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Université d’Oran
Faculté des Sciences
Département de Physique
Thèse de Doctorat
Adsorption of BSA Protein on Silicon, Germanium and
Titanium Dioxide Investigated by In Situ ATR-IR
Spectroscopy
Présentée par
Ahmed Bouhekka
Acceptée sur Proposition du Jury :
Soutenue le 11 Mars 2013
Président, Prof. Aissa Kebab, Département de Physique, Université d’Oran, Algérie
Directeur, Prof. Jamal Dine Sib, Ecole Préparatoire en Sciences et Techniques d’Oran, Algérie
Co-directeur, Prof. Thomas Bürgi, Département de Chimie Physique, Université de Genève, Suisse
Examinateur, Prof. Saad Hamzaoui, Département de Physique, Université USTO, Algérie
Examinateur, Prof. Djamel Saidi, Département de Biologie, Université d’Oran, Algérie
Examinateur, Prof. Mohamed Kechouane, Faculté de Physique, Université USTHB d´Alger Algérie
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Adsorption of BSA Protein on Silicon, Germanium and
Titanium Dioxide Investigated by In Situ ATR-IR
Spectroscopy
Ahmed Bouhekka
Abstract
The adsorption of protein onto surfaces is not yet well understood. This phenomenon
is a very complex process, which is driven by different protein-surface forces. It is very
important to understand the behavior of protein on the solid surfaces especially under
different conditions because knowledge about it is crucial for many disciplines especially for
biocompatibility, food storage, medicine and biology.
Several techniques have been used to study this kind of adsorption in the last few
years and Fourier transformation attenuated total reflection infrared spectroscopy (ATR-
FTIR) is one of the most important tools used to investigate the solid-liquid interface. The
application of ATR-FTIR spectroscopy to protein analysis is based on the assessment of the
amide bands. ATR-FTIR is a powerful technique to determine the secondary structure of
adsorbed globular proteins.
The overall goal of this research effort was to the study the adsorption of Bovine
Serum Albumin protein onto different surfaces like: Silicon, Germanium and Titanium
Dioxide using in situ attenuated total reflection spectroscopy. Important parameters for
adsorption like pH, temperature and ionic strength are studied to evaluate their effect on
adsorption. One of the most important points in this research was to study the photo-
degradation of BSA onto TiO2 surface by UV illumination and to examine the changes in the
secondary structure of adsorbed BSA using curve fitting of the second derivative which
allows a direct quantitative analysis. Another point was to study the effect of visible light
irradiation on the secondary structure of BSA during rinsing with water. This latter has a
peculiar behavior especially on the interface between BSA and TiO2 surface that can affect
somehow the protein structure. In particular during irradiation with light the surface
iv
morphology is destroyed leading to the increase in the number of OH groups at the surface of
the adsorbed water.
This thesis consists of four chapters including necessary background, motivation and
the objectives of the research effort in Chapter 1 where we show the important properties and
applications of some well known semiconductors used in this work.
The second Chapter of the present manuscript deals with attenuated total reflection
infrared spectroscopy as a promising technique to probe solid-liquid interfaces in order to give
insight of the reactions occurring.
Results and discussion are divided into two Chapters: Chapter 3 provides the effect of
the environment on the adsorption of BSA onto solid-surfaces. Finally, the photo-degradation
and the denaturation of adsorbed Bovine Serum Albumin protein onto the surface of titanium
dioxide and the changes in the secondary structure are presented in Chapter 4.
v
Adsorption von BSA Protein auf Silizium, Germanium und
Titanium Dioxide Untersuchte In-situ-ATR-IR-
Spektroskopie
Ahmed Bouhekka
Kurzfassung
Die Adsorption von Proteinen an Oberflächen ist noch nicht gut verstanden. Dieses
Phänomen ist ein sehr komplexer Vorgang, der von verschiedenen Protein-Oberflächen
Kräften angetrieben wird. Es ist sehr wichtig das Verhalten von Proteinen auf festen
Oberflächen zu verstehen vor allem unter verschiedenen Bedingungen, da dieses Wissen von
entscheidender Bedeutung für viele Disziplinen ist speziell für Biokompatibilität, Lagerung
von Lebensmitteln, Medizin und Biologie.
Verschiedene Techniken werden in den letzten Jahren verwendet, um diese Art der
Adsorption zu untersuchen und Fouriertransformation abgeschwächte Totalreflexion
Infrarotspektroskopie (ATR-FTIR) ist eines der wichtigsten Hilfsmittel zur Untersuchung von
fest-flüssig Grenzflächen. Die Anwendung der ATR-FTIR-Spektroskopie zur Protein-Analyse
beruht auf der Einschätzung der Amid-Banden. ATR-FTIR ist eine wervolle Technik, um die
Sekundärstruktur der adsorbierten globulären Proteine zu bestimmen.
Das übergeordnete Ziel dieser Forschungsarbeit war es, die Adsorption von
Rinderserumalbumin Protein (BSA) auf verschiedenen Oberflächen wie Silizium, Germanium
und Titandioxid mit in situ abgeschwächter Totalreflexion-Spektroskopie zu studieren.
Wichtige Parameter für die Adsorption wie pH-Wert, Temperatur und Ionenstärke wurden
untersucht, um ihre Wirkung auf diese Art von Adsorption zu bewerten. Einer der wichtigsten
Punkte in dieser Untersuchung war es, den Foto-Abbau von BSA auf TiO2 Oberflächen durch
UV-Beleuchtung zu untersuchen und die Veränderungen in der Sekundärstruktur der
adsorbierten BSA mit Kurvenanpassung der zweiten Ableitung, die eine direkte quantitative
Analyse ermöglicht, zu studieren. Ein weiterer Punkt war, die Wirkung von Bestrahlung mit
sichtbarem Licht auf die Sekundärstruktur von BSA beim Spülen mit Wasser zu studieren.
vi
Diese Bestrahlung hat ein eigenartiges Verhalten insbesondere der Zwischenschicht zwischen
adsorbierten BSA und der TiO2-Oberfläche zur Folge, welche die Proteinstruktur beeinflussen
kann. Die Bestrahlung zerstört die Oberflächenmorphologie und führt zu einer Erhöhung der
Anzahl der OH-Gruppen an der Oberfläche.
Diese Arbeit besteht aus vier Kapiteln einschließlich dem erforderlichen Hintergrund,
der Motivation und der Formulierung der Ziele der Forschung in Kapitel 1, wo wir die
wichtigsten Eigenschaften und Anwendungen von einigen bekannten Halbleitern zeigen,
welche in dieser Arbeit verwendet wurden.
Das zweite Kapitel des vorliegenden Manuskripts beschäftigt sich mit abgeschwächter
Totalreflexion Infrarot-Spektroskopie als eine viel versprechende Technik zur Untersuchung
von fest-flüssig Grenzflächen, um Einblick in die ablaufenden Reaktionen geben.
Ergebnisse und Diskussion sind in zwei Kapitel unterteilt: Kapitel 3 befasst sich mit
der Wirkung der Umwelt auf die Adsorption von BSA auf feste Oberflächen. Schließlich
werden die Photodegradation und die Denaturierung von adsorbiertem Rinderserumalbumin
Protein auf der Oberfläche von Titandioxid und die Veränderungen in der Sekundärstruktur in
Kapitel 4 dargestellt.
vii
Adsorption de la Protéine BSA sur Silicium, Germanium et
Dioxyde de Titane Etudiée par Spectroscopie In Situ ATR-
IR
Ahmed Bouhekka
Résumé
L’adsorption de la protéine sur les surfaces n’est pas encore maitrisée. Ce phénomène
est assez complexe et contrôlé par différentes forces de protéine-surface. Il est très important
de comprendre le comportement de protéine sur la surface essentiellement sous différentes
conditions car sa connaissance est cruciale dans beaucoup de domaines notamment la
médecine, la biologie, la biocompatibilité.
Dans les dernières années, plusieurs techniques ont été utilisées pour étudier ce genre
d’adsorption dont l’atténué spectroscopie infrarouge de réflexion totale à transformé de
Fourier (ATR-FTIR) est la technique de choix pour étudier l’interface solide-liquide.
L’application de la spectroscopie ATR-FTIR pour analyser la protéine est basée sur
l’évaluation des bandes des amides. ATR-FTIR est une bonne technique pour déterminer la
structure secondaire des protéines globulaires.
Le but de cette recherche est l’étude de l’adsorption de la protéine Sérum Albumine
Bovine sur des surfaces différentes comme : Silicium, Germanium et Dioxyde de Titane en
utilisant l’atténué spectroscopie infrarouge de réflexion totale. Les paramètres importants de
l’adsorption comme : pH, température, force ionique sont étudiés pour évaluer leurs effets sur
ce genre d’adsorption. Le point le plus important dans cette recherche est la photo-
dégradation par la lumière ultraviolette (UV) de BSA adsorbée sur la surface de TiO2 pour
examiner les changements dans la structure secondaire en utilisant le fit de la dérivée seconde
qui permet une analyse quantitative. Un autre point est l’étude de l’effet de la lumière visible
sur la structure secondaire de BSA pendant rinçage par l’eau. Cette dernière a un
comportement curieux et surtout les monocouches adsorbées entre la BSA et la surface de
TiO2 qui peuvent perturber la structure de la protéine pendant illumination par une lumière
viii
extérieure ce qui va détruire la morphologie de la surface en augmentant les groupes OH de la
quantité d’eau adsorbée.
Cette thèse se compose de quatre chapitres, y compris de base nécessaire, la
motivation et les objectifs de l'effort de recherche dans le chapitre 1 où nous montrons les
propriétés et les applications importantes de certains semi-conducteurs bien connus et utilisés
dans ce travail.
Le deuxième chapitre du présent manuscrit traite la spectroscopie infrarouge de
réflexion totale atténuée comme une technique prometteuse pour sonder les interfaces solide-
liquide afin de donner un aperçu des réactions qui se produisent.
Les résultats et discussion sont divisés en deux chapitres: Le chapitre 3 présente l'effet
de l'environnement sur l'adsorption de BSA sur la surface du solide. Enfin, la photo-
dégradation et la dénaturation de la protéine Sérum Albumine Bovine adsorbée sur la surface
du dioxyde de titane et les changements dans la structure secondaire sont présentés au
chapitre 4.
ix
Keywords
Semi-conductors, Silicon, Germanium, Titanium Dioxide, Photocatalysis, Attenuated Total
Reflection Infrared Spectroscopy, Protein Adsorption, Solid-Liquid Interface, Protein
Denaturation, Protein Secondary Structure.
x
Acknowledgements
Any thesis demands a lot of effort and patient especially in working in different topics
between different groups and countries. The present manuscript is only a little of the whole
work we have done for more than seven years of research between the University of Oran
(Algeria), the University of Heidelberg (Germany) and the University of Geneva
(Switzerland). I believe that I will never be able to thank all the people who helped me during
these years but I am sure they understand me even I forget to mention their names here. I
would like to thank all people who helped and inspired me through this work.
I especially want to thank my two supervisors Professor Jamal Dine Sib and Professor
Thomas Bürgi. I am honestly very lucky to work with Prof. Sib for a long period of time and I
have to thank him for everything he has done for me since 2002, the first year of Magister
thesis, where he really gave me the independence in doing research. His encouragements and
supports during all the time I spent far from Algeria especially with the administration will
never be forgotten. I was always very happy to help people but I am sure I have never done
with them as my supervisor Prof. Thomas Bürgi did with me. He really believed in my
capacities since the first contact at the University of Neuchâtel (Switzerland) in my training
period and gave me the chance to finish this work in his group. His guidance during my
research and study at the University of Neuchâtel, Heidelberg and Geneva will stay in my
heart. His patience, enthusiasm and unfailing energy made this work possible, keeping me on
the right track all the time. I know that the words are not enough to thank you especially for
your humanity and simplicity…!
Thanks too much to all the members of the jury for accepting and contributing in the
exam of this simple research work. I would like to thank the president of the jury Prof. Aissa
Kebab from the department of physics (University of Oran Es-Senia) and the referees Prof.
Djamel Saidi from the department of Biology (University of Oran Es-Senia), Prof. Saad
Hamzaoui from the department of physics (University of Sciences and Technology-Oran) and
Prof. Mohamed Kechouane from the faculty of physics (University of Sciences and
Technology Houari Boumadian-Algiers).
I am grateful to the head of LPCMME laboratory at the University of Oran Prof.
Abdessamed Khlil for accepting me in his lab since 2001 and to all my professors especially
xi
Prof. Larbi Chahed and Prof. Yahia Bouizem for everything they have done for us during our
studies and research in the laboratory.
Many thanks to all my colleagues at the University Hassiba Ben Bouali of Chlef in
Algeria: Mahmoud, Khlifa, Mohamed, Abdelatif… especially Dr. H. Khalfoun and Dr. H.
Mahmoudi for their encouragements and for solving some of my problems with the
administration. Here I have special thank to M. Belaid who really did the best with me by
explaining all the rules and finding the best solution at the last time.
I would like to thank the University of Chlef for funding some parts of my research for
one month at the University of Neuchâtel and two months at the University of Heidelberg.
Also, I would like to thank all the members of DAAD -Deutscher Akademischer
Austauch Dienst- for giving me the opportunity to study in Germany by giving me DAAD
scholarship and funding my intensive German course at the Goethe Institute in Mannheim for
04 months and supporting my research at the Physical Chemistry Institute in Heidelberg for
around one year and half. Thanks a lot lady Anke Bahrani for everything you have done for
me.
Living in Germany gave me the chance to learn German language and to improve my
English. I would like to thank all my teachers at Goethe Institute in Mannheim, Max-Weeber
Haus and zentral Sprachlabor in Heidelberg especially Mr. Marco Kollemann who made
German very simple for me even if it was complicated.
I gratefully acknowledge the University of Geneva for financial support during my
studies in Switzerland and a lot of thanks to Isabel Garin for her efforts and helps by solving
all the administration problems for getting all the necessary papers.
Many thanks to all my friends in LPCMME laboratory in Oran: Hadj, Mokhtar,
Rachid, Djamel, Charef, Fouzia, Myrieme, Saadia…for many nice and funny moments we
spent together.
I am grateful to Leo, Thomas and Moutalib in Heidelberg and all my colleagues in
Geneva: Alastair, Stefan, Andrea, Patrick, Gerard, Igor, Harikrischna, Maschid, Birte,
Ugo…especially Alastair for giving me 10 minutes English every day and correcting my
pronunciation.
Finally, thanks a lot to all my friends especially Zahir and Miloud and all the members
of my family who were living for around three years in my small village -Melaab- far from
me in the space but you are always in my heart. Thanks for supporting me during all my
studies especially in the difficult periods.
xii
Table of Contents
Abstract..........................................................................................................................................iii
Keywords.......................................................................................................................................ix
Acknowledgments...........................................................................................................................x
Table of Contents..........................................................................................................................xii
General Introduction.....................................................................................................................15
References............................................................................................................................19
1 Properties, Characterization and Applications of Silicon, Germanium and Titanium
Dioxide................................................................................................................................21
1.1 Introduction...........................................................................................................22
1.2 Electronic Band Structure...... ...............................................................................22
1.3 Materials in Solid State..........................................................................................24
1.4 Bio-molecules Adsorption on Solid Semiconductors Surfaces.............................43
1.5 Conclusion.............................................................................................................46
References............................................................................................................................48
2 Liquid-Solid Interface and In Situ Attenuated Total Reflection Infrared Spectroscopy:
Case of BSA Adsorption onto surface.................................................................................53
2.1 Introduction...........................................................................................................54
2.2 Probing the Solid-Liquid Interface........................................................................54
2.3 Motivations and Principles of in Situ Study..........................................................55
2.4 Theory of Attenuated Total Reflection Infrared Spectroscopy.............................55
2.5 Modulation Excitation Spectroscopy.....................................................................60
2.6 Experimental Section.............................................................................................62
2.7 Proteins Structure..................................................................................................68
2.8 Protein Solid-Surface Interaction..........................................................................79
2.9 Conclusion.............................................................................................................79
References............................................................................................................................80
3 Environment Effect on the Adsorption of BSA Protein onto Solid Surface.......................87
3.1 Introduction...........................................................................................................88
3.2 Importance of Protein and Surface Properties.......................................................88
3.3 Thermal Modelling of Active IPEM ....................................................................55
4 Photodegradation and Denaturation by Light Illumination of Adsorbed BSA on the
Surface.................................................................................................................................49
4.1 Introduction...........................................................................................................50
xiii
3.3 Solutions and ATR-IR Study of Proteins..............................................................89
3.4 BSA in Different States.........................................................................................90
3.5 TiO2 Surface Characterized by SEM and AFM....................................................93
3.6 Adsorption of BSA onto TiO2 Coated Surface......................................................94
3.7 Adsorption of BSA onto Different Surfaces..........................................................97
3.8 pH Effect on the Adsorption of BSA onto TiO2..................................................100
3.9 Salt Effect on the Adsorption of BSA.................................................................104
3.10 Warm Water Effect on Adsorbed BSA...............................................................107
3.11 Water Interaction with TiO2 Surface and Adsorbed BSA...................................113
3.12 UV Modulation of Adsorbed BSA......................................................................115
3.13 Conclusion...........................................................................................................116
References..........................................................................................................................117
4 Photodegradation and Denaturation by Light Illumination of Adsorbed BSA on the
Surface of TiO2..................................................................................................................121
4.1 Introduction.........................................................................................................122
4.2 UV Photo-degradation of BSA over TiO2 Anatase.............................................122
4.3 Visible Light Denaturation of Adsorbed BSA onto TiO2....................................130
4.4 Conclusion...........................................................................................................140
References..........................................................................................................................142
General Conclusion and Recommendations...............................................................................145
References...................................................................................................................................148
Vita..............................................................................................................................................150
xiv
“It is not so very important for a person to learn facts. For that he does not really need
a college. He can learn them from books. The value of an education…is not learning
of many facts but the training of the mind to think something that cannot be learned
from textbooks.”
-Albert Einstein-
15
General Introduction
Knowledge is acquired through experience, everything else is just information!
Starting from this idea of the physicist Albert Einstein, we know that the human needs
increase from day to day and to improve our life we should solve the problems: socials,
economics, education,…The search for knowledge to establish novel facts, solve new or
existing problems, prove new ideas, or develop new theories is called research.
Scientific research provides scientific information and theories for the explanation of
the nature and the properties of the world. It makes practical applications possible. Scientific
research is funded by public authorities, by charitable organizations and by private groups,
including many companies. Scientific research can be subdivided into different classes
according to their academic and application disciplines.
Energy and health are critical determinants of human being life. Health is an important
enough aspect of energy policy to deserve a much greater influence on decisions about our
future personal, national, and global energy strategies. 2 billion people live in energy poverty
and insecurity. International institutions, such as the World Bank and WHO [1]!, have
repeatedly failed to make the connection between energy and health in their country work.
During the last decades, the energy requirements of our technical civilization have drastically
increased.
One of the most important huge future energy projects is DESERTEC between
Europe, North Africa and Middle East countries [2]. This project will provide Europe
countries with electricity from the huge deserts in Africa and Asia. The matter used to convert
solar energy into electricity is semiconductor especially silicon which is one of the most well
studied material in the field of solid materials. Other semiconductors can be used in the same
field like germanium and titanium dioxide. Research is developing and scientific fields of
Physics, Chemistry and Biology become more near to each other especially when considering
surfaces and interfaces. The interactions between bio-molecules like proteins and
semiconductors surfaces are a big challenge for scientists. Our work discusses the behavior of
BSA protein on different semiconductors surfaces but before giving more details what does
mean semiconductors and how about their importance in our daily life?
To understand the importance of semiconductors [3-5] let's first understand the
difference between electricity and electronics. Both are concerned with generating,
16
transferring, and utilizing electrical energy. The chief difference is that electricity is
concerned with using that electrical energy in power applications for heat, light, and motors
while electronics is concerned with power control and communications applications such as
electronic thermostats, electric motor speed control and radio. Engineering importance of
semiconductors results from the fact that they can be conductors as well as insulators.
Semiconductors are especially important because varying conditions like temperature and
impurity content can easily alter their conductivity. The combination of different
semiconductor types together generates devices with special electrical properties, which allow
control of electrical signals. Semiconductors are employed in the manufacture of electronic
devices and integrated circuits. Imagine life without electronic devices. There would be no
radios, no TV's, no computers, no video games, and poor medical diagnostic equipment.
Semiconductors can be used in a lot other areas especially in the environment. One of the
most useful materials, among many candidates for photocatalysts, is titanium dioxide which is
almost the only material suitable for industrial use at present and probably in the future. This
material can be also used in water cleaning and as a pigment [6-9].
Semiconductors solid-liquid interfaces especially in the presence of bio-molecules
play a fundamental role in nature and technology. Despite great importance in science and
industry, solid-liquid interfaces were always less studied than vacuum-solid interfaces. The
fundamental understanding of such processes requires information from different physical,
chemical and biological points of view and application of a wide variety of techniques [10,
11]. There are a lot of motivations and principles regarding the technique used and the
objective of the study. Probing the solid-liquid interface in the presence of proteins is a very
complex process that can give much important information about the behavior of these huge
molecules and their contact with the semiconductors surfaces under light shining using in situ
techniques. Protein adsorption on TiO2 is important because titan, which is covered by a TiO2
layer, is used as material for implants in medicine due to its inertness. TiO2 is also an efficient
photocatalyst and can be used for the decomposition of organic compounds under UV
irradiation.
Two public health interventions, clean water and vaccines, have had the greatest
impact on the world’s health. Vaccines prevent disease or death for millions of individuals
every year. Vaccine manufacturers and public authorities, e.g. World Health Organization
(WHO) [12], have established ambitious goals for enhancing present vaccines and for
developing new ones. New vaccine candidates have emerged over the past years against
17
allergic, infectious, autoimmune diseases, and for cancer and fertility treatment as well. In
most cases disease is the result of changes in the proteins structure of the cell and other human
organism. The diseases that arise from deposits of misfolded proteins are referred to as
protein-folding diseases. Neurodegenerative diseases are the third major group of age-related
diseases. This disease increases sharply with age and the most prominent and widespread of
these age-dependent disorders are Alzheimer’s disease and Parkinson’s disease [13-16].
The causal agent of the disease is a misfolded protein that is for one of a number of
reasons has altered its three-dimensional folded shape from one that supports its normal
function in healthy tissue to one that leads to disease. So, it is very important to understand
the changes (folding, unfolding, aggregation and denaturation) of the protein! [17-22].
The aim of this thesis is to study using in situ attenuated total reflection infrared
spectroscopy the adsorption and the behavior of biomolecules like Bovine Serum Albumin
protein on the surface of Silicon, Germanium and Titanium Dioxide. Proteins are huge
molecules that have so many functional groups and their adsorption is driven by different
forces including van der Waals, electrostatic and hydrophilicity. This kind of adsorption
happens on the surface and is still not well understood.
Besides this introduction and the final part containing the conclusions, this thesis is
divided into four main parts:
The first part (Chapter 1) deals with the properties, characterization and applications
of the semiconductors (silicon, germanium and titanium dioxide) used to study the
protein adsorption. We will focus more on the electronics properties and the states of
surface and defects that perturb somehow the electron transfer from the surface to the
adsorbed material.
The second part (Chapter 2) is focused on the detailed description of the in situ
attenuated total reflection spectroscopy used as a main technique in our work. After
introducing the essential part of the instrument and the theory of phase sensitive
detection, we will describe the technique used to prepare our samples of titanium
dioxide on silicon and germanium substrates. We will present the structure of the
protein used (BSA). Using water as a solvent is really a challenge for infrared
spectroscopy technique that is why we will mention the way to correct protein spectra
18
from water before doing analysis in order to get information about the changes in the
secondary structure of the protein.
The third part (Chapter 3) is devoted to present the results of the adsorption of
Bovine Serum Albumin on different surfaces: Silicon, germanium and titanium
dioxide. First, we will briefly explain the mechanism of adsorption of BSA onto solid-
surfaces where we show the effect of the surface. Afterwards, we will discuss the
effect of environment parameters including pH, temperature and the NaCl salt on the
adsorption of BSA. In particular, we will show the conformation of the protein under
varying the adsorption conditions.
The last part (Chapter 4) covers the important results of irradiating adsorbed BSA
using UV and visible light. The photo-degradation of BSA using UV is discussed in
the first part of this chapter. Then, we will elucidate the visible light denaturation of
the adsorbed BSA and the changes in the secondary structure using second derivative
fitting technique. An electronic explanation of the shift in infrared spectra during
visible light illumination is given in the last part of this chapter.
It is very important to conclude our manuscript by a general conclusion in which we
resume the most important results of our research contribution and the recommendations for
future research in the field of bio-macromolecules adsorption onto solid surfaces.
19
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[20] G. Hüttmann, R. Birngruber, On the possibility of high-precision photothermal
microeffects and the measurement of fast thermal denaturation of proteins. IEEE
Journal of Selected Topics in Quantum Electronics 5 (4) (1999) 954-962.
[21] T.Y. Tsong, R.L. Baldwin, Kinetic evidence for intermediate states in the unfolding of
Chymotrypsinogen A. Journal of Molecular Biology 69 (1972) 145-148.
[22] B.J. Bennion, V. Daggett, The molecular basis for the chemical denaturation of proteins
by urea. Proceedings of the National Academy of Sciences 100 (9) (2003) 5142-5147.
21
Chapter 1
Properties, Characterization and
Applications of Silicon, Germanium
and Titanium Dioxide
Semiconductors are very important in the field of electronics and physics because
changing temperature and impurities can easily alter their conductivity. They are used in
electronic devices and integrated circuits that improve our daily life. In this chapter, we give
an overview about the properties and characterization of semiconductors and their
applications. We focus especially on silicon, germanium and titanium dioxide used in our
work to study the adsorption of protein and its behavior under varying conditions.
22
1.1 Introduction
The knowledge in all the scientific fields is the power that can prove new ideas and solve
new or existing problems in order to improve our daily life. Research on semiconducting
materials started in the early nineteenth century. Since then, many semiconductors have been
investigated. The well known semiconductors are silicon (Si) and germanium (Ge). The bulk
crystal of Si or Ge consists of the periodic arrangement of a single atom whereas other
semiconductors like titanium dioxide TiO2 are built from two different elements. Such
composite materials have electrical and optical properties different from pure semiconductors
made of only one type of atom [1-4].
The periodicity of any crystal finishes at the surface. Thus the surface of any
semiconductor is different from the volume because of the dangling bonds and the defects.
The interface formed between semiconductor surface and the bio-molecules phases usually
has a higher standard free energy than the bulk phase. As a result, the interface is apt to be
thermodynamically stabilized by adsorbing any substances that are different from the solvent
molecules. The bio-molecules are more or less altered in their structures upon adsorption on
the surface of semiconductors and sometimes change their functions. The adsorption of
proteins on a solid semiconductor surface is a generally observed phenomenon in various
fields and the changes in their structures and functions upon adsorption as well as the
adsorbed amounts sometimes have a very important consequence. The adsorption behavior of
proteins at the solid/liquid interface and the mechanisms of interaction are very important
because of their applications in biofoulding, food processing and the construction of
biocompatible materials [5].
1.2 Electronic Band Structure
In an isolated atom, the electrons occupy atomic orbitals, which form a discrete set of
energy levels. When a large number of atoms are brought together to form a solid, the number
of orbitals becomes large and the energy difference between them becomes very small. Thus,
in solids the levels form continuous bands of energy rather than the discrete energy levels of
the atoms in isolation. These continuous bands are known as conduction band CB and valance
band VB separated by a forbidden band Eg as shown in figure 1.1 [6]
23
Figure 1.1 Formation of energy bands for electrons in a crystal (HOMO: Highest Occupied
Molecular Orbital, and LUMO: Low Unoccupied Molecular Orbital) [6]
The forbidden region is not accessible for electrons and it is called the gap Eg. The lowest
energy level of the conduction band is denoted EC and the highest energy level of the valance
band is named EV and Eg = EC-EV and its value is a characteristic of the material. Eg allows
the classification of solids as conductors, semiconductors and insulators.
24
1.3 Materials in Solid-State
Depending on the forbidden band, we can classify the materials in solid-state into three
groups: insulators, semiconductors and conductors. The semiconductors are materials having
an electrical conductivity in between the electrical conductivity of conductors and insulators
which means a forbidden band Eg larger than the Eg of conductors and smaller than Eg on
insulators (Figure 1.2) [7].
Figure 1.2 Representation of energy bands for insulator, semiconductor and conductor
At room temperature the conductivity of a semiconductor is very small. For a conductor, there
is no separation between valance band and conduction band (no energy gap). The conduction
band is partially occupied, resulting in a high electrical conductivity.
Table 1.1 shows the classification of conductor, semincoductor and insulator according to
their resistivity.
Table 1.1 Classification of materials according to their resistivity [7]
Material Conductor Semiconductor Insulator
Resistivity ρ (Ω.cm) 10-7
10-2
-109 10
14-10
19
~ 1 eV
Empty Conduction Band
Full Valence Band
Almost Empty
Conduction Band
Almost Full Valence
Band
Insultor Semi-conductor Conductor
Almost Full Conduction
Band
Valence Band
> 9 eV
25
1.3.1 The Semiconductors
A semiconductor [8, 9] that has no impurities is called intrinsic which means the holes
in the valance band are vacancies created by electrons that have been excited into the
conduction band.
The density of state N(E) gives the number of states (per unit volume and per unit energy)
between E and E+dE and represents the room available for electron in the conduction band
NC(E) and for holes in the balance band NV(E) given by:
Nc(E) = (1/2π2)(2mc/ ħ2)3/2(E-Ec)
1/2
[cm-3
/eV] Eq 1.1
Nv(E) = (1/2π2)(2mv/ ħ2)3/2(Ev-E)1/2
Where ħ = h/(2π) is the normalized Planck constant (h=6,626.10-34
Js) and mc (resp. mv) is the
average effective mass of the conduction band (resp. of the valence band). For a direct gap
semiconductor, mc (resp. mv) is the effective mass of an electron me (resp. a hole mh) in the
crystal.
The probability for an electron to occupy a level with a given energy E is given by the Fermi-
Dirac distribution function [7]:
F(E) = [1+exp((E-EF)/(kT))]-1/2 Eq 1.2
Where k=1,38.10-23
JK-1
is the Boltzmann constant, T the temperature and EF the Fermi
energy, which is the chemical potential for semiconductors.
The electrons density n [cm-3
] in the conduction band is given by:
n = Ecʃ+∞
Nc(E).f(E)dE Eq 1.3
And the hole density p [cm-3
] in the valence band writes:
p = - Evʃ-∞
Nv(E).[1-f(E)]dE Eq 1.4
The product of the two densities is:
np = n2 = p2 = ni2 Eq 1.5
26
Where ni is the density of intrinsic carriers (for Si at 300 °K, ni=1010
cm-3
).
For an intrinsic semiconductor (without impurities), each electron in the conduction band is
associated with a hole in the valence band. We conclude that the electron and hole densities
are equal as shown in figure 1.3
n = p = ni
Figure 1.3 Diagram showing the electronic bonds in an intrinsic semiconductor (Si)
An extrinsic semiconductor is a material that has impurities (doped) and electrons and holes
are supplied by these foreign atoms. These impurities modify the properties of this material,
making it suitable for electronic or optoelectronic applications.
The impurities are acceptors if they lack one or several electrons to realize a full bonding with
the rest of the crystal and the semiconductor is p-type. Electrons are said to be the minority
carriers whereas holes are the majority carriers.
In p-type material, extra holes in the band gap allow excitation of valence band
electrons, leaving mobile holes in the valence band.
In a n-type semiconductor, the impurities are called donor impurities since they have to give
an extra electron to the conduction band in order to make all the bonds with neighboring
atoms. Holes are said to be the minority carriers whereas electrons are the majority carriers.
In n-type material there are electrons energy levels near the top of the band gap so that they
can be easily excited into the conduction band.
27
1.3.1.1 Germanium
In the nineteen fifties, germanium, discovered by Clemens Winkler in 1886, used to be the
most frequently employed material. It has four valence electrons and it will at a given
temperature have more free electrons and a higher conductivity than silicon.
Germanium is the material of choice for systems operating in the far infrared wavelength
region, 8 to 12 microns. It is also useful at wavelengths down to 2 microns. This material is
quite stable in air up to 400°C when slow oxidation begins. Oxidation becomes noticeably
more rapid above 600°C.
Germanium is not toxic but broken Germanium is sharp and can easily cause cuts. The prism
of Germanium used as a substrate and as an infrared refractive element has a good
transmission in the middle infrared region as shown in figure 1.4
Figure 1.4 Transmittance spectrum of Germanium prism (5 cm x 2cm x 0.1cm)
Germanium exhibits low absorption of infrared radiation in the usable wavelength range of 2
to 12 μm. The band gap of 0.67 eV in Germanium is responsible for the increase in absorption
in the short wavelength region. The lattice (phonon) absorption bands are responsible for the
long wavelength absorption [10, 11].
One of the most important properties of Germanium is its high refractive index, making it a
very useful imaging component of IR systems operating in the 2 to 12 μm range (Figure 1.5).
28
Figure 1.5 Refractive index of Germanium
1.3.1.2 Silicon
Silicon was discovered in 1824 by J.J. Berzelius and has started to overcome all other
semiconductors since 1960, because it was significantly cheaper and less power-consuming.
Silicon is the most used semiconductor for electronics, partly because it can be used at much
higher temperatures than germanium. Silicon atoms form covalent bonds and can crystallize
into a regular lattice. The illustration below is a simplified sketch; the actual crystal structure
of silicon is a diamond lattice. This crystal (Figure 1.6) is called an intrinsic semiconductor
and can conduct a small amount of current [12].
Figure 1.6 Lattice and unit cell of crystal silicon
29
The main point here is that a silicon atom has four electrons which it can share in covalent
bonds with its neighbors. These simplified diagrams do not do justice to the nature of that
sharing since any one silicon atom will be influenced by more than four other silicon atoms,
as may be appreciated by looking at the silicon unit cell. Silicon crystallizes in the same
pattern as diamond, in a structure which Ashcroft and Mermin call "two interpenetrating face-
centered cubic" primitive lattices [12]. The lines between silicon atoms in the lattice
illustration indicate nearest-neighbor bonds (Figure 1.6). The cube side for silicon is 0.543
nm. Germanium has the same diamond structure with a cell dimension of 0.566 nm.
The crystalline structure of silicon gives continuous energy levels called conduction band and
valence band separated by a gap of 1.12 eV as shown bellow in figure 1.7.
Figure 1.7 Energy Band Diagram of crystal silicon
The silicon prism used in our work as substrate has a transmission in the middle infrared
shown in figure 1.8
30
Figure 1.8 Transmission spectrum of silicon prism (5 cm x 2 cm x 0.1 cm)
The crystal silicon is very expensive to be made that is why in the last years research is
focused on other structure of this material especially amorphous and microcrystalline silicon
for solar cells applications. The structure of any material strongly depends on the technique of
preparation. There are several methods used to prepare this material like: thermal evaporation,
sputtering and the most used is plasma enhanced chemical vapor deposition (PECVD) [13].
Amorphous silicon (a-Si) is a solid-state material made of silicon atoms which are arranged
on a lattice that has a certain short range order, but no long range order. Compared to
crystalline silicon (c-Si), the average bond angles between neighbouring atoms are distorted.
This material introduces a high level energy states in the forbidden gap and to reduce these
states the incorporation of hydrogen is suggested and the material is called hydrogenated
amorphous silicon (a-Si:H). In both materials some bonds are even broken and result in so-
called "dangling bonds". The presence of hydrogen atoms during the fabrication of
amorphous silicon material enables one to passivate a large part of these dangling bonds.
These two main "defects" of the lattice of a-Si:H – bond distortion and dangling bonds-give
rise to an electronic band structure containing localised states within the so-called "mobility"
bandgap (Figure 1.9) [14].
31
Figure 1.9 Density of states N(E) for intrinsic a-Si:H. Within the mobility bandgap (delimited
by EC and EV), the states are localised (dangling bonds and bandtails) [14]
The distortion of the bond gives a bandtails near the conduction and valence bands. The
electrons in the bandtails do not participate directly in the electronic transport because they
are localized. The dangling bonds (non-passivated) introduce deep levels (states) near the
middle of the bandgap. These dangling bonds (D.B) can be positive D+ (absence of electron),
neutral D0 (one electron) or negative D
- (two electrons). These bonds (D.B) behave as
recombination centers for free electrons and holes over D0/D
- and D
+/D
0. Thus, they affect the
electronic transport by influencing the total electric charge. In hydrogenated amorphous and
microcrystalline silicon (µc-Si:H) hydrogen atoms are very important to reduce the number of
defects (dangling bonds), in order to make this material suitable for use in optoelectronic
devices like solar cells and detectors.
32
The µc-Si:H and a-Si:H are related to each other. They are prepared by the same technique of
deposition. Only the deposition parameters (Temperature, silane concentration, plasma
frequency, pressure…) are adjusted to obtain one or the other.
µc-Si:H is considered as a (complex) mixture between crystalline silicon (c-Si) and a-Si:H
(figure 1.10) [15]. This material has characteristics different from those of a-Si:H.
Figure 1.10 Schematic representation of a µc-Si:H layer: pencil-like conglomerates ()
formed by a multitude of nanocrystals (), plus their corresponding boundaries [15].
Exposing hydrogenated amorphous silicon to light changes its electrical properties because
light induces a degradation of this material. One of the main advantages of µc-Si:H is its
stability under light exposure. On the other hand µc-Si:H is very sensitive to oxygen
compared to a-Si:H.
The forbidden band gives important electronic properties of any semiconductor. This gap is
affected by temperature as:
Eg (T) = Eg (0)-(αT2/(β+T)) Eq 1.6
Where Eg(0) is the gap at 0°K, T: temperature in °K, α and β constant (Table 1.2)
33
Table 1.2 Parameters of variation of the silicon and Germanium gap versus temperature [12]
Semiconductor Eg(0) (eV) α (eV/K) β (K)
Silicon 1.170 4.73 .10-4
636
Germanium 0.7437 5.405 .10-4
235
The figure 1.11 clearly shows the changes of the silicon and germanium gap as a function of
temperature. It is clear that at room temperature the gap of silicon is around 1.12 eV and the
one of Germanium is around 0.67 eV.
Figure 1.11 Variation of Si and Ge gap versus temperature
At higher temperature, we observe a significant decrease in the width of the forbidden band of
germanium and silicon.
For more information, we summarize in the following table 1.3 the physical and electrical
properties of silicon and germanium.
34
Table 1.3 Physical and electrical properties of Si and Ge at 300 °K
Semiconductor Silicon Germanium
Structure Diamond Diamond
Density (g/cm3) 2,328 5,3267
dielectric constant 11,9 16,0
Nc (cm-3
) 2,8.1019
1,04.1019
Nv (cm-3
) 1,04.1019
6,0.1018
Electron affinity 4,05 4,0
Gap energy at 300 °K (eV) 1,12 0.66
Intrinsic concentration of carrier (cm-3
) 1,45.1010
2,4.1013
Electron mobility (cm2.V
-1.s
-1) 1500 3900
Hole mobility (cm2.V
-1.s
-1) 450 1900
Refractive index 3,44 3,97
Atom concentration (cm-3
) 5. 1022
4,42.1022
1.3.1.3 Titanium Dioxide
Titanium dioxide is one of the most basic materials in our daily life. It is found in three
different crystallographic structures; rutile, anatase, and brookite [16]. Only rutile and anatase
surfaces have been studied in detail; brookite transforms into rutile at quite low temperatures.
Rutile has a primitive tetragonal unit cell and it has a gap of around 3 eV and anatase
crystallizes in the tetragonal system and it has a gap of 3.2 eV. Figure 1.12 shows the three
structures of TiO2.
Figure 1.12 The three structure of TiO2 a-Rutile, b-Brookite, c-Anatase
-a- -b-
-c-
35
Titanium dioxide (TiO2) has been studied extensively in the field of surface science due to the
possible applications in photocatalysis [17]. The photocatalytic activity of TiO2 surface is
mainly governed by the bridging oxygen vacancies which act as the adsorption sites for
different molecules. Liu et al. [18] have shown that the dominant defects in TiO2 surfaces are
Ti3+
defects and oxygen vacancies. TiO2 is a photocatalyst with high efficiency for the
decomposition of water [19-23] and the degradation of organic molecules [24-28].
TiO2 is inert, non toxic and cheap material. These advantages make it one of the most used
semiconductors in photocatalysis. One of its disadvantages is that this material does not
absorb the visible light because of its bandgap of around 3 eV [29]. It was reported that
anatase shows the better photocatalytic properties [30-32]. There are researches that classify
rutile as the most active photocatalyst [29].
To investigate the adsorption of protein in our present study, TiO2 anatase and
commercial type P25 TiO2 were used. This latter contains two crystallite forms anatase 80%
and rutile 20%. This material is very well characterized and is a standard material for many
applications. Figure 1.13 shows wide range of application of photoactivated TiO2.
Figure 1.13 Light-induced processes on TiO2
Activated TiO2
Photovoltaic
Phtoinduced Hydrophilicity
Special
Reactions
Phtocatalysis
Organic
Synthesis
Degradation of
Polluants
36
TiO2 Photocatalysis Mechanism
Photocatalysis is the acceleration of a photoreaction in the presence of a catalyst [33].
It is the ability of the semiconductor to stabilize the holders of photogenerated charges [34-
35] so they can react to the surface by reducing or oxidizing surrounding species. The
photocatalysis is used to reduce water to hydrogen, or to oxidize pollutants in the water, air, or
on the surface of self-cleaning systems.
Creation and evolution of electron-hole
Photodegradation of organic molecules by TiO2 is based on the absorption of an
ultraviolet radiation (a photon with energy bigger than the band gap of TiO2). This absorption
leads to the generation of electron hole pairs (equation 1). The excitation of the electron from
the valence band to the conduction band will create a hole in the valence band (figure 1.14)
[36-37].
Semiconductor + hν → é, h+ (≈fs time-scale) Eq 1.7
Figure 1.14 Scheme of photocatalysis process in a semiconductor like TiO2
Energy
37
Photoexcitation on semiconductor surfaces-basic principles
The photogenerated electrons and holes will then be able to evolve in several ways.
Figure 1.15 shows a schematic of the photoexcitation of a semiconductor solid particle by
exposure to radiation with energy above the bandgap [38]. The absorption of a photon
produces an exciton as shown by the star symbol in the figure 1.15. This phenomenon is
followed by charge separation (production of an electron–hole pair). The electron and the hole
can be recombined at the surface or in the bulk of the semiconductor (processes A and B in
figure 1.15). The charge carriers (electron and hole) can migrate to the surface of the particle
(processes C and D). This step is very important in the photocatalytic process because it leads
respectively to desirable reduction and oxidation reactions at the surface [39].
Figure 1.15 Schematic photoexcitation in a semiconductor particle followed by later events
[38]
The number of electron hole created depends strongly on the light flux used in the irradiation.
CB
é
h+
VB
hv
A
hv
+
-
-
-
+
+
Surface
Recombination
- +
Volume Recombination
A-
D
D+
C B
A
D
Hole
Electron
38
Surface recombination
The surface of any material represents an abrupt discontinuity from the bulk (lattice).
This discontinuity provides a high density of energy state in the surface. The defects present
(irregularities of the crystal) in the bulk and at the surface of the semiconductors are
associated with electronic states with an energy that differs from the one of pure
semiconductor [38].
The energy level of the states localized in the bandgap of the semiconductor. These states are
traps to charge transfer and avoid the recombination of charge carriers as shown in figure 1.16
[40]. Defects in the crystal structure are approximately of 1012
per cm-3
in the commercial
semiconductors. The nature and number of defect sites is a parameter which is difficult to
control and strongly depends on the method of synthesis of the material [37].
Figure 1.16 shows a schematic energetic picture of surface and bulk electron trap states.
These states exist in crystalline and amorphous materials. Surface oxygen vacancy defects and
defects in the crystalline lattice, in the case of colloidal TiO2, provide new localized energy
states not available in the perfect crystal.
Figure 1.16 Surface and bulk electron carrier trapping leading to an enhanced charge carrier
recombination rate and shorter hole lifetimes [38]
The recombination on the surface can be given by:
S = S0(N/Nset)α Eq 1.8
+
-
hv≥ Eg
Energy
Bulk Trap
Eg
+
Surface
Trap
39
Where the constant S0, Nset and α depend on the surface treatment and the passivation, which
means that addition of another thin film like SiO2 on the surface of semiconductor that
reduces the dangling bands will lead to a reduction of surface recombination [12, 41].
In the case of TiO2, the trapping of the carrier on the surface will lead to new defects,
Ti(OH)0+
and Ti3+
. These defects can trap other carriers as shown in the following equations
[36]:
Ti4+
(OH)o+
+ é→Ti4+
OH
Ti3+
+ h+ →Ti
4+
Ti3+
(OH) + h+ →Ti
4+OH
Under low light flux, the photocatalytic activity of TiO2 will be proportional to the intensity
of the irradiation [42]. A large luminous flux will in turn cause a high density of charge
carriers, leading to increased recombination rates: the photocatalytic activity then evolves as
the root of the intensity of irradiation [42].
Trapping of carriers at the surface
The surface of TiO2 presents some defects and end groups that modify the energy of the
crystal allowing trapping carriers. On the hydroxyl groups of the surface, the following
reactions can take place [36].
Ti4+
(OH) + h+→Ti
4+(OH)
o+ (10 ns)
Ti4+
(OH) + é →Ti3+
OH
(100 ps)
Recombination of charge carriers in the volume
The charge carriers, once generated, can recombine in the volume of the material
according to the equation [36]:
TiO2 + é + h+→TiO2 (200 ns)
40
Trapping of charge carriers in the volume
The trapping of electrons and holes in the volume is done according to different mechanisms.
Thus, the electron reacts with titanium in the crystal lattice:
Ti4+
+ é →Ti3+
(10 ns)
These centers Ti3+
are the source of the blue color that TiO2 takes when it is irradiated
by UV in an environment where no electron trap is available [43].
Moreover, these Ti3+
centers will themselves be able to serve as traps for holes:
Ti3+
+ h+ →Ti
4+
A solution to increase the lifetime of the carriers is to improve the crystallinity
of the material [44], which reduces the number of defects, and therefore the number of
recombination centers. Therefore, the photocatalytic activity is increased [45].
Shockley-Read-Hall recombination model
Figure 1.17 Schematic of four electronic transition processes that may occur and which relate
to charge carrier recombination at trap sites. CB: conduction band, VB: Valence band and E t
is an energy level in the gap
The trapping of electrons and holes in the semiconductor was studies by Shockley, Read and
Hall [46]. Figure 1.17 shows four indirect electronic transition processes.
Electron Capture Electron Emission Hole Capture
VB
CB
Et
VB
CB
Et
VB
CB
Et
VB
CB
Et
-
- -
+ +
Hole Emission
41
Process 1: illustrates electron capture from the conduction band by a recombination center.
Process 2: indicates the rate of emission of electrons from the recombination center; under
equilibrium conditions this rate will be equal to the electron capture rate.
Process 3: represents a hole capture process where a trapped electron recombines with a hole
in the valence band.
Process 4: is termed hole emission and describes the excitation of an electron from the
valence band to an electron trap state, leaving a hole in the valence band [39].
Reactions between the trapped carriers at the surface and species outside
The mechanism that we seek to exploit is the reaction of the radicals formed
on the TiO2 surface with their environment, namely the adsorbed molecules. Thus, these
chemical species will undergo oxidation-reduction reactions that may, in the case of
organic molecules, lead to their degradation.
Thus, the electrons will react with electron acceptors, such as dioxygen, to
form superoxide radicals, or even hydrogen peroxide:
Ti3+
+ O2 →Ti4+
+ O2
o- [47]
Ti4+
O2o-
+ H2O →Ti3+
(OH)+HO2o
[47]
2HO2o → H2O2 + O2
[36]
The holes for their part will react with electron donors, as the organic compounds
noted here R, or water. The radicals OH° and R°+
are formed on the surface of
TiO2 and may spread into the environment. The detection of these species need
traps using molecules, which facilitate the observation of radical species [48].
Ti4+
(OH)o+
+ R → Ti4+
OH + Ro+
Ti4+
(OH)o+
+ H2O → Ti4+
OH + HOo + H
+
The two equations above show two degradation processes:
42
A direct degradation, by oxidation of an organic molecule adsorbed at the
surface of TiO2
An indirect degradation, where the oxidation of pollutants is performed through
hydroxyl radicals, very strong oxidants, formed on the surface of the semiconductor.
If both types of charge carriers can lead to mineralization of organic molecules, degradation
primarily involves the holes: their transfer time is much shorter than the reactions involving
electrons. Moreover, the existence of these two degradations processes implies that the
mechanisms of photocatalysis will not be the same throughout the systems studied. Indeed,
if the molecules to degrade are distant from the photocatalytic material (as in the case of
the air pollution control), the indirect mechanism via hydroxyl radicals will be
preponderant, while if the pollutant is in contact with TiO2 (by example during the
water pollution control), the share of direct degradation mechanism will be significant [36].
One of the disadvantages of TiO2 is its absorption in the only UV region. To increase the
photocatalytic activity of this material doping is a suggested solution.
Doping of TiO2
We can modify the physicochemical properties of TiO2 by doping this material with other
different material. This will increase the absorption region towards the visible spectral range
and enables a more efficient use of sun light as a source of irradiation.
Doping TiO2 with another semiconductor will increase the spectral range of irradiation.
Depending on the illumination radiation we can distinguish two cases. If the energy is larger
than the band gap of both semiconductors we can excite both. For example, using CdS (gap of
2,4 eV) as a doped material and shining with an energy more than 3 eV we can excite TiO2
and Cds as shown below [37]:
CdS (é + h+) + TiO2 (é + h+) →CdS (h+, h+) + TiO2 (é, é) Eq 1.9
This behavior is summarized in the figure 1.18-a
43
Figure 1.18 Exciting two semiconductors in the composite of TiO2: a-Using UV light and b-
Using visible light [37]
In this case the photocatalytic activity of some organic molecules will be increased by a factor
of 1.5 to 4 compared to the case of TiO2 alone [49-51].
Using an irradiation energy corresponding to the semiconductor used for doping will lead to
exciting only this latter as shown in the figure 1.18-b. There are a lot of materials that can be
used for doping TiO2 like Gold, metal, nitrogen depending on the objective and applications.
The properties mentioned above of any semi-conductor solid-surface clearly show that it is
very difficult to control well the processes happening on the surface, which is particularly also
true for the behavior of bio-molecules like proteins adsorbed on these surfaces.
1.4 Bio-molecules Adsorption on Solid Semiconductors Surfaces
To explain the adsorption phenomenon, it is easy to imagine a simple fixation of gas
molecule on a solid surface. This molecule can maintain a fixed neutral towards its support or
react with it [7, 52, 53].
Gas Gas
............ ..............
Solid 1 Solid 1
Gas Gas
............. ................. ...............
Solid 1 Solid 1 Solid 2
Adsorption-Desorption
Adsorption-Desorption Reaction
44
We can distinguish two types of adsorption phenomena: Physisorption and chemisorption.
These processes are successive and well controlled by the thermodynamic conditions. There is
no electron transfer in the case of physisorption, however an electron can be transferred from
the gas to the solid or the opposite in the case of chemisorption [7].
Gas Gas+
Gas-
............ ............... ................ or ...............
Solid Physisorption Gas-Solid Chimisorption Solid-
Solid+
The adsorption process happens on the surface which plays an important role in understanding
the reversibility or no of such behavior. The huge molecules like proteins can also get
adsorbed on the solid surface but the mechanism of adsorption is not really clear because of
their complex structure.
1.4.1 Kinetic of Proteins Adsorption on Semiconductors Surfaces
Before adsorbing onto a solid surface, the protein can undergo several steps [54].
These steps are schematically presented in the figure 1.19.
Step 1: Random transport of a protein from solution towards solid surface by diffusion and
convection.
Step 2: Attachment of a protein at a surface which is driven by a decrease of the Gibbs energy
in the system.
Step 3: Conformational changes of the protein at the surface. Over time, the number of
interaction points is further increased. Changes in conformation can occur immediately during
adsorption or slowly over time after the protein has attached to the surface. The changes of
conformation depend strongly on the concentration of protein at the surface. These changes
are suggested to be higher at low surface-coverage, where the protein has enough free space,
because other molecules are believed to have an effect on the protein to adopt different
conformation.
45
Step 4: This step describes detachment of a protein from the surface. Due to a high number of
interaction points with the surface, this phenomenon is less probable for unfolded proteins
than for native ones. An important force is required for breaking all the segments formed
between the adsorbed protein and the surface. The adsorption of protein is only partial
reversible. Proteins undergo structural changes due adsorption and they get attached with
many segments to the surface. Changing pH or increasing ionic strength may lead to
desorption of proteins.
Figure 1.19 Schematic representation of the protein adsorption process. Hydrophobic (///) and
hydrophilic (...) and charged groups of the protein molecule and on the sorbent surface are
indicated. Taken from [55]
Step 5: Transport away from the surface to the bulk of the solution is just the reverse of step
one. After desorption, the protein goes away into the solution and it might have similar
behavior of all the steps mentioned here. It is possible that the desorbed proteins have an
altered structure compared with the native states. In many cases the desorbed proteins may
adsorb again. In other cases the protein can recover their native conformation [55, 56].
46
Not all protein molecules that arrive the surface adsorb. Consequently, slower than expected
rates indicate that there is an energy barrier for adsorption. Before the protein can be adsorbed
onto the surface both, protein and the surface, have to be dehydrated because the
hydrodynamic affect might create a barrier. More addition to this, the protein has to be in the
proper spatial orientation towards the surface, and the electrostatic interaction (repulsion and
attraction) needs to be overcome. If there is no barrier for the adsorption, diffusion can be rate
determining.
The driving forces which facilitate protein adsorption on a surface were discussed by many
authors [43-45]. But till our days this behavior is still not really well understood.
1.5 Conclusion
Semi-conductors are very important in the electronic field because of their special
properties under varying conditions like temperature. Irradiating the semi-conductor with light
of energy more or equal to its forbidden band leads to the photogeneration of electron hole
pair. All the applications of these materials are based on this photogenration especially in the
solar cell applications. The defects coming from the environment and the technique used to
prepare these materials perturb somehow their properties. Defects present in semiconductors
introduce energy levels in the gap that can capture electrons. More addition to this the surface
of any material is a special case because it is different from the bulk and all the reactions
taking place on this surface are difficult to be controlled in a good way.
Photocatalysis based on titanium dioxide consists of the oxidation of organic
molecules until the formation of H2O, CO2, of volatile compounds or salts. This reaction is
possible due to the photogeneration of carriers in TiO2 that may be involved in redox
reactions.
The mechanism of photocatalysis, step by step, is as follows:
TiO2 absorbs light radiation (UV) of energy greater than its energy bandgap, which
allows to generate electron-hole pairs;
A portion of the charge carriers migrate in the crystal until it reaches the surface.
During the diffusion processes the charge carriers can be trapped, recombine with each
other (in the bulk or at the surface) and the rest react with the adsorbed species.
47
The holes oxide directly the adsorbed molecule on the surface of TiO2, or indirectly
via the formation of HO° molecule that diffuse to the organic molecule.
The best way to control surface reactions is in situ measurements because ex situ ones can
give us only the final results of this behavior and it is therefore difficult to learn something
about the real processes. The next chapter will deal with in situ attenuated total reflection
spectroscopy used for investigating the adsorption of BSA protein onto different surfaces.
48
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53
Chapter 2
Liquid-Solid Interface and in Situ
Attenuated Total Reflection Infrared
Spectroscopy: Case of BSA adsorption
onto Surface
There are four well known states of matter that are commonly recognized in the
Universe, solid, liquid, gas and plasma. However, on earth only solid, liquid, and gas are
common. The solid-liquid interface has different properties from the two phases (solid,
liquid). How can we study this contact? In this chapter, we will give an overview about the in
situ investigation of solid-liquid interfaces using attenuated total reflection spectroscopy.
54
2.1 Introduction
Infrared spectroscopy is a widely used technique that for many years has been an
important tool for investigating chemical processes and structure [1]. Knowledge about solid-
liquid interface is so important and plays a fundamental role in nature and technology
especially in the field of metal oxide-aqueous solution interfaces that have attracted great
attention because of their importance in several fields ranging from heterogeneous catalysis,
atmospheric chemistry, corrosion, implants and adhesion to metal oxide crystal growth [2].
Despite great importance in science and industry, solid-liquid interfaces were always less
studied than vacuum-solid interfaces. The choice between these two was very often explained
by limitation of tools suitable for exploring solid-liquid interfaces and not so much by
scientific arguments that solid-liquid interface are more complex and therefore more difficult
to understand. The fundamental understanding of such processes requires information from
different physical and chemical points of view and application of a wide variety of techniques
[3].
Attenuated total reflection spectroscopy (ATR-FTIR) is an important tool for
investigating solid-liquid interface to analyze the chemical reactions taking place at the
interface [4]. ATR-IR allows us working under different conditions like: pH, temperature,
different concentrations, following the photo-degradation of molecules using an exterior UV
illumination and the visible light irradiation. This technique allows doing measurements
during the real conditions of the experiment that can easily be changed in the ex situ
measurements. However using ATR-IR for investigating proteins adsorption in water solution
is a challenge because of the absorption of water in the same region of proteins as we will see
in the next parts of this manuscript.
2.2 Probing the Solid-Liquid Interface
The fundamental understanding of the processes occurring at the solid-liquid interface
needs analytical techniques which are sensitive towards the interface. Several techniques are
available to study surfaces ex situ after removing the liquid, rinsing the surface and subjecting
the solid to ultra-high vacuum prior to analysis like, X-ray (XPS) and ultra violet
photoelectron spectroscopy (UPS), electron energy loss spectroscopy (EELS), Auger electron
spectroscopy (AES), low-energy electron diffraction (LEED), field emission microscopy
55
(FEM), scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The big
problem of these techniques is the dramatic conditions needed for the analysis which are far
away from the conditions where the process under investigation is observed normally. More
addition to this, the surface can be changed between the real experiment and the analysis. The
best solution is to study this behavior under real conditions during the same experiment itself
using in-situ techniques [2, 3].
2.3 Motivations and Principles of in Situ Study
One of the most important motivations to use in situ techniques is the fact that the
properties of an interface during the process of interest may be different from those
determined during analysis beforehand or afterwards. Therefore, information we obtain in situ
is more exact. Applying infrared spectroscopic methods to solid–liquid and in particular
solid–water interfaces is a big challenge due to the strong absorption of the solvent. In situ
investigation allows us to follow the real processes occurring at the interface and its evolution
during measurements because there are some molecules that can be found only on the surface
[5-8].
To probe solid-liquid interfaces in situ the following techniques have found
application: Infrared spectroscopy (IR), X-ray absorption spectroscopy (XAS), sum frequency
generation (SFG) and surface enhanced Raman spectroscopy (SERS). A promising method to
identify species at the interfaces in situ during reactions and to unravel their fate is attenuated
total reflection infrared (ATR-IR) spectroscopy [3].
ATR-IR spectroscopy is a good technique to study interfaces [7-8]. The vibrational
spectrum contains detailed information about interaction modes between surface and
adsorbate, orientation of the molecules and intermolecular interactions within the adsorbate
layer [3]. The contact between semiconductors surfaces like TiO2 and liquid phase play an
important role in photocatalysis especially in the presence of proteins which are considered in
this work.
2.4 Theory of Attenuated Total Reflection Infrared Spectroscopy
Jacques Fahrenfort and N.J. Harrick [9, 11] devised the theories of attenuated total
reflection (ATR) spectroscopy and suggested a wide range of applications.
56
In the presence of two different mediums, internal reflection can occur when the angle of the
refracted beam θt is larger than the angle of incidence θi. According to Snell’s law the
refractive index of the medium two must be smaller than that of medium 1 (refractive index n2
< n1) see Figure 2.1. Total internal reflection occurs when the θi of the beam exceeds the
critical angle θc. Equation 2.1
sin(θc) = n2/n1 = n21 Eq 2.1
n1>n2
θ>θc
Figure 2.1 Schematic representation of the path of a ray of light for total internal reflection
where n1 and n2 represent refractive index of dense and rare medium respectively, θi is the
angle of incidence and θc the critical angle.
When the light undergoes total reflection at the interface of the two mediums (θ > θc), an
electric field is formed at the reflection points that penetrate into the rare medium. This
electric field is referred to as evanescent field and is derived from the Latin root evanescere,
meaning “to tend to vanish or pass away like a vapor” [12].
Figure 2.2 shows a transverse standing wave totally reflected at the interface. This wave is
best described as the interference wave of the incident and reflected waves [13]. The most
important characteristic properties of the evanescent field are:
1) The field intensity in the medium of lower refractive index is nonzero, and there is an
instantaneous normal component of energy flow into this medium whose time average is zero.
Thus, there is no energy loss and the propagating radiation in the denser material is totally
internally reflected.
2) The evanescent field is a nontransverse wave that has vector components in all spatial
directions. This is a very unique feature and has many implications.
3) Intensity of the field decreases (exponentially) with increasing distance into the medium,
normal to its surface. Therefore, the field exists only near the vicinity of the surface.
n2
n1 θ
IR beam
57
4) A nonzero energy flow parallel to the interface results in a displacement of the incident and
reflected waves. This is known as the Goos-Hanchen shift [12, 14]. Many efforts have been
made to investigate the correlations between this shift and the depth of penetration or
effective thickness, but nothing conclusive has resulted.
Figure 2.2 Schematic of the evanescent wave formed at the internal reflection element sample
surface. dp is defined as the penetration depth
The amplitude of the evanescent electric field decays exponentially with the distance z from
the interface as shown in Equation 2.2
E = E0exp(-z/dp) Eq 2.2
Where, E0 is the electric field amplitude at the interface which depends on the angle of
incidence, polarization of the field and refractive index. The depth of penetration dp is defined
as the distance required for the electric field amplitude to fall to e-1
of its value at the surface.
dp = λ1[2π (sin2(θ)-n221)
1/2]-1 Eq 2.3
58
λ1 = λ/n1 wavelength in the optical denser medium, θ angle of incidence and refractive index
n21 = n2/n1. Depending on the refractive index of the IRE and medium 2 and the angle of
incidence the values of dp is in the range of 0.1-1.3 μm for infrared radiation [3].
For attenuated total reflection (ATR)-IR spectroscopy internal reflection elements (IREs) like
germanium and silicon are used as the denser medium that has a high refractive index. The
IRE should be transparent in the infrared region to allow the beam arriving to the interface.
The light propagates through the IRE striking the element at θi. At the point of reflection in
between the two media, an evanescent electromagnetic field is generated that penetrates into
the sample. If the medium adjacent to the IRE (the sample) is absorbing the reflection is not
total anymore, since the reflected beam is attenuated. Therefore a spectrum can be obtained
when the light is attenuated by the sample.
Figure 2.3 shows clearly the principle of this phenomenon where a powder thin film catalyst
was deposited on Ge internal reflection element in contact with liquid phase.
Figure 2.3 Principle of attenuated total reflection (ATR) spectroscopy. The evanescent field
formed at the interface between internal reflection element and adjacent medium is used for
spectroscopy [15]
Attenuated total reflection infrared (ATR-IR) spectroscopy is an ideal tool that allows
studying the processes which take places at the catalytic interfaces which are the key of
heterogeneous catalysis. This technique can be used to study thick or highly absorbing solid
and liquid materials, including films, coatings, powders, threads, adhesives, polymers and
aqueous samples. Increasing the number of reflections at the interface leads to achieve the
59
sensitivity of ATR-FTIR technique. ATR-IR has a lot of applications in the fields of catalysis,
polymers, environmental science biology and pharmacy [16-20]. It has been used to study
solid-liquid and gas-solid interfaces, even at high pressures and temperatures.
A summary of different kinds of materials used as infrared element (IRE) is shown in table
1.1. Depending on the geometry of the IRE, the beam of infrared can be totally reflected once
or several times before it leaves the IRE. If the sample does not absorb IR then no energy is
lost, but when absorption occurs at the interface, the evanescent field is attenuated and the
infrared spectrum of the sample is generated.
The penetration depth (dp) of the IR beam determines the volume (small) that can be probed
near IRE’s surface. This volume gives advantages to IR spectroscopy in the ATR mode
compared to the transmission mode where the beam goes directly through the sample.
Especially in studying liquid systems where the solvent (like water) has a high absorption in
the IR range. The advantages of the ATR-IR technique makes it a promised tool to investigate
the solid-liquid and solid-gas interfaces in the fields of catalysis, biochemistry,
electrochemistry...
Table 2.1 Physical properties of the most common materials used as internal reflection
elements in ATR-IR spectroscopy [2]
1) The critical angle was calculated assuming a refractive index of 1.4 for the optical thinner
medium.
Material Mean Refractive
Index (n1) at 1000
cm-1
Useable
Transmission
Range (cm-1
)
Critical
Angle (θc)
Chemical
Properties
Diamond, C 2.4 2500-45000 36 Insoluble in
water, acids and
bases
Ge 4 870-5500 21 Hard, brittle,
chem. stable
ZnSe 2.4 650-20000 36 Hard, brittle,
soluble in acids
ZnS 2.2 950-17000 40 Soluble in acids
Si 3.5 4000-1000 - Hard, brittle,
chem.stable
60
2.5 Modulation Excitation Spectroscopy
Our system (Ge, water, TiO2, Protein) is very complex especially under shining light.
Investigating such a system using ATR-FTIR is a challenge because many species are present
simultaneously and the technique is not intrinsically selective. All the components that absorb
IR irradiation give rise to signal in the spectrum. This problem is particularly pronounced in
photocatalysis where a lot of species exist during the reaction taking places at the interface.
The number of components is not the only problem in this study. But it is very important to
distinguish between active species (involved in the reaction) and the inactive ones
(spectators). To solve this problem, one should improve the sensitivity and the selectivity and
the promising method is modulation excitation (ME) spectroscopy. This method (ME) is a
sensitive technique that can be applied for the investigation of reversible systems periodically
stimulated by modulating an external parameter [3].
2.5.1 Theory of Phase Sensitive Detection
Using modulation excitation (ME) technique we can distinguish between active and
spectator species present in the physical/chemical phenomenon to be investigated. To achieve
this objective, it is very important to perturb periodically the system with an external
parameter. This perturbation is called stimulation which is chosen so that it can influence the
kinetics of the species in the system we want to see.
Stimulating a system by an external periodic perturbation (concentration, temperature,
light, pH…) will lead to some changes in the behavior of the components present in this
system. All the active species that are affected by this stimulation will change periodically at
the same frequency as the perturbation (ω) or harmonics therefore (2ω, 3ω…). In the
beginning of the stimulation, the affected species relax to new quasi steady-state values
around which they oscillate. The response of these active components shows a frequency-
dependent amplitude and phase delay with respect to the stimulation.
When the response of the periodically oscillating parameter is denoted as A(t), the
phase-domain response at the fundamental ( k =1, k : demodulation index) and harmonic ( k =
2,3,…) frequencies are obtained by phase sensitive detection (PSD), or so-called
demodulation, according to eq. (1.1) [21, 22].
Ak(ϕkPSD) = (2/T) 0ʃ
TA(t)sin(kωt+ ϕkPSD)dt Eq 2.4
61
Where k determines the demodulation frequency, T is the duration of the modulation period,
ω = 2π/T denotes modulation frequencies A(t) is the time dependent absorbance at
wavenumber ν, ϕkPSD
is the demodulation phase angle.
Figure 2.4 illustrates clearly the typical outputs of a MES technique and the PSD
principle. If the perturbation is given by a sinusoidal stimulation A(t) (e.g. periodic change of
light illumination) then the response of a system is measured over a number of periods and
averaged into one period. The response of the system signal can be written as the sum of three
components B(t) + C(t) + D(t) , where B(t) is active species response (affected by the
stimulation), C(t) is spectator species response (not affected) and D(t) is noise.
Figure 2.4 Phase sensitive detection principle. A(t): Stimulation function, B(t): response of
‘active’ species perturbed by the stimulation A(t) where ϕ is the phase delay with respect to
the stimulation, C(t): response of ‘spectator’ species which does not respond to the
perturbation, D(t): Fourier-decomposed noise. The sum of the response components, B(t) +
C(t) + D(t), is the actual experimental response [21]
The active species respond at the same frequency ω with a phase delay ϕ with respect to the
stimulation. The kinetic information of the active species are contained in the amplitude B and
the phase delay ϕ. The response of spectator species is constant because they do not respond
to the stimulation. The noise D(t) can be decomposed into different frequency terms by the
Fourier decomposition.
62
After the PSD of the time-domain total response signal B(t) + C(t) + D(t) using eq. (2.4) at k =
1, the corresponding phase-domain total response signal B(φPSD
) + C(φPSD
) + D(φPSD
) is
obtained. The active species term B(φPSD
) is nearly identical to that in time-domain, B(t),
keeping the kinetic information, (amplitude and phase delay) inside. The spectator term
C(φPSD
) and the noise term D(φPSD
) are zero. Brief, PDS allows removing the spectator and
noise components from the signal. The PSD can greatly enhance the signal-to-noise ratio and
allows selective detection of the interesting active species by carefully choosing the type of
stimulation.
The combination of modulation excitation spectroscopy (MES) with another analytical
method like Fourier transform infrared is very important. This combination makes MES a
powerful technique that allows separation of overlapping bands of different kinetic behavior.
These advantages led to a wide range of applications of MES in a lot of fields.
2.6 Experimental Section
To study the adsorption of proteins onto surfaces and solid liquid interfaces in situ a
home built flow through cell was designed and applied in ATR-IR spectroscopy
measurements and in combination with modulation excitation (MS) spectroscopy. The design
of the cell used in these experiments is an appropriate solution, allowing different types of the
measurements in photocatalysis.
2.6.1 ATR-IR Setup and Application of Modulation Excitation
Spectroscopy
Infrared spectra were recorded on a Bruker Equinox-55 FTIR spectrometer, equipped
with a narrow band MCT detector and attachment for ATR measurements (Wilks Scientific).
Another FTIR spectroscopy Vertex 80V under vacuum (sample compartment and optics
bench vacuum was around 1 hpa) was used in this work. All spectra were measured at room
temperature at a resolution of 4 cm-1
. The sample was placed on a Ge IRE (52 mm x 20 mm x
1mm; KOMLAS), (50 mm x 20 mm x 1mm; KOMLAS), and Si IRE (50 mm x 20 mm x
1mm; KOMLAS) and fixed inside the home built flow-through cell. The cell was made from
a Teflon piece, a fused silica plate (45 mm x 35 mm x 3mm) with holes for the outlet and inlet
36 mm apart and a flat seal (1mm). The fluid compartment had a volume of 0.5ml. The
63
solution was passed through the cell at a flow rate of around 0.2 ml/min by means of a
peristaltic pump (Ismatec, Reglo 100). A scheme of the experimental setup is shown in the
Figure 2.5
Figure 2.5 Schematic setup for in situ ATR-IR spectroscopy of photocatalytic reactions in a
small volume flow-through cell [3] (Equinox 55)
Such cell model allowed different types of modulation excitation experiments: light
modulation, concentration modulation or gas modulation experiments. In light modulation
experiments irradiation of the sample was carried out by a 75 W Xenon arc lamp. Schott UG
11 and BG 42 (50 mm x 50 mm x 1mm) broadband filters from ITOS were used to remove
visible light. An electronic shutter (Newport model 71445) was used to achieve UV light
modulation. The UV light from the source was guided to the cell via two fiber bundles. For
concentration modulation experiments two glass bubble tanks were used. In our investigation,
we tried to modulate the adsorbed protein using UV light periodic stimulation. To do that a
shutter (light modulation) was triggered by the FTIR spectrometer. This ensures the
synchronization of modulation and data acquisition.
64
Figure 2.6 The Bruker Equinox 55 ATR-IR spectrometer equipped with ATR-IR attachment
and flow-through cell for in situ photocatalytic measurements [3]
The Equinox 55 is a compact, rugged FT-IR spectrometer designed for demanding analytical
laboratory applications. OPUS/IR software is used to control the spectrometer and for spectra
manipulation. The physical dimensions (depth x width x height) of the Equinox 55 (figure
2.6) and sample compartment are as follows:
61 x 70 x 27 cm physical dimensions of the EQUINOX 55
25.5 x 26 x 19 cm sample compartment of the spectrometer
The frequency range of the spectrometer is 7500-370 cm-1
. Figure 2.7 shows sample
compartment of the Equinox 55 spectrometer equipped with ATR-IR attachment and flow
through cell for in situ photocatalytic measurements.
65
Figure 2.7 Setup for photocatalytic measurements in situ: (A) IR beam, (B) optical fibers from
UV source, (C) flow-through cell with Ge internal reflection element (IRE) and fused silica
window, (D) mirrors [3]
2.6.2 Catalyst and Chemicals
Only commercial TiO2 anatase (with an average particle size less than 25 nm, spec.
surface area 200-220 m2/g, density of 3.9 g/ml at 25 °C(lit.)) and Degussa P25 TiO2,
containing 80% anatase and 20% rutile with a surface area of 51 m² g-1
and average primary
particle size of 21 nm were used in the photo-assisted reactions. Sodium hydroxide (Sigma-
Aldrich, 97%), deuterium oxide (Aldrich, D-99, 9%) were used as received. Bovine serum
albumin (BSA) (68 kDa, solubility 1g in 25 ml of H2O, and pH = 6.5-7.5) from Sigma-
Aldrich is used to prepare a BSA solution of 10-6
mol/l in all experiments.
66
2.6.3 Thin Film Preparation
Commercial type TiO2 anatase (Sigma-Aldrich Chemie GmbH) with an average
particle size less than 25 nm (specific surface area 200-220 m2/g, density: 3.9 g/mL at 25 °C)
was used in the photocatalytic experiments. The catalyst films were prepared by suspending
20 mg of TiO2 anatase in 10 ml of purified water (Milli-Q, Millipore) water (18 MΩ.cm). The
slurry was sonicated for 30 min. The film was formed by dropping the slurry onto a Ge
internal reflection element (IRE, 52 mm x 20 mm x 1 mm, Komlas GmbH). Before film
deposition the IRE was first cleaned with ethanol and then put in air plasma for around 15
min. The solvent was evaporated using the spin coating method (1000 rotation per minute)
twelve successive spin coatings were applied with 2.25 minutes between the individual
coating steps. Then the samples were dried at 80°C for some hours in an oven. After drying
the film was ready for use. For every experiment a fresh catalyst film was prepared, and
results were reproducible on different catalyst films. Bovine serum albumin (BSA, Sigma-
Aldrich) (68 kDa, solubility 1g in 25 ml of H2O) was used to prepare a BSA solution of 10-6
mol/l in all experiments.
Commercial P25 TiO2 containing 80% anatase and 20% rutile from Sigma-Aldrich
Chemie GmbH, with an average particle size of 21 nm (specific surface area 51 m2/g) was
used in the work also. Instead of spin coating, the solvent was evaporated after drying on a
heater for several minutes at around 35°C then the samples were dried again at 80°C for some
minutes on a heater.
2.6.4 ATR-FTIR Spectroscopy Measurements
We used two FTIR instruments, Bruker Equinox 55 FTIR spectrometer and Bruker
Vertex 80V. This latter was under vacuum of around 1hpa (sample compartment and optical
bench). ATR spectra were recorded with a dedicated flow-through cell, made from a Teflon
piece, a fused silica plate (45 mm x 35 mm x 3 mm) with holes for the inlet and outlet (36 mm
apart), and a flat (1 mm) viton seal. The cell was mounted on an attachment for ATR
measurements within the sample compartment of a Bruker (Equinox 55/Vertex 80V) FTIR
spectrometer equipped with a narrow-band MCT detector. Spectra were recorded at a room
temperature at a resolution of 4 cm-1
67
The aqueous BSA solution can pass through the cell and over the sample by means of
peristaltic Pump (Ismatec, Reglo 100) located after/before the cell in Equinox 55/Vertex 80V
respectively. A constant flow rate of about 0.2 ml/min was used.
In ATR-IR, a beam of infrared light is passed through the ATR crystal (Ge) as shown
in Figure 2.8, in such a way that it reflects at least once off the internal surface in contact with
the sample. This reflection leads to an evanescent field which extends into the sample. The
penetration depth into the sample is typically between 0.5 and 2 micrometers, with the exact
value being determined by the wavelength of light, the angle of incidence and the indices of
refraction of the ATR crystal and the medium being probed. The beam is then collected and
guided to a detector as it exits the crystal. Illumination of the sample with UV light was
carried out using a 75 W Xe arc lamp in Equinox 55 and 300 W Xe lamp in Vertex 80V. The
UV light from the source is guided to the cell via two fiber bundles and mirrors. The light was
passed through a 5 cm water filter to remove any infrared radiation. A Schott UG 11 (50 x
mm x 50 mm x 1 mm) broadband filters from ITOS were used to remove visible light
(transmission between 270 and 380 nm) or the UV one (transmittance between 800 and 400
nm). The intensity of the UV light at the surface is measured to be (2-4) mW/cm2 and the
temperature is increased by around 2°C. The visible light intensity at the surface of the sample
using Brucker Vertex 80V is calculated to be around 80 mW/cm2.
Figure 2.8 Schematic set-up for in situ ATR-IR spectroscopy of photo-assisted reactions in a
small volume flow-through cell in Vertex 80V FTIR
68
2.7 Proteins Structure
The amino acids (Figure 2.9) are the monomeric units in the formation of proteins
which are linear biological polymers. There are twenty different amino acids that are used to
form proteins. The amino acids are distinguished from each other by the identity of the “R”
group in each one. The amino acids are linked to each other by an amide bond (called also
peptide bond by protein chemists), which is the link between amino group on one amino acid
and the carboxylic acid group on another amino acid as shown in Figure 2.10.
Figure 2.9 The structure of an amino acid
Linking two amino acids together by an amide bond (peptide bond) is called a dipeptide as
shown in figure 2.10; and when many dipeptides are linked together it is called a polypeptide
that has backbone and side chains. The backbone or main chain contains the amide nitrogen,
the alpha carbon and the carbonyl carbon that are contributed by each amino acid unit. The
side chains comprise the “R” groups of amino acids and it arises in the amide I band.
Figure 2.10 Linking two amino acids together by an amide bond to form a dipeptide
The difference between proteins and their functions are related to the sequence (order) of
amino acids units and their number. This latter varies from 50 to hundreds of amino acid
units, making the number of possible sequences astronomical. Few polypeptides that are able
69
to fold into a well-defined 3-dimentional structure are considered as proteins. The function of
each protein is controlled by its 3-dimensional structure.
The protein can fold to form a well defined 3-dimensional structure only if it exhibits
three levels of structure: primary, secondary and tertiary. The primary structure is the simple
sequence of amino acids. One can model the primary structure as beads on a string and each
bead represents one amino acid. The tertiary structure of protein can be represented as a
tightly-packed snowball, where each atom in the protein has a well defined location. Protein
folding might be likened to scrunching up the string of beads (the primary structure) into a
tightly-packed ball (the tertiary structure) as shown in Figure 2.11.a. During the formation of
the tertiary structure of the protein, some amino acids find themselves inside of the tightly-
packed ball where they cannot interact directly with water molecules. These molecules of
water can form hydrogen bonds with amides (Figure 2.11.b). The amino acids located inside
the tightly-packed ball provide alternative ways for the buried amides to hydrogen bond. They
form the amide bonds hydrogen bond to each another (Figure 2.12).
Figure 2.11 a. The crudest of protein folding models: scrunching up a string of beads. b. The
hydrogen bonds that form between the amide bond and water
The formation of hydrogen bonds between the amino acids leads to an intermediate
level of protein structure called secondary structure. This structure includes the α-helices and
β-sheets, which are periodic structures and allow the amides to hydrogen bond very efficiently
with one another. Figure 2.13 (left) shows a segment of α-helix from the small protein bovine
pancreatic trypsin inhibitor (BPTI). It is clear that in this structure the polypeptide backbone is
coiled in a right-handed helix where the hydrogen bonding occurs between successive turns of
the helix. As α-helix, β-sheets structures are stabilized by hydrogen bonds where strands of
protein are stretched out and lie either parallel or antiparallel to one another. Figure 2.14
(right) illustrates this configuration with a piece of antiparallel β-sheet from BPTI. It is clear
70
from this figure that the strands interact laterally via hydrogen bonds between backbone
carbonyl oxygen and amino H atoms.
Figure 2.12 The hydrogen bonds that form between amide bonds buried inside a folded
protein
The other components of secondary structure include β-turns and unordered structure. β-turns
are sharp turns that connect the adjacent strands in an antiparallel β-sheet. Unordered structure
is generally a random structure and catch-all for regions that do not fall into one of the other
categories. These are often loops which form near the surface of proteins and join the other
elements of secondary structure.
Figure 2.13 α-helix structures [23]
71
Figure 2.14 β-sheet structures [23]
Figure 2.15 Interections in the same chain of protein [24]
Figure 2.15 shows the interaction in the same chain of the protein as S-S bridge, hydrophobic
effect, hydrogen bond and ioic bond. All these interactions stabilize the tertiary structure of
protein.
72
2.7.1 Bovine Serum Albumin Structure
Proteins are very important in the body and they make up to half of cell dry mass [25].
Albumin proteins are a major constituent of blood serum and play a significant role in a lot of
disciplines like biocompatibility [26]. The polypeptide backbone of proteins fold, due to
hydrogen bonding, into stereotypical configurations giving secondary structure, including α-
helices, β-sheets, β-barrel, reverse turns and omega loops [27]. The albumin proteins have
secondary structures of: α-helices, β-sheets and turns [28, 29]. The structures of α-helices and
β-sheet are very organized using highly favored rotational angles and tight packing of atoms
in well defined positions in the space [27]. Due to its relatively flexible structure, albumin is
classed as a soft protein. This protein can undergo conformational changes [26]. The three
dimension image of bovine serum albumin (BSA) molecule is shown in Figure 2.16. BSA is
widely used as a model globular albumin protein that has stability in biochemical reactions
[30]. BSA has 604 peptide units with a molecular weight of 66462 g/mol [31]. According to
literature, various methods of measurement, BSA molecule consists of 55-65% α-helices,
21% β-sheet, and the rest are turns [28, 29, 32]. The components of the secondary structures
of BSA depend on pH [33]: in the pH range 4.3 to 8.0, BSA protein keeps a triangular or
heart-shaped structure [31] with the normal form (native state), that comprises around 60% α-
helix structure and the remainder being β-sheet and turns. The BSA molecule unfolds into the
fast form with 45% α-helix at pH less than 4.3 and further unfolding to the expanded form
was observed, with 35% α-helix at pH below 2.7 [34] where this protein loses its structure. In
basic solutions with pH more than 8, the BSA adopts a basic from that has 47% α-helix
[35].The changes in the secondary structure of protein lead to a transformation from one
structure to another or a creation of new one. The reduction of α-helix structure results from
unfolding of the domains with consequent loss of intradomain helicity [31].
73
Figure 2.16 Three dimension image of serum albumin molecule.(A) Side view, (B) Front
view, (C) Cavities in BSA. (D) α-helix structure colored in red; loop in white. Figure from
Protein Database [36]
According to the studies done by Brown et al in 1975 [37] and Hirayama et al in 1990 [38],
the composition of BSA protein is shown in the following tables (Table 2.2 and 2.3).
From these tables, we note that all the amino acids have different properties that give a
complex structure to any protein. We see also that BSA has two amino acids that have S
atom. This atom is very important in building the S-S bridge in the secondary structure of
BSA protein.
-a- -b-
Cavities in BSA
-c-
-D-
74
Table 2.2 The composition in amino acids of BSA protein (*presence of S in the molecule)
[24]
Table 2.3 Atomic composition of BSA protein [24]
Number of atoms C N O S
Brown et al. 1975 2926 779 897 39
Hirayama et al. 1990 3030 841 947 40
2.7.2 FTIR Spectroscopy and Protein Structure
Fourier transform infrared spectroscopy is a good technique that can be applied to
study the secondary structure of globular proteins [39-41]. Its applications to determine the
75
structure of the protein is based on the assessment of amide bands. Kumosinski et al. [42]
demonstrated that the results concerning the secondary structure of 14 proteins using FTIR
spectroscopy and X-ray crystallographic data were in good agreement. The FTIR has many
advantages, over other techniques, in studying protein and the major one is the lack of
dependence on the physical state of the sample (gas, aqueous or organic solution, hydrated
film, inhomogeneous suspension, or solid). The FTIR spectroscopy has been used several
years ago to analyze the proteins structure and it is extensively reviewed [41, 43-46]. This
method (FTIR) is particularly suitable for the study of adsorbed proteins on surfaces [47, 48].
FTIR was applied to investigate the loss of secondary structure during insulin unfolding on a
model lipid-water interface [49], adsorbed proteins on silica surfaces [50-52], different clay
surfaces [53], interface of oil-water [54], air water interface [55], and brushite [56]. The
conformation of adsorbed protein depends on properties of the surface.
2.7.2.1 FTIR Spectrum of Protein
The absorption bands in FTIR spectroscopy measurements are sensitive to bond angles
in the molecules and hydrogen bands. Theoretically, any changes (intensity, shift in peak
position…) are caused by an alteration (conformation) in the secondary structure of the
protein. Each type of secondary structure absorbs at a specific frequency in the FTIR
spectrum [43]. The amide groups of proteins exhibit vibrational modes in the infrared region,
which give rise to the amide bands A, B, and I-VII [45, 57]. The amide IV-VII bands are not
very important in mid-infrared region due to their low intensities. The amide I, amide II and
amide III are the most important bands that can be applied to determine secondary structure of
proteins [58].
Amide I
The infrared band that corresponds to amide I vibration in FTIR spectroscopy absorbs
from 1600 to 1700 cm-1
. The C=O stretching vibration with some minor contributions from
the out-of-phase CN stretching vibration, the CNN deformation and the NH in-plane bend are
located around 1650 cm-1
[39, 57]. The high signal of intensity (70-85 %) in the amide I band
is from C=O stretching and (10-20 %) is from CN stretching [39]. The nature of the amino
acid side-chain strongly affects the amide I which only depends on the secondary structure of
the backbone. Thus, the amide I band is best suited to determine the secondary structure of
proteins.
76
Amide II
The Vibration of amide II band in FTIR absorbs from 1500 to 1600 cm-1
. The peak of
Amide II is located around 1550 cm-1
and it has the contributions of the out-of-phase
combination of the NH in-plane bending (40-60 %) and the CN stretching vibration (18-40 %)
with smaller contributions from the CO in-plane bend and the CC and CN stretching
vibrations [39]. Amide II is affected by side-chain vibrations but the correlation between
secondary structure and frequency is less straightforward as compared to the amide I region.
Amide III The signal of Amide III vibration can be seen in the infrared region from 1200 to 1400
cm-1
. This vibration is the combination of the NH bending and the CN stretching with small
contribution from the CN in-plane bending and CC stretching vibration [39]. Amide III is less
suitable for studying the secondary structure of proteins, even water effect is reduced, because
it is affected by side-chain and the backbone vibrations vary considerably. Amide III signal in
FTIR is weaker than amide I and amide II. However previous studies demonstrated that the
amide III region can be used to calculate the secondary structures for various proteins [59-62].
Griebenow and Klibanov showed that the amide III results and X-ray data were in good
agreement for most proteins [63].
2.7.2.2 The Correspondence between Protein Secondary Structure and Amide
Bonds
The most important point in the interpretation of proteins infrared spectra is to involve
the component bands to different types of secondary structures. A lot of theories and
experiments have been done to correlate FTIR absorption bands of protein to the secondary
structure of protein in its different states. The bands in the range 1650-1658 cm-1
is associated
to α-helix conformers in aqueous environments. The α-helical structures overlap with those
from random (unordered structure) (1645-1652 cm-1
) [64] and loops (1658-1665 cm-1
) [65],
and they occur from 1650 to 1655 cm-1
in soluble proteins [66].
The vibration of β-sheet can be seen in the region from 1620 to 1640 cm-1
and its
position can be affected by varying strengths of the hydrogen bonding and transition dipole
coupling in different β-strands [64]. β-turn (turn) vibration is around 1662-1690 cm-1
. The
secondary structure of protein can be also determined from the amide II band, but the
correspondence between FTIR spectra and secondary structure is more complex than in the
77
amide I region because bands in the amide II region have not been well studied. In amide II
region, bands in the range 1540-1550 cm-1
are regarded as α-helix and the β-sheet vibration is
at the range 1520-1530 cm-1
[68, 69]. β-turns can be seen around 1568 cm-1
[70, 71].
Due to their low intensity and the contribution of the side chain vibration, the FTIR
spectra of the protein in the amide III region have not been fully understood yet. It was
reported by Cai and Singh [61] that α-helix bands usually appear in range 1295-1340 cm-1
; β-
turns gives rise to bands around 1270-1295 cm-1
; random structure is at 1250-1270 cm-1
; and
β-sheet is assigned from 1220 to 1250 cm-1
. The important assignments of FTIR bands are
given in Tab. 2.4.
Table 2.4 Band assignments in the amide I region of FTIR spectrum [72]
FTIR region Wavenumbers (cm-1
) Secondary structure References
Amide I
1620-1640 β-sheet [64]
1645-1652 Random or unordered [64]
1650-1658 α-helix [64]
1662-1690 β-turn [64,73]
Amide II
1520-1530 β-sheet [68,69]
1540-1550 α-helix [68,69]
1568 β-turn [70,71]
Amide III
1220-1250 β-sheet [61]
1250-1270 Random [61]
1270-1295 β-turn [61]
1295-1340 α-helix [61]
2.7.2.3 Water Absorption in FTIR and Protein Spectra Correction
The protein FTIR spectra can be influenced by the contribution of water and other
components. Although water (H2O) is challenging for IR spectroscopy, it is much preferable
than D2O for studying protein structure because it has the advantage of providing a more
native environment [74, 75]. The structure of the protein can be changed somewhat by D2O
with respect to the native state because the amide I bands are strongly affected by the H–D
exchanges in the peptide linkages [76, 77]. In H2O solution, the bands between 1654 cm−1
and
1658 cm−1
are assigned to α-helix. The unordered conformation (random coil) is usually
associated with the IR band between 1640 cm−1
and 1648 cm−1
, β-turn is between 1675 cm−1
and 1685 cm−1
, β-sheet is from 1690 cm−1
to 1696 cm−1
and from 1624 cm−1
to 1642 cm−1
,
and intermolecular β-sheet is around 1615 cm−1
[74]. The contribution of water (IR
absorption) needs to be removed before further analysis of the protein spectrum. H2O has a
strong IR absorbance around 3400 cm−1
(O-H stretching), 2125 cm−1
(water association
78
combination band) and 1640 cm−1
(H-O-H bending). The amide I mode of proteins absorbs
between 1600 cm−1
and 1700 cm−1
, overlapping directly with the H2O bending vibrational
mode at 1640 cm−1
[36]. For ATR-IR study of proteins in H2O solution, water absorption in
the region 1600–1700 cm−1
is the biggest problem whereas D2O has no absorption band in the
region where the amide I and amide II bands are observed [74]. The water contribution can be
eliminated using digital subtraction by measuring water and the protein in water at identical
conditions. Two important criteria allow a good subtraction of absorption bands due to liquid
water and gaseous water in the atmosphere. First, the bands originating from water vapor
must be subtracted accurately from the protein spectrum between 1800 and 1500 cm−1
. To do
so we measured a gas phase water spectrum just before the actual experiment and used this
for the subtraction. However, gas phase signals were very small or absent since the sample
compartment was under vacuum during the experiments (in the case of FTIR under vacuum).
Second, a straight baseline must be obtained from 2000 to 1750 cm−1
. Using these two criteria
to judge the successfulness of water subtraction leads to higher quality protein spectra [78,
79]. Some external stimuli (visible light irradiation) might also affect the water spectrum. In
order to eliminate such effects experiments with BSA were repeated in an identical way but
without BSA (i.e. Ge IRE, TiO2 and water). Corresponding spectra (time, visible light
irradiation) for the experiment without BSA were subtracted from the spectra with BSA.
It was reported [80] that 2125 cm-1
water association band is not affected by the presence of
proteins that are not highly charged.
2.7.2.4 Resolution-Enhancement
In MIR-FTIR spectra and especially for complicated samples like protein, it is difficult
to perform a straightforward analysis of the spectra. To solve this problem, resolution-
enhancement methods are applied. Fourier self-deconvolution (FSD) and derivation have been
used for many years to allow visualization of overlapping bands [81, 82].
Knowledge about the number of bands, their shapes and parameters, such as position, band
width and intensity is required for FSD. FSD reduces the width of water vapour absorption
and enhance noises which are a problem in deconvoluted spectra. Thus, FSD should only be
performed on spectra with a high signal-to-noise ratio (SNR) and a low contribution from
water vapour [43].
79
Dong et al. [83] introduced derivative spectroscopy to analyze the secondary structure
of proteins in aqueous solution. This method is mainly important to identify the number of
bands and their position and it can be used for quantitative studies.
2.7.2.5 Disadvantages of FTIR for Protein Structure Analysis
According to the information mentioned above, FTIR spectroscopy cannot provide full
three-dimensional structural information of proteins. The contribution of water and other
components can influence the spectrum of a protein. High protein concentration (10-20%
w/w) is needed for FTIR measurements for obtaining the SNR necessary for conformational
analyses [84].
2.8 Protein Solid-Surface Interaction
As it is shown in the last part of Chapter 1, BSA protein can undergo several steps to
be adsorbed onto the semiconductor solid-surface. This kind of interactions is driven by a lot
forces including Van der Waals and electrostatic ones. This adsorption can perturb even
slightly the structure of BSA because of the interaction between this latter and the surface.
Some steps of the adsorption are a time-depended structural re-arrangements of the protein on
the surface. Changes in conformation can occur immediately during adsorption or slowly over
time after the protein has attached to the surface.
2.9 Conclusion
In this chapter we present the attenuated total reflection spectroscopy and its
importance for investigating the solid-liquid interface. We show also the theory of phase
sensitive detection and the composition of proteins and their interactions with surfaces.
It is clear that using ATR-IR for studying the contact between semiconductors-
surfaces and the protein especially in water solution is a big challenge. The results of this
study are shown in the Chapters 3 and 4.
80
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87
Chapter 3
Environment Effect on the Adsorption
of BSA Protein onto Solid-Surfaces
The adsorption of protein is controlled by many important environment parameters
like: Temperature, Concentration, pH, Ionic strength. The mechanism of adsorption and the
bond between the protein and the surface is still not well understood right now. The form of
the protein and the properties of the surface play also a fundamental role in the adsorption. In
this Chapter, we will present the results of the environment effect on the protein adsorption.
88
3.1 Introduction
The adsorption of protein onto a solid surface is a far-ranging and complex problem
and its function is highly related to the structure that can be affected by changes in the
environment [1-6]. Issues of electrostatics, conformation and topography, and of course,
thermodynamics, all factor significantly into the process [7-9]. Solution chemistry in all its
details including pH and ionic strength can be used to control the adsorption process, but can
also confound it if not properly considered [10-12]. In the following section, we will
introduce some of our results of these major considerations, and how understanding of protein
adsorption can be controlled under varying the adsorption conditions.
3.2 Importance of Protein and Surface Properties
The properties of the protein and the surface where biomolecules are interacting
influence the interfacial behavior. Figure 3.1 clearly shows the most important protein and
surface properties, respectively.
Figure 3.1 Schematic view of a protein and a well characterized surface [13]
Once a protein molecule has reached the surface, complex dynamic processes occur (Figure
3.2). First, the protein molecules approach the surface in random orientations. Since the
protein molecule is irregular and heterogeneous with respect to surface polarity and charge,
several orientations may occur with different strength. As the residence time at the surface
89
increases, the number of contact points between the protein and the surface might increase.
Over time this can lead to irreversible adsorption and conformational changes such that a
larger area is occupied by each protein.
Figure 3.2 Protein adsorption characterized by two states: A1 is a reversible state but can lead
to an irreversible state as A2. The Kon and the Koff represent the probability of a protein
molecule to attach or detach from the surface [13]
3.3 Solutions and ATR-IR Study of Protein
Using water as solution is really a challenge for ATR-IR study. The best solution to do
such kind of studies is D2O. Figure 3.3 shows the absorption spectra of H2O and D2O. This
figure indicates the important regions of absorptions. We see that water has a strong
absorption in the region between 3000-4000 cm-1
and 1700-1600 cm-1
that correspond to the
region of NH absorption and Amide I in the protein respectively.
90
Figure 3.3 Absorbance spectra of H2O and D2O
3.4 BSA in Different States
The following figures (3.4-6) show the spectra of BSA protein in its three different
states: solid, liquid and deposited onto a silicon solid surface at 50 °C. It is very clear from
these graphs that the most important absorption peaks of the BSA are the amides I and II
between 1700 and 1500 cm-1
and the N-H vibration at around 3300 cm-1
. To get a high quality
spectra of BSA it is better to use D2O as a solvent but this latter can alter somehow the
structure of the protein because of the H-D exchange. According to figure 3.3 D2O has no
absorption in the region of amide and NH region whereas water (H2O) has a strong absorption
in the region of amide (1640 cm-1
) due to the bending vibration mode. Furthermore, because
the exchange of D for H can affect the strength and length of hydrogen bonds, it is possible
that protein secondary structures might be altered by the replacement of H2O by D2O.
Therefore, H2O-based media have the advantage of providing a more native environment
[14].
91
Figure 3.4 BSA powder spectrum
Figure3.5 BSA spectrum in water solution at a concentration of 10-4
mol/l
92
Figure 3.6 BSA Spectrum deposited at 50 °C on Silicon surface
Protein adsorption and biotechnology can be used to help improve products during the
product development cycle. Modifying solution chemistry and surface environment are ways
to improve performance of adsorbed protein layers. Before discussing the adsorption behavior
of protein onto solid surfaces, it is very important to take an idea about the surface
morphology of our porous TiO2 thin films spin coated onto germanium surface.
93
3.5 TiO2 Surface Characterized by SEM and AFM
Our porous TiO2 anatase film prepared by spin coating technique and used in the most part of
our work are characterized by SEM and AFM as shown bellow.
Figure 3.7 SEM and AFM pictures of the TiO2 porous thin film 10 times spincoated on Ge
surface at a concentration of 100 mg/10 ml at 1000 rotations per minute (rpm). a, b- The
morphology of the film (taken by SEM). c- The film thickness (taken by AFM)
-c-
-a- -b-
94
According to the pictures of SEM shown here, it is very clear that our surface is porous and
the thickness is around nm. This thickness is suitable for the penetration depth of infrared
beam that interacts with the molecules adsorbed onto this surface giving the signal to analyze.
3.6 Adsorption of BSA onto TiO2 coated surface
The figure 3.8 illustrates the time evolution of in situ ATR spectra of adsorbed BSA at 10-6
mol/l in water solvent taken as a background (base line) during flowing over TiO2 surface at a
constant velocity.
Figure 3.8 Time evolution, around 10 min between two successive spectra, of In Situ ATR
spectra of adsorbed BSA on TiO2 anatase surface (spin coated on Ge and water is used as
solvent at 10-6
mol/l of concentration) -equilibrium is reached after 80 min-
95
It is very clear that adsorption of BSA biomolecules onto the TiO2 surface is accompanied by
releasing of water from the surface as negative water bands in between 3000-3600 cm-1
are
observed in figure 3.8. The kinetics of this kind of adsorption is demonstrated in figure 3.9.
Figure 3.9 Maximum of amide I band versus time
It is clear from this figure that the adsorption of BSA onto the surface of TiO2 anatase coated
around 10 to 12 times on the surface of Ge follows three important stages. The first one is the
strong and fast adsorption. At this stage there is enough space on the surface. With time the
space will be reduced and the protein follow a second regime which is slower than the first
one. And the last one is the multilayer adsorption regime. After reaching the equilibrium of
adsorption, rinsing with water does not affect the signal strength, which means that this kind
of adsorption is strong and not reversible by water rinsing.
The mechanism of BSA adsorption onto TiO2 surface is very complicated to be well
explained. This adsorption is driven by different forces as mentioned in the first sections of
this manuscript. The chemical bonding process has been studied: (1) the TiO2 particle surface
is non-charged at around pH 5 [15], so under their experimental pH of 7.4, the predominant
TiO2 surface groups are Ti2=O− and Ti−OH, with few Ti2=OH; and the main protein
96
functional groups are R−COO− and R−NH3+ [16, 17]. (2) Electrostatic interaction occurs
between these groups on the surfaces of both TiO2 and protein:
Ti−OH2+: NH2−R (electrostatic interactions)
Ti2=O− +NH3−R (electrostatic interactions)
Ti−OH…−COO−R (hydrogen bonding interaction) [18]
Chemical bonding between BSA protein and TiO2 particles would be similar as the
bonding between fibrinogen and TiO2, but may not be the same. The form of BSA is one of
the important parameters that control the adsorption and its contact with the surface. In figure
3.10, we show the possibilities of contact between HAS (very similar to BSA) and the
surface.
Figure 3.10 Protein-surface interactions: A1- a demonstration of the preferred adsorption
orientations clustered from the results of Monte Carlo simulations. B1-The detailed
configurations of the top three most favorable adsorption orientations: (a) back-on, (b) front
slant-on, and (c) edge A-on orientations. The hydrophobicity distribution of HSA was mapped
by Insight II and the hydrophilic residues are coded blue [19]
-A1- -B1-
97
From figure 3.10, we can easily conclude that the adsorption of globular protein like BSA
onto a solid-surface lets some voids between adsorbed BSA molecules. These voids strongly
depend on the form of any protein and they can be filled by water molecules attached onto the
surface and between the adsorbed proteins themselves. If the shape of the protein is important
so what about the surfaces of different materials?
3.7 Adsorption of BSA onto Different Surfaces
The figure 3.11 bellow shows the effect of different surfaces on the equilibrium adsorption of
BSA protein at a concentration of 10-6
mol/l.
Figure 3.11 Equilibrium adsorption of BSA onto different surfaces in water solution at 10-6
mol/l (TiO2 adsorbed on Ge was prepared by adsorbing in situ TiO2 particles from solution on
the Ge element. A homogenous thin film on the surface was obtained in this way)
98
The equilibrium on the surfaces is not the same which is a strong indication that the amount of
BSA adsorbed is not the same too. The most important information about the structure of any
protein can be taken from the amide I and II bands. These amides shown in the figure 3.12
where the base line is corrected indicate that the amount of adsorbed BSA onto the surface of
deposited TiO2 P25 onto a germanium surface is higher than for the other surfaces. This
phenomenon can be explained by the effect of the roughness of each surface [20]. For smooth
surfaces like Ge and Si, the BSA cannot attach too much. We have to mention that the
releasing of water from the surface is not the same (Figure 3.11 region 3000-3600 cm-1
) and
this behavior could be explained by the fact that water molecules adsorbed on the surfaces
depend also on the roughness and this latter controls both kind of adsorption.
Figure 3.12 Equilibrium spectra of amide I and II regions of adsorbed BSA onto different
surfaces in water solution at 10-6
mol/l (the base line is corrected)
Not only the equilibrium of adsorbed BSA is not the same on different surface, but also the
kinetic of adsorption is not the same. Figure 3.13 clearly demonstrates the adsorption isotherm
of each surface. We see that adsorption is fast on the surface of silicon and reaches an
99
equilibrium after around fifteen minutes (15 min). On the surface of germanium, the
equilibrium can be reached after around half an hour (30 min) and it takes much time in the
case of TiO2 surfaces. The film of TiO2 P25 deposited on germanium surface at around 40 °C
in air is surely very roughness compared to the others surfaces. This is a reason why it reaches
equilibrium after more than 80 minutes. In all the experiments done using any surface, rinsing
with water has no effect on the last equilibrium which indicates that BSA is strongly adsorbed
onto most solid-surfaces and it is an irreversible phenomenon.
Figure 3.13 Kinetic adsorption of amide I maximum position of adsorbed BSA onto different
surfaces (10-6
mol/l in water)
The folding (spreading) of the adsorbed protein on the surface strongly depends on the
amount of protein adsorbed. If there is enough space on the surface, the protein can spread
and the contact points between it and the surface will increase and the adsorption will be
strong. The figure 3.14 shows the spreading of adsorbed protein onto a surface.
100
Figure 3.14 Time-dependent molecular spreading of a protein on a surface
3.8 pH Effect on the Adsorption of BSA onto TiO2
The pH of any solution used as a solvent for the protein is one of the most important
environment parameters that affect the structure of the protein. Thus the adsorption is affected
too.
Figure 3.15 clearly elucidates the effect of pH on the adsorption of BSA onto TiO2 anatase
surface. For a pH around 10 the adsorption is very weak even it does not exist. At this basic
pH the shape of the BSA is not well known. But one of the most important factors that can
affect the adsorption is the electrostatic forces between TiO2 surface and BSA [16, 21]. Using
an acidic solution of pH lower than 2 there is a weak adsorption because the protein changes
its structure and it takes more space on the surface (Figures 3.16 and 3.17) that is why the
amount of adsorbed protein is reduced.
101
Figure 3.15 Effect of pH on the BSA adsorption onto TiO2 spin coated thin film (water is used
as a solvent at 10-6
mol/l of BSA concentration)
Figure 3.16 Different conformational isomerisation of BSA as a function of pH (N form =
Native, F form = Fast and E form = Expanded) [22, 23]
The unfolding of protein adsorbed on a solid-surface under varying pH leads to more contact
between the protein and the surface as shown in figure 3.17. This means that the footprint of
protein is increased and the space between adsorbed proteins will be reduced.
102
Figure 3.17 Effect of protein unfolding on interaction with a surface
The pH changes the protein structure and its charge. It changes also the structure and the
charge of the TiO2 surface as shown in figure 3.18.
Figure 3.18 Charge surface of TiO2 at different pH. (a) pH<pHpzc (b) pH=pHpzc and (c)
pH>pHpzc [24]
The highest adsorbed amount of protein on TiO2 surface is observed using a pH between 4,5
and 5 which is very near to the zero charge of the BSA protein (4.7-4.9). Here the total charge
103
of BSA is zero which will decrease the electrostatic interactions between BSA and titanium
surface.
The following figure shows the relation between the maximum signal of amide I and
the pH. There is no adsorption of BSA in a pH of 10 which is not well understood right now
because the effect of a basic solution on the structure of the protein is not clear but it could be
explained by the saturation of the actives group in the protein and the increasing of the
electrostatic forces between BSA and TiO2 surface.
Figure 3.19 Effect of pH on the adsorption of BSA onto the surface of TiO2 anatase at room
temperature. The concentration of BSA in water was 10-6
mol/l in each experiment
In the figure 3.20, we clearly summarize the interactions between the surfaces of TiO2 and
human serum albumin (HAS) which is recognized as a principal component of blood and the
most abundant protein (very similar to BSA [16]) and the effect of pH. It is very clear that the
optimal conditions to get a maximum of adsorbed amount of protein is to use a solution of pH
ranges between 4.6-5 which is near to the point of zero charge of BSA. This will strongly
reduce the effect of electrostatic forces between the BSA and the surface. More addition to
this pH value will not change too much the structure of BSA.
104
Figure 3.20 Schematic representation of: The theoretical variation of the surface charge (σs) vs
pH curves for (a) HAS in solution and (b) TiO2 colloidal particles. (c) Representation of
protein molecules adsorbed under different electrostatics conditions [16]
3.9 Salt Effect on the Adsorption of BSA
J. Chen et al. modeled the salt effects on the adsorption of protein [25]. The figure 3.21
bellow shows the effect of salt concentration on the adsorption of BSA onto TiO2 surface. We
studied the adsorption of BSA using three different concentrations of salt (10-5
, 10-4
and 10-2
mol/l) and we have seen that the concentration does not change too much the adsorbed
amount of BSA whereas it changes the kinetic of adsorption. It was observed that at 10-2
mol/l
of NaCl salt increased a bit the adsorption of BSA. It has been documented that increasing salt
concentration has a positive effect on protein adsorption to hydrophobic adsorbents.
105
Figure 3.21 Adsorption equilibrium spectra of BSA onto TiO2 anatase using 10-2
mol/l of
NaCl salt concentration in water solvent at 10-6
mol/l of BSA concentration. Time between
spectra is around 10 min.
Figure 3.22 Second derivative of adsorbed BSA spectra with salt concentration of 10-2
mol/l.
around 10 min between two spectra
106
This is very obvious in terms of our experimental data shown in figure 3.21.The salt can not
affect the secondary structure of protein as shown in figure 3.22.
It is well known that protein molecules are associated with a hydration shell in solution. The
bound water prevents protein molecules from binding to the hydrophobic ligands on an
adsorbent surface. However, in the presence of a salt, the protein will be dehydrated due to the
hydration effect of the salt molecules surrounding the protein (Figure 3.23). Thus, the
hydrophobic zones of the protein will be naked gradually with increasing salt concentration.
Figure 3.23 Schematic presentation of the two-state protein model. The hydrated protein
molecule is associated with a hydration shell, so its hydrophobic zones are completely
covered by water, preventing it from binding to any hydrophobic ligand. The hydrophobic
zones of dehydrated-state protein are exposed due to the hydration effect of salt in solution, so
it can bind to hydrophobic ligands through the exposed hydrophobic zones. Note that the
hydration shell on the hydrophilic and charged surfaces (white area) is not indicated [25]
107
3.10 Warm Water Effect on Adsorbed BSA
Heating BSA in aqueous environment induce gels [26-28] which have great interest for
protein science [29, 30]. According to the results presented in this section, it is very clear that
rinsing with normal water at room temperature does not affect the equilibrium any somehow.
This equilibrium is affected by rinsing with warm water (heated at 50, 70 and 100 °C) as
shown in figure 3.24.
Figure 3.24 In Situ spectra of BSA adsorption equilibrium affected by rinsing with warm
water
This graph demonstrates that warm water has an effect on the equilibrium especially in the
region between 3000 and 3600 cm-1
where the peak at 3200 cm-1
is decreased which means
that the ice water detected in the first equilibrium is perturbed somehow and it shows another
sign at around 864 cm-1
.
There is also a shift in the region of amide I band from 1653 cm-1
to 1648 cm-1
as shown in
the figure 3.25. This shift as function of time is represented in the figure 3.26.
108
Figure 3.25 Shift in amide I band due rinsing with warm water
Figure 3.26 The shift from 1653 to 1648 cm-1
as a function of time
109
Both figures (3.25 and 3.26) clearly show that the shift is fast in the first 15 minutes and then
it goes to reach equilibrium. The two equilibria are shown in the figure 3.27 and the
difference between them is illustrated in the figure 3.28.
Figure 3.27 Equilibrium after rinsing with normal water and warm water
Figure 3.28 Difference between equilibrium spectrum after rinsing with warm water and after
rinsing with normal water
110
From the figure 3.28, we can conclude that rinsing with warm water of the adsorbed protein
leads to decrease ice water detected at around 3200 cm-1
and the appearance of free water at
around 3600 cm-1
. In order to show if this warm water rinsing affects the structure of adsorbed
protein at least qualitatively, we present in the figure 3.29 the second derivative evolution of
amide I band of the equilibrium after rinsing with normal water (at room temperature) and
warm water.
Figure 3.29 Second derivative of amide I spectrum of adsorbed BSA equilibrium on TiO2
anatase. Spectra are corrected from water liquid (normal/warm) and gaseous water
In order to avoid any perturbation of water spectrum changes on the quality of our adsorbed
BSA spectrum, we corrected the spectra using a normal water spectrum and a warm water
spectrum at the same conditions of the experiment. The water vapor is also corrected from our
spectra. It is very clear that the second derivatives of the two equilibria are not the same.
There is an appearance of new peak at around 1646 cm-1
. This peak refers to a random coil in
the secondary structure of the adsorbed BSA protein. More addition to this there is an increase
in the peak at around 1630 cm-1
. These observations strongly indicate that heating the surface
where the protein is adsorbed will change the secondary structure of the protein.
To check the effect of temperature on the adsorption kinetic of BSA, we present in the figure
3.30 the changes in the background (base line) using water at room temperature and warm
water at a defined temperature of 50 °C in the beaker outside the FTIR instrument.
111
Figure 3.30 Changes in the background after rinsing with warm water
Figure 3.31 BSA adsorption with normal water and warm water as solvents (background is
taken with normal water and warm water, respectively)
Figure 3.30 indicates that water spectrum is changed under heating. We can see the free water
at around 3620 cm-1
and the reducing of ice water that gives a sign at 864 as a negative peak.
Two measurements are done using the same concentration of BSA (10-6
mol/l), the first is
done with warm water and the second using normal water at room temperature. The two
112
equilibrium spectra are shown in the figure 3.31 that demonstrates that the signal in the case
of BSA adsorption using warm water is much higher than in the case of normal water. This
means that BSA adsorption is higher at high temperatures than at room temperatures.
The adsorption kinetics does not change until the temperature gets to the point at which the
protein denatures. Beyond this temperature, adsorption increases quickly.
When denaturation occurs the adsorption will abruptly and dramatically increase. The layer,
which gets formed, was considerably thicker than a monomolecular layer. An explanation for
this behavior could be at high temperatures some amino acids from the protein are exposed to
the surface and can be linked to other molecules by S-S bridges as shown in the figure 3.32.
Figure 3.32 Aggregation of β-lactoglobulin protein on the solid surface at a high temperature.
Taken from [31]
This behavior under high temperatures (denaturation) strongly depends on the exact
temperature and it can be reversible or irreversible. Other authors found a decreasing in
adsorption amount by rising temperature [32].
Water molecules have interactions with BSA molecules and can also play a fundamental role
in this denaturation under high temperature so it is important to know how water interacts
with the protein.
Surface
Denaturated Aggregated
Native Protein
Heat
Heat Heat
113
3.11 Water Interaction with TiO2 Surface and Adsorbed BSA
The interaction of water with the protein in solution or at the surface is still one of the biggest
difficulties that are not very clear right now. Water molecules can interact with BSA
molecules by Van der Waals or hydrogen bonds because the BSA has nitrogen and oxygen
atoms. Using ATR in situ spectroscopy we proved that water can adsorbed onto the surface of
BSA.
Figure 3.33 In situ ATR spectra evolution of normal drop water (not heated) deposited on
BSA deposited onto TiO2 (spectra and background are collected in 1000 hpa of vacuum in the
sample compartment. BSA spectrum is corrected from water gaseous)
Figure 3.33 shows the in situ time evolution of spectra after a drop of water was deposited on
the surface of BSA deposited on TiO2 at a temperature of 50 °C. The evaporation of this water
drop from the surface of BSA is shown in figure 3.34. In the beginning water is slightly stable
then is begins to evaporate and that is the reason why we see the peak at 3380 cm-1
reducing
as a function of time.
114
Figure 3.34 Evolution of the maximum position at 3380 cm
-1 in water spectrum versus time
In order to elucidate the conformational changes induced of water adsorbed on the surface of
BSA as a function of time, we show the second derivative FT-IR spectra in the ice and liquid
water region between 3000-3600 cm-1
in figure 3.35.
Figure 3.35 Second derivative of water spectrum in the region of ice and liquid water
115
We have seen the same behavior of water drop on the surface of TiO2 without protein.
According to this graph, it is clear that the water molecules can easily adsorb onto BSA
surface as presented on the picture of the figure 3.36.
Figure 3.36 Water-protein interaction a- a schematic illustration of the hydration structures
on the protein surface, b- distribution of hydration water molecules around protein: The green,
yellow and purple spheres (diameter of 3A°) are the hydration water molecules in the ‘first-
layer’, the ‘second-layer’ and the ‘contact’ classes, respectively [33]
The interaction between water and protein (love-hate relationship) are still a mystery in the
field because of the shape and cavities in the protein and the hydrogen bonding between the
two systems [34-41].
3.12 UV Modulation of Adsorbed BSA
Varying periodically any exterior parameter (UV/VL light, concentration, pH) that can affect
somehow the system (adsorbed BSA on the surface) will lead to a periodic behavior of the
system itself or some of its molecules that can follow the variation of the exterior parameter.
The in situ modulation using ATR spectroscopy is done using Xenon lamp and the OPUS
program that controls the period of modulation. The figure 3.37 shows the 3D spectrum of the
modulation where the first thirty (30) spectra are collected under UV irradiation and the
second thirty spectra (30) are collected in dark.
Using another developed program we can do the demodulation of 3D spectra. This
demodulation will show the little changes in the system during and before UV irradiation.
-a- -b-
116
Figure 3.37 UV modulation of adsorbed BSA onto TiO2 anatase (40 scan)
3.13 Conclusion
The results presented in this chapter concerning the effect of environment on the
adsorption of BSA on different surfaces are important. This study allows us choosing good
conditions for adsorption depending on the characterizations wanted like the amount of
adsorbed BSA, the conformation and the kinetics of adsorption (slowly or quickly). We have
seen that the properties of the surface control somehow the adsorption. pH of the solvent
denaturate the BSA protein and the high amount of adsorption is found to be at a pH near to
the isoelectric point of BSA or the surface. The kinetics of adsorption of warm BSA is
different from the normal adsorption at room temperature. NaCl does not affect too much the
kinetic of adsorption and the secondary structure of BSA.
In the next chapter we will present the results of the behavior of adsorbed BSA under
light shining that can affect the secondary structure component of this protein. The interaction
between the protein and the surface under exposure to light needs to be understood because of
the important applications of this kind of adsorption in a lot of disciplines.
117
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121
Chapter 4
Photo-degradation and Denaturation by
Light Illumination of Adsorbed BSA on
the Surface of TiO2
The behavior of adsorbed protein on solid-surface under shining light is very
important. In the first part of this chapter, we will present the most important results of the
photo-degradation by UV light of adsorbed BSA on TiO2 anatase. In the second part we show
the denaturation of adsorbed BSA on TiO2 P25 using visible light illumination. These results
will be discussed on the basic of previous studies.
122
4.1 Introduction
The irradiation of adsorbed BSA on solid-surface will lead to some changes in the
protein. It is well known that TiO2 has a band gap around 3 eV that allows this material
absorbing UV light [1]. This absorption leads to the creation of electron hole pair which
migrates to the surface of TiO2. At the surface where BSA is adsorbed, it might react with the
protein molecules. This photo-degradation process can produce new products and affect
somehow the secondary structure of the adsorbed protein. Using visible light illumination, the
protein can with the light and its structure is affected. It is very important to take into account
the changes in the water spectrum during light irradiation before doing second derivative
analysis that enables the details about the quantitative amount of the secondary structure of
adsorbed BSA. In general the exposure of proteins to light can lead to changes in primary,
secondary and tertiary structure of protein and these changes, while not well established,
could lead to differences in long-term stability and bioactivity [2]. In the following parts of
this chapter we will present the most important results of the behavior of adsorbed BSA on
TiO2 under shining with UV light and visible light. These results indicate that ATR-FTIR
spectroscopy is a powerful technique that can give important information about the changes in
the secondary structure of adsorbed BSA.
4.2 UV Photo-degradation of BSA over TiO2 Anatase
Figures 4.1 and 4.2 show the equilibrium of the BSA adsorption onto TiO2 and the effect of
UV light on this equilibrium, respectively. It is clear that UV light decreases the adsorbed
amount of BSA. Due to its stability and non-toxicity TiO2 has been the most investigated in
detail as photocatalyst [3]. Its band gap is ca. 3 eV which corresponds to wavelength in the
UV region [1]. Therefore, a wavelength with energy equal or bigger than the band gap of this
semi-conductor can excite electrons in the valence band to conduction band [4-8]. The
electron-hole pair generated by light serves as the oxidizing and reducing agents [9] of
adsorbed BSA. This phenomenon is complex and still not very well understood right now.
The efficiency of TiO2 was reported to be influenced by many factors [10-14] and the
hydrophilicity was found to play an important role in the photo-degradation activities.
123
Figure 4.1 ATR-IR spectra recorded during adsorption of BSA onto TiO2. Equilibrium was
reached after about 80 minutes. a- Absorbance spectra collected in situ, b- variation of amide I
band versus time
Figure 4.2 The effect of UV light on the adsorbed BSA. a- Absorbance spectra collected in
situ (spectra were collected while flowing water and illuminating with UV light), b- variation
of amide I band versus time
-a-
-b-
-b-
-a-
124
Figure 4.3 shows the signal at 1654 cm-1
as a function of time for BSA adsorption on TiO2
followed by rinsing by water and UV illumination. The adsorption is initially very fast and
then turns over in a linear regime. By UV illumination one part of the BSA is removed but the
removal seems to stabilize at some point, indicating that some of the BSA molecules are
“irreversibly” bound to the surface. This behavior is still not well understood and it could be
caused by the changes of the surface properties during UV illumination for long time.
According to literature, the photocatalytic activity of TiO2 has been found to be tied to the
surface properties of the catalyst. Some of the particle properties which are known to affect
the photocatalytic activity are particle size, crystal structure, amounts and the identity of
defects and preparation method [15-18]. It has been found that UV irradiation of the titanium
dioxide surface will induce superhydrophilicity, which changes the nature of a surface from
hydrophobic to hydrophilic by removing organic compounds, inducing oxygen vacancies and
breaking bonds [19-24].
Figure 4.3 Absorbance at 1654 cm-1
corresponding to the amide I band of BSA adsorbed on
TiO2 as a function of time during adsorption, rinsing by water and UV illumination
We have seen also the appearance of a peak at 2341 cm-1
(Figure 4.4) characteristic of
dissolved CO2. This is a strong indication that mineralization of BSA takes place upon
illumination of the surface by UV (light).
125
Figure 4.4 The appearance of a peak at 2341 cm-1
after UV illumination for 5 hours and
during the whole night. Spectra were smoothed using smooth function (Savitzky-Golay
algorithm) in OPUS program to reduce the noise
Figure 4.5 Absorbance of pure BSA in water at a concentration of 10-6
mol/l
The figure 4.5 obtained by spectrophotometer (UV/vis Jasco 650) shows the absorbance of
the pure BSA in water (10-6
mol/l) where it is clear that there are two peaks of absorption
126
around 280 nm (less intense) and around 210 nm (more intense) are due the aromatic groups
and amide groups, respectively, in BSA.
A pure solution of BSA in water was irradiated by UV light for more than two hours and after
measuring the absorbance we could not see any difference before and after illumination.
We have collected the solution of BSA during illumination by UV and we measured by
spectrophotometer the absorbance which is shown in figure 4.6. It is very clear that there is a
big difference between this spectrum and the one shown on figure 4.5.
Figure 4.6 Absorbance of solution obtained during illumination of adsorbed BSA by UV
We adsorbed the same solution (obtained after irradiation by UV) on a fresh TiO2 surface and
the spectrum is different from that of pure BSA in water.
These two observations confirm that the UV light does not desorb the whole protein from the
surface of TiO2 but it may cut it into small pieces that produce other products such as CO2, as
it is expected by other authors [25], which was indicated by the appearance of a peak around
2341 cm-1
, after irradiating for long time, characteristic to dissolved CO2 in water.
To study the effect of UV irradiation on the secondary structure of BSA we calculated the
percentage of this structure of BSA in water solution. We prepared three solutions of high
concentration of BSA (1.5x10-4
, 2x10-4
and 3x10-4
mol/l) and we adsorbed each solution on
ATR crystal of Germanium (Figure 4.7) and after rinsing by pure water the difference
between the two spectra is corrected from water using the two criteria mentioned before.
127
Figure 4.7 Corrected spectrum of pure BSA in solution at a concentration of 2*10-4
mol/l from
water using the two criteria described in the text
Figure 4.8 Curve fitting of negative second derivative of pure BSA in water solution at
concentration of 2*10-4
mol/l
The curve fitting of negative second derivative (Figure 4.8) of corrected spectra reveals the
percentage of the secondary component as follows:
128
Intermolecular β-sheet at 1613.8 cm-1
1.5%
β-sheet at 1632.0 cm-1
22.5%
α-helix at 1655.1 cm-1
66.6%
Turn at 1680.2 cm-1
9.2%
The same procedure is taken to study the secondary structure of BSA under and before UV
irradiation. The table 4.1 shows the result of negative second derivative fitting of adsorbed
BSA spectra onto TiO2 before UV irradiation.
Table 4.1 Curve fitting results of negative second derivative of adsorbed BSA on TiO2 before
UV irradiation (% is the percentage corresponds to each peak at different times)
Time min
Peak 7.4 14.6 28.9 43.6 58.4 79.9
1614 cm-1
% 1.5 2.0 1.7 2.2 2.2 2.5 1634 cm
-1 % 25.5 24.5 25.1 24.3 24.7 24.9
1655 cm-1
% 64.1 63.5 62.7 63.9 63.1 62.5 1680 cm
-1 % 8.7 9.8 10.3 9.6 9.9 10.0
Table 4.2 Curve fitting results of negative second derivative of adsorbed BSA on TiO2 after
UV irradiation (% is the percentage corresponds to each peak at different times)
Time min
Peak 0.7 36.4 72.6 108.8 145.4 181.6 218.3 254.6
1614 cm-1
% 2.0 2.4 2.2 1.8 1.5 2.0 2.0 2.4
1634 cm-1
% 25.1 24.2 26.1 26.1 26.6 26.6 27.1 26.7 1654 cm
-1 % 62.5 60.2 58.1 57.3 56.7 55.8 54.7 54.0
1680 cm-1
% 10.4 12.8 13.3 14.7 15.1 15.5 16.0 16.5
According to tables 4.1 and 4.2, the percentages of the secondary structure before UV
irradiation are still constant, slight change, but after UV irradiation the percentage of α-helix
decreases.
The figure 4.9 and 4.10 show the decreasing of α-helix from around 62% before UV
irradiation to around 54% and the increasing of β-turn from around 10% to 16% after UV
illumination respectively.
129
Figure 4.9 The variation of the percentage of α-helix versus time
Figure 4.10 The variation of the percentage of β-turn versus time
130
4.3 Visible Light Denaturation of Adsorbed BSA onto TiO2
Figure 4.11 shows the amide I region in the corrected equilibrium spectrum of
adsorbed BSA on TiO2 after rinsing with water and the same region under visible light
irradiation with rinsing water for around 01 hour and (corrected also taking a liquid spectrum
of water irradiated around 1 hour in the same background). The amide I mode consists of
C=O stretching and C-N stretching. It appears in the region from 1600 to 1700 cm-1
and it is
highly sensitive to the secondary structure of proteins, so that it has served as an indicator of
α-helix and/or β-sheet conformations. The dominant peak at around 1653 cm-1
of the adsorbed
BSA in water is characteristic for the helical secondary structure which is the major structural
component of BSA. This peak is shifted from 1653 cm-1
to around 1648 cm-1
under visible
light illumination and it indicates an increase in population of disordered structure.
Figure 4.11 Amide I region of the ATR-IR spectrum (water corrected) of BSA adsorbed on
TiO2 before and after irradiation with visible light for around one hour. The successful water
correction is indicated by the straight baseline above 1720 cm-1
Figure 4.12 shows the peak position of the dominant amide I band plotted as function of time
during visible light irradiation and rinsing with water
131
Figure 4.12 The position of the maximum of the amide I band versus time for adsorbed BSA
on TiO2 during visible light illumination. Illumination was performed while rinsing the
sample with water
The evolution of peak position clearly shows that the denaturation of adsorbed BSA
during illumination with visible light is fast in the beginning and it becomes stable by the end
of the first half an hour where the denaturation reaches its maximum.
For interpreting this shift and in order to elucidate the conformational changes induced
by visible light, we show in Figures 4.13 and 4.14 the second derivative of the corrected
spectra before and after illuminating without and with visible light respectively.
As a comparison between these two figures, we see that the peak at 1654 cm-1
is
decreased, whereas the one at 1631 cm-1
is shifted to 1628 cm-1
and increased which is
characteristic to β-sheet conformation. There is an appearance of a new peak between 1645
cm-1
and 1642 cm-1
which refers to a random coil structure.
Following iterative fitting of Gaussian curves to the observed bands in the second derivative,
the relative amounts of secondary structure were determined from areas under bands assigned
to a particular structure.
132
Figure 4.13 Negative second derivative of amide I region for corrected equilibrium adsorption
spectrum of BSA on TiO2 before irradiating with visible light (VL)
Figure 4.14 Second derivative of amide I region for corrected equilibrium adsorption
spectrum of BSA on TiO2 under rinsing by water and irradiation with visible light (VL) for
around one hour. Intervals between spectra are around 10 minutes
133
The fit of the second derivative spectra before (figure 4.13) and during visible light
irradiation (figure 4.14) allows us to follow the variation and changes of the secondary
structure of adsorbed BSA on TiO2 induced by light.
The results of fitting before light (Figure 4.13) irradiation show:
Peak at 1615 cm-1
%: 1.6%
Peak at 1631 cm-1
%: 22%
Peak at 1653 cm-1
%: 66%
Peak at 1680 cm-1
%: 10%
which are in well agreement with literature [26-32].
Figure 4.15 Fitting of negative second derivative spectrum of adsorbed BSA after one hour of
visible light irradiation
The results of fitting during illumination (after around 30 minutes, Figure 4.15) show
Peak at 1614 cm-1
%: 2.3%
Peak at 1629 cm-1
%: 18.7%
Peak at 1643 cm-1
%: 21.3%
Peak at 1656 cm-1
%: 40.2%
Peak at 1678 cm-1
%: 9.7%
Peak at 1690 cm-1
%: 7.5%
134
Figures 4.16 and 4.17 show the variation of α-helix percentage at around 1653 cm-1
under visible light illumination and the variation of random coil percentage at around 1643
cm-1
respectively. It is very clear that the behaviour of the changes in the secondary structure
of adsorbed BSA on TiO2 (reducing α-helix and increasing random coil structure) follows the
shift in peak position shown in Figure 4.12.
Figure 4.16 Variation of α-helix structure percentage of adsorbed BSA during visible light
irradiation (corrections were made in the original spectra)
The visible light decreases the percentage of α-helix from around 66% to 34% because
of the destruction of hydrogen bonds that give a perturbation of the secondary structure of
adsorbed BSA by the creation of new structure such as β-sheet around 1690 cm-1
and random
coil around 1645 cm-1
. During this behaviour we have seen a new peak at 3600 cm-1
(not
shown here) which is a strong indication of free water (OH) that can come from adsorbed
water, protein or both.
135
Figure 4.17 Variation of random coil structure percentage of adsorbed BSA during visible
light irradiation (corrections were made in the original spectra)
Figure 4.18 Negative second derivative spectrum of amide I region for BSA on TiO2. The
spectrum was recorded in dark (without visible light) and during rinsing with water after
visible illumination for around one hour
136
We did the same analysis in dark after correcting the spectra and it is clear that the
percentages of the secondary structure is more or less stable as shown in Figure 4.18 which
indicates that the denaturation of the protein on the surface of TiO2 by visible light is an
irreversible phenomenon.
The visible light changes also the behavior of adsorption as shown in figure 4.19
which clearly indicates that adsorption of BSA with visible light is faster than without
illumination. It could be explained by an influence of visible light illumination on the
properties of the surface of TiO2 and BSA during adsorption because it is known that higher
temperatures increase the amplitude flexibility [33-34]. As a result, the hydrophobic core of
protein possibly exposes, and therefore the protein at high temperature tends to aggregate
more easily. So, we expect that BSA under visible light has flexible structure, easily entwines
with each other and form intermolecular β-sheet structure.
Figure 4.19 Variation of the intensity of amide I band of adsorbed BSA on TiO2 with and
without visible light illumination (VL)
We note that illumination of BSA adsorbed on TiO2 by light changes more the
secondary structure than adsorbing the protein during illumination.
Shining the surface with light especially visible will change the morphology of the water layer
adsorbed on the surface and in contact with the protein. Germanium is good material for
infrared application but it has a gap of around 0.7 eV. This allows it to absorb in the visible
137
light region. To elucidate the change in the spectrum of germanium during visible light
illumination, we illuminated the pure germanium with visible light. The figure 4.20 shows
this behavior under illumination. This graph strongly shows that germanium spectrum
changes during visible light irradiation. The base line shifts between around 1700 cm-1
to
around 900 cm-1
. This shift is faster in the beginning then it reaches equilibrium. This
phenomenon is reversible because in dark we can return to the initial state before shining the
surface with visible light. The same behavior has been seen on the surface of germanium and
silicon without TiO2 porous film. We mention also here that increasing the velocity of
pumping during illumination affects also the behavior of water on the surface of TiO2.
Figure 4.20 Absorbance spectra during visible light illumination of pure germanium surface,
around 4 minutes between two successive spectra.
The previous results presented in this work show how visible light change the spectrum of
germanium, used as a substrate, which perturb the spectra of adsorbed protein. The
deconvolution technique and the fitting can give important information about the secondary
structure of the protein especially on the surface. Water envelopes the region of amide I (1640
cm-1
) and the region of the NH vibration. Before studying the secondary structure of the
138
protein it is very important to take into account the changes in water spectrum before doing
corrections in adsorbed protein spectra.
Light disturbs somehow the equilibrium of the surface especially the molecules of water in
the bulk and at the surface.
The same experiment was done in the presence of TiO2 P25 on the surface of Germanium.
The shift in the base line is shown in the figure 4.21. It is very clear that this shift is bigger
than the first one (with only germanium) even it has the same behavior as before concerning
the kinetic.
Figure 4.21 Absorbance spectra of TiO2 deposited on germanium during visible light
illumination (around 4 minutes between two successive spectra)
Nakamura et al. have reported that UV irradiation of the TiO2 surface thin film in air increases
the 3270 cm-1
IR absorption peak, which is assigned to the O-H stretching band [36].
Figure 4.21 clearly indicates an important shift in the ATR-IR absorption spectra between
1750 cm-1
and 1000 cm-1
. The increase in the adsorption in this region is due to the shallow
trap electrons excitation as explained by many authors [37, 38].
139
Figure 4.22 Processes involving electrons after optical promotion into CB [37] (a) Intraband
excitation. (b) Electrons trapped in shallow traps. (c) Excitation of shallowly trapped electrons
into CB
Figure 4.23 Possible optical transitions in a band gap with shallow traps irradiated: (a) the
intra-band transition of UV-excited electrons; (b) Electrons trapped in shallow traps. (c) the
trapped-electron excitation to the CB; (E) the intra-band transition of UV-excited holes and
(d) the trapped hole excitation to the VB. ΔE1 and ΔE2 represent the gap between the mid-gap
states and the CB, VB bands respectively [38]
140
Figure 4.22 and 4.23 show the possible transitions in a TiO2 film with two energy levels in the
band gap. Using UV light will create generate an electron from the valence band to the
conduction band. This electron can be moved in the same band (CB or VB) by absorption of
infrared (Figure 4.21) radiation and this is one of the explanations of the increase in the region
between 1750 cm-1
to 1000 cm-1
. The existence of an energy level near to the CB or VB can
also have a fundamental role in the infrared absorption.
In the previous studies, the authors did not mention any relationship between the shift in the
region of 1750-1000 cm-1
and temperature. The energy we put onto the surface during light
illumination is not exactly known to us. Furthermore, the gap of any semiconductor decreases
with increasing temperature (Chapter 1). Temperature can therefore be responsible for
decreasing the difference in energy between the shallow traps of electrons in the band gap and
the CB/BV that leads to an increase in the infrared absorption.
Commercial semiconductors, such as our TiO2, have a lot of defects. The gap of TiO2 thin
films is affected by the defects especially from the oxygen and the carbonic molecules coming
from the air and which are incorporated in the film during preparation stage. If there are so
many defects in the material this can also introduce many energy levels in the gap of TiO2. If
the difference between the levels of the defects is on the order of infrared energy, it can
contribute to the absorption of infrared, even a bit, which leads to the increase of the
absorption in the region concerned.
4.4 Conclusion
The results presented in this chapter clearly show that using UV light for a long time
(around 05 hours) to irradiate the surface can reduce the amount of adsorbed BSA which is
irreversibly adsorbed under rinsing only with water. Our results show that UV illumination
might cut the protein into small compounds that react between them to give final products like
CO2 detected in our experiments. The quantitative analysis of the secondary structural
components of adsorbed BSA clearly demonstrates the decrease of the percentage of α-helix
under UV light illumination. This behavior is totally different from the effect of pH and
temperature.
Using visible light can easily denaturate the protein and we have seen that the α-helix
structure is decreased whereas the new structure (random coil) is increased. This behavior is
similar to the temperature effect but might be different because visible light can create the
141
electron hole pair from the defects of TiO2 that can react with the adsorbed BSA at the
surface.
The behavior of protein adsorbed onto semiconductor surfaces especially under light
shining is still not really well understood right now. One of the biggest problems in
understanding protein behavior under light illumination is the effect of small molecules
attached to the surface of the protein and filling the space in between the protein molecules.
Water molecules are interacting with the BSA protein and they can be found between
adsorbed BSA and the surface of TiO2 and the transfer of electron and hole to the surface
crossing this adsorbed water layer is really a topic of research that can offer another
possibility for explaining and understanding more the behavior of protein adsorbed on the
surface under illumination. Understanding this complex phenomenon will be very useful in
medicine and other disciplines like biology.
142
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[17] M. Kang, S. Y. Lee, C. H. Chung, S.M. Cho, G.Y. Han, B. W. Kim, K.J. Yoon,
Characterization of a TiO2 photocatalyst synthesized by the solvothermal method and its
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145
General Conclusion and Recommendations
The solid-liquid interface is one of the most important topics of physics and chemistry
of surfaces. This interface is very important in many fields such as in the field of biology and
biocompatibility. There are several techniques useful for investigating this interface and in
situ attenuated total reflection spectroscopy is a powerful technique. ATR-IR allows working
under different conditions by changing parameters as: Temperature, pH, concentration,
light…
Knowledge about protein adsorption to solid surfaces especially in aqueous
environments (H2O) is crucial for many disciplines including environmental science. This
kind of adsorption is a very complex phenomenon and it is driven by different protein-surface
forces including van der Waals, hydrophobic and electrostatic forces. The behavior of protein
on the surface especially under varying the adsorption conditions is still not well understood.
In this research, we managed to achieve a few goals discussed in the general
introduction. The contributions of this thesis are discussed in the following:
Environment and protein adsorption. Well known small molecules are always easy to
be studied using in situ ATR-IR spectroscopy. Getting knowledge and information about
protein adsorption especially in water solution is a challenge for this technique. Important
parameters for adsorption are pH, temperature, the ionic strength, the properties of the protein
and the surface and also the nature of the solvent. The effect of these parameters and the
important results are discussed in the Chapter 3 of the present manuscript. The adsorption of
Bovine Serum Albumin (BSA) onto the different surfaces silicon, germanium and titanium
dioxide is an irreversible phenomenon after rinsing with water. The results show that the
amount of adsorbed BSA on TiO2 is much larger than the adsorbed amount of this protein on
silicon and germanium. This is a strong indication that the surface plays a role in controlling
protein adsorption. The reason is that the titania surface is porous and the roughness is bigger
than for silicon and germanium. The analysis of our results clearly shows that the adsorption
affects (slightly) somehow, in the very beginning of adsorption, the secondary structure of
adsorbed protein. The percentage of α-helix of BSA in water is around 65%. The influence of
pH is also studied and we found that the adsorption of BSA using a pH near to the isoelectric
point of protein or the surface increases the amount of adsorbed protein because the effect of
146
electrostatic forces will be decreased. The NaCl has an effect on the adsorption because there
is an interaction with the molecules of BSA.
Light effect on BSA. In Chapter 4, the most important results of photo-degradation and
denaturation of adsorbed BSA are discussed. In the first part of this chapter we showed that
the irradiation with UV light decreases the amount of adsorbed BSA. Reducing adsorbed BSA
does not mean releasing the whole protein from the surface but it might be cut into small
pieces that react between them and one of the final results of this photo-degradation is the
production of CO2 gas. The fitting technique of the second derivative of adsorbed BSA
spectra during UV illumination clearly shows that the percentage of α-helix is reduced from
around 65% to reach around 54% whereas the percentage of β-turn is increased from around
10% to 16%. The results concerning the denaturation of adsorbed BSA on TiO2 surface using
visible light irradiation are shown in the second part of Chapter 4. Irradiating the surface with
visible light shifts the maximum position of the amide I from 1654 cm-1
to 1648 cm-1
which
indicates changes in the secondary structure of adsorbed BSA. Our analysis clearly
demonstrates the creation of random coil structure which affects the percentages of other
structure by decreasing α-helix from around 65% to around 34% and increasing random coil
till around 30%. This denaturation by visible light is irreversible which indicates that the
adsorbed protein is strongly denaturated therefore the possibility is smaller that it refolds to
native conformation.
Future recommendations. Many studies were done for investigating the adsorption of
proteins on different surfaces and our present manuscript is one of them. This study confirms
that adsorption of protein is still a very complex process especially in water solution. The
interaction of water molecules and their effect on the adsorption mainly on the correction of
gaseous water and liquid from protein spectra before doing analysis is very important. Under
light shining the nature of the adsorbed water layer on the surface is perturbed and this leads
to changes in the structure of protein which strongly depends on the position of adsorption
and the mechanism that determines the bonds between the protein and the surface. Another
important research question would be whether adsorbed proteins are bioactive or not. It was
found that some adsorbed proteins have a stable structure on the sorbent but they are less
bioactive than less stable ones. Perhaps the bioactive center of a stable protein is hindered to
operate but the bioactive center of a less stable protein may still operate. The transfer of
electrons in the protein is controlled by a lot of parameters [1-6] and one of the most open
questions in studying protein-solid surface interactions especially in the photo-degradation is
the transfer of the electron from the surface to the protein. This behavior study is really
147
recommended because it will allow a certain control over the electron hole pair generated by
light. This control enables to improve the photo-catalysis efficiency by doping the material or
looking for another new material that has more important characteristics. Using hydrogenated
amorphous or microcrystalline TiO2 can be of interest because this will increase the
absorption spectrum of light. Finding such material can open a lot of applications of photo-
catalysis in the industry field.
At the end of this thesis and according to the efforts discussed and the suggested
recommendations, it is very clear that the adsorption of biology macromolecules on surfaces
still remain one of the very complex processes. This behavior can only be well studied when
Physics and Chemistry meet Biology! Because “the most beautiful thing we can experience is
the mystery of things” and “The joy of looking and understanding is the greatest gift of
nature” Albert Einstein.
148
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149
Papers Related to this Research
[1] M. Chahi, A. Bouhekka, J.D. Sib, A. Kebab, Y. Bouizem, L. Chahed, Optoelectronic
properties simulation of hydrogenated microcrystalline silicon Schottky diode. Physica
Status Solidi (c) 7 (3-4) (2010) 640-645.
[2] A. Bouhekka, A. Kebab, J.D. Sib, Y. Bouizem, M. Benbekhti, L. Chahed, Monte-Carlo
simulation of hydrogenated amorphous silicon growth. Journal of the Association of
Arab Universities for Basic and Applied Sciences 12 (2012) 11-16.
[3] A. Bouhekka, T. Bürgi, In situ ATR-IR spectroscopy study of adsorbed protein: Visible
light denaturation of bovine serum albumin on TiO2. Applied Surface Science xxx
(2012) xxx-xxx.
[4] A. Bouhekka, T. Bürgi, Photodegradation of Adsorbed Bovine Serum Albumin on TiO2
Anatase Investigated by In-Situ ATR-IR Spectroscopy. Acta Chimica Slovenica 261
(2012) 369-374.
150
Curriculum Vitae
Name Ahmed Bouhekka
Date of Birth March 20.02.1977
Place of Birth Melaab-Tissemsilt, Algeria
Nationality Algerian
Education
Since 2005 Doctoral studies under the supervision of Prof. Sib Jamal
Dine at LPCMME laboratory, University of Oran,
Algeria, and Professor Thomas Bürgi at the Institute of
Physical Chemistry, Heidelberg, Germany and Physical
Chemistry department at the University of Geneva,
Switzerland
2001-2005 Magister in Physics, University of Oran Es-Senia,
Algeria
1997-2001 Diploma graduate studies in physics (D.E.S), University
Ibn Khaldoun, Tiaret, Algeria
1994-1997 Secondary school studies (Bacalaureat Diploma),
Mohamed Seray Secondary school, Lardjem-Tissemsilt,
Algeria
1994-1985 Primary and fundamental school studies, Melaab,
Tissemsilt, Algeria
Conferences/Workshops
23-28.08.2009 23
rd International Conference on Amorphous and
Nanocrystalline Semiconductors, Utrecht-the
Netherlands
13-14.10.2011 Bunsen lnternational Discussion Meeting, Heidelberg,
Germany
27.01.2012 Groupe Suisse de Travail Surface/Interface GSSI,
Fribourg, Switzerland
06-07.02.2012 Molecular Recognition: When Biology meets Chemistry,
USGEB at the Univesrity of Lausanne, Swilzerland
20-24.08.2012 Summer School Villars 2012 "Hydrogen Bonding" at
EurotelVictoria in Villars, Switzerland
151
13.09.2012 Swiss Chemical Society (SCS) Fall Meeting, Ziruck,
Switzerland
Scholarships
16.09.2007-13.01.2008 Training period at Professor Bürgi group at the
University of Neuchatel, Switzerland
01.03.2009-01.05.2009 Training period at Professor Bürgi group at the
University of Heidelberg, Germany
01.09.2009-01.07.2011 DAAD Scholarship, German course and research in
Mannheim and Heidelberg, Germany
01.07.2011-30.10.2012 Funding from Geneva University, Switzerland
Professional Experience
2005-2006 Travaux pratiques, University of Oran, Algeria
2006-2008 Maitre Assistant (B), University of Chlef, Algeria
Since 2008 Maitre Assistant (A), University of Chlef, Algeria
01.10.2011-20.09.2011 Thermodynamics Exercises for the first year bachlor
students, University of Geneva, Switzerland
20.09.2012-30.10.2012 Thermodynamics Exercises for the first year bachlor
students, University of Geneva, Switzerland
Languages Spoken
Arabic Native speaker
French Fluent
English Fluent (C1 Level from Zentral sprachlabor Institute in
Heidelberg, Germany)
German B2 Level from Goethe Institute, Mannheim, and Max-
Weber-Haus Institute, Heidelberg, Germany
