Characterisation of polydiacetylene for the detection of forces in membranes

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Thesis

Reference

Characterisation of polydiacetylene for the detection of forces in membranes

ORTUSO, Roberto Diego

Abstract

The thesis will discuss the main aspects of polydiacetylenes. This initial discussion starts from the general aspects known in the field with examples of applications for polydiacetylenes. It then continues into the polymerisation observed through infrared spectroscopy, the study of a novel type of polydiacetylene to peptides and terminates with the resolve of a technological limitation which could have had the potential of limiting our progress.

ORTUSO, Roberto Diego. Characterisation of polydiacetylene for the detection of forces in membranes. Thèse de doctorat : Univ. Genève, 2019, no. Sc. 5351

DOI : 10.13097/archive-ouverte/unige:120401 URN : urn:nbn:ch:unige-1204019

Available at:

http://archive-ouverte.unige.ch/unige:120401

Disclaimer: layout of this document may differ from the published version.

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Section de Chimie et Biochimie Département de Chimie Physique

Professeure Kaori Sugihara

Characterisation of polydiacetylene for the detection of forces in membranes

THÈSE

Présente à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès Sciences, mention Chimie

par

Roberto Diego Ortuso

D’Italie Thèse N° 5351

Genève, 2019

Atelier d’impression RepoMail

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Acknowledgments

Some life occasions are there to test us, push our limits and make us discover the small truths about life. Early encounters forge us. In the years leading up to this moment, I, like many others, wavered in time. Uncertainty and decisions are necessary, this is no mystery, and however living with the decisions made might result into a burden to bear through life.

Nevertheless, my path has forged me into the person who writes this conclusion to a life chapter.

An affectionate thanks goes to Prof. Dr. Sugihara, for the path we together walked on.

Remembrance goes also to others. This brings me to thank Prof. Dr. Bürgi, Prof. Dr.

Wesolowski, and Prof. Dr. Hagemann for the support they have all given me in many of the occasions in which a piece of the puzzle was added to the final composition, and Prof. Dr.

Weder who dedicated time to my efforts by reading, travelling to Geneva and discussing the work done in these past few years.

Every day in these corridors, I grew. Frowning on the same scenery, of these grey corridors decorated with doors to mysterious cabinets, and surmounted by the crackling tubing that adorn the repetitive ceilings. Alas the end; to new surroundings.

To this, I turn my thought to many other people. To this I must add many other thoughts that go to other people, too many, some of whom present a face but no name.

Overall, these almost five years have been a wonderful opportunity. Day in and day out, smiles, waves, jokes. In some occasions, more than once a day, knowing that these relationships would last outside these walls.

Diving into science is not for the faint hearted. It is often difficult. Frustrating at times.

Enthusiastic at others. Taunting. Tackling. Inventive; you never have too much of this one.

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My special thanks need to be extended also to my group colleagues, the current and the former, who, with their always positive and friendly attitude, allowed me to find myself over time. They made each day here more memorable.

Let me also mention the whole Bürgi group, the whole Hagemann group, the whole Wesolowski group, and the whole Vauthey group scientists, the present and erstwhile, with which a coffee was always too little. Without them, these past years would have been empty of memories, some of which I treasure. These thanks are to you: Thank you!

Last, but by far not the least, Rocco, Anna, Serena and Lucky, which well, no words can express my feelings for them. I may write many words, however none would suffice.

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Abstract

Investigation into the unknown has been one of the main purpose of science since the beginning of time. The early days of chemistry can be identified in the alchemists whose aim was to improve the understanding of how materials transform under different conditions. Chemistry has evolved into a tightly woven network of different branches, some regarding the investigation into studies of observable phenomena, others focused on using material properties to identify the presence of chemical species and physical entities. One thing is certain though;

today’s society is found in need of new ways to detect and quantify the variations of physical and chemical properties into a measurable entity. In the early 70’s Wegner et al. first discovered that when a particular class of molecules, with an embedded diacetylene moiety, was exposed to UV radiation it changed colour¹. This was attributed to the polymerisation of the molecules^1,2,3. This polymer was named polydiacetylene (PDA) ^1,4. However, this

L'enquête sur l'inconnu est l'un des principaux objectifs de la science depuis la nuit des temps. Les premiers jours de la chimie peuvent être identifiés chez les alchimistes dont le but était d'améliorer la compréhension de la transformation des matériaux dans différentes conditions. La chimie a évolué pour devenir un réseau étroitement tissé de différentes branches, certaines concernant la recherche sur des études de phénomènes observables, d'autres axées sur l'utilisation des propriétés des matériaux pour identifier la présence d'espèces chimiques et d'entités physiques.

Une chose est certaine cependant; La société actuelle a besoin de nouveaux moyens pour détecter et quantifier les variations des propriétés physiques et chimiques en une entité mesurable. Au début des années 70, Wegner et al. On a découvert pour la première fois que lorsqu'une classe particulière de molécules, avec une fraction de diacétylène incorporée, était exposée aux rayons UV, elle changeait de couleur¹. Ceci a été attribué à la polymérisation des molécules^1,2,3. Ce polymère a été appelé polydiacétylène (PDA)^1,4. Cependant, ce n’était pas la seule observation; à mesure

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12 | 212 was not the only observation; as heat was

applied, the polymer went from a visible blue colour to a red colour^1,5,6. The sensitivity of PDA to heat opened a wide set of doors into the use of PDA for sensing applications⁷. As transition electronic states were studied and the transient optical capability of some PDAs was observed, further interest in the optical properties of PDAs arose⁸. Having understood the potential of PDA, efforts were invested in developing PDAs with multiple shapes and structures. The step to the creation of diacetylene lipid-like structures was very short⁹. Subsequently PDA leaked into the biological domain in which still to date it is widely investigated.

The focus of this text will be mostly on lipid-like monomers, which will be further referred to as diacetylene molecules. Strictly speaking this definition is incorrect as the family of diacetylene monomers also comprehends monomers that may include other groups within their structures like benzene rings¹⁰, pyridine

que la chaleur était appliquée, le polymère passait d'une couleur bleue visible à une couleur rouge^1,5,6. La sensibilité de la PDA à la chaleur a ouvert de nombreuses portes à l'utilisation de la PDA pour les applications de détection⁷. Alors que les états électroniques de transition ont été étudiés et que la capacité optique transitoire de certains PDA a été observée, les propriétés optiques des PDA ont suscité un intérêt supplémentaire⁸. Ayant compris le potentiel des PDA, des efforts ont été déployés pour développer des PDA aux formes et structures multiples. La création de structures de type diacétylène lipidique a été très brève⁹. Par la suite, la PDA s'est infiltrée dans le domaine biologique dans lequel elle fait encore l'objet

de nombreuses recherches.

Ce texte sera principalement axé sur les monomères analogues aux lipides, appelés molécules de diacétylène. À proprement parler, cette définition est incorrecte car la famille des monomères de diacétylène comprend également les monomères qui peuvent inclure d'autres groupes dans leurs structures, tels que les cycles benzéniques¹⁰, les substituts pyridine, les macrocycles¹¹ ou d'autres groupes fonctionnels¹².

Dans ce texte, nous allons nous

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functional groups¹².

In this text, we are going to concentrate on part of the research that has been done over the past few years in the field of polydiacetylenes by other groups. We will start by focusing on the monomer structures, and on how the different assemblies will be influenced by the different intrinsic characteristics of the monomers at hand, like tail length, position of the diacetylene moiety and head dimensions. Following this introduction to the monomer characteristics, we are going to journey through a number of PDA applications reported in literature. These spaces from bio-detection to their application in regenerative medicine.

Our journey will then take us to the scientific process that was done during my years as an early stage researcher.

I started my investigation with the detailed study of infrared spectroscopy.

In depth signal processing applied to infrared spectroscopy will elucidate the origin of the a triple carbon bond peak,

concentrer sur une partie des recherches effectuées ces dernières années dans le domaine des polydiacétylènes par d'autres groupes. Nous commencerons par nous concentrer sur les structures des monomères et sur la manière dont les différents assemblages seront influencés par les différentes caractéristiques intrinsèques des monomères disponibles, comme la longueur de la queue, la position de la fraction diacétylène et les dimensions de la tête.

Après cette introduction aux caractéristiques des monomères, nous allons parcourir un certain nombre d’applications pour PDA rapportées dans la littérature. Ces espaces de la biodétection à leur application en

médecine régénérative.

Notre voyage nous mènera ensuite au processus scientifique qui a été fait au cours de mes années en tant que chercheur débutant.

J'ai commencé mon enquête par l'étude détaillée de la spectroscopie infrarouge. Le traitement du signal en profondeur appliqué à la spectroscopie infrarouge élucidera l'origine du pic de triple liaison carbone, qui a échappé aux chercheurs pendant quarante ans, nous offrant un potentiel sans précédent pour corréler la polymérisation et l'assemblage des diacétylènes au signal

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giving us unprecedented potential to correlate the polymerisation and assembly of diacetylenes to the infrared signal. These mathematical techniques, coupled with the infrared signal allows us to follow the polymerisation even when other more commonly used techniques, like UV/Vis and microscopy, fall short for this purpose. We also confirmed the signals arising from the anisotropic packing during polymerisation, which agrees with previously reported X-ray studies.

I will then continue the discussion to the stepping-stone for developing an unparalleled mechanosensors that can be potentially introduced in cellular membranes with minimal disruption to the membranes function.

Phosphocholine headgroup diacetylenes result being more biocompatible than other types of diacetylenes. The reaction of this phosphocholine headgroup diacetylene was compared with the “golden standard” carboxyl headgroup polydiacetylene used for bio-detection

infrarouge. Ces techniques mathématiques, associées au signal infrarouge, nous permettent de suivre la polymérisation même lorsque d’autres techniques plus couramment utilisées, telles que l’UV/Vis et la microscopie, se révèlent inefficaces. Nous avons également confirmé les signaux provenant du tassement anisotrope lors de la polymérisation, ce qui concorde avec les études par rayons X précédemment rapportées.

Je poursuivrai ensuite la discussion en guise de tremplin pour la mise au point de mécanosenseurs inégalés pouvant potentiellement être introduits dans les membranes cellulaires avec une perturbation minimale du fonctionnement

de la membrane.

Les diacétylènes du groupe de tête phosphocholine sont plus biocompatibles que d'autres types de diacétylènes. La réaction de ce diacétylène du groupe de tête phosphocholine a été comparée au polydiacétylène du groupe de tête carboxyle

«standard» utilisé à des fins de détection biologique. Ces expériences nous ont permis de conclure que la réponse du diacétylène du groupe de tête phosphocholine au peptide était plus forte que celle du carboxy- polydiacétylène bien que la sensibilité soit

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to conclude that the response of the phosphocholine head group diacetylene to the peptide was stronger than the carboxyl polydiacetylene even though sensitivity was comparable.

As our ultimate goal is the development of a mechanosensor, the next step is the application of controlled forces. AFM provides the means to do so we require identifying the limits of the technique. The calibration process of AFM is highly dependent on the mathematical model used for the calibration itself. In calibrating our system, we identified the calibration limitations, pinpointed the source of the errors and warned the field of the errors the method presents.

The future steps will be to correlate the fluorescence response of polydiacetylenes to applied forces, the last milestone for the development of an effective membrane force sensor.

comparable.

Notre objectif ultime étant la mise au point d'un mécanocapteur, la prochaine étape consiste à appliquer des forces contrôlées.

AFM fournit les moyens de le faire, nous devons donc identifier les limites de la technique. Le processus d'étalonnage de l'AFM dépend fortement du modèle mathématique utilisé pour l'étalonnage lui- même. Lors de l'étalonnage de notre système, nous avons identifié les limites d'étalonnage, identifié la source des erreurs et averti le champ des erreurs présentées par

la méthode.

Les prochaines étapes consisteront à établir une corrélation entre la réponse en fluorescence des polydiacétylènes et les forces appliquées, dernière étape du développement d’un capteur de force membranaire efficace.

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List of abbreviations and symbols

Abbreviation Expansion

µ Sloped friction coefficient µ^flat Flat friction coefficient

AC Asymmetric carbenes

C=C Double carbon bond

C=O Carbon-oxygen double bond

C≡C Triple carbon bond

CBH Correlated barrier hopping

CD Circular dichroism

CDD Controlled drug delivery

CR Colorimetric response

DA Diacetylene monomers

DC Dicarbenes

DC(8,9)PC 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine DNA Deoxy ribonucleic acid

DOPC 2-((2,3-Bis(oleoyloxy)propyl)dimethylammonio)ethyl hydrogen phosphate

DR Diradicals

DSC Differential scanning calorimetry EC50 Half maximum response

ED Electron diffraction

ELISA Enzyme-linked immunosorbent assay EPR Enhanced permeability and retention ESR Electron spin resonance

FA Adhesion force

FII Parallel to substrate forces

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18 | 212 FN Perpendicular to substrate forces FRP Free-radical polymerisation FTIR Fourier transform infrared

GIXD Grazing incidence X-ray diffraction GMO(s) Genetically modified organism (Plural) GNP(s) Gold nanoparticle (Plural)

HOPG Highly oriented pyrolytic graphite IC(s) Integrated circuit (Plural)

IR Infrared Spectroscopy

KN Vertical Stiffness

Kθ Torsional Stiffness

L Load

LBt Langmuir-Blodgett trough LED(s) Light emitting diode (Plural) LFM Lateral force microscopy

LSPR Local surface plasmon resonance LSt Langmuir-Sheffield trough

M0 Monomer 0

M1 Monomer 1

NIR Near infrared

NMR Nuclear magnetic resonance OCD Oriented circular dichroism

OFET(s) Organic field-effect transistor (Plural)

OPA One-photon absorption

PDA Polydiacetylene

POPC 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine

PP Packing parameter

PSD Position sensitive detector

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RI Radical-initiator

SEM Scanning electron microscope SN Vertical sensitivity

SO Stable state oligomer SPR Surface plasmon resonance ssDNA Single-stranded DNA

STM Scanning tunnelling microscopy Sθ Lateral sensitivity

TEM Transmission electron microscope

Tm Transition temperature

TPA Two-photon absorption

TRCDA 10,12-Tricosadiynoic acid VCF Vertical calibration factor

W^slope Half-loop width

XRD X – ray diffraction α Calibration coefficient αHill Hill coefficient

Δ^flat Flat loop offset ΔH Specific heat capacity

Δ^slope Slope loop offset

𝑅𝐴𝐼 Relative absorption intensity

𝜃 Slope of substrate

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List of figures

Figure Caption Page

Figure 1 Schematic representation of one type of lipid-like diacetylene monomer: 10,12- Tricosadiynoic acid (TRCDA). tl) Highlights the number of carbons that make up the alkyl tail, hg) Highlights the head group, n) highlights the number of methylene groups that separate the diacetylene moiety from the head group, m) highlights the number of methylene groups and the methyl group that separate the diacetylene moiety from the

termination of the alkyl chain. 30

Figure 2 Molecular shapes and packing parameter of lipid structures. On the top the packing shape, which will influence the packing parameter value. In the middle packing parameter key values. On the bottom schematics of the structures formed. Adapted with

Figure 3 Schematic representation of initial stages of PDA polymerisation. The head group is located on the top side of the diacetylene moiety (towards R0, R1 and R2). 1) Diacetylene monomers, 2) exposure to UV radiation, two photons are absorbed by the diacetylene moiety, 3) schematic representation of the radicalic monomer with eight unpaired electrons in green. The radical electrons derive from the break of the two triple bonds.

Please note that the time for intramonomer bond formation is less than 1ps³⁹. 4) Radicalic monomer with formation of the (σ+π)-orbitals of the two central carbon atoms, and the four unpaired electrons that will then take part into the crosslinking of other

monomers, forming the PDA conjugation (continues to Figure 4). 41

Figure 4 Schematic representation of final PDA polymerisation. 4) (Continued from Figure 3), 5) neighbouring monomers to the radicalic-initiator find themselves in a radicalic state. The two arrows highlight the two carbons that will bind to form the polymer. 6) Structure of

the stable oligomer. The two arrows highlight the two carbons also highlighted in 5). 43

Figure 5 Intermediate structures of PDA backbone antecedent to the stable oligomer backbone structure. The structures shown are schematic and simplified. The grey notations report the bond lengths found in¹². Red dots represent radical electrons. A) Diradical structure, B) dicarbene structure, C) asymmetric carbene structure, D) stable oligomer. Adapted

Figure 6 Schematic representation of the two terminal structures hypothesis. On the left hand side the pseudo-cyclopropane structure¹. On the right hand side the formation of a

R=CHR’ group type, with the radical that extinguishes further down the chain⁵⁰. 48

Figure 7 Exemplary kinetic curves generated from the Avrami equation (please refer to Eq. 2) for different Avrami exponents (n). In this example the kinetic parameter was set to unity (k=1). Step size of n between [0; 1] is of 0.2 units. The data was generated with the use

of Matlab® software (MathWorks, Inc., USA). 50

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Figure 8 A) Microscopy image of 10,12-tricosadiynoic acid (TRCDA) multilayers obtained by Langmuir-Blodgett deposition. Brightfield image captured with colour camera. In the outer part blue state PDA, in the inner part red state PDA obtained through electromagnetic irradiation (Laser wavelength of 690 nm). B) Fluorescent image of sample displayed in A), red state PDA shows fluorescent emission. C) UV/VIS spectra of blue and red state TRCDA-PDA. Adapted with permission from⁶⁹. Copyright 2001 Springer

Nature. 53

Figure 9 Schematic drawing of PDA phase transition according to⁵⁸. A) Monomer assembly B) blue PDA C) red PDA. Note (i) the different conformation of the methyl versus carboxyl terminated chains, (ii) the arced methyl-terminated chains in A) and B), and (iii) the different tilt angle of the chains in the B) versus C). This drawing is from the direction perpendicular to the polymer backbone, but the chains tilt has a diagonal component into the drawing plane. Although one monolayer is presented here, this structure is repeated to form a multilayer organisation known for PDA films. Adapted with

Figure 10 Schematic drawing of PDA phase transition according to the rotation of the single carbon bond. A) Schematic representation of one PDA repeat unit. Grey dashed box represents the bond along which the rotation occurs. B) & C) schematic representation of the orbitals of the two carbons along which the rotation occurs. B) When the PDA is in blue state the sp and sp2 π-orbitals are on the same plane. C) When the PDA is in red state the sp and sp2 π-orbitals are on different planes. This causes a break the backbone

conjugation. 56

Figure 11 A) & B) Representative single-particle state of alternating chains. A) Referred to blue state PDA. B) Referred to red state PDA in which fluorescent pathway relaxation is allowed. Adapted with permission from⁸⁹. Copyright 1991 American Physical Society. C) Microscopic fluorescent imaging spectroscopy of an isolated red state PDA single chain.

Arrow indicates the laser excitation spot centred at 32.8 μm. Adapted with permission from⁹⁰. Copyright 2005 Nature Physics. D) Excitation sequence and simplified structure of polymer backbone. Adapted with permission from⁹¹. Copyright 1985 Chemical physics

letters. 58

Figure 12 Schematic molecular structure of 10,12-Tricosadiynoic acid films. A) Monomer structure of non-annealed film, B) monomer structure of annealed film. C), D) E) & F) orange dots symbolise the conjugated backbone. C) Blue state PDA structure of non-annealed film, D) blue state PDA structure of annealed film. Adapted with permission from⁹⁹. Copyright 1996 America Chemical Society. E) Red state PDA structure of non-annealed film, F) red state PDA structure of annealed film. Adapted with permission from¹⁰¹. Copyright 1998

America Chemical Society. 62

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Figure 13 A) Photograph of a portion of a 96-well plate depicting the colours of DMPC–PDA solutions after additions of the following native peptides and peptide analogues. (I) Melittin analogues: a, Control (no peptide added to vesicle solution); b, Native melittin;

c, K7L-melittin (Lys7 substituted with leucine); d, ΔW19-melittin (tryptophan omitted at position 19); e, K19E-melittin (Lys19 substituted with glutamic acid). (II) Magainin II analogues: a, Control (no peptide added to vesicle solution); b, Native magainin II; c, bK10E-magainin II (Lys10 substituted with glutamic acid); d, K10E,K11E-magainin II (Lys10 and Lys11 substituted with glutamic acids); e, F12W-magainin II (Phe12 substituted with tryptophan). (III) Alamethicin analogue: a, Control (aqueous vesicle solution + 10 ml trifluoroethanol, TFE); b, Native alamethicin (dissolved in 10 ml TFE); c, P12-alamethicin, dissolved in 10 ml TFE (proline moved from position 14 to position 12 in the alamethicin sequence). All cells contained 100 μl solutions of 1 mM DMPC–PDA vesicles (4:6 mole ratio) and 2 mM tris(hydroxymethyl)aminomethane (Tris or THAM) at pH 8.5. Peptide concentrations were adjusted to 0.1 mM. Adapted with permission from⁹⁹. Copyright 2000 American Chemical Society. B) Colorimetric bacterial fingerprinting schematic. The colour combination for each bacterium reflects the RCS values recorded 7 h after the start of growth at a bacterial concentration of 109/mL. The relative colour scale is shown on the left. (I) DOPE/PDA (1:9mole ratio); (II) sphingomyelin/cholesterol/PDA (7:3:90); (III) DMPC/ PDA (1:9); and (IV) POPG/PDA (1:9).

a, E.coli XL1; b, E.coli C600pMRInv; c, B. cereus; d, S. typhimurium. Adapted with permission from¹³¹. Copyright 2007 Langmuir. C) Spectroscopic changes of PDA vesicles caused by the interaction with nucleic acids in aqueous solution reported in insert. Insert:

Photograph of the PDA liposomes after the reaction with the nucleic acids for 15 min.

The PDA sensor was composed of PCDA/PCDA-EDEA with a molar ratio of 5:5. a) a control sample without PCR amplification; b) a sample containing amplified nucleic acids with 605 base pairs (100 nM); c) a sample with a 26-mer oligonucleotide (100 nM). Adapted

Figure 14 A) Conformational changes as recognition of the target mercury ions occur, this results in red fluorescent emission. B) Correlation curve between the fluorescence intensity and the concentration of Hg2+. Adapted with permission from¹³⁸. Copyright 2009 Advanced materials. C) Structure of PDA studied in¹³⁹, and proposed origin of the CO2-induced colour change. D) UV/vis spectral changes of PDA in aqueous solution (25 μM, containing 0.5% TEA) after bubbling with volumes of CO2 going from 0 mL to 1.50 mL. Adapted with

Figure 15 A) Schematics of organic field-effect transistors (OFETs) with bottom-contact and top- contact configurations. Adapted with permission from¹⁴⁸. Copyright 2006 Advanced Materials. B) Transfer characteristics with bi-directional scan of gate voltage as a function of the PDA thickness on bare silica surface. The grey arrows indicate the direction of the sweep. Adapted with permission from¹⁴¹. Copyright 2010 Applied Material and Interfaces. C) Molecular Structures of the Diacetylene Monomers: (I) 10,12- Tricosadiynoic Acid; (II) 10,12-Pentacosadiynoic Acid; (III) Docosa-10,12-diynedioic Acid;

(IV) N-(2-((4,6-Diamino-1,3,5-triazin-2-yl)amino)ethyl)tricosa-10,12-diynamide; (V) 1,6- Diphenoxy-2,4-hexadiyne. D) Capacitive response curves of (IV)-PDA following addition of different vapours. E) Array-based colour code identification of different vapours using (I)-(IV) PDA and (V) DA capacitive sensors; the colour code on the right indicates the degrees of capacitance change. Adapted with permission from¹⁵⁰. Copyright 2019

American Chemical Society. 76

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Figure 16 A) Top row, confocal fluorescence images of MDA-MB-231 cells incubated with doxorubicin-partially polymerised liposomes / GNPs (Dox-PPL/GNP) complexes. I) DAPI stain, II) doxorubicin, I+II) overlay of I) and II). Bottom row, free doxorubicin shown in III) DAPI stain, IV) doxorubicin, III+IV) overlay of III) and IV). B) Viability study of MDA-MB- 231 cells incubated with GNPs, Dox-PPLs, and Dox-PPL/GNP with or without laser

Figure 17 A) Various examples of polymerised PDA shapes to various microstructures which can be used as scaffolds for cellular growth. B) Morphological characteristics of cells on various PDA substrates. Cell growth is highly dependent on the topography of the structure on which they grow. I) Cells on Smooth-PDA exhibited broad flattened shape with randomly oriented processes. II) Cells grown on grooved substrates exhibited narrower cell bodies that aligned along the microchannel axis. III) Cells grown on a pours structure tend to migrate and spread inside the grove. Adapted with permission from¹⁸¹. Copyright 2008

Royal Chemical Society. 83

Figure 18 UV-VIS spectra. A) UV-VIS spectra of TRCDA after different UV areal doses. B) Position of the red and the blue peak as a function of the UV doses. C) Blue to red ratio, calculated with the height of the peaks. The vertical solid line highlights the point, in which the blue and the red polymers are at a 1:1 ratio (blue to red ratio 100%). D) Variation of the Area under the absorption spectra between 480 nm and 710 nm over the amount of the UV

dose. The vertical dashed line identifies the point, where 10% PDA was degraded. 91

Figure 19 ATR-FTIR spectra of TRCDA at 0 J/cm2 (black), blue (blue), red (red), and degraded (grey) states. The blank ATR-FTIR ZnSe element was used as reference spectra and the baseline was subtracted. All the peaks that are identified and will be analysed later are indicated by wavenumbers. Note that C≡C peaks (2181, 2167, 2139 cm^-1) are not visible in the

Figure since their intensities are small. See the zoom-in Figure 20 for the detail. 93

Figure 20 C≡C stretching peaks. A.I) ATR-FTIR spectra for C≡C stretching at different UV areal irradiation doses, where the results from the deconvolution are superposed. The regions within the two vertical dashed lines were used for the deconvolution. The characteristic triple peaks from C≡C stretching that have been reported before are highlighted by violet, green and yellow. A.II) The normalised area underneath the peaks and A.III) Δ peak position versus areal irradiation doses. B) The characteristic triple peaks changed

their intensities and positions, depending on their solvent environments. 96

Figure 21 C=C and C=O stretching modes. A.I) ATR-FTIR spectra for C=C and C=O stretching at different UV areal irradiation doses, where the results from the deconvolution are superposed. A.II) The normalised area underneath the peaks and A.III) Δ peak position versus areal irradiation doses for characteristic peaks at 1539, 1692, 1698, 1736 cm-1. B) Simulated IR spectra from C=O stretching mode, where hydrogen bonds exist intra or

inter layers. 99

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Figure 22 CH2 in-plane bending mode. A.I) ATR-FTIR spectra for CH2 in-plane bending mode at different UV areal irradiation doses. A.II) The area underneath the peaks versus UV areal irradiation doses and A.III) Δ peak position versus areal irradiation doses. AI) ATR-FTIR spectra, II) the analysis of the area underneath the peak versus UV irradiation energies, where arrows indicate irradiation energies at which the deconvolution was studied, and III) the peak shift versus UV irradiation energies are shown. The region within the two vertical dashed lines in A.I indicates the wavenumber areas that were interpreted as one peak. B) Overlaying plot of the original spectra and the sum of the deconvoluted spectra, where the good overlap ensures the successful deconvolution. C) Deconvoluted spectra of CH2 in-plane bending ATR-FTIR signal at 0 J/cm² (in black), blue polymer (40.6 J/cm² in

blue), and red polymer (162.5 J/cm² in red). 102

Figure 23 CH2 symmetric stretching mode. A.I) ATR-FTIR spectra for CH2 symmetric stretching mode at different UV areal irradiation doses. A.II) The area underneath the peaks versus

UV areal irradiation doses and A.III) Δ peak position versus areal irradiation doses. 104

Figure 24 The chemical structures of DC(8,9)PC, TRCDA monomers and polydiacetylene. 109

Figure 25 UV/VIS spectra of PDA vesicle suspension made of 75% DC(8,9)PC and 25% DOPC for

different UV doses. 112

Figure 26 (A) Photos showing the appearance of the vesicle suspension before and after the addition of melittin. UV/VIS spectra of blue-state PDA vesicle suspension made of (B) 75% DC(8,9)PC + 25% DOPC and (C) 75% TRCDA + 25% DOPC upon addition of melittin

at 0.285 mg/ml. 113

Figure 27 Colorimetric response (CR) versus incubation time after the addition of melittin for PDAs made of 75% DC(8,9)PC + 25% DOPC (red) and 75% TRCDA + 25% DOPC (black). Melittin

concentration in the samples is 0.285 mg/ml. 115

Figure 28 Fluorescence spectra of PDA suspension made of (A) 75% DC(8,9)PC + 25% DOPC and (B) 75% TRCDA +25% DOPC at different time points after the addition of melittin. These spectra were obtained at the excitation and emission wavelength of (A) 530nm and 545 nm for DC(8,9)PC-PDA, (B) 500 nm and 620 nm for TRCDA-PDA. The final melittin

concentration is 0.285 mg/ml. 117

Figure 29 The time evolution of the fluorescent excitation/emission peak heights with 75%

DC(8,9)PC + 25% DOPC PDA vesicles after the addition of melittin at 0.285 mg/ml. 118

Figure 30 The particle size estimated by dynamic light scattering (DLS) over time. The PDA vesicles were made of 75% DC(8,9)PC + 25% DOPC. Melittin was added at the concentration of

0.285 mg/ml at 0 h. 119

Figure 31 A scheme showing the interaction between DC(8,9)PC-PDA suspension and melittin. 120

Figure 32 UV/VIS spectra of blue-state PDA vesicle suspension made of 75% DC(8,9)PC + 25% DOPS

upon addition of melittin at 0.285 mg/ml. 122

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Figure 33 (A) UV/VIS spectra of 75% DC(8,9)PC + 25% DOPC at different melittin concentrations.

(B) Colorimetric Response (CR) versus melittin concentration (dose curve). All data were

taken after 5h incubation to assure saturation of the signal. 124

Figure 34 Schemes of the tip movement on a sloped and flat surface (grating) (A) during uphill cantilever movement (from left to right) and (B) downhill cantilever movement (from right to left). (C) Ideal lateral deflection signals and (D) height profile, while scanning a

sloped surface. Colour code corresponds to the schemes. 134

Figure 35 (A-C) Rate at which μ is in real domain versus setpoint. To simplify the figure, the value is averaged by a grouped setpoints. (A) Low stiffness tips (K_N = 0.03 N/m). Grouping interval: 0.5 nN. (B) Medium stiffness tips (KN = 0.09 N/m). Grouping interval: 1.5 nN. (C) High stiffness tips (KN = 0.3 N/m). Grouping interval: 4.5 nN. (D-F) Calibration coefficients (α) versus setpoints. (D) Low stiffness tips (K_N = 0.03 N/m). (E) Medium stiffness tips (K_N

= 0.09 N/m). (F) High stiffness tips (KN = 0.3 N/m). (G-I) Friction coefficients versus setpoints. (G) Low stiffness tips (KN = 0.03 N/m). (H) Medium stiffness tips (KN = 0.09 N/m). (I) High stiffness tips (K_N = 0.3 N/m). For all the plots, vertical dashed lines indicate the adhesion forces. Grey areas indicate the region, where setpoints are lower than adhesion forces (setpoint < adhesion). Horizontal areas with a diagonal grey pattern in

(G-I) highlight friction coefficients’ range reported in literature (0.1 – 0.37)265,267,268,269,270. 137

Figure 36 Friction forces versus setpoints. (A) Low stiffness tips (KN = 0.03 N/m). (B) Medium stiffness tips (KN = 0.09 N/m). (C) High stiffness tips (KN = 0.3 N/m). Lines indicate linear

fitting with fixed friction coefficient. 140

Figure 37 (A) μ calculated from the experimentally determined parameters versus load. Grey area indicate the region where setpoints are lower than adhesion forces (setpoint <

adhesion). (B) Ideal Υ (Υ =^Δ^slope_W_slope^-Δ^flat), where μ = 0.322 (the “correct” high-force-end value) and the measured adhesion force (FA) were used, versus setpoints (in red) together with experimentally-measured Υ (in light blue). Grey area indicate the region where setpoints are lower than adhesion forces (setpoint < adhesion). (C) μ for Υ with different errors (0%, ± 1%) versus setpoint. Grey area on real plain indicates the region where setpoints are lower than adhesion forces (setpoint < adhesion). (D) Adhesion force versus setpoint. The adhesion experimentally obtained (in blue) and the ideal F_A (FA for μ = 0.322, in red) that would have yielded μ = 0.322 throughout the whole

setpoint range. E) μ for FA with different errors (0%, ± 1%) versus setpoint. 142

Figure 38 A) Loop offset (Δ) for the left slope, the flat part and the right slope. B) Half-loop width (W) for the left slope, the flat part and the right slope. For all the data linear fittings are

shown. 145

Figure 39 Root mean square roughness versus setpoints. A) Rrmsfor left slope. B) Rrmsfor flat

region. C) Rrmsfor right slope. 147

Figure 40 A) Actual measured force curve with indication of the attraction force of 0.106 nN. The inset shows the zoom-out plot, where adhesion is extracted. B) Attractive force between the AFM tip and the grating at various setpoints. Error bars indicate the standard deviation. C) Adhesive force between the AFM tip and the grating at various setpoints.

Error bars indicate the standard deviation. Note that the values of the attractive force (B

in pN) and the adhesion force (C in nN) are different by orders of magnitude. 148

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Figure 41 α_F_A_{< L} versus vertical calibration factors with linear fitting. Colour code corresponds to

the one used for the tips from Figure 35D-I. 149

Figure App.A1

Both samples A and B were stored in dry form at -27°C for around four years. Sample A:

chloroform was added less than 1h prior to measurement. Sample B: was left approximately two months in chloroform before measurement. A.I) ATR-FTIR spectra for C≡C region. A.II) ATR-FTIR spectra for C=O and C=C region. Insert: photograph of the

samples. 158

Figure App.A2

Simulated IR spectra of TRCDA-monomer, trimer and tetramer. Please note that a dimer

yielded no IR active mode. 159

Figure App.B1

A) Cross section of the height profile for the extraction of the angle. B) Actual measured force curve, from which the adhesion F_A value of 6.7 nN was extracted. C) Lateral deflection (LD, trace in light grey and retrace in dark grey) with focus on the left part of the slope. Sloped loop offset (∆^slope), sloped half-loop width (W^slope), flat loop offset (∆^flat) and flat half loop width (W^flat) are extracted from these line scan as indicated. D) Lateral deflection (LD) trace with focus on the right part of the slope. Sloped half-loop width (W^slope), sloped loop offset (∆^slope), flat half loop width (W^flat) and flat loop offset

(∆^flat) are extracted as indicated. 167

Figure App.B2

μ versus setpoints for the ideal Υ that yields the correct μ for the entire load range (in red) and Υ with +1% error (in grey). As indicated with the orange lines, the threshold load (LTh), at which μ starts to deviate by 2%, can be obtained from the analytical shape of this function μ. Grey area indicates the region where setpoints are lower that adhesion

forces (setpoints < adhesion). 170

Figure App.C1

TRCDA spin-coated on the ATR element imaged with A) bright field, B) confocal laser scanning and C) atomic force microscopy. A) A bright-field microscopy image with a 60x oil immersion objective lens of a spin-coated sample after polymerisation. B) A 3D reconstituted confocal laser scanning microscopy image of the sample in the area indicated with a dotted red square in A). The vertical step size is 100 nm and a TRITC filter was used. C) Topology of the same sample imaged by atomic force microscopy in non-contact mode, where (α) amorphous-like areas and (β) micro-crystal domains were

observed. 173

Figure App.E1

A scheme, showing ideal force curves for approach (blue) and retract (red), from which

adhesion (F_A) is extracted. 186

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List of tables

Table Description Page

Table 1 Number of carbon atoms and their influence on diacetylene monomer aggregation as

reported by Löwik et al.¹⁹. 33

Table 2 Position of diacetylene moiety and the collapse pressures measured with LBt at

T=25°C as studied by Tieke et al.²³. 35

Table 3 Thermal properties of different monomer compounds with different position of the diacetylene moiety as studied by Khanantong et al.²⁴. *Highlights the alkyl chains with 20 carbon atoms independently of the position of the moiety. ^‡Highlights the alkyl chains with 18 carbon atoms independently of the position of the moiety. 36

Table 4 Angles of backbone tilt and thickness of diacetylene monolayers as measured by Tieke et.al.²³, and Khanantong et.al.²⁴. All molecules had a carboxyl head group, and were

deposited by LBt²³ or drop casting²⁴. 37

Table 5 Visible colour of PDA in function of UV exposure as measured by Tieke et.al.²³, and

Tachibana et.al.²⁵. 38

Table 6 Spectral interpretation of ATR-FTIR signal in terms of the functional group. 93

Table 7 Dynamic light scattering (DLS) determined particle sizes estimates at different

preparation steps. 121

Table 8 Zeta potential measurements of different samples after sonication. 121

Table 9 Friction coefficients between the tips and the substrate obtained by averaging μ^flat (Figure 35G-I) in the range, where setpoints are higher than adhesion forces (adhesion

< setpoint). All the μ^flat values are in agreement with the reported values in literature

for gold – silica interface (0.1 – 0.37)265,267,268,269. 139

Table 10 y-intercepts extracted from the linear fittings in Figure 36. 141 Table

App.B1 Different values of C (moment scaling factor) for the different cantilevers used.

162

Table

App.B2 μ calculated through Eq.2 for left and right slope.

168

Table

App.E1 Tip specifics, declared and measured.

184

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List of equations

Equation Expressed parameter and dependence Page

Eq. 1 Expresses the packing parameter (PP); function of the surface area of the lipid head group (𝑎_𝑜), the volume in which the alkyl lipid tail can arrange (𝑣₀) and the alkyl tail

length of the lipid (𝑙0) in length units. 31

Eq. 2 Expresses the fraction of polymer formed (𝑋_polymer); function of the kinetic parameter (𝑘) (which in turn is function of temperature (𝑇), the nucleation rate (𝑁_rate), crystal growth rate (𝐺_rate)), the photo-irradiation time (𝑡), and the Avrami

exponent (𝑛). 50

Eq. 3 Expresses the relative absorption intensity (𝑅𝐴𝐼); function of the value of the absorption spectra of red state PDA (𝐼_λ^Red) at the characteristic red absorption peak and the value of the absorption spectra of blue state PDA (𝐼_λ^Blue) at the characteristic

blue absorption peak. 64

Eq. 4 Expresses the colorimetric response (𝐶𝑅); function of relative absorption intensity (𝑅𝐴𝐼) and the relative absorption intensity in the initial blue state PDA (𝑅𝐴𝐼Blue). 65 Eq. 5 Force and momentum balancing equation around the AFM tip, where the

momentums are linked to the measured lateral deflection values (Δ^slope, Δ^flat,

𝑊^slope). 135

Eq. 6 Correlates the forces acting on the tip, friction coefficient on the sloped part of the substrate (𝜇), and angles of the substrate (𝜃) with the calibration factor (𝛼) and

measured lateral deflection value (𝑊^slope). 135

Eq. 7 Expresses the friction coefficient (𝜇^flat); function of the measured calibration

coefficient, lateral deflection value and the forces acting on the AFM tip. 135

Eq. 8 Defines the ratio of the measured values (𝛶); function of the measured lateral

deflection values (Δ^slope, Δ^flat, 𝑊^slope). 146

Eq. 9 Expresses the calibration factor (𝛼); function of the sensitivity of the AFM, geometrical sizes of the AFM cantilever, and characteristic mechanical values of the

cantilever. 150

Eq. App.B1 to Eq. App.B30

Mathematical detail of the conventional wedge calibration method developed by Varenberg et al.²⁶⁵.

160 to 169

Eq. App.B31 Defines the ratio of the measured values for imposed conditions (𝛶); function of friction coefficient on the sloped part of the substrate (𝜇), angles of the substrate (𝜃),

the load forces (𝐿) and attractive forces (𝐹A) acting on the AFM cantilever. 170

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Eq. App.B32 to Eq. App.B35

Mathematical solutions to imposed conditions of (𝛶), for extracting the threshold load from which the results returned by the wedge calibration technique gives

sufficiently accurate calibration. 171

Eq. App.D1 Expresses the relative fluorescence of the PDA sample (𝑧); function of the fluorescence emission for a specific analyte concentration (𝐼λ) and the reference

emission (𝐼_Ref.Blueλ). 181

Eq. App.D2 Expresses the function for fitting the hill equation (𝑦); function of the maximum colorimetric response (𝐶𝑅Max), half maximum response value 𝐸𝐶50 and hill

coefficient (𝛼_Hill). 182

Eq. App.E1 First order polynomial fit equation. 186

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Chapter 1: Diacetylenes and polydiacetylenes properties

Introduction to chapter

In this section we will go through the main aspects of diacetylene containing molecules. Diacetylene monomers come in a variety of shapes and structures. The focus of this text will be mostly on lipid-like monomers, which will be further referred to as diacetylene molecules. Strictly speaking this definition is incorrect as the family of diacetylene monomers also comprehends monomers that may include other groups within their structures like benzene rings¹⁰, pyridine substitutes macrocycles¹¹ or other functional groups¹².

We will start by focusing on the monomer structures, and on how the different assemblies will be influenced by the different intrinsic characteristics of the monomers at hand; like tail length, position of the diacetylene moiety and head dimensions. We will then focus on the polymerisation of the PDA, continuing onto the chemistry behind the polymerisation, the role of the monolayer assembly on the polymerisation and the properties of the system once polymerised. We will then conclude the discussion reporting some examples of PDA applications in different fields.

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32 | 212 Diacetylene monomers

The lipid-like diacetylenes can be subdivided in two different substructures.¹² The first is the alkyl tail and the second is the head group, which is located at one of the two extremities of the tail. Four main parts will influence the behaviour of the single molecule among the assembly. Each monomer type, with distinct subparts, will result in a different self-assembly that will be reflected on the conjugated polymer.

Figure 1: Schematic representation of one type of lipid-like diacetylene monomer: 10,12-Tricosadiynoic acid (TRCDA). tl) Highlights the number of carbons that make up the alkyl tail, hg) Highlights the head group, n) highlights the number of methylene groups that separate the diacetylene moiety from the head group, m) highlights the number of methylene groups and the methyl group that separate the diacetylene moiety from the termination of the alkyl chain.

The different molecular aspects that impact the assembly are the number of carbons that make up the alkyl tail (please refer to Figure 1 highlight tl), the head group chemical type and size (please refer to Figure 1 highlight hg), the number of methylene groups that separate the head from the diacetylene moiety (please refer to Figure 1 highlight n) and the number of methylene groups and the methyl group that compose the terminal part of the tail (please refer to Figure 1 highlight m).

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33 | 212 Role of the alkyl tail and head group

The area occupied by the head group, the alkyl chain volume and the alkyl tail length play a key role in establishing the assemblies of the monomers. These three parameters have been included in a mathematical predictive value that forecast the assembly of lipid structures. This parameter, known as packing parameter (PP), has been algebraically defined^13,14 as:

𝑃𝑃 = 1 𝑎₀

𝑣₀

𝑙₀ . Eq. 1

The term 𝑎_𝑜 is a thermodynamically relevant quantity related to the surface area of the head group. Its value depends on the considered solvent, or gas, and geometrical conformation that the head group assumes within the assembly¹³. It might further change its amount based on temperature, salt concentrations, additives and other factors^13,15. 𝑣₀ is defined as the volume of the diacetylene tail and 𝑙₀ the length of the tail in length units (such as Å and nm)^13,14.

Figure 2: Molecular shapes and packing parameter of lipid structures. On the top the packing shape, which will influence the packing parameter value. In the middle packing parameter key values. On the bottom schematics of the structures formed. Adapted with permission from¹⁶. Copyright 2014 Royal Society of Chemistry.

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Based on the values of the PP we can predict how the monomer structure will assemble (please refer to Figure 2)¹⁷. For PP values close to unity, the structures formed will be planar. When the stylised shape of the lipid will resemble a conical shape with the apex oriented towards the end of the alkyl tail the PP value will be below unity. This will ultimately lead to the assembly of spherical assemblies. The more the stylised shape of the lipid will resemble a conical shape with the apex oriented towards the head of the lipid the more the PP value will be higher than unity. This will lead to an inverted micelle-like assembly¹⁶.

Theoretical thermodynamic investigations pointed out how the parameter 𝑎_𝑜 is mainly responsible for the initial aggregation of the lipid structures and its stability¹⁸, but plays a less relevant role in the final assembly structure¹³. The main molecular component that influences the final lipid assembly has been pinned on the alkyl chain part¹³. The alkyl chain makes up a large part of the molecular weight therefore a thermodynamic reduction of the energy of the alkyl chain plays a more pronounced role in the final assembly¹⁴. The influence of the tail on aggregation was shown experimentally with the use of different acetylated lipids. A nucleotide sequence was chemically clicked-on to the head group of different length alkyl chain tail lengths to demonstrate its influence in aggregation¹⁹. The aggregation of monomers, measured through circular dichroism spectra’s (CD) have shown how the conformational behaviour of the monomers was affected by the number of methylene groups within the tail¹⁹. They further resorted to anneling¹⁹. The annealing treatment had the objective of changing the lipid interdigitation reducing defects and minimising the energy of the assembly^20,21. The process of heating up caused the braking-up of the lipid assembly and the slow cooling its re-aggregation with more time for the zwitterionic molecules to assemble¹⁹. This step increases the effect that the alkyl tails have on the assembly as heating does not influence significantly the inter-lipid head group interactions; if carried out below denaturation temperatures¹⁹.

Characterisation of polydiacetylene for the detection of forces in membranes

Thesis

Reference

Characterisation of polydiacetylene for the detection of forces in membranes

Characterisation of polydiacetylene for the detection of forces in membranes

THÈSE

Présente à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès Sciences, mention Chimie

par

Roberto Diego Ortuso

D’Italie Thèse N° 5351

Genève, 2019

Table of contents

Acknowledgments

Abstract

List of abbreviations and symbols

List of figures

List of tables

List of equations

Chapter 1: Diacetylenes and polydiacetylenes properties