Marco Morani

Myriam Taverna

Zuzana Krupova

Lucile Alexandre

Pierre Defrenaix

Thanh Duc Mai
email: thanh-duc.mai@u-psud.fr

Development of a microfluidic droplet platform with an antibody-free magnetic-beadbased strategy for

Keywords: Vésicules Extracellulaires, Méthodes de séparations Électrocinétiques, Microfluidique, Billes Magnétiques, Électrophorèse Capillaire, Biomarqueur Novel electrokinetic and analytical Extracellular Vesicles, Electrokinetic separation techniques, Microfluidic, Magnetic Beads, Capillary Electrophoresis, Biomarker extracellular vesicles, isolation, magnetic beads, polymer precipitation, microfluidic droplets

Les vésicules extracellulaires (EVs) sont d'importants messagers intercellulaires qui peuvent être exploités comme biomarqueurs pronostiques ou diagnostiques. Cependant, isoler, enrichir et caractériser les EVs à partir de fluides biologiques reste un défi. L'objectif de la thèse est de développer de nouvelles approches analytiques pour l'isolement et la détection des EVs, et l'analyse de protéines internes.

Dans un premier temps, nous présentons une nouvelle méthode d'électrophorèse capillaire (CE), couplée à une détection fluorescente induite par laser (LIF) pour l'identification et la quantification des EVs. Ensuite, l'analyse de dispersion de Taylor (TDA) a été étudiée pour déterminer la taille des EVs.

Enfin, nous présentons une nouvelle approche pour l'isolement des EVs à l'aide de polyéthylène glycol (PEG) pour précipiter les EVs à la surface de billes magnétiques. Pour améliorer les performances, nous avons transféré cette méthode dans un système microfluidique de gouttes.

Résumé de la thèse en français

Les vésicules extracellulaires (EVs) sont une famille de particules délimitées par une double couche phospholipidique et sécrétées par tous types de cellules. Les EVs transportent des protéines, lipides, acides nucléiques vers des cellules cibles et sont donc des acteurs clés de la communication intercellulaire dans des conditions normales ou pathologiques. Par conséquent, le potentiel des EVs à fournir des biomarqueurs moléculaires pronostiques ou diagnostiques de pathologies attire une attention croissante ces dernières années. L'isolement et l'enrichissement des EVs à partir de fluides biologiques reste un préalable difficile avant d'explorer les EVs. L'ultracentrifugation est la méthode la plus utilisée dans toutes les applications sur les EVs. Cependant, cette technique présente de nombreux inconvénients. En conséquence, des technologies pouvant extraire et garantir la pureté et l'intégrité des EVs isolées de manière reproductible sont toujours recherchées. De plus, pour fournir des informations physicochimiques et biologiques suffisantes sur les EVs isolées, de nombreuses techniques complémentaires doivent être mises en oeuvre. Bien que robustes et efficaces, la plupart des plates-formes analytiques fournissent des informations sur la population des EVs dans son ensemble, masquant l'hétérogénéité des populations. L'objectif de la thèse a été de développer de nouvelles approches analytiques pour l'extraction et la détection d'EVs intactes, et l'analyse de protéines internes et ciblées d'EVs pouvant servir de futurs biomarqueurs de maladies. La première partie expérimentale du projet de thèse est dédiée au développement de méthodes basées sur l'électrophorèse capillaire (CE) pour analyser des EVs ou des protéines. Pour la première fois, nous présentons la CE couplée à la détection par fluorescence induite par laser (LIF) pour l'identification et la quantification des EVs. Cette méthode repose sur un marquage fluorescent des membranes des EV par clivage enzymatique intravésiculaire d'un précurseur. Ce marquage n'affecte ni la taille moyenne ni la distribution de charge des vésicules, indiquant qu'elles ont été préservées. Pour y parvenir des électrolytes moins classiques ont été utilisés comme tampon de séparation. Ces électrolytes, qui contiennent de grosses molécules faiblement chargées à des concentrations extrêmement élevées, ont permis d'éviter/minimiser l'adsorption des EVs sur la paroi du capillaire en silice et la lyse des EVs lors de l'électrophorèse. Pour éviter l'agrégation des EVs, une substitution de tampon des échantillons analysés a été effectuée pour diminuer la conductivité de la matrice d'échantillon. La CE-LIF a ainsi permis de différencier des EVs d'origine variée (sérum, plasma, lait) ou obtenues par différentes méthodes d'isolement et de vérifier leur qualité. Comme alternative à la DLS ou à la NTA, nous avons également exploré l'intérêt de la dispersion de Taylor (TDA-UV et TDA-LIF) pour estimer la taille des EVs isolées. Il s'agit de la première preuve de concept de l'applicabilité de la TDA à ce type de bionanoparticules. La deuxième partie du travail a consisté à développer une technique pour l'isolement des EVs à partir de fluides biologiques reposant sur l'utilisation de billes magnétiques dans une plateforme de gouttelettes microfluidique. Grâce à des EVs de haute qualité et bien caractérisées, isolées à partir de lait bovin, nous avons pu évaluer les performances d'extraction des EVs. Le polyéthylène glycol (PEG), un polymère d'exclusion d'eau a été utilisé pour précipiter des EVs à la surface des billes magnétiques. Par rapport au format d'extraction en tube, une nette augmentation du taux de récupération (jusqu'à 50 %) est obtenue avec une réduction des quantités d'échantillons et de réactifs (x10) et du temps d'extraction (x3). En conclusion, ces recherches contribuent au développement de méthodes innovantes, fiables et compactes pour la séparation, la purification et la caractérisation des EVs.

First of all, I would like to thank my thesis director, Professor Myriam Taverna, for welcoming me to her laboratory, but also for her great availability, her attentive listening and her undeniable scientific rigour. Thank you for your valuable advice, both scientifically and personally, and thank you for giving me the opportunity to present my work at several international conferences. A big thank you also to my thesis co-supervisor, Doctor Duc Thanh Mai, for his advice and the more global vision of the project that he was able to bring me during these three years, for his great listening, his availability but also for his unfailing optimism. Thanks to your extensive scientific knowledge and skills, I was able to learn and evolve on a daily basis within the laboratory. It was a great pleasure to be able to discuss with you both on scientific subjects and on life in general.

I would like to thank Professor Anne Varenne, Doctor Céline Elie-Caille, Professor Guillaume Van Niel and Professor Bruno Le Pioufle who do me the honor of being members of my thesis jury. I would also like to thank Doctor Christophe Junot for agreeing to be the external member of my thesis committee and for advising me throughout this project.

I also wanted to express my thanks to Doctor Zuzana Krupova and Doctor Pierre Defrenaix with whom we had the chance to collaborate during these three years of thesis. Thank you for your availability but also for your precious advice to carry out my thesis project. I also thank you for always providing us with high-quality extracellular vesicle samples for our experiments. This thesis project was able to progress in large part thanks to you. I would like to thank Professor Hervé Cottet and Doctor Joseph Chamieh for their invaluable assistance with Taylor dispersion analysis. You have been a great help to us over the last few months of manipulations and we could not have gone this far in the project without you.

I would also like to thank Professor Marja-Liisa Riekkola and Doctor Evgen Multia for their collaboration and for their precious extracellular vesicles samples they extracted from human plasma using their novel immunoaffinity chromatography.

Thank you also to the PNAS (Proteins and Nanotechnology in Analytical Sciences) team members:

Professor Claire Smadja and Professor Thuy Tran. It has been an honor to spend these three years with you, both scientifically and personally. Your teachings have been very influential in my life.

I would also like to thank Marie-Claude, Sylvie Z., Patricia, Fatiah, Maria and Orane for their daily logistical assistance. Thank you for your promptness and assistance. Thank you very much for the several cake sessions and other goodies.

I would also like to thank the old and new doctoral students as well as the interns I had the chance to meet during my thesis: Corentin, Jeanne, Cédric, Seray, Etienne, Ayoub, Balazs, Emmanuel, Gregory, Camille, Clara, Lucile, Théo, Van Thanh and Bin. Special thanks to Doctor Coralie Ruel who introduced me to the PNAS team during my 6 months of Erasmus even before I started my thesis and for his training on capillary electrophoresis, you have helped me save valuable time. It was a real pleasure to work with you.

A special thank you to Doctors Emilie and Minh, with whom I had the chance to share my office at the start of my thesis. Every day, I laughed a lot thanks to you and all your good jokes.

Thanks to Sylvie Liu, for being my personal French teacher when I arrived, for all the discussions we had, thank you for all the good times we shared in the laboratory and thank you for your good mood.

You helped me a lot on a daily basis, and I wish you all the best for your new adventure.

Many thanks to Joanie and Cecile, with whom I shared my office at the end of my thesis. I laughed a lot by your side, and I really appreciated all the moments we spent together in the laboratory but also outside. Thank you for letting me also decorate our office with some Italian flags (just a few) to celebrate the victory of the European Championship. I wish you much success in your thesis. You have become friends outside the lab, and I know that we will continue to see each other in the future.

Thank you to all my friends for staying close despite the distance: Bale, Lori, Fra, Ale, Ciccio, Fabio, Signo, Teo C., Teo D., Agui. Thank you for always being there for me even though my time in Italy has been more than limited over the last three years. Thanks also to my friends I met in Paris: Dome, Delia, Fede, Needhi, Ste, Romane, Fra, Edo. I've always spent amazing weekends or weeks of vacation with you, and I'm certain that will continue.

Thanks to Anja for your presence by my side, your encouragement and your daily support.

Infine, vorrei fare un enorme ringraziamento ai miei genitori e alla mia sorellona. Grazie per avermi permesso di evolvere liberamente fino a questo dottorato e per avermi dato fiducia. Grazie per il vostro supporto. Tutto questo è stato possibile grazie a voi.

Curriculum Vitae

Education:

• Single-cycle Master Pharmacy and Industrial Pharmacy at Università degli Studi di Milano (SEP 2012-JUL 2018) • PhD in Analytical Chemistry at the Faculty of Sciences of Orsay, University of Paris-Saclay (2018-2021) Oral communication:

Résumé de la thèse en français

Summary of the thesis in English Errore. Il segnalibro non è definito.

General introduction

Extracellular vesicles (EVs) are a family of heterogeneous lipid bilayer-delimited particles secreted into the extracellular space by all types of cells and thus found in various human body's physiological fluids (plasma, saliva, urine, cerebrospinal fluid, etc.). Circulating EVs may transport proteins, lipids, and nucleic acids that mirror the producing cells; therefore they can reflect a broad spectrum of biological activities, both normal and pathological. Consequently, open up new possibilities for noninvasive liquid biopsy. Several characteristics make them particularly interesting in clinical diagnosis, including their high concentration in biological fluids in comparison to circulating tumor cells (of the order of 10 10 ) and their stability, which distinguishes them from other soluble RNA-type biomarkers, for example [1].

Finally, because of their inherent biological properties, EVs may be engineered with a specific active component as well as therapeutic and vectorization properties. Furthermore, in recent years, they have emerged not just as novel potential drug delivery vehicles, but also for their intrinsic therapeutic properties, particularly in the context of regenerative medicine [2].

Nevertheless, the isolation and enrichment of EVs from biological fluids remains a difficult prerequisite before exploring EVs. Ultracentrifugation is the most used method in all EV applications. However, this technique presents many drawbacks. As a result, developing EVs separation or extraction technologies that may guarantee EV purity and integrity in a repeatable way is still in great demand. Furthermore, EVs are quite heterogeneous, with a certain heterogeneity in structure and molecular content resulting in distinct EV subpopulations. For this reason, numerous complementary approaches must be used to provide adequate physical and biological information on the isolated EVs. A good approach should fulfill all of the requirements for EV profiling, including the capacity to distinguish between various subpopulations and to maintain EV integrity throughout analysis. Faced with this limitation, the International Society for Extracellular Vesicles (ISEV) has brought together researchers from diverse backgrounds to give recommendations in order to homogenize existing protocols for isolation and characterization of EVs as well as foster the development of new reproducible and standardized methods [3].

In this context, microfluidic systems are particularly attractive for enhancing the isolation performance of EVs, in part because new mechanisms may be applied and be more efficient in a microfluidic format.

Microfluidic devices aimed to reduce the time required for the processing of EV samples by making the isolation system portable, efficient, and simple to use. One part of the present microfluidic-based systems for EVs isolation is based on the use of capture molecules (e.g., antibodies) capable of binding specific markers (called Labels here), that are either (i) constantly found surface proteins (like CD63)

or specific ones that are overexpressed during development of tumors for instance (like EpCAM). These approaches ensure labeled EV separation purity, but they need to be enhanced for greater throughput and targeting numerous EV populations with different labels. Alternative approaches focusing on the physical properties (e.g., particle size and electric charge) of EVs, also in combination with the use of external forces such as hydrodynamic force, electric field, or acoustic field have been developed to allow isolation without the use of labels (label-free) [4]. Despite significant advances in label-free isolation, existing microfluidic technologies are limited in their ability to separate certain EV subpopulations and remove undesirable components such aggregating proteins and lipoproteins.

Among the various microfluidics technologies, an interesting but unexplored option for EV isolation is Droplet-based Microfluidics, which is broadly defined as the science and technology of generating and manipulating micro to femtoliter microreactors (droplets). This enables constant sample and reagent compartmentalization while reducing contamination risks with the surrounding environment. In particular, Droplet-based microfluidic devices utilizing magnetic beads (MB) as solid support have already been demonstrated for the extraction and purification of different kind of target analytes (such as proteins, nucleic acids and viruses) from complex matrices, but they have not yet been demonstrated for the isolation of EVs.

In parallel, capillary electrophoresis (CE), which may be defined broadly as a micro technique allowing the migration and therefore the separation of charged compounds in solution under an electric field, is a separation and quantitative technique which format could be compatible with the microdroplet volume produced in a microfluidic droplet system. This technique is capable of analyzing small molecules as well as particles, virus and bacteria but was not yet explored for intact EVs analysis when I started my thesis. While electric field have been used to sort and drive EVs in different microdevices, capillary electrophoresis to assess EVs or discriminate EV subtypes based on electrophoretic migration was never reported. Moreover, Taylor dispersion analysis (TDA), which uses pressure rather than an electric field to determine the hydrodynamic radius from the observed peak dispersion, makes CE equipment especially appealing for profiling the distribution and quality of isolated EV populations.

The objective of this thesis is therefore to combine a microfluidic droplet technology that can allow the extraction of specific EVs or whole EVs present in a biological sample, with multiple capillary electrophoresis modes for analysis of intact EVs, which would result in a new automated device, potentially reproducible and standardizable, for EVs users.

The first chapter presents an overview on EVs, their biogenesis and molecular composition, their biological and pathological role in order to emphasize their diagnostic potential for monitoring human health as well as carriers or delivery nano systems for therapeutic applications. Chapter II presents an overview of the different conventional techniques currently available for isolating and characterizing EVs and their associated limits. The third chapter focuses on the advantages of emerging methods applied to EVs including microfluidic-based technologies and in particular electrokinetically driven methods for isolation, enrichment and characterization of EVs. The objectives of the thesis are detailed at the end of this chapter.

The experimental part of the manuscript is divided into two chapters that describe the two main strategies that have been investigated to achieve the objective of our project.

The fourth chapter focuses rather on the development of innovative methods all based on the technique of CE for characterizing intact EVs and also specific intravesicular proteins. Its multiple modes of separation and analysis and the possibility to run analyses under voltage or pressure allowed to investigate several different approaches, e.g., Capillary zone electrophoresis method coupled with laser induced fluorescence detection (CE-LIF) for analyzing intact EVs or the internal protein content of them, and Taylor dispersion analysis (TDA) for size estimation of isolated intact EVs. Additionally, strategies for in-capillary sample preconcentration of EVs are discussed in order to improve the sensitivity of conventional LIF detection.

The fifth chapter presents the development of the first module of the platform relying on a microfluidic droplet system by integration of magnetic beads for EVs manipulation and handling, during the multistep sample treatment. Different kind of magnetic beads-based approached are compared using already purified EVs samples before transferring the protocol to droplet system and applying it to more complex biofluids (e.g., pony serum and plasma).

Bibliography Chapter I: Extracellular vesicles

EVs are a family of cell-derived membrane vesicles which can transport their cargo to target cells acting as mediators of key functions in numerous patho-physiological conditions. However, their properties were not readily known when they were first observed more than 50 years ago. Throughout these years of studies and discoveries, their role has been increasingly clarified and a wide terminology has been proposed to identify or class EVs. As a result, the scientific field of EVs required harmonization.

EVs can be narrowly classified into two major groups: exosomes and microvesicles. The difference between these vesicles lies in their biogenesis, size, content, and functions. Despite their great potential for pathology diagnosis or treatment, the use of exosomes in the clinic is currently limited due to a lack of standardization of separation, isolation and analysis or characterization methods.

Misuse of terminology is common in the literature due to the difficulty of distinguishing exosomes from microvesicles. As a result, the word exosome is frequently used to refer to a heterogeneous sample comprising exosomes and microvesicles. Unless otherwise specified, the term EV will refer to exosomes and microvesicles throughout this thesis.

In this chapter I will briefly present the extracellular vesicles, their physical and biochemical characteristics, their physiological roles, their implications in various pathologies as well as the recent advances of EV-based therapies, before detailing current and developing methods for EVs isolation and analysis or characterization

I.1.General aspects I.1.1. Historical background

As often reported by researchers working on EVs, the early evidence for the existence of EVs have been firstly reported in 1946 by Chargaff and West [5] when they described the presence of a precipitable factor in platelet-free plasma (PFP) that can accelerate the generation of thrombin. These results showed, for the first time, the presence of factors other than platelets, which were responsible for blood clotting. Subsequently, in 1967, Wolf et al [6], with the help of electron microscopy, similarly observed small lipid-rich particles released from platelets with procoagulant activity, referring to them as "platelet dust". The term "extracellular vesicle" was first used in 1971 by Aaronson [7] for EVs released from Ochromonas Danica, a flagellated alga.

In 1981, Trams [8] was the first to use the term "exosomes" to describe a large panel of vesicles ranging in size from 40 nm to 1 µm that were released from cultured cells.

However, a turning point arrived in 1983, when the two groups of Stahl and Johnstone reported almost simultaneously that transferrin receptors associated with small vesicles of around 50 nm were released by reticulocytes into the extracellular space [9] [10]. Later in 1987, Johnstone discovered that such release took place through the endosomal route with formation of intraluminal vesicles (ILVs) in the endosome, followed by fusion of MVBs with the plasma membrane and extracellular release of these ILVs [11]. This time, the term "exosomes" previously coined by Trams, was used to describe these ILVs.

A decade later, interest in vesicles increased in the field of immunology. A breakthrough article appeared in 1996, when Raposo's team demonstrated that B lymphocytes release exosomes expressing the Class II Major Histocompatibility Complex (MHC-II) and thus, induce a response in T cells [12].

In 2007, Valadi and his colleagues reignited the interest in research on EVs by demonstrating for the first time the presence of messenger ribonucleic acids (mRNA) and microRNAs (miRNA) in the exosomes isolated from murine and human mast cells, recognizing them as cell-cell communication mediators [13]. Over the last decade, the number of studies on EVs has increased significantly, as

shown by the number of publications per year (Fig. 1), found with the key words "extracellular vesicles"

in Web of Science database. The first meeting bringing together EVs scientists was held in Montreal (Canada) in 2005. Six years later, in 2011, an international conference on EVs was organized at the Institut Curie in Paris, France.

In the same year, the International Society for Extracellular Vesicles (ISEV, https://www.isev.org/) was established, aiming at standardizing the field, and this also led to the creation of the Journal of Extracellular Vesicles (JEV). Currently, the society has over 1500 members, but a plenty of national EV societies have also been formed. The French Society of Extracellular Vesicles (FSEV, https://www.fsev.fr/) was officially founded on April 21, 2018.

I.1.2. Nomenclature and classification

Over the years, the nomenclature for extracellular vesicles has become highly muddled. The accumulated data show that content, size, and membrane composition of the EVs are highly heterogeneous and complex, depending on the pathophysiological state of the origin cell. Several terms have been used to describe vesicles released by cells. Vesicles have been named after the cells or tissues from where they are released. Thus, terms like oncosomes, for EVs released by cancer cells [14] [15]; prostasomes, for those released by prostate epithelial cells [16] [17] or dexosomes, for those released by dendritic cells (DCs) [18] have been employed. When nomenclature is based on the mechanism of generation, terms like exosomes (endosomal vesicles) [19], ectosomes (vesicles from membrane budding) [20], or apoptotic bodies (vesicles formed following cell death) [21] have been also used. Alternatively, depending on their biological role, we can find terms like calcifying matrix vesicles [22], tolerosomes [23] or argosomes [24]. Although specific characteristics have been proposed for each EVs subpopulation, at present there is still no specific identification of each subgroup that is widely accepted and it remains a matter of debate [25][26][27]. For such reason, according to the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines from ISEV, authors are recommended to use "extracellular vesicle" as an umbrella term to represent all types of cellsecreted vesicles, except if authors may identify remarkable markers that are reliable within their experimental system [28]. However, the most commonly used terminology is based on two key criteria: the mode of biogenesis and the biochemical markers. Then, EVs can be narrowly classified into two major groups: exosomes and microvesicles which exhibit moreover, size differences.

I.1.2.1. Biogenesis of Exosomes

Exosomes are vesicles surrounded by a lipid bilayer, having a size between 30 and 100 nm, even though they can reach 150 nm according to some authors [29,30], and density ranging from 1.13 to 1.19 g/mL [31]. Exosomes are produced via the endosomal pathway. They are first generated as intraluminal vesicles (ILVs) in the lumen of early endosomes, which mature into late endosomes, forming multivesicular bodies (MVBs). Before being secreted, exosomes go through a "sorting" step.

Indeed, they are either degraded in the lysosome or sent to the plasma membrane to be secreted in the extracellular medium [32]. The causes that determine the fate of these multivesicular bodies are not yet known, but it seems that cholesterol levels in MVBs play a role. Actually, some studies have shown that cholesterol-rich vesicles are secreted, whereas cholesterol-poor vesicles are degraded by the lysosome [33].

The ESCRT machinery (Endosomal Sorting Complex Required for Transport) is the most well-studied pathway for inducing the formation of ILVs and subsequently of MVBs [34,35]. Reprinted from [36].

The biogenesis of exosomes can also occur in an ESCRT-independent pathway which involves the formation of lipid rafts, in which sphingomyelin is transformed into ceramide under the action of sphingomyelinase [37]. These ceramide-rich domains cause invagination of the endosome membrane and the generation of intraluminal vesicles.

Along with this mechanism of exosome biogenesis, exosomes can be formed by direct budding from domains of the plasma membrane enriched in endosomal and exosomal proteins, called "endosomelike domains" [38]. On the other hand, actin-myosin binding has been shown to play a role in the formation of MVBs and the biogenesis of exosomes [39]. However, more evidence is needed to support exosome biogenesis at the plasma membrane.

From these observations, we can therefore note that the exact mechanism responsible for the formation and release of exosomes remains discussed in the literature, and it seems that the presence of different mechanisms of exosome biogenesis depends on the type of cell and / or intracellular conditions.

I.1.2.2. Biogenesis of Microvesicles

Microvesicles are delimited by a lipid bilayer as well, but their size range from 100 to 1000 nm, and their density is less than 1.10 g/mL [40]. They are produced as a result of direct outward blebbing and pinching of the cell membrane, following activation of cells by various physicochemical stimuli. The exact mechanism of formation and release are still not fully understood, but it seems that it involves redistribution of membrane lipids and the use of contractile machinery located at the level of the plasma membrane like actin and microtubules, as well as molecular motors like kinesis and myosins [30]. In general, a stimulus in the cell causes an increase in the intracellular concentration of calcium which inhibits the aminophospholipid translocase and activates the scramblase, resulting in the loss of lipid asymmetry. All these events lead to membrane budding and the release of microvesicles exposing phosphatidylserine (PS) on their outer leaflet (Fig. 3) [41] .

I.2. EV composition

EVs are composed by a lipid bilayer that encloses a variety of soluble or membrane proteins, lipids, metabolites, and nucleic acids. Numerous studies on the lipidome, proteome and transcriptome of EVs have been conducted and the results have been compiled in web-based catalogues such as ExoCarta (http://exocarta.org) and Vesiclepedia (http://microvesicles.org) which are constantly updated with the help of the scientific community. According to these findings, the type of the EV content is largely related on the donor cell and its physiological or pathological status.

I.2.1. Lipids

EVs are mainly composed of phosphatidylserine (PS), phosphatidyl-ethanolamine (PE), sphingomyelin (SM), phosphatidylinositol (PI), phosphatidylcholine (PC), cholesterol and ceramides [42][43][START_REF] Lydic | Rapid and comprehensive 'shotgun'lipidome profiling of colorectal cancer cell derived exosomes[END_REF][START_REF] Skotland | An emerging focus on lipids in extracellular vesicles[END_REF]. It is well established that exosomes exhibit greater rigidity than the plasma membranes of cells. This rigidity would be the result of a pH-dependent mechanism. Indeed, the membrane rigidity of exosomes increases between pH 5 and 7 [START_REF] Laulagnier | Mast cell-and dendritic cell-derived exosomes display a specific lipid composition and an unusual membrane organization[END_REF]. This finding could explain why a lower pH such as those of the tumor microenvironment enhances cellular absorption of EVs [START_REF] Parolini | Microenvironmental pH is a key factor for exosome traffic in tumor cells[END_REF]. The increased acidity makes the membranes of EVs more fluid thereby promoting their fusion with cell plasma membranes. Finally, the greater rigidity of EV membranes comes from their high content of sphingomyelin, desaturated lipids, and cholesterol [START_REF] Wubbolts | Proteomic and biochemical analyses of human B cell-derived exosomes: potential implications for their function and multivesicular body formation[END_REF][START_REF] Brouwers | Distinct lipid compositions of two types of human prostasomes[END_REF] which might contribute to their resistance to degradation and, as a result, their stability as carriers of different biomolecules through bodily fluids [START_REF] Alvarez-Erviti | Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes[END_REF][START_REF] Ridder | Extracellular vesicle-mediated transfer of genetic information between the hematopoietic system and the brain in response to inflammation[END_REF]. Consistently, exosomes have been shown to be more resistant to detergent treatments than microvesicles, indicating a greater membrane lipid order [START_REF] Osteikoetxea | Differential detergent sensitivity of extracellular vesicle subpopulations[END_REF]. Furthermore, EVs' high cholesterol and sphingomyelin content might lead to apoptosis of receptor cells [START_REF] Ristorcelli | Human tumor nanoparticles induce apoptosis of pancreatic cancer cells[END_REF][START_REF] Ristorcelli | Essential role of Notch signaling in apoptosis of human pancreatic tumoral cells mediated by exosomal nanoparticles[END_REF]. Indeed, synthesized nanoparticles imitating the lipid content of EVs decrease the survival of pancreatic tumor cells [START_REF] Beloribi | Exosomal lipids impact notch signaling and induce death of human pancreatic tumoral SOJ-6 cells[END_REF]. On the other hand, such synthetic exosome-like nanoparticles induced the activation of the survival pathway in a different human pancreatic tumoral cell line [START_REF] Beloribi-Djefaflia | Exosomal lipids induce human pancreatic tumoral MiaPaCa-2 cells resistance through the CXCR4-SDF-1α signaling axis[END_REF]. Taken together, these data show that lipids not only convey stability and structural rigidity to the vesicular membrane, but also take part in cellular signaling pathways [START_REF] Record | Extracellular vesicles: lipids as key components of their biogenesis and functions[END_REF].

I.2.2. Proteins

Proteins are important components of EV cargo, and their expression is frequently employed for characterization purposes. Other than being used as a quality control tool, they can be used to detect exogenous or disease-specific proteins. Although proteins can be typical of different EVs, they cannot be considered real specific markers of the various types of EVs. In this context, the ISEV provided the minimal experimental requirements for the definition of EVs. The specific categories of proteins to consider for EV characterization are listed in the table 3 from the Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018) guidelines [3]. EVs proteins include proteins of endosomal and cytosolic origin (annexins, ALIX, TSG101, flotillins) [START_REF] Van Niel | Exosomes: a common pathway for a specialized function[END_REF], tetraspanins (CD9, CD63, CD81) that are among the most prevalent membrane proteins on EVs and are often utilized to characterize them [START_REF] Kleijmeer | Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes[END_REF][START_REF] Andreu | Tetraspanins in extracellular vesicle formation and function[END_REF],

heat shock proteins (HSP70, HSP90) [11,[START_REF] Théry | Molecular characterization of dendritic cell-derived exosomes: selective accumulation of the heat shock protein hsc73[END_REF] and cytoskeletal ones (tubulin, actin). These molecules are generally present in EVs regardless of their cellular origin (Fig. 4). However, there are proteins found in relatively low proportions in EVs that are negative markers of EVs: when compared to cell lysate. This is the case, for example, of calnexin [30]. EVs also contain cell-type specific proteins like MHC class II on antigen presenting cells (APCs)-derived vesicles [12]. Likewise, milk fat globuleepidermal growth factor 8 (MFG-E8), also known as lactadherin, is present only in EVs originating from immature dendritic cells [START_REF] Véron | Accumulation of MFG-E8/lactadherin on exosomes from immature dendritic cells[END_REF]. These observations highlight the specificity of formation of these vesicles. Furthermore, EVs include glycosylated proteins that play an important role in cellular targeting of EVs and their interactions [START_REF] Liang | Complex N-linked glycans serve as a determinant for exosome/microvesicle cargo recruitment[END_REF][START_REF] Menck | Tumor-derived microvesicles mediate human breast cancer invasion through differentially glycosylated EMMPRIN[END_REF][START_REF] Williams | Glycosylation of extracellular vesicles: current knowledge, tools and clinical perspectives[END_REF]. Indeed, the protein profile of EVs reveals the presence of many glycan binding proteins that control which cells would be targeted by EVs and how [40]. For example, glypican-1, a heparan sulfate-containing cell surface proteoglycan is implicated in exosome docking [START_REF] Melo | Glypican-1 identifies cancer exosomes and detects early pancreatic cancer[END_REF]. P-selectin (CD62p) present on the surface of EVs produced by activated platelets permits EVs' attachment to target cells [START_REF] Han | Platelet P-selectin initiates cross-presentation and dendritic cell differentiation in blood monocytes[END_REF]. Therefore, the protein composition of EVs influences their roles, adhesion capacities and cell targeting.

I.2.3. Nucleic acids

Significant advancements in the characterization of EVs have revealed that they can also transport nucleic acids. The majority of studies have described RNA, including mRNA and miRNA which are often smaller in size than the RNA found in cells [13,[START_REF] Ratajczak | Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication[END_REF]. Aside from transporting RNA, the presence of deoxyribonucleic acid (DNA) in EVs has been less studied and is still a matter of debate as DNA might be considered a contaminant from improper isolation [START_REF] Mateescu | Obstacles and opportunities in the functional analysis of extracellular vesicle RNA-an ISEV position paper[END_REF][START_REF] Jeppesen | Reassessment of exosome composition[END_REF]. Therefore, further research is required to determine whether DNA is present and, if so, what its real significance in EVs is. The RNA content of EVs varies depending on the cell type. For instance, EVs produced by certain cancer cells contain more RNA than those produced by healthy cells [START_REF] Balaj | Tumour microvesicles contain retrotransposon elements and amplified oncogene sequences[END_REF]. The RNA content of EVs reflects the RNA content of the parent cell, suggesting that RNA molecules are selectively integrated into EVs. However, the mechanism involved in the selection of RNA to be transferred in EVs has not yet been explained.

Furthermore, it has been shown that the extracellular miRNAs encapsulated inside the EVs can be transferred to the recipient cells, altering their gene expression and mediating their functional effects [START_REF] Camussi | Exosomes/microvesicles as a mechanism of cell-to-cell communication[END_REF][START_REF] Kosaka | Secretory Mechanisms and Intercellular Transfer of MicroRNAs in Living Cells*[END_REF].

I.3. Extracellular vesicles: new sources of human health information?

Initially, EVs were thought to be membrane detritus with little biological significance. However, Raposo et al. demonstrated in 1996 that these vesicles might trigger the adaptive immune response [12].

Thereafter, several articles have established the relevance of EVs in intercellular communication via the transfer of proteins, lipids, and nucleic acids. The use of EVs for pathology diagnosis or therapy is extremely promising due to their unique biological characteristics and endogenous source. As a result, interest in these vesicles grows year after year. In this section, I will briefly discuss the significance of EVs in the maintenance of normal physiology, as well as their potential clinical use for new perspectives in medicine, both diagnostically and therapeutically.

I.3.1. Biological role

The biological functions of EVs differ depending on the cell type from which they are derived, and hence on what they carry. In fact, they can operate as a signaling complex, delivering membrane receptors and other proteins to target cells, or altering their phenotypic via horizontal genetic information transfer. Inflammation and immunological response, intercellular communication, cell survival, angiogenesis, coagulation and waste disposal represent the major biological activities reported on EVs [START_REF] Van Niel | Shedding light on the cell biology of extracellular vesicles[END_REF]. An implication in stem cell biology and different aspects of early development have also been confirmed quiet recently [START_REF] Hur | Extracellular vesicles and their roles in stem cell biology[END_REF]. Despite these findings, several physiological functions of EVs remain unclear due to the difficulties in investigating endogenous vesicles in vivo. However, significant attempts have been undertaken to develop in vivo models to better understand EV physiology [START_REF] Verweij | Live tracking of inter-organ communication by endogenous exosomes in vivo[END_REF].

I.3.2. Pathological role

EVs play an important and complex role also in some pathologies. The role of EVs in cancer biology has been widely investigated. Cancer cells are able to release EVs that promote tumor growth by boosting angiogenesis [START_REF] Webber | Extracellular vesicles as modulators of the cancer microenvironment[END_REF]. They can promote invasive growth of the tumor mass by degrading the extracellular matrix [START_REF] Shimoda | Metalloproteinases in extracellular vesicles[END_REF], and carry oncogenes that induce the transformation of the phenotype of the target cells, favoring metastasis formation [START_REF] Becker | Extracellular Vesicles in Cancer: Cell-to-Cell Mediators of Metastasis[END_REF]. Moreover, cancer EVs protect cancer cells by allowing them to evade apoptosis and develop resistance to chemotherapy [START_REF] Safaei | Abnormal lysosomal trafficking and enhanced exosomal export of cisplatin in drug-resistant human ovarian carcinoma cells[END_REF][START_REF] Nehrbas | Extracellular vesicles and chemotherapy resistance in the AML microenvironment[END_REF] , and finally they have the ability to escape the immune system [START_REF] Wieckowski | Tumor-derived microvesicles promote regulatory T cell expansion and induce apoptosis in tumor-reactive activated CD8+ T lymphocytes[END_REF].

Beyond tumors, EVs have also been linked to the spread of neurological disorders. In Alzheimer's disease, EVs carrying amyloid beta (Aβ), the toxic protein responsible for the formation of amyloid plaques, as well as hyperphosphorylated protein Tau, were shown to promote their deposition in several areas of the brain and to hijack the secretory endosomal pathway in the recipient neurons [START_REF] Bellingham | Exosomes: vehicles for the transfer of toxic proteins associated with neurodegenerative diseases?[END_REF][START_REF] Polanco | Exosomes taken up by neurons hijack the endosomal pathway to spread to interconnected neurons[END_REF]. Similarly, α-synuclein monomers, oligomers and fibrils can be found in the lumen as well as on the surface of EVs, contributing to the progression of Parkinson's disease [START_REF] Stuendl | Induction of α-synuclein aggregate formation by CSF exosomes from patients with Parkinson's disease and dementia with Lewy bodies[END_REF]. Other studies have shown that EVs have a role in the development of infectious diseases by increasing the movement of receptors, virulence factors, and genetic material across cells, resulting in pathogen spread and harmful effects [START_REF] Pegtel | Functional delivery of viral miRNAs via exosomes[END_REF][START_REF] Schorey | Extracellular vesicles and infectious diseases: new complexity to an old story[END_REF].

I.3.3. EVs as a source of promising biomarkers

A biomarker is defined as "a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic intervention" [START_REF] Group | Biomarkers, EndpointS, and other Tools) Resource[END_REF]. The use of a biomarker therefore offers information about a patient's medical status, allowing for more precise diagnosis and prognosis, as well as assessing the success and compatibility of therapeutic treatments. As a result, it must be pathological specific, i.e. capable of identifying only patients presenting a given disease, as well as sensitive, i.e. capable of detecting as many ill patients as possible. A biomarker must also be robust (show a link between the biomarker and the physiopathology of the disease) and ideally predictive (the biomarker must be correlated with the severity of the pathology) [START_REF] Stremersch | Therapeutic and diagnostic applications of extracellular vesicles[END_REF]. When a diagnostic test is based on a biomarker assay, a cut-off value is established. This is the value used to classify subjects as either healthy or sick. With the growing evidence that EVs contain a biologically active cargo that reflects the pathophysiological state of the parent cell, their potential use as a source of biomarkers in the identification of diseases in a noninvasive manner has increased significantly.

Many EVs studies focused on their application in identifying different cancers at their earliest stages and understanding their progression [START_REF] Taylor | MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer[END_REF][START_REF] Rabinowits | Exosomal microRNA: a diagnostic marker for lung cancer[END_REF][START_REF] Wang | Peripheral blood microvesicles are potential biomarkers for hepatocellular carcinoma[END_REF][START_REF] Rahbari | Exosomes: novel implications in diagnosis and treatment of gastrointestinal cancer[END_REF][95][START_REF] Lane | Extracellular vesicles as circulating cancer biomarkers: opportunities and challenges[END_REF]. Exploiting EVs' content has resulted in the identification of different potential biomarkers.

Pathology

Biofluids Identified biomarker References

Ovarian cancer Ascitic fluid HNRHPU, U2AF2

TGM2 and U2AF1

CD 24 [START_REF] Shender | Proteome-metabolome profiling of ovarian cancer ascites reveals novel components involved in intercellular communication[END_REF] [98] Urine miR-30a-5p [START_REF] Zhou | Urinary microRNA-30a-5p is a potential biomarker for ovarian serous adenocarcinoma[END_REF] Plasma TGF-β1 et MAGE3/6

Claudin-4

[100]

[101] Serum miR -21, miR-141, miR-200a, miR200c, miR-200b, miR-203, miR-205 and miR-214 [91] Blood miR- The "SeleCTEV" technology captures EVs by an affinity method with peptides while the "SoRTEV" system captures them by beads coated with specific antibodies against surface antigens. Exosomics also markets analytical assays, based on ELISA or PCR (Polymerase Chain Reaction) type platforms.

Caris Life Sciences (Dallas, USA) has also developed an immunocapture method called "Carisome" that allows for the capture of EVs from blood onto coated beads and the detection of specific fluorescently tagged surface proteins. A preliminary test based on a blood sample is now available for the diagnosis of prostate cancer and offers higher sensitivity and specificity than the currently used prostate-specific antigen (PSA) test. Finally, Exosome Sciences (San Diego, USA) is developing an ELLSA (Enzyme-Linked Lectin-Specific Assay) test to isolate biomarkers such as TauSome, a non-invasive marker of chronic traumatic encephalopathy from a blood sample.

However, transitioning from basic research to clinical validation and industrial manufacturing is still challenging. The primary issue is the lack of standardization of EV isolation procedures. This has an effect on the repeatability of the results, as well as the identification and confirmation of the discovered biomarkers. Beside the purification protocols, the procedure utilized to collect the biological fluid containing the EVs, as well as the storage conditions, can also have an impact on the quality and purity of the isolated EVs and therefore on biomarker identification. Indeed, they can alter the protein content of isolated vesicles, but they can also cause EV aggregation. As a result, the diagnostic test's performance is affected [125]. Moreover, it is critical to identify specific subtypes of EVs because distinct vesicles might have diverse biological effects. Several molecules transported by

EVs have therefore been identified as potential biomarkers, but further studies are required before they can be used to diagnose diseases.

I.3.4. Therapeutic potential of EVs

Considering that EVs have an inherent biological impact that modifies the phenotypic of the receiving cell, the scientific community is increasingly trying to exploit EVs for therapeutic purposes. One area where EVs have found application is regenerative medicine. The objective of regenerative therapy has always been to find stem cells that can be safely transplanted into a patient and successfully repair the injured organ, freeing it from the difficulties of the immune response. Furthermore, MSC-derived EVs are being studied for a variety of inflammatory diseases due to their immunomodulatory characteristics. Several papers discuss also the use of EVs in cancer therapy [133].

These vesicles have several advantages: they have no tumorigenic potential since they cannot replicate unlike cells they are easy to handle and they can be sterilized by filtration [134].

EVs have also been exploited as vehicles to deliver small molecules and drugs. However, the use of EVs in the treatment of pathologies brings also several drawbacks. A large number of EVs is required for the treatment of a single patient. This implies that a high number of cells are required, followed by the ability to separate EVs in accordance with acceptable laboratory procedures.

EVs have also been explored as a platform for vaccination, by delivering disease-associated antigens

I.4. Concluding remarks

As mentioned in the preceding paragraphs, EVs are very promising biological objects for diagnostic and therapeutic purposes. However, scientific interest in EVs is still relatively new, and current research presents several hurdles. Different aspects must be optimized in order to increase the knowledge on the functions of EVs and their potential. The nomenclature of EVs must be standardized to clarify the different types of EVs, which are very complex to identify with precision. The biogenesis of EVs must therefore be better understood. Additionally, current isolation and quantification protocols are not considered fully robust, therefore new techniques need to be implemented and standardized. These techniques must also be developed in such a way that they enable for the isolation of distinct EV subpopulations. To get pure EV samples and offer a thorough investigation of the exosomal signature, robust isolation and characterization approaches are required. The next section discusses isolation and characterization approaches, as well as developing technologies to address the limits of existing methods.

Chapter II: Current methods for EVs isolation and characterization

II.1. EVs extraction and isolation

The high reproducibility we will be able to get representative batches of the whole EV population and prevent unacceptable batch-to-batch fluctuations. When dealing with such vesicles, however, a number of technical considerations must be considered, including their small sizes, their fragility, as well as their propensity to aggregate or lyse. Since the separation of EV subpopulations from each other is still very complicated with existing techniques and reported works, the terms exosomes and extracellular vesicles will be used interchangeably throughout the rest of this chapter. In this section, we will go through the most common macroscopic techniques reported for preparing and isolating EVs, presenting for each of them their advantages and disadvantages. It will be addressed the many aspects of distinct techniques, including separation efficiency, EV yield and the qualities of isolated EVs.

II.1.1 Ultracentrifugation (UC)

According to a global survey of techniques used for EV isolation made in 2016 and submitted to all ISEV members, ultracentrifugation is the most widely used primary isolation method (> 80 % of respondents) [139]. When a heterogeneous suspension is subjected to centrifugal force, the particles it contains sediment according to their density, size and shape. There are 2 types of ultracentrifugationbased techniques (Fig. 6), differential ultracentrifugation and density gradient ultracentrifugation.

II.1.1.1. Differential ultracentrifugation

Differential centrifugation consists in applying several cycles of centrifugation at different acceleration and duration. Théry et al proposed the approach shown in figure 7 for the isolation of exosomes. First, to eliminate dead cells and cellular debris, the culture supernatant or biological fluid containing the exosomes is centrifuged at 300xg and again at 2,000xg. After that, the supernatant is centrifuged at 10,000xg to eliminate bigger vesicles, then the supernatant is centrifuged at 100,000xg for at least 1 hour. This protocol and variation of it (centrifuge time, speeds) have been extensively utilized by different teams. A rinse procedure is recommended to remove any potential protein aggregate that may have co-precipitated with EVs. Exosomes are typically washed in a PBS solution before being centrifuged at 100,000xg. Although this approach is widely used, it is still highly reliant on the viscosity of the medium. As a result, differential ultracentrifugation is less efficient when dealing with viscous samples like serum and plasma [141]. This method offers however several advantages. Indeed, this approach is quite simple to use because it requires minimal technical skill and does not necessitate pre-processing of the samples. However, we must keep in mind that it is time consuming, impurities (such as lipoproteins, aggregates, microparticles, MVs etc.) may still be present, and EVs can cluster together or deteriorate, resulting in artefacts during analysis [142].

Figure 7: Workflow for the differential ultracentrifugation-based exosome purification method. Pellets (cells, dead cells, and cell debris) are eliminated after the first three centrifugations, and the supernatant is kept for the following step. After the two 100,000 g centrifugations, the pellets (exosomes + contaminating proteins, exosomes) are preserved, while the supernatants are discarded.

Reprinted from [143].

II.1.1.2. Density gradient ultracentrifugation

II.1.2. Ultrafiltration (UF)

Ultrafiltration may be used to separate EVs from their biological fluid based on their size and molecular weight utilizing semi-permeable membranes coupled with small centrifugation cycles [152,153]. In comparison to ultracentrifugation procedures, ultrafiltration is quicker and do not require specific equipment. The molecular weight cut-off (MWCO) ranges from 10 to 100 kDa for ultrafiltration, and the most often utilized membrane is regenerated cellulose with a 100 kDa pore size [154]. Vergauwen et al., on the other hand, revealed that regenerated cellulose membranes with pore sizes of 10 kDa recovered the most EVs, whereas other tested filters recovered less than 40%, when using plasma, urine, and EV-spiked PBS [155].

Tangential flow filtration (TFF), also known as cross-flow filtration, is a more advantageous alternative ultrafiltration technique to classical centrifugal one, in which the fluid flows directly through the membrane, resulting in cake formation that clogs the pores and, as a result, EV loss [156]. In TFF EVcontaining fluids moves tangentially over the membrane and smaller molecules which pass through the membrane are eliminated, whereas bigger molecules, like EVs, stay above membrane and are recirculated and concentrated (Fig. 8).

II.1.3. Size-exclusion chromatography (SEC)

Using size exclusion chromatography (SEC), EVs can be separated from biological fluid based on their hydrodynamic diameter. First, the material is centrifuged to eliminate dead cells and debris that might clog the column. The medium is then pushed through a column packed with porous spherical polymer beads whose pores have a specific calibrated size (Fig. 9). Larger particles, such as EVs, elute first because they cannot penetrate the pores and hence flow around the resin. Particles smaller than the isolation range penetrate the pores and elute later. Cross-linked agarose beads (commercially known as Sepharose® (CL-2B and CL-4B) and Sephacryl® S-400) are the most often utilized stationary phase materials [154].

II.1.5. Immunoaffinity capture-based methods

Immunocapture is the most specific and effective approach to isolate EVs [170]. In contrast to previous techniques based on physical properties, the affinity-based technique are based on EVs capture using antibodies against EV surface proteins, which are often covalently linked to magnetic beads. The most widely utilized antibodies target tetraspanin proteins often found on exosome and EV surfaces, including CD9, CD63, and CD81 [START_REF] Andreu | Tetraspanins in extracellular vesicle formation and function[END_REF], resulting in kits made by companies like ThermoFisher, Miltenyi Biotec, and Abcam (Fig. 10A). Immunoaffinity chromatography using monolithic materials have been reported as an alternative to magnetic beads to isolate EVs [171,172]. In particular, Multia et al. Affinity-based approaches have the advantage of being rapid, simple, and compatible with laboratory practice. However, one of the major drawbacks is that it could only capture EVs expressing a given specific antigen, potentially excluding other EVs subpopulations concurrently present in a biofluid.

Moreover, the sample volume must be small, and it must first be enriched in EVs through ultracentrifugation or ultrafiltration. Furthermore, the strong antibody-antigen affinity may be a limiting factor that prevents the release of pure EVs for further applications. Only a few works on capture and eventual elution of EVs on magnetic beads have been reported. than that of UC and more than twice as high as that of PEG-based precipitation. Although these procedures effectively release intact EVs, they may have an effect on their physicochemical qualities since they will bear the antibody used to capture them on their surface.

II.1.6. Asymmetric flow field-flow fractionation (A4F)

Another size-based fractionation technique for EV separation is flow field-flow fractionation (FFF), with asymmetrical flow field-flow fractionation (AsFlFFF or AF4). It comprises a small channel with a diameter ranging from 5 to 500 µm and a permeable membrane at the bottom (accumulation wall).

The sample is carried along the channel by a laminar flow to detectors. To accomplish size-based fractionation, a perpendicular cross flow to the parabolic one is used to separate EVs on the accumulation wall (Fig. 11). Then, due to their greater diffusion coefficient, small particles remain far from the accumulation bottom, while larger particles accumulate near to the wall. As a result, the parabolic flow profile transports small particles first, followed by bigger ones, contrary to SEC, which elutes larger particles first. This technique has the advantage of being compatible with several type of detectors that can even be placed in series, such as ultraviolet (UV), multi-angle light scattering (MALS), and refractometer, providing information on EV size distribution, size morphology and aggregation or agglomeration states. EV subpopulations can also be collected for further studies when combined with a fraction collector. AsFlFFF has proven particularly significant in the separation of EV subpopulations, culminating in the separation of a novel EV subpopulation known as exomeres (~35 nm) (see Fig. 12) [180,181]. However, AsFlFFF has several disadvantages, which explains why it is not widely used. Indeed, this technique cannot separate particle with the same hydrodynamic radius but having different morphology, surface molecules or other physical properties, its resolution is not high. Only little amounts of sample can be injected to minimize self-association and overloading effects. Furthermore, fractionated EVs collected by AsFlFFF are largely diluted and may necessitate a further preconcentration step.

II.1.7. Concluding remarks

If many isolation techniques based on different mechanisms are already, none of the above-mentioned EV isolation techniques can combine effectiveness, specificity, good reproducibility and high yield and purity. The choice of one method is mostly determined by the analysis that will be used following the isolation of the vesicles as well as the availability of the necessary equipment.

In conclusion to this presentation on EV isolation strategies, a table comparing the most commonly used procedures is included in our review paper 1 in subchapter III.2. of this thesis (Table 1).

Ultracentrifugation remains the reference method of isolation despite the vesicular pellet is often contaminated with aggregated proteins and lipoproteins, and high-speed centrifugation may cause EV mechanical damage. When the column is commercial, size exclusion chromatography is relatively fast; nevertheless, the available phases are not yet sufficiently resolutive to remove lipoproteins, which have a retention time similar to that of EVs. Polymer-based precipitation using commercial kits is unquestionably the approach with the lowest EV purity. Immunocapture can be used to isolate subpopulations of EVs depending on the marker targeted by the beads. However, the strength of antibody-antigen binding makes dissociation difficult, limiting EV recovery for other uses or analyses.

Because different approaches and technologies have benefits and drawbacks and distinct principles for EV isolation, combination of two techniques, like UC with UF or SEC, or even more sophisticated combination of three different methods (UF, SEC, and AsFlFFF), have been recently proposed to reach EV purity while preserving their integrity in a straightforward and high-throughput manner. Among the other new approaches are microfluidic-based tools for fast and efficient EV processing, which will be discussed in depth in Chapter III.. Towards this direction, one development of my thesis was toward a novel method for isolating EVs from pre-purified and biofluid samples using microfluidic droplets.

II.2. EVs analysis and characterization

EVs must be characterized once they have been isolated. This step verifies that the isolated vesicles are EVs, as well as that the population is homogenous and that the vesicles have not been lysed or aggregated. Today, isolated vesicles are often subjected to two distinct kinds of investigation: physical and chemical/biochemical/compositional analysis. Physical analysis provides information on the size and/or concentration of particles. Chemical/biochemical/compositional analysis provides information on the separated vesicles' contents. According to a recent study conducted by Gardiner et al. [139],

the most commonly used techniques to study EVs are: Western-blot, particle size measurement techniques (nanoparticle tracking analysis (NTA), tunable resistive pulse sensing TRPS, and dynamic light scattering (DLS)), electron microscopy, and flow cytometry, which represents the gold standard because it is used by nearly 40% of laboratories. There are currently no uniform rules for performing EV characterization. However, in 2018, the International Society for Extracellular Vesicles (ISEV) published a guide to help research teams characterizing EVs [28]. As a result, the following are the primary recommendations:

-Provide an analysis of the protein composition of the EV preparation. The identification and relative quantification of several proteins (at least three) is required since no specific marker for the presence of EVs has been established so far.

-Characterize the heterogeneity of the EV population using at least two distinct methods, in particular techniques for assessing size distributions that must be compared to microscopic observations.

-After demonstrating the function of vesicular proteins or particular RNA species, it is crucial to confirm that these components are derived from EVs and are not co-isolated soluble molecules.

In this section, we will discuss the most often used approaches, as well as their benefits and drawbacks. Therefore DLS is more effective technique for measuring monodisperse sample [183]. The evolution of the size distribution towards a higher diameter range for the EV populations was observed by Van der Pol et al. [184]. This bias can even render small vesicles undetectable. This is why a preliminary preparation of the samples of EVs aiming to split them into subpopulations is necessary if one wishes to use the DLS.

II

II.2.1.2. Nanoparticle tracking analysis (NTA)

Nanoparticle tracking analysis (NTA) is a more recent technology that has recently gained popularity.

Like DLS, it is based on light scattering and the Brownian motion of suspended vesicles, but this approach allows for individual monitoring of all nanoparticles present in the medium. The sample is loaded in a chamber through which a light beam pass. When moving particles cross the path of the beam, they scatter light. By live tracking the mean squared displacement of a single particle using an optical microscope coupled to a camera, hydrodynamic diameters (Dh) can be determined using the Stokes-Einstein equation (Fig. 13A). As a result, NTA is more suited to measure the size of particles in a polydisperse sample. The non-symmetric profile of the size distribution in Figure 13B, for example, clearly suggests that the examined solution comprises a heterogeneous population of particles [183].

Furthermore, it provides quantitative information because it is possible to know the concentration, namely the number of particles per milliliter present in the population examined. NTA gives information on the size distribution of vesicles between 50nm and 1µm, and the ideal particle concentration is advised to be in the range of 2x10 8 to 2x10 9 EVs/mL [185]. If necessary, a fluorescence mode (F-NTA) is also available for the detection of antigens present on EVs surface using specific antibodies coupled to a fluorochrome. This can be particularly interesting in applications involving the characterization of vesicular subtypes or when looking for phenotypic changes in EVs passing from a physiological state to a pathological state [186]. The benefit of using this NTA technique is that the sample can be recovered, the analysis is performed in liquid media with no EV morphological changes. The sample preparation is easy and fast. However, this particle counterbased technique cannot discriminate extracellular vesicles from aggregated proteins. Accurate quantitative measurement may be a challenge that require, sample dilution, camera detection threshold, and focus to be precisely optimized.

A B

II.2.1.3. Tunable resistive pulse sensing (TRPS)

Tunable resistive pulse sensing (TRPS) may be used instead of NTA to determine the concentration and size distribution of EVs. It is based on measuring the electrical resistance generated by the passage of individual EVs through a pore within an insulating membrane (Fig. 14). This membrane separates two chambers, each of which contains a single electrode immersed in an electrolyte solution. In this configuration, an ionic current flow between the two chambers, and each particle passing through this pore generates a resistivity peak which amplitude is proportional to the particle's size, while the blockade rate is related to the concentration. In order to measure a polydisperse population of EVs, several pores must be cascaded with different detection limits. Maas et al. demonstrated the influence of pore size on the measured particle concentrations [188].

Depending on their pore size, TRPS instruments can measure particles from 50 nm to 10 µm [189] and recent developments have shown further improvements in measurement stability and sensitivity

[190], although lower detection limit are still needed in order to analyze all sizes of EVs. The main problem with using TRPS to characterize EVs is the risk of many particles passing through the pore at the same time (which are identified as a single large particle), and the risk of pore clogging or protein adhesion on the pore surface. Izon Science Ltd was the first company to market the qNano instrument, a bench-top device enabling TRPS application in practice [191]. Thanks to stretchable pores in polyurethane membrane, this equipment can characterize particles ranging in size from 40 nm to 10 µm. TRPS and NTA were recently compared by Akers et al. [192], showing that the NTA detected more EVs smaller than 150 nm, while the TRPS system was more efficient in detecting large EVs.

II.2.1.4. Flow cytometry

Flow cytometry has always been regarded as a robust technique for characterizing cells or micrometric entities. This method employs a laser beam in front of which the EVs pass one by one. The light scattered by the EVs which is proportional to particle size is measured by the flow cytometer. Axial (Forward Scatter or FSC) and orthogonal light scattering (Side Scatter or SSC) light are available, the latter being more sensitive. The principle is shown schematically in Figure 15. comparative study showed that the VFC, however, had two to three times fewer EVs than the NTA and the TRPS, perhaps because this system makes it possible to exclude protein aggregates or other particles of similar size [192]. Apogee Flow Systems (Northwood, UK) currently sells a microflow cytometer dedicated to the identification of nanometric particles such as EVs or protein aggregates. A comparative study showed that conventional flow cytometry gave EV concentrations 300 times lower than the comparative analyzes by NTA and TRPS. In parallel, the dedicated Apogee A50 Micro cytometer gave an underestimation by a factor of fifteen of the concentration of EVs . However, as with TRPS, overlapping signals can occur when multiple vesicles are contained in the detection volume concurrently [196]. As a result, the particle concentration range that may be employed to identify individual EVs is limited to between 10 7 and 10 10 particles/mL [197]. Flow cytometry is still one of the most used approaches because of its flexibility, but it needs specific equipment, which raises costs and limits accessibility [198].

II.2.1.5. Electron microscopy (EM)

Images of EVs with a resolution of 1 angstrom (Å) may be obtained using electron microscopy (EM).

EVs have a distorted cup-shaped morphology, similar to that of a red blood cell (Fig. 16). The images are obtained either by transmission of the electron beam through the sample (transmission electron microscopy, TEM) or by inelastic scattering of the electron beam on the surface of the sample (scanning electron microscopy, SEM). Because of its higher resolution, TEM is the most often used EM method for characterizing EVs. As EM observations are performed under vacuum, EVs must first be chemically fixed and dehydrated to be observed. The most common sample preparation protocol consists of depositing a drop of liquid sample on the grid, fixing it by chemical treatment with paraformaldehyde

[150] or glutaraldehyde [199] and then dehydrating it (Fig. 16A). Sometimes, these chemical treatments are responsible for artifacts on the morphology and size of EVs. Cryogenic freezing, preceding the EM observations, is a preparation technique that is increasingly being used to replace the chemical fixing procedures (cryo-TEM) [199]. However, changes in the size and natural structure of the vesicles may still occur in cryo-TEM owing to the thickness of ice (typically a few hundred nanometers), which can compress the vesicles together. However, changes in the size and natural structure of the vesicles may still occur in cryo-TEM owing to the thickness of ice (typically a few hundred nanometers), which can compress bigger vesicles and make them appear larger. Moreover, the strong intensity of the electron beam can be responsible for damage caused to the EVs [200] (Fig. 16B). However, at the moment, cryo-TEM is the most powerful method for analyzing the morphology of EVs. Finally, TEM may be integrated with immunodetection utilizing antibodies linked to gold particles to detect subpopulations of EVs, as reported by Melo et al. who specifically imaged EVs from breast cancer cells expressing the CD9 protein on their surface [201].

II.2.1.6. Atomic force microscopy (AFM)

Another approach for viewing the surface of EVs and consequently analyzing their morphology is atomic force microscopy (AFM) [202,203]. It enables the visualization of EVs with nanometric resolution whether they are deposited on a flat surface, in a specific way (captured by specific ligands)

or non-specifically (adsorption), in a liquid medium, at atmospheric pressure, and without sample preparation or labeling. AFM is based on the employment of a flexible cantilever with a tip that scans the surface of the sample to produce a topographic picture of the surface (Fig. 17). Other nano-plasmonic devices have been developed, including the one described by Liang et al. [210].

By functionalizing a microarray with anti-CD81 antibodies, EVs were captured and then detected using anti-CD63 and anti-CD9 antibodies coupled to easily identifiable gold nanospheres and nanorods, as

shown in Figure 18. This combination produced a local plasmon effect that improved the sensitivity and specificity of EV detection. This technique has been demonstrated on EVs derived from pancreatic cancer and has outperformed ELISA tests in terms of LOD, requiring only 1 µL of biological sample and 5 hours of analysis, making this device a promising diagnostic tool. This device is now being developed for commercial use.

II.2.2. Molecular characterization

II.2.2.1. Western blot (WB) and ELISA

Western blot (WB) is a biochemical method for detecting particular proteins in a sample containing different proteins. WB is frequently used to verify the expression of EV-associated proteins (e.g. CD9, CD81, CD63, Alix, Tsg101) and the lack of contaminating proteins (e.g. Calnexin, Albumin, Fibronectin) in isolated samples [28]. Following EV lysis, the proteins are separated by gel electrophoresis (generally SDS-PAGE) and transferred onto a membrane (often nitrocellulose) before being revealed by specific antibodies. Because contaminating proteins cannot be differentiated from exosomal proteins, WB necessitates meticulous previous purification of EVs. This is why WB identification of the proteins on the surface of the EVs is required but not sufficient to verify the existence of the vesicles in the sample.

In addition, the considerable quantity of EVs used in this type of analysis and the duration of manipulation (2 days) do not allow considering the WB as a routine characterization.

Enzyme-Linked Immunosorbent Assay (ELISA) can also be used to analyze specific proteins under different configurations: direct, indirect, in a sandwich, or via competition. The sandwich configuration is often used for EVs. Isolated EVs are captured by an antibody immobilized on a solid support allowing additional washing procedures to remove non-EV-associated proteins. A second antibody that recognizes trapped EVs is then employed [171]. The use of the ELISA test as a reference for the development of EV detection technologies has been widely demonstrated [210,211].

Commercial ELISA kits are available, such as ExoElisa-ULTRA (System Biosciences) and Exo-TEST (HansaBioMed) which are designed for the detection of EVs using particular tetraspanin probes (CD9, CD81 ...).

In terms of detection limit and outcomes, the two approaches presented here are similar. They are mostly determined by the efficacy and specificity of the used antibodies. ELISA is quicker and seems to be more suited for routine use. However, it is still difficult to find specific antibodies for each subcategory of EVs. Moreover, the ELISA does not directly measure the number of vesicles in the sample and does not give any information on the physical characteristics (size and morphology) of the EVs.

II.2.2.2. Mass spectrometry (MS)

The protein composition of EVs may be also investigated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The proteins of the isolated EVs are digested enzymatically and the peptides are then separated using liquid chromatography and ionized before being analyzed by mass spectrometry. Several steps and parameters in this process have a significant impact on the proteome profile of EVs. In addition to efficient EV purification by selecting a good technique to minimize contamination by non-vesicular proteins, the choice of mass analysis and the performance of the mass spectrometer used are also very important. Because mass spectrometry analyzes peptide fragments, identification, quantification, and proper validation of the peptides produced are required. The relevance of proteomic data is determined not only by the stringency of the filters used, but also by the databases consulted during the raw data analysis. Indeed, not all of the databases are updated on a regular basis. Although mass spectrometry necessitates substantial data preparation and processing

II.2.3. Concluding remarks

In conclusion, each of the approaches presented has advantages but also limits, and none of these techniques allows us to assess simultaneously the concentration, size, and biochemical properties of EVs. A comparison table of the most frequently used procedures for EV characterization is presented in our review paper 1 in subchapter III.2. of this thesis (Table 2). At the present, there is an agreement that multiple of these methodologies should be used in order to obtain an accurate and complete characterization of EVs. Moreover, the most appropriate analysis method for studying EVs must meet several conditions:

• Ensure an analysis of EVs directly in their raw sample or with the minimum possible pre-analytical interventions.

• Enable the biochemical and physical characterization of EVs

• Give access to a quantification / assay of EVs.

• Characterize the vesicles on an individual scale.

• Ensure reproducibility and make automation possible However, because of their small size, heterogeneity, the presence of contaminants, and the low sensitivity of existing methods, EV characterization remains a challenge.

Chapter III: Emerging methods for EVs isolation, analysis and detection

III.3. Microfluidic-based technologies

In recent years, researchers have developed numerous microfluidic systems for the isolation, detection and analysis of EVs, utilizing both physical and biological characteristics of EVs at the microscale level.

Microfluidics has enormous potential by reducing sample volume, cost, and reagent consumption, completing reactions faster, and running multiple assays in small devices at the same time.

Additionally, microfluidic systems, on the other hand, are especially appealing for improving the isolation performance of EVs, in part because novel processes may be used and are more efficient in a microfluidic format. In this part, several representative examples of microfluidic-based devices are given, which are categorized into two primary approaches: affinity-based (label-based) and label-free isolation methods.

III.1.1. Immunoaffinity-based microfluidics for EV isolation

Like the widely used immunoaffinity-based EVs separation approach described above, the immunomicrofluidic-based EV separation devices employ antibodies to recognize specific EVs markers. This may be done by covering the inner wall of the micro-channels with antibodies or employing magnetic beads coated with antibodies inside the microfluidic system [221]. Chen et al. pioneered the use of microfluidic affinity methods to extract EVs from human serum or cell culture media using anti-CD63coated microchannels. Furthermore, the researchers were able to recover RNA by adding a lysis solution to the collected EVs on the microchannel surface and flushing with air, demonstrating the method's potential for cancer diagnostics. [222]. Many microfluidic immuno-affinity systems have been developed since then to enhance interactions between EVs and antibodies and they can be divided into two main types: those with an internal surface covered with antibodies and those that use capture beads. Table 2 lists the main immunoaffinity-based microfluidic approaches. Here, we provide two examples to demonstrate the two alternative methods (Fig. 19).

The higher the microchannel's surface area, the more antibodies may be immobilized on the surface, resulting in a higher EVs isolation recovery rate. When Zhang et al. came up with this concept, they decided to utilize graphene oxide and polydopamine (GO/PDA) to create a larger nanoporous structure and greater surface area to immobilize more anti-CD81 antibodies and so capture more EVs (Fig. 19A)

[231]. This device, known as nano-IMEX, exhibited on-chip measurement of ovarian cancer-derived exosomes with a plasma sample volume of 2 µL and a LOD 10 3 -fold greater than benchtop chemiluminescence ELISA. Although immunoaffinity-based EV isolation has been proven to be simple and efficient, these techniques may only capture EVs expressing a particular antigen, possibly ignoring other EV subpopulations present in a biofluid. Furthermore, the binding's high antibody-antigen affinity, which is difficult to break, may be a limiting factor that prevents the release of pure EVs for later usage.

III.1.2. Label-free microfluidic strategies for EV isolation

Many label-free microfluidic approaches for EV isolation have been developed [237,238] The trapping process was relatively fast (~10 min), with a higher yield than ultracentrifugation.

However, the device's recovery performance was shown to decrease with greater sample volumes, most likely due to saturation effects (around 10 10 EVs/mL from 500 μL of human plasma)

III.1.2.2. Viscoelastic-flow sorting

When particles of different sizes move through channels in a viscoelastic medium, they migrate in a as the separation medium and found that exosomes smaller than 200 nm were moved by elastic lift forces along the microchannel sidewalls, but bigger vesicles tend to migrate towards the channel midline (Fig. 21). The authors demonstrated a high separation purity (>90%) and higher recovery (>80%) of exosomes than that of ultracentrifugation (5-25%) from cell culture media [240]. Later, to increase separation efficiency and recovery, they employed λ-DNA as a viscoelastic medium and an aptamer-mediated method to fractionate three distinct EV subtypes (30-200 nm, 200-1000 nm and >1000 nm) [241].

III.1.2.3. Deterministic lateral displacement (DLD)

DLD is a passive microfluidic separation approach that employs a gradient of nano-pillar arrays placed within microfluidic channels to geometrically define the critical diameter (Dc). However, the device requires high pressure due to the dense structure of the network and the restricted experimental time. In fact, the device can function for more than 2 hours before the accumulation of particles at the interface of the input pillar obstructs it and prevents substantial data acquisition.

III.1.2.4. Acoustic separation

Acoustics technique enables a contact-free manipulation of particles on microfluidic devices. This The development of acoustics-based techniques is still in its early stages, but it offers tremendous promise for future applications requiring quick and high-resolution sorting of undamaged EVs.

However, biological fluids include several components with comparable size and acoustic properties to EVs, making acoustic separation difficult.

III.1.2.5. Thermophoresis technology

Thermophoresis is a phenomenon observed in mobile particle mixture in which various particle types respond differently to the force induced by a temperature gradient. Liu et al. were the first to use thermophoresis to create a thermophoretic aptasensor (TAS) to combine thermophoretic enrichment and aptamer-based fluorescence labeling to achieve effective EVs profiling [247]. They created a hightemperature zone using a microfluidic system that can be heated by a laser (Fig. 24). The high charge aptamers had a substantial thermophoretic impact. This technique was able to enrich the aptamer bounded EVs in a matter of minutes while also demonstrating device economy due to the low cost of aptamer production and ease of device manufacturing. The thermophoretic aptasensor was then utilized by the same team to profile cancer-associated protein markers from plasma EVs and forecast the progression of metastatic breast cancer [248]. Recently, Yang et al. also reported PD-L1

(programmed death-ligand 1) aptamer to recognize exosomal PD-L1 indicating significant potential as non-invasive tests for early cancer screening [249].

Microfluidic systems can combine the advantages of portability, cheap cost, small sample volume, high throughput, and high sensitivity into a single device. Despite these benefits, microfluidic devices for EV application are still uncommon. This is due to the numerous difficulties in microfluidic control and microfabrication encountered while creating integrated systems capable of multistep processes.

Researchers have given more an interest in the immuno-affinity technique because of its high specificity thanks to antibody-antigen interaction and wide range of potential applications. These techniques, however, depend substantially on the development of antibodies, which must be both specific to the target of interest and non-interfering with the downstream analysis. Furthermore, not only is it difficult to find specific antibodies for each specific analyte, but it is also necessary to thoroughly investigate the stability of the antibodies and the reusability of the device. Finally, the binding of antibodies to EVs' surface may adversely impact the biological properties of EVs. In parallel

III.2. Electrokinetic-based technologies

The ability of an electric field to isolate and control EVs has recently been used as a contactless particle sorting process in various microdevices. Indeed, when suspended in a neutral medium, EVs have a negative surface charge [250]; thus, negatively charged EVs may migrate at different velocity based on their size, charge, and shape when a voltage is applied. This approach may be used to isolate EVs (for example, through a selective membrane) from other species (e.g. proteins, cell membrane debris) or to separate EVs via a microchannel (capillary or microchip) (see Fig. 25 for schematic example). The following paper (Morani et al., Trends in Analytical Chemistry, 2021 [251]) provides a comprehensive review of electrokinetic-based methodologies for EV isolation, separation, and quantification up to 2020. In this paper, we discussed several instrumental and methodological advancements for such aims, as well as their advantages and disadvantages. was employed to stain both intravesicular and free nuclei acids (NAs). The EVs and free NAs were subject to electrophoresis and separated into two distinct zones. After EVs lysis by SDS, the peak indicating the EV percentage disappeared, and the peak representing NAs increased. This increment represented the total quantity of intravesicular NAs released from EVs. This method offered good sensitivity achieving a detection limit of approximatively 10 6 EVs/mL. However, this method did not provide any electrokinetic information on intact EVs profile.

III.3. Concluding remarks

Microfluidic systems can combine the advantages of portability, cheap cost, small sample volume, high throughput, and high sensitivity into a single device. Despite these benefits, microfluidic devices for EV application are still uncommon. This is due to the numerous difficulties in microfluidic control and microfabrication encountered while creating integrated systems capable of multistep processes.

Researchers have given more an interest in the immuno-affinity technique because of its high specificity thanks to antibody-antigen interaction and wide range of potential applications. These techniques, however, depend substantially on the development of antibodies, which must be both specific to the target of interest and non-interfering with the downstream analysis. Furthermore, not only is it difficult to find specific antibodies for each specific analyte, but it is also necessary to thoroughly investigate the stability of the antibodies and the reusability of the device. Finally, the binding of antibodies to EVs' surface may adversely impact the biological properties of EVs. In parallel to immune-capture based approaches, other techniques focusing on the physical features of EVs have been developed to explore EVs separation. However, passive size-based approaches such as membrane filtration have a low selection resolution. Although, active approaches have the potential to alleviate this issue. Among them, we have emphasized how an external electrical field has been employed for EV isolation, enrichment, and characterisation. Although electrokinetic strategies are not yet capable of fully integrating EV isolation and characterization into a single system, they can do it rather successfully as distinct modules.

Objective of the thesis

As previously stated in the preceding bibliography chapters, purification of EVs from biological fluids with good yields and reproducibility, and production of pure EVs to be used for diagnostic and pharmaceutical purposes remains a key challenge. This is mainly due to the technological difficulties to isolate and characterize such nanometric vesicles. Standardized approaches for accurately quantifying isolated EVs or characterize them in-depth, from biological fluids are currently required for clinical applications. The difficulties that must be overcome for the creation of diagnostic tools based on EVs are significant. It is necessary to standardize and simplify the isolation of EVs from undiluted biological fluids, to detect and quantify all relevant EV biomarkers with high sensitivity and specificity, and finally to passively collect intact vesicles in order to study their structural and biophysical properties in more detail in parallel with proteomic and genomic analysis [254].

In all EV applications, ultracentrifugation is considered the gold standard for their isolation. However, this method has several downsides, including lengthy processes, contamination of EV populations by protein aggregates and other particles, membrane damage, and significant loss of EVs. As a result, developing EVs isolation approaches that may guarantee EV purity and integrity in a repeatable and high-throughput way are still in great demand. Microfluidic systems look especially appealing in this context because they bring new functionalities and new mechanisms to sort or isolate EVs together with a capacity to reduce sample volume, costs, and reagent usage, speed up processes, and run several experiments in small devices at the same time.

Furthermore, the great heterogeneity of EV populations in terms of size and protein content complicates not only their isolation but also their physical and biochemical characterisation. Indeed, various complementary techniques must be carried out to ensure adequate physical and biological information are provided from the isolated EVs or that the extracted population is pure and has expected characteristics. Thus, new EV analysis methods or techniques are still searched to guarantee consistent EV purity and integrity.

In this regard, the goal of the thesis is to create a unique integrated analytical platform able to:

• allow effective extraction of EVs from biological fluids

• profile the distribution and quality of isolated of EV populations

• separate precise EV subpopulations

• extract specific intravesicular proteins of EVs that might be relevant as biomarkers in future diagnosis application and quantify or analyse them.

To achieve these objectives, different methodologies/techniques were developed throughout the course of this thesis with the purpose of ensuring the smooth progression toward the conception of the integrated analytical platform dedicated to EVs. The first panel of investigated methods relies on capillary electrophoresis (CE) which separates charged substances in solution under an electric field and has previously been reported for assessing the physical properties of nanoparticles such as their ζ-potential, surface charge density, particle radius, and size distribution. All the results of these developments are gathered in chapter IV.. The second category of techniques explored the capture and intact release of EVs using both immunoaffinity bead-based kits that utilize specific antibodies against EVs surface proteins and antibody-free magnetic beads that take advantage of their physical features rather. After refining the batchwise technique, it was converted to a microfluidic droplet procedure. Chapter V. compiles all of the outcomes of these advancements.

Faced with the challenge of isolating better EVs, the area of microfluidics as a tool for biological sample preparation is expanding. In order to develop the first module of the platform dedicated to EVs isolation, among all branches of microfluidics, we paid our particular attention to microfluidic droplets.

Droplet-based microfluidics, which consists in manipulating discrete volumes of fluids in an immiscible phase, was chosen to ensure consistent compartmentalization of sample and reagents while simultaneously reducing both the amount of liquid required for analysis and the risk of contamination with the surrounding environment through the use of droplets. Microfluidic droplet systems with magnetic beads as solid support for extracting and purifying target analytes from complex matrices, in particular by using magnetic tweezers technology to transfer the magnetic beads from one droplet to another, have been demonstrated as promising biological sample preparation devices. Traditionally, EVs have been extracted using magnetic beads coated with specific antibodies against EV surface proteins. However, after isolation, such commercial kits, which are more often used for further EVs downstream lysis and analysis, do not provide any effective elution option for recovering intact EVs. In order to overcome this problem, we investigated different antibody-free magnetic beads strategy for capturing and eluting of EVs and in particular different surface chemistries of magnetic beads surface able to retain EVs. These developments relied on a microfluidic droplet technology already available in our laboratory. Furthermore, droplet microfluidics was only reported twice during the course of my thesis for immunoassay-based EV detection and never for high performance and high throughput isolation of EVs.

To develop the platform, we had then to combine the microfluidic collection of EVs to a characterization technique capable of distinguishing and detecting the different EV subpopulations as well as profiling the quality of isolated EVs. To facilitate an easier integration to the little amount of volume generated by the droplet module, we chose CE as it appears to be quite powerful and unexplored option to address this challenge either to analyze intact EVs or specific intravesicular proteins. Furthermore, CE provides several options for efficient analyte preconcentration and, thanks to its varied modes/mechanisms of performing separation and analyses under voltage or pressure, may be tuned to give complementary analytical modes. For the first time, we thus explored one mode of CE: Capillary Zone Electrophoresis (CZE) for profiling intact EVs but also for intravesicular protein analysis. Since conventional DLS and NTA for EVs size measurements are very sensitive to the presence of aggregates, Taylor dispersion analysis (TDA) which has emerged as a promising approach due to its ability to attain size scales ranging from molecule to nanoparticle was also explored for the first time for intact EVs size characterization. TDA can indeed be performed with a conventional CE. This CE-TDAbased characterization module was anticipated to be in the future either directly hyphenated to the isolation module (Droplet-TDA) or on-line to the CZE method, already hyphenated to the isolation module (Droplet-CZE-TDA). A summary scheme of the thesis project is presented below (Fig. 25)

Combining a micro-sized instrument for the controlled capture and release of EVs through magnetic beads with a promising methodology for electrokinetic profiling of EVs and intravesicular proteins, as well as EV size characterisation using CE technology, looks to be an attractive strategy to pursue as an innovative and promising project in terms of diagnosis application.

For the realization of this platform, another thesis project led by Théo Lienard-Mayor (Institut Galien Paris Saclay), aims to develop the instrumentation components, which will require i) a dropletinterfaced capillary electrophoresis (Digital CE) and ii) a system to interface the microfluidic droplet module to digital CE. It has been proven before that protein alterations, exhibited on their surfaces or in their content, may reflect the state and the nature of the generating cells. As a result, EVs are regarded as suitable source of biomarkers in a variety of diseases. Given the focus placed on liquid biopsy in recent years, this characteristic offers up new avenues for EV clinical applications. However, since none of the EV isolation procedures discussed above can combine repeatability, high yield, and high purity, the quality and concentration of isolated EVs must be validated before target intravesicular molecules can be evaluated for diagnostic and prognostic purposes. That is why, studies which investigate the potential of EVs as biomarkers are restricted by the characterization tools currently available, and one of the challenges of this thesis is to contribute to the creation of an appropriate technology or approach. Our analytical platform is indeed intended to allow effective extraction of EVs from biological fluids not only for intracellular protein analysis, but also for characterization of isolated EVs through differentiation of EV subpopulations, as well as to ensure their quality through the detection of potential contaminants.

One challenge with intact EV analysis is related to their very small size, the polydispersity of the EV population, the low refractive index of EVs, and the presence of protein aggregates and other particles contaminants in most isolated EVs samples. Single particle tracking (72%, mostly with NTA) and electron microscopy (60%) are the most often utilized traditional techniques for intact EV analysis [139]. Although robust and effective, they present some disadvantages that have been discussed in the bibliography section. A good approach should be able to identify and characterize EVs reliably and quickly, while also satisfying all of the requirements for EV phenotyping, such as the capacity to distinguish between various size sub-populations and potential contaminants, as well as maintaining

EV integrity throughout analysis. In order to separate such close bioentities, the use of a separative analytical method with great resolving power is essential.

CE is an analytical method for the separation and quantification of a wide range of compounds, which can be described in general as the movement of charged substances in solution using an applied voltage. The narrow tubes (generally between 20 and 100 µm in diameter) that are employed in CE contribute to the high resolution of the method. The use of narrow capillary tubes has the benefit of reducing lateral diffusion effects as well as reducing Joule heating effect upon application of a high voltage, and thus band-broadening. Compared to high-performance liquid chromatography (HPLC), CE does not suffer from band-broadening and wider peaks due to the parabolic velocity profile, as the liquid's velocity through the CE capillary is flat and uniform across the tube. Moreover, the use of CE silica capillaries without packing materials simplifies instrument design and generate less fluidic resistance then packed columns as in HPLC. Additionally, in our project, where we aim to develop a downstream separation approach to be subsequently integrated to the forefront droplet platform for EVs isolation (which will be discussed in the next chapter), CE is the separation approach that is most compatible to the limited sample volumes generated by the droplet platform. For all these reasons, CE was chosen as an intriguing and never explored option for potentially analysing EVs and their subpopulations as well as for a quality control of isolated EVs. In parallel, thanks to the ease with which

Taylor dispersion analysis (TDA) can be set up on a commercial CE device by applying pressure to the capillary's inlet rather than an electric field, and the ability to determine the size of ultra-small NPs (< detection was investigated rather than UV, to bring more specificity in the obtained profiles but also sensitivity. As CE is already considered as a powerful techniques for protein analysis [256,257], we aimed to use it also for potential analysis of specific intravesicular proteins. We used LIF for both objectives in order to get high sensitivity in both the proteins and EVs that were analyzed. However, proteins must be fluorescently labelled; the most common strategy to label proteins is via their The first part of this experimental chapter IV., which is dedicated to all investigated strategies relying on CE, will briefly present the fundamentals and some theoretical aspects of CE. The second part will present our achievements in improving the performance of CE-LIF in terms of stacking preconcentration and separation resolution of fluorescently labelled proteins and peptides. In the third part, our first achievements in establishing a solid background for the electrokinetic profiling of EVs is presented. In the fourth section, effective strategies for in-capillary sample preconcentration for EVs are explored in order to further increase the sensitivity of regular LIF detection. Finally, the TDA applicability to EVs was explored for precise size evaluation and our preliminary and encouraging results are presented in the last section of this chapter.

IV.2. Principle of capillary electrophoresis (CE)

EVs have been shown to be high in negatively charged phosphatidylserine [261,262]. As a result, by applying a voltage, negatively charged EVs may migrate at varying velocities depending on their size, charge, and shape. In chapter III. of the bibliographic part of this thesis, I highlighted how this mechanism has lately been exploited, most notably as a particle sorting method in various microdevices to separate and drive the EVs. However, when I began my thesis, no publication had mentioned the possibility of using CE to analyze EVs or distinguish EV subtypes based on their electrophoretic migration. During the course of my thesis, four CE methods for analyzing and quantifying EVs have been disclosed [252,253,255,263]. However, only two of these have been proposed for the investigation of intact EVs. Before investigating the power of CE to address two of our aims (i) develop a CE approach capable of assessing intact EVs after their isolation in order to deliver information on the diverse subpopulations of EVs and (ii) assess their protein internal content, which may be employed as biomarkers in the future once validated; this first part is intended to provide the fundamentals of electrophoretic migration and some theoretical aspects of CE, which will be necessary in order to better understand the achievements achieved and show the potential for CE in the area of EVs.

Typically, in CE (see Fig. 26) performed with voltage application, the sample is injected into a capillary filled with the background electrolyte (BGE) serving as the separation medium and then a high voltage is applied over two ends of the capillary (immersed in a BGE containing reservoir) to trigger the separation of target molecules. On-capillary detection via an optical window created directly on the capillary contributes to the great efficiency found in CE because the separation continues to occur while analytes passes through the detection window, and there is no broadening of the zone due to dead volume, as otherwise encountered in post-column detection.

IV.2.1. Electroosmotic flow (EOF)

The fused silica capillaries that are typically used for separations have ionizable silanol groups (Si-OH) which are negatively charged (SiO -) when the pH of the BGE filled inside the capillary is superior to 3.0 (see Fig. 27) [265]. Under the action of an electric field, the cations of the diffuse layer are attracted to the cathode, while the cations of the compact layer are retained by strong electrostatic interactions binding them to silanol groups. This movement of the outer layer, which causes all the compounds present in the electrolyte to move toward the cathode, is called the electroosmotic flow (EOF). This displacement has a flat profile, unlike pressure separations which have a parabolic velocity one (liquid chromatography) that limits diffusion during separations and allows CE to achieve high efficiencies. The electroosmotic flow, can be characterized according to equation 2 by the electroosmotic velocity νeo (m.s -1 ), dependent on the electroosmotic mobility μeo(m 2 .V -1 .s -1 ) and the applied electric field E (V.cm -1 ):

(

) 2
Where η corresponds to the viscosity of the medium (Pa.s). The electroosmotic mobility depends on the medium (composition, pH, ionic strength, viscosity of the BGE) as well as the surface chemistry and nature of the capillary. Note here that it is possible to modify this surface by specific treatments, such as covering with polymers modifying the surface properties.

IV.2.2. Electrophoretic mobility (μep)

Electrophoretic mobility corresponds to the displacement of a charged species under an electric field

[266]. This species will be attracted towards the electrode of polarity opposite to its charge at the velocity νep (m.s -1 ), dependent on the electrophoretic mobility of the species μep (m 2 .V -1 .cm -1 ) and on the electric field applied E (V.cm -1 ) described in equation 3:

(3)

The electrophoretic mobility is specific to each compound and the physicochemical properties of the electrolyte. In case of infinite dilution, the electrophoretic mobility μep of a species is dependent on its hydrodynamic radius Rh as well as on its charge q, therefore more generally on its charge density (equation 4). At equal charge, for example, a small cation will move faster than a large one.

(

) 4
CZE is one of the most frequent and simplest of the CE modes and is often used for the study and separation of a wide variety of compounds, including proteins and NPs. The migration of the species to be separated under the effect of the electric field is governed by their charge density. The higher this density, the faster the species will be attracted towards the electrode of opposite polarity. In the case of the analysis of peptides and proteins having ionizable groups, the pH of the electrolyte therefore plays a major role since it determine the charge state of the peptide/protein according to titration curve. At the same time the pH impacts the electroosmotic mobility. The EOF is substantially bigger at high pH, where the silanol groups are largely deprotonated, than at low pH, when they get protonated. Thus, at high pH, the EOF may be excessively strong, resulting in species elution before separation. In CZE, the migration of a species is characterized by its apparent mobility μapp, the sum of electroosmotic mobility and electrophoretic mobility [267] practically measured using Eq5.

(

) 5
Where L is the total length of the capillary (cm), l the length to the detector (cm), V the applied voltage (V) and tm the migration time of the species (s). Figure 28 shows the direction and intensity of mobility of each type of species during separation. Note that it will only be possible to detect an anion if the electroosmotic mobility is greater in absolute value than its electrophoretic mobility and that neutral compounds or particles cannot be easily discriminated from each other. However, electrophoretic methods have some drawbacks. Adsorption onto the walls of fused silica capillaries owing to electrostatic or hydrophobic interactions is a well-known limitation in CZE protein analysis. Protein adsorption causes poor repeatability, lower detector response, decreased resolution and separation efficiency. Similar to proteins, the problem with EVs may be exacerbated by the presence of various amine functionalities present on EVs membrane proteins which can interact with negatively charged silanol groups on the capillary surface. Another issue of CE is its poor detection sensitivity due to small sample injection volumes (some nL ranges in CZE mode) and limited detection pathlengths across the capillary section when working with optical detection modes, which may be troublesome when working with biological materials such as EVs or their protein contents at low concentrations. In the following section, I will present a strategy based on the selection of appropriate BGE to overcome these issues as well as the results achieved for CE-LIF analysis of proteins and, subsequently, EVs.

IV.3. "Inorganic-species-free" BGE (ISF BGE) for capillary electrophoresis-laser induced fluorescence (CE-LIF) performance improvement

In this thesis project and for all CE-based developments, LIF detection was preferred over commonly used UV detection because of its higher sensitivity and selectivity for the detection of target analytes, which is especially crucial for achieving our final goal to analyze and investigate intravesicular molecules (mostly proteins) in the pM range. The detection sensitivity for target proteins, is heavily dependent on their amino acid composition, and in part on the intrinsic fluorescence of tryptophan, tyrosine, and phenylalanine. As these amino acids with intrinsic fluorescence are most often not abundant in a protein sequence, proteins and peptides must be labeled with a fluorescent tag to allow for sufficient sensitivity of detection. Depending on the desired applications, there are many markers that can be used to derive proteins [258]. The choice of a fluorophore to label target proteins/peptides is normally based on its extinction coefficient, emission efficiency, excitation/emission wavelengths, as well as the functional group present on the protein for covalent attachment. The most popular technique is to target cysteine and amine groups with direct chemical labelling. Cysteines provide the most flexibility in terms of labeling position. Lysine residues or N-terminal amines may be labeled using amine-reactive conjugates. Labeling a particular protein which bears multiple target residues, on the other hand, results in a number of distinct labelled protein species, each with a different number of tags and consequently a varied electrophoretic mobility. As a result, it is critical that the CE conditions employed should ensure efficient separation of bound and non-bound labeled reagents (i.e., tagged target proteins and residual fluorophores in our particular case) as well as simultaneous analysis of several proteins and their possible multiple tagged species. We chose rather to reinvestigate BGE compositions to achieve this goal. Indeed, BGEs for CE-LIF separation are often derived from those optimized for CE-UV separation, and little attention has been devoted to the development of proper BGEs for CE-LIF. Based on this logic, we investigated a new BGE selection strategy to improve the performance of CE-LIF in terms of stacking preconcentration and separation resolution of fluorescently labeled proteins and peptides. Beta amyloid peptide (Aβ 1-42) and trypsin inhibitor (TI) were utilized as model of peptides and proteins, respectively, for this study. Following that, to assess the resolution enhancement, we worked on ovalbumin, which may be tagged at several spots and to varied degrees and is also a more complex protein, being glycosylated and so existing as a mixture of glycoforms.

Actually, one of the long-term goals of this thesis was to bring new information and also to possibly

IV.4. CE-LIF method development to study the electrokinetic distribution of EVs

CE appears as an intriguing and little explored option for high performance profiling EVs and possibly distinguishing EV subtypes based on their electrophoretic behavior. When I started my thesis CE was not yet explored for intact EVs analysis at all. Furthermore, the volume of sample solution generated by the droplet platform intended to isolate the EVs at the forefront, which will be detailed in the next chapter, is compatible and combinable with this microscale electrophoresis technique. The main challenges encountered with CZE analysis of EVs is to avoid during the electrophoretic process their lysis, their adsorption to the capillary wall and formation of cluster or aggregates. Furthermore, when dealing with EVs extracted from CSF or plasma fluids, a high detection sensitivity is desirable, since their concentrations might be very low. For this reason, LIF was selected as the detection mode for

EVs. The solutions that we developed to solve these difficulties, as well as the accomplishments gained using CZE for isolated EVs, will be discussed in detail in this part.

First, the above-presented ISF BGEs , which we demonstrated to improve the performance of CE-LIF for proteins and peptides in terms of improved peak resolution, sharper and higher peaks, and low electric conductivity [268], were further exploited to develop a CZE method prior to LIF detection of EVs. These BGEs were expected to satisfy several requirements for CE analysis of EVs, such as avoiding lysis during the electrophoretic process, avoiding adsorption of EVs to the silica capillary wall, and avoiding the formation of numerous spikes due to unwanted EV clusters or aggregates.

Prior to CE-LIF, EVs must be labeled with a fluorophore. In this context, it is important that the fluorescent dye used, label exclusively EVs, has a low background signal in its free form, lead to a high signal-to-noise ratio, and can preserve EVs' innate biological activity as well as their physical properties and integrity. EVs may be tagged with a variety of fluorescent dyes in different ways: by labeling proteins on the membrane surface, by intercalating dyes into the EVs lipid structure, or by labeling the EVs lumen [269]. Among these, we exploited the fluorescent luminal labelling using a membrane permeable dye that become fluorescent and EV-impermeable in its hydrolyzed forms following cleavage by internal EVs enzymes. Our objective was to develop a EVs labeling strategy while maintaining the EVs' natural physical characteristics by preventing size and charge modification after EVs fluorescent tagging, which otherwise would result in unwanted changes in their original electrophoretic mobilities.

The EVs to a certain extend can be considered (bio)nanoparticles. Therefore, before going into the details of the method's development for electrophoretic separation of EVs shown in the published article 3 (Morani et al., Analytica Chimica Acta, 2020 [270]), the relation between the electrophoretic migration behavior of nanoparticles (NPs) and their physical and chemical characteristics is discussed here, based on the background already established for CE of NPs.

Nanoparticles are generally defined as particles having one or more dimensions in the nanoscale (around 1 -100 nm). CE is a relevant method for analyzing and characterizing NPs (e.g. noble-metal, metal oxide and polymeric NPs) [271]. It allows separation of particles according to their size and zeta

where e is the elementary electric charge, NA the Avogadro's number, I the ionic strength of the medium, εr the relative electric permittivity of the electrolyte, ε0 the electric permittivity of vacuum, kB the Boltzmann constant, T the absolute temperature. κ represents the thickness of the electronic double layer that forms on contact between the charged surface of the NP and the ions present in the electrolyte.

In the case where κRh ≪ 1, in example when the size of the particle is small compared to the thickness of the double layer, the Hückel equation is valid:

(7)

In the case where κRh ≫ 1, i.e., when the electronic double layer is negligible compared to the size of the NP, the Smoluchowski equation is reached: However, the dependence of NP µep on size, charge, or even charge-to-size ratio cannot be generalized.

Before establishing a clear association between electrophoretic mobilities of NPs and one of their

IV.5. Development of in-line electrokinetic preconcentration prior to CE-LIF separation of EVs

In comparison to the CE-UV methods recently developed for the electrophoretic profile of EVs [252,255], our proposed CE-LIF approach provided superior detection limits. However, improved detection limits are still needed when dealing with low concentrated EVs samples. This section therefore focuses on our efforts to develop in-capillary, online, and automated electrokinetic preconcentration of EVs.

In-line electrokinetic preconcentration of analytes is based on variations in the analyte migration velocity across discontinuous solution/electrolyte interfaces that are created inside the capillary. As a result, the analytes dissolved in a sample matrix distinct from the BGE are accumulated (stacked) at the boundary of the two solutions, resulting in electrokinetic preconcentration of target analytes.

Numerous in-line preconcentration approaches have been developed to date, notably large volume sample stacking (LVSS), isotachophoresis (ITP), Field amplified sample stacking (FASS), field-amplified sample injection (FASI) and dynamic pH-junction [282,283]. However, special attention should be given to EVs when considering the appropriate preconcentration strategies due to their possible instability. We have indeed to preserve their integrity and avoid any agglomeration/aggregation or lysis of EVs during the preconcentration process under high electric fields. Furthermore, for some of these methodologies to be applied to EVs, a deeper understanding is necessary. For example, ITP concentrates the sample between a leading electrolyte (LE) and the terminating electrolyte (TE), which have higher and lower mobility, respectively. However, predicting the precise electrophoretic mobility of migrating EVs at the stacking zone is challenging. Dynamic pH-junction, which operates when an analyte's ionisation state changes when it is moved to a different pH zone in the capillary, necessitates a thorough investigation of the surface charge of EVs as pH varies. Furthermore, a sudden rise or decrease in pH might affect the stability of the EV membrane. While electrokinetic preconcentration of NPs have been reported [284], this approach has never been applied to EVs which are more challenging and fragile entities than NPs. As field-amplified stacking and LVSS approaches were already successfully applied for NPs [284], we first explored electrokinetic preconcentration of EVs using these two strategies. Field-amplified stacking methods (FASS, FASI) rely on the utilization of samples prepared in a matrix (e.g, water or diluted BGE) having lower ionic strengths (thus lower conductivity) than that of the BGE. Due to the inverse relationship between the electric field (E) and conductivity (σ)

(𝜎 = 𝐽 𝐸
where J is the magnitude of the current density), and considering Eq. 3 from the introduction of this chapter (ν = µ x E), analytes move quicker inside the sample zone where the electric field is greater and are slowed down when they approach the sample/BGE interface. A decrease in buffer conductivity may be accomplished by lowering the ionic strength (IS =1/2 ∑i ci zi 2 where zi is the valence of ion i and ci is the concentration). Our preliminary tests with FASS of EVs did not lead to significant signal improvement (article 3, paragraph 3.3 in the publication). Indeed, we demonstrated the need of maintaining physiological and isotonic conditions during electrokinetic preconcentration (FASS) and separation in order to minimize EV lysis and aggregation/collision. Such conditions are nevertheless not favourable EVs stacking preconcentration.

We then diverted our effort to develop an in-line preconcentration technique based on larger sample volume injection (LVSS). In general, in CZE, the injected sample volume should not exceed 1 to 3 % of the capillary volume in order to obtain a good separation efficiency. During the method development for CE-LIF of EVs, we indeed employed large sample volume injection (10% of total capillary volume)

to boost the sensitivity of EVs detection (paper 3, paragraph 3.2). We then explored further this direction with single and multiple cycles of LVSS and provide here our pioneering results in the LVSS preconcentration of EVs.

IV.5.1. Large volume sample stacking (LVSS) with polarity switching of EVs

First, we chose to test the LVSS approach, which allows for the injection of very large sample volumes, up to 100 % of the capillary volume when the electrophoretic mobility of the analytes is opposed to the EOF [283], which is the case in the CZE-LIF method that we developed (Figure 29). The capillary is filled with a large volume of sample having a low conductivity and then voltage is applied so that the elevated EOF in the sample zone is directed towards the injection side of the capillary, which will drive the sample matrix out of the capillary. At the same time, the difference in conductivity between the sample matrix and the BGE will induce an enrichment of analytes at the boundary of the two media, as in FASS. During this step, the current increases as the sample matrix is gradually replaced by the BGE and the analytes are pushed towards the entrance of the capillary. When almost all the sample matrix is removed from the capillary, i.e. when the current intensity is between 90% and 95% of the maximum intensity obtained for a capillary filled with 100 % BGE, the voltage is reversed and the classical separation method is then applied. In this study, the LVSS procedure was applied to a sample of bovine milk-derived EVs previously purified by Excilone and considered here as a standard. Details for EVs analysis can be seen in our previous article 3 (paragraphs 2.3.4, 2.3.5, 2.3.6). Briefly, fluorescently labelled EVs were prepared using the 5 As a reminder, the EOF is considerably influenced by the counter ions in the buffer electrolyte, since the ionic strength of the counter ions affects the zeta potential (see Eq. 1, subchapter IV.2.1. of this chapter). As a result, when working with low IS sample matrix, the local generated EOF is high, and this is required to achieve preconcentration. But at the same time lysis of EVs may occurred due to the too low ionic strengths (lower than isotonic conditions). This may be the origin of unwanted spikes induced by EVs lysis, and thus poor EVs signal in Fig. 30. Then, sample matrices composed of Tris/CHES at higher IS (up to 50 mM) were also examined, but unsatisfactory data were obtained (data not shown). On the other hand, the BGE employed (Tris/CHES, IS 140 mM, pH 8.4), which may be advantageous in producing high IS without generating high current as previously showed in our paper 2 (see subchapter IV.3. of this chapter) cannot be used to generate a high enough EOF required to implement successfully LVSS. Indeed, the high concentrations of the large weakly charged molecules constituting the BGE led to significant reduction of EOF. As a consequence, Fig. 30 shows that the EVs' peaks are still wide and that the longer the EVs are exposed to high voltages, the more the spikes on the EVs' peaks may be seen, reflected by more spikes observed on the second haft of EVs peak. In an effort to address these challenges, we tested two strategies: i) the addition of sugars to the sample matrix (i.e., diluted BGE to maintain a low ionic strength required to generate a high EOF magnitude) with the goal of stabilizing the EVs without incurring high conductivity of the sample zone; and ii) the use of the optimized ISF BGE Tris / CHES (IS 90 mM, pH 8.4) for both preconcentration and CZE steps which was demonstrated in our paper 3 to achieve a higher EOF while maintaining a sufficient resolution between the EVs and the CFSE fluorophore. A combination of sucrose and dextrose was recently demonstrated to maintain the osmolarity and physiological condition in a microdevice to capture EVs across a porous dialysis membrane under application of electric field [285]. From this rationality, such composition was tested for our LVSS study. Thus, with the use of commercial Exosome Spin Columns, the EVs were resuspended in Tris / CHES 5 mM with the addition of i) sucrose (30 mM) in combination with dextrose (145 mM); ii) mannose (from 100 to 200 mM) and iii) trehalose (from 25 to 200 mM). As can be seen in Fig. 31A for preliminary test in this direction using the combination of sucrose and dextrose EVs peak was still broad, but in turn provokes much less spike-reflected aggregation. Fig. 31B shows the results obtained using a mannose concentration of 100 and 200 mM. Also here, less spike-reflected aggregation was observed but EV peak was still broad. A concentration of 200 mM was found to slow down the rate of EVs lysis probably thanks to the sugar concentrations being closer to isotonic conditions, resulting in a higher signal for EVs comparing to that obtained with 100 mM of mannose.

We then extended our study with the addition of trehalose (consisting of two molecules of glucose)

since it has been shown to minimize EV aggregation and maintain EV integrity throughout freezing and thawing [286]. As clearly seen in Fig. 31C, the EVs signal increased significantly with much less spikes when the trehalose concentration was increased from 25 to 200 mM. The best signal for EVs was obtained using a sample matrix comprised of Tris/CHES IS 5 mM and trehalose 200 mM and the BGE composed of Tris/CHES (IS 90 mM, pH 8.4). Peak intensity was increased in comparison to our previously published CZE-LIF approach that did not include a preconcentration phase (see the dashed electropherogram in Fig. 31C). In terms of peak heights, trehalose enhanced the EVs signal by nearly 7-fold, and the enhancement was only 2-fold using sucrose/dextrose or mannose. Thus, trehalose proved to be the best likely owing to its better ability to maintain sufficient repulsion by shielding attractive interactions between vesicles [286]. Note also that when using the optimised sample matrix containing trehalose, the EVs peak obtained was still much broader than that obtained with CE-LIF of EVs without the preconcentration. This may be due to residual trehalose that may increase buffer viscosity, impairing the hight EOF mobility required to assure baseline separation of enriched EVs.

IV.2. Online Preconcentration in Capillaries by Multiple LVSS of EVs

Following a single LVSS, the approach was extended to multiple cycles of electrokinetic preconcentration as shown in figure 32, in order to further increase detection sensitivity, as inspired by a similar approach previously described by our group [287]. This protocol, which relies on repeated cycles of injection of very large sample volumes for analyte stacking and matrix removal using an EOF directed to the inlet capillary, was investigated to enable in-capillary enrichment and separation of EVs from unlimited sample volumes. For multiple LVSS, we performed the same process for LVSS for the first injection (100 % of total capillary volume) and the applied voltage was kept the same (25 kV). In this case, when almost all the sample matrix is removed from the capillary, unlike classic LVSS, a second sample injection is carried out, followed by another step of matrix sample removal using an EOF toward the inlet capillary. These steps in theory may be repeated several times (cycles) for further The obtained results indicates that the detection sensitivity was comparable to that of a single LVSS.

This may be attributed to sample loss caused by the repetition of cycles. Indeed, it is possible that during the preconcentration step, i.e. the reverse polarity is applied when the current intensity reaches 90-95 % of its maximum value obtained when the capillary is completely filled with buffer (namely 21 µA), part of the EVs inside the capillary are not fully preconcentrated at the capillary inlet due to their lower µep. Moreover, the profile obtained shows a loss of resolution between the peaks of the EVs and excess fluorophore (CFDA-SE). To better understand this observation, a second test was conducted using just two cycles. We performed a sample injection, filling 100% of the capillary and applied the process of LVSS. Then a second injection of the sample but this time of 50% of the capillary volume was applied and the same process of preconcentration was 4done before CZE separation. However, as can be seen in Fig. 33B, there was no difference between a 2 or a 3 cycle LVSS preconcentration in term of signal intensity for the EVs peak. This confirmed our observation that a portion of the sample is stacked from the second cycle of preconcentration.

A loss of resolution was noted once again. When using the optimised sample matrix containing trehalose, the EVs peak obtained was still much broader than that obtained with CE-LIF of EVs without the preconcentration. This loss of resolution is most likely caused by the existence of a rather significant electroosmotic flow (EOF) (2 x 10 -4 cm 2 .V -1 . s -1 ), during electrophoresis, which requires manual switching of polarity to trigger the electrophoretic separation of enriched EVs. This resolution lost is expected to alleviate if EOF can be quasi-totally suppressed to allow EVs to electrokinetically migrate against the EOF, thus does not require polarity switching between the LVSS and CE separation steps. Furthermore, the presence of a significant amount of free fluorescent dye (CFDA-SE) was indicated in these studies by the reappearance of fluorophore signals in the electropherograms as a consequence of sample degradation over time. Indeed, this behaviour was already documented in our Paper 3. As a result, for the moment unsatisfactory results were obtained with our preliminary tests on EVs electrokinetic preconcentration with LVSS.

IV.3. Concluding remarks

Among current CE methods for EV electrophoretic profiles, our CE-LIF approach has the best limit of detection to date (around 10 10 EVs/mL). Efforts were made to improve the sensitivity of EVs detection via development of a forefront in-line electrokinetic preconcentration step. However, all the electrokinetic methods for in-capillary preconcentration that we developed have not yet overcome the hurdles encountered when working with EVs. Indeed, as previously noted, finding a compromise between high performance EVs stacking (via an EVs sample matrix with a conductivity significantly lower than that of the BGE), and avoidance of EVs lysis under such extreme conditions (low or high conductivities) remains a difficult requirement. We have to keep in mind that if low conductivities can destroy the EVs, high conductivity media can induce joule heating during electrophoresis that leads to damage of EVs as well. From this rationality, capillary isoelectric focusing (cIEF, for separation of target analytes into bands along a pH gradient in a capillary induced by an electric field in the presence of ampholytes) is expected to be a more compatible separation and preconcentration approach for EVs.

Exploration of cIEF for EVs enrichment and separation is envisioned in the next phase of the project.

Furthermore, other principles might be investigated, notably ITP that concentrates the sample between a leading electrolyte (LE) and the terminating electrolyte (TE), which have higher and lower mobility, respectively. However, predicting the precise electrophoretic mobility of migrating EVs at the stacking zone might be challenging.

IV.6. Size-based characterization of EVs by Taylor Dispersion Analysis (TDA)

It is generally established that EVs are highly heterogeneous in size, but it is difficult to distinguish between true heterogeneity and that caused by impurities, aggregated or debris of EVs, and this is strongly related to their isolation mode, which is addressed in the second chapter of the thesis, but also to the characterization methods. Most EVs have a diameter of 100 nm or more, but exosomes are considered to have a smaller size between 30 and 100 nm. Nevertheless, the top size limit of EVs is still unknown [288]. As previously indicated in Chapter II.2. of this thesis, employing DLS and NTA to determine the distribution of EVs is a common method. While these methods are simple to apply, the results may be deceptive, since NTA and DLS are very sensitive to the presence of aggregates. previously. Based on this logic, this technique, which can be implemented on a CE apparatus, is particularly compatible with our aim to build a micro platform for the separation and analysis of EVs.

Indeed, following the isolation of the EVs via the droplet system module, that will be described in the following chapter, this technique could represent a characterization module to be coupled either directly to the isolation module (Droplet-TDA) or on-line to the CZE method (Droplet-CZE-TDA). We have indeed seen that the CZE method is currently limited for the size elucidation of the separated subpopulations. The TDA-CZE hyphenation would allow the determination of the diffusion coefficient (or hydrodynamic radius) of each EV zone previously separated by CZE. Furthermore, the amount of sample solution required with TDA is substantially small (2 μL for TDA versus 1 mL for DLS/NTA), which represents an advantage for sample volumes of EVs generated by the droplet platform (only some μL range).

In this section the principle of TDA will be given, followed by preliminary experiments to explore, for the first time, TDA combined with UV and LIF detection to estimate the size of standard EVs. A comparison of data obtained for different EVs sources to NTA results is also performed and discussed.

TDA experiments were carried out in collaboration with Hervé Cottet and Joseph Chamieh (Institut des Biomolécules Max Mousseron, Montpellier).

IV.6.1. Principle of Taylor Dispersion Analysis (TDA)

The source for the theoretical portion of this part regarding the TDA is Lucie Labied's doctoral thesis, publicly defended on 17/12/2021 (http://www.theses.fr/s218954). Recently, Moser and Baker also provided an intriguing instructional tutorial review of TDA, aimed to help analytical chemists and separation scientists better understand and use this sizing approach [294].

Taylor dispersion is a phenomenon initially reported by Sir Geoffrey Taylor in 1953 [289, 295, 296]. It Taylor dispersion analysis is valid if the axial diffusion can be neglected [296]. The Péclet number (Pe), which describes the relative rates of mass transfer along the axis of the capillary due to convection and diffusion [291,297], must be chosen such that [298]: (9) where u is the linear velocity of the fluid, Rc is the capillary radius, D is the molecular diffusion coefficient. The dimensionless diffusion time τ, defined as the ratio of the mean solute residence time to the time necessary for a solute to diffuse over a distance equal to the capillary's radius [291,297], must be chosen in such a way that [298]: (10) where t0 is the residence time of the peak in the capillary.

Only radial diffusion occurs under these circumstances and relative error on the determination of D is lower than 3% [298]. The species in the capillary's core disperse towards the walls, where their speed decreases. Similarly, species near the walls disperse and accelerate towards the centre. These diffusion phenomena cause the speed of the species in solution to be distributed around the mean speed. The signal detected at the output of the capillary (typically at the detection window of one capillary when using a CE instrument) as a function of time can then be assimilated to a Gaussian peak and is called taylorgram. A schematic representation is given in figure 35. Small molecules diffuse rapidly, resulting in a small deviation around the average speed, and therefore t0. The detected peak is then thin. In contrast, the diffusion of a bigger analyte, such as a protein or NP, is slower, thus the observed peak is wide. The peak of a taylorgram for a solution containing a molecule with a diffusion coefficient D can be modelled by a Gaussian: (11) where S is the signal as a function of the analysis time t, y0 the baseline, A the area of the peak, which depends on the detection and the concentration, σ the temporal variance of the peak, t0 the average residence time peak.

The molecular diffusion coefficient D may thus be represented as a function of the Gaussian parameters t0 using the following equation: ( 12)

IV.6.2. From diffusion to size determination: experimental considerations

TDA was originally proposed as a method for determining the diffusion coefficients of small molecules, however the Stokes-Einstein equation allows for a direct relationship between D and the size of a given spherical analyte present in a diluted sample: (13) where kB is Boltzmann constant, T the temperature, η the fluid's viscosity, Rh the hydrodynamic radius.

The hydrodynamic radius is defined as the radius of a theoretical sphere with the same diffusion coefficient as the object under consideration (molecule, NP, protein, etc.).

In the 50's and 60's, analytical tools did not meet the criteria of validity to study analytes or objects bigger than molecules. However, thanks to improvements in capillary tube fabrication and in instrumentation, the validity conditions of equations ( 9) and ( 10) could be met for macromolecules, particles or assemblies with smaller diffusion coefficients, and thus bigger objects. Nowadays, TDA may be set up simply on a commercial CE device equipped with UV-vis detection by applying pressure to the capillary's inlet instead of applying an electric field.

The experimental limits of TDA have been explored and deepened by the work of Chamieh et al. [299].

For a given hydrodynamic radius Rh, the authors summarized the conditions of equations (9 and 10) in a unique inequality that relies solely on the applied pressure and capillary dimensions: (14) where Lc is the total length of the capillary and Ld the capillary length to the detector, ∆P the mobilization pressure, Rc is the capillary radius and Rh the hydrodynamic radius. This inequality makes it possible to draw graphs of log∆P = f (logRh) for a given type of capillary. Figure 36 shows an example of a capillary with an internal diameter of 75 µm and a length of 70 cm, where the green zone indicates zone where the Taylor regime occurs meaning that the conditions of the equations ( 9) and ( 10) are respected. This working zone may be changed by adjusting the capillary's geometric parameters: a larger object can be measured with a thinner or longer capillary. However, there is a limit for oversized objects. If the chosen capillary diameter is too small for the species considered (Rh / Rc ≥ 0.0051), the Taylor dispersion takes place in a hydrodynamic chromatography regime. Large species are excluded by the capillary walls under these circumstances, and the sample "sees" a smaller capillary, which distorts the size predicted from equations ( 12) and ( 13). For a capillary with an internal diameter of 75 µm, it is possible to measure sizes up to a diameter of the order of 382 nm with less than 3% error due to the hydrodynamic chromatography regime [299].

TDA was used by Belongia and Baygents to determine the diffusion coefficients of large species like tobacco mosaic virus (TMV), a rodlike virus with a length of 300 nm and a diameter of 18 nm, as well as hydrophilic and hydrophobic polystyrene latex and hydrophobic polymethyl methacrylate particles ranging in size from 0.19 μm to 0.83 μm [290]. The authors demonstrated, using a wax-coated (50 μm diameter) and a polyethylene glycol-coated (50 μm diameter) capillary, all the care that must be taken to suppress interactions between the particle and the capillary walls if the dispersion results are to be interpreted using traditional TDA theory. Using didodecyldimethylammonium bromide (DDAB) coated technique for the analysis of mixtures of nanoparticles of comparable size. Furthermore, Labied et al.

[304] recently established a unique coupling, TDA-inductively coupled plasma-mass spectrometry (ICP-MS), that enabled the direct determination of the size and stability of ultra-small Gadolinium NPs (sizes less than 5 nm) in complex biological fluids (i.e., blood serum, urine, cerebrospinal fluid)

In view of the literature reporting successful TDA analysis of virus, organic or hybrid or inorganic particles, TDA appears as a promising alternative for the size characterization of EVs. Indeed, TDA is an absolute approach (no calibration required) for determining the molecular diffusion coefficient, D, and the hydrodynamic radius, Rh, with no size bias because the contribution of small and large solutes is proportional to their mass abundance in the mixture.

Herein, TDA was tested for the first time for the size characterization of our high-quality EVs isolated from bovine milk with narrow size distribution, which were given by our commercial partner Excilone (France). As a reminder, NTA, DLS, LC-MS / MS, and TEM data are available in the supplementary information of our CZE-LIF paper in section IV.4. of this chapter. TDA-UV and TDA-LIF using CE were both investigated for this purpose for native EVs and EVs fluorescently labelled using the method reported in our paper in section IV.4. of this chapter. with the following sequence: water, 1 M NaOH, 1 M HCl and then water, for 10 min each. Between the measurements, the capillaries were flushed for 5 min each with 50 mM SDS, 1 M NaOH, deionized water, and finally the running buffer using a pressure of 30 psi. EVs were introduced by pressure injection (0.4 psi) for 9 s (0.96% Vol to detector). Experiments were performed using a mobilization pressure of 0.4 psi. TDA-UV and TDA-LIF were performed with bovine milk-derived EVs isolated with sucrose gradient ultracentrifugation and human plasma EVs with monolithic disks via immunoaffinity chromatography. The techniques for EV separation and fluorescent labelling, as well as the matrix replacement approach for recovering EVs in PBS 1X and removing excess residual fluorophores, can be found in our paper, which is described in section IV.4. of this chapter.

IV.6.3. CE procedure

Taylorgrams were recorded using karat 32 software and then were exported to Microsoft Excel for subsequent data processing. The deconvolution of the taylorgrams with a finite number of Gaussian functions and the data fitting were conducted using the Least Significant Difference method using the "GRG nonlinear" in Microsoft Excel.

IV.6.4. Results

IV.6.4.1. TDA-UV and TDA-LIF of bovine milk derived EVs

To evaluate the performance of TDA under physiological conditions (i.e., neutral pH with high salt concentrations), the TDA-UV approach was initially tried on high pure EVs extracted from bovine milk using sucrose gradient ultracentrifugation and suspended in PBS 1X buffer. Unmodified silica capillary were used with 1X PBS as the mobilization buffer. Figure 37 shows the taylorgram obtained for bovine milk-derived EVs diluted to a concentration of 2 x 10 12 EV/mL in PBS 1X. Interestingly, here no adsorption onto the capillary wall was detected, as evidenced by the symmetric signal. Diffusion coefficients were provided by inserting σ and tR values into eq 12, and hydrodynamic radii were calculated using the Stokes equation (eq13). The Taylor dispersion analysis showed a Rh of 96.5 nm. The agreement between our experimental results and NTA values was satisfactory (see table 3).

In addition to UV, TDA was coupled to LIF to detect fluorescently labelled bovine milk-derived EVs using the CFDA-SE dye already reported for the CZE-LIF method presented in section IV.4. of this chapter.

According to NTA data, the intra-membrane labeling approach, resulted in no significant changes in the size distribution of EVs. Indeed, NTA data showed only a very slight size shift towards smaller particles when EVs were labelled (Dh: 171 nm ± 42 nm before vs Dh: 157 nm ± 37 nm after the labelling; see Fig. S2 from paper 3) that was rather attributed to partial EVs lysis or leakage of the covalently bound fluorescent dye over long time, as previously evidenced with the CE-LIF method (see Fig. S6 from paper 3). TDA-LIF, on the other hand, was able to distinguish two distinct Rh populations. The taylorgram for fluorescently labelled bovine milk-derived EVs diluted to a concentration of 1.05 x 10 12

EV/mL in PBS 1X is shown in Figure 38. By performing a fit of the experimental trace with a sum of two Gaussian functions, two radii could be simultaneously determined. A first population was sized at 111 nm Rh (~ 75% of the signal), which corresponds to the EVs, while a second smaller population was sized at 0.74 nm Rh (~ 25% of the signal), which most likely corresponds to the released or excess of CSDA-SE. Based on these results, the TDA has a substantial better potential for discriminating EVs from smaller subpopulations that would not be identified by conventional techniques such as DLS and NTA due to their detection limits.

Interestingly, TDA results also showed a size shift toward bigger particles with fluorescently labeled EVs (Rh: 96 nm before vs Rh: 111 nm after labeling). While a conclusion on the reason behind such shift observed with TDA cannot be made at this stage of proof-of-concept, we hypothesized that this was due to labeling occurring on the outer membrane of EVs. However, RSD of the TDA measurements by performing a reproducibility study from a given sample would help determining if those differences are significant or not.

IV.6.4.2. TDA-UV and TDA-LIF of human plasma derived EVs

Preliminary studies on uncoated silica capillaries were also performed on more complex biological samples. For this, we used human plasma EVs isolated via immunoaffinity chromatography using monolithic disk immobilized with monoclonal anti-human CD9 [172] and human plasma EVs found in an ELISA kit provided by HansaBioMed and extracted from human plasma by SEC and concentrated by tangential flow filtration. Experiments were conducted under the same settings as mentioned above.

According to preliminary NTA data, the final concentration before TDA-LIF analysis for EVs separated via immunoaffinity chromatography and by HansaBioMed should be roughly 1.9 x 10 11 EV/mL and 9 x 10 11 EV/mL. The resulting taylorograms are given in figure 39. The peaks obtained with both EVs samples were much sharper than the peak obtained with bovine milk-derived EVs. The hydrodynamic radii resulting from Gaussian corrections were 5.4 nm (UV) and 4.2 nm (LIF) for the isolated EVs with monolithic disks, and 6.6 nm (UV) and 4.3 nm (LIF) for the conventional EVs. These values are not in agreement with NTA data, where the radius measurement was at least 13 times greater than that of the TDA (see table 3). In an attempt to explain this phenomenon, some aspects have been considered. Given that the silica capillary surface contains negatively charged silanol functions and that the surface of EVs at pH 7.4 may contain some positive charges due to the presence of various amine functions on EVs membrane proteins or, to a lesser extent, amines on the head groups of the membrane lipid phosphatidylethanolamine, we first suspected a possible EV adsorption with the wall of the capillary. However, if this occurs, the resultant taylorgram will be broader and the diameter obtained by TDA would give larger sizes. Another reason might be because samples from plasma include contaminants associated with the mode of isolation used, which interact with the capillary wall and the EVs too, adding an additional dispersion phenomenon to Taylor's one. Moreover, these little entities detected by TDA, but invisible to NTA due to its detection limit at 40-50 nm, would represent the majority of the species in these samples. Further techniques to confirm this are then envisaged (e.g., cryo-TEM). Finally, further TDA investigations on EVs isolates with better purity from different origins and provided by our commercial collaborator Excilone, are envisaged. TDA performed with a capillary electrophoresis equipment proved to be a quick, straightforward, and reliable approach for measuring EV diffusion coefficients, providing access to particle Stokes radii through the Stokes-Einstein equation. We effectively demonstrated a unique approach and viable alternative to DLS, NTA, and cryo-transmission electron microscopy that are commonly used for EVs size characterization, providing the first evidence that TDA can be applied to this kind of bionanoparticles for precise size evaluation. Furthermore, TDA-LIF seemed to be capable of detecting minor variations in hydrodynamic radius that might be caused by fluorescent labelling at the outer membrane of EVs. However, knowing the RSD of the TDA measurements via a reproducibility study on a specific sample might aid in assessing whether or not such variations are significant. While TDA was found to characterize the size of highly pure EVs (i.e., bovine milk derived EVs) with satisfactory results, this was not the case for other source of EVs. Since a logical explanation cannot be given at this time, this could be due to the high presence of small impurities in EV samples, such as plasma proteins and lipoprotein particles, which may overlap with the Taylor diffusion.

Finally, including a first step of capillary zone electrophoresis (CZE) to differentiate EV subpopulations based on their electrophoretic mobilities, followed by a second step of TDA-LIF/UV measurements to determine the diffusion coefficients of each EV subpopulation could be a perspective of this work (CZE-TDA-LIF/UV) in order to provide a platform with dual characterization potential. Furthermore, it may constitute an attractive option for EV quality control by allowing the detection of contaminants caused by different isolation techniques.

Chapter V: New micro-sized method for the isolation of EVs

V.1. Introduction

In Recently, droplet-based microfluidic devices that used micro or nanometric magnetic beads (MB) as solid support to transfer target analytes (e.g. nucleic acids, proteins, cells and viruses) between different droplets , serving for extraction and purification and detection of target analytes have been reported [306]. Indeed, MB have the advantages of:

i) being able to be functionalized on their surface with a wide range of surface chemistries, allowing various strategies for the covalent linking of ligands ii) having a high surface to volume ratio due to their micro/nanometric sizes, which guarantees an improved performance of target analyte capture iii) having the possibility to be controlled with an automatic and high-throughput manner by an external magnetic field from a distance.

Given the versatility and widespread use of functionalized MB for EV isolation and detection in both conventional macroscopic protocols, their integration in droplet-based microfluidic systems for EV isolation holds considerable promise, in our opinion. When I started the work on EVs isolation, droplet microfluidics was reported twice for immunoassay-based EV detection [308,309]. However, in both cases it was not for extracting EVs but rather to quantify them or to determine surface EV protein composition and has never been explored for high performance and high throughput isolation of EVs.

Droplet microfluidics fits well with our aim to develop a platform (Droplet-CE), in which micro/nanometric droplets are utilized as carriers of target analytes (EVs) to hyphenate droplet-based sample preparation and handling with electrophoretic separation (i.e., capillary electrophoresis) [310].

Our group recently presented an in-house droplet-based sample handling system in which micro/nanometric droplets serve as a bridge for MB-based immunoassays for the sequential identification of amyloid-beta (Aβ) peptide biomarkers for Alzheimer's disease molecular diagnosis

[311]. Based on these first achievements, in this work we developed and evaluated different beadbased strategies for isolating and recovering intact EVs, and then to adapt the most performing one to the microfluidic droplet platform that we developed.

EVs may be isolated using immunoaffinity bead-based kits, which employ specific antibodies for EV surface proteins (transmembrane proteins such as CD81, CD63, CD9, annexin, and Alix are among the most often used). However, after separation, such commercial kits do not provide any elution protocol to recover intact EVs. In this regard, we first investigated conditions compatible with the elution of EVs from commercial immunoaffinity MB. Some common conditions reported to break antibody/antigen affinity were tested to elute EVs after their immunocapture. However, classical protocols usually require extreme pH to break the antibody-antigen bond which could damage the recovered EVs. We therefore investigated alternative and antibody-free strategies reported in a few recent studies to capture and eventually elute EVs using MB. Once in tube optimized and validated, we then translated this in-tube protocol to the microfluidic droplet platform. At the same time, attention had to be paid on possible beads clustering and poor recirculation during in-droplet incubation with regular droplet back and forth movements. As a consequence, we studied multiple approaches to address these challenges.

The first part of this chapter presents some principle of microfluidic droplets and MB manipulation.

The second one will present our efforts for the development of an intact EVs capture and release using immunocapture bead-based approaches, while the third one focus on antibody-free approaches and achievements obtained with our microfluidic droplet platform for EVs isolation from complex matrices.

V.2. Principle of microfluidic droplets and their combination with MB V.2.1. General statements on Droplet Microfluidics

V.2.2. Magnetic beads as cargos: principle and theory

When MB are used in combination to a droplet train, they need to be extracted from one droplet and then transferred into the subsequent droplet (see Fig. 40). The extraction of MB contained in a droplet is primarily influenced by two forces: the magnetic force (Fmag) acting on the beads through an external magnetic field and the interfacial force (Fint) associated with the droplet interface [306]. The force acting on the beads can be approximated by

Fmag ≈ (Q / ρ)•M•∇B ( 15
)
where Q is the quantity of particles, ρ is their density, M is the magnetization of a particle and ∇B is the gradient local magnetic field [313].

On the other side, the presence of an interface between two immiscible fluids is involved for multiphase microfluidics. This interface is defined by a capillary interfacial force, which for a spherical meniscus may be expressed as Fint ∝ γL, where γ is the interfacial tension between the immiscible fluids and L is a representative length of the system, such as the radius of curvature of the meniscus [306].

As a result, a magnetic force greater than the interface's interfacial force (Fmag > Fint) is needed to extract the MB from a given droplet. Implementation of a multistep procedure such as the one required to capture one specific analyte, wash nonspecific capture and elute the captured analyte, requires successive extraction of beads from droplets and redispersion of them in a different droplet.

A first strategy for MB extraction from the droplet is based on the hydrodynamic splitting of droplets containing magnetic particles, with one of the two resultant droplets possessing a high concentration of magnetic particles due to its proximity to the magnet present in the microfluidic chip (see Fig. 41A) [314]. Despite the fact that this method provides for high throughput handling, it has a poor purification yields owing to inadequate supernatant removal. Other techniques based on the same hydrodynamic separation concept but with different geometries have been developed to improve the beads capture rate [306]. However, the redispersion of the extracted magnetic cluster into the next droplet to perform subsequent typical biological operations, such as rinse steps or immunoassays, can be difficult to integrate, since additional microchannel circuitry is required. Another method, which was utilized in the system I used in the thesis is the direct magnetic extraction of the MB. The separation is no longer caused by the splitting of the initial droplet at the junction of two channels, but by the balance of the viscous drag force that pushes the drop forward in the channel, the capillary forces that tend to keep the drop entire, and the magnetic force which retains magnetic particles as a cluster. This strategy allows a very high purification yield rate compared to other techniques because the second droplet created by the extraction contains the aggregate of MB and only a small amount of initial solution. Furthermore, using the magnetic tweezers system employed in our laboratory, we can sequentially capture and release the beads in droplets for multistep operation. Indeed, the magnetic field is generated by current flowing through a coil, generating a local magnetic field, and is concentrated close to the capillary through which the droplets travel due to the presence of a paramagnetic tip. When a droplet containing MB approaches, the current is activated, the magnetic field is triggered on, and the beads join together to form a compact aggregate (or cluster) that is removed from the parent droplet (Fig. 41B). Because this cluster is confined in an aqueous sub-droplet, it is never in contact with the oil or the tube walls. However, if high flow rates or droplet viscosity are used, partial extraction or trapping failure of magnetic particles may occur. first did some in-tube preliminary experiments in order to select the best MB and affinity strategy to be used. We investigated antibody-based approach and also antibody-free ones.

Accordingly, in our preliminary experiments, a combination of beads coated with a varied portfolio of EVs-specific antibodies, such as anti-CD9, anti-CD63, and anti-CD81, was planned in order to collect the highest amount of EVs in the most precise manner possible. For preliminary results, we selected to work exclusively on commercially available Exosome-Human CD9 Isolation Reagent kit purchased from Thermo Fisher Scientific (Waltham, MA, USA). This kit was tested for EVs capture and subsequent According to the NTA analysis (Fig. 43), regardless of the type of elution used, the quantity of EVs captured by this Exosome-Human CD9 Isolation commercial kit compared to the total population of EVs in the initial sample was higher with human plasma-derived EVs (from 9 to 36 %) than with bovine milk-derived EVs (from 2 to 8 %). This is most likely due to the kit's increased specificity for EVs of human origins. The maximum EV recovery, on the other hand, was obtained when elution from MB was done using ammonium hydroxide (8.16 x 10 10 particles / mL). EVs concentration was four times higher with this ammonium solution than the commercial solution proposed by Ozyme and more than two times higher than the sodium carbonate-bicarbonate solution. Furthermore, when the EVs were eluted with the EXOFLOWBUFR-2 commercial solution, the size (135nm) differed from the two other alkaline solutions (170 and 180 nm for Na2CO3/NaHCO3 and NH4OH respectively). These latter alkaline solutions provide a mean size distribution closer to the one of EVs starting diameter of 172 nm. To better understand the origin of these results, NTA was utilized to analyse a blank containing just the solutions used for the different elutions. Surprisingly, the EXOFLWBFR-2 solution included particles with a size distribution of 125 ± 57 nm and a concentration of 1.64 x 10 10 particles / mL, while the two alkaline solutions contained no particles. Since the composition of this solution is confidential, it was impossible to determine their nature. However, given their high concentration, they might explain why the EVs eluted with this kind of solution shifted toward lower size values. In an effort to improve the performance of EVs capture and thus isolation yield with ammonium hydroxide elution, higher beads concentrations were also tested (20 and 40 µL of MB in 100 µL final volume). However, they did not result in significantly higher EV isolation yields (see Fig. 44). While the specificity isolation methods, this strategy may also be restrictive since it is likely to extract just one sub-population of EVs. There is currently a lack of sufficient knowledge on which EVs markers are present on distinct EV subpopulations, or which markers are constantly present on all EVs surface (even though their proportion may vary for different sub-population) as well as the subcellular and cellular origins of these EVs. Based on NTA analysis, the CD9+ population profile of pre-purified EVs samples seems to be identical to that of the original sample when the CD9 protein used for their capture. The ammonium hydroxide solution (pH 11.3) provided the highest recovery yield for this isolation method. A combination of coated beads with a varied range of EVs-specific antibodies such as anti-CD9, anti-CD63, and anti-CD81 might be interesting. Although this experiment could only be performed with anti-CD9 coated beads due to time constraints for the thesis, it gives early information on possible elution of EVs from MB functionalized with specific antibodies under alkaline conditions.

However, further research is required to determine the influence of these elution conditions on EVs integrity. Indeed, the extreme pH levels required to break the antibody-antigen bond may cause considerable damage to EVs which could be the reason of unsatisfied results obtained. These might impair the biological activity of the recovered EVs and cause a misleading conclusions in the subsequent function analysis.

V.4. Alternative MB-based strategies to extract EVs

When using the antibody-antigen strategy, we encountered two major problems: i) the harsh elution conditions that can damage EVs and ii) the lack of universal EVs markers to ensure entire capture of all EVs. As a result, we have reoriented our strategy and explored other and current methodologies that employ MB for non-specific capture and release of EVs.

We therefore compared some alternative approaches reported in the literature to capture EVs: via phosphatidylserine (PS) displayed on the EVs surface [177], via lipid-based nanoprobes [316], via anionic exchange [317] and via selective adsorption [318]. We choose among them, those that could be easily adapted to droplet operations and to our microfluidic system. The first is the only commercially available alternative (ExoCAS-2), based on the electrostatic interaction [317]. The second method which relies on EVs precipitation on MB in the presence of PEG as it was considered the most cost-effective and did not require any functionalization on the surface of the MB [318]. This study constituted the basis for a manuscript, which was submitted to Talanta (manuscript under peerreview) and is discussed in the second section of this subchapter.

contaminations and enriched in EVs proteins. However, this approach was only validated with cultured cell supernatants and not with more complicated biological fluids like as human plasma, where the widely varying amount of phosphatidylserine may impair EV purity. Furthermore, there is no logical explanation for the removal of contaminants by this sort of capture. of ExoCAS-2. However, the ExoCAS-2 approach produced less protein contamination than ExoQuick (20 times lower), but it was still roughly 2.3-fold greater than ultracentrifugation. Among these methods, the ExoCAS-2 approach showed the greatest purity ratio (particle concentration to protein concentration). Western blot analysis also exhibited the strongest bands for the reference EV protein markers (ALIX, TSG101, CD9, and CD81) when ExoCAS-2 was used.

As described in Section II. Among the various strategies outlined above, we investigated two newly disclosed batchwise EVenrichment processes that employ MB and successfully elute intact EVs and have the highest potential for eventual downscaling to microfluidic droplet format, from our viewpoint. The first, known as ExoCAS-2, uses an ionic exchange mechanism to rapidly capture negatively charged EVs through electrostatic contact, employing MB covered with a polycationic polymer called poly-L-lysine (PLL)

[317]. This one was chosen because it is the only one that is currently on the market (sold by Microgentas Inc., Republic of Korea). The second is a MB-mediated selective adsorption strategy known as MagExo, which takes advantage of the presence of a water-excluding polymer, polyethylene glycol (PEG), to lock up a significant number of water molecules, forcing EVs to aggregate and precipitate on the surface of MB [318]. We used EV standards derived from bovine milk with a narrow size range (provided by Excilone), allowing allows for more precise EV recovery evaluation. The amount of isolated EVs with the selected MB-based methods were analyzed using NTA and our novel CE-LIF characterization tool. The refined batchwise approach was then turned and adapted into a microfluidic droplet procedure to give high automation and integration, lower sample/reagent quantities, and better isolation efficiency. This was then utilized to separate EVs from more complicated matrices. The details of this work are described in the following manuscript that has been submitted to Talanta for publication (Morani, Talanta, 2022).

Introduction

Extracellular vesicles (EVs) are phospholipid bilayer-delimited particles produced by most cell types and present in many body fluids [1,2]. EVs contain and carry diverse biomolecules that are specific to the mother cells from which they are secreted, allowing them to transmit a variety of essential signals under both normal and pathological conditions. Hence, the potential of EVs as prognostic or diagnostic biomarkers has attracted significant attention in recent years [3][4][5].

Furthermore, due to their high specific targeting ability, EVs have gained much interest as engineered drug delivery systems for clinical and pharmaceutical applications [6,7]. However, there are still technological hurdles to purify, analyze and characterize such nanometric bioentities. Many methods for isolating EVs have been developed so far, including ultracentrifugation (UC), gradient ultracentrifugation, ultrafiltration (UF), polymer coprecipitation, size-exclusion liquid chromatography (SEC), immuno-extraction [8]. Among these, ultracentrifugation is widely considered as the gold standard in all EV applications.

However, this technique presents many drawbacks, such as time-consuming procedures, contamination of EV populations by protein aggregates and other particles, damage to the EVs membrane structure and possible considerable loss of EVs (EVs yield may drop to 2%) [9,10].

Thus, there is still an urgent need for emerging EVs isolation approaches that can provide EV purity and integrity in a reproducible and high-throughput manner. Many modern isolation methods have been developed in this direction, such as flow field-flow fractionation, ionexchange, electrokinetic approaches as well as the combination of multiple techniques, like UC with UF or SEC [11][12][13]. In parallel, microfluidic technologies have made significant progresses for such purpose, exploiting both physical and biochemical properties of EVs at micro/nanoscale level for their capture and/or detection [14][15][16][17][18][19]. The majority of microfluidic approaches rely on immunoaffinity to selectively capture EVs. Immunoaffinity bead-based kits allow highly selective isolation of EVs through antibodies specific for target EVs surface proteins [20][21][22][23]. However, following isolation, those commercial kits, which are rather used for subsequent EVs downstream lysis and analysis, do not provide any efficient elution possibility to recover intact EVs. Moreover, the main disadvantage of those strategies is the absence of universal EVs markers to ensure total capture of all EVs. Few recent works on capture and eventual elution of EVs on magnetic beads have been reported, using either electrostatic interaction [24], polymer mediated adsorption of EVs on magnetic beads [25], a DNA aptamer-based system [26] or DNA linker spacers [27]. No EVs recovery efficiency was reported in these works that used cell culture media and/or plasma samples as starting materials from which accurate EVs quantification is not trivial. At the actual stage, these works had to be realized batchwise with multiple in-tube steps without automation.

The goal of this study was to investigate bead-based strategies for isolating and recovering intact EVs without the use of immunoaffinity recognition and to adapt them to droplet microfluidics. For such purposes, well-characterized high-quality EVs isolated from bovine milk were used as EVs standards rather than non-quantified EVs from cell culture and plasma samples. Then, we transferred the more performing batchwise approach into an automated and high-throughput protocol relying on a microfluidic droplet train. Different operations in microfluidic droplets were developed and optimized to overcome the challenges of beads clustering and poor recirculation in droplets in the presence of viscous polymers, allowing to realise EVs capture on beads, washing and elution with a droplet sequence. So far, droplet microfluidics has been communicated only twice for immunoassay-based detection of EVs [28,29] and has never been exploited for high performance and high throughput isolation of EVs.

Materials and methods

Chemicals and reagents

Apparatus and Material

For macroscale protocols, all magnetic-bead-based assays in batch were carried out in protein LoBind 1.5 mL tubes purchased from Eppendorf (Hamburg, Germany). For retaining magnetic beads, a neodymium magnet purchased from Ademtech (Adem Mag MSV, Bessac, France) was used. Shaking of magnetic bead suspensions during the incubation and washing steps was realized with a mixer (Eppendorf ThermoMixer C).

For droplet microfluidic operations, a microfluidic droplet platform was constructed and was inspired from our previous configuration [30]. were employed for droplet observation.

The CE-LIF analyses were performed with a PA 800 Plus system (Sciex Separation, Brea, CA) equipped with a solid-state laserinducedfluorescence detector (excitation wavelength of 488 nm, emission wavelength of 520 nm) purchased from Integrated Optics (Art. No. 40A-48A-52A-64A-14-DM-PT, distributed by Acal BFi, Evry, France). Uncoatedfused silica capillaries were purchased from CM Scientific (Silsden,UK). Data acquisition and instrument control were carried out using Karat 8.0 software (Sciex Separation, Brea, CA).

Methods

Isolation of bovine milk-derived EVs with sucrose gradient ultracentrifugation

Skimmed bovine milk samples were obtained by centrifugation of 50 ml whole milk at 3,000 g for 30 min at 4°C (Allegra X-15R, Beckman Coulter, France).The whey was obtained after acid precipitation with 10% (v/v) acetic acid, incubation at 37 °C for 10 minutes and 10% (v/v) 1M sodium acetate for 10 minutes at RT followed by centrifugation at 1500g, 4°C for 15 min and filtration using vacuum-driven filtration system Millipore Steritop, 0.22 μm. The whey supernatants were concentrated using Amicon 100kDa centrifugal filter units (Merck Millipore) at 4,000 g and 20°C up to final volume of 6 mL. The obtained retentate was ultra-centrifuged for pelleting the EVs at 100 000 g for 1h10 at 4°C (Beckman Coulter, Optima XPN-80, 50TI

rotor). The pellets were solubilized in 500 μL of PBS then added to 11 ml of pre-prepared sucrose gradient 5-40% and ultra-centrifuged at 200 000 g for 18h at 4°C (Beckman Coulter, Optima XPN-80, SW41 rotor). Fractions of 1 mL were collected and the selected ones containing targeted exosome population were diluted in 6 ml of PBS 1X and finally centrifuged at 100 000 g for 1h10 at 4°C (Beckman Coulter, Optima XPN-80, 50TI rotor). The pellets were resuspended in 50 μL of PBS 1X and stored at -80°C, until further analyses.

Isolation of pony plasma and serum derived EVs with size exclusion chromatography (SEC)

Preparation of plasma: Peripheral blood was collected into EDTA-coated vacutainer tubes.

After ten-time inversion, samples were processed within the 60 minutes of collection.

Consecutive centrifugation steps at 2,500 g, 4°C for 15 minutes and then at 15 000 g for 10 minutes were performed followed by filtration of the supernatant through 0.22µm filters.

Preparation of serum: Whole blood was collected into anticoagulant-free tubes and allowed to clot at room temperature for 45 minutes. The clot was removed by centrifuging at 3 200 g, 4°C for 15 minutes, followed by centrifugation at 15 000 g, 4°C for 10 minutes and filtration of the supernatant through 0.22µm filters. 500 µl of pre-treated plasma/serum was loaded onto a qEVoriginal SEC column (Izon Science, New Zealand) previously washed and equilibrated with PBS. Fraction collection (0.5 mL per fraction) was carried out immediately using PBS 1X as elution buffer. The selected elution fractions were pooled and were subsequently concentrated using 100 kDa Amicon centrifugal filter units (Merck Millipore). Post-treatment processing with several washing steps with PBS was applied to obtain highly pure EV fractions highly enriched with exosomes.

EV isolation using commercial kits

ExoCAS-2 magnetic bead-based ion exchange kit was used for the study of EV yield performance. The experimental procedure is described in a paper recently published [24].

Briefly, PLL-coated beads were mixed with our standard EVs (bovine milk derived EVs), followed by incubation of the mixture for 30 min at 4° C in a rocking platform mixer. After incubation, the EVs-bound beads were carefully washed with 2 mL of ExoCAS-2 washing solution and then re-suspended in ExoCAS-2 elution solution by shaking for 5 min at 1000 rpm.

Finally, a magnet was employed to retain magnetic beads and the supernatant containing yielded EVs was collected.

PEG-based EVs precipitation on magnetic beads in batch mode

For PEG-based EVs precipitation on magnetic beads in batch, a volume of 200 µL of PEG ( 25% m/v), 150 µL of carboxylate functionalized magnetic beads (10 mg/mL) and 250 µL of deionized water was incubated with 400 µL of standard EVs or biological fluid (simulated human serum and pony plasma and serum, 5 times diluted with deionized water) on a mixer at 25 ° C for 1 h at 700 rpm. Beads were then carefully washed twice with PEG 5%/ NaCl 0.2 M.

After removal of washing solution, the magnetic beads in tubes were re-dispersed in 400 µL of PBS 1X solution and then incubated on shaking for 5 min at 25 °C for EV elution. A magnet was used to remove the magnetic beads, and EVs were recovered in the supernatant.

The Zetaview system (Particle Metrix, Germany) was equipped with a 488 nm laser.

CE-LIF of fluorescent labelled EVs

Details on the CE-LIF method for EVs analysis can be seen in our recent work [31].

Results and Discussion

Batchwise EVs isolation development

With the aim to establish a high-performance and high-throughput microfluidic droplet system for EVs isolation, we evaluated two recently communicated batchwise EV-enrichment strategies that are alternative approaches to the immunoaffinity-based ones. They hold high potential for subsequent translation into a microfluidic format in terms of minimal forefront preparations, non-laborious operations, as well as ease of manipulation and step transition thanks to the use of magnetic beads as cargos. The first one is a magnetic bead-mediated selective adsorption strategy (MagExo), exploiting the presence of a water-excluding polymer, polyethylene glycol (PEG), to lock up a significant number of water molecules, forcing thereby the EVs to aggregate and precipitate on the surface of magnetic beads [25]. The second strategy called ExoCAS-2 relies on an ionic exchange mechanism, using magnetic beads coated with a polycationic polymer, poly-L-lysine (PLL), to quickly trap negatively charged EVs via electrostatic interaction [24]. For performance comparison, EVs standards derived from bovine milk having narrow size distribution, with well-defined concentrations and exhaustive characterizations by NTA, DLS, LC-MS/MS and TEM, were used as starting sample. TEM images revealed the absence of contaminating protein residues in these EVs standards (Fig. S1A in the supporting information ESI), and major protein contaminants (e.g. α-s1 casein, βcasein, α-Lactalbumin and serum albumin) were not found according to the LC-MS/MS analysis (Fig. S1B). The use of EV standards of high purity allowed to evaluate the EVs recovery more accurately. In our case, quantification of EVs isolation yields obtained with MagExo and ExoCAS-2 methods was possible by comparing the EVs concentrations before and after isolation processes (see Fig. 1). Unsatisfactory EVs recovery (less than 10%) was obtained with commercial PLL-coated beads, compared to that achieved when using PEG and silica magnetic beads (34 %). NTA data also showed a size shift towards smaller particles when EVs were eluted from PLL beads. This could be a consequence of PLL release from the beads during the elution process. This was confirmed by the CE-LIF analysis (discussion below). This led to a critical PLL concentration in the eluent that is high enough to cause EVs lysis. Indeed, several studies have shown that PLL can penetrate through vesicles via interaction with the lipid membrane [32][33][34], provoking vesicle lysis from a certain threshold concentration of PLL [35]. In parallel to NTA measurements for EVs recovery evaluation, we used our recently developed CE-LIF approach [31] to validate the identity of EVs collected with MagExo and ExoCAS-2 methods (see Fig. 2). The peak profile of EVs collected with PEG-based protocol corresponds well to the fingerprint of bovine milk-derived EVs, confirming the presence of intact EVs in the eluent (Fig. 2A). On the contrary, with the ion exchange method, multiple tagged species were detected. To understand the origin of these peaks, solutions containing different PLL concentrations without the presence of EVs were analyzed. As can be seen in Fig. 2B, many peaks were still detected, whose intensities were related to the PLL concentrations. These results confirmed the hypotheses that the peaks observed come from PLL leakage from the magnetic beads, which could not be visualized with conventional NTA. The released PLL which can be labelled by the residual CFDA-SE dye through its amino groups lead to unwanted products in the eluent. The ExoCAS-2 method was therefore not further considered and the PEG-based method (MagExo) was chosen for further optimisation.

Besides the preservation of intact EVs after purification, protocol reproducibility is another important point to consider to guarantee consistency of isolated EVs population and quality.

Indeed, by reproducing the MagExo procedure described in reference [25], we observed significant batch-to-batch variation in vesicle concentration (RSD 30 %). The lack of EVs washing between the capture and elution steps in the original protocol led to misleading data.

To minimize this cross contamination and to recover intact EVs for further characterization, we droplet to another without any risk of cross contamination. This was expected to be overcome with our setup through the use of magnetic beads as the controllable carrier of target analytes between droplets. A purpose-made magnetic tweezer, composed of a paramagnetic tip activated by an electrical coil [30,36], was employed to manipulate magnetic beads between droplets via application of an external electrical field. Table 1 provides an overview of the operation sequence. A train of droplets containing different solutions and sample in a defined order was delivered through the magnetic tweezer where the beads were extracted from the first droplet and transported into the sample one containing EVs. The incubation was subsequently performed by pushing the droplet train back and forth inside the tubing. The magnetic beads that retain EVs on their surface were then trapped by the magnetic tweezer and the supernatant droplet was washed away, followed by flushing of the trapped magnetic beads with two washing droplets. The washed beads were finally released into an elution droplet that was finally collected in an oil-containing tube for further analyses.

Several problems were however encountered that led to failure of such droplet protocol in our first experiments, notably beads clustering and poor recirculation in droplets in the presence of PEG. Indeed, the beads often stayed at the rear of moving droplet, forming undesired bead clusters when the droplet moved inside the tubing over an extended distance. This was ascribed to come from the presence of PEG which significantly increased droplet viscosity [37], which in turn impaired the effective recirculation of the beads (see Fig. S3 in the ESI). To enhance recirculation of the beads within a droplet, we put our efforts on optimizing the droplet movement pattern and travel volume (i.e., the oil volume required to push and pull the droplet)

during the back-and-forth movement. Chaotic trajectories are known to enhance mixing efficiency [38,39]. Consequently, different droplet movement patterns were also tested, including the straight, the U-shaped patterns (i.e., the tubing was shaped over a metallic guideline) and the spiral-formed one (i.e., the tubing was coiled around a cylindrical support) (see Fig. S4 in ESI). With the straight pattern, bead aggregation was always observed in the presence of PEG, regardless of the travel volume (Fig. S3A). For other movement patterns, bead clustering was alleviated but still visible when working with a large travel volume of 10 µL (Fig. S3 B andC). This undesired phenomenon was finally avoided when employing the spiral or U-shaped patterns with a short travel volume of 6 µL (equivalent to a droplet volume).

By keeping these optimized setups and conditions, we further investigated the in-droplet incubation duration (from 5 to 45 min) during the EVs capture step (Fig. S5 in the ESI). The best EVs recovery of 27 % was found for 25 min incubation when using the spiral-formed pattern. Longer incubation time did not result in any improvement of EVs recovery. Under the same optimized conditions, the U-shaped pattern offered the best EVs recovery rate of 39%.

Note that the droplet system offers higher EVs isolation yields than that obtained with the batchwise setup, regardless of the droplet movement pattern used. To obtain more precise information on the quality of the isolated EVs, the samples were analyzed with Particle Metrix'

ZetaView [40]. As revealed by Zetaview data (Fig. 4B), 80% of the recovered EVs fell within the range of the initial size distribution, while the remaining 20% were represented by larger aggregates. These aggregates are presumably formed due to the PEG capacity to wrap and condense hundreds of EVs, making them hard to resuspend in the absence of PEG during the elution step.

Microfluidic droplet-based isolation of EVs from biofluids

The developed microfluidic droplet instrument and protocol were then used to isolate EVs from more complex matrices. First, we used PBS mixed with three main extracellular matrix proteins, including albumin, IgG and transferrin to imitate human extracellular fluids [41]. This simulated human serum was then spiked with standard EVs (i.e., bovine milk derived EVs at 1.02 x 10 11 particles/mL) and passed through the microfluidic droplet system to evaluate the EVs isolation performance. As indicated in Fig. 5, the concentration of collected EVs was 3.69

x 10 10 particles/mL, giving an EVs recovery rate of 36% with spiked simulated human serum, which was not far from that obtained with pure standard EVs (39%). We then applied the droplet protocol to pony plasma and serum (Fig. S6 in ESI). When dealing with such complex biofluid matrices, the droplet approach gave an overall NTA-measured particle concentration eight-time higher than that obtained with the in-tube method. To confirm the identity of EVs isolated from plasma and serum samples, the elution droplets were also analysed with CE-LIF to reveal different EVs subpopulations (Fig. 6). Based on the peak intensities in each electropherogram, the highest concentration was found for the fraction with the shortest migration time (9-12 min) whereas the signal of the second peak zone (12-15 min) was less intense and this is more remarked for the serum samples. This kind of EVs fingerprints was found similar to those obtained in our previous work with EVs from pony plasma and serum isolated by SEC [31]. Interestingly, the third peak zone (15-20 min) appeared in the electropherograms obtained with both in-batch mode and the droplet system, suggesting that a distinct subpopulation emerges when using the PEG-based isolation methods. Indeed, different isolation methods may lead to the shift or differences in size distributions of the collected EVs, as already evidenced for SEC and UC isolation methods [42,43]. In our case, the PEG-based method is expected to capture non-selectively all EVs subpopulations, leading to more EVs fractions being visualized with CE-LIF as seen in Fig. 6. NTA measurements for both pony serum and plasma revealed a shift towards smaller size distributions when using the droplet approach (Fig. S6 in ESI). This may correspond to an increased concentration of the smallsized subpopulations, which may correlate to the more pronounced appearance of the third peak zone (15-20 min) in the CE-LIF electropherograms. Nevertheless, no further speculation was made to interpret the presence of these three subpopulations observed by CE as no clear relationship between size and charge properties of EVs and their electrophoretic mobilities can be stated at this stage.

Conclusion remarks

We successfully developed a new approach (instrumentation and methodology) for EVs isolation from both pre-purified standards and biofluid samples, for the first time in microfluidic droplet format. Using a train of micrometric droplets containing respectively magnetic beads, sample, washing and elution solutions, we allowed significant sample and reagent volume reduction (by 5 times), minimization of manual operations, diminution of operation time (by twice) and improvement of EVs recovery rate by almost 2 folds. Thanks to automatization and miniaturization that we achieved with droplet microfluidics higher throughput can now be expected. Integration of this microfluidic EVs isolation module as a forefront of downstream EVs analysis and characterization is now envisaged.

A) B)

Conclusion and perspectives

For some years, EVs have been identified as a possible source of biomarkers since their internal RNA, internal or membrane proteins derive directly from the emitting cells and reflect the latter's physiological or pathological condition. Thus, scientists are developing novel diagnostic methods based on the utilization of EVs either for identification of specific pathology-indicating vesicles or as a source of biomarker, notably in the area of cancer. However, only a few biomarkers have been validated till now for cancer diagnosis. The obstacles associated with standardizing this liquid biopsy approach for diagnosis are enormous. To be effective in the clinic, an EV-based biomarker must be extensively validated, and a high degree of repeatability throughout the process is needed. On the other hand, owing to their capacity to transmit bioactive components and cross biological barriers, EVs are also being investigated as possible therapeutic vectors or agents. For future clinical trials and for the transition to regulatory-approved clinical therapy, it is necessary to have not only higher purity, retained integrity and functionality but also standard operating procedures (SOPs) for reproducibility, quality control criteria. Because of this, ISEV has produced recommendations on a regular basis since its creation in 2011 in order to best define practices in the area of EVs and promote the development of new isolation and characterisation strategies. However, the field is still in its early stages.

The state of art on emerging microfluidic technologies has permitted to identify a number of problems that must be solved before EVs can become widely used. The development of a new platform based on a complete microfluidic system capable of extracting EVs from biological fluids, in high quality control standards, with a technique capable of simultaneously providing physical and biological information on isolated EVs, emerged as a major objective of my thesis but as a difficult challenge. The idea was to avoid the numerous complementary techniques that must be used nowadays and to propose an automated method of EV isolation and multi-characterization that can be reproducible and standardizable.

Several separation modes/mechanisms of Capillary Electrophoresis for EVs characterisation were thus evaluated in this thesis. A solid knowledge of this technique has allowed for a better understanding of the difficulties surrounding the development of novel systems for the separation and analysis of EVs.

The CZE method we developed was able to evidence differences in electrophoretic mobilities from EVs isolated from different animal origins (e.g. bovine milk, pony serum and pony plasma derived EVs).

Furthermore, when applied to human plasma-derived EVs isolated via immunoaffinity chromatography using monolithic disk columns selecting either CD61 or CD9 surface marker of EVs, our method revealed three distinct peak zones with varying signal intensities depending on the targeted marker. However, at this stage no clear relationship between the size and/or charge characteristics of EVs and their electrophoretic mobilities can be defined. The translation of CE theory and experience on nanoparticles to such heterogeneous bioentities seems indeed not trivial. To understand the electrophoretic behaviour of EVs, a deeper characterisation study utilizing EVs with a lower degree of polydispersity and narrow size distribution or well defined zeta potential would be required. However, such pure EVs are not currently obtainable using current EV purifying technologies.

Another alternative could be to collect the subfractions from CZE runs and analyze them. However, subfraction analysis from CZE would need a higher resolution in order to collect only a specific subfraction and several injection/collection cycles due to the low injected volumes used in CE (around 125 nL, for a 60-cm capillary and 50-μm id). The resolution will be maximum with values of EOF close to those of the electrophoretic mobilities (µep) of the EVs. We could therefore try to better tune the EOF to improve the resolution between subfractions and collect this way purer fractions by adding EOF modulators such as detergents or alkylamines.

When compared to CE-UV approaches developed by other groups throughout the course of my thesis, the LIF detection used in our work enabled us to achieve superior LODs (on the order of 10 10 EVs/mL).

However this is still not sufficient to address low abundant EVs samples, a good target would be to decrease this limit to about 10 8 EVs/mL. We have conducted some pioneering research on viable ways for in-capillary electrokinetic preconcentration for EVs in order to boost the sensitivity of our CE-LIF detection). All of the electrokinetic approaches tested for in-capillary preconcentration (FASS, LVSS and multiple cycle-LVSS), however, were found unsuitable for EVs. It has been indeed challenging to find a balance between an EVs sample matrix with a conductivity significantly lower than that of the BGE while limiting or eliminating EV lysis during analysis under these extreme conditions (low or high conductivities). Actually, we must bear in mind that, low conductivities may harm EVs whereas high conductivities can cause joule heating and damage EVs. Alternative electrokinetic preconcentration modes that might preconcentrate EVs that I have not yet attempted (for example, capillary isoelectric focusing (cIEF) or isotachophoresis (ITP)) could be an interesting possibility to consider in future.

Beside CZE developments, we showed that TDA-UV performed with a commercial CE device by using pressure to the capillary's inlet could successfully estimate the size of bovine milk EVs in accordance with those evaluated by NTA. Additionally, TDA-LIF seemed capable of detecting very subtle changes in hydrodynamic radius that might be produced by fluorescent labeling at the outer membrane of EVs and also to detect the presence of smaller entities (Rh 20 nm) most likely due to either the release of fluorescent dye (CFDA-SE) from EVs over the time (phenomenon also observed in CE) or excess of CSDA-SE that contaminated the samples. Conversely these features were not detected by NTA.

While TDA proved out to be an excellent method for determining the size of extremely pure EVs (i.e., EVs obtained from bovine milk), this was not the case for human plasma-derived EVs. This is possibly due to a high concentration of small contaminants in those plasma EV samples, such as plasma proteins and lipoprotein particles, which may overlap with the Taylor diffusion phenomenon. To confirm this it would be worth investigating TDA on other EV isolates of higher purity for instance and also derived from different biological sources.

This TDA-based characterisation method is intended to be integrated to the isolation module in the future, either directly for direct EVs sizing (as an alternative to the CZE profiling) or on-line to CZE (CZE-TDA) to calculate the diffusion coefficients of each previously separated EV subpopulations.

With the objective of realizing a complete and integrated platform for EVs isolation and analysis, the development of a microfluidic droplet system combined to magnetic beads used as cargo between each step of the EV capture process was then investigated.

The use of PEG to precipitate EVs onto magnetic beads, as an alternative to antibodies, has the advantage of being less expensive and not requiring any special functionalization of the magnetic beads. After having optimized the extraction condition in tube, its further translation into a droplet microfluidic procedure was successful to enable EVs recovery from more complex biofluids such as diluted pony serum and plasma. When dealing with such complex biofluid matrices, the droplet technique resulted in an 8-fold increase in the total NTA-measured particle concentration compared to the in-tube method (1.7E10 11 particles/mL for pony plasma, for example). The PEG-based method is expected to capture non-selectively all EV subpopulations, but it is also possible that contaminants (e.g. aggregated proteins and lipoproteins) may co-isolate with them. Because no further interpretation of the profiles obtained by our CZE approach is possible, LC-MS or western blot should be investigated in the future to identify total proteins of these separated EVs.

The natural next step in this study will be to connect the function of the microfluidic droplet-based technique to the CE system, with the goal of developing a full microfluidic instrument. However, droplet-interfacing for CE is only in its infancy at the moment, and its coupling is only possible via yetto-be-developed interfaces. This means that at the moment manual transfer between the dropletbased procedure and CE system of the final droplet containing eluted EVs is required before complete automation and high-throughput are attainable.

Figure 1 :

 1 Figure 1 : Evolution of the number of publications dealing with EVs between 2010 and 2021. The graph shows the number of publications with the term "extracellular vesicles" in the title, between 2010 and 2021 (October), obtained from the entire Web of Science (WoS) database.

  ESCRT is a complex composed of four sub-complexes (0 , I, II and III) and other associated proteins such as VPS4 (Vacuolar Protein Sorting-associated protein 4), ALIX (Apoptosis-Linked gene 2-Interacting protein X), TSG 101 (Tumor Susceptibility Gene 101 protein) and ATPase (adenosine triphosphatase), playing a crucial role in the transport of ubiquitinylated proteins to endosomes and the formation of MVBs. ESCRT-0 is recruited at the endosomal membrane boundary by interacting with the phospholipid PI3P (Phosphatidylinositol-3 phosphate) present in the endosome membrane. ESCRT-0 recruits ESCRT-I and together they group the ubiquitinylated cargoes into microdomains. ESCRT-I recruits ESCRT-II and starts creating involution of the membrane. Finally, ESCRT-I/II in turn recruits ESCRT-III which is involved in the detachment of the formed vesicle. ESCRT-III is then dissociated and recycled by the action of ATPase VPS4 (Fig. 2).

Figure 2 :

 2 Figure 2: Representation of the ESCRT complex. ESCRT machinery is first set up by the ESCRT-0 complex. This complex can divide proteins into microdomains and interact with the ESCRT-I complex's TSG101 subunit, which, in turn, recruits ESCRT-II subunits. The inner budding of the ILVs into the lumen of the MVBs is then triggered by ESCRT-I and ESCRT-II. ESCRT-I and ESCRT-II initiate the inward budding of the ILVs into lumen of the MVBs. Subsequently, ESCRT-III subunits bind to the neck of the early ILVs and cleavage them into free vesicles. Reprinted from [36].

Figure 3 :

 3 Figure 3: Mechanism of microvesicles biogenesis from plasma membrane of cells. Reprinted from [41].

Figure 4 :

 4 Figure 4: A graphical illustration of common proteins found in EVs. The molecules depicted here are classified according to their function or protein class: Cytoskeletal proteins, Membrane trafficking proteins, Immunoregulator molecules, Adhesion molecules, Signal transduction molecules and Tetraspanins. Readapted from [68].

5 )

 5 . In 2019, Avalon GloboCare developed the world's first saliva-based exosomal microRNA biomarker, miR-185, as a dual diagnostic and therapeutic target for oral cancer. Exosomics (Siena, Italy) also provides RNA and DNA extraction kits from intravesicular contents for liquid biopsy and cancer screening. A few milliliters of plasma are collected for the isolation of EVs by two different methods.

Figure 5 :

 5 Figure 5: A graphical illustration of ExoDx Prostate Test (EPI) workflow. RNA is isolated from urine exosomes to examine the expression of three genes (PCA3, ERG, and SPDEF) linked to high-grade, aggressive prostate cancer. The RNA expression levels of these three genes are then included into an equally weighted proprietary algorithm and a personalized risk score. Reprinted from (https://www.urologytimes.com/view/the-role-of-the-exodx-prostate-test-in-early-detection-ofprostate-cancer)

[

  137]. This fundamental approach was evaluated in vitro with EVs loaded with the viral spike S protein against the SARS-CoV-2 coronavirus, offering insight into the possible use of EVs for SARS-CoV-2 vaccination [138].

Figure 4

 4 Figure 4 highlights the major centrifugation and ultracentrifugation steps; however, specific

Figure 6 :

 6 Figure 6: Ultracentrifugation-based isolation methods used for EVs. (1) First successive rounds of centrifugation are intended to pellet consecutively cells (alive and dead) and debris. EVs are then separated and enriched using either several cycles of centrifugation (2) or after prolonged centrifugation in a gradient medium (3). Readapted from [140].

Figure 8 :

 8 Figure 8: Schematic representation of TFF. Biological fluids travel over the ultrafiltration membrane. Smaller molecules pass through the membrane while larger molecules, such as EVs, remain on the membrane. Reprinted from [156].

Figure 9 :

 9 Figure 9: Schematic representation of size-exclusion chromatography principle. From a heterogeneous sample comprising EVs and other molecules, the small contaminating molecules, such as protein aggregates, seep into the pores and are eluted later, whilst EVs are eluted in the early fractions. Reprinted from [161].

Figure 10 :

 10 Figure 10: Schematic representation of (A) magnetic bead-based capture with specific antibody against EVs and (B) whole EV separation technique from plasma using the anti-CD61 disk developed by Multia 2019. Diluted human plasma samples are percolated through monolithic disk columns. The enriched EVs are eluted with either ammonium hydroxide (pH 11.3) or carbonate-bicarbonate (pH 11.3). Reprinted from [172].

Figure 11 :

 11 Figure 11: Schematic representation of Asymmetrical flow field-flow fractionation (AsFlFFF) principle. Reprinted from [179].

Figure 12 :

 12 Figure 12: AF4 fractograms of EVs from cultured murine melanoma cell line. Three major peaks (P2, P3 and P4) represented the exomeres and two exosome subsets (small exosomes (Exo-S) and large exosomes (Exo-L) respectively. P0 is the void peak. P5 is due to larger microparticles. Reprinted from [181].

  Dynamic light scattering (DLS) is a quite simple technique to determine the size of the particles in a sample ranging from nm to µm size. The vesicles in suspension are subjected to a random Brownian motion whereby small particles move faster than larger ones. A light beam passing through the suspension causes the scattering of laser light at different intensities. The analysis of these intensity fluctuations gives the velocity of the Brownian motion and hence the particle size using the Stokes-Einstein equation. In the case of a polydisperse population, such as EVs, the size distribution is strongly influenced by the large particles, since the intensity of scattered light increases with particle size[182].

Figure 13 :

 13 Figure 13 : (A) Scheme of NTA measurement set-up. (https://www.azonano.com/). (B) NTA results of EVs released by tumor cells of the gastric cancer cell line GC1401. Reprinted from [183].

Figure 14 :

 14 Figure 14: Schematic representation of tunable resistive pulse sensing (TRPS) measurement set-up. Reprinted from [187].

Figure 15 :

 15 Figure 15 : Schematic representation of flow cytometry system. Reprinted from https://www.semrock.com/flow-cytometry.aspx

Figure 16 :

 16 Figure 16: (A) TEM observations of the cup-shaped structure of EVs after chemical fixation. Magnification: Labeling of exosomal MHC class II surface antigens with 10 nm gold particles. Reprinted from [199]. (B) TEM observations of EVs after cryogenic fixation and rupture of the vesicle membrane. Reprinted from [200].

Figure 17 :II. 2 . 1 . 7 .

 17217 Figure 17 : (A) Schematic representation of the AFM setup. AFM involves the interaction of a cantilever with the sample, and the reflected laser beam is monitored by a photodiode. Reprinted from [204]. (B) AFM image of salivary exosomes from healthy individuals. Reprinted from [205].

Figure 18 :

 18 Figure 18: Capture and detection of EVs on the surface of a nanoplasmon-enhanced scattering (nPES) device using antibodies directed against the three tetraspanins. The signal is amplified when the CD63 and CD9 proteins are present on the EVs (yellow signal). Scale bars: main images, 2 μm; magnified images, 100 nm. Reprinted from [210].

Functionalized

  beads are easier to manipulate than the functionalized microchannels mentioned earlier. For a typical example, Zhao et al. recently developed a microfluidic chip, called ExoSearch, that allows continuous isolation of EVs using immunomagnetic beads[230]. The chip consists of a Y-shaped injector and a serpentine fluidic mixer that allows the incubation of three probing antibodies (against CA-125, antiCD-24, and anti EpCAM) labeled with various fluorescent dyes (Fig.19B). This technique was capable of quantifying ovarian cancer-derived exosomes in patient plasma samples with a LOD of 750 particles per µL, a sample volume of 20 µL, and a 40-minute processing period.

Figure 19 :

 19 Figure 19 : Examples of immunoaffinity-based microfluidic approaches for EVs isolation. (A) Schematic of the nano-IMEX platform. The surface of the microchannel and micro posts were coated with graphene oxide and polydopamine to capture EVs using anti-CD81 antibody immobilized on protein G. Isolated EVs were then detected using ELISA. Reprinted from [231]. (B) Schematic workflow of the ExoSearch platform continuous mixing, separation, and in-situ, multiplexed detection of EVs. Reprinted from [230].

Figure 20 :

 20 Figure 20 : ExoTIC's scheme. The modular components, which have pore diameters of 200, 100, 50, and 30 nm, are linked in series to separate exosomes of various sizes. Reprinted from [239].

  size-dependent way due to the effect of elastic lift. Viscoelastic fluids are non-Newtonian fluids that show nonlinear viscosity and elasticity under the effect of shear stress. The impact of viscoelastic flow sorting is determined by the viscoelastic medium. For example, Liu et al. used poly-(oxyethylene) (PEO)

Figure 21 :

 21 Figure 21 : (A) Schematic of a viscoelastic separation of exosome from large EVs. (B) Separation mechanism in viscoelastic microfluidics. The sheath fluid aligns the sample fluid along the sidewalls. Exosomes and large EVs are driven toward the microchannel centerline by elastic lift forces caused by fluid viscoelasticity, resulting in their size-dependent separation. Reprinted from [240].

  The particle separation concept in a DLD chip is that the flow path of particles bigger than the Dc is altered while the path of smaller particles remains unchanged. Inspired by the DLD technology introduced by Huang et al. in 2004 [242], Santana et al. developed a device to extract exosomes from a heterogeneous EV sample pre-purified from cell culture media [243]. In another paper, Wunsch et al. showed the enrichment and separation of EVs with diameters down to 20 nm at the nanoscale using DLD (Nano-DLD) which yielded rates of 10 4 -10 6 particles per minute. [244] (Fig. 22).

Figure 22 :

 22 Figure 22 : Schematic representation of the nano-DLD device which is in the form of a matrix formed by a network of pillars spaced from 25 to 235 nm and a maximum migration angle θmax = 5.7 °. Trajectories for particles with a particle diameter (DP) less than the nominal critical diameter (Dc) follow laminar flow in zigzag mode (red), while larger ones with DP ≥ Dc follow a deviation θmax in a bumping mode (blue). Reprinted from [244].

  approach employs ultrasonic waves to provide differential acoustic stress on particles in order to separate them depending on size and density. Based on this concept,Lee et al. employed acoustic waves to separate EVs from cell culture media[245]. The device consists of a pair of interdigital transducers to generate a standing surface acoustic wave. Particles in the acoustic region experience radial pressure and migrate. Larger vesicles move faster because the acoustic force is proportional to the volume of the vesicle. Wu et al. described an integrated acoustofluidic device that included a microscale cell-removal module, which eliminates bigger blood components [246] (Fig.23). In an automated way, this platform was able to isolate EVs directly from undiluted blood samples with both high purity (98.4%) and efficient recovery (82.4%).

Figure 23 :

 23 Figure 23 : Schematic of the acoustofluidic module for separation of EVs from whole blood. The cellremoval module removes red blood cells (RBCs), white blood cells (WBCs), and platelets (PLTs), and the EVs-isolation module separates them. Reprinted from [246].

Figure 24 :

 24 Figure 24 : Schematic illustration of the thermophoretic mechanism of accumulation of EVs. Reprinted from [247].

Figure 25 :

 25 Figure 25 : Electrokinetically driven techniques for (A) EV isolation through a selective membrane or monolithic support; and (B) EV separation and characterization employing either non-uniform (dielectrophoresis) or uniform electric fields (for microscale electrophoresis). Reprinted from [251].

Figure 25 :

 25 Figure 25: Schematic representation of the thesis project and on the two modules I focused on during my thesis

  nm), we found it interesting to test the possibility to perform TDA either directly after EVs isolation or for subsequent coupling of CZE-TDA in the same capillary, allowing for the determination of the hydrodynamic radius of each sample zone previously separated by CE. Indeed, when I started my thesis, no work reported the use of CE for the identification and quantification of EVs after their isolation. During the course of my thesis, only another CE-based method [255] was disclosed on bacterial EVs and shortly before the publication of our research. In this work authors did not propose to separate and distinguish EVs from different origins. In our hands, laser induced Fluorescence (LIF)

  cysteine and amino groups[258]. Then, being the first group to explore CE on EVs, I had to figure out how to avoid EV lysis and prevent the formation of EV aggregates under high voltages. At the same time, attention had to be paid on possible EV adsorption to the silica capillary wall that could result in some resolution loss. Moreover, even though LIF is still one of the most sensitive and selective detection methods in CE, various protein biomarkers may be visualized at low amounts in an EVs sample[259]. In addition, EVs concentration after extraction from complex biofluids may be very low and vary significantly among biofluids, ranging from 10 7 particles/mL for cerebrospinal fluid (CSF) to 92 10 8 -10 9 particles/mL for plasma and blood[260]. As a result, we investigated several ways to overcome these difficulties and in particular to try to gain sensitivity. The first one relied on the use of new BGEs and the another on investigating electrokinetic preconcentration techniques which are based on differences in the analyte migration velocity across discontinuous solution interfaces. The analytes dissolved in a different sample matrix than the BGE are accumulated (stacked) at the boundary of the two solutions.

Figure 26 :

 26 Figure 26: Schematic representation of the instrumentation used in CE.

Figure 27 : 1 ) 2 )

 2712 Figure 27: Electroosmotic flow of positive ions through the capillary with negative charged walls.

Figure 28 :

 28 Figure 28 : Principle of separation in capillary zone electrophoresis.

  (ζ) potential. When a rigidly charged sphere is immersed in an electrolyte solution, an ion cloud forms around it, perfectly compensating for its charge. The potential ζ is defined by the potential difference between the charged surface of the NP and the solvent molecules far from the particle. This can be determined from the measurement of electrophoretic mobility µep [272, 273] depending on the value of κRh, with Rh being the hydrodynamic radius of NP and κ the inverse of Debye's length (equation 6):

( 8 )

 8 Between the two, analytical approximations have been developed to obtain the potential ζ [274, 275].

Figure 29 :

 29 Figure 29: Principle of preconcentration by LVSS in a silica capillary (in the presence of EOF) for EVs. 1. Hydrodynamic injection of a very large sample volume (100%) with low conductivity. 2. Application of a voltage (25 kV) and creation of a discontinuous electric field in the capillary with accumulation of EVs at the sample/BGE boundary. The generated EOF begins to eliminate the matrix 3. Elimination of almost all of the matrix 4. Reversal of polarity. 5. Conventional separation by CZE.

Figure 30 :

 30 Figure 30: LVSS-CE-LIF of fluorescently labelled EVs after matrix substitution in Tris/CHES 5 mM, using ISF BGE Tris/CHES (IS 140 mM, pH 8.4) for both preconcentration and CZE steps. Other CE conditions: uncoated fused silica capillary with I.D. of 50 mm, effective length (leff) of 50.2 cm and total length (Ltot) of 60.2 cm; Applied voltage: 25 kV; LIF detection λex 488 nm, λem 520 nm.

Figure 31 :

 31 Figure 31: LVSS-CE-LIF of fluorescently labelled EVs after matrix substitution in Tris/CHES 5 mM in combination with (A) sucrose (30 mM) + dextrose (145 mM); (B) mannose at 100 mM and 200 mM; (C) trehalose at 25 mM, 75 mM, 100 mM and 200 mM, using ISF BGE Tris/CHES (IS 90 mM, pH 8.4) for both preconcentration and CZE steps. The dashed lines represent CE-LIF of fluorescently labelled EVs (see conditions in paper 3). Other CE conditions as described in Fig. 26.

  preconcentration of freshly injected EVs during each cycle. As for classic LVSS, multiple LVSS procedure was applied to a sample of fluorescently bovine milk-derived EVs resuspended in in Tris/CHES 5 mM and trehalose 200 mM. ISF BGE Tris/CHES (IS 90 mM, pH 8.4) was used for all preconcentration cycles and CZE steps. Labelled EVs were analyzed in a fused silica capillary having ID of 50 µm, effective length (Leff) of 50.2 cm and total length (Ltot) of 60.2 cm.

Figure 32 :

 32 Figure 32: Schematic principle of a multiple LVSS for EVs. (1) Sample injection equivalent to 100% of the capillary volume, (2) application of preconcentration potential (reverse polarity), (3) Second hydrodynamic injection, (4) Second process of preconcentration, (5) Separation and detection of EVs. Steps 3 and 4 may be repeated several times (cycles) for further preconcentration of freshly injected EVs during each cycle. Other CE conditions: uncoated fused silica capillary with I.D. of 50 mm, effective length (leff) of 50.2 cm and total length (Ltot) of 60.2 cm; Applied voltage: 25 kV; LIF detection λex 488 nm, λem 520 nm.

Figure 33 :

 33 Figure 33: Multiple LVSS-CE-LIF of fluorescently labelled EVs after matrix substitution in Tris/CHES 5 mM and trehalose 200 mM. For the first injection, the capillary is totally filled with the sample. For the subsequent cycles, (A) 90% and 50% of sample are injected for second and third cycles respectively. (B) 50% of sample is injected for second cycle of preconcentration. ISF BGE Tris/CHES (IS 90 mM, pH 8.4) for both preconcentration and CE steps. CFDA-SE indicate the region of migration of the dye. Other CE conditions as described in Fig. 28.

Furthermore

  NTA can hardly detect particles of sizes smaller than 50 nm. Other methods like electron microscopy, flow cytometry, and atomic force microscopy should be used to verify the data obtained by these systems.TDA is an absolute and simple separative technique based on dispersion for calculating the diffusion coefficient (or hydrodynamic radius) from the peak dispersion observed in an open tube underPoiseuille laminar flow conditions[289]. The diffusion coefficients of a broad range of structures, including viruses and NPs, have been determined using TDA[276,[290][291][292][293]. However, determining the mean collective diffusion coefficients (and mean hydrodynamic radii) of EVs has never been explored

  refers to the dispersion that occurs when a molecule is exposed to laminar flow in a tube with little diffusion along the tube. Its principle is shown schematically in figure34. The velocity profile of laminar flow in a circular section tube is parabolic (represented by blue arrows). The fluid's speed is the strongest in the middle of the tube and almost null towards the walls. When a sample is injected by pressure, the species in the middle of the tube move faster than the species at the walls, resulting in the formation of a concentration gradient.

Figure 34 :

 34 Figure 34 : Principle of Taylor dispersion analysis in cylindrical microchannel with parabolic flow profile. The distribution D moves at velocity u between the center of the capillary and the walls. u0 is the mean fluid velocity defined as the fluid velocity averaged over the cross section of the capillary.

Figure 35 :

 35 Figure 35: Schematic form of a taylorgram according to the size of the species detected. dh indicates hydrodynamic diameter, σ the temporal variance of the peak and t0 the average residence time peak.

Figure 36 :

 36 Figure 36: Representation of the experimental parameters adapted to a capillary with an internal diameter of 75 µm and a length of 70 cm. The area coloured in green represents the theoretically measurable hydrodynamic radii for a given mobilization pressure. From Labied's thesis (http://www.theses.fr/s218954).

capillaries, d '

 d Orlyè et al. determined the mean collective diffusion coefficients of maghemite (γ-Fe2O3) NPs in the range of 6-10 nm diameter [291]. Compared to DLS data, TDA particle mean solid radii were lower owing to more particle contribution of the largest particles in light scattering. Furthermore, several research groups have achieved successful on-line CE-TDA integration for nanoparticle characterisation [300-303]. Oukacine et al., for example, used CE-TDA to determine the Rh of a bimodal mixture of nanolatexes (56 and 70 nm in size) [302]. Before TDA could be done, a baseline separation of the two nanolatexes using CE was necessary. In this investigation, a UV detector and capillary with three detection windows in a looping design were employed. The reported values of D agreed well with the values obtained by TDA of the two nanolatexes separately, confirming CE-TDA as a feasible

Figure 37 :

 37 Figure 37: TDA-UV: Representative taylorgram (black line) and corresponding fit (red line) for bovine milk-derived EVs. Experimental conditions: uncoated fused silica capillary (50 μm i.d., length 60 cm); buffer PBS 1X, pH 7.4; injection 0.4 psi 9 s; applied pressure 0.4 psi; the wavelength used was 200 nm.

Figure 38 :

 38 Figure 38: TDA-LIF: Representative taylorgram (black line) and corresponding fit (red line) for bovine milk-derived EVs. Experimental conditions: uncoated fused silica capillary (50 μm i.d., length 60 cm); buffer PBS 1X, pH 7.4; injection 0.4 psi 9 s; applied pressure 0.4 psi; λex 488 nm, λem 520 nm.

Figure 39 :

 39 Figure 39: TDA-UV (on the left) and TDA-LIF (on the right): Representative taylorgram (black line) and corresponding fit (red line) for (A)(B) human plasma EVs isolated with affinity monolithic disks; (C)(D) standard human plasma EVs from HansaBioMed. Experimental conditions: uncoated fused silica capillary (50 μm i.d., length 60 cm); buffer PBS 1X, pH 7.4; injection 0.4 psi 9 s; applied pressure 0.4 psi; UV: 200 nm; LIF: λex 488 nm, λem 520 nm.

  the Chapter IV, I explored and developed several CE-based approaches, serving as the downstream microscale separation module of the analytical droplet platform for EVs. These methods allow now to characterize EVs profile, serving for EVs identification and representativeness for further diagnostic and theranostic applications. In the second part of my thesis, I focused on the development of the first module of this platform in order to isolate and purify EVs from complex matrices (typically biological fluids) and concentrate them to a sufficient extend for downstream EVs analysis and / or characterization. Several isolation strategies using various mechanisms have already been discussed in chapter II.1.. Briefly, the four most popular isolation procedures are ultracentrifugation (UC), sizebased separation (such as size exclusion chromatography or ultrafiltration), immunoaffinity, and precipitation-based methods. However, none of existing approaches can satisfy all criteria of high purity, high isolation yield and high throughput (via automation).The use of microfluidic techniques has gained more and more attention, as it brings new functionalities, not available with macroscopic techniques, and offers the ability to minimize sample amount, cost, reagent consumption, speed up operations, and execute several tests in small devices at the same time. Microfluidic approaches hold the potential to meet all aforementioned requirements for EVs purification. Several examples of microfluidic-based devices for such purpose were covered in Chapter III. Among all branches of microfluidics, we paid particular attention to droplet-based microfluidic platforms. Droplet-based microfluidics may be defined as the science and technology of creating, manipulating, reacting, analyzing, and screening micro to femtoliter microreactors that are employed as discrete functional units[305, 306]. Such microreactors (typically in aqueous phases) are confined and separated by immiscible phases (typically with fluorinated oil for different bioapplications). This technology can be divided into two main categories: "Stationary Droplet Microfluidics", in which the solutions are typically manipulated by pipetting, without the use of external pressure or flow control systems, and "Dynamic Droplet Microfluidics," in which electrical potentials or pressure-driven flows can be used to actuate the droplets[306]. For the work in this thesis, we focused on pressure-driven flows, often known as "Droplet Microfluidics" [307] or also renamed "Continuous Droplet Microfluidics"[306]. Droplet microfluidics systems generally mix two (or more) immiscible fluids in microfluidic channels, resulting in controlled emulsions of one phase within the other. This ensures consistent compartmentalization of sample and reagents while decreasing, in comparison to standard lab-scale techniques, both the amount of liquid required for analysis and minimizing the risks of contamination with the surrounding environment.

  Droplet microfluidics is a very effective technique for creating and controlling very small quantities of fluid (μl to fl). Droplet microfluidics is based on the use of two immiscible fluid, must often oil and aqueous solutions. These droplet emulsions are often generated by specific channel geometries, such as T-junctions and flow-focusing, or by pipetting robots (as employed in the present work), and are conveyed in a pressure-driven flow[312].Droplet microfluidics was selected in this work to enable the development of our platform for the separation, preconcentration, and characterization of EVs (Droplet-CE). Unlike conventional continuous flow microfluidic systems, which are limited by the number of steps and operations that can be made in series because it implies necessarily a device size increases (almost linearly with the number of operation needed for a sample treatment/preconcentration/enrichment for instance), droplet-based systems may execute a high number of steps without increasing the device size nor its complexity. Moreover, droplet-based microfluidics allows for independent control of each droplet, resulting in individual micro-reactors that may be transported, mixed together, split, and analyzed separately. Droplets that act as bridges and carriers of EVs from the sample, where they are diluted and confined with other species, until their injection into CE systems, as isolated and enriched EVs sample, may overcome the limitations of working dimension mismatch and low module integration.

Figure 40 :

 40 Figure 40: On the left, schematic drawing of droplet train containing solution of B: magnetic beads; S: sample; W: washing and E: elution. On the right, schematic drawing showing the magnetic tweezers technology.

Figure 41 :

 41 Figure 41: (A) Schematics of the device proposed by Lombardi and Dittrich. Emulsified in oil is an aqueous solution comprising magnetic beads coated with human serum albumin (HAS_MBs) that bind warfarin. The droplets are symmetrically divided at a T-junction, but the addition of an external magnet causes the magnetic beads to be concentrated in half of the initial volume. Reprinted from [314] with permission. Copyright (2011) Analytical and bioanalytical chemistry. (B) Sequence of images showing the magnetic tweezers technology. a) Photos of the paramagnetic tip placed in the coil and close to the capillary, b) to h) sequence of extraction and redispersion of magnetic beads in droplets by turning on and off the magnetic field while droplets are passing in front of it. MB act as carriers to transport analytes from one droplet to another. Reprinted from [315].

Figure 42 :V. 3 .V. 3 . 1 .

 42331 Figure 42: Schematic drawing of the microfluidic droplet configuration. D is for detection droplet; W stands for washing droplet; S stands for sample droplet; B stands for magnetic bead droplet; and A stands for air bubble. Oil separates the droplets. Reprinted from [311].

Figure 43 :

 43 Figure 43: NTA profiles for (A) bovine milk-derived EVs and (B) human plasma-derived EVs. (a) Starting pre-purified EVs sample profiles. EV elution from Dynabeads-CD9 with EXOFLOWBUFR-2 solution, and under alkaline condition (pH 11.3) with Na2CO3/NaHCO3 or NH4OH. PSD: particle size distribution. NTA histograms represent the mean of three replicate measurement of the same sample and SD in red.

Figure 44 :

 44 Figure 44: NTA profiles for human plasma-derived EVs isolated with Dynabeads-CD9 from pre-purified samples. Incubation with (A) 20 µL and (B) 40 µL of magnetic beads in 100 µL final volume. Elution under alkaline condition (pH 11.3) was performed with ammonium hydroxide. NTA histograms represent the mean of three replicate measurement of the same sample and SD are indicated by the red lines.

  Rather than focusing exclusively on one type of phospholipid,Wan et al. developed a lipid nanoprobe composed of DSPE (1,2-distearoyl-snglycero-3-phosphethanolamine)-PEG-desthiobiotin, which contains two hydrophobic fatty acid tails, for the rapid isolation of EVs from cell culture supernatant and plasma [316]. Non-covalent interactions between the two hydrophobic fatty acid tails of DSPE and the lipid membranes of EVs allow DSPE to be introduced into EV membranes. Following labelling with the lipid nanoprobe, EVs were collected by Neutravidin-coated magnetic particles within 15 min and released by DSPE-PEG-desthiobiotin displacement with biotin. There were no discernible variations in the contents of EVs (DNA, RNA, and proteins) recovered by ultracentrifugation against those obtained by the lipid nanoprobe. This method eliminated the need for recurrent ultracentrifugation purification stages since the one-step separation procedure removes 68 % of proteins. However, this technique might affect the physicochemical properties of separated EVs since released EVs will bear the lipid nanoprobe employed to capture them on their surface. Regarding the approaches using biophysical or chemical properties: Kim and Shin developed a MBbased ion exchange commercial kit (ExoCAS-2) for the isolation of EVs from blood plasma within 40 min [317]. The EVs were separated by electrostatic interactions between negatively charged EVs and positively charged poly-L-lysine-coated cationic beads. The EVs bonded to the surface of the coated beads and are then flushed using an elution solution with varying ionic strengths to break the electrostatic interactions. When compared to ExoQuick (exosome commercial precipitation kit), NTA indicated that the EVs concentration for ExoQuick (2.25x10 11 EVs/mL) was approximately two-fold that

  Measurement concentrations were obtained by pre-diluting the samples to the ideal particles/frame. Each experiment was performed in duplicate on 11 different positions within the sample cell with following specifications and analysis parameters: cell temperature 25°C, sensitivity 70, shutter 100, Max Area 1000, Min Area 10, Min Brightness 25. The results were validated while obtaining at least 1 000 valid tracks for each run. For data capture and analysis, the Nanoparticle Tracking Analysis Software (ZNTA) vs 8.05.04 was used.

  Briefly, fluorescently labelled EVs were prepared using the 5-(and-6)-Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE). After removal of residual CFDA-SE via filtration with commercial Exosome Spin Columns (MW 3000, Thermo Fisher Scientific Waltham, MA USA), labelled EVs were analysed with CE-LIF using a fused silica capillary having I.D. of 50 µm, effective length (Leff) of 50.2 cm and total length (Ltot) of 60.2 cm. The capillary was preconditioned with water for 10 min, 1 M NaOH for 10 min, 1 M HCl for 10 min and then water for 10 min. The rinsing between two analyses was carried out with 50 mM SDS for 5 min, 1 M NaOH for 5 min, deionized water for 5 min, and finally the running BGE composed of Tris / CHES (IS 90 mM, pH 8.4) for 5 min using a pressure of 30 psi. EVs samples were injected hydrodynamically from the inlet end by applying a pressure of 0.5 psi for 2 min. The separation was carried out under 25 kV (normal polarity) at 25 °C and the samples were kept at 5 °C with the sample storage module of the PA 800 Plus equipment.

Figure : Fig. 1 .

 :1 Figure:

Fig. 3 .

 3 Fig. 3. Schematic drawing of microfluidic droplet system. B: droplet containing magnetic

Fig. 4 .Fig. 5 .Fig. 6 .

 456 Fig. 4. Comparison between (A) Nanosight NS300 (Malvern) vs (B) Zetaview NTA (Particle

Figure S1 .

 S1 Figure S1. (A) Transmission Electron Microscopy with negative staining by uranyl acetate of bovine

Figure S2 .

 S2 Figure S2. NTA profiles for optimization of PEG-based EVs precipitation method in tube. I:

Figure S3 .Figure S5 .

 S3S5 Figure S3. Photos of beads behavior inside a droplet during incubation with back-and-forth motions in

Figure S6 .

 S6 Figure S6. NTA measurements for EVs isolated from (A) pony serum and (B) pony plasma with the

Table A :

 A

Representative table showing a selection of completed and ongoing clinical trials with the term "exosome" (from http://clicinicaltrials.gov, November 2021). Table B: Representative table showing a selection of completed and ongoing clinical trials with the term "extracellular vesicle" (from http://clicinicaltrials.gov, November 2021).

Table 1

 1

provides a list of relevant biomarkers from EVs found in different biological fluids, which have been investigated in individuals with different pathologies. It is interesting to note that EVs are currently being explored mostly in cancerology and neurology. In addition, it can be noted that the EVs markers used are, in most cases, genetic material and more particularly microRNAs.

Table 1 :

 1 Biomarkers from EVs used in the diagnosis of different pathologies.

  Busatto et al. reported that TFF resulted in a one to two orders of magnitude improvement in EV

	recovery per million cultivated cells (~ 10 10 EVs/10 6 cells vs 10 8 EVs/10 6 cells) and improved batch-to-
	batch repeatability when compared to ultracentrifugation. Albumin removal was also superior by a
	factor of 40 [156]. Sequential centrifugal ultrafiltration may also fractionate EVs. In the procedure
	reported by Xu et al., two EV subtypes were identified from a human colon cancer cell line by using
	consecutive hydrophilic polyvinylidene difluoride membranes with 0.65, 0.45, 0.22, and 0.1 μm pores
	[157]. Finally, while ultrafiltration alone may be adequate for EV separation in certain circumstances,
	combined to other methods (such as size exclusion chromatography), it may bring extra purity and
	selectivity [158-160].

II.1.4. Polymer-based precipitation

	Isolation of EVs by precipitation is a preferred solution for users with limited technological resources
	in the laboratory. Water excluding polymer, mostly polyethylene glycol (PEG), have recently been
	employed to provide an affordable and efficient alternative for EV isolation. Numerous commercial
	exosome isolation kits are now available, such as ExoQuick (System Biosciences), Total Exosome
	Isolation (TEI) Reagent (Life Technologies), ExoPrep (HansaBioMed) and Exosome Purification Kit
	(Norgen Biotek), for EVs isolation from plasma, serum, cerebrospinal fluid and cell culture medium.
	These commercial reagents generally reduce the hydration of EVs (and therefore their solubility) to
	cause their precipitation. Next, the precipitated EVs can be isolated easily and reproducibly with low
	centrifugal forces, avoiding long ultracentrifugation. PEG with molecular weights ranging from 6000 to
	20000 Da are commonly used in existing polymer-based EVs precipitation techniques [164-167]. From
	a technical point of view, PEG-based technique is relatively simple to use, quick and do not require
	special equipment. PEG demonstrated 10 times higher yield than ultracentrifugation starting from 2
	mL of human serum volume (4x10 10 particles/mL with PEG) [164]. However, its main disadvantage is
	the co-precipitation of non-vesicular components and the presence of polymers in the final solution
	[144]. Polymer-based precipitation of EVs is therefore limited if it is not coupled with more resolving
	techniques. The aqueous two phase system (ATPS) has been shown to isolate EVs with higher purity.
	Once the sample has been thoroughly mixed with the ATPS solution containing PEG and dextran, the
	mixture is centrifuged [168]. The EVs then migrate to the dextran phase, which has surface
	characteristics that are more favorable to EVs. However, because EVs are recovered in a dextran-
	containing solution that may interfere with standard analytical methods like Western Blotting or RNA
	Takov et al. recently evaluated ultracentrifugation and qEV using rat plasma and found that separation, further washing steps are required to decrease the amount of dextran in the final
	SEC achieves a greater particle yield based on the estimation of the concentration of two EVs markers suspension [168, 169].
	(CD81 and HSP70) than ultracentrifugation (ultracentrifugation yield was barely a fourth of the EVs-
	containing SEC fraction yield). However, the SEC-derived EVs exhibited much worse purity, resulting in
	a 30 times higher concentration of a lipoprotein marker (APOB) compared to ultracentrifugation
	samples [162]. Purity was increased by using two Sepharose SEC columns with identical stacking
	volumes but differing column lengths (56 mm and 222 mm). While EV particle size distribution and
	yields were the same, protein and immunoglobulin contamination was reduced by 90% [163]. It is
	worth noting that SEC protects more EV functionality and integrity thanks to the use of gentle mobile
	phases like PBS. In this thesis we have principally worked on EVs provided by Excilone, which employs
	qEV columns to isolate EVs from pony plasma / serum and human plasma. Paper 3 in Chapter IV.4. will
	go into further depth.

  of EV-associated RNA, and numerous approaches have been used to measure and identify distinct types of extracellular RNA in EVs[218, 219]. Conventional nucleic acid extraction and analysis methods have been utilized successfully to establish a solid foundation for our understanding of EV nucleic acids. This analysis consists of the extraction of vesicular RNA using cell-like techniques, followed by conversion to DNA, amplification, and sequencing. A target sequence can be amplified selectively using polymerase chain reaction (PCR) and identified using end-point electrophoresis or real-time fluorescence techniques (RT-PCR) [220]. With a rising interest in using EV nucleic acids as non-invasive diagnostic markers, new biosensor technologies have been developed to allow for more efficient and faster extraction and analysis. Taller et al. developed an ion exchange nano sensor. It consists of two microfluidic systems connected in series. The first enables EV lysis through surface acoustic vibrations. The second uses an ion exchange nanomembrane to detect RNA. By applying an

	time, it may enable high throughput, quantitative, and comparative proteomic analyses of EVs. Mass
	spectrometry has been used to characterize thousands of vesicular proteins [212], which have been
	compiled in databases such as EVpedia [213], ExoCarta [214] and Vesiclepedia [215]. In addition, other
	MS-based workflows have been conducted to investigate the lipid and phospholipid content of EVs
	[216, 217].
	II.2.2.3. Nucleic acid analysis
	In addition to proteins, EVs include several types of RNAs (messenger RNAs, microRNAs and small non-

coding RNAs). The amounts of RNA in biofluids are relatively low, 20-50 ng/mL of total plasma and 5 ng/mL electric current across the membrane, anions are forced through the membrane, resulting in a corresponding current-voltage characteristic (CVC) recorded across the membrane. By functionalizing the membrane surface with capturing oligonucleotide probes, changes in CVC measures may be sensitively and quantitatively detected.

Table 2 :

 2 Immunoaffinity-based microfluidic platforms for isolation of EVs referenced in the literature

	from 2010.
	Method	Sample	Volume	Recovery yield (%)	Ref
			(μL)
	Micro-channels coated with anti-	Serum	400	42 -94	[222]
	CD63
	Mica surface coated with anti-	Plasma	150	Not communicated [223]
	CD41
	ExoChip: multi-channel coated	Serum	400	Not communicated [224]
	with anti-CD63
	Capture via functionalized	Serum	500	Not communicated [225]
	electrodes (CD9, HER2)
	Filter paper coated with anti-	Serum	72	Not communicated [226]
	CD63 antibodies and annexin V
	Microchannel to mix with	Plasma	30	Not communicated [227]
	magnetic beads conjugated with
	anti-EpCAM, IGF-1R, CA125,
	CD9, CD63, CD81
	surface plasmon resonance	Ascetic	150	Not communicated [98]
	platform (nPLEX) functionalized	fluid
	with anti-CD63
	RInSE: anti-EpCAM coated	Plasma Continuous	Not communicated [228]
	polystyrene beads		flow
	iMER: magnetic microbeads	Serum	100	> 93	[229]
	coated with anti-EGFR
	ExoSearch: micro-sized magnetic	Plasma	20	72	[230]
	beads coated with anti-CD9
	Nano-IMEX: Y-shaped micro rods	Plasma	20	Not communicated [231]
	coated with anti-CD81
	Functionalized SPR chip with anti-	Serum	250	Not communicated [232]
	CD9 and/or anti-CD63

1.2.1. Microfluidic filtration Isolation

. They can be divided into two broad categories: active and passive modes. Passive techniques depend mostly on vesicle intrinsic properties (e.g. size or electrical properties). On the contrary, active separation techniques rely on the interaction of intact vesicles with an external force field. Different typical examples of passive techniques are given below. Typically, electrical fields and magnetic fields can be used to manipulate EVs. Readers can refer to section III.2. of this chapter to have some examples of these passive technologies. For exhaustive view of all passive and active strategies, readers can glean from recent reviews [221, 237, 238]. III.based on size is an appealing method for separating EVs from big cellular detritus. Several microfluidic filter methods have been designed to separate EVs from big cellular detritus and protein aggregates. For example, exosome complete isolation chip (ExoTIC) developed by Liu et al. is made up of several plastic layers and nanoporous filter membranes with different pore sizes [239] (Fig. 20).

  specific properties, numerous criteria must be evaluated and determined. D'Orlyé et al. demonstrated some dependence of µep to maghemite (γ-Fe2O3) NPs size in a particular range of 6-10 nm, assuming a uniform zeta potential, at a given electrolyte ionic strength[276]. Charge-based characterization of cationic bifunctional maghemite/silica core/shell NPs by CZE via their µep was possible if NPs of homogeneous sizes could be obtained [277]. Liu et al. demonstrated that the geometry of NPs may affect their electrophoretic mobilities when they used CE to separate a mixture of silver nanorods and nanoparticles [278]. Vanifatova et al. observed that coexistence of different iron oxide NPs populations influenced their electrokinetic migration [279]. Other factors such as BGE ionic strength, pH and composition, applied electrical field, injected NPs quantity have been reported to influenceelectrophoretic mobilities[275]. Of course, the theory on CE of NPs cannot be applied directly to EVs, as the scenario is significantly more complex in the case of EVs. Their size distribution, in particular, is more heterogeneous, ranging from about 30 nm to a few micrometres. Additionally, EVs might differ significantly in terms of their membrane composition. As a consequence, the zeta potential of EVs,

which is determined by their molecular surface composition (lipids, membrane proteins, etc...), may vary within a single EV population. Furthermore, EVs, like cells, may display shape, size and zeta potential variations in different buffer conditions

[280, 281]

. Considering all these challenges, we put our efforts to explore for the first time the power of CE-LIF for elucidation of electrokinetic distribution of EVs. The main achievements are described in the following article 3. To accomplish so, our commercial collaborator Excilone (Elancourt, France) supplied us with bovine milk-derived EVs with narrow size distribution, well-determined concentrations, and extensive characterizations by NTA, DLS, LC-MS / MS, and TEM. These EVs isolated by sucrose gradient ultracentrifugation were considered as EV standards for our CE-LIF method development. Excilone also provided us with samples of EVs from other sources, such as pony plasma / serum and human plasma that were isolated by SEC. In addition a collaboration with the Marja-Liisa Riekkola team (INAR Analytical Chemistry) from the University of Helsinki (Finland), enabled us to test our CE-LIF method on human plasma EVs isolated using their newly developed immunoaffinity chromatography technology

[172]

.

  MW 3000) (Thermo Fisher Scientific) were used here both for removal of residual CFDA-SE and for matrix substitution in different low conductivity solutions. Labelled EVs were analyzed in a fused silica capillary having ID of 50 µm, effective length (Leff) of 50.2 cm and total length (Ltot) of 60.2 cm. Our first test was realized with fluorescently labelled EVs substituted in Tris/CHES(IS 5 mM, pH

	8.4) with low conductivity, and ISF-BGE composed of Tris/CHES (IS 140 mM, pH 8.4) with a 28 times
	higher conductivity. As shown in Figure 30 we observed insufficient EV stacking, as well as appearance
	of multiple spikes that were attributed to EVs aggregation/collision during electrophoresis.

-(and-6)-Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE). Commercial Exosome Spin Columns (

  : 520 nm). The fused silica capillary (I.D. of 50 μm, O.D. of 375 µm, effective length (Leff) of 50.2 cm and total length (Ltot) of 60.2 cm) was preconditioned (using a pressure of 25 psi at the capillary inlet)

TDA-UV experiments were carried out with a P/ACE MDQ (Beckman Coulter, Brea, CA, USA) equipped with a UV detection at 200 nm. TDA-LIF studies were performed with a PA 800 Plus system (Sciex Separation, Brea, CA) equipped with a solid-state laser induced fluorescence detector (λex: 488 nm, λem

Table 3 :

 3 Mean hydrodynamic radius of bovine milk-derived and human plasma EVs measured by TDA-UV/LIF by NTA.TDA -UV (Rh in nm) TDA -LIF (Rh in nm) NTA (mean Rh ± SD in nm)

	Bovine milk derived EVs	96.5	111 (~ 75%)	87 ± 26
			21 (~ 25%)
	EVs -affinity monolithic disks	5.14	4.2	64 ± 21
	EVs -HansaBioMed	4.2	4.3	67 ± 22
	IV.6.5. Concluding remarks

  1.4. of Chapter II, PEG is extensively used as a precipitating agent to extract EVs. A MB-mediated selective adsorption technology (MagExo) has recently enhanced the PEG-based isolation approach[318].Fang et al. discovered that EVs could preferentially precipitate on MB with 5% of PEG added to the EVs solutions, whereas most proteins remained in the supernatant. The significant hydrophilic impact of PEG might explain this phenomenon. Indeed, PEG locks up a lot of water molecules in a solution. As a result, the EV dispersion stability changes, forcing them to aggregate and precipitate on the surface of silica MB. The best EV yield and lowest protein residues under optimal conditions were 79 and 12 %, respectively. When EVs extracted from plasma using MagExo were compared to gold standard techniques and other commercial kits, they had the greatest particle yield (103, 3.04, and 2.01 times higher than those of differential ultracentrifugation (DG-UC), SEC, and ExoQuick Ultra, respectively). The second greatest purity (19.2 x 10 10 particles/mg protein) was reached by repeating the full technique twice (two-step MagExo), while the yield was1.60, 2.43, and 82.4 times higher than ExoQuick Ultra, SEC, and DG-UC, respectively.

  It comprises a syringe pump (from Nemesys,

	Cetoni GmbH) equipped with 1 mL glass syringes (purchased from SGE) and a motorized
	pipettor arm (Rotaxys, Cetoni GmbH), used for droplets generation. A 96-well plate (Thermo
	Scientific) was used for sample storage and reagent solutions. The plate was mounted on a
	custom-made holder that can be moved in the X, Y, and Z directions. PTFE tubing with ID of
	0.3 mm and OD of 0.6 mm (Z609692-1PAK, Sigma Aldrich) was used to conduct the droplet
	trains. Fluorinated oil FC-40 mixed with the surfactant (1H, 1H, 2H, 2H -perfluoro-1-decanol,
	2% w/w) was used to separate aqueous droplets inside PTFE tubing. In the protocol, a magnetic
	tweezer, prepared in-house was used. It is composed of a paramagnetic tip activated by a
	magnetic coil. Macro Objective (MLH-10X) mounted on a low-cost CMOS Cameras
	(acA1300-60 gm, Basler) and white LED back light illumination (Schott Lighting and Imaging)

Table 1 :

 1 Operation sequence of the microfluidic droplet protocol using droplets of 6 µL each

	Operation	Droplet composition	Flowrate	Incubation	Back-and-forth
			(μL/s)	time (min)	droplet	travel
					distance (+/-μL)
	Sample incubation	EVs sample + PEG 5%	4	25	6
		(w/v) + magnetic beads (1.5
		mg/mL
	Washing 1	PEG 5% / NaCl 0.2 M	0.5	Flush	-
	Washing 2	PEG 5% / NaCl 0.2 M	0.5	Flush	-
	Sample elution	PBS 1X	4	5	6

  NCT04720599 Clinical Evaluation of ExoDx Prostate(IntelliScore) in Men Presenting for NCT03415984 Prevalence of Age Related Macular Degeneration (ARMD) in Parkinson's Patients and Assesment of the Role of L-DOPA NCT02862470 Anaplastic Thyroid Cancer and Follicular Thyroid Cancer-derived Exosomal Analysis Via Treatment of Lovastatin and Vildagliptin and Pilot Prognostic Study Via Urine Exosomal Biological Markers in Thyroid

	NCT03995836 Epigenetics Modifications in Morbid Obesity and Obstructive Sleep NCT04164134 New Strategies to Detect Cancers in Carriers of Mutations in RB1	Completed Completed Completed Completed Recruiting	•Urologic Cancer •Parkinson Disease •Thyroid Cancer •Obstructive Sleep Apnea •Retinoblastoma	•Diagnostic Test: ExoDx Prostate(IntelliScore) •Diagnostic Test: Color retinography •Device: CPAP •Other: blood draw
	Initial Prostate Biopsy NCT03236675 Detection of Either the EML4-ALK Gene Rearrangements or the T790M NCT04100811 Identification of Clinically Insignificant or Significant Prostate Cancer With the miR Scientific Sentinel™ Platform Cancer Patients Apnea Patients: The EPIMOOSA Study	Active, not Recruiting	•Carcinoma, Non-Small-Cell Lung •Prostate Cancer •Age Related Macular Degeneration •Morbid Obesity •Secondary Primary Malignancies •Epigenetic Disorder After Retinoblastoma	•Diagnostic Test: Optical coherence tomography •Diagnostic Test: Fundus autofluorescence •Procedure: Bariatric Surgery
	EGFR Mutation in the Plasma of Advanced NSCLC Patients NCT04315753 Circulating and Imaging Biomarkers to Improve Lung Cancer Management and Early Detection NCT04357717 ExoDx Prostate Evaluation in Prior Negative Prostate Biopsy Setting NCT03694483 Prostasomes as Diagnostic Tool for Prostate Cancer Detection NCT04323579 Validation of Multiparametric Models and Circulating and Imaging NCT03911999 Exosomal microRNA in Predicting the Aggressiveness of Prostate Cancer NCT03791073 New Biomarkers in Pancreatic Cancer Using EXPEL Concept NCT03941210 Micro RNA as Prediction and/or Prognostic Markers of IRIS in TB-HIV NCT03957252 Validation of ClarityDX Prostate as a Reflex Test to Refine the Prediction of Clinically-significant Prostate Cancer Coinfected Patients in Chinese Patients NCT02063464 Blood Collection From People With Ovarian Cancer NCT04633109 The Obese Taste Bud Study Biomarkers to Improve Lung Cancer EARLY Detection. NCT04617405 Hormonal and Inflammatory Changes During Pregnancy in Women With NCT04921774 Research on Patients With Heart Transplantation	recruiting Recruiting Recruiting Recruiting Recruiting Completed Active, not recruiting Recruiting Recruiting Completed Recruiting Recruiting Recruiting	•Lung Cancer •Prostate Cancer •Prostate Cancer •Lung Cancer •Prostate Cancer •Oncology •HIV Infection •Prostate Cancer •Tuberculosis Infection •Ovarian Cancer •Obesity •Diabetes Mellitus, Type 2 •Cancer of the Ovary •Heart Transplant Rejection	•Other: LDCT (Low Dose CT) •Diagnostic Test: ExoDx Prostate imaging •Diagnostic Test: Genetic analysis for the detection of prostasomes •Diagnostic Test: Blood test: ClarityDX Prostate •Other: Detection of molecular Biomarkers •Procedure: taste bud biopsy •Other: No interventions •Diagnostic Test: oral glucose tolerance test
	NCT04227886 Study on Predictive Biomarkers of Neoadjuvant Chemoradiotherapy for Rectal Cancer NCT03031418 Clinical Evaluation of the 'ExoDx Prostate IntelliScore' (EPI) Glucose Metabolic Disorders. NCT03250078 A Pancreatic Cancer Screening Study in Hereditary High Risk Individuals NCT04661176 Evaluation of the Sentinel™ PCC4 Assay for Diagnosis, Prognosis and NCT03317080 Dynamic Monitoring Circulating Tumor DNA in Surgical Patients With Lung Cancer NCT03334708 A Study of Blood Based Biomarkers for Pancreas Adenocarcinoma Monitoring of Prostate Cancer in Puerto Rico NCT04924504 Mechanisms Behind Severe Insulin Resistance During Pregnancy in Women With Glucose Metabolic Disorders (SIR-MET) NCT03049202 BROnchoalveolar Investigations of Never-smokers With Chronic Obstruction From the Swedish CardioPulmonary bioImage Study	Recruiting Completed Recruiting Recruiting Active, not recruiting Recruiting Recruiting Recruiting	•Rectal Neoplasm Malignant Carcinoma •Chemoradiotherapy •Neoadjuvant Therapy •Predictive Biomarkers •Adenocarcinoma •Cancer of Prostate •Gestational Diabetes •Ovarian Neoplasms •Pancreatic Neoplasms •Prostate Cancer •Overweight and Obesity •Pregnancy in Diabetic •Lung Cancer •Pancreatic Cancer •Diabetes Mellitus, Type 2 •Gestational Diabetes •Overweight and Obesity •Pancreatic Diseases •Insulin Resistance •Insulin Sensitivity •Pregnancy, High Risk •Chronic Obstructive Pulmonary •Pancreatitis Disease •Emphysema •Pancreatic Cyst	•Diagnostic Test: ExoDx Prostate Intelliscore •Diagnostic Test: Taste sensitivity •Diagnostic Test: MRI/MRCP •Radiation: Radiation •Drug: Capecitabine-Irinotecan Combination •Diagnostic Test: miR Sentinel™ PCC4 Assay •Diagnostic Test: olfactory function •Diagnostic Test: Blood Draw •Diagnostic Test: taste bud density •Other: No interventions •Diagnostic Test: Tumor Tissue Collection •Diagnostic Test: anthropometry •Diagnostic Test: Cyst Fluid •Other: biospecimen collection •Other: questionnaires
	NCT04120272 Discovery for Biomarkers and Risk Factors for Postoperative Delirium in Elderly Patients With Spine Surgery NCT03488134 Predicting Prognosis and Recurrence of Thyroid Cancer Via New Biomarkers, Urinary Exosomal Thyroglobulin and Galectin-3 NCT04164966 Development of Novel Biomarkers for the Early Diagnosis of Type 1 Diabetes NCT02226055 An Investigation Into the Cardiovascular Risk and Aetiology of CKDu in Sri Lanka NCT04183530 The Individualized Accurate Diagnosis and Treatment of Chronic NCT03432806 A Study of Imaging, Blood, and Tissue Samples to Guide Treatment of NCT04928534 Cohort Study of Blood Biomarkers for TES Objective Pulmonary Disease(COPD) Patients Based on Multidimensional Data NCT04349189 Venous Thrombosis Biomarkers in Sickle Cell Disease and Sickle Cell Trait NCT03280576 Validation of Progranulin as a Biomarker for Sepsis NCT03984006 Early Detection of Autoimmune Thyroid Heart Disease Via Urinary Exosomal Proteins NCT04948437 Urinary Exosomal Biomarkers of Thyroglobulin and Galectin-3 for Prognosis and Follow-up in Patients of Thyroid Cancer Colon Cancer and Related Liver Tumors NCT04534647 Serological and Urinary Biomarkers in Latin American Patients With Systemic Lupus Erythematosus: GLADEL 2.0 Cohort NCT03971955 CharacterIzation of Adult Onset Autoimmune Diabetes NCT03944603 Longitudinal Innate Immunity and Aging Study NCT04792983 Cognition and the Immunology of Postoperative Outcomes	Recruiting Active, not recruiting Recruiting Completed Recruiting Recruiting Recruiting Recruiting Completed Completed Recruiting Recruiting Recruiting Recruiting Recruiting	•Spinal Disease •Thyroid Cancer •Type 1 Diabetes •CKDu •Arterial Stiffness •Chronic Bronchitis •Copd •Pregnancy in Diabetic •Insulin Resistance •Insulin Sensitivity •Pregnancy, High Risk •Colon Cancer •Chronic Traumatic Encephalopathy •Airway Obstruction •Sickle Cell Disease •Acute Exacerbation of Chronic Obstructive Pulmonary Disease •Venous Thrombosis •Smoking, Tobacco •Traumatic Encephalopathy, Chronic •Traumatic; Encephalopathy, Postcontusional •Sickle Cell Trait •Gender •Thyroid Diseases •Thyroid Cancer •Hypercoagulable State •Papillary Thyroid Cancer •Venous Thromboembolism •Heart Failure •Follicular Thyroid Cancer •Sepsis •Liver Tumors •Lupus Nephritis •Diabetes •Frail Elderly Syndrome •Healthy Older Adults Ages 60-89 •Cognitive Impairment	•Procedure: Spine surgery •Device: Arterial Stiffness Assessment •Other: blood draws •Diagnostic Test: Blood tests, Cognitive function tests, head MRI (plain scan and DTI sequence) examination and head PET (FDG-PET, Tau-PET and Amyloid-PET) examination •Procedure: colectomy or hepatectomy •Diagnostic Test: Fibroscan test •Diagnostic Test: different serum and urine biomarkers •Other: Mixed Meal Test •Other: Dual Energy X-Ray Absorptiometry •Other: No intervention
	NCT03034265 New Biomarkers and Difficult-to-treat Hypertension NCT05004090 Emotional Regulation in Children With ND: the Role of Genomic NCT00331331 The Vitreous Proteome and Inflammatory Mediators in Ocular NCT03870542 Multicenter, Prospective Study for Urinary Exosomal Biomarkers of Variation, Proteomic Patterns, and Early Experience Kidney Allograft Tubulointerstitial Fibrosis Inflammatory Disease Blood Plasma Derived Exosome NCT04529915 Multicenter Clinical Research for Early Diagnosis of Lung Cancer Using	Completed Recruiting Completed Recruiting recruiting Active, not	•Proteinuria •Serum Creatinine •Neurodevelopmental Disabilities •Delirium •Hypertension •Uveitis •Renal Fibrosis •Emotional Regulation •Surgery •Kidney Transplant Failure •Epigenetics •Urinary Biomarkers •DNA Adducts •Vasculitis •Parental Factors •Lung Cancer	(DEXA) •Diagnostic Test: DNA methylation analysis •Procedure: kidney transplantation •Procedure: Adipose Tissue Biopsy •Diagnostic Test: Proteomics analysis •Diagnostic Test: Still Face Paradigm •Diagnostic Test: Exosome sampling
			•Ocular Inflammatory Disease
		237 238 239 240

reveal interesting post-translational modifications of specific glycoproteins contained within EVs extracted from CSF and serum for diagnostic purpose. Although this experiment was unable to be carried out due to time constraints for the thesis, the data reported in the following paper(Morani et al., Electrophoresis, 2019 [268]) highlighted the difficulties when dealing with multi-tagged proteins that could be assimilated to intravesicular proteins.

Acknowledgements

Acknowledgement This work has been financially supported by the Institut Universitaire de France (for M. Taverna, senior member) and the Agence Nationale de la Recherche (ANR, France) with the grant no. ANR-18-CE29-0005-01. The European fellowship for Lucile Alexandre was supported by the project H2020-MSCA-IF-2019 (IMPED 896313). The doctoral scholarship for Marco Morani was supported by the doctoral school 2MIB (Sciences Chimiques: Molécules, Matériaux, Instrumentation et Biosystèmes) -University Paris Saclay. The authors have declared no conflict of interest.

Experimental part

elution, using standard EVs (bovine milk-derived EVs), provided by our commercial collaborator Excilone (Elancourt, France), and human plasma EVs isolated with monolithic disks via immunoaffinity chromatography from a collaboration with the Marja-Liisa Riekkola team (INAR Analytical Chemistry) from the University of Helsinki (Finland). The way these two EVs preparation were obtained are fully detailed in paper 3 (Morani et al., Analytica Chimica Acta, 2020) in chapter IV.4.. Inspired by the method reported by Multia et al. for elution of EVs from a monolithic disk immobilized with anti-human CD9 antibody [172], I tested the same high alkaline conditions to elute EVs from MB (see Materials and methods part of paper 3 in chapter IV. 4. for details). These were compared to a commercial elution solution (EXOFLOWBUFR-2), purchased separately from Ozyme (France), intended to allow elution from MB grafted with either anti-CD9, CD63, or CD81.

V.3.2. Preliminary in-tube experiments to select MB capture and elution conditions

Following the manufacturer's instructions (Invitrogen), 10 µL Dynabeads-CD9 from (2.7 µm dia.) were incubated in 100 µL of bovine milk-derived or human plasma EVs samples overnight at 4°C with gentle shaking to promote attachment of EVs. Next, a magnetic separator was used to remove the supernatant and collect the captured EVs. After washing 3 consecutive times with 300 µL of PBS, the enriched EVs were eluted in 100 µL of either ammonium hydroxide (NH4OH,pH 11.3) or carbonatebicarbonate (Na2CO3/NaHCO3, pH 11.3) solution and neutralized immediately with the addition of 10 μL 1 M HCl [172]. EXOFLOWBUFR-2 was also tested for intact EVs elution. NTA was used to assess the supernatant containing the collected EVs from both pre-purified bovine milk-derived and human plasma-derived EVs (see Fig. 43). The video acquisition was performed using a camera level of 14. Per sample, 3 videos of 90 seconds with a frame rate of 30 frames/sec were captured at 25°C and subsequently analyzed with a threshold set up at 5. Tests were conducted using pre-diluted samples in PBS resulting in particle concentrations between 10 6 and 10 9 particles per ml for best analysis.

V.4.1. Recent alternative MB-based methods to extract intact EVs: a brief revision of the literature

Aside from immunoaffinity MB that target specific protein markers, lipid-related proteins and phospholipids may be regarded "universal" markers and might potentially be used to develop MBbased EV isolations. Additionally, EVs have specific biophysical/chemical characteristics that may exploited to capture them with adapted MB. For instance EVs have a net-negative surface charge owing to the composition of their surface molecules, which include glycans, phospho and sulpho groups (see chapter I.) that can be used for extracting them with an ion exchange mechanism. These kind of characteristics have been exploited to develop recent antibody-free MB-based isolations of EVs, which are summarized in Fig. 45.

Figure 45:

Principle of the two categories of antibody-free MB-based EV capture and elution. It may be done using "universal" markers. Isolation may also be accomplished using biophysical or chemical properties, such as surface charge or EV dispersion stability linked to their solubility.

Regarding the universal markers approaches: In addition to specific surface proteins, phospholipids, which form the basis of EV membranes, might be considered as "universal" markers for EV isolation. This kind of approach was developed by Nakai et al. who employed T cell immunoglobulin and mucin domain protein 4 (Tim 4), a transmembrane protein expressed on macrophages and phosphatidylserine receptor that is Ca 2+ -dependent in its ligand binding [177]. For EV capture in the presence of Ca 2+ , the authors employed the extracellular domain of murine Tim4 fused to the Fc segment of human IgG and immobilized Tim4-Fc protein on MB. By adding a buffer containing a Ca 2+ chelating agent (EDTA), the collected EVs were easily released from the beads. NTA revealed lower mean size for EVs isolated using Tim4-affinity technique compared to ultracentrifugation or polymer precipitation, due to increased population of aggregated or fused EVs. The determination of the Total protein content revealed a two-fold higher yield with polymer precipitation compared to the Tim4affinity approach. Tim4-affinity isolated EVs, on the other hand, were mostly devoid of protein

Abstract

In this study, we present a novel microfluidic droplet-based strategy for high performance isolation of extracellular vesicles (EVs). For EVs capture and release, a magnetic bead-based approach without having recourse to any antibody was optimized in batch and then adapted to the microfluidic droplet system. This antibody-free capture approach relies on the presence of a water-excluding polymer, polyethylene glycol (PEG), to precipitate EVs on the surface of negatively charged magnetic beads. We significantly improved the reproducibility of EV recovery and avoided positive false bias by including a washing step and optimizing the protocol. Well-characterized EV standards derived from pre-purified bovine milk were used for EVs isolation performance evaluation. An EVs recovery of up to 25% estimated with nanoparticle tracking analysis (NTA) was achieved for this batchwise PEG-based approach.

The confirmation of isolated EVs identity was also made with our recently developed capillary electrophoresis (CE) method coupled with laser-induced fluorescent (LIF). In parallel, a purpose-made droplet platform working with magnetic tweezers was developed for translation of this PEG-based method into a droplet microfluidic protocol to further improve the performance in terms of EVs capture efficiency and high throughput. The droplet-based protocol offers a significant improvement of recovery rate (up to 50 %) while reducing sample and reagent volumes (by more than 10 folds) and operation time (by 3 folds) compared to the batch-wise mode.

PEG-based EVs precipitation on magnetic beads using the microfluidic droplet platform

A robotic arm and a 500 μL syringe were utilized to pipette the droplets from separate reservoirs into a PTFE tube in a fully automated manner. To complete one PEG-based EVs precipitation protocol, a train of 5 droplets (each containing 6 μL) confined and separated by oil was required.

It includes: i) one droplet of carboxylate functionalized magnetic beads (1.5 mg/mL), ii) one droplet of EV sample in PEG 5%, iii-iv) two droplets of washing solution (PEG 5%/NaCl 0.2 M) and v) one droplet of elution solution (PBS 1X). Beads were trapped out of one droplet and released into another by electronic triggering of the magnetic tweezer. The incubation was carried out with regular droplet back and forth movements at RT for 25 min. After incubation, two washing droplets are flushed over EVs-bound beads. The EVs-bound beads were then dispersed into the elution droplet with regular droplet back and forth movements at RT for 5 min. The elution droplet containing released EVs was then collected in a tube for downstream analysis.

Nanoparticle Tracking Analysis (NTA) of EVs

Particle concentration and size distribution were determined with a Nanosight NS300 instrument (Malvern,version NTA 3.2 Dev Build 3.2.16) equipped with a 405 nm laser, sCMOS camera type and the NTA software v3.1. The video acquisition was performed using a camera level of 14. Per sample, 3 videos of 90 seconds with a frame rate of 30 frames/sec were captured at 25°C and subsequently analyzed with a threshold set up at 5. The results were validated with at least 2,000 valid tracks for each triplicate. All experiments were carried out with pre-diluted samples in PBS according to input sample concentrations, leading to particle concentration within the 10 6 -10 9 particles per ml range for optimal analysis. developed a washing protocol after the EVs capture step. By adding two consecutive washing steps with PEG 5% w/v to remove residual unbound EVs while maintaining captured EVs on magnetic beads, we significantly improved the repeatability (RSD) to 8 %. This came with some penalty, as the EV isolation yield fell to 17%. To further improve the performance of EVs capture and thus isolation yield, we carried out different optimizations on magnetic bead concentrations (0.5 -1.5 mg/mL), bead chemical surface (with carboxylic or silane groups), incubation temperature for EVs capture (4 -25 °C) as well as PEG concentrations (5-15 % w/v) (see Fig. S2 in the ESI). The highest EVs recovery rate (after 2 washing steps) of 25 % was achieved with the optimized conditions using PEG concentration of 5% and carboxylic magnetic beads' concentration of 1.5 mg/mL with 1 h incubation at 25 °C. Higher PEG or beads' concentrations were also tested but did not lead to better EVs isolation yields.

Conveniently, the working temperature of 25°C is well adapted for subsequent translation of batchwise protocol into a microfluidic format where cooling function is not readily available.

Droplet microfluidics for EVs isolation: proof of concept

The optimised batchwise PEG-based method was subsequently converted into a microfluidic droplet protocol in order to provide a high level of automation and integration, significant reduction in sample/reagent amounts, and a higher performance in terms of isolation efficiency.

The instrumental setup of the purpose-made microfluidic platform, composed of a syringe pump, a motorized pipettor arm for droplet production, a 96-well plate for sample and reagent storage and a magnetic tweezer for manipulation of magnetic beads, is shown in Fig. 3. With this system, we used a train of 6µL droplets containing in a defined sequence (i) the magnetic bead suspension, (ii) EVs sample, (iii) washing and (iv) elution solutions to replace different tube-based steps. When working with the droplet-wise multi-step protocol, one hurdle encountered is the difficulty to efficiently transfer the target species (EVs in this case) from one The many conceivable combinations of this integrated platform's capabilities may allow for a broad range of applications. For example, if magnetic beads were used for non-specific EV isolation, when coupled to CZE-TDA, a screen of the totality of the isolated EVs and potential additional information based on the origin of the sample as well as a quality control would be possible. On the other hand, if one wants to employ particular magnetic beads for capturing specific EVs surface proteins identified as biomarkers of one disease, when coupled to the CZE-TDA, it will provide fresh information on precisely extracted EVs. Again, once the EVs have been isolated using either non-specific or specific magnetic bead-based approaches, a lysis step of the EVs prior characterization is conceivable. If this approach is chosen, the CZE would enable, for example, a screening of a selected intravesicular protein, which may result in the identification of novel potential biomarkers that may serve in determining specific disease progression. It should be noted that CZE-LIF for internal proteins may be somewhat challenging owing to the derivatization phase, which may result in many tagged species.

Therefore, it may be worthwhile to target just one protein or glycoprotein and attempt to profile it, for example. Thus, our platform's potential applications could be useful for the diagnosis or discovery of potential biomarkers for cancer, but as mentioned in the previous bibliographic section, the study of EV alterations, whether on their surfaces or in their content, represent a promising and potential source of biomarkers for a wide variety of diseases such as neurodegenerative diseases, arthritis or diabetes.

APPENDIX

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