Keywords: ε molar extinction coefficient. 74 SPM scanning XRD X-ray diffraction. 14, 23, 25 Acronyms

NPs nanoparticles. 46

Firstly, I would like to deeply thank my supervisor Dr. Clémence Allain and my co-supervisor Dr. Rémi Métivier for entrusting me with this exciting project, for their constant support, patience and immense knowledge. I could have never hoped for a better guidance during these years.

Besides my advisors, I would like to express my deepest gratitude to Arnaud Brosseau, my "guardian angel" next to the microscope. Without his immense knowledge in microscopy, spectrocscopy and his natural talent in coming up always with the best technical solution, this whole project wouldn't have been possible.

I thank my fellow labmates and all my colleagues in PPSM for the time spent together, the long scientific discussions and the nights out to forget the sorrows of being a PhD student. I also thank all my other friends in whom I have always found comfort through the darkest moments and companionship through the happiest ones. My deepest gratitude also goes to Dr. Rachel Meallet-Renault for enlightening me the first glances of research during her captivating lectures in M1 and M2 and for directing me to PPSM for my PhD.

I also want to thank the European Research council for funding, Ecole Normale Supérieure Paris-Saclay and Centre National de la Recherche Scientifique (CNRS) for supporting this exciting project.

Last but not least, I would like to thank my parents and my sister for their spiritual support throughout writing this thesis and my life in general.

General introduction

This work concerns the study of the so-called mechano-responsive luminescent materials. They are fluorescent systems -capable of absorbing and emitting lightwhose fluorescence intensity is modulated through mechanical stress (friction, compression, stretching etc.). The discovery of this phenomenon dates back to the 1980s, but it is only in the past decades that it has gained a lot of attention among researchers. Many varieties of systems have proven to show such properties, and in the latest years increasing efforts have been invested in defining general structural characteristics that lead to mechanofluorochromic systems. Specifically, the objective of the present work is to give new insights on the mechanisms of this phenomenon and to optimize its properties to quantify the response to well-calibrated stimuli to lay the foundations for the development of reliable, versatile and ultrasensitive fluorescent force sensors.

To do so, we chose the well-known π-conjugated polymers -polydiacetylenes (PDAs) -which are widely used as fluorescent sensors thanks to their unique optical properties that can be modulated by a wide variety of external stimuli. Herein, we carried out an initial synthetic work to prepare four PDA-derivatives, to assess the role of the self-assembling group on the lateral monomer chain and the presence of a fluorophore on their optical properties, polymerization efficiency and on their response to mechanical and thermal stimulations. The study of mechanofluorochromism (MFC) was carried out on different scales. First of all, we determined qualitatively if these materials responded to mechanical stress at the macroscale. At a later stage, we tackled the task from a nanoscale point of view, relying on a microscopy set-up composed of an atomic force microscope coupled with a fluorescence microscope, with the aim of evaluating: the force intensity that is required to trigger a fluorescence activation; the type of force that is more adapted; the aggregation state in which the PDAs are arranged that is more convenient for the design of force sensors; if fluorescence response intensity and applied force intensity are correlated.

Chapter 1 is a bibliographic review on the main recent advancements in mechanofluorochromic compounds, specifically focusing on the correlation between structure and emission properties. Particular attention is dedicated in retracing the key points in PDA-based materials and their application as fluorescent or chromatic sensors.

Chapter 2 deals with the synthesis of four novel diacetylenes (DAs), where the self assembling group on the side of the chain is modified and a tetrazine fluorophore is covalently attached to the other end of the chain. The polymerization properties of the four compounds are studied by steady-state spectroscopy methods. The energy transfer process that occurs in tetrazine-functionalized PDAs is studied by steady-state and time-resolved spectroscopy. At last, the mechanofluorochromic and thermofluorochromic properties of PDA films are evaluated at the macroscale, in order to answer the question: Does a mechanical stimulation produce a fluorescence signal?

Chapter 3 digs into the nanoscale study of the same thin films, by means of an atomic force microscope coupled to a fluorescence microscope. First of all, topographic atomic force microscopy (AFM) images are used to trace the morphological peculiarities of each compound. In addition, AFM is used to mechanically stimulate the films with a nanometric sized tip. The fluorescence response is probed consecutively by fluorescence microscopy. A systematic study on the AFM parameters adjustments is carried out to conclude on the best conditions to apply friction forces and activate PDA fluorescence. Multiple tests are then performed on different zones of the samples, at different forces, to answer the questions: What is the needed applied force intensity that triggers a fluorescent response? Are fluorescence intensity and force intensity correlated?

Chapter 4 focuses on the same PDA derivatives in the form of aggregates in suspension and isolated single crystals deposited on glass slides. Suspensions are prepared by different methods and the spectroscopic properties that arise from polymerization are attentively assessed as a function of compound, preparation method and preparation parameters. Thermal stimuli are also tested on some of these suspensions. Isolated single crystals are obtained by deposition of the prepared suspensions on glass slides. The study is then extended to the nanoscale, analysing the morphological features of these objects by AFM and their dependence, again, on compound type, preparation method and preparation parameters. Friction forces and pressures are applied by AFM on the crystals surfaces and the fluorescence response is probed by fluorescence microscopy. 10 General introduction

Chapter 1

Bibliographic review 1.1 Brief history of materials science

Since the beginning of time, humankind looked at science for finding answers to practical needs. The discovery of new materials and the knowledge on how to use them shaped the development of civilization. Human history, especially at its beginnings, is so dependent on materials that historians classify entire periods of time in relation to the main material that humans used the most (stone, bronze and iron age). The accessibility of particular metal resources indeed influenced the rate at which specific cultural groups developed more sophisticated technologies in respect to the others, and eventually determined their technical superiority. A faster technological development allowed these groups to establish complex social and institutional fabrics, which resulted in the modern repartition of power and wealth. 1 The approach of studying new materials changed in the XV and XVI century, when the first monographs on metallurgy were published: De re metallica by G. Agricola and De Pirotechnia by V. Biringuccio. 2 The process of discovering is progressively converted into designing, where man plays an active role. Until recently, how we made use of these materials has been limited by what we could see with our eyes, and what we could touch with our hands. Natural resources were exploited to extract materials to shape tools, but when atoms were discovered, everything changed. Over a period of a few decades at the beginning of the XX century, scientists and engineers were not limited by what they could see and touch any more, but could play with the basic building blocks of all materials. The growing scientific knowledge in physics and chemistry led to the birth of materials science as we know it today: an interdisciplinary field that intertwines concepts from solid state physics, chemistry, crystallography, optics and engineering, for the design and production of new materials. The correlation between molecular structure and physical properties becomes crucial to the understanding of how matter behaves to further exploit such behaviours in real life applications or mimic and ameliorate specific characteristics in new systems.

In the past decades, scientific research has led to the development of new advanced materials that had deeply affected our way of living and our standards. We live in the so-called silicon age, which began with the invention in 1959 of new generation transistors based on semi-conducting silicon materials that allowed the development of modern computers and mobile phones, marking the entering in the so-called information era. It is in the same year that the renowned physicist Richard Feynman gave a speech called "There is plenty of room at the bottom [for miniaturization]", 3 implying that the field of physics (and science in a broader sense) was on the way to a big revolution where the "infinite small" was not a secret any more. In his vision, it would have soon be possible to control matter at the molecular level to build nanoscale devices that do exactly what we want, which are what we call now smart materials.

Intelligent materials are designed materials that respond in a controlled fashion to external stimuli, producing a change in its properties. Temperature, mechanical stress, pH, light, electric field are all external inputs that can affect in a controlled way a specific material property. Here we focus on what are called chromogenic fluorescent materials, i.e. materials that are capable to absorb and emit light (fluorescence) and that, when stimulated, change properties (color or intensity) of light emission.

Mechanofluorochromic compounds

Among the wide variety of stimuli that smart materials respond to, mechanical stress is one of the simplest and most important that control their properties. Mechanical stimuli as grinding, shearing, pressing, rubbing, stretching or bending can all provide controlled functions. Mechanochromism is the phenomenon of color change upon mechanical grinding, crushing or rubbing, of a solid compound and potential reversion to the original color by, for example, heating or recrystallization.

Mechanofluorochromism is defined as a fluorescence modulation of color or intensity in the solid state by mechanical stress (grinding, shearing, pressing etc.). In literature, we find different terms around this phenomenon that are used interchangeably, like piezofluorochromism or piezochromic luminescence, mechanofluorochromism and mechanochromic luminescence. To avoid confusion, "piezo" comes from the greek "πιεζειν", which means to squeeze or press. It is thus preferable to use piezofluorochromic for compounds that change fluorescence properties upon pressure forces. It is important also to distinguish those so-called ON-OFF switching mate-rials that respond to mechanical stress by an extinction or activation of fluorescence emission (intensity modulation), for which the term mechanoresponsive luminescence is more appropriate, as discussed in the review of 2016 by Sagara et al. 4 The mechanism behind the fluorescence modification is still not well understood at the molecular level. However, it is known that the fluorescence properties of a solid material strongly depend on the molecular arrangement and intermolecular interactions between molecules. Any alteration of the molecular packing between fluorophores triggers a modification in molecular energy levels, thus a modified fluorescence signal. Controlling the aggregation state is in fact a way to achieve mechanofluorochromism as well as applying a mechanical force on a π-conjugated system.

The discovery of the phenomenon dates back to the 1970s, when spectroscopic changes due to pressure applications were recorded for the first time on conjugated polymers. 5 It is only in the past two decades that these compounds gained a lot of attention. In this section, we will briefly review the main examples of mechanofluorochromic compounds that have been discovered in the last years.

Organic molecules

The pioneer study on pure organic mechanofluorochromic small molecules was carried out by Araki et al. on tetraphenylpyrene-based dyes. Compound 1 in Figure 1.1a consists in a tetraphenylpyrene backbone with four hexyl amide groups at the para position of the phenyl units. By adding methanol to a solution of this compound in chloroform, a white powder as shown in Figure 1.1b-left is obtained and it shows a blue fluorescence ("B-form"). After grinding, the powder switches to an intense yellow color with a strong green fluorescence ("G-form") in Figure 1.1b-right, which can be backconverted to the B-form by heating. The process is schematized in Figure 1.1c: mechanical stimulation interferes with the molecular packing of the B-form, resulting in a random distribution of hydrogen bonding in the G-form. The obtained metastable form has different fluorescence properties due to the modified π-conjugation and can be converted back to the B stable form by heating. 6,7 This first example led the group to inquire further on this family of compound and especially on the role of the amine side chains. 8,9 The series of TPPy-based dyes shown in Figure 1.1d is synthesized and studied in respect to the mechanofluorochromic behaviour of these molecules. All of them presented MFC properties similar to the precursor, except for compounds 2a and 2d. It is concluded that hydrogen bonding between amines is crucial in establishing the pyrene-pyrene molecular packing to have MFC properties. The ester function in compound 2e does not allow H-bond formation, therefore it does not form the same molecular assembly. If compound 1 in Figure 1.1a shows a progressive red-shifted fluorescence when the Chapter 1. Bibliographic review applied pressure is increased from 2.0 MPa to 9.8 MPa, the TPPy substituted with an ester 2e needs higher pressures to show a mild fluorescence shift. In addition, compound 1 fluorescence modification is reversible by heating at 112 • C while compound 2e can be regenerated to the initial form by heating at only 40 • C, indicating that the pressed sample is unstable at atmospheric pressure.

Strong π-π interactions are also encountered in oligo(p-phenylenevinylene) (OPV) derivatives, whose crystal structures consist in planarized molecules stacked in a cofacial arrangement, producing excimer emission (see Box 1.2.1 for definition). Weder et al. studied a series of these derivatives with variable substituent groups (Figure 1.2a). 10 Among these, only C12-YB and C18-YB show evident piezofluorochromic behaviour as pure solids from yellow to blue fluorescence. Figure 1.2b shows then normalized fluorescence spectra of C18-YB as prepared (solid line) and after pressure (dashed line), which is bathochromically shifted by 26 nm and corresponds to the excimer form emission. Heating the compressed material to 130 • C restores completely the monomer form emission (dashed-dotted line in Figure 1.2b). Figure 1.2c shows that this cycle between the two forms can be repeated. Pressing for longer times yield to the same excimer form, as shown by the dotted line in Figure 1.2b. X-ray diffraction (XRD) analysis of the forms, which are both stable in time, showed the appearance of new peaks after grinding, and the same diffraction pattern after heating, concluding that the process is the interconversion between the same forms. An excimer (originally short for excited dimer ) is a short lived dimeric molecule in the excited state, generated from two identical molecules M, one in its excited state and the other in its ground state. Both singlet and triplet excimers exist and are noted 1 (MM) * and 3 (MM) * respectively. Excimers form within the lifetime of the excited state of the monomer, and they have their own radiative and non-radiative deactivation fate.

Excimer emission is usually characterized by a broad emission spectrum and is frequent in aromatic hydrocarbons giving rise to π-π stacking. Excimers are usually formed at high concentration, since a certain vicinity between monomers is needed.

Mechanofluorochromic properties are also found in liquid crystalline materials, which are functional, soft materials with ordered, dynamic and metastable phases. Kato et al. studied in depth the MFC behaviour of liquid crystalline materials. [11][12][13] Chapter 1. Bibliographic review

In the example we show here, two liquid crystalline dyes with structure shown in Figure 1.3a are studied. Compounds 1a and 1b, respectively pyrene and anthracenebased, initially show yellow fluorescence. Upon mechanical shearing, the pyrene derivative switches to green and the anthracene derivative to light blue. In both cases, the materials are initially in a metastable cubic phase, where both hydrogen bonding and π-π interactions in either the pyrene or the anthracene moieties are present. Mechanical stress induces a conversion to a columnar phase where no Hbonds are established (Figure 1.3b). π-π stacking in the cubic phase produces the excimer emission, that is lost in the columnar phase (hypsochromic shift). [7]. Another worth-noting example of organic molecules that show outstanding optical properties in the solid state are difluoroboron β-diketonate derivatives. The classical dye of this family that is brought as one of the best examples of mechanofluorchromism was reported by Fraser et al. in 2010. 14 Millimetric crystals of the dye shown in Figure 1.4a present different fluorescence colors, depending on the crystal type. Prism crystalline structures show green luminescence, while needles and dendritic crystals show cyan and blue luminescence (Figure 1.4b1, b2, b3). Spectra of the corresponding crystal structures can be seen in Figure 1.4c. Fluorescence of a thermally annealed solid film of the same compound show blue-green fluorescence. When sheared with a cotton swab tip, the emission switches to yellow. Annealing with a heat gun restores the blue-green initial fluorescence and cycles of writingerasing can be thus performed (Figure 1.4d).

The polymorphism of these derivatives with different functional groups have been studied further in the group 15 and their properties at the nanoscale have been inquired. 16 These studies put the stress on the different emission between the crystalline and amorphous phase of these compounds.

In the recent years, many other families of organic molecules showing MFC properties have been reported. Different polymorphs of triphenylamine derivatives show different wavelengths of emission. 17 Pyran derivatives also have been reported [14].

to show aggregation induced emission (AIE) and polymorphism that lead to MFC properties. 18 Small organic molecules that can probe as well mechanical constraints in their environments are the so-called rigidochromes: fluorescent dyes whose emission intensity depends on the viscosity of their milieu. The mechanism behind this modulation lays on the rapid non-radiative relaxation of such molecules when rotation on specific bonds is allowed (low viscosity). 19,20 This type of smart-materials have been used to measure local viscosities in polymer films and study their free volume and glass transition 21,22 and to investigate the viscosity of membranes and intracellular media. 23,24

Organometallic complexes

Mechanochromic luminescence of organic-inorganic complexes have also shown to be possible and gained a lot of attention recently. 25 Mechanical treatment of certain metal complexes has been shown to produce luminescence changes, but the mechanism is still under debate. It is believed that luminescence variation, similarly to pure organic molecules, generate from intramolecular conformational distortions, or intermolecular variations in the π-π stacking, metal-metal or H-bonding interactions. Different metal ions have been found to yield MFC complexes: Zn(II), 26,27 Au(I), [28][29][30][31] Pt(II), [32][33][34] Cu(I), [35][36][37] Ag(I), 38 Al(III), 39 Ir(III). 40 Dinuclear complexes have shown such properties as well. 41 We report here one particular example of a gold complex reported in 2013, Chapter 1. Bibliographic review that showed mechanically induced molecular domino transformations that propagate from crystal to crystal. 42 The Au complex has the structure shown in Figure 1.5a. Depending on the preparation method, different polymorphs are obtained (I b and II y ) that have different spectroscopic properties. I b has several absorption bands in the range 300 -400 nm and several emission bands in the range 400 -450 nm. II y has a strong absorption band at 432 nm and an emission band at 567 nm. The different spectroscopic properties of the two polymorphs stem out of different intermolecular interactions. From crystal X-ray analysis it is found that II y head-to-tail dimers have a short Au-Au distance of 3.177 Å, suggesting the presence of strong aurophilic interactions that are not present in I b polymorph. Interestingly, it was found that mechanical shearing of form I b produced the II y form, as confirmed by the emission spectra shown in Figure 1.5b. A smaller force was applied with a needle on I b crystal. A small pit was formed on the surface and yellow luminescence spots appeared. Acquisition of images in time, showed that the conversion is extended to other portions of the crystal, yielding to yellow luminescent II y form (Figure 1.5c).

Polymer composites

Dye-doped polymers are a representative family of mechanofluorochromic materials. A fluorophore is thus dispersed in the polymer matrix, where it is considered to aggregate in supramolecular arrangements through non-covalent interactions like π-π interactions between planar aromatic backbones. It is likely that upon fluorescence excitation, emission of the excited dimer is recorded. Tensile deformation of the polymer matrix breaks the non-covalent interactions between fluorophores, which are then dispersed in the matrix (excimer-to-monomer transformation). The monomer fluorescence is usually blue shifted compared to the excimer emission. Of course, not all dyes give such mechanofluorochromic behaviours when dispersed in a polymer matrix. Plus, different requirements of dye-polymer compatibility need to be taken into account to form the composite. The fluorophore has to be highly aromatically conjugated and free from bulky substituent groups to promote π-π stacking. The polymer should be soluble in solvents to be easily mixed with the emitting unit, or high temperatures must be used to mix the dye in the melted polymer. 43,44 The first example of a dye-doped polymer was reported by Weder et al. in 2002. 45 They prepared two blends of OPV derivatives, substituted by cyano groups, in linear low density polyethylene (LLDPE). The structures are shown in Figure 1.6a and b (BCMDB and BCMB). Both compounds show increasing excimer emission for concentrations higher than 0.02% w/w and only monomer emission for lower concentrations. In Figure 1.6a and b, images of the polymer blends subjected to tensile stress are shown. BCMDB's emission switches from orange to yellow and BCMB's from yellow to blue in the part that has been stretched. Monomer emission grows back progressively when the tensile stress is increased from 0 to 500%, as proven by the fluorescence spectra in Figure 1.6. 46 Figure 1.6: Structure, fluorescence images after tensile stress when dispersed in LLDPE (0.20% w/w ) and fluorescence spectra for progressive tensile stress until 500% of compound BCMDB (a); structure, fluorescence images after tensile stress when dispersed in LLDPE (0.20% w/w ) and fluorescence spectra for progressive tensile stress until 500% of compound BCMB (b). Figure adapted from reference [46].

Other examples using other dyes and other polymers exist. An extensive review lists all the MFC polymers, including dye-sensitized polymers. 47 Pucci et al. used bis(benzoxazolyl)stilbene (BBS) as fluorescent moiety, dispersed in poly-(propylene) (PP), 48 LLDPE, 49 poly-(1,4-butylene succinate) (PBS). 50 Another approach to build mechanoresponsive luminescent (MRL) polymers has been recently proposed by Weder et al. and it consists in the derivatization of MRL chromophores with supramolecular binding motifs. An OPV derivative is functionalized with selfcomplementary hydrogen-bonding ureido-4-pyrimidinone (UPy) groups. As prepared, the polymer has orange emission. If subjected to a process of heating and cooling, the fluorescence switches first to green and then to red. Scratching on the Chapter 1. Bibliographic review polymer surface of either the red or orange form shifts the fluorescence to yellow. 51 The great disadvantage of these examples is that generally a plastic deformation of the system (i.e. irreversible) is needed to cause fluorescence modifications. This represents indeed a great limitation for their applicability as force sensors.

Intrinsic mechanofluorochromic polymers

The possibility of having intrinsinc mechanofluorochromic polymers is very interesting from an industrial point of view, because of their better processability and wider applications. However, thermoplastic polymers are composed of long chains of atoms usually connected by single bonds. In fact, the electrons of these bonds reside in low energy orbitals that are separated by a large energy gap from the corresponding anti-bonding molecular orbitals, which does not allow light absorption or emission. For this reason, to achieve optically active polymers, it is necessary to have highly conjugated polymer chains, which result in rigid structures. Viscoelasticity, however, is an essential property to apply mechanical tensile stress. One of the approaches to overcome these limitations consists in dispersing a coloured dye in the polymer of interest, as discussed in the previous section. A second approach is to covalently link a mechanofluorochromic moiety (mecanophore) to the macromolecular chains by co-polymerization. The emission change can be issued either by an alteration of the intermolecular interactions or by covalent bond cleavage.

In 2009, a spiropyran (SP) was incorporated by copolymerization into an elastic polymer, poly-(methyl acrylate) (PMA) or used as coupling agent in the synthesis of poly-(methyl methacrylate) (PMMA) beads. [START_REF] Davis | Force-Induced Activation of Covalent Bonds in Mechanoresponsive Polymeric Materials[END_REF] The spiropyran mechanophore, whose structure is presented in Figure 1.7a, reversibly transforms to a coloured and fluorescent merocyanine form via rupture of the spiro group carbon-oxygen (C-O) bond when it is subjected to mechanical stress (Figure 1.7b).

It is crucial that the stress applied on the SP -linked polymer is efficiently transferred on this specific bond to promote the chemical reaction. It was shown that efficient conversion is obtained when the SP moiety is linked to the polymer chain through the 5' and 6' positions of the indole side and through positions 7 and 8 of the benzopyran side. Figure 1.7b shows that the application of a mechanical tensile stress on spiropyran-functionalized PMMA films promotes the spiropyranmerocyanine conversion. Fluorescence anisotropy measurements on the same system concluded that the first spiropyran moieties that undergo conversion to merocyanin are those oriented along the traction force axis. When traction force is increased, the mechanophores tend to align on the force axis.

Mechanofluorochromism at the nanoscale

The majority of studies on mechanofluorochromism, as shown in the previous section, are carried out at the macroscale. Powders, drop casted films, dye-doped polymer matrices are the usual supports on which MFC compounds properties are characterized. However, Carpick et al. showed in 2000 that it is possible to stimulate mechanically a solid film at the nanoscale by means of an AFM tip and probe the mechanoresponsive luminescence by fluorescence microscopy. [START_REF] Carpick | First Observation of Mechanochromism at the Nanometer Scale[END_REF] This first report on polydiacetylene trilayers, which will be reviewed in the dedicated section (Section 1.3), opened up a new approach to inquire on the mechanisms behind mechanochromic luminescence and mechanofluorochromism in general. In the past years, a number of studies have been carried out on different families of MFC molecules and aggregation states.

Vacha et al. reported in 2013 some interesting results on simultaneous AFM and fluorescence microspectroscopy on single nanoparticles of the conjugated polymer poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV). [START_REF] Kobayashi | Mechanical Manipulation of Photophysical Properties of Single Conjugated Polymer Nanoparticles[END_REF] Each MEH-PPV nanoparticle is composed of randomly oriented fluorophores, with a broad distribution of segment lengths, where the absorbed energy is transferred to the lowest energy segments that emit fluorescence. Mechanical pressure of an isolated MEH-PPV nanoparticle has a double effect: first it impacts the size of the particle, secondly it causes local conformational disruption of the MEH-PPV chains near the tip (with the tendency to break long segments that serve as energy traps), as shown Chapter 1. Bibliographic review in the schematics in Figure 1.8a. Since the fluorescence signal is linearly correlated to the size of the particle, the pressure-induced modifications lead to an activation of the fluorescence signal, as can be seen in Figure 1.8b. In addition, long red-emitting segments are disrupted by the AFM tip, causing a blue shift in fluorescence, as can be seen in Figure 1.8b. Always in Figure 1.8, it is interesting to notice that these spectroscopic modifications are reversible, and the initial state is restored when the tip is retracted.

A similar fluorescence modulation has been recently reported by the same group. Completely reversible ON-to-OFF mechanism is achieved by applying a pressure (for a wide range of vertical forces, from 100 nN to 1 µN) on single nanoparticles of poly(9,9-dioctylfluorene) (PFO). The nanoparticles show an intense emission at 429 nm, characteristic of a well-ordered β phase.

As shown in Figure 1.9a, the fluorescence signal drops when a 1 µN pressure is applied to the particle. Degradation does not occur, as shown by the normalized fluorescence spectra in Figure 1.9b which keep the same spectral shape before, during and after pressure. Polarization experiments confirmed that the β phase segments remain intact and do not change their orientation after stimulus. The fluorescence change is therefore attributed to a more dense chain packing of the soft amorphous

22

Chapter 1. Bibliographic review PFO chains, resulting in strong interchain interaction. When the pressure is increased to 5 µN, an increase compared to the initial state is recorded (Figure 1.9c).

The pressure causes breaking of the β-phase conformation and generation of the glass phase. The fraction of the glass phase further increases after retraction of the tip from the NP, as can be seen from the fluorescence spectra in Figure 1.9d.

Another remarkable example of mechanofluorochromism recorded at the nanoscale was reported by Genovese et al. in 2016. A platinum complex, whose structure is shown in Figure 1.10a, shows mechanofluorochromic properties at the nanoscale. The powder, as synthesized, shows bright blue fluorescence that switches to an intense yellow fluorescence upon grinding as shown by the pictures under UV light in Figure 1.10b and the fluorescence spectra in Figure 1.10c. Fluorescence lifetime measurements and XRD experiments confirm that the Pt(II) complex arrange in ultralong ribbons via weak supramolecular interactions (π-π interactions) and not by Pt-Pt interactions. Plus, it was found that grinding is responsible of the transition of a highly crystalline phase (blue-emitting) to an amorphous (yellow emitting) phase, where the long order is broken. A schematized mechanism of the process is Chapter 1. Bibliographic review proposed by the authors and shown in Figure 1.10d. Long crystals of the Pt(II) complex are subjected to friction forces at the nanoscale, using an AFM tip. The fluorescence is recorded simultaneously. A constant force of 24 µN is used to engrave the word "mechano", at a scan rate of 10 µm•s -1 . The fluorescence image after engraving is shown in Figure 1.10e. The mechanical stimulation induces the same conversion from a highly crystalline phase to an amorphous state, causing a fluorescence switch from blue to yellow. In our group, Louis et al. reported in 2019 a similar mechanofluorochromic behaviour on nanoparticles prepared from a difluorobon-β-diketonate derivative. The structure of the studied compound (DFB-Bu-Amide) is shown in Figure 1.11a, along with a schematics of the reprecipitation process from a tetrahydrofurane (THF) solution of DFB-Bu-Amide in water to form nanoparticles. The suspension is then deposited on glass slides, and studied by a home-made set up that couples AFM and fluorescence microscopy, shown in Figure 1.11b. The objects morphology is studied by AFM, as shown in Figure 1.11c, and the fluorescence of the same zone of the sample can be recorded, as shown in Figure 1.11d. Fluorescence spectra can also be recorded and can be centred on the single objects on the substrate. Therefore, a fluorescence spectrum is recorded before and after consecutive mechanical stimulations performed in contact mode with the AFM. Before mechanical stress, a large emission band centred at 533 nm is recorded. This band shifts progressively towards lower wavelengths when frictions at normal force of 130 nN (two times) and 260 nN are applied, as shown in Figure 1.11e. The morphology is probed by AFM after each stimulation. Morphological modifications are recorded for each force application, until disruption of the nanoparticle. The change in fluorescence of DFB-Bu-Amide is reconducted to a progressive transition from a crystalline to an amorphous phase, as it was confirmed by XRD measurements at the macroscale. 16

Polydiacetylenes

Since their discovery by Wegner et al. in 1969, 58 PDAs have attracted a lot of attention over the years because of their unique photophysical properties. PDAs are prepared by topochemical polymerization of ordered diacetylenes, to form long πconjugated chains (general structure shown in Figure 1.12a). Polymerization usually occurs via 1,4-addition of diacetylene monomers triggered by UV (254 nm), γ light irradiation, X-rays or e -beams, that results in alternated triple and double bonds Chapter 1. Bibliographic review (ene-yne) polymer chains. The peculiarity of diacetylene (DA) polymerization is that it occurs in the solid state. Therefore, unlike the vast majority of polymeric materials prepared by chemical reactions, there is no need of using high temperatures, catalysts or chemical initiators. The first studies on the polymerization properties of this family of molecules appear in the 1970s and are developed over the years. [START_REF] Wegner | Topochemical Polymerization of Monomers with Conjugated Triple Bonds[END_REF] In the 1980s, a comparative study on differently functionalized di-alkynes allowed to designate the general requirements for successful polymerization. [START_REF] Volker Enkelmann | Structural Aspects of the Topochemical Polymerization of Diacetylenes[END_REF][START_REF] Bässler | Photopolymerization of Diacetylenes[END_REF] The monomer units must be self-assembled in a specific geometrical and packing order to favour reaction between two diacetylenes. It was shown that for the majority of monomer types under study, the maximal reactivity between two neighbouring units is achieved for a stacking distance d <5 Å and φ ≈ 45 • . See Figure 1.12b for a graphical representation. Unlike usual chemical polymerizations, topochemical reactions occur in the solid state by diffusionless motions, meaning that in general the crystalline features of the self-assembled monomers are retained in the polymer crystals (see Figure 1.12b). Figure 1.12: General structure of a polydiacetylene (a); schematics of the topochemical polymerization of PDAs, where d is the packing parameter, ϕ is the monomer angle with respect to the packing axis and R v is the Van der Waals distance (minimal allowed distance) between monomers (b). Figure adapted from reference [60].

The vast majority of PDAs are insoluble in any solvent, which complicates their structural analysis by conventional methods. A detailed study of the reaction mechanism of diacetylene photopolymerization is conducted in 1984 by H. Sixl by means of optical absorption and electron spin resonance measurements at low temperature on a TS-6 (structure shown in Figure 1.13a). The individual reaction steps are given by the photoinitiation reaction (formation of reactive dimers), the photoinduced chain propagation (addition of monomer molecules to the reactive oligomers), and the photoinduced chain termination reaction (formation of unreactive chain ends). Chain initiation consists in the photoexcitation of two monomer units, which then excite by energy transfer the neighbouring molecules M'. The first reactive centre is proven to have radical nature, while the second excitation does not need to be of radical nature. From the excited monomers, reactive dimers are obtained: diradicals (DR 2 ) and asymmetric carbenes (AC 2 ), see Figure 1.13b. Parallel to the photoinitiation processes (with hν), photoaddition processes are observed. After dimer initiation, trimer formation from the dimer is possible etc. The monomer molecules adjacent to the reactive centres form a trap for the optical excitation. They can be either excited directly by the irradiation, or by energy transfer from neighbouring photoexcited monomer. The photoaddition reaction, through diradicals or asymmetric carbenes, is in competition with the chain initiation reaction. This is why it is common to many PDAs to slow down their polymerization kinetics when the polymer content increases. Simultaneously, chain termination reactions lead to stable oligomers (SO), of variable lengths. All the processes are outlined in the scheme in Figure 1.13c. [START_REF] Sixl | Spectroscopy of the Intermediate States of the Solid State Polymerization Reaction in Diacetylene Crystals[END_REF] By a concomitant study of a similar PDA derivative, TS-12 (structure shown in Figure 1.13a), conducted at room temperature, V. Enkelmann showed that the final chain length distribution in photopolymerized TS-12 is dependent on the monomer-to-polymer conversion rate (X). Figure 1.13d shows the distribution curve of the chain length as a function of conversion (X). The lengths can thus vary a lot, from 10 to 1000 monomer units depending on the extent of polymerization.

Concerning polymerization quantum yields, the efficiency of such processes is far to be 100% and depends on the energy trigger for the initiation step. As it was shown in recent studies by Schott et al. on the 3BCMU and 4BCMU derivatives, [START_REF] Schott | Photopolymerization Quantum Yields in Two Reactive Diacetylenes, 3BCMU and 4BCMU, and Relation to γ-Ray Induced Polymerization[END_REF] the polymerization quantum yield is lower than 100% and the reaction kinetics is significantly slowed down by the formation of new polymer chains. In a more recent study, [START_REF] Spagnoli | Photopolymerization of Thin Polycrystalline Diacetylene Films and Quenching of the Precursor Excited State[END_REF] a general method for determining the polymer content in PDA crystalline films is proposed and used to conclude that UV irradiation does not lead to completely polymerized samples for different reasons: existing chains may block chain propagation; the excited state from which polymerization is triggered may be quenched by neighbouring chains; side groups influence the packing of the monomers, thus may also affect the reaction efficiency; when using UV light trigger, the formation of new chains may act as a light filter for monomers that have not reacted yet. At the end of the polymerization process by UV irradiation on 253 nm thick films of 3BCMU and 4BCMU, total conversions to polymer of around 35 and 50% are obtained. γ rays triggered-polymerization of 4BCMU gave almost completely polymerized films.

Thanks to the alternation of ene-yne motifs, electrons in the main backbone are highly delocalized, giving the polymer interesting optical characteristics. The crystalline powder, film or aggregate shows an intense blue color appearing when the sample is polymerized, with a characteristic absorption band centred at 640 nm. [START_REF] Tieke | Polymerization of Mono-and Multilayer Forming Diacetylenes[END_REF][START_REF] Patel | Structure-Property Relationships of Diacetylenes and Their Polymers[END_REF] More interestingly, another chromogenic transition occurs when the blue-PDA is Chapter 1. Bibliographic review exposed to external stimuli: the sample turns red and the absorption spectrum shifts towards lower wavelengths, with a characteristic absorption band centred at 540 nm. This transition is visible to the naked eye. However, the most promising feature of these molecules in the context of sensing is that the blue phase is non fluorescent, while the red phase of the polymer exhibits fluorescence with minimal bleaching. [START_REF] Baughman | Comments on the Optical Properties of Fully Conjugated Polymers: Analogy between Polyenes and Polydiacetylenes[END_REF][START_REF] Olmsted | Fluorescence of Polymerized Diacetylene Bilayer Films[END_REF][START_REF] Carpick | Polydiacetylene Films: A Review of Recent Investigations into Chromogenic Transitions and Nanomechanical Properties[END_REF][START_REF] Sun | Chromatic Polydiacetylene with Novel Sensitivity[END_REF] The exact mechanism behind the electronic transition between blue and red phase is still not well understood and under debate. However, it is presumed to be connected with the supramolecular arrangement of the polymer that goes from strictly planar (blue-phase) to non-planar (red-phase), causing a blue shift in the absorption spectrum. [START_REF] Carpick | Polydiacetylene Films: A Review of Recent Investigations into Chromogenic Transitions and Nanomechanical Properties[END_REF][START_REF] Filhol | Polymorphs and Colors of Polydiacetylenes: A First Principles Study[END_REF] The interaction between the side chains is crucial in determining the spectroscopic properties of the polymer and its sensing properties through phase change. No general guidelines of synthesis are given to predict them, but a large amount of diacetylene derivatives have been synthesized and compared. Chain-chain intermolecular interactions may be manipulated to design a system with specific properties by adjusting the topological structure of the side chains like alkyl chain length, position of the diacetylene moiety in the backbone and the nature of the self-assembling groups. [START_REF] Tachibana | Effect of Position of Butadiyne Moiety in Amphiphilic Diacetylenes on the Polymerization in the Langmuir-Blodgett Films[END_REF][START_REF] Huo | Advances in Polydiacetylene Development for the Design of Side Chain Groups in Smart Material Applications -a Mini Review[END_REF][START_REF] Yu | A Spectroscopic Analysis of the Role of Side Chains in Controlling Thermochromic Transitions in Polydiacetylenes[END_REF][START_REF] Pattanatornchai | Influences of Structural Mismatch on Morphology, Phase Transition Temperature, Segmental Dynamics and Color-Transition Behaviors of Polydiacetylene Vesicles[END_REF][START_REF] Khanantong | Influences of Bulky Aromatic Head Group on Morphology, Structure and Color-Transition Behaviors of Polydiacetylene Assemblies upon Exposure to Thermal and Chemical Stimuli[END_REF] In Figure 1.14a, absorption and fluorescence spectra of 10,12-pentacosadiynoic acid (PCDA) are shown. Blue-poly-PCDA absorbs at 640 nm and has a vibronic peak at 580 nm, red-poly-PCDA absorbs at 540 nm and has a vibronic peak at 480 nm (Figure 1.14a). The fluorescence signature of red-poly-PCDA is shown in Figure 1.14b, where only the red phase has a detectable fluorescence signal. Polymerization of DAs, however, is a complicated process that often in literature is reduced to the formation of two distinguishable and spectroscopically different phases, blue and red. A recent study by Lifshitz et al., showed for the same compound that irradiation in the UV of differently prepared samples (Langmuir films polymerized in water -LF W -; Langmuir films polymerized on solid support -LF S -; spin-coated films -SC -) is a much more dynamic process than it is usually assumed to be. In fact, polymerization in films indeed induces deformations in the sample, which are responsible of progressive shifts in the polymer exciton. As shown in the absorption spectra in Figure 1.14c, the blue-poly-PCDA absorption band evolves and shifts towards lower wavelengths along irradiation, until sometimes a complete conversion to the red phase. Therefore, it is incorrect to speak dichotomically of blue and red PDA phases, since intermediated states in between the two do exist. The intermediate phase that absorbs around 600 nm can also be called purple phase (in between blue and red) and depending on the monomer structure and side chains, it can be obtained at the very beginning of the polymerization, by progressive irradiation or not witnessed at all. [START_REF] Lifshitz | Phase Transition Kinetics in Langmuir and Spin-Coated Polydiacetylene Films[END_REF] Since the first report by Charych et al. in 1993 on a sialic-modified PDA for selective detection of influenza virus, exploiting its blue-to-red chromatic transition, [START_REF] Charych | Direct Colorimetric Detection of a Receptor-Ligand Interaction by a Polymerized Bilayer Assembly[END_REF] the literature on PDA sensing applications has been flourishing with new sophisticated systems to sense a large amount of different stimuli. Amongst them we find bio-or chemo-specific binding for microorganisms and biomolecules of interest, [START_REF] Ma | Colorimetric Detection of Escherichia Coli by Polydiacetylene Vesicles Functionalized with Glycolipid[END_REF][START_REF] Pindzola | Antibody-Functionalized Polydiacetylene Coatings on Nanoporous Membranes for Microorganism Detection[END_REF][START_REF] Scindia | Colorimetric Detection and Fingerprinting of Bacteria by Glass-Supported Lipid/Polydiacetylene Films[END_REF][START_REF] Cheol | A Direct, Multiplex Biosensor Platform for Pathogen Detection Based on Cross-Linked Polydiacetylene (PDA) Supramolecules[END_REF][START_REF] Lee | Recent Progress in Stimuli-Induced Polydiacetylenes for Sensing Temperature, Chemical and Biological Targets[END_REF][START_REF] Kyung | Specific Colorimetric Detection of Proteins Using Bidentate Aptamer-Conjugated Polydiacetylene (PDA) Liposomes[END_REF][START_REF] Do | Biomimetic Detection of Aminoglycosidic Antibiotics Using Polydiacetylene-Phospholipids Supramolecules[END_REF][START_REF] Jeon | Conjugated Polydiacetylenes Bearing Quaternary Ammonium Groups as a Dual Colorimetric and Fluorescent Sensor for ATP[END_REF] temperature, [START_REF] Chance | Thermochromism in a Polydiacetylene Crystal[END_REF][START_REF] Chen | A Thermally Reversible Temperature Sensor Based on Polydiacetylene: Synthesis and Thermochromic Properties[END_REF][START_REF] Xu | Thermochromism and Supramolecular Chirality of the Coumarin-Substituted Polydiacetylene LB Films[END_REF] pH, [START_REF] Song | Modulating Artificial Membrane Morphology: pH-Induced Chromatic Transition and Nanostructural Transformation of a Bolaamphiphilic Conjugated Polymer from Blue Helical Ribbons to Red Nanofibers[END_REF][START_REF] Kew | pH Response of Carboxy-Terminated Colorimetric Polydiacetylene Vesicles[END_REF] metal ions, [START_REF] Lee | Polydiacetylene Liposome Arrays for Selective Potassium Detection[END_REF][START_REF] Lee | Polydiacetylene-Liposome Microarrays for Selective and Sensitive Mercury(II) Detection[END_REF][START_REF] Wang | Polydiacetylene Liposome-Encapsulated Alginate Hydrogel Beads for Pb2+ Detection with Enhanced Sensitivity[END_REF] solvent detection 97 and mechanical forces. [START_REF] Carpick | First Observation of Mechanochromism at the Nanometer Scale[END_REF][START_REF] Sik | Shear-Induced Color Transition of PDA (Polydiacetylene) Liposome in Polymeric Solutions[END_REF][START_REF] Feng | Force Sensors: Hybrid Mechanoresponsive Polymer Wires Under Force Activation[END_REF][START_REF] Chae | Mechanically Drawable Thermochromic and Mechanothermochromic Polydiacetylene Sensors[END_REF][START_REF] Terada | Visualization and Quantitative Detection of Friction Force by Self-Organized Organic Layered Composites[END_REF][START_REF] Pil | A Reversibly Mechanochromic Conjugated Polymer[END_REF] Manipulation of the chemical structure of such compounds can ensure the recognition of a particular analyte to serve as sensor. However, when playing with the monomer nature and functionalization of its side chains, it is of crucial importance to consider the effect that these modifications have on the supramolecular arrangement of the polymer and ultimately on its spectroscopic properties. The functionality of a material is not determined exclusively by its chemical structure, but also by the way the components of such a material arrange. Efficient polymerization has been shown to be possible in different aggregation states, as long as the monomers are well packed: thin crystalline films, [START_REF] Spagnoli | Photopolymerization of Thin Polycrystalline Diacetylene Films and Quenching of the Precursor Excited State[END_REF][START_REF] Thierry Barisien | Reversible Quenching of a Chromophore Luminescence by Color Transition of a Polydiacetylene[END_REF][START_REF] Polacchi | Mechano-Responsive Fluorescent Polydiacetylene-Based Materials: Towards Quantification of Shearing Stress at the Nanoscale[END_REF] Langmuir-Blodgett (LB) [START_REF] Carpick | Polydiacetylene Films: A Review of Recent Investigations into Chromogenic Transitions and Nanomechanical Properties[END_REF][START_REF] Xu | Thermochromism and Supramolecular Chirality of the Coumarin-Substituted Polydiacetylene LB Films[END_REF]104 or Langmuir-Schaefer (LS) [START_REF] Alekseev | Stable Blue Phase Polymeric Langmuir-Schaefer Films Based on Unsymmetrical Hydroxyalkadiynyl N-Arylcarbamate Derivatives[END_REF] films, aggregates (nanoparticles) in solution, [START_REF] Chu | Chromatic Transition of Polydiacetylene in Solution[END_REF][START_REF] Okada | Color and Chromism of Polydiacetylene Vesicles[END_REF][START_REF] Pevzner | Giant Chromatic Lipid/Polydiacetylene Vesicles for Detection and Visualization of Membrane Interactions[END_REF][START_REF] Kim | Polydiacetylene (PDA) Liposome-Based Immunosensor for the Detection of Exosomes[END_REF] nanopatterned on other substrates [START_REF] Lee | Network Polydiacetylene Films: Preparation, Patterning, and Sensor Applications[END_REF] or embedded in other matrices. [START_REF] Feng | Force Sensors: Hybrid Mechanoresponsive Polymer Wires Under Force Activation[END_REF][START_REF] Chae | Mechanically Drawable Thermochromic and Mechanothermochromic Polydiacetylene Sensors[END_REF] Because of their ease of preparation, nanoparticles in solution have been widely

Chapter 1. Bibliographic review studied and a vast number of sensors have been developed. Particular interest falls in those derivatives with a polar head and an hydrophobic tail (amphiphiles), which can easily arrange in Langmuir-Blodgett films or in vesicles. Vesicle formation is achieved by simple sonication in water of diacetylene monomers, which forces the aggregation in circular double layers. For this reason, they are often called micelles, liposomes or more generally vesicles. A schematics of PDA aggregation is proposed by Reppy and Pindzola in reference [111] and it is shown in Figure 1.15. In a number of studies, these aggregates have shown to polymerize efficiently and to be particularly well suited for biochemical essays thanks to the biocompatibility of their milieu.

We will try now to review the main sensing mechanisms that produce a signal in PDA-based sensors.

Thermochromism

Thermochromism is by far the most explored mechanism of PDAs sensing since the first report dating back in 1976. [START_REF] Exarhos | Resonance Raman Study of the Thermochromic Phase Transition of a Polydiacetylene[END_REF] An increase of the ambient temperature can lead to the blue-to-red chromogenic transition of PDAs, in a range of temperature that is specific for the system, and with different chromatic responses that depend on its 30

Chapter 1. Bibliographic review C to red to finally be converted to a yellow phase (probably corresponding to the melted PDA) at 150 • C and higher.

Several compounds with different molar fraction of organic silicon to the total Si were prepared. The study is very thorough in showing how the percentage of organic silicon influences the color change, with the general trend that a higher percentage causes a blue shift of the absorption for each phase. It was also shown that for PDAPMO-100%, the conversion between purple and red phase is completely reversible, with high fatigue resistance. Indeed, the reversibility of the thermochromic response is an interesting property that is not common to all other stimulation mechanisms, which are typically irreversible. It has been shown in different studies how the reversible behaviour of PDAs is connected to non-covalent intermolecular force like hydrogen bonding, π-π stacking and Van der Waals forces. Generally, the stronger these interactions are, the easier is to achieve fully reversible thermochromic systems. [START_REF] Chen | A Thermally Reversible Temperature Sensor Based on Polydiacetylene: Synthesis and Thermochromic Properties[END_REF][START_REF] June Ahn | Colorimetric Reversibility of Polydiacetylene Supramolecules Having Enhanced Hydrogen-Bonding under Thermal and pH Stimuli[END_REF][START_REF] Ho | Optimal Photoluminescence Achieved by Control of Photopolymerization for Diacetylene Derivatives That Induce Reversible, Partially Reversible, and Irreversible Responses[END_REF][START_REF] Dong | New Dendritic Polydiacetylene Sensor with Good Reversible Thermochromic Ability in Aqueous Solution and Solid Film[END_REF] Chapter 1. Bibliographic review A remarkable reversible high-temperature thermofluorochromic PDA system is synthesized and the colorimetric response (CR) (definition in Box 1.3.1) and fluorescence response to thermal stimulation are studied in a recent report by Huo et al. [START_REF] Huo | High Temperature Thermochromic Polydiacetylenes: Design and Colorimetric Properties[END_REF] The importance of this study resides in the correlation between structure and spectroscopic response. In fact, three PDA-derivatives are prepared (structures shown in Figure 1.17a). The presence of multiple H-bonds in the structure ensures a reversible thermochromism and a high temperature resistance (need of higher energy to perturb the molecular packing). Plus, adding pyrene moieties increases the inter-chain supramolecular interactions by π-stacking.

A fully reversible thermochromism is found only for compound c, where the absorption spectra perfectly overlap in wavelength maxima and intensity before and after heating. The authors only provided pictures of the heated and cooled films, shown in Figure 1.17b. The CR of the three compounds is shown in Figure 1.17c. The derivative with two pyrene moieties (c) has higher resistance to temperature (up to 300 • C) and can sense with higher accuracy a wider range of temperatures. Plus, its thermochromic behaviour is perfectly reversible since the absorption spectra before heating and after a heating-cooling cycle perfectly overlap. The fluorescence spectra of the films for increasing temperature are shown in Figure 1.17d. In all cases, increasing the temperature corresponds with an increase of fluorescence signal. A remarkable shift towards higher wavelengths is registered for the three compounds, because of the increasing emission of the progressively formed red fluorescent polymer that covers pyrene emission.

BOX 1.3.1. Colorimetric response (CR)

The colorimetric response is a way to express the color changes that have occurred after stimulus of PDA material, and it has been largely used by several research groups to quantify the PDA response to a specific stimulus. It was defined by Okada et et al. in 1998 to characterize the color change upon thermal stimulus of PDA vesicles. [START_REF] Okada | Color and Chromism of Polydiacetylene Vesicles[END_REF] The CR reports the relative change in percent blue form PDA in the sensing material. The initial percent blue PB(0) is defined as:

P B(0) = A blue [A blue + A red ] • 100%
where A is the absorbance at the wavelength of either the blue or red form. This same formula can be used for any time point in the process, taking the corresponding absorbance. The colorimetric response characterizes the percent conversion to the red phase after conversion:

CR = P B(0) -P B(f ) P B(0) • 100%
Chapter 1. Bibliographic review

Mechanochromism

Mechanical stress, as thermal stimuli, can also induce the PDA blue to red transition, when an appropriate amount of energy is transmitted to the substrate and causes a distortion in the π-orbitals conjugation of the polymer chain. Mechanochromism on polydiacetylenes was reported for the first time in 1978 by Cottle et al. 5 Single millimetric monocrystals were prepared from bis (p-toluene sulphonate) of 2,4-hexadiyne 1,6-diol (TSHD), which was the reference polydiacetylene system at the time, synthesized by Wegner et al. in the same years. The crystal is subjected to pressure stress and the reflectivity is recorded for increasing pressure. The maximum absorption band shifts gradually towards lower wavelengths, corresponding to the blue-to-red transition of the polymer.

A similar experiment was conducted few years later, in 1989, by Tomioka et al., who reported for the first time a pressure-induced color change of poly-(heptacosa-10,12-diynoic acid) monolayers, at a gas-water interface. [START_REF] Tomioka | Surface-pressure-induced Reversible Color Change of a Polydiacetylene Monolayer at a Gas-Water Interface[END_REF] When applying an increasing pressure from 3 to 100 mN•m -1 , a progressive shift towards lower wavelengths of the reflectance spectrum is witnessed, corresponding to the blue-to-red transition of the polymer. The stimulus causes a decrease in the blue-PDA absorption (640 nm) and an increase in the red-PDA absorption band (540 nm), as it can be seen in Figure 1.18a.

In 1986, Rubner synthesized and characterized the first example of polyurethanediacetylene segmented copolymer. Cross-polymerization of the diacetylene moieties was successful, and modified the thermal-mechanical properties of the polymer, which can thus be tuned by controlling the extent of diacetylene cross-polymerization. [START_REF] Rubner | Synthesis and Characterization of Polyurethane-Diacetylene Segmented Copolymers[END_REF] In the same year, the chromatic properties of this derivative were studied in detail. Diacetylene polymerization was successful in situ, by UV irradiation. Plus, the chromatic response of the PDA moiety was studied by UV-vis absorption in respect to thermal and strain stress. Both stimuli induce a blue-to-red transition. The conversion by strain occurs for different polymerization ratios as can be seen in Figure 1.18b and 1.18c. [START_REF] Rubner | Novel Optical Properties of Polyurethane-Diacetylene Segmented Copolymers[END_REF] A more extensive study on the mechanochromic properties of this system is published in 1991 by Nallicheri and Rubner. [START_REF] Nallicheri | Investigations of the Mechanochromic Behavior of Poly(Urethane-Diacetylene) Segmented Copolymers[END_REF] Recent reports on mechano(fluoro)chromism at the macroscale use PDAs as sensing platform for direct measurement of applied mechanical stress. Amineintercalated PDA films were used by Ishijima et al. to probe the accumulation of shearing stress on solid substrates. [START_REF] Terada | Visualization and Quantitative Detection of Friction Force by Self-Organized Organic Layered Composites[END_REF] A schematics of the intercalation of PCDA molecules and alkyl-amines is presented in Figure 1.19a. Friction is applied on the films by means of weight-loaded cage that rasters the sample at constant speed, as shown in the model scheme in Figure 1.19a. The sheared area turns to red-PDA, as shown in Figure 1.19b. By changing the mass of the cage, the actual load transmit- [123, 125]. ted to the sample can be varied. This way, a first quantification of friction force by colorimetric response of the PDA was achieved. Forces range between 3 N and 25 N. Intercalation of amines of variable length was proven to be an effective method to tune the colorimetric response of PDAs films both to thermal and mechanical stimuli. [START_REF] Ishijima | Tunable Mechano-Responsive Color-Change Properties of Organic Layered Material by Intercalation[END_REF][START_REF] Watanabe | A Paper-Based Device of a Specially Designed Soft Layered Polymer Composite for Measurement of Weak Friction Force[END_REF] Reversible mechanochromism is also an important research prerogative on these kind of systems. However, not many examples exist in the literature, contrarily to small organic molecules. Lee et al. recently reported a reversible pure PDA system, made of a novel DA monomer, PCDA-4APDS, which contains a diphenyl disulfide moiety located in the middle of a structure containing two covalently linked and diametrically opposed DA-containing alkyl chains. Monomers are expected to self-Chapter 1. Bibliographic review assemble by engaging extensive hydrogen-bonds through the amine groups and by π-stacking though the central diphenyl moieties (structure shown in Figure 1.20a). This ensures a tight packing and a certain structural rigidity, which are essential for chromatic reversibility. In fact, grinding the polymerized blue powder switches the color to green (temperature is below the chromatic transition characteristic temperature), for which absorption spectra are not provided. No report on a green PDA form exists in the literature, making the understanding of the process quite obscure. Thermal annealing of the green-PDA leads to the classical red-PDA phase that upon cooling turns back to blue, as can be seen in Figure 1.20b. Despite the doubtful conversion to a green-PDA form, the system also needs to be converted to the more classical red-PDA phase, which upon cooling backconverts to the blue phase, as most of the thermochromic systems. Another interesting characteristic of certain PDA-based materials is the pressureinduced photopolymerizability. Kim et al. synthesized bisamide substituted DAs, with variable alkyl chain lengths. Interestingly, the powder obtained for the derivatives with 8 and 14 carbons alkyl chains by recrystallization with methanol were not polymerizable by UV irradiation at 254 nm. However, when these powders were subjected to mechanical pressures from 10 to 150 MPa, UV irradiation causes the classical chromogenic transition due to the formation of blue-PDA. This property was then used on DA-C8 coated PET films, whose structure is shown in Figure 1.21a, to pattern an acronym by selective force transfer in certain zones of the film. This way, UV irradiation triggers the polymerization exclusively in the zones that have been subjected to pressure (Figure 1.21b). [START_REF] Kim | Pressure-Induced Transition of Bisamide-Substituted Diacetylene Crystals from Nonphotopolymerizable to Photopolymerizable State[END_REF] An important breakthrough in mechanochromic materials and polydiacetylene chromatic characterization was made by Carpick et al. in 2000. The group studied the fluorogenic response of a PCDA acid trilayer thin film at the nanoscale (structure in Figure 1.22a). They managed to stimulate mechanically the film both by nearfield scanning optical microscopy (NSOM) and to probe the blue-to-red transition by optical fluorescence microscopy (Figure 1.22b). Plus, the first stimulation creates a nucleation point for the red phase that expands spatially in time, as shown by the topography images in Figure 1.22c, where a trilayer island has grown as a consequence of force application. [START_REF] Carpick | First Observation of Mechanochromism at the Nanometer Scale[END_REF] This pioneer experiment was repeated also on PDA monolayers, where the transition was also probed, [START_REF] Burns | Shear-Induced Mechanochromism in Polydiacetylene Monolayers[END_REF] and inspired our group and others to investigate the mechanofluorochromic behaviour at the nanoscale. [START_REF] Polacchi | Mechano-Responsive Fluorescent Polydiacetylene-Based Materials: Towards Quantification of Shearing Stress at the Nanoscale[END_REF]104 When driven by a direct application, mechanical stimulation is interesting in the context of sensing analytes that do not have a specific affinity with a recognition site. This is the case for example of saturated alyphatic hydrocarbons (SAHCs), which are extremely non-polar and do not have any functional groups that can interact with probes. The work of Park et al. consisted in exploiting the blue-to-red color transition of PDAs upon external mechanical stimuli and the swelling properties of polydimethylsiloxane (PDMS). [START_REF] Park | A Colorimetric Hydrocarbon Sensor Employing a Swelling-Induced Mechanochromic Polydiacetylene[END_REF] PCDA is mixed with PDMS precursor, irradiated at 254 nm to trigger DA polymerization and cured. The result is a flexible and transparent-blue 600 µm thick film, where PDA is homogeneously distributed. When the film is exposed to SAHCs of different lengths, the films turns red for pentane, purple for heptane and deep purple for nonane (as can be seen in the absorption spectra in Figure 1.24a). The color change is connected with the degree of swelling of PDMS, and the authors concluded that the shorter the hydrocarbon chain is and the more the film will swell, causing a higher blue-to-red conversion (see graph in Figure 1.24b). When looking at the microscopy images in Figure 1.24c of the embedded crystals of PCDA, swelling of the PDMS matrix causes mechanical strain on the crystal that start turning red from the weak edges and is progressively converted in its entirety. To confirm that the the blue-to-red PDA transition driving force is actually the strain forces caused by swelling of the matrix and not the interaction of the PDA crystals with solvents, exposure of these crystals to solvents without the PDMS matrix is necessary. In fact, some PDAs are naturally solvatochromic, as it will be review later.

Solvatochromism

Solvatochromism defines a color change of a system depending on the solvent polarity it is exposed to. PDAs, as many other molecules, undergo conformational changes in the supramolecular structure that are responsible of the blue-to-red chromatic transition when exposed to solvents. [START_REF] Lee | Recent Progress in Stimuli-Induced Polydiacetylenes for Sensing Temperature, Chemical and Biological Targets[END_REF] Chemical sensors for specific solvents can circumvent the use of expensive machinery like gas chromatography and most of all can give a direct response. However, distinguishing solvents with similar polarities can be extremely challenging for a single component sensor, since absorption and emission bands overlap to a certain extent in different solvents. Specific color transitions for different solvents is pretty rare, but a sensor composed of multiple Chapter 1. Bibliographic review probes with one-solvent specificity can solve the problem. A particular effective system was proposed in 2018 by Park et al., using an array of four different PDAs on conventional paper. [START_REF] Park | Smartphone-Based VOC Sensor Using Colorimetric Polydiacetylenes[END_REF] The monomers are inkjet printed in four spots of the paper substrate and polymerized by UV irradiation. The structures of the monomer units are shown in Figure 1.25a. Each polymerized form reacts slightly different to 11 volatile organic compounds, creating a different 4-well pattern depending on which solvents the sensor has been exposed to, as shown in Figure 1.25b. A smartphone app is also developed to have a direct output by taking a picture of the result.

Hydrochromism

Hydrochromism refers to a color change in response to water exposure. Some PDAs show interesting hydrochromic properties that can be applied for human sweat pore mapping, as reported by Park et al. in 2016. [START_REF] Park | Inkjet-Printable Amphiphilic Polydiacetylene Precursor for Hydrochromic Imaging on Paper[END_REF] The group synthesized three novel DA moieties, among which two show hydrochromic properties: the blue-to-red transition is triggered by exposure to water (structure shown in Figure 1.26a). The system, compared to previous ones studied in the same group, [START_REF] Yoon | Inkjet Printing of Conjugated Polymer Precursors on Paper Substrates for Colorimetric Sensing and Flexible Electrothermochromic Display[END_REF]134 showed a temperature dependent hydrochromic response that prevents the sensor to activate under atmospheric humidity, as shown in Figure 1.26b, where water coming from an ice cube (at low temperature) does not activate the transition while human hand does.

The process works efficiently when the molecules are inkjet printed on common paper, which allows to produce cheap, disposable and user-friendly paper sensors. Furthermore, the polymer conversion occurs very efficiently close to body temperature, which is very convenient for sweat pores mapping. As shown in Figure 1.26c, the sweat pores of a human hand produce the expected blue-to red color transition activating the fluorescence or red-PDA.

Solution-based sensors

DA amphiphiles self-assemble in water solution forming nanoparticles of bilayers with an enclosed volume, which in the literature are called indistinguishably vesicles or liposomes. Vesicles prepared by self-assembly in water found extensive use as sensors, based on the chromatic blue-to-red transition upon exposure to stimuli and on the fluorescence signature of the red-PDA. PDAs in this aggregation state found various applications in biological essays thanks to their relatively easy structure modulation that allows the incorporation of specific recognition sites and their stability in water (biocompatibility). In this section we will briefly review the main breakthroughs in PDA vesicles-based sensors.

Influenza virus is probably the most common virus that we are brought to deal with every year. The virus has a lipid bilayer structure. The viral envelope, the outermost layer, is composed mainly of phospholipids, proteins and glycoproteins that serve to identify and bind to receptor sites on the host's membrane. The most common glycoprotein is hemagglutinin (HA), which binds to sialic acid sugars on the surfaces of epithelial cells in the lung and throat of the host organism to initiate the viral infection. Therefore, the first approach to probe the presence of influenza virus is to functionalize the DA heads with sialic acid, as it was done first with bilayer films by Charych et al. [START_REF] Charych | Direct Colorimetric Detection of a Receptor-Ligand Interaction by a Polymerized Bilayer Assembly[END_REF] and with vesicles by the same group. [START_REF] Reichert | Polydiacetylene Liposomes Functionalized with Sialic Acid Bind and Colorimetrically Detect Influenza Virus[END_REF] An alternative to sialic acid includes DA functionalization with virus-specific antibodies for antigen recognition. Jeong et al. realized hybrid nano-vesicles made of M149 antibody for antigen recognition, PCDA for chromatic sensing and dimyristoylphosphatidylcholine (DMPC) for fluidifying the membrane (structure shown in Figure 1.27a. The vesicles polymerized by irradiation at 254 nm in the blue form and turned red upon influenza H1N1 antigen exposures. Different ratios of DMPC were tested, as well as a control system without antibody. As shown by the col-Chapter 1. Bibliographic review orimetric response plot in Figure 1.27b, the chromatic and fluorogenic transition of PDA occurs thanks to antibody-antigen reaction. [START_REF] Jeong | Label-Free Colorimetric Detection of Influenza Antigen Based on an Antibody-Polydiacetylene Conjugate and Its Coated Polyvinylidene Difluoride Membrane[END_REF] The authors do not inquire in detail on the driving force that triggers the PDA blue-to-red transition. We believe that the antibody-antigen reaction alone can not be responsible alone for the sensor activation. The binding to the recognition site probably triggers a mechanical force of traction on the blue vesicle, which eventually turns red. Bacteria are also biological organisms of great importance in the modern world, for which there is a high demand of specific and efficient sensors. In 2006, Silbert et al. prepared hybrid vesicles made of PDA for the chromatic response and phospholipids to mimic a membrane, dispersed in an agar-agar matrix. DMPC and 10,12-tricosadiynoic acid are mixed together, dried and sonicated in water to form vesicles, whose structure is outlined in Figure 1.28a (in black the DMPC, blue the PDA before bacteria exposure, yellow agar-agar gel and in red the PDA after bacteria exposure). The unpolymerized DMPC-DA vesicles are poured in hot agar, cooled down and stored at 4 • C for two days. Polymerization of the DA derivatives is performed by UV irradiation, and a blue color appear. Figure 1.28b shows the pictures of the resulting vesicles embedded in agar-agar gel when exposed for Bacillus Cereus (center) and to Escherichia Coli. Interestingly, red spots appear in correspondence to bacterial growth. Similarly, a fluorescence signal in correspondence of Salmonella enterica growth is recorded (Figure 1.28c). Both the chromatic change and the fluorescence enhancement come from the blueto-red transition of the PDA moiety. In the same study, it was proven that the transition is triggered by bacterially released substances. Therefore, this method consists in an efficient but non-specific bacteria detection sensor. [START_REF] Silbert | Rapid Chromatic Detection of Bacteria by Use of a New Biomimetic Polymer Sensor[END_REF] A remarkable advancement in the context of bacteria detection is achieved by Lee et al. in 2017, by conceiving a dual system that detects and inhibits bacteria at the same time. Two monomers functionalized with imidazolium salt and imida- zole (structure in Figure 1.29a) are used to form vesicles. When the polymerized vesicles are brought in contact with MRSA (methicillin-resistant Staphylococcus aureus) and ESBL-EC (extended-spectrum β-lactamase-producing Escherichia coli), a blue-to-red transition of the PDA is witnessed by naked eye along with a fluorescence enhancement (Figure 1.29b left and right respectively). Furthermore, when comparing the transmission electron microscopy (TEM) images of ESBL-EC cells in absence of PDA vesicles (left) and treated with 10 µM PDA-vesicles for 30 min (center and right) we can suppose that some damage in correspondence of the bacterial membranes occur. In fact, it is believed that an antibacterial action is achieved through electrostatic interactions between the negatively charged bacterial surface and the imidazolium moiety in the vesicles that cause irreparable damages on the bacterium. [START_REF] Lee | Sensing and Antibacterial Activity of Imidazolium-Based Conjugated Polydiacetylenes[END_REF] Another interesting application of solution-based sensors made of PDAs is metal ions detection. Wang et al. proposed in 2015 an efficient sensor for Pb 2+ detection. [START_REF] Wang | Polydiacetylene-Based Sensor for Highly Sensitive and Selective Pb2+ Detection[END_REF] A PCDA monomer is mixed with a crown ether modified PCDA moiety (PCDA-L) in a 7:3 ratio to form vesicles (structure shown in Figure 1.30a). Because of the presence of a bulky functional group, it is important to keep PCDA-L in a lower proportion in respect to PCDA for ensuring an efficient self-assembly and polymerization. The crown-ether is known to bind ions like Pb 2+ . When Pb 2+ is injected in the vesicle, the solution turns to red. Interestingly, this system showed an outstanding selectivity for Pb 2+ , as it can be seen from the specific response in absorption (Figure 1.30b) and fluorescence (Figure 1.30c) that this ion produced. [START_REF] Wang | Polydiacetylene-Based Sensor for Highly Sensitive and Selective Pb2+ Detection[END_REF] Other PDA-based systems have been developed for selective Hg 2+ and Cs + detection. [START_REF] Gyu | A Colorimetric and Fluorescent Chemosensor for Detection of Hg2+ Using Counterion Exchange of Cationic Polydiacetylene[END_REF][START_REF] Jin Gwon | Chromatic Detection of Cs Ions Using Polydiacetylene-Based Vesicles Containing Crown-Ether-like Ethylene Glycol Units[END_REF] Vesicles in solution have been largely used for a wide variety of application, but a Chapter 1. Bibliographic review valuable alternative to overcome problems of nanoparticle stability, poor portability and low sensitivities connected to the solution-based sensors is to graft the aggregates on a substrate. Many different substrates have been used for this purpose: glass, hydrogel, paper, conductive gold surfaces etc.

Force sensors

Probing and quantifying mechanical forces is a key-topic in multiple fields. Structural materials used in construction or vehicles, for instance, are constantly subjected to external mechanical stress. Therefore, it is necessary to determine the type of force they are subjected to and how intense this force is, in order to design a component that has the appropriate mechanical properties to serve its application. Mechanical engineers nowadays rely mostly on piezoelectric sensors. The piezoelectric effect is that phenomenon of internal generation of electrical charge resulting from an applied mechanical force. It is a reversible process, meaning that applying a voltage to a piezoelectric unit causes the generation of internal mechanical strain that results in a compression or expansion of the material. The nature of the piezoelectric effect is closely related to the occurrence of electric dipole moments in solids. Applying a mechanical stress to these materials causes a reorganization of the dipolemoments or a re-configuration of the dipole-inducing surrounding. The discovery of this phenomenon was first discovered in the mid-XVII century on a number of solid crystals with no inversion symmetry, like quartz, topaz or lead titanate. Piezoelectricity is not limited to inorganic crystals but it is also found in ceramics with randomly oriented grains, semiconducting crystals and crystalline polymers. When a mechanical force is applied on a piezoelectric gauge, the compression or strain of the crystal will generate a charge, that is measured by a charge amplifier. Since the 1950s, this technology has been developed for a vast variety of applications, for strain, compression or elongation. However, miniaturization of these systems is still quite limited (micrometer scale) and the need of an output reader (computer), limits their application. The stress that is probed is the one applied on the gauge and not, for example, inside a test material, at the molecular level. Furthermore, truly static measurements are limited by the electronics, which always suffer from electron-loss, underestimating output values.

In biological systems, cells and tissues are constantly subjected to mechanical forces. Cells can sense and respond to external mechanical stimulations and adapt by changing the mechanical properties of their environment. Moreover, cells are not rigid systems themselves, but can move and apply forces to the external environment through their actomyosin cytoskeleton. It is believed that mechanical stimuli, along with associated biochemical processes, play an important role in the regulation of different cellular activities. [START_REF] Nguyen | Physicochemical Tools for Visualizing and Quantifying Cell-Generated Forces[END_REF] On one hand, it is important to characterize cells response to mechanical stimuli, on the other hand a great challenge of the last decade of mechanobiology research is to map and quantify the mechanical forces that cells exert on their external environment. In fact, force generation is essential in mechanosensing, cell migration and cell adhesion.

The most popular technique to probe traction and shear force exerted by cells on their substrate is traction force microscopy (TFM). [START_REF] Dembo | Stresses at the Cell-to-Substrate Interface during Locomotion of Fibroblasts[END_REF] Cells are grown in soft gel matrices like polyacrylamide (PAA) or PDMS, in which fluorescent reference beads are homogeneously dispersed forming a pattern visible by fluorescence microscopy. When the cell spreads and migrates on this substrate, the gel is deformed by the exerted traction and shear forces. The deformation of the matrix is deducible from Chapter 1. Bibliographic review the displacement of the reference beads. Knowing the mechanical properties of the matrix and the displacement of the beads, the intensity of the applied forces can be calculated. Conventional TFM, however, is limited to ≈1 nN force sensitivity and ≈1 µm spatial resolution. This is because the displacement field depends on the density of the markers, limiting the smallest detectable source of force (spatial resolution), and because there is an uncertainty in the precise localization of the beads, limiting the sensitivity. To overcome this problems, many variants of classical 2D-TFM have been developed, like the use of confocal microscopy to probe the beads displacement in 3D, or the implementation of a super-resolution stimulated emission depletion microscopy (STED) for a precise localization of the beads position. Figure 1.31a shows a schematic of a cell exerting traction forces on a PAA matrix, causing the fluorescent beads to displace. Figure 1.31b shows in the left the schematics of a TFM-STED set up and comparison with confocal microscopy of the obtained images on the right. In the past few years, new approaches to sense mechanical forces exerted by cells that rely on molecular tension probes have been developed. [START_REF] Liu | Nanoparticle Tension Probes Patterned at the Nanoscale: Impact of Integrin Clustering on Force Transmission[END_REF][START_REF] Chang | A General Approach for Generating Fluorescent Probes to Visualize Piconewton Forces at the Cell Surface[END_REF][START_REF] Freikamp | The Piconewton Force Awakens: Quantifying Mechanics in Cells[END_REF][START_REF] Freikamp | Investigating Piconewton Forces in Cells by FRET-Based Molecular Force Microscopy[END_REF] Molecular tension probes consist of a flexible tension-sensitive spring-like moiety, a distant-dependent fluorescent system, an anchor moiety to bind the substrate and an anchor moiety to bind the integrin sites on the cell membrane that enable cell movement. Particular effort in the field was promoted by the group of Salaita, which developed several Förster resonant energy transfer (FRET)-based tension sensors. The example that we report here consists of a polyethyleneglycol (PEG) flexible linker on which an anchor for cellular integrins (cyclized Arg-Gly-Asp-dPhe-Lys (c-RGDfK) peptide) is attached, which is functionalized by a fluorescent Cy3B dye. On the other end of the PEG chain, a thiol function serves as anchor for 15 nm Au gold nanoparticles (NPs) patterned on a substrate (anchor to the substrate), as shown in Figure 1.32a. The Au NPs serve both as a physical binding site to the substrate and as fluorescent quencher for the Cy3B dye, as shown in Figure 1.32b. Therefore, when the PEG linker is in default state (folded), the fluorescent dye is in its OFF state (quenched), while when the linker is extended by traction forces exerted by the cell, the fluorescent dye is brought far from the Au quencher and its fluorescence is activated (ON state). [START_REF] Liu | Nanoparticle Tension Probes Patterned at the Nanoscale: Impact of Integrin Clustering on Force Transmission[END_REF] What is interesting about these systems, is that the extent of the linker stretching, i.e. the distance between donor and acceptor in the FRET couple, is proportional to the fluorescence signal. From the fluorescence signal we can indirectly calculate the elongation of the linker and ultimately the force that the cell has produced to achieve such an elongation. Indeed, previous calibration of such systems is needed. Optimization of FRET-based systems as well as microscopy techniques are now ongoing. However, all these methods rely on indirect measurements of exerted force and most of all they rely on sophisticated preparation methods and expensive equipments. Contrariwise, mechanofluorochromic compounds, which are naturally sensitive to mechanical forces and produce a detectable fluorescence change, may represent a valid and more direct alternative to build reliable force sensors. Based on the assumption that for higher forces a greater change in the molecular structure is obtained, we can expect that a greater modification of the fluorescence properties of the compound are also registered.

Chapter 1. Bibliographic review

The challenge and the plan

On the light of the bibliographic examples on the recent advances in mechanofluorochromic molecular probes and force sensors, the present work is aimed at taking up the challenge of developing new molecular force sensors based on the mechanofluorochromic response of novel polydiacetylene-based materials.

Mechanofluorochromism is a relatively new phenomenon that is under study by many different research groups. Every year, new compounds are discovered and characterized, but not much effort yet is put in quantifying the necessary applied force to trigger a fluorescence modification. Furthermore, most of the studies are limited to the macroscale properties of such materials, with inevitable limitations of their applications in precise sensors for mechanical engineering or mechanobiology. The systems that exist now for these purposes are either limited by the need of a computerized system for reading the output (piezoelectric gauges) or by sophisticated designs that provide indirect force measurements (molecular FRET-based systems in mechanobiology). Mechanofluorochromic compounds, on the contrary, are naturally sensitive to mechanical stress, they can provide direct measurement of the applied force by recording the fluorescence signal and may be used for portable and inexpensive devices.

However, the exploitation of such materials for actual force sensor is still far from seeing an actual application. First of all, a full characterization of their response is needed with model systems. Among the wide variety of MFC materials, we chose polydiacetylenes, since they are well known polymers and, most of all, are very versatile. Chain functionalization opens easily new leads for specific sensing, and the different self-assembled forms in which they can be arranged widen the possibilities of their application.

In the Chapter 2 of the manuscript, the macroscale properties of four novel PDAs thin films are presented. Their polymerization properties are characterized by UV-Vis absorption, steady-state and time-resolved fluorescence spectroscopy. Furthermore, the response to thermal and mechanical stimulations are assessed.

In the Chapter 3 of the manuscript, the same systems are analysed at the nanoscale. Morphology and mechanical properties upon polymerization are studied by means of an AFM. In addition, the response to nanoscale mechanical forces, applied by the AFM tip, is probed by fluorescence microscopy.

In the Chapter 4 of the manuscript, at last, macroscale and nanoscale world come together in the characterization of PDA aggregates in suspension (macro) and deposited on glass slides as single crystals (nano). Response to mechanical forces on solid single crystals are studied again by the combination of AFM and fluorescence microscopy.

Chapter 2 New polydiacetylenes as force sensors

Two main types of diacetylenes have been studied: simple chain DAs and fluorophoresubstituted DAs. As discussed in detail in Chapter 1, PDAs undergo dramatic chromogenic and fluorogenic transitions when an external stimulus is applied, converting the so called blue-PDA into red-PDA. [1][2][3][4] However, these sensors can not rely on high sensitivities since the red form is only weakly fluorescent and the chromogenic transition is based on absorbance measurements. The sensitivity of these systems is limited by the low fluorescence quantum yields of the stimulated PDA red-form, that can be overcome by adding an appropriate fluorophore to allow energy transfer. Lipophilic BODIPY has been successfully incorporated in PDA liposomes and used as donor to thermally-induced red chains acceptors, whose fluorescence signal is thus amplified. 5 Similarly, an efficient FRET mechanism is achieved with red-PDA chains, arranged as liposomes in solution, with dansyl fluorophore covalently attached to the DA unit. 6 Alternatively, 1,2,4,5-tetrazine can be used as donor uniquely to blue chains acceptors, resulting in a fluorescence recovery when the polymer is heated and converted to the red form in drop-casted films, as shown in a collaborative study between PPSM and INSP. 7 This second approach leads to more versatile systems, where the fluorophore interchange between three emission states: it is highly fluorescent in the monomer form, it goes to non-emissive with photopolymerization, and goes back to the ON state when the material has been stimulated. Therefore, in addition to higher fluorescence quantum yields, it is also possible to study the dynamics of the photopolymerization by fluorescence spectroscopy. See Figure 2.1 for a graphical representation of the mechanism.

Most of the experiments have been performed on the first synthesized compound, TzDA1, whose di-alkyl backbone bears a urethane moiety as self-assembling unit, and a tetrazine fluorophore bearing a C12 alkyl chain on the other end. This compound showed very promising mechanofluorochromic properties, which made it a good candidate for fluorescence-mechanical stress correlation at the nanoscale. In order to investigate properly the role of the tetrazine in the system, a simple chain analogue was prepared. DA1 has the same urethane self-assembling unit, and just an alkyl chain on the other end of the backbone. As it will be shown, the absence of tetrazine largely affects two important parameters: firstly the thin film morphology and thus its response to mechanical friction forces; secondly the brightness of the stimulated PDA is reduced by a factor 10, which can be problematic in fluorescence microscopy. Using again TzDA1 as a starting point, TzDA2 was synthesised, another tetrazine substituted DA, with a different self-assembling group. As expected, this modified urethane moiety largely affected the morphology of the vacuum evaporated thin films which limited their applicability for microscopy experiments. At last, the self assembling unit was changed for an amide group in TzDA3, which is chemically more interesting. In fact this could be the starting block to build bigger covalent DA-based systems, for example with proteins (biosensing) or lipids (colloidal stimulus responsive assemblies).

In this chapter, the synthesis, the mechanofluorochromic properties at the macroscale and the photopolymerisation dynamics study of the four polydiacetylenes are presented.

Molecular design, synthesis and X-ray crystal structure 2.1.1 Molecular design

Four polydiacetylene-based mechano-responsive systems have been designed and prepared. DA1 (Figure 2.2a) is a simple chain diacetylene with a C12 chain on one side of the acetylene unit, and a urethane moiety on the other. This stands as homologue of the second type of system, TzDA1 (Figure 2.2b), in which a fluorophore is covalently attached. As previously reported in [7], tetrazine was found to be a suitable fluorophore to covalently attach to a diacetylene moiety because of its small size and appropriate fluorescence band (λ em = 560 nm) that overlaps with the absorbance of the blue form of the PDA. The appropriate distance between the monomers for the topotactical polymerization to occur is not exceeded and is ensured by the presence of the same self-assembling group as DA1, that allows the formation of inter-chain H-bonds, and the alkyl chain. In TzDA2 (Figure 2.2c) the terminal group has been modified adding a butyl-ester, similar to the one encountered in the widely studied 3-and 4-BCMU polydiacetylenes. [8][9][10][11] A further modification was made on the self-assembling group in order to have a simple amide group. This led to a third Tz-DA derivative, TzDA3, whose structure is shown in Figure 2.2d. This new derivative was conceived for two reasons. The former is for pure synthetic ease, since TzDA1 and TzDA2 functional groups come from the condensation of an alkyl alcohol and an isocyanate, whose commercial availability is fairly limited, the cost can be elevated, they are usually toxic and very reactive (not very stable). The latter is more application oriented, since an amide group consists in an easier group to modify for a possible further functionalization.

Playing with the chemical structures of these diacetylene derivatives is expected to influence the sample morphology thus to potentially tune their mechanical and photophysical properties, that affect the response to mechanical stimulation at the nanoscale. Simple diacetylenes usually polymerize in a blue non-fluorescent form (λ abs ∼ 630 nm). When an external stimulus is applied, the blue form is converted to a red form (λ abs ∼ 540 nm), which is weakly fluorescent (λ em = 640 nm, plus a distinguishable vibronic peak at λ em = 560 nm). 12 They constitute thus OFF-to-ON sensors. Tetrazine-substituted diacetylenes are expected to be instead ON-to-OFF-to-ON systems. In fact, because the fluorescence emission band of tetrazine overlaps with the blue-poly-PDA absorbance, an energy transfer is expected to occur exclusively with the blue form of the PDA. Therefore, the tetrazine fluorescence could switch from the ON state of the monomer to the OFF state of the blue polymer, and back to the ON state when the blue form is converted to the red form and the spectral overlap is lost or strongly decreased (see Figure 2.1).

Synthesis

As general procedure, diacetylenes are obtained by coupling of two terminal alkynes through a copper mediated coupling reaction. The symmetric oxidative coupling of terminal acetylenes in the presence of a cuprous salt (oxidative agent) was discovered by Glaser in 1869 and it is the most investigated method to produce diacetylenes. 13,14 However, it was shown that the Glaser method gives unsatisfactory results in unsymmetrycal coupling, because of the predominant formation of the two symmetrical diacetylenes. A more versatile method that works well for symmetrical and asymmetrical diacetylenes is the Cadiot-Chodkiewicz coupling, proposed in 1957. It consists of the condensation of terminal acetylenes with 1-bromoacetylenes in the presence of a cuprous salt and a suitable amine. 15,16 The cuprous salt is used in catalytic amounts and forms a complex with the terminal alkyne, which is the reaction intermediate. A low concentration of Cu(I) reduces the self-coupling of the bromoalkyne (symmetrical diacetylene). The amine is used to both neutralize the resulting bromic acid from the condensation, and as a ligand for the Cu(I) metallic center. A good solubility of the terminal acetylene and the cuprous ion is essential. For this reason, it is not uncommon to employ mixtures of organic solvents and water. 17 In all the syntheses the two alkyne moieties are prepared separately and coupled; for tetrazine-substituted DAs, the fluorophore is added as a last step.

Chapter 2. New polydiacetylenes as force sensors 5) is obtained by the reaction of 6-hexynol (3) with propyl isocyanate (4) in refluxed toluene with triethylamine. The terminal alkyne (5) and the bromoalkyne (2) are then coupled unsymmetrically by a Cu(I)-catalysed Cadiot-Chodkiewicz coupling reaction, to obtain DA1 (6). 17 Ethylamine is used to both neutralise the resulting bromic acid from the condensation, and as a ligand for the Cu(I) metallic center. A mixture of methanol (MeOH) and water is used to ensure a good solubility of both the acetylenes (organic phase) and the cuprous derivative (aqueous phase). All these steps occur with reaction yields higher than 70%.

TzDAs

Three tetrazine-substituted diacetylene compounds differing for the self-assembling group on one side of the diacetylene unit have been synthesised. TzDA1 (R1 in Figure 2.2) has the same urethane moiety of DA1; in TzDA2 the urethane group was modified adding a butyl-ester end (R2 in Figure 2.2); TzDA3 has a simple amide group (R3 in Figure 2.2).

70

Chapter 2. New polydiacetylenes as force sensors

The procedure shown in Scheme 2.2 sums up the common steps of the syntheses of TzDA1, TzDA2 and TzDA3. The three compounds bear a different substituent group R and have a linker length between the acetylene moiety and the substituent R: n = 4 for TzDA1 and TzDA2; n = 5 for TzDA3. Scheme 2.2: Synthesis steps for obtaining TzDAs.

For the three compounds, 6-bromohex-5yn-1-ol ( 2) is obtained by Strauss bromination of 6-hexyin-1-ol (1) with Br 2 in alkaline water (KOH) in the dark. 18 The synthesis of the terminal alkyne (3) differs for the three compounds. For TzDA1, the terminal alkyne hex-5-yn-1-yl propyl carbamate (3) is obtained by the reaction of 6-hexynol (1) with propyl isocyanate (9) -both commercially available -in refluxed toluene with triethylamine, as in DA1 synthesis.

Scheme 2.3: Synthesis of TzDA1 terminal alkyne

For TzDA2, butyl(hex-5-yn-1-yloxy)carbonyl)glycinate ( 3) is obtained by the reaction of 6-hexynol (1) with butyl-2-isocyanoacetate (10) -both commercially available -in refluxed toluene with triethylamine.

Chapter 2. New polydiacetylenes as force sensors The reactions of alcohols with isocyanates are usually well-known and efficient, however, the commercial options for the latter are fairly limited and sometimes expensive. TzDA3 has a simple amide group, which constitutes a simple and valid alternative for ulterior group modifications. 7-octyn-1-ol ( 11) is oxydized to oct-7ynoic acid ( 12) by chromic acid in acetone. Degradation over time of sulfochromic solution is quite recurrent, and the quality of the reagent may influence the effectiveness of the oxidation. For this reason, the sulfochromic solution was prepared starting from chromium trioxide solid, dissolved in water and sulfuric acid (see Section 6 for the preparation protocol). 19 It is also crucial to dissolve 7-octyn-1-ol in acetone and add it dropwise to the oxidizing solution. Fast addition favours the esterification of 7-octyn-1-ol by reaction with oct-7-ynoic acid resulting from the oxidation. Oct-7-ynoic acid ( 12) is isolated by column chromatography and sequentially chlorinated with thionyl chloride and made react with N-propylamine to obtain N-propyloct-7-ynamide (3) in one step.

Scheme 2.5: Synthesis of TzDA3 terminal alkyne

The bromoalkyne ( 2) is coupled with the terminal alkyne (3) through the Cadiot-Chodkiewicz Cu(I)-catalysed reaction, performed in a mixture of dichloromethane (DCM)/distilled water (H 2 O). N-butylamine is used to both neutralize the resulting bromic acid from the condensation, and as a ligand for the Cu(I) metallic center. The reaction yield can vary according to the terminal group and linker length, from around 30% (R 3 ), up to around 60% (R 1 , R 2 ). Chloroalkyloxytetrazine ( 7) is obtained with 83% yield from commercially available dodecanol (6) and dichlorotetrazine (5), which is prepared regularly for further uses in the laboratory, by aromatic nucleophilic substitution, using conditions previously optimized in the lab-oratory. 20 The final TzDA ( 8) is obtained in acceptable yields (24-34%) by nucleophilic substitution of the diacetylene (4) on the electrondeficient fluorophore (7). 4-dimethylaminopiridine (DMAP) is used as non-nucleophilic base to activate the alcohol group on compound (7). The reaction is performed under Ar atmosphere, taking care of drying under vacuum the glassware to avoid any presence of water that could degrade tetrazine, in DCM at 3-7 • C overnight. Relatively low yields can be explained by the formation of symmetrically substituted tetrazine dyes.

TzDA1 crystal structure

The X-ray crystal structure of TzDA1 was obtained on isolated crystals grown by liquid-liquid diffusion method. The distance between C1 and C4', which are the carbons engaged in the photopolymerization process (1,4'-addition), is 4.00 and 3.71 Å in the asymmetric cell, the packing distance d between monomer units is averaged at 4.78 Å and the angle φ between the diacetylene rod and the stacking axis is 59 • (see Figure 2.3a). Ac-Chapter 2. New polydiacetylenes as force sensors cording to the model proposed by Enkelmann in 1984, the essential requirements for efficient polymerization are satisfied. 8 The packing distance is surely affected by the presence of H-bonds between oxygens and hydrogens of the urethane moiety as common in BCMU structures, 11 with two characteristic lengths in the asymmetric cell of 1.93 and 2.03 Å (Figure 2.3a). Interestingly, on the other side of the diacetylenic unit, tetrazines do not interact by π-stacking and are tilted by 69.20 • (Figure 2.3b), weakening the packing tightness. The C12 alkyl chains are linear and lay on planes tilted by 76.98 • (Figure 2.3c). Please refer to Section for crystallographic data and structure refinement details.

Preliminary characterisations

The photophysical properties, fluorescence quantum yield (Φ F ) and molar extinction coefficient (ε), of the monomeric forms of TzDAs were tested in acetonitrile solution and are shown in Table 2.1. The ε was measured at the visible absorption peak of tetrazine at 520 nm, which corresponds to a n-π* transition. Tetrazine's Φ F measurements were measured using Rhodamine 6G as standard reference, the maximum wavelength of tetrazine emission being centred at 560 nm. For supplementary information on calculations see Section 6.3. The ε in the range of 500 -650 L•mol -1 •cm -1 are in good accordance with 74 Chapter 2. New polydiacetylenes as force sensors alkoxy-substituted tetrazines found in litterature, as well as for Φ F between 5 and 10 %. 20 As shown in the absorption and fluorescence spectra of TzDA1 and TzDA2 in acetonitrile (MeCN) in Figure 2.4a and 2.4b respectively, the spectroscopic features of tetrazine are maintained.

Compound ε Φ F TzDA1 500 L•mol -1 •cm -1 8.5% TzDA2 650 L•mol -1 •cm -1 5.4%

Polydiacetylene thin films

PDAs have attracted particular interest for their chromogenic and fluorogenic properties at the solid state. Langmuir-Blodgett (LB) and Langmuir-Schaefer (LS) thin films are among the first methods to be used to study well-ordered polydiacetylenes at the solid state, and still remain widely used by a number of research groups for very different applications. The first studies on polymerisation of LB films appear in the 80s and attract attention for their applications mainly focusing on the response to temperature. 12,21 The first microscopy studies on such samples were performed by AFM 22 or near-field optical microscopy, 23 and their morphology coupled to their fluorescence properties still attract wide interest in the community. 24,25 From these first thermochromic characterisations, more sophisticated systems have been conceived for virus detection, 26 with reversible thermochromism, 27 or with chiral properties. [28][29][30] However, these methods require a specific chemical structure with an hydrophobic and an hydrophilic end and they produce only mono-, bi-or tri-layers films. Therefore, these techniques are circumscribed to a limited range of DAs and produce very thin films with low brightness. 4 Wider applications are found for immobilised PDA vesicles, previously prepared and then grafted to a substrate to form a film. Simple grafting on functionalised glass was proved succesful by Kim et al. 31 , and more sophisticated sensors were then built based on micropatterned liposomes on a glass surface for temperature detection 32 , bio-recognition assays and even label free cyclodextrin recognition. 33 Stable grafting on conductive gold surfaces was also shown for the first time by Singh's group in 2001 34 , which is interesting if electrical or magnetic induced signals transduction wants to be implemented in the sensor.

Therefore, polydiacetylene films have been widely exploited in the past and continue to be developed for their applications. However, for the majority of these studies, the focus is centred on the outcome of the assays and less on the properties of the PDA film and on their modulation. Moreover, LB or LS thin films have in general low fluorescence emission due to their small thickness, making eye-detection in fact impossible, while grafted PDA liposomes present important stability issues (they tend to unravel and form a monolayer or bilayers on solid surfaces). For these reasons, it is important to address PDA-only thin films deposited on glass or solid surfaces, to study their solid-state polymerisation properties, and direct response Chapter 2. New polydiacetylenes as force sensors to stimuli. In this regard, particular efforts were made by the group of M. Schott, which carried out a massive number of physical characterisations on PDA isolated polymer chains dispersed in monomer matrix [35][36][37][38][39][40][41] and on thin films 42,43 .

Herein, the photopolymerization dynamics and the mechanofluorochromic behaviour at the macroscale of four PDA thin films are presented.

Thin film preparation

The spectroscopic studies shown in this chapter as well as the microscopy studies that will be shown in Chapter 3 are performed on vacuum evaporated thin films (if not specified otherwise). Vacuum evaporation is a useful method to produce thin films and control their thickness. A known amount of powder is introduced in a crucible, which is placed in a high vacuum chamber (∼ 1 • 10 -5 mbar). Clean microscope glasses are fixed on top of the chamber. When a stable pressure is reached, a 10 • C • min -1 ramp is applied and reduced to 4 • C • min -1 when the powder starts to evaporate and re-condensate on the glass slides, which are at ambient temperature. This ramp is applied until the speed of deposition reached the maximal value of 2 Å•s -1 then the temperature was manually adjusted to keep the deposition rate as constant as possible (for detailed evaporation protocols see Section 6.4.2). It is possible to measure the speed of deposition thanks to the presence of a piezoelectric crystal, placed right next to the glass slides. The thickness of the obtained films was measured by AFM and is 80 ± 12 nm for 12 mg of TzDA1 and 313 ± 20 nm for 20 mg of DA1. Film thickness for 11.2 mg of TzDA2 and 10.2 mg of TzDA3 is estimated at 80 and 100 nm respectively from optical density measurements, as it will be explained in Section 3.2.1 (see Section 6.3.6 for details on the calculation).

Solid state photopolymerization

Self-assembled, crystalline or simply ordered diacetylenes are well known to polymerize in the solid state when irradiated in the UV at 254 nm. However, the kinetics of polymer formation and the total polymer quantity can vary quite a lot according to the specific chemical structure, which in its turn determines a specific supramolecular arrangement and film morphology and eventually influences the response to mechanical stimulations. The capability of one compound to establish intermolecular H-bonds between monomer units is crucial in determining the molecular packing and morphology, and to ensure the minimal distance between reactive centres to favour polymerization. To track the polymerization progress by spectroscopy, a UV-Vis spectrum is recorded after each subsequent irradiation at 254 nm and to each the initial spectrum is subtracted to exclude the contribution of thermally polymerized PDA and tetrazine absorption band in TzDA1, TzDA2 and TzDA3 (Figure 2.5). When looking at the spectral shape of the solid films (Figure 2.5a for DA1, 2.5b for TzDA1, 2.5c for TzDA2 and 2.5d for TzDA3), it is confirmed that the presence of tetrazine and the modifications in the self-assembling group result in different supramolecular arrangements. When irradiating DA1 thin films at 254 nm, a structured absorption band centered at 635 nm and a vibronic band centred at 580 nm is produced and increases with irradiation, corresponding to the formation of the "classical" blue PDA form (light blue arrow in Figure 2.5a). 33,44 After 14 s of irradiation at 0.33 mW•cm -2 (4.62 mJ•cm -2 ) a progressive distortion of this band towards the low energies (650 nm) is witnessed (see dark blue arrow in Figure 2.5a). In addition, from 6 mJ•cm -2 irradiation energy, a distortion of the band in the low wavelengths is witnessed and is more pronounced for the final steps of the irradiation (high polymer content), leading to a large absorption band covering the range 450 -650 nm at the end of the polymerization process. This is due to the production of red-poly-DA1 chains, as it is common to PDAs with fast polymerization kinetics. 45 On the other hand, the photopolymerization of TzDA1 (Figure 2.5b) and TzDA2 (Figure 2.5c) begins with a large and unstructured absorption band centred at 600 nm along with the characteristic vibronic band at shorter wavelength. This high-energy form is what in Chapter 2. New polydiacetylenes as force sensors literature is referred to as "purple form". 44,46,47 Along the UV irradiation, the polymer fraction in the film increases and the corresponding absorption band increases. Starting at 196 mJ•cm -2 in TzDA1 and at 38 mJ•cm -2 in TzDA2, it progressively shifts towards higher energies. This effect could be due to the formation of a new PDA form that coexists with the already present "purple" chains, or to a conversion of the "purple" chains to a new PDA form by plastic deformation of the polymer film that steps in beyond a certain polymer fraction. 42 It is however impossible to confirm with certainty one of the two hypothesis from steady state absorption spectra. TzDA2 shifts sooner than TzDA1 but shows a smaller shift in the final polymer form of 16 nm (λ f inal = 584 nm) against the 30 nm in TzDA1 (λ f inal = 570 nm).

TzDA3's lateral chain is functionalized with a simple amide group, which presumably interconnects with neighbouring chains by H-bonds. This modification produces a different morphology (as shown in Section 3.2.1) and clearly different spectroscopic features as shown in Figure 2.5d. TzDA3 polymerizes forming a small quantity of blue-poly-TzDA3 with an absorption band centred at 600 nm (purple form) and mostly produces a well structured absorption band at 554 nm with a vibronic peak at 534 nm, corresponding to red-poly-TzDA3.

Blue polymer formation kinetics can be analyzed and compared looking at the evolution of the normalized blue polymer absorption band versus irradiation energy. The curves obtained for DA1, TzDA1 and TzDA2 are superimposed in Figure 2.6a. It has to be noted that the total irradiation energy is different for each compound because the irradiation was stopped when a plateau is reached: the last point in the kinetics curve corresponds to the maximum blue polymer quantity that the compound forms in these conditions. It is clear then that DA1 polymerizes faster than the tetrazine-substituted DA derivatives, since it reaches the maximum blue polymer quantity (at λ abs = 635 nm) with only 90 mJ•cm -2 , while TzDA1 and TzDA2 (λ abs = 600 nm) require 1.8 and 1.4 J•cm -2 , respectively. Therefore, the presence of tetrazine in the structure probably perturbs the efficient packing of DA simple chains which undergo fast and easy photopolymerization. The selfassembling group also plays a role, resulting in a slower photoinduced polymerization for a simple urethane moiety in TzDA1. The ratio of blue polymer absorption and film thickness versus the irradiation energy gives a mean to compare the maximum amount of blue polymer formed by UV irradiation. The curves are traced in Figure 2.6b, where the maximum amount of formed blue polymer is represented by the height of the plateau. Therefore, DA1 approximately forms two times more blue polymer than TzDA2 and almost 1.5 times more than TzDA1, meaning that the presence of the tetrazine not only slows down the polymerization rate, but also reduces the total amount of polymer formed. Concerning the self-assembling group in tetrazine-substituted derivatives, the simple urethane of TzDA1 resulted in a globally slower photopolymerization, but in a higher monomer-to-polymer conversion. It is important to highlight that the maximum polymer fraction is far to be 100% for any polydiacetylene, as utterly explained in reference [42]. In fact, if we assume an absorptivity coefficient α= 2 • 10 5 cm -1 for a generic polydiacetylene chain, the monomer-to-polymer conversion ratio is around 10% for TzDA1 and 7% for TzDA2. Assuming the same absorptivity coefficient, a conversion ratio of 15% is obtained for DA1 (more details on the calculation are shown in Section 6.3.7). When looking at the evolution of the spectral shape of the subsequent absorption spectra of DA1 photopolymerization (Figure 2.5b), it is clear that such an analysis does not take into account the whole complexity of the system. In fact, this estimates only the quantity of one blue polymer form, excluding the 650 nm form and the 540 nm red-poly-DA1.

As previously mentioned, TzDA3 shows different photopolymerization properties compared to DA1, TzDA1 and TzDA2. It was shown in Figure 2.5d that mainly red-poly-TzDA3 (λ abs = 554 nm) is formed by the very first irradiations at 254 nm. In Figure 2.7a the evolution of the normalized absorbance at 554 and 600 nm as a function of the irradiation energy is presented. Blue-poly-TzDA3 grows faster than red-poly-TzDA3 between 0 and 200 mJ•cm -2 , but it reaches the plateau when red-poly-TzDA3 is still increasing. In addition, from 400 mJ•cm -2 , it seems that the quantity of blue-poly-TzDA3 actually decreases: probably plastic deformations in the film due to the polymerization process cause the conversion of the blue form to the red form. This is confirmed by the evolution of the polymer quantities in the film, shown in Figure 2.7b. Until 200 mJ•cm -2 the same quantity of blue and red-poly-TzDA3 are formed approximately at the same rate, but from this point Chapter 2. New polydiacetylenes as force sensors only red-poly-TzDA3 is produced while the blue phase actually starts decreasing. It is also interesting to notice that, compared to other TzDAs, the abs/thickness values are generally one order of magnitude lower, meaning that TzDA3 forms less polymer. In fact, when we estimate the polymer content in the film at the end of the irradiation using α = 2 • 10 5 cm -1 , we obtain a 1% polymer ratio, which is significantly lower compared to the other analyzed compounds. Even though in general the absorptivity coefficients of the blue and red phases is of the same order of magnitude, 42 it is important to remind that the polymerization of TzDA3 was stopped earlier and that this calculation is just an estimation.

Fluorescence and energy transfer study in TzDAs

The progress of photopolymerization of DA1 thin films cannot be directly followed by fluorescence spectroscopy since the blue phase of the polymer is non-fluorescent.

When the polymerized film is heated on a heating plate at around 80 • C, the blue to red conversion activates the fluorescence of the system (absorption and fluorescence spectra in Figure 2.8a and 2.8b respectively). The fluorescence quantum yield in the solid state was measured by means of an integrating sphere and is less than 1%, which is more reliable than the value of 2-3% obtained by means of comparison to a standard fluorophore dissolved in nail polish and painted on a glass slide. 12 These values are consistent if we consider that solid state measurements performed at 15 and 40K gave 0.30±0.05 and 0.20±0.05 respectively, showing a decrease of Φ F with temperature. 37 At low temperatures, the fluorescence lifetime is fitted by a single exponential function with τ = 94 ps, which becomes shorter with increasing temperature. 48 In fact, the fluorescence lifetime of DA1 thin films at room temperature was found to be shorter than 10 ps, according to the instrument resolution. Inserting a tetrazine in the chemical structure is therefore a valuable strategy to ameliorate the brightness of the system.

Tetrazine fluorescence in TzDAs is modulated accordingly with the state of the chain. The tetrazine chromophore is highly fluorescent in monomeric form (ON state), quenched when the DA is polymerized to the blue form (OFF state) and recovers the emission when the polymer is stimulated (thermally or mechanically) and converted to the red form (ON state). We believe this is due to an energy transfer process occurring between the tetrazine fluorophore and the blue PDA. When the polymer backbone is converted to the red form, the spectral overlap between tetrazine (donor) fluorescence and PDA (acceptor) absorption is strongly reduced, resulting in a fluorescence restoration. The overlaps are shown in Figure 2.9 for TzDA1 (a), TzDA2 (b) and TzDA3 (c). This ON-to-OFF-to-ON mechanism is very efficient in TzDA1 and TzDA2, where a good spectral overlap is achieved only with the blue form of the polymer and it is strongly reduced when the polymer is converted to the red form (blue and red lines respectively in Figure 2.9a for TzDA1 and 2.9b for TzDA2). On the other hand, TzDA3 has a limited modulation capability since it polymerizes mainly in the red form and forms only very small quantities of blue polymer. Red-poly-TzDA3 has a maximum of absorbance at 554 nm, which overlaps very efficiently with tetrazine fluorescence (λ em = 560 nm), resulting in an efficient quenching. Heating the thin film generates an unusual absorption profile, probably due to degradation of the film, where the absorption bands of red-poly-TzDA3 are still recognizable but lower in intensity (red line in Figure 2.9c).

Fluorescence spectra at ambient and low temperature on TzDA1 thin films are Chapter 2. New polydiacetylenes as force sensors Freezing the system strongly decreases vibrational relaxation and all deactivation processes that depend on spatial rearrangements of the molecule in the excited state. Figure 2.10 shows that the increase of fluorescence intensity in the poly-TzDA (blue and light-blue lines) is comparable and even lower to the monomer case (purple and pink lines), excluding a quenching by electron transfer thus confirming an energy transfer mechanism.

It is possible to follow the emission quenching by steady-state fluorescence spectroscopy, recording a spectrum after subsequent irradiations at 254 nm which gradually polymerize the thin films (Figure 2.11). The maximum of emission is located at 560 nm for TzDA1 and TzDA2, while in TzDA3 it is lightly blue shifted to 553 nm. The spectral shape is not affected by the photopolymerization process, but only the intensity is quenched. When photopolymerization is achieved, 91% of the initial tetrazine fluorescence is quenched in poly-TzDA1 (Figure 2.11a), 97% in poly-TzDA2 (Figure 2.11b) and 87% in poly-TzDA3 (Figure 2.11c). The percentage quenching is calculated with the following formula:

Chapter 2. New polydiacetylenes as force sensors

A mono -A poly A mono • 100%
where A is the area of the fluorescence spectrum of the monomer (A mono ) and of the polymerized sample (A poly ).

To evaluate the tetrazine quenching efficiency by poly-TzDA1 and poly-TzDA2 along the photopolymerization reaction, the maximum of fluorescence emission (560 nm) is plotted as a function of the UV-vis absorption at 600 nm divided by the film thickness for the two samples, corresponding to the amount of blue polymer formed. The curves are shown in Figure 2.12a. The quenching efficiency of poly-TzDA3 is evaluated in Figure 2.12b, by plotting the maximum of fluorescence emission (553 nm) as a function of UV-vis absorption at 554 nm divided by the film thickness, which corresponds to the amount of the polymer that is mainly formed by photopolymerization (red-poly-TzDA3). Both in TzDA1 and TzDA2 solid films, the decrease in fluorescence emission is more pronounced than the increase in absorption at 600 nm, resulting in a nonlinear correlation proving an efficient energy transfer from multiple tetrazine donors to blue-PDA acceptor. The dotted lines in Figure 2.12a underline how the efficiency in Tz-quenching is more pronounced in TzDA2 than in TzDA1, showing that 50% of fluorescence quenching is reached for a lower blue polymer quantity (lower blue-PDA absorption) in TzDA2. In absolute terms, an extraordinary energy transfer efficiency is achieved in both systems, where the tetrazine fluorescence signal is quenched by more than 90% of its initial intensity by less than 2% of blue-PDA. Since the energy transfer processes are strongly driven by spatial parameters (distance and relative orientation between the emitter and the quencher species), the different fluorescence quenching profiles of TzDA1 and TzDA2 as a function photopolymerization conversion (Figure 2.12a) is a strong indication that the supramolecular arrangement of the tetrazine dyes and PDA molecules, as well as the polymerization distribution within the material, are not identical for the two compounds.

The same non-linear effect of fluorescence quenching is found in TzDA3 (Figure 2.12b). Even though TzDA3 forms only red-poly-TzDA3, the absorption band efficiently overlaps with the tetrazine fluorescence (Figure 2.9c) and favours energy transfer, leading to a very efficient tetrazine quenching.

To investigate the dynamics of the energy transfer processes upon photopolymerization, fluorescence decay curves and solid state fluorescence quantum yields for subsequently UV-irradiated films of TzDA1 were recorded. The monomer in acetonitrile presented in Figure 2.13 shows the typical profile of a tetrazine fluorescence decay, with a mean lifetime of 35.6 ns. 20 The decay is fitted by two discrete exponentials, a short (4.6 ns) and a long one (36.3 ns), the short being less than 1% in the fractional intensity (probably due to the presence of some unidentified impurity).

I(t) = a 1 • exp - t τ 1 β + a 2 • exp t τ 2
Chapter 2. New polydiacetylenes as force sensors performing a global analysis where the discrete component was fixed for all the decays, while the stretched component was left free. The stretched exponential 49,50 can efficiently reflect an underlying distribution of time-decays in such a complex (heterogeneous) system, evolving with the irradiation time, whereas the discrete component can take into account a minor emissive contribution which stays constant under irradiation. The global χ 2 R of the analysis is 1.17 The discrete decay-time was determined at 0.22 ns. Along polymerization, the tetrazine lifetime is expected to decrease progressively because of energy transfer with increasing quantities of acceptors (blue-poly-TzDA1), which is what qualitatively can be concluded from Figure 2.14a, with a progressive shortening of the fluorescence decays upon UV irradiation. As shown in Figure 2.14d, the stretched exponential parameters τ 1 and β decrease with increasing irradiation time (increasing quantity of blue-poly-TzDA1), which qualitatively represents a widening and a shortening of the overall decay-time distribution. Indeed, the stretched exponential that is used to fit the fluorescence decay profile I(t) can be treated as a distribution of rate constants H(k) as expressed by the following equation: ∞ 0 H(k)e -kt dk, H(k) being the inverse Laplace transform of I(t). The normalized distributions of decay rates were numerically calculated from a mathematical treatment described by M.N. Berberan-Santos et al., 51 and the corresponding H(k) curves are shown in Figure 2.14e. They reveal a progressive shortening of the decay time along the polymerization process: at 0 s irradiation time, k is comprised between 0.05 and 0.2 ns -1 (5-20 ns decay time), while at 560s irradiation time, the k distribution is much larger, comprised between 0.1 and 2 ns -1 (0.5-10 ns decay time).

To confirm that the fluorescence quenching mainly corresponds to a quenching of the stretched component, it is necessary to evaluate the intensity fractions of the discrete and stretched components in the overall decay function and their evolution with UV irradiation time. Figure 2.15a and 2.15b show the plots of the total exponential function (sum of the two components) and the single stretched and discrete components of 0 s and 560 s irradiations respectively. The ratio of the numerical integration of the single components (stretched exponential and discrete exponential, respectively) and the integration of the total exponential function gives a relative fraction of intensity (f stretched and f discrete), underlining that the discrete component has a minor (<4%) contribution to the overall fluorescence emission. The sum of these values is then normalized to the steady-state fluorescence intensity. The table in Figure 2.15c sums up all the values (f stretched and discrete, normalized or not), and Figure 2.15d displays the evolution of the normalized intensity fraction of the stretched exponential with irradiation time, confirming that fluorescence quenching mainly corresponds to a quenching of the stretched component. Therefore, the long stretched component is assigned to the tetrazine fluorophores that are involved in the energy transfer to the blue-poly-TzDA1, with a wide distribution of efficiencies. On the other hand, the short discrete component could represent a small portion of the tetrazine that has been degraded, probably during vacuum evaporation, and does not take part in the quenching mechanism, with a larger relative contribution at longer irradiation time, when the tetrazine fluorophores are quenched in larger extents (Figure 2.15d). An ulterior proof of the energy transfer mechanism is constituted by the fluorescence quantum yields that were calculated on the first and last step of the photopolymerization process, by means of an in-Chapter 2. New polydiacetylenes as force sensors tegrating sphere. It was found for the monomeric film Φ F = 17.3%, while for the polymeric film Φ F = 1.2%.

Mechanofluorochromism and thermofluorochromism at the macroscale 2.3.4.1 TzDAs

Poly-TzDA1 responsiveness to heating and mechanical stress was investigated qualitatively by UV-Vis and fluorescence spectroscopy on vacuum evaporated thin films on glass slides (drop casted films on a paper substrate were used to monitor the fluorescence recovery after mechanical stimulation). By heating the polymerised thin film, the typical absorption spectrum of a red-PDA is obtained (Figure 2.16a, blue and red curves respectively). Simultaneously, Figure 2.16b shows that the ON state of the monomeric form (black curve) is efficiently switched to the OFF state where the tetrazine fluorescence is quenched by energy transfer towards the blue-PDA (blue curve). Upon heating, the conversion to the red-PDA causes a strong weakening of the energy transfer, resulting in the efficient restoration of the tetrazine fluorescence (red curve). Similarly, when the blue polymer film is subjected to mechanical stress, here performed by hand with a spatula, the red-PDA is obtained, which is confirmed by the absorption spectra shown in Figure 2.16c. Therefore, mechanical stress, like heating, restores tetrazine fluorescence as shown in Figure 2.16d. Additionally, this mechanism is pseudoreversible if cycles of UV irradiation and mechanical stimulation are performed. Figure 2.16d shows that a second irradiation forms more blue polymer and thus the fluorescence intensity decreases (purple curve), and that a subsequent mechanical stimulation restores again the quenched fluorescence intensity (brown curve). This qualitative spectroscopic study constitutes also a strong proof of the quenching by energy transfer mechanism that occurs efficiently towards the blue PDA only. Fluorescence is quenched by polymerization and switched back on after heating. It has to be noticed that the fluorescence is higher for the heated polymer than for the initial monomeric form, which can be explained considering the presence of some blue-poly-TzDA2 in what is considered the "monomeric" film (see the presence of an absorption band at 600 nm in Figure 2.17a, black line). Similarly, when applying a mechanical stress to the solid sample, the blue-to-red conversion occurs. Figure 2.17d shows the absorption spectra before polymerization (black), after polymerization (blue) and after rubbing on the sample with a spatula (red). It is confirmed that blue-poly-TzDA2 switches to red-poly-TzDA2 when submitted to mechanical stress. At the same time, fluorescence is quenched by polymerization, and restored when mechanical stress is applied (Figure 2.17d). Cycles of UV irradiations and mechanical stimulations result in the same pseudo-reversible behaviour that was found for TzDA1, with less fatigue resistance (loss of fluorescence at the second mechanical stimulation, brown line in Figure 2.17d).

Mechanofluorochromism of DA1

The mechanofluorochromic behaviour of DA1 is shown in Figure 2.18. When applying a mechanical stress to a polymerized DA1 thin film, the classical red PDA is produced. Absorption spectra are shown in Figure 2.18a, being blue-poly-DA1 the blue line and red-poly-DA1 the red one. Simultaneously, fluorescence spectra were also recorded and shown in Figure 2.18b. From the non-fluorescent blue form, poly-blue-DA1 is converted to its fluorescent red counterpart (blue and red line respectively). The responsiveness to temperature will be assessed in the following dedicated section.

The reversible thermofluorochromism of DA1

DA1 thin films showed an interesting behaviour when polymerized and exposed to thermal stimulus. A thin film that was polymerized in different areas for previous experiments was tested to temperature. Three distinguishable zones can be identified in this sample. Zone 1 is where the irradiation at 254 nm is not sent intentionally, Chapter 2. New polydiacetylenes as force sensors but it is accidentally received when irradiating directly zone 2 for tracking polymerization extent by UV-Vis spectra. If zone 2 has received 90 mJ•cm -2 , zone 3 corresponds to a polymerized spot under the microscope set-up (see Chapter 3) and has thus received much lower doses of irradiation, around 2 mJ•cm -2 . When heating to 60 • C, all the blue-poly-DA1 that was formed in the three zones is converted to red-poly-DA1 indistinguishably, with the difference that zone 2, when brought back spontaneously to ambient temperature, partially backconverts to the blue form instantaneously (see zone 2 in Figure 2.19a and 2.19b). UV-Vis absorption spectra shown in Figure 2.19c are taken at T amb on zone 2 at the end of the photopolymerization process (blue line), after heating (red line) and after multiple cycles of heating and resting at ambient temperature (grey lines). As explained in Section 2.3.2, DA1 absorption spectrum evolves along photopolymerization: a first increase in 635 nm absorbance is followed by a 650 nm distortion of the spectrum for long exposures to UV irradiation. At the end of the polymerization DA1 has formed at least three distinguishable forms of polymer: a red phase (λ abs = 540 nm) and two blue phases (λ abs = 635 and 650 nm). After heating the film (red and grey spectra in Figure 2.19c), a new band centred at 530 nm along with the vibronic bands appears and it corresponds to the red-poly-DA1. Unexpectedly, the band centred at 650 nm, corresponding to a form of blue-poly-DA1, does not disappear (blue-to-red conversion) despite the stimulation. Therefore, either this form is not responsive to temperature whatsoever, either it is intrinsically reversible, meaning that it converts to the red form while heating and goes back to the blue form at room temperature. On the contrary, the form absorbing at 635 nm completely disappears upon heating (complete blue-to-red conversion). If DA1 thermofluorochromism reversibility is exclusively due to this 650 nm form, the fact that this low energy form appears at high UV doses in the photopolymerization process explains why zone 3 stays red, while zone 2 goes back to blue.

To verify this hypothesis on the reversible thermochromism of DA1, a thorough study was conducted on a new vacuum evaporated sample that was exposed to a low dose of UV irradiation and its response to temperature was followed by fluorescence spectroscopy (Figure 2.20). A fluorescence signal is expected to appear as the blue-poly-DA1 is converted to red-poly-DA1, and this same signal is expected to decrease if red-poly-DA1 goes back to the blue form. However, if only the supposedly non-reversible form (λ abs = 635 nm) is present, the fluorescence signal should not change over time (no reversible phase). Figure 2.20a shows that a small quantity of blue-poly-DA1 is already present Chapter 2. New polydiacetylenes as force sensors in the sample (black line) because of the presence of an absorption band at 635 nm. Blue-poly-DA1 quantity is increased by irradiating the film at 254 nm (grey lines) and the polymerization is stopped when only this form is present (blue line) at 1.46 mJ•cm -2 . Heating the sample produces red-poly-DA1 whose spectrum is shown by the red line in Figure 2.20a. The heating process is performed in a temperature controlled cell and the process is followed by fluorescence spectroscopy. As expected, when temperature is increased from 23 to 80 • C, a fluorescence signal appears (Figure 2.20b). The plot of fluorescence intensity at λ max = 540 nm versus temperature (Figure 2.20d) shows that the blue-to-red conversion occurs suddenly between 65-70 • C. Figure 2.20c shows the evolution of the fluorescence when temperature is let go back to ambient. Surprisingly, even though the supposed reversible form absorbing at 650 nm is apparently absent, the fluorescence signal decreases anyway (red and light-blue line in Figure 2.20c). Therefore it is not possible to establish the reason behind this reversibility at present, and additional experiments would be needed.

Conclusion

In this chapter, the design, synthesis and spectroscopic properties of four novel DAs have been presented and discussed. Despite their large success over the decades, the possibilities of modulating their molecular design to tune their photopolymerization, spectroscopic properties and response to external stimuli still remain partially unexplored.

From a synthetic point of view, the four compounds here presented constitute fairly accessible systems. DA1, a simple chain DA, is in fact prepared via a three steps synthesis, while tetrazine-substituted DAs via a four steps synthesis. It was shown that modifying the functional parts of the chain is feasible and easy.

The interest in playing with the chemical structure of this family of compounds is translated in unique spectroscopic properties and different photopolymerization dynamics, which were studied on vacuum evaporated thin films. Simple chain DA1 polymerizes in a blue phase (λ abs = 635 nm) generally faster and more efficiently than TzDAs. It is estimated that TzDA1, that bears the same urethane moiety as DA1 but 1,2,4,5-tetrazine on the other side, forms approximately 10% of blue-poly-TzDA1 at the end of the polymerization process. When the self-assembling group is modified by adding a butyl-ester, the monomer-to-polymer conversion remains in the same order of magnitude, 7% (TzDA2). Compared to DA1, the blue-PDA phase that is formed in these two compounds is shifted towards the higher energies and is called "purple form" (λ abs = 600 nm). On the other hand, when the functional group is simplified to a propyl-amide (TzDA3), the main polymer phase that is produced is the red-poly-TzDA3 (λ abs = 554 nm) and the conversion ratio drops to 94 Chapter 2. New polydiacetylenes as force sensors approximately 1%.

As explained, simple PDAs undergo a fluorogenic transition when they are converted from blue to red phase. However, red-poly-DA1 has a fluorescence quantum yield of less than 1%, which is why the introduction of a fluorophore represents a valid way to optimize the fluorescent response (Φ F = 17% in the monomeric solid film). In this regard, it is important that the fluorophore emission is quenched uniquely by one polymer phase only (blue). Therefore, the quenching dynamic is studied by stationary fluorescence spectroscopy for all TzDAs and by time-resolved fluorescence spectroscopy for TzDA1. It is concluded that all compounds quench the tetrazine by an energy transfer mechanism. For the three compounds, one polymer chain (acceptor) is able to quench multiple tetrazine emitters (donor), which results in a non linear correlation between blue-polymer absorption and fluorescence intensity with an amplification effect of fluorescence extinction. Fluorescence lifetimes for progressively polymerized thin films of TzDA1 revealed a certain complexity and heterogeneity of energy transfer efficiency from tetrazine to blue-poly-TzDA1. For each decay profile, a discrete exponential component is fixed and a stretched exponential component evolves along the irradiation. The characteristic parameters β and τ 1 of the stretched exponential decrease progressively as a function of irradiation time. Plus, the distribution of decay times from the beginning to the end of the polymerization process shorten, from 5 -20 ns to 0.5 -10 ns.

The spectroscopic characteristics of the polymerized solid thin films and their ability of efficiently quench tetrazine fluorescence (if present) are crucial for the applications that can be foreseen for these materials. The mechanofluorochromic behaviour of these PDAs are here treated from a macroscopic point of view, to answer the key question in the field: Does a mechanical stimulation produce a fluorescence signal? All the studied compounds, in fact, respond to mechanical and thermal stimulation, producing the red phase and an enhanced fluorescence signal. It was demonstrated that ON-OFF-ON-OFF cycles are possible by subsequently rubbing and irradiating the film multiple times. The peculiar pseudo-reversible thermofluorochromic behaviour of DA1 was also inquired by fluorescence spectroscopy, proving that the blue-to-red transition occurs suddenly between 65 and 70 • C and that, to a certain extent, red-poly-DA1 goes back to its blue counterpart. These studies represent an important proof of concept that the systems actually serve their purpose, nonetheless, they do not respond to the ambitious question:

What is the needed applied force intensity that triggers a fluorescent response? that will be assessed in Chapter 3. Scanning probe microscopy includes all related technologies for measuring and imaging surfaces down to the molecular or atomic level by means of a physical probe. As general common characteristics, these techniques rely on the use of very sharp tips, modelled in such a way to be as close as possible (few nanometers) to the surface of the probed substrate. The scanning process will be affected by the surface morphology, which interacts directly with the tip and produces a signal proportional to the tip-surface distance.

The pioneer technique of this family of microscopes is scanning tunnelling microscopy (STM). STM first and AFM later owe their development to mainly two German physicists, Gerd Binnig and Heinrich Rohrer, who massively contributed to the understanding of the fundamental physics behind scanning probe microscopy (SPM) and to the technical implementation of this technique between 1981 and 1986. [1][2][3][4][5][6][7][8][9] Tunnelling current is the quantum mechanical principle at the base of STM. A sharp metal tip in close vicinity of a conducting plate can be treated as two quantum wells separated by a potential barrier (tip-surface distance). When no voltage difference is applied between the two wells, no electron will flow. However, when a voltage is applied to one of the two wells, either the tip or the conducting plate, electrons will start crossing the potential barrier (tunnelling current). Firstly, this current depends on the tip-substrate separation. When the surface barrier gets larger, the probability of finding an electron across the barrier decreases. The tunnelling current is thus also called probability current (J), and can be determined by solving the quantum barrier problem with the Schroedinger equation. It is found that the wavefunction in the barrier decays exponentially, and enlarging the width of the barrier affects the decay. The tip geometry, in this regard, is crucial. If the tip is large, many gaps are present between tip and substrate, and every gap is responsible for a part of the resulting tunnelling current. On the contrary, if the tip is very sharp, the number of gaps is ideally reduced to one (one atom only on the apex of the tip), and the current varies exponentially with the distance between tip and sample. If this is true, the tunnelling current can be used as a height indicator. A setpoint current can be selected for a tip scanning a surface and when something changes on the surface (height discontinuities due to morphological peculiarities of the sample), the setpoint is disturbed. One can measure the current that has to be applied to keep it constant throughout the scan, which is related to the distance the tip has to be moved. This is the founding principle of a scanning tunneling microscope. The microscope can be operated substantially in two modes: constant height mode -where a setpoint current is selected for a tip scanning a surface and when something changes on the surface (height discontinuities due to morphological peculiarities of the sample), the setpoint is disturbed -; constant current modewhere one measures the tunnelling current variations compared to the chosen setpoint according to the surface modifications (good for flat surfaces) or the height of the tip is adjusted to keep the current constant (better for rougher surfaces). The current flowing between the tip and the substrate is proportional to the local density of states of electrons (LDOS, which describe the number of states that the system can occupy at a certain energy level) and to the applied voltage. LDOS of electrons are thus affected by the surface properties (atom positions, step edges, terraces), grain boundaries, the presence of adsorbates, impurities, dislocations and vacancies. To have atomic resolution, it is essential to have a single atom at the apex of the tip in order for the wavefunction to protrude in a very sharp way. The nature of this atom is also crucial, since the electron can be placed in a different orbital, with different geometries. It is impossible in practice to control which atom is at the apex, but we can model to know what to expect, considering the wavefunction that the state is generating. From a practical point of view, it is therefore fundamental to have a tip that ends in one atom (atomic resolution).

In a general scanning probe microscope set-up (Figure 3.1a), the tip is mounted on a piezoelectric scanner that allows position controlling through vibrationless motions in the three directions of space, X, Y (lateral) and Z (height). When a voltage is applied to a piezoelectric bar, the generation of an internal electrical field causes a mechanical deformation ∆l (inverse piezoelectric effect): ∆l = d • l h • V 0 , where d is the piezoelectric coefficient (characteristic of each material), l is the length of the bar, h is the height of the bar, V 0 is the applied voltage and E is the resulting internal electric field (Figure 3.1b). In order to control the displacement in a specific Chapter 3. Force quantification by fluorescence at the nanoscale direction, quadranted piezoelectric tubes are used and the voltage is applied specifically to a quadrant of the tube (X, Y, Z). The bending of the piezoelectric quadrant as a response to the applied voltage is not instantaneous, but takes a certain amount of time (creep, Figure 3.1c), plus in all piezoelectric material the bending response is not linear with the applied voltage (Figure 3.1d), which affect the quality of the scan and needs to be taken into consideration in the design of the set-up. Strain gauges that track the real piezo-position are used to counterbalance the non linearity effect, even if for small scans this effect is negligible and strain gauges are not needed.

The key part of a generic SPM set-up is the feedback circuit, shown in Figure 3.2. An operational amplifier compares the output signal with the reference setpoint. The height of the tip is controlled by the piezoelectric Z-scanner in such a way to match the output and input signals. In STM, the output signal is the tunnelling current (which is converted into a voltage by an I-V converter) generated between tip and substrate when a voltage is applied and the input signal is the reference voltage that is applied initially to the Z-scanner. As it will be discussed, in AFM, the output signal is the deflection amplitude of the cantilever mounting the tip (height or applied force) and the input signal is the voltage that is applied initially to the 104

Chapter 3. Force quantification by fluorescence at the nanoscale Z-scanner. Therefore, the final signal that is processed by the computer system for image reconstruction is the voltage needed to match the output and input signals, given by the feedback loop. It is evident, then, that the feedback system needs to be as fast as possible to favour precise surface imaging in reasonable times. The great limitation of STM technique is that it requires a conductive substrate to allow tunnelling current transmission. This limitation is overcome few years later the first publications on STM, with the implementation of AFM. A silicon tip is mounted on a cantilever, whose behaviour can be treated as a spring following Hooke's law in first approximation. When the tip rasters the surface, the cantilever is bended according to the attraction and repulsion forces that the tip experiences. No current is flowing between the tip and the sample, what governs AFM is two main kinds of forces: atom-atom interactions (short range, 2-3 Å) and collective tipsubstrate interaction (Van der Waals type, long range, 1 nm). If it is assumed that the nature of tip and substrate atoms is the same, when they are brought very close together, the main forces of interaction are at first Van der Waals attracting forces and Pauli repulsion forces at very small inter-atomic distances, where the electron clouds of the two approaching atoms start to overlap and repulse. Van der Waals forces govern tip-substrate interaction, with polarisation forces (Keesom), dipoleinduced dipole interactions (Debye) and dispersion interaction (London). These are crucial in AFM since they can be present between two neutral objects (tip-substrate) thanks to the presence of fluctuating dipoles. See Figure 3.3a for a graphical representation of the potential energy profile with respect to tip-substrate distance and interaction.

The first appearance of AFM dates back in 1986. 10 An STM is used to measure the rise and fall of a tip mounted on a flexible gold coated cantilever when rastered Chapter 3. Force quantification by fluorescence at the nanoscale across an insulating substrate. The first examples of AFM are operated in contact mode, where the tip is in close contact with the surface. It is in 1987 that noncontact scanning mode was born. 11 The year after, between 1988 and 1989, AFM gains a lot of attention, and great advancements are made in its technical implementation. Cantilever position sensing through optical beam bounce on a photodiode is developed, 12 which is the first step towards a stand-alone technique, independent from STM. In 1991, the first microfabricated tips appear, which improved remarkably the spatial resolution. 13 In 1993, thin films of piezoelectric materials are used to cover cantilevers and allow the measurement of the cantilever bending through an optic-free system, relying on the piezoelectric current generated by the mechanical distortion. 14 It is in 1993 that AFM technique reaches a turning point with the development of intermittent contact mode (or tapping mode). 15 In modern systems, the measurement of cantilever bending is usually done by a beam deflection method on a quadrant photodiode, as depicted in Figure 3.3b. A laser beam is sent on the top of the cantilever end, which is coated with a reflecting foil (usually gold or silver) that sends back the light beam to the photodiode. Before scanning, the laser is centred in the quadrant. While scanning, the cantilever deflection causes a redirection of this beam on the quadrant, according to the motion. Vertical and lateral deflection are detected and correspond respectively to bending and torsion of the cantilever. The readout voltage of the photodiode constitutes the output signal that enters the feedback loop, already described for STM. Either the cantilever itself or the sample stage are mounted on a piezoelectric tube scanner, that controls the position in the three directions of space, lateral (X and Y) and vertical (Z). The Z position is determined pixel by pixel by the input signal coming from the feedback loop, to match the reference set voltage (setpoint). The "nature" of the setpoint depends on the mode of scan that is chosen:

Contact. The tip is in contact with the substrate surface and applies a certain vertical force (Figure 3.4a). The contact is kept along the scan. The setpoint voltage is here converted to a vertical applied force, usually expressed in nN, depending on calibration and on the cantilever specifications (stiffness, force constant).

Tapping. The cantilever is in constant oscillation, "tapping" on the substrate with a specific oscillation frequency, that is perturbed when the morphology of the substrate surface changes (Figure 3.4b). This perturbation is compensated by the feedback piezoelectric system to match the reference oscillation frequency (resonant frequency, specific for the type of cantilever). Quantitative Imaging (QI). The tip is displaced from pixel to pixel and in each point a force curve is obtained at a certain indentation force (Figure 3.4c). This mode allows to map at the same time the topography of the sample and its mechanical properties. In fact, in each pixel both a height value and a force curve is obtained. A force curve is the plot of the cantilever deflection (V) Chapter 3. Force quantification by fluorescence at the nanoscale versus the measured height (nm). Three main regions can be distinguished, as shown in Figure 3.4d. In the approach region the tip is distant from the surface and no attraction or repulsive forces are experienced. When the tip gets closer to the substrate, long range attractive Van der Waals forces come into play and the tip "jumps to contact" with the substrate. When the tip is pushed further to the surface (contact), short range repulsive inter-atomic forces start acting and define the indentation region. The slope of the indentation segment gives information on the stiffness of the surface. If a small indentation height causes a high cantilever deflection, the sample is stiff. By contrast, the surface is softer if for the same indentation depth the cantilever does not need to deflect as much. The retract segment usually follows the same trace of the approach segment, with the difference that, according to the sample properties, the tip can be pushed back by adhesion forces while trying to retract from contact. The adhesion data is thus obtained from the deflection difference between the lowest point in the retract curve and the baseline. The monomer sample is deposited on clean glass slides and can be illuminated from the top through a condenser or from the bottom through the microscope objective (60X objective, air, NA = 0.95). A white light lamp is used as source to illuminate the sample from the top to take wide-field transmission images and perform in-situ photopolymerization. In the first case, a long-pass filter is placed after the condenser to cut wavelengths lower than 400 nm and avoid unwanted photopolymerization; in the second, a Semrock interferential filter is placed before the condenser to select exclusively the 254 nm wavelength that is needed to trigger the photopolymerization process in the PDA sample. A Lumencor (Semrock) multiwavelength LED source is used instead to illuminate the sample from the bottom and excite its fluorescence (λ exc = 488 nm). A dichroic mirror is placed between the objective and the detector, to reflect the excitation light to the sample and let pass to the detection unit the sample emission light exclusively. An EM-CCD camera allows to record transmission and fluorescence images. Alternatively the light path can be redirected to a spectrograph, which allows to record transmission and fluorescence spectra. The advantage of this set-up is the possibility of installing an AFM instrument on top of the sample, which is optically aligned with the fluorescence microscope. Therefore, it is possible to spatially correlate morphology and fluorescence images. Furthermore, the AFM can operate in different modes: tapping and QI modes are used for the topography images shown in Section 3.2.1 while contact mode is used to mechanically stimulate the sample at a specific vertical force (nN). Fluorescence response can be probed sequentially. The statistical distributions of crystal length and width are shown in Figure 3.7a and 3.7b respectively; distribution of the crystal orientations for the two samples are shown in Figure 3.7c (TzDA1) and 3.7d (DA1). When looking at the sizedistributions, DA1 films form long and large surface crystals that can often exceed 1 µm length and 400 nm width, whereas most of surface crystals are much smaller for TzDA1 films, ranging in the 160 -880 nm interval (in length) and 80 -240 nm (in width). Moreover, DA1 surface crystals orientate in specific directions, forming visible domains, as shown in Figure 3.7d. The photopolymerization, performed under the microscope, does not affect the morphology in accordance with the topotactical polymerization model mechanism. [16][17][18] Even though the position and dimensions of the crystals are retained in the polymer films, when DA1 films are exposed to high power UV light or long irradiations, the film cracks along the domains (see Figure 3.6d, green arrows). The insets are detailed morphology images in smaller scales of the white square areas, and show more clearly that surface crystals do not change either in position or in dimension. Vacuum evaporated thin films of TzDA2 show instead a very different morphology. AFM images before (Figure 3.8a) and after (Figure 3.8b) photopolymerization show that the compound does not form well-defined crystals. The introduction of an ester function in the self-assembling group results thus in wide arranged aggregates, spaced by empty zones. This inhomogeneous morphology prevents accurate thickness measurements, which could only be estimated by optical UV-Vis absorbance measurements around 80 nm (see section 6.3.6) for 12 mg of TzDA2 powder used for the evaporation. However, polymerization was still effective on these samples despite their morphology, as detailed in section 2.3.2.

Vacuum evaporated thin films of TzDA3 also are not crystalline as TzDA1 and DA1. Morphology AFM images are shown in Figure 3.8, before (3.8c) and after (3.8d) photopolymerisation. The film is continuous and no big aggregates are found over large areas. Some material accumulates however to form some discontinuities in the film, which are not exceeding 100 nm height. Polymerisation, as for the other compounds, does not affect visibly the morphology of the film: all the morphological Chapter 3. Force quantification by fluorescence at the nanoscale features are retained in the polymeric film. The thickness of the film is estimated at 100 nm by the optical density (details can be found in Chapter 6).

Mechanical properties of non-polymerized and polymerized PDA thin films

Atomic force microscopy is a useful tool to map the morphological features of a non conductive substrate surface as well as its mechanical properties. In imaging modes, the tip is rastered across the surface to reconstruct a 3D image based on the tip-sample interaction forces that affect the chosen set point (see Section 3.1.1). Force spectroscopy measurements allow instead to probe the interaction forces in the tip-sample contact area, in different areas of the substrate. Mechanical properties like stiffness (Young's modulus), viscoelastic response and adhesion can be deduced from the force curves shown in Section 3.1.1, Figure 3.4d. In the approach segment, the tip starts interacting with the sample first by long-range electrostatic forces and, when it gets to few microns from the surface, by Van der Waals, capillary (if in air) and solvation forces. In the crucial moment of contact, the tip indents the surface and the cantilever is deflected. The repulsive contact can provide useful information on sample stiffness (Young's modulus) and on the viscoelastic response. In the retract segment, the tip is pulled from the surface and adhesion forces hold it back and cause a downward deflection of the cantilever. When working in air, the sample is usually covered by a thin layer (few nanometers) of water that creates a capillary neck between tip and sample, thus strong adhesion forces.

In this section, the attention will be focused on the study of sample hardness and Young's modulus of elasticity calculation, achieved by AFM nano-indentation measurements. Different methods can be implemented to treat force curves and they have largely been used in the past to characterize mechanically different materials (nanoparticles, polymers, biological tissues etc.). [19][20][21][22][23][24] The simplest and most used method to analyse force spectroscopy data is the Hertz model. Force curves are usually plotted as cantilever deflection (V) versus height measured (nm), and are converted into force (nN) versus tip sample separation (nm) for further analyses. The measured height corresponds to the piezo displacement z (filled line in Figure 3.9a), and needs to be corrected by the cantilever bending (x in Figure 3.9b) to derive the tip-sample separation. It is of great importance to calibrate the cantilever before performing any measurement and conversion, to make sure that a deflection voltage correctly corresponds to the indentation force. This calibration is performed by thermal noise method, provided by the constructor. The cantilever is moved towards the sample by a distance z (height measured). When the tip gets in contact with the surface, the cantilever bends into the opposite direction of approach (x) and the sample is indented by δ. δ is calculated by subtracting the cantilever deflection (x) from the height measured (z). See Figure 3.9b for a scheme of the indentation experiment. Hertz model approximates the sample as an isotropic linear elastic solid, occupying an infinite half-space. It also assumes that the indenter is not deformable (using diamond tips is therefore advised) and that no additional interaction between sample and indenter occurs apart from indentation. For a spherical indenter Young's modulus of elasticity is given by:

F = E 1 -ν 2 • a 2 + R 2 2 ln R + a R -a -aR
Where E is the Young's modulus, ν is the Poisson's ratio, a is the radius of the contact circle and R is the radius of the sphere. Extensions to parabolic and conical tip geometries are available, but for simplicity reasons, we assumed a spherical geometry. Therefore, this model provides a simple and direct way of treating indentation data but in the present case Young's modulus values are not fully reliable. Firstly because the analysed PDA thin films are far to be absolutely elastic, so part of the energy delivered by the indenter dissipates in the film. Secondly, because we had to make approximations on Poisson's ratio values. In fact, the value of Poisson's ratio is essential to perform the calculation, and they are tabulated for a great number of polymers but not specifically polydiacetylenes. It was taken ν = 0.4, which is a reasonable value between polystyrene (PS), 0.34, and rubber, 0.5. The size (R) of the tip is also a crucial parameter that may change from one acquisition to another, due to tip pollution and deformation. For this reason, before and after each measurement, a PS reference sample is imaged. Knowing the tabulated PS's Young's modulus is 2.5 GPa and that the Poisson's ratio is 0.34, the QI images can be used to derive the size of the tip. TzDA1 and DA1 thin films have been tested before and after polymerization. Due to the experimental limitations explained above, it is not cautious to compare the results obtained in different samples, since the experiments were performed with different tips (properly calibrated). It is possible, however, to compare the stiffness of the thin film before and after polymerization.

Experimental procedure. QI images of a reference polystyrene film and gold nanoparticles are acquired to derive information on tip size and shape. PS testing and analysis through Hertz model provides with the tip size, while gold nanoparticles testing is performed to make sure that the tip is not polluted (step 1 in Figure 3.10). QI images of the samples are recorded (step 2) and reference samples are tested again (step 3) to make sure that the tip has not been modified during the measurement (or to evaluate this modification and take it into account for the next analyses).

The sample is then polymerized by UV irradiation at 254 nm (step 4) and tested again in QI mode (step 5). At last, reference samples are imaged (step 6). Figure 3.11a and d are the topography images and Figure 3.11b,c and e,f are the adhesion and the Young's modulus maps of non-polymerized and polymerized thin films. Polymerization does not affect the topography features of either the crystals laying on the surface or the continuous film in the background (Figure 3.11a,d) as discussed in Section 3.2.1. However, the polymerized film shows globally a higher adhesion force (2 nN in the monomer and up to 6 nN in the polymer) when the tip is retracting from the surface, as it can be concluded comparing Figure 3.11b and e. Analysis of the force curves acquired in each pixel of the image through Hertz model leads to the Young's modulus map shown in Figure 3.11c and 3.11cf (before and after polymerization respectively). The crystalline domains that constitute the surface background globally show a higher Young's modulus when they have been polymerized (from 1 GPa in the monomer to 1.8 GPa in the polymer, in the background zones of the sample). Concerning the crystals laying on the surface, the measurements are not reliable considering the dimension of the tip. From reference PS measurement, it was found 8 nm radius before the non polymerized measurement and 6 nm radius after the measurement. The contours of the crystals appear less stiff Chapter 3. Force quantification by fluorescence at the nanoscale than the background and the center of the crystal. The reason may be reconducted to the conical shape of the tip, meaning that on the crystal edges the sample is in contact with a side of the tip, which can not be treated as a sphere (according to Hertz model).

DA1 thin films

DA1 thin films were prepared by vacuum evaporation as described in Section 2.3.1. Figure 3.12 shows 10x10 µm QI images acquired before (a,b,c) and after (d,e,f) polymerization. Therefore, polymerization of DA1 causes structural modifications with the ap-parition of cracks in the film (Section 3.2.1) that soften the film increasing its elasticity. It is interesting to notice that polymerization does not have the same effects on TzDA1 and DA1, since in the former adhesion and Young's modulus increase in the polymer, while in the latter they both decrease in the polymer.

Application of a friction force to PDA thin films: force quantification

In the past years, mechanoresponsive fluorescent materials have been widely used to evaluate mechanical forces through the change in the emission properties that occur under mechanical stress. A wide variety of compounds has been proven to be mechanofluorochromic: single organic molecules, [25][26][27] organometallic complexes 28,29 and polymer composites. 30,31 Recently, researchers have tried to understand the mechanism behind mechanoresponsive luminescence, with the objective of building reliable fluorescent force sensors, investigating their response to various types of force and different force intensities. The type of force that is applied has an important influence on the compound structural changes that determine the fluorescence modification. Distinct responses are to expect depending on the type of force that is applied, as it was shown for tetrathiazolylthiophene, 32 triphenylamine 33 and boron diketonate 34 crystals in respect to mechanical grinding (friction force), hydrostatic pressure and smashing. Application of a hydrostatic pressure on a crystal is a convenient way of applying a controlled pressure and monitor simultaneously the fluorescence signal modifications. Pressure and fluorescence can be quantitatively correlated, showing that a greater pressure is followed by a greater fluorescence modification. [34][35][36] Mechanical friction has also been tested on PDA coated paper substrate by means of weightloaded cage that is moved at constant speed on the sample. The resulting shear stress is changed using the mass of the weight loaded on the cage, which controls the normal applied force. The applied friction force is correlated with the colorimetric response of the PDA film, showing an increasing blue-to-red conversion rate when increasing the applied force. 37 These examples show indeed that mechanical force can be correlated to either the fluorescence change in mechanofluorochromic systems or the colorimetric response of mechanochromic compounds.

To have a better insight on the mechanofluorochromic mechanism, nanoscale studies have been carried out. The first observation of mechanochromism at nanoscale was achieved by Carpick et al. in 2000, 38 where PDA trilayer thin films (10,12pentacosadiynoic acid) were stimulated by NSOM and AFM and the blue-to-red transition was probed by fluorescence microscopy, thanks to the fluorescent signa-Chapter 3. Force quantification by fluorescence at the nanoscale ture of red-PDA. AFM constitutes a valuable technique to apply a controlled force at the nanoscale. When coupled to a fluorescence microscope, mechanical stimulation and simultaneous fluorescence probing is possible. Isolated nano-objects have attracted great interest in the past few years. Compressive stress applied by an AFM tip was tested on single molecules, 39 and on single semiconductor nanocrystals. 40 Shear stress can also be applied by AFM and the fluorescent change probed by confocal microscopy, as shown on single nanoscrystals of a difluoroboron-β-diketonate derivative. 41 When applying a mechanical shear stress by means of an AFM, a number of experimental parameters are crucial in determining the effectiveness of the stimulation for fluorescence recovery purposes. Moreover, it is crucial to establish a general modus operandi to be used with all the compounds for comparability. The sample is first observed by wide-field optical microscopy (transmission and fluorescence images) to individuate an area to work with, carefully checking that no big inhomogeneities in the film are present (large aggregates, deteriorated areas, presence of dust, etc.). Photopolymerization of the DA thin film is carried out in situ as explained in Section 3.1.2 directly on the microscope with irradiation times that vary according to the sample. Time of irradiation is chosen according to the polymerization kinetics of the compound under study. Looking at the polymerization kinetics in Figure 2.6, the total irradiation energy is chosen in such a way to produce enough blue-polymer to efficiently quench the tetrazine fluorophore but far from saturation, that being before the plateau. The irradiation power is measured every day and adjusted to be constant throughout the set of experiments.The AFM is used in tapping mode or quantitative imaging mode to map the morphology of a part of the zone imaged by the optical microscope (60X objective) after photopolymerization (and possibly also before) and after mechanical stimulation, which is performed operating the AFM in contact mode. Different types of tips are used for these two applications: for imaging purposes a standard silicon tip is used, while for mechanical stimulation a diamond-like carbon coated tip is used (see specifications in Section 6.4.4). The mechanical stimulation consists in a squared 3x3 µm 2 scan performed in contact mode at constant setpoint (normal load, given in nN after calibration) with a resolution of 128x128 px. The calibration of the spring constant is performed by thermal noise method provided by the JPK software, in ambient conditions, before each set of measurements. The scan consists in a "trace" and "retrace" segment for each pixel of the slow axis, from bottom to top of the area.

The tip starts from point A and goes to point B at 6.1 µm•s -1 (trace). It goes back from point B to point A (retrace) and moves to the next line towards the top of the selected area. Different scan rates have also been tested, ranging from 0.7 to 10.2 µm•s -1 .

Fluorescence images are taken with the same excitation power and same exposure time of 1 s after each scan/stimulation is completed. Both tetrazine (in TzDAs) and stimulated poly-DAs are excited by a 488 nm LED light.

To assign a numerical value to the fluorescence restoration after mechanical stimulations, differential images are taken of consecutive fluorescence images corresponding to subsequent stimulated areas and the signal is integrated over the whole stimulated area. The subtraction is performed by a LabView program developed in the laboratory, that allows image repositioning and subtraction of each pixel value. The result is a delta image where the background signal is levelled to zero and where only the signal coming from the stimulated material is present. Finally, the numerical values obtained from the integration can be plotted versus the applied normal force (setpoint) of the corresponding area to build the fluorescence recovery-force correlation curves that will be shown here (Figure 3.23).

TzDA1

Standard silicon tip (PPP-NCHR), 4.04 µm•s -1 scan rate. The first nanoscale tests on mechanofluorochromism of TzDA1 were performed with a standard silicon tip at a scan rate of 4.04 µm•s -1 , which are the same scanning conditions used for topography imaging in tapping mode. As detailed in Section 3.2.1, the sample surface topography is not affected by polymerization. Optically, a quenching of the monomer fluorescence signal is expected to occur when mono-TzDA1 is converted to blue-poly-TzDA1 as shown in the fluorescence spectra in Section 2.3.3. Figure 3.13a shows that the sample is Chapter 3. Force quantification by fluorescence at the nanoscale highly fluorescent in the monomer form, and the fluorescence is quenched homogeneously when polymerization is achieved by irradiation at 254 nm directly on the microscope set-up (Figure 3.13b). When mechanical stimulations at the same nominal vertical force of 125 nN were applied in different areas, the fluorescence signal is restored locally in those areas (Figure 3.13c). In addition, the integration over these areas of the signal resulted in fluorescence recoveries of the same order of magnitude, as shown in Figure 3.13d. Values range between 1.85•10 5 and 4•10 5 , which is consistent to the correlation curve that will be established at the end of the chapter (Figure 3.23). This first experiment stood as a strong proof that nanoscale mechanofluoroschromism of tetrazine-substituted diacetylene is in principle possible and measurable. In order to get to an exhaustive correlation between mechanical stimulation intensity and fluorescence recovery, additional testing was needed. Different parameters were adjusted to optimize this response, with special attention to the scan rate during stimulation and tip type (material, shape, force constant etc.).

More rigorous testing was performed on vacuum evaporated sample of TzDA1, changing the vertical normal force in each scanned area, to check if a higher applied force produces a higher fluorescence recovery signal. Figure 3.14a shows the morphology image of the chosen polymerized zone of the sample for mechanical stimulation. As explained in Section 3.2.1, rod-like crystals distribute homogeneously over a continuous film. The homogeneity of the film surface is one key parameter for comparing different areas of the sample zone, thus assuming that the mechanical stimulations are performed on morphologically equivalent areas. Figure 3.14b is the corresponding fluorescence image. The system is in its OFF state, thus the signal does not exceed 500 counts in any pixel. Mechanical stimulation is applied in 30 different areas with normal forces ranging from 50 to 610 nN. Figure 3.14c shows the sample morphology after stimulus. In each area, the sample surface has been modified. This modification leads to the conversion of the blue phase of the polymer to the red phase, restoring the tetrazine fluorescence signal, similarly to what it is achieved at the macroscale in Section 2.3.4. The corresponding fluorescence image is shown in Figure 3.14d. A higher signal intensity is witnessed for higher applied forces as expected, however the restored fluorescence is concentrated on the sides of the stimulated area, leaving a non-fluorescent part in the middle. As it can be noticed in Figure 3.14c, all applied normal loads displace part of the surface material on the sides of the scan area. It is common, in fact, that some of the material that is stimulated along the experiments sticks to the tip surface (polluted tip) and is released in another area. Usually, it detaches in the top left corner of the scan, which is the last pixel before detachment from the surface (350 nN scan in Figure 3.14). This phenomenon decreases the reproducibility of the experiment and the reliability of the integrated signal. Material displacement is an unavoidable phenomenon when mechanical stimulation is performed by AFM. Mechanically, this is understandable for high normal forces, where the tip indents deep in the film and displaces the material on the sides while scanning. However, there are some parameters that, if well-adjusted, can milden this effect. Firstly, the scan rate can influence the probability of tip pollution by the sample (a low tip velocity will increase the chance of pollution) and secondly, the material of the tip itself also affects the affinity for the sample, thus the probability of tip pollution. In the following paragraphs, tip type and scan rate were modified in order to optimize TzDA1 fluorescence response. mal forces between 20 and 200 nN, meaning that the AFM tip equally stimulates each scan pixel by applying a constant force. For higher forces, fluorescent material accumulates on the borders of the areas. The corresponding AFM morphology images are shown in Figure 3.16b-c-d, and confirm that material displacement still occurs, but is more pronounced for forces higher than 200 nN. Indeed, carbon coated tips resulted less prone to pollution and deformation, ensuring a longer-lasting performance of shearing.

DA1

Vacuum evaporated DA1 thin films present a different morphology (Section 3.2.1) and mechanical properties (Section 3.2.2) than TzDA1 thin films due to the absence of tetrazine in the chemical structure. In addition, the spectroscopic properties and especially the photoluminescence quantum yield is highly affected, as explained in Section 2.3.3. A different response to nanoscale mechanical stimulation is therefore expected.

Standard silicon tip, 6.1 µm•s -1 . At first, standard silicon tips were used to perform the mechanical stimulations, at 6.1 µm•s -1 scan rate that was found optimal for TzDA1 thin films.

Polymerization of DA1 does not affect the sample morphology, but long irradiations may lead to cracks in the continuous film on which rod-like crystals lay (Section 3.2.1). Figure 3 The impact on the sample morphology seems to be smaller after that several stimulations have been performed, causing the corresponding fluorescence response to fade as well, even though the applied normal load is increasing. It is evident that morphology perturbation and fluorescence activation are strictly interconnected. Such a drop in fluorescence response due to a gradual weakening of the efficacy in perturbating the morphology by AFM may be explained by a deterioration of the tip surface, that worsen from one stimulation to the next.

Carbon coated diamond-like tip, 6.1 µm•s -1 . Vacuum evaporated samples of DA1 with the same morphology were tested to nanoscale mechanical forces with a carbon coated diamond-like tip, which is more inert than standard silicon ones, thus should be less prone to deterioration and tip pollution. In fact, Figure 3.18 shows a complete test to mechanical forces ranging from 25 to 600 nN normal loads applied in two zones of the sample, and no evident drop in fluorescence signal is witnessed along the experiment. No clear increase of fluorescence signal with increasing applied normal force is witnessed either, suggesting that DA1 thin films do not respond to mechanical forces the same way as TzDA1 thin films.

TzDA2 and TzDA3

TzDA2 and TzDA3 thin films were tested to mechanical forces at the nanoscale, even though the former has an irregular surface morphology (Section 3.2.1) and the latter has limited fluorescence recovery properties since photopolymerization produces mainly red-poly-TzDA3 (Section 2.3.2).

It is shown in Section 3.2.1 and 2.3.2 how TzDA2 arranges differently, photopolymerizes faster but forms globally less blue polymer than TzDA1. Nonetheless, this arrangement is responsible for a more efficient quenching of the tetrazine fluorophore, as shown in Section 2.3.3. Extending the quenching capability is expected to result in a wider range of forces that can be probed, but the resulting inhomogeneous layer deposition is not well adapted to the experiments shown in this work, whose reproducibility depends on the homogeneity of the sample surface, that ensures the possible comparison between different stimulated areas. Figure 3.19 shows the Chapter 3. Force quantification by fluorescence at the nanoscale first tests that were performed on TzDA2 thin films. Topography and fluorescence images of the monomer (3.19a and 3.19b), polymer (3.19c and 3.19d) and stimulated polymer (3.19e and 3.19f) show that UV irradiation at 254 nm quench the fluorescence as expected and does not affect the topography, and that mechanical stimulation switches the fluorescence back on. However, it is evident that the optical response is not correlated to the intensity of the applied vertical force: the three central stimulated areas have been subjected to the same force, but clearly fluoresce with different intensities. This lack of reproducibility is due to the fact that the morphology varies a lot according to the zone of the sample. Crystalline aggregates are spaced by empty zones, thus to actually stimulate mechanically the material, it is necessary to position the AFM tip on the aggregates, as depicted in Figure 3.19 by the blue dotted lines. Green dotted lines, instead, show the zones where the fluorescence restoration was less effective, and they correspond to empty zones or mixed morphologies (empty zones and portions of crystalline aggregates). When TzDA3 thin films are polymerized, the main polymer phase that is produced is red-poly-TzDA3 (λ abs = 554 nm) as shown in Section 2.3.2. An efficient tetrazine fluorescence quenching is achieved, so a mild recovery is expected from mechanical stimulation. Red-poly-TzDA3 efficiently quenches the fluorescence and cannot be converted to any other polymer phase to decrease the spectral overlap (see Section 2.3). Therefore, even the blue-to-red conversion of the small blue phase quantity that is formed is not expected to have particular effects on the fluorescence quenching efficiency. Topography and corresponding fluorescence images of the monomeric film are shown in Figure 3.21a and b respectively. When photopolymerization is performed, the surface topography is not affected (Figure 3.21c) and the fluorescence is efficiently and homogeneously quenched (Figure 3.21d). Nine areas of the sample are stimulated by AFM in contact mode, ranging vertical forces from 20 to 300 nN. Topography and fluorescence images after mechanical stress are shown in Figure 3.21e and 3.21f. All the vertical forces induce a change in the surface morphology, perturbation that seems more pronounced for high forces. However, the corresponding fluorescence image shows that the fluorescence recovery caused by mechanical stress is barely visible by eye, confirming the expectations. For this reason, no further testing was performed on TzDA3.

Correlation of fluorescence intensity and applied normal force

The aim of these experiments is to quantify the fluorescence activation (DA1) or recovery (TzDAs) induced by a mechanical stimulation at the nanoscale. Qualitatively, Section 3.3 showed that when the AFM tip scans the sample surface in contact mode at a certain vertical force, the morphology is usually perturbed. This modification is localized in the area of interest and the fluorescence signal is activated exclusively in this same area. The starting morphology is crucial in determining the efficiency of the mechanical stimulation: irregularities in the film prevent an homogeneous application of the friction force in all the pixels of the selected scan area, which is the case for TzDA2 thin films. The mechanical properties of the films have an influence in the phenomena of tip-pollution and degradation, which eventually affect the actual mechanical stress received by the sample: TzDA1 thin films are easily deformed, with important displacement of material by the tip and tip-pollution effects, while DA1 thin films are less affected by tip scanning. The resulting efficiency in the morphology modification is reflected in the fluorescence response.

To numerically evaluate the fluorescence signal activation, fluorescence images of two consecutive mechanical stimulations are subtracted to exclude the background signal and "isolate" the signal coming from the mechanical stress only. Delta fluorescence images are obtained. This procedure is performed using a LabView software conceived in the laboratory, that allows image repositioning (consecutive images can be shifted by few pixels) and pixel by pixel subtraction. Next, the fluorescence signal is integrated by Igor Pro software over a fixed area corresponding to the stimulated one in each delta image. Therefore, each nominal applied force can be correlated to a numerical value of fluorescence signal. Figure 3.22 explains the process of fluorescence signal quantification, through subtraction and integration. This procedure is applied to a set of experiments performed in the same experimental conditions for stimulation (same tip type, same scan rate), fluorescence images acquisition (integration time, excitation power) and sample preparation (vacuum evaporated thin films, exposure time to UV irradiation for polymerization). The obtained numerical value is then plotted versus the nominal applied force to build a correlation curve. As reported in [42], the fluorescence signal of the tetrazine dye in TzDA1 increases along with the intensity of vertical force applied to the sample by the AFM tip during the friction, until a plateau is reached around a nominal vertical force of 200 nN (Figure 3.23, black dots). Higher forces produce the same fluorescence signal activation. The lack of fluorescence variation at high force range may be ascribable either to blue-to-red PDA conversion saturation or, most probably, to a constant applied mechanical stress. In this range, the tip indentation in the sample surface is deep, and thus the actual applied force decorrelates from the vertical piezo-stage displacement, resulting in the same applied friction force. In the case of poly-DA1, the fluorescence signal is also increasing under AFM-based mechanical trigger. However, this tendency is not as straightforward as it was found to be for poly-TzDA1 (Figure 3.23). In addition, the Δfluo signal of the system is 7-fold lower, which could be due to a lower amount of converted chains (blue-to-red) or to the absence of the tetrazine fluorophore. Taking into consideration that DA1 thin films are 4 times thicker than TzDA1 films thus absorb 3 times more at the excitation wavelength (488 nm), the resulting fluorescence signal upon mechanical stimulation is actually 20-fold lower. It is found that this same factor corresponds to the solid state fluorescence quantum yields of red-poly-DA1 and tetrazine (0.008 vs 0.17, see Section 2.3.3), meaning that the introduction of the fluorophore accounts for most of the fluorescence activation enhancement. Attenuation through the film of excitation light and fluorescence reabsorption phenomena can be reasonably neglected since the absorption at these wavelengths is lower than 0.1. Compared to poly-TzDA1, poly-DA1 thin films are less deformable. If in the former forces higher than 200 nN deeply perturb the film surface topography, vertical forces up to 600 nN do not seem to affect noticeably the surface topology of poly-DA1 thin films (see Figure 3.18). An important factor that comes into play in these experiments is the quality of the AFM tip after one or more stimulations. In fact, the friction between the tip and a stiff sample surface like poly-DA1 can deform it, while in softer films like poly-TzDA1 some material can be detached from the sample surface and stick to the tip. When the AFM tip is deformed, damaged or polluted, the correlation between applied vertical force and the actual mechanical stress experienced by the sample is reduced. This may explain why in the same experiment it is not rare to witness a milder morphological change for consecutive increasing mechanical stimulations. The correlation curves shown in Figure 3.23 allow us to conclude that TzDA1 is a promising candidate for building a reliable force sensor for an interesting range of probed forces from 0 to 200 nN. In fact, in this range, even small force variations small as 50 nN induce a detectable fluorescence activation of 100000 counts.

Conclusion

In this chapter, the study of PDAs thin films is extended to the nanometer scale. Atomic force microscopy and fluorescence microscopy represent valuable tools to correlate morphology, mechanical properties and fluorescence characteristics, with the aim of finding the most suitable derivative to probe friction forces applied by AFM.

Morphology of the four PDA-derivatives were studied in detail by AFM, with particular attention to DA1 and TzDA1 to evaluate the influence of tetrazine in the molecular structure on the film arrangement. Both compounds give mixed morphologies of rod-like crystals laying on a continuous film. A statistical analysis allowed us to conclude that DA1 forms generally longer crystals (>1 µm length) than TzDA1 (<1 µm) and orientate in specific directions, while TzDA1 crystals do Chapter 3. Force quantification by fluorescence at the nanoscale not. TzDA2 forms big aggregates spaced by empty zones and, similarly, TzDA3 forms discontinuous aggregates. Adhesion and stiffness properties of polymerized and non-polymerized DA1 and TzDA1 thin films are determined by advanced force spectroscopy measurements. Taking all precautions in relying in the absolute values of Young's modulus that are obtained by treatment via Hertz model, DA1 thin films appeared to be globally stiffer than TzDA1. In addition, it was shown how polymerization affects the elasticity of the films, which is increasing in TzDA1 and decreasing in DA1.

The main goal of the chapter is to characterize the mechanofluorochromic properties of these compounds at the nanoscale. Polymerized thin films are thus subjected to mechanical stimulations using an AFM tip. Tip specifications, such as size, material and shape, deeply affect the effectiveness of a mechanical stress in perturbing the local morphology and switch fluorescence ON. Also the scan rate has an influence on the morphological modification. Shearing forces are applied in contact mode at a chosen vertical normal force, on squared areas of the same dimension. It is concluded from these experiments that carbon coated ones are the most efficient tips in applying friction forces and that the best results in terms of morphology modification and fluorescence activation were obtained using a scan rate of 6.1 µm•s -1 . The fluorescence response is visualized by fluorescence microscopy.

To numerically evaluate the emission switching, delta images of consecutive stimulations are obtained and the signal is integrated over a fixed area. Force -fluorescence activation correlation curves are obtained for DA1 and TzDA1 thin films (Figure 3.23), showing that both compounds' fluorescence increases with the applied force. TzDA1 shows a characteristic trend, where fluorescence increases in the 0 -200 nN force span to reach a constant activation signal for higher stress, which is a very promising result for further force sensors applications. On the contrary, DA1 does not present such a straightforward trend. The film is in fact less deformable, and for the same force, the morphology change in DA1 respectively to TzDA1 are much less pronounced. TzDA2's discontinuous morphology is not optimal for such experiments, where morphological equivalence of different zones is essential. TzDA3 application as force sensor is limited by its spectroscopic properties. When polymerized, the main polymer phase that is formed is red-poly-TzDA3, which is the irreversible final phase corresponding to tetrazine ON state.

In summary, we have discussed how the molecular design of PDA derivatives influence the resulting morphology of vacuum evaporated thin films, their mechanical properties and ultimately their applicability as friction force sensors. It is found that the presence of tetrazine has a double positive effect: it loosen the supramolecular arrangement, making the film more prone to be morphologically modified by the AFM tip and increases the fluorescence quantum yield, leading to better contrast fluorescence images and higher fluorescence brightness. In the next chapter, the PDA derivatives will be studied in the form of suspensions in solution and as isolated single crystals to broaden the discussion of force sensing in multiple aggregation states.

Chapter 3. Force quantification by fluorescence at the nanoscale Chapter 4

Polydiacetylene microcrystals

Polydiacetylenes are versatile molecules that can be exploited in different aggregation states. Chapters 2 and 3 focused on the analysis of different PDA derivatives deposited on glass slides as thin films. In this chapter, the study of these PDA derivatives is extended to aggregates suspensions and isolated single crystals deposited on glass slides. PDAs aggregates in suspension are the most explored systems for their application. The possibility of having a system that is responsive to different stimuli in aqueous solutions is a great advantage for biological applications. 1 Therefore, in the present chapter a full characterization of the spectroscopic properties of these systems and of the influence that the preparation method has on these properties are assessed, which is necessary before conceiving any application. Apart from the biological field, another possible application of PDA aggregates is to incorporate them in elastomers, to probe mechanical tension that is produced by the elongation of the substrate. Incorporation of self-assembled aggregates in a polymer matrix (PDMS) was used by Park et al. to build a colorimetric hydrocarbon sensor. The film swells upon hydrocarbon exposure, causing a mechanical stress on the embedded PDA aggregates that convert from the blue to the red phase. 2,3 Polydiacetylenes are known to produce plate-like and needle-like single crystals since the first report on these molecules by Wegner et al. in 1969. 4 These objects attracted a lot of attention immediately for their optical properties and especially for their responsiveness to temperature. [5][6][7] Single crystals constitute a good model to study polymerization kinetics of PDAs, which were characterized by time-resolved absorption spectroscopy 8,9 and by time-resolved electron spin resonance (ESR). 10 Morphology and structure of these aggregates are studied for the first time in 1979 by TEM, 11 and the effects of mechanical stimulation on the electronic properties and the morphology of TCDU crystals were reported by Müller and Eckhardt in 1978. 12 In the 1990s, it was shown by Katagi et al. that PDA crystals could be obtained by a simple reprecipitation method and that the size and shape of the resulting crystals could be more or less controlled by modifying certain preparation parameters. [13][14][15] Nowadays, these systems are often used as models for spectroscopic studies on PDA polymerization and red-phase characterization. [16][17][18] In this present research, we propose a complete morphology study of these objects, prepared via different methods, i.e. reprecipitation, sonication and recrystallization. Furthermore, these objects constitute a good model to study force stimulation at the nanoscale. They are, in fact, better defined than thin films, may present interesting optical properties like spatial 19 or spectroscopic 20 fluorescence amplification and may also respond to different kinds of mechanical stimulations like friction and pressure.

Preparation methods

Reprecipitation

Reprecipitation is by far the simplest and most direct method to obtain aggregates in suspension. It consists in dissolving the monomeric diacetylene in a "good" solvent (organic), which is then injected in a stirring volume of "bad" solvent (usually water) in which the compound is not soluble and the "good" solvent is miscible with ( Different parameters can be adjusted to potentially achieve different shapes and morphologies of aggregates in solution: the nature of the organic solvent, especially its polarity and its partial miscibility with water; the concentration (C ) of the diacetylene in the starting solution; the proportion of the initial solution and the bad solvent. 13 Temperature and stirring speed also play an important role, and they were kept constant throughout the experiments to 25 • C and 2000 rpm.

Sonication

The number of diacetylenes that is soluble in water is pretty limited and those molecules usually bear highly hydrophilic or charged side groups. This however, does not ensure the solubility of the polymerized diacetylene. The molecules under study are not soluble in water. Nonetheless, it is possible to force aggregation and suspension formation in water by ultrasound under heating, as it was shown for a number of DA molecules in literature. 1,21 The DA unit is dissolved in a small volume of a volatile solvent, like chloroform, and dried by rotavapor to form a thin film of DA in the flask. Water is added to the flask, which is then sonicated for a certain time at a constant temperature (Figure 4.2). The suspension is then let rest overnight at 4 • C for stabilization. The final concentration is calculated directly with the injected volume of water, as if the DA was completely soluble. Sonication time, temperature and DA final concentration are the parameters than can be varied.

Recrystallization

Crystal growth was achieved by liquid-liquid slow diffusion method. It consists in choosing a binary solvent system, with different specific densities. A small volume of whichever liquid has the higher specific density is transferred in a small receptacle and is layered with the other liquid. The compound to recrystallize is concentrated in the more suitable solvent. Over time, the two solvents mix and crystals form (Figure 4.

3).

A number of parameters influences the crystal growth process: nature of the organic solvent, concentration of DA in the organic solution, time of rest before deposition Chapter 4. Polydiacetylene microcrystals and temperature at which the solution is let rest. For the molecules under study, the chosen solvents are water and MeCN.

Prepared and analysed suspensions

Suspensions prepared by reprecipitation and sonication were studied in the liquid phase and as deposited crystals (Sections 4.2 and 4.3), while recrystallization was used to prepare isolated single crystals on glass slides only (Section 4.3). Different parameters can be adjusted in each preparation method in order to potentially control size, shape and ultimately spectroscopic properties of DA aggregates. 14,15 4.2 Spectroscopic studies in suspension

Photopolymerization in suspension

As for thin films, absorption spectra for progressively UV-irradiated (photopolymerized) aggregates in solution are expected to evolve because of the formation of polydiacetylene chains inside the aggregate. The formation of blue polymer corresponds to an increase of the absorption band centred around 600 nm. A similar mechanism of tetrazine fluorescence quenching by the blue-PDA that is found for PDA thin films is likely to occur in this aggregation state.

TzDA1

Table 4.1 sums up the tested TzDA1 suspensions. For reprecipitation method, solvent and initial TzDA1 concentrations are listed as well as the proportions between DA solution and water. For sonication in water, final DA concentration in the solution, temperature and time of sonication are listed.

Reprecipitation THF -water

TzDA1 aggregates in suspension were prepared by injecting with a syringe a TzDA1 solution in THF at variable concentrations into a volume of stirring distilled water, at 8:2 water:THF solution proportions. The absorbance of the resulting suspensions of two different concentrations was recorded and their response to UV irradiation at 254 nm for polymerization was tested. 4.4a shows the absorption spectra of a 0.1 mM suspension of TzDA1. It is evident that irradiation at 254 nm produces the absorption band corresponding to blue-poly-TzDA1, centred at 615 nm, and it increases when the sample is exposed to subsequent irradiations (grey lines in Figure 4.4a). To better visualize the polymer absorption band, the first absorption spectrum, corresponding to the monomer form, has been subtracted to cancel out the contribution of tetrazine absorption to the visible range of the spectrum (λ abs = 520 nm). The resulting spectra are shown in Figure 4.4b. The band at 615 nm increases in the same way, plus the side bands at lower wavelengths become visible. They probably correspond to a vibronic band of the blue phase (580 nm) and to the absorption band of red-poly-TzDA1 (540 nm). Tetrazine fluorescence signal (Figure 4.4c) decreases as expected, according to the energy transfer mechanism that is discussed for thin films in Chapter 2. The emission is quenched progressively as the amount of blue-poly-TzDA1 increases in the aggregates (black to grey to blue lines in Figure 4. 4a andb).

The same behaviour is found when the initial concentration of TzDA1 in THF is increased to 0.5 mM. Figure 4.4d shows the raw absorption spectra for subsequent irradiations at 254 nm. The subtraction of the monomer spectrum is here inconvenient due to a baseline shift between irradiated (grey and blue lines) and non These experiments stand as a proof of concept that TzDA1 reprecipitation in THF -water gave polymerizable aggregates in solution. Since the irradiation power at 254 nm has not been measured, polymerization kinetics and energy transfer efficiency can not be compared (thus not shown). Further and more accurate studies are necessary to complete the discussion. solution as proven by the absorption spectra in Figure 4.4. Two initial concentrations of the TzDA1 solution in THF were used (0.1 and 0.5 mM) to test its influence on the spectroscopic properties and on the polymerization process. Both concentrations lead to scattering objects in suspension, as can be seen by the continuous increase in extinction towards the shorter wavelengths in the absorption spectra (Figure 4.4a and d). The 0.5 mM solution leads to a 7-fold more absorbing solution, as proven by the maximum of absorbance at the tetrazine absorption band in the visible (530 nm). Polymerization by UV irradiation at 254 nm is efficient for both samples and the spectral shape is similar: an increasing band at 615 nm (blue-poly-TzDA1) with its vibronic peak at 580 nm, plus a small band at 540 nm (red-poly-TzDA1). Polymerization of the aggregates causes tetrazine fluorescence quenching in both cases, but more efficient for the 0.5 mM case. In fact, the fluorescence signal is quenched by approximately 55% in aggregates prepared from a 0.1 mM solution of TzDA1 in THF, and by approximately 80% in aggregates prepared from a 0.5 mM solution of TzDA1 in THF when the suspensions are polymerized by 254 nm irradiation for 110 s.

Reprecipitation MeCN -water

TzDA1 reprecipitation was tested as well starting from solutions in MeCN. A 0.25 mM TzDA1 solution in MeCN is mixed with water (proportion of water:MeCN is 8:2 as shown in Table 4.1) to give aggregates in solution that polymerize with UV irradiation at 254 nm as proven by the absorption spectra shown in Figure 4.5a and fluorescence spectra shown in Figure 4.5b. The absorption band at 615 nm corresponding to blue-poly-TzDA1 increases with UV irradiation (black to grey to blue line in Figure 4.5a) and the tetrazine fluorescence intensity (λ em = 560 nm) decreases consistently when the sample is polymerized. Polymerization kinetics are not shown since polymerization causes again a baseline shift for the first irradiations. However, the final polymerized suspension is tested to temperature, by placing the cuvette on a heating plate for 3 min at 100 • C. The blue solution is converted to a bright red solution, as confirmed by the absorption spectra before (blue line) and after (red line) heating in Figure 4.5c. The fluorescence intensity, as discussed previously, is quenched when polymerization is performed (black to blue line in Figure 4.5d). When the blue-poly-TzDA1 is converted to red-poly-TzDA1 by heating, the fluorescence signal grows back, as shown by the red line in Figure 4.5d. It is worth noticing that the spectral shape of tetrazine fluorescence is affected by heating, resulting in a narrower band at 560 nm, plus a large shoulder at higher wavelength. This is probably due to a rearrangement of the polymer chains coming from blueto-red conversion and a contribution of the intrinsic fluorescence of red-poly-TzDA1 in the same range. Time-resolved fluorescence spectroscopy measurement would be needed to conclude on the nature of this spectral shoulder. A shoulder around 650 nm is present along with the band at 560 nm proper of tetrazine emission. The nature of this band is still unclear, but it is probably coming from tetrazine as well, since blue-poly-TzDA1 fluorescence is too low to be detected. If it was generating from red-poly-TzDA1 fluorescence, the band would increase along polymerization, while it would be constant if it was coming from an impurity in the sample. Using a more concentrated initial solution of TzDA1 in MeCN (0.5 mM instead of 0.25 mM), leads to a more scattering solution, as can be seen by the absorption spectra of the two concentration in Figure 4.5a and 4.6a. This could represent the formation of bigger objects in suspension or simply more objects of the same size. However, it is certain that in both concentrations, the first irradiation has an influence on the size or optical response of the objects. In fact, an absorbance baseline shift is recorded for both samples after the first UV irradiation. It is likely that polymerization, especially at the beginning when the longer chains are formed, 22 induce a change in the size, shape or index of refraction of the particles.

Both THF and MeCN TzDA1 initial solutions give aggregates in suspension for a range of different initial concentrations. These aggregates all polymerize efficiently when irradiated at 254 nm. The blue-poly-TzDA1 absorption band is centred at 615 nm in both cases and in both cases some red-poly-TzDA1 formation is observed (λ abs = 540 nm). These absorption bands are much more well distinguishable when compared to the solid state TzDA1 thin films (Figure 2.5b in Section 2.3.2). Plus, TzDA1 thin films form a blue phase that absorbs at 600 nm (defined as "purple" phase in the literature), while the aggregates polymerize in a more classical blue phase, absorbing at 615 nm. Plus, in all these systems, tetrazine showed efficient fluorescence quenching under UV irradiation at 254, which is qualitatively reversible by heating. Sonication in water A 0.5 mM solution of TzDA1 aggregates in water is prepared by sonication at 60 • C for 15 min. Absorption spectra of the resulting solution exposed to irradiation at 254 nm for photopolymerization are shown in Figure 4.7a. The monomeric suspension spectrum (black line) shows the characteristic features of tetrazine (two absorption bands at 350 and 520 nm). When the sample is irradiated in the UV Chapter 4. Polydiacetylene microcrystals (grey lines), the absorption band between 480 and 560 nm increases progressively, to give the blue spectrum at the end of the photopolymerization process (2 J•cm -2 ). No absorption band corresponding to blue-poly-TzDA1 is observed. To better visualize the evolution of the absorption spectral shape along polymerization, the monomeric suspension spectrum has been subtracted to the final polymerized suspension absorption spectrum in Figure 4.7b (red line). This way tetrazine's contribution to the visible absorption is suppressed, and only the polymer absorption is visible. It is clear that in this specific aggregation state, at these preparation conditions, TzDA1 polymerizes directly to the red phase with the characteristic spectrum shown by the red line in Figure 4.7b, with a maximum absorption band at 540 nm and a vibronic peak at 500 nm. Even though blue-poly-TzDA1 is not formed, tetrazine fluorescence intensity at 560 nm progressively decreases as shown in Figure 4.7c. The spectral overlap between tetrazine fluorescence and red-poly-TzDA1 is lower compared to blue-poly-TzDA1 but still somewhat present, causing a similar quenching mechanism. TzDA1 self-assembles efficiently when reprecipitated from THF and MeCN solu-tions in water for a range of different initial concentrations. Irradiation in the UV was effective in forming a blue polymer phase absorbing at 615 nm, which is converted to the red counterpart by heat. Sonication in water at 60 • C for 15 min successfully produced aggregates in solution as can be deduced from the obtained translucent solution and the scattering contribution in the absorption spectra (Figure 4.19a). However, UV irradiation of these samples produce exclusively the red phase of the polymer. Therefore, the so-prepared suspensions have no use in the context of sensing, since the red form is not responsive to stimuli and it is back-converted to its blue counterpart only in very specific systems. However, it is interesting to remark that indeed the preparation method of TzDA1 suspensions has an influence on the geometrical disposition of the self-assembled DAs.

TzDA2

Table 4.2 sums up the tested TzDA2 suspensions. For reprecipitation method, solvent and initial TzDA2 concentrations are listed as well as the proportions between DA solution and water. For sonication in water, final DA concentration in the solution, temperature and time of sonication are listed.

Reprecipitation THF -water

TzDA2 aggregates in suspension were prepared by mixing a TzDA2 solution in THF at variable concentrations with distilled water, at 8:2 water:THF solution proportions. The absorbance of the resulting suspensions of three different concen-Chapter 4. Polydiacetylene microcrystals trations were acquired and their responses to UV irradiation at 254 nm for polymerization were tested. Figure 4.8a shows the absorption spectrum of a 0.05 mM TzDA2 suspension (black line) and its corresponding fluorescence (yellow line). The characteristic absorption bands of tetrazine can be identified: in the UV at 350 nm and in the visible at 520 nm. The fluorescence band is centred at 580 nm, which is shifted bathochromically by 20 nm compared to the wavelength of emission in the solid state (560 nm). The obtained spectra are noisy and the fluorescence signal is low. Therefore, no UV irradiation testing was performed on this sample. When starting from a 0.1 or 0.5 mM TzDA2 THF solution, the obtained spectra are less noisy (Figure 4.8b and 4.8c respectively) and the fluorescence signal is intense. The spectroscopic features of tetrazine are the same described for the 0.05 mM TzDA2 suspension. UV irradiation at 254 nm is sent on the cuvette, and UV-vis and fluorescence spectra are recorded. In both concentrations, no clear sign of polymerization is detected: neither an increase of the blue-poly-TzDA2 absorption band (around 600 nm) nor a significant decrease in tetrazine fluorescence. The same experimental conditions of preparation were used for TzDA1, which self-assembled properly to give UV-polymerized aggregates in suspension. If no blue or red-poly-TzDA2 absorption band is formed upon irradiation, it means that most probably TzDA2 does not selfassemble properly to polymerize, which could be due to the polarity of the solvent. It is concluded that THF is not the suitable solvent to produce TzDA2 aggregates in suspension.

Reprecipitation MeCN -water

TzDA2 is reprecipitated from a 0.1 mM MeCN solution in water. In Figure 4.9 absorption (4.9a) and fluorescence (4.9b) of the suspension progressively irradiated in the UV at 254 nm are shown. Black lines correspond to the monomer suspension. The spectroscopic features of tetrazine are comparable to the solid state case: two absorption bands at 350 and 530 nm and emission centred at 560 nm. The first irradiations (grey lines) cause an increase in the blue-poly-TzDA2 absorption band at 605 nm (black-grey arrow) and a decrease of the tetrazine emission intensity, progressively quenched by the increasing quantity of blue-poly-TzDA2. Starting from 350 s of UV irradiation, the absorbance at 605 nm decreases (orange-blue arrow) while a new band at 540 nm and a small vibronic band at 480 nm appears. When comparing to the photopolymerization in the solid state (see Section 2.3.2), such an absorbance shift could correspond to blue-to-red polymer conversion. When reaching 1440 s of UV irradiation, the band at 605 nm is drastically reduced while the band at 540 nm is still visible. This conversion is represented by an increase in the emission intensity (Figure 4.9b, blue line), which can be explained by the reduced energy transfer efficiency of the red-poly-TzDA2 compared to the blue one. Compared to the solid state thin films, where the blue-poly-absorption band progressively shifts towards lower Chapter 4. Polydiacetylene microcrystals wavelengths (Figure 2.5c in Section 2.3.2), the conversion to the red phase in the aggregates prepared by reprecipitation of a 0.1 mM TzDA2 MeCN solution in water is less gradual. Such a conversion for long irradiation times is common in systems that have a fast polymerization kinetics. 23 We could then hypothesize that the polymerization rate in aggregates is faster than in thin films, but more accurate kinetics studies are needed to make such a conclusion. Figure 4.10 shows the spectroscopic study of photopolymerization of a TzDA2 suspension prepared starting from a 0.25 mM solution of TzDA2 in MeCN reprecipitated in water. Irradiation at 254 nm of the suspension produces an absorption band at 605 nm corresponding to blue-poly-TzDA2 (Figure 4.10a). Subtracting the monomer spectrum to the irradiated ones allows to see more clearly the absorption bands corresponding to the PDA (Figure 4.5b). This polymer form has an absorption band centred at 605 nm, which resembles the one that is formed in TzDA2 thin films (λ abs = 600 nm, see Section 2.3.2). It is defined in the literature as "purple" phase rather than blue. 24 A pure blue form has a strong absorption around 640 nm, while a purple form absorbs at lower wavelengths. To avoid confusion, we will keep using the term blue-poly-TzDA2 to designate any form that is different from redpoly-TzDA2 (λ abs = 540 nm). The band at 605 nm increases progressively along the photopolymerization process and, as depicted in Figure 4.10d, the increase is very steep at the beginning (0 -1000 mJ•cm -2 ) until the maximum polymer quantity is obtained around 1500 mJ•cm -2 and the curve reaches a plateau. Blue-poly-TzDA2 absorbance seems to decrease in the last irradiations, at irradiation doses higher than 4000 mJ•cm -2 . The maximum of absorbance is at first centred at 605 nm and shifts progressively towards lower wavelengths until 585 nm (25 nm shift) in the last steps of the photopolymerization (Figure 4.10a and b). It is impossible to determine by steady-state measurements if from this point different forms of poly-TzDA2 coexist or if the initial blue-poly-TzDA2 is converted to another phase. Consistently with the energy transfer mechanism that was discussed for the solid state case, the production of blue-poly-TzDA2 in suspension causes a quenching of tetrazine's fluorescence intensity, as shown in Figure 4.10c. The decrease reflects the blue polymer formation, being very fast between 0 and 1000 mJ•cm -2 and reaching a plateau around 1500 mJ•cm -2 (Figure 4.10d). It is also worth noticing that the fluorescence intensity at the final step of the polymerization has been quenched by only 70% of the initial value, against 97% in TzDA2 thin films. The residual fluorescence signal at the end of the photopolymerization process may be due to the presence of nonaggregated TzDA2 molecules that do not take part in the polymerization process, therefore their emissive unit is not quenched by the formation of blue-poly-TzDA2. A suspension prepared from a 0.5 mM TzDA2 solution in MeCN (H 2 O:MeCN 8:2) reprecipitated in water is subjected to consecutive irradiations at 254 nm and for each irradiation an absorption and fluorescence spectrum are recorded (shown in Figure 4.11). The same blue-poly-TzDA2 absorption band at 605 nm that is formed for lower concentrations is observed when the suspension is irradiated in the UV, as can be seen in Figure 4.11a. To visualize better this increasing band, the monomer spectrum is subtracted to the consecutive spectra, as shown in Figure 4.11b. Along the polymerization process, the band shifts towards the shorter wavelengths, as in the 0.25 mM suspension and the thin films. The blue-poly-TZDA2 band is first centred at 605 nm and is shifted to 590 nm. Compared to the 0.25 mM suspension, the total band shift is smaller and equal to 15 nm, which is not surprising knowing that the irradiation dose for the 0.5 mM suspension was lower (≈ 1600 mJ•cm -2 versus 6000 mJ•cm -2 ). If the aggregates formed from a 0.25 mM solution show no sign of degradation up to 6000 mJ•cm -2 , the tetrazine in 0.5 mM suspensions seems to undergo photobleaching, represented by a decrease in the band at 350 nm in Figure 4.11a. Blue-poly-TzDA2 increases progressively with irradiation and reaches a plateau around 800 mJ•cm -2 (Figure 4.11d), similarly to the thin films case Chapter 4. Polydiacetylene microcrystals (around 1000 mJ•cm -2 ), while it was almost the double for 0.25 mM suspensions (1600 mJ•cm -2 ). Concerning tetrazine fluorescence evolution with polymerization, the intensity at 565 nm is quenched progressively as expected (Figure 4.11c). The total fluorescence quenching is equal to 95% at the end of the photopolymerization process (Figure 4.11d), and it is difficult to say how much tetrazine photobleaching takes part in this reduced fluorescence signal. In conclusion, the polymerization process approaches the solid state case in terms of blue polymer formation (similar irradiation energy to reach blue-poly-TzDA2 maximal quantity) and tetrazine quenching (95% in suspension versus 94% in solid films). When substituting THF with a more polar solvent like MeCN, UV irradiation at 254 nm produces polymerized aggregates in suspensions. The polarity of the solvent has thus an influence on how the DAs self-assemble in the aggregate, giving non-polymerizable aggregates in THF and polymerizable aggregates in MeCN.

TzDA2 suspensions are prepared by reprecipitation in water from a 1 mM solution in MeCN, in H 2 O:MeCN 9:1 and 8:2 proportions. The absorption spectra of the two suspensions are shown in Figure 4.12a and 4.12b respectively. Both preparations give scattering suspensions with the characteristic absorption peaks of tetrazine at 530 and 350 nm. However, 9:1 reprecipitation gave aggregates that do not polymerize under UV irradiation, as shown in Figure 4.12a. On the contrary, 8:2 proportion gave aggregates that form blue-poly-TzDA2, absorbing at 605 nm, as shown in Figure 4.12b (yellow curve). Because of the high concentration, scattering deeply affects the absorption spectra, therefore no further testing was performed on such samples. As a conclusion, aggregates in suspension reprecipitated from TzDA2 solutions in MeCN were studied for different initial concentrations in the same H 2 O:MeCN proportion 8:2. The initial concentration has an interesting influence on the polymerization properties of the aggregates. When preparing them from 0.1 mM solution, the UV irradiation produces initially an absorption band at 605 nm that suddenly decreases at long irradiation times to leave space to the red-poly-TzDA2 absorption band at 540 nm. The same absorption band at 605 is recorded for suspensions prepared from more concentrated solutions of TzDA2 (0.25 and 0.5 mM). With UV irradiation, the band increases and progressively shifts towards shorter wavelengths. However, no clear blue-to-red conversion is achieved by irradiation only despite the long irradiation energies supplied to the sample (≈ 1600 mJ•cm -2 and 6000 mJ•cm -2 respectively). It is interesting to notice that a similar behaviour is observed in TzDA2 thin film polymerization, where the band at 600 nm shifts progressively. Also suspensions prepared from 1 mM TzDA2 solutions in 8:2 H 2 O:MeCN proportions gave rise to an absorption band at 605 nm. On the contrary, the same solution reprecipitated in 9:1 H 2 O:MeCN proportions did not give any sign of polymer formation upon UV irradiation. When looking at tetrazine fluorescence of the three samples (1 mM excluded), the intensity is decreased by the formation of poly-TzDA2. It is found that aggregates prepared from a 0.5 mM solution approach the quenching efficiency achieved in the solid state (95% in suspension versus 94% in solid films), which is 20% higher for aggregates prepared from a 0.25 mM TzDA2 solution. Sonication in water A 0.1 mM suspension of aggregated TzDA2 monomers in water is obtained by sonication at 40 • C for 30 min. The obtained suspension, after resting at 4 • C for 12 h, is translucent, suggesting the successful formation of aggregates. The absorption spectrum of the resulting solution is shown in Figure 4.13a (black line). The absorption bands at 530 and another at 350 nm correspond to the characteristic absorption bands of tetrazine fluorophore. Even though the spectra are highly affected by light scattering phenomena by the particles in suspension, the increase of the absorption band of blue-poly-TzDA2 at 605 nm is visible for consecutive irradiations at 254 nm (grey lines in Figure 4.13a). At the end of the polymerization process (0.7 J•cm -2 ), particles of blue-poly-TzDA2 are formed (blue spectrum in Figure 4.13a). Because of the impact of light scattering that causes a baseline shift, it is impossible to track the polymerization kinetics and to determine if the tetrazine has bleached during UV exposure (usually visible at the 350 nm absorption band). Probably, polymerization causes a modification in the arrangement of the self-assembled monomers, which result in a modification of shape, size or index of refraction that influence the scattering properties of the aggregates in suspension. For the same sonication parameters, a 0.5 mM suspension of TzDA2 was prepared. Figure 4.14 shows absorption (4.14a) and fluorescence (4.14b) spectra when the suspension is exposed to UV irradiation at 254 nm. An important baseline shift occurs especially between the monomeric phase and the polymerized sample at the very beginning of the polymerization process, which results in the gap between the black spectrum (monomer) and the first grey spectrum (polymer) in Figure 4.14a.

Blue-poly-TzDA2 absorbs at 605 nm, and the absorption band increases progressively with UV irradiation and progressively shifts towards lower wavelengths for higher polymer content. The same plastic deformations that led to a purple phase in solid state are thus present also in solid aggregates in solution. Along with the blue phase, also red-poly-TzDA2 is formed, especially at the end of the polymerization process (0.6 J•cm -2 ) where the absorption band around 540 nm is clearly visible. Fluorescence spectra shown in Figure 4.14b prove that tetrazine emission is quenched by around 80% of its initial intensity, by formation of blue-poly-TzDA2. A 0.5 mM suspension obtained by sonication at 70 • C for 15 min is exposed to subsequent UV irradiation at 254 nm. The absorption spectra after each irradiation are shown in Figure 4.15a. In Figure 4.15b, the monomer spectrum has been subtracted to cancel out the contribution of tetrazine thus to highlight the progressive increase of blue-poly-TzDA2 absorption. The absorbance maximum starts at 605 nm increases along the irradiation and shifts progressively towards lower wavelengths, as already witnessed in the previous experiments. Therefore, sonication time can be reduced to 15 min if the temperature is increased to 70 • C, which successfully produced aggregates in water. When the solution is heated on a heating plate, the suspension turns from purple-blue (blue spectrum in Figure 4.15c) to deep red (red spectrum in Figure 4.15c), demonstrating that also PDA aggregates in suspension respond to external stimuli like thin films. Tetrazine emissive state switches from an ON state in the monomeric form (black line), to an OFF state in the polymerized sample (blue line) and back to the ON state when blue-poly-TzDA2 is converted by heating to red-poly-TzDA2 (red line), as shown in To summarize, TzDA2 self-assembles efficiently only when reprecipitated from MeCN solutions in water for a range of different initial concentrations. Irradiation in the UV was effective in forming a blue polymer phase absorbing at 605 nm, very similar to the blue polymer absorption recorded in solid thin films. When starting from low concentrations, long UV irradiations cause a blue-to-red conversion, which is not observed in more concentrated suspensions. However, the absorption band at 605 nm in the 0.25 and 0.5 mM suspensions shifts towards shorter wavelengths, as it was observed in thin films. The initial concentration is thus supposed to have an influence on the arrangement of self-assembled DAs, that behave differently when exposed to UV light. When THF is used as starting solution for reprecipitation of TzDA2 in water, aggregates are formed but do not polymerize under irradiation at of the DAs in the aggregates that for TzDA2 do not self-assemble properly to polymerize. It is worth noting that the same experimental preparation conditions are used for TzDA1, which on the contrary could be polymerized efficiently. Sonication in water at 40 • C for 30 min and at 70 • C for 15 min successfully produced aggregates in solution as can be deduced from the obtained translucent solution and the scattering contribution in the absorption spectra (Figure 4.13a, 4.14a and 4.15a). Contrarily to TzDA1 that formed only the red phase, irradiation in UV of these samples produce blue-poly-TzDA2 with the usual absorption band centred at 605 nm (like in thin films and reprecipitated aggregates). Thermal stimulus was tested on a polymerized 0.5 mM suspension of TzDA2 prepared by sonication at 70 • C for 15 min. The blue-to-red conversion was achieved and the tetrazine fluorescence was also restored.

Isolated deposited crystals

Solution-based PDAs are very useful for a number of applications as sensors for different external stimuli, but their applicability as friction forces sensors is hardly conceivable in liquid state. There has been few attempts of applying mechanical stress to solution based sensors, 25,26 but solid samples are indeed more adapted to this purpose, and isolated single crystals represent good models to study and quantify the mechanofluorochromic properties. The suspensions are prepared by the methods described above and the solution is then deposited on a clean coverslip and dried under vacuum. Indeed the preparation method and the chosen parameters influence the morphology and shape of the resulting aggregates, more or less adapted for sensing AFM-applied mechanical forces.

Morphology study

As reviewed in Chapter 1, the literature has been flourishing with a vast amount of reports on PDA-based vesicles for sensing. [27][28][29] However, the structure and morphology of the vesicles is rarely studied. Optical microscopy along with scanning electron microscopy (SEM) images of pure PDA vesicles and mixed PDA-phospholypids vesicles were reported by Pevzner et al., showing the formation of micrometer sized objects, where mixed PDA-phospholypids vesicles clearly assemble in double-layered aggregates (vesicles). Pure PDA vesicles are characterized by a sheet-like arrangement on the surface of the object. 30 We prepared aggregates in suspension by the methods presented above (reprecipitation, sonication and recrystallization). After deposition and drying, the obtained Photopolymerization is performed in situ irradiating for 30 min at 254 nm with an irradiation power of approximately 1.4•10 -4 W•cm -2 (the irradiation area is not easily accessible as for thin films, thus an area of 2 mm 2 is assumed by comparison with thin films experiments). In conclusion, if for TzDA1 the H 2 O:MeCN proportions in the preparation of the suspension had a major role in determining the size and shape of the crystals, H 2 O:MeCN 9:1 and 8:2 for TzDA2 (same initial concentration of 0.5 mM) gave crystals with the same morphology. Both proportions produce needle-like crystals that can extend up to few µm in length. Fluorescence and polymerization properties of these aggregates were not probed, thus additional experiments to characterize TzDA2 reprecipitated crystals are needed.

Single crystals obtained by sonication

TzDA1

A 0.5 mM suspension of TzDA1 in water is obtained by sonication for 15 min at 60 • C. The suspension is then deposited on clean glass slides and dried under vacuum. Isolated objects of variable dimensions are obtained as it can be seen in the optical transmission images shown in Figure 4.19a and d. Aggregates of variable sizes are obtained. Mainly two populations of "small" and "large" aggregates are obtained. Small aggregates are comparable to crystals obtained by 8:2 reprecipitation and large ones are comparable to 5:5 reprecipitation. For instance, the crystal in Figure 4.7a and b is 4x3 µm, plus the shape of the crystal has less defined edges, but a rather flat surface, on top of which other smaller objects are superimposed as it can be seen in the line profile in Figure 4.19b. In the same conditions of fluorescence excitation (excitation power and 1 s integration time), aggregates prepared by sonication are more intensely fluorescent than aggregates prepared by reprecipitation, as it can be noticed in Similarly to reprecipitation, sonicated TzDA2 suspensions gave long fiber-like crystals that are fluorescent. Depending on the zone of the sample and on the concentration of the aggregates in that specific zone, the fluorescence intensities can differ by 10-fold: maximum intensity recorded in Figure 4.20b is 1500 counts, while in Figure 4.20e is 15000 counts. At present, polymerization has not been studied on this compound and further investigations will be carried out in the future. From the morphology studies that were performed on crystals prepared by reprecipitation or sonication of two PDA derivatives, TzDA1 and TzDA2, we can conclude that only for the former the preparation conditions had a clear influence on the shape and size of the resulting crystals. Small rod-like or large square plate-like objects are obtained with reprecipitation in different H 2 O:MeCN proportions. Sonication gave instead large crystals with less defined edges. On the contrary, both preparation methods for TzDA2 gave the same needle-like shape. Polymerization with UV light at 254 nm was efficient for every shape and size of TzDA1 crystals, which is an urgent future investigation that needs to be done on TzDA2. Finally, aggregates in suspension were prepared by reprecipitation, sonication and recrystallization. The morphological study here presented shows that the preparation method plays a crucial role in determining size and shape of the resulting Chapter 4. Polydiacetylene microcrystals objects in TzDA1. Several hundreds of nm long rod-like crystals are obtained by 8:2 reprecipitation, while 5:5 reprecipitation gives large crystals that extend up to 20 µm in length. Sonication gives crystals of comparable size to 5:5 reprecipitation, but with less defined edges. At last, recrystallization can give either plate-like crystals or needle-like crystals depending on the temperature at which the slow diffusion is performed. All these aggregates show different fluorescence intensities and tetrazine quenching efficiencies upon UV-polymerization. Analysing qualitatively the fluorescence images, recrystallization and sonication gave the most intensely fluorescent objects, and recrystallization gave crystals that quench tetrazine the most efficiently. In the near future, comparative and quantitative fluorescence examinations will be performed, to conclude more rigorously the discussion.

On the other hand, TzDA2 crystals are not affected the same way by the preparation method. Needle-like crystals are obtained with every protocol. The study on this compound is still ongoing. Fluorescence and polymerization studies need to be performed to allow a comparison with TzDA1.

Application of friction forces and pressures on isolated single crystals of TzDA1

In the previous section, morphology and ability to polymerize of isolated aggregates deposited on glass slides were discussed. Different morphologies and especially sizes of crystals can be obtained according to the preparation method and chosen parameters. The morphological characteristics are crucial in determining if the friction force application experiments by AFM are feasible. In this section, single crystals prepared with the methods described above are subjected to friction forces and pressures by AFM, and their fluorescence response is analysed by fluorescence microscopy. Regular silicon nitride tips are used to apply frictions, while spherical tips with radius 500 nm are used to apply pressures. A morphology change is registered also on a second tested aggregate (aggregate 2), when a mechanical stimulation of 100 nN is performed. Despite the higher normal applied force, the particle is not completely disrupted, but some material is indeed displaced on the sides of the rod as it can be seen in AFM topography images in The same analysis is performed on another sample prepared by the same method, using fluorescence spectra instead of images. Four aggregates are analysed and the corresponding fluorescence spectra are recorded on a 10x10 pixels area centred on the aggregate (Figure 4.24). Spectra are acquired before (black lines) and after (green lines) polymerization and after mechanical stimulation (red lines) and are shown in Fluorescence is excited at 488 nm. 8:2 reprecipitation in water from a 0.5 mM TzDA1 solution in MeCN gives small rod-like objects that polymerize by exposure to UV irradiation (as proven by fluorescence images and spectra), but do not show any evident fluorescence activation upon mechanical friction applied by AFM. The object size surely limits the accuracy both in the detection (difficult to visualize fluorescence changes in images and difficult to set the correct ROI for spectra acquisition) and in the force application by AFM. Larger crystals may be more well-adapted for force application experiments. No evident change in fluorescence intensity is registered. On the contrary, when a shear force (friction) at the same normal force is applied on a zone of the crystal, a fluorescence recovery in this same area is obtained, as shown in Figure 4.26d. The maximum fluorescence signal intensity recorded in the stimulated area is 600 counts, meaning an activation by 2-fold the maximum value of the polymerized crystal. Figure 4.26e shows the morphology AFM image after mechanical stimulation. The global rectangular morphology is retained even after stimulation and the crystal is not disrupted by the AFM tip. However, some PDA material has clearly been displaced in the stimulated area, as is highlighted by the 3D view in the inset of Figure 4.26e. It is concluded that a localized stimulation produces a localized fluorescence recovery signal, from the same area that has been morphologically perturbed. Reprecipitation in water of a 0.5 mM TzDA1 solution in MeCN, with 5:5 H 2 O:MeCN proportions, gives larger objects in the micrometer range with rather flat surfaces that are much more well adapted to be mechanically sheared by an AFM tip than the small objects obtained by the same reprecipitation method in 8:2 H 2 O:MeCN proportions. It is possible to select a specific area of these crystals and launch a scan in contact mode at a certain vertical force and the fluorescence modifications are easily observed by fluorescence microscopy thanks to their size. As a general trend, when the morphology of the aggregate is affected by the stimulation, a fluorescence recovery is recorded. Plus, localized fluorescence activation is possible when only a section of the surface is stimulated by friction. Pressure forces applied by AFM were also tested, using a sharp (R = 10 nm) silicon tip. The tip is placed on the crystal surface at a certain vertical force for a few seconds, then retracted and a fluorescence image is acquired. No visible change in fluorescence intensity is observed. Such a mechanical stimulation does not transfer enough energy to the system to cause a morphology change i.e. the blue-to-red PDA transition. One of the reasons may be the size and shape of the indenting object (AFM tip), which is very sharp and small compared to the imaged surface. Larger tips with a different geometry may be more effective for this purpose. Objects of variable size are obtained, and all of them are fluorescent when excited at 488 nm. Compared to the morphology studies shown in Section 4.3.1.2, the chosen zone is populated by smaller objects. Plus, they are gobally less fluorescent considering a maximum fluorescence value of 2000 counts (compared to 20000 counts), which is logical for smaller objects. Photopolymerization is performed in situ by irradiating at 254 nm for 30 min (P = 1.4•10 -4 W•cm -2 ). The fluorescence is quenched, as shown in Figure 4.27c, with a quenching efficiency estimated to 2.5-fold the initial intensity value (I F (mono) = 1900 counts, I F (poly) = 800 counts). AFM topography images of the two chosen aggregates are shown in Figure 4.27e and f, showing that they are constituted by several particles sticking together. Aggregate 1 is stimulated by AFM with a normal force of 100 nN, while particle 2 with a normal force of 200 nN. Figure 4.27d shows that the fluorescence is evidently not recovered, but the emitting area has been modified, as it was the case for small aggregates formed by reprecipitation in 8:2 proportions of H 2 O:MeCN. The stimulation, in fact, displaces the single particles, without modifying their morphology, as shown in Figure 4.27g and h. 500 nm long rod-like aggregates prepared by sonication in water were subjected to friction forces by means of an AFM tip. They are all fluorescent and they all quench tetrazine when UV-polymerized. However, because of the small size of the objects, as already discussed for 8:2 reprecipitation, localizing the mechanical stimulation exactly on the surface of these objects is not trivial. In most experiments, instead of a shear force that perturbs the morphology of the aggregate, a displacement of the single particles composing the aggregates occurs. Displacement alone is not effective in perturbing a morphology change, thus no fluorescence activation is recorded.

Reprecipitation

Transmission and fluorescence images of a new zone of the sample are shown in Figure 4.28a and b respectively. The aggregate highlighted by the yellow square is selected to perform mechanical pressures on top of its surface. Polymerization is performed in situ by irradiating at 254 nm for 30 min at a power of 1.4•10 -4 W•cm -2 . Fluorescence is quenched by approximately 2-fold the initial value (I F (mono) = 750 counts, I F (poly) = 325 counts, I F (background) = 230 counts) as it can be seen in Figure 4.28d. AFM morphology of the chosen aggregate is shown in Figure 4.28c. The aggregate is composed of different particles sticking together, to give a 400 nm long, 200 nm large and 300 nm high object. Since sharp 10 nm tips did not cause visible morphological modifications to the aggregate, an AFM tip with spherical geometry and radius of 500 nm is chosen to apply pressure forces on this aggregate. The tip is placed on top of the aggregate surface and the setpoint is set to 200 nN. The tip is left in place for a few seconds, then retracted. A consecutive pressure at 500 nN is applied for a few seconds, then the tip is retracted again. Fluorescence images are acquired after each pressure application and are shown in Figure 4.28e (200 nN) and 4.28f (500 nN). No evident fluorescence restoration is observed after the stimulation, as can be seen by comparing the fluorescence images before (Figure 4.28d) and after the stimulations (Figure 4.28e and f). In fact, when looking at the topography image after pressure (Figure 4.28g), the crystal's morphology has not been visibly affected. Such a compression probably does not transfer enough energy to the system to promote the blue-to-red PDA conversion to re-activate tetrazine fluorescence. Another possible explanation could be that, as it was happening in suspensions (Section 4.2), the aggregates polymerize directly to the red phase, making any further transition and fluorescence activation impossible. Without absorption spectra of the polymerize objects, it is impossible to confirm this hypothesis, therefore further investigations are required. All in all, aggregates prepared by sonication of TzDA1 in water, deposition and drying under vacuum are usually constituted by agglomerates of small particles sticking together. The dimensions of the aggregates rarely exceeds the µm size, which is pretty limiting for shear force applications. In fact, as discussed for the small objects obtained by 8:2 reprecipitation of TzDA1, it is difficult to place the tip exactly on the surface of the objects, plus fluorescence changes in wide-field images as the ones shown are hard to be detected. For this reasons, pressure forces have also been tested. A tip with spherical geometry and a radius of 500 nm is chosen, since previously sharp tips did not give satisfactory results. However, even pressures of 500 nN do not cause any evident fluorescence activation. Fluorescence is activated uniquely in the areas that have been rastered by the tip, with intensities up to 800 counts. No clear effect of the direction of scan can be noticed. Morphology AFM images were not acquired on this samples, but from the fluorescence images it can be deduced that some material on the surface of the crystal has been displaced on the sides of the selected area. Large single crystals, prepared by 5:5 reprecipitation or by 8:2 recrystallization methods, respond locally to fluorescence in correspondence with the area that has been mechanically stimulated (Figure 4.26d and 4.29b). Therefore, nanolithography on single crystal surface could be an interesting field of application of such materials, which could reveal a specific morphological pattern by fluorescence. We chose an isolated crystal, whose transmission optical image is shown in Figure 4.30a. The crystal is approximately 6x5 µm wide and 800 nm in height as can be seen in the AFM topography image shown in Figure 4.30b, which leaves enough space for force manipulation. UV polymerization is performed in situ by irradiating the sample at 254 nm for 30 min (P = 1.3•10 -4 W•cm -2 ). The fluorescence image of the UVpolymerized crystal is shown in Figure 4.30c, where the maximum recorded intensity is around 700 counts. The tip is placed in the bottom left corner of the crystal and the AFM is programmed to perform a scan in contact mode at a nominal force of The acronym PPSM is well readable in the morphology images, while the flu-Chapter 4. Polydiacetylene microcrystals orescence activation does not seem to be constant in each point of the engraved letters. Interestingly, a bias towards the direction of scan can be noticed. Friction forces applied along the vertical axis of the image seem to be more efficient in recovering tetrazine quenched fluorescence than forces applied horizontally (visible in the letter P ). Probably, the disposition of the polymerized PDA chains in the crystal lattice influence their responsiveness to external forces, as it was witnessed already in PCDA trilayers by Carpick et al. 31 Further testing is necessary to check if the same behaviour is found in single crystals.

So far, different preparation methods have been utilized to assemble TzDA1 derivative in isolated objects. Mechanical forces at the nanoscale like friction and pressure have been tested on polymerized aggregates and crystals, and their fluorescence response is probed before and after stimulation. Analysis of AFM morphology images allows to establish a general trend according to which a fluorescence activation is given by a morphology modification on the surface of the crystal. Small objects, like the ones prepared by 8:2 reprecipitation or sonication, present several limitations for this kind of experiments due to their size. Shear forces are difficult to apply exactly on the surface of the aggregates and eventual fluorescence modifications are difficult to detect by wide-field fluorescence images or fluorescence spectra. Also pressures with large squared tips did not induce considerable fluorescence activation. On the other hand, large micrometer sized objects obtained by 5:5 reprecipitation or recrystallization, are well-adapted to be sheared or pressed by an AFM tip. The sizes of these objects allow the posibility of performing nanolithography on the crystal surfaces, by shearing with the AFM tip following a specific path. Friction forces usually induce morphological changes (for 50 and 100 nN vertical force) that result in tetrazine fluorescence re-activation. On the contrary, pressure forces with sharp silicon tips did not cause any fluorescence activation.

These experiments stand as a proof of concept that mechanical force applied on isolated polymerized TzDA1 single crystals leads to a fluorescence restoration. In the next Section, quantification of the restored signal is assessed for a wider range of applied nominal forces.

Quantification of fluorescence activation by friction forces on isolated single crystals of TzDA1

The experiments presented in the previous Section showed how the preparation method has an influence on the size, shape and morphology of the aggregates deposited on a solid substrate. In addition, friction forces and pressures have been applied on polymerized isolated objects and the fluorescence response upon stimulation was probed. Indeed, when the stimulation applies enough energy to system, it promotes the blue-to-red conversion that is responsible for the fluorescence activation. In general, this process corresponds to a visible morphology modification on the crystal surface. Micrometric single crystals represent a valid alternative to vacuum evaporated thin films that were largely discussed in Chapter 3. The flat and rigid crystalline surface is more adapted than films for mechanical friction forces. As shown in the previous section, nano-shearing causes less material displacement so a smaller contribution to tip-pollution effects is also expected.

In this section, we will dig into the quantification of the mechanically-activated fluorescent single crystals.

In order to quantify the fluorescence signal that is restored when mechanical friction is applied on isolated single crystals, a similar procedure that was used for thin films, explained in Section 3.3, was developed. First of all, delta fluorescence images are obtained by subtraction of the fluorescence image after and before mechanical stress. This way, the fluorescence signal coming from the stimulation is isolated, and the background signal is levelled to zero. Since crystals orientate in random directions and have different sizes, squared integration of fluorescence signal may be complicated. For this reason, a threshold value of intensity is set on the delta image, and all the pixels with intensity lower than that value are excluded. An average value is then calculated with the remaining pixels to assign a numerical value to the fluorescence restoration. The procedure is summed up in areas of their surface, as can be see in Figure 4.32b. The area of stimulation is kept constant at 1.5x1.5 µm 2 and the acquisition parameters of fluorescence images are kept the same as well (excitation power and 1 s integration time). It has to be noticed that crystals have not exactly the same size, their surface morphology may also be different and not completely flat. We compare results making the assumption that the stimulated areas are morphologically equivalent. The fluorescence images are treated with the procedure explained above, which gives a mean value of fluorescence signal activation. The average is calculated on all the pixels with signal higher than a chosen threshold. The number of pixels may differ from one crystal to another or from one zone of the crystal surface to another. The average fluorescence values are plotted against the corresponding vertical applied force. The resulting correlation curve is shown in Figure 4.33, where the black dots correspond to the mean value of fluorescence activation resulting from several experiments on different crystals and the error bars are the standard deviation. The tetrazine fluorescence increases along with the applied normal force, but compared to thin films, this trend is less obvious. Each consecutive fluorescence activation for a higher force always falls in the error bars of the adjacent measurements, which could indicate a more or less constant fluorescence recovery. These results reveal some very interesting and original effects for both the study on isolated PDA microcrystals and the applicability of unidimensional force sensors. Sensibilities to forces down to the nN scale are in principle attainable, different fluorescent responses may be expected according to the type of force that is applied on the crystal surface (friction or pressure). Adding up the preliminary results obtained on the dependence of the fluorescence response according to the direction of friction opens up interesting perspectives in the design of versatile force sensors, whose response is modulated by 180

Chapter 4. Polydiacetylene microcrystals force intensity, force type and force direction. In this regard, AFM is confirmed to be a powerful tool to study these effects. Additional tests are required to validate the general behaviour described above and conclude on the performance of single crystals as friction force sensors.

Conclusion

In this chapter, PDA derivatives TzDA1 and TzDA2 were studied as self-assembled aggregates in suspension and in the form of isolated crystals/aggregates deposited on glass slides. This nanoscale study relies on the use of steady-state spectroscopy and AFM coupled with fluorescence microscopy, which allowed us to map the morphology of the solid aggregates, apply forces on their surface and probe their fluorescence response.

In the first part, suspensions of TzDA1 and TzDA2 were prepared by different methods: reprecipitation in water from differently concentrated DA solutions in either THF or MeCN; sonication in water; recrystallization in water of the DA dissolved in MeCN by slow liquid diffusion. For both compounds, we tested different parameters in the preparations and studied how they affected the polymerization by UV irradiation in suspension. TzDA1 efficiently polymerizes in suspensions prepared from both THF and MeCN solutions. Absorption spectra upon irradiation of these suspensions have all the same spectral features that evolve consistently upon photopolymerization. An absorption band at 615 nm, corresponding to bluepoly-TzDA1, is formed and evolves with increasing irradiation. For all suspensions, red-poly-TzDA1 is also formed along polymerization, with a characteristic absorption band at 540 nm. Accordingly with the energy transfer mechanism that was described for solid thin films (Section), blue-poly-TzDA1 formation quenches tetrazine fluorescence signal. In addition, when heating a suspension prepared from a 0.5 mM TzDA1 solution (H 2 O:MeCN 8:2), the blue phase is converted to the red phase of the polymer, restoring the quenched tetrazine fluorescence due to a reduced energy transfer efficiency between red-PDA and tetrazine. Sonication of TzDA1 in water for 15 min at 60 • C successfully produced aggregates in suspension that polymerize upon irradiation at 254 nm. However, the polymer phase that is formed is redpoly-TzDA1. Since the blue-to-red conversion is a one-way irreversible transition, these aggregates do not have any possible application in the context of sensing. TzDA2 suspensions were prepared with the same methods. Contrarily to TzDA1, reprecipitated suspension in THF did not give UV-polymerizable aggregates for the three tested initial concentrations of 0.05, 0.1 and 0.5 mM. We believe that the reason must be connected with the polarity of the solvent. In fact, when using a more polar solvent for reprecipitation like MeCN, polymerization efficiently occur for (almost) all initial concentrations and proportions. Interestingly, suspensions prepared from a 0.1 mM showed different polymerization properties than suspensions prepared from 0.25 or 0.5 mM solutions. In the former, the aggregates start polymerizing in the blue phase (λ abs = 605 nm) and suddenly shift to the red phase (λ abs = 540 nm) from a certain irradiation time. On the other hand, when starting from a more concentrated solution, the only polymer phase is blue-poly-TzDA2 absorbing at 605 nm. In both concentration, this band shifts gradually with irradiation towards lower wavelengths, as it was already recorded for solid thin films of the same compound. Increasing the initial concentration from 0.25 to 0.5 mM also increases tetrazine fluorescence quenching efficiency, that passes from 70% to 95%). Therefore, we believe that the initial concentration has an influence on the arrangement of self-assembled DAs that behave differently to 254 nm irradiation. Plus, 9:1 H 2 O:MeCN 8:2 contrarily to 8:2 proportion did not give UV-polymerizable aggregates. Sonication of TzDA2 in water was tested at 70 • C for 15 min and at 40 • C for 30 min. Interestingly, both preparations gave aggregates in solution that polymerize both upon irradiation at 254 nm. Contrarily to TzDA1, TzDA2 sonicated suspensions form blue-poly-TzDA2, absorbing at 605 nm, as thin films and the other suspensions. Tetrazine fluorescence is also quenched as expected, and thermal stimulus recovers the fluorescence signal by blue-to-red conversion of poly-TzDA2. We can conclude that indeed the preparation method and parameters have an influence on the arrangement of DAs in suspension that eventually have different polymerization behaviours. Plus, the choice of the solvent is crucial in determining the polymerizability of a specific compound by irradiation at 254 nm. Further characterizations by means of powder X-ray diffraction (PXRD) or TEM on the structure of these objects are envisaged to help us understand better and correlate the variability of PDA aggregates spectroscopic behaviours as a function of their structure and preparation method.

In the second part of the chapter, suspensions of TzDA1 and TzDA2 are deposited on glass slide and the resulting aggregates and crystals are studied by AFM and optical microscopy. Different sizes and morphologies are obtained according to the chosen preparation method. Small nanometric aggregates are obtained by reprecipitating a 0.5 mM solution of TzDA1 in MeCN with 8:2 H 2 O:MeCN. Sonication 60 • C for 15 min gave aggregates with mixed morphologies and sizes, going from few hundreds of nanometers up to several µm. On the other hand, reprecipitation of a 0.5 mM solution of TzDA1 in MeCN with 5:5 H 2 O:MeCN gave large plate-like crystals of micrometric size. Similar results are obtained by recrystallization of a 0.5 mM TzDA1 MeCN solution in water by slow diffusion. Lowering the diffusion temperature produces the coexistence of plate-like crystals and fiber-like crystals that extend up to several mu in length. Irradiation in situ at 254 nm produces a fluorescence quenching of the aggregates, no matter their size and shape. With respect to mechanical friction application, larger crystals are better adapted than small aggregates, on which positioning the AFM can be hard and especially the fluorescence probing either by images or spectra is less accurate compared to larger surfaces. UVpolymerized single crystals prepared by 5:5 reprecipitation and by liquid diffusion recrystallization are subjected to shear forces and pressures by an AFM tip. The fluorescence is probed consecutively. Mechanical friction, contrarily to pressures, produces a fluorescence activation exclusively in the stimulated zone. This allowed us to perform nanolithography experiments, where an acronym is engraved on the crystal surface and visualized by fluorescence. Quantification of the fluorescence activation signal is performed on these samples, and correlated to the normal applied force in the sheared area. Increasing the force produces a higher fluorescence activation signal, but the found trend is not as straightforward as it was found for TzDA1 thin films. Further testing and analysis at this purpose are needed. At last, TzDA2 deposited aggregates morphology was also tested. Compared to TzDA1, this compound have less variability in the size and shape of the formed aggregates, that seems to form the same way no matter the preparation method or parameters. Both reprecipitation and sonication gave long fiber-like aggregates.

In conclusion, the results obtained on isolated single crystals stand out for their novelty in the context of unidimensional force sensors. PDA are confirmed as great models for this purpose, showing very high sensitivities down to the nN and a specific Chapter 4. Polydiacetylene microcrystals fluorescent response according to the type of applied force and to the direction of force application. It would be interesting to incorporate these objects in polymer matrices, like PMMA, PEO or stretchable LLDPE and test their response to matrix deformations. Actin gels may also be a fascinating option for PDA incorporation that could be used to probe mechanical forces upon the actin polymerization process.

Chapter 5 Conclusion & perspectives

The objective of this work is to give new insights on the mechanofluorochromic properties of polydiacetylenes, to understand how the chemical structure of these derivatives influences their polymerization dynamics, morphology and response to mechanical stimulation. The study is carried out both at the macro-and the nanoscale, on thin films and isolated single crystals of the prepared derivatives, with the aim of evaluating qualitatively and most of all quantitatively the forces that need to be applied to trigger a fluorescence modification.

Chapter 1 is a bibliographic review on the main recent advancements in the development of mechanofluorochromic compounds, focusing on the correlation between structure and emission properties. Specifically, the family of PDAs is reviewed attentively from a fundamental and applicative points of view. We presented the main spectroscopic and microscopy studies that have characterized the optical properties of these compounds in respect to their polymerization and their chromo-and fluorogenic transitions when exposed to external stimuli. At a further stage, we reviewed the main recent progresses in the design of PDA-based materials in the context of sensing, demonstrating the impressive versatility of these molecules to assemble in different aggregation states and to probe a wide range of different stimuli (temperature, solvents, water, mechanical stress etc.).

Chapter 2 deals at first with the molecular design and synthesis of four novel DAs: a single chain DA bearing a urethane moiety at one end (DA1) and three tetrazine substituted DAs, where the self-assembling group is modified. TzDA1 has the same urethane moiety as DA1. TzDA2 has an additional ester function and TzDA3 has a simple amide group. All compounds are synthesized with acceptable yields.

Vacuum evaporated thin films of the four compounds were successfully obtained and used to characterize the macroscale spectroscopic properties in respect with their UV-polymerization and response to temperature/mechanical stress after polymerization. Different kinetics of polymerization are obtained for the four derivatives, pointing out how the different structure influences their arrangement. DA1 polymerizes to a classical blue phase (λ abs = 635 nm) faster and more efficiently than any other tetrazine-substituted system. TzDA1 and TzDA2 both polymerize to a purple phase (λ abs = 600 nm), while TzDA3 polymerizes directly to the red phase (λ abs = 554 nm).

Tetrazine in TzDAs is quenched by the formation of the polymer in the films through an energy transfer mechanism. A comparative spectroscopic study was performed on the three compounds. For the three, one polymer chain (acceptor) is able to quench multiple tetrazine emitters (donor), which results in a non linear correlation between blue-polymer absorption and fluorescence intensity with an amplification effect of fluorescence extinction. It was concluded that that TzDA2 polymerizes less but quenches more efficiently the emitting unit. Due to the important dependence of energy transfer on spatial parameters, this stands as an ulterior proof of a different arrangement of DAs in the film. Tetrazine is also quenched efficiently in TzDA3. Time-resolved fluorescence measurements on progressively polymerized TzDA1 films allowed us to trace the fluorescence decays for each stage of the polymerization process. The decay profiles were fitted by a fixed discrete exponential and a stretched exponential components. The characteristic parameters β and τ 1 of the stretched exponential decrease progressively as a function of irradiation time. Plus, the lifetime distribution progressively shortened along the polymerization (5-20 ns to 0.5-10 ns), confirm such an energy transfer mechanism.

All the studied compounds responded to mechanical and thermal stimulation, producing the red phase and an enhanced fluorescence signal.

Chapter 3 extends the study of PDA thin films to the nanoscale, by the use of an AFM coupled with a fluorescence microscope.

First of all, AFM topography images are used to trace the morphological peculiarities of each compound. DA1 and TzDA1 forms continuous films on which rod-shape crystals of variable size are deposited. A statistical analysis on the crystal dimensions allowed us to conclude that DA1 formed longer objects compared to TzDA1, which orientated in specific directions on the film. TzDA2 and TzDA3 gave big aggregates either spaced by empty zones or by a discontinuous film. The mechanical properties in respect with polymerization of DA1 and TzDA1 films were characterized by advanced force spectroscopy measurements, to conclude that DA1 is stiffer than TzDA1. Plus, TzDA1 became stiffer upon polymerization (Young's modulus from 1 GPa to 1.8 GPa) while DA1 became softer (Young's modulus from 2 GPa to 3 GPa).

The atomic force microscope was used in contact mode to apply shearing stress on the polymerized thin films and fluorescence microscopy was used to probe the mechanically-induced fluorescence activation. Fluorescence activation was observed for all the compounds. However, the irregular morphology of TzDA2 and TzDA3 did not allow comparative studies of different stimulated zones of the sample. Contrarily, TzDA1 and DA1 samples could be assumed to be morphologically homogeneous in different zones, which is a crucial prerogative when we want to compare different stimulated zones. On these two derivatives, we performed a rigorous testing of friction applied in different zones of 3x3 µm 2 , varying the normal applied force. In addition, different tip types and scan rates were tested. It was concluded that an optimal fluorescence restoration was achieved when using carbon coated diamondlike tip at a scan rate of 6.1 µm•s -1 . At a second stage, the fluorescence signal coming from the mechanical stimulation was evaluated by integrating on the sheared area and the resulting value was correlated to the normal applied force. TzDA1 showed an increasing fluorescence signal for forces ranging from 0 to 200 nN, while the response of DA1 did not seem as clearly correlated to the applied force as TzDA1.

These results represent the first straightforward correlation of applied force and fluorescence activation of a mechano-responsive luminescent compound at the nanoscale, which hopefully will pave the way for the design of reliable and ultrasensitive force sensors.

Chapter 4 deals with the study of the same PDA derivatives in the form of aggregates in suspension and isolated crystals deposited on glass slides. Different methods were used to prepare TzDA1 and TzDA2 suspensions: reprecipitation in water of solutions in MeCN and THF, sonication in water and recrystallization by slow diffusion in water from solutions in MeCN. Apart from reprecipitation from THF for TzDA2, both reprecipitation and sonication gave rise to aggregates in suspension. Indeed, the preparation method and the condition parameters gave rise to aggregates that do not polymerize the same way by UV-irradiation. 254 nm irradiation on TzDA1 gave rise to an absorption band at 615 nm, shifted hypsochromically by 15 nm compared to thin films. TzDA2, instead, formed the same absorption band at 600 nm as in thin films. In both cases, when polymerization was successful, tetrazine fluorescence was quenched. Quenching efficiencies could be evaluated precisely for reprecipitated TzDA2 only, and were comparable to thin film efficiencies when the initial concentration of the starting solution is 0.5 mM (95% in suspension versus 94% in solid films). These same suspensions were then drop-casted on glass slides and dried. Objects of different shapes and sizes were obtained for the two compounds. TzDA2 resulted in long fiber-like crystalline objects, no matter the preparation method and prepa-ration conditions. On the contrary, for TzDA1 reprecipitation at 8:2 H 2 O:MeCN proportions gave nanometric rod-like objects while at 5:5 proportions large platelike micrometric crystals were obtained. Recrystallization by slow diffusion also gave large parallelepipedic crystalline objects.

Mechanical frictions and compressions were then applied on the surface of polymerized crystals of TzDA1. When a shearing force was applied, the fluorescence was recovered in correspondence with the stimulated zone from 20 to 300 nN vertical forces. Compressions, instead, did not seem to trigger any visible fluorescence response. Different vertical forces ranging from 0 to 300 nN were applied on different crystals surfaces of TzDA1. The fluorescence activation was evaluated by averaging the signal in the sheared area and the values were plotted against the applied normal force. Indeed, increasing the force produced an increase in the fluorescence signal. However, further testing is necessary to conclude on the real performance of isolated single crystals as force sensors. Interestingly, we noticed in these objects a fluorescence activation bias towards the direction of scan, depending on whether the scan follows the crystal longitudinal axis or not. Such a property could be interesting for the fabrication of fluorescent force sensors that could probe not only if a force is applied but also its direction.

The results obtained in this work represent a remarkable advancement in the study of mechanofluorochromism and its possible application in the context of force sensing. Raman spectroscopy could be an interesting way to characterize the blueto-red PDA conversions, since the two phases have different spectral signatures.

PDA isolated crystals and aggregates probably represent the most promising applicative perspective of this family of systems. Incorporation of these objects in different matrices may have interesting applicative imports. Stretchable polymer matrices like LLDPE are currently in use in the laboratory for incorporation of difluoroboron β-diketonate complexes and we envisage to perform similar tests using single chain DAs or tetrazine-substituted DAs. Additional experiments of force application at the nanoscale are also envisioned, to check, for example, real-time fluorescence changes while a compression is applied by the AFM tip.

In order to approach an application in mechano-biology, PDAs suspensions in water represent probably the best option in terms of biocompatibility. We plan, in fact, to incorporate these systems in actin filaments networks, which are known to apply mechanical forces when they polymerize. The insertion of PDA vesicles in such networks could lead to force sensing and, hopefully, to force quantification. A synthetic work to improve the stability, the bio-compatibility and the specificity of these sensors is also foreseen.

TzDA3 is a valid candidate to be easily substituted on its amide group with 192

Conclusion & perspectives specific recognition sites that bind to proteins on the cellular membrane. This way it could be possible to track the cellular tractions directly by PDA conversion. In a round bottom flask 0.900 mL (3.43 mmol) of 1-pentadecyne are dissolved in 50 mL of acetone and 1.22 g (6.86 mmol) of NBS are added to the solution. The solution turns immediately yellow, and a yellow precipitate corresponding to the products of degradation of NBS deposit at the bot-tom of the flask. 0.175 g of silver nitrate AgNO 3 are added, the solution turns to milky white and the flask is protected from light with aluminum paper. The mixture is let react for 40 minutes and TLC in petroleum ether (PE) shows the complete disappearance of 1-pentadecyne and the appearance of a new spot under UV at 254 nm, corresponding to 1-bromopentadec-1-yne. The reaction mixture is filtered on celite and concentrated on rotavapor. A yellow powder is obtained, due to the presence of residual NBS salts. The powder is dissolved in DCM and filtered on SiO 2 to remove the NBS salts. The solvent is dried to obtain 0.932 g of a yellow-brown oil, (yield = 94.6 %). Temperature is controlled to 0-5 • C with an ice bath. Under N 2 , a catalytic amount of CuCl (0.270 g, 2.72 mmol, 0.25 eq.) and 40 mL of DCM are added to the solution. Hydroxylamine is added until complete disappearance of the blue colour due to the presence of cupric ions. 2.00 g (10.9 mmol) of hex-5-yn-1-yl propylcarbamate are added to the reaction solution and 1.93 g (10.9 mmol) of 6-bromohex-5yn-1-ol are first dissolved in 15 mL of DCM and added dropwise to the reaction mixture. The system is let at 0-5 • C under stirring for 1h and let react at room temperature overnight. TLC (8/2 PE/EtOAc) shows the presence of a new UV-active product. The reaction solution is diluted with 50 mL of DCM and washed 3 times with 50 mL of NH 4 Cl sat . The organic phases are joined, dried on MgSO 4 and concentrated. The yellow-brownish oily solution obtained is purified by SiO 2 column chromatography with 8/2 PE/EtOAc, to give 1.94 g of a blue powder (yield = 63.7%) that should be stored at low temperature to avoid thermal polymerization.

1 H NMR (CDCl 3 ): δ = 0.92 (3H, t, J = 7.3 Hz); 1.45 -1.75 (10H, m); 2.30 (4H, m); 3.13 (2H, q, J = 6.6 Hz); 3.66 (2H, t, J = 6.2 Hz); 4.06 (2H, t, J = 6.2 Hz); 4.65 (1H, s) ppm The synthetic protocol of 6-bromohex-5-yn-1-ol is described in Section 6.1.3.1.

Butyl (((12-hydroxydodeca-5,7-diyn-1-yl)oxy)carbonyl)glycinate 30 mL of distilled water and 5.80 mL (58.8 mmol, 10 eq.) of n-butylamine are mixed in a 3-neck round bottom flask. Nitrogen is bubbled in the solution and the system is thermostated at 0 • C with an ice bath. 0.146 g (1.47 mmol, 0.25 eq.) of CuCl are added along with 20 mL of DCM. The solution turns to a deep blue color due to the presence of cupric ions. Hydroxylamine is added until the blue color of the solution disappears (reduction of Cu 2+ ). 1.50 g (5.88 mmol, 1 eq.) of butyl (((12-hydroxydodeca-5,7-diyn-1yl)oxy)carbonyl)glycinate are added and the solution turns to pale yellow. 1.04 g (5.88 mmol, 1 eq.) of 6-bromohex-5-yn-1-ol are diluted in 10 mL of DCM and added dropwise to the reaction mixture (the solution turns blue again, so more hydroxylamine is added until the blue color disappears). The mixture is let react under stirring, in N 2 atmosphere, at 0-5 • C for 5 hours. TLC (7/3 PE/EtOAc) shows the total consumption of starting products and the appearance of a new 254 nm UV-Chapter 6. Methods active spot. The solution is washed 4 times with 30 mL of NH 4 Cl sat , the organic phases are joint together, concentrated on rotavapor. The resulting yellow-brown oil is purified on SiO 2 colum chromatography 9/1 PE/EtOAc to give 1.66 g of a viscous yellow oil (yield = 80%). Butyl (((12-((6-(dodecyloxy)-1,2,4,5-tetrazin-3-yl)oxy)dodeca-5,7-diyn-1yl)oxy)carbonyl)glycinate (TzDA2) 0.866 g (2.46 mmol, 1 eq.) of butyl (((12-hydroxydodeca-5,7-diyn-1-yl)oxy)carbonyl) glycinate, 0.814 g (2.71 mmol, 1.1 eq.) of 3-chloro-6-(dodecyloxy)-1,2,4,5-tetrazine and 0.331 g (2.71 mmol, 1.1 eq.) of DMAP are added in a Schlenk that is previously dried under vacuum to remove any trace of humidity. The system is brought under nitrogen and 20 mL of DCM are introduced in the flask through a syringe. The reaction is let run under inert atmosphere at thermostated 7 • C over night. The product solution is concentrated on rotavapor and purified by SiO 2 column chromatography 8/2 PE/EtOAc to give 0.356 g of TzDA2 (yield = 24%).

1 H NMR (CDCl 3 ): δ = 0.87 (3H, t, J = 6.8 Hz); 0.94 (3H, t, J = 7.0 Hz); 1.26 -1.37 (16H, m); 1.50 (2H, m); 1.61 (6H, m); 1.75 (4H, m); 1.90 (2H, m); 2.04 (2H, m); 2.30 (2H, t, J = 6.8 Hz); 2.37 (2H, t, J = 7.0 Hz); 3.95 (2H, d, J = 5.2 Hz); 4.10 (2H, t, J = 6.4 Hz); 4.16 (2H, t, J = 6.8 Hz); 4.54 (2H, t, J = 6.4 Hz); 4.57 (2H, t, J = 6.4 Hz); 5. 13 1 H NMR (CDCl 3 ): δ = 1.47 (2H, q); 1.55 (2H, q); 1.66 (2H, q); 1.95 (1H, t); 2.21 1 H NMR (CDCl 3 ): δ = 0.92 (3H, t, J = 7.6 Hz); 1.41 -1.59 (4H, m); 1.66 (2H, q, J = 7.2 Hz); 1.94 (1H, t, J = 2.6 Hz); 2.15 -2.23 (4H, m); 3.22 (2H, d(t)); 5.46 (1H, s) ppm 13 C NMR (CDCl 3 ): δ = 172.9; 84.6; 68.4; 41.3; 36.8; 28.5; 28.3; 25.4; 23.1; 18.4; 11.5 204 Chapter 6. Methods

6-bromohex-5-yn-1-ol

The synthetic protocol of 6-bromohex-5-yn-1-ol is described in Section 6.1.3.1.

14-hydroxy-N-propyltetradeca-7,9-diynamide 1.210 g (16.5 mmol, 9.9 eq.) of N-butylamine are diluted in 8.84 mL of distilled water and 5 mL of DCM, then 0.048 g (0.485 mmol, 0.29 eq.) of CuCl are added. The solution turn blue due to the presence of cupric ions, that are then reduced by adding hydroxylamine until the color disappears. 0.303 g (1.67 mmol, 1 eq.) of N-propyloct-7-ynamide are added and the solution turn to yellow. 0.326 g (1.84 mmol, 1.1 eq.) of 6-bromohex-5-yn-1-ol are dissolved in 4 mL of DCM and added dropwise to the stirring solution at room temperature. The rection is let run overnight. TLC (5/5 PE/EtOAc) shows the appearance of a new é(' nm UV-active spot. The reaction mixture is diluted with DCM, washed 3 times with 20 mL NH 4 Cl sat , and the organic phase is driend on MgSO 4 and concentrated on rotavapor. The resulting mixture is purified on SiO 2 column chromatography 6/4 EtOAc/PE to give 0.160 g of crystalline whiteblue paste corresponding to 14-hydroxy-N-propyltetradeca-7,9-diynamide, (yield = 34.5%). The UV/Vis absorption spectra were recorded on a double beam spectrophotometer Cary 5000 equipped with deuterated/halogen lamps for solid samples and on a double beam spectrophotometer Cary 100 for solutions, both from Agilent technologies. The emission spectra were recorded on the Fluorolog FL3-221 with xenon light source for solid samples and on a Fluoromax 3 for solutions, both from Horiba Jobin-Yvon. Irradiation at 254 nm was performed by a Hg/Xe LC8 light source from Hamamatsu. An interferential filter from Semrock is added to select the 254 nm mercury line. The power of the irradiation was calculated with an Ophir powermeter (PD300-UV), using a Schott Long Pass (LP) 385 nm to measure IR irradiation that is let through the Semrock filter, considering a 90% transmittance: 208 Chapter 6. Methods Titre:

Développement de matériaux mécanouorochromes à base de polydiacétylènes comme capteurs de force ultrasensibles Mots clés: Mechanouorochromisme, polydiacétylènes, microscopie à force atomique, microscopie de uorescence, capteurs de force Résumé: Dans le cadre de la détection de forces mécaniques, le développement de sondes uorescentes ultra-sensibles représente une perspective intéressante dans des domaines variés, de la mécano-biologie à la mécanique des structures. Dans ce contexte, les matériaux mécanouorochromes capables de changer leurs propriétés de uorescence sous contraintes mécaniques pourraient se révéler de bons candidats pour la conception de telles sondes. Nous présentons dans ces travaux l'étude de quatre dérivés inédits de matériaux uorescents sensibles aux forces à base de polydiacetylènes. Les quatre systèmes ont été conçus spéciquement pour évaluer l'inuence de certains changements structuraux sur leurs dynamique de polymérisation, morphologie, propriétés photophysiques et leur réponse uorescente aux stimulations mécaniques. L'étude a été menée à la macro échelle aussi bien qu'à la nano échelle, à l'aide d'un microscope à force atomique couplé à un microscope de uorescence pour à la fois appliquer des forces à l'échelle du nN et pour évaluer l'activation de uorescence correspondante. Les nouveaux polydiacétylènes substitués par le uorophore tétrazine se sont montrés de bons candidats pour répondre optiquement de façon dénie aux forces jusqu'à l'échelle du nN.

Title:

Elaboration of mechanouorochromic materials based on polydiacetylene as ultrasensitive force probes Keywords: Mechanouorochromism, polydiacetylenes, atomic force microscopy, uorescence microscopy, force sensors Abstract: In the context of mechanical force sensing, the development of uorescent ultrasensitive probes is an intriguing perspective for various elds of application, like mechano-biology and mechanical engineering. In this regard, mechanouorochromic materials that change their uorescence properties when subjected to external mechanical stimulation may be a valid option for the design of such sensors. We present here the study of four novel mechanoresponsive luminescent polydiacetylene derivatives. The systems were accurately designed to evaluate how structural changes inuence their polymerization dynamics, morphology, photophysical properties and uorescence response upon mechanical stress application. The study is carried out at the macro and nanoscale, where an atomic force microscope coupled to a uorescence microscope were used to apply forces in the nN range and evaluate the uorescence activation. Original tetrazine-substituted diacetylenes in the form of thin lms and single crystals are conrmed to be good candidates to optically respond to forces down to the nN scale.

Université Paris-Saclay PPSM / Ecole Normale Supérieure Paris-Saclay 4 Avenue des Sciences / 91190 Gif-sur-Yvette, France

Figure 1 . 1 :

 11 Figure 1.1: Structure of the studied tetraphenylpyrene derivative (a); images of the compound powder under visible and UV light before (left) and after (right) grinding (b); schematization of the two aggregation forms before and after grinding (c); series of studied TPPy derivatives (d) Figure adapted from references [7, 8].

Figure 1 . 2 :

 12 Figure 1.2: Series of studied OPVs (a); normalized fluorescence spectra of C18-YB as-prepared (solid), pressed for 1 min at 1500 psi -dashed -, pressed for 19.5 h at 1500 psi -dotted -, and pressed for 1 min at 1500 psi/heated to 130 • C for 5 min, and quenched to RT by placing on a cold surface -dashdotted -(b); Pictures of a C18-YB/KBr mixture pressed for 1 min at 1500 psi -top left -, the same sample subsequently heated to 130 • C and quenched to RT by placing on a cold surface -top right -, the same sample pressed again as before -bottom right -, and the same sample heated and cooled as before -bottom left -(c).Figure adapted from reference [10].

Figure 1 . 3 :

 13 Figure 1.3: Structure of the studied liquid crystal materials a; scheme of the phase transition mechanism upon mechanical shearing (b).Figure adapted from reference [7].

  Figure 1.3: Structure of the studied liquid crystal materials a; scheme of the phase transition mechanism upon mechanical shearing (b).Figure adapted from reference [7].

Figure 1 . 4 :

 14 Figure 1.4: Structure of the difluoroboron-β-diketonate derivative under study (a); emission of prism (a1), needle (a2) and dendritic (a3) crystals; absorption and fluorescence spectra of the different crystal polymorphs (b); rewritable mechanofluoroschormism of a thermally annealed film (c). Figure adapted from reference [14].

Figure 1 . 5 :

 15 Figure 1.5: Structure of the MFC Au complex (a); excitation and fluorescence spectra of the crystal polymorphs and ground crystals (b); fluorescence images of a needle-indented single crystal (c). Figure adapted from reference [42].

53

 53

Figure 1 . 7 :

 17 Figure 1.7: Schematics of a spiropyran-mercoyanin conversion in a spiropyransubstituted PMMA substrate under tensile force (a); picture of the same substrate before and after tensile stress (b). Figure adapted from reference [52].

Figure 1 . 8 :

 18 Figure 1.8: Schematics of AFM-induced deformation of MEH-PPV nanoparticles (a); force profile and simultaneous fluorescence signal recording (b).Figure adapted from reference [55].

  Figure 1.8: Schematics of AFM-induced deformation of MEH-PPV nanoparticles (a); force profile and simultaneous fluorescence signal recording (b).Figure adapted from reference [55].

Figure 1 . 9 :

 19 Figure 1.9: Fluorescence signal monitoring upon 1 µN pressure (a); normalized fluorescence spectra before, during and after mechanical stimulation (b); fluorescence signal monitoring upon up to 5 µN pressure (c); normalized fluorescence spectra before, during and after consecutive mechanical stimulations up to 5 µN (d). Figure adapted from reference [56].

57

 57

Figure 1 . 10 :

 110 Figure 1.10: Structure of the Pt(II) under study (a); pictures of the compound powder under UV before and after grinding (b); absorbance and fluorescence spectra before and after grinding (c); schematics of the MFC mechanism (d); nano-engraving of the word "mechano" by AFM on the crystal surface (e).Figure adapted from reference [57].

Figure 1 . 11 :

 111 Figure 1.11: Structure of DFB-Bu-Amide and nanoparticles preparation (a); schematics of the microscopy set-up (b); topography image of the deposited nanoparticles (c); corresponding fluorescence image (d); fluorescence spectra and AFM image of a mechanically stimulated nanoparticle (e). Figure adapted from reference [16].

Figure 1 . 13 :

 113 Figure 1.13: Structures of two PDA derivatives, TS-6 and TS-12 (a); photoinduced formation of reactive diradicals and asymmetric carbenes (b); schematics of the photoinduced polymerization steps in PDAs (c); chain length distribution as a function of conversion rate (d). Figure adapted from references [60, 62].

Figure 1 .

 1 Figure 1.14: Absorption (a) and fluorescence (b) spectra of PCDA; absorption spectra of differently prepared PCDA films (Langmuir films polymerized in water -LF W -; Langmuir films polymerized on solid support -LF S -; spin-coated films -SC -) upon photopolymerization by UV light at 254 nm (c). Figure adapted from references [77-79].

Figure 1 . 15 :

 115 Figure 1.15: Schematics of PDA amphiphiles aggregation. Figure adapted from reference [111].

Figure 1 . 16 :

 116 Figure 1.16: Structure of PDAPMO, polydiacetylene-based periodic mesoporous organosilica film (a); Different colors obtained by heating at increasing temperatures (b); absorption spectra of the colored polymer phases obtained (c).Figure is adapted from reference [118].

Figure 1 . 17 :

 117 Figure 1.17: Structure of three PDA-derivatives (a); color change upon heating of the three films (b); colorimetric response upon heating of the three derivatives (c); fluorescence spectra for the three compounds upon heating (d).Figure adapted from reference [122].

  Figure 1.17: Structure of three PDA-derivatives (a); color change upon heating of the three films (b); colorimetric response upon heating of the three derivatives (c); fluorescence spectra for the three compounds upon heating (d).Figure adapted from reference [122].

Figure 1 . 18 :

 118 Figure 1.18: Absorption spectra of TSHD monolayer under compression (a); absorption spectra of different polymerization extents of PCDA copolymerized in polyurethane after increasing strain application (b and c).Figure adapted from references [123, 125].

  Figure 1.18: Absorption spectra of TSHD monolayer under compression (a); absorption spectra of different polymerization extents of PCDA copolymerized in polyurethane after increasing strain application (b and c).Figure adapted from references [123, 125].

Figure 1 . 19 :

 119 Figure 1.19: Schematics of intercalated PCDA with alkyl amines films and set-up for shearing forces application and sensing (a); intercalated PCDA with C6-alkyl amine films after shearing force applied (b). Figure is adapted from references [101].

  Figure 1.19: Schematics of intercalated PCDA with alkyl amines films and set-up for shearing forces application and sensing (a); intercalated PCDA with C6-alkyl amine films after shearing force applied (b). Figure is adapted from references [101].

102

 102

Figure 1 . 20 :

 120 Figure 1.20: Structure of PCDA-4APDS (a); reversible color change of PCDA-4APDS upond grinding and heating (b). Figure adapted from reference [102].

Figure 1 . 21 :

 121 Figure 1.21: Structure of DA-C8 (a); patterned PDA-C8 film on PET through pressure-induced (50 MPa) UV polymerization (b).Figure adapted from references [129].

  Figure 1.21: Structure of DA-C8 (a); patterned PDA-C8 film on PET through pressure-induced (50 MPa) UV polymerization (b).Figure adapted from references [129].

Figure 1 . 22 :

 122 Figure 1.22: Structure of PCDA (a); NSOM and simultaneous fluorescence scans of a PCDA trilayer film (b); topography evolution after mechanical stimulation (c).Figure is adapted from reference [54].

  Figure 1.22: Structure of PCDA (a); NSOM and simultaneous fluorescence scans of a PCDA trilayer film (b); topography evolution after mechanical stimulation (c).Figure is adapted from reference [54].

Figure 1 .

 1 Figure 1.23: Dispersed PCDA in PEO wires (a); Raman spectra of a PEO -PDAembedded polymer wire under force activation (b). Figure is adapted from reference [99].

Figure 1 . 24 :

 124 Figure 1.24: Absorption spectra of PCDA dispersed in PDMS upon exposure to SAHCs of different lengths (a); plots of red intensity and degrees of swelling of a PDA embedded PDMS film after 10 min incubation in linear alkanes (b); optical microscopy images of the embedded PCDA crystal upon swelling of the matrix (c). Figure adapted from reference [131].

  Figure 1.24: Absorption spectra of PCDA dispersed in PDMS upon exposure to SAHCs of different lengths (a); plots of red intensity and degrees of swelling of a PDA embedded PDMS film after 10 min incubation in linear alkanes (b); optical microscopy images of the embedded PCDA crystal upon swelling of the matrix (c). Figure adapted from reference [131].

Figure 1 . 25 :

 125 Figure 1.25: Structure of the 4 monomer units composing the sensor chip (a); different color patterns when the PDA sensors were exposed to various solvents (b).Figure adapted from reference [97].

  Figure 1.25: Structure of the 4 monomer units composing the sensor chip (a); different color patterns when the PDA sensors were exposed to various solvents (b).Figure adapted from reference [97].

Figure 1 . 26 :

 126 Figure 1.26: Structure of the DA monomer (a); inkjet printed PDA on paper upon water exposure at low temperature with ice and human body temperature (b); Fluorescent image of sweat pores on fingertips and the palm deposited on the handshape region in panel (c). Figure adapted from reference [132].

Figure 1 . 27 :

 127 Figure 1.27: Structure of the 3 components of the hybrid nano-vesicles (a); colorimetric response of the nano-vesicles when exposed to the antigent (b). Figure adapted from reference [136].

  Figure 1.27: Structure of the 3 components of the hybrid nano-vesicles (a); colorimetric response of the nano-vesicles when exposed to the antigent (b). Figure adapted from reference [136].

Figure 1 . 28 :

 128 Figure 1.28: Chromatic sensor concept, structure and mechanism (a); colorimetric response of the nano-vesicles when exposed for 18h to different bacteria (b); fluorescence response of the system before (left) and after (right) exposure to bacteria (c). Figure adapted from reference [137].

Figure 1 . 29 :

 129 Figure 1.29: Structure of the monomers composing the vesicles (a); blue-to-red chromatic transition (left) and fluorescence activation (right) upon bacterial exposure (b); TEM images of non treated (left) and treated with PDA vesicles (center and right) ESBL-EC cells (c). Figure adapted from reference [138].

  Figure 1.29: Structure of the monomers composing the vesicles (a); blue-to-red chromatic transition (left) and fluorescence activation (right) upon bacterial exposure (b); TEM images of non treated (left) and treated with PDA vesicles (center and right) ESBL-EC cells (c). Figure adapted from reference [138].

Figure 1 . 30 :

 130 Figure 1.30: Structure of a portion of polymer unit composing the vesicles and mechanism of Pb 2+ binding (a); absorption (b) and fluorescence (c) spectra upon metal ions exposure. Figure adapted from reference [139].

144

 144

Figure 1 . 31 :

 131 Figure 1.31: Schematics of a cell exerting traction force on a PAA matrix (a); schematics of a TFM-STED set up and comparison of obtained images with confocal microscopy (b). Figure adapted from reference [144].

  Figure 1.31: Schematics of a cell exerting traction force on a PAA matrix (a); schematics of a TFM-STED set up and comparison of obtained images with confocal microscopy (b). Figure adapted from reference [144].

Figure 1 . 32 :

 132 Figure 1.32: Molecular structure of the tension sensor (a); schematics of the forcesensing mechanism (b); general view of the traction-sensing experiment (c). Figure adapted from reference [145].

Figure 2 . 1 :

 21 Figure 2.1: Scheme of fluorescence signal modulation according to the PDA state. Simple chain DAs are non-fluorescent in monomer state and blue-polymer phase. The fluorescence is activated by the blue-to-red PDA conversion. The tetrazine fluorophore in TzDAs monomers is fluorescent, quenched when the DAs are polymerized to the blue form and fluorescent again when the blue-PDA is converted to red-PDA.

Figure 2 . 2 :

 22 Figure 2.2: Synthesized DA molecules and their functional groups.

Scheme 2 . 1 :

 21 Scheme 2.1: Synthesis of DA1

Scheme 2 . 4 :

 24 Scheme 2.4: Synthesis of TzDA2 terminal alkyne

Figure 2 . 3 :

 23 Figure 2.3: Key distances and angles in the structure (packing distance d, H-bond lengths, C1-C4' distance lengths and the angle between the diacetylene rod and the stacking axis φ) (a); mean planes calculated on two tetrazine rings on two adjacent DA units tilted by 69.2 • (b); mean planes calculated on two alkyl chains C12 on two adjacent DA units, which are tilted by 76.98 • (c).

Figure 2 . 4 :

 24 Figure 2.4: Absorption (filled lines) and fluorescence spectra (dotted lines) of a 0.148 mM solution of TzDA1 in MeCN (a) and a 0.072 mM solution of TzDA2 solution in MeCN (b) Fluorescence is excited at 488 nm.

Figure 2 . 5 :

 25 Figure 2.5: Absorption spectra for consecutive irradiations at 254 nm of vacuum evaporated DA1 (a), TzDA1 (b), TzDA2 (c) and TzDA3 (d).

Figure 2 . 6 :

 26 Figure 2.6: Photopolymerization evolution of DA1, TzDA1 and TzDA2. Kinetics of the blue polymer formation by plotting normalized absorbance of the blue polymer form versus the irradiation energy (a); Total blue polymer quantity that is formed by plotting the absorbance of the blue polymer normalized by the thickness of the film versus the irradiation energy (b).

Figure 2 . 7 :

 27 Figure 2.7: Photopolymerization evolution of TzDA3. Kinetics of the blue (purple line) and red (red line) polymer formation by plotting normalized absorbance of the polymer form versus the irradiation energy (a); Total polymer quantity that is formed by plotting the absorbance of the polymer normalized by the thickness of the film versus the irradiation energy (b).

Figure 2 . 8 :

 28 Figure 2.8: Absorption spectra of mono-DA1 (black line), blue-poly-DA1 (blue line) and red-poly-DA1 (red line) (a); Fluorescence spectra of blue-poly-DA1 (blue line) and red-poly-DA1 (red line) (b). Fluorescence is excited at 488 nm.

Figure 2 . 9 :

 29 Figure 2.9: Tetrazine fluorescence and poly-TzDA absorbance spectral overlap: TzDA1 (a); TzDA2 (b); TzDA3 (c). Red-poly-TzDA is obtained by heating the glass slide on a heating plate at around 80 • C. Fluorescence is excited at 488 nm.

Figure 2 .

 2 Figure 2.10: Fluorescence spectra of vacuum evaporated TzDA1 monomer at T amb (purple) and at -175 • C (pink); fluorescence spectra of the same TzDA film, polymerized by UV irradiation at 254 nm for 10 min (P 254 = 1.37 mW•cm -2 ), at T amb (dark blue) and at -180°C (light blue). λ exc = 488 nm

Figure 2 . 11 :

 211 Figure 2.11: Fluorescence spectra after subsequent irradiations at 254 nm of thin films of: a. TzDA1, P 254 = 1.4 mW•cm -2 , λ exc = 510 nm; b. TzDA2, P 254 = 0.35 mW•cm -2 , λ exc = 500 nm; c. TzDA3, P 254 = 1.20 mW•cm -2 , λ exc = 488 nm .

Figure 2 . 12 :

 212 Figure 2.12: Correlation of normalized fluorescence intensity with blue polymer relative absorbance (Abs(600nm)/thickness) of TzDA1 and TzDA2 (a) and with red polymer relative absorbance (Abs(554nm)/thickness) of TzDA3 (b) for progressive irradiation at 254 nm.

Figure 2 . 13 :

 213 Figure 2.13: Fluorescence lifetime of TzDA1 monomer in MeCN. λ exc = 520 nm.

  . The fitting parameters are shown in the table in

  Figure 2.14c.

Figure 2 . 14 :

 214 Figure 2.14: Lifetime profiles of progressively photopolymerized thin films of TzDA1 (a) with their corresponding steady-state fluorescence spectra (b). The decays were fitted using the sum of a stretched and a discrete exponential performing a global analysis that fixes the discrete component. The table in (c) shows the parameters of the fit; stretched exponential parameters τ 1 and β versus increasing UV irradiation (d); distribution of rate constants H(k) of the stretched exponential compnent of each irradiation step (e). λ exc = 360 nm.

Figure 2 . 15 :

 215 Figure 2.15: Intensity fractions of single component exponentials of TzDA1 thin films in the monomer state (a) and irradiated at 254 nm for 560 s (b). The fractions for all stages of polymerization are reported in the table (c) and the normalized intensity fraction of the stretched exponential component is plotted versus the irradiation time (d).

Figure 2 . 16 :

 216 Figure 2.16: Absorption spectra before (blue) and after (red) heating at 70 • C (a); fluorescence spectra before polymerisation (black), after irradiation (blue) and after heating (red), λ exc = 500 nm. (b); absorption spectra before (blue) and after (red) mechanical shearing with a spatula (c). In (a-c) TzDA1 is deposited on a glass slide support; fluorescence spectra of a film on paper before polymerisation (black), and after two consecutive cycles of irradiation (blue, purple)/shearing (red, brown) (d), λ exc = 488 nm.

Figure 2 . 17 :

 217 Figure2.17: Absorption spectra of mono-TzDA2 (black) and poly-TzDA2 before (blue) and after (red) heating at 80 • C (a); fluorescence spectra before polymerisation (black), after irradiation (blue) and after heating (red) (b), λ exc = 488 nm; absorption spectra before (blue) and after (red) mechanical rubbing with a spatula (c); in (a-c) TzDA2 is deposited on a glass slide support; fluorescence spectra of a film on paper before polymerisation (black), and after two consecutive cycles of irradiation (blue, purple)/shearing (red, brown) (d), λ exc = 488 nm.

Figure 2 . 18 :

 218 Figure 2.18: Absorption spectra of poly-blue-DA1, obtained by irradiation at 254 nm for 30 s at 0.34 mW•cm -2 (blue line) and of red-poly-DA1 obtained by rubbing on the substrate (red line) (a); fluorescence spectra of blue-(blue line) and red-(red line) poly-DA1 (b), λ exc = 488 nm.

Figure 2 . 19 :

 219 Figure 2.19: Scheme of the heating effect on DA1 thin films previously polymerized for 5 s at 292 µW•cm -2 (a); Picture of the thin film after heating and resting at 25 • C (b); UV-Vis absorption spectra of the initial polymerized DA1 thin film (zone 2, blue line), after heating (zone 2, red line) and after multiple cycles of heating and resting at ambient temperature (zone 2, grey lines) (c).

Figure 2 . 20 :

 220 Figure 2.20: Absorption spectra of initial DA1 thin film (black line), UV irradiated (grey lines), final blue-poly-DA1 (blue line) and heated (red line) (a); Fluorescence spectra of blue-poly-DA1 under increasing temperature (23-80 • C) (b); Fluorescence spectra of red-poly-DA1 when temperature is decreased (c), λ exc = 488 nm; fluorescence intensity at 540 nm evolution in respect to increasing temperature (d).

1

 1 Atomic force microscopy: a brief review

Figure 3 . 1 :

 31 Figure 3.1: Scheme of an SPM microscope (a); piezo-electric bar constituting the scanner (b); creep phenomenon in piezoelectric materials (c); non-linear bending upon applied voltage (d)

Figure 3 . 2 :

 32 Figure 3.2: Scheme of an SPM feedback circuit, in the specific case of an STM microscope.

Figure 3 . 3 :

 33 Figure 3.3: Potential energy curve with respect to tip-substrate interaction (a); scheme of an AFM microscope (b).

Figure 3 . 4 :

 34 Figure 3.4: Graphical representation of AFM scanning modes: contact (a); tapping (b); quantitative imaging (c); typical force curve obtained by force mapping (d).

3. 1 . 2

 12 Figure 3.5 represents the scheme of the home-built microscopy set-up that is composed of an AFM coupled to a fluorescence microscope. For the same zone of the sample, topography images, mechanical stimulations and fluorescence probing are possible. Refer to Chapter 6.4 for set-up specifications and glass cleaning protocol.

Figure 3 . 5 :

 35 Figure 3.5: Scheme of the microscopy set-up: atomic force microscope coupled with a fluorescence microscope.

Chapter 3 .

 3 Force quantification by fluorescence at the nanoscale 3.2 Microscopy on PDA thin films 3.2.1 Morphology study AFM topography images of TzDA1 and DA1 thin films before (3.6a and 3.6c respectively) and after (3.6b and 3.6d respectively) photopolymerization are shown in Figure 3.6. In both cases, rod-shaped crystals lay on a continuous film of deposited diacetylene and this same morphology is found in different zones of the same sample.

Figure 3 . 6 :

 36 Figure 3.6: AFM morphology images of TzDA1 and DA1 before (a and c respectively) and after (b and d respectively) photopolymerization performed under the microscope at 1.3•10 -4 W•cm -2 for 70 min and 30 s on TzDA1 and DA1 respectively. The green arrows show the cracks on DA1 films issued by the photopolymerization process.

Figure 3 . 7 :

 37 Figure 3.7: Statistical analysis of surface crystal lengths (a), widths (b). Blue bars correspond to DA1 films and red bars to TzDA1 films. Bin width was set to 80 nm according to AFM images resolution. The analysis was performed on Figure 3.6b for TzDA1 and on Figure 3.6d for DA1. Statistical analysis of surface crystal orientations, showing random orientations in TzDA1 (c) and two main preferred orientations in DA1 (d). Bin width was set to 0.04π rad. The analysis was performed on Figure 3.6b for TzDA1 and on Figure 3.6d for DA1.

Figure 3 . 8 :

 38 Figure 3.8: AFM morphology images of TzDA2 and TzDA3 before (a and c respectively) and after (b and d respectively) photopolymerization performed under the microscope at 1.3 • 10 -4 W•cm -2 for 70 min.

Figure 3 . 9 :

 39 Figure 3.9: Schematic of the correction of the measured height by the cantilever bending (a); schematic of indentation experiment (b).

Figure 3 . 10 :

 310 Figure 3.10: Schematic of the experiment procedure for mechanical tests on PDA thin films.

Figure 3 . 11 :

 311 Figure 3.11: Topography image (a), adhesion map (b) and Young's modulus map (c) of non polymerized TzDA1 thin film. Topography image (d), adhesion map (e) and Young's modulus map (f ) of polymerized TzDA1 thin film. Polymerization is achieved by irradiation at 254 nm for 70 min at P = 1.3•10 -4 W•cm -2 .

Figure 3 .

 3 12a and d are the topography images and Figure 3.12b,c and e,f are the adhesion and the Young's modulus maps of non-polymerized and polymerized thin films. Polymerization does not affect the morphology features of either the crystals laying on the surface or the continuous film in the background (Figure 3.12a,d) as discussed in Section 3.2.1. However, polymerization has an influence on the mechanical properties of the film. The polymerized continuous film shows globally a lower adhesion force while retracting the tip (Figure 3.12e) compared to the non polymerized one (Figure 3.12b), going from approximately 1.5 nN to 1 nN in the film background. Concerning the Young's elasticity, the poly-DA1 (Figure 3.12c) has globally lower Young's moduli than non-polymerized DA1 thin films (Figure 3.12f), up to 3 GPa in the monomeric film background compared to 2 GPa in the polymeric film.

Figure 3 . 12 :

 312 Figure 3.12: Topography image (a), adhesion map (b) and Young's modulus map (c) of non polymerized DA1 thin film. Topography image (d), adhesion map (e) and Young's modulus map (f ) of polymerized DA1 thin film. Polymerization is achieved by irradiation at 254 nm for 30 s at P = 1.3•10 -4 W•cm -2 .

Figure 3 . 13 :

 313 Figure 3.13: Fluorescence images of mono-TzDA1 (a); blue-poly-TzDA1 (b), 70 min irradiation at 254 nm at P = 3.9 mW•cm -2 , white dotted squares show where the mechanical stimulation is performed; red-poly-TzDA1 (c), the fluorescent areas correspond to where the mechanical stimulation has been performed by AFM contact mode scans (3x3 µm 2 ), λ exc = 488 nm; integration values of fluorescent zones (d).

Figure 3 . 14 :

 314 Figure 3.14: AFM topography image of blue-poly-TzDA1 (a); corresponding fluorescence image (b); AFM topography image of the same zone after mechanical stimulation (c); corresponding fluorescence image (d). AFM images were taken in tapping mode, with a silicon tip at 4.04 µm•s -1 . Fluorescence images are taken over 1 s integration time at λ exc = 488 nm.

Figure 3 . 16 :

 316 Figure 3.16: Fluorescence image of the response to mechanical stimulations performed on TzDA1 thin films with carbon coated diamond-like AFM tips, at a scan rate of 6.1 µm•s -1 (1 s integration time, λ exc = 488 nm). The sample was polymerized under the microscope by irradiation at 254 nm for 70 min, P = 0.16 mW•cm -2 (a); AFM topography images of the stimulated zones (b-c-d).

  .17a shows the topography image of the chosen polymerized zone of the sample. The corresponding fluorescence image (Figure 3.17b) shows that the Chapter 3. Force quantification by fluorescence at the nanoscale 123 sample is in the OFF state. Friction forces with nominal vertical force ranging from 300 to 460 nN are applied in 9 different areas. Despite the high applied load (compared to TzDA1 experiments), the surface morphology is slightly affected (Figure 3.17c): the crystals are bent but their initial profile is basically retained and the continuous film in the background remains intact. The fluorescence response resulting from the stimulation is shown in Figure 3.17d. The signal is homogeneous over the scanned area and approximately two-fold less intense than the one registered for TzDA1 thin films. It can be concluded that a small morphology modification (small quantity of blue polymer converted to the red form) results in a low fluorescence activation.

Figure 3 . 17 :

 317 Figure 3.17: Topography (a) and corresponding fluorescence (b) images of polymerized DA1 thin film. The sample was polymerized under the microscope by irradiation at 254 nm for 30 s, P = 1.3•10 -4 mW•cm -2 ; topography (c) and corresponding fluorescence (d) images of mechanically stimulated DA1 thin films. Fluorescence images are taken over 1 s integration time at λ exc = 488 nm.

Figure 3 . 18 :

 318 Figure 3.18: Mechanical stimulation of polymerized DA1 thin films by an AFM mounting a carbon coated tip in two different zones of the sample. Topography images before (a,e) and after (c,g) mechanical stimulation; corresponding fluorescence images before (b,f ) and after (d,h) stimulation. The sample was polymerized under the microscope by irradiation at 254 nm for 70.4 s, P = 7.2•10 -5 W•cm -2 . Fluorescence images are acquired over 1 s integration time at λ exc = 488 nm.

Figure 3 . 19 :

 319 Figure 3.19: Mechanical stimulation of polymerized TzDA2 thin films by an AFM mounting a carbon coated tip. Topography (a) and corresponding fluorescence (b) of mono-TzDA2 thin films. Topography images before (c) and after (e) mechanical stimulation; corresponding fluorescence images before (d) and after (f ) stimulation. Fluorescence images are acquired over 1 s integration time at λ exc = 488 nm.

Figure 3 . 126 Chapter 3 .

 31263 Figure 3.20 shows AFM images of poly-TzDA2 before (3.20a) and after (3.20c) mechanical shearing and their corresponding fluorescence images (3.20b and 3.20d respectively). Vertical applied force was varied from 180 to 340 nN in 9 different areas. The ones corresponding to 200, 240 and 260 nN were not as morphologically affected as the others and as a consequence they did not restore as much the fluorescence. When looking at the corresponding morphology before stimulation (Figure

Figure 3 . 20 :

 320 Figure 3.20: Mechanical stimulation of polymerized TzDA2 thin films by an AFM mounting a carbon coated tip. Topography images before (a) and after (c) mechanical stimulation; corresponding fluorescence images before (b) and after (d) stimulation. Fluorescence images are acquired over 1 s integration time at λ exc = 488 nm.

Figure 3 . 21 :

 321 Figure 3.21: Mechanical stimulation of polymerized TzDA3 thin films by an AFM mounting a carbon coated tip. Topography (a) and corresponding fluorescence (b) of mono-TzDA3 thin films. Topography images before (c) and after (e) mechanical stimulation; corresponding fluorescence images before (d) and after (f ) stimulation. Fluorescence images are acquired over 1 s integration time at λ exc = 488 nm.

Figure 3 . 22 :

 322 Figure 3.22: Scheme of fluorescence signal quantification process. First, two consecutive fluorescence images (Fluo 01 and Fluo 02) are subtracted to produce the delta image, Delta fluo 02-01. Here only the fluorescence that has been activated through mechanical stimulation is visible, and the background is levelled to 0. The signal is integrated over the red striped area to get a numerical value.

Figure 3 .

 3 23 shows the resulting fluorescence -force correlation curves obtained for TzDA1 and DA1. Black dots (TzDA1) and blue triangles (DA1) correspond to the mean value of fluorescence activation resulting from several Chapter 3. Force quantification by fluorescence at the nanoscale experiments in different areas of the sample and the error bars correspond to the standard deviation. Points without error bars correspond to single measurements that were not repeated. The orange and blue dotted lines are the observed trends of TzDA1 and DA1 respectively and they are used as guides to the eye.

Figure 3 . 23 :

 323 Figure 3.23: Graphical representation of the fluorescence signal in each stimulated area versus the corresponding applied vertical force for TzDA1 (black points) and DA1 (blue points). Each point corresponds to the mean value of different measurements, and the error bars correspond to the standard deviation. The orange and blue dotted lines are the generic trends that are found in TzDA1 and DA1 respectively, and they are used as guides to the eye.

Figure 4

 4

Figure 4 . 1 :

 41 Figure 4.1: Scheme of aggregates formation by reprecipitation method.

Figure 4 . 2 :

 42 Figure 4.2: Scheme of aggregates formation by sonication method.

Figure 4 . 3 :

 43 Figure 4.3: Scheme of DA recrystallization by liquid diffusion method.

Figure 4 .

 4 Figure 4.4 shows the absorption and fluorescence spectra of UV irradiated suspensions of TzDA1, prepared by reprecipitation in THF -water for two different initial concentrations (0.1 and 0.5 mM). Figure4.4a shows the absorption spectra of a 0.1 mM suspension of TzDA1. It is evident that irradiation at 254 nm produces the absorption band corresponding to blue-poly-TzDA1, centred at 615 nm, and it increases when the sample is exposed to subsequent irradiations (grey lines in Figure4.4a). To better visualize the polymer absorption band, the first absorption spectrum, corresponding to the monomer form, has been subtracted to cancel out the contribution of tetrazine absorption to the visible range of the spectrum (λ abs = 520 nm). The resulting spectra are shown in Figure4.4b. The band at 615 nm increases in the same way, plus the side bands at lower wavelengths become visible. They probably correspond to a vibronic band of the blue phase (580 nm) and to the absorption band of red-poly-TzDA1 (540 nm). Tetrazine fluorescence signal (Figure4.4c) decreases as expected, according to the energy transfer mechanism that is discussed for thin films in Chapter 2. The emission is quenched progressively as the amount of blue-poly-TzDA1 increases in the aggregates (black to grey to blue lines in Figure4.4a and b).

Figure

  Figure 4.4 shows the absorption and fluorescence spectra of UV irradiated suspensions of TzDA1, prepared by reprecipitation in THF -water for two different initial concentrations (0.1 and 0.5 mM). Figure4.4a shows the absorption spectra of a 0.1 mM suspension of TzDA1. It is evident that irradiation at 254 nm produces the absorption band corresponding to blue-poly-TzDA1, centred at 615 nm, and it increases when the sample is exposed to subsequent irradiations (grey lines in Figure4.4a). To better visualize the polymer absorption band, the first absorption spectrum, corresponding to the monomer form, has been subtracted to cancel out the contribution of tetrazine absorption to the visible range of the spectrum (λ abs = 520 nm). The resulting spectra are shown in Figure4.4b. The band at 615 nm increases in the same way, plus the side bands at lower wavelengths become visible. They probably correspond to a vibronic band of the blue phase (580 nm) and to the absorption band of red-poly-TzDA1 (540 nm). Tetrazine fluorescence signal (Figure4.4c) decreases as expected, according to the energy transfer mechanism that is discussed for thin films in Chapter 2. The emission is quenched progressively as the amount of blue-poly-TzDA1 increases in the aggregates (black to grey to blue lines in Figure4.4a and b).

Chapter 4 .

 4 Polydiacetylene microcrystals irradiated sample (black line). The absorption band of blue-poly-TzDA1 at 615 nm increases along with a small contribution of red-poly-TzDA1 (Figure 4.4d), represented by a an increasing absorption band at 540 nm. Tetrazine fluorescence (λ em = 565 nm) consistently decreases along irradiation (Figure 4.4e), due to quencher formation in the aggregate (blue-poly-TzDA1).

Figure 4 . 4 :

 44 Figure 4.4: Absorption (a), ∆Absorption (b) and fluorescence (b) spectra of a 0.1 mM TzDA1 suspension progressively irradiated at 254 nm. Absorption (d), and fluorescence (e) spectra of a 0.5 mM TzDA1 suspension progressively irradiated at 254 nm. Both suspensions were prepared by reprecipitation method in THF with H 2 O:THF proportion 8:2. λ exc of fluorescence is 500 nm.

Figure 4 . 5 :

 45 Figure 4.5: Absorption (a) and fluorescence (b) spectra of TzDA1 suspensions progressively irradiated at 254 nm, prepared by reprecipitation method in MeCN at 0.25 mM initial concentration. The proportion of H 2 O:MeCN is 8:2. Absorption (c) and fluorescence (d) of monomer aggregates (black line), polymerized aggregates (blue line) and heated aggregates (red line). λ exc of fluorescence is 488 nm.

Figure 4 .

 4 Figure 4.6 shows absorption (4.6a) and fluorescence (4.6b) spectra of progressively polymerized aggregates of TzDA1 prepared by reprecipitation in water starting from a 0.5 mM solution in MeCN. Irradiation at 254 nm produces blue-poly-TzDA1 as confirmed by the growing absorption band at 615 nm in Figure 4.6a. A baseline shift caused by polymerization prevents us to trace the kinetics profile of blue polymer formation. The successful formation of blue-poly-TzDA1 is confirmed by the decrease in fluorescence signal intensity at 560 nm with UV irradiation shown in Figure 4.6b.A shoulder around 650 nm is present along with the band at 560 nm proper of tetrazine emission. The nature of this band is still unclear, but it is probably coming from tetrazine as well, since blue-poly-TzDA1 fluorescence is too low to be detected. If it was generating from red-poly-TzDA1 fluorescence, the band would increase along polymerization, while it would be constant if it was coming from an impurity in the sample.

Figure 4 . 6 :

 46 Figure 4.6: Absorption (a) and fluorescence (b) spectra of progressively irradiated at 254 nm TzDA1 suspensions, prepared by reprecipitation method in MeCN at 0.5 mM initial concentration. The proportion of H 2 O:MeCN is 8:2. λ exc of fluorescence is 500 nm.

Figure 4 . 7 :

 47 Figure 4.7: Absorption spectra of TzDA1 suspension, progressively irradiated at 254 nm (a); absorption spectra of monomeric (black line) and polymerized suspension (red line) after substraction of monomer suspension absorption (b); fluorescence spectra of TzDA1 suspension, progressively irradiated at 254 nm (c). The suspension is prepared by sonication in water at 60 • C for 15 min. The final concentration is 0.5 mM. λ exc of fluorescence is 488 nm.

Figure 4 . 8 :

 48 Figure 4.8: Absorption and fluorescence spectra of progressively irradiated at 254 nm TzDA2 suspensions, prepared by reprecipitation method in THF. Filled lines correspond to absorption spectra; dashed lines to fluorescence spectra. Different initial concentrations have been explored: 0.05 mM (a); 0.1 mM (b); 0.5 mM (c). The proportion of H 2 O:THF is always 8:2. λ exc of fluorescence is 500 nm.

Figure 4 . 9 :

 49 Figure 4.9: Absorption (a) and fluorescence (b) spectra of progressively irradiated at 254 nm TzDA2 suspensions, prepared by reprecipitation method in MeCN at 0.1 mM initial concentration. The proportion of H 2 O:MeCN is 8:2. λ exc of fluorescence is 500 nm.

Figure 4 .

 4 Figure 4.10: Absorption (a) and fluorescence (c) spectra of progressively irradiated at 254 nm TzDA2 suspensions, prepared by reprecipitation method in MeCN at 0.25 mM initial concentration. The proportion of H 2 O:MeCN is 8:2. Increase of blue polymer absorption at 605 nm (b) and decrease of tetrazine emission intensity at 560 nm (d). λ exc of fluorescence is 500 nm.

Figure 4 .

 4 Figure 4.11: Absorption (a), ∆Absorption (b) and fluorescence (c) spectra of progressively irradiated at 254 nm TzDA2 suspensions, prepared by reprecipitation method in MeCN at 0.5 mM initial concentration. The proportion of H 2 O:MeCN is 8:2. Increase of blue polymer absorption at 605 nm and decrease of tetrazine emission intensity at 565 nm (d). λ exc of fluorescence is 500 nm.

Figure 4 . 12 :

 412 Figure 4.12: Absorption spectra of progressively irradiated at 254 nm TzDA2 suspensions , prepared by reprecipitation method in MeCN at 1 mM initial concentration for two H 2 O:MeCN proportions, 9:1 (a) and 8:2 (b).

Figure 4 .

 4 Figure 4.13: Absorption (a) and fluorescence (b) spectra of progressively irradiated at 254 nm TzDA2 suspensions, prepared by sonication in water at 40 • C for 30 min. The final concentration is 0.1 mM. λ exc of fluorescence is 500 nm.

Figure 4 .

 4 Figure 4.14: Absorption (a), ∆Absorption (b) and fluorescence (c) spectra of progressively irradiated at 254 nm TzDA2 suspensions, prepared by sonication in water at 40 • C for 30 min. The final concentration is 0.5 mM. λ exc of fluorescence is 488 nm.

  Figure 4.15d.

Figure 4 .

 4 Figure 4.15: Absorption (a), ∆Absorption (b) and fluorescence (c) spectra of progressively irradiated at 254 nm TzDA2 suspensions, prepared by sonication in water at 70 • C for 15 min. The final concentration is 0.5 mM. λ exc of fluorescence is 488 nm.

Chapter 4 .

 4 Polydiacetylene microcrystals single crystals are imaged by optical microscopy and AFM. 4.3.1.1 Single crystals obtained by reprecipitation TzDA1 H 2 O:MeCN 8:2, C = 0.5 mM A suspension of TzDA1 is prepared by mixing a starting solution of TzDA1 in MeCN at 0.5 mM with stirring water, in H 2 O:MeCN 8:2 proportions. The suspension is deposited on a clean glass slide and dried under vacuum at 50 • C. Transmission optical images are acquired on the dried sample and are shown in Figure 4.16a-d. Crystalline rods of variable dimensions are formed and distributed on the substrate.From image 4.16b we can conclude that these objects are fluorescent independently of their specific shape. To have a closer look into the morphology of these aggregates, AFM images of the area shown in Figure4.16d are acquired before (4.16e) and after (4.16g) photopolymerization. It is confirmed that rod-like crystals are formed, with lengths that range between 50 nm and 1 µm and heights globally constant around 200 nm and that the size of the objects is not affected by polymerization.

Figure 4 .

 4 Figure 4.16: Transmission (a) and fluorescence (b) images of deposited TzDA1 suspensions, prepared by reprecipitation in water from a 0.5 mM solution of TzDA1 in MeCN. The proportion H 2 O:MeCN is 8:2. Transmission image of another zone of the sample (c); transmission image detail (d); AFM morphology and fluorescence images of this small area of sample before (e and f ) and after (g and h) in situ photopolymerization at 254 nm. Fluorescence is excited at 488 nm.

H 2 O

 2 :MeCN 5:5, C = 0.5 mM When the same starting solution of 0.5 mM TzDA1 in MeCN is mixed with water in equivolumic proportions, the crystals that are obtained present different shapes and morphology compared to the previous case. In fact, as shown in Figure 4.17a and c, larger parallelepipedic crystals are obtained after deposition and drying on a glass slide. Crystals are also fluorescent, as shown in Figure 4.17b. A single crystal, shown in Figure 4.17d, is imaged by AFM before (4.17e) and after (4.17g) polymerization. If length and width of the object are in the micrometer size (5 x 4 µm), the height is only 120 nm as shown in the line profiles in the insets, meaning that TzDA1 arranges in rectangular plate-like crystals.

Figure 4 .

 4 Figure 4.17: Transmission (a) and fluorescence (b) images of deposited TzDA1 suspensions, prepared by reprecipitation in water from a 0.5 mM solution of TzDA1 in MeCN. The proportion H 2 O:MeCN is 5:5. Transmission image of another zone of the sample (c); transmission image detail (d); AFM morphology and fluorescence images of this small area of sample before (e and f ) and after (g and h) in situ photopolymerization. Fluorescence is excited at 488 nm.

Figure 4 .

 4 19e. A different aggregation state, with a different number of molecules and packing could be the reason of this enhanced fluorescence signal for the same object size. Despite the different morphology, when they are exposed to irradiation at 254 nm, they polymerize and quench tetrazine fluorescence quite effectively as shown in Figure 4.19f. Fluorescence quenching can be estimated to 5-fold the initial value (I F (mono) = 10000 counts, I F (poly) = 2200 counts, I F (background) = 600 counts). Chapter 4. Polydiacetylene microcrystals

Figure 4 .

 4 Figure 4.19: Transmission (a) and fluorescence (c) images of deposited TzDA1 suspensions, prepared by sonication in water for 15 min at 60 • C. The final concentration is 0.5 mM. AFM topography image of an isolated object (b). Transmission image of another zone of the sample (d) and corresponding fluorescence images before (e) and after (f ) in situ photopolymerization. Fluorescence is excited at 488 nm.

Figure 4 .

 4 20c and d are AFM topography images of these objects, and confirm the fiber-like morphology.

Figure 4 .

 4 Figure 4.20: Transmission (a, d) and fluorescence (b, e) images of deposited TzDA2 suspensions, prepared by sonication in water for 15 min at 70 • C. The final concentration is 0.5 mM. AFM topography images of two zones of the sample (c, f ).Fluorescence is excited at 488 nm.

4. 3 . 1 . 3

 313 Single crystals obtained by recrystallization Single crystals obtained by liquid-liquid diffusion from a 0.5 mM solution of TzDA1 in MeCN with water were studied by optical transmission microscopy (Figure 4.21). Plate-like crystals are obtained when the recrystallization is performed at ambient Chapter 4. Polydiacetylene microcrystals temperature at 8:2 H 2 O:MeCN proportion, as shown in Figure 4.21a. Dimensions of the crystal can vary quite a lot, from few µm up to 20 µm length and width, as depicted in the histograms in Figure 4.21g for length distribution and 4.21h for width distribution. Decreasing the temperature to 5 • C leads to the coexistence of two distinct morphologies. As shown in Figures 4.21b and c, fiber-like aggregates of tens of µm length appear. Modifying the H 2 O:MeCN proportions does not affect much the final morphology, leading again to micrometric plate like crystals, as shown for 7:3 (Figure 4.21d) and 6:4 (Figure 4.21e). Using an equivolumic proportion of water and starting MeCN solution leads to big aggregates of multiple crystals of different morphology that become indistinguishable (Figure 4.21f).

Figure 4 . 21 :

 421 Figure 4.21: Transmission images of TzDA1 single crystals prepared by liquid-liquid diffusion recrystallization method. 8:2 H 2 O:MeCN for 3 days at T amb (a), 3 days at 5 • C (b and c); 7:3 H 2 O:MeCN for 3 days (d); 6:4 H 2 O:MeCN for 3 days (e); 5:5 H 2 O:MeCN for 3 days (f ); length (g) and width (h distributions. Analysis performed on 32 crystals in a.

Figure 4 .

 4 22a and d show the transmission images of this new sample and demonstrate that the crystals are reproducible in the same morphology as shown above. The crystals are highly fluorescent when excited at 488 nm, as shown in Figure 4.22b and e, with maximum recorded intensity up to 20000 counts where large crystals are present (Figure 4.22b). The topography AFM image in Figure 4.22c shows that the crystals are indeed in the micrometer range of sizes and that their height can vary from few hundreds of nanometers up to 2 µm, as shown by the line profiles in the inset (crystal 1, 500 nm; crystal 2, 800 nm; crystal 3, 350 nm; crystal 4 and 5, 2000 nm). Crystals that are found in another zone of the same sample (Figure 4.22d and e) are exposed to UV irradiation at 254 nm in situ, with an irradiating power of approximately 4.5•10 -5 W•cm -2 . Tetrazine fluorescence is homogeneously quenched in all crystals, as shown in Figure 4.22f. Tetrazine quenching can be estimated to 15-fold the initial value of intensity (I F (mono) = 6000 counts, I F (poly) = 400 counts), standing as the most efficient so far.

Figure 4 . 22 :

 422 Figure 4.22: Transmission images of TzDA1 single crystals prepared by liquid-liquid diffusion recrystallization method for 3 days at T amb with 8:2 H 2 O:MeCN proportions. Transmission (a), fluorescence (b) and topography AFM (c) images of a zone of the sample with line profiles of 5 crystals (inset); transmission image (d), fluorescence images before (e) and after (f ) UV irradiation at 254 nm. Fluorescence is excited at 488 nm.

H 2 O

 2 :MeCN 8:2, C = 0.5 mM Deposition of a suspension of TzDA1 prepared by reprecipitation in proportions H 2 O:MeCN 8:2 produces isolated small crystals of few hundreds of nm length, as explained in Section 4.3.1. The fluorescence image corresponding to the chosen area of the sample is shown in Figure 4.23a. The AFM tip is placed in contact with the surface of an isolated object, and a scan in contact mode at a chosen normal force (setpoint) is launched. A first aggregate (aggregate 1) is stimulated with a normal force of 25 nN. By comparing the fluorescence images before (Figure 4.23b) and after (Figure 4.23c) mechanical stimulation, we do not observe an evident 168 Chapter 4. Polydiacetylene microcrystals fluorescence activation, but the fluorescent area has increased. In fact, the particle has been disrupted and a portion is displaced next to the core, as confirmed by AFM topography images before (Figure 4.23d) and after (Figure 4.23e) stimulation.

Figure 4 .

 4 23h (before) and i (after). Fluorescence activation is again hard to observe and no apparent change is registered as shown by the fluorescence images before (Figure4.23f) and after (Figure4.23g) mechanical stimulation.

Figure 4 . 23 :

 423 Figure 4.23: Fluorescence image of TzDA1 single crystals prepared by reprecipitation from a 0.5 mM solution of TzDA1 in MeCN. H 2 O:MeCN proportion is 8:2 (a); fluorescence and AFM morphology images of particle 1 before (b and d respectively) and after (c and e respectively) mechanical stimulation; fluorescence and AFM morphology images of particle 2 before (f and h respectively) and after (g and i respectively) mechanical stimulation. Fluorescence is excited at 488 nm.

Figure 4 .

 4 photopolymerization causes a decrease in tetrazine fluorescence intensity (black to green lines). However, after stimulating mechanically with a normal force of 50 nN (aggregates 1 and 2) and of 30 nN (aggregates 3 and 4) TzDA1 does not restore the tetrazine fluorescence signal. Stimulated aggregates 1 and 2 (red lines in Figure 4.24b and c) have a lower fluorescence signal than blue-poly-TzDA1 (green line) while for aggregate 3 (Figure 4.24d) the signal remains constant and for aggregate 4 (Figure 4.24e) slightly increases. A loss in signal is probably due to a loss of fluorescent material at the moment of the stimulation. Because of the object size, it is hard to individuate a fluorescence change on images; because of material displacement by mechanical stimulation it is fairly complicated to center spectra acquisition in the correct region of interest (ROI).

Figure 4 . 24 :

 424 Figure 4.24: Transmission image of TzDA1 single crystals prepared by reprecipitation from a 0.5 mM solution of TzDA1 in MeCN. H 2 O:MeCN proportion is 8:2 (a); fluorescence spectra before photopolymerization (black lines), after polymerization (green lines) and after mechanical stress (red lines) of four particles (b, c, d and e).Fluorescence is excited at 488 nm.

  H 2 O:MeCN 5:5, C = 0.5 mM As explained in Section 4.3.1, equivolumic proportions of H 2 O:MeCN for reprecipitation lead to larger isolated crystals, on which mechanical stimulation effects on fluorescence are expected to be more visible.

Figure 4 .

 4 25a shows the fluorescence image of two UV-polymerized crystals (30 min at 4.6•10 -4 W•cm -2 ) that are chosen for mechanical stimulation. The topography images before stimulation are shown in Figure4.25b and c. Parallelepipedic plate-like crystals of micrometric size are formed, consistently with the preparation method, as described in Section 4.3.1. The AFM tip is then placed on the surface of the crystals, and a scan in contact mode is launched, with normal applied force of 100 and 50 nN for what we called crystal 1 and 2 respectively. A scan rate of 0.9 Hz was used for the two stimulations. After stimulation, tetrazine fluorescence is switched back on, as it can be seen by comparing fluorescence images before (Figure4.25a) and after (Figure4.25b). The fluorescence activation corresponds to a morphological modification of the crystals as can be seen in the morphology AFM images taken after stimulation (Figure4.25e) and Figure4.25f. Crystal 1 has been stimulated with a 100 nN shearing force that engraves lines on the crystal surface and disrupts a portion. Similarly, crystal 2, even if stimulated at a lower vertical force of 50 nN, is partially disrupted and its surface morphology is clearly modified.

Figure 4 . 25 :

 425 Figure 4.25: Fluorescence images before (a) and after (b) mechanical stimulation of two TzDA1 single crystals prepared by reprecipitation method in H 2 O:MeCN equivolumic proportions. AFM images of the two crystals are recorded before (c, d) and after (e, f ) mechanical stimulation. Fluorescence is excited at 488 nm.

Figure 4 .

 4 26a is the fluorescence image of the polymerized crystal and Figure4.26b is the corresponding AFM morphology image. The crystal is rectangular with edges of 4 and 2 µm. The surface is not completely flat but has a step that runs through the whole length of the crystal, as can be noticed in the 3D morphology image in the inset of Figure4.26b. The height is approximately 120 nm, confirming a plate-like structure. UV polymerizationperformed in situ for 30 min at a power of 4.6•10 -4 W•cm -2 -efficiently turns off Chapter 4. Polydiacetylene microcrystals tetrazine fluorescence, for a final average intensity value of 300 counts, while the background is around 220 counts. To test a pressure force, the tip is placed on the surface of the sample surface at 100 nN normal force for a few seconds and then retracted. A fluorescence image is acquired afterwards and is shown in Figure4.26c.

Figure 4 . 26 :

 426 Figure 4.26: Fluorescence image of a polymerized TzDA1 single crystal prepared by reprecipitation from a 0.5 mM solution of TzDA1 in MeCN. H 2 O:MeCN proportion is 5:5. (a); corresponding AFM morphology image (b); fluorescence images after 100 nN pressure (c), and after 100 nN friction force (d); AFM morphology image after mechanical stimulation (e). Fluorescence is excited at 488 nm.

4. 3

 3 .2.2 Sonication Mechanical friction forces and pressures have been tested on TzDA1 aggregates prepared by sonication in water as well. After sonicating for 15 min at 60 • C, the suspension is drop-casted on a glass slide and dried under vacuum. Transmission and fluorescence images of the resulting sample are shown respectively in Figure 4.27a and b.

Figure 4 .

 4 Figure 4.27: Transmission (a) and fluorescence (b) image of TzDA1 aggregates prepared by sonication in water for 15 min at 60 • C and deposited on glass slides; fluorescence image (c) of two polymerized particles; fluorescence image (d) after mechanical stimulation; AFM images of the two aggregates before (e and f ) and after (g and h) mechanical stimulation. Fluorescence is excited at 488 nm.

Figure 4 .

 4 Figure 4.28: Transmission (a) and fluorescence (b) image of TzDA1 aggregates prepared by sonication in water for 15 min at 60 • C and deposited on glass slides; AFM image of the chosen particle before mechnical stimulation (c); fluorescence image of the polymerized particle (d); fluorescence images for consecutive applied pressures of 200 and 500 nN normal load (e and f respectively); AFM image after mechanical stimulation (g). Fluorescence is excited at 488 nm.

Chapter 4 .

 4 Polydiacetylene microcrystals 4.3.2.3 Recrystallization TzDA1 single crystals are prepared by recrystallization via liquid diffusion from a 0.5 mM solution of TzDA1 in MeCN with water. H 2 O:MeCN proportion is 8:2. Micrometer sized crystals are obtained, consistently with previous preparations shown in Section 4.3.1.3. UV polymerization is performed in situ by irradiating the sample for 30 min at 1.3•10 -4 W•cm -2 . The fluorescence image of the chosen polymerized crystals is shown in Figure 4.29a, with I F (poly) = 450 counts. Friction forces on two crystals are applied by means of an AFM tip, for a total of 3 squared stimulations, at a vertical force of 350 nN, changing the direction of scan in each area, parallel and perpendicular to the crystal long axis. The direction of scan is shown by the yellow arrows in Figure 4.29b, where the double arrow is the fast axis and the single arrow is the slow axis (that finally indicates the direction of scan). The resulting fluorescence image, after application of mechanical friction, is shown in Figure 4.29b.

Figure 4 . 29 :

 429 Figure 4.29: Fluorescence images before (a) and after (b) consecutive applications of 250 nN friction forces on a polymerized TzDA1 single crystal prepared by recrystallyzation via 3 days liquid diffusion from a 0.5 mM solution of TzDA1 in MeCN with water. H 2 O:MeCN proportion is 8:2. Fluorescence is excited at 488 nm.

  500 nN at a scan rate of 0.8 µm•s -1 following a hand-drawn path that consists in the acronym PPSM. As shown in the resulting fluorescence image in Figure 4.30d and in the delta image obtained by subtraction of image d and c in Figure 4.30e, the signal has been partially recovered along the AFM scan.

Figure 4 .

 4 30f is the topography image after force manipulation and Figure4.30g is a detail image of the engraved acronym.

Figure 4 .

 4 Figure 4.30: Transmission (a), AFM toporaphy (b) and fluorescence (c) images of a polymerized TzDA1 single crystal prepared by recrystallyzation via 3 days liquid diffusion from a 0.5 mM solution of TzDA1 in MeCN with water. H 2 O:MeCN proportion is 8:2. Fluorescence (d), delta fluorescence (e) and topography (f ) images of the crystal after AFM engraving; zoom on the stimulated area of the crystal imaged by AFM (g).

  Figure 4.31.

Figure 4 . 31 :

 431 Figure 4.31: Scheme of averaging procedure. First, two consecutive images (Fluo 01 and Fluo 02) are subtracted to produce the delta image, Delta fluo 02-01. A threshold value is set to the delta image and all pixels with intensity higher than the threshold are counted to produce the average image on the right. The average signal is then extracted.

Figure 4 . 32 :

 432 Figure 4.32: Fluorescence images of three crystals of TzDA1 (yellow boxes), before (a) and after (b) friction forces application. Fluorescence is excited at 488 nm.

Figure 4 . 33 :

 433 Figure 4.33: Graphical representation of the fluorescence signal in each stimulated area versus the corresponding applied force on TzDA1 single crystals. Each point corresponds to the mean value of different measurements, and the error bars correspond to the standard deviation.

Chapter 6 Methods 6 . 1 6 . 1 . 2

 661612 Details of synthetic procedures 6.1.1 General syntheses, purification, and characterisation All reagents purchased from commercial supplier were used without further purification. Anhydrous solvents were dried and purified by passage through alumina columns. All reactions were monitored by thin layer chromatography (TLC) using E Merck TLC silica gel 60 F254, thickness 0.2 µm, and visualized under UV 254 nm or KMnO 4 revelation. 1 H and13 C nuclear magnetic resonance (NMR) spectra were recorded on a JEOL JMS ECS 400 MHz spectrometer (100 MHz for 13 C) at 25 • C. Chemical shifts are expressed in parts per million (ppm) and defined in respect to tetramethylsilane (TMS) = 0 ppm for 1 H and 13 C. The following abbreviations are used to describe the signals: s for singlet; d for doublet; t for triplet; q for quadruplet; m for multiplet. High resolution mass spectrometry was performed at the CNRS Imagif platform. Melting points were measured with a Stuart apparatus SMP10. Synthetic procedure for a single chain diacetylene: DA1 1-bromopentadec-1-yne

1 H

 1 NMR (CDCl 3 ): δ = 0.88 (3H, t, J = 7.0 Hz); 1.26 (20H, m); 1.5 (2H, m); 2.19 (2H, t, J = 7.2 Hz) 13 C NMR (CDCl 3 ): 80.7, 37.5, 32.1, 29.83, 29.80 (2C), 29.76, 29.6, 29.5, 29.2, 28.9, 28.4, 22.9, 19.8, 14.3 hex-5-yn-1-yl-propylcarbamate 2.21 mL (1.96 g, 20 mmol) of 5-hexyn-1-ol are dissolved in 75 mL of toluene and 3.20 mL (2.32 g, 23 mmol, 1.15 eq.) of triethylamine are added to the solution. 2.20 mL (2.00 g, 23.4 mmol, 1.17 eq.) of propylisocyanate are added afterwards. The solution is brought to reflux under stirring overnight. The product solution is washed 3 times with 20 mL of H 2 O and the water phase is washed 3 times with 20 mL of ethyl acetate (EtOAc). The organic phases are joined, dried on Na 2 SO 4 and concentrated. The brown oily solution obtained is purified by SiO 2 column chromatography with 9/1 PE/EtOAc, to give 3.01 g of hex-5-yn-1-yl propylcarbamate, (yield = 82%).

1 H

 1 NMR (CDCl 3 ): δ = 0.89 (3H, t, J = 7.2 Hz); 1.43 -1.54 (2H, m); 1.54 -1.62 (2H, q, J = 7.2 Hz); 1.65 -1.77 (2H, m); 1.94 (1H, t, J = 2.3 Hz); 2.20 (2H, t(d), J1 = 7.2 Hz, J2 = 2.3 Hz); 3.1 (2H, q, J = 6.4 Hz); 4.04 (2H, t, J = 6.2 Hz); 4.73 (1H, s) ppm.

13 C 3 196

 133 NMR (CDCl 3 ): δ = 11.3; 18.2; 23.5; 25.0; 28.3; 42.8; 64.2; 68.8; 84.1; 156.9 ppm. henicosa-5,7-diyn-1-yl propylcarbamate (DA1) 0.020 g (0.20 mmol) of copper chloride CuCl are introduced in a round bottom flask under argon flux. 0.47 mL of methanol, 1.35 mL of distilled water and 0.730 mL (1.32 mmol) of ethylamine are added. The solution takes an intense blue colour due to the presence of Cu 2+ ions in solution. 0.132 g (1.90 mmol) of hydroxylamine chloride are added to reduce Cu(II) ions to Cu(I), and the solution colour disappears. Under Chapter 6. Methods Ar atmosphere, 0.464 g (2.53 mmol) of hex-5-yn-1-yl propylcarbamate are added to obtain a bright yellow solution. 0.800 g (2.78 mmol) of 1-bromopentadec-1-yne are added slowly to the mixture and a foamy bluegreen precipitate appears. The reaction mixture is heated to 40 • C and let react overnight under stirring. TLC 9/1 PE/EtOAc shows of a new spot active in the UV 254 nm corresponding to DA1. The reaction solution is diluted with 10 mL of DCM and washed 4 times with 20 mL of NH 4 Cl sat . The organic phases are joined, dried on MgSO 4 and concentrated. The solid paste that is obtained is purified by SiO 2 column chromatography with 9/1 PE/EtOAc, to give 0.724 g of a white powder (yield = 73.5%) that polymerises very easily under ambient light, giving a blue powder. 1 H NMR (CDCl 3 ): δ = 0.90 (6H, m); 1.26 (18H, m); 1.52 (6H, m); 1.61 (2H, m); 2.24 (2H, t, J = 7.2 Hz); 2.29 (2H, t, J = 7.0 Hz); 3.13 (2H, t(d), J = 6.3 Hz); 4.06 (2H, t, J = 5.6 Hz); 4.65 (1H, s) 13 C NMR (CDCl 3 ): 156.8; 78.0; 65.9; 65.3; 64.2; 42.8; 32.1; 29.8; 29.6; 29.5; 29.2; 29.0; 28.5; 28.3; 25.0; 23.4; 22.8; 19.3; 19.1; 14.3; 11.Chapter 6. Methods HRMS: Chemical formula: C 25 H 43 NO 2 [M + H] + calculated = 390.3372 [M + H] + found = 390.3362 mp = 74.4 • C 6.1.3 Synthetic procedure for tetrazine substituted diacetylenes 6.1.3.1 TzDA1 hex-5-yn-1-yl propylcarbamateThe synthetic protocol of hex-5-yn-1-yl propylcarbamate is described in Section 6.1.2.6-bromohex-5-yn-1-ol 3.12 mL (3.58 g, 22.4 mmol, 1.1 eq.) of Br 2 are added to a thermostated at 0-5 • C solution of 4.50 g (80 mmol, 3.9 eq.) of KOH in water. 0.89 mL (2.00 g, 20.4 mmol) of 5-hexyn-1-ol are added dropwise to the solution. The reaction is let run for 2h protected from light. TLC shows the complete consumption of the starting compound. The product solution is extracted 3 times with diethylether, the organic phases are joined, washed 3 times with NaCl sat , dried on MgSO 4 and concentrated to give 3.50 g of 6-bromohex-5yn-1-ol (yield = 95%).

1 H

 1 NMR (CDCl 3 ): δ = 1.50 -1.70 (4H, m); 2.22 (2H, t, J = 6.9Hz); 2.30 (1H, s); 3.61 (2H, t, J = 6.0 Hz) ppm13 C NMR (CDCl 3 ): δ = 80.1, 62.0, 38.1, 31.6, 24.6, 19.5 ppm 12-hydroxydodeca-5,7-diyn-1-yl propylcarbamate 10.8 mL (7.97 g, 109 mmol, 10 eq.) of n-butylamine are dissolved in 55 mL of water and N 2 is bubbled in the solution.

13 C

 13 NMR (CDCl 3 ): δ = 11.4; 19.0, 19.1; 23.4; 27.4, 24.9; 28.3; 31.9; 42.7; 62.5; 64.2; 65.7; 77.4; 156.8 ppm 3-chloro-6-(dodecyloxy)-1,2,4,5-tetrazine 1.863 g (10 mmol) of dodecanol and 1.510 g (10 mmol) of dichlorotetrazine are dissolved in 70 mL of DCM. 1.59 mL (1.47 g, 11 mmol) of collidine are added under stirring. After 3h, TLC 9/1 PE/DCM shows the complete disappearance of dichlorotetrazine. The solution is concentrated and purified by SiO 2 column 198 Chapter 6. Methods chromatography 9/1 PE/DCM, to give 2.497 g of 3-chloro-6-(dodecyloxy)-1,2,4,5tetrazine, (yield = 83%).

1 H

 1 NMR (CDCl 3 ): δ = 0.88 (3H, t, J = 6.7 Hz); 1.26 (16H, m); 1.37 (2H, m); 1.94 (2H, m); 4.65 (2H, t, J = 6.6 Hz) ppm 13 C NMR (CDCl 3 ): δ = 14.3; 22.8; 25.8; 28.6; 29.3; 29.5; 29.6; 29.7; 29.8 (2C); 32.1; 71.3; 164.3; 166.9 ppm 12-((6-(dodecyloxy)-1,2,4,5-tetrazin-3-yl)oxy)dodeca-5,7-diyn-1-yl propylcarbamate (TzDA1)0.150 g (0.537 mmol) of 12-hydroxydodeca-5,7-diyn-1-yl propylcarbamate, 0.177 g (0.591 mmol, 1.1 eq.) of 3-chloro-6-(dodecyloxy)-1,2,4,5-tetrazine and 0.072 g (0.591 mmol, 1.1 eq.) of DMAP are added in a Schlenk that is previously dried under vacuum to remove any trace of humidity. The system is brought under nitrogen and 8 mL of DCM are introduced in the flask through a syringe. The reaction is let run under inert atmosphere at thermostated 7 • C over night. The product solution is concentrated on rotavapor and purified by SiO 2 column chromatography 9/1 PE/EtOAc to give 0.990 g of TzDA1, (yield = 33.9%).

6. 1 . 3 . 2

 132 TzDA2butyl ((hex-5-yn-1-yloxy)carbonyl)glycinate 2.20 mL (20 mmol, 1 eq.) of 6-hexyn-1-ol and 3.07 mL (22 mmol, 1.1 eq.) of triethylamine are diluted in 75 mL of toluene. 4.08 mL (27 mmol, 1.35 eq.) of butylisocyanoacetate are added to the reaction mixture which is heated to reflux and let react under stirring overnight. TLC (7/3 PE/EtOAc) reveals the absence of starting product. The reaction mixture is then washed 3 times with distilled water and the aqueous phase is washed 3 times with EtOAc. The organic phases are joint together, concentrated on rotavapor and purified by SiO 2 colum chromatography 7/3 PE/EtOAc to give 5.10 g of a yellow-brown oil (yield 84%).

1

  H NMR (CDCl 3 ): δ = 0.89 (3H, t); 1.34 (2H, m); 1.57 (2H, m); 1.71 (2H, m); 1.92 (1H, t); 2.18 (2H, d(t)); 3.91 (2H, d); 4.09 (4H, m); 5.26 (1H, t) 6-bromohex-5-yn-1-ol

1 H

 1 NMR (CDCl 3 ): δ = 0.91 (t, J = 7.3 Hz, 3H); 1.41-1.31 (m, 2H); 1.76-1.52 (m, 10H); 2.27 (t, J = 6.8 Hz, 2H); 2.28 (t, J = 6.8 Hz, 2H); 3.64 (t, J = 6.2 Hz, 2H); 3.93 (d, J = 5.5 Hz, 2H); 4.15-4.06 (m, 4H); 5.21 (br s, 1H, NH) ppm The characterizations match with the previously published compound, Barisien et al., ACS Appl. Mater. Interfaces, (2013), 21, 10836. 3-chloro-6-(dodecyloxy)-1,2,4,5-tetrazine The synthetic protocol of 3-chloro-6-(dodecyloxy)-1,2,4,5-tetrazine is described in Section 6.1.3.1.

  H 53 N 5 O 6 [M + H] + calculated = 616.4074 [M + H] + found = 616.4103 mp = 64.6 • C g (15 mmol, 1.5 eq.) of CrO 3 are added at 0 • C to 6 mL of distilled water in a round bottom flask. 1.130 mL (20 mmol, 2 eq.) of H 2 SO 4 95% w are then slowly added keeping the flask in the ice bath. 1.430 mL (10 mmol, 1 eq.) of 7-octyn-1-ol are dissolved in 11 mL of acetone and added dropwise to the stirring cromium solution at 0 • C. When finished, the mixture is let react at room temperature for 1h. TLC (9/1 PE/EtOAc) shows the absence of starting product and the appearance of two new spots. The reaction mixture is diluted with 10 mL of EtOAc and 20 mL of water, then washed 3 times with EtOAc. The organic phase is washed 4 times with 30 mL of NH 4 Cl sat , dried on MgSO 4 , concentrated on rotavapor and purified by SiO 2 column chromatography 9/1 PE/EtOAc, to give 0.692 g of oct-7-ynoic acid, (yield = 49%).

(

  2H, d(t)); 2.37 (2H, t); 11.2 (1H, s) ppm13 C NMR (CDCl 3 ): δ = 179.7; 84.4; 68.5; 33.9; 28.23; 28.19; 24.3; 18.4 ppm N-propyloct-7-ynamide 0.455 g (3.22 mmol, 1 eq.) of oct-7-ynoic acid and 0.280 mL (3.54 mmol, 1.1 eq.) of SOCl 2 are diluted in 20 mL of toluene. The reaction mixture is heated to reflux, let react for 3 hours and eventually brought back under Ar atmosphere to room temperature. 0.700 g (11.8 mmol, 3.7 eq.) of N-propylamine are added to the mixture under stirring. After 3h, TLC (8/2 PE/EtOAc) shows the complete disappearance of the starting compound and the presence of a new spot. The reaction mixture is then washed 3 times with distilled water and the resulting aqueous phase is washed 3 times with DCM. The organic phases are joint together, dried on MgSO 4 and concentrated on rotavapor to give 0.516 g of a yellow-brown paste corresponding to N-propyloct-7-ynamide, (yield = 88.5%).

1 H

 1 NMR (CDCl 3 ): δ = 0.92 (3H, t, J = 7.6 Hz); 1.25 (1H, s); 1.4 -1.7 (12H, m); 2.16 (2H, t, J = 7.4 Hz); 2.21 -2.33 (4H, m); 3.2 (2H, m); 3.67 (2H, m); 5.42 (1H, s) ppm 13 C NMR (CDCl 3 ): δ = 172.9; 65.8; 65.5; 41.3; 36.8; 31.9; 28.5; 28.1; 25.6; 24.7; 23.1; 19.2; 19.1; 11.5 ppm 3-chloro-6-(dodecyloxy)-1,2,4,5-tetrazine The synthetic protocol of 3-chloro-6-(dodecyloxy)-1,2,4,5-tetrazine is described in Section 6.1.3.1. 14-((6-(dodecyloxy)-1,2,4,5-tetrazin-3-yl)oxy)-N-propyltetradeca-7,9-diynamide (TzDA3)

Table 2 .

 2

1: Photophysical data of monomeric TzDAs in acetonitrile

Table 4 .

 4

	1: Parameters for TzDA1 reprecipitation and sonication
	Reprecipitation
	Solvent	Initial	Proportions H 2 O/solvent
		concentration (mM)
	THF	0.1 0.5	8:2 8:2
	MeCN	0.25 0.5	8:2 8:2
	Sonication in water
	Final	Temperature ( • C)	Time (min)
	concentration (mM)
	0.5	60	15

Table 4 .

 4

	2: Parameters for TzDA2 reprecipitation and sonication
	Reprecipitation
	Solvent	Initial	Proportions H 2 O/solvent
		Concentration (mM)
		0.05	8:2
	THF	0.1	8:2
		0.5	8:2
		0.1	8:2
		0.25	8:2
	MeCN	0.5	8:2
		1	9:1 8:2
	Sonication in water
	Final	Temperature ( • C)	Time (min)
	Concentration (mM)
	0.1	40	30
	0.5	40	30
	0.5	70	15

  (1H, s) ppm 202 Chapter 6. Methods 13 C NMR (CDCl 3 ): δ = 170.3; 166.3; 166.1; 156.6; 70.2; 69.3; 66.1; 65.8; 65.5; 64.8;

42.8; 32.0; 30.7; 29.8; 29.7; 29.6; 29.5; 29.4; 28.8; 28.2; 27.9; 25.9; 24.8; 24.7; 22.8; 19.2; 19.0; 14.2; 13.8 ppm HRMS: Chemical formula: C 33

Table 6 .

 6 1: Crystal data collection and refinement parameters Empirical Formula C 30 H 49 N 5 O 4

	M r	543.74
	Crystal size, mm 3	0.010 x 0.030 x 0.040
	Crystal system	triclinic
	Space group	P1
	a, Å	7.1986(18)
	b, Å	7.5910(18)
	c, Å α, °84.702(17) 28.718(7) β, °89.324(18) γ, °80.32(2) Cell volume, Å3 1540.4(7)
	Z ; Z'	2 ; 2
	T, K	100 (1)
	Radiation type ; wavelength Å	CuKα; 1.54178
	F000 µ, mm -1 Range, °3.091 -59.739 592 0.625
	Reflection collected	11 010
	Reflections unique	7 342
	R int	0.2487
	GOF	0.984
	Refl. obs. (I>2(I))	1 932
	Parameters	306
	wR 2 (all data)	0.3950
	R value (I>2(I))	0.1715
	Largest diff. peak and hole (e-. Å-3)	0.327 ; -0.298
	6.3 Photophysical studies and characterisations
	6.3.1 Steady-state spectroscopy

Chapter 1. Bibliographic review

Chapter 1. Bibliographic review

Chapter 2. New polydiacetylenes as force sensors

Chapter 2. New polydiacetylenes as force sensors

Chapter 4. Polydiacetylene microcrystals

Conclusion & perspectives

Acknowledgements

General introduction

Acknowledgements

Standard silicon tip, variable scan rate. In order to optimize the contact mode scan rate, a range of tip velocities was tested at the same nominal force of 150 nN. Figure 3.15a shows the resulting fluorescence image and Figure 3.15b shows the conversion from Hz (trace + retrace line per second) to µm•s -1 . Scan rate ranging from 0.1 to 0.7 Hz produces a high fluorescence signal on the borders of the scanned area, leaving a non fluorescent zone in the middle. These slow scan rates favour material displacement and tip pollution, which is proven by the highly fluorescent spot on the top left corner of the scanned area corresponding to 0.3 Hz, where all the material previously attached is released. On the contrary, fast rates from 1.1 to 1.5 Hz do not ensure a homogeneous application of the nominal force along the scan lines, since the feedback loop of the Z piezo needs to be very fast as well. The most homogeneous response in fluorescence along with the certainty of applying the chosen vertical nominal force was found for scan rates between 0.8 and 1.0 Hz. The intermediate value 0.9 Hz (corresponding to 6.1 µm•s -1 for a 3x3 µm 2 scan area) was then chosen as default speed of scan.

Carbon coated diamond-like tip, 6.1 µm•s -1 scan rate. The tip material, as explained, also influences the quality of the mechanical stimulation, affecting the probability of tip pollution. For this reason, a carbon coated diamond-like tip was used, which is more chemically inert and mechanically resistant compared to the standard silicon ones previously used. Figure 3.16a shows the fluorescence image obtained after mechanical stimulation performed with the diamond-like tip at 6.1 µm•s -1 . The response is homogeneously distributed over the sheared area, for nor-highly fluorescent in the monomeric state (as deposited) as shown in Figure 4.17f, and switch to an OFF state when TzDA1 is polymerized as shown in Figure 4.17h. The fluorescence is estimated to be quenched by 4-fold the initial value (I F (mono) = 1200 counts, I F (poly) = 300 counts, I F (background) = 220 counts).

Summarizing, changing the H 2 O:MeCN proportions in the reprecipitation of a TzDA1 0.5 mM solution has a great impact on the size and shape of the crystals that are formed after deposition and drying. H 2 O:MeCN 8:2 produces rod-like crystals that range between 50 nm and 1 µm in length and approximately 200 nm in height.

On the other hand, H 2 O:MeCN 5:5 leads to much bigger crystals that do not exceed few hundreds of nm in height but can extend up to several µm in length and width, confirming a squared plate-like shape. No matter the size and shape of the crystals, they are all fluorescent in the monomeric form, and they all quench tetrazine emission after UV light irradiation, which stands as a proof of successful polymerization of the crystals. The large crystals obtained with 5:5 proportion fluoresce more intensely than the rod-like structures obtained by 8:2 proportion, with maximum recorded intensities of respectively 700 and 1200 counts.

Morphology before and after polymerization is studied by AFM microscopy: the same morphological features of the monomer crystal are retained after polymerization, independently of size and shape of the crystal. Suspensions of TzDA2 are prepared by mixing a starting solution of TzDA2 in 0.154 g (0.55 mmol, 1 eq.) of 14-hydroxy-N-propyltetradeca-7,9-diynamide, 0.196 g (0.65 mmol, 1.2 eq.) of 3-chloro-6-(dodecyloxy)-1,2,4,5-tetrazine and 0.080 g (0655 mmol, 1.2 eq.) of DMAP are added in a Schlenk that is previously dried under vacuum to remove any trace of humidity. The system is brought under nitrogen and 5 mL of DCM are introduced in the flask through a syringe. The reaction is let run under inert atmosphere at thermostated 3 • C over night. The product solution is concentrated on rotavapor and purified by SiO 2 column chromatography 8/2 PE/EtOAc to give 0.303 g of TzDA3, (yield = 26.7%).

1 H NMR (CDCl 3 ): δ = 0.90 (6H, m); 1.26 (16H, m); 1.57 (10H, m); 1.77 (2H, q, J = 7.3 Hz); 1.90 (2H, q, J = 7.1 Hz); 2.03 (2H, q, J = 7.0 Hz); 2.17 (2H, t, J = 7.4 Hz); 2.26 (2H, t, J = 7.0 Hz); 2.37 (2H, t, J = 6.8 Hz); 3.22 (2H, q, J = 6.7 Hz); 6.2 TzDA1 X-ray crystal structure X-ray diffraction data for compound TzDA1 was collected by using a VENTURE PHOTON100 CMOS Bruker diffractometer with Micro-focus IuS source Cu Kα radiation. Crystals were mounted on a CryoLoop (Hampton Research) with Paratone-N (Hampton Research) as cryoprotectant and then flashfrozen in a nitrogen-gas stream at 100 K. For compounds, the temperature of the crystal was maintained at the selected value by means of a N-Helix Cryostream cooling device to within an accuracy of ±1K. The data were corrected for Lorentz polarization, and absorption effects.

The structures were solved by direct methods using SHELXS-97 and refined against F2 by full-matrix least-squares techniques using SHELXL-2018 with anisotropic displacement parameters for all non-hydrogen atoms. Hydrogen atoms were introduced 206

Chapter 6. Methods into the calculations as a riding model with isotropic thermal parameters. All calculations were performed by using the Crystal Structure crystallographic software package WINGX. The crystal data collection and refinement parameters are given in Table 6.1 and the asymmetric cell representation is shown in Figure 6.1.

Fluorescence quantum yield and molar extinction coefficient in solution

Fluorescence quantum yields in solution were measured using rhodamine 6G in ethanol (literature Φ F = 95%) as the reference. All solutions were diluted to have an OD < 0.1. The fluorescence quantum yield of the samples Φ F (x) were calculated using the following equation:

with Φ F (0) and Φ F (x) the fluorescence quantum yield of the reference and of the sample respectively; S 0 and S x are the integrated fluorescence spectra of the reference and the sample; A 0 and A x are the values of the absorbance of both samples at the chosen excitation wavelength; n 0 and n x are the refraction index of the solvent and the solution, respectively.

Time-resolved spectroscopy

Emission lifetime measurements were performed via Time-Correlated Single Photon Counting (TCSPC) method. Excitation was done via Titanium:Sapphire femtosecond oscillator laser (Tsunami, Spectra Physics) pumped by a double intra-cavity Nd:YVO4 laser (Millenia Xs, Spectra Physics). The wavelength was adjusted at 720 nm at the output of the oscillator. Repetition rate was reduced to 4 MHz by a pulse selector (model 3980, Spectra Physics). The wavelength at 360 nm was generated by focalizing the fundamental beam at 720 nm inside a nonlinear SHG crystal. The pulse duration of the excitation laser at 360 nm was less than 500 fs. The emission was detected perpendicular to the excitation beam through a low pass filter (to remove the excitation light), an analyzer at magic angle, a monochromator, and a MCP-PMT detector (R3809U-50, Hamamatsu) connected to a TCSPC card (SPC 630, Becker & Hickl). The instrumental response function was recorded for each decay curve. The data were analyzed with multiexponential functions including reconvolution analysis, using the Globals software (Laboratory for Fluorescence Dynamics at the University of California, Irvine).

Solid state fluorescence quantum yield

Absolute solid state Φ F were obtained by means of a Quanta-φF-3029 integrating sphere by Horiba scientific. Fluorescence was excited at 360 nm in TzDA1 and at Chapter 6. Methods 209 488 nm in DA1. The ratio of emitted and absorbed photons (quantum yield) was obtained using the calculation sheet provided by the constructor.

Low temperature fluorescence spectra

The measurements were performed on vacuum deposited TzDA1 on glass slides inserted in an optical cryostat by Oxford instruments (Optistat DN) on the Fluorolog FL3-221 spectrofluorometer.

Estimation of film thickness by UV-Vis absorption

For an absorbing thin film

where αis the absorptivity of the film, the OD is the absorbance at the S 1 transition of the tetrazine at 530 nm and d is the thickness of the film. For an 82 nm thick film of TzDA1 it is found α= 1.5 • 10 4 cm -1 . Using this value for TzDA2 thin film gives a thickness d = 80 nm, while for TzDA3 thin films gives a thickness of d = 100 nm.

Estimation of polymer content by UV-Vis absorption

Assuming an absorptivity coefficient for a general PDA chain, at the absorption band of the blue phase α= 2 • 10 5 cm -1 , we know that:

where OD 100 is the optical density at 100% polymer content. Knowing the thickness of the film d, it is possible to calculate the hypothetical OD 100 (absorbance as if the film had completely polymerized. It is obtained for TzDA1 and TzDA2 (80 nm thick), OD 1 00 = 0.7; for DA1 (300 nm thick), OD 1 00 = 2.6. Using the real OD at 600 nm for TzDA1 and TzDA2, and at 635 nm for DA1, the real polymer is calculated with the following formula:

It is obtained:

TzDA1: 0.0644 0.7 • 100 = 9.2% TzDA2: 0.0495 0.7 • 100 = 7.1% DA1: 0.350 2.6 • 100 = 13.5% 210 Chapter 6. Methods

Microscopy study 6.4.1 Coverglass cleaning

The coverglasses were preliminary cleaned before film or suspension deposition by several steps of sonication in the following solvents: 1% Hellmanex water solution, acetone, ethanol, 1M sodium hydroxide water solution and distilled water, then dried with a heat gun.

Thin film preparation

The deposition of thin films was carried out using a Leybold Univex 300 vacuum evaporator. The pure organic powder obtained from the synthesis was introduced in the crucible (20 mg of DA1, 11.2 mg of TzDA1, 12 mg of TzDA2) and thin films were obtained. The crucible, placed in a vacuum chamber (2•10 -5 mbar), was heated by means of a tungsten filament source and the temperature was controlled by a thermocouple. Film thickness and growth rate were monitored in situ by a piezoelectric quartz crystal system. Film thickness was measured by manually performing a scratch and measuring the step height in 5 different points along the scratch.

DA1

Fluorescence microscopy

The images acquired by fluorescence microscopy were recorded on an inverted microscope (Nikon, Eclipse Ti-U) used in wide field. The samples were excited by a Lumencor Spectra X light engine at 488 nm. The fluorescence signal is collected in epifluorescence by the objective lens Nikon Plan Apo λ 60x/0.95. The fluorescence signal is separated and filtered from the incident excitation beam by Semrock optics (LF488/LP-B-000 dichroic mirror, BLP01-488R emission filter) and directed to an EMCCD (Andor; iXon Ultra 897). All the images were analysed by the Solis software of Andor. Polymerisation by irradiation at 254 nm was performed in situ, by selecting the mercury line of Hg/Xe light source.

Measurement of irradiation area at 254 nm was imaged by optical fluorescence microscopy on polymerized films, where fluorescence is quenched in the irradiation spot.

AFM measurements

AFM images were acquired with a JPK NanoWizard® 3 AFM. The head of the AFM was designed to be coupled with the Nikon microscope, allowing the correlation of object position, size and shape with its photophysical properties. AFM images were recorded in tapping mode or in QI mode under ambient conditions. The thermal tune calibration, included in the JPK software, was used to make the calibration of the spring constant. Image analysis was performed using the JPK analysis software. Statistical analyses of topography images were performed using Gwyddyon software. Chapter 6. Methods