of adsorbed proteins on Ta-Zn binary oxide film is measured by using ellipsometry. This optical technique is frequently used for measuring amount of adsorbed proteins because it is fast and non-destructive. The thickness comparison of adsorbed proteins with pure oxide films shows how properties of film affect adsorption behaviors and how effective our approach is for reducing adsorption of proteins. 6.2 Experimental 6.2.1 Preparation of Binary Oxide Thin Films Binary Ta-Zn (TZ) oxide thin film was deposited on Si (001) substrate by pulsed laser deposition (PLD). The substrates were ultrasonically cleansed in acetone, methanol and deionized water for 5 minutes, respectively, and then HF (10%) was used to remove natural oxide layer on silicon surface. PLD target was prepared by mixing and pressing two oxide powders (99.99%), which are composed of Ta 2 O 5 (50 wt%) and ZnO (50 wt%). And then, pressed target was sintered at 1300 C for 3hr. During deposition, an oxide target is rotated with an angle 45 to the normal surface and a pulsed KrF excimer ( = 248 nm) laser was used with laser fluence of 3.3 J/cm 2 and frequency of 5 Hz. No intentional substrate heating was employed and substrate was rested on platter during the film growth. Ultra-high-purity (99.999%) O 2 gas was introduced to kept constant vacuum 78

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level of 0.3 Torr throughout the film growth. The ablated material was deposited on Si substrate, which is located from the target with a distance of 6 cm. The deposited films were analyzed using various characterization techniques. The crystallinity of films was studied using X-ray diffraction (XRD), and X-ray photoelectron microscopy (XPS) was carried out to analysis the chemical composition of binary oxide film. The morphology, topography and roughness of film surface were examined by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The zeta potential is calculated based on the flow of the solvent (1 mM KCl) to obtain isolectric point of TZ binary oxide film. Surface wettability of oxide film was also determined by measuring contact angle of water droplet. 6.2.2 Protein Adsorption Experiment Fibrinogen from bovine plasma (A4753, MW 340 kDa), albumin from bovine serum (A1933, MW 66kDa) and lysozyme from chicken egg white (L6876, MW 14.3kDa) were purchased from Sigma and used without further purification. All protein solutions (0.1mg/mL concentration) were prepared by dissolving respective protein powder in phosphate buffer saline (PBS) solution (pH 7.4). Fibrinogen and BSA are negatively charged in PBS because of their inherent isoelectric points, while lysozyme is positively charged. All adsorption experiments were performed with dropping protein solutions on TZ oxide film surfaces at room temperature. Protein solution-dropped samples were remained to 10, 30, 60, and 120 min, respectively. After finishing adsorption, samples were rinsed by deionized water and PBS solution. Subsequently, they were dried with blowing of nitrogen gas. 6.3 Results and Discussion 6.3.1 Characterization of Ta-Zn Oxide Thin Films Prior to film deposition, PLD target was examined by conventional XRD (Cu K, =1.5406) to analyze material structure. Because PLD system provides stoichiometric transfer 79

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of molecules of the target into the substrate, the information of target is important for studying the cyrstallinity and structure of deposited films. In the XRD pattern of Figure 6-1(a), the spectra showed that this target material has crystalline structure, resulting in the formation of a compound Ta 2 Zn 3 O 8 (JCPDS card #20-1237). It is believed that new phase resulted from sintering process during the preparation of target. Figure 6-1(b) shows grazing incidence XRD pattern of TZ binary oxide film with thickness of 10-12 nm. As shown in this figure, this deposited film is amorphous, showing no diffraction peak. The formation of a new compound examined by XRD technique can be further confirmed by XPS. In order to investigate the chemical composition and valence states of binary oxide films, Ta 4f and Zn 2P spectra were obtained by XPS measurement. In Figure 6-2 A, Ta 4f peak of TZ oxide film was shifted to the higher value by 1 eV compared to that of pure Ta 2 O 5 thin film, indicating the change of Ta chemical valence due to addition of Zn. In addition, XPS curves of Zn 2p reveal that two peaks of TZ film also moved into the position of higher binding energy value. This is in agreement with Ta 4f peak, indicating the variation of Zn chemical state. The surface morphology, topography and roughness of deposited TZ film were determined from an analysis of scanning electron micrograph (SEM) and atomic force micrograph (AFM) results (Figure 6-3). The SEM picture (X 100,000) of film reveals the formation of 50-80 nm large particulates with relatively porous structure. Three dimensional (3-D) AFM image also confirms that the surface of binary film is much rough, showing a RMS roughness of 9.19nm. Figure 6-4 A shows the zeta potential () variation with pH for TZ binary oxide film. From this measurement, we found that the isoelectric point is approximately pH 7.1, which is indicated that this film is nearly closed to neutral charge when it is in pH 7.4 PBS solution. According to previous data in chapter 4&5, isoelectric points of pure Ta 2 O 5 and ZnO oxide film were pH 2.9 80

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and pH 8.5, respectively. The value of isoelectric point of TZ film may be reasonable because pH 7.1 is less than ZnO and more than Ta 2 O 5 Therefore, TZ oxide film has weak negative surface charge which is nearly closed to neutral. The contact angle measurement was performed to investigate surface wettability of binary oxide film. The droplet image was in Fig. 6-4 B. The angle between water droplet and oxide film was 19.1 2, indicating TZ oxide film is hydrophilic. The oxide film properties of the pure Ta 2 O 5 ZnO and TZ binary film are summarized and compared in Table 1. 6.3.2 Adsorption of Proteins on TZ Oxide Film De Feijters method [121-122] was applied to obtain an amount of adsorbed protein per unit area. For this method, adsorbed protein thickness was measured by using ellipsometric method at solid-liquid interfaces. )/()()/(2cnmffddnndmmng ....................6-1 where d f is the thickness of protein (nm), n f is the refractive index of proteins, n m is the refractive index of PBS, and d n /d c is the refractive index increment. It is assumed that the refractive index of a protein in PBS is a linear function with its concentration. In the Si substrate system, equation 6-1 can be modified by (ng / mm 2 ) = K adsorbed protein thickness (nm) where K is approximately 1.2 [62]. The graph of adsorbed protein density in Figure 6-5 was obtained in order to characterize quantitatively and predict protein adsorption on TZ oxide film. As shown in this figure, adsorption of fibrinogen on TZ film is the highest, followed by BSA and lysozyme. Fibrinogen adhered to hydrophilic surface greater than two other proteins. Furthermore, we found that over 70-85% of total adsorption was occurred within 10 min like commonly observed results and 81

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steady state of adsorption is gradually obtained with time decreasing adsorption rate. It is generally accepted that adsorbed protein onto solid surface undergoes denaturation, which is a change in conformation and orientation [123-126]. This structural change usually results in increasing an occupied area of surface due to larger contact region and decreasing adsorption of proteins on surface, followed by saturation of adsorbed layer [127]. 6.3.3 Comparison of Protein Adsorption In order to investigate improved behaviors of binary TZ oxide film, ellipsometrical thickness of adsorbed proteins was compared with pure Ta 2 O 5 and ZnO. As shown in Figure 6-6, negatively charged fibrinogen in PBS had stronger affinity to hydrophobic and positively charged ZnO, but less adsorbed onto hydrophilic and negatively charged Ta 2 O 5 The hydrophilic, almost neutral charged TZ showed higher adsorption over Ta 2 O 5 during early adsorption, but smaller adsorption by 5 after 120 min. Another negatively charged BSA showed same adsorption trend (Figure 6-7). Adsorption on TZ was also the smallest. The difference of adsorption between BSA and fibrinogen is that final thickness of BSA on all oxide films is greater than fibrinogen. This may be influenced by strength of protein charge and molecular weight of proteins. This trend is completely different in the case of lysozyme (Figure 6-8). Positively charged protein has stronger affinity to Ta 2 O 5 with negative charge during initial time. After 1hr adsorption, thickness of lysozyme to ZnO became greater than Ta 2 O 5 due to hydrophobic interaction. TZ oxide film exhibited the least adsorption of lysozyme during all time of adsorption. 6.4 Summary We prepared binary Ta-Zn oxide thin film by pused laser ablation method. Several characterization of deposited film revealed that this TZ film is amorphous, hydrophilic and 82

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almost neutrally charged in pH 7.4 PBS solution. To investigate improved adsorption behaviors of TZ film, the thickness of adsorbed proteins was measured by ellipsometry and compared with pure Ta 2 O 5 and ZnO. The ellopsometrical results showed that the hydrophilic and neutrally charged TZ film has the least affinity to all proteins and this film is more resistance to protein adsorption than two pure thin films. 83

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Table 6-1. Summarized properties of three oxide thin films TZ Ta2O5 ZnO Crystallinity amorphous crystalline crystalline RMS roughness 9.19 nm 7.86 nm 1.55 nm Isoelectric point pH 7.1 pH 2.9 pH 8.5 Surface charge in pH 7.4 PBS near to neutral negative positive Contact angle 19.1 10.1 75.6 84

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Figure 6-1. X-ray diffraction patterns of (a) target material and (b) binary Ta-Zn oxide film. 85

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A B Figure 6-2. The XPS spectra of as grown binary Ta-Zn oxide thin films.A) Ta 4f and B) Zn 2p 86

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A B Figure 6-3. The SEM (A) and AFM (B) images of binary TZ oxide films 87

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A B Figure 6-4. Zeta potential vs pH curve (A) and water drop images (B) of TZ oxide films 88

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Figure 6-5. Adsorption density of proteins vs time on Ta-Zn binary oxide film Figure 6-6. Comparison graph of Fibrinogen thickness onto three oxide films in pH 7.4 solution 89

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Figure 6-7. Comparison graph of BSA thickness onto three oxide films in pH 7.4 solution Figure 6-8. Comparison graph of lysozyme thickness onto three oxide films in pH 7.4 solution 90

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CHAPTER 7 PULSED LASER ABLATED NANOSTRUCTURED OXIDE FILMS-MEDIATED ZERO ADSORPTION OF LYSOZYME 7.1 Introduction Since the spontaneous adsorption of proteins to biomaterials can damage their functions and properties, resistance of protein adsorption is an important process for developing blood contacting applications [65, 128-130]. In order to obtain surfaces resisting protein adsorption, investigators tried many new techniques [131-133] using hydrophilic polymeric materials [134-138]. However, even though these polymers showed excellent resistance, several drawbacks are still unsolved; oxygen sensitivity of some polymers and polymer degradability with time and chemical modification of substrate. Considerable attentions on metal oxide thin films have been increased for wide ranges of applications. Especially, Ta 2 O 5 and ZnO thin films have been also extensively investigated for their excellent electrical, magnetic and optical properties, which can be used in many possible areas [92, 139-140]. Recently, these oxide materials have been used in the bio-related researches such as coating of implants, biosensor and biomedicine due to their good bio/ blood compatibility and chemical stability [141-142]. However, study on resistance of protein adsorption using metal oxides is still much to be done. It has been widely known that the adsorption process much depends on the properties of substrate such as surface charge, wettability, roughness, surface chemistry and so on [34,68,69,71]. According to research papers, Ta 2 O 5 and ZnO thin films have opposite surface charge in pH 7.4 solution, due to the isoelectric points of these materials which are approximately pH 2.8-2.9 and pH 8.7-9.3, respectively [81]. Furthermore, water wetting property is also different, since Ta 2 O 5 is hydrophilic and ZnO is hydrophobic [143]. Therefore, the fabrication of well controlled nanostructured oxide film where opposite propertied materials 91

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coexist would be a promising method for reducing the protein adsorption. Pulsed laser ablation method is employed for preparation of nanostructured oxide thin films. This technique has been widely used for the growth of many oxide thin films because it provides stoichiometric deposition from target materials and good control of film morphology [144-145]. 7.2 Experimental In this letter, oxide thin film was grown on silicon (001) substrate using a pulsed KrF laser (248 nm wavelength, 3.3 J/cm 2 energy density, 5 Hz repetition frequency of laser). The deposition was performed under 3.0 X 10 -1 Torr oxygen pressure without intentional substrate heating. Subsequently, the second oxide was deposited ultra thinly on first grown oxide film to form the nanostructured oxide film. The contact angle measurements were carried out using a Rame-Hart 100 goniometer under an ambient condition in order to study the surface wettabilty of the nanostructured oxide films. The information of chemical state and phase identification was also obtained using X-ray photoelectron spectroscopy (XPS). For the protein adsorption test, 0.1mg/mL of lysozyme from chicken egg was prepared by dissolving the protein in PBS (phosphate buffer saline, pH 7.4) solution. This lysozyme solution was dropped onto the nanostructured oxde surface at a room temperature and remained for 1hr and 2hr, respectively. After the adsorption, samples were cleaned 2 times with deionized water and, PBS solution and then were blown dry with a stream of nitrogen gas. Thickness of the adsorbed lysozyme on the oxide films was measured using a variable angle spectroscopic ellipsometer from J.A. Woollam. Ellipsometry is a nondestructive method for determining thickness of organic thin films and is widely used for fast characterization with an angstrom level [146]. 92

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7.3 Results and Discussion Structures of the nanostructured oxide thin films are shown in the Figure 7-1. The process of nanostructured oxide thin film includes deposition of two target materials by PLD. As seen in this figure, oxide I layer, firstly, are grown on a Si substrate, and then very short time deposition of oxide II are performed without a complete formation of monolayer. In order to prepare ZnO-based oxide film (T_ZnO), ZnO thin film is firstly grown on a Si substrate, followed by a deposition of Ta 2 O 5 Ta 2 O 5 -based oxide film (Z_Ta 2 O 5 ) is also composed of deposition of Ta 2 O 5 film on a Si substrate and subsequent very quick deposition of ZnO. As varying deposition time and amount of oxide II films, nanostrucuted oxide samples are divided into A and B. To investigate the wettability variation of surface with the deposition of oxide II static contact angles of nanostructured oxide films were measured using a sessile drop method and compared with a pure oxide film (Figure 7-2). In case of T_ZnO surface (red line), pure ZnO had a contact angle of approximately 75.6 2 indicating a relatively hydrophobic material. As depositing Ta 2 O 5 on ZnO layer, contact angle of T_ZnO surface was decreased to 50.7 2 for #a sample and 33.8 2 for #b sample, respectively. This trend is reasonable when Ta 2 O 5 is an intrinsically hydrophilic material. In the case of Z_Ta 2 O 5 (black line), the contact angle of the nanostructured surface was increased to 18.8 2 and 39.2 2 due to the addition of hydrophobic ZnO, even though pure Ta 2 O 5 has a CA of about 10.0 2. The changes of surface wettability reveal that oxide II is dispersedly deposited on oxide I layer and does not form a complete layer. Figure 7-3 A and B show that Zn 2p and Ta 4f peaks of bilayer oxide films and pure oxide films. As can be seen in Fig. 7-3A, peaks of Zn 2p in Ta 2 O 5 -based (Z_Ta 2 O 5 ) sample have much smaller intensity while peaks of ZnO-based (T_ZnO) sample is similar to those of pure ZnO film. 93

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However, Zn peaks of two nanostructured samples have the same positions, which are located at 1022.5 eV for Zn 2p3 and 1045.5 eV for Zn 2p1 in pure ZnO thin film. In addition, XPS curves of Ta 4f in Z_Ta 2 O 5 and T_ZnO oxide samples reveal that their intensity is much smaller than that of Ta 2 O 5 thin film and maximum peak location of two nanostructured samples are also same with Ta2O5 film. Same positions of Zn and Ta peaks indicate that nanostructured oxide films with two oxide materials does not form any new phase in the middle of deposition. An ellipsometrical analysis of the lysozyme thickness on nanostructured oxide films are shown in Figure 7-4. These diagrams also include adsorption data of pure Ta 2 O 5 and ZnO thin films. Figure 7-4 A reveals that sample #a and #b of T_ZnO film have similar affinity to lysozyme compared with two pure oxide films. In addition, the lysozyme thickness of sample #a is greatest after 2hr adsorption. Interestingly, thickness of sample #b decreases from 105.0 to 77.2 after 2hr. Adsorption data on Z_Ta 2 O 5 film are presented in Figure 7-4 B. Unlike T_ZnO film, Z_Ta 2 O 5 films show an excellent resistance of lysozyme adsorption. Thickness of lysozyme on sample #a is below 1 and no adsorption behavior was remained to 2hr. Even though pure Ta 2 O 5 film is more hydrophilic, Ta 2 O 5 -based nanostructured film, which is composed of two oxide materials with opposite surface charge and wettability had better resistant property of adsorption. Sample #b shows lysozyme thickness of 29.6 and 64.1 after adsorption, respectively. These values are bigger than that of sample #a, but smaller than pure oxide films. Furthermore, even though the contact angles of sample #b in T_ZnO and Z_Ta 2 O 5 films are similar, the adsorption resistance of lysozyme on the latter film is better. Through these results, we find that Ta 2 O 5 -based film is more effective against resisting lysozyme adsorption. 94

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7.4 Summary In summary, lysozyme adsorption was conducted on nanostructured oxide thin films prepared by laser ablation method in 0.3 Torr oxygen ambience. Water contact angle measurements revealed that the deposition of oxide II did not form a complete layer. The analysis using XPS also showed that two oxides (Ta 2 O 5 and ZnO) on nanostructured surface existed separately with no formation of new phase due to chemical reaction. After measuring thickness of lysozyme, Ta 2 O 5 -based films (Z_Ta 2 O 5 ) showed better resistance to lysozyme adsorption than ZnO-based films. Especially, sample Z_Ta 2 O 5 _#a showed an excellent resistance ability indicating no adsorption of lysozyme. 95

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A B Figure 7-1. Structures of nanostructured oxide thin films. Samples are divided into A) and B) as amount of oxide II Figure 7-2. Contact angle variation of nanostructured oxide films 96

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A B Figure 7-3. The XPS spectra of nanostructured oxide films. A) Zn 2p peaks and B) Ta 4f peaks 97

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A B Figure 7-4: Thickness diagrams of adsorbed lysozyme on nanostructured oxide films.A) is for Zno-based films and B) is for Ta 2 O 5 -based films. 98

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CHAPTER 8 CONCLUSION The research presented in this dissertation focused on metal oxide thin films growth by pulsed laser deposition for confirming their versatility of controlling protein adsorption. Firstly, the physical and chemical properties of as grown and annealed oxide thin films were analyzed to understand the influence of adsorption behaviors. In order to obtain several types of oxide film, binary Ta-Zn oxide films (TZ) and ultrathin bilayer oxide films as well as pure Ta 2 O 5 and ZnO thin films were employed. The thickness of fibrinogen, BSA and lysozyme on these oxide films were measured in order to reveal the adsorption relationship with the property of metal oxide thin films. 8.1 Effect of Surface Charge on Protein Adsorption Prior to protein adsorption, understanding the characteristics of metal oxide films is very important because surface properties of the films are determining factors for controlling adsorption. As grown Ta 2 O 5 and ZnO thin films have opposite surface charges in pH 7.4 PBS, and proteins also have different net charges as their inherent IEP. Negatively charged Ta 2 O 5 has a greater adsorption onto positively charged lysozyme, and positively charged ZnO shows same trend. During protein adsorption in solution, attraction force due to opposite surface charge between oxide substrate and proteins is one of main factors in adsorption behaviors. The repulsion force between same charged materials can be used for controlling or reducing adsorption of proteins. 8.2 Annealing Effect on Fibrinogen Adsorption Annealing process (800C, 30 min) for as grown oxide films resulted in the property changes of Ta 2 O 5 and ZnO. The annealed Ta 2 O 5 were switched from amorphous to crystalline and the contact angle was decreased. Whereas ZnO was still crystalline structure and contact 99

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angle was decreased from 75.6 to 39.4. The effects of annealing treatment on fibrinogen adsorption onto oxide films have been studied using spectroscopic ellipsometry and AFM. Annealed Ta 2 O 5 film showed higher adsorption of fibrinogen compared with as grown Ta 2 O 5 while the adsorption on annealed ZnO film was much decreased compared with as grown ZnO because of surface wettability change. 8.3 Effect of Binary Ta-Zn Oxide Films on Protein Adsorption Pulsed laser deposited binary Ta-Zn oxide thin films (TZ) were amorphous and its surface charge was nearly neutrality in pH 7.4 solution. Furthermore, the wettability of TZ was hydrophilic becaused of 19.1 of contact angle. As results of ellipsometical measurements for protein adsorption, thickness of adsorbed proteins (fibrinogen, BSA, and lysozyme) onto TZ surface was the lowest compared with Ta 2 O 5 and ZnO surface. These results are influenced by neutral charge and hydrophilicity of TZ oxide film. 8.4 Influence of Nanostructured Oxide Films on Lysozyme Adsorption Nanostructured oxide thin films (T_ZnO and Z_Ta 2 O 5 ) were fabricated by deposition of oxideI and oxide II. Oxide II was deposited on oxide I and it was not a complete monolayer. This was confirmed by the measurement of contact angle. XPS analysis also showed that no new phase was formed except Ta 2 O 5 and ZnO during the growth of nanostructured oxide films. Results showed that Ta 2 O 5 -based films (Z_Ta 2 O 5 ) had better resistance to lysozyme adsorption than ZnO-based films. Especially, sample Z_Ta 2 O 5 _#a showed an excellent resistance ability indicating no adsorption of lysozyme. 100

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BIOGRAPHICAL SKETCH Se Jin Kim was born in 1973, in Kim Cheon, South Korea. In 1992, he entered the Department of Metallurgical Engineering at Korea University. After earning a Bachelor of Science degree, he continued his graduate study and earned a Master of Science degree in 2001. His research topic was a study on preparation and physical properties of carbon /carbon composite from mesophase pitch precursor under the supervision of Dr. Moo Young Huh. In August 2002, he enrolled at the University of Florida in the Department of Materials Science and Engineering to pursue his Ph.D under the advisement of Dr. Rajiv.K. Singh. His main research was involved in growth and characterization of oxide thin films by pulsed laser deposition (PLD). He also focused on control of protein adsorption using laser ablated engineered oxide thin films.