CONJUGATED POLYMER SURFACE SWITCHES
FOR ACTIVE CELL CONTROL
Maria Helene Bolin
Norrköping 2011
CONJUGATED POLYMER SURFACE SWITCHES
FOR ACTIVE CELL CONTROL
Maria Helene Bolin
During the course of research underlying this thesis, Maria Bolin was enrolled in
Forum Scientium, a multidisciplinary doctoral programme at Linköping University,
Sweden
Linköping Studies in Science and Technology. Dissertation No. 1398
Electronic publication: http://www.ep.liu.se
Printed in Sweden by LiU-Tryck, 2011
ISBN 978-91-7393-063-5
ISSN 0345-7524
Cover by Maria Helene Bolin
© Maria Bolin 2011, unless otherwise noted
ABSTRACT
Conjugated polymers have been found useful in a wide range of applications such as
sensors, electrochemical transistors, solar cells, and printed electronics due to their
mechanical, optical and electronic properties. An amazing research field has grown during
the last three decades since the discovery of conducting polymers in 1976. Since the
materials can be made from solutions, different processing methods such as spin coating
and vapor phase polymerization can be used to coat a huge variety of substrates. The choice
of method depends mainly on monomer solubility and kind of substrate to be coated.
During the synthesis the polymers can be chemically modified to tailor their functionalities.
Due to this variability in materials and the processability, electronics can be achieved on
unconventional substrates such as flexible plastic foils and cell culturing dishes. As a
contrast to inorganic, usually metallic materials, conducting polymers are built up from
organic compounds in a molecular structure with soft mechanical properties that have
shown to be a benefit in combination with biology, ranging from interactions with cells to
interactions with advanced biological species such as tissues. This combination of research
fields and the possible applications are merged within the field of organic bioelectronics.
The primary purpose of this thesis is to give a background to organic electronics in general
and how electrochemical devices can be processed and developed for biological applications
in particular. An organic electronic surface switch is introduced to control cell adhesion and
proliferation as well as an electrochemical transistor to spatially tune the cell adhesion along
an electrochemical gradient. To mimic a more natural cell environment a three dimensional
fiber substrate was used to design an electronically active matrix to promote nerve cell
adhesion and communication. By combining standard microfabrication techniques and
conjugated polymers desired patterns of electroactive polymer were created to enable active
regulation of cell populations and their extracellular environment at high spatial resolution.
Finally, a brief look into future challenges will also be presented.
POPULÄRVETENSKAPLIG SAMMANFATTNING
Vetenskapen strävar hela tiden efter ökad kunskap om det okända för att öka förståelse och
förmågan att kunna påverka det som händer omkring oss. Områden som medicinsk teknik
och materialvetenskap är inget undantag. Här har forskningen inom implantat, drug
release, kontroll av stamceller så väl som vävnadsodling förändrat våra medicinska
möjligheter markant. Den här avhandlingen handlar om plaster som kan leda ström och
hur dessa kan användas för att tillverka elektroniska ytor för att aktivt kunna kontrollera
hur celler växer samt studera deras beteende, in vitro. Plaster är organiska material
bestående av långa polymerkedjor som i sig är uppbyggda av många mindre repeterande
enheter så kallade monomerer. Generellt sett är plaster icke-ledande och används flitigt för
att isolera material i vår omgivning. Men om den långa polymerkedjan består av ett
konjugerat system dvs. omväxlande dubbel- och enkelbindningar så kan materialet fungera
som en halvledare. Elektronerna som bygger upp de mindre stabila dubbelbindningarna är
rörliga och bidrar till en viss ledningsförmåga. För att öka rörligheten hos dessa elektroner
och därmed öka ledningsförmågan i materialet kan polymeren dopas vilket innebär att
ämnen som drar till sig eller donerar elektroner tillförs för att ytterligare störa stabiliteten i
dubbelbindningarna. Om ett spänningsfält läggs över polymeren så kommer elektronerna
att röra sig snabbt längs med kedjan eller hoppa mellan olika polymerkedjor och därmed ge
materialet näst intill metallisk ledningsförmåga. Den här upptäckten, som belönades med
Nobelpriset i Kemi 2000, har skapat stort industriellt intresse och spekulationer om möjliga
tillämpningar och gett upphov till begreppet organisk elektronik. Materialet kan förutom
att leda ström ge upphov till elektriska fält och jontransport. Det här är fysikaliska
egenskaper som givetvis går att uppfylla med befintliga metaller men där ledande polymerer
generellt erbjuder betydligt mera biovänliga kontaktytor till sin omgivning. Ledande
polymerer är även relativt billiga, enkla att processa på önskade substrat, kräver låg
styrspänning, har uppskattade mekaniska och fysikaliska egenskaper samt att deras
funktioner går att skräddarsy med traditionell synteskemi.
Mycket har hänt inom området för organisk elektronik i kombination med biologiska
system sedan tidigt 90-tal och ännu mera kommer att hända framöver. Kunskaper inom
traditionell halvledarteknik och elektronik kombineras med naturvetenskap vilket öppnar
upp för spännande möjligheter. Här i gränslandet möts fysiker och kemister som utvecklar
material och skräddarsydda strukturer med biologer och medicinare som studerar cell
system. Cellers naturliga mikromiljö utgörs av mönster, topografier samt kemiska och
biofysikaliska ledtrådar som är väl definierade i tid och rymd. Inom medicinsk teknik är det
en utmaning att förstå och härma de biofunktionaliserade ytorna in vivo för att kunna
kontrollera celladhesion och differentiering genom att isolera enskilda celler såväl som
multicellulära system in vitro. Här vore det av intresse att kunna skapa funktionella och
dynamiska material vars ytegenskaper är lätta att förändra och anpassa. I denna avhandling
undersöks hur organisk elektronik kan användas för att fylla ett hålrum i detta växande fält
av efterfrågad kontroll på protein adhesion, cell mobilitet, kontrollerad signalering och
celldifferentiering. Genom att lägga på en spänning över en ledande polymeryta kan vissa
av dess egenskaper ändras på ett kontrollerat och reversibelt vis. I samtliga experiment i
avhandlingen används den ledande polymeren PEDOT som har visat sig vara ett lämpligt
material för bioapplikationer. Den uppvisar god ledningsförmåga, fungerar med ett
spektrum av olika dopjoner samt är lätt att processa med olika metoder för att passa diverse
typer av substrat. PEDOT är stabil i luft och arbetar i jonlösning vilket är den naturliga
miljön för de flesta biosystem. Med hjälp av diverse mikrofabrikationsmetoder går det att
skapa smarta ytor med reproducerbara geometrier samt funktionaliteter med god
upplösning för cell system in vitro. Metoder som är väl utvecklade och etablerade för
traditionell halvledarteknologi. Här kan t.ex. fotolitografi, plasma etsning, mjuk litografi,
ink-jet och olika tryckmetoder nämnas. Med dessa metoder går det att tillverka strukturer i
mikro- och submikrometer skala för spatiell kontroll av protein- och celladhesion. För att
modifiera ett materials biokompabilitet kan statiska ytegenskaper mönstras genom att fästa
naturliga eller syntetiska biomolekyler som ankare alternativt genom att kemiskt ändra
ytegenskaperna på materialet. Här presenteras metoder för att tillverka och mönstra ledande
polymerer på substrat där ytfunktionen, likväl som cellens microsystem, kan ändras
dynamiskt och reversibelt genom att lägga på en spänning över materialet. Förhoppningen
är att fundamental information från väl kontrollerade cell system in vitro ska leda till ökad
förståelse och framgångar inom utvecklingen av medicinska implantat och kretsar med
förbättrad läkning och beteende in vivo.
ACKNOWLEDGEMENTS
I wish to express my sincerest gratitude to all colleagues, co-workers and people I know
who have contributed to this thesis, directly or indirectly. In particular, I would like to
thank:
My supervisors and co-supervisors that have kept me on track throughout the whole of this
inspiring journey even though I got the opportunity to walk my own paths.
Magnus Berggren, my supervisor, for inspiring enthusiasm, creative curiosity, a vivid
imagination and never ending optimism.
Edwin Jager, my co-supervisor, for invaluable input on my texts during the years and for
all encouraging support and patience. Proost!
Nathaniel Robinson, my first co-supervisor, for inspiring discussions when I was new
within the scientific field.
Sophie Lindesvik, for knowing everything. You make our employment so much easier.
Agneta Richter-Dahlfors , for giving me the opportunity to work with biological systems.
Kalle Svennersten, for so many hours in front of the microscope and for long time of nice
collaboration.
Lars Faxälv, for fun experiments and for our afternoon project that became so much more.
Abeni Wickham, for heaps of samples and your infectious smile and energy.
Emilien Saindon, for the fun and energizing kick start into surface switches.
Co-authors of the included papers, for contribution of your expertise and to facilitate the
research within this boundary spanning field.
All the members of the Organic Electronics group that has come and gone throughout the
years. For all the discussions concerning scientific matters as well as everything else between
heaven and earth and for cheering up the weekdays and being patient with all my
humming. I do really like you guys. I would especially like to thank: Xiangjung, for all the
fun and interesting work and scientific discussions we had during the fiber work. I really
appreciated it. Oscar and Lars, for all fun conversations, support and being great friends
both at work and in private. Kristin, for all valuable discussions and support regarding
work and for all good laughs and strategic shopping company wherever we go. Klas, for
being such an amazing friend and for all fun and valuable scientific discussions and
argumentations. You will never beat me.
The personnel at Acreo, especially David Nilsson, Bengt Råsander, Anurak Sawatee,
Mats Sandberg, and Annelie Eveborn for all discussions and help in the lab and for
contributing with valuable experiments to this thesis.
Olle-Jonny Hagel and Anders Hägerström, for all the discussions and expertise during
processing problems.
Forum Scientium and Stefan Klintström, for all the knowledge and friendship and all
great research trips we have experienced.
Anna, Linda, Mari, Marie, Lisa, and Badbrudarna, for the support, many laughs and for
being such good friends for so many years.
My mum, dad , and family , for all love, and for encouraging, supporting, and always
believing in me.
Finally, I would like to thank Magnus and Malte for all the joy, patience and boundless
love.
Tack!
Maria, 17 september 2011
PAPERS INCLUDED IN THIS THESIS
PAPER I: CONTROL OF NEURAL STEM CELL ADHESION AND DENSITY BY AN ELECTRONIC
POLYMER SURFACE SWITCH
Carmen Salto, Emilien Saindon, Maria Bolin, Anna Kanciurzewska, Mats Fahlman, Edwin W.
H. Jager, Pentti Tengvall, Ernest Arenas, and Magnus Berggren
Langmuir 2008, 24, 14133-14138
Contribution: About half of the experimental work, except protein absorption studies and cell
experiments. Was involved in the final editing of the manuscript together with the co-authors.
PAPER II: ELECTROCHEMICAL MODULATION OF EPITHELIA FORMATION
Karl Svennersten, Maria H. Bolin, Edwin W. H. Jager, Magnus Berggren, Agneta RichterDahlfors
Biomaterials, 30, 6257-6264 (2009)
Contribution: About half of the experimental work, except cell experiments. Was involved in
writing the first draft of the manuscript and was involved in the final editing of the manuscript
together with the co-authors.
PAPER III: ACTIVE CONTROL OF EPITHELIAL CELL DENSITY GRADIENTS GROWN ALONG THE
CHANNEL OF AN ORGANIC ELECTROCHEMICAL TRANSISTOR
Maria H. Bolin, Karl Svennersten, David Nilsson, Anurak Sawatdee, Edwin W. H. Jager, Agneta
Richter- Dahlfors and Magnus Berggren
Advanced Materials, 21, 4379 - 4382 (2009)
Contribution: Most of the experimental work, except cell experiments. Wrote the first draft as
well as most of the manuscript and coordinated the final editing and submission of the
manuscript in cooperation with the co-authors
PAPER IV: NANO-FIBER SCAFFOLD ELECTRODES BASED ON PEDOT FOR CELL STIMULATION
Maria H. Bolin, Karl Svennersten, Xiangjun Wang, Ioannis S. Chronakis, Agneta RichterDahlfors, Edwin W. H. Jager, Magnus Berggren
Sensors and Actuators B, 145, 451-456 (2009)
Contribution: A majority of the experimental work, except some of the cell experiments. Wrote
the first draft as well as most of the manuscript and coordinated the final editing in cooperation
with the co-authors.
PAPER V: ELECTROACTIVE CONTROL OF PLATELET ADHESION TO CONDUCTING POLYMER
MICROPATTERNS
Maria H. Bolin, Lars Faxälv, Edwin W. H. Jager, Tomas Lindahl and Magnus Berggren
Manuscript
Contribution: All experimental work regarding material matters and being involved in some of
the cell experiments as well. Wrote the first draft and coordinating the writing.
CONFERENCE CONTRIBUTIONS NOT INCLUDED IN THE THESIS
POSTER: CONTROL OF CELL ADHESION USING CONDUCTING POLYMERS.
Maria Bolin, Kalle Svennersten, Emilien Saindon, Agneta Richter-Dahlfors and Magnus
Berggren, Material Research Symposium, 2007, San Francisco, USA
POSTER: COMPLEX PATTERNING OF ELECTROACTIVE SURFACES BASED ON CONDUCTING
POLYMERS TO CONTROL CELL ADHESION AND GROWTH.
Maria H. Bolin, Karl Svennersten, Edwin W. H. Jager, Agneta Richter-Dahlfors and Magnus
Berggren, Micronano System Workshop, 2010, Stockholm, Sweden
POSTER: COMPLEX PATTERNING OF ELECTROACTIVE SURFACES BASED ON CONDUCTING
POLYMERS FOR CONTROLLING CELL ADHESION AND GROWTH.
Maria H. Bolin, Karl Svennersten, Edwin W. H. Jager, Agneta Richter-Dahlfors and Magnus
Berggren, SPIE Optics + Photonics, 2010, San Diego, USA
ORAL COMMUNICATION: ADDRESSABLE MATRICES OF ELECTRO-ACTIVE SURFACES FOR
SPATIALLY CONTROLLED ADHESION OF PROTEINS AND CELLS.
Maria H. Bolin, Karl Svennersten, Edwin W. H. Jager, Agneta Richter-Dahlfors and Magnus
Berggren, Material Research Symposium, 2011, San Francisco, USA
OTHER CONTRIBUTIONS NOT INCLUDED IN THE THESIS
PATENT: METHOD AND SYSTEM FOR EXTRACTION OF CELL TYPES USING CELL SURFACE
RECEPTOR-SPECIFIC ATTACHMENT MOLECULES
WO2010072256
Berggren, Rolf Magnus; Richter-Dahlfors, Agneta; Bolin, Maria; Svennersten, Karl; Jager, Edwin;
Wang, Xiangjun
CONTENTS
1
Introduction to Organic Electronics ................................................................................ 1
2
Conducting Polymers ........................................................................................................ 3
Electronic Structure of Conjugated Polymers ....................................................................... 3
Charge Carriers .................................................................................................................... 6
Doping of Conjugated Polymers .......................................................................................... 7
Electrochromism ................................................................................................................ 10
3
Electrochemical Devices .................................................................................................. 11
Reduction and Oxidation ................................................................................................... 11
Devices............................................................................................................................... 14
Structure 1 ......................................................................................................................... 14
Structure 2 ......................................................................................................................... 15
The electrochemical transistor ............................................................................................ 16
4
Processing of Polymer Films and Microfabrication ....................................................... 19
Chemical Polymerization ................................................................................................... 19
Spin Coating ...................................................................................................................... 21
Barcoating .......................................................................................................................... 22
Vapor Phase Polymerization ............................................................................................... 23
Electropolymerization ........................................................................................................ 24
Micropattering ................................................................................................................... 25
5
Cell Adhesion ................................................................................................................... 27
The cell membrane and cell communication ...................................................................... 27
The extracellular matrix and cell adhesion .......................................................................... 29
Protein adsorption.............................................................................................................. 30
the role of conducting polymers in biomedical applications................................................ 31
Surface modifications for controlled cell adhesion .............................................................. 33
6
Major Results and Conclusions of the Thesis ................................................................ 37
Stem Cell Switch ................................................................................................................ 38
The Electronic Surface Switch to Regulate MDCK Cell Adhesion ..................................... 39
Gradient............................................................................................................................. 40
Nano-Fiber Scaffold Electrodes Based on PEDOT for Cell Stimulation ............................ 41
Electroactive Control of Platelet Adhesion to Conducting Polymer Micropatterns ........ 43
7
Future Outlook and Challenges in Organic Bioelectronics and Surface Switches ....... 45
References ................................................................................................................................ 47
The Papers ............................................................................................................................... 53
Introduction to Organic Electronics
1 INTRODUCTION TO ORGANIC ELECTRONICS
Organic electronics denotes the science and technology area in which hydrocarbon-based
semiconductors and conductors are utilized as the active materials in electronic devices.
These organic molecular compounds might also contain other elements such as sulfur,
oxygen, nitrogen, and iodine that together provide a wide diversity of material properties.
The possibility to design flexible, tailor-made and cheap electronic devices became a reality
thanks to a discovery made by Alan J. Heeger, Alan G. MacDiarmid and Hideki Shirakawa
in November 1976. Together they found that an organic polymer, known as polyacetylene,
could be doped through chemical oxidation to achieve metallic conductivity [1]. This
discovery was awarded the Nobel Prize in chemistry year 2000 “for the discovery and
development of conductive polymers”. Until that day, in 1976, plastic materials had always
been considered as insulating materials that can be utilized in a broad spectrum of
applications in our daily life. Today, polymer-based organic electronics is a well-established
research and technology field, which now also has reached a point where the first products
are found on the market [2]. Due to the possibility to easily tune their processing and
chemical functionality the polymers can be synthesized to express certain features and
properties dedicated for specific applications and also to control compatibility
characteristics for a variety of substrates. The combination of the unique processing features
of polymers and their (semi-)conducting characteristics enable a completely new type of
ultra-low cost devices with tailor-made device functionality. This suggests developing of
novel electronics on unconventional substrates, such as on flexible plastic foils, on paper
etc, at which traditional inorganic semiconductor technology come short [3].
For a long time polymer coatings have been used within biology and medicine to promote
biocompatibility of various surfaces and to create substrates for cell analysis. The branch of
microtechnology is beginning to impact on this field as well. [4] Research in this area has
1
Introduction to Organic Electronics
shown that conducting polymers can be used in sensors, as the cell communication
interface, and also to control cell adhesion [5-7]. The possibility to spatially control cellular
and sub-cellular entities using patterned surfaces as smart bio-adhesive and non-adhesive
areas has caused augmenting attraction over the past few decades and has contributed to
substantial progress achieved in a number of biology-related research areas [8]. Developing
a next generation of biosensors and bio-microelectronic devices involves addressing the
following issues: improving the control of the binding between biomolecules and the
carrying substrate, increasing the understanding of the interfacial mechanism during
adhesion, enhancing the chemical and physical stability of the bio-interface, and better
understanding of the impact of the electronic properties of the solid substrates. The
primary achievements of this thesis include taking use of fundamental features of
conducting polymers, in the form as organic bioelectronics surfaces, to control protein and
cell adhesion.
The thesis is divided into two parts, in which the first part provides an introduction and
review of the subjects and theory behind the topics of the science of the thesis. The second
part of the thesis is devoted to the achieved science and technology results, which are
summarized in five separate papers. A shorter presentation and discussion of the results are
given in the chapter Major Results and Conclusions of the Thesis.
The first chapters in the introduction part present an overview to the area of conducting
polymers and their physical and chemical properties in general. Thereafter, the classical and
basic principles of electrochemistry and electrochemical device structures are described as
well as the processing methods that have been used to fabricate the devices that are
included in the scientific reports included in the thesis. The final chapter reviews and treats
the area of surface-bio interfaces with the aim to give some clues about protein- and cell
adhesion as well as surface modifications for biological applications. The introductory part
ends with some overall conclusions drawn from the projects and finally a reflection for
future progress in the field. This part is based on a previous published Licentiate thesis,
Organic Electronic Surface Switches to Control Adhesion and Growth of Cells by Maria
Helene Bolin, Licentiate Thesis No. 1405, 2009 at Linköping University. All parts have
went through a slightly or major modification and this thesis is also complimented with
some new parts.
2
Conducting Polymers
2 CONDUCTING POLYMERS
Polymers are carbon based macromolecules that consist of many repeated molecular units, called
monomers, which builds up a long chain. The repeating units are coupled to each other by
covalent bonds. Polymers exist naturally in a wide range of systems in nature such as in
biomolecules (DNA, cellulose, collagen etc.). Polymers can also be manufactured by synthetic
processing. Since the materials can be chemically synthesized, properties such as solubility, optical
characteristics, reactivity etc. can be tailor-made for certain applications and processing
conditions. By varying the substituents attached to the conjugated system a vast array of different
functionalities can be achieved within the polymer chain. This designability and processability
make polymers an interesting group of materials for industry and research. A polymer with
extended conjugation will exhibit electronic conductivity when suitable doped with a counterion. The electronic conductivity of an organic semiconductor is influenced by many factors
including the choice of synthesis method, temperature, chemical structure, film morphology,
doping concentration, and dopant ion. There are many classes of organic conducting polymers,
where perhaps the most fascinating ones are the polyheterocycles such as polypyrroles,
polythiophenes and polyanilines, which typically exhibit good stability and reaches electrical
conductivity in the range from 1 to 103 Scm-1. Compare with metallic silver that has a
conductivity that reaches 5*105 Scm-1.
ELECTRONIC STRUCTURE OF CONJUGATED POLYMERS
The electron distribution in a material determines how bonds are formed and how they
interact with the surrounding matter, thus giving the material specific chemical, electrical
and optical properties. In organic polymers the backbone of the macromolecule consists of
covalently bonded carbon atoms. The carbon atom has 4 valence electrons available for
bonding which make the atom multifaceted since it has the ability to form single, double,
3
Conducting Polymers
and triple bonds. The distribution of electrons in space and time can in a simplified way be
described by atomic orbitals. When two carbon atoms are brought together the valence
electrons in their atomic orbitals are affected by the surrounding i.e. distorted and can be
described with hybridized orbitals. The configuration and characteristics of the formed
molecule are defined by those hybrid orbitals where only specific angle orientations are
possible between the orbitals. Here, two different polymer systems are selected in order to
explain the consequences of the bond orientation and hybridization. Polyethylene and
polyacetylene are examples of a saturated and conjugated polymer, respectively. The
physical properties of these two polymers are described below.
Plastics in general are good insulators, which in part can be explained by their chemical
structure and the properties of the bonds in particular. The bond characteristics of the
carbon atoms in an insulating polymer, such as in polyethylene, are hybridized including
the s- and px-, py-, pz-orbitals. This results in four equivalent sp3-hybrid orbitals and hence
four homologous σ-bonds to the surrounding atoms. The sp3-orbitals have a tetrahedral
arrangement with an angle of approximately 109.5° between the orbitals. Since all the
valence electrons in the carbon are strongly localized in these molecular bond orbitals there
are no free electrons that can be transported along the polymer chain, which is required for
electric conductivity of a bulk. The energy gap between the HOMO (the orbital with
highest energy level that is occupied with electrons) and LUMO (the orbital with the
lowest energy that is unoccupied) levels is invincible large in such materials (Figure 2).
The essential characteristic of conjugated polymers is the alternating single and double
carbon-carbon bonds along the polymer backbone. Generally, the chain is based on carbon
but there is a growing number of material systems including for example nitrogen and
sulfur atoms incorporated in the conjugated backbone. Since the backbone in transpolyacetylene is conjugated each carbon binds to three other neighboring atoms compared
to the four bonds formed in polyethylene. This results in three sp2-hybridized orbitals
which forms three σ-bonds that all are located in the same plane rotated 120° between each
other. When a carbon atom binds to three other atoms one valance electron is left unbound
in a non-hybridized p-orbital. These repeated atomic p-orbitals are oriented perpendicular
to the plane of the σ-bonds and the extension of the polymer backbone. The electrons in
these p-orbitals are not associated with any specific atom or bond which results in
delocalized electron clouds along the entire polymer chain. This builds up the conjugated
π-system and allows charge mobility along the polymer backbone and also in between
adjacent chains (Figure 1).
4
Conducting Polymers
Figure 1 Basic chemical and electronic structure of trans-polyacetylene. a) Conjugated segment of the
trans-polyacetylene backbone. b) Overlapping pz orbitals forming theπ -orbitals that are oriented
perpendicular to the σ-bond plane.
In a molecule with two sp2-hybridized carbon atoms, the interaction between the p-orbitals
results in two π bonds, one bonding (π) with lower energy and one antibonding (π*) with
higher energy. As the number of carbon atoms in the conjugated system increase there is a
proportional increase of the number of bonding π and antibonding π* orbitals. For an
infinitely long conjugated chain the energy difference between the levels decreases until
they merge into continuous bands. If all carbon-carbon bonds were equally long the
polymer would gain metallic behavior. Due to Peierls´ theorem, it turns out that the lowest
energy, and hence most stable configuration, of the conjugated system is reached when the
π-orbitals are unevenly distributed over the two carbon atoms due to the formation of
alternating single bonds (1.47 Å) and slightly shorter double bonds (1.34 Å). Thanks to
this bond alternations the π band is stabilized while the π* bands is destabilized resulting in
an energy gap, Eg, between the bands which corresponds to a semiconducting polymer,
which is shown in Figure 2. The π and π* bands are commonly described as the valence
band and conduction band in classical solid state physics.
The band gap in conjugated polymer films is not well defined since the film consists of
several chain segments that correspond to a distribution of band gaps. This is due to
variations in chain lengths of the polymers or defects related to morphological disorder,
chemical defects, interaction with the solution or carrying surface etc. The band gap value
is therefore an average of all polymer chains that together are building up the solid film or
solution. The energy gap in a conjugated polymer typically ranges from 1 to 4 eV [9].
5
Conducting Polymers
Figure 2 Band structure for a conductor, semiconductor and insulator.
CHARGE CARRIERS
The conductivity of a solid state semiconducting conjugated polymer typically increases if
the temperature increases. This is in part due to an enhanced hopping probability within,
and between, the polymer chains of the film. In organic materials there is a strong coupling
between the electronic and molecular structure of the material. Introduced charges
(electrons or holes) in combination with distortion of the polymer structure define the
properties for the charge carriers of the material [10].
Depending on the associated disorder different kinds of charge carrier can be formed: a
soliton, a polaron, or a bipolaron. Further, depending on if the polymer has a degenerated
or non-degenerated ground state its charge carrier species are either solitons or polarons
[11]. A degenerated ground state implies that there is zero difference in energy if the
position of the double bond and the single bond is interchanged. The two alternatives are
equally likely to be found and the boundary between these two is called a soliton. The
soliton will result in an electronic level in the middle of the bandgap. Trans-polyacetylene,
previously mentioned, exemplifies a polymer with a degenerated ground state. Most other
conjugated polymers have a non-degenerated ground state. This means that there is only
one energy ground state and that the single and double bonds cannot be interchanged
without an expense in energy, causing a destabilization of the polymer chain. An example is
the frequently used polythiophene family where a bond distortion of the aromatic structure
will result in a quinoid structure causing a higher energy.
In non-degenerated conjugated systems a geometric perturbation, caused by the
introduction of a charge, induces a state of higher energy. This perturbation, which can be
seen as an interruption of the π-bonds, is referred to as a polaron and is distributed over the
length of a few monomers. Since the polarons can move along the polymer chain, or jump
in between different chains, they represent the electronic conductivity of the conjugated
polymer bulk. When a polaron travels within the polymer it can be seen as a domain of a
delocalized charge that reconfigure the conjugated structure while it is transported. If the
concentration of polarons is increased in the polymer system it is energetically favorable to
6
Conducting Polymers
form a so-called bipolaron, which is a double-charged polaron species. The band diagrams
of polarons are illustrated in Figure 3.
Figure 3 Band diagrams for polarons and bipolarons
DOPING OF CONJUGATED POLYMERS
Conjugated polymers are generally poor electronic conductors or insulators, due to a low
concentration of free charge carriers in the pristine state. Upon doping the polymer, mobile
charge carriers are generated and the conductivity can be modified from as low as 10-10 to
104 Scm-1. Hence, semiconducting conjugated polymer can be doped close to a
conductivity level of those of true metals [1]. Generation of charge carriers can be achieved
by for instance a redox reaction [12]. The doping can be made in different ways, where
chemical and electrochemical doping are commonly used. Oxidative (p) doping is more
commonly used and studied in conjugated polymers since reductive (n) doping requires
polymer materials that are typically unstable in ambient conditions. Such reaction is not
localized at a specific atomic site along the polymer but rather delocalized over a number of
conducting polymer monomers [13]. Simplistically, p-doping (oxidation) can be viewed as
the creation of mobile holes within the valence band, while n-doping (reduction) as the
addition of mobile electrons in the conductivity band. These modifications change the
electronic structure of the polymer system and hence creates mid gap states, which can be
seen in Figure 3. By removing an electron (p-doping) from the conjugated polymer a
positively charged polaron is generated. A bipolaron is established by removing an
additional electron. Illustration of the geometrical structures of a positive polaron and
bipolaron in thiophenes in Figure 4.
7
Conducting Polymers
Figure 4 Geometric structure of a positive polaron and a further oxidized bipolaron.
In the doped system the polarons and bipolarons are charge-neutralized by counter ions
distributed in the polymer system, as shown in Figure 5.
Figure 5 Doping of the conjugated polymer PEDOT. A bipolaron is established when two electrons are
removed. The Tosylate acting as a counter ion to maintain charge neutrality within the polymer
system.
8
Conducting Polymers
In a chemical doping process electrons will spontaneously be transferred from the HOMO
levels of the conjugated polymer to the oxidizing agent (dopant), which is driven by a
mismatch of the energy levels. P-doping occurs and a positively charge and mobile polaron
(radical cation) is formed. If the HOMO of the dopant is close to the LUMO of the
polymer, an electron is transferred from the dopant to the host which results in a negative
polaron, i.e. n-doping. The reactive species that are responsible for the doping of the
conjugated polymer will be charged (anion or cation) and may in some cases act as a doping
agent as well. [14]
To maintain an electrochemical doping the polymer needs to be in contact with a working
electrode and an ionically conducting electrolyte which in turn is connected to a counter
electrode. The injection of charges from the working electrode is controlled by an applied
potential difference between the electrodes while ions from the electrolyte are acting as
counter ions resulting in a charge neutralization of the system.
Introduced charges can then migrate along the polymer film, under the influence of an
electric field or via diffusion. This cause local distortion of the geometry of the molecular
structure and travels throughout the bulk, i.e. an electrical current is generated. The
conductivity of the material is confined by the number of excited or injected charge carriers
and their associated mobility [15]. Due to the attraction between an electron and
neighboring nuclei the electron starts to move along the polymeric chain or between chains,
so called electron hopping. The charge mobility is limited by disorder in the polymeric
system and Coulombic interactions between electrons and holes. A relatively high density
of traps for charges in combination with a high energy barrier for charges to migrate from
one chain to another, results in small intrinsic bulk conductivity. If one compares doping of
an organic semiconductor, such as a conjugated polymer, with the doping of inorganic
semiconductors, such as Si, the organic material requires approximately 25% (by volume)
of dopant concentration to increase the conductivity so that the material can be used as a
conductor. In comparison, only a few ppm of doping concentration is needed to
considerably impact the conductivity of for instance Si. Further, the doping process in
organic systems is caused by ionic interaction while in inorganic crystals the doping process
is due to that the dopant is included in the crystal lattice via covalent bonds.
For solid state films in particular, the size of dopant is of great importance in order to
control the function of for instance a device. A large dope ion will be physically trapped
within the polymer film and becomes more stably integrated in the conjugated polymer
film while a smaller dopant may diffuse out of the polymer bulk and will be exchanged
with other ions in the surrounding environment.
9
Conducting Polymers
ELECTROCHROMISM
Many conjugated polymers absorb light in the visible region, and are colored. Thus, their
optical band gap agrees with visible wavelengths (λ = 400-800 nm corresponding to a band
gap energy of 1.5-3.0 eV). Electrochemical switching of the conjugated polymer causes
changes of the electronic structure, which then control the absorption of light (Figure 6).
PEDOT: Tosylate [16], the prime conjugated polymer system of this thesis, belongs to the
group of electrochromic polymer materials exhibiting a visible change of color from pale
blue to dark blue upon electrochemical doping mechanisms [17]. By doping a conjugated
polymer, new energy levels are created within the band gap due to generation of polarons in
the polymer bulk. In its original pristine state, i.e. in its partly oxidized state, the PEDOT
material appears as pale with a light-blue hue and is fairly transparent. When the polymer is
further oxidized it becomes even more transparent. Conversely, reduction of the pristine
state towards a more neutral PEDOT film decreases the number of polaronic and
bipolaronic states within the band gap and hence modifies the optical absorption from the
near-infrared region towards the visible region. This will be seen as a clear chromatic switch
as the film now becomes dark blue and the absorbance maximum occurs at around 640
nm. Electrochromic organic materials have several feature advantages in applications related
to color switching.
Figure 6 Adsorption emission energy diagram
10
Electrochemical Devices
3 ELECTROCHEMICAL DEVICES
The physical material properties such as color, conductivity, and surface energy can be controlled
by electrochemical doping in a conjugated polymer. These features make these materials
promising and unique for various electrochemical device applications. As mentioned in the
previous chapter, the doping level of conjugated polymers can be altered through redox reactions
which can be carried out in an electrochemical cell [18]. All experiments included in this thesis
have been performed utilizing an electrochemical device configuration based on the structure 2
or the three-terminal electrochemical transistor device architecture. Those device architectures,
and their particular device characteristics, will be explained and treated in the present chapter.
REDUCTION AND OXIDATION
An electrochemical reaction is a chemical process regulated by a potential applied to
electrodes that involves an exchange of electronic charges and material at the electrodes.
Conducting polymers generally exhibit both oxidative and reductive electrochemistry
relative to the neutral states. The redox process can usually be reversed, although the
oxidized and/or reduced states of many polymers have limited stability. [19] Cyclic
voltammetry (CV) is a technique to characterize a material’s redox properties such as
potential levels, stability and reversibility. The average of the oxidation and reduction
potential for p- and n-doping can provide estimates of the energies of the HOMO and
LUMO bands of the conjugated polymer, and its corresponding band gap [19]. The
electrochemical cell is often divided into two half cells each composed of one electrode and
the common surrounding electrolyte. The two cells are connected by the electrolyte (ionic
conductivity) and wires (electronic conductivity) which provide that both ions and
electrons can be transported to the sites of reactions and the two electrodes (Figure 7).
11
Electrochemical Devices
Figure 7 Sketch of electrochemical cells. a) The cell consists of two electrodes that are connected,
through the salt bridge enabling ion transport, and electronically conducting wires. Anions (-) are
electrostatically attracted to the positive anode (+) and cations (+) to the cathode (-). b) A threeelectrode set up.
If a potential difference is applied between the two electrodes ions of the electrolyte will
start to migrate in the electric field, diffusion and/or drift, in the direction towards the
electrode in which they are involved in electrochemical reactions. This ionic charge
transport results in a net flow of charge within the electrolyte. The electrochemistry of
conducting polymer films involves ion expulsion or insertion to maintain electroneutrality,
when the film is cycled between the doped and undoped states, both types of ions will be
involved to some extent. [19] The electrodes, which are electronic conductors, provide or
receive electrons in the system to balance the ionic charges that are collected at the electrode
surface. To achieve an electrical current the reactions at each half-cell, respectively, need to
be complementary.
In a conjugated polymer one typical half-cell reaction will result in a positive polaron in
case of oxidation (electrochemical p-doping) as shown in reaction (1).
P0 + X- →P+: X- + e-
(1)
The P0 corresponds to the neutral polymer and X- is the anion. The anion enters the
polymer film to charge neutralize the positive charge that is formed when an electron is
removed. This reaction can be reversed which means that the oxidized polymer can be
reduced to the neutral state or to reduced state (n-doping). This will be a similar reaction
12
Electrochemical Devices
but instead cations will react with the polymer film to charge compensate for the added
electrons as in reaction (2).
P0 + M+ + e- →P- : M+
(2)
As mentioned in the previous chapter p-doping of a conjugated polymer is the most
common type of electrochemical doping process since it involves and produces more stable
species. As compared to n-doping of conjugated polymers. Films of PEDOT: PSS include
immobilized anions (here PSS-) where mobile cations diffuse or migrate in and out of the
film. In its pristine state this material is partially oxidized and the conducting polymer can
thus be further oxidized to even further increase conductivity or it can be reduced to the
neutral semiconducting state, see reaction (3). This reaction is reversible in both directions
and can be described as
PEDOT+ : PSS- + M+ + e- →PEDOT0 + PSS- : M+
(3)
The conducting polymer used in this thesis is PEDOT and doped with the negative iron
(III) tosylate (or p-toluenesulfonate). The material system behaves similarly to the wellknown PEDOT: PSS in electrochemical cells (Figure 8).
When a potential is applied between the electrodes, ions start to migrate in the electric field
or diffuse which results in that a redox reaction takes place within the electrodes of the cell.
13
Electrochemical Devices
Figure 8 Schematic cross section of an electrochemical cell, including PEDOT: Tosylate as the electrode
material, in a lateral configuration. The cell consists of two adjacent electrodes of the conducting
polymer that are separated by an electronically insulated area. The two electrodes are in connection
by the ionically conducting electrolyte. When potential is applied redox reactions takes place within
the two electrodes.
DEVICES
From a practical application point of view, it is sometimes desired to create an
electrochemical cell that is easy to manufacture and that includes all parts integrated into a
self-supporting monolithic component. Due to common process and manufacturing
methods the devices have often a lateral configuration, i.e. the electrodes are in the same
plane with the electrolyte on top, as illustrated in the example shown in Figure 8. The
electrolyte is an ionically conducting material and it can be represented by a liquid, solid, or
gelled phase depending on the choice of materials and applications. In order to define
electrodes, the polymer film needs to be patterned. This can be done in several ways such as
by simply removing conducting material with a scalpel or by using an electrical method
involving over-oxidation of the PEDOT phase. Over-oxidation by chemical (or
electrochemical) means destroys the conductivity of the material by breaking the
conjugation along the polymer chains [20]. For smaller features this can be done by simply
applying the over-oxidation solution by a pen or using screen printing meshes using the
squeegee as the one electrode and the PEDOT electrode (anode) as the other. For even
smaller dimensions photolithography may be a more suitable way to pattern the electrodes.
The PEDOT-based electrochemical devices can be classified with respect to their different
geometries. Some of these designs, called structure 1, structure 2 and the EC transistor will
be described here.
STRUCTURE 1
The simplest design, called structure 1, is an architecture in which the conducting polymer
film is made as one electrode covered by an electrolyte (Figure 9). In this configuration
both ion and electron transport occurs in parallel. Since the conjugated polymer film is a
continuous piece electronic charges will be conducted through the electrode material.
14
Electrochemical Devices
When the voltage is applied, a potential difference will be established along the polymer
film due to the resistivity along the thin film. The negatively biased side of the film will
start to reduce while the positive side will become further oxidized when an electrolyte is
being placed on top. Ions from the electrolyte will work as the counter charges and will
thus move in and out from the electrode film. A redox- as well as a color gradient will
appear along the polymer film.
Figure 9 Schematic cross section of an electrochemical cell in a structure 1 design. Both electronic and
ionic conductivity will occur along the polymer film.
STRUCTURE 2
In structure 2, given in Figure 8, the conjugated polymer is divided in two adjacent
electrodes separated by an electronically insulating line. The electrochemical behavior in
this device is equivalent to the simple two-electrode electrochemical cell given in Figure 7.
Since the two electrodes are electronically isolated from each other the only possible path
for charges is through the electrolyte. As the voltage is applied, the electronic current in the
polymer is converted into ionic current via the electrolyte. This occurs thanks to
electrochemistry, which results in that the negatively addressed electrode becomes reduced
while the positively addressed one becomes oxidized. When the positively addressed
electrode oxidizes further beyond its semi-oxidized state, the number of positively charged
bipolarons/polarons increases and hence gives rise to an increased level of conductivity. To
balance the reaction anions have to migrate into the polymer film. A complementary
electrochemical reaction will take place on the opposite electrode. The modified number of
bipolarons results in a change of the optical absorption which becomes evident as a
chromatic switch at the two electrodes. As the biasing of the electrodes is interrupted, e.g.
by disconnecting the wires, the redox state at the electrodes, respectively, remains for a
certain period of time since the electrodes are electronically isolated from each other. The
surrounding factors such as the oxygen content inside the electrolyte and in the atmosphere
will eventually force the two electrodes back to their semi-oxidized pristine state.
15
Electrochemical Devices
THE ELECTROCHEMICAL TRANSISTOR
In an electrochemical transistor both ions and electrons serve as charge carriers. The
potential difference that is required to drive the device depends on which electrolyte that is
used and on the specific device configuration. But the driving voltages usually are found to
vary between one and four volt. The operation principle of the electrochemical transistor is:
the transistor drain-source current is modulated by the redox state within the conjugated
polymer transistor channel, which is dictated by the gate voltage.
The three-terminal transistor is built up from a combination of structure 1 and structure 2,
as illustrated in Figure 10. In this conjugated polymer-based electrochemical transistor a
continuous potential gradient is established along the channel, i.e. structure 1, between the
two electrodes (called drain and source). At the positive biased side (source) the cannel will
become more oxidized while at the more negatively biased side the channel closest to drain
will be more reduced. Reduction of the polymer film gives rise to a local increase of the
impedance in the channel. Hence, a larger resistance to electrical currents passing within
the film is established. This current versus voltage feature of structure 1 is responsible for
the current saturation of the drain-source current (the so-called pinch-off behavior) at high
drain-source voltages [18, 21, 22]. The channel between source and drain is ionically
bridged via an electrolyte to an electronically separated third electrode, which defines the
gate electrode, in order to create the transistor modulation of the channel impedance. This
geometry is in principal the structure 2 configuration, as shown in Figure 8. Before any
voltage is applied, the conducting polymer in the electrochemical transistor is in its pristine
state, which for PEDOT implies that the material is partially oxidized within both the
channel and gate. As the gate is positively addressed, as compared to the drain contact
potential, homogenous reduction of PEDOT inside the channel towards a relatively more
neutral state occurs, as shown in reaction (4).
PEDOT+ :Tosylate- + e- + M+ →PEDOT0+ M+:Tosylate-
16
(4)
Electrochemical Devices
Figure 10 Top view of an electrochemical three-terminal transistor design. The drain and source
electrodes are denoted D and S respectively and the gate is indicated with G. The electrodes can be
made of conducting polymers or inert metals. The electrolyte is an ionically conducting material
and can be liquid, solid or gel-like depending on applications.
As the gate electrode is changed the electronic current between drain and source is
modulated since the reduction level inside the channel is altered. The associated typical
current-voltage characteristics for a p-doped polymer material are given in Figure 11. Here,
the transistor operates in the first (VD > 0, ID > 0) and third (VD < 0, ID < 0) quadrant,
which means that the drain-source voltage is swept from negative to positive voltages [23].
When a zero gate voltage is applied the transistor has a linear behavior in the first quadrant,
corresponding to the structure 1. An increase in drain voltage forces the polymer in the
channel to become more conducting. In the third quadrant the transistor reaches a current
saturation behavior. The reduction in the channel is increased which is promoted by the
fixed gate potential.
17
Electrochemical Devices
Figure 11 IV characteristics at different gate voltages in an electrochemical three-terminal transistor.
From ref D. Nilsson et al 2002 [18]. The p-doped electrochemical transistor operates in the first
and third quadrant while the drain-source voltage is swept in a range from negative voltages to
positive voltages.
18
Processing of Polymer Films and Microfabrication
4 PROCESSING OF POLYMER FILMS AND MICROFABRICATION
Conducting polymers have the advantage that they can be processed on flexible substrates, to form
organic plastic foil coatings, with low-cost techniques such as spin coating, printing, and bar
coating. The choice of fabrication method depends on the character of the substrate and the film
quality requirements.
All experiments included in this thesis have utilized electrochemical devices in which chemically
polymerized poly (3, 4-ethylenedioxythiophene) (PEDOT) doped with iron (III) tosylate, see
Figure 19, has been used as the conducting polymer. Due to its rigid conjugated bond structure
PEDOT is an environmentally and thermally stable and highly conducting polymer that is
possible to process with a broad variety of methods [24-26]. Its polymer properties can be tailored
by including different dopants [27]. The polymer film can be formed on substrates such as
plastics or glass and the shape of the substrate itself is not a limitation. Since applications require
different kind of substrates, a variety of fabrication methods have been used. In this chapter a
brief orientation of the coating and synthesis methods will be presented as well as the patterning
process to define electroactive areas of the PEDOT: Tosylate.
CHEMICAL POLYMERIZATION
Polymerization is a process of reacting monomer molecules together in a chemical reaction
to form polymer chains, as shown in Figure 12. This can be achieved by different methods
including condensation polymerization or addition polymerization. During condensation
polymerization the process is driven by the loss of small molecules such as water while the
addition process requires a reactive radical, cation or anion intermediate state during the
synthesis. These polymerization methods are powerful tools to synthesize different kinds of
conjugated polymers in large amounts. [28]
19
Processing of Polymer Films and Microfabrication
Polymer synthesis by oxidative polymerization of the monomer is generally a facile process
that can be achieved chemically by mixing a soluble monomer, an oxidation agent, a
dopant and if required an inhibitor in a solution prior to casting onto a substrate. It is well
known that choice and concentration of dopant and oxidation agent, solvent, temperature
and time are conditions that have great impact on the film properties such as electrical
conductivity, morphology, and stability. To achieve a homogenous highly conductive film
it is important to inhibit potential agglomerate formation of polymer chains in the solution
prior to casting. By adding a strong base to the oxidation solution any possible acidicpromoted side reactions that may result in poor conjugation of the polymer and hence poor
film conductivity, are inhibited [29, 30]. The chemical polymerization can be combined
with spin coating, bar coating and printing methods to achieve a thin and even conducting
polymer coating on a substrate. After casting the substrate is transferred to a heated
environment in order to facilitate the polymerization process and evaporation of solvents.
By controlling the temperature during the polymerization process the physical properties,
such as film thickness, conductivity, optical properties (transmittance) and film coverage
can be controlled [31, 32]. After the polymerization process the polymer film has to be
carefully cleaned in order to remove non-reacted monomers, oxidant, salt crystals, and any
side products before it can be dried and used in devices. This may be done by sequential
washing step using organic solvents such as butanol, isopropanol and DI water.
Another way to synthesize a conducting polymer film is by electrochemical synthesis.
Common conducting polymers such as PEDOT, PPy and PANI can be synthesized both
chemically and electrochemically.
20
Processing of Polymer Films and Microfabrication
Figure 12 Scheme for oxidative polymerization mechanism through the coupling of radical cations
SPIN COATING
A well established method, especially in the semiconductor industry, to apply thin films
from a solution is spin coating. Here the solution, of dissolved polymers or as in our case
the oxidant-monomer solution, is cast on a rotating substrate, such as a silicon wafer. The
substrate onto which the film will be applied is attached onto a chuck using vacuum, as
shown in Figure 13. The oxidant-monomer solution is applied onto the substrate, which
then is rotated at a desired speed. The spinning motion distributes the polymerization
solution over the entire substrate, resulting in a homogenous polymer film. The spin speed,
polymer concentration, and characteristics of the solvent affect the thickness and
morphology of the resulting polymer film. This method can be used on flat and non-flat
substrates such as the entire inside of cell culture dishes. To achieve a well-formed film, of a
desired smoothness, it is necessary that the polymerization reaction does not occur faster
than it takes for the mixture to spread along the surface. One practical drawback of this
manufacturing method is that the polymerization can already initiate in the mixture, which
causes a short pot lifetime and may result in uneven film thickness and rough film
morphology. This can be avoided by spin casting the monomer and oxidant sequentially
onto the substrate [33]. By adding a strong base the rate of pre-polymerization can be
controlled, as described above. Using this base approach to polymerize films, spin coating
results in a thin and homogenous film, as shown by De Leeuw et al. [29]. Spin coating is a
deposition method that can be applied to large areas although in chemical polymerization it
may be difficult to reproduce homogenous films and it produces a lot of waste materials.
21
Processing of Polymer Films and Microfabrication
Figure 13 Simplified sketch of the chemical polymerization using spin coating setup. The added
monomer solution is distributed onto the substrate due to rotating movement.
BARCOATING
From a manufacturing point of view, there is a desire to coat large area substrates. This has
encouraged engineers to develop suitable large area processing methods. Barcoating is a
deposition method that utilizes an all-liquid process, in the same way as for spin coating,
where all the polymerization components are mixed into one solution prior to the coating
step. The substrate is coated by a horizontal movement of a wire wound rod that distributes
the solution along the substrate (Figure 14). The velocity of the moving the bar as well as
the characteristics of the solvents influence the physical properties of the polymer film. In
similarity to the spin coating processing it is required to control and suppress any possible
pre-polymerization that might occur in the mixture before actual coating. The bar that is
used is wrapped by a tapering thin metallic thread and as the thread gets smaller, the film
becomes thinner, smoother, and more homogenous. This coating achieved by movement of
a rod may be carried out both manually as well as by an automatic film applicator. In
general, this bar coating method is an easy way to rapidly coat larger surfaces even though it
is restricted to flat substrates.
22
Processing of Polymer Films and Microfabrication
Figure 14 Simplified sketch of the bar coating setup. The added monomer solution is distributed
along the substrate by the bar moving horizontally over the substrate.
VAPOR PHASE POLYMERIZATION
Vapor phase polymerization (VPP) is a synthesis method that can be used to form thin
homogenous, highly conducting polymers film on various substrates and geometries and
was originally described by Mohammadi et al. [34]. It is a chemical polymerization method
similar to the above described methods. An oxidant solution is applied onto a clean
substrate using a solvent-coating method such as using spin or dip coating. Next, the
substrate is exposed to a monomer vapor (Figure 15). As a contrast to the previous
mentioned casting methods the VPP method is possible to implement independently of the
monomer solubility [35]. Since this method is based on the use of a vapor, the geometry
and topography of the substrate to be coated, is less critical as compared to many other
coating methods [36]. The substrates can for instance be foils, fibers, dishes, or tubes, and
may be made of glass, polystyrene, polyvinylchloride, polyethyleneterephthalate, etc. On
the other hand, this method suffers from some major drawbacks; it is time consuming and
is less suitable to use on very large substrates or surfaces.
23
Processing of Polymer Films and Microfabrication
Figure 15 Simplified sketch of the vapor phase polymerization (VPP) setup. The precoated substrate is
placed in a temperate-adjusted water chamber where a controlled environment facilitates the
oxidative polymerization process. A smaller beaker with the monomer and substrate is placed inside
the larger chamber to give a homogenous covering of polymer film onto the substrate.
ELECTROPOLYMERIZATION
Another way to manufacture thin homogenous conducting polymer films is to use an
electropolymerization procedure, as can be seen in Figure 7. This procedure gives good
control of the reaction conditions and enables the use of a wide range of molecular dopants.
This synthesis is performed in an electrochemical cell comprising solution of the monomer,
electrolyte (dopant), solvents and two or three electrodes, as illustrated in Figure 16. By
using a potentiostat a potential (or current) is applied to the conducting substrate (the
working electrode, WE). The electrode material or substrate can for instance be a metal or
another conducting polymer. In this method the monomer is present in the electrolyte and
when a potential is applied, oxidation of the monomers creates reactive radicals. These
radicals can combine to form dimers, which in turn form oligomers and finally polymer
chains are formed that precipitate on the WE surface. This process will result in a film that
covers the part of the electrode that has been in contact with the electrolyte. A second
electrode (the counter electrode, CE) is used to supply the other half reaction, hence
completing the electrochemical circuit (Figure 16). Preferably, a third electrode, i.e. a
reference electrode may be used to achieve a well-defined potential on the working
electrode. The properties of the electropolymerized polymer films are sensitive to the
electrode material and the different deposition conditions, including electrolyte, solvent
and applied potential. [29, 33, 37, 38] The polymerization process is dependent on
diffusion, adsorption, and charge transfer [39]. Electropolymerization is a widely used
method to manufacture thin films. However, the process requires a conducting substrate,
24
Processing of Polymer Films and Microfabrication
such as a metal, carbon electrode, or another conducting polymer, thus limiting the choice
of substrates and device configurations [2].
Figure 16 Electropolymerization at the electrode. The monomer oxidizes at the electrode surface and
become a radical cation which quickly forms polymer chains together with other radical cations at the
electrode.
MICROPATTERING
The utilization of microscale technologies traditionally used in microelectronics is also
useful in the development of biomaterials and devices since they enable fabrication of small
features at a low cost in a reproducible manner [40]. Polymers can be patterned using a
variety of patterning techniques [41]. Over the past three decades, photolithography has
been one of the main methods used for patterning surfaces of functional polymers.
Photolithography involves selectively exposing of a mono- or polymer coated surface to a
high energy beam, typically UV-light, through a photo mask that contains a positive or
negative image of the pattern to be transferred to the substrate. [42] The irradiation results
in a photopolymerization, photocrosslinking, functionalization or decomposition reaction
in the exposed areas. Photolithography enables the fabrication of high-resolution patterns
in the range from micrometers to sub -100 nanometers dimensions. This method is suitable
for large area patterning with very good alignment to previously patterned features on the
substrate. There are some challenges for patterning conjugated polymers without
destroying their properties. Direct photo irradiation may cause polymer degradation due to
weak π -bonds and hence affect the electronic and optical properties of the material [43,
44]. A drawback is that it requires clean room facilities, rigorous experimental protocols
and rather expensive equipment.
25
Processing of Polymer Films and Microfabrication
To overcome the requirement of expensive clean room equipment, new microscale
technologies to fabricate patterning of conducting polymers have appeared. One
increasingly used method is the soft lithography were an elastomeric stamp is used to stamp
or mold materials [45]. Micro- and nanostructures are achieved by curing a pre-polymer on
a well-defined prefabricated master. By using a mask created by photolithography the
acquired patterns can get resolutions down to tens of nanometers. [46] Soft lithography can
be used to fabricate microfluidic channels and to introduce topographical features, as well
as control spatial distribution of molecules on a surface. [47, 48]
After the surface has went through lithography patterning methods, a number of
subtractive and additive processes are required to develop the actual structures which means
that materials are either removed or added to the substrate. Generally spoken etching can
be seen as a transfer of patterns, through a patterned protective layer, by chemical or
physical removal of material from the substrate. Reactive ion etching (RIE) is one of the
most commonly used subtractive dry etching processes for microfabrication. Additive
techniques involve that solids are deposited onto a substrate. This may be done from
liquids, plasma, gas or solid state where two commonly used methods that uses gas phase
are chemical vapor deposition (CVD) and physical vapors deposition (PVD).
Metalloorganic (MOCVD) is a kind of CVD method while evaporation and sputtering are
examples of PVD techniques. [42, 49]
Another way to pattern the conducting polymer without removing any material is to
inactivate defined parts of the film. This may be achieved by overoxidization of the
conducting polymer resulting in a non-reversible loss of conductivity due to breakage of the
conjugation in the backbone. [20] The overoxidization can be done by exposing the
substrate to chemicals such as a sodiumhypochloride solution through patterned gaps in a
protecting layer or by using a screen printing technique. An even faster method which also
may require less hazardous reagents is the use of electrochemical overoxidization by a
plotter pen, screen printing or by dipping in an electrolyte while applying potential
between the substrate and a counter electrode. The advantages of utilizing over oxidization
methods is that the conducting films become more homogenous and smaller topographical
steps are introduced than when using an additive or subtractive method.
Other methods that have been developed for the patterning of conducting polymers but
were not used in this thesis are among others lift-off, ink-jet, electropolymerization, and
micro-contact printing [42].
26
Cell Adhesion
5 CELL ADHESION
In order to survive most eukaryotic cells need to interact with their surroundings by
adhesion either to adjacent cells or a surface. While adhered to the surface they can start to
grow, change their morphology, differentiate and communicate with other cells. When cells
are attached to a surface they interact with each other to form tissues possessing advanced
functionalities. Examples of such tissues are the blood vessels, the skin etc. The scientific
field exploring cell adhesion onto artificial surfaces is giant and complex due to the diversity
of cell types, adhesion proteins and variety of surfaces that are involved in this process. In
particular the conformational changes in an adsorbed protein are considered to be one of
the most important aspects affecting cell response. Polymer systems are soft and have an
organic composition that chemically resembles of the materials in our body which makes
them an interesting material for biological applications. Conducting polymers do not only
have the biocompatible aspects of polymeric structures but that they have the properties to
function as a traditional metal or inorganic semiconductor in order to introduce electric
fields as well as electrical stimuli to cells and tissues. The work of this thesis has primarily
been directed towards electronic control of protein and cell adhesion. This chapter will give
a brief background of protein adsorption, surface properties and modifications responsible
to affect cell adhesion.
THE CELL MEMBRANE AND CELL COMMUNICATION
The eukaryotic cell is defined by a double layer of lipids which is called the cell membrane.
This membrane acts as a boundary between the advanced machinery inside the cell and the
surrounding world outside the cell (Figure 17). Due to its semipermeable functionality it
also controls the transportation in and out from the cell. Besides the lipids the membrane
27
Cell Adhesion
also hosts other molecules such as membrane proteins that are involved in ion transport,
cell communication and cell adhesion. [50, 51] One example of the membrane proteins are
the transmembrane receptors called integrins that interact with the extracellular matrix and
the cell to cell binding. [52] The family of integrins is very diverse and many cell types can
express several kinds of integrins at the cell membrane [53]. A similarity between all
integrins is that they are heterodimers which means that they consist of two different
subunits denoted α and β.
The cell membrane also acts as an anchoring point for the cytoskeleton which provides the
shape of the cell and enables cell motion. The cytoskeleton is composed of three different
kinds of filaments, microfilaments, intermediate filaments, and microtubules. The
microfilament, also known as actin filaments, is the most studied form and is mainly
responsible for cell morphology and participates in cell-cell or cell-extracellular matrix
interactions through the integrin receptors.
Cell communication is very complex and involves both inter- and intracellular signaling
pathways. The cells can signal to each other in many different ways, mainly dependent on
the distance between the signaling cell and the target cell. [54] Cell signaling can be
triggered by cell-cell interactions or cell-extracellular matrix interactions. The
communication is achieved by the release and detection of signaling molecules such as
hormones and neurotransmitters. A molecule that is specifically recognized by a receptor is
called a ligand and can transport the signal between neighboring cells over a short distance
as well as between remote cells within for instance the human body to elicit a physiological
response. When it comes to nerve signaling this is induced by synaptic signaling, i.e.
changes in the electrical potential across the cell membrane.
28
Cell Adhesion
Figure 17 Sketch cellbinding compartments adhesion to ECM
THE EXTRACELLULAR MATRIX AND CELL ADHESION
Outside the cell membrane, but still associated with the cell, an active and dynamic
extracellular matrix (ECM) creates a complex environment. This matrix can be seen as a
three-dimensional fibrous connective tissue formed by biomolecules (Figure 18). Due to its
composition the ECM functions as a guiding scaffold for cell adhesion, morphogenesis, and
plays an important role when injury reparation takes place inside a tissue [55]. The ECM
mainly consists of specialized proteins such as fibronectin and laminin, structural
supporting proteins such as collagen, signal molecules and a wide range of growth factors
[56]. ECM proteins interact directly with the cell surface receptors to initiate signal
transduction pathways. The amount and type of these components vary depending on the
kind of tissue as well as the developmental stage of the organism. Hence, modulation of the
ECM, by changing its properties and activity has profound effects on its function and the
consequent behavior of cells living on or within it. [57] The basement membrane that is a
part of the ECM is mainly composed of collagen and its major role is to regulate the cell
polarity and creates an adhesion sheet for cells, such as epithelial cells. The fibronectin
proteins regulate the cell adhesion and act as an intermediate between collagen fibers and
the integrins of the cell membrane. When the extracellular domain of an integrin binds to
fibronectin an intracellular signaling event is initiated. The strength of cell adhesion,
partially due to the binding between integrins and fibronectins, is affected by the chemical,
physical and mechanical properties of the environment. When a cell is seeded onto an
artificial surface, proteins such as fibronectin will act as an anchor between the cell and the
surface. When the cells have adhered to the surface their actin filaments activates through
integrin signaling, which further affects the cell morphology and organization along a
29
Cell Adhesion
carrying substrate [58]. The ECM may play a more complex role in cell biology than solely
acting as an anchor for the attachment. The ECM presents both chemical signals and
topographical mechano-transductive cues to the surrounding cells that will affect the
attachment, proliferation, polarization as well as migration [59, 60]. The cell morphology
and cell mechanics are both regulated by the binding to other cells as well as to a substrate.
This generates special cell signals, such as phosphorylation cascades, that will control the
cellular function and further cell-cell or cell-surface interactions [53]. Proper interactions
between cells and their substrates are important and regulate the overall survival and
proliferation of cells, as well as differentiation of stem cells. Signaling molecules, the
extracellular matrix and the surrounding cells builds together up a complex
microenvironment in which the cells reside and any small changes in the vicinity are
recorded by the cells [61].
Figure 18 Simplified sketch of the extracellular matrix and cell components of a tissue. The fibrous
connective tissue that surrounds the cell and different cell types combined to create functional
tissues.
PROTEIN ADSORPTION
It is widely recognized that proteins adsorbing onto material surfaces mediate subsequent
biological responses and cellular interactions to the surface. These surface adsorbed proteins
may be from serum containing cell media, or a pre-conditioning step with a cell adhesive
protein such as fibronectin. [62-64] Inorganic, usually metallic, electrodes are inherently
incompatible with biological systems. The absence of strong interactions between proteins
and the electrode surface causes desorption of biomolecules for many electrode materials
such as Pt, Au or Carbon. [65, 66] Organic electroactive polymers perform well in
comparison to conventional electrodes since they are dynamic at an ionic level hence
creating an interesting material for the study of cellular communication and surface
interactions. Proteins adsorb in different quantities, densities, conformations, and
orientations, depending on the chemical and physical properties of the surface. The process
is complex and involves van der Waals, hydrophobic and electrostatic interactions, and
30
Cell Adhesion
hydrogen bonding. [67-70] Proteins adsorbed to a polymeric film show good stability and
may work as a functionalized electrode. The ability to control protein interactions on
conducting polymers can be used to design active surfaces for cell culturing, enzymatic
biosensors and implantable biomaterials. [3, 66] Conducting polymers act as inert and
stable electrodes in aqueous environments. By applying a positive potential (0.4-1.0 V) to
polyanion-doped polypyrrole the protein adsorption increases both in time and amount
compare to the process without potential. [65]
Changes in cellular adhesion, morphology, motility, gene expression and differentiation are
all affected by the properties of the material on which the cells have been cultured, however
the mechanisms behind are not well understood. Properties that have shown to influence
the protein adsorption are among others chemical structure, surface energy, type and
density of surface charges, molecular weight of the polymer, surface topography and
roughness. [64, 71-73] Proteins undergo conformational changes from their native
structure in order to adsorb to a surface. One of the main reasons to these conformational
changes of the adsorbed protein is the interfacial free energy that builds up between a
protein rich environment and the material. The conformational changes become more
irreversible upon closer exposure to an interface. In general, hydrophobic surfaces tend to
adsorb larger amounts of proteins than hydrophilic ones. A surface with high water content
and polar surface will result in lower interfacial energy and hence result in less protein
adsorption and cell adhesion to the material. [74] Lee et al. created a wettability gradient to
study the behavior of platelets in terms of the surface energy. In the absence of plasma
proteins the platelet adhesion increased as the wettability increased while in the presence of
plasma proteins the cell adhesion decreased with increasing wettability. [75, 76] The
hydrophobic surface shows properties that gain protein adsorption and interactions that
retain the proteins at the surface. [65] Roach et al investigated fibrinogen and albumin
adsorption to hydrophobic and hydrophilic surfaces. The albumin showed a strong affinity
to the hydrophobic surface while fibrinogen adhered rapidly to both surfaces though having
a slightly higher affinity to the hydrophobic surface as well. Conformational studies on
those two systems showed that both proteins exhibit a less organized secondary structure
upon adsorption onto a hydrophobic surface than onto a hydrophilic surface. [68] Deeper
understanding of the mechanisms of protein adsorption and cellular response in relation to
the surface characteristics of polymeric materials is of great importance for the development
of biomedical materials, regenerative medicine, tissue engineering and biosensors. [64, 68]
THE ROLE OF CONDUCTING POLYMERS IN BIOMEDICAL APPLICATIONS
Synthetic polymers have been established as an important material for medical use in areas
such as wound healing, drug delivery systems and long term implantable devices. The
reason why polymeric materials have got this spot in biomaterials is because of that they are
relatively easy to manufacture into different shapes as products and devices at a reasonably
31
Cell Adhesion
cost and with tailored mechanical, physical and chemical properties. Important factors such
as chemical structure and surface properties affect the biocompatibility (protein adsorption,
cell adhesion and cytotoxity etc) of the material and need to be understood to improve
existing polymers as well as the development of new biocompatible polymers.
Biocompatible synthetic surfaces that are able to control the interaction between a living
system and an implanted material remain a major theme for biomaterial applications in
medicine. In the recent 30 years the experience and research within the organic
bioelectronics field has emerged since the enlightenment of the potential of combining
organic electronics and biology. The electrical activity in conducting polymers render the
possibilities for novel advantages in biomedical applications compared to the inactive
synthetic polymers. Both conducting polymers and biomolecules have an organic
composition and a polymeric structure which makes it interesting to combine conducting
polymers with biological systems. Conducting polymers have the ability to be rapidly and
reversible switched between different oxidation states, which make it possible to
dynamically alter the electrical, chemical and physical properties of the material. Many
types of cells, such as cardiac, nerve, bone and muscle cells, respond to and use electrical
stimulation to maintain their function. The fact that several tissues are sensitive for electric
fields and electrical stimuli has made conjugated polymers utilizable for a number of
biological and medical applications. [3] The applied electrical stimuli and electrically
altered polymer properties influence the ability of conducting polymers to interact with
biomolecular systems. In the 1980s it was shown that conducting polymers were
compatible with many biological materials and molecules for the applications within
biosensors [77]. These features are useful in many biomedical applications such as neural
probes [78], actuators , and tissue engineering. Further, the material has the capability to
transfer charges from biochemical reactions which can be used in biosensors [6, 79], and
cellular communications (ref), as well as the polymer can entrap and release molecules of
biological relevance that could be used in controlled drug release [80, 81] In the mid-1990s
the electrical activity of conducting polymers was introduced with the aim to modulate the
cellular activity. It was shown that an applied potential could affect protein and cell
adhesion, protein production, cell migration, and DNA synthesis in mammalian cells [82].
Direct electric stimuli has been found to stimulate bone regrowth, enhance wound healing,
control mammalian cell cultures and promote nerve degeneration [66, 83, 84]. The role,
and amount, of fibronectin was shown to be adjusted by electrochemical control of the
oxidation state of the polymer during cell culture and growth. [85-87]
Different polymers induce different degree of conformational changes in proteins which
means that the protein undergoes structural changes to adsorb to the surface. [74]
Interaction of proteins with conducting polymer films is greatly dependent on the
oxidation state of the polymer and hence on the potential applied. For instance Khan and
Wernet showed that the adsorption of proteins accelerated and enhanced on the conductive
32
Cell Adhesion
polyanion-doped polypyrrole film in the more conducting state [65]. An electrochemical
switch between oxidation states within a conducting polymer will change the surface
properties, such as hydrophobic characteristics, and thus affect the protein conformation
and adhesion. There are several molecular interactions that can promote cell attachment,
however much of the research in this area has focused on utilizing adhesive peptides that
engage and activate integrin adhesion receptors on the cell surface [88, 89] By
electrochemically changing the redox state, the conformation of fibronectin can be
modified and hence the presentation of the RGD site is altered which results in that the
integrins cannot bind and thus a loss of cell attachment [90, 91]. Material properties of
conducting polymers that are of importance for medical applications such as tissue
engineering are among others, conductivity, reversible oxidation, redox stability,
biocompatibility, hydrophobicity (since 40-70° water contact angle promotes cell
adhesion), three-dimensional geometry, and surface topography [92]. However, there
remain many unanswered questions, particularly regarding the mechanism by which
electrical stimulation through conducting polymers affect the cells. The ability to
manipulate growth, differentiation and eventually the performance of the complex
molecular machinery is an important and promising level of biomolecular communication.
SURFACE MODIFICATIONS FOR CONTROLLED CELL ADHESION
Utilization of traditional semiconducting microelectronic and plastic electronic fabrication
is a powerful tool to understand biological context and medical phenomenon including the
study of in vitro cell culture studies and tissue engineering. [93, 94] The fundamental
insights will affect the research within surface science and development of biomedical
devices. [44] Surface modification using thin polymer layers has been used to avoid
uncontrolled interactions with cells and proteins in biomedical applications and clinical
device. [95] In general cells respond to numerous chemical, physical, mechanical, and
electrical stimuli which all can be individually engineered to control cell functioning. In
vivo cell attachment to the tissue is conveyed by proteins that are present in the tissue or
that are produced by the cell itself. Most cell-surface interactions depend on the presence of
an intermediating biomolecule layer where proteins are the most important components
[96]. These interactions have to be mimicked when working with solid/liquid interfaces to
control cellular events in vitro. Surface modifications can be done to indirectly modulate
the cell attachment by influencing the adsorption of proteins onto the surface when
introduced to a protein rich environment. The possibility to control the behavior of
biomolecules at a surface in both space and time has an impact on the ability to design
advanced medical devices and technologies for cell culturing and implantable biomaterials.
Also, by mimicking the in vivo events and interactions researchers will gain a lot of
biological understanding.
33
Cell Adhesion
Surface modification involving patterning of surfaces into bio-adhesive and non-adhesive
areas has gained increasing interest over the past few decades. The possibility to spatially
control the organization of cells and subcellular entities at functional surfaces has started to
make a significant impact on the biomedical field. [96, 97] The ability to control the
biomolecules that act as anchors for the cell is a requirement for manipulation of living cells
at a surface in vitro. Anchors of interest cover the span from peptides, proteins, DNA all the
way to arrangements such as complex living cells [98]. It is of interest to create patterns and
structures with dimension down to that of a single cell, i.e. 5-50 micrometer to create the
possibilities to affect clusters of cells down to single cell or cell membrane components.
Chemical patterns have attracted focus for applications in the field of biosensors,
fundamental cell-surface interaction studies, tissue engineering, and biomaterials to induce
rapid healing of both implants and wounds [99]. A broad array of techniques developed
and designed to be used in the semiconductor industry has been modified for design and
fabrication of a wide spectrum of materials within the biomaterial research field. This
variety of micro- and nano-patterning techniques to create chemical, biological and
topographical characteristics renders the possibility to create advanced biomaterials. [74]
Since polymers are compatible with most patterning techniques methods such as standard
photo- and electron-beam lithography, soft-lithography, ink-jet printing, screen printing,
dip-pen lithography, microfluidics, microcontact printing etc can be used to directly or
indirectly pattern biomolecules onto a surface with the aim of creating micropatterns and
microarrays. These methods results in cell arrays and pattern functionalities with high
resolution while maintaining a good biomolecule conformation and function. [96]
Direct surface modifications may also involve techniques such as self-assembled monolayers
(SAM) to covalently incorporate signals such as patterned biomolecules etc [100-102].
Other methods create chemically or electrochemically modified surfaces or polymers to
influence binding rates and conformation of bound proteins and cells. [103-106]
Temperature-responsive interfaces for biomedical separation and cell sheet engineering has
been shown as an alternative to control cell adhesion and protein adsorption. Here,
temperature changes induce hydrophilic/hydrophobic alterations, which mean that the
surfaces could be thermally adjusted to modulate interactions with biomolecules and cells.
[95] Independent of the patterning method the behavior and function of the proteins at the
surface interface is strongly affected by electrostatic and hydrophobic interactions.
In parallel to chemically controlled adhesion of cells there is evidence that topographical
signals have an impact on attachment, proliferation, signaling and migration of cells and
tissue as well [107]. The ability to tailor substrate topography is of great interest since it
among others enables the production of surfaces that resemble the ECM construction onto
which the cells grow in vivo as a contrast to the 2D cell cultures that are routinely used as
models within cell biology. [108] With the aim to create more accurate models of the
natural condition several researchers have started to develop 3D structures where the cells
34
Cell Adhesion
not are forced to adapt to the flat substrate [109-111]. Such 3D substrates that provide an
environment that resembles of the in vivo conditions can be created by functionalized
nanofibers, microstructured thermoformable polymer films and soft lithography etc.
Fundamental understanding gained from these well controlled in vitro artificial scaffolds
would finally contribute to the development of biomedical implants with improved healing
and performance in vivo, more reliable cell-based sensors for drug screening and
development, and better tissue engineering scaffolds.
35
Major Results and Conclusions of the Thesis
6 MAJOR RESULTS AND CONCLUSIONS OF THE THESIS
This chapter gives a brief overview of the projects that have been carried out within the frame
this thesis, which are reported in the manuscripts, as well as the major conclusions and
discussions of the results achieved.
In general, conjugated polymers are soft and flexible and since they are organic compounds they
comprise chemical, physical and structural properties very similar to those of biological systems.
These properties together with the eletroactive features make them interesting as active and
updateable materials for bioapplications where cell and tissue interactions are of importance.
There is a broad spectrum of polymers that potentially could be of great interest for such research
[3]. In 1994, Wong et al [7] showed that a film of polypyrrole could be used to electronically
control cell adhesion since the surface properties such as wettability and surface chemistry can be
controlled reversibly, simply by applying a potential to the conducting polymer. Until now,
polypyrrole (PPy) has been one of the most commonly studied polymers as active biomaterials, so
far, and has proven to be biocompatible, highly conductive, and also easy to manufacture
through electro-polymerization. However, it comprise one drawback from a bioapplication point
of view; PPy can easily become reduced by biological electrolytes and hence lack long-term
biostability during applied cathodic potentials. The increasing interest for using conducting
polymers within the field of bioelectronics has forced scientists to develop new more stable
polymers [24, 26]. Several reasons have led us to the choice of using PEDOT as the conducting
polymer interface i) several polymerization methods are possible ii) the resulting film is typically
very robust and is relatively homogenous with a high conductivity iii) in the thin film state the
material appears as highly transparent, and iv) exhibiting good biocompatibility characteristics
for the applications studied in this thesis. Hence, PEDOT fulfills many of the crucial criteria in
order to become a suitable conjugated polymer for bioapplications within the field of organic
bioelectronics.
37
Major Results and Conclusions of the Thesis
The focus of this thesis has been to study the use of PEDOT: Tosylate to design a smart surface
switch serving as an active cell hosting layer that can be electronically controlled in order to
regulate cell adhesion and cell response. The goal has been to derive surfaces and scaffolds of
which the cell-hosting characteristics can be electronically controlled with desired temporal
control. Electrochemical addressing of patterned surface switches controls the state and
conformation of crucial biomolecules, which then affects the surface properties that regulate cell
adhesion. If adsorbed proteins shows the correct conformation they will act as anchors to mediate
cell attachment. Integrin binding sites on the cell membrane interacts with specific peptide
regions within the proteins e.g. RGD in fibronectin. The results would be of great interest for
clinical cell culture studies as well as for regenerative medicine and tissue engineering. The work
have included surface engineering, process development and cell experiments in order to study the
surface switches to control the spatial distribution and also density of cells.
Figure 19 Chemical structure of 3, 4-ethylenedioxythiophene (EDOT) and iron(III)tosylate.
STEM CELL SWITCH (PAPER I)
To improve cell spreading, proliferation and differentiation within stem cell biology, a
deeper understanding regarding the interaction between stem cells and the extracellular
matrix is essential [55]. Today a commonly used method to achieve stem cells viability is to
create a supporting surface for cell adhesion by precoating the cultivation dish with proteins
typical for the extracellular matrix.
The objective of this project was to create a switchable surface that controls the cell
adhesion and cell density of the neural stem cells c17.2 by changing the redox state of the
surface switch based on PEDOT: Tosylate.
As the redox state is switched of the PEDOT: Tosylate surface, its chemistry is altered and
hence the wettability and adhesive properties are modulated as well. Previous studies have
shown that the cell binding and spreading strongly depends on surface properties such as
the structure, chemistry, wettability and elasticity. By modifying the surface properties the
conditions for protein binding will change. Hence, a change in redox state would render
control of possible protein binding pathways.
38
Major Results and Conclusions of the Thesis
To facilitate the cell experiments the first step includes coating a substrate suitable for cell
culturing procedures with the conducting polymer PEDOT: Tosylate. Due to the geometry
of a petri dish, a vapor phase polymerization procedure proved to be a suitable method to
coat a thin and homogenous film. By introducing an insulating line the coated dish was
divided into two adjacent electrodes. With an aqueous NaCl electrolyte and an applied
potential, the film was electrochemically switched which also is evident by that a strong
electrochromic response. Surface energy measurements, in the form of water contact angle
measurements, indicated that the pristine PEDOT surface coated in a petri dish is relatively
more hydrophilic. After a complete redox switching, the reduced electrode becomes slightly
more hydrophilic than the oxidized electrode. The difference disappeared as the biased
surfaces were immersed into an electrolyte for extended time periods. This could be
explained by that there is an extensive exchange of doping ions (tosylate) and counter ions
from the electrolyte along the surface. Protein binding studies of Human Serum Albumin
(HSA) showed that less and weaker bonded proteins were attached to the oxidized electrode
as compared to the reduced electrode surface.
In the next step of this project cell viability experiments were carried out. Cell media was
used as the electrolyte and no cell-adhesion promoters were pre-coated before the stem cells
were seeded. Due to the transparency of the thin films of PEDOT standard transmission
microscopy methods could be utilized to analyze the cultured cells. The substrate was
switched simultaneously as the cells were seeded. After incubation the cells were fixated,
stained and then counted. Quantification of the c17.2 stem cells showed that the oxidized
PEDOT increased the cell adhesion twofold as compared to the reduced PEDOT electrode
immersed into the same solution. This result advocates that the redox switch in PEDOT
can be used to electronically control the stem cell density by altering the protein adsorption
onto the substrate without any precoating processes.
THE ELECTRONIC SURFACE SWITCH TO REGULATE MDCK CELL ADHESION (PAPER II)
This project can in one way be seen as a compliment to the Paper I but with a focus
towards deeper understanding of the molecular aspects of cell interfacing occurring along
the surface switch. By using the reversible redox properties of the PEDOT: Tosylate surface
switch dynamic control over the binding characteristics of extracellular proteins, and thus
cell adhesion, is accomplished. A similar two electrode device as used in paper I was utilized
but instead of vapor phase polymerization spin coating and barcoating methods were
explored. The major advantages with these processes are that they are much faster to
manufacture and that films can more easily be applied on larger substrates. Dynamic
control of cell adhesion along the substrates during cell cultivation is of great interest in
various biomedical applications, including fundamental cellular studies, tissue engineering,
and cell analysis.
39
Major Results and Conclusions of the Thesis
The cell culture dish of polystyrene was coated by a spin coating process in which all the
polymerization components were mixed into one common solution before applying onto
the surface. Since each sample was spun for a long time the films became thin and
homogenous. In parallel to the spincoating, larger substrates of planar plastic foils of PET
were coated by a barcoating method.
The coated film was patterned forming two adjacent electrodes to enable simultaneous
study of the response of to the two redox states in the same cell culturing media. AFM
measurements were carried out in order to investigate if there were any topography
differences, due to redox switching, between the two electrodes that potentially could affect
the cell response. Due to the inherent roughness of the soft polystyrene substrate itself and
eventually rest products from the electrolyte it was impossible to see any hypothetical
topography difference between the two redox states of the electrodes.
MDCK epithelial cells were cultivated on the surface switch as a potential difference was
applied to the electrodes. No differences regarding cell attachment could be seen between
the two electrodes when cells were seeded before switching the substrate. However, when
the electrodes were switched just before the cell seeding step, it resulted in a clear difference
in cell density on the different biased electrodes. In the contrary to the c17.2 stem cells the
MDCK cells favor the reduced PEDOT electrode. The cell proliferation along the two
electrodes upon simultaneous switching was investigated. The cells on the reduced
electrode showed a relatively much more healthy morphology, good proliferation and
formed polarized monolayers. The cells on the oxidized electrode showed a typical rounded
shape due to poor adhesion. These differences were further investigated by molecular
studies of the anchoring protein fibronectin. The reduced electrode presented adsorbed
fibronectin in its native conformation, thereby promoting cell adhesion. On the oxidized
PEDOT: Tosylate electrode the surface chemistry affected the fibronectin conformation in
a way that MDCK cell adhesion was suppressed. These experiments shows that an
electrochemical PEDOT: Tosylate surface switch can be used as a dynamic cell seeding
surface of which the cell hosting characteristics can be controlled by electronic modulation
of surface properties.
GRADIENT (PAPER III)
The surface switches described in previous experiments only present two distinct redox
states, oxidized and reduced, simultaneously. By modifying the organic bioelectronic cell
culture dish into an electrochemical transistor a tunable redox gradient to regulate cell
growth can be introduced. Chemical and molecular gradients are well studied and are of
great importance in cell systems both in nature and in medical technology applications.
A flat PET substrate was used as the substrate, onto which a bi-stable electrochemical
transistor structure based on PEDOT: Tosylate was manufactured. To evaluate the
40
Major Results and Conclusions of the Thesis
transistor functions, current-voltage characteristics were recorded and they showed that the
transistor switched easily by using an aqueous NaCl electrolyte. The transistor operates in
the first and third quadrant. The electrochemical transistor includes a structure 1
configuration which means that a redox gradient is built up along the defined polymer
channel area. Due to the electrochromic properties of PEDOT this redox gradient can also
be seen as a color gradient that gradually changes from transparent light blue, at the
oxidized side, towards darker blue, at the reduced side. By tuning the applied gate potential
the scalar value, i.e. the overall magnitude, of the electrochemical gradient is changed along
the channel.
Since it was already known that adhesion of MDCK epithelial cells is sensitive upon redox
switching, see paper III, the possibility to seed the cells onto a redox gradient provide us a
chance to gain further understanding about their response to the electrochemical states of
PEDOT. When cells were seeded on the gradient of the channel that was simultaneously
switched the cells showed a clear distribution pattern along the channel. For instance, it was
possible to identify which potential, corresponding to a certain surface state, favored cell
adhesion the most. When the gate voltage was modulated, which resulted in a change of
the redox gradient, the cell density distribution was also changed along the same direction
which clearly shows that the cells favor a specific redox potential to achieve a healthy
morphology and to form cell monolayers.
NANO-FIBER SCAFFOLD ELECTRODES BASED ON PEDOT FOR CELL STIMULATION
(PAPER IV)
A common problem today with biomaterials is that they are stiff and that they are typically
a two-dimensional surface, in great contrast to the elastic three-dimensional tissues found in
the human body. For this reason a nano-fibrous material would be of great interest as a
substrate and template to mimic the natural environment for cell adhesion and tissue
engineering [112]. The specific target in this nano-fiber project was the fact that nerve cells
typically require a porous environment to facilitate a healthy morphology and that they can
be electrically stimulated via their substrate.
Randomly electrospun PET (polyethyleneterephthalate) nano-fibers with a diameter of
150-400 nm and with a pore size from 5 to 10 micrometers were supplied by Swerea IVF
Institute, Mölndal, Sweden. Vapor phase polymerization (VPP) and spin coating were
carried in parallel in order to enable proper evaluation of two kinds of method to coat the
fiber mats with PEDOT:Tosylate. Both methods showed good adhesion to the substrate
and from electron microscopy studies it was found that the VPP method was more suitable
since it preserves the desired nano-fiber structures of the mats. While the VPP method
resulted in approximately 100 nm thin conformal polymer films around the fibers, the spin
coating method generated more inhomogeneous films that covered both the fibers and
41
Major Results and Conclusions of the Thesis
clogged the voids in between. The VPP:PEDOT coated nano-fiber mat was divided into
two electrodes and was electrochemically switched by applying a potential difference
between two electrodes including an aqueous NaCl electrolyte. The switch could be
reversed several times within the potential range of 1.5 V – 3.0 V, which corresponds to the
range where the electrochemical switch could be performed without risk for overoxidization of the PEDOT material. This could also be seen as a clear color switch due to
the electrochromic properties in PEDOT. Contact angle measurements were run on
uncoated PET nano-fibers, VPP:PEDOT coated nano-fibers and flat PEDOT coated PET
foil substrates. By coating the fibers with PEDOT the contact angles were switched from
super-hydrophobic to super-hydrophilic while the flat substrate showed hydrophilic
properties both with and without the PEDOT coating. This giant change in effective
surface tension could be explained be the impact of substrate topography.Since it was
shown that the fibers could be conformally coated by the VPP: PEDOT process and that
they also showed excellent electrochemical behavior, the coated fibers were chosen for cell
experiments. SH-SY5Y nerve cells were successfully seeded onto the VPP: PEDOT nanofibers for further testing. The cell viability experiments showed that the cells adhered and
formed actin filament that proved a healthy condition of the cells and that also indicated a
good host characteristics of the scaffold material. Electron microscopy pictures showed that
nerve cells adhered well to the substrate.
Electrical stimulation was then run on the cells that were seeded onto the coated fibers and
quantification of Ca2+ signaling was used as a measurement of electrical response. When 3
V was applied to the substrate the voltage operated calcium channels (VOCC: s) were
activated and the response to the applied potential stimulus could be seen as a clear peak in
the intracellular Ca2+ level. This indicated that the nerve cells could be triggered upon
electrical stimulation. To eliminate the probability of cell-induced Ca2+ signaling caused
from other pathways than through the voltage sensitive channels, a parallel experiment in
which the channels were blocked was also performed. In this control experiment no
calcium peaks were observed.
It would be of great interest to test these porous fiber substrates on relatively smaller and
more mobile cells to see if they can migrate within the fiber mat scaffold and if their
spreading can be controlled by the signal applied. Copolymerization with another polymer,
such as PPy, to create mechanically switchable fiber structures, could be another step
beyond the present scope.
42
Major Results and Conclusions of the Thesis
ELECTROACTIVE CONTROL OF PLATELET ADHESION TO CONDUCTING POLYMER
MICROPATTERNS (PAPER V)
In this project we report an addressable matrix electroactive surface based on a conducting
polymer to gain control over the spatial distribution of cell adhesion and proliferation, in
vitro.
Microscale technologies can be used to create miniaturized surfaces for in vitro cell culture
studies and to address and control the cellular microenvironment in a culture. A commonly
utilized method to pattern substrates for bioapplication is by direct patterning with active
biomolecules at certain areas, leaving the surrounding intact. In our case the entire surface
is indirectly coated but the patterned properties is dynamically controlled by
electroactivation to affect cell response.
Switchable devices were manufactured using barcoating and photolithography to create
desired patterns of PEDOT on flexible PET foils. The conductive layer of PEDOT:
Tosylate was patterned into electroactive structures, spatially separated by insulating lines.
Chemical over oxidization of the PEDOT: Tosylate film was accomplished by exposing the
unsealed polymer to a low concentration of oxidant (1% NaOCl) for a short time. This
process is called deactivation or passivation since it will destroy the conductivity of the
substrate without removing the film. Hence, this method will change the polymer
properties without introducing a large topographical step and therefore leaving a smooth
patterned surface with conducting and non-conducting areas.
The patterns consist of individually switchable finger structures and pixels down to a size of
10 micrometer that can be addressed in a parallel, or separate, manner. Micro-patterning
was achieved at the scale of individual cells, or subculture of cells, to enable active
regulation of cell populations and their extracellular environment at high spatial resolution.
We decided to work with platelets since they are a suitable model system for our switchable
device. The cells are easy accessible, adhere quickly and are rather easy to evaluate in terms
of cell density etc. Platelets rich plasma was used as the electrolyte during the
electrochemical switch. The adhesion of platelets showed a clear preference for the reduced
side and followed the patterned structures well down to dimensions of 10 micrometers.
While analyzing the amount of anchor protein i.e. fibrinogen bound to the surfaces a small
quantitative difference was demonstrated but not significantly enough to correspond to the
differences in cell response. These results strengthen our earlier results how the redox
potential dictates changes in the conformation and/or orientation of surface proteins thus
indirectly regulating the cell adhesion to the electrodes. Our electrode matrix system opens
up for fundamental studies of cell growth behavior and complex manipulation of the
growth characteristics in a highly parallel manner. Our findings promise for a future e-dish
technology to regulate the fate of cell cultures.
43
Future Outlook and Challenges in Organic Bioelectronics and Surface Switches
7 FUTURE OUTLOOK AND CHALLENGES IN ORGANIC BIOELECTRONICS
AND SURFACE SWITCHES
In recent years, the biomaterial society has changed the perception about implanted
substrates. The material should be active, integrate with surrounding tissue, and promote
regeneration besides exclusively contributing as structural support for damages tissue, as
previously believed. This evolution in thinking has driven the development and engineering
of new smart materials and the willing to understand the interactions between surfaces and
biological systems.
The performed experiments covered in this thesis, among others, shows that PEDOT
works very well as a biocompatible material and that electronically control of the redox
state of the polymer can be used to achieve a dynamically cell adhesion device. By varying
substrate for polymer coating as well as dope ions the polymer can be tailor made to suit
different cell types and biomedical applications. The work reported in this thesis covering
the utilization of different coating methods and substrates to show that a certain cell type
response in a similar way independently of coating method. These results hint that the
electronically changed surface properties that steer the cell (protein) adhesion are more
sensitive to the degree of applied potential rather than differences in film topography and
morphology due to coating method.
As mentioned in the previous chapter a lot of interesting areas have just been initiated and
future work within surface switches to control adhesion as well as release of cells and
proteins will be achieved through collaborations with research groups at the Karolinska
Institute and The Health University in Linköping, among others.
Modification of the fiber substrate as well as the polymer coating should be investigated
with the aim to create substrate that can be adjustable for specific cell types and
45
Future Outlook and Challenges in Organic Bioelectronics and Surface Switches
experiments. One task could be to modify the dimensions and arrangements of fibers, as
well as the cavities in between, to promote cell migration within the porous structure which
would be a requirement for a 3D implant. It would also be of great interest if
copolymerization can be used to create a dynamically fiber structure in purpose to design a
mechanically switchable cell device. Another interesting aspect would be to incorporate
functional dope ions into this fiber structures to chemically control cell behavior and drug
release.
When it comes to the switch project this has come to a point where the next step would be
to develop material and patterning processing and device architecture to create more
advanced active matrixes. In my research I have used photolithography patterning to create
electrically controllable substrates to indirectly control attachment and conformation of
biomolecules to the substrate. This can be further developed to comprise several electrodes
where addressing, patterning and contacting would be the ingenious part of the project. It
would also be of great interest to introduce different biomolecules as dope ions and
moreover to perform electropolymerization of various polymers and dope ions to
individually addressed pixels. Dynamic patterns in a multicellular system should be of
considerable interest in tissue engineering as well as fundamental studies within cell
functions, cell-cell interactions, and cell-surface interactions. By spatially regulating the
attachment of biomolecules to a surface it is possible to modify the microenvironment and
creating well defined cell patterns. By develop the addressing and patterning resolution it
would be possible to affect a single cell that is part of an environmental community.
Microfabrication: would open up for the design and fabrication of structures on which
thousands of parallel experiments with single cells-or small groups of cells-can be carried
out under identical or tailor made conditions. Organic electronics offers a unique
opportunity to bridge between bio-and electronics from the nano to microscale.
46
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