Acknowledgement

The real friendship is like fluorescence; it shines better when everything has darkened. -Rabindranath Tagore On the 6th of February 2023, Prof. Dr. Ben L. Feringa, the 2016 Laureate of the Nobel Prize in Chemistry, said to me: "It is my great honor and pleasure to be the first to congratulate you with this achievement, this title. I wish you all the best and success in the future." That day, the day I obtained my doctorate, was one of the most beautiful days of my life. I couldn't have achieved this without the support of many people. These past three years have been full of ups and downs, successes and failures, which have all shaped me into the person I am today. I want to express my sincere gratitude to all the individuals who have directly and indirectly, near and far, played a part in the success of this thesis work.

First and foremost, I would like to thank the members of my thesis jury for having carefully read my work and to have evaluated it. I thank Prof. Dr. Ben L. Feringa, president of the jury, for the numerous feedback and (also the compliments) given during my defense. It is one of the biggest honors of my life to have him as a member of my jury. My sincere gratitude to Dr. Fabienne Gauffre and Prof. Dr. Hugues Bodiguel for agreeing to examine my work. It means a lot to me that they were so understanding and willing to take on the task in such a short period of time. I appreciated very much our exchanges as well as their constructive remarks, which allowed me to have a critical look at my work. My heartfelt thanks to Prof. Dr. Sebastien Lecommandoux for his valuable remarks. It is truly an honor to have had the opportunity to integrate the LCPO for a period of two years, and I am grateful for his welcome and support throughout my time there.

My thesis was mainly carried out at the Laboratory of the Future (LOF), a joint research unit between the Solvay group, the University of Bordeaux, and the CNRS. It was indeed an incredible chance, and for that, I would like to thank Mr. Kazuhiko Yokota, Dr. Patrick Maestro, and Dr. Mikel Morvan, the successive directors of the laboratory, for allowing me to carry out this thesis in such good, conducive conditions. Patrick, thank you for believing in me and giving me a chance to grow. Your support and encouragement have been invaluable, and I am grateful for the advice and ideas you have shared with me. Mikel, thank you for the constant motivation (especially during the days before my defense) and the many reminders to return home.

A special thanks to Dr. Pierre Guillot, the UMR team manager, for being a great team leader, especially during the tough Covid-19 crisis times. Pierre, I truly appreciate your kindness, patience, and availability. It means a lot that you always check on me, not just when things are going well but especially during hard times. I am grateful for all the support and guidance you've given me. Thank you for everything! Now, I would like to extend my warmest thanks to my thesis director, Dr. Yaocihuatl Medina-Gonzalez. Yao, thank you for choosing and trusting me to be a part of this project.

Your constant support, advice, and help have been invaluable to me, and I wouldn't be the person I am today without it. I also appreciate your patience with me throughout the process. I have learned so much from you, especially when it comes to defending myself and navigating the academic world. You are one of the strongest women I know; working with you has been a good experience! Next, I would like to express my sincere gratitude to the co-supervisors of my thesis: Dr. Pierre Lidon and Dr. Simon Harrisson. Pierre, thank you so much for sharing your knowledge and experience with me. I appreciate you taking the time to teach me how to code in Python and MATLAB and for being so patient with me. It means a lot that you were always available to help me despite your busy schedule. But even more than that, I want to express my gratitude for your passion for teaching. You make learning fun, and it's been inspiring to see how enthusiastic you are about helping others learn and grow. Simon, thank you for being extremely patient with me; your kindness and encouragement have been a constant source of motivation for me.

Throughout my time at the LCPO, you have shown me remarkable understanding, always taking the time to explain difficult concepts (repeatedly!), providing me with constructive feedback, and finding solutions to my many unsuccessful experiments. Thank you for always checking on me while I was "cooking" in the lab. I truly believe that I would not have survived this whole new polymer world without your guidance.

There are indeed not enough words to thank all three of you. As all of you know, the writing process was very complicated for me, but I made it through it thanks to your firm support. You have made an immeasurable impact on my life, and I will always look back on this experience with gratitude.

Next, I thank my MicroVISCOTOR project partner, Florence Gibouin, for developing an awesome MATLAB GUI that has made my job a breeze. I also really appreciate how supportive and helpful you have been throughout this project -you are a great teammate! Also, my sincere gratitude towards my amazing lab mate, Gerald Clisson. He has been an exceptional support system for me whenever I face any challenges in the lab. I am truly grateful for everything he has taught me about microfabrication and microfluidics and for his help and support throughout my research journey. I couldn't have made it this far without him. Gerald, you're my lab lifesaver! I would also like to express my appreciation to my interns, Julien Renaudeau and Maxime Zitouni, for their contributions to my thesis project. They worked hard, shared valuable ideas, and helped me achieve my research goals. Both of you made a big difference, and I am glad to have worked with you! Having a supportive environment is really important when it comes to personal and professional growth. Luckily, the LOF is just the place for that! It is heartening to see that everyone from permanent staff to Ph.D. candidates, Post-docs, and interns contribute to a positive atmosphere with their good mood and benevolence.

I would like to thank my colleagues at the LOF in no particular order: Alexandra, Marie, Léa, Célia, Tiphaine, Samantha, Gaelle, Emie, Ophelie, and Jamilla for always greeting me with a big bright smile and a warm "Coucouuu/Salut, Dharshana!", Maewenn (for our shared passion and love for the little things in life such as pâquerette), Arnaud (for being extremely nice to me, for teaching me many things on French culture, for your little gifts and sweet gestures), Charles (for your jokes, for your fun facts and for being a very good listener. Thank you for always being there for me!), Sabrina (a constant motivator, I'm grateful for our lunch dates and coffee machine discussions), Pierre Legout (for your insightful suggestions and help during the writing period), Celeste (for your constant moral support and for forcing me to go back home), Thomas (for believing in me and for your many bits of help); Cedric, Matthieu, Julien, Roberto, and Damien (for your helping hand and good humor). A special mention to occupants of the Petanque's Open Space for your ever-present good mood: Jordy, Roman, Baptiste, Celine Guillaume (for your love towards Malaysia and 100Plus as well as our numerous discussions on that matter), and Jean Gimenez (for your help and discussions on a variety of topics). I am also thankful to Dr. Jacques Leng, Dr. Guillaume Ovarlez, Dr. Jean-Baptiste Salmon, Dr. Steve Meeker, and Dr. Emmanuel Mignard for their perspectives, ideas, and constant encouragement throughout my 4-year journey at the LOF. They have been instrumental in helping me achieve my goals.

Not forgetting the people of St.Estephe: Camille (who taught me many kinds of stuff on microfluidics and life), Moncef (my MATLAB guru and a dear Fresnel lab mate), Nicolas Belicard (a beloved member of Docteurs Survivors, a role model of calmness and perseverance), Djibrilla (for being the wise man of the Open Space, for patiently answering my many, many questions about life), Marleine (for being the sweetest big sister, for all your help and for taking care of me), Margot (for our shared passion in cooking and for many other interesting things that you taught me), and the newcomer, Mingshan (for your calm attitude. All the best to you!), Dmytro, Justine, and Thanh (for your kindness, for sharing your knowledge and experience), and Shi Zhang (a fellow Asian at the lab, thanks for your suggestions for good food).

On top of that, I would like to extend my sincere gratitude to everyone at the LCPO for their warm hospitality and incredible kindness. In particular, I am incredibly grateful to Hanaé Dupont, Valentin Maingret, Samir Zaky, Assia Stiti, Nadia Singraff, Karine Abou Ezza, Barbara Farias, Alban Charlier, Chloé Pascouau, Peter McMichael, Dijwar Yilmaz, Margaux Haurat, Manon Carré, Péroline Helbing, Simon Colanges (also an ex-Lofeur), Vaiana Royer, Abang Azmil and Corentin. As someone who came into the lab without any prior knowledge of polymer synthesis, I truly appreciate how patient and understanding everyone has been with me. Your willingness to guide me through the process has made all the difference, and I couldn't have made it this far without your support. Also, your constant encouragement has meant the world to me, and I truly appreciate all of the insightful discussions we've had together. I will always cherish my time at LCPO and the memories we've made together. Thank you all so much for everything! "There are friends, there is family, and then there are friends that become family."

A special shout-out to my dear friends, Chloé Garat and Leila Khacef! Your support and friendship mean the world to me. Leila, thank you for being there for me like a sister, always checking up on me and making sure I am doing alright. Chloé, thank you for always being there for me, reminding me to take care of myself, and showing me what it means to be human. Aymerick, thank you for being a motivational force and a big brother figure (even though you're younger than me); I learned so much from you. Thank you for the unlimited glasses of Gin-Tonic, and I will never forget the chicken McNuggets; they will always be the best. Alexie, thank you for your lovely friendship and for always being a constant motivator. Your kindness during the last week of the writing period will never be forgotten. You are one of the kindest people I know; keep spreading love, sweetheart.

Nizar, thank you for being my emergency MATLAB rescuer and always entertaining me with your unique statements and second-degree humor. You truly know how to keep me entertained. Margaux, my microfluidic (and sometimes drinking) compatriot, thank you for being a wonderful colleague, always supporting me, and of course, for organizing my gifts. You did a great job! Next, I would like to express my sincere gratitude to my foster family members: Isabelle Michel, Patrick Ellul, Joanna Ellul, Hélène Caputo, and Clelia Caputo, for everything you have done for me. From the moment I arrived, you have provided me with support, care, and kindness, and I feel incredibly fortunate to have you as my French family. I will always cherish our memories together. To Grandpa and Ma of the Michel family, thank you so much for driving all the way from St. Nazaire to Bordeaux for my thesis defense. I want to express my heartfelt appreciation to Agnes and Thibault for everything they have done for me. Both of you and my sweet boiii, Biscuit, are like family to me, and I am so grateful for your endless support and kindness that have made my time in Bordeaux much more enjoyable. Many thanks to my ENSIACET girl gang: the sweetest Angie, Audrey, Estelle, Marie, Marie-Ange, Viena, and Zeying for their constant support and for the beautiful friendship we share. Thanks a bunch to my friends in Malaysia and France: Abishek, Arvinderan, Dhivina, Heeran, Cass, Sharviin, Thanusha, Kiridhran, Rajanaandini, Geo, Vincent, Borhan, Aaron, Jiayau, Clement, Peggy, Cheng Kuan, Jessica, Nabilla, Yvonne, Hasanathull, Amalina, Thaayaleni, Leeshaalenee, and Dahrshini; for the constant moral support and steadfast friendship they have shown me.

All of you have been such amazing friends and have always had my back; I am indeed blessed to have you in my life.

A big shout-out goes to my friend, Seema, for always being there for me. She's seriously been a lifesaver, and I can't thank her enough for all the love and support she's given me over the years. I couldn't have made it this far without her help, and I just want her to know how much I appreciate her. So, Seema, this one's for you -thanks for being an awesome friend and always having my back! I want to express my sincere gratitude to my dear friend, Nivetha, as she's been a wonderful source of motivation and support for me all the way from Malaysia! I appreciate our healthy sisterhood and the fact that we can be understanding (amazing!) pen-pals. Not forgetting, Theju for always being there for me since 2005.

Goundamani, my sweet little darling hamster, thank you for being a part of my life; you were a blessing. During my lowest point, my siblings, Vhovignesh (Aiyaa) and Rivaseni, provided me with constant support; they never let me give up and were always there for me. I couldn't have asked for better siblings, and I am so thankful for them. Our bond is something that I will always cherish and hold close to my heart. I love both of you so much! Last but not least, I want to express my gratitude to my parents, Mr. Nalatamby and Mrs. Ramayee, for their unwavering support and motivation throughout this thesis and throughout my life. They have made so many sacrifices to ensure that I have had the best opportunities and experiences possible, and I cannot thank them enough for their selflessness. Above all, their love has always been a guiding light and source of strength for me. I am truly blessed to have such amazing parents. I love you very much, and I dedicate this thesis to you. De plus, une étude détaillée du processus photochimique des rotors doit être réalisée afin de mieux comprendre le comportement d'un polymère intégré dans un FMR et greffé sur une surface. D'autre part, un capteur polymère à l'état solide basé sur la FMR à l'état solide pourrait également être développé via la technique de "grafting-from", par exemple, via la polymérisation radicale par transfert d'atomes médiée par cuivre, qui peut fournir une liaison plus forte à la surface par rapport à la méthode proposée dans ce travail. En résumé, le travail présenté dans ce manuscrit a prouvé que la FMR est un outil efficace pour étudier quantitativement et localement, l'écoulement des fluides dans des géométries confinées à l'échelle de la micro-longueur.

General Conclusion

Bibliography

List of Abbreviations

Introduction

Through thick and thin. . . Viscosity plays a crucial role in many industrial processes involving fluid flow, and a local viscosity measurement is important because the fluid dynamics at the microscale and the microrheological behavior are often different from their bulk characteristics. The most common tool to measure this quantity is viscosimeters; however, their use requires great care.

More importantly, viscosimeters only measure a viscosity averaged over macroscopic volumes and are challenging to implement online. This strongly limits their application in industrial processes, small-volume systems, and confined systems. The impossibility of obtaining local data also impedes their use with complex flows involving spatial and/or temporal heterogeneity, as is commonplace both in macroscopic flows (e.g., in industrial systems like paint manufacturing or food processing) and at small scale (e.g., in microor millifluidics multiphase flows or porous media, with applications in enhanced oil recovery). Designing tools for small-scale viscosity measurement is thus important both for fundamental studies and industrial applications.

On the other hand, microfluidic systems have been widely used as an essential tool in numerous applications such as high-throughput screening and chemical reaction analysis, leaving open the possibilities for viscosity measurements. This thesis develops a straightforward, innovative methodology for local viscosity measurements in microfluidic devices, and the corresponding research and development works are presented in four chapters. The first chapter is dedicated to general explanations in order to introduce and elucidate the term: Fluorescent Molecular Rotor(s) (FMR) and the main concepts addressed in this work. Previous works on microviscosity measurements using FMR in several domains are also provided.

Chapter 2 is consecrated to the quantitative local viscosity mapping in microfluidic flows with a newly characterized BODIPY-based FMR using a fluorescence lifetime imaging microscope (FLIM). First, these rotors were utilized in the well-controlled situation of a co-flow consisting of two miscible fluids in a Y-mixer. In such a configuration, mixing occurs only via transverse diffusion. The retrieved experimental viscosity profiles and interdiffusion coefficient were in agreement with the model and experiments from the literature. Our method was then applied to characterize more complex flows, namely the chaotic mixing of two streams in a passive micromixer. The presented findings prove that the developed method can be used quantitatively for complex microfluidic systems.

The third chapter is focused on the development of an FMR-functionalized statistical copolymer as a viscosity-sensitive polymeric material. Viscosity-sensitive monomers were synthesized via condensation of cyanoesters and julolidine aldehyde to achieve this goal. The FMR-functionalized monomer was copolymerized with dimethyl acrylamide (DMA) via Reversible Addition-Fragmentation chain Transfer (RAFT) polymerization. The polymer's fluorescent response to changes in viscosity in solution is evaluated.

Following up on the work described in Chapter 3, a detailed investigation of grafted viscosity-sensitive fluorescent polymers to obtain solid-state FMR is presented in Chapter 4. This study eventually led to the development of a viscosity-sensitive microfluidic chip by attaching a polydimethylsiloxane (PDMS) microchannel onto a polymer-grafted surface. The engineered device is capable of measuring and mapping online and in real-time local viscosities of fluid using less than 50 µL of a sample. While still a simple proofof-concept, after optimization, this device could be used in the long run as an original local viscosity diagnosis tool as it would be a portable, fast, low-cost probe that facilitates high-throughput screening of local viscosity. Applications of this prototype could also be proposed in organs-on-chips, advanced materials, and online measurement fields.

The manuscript comes to a completion with the General Conclusion, summarizing the developed methodology and obtained results. Several research outlooks and recommendations are proposed as well. In a nutshell, the work presented in this manuscript proved that FMRs are an effective tool for quantitatively and locally studying fluid flow in confined micro-length scale geometries.

The History of Fluorescence

Once upon a time, in a land not too far away 1 , Sir George G. Stokes was observing the ability of uranium glass and fluorspar to change ultraviolet (UV) light into radiation of longer-wavelength blue light. In his well-known paper, "On the Refrangibility of Light," Stokes wrote, in a footnote on page 479: I am almost inclined to coin a word, and call the appearance fluorescence, from fluorspar, as the analogous term opalescence is derived from the name of a mineral [START_REF] Stokes | On the Change of Refrangibility of Light[END_REF]. Stokes was inspired by the observation reported by Sir John Frederick William Herschel in 1845, who described the blue light emitted from the quinine in transparent tonic water when exposed to sunlight [START_REF] Herschel | IV.'aµóρφωτ a, no. I.-on a case of superficial colour presented by a homogeneous liquid internally colourless[END_REF]. Stokes also noted that the fluorescence emission always happens at a longer wavelength, this change is known as the Stokes shift. 2 Interestingly, one of the earliest known records of fluorescence can be dated back to mid 16 th century when Nicolás Monardes observed this phenomenon in an infusion of a specific wood from Mexico, used to treat kidney and urinary diseases [START_REF] Valeur | A brief history of fluorescence and phosphorescence before the emergence of quantum theory[END_REF]. This could be the first application of the phenomenon that Stokes would later call fluorescence. Since then, fluorescence has found many applications from daily life products such as washing powder 3 , to decorative items, jewelry, and many more. 1 In England in 1852. 2 This will be further discussed in Section 1.2.

3 Through the addition of optical brighteners or fluorescent whitening agents.

Fluorescence in General

Fluorescence, a subgroup of luminescence, is the characteristic of an atom or molecule to emit light at a specific wavelength after absorbing light at a shorter wavelength (i.e., with a different color than the emitted light). Fluorescence-emitting molecules are referred to as fluorophores, fluorescent probes, or fluorochromes, and these molecules are generally composed of a few aromatic groups, planar or cyclic compounds with several π-bonds [START_REF] Lakowicz | Principles of Fluorescence Spectroscopy[END_REF]. Essentially, the fluorescence mechanism depends on various occurrences happening at different timescales, as shown in proposed the schema to illustrate light absorption and emission [START_REF] Lakowicz | Principles of Fluorescence Spectroscopy[END_REF]. The significant contributions to the theory of fluorescence by French physicists Jean Baptist Perrin, and his son Francis Perrin, resulted in their name being associated with the well-known diagram.

Fluorescence is observed when a molecule in the first electronically excited singlet state returns to the ground state by emitting a photon [START_REF] Lakowicz | Principles of Fluorescence Spectroscopy[END_REF]. The total fluorescence efficiency is lowered by other competing processes, for instance, internal conversion or intersystem The molecule would then undergo rapid internal conversion, vibrational relaxation, or both to the lowest energy level of S 1 . In general, fluorescence takes place from there to the higher vibrational levels of S 0 in a matter of nanoseconds. Thus, the energy of fluorescence emission is lower than the absorption energy, and as a result, the wavelength of the emitted photon is longer than that of the absorbed photon. The difference in absorption and emission wavelength is known as the Stokes shift. Also, there is a transition where molecules in excited singlet states shift into the triplet state, T 1 , and this is known as intersystem crossover. The photon emission from the T 1 state to the S 0 is called phosphorescence. As the energy in the T 1 state is lower than the S 1 state, phosphorescence takes place with a longer emission wavelength than the fluorescence emission.

In addition to the presented transitions, the fluorescence emission of certain fluorophores is greatly affected by the internal conversion from the lowest energy of S 1 state to the ground state. This relaxation usually occurs without any photon emission. The following section will be focused on this unique group of fluorophores and their fluorescence mechanism.

What are Fluorescent Molecular Rotors?

Since the mid-90s, single-molecule rotors or more sophisticated nanomachines that could induce unidirectional movements have garnered interest in the domain of scientific research. So much so that the development of "Molecular Machines" has led Jean-Pierre Sauvage, Sir J. Fraser Stoddart, and Bernard L. Feringa to receive the 2016 Nobel Prize in Chemistry. These molecules are a million times smaller than a hair strand, and yet they were able to rotate a 28 µm-long glass cylinder while being embedded in a liquid crystal film [START_REF] Eelkema | Nanomotor rotates microscale objects[END_REF].

The backbone of this thesis, the fluorescent molecular rotors (FMR), belong to a similar molecular-nanostructure family. The fluorescence quantum yield of FMR depends on its local microenvironment. They are part of a fluorescent molecules category that could adopt the Twisted Intramolecular Charge Transfer, (TICT) complexes [START_REF] Rettig | Probe design and chemical sensing[END_REF]. The TICT hypothesis was first suggested by [START_REF] Rotkiewicz | Reinterpretation of the anomalous fluorescence of p-n,n-dimethylamino-benzonitrile[END_REF], and it still holds true today [START_REF] Rotkiewicz | Reinterpretation of the anomalous fluorescence of p-n,n-dimethylamino-benzonitrile[END_REF][START_REF] Haidekker | Molecular rotors -Fluorescent biosensors for viscosity and flow[END_REF].

These molecules are composed of an electron donor group which is in conjugation with an electron acceptor group, as shown in Figure 1.3 in a simple manner [START_REF] Haidekker | Ratiometric mechanosensitive fluorescent dyes: Design and applications[END_REF].

Figure 1.3: General configuration of a fluorescent molecular rotor, inspired by [START_REF] Haidekker | Dyes with Segmental Mobility: Molecular Rotors[END_REF].

Principle of Molecular Rotors

The principal property of an FMR is its sensitivity to the microviscosity of the environment, which is due to the competition between different relaxation pathways from the excited state to the lower-energy ground state as illustrated using an abridged Jablonski Upon photoexcitation, an electron transfer occurs from the electron-donor to the electron-acceptor of an FMR through the conjugated bonds. This subsequently leads to the formation of the TICT complexes in the excited state, accompanied by a rotation around the single bond linking the two substructures [START_REF] Li | Design , Synthesis , and Photodynamics of Light-Harvesting Arrays Comprised of a Porphyrin and One , Two , or Eight Boron-Dipyrrin Accessory Pigments[END_REF][START_REF] Kottas | Artificial molecular rotors[END_REF]. The relaxation of the molecule from the excited state to its ground state can occur either through conventional fluorescent photoemission or via a non-radiative relaxation from the TICT state. The deactivation in the twisted conformation occurs without a photon emission as the difference in the energy level is low between the excited state and the ground state [START_REF] Allen | The photophysical properties of a julolidene-based molecular rotor[END_REF].

The competition of these deactivation pathways is the reason behind the viscositysensitive property of FMR. The energy absorption of an FMR, as well as the refractive index of its surroundings, affect the radiative rate, k r while the non-radiative rate, k nr depends on the viscosity of the microenvironment of the molecule [START_REF] Strickler | Relationship between Absorption Intensity and Fluorescence Lifetime of Molecules[END_REF][START_REF] Haidekker | Environment-sensitive behavior of fluorescent molecular rotors[END_REF]. As shown in julolidine, (DCVJ), in high-viscosity medium, the ability of an FMR to relax via a change in conformation (via intermolecular rotation) is hampered. This results in an increase in fluorescence quantum yield [START_REF] Kung | Fluorescent Molecular Rotors : A New Class of Probes for Tubulin Structure and Assembly[END_REF]. The fluorescence quantum yield of FMR, defined in Equation 1.1 can be deduced by measuring the fluorescence intensity or lifetime. It is the ratio of the radiative rate to the sum of all the possible rates of deactivation processes.

φ F " k r k r `knr « k r k or (1.1)
where k or represents the intramolecular reorientation rate. The approximation is wellfounded for molecular rotors as k or is the main nonradiative relaxation pathway and k or " k r [START_REF] Haidekker | Dyes with Segmental Mobility: Molecular Rotors[END_REF][START_REF] Haidekker | Environment-sensitive behavior of fluorescent molecular rotors[END_REF]. In spite of that, there are also FMR with dual emission bands with a red-shifted emission generated from the TICT state, such as 4,4-dimethylaminobenzonitrile (DMABN) [START_REF] Haidekker | Effects of solvent polarity and solvent viscosity on the fluorescent properties of molecular rotors and related probes[END_REF].

Apart from having sensitivity to viscosity, there are FMR whose fluorescence quantum yield rather depends on the change in polarity, such as 8-(phenylamino)-1-naphthalenesulfonates (ANS) [START_REF] Kosower | Intramolecular donor-acceptor systems. 9. photophysics of (phenylamino) naphthalenesulfonates: a paradigm for excited-state intramolecular charge transfer[END_REF], and Nile Red [START_REF] Dutta | Spectroscopic studies of nile red in organic solvents and polymers[END_REF]. Hence, for viscosity probing, the fluorescence emission of molecular rotors must selectively respond to solvent viscosity and needs to be unaffected by solvent polarity. The FMR chosen for this thesis are only sensitive to viscosity with a single emission band.

Förster-Hoffmann Relationship

The core of the most widely applied theories of FMR-solvent interaction is diffusion [START_REF] Haidekker | Environment-sensitive behavior of fluorescent molecular rotors[END_REF] and dissipation at the molecular level. In 1971, Förster and Hoffmann proposed an original approach to solvent microfriction, κ, which is proportional to viscosity, based on the classic Debye-Stokes-Einstein, (DSE) diffusion equation. Their theory shed light on the quantitative relationship between solvent microviscosity and fluorescence quantum yield Chapter 1 of a molecular rotor [START_REF] Förster | Die viskositätsabhängigkeit der fluoreszenzquantenausbeuten einiger farbstoffsysteme[END_REF]. Three different conditions were studied by Förster and Hoffmann using a few triphenylamine rotors with TICT attributes. [START_REF] Förster | Die viskositätsabhängigkeit der fluoreszenzquantenausbeuten einiger farbstoffsysteme[END_REF] The first condition was for low viscosity in which the quantum yield of the FMR attains an independent, minimum plateau value:

φ F,min " 1 β τ 0 δ 2 (1.2)
where β is the constant of proportionality, δ is the angle between the conformations of lowest energy in the ground state and the excited state, and τ 0 the natural lifetime of an FMR, i.e., the lifetime without any non-radiative deactivation. [START_REF] Haidekker | Environment-sensitive behavior of fluorescent molecular rotors[END_REF] A comprehensive discussion on the lifetime parameter is available in Section 1.3.3.

High viscosity environment was then addressed in the second case where the fluorescence quantum yield can be estimated as shown in Equation 1.3. In this context, the non-radiative relaxation due to the reorientation of FMR is negligible.

φ F,max " τ s τ 0 " ˆ1 ´6σ 2 η 2 ˙(1.3)
where τ s is the lifetime in the absence of relaxation pathway due to conformational changes and the constant σ depends on the FMR and has the unit of viscosity.

Last of all, the third case is mentioned to be the most important condition [START_REF] Haidekker | Dyes with Segmental Mobility: Molecular Rotors[END_REF][START_REF] Haidekker | Environment-sensitive behavior of fluorescent molecular rotors[END_REF] where the equation for intermediate viscosity, η ! σ can be expressed as follows:

φ F " ψ ¨τs τ 0 " ´η σ ¯2 3 (1.4)
where ψ is a constant, which equals 0.893. Almost all the constants in Equation 1.4 are rotor-dependent and could be determined via experiments. Equation 1.5 is obtained when these constants are combined into a unique dye-dependent parameter,

C φ F " C ¨η 2 3 . (1.5)
The value of the exponent, 2 {3 is due to the integration calculation and can be defined as the maximum theoretical value when η ! σ. This is done under the assumption that the rotating mechanism of the FMR is perfectly elliptic and in accordance with the secondorder diffusion equation [START_REF] Kuimova | Mapping viscosity in cells using molecular rotors[END_REF]. It is possible to obtain a lower value experimentally. Thus, in general, the relationship between fluorescence quantum yield, φ F and the viscosity of the solvent, η is described by the following equation [START_REF] Förster | Die viskositätsabhängigkeit der fluoreszenzquantenausbeuten einiger farbstoffsysteme[END_REF]:

log φ F " α log η `C (1.6)
where φ F and η represent the fluorescence quantum yield and the local viscosity respectively; whereas α is a dye-dependent constant and C is a proportionality constant.

Besides that, Loutfy and coworkers perceived free volume as an essential parameter in determining the quantum yield of FMR [START_REF] Loutfy | Effect of viscosity and temperature on torsional relaxation of molecular rotors[END_REF][START_REF] Loutfy | Fluorescence probes for polymer free-volume[END_REF][START_REF] Loutfy | Electrochemistry and spectroscopy of intramolecular charge-transfer complexes. pn, n-dialkylaminobenzylidenemalononitriles[END_REF]. Their works were principally based on the Equation 1.7, proposed by Doolittle which shows the empirical relationship between free volume and viscosity [START_REF] Doolittle | Studies in newtonian flow. iii. the dependence of the viscosity of liquids on molecular weight and free space (in homologous series)[END_REF].

η " A ¨exp ˆB ν 0 ν f ˙(1.7)
where ν 0 and ν f correspond respectively to the occupied volume and the temperaturedependent free volume, the empirical constants A and B are solvent-dependent with B « 1. Also, experimental data in the work of Loutfy and Arnold [START_REF] Loutfy | Effect of viscosity and temperature on torsional relaxation of molecular rotors[END_REF] proved that the quantum yield of FMR is governed by a relationship similar to the Equation 1.7:

φ f " k r k nr,0 ¨exp ˆα ν 0 ν f ˙(1.8)
where k nr,0 is fluorescent dye-dependent constant. By substituting Equation 1.1 into Equation 1.8, the rotational relaxation rate can be expressed as a function of free volume, as shown in Equation 1.9:

k or " k nr,0 ¨exp ˆ´α ν 0 ν f ˙. (1.9)
On the other hand, the term ν f in Equation 1.7 can be expressed as a function of viscosity, η and ν 0 as follows:

ν f " ν 0 ln `η A ˘. (1.10)
The substitution of the above-mentioned free-volume term in Equation 1.8 and the eventual introduction of η resulted in Equation 1.11:

Chapter 1 φ F " k r k nr,0 ´η A ¯α . (1.11)
Equation 1.6 is eventually obtained by merging the dye-and solvent-dependent constants k r , k nr,0 and A α into one constant, Ĉ φ F " Ĉ ¨ηα .

(1.12)

Unlike the Förster-Hoffmann derivation with Equation 1.5, the value of exponent α in Equation 1.12 changes with the solvent and the chemical structure of FMR. It has to be noted that the results presented in this work are in agreement with this empiric derivation of Loutfy and Arnold.

In essence, an identical relationship between fluorescence quantum yield and bulk viscosity is obtained by both theories: i) the detailed derivation by Förster and Hoffmann, which assumes rotational friction based on the DSE model, and ii) the empirical derivation by Loutfy and Arnold with the assumption of a power-law microfriction behavior.

Intensity, Lifetime, and Viscosity: a luminous relationship

The fluorescence phenomenon can be quantified with fluorescence intensity or fluorescence lifetime. Fluorescence intensity, I F is the extent of emission whereas fluorescence lifetime, τ F is the average time a molecule remains in an excited state of higher energy upon photoexcitation [START_REF] Kafle | Chapter 9 : Molecular luminescence spectroscopy[END_REF]. As detailed earlier in 1.2, fluorescence occurs as the excited molecule loses energy when returning to the ground state by emitting a photon. Emitted fluorescence intensity and quantum yield are correlated with the intensity of absorbed light, I abs through Correlation 1.13 [START_REF] Viriot | Molecular rotors as fluorescent probes for biological studies[END_REF].

φ F ¨Iabs ∝ I F . (1.13)
Consequently, the emitted intensity follows the Förster-Hoffmann relationship (Equation 1.6) and can be expressed as follows:

log I F " α I F log η `CI F . (1.14)
Corrections for light absorption and scattering and, most importantly, the dye concentration has to be taken into consideration for C I F . Also, it strongly depends on the parameters of the instrument used to measure the fluorescence intensity, including excitation intensity, emission collection efficiency, and fluorescence amplifiers [START_REF] Haidekker | Dyes with Segmental Mobility: Molecular Rotors[END_REF]. A calibration with fluids of known viscosity has to be carried out to determine its value [START_REF] Haidekker | Molecular rotors -Fluorescent biosensors for viscosity and flow[END_REF].

On the other hand, the relationship between fluorescence lifetime and quantum yield is defined as follows:

φ F " k r k r `knr " k r τ F (1.15)
where τ F is the average excited-state lifetime of an FMR, the radiative rate constant is represented by k r , and k nr is the non-radiative rate constant [START_REF] Kuimova | Mapping viscosity in cells using molecular rotors[END_REF][START_REF] Kuimova | Molecular rotor measures viscosity of live cells via fluorescence lifetime imaging[END_REF].

Accordingly, by replacing the quantum yield in Equation 1.6 with fluorescence lifetime, a similar equation to Equation 1.14 can be obtained:

log τ F " α τ F log η `Cτ F (1.16)
It is noteworthy that the viscosity determination by lifetime measurements is neither influenced by the concentration-dependent nor the instrument-dependent factors [START_REF] Haidekker | Dyes with Segmental Mobility: Molecular Rotors[END_REF][START_REF] Kuimova | Mapping viscosity in cells using molecular rotors[END_REF].

Intensity is an easy-to-measure parameter for an FMR; however, it does not only depend on the viscosity of its environment but also the concentration of the dyes. The excitation intensity, absorption by the sample, the geometries of experimental designs and parameters, as well as photo-bleaching also play a role in the fluorescence emission. Due to these drawbacks, accurate calibrations are needed to acquire quantitative measurements in microscopy from measurements of fluorescence intensity. Hence, the lifetime measurement of FMR is usually preferred as it solely depends on the local viscosity of a medium and is highly sensitive to its changes [START_REF] Kafle | Chapter 9 : Molecular luminescence spectroscopy[END_REF][START_REF] Ponjavic | In situ viscosity measurement of confined liquids[END_REF][START_REF] Michael | Imaging phase separation in model lipid membranes through the use of BODIPY based molecular rotors †[END_REF]. Furthermore, viscosity mapping can be performed with high spatial resolution using lifetime-based measures [START_REF] Ponjavic | In situ viscosity measurement of confined liquids[END_REF]. The variation in fluorescence lifetimes can be mapped using Fluorescence Lifetime Imaging Microscopy, FLIM (further explained in section 1.4.3).

Fluorescence Measurements of Molecular Rotors

The works carried out in this thesis focused on steady-state fluorescence measurement ter Photobleaching (FRAP), Fluorescence Correlation Spectroscopy (FCS) and particle tracking using a simple fluorescent dye. In general, these methods relate the diffusion of a fluorophore to the viscosity of its surrounding medium. However, these methods are more complicated than the presented FMR-coupled techniques in this section.

Absorption and Fluorescence Spectroscopy

In this work, absorption spectroscopy was used to determine the absorption or excitation wavelength peak of analyzed fluorophores in different solvents. This technique could also be used to quantify the amount of fluorophore present in a sample. Additionally, fluorescence spectroscopy was used for the determination of the emission wavelength peak and to confirm the viscosity-sensitivity attribute of a new FMR. Both techniques belong to the general electromagnetic spectroscopy category.

Absorption spectroscopy is a method that measures the absorption energy during irradiation as a function of wavelength or frequency. It is carried out across the electromagnetic spectrum. The intensity variation of absorption as a function of wavelength is called an absorption spectrum. According to Planck's Law, the absorption of a photon with energy, E supplied by an external source is defined as follows:

E " hν " hc λ (1.17)
where h and ν represent the Planck's constant and the frequency respectively; c is the speed of light and λ, the wavelength. The energy of an absorbed photon is directly proportional to the frequency and inversely proportional to the wavelength. Hence, shorter excitation wavelengths have a higher quantum of energy.

The presence of different excited states (as explained in Section 1. Concerning the viscosity measurement by using FMR, Haidekker, and co-workers have indicated in their many works that the accuracy of measurement via steady-state fluorescence spectroscopy can match or surpass the analysis with mechanical rheometers [START_REF] Haidekker | Environment-sensitive behavior of fluorescent molecular rotors[END_REF][START_REF] Akers | Precision assessment of biofluid viscosity measurements using molecular rotors[END_REF].

Epifluorescence Microscopy

Fluorescence microscopy belongs to the family of optical microscopy, which utilizes fluorescence emission rather than visible light in order to observe a specimen. Epifluorescence microscopy, on the other hand, is a subcategory of fluorescence microscopy wherein the excitation beam and the emitted fluorescence of a fluorophore take place through the same objective lens. The excitation beam is reflected onto the sample via the objective, therefore explaining the Greek word "epi", which translates to "upon" [START_REF] Rost | Fluorescence microscopy, applications*[END_REF]. As shown in intensity of which is related to the amount of light it captured.

Fluorescence Lifetime Imaging Microscopy (FLIM)

The FLIM technique has been widely used to map viscosity using FMR in heterogeneous environments in biomedical and life sciences [START_REF] Kuimova | Molecular rotor measures viscosity of live cells via fluorescence lifetime imaging[END_REF][START_REF] Wu | Molecular rheometry: direct determination of viscosity in Lo and Ld lipid phases via fluorescence lifetime imaging[END_REF][START_REF] Levitt | Membrane-Bound Molecular Rotors Measure Viscosity in Live Cells via Fluorescence Lifetime Imaging[END_REF]. Moreover, this imaging method has been adapted in the examination of artwork pieces by Comelli et al., [START_REF] Comelli | Fluorescence lifetime imaging and spectroscopy as tools for nondestructive analysis of works of art[END_REF] in forensic science for fingerprint detection by Seah and coworkers, [START_REF] Seah | Application of fluorescence lifetime imaging (flim) in latent finger mark detection[END_REF] as well as in the study of microfluidics [START_REF] Magennis | Quantitative Spatial Mapping of Mixing in Microfluidic Systems[END_REF][START_REF] Robinson | Three-Dimensional molecular Mapping in a Microfluidic Mixing Device Using Fluorescence Lifetime Imaging[END_REF][START_REF] Benninger | Time-resolved fluorescence imaging of solvent interactions in microfluidic devices[END_REF][START_REF] Elder | The application of frequency-domain Fluorescence Lifetime Imaging Microscopy as a quantitative analytical tool for microfluidic devices[END_REF].

The Lambert Instruments FLIM Attachment (LIFA)-FLIM used in this work is a wide-field method where the time-resolved image information is acquired in the frequency domain via continuous sinusoidal modulation of the excitation source and the detector.

The fluorescence lifetime is calculated from the decrease in modulation, M , and the change in phase shift of the fluorescence emission compared to the excitation source, φ, as illustrated in Figure 1.7 [START_REF] Suhling | Time-resolved fluorescence microscopy †[END_REF][START_REF] Munster | Fluorescence Lifetime Imaging Microscopy (FLIM)[END_REF].

When the modulation of an excitation light intensity follows a sinusoidal function of time, Eptq with a frequency f , the fluorescence emission intensity, F ptq is a sinusoidal function with the same frequency [START_REF] Gadella | Fluorescence lifetime imaging microscopy: Pixel-by-pixel analysis of phase-modulation data[END_REF]:

Eptq " E 0 r1 `ME,ω sin pωt ´ϕE qs (1.18)

F ptq " F 0 r1 `MF,ω sin pωt ´ϕF qs (1.19) where E 0 and F 0 are time-independent offsets, M E,ω the relative modulation of the excitation light and M F,ω for the fluorescence emission, ϕ E,ω and ϕ F,ω represent the arbitrary phase delays. In these equations, ω " 2πf is the radial frequency. From now on, subscript ω will be forgotten, but one should keep in mind that phases φ and modulation M parameters depend on it.

According to Seah et al., the suitable range of modulation frequencies is from 20 to 100 MHz for lifetimes of 1-10 ns. Direct measurement of phase shift and demodulation is not possible at these frequencies. Hence, the high-frequency signal is converted via homodyne detection or heterodyne detection with very low frequencies to facilitate these measurements. Homodyne measurement was applied for the LIFA-FLIM used in this work as it is relatively easy compared to heterodyne measurement. The frequency of the source and the detector is the same for homodyne detection [START_REF] Seah | Application of fluorescence lifetime imaging (flim) in latent finger mark detection[END_REF]. are valid for both the reference fluorophore and the analyzed samples.

φ " ϕ F ´ϕE (1.20) M " M F M E (1.21)
Subsequently, the fluorescence lifetime values can be determined from φ along:

τ φ " 1 ω tan φ (1.22)
or from M along:

τ M " 1 ω c 1 M 2 ´1. (1.23)
The values of τ φ and τ M are similar in a homogeneous sample, and a system can be considered heterogeneous if they are different [START_REF] Seah | Application of fluorescence lifetime imaging (flim) in latent finger mark detection[END_REF]. In this work, the average lifetime taken into account is the τ φ . The τ M is less accurate than τ φ because it is much more complicated to measure demodulation with two different fluorescence intensity signals compared

to the measurement with a phase shift. This is because the fluorescence intensity depends on the concentration of the fluorophore, and it is not necessarily identical in the analyzed sample and the reference specimen.

However, the excitation-related parameters (ϕ E and M E ) are unavailable for this setup.

This explains the importance of a reference fluorophore in the FLIM method, as the unknown values for a sample have to be corrected with the parameters of the reference.

In the end, by applying the Equation 1.20 to both reference fluorophore and sample and then combining these equations to Equation 1.22, Equation 1.24 is obtained with which a sample's lifetime can be determined.

τ sample " 1 ω tan " ϕ sample ´ϕref `tan ´1 pω ¨τref q ‰ (1.24)
With the LIFA-FLIM setup, fluorescence lifetimes across the field of view of the microscope were mapped within a few seconds [START_REF] Festy | Imaging proteins in vivo using fluorescence lifetime microscopy[END_REF].

Microviscosity and Molecular Rotors

As mentioned in Section 1.3.2, the theory of fluorescence dependency of FMR on viscosity was first suggested by Förster and Hoffmann more than 50 years ago, and its use for viscosity measurements kick-started around ten years later. It is worth reminding that the use of FMR for more sophisticated viscosity mapping rather than a simple contrast marker is well-recognized and has been growing fast in past years. FMR are indeed an excellent choice for local viscosity probes, especially in the confined system, thanks to their high spatial resolution and real-time response to viscosity changes [START_REF] Haidekker | Molecular rotors -Fluorescent biosensors for viscosity and flow[END_REF][START_REF] Ponjavic | In situ viscosity measurement of confined liquids[END_REF]. One of the first use of FMR was to observe in real-time the processes of aggregation and polymerization of poly(methyl methacrylate) and polystyrene by Loutfy and co-workers [START_REF] Loutfy | Effect of polymer chain tacticity on the fluorescence of molecular rotors[END_REF][START_REF] Loutfy | Effect of polystyrene molecular weight on the fluorescence of molecular rotors[END_REF]. At the same period of time, phospholipid bilayers systems were examined using the malononitrilebased rotors, e.g., DCVJ by Kung and Reed in [START_REF] Kung | Microviscosity Measurements of Phospholipid Bilayers Using Fluorescent Dyes That Undergo Torsional Relaxation[END_REF] and by Lukac in [START_REF] Lukac | Thermally Induced Variations in Polarity and Microviscosity of Phospholipid and Surfactant Vesicles Monitored with a Probe Forming an Intramolecular Charge-Transfer Complex[END_REF]. In his work, Lukac employed FMR to probe temperature-induced changes of microviscosity and eventually the phase transformation in phospholipids vesicles from gel to liquid-crystal.

The chemical structure of an FMR can be engineered to modify its photophysical characteristics for different applications, such as for specific protein binding [START_REF] Hwang | Protein induced fluorescence enhancement for probing protein-nucleic acid interactions[END_REF][START_REF] Goh | Molecular rotors as conditionally fluorescent labels for rapid detection of biomolecular interactions[END_REF] and membrane integration [START_REF] Haidekker | New fluorescent probes for the measurement of cell membrane viscosity[END_REF]. The employment of FMR is more prominent in biology and biochemistry, where they became a game-changer in the study of microviscosity in living cells. This is because FMRs are capable of differentiating viscosity between the subcellular components [START_REF] Kuimova | Mapping viscosity in cells using molecular rotors[END_REF]. This highlights another advantage of FMR as a non-invasive technique where samples could be analyzed without the involvement of fluid shear that could damage the biological sample. This is not feasible with conventional viscosimeters. The viscosity fields in both cases were reconstructed using a pre-established calibration curve.

In addition, well-established research works on viscosity measurement using FMR were carried out by Kuimova and her collaborators, mainly in physical chemistry and biology.

Most of their works were carried out with FMR belonging to the BODIPY molecular family, as described in the work of Vyšniauskas et al., where several new fluorophores were synthesized and analyzed to enhance the viscosity probing ability of existing FMR.

The chemical structural effect on its function was studied as well by imaging live cells, as shown in Figure 1.10b [START_REF] Kuimova | Exploring viscosity, polarity and temperature sensitive of BODIPY-based molecular rotors[END_REF]. A conjugated porphyrin dimer, also a ratiometric FMR, was used in Kuimova's work [START_REF] Kuimova | Mapping viscosity in cells using molecular rotors[END_REF] to perform quantitative viscosity measurements during Photodynamic Treatment (PDT) of a cancerous cell as shown in Figure 1.10a. Apart Chapter 1

The research group of Brouwer worked on the visualization of micro-contacts between solid objects in Suhina et al. [START_REF] Suhina | Fluorescence Microscopy Visualization of Contacts Between Objects[END_REF]. The contact area between a plastic sphere and a silanized flat glass surface was determined by imaging fluorescence lifetimes of surface-attached rigidochromic probe: a newly-synthesized dicyanomethylene dihydrofuran (DCDHF)-based rotors. The fluorescence responses were measured when the two objects in contact were pressed with different weights; the heavier the load applied, the higher the fluorescence intensity.

A model viscosity probe, Thioflavin T, was used to measure high-pressure and highshear in-situ viscosity in a confined liquid. [START_REF] Ponjavic | In situ viscosity measurement of confined liquids[END_REF]. The differences in rheological responses of a Newtonian and non-Newtonian fluid could be detected by this technique. Also, spatial viscosity variation generated by the pressure difference within a thin fluid film was observed. These results show the robustness of in-situ viscosity measurements performed by using FMR.

Thesis Objectives

This first chapter has allowed us to understand the fundamental theories of fluorescence and, most importantly, the viscosity-sensitive fluorescent molecular rotors. The Förster-Hoffmann model, which can be considered one of the most important relationships of this work, was defined and well-studied. The relation between the fluorescence intensity and lifetime of an FMR and its surrounding viscosity was also thoroughly elucidated. Also, two practical and theoretical basis of the main methods of fluorescence measurement employed in this work were explained. While it is clear that the microviscosity measured by the rotor is correlated to the regular viscosity, their precise link remains unclear. Yet, for practical applications, a precise understanding is not needed as long as the response of the rotor is calibrated.

Viscosity plays a significant part in many industrial processes, especially in continuously evolving fluid flow systems. Generally, viscosimeters are used to measure this quantity, but their use is limited to macroscopic volumes and poses challenges for online implementation. This restrains their application in small-volume systems. Moreover, spatial heterogeneities are usually present in complex flows, on the macroscopic scale, for instance, in paint manufacturing or food processing; and at small-scale flows, such as in porous media, with applications in enhanced oil recovery. Bulk viscosimeters are incapable of obtaining local data in these given examples. Therefore, developing tools for small-scale viscosity measurement is crucial for both fundamental studies and industrial applications [START_REF] Arnes | On-line or process viscometry -a review[END_REF].

Microfluidics systems have been widely used as an essential tool in numerous applications such as high-throughput screening in the research and development domain [START_REF] Dunn | Challenges and solutions to ultra-high-throughput screening assay miniaturization : submicroliter fluid handling handling[END_REF] and chemical reactions analysis [START_REF] Losey | Microfabricated Multiphase Packed-Bed Reactors: Characterization of Mass Transfer and Reactions[END_REF]. Also, they provide interesting opportunities for viscosity measurements. As elucidated in this chapter, FMRs offer a path for direct local viscosity measurements on a micro-meter scale. FMRs have been widely used for fluid viscosity distribution mapping in cells and biological membranes; however, the utilization of these fluorophores outside biology-based fields remains scarce.

The heart of this thesis lies in the integration of FMR into the domain of microfluidics for the local viscosity analysis in microfluidic devices and, ultimately, the conception of a viscosity-sensitive microfluidic chip. The next chapter describes in detail the microfabrication of the microfluidic devices developed during this thesis and quantitative viscosity mapping, as well as the flow characterization in these devices.

State of the art: Do we need a microviscosimeter?

Microfluidics can be defined as the science and field of technology that is based on the study of simple and/or complex fluids, mono-or multiphasic, contained in geometries where at least one dimension is submillimetric (1 to 999 µm) [START_REF] Tabeling | Introduction to microfluidics[END_REF][START_REF] Whitesides | The origins and the future of microfluidics[END_REF].

One of the principal advantages of microfluidic systems is the possibility to perform measurements of good precision, allowing highly reproducible tests [START_REF] Whitesides | The origins and the future of microfluidics[END_REF]. This directly leads to a significant increase in the reliability of results. In addition, the costs associated with experimental materials are greatly reduced as the analysis involving microfluidics requires only small volumes of samples. Experiments can be carried out in a short duration while achieving relatively fast system stabilization. The acceleration of the experimentation time thus offers the possibility to screen fluids in a high throughput manner.

The earliest application of microfluidic technologies was in the field of molecular analysis. The intensive advancement in microfluidics can be considered an evident sequel to the success of several microanalytical methods in a capillary format, such as gas-phase chromatography, capillary electrophoresis, as well as high-pressure liquid chromatography, which transformed the domain of chemical analysis [START_REF] Whitesides | The origins and the future of microfluidics[END_REF].

As briefly mentioned earlier, viscosity at the micro-or nanoscale is hard to measure with mechanical viscosimeters. Mechanical measurements have limitations as they require a relatively large volume of samples, have bulky configurations, limited to global viscosity measurement, and have to be managed with great care, including being placed on a flat, vibration-free table [START_REF] Puneeth | Microfluidic viscometers for biochemical and biomedical applications: A review[END_REF]. More importantly, it is difficult to measure local viscosities in real-time conditions [START_REF] Dragan | Fluorescence-based Broad Dynamic Range Viscosity Probes[END_REF]. Time-and spatially-resolved mapping of the local viscosities in confined systems can be challenging as well.

The utilization of microfluidic devices as an analytical tool for small-scale viscosity measurement has been applied in several research and development fields such as in the food industry [START_REF] Venkateswaran | Rapid and automated measurement of milk adulteration using a 3d printed optofluidic microviscometer (omv)[END_REF][START_REF] Puneeth | Novel 3d printed microfluidic paper-based analytical device with integrated screen-printed electrodes for automated viscosity measurements[END_REF], disease diagnosis [START_REF] Duan | Optimizing multiplexed detections of diabetes antibodies via quantitative microfluidic droplet array[END_REF] and for the in-depth study of biofluids [START_REF] Nguyen | Microfluidic rheometer based on hydrodynamic focusing[END_REF].

The study of viscosity is essential in these domains; thus, development in microfluidics, where fluid flows can be well controlled, helps to perform the necessary local analysis in Chapter 2

an easy-to-use way, just by using a simple, stable microdevice [START_REF] Pipe | Microfluidic rheometry[END_REF].

Some strategies have been implemented to measure viscosities at the micrometer length scale, for instance, by measuring pressure drop along a microchannel for an applied flow rate [START_REF] Kang | High shear microfluidics and its application in rheological measurement[END_REF]. Fluid viscosity has been measured using capillary-pressure-driven flow in a microfluidic channel using only a sample volume of 600 nL. This method, however, is limited to low viscosities (lower than 5 mPa s) [START_REF] Srivastava | Nanoliter Viscometer for Analyzing Blood Plasma and Other Liquid Samples[END_REF]. Fluorescence microscopy can be adapted as another approach for the determination of local viscosity. In the work of Galambos and

Forster, an unknown sample's viscosity was calculated by knowing the flow rates and the viscosity of a reference fluid [START_REF] Galambos | An optical micro-fluidic viscometer[END_REF]. This was done by measuring the interdiffusion interface position in a microfluidic co-flow with the reference fluid. This strategy also worked well for immiscible fluids even with very low shear rates in the study of Guillot et al. [START_REF] Guillot | Viscosimeter on a Microfluidic Chip[END_REF].

However, even if performed at a small scale, these techniques only give access to the viscosity averaged over the whole volume of the samples. More local measurements can be done by imaging the local velocity field within the microchannel with micro-PIV techniques [START_REF] Cuenca | Submicron flow of polymer solutions: Slippage reduction due to confinement[END_REF][START_REF] Bodiguel | Flow enhancement due to elastic turbulence in channel flows of shear thinning fluids[END_REF]. Nonetheless, these works are limited to simple microfluidic configurations.

On the other hand, as explained in Section 1.5, FMR makes viscosity measurements at the molecular scale in a small confined area possible. Benninger et al. used an FMR, DASPI, for the high-speed imaging of real-time changes in viscosity during dynamic fluid mixing in microdevices. Rotation correlation time that depends on the solvent viscosity was deduced via the FLIM technique in time domain [START_REF] Benninger | Time-resolved fluorescence imaging of solvent interactions in microfluidic devices[END_REF]. Nevertheless, this method remains challenging to apply by non-specialists as its optical setup is sophisticated and expensive.

Even if the works and techniques described above allow precise measuring of the local viscosity in a more or less complex system, a limitation remains constant: that of not being able to map in situ the local viscosity during fluid flow in any microfluidic system configuration in an easy, straightforward way. Considering these limiting conditions, the works presented in this chapter were consecrated to the development of a new quantitative, in-situ and local viscosity measurements in heterogeneous microfluidic flows. The developed method is versatile and can be accommodated in any microfluidic configuration. It is worth mentioning that a scientific article based on the research work presented in this chapter has been submitted and is under review [START_REF] Nalatamby | Molecular rotors for in situ local viscosity mapping in microfluidic chips[END_REF]. Based on previously reported rotors, BODIPY-2-OH was precisely designed to ensure its viscosity-sensitive characteristics [START_REF] Kuimova | Exploring viscosity, polarity and temperature sensitive of BODIPY-based molecular rotors[END_REF]. The phenyl unit of this molecule is an electron donor group which is in conjugation with the BODIPY core, an electron acceptor group.

BODIPY-2-OH as fluorescence lifetime probe

As mentioned in Section 1.3.1, after photon absorption, the molecule relaxes to its ground state via two deexcitation pathways: i) through fluorescence emission or ii) through nonradiative relaxation from the TICT state succeeding the rotation around the single bond linking phenyl and BODIPY groups [START_REF] Li | Design , Synthesis , and Photodynamics of Light-Harvesting Arrays Comprised of a Porphyrin and One , Two , or Eight Boron-Dipyrrin Accessory Pigments[END_REF].

Photophysical characterization of BODIPY-2-OH

The preliminary step to a quantitative viscosity mapping using an FMR is the analysis of the probe's sensitivity to media of different viscosity. Hence, the fluorescence responses of BODIPY-2-OH to viscosity were studied. The absorption and emission spectra of BODIPY-2-OH were measured respectively using the Agilent Technologies Cary Chapter 2

UV-Visible Spectrophotometer and the Agilent Technologies Cary Eclipse Fluorescence Spectrophotometer. Dimethyl sulfoxide (DMSO)-glycerol mixtures were prepared from a dye stock solution prepared beforehand in DMSO. The mixtures of DMSO and glycerol were chosen as the working fluids in this study due to the limited solubility of the fluorescent molecule in water. Also, BODIPY-2-OH is highly soluble in both solvents, and the viscosity of the mixtures varies significantly with their composition, with comparatively small polarity changes [START_REF] Angulo | Characterization of dimethylsulfoxide/glycerol mixtures: A binary solvent system for the study of "friction-dependent" chemical reactivity[END_REF].

The obtained spectra are depicted in Figure 2.2a. The maximum excitation and emission wavelengths of BODIPY-2-OH in glycerol were respectively found to be 500 nm and 515 nm. These spectra are similar to those previously reported for BODIPY-based molecular rotors, which proves that the fluorescence characteristics are not affected by the different groups attached to the BODIPY base [START_REF] Wu | Molecular rheometry: direct determination of viscosity in Lo and Ld lipid phases via fluorescence lifetime imaging[END_REF][START_REF] Kuimova | Exploring viscosity, polarity and temperature sensitive of BODIPY-based molecular rotors[END_REF].

BODIPY molecules are susceptible to photodegradation after constant irradiation [START_REF] Rybczynski | Photochemical Properties and Stability of BODIPY Dyes[END_REF].

The samples were stored in amber vials to limit bleaching by ambient light, even though the degradation process is slow enough to be negligible over the course of our exper- Measurements were taken for 60 s for each shear rate with an acquisition rate of 1 point/s and were repeated thrice to ensure good repeatability. The final viscosity value is an average taken over all the applied shear rates.

Calibration of the viscosity response of BODIPY-2-OH

It is important to note that the fluorescence response of an FMR to viscosity depends on the nature of the solvent environment and has to be carefully calibrated in order to map viscosity quantitatively [START_REF] Haidekker | Dyes with Segmental Mobility: Molecular Rotors[END_REF][START_REF] Akers | Precision assessment of biofluid viscosity measurements using molecular rotors[END_REF]. Hence, calibration curves were established by measuring BODIPY-2-OH response in mixtures of known viscosity prior to the use in microdevices.

The emission intensity under steady illumination and fluorescence lifetime of the FMR were characterized. The LIFA-FLIM, presented in Section 1.4.3 in Chapter 1, was used to measure fluorescence lifetimes, and the bulk viscosity measurements of the samples were performed using the protocol described previously.

Settings of LIFA-FLIM for lifetime measurement

The FLIM experiments were performed with a LIFA device mounted on an Olympus

Results and Discussion

As displayed in

Viscosity mapping of diffusive mixing

In this work, quantitative viscosity measurement and eventual in-situ mapping were initially conducted via the measurement of the fluorescence intensity during diffusive mixing.

The FMR used for these experiments was DCVJ (the chemical structure can be found in Figure 1.5) and it was chosen for its viscosity sensitivity that follows well the Förster-Hoffmann model [START_REF] Haidekker | Molecular rotors -Fluorescent biosensors for viscosity and flow[END_REF], as depicted in Figure 2.4b. The same α-exponent (α = 0.6) was obtained for calibration experiments conducted in our work for mixtures of DMSO-glycerol, as found in the literature [START_REF] Haidekker | Molecular rotors -Fluorescent biosensors for viscosity and flow[END_REF][START_REF] Haidekker | New fluorescent probes for the measurement of cell membrane viscosity[END_REF]. In addition to being very little affected by changes in polarity [START_REF] Allen | The photophysical properties of a julolidene-based molecular rotor[END_REF], DCVJ is also one of the first and well-characterized as well as commercially available FMR. It is important that viscosity-sensitive molecular rotors are used and not polarity dependent because in this way, the fluorescence variations are only due to local viscosity changes [START_REF] Akers | Precision assessment of biofluid viscosity measurements using molecular rotors[END_REF].

Nevertheless, the image analysis following the fluorescence intensity measurement was tedious, and great precautions had to be taken into account to obtain reliable results with good repeatability. The concentration of the probe has to be diligently kept constant, and the obtained intensity signals were noisy. In short, the local viscosity mapping with It should be noted that DCVJ is not a suitable FMR for fluorescence lifetime measurements as it exhibits very short lifetimes, from 10 to 100 ps in a range of viscosity from 50 to 1000 mPa s [START_REF] Kuimova | Mapping viscosity in cells using molecular rotors[END_REF][START_REF] Levitt | Fluorescence anisotropy of molecular rotors[END_REF]. The available experimental setup in our research laboratory cannot be used to measure such short lifetimes.

Fabrication of PEGDA microfluidic chips

Viscosity mapping during a diffusive mixing was carried out in a simple microfluidic Yjunction. It is worth mentioning that polydimethylsiloxane (PDMS) chip was chosen in the early stages of this work for viscosity mapping using DCVJ. However, DCVJ was found to be adsorbed to the surface of the PDMS microchannel. It is known in the literature that PDMS is prone to non-specific adsorption [START_REF] Sia | Microfluidic devices fabricated in poly (dimethylsiloxane) for biological studies[END_REF]. Hence, a poly(ethylene glycol) diacrylate (PEGDA)-based microdevice was eventually selected as non-specific molecular adsorption is less likely to occur within a PEGDA microchannel. PEGDA also possesses the good qualities of PDMS, such as optical clarity and low background fluorescence [START_REF] Rogers | Single-Monomer Formulation of Polymerized Polyethylene Glycol[END_REF].

The use of the PEGDA microfluidic chip was continued for the eventual viscosity mapping study with the FMR, BODIPY-2-OH. Furthermore, the PEGDA chip was chosen for this study due to its fast microfabrication method, which only takes around five minutes to make a functional chip [START_REF] Decock | In situ photo-patterning of pressureresistant hydrogel membranes with controlled permeabilities in PEGDA microfluidic channels[END_REF].

Different steps of the microfabrication of the PEGDA microfluidic chip are shown in

Microfluidics Experiments

Local viscosity mapping in a simple Y-mixer

For the co-flow experiments with two miscible solutions of different viscosities, the flow was laminar inside the microchannel as small-scale lengths and velocities were applied.

The liquids were thus mixed only by transverse diffusion, perpendicular to the flow direction [START_REF] Salmon | In situ raman imaging of interdiffusion in a microchannel[END_REF][START_REF] Salmon | Transverse transport of solutes between co-flowing pressure-driven streams for microfluidic studies of diffusion/reaction processes[END_REF]. It is worth noting that this is a well-characterized configuration in microfluidics, and the evolution of the fluid composition along the microchannel has been thoroughly studied. Similar studies on spatial mapping in microfluidic devices with the FLIM method have been reported based on changes in the lifetimes of fluorescent dyes through quenching by a diffusing ion [START_REF] Elder | The application of frequency-domain Fluorescence Lifetime Imaging Microscopy as a quantitative analytical tool for microfluidic devices[END_REF] and by changes in solvent polarity [START_REF] Magennis | Quantitative Spatial Mapping of Mixing in Microfluidic Systems[END_REF].

The mapping of fluorescence lifetime was subsequently carried out using the LIFA-FLIM, and a x ´y microactuator was used to move the chip above the microscope objective, and images were taken along the main microchannel. Fluorescence data postprocessing with MATLAB led to the mapping of viscosity in the microchannel. It is to be noted that the background signal was subtracted from the fluorescence intensity signal for both the reference and the sample in order to increase the accuracy of our results. for two different sets of flow rates. It is to note that a few outliers were observed on images, corresponding to computed lifetimes below the detection limit of LIFA, which is 0.15 ns. However, these are observed in limited numbers and were not taken into account for further data analysis.

The two streams of initially different viscosities progressively mixed with an interdiffusion layer that widened along the channel. As can be seen by comparing Figure 2.11(a)

and (b), a slower flow led to better mixing at the end of the channel, as it corresponded to a longer time of contact between the solutions. The interdiffusion layer was closer to the center of the channel for a ratio of flow rates close to the ratio of viscosity, as would be the case for immiscible liquids. As already reported in the literature, a progressive drift of the interdiffusion zone towards the higher-viscosity fluid was also observed. This latter is due to the coupling between hydrodynamics and the mixing through the dependence of the viscosity with the glycerol concentration along the mixing channel [START_REF] Dambrine | Interdiffusion of liquids of different viscosities in a microchannel[END_REF].

Measurement of Effective Diffusion Coefficient (D eff )

The validity of the viscosity measurements was assessed by analyzing the local viscosity maps in further detail to compare with a model from the literature. Transverse viscosity profiles ηpyq can be first extracted, with y P r0, ws corresponding to the coordinate perpendicular to the flow direction, from the experimental viscosity maps at different positions x in the channel shown in Figure 2.11. The viscosity profiles on a single image did not significantly evolve with x as molecular diffusion was slow enough. Hence, the profiles were averaged in all pictures to reduce the noise level. The obtained results are displayed in Figure 2.12.

Fluctuations around 8% were observed for the local viscosity value of S2 along the channel in Figure 2.12: η " 74 mPa s to 80 mPa s whereas the average viscosity measured with the rheometer was 74 mPa s. This is an acceptable fluctuation as a deviation of 10% is considered normal for rheological measurement using a mechanical viscosimeter with cone-plate geometry, especially after multiple reloading steps. The evolution of viscosity across the channel was observed more clearly with the profiles where the two streams were separated by an interdiffusion layer that widened along the channel. This situation of diffusive mixing in a microfluidic co-flow can be modeled by an advection-diffusion problem. In the case of an infinitely wide channel, analytical solutions have been obtained for the volume fraction φ of glycerol in the study of Salmon et al. [START_REF] Salmon | Transverse transport of solutes between co-flowing pressure-driven streams for microfluidic studies of diffusion/reaction processes[END_REF]:

φ px, yq " φ 0 2 ˆy ´ym pxq 2σ d pxq ˙`φ m . (2.1)
At a given position x in the channel, φ varies from φ min " φ m ´φ0 {2 to φ max " φ m `φ0 {2, with a transition described by an error function, of center y m pxq and characteristic width σ d pxq.

Equation 2.1 has been validated in the study of Dambrine et al. [START_REF] Dambrine | Interdiffusion of liquids of different viscosities in a microchannel[END_REF], and the results from this study were taken as a reference to validate our measurement method notably as a similar microfluidic design was used. A similar aspect ratio of the channel was kept to ensure a unidimensional flow profile, and flow rates were adapted to have small Reynolds number, typically between 10 ´2 and 10 ´1, in order to obtain laminar flow in the microchannels. However, for practical reasons, a narrower channel of a factor of about 2 was used. Finally, the entrance length L e , of about 0.4 mm, was small enough to consider that the flow reached a fully developed Poiseuille profile in the analyzed pictures [START_REF] Yager | Transverse diffusion in microfluidic systems[END_REF][START_REF] Stiles | Hydrodynamic control of the interface between two liquids flowing through a horizontal or vertical microchannel[END_REF]. The continuous line corresponds to application of a lnpη{η 0 q model, with a " 0.16 and η 0 " 1.87.

In order to use Equation (2.1), the measured viscosity η was first converted into glycerol volume fraction φ by using a pre-established calibration curve shown in Figure 2.13.

The corresponding profiles at different positions x are displayed in Figure 2.14a and were fitted using Equation (2.1). Moreover, as observed on the viscosity profiles, the minimum and maximum volume fractions at both sides were not constant along the microchannel:

this was due to the finite width w of the channel, which induced side effects not considered in the model. Parameters φ 0 , φ m , σ d , and y m were thus taken as free fit parameters at every position x. The resulting fits are superimposed to experimental data in Figure 2.14a. The variations of the fitting parameters with the position x along the microchannel could be characterized in more detail. As commonly performed in the field of microfluidics, the average time τ spent by the fluid in the channel at a given position x was used to describe the evolution of the system:

τ " 2xhw Q S1 `QS2 " 2x v 1 `v2 (2.4)
with v 1 and v 2 representing the average velocities of the incoming streams.

The evolution of the amplitude φ 0 of the profile and the volume fraction at the middle of the interdiffusion layer φ m are given in Figure 2.15b and Figure 2.15c. These parameters are difficult to interpret, but they are found to be roughly constant within a 10 % variation. This may seem contradictory with the clear evolution of the viscosity on the sides of the channel: this is, however easily explained by the strongly non-linear evolution of the viscosity of glycerol-DMSO mixture with glycerol concentration. Due to the finite width of the channel, the volume fractions at the walls of the channel slightly evolve, which induces a strong viscosity variation for the stream highly concentrated in glycerol.

More interestingly, the model well predicted the evolution of width σ d of the interdiffusion layer, which is displayed in Figure 2.16. As the spreading of this layer is driven by transverse diffusion, the squared width σ 2 d pxq at a given position x was expected to evolve linearly with the time of diffusion τ pxq along:

σ 2 d pxq " D eff τ pxq. (2.5)
Experimental observations were in agreement with this relation and allowed to retrieve an effective diffusion coefficient D eff " 2 ˆ10 ´10 m 2 s ´1, to be compared with the reference value D ref " 7 ˆ10 ´10 m 2 s ´1 from the literature [START_REF] Dambrine | Interdiffusion of liquids of different viscosities in a microchannel[END_REF].

These two values have a similar order of magnitude, and this agreement can be considered satisfactory. This difference can be explained by the fact that the reference value was obtained for water-glycerol mixtures, while DMSO-glycerol mixtures were used for this work. Also, no systematic determination of experimental uncertainties was performed, but considering the number of steps to retrieve this diffusion coefficient, uncertainties are likely to be significant. Finally, the existence of a finite-size effect due to the narrower channels employed in this study can also slightly modify the effective diffusion coefficient.

Viscosity mapping during chaotic mixing

After the verification of the proposed methodology with a well-controlled system, the next part of this work emphasized a more complex flow configuration in which only the basic Prior to this work, different methods were utilized to characterize micromixers, such as via a chemical-reaction-based strategy where streams of sodium hydroxide (NaOH) and phenolphthalein solutions were injected respectively into two inlets of a microchannel. The reaction of these streams changes the color of the fluids from colorless to red upon mixing, and the degree of mixing was analyzed [START_REF] Enders | 3D Printed Microfluidic Mixers -A Comparative Study on Mixing Unit Performances[END_REF]. The pH-sensitivity property of fluorescein could also be exploited as the fluorescence quantum yield of fluorescein is higher in an alkaline medium than in an acidic medium [START_REF] Yager | Transverse diffusion in microfluidic systems[END_REF]. Or, the mixing quality in a micromixer can be examined simply by using different streams of fluorescent dye and a clear solution [START_REF] Stroock | Chaotic mixer for microchannels[END_REF][START_REF] Liao | A millisecond passive micromixer with low flow rate, low sample consumption and easy fabrication[END_REF]. For a quick optical characterization, a simple qualitative mixing analysis can be performed using a non-fluorescent dye as shown in Figure 2.17. Image analysis can be carried out to evaluate the quality of mixing.

Microfabrication of PDMS micromixer

A 2-level microfluidic chip was made using a negative photoresist mold (SU-8 3050, MicroChem) with classic soft lithography techniques. PDMS microfluidic devices were chosen for this study as the microfabrication process of a 2-level PEGDA microfluidic chip was difficult, and good-quality devices were not obtained after several attempts.

A normal glass slide was sealed to the PDMS microfluidic chip after undergoing air plasma treatment for 2 minutes. This step was necessary to ensure covalent anchoring of the PDMS block to the glass slide [START_REF] Whitesides | Poly ( dimethylsiloxane ) as a Material for Fabricating Microfluidic Devices[END_REF]. This was possible due to the generation of silanol groups (Si-OH) on the PDMS surface after the oxidation of methyl groups. The chip was then placed in an oven at 65 °C for 10 minutes to strengthen the seal. In order to quantify the evolution of the mixing along the channel, zones, including micromixers, were separated by zones of free diffusion with the design depicted in The design of asymmetrical herringbone grooves that was taken into account helped to develop laminar chaotic flows in the microchannel by stretching and folding the two incoming steady streams into alternate thin liquid sheets to enhance diffusion efficiency [START_REF] Stroock | Chaotic mixer for microchannels[END_REF][START_REF] Stone | Engineering Flows in Small Devices : Microfluidics Toward a Lab-on-a-Chip[END_REF].

Local viscosity mapping in a passive micromixer

For chaotic mixing studies, glycerol and DMSO mixtures of distinct viscosities were introduced at constant flow rates with a neMESYS syringe pump into the two sleeves of the Y-shaped PDMS micromixer. In this experiment, the mixing of streams of initial viscosity η 1 " 9 mPa s and η 2 " 206 mPa s in such a system was studied. The lifetimes measured by LIFA-FLIM were converted into viscosities by using the calibration equation for the PDMS microchannel displayed in Figure 2 The continuous line corresponds to application of a lnpη{η 0 q model, with a " 2.17 and η 0 " 1.41.

final glycerol concentration of the homogeneous mixture rGly calc s was related to the flow rates, Q Si and concentrations, rGly Si s of the two incoming streams through:

rGly calc s " rGly S1 s.Q S1 `rGly S2 s.Q S2 Q S1 `QS2 (2.6)
In order to assess the validity of these measurements, the final concentration of glycerol rGly exp s was measured by converting the measured viscosity into glycerol concentration with a pre-established viscosity-concentration curve as depicted in Figure 2.19b. The corresponding results for different sets of incoming flow rates are gathered in Table 2.1. The measured and calculated value are in good agreement, as can be seen, more quantitatively through their relative discrepancy σ rel remaining below 10 %.

Efficiency of passive micromixers

The obtained viscosity maps could be analyzed more quantitatively to determine the efficiency of the micromixer. It should be noted that the objective of this study is not to provide an efficient micromixer design but rather to propose and test a methodology that can be used to evaluate the efficiency of micromixers. As already mentioned, the flow in the SHM parts of the channel was three-dimensional and difficult to analyze: it was thus more relevant to focus on images taken before each of the SHMs groups, where flow returned to its laminar state. In the following discussion, the mixer design was kept similar, and the effect of injection flow rates is discussed.

Q S1 (µL{minq Q S2 (
The standard deviation of the lifetime (σ) was measured over the different images taken along the microchannel to characterize the mixing efficiency based on previous approaches found in the literature [START_REF] Enders | 3D Printed Microfluidic Mixers -A Comparative Study on Mixing Unit Performances[END_REF][START_REF] Stroock | Chaotic mixer for microchannels[END_REF]. In an ideal situation, σ should be maximal at the entrance of the microchannel and associated with a stepwise profile of lifetime, and decay to zero when the two fluids are homogeneously mixed. Nevertheless, in practice, due to experimental noise, σ reaches a plateau value at the end of the microfluidic chip.

For better comparisons of the situations obtained with different flow rates, it is convenient to study the normalized parameter (σ) defined by:

σ " σ ´σmin σ max ´σmin (2.7)
where σ min is the minimum standard deviation measured at the outlet of the chip, and σ max is its maximum value measured at the entrance of the chip. Values of these extrema were similar within a 10 % variation for the different flow rates considered here.

As demonstrated in Figure 2.21, the mixing performance of the Y-mixer strongly depended on the flow rate, which is in agreement with previous studies [START_REF] Stroock | Chaotic mixer for microchannels[END_REF]. The flow rates were set in random order to account for a possible influence of the previous flow rate before taking the measurement. When the normalized standard deviation was less than 0.2 and remained early constant for a long distance, the two streams were considered to be thoroughly mixed. The colored continuous lines were added for visual clarity only.

The black dashed line represents the evolution that would be obtained for purely diffusive mixing in a simple Y-mixer.

With this design, the mixing length in the microfluidic channel increased with the applied flow rates. Also, the number of SHM groups needed to mix the incoming streams increased as the flow rate increased. The mixing remained minimal before the entry of MM1, the first group of SHMs, which can be observed in Figure 2.20. A drastic drop in standard deviation value was observed after MM2 for all configurations except those with the highest flow rates. Mixing was completed after MM5 at the highest flow rates configuration, while it took four groups of SHM to complete the mixing in other configurations.

At the lowest flow rates Q S1 " 3.5 µL{ min, Q S2 " 0.25 µL{ min, which corresponded to Re = 0.02 in the mixing channel, the micromixer reached the lowest standard deviation first. Mixing for the lowest flow rates improved after MM1 but stayed minimal for other configurations. This is an anticipated result as at low Re (lesser than 10), the slow mixing of two straight, stable streamlines is only due to diffusion [START_REF] Dreher | Characterization of laminar transient flow regimes and mixing in t-shaped micromixers[END_REF]. This can be attributed to Chapter 2 the three-dimensional flow in SHMs, which decomposes the streams into alternated thin sheets resulting in a more efficient diffusion [START_REF] Enders | 3D Printed Microfluidic Mixers -A Comparative Study on Mixing Unit Performances[END_REF][START_REF] Dreher | Characterization of laminar transient flow regimes and mixing in t-shaped micromixers[END_REF].

Conclusion

In conclusion, a relatively easy analytical method was proposed in this chapter to map local viscosity in two microfluidic configurations; firstly, a simple Y-mixer where the mixing of two fluids occurs only via transverse diffusion, and secondly, a passive micromixer in which the mixing of injected streams was aided by chaotic, multi diffusion mixing. To the best of our knowledge, this is the first time that local viscosity mapping has been performed with a FLIM, by measuring the fluorescence lifetime of FMRs in microfluidic chips.

In the first case study, the obtained viscosity maps were in quantitative agreement with models and experiments previously proposed in the literature for a similar system, and diffusion-related parameters, such as the effective diffusion coefficient, were extracted.

Secondly, the study involving complex flow with the passive micromixer allowed us to obtain essential parameters governing micro-scale fluid flow and mixing efficiency. Once the system was well-calibrated, less than 50 µL of sample is needed for local viscosity measurement and mapping. It is important to note that this strategy allowed for measurement of up to three decades in viscosity, nearly up to 1 Pa s. These results validate the possibility of using FMRs for quantitative measurement beyond their previously reported use as contrast agents in bioimaging.

Additionally, the photophysical characterization of a new BODIPY-based molecular rotor for fluorescence lifetime measurements is reported. The fluorescence quantum yield of this FMR was found not to be affected by polarity changes and was exclusively dependent on the medium viscosity.

The results presented in this chapter substantiate the potential of FMRs for quantitative and local characterization of confined flows. Notably, this technique can be adapted to a different system as FMRs can be synthesized to be tailored to a specific viscosity range and solvent environment. This would eventually result in an alternative flow characterization in various situations, from industrial processes to natural flows. Local viscosity measurement in microdevices using FMR could also be an excellent way to characterize fluids that cannot be subjected to shear forces, for instance, biofluids in an organ-on-achip. Nevertheless, it is worth reminding that the local environment of FMR can impact the Förster-Hoffmann coefficients. Hence, it is essential to emphasize the need to perform in situ calibrations for precise quantitative viscosity measurements.

In this thesis work, all fluids employed were Newtonian and confined at the microscale.

This study can be pushed further by focusing on the characterization of complex fluids and highly confined flows close to the molecular scale. This could eventually lead to the determination of the spatial scale over which the viscosity of the microenvironment influences the response of FMRs.

Molecular Rotors and Polymers

One of the main pillars of this thesis is the engineering of a viscosity-sensitive fluorescent polymer that can be grafted on a surface with the aim of developing a solid-state viscosity probe. In order to achieve our goal, a new viscosity-sensitive monomer was synthesized.

This monomer was then integrated into a statistical copolymer before being grafted onto a surface to obtain a viscosity-sensitive surface.

The motivation behind the strategy came from many successful works on the grafting of thermosensitive polymers. Photochromic dyes were grafted to polymers with a low T g to improve the decolorization reaction's kinetics. The mechanism involves the twisting of a bulky group and is strongly affected by the viscosity of the solution [START_REF] Ercole | Comprehensive modulation of naphthopyran photochromism in a rigid host matrix by applying polymer conjugation[END_REF][START_REF] Ercole | Photochromic polymer conjugates: The importance of macromolecular architecture in controlling switching speed within a polymer matrix[END_REF][START_REF] Evans | The generic enhancement of photochromic dye switching speeds in a rigid polymer matrix[END_REF][START_REF] Such | Control of photochromism through local environment effects using living radical polymerization (ATRP)[END_REF][START_REF] Such | Tailoring photochromic performance of polymer-dye conjugates using living radical polymerization (ATRP)[END_REF]. Also, in a similar study, the kinetics of decolorization were used to investigate the self-assembly of a thermosensitive block copolymer [START_REF] Ercole | The application of a photochromic probe to monitor the self-assembly of thermosensitive block copolymers[END_REF]. As can be perceived, the above-mentioned research works employed FMR as probes in polymer environments, however, very few works have addressed the amalgamation of an FMR-polymer system capable of measuring the viscosity of the surrounding environment.

Viscosity-sensitive fluorescent monomer

The focus of this section is the synthesis and characterization of a monomer possessing the viscosity-sensitive characteristics of an FMR. The newly-engineered monomer was named A relatively large amount of monomer was required; nonetheless, the commercialized FMRs are expensive, for instance, 152 € for 5 mg of CCVJ and 116€ for 5 mg of DCVJ. 1Hence, the principal reasoning behind this approach was to acquire the necessary quantity of starting material for the polymerization steps.

Synthesis of Julolidine Aldehyde

The protocol used for the synthesis of julolidine aldehyde was adapted from the work of Haidekker et al. [START_REF] Haidekker | New fluorescent probes for the measurement of cell membrane viscosity[END_REF] and the synthesis route is presented in Figure 3 In this work, the 1 H-NMR analyses were performed using a Bruker Avance III HD 400 spectrometer with a field frequency of 400.2 MHz. The analyzed samples were solubilized in deuterated chloroform (CDCl 3 ) at a concentration of about 10 -12 mg mL ´1.

The samples were referenced to an internal standard, tetramethylsilane (TMS) at 0 ppm.

Deuterated solvents are used to ease locking for the compensation of the natural drift effect of the NMR's magnetic field. The solvents are also necessary for the shimming processes, as well as to eliminate the interference of solvent's protons in the proton NMR spectrum.

In the NMR spectra shown in Figure 3.2, the characteristic peak for aldehyde appears at 9.53 ppm (s, 1H), and the corresponding peak for -CH= band of julolidine at 6.53-6.57 ppm (t, 1H) was absent [START_REF] Haidekker | New fluorescent probes for the measurement of cell membrane viscosity[END_REF]. This confirms the absence of the starting material, julolidine, in the final product, and the purity of julolidine aldehyde was found to be approximately 97 %. The final product contains traces of the solvent, DCM (peak at 5.22 ppm) [START_REF] Babij | NMR chemical shifts of trace impurities: Industrially preferred solvents used in process and green chemistry[END_REF]; however, this does not pose a problem for the next synthesis as it would be easily removed during the work-up. The aldehyde was synthesized several times during this thesis work, and the best yield obtained was 67 %. Even though the first reaction was quenched after the complete conversion of julolidine, the yield is low as a considerable amount of product was lost during the work-up.

Synthesis of Methacrylate Ester

The initial strategy chosen for the synthesis of the desired methacrylate ester was via Steglich esterification, where dicyclohexyl carbodiimide (DCC) was used as a coupling agent to mediate the reaction between a carboxylic acid and an alcohol [START_REF] Neises | Simple method for the esterification of carboxylic acids[END_REF]. During the reaction, which is usually carried out in DMF or a chlorinated solvent system, DCC forms an activated acid derivative to undergo a nucleophilic acyl substitution with the alcohol. Most importantly, mild coupling of complex carboxylic acids and alcohols, along with secondary and tertiary alcohols that are sterically hindered, could be facilitated by

Steglich esterification [START_REF] Tsakos | Ester coupling reactions-an enduring challenge in the chemical synthesis of bioactive natural products[END_REF].

The first synthesis route of the desired ester, DCC-mediated esterification, is shown in Figure 3.3. In this work, the strategy is to synthesize an acrylate ester, 2-cyanoace- to eliminate unreacted DCC, and with brine to remove traces of water. The organic phase was dried with MgSO 4 and concentrated to afford the methacrylate ester.

Cyanoacetic acid and DCC were added in excess to obtain complete conversion of HEMA to methacrylate ester. Nevertheless, this synthesis route was eventually discarded because, firstly, without the catalyst, a long reaction time (more than 48 hours) was required to reach the expected conversion of HEMA. Secondly, the removal process of white-solid DCU was tedious, and the anticipated result was not achieved even with a thorough workup. Removal of DCU was expected to be simple as it is insoluble in DCM and other organic solvents [START_REF] Tsakos | Ester coupling reactions-an enduring challenge in the chemical synthesis of bioactive natural products[END_REF]. Different purification methods were performed, such as multiple liquid-liquid separations (up to 5) and flash chromatography; nonetheless, it was impossible to separate the last traces of DCU from the final product, the methacrylate ester.

The presence of DCU in the final product after a thorough purification is shown in the purple spectra in Figure 3.5. The corresponding peaks for DCU were compared and justified with results found in the literature [START_REF] Li | 1 H NMR determination of 1,3dicyclohexylurea, glutaric acid and triethylamine in medical four-arm poly(ethylene glycol)-N-hydroxysuccinimide-glutarate for better quality control[END_REF]. The peak in the dashed circle corresponds to N -acylurea, the by-product of the reaction between cyanoacetic acid and DCC.

After several failed attempts to synthesize the methacrylate ester without traces of by-products, an alternative strategy was taken to achieve our goal. It was based on the work of Lutjen et al., in which a different synthesis route for Steglich esterification using acetonitrile was proposed: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)-induced esterification [START_REF] Lutjen | Synthesis of esters via a greener steglich esterification in acetonitrile[END_REF]. This synthesis pathway was chosen mainly because of the easy removal of by-products and residual reagents via acidic and basic wash steps [START_REF] Tsakos | Ester coupling reactions-an enduring challenge in the chemical synthesis of bioactive natural products[END_REF][START_REF] Lutjen | Synthesis of esters via a greener steglich esterification in acetonitrile[END_REF]. This is possible thanks to the presence of a basic amine functional group in the chemical structure of EDC, as can be seen in Figure 3.6. Mild coupling of large, complex carboxylic acids and alcohols is enabled with EDC-induced esterification, just like the DCC-induced esterification.

To a solution of 2.8 g cyanoacetic acid (33 mmol) and 6.3 g EDC (33 mmol) was added The reaction was stopped once the intended conversion of HEMA was obtained, and acetonitrile was removed using a rotary evaporator. This is a crucial step because acetonitrile is miscible with water, and its presence with the desired product will decrease the efficiency of the work-up process. The crude solid obtained was then solubilized in orescent monomer, MECVJ. The MECVJ was prepared by the synthetic route shown in It is worth noting that the first synthesis of MECVJ was carried out with julolidine aldehyde as the limiting agent, as described in the literature [START_REF] Haidekker | New fluorescent probes for the measurement of cell membrane viscosity[END_REF]. However, the separation Chapter 3

of the unreacted ester from the final product was tedious and unfruitful. For this reason, julolidine aldehyde was introduced in excess in the earlier described (finalized) reaction route. Furthermore, the synthesis of MECVJ was also performed with the methacrylate ester that was "contaminated" with DCU. Nevertheless, it was impossible to separate DCU from the final product, even via flash chromatography. The solid particles of residual DCU present at the end of the condensation reaction between the ester and aldehyde can be observed in Figure 3.9b. On the other hand, the absence of visible solid by-products can be noticed in the reaction mixture of MECVJ synthesized with ester obtained via EDC-induced esterification. The total amount of MECVJ synthesized in the span of this thesis is around 1.6 g, which is worth more than 37000 € (calculated based on the average selling price of commercialized DCVJ). More than 90 % of the synthesized MECVJ was used for photophysical characterization and eventual polymerization. This evidently shows the necessity of the different syntheses presented in this part.

Photophysical characterization of MECVJ

The experimental protocols for the characterization of MECVJ were the same as those described in Chapter 2 for the characterization of BODIPY-2-OH; refer to Section 2.2.1.

As illustrated in Figure 3.10, the absorption and emission maxima of the MECVJ in glycerol are, respectively, in the blue and green range. The absorption maximum lies at about 467 nm with emission maximum found at 508 nm. These spectra are similar to those previously reported for julolidine-based FMR, which proves that the fluorescence characteristics are not affected by the addition of the methacryloyloxyethyl group to the (cyano-vinyl)julolidine base. The sensitivity of MECVJ to viscosity was verified after the validation of the fluorescence characteristics. In Figure 3.11, viscosity-dependent fluorescence emission was observed for MECVJ in different mixtures of DMSO and glycerol. The concentration of MECVJ in these samples was kept constant at 10 ´5 mol L ´1. The black vertical line at 508 nm shows that the emission maximum is independent of the viscosity of the samples. These results validate our strategy and prove that MECVJ can be used as a local viscosity probe. In addition, the relationship between the fluorescence response of MECVJ and viscosity was studied using media with varying concentration in glycerol and DMSO. and C τ " ´2.1 for lifetime measurement when η is expressed in mPa s and τ in ns.

The exponent α obtained is comparable to the results presented in the literature, for example, 0.52 for CCVJ in glycerol/methanol mixtures [START_REF] Levitt | Fluorescence anisotropy of molecular rotors[END_REF], 0.58 and 0.6 for DCVJ in mixtures of glycerol/ethylene glycol [START_REF] Kung | Microviscosity Measurements of Phospholipid Bilayers Using Fluorescent Dyes That Undergo Torsional Relaxation[END_REF][START_REF] Haidekker | Fluid shear stress increases membrane fluidity in endothelial cells: a study with dcvj fluorescence[END_REF] as well as 0.51 for DCVJ in mixtures of glycerol/methanol [START_REF] Levitt | Fluorescence anisotropy of molecular rotors[END_REF]. These findings demonstrate the validity of the newly synthesized julolidine-based MECVJ as an FMR that can be used for quantitative viscosity mapping.

Viscosity-sensitive Fluorescent Polymers

What is RAFT Polymerization?

In this work, viscosity-sensitive fluorescent polymers were synthesized via Reversible Addition Fragmentation Chain Transfer, RAFT polymerization. RAFT was discovered at the Commonwealth Scientific and Industrial Research Organisation (CSIRO) Australia in 1998 by a team led by Ezio Rizzardo [START_REF] Chiefari | Living freeradical polymerization by reversible addition -Fragmentation chain transfer: The RAFT process[END_REF]. Similarly to other methods of free radical polymerization, the activation step of a photo-or thermal initiator is the first step in the mechanism of RAFT polymerization, as illustrated in Figure 3.13. Next, the initiatorderived radicals, (I ' ) react with a single monomer molecule (M), yielding a propagating polymeric radical, (P ' n ). The particularity of this controlled radical polymerization technique is the involvement of the reversible addition-fragmentation step with the use The thiocarbonylthio group of RAFT agents, shown in Step III has a free radical leaving group, -R, which is capable of reinitiating polymerization (Step IV). The Z-group influences the polymerization reaction kinetics and the degree of structural control that can be obtained. The R and Z groups determine the solubility and reactivity of a RAFT agent; hence, the choice of a RAFT agent has to be adapted to a particular class of monomers. The main categories of RAFT agents are dithioesters, dithiocarbamates, trithiocarbonates, and xanthates [START_REF] Perrier | 50th Anniversary Perspective: RAFT Polymerization -A User Guide[END_REF].

In RAFT pre-equilibrium and equilibrium steps (steps III and V, respectively), "living" polymer chains are generated from dormant chains (macroRAFT agent, refer to Figure 3.13), i.e., chain extension via further addition of monome [START_REF] Perrier | 50th Anniversary Perspective: RAFT Polymerization -A User Guide[END_REF]. In Step III, the intermediate radical is formed when a RAFT agent reacts with a polymeric radical with n-monomer units (P ' n ). The intermediate radical then goes through a reversible fragmentation reaction in either direction, either regenerating the starting species either the (P ' n ) and RAFT agent or expelling the radical leaving group (R ' ) to generate a dormant macroRAFT agent. The macroRAFT agents thus generated continue to undergo Chapter 3 Figure 3.13: Mechanism of RAFT polymerization; scheme taken from [START_REF] Perrier | 50th Anniversary Perspective: RAFT Polymerization -A User Guide[END_REF]. exchange reactions with propagating polymeric radicals (Step V), with the result that each chain undergoes brief periods of activity alternating with long periods of dormancy.

In the ideal case of rapid exchange between active and dormant chains, all chains grow at a similar rate throughout the reaction, leading to control over molecular weight and a narrow chain length distribution.

The Design Criteria of poly(DMA-s-MECVJ)

The newly engineered statistical copolymer in this work consists of two monomers: the previously described MECVJ and dimethylacrylamide, DMA. This copolymer is designed to ensure that the photophysical characteristic of the MECVJ is not affected. The choice of DMA is due to its hydrophilic property, which is compatible with solvents such as glycerol, DMSO, and water. These green solvents are prudently selected to accommodate the health and safety regulation of the laboratory. DMA makes up at least 90% of the polymer composition. As DMA swells well in the targeted solvents, the viscosity-sensitive part of MECVJ would be brought further into the analyzed samples.

In most cases, polymerization is quenched before the full conversion of all the monomers present in the reaction mixture in order to avoid side reactions and maintain good control of the polymerization. Considering this work, the methacrylate-based MECVJ was designed as a comonomer, as when methacrylates and acrylamides are copolymerized, the methacrylate is typically consumed more rapidly. In contrast, an acrylate-based viscositysensitive monomer would react at a similar rate to the DMA. At the end of polymerization with an acrylate-based monomer and DMA, a considerable amount of the unreacted "expensive" monomer would need to be removed during the purification steps. This would be an unwise strategy as the monomer synthesized via several time-and energy-consuming reactions would be disposed of as waste. Also, the presence of an unreacted fluorescence monomer in the final reaction mixture would be problematic as it would be tricky to differentiate the fluorescence property of both the monomer and polymer, considering that traces of monomers could be found in the purified polymer even after a thorough work-up step. This approach was also used in the study of Ercole et al. as a strategy to ensure the efficient use of photochromic monomers [START_REF] Ercole | Photochromic polymer conjugates: The importance of macromolecular architecture in controlling switching speed within a polymer matrix[END_REF].

RAFT polymerization was chosen to synthesize this copolymer as it contributes to narrow dispersity and gives a well-defined end group. The RAFT agent used was S -cyanomethyl S'-dodecyl trithiocarbonate, depicted in Figure 3.14. The trithiocarbonate part of the synthesized polymer will be the essential tool for grafting onto a surface; a detailed discussion can be found in Chapter 4. MECVJ and DMA are considered "more activated" monomers, with substituents that stabilize the radicals formed during polymerization. As a result, a Z-group is required to enhance the stabilization of the intermediate radical to favor radical addition to the C=S double bond. Therefore, trithiocarbonate or dithiobenzoate RAFT agents are widely preferred to control the polymerization of "more activated" monomers [START_REF] Perrier | 50th Anniversary Perspective: RAFT Polymerization -A User Guide[END_REF]. ization, the rate of the addition-fragmentation equilibrium is greater than the propagation rate. As a result, all polymeric chains grow at the same rate and will have a similar degree of polymerization [START_REF] Perrier | 50th Anniversary Perspective: RAFT Polymerization -A User Guide[END_REF]. The general structure of the proposed polymer is illustrated in

Synthesis of poly(DMA-s-MECVJ)

For the first synthesis of poly(DMA-s-MECVJ)-P3, the required quantities of monomers, initiator (I), RAFT agent were calculated from the following ratio of numbers of equivalents: [MECVJ]/[DMA]/[I]/[RAFT] = 1/100/0.2/1. The initiator used for all the polymerizations was azobisisobutyronitrile (AIBN). Firstly, 31.9 mg of RAFT agent (0.1 mmol) and 3.8 mg of AIBN (0.02 mmol) were added to 9 mL of dioxane in a twin-neck round bottom flask equipped with a magnetic stirrer. Then, 994 mg of DMA (10 mmol) and 38.9 mg of MECVJ (0.1 mmol) were introduced. Next, the reaction medium was degassed via bubbling with N 2 for 30 minutes. The polymerization reaction began when the flask was put in the oil bath at 60 ℃, under stirring. Different samples were taken from the reaction flask via cannula transfer under a current of N 2 for 1 H-NMR analysis. This was done in order to determine the conversion of the MECVJ and DMA over time. The polymerization was quenched by placing the reaction flask in an ice bath at 0 ℃. Four different polymers were synthesized with the above-described protocols with varying ratios of starting materials and reaction time. Details of these reactions are shown in Table 3 The workup started with the removal of dioxane under reduced pressure to afford darkyellow viscous liquid. The product was then solubilized in around 10 mL of DCM. The polymer mixture was added dropwise in a cold conical flask containing 200 mL of diethyl ether, resulting in precipitation of poly(DMA-s-MECVJ). The precipitation medium was centrifuged at 5 ℃ for 30 minutes (at 2500 rpm). Finally, dark orange-yellow solids were obtained at the end of the purification steps, as presented in Figure 3.17. The 1 H-NMR and SEC analyses were performed to obtain data on monomer conversion and molar mass.

Characterization of Polymers

Size Exclusion Chromatography (SEC)

The principle of the Size Exclusion Chromatography (SEC) is based on the separation of macromolecules such as proteins and polymers according to their sizes and molar mass.

The separation is performed using a column containing pre-calibrated porous gel. The macromolecules with higher molar mass are eluted first as they cannot pass through the pores. Conversely, smaller components or lower-molar-mass molecules are able to move through the pores, which increases their residence time in the column, and they are eventually eluted later.

The injection into the chromatography column is automated and aided by a pump.

Different detectors are concatenated in series at the column's exit: a differential refractometer, a multi-angle light scattering detector (MALS), and an UV detector. The MALS detector allows the determination of the absolute molar mass of polymer chains, if their refractive index increment (dn/dc) is known. The dispersity (Ð), which is the ratio of the weight average molar mass (M W ) to the number average molar mass (M n ), can also be estimated.

The measurements in this work were carried out using an UltiMate 3000 SEC from Thermoscientific in DMF/Lithium bromide (LiBr) (1 g L ´1). The separation was performed using Shodex Asahipack columns (GF-310 and GF-510) of 7.8 ˆ300 mm. 0.45 µm Teflon syringe filters were used to filter around 200 µL of polymer solution of 2 -4 mg mL ´1, which were prepared in DMF/LiBr, before the sample injection. The samples were injected with a flow rate of 0.5 mL{ min at 50℃. The detection system consists of an 8-angle Dawn Heleos MALS detector as well as a Wyatt Optilab rEX differential refractive index detector. It should be stated that only qualitative analysis was done for all the copolymers by using a pre-established calibration with polystyrene standards. This is due to the concern that the low-molecular-weight polymers could not scatter well, resulting in a low signal with the light scattering detection. A pre-established calibration using polystyrene standards of known molecular weight was used to provide approximate molecular weights of the analyzed polymers.

Results and General Discussion

As indicated in Table 3.1, polymerization reactions were carried out for more than 10 hours for all the polymers except for P3. The higher the amount of MECVJ added to the polymerization reaction, the longer it took for MECVJ to integrate entirely into the polymer. This was evident for the polymer with the highest amount of MECVJ, P4, where the fluorescent monomer was only completely consumed after 54 hours with the conversion of DMA at 86% of the initial amount. This might be due to the slow reaction of MECVJ coupled with the eventual consumption of AIBN.

The half-life of AIBN, which is the time required to reduce the initial concentration of the initiator by 50%, is roughly 10 hours at 60℃. Thus, within 40 hours, almost 95%

of AIBN was depleted. Taking this into account, for P4, an additional amount of AIBN was added into the reaction flask during the course of the polymerization reaction.

Also, the higher amount of RAFT agent added in the reaction mixture for the purpose of obtaining short polymers (low degree of polymerization) decelerates the polymerization reaction. This effect can be observed by comparing the polymerization time for P1 and P3, where the ratio of initial monomers are the same for both polymers.

The combination of methacrylate-based MECVJ and DMA was chosen, as explained earlier in Subsection 3.3.2, in order to take advantage of the higher reactivity of methacrylates in this copolymerization. The evolution of MECVJ and DMA polymerization for P2 is shown in Figure 3.16. MECVJ can be seen to be fully incorporated into the copolymer before full conversion of DMA is reached, with the absence of two characteristic proton peaks of MECVJ at 5.5 and 6.1 ppm after 13 hours of reaction. Due to time constraints, a quantitative kinetic study was not carried out for all the polymerizations. However, this does not affect our study as this part aims to acquire viscosity-sensitive fluorescent polymers.

Furthermore, the quality of the retrieved polymers was analyzed with 1 H-NMR and SEC, and the results obtained for P3 are portrayed in Figure 3.17. An absence of starting monomers and solvent can be perceived, and the narrow differential refractive index signal in the chromatogram confirms the low dispersity of the synthesized polymer. Nevertheless, as listed in Table 3.1, a low dispersity value was not achieved for P4, for which MECVJ makes up around 10% of its composition. Cyanomethyl dodecyl trithiocarbonate is a mediocre RAFT agent for methacrylate monomers, it could be that there was too much MECVJ in this polymerization, and thus, control was lost. However, it would be more likely that the fluorescent part of the MECVJ interacts with the propagating radicals in some way. Lower than expected molecular weights and higher than expected disper-

Photophysical Properties of Polymers

The photophysical features of the synthesized polymers were examined after obtaining polymers of good purity (> 95%). The absorption and emission maxima were determined for all the polymers, and they fall in the blue and green visible ranges, respectively. As illustrated in Figure 3.18 for polymer P2 in glycerol, the wavelengths of the maxima of absorption and emission were respectively found to be 461 nm and 510 nm. The details on the absorption and emission maxima for the polymers are listed in Table 3.2. These data are similar to the fluorescent monomer, MECVJ, proving that the fluorescence characteristics of MECVJ are not affected while being integrated into a polymer.

During his L3 internship at the Laboratory of the Future (LOF) in Pessac, Mr. Maxime Zitouni conducted a part of the experimental studies described in this subsection. thesized polymers in this work within a viscosity range between 1 mPa s and 820 mPa s.

The detailed experimental protocol for this part can be found in Section of Chapter 2.

Viscosity measurements were carried out using cone-plate geometries for different mixtures of glycerol and DMSO (green lines) as well as for water (magenta lines). The concentration of polymers in these samples was kept constant at 10 ´5 mol L ´1 except for samples of P4, in which the polymer concentration was reduced to 10 ´6 mol L ´1 due to the much higher concentration of fluorophores in this copolymer. This was done to adapt to the limitations of the spectrophotometer. Smaller α-coefficients were obtained for all the polymers compared to MECVJ (α MECVJ = 0.6) when η is expressed in mPa s and τ in ns. This indicates lower sensitivity of the fluorescent polymers to viscosity. In the case of P1 and P3, the α-coefficients were found to be similar for both fluorescence lifetime and intensity measurements. However, a difference in this value was observed between intensity and lifetime measurements regarding polymers with a higher amount of incorporated MECVJ. This dissimilarity is mainly due to the multiexponential decay behavior of polymers that can be detected in lifetime measurement [START_REF] Haidekker | Environment-sensitive behavior of fluorescent molecular rotors[END_REF].

Most likely, the interaction between multiple MECVJ that are close to each other in It is worth highlighting that even though the synthesized polymers are slightly less sensitive to viscosity than the MECVJ, the sensitivity is still adequate for quantitative viscosity measurements. The polymers, P2 and P4 do not follow the Förster-Hoffmann relationship, which should give the same α exponent, as observed for the monomer, MECVJ.

However, this is not important from the application point of view, as the obtained calibration curves can still be used to obtain a viscosity value from the fluorescence response. As explained previously in Chapter 2 and as indicated in the literature, julolidine-based FMR cannot be used for quantitative lifetime measurement for low-viscosity samples due to very short corresponding fluorescence lifetimes [START_REF] Kuimova | Mapping viscosity in cells using molecular rotors[END_REF]. Based on these findings, it can be concluded that poly(DMA-s-MECVJ) can be used as a viscosity probe in compatible solvents/mediums for quantitative viscosity measurements.

Conclusion

In summary, the first objective of this chapter was the synthesis of a new viscosity-sensitive fluorescent monomer, the MECVJ. The starting materials of this synthesis, the julolidine aldehyde and a methacrylate ester, were also synthesized to have good control of the ensuing polymerization reaction. Following the synthesis, the photophysical properties of MECVJ in solution were analyzed and compared to those of commercially available The results obtained and presented in this chapter show that FMRs can be incorporated into polymer chains while maintaining their sensitivity to the viscosity of their local environment. It was found that at a given viscosity value, the fluorescence lifetimes of an FMR-integrated polymer are longer than that of the julolidine-based monomer used for its synthesis. This is an interesting finding, as the lifetimes of julolidine-based FMRs are known in the literature to be too short for measurements with a FLIM setup. This also raises questions about the interaction of the FMR with the polymer chains and enabling, at the same time, the engineering of materials with a desired response in terms of lifetime.

In this thesis, the integration of a julolidine-based FMR into a polymer allowed obtaining material in the good working range of a standard FLIM setup. Further studies need to be conducted to comprehend the effect of the polymer's molecular weight on the fluorescence quantum yield.

Nevertheless, there are still some crucial questions concerning the effect of the polymer environment on the behavior of the rotor. Concerning the experiments carried out throughout this thesis, the fluorescence response of the developed polymeric materials increases with increasing viscosity. Even though some of the obtained polymers did not follow the Förster-Hoffmann model, an evident trend was observed, and this property is satisfactory enough to validate its utilization as viscosity-sensitive materials. More Chapter 4

In Chapter 3, it was demonstrated that viscosity-sensitive fluorescent polymers can be engineered by integrating FMR into the polymer chains. These polymers were found to be slightly less sensitive to local viscosity compared to the monomer, MECVJ, yet, they exhibited longer fluorescence lifetimes at a given viscosity value. In this final chapter, the utilization of the obtained polymers with different percentages of MECVJ for in-situ viscosity measurement was further explored. A microfluidic strategy was developed by integrating a surface incorporating the poly(DMA-s-MECVJ) as a wall of the microchannels.

This innovative microdevice presented many advantages, most importantly, the possibility to observe in-situ the viscosity changes occurring within the system and to perform its eventual mapping, as demonstrated in Chapters 2 and 3. Nonetheless, it also comes with several technical challenges. The main difficulty of the fabrication of this microviscosimeter was the integration of a viscosity-sensitive polymeric material within a microfluidic device and its exploitation to set up microfluidic experiments. As a last part of this thesis, part the results presented in this chapter are still under investigation and not fully understood: they should be taken as a proof-of-concept for the realization of a viscosity-sensitive microfluidic chip, opening paths for future investigations.

State of the Art: Solid-State Molecular Rotors

Fluorescence dyes have been employed in some research works to develop solid-state sensors for various applications, for instance, the self-erasable and reversible solid-state platform using crystallochromic fluorophores for anti-counterfeiting applications was demonstrated in the work of Bhuin et al. [START_REF] Bhuin | Acceptor-donor-acceptor-linked triphenylamine and phenothiazine motifs as cousin molecules: Methyl-effect on stimuli-responsiveness, crystallochromism and dual-state emission[END_REF].

Grafting of viscosity-sensitive polymers onto surfaces

Hence, we chose to graft the rotor in the form of polymers with the aim of developing viscosity-sensitive microfluidic chips. We hypothesized that collapsing of the fluorescent probe could be prevented in this way. Surface-grafted polymers can be obtained via two techniques: the "grafting-from" technique involving the use of a surface-immobilized initiator to generate polymer brushes, and the "grafting-to" technique, where presynthesized polymer chains are covalently attached to a surface. In this work, the "grafting-to" strategy was chosen as it is easier to control the polymerization and the polymer growth as well as to characterize the polymers, for example, with liquid 1 H-NMR and SEC techniques presented in Chapter 3. The exact chemical composition of the grafted polymer would be known. Also, the trithiocarbonate part of the RAFT agent in our polymer is ideal for grafting onto a surface.

"Grafting-to": "Thiol-ene Click" Chemistry/Michael addition

The initial strategy chosen to achieve the goal, as mentioned earlier, was the technique of "Grafting-to" of poly(DMA-s-MECVJ) via Michael addition or "thiol-ene click" chemistry on a silanized glass slide. The experiments carried out were based on a simple technique developed by the Gibson group [START_REF] Biggs | Thiol-ene immobilisation of carbohydrates onto glass slides as a simple alternative to gold-thiol monolayers, amines or lipid binding[END_REF][START_REF] Biggs | Grafting to" of RAFTed Responsive Polymers to Glass Substrates by Thiol-Ene and Critical Comparison to Thiol-Gold Coupling[END_REF]; an example of the applied strategy is depicted in

Grafted surface characterization: Contact-angle measurements

The outcome of the grafting method was verified qualitatively via contact angle measurements. As poly(DMA-s-MECVJ) is a hydrophilic polymer, successful grafting should be associated with a decrease in the contact angle of a drop of water, when compared to a silanized glass slide. The corresponding experiments were performed with the Attension Theta Optical Tensiometer using the sessile drop method. This technique is mainly used to measure the wetting of a liquid on a flat, horizontal solid surface. The analysis method consists in depositing a drop of water on a surface and measuring its contact angle. A direct result on the relative hydrophilicity or hydrophobicity characteristics of the ana-

"Grafting-to": Au-S Covalent Attachment

This second strategy, thiol-gold immobilization, was chosen for the grafting of poly(DMAs-MECVJ) not only for its easy, straightforward execution to obtain grafted layers but also because it is one of the most extensively studied techniques of molecule immobilization. This had served as an inspiration for the group of McCormick, who used polymers prepared via RAFT for immobilization on gold surfaces. The dithioester end groups of the RAFT agent were reduced to thiols in the presence of the reducing agent, sodium borohydride (NaBH 4 ) before being attached to gold [START_REF] Sumerlin | Modification of gold surfaces with water-soluble (co) polymers prepared via aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization[END_REF]. However, this method was not adopted in our work as the reducing agents may react with the MECVJ causing the loss 4.2. GRAFTING OF VISCOSITY-SENSITIVE POLYMERS ONTO SURFACES 121 of its fluorescence and viscosity-sensitivity characteristics.

On the other hand, the Duwez research group has shown in their work that a similar grafting efficiency can be achieved with dithioesters and trithiocarbonate groups, resulting in monolayers on gold [START_REF] Duwez | Dithioesters and trithiocarbonates as anchoring groups for the "grafting-to" approach[END_REF]. The schematic representation of the trithiocarbonate group on gold is depicted in Figure 4.4 (far-right). Since then, many research work on the attachment of RAFT-ed polymers with single and multiple trithiocarbonate groups to gold surfaces before, particularly onto the surface of gold nanoparticles, have been carried out [START_REF] Ebeling | RAFT-polymers with single and multiple trithiocarbonate groups as uniform gold-nanoparticle coatings[END_REF][START_REF] Fustin | Tuning the hydrophilicity of gold nanoparticles templated in star block copolymers[END_REF]. This method was adapted for this thesis as well.

Experimental Protocols

Some experimental works presented in this section were carried out by Mr. Julien Renaudeau during his M1 internship at the Laboratory of the Future (LOF) in Pessac. Prior to the polymer grafting, a thin gold film of 30 nm (metal purity = 99.9 %) was deposited on regular glass slides via metal evaporation. This was carried out in a vacuum pressure at around 10 ´6 mbar (glove box). The glass slides were cleaned with isopropanol and dried using Kimtech wipes to remove impurities such as stains or dust specks in order to obtain a clean, flat glass surface. Also, a 5 nm-thick chromium film was deposited beforehand to provide good adhesion of the gold coating to the glass surface.

Firstly, polymer solution in water of 14.3 mg mL ´1 was deposited on the gold-coated glass slide. The glass slides were kept in a closed Petri dish and left at room temperature, in the dark, for at least 16 hours. The glass pieces were then rinsed with distilled water (5 ˆ2 mL) and dried delicately under a stream of N 2 . Photographs were taken before analyzed by using VG ESCALAB 220i-XL X-ray photoelectron spectrometer with nonmonochromatic Mg Kα radiation (hν " 1253.6 eV) and a pass energy of 50 eV. An internal reference of C1s binding energy (285.0 eV) was used. The setup allows measurement with a lateral resolution of 150 µm in routine in an ultra-high vacuum condition, approximately at 10 ´8 mbar.

A small piece of polymer-grafted gold-coated glass slide was analyzed alongside a regular gold-coated glass slide which acts as a reference. The grafting was carried out with a solution of poly(DMA-s-MECVJ), precisely P3, at a concentration of 5 mg mL ´1 in water.

As a reminder, details of the different polymers synthesized in this work can be found in Table 3. Also, a decrease in the atomic percentage of gold (Au) was noted for the polymergrafted surface, signaling that the surface was well-grafted with poly(DMA-s-MECVJ). A large characteristic peak for nitrogen N 1s, absent for non-grafted spectra, was observed for the polymer-grafted surface around 400 eV [START_REF] Beamson | High resolution XPS of organic polymers[END_REF]. The presence of sulfur (S) was observed on the polymer-grafted surface. The S2p peak at 163.6 eV is representative of the C-S bond that is not interacting with Au, whereas the peak at 162.4 eV represents the chemisorbed sulfur. These results are in accordance with data presented in the work of Fustin and Duwez [START_REF] Fustin | Dithioesters and trithiocarbonates monolayers on gold[END_REF].

It can be confidently said that the polymers were indeed grafted to the surface based on the change of contact angle and changes in the XPS signal. Subsequently, the fluorescence characteristic of the grafted polymer was verified with the FLIM-LIFA. As a reminder, the α-exponent found for polymer P4 in solution is 0.42 for fluorescence lifetime measurements. The sensitivity of the probe to local viscosity was thus reduced when being grafted onto a surface. Most likely, the lower sensitivity of the probe is due to covalent linking. Covalent tethering of polymer chains can decrease the intramolecular twisting ability of the integrated FMR and directly reduce the solvent viscosity effect on the quantum yield. Another reason could be the close distance of the hydrophobic julolidine-end of MECVJ to the surface, which might provoke the collapsing of the molecule onto the hydrophobic surface [START_REF] Lichlyter | Immobilization techniques for molecular rotors-Towards a solid-state viscosity sensor platform[END_REF]. However, sensitivity remained sufficient to observe changes in viscosity, as will be shown in part 4.3. Future work will be devoted to understanding this reduction of sensitivity and finding ways to minimize or avoid this effect.

Further control of the surface

During the characterization experiments, the grafted surface was washed abundantly yet delicately with water after each lifetime measurement of a glycerol-water solution. This was done to ensure the removal of previously tested samples. However, it was observed that the grafted polymer eventually detached after a day of continuous experiments. Following this observation, the effect of continuous washing with water was studied, and the results are plotted in Figure 4.7b. The objective was to observe the effect of constant fluid contact on the probe's sensitivity to viscosity. After prolonged washing for 63 hours, the grafted surface was found to be highly heterogeneous yet, still sensitive to changes in viscosity with a value of α-exponent similar to these for previously analyzed grafted samples Chapter 4

(cleaned with overnight washing). It was concluded that an extended liquid contact detaches the deposited gold along with the polymer onto it but does not affect its sensitivity.

Moreover, a control experiment of polymer grafting was carried out using a regular glass slide. Unexpectedly, the polymer was attached to a non-gold glass slide when the previously described grafting protocol was applied. The polymer film was still present after thorough washing in water. The viscosity-sensitivity of the polymer film was evaluated with different solutions of glycerol-water, and the obtained data is plotted in Figure 4.7c.

During the lifetime measurement, it was observed that the tested fluids were stained with the polymer present on the surface. (the colorless mixture turned yellow). This was not observed for the polymer-grafted gold glass slide. The surface was not sensitive to viscosity as the polymer detached into the tested samples. It can be concluded that attaching the polymer to gold film is required in order to prevent the leaching of the polymer into the sample. However, the gold surface itself is prone to detach from the glass slide after prolonged use. The effect of heat on the polymer-grafted surface was investigated as well. Immediate surface alteration upon contact on a hot plate at 95 °C was observed, as portrayed in The surface alteration is most likely due to the thermal expansion of glass which is different from the thermal expansion of the deposited metals. This quick analysis could be helpful in the conception of other types of FMR-integrated microfluidic chips, such as OS-TEMER, which requires exposure at high temperatures during the microfabrication steps.

From these results, we can conclude our strategy for grafting the synthesized viscositysensitive polymers on solid surfaces. A more systematic study is obviously required to understand and ideally control the changes of sensitivity upon attachment on a surface, however, due to time constraints, this will be left for future investigation. However, even if not optimized, we managed to design a viscosity-sensitive surface as intended.

Viscosity-sensitive Microfluidic Chip

Once acceptable parameters were determined for the preparation of a polymer-grafted surface, the next part of this work was consecrated to the development of a viscosity-sensitive microfluidic chip prototype. A simple, straight microfluidic channel with a width of 2 mm and height h of 100 µm with one inlet and outlet was chosen for basic local viscosity measurement. Considering the time remaining for this thesis, PDMS was selected as the material for the microchannel as its microfabrication protocol is relatively easy and rapid.

The PDMS block was then sealed with a poly(DMA-s-MECVJ)-grafted glass slide using foldback clips, as shown in Figure 4.9. The common sealing protocol via plasma treatment did not work for this setup, thus explaining the use of the clips. Care should be taken to ensure that the pressure exerted by these clips does not deform the microfluidic channel. This can be checked by measuring the hydraulic resistance of the channel. As it depends on the section of the channel, any change would indicate a deformation. This was not a problem for the experiments conducted in this work as the aim was merely to demonstrate that the microfluidic device is sensitive to viscosity by measuring the lifetime, and the latter does not depend on the section of the channel. Only a small section of the glass base of the PDMS chip was deposited with gold and grafted with polymer.

A polyimide film (Kapton) was used to cover the rest of the glass surface during gold deposition. The current prototype can be improved in several ways, such as adapting the width of the channel to match the width of the deposited gold on the glass slide. This is because the grafted surface is fragile and breaks easily with the pressure exerted by the clips. Due to time constraints, this had not been done in this work.

In the context of this work, a PEGDA microfluidic chip would not be ideal: in addition to the complicated protocol of surface silanization on a gold-coated surface, the microfluidic device has to be continuously exposed to UV during the microfabrication step, which would lead to photobleaching of MECVJ. For the conception of the microfluidic chip, the concentration of polymer solution for grafting was doubled to 28.6 mg mL ´1 to improve the fluorescence signal of the device.

Conclusions and Prospects

In summary, this chapter was devoted to presenting the results concerning the grafting of the poly(DMA-s-MECVJ) presented in Chapter 3 onto glass surfaces with the aim of developing a viscosity-sensitive surface and integrating it in a microfluidic chip. Qualitative verification of the success of the grafting was carried out with contact-angle measurements and XPS. Also, the photophysical properties of the surface were verified with FLIM-LIFA.

When grafted on a surface, FMR-integrated polymer sensitivity to viscosity decreased compared to the polymer in solution. However, it remained sufficient for practical applications. As a perspective, in order to limit this loss, we could increase the polymer length to have more space between the FMR from the surface. One could also imagine working with a more flexible polymer, such as polyethylene glycol, or changing the type of FMR.

For the integration of the polymer in a microfluidic chip, different strategies have to be implemented for different types of FMR.

Further quantitative measurements may be performed to characterize the polymer grafting onto surfaces. First, the thickness of the grafted polymer layer should be measured, for instance, by ellipsometry. XPS measurements should also be more quantitatively exploited. Finally, information on the grafting density of the poly(DMA-s-MECVJ) could be obtained using a quartz crystal microbalance to compare the gold immobilization method.

The presented works and results in this chapter on the viscosity-sensitive microfluidic device were developed as a proof-of-concept. We were able to design viscosity-sensitive surfaces and successfully integrate them into a microfluidic chip. The simplicity of the integration methodology makes our technique very versatile and easily adapted to any flow configuration. This thus paves the way for developing in situ and inline analytical tools for the characterization of fluids with evolving viscosity relevant to industrial processes.

It is to remark that this is the first example of a microfluidic chip capable of performing measurements of viscosity online, allowing viscosity mapping of fluids.

General Conclusion

The aim of this thesis was to incorporate viscosity-sensitive fluorescent molecular rotors in the domain of microfluidics for local viscosity measurements. For a better understanding of the concepts and results presented in this manuscript, the key ideas covered in this work, such as fluorescence molecular rotors, fluorescence intensity, fluorescence lifetime, and the Förster-Hoffmann model, were introduced and clarified in the first chapter.

The second chapter of this thesis manuscript focused on the quantitative local viscosity mapping in microfluidic flows using the FLIM technique. The viscosity mapping experiments in this chapter were carried out with a newly-reported BODIPY-based FMR.

The photophysical characteristics and the sensitivity of this molecule to viscosity were analyzed thoroughly. Two microfluidic configurations were studied: a common Y-mixer in which the mixing of two miscible fluids occurs only via transverse diffusion and is precisely modeled, and secondly, a passive micromixer in which the mixing of injected streams was aided by chaotic mixing. Diffusion-related parameters, such as the effective diffusion coefficient, were retrieved from the viscosity maps performed in the first case study. Next, parameters affecting mixing effectiveness and micro-scale fluid flow were obtained in the study on a more complex flow using the passive micromixer. It is worth mentioning that the proposed method enabled measurements of viscosities up to three decades, ranging from 9 mPa s up to almost 1 Pa s. The obtained results for both case studies were quantitatively in accord with models found in the literature for a similar system.

In Chapter 3, a comprehensive report on the synthesis of different statistical copolymers with various ratios of comonomers: MECVJ and DMA were presented. MECVJ is a new viscosity-sensitive fluorescent monomer synthesized in this work. The reported findings in this chapter demonstrate that FMRs can be included in polymer chains while retaining their sensitivity to the local environment's viscosity. It was discovered that the fluorescence lifetimes of the synthesized FMR-integrated polymer are longer than these of the julolidine-based monomer utilized in its manufacture at a given viscosity value. This is an intriguing discovery because it has been shown in the literature that julolidine-based FMR lifetimes are too short to be measured using a FLIM system.

The main takeaway from Chapter 4 is the development of a novel viscosity-sensitive microfluidic device via the incorporation of a polymer-grafted surface with a PDMS microchannel. When grafted on a surface, FMR-integrated polymer sensitivity to viscosity decreased compared to the polymer in solution. Nevertheless, it can be asserted that the obtained sensitivity remained sufficient for practical applications. The presented proofof-concept will be a stepping-stone to the development of a reliable FMR-integrated microfluidic device that could perform precise quantitative viscosity measurement. This device could be a new approach to measuring viscosity in large series as it would be a portable tool with real-time response in viscosity changes. It also opens the possibility for inline viscosity measurement, which would be beneficial for continuous process control.

The developed methodology and the obtained results in this thesis work open an avenue of ideas and possibilities for future research. Firstly, it is still unclear what the FMR is measuring exactly. We can be sure that it is sensitive to a parameter correlated to local viscosity but defined at the molecular scale, while macro-viscosity is averaged on mesoscopic volume. Working with confined systems such as thin liquid films, nanofluidic channels, or non-newtonian materials with a mesoscale structure like micelle solutions or polymer solutions could be a way to tackle this question. Next, all fluids examined in this thesis work were Newtonian and confined at the microscale. Future research should be pivoted to characterizing highly confined flows and complex fluids that are closer to the molecular scale. It will be easier to establish the spatial scale at which the microenvironment's viscosity affects the FMR response. Study and eventual local viscosity mapping of non-Newtonian fluids, for example, shear-thinning fluids, is possible as well. Analyzing these fluids on an FMR-integrated viscosity-sensitive surface would also be interesting.

The effect of the polymer architecture on its sensitivity to viscosity can also be studied.

It would be interesting to incorporate FMRs in block copolymers and analyze the changes when the copolymers self-assemble. FMRs could also be integrated into thermoresponsive polymers to investigate their phase transitions. New polymer designs for specific reactions are worthy of further exploration. Polymer engineering with BODIPY-based FMRs would be a good choice as these molecules are cheaper, commercially available/easier to synthesize. Moreover, a detailed investigation of the rotor's photochemical process needs to be carried out in order to have a better understanding of the behavior of an FMRintegrated polymer grafting to a surface. On the other hand, a solid-state FMR-based polymeric sensor could also be developed via the "grafting-from" technique, for instance, via Cu-mediated Atom transfer radical polymerization, which can provide a stronger bond to the surface compared to the method proposed in this work.

2. 3 . 1

 31 Fabrication of PEGDA microfluidic chips . . . . . . . . . . . . . . . 2.3.2 Microfluidics Experiments . . . . . . . . . . . . . . . . . . . . . . . 2.3.3 Local viscosity mapping in a simple Y-mixer . . . . . . . . . . . . . 2.3.4 Measurement of Effective Diffusion Coefficient (D eff ) . . . . . . . . . 2.4 Viscosity mapping during chaotic mixing . . . . . . . . . . . . . . . . . . . 2.4.1 Microfabrication of PDMS micromixer . . . . . . . . . . . . . . . . 2.4.2 Local viscosity mapping in a passive micromixer . . . . . . . . . . . 2.4.3 Efficiency of passive micromixers . . . . . . . . . . . . . . . . . . .2.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Synthesis of viscosity-sensitive fluorescent polymers 3.1 Molecular Rotors and Polymers . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Viscosity-sensitive fluorescent monomer . . . . . . . . . . . . . . . . . . . . 3.2.1 Synthesis of Julolidine Aldehyde . . . . . . . . . . . . . . . . . . . . 3.2.2 Synthesis of Methacrylate Ester . . . . . . . . . . . . . . . . . . . . 3.2.3 Photophysical characterization of MECVJ . . . . . . . . . . . . . . 3.3 Viscosity-sensitive Fluorescent Polymers . . . . . . . . . . . . . . . . . . . 3.3.1 What is RAFT Polymerization? . . . . . . . . . . . . . . . . . . . . 3.3.2 The Design Criteria of poly(DMA-s-MECVJ) . . . . . . . . . . . . 3.3.3 Synthesis of poly(DMA-s-MECVJ) . . . . . . . . . . . . . . . . . . 3.3.4 Characterization of Polymers . . . . . . . . . . . . . . . . . . . . . 3.3.4.1 Methods of Characterization . . . . . . . . . . . . . . . . . 3.3.4.2 Results and General Discussion . . . . . . . . . . . . . . . 3.3.5 Photophysical Properties of Polymers . . . . . . . . . . . . . . . . . 3.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Grafting FMRs-Polymer materials on surfaces: Towards a Solid-state Viscosity Probe 4.1 State of the Art: Solid-State Molecular Rotors . . . . . . . . . . . . . . . . 4.2 Grafting of viscosity-sensitive polymers onto surfaces . . . . . . . . . . . . 4.2.1 "Grafting-to": "Thiol-ene Click" Chemistry/Michael addition . . . . 4.2.2 "Grafting-to": Au-S Covalent Attachment . . . . . . . . . . . . . . . 4.2.2.1 Qualitative evidence of polymer attachment . . . . . . . . 4.2.2.2 Viscosity-sensitivity of polymer-grafted surfaces . . . . . . 4.2.2.3 Further control of the surface . . . . . . . . . . . . . . . . 4.3 Viscosity-sensitive Microfluidic Chip . . . . . . . . . . . . . . . . . . . . . . 4.4 Conclusions and Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . .

MW 4 2 POCl 3

 423 Weight average molar mass M n Number average molar mass CDCl 3 Deuterated Chloroform MgSO Magnesium sulfate N Nitrogen Phosphorus oxychloride 4-DASPI trans-4-[4-(dimethylamino)styryl]-1-methyl-pyridinium iodide AIBN Azobisisobutyronitrile CCVJ 9-(2-Carboxy-2-cyanovinyl)-julolidine DCC Dicyclohexyl carbodiimide DCM Dichloromethane DCU Dicyclohexylurea DCVJ 9-(2,2-Dicyanovinyl)-julolidine DMAP 4-dimethylaminopyridine DMF N,N-Dimethylformamide DMSO dimethyl sulfoxide EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride FCS Fluorescence Correlation Spectroscopy FLIM Fluorescence Lifetime Imaging Microscopy FRAP Fluorescence Recovery after Photobleaching HCl Hydrochloric Acid HEMA Hydroxyethylmethacrylate I Initiator LiBr Lithium bromide LIFA Lambert Instruments FLIM Attachment M Monomer MALS Multi Angle Light Scattering MECVJ 9-(2-methacryloyloxyethyloxycarbonyl-2-cyanovinyl)-julolidine NaOH Sodium hydroxide NMR Nuclear Magnetic Resonance PDMS Polydimethylsiloxane PEGDA Poly(ethylene glycol) diacrylate RAFT Reversible Addition Fragmentation Chain Transfer SEC Steric Exclusion Chromatography T g Glass Transition Temperature THF Tetrahydrofuran TICT Twisted Intramolecular Charge Transfer TMS Tetramethylsilane UV Ultra-violet XPS X-ray photoelectron spectroscopy Ð Dispersity Résumé de Thèse La viscosité joue un rôle crucial dans de nombreux processus industriels impliquant l'écoulement de fluides, et une mesure locale de la viscosité est importante car la dynamique des fluides à l'échelle microscopique et le comportement microrhéologique sont souvent différents de leurs caractéristiques globales. Les outils les plus courants utilisé pour mesurer cette quantité sont les viscosimètres, mais leur utilisation nécessite une grande prudence. En plus, les viscosimètres ne mesurent qu'une viscosité moyenne sur des volumes macroscopiques et sont difficiles à mettre en oeuvre en ligne. Cela limite fortement leur application dans les processus industriels, les systèmes de petit volume et les systèmes confinés. L'impossibilité d'obtenir des données locales empêche également leur utilisation dans le cas d'écoulements complexes impliquant une hétérogénéité spatiale et/ou temporelle, comme c'est souvent le cas dans les écoulements macroscopiques (par exemple, dans les systèmes industriels tels que la fabrication de peintures ou la transformation des aliments) et à petite échelle (par exemple, dans les écoulements multiphasiques micro/millifluidiques ou dans les milieux poreux, avec des applications dans la récupération améliorée du pétrole). La conception d'outils pour la mesure de la viscosité à petite échelle est donc importante tant pour les études fondamentales que pour les applications industrielles. D'autre part, les systèmes microfluidiques ont été largement utilisés comme un outil essentiel dans de nombreuses applications telles que le criblage à haut débit et l'analyse des réactions chimiques, laissant ouvertes les possibilités de mesure de la viscosité. Cette thèse développe une méthodologie simple et innovante pour les mesures de viscosité locale dans les dispositifs microfluidiques, et les travaux de recherche et de développement correspondants sont présentés dans quatre chapitres. Le premier chapitre est consacré aux explications générales afin d'introduire et d'élucider le terme : Rotor(s) moléculaire(s) fluorescent(s) (FMR) et les principaux concepts abordés dans ce travail tels que l'intensité de fluorescence, le temps de vie de fluorescence et le modèle de Förster-Hoffmann. Des travaux antérieurs sur les mesures de microviscosité utilisant le FMR dans plusieurs domaines sont également présentés. La fluorescence, un sous-groupe de la luminescence, est la caractéristique d'un atome ou d'une molécule d'émettre de la lumière à une longueur d'onde spécifique après avoir absorbé de la lumière à une longueur d'onde plus courte (c'est-à-dire avec une couleur différente de la lumière émise). La fluorescence est observée lorsqu'une molécule dans le premier état singulet excité électroniquement retourne à l'état fondamental en émettant un photon. L'efficacité totale de la fluorescence est diminuée par d'autres processus concurrents, par exemple la conversion interne ou le croisement intersystème. Les molécules émettant de la fluorescence sont appelées fluorophores, sondes fluorescentes ou fluorochromes, et ces molécules sont généralement composées de quelques groupes aromatiques, de composés planaires ou cycliques avec plusieurs liaisons π .

Figure 1 :

 1 Figure 1: Schéma décrivant le mécanisme de fluorescence dépendant de la viscosité lors de la photoexcitation d'un rotor moléculaire, DCVJ.

Figure 2 :

 2 Figure 2: Cartographie de la viscosité lors d'un simple co-flow entre un mélange (viscosités initiales) de 9 mPa s (S1-courant en dessus) et 74 mPa s (S2-courant au dessous). Les débits appliqués (S1, S2) pour (a) sont de (3,1) µL{ min et pour (b) de (7,1) µL{ min. La flèche représente la direction de l'écoulement et une longueur d'échelle de 150 µm tandis que w représente la largeur du canal. Les images ont été prises sur la longueur du microcanal, et x représente la position où l'image a été prise. Le premier point de contact des deux solutions est défini comme x = 0 mm, le début de la distance le long de la direction de l'écoulement.

Figure 3 :

 3 Figure 3: (En haut) Schéma d'un mélangeur microfluidique en Y avec des micromélangeurs passifs à chevrons en quinconce (en anglais: staggered herringbone passive micromixers (SHM)). (En bas) Cartographie de la viscosité dans un mélangeur en Y avec SHM pendant un co-flow avec un mélange de 9 mPa s (S1) et 206 mPa s (S2). Les débits appliqués (S1, S2) pour (35, 0,25) µL{ min. La flèche représente la direction du flux et une longueur d'échelle de 200 µm. Les images ont été prises sur la longueur du microcanal et E représente l'"entrée", S la "sortie" et M la zone en milieu d'un micromélangeur.

Figure 4 :

 4 Figure 4: Voie de synthèse du MECVJ (3) par condensation de l'aldéhyde de julolidine (1) et d'un cyanoester (2).

Figure 5 :

 5 Figure 5: Structure chimique générale du polymère sensible à la viscosité, le poly(DMA-s-MECVJ). La flèche en pointillé représente la rotation du rotor autour de la liaison simple, qui relie les groupes donneur et accepteur d'électrons. Le nombre total de monomères (MECVJ et DMA) ciblés dans une chaîne polymère est noté n (pour plus de détails, voir le chapitre 3).

Chapter 1 Fluorescence 1 . 1 32 1. 2 33 1. 3 35 1. 3 . 1 40 1. 4 Fluorescence 41 1. 4 . 1 44 1. 5 47 1. 6

 11132233335314044141445476 and Molecular Rotors "I will love the light for it shows me the way, yet I will endure the darkness because it shows me the stars." -Og Mandino, A Better Way to Live Contents The History of Fluorescence . . . . . . . . . . . . . . . . . . . . Fluorescence in General . . . . . . . . . . . . . . . . . . . . . . What are Fluorescent Molecular Rotors? . . . . . . . . . . . . Principle of Molecular Rotors . . . . . . . . . . . . . . . . . . . 36 1.3.2 Förster-Hoffmann Relationship . . . . . . . . . . . . . . . . . . 37 1.3.3 Intensity, Lifetime, and Viscosity: a luminous relationship . . . Measurements of Molecular Rotors . . . . . . . Absorption and Fluorescence Spectroscopy . . . . . . . . . . . 42 1.4.2 Epifluorescence Microscopy . . . . . . . . . . . . . . . . . . . . 43 1.4.3 Fluorescence Lifetime Imaging Microscopy (FLIM) . . . . . . . Microviscosity and Molecular Rotors . . . . . . . . . . . . . . Thesis Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Chapter 1

Figure 1 . 1 :

 11 Figure 1.1: (a) Sir George G. Stokes, 1st Baronet. He was a British physicist and mathematician. (b) Fluorspar, the commercial name for the mineral fluorite, taken from [4].

Chapter 1 Figure 1 . 2 :

 112 Figure 1.2: Jablonski energy diagram depicting the possible radiative and non-radiative transitions that can occur upon photoexcitation.

diagram in Figure 1

 1 .4.

Figure 1 . 4 :

 14 Figure 1.4: A simplified Jablonski Diagram focusing on the fluorescence mechanism of FMR.

Figure 1 . 5 ,

 15 Figure 1.5, by taking an example of a single-emission model FMR, 9-(2,2-Dicyanovinyl)-

Figure 1 . 5 :

 15 Figure 1.5: Scheme depicting the mechanism of viscosity-dependent fluorescence upon photoexcitation of a molecular rotor, DCVJ.

  using fluorescence spectroscopy and steady-state fluorescence microscopy and on timeresolved fluorescence measurement with the FLIM. Two-dimensional fluorescence map-Chapter 1 pings were also performed with fluorescence microscopy. Measurements of viscosity have been carried out via fluorescence-based techniques such as Fluorescence Recovery af-

2 )

 2 can be detected via fluorescence spectroscopy with the emission spectrum of a fluorophore. Fluorescence spectroscopy was performed during this thesis with a spectrophotometer that uses a xenon flash lamp technology as the light source. In this technique, the sample is irradiated by the light from the excitation source after passing through a monochromator. The incident light and emitted fluorescence light are separated by the monochromator. After a rapid photoexcitation, fluorophores, including FMR, emit light in all directions in the spectrometer. A fraction of the fluorescence light reaches the detector after traversing a monochromator or a filter. A detector, in most cases, is positioned 90°from the excitation light beam to reduce the possibility of incident light being transmitted or reflected reaching the detector. At this angle, the ratio of stray light (different wavelengths of light than the targeted ones) is significantly reduced compared to a measurement at 180°, eventually improving the signal-to-noise ratio. Comprehensive scans within the range of wavelength of 190-1100 nm were done at a specific excitation wavelength for all the analyzed FMRs.

Figure 1 . 6 , 1 Figure 1 . 6 :

 16116 Figure 1.6, in a classic configuration, an epifluorescence microscope consists of a light source, an excitation filter, a dichroic mirror, an emission filter, and a detector. The light source for the excitation beam could be a mercury-vapor lamp, xenon arc lamp, highpowered light-emitting light-emitting diodes, LEDs, or lasers. A customized combination of an excitation filter, dichroic mirror, and emission filter has to be employed depending on the fluorescent dyes used for the microscopic examination.

Figure 1 . 7 :

 17 Figure 1.7: Phase shift, φ, and demodulation, M of fluorescence signal relative to the excitation light. Blue and green sinusoidal curves represent the excitation and fluorescence intensity signals, respectively.

Figure 1 . 8 :

 18 Figure 1.8: Fluorescence lifetime mapping of microviscosity in different parts of a model plant, Arabidopsis root. A BODIPY-based FMR was used to perform the mapping. The grey line on each image represents a scale bar of 100 µm; the figure was taken from [53].

Figure 1 .

 1 Figure 1.9: (a)-(c): Pure glycerol penetrating into glycerol-water solution (85% glycerol in mass) (d)-(f): Glycerol-water solution (85% glycerol in mass) displacing pure glycerol. Images (c) and (f) are reconstructed viscosity fields on a logarithmic scale. The placement of the thermoelectric heater is shown by the white dashed squares in (a), (b), (d), and (e), whereas the white line in (b) represents the scale bar of 5 cm. The figure is sourced from [59].

Figure 1 .

 1 Figure 1.10: (a) Before and after comparison of microviscosity cartography in a CHO cell undergoing PDT; extracted from [22] (b) Fluorescence image (E) and fluorescence lifetime map (F) of live epithelial cells; taken from [60].

A

  new viscosity-sensitive fluorescent boron-dipyrromethene (BODIPY)-based probe, BODIPY-2-OH, as shown in Figure 2.1, was specifically developed for this study. This FMR was synthesized by the group of Pr. Norberto Farfan, at the Facultad de Química of the Universidad Nacional Autónoma de México (UNAM). Synthesis details of BODIPY-2-OH are not presented in this manuscript as it is out of the scope of this thesis, where the aim is to employ the FMR as a local viscosity probe in microfluidic systems. Exhaustive synthesis protocol and molecule characterization can be found in Nalatamby et al. [83].

Figure 2 . 1 :

 21 Figure 2.1: The chemical structure of BODIPY-2-OH synthesized in this work.

  iments. The concentration of BODIPY-2-OH in the analyzed mixtures was kept constant at 10 ´5 mol L ´1 to avoid changes in fluorescence intensity due to dye concentration. Single-use cuvettes (BRAND GMBH 70 µL UV-Cuvette micro) were used for excitation and emission spectra measurement. Viscosity data were acquired for the photophysical analysis of BODIPY-2-OH as well as the Förster-Hoffmann calibration before the microfluidic experiments. The mixtures of DMSO-glycerol analyzed in this work were Newtonian fluids. Bulk viscosity measurements were performed using a Kinexus Ultra+ rheometer (Netzsch) with a Double-Gap geometry, DG24/27 SS CUP for samples of low viscosity and 1°, 60 mm-diameter, coneplate geometry for viscous samples (for viscosity more than 15 mPa s). The experimental temperature was controlled by a Peltier module and set to T " 25 °C. The shear viscosity of every sample was measured by applying 4 different shear rates: 1 ;10; 100; 1000 s ´1.

Figure 2 . 2 :

 22 Figure 2.2: (a) Excitation spectra (blue line) and emission spectra (green line) for BODIPY-2-OH in glycerol. (b) Emission spectra of BODIPY-2-OH in DMSO-glycerol mixtures of different viscosities. The black vertical line shows that the emission maxima are independent of the viscosity of the samples.

Figure 2 .

 2 Figure2.2b shows the viscosity-dependent fluorescence emission property of BODIPY-2-OH. The maximum intensity was observed to increase with viscosity, proving that BODIPY-2-OH can be used as a viscosity probe. Moreover, the emission wavelength was not affected by the composition of the different DMSO-glycerol solutions, which proves that fluorescence response is not affected by polarity changes in different DMSO-glycerol mixtures within a viscosity range between 9 mPa s to 800 mPa s[START_REF] Michael | Imaging phase separation in model lipid membranes through the use of BODIPY based molecular rotors †[END_REF].

  IX71 inverted microscope. The image acquisitions were carried out at LED modulation frequency of 40 MHz and with 12 acquisition phases and 1x CCD gain. A 10 µmol L ´1 fluorescein solution with a known lifetime of 4.02 ns was taken as reference. For lifetime calibration, cavity slides were used to hold the DMSO-glycerol solutions containing BODIPY-2-OH at a fixed concentration of 10 ´5 mol L ´1 and glass coverslips were used to obtain a flat layer of liquid with even thickness. The samples were illuminated with a LED beam at 451 nm through the dry microscope objectives lenses (20X Olympus), using a dichroic mirror. The fluorescence emission of the molecular rotor was collected by the same objective and transmitted to the cooled detector after passing through an Olympus 440x-490 nm long pass filter. The retrieved data were then analyzed using a GUI MATLAB application developed by Dr. Florence Gibouin, a postdoctoral researcher at the Laboratory of the Future.

Figure 2 . 3 ,

 23 fluorescence intensity and lifetime evolve with viscosity in a power-law manner with a similar exponent α " 0.6. These are linear evolutions on a double logarithmic scale for a viscosity varying over three orders of magnitude. This observation is in line with the results found in the literature for other BODIPY-based FMRs[START_REF] Kuimova | Mapping viscosity in cells using molecular rotors[END_REF][START_REF] Wu | Molecular rheometry: direct determination of viscosity in Lo and Ld lipid phases via fluorescence lifetime imaging[END_REF][START_REF] Kuimova | Exploring viscosity, polarity and temperature sensitive of BODIPY-based molecular rotors[END_REF]. The result confirms that BODIPY-2-OH can be used as a local viscosity probe. Unlike the α constant, the value of C depends on the instrument used to measure the fluorescence quantum yield. Hence, the difference in this constant, as demonstrated in Figure2.3, is inconsequential. Lifetime values of the probe measured for viscosity above 9 mPa s are within the detection limit of the LIFA-FLIM used for this work. The power-law increase of fluorescence properties with ambient viscosity is usual with FMRs and derives from the Förster-Hoffmann equation (Equation 1.6)[START_REF] Förster | Die viskositätsabhängigkeit der fluoreszenzquantenausbeuten einiger farbstoffsysteme[END_REF].

Figure 2 . 3 :

 23 Figure 2.3: Calibration curve in logarithmic scale of BODIPY-OH-2 fluorescence intensities, I F (blue dots) and fluorescence lifetimes, τ F (green dots) versus DMSO-glycerol mixture viscosity, η. The straight lines correspond to a fit with Förster-Hoffmann model (Equation 1.6) with dye-dependent coefficient, α " 0.6 for both fluorescence lifetime and intensity measurements. The proportionality constant, C was found to be 1.3 for fluorescence intensity and -1.1 for lifetime measurement.

Figure 2 . 4 :

 24 Figure 2.4: (a) Excitation (dashed lines) and emission (solid lines) spectra of DCVJ in various mixtures of ethylene glycol and glycerol (b) Förster-Hoffmann model fit of DCVJ in mixtures of ethylene glycol and glycerol with α " 0.59. These figures are taken with permission from [9].

Figure 2 . 6 .Figure 2 .Figure 2 . 5 :

 26225 Figure 2.6. First of all, two elements have to be prepared prior to the microfabrication steps; the first is a mixture of 95 % PEGDA-250 and 5 % of photoinitiator, 2-hydroxy-2methylpropiophenone; the chemical structures are shown respectively in Figure 2.5a and Figure 2.5b. Second are the silanized glass slides, on which the PEGDA stamps would be attached to form a microfluidic channel. The glass slides are silanized with silane acrylate, 3-(Trimethoxysilyl)propyl acrylate (Figure 2.5c) via chemical vapor deposition at 65 ℃ for at least 24 hours. This surface treatment is necessary as PEGDA has a weak adhesion on a glass surface.

Figure 2 . 6 :

 26 Figure 2.6: Scheme of different steps in the microfabrication of PEGDA microfluidics chip, adapted from the thesis of Dr. Camille Keita [91].

Figure 2 . 7 :

 27 Figure 2.7: Photograph of a functional PEGDA Y-junction microfluidic chip with flatbottomed NanoPorts attached on the inlets and outlet.

A

  Y-junction microfluidic chip was chosen for mapping because, with this design, two fluids flow side by side in a channel and mix through molecular diffusion. The dimensions of the main microchannel in Figure 2.8 were measured after classic photolithography of the SU-8 mold using a Sensofar Non-contact 3D Optical Profiler. Height and width of the channel were respectively h " 40 µm, and the width of the channel, w " 500 µm. Mixtures of glycerol and DMSO of different viscosities were injected at constant flow rates with a neMESYS syringe pump into the two sleeves of the Y-shaped PEGDA microchannel for simple diffusion experiments.

Figure 2 . 8 :

 28 Figure 2.8: Scheme of a PEGDA microfluidic Y-junction of height h and width w. The mixing of two fluids flowing side by side in the microchannel occurs through transverse molecular diffusion.

Figure 2 . 9 :

 29 Figure 2.9: Schematic representation of fluorescence lifetime measurements of a fluorescein solution; C=10 ´6 mol L ´1 in flow along a PDMS microchannel. (i) represents the image taken in greyscale, and (ii) represents the color-scaled image obtained after image analysis using MATLAB.

Figure 2 . 10 :

 210 Figure 2.10: Logarithmic-scale calibration curve of BODIPY-2-OH fluorescence lifetimes, τ F versus DMSO-glycerol mixture viscosity, η in a PEGDA microchannel. Straight lines correspond to a fit with the Förster-Hoffmann model (Equation 1.6) with exponent α = 0.5. Error bars for τ F values are smaller than the size of data points.

Figure 2 . 11 :

 211 Figure 2.11: Viscosity mapping during a simple co-flow between a mixture (initial viscosities) of 9 mPa s (S1-upper stream) and 74 mPa s (S2-lower stream). The flow rates applied (S1, S2) for (a) is (3,1) µL{ min and for (b) is (7,1) µL{ min. The arrow represents the flow direction, and a scale length of 150 µm whereas w represents the width of the channel. Images were taken along the length of the microchannel, and x represents the position where the image was taken. The first point of contact of the two solutions is defined as x = 0 mm, the beginning of the distance along the flow direction.

Figure 2 . 12 :

 212 Figure2.12: Viscosity profiles η versus y, for different positions x along the microchannel (color-coded: darker to lighter shades represent increasing x direction, downstream), in a simple Y-mixer with flow rates pQ S1 , Q S2 q " p7, 1q µL{ min. The arrow represents downstream evolution, with growing x.

Figure 2 . 13 :

 213 Figure 2.13: Glycerol volume fraction variation in function of the viscosity of the mixture.The continuous line corresponds to application of a lnpη{η 0 q model, with a " 0.16 and η 0 " 1.87.

First, in orderFigure 2 . 14 :

 214 Figure 2.14: (a) Profiles of glycerol volume fraction φ exp pyq at different positions x along the micro-channel (color-coded: darker to lighter shades represent increasing x direction, downstream) obtained from viscosity profiles obtained in Figure 2.12. The continuous lines represent the fits according to Equation 2.1. (b) Normalized glycerol volume fraction p hi versus normalized position ỹ as defined in Equation2.2. Data obtained for different flow rates pQ S1 , Q S2 q " p7, 1q (brown), p3, 1q (blue) and p11, 1q µL{ min (blue). The black dashed line represents the theoretical master curve from Equation 2.3.

Figure 2 . 15 :Figure 2 . 16 :

 215216 Figure 2.15: Variation of fitting parameters (a) y m , (b) φ 0 , and (c) φ min versus characteristic time τ obtained during the co-flow experiments in a simple Y-mixer with (Q S1 , Q S2 ) = p7, 1q (brown); p3, 1q (blue); p11, 1q µL{ min (green).

Figure 2 . 17 :

 217 Figure 2.17: Passive micromixer characterization using a non-fluorescent blue dye. Images were taken with a digital microscope color camera.

Figure 2 . 18 :

 218 Figure 2.18: Schematic diagram of a microfluidic Y-junction with staggered herringbone passive mixer (SHM). The mixing of two fluids of different viscosities in the microchannel occurs via chaotic advection.

Figure 2 . 18 .

 218 Figure 2.18. Groups of three staggered herringbone passive micromixers (SHM) grooves occupying a length of 1.95 mm were separated by free intervals of length 9.40 mm. The distance from the beginning of the Y-junction to the first group of SHM is about 7620 µm.

Figure 2 . 19 :

 219 figure: in particular, images designated by E correspond to the entrance of SHMs zones, where the flow is laminar. In these images, after every SHM, the mixing degree increased, which was characterized by the widening interdiffusion layer and the homogenization of the viscosities of the two streams. Images designated by M and S were taken in and after SHMs zones, respectively, where we can observe destabilization of the interdiffusion layer, leading to enhanced mixing.

Figure 2 .

 2 Figure 2.20: (Top) Schematic diagram of a microfluidic Y-mixer with staggered herringbone passive micromixers (SHM). (Bottom) Viscosity mapping in a Y-mixer with SHM during a co-flow with a mixture of 9 mPa s (S1) and 206 mPa s (S2). The flow rates applied (S1, S2) for (35, 0.25) µL{ min. The arrow represents the flow direction and a scale length of 200 µm. Images were taken along the length of the microchannel and E stands for "inlet", S for "outlet" and M for the middle zone of the micromixer.

Figure 2 . 21 :

 221 Figure 2.21: Normalized standard deviation, σ, of the lifetime maps such as in Fig 2.20 defined in Equation (2.7) as a function of the distance downstream from the entrance of the channel, x, at the beginning of every SHMs group. Data shown in this figurewere generated from a single microfluidic device during the mixing of DMSO-glycerol solutions of different viscosity, η 1 " 9 mPa s (S1) and η 2 " 206 mPa s (S2). Applied flow rates pQ S1 , Q S2 q were p3.5, 0.25q (˛, average Reynolds number Re " 2 ˆ10 ´2); p7.0, 0.5q (', average Reynolds number Re " 5 ˆ10 ´2); p14.0, 1.0q (', average Reynolds number Re " 0.1); p35.0, 2.5q µL min ´1 ( , average Reynolds number Re " 0.2).

" 43 Contents 3 . 1 3 . 2

 433132 If you found the light once, you can find it again." -The Good Doctor, S4 Ep.18, @11:Molecular Rotors and Polymers . . . . . . . . . . . . . . . . . . Viscosity-sensitive fluorescent monomer . . . . . . . . . . . . .

FMRs have been associated

  with the study of polymers in some research works, and in addition to the examples presented in Section 1.5 in Chapter 1, some of the most widely-cited, earliest reported work on the incorporation of FMR with polymer is the work of Loutfy et al. In these works, the progress of polymerization reactions of methyl methacrylate, styrene, ethyl acrylate, and many more were monitored with the change in fluorescence intensity of dimethylaminobenzylidene malononitrile and DCVJ. The results obtained in this study led to the development of a method to study the distribution and changes in free volume in polymer systems [23, 24]. Most recently, the group of Daniel Bonn presented several works on polymer analysis by taking advantage of the environment-dependent fluorescence trait of FMR. Bittermann et al. reported the study of the local viscosity of aqueous poly(ethylene oxide) solutions at the nanometer scales using a water-soluble FMR, trans-4-[4-(dimethylamino)styryl]-1methyl-pyridinium iodide (4-4-DASPI). A correlation between the fluorescence response of 4-DASPI and the polymer chain length was determined in addition to the verification of the Förster-Hoffmann equation (Equation 1.6). The results found in this paper led to a conclusion that in an aqueous polymer solution, FMR is not only sensitive to the macroviscosity but also to the molecular weight of the analyzed polymer[START_REF] Bittermann | Disentangling Nano-and Macroscopic Viscosities of Aqueous Polymer Solutions Using a Fluorescent Molecular Rotor[END_REF]. In the work of Mirzahossein et al., the local viscosity heterogeneity near the glass transition temperature (T g ) of a polymer glass was examined by embedding FMR in the polymer matrix. Dicyanodihydrofuran-embedded polyvinyl acetate polymer glass was examined locally with confocal microscopy for intensity measurements and FLIM for lifetime measurements. Similar behavior observed in both techniques proves that FMR is an excellent probe of free volume at the nanoscale[START_REF] Mirzahossein | Molecular rotors to probe the local viscosity of a polymer glass[END_REF].In a very recent study published in December 2022 by Garcia and Blum, FMR were used to determine one of the essential properties of a polymer, the weight average molar mass (M W ). Analyzed polymers were incorporated with FMR by adding 1 in 10 7 monomer labeled with a BODIPY-based FMR into the reaction mixture. A calibration was established beforehand for polymers ranging from 35 to 570 kg mol ´1 with molecular weight data obtained from gel-permeation chromatography and fluorescence lifetime of FMR, measured with the FLIM technique. The continuous evolution of the molecular weight of polynorbornene and polydicyclopentadiene was then determined within 1 s (per measurement) by correlating the changes in the fluorescence lifetime of the polymer. The quantitative analysis is independent of polymer solubility and could be performed without removing the sample from the reaction mixture[START_REF] Garcia | Polymer molecular weight determination via fluorescence lifetime[END_REF].

9-( 2 -

 2 methacryloyloxyethyloxycarbonyl-2-cyanovinyl)-julolidine (MECVJ), and it was obtained via condensation of an aldehyde and a methacrylate ester. These starting materials were synthesized in this work as well. This study was developed in collaboration with the Organic Polymer Chemistry Laboratory (LCPO-UMR 5629) in Bordeaux, France, under the supervision of Dr. Simon Harrisson.

. 1 .

 1 In a roundbottom flask of 50 mL, 0.5 g (2.89 mmol) of julolidine (1) was added to a mixture of N, N-dimethyl formamide (DMF) (0.54 mL, 6.97 mmol) and 7 mL of dichloromethane (DCM).Then, 0.58 mL (6.2 mmol) of phosphorous oxychloride (POCl 3 ) was gradually added dropwise, and the mixture was stirred at 25 ℃ for 12 hours. The reaction flask was placed in an ice bath at 0 ℃ for 15 minutes before the addition of 28 mL (2 M) sodium hydroxide (NaOH) aqueous solution. The mixture was stirred at 0 ℃ for four hours.The first step of the work-up was the extraction of the organic layer with diethyl ether (3 ˆ10 mL). The retrieved organic phase was dried with magnesium sulfate (MgSO 4 ), and the diethyl ether was removed under reduced pressure. The residue was crystallized twice in DCM/heptane (1:10) mixture to obtain long pale yellow crystals. Lastly, the obtained crystals, julolidine aldehyde (2), were filtered via vacuum filtration (0.25 mg, 1.25 mmol, 43 % yield).

Figure 3 . 1 :

 31 Figure 3.1: Synthesis route of compound (2), Julolidine Aldehyde

Figure 3 . 2 :

 32 Figure 3.2: 1 H-NMR spectra of the starting material, julolidine, and the desired product, julolidine aldehyde.

Figure 3 . 3 :toxyethylmethacrylate ( 5 )

 335 Figure 3.3: Synthesis of 2-cyanoacetoxyethylmethacrylate (5) via DCC-mediated esterification.

Figure 3 . 4 :

 34 Figure 3.4: Chemical structure of 4-dimethylaminopyridine (DMAP).

Figure 3 . 5 :

 35 Figure 3.5: Qualitative comparison of 1 H-NMR spectra of 2-cyanoacetoxyethylmethacrylate (5). Top-purple spectra correspond to the final product obtained via DCC-mediated esterification, and bottom-blue spectra correspond to the product synthesized via EDCmediated esterification. The peak encircled with dashed circle corresponds to N -acylurea, the by-product of the reaction between cyanoacetic acid and DCC.

Figure 3 . 6 :

 36 Figure 3.6: Chemical structure of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, EDC

Figure 3 . 7 .

 37 Figure 3.7. Firstly, 0.51 g of 2-cyanoacetoxyethylmethacrylate (2.57 mmol) and 0.59 g of julolidine aldehyde (2.96 mmol) were added into a round-bottom flask containing 15 mL of tetrahydrofuran (THF). The flask was then placed in an oil bath at 50 ℃, and 1.6 mL of triethylamine (11.5 mmol) was added drop-wise. The mixture was continuously stirred for 96 hours.

Figure 3 . 8 :

 38 Figure 3.8: 1 H-NMR spectra of viscosity-sensitive monomer, MECVJ.

Figure 3 . 9 :

 39 Figure 3.9: (a) Mixture of MECVJ synthesized with DDC-induced esterification in the reaction solvent, THF, with the presence of DCU fragments and (b) Mixture of MECVJ synthesized with EDC-induced esterification in THF. Both images were taken before the evaporation of the solvent.

Figure 3 . 10 :

 310 Figure 3.10: Absorption (blue line) and emission spectra (green line) of MECVJ in glycerol. Maximum absorption and emission wavelengths of MECVJ in glycerol are respectively 467 nm and 508 nm.

Fig- ure 3 .Figure 3 . 11 :

 3311 Figure 3.11: Emission spectrum of MECVJ in different DMSO-glycerol mixtures of varying viscosity.

Figure 3 . 12 :

 312 Figure 3.12: Calibration curves in logarithmic scale of MECVJ fluorescence intensities, I F (green points) and fluorescence lifetimes, τ F (purple points) versus DMSO-glycerol mixture viscosity, η. Error bars for I F and τ F are smaller than the size of data points.

Figure 3 . 14 :

 314 Figure 3.14: Chemical structure of the RAFT agent used in this work : S -cyanomethyl S'-dodecyl trithicarbonate

Figure 3

 3 Figure 3.15.

Figure 3 . 15 :

 315 Figure 3.15: General chemical structure of the viscosity-sensitive polymer, poly(DMAs-MECVJ. The dotted arrow depicts the rotation of the rotor around the single bond, which links the electron donor and acceptor groups.

3. 3 . 4 . 1

 341 Methods of Characterization Proton Nuclear Magnetic Resonance Spectroscopy ( 1 H-NMR)As explained earlier, the 1 H-NMR spectroscopic analyses were performed with a Bruker Avance III HD 400 spectrometer with a field frequency of 400.2 MHz. The analyzed polymers were dissolved in approximately 0.5 mL of CDCl 3 , and the concentration of the polymer solutions was kept between 10 -12 mg mL ´1. The samples were referenced to Chapter 3 TMS (0 ppm).

Figure 3 .

 3 Figure 3.16: 1 H-NMR monitoring during the synthesis of poly(DMA-s-MECVJ), P2, in CDCl 3 . The inset zoomed spectra focus on the over-time disappearance of the characteristic peaks of MECVJ.

  sities may indicate irreversible chain transfer reactions, while retardation may indicate increased chain termination. This reasoning could explain the lower than the expected M n obtained for P2 and P4. It is worth reiterating that the essential part of this study is to synthesize viscosity-sensitive fluorescent polymers, and these less-understood results did not pose any problem to the upcoming experiments and the continuity of this thesis work.

Figure 3 . 17

 317 Figure 3.17: 1 H-NMR spectra of poly(DMA-s-MECVJ P3. (a), (b), and (d) represent the characteristic proton peaks of the RAFT agent, (c) the polymerized DMA, and (e) the polymerized MECVJ. The inset chromatogram obtained via SEC in DMF represents the differential refractive signal of the copolymer. Top-right inset: an image of P3 synthesized in this work.

  Figure 3.17: 1 H-NMR spectra of poly(DMA-s-MECVJ P3. (a), (b), and (d) represent the characteristic proton peaks of the RAFT agent, (c) the polymerized DMA, and (e) the polymerized MECVJ. The inset chromatogram obtained via SEC in DMF represents the differential refractive signal of the copolymer. Top-right inset: an image of P3 synthesized in this work.

Figure 3 .Figure 3 . 18 : 2 :

 33182 Figure 3.19 shows the viscosity-dependent fluorescence emission property of all the syn-

Figure 3 . 19 :

 319 Figure 3.19: Emission spectrum of (a) P1, (b) P2, (c) P3, and (d) P4 in different mixtures of varying viscosity.

Figure 3 .

 3 Figure 3.20 shows the compilation of double logarithmic plots of fluorescence intensity and lifetime with viscosity established for these polymers. The straight lines correspond to fits with the Förster-Hoffmann model (Eq. 1.6). Error bars for I F and τ F are smaller than the size of data points.

  P2 and P4, is the reason behind the multiexponential decay in lifetime measurement with the FLIM-LIFA. The corresponding lifetime values were then averaged before being used for Förster-Hoffmann calibration. It is not possible to detect the multi-exponential behavior in intensity measurement, where the retrieved data used for the plots of Figure3.20 corresponds to the brightest intensity peak belonging to one polymer species. Intensity also depends on the relative amount of MECVJ incorporated in the different polymer

Figure 3 . 20 :

 320 Figure 3.20: Calibration curves in logarithmic scale of (a) P1, (b) P2, (c) P3, and (d) P4. Fluorescence intensities, I F are represented with green points and fluorescence lifetimes, τ F with purple points.

Figure 3 . 21 :

 321 Figure 3.21: Lifetime comparison of P3 with MECVJ

FMRs

  such as DCVJ and CCVJ. The focal point of the second part of Chapter 3 was the synthesis of different statistical copolymers with various ratios of comonomers: MECVJ and DMA. The polymerization processes were constantly improved based on results and observations made with the precedent experiments. Subsequently, the photophysical properties of the different polymers were evaluated.

Figure 4 .

 4 1a. The principle of this strategy is the in-situ generation of polymer-SH by aminolysis of the RAFT agent part of poly(DMA-s-MECVJ) via a reaction with ethanolamine, presented in Figure 4.1b. The chemical structure of the RAFT agent can be found in Figure 3.14. The resulting polymer-SH chains were then attached with the acrylate groups on the silanized glass surface.

Figure 4 . 1 :

 41 Figure 4.1: (a) Schematic protocol of "grafting-to" of a RAFT polymer via "thiol-ene click" chemistry; modified figure taken from the work of Biggs et al. in [126]. (b) Simplified reaction of in-situ aminolysis of RAFT-ed polymer with ethanolamine.

4. 2 .

 2 GRAFTING OF VISCOSITY-SENSITIVE POLYMERS ONTO SURFACES 119 lyzed surface can be acquired by comparing the measured angles. For each measurement, a volume of 100 µL of water was deposited on the analyzed surface. The measurements were repeated for each analyzed surface three times, and an average value was taken.

Figure 4 . 2 :Figure 4 . 3 :

 4243 Figure 4.2: Contact angle measurement with a water droplet for different steps of surface modification performed during Michael addition of viscosity-sensitive polymers.

Figure 4 . 4 :

 44 Figure 4.4: Grafting-to of poly(DMA-s-MECVJ) onto gold surface. Grafting was carried out at least overnight, washed abundantly with water and N 2 -dried.

1 .

 1 The essential elemental peak signals for this work are compiled in Figure 4.6. Apparent differences were observed between the XPS spectra of the non-grafted gold surface (blue spectra) and these of the polymer-grafted gold-coated glass surface (red spectra). Quantitatively, there was an increase in the atomic percentage of carbon (C) and oxygen (characteristic peak not represented here), indicating the presence of polymer on the analyzed surface as a large part of the polymer chains is constituted of carbon and oxygen. The tiny shoulder observed on the C 1s spectrum, circled with a dashed line, indicates the presence of an oxidized form of C.

Figure 4 . 6 :

 46 Figure 4.6: XPS spectroscopy analysis of grafted poly(DMA-s-MECVJ) on a gold-coated glass slide is represented with red spectra. The blue spectra represent the chemical analysis for a non-grafted gold glass slide. (a) Representative C 1s spectrum. (b) Representative N 1s spectrum. (c) Representative S 2p spectrum. The significance of the dashed lines is explained in the accompanying text.

Figure 4 . 7 :

 47 Figure 4.7: (a) Fluorescence lifetime response of grafted viscosity-sensitive polymer to mixtures of different viscosity for various grafting times. (b) Fluorescence lifetime response of grafted viscosity-sensitive polymer to mixtures of different viscosity after continuous washing for 63 hours. (c) Fluorescence lifetime response of attached viscosity-sensitive polymer on a regular glass slide to mixtures of different viscosity. Data are plotted in logarithmic scale with η expressed in mPa s and τ F in ns the straight line represents the Förster-Hoffmann model fit.

Figure 4 . 8 :

 48 Figure 4.8: (a) Difference observed of a polymer-grafted (P4) gold-coated surface after heat exposure at 95 °C. (b) Verification of viscosity-sensitivity property of the altered polymer-grafted (P4) surface after the heat exposure; data are plotted in logarithmic scale with η expressed in mPa s and τ F in ns the straight line represents the Förster-Hoffmann model fit.

Figure 4 .

 4 Figure 4.8a. The response of the altered surface to viscosity was evaluated, and a slightly smaller α-exponent was obtained, as shown in Figure 4.8b indicating reduced sensitivity.

Figure 4 . 9 :

 49 Figure 4.9: Photograph of the viscosity-sensitive PDMS microfluidic chip prototype developed in this thesis.

Figure 4 .

 4 Figure 4.10a shows the variation of fluorescence lifetime measured for different mixtures of water-glycerol of varying viscosity using the FMR-integrated microfluidic chip developed in this thesis. In order to check for the reproducibility of the process, the experiments were repeated with two chips prepared following the same protocol. For both, an exponent α » 0.1, similar to the values obtained with bare surfaces. The viscosity-sensitivity of the polymeric surface was thus not modified with its integration into the microfluidic device.

Figure 4 .

 4 Figure 4.10: (a) Calibration curves in logarithmic scale of fluorescence lifetimes, τ F in two different poly(DMA-s-MECVJ)-grafted microfluidic chips versus water-glycerol mixture viscosity, η. The straight lines correspond to fits with the Förster-Hoffmann model (Equation 1.6) with similar exponent α « 0.1 for both microdevices when η is expressed in mPa s and τ F in ns. (b) Real-time fluorescence response of viscosity-sensitive microfluidic chip developed in this thesis to changes in viscosity; a further explanation on points (a,b, and c) is provided in the accompanying text. (c) A plot of fluorescence lifetime values obtained for two different mixtures of glycerol-water tested multiple times on the poly(DMA-s-MECVJ)-grafted surface. The average lifetime values for both mixtures are represented by the dotted lines. (d) Comparison of exponent-α of the Förster-Hoffmann model (slope-value) obtained for a polymer-grafted surface. The dotted line represents the average value of α for all the represented data.

Figure 4 .

 4 Figure 4.10c shows the successive lifetimes measurements taken at a specific point in the microchannel for two different mixtures of water-glycerol. It is interesting to note that photobleaching was not observed during the experiment and that there is no systematic variation in the lifetime value for both fluids, showing that the developed device remains functional upon continuous photoexcitation, at least for a few hours.

Table 1

 1

	.1. The absorption of photon following

Deactivation Pathways Timescale (s) Transitions

	Absorption	10 ´15	radiative
	Fluorescence	10 ´10 ´10 ´7	radiative
	Phosphorescence	10 ´3 ´10 2	radiative
	Internal conversion	10 ´14 ´10 ´10 non-radiative
	Vibrational relaxation	10 ´12 ´10 ´10 non-radiative
	Intersystem crossing	10 ´4 ´10 ´12	non-radiative
	Table 1.1: The properties of various deactivation pathways of a fluorophore upon absorp-
	tion of a photon.
	The Jablonski diagram, also known as the Perrin-Jablonski diagram, is widely used
	to visualize the excited states of a molecule and the possible radiative and non-radiative
	transitions. It was initially named after Aleksander Jabłoński, a Polish physicist who first

Table 2 .

 2 µL{minq rGlycerols calc mol L ´1 rGlycerols exp mol L ´1 σ rel p%q 1: Relative difference between the calculated glycerol concentration rGly calc s and experimental values rGly exp s after full mixing of the two streams in a SHM, for different applied flow rates, Q S1 and Q S2 .

	7.0	0.5	4.28	4.21	1.5
	16.0	0.5	4.02	4.10	1.9
	4.0	0.5	4.59	4.19	8.6

3 Viscosity-sensitive Fluorescent Polymers . . . . . . . . . . . .

	3.3.4 Characterization of Polymers . . . . . . . . . . . . . . . . . . .
	3.3.4.1 Methods of Characterization . . . . . . . . . . . . . .
	3.3.4.2 Results and General Discussion . . . . . . . . . . . . .

3.2.1 Synthesis of Julolidine Aldehyde . . . . . . . . . . . . . . . . . 3.2.2 Synthesis of Methacrylate Ester . . . . . . . . . . . . . . . . . . 3.2.3 Photophysical characterization of MECVJ . . . . . . . . . . . . 3.3.3.1 What is RAFT Polymerization? . . . . . . . . . . . . . . . . . 3.3.2 The Design Criteria of poly(DMA-s-MECVJ) . . . . . . . . . 3.3.3 Synthesis of poly(DMA-s-MECVJ) . . . . . . . . . . . . . . . . 3.3.5 Photophysical Properties of Polymers . . . . . . . . . . . . . . 3.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Table 3 .

 3 .1. 1: Details of poly(DMA-MECVJ) statistical copolymer synthesis at 60 ℃ and their macromolecular characteristics. The total number of monomers (MECVJ and DMA) targeted in a polymer chain is denoted as n targeted , t is the polymerization reaction time in hours, and Ð represents the dispersity of the obtained polymers. The conversion of DMA (%) were ˚determined from 1 H-NMR. The number average molar mass were ˚˚determined by SEC in LiBr/DMF.

		n targeted	DMA/MECVJ (mol %) initial	t(h) Conv.DMA ˚(%) M n	[SEC]˚˚( g/mol) Ð
	P1	20	100/1	27	93	4575	1.19
	P2	50	95/5	16	85	3697	1.21
	P3	100	100/1	6	81	9240	1.18
	P4	100	90/10	54	81	4742	1.76

  Measurements were carried out at a constant temperature, T " 25 °C. The shear viscosity of every sample was measured by applying a logarithmic shear-rate ramp from 9 γ " 1 s ´1 to 1000 s ´1.

			(a)					(b)
	800
				1 mPa.s Viscosity,		800			1 mPa.s Viscosity,
				2 mPa.s					2 mPa.s
	600			4 mPa.s					5 mPa.s
				7 mPa.s		600			8 mPa.s
				12 mPa.s					13 mPa.s
				40 mPa.s					41 mPa.s
	400			78 mPa.s					72 mPa.s
				132 mPa.s		400			145 mPa.s
				305 mPa.s					330 mPa.s
				593 mPa.s					569 mPa.s
	200					200
	0					0
	470	515	560	605	650	470	515	560	605	650
			(c)					(d)
	1250					800
				Viscosity,					Viscosity,
				2 mPa.s					3 mPa.s
	1000			8 mPa.s 15 mPa.s 18 mPa.s		600			6 mPa.s 28 mPa.s
				30 mPa.s 89 mPa.s					51 mPa.s
	750			358 mPa.s 606 mPa.s					204 mPa.s 428 mPa.s
				623 mPa.s 818 mPa.s		400			745 mPa.s
	500
	250					200
	480 0	500	520	540		470 0	515	560	605	650

2.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

Average selling price with Sigma-Aldrich in

A slight variation in the prices is always present over time.

Chapter 3 DCM, and the solution was transferred into a separatory funnel. The solution was washed twice with 50 mL of HCl to remove the basic impurities, such as the unreacted EDC and urea by-products. This was followed by washing with water (2 ˆ60 mL) and with saturated sodium bicarbonate solution (2 ˆ60 mL) as well as with saturated sodium chloride solution (60 mL). This step ensures the removal of residual cyanoacetic acid. The organic layer was retrieved and dried with MgSO 4 . The absence of residual reagents and by-products was verified with 1 H-NMR analysis upon obtaining a "dry" mixture after the vacuum filtration. Finally, DCM was evaporated under reduced pressure, and the methacrylate ester was obtained in a pale yellow oil form, as observed in the inset of Figure 3.5. The yield of this reaction was 77 %, and the purity of the final product is approximately 98 %. As portrayed with blue 1 H-NMR spectra in Figure 3.5, only traces of solvent were present, and no residual reagents or by-products were observed. This long-awaited successful synthesis strategy was applied for the eventual scale-up synthesis of our methacrylate ester.

The protocol described is a modified version of the synthesis route presented in the reference work [START_REF] Lutjen | Synthesis of esters via a greener steglich esterification in acetonitrile[END_REF]. The exact ratio of reagents in the work of Lutjen et al. was applied first in the initial experiment. However, the final yield was around 36 %, which is considerably lower. This might be due to the loss of ester during purification and the formation of by-products. Hence, cyanoacetic acid was added in excess (1.5 ˆHEMA) for the following synthesis. Also, DMAP was not used for the previously explained reason. Once the julolidine aldehyde (3) and methacrylate ester (5) of good purity (> 95 %) were obtained, the next step of this work was the synthesis of the viscosity-sensitive flu-Chapter 3 profound studies on the interaction between the polymer chain and the response of the viscosity-sensitive probe would be judicious.

A further study on the effect of different solvents on the photophysical property of the synthesized polymer could be performed. The presented results open a path for the engineering of viscosity-sensitive polymers where different types or structures of polymer could be analyzed: e.g., block copolymers and thermo-responsive polymers.

Following the works and results presented in this chapter, the focus in the next chapter will be integrating these polymeric materials on a surface for the conception of a viscositysensitive microfluidic probe.

X-ray photoelectron spectroscopy (XPS)

X-ray photoelectron spectroscopy (XPS) is a type of surface analysis technique that provides quantitative elemental and chemical state information. When a surface is exposed to X-ray, photoelectrons are released upon the interaction between the X-ray and atoms. Information about the surface of a material can then be retrieved via eventual analysis of the energy of these photoelectrons. Data acquisition is possible from the surface to a depth of approximately 10 nm. The surface composition of the catalysts was

Viscosity-sensitivity of polymer-grafted surfaces

All four synthesized polymers in this work were tested for grafting. The grafting-to of polymers onto gold surfaces with 1 % MECVJ, P1, and P3 did not render satisfactory results; refer to Table 3.1 in Chapter 3 for details on different polymers. The grafted surface did not exhibit sufficient fluorescence to allow viscosity measurements with fluorescence intensity and lifetime response. With P2, fluorescent viscosity-sensitive surfaces were obtained after a long grafting time, around 66 hours. However, the grafted surfaces were heterogeneous. The best results were obtained with P4. As the ultimate aim of this work is to establish the proof-of-concept of a viscosity-sensitive surface, only P4 was selected for further study in this chapter. Due to time-constraint, no further study was performed to improve the grafting of other synthesized polymers in this work.

Hence, from here on, P4 will be referred to as the polymer.

During the preliminary characterization, it was observed that the quality of the polymergrafted surface improves with a longer grafting time. A thorough analysis was then conducted to study the effect of grafting time on the viscosity-sensitivity of the polymergrafted surface. This can be evaluated by determining the α-exponent of the corresponding Förster-Hoffmann model fit. Fluorescence response to viscosity for three gold-coated glass slides grafted with polymer for various grafting times were evaluated.

The concentration of the polymer solution used for grafting was 14.3 mg mL ´1. After being grafted for a specific duration, as indicated in Table 4.1, the glass slides were washed for 24 hours in a beaker of distilled water (1 liter, under constant agitation) in order to remove non-grafted polymer chains. The polymer-grafted surfaces were analyzed with mixtures of water-glycerol of varying viscosity. The grafted surfaces were excited at 451 nm, and experiments with the FLIM-LIFA were carried out at room temperature.

The corresponding results are plotted in Figure 4.7a, and the α-exponents of the Förster-Hoffmann model fit for the polymer-grafted surfaces are tabulated in Table 4.1.

Results from initial experiments showed that the polymer solution should be left to graft for at least 16 hours to obtain an adequate fluorescence response for viscosity measurements. Based on the results shown in Figure 4.7a, it can be said that the prolonged grafting time (more than 16 hours) does not influence the sensitivity of the grafted probe. Also, longer grafting time resulted in a decrease in the signal-to-noise ratio due to the Fluorescent molecular rotors (FMRs) are an emerging tool that can be used to probe viscosity at a microscopic scale. Increasing viscosity inhibits their ability to relax via a change in conformation, which leads to an increase in fluorescence quantum yield. In this work, FMRs were used for in situ measurements of local viscosity in a microfluidic co-flow of Newtonian fluids. A calibration curve based on the Förster-Hoffmann equation allowed to retrieve the local viscosity and to perform its mapping. This strategy allowed the development of a novel method to study the efficiency of diffusive mixing between two fluids inside a micromixer. In addition, an FMR-functionalized statistical polymer was developed as a viscositysensitive polymeric material. Finally, a viscosity probe was engineered by attaching a microchannel onto a viscosity-sensitive surface. The viscosity measurement of solutions flowing through the channel was made possible. This device could be used as an innovative local viscosity diagnosis tool as it would be a portable, fast, low-cost probe that facilitates high-throughput screening.