5. [7] M.A. Arnold, M.E. Meyerhoff, Crit. Rev. Anal. Chem., 20 (1988) 149â196. [8] S. Belkin, Curr. Opin. Microbiol., 6 (2003) 206â212. [9] B. R. Eggins, Chemical ...
Protein Adsorption on Hydrogenated Silicon Surfaces PhD Thesis
Larbi Filali University of Oran 1, Algeria Physics Department February 2018
Acknowledgements This dissertation evolved with the generous help of many people to whom I feel greatly indebted. I must extend my profound gratitude first and foremost to my thesis advisor Prof. Jamal Dine Sib for his unlimited guidance, encouragement, help with all the administration papers, and kindness during the course of this work. I would also like to thank Prof. Bouizem for presiding over the committee that reviewed this thesis. I would also like to express my sincere gratitude to committee members: Prof. M. Kechouane (USTHB University of Algiers), Prof. A. Khelil (University of Oran1), Prof. Y. Ghamnia (University of Oran1), and Prof. Y. Kail (University of Oran1). This dissertation is the result of their insightful comments, unreserved support, and kind patience. Thanks are also due to the administrative staff of the Physics department at the University of Oran1, and especially Prof. A. Kebab for his help throughout my graduate studies. I would like to thank again Prof. Khelil for accepting me at the LPCMME laboratory and Prof. L. Chahed for giving me the opportunity to do research among his team since 2012. Very special thanks to Dr. J. Benlakehal (University of Oran 1) who helped me readily during the deposition of the silicon thin films and many crucial measurements and for his constant support. I would like to thank Dr. A. Bouhekka (University Chelif) and Dr. M. Chahi (University of Oran 1) for their kindness and help throughout this work. Their contributions impacted massively this project. I am very grateful for the assistance of Dr. N. Hamzaoui (University of Oran 1) in AFM measurements and Pr. Kechouane and his team, especially Dr. Iness Lachebi, for the help with Ellipsometry measurements. Last but not least, I would like to thank Dr. Asmaa Argoub (University of Oran 1) for her assisting in preparing BSA solutions. Many thanks to my colleagues at the lab and friends Yamina Brahmi, Zeudmi Fouzya, Sid Ahmed Memchout, and Hadj Benhabara for sharing with me this experience and I‟m forever grateful for their support. At last I would like to thank all my family members in Oran and abroad and all my friends for their emotional support during these 5 years. This work is dedicated to my mother who stood behind me and my crazy time schedule with unwavering support, and my sister Amina who supported me unreservedly throughout this journey.
Abstract In this work, the adsorption of proteins on hydrogenated silicon surfaces has been studied by various experimental techniques, such as infrared spectroscopy in ATR mode (FTIR/ATR), ellipsometric spectroscopy, atomic force microscopy (AFM), and water contact angle measurements. Understanding protein adsorption at the solid / liquid interface involves a multitude of research fields across biology, medicine, biotechnology and food processing. On the other hand, hydrogenated amorphous silicon (a-Si: H) has many physical properties which would make this material an ideal candidate for biological applications such as biosensors. The aim of this research is to illustrate the role of the hydrogenation of amorphous silicon in the adsorption of proteins. The work was divided into two parts; first, surfaces of amorphous silicon (deposited by the sputtering technique) were treated with molecular hydrogen with varying flows. These surfaces have been characterized by FTIR / ATR to identify chemical species at the surface, by ellipsometry to measure the deposited film thicknesses and roughness, by AFM to illustrate the topographic changes before and after hydrogenation, and finally measuring the contact angle to quantify the wettability of the surfaces. The conclusion which has been drawn about the hydrogen treatment of amorphous silicon surfaces is that this it reduces the surface roughness and, on the other hand, increases the wettability of the surfaces. Therefore the hydrogenation renders the amorphous silicon surfaces flatter and more wettable, which should be a disadvantage for the adsorption of proteins which have more affinity for rough and hydrophobic surfaces.
The second part is devoted to the adsorption of Bovine Serum Albumin (BSA) proteins at the solid/liquid interface. For the un-treated „hydrophilic‟ substrate the protein molecules are adsorbed on sparse “hydrophobic” sites, with minimal protein-protein interactions. For the hydrogenated surfaces, the SiHx covalent (non-polar) molecules attract the BSA molecules through hydrophobic forces that are energetically able to displace water molecules at the vicinity of the surface. The proteins are adsorbed close to each other witch leads to a densely packed layer prompting neighboring proteins to change their orientation to minimize the repulsive forces between the molecules. The adsorbed proteins, however, lose their α-helical structure and thus they become denaturated.
Résumé Dans ce travail, l‟adsorption de protéines en solution liquide sur des surfaces solides de silicium hydrogéné a été étudiée par diverses techniques expérimentales, telles que la spectroscopie infrarouge en mode ATR (FTIR/ATR), la spectroscopie ellipsometrique, la microscopie à force atomique (AFM), et les mesures de l‟angle de contact de l‟eau. La compréhension de l‟adsorption des protéines à l‟interface solide/liquide intéresse une multitude de domaines de recherche à travers la biologie, la médecine, la biotechnologie et la transformation des aliments. D‟autre part, Le silicium amorphe hydrogéné (a-Si:H) possède beaucoup de propriétés physiques qui le rendraient un candidat idéal pour des applications biologiques telles que les biosensors. Le but de cette recherche est d‟illustrer le rôle de l‟hydrogénation du silicium amorphe dans l‟adsorption des protéines à l‟interface solide/liquide. Le travail a été divisé en deux parties; en premier lieu, des surfaces de silicium amorphe (déposé par la technique de pulvérisation cathodique) ont été traitées par de l‟hydrogène moléculaire avec des flux variés. Ces surfaces ont été caractérisées par FTIR/ATR pour identifier les espèces chimiques au niveau de la surface, par l‟ellipsométrie pour mesurer les épaisseurs des couches déposées et leurs rugosités, par l‟AFM pour illustrer les changements topographiques avant et après l‟hydrogénation, et enfin l‟angle de contact a été mesuré afin de quantifier la mouillabilité des surfaces. La conclusion qui a été tiré à propos du traitement des surfaces de silicium amorphe par de l‟hydrogène est qu‟elles deviennent moins rugueuses et, d‟autre part, plus mouillables, ce qui devrait être à priori un inconvénient pour l‟adsorption des protéines qui ont plus d‟affinités pour les surfaces rugueuses et hydrophobes. La deuxième partie concerne l‟adsorption des protéines de Bovine Sérum Albumin (BSA) à l‟interface solide/liquide. Pour les surfaces non hydrogénés et donc hydrophiles, les molécules sont adsorbées sur des sites éloignés avec un changement minimal de conformation. Pour les surfaces hydrogénées, les molécules covalentes SiHx (non polaires) attirent les molécules de BSA par des forces hydrophobes qui sont capables de déplacer rigoureusement les molécules d'eau au voisinage de la surface. Les protéines sont adsorbées les unes près des autres ce qui conduit à une couche dense qui incite les protéines voisines de changer leur orientation afin de minimiser les forces de répulsion entre les molécules. Les protéines adsorbées perdent cependant leur structure hélicoïdale et sont donc dénaturées.
ملخص
فً هذه األطروحة ،تمت دراسة امتزاز البروتٌنات من طرف سطوح السٌلٌكون المهدرج عن طرٌق عدد من التقنٌات التجرٌبٌة ،مثل التحلٌل الطٌفً باألشعة تحت الحمراء ،التحلٌل الطٌفً االهلٌلجً ،مجهر القوة الذرٌة ،وقٌاس زاوٌة االلتماس للماء. فهم امتزاز البروتٌن فً الواجهة صلب /سائل ٌخص عدة مجاالت بحثٌة كالبٌولوجٌا والطب والتكنولوجٌا الحٌوٌة والصناعات الغذائٌة .من ناحٌة أخرى ،السٌلٌكون المهدرج غٌر المتبلور ٌحتوي على العدٌد من الخصائص الفٌزٌائٌة التً من شأنها أن تجعل هذه المادة المرشح المثالً للتطبٌقات البٌولوجٌة مثل أجهزة االستشعار .الهدف من هذا البحث هو توضٌح دور هدرجة السٌلكون غٌر المتبلور فً امتزاز البروتٌنات. قسم العمل إلى قسمٌن؛ أوال تمت معالجة سطوح السٌلكون غٌر المبلور ،الذي صمم عن طرٌق تقنٌة الترذٌذ، بالهٌدروجٌن الجزٌئً بتدفقات متفاوتة .تم تشخٌص هذه السطوح بتقنٌة التحلٌل الطٌفً باألشعة تحت الحمراء لتحدٌد األنواع الكٌمٌائٌة المتواجدة على السطح ،بتقنٌة القٌاس االهلٌلجً لحساب سمك الطبقة المصممة وخشونتها ،بتقنٌة مجهر القوة الذرٌة لتوضٌح التغٌرات الطبوغرافٌة قبل وبعد الهدرجة ،وأخٌرا قٌاس زاوٌة االلتماس لمعرفة قابلٌة ترطٌب األسطح .االستنتاج الذي تم استخالصه حول معالجة أسطح السٌلكون غٌر المتبلور بالهٌدروجٌن هو أن هذا العالج ٌقلل من خشونة السطح من ناحٌة ،وٌرفع قابلٌة ترطٌبه من ناحٌة أخرى.بذلك فإن الهدرجة تجعل األسطح السٌلكونٌة غٌر المتبلورة مستوٌة وأكثر رطوبة ،والذي من المفروض أن ٌكون عائقا أمام امتزاز البروتٌنات التً تنجذب أكثر لألسطح النافرة للماء. خصص الجزء الثانً المتزاز بروتٌنات ألبٌومٌن المصل البقري على واجهة صلب/سائل .بالنسبة للسطوح السٌلكونٌة التً لم تتم هدرجتها أي الرطبة ،فقد امتزت جزٌئات البروتٌن على مواقع متباعدة ،مما قلل من التفاعالت بٌن البروتٌنات .،أما على السطوح المهدرجة ،فقد جذبت جزٌئات البروتٌن بقوى نافرة للماء ،مما أدى إلى تكتل البروتٌنات وبذلك تشكلت طبقة بروتٌنٌة كثٌفة على السطح مما دفع البروتٌنات المتجاورة لتغٌٌر توجهها على السطح .مع ذلك ,فان البروتٌنات المبتزة فقدت تركٌبتها الحلزونٌة أي أنها فسدت.
Summary General Introduction ............................................................................................................................... 1 References ........................................................................................................................................... 7 Chapter I. Hydrogenated amorphous Silicon .......................................................................................... 9 1. Crystalline Silicon ......................................................................................................................... 10 2. Amorphous silicon ........................................................................................................................ 11 3. Hydrogen adsorption on Silicon surfaces ...................................................................................... 14 4. Silicon film deposition techniques ................................................................................................ 14
Chemical vapor deposition (CVD) .......................................................................................... 14
Sputtering .............................................................................................................................. 17
5. Characterization techniques........................................................................................................... 23
Infrared Spectroscopy ........................................................................................................... 23
Vibration modes of the Si-H bonds ....................................................................................... 24
Attenuated Total Reflectance FTIR-ATR Spectroscopy ......................................................... 25
Optical (UV-Vis-NIR) transmission ......................................................................................... 28
Determination of the refractive index and the thickness of the films .................................. 30
Determination of the absorption coefficient ........................................................................ 31
Spectroscopic Ellipsometry ................................................................................................... 32
Effective medium approximations ........................................................................................ 35
Contact Angle ........................................................................................................................ 37
Atomic Force Microscopy ...................................................................................................... 39
6. Rough surfaces .............................................................................................................................. 41
Mechanisms in surface growth ............................................................................................. 41
References ......................................................................................................................................... 42 Chapter II. Protein adsorption on solid surfaces ................................................................................... 45 1. Protein structure ........................................................................................................................... 46
Intra-protein Forces............................................................................................................... 50
Bovine Serum Albumin (BSA) ................................................................................................ 51
2. Protein adsorption at the solid/liquid interface .............................................................................. 53
Phenomena of protein adsorption ........................................................................................ 53
Parameters affecting protein adsorption .............................................................................. 55
Physiochemical properties of proteins.................................................................................. 56
Protein-Surface Interactions ................................................................................................. 57
Surface hydrophobicity ......................................................................................................... 58
Techniques for measurement of protein adsorption ............................................................ 59
FTIR/ATR ................................................................................................................................ 60
Ellipsometry ........................................................................................................................... 62
References ......................................................................................................................................... 63 Chapter III. Results and discussion ....................................................................................................... 67 1. Experimental protocol to grow and analyze the surfaces .............................................................. 68 2. Surface hydrogenation analysis ..................................................................................................... 69
Optical Transmission results.................................................................................................. 69
ATR-IR results ........................................................................................................................ 73
Ellipsometry results ............................................................................................................... 76
Atomic Force Microscopy results .......................................................................................... 80
Contact Angle Measurements ............................................................................................... 83
3. Protein adsorption analysis ............................................................................................................ 86
ATR-IR results ........................................................................................................................ 86
Ellipsometry results ............................................................................................................... 93
4. Conclusion ..................................................................................................................................... 98 References ....................................................................................................................................... 100 General Conclusion ............................................................................................................................. 103
List of Figures Chapter I Figure 1. Crystalline silicon 3D structure, called diamond. .................................................................. 10 Figure 2. Energy Band Diagram of crystalline Silicon. ........................................................................... 11 Figure 3. 2D representations of a) crystalline silicon and b) hydrogenated amorphous silicon. .......... 12 Figure 4. Schematic representation of the density of states of C-Si, a-Si, and a-Si:H ........................... 13 Figure 5. General scheme of the RF discharge ..................................................................................... 16 Figure 6. Sputtering general scheme .................................................................................................... 18 Figure 7. Ion attraction and sputtering ................................................................................................ 19 Figure 8. Self-bias formation ................................................................................................................. 20 Figure 9. Circular magnetron cathode scheme .................................................................................... 21 Figure 10. Scheme of the magnetron assisted sputtering reactor ........................................................ 22 Figure 11. Vibration modes of a-Si:H..................................................................................................... 24 Figure 12. Schematic of the evanescent wave formed at the internal reflection element sample surface. .................................................................................................................................................. 25 Figure 13. Transmission and reflection of a thin film sandwiched between two semi-infinite mediums (air and substrate) ................................................................................................................................. 28 Figure 14. Optical transmission spectrum of an a-Si:H film .................................................................. 29 Figure 15. Transmission spectrum provided with the envelopes ......................................................... 31 Figure 16. Polarization modification after reflection ............................................................................ 33 Figure 17. Types of structures (left) and the corresponding models (right) ......................................... 36 Figure 18. Illustration of contact angles formed by sessile liquid drops on a smooth homogeneous solid surface........................................................................................................................................... 37 Figure 19. A contact angle goniometer. ................................................................................................ 38 Figure 20. Principle of AFM ................................................................................................................... 39 Figure 21. Contact Mode. ...................................................................................................................... 40 Figure 22. Intermittent-Contact Mode.................................................................................................. 40 Figure 23. Non-Contact Mode. .............................................................................................................. 40 Figure 24. Schematic illustration of a rough surface. ............................................................................ 41
Chapter II Figure 1. The structure of an amino-acid. ............................................................................................. 46 Figure 2. Polypeptide showing peptide bond, and dipole moment of bond. ....................................... 47 Figure 3. Polypeptide indicating coplanar bonds (shaded region) and rotation bonds φ and ψ.......... 47 Figure 4. α-helix structure ..................................................................................................................... 48 Figure 5. Antiparallel beta sheet
Figure 6. Parallel beta sheet....................................................... 49
Figure 7. β-sheet structure .................................................................................................................... 49 Figure 9. 3D structure of Hen Egg
Figure 8. 3D structure of bovine lactoferrin ...................... 50
Figure 10. Basic steps of protein adsorption on a solid surface............................................................ 54 Figure 11. Schematic to show (a) a globular protein, whose conformation may be distorted on interaction with the surface and (b) a rod-like protein undergoing multistage adsorption process. .. 55 Figure 12. Illustration of protein-surface interaction in monolayer and multilayer ............................. 57 Figure 13. Illustration of adsorption from higher concentration and from low concentration onto surface with the same area ................................................................................................................... 57 Figure 14. Schematic diagram of FTIR/ATR flow cell. ............................................................................ 62
Chapter III Figure 1. Refractive index dispersions of all samples ............................................................................ 71 Figure 2. Measured thicknesses of all the films ................................................................................... 71 Figure 3. Static index and optical gap of all samples............................................................................. 72 Figure 4. IR-ATR Transmittance spectra of all samples. ........................................................................ 74 Figure 5. Areas under the wagging peaks in Fig. 4. ............................................................................... 74 Figure 6. Decomposition of the stretching band for the three hydrogenated substrates .................... 75 and the 2144 peak of the Si2H and Si3H substrates. ............................................................................ 75 Figure 7. Real (top) and imaginary (bottom) parts of pseudo-dielectric functions of the untreated and the hydrogenated substrates. ............................................................................................................... 77 Figure 8. Model used to analyze ellipsometry data. All layers are considered to have flat thickness. 78 Figure 9. Bulk (Db) and surface (Dr) thicknesses of all the films ........................................................... 78 Figure 10. Example of the fitted and experimental ellipsometry data of Si3H substrate. .................... 79 Figure 11. 2D cross sectional height profiles of the different substrates. ............................................ 80 Figure 12. The topographical AFM 2D images and the lines where the cross sections were taken ..... 81 Figure 13. RMS roughness values of all the surfaces ............................................................................ 82 Figure 14. Hydrogen atoms arriving on a rough surface target mainly the peaks, creating a shadow effect. .................................................................................................................................................... 83 Figure 15. Typical images of water drops on the surfaces .................................................................... 83 Figure 16. Contact angle values of all the surfaces ............................................................................... 84 Figure 17. Liquid droplet on a rough substrate. .................................................................................... 85 Figure 18. IR-ATR absorbance spectra of the amide I and amide II region, of all the substrates. ........ 87 Figure 19. the maximum of Amide II for the different samples. ........................................................... 89 Figure 21. Curve fitting of the amide I band of all the samples. ........................................................... 91 Figure 22. The β-sheet and the α-helix proportions of amide I peak on Fig. 21 ................................... 92 Figure 24. Model used to measure the adsorbed protein film thickness ............................................. 94 Figure 23. Ellipsometry data of all substrates before (black) and after (red) adsorption. .................... 95 Figure 25. The protein surface concentration for all the samples ........................................................ 96 Figure 26. Schematic representation of protein adsorption on a hydrophilic surface. ........................ 98 Figure 27. Protein adsorption on hydrophobic surfaces. ...................................................................... 98 Figure 28. Schematic representation of the amount of proteins adsorbed on the surface and the change of their shape as a result of the conformational change.......................................................... 99
List of tables Chapter II Table 1. The composition in amino acids of BSA protein (*presence of S in the molecule) ................ 52 Table 2. Atomic composition of BSA .................................................................................................... 52 Table 3. Band names and their corresponding wave numbers and assignments ................................ 60 Table 4. Assignment of bands associated with secondary components of amide I ............................. 61
Chapter III Table 1. Optical transmission results: Thickness (d), static index (n0), and optical gap (ET) ................. 70 Table 2. Post-deposition hydrogen pressure, areas under each peak .................................................. 75 Table 3. Root mean square roughness, roughness average, peak to valley and contact angle results 82 Table 4. Analysis of the secondary structure of proteins after adsorption on all substrates. .............. 90 Table 5. Cauchy fit parameters, thickness δ and surface concentration . ............................................ 94
General Introduction
General Introduction Protein adsorption on surfaces is not only a fundamental phenomenon, but it is also key to many bio-chemical and process industries. Adsorbed proteins form a bio-layer that is detrimental to the quality standards in industrial production, and are often associated with biological contamination as well as efficiency loss in industrial equipment. Considerable effort and costs are devoted to cleaning and maintenance of fouled process equipment. Therefore, knowledge of the mechanism of adsorption and the structure of the adsorbed
protein layers is important across diverse areas covering biology (protein chromatography, cellular adhesion), medicine (biomedical materials), food processing (stabilization of foams and emulsions, fouling of equipment) and biotechnology [1]. Wherever proteins come into contact with a solid surface, they are very likely to adsorb to it. This phenomenon includes a number of interactions at the solid–liquid interfaces [2, 3]. Protein‐surface interactions are fundamentally responsible for the biocompatibility of medical devices, or the lack thereof. When a solid material (e.g., a catheter, stent, hip joint replacement, or tissue engineering substrate) comes in contact with a fluid that contains soluble proteins (e.g., blood, interstitial fluid, cell culture media), proteins rapidly adsorb onto the surface of the material, saturating the surface within a time frame of seconds to minutes [4]. Therefore, when living cells (which are much larger than proteins and thus move much slowler) approach the material surface, they do not actually contact the molecular structure of the material itself, they rather contact and interact with the molecular structure of the adsorbed protein layer [4]. Such material is called a biomaterial which means a material (other than food or drugs) that is used and adapted for therapeutic and diagnostic application [5]. The areas that are covered by the use of biomaterials are orthopedics, cardiovascular systems, ophthalmics, dental restoration, and drug delivery systems [6]. The application that is relevant to this study is the detection of biomolecules through the use of biosensors. The most widely accepted definition of a biosensor is that it is an analytical device made up of a biologically active element, which can be an enzyme, an antibody, or a nucleic acid, with an appropriate physical transducer to generate a measurable biological response proportional to the concentration of chemical species in any type of sample, which is then converted to an electric signal by the transducer [7-12]. A biosensor consists of three main elements: a bioreceptor, a transducer, and a signal processing system. It can be classified by its bioreceptor or its transducer type. Biosensors can also be classified either by the type of biological signaling mechanism they utilize or by the type of signal transduction they employ. Transduction can be accomplished via a great variety of methods, 2
General Introduction which can be categorized in one of three main classes: Electrochemical Biosensors, Optical Biosensors, and mass detection methods [13].
Electrochemical Biosensors are the ones that are targeted by this study. Electrochemical sensors for multiple analytes were the first scientifically proposed (as well as successfully commercialized) biosensors [14-17]. At present, there are many proposed and already commercialized devices based on the electrochemical principle including those for pathogens and toxins [18]. The basic principle for this class of biosensors is that chemical reactions between an immobilized biomolecule and a target analyte produce or consume ions or electrons, which affects measurable electrical properties of the solution, such as an electric current or potential [15, 16]. The produced electrochemical signal is then used to relate quantitatively to the amount of analyte present in a sample solution. Potentiometry, amperometry, voltammetry, and, more recently, electrochemical impedance spectroscopic measurements are among the electrochemical detection techniques often used in conjunction with immunoassay systems and immunosensors, leading to their respective categories according to the type of signal measured.
The success of a biosensor depends on molecular recognition, which is the ability of one molecule to “recognize” another through bonding interactions and molecular geometry. It refers to the specific interaction between two or more molecules through both covalent and noncovalent binding such as hydrogen bonding, hydrophobic forces, Van Der Waals forces, and electrostatic Effects. The electrostatic interaction between charged molecules and oppositely charged surfaces is an effective approach exploited in molecular recognition phenomenon. Similar to this, hydrophobic interaction is a good alternative for the recognition of biomolecules with a lipophilic property [12]. The search for a suitable material, which has a high affinity for biomolecules and whose electronic and optical properties are well known, has motivated us to undergo this study.
Hydrogenated amorphous silicon (a-Si:H) and its alloys with oxygen, nitrogen, or carbon possess a lot of physical properties which would make these materials ideal candidates for biological applications such as advanced biosensors:
3
General Introduction (i)
They can be deposited on large areas and at low temperatures on almost any substrate material by standard plasma-enhanced chemical vapor deposition (PECVD) or sputtering processes.
(ii)
They are very flexible with respect to processing and structural modifications, including nanopatterning, and enjoy an overall compatibility with the established silicon technology.
(iii)
Their electronic and optical properties can be defined by doping and alloying to meet a broad range of requirements concerning electrical conductivity and optical transparency.
(iv)
Advanced applications such as photosensors, photovoltaic cells, thin film transistors in active matrix displays, or micro-electro-mechanical systems are by now well established on an industrial scale or are under investigation at the prototype level.
(v)
Compared to crystalline silicon, a-Si:H has the additional advantage of a strongly reduced thermal carrier generation under normal biocompatible conditions because of its larger band gap [19].
Silicon-based microelectronics and biosensors have undergone tremendous technical development but the bioactivity and biocompatibility of silicon is relatively not well understood. In fact, the surface biocompatibility of silicon is usually poor and the interaction between silicon-based biosensors and the human body may not be desirable [20, 21]. Longterm issues associated with the packaging and biocompatibility of Si chips has been identified to be a major hurdle [22].
Therefore, it is necessary to improve the bioactivity and biocompatibility of siliconbased microelectronic devices and biosensors to meet clinical needs. Some attempts have been made to improve the bioactivity and biocompatibility of silicon wafers. For instance, Canham reported that the wet etching of the surface of micro-porous silicon films could induce the formation of apatite [23].
Dahmen et al. have shown that surface functionalization of hydrogenated amorphous silicon (a-Si:H) and amorphous silicon suboxide films (a-SiOx:H) produced by a hydrosilylation reaction are largely biocompatible [24]. The samples of a-Si:H or (a-SiOx:H) were deposited by R.F. PECVD in a capacitively coupled reactor. The ability to form apatite 4
General Introduction on materials soaked in a simulated body fluid (SBF) is commonly used by biomedical researchers to evaluate its bioactivity. If a positive response can be induced, biocompatible Si biochips can be developed to bond directly with both living tissues and bone [22]. Moreover, a basic understanding of bioelectronic processes at the a-Si:H/organic interface is still lacking.
Proteins are present in all bodily fluids and thus coat materials regardless of the location of implantation. The dynamics and specificity of protein adsorption is influenced by the properties of the material surface such as charge, hydrophobicity, and roughness [25-28]. Adsorbed biofilms consequently function as a mechanism of recognition for the direction of biological responses to foreign materials. Proteins are well suited for this purpose as they contain a wide range of properties incorporated within hydrophobic, hydrophilic, cationic, and anionic domains. The proteins found in blood contain a diversity of chemical properties extensive enough to interact with all known materials surfaces. Detailed knowledge of the process of protein adsorption is therefore essential to the design of biosensing devices, and extensive research has been performed in this area [29].
Thus, this thesis hopes to achieve three objectives: find a way to improve the affinity of a-Si surfaces for protein molecules through the treatment of these surfaces by hydrogenation, study the effect of this treatment on protein adsorption at the solid/liquid interface, and show how the structure of the adsorbed proteins is modified, compared to their native state, by hydrogenation.
The first chapter of this thesis is devoted to amorphous silicon this films. It presents its characteristics, compared with crystalline silicon, the improvement of its properties by hydrogenation, its deposition techniques, and the characterization techniques that are used to analyze it.
The second chapter introduces the structure and the physical characteristics of proteins. It presents also the phenomenon of protein adsorption on solid surfaces and the parameters that influence it. Two analysis techniques that measure the adsorbed protein amount and analyze the structure of the biofilm on the surface will also be presented.
5
General Introduction The third and final chapter will regroup the results of the study of the effect of the hydrogenation of the a-Si surfaces on a multitude of parameters that play an important role in protein adsorption. It also reveals the results of the study of protein adsorption on a-Si surfaces in a comparative way which will show which is the most important factor in improving the surface affinity for protein molecules.
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General Introduction References [1] S. Seeger, M. Rabe, D. Verdes, Adv. Colloid Interface Sci. 162 (2011) 87–106. [2] W. Norde, T.A. Horbett, J.L. Brash, In: Proteins at Interfaces III: Introductory Overview, Chapter 1, American Chemical Society (2012). [3] A.G. Richter, I. Kuzmenko, Langmuir 29 (2013) 5167–5180. [4] A. Tathe, M. Ghodke A. Pratima Nikalje Int. J. Pharm. Pharm. Sci., 2 (2010) 19-23. [5] N. Peppas, R. Langer, Science, 263 (5154) (1994) 1715-1720. [6] Handbook of Materials for Medical Devices, ed. J.R. Davis, ASM International, (2003) 15. [7] M.A. Arnold, M.E. Meyerhoff, Crit. Rev. Anal. Chem., 20 (1988) 149–196. [8] S. Belkin, Curr. Opin. Microbiol., 6 (2003) 206–212. [9] B. R. Eggins, Chemical Sensors and Biosensors, Wiley (2002). [10] G. S. Wilson, R. Gifford, Biosens. Bioelectron., 20 (2005) 2388–2403. [11] J. S. Wilson, Sensor Technology Handbook, Elsevier, Amsterdam/Boston (2005). [12] K. Chandran, R. Rajkumar, K. Bhagrava, in: Biosensors and Bioelectronics, Chapter 1, Elsevier (2015) 2-66. [13] A. Touhami, in: Biosensors and Nanobiosensors: Design and Applications, (2014) 374403. [14] J. Wang, Chem. Rev., 108 (2008) 814-825 [15] D. R. Thevenot, K. Toth, R. A. Durst, G. S. Wilson, Biosensors & Bioelectronics, 16(12) (2001) 121–131. [16] X. Zhang, H. Ju, J. Wang, Electrochemical Sensors, Biosensors and their Biomedical Applications, Academic Press (2008) [17] J. Ding, W. Qin, Biosensors and Bioelectronics, 47 (2013) 559–565 [18] J. C.Vidal, L. Bonel , A. Ezquerra , S. Hernández , J. R. Bertolín, C. Cubel , J. R. Castillo, Biosensors and Bioelectronics, 49 (2013) 146–158 [19] X. Liu, R. K.Y. Fu, C. Ding, P. K. Chu, Biomolecular Engineering 24 (2007) 113–117. [20] K.D. Wise, K. Najali, in: VLSI in Medicine, Chapter 10, Academic Press, New York (1989). [21] M. Madou, M. J. Tierney, Appl. Biochem. Biotechnol., 41 (1993) 109. [22] L. Bowman, J. D. Mendl, IEEE Trans. Biomed. Eng., 33 (1986) 248. [23] L.T. Canham, Adv. Mater., 7 (1995) 1033.
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General Introduction [24] C. Dahmen, A. Janotta, D. Dimova-Malinovska, S. Marx, B. Jeschke, B. Nies, H. Kessler, M. Stutzmann, Thin Solid Films, 427 (2003) 201. [25] M. Luck, B. R. Paulke, et al. J Biomed. Mater. Res., 39(3) (1998) 478-85. [26] Q. Qiu, M. Sayer, et al., J Biomed. Mater. Res., 42 (1) (1998) 117-27. [27] Y. F. Dufrene, T. G. Marchal, et al., Langmuir, 15(8) (1999) 2871-2878. [28] S. L. McGurk, R. J. Green, et al., Langmuir, 15(15) (1999) 5136-5140. [29] S. Evan, Improving Biocompatibility by Controlling Protein Adsorption, University of Washington, USA (2009).
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Chapter I. Hydrogenated amorphous Silicon 1. Crystalline Silicon ......................................................................................................................... 10 2. Amorphous silicon ........................................................................................................................ 11 3. Hydrogen adsorption on Silicon surfaces ...................................................................................... 14 4. Silicon film deposition techniques ................................................................................................ 14
Chemical vapor deposition (CVD) .......................................................................................... 14
Sputtering .............................................................................................................................. 17
5. Characterization techniques........................................................................................................... 23
Infrared Spectroscopy ........................................................................................................... 23
Vibration modes of the Si-H bonds ....................................................................................... 24
Attenuated Total Reflectance FTIR-ATR Spectroscopy ......................................................... 25
Optical (UV-Vis-NIR) transmission ......................................................................................... 28
Determination of the refractive index and the thickness of the films .................................. 30
Determination of the absorption coefficient ........................................................................ 31
Spectroscopic Ellipsometry ................................................................................................... 32
Effective medium approximations ........................................................................................ 35
Contact Angle ........................................................................................................................ 37
Atomic Force Microscopy ...................................................................................................... 39
6. Rough surfaces .............................................................................................................................. 41
Mechanisms in surface growth ............................................................................................. 41
References ......................................................................................................................................... 42
1. Crystalline Silicon Studies on protein adsorption on solid surfaces have shown that structural changes of the proteins are induced by the physiochemical nature of the surface. Surface properties like its chemistry and topography are hugely important for the biological performance of materials [1,2]. Thus, the growth and characterization of surfaces are very important for this work. Hence this chapter will be devoted to the deposition and characterization techniques of silicon films, which was the chosen material for this work, with an emphasis on the techniques that probe the surface of the films.
1. Crystalline Silicon Silicon is the most widely used semiconductor in the electronics industry. First discovered by J. J. Berzelius in 1824, it has been the go to semiconductor since 1960 [3]. Silicon atoms organize themselves in a structure called “two interpenetrating face-centered cubic” primitive lattices, also called diamond lattice (Figure 1). The Si-Si covalent bond has a 2.5 eV energy with a length of 0.273 nm. The tetrahedral structure is imposed by the sp3 hybridization of each atom orbitals. It is formed with an atomic mass of 28.08 amu and can conduct a small amount of current [4].
Figure 1. Crystalline silicon 3D structure, called diamond.
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Chapter I. Hydrogenated amorphous Silicon The cube side for the crystalline silicon structure is 0.543 nm and the angle between two Si-Si bonds is 109.5° [3]. This type of bonds generate electronic states coiled by a periodic potential, leading to two continuous energy bands, a valence band VB and a conduction band CB, separated by a gap of about 1.12 eV, which is characteristic of crystalline silicon, see Figure 2.
Figure 2. Energy Band Diagram of crystalline Silicon.
2. Amorphous silicon Just like crystalline silicon, amorphous silicon is organized in a tetrahedral network and each atom is linked to four neighboring atoms by covalent bonds. Unlike the atoms of the crystal lattice, which occupy well-defined and periodic positions, amorphous silicon presents small variations in the length and the bond angle which will have the effect of destroying the lattice order after a few atomic distances. These variations will result in the breaking of the bonds and to defects in the atomic structure of the material. Dangling bonds, or uncoordinated silicon atoms, will form a continuous random network structure [5]. Figure 3 below represents a graphical representation of the difference between the structures of a-Si and c-Si.
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2. Amorphous silicon
Figure 3. 2D representations of a) crystalline silicon and b) hydrogenated amorphous silicon. The closed circles are silicon atoms and the open circles are hydrogen atoms [5, 6].
The concentration of these dangling bonds in a-Si is about 1019-1020 cm-3, which will have the effect of decreasing the photons absorption. The existence of these defects in the structure (dangling bonds) is manifested by the appearance of deep states in the gap. The loss of order cannot lead to a complete localization of all the states of the conduction or valence bands, and the fluctuation of the angles and length of bonds leads to the formation of band tails separated by the gap.
It was soon realized that the absorption of a-Si could be improved by introducing hydrogen to saturate these dangling bonds [7]. This was done by the dilution of silane (SiH 4) gas with hydrogen (H2) gas during plasma-enhanced chemical vapor deposition (PECVD) of a-Si, resulting in the deposition of hydrogenated amorphous silicon (a-Si:H) [8]. Thus, a SiH alloy is formed, with the Si-Si bonds being replaced by Si-H bonds. The Breaking of each SiSi bond results in formation of two dangling bonds which will then be passivated by hydrogen. This breaking decreases the structural disorder and thus the internal stresses of the material. Moreover, the incorporation of hydrogen lowers the average coordinance and makes
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Chapter I. Hydrogenated amorphous Silicon the amorphous network more flexible, resulting in a partial relaxation of the stresses and a decrease disorder. Hydrogen can be incorporated in a-Si in multiple forms:
Isolated Si-H bonds
A pair of hydrogen atoms interrupting a weak Si-Si bond (Si-H H-Si)
In the form of hydrogen clusters
The incorporation of hydrogen results in the replacement of the weak Si-Si bonds (2.5 eV) by stronger Si-H bonds (3.4 eV). This results in a larger separation between the bonding and anti-bonding electronic states, broadening both the conduction band and valence band. Thus, hydrogenated amorphous silicon will have an energy gap of 1.8-1.9 eV. The density of these band tails decrease as it extends deeper into the gap as seen in Figure 4, depicting the cSi, a-Si, and a-Si:H band structures [5, 9].
Figure 4. Schematic representation of the density of states of C-Si, a-Si, and a-Si:H
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3. Hydrogen adsorption on Silicon surfaces 3. Hydrogen adsorption on Silicon surfaces The interaction of hydrogen with Si surfaces is of considerable importance, e.g. for the growth of epitaxial Si layers by chemical vapor deposition [10]. The adsorption of atomic hydrogen on these surfaces is straightforward and well understood [11–14]. At low coverages, hydrogen atoms simply stick to the Si dangling bond and do not cause a breaking of Si–Si bonds. The induced structural changes of the surface, although important, can thus still be treated in terms of distortions from the equilibrium structure.
Due to the small sticking probability of H2/Si, quantitative adsorption experiments only started in 1994. The experiments clearly showed that the reaction dynamics of H2/Si involves an interesting new aspect. In the case of H2 adsorption on metal surfaces, the substrate was typically considered to be static, at least in a first approximation. However, this is not appropriate in the case of H2/Si because of the directionality of the covalent Si–Si and Si–H bonds. A very basic understanding of energy barriers and transitions states makes it necessary to take possible rearrangements of substrate atoms into account. For some experimental parameters, the reaction dynamics is even dominated by the substrate degrees of freedom [15].
4. Silicon film deposition techniques Various techniques for depositing thin amorphous and polycrystalline silicon films have been investigated, with chemical vapor based depositions currently proving popular. However, silicon films produced by direct current sputtering offer safety advantages, avoiding the use of toxic gases, and can be better suited to industrial scale, large area depositions. Sputtering also offers improved adhesion to the substrate as during the deposition the atoms have much greater kinetic energy than that of other processes [16]. The various deposition techniques will be presented and a thorough discussion of the technique that was used in this work will be provided below.
Chemical vapor deposition (CVD) This technique consists of putting a volatile compound of the materials on the substrate, with or without the presence of an external gas. A number of chemical reactions are then provoked, yielding at least one solid product on the surface. The other products must be in their gaseous form so that they can be phased out of the deposition chamber.
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Chapter I. Hydrogenated amorphous Silicon A number of constituents of a gas (SiH4, SiH6, SiHCl6…) could thus be thermally dissociated, and then react to form a solid film on the substrate. This technique is called Thermal CVD, in which a film is obtained by a chemical reaction between the vapor phase and the heated substrate. The CVD is a multidisciplinary field, bridging a number of chemical reactions, a thermodynamic process, and a transport mechanism. The chemical reaction is the central part; it determines the nature, the type, and the present species. There are two types of reactors: a hot wall reactor and a cold wall reactor. In the former, the wall is directly heated (>500°) and because the dissociation temperature of silane (SiH4) is superior to the exodiffusion of hydrogen we obtain films of poor quality. To solve this problem, it was found that the use of disilane or trisilane is more advantageous since their dissociation temperature is in the range of 350°C to 450°C [17]. The pressures are lowered as well to 75 mTorr, for which the depositions occur on the substrate as well as on the walls [18]. In the case of a cold wall, only the substrate is heated, so that the reaction is only effective on the heated substrate, and it‟s produced at atmospheric pressures (APCVD) which will allow for only a minimal use of gas and will yield films with few defects. The major drawback of this technique is the low deposition rate (1μm) [19]. The gas decomposition could be catalyzed by a light source that provides infrared or ultraviolet photons. This technique is called Photo-CVD, in which IR photons are generally produced by CO2 laser with a low energy (0.1 eV) which is not enough to dissociate the gas [20,21]. The rare species produced by this dissociation react with each other yielding a silicon powder deposited on the surface. The obtained films are porous and present poor electronic properties. With UV light, the molecules are dissociated either by direct photon absorption, or through collisions with excited particles [22]. The high cost and the low deposition rate are major drawbacks for this technique [23]. The most wide spread technique used in the deposition of hydrogenated silicon films is the Plasma Enhanced Chemical Vapor Deposition (PECVD). The film deposition takes place in a relatively high vacuum chamber (
