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Linköping Studies in Science and Technology
Dissertation No. 1446
Structural Studies of
Oligo(ethylene glycol)-Containing
Assemblies on Gold
Hung-Hsun Lee
Division of Molecular Physics, Department of Physics, Chemistry and Biology
Linköping University, SE-581 83 Linköping, Sweden
Linköping 2012
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Cover: The front cover is optimized geometries of the OEG-containing alkanethiols on a
gold surface. The back cover is an IR-RA spectrum in 3000-2800 cm-1 region obtained by
pairwise subtraction of an alkanethiolate SAM and a deuterated alkanethiolate SAM.
During the course of the research underlying this thesis, Hung-Hsun Lee was enrolled in
Forum Scientium, a multidisciplinary doctoral programme at Linköping University, Sweden.
© Copyright 2012 Hung-Hsun Lee, unless otherwise noted
Hung-Hsun, Lee
Structural Studies of Oligo(ethylene glycol)-Containing Assemblies on Gold
ISBN: 978-91-7519-898-9
ISSN 0345-7524
Linköping Studies in Science and Technology, Dissertation No. 1446
Electronic publication: http://www.ep.liu.se
Printed in Sweden by LiU-Tryck, 2012
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For we walk by faith, not by sight
II Corinthians 5:7
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Abstract
The work presents in this thesis has been focused on structural characterization of a series of
selected well-defined molecular architectures for the application as biomimetic membranes. The
molecular architectures were prepared by self-assembly from dilute solution onto gold substrates,
so called self-assembled monolayers (SAMs).
Biological membranes are essential components for all living systems; their molecular
organizations and interactions with intra- and extracellular networks are key factors of cell
functions. Many important biological processes are regulated at membrane interfaces via
interactions between membrane proteins. Therefore, identification of the cell structures and
understanding of the processes associated with membranes are crucial. However, the intrinsic
complexity of the cell membrane systems makes direct investigation extra difficult. Based on this
reason, artificial model membranes have become a useful strategy. Especially, solid supported
tethered lipid membranes on SAMs allow for controlling the composition and geometry of
biomimetic assemblies on molecular scale. However, the underlying mechanisms of lipid vesicle
fusion on SAMs remain unclear. In this thesis, a series of thiolate SAMs containing alkyl chains
and oligo(ethylene glycol) (OEG) portions of different length as well as amide linking groups
were prepared and characterized in detail by employing a number of surface analyzing methods.
In parallel, a set of ab initio modeling was undertaken for the best interpretation of the
experimental infrared spectra. Investigation of small unilamellar vesicles interact with such
SAMs is included as well.
The results show this type of assemblies forms highly ordered and oriented SAMs regardless of
the length of the extended alkyl chains. The two layers of lateral hydrogen bonding networks
through the two amide linking groups improve further the structural robustness of the assemblies.
Furthermore, the use of deuterated terminal alkyl chains enables a direct relation between the
surface density of the anchor molecules and the properties of the lipidbilayers. IRRAS data and
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ab initio modeling confirm that orientation of the helical OEG is affected by the second
hydrogen bonding layer rather than the extended alkyl tails. Nanopatterns consisting of such
SAMs with different extended alkyl chains can be employed as supports for the assembly of
artificial cell membranes.
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Populärvetenskaplig Sammanfattning
Biologiska cellmembran är centrala beståndsdelar i alla levande system. Deras molekylära
organisation och växelverkan med intra- och extra-cellulära nätverk är viktiga faktorer för cellers
funktion. En annan viktig uppgift är hålla giftiga ämnen utanför cellen. De styr också selektiv
transport av specifika näringsämnen, molekyler och joner via öppningar och kanaler. Biologiska
cellmembran består av ett dubbelt lipidskikt, som befinner sig i ett väskeliknande tillstånd;
membranproteiner vars uppgift är att styra cellsignalering och reglera cellulära reaktioner;
kolhydrater på ytan som kommunicerar med omgivningen. Biologiska cellmembran är
strukturellt mycket komplexa och dynamiska supramolekylära system vars huvudsakliga uppgift
är att skydda, innesluta, organisera funktioner i levande celler. Intresset för att studera olika
processer och funktioner hos biologiska cellmembran har ökat markant de senaste åren. Som ett
led i denna forskning har man utvecklat biomimetiska lipidskikt, en typ av konstgjorda
cellmembran. Den stora fördelen med biomimetiska modellskikt är att de kan studeras med
avancerade ytanalytiska och spektroskopiska metoder. Dessutom har biomimetiska cellmembran
funnit en rad praktiska tillämpningar, t.ex, som biosensorer, elektroniska tungor och näsor,
komponenter för artificiell fotosyntes för att nämna några.
För att kunna studera sammansättning och interaktioner på och över cell membran är det viktigt
att lipidbilagret behåller sin ”vätskeliknande” karaktär så att de membranbundna proteinerna kan
röra sig fritt i membranet. Det är också viktigt att proteinerna i membranet ej kommer i direkt
kontakt med den fasta ytan eftersom detta ofta leder till konformationsändringar och i vissa fall
till total deaktivering av proteinets funktion. För att undvika detta deponeras lipidskiktet på ett
skyddande lager bestående av hydratiserade molekyler eller polymerer. I avhandlingen har jag
studerat en serie av sådana lager baserade på oligo(etylen glykol)-tioler med spektroskopiska och
ytanalytiska metoder. De molekylära lagren har tillverkats genom självorganisering från utspädd
lösning på guldsubstrat, sk ”self-assembled monolayers” (SAMs). Jag diskuterar lagrens kvalitet,
konformation, orientering och defektstruktur, liksom vikten av teoretiska beräkningar och
modellering för att få en förbättrad strukturell förståelse av denna typ av SAMs.
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Mina resultat visar på den här typen av oligo(etylen glykol)-tioler bildar mycket välorganiserade
och elektriskt isolerande SAMs på guld. I avhandlingen beskrivs också två olika tillämpningar av
dessa SAMs, dels inom marin biofouling och dels inom vesikelfusion på guld.
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List of publications (Papers included in the thesis)
Paper I
Hung-Hsun Lee, Živilė Ruželė, Lyuba Malysheva, Alexander Onipko, Albert Gutės, Fredrik
Björefors, Ramūnas Valiokas and Bo Liedberg,
“Long-Chain Alkylthiol Assemblies Containing Buried In-Plane Stabilizing Architectures”
Langmuir, 2009, 25(24), 13959-13971
Paper II
Hung-Hsun Lee, Martynas Gavutis, Živilė Ruželė, Ramūnas Valiokas and Bo Liedberg,
“Mixed Self-Assembled Monolayers with Deuterated Terminal Anchors for Tethered Lipid
Membrane Formation”
Manuscript, 2012
Paper III
Ramunas Valiokas, Lyuba Malysheva, Alexander Onipko, Hung-Hsun Lee, Živilė Ruželė, Sofia
Svedhem, Stefan C.T. Svensson, Ulrik Gelius and Bo Liedberg,
“On the quality and structural characteristics of OEG assemblies on gold: An experimental and
theoretical study”
J. Electron Spectroscopy and Related Phenomena. 2009, 172, 9-20
Paper IV
Lyuba Malysheva, Alexander Onipko, Timmy Fyrner, Hung-Hsun Lee, Ramūnas Valiokas, Peter
Konradsson and Bo Liedberg,
“Spectroscopic characterization and modeling of methyl- and hydrogen-terminated oligo
(ethylene glycol) SAMs”
J. Physical Chemistry A, 2012, submitted
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Paper V
Martynas Gavutis, Hung-Hsun Lee, Živilė Ruželė, and Bo Liedberg and Ramūnas Valiokas,
“Tethered Lipid Membrane Formation on Assemblies with Controlled Presentation of Anchor
Molecules: A QCM-D study”
Manuscript, 2012
Paper VI
Timmy Fyrner, Hung-Hsun Lee, Alberto Mangone, Tobias Ekblad, Michala E. Pettitt, Maureen
E. Callow, James A. Callow, Sheelagh l. Conlan, Robert Mutton, Anthony S. Clare, Peter
Konradsson, Bo Liedberg and Thomas Ederth,
”Saccharide-Functionalized Alkanethiols for Fouling-Resistant Self-Assembled Monolayers:
Synthesis, Monolayer Properties, and Antifouling Behavior”
Langmuir, 2011, 27, 15034-15047
Publication NOT included in the thesis
Paper VII
Pinar Şen, Catherine Hirel, Chantal Andraud, Christophe Aronica, Yann Bretonnière,
Abdelsalam Mohamme , Hans Ågren, Boris Minaev, Valentina Minaeva, Gleb Baryshnikov,
Hung-Hsun Lee, Julien Duboisset and Mikael Lindgren
“Fluorescence and FTIR Spectra Analysis of Trans-A2B2-Substituted Di- and Tetra-Phenyl
Porphyrins”
Materials, 2010, 3(8), 4446-4475
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Contents
Preface ……………………………………………………………………………………………………..…….1
Acknowledgement…………………………………………………………………………….….…………3
1. Introducion………………………………………………………………………..……………………….5
1.1. Self-Assembled Monolayers (SAMs) ……………………………………………………………………….…...7
1.2. Manipulation and Structuring of Surfaces using SAMs……………………………………………….14
1.3. Poly(ethylene glycol) and Oligo(ethylene glycol)-Containing SAMs……………………………..17
2. Experimental and Theoretical Background……………………………………………...29
2.1. SAMs and IRRAS………………………………………………………………………………………………………….30
2.2. Ab-initio Calculations ………………………………………………………………………………………………….34
3. Experimental Techniques…………………………………………………………..…………….37
3.1. Preparation of Gold Surfaces………………………………………………………………………………………37
3.2. Preparation of SAMs………………………………………………….………………………………………………..38
3.3. Null-Ellipsometry……………………………………………………………………………………..………………….38
3.4. Contact Angle Goniometry ……………………………………………………………….………………………..40
3.5. Infrared Reflection-Absorption Spectroscopy (IRRAS)…………………………….…………………..42
3.6. X-ray Photoelectron Spectroscopy (XPS)………………………………………..……………………………42
3.7. Cyclic Voltammetry (CV)……………………………………………………………………………………………..44
3.8. Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D)…………………..……….44
4. Future Outlook…………………………..…….………………………………………………………45
5. References…………………………..……………………………………………………………………47
6. Summary of the Included Papers…………………………………………...…………………57
Publications
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1
Preface
The work described in this dissertation was carried out between late 2007 and 2011 at the
laboratory of Molecular Physics, Department of Physics, Chemistry and Biology, Linköping
University. In this thesis, molecular structures of well-defined, adsorbed organic thin layers on
metal surfaces were investigated both experimentally and theoretically. Infrared reflection-
absorption spectroscopy (IRRAS), null-ellipsometry and contact angle goniometry have been
used as main surface analytical methods and were all executed by me. The experimental work
has been complemented with theoretical modeling to explore and optimize the infrared
signatures of the molecules. All calculations were performed using a set of ab initio methods by
Dr. L. Malysheva and Dr. A. Onipko (members of the Ukrainian team within the preceding and
current Visby projects). All investigated chemical compounds were designed and synthesized by
Dr. Ž. Ruželė and Dr. T. Fyrner.
Paper I is a study of the unique structural properties of the HSC15CONH(EG)6CONHC16H33
compound assembled on Au(111). We undertook a systematic investigation of the SAMs formed
by a series of this type of molecules with varying lengths of extended alkyl chains. Our purpose
2
was to reveal the role of the additional amide linkage and the extended alkyl chain on the
structure and blocking properties. In Paper II, we utilized the molecules that investigated in the
paper I to build a stable mixed self-assembled monolayer (SAM) as an underlying flexible
surface for tethered lipid membranes application. A demonstration using quartz crystal
microbalance with dissipation monitoring (QCM-D) to study small unilamellar vesicle
interaction with such SAMs is included. Paper III is a summary of the main results from our
experimental and theoretical work over the years based on 15 different alkanethiolate SAMs on
gold. All these SAMs contain alkyl chains, OEG portions of different length, and amide linking
groups. We discussed the quality, conformation, orientation, defect structure and IR spectra of
these OEG-containing SAMs, as well as the importance of theoretical modeling in order to
obtain a comprehensive understanding of the origin of the IR signatures. In Paper IV, we aimed
to extract fundamental information from alkyl-free oligo(ethylene glycol) (OEG) SAMs through
infrared reflection-absorption spectroscopy and ab initio calculations. We hoped to gain deeper
understanding of the spectral appearance of these molecules in terms of chain lengths, terminal
groups and chain orientations. Paper V and paper VI are two typical applications in using OEG
related SAMs as bases. In Paper V, we investigated interactions of the adsorbed vesicles on the
OEG-containing SAMs by employing QCM-D technique. Our SAM models are based on the
results from Paper II. This approach provides information concerning the relationship between
lipid bilayer formation and subtle changes of the surface properties of the OEG-containing
SAMs. The collected data can be useful in development of patterned bilayer lipid membranes on
solid support as biomimmetic model membranes in nano- and micro-scales. Paper VI, the
intention of this study was to investigate structural properties of mono- and oligosaccharide
SAMs as well as nonspecific protein adsorption and marine fouling properties on the monolayer
surfaces consisting of saccharide-terminated alkanethiols. Further dilution of the saccharides on
the surface by adding filler molecules with a short EG tail is a useful strategy for tuning the
exposure of the sugars to the environment and thus enables specific interactions of proteins and
cells with the surface-bound sugars.
3
Acknowledgement
First and foremost, I would like to thank my surpervisor, Prof. Bo Liedberg who gave me the
opportunity to join molecular physics group and to accomplish my initial motivation to come to
Sweden, although this achievement has been delayed for 20 years. Beside the scientific
discussions, I would like to express my gratitude to Dr. Ramunas Valiokas, it was he at the very
first time showed me how to prepare a high quality self-assembled monolayer (SAM) in an
ambient environment. His skill and extremely careful to any possibly tiny factor that might cause
contamination to the sample surface really impressed me. My high reproducibility and “good-
looking” IR spectra are definitely related to what he taught me the preparation of a SAM.
Writing this thesis has been hard, however during the process of writing I have learned a lot. I
sincerely thank to Dr. Thomas Ederth, his willing to take his time and discuss with me
concerning my work, and Dr. Lyuba Malysheva kindly explained me the basic concept of the
modeling. I would also like to give my appreciation to our technician Bo Thuner and Dr.
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Fredrik Eriksson who helped me to calibrate the new evaporation system and thicknesses
calculation of the evaporated gold layers via XRD measurements. I am grateful to thank Stefan
Klinström, director of Forum Scientium (FS) at LiU, and his team. I did during these PhD-years
broaden my knowledge, exchanged ideas with other FS members, and had fun through the
activities hosted by FS. In addition, I´d like to thank Prof. Kajsa Uvdal for her advice and
encouragement during this thesis-writing period.
The informal support and encouragement from my colleagues, fb-friends, dumpling-friends and
brownie-friends have been indispensable. Thanks dear friends, you guys make my PhD student
life colorful. At last, I want to thank my daughter, Ylva L Lindgren: while I try to teach you all
about life, your young spirit tells me what life is all about. Thanks for all your concern to me
during the late years.
李 鴻 薰 April, 2012
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1. Introduction
Biological membranes are essential components for all living systems; their molecular
organizations and interactions with intra- and extracellular networks are key factors of cell
functions. They act as filters keeping toxic substances out of the cells and specific nutrients,
wastes and metabolites can pass through the membranes to reach their destinations. The
membrane proteins within the lipid bilayers serve to control cell signaling and the cellular
response to a range of external factors. Many important biological processes are regulated at
membrane interfaces via interactions between membrane proteins. Therefore, identification of
the cell structures and deeper understanding of the processes associated with membranes such as
energy conversion, transport of signals and catalysis of cellular reactions are crucial [1-4].
However, the intrinsic complexity of the cell membrane systems makes direct investigation extra
difficult. Based on this reason, model systems prepared by stabilizing biomimetic lipid
membranes on a solid support, following incorporation of membrane proteins on it have become
a strategy [5-6]. If proteins which attached on such a supported membrane retain their lateral
mobility while avoiding non-specific van der Waals attraction with the model membrane, then
6
their assembly and interactions can be studied. Artificial model membranes have been frequently
used not only for unraveling the physical and chemical properties of membranes but also for
probing the dynamic membrane functions. Analytical methods such as total interference
fluorescence, nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy
(FTIR), surface plasmon resonance (SPR), x-ray or neutron reflection (NR) can all be used to
probe the structural and dynamic properties of supported membranes.
One of fundamental drawbacks of artificial membranes is probably from the bare solid surfaces
which they deposited on. The space between the membranes and substrates is usually not enough
large to avoid direct contact of the incorporated proteins and the solid surface, especially for
large proteins or protein complexes which their extracellular domains can extend to several tens
of nanometers. An elegant way to solve this problem might separate the membrane from the
solid substrate using soft polymeric materials that cover on the substrate and support the
membrane [7]. As a result, such a layer may significantly reduce the frictional coupling between
the incorporated proteins and the solid substrate and hence the risk of protein denaturation.
Alternative is to incorporate lipopolymer tethers into the membrane where their head groups act
as spacers that control the membrane-substrate distance [8]. Mixed monolayers consisting of two
compounds which one terminated with hydroxyl group and the other, for example, cholesterol
derivatives have also been suggested as a suitable interface for supported lipid membrane [9]. In
this system, the terminal cholesterol group penetrates into the hydrophobic region of the bilayer
and act as hooks to anchor the bilayer to the solid support. This type of interface has a
hydrophilic region close to the substrate which is able to prevent denaturation of incorporated
membrane proteins, and subsequently improve the efficiency of protein incorporation.
In this thesis, I have been focusing on the structural characterization of selected well-defined
molecular interfaces for the integrating biomimetic membranes. These molecular interfaces were
prepared by self-assembly from dilute solution onto gold substrates, so called self-assembled
monolayers (SAMs). A number of surface analyzing methods were used for characterization of
such surfaces, including Infrared reflection-absorption spectroscopy (IRRAS), Null-Ellipsometry
7
and Contact Angle Goniometry. In parallel, a set of ab initio modeling has been proceeded for
the best interpretation of experimental data obtained from IR-RA spectra. Hopefully in the long
run, this study will contribute to a deeper understanding of the mechanisms behind the
interactions between proteins and interface molecular layers.
1.1. Self-Assembled Monolayers (SAMs)
Amphiphilic molecules can spontaneously organize themselves into assemblies at a variety of
interfaces, Langmuir– Blodgett (L–B) film formation is one of examples [10-11]. However, L–B
films are known to be chemically and mechanically unstable, therefore researchers have pointed
to new types of assemblies on solid surface that are more environmentally and chemically stable.
By placing a metallic (often gold) substrate into a milli- or micro-molar molecular (e.g.
alkanethiol) solution in ethanol (or water), a spontaneous assembly process occurs. This results
in a crystalline-like monolayer formed on the metal surface, called self-assembled monolayer
(SAM) [12].
SAMs provide a convenient, flexible and ease of preparation system with which to tailor the
interfacial properties of metals, metal oxides and semiconductors. There are a number of
advantages of SAMs, for example: (i) its surface properties can be selectively modified by
changing specific functional groups while leaving the rest of the molecule unchanged, (ii) it can
be used to build desired structures and functions on many substrates not only on planar surfaces,
(iii) self-assembly is one of the most important strategies used in biology for the development of
complex, functional structures.
The first systematic research related to SAM systems was performed by Zisman et al in 1946
[13]; they reported the preparation of a monomolecular layer by adsorption of a surfactant onto a
clean metal surface. However, the potential of self-assembly was not recognized at that time, and
this publication did not bring much attention. Not until 1983, Nuzzo and Allara “discovered”
8
that SAMs of alkanethiolates on gold can be prepared by adsorption of di-n-alkyl disulfide
solutions [14], these ordered organic monolayers opened the study of organic surfaces for real
exploitation. Here I list some of examples: (i) structure and reactivity of alkyltrichlorosilanes
adsorb from solution onto silicon substrates [15], (ii) preparation and properties of solution
adsorbed organosulfur compounds on gold, silver or copper substrates [16-20], (iii) interaction of
anhydride and carboxylic acids on aluminum oxide or silver oxide [21-22]. Among these,
however, monolayers of alkanethiolates on gold are probably the most studied SAMs to date. A
major advantage of using gold as substrate material is that gold does not form a stable surface
oxide, and thus can be handled in ambient environments. The high affinity of thiol head-groups
to gold makes it possible to generate well-defined organic surfaces, and it is this spontaneous
chemisorption of the head-group to the gold surface which drives the formation of SAMs.
Formation of SAMS
The mechanism of the self-assembly process has been well studied and elucidated [23-24].
Adsorption of a SAM on solid surfaces is usually divided into two steps: a lying-down phase
which is a fast step and a standing-up phase, a slow step. In the fast step, the molecules in
solution are transported through diffusion and convection to the solid-liquid interface,
immediately following an adsorption which the head-group of the molecules assembles together
on the surface of the substrate. The molecules at this stage are at a low density, conformationally
disordered and randomly lying-down phase on the surface. This fast step takes up to a few
minutes, depending on the adsorption rate (the head group-substrate interaction). The slow step,
on the other hand, the molecules begin to form ordered islands which grow slowly until a two
dimensional close-packed and conformational ordered standing-up phase fully covers the
substrate. This step can be described as a surface crystallization process, relying on the chain
conformations, chain-chain interactions, and surface mobility of chains; it might last from
several hours to several days (Figure 1).
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Figure 1. Scheme of the two steps taking place during the self-assembly of alkanethiols on
Au(111): (i) fast step, lying down phase, (ii) slow step, standing-up phase.
Au(111) surface is the lowest energy surface which is therefore preferred in the growth of thin
Au films. Electron diffraction [25] and atomic force microscopy (AFM) studies [26] proved the
symmetry of sulfur atoms of long chain alkanthiols is placed in a hexagonal ( ) R30o
structure with a nearest S∙∙∙∙S spacing of 4.97Å on top of the Au(111) lattice (Figure 2). This
leads to an area/molecule of 21.4 Å2 which is ~15% greater than the cross-sectional area (18.4 Å
2)
of each alkyl chain onto the 2-D plane. As a result, the axis of the alkyl chain must tilt away the
surface normal in order to maximize van der Waals interaction between the chains. The exact
structure of a monolayer depends on the chemistry of the chain [16-17, 27]. On the other hand,
long chain alkanethiols adsorbed on an Ag(111) surface is distinct from the structure of that on
Au(111). Although the film on Ag showed dense and highly ordered, x-ray diffraction indicated
the monolayer is both incommensurate and rotated with respect to the Ag lattice. The alkyl chain
is less tilt on Ag than on Au substrate, and the nearest S∙∙∙∙S spacing is of 4.6 - 4.7 Å. Besides, it
was found that the outermost surface of the monolayer is less ordered than the interior [20, 28].
10
Figure 2. Scheme of sulfur atoms of a SAM adopt a hexagonal ( ) R30o
structure with a
nearest S∙∙∙∙S spacing of ~5Å on Au(111) surfaces.
Contributing Interactions
Self-assembly can be found in many natural processes such as protein folding, DNA transcribing
and formation of cell membranes. The process of self-assembly in nature is governed by inter-
and intramolecular interactions that drive the molecules into a stable structure at thermodynamic
minimum state. Several parameters related to the interaction energies include electrostatic
interaction, hydrophobic interaction, van der Waals force and hydrogen bonding. There are
several driving forces for the assembly of alkanethiols onto metal surfaces: the head group-
substrate interaction, the chain-chain interaction, and the end group-end group interaction. The
head group-substrate binding energy, S-Au, is the strongest of all interactions and is the primary
driving force for the self-assembly process. It is this spontaneous self-assembly that brings
molecules close enough together and allows the short range van der Waals force to become
important. The binding energy of S-Au is approximately 44 kcal/mol [29]. In comparison, the
bond strength of a C-C is of 83 kcal/mol.
During growth of the surface coverage, the chain-chain interaction become more and more
important; the weak van der Waals force dominates the conformation of individual alkyl chain as
11
well as their packing and ordering within the SAM. Obviously, only certain combinations of
chain-chain separation and tilt angle are allowed for effective packing of alkyl chains. The
spacing between molecular head groups has an impact on the chain-chain interactions, and is
dependent of types of head groups and substrates. FTIR studies reveal a well-ordered
alkanethiolate SAM is of at least 10 carbons [30]. The optimum condition of a close-packed
alkanethiolate SAM on Au(111) surface is the tilt angle of the alkyl chains at about 26o to 28
o
away from the surface normal and the rotation angle at about 52o to 55
o around the molecular
axis [28]. This tilt angle is a result of the optimizing van der Waals interaction between the
chains with the head group S∙∙∙∙S distance about 5Å. The energy associated with the van der
Waals intermolecular interaction depend on both the chain length and the packing density and is
in the range of 0.8 -1.8 kcal/mol per CH2 [31]. The end group-end group interaction is however
only a few kT of energy [28].
Many studies have been pointed out that incorporation of different heteroatoms or chemical
groups such as –SO2-, -NO2- and -COO- into mid-interior portion of the monolayer might cause
a dipole-dipole type interaction within the assembly [32-34]. Depending on the magnitude and
direction, the dipole-dipole force has an influence on the packing and ordering of the monolayer.
Hydrogen bonding is a type of dipole-dipole interaction that involves molecules with –OH and -
NH groups. It arises between a hydrogen donating and a hydrogen accepting groups. The energy
of the hydrogen bond is approximately proportional to
which r denotes the distance between
two atoms. It has been reported the formation of hydrogen bonding network within the SAM
increases the stabilization of the assembly and has impact for long-range electron transport in
biological systems [35-38].
Defects
The purity of the alkanethiols being used can affect the self-assembly process. Even the
existence of low degrees of contaminants can result in a disordered monolayer. Other external
factors affect the quality of SAMs including smoothness and cleanliness of the substrate and the
preparation method (immersion time, solution concentration, solvent and temperature) [39]. It
12
has been detected vacancies formation on the gold surface in ethanolic thiol solution [40]. This
result suggests that the thiol molecule itself is an active oxidant, and depends on the experimental
conditions; some of the gold on the topmost layer may be corroded during the self-assembly
process. Intrinsically, SAMs form defects under the growth process and can be detected using
scanning tunneling microscope (STM) as a local probe. For a thiolate SAM on gold, typical
defects are single-atom vacancies, gold vacancy islands and adsorbate vacancies, including
molecular lattice and domain boundaries [41-42]. It has been proposed that molecules adsorbed
at these defect sites are conformational less constrained and are more accessible by solvent than
their neighbors with a close-packed structure, therefore promoting the exchange of the adsorbed
molecules to other new species [43]. Step edges where one atomic layer of gold mismatches the
gold terraces from each other are also likely accessible to the solvent and present the substrate
atoms with low coordination number.
Surface Modifications
The surface properties of a SAM can be modified by simply changing tail groups of the alkyl
chain with polar groups, ionic groups, hydrophobic or hydrophilic groups, groups that resistant to
protein adsorption and groups that have the ability to perform chemical reactions. Additionally,
SAMs can have terminal groups with conformation switchable oligo(ethylene glycol)
compounds. Changing the functionality of the exposed terminal group at the air-film surface is
critical for determining and designing the interaction strengths in sensing, electron transfer, cell
adhesion, protein adsorption and other areas. Table 1 lists some examples of various functional
groups that have been incorporated into thiolate SAMs [44].
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Table 1. Various functional groups that have been incorporated into alkanethiols, HS(CH2)nR,
where n is the number of methylene units and R represents the end group of the alkyl chain
(adopted from Smith et al [44])
Functional group (R) Name
-CH3, -CH2- Alkyl
-CF3, -CF2- Trifluoromethyl,
Difluoromethylene
-CH2OH, CH2OCH2- Hydroxyl, Ether
-COOH, -COO- Carboxylic acid
-CO2CH3, -CO2CH2- Ester
-CONH2, -CONH- Amide
-Cl, -Br Chloro, Bromo
-CN Nitrile
-NH2, -NH3+ Amine
-B(OH)2 Borate
Aryl
Quinone
Oligo(ethylene glycol)
Epoxide
Pyrene
14
Applications of SAMs are very diverse; their densely packed, well ordered, flexible and
chemically stable properties have made them ideal as components in surface coating, such as
adhesion improvement [45], protection against corrosion [46] and friction control [47]. They are
suitable materials for studying interfacial properties and for fabricating micro- and nanostructural
patterns. SAMs are also popular for bio-related applications due to their biocompatible nature
and the possibility to modify a SAM surface with biologically relevant functionalities allow them
being served as a binding site of other target biomolecules [39, 48-49]. In the design of interfaces
for molecular recognition and biosensors, mixed SAMs are often introduced with one component
is present at the surface and the other adsorbs from solution by means of “host-guest”
interactions [50].
1.2. Manipulation and structuring of surfaces using SAMs
SAMs are of primarily technological interest, therefore, as the increasingly understanding and
better control of the SAMs, researchers have begun to pattern and manipulate this type of
assemblies with more complex and functional architectures. Technology to engineer the
properties of SAM-coated substrates has grown rapidly. SAMs have been used in some etching
processes as physical barriers, protecting particular areas from underlying metal. They have also
been used to control nucleation of crystals [51] and for other purposes that require control of
surface composition. The development of patterned SAMs is critical as they can serve as final
structures or supports. This powerful class of nano- and micro-fabrication techniques makes it
possible to pattern surfaces with varying degrees of precision, depends on the methods used.
Mixed SAMs
Monolayers comprising a mixture of organic functional groups by co-adsorption of thiols from
solutions are called mixed SAMs. This co-adsorption process can be controlled by varying the
relative concentration of the two or more thiols in solution. However, the mole fraction of a
specific adsorbate in the mixed SAM is not necessary the same as the mole fraction of the
adsorbate in solution through all ranges of concentration. By changing the nature of the tail
15
groups or by introducing functional groups into the hydrocarbon chains of the thiols, the surface
chemistry and structure of the multicomponent interface can be predetermined precisely. Bain
and Laibinis et al [52-57] studied the influence of tail group, chain length, solvent, and phase
behavior on the structure of two-component monolayers on gold. Their results led to conclusions:
(i) co-adsorption process is preferential the longer chain over the shorter chains. (ii) monolayers
composed largely of a single component is energetically favored in relation to that of containing
comparable amounts of the two components in mixed SAMs. However, phase-separated domains
are often present in these binary-component systems. Nano-scale phase segregation in a mixed
SAM might occur (i) if the tail groups of the components are vastly different in polarity [55], (ii)
if the molecules possess differing functional groups that are buried in the film near the metal
surface [58], (iii) if the chain length of the two components are very different [59]. Furthermore,
the two components can be separated into their homogeneous domains by post-adsorption
techniques [43]. The principles of phase segregation have been utilized to make SAM-based
sensors, for example, sensors for cholesterol can be created by capitalizing on the phase
separation between such analytes and alkanethiolates [60]. However, phase separation seems to
be hindered at once the SAM forms because it is not observed on a macroscopic scale.
Microcontact printing (μCP)
Microcontact printing (μCP) is a flexible technique that forms patterned SAMs terminated by
different chemical functional group with defined regions. It does not require a dust-free
laboratory environment and can produce patterned gold substrates at relatively low cost. Patterns
of SAM are formed using an alkanethiol molecule as an ‘ink’ and printing it on a metal surface
with a polymeric ‘stamp’. Only the regions that come into contact with the stamp are covered
with the SAM. The size of the stamped regions can be defined arbitrarily with dimensions from
50 nm to 1000 μm and line widths less than 100 nm. However, alkanethiols with more than 20
carbons are less soluble in ethanol and the PDMS stamp, and therefore are not suitable for μCP
patterning. Applications of the μCP patterned SAMs including microfabrication, studies of
wetting and nucleation phenomena as well as protein and cellular adsorptions. However, the
resolution of the resulting patterns is limited by the material and dimensions of the stamp. Mixed
16
SAMs patterned by μCP technique can also be used as templates for the selective deposition of
other nanoscale materials [61] and periodic arrays of oriented crystals [62].
Dip-pen nanolithography (DPN)
Dip-pen nanolithography (DPN) is amenable to printing molecules with a variety of functional
groups where alkanethiol molecules suspended in droplets at the end of an AFM tip as a
molecular “ink” [63]. By rastering the probe tip close to a gold surface, the alkanethiol molecules
are transported to the surface via capillary action in ambient conditions. Dip-pen resolution can
be down to linewidths of 15 nm; however, tip-substrate contact time, scan speed, relative
humidity and solubility of the molecule in the water meniscus all affect the resolution of
patterned features. Generally, the alkanethiolate SAMs that form via DPN are well ordered.
Most DPN work has been done with the alkanethiolate on gold model system, including n-
alkanethiols, arylthiols, thiol-functionalized proteins and alkanethiol-modified oligonucleotides
for gold substrates. However, alkylsilazanes and inorganic salts for SiO2 surfaces and
alkylsilazanes for oxidized GaAs have also been investigated [64]. DPN has been used to, for
example, (i) pattern thiols on gold substrates where the partial SAMs were utilized as etch resists
[65], (ii) pattern periodic arrays of differently sized nanoparticles, proteins, cells, and inorganic
films [66].
Molecular Gradients
A surface whose physical-chemical properties gradually change as a function of the position is
called a gradient surface. The formation of two-component molecular gradients was first
reported by Liedberg et al [67]. By cross-diffusion of two differently terminated thiols through
an ethanol-soaked polysaccharide gel that is covering the gold substrate, a continuous gradient
of 10-20 mm length may be formed. These gradient surfaces are applicable for studying
wetting and adhesion [68] as well as neuron growth in cell biology [69].
17
1.3. Poly(ethylene glycol) and Oligo(ethylene glycol)- containing SAMs
Poly(ethylene glycol) (PEG), also known as poly(ethylene oxide) (PEO), is a synthetic polyether
with a general formula HO(CH2CH2O)xH. PEG can be dissolved in water so as in many organic
solvents. PEGs have been proven to be nontoxic and are commonly used as carriers in different
pharmaceutical formulations, foods, and cosmetics [70-71]. In the coating industry, adsorption of
PEGs onto colloidal particles is used for stabilizing the dispersion against flocculation [72]. The
famous Vasa warship has been sprayed with PEG as a preserved agent for many years. PEGs are
also widely used in biomedical research such as drug delivery, tissue engineering scaffolds, and
surface functionalization [73-74]. In drug delivery systems, for example, PEG is used to improve
the solubility of the drugs and to help stabilize unstable protein drugs, enhance the circulation
times of drugs in the body [75]. PEG hydrogels are attractive candidates being employed as
scaffold materials because they are degradable, have mechanical and structural properties similar
to the extracellular matrix of many tissues, and can often be processed under relatively mild
conditions [76]. Furthermore, PEG molecules attached to surfaces have been shown high
resistance to protein adsorption [77-78] due to their unique solution properties and their
molecular conformations in aqueous environments. It is now an attractive approach that PEG
grafted into polymer backbones or immobilized onto polymeric biomaterial surfaces for
nonfouling application.
Jeon et al [79-80] considered the balance forces between a protein and PEG surface in water.
According to their calculations, protein resistance is due to the net force of steric repulsion and
hydrophobic interaction between the PEG surface and proteins under the assumption of low
hydrophobicity of PEG. Adsorption and concomitant compression of the PEG-layer upon protein
adsorption result in a reduction of the conformational freedom of the PEG chains. This leads to a
loss in entropy as well as desolvation of the PEG due to the increasing hydrophobicity of the
surface. The effect is referred to as steric repulsion and it offers one plausible explanation the
protein-resistant properties of PEGs. It has been shown that longer chain lengths and higher
surface density of the PEG segments lead to better protein resistance [81]. However, this model
is more applicable for polymer chains having more than thousand segments and for larger size of
18
proteins. Szleifer [82-83] later proposed a so called SCMF theory for study the interaction
between proteins and polymers with short to intermediate chain lengths using small proteins as
lysozyme. This approach suggests that the existence of conformational freedoms of PEG chains
is not a necessary condition for protein resistance. Above a molecular weight threshold, the
amount of protein adsorption onto the surface becomes independent of the chain length of the
polymer. He concluded that the main factor in determining the protein adsorption behavior is to
prevent proteins from reaching the interface and therefore dominated by the density of polymer
close to the surface region. The thickness of the polymer layer, on the other hand, contributes
further to the repulsive interactions between the protein and the polymer since the proteins must
pass through before they reach the surface.
However, the ability to resist protein adsorption was also found in a short fragment of PEG by
Prime and Whitesides [84-85]. They compared and correlated the surface composition of
different functionalized long chain alkanethiolate SAMs on gold with the amount of proteins
adsorbed onto the surfaces. To their observations, the SAMs containing a sufficiently large
amount of OEG-terminated alkanethiolates (OEG-SAMs) are the most effective in preventing
nonspecific protein adsorption. The efficiency of protein resistance in the OEG-SAMs increases
with the length of the OEG chains, and with a minimum of two EG units is necessary under
standard experimental conditions. The hydroxyl-terminated or methoxy-terminated OEG-SAMs,
however, seem to no difference on protein adsorption behavior. It is for sure that the interfacial
properties of each SAM determines the effect of protein adsorption but not protein itself since
similar behavior of different proteins in the same surface was detected. These results imply that
surface density of the OEG might not be that sensitive; other factors such as composition and
conformation than steric repulsion might involve in terms of protein adsorption.
In a following study, Harder et al [86] found a correlation between the molecular conformation
of the OEG segments and the protein repellant properties of the OEG-SAMs. Their experimental
data clearly indicated that the protein repellant properties in the liquid state were strongly
correlated on the OEG conformation in a SAM. Their results showed that SAMs prepared on
19
gold substrates from HSC11(EG)6OH and HSC11(EG)3OCH3 displayed a helix-like OEG
conformation that rendered the surface protein resistant, whereas those self-assembled on silver
surfaces adopted an all-trans conformation and were not protein resistant. Figure 3 presents an
example of helical and all-trans (EG)3 conformation. The conformational transitions between
different metal surfaces are believed due to a higher packing density on Ag which leads to a
lateral compression of the (EG)3 chains. In addition, IR-RA spectra show the helical OEG-SAMs
change their conformations to a predominantly amorphous phase when exposed to aqueous
solutions. Based on these observations, Grunze and coworkers [87-89] further simulated the
behavior of water molecules near helical and all-trans conformers of (EG)3OCH3 terminated
SAMs. A brief summary to these reports: (i) the planar all-trans and helical conformers show
different response in contact with water. Only the helical OEG provides effective adsorption sites
for liquid water, forming hydrogen bonds, (ii) the helical OEG undergoes a comformational
disordering (amorphization) when contact with water; this disordering may affect the structure
even deeper into the alkyl chains. The result is in agreement with the experimental observation
from sum frequency spectroscopy [90], (iii) by contrast, the all-trans OEG-SAMs retain a good
conformational order when exposed to water and is affected mostly only within the methoxy
terminal group of the OEG chains, (iv) compared to the helical conformer, the all-trans OEG
shows a significantly lower surface density of hydrogen bonding sites for interaction with water
molecules, hence lower hydrophilicity. In conclusion, they suggested that the OEG conformation
is crucial for the interaction with water. The helical OEG surface provided sites for strong
interaction to water molecules, whereas no or little water molecules were found to bind to/within
the all-trans OEGs.
20
Figure 3. (a) Helical and (b) all-trans (EG)3 conformation of the HSC11(EG)3OCH3 molecules.
Furthermore, the effect of external electrostatic fields on the helical and planar all-trans
conformers of OEG-SAMs has also been investigated [91-92]. The helical (EG)3 in an
electrostatic field undergoes distortion, length shortening and conformational disordering
(gauche formation, amorphization) of the O-C-C-O bonds whereas the relative rigid planar OEG
segments only display subtle changes of the orientation in tail groups under the same conditions.
The structural changes of the helical conformer start already at a field of 0.5 V/Å with
redistribution of the electron density and atomic displacements. At higher field strengths (0.5 –
1.5 V/Å) the effect is comparable to what is happening in polar solvents, e.g. H2O, as observed in
vibrational sum frequency spectroscopy [90]. On the other hand, the all-trans structure remains
intact up to a field of about 1 V/Å. At this strength of the field, the terminal methoxy groups
might reorient, making the outermost oxygen atom either stretched out or bent into the surface.
This result indicates that it might possible to modify the surface properties of OEG-SAMs from
being more hydrophilic to more hydrophobic by applying an electrostatic field relying on the
field polarity.
A broader investigation of the factors involved in the protein resistance of OEG-SAMs was
reported by Herrwerth et al [93]. They examined different types of oligoether alkanethiolate
21
SAMs on Au and Ag by means of contact angle measurements, XPS, IRRAS and protein
adsorption tests. Their results show that SAMs terminated with hydroxyl groups are all protein
resistant both on Au and Ag, whereas those (EG)xOCH3-terminated SAMs are only protein
resistant on Au. When these molecules self-assembled on Ag, higher lateral packing densities of
the monolayers are formed and they are no longer protein resistant. For example, the
HSC11(EG)6OCH3 SAM form helical OEG conformation on Au is fully protein resistant,
whereas the SAM formed on Ag adsorbs protein, although it retains its helix-like conformation.
In fact, the two monolayers are found vary in their relative surface coverage but show no
difference in their contact angle values. The RA spectra on Au and Ag reveal also structural
differences. The film on Ag shows sharper and higher intensities of the helical OEG
characteristic bands, as compare to the film forms on Au. They therefore conclude that a SAM
with relaxed lateral packing density, containing some disorder or defects is necessary for high
protein resistance. The hydrophilicity of the interior and the end group of the oligoether units are
also important and only the combination of these factors allows the achievement of high protein
repelling capabilities. If one of the factors, for example, the ability of an oligoether SAM to
coordinate with water both in its interior and on its surface is reduced or absent, the overall
protein resistance decreases.
Pertsin et al [94] calculated low energy conformers of an isolated HSC11(EG)3OCH3 molecule
on Au and Ag in order to understand the conformational transition of the OEG. The OEG
conformers are designated based on the dihedral angle between two planes from O-C-C-O bonds.
The helical conformer (g+ or g
-) is obtained when all dihedral angles about C-O bonds are ~180
o
and those about C-C bonds are 60-70o, clockwise (g
+) or anticlockwise (g
-), whereas the trans
conformation (t) is denoted when all dihedral angles about C-O and C-C bonds are close to 180o.
Their results show that the lowest energy of OEG conformation on Au does adopt a helix-like
conformer. The helical conformer is stabilized by intra- and intermolecular interactions between
the EG units. However, when the monolayer self-assembled onto a Ag(111) substrate, due to the
smaller S-S lattice spacing, the helical conformer encounters conformational strains and loses its
stability, subsequently transfers into a higher packing density with nearly upright chains, planar
all-trans phase. In a SAM system, on the other hand, the substrate-head group interactions and
22
the packing interactions between the chains must also be taken into consideration. Although the
best packing energy of the (EG)3OCH3 was found in an all-trans conformation, the loss in
packing energy of the helical conformer was however compensated by an even larger gain in the
electrostatic interaction energy (see in Table 2). Thus the most stable conformer is the lowest
energy conformer found in an isolated HSC11(EG)3OCH3 molecule on Au. This work support the
proposal that the origin of the gauche conformer about the C-C bond (so called gauche effect)
observed in OEG is a self-stabilization effect but not intrinsic properties of the OEG itself. Table
2 lists the calculated low-energy conformers and parameters of the HSC11(EG)3OCH3 SAM on
gold within 1 kcal/mol range. All energies are given relative to the respective values for the all-
trans conformer.
Table 2. Low-energy conformations of the HSC11(EG)3OCH3 SAM on Au(111) (adopted from
Pertsin et al [94]) Etot: total lattice energy, Econf: conformational (intramolecular) energy, Einter:
intermolecular energy, Epack inter: packing (van der Waals) contribution to intermolecular energy,
Eelst inter: electrostatic contribution to intermolecular energy. All energies are in kcal/mol relative
to the respective values for the all-trans conformer. The rotation and twist angles, ψ and φ are in
degrees. The tilt angle is ranged between 35-37o which not present here.
(EG)3 Etot Econf Einter Epack inter Eelst inter φ
t, t, t 0.0 0.0 0.0 0.0 0.0 39 131
g+, g
+, g
+ -0.5 -0.3 -0.2 2.5 -2.7 42 129
g+, g
+, g
+
g-, g
-, g
-
0.0 0.0 0.0 3.0 -3.0 88
91
-135
134
g+, g
+, g
+
g-, g
-, g
-
0.0 1.1 -1.1 2.1 -3.2 55
56
-73
108
t, t, t 0.3 -0.3 0.6 1.0 -0.4 37 18
g-, t, t 0.5 1.9 -1.4 0.4 -1.8 40 130
g+, g
+, t 0.5 3.6 -3.2 0.3 -3.5 39 132
23
Table 3. Low-energy conformations of an (EG)3 without water and in contact with one and two
water molecules E(no H2O): ground state energies, given in kcal/mol, E(+1 H2O): energies with
one water molecule, E(+2 H2O): energies with two water molecules. The energies are relative to
that of the helical conformer (adopted from Wang et al [89])
(EG)3 E(no H2O) E(+1 H2O) E(+2 H2O)
t, t, t 0.17 -7.87 -9.58
t, t, g± 0.13 -5.57
t, g±, t 0.04 -5.49
g+, t, g
+ 0.18 -5.14
g+, t, g
- 0.11 -5.76
t, g+, g
+ 0.02 -6.38 -13.35
g+, g
+, g
+ 0.00 -6.93 -13.21
t, g+, g
- 0.86 -7.88
g+, g
+, g
- 0.48 -8.29 -15.93
g+, g
-, g
+ 1.07 -8.24 -16.43
There are ten energetically different groups of conformers for an isolated (EG)3OCH3 molecule
as listed in Table 3. Many of these conformers are energetically almost identical. Two of the
conformers having C-C gauche rotations of opposite directions around neighboring EG units are
energetically most unfavorable, i.e. (g+, g
+, g
-) and (g
+, g
-, g
+) especially the latter one. In this
conformer, the first and third units of EG fold back toward the second unit, this reduces the
distances between the oxygen atoms of O1 and O3 as well as O2 and O4, resulting in a stronger
repulsion between these oxygen atoms and reoriented methoxy end groups [89]. It is clear that
OEG tails with this conformer would hinder the formation of an ordered SAM due to the strong
repulsions between the bulky OEG tails as illustrated in Figure 4. In fact, simulation data show
that the least favored conformations of the (EG)3OCH3 in ambient turn out to be the most stable
conformers in water because these conformers with subsequent EG gauche units (g+, g
-) gain the
most energies upon solvation as shown in Table 3. This distorted OEG tails provide acceptor
sites and facilitate water penetration into the chains, forming double or triple hydrogen bonds
with two or more oxygen atoms along the EG chains. Experimentally, Zolk and coworkers [90]
24
has been demonstrated that the stable helical (EG)3 conformer in a HSC11(EG)3OCH3 SAM is
not stable in the presence of bulk water. Solvation of the SAMs causes reorganization of the
OEG conformations. As seen in SFG spectra, the originally strong, well-ordered methoxy
vibrations become disordered and nearly disappeared; methylene vibrations of the OEG moieties
exhibit not only decrease in intensities but also blue-shift in frequencies, indicating the OEG
portions undergo conformational changes when in contact with water. Similar results were
observed for SAMs with longer EG units [95] where the same interactions between
HSC11(EG)6OCH3 SAM and water molecules clearly exist. IR-RA spectra show that the helical
conformation of the (EG)6OCH3 remains the dominating phase and the terminal methoxy groups
reappear, showing distinct conformational changes after removal of water. This indicates that
those bound water most likely interacts with the outermost ether oxygen atoms of the monolayer
but keeps the lower part of the OEG in a helical conformer. The experimental results agree well
with the simulations which show that water molecules can only penetrate into the near surface of
the helical OEG-SAMs, resulting in conformational disordering of the films [88]. Furthermore,
the underlying alkyl chains seem to be rigidly attached to the substrate and are nearly unaffected
by the conformational changes of the OEG moieties. This is consistent with the calculations by
Malysheva et al [96] that suggest the underlying alkyl chains are in an all-trans form and have
very little effect on the conformational properties of the OEG parts.
Figure 4. Schemetic illustration of HSC11(EG)3OCH3 SAMs on Au (a) the well-ordered SAM
with all-trans alkyl chains and (EG)3 segments adopt a helical conformation (g+, g+, g+) (b) the
energetically unfavarable (g+, g-, g+) conformer of the EG units hinders the formation of an
ordered SAM on the Au surface
25
The use of OEG-SAMs in more complex studies requires a deeper understanding of inter- and
intramolecular interactions. By controlling the conformational order of OEG segments, the
OEG-SAMs can be tuned to obtain desirable biocompatibility properties. Vanderah and
coworkers [97-101] systematically examined the OEG-containing alkanethiolate SAMs on gold.
They synthesized and characterized different series of SAMs and studied their molecular
conformations as well as packing conditions with varying length where the OEG segment was
either directly linked to a sulfur atom or separated by an alkyl chain. They observed that: (i)
OEG-SAMs which the EG moieties directly linked to the substrate via sulfur were found to
require at least five EG units to adopt an ordered (helical) OEG conformation (ii) the
conformation and packing order of the HS(EG)6CH3 SAMs was dependant on the solvent used.
The monolayers assembled from 95% and 100% ethanol gave the best ordered 7/2 helical
conformation, as revealed by IRRAS. However, the latter SAMs show slightly less insulating
properties as measured by electrochemical impedance spectroscopy. This result indicates that the
SAMs made by 100% ethanol are somehow less ordered and/or contain defect sites as compared
to the SAM formed from 95%. In contrast, SAMs made by tetrahydrofuran (THF) are disordered;
(iii) A short alkyl spacer between the sulfur atom and the OEG facilitates the formation of helical
OEG structure. It was found that the HSC3O(EG)4CH3 SAM is almost perfectly ordered whereas
the HS(EG)4CH3 is largely disordered. In addition, with an alkyl spacer, the OEG-SAMs
ordering process is faster and allow wider range of assembly conditions, (iv) SAMs of
HSC3O(EG)xCH3 correspond to SAMs of [S(EG)x+1C18H37]2, where x=5-7. These films are
characterized as anisotropic, isostructural films where their structural order is independent of x.
Table 4 lists OEG conformations in the OEG-containing SAMs on gold that have been reported
in the literatures.
26
Table 4. Chemical structures of OEG-SAMs and conformations of the OEG portion on Au
(EG)x = (CH2CH2O)xH
Chemical Structure Ref OEG conformation on Au
HSC11(EG)6OH [86] Helical (helical on Ag)
HSC11(EG)6OCH3 [93] Helical (helical on Ag)
HSC11(EG)3OCH3 [86, 94] Helical (all-trans on Ag)
HS(EG)xC10H21, x=5-7 [98] Helical
HS(EG)4C10H21 [98] All-trans
HS(EG)8C10H21 [98] Disordered/amorphous + helical
HS(EG)6C18H37 [97] Helical
HS(EG)xCH3, x=3-4 [100] Disordered/amorphous + all-trans
HS(EG)xCH3, x=5-6 [100] Helical
HSC3O(EG)xCH3, x=4-9 [101] Helical
HSC3O(EG)xCH3, x=3 [101] Disordered/amorphous + all-trans
HSC15CONH(EG)x, x=2,4 [37, 102] All-trans
HSC15CONH(EG)6 [37, 102] Helical
HSC11CONH(EG)6 [37] Helical
HSC15COO (EG)4 [37] Disordered/amorphous
HSC15COO (EG)6 [37] Helical
HSC11O(EG)6 [37] Helical
[S(EG)xC18H37]2, x=5-8 [103] Helical
[S(EG)4C18H37]2 [103] All-trans
HSC15CONH(EG)6CONHC16H33 [104] Helical
27
Valiokas et al [105] studied the phase behavior of a new class of OEG-SAMs containing an
amide group between the alkanethiol and the OEG segments. This combination resulted in
formation of lateral hydrogen bonds within the SAMs. These hydrogen bonds influenced the
stability of the SAM and enabled the investigators to fine-tune the conformation of the OEG
moieties as well as the overall packing of the alkyl chains [102]. They also observed a fully
reversible phase transition of the HSC15CONH(EG)6H SAM from a helical phase of the (EG)6
portion at room temperature to all-trans conformation at elevated temperatures. This transition
between two ordered conformations was somewhat unexpected since PEG normally undergoes a
sort of melting transition at elevated temperatures (around 60-70 oC). On the other hand, the
(EG)4 containing SAM that has an all-trans conformation shows no structural change between 20
and 75oC, indicating a stable and good packing on both the alkyl and the OEG segments. This
phase behavior seems to be a result of the particular molecular design, including the OEG length,
the alkyl chain length and the specifically built-in lateral hydrogen bonds [37, 106]. In a further
study, they explored an extra long OEG-SAM containing double amide linkages and extended
alkyl tails on gold [104]. The superior thermal stability of this type of SAM can be applied in the
fabrication of protein-arrays where the extended terminal alkyl chains might act as hydrophobic
barriers separating from the hydrogel-like domains, for example, a long
HSC15CONH(EG)6CONHC16H33 and a shorter compound of HSC15CONH(EG)6H. For example,
it is possible to build biomimetic cell membranes, such as lipid bilayers that can be supported by
a soft (EG)6 cushion and partly interpenetrating alkyl chains from such a mixture SAM
architecture.
Theoretical calculations for OEG-SAMs contain amide linking groups have been studied in
detail by Malysheva et al [96, 107]. They are the first who used first-principle modeling to
simulate infrared spectra and compared straightforward with measured spectra of OEG-SAMs on
gold. According to their calculations, the main peak near 2890 cm-1
which previously assigned to
the CH2 symmetric stretching is most likely due to the asymmetric mode of helical OEG chains
[108]. Besides, the intense peak attributed to C-O-C asymmetric skeletal stretching vibration was
found in fact a superposition of several strong C-O-C stretching modes that forms a peak
centered at ~1110 or ~1140 cm-1
, depends on the OEG conformations. The distribution of this
28
multimode intensity is proved to be very sensitive to whether the terminus is –CH3 or -OH.
Simulated RA spectra reveal the presence of the C-O-C band shoulder at higher frequency
(~1130 cm-1
) is associated with the -OH terminus but not the -CH3 terminus [109]. In addition,
the incorporated amide group couples strongly with the OEG and enhances the C-O-C band
shoulder. They also studied the defect structure in helical OEG SAMs and suggested that such
defects exist as intact chains adopting exclusively the all trans conformation rather helical chains
with one or two an all-trans defects [109]. The theoretical modeling has provided a deeper
understanding about the conformation and packing properties of the amide and OEG-containing
alkanethiolate assemblies.
29
2. Experimental and Theoretical Background
This thesis concerns mainly the use of Infrared reflection-absorption spectroscopy (IRRAS) as an
analytical method for structural characterization of adsorbed thin organic layers on metal
substrates. IRRAS is an external reflection method; as molecular vibrations are excited by
incoming photons in the infrared region at specific frequencies, corresponding absorptions occur.
Reflection-absorption (RA) spectra are obtained by calculating the -log
, where represents
the reflectance from the thin layer coated substrate, and is the reflectance from a reference.
The two spectra are ratioed in order to cancel all contributions from the spectrometer optics. IR-
RA spectra can give detailed information about the geometric structure of the molecules and
their inter- and intra-molecular interactions between adsorbed species, identification of
functional groups in the molecules as well as their orientations relative to the solid substrates.
30
2.1. SAMs and IRRAS
In the early work, infrared spectra of adsorbed molecules on metal surfaces were studied using
transmission infrared spectroscopy. The molecules were chemisorbed on tiny metal particles
which were dispersed in an infrared transparent matrix [110], however, the surfaces with this
sampling were usually poorly characterized. In the 1950s and 1960s, developments of infrared
techniques for obtaining infrared spectra of one or a few molecular layers adsorbed on metallic
substrates were intensively proceeded. The main challenge in such a system at that time was to
enable the weak absorptions of the monolayer on a reflecting metal surface to be observed. In
1959, Francis and Ellison used multiple reflection IRRAS to overcome this sensitivity problem
[111]. They demonstrated that absorption of infrared radiation is enhanced at high angles of
incidence and only the p-polarized radiation is involved. Based on a three-phase model,
Greenler and coworkers reported a series IRRAS investigation of isotropic thin films on
metallic substrates [112-115]. They pointed out that the absorption factor for p-polarized infrared
radiation at an incident angle of ~88o is 5000 times greater than that at normal incidence.
Additionally, the absorption factor under the chosen conditions shows the reflection sampling
has a factor of 25 more sensitive than the transmission sampling. A more thorough theoretical
prediction for isotropic films in a two-phase, three-phase, and N-phase systems was later
presented by Hansen in 1968 [116]. Additionally, IR detectors have been extensively developed
to improve sensitivity and response time since the 1940´s; the RA technique is now a powerful
tool for structural studies of thin films on both metallic and nonmetallic substrates.
Principle of IRRAS
Light is an electromagnetic wave which can be described in terms of intensity of the electric field.
Consider a plane wave as a superposition of two orthogonal linearly polarized waves, thus the IR
beam can be divided into a parallel, p-, and a perpendicular, s-, components of light to the plane
of incidence as shown in Figure 5. Thus one can apply boundary conditions at the interface
between two media, i.e. the electric field at the interface must be continuous across the boundary.
When an IR beam strikes on a metal surface, depending on the angle of incidence, Ѳ, and
polarization state of the incident light, its electric field will undergo a phase shift, δ, upon
31
reflection. At normal incidence (Ѳ=0), for example, the s- polarized component goes through a
phase shift on reflection by about 180o. This implies the incident and reflected light interfere
destructively to each other, resulting in the electric field right at the metal surface to become zero.
Thus, no or very small contribution in the resulting electric field comes from the s-component.
Note that at Ѳ=0, the p- and s-polarized light cannot be distinguished; therefore the δ must be
equal ~180o for both polarization. When Ѳ increases from 0
o to 90
o, the δ of the s-polarized
component remains close to 180o for all angles of incidence. However the δ of the p-polarized
component behaves very differently and is strongly dependent on Ѳ. In Figure 6, when Ѳ
approaches about 87o, the δ of p-polarized component is near 90
o. At this point the incident and
reflected light interfere constructively, and the resulting electric field at the metal surface is
enhanced. In this case, the IR radiation can interact strongly with material right at the surface of
the metal.
Figure 5. The principle of IRRAS. The incident IR beam can be divided into two linearly
polarized light waves, Eip parallel and Eis perpendicular to the plane of incidence. Ѳ is the angle
of incidence. Upon reflection, the electric vector experiences a phase change where the s-
polarized radiation is denoted Ers and p-polarized radiation, Erp. It is the absorption of the
resulting electric field, ER, at the surface that is measured in IRRAS.
32
Figure 6. The calculated phase shift, δ, of s- and p- polarized light as a function of the angle of
incidence on a metallic substrate. (Reprinted with permission from [117], John Wiley & Sons,
Inc; copyright © 2007 )
Surface Dipole Selection Rule
All molecules vibrate; however, not every molecular vibration can be excited by IR radiation.
The necessary condition for a molecule to absorb infrared light is that the molecule must have a
vibration during which its change in dipole moment is non-zero. This can be described by the
term
Mi =
where µ is the dipole moment and Mi denotes the transition dipole moment (TDM) for the
vibration mode i with the coordinate Qi. Vibrations that satisfy this condition are said to be
infrared active, causing the absorptions seen in an IR spectrum. Besides, because the intensity of
the absorption peak Ai which appears in the IR spectrum is proportional to the square of the
electric field (E) amplitude, there is an enhancement of the field intensity at the surface nearly
four times the incident field intensity. The following term expresses the relation in terms of the
absorption peak and the angle Ѳ between E and Mi.
Ai ∞ Ι Mi • E Ι2 = Ι Mi Ι
2 Ι E Ι2 cos
2 Ѳ
33
In IRRAS, since the resulting electric field ER at a thin layer coated metal surface is
perpendicular to the substrate, which means that if the Mi for any vibration mode has a
component in the direction of the resulting electric field, absorption occurs. This is so called
surface dipole selection rule which states only vibrations with components transition dipole
moment perpendicular to the surface can interact with incident light whereas components with
dipole moment change parallel to the surface are invisible in the RA spectrum. The maximum
intensity of absorption is obtained when the Mi is oriented parallel to ER and the beam area, 1 /
cos Ѳ, over which the ER is exerted on the surface, is enlarged to the maximum. Thus the surface
dipole selection rule can be used to determine the orientation of adsorbed molecules, that is, only
those vibrations with TDM components that are aligned perpendicular to the substrate will
appear with enhanced intensities in the RA spectrum, while vibration modes that are aligned
parallel to the substrate will appear weak or no absorption in the spectrum. Figure 7 shows the
variation of absorbance of a thin film, Ap and As, as a function of the incident angle (Ѳ). It can be
seen that the absorbance remains small until Ѳ exceeds ~45o and then increases rapidly for p-
component radiation, reaching a maximum value at ~87o whereas the s- component has little
contribution on the absorbance for all angles of incidence.
Figure 7. The calculated absorbance of Ap and As as a function of the angle of incidence of a
typical thin film on a metallic substrate. (Reprinted with permission from [118], Academic Press;
copyright © 1985 )
34
Allara et al [119] established a method to determine the orientation for ordered, yet anisotropic
monolayers adsorbed onto a smooth reflective substrate. The relationship between the theoretical
isotropic molecular intensity and the observed anisotropic intensity is described as follows:
Where and are the intensities of the experimental and calculated data, respectively, and
is as defined above. Note that the calculation of is obtained based on the transmission
spectrum of the same compound in bulk such as KBr pellets. More complicated quantitative
simulations of reflection spectra from multilayered, anisotropic films on both metallic and
nonmetallic substrates were later developed by Parikh et al [120].
2.2. Ab-initio Calculations
Ab-initio calculations are a method of calculating atomic and molecular structure directly from
the first principles of quantum mechanics without using quantities derived from experiment as
parameters. Ab-initio calculations are used to determine the bond lengths and bond angles of
molecules by calculating the total energy of the molecule for a variety of molecular geometries
and finding which conformation has the lowest energies. Since the calculations require a large
amount of numerical computation, the computing time increases dramatically as the size of the
atom or molecule increases.
There are many program packages can be used to calculate molecular structural and spectral
properties. In the Born-Oppenheimer approximation, the nuclear and electronic motions are
treated separately. Thus, each electronic structure calculation is performed for a fixed nuclear
configuration, and therefore the positions of all atoms must be specified in an input file. The ab-
initio program then computes the electronic energy by solving the electronic Schrödinger
equation for this fixed nuclear configuration. The electronic energy as function of the 3N-6
internal nuclear degrees of freedom defines the potential energy surface. However, the electronic
35
Schrödinger equation cannot be solved exactly, except for a very simple system like the
hydrogen atom. Therefore, the electronic wave-function is represented in certain finite basis sets,
and the Schrödinger equation is transformed into an algebraic equation which can be solved
using numerical methods. In most programs, Gaussian basis functions are applied to approximate
the molecular orbital since the required integrals can be computed very quickly in this basis. The
challenge, however, is to know which basis set and method to use for a particular problem in
order to obtain an accurate result for a minimum possible cost. This is something which needs a
lot of experience. These simulated RA-spectra played an important role in better understanding
of the experimental results.
Our simulated RA-spectra are based on the assumption that the molecular vibrations are weakly
perturbed by the substrate and intermolecular interactions. The TDM of each mode for a single
molecule was projected onto Cartesian coordinate axes which chosen to be connected with the
alkyl plane. The arbitrary TDM component of
which is perpendicular to the substrate is
given by the following relation where and ψ represent the tilt and rotation angles of the
alkyl plane, as shown in Figure 8(a).
ψ
ψ
The calculated RA spectra are obtained as the sum of Lorentzian-shaped peaks, each centered at
the fundamental mode frequency having the half width at half-maximum determined by a
parameter σ in cm-1
. Thus the frequency dependence of a RA spectrum can be expressed by
Where =
is for the molecules self-assembled on gold and =
, for the randomly
oriented molecules. Note that the squared TDM,
for modeling the RA spectra of
randomly oriented molecules is defined as
36
For example, in our HS(CH2)15CONH(EG)6H SAM system, the step-by-step RA-modeling
procedure is as following: (i) find optimized geometry of the components, (ii) determine a range
values of and ψ
by comparing the measured and calculated vibrational spectra of
alkanethiols, (iii) find specific values of and ψ
within the range of C=O and N-H angles are
not too far from 90o, and (iv) find the values of azimuthal angle φA that would provide the
minimum distance of H∙∙∙∙O between the nearest-neighbor amide group. The optimized
geometries, vibrational frequencies and transition dipole moments were calculated using DFT
methods from Gaussian-03 package. By comparing with experimental data, the averaged
molecular orientation can be determined by the Euler angles ( , ψ, φ) of each component, e.g.
alkyl part, OEG and amide group. Details of calculations can be found in the reference [96].
Figure 8. Definition of Euler angles and the axes of (a) alkyl segment, (b) OEG helix, and (c)
amide group (adopted from Malysheva et al [96])
37
3. Experimental Techniques
3.1. Preparation of gold Surfaces
Our flat, gold surfaces were prepared from standard silicon (100) wafers and used as substrates
for SAM formation. The silicon wafers were cut in desired sizes and cleaned before deposition
processes. A Balzers UMS 500 P system has been employed for operating electron beam
evaporation of metals at a base pressure of 10-9
mbar and an evaporation pressure of about 10-7
mbar. After deposition of a 25 Å titanium adhesion layer on the silicon substrate, an additional
2000 Å thick gold film was deposited on top of the surface. A constant evaporation rate of 10
Å/s was set for the gold layer deposition. The resulting polycrystalline gold film has been shown
heavily dominated with (111) oriented terraces as this is the lowest energy surface of Au. It is
worth to mention that the crystalline quality, the number of non-(111) oriented crystallites and
the density of defects can vary significantly depending on the evaporation condition.
38
3.2. Preparation of SAMs
Thiol solutions (20 μM) were prepared in plastic jars from 1 mM stock solutions in ethanol
(99.5%) and stored in glass vials at room temperature. Prior to SAM adsorption, the gold
surfaces were cleaned in a 5:1:1 mixture of deionized water (Milli-Q), 25% hydrogen peroxide,
and 30% ammonia for 5 to 10 min at 80oC and then rinsed thoroughly in deionized water. In
order to check the efficiency of cleaning, the optical characteristics of a gold surface that was
taken from the cleaned batch were measured before incubation of the SAMs. The rest of the
cleaned gold surfaces were soaked in ethanol and then transferred into the incubation thiol
solutions. After at least 24 h of adsorption, the samples were rinsed in ethanol, ultrasonicated,
and rinsed again. Finally, they were dried in a flow of nitrogen gas and immediately analyzed.
Solutions for mixed SAMs were prepared by volumetrically mixing the thiol solution (20 μM or
50 μM) of individual components to the desired molar ratio. Adsorption procedures are the same
as mentioned above.
3.3. Null-Ellipsometry
Null-ellipsometry is a quick and simple technique for obtaining optical parameters of the
substrates and thicknesses of the adsorbed layers. Depending on the substrate and the thin film
properties, the change in polarization of a light beam reflected from the sample surface is
measured at null condition which the reflected beam is extinguished on the detector. Such
measurements allow determination of optical constants of the substrates, and refractive indices
and thicknesses of thin layers covered on the substrates.
A basic null-ellipsometric system consists of a polarizer, a compensator, a sample, and an
analyzer. Due to its robustness, simplicity and high precision, it is widely used in numerous
39
fields (Figure 9). In this system, a collimated unpolarized light from the source passes through a
rotatable polarizer, being converted to linearly polarized light, and to elliptically polarized light
by passing a quarter-wave compensator. The elliptically-polarized beam strikes the sample
surface at a desired angle of incidence is reflected at the same angle, and passes through a
rotatable analyzer, and finally reaches the detector. When a monochromatic collimated beam of
polarized light reflected from a surface, it will bring about changes in the relative phases and the
relative amplitudes of the p- and s- components. These changes determine the two parameters, ψ
and Δ in angles which measured by ellipsometry. The equation of ellipsometry, ρ, is defined as:
Where represents the change in the amplitude ratio upon reflection and Δ represents the
change in the phase difference between the p- and s- components caused by reflection. The two
optical angles are then applied to the Fresnel equations and convert into the refractive index of
the substrate.
Figure 9. The principle of a null-ellipsometer system
An ideal substrate is optically flat, specular and highly reflective in order to obtain uniform
changes in polarization state over the entire cross section of the reflected beam. The film covered
on the substrate is assumed to be homogeneous and isotropic, and is preferable large differences
in refractive indices from the substrate. On the other hand, uneven surface causes scattering of
40
the reflected beam, causing either loss of light energy or changing polarization states of the beam,
resulting degradation of measuring accuracy. A typical light source is a HeNe laser with λ =
632.8nm at an angle of incidence of 70o.
In this thesis, I applied the automatic Rudolph Research AutoEL ellipsometer software to
determine thicknesses of the SAMs. Before SAM preparation, the refractive index of a cleaned
gold surface was measured. The thickness of the film was calculated based on the
ambient/organic film/gold model, assuming an isotropic, transparent organic layer with a
refractive index of 1.5 on the gold surface. The thickness of the gold (2000Å) is considered to be
optically infinitely thick, so that no considerable amount of light returned from the back. Note
that any difference between the real refractive index of the adsorbed film and 1.5 would result in
a systematic error in the calculated thickness but would not affect the relative values.
3.4. Contact Angle Goniometry
Wetting is a surface property which is sensitive to microscopic changes in composition,
morphology and functionality of the surface. Thus, the microscopic structure of a surface is
directly linked to the wettability. Contact angles help to understand the structure and chemistry
of the outermost few angstroms of a surface. Measurement of contact angles is a measure of the
wetting at which the angle formed by the solid and the tangent line to the liquid drop interface, as
shown in Figure 10. The shape of the droplet is determined according to the Young-Laplace
equation given below, where is measured contact angle, is solid surface free energy, is
liquid surface free energy and is solid/liquid interfacial free energy.
A low contact angle means high wettability and a high contact angle means poor wettability. For
example, solid materials like metals and glasses generally have large surface free energy (large
), therefore water droplets on these materials should form a small contact angle.
41
Figure 10. Schematic of a liquid contact angle on a solid surface, is the measured contact angle,
is solid surface free energy, is liquid surface free energy and is solid/liquid interfacial
free energy
For dynamic contact angles, the advancing (θa) and receding (θr) contact angles are measured
upon expanding and reducing the volume of the formed droplet, and a hysteresis H = θa – θr is
obtained. Hysteresis provides information about the heterogenity and flexibility of the outermost
layer of the SAM. In our lab, a semiautomatic optical contact angle meter, KSV CAM 200, was
used to determine advancing and receding contact angles of water on the SAMs. A manual
dispenser was used to expand or retract a droplet. As the drop was dispensed onto the surface and
expanded, the advancing contact angle was measured quasi-statically. Similarly, the receding
contact angle was measured by partially withdrawing the drop back into the syringe. During the
measurements, the syringe needle was kept attaching onto the drop, and a high-speed CCD
camera was used for image capturing. The images of the drop shape were analyzed by KSV
CAM software to generate consistent contact angle data. The accuracy of the contact angles is at
about ±1o.
42
3.5. Infrared Reflection-Absorption Spectroscopy (IRRAS)
In this thesis, all RA spectra were recorded on a Bruker IFS 66 FTIR system equipped with a
grazing angle (85o) and a liquid nitrogen cooled MCT detector. The sample chamber was purged
continuously with nitrogen gas during the measurements. A home-made sample holder fixed to
2x4 cm2 surface was used and all spectra were acquired 3000 scans at a resolution of 2 cm
-1
between 4000 and 400 cm-1
. A three-term Blackmann-Harris apodization function was applied to
the interferograms before Fourier transformation. Depending on the spectral range of interest, a
deuterated hexadecanethiolate (HS(CD2)15CD3) SAM or a non-deuterated hexadecanethiolate
(HS(CH2)15CH3) SAM on gold was used as background spectra ( ), The sample spectra ( )
were recorded under identical conditions.
3.6. X-ray photoelectron spectroscopy (XPS)
X-ray photoelectron Spectroscopy (XPS) is a surface sensitive technique which the sample is
irradiated by X-rays; electrons of the atoms are ejected out off the sample and their kinetic
energies ( ) are measured (Figure 11). The electron binding energy ( ) is determined by the
following equation:
where is the X-ray radiation and is the work function of the spectrometer. For every
element, there is a characteristic binding energy associated with each atomic orbital, i.e. each
element will give rise to its characteristic peaks in the photoelectron spectrum. The binding
energy might slightly shift due to the chemical state of the emitting atom; hence it is possible to
obtain information about the chemical bonds between neighboring atoms.
43
Figure 11. A schematic diagram of the principle of x-ray photoelectron spectroscopy
XPS instruments consist of an X-ray source, an energy analyzer for the photoelectrons, and an
electron detector. The XPS experiments in this thesis were carried out on a SCIENTA ESCA 300
spectrometer. A monochromatized Al Kα radiation (1486.6 eV) was used and the photoelectron
take-off angles were 90o and 10
o relative to the surface. The power of the X-ray gun was 8 or 6
kW, the pass energy is 300 eV and the width of the analyzer entrance slit is 0.8mm, giving a 0.5
eV resolution of the spectrometer. The ultra high vacuum chamber was held at the ~10 mbar
scale and the temperature, ~303 K. Several spots on each sample were investigated where the
spots were selected so that the possibility of X-ray induced damage of the sample was minimized.
44
3.7. Cyclic Voltammetry (CV)
Cyclic voltammetry (CV) is an electrochemical technique which has been widely used to probe
the packing density and distribution of pinhole defects in the monolayer. It provides information
concerning dynamic of electron transfer at the electrochemical interface.
Our voltammetric experiments in this thesis were performed in an Autolab PGSTAT20 (Eco-
Chemie, The Netherlands). A three-electrode mode, consisting of an Ag/AgCl reference
electrode (RE-5B, BAS) and a platinum wire as a counter electrode was used. SAM-coated gold
surfaces were brought into contact with the electrolyte via press fitting to an O-ring on the side
of a Kel-F-based electrochemical cell. A 1mM K3Fe(CN)6 was used as redox species and a 100
mM KNO3 solution, as supporting electrolyte.
3.8. Quartz Crystal Microbalance with Dissipation monitoring (QCM-D)
QCM-D is a label free, real time measurements of molecular adsorption and interactions on solid
surfaces. The adsorbed mass (ng/cm2) is measured as changes in frequency (ΔF) of the quartz
crystal and the dissipation parameter (ΔD) provides information regarding structural properties
of the adsorbed layers.
Here we used QCM-D (Q-sense E4) and Au coated (100nm) sensors from Q-sense to study
vesicle fusion on the surfaces. The cleanliness and formation of SAMs on the sensors are the
same procedures as described in 3.2 preparation of SAMs. Small unilaminar vesicles (SUVs)
were prepared by probe sonication and a Hepes buffer with high salt concentration (20mM
Hepes + 1M NaCl, pH= 7.5) was used as the running buffer.
45
4. Future Outlook
Solid supported lipid membranes (SLMs) have been investigated for the applications in
membrane-based biosensing and pharmaceutical screening for several decades. These systems
are relative robust, stable, keeping fluidic between the substrate and the bilayer and mostly, they
can be patterned. Tethered lipid membranes (TLMs) on self-assembled monolayers (SAMs) are
one of the approaches, e.g. mixed SAMs that consist of a hydrophobic anchor compound
dispersed in a hydrophilic matrix served as underlying layer between the solid support and the
bilayer. The underlying SAM can decouple effectively the membrane from solid support, thus
prevent the protein being immobilized and losing its function in the membrane. Additionally, due
to the ease of preparation, control over the surface composition, stability and applicability of
variety of analytical techniques, these self-assembled, substrate-supported lipid membranes have
become a center role in the field of membrane research.
46
It is clear that understanding of the underlying processes and mechanisms of such surface-based
assemblies would enable more general patterning capabilities, reaching down to the nanometer
scale. In addition, exploiting the interactions between neighboring molecules has led to self-
assembled nanostructures being applicable in biomolecular arrays and scaffolds, lithography
resists and molecular nanoelectronic assemblies. By controlling the molecular interactions, the
patterning of SAMs and the related technologies holds promise for obtaining the selective
functionality at the molecular scale. SAMs are one of the best systems for studying the
contributions of molecular structures and compositions to the macroscopic properties of
materials. The extensive characterization of SAMs, especially OEG-containing alkanethiolate
SAMs on gold, has provided a broad understanding of several aspects of these systems.
In this thesis, I have put effort on investigation of the molecular assemblies consisting of a
matrix molecule and a long chain OEG-contaning alkanethiol with an additional amide-bridged
alkyl chain as anchors. My approach was aimed at developing a stable and flexible SAM with
controlled chemical composition for investigations of the TLM formation on solid support.
Based on the knowledge we collected from experimental and theoretical studies, hopefully this
can contribute to the construction of new, advanced molecular architectures.
Hopefully, the field of supported lipid bilayer technology will continue to expand with new
platforms of sensors and nanodevices, e.g. DNA microarrays. By combination of microfluidic
platforms and array-based systems with solid support for membranes, one may develop early
warning biological sensors for warfare agents, and discover novel drugs for viral and neural
degenerative diseases in the field of medicine. Moreover, by introducing proper ligands and
proteins onto the surface of supported membrane-coated nanodevices, it might possible to detect
a specific location of a tumor cell, subsequently control the delivery of chemotherapies.
47
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Summary of the Included Papers
Paper I
“Long-Chain Alkylthiol Assemblies Containing Buried In-Plane Stabilizing Architectures”
H-H. Lee, Ž. Ruželė, L. Malysheva, A. Onipko, A. Gutės, F. Björefors, R. Valiokas and B.
Liedberg, , Langmuir, 2009, 25(24), 13959-13971
My Contribution: Planning and performing all experimental work regarding IRRAS, Null-
Ellipsometry and Contact Angle measurements; gold and SAM surfaces preparation; evaluation
of the experimental data; manuscript writing related to these issues.
A series of complex compounds were synthesized and structural characterized on their SAM
assemblies consisting of interchanging structural modules that stabilized by intermolecular
hydrogen bonds. The experimental data strongly indicate the formation of highly ordered and
oriented assemblies regardless of the length of the extended alkyl chain. In addition with
theoretical calculation, it was shown that the lower alkyl portion and the hexa(ethylene glycol)
portion are stabilized by the two layers of lateral hydrogen bonding networks through the two
amide linking groups. These hydrogen bonding networks further improve the structural
robustness of the extended chains. The present system demonstrates the possibility to obtain
highly ordered, electrically isolating SAMs in the thickness range of at least up to 60Å.
Nanopatterns consisting of SAMs with different extended alkyl chains can be employed as
supports for the assembly of artificial cell membranes.
58
Paper II
“Mixed Self-Assembled Monolayers with Deuterated Terminal Anchors for Tethered Lipid
Membrane Formation”
H-H. Lee, M. Gavutis, Ž. Ruželė, R. Valiokas and B. Liedberg, Manuscript, 2012
My Contribution: Planning and performing all experimental work regarding IRRAS, Null-
Ellipsometry and Contact Angle measurements; gold and SAM/mixed-SAM surfaces preparation;
evaluation of the experimental data; manuscript writing related to these issues.
Model SAM surfaces consisting of a mixture of a filling molecule, HS(CH2)15CONHCH2CH2OH,
and an anchor molecule, HS(CH2)15CONH(CH2CH2O)6CH2CONH-X, where X is either –
(CD2)7CD3 or –(CD2)15CD3 were prepared. Their surface structures and chemical compositions
as well as the adsorption of lipids on the SAMs were characterized. The use of deuterated
terminal alkyl chains enabled a direct relation between the surface density of the anchors and the
properties of the lipidbilayers. We found that the relative surface density of the shorter anchor
molecules is higher than that of the longer anchor molecules for SAMs prepared at the same
mole fractions of the anchors. The longer anchor molecule was found to phase segregate upon
co-assembly with the matrix molecule, whereas the shorter molecule did not. This behavior of
the longer molecule seemed be of less importance for the interaction with the lipid material as
the infrared RA spectra of the two different SAMs, after SOPC assembly, look more or less the
same. QCM-D indicated that SUV fuse efficiently on 5 mol% mixed SAMs. The described SAM
system will be used in our subsequent studies of cell membrane-mimicking molecular
architectures.
59
Paper III
“On the quality and structural characteristics of OEG assemblies on gold: An experimental and
theoretical study”
R. Valiokas, L. Malysheva, A. Onipko, H-H. Lee, Ž. Ruželė, S. Svedhem, S.C.T. Svensson, U.
Gelius and B. Liedberg, J. Electron Spectroscopy and Related Phenomena, 2009, 172, 9-20
My Contribution: Planning and performing the experiments of long thiol-molecules regarding
IRRAS and Null-Ellipsometry measurements; gold and SAM surfaces preparation.
We investigated the quality, conformation, orientation, defect structure and infrared (IR)
signatures of different OEG-containing SAMs. XPS data strongly suggests that compounds with
structurally complex tails as the helical OEG conformer should be prepared keeping the
incubation solution concentrations as low as possible (typically ∼20μM) especially for those
containing long OEG chains. Further on, the supporting alkyl spacer should be long enough to
provide maximal van der Waals interactions, forming a densely packed alkanethiolate overlayer
on Au(1 1 1) surface. With this condition fulfilled, we are able to vary the length of the terminal
OEG portion from 1 to at least 12, without affecting the integrity and conformational
characteristics of the supporting alkyl part of the SAMs. IRRAS and ab initio modeling
confirmed that the orientation of the helical OEG is affected by the second hydrogen bonding
layer rather than the extended alkyl tails.
60
Paper IV
“Spectroscopic characterization and modeling of methyl- and hydrogen-terminated oligo
(ethylene glycol) SAMs”
L. Malysheva, A. Onipko, T. Fyrner, H-H. Lee, R. Valiokas, P. Konradsson and B. Liedberg, J.
Physical Chemistry A, 2011, submitted
My Contribution: Planning and performing all experimental work including gold and SAM
surfaces preparation, IR-RA measurements, thicknesses of the films and contact angles
measurements, as well as evaluation of the experimental data.
Two series of alkyl-free HS-(CH2CH2O)nR with R = CH3, H and n = 5, 6, 7, have been
synthesized and used to study their SAM structures on Au(111) in detail by means of IRRAS and
ab intio modeling. The optimized geometries of these SAM constituents their vibrational
frequencies and transition dipole moments were obtained by the BP86 DFT method using the 6-
31G* basis set. Our data show the OEG helix axis tilted by about 20o with respect to the surface
normal and by adding one EG unit, the rotational angle (ψ) changes by about -100o preserves the
head group orientation. A new spectral feature has been observed at 2947 cm-1
as localized
vibrations that are specific for hydrogen-terminated OEG thiolate SAMs. This band can be used
as an indicator of a high crystalline like ordering.
61
Paper V
“Tethered Lipid Membrane Formation on Assemblies with Controlled Presentation of Anchor
Molecules: A QCM-D study”
M. Gavutis, H-H. Lee, Ž. Ruželė, B. Liedberg and R. Valiokas, Manuscript, 2012
My Contribution: Preparing SAMs/mixed-SAMs on Au(111) surfaces in parallel with the
preparation of QCM samples; IR-RA, thickness and contact angle measurements for the quality
control of these SAMs and mixed-SAMs.
We investigated how the density of anchor molecules on the surface affects formation of lipid
bilayer. SAMs were prepared with various molar ratio of solution concentration composing of an
anchor molecule (EG6AC8D and EG6AC16D) and an inert molecule (EG1H). Vesicle adsorbtion
on the surface was followed by incorporating anchor molecules into the vesicles. QCM-D
analysis revealed that short anchors incorporate faster and easier than long ones. Furthermore, a
critical concentration of the small unilamellar vesicles (SUVs) on the surface is necessary for
vesicle rupture and bilayer formation. The critical SUV concentration decreased with increasing
density of the anchors. In addition, the anchor molecules become an integral part of the tethered
bilayer lipid membrane (tBLM) was observed. Based on the obtained results, we are able to
develop micro- and nanopatterned tBLMs as biophysical models of complex cell membrane
assemblies.
62
Paper VI
”Saccharide-Functionalized Alkanethiols for Fouling-Resistant Self-Assembled Monolayers:
Synthesis, Monolayer Properties, and Antifouling Behavior”
T. Fyrner, H-H. Lee, A. Mangone, T. Ekblad, M.E. Pettitt, M.E. Callow, J.A. Callow, S.l. Conlan,
R. Mutton, A.S. Clare, P. Konradsson, B. Liedberg and T. Ederth, Langmuir, 2011, 27, 15034-
15047
My Contribution: Planning and preparing SAMs/mixed-SAMs on Au(111) surfaces as well
as providing the structural properties of these monolayers using IRRAS, Null-Ellipsometry and
Contact Angle Goniometry.
We synthesized a series of mono-, di-, and trisaccharide-functionalized alkanethiols and studied
their formation of self-assembled monolayers (SAMs). Our results indicate that upon increasing
the size of the saccharide terminus, increase disorder in the SAMs and therefore the structural
qualities of the monolayers decreased. For the trisaccharide, a slow reorganization dynamics in
response to changes in the environmental polarity was observed. These SAMs show in general
good protein resistance except for the fibrinogen. However, there is a clear trend that the more
glucose units in the chain, the higher resistance to fibrinogen adsorption. In addition, marine
organism tests show significantly lower attachment on the SAMs than on the glass and
polystyrene. In order to improve the packing order of the SAMs, we employed mixed-SAMs
where the saccharide-terminated alkanethiols are diluted with filler molecules having short EG
tail groups. With this possibility, we´ll be able to study specific interactions of proteins/cells with
sugar groups on the surface by means of adjusting exposure of the saccharide moieties to the
environment.
