Doctoral thesis Fredrik Bäcklund

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Linköping Studies in Science and technology

No. 1695

Preparation and Application of

Functionalized Protein Fibrils

Fredrik Bäcklund

Division of Biomolecular and Organic Electronics

Department of Physics, Chemistry and Biology

Linköping University, SE-581 83 Linköping, Sweden

Linköping 2015

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During the course of the research underlying this thesis, Fredrik Bäcklund was enrolled in Forum Scientium, a multidiciplinary doctoral program at Linköping University, Sweden.

Preparation and Application of Functionalized Protein Fibrils

ISBN: 978-91-7685-978-0

ISSN: 0345-7524

Copyright © 2015, Fredrik Bäcklund

Printed in Sweden by LiU-Tryck, Linköping 2015

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ABSTRACT

Many proteins have an innate ability to self-assemble into fibrous structures known as amyloid

fibrils. From a material science perspective, fibrils have several interesting characteristics,

including a high stability, a distinct shape and tunable surface properties. Such structures can be

given additional properties through functionalization by other compounds such as fluorophores.

Combination of fibrils with a function yielding compound can be achieved in several ways.

Covalent bond attachment is specific, but cumbersome. External surface adhesion is nonspecific,

but simple. However, in addition, internal non-covalent functionalization is possible. In this

thesis, particular emphasis is put on internal functionalization of fibrils; by co-grinding fibril

forming proteins with a hydrophobic molecule, a protein-hydrophobic compound molecule

composite can be created that retains the proteins innate ability to form fibrils. Subsequently

formed fibrils will thus have the structural properties of the protein fibril as well as the

properties of the incorporated compound. The functionalization procedures used throughout

this thesis are applicable for a wide range of chromophores commonly used for organic

electronics and photonics. The methods developed and the prepared materials are useful for

applications within optoelectronics as well as biomedicine.

Regardless of the methodology of functionalization, using functionalized fibrils in a controlled

fashion for material design requires an intimate understanding of the formation process and

knowledge of the tools available to control not only the formation but also any subsequent

macroscale assembly of fibrils. The development and application of such tools are described in

several of the papers included in this thesis. With the required knowledge in hand, the possible

influence of fibrils on the functionalizing agents, and vice versa, can be probed. The

characteristic traits of the functionalized fibril can be customized and the resulting material can

be organized and steered towards a specific shape and form. This thesis describes how control

over the process of formation, functionalization and organization of functionalized fibrils can be

utilized to influence the hierarchical assembly of fibrils – ranging from spherical structures to

spirals; the function – fluorescent or conducting; and macroscopic properties – optical

birefringence and specific arrangement of functionalized fibrils in the solid state. In conclusion,

the use of amyloid fibrils in material science has great potential. Herein is presented a possible

route towards a fully bottom up approach ranging from the nanoscale to the macroscale.

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Populärvetenskaplig sammanfattning

Proteiner är en typ av molekyler som återfinns i levande organismer. De har en mängd olika

funktioner och former, alltifrån transportörer av andra föreningar till byggnadsmaterial för

vävnad. En egenskap som många har gemensam är dock att de vid särskilda förhållanden

m.a.p. t.ex. temperatur och koncentration kan bilda långa fiberliknande strukturer kallade

amyloida fibrer. Dessa fiberstrukturer har vanligen en diameter i nanometerskalan och en

längd på några mikrometer. Amyloida fibrer är intressanta för materialvetenskap eftersom

de är stabila, har en förutsägbar och regelbunden uppbyggnad och en specifik struktur. I den

här avhandlingen beskrivs metoder för kontroll av den intrikata process som fiberbildningen

utgör och för att styra bildningen mot specifika strukturer och former. Särskilt fokus har lagts

på att ge fibrerna skräddarsydda funktioner genom att kombinera dem med andra

föreningar – att skapa funktionaliserade fibrer.

I organisk elektronik designar man elektroniska och optiska komponenter baserade på

organiska föreningar som t.ex. ledande polymerer eller specifikt designade molekyler. Dessa

kategorier av föreningar har tidigare studerats ingående var för sig och används mer eller

mindre rutinmässigt inom organisk elektronik för tillverkning av t.ex. solceller, transistorer

eller lysdioder. Biomolekyler kombinerade med sådana föreningar är dock mindre vanligt, -

trots att deras unika strukturer i princip möjliggör för dem att agera som templat för

organisering av andra molekyler. Detta beror huvudsakligen på att de flesta biomolekyler har

en begränsad stabilitet och att många av de föreningar som används inom organisk

elektronik inte är vattenlösliga – de är hydrofoba. Stabilitet är dock inte ett problem för just

amyloida fibrer, och vi har använt den hydrofoba egenskapen för organiska föreningar för att

tvinga dem att bilda komplex med proteinfibrer.

Att kombinera amyloida fibrer med polymerer och/eller andra mindre molekyler med en

specifik funktion kan resultera i material som har en kombination av proteinfiberns

strukturella egenskaper och funktionen hos föreningen den funktionaliserats med. Den här

avhandling tar upp flera exempel på hur fibrer kan ges specifika egenskaper m.h.a. andra

föreningar så att de blir fluorescenta eller ledande. Hur funktionalisering kan påverka

fibrerna och hur fibrerna påverkar de funktionaliserande föreningarna har studerats för ett

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flertal fall. Fibrernas struktur har t.ex. utnyttjats för att ordna upp linjära hydrofoba

molekyler längs med fibrernas långa axel.

Avhandlingen behandlar slutligen även hur kombinationen av en kontrollerad fiberbildning,

specifikt vald funktionalisering och efterbehandling av det funktionaliserade materialet kan

påverka egenskaper och funktioner hos material gjorda med funktionaliserade fibrer från

nanometer-nivå ända upp till en makroskopisk (cm) storleksskala. .

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Acknowledgement

To my supervisors Niclas Solin and Olle Inganäs, to the co-authors of the papers included in

this thesis, to all of my close colleagues and friends in the Biorgel research group - past and

present, to my colleagues at IFM, to the members of Forum Scientium, to the members of

the coffee club, to friends and family, for sharing your ideas, expertise and support without

which this thesis would not be;

Thank you!

Linköping, September 2015

/Fredrik Bäcklund

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Paper 1 - Controlling amyloid fibril formation by partial stirring

Bäcklund F.G., Pallbo J., Solin N. Submitted

Paper 2 - Amyloid fibrils as dispersing agents for oligothiophenes: control of photophysical properties through nanoscale templating and flow induced fibril alignment

Bäcklund, F. G., Wigenius, J., Westerlund, F., Inganäs, O., Solin, N. J. Mater. Chem. C 2, 7811 (2014). Figures included in thesis adapted with permission from the Royal Society of Chemistry

Paper 3 - Development and application of methodology for rapid screening of potential amyloid probes

Bäcklund, F.G., Solin, N. ACS Comb. Sci. 16, 721–729 (2014).

Figures included in thesis adapted with permission from the American Chemical Society © 2014 American Chemical Society

Paper 4 - Tuning the aqueous self-assembly process of insulin by a hydrophobic additive

Bäcklund F.G., Solin N. Submitted

Paper 5 - Convection induced air-water interface assembly of amyloid fibrils

Bäcklund F.G., Ajjan F.N., Solin N. Manuscript

Paper 6 - Protein nanowires with conductive properties

Elfwing, A., Bäcklund, F. G., Musumeci, C., Inganäs, O., Solin, N. J. Mater. Chem. C 3, 6499–6504 (2015).

Figures included in thesis adapted with permission from the Royal Society of Chemistry

Paper 7 - PEDOT-S coated protein fibril microhelices

Bäcklund F. G., Elfwing A., Ajjan F.N., Babenko V., Dzwolak W., Solin N., Inganäs O. Manuscript

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Author contributions:

Paper 1

Involved in all experiment planning.

Performed the majority of experiments.

Supervised experiments performed by project worker.

Part of writing and reviewing.

Paper 2


Performed all experiments, some together with co-authors,


Paper 3


Performed all experiments.


Paper 4


Performed all experiments.


Paper 5


Performed majority of experiments.

Part of writing and reviewing

Paper 6


Performed all experiments, except C-AFM studies, together with co-writers.

Minor part of writing and reviewing.

Paper 7


Performed the majority of experiments.

Majority of writing and part of reviewing.

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Abbreviated compounds:

6T α-sexithiophene

6P para-sexiphenyl

BMSBP 4,4-bis(2-methoxystyryl)-biphenyl

DCM 4-(Dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran

PCBM Phenyl-C61-butyric-acid-methyl ester

Perylene bisimide derivative N,N′-Bis(3-pentyl)perylene-3,4,9,10-bis(dicarboximide)

PEDOT poly(ethylenedioxythiophene)

PEDOT-S Alkoxysulfonated poly(ethylenedioxythiophene)

PEDOT:PSS poly(3,4-ethylenedioxythiophene)- poly-(styrenesulfonate)

Tinopal CBS Disodium 4,4′-bis(2-sulfonatostyryl)-biphenyl

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Table of Contents Introduction ............................................................................................................................................. 1

Chapter 1 – general concepts .................................................................................................................. 3

1.1 Connecting nano to macro ............................................................................................................ 3

1.2 Supramolecular interactions ......................................................................................................... 4

1.3 Self-assembly ................................................................................................................................. 7

1.4 Mechanochemistry ........................................................................................................................ 7

1.5 Polarized light as a tool for investigating molecular orientation .................................................. 8

1.6 The native fold of insulin ............................................................................................................... 9

1.7 Amyloid fibrils .............................................................................................................................. 10

1.7.1 Amyloid fibrils – in disease and in health ............................................................................. 11

1.7.2 Amyloid fibrils – the structure .............................................................................................. 12

1.7.3 Amyloid fibrils – interactions with other compounds .......................................................... 13

1.7.4 Amyloid fibrils – mechanism of formation ........................................................................... 14

1.7.5 Spherulites ............................................................................................................................ 16

1.7.6 Amyloid fibrils as material components ............................................................................... 16

1.8 Conjugated π-electron systems ................................................................................................... 17

1.9 Photophysics................................................................................................................................ 18

1.10 Conducting materials in organic electronics ............................................................................. 22

1.11 Standard film preparation methods for organic materials ....................................................... 24

Chapter 2 – Morphology control of amyloid fibrils, paper 1 ................................................................. 26

2.1 The effect of agitation on amyloid fibril formation ..................................................................... 26

2.2 Influencing the reaction kinetics and morphology of internally functionalized fibrils ............... 28

Chapter 3 – The influence of fibril formation on functionalizing compounds, papers 2 & 3 ................ 32

3.1 Dispersion and organization of internalized oligothiophenes .................................................... 33

3.1.1 Optoelectronic properties of oligothiophenes ..................................................................... 33

3.1.2 Dispersion of oligothiophenes in amyloid fibrils .................................................................. 35

3.1.3 Nanoscale templating of oligothiophenes ........................................................................... 36

3.1.4 Transferring nanoscale organization to the macroscale ...................................................... 38

3.2 Evaluating the effect of fibrillation on hydrophobic compounds ............................................... 39

3.2.1 Amyloid probe characteristics .............................................................................................. 39

3.2.2 Identifying hydrophobic compounds as amyloid probes ..................................................... 40

Chapter 4 – The impact of functionalizing compounds on the fibril reaction pathway, paper 4 ......... 43

4.1 Particle size and implications for alternative aggregation routes ............................................... 44

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4.2 Characteristics of insulin-6T spherulites..................................................................................... 45

4.3 Inhomogeneous dispersion of 6T in spherulites ......................................................................... 45

Chapter 5 – Air-water interface assembly of functionalized amyloid fibril films, paper 5 ................... 47

5.1 Air-water interface assembly of proteins .................................................................................... 47

5.2 Factors governing air-water protein fibril interface assembly .................................................... 48

5.3 Characteristics of air-water interface assembled insulin fibril films ........................................... 49

5.4 Control of film formation anisotropy .......................................................................................... 51

Chapter 6 – External functionalization of amyloid fibrils, papers 6 & 7 ............................................... 53

6.1 PEDOT-S ....................................................................................................................................... 53

6.2 Interaction of internalized and externally added functionalizing compounds ........................... 54

6.2.1 Energy transfer and quenching, the Förster distance .......................................................... 54

6.2.2 Non radiative energy transfer between fibril-internalized 4,4’-bis(2-methoxystyryl)-biphenyl (BMSBP) and externally added PEDOT-S ....................................................................... 55

6.3 PEDOT-S induced conductance of amyloid fibrils ....................................................................... 56

6.4 External functionalization of fibril superstructures .................................................................... 57

6.4.1 A stabilizing effect of PEDOT-S adherence to insulin fibril superstructures ........................ 59

Future and outlook ................................................................................................................................ 62

References ............................................................................................................................................. 63

1

Introduction Consider the following: DNA could rightly be called a blueprint of life as within its structure

is encoded the information needed to construct virtually every living system known to date.

However, what DNA is a blueprint of is mainly the construction of proteins. In other words, if

DNA is the blueprint of life, proteins constitute the actual building material for life itself. In

humans, there are approximately 20 000 genes whose readout will result in a protein1.

However, due mainly to so called post translational modifications, more than 80 000 protein

isoforms have been documented for humans2. Thus, in terms of number of genes, humans

are not that different from the well-studied worm C. Elegans that also has about 20 000

genes3. More complex organisms such as humans differ from life forms closer to a worm not

by the number of genes but rather by the number and complexity of proteins. Proteins are

involved in virtually every chemical process in any biological system; they facilitate the

transport of information (in the form of hormones) and transport other molecules (such as

oxygen), they are an integral part of the human immune system (in the form of antibodies),

they constitute a main building material for tissue (muscle tissue) and they provide chemical

reaction catalysis (as enzymes). However, in terms of stability, in their normal functional

form - the native state, proteins are as a rule too unstable for use in devices. In stark

contrast, the special kind of protein aggregates used and studied in this thesis are

surprisingly stable and present a very similar architecture even if made from functionally

very different proteins.

A fundamental part of the work in this thesis revolves around mechanochemistry, of which

the first known documentation in writing can be found in the book “De Lapidibus” (on

stones) by Theophrastus of Ephesus (371-286 B.C.).4 In the case of Theophrastus, the

mechanochemistry performed involved the breaking and reforming of covalent bonds

(cinnabar was reduced to mercury while forming coppermonosulfide by grinding with a

copper mortar and pestle). In this thesis mechanochemical methodology has been

implemented through the grinding with mortar and pestle of proteins and various

hydrophobic compounds. Thus, like Theophrastus we have used a mortar and pestle to

promote an event not otherwise easily attained.

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The mechanochemistry approach through grinding has been employed here mainly for

convenience. To achieve the composite materials we have strived to prepare, we have when

possible chosen a simplistic preparatory approach rather than a more complex, but more

controlled, scheme of material preparation. The goal in preparatory terms have been to

work with a bottom up approach were our materials are designed at and built from a

molecular level and then realized up to a macroscopic scale. Self-assembly and

supramolecular interactions have thus been instrumental for our work. We have not been

attempting to predesign a scheme for the chemical interactions needed to construct our

materials as one would with traditional organic chemical synthesis - involving stepwise

covalent bond linkages. Rather, we have attempted to utilize non-covalent interactions and

to guide the making and braking of such interactions by external stimuli such as temperature

and pH levels. Although the preparation methods were often simplistic, the chemistry

occurring has involved a high level of complexity and a large scope of possible preparation

routes to investigate.

A definite advantage of the preparation procedures we have used is the relative ease for up

scaling. Ultimately, we would like to implement our materials in devices such as OLEDS

(organic light emitting diodes). To do this and to test a library of different prepared materials

to be used for such applications having ml amounts and more even for only exploratory

research is highly desirable.

We have chosen an approach were the compounds used to create our composite materials

could roughly be divided into two categories; function yielding and structural scaffolding.

The scaffolding has been provided by in vitro prepared protein amyloid fibrils and the

function by compounds attached non-covalently to the proteins. This has provided a simple

way of scanning through a large number of possible composites by simply changing the

function yielding part of the composite. Since the materials have to a large extent been

forming, although directed, through self-assembly, a significant part of this work has been

the characterization of our materials and an array of techniques has been employed to this

end. During this process, new discoveries have been made regarding the potential of

amyloid fibrils as structural scaffolds and their interaction with and effect on various

luminescent and conducting compounds. In short; a new set of tools creating functional

materials for organic electronics and photonics applications has been developed.

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Chapter 1 – general concepts

1.1 Connecting nano to macro In order to fully grasp the fundamental tools, phenomena and techniques that have become

the foundation of this thesis, an overview of said concepts will now follow, starting with a

perspective on size; an example spanning the full conceptual size range of this thesis is the

components of a film made from amyloid fibrils (figure 1.1), a structure discussed further in

chapter 5. This size range covers the macroscopic square centimeter regime all the way

down to the individual molecular components that make up the smallest units of the film.

Working our way downwards in size hierarchy, the film itself represents the upper size limit

being in the square cm range. Such a film is in turn made up of hundreds of thousands of

amyloid fibrils that have clustered together to form a continuous sheet. Each of those fibrils

is in turn typically some micrometers (0.000001 m, the diameter of an average sized

bacterium) in length, and a few nanometers in diameter (0.000000001 m, the distance

required to traverse a single cell membrane). A single amyloid fibril is itself composed of a

huge number of protein monomers that have been reorganized from their native state and

assembled to form the elongated fibril strand. The basis of this assembly can be found in the

interactions between individual molecules and atoms that hold the entire structure

together. These chemical bonds go down to tenths of nanometers in length, the typical size

of a single carbon-carbon covalent bond being about 1.5 Ångström (0.00000000015 m).

Thus, each time we made these films we used and to some extent exerted control over a

process that started out on a nanometer scale and ended up becoming a solid macroscopic

object clearly visible to the naked eye.

Figure 1.1 – Schematic components of a film made from amyloid fibrils. a, The fully formed film. b, A typical sized single functionalized amyloid fibril. c, A single monomer of bovine insulin. d, Two carbon atoms linked by a covalent bond.

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1.2 Supramolecular interactions

Supramolecular, or non-covalent, interactions are crucial for keeping together the

conformations of biomolecules such as proteins. Thus they are often present as

intramolecular interactions for a single molecule. However, non-covalent interactions are

also the basis of intermolecular interactions between different individual molecules. In this

context, such intermolecular interactions are termed supramolecular interactions (see figure

1.2).

For reference, covalent bonds, the sharing of one or more electrons between atoms (figure

1.2 a), have an energy value per mole of about 350-450 kilojoule (kJ). Hydrogen bonds, an

attractive force involving a hydrogen atom and an electronegative atom such as oxygen, are

the strongest and most prevalent non covalent interactions found in proteins with an energy

value per mole of 4-120 kJ. In this thesis, hydrogen bonds have played a major role in the

part they play in stabilizing the structure of amyloid fibrils; for amyloid fibrils, hydrogen

bonds lock the β-strands in place within the sheet. Such hydrogen bonds can be formed

between amide groups of the backbone primary sequence, as the amide group can act both

as a hydrogen donor and acceptor. Moreover, additional hydrogen bonds can form if the

residues of the peptide sequence contains side chains with a capacity for hydrogen

bonding.5 In contrast, the hydrogen bonds holding together an α-helix structure form

primarily between the backbone amide carbonyl oxygens and N-H groups (see figure 2 b).6

Figure 1.2 - Various types of chemical interactions. a, A covalent bond linking two hydrogen atoms. b, Schematic depiction of backbone hydrogen bonding in an α-helix. c, π-π interactions between two benzene rings. d, solid particles of 6T in acidic water. e, 6T incorporated into proteins, dissolved in acidic water.

Not as strong, but important for our studies involving molecules such as α-sexithiophene,

are π-π interactions where the π-electrons of different conjugated ring structures interact

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with each other (figure 1.2c). 7 π-π interactions can have a significant influence on the

spectral properties of fluorescent compounds in a solid state, as exemplified by the case of

H-aggregated of α-thiophene observed in paper 2 (section 3.1.1).

A type of interaction that has played a particularly central role in our studies is the

hydrophobic interaction. This phenomenon originate with polar water molecules repelling

non-polar entities, since clustering of nonpolar solute molecules will put structural

constraints on fewer solvent molecules and increase entropy of the solvent.8 Consequently;

non-polar hydrophobic molecules will when put into an aqueous environment have a strong

tendency to seek seclusion from surrounding water molecules. Because of this, if a highly

hydrophobic compound is put by itself into water, it will remain in a solid state and resist

dispersion/solvation, as evidenced by the behavior of the highly hydrophobic molecule α-

sexithiophene (6T) in aqueous solvent (figure 1.2 d). However, as we have shown, if

hydrophobic compounds are first mechanically forced to intermingle with proteins by

grinding, they will stay encapsulated by surrounding protein molecules upon subsequent

dissolving of the hydrophobic compound-protein complex in water, (figure 1.2 e). Thus, for

the studies presented in this thesis, the apparent adverse combination of hydrophobic

compounds and an aqueous solvent - via internalization in proteins, has actually worked in

our favor.

An important special case of non-covalent interactions are electrostatic interactions. They

can vary greatly in strength and the essential features can be explained by Coulomb’s law.

From this classic equation it follows that the strength of the electrostatic interaction

between two charges depends not only on the charges themselves but also on the distance

between the charges. Typically, direct chemical interactions occur over distances measuring

a few Ångströms.9 The potential energy of interaction between charges in a solvent must,

however, also take into account the dielectric constant of the medium (also called the

relative permittivity, ε); while its value is by definition set to 1 in vacuum, in water it is 78 at

25o C. The value of the dielectric constant increases with the degree of polarity of the

solvent.8 Thus, two charges of opposite sign will attract each other much less strongly if in

water than in a more non-polar hydrophobic environment.

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Equation 1.1 – Potential energy U(r) between two charges q1 and q2 in a medium with a dielectric constant ε separated by the distance r.

A phenomenon related to electrostatic interactions is the dispersion of colloids in solution.

Colloids can be defined simply as particles within the size range of 1 nm to 1 µm which are

dispersed in a solution.10 However, how such particles interact is far from simple. An attempt

at a systematic description of colloid interaction is given by the DLVO (from the names of

developers Derjaguin, Landau, Verwey and Overbeek) theory. The theory is based on the

assumption that the total energy of interaction between two particles can be expressed as a

sum of attractive and repulsive contributions. The contributors to this interaction are

identified as a combination of electrostatic interactions through the overlap of electric

double layers and van der Waal forces.11,12 According to the model, van der Waal forces

between particles of the same kind will act as an attractive force e.g. between two

temporary dipoles. In contrast, the electrostatic repulsion between particles of the same

charge act to push the particles apart. Since charged particles in a solution containing salt

will become surrounded by oppositely charged ions, an electric double layer will form that

can carry the effect of the electric charge of the particle farther into the solution and allow

for repulsive interaction over a distance.

It is quite natural to consider the DLVO theory for aggregation of protein fibrils since they

can interact with each other and their surrounding electrostatically, through hydrophobic

interactions and, locally, by van der Waals interactions such as local dipole-dipole

interactions. However, the DLVO theory has its shortcomings; it does e.g. not take into

account specific interactions between individual molecules and the effect that the shape and

size of interacting particles can have in close proximity. When considering the self-assembly

of protein fibril films in chapter 5, while the principles of the DLVO theory stands, the size

and shape of the functionalized fibrils are very likely to have a significant effect on any self-

assembly process in between such fibrils. In addition, for the self-assembly of fibrils into film

the hydrophobic effect is likely to be of importance. The attractive force between

hydrophobic surfaces has been shown to be orders of magnitude stronger than van der

Waals forces13 and to reach as far as 80 nm.14

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1.3 Self-assembly Self-assembling systems are abundant in nature and provide a rich source for creating novel

materials through directed self-assembly processes.15,16 In this thesis, the main self-assembly

process we have studied is the formation of amyloid fibrils. Two things that characterizes

this particular self-assembly process is the specificity of how the smaller components form

the larger aggregate and the strength with which the formed aggregates are held together.

We have also studied self-assembly of fibrils with other compounds such as poly-electrolytes

involving electrostatic interactions as a main driving force, and the complexation of fibrils

with other fibrils to form fibril superstructures. During a single reaction, there can be several

different, sometimes competing, self-assembly processes taking place simultaneously. By

changing the environment in which the self- assembly takes place, it is possible to steer the

self-assembly pathway towards different end structures and conformations. We have

focused in particular on the effects on various self-assembly processes due to changes in

agitation, temperature, hydrophobic additives and pH-levels.

1.4 Mechanochemistry

Mechanochemistry is commonly used to describe a process affecting covalent bonds; a

mechano-chemical reaction is, as defined by IUPAC, ‘‘a chemical reaction that is induced by

the direct absorption of mechanical energy”.17 However, mechanochemistry as a term can

be broadened to include also changes in supramolecular interactions, i.e. non-covalent

chemical interactions such as hydrogen bonds and hydrophobic interactions. This particular

phenomena was first touched upon in 1893.18 However, the writers of the 1893 report

lacked the knowledge that the recent decades of development in analytical equipment and

science in general has provided regarding chemical bonds and specific molecular

interactions. Such knowledge is integral to a controlled and purpose-designed utilization of

mechanochemistry, and mechanochemistry methodology has been applied to e.g.

polymers19 and fullerene based compounds.20 Covalent bond cleavage has been

demonstrated for polymers using mechanophores – molecules within a polymer sensitive

to mechanical force, to selectively cleave polymeric units using ultrasound,21 whereas

mechanical milling has been used to induce the covalent bonding of various molecules such

as e.g. aromatic hydrocarbons to C60.22 Furthermore, mechanochemistry has been shown to

be of value for supramolecular synthesis.18 In addition, for milling/grinding, a fundamental

8

application in and of itself is the fine mixing of different components.23 In reactions between

solids, the area of contact between reactants can be more important than the amount

present in the mix.24 The solid state dispersion and large scale intermingling of multiple

compounds that in the absence of grinding would remain fundamentally separated is the

basis for the preparation of internalized functionalized fibrils as prepared in this thesis.

1.5 Polarized light as a tool for investigating molecular orientation

Linearly polarized light is light that oscillates in a single plane, this being the direction of

polarization. Although most light sources produce light whose electric component oscillates

in more than one plane (see figure 1.3 a and b), by placing an polarizer that transmits only

light oscillating in one specific direction between a light source and a sample, the light

reaching the sample will be polarized in one plane (as shown in figure 1.3 c).25 Turning the

filter will then change the direction of the polarization relative to the sample. Using

polarizing filters it is also possible to achieve circularly polarized light, achieved by setting the

relative phase difference of two orthogonally oriented waves of light of equal magnitude to

π/2.26

If two polarizing filters are set perpendicular to each other, i.e. in a crossed polarizer setting

(figure 1.3d), light will not be able to pass through both polarizers and the view through the

set of polarizers will be black. However, if a sample is placed between crossed polarizers, this

can sometimes result in a part of the polarized light coming from the first polarizer changing

its plane of oscillation so that it can pass through the second polarizer (termed the analyzer).

Isotropic (randomly organized) materials will not affect the polarization of the light as it

traverses the bulk material and the light transmitted through the sample will not pass the

second polarizer. In contrast, for the same analytical setup anisotropic (not randomly

organized) materials can change the polarization of some of the light during its passage

through the sample. This is because these materials contain a well-defined symmetrical

orientation along a certain axis. If light travels in parallel to this axis the polarization will

remain unaltered. However, if light passes through the material by a route different from

that of the axis of symmetrical orientation, the result will be a double refraction into two

light beams with perpendicular polarizations – a phenomenon known as birefringence.

9

Figure 1.3 – Schematic illustration of polarized light. a, The electric fields of unpolarized light traveling out of the page represented as double arrows. b, The light in a represented as the superposition of two polarized waves with perpendicular planes of oscillation. c, The plane of oscillation (red) and direction of propagation (blue) of linearly polarized light. d, A crossed polarizer setup.

Birefringence can occur for molecules with a large aspect ratio and is then directly related to

the long axis of the molecule.27 This becomes important when considering the structures

with a long aspect ratio, such as amyloid fibrils. Amyloid fibrils can form liquid crystalline

phases (i.e. loose aggregates of long linear structures aligned in parallel to each other) in

solution - visible when studied in between crossed polarizers. Here, the degree of molecular

orientation is related to the degree of birefringence. It should be noted, however, that while

probing the birefringence of a sample using crossed polarizers, the outcome is dependent on

an average and thus individual molecules within the sample may have different

orientations.26 Depending on the direction of the optical axis of the sample relative to the

second polarizer (the analyzer), light that has passed through the sample can become

polarized along the polarization direction of the second polarizer and thus pass through. We

have used polarized light to study materials since the appearance of birefringence has been

useful to characterize the large scale structural organization of our materials.

1.6 The native fold of insulin Proteins in their native state are classified to have different levels of structural ordering, the

simplest being the sequence of residues termed the primary structure. The primary structure

can fold into certain favored geometries, known as the secondary structure. The three

dominating secondary folds found in native state proteins are the random coil, the α-helix,

and the β-sheet. The secondary structure elements are then organized relative to each other

forming the tertiary structure (figure 1.4). Taken together, these structural patterns make

up the native fold of a single protein and facilitate the huge structural variety of native

protein folds.

10

In this thesis, we have used insulin as model protein for forming amyloid fibrils. Insulin in its

native state, as shown in figure 1.4, consists mainly of α-helix secondary structure. Thus, to

form amyloid fibrils, this structure must first be at least partially unfolded and then

reorganized into the predominant β-sheet fold of amyloid fibrils. From a physiological

standpoint, the conditions required for this to readily occur for insulin are extreme although

easy to handle from a preparative standpoint; we have routinely used a pH of 1.6 and

heating at 65o C to induce in vitro the structural conversion of insulin into fibrils within

hours. Inducing fibril formation from insulin in vitro is facilitated by the propensity of small

proteins to form amyloid fibrils. Proteins with shorter peptide chain lengths have been found

to form amyloid fibrils more easily, due in part to a higher number of intermolecular

interactions within the resulting fibril structure.28 Unlike the native fold of proteins, the

thermodynamic stability of fibrils as compared to the fully unfolded state is less dependent

on the specific amino acid sequence.28

Figure 1.4 – The native structure fold of a bovine insulin dimer. An α-helix region is highlighted in red and a section of β-sheet fold in blue.

1.7 Amyloid fibrils Considering the huge variety in structural composition, shape and size, it is far from self-

evident that many functionally diverse proteins seem to have in common the generic

structural form of amyloid fibrils, to which they will transform given the appropriate

environmental conditions. It has been proposed that fibrils may represent an ancestral form

of protein fold that predates the huge number of different folding patterns that has since

evolved over the ages.29 This structural state has remarkable properties, it has strength

comparable to that of steel16 and has a resistance to chemicals, temperatures and pH-levels

that would leave other forms of biomolecules to be more or less completely disintegrated.

Although the shared properties in overall structure far exceed the differences, amyloid fibrils

11

can have subtle but distinct differences - different morphology, observed as e.g. differences

in the degree of twist,30 thickness or length.31 The term amyloid (derived from the Greek

word for starch; ”amylon”) originates from that fact that amyloids can be stained with

iodine. In 1854, when this was first discovered, it was taken as an indicator of the stained

substance being a form of starch, which was known to react with iodine. Although it was

determined a few years later that the amyloids were more likely to be protein based rather

than starch32, it took until the advent of electron microscopy to reveal that the amyloid

structure was composed of fibrous units typically a few nanometers in length.33 Studies with

X-ray diffraction,34 infrared spectroscopy35,36 and circular dichroism37 has since then revealed

that amyloid fibrils are composed mainly of β-sheet structure. The fibrous nature of what

has come to now be known as amyloid fibrils also results in specific optical characteristics

which enables the birefringent staining with the dye Congo red that since its conception in

192713 has been established as a benchmark method of amyloid detection. As noted in

section 1.5, the long aspect ratio of fibrils is convenient for analysis since it is possible to

investigate the large scale organization of fibrils by using polarized optical light microscopy

(POM) even without staining; if enough fibrils are aligned parallel to each other, this

collection of fibrils will create an anisotropic region that becomes visible with POM.

1.7.1 Amyloid fibrils – in disease and in health

The accumulated knowledge about amyloid fibrils to a large extent originates from research

performed with a goal of understanding various diseases that have been linked directly or

indirectly to such structures, notably the neurodegenerative disorders of Alzheimer’s disease

and Parkinson’s disease38,39, type II diabetes,40 and the prion coupled Creutzfeldt-Jacobs

disease and its bovine counterpart bovine spongiform encephalopathy (“mad cow

disease”).41 It is to date not fully known as to what extent amyloid fibrils are the cause or

effect of these and other diseases as well as what makes up their toxicity. However, a

consensus appears to be forming in the amyloid research community that it is the precursors

to rather than the mature form of fibrils that can be linked to toxicity.42–44 Prion proteins

represent a special category in the ability of the fibril state to rapidly transform normally

folded prion proteins to the disease linked fibril state in vivo, thereby in effect being

contagious.45,46 This feature is not, however, a feature inherent of all amyloid fibrils.46 In

short, that amyloid fibril structures can be found in and linked to several diseases is not in

12

doubt; however, a growing amount of data is being accumulated showing that amyloid fibrils

can also be found in numerous benign settings with vital functions for the organism housing

them. Amyloids have been identified as being part of protective coatings for bacteria,47,48

nitrogen catabolism and heterokaryon formation in fungi and DNA transcription in yeast,49

immune response of insects,50 and as the major component of the silkmoth eggshells.51 In

humans amyloid fibrils have been found to sequester toxic melanin precursors during

melanin synthesis52 and pituitary secretory granules store peptide hormones in an amyloid

like structural state.53

1.7.2 Amyloid fibrils – the structure

As mentioned in section 1.7, amyloid fibrils are composed mainly of a characteristic β-sheet

structure.54 As with β-sheets found in native protein folds, hydrogen bonds between the β -

strands (the part of the primary sequence that make up part of a β-sheet) are the main force

keeping the sheet together also for amyloid fibrils.5 The individual β-strands that make up

the sheets are fixed perpendicular to the long axis the fibril (see figure 1.6 a). Furthermore,

the sheets that make up the structure are paired together in protofilaments to form an

interlaying hydrophobic interface stabilized by hydrophobic interactions as well as van der

Waals interactions between interdigitated amino acid side chains (a “steric zipper”).5 The

sheets are slightly twisted around each other in a regular fashion along the length of the

protofilament, although to a lesser extent than what is commonly seen for β-sheets in native

protein folds.55 Typically, the strands of the sheets have a separation of 4.7-4.9 nm and the

sheets are themselves spaced 10 nm apart.5,54 Protofilaments combine to form the final

mature fibril. Fibrils can themselves further intertwine to form even thicker aggregates.

The diameter of fibrils made in vitro under a given set of reaction conditions from the same

protein tends to be of similar diameter and thus multiple intertwining of protofilaments is

typically limited. For the in vitro amyloid forming protein insulin, supported by AFM studies,

the fibrils have been suggested to be composed of 4-8 protofilament segments resulting in

fibrils of 3-5 nm in diameter, whereas lengthwise the fibrils extend up to several µm 31. Due

to the generic dominant features of amyloid fibrils, although the exact dimensions vary, even

for different proteins the diameter is typically in the range of 3-10 nm and the length in the

single digit µm range.55,56 In accordance with the previously well-established dimensions of

13

amyloid fibrils, in this thesis the observed length of insulin fibrils formed with or without

functionalizing agents have been between 1 µm up to approximately 10 µm depending on

the applied reaction conditions.

1.7.3 Amyloid fibrils – interactions with other compounds

The study of fibril formation has gathered considerable interest over the years and a

methodology for finding new amyloid probes is presented in this thesis. Probably the most

well-known compound that have been shown to interact with amyloid fibrils is Congo red;

when attached to fibrils it shows a yellow-green birefringence when viewed with cross

polarizers.57 Thioflavine T (ThT) increases its fluorescence intensity by orders of magnitude

when attached to fibrils58 and thiophene based compounds have been shown to change

their photoluminescent properties upon binding to fibrils,59,60 as has the dye Nile red (see

figure 1.5 for the chemical structure).61

Figure 1.5 - The chemical structure of three established amyloid probes.

The structure of amyloid fibrils, as described in the previous section, consists mainly of long

stretches of β-sheets with the individual β-strands being perpendicular to the long fibril axis

(figure 1.6 a). The interlaying hydrophobic interface thus created between filaments will

form, for the lack of a better word, grooves that run along the fibrils entire length (figure 1.6

b). In addition, since the primary structures of proteins generally include many sites that will

be charged either positively or negatively depending on the pH levels in the surrounding

water solvent, the exposed surface of the fibrils will

present a charged surface towards the surrounding

water solution. Thus, fibrils have a tunable charged

surface and an elongated hydrophobic core region.

Figure 1.6 – a, A schematic representation of pair of β-sheets paired together to form a protofilament. b, A schematic depiction of the

grooves created by an elongated β-sheet, highlighted by a double arrow.

14

We have used the structural characteristics of fibrils to promote interaction between fibrils

and hydrophobic compounds as well as charged compounds. We have also shown in paper

2 that the most obvious feature of the fibrils, the extreme aspect ratio of several hundred

times longer length than width, can have a profound effect on the function of amyloid fibrils

as templates for organization of functionalizing compounds.

1.7.4 Amyloid fibrils – mechanism of formation

As mentioned in section 1.6, preparing amyloid fibrils from bovine insulin represents a

simple preparatory procedure; insulin dissolved in acid water will rapidly form fibrils once

exposed to heat. However, the self-assembly process occurring during the formation of the

fibrils is highly complex. The process of amyloid fibril formation has been followed in

numerous studies using compounds that interact specifically with amyloid fibril structure. In

such studies, the fibril formation has been shown to consistently follow a sigmoidal growth

curve with three distinct phases; a lag phase, a growth phase and a stationary phase (see

figure 1.7). In the lag phase, the native protein is converted to a partially unfolded state that

enables conversion into a predominant β-sheet fold upon incorporation into a growing fibril

structure. The growth initiation, i.e. the nucleation, of the fibrils then ensues from the

monomeric units these intermediary structures represent. As discussed in coming chapters,

if hydrophobic compounds are included in the original mix by grinding they will become

incorporated into the mature fibril structure.

15

Figure 1.7 – Growth of amyloid fibrils. Protein structures are depicted in green and functionalizing compounds in red. a, The sigmoidal growth curve and the three main phases. b, Processes of nucleation.

The nucleation and growth of fibrils can occur by different processes; primary nucleation -

whereby monomeric units form small prefibrillar species; elongation – when monomeric

units attach to the ends of existing fibrillar structures; and secondary nucleation - when

growth is catalyzed by monomeric units reversibly adhered alongside a surface such as that

of a previously formed fibrillar structure.62 In addition to these three main growth

mechanisms, fragmentation of fibrils can also be a contributing factor as this leads to an

increase in the number of ends where monomers can attach, thus increasing the rate of

elongation.

Previous studies by modeling indicate that the three main mechanisms of fibril growth do

not affect the fibril growth in the same manner.62 While changes in primary nucleation has

an impact mainly on the lag phase, the effects of secondary nucleation and changes in

elongation can alter the appearance of both the lag and the growth phase of the sigmoidal

growth curve.

16

1.7.5 Spherulites

When making amyloid fibrils in vitro under quiescent conditions, there can be an additional

reaction pathway available for the protein leading to spherical structures called protein

spherulites. This is by no means an unusual type of structure; spherulites in the form of

radially grown polycrystalline aggregates formed by successive branching of a nucleus are

known to form in two or three dimensions from e.g. polymers such as polyethylene, from

silicates crystallizing in magma, from graphite, and from iron.63 Protein spherulites are

thought to be formed from an amorphous nucleus, formed during the lag phase, on which

subsequent radial growth of fibrils occurs, eventually forming a sphere (figure 1.8 a).

Figure 1.8 –Spherulite formation. a, Schematic depiction of the formation and structural composition of a protein spherulite with an amorphous core as base for radial growth of fibrils. b, A schematic image of a Maltese cross extinction pattern. c, POM image of insulin spherulites.

In the case of insulin, the main protein model for fibril formation used in this thesis, the

spherulites typically have a diameter of 50 µm, although some may grow up to 150 µm in

diameter.64 In terms of yield for the self-assembly reaction spherulites can be a significant

problem since the volume fraction of spherulites relative to fibrils can actually be dominated

by spherulites.65 Because of the symmetric radial fibril growth, spherulites give rise to a

Maltese cross light extinction pattern when viewed with crossed polarizers (see figure 1.8 b

and c). Polarized optical microscopy is therefore a convenient way of studying spherulites

and was used extensively for this purpose in chapters 2 and 4.

1.7.6 Amyloid fibrils as material components

A scalable bottom up approach is an attractive alternative to a top down strategy such as

lithography for achieving miniaturized organic electronic devices and components due to its

17

ability to offer a high degree of control on the individual building blocks of the architecture

of a device.66 Biomolecules such as proteins are interesting to investigate for the design of

bottom up preparation schemes because of their inherent ability to self-organize into

structures with specific shapes and orientation as evidenced by their proof of concept usage

in e.g. the design of electrical connections67 or as templates for creating arrays of quantum

dots.68 In such a context, amyloid fibrils have several distinct features that are of interest for

their use as material components; they are highly stable and resistant to chemical

degradation (as mentioned in section 1.6, in this thesis the standard procedure used to

induce self-assembly of insulin fibrils involved a pH of 1.6 and heating at 65o C), they can be

used as long linear templates (as described in section 3.1) and as shown in this thesis they

can be easily functionalized with multiple different compounds (see chapter 3). The

interaction between the function yielding compounds and the structure defining fibrils of

the functionalized fibrils we have prepared and studied can be very specific, as seen in

chapter 3 for the specific dispersion and alignment of fibril internalized α-sexithiophene.

Using fibrils to organize chromophores have previously been shown to have distinct

beneficial effects related to the performance of the chromophores such as e.g. improved

triplet emission for phosphorescent Iridium-complexes.69

Many chromophores such as α-sexithiophene, studied in chapters 3, 4 and 5, behave very

differently depending on their organization in a solid state form, or if dissolved in different

solvents. We have shown that it is possible to promote a specific organization of the

functionalizing compounds enabled by the interaction with the fibril as a template. Finally,

the functionalizing agents can sometimes influence the proteins in turn; in chapter 4 the

self-assembled spherulites becomes a new form of scaffold for 6T organization and in

chapter 6 for hierarchical helical superstructures of amyloid fibrils are stabilized by the

external adhesion of the oligomeric compound PEDOT-S.

1.8 Conjugated π-electron systems Organic compounds with properties such as conductivity or fluorescence typically contain π-

electron systems. A particularly common motif is the conjugated system – a repeated

sequence of one single bond followed by one double bond. The double bonds are

delocalized along the conjugated system (see figure 1.9). The electrons forming the bonds in

a conjugated system can be divided into two categories; electrons forming σ-bonds and

18

those forming π-bonds. When depicting the electron distribution for a molecule as sp2

hybridized molecular orbitals, in becomes evident that the π-bonding electrons are in a

geometrical plane different from that of electrons in σ-bonds. The out of plane orientation

of the π-bonding orbitals relative to that of the σ-bonds makes a conjugated system such as

that of polythiophene sensitive to σ-bond rotation, but also enables intermolecular

interaction through orbital overlap between neighboring π-electron systems and charge

transport within conjugated structures.

Figure 1.9 – Schematic depiction of a thiophene conjugated system as well as an illustration of the difference between σ- and π-bonds.

The conjugation length of fluorescent compounds can be directly linked to spectral features

since an increase in the conjugation length can lead to a corresponding increase in

wavelength of absorption for that compound, because of a decreased energy gap between

the HOMO-LUMO energy levels, or vice versa.

1.9 Photophysics Most of the compounds we have used for adding function to fibrils, such as α-sexithiophene,

DCM, 4,4-bis(2-methoxystyryl)-biphenyl and Nile red, are luminescent. Luminescence from

organic compounds can be achieved through either electroluminescence or

photoluminescence. Electroluminescence produces light through the recombination of

charges and is the basis for organic light emitting diodes (OLEDS), previously investigated in

relation to functionalized amyloid fibrils as an integral material component.69 However, in

the papers included in this thesis, we have focused on processes where molecules absorb

photons, leading to excited states that can relax radiatively - a process known as

photoluminescence.

19

To fully understand a process such as photoluminescence, it helps to first consider that an

intrinsic property of electrons is that they have a spin angular momentum, a corresponding

spin quantum number of ½, and a spin magnetic quantum number of either -½ (spin up) or

+½ (spin down). Furthermore, it is a fundamental principle that electrons within a given

atom can have different energies, i.e. occupy different energy levels. However, if an energy

level of that atom is already occupied by an electron with a magnetic spin of +½, a second

electron placed in that same level would assume an opposite spin value of -½.25

When a molecule is put under illumination, electrons in the atoms of the molecule may

absorb energy from the incoming light if the light contains photons of an energy

corresponding to the difference between two of the possible energy levels of the molecule.

I.e. energy thus absorbed will cause an electron to move from its original energy level - its

ground state, to a higher energy level - an excited state, a process occurring in about 10-15 s.

In molecules, each electronic energy state will also contain several vibrational energy levels.

Because electronic excitation occurs without affecting the internuclear separation, but

rather represents a change to a less energetically favored electron density distribution, the

spacing of the vibrational energy levels of an excited state is similar to that of the ground

state. This explains why spectral patterns, arising from transitions to different vibrational

levels, are often similar in corresponding absorption and emission spectra. Typically, after

transition to an excited state, relaxation to the lowest excited energy level follows through

vibrational relation in about 10-12 s. The average time spent in an excited state before

relation to the ground state (i.e. the fluorescence lifetime) is close to 10-8 s. After transition

to an excited state, an electron will return back to the ground state either by non-radiative

relaxation - dissipating heat and/or altering chemical interactions, or by emission of a

photon through fluorescence or phosphorescence.9 Because of vibrational relaxations,

emissive transition to the ground state typically represents a lower energy difference than

the corresponding transition for absorbed light.

The process of photoluminescence can be illustrated graphically in the form of an energy

diagram such as that in figure 1.10 (known as a Jablonski diagram), with the ground state

being termed S0, the first excited singlet state S1, the second excited singlet state S2 and the

exited triplet state T1.

20

Figure 1.10 – An energy diagram depicting various energy transitions.

An excited electron in a singlet state (Sn, n = integer), has a spin direction opposite to that of

an unpaired (by spin) electron in the ground state. However, spin conversion can also occur

to the triplet energy state T1 (a conversion termed intersystem crossing). Here, the excited

electron has a spin in parallel to its counterpart in the ground state. Transition can then

occur to the ground state resulting in phosphorescence. However, because of the similar

spin direction such a transition is highly unfavorable (the transition is said to be forbidden)

and fluorescence is considerably faster than phosphorescence with the relaxation occurring

on a timescale of nanoseconds, whereas phosphorescence occurs over milliseconds to

seconds.70

When considering excitation leading to photoluminescence, it can be important to note that

a given molecule can have a certain axis along which a maximum interaction with an

electromagnetic wave (i.e. light) will occur. Such an axis - the direction of an electric dipole

transition or the electric symmetry axis, is usually along the long axis of the molecule.27

Although of less significance in solution, where random motion will nullify this effect, in a

solid state this effect can lead to stronger emission along a certain axis of orientation within

the solid if a large enough number of emissive molecules are oriented in parallel to each

other. This effect was observed in the form of polarized emission in chapters 2, 3 and 5.

21

The different possible energy states that an electron can occupy can be directly linked to the

appearance of absorbance as well as photoluminescence spectra. Since transitions to

different energy levels including vibrational levels occur with a specific probability for any

given compound, different compounds absorb different wavelengths of light to a varying and

compound-specific degree. The result is the characteristic set of absorbance maxima

constituting an absorbance spectrum. However, the characteristics of photophysical

processes can also vary depending on the interaction of a molecule with its surroundings and

related conformational changes such as twisting or bending of the molecular structure. This

link between structural distortions and spectral data is key for explaining the interaction of

chromophores with amyloids (see e.g. section 3.2.1) and in addition to spectral intensity

changes, shifts of one or more peaks in the absorbance and/or fluorescence spectra can

occur towards both longer wavelengths (bathochromic/red shift) or shorter wavelengths

(hypsochromic/blue shift).71 Such effects can be clearly observed in papers 2 and 3 were the

fluorescent compounds incorporated into insulin dramatically changes their fluorescence

spectra during fibril formation.

The probability distribution of transitions for absorption, in effect the relative peak intensity,

is often reflected in the appearance of a corresponding fluorescence spectrum. However,

fluorescence decay typically occurs from the lowest vibrational level of the S1 excited state.

This, together with the relaxation sometimes going from S1 to one of the higher vibrational

levels of S0, causes a wavelength shift to lower energies (higher wavelengths) for the

fluorescence spectra compared to the absorbance spectra – termed a Stokes shift (from its

discoverer G. G. Stokes).70 Further deviances from a simple mirror image of the absorbance

spectra can occur due to one or more transitions in the absorbance spectra of a fluorescent

compound being non emissive. This phenomenon where the wavelengths absorbed do not

correlate fully with the excitation wavelengths leading to emissive relaxation can be

observed in section 3.1.2.

22

1.10 Conducting materials in organic electronics

Electronics is by default all about the ability of a material to carry current – the conductivity.

Current is in effect a biased movement of electrons and therefore requires available

electrons free to move. The number of electrons available for movement, and thus the

conductivity, is related to the atomic composition of a given material. As mentioned in

section 1.9, interaction between neighboring atoms and molecules will create energy levels

which can become occupied by electrons. In a solid material, the high number of

neighboring atoms will cause so many energy levels to form that it becomes more

convenient to call a cluster of energy levels an energy band. Individual bands will be

separated by energy levels that electrons cannot possess – an energy gap, resulting in a

band-gap pattern. In an insulator, the highest occupied energy level of the valence band is

fully occupied and separated by an energy gap EG to the next energy level of higher energy

(figure 1.11 a). This material-specific energy gap, corresponding to the difference between

the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital

(LUMO) of individual molecules, prevents movement of electrons across the bandgap EG to

higher energy levels. Amyloid fibrils are typically non conducting, as demonstrated clearly for

insulin fibrils in paper 6, although conductance have been reported from amyloid like fibrils

made from a short peptide sequence.72 In contrast to insulators, metals i.e. good

conductors, have their highest occupied energy level – termed the Fermi level, situated

inside an energy band with no significant energy barrier blocking electrons from moving to

higher energy levels (Figure 1.11 a). Semi-conductors have a distinct energy gap in the same

manner as insulators, but the gap is small enough that there is a real possibility that

electrons from the lower energy band – the valence band, can move across the band gap to

the next energy band –the conduction band (Figure 1.11 a). The conductivity of semi-

conductors can be improved by the addition (n-doping) or removal (p-doping) of electrons,

leading to either electrons or holes as the main type of charge carriers. Semi-conductors are

particularly useful since their conductivity can be altered; in effect they can be switched on

or off and are thus an integral component of transistors. Amyloid fibrils functionalized with

the self-doped PEDOT-derivative PEDOT-S have previously been used to design

electrochemical transistors.73

23

Figure 1.11 – Conductivity and hopping, Energy bands and the band gap EG for a conductor, an

insulator and a semi-conductor. b, The chemical structure of the organic conductor PEDOT. c,

Intermolecular transfer of charge - hopping.

Semi-conductors are important not the least because they are at the heart of the field of

organic electronics. Conducting polymers such as PEDOT (a compound related to PEDOT-S –

used in chapter 6) and semi-conductors such as α-sexithiophene enable electronic

components to be designed without the use of metals. For conducting polymers made from

e.g. PEDOT (see figure 1.11 b), the delocalization of π-electrons throughout the π-conjugated

system is the key feature that allows for the movement of electrons, and thus the

conductivity. Although PEDOT is by itself is insoluble in water this can be circumvented by

using the PEDOT derivate poly(3,4-ethylenedioxythiophene)- poly-(styrenesulfonate)

(abbreviated PEDOT:PSS) – a p-doped water soluble complex where positive charges on the

PEDOT backbone structure is balanced by counter ions on the p-doping sulfonic acid PSS.

It should be noted that, although the transportation of electrons or holes in conjugated

systems is important for the conductivity of the material, the conductivity of e.g. a film made

from conducting polymers will also depend on the mobility of charge carriers throughout the

film, between polymers. A surplus charge (an electron or a hole) can also be considered in

the form of a local charge disruption – termed a polaron.74 The polaron can move not only

within one molecule, but also “hop” to an overlapping molecular orbital of an adjacent

molecule (see figure 1.11 c). Since a film of polymers is typically made from a dense network

of many polymer units, the transportation of charge will be heavily influenced by the

possibility of polaron intermolecular hopping and by extension the molecular packing of

polymers within the film.

24

In conclusion, for organic materials, the ability of a material to conduct current is intimately

linked to the presence of a conjugated π-electron system. This structural feature is also

commonly found for compounds with photophysical properties such as fluorescence. In

organic electronics, one feature often follows the other.

1.11 Standard film preparation methods for organic materials Most electronic devices such as e.g. organic light emitting diodes (OLEDS) are in a solid state

form, whereas materials based on organic compounds are prepared in dispersion. A

straightforward way of preparing solid state devices from one or more solution based

material components is film formation. There are numerous methods available for making

films. Although vapor deposition and variants thereof can be employed to good effect for

controlled film formation of small organic compounds, we have chosen in our work to focus

on solution based processes. The simplicity and potential for upscaling makes solution based

processes an attractive option for preparing material for organic electronics. Furthermore, as

vapor deposition by default involves vaporization as well as processing during vacuum, it can

be a (too) harsh treatment for many compounds. The simplest process for making a film

from solution is probably drop-casting (see figure 1.12 a), whereby a liquid is placed on a

surface and left to dry. Spin-coating, whereby a liquid sample is spun with a precise rotation

speed after being placed on a substrate (figure 1.12 b) is effective in making homogenous

and in terms of thickness well defined films, however, like drop-casting spin coating is

unsuitable for large scale film production. Other methods include dip coating –repeated

immersion/withdrawal of substrate into the sample solution (figure 1.12 c), numerous

printing techniques employed in conventional printing industry, and blade coating (figure

1.11 d).74

Figure 1.12 – Schematic representation of selected film preparation techniques. a, Drop casting b, Spin coating c, Dip coating d, Blade coating

25

Blade coating is also called doctor blading or knife coating. Several variants, with or without

heating, different types of “blades” etc. exist. However, the common feature is that a

solution is dragged across a substrate by a blade at fixed height with a set speed. Unlike e.g.

spin-coating, blade coating can easily be applied to large scale production. When applying

the standard techniques described above to aqueous solutions of amyloid fibrils, careful

consideration is required regarding e.g. the viscosity of the solution – which increases with a

higher fibril concentration. In fact, as described in chapter 5, a more suitable process for

achieving well defined and scalable films of functionalized amyloid fibrils is fine-tuned air-

water interface assembly.

26

Chapter 2 – Morphology control of amyloid fibrils,

paper 1

Material preparation using self-assembly systems will commonly result in the formation of

multiple end products. However, for processing and applications, a more selective process

resulting in a less heterogeneous end product is often desirable. Thus, it is advantageous to

be able to direct a self-assembly process towards a specific end product. In this thesis, the

work has been focused on the self-assembly of amyloid fibrils and their functionalization. As

described in section 1.7.2, the self-assembly of amyloid fibrils results in fibrous structures

with a diameter in the range of 3-10 nm and a length in the single digit µm range. However,

the final morphology can be significantly altered by the environment during formation and

the protein from which the fibrils are made. Furthermore, as mentioned in section 1.7.5, the

formation of protein spherulites acts as a competing reaction pathway to the formation of

fibrils. In fact, under many conditions spherulites rather than fibrils can constitute the main

product.65

2.1 The effect of agitation on amyloid fibril formation It has been established previously by others that agitation can influence in vitro fibril

formation.75 Specifically how depends on the nature of agitation. Under quiescent

conditions, fibril formation processes are limited by diffusion. Agitation by e.g. stirring can

increase the speed of fibril nucleation by increasing the frequency at which particles

encounter one another. The shear forces caused by stirring can also cause fragmentation of

fibrils, increasing the number of available fibril ends and thus increasing the active volume

where fibril growth occurs (see section 1.7.4). It has furthermore been shown that vigorous

stirring can prevent the formation of spherulites.76 This effect likely occurs mainly due to the

induced shear flow preventing the formation of spherulites. I.e. the aggregates representing

the early stages of spherulites growth are less stable towards shear forces than the

corresponding intermediates for fibril formation. An additional effect of stirring is a

limitation on the length of the formed fibrils. Longer fibrils will be prone to an alignment

along the direction of a shear flow (a fact utilized in chapter 3 for the application of flow

linear dichroism spectroscopy studies). Although a shear flow may allow smaller monomeric

units to move more freely and find their way to each other and to small fibril precursors, it is

27

likely that larger fibril fragments present greater resistance to the shear flow and thus the

probability of further growth is reduced and fragmentation increased. If so, continuous

stirring will prevent both the formation of spherulites as well as long fibrils.

Based on the facts and reasoning stated above we investigated the effect of partial stirring

on fibril formation. We put particular emphasis on studying the initial lag phase of the fibril

formation process, since, as mentioned in section 1.7.5, this coincides with the formation of

the spherulite precursors. Polarized optical microscope studies show that for quiescent

conditions, an abundance of spherulites can be observed by their distinctive Maltese cross

pattern (figure 2.1 a), in accordance with previous studies.65 Stirring at a late stage of the

reaction, after spherulites have been formed, causes visible fragmentation of spherulites

(figure 2.1 b). Stirring for the first 10 min of a 24 h reaction, spectacularly reduces the

amount of spherulites (figure 2.1 c) and stirring during the initial hour of reaction is sufficient

for a complete removal of spherulites (figure 2.1 d).

Figure 2.1 – POM-images of insulin samples after 24 h of heating for various conditions of stirring. a, Spherulites formed in the absence of stirring. b, Stirring employed for 1 h after 7 h of heating. c, Stirring employed for the first 10 minutes of the reaction. d, Stirring employed for the initial hour of the reaction. An additional feature observed when studying a sample with employed partial stirring as for

the samples of figure 2.1 c and d, is the emergence of liquid crystalline phases, appearing as

bright streaks in the backdrop. Liquid crystalline phases are composed of high aspect ratio

objects stacked parallel to each other. Liquid crystalline phases are visible with POM due to

being an anisotropic region, as described in section 1.5. Long amyloid fibrils will readily form

such loose aggregates at high concentration; however, because of the low concentration of

fibrils (due to spherulite formation) they are typically not formed for samples prepared

under quiescent conditions (as seen for figure 2.1 a).

28

Our studies indicate that partial stirring increase the amount of fibrils relative to spherulites.

Furthermore, partial stirring results in a more significant presence of longer fibrils as

compared to a corresponding sample stirred continuously. If studied by atomic force

microscopy (AFM), the difference in length between the shorter fibrils from samples stirred

continuously and the longer fibrils from samples stirred for the initial hour of reaction

becomes evident. Continuous stirring results mainly in fibrils roughly 1 µm long (figure 2.2 a)

while partial stirring will result in fibrils with a variety of longer lengths that by a cautious

estimate, barring full image analysis, reach up to 7 µm in length (figure 2.2 b).

Figure 2.2 – The impact of partial and continuous stirring on fibril morphology. a, A sample stirred continuously for 24 h. b, A sample stirred for the initial hour out of 24 h.

2.2 Influencing the reaction kinetics and morphology of internally

functionalized fibrils Building on a protocol for functionalization of protein fibrils previously developed by Solin et

al.,77 we have prepared numerous differently functionalized fibrils. The methodology is

based on grinding of an amyloid forming protein with a hydrophobic compound in solid

state. The grinding and the shearing forces this entails mechanically force the hydrophobic

compound to mix with the protein. After subsequently dissolving the compound-protein

composite in acidic water, the hydrophobic compound remains associated with the protein

molecules – protected from the water, as the protein assembles into fibrils upon heating

(see figure 2.3).

29

Figure 2.3 – Schematic drawing of the preparation procedure for internally functionalized fibrils.

Although no thorough statistical analysis has been performed, during the preparation and

analysis of the functionalized fibrils studied in this thesis, we have observed a limited effect

on the fibril morphology due to the compounds we have incorporated into fibrils. In

addition, we have observed a significant change in reaction kinetics; when incorporating the

laser dye Nile red into fibrils, the lag phase of the fibril formation is prolonged by more than

a factor of 2. Nile red has in other studies been shown to act as a probe of amyloid

formation,61 and we utilized this to use Nile red as an internal probe for fibril formation and

follow the reaction kinetics for Nile red functionalized fibrils. For the reaction conditions

studied, with an insulin protein concentration of about 2.5 g/l and without any

functionalizing compounds added the lag phase for fibril formation was determined to be

about 4 hours long, as followed by ThT.

In contrast to a reaction with only insulin, for insulin functionalized by grinding with Nile red,

the fibril formation process has a lag phase of about 10 hours (figure 2.4 a). Even with a two

fold increase of the protein-Nile red composite concentration, the lag phase as well as the

time frame of the entire reaction remains extended relative to a reaction with 2.5 g/l of only

insulin, as seen in figure 2.4 b. However, employing stirring drastically reduces the reaction

time and nullifies the lagging effect Nile red functionalization has on fibril formation kinetics

(figure 2.4 c).

30

Figure 2.4 - The kinetics of formation for Nile red functionalized fibrils. Fibrillation kinetics followed by Nile red fluorescence for insulin functionalized with Nile red in a 50:1 weight ratio. a, 2.5 g/l insulin functionalized with Nile red. b, 5 g/l insulin functionalized with Nile red. c, 5 g/l insulin functionalized with Nile red and continuous stirring at 1000 rpm.

When employing continuous or partial stirring with functionalized fibrils, we have observed

the same trends as for protein-only samples, i.e. a reduction of spherulite content and

relatively short or long fibril lengths respectively. The benefit for film preparation of

removing spherulites also becomes evident for Nile red functionalized fibrils when studying a

drop casted film with fluorescence microscopy (see figure 2.5 a and b). Thus, for Nile red

functionalized fibrils as with insulin-only fibrils, partial stirring can simultaneously speed up

the reaction kinetics and increase the yield of fibrils.

Figure 2.5 – Fluorescence microscopy images of drop casted Nile red functionalized insulin solutions after 24 h of heating without (a) and with (b) 1 h of initial stirring.

As mentioned in section 1.11, there are numerous methods available for making films from

organic materials. Blade coating spreads a liquid onto a substrate by slowly passing the

blade across the substrate. While doing so, the induced drag in the solution can cause

anisotropic objects to preferentially align along the direction of the propagation of the

blade. However, as with solution shear flow induced by spinning (see section 3.1.3), the

propensity to align in a shear flow is related to the aspect ratio of the dispersed objects in

the flow.76 In other words, longer (high aspect ratio) objects align more readily than shorter

31

(low aspect ratio) objects. We have demonstrated for samples of Nile red functionalized

fibrils that the difference in lengths related to continuous or partial stirring has a tangible

effect on the alignment of the fibrils in films prepared by blade coating. This can be done by

comparing the extent of polarization of the emitted light from Nile red functionalized fibrils

prepared with continuous or partial stirring, respectively. The experimental setup used for

such an analysis is outlined in figure 2.6 a. Fibrils prepared with partial stirring exhibited a 50

% (± 4 %) difference in emission intensity for perpendicular orientations of polarization (see

figure 2.6 b). In comparison, fibrils prepared with stirring showed a 7 % (± 3 %) difference for

the corresponding emission intensity (see figure 2 .6 c). That the polarization of the emitted

light from Nile red can be affected in this way implies a close relationship between the

orientation of the flat linear shaped Nile red and the fibril structure, a feature investigated in

detail for the compound α-sexithiophene – also a flat linear molecule, in section 3.1 and in

previous studies by others for Thioflavine T.78

Figure 2.6 – Nile red polarized light. a, Schematic drawing of the setup used to evaluate the extent of polarized emission from blade coated films of Nile red functionalized fibrils. b, The emission from films made from partially stirred samples. c, The emission from films made from continuously stirred samples.

32

Chapter 3 – The influence of fibril formation on

functionalizing compounds, papers 2 & 3

Amyloid fibrils have since their discovery been intensively studied by a range of analytical

techniques. However, using organic dyes that change their photophysical properties upon

attaching to fibrils remain a benchmark method for following the growth in vitro and to

identify and localize fibril deposits in tissues. As mentioned in section 1.7.3, compounds

known to interact with fibrils include ThT, Congo red, Nile red and oligothiophene-based

structures. Such structures have a structural commonality in being rather flat and linear

rather than bulky and/or branched. As it turned out during our studies of functionalized

amyloid fibrils, the shape and dimension of compounds interacting with fibrils may be a

crucial factor in their functionality as probes as well as for using fibrils as a template for

organizing a functionalizing agent. For reference, figure 3.1 lists all compounds employed as

internal functionalizing agents in this thesis.

Figure 3.1 – Compounds investigated for internal functionalization.

33

3.1 Dispersion and organization of internalized oligothiophenes Oligothiophenes are short oligomeric chains of thiophene units. By their nature, they are

linear in shape and have a flat aromatic π-conjugated backbone structure stretching along

the long axis of molecule. Other common characteristic traits of these compounds are their

semiconducting properties79, fluorescence and a strong hydrophobicity. α-sexithiophene (6T,

see figure 3.1) is a particularly well studied oligothiophene.

3.1.1 Optoelectronic properties of oligothiophenes

As with other thiophene compounds, the optoelectronic characteristics of 6T are related to

the effective conjugation length79 as well as the type of solid state crystal packing.80 The

effective conjugation length can be influenced by rotation between the individual thiophene

rings since this can bring π-electrons out of plane with each other and thus reduce orbital

overlap (see section 1.8).

Considering the possible effects of structural distortions on the photophysics of

oligothiophenes, it becomes evident that 6T will be highly sensitive to its environment. In

general, going from a solid crystalline state to a molecularly dissolved state will entail a blue

shift of absorption as well as fluorescence. Rotation of σ-bonds will also cause a general

broadening of the absorbance bands because of the constantly varying conjugation length.

In addition, although not studied herein, a broadening of absorbance bands will also follow

with a rise in temperature due to increased population of multiple vibronic states.81

In the solid state, oligothiophenes can stack in various ways. Thiophenes crystals commonly

stack in a herringbone pattern, promoting an edge to face interaction.79 However, π-stacking

of compounds such as thiophenes can sometimes take the form of so called H- or J-

aggregates where the ring systems of the interacting molecules are interacting face to face.

H-aggregates represent a face to face stacking as shown in figure 3.2 a, whereas J-

aggregates represent a head to tail pattern (see figure 3.2 b).82 H- and J-aggregates results in

an interaction between neighboring molecules that has a significant effect on the resulting

absorption spectra of compounds through dipole-dipole interactions; compared to a

monomeric state (i.e. molecularly dissolved), solid state H-aggregates characteristically

34

cause a blue shifted absorption with a low fluorescence yield. In contrast, J-aggregates cause

a red shift in absorption and a high fluorescence yield.83

The theoretical origin of the effects H- and J-aggregates have on the absorption spectra of

e.g. 6T can be explained by considering the stacking molecules as interacting dimers of

temporary dipoles.84 Going first from a single dipole to a dimer of two dipoles, the required

electronic transition energy to go from the ground state to an excited state is lessened

somewhat due to an attractive interaction between the two molecules. Considering next the

oscillating electronic dipole transition moments µ1 and µ2 (typically, and in the case of 6T,

along the long axis of the molecule) of the two molecules constituting the dimer; these can

interact with each other in either a repulsive or attractive manner depending on if the

oscillations of the dipoles are in phase or not. In other words, two neighboring dipoles in an

H-aggregate (parallel) configuration facing the same direction will interact repulsively, and

vice versa.

Taken together, of the two excited states available to a fluorescent dipole dimer in an H-

aggregate configuration, the upper excited state E2 will be very sparsely populated due to

rapid relaxation to the lower excited state E1. In contrast, since the resulting dipole transition

moment (µ1 + µ2) for the lower excited state E1 is dipole forbidden (the sum is zero), no

emission will occur from this state given perfectly aligned dipoles. In effect (because of

slightly misaligned dipoles), as predicted by this model, H-aggregates of 6T will have a very

weak emission and the absorption will be shifted to higher energies – a blue shift (see figure

3.2 a). For J- aggregates, i.e. dipoles with a head to tail configuration, the situation will be

reversed. An attractive interaction between two neighboring transition dipole moments will

occur for dipoles that are facing opposite directions (see figure 3.2 b). This generally results

in strong emission from the lower excited state E1 and a red shifted absorption.

35

Figure 3.2 – Schematic depiction of the dipole excitation model applied to H-aggregates (a) and J-aggregates (b) of 6T.

3.1.2 Dispersion of oligothiophenes in amyloid fibrils

After mixing 6T with protein by grinding, followed by dissolution of the resulting composite

in aqeous acid, 6T molecules are in a restricted environment and a significant population of

6T is likely to exist in a crystalline state packed together with neighboring 6T molecules.

When comparing the absorption maximum of 6T dissolved in chloroform – 436 nm, and that

of a 6T-insulin mixture dissolved in water – 397 nm (see figure 3.3 a, 0 h), there is a relative

blueshift in absorption. This shift can be explained by a strong presence of H-aggregates. In

previous studies, a characteristic emission spectrum of H-aggregated 6T has been shown to

have emission peaks at approximately 539 nm, 587 nm and 640 nm, the dominant peak

being 587 nm corresponding to the 0-1 vibronic transition.83 This correlates very well with

the emission from an insulin-6T mixture (figure 3.3 b, 0 h). Considering also the low

fluorescence yield and the blue shifted absorption, a significant presence of H-aggregates is

likely.

Intriguingly, when an insulin-6T mixture is subjected to heat, resulting in the conversion of

insulin into fibrils, the emission shows a significant blue shift and intensity increase (figure

3.3 b). Meanwhile, the absorption maximum undergoes a red shift (figure 3.3 a). Taken

together, these spectral changes indicate a significant change in the aggregation state of 6T

during fibrillation. By comparing the fluorescence decay time of an insulin-6T mix, fibrillated

insulin-6T and 6T dissolved in chloroform, the conclusion then becomes that the state 6T has

adapted in mature fibrils closely resembles that of molecularly dissolved 6T (see figure 3.3 c).

In other words; 6T becomes dispersed within the fibril structure.

36

Figure 3.3 – Spectral changes for a 6T-insulin mixture during 24 h of heating. a, Absorbance spectra. b, Photoluminescence spectra. c, Fluorescence decay curves.

3.1.3 Nanoscale templating of oligothiophenes

That oligothiophenes can disperse within amyloid fibrils raises an obvious question; how?

The likely answer lies with the core structure of the fibrils. As described in section 1.7.2,

fibrils have a main structural element consisting of elongated β-sheets. These form a

hydrophobic core region running along the fibril length creating elongated grooves. The

dimensions of such grooves make it feasible that molecules such as 6T can fit into these

regions (figure 3.4).

Figure 3.4 - A schematic drawing of the intercalation of 6T in amyloid fibrils. a, A fibril and a representative protofilament component. b, 6T intercalated in part of the groove structure of the protofilament component in a.

This assumption is supported by studies made on the well-established amyloid interacting

compound ThT indicating a similar binding mode with its long axis along the long axis of the

fibril.78 Based on molecular dynamics simulations, the main binding site of ThT to amyloid

fibrils has been suggested to be a type of groove as that described in section 1.7.3.85 X-ray

37

crystallography studies on small peptide fragments designed to mimic the amyloid fibril

structure supports this statement.86 The dimensions of the extended groove region further

imply that a long linear molecule such as the oligothiophene 6T, internalized into the fibrils

by grinding prior to fibril formation, will be prone to end up oriented along the long fibril

axis, along the groove.

An alignment such as that proposed in figure 3.4 for 6T is conveniently studied by flow linear

dichroism (LD) - a technique where a liquid sample is exposed to a strong shear flow, aligning

high aspect ratio objects along the flow direction. The shear flow is achieved by placing the

sample solution in a rotating cylindrical Couette cell. There, the solution is trapped between

two transparent surfaces – one fixed while the other is moving, creating a laminar shear flow

(see figure 3.5 a). Simultaneous application of linearly polarized light on the sample allows

for determining whether the long axis of amyloid fibrils and that of incorporated 6T

molecules, are aligned. The high aspect ratio protein fibrils will be aligned by the shear flow.

Since, by definition, the output signal in flow linear dichroism is a sum of light absorbed in

parallel to the direction of the shear flow subtracted by light absorbed perpendicular to the

direction of the shear flow, a positive signal indicates material with its mean transition

moment aligned along the flow. Thus, since the LD absorbance of 6T is positive, both 6T and

the fibrils must be aligned in the same direction (see figure 3.5 b). This implies strongly that

the dispersion of 6T throughout the fibril structure occurs with the long axis of 6T along the

direction of the grooves, i.e. along the long fibril axis.

Figure 3.5 – Flow linear dichroism. a, Schematic illustration of the Couette measurement setup. b, LD spectra for a 6T-insulin composite mixture exposed to heating for 24 h. Aliquots were removed at the indicated times.

38

3.1.4 Transferring nanoscale organization to the macroscale

The organization of oligothiophenes along the long fibril axis implies not only that this is a

general binding mode for linear hydrophobic molecules to amyloid fibrils, but also that the

nanoscale organization of dyes can be transferred to the macroscale by in turn aligning the

templating objects, i.e. the fibrils. This was achieved in chapter 2 by using blade coated

dispersions of amyloid fibrils functionalized with Nile red. An alternative and modular way of

achieving such macroscale organization is by utilizing photolithography to design patterned

PDMS stamps. Photolithography is a standardized set of methodologies useful for preparing

PDMS (polydimethylsiloxane, in effect, transparent rubber) stamps with designed patterns,

e.g. in the shape of channels. Thus, a PDMS stamp placed against a glass surface can be used

to form µm-wide channels. Due to capillary forces, a liquid dispersion of 6T functionalized

fibrils placed at the orifice of such a channel will be dragged along the channels length to fill

up the channel. After subsequent removal of the stamp, it is possible to achieve large scale

patterning of 6T functionalized fibrils (see figure 3.6 a). Since 6T is fluorescent, the

patterning is conveniently demonstrated by polarized fluorescence microscopy as seen in

figure 3.6 b and c. With the polarized optical microscopy setup used here, light polarized

horizontally and vertically, respectively, is collected and displayed as two corresponding

images of the same area. When comparing the images generated in this fashion in figure 3.6

b and figure 3.6 c, it becomes evident that the maximum brightness (i.e. the highest light

intensity) is achieved for sample lines aligned in parallel with the direction of polarization

(figure 3.6 b).

Figure 3.6 – Macroscale patterning of 6T functionalized fibrils. a, Schematic drawing of the patterning procedure. b, Polarized fluorescence microscope images of aligned 6T-functionalized fibrils. The polarization direction is indicated by double arrows.

39

3.2 Evaluating the effect of fibrillation on hydrophobic compounds As mentioned previously, in chapter 1, amyloid fibrils have been studied to a large extent

due to connections with various diseases. In such studies, a central part of the research is

the ability to identify amyloid deposits in tissue. Commonly done by staining with

compounds acting as probes for the presence of amyloid fibrils, improving existing and

identifying new probes is an active area of research.60,61,87–92 Such probes are in effect

compounds that have a specific interaction with amyloid fibrils and thus, identifying probes

goes hand in hand with identifying functionalizing compounds of particular interest to

incorporate into functionalized amyloid fibrils in vitro for organic electronics and photonics

applications. Finding new compounds that act as probes is, however, not trivial. One

problem is that most commercially available compounds are synthesized in a hydrophobic

setting and as a result are poorly water soluble. To apply and test such compounds they

would first have to be made water soluble by e.g. adding charged and/or polar substituents.

Internalization of hydrophobic probe candidates into amyloid fibrils and screening for probe

functionality of hydrophobic compounds represents a time saving alternative. Moreover, the

resulting functionalized fibrils may find applications within organic electronics and

photonics.

3.2.1 Amyloid probe characteristics

The studies described in section 3.1 on the organization of internalized 6T in amyloid fibrils

hint that the photophysical properties of a fluorescent compound with a shape fitting the

hydrophobic groove pattern of amyloid fibrils is likely to be affected by attachment to the

fibrils. This is further supported by a glance at the linear flat dimensions of the established

amyloid probes of ThT, congo red and Nile red (see figure 1.5). Also, as mentioned in section

3.1.3, previous studies on the well-established probe ThT suggest a similar mode of binding.

It is a significant advantage if the photo physical changes upon incorporation of a dye into

fibrils are characteristic and easily identifiable. As mentioned in section 1.7.3, established

probes such as ThT typically present a significant intensity increase in fluorescence upon

binding to amyloid fibrils. Another common property is a shift in the wavelength of emission

maximum. For any such changes to occur, however, the compound must be sensitive to

changes in its environment. In the case of the well-studied compound ThT, it has been

40

suggested, based on computational studies, that the free bond rotation in solution quenches

the fluorescence by presenting an alternative non-radiative route of relaxation for excited

electrons.93 Upon binding to fibrils, the restriction in rotation prevents the non-radiative

relaxation and results in an increase in fluorescence intensity for amyloid bound ThT.

Compounds such as Nile red on the other hand are sensitive to a change in the polarity of

the environment and adhering to the hydrophobic groove of amyloid fibrils represents a

significant change in polarity compared to that of water. For thiophenes, the conjugated

backbone structure is highly sensitive to conformational changes and results in specific

spectral changes for thiophene emission upon association with fibrils.94

3.2.2 Identifying hydrophobic compounds as amyloid probes

Using the same functionalization procedure as described in section 2.2, evaluation of

amyloid probe functionality can be performed for hydrophobic compounds. By evaluating

the fluorescence spectra of a given set of compounds, protein-incorporated compounds that

exhibit a significant change in fluorescence upon fibrillation can be identified. Two examples

are shown in figure 3.7.

Figure 3.7 – Fluorescence spectra before and after 24 h of heating. a, BMSBP b, DCM. Chemical structures of the fluorescent compounds are included as insets. 0 h refers to the sample before fibrillation. 24 h refers to the sample after fibrillation.

Based on the significant fluorescence intensity increase upon fibrillation seen in figure 3.7,

DCM and BMSBP can be identified as potential amyloid probe candidates. However, to test a

hydrophobic probe candidate for functionality in an aqueous solvent requires the compound

to be tested in a water soluble form. The full probe evaluation procedure is illustrated in

41

figure 3.8. In a case such as DCM, due to its slight polarity, water solubility can be achieved

by using a co-solvent such as methanol. For 4,4-bis(2-methoxystyryl)-biphenyl (BMSBP), a

water soluble structural analog is the commercially available Disodium 4,4′-bis(2-

sulfonatostyryl)-biphenyl (also known as Tinopal CBS).

Figure 3.8 - Schematic illustration of the methodology for rapid amyloid probe screening. In step 1, a hydrophobic compound is ground with bovine insulin and after solvation and amyloid formation spectral changes are evaluated. For promising candidates, the corresponding hydrophobic compound substituted with polar groups is tested in step 2, by addition of the potential probe to preformed amyloid fibrils. Alternatively, if the probe is sufficiently hydrophilic a co-solvent miscible with water can be employed.

Using the water soluble forms of probe candidates, it is possible to test the probe

functionality by following the kinetics of amyloid fibril formation. A comparison of the

kinetics curves obtained by using DCM, Tinopal CBS and the established probe ThT,

respectively, reveals a striking similarity and thus the amyloid probe functionality of the

investigated compounds (see figure 3.9).

42

Figure 3.9 - Insulin amyloid fibril formation kinetics followed by ThT (squares), Tinopal CBS (filled

circles), and DCM (triangles) fluorescence measured at pH 7.4.

43

Chapter 4 – The impact of functionalizing compounds

on the fibril reaction pathway, paper 4

In our studies of amyloid fibrils, the presence of an internalized functionalizing compound, i.e. a compound that has been incorporated into fibrils by the grinding methodology outlined in section 2.2, has not resulted in any dramatic changes in the morphology of the functionalized fibrils However, the presence of a hydrophobic compound may alter the possible aggregation pathways involved in amyloid formation. With either partial or continuous stirring, this alteration has a limited effect as observed during our studies during standard reaction conditions (with one notable exception regarding the air-water interface assembly of films, see chapter 5). However, during quiescent conditions, when spherulites will form, the presence of a hydrophobic additive can be dramatic both with respect to an effect on protein spherulite formation as well as to a highly specific ordering of the additive within such aggregated species as schematically represented in figure 4.1.

Figure 4.1 - Schematic representation of the protein spherulite formation occurring upon heat treatment of insulin in acidic water. a, insulin with 6T b, insulin-only.

44

4.1 Particle size and implications for alternative aggregation routes When studying insulin particle size with dynamic light scattering (DLS), the hydrodynamic

radius of insulin in an aqueous solution with a pH close to pH 2 is about 1.5 nm.95 It should

be noted that hydrodynamic radius is an estimate that is based on the calculated volume

displaced by a particle in the shape of a sphere. Put another way, a spherical particle A and a

rod B may have the same hydrodynamic radius if the rod rearranged into a spherical shape

would have the same volume as the sphere A. None the less, the hydrodynamic radius is a

convenient way of quantifying the size of small particles. Estimation of the hydrodynamic

radius with DLS is based on the recording of a correlation curve – a measure of how fast

particles move in a solution which can be directly linked to particle size. Differently sized

particles can thus be detected by a relative change in their corresponding correlation curves,

as seen in figure 4.2. When incorporating 6T into insulin by grinding, the mixture shows a

distinct increase in particle size relative to insulin only. This is logical given that 6T is present

in a clustered form and most likely encapsulated by surrounding insulin molecules –forming

a larger sized aggregate. In addition, since spherulites, as mentioned in section 1.7.4, form

from an amorphous aggregate, the presence of large 6T-insulin aggregates at the start of a

reaction may have a significant influence on spherulite formation.

Figure 4.2 – DLS data for samples heated for 4 hours at 65o C. a, Correlation curves of insulin only (solid line) and 6T-insulin (dashed line). b, Estimated hydrodynamic radius for insulin only (solid line) and 6T-insulin (dashed line).

45

4.2 Characteristics of insulin-6T spherulites When spherulites are formed from a mixture of 6T and insulin, in addition to spherulites of

the typical size of 50 µm, the sample is dominated by spherulites typically around 400 µm,

but reaching up to as much as 1.4 mm in diameter (figure 4.3 a). Containing 6T, these

structures have a strong orange color, and are strongly fluorescent when photoexcited

(figure 4.3 b). Polarized light microscopy reveals the radially oriented structure component

characteristic of spherulites (figure 4.3 c), linking them to the structural composition of

insulin-only spherulites described in section 1.7.5.

Figure 4.3 – Large insulin-6T spherulites. a, Photograph of a macroscopic spherulite with a diameter of 1.4 mm. b, Surface view of the sphere in a under 420 nm illumination. c, POM-image of a large insulin-6T spherulite. d, Fluorescence microscope image of Spherulites fused together by merging growth excited at 405 nm.

The presence of 6T in an insulin-6T mixture not only has an effect on particle size; 6T is an

extremely hydrophobic compound. The hydrophobicity of 6T spherulites is likely to be

significantly higher than their insulin-only counterparts. This is illustrated by the ability of 6T

spherulites to merge together to form larger structures. Figure 4.4 shows several spherulites

that have merged together into one unit. In addition to promoting the irreversible merging

growth of spherulites, the apparent amplified hydrophobic interaction of insulin

functionalized with 6T can be exploited for the formation of air-water interface assembled

films, as detailed in Chapter 5.

4.3 Inhomogeneous dispersion of 6T in spherulites During the formation of 6T functionalized spherulites, 6T that has not been intercalated in

the hydrophobic grooves of fibrils becomes enriched at the surface region of the spherulites.

Since water is present in the interior of spherulites, there is likely a significant driving force

for the highly hydrophobic 6T molecules not already incorporated in fibrils to crystallize.

Assuming for the formation of 6T functionalized spherulites a radial fibril growth extending

46

out from an amorphous core, the area close to the surface will be less dense than closer to

the core. The larger crystalline structures may thus be more likely to accumulate at the

exterior region of the spherulite. The extent of inhomogeneity in the state of 6T within a

spherulite can be visualized by fluorescence microscopy and fluorescent lifetime imaging

microscopy (FLIM). Fluorescence microscopy performed on a slice cut out from a spherulite

results in an image where the central region of the slice has a strong green emission and an

outer region of weaker emission (figure 4.4 a).

As with conventional fluorescence lifetime spectroscopy, FLIM detects the fluorescent decay

over time for fluorescent compounds. This can then be used to distinguish regions in a

fluorescent sample that contain fluorophores with different lifetimes.96 As seen previously in

figure 3.3 c, fluorescence decay time can be used to distinguish between dispersed 6T

(longer lifetime) and 6T in a crystalline form (shorter lifetime). FLIM images displaying the

distribution of recorded lifetime notably show two distinct lifetimes, corresponding to

dispersed and crystalline 6T respectively and their inhomogeneous distribution within a slice

of a spherulite (figure 4.4 b and c).

Figure 4.4 – FLIM data for a slice of an insulin-6T spherulite. a, For reference, a fluorescence microcopy image. b, A FLIM image depicting the lifetime distribution of the slice in a, color coded from red (300 ps) to dark blue (1100 ps). c, Intensity weighted mean lifetime of the slice in a.

47

Chapter 5 – Air-water interface assembly of

functionalized amyloid fibril films, paper 5

From our studies of functionalized fibrils in Chapters 2-4, it is possible to conclude that

amyloid fibrils have a distinct propensity to align along each other and that the aggregation

of fibrils into larger aggregates can be heavily influenced by the presence of hydrophobic

additives. In contrast, electrostatic interactions between fibrils tend to discourage

aggregation and maintain fibrils in dispersion. As indicated by the results discussed in

chapter 4, 6T functionalization can modify the interaction balance during self-assembly. This

can be utilized to promote the formation of highly anisotropic and nm-µm thick films of

functionalized fibrils measuring several cm2, as shown schematically in figure 5.1. We have

focused our studies on films made from 6T-functionalized fibrils, although films were made

also from fibrils functionalized with a perylene bisimide derivative.

Figure 5.1 – Schematic representation of air-water interface assembly of protein fibril films and

chemical structures of the functionalizing compounds used.

5.1 Air-water interface assembly of proteins A well-established phenomenon is that amphiphilic molecules, of which a well-known

example is phospholipids, can readily form films at an air-water interface. This process is

driven by the assembling molecules having two distinct parts; one hydrophilic and one

hydrophobic, were the assembling molecules are facing the (hydrophobic) gas with their

48

hydrophobic end, while the hydrophilic part is turned into the aqueous solution.97 This type

of self-assembly has been observed also for proteins98 and amyloid fibrils have been shown

to form monolayers of amyloid fibrils at an air-water interface as a part of amyloid β-sheet

formation.99 For proteins, large scale self-assembling films at the air-water interface can be

observed for food preparation of Yuba – made by heating soy protein and inducing a

convection flow that drives the protein towards the interface where it aggregates into a solid

state film.100 The air-water interface assembly of amyloid fibrils studied in this thesis is, like

Yuba, facilitated by an induced convection flow. However, the controlled air-water interface

assembly of amyloid fibrils into thin films is governed by a complex set of interactions and

require precise control of reaction conditions.

5.2 Factors governing air-water protein fibril interface assembly As already stated, electrostatic interactions between insulin fibrils will inhibit large scale

aggregation. In contrast, the increased hydrophobicity of 6T-functionalized fibrils appears to

promote aggregation, as indicated in section 4.2 for 6T functionalized spherulites. Insulin

fibrils functionalized with a hydrophobic additive are thus likely to be more attracted to a

hydrophobic air interface, as well as to each other, than their insulin-only counterparts.

From our studies of the film formation at the air-water interface, we propose the following;

by lowering the fibril concentration, it is possible to promote the interaction with the

(hydrophobic) air interface at the expense of fibril-to-fibril hydrophobic interactions.

However, the effect of the air interface is only valid for fibrils in its vicinity; the bulk of a

solution will be unaffected. Thus, promoting a strong convection flow through a vertical

thermal gradient benefits air interface film formation by gradually exposing all the fibrils in

the bulk to the presence of the hydrophobic air interface. Lastly, for a preferential assembly

at the air interface to occur, the repulsion between fibrils must be high enough to prevent

the spontaneous assembly of aggregates in the bulk solution. In other words; the self-

assembly of amyloid fibril films can in effect be viewed as a that of a system of anisotropic

colloidal particles governed by a DLVO-model type self-assembly (see section 1.2) and driven

by a heat induced convection flow to form films at the air-water interface. In addition, the

convection flow can likely disrupt the formation of fibril clusters in the bulk.

49

5.3 Characteristics of air-water interface assembled insulin fibril films Given a sufficiently low protein concentration, a convection flow to promote mixing, and a

suitable balance between fibril-to-fibril repulsion and attraction, insulin fibrils will self-

assemble into a film at the air-water interface. If a dispersion of 6T functionalized fibrils

(figure 5.2 a) is left under such conditions over time, fibrils in the bulk solution will become

incorporated into the surface film leaving the bulk devoid of fibrils, as shown in figure 5.2 b.

The film in its entirety can then be transferred to e.g. a glass slide (figure 5.2 c). Fibrils

functionalized with a perylene bisimide derivative gives a similar result (figure 5.2 d-f). In

addition, fibrils functionalized with different hydrophobic compounds can be made to form

sequential multilayer films (see figure 5.2 g). The not insignificant cohesive strength of the

film is demonstrated visually by a free hanging film in figure 5.2 h.

Figure 5.2 - Photographs showing the assembly process of air-water interface assembled films made at a protein concentration of 0.5 g/l. a, A solution of 6T functionalized fibrils. b, The solution in a after 72 h. c, The extracted film on a glass slide. d, A solution of perylene bisimide derivative - functionalized fibrils. e, The solution in d after 72 h. f, The extracted film in e transferred to a glass slide. g, A double layer film consisting of 6T-functionalized fibrils and fibrils functionalized with the perylene bisimide derivative. h, A film made with a fixed syringe present during the film formation, enabling removal of the underlying water.

The thickness of an air-water interface assembled fibril film can be varied by altering the

amount of fibrils available for incorporation into the film. A smaller volume or lower

concentration of fibrils will thus result in a thinner film. In addition, the resulting film

50

thickness can be controlled simply by extracting the film at different time points during its

formation. A summary of the thickness variations dependent on incubation time and

concentration for 6T functionalized films is shown in table 1. Films made at a protein

concentration of 6,25 mg/l notably exhibit a thickness of approximately 85 nm, with a

surface roughness Ra of 2 nm estimated over 200 µm.

Table 1 –Thickness of films formed from 6T-functionalized fibrils at various concentrations and

reaction times.

When considering the thickness variation over time for film formation in table 1, the values

hint at a non-linear growth process. This observation is supported by extracting samples at

selected time points and plotting the resulting absorbance at 278 nm; the decay of the

amount of fibrils in the solution can be fitted to a standard exponential decay curve (figure

5.3 a). This holds for both 6T functionalized fibrils as well as fibrils functionalized with the

perylene bisimide derivative. In addition, the data of the film thickness over time appears to

follow an exponential increase (figure 5.3 b).

Figure 5.3 – Film formation over time. a, Absorption decay for bulk solution. b, The plotted

thickness variation of films removed after 4, 12 and 72 h seen in table 1.

51

Furthermore, the same procedure shows that an existing film can increase the speed of

subsequent film formation for fibrils in the bulk solution. In contrast, insulin without any

functionalizing compound shows only a minor change in absorption after 72 hours (see

figure 5.3 a), indicating that film formation is greatly enhanced for fibrils functionalized with

the hydrophobic compounds.

5.4 Control of film formation anisotropy A film such as the one shown in figure 5.4 a, when viewed through crossed polarizers after

placement on a glass slide, shows an extinction pattern reminiscent to that expected from

two dimensional radially oriented anisotropic structures (figure 5.4).63 This indicates that

such films have a significant component of anisotropy.

Figure 5.4 – Air-water interface assembled films made in round vials. a, A film with a

diameter of 1 cm. b, The film in a viewed with crossed polarizers. c, A film with a diameter of

1 cm after drying, viewed with crossed polarizers.

The existence of an extinction pattern such as the one visible in figure 5.4 b indicates that

functionalized fibrils attaching to a growing film do so preferentially in alignment with

already film-associated fibrils. The structural organization of films made at the air-water

interface can, in fact, be controlled by simply altering the type of vial used for the reaction. A

rectangular shaped glass container (i.e. a TLC well) will not only result in cm2 sized films. The

films will have the characteristics of linearly polarized sheets. The extent of anisotropy is

demonstrated by viewing such a film with crossed polarizers (figure 5.5); by turning the

sample table relative to the crossed polarizers, the film shows a 45 degree periodicity in

maximum brightness – as expected from a surface with high anisotropy. The area

52

showcasing the behavior of figure 5.5 c and d was determined to be in excess of an area of 4

cm2 – an area too large to capture fully within the scope of a standard polarized optical

microscope.

Figure 5.5 – Selected POM-images of a highly anisotropic fibril film. The sample was turned by a

number of degrees relative to the crossed polarizers as indicated in the images.

The connection between the orientation of internalized linear hydrophobic molecules such

as 6T and the direction of the fibrils hosting them was described in detail in section 3.1.

Performing the same type of experiment on polarized emission from a solid state film as was

used for blade coated Nile red fibrils described in section 2.2, the high degree of anisotropy

of an air-water interface assembled film prepared in a TLC well was shown to result in a 70 %

difference in emission intensity for perpendicular orientations of polarization (figure 5.6). In

conclusion, the air-water interface assembly of protein fibrils can be utilized to prepare

highly ordered films with tunable dimensions and optical properties.

Figure 5.6 – Fluorescent films. a, A fluorescence microscope image of an air-water interface assembled film functionalized with 6T excited at 405 nm. b, Polarized emission; altering the direction of a polarizer by 90 degrees relative to a film will significantly alter the recorded emission intensity, indicating a high level of anisotropy for the fluorescent 6T-molecules in the film.

53

Chapter 6 – External functionalization of amyloid

fibrils, papers 6 & 7

External functionalization of amyloid fibrils can be achieved by the adherence to fibrils of

compounds from solution. Polyelectrolytes with charged side groups are a natural choice for

external functionalization as they will adhere given a charge complementary to that of the

amyloid fibrils intended for functionalization. One example of a compound suitable as

functionalization agent is thus the negatively charged conducting compound PEDOT-S,

described in section 6.1. Combining PEDOT-S with amyloid fibrils is of particular interest

since although amyloid fibrils have a wire-like shape desirable for a conductor, they are

inherent insulators. In this manner, the structural properties of amyloid fibrils can be

combined with the conducting capability of PEDOT-S.

6.1 PEDOT-S Conjugated systems made up of a sequence of thiophene rings represent a well-established

motif for semiconducting compounds used in organic electronics. An example being

conducting polymers such as the derivatives of poly(ethylenedioxythiophene) (PEDOT).101

However, as mentioned in section 1.10, since PEDOT is insoluble in water, PEDOT is often

combined with PSS – a negatively charged insulating polyelectrolyte. Although PEDOT:PSS is

dispersible in water, functionalization of amyloid fibrils with PEDOT:PSS is problematic,

possibly due to the presence of the bulky PSS component preventing effective adherence to

amyloid fibrils and/or electrostatic interactions by charged side chains.

Unlike PEDOT:PSS, Alkoxysulfonated poly(ethylenedioxythiophene), PEDOT-S for short, is a

self-doped (via the sulfonate groups on the side chains) oligomeric compound. Because of

the negatively charged sulfonate groups on the side chains, PEDOT-S readily attaches to

insulin amyloid fibrils at low pH; with the isoelectric point of bovine insulin being 5.3, insulin

fibrils will be positively charged at pH 1.6 and thus favor an attractive electrostatic

interaction with PEDOT-S. With a high water solubility and a conductivity of 30 S cm-1,

PEDOT-S has previously been shown to act as a functionalizing agent for amyloid fibrils

resulting in a conductive biomaterial.73

54

6.2 Interaction of internalized and externally added functionalizing

compounds There are a number of different processes that can affect the excited state of a given

molecule and as exemplified in chapter 4 for 6T in the form of H-aggregates, not all are

intramolecular. However, intermolecular interactions are limited by distance. Since amyloid

fibrils typically have a diameter of 3-10 nm, internalized compounds are within the

geometric bounds of direct interaction with molecules on the fibril surface. In other words;

compounds adhering to the surface of fibrils may interact with compounds incorporated into

the fibrils and influence their photophysical properties.

6.2.1 Energy transfer and quenching, the Förster distance

When a molecule in an excited state is in close proximity to a neighboring molecule,

alternative routes for relaxation of the excited state (other than through emission) may

become available. The excited state of a donor molecule can be transferred to another

molecule - an acceptor, in an energy transfer process with (emissive) or without (non-

radiative, quenching) the release of a photon. In the case of emissive energy transfer, the

excited state of the acceptor may decay by fluorescence. In contrast, if alternative relaxation

processes are available to the acceptor, the energy transferred from the donor will not result

in emitted light. The energy transfer will then instead result in quenching of the donors

emission.

Whether the transfer of energy result in radiative decay or not, there are two factors limiting

the extent of energy transfer between a donor and an acceptor; the distance separating the

two compounds, and the degree of spectral overlap between the emission of the donor and

the absorption of the acceptor. A common way of quantifying the impact of distance on

energy transfer is by the Förster distance for resonance energy transfer (RET). A typical

Förster distance for a donor-acceptor pair is in the range of 3 - 6 nm and the efficiency of

energy transfer for a single donor-acceptor pair at a fixed distance r is described by equation

6.1.70 The efficiency of energy transfer E thus changes by the intermolecular distance r to the

power of 6, and the Förster distance R0 can be defined as the intermolecular separation that

yields 50 % energy transfer efficiency.

55

Equation 6.1 - Efficiency of energy transfer.

6.2.2 Non radiative energy transfer between fibril-internalized 4,4’-bis(2-

methoxystyryl)-biphenyl (BMSBP) and externally added PEDOT-S

The absorption spectra of the non-emissive PEDOT-S overlaps significantly with the emission

spectra of the dye BMSBP (see figure 3.1 for the chemical structure and figure 3.7 a for the

emission spectrum), and the radius of amyloid fibrils falls within the range of a likely Förster

radius, thereby in principle enabling the intermolecular non radiative energy transfer

between BMSBP and PEDOT-S. Furthermore, fibrils functionalized with the fluorescent dye

BMSBP can be easily coated with PEDOT-S (figure 6.1 a) due to favorable electrostatic

interactions (see figure 6.1 b).

Addition of PEDOT-S to BMSBP functionalized fibrils results in a quenching of the

fluorescence from BMSBP (figure 6.1 c). The quenching effect of an increasing ratio of

externally added PEDOT-S to fibril internalized BMSBP can be seen in figure 6.1 c. At a 1:1

weight ratio, efficient quenching is achieved.

Figure 6.1 – PEDOT-S quenching of BMSBP. a, The chemical structure of PEDOT-S. b, Schematic

representation of PEDOT-S association to BMSBP functionalized protein fibrils. c, BMSBP

fluorescence as a function of PEDOT-S concentration.

As the quenching effect of PEDOT-S on fibrils functionalized with BMSBP is dependent on

proximity between the donor (BMSBP) and the acceptor (PEDOT-S), the quenching is

56

dependent on adherence of PEDOT-S to the fibrils. Furthermore, since the negative charge

of PEDOT-S promotes electrostatic attraction to positively charged fibrils, the addition of salt

to a solution containing positively charged functionalized fibrils will screen charges and

reduce the electrostatic attraction between positively charged insulin fibrils and the

negatively charged PEDOT-S. In paper 6, we have shown that a 0.5 M concentration of NaCl

will reduce the quenching efficiency of PEDOT-S by 30 %. However, failure to achieve a

complete release of PEDOT-S purely by salt aided screening indicates that electrostatic

interaction is not the only driving force for PEDOT-S adhesion to fibrils. It is likely that the

hydrophobic backbone structure of PEDOT-S also has a significant role to play in the

adhesion of PEDOT-S to amyloid fibrils.

6.3 PEDOT-S induced conductance of amyloid fibrils Since PEDOT-S is capable of forming a conductive film in and of itself, it is crucial to

determine the origin of conductance when studying fibrils functionalized with PEDOT-S. This

can be done by conductive AFM (C-AFM), whereby the AFM-tip acts as an electrode

complementary to a second electrode on the substrate on which the presumptive

conducting sample is placed. By comparing a topography image with the corresponding

current map, conducting areas can be linked to structures seen in the topography image.

In paper 6, we used C-AFM to verify that the conductance of a sample of PEDOT-S mixed

with fibrils did indeed originate with the fibril structures; a comparison of a topography

image (figure 6.2 a) and the corresponding current map (figure 6.2 b) shows current

originating from the PEDOT-S coated fibrils. In addition, the absence of background current

verified that the rinsing procedure we developed to remove PEDOT-S not associated with

the fibrils was successful; utilizing the strong interaction of PEDOT-S with fibrils, repeated

centrifugation and subsequent water dilution and resuspension of the resulting fibril-PEDOT-

S pellets allows for an apparent complete removal of excess PEDOT-S not associated with

fibrils.

57

Figure 6.2 – C-AFM performed on PEDOT-S coated fibrils. a, AFM topography image. b, Current map of PEDOT-S covered fibrils.

6.4 External functionalization of fibril superstructures A fundamental electronic component is that of the electromagnetic coil. It is, literally, at the

core of components such as electromagnets, inductors and transducer coils. Although

routinely produced at the macroscale, miniaturizing such components to the µm size range

is considerably less trivial.102 However, superstructures of insulin amyloid fibrils can be made

to form helical superstructures. Such superstructures can then be used as templates to

organize PEDOT-S in a helical fashion at the microscale.

Starting from helical insulin superstructures prepared, and in previous studies thoroughly

investigated, by Dzwolak et al.,103–105 PEDOT-S can be added to achieve a coating of PEDOT-S,

schematically depicted in figure 6.3. The superstructures can be prepared in either “right

handed” or “left handed” helical orientation. The bifurcation during preparation as

categorized by Dzwolak et al. results in structures termed either –ICD (left handed) or +ICD

(right handed), based on the induced CD signal of adhered ThT.104

58

Figure 6.3 – PEDOT-S coating of insulin fibril superstructures.

Due to the chiral nature of the insulin superstructures, the normally achiral PEDOT-S will

become organized by the protein superstructure template into a chiral helical form. The

induced chirality of PEDOT-S can be confirmed by CD spectroscopy.

As shown in equation 6.2, the output signal of the CD spectrometer results from the

differential absorbance (ΔA) of right handed (RA) and left handed (LA) absorbed circularly

polarized light. Therefore, a sample only absorbing right handed polarized light will yield a

signal with opposite sign to that of a sample absorbing only left handed polarized light.

ΔA = LA – RA

Equation 6.2 – Differential absorbance.

Thus, as expected from different orientations of helicity, PEDOT-S adhered to –ICD

structures yields an induced CD signal with an opposite (positive) sign to that of PEDOT-S

adhered to +ICD structures (see figure 6.4 a). The signal below 300 nm originates from the β-

sheet secondary structure of protein whereas the signal seen above 300 nm originates with

PEDOT-S, the latter being the induced CD signal due to helical organization of PEDOT-S by

the superstructures. A UV-vis absorbance spectrum of PEDOT-S is shown for reference in

figure 6.4 b.

59

Figure 6.4 – Induced CD from PEDOT-S covered superstructures. a, CD spectra of Insulin fibril superstructures (dashed) and PEDOT-S covered insulin fibril superstructures (solid lines) in 100 mM NaCl. b, PEDOT-S absorption curve.

6.4.1 A stabilizing effect of PEDOT-S adherence to insulin fibril superstructures

Insulin amyloid fibril superstructures of the kind prepared by Dzwolak et al. are ordered

aggregates of fibrils. In the absence of salt, charge repulsion between fibrils will have a

highly destabilizing effect on the aggregates. However, the same washing procedure

developed for PEDOT-S covered fibrils and described in section 6.3 can be used for insulin

superstructures. Figure 6.5 illustrates the effect water immersion has on the ability of

superstructures to helically organize PEDOT-S; if PEDOT-S is added to insulin fibril

superstructures while in a salt solution, and the salt is later removed, the PEDOT-S coverage

results in an induced CD signal (see figure 6.5 a). In contrast, if PEDOT-S is added to helical

superstructures that have first been incubated in water without salt, the ability of the

superstructures to organize PEDOT-S is lost (figure 6.5 b).

Figure 6.5 - CD spectra on PEDOT-S covered superstructures diluted in water. a, Solid lines - centrifuged washed PEDOT-S-covered insulin fibril superstructures. b, Water incubated insulin fibril superstructures with subsequently added PEDOT-S.

60

Viewed with scanning electron microscopy (SEM), diluted samples of washed PEDOT-S

covered superstructures contain numerous elliptical objects of dimensions of about 5 µm in

length and 2 µm in width (figure 6.6 a and b). In contrast, a sample of water incubated naked

superstructures is dominated by the apparent fractured remnants of the type of objects that

are observed for washed samples (figure 6.6 c and d).

Figure 6.6 - SEM images on superstructures diluted in water. a, The centrifuged washed –ICD PEDOT-S-covered insulin fibril superstructures of figure 2a. b, Close up of one of the structure in figure 3a. c, Water incubated insulin fibril superstructures. d, Close up of the largest structure found in figure 3c.

Figure 6.7 - Polarized optical microscopy images of a birefringent superstructure. The direction of the object relative to the crossed polarizers (represented by double arrows) was changed gradually by 15 degree increments. The stabilizing effect of PEDOT-S on insulin superstructures is further verified by polarized

light microscopy (POM) as seen in figure 6.7; when studying a sample of washed PEDOT-S

covered superstructures with crossed polarizers a high number of bright objects of

homogenous size can be seen. Individually, these objects behave as anisotropic crystals with

an optical axis along their long axis. Such objects are absent in a sample of superstructures

61

not treated with PEDOT-S when viewed with crossed polarizers. Given the dimensions, it is

highly likely that the elliptical objects seen with SEM and POM are one and the same.

Lastly, a further benefit of the stabilizing effect of PEDOT-S is the possibility of making

continuous solid state films of superstructures without the highly disruptive presence of

NaCl salt crystals (figure 6.9 a). Such films retain the ability to produce an induced CD signal

from PEDOT-S indicating that the helicity is retained in a solid state. Taken together, it is thus

possible to obtain PEDOT-S in a stable helical conformation which is transferrable to the

solid state using insulin fibril superstructures as templates.

Figure 6.9 – Solid state PEDOT-S coated insulin fibril superstructures. a, SEM image of a film of

PEDOT-S covered insulin fibril superstructures. b, CD spectra on PEDOT-S covered superstructures in

a solid state.

62

Future and outlook

There remains of course much to be investigated for the use of functionalized amyloid fibrils

as material components. Notably, insulin has been used as the protein of choice throughout

this thesis due to its excellent capacity for fibril formation. However, insulin is a relatively

expensive protein and for future applications of functionalized amyloid fibrils, cheaper

proteins would need to be investigated as the basis for the incorporation of functionalized

fibrils into devices on a larger scale. Finding such candidates has a significant probability of

success given the large number of proteins that could potentially form amyloid fibrils in

vitro, although it was not a topic pursued in this thesis. Furthermore, on the level of

fundamental research, we have only investigated a small number of the possible

combinations of functionalizing agents and protein fibrils. If investigated further, this will

likely result in more possibilities for utilizing functionalized fibrils as material components in

organic electronics applications.

An evident next step for the tools and the information gathered herein is to be implemented

for the incorporation of functionalized fibrils into electronic devices. The structural

properties and functionally diverse possibilities of functionalized fibrils we have touched

upon in this thesis make this a promising pursuit. The preparation of films presented in

chapter 5 in particular is promising for applications such as OLEDS, where the ability to

prepare well defined films is a key factor. Lastly, this thesis has hopefully played a part in

identifying a promising direction for further studies on the interplay between functionalizing

compounds and fibrils - whether that may be for applications within electronics or medicine,

or simply to gain a more complete understanding of self-assembling systems involving

biomolecules at the interface to electronics and photonics.

63

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Doctoral thesis Fredrik Bäcklund

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