Keywords: Surfaces luminescentes pour la detection Fluorescence, Surfaces, Bactéries, Nanoparticules, Microscopie Luminescent Surfaces to Kill or Detect Bacteria Fluorescence, Surfaces, Bacteria, Nanoparticles, Microscopy

ou la lutte contre les bacteries

L'Organisation mondiale de la santé (L'OMS) a récemment signalé que «la résistance aux antibiotiques représente une menace croissante pour la santé et la sécurité publique dans le monde». Au moins 50000 décès annuels, uniquement en Europe et aux États-Unis, sont dus à des infections résistantes aux antibiotiques. Comme proposé par le Centre de contrôle et de prévention des maladies (CDC), quatre actions essentielles sont nécessaires pour prévenir la résistance aux antibiotiques: premièrement, la prévention des infections qui permet la prévention de la propagation de la résistance; deuxièmement, suivre et rassembler des données sur les infections résistantes aux antibiotiques; troisièmement, améliorer la prescription et l'utilisation des antibiotiques. Et enfin, développer de nouveaux médicaments et tests de diagnostic. Afin de participer à l'effort mondial de lutte contre la résistance, deux stratégies différentes seront explorées dans ce manuscrit en suivant deux des quatre actions principales proposées par le CDC: premièrement, par le développement et la caractérisation d'un capteur pour la détection de bactéries; deuxièmement, par l'étude et la caractérisation de nouveaux matériaux antibactériens utilisés pour éviter l'adhésion des pathogènes ou tuer les bactéries, afin de prévenir les infections associées aux biofilms.

Le manuscrit est organisé comme suit:

Première partie:

La croissance bactérienne est souvent associée à une diminution du pH du milieu de croissance en raison de la libération de métabolites acides tels que l'acide acétique, l'acide lactique et le CO2. Par conséquent, différents types de capteurs de pH ont été utilisés pour mesurer la croissance des bactéries. Grâce à des travaux précédents, des nanoparticules fluorescentes (FNP) auto-stabilisées, solubles dans l'eau et ultra-brillantes, facilement synthétisables ont été mises au point. Récemment, ces FNP ont été utilisées pour préparer un nouveau nanocapteur ratiométrique du pH. Notre objectif est de développer des surfaces sensibles au pH, offrant une configuration plus robuste, technologique, transportable et facile à manipuler, capable de détecter les bactéries en quelques heures à l'aide d'un signal de fluorescence ratiométrique. La partie centrale de l'appareil utilisera les nanocapteurs immobilisés préparés à partir de nos nanoparticules fluorescentes (FNP). La synthèse, la caractérisation et la réponse au pH d'un nouveau type de FNP seront présentées (chapitre 2). Par la suite, comme preuve de concept, nous avons commencé à mettre au point des surfaces qui pourront par la suite être embarquées dans un microdispositif : la préparation, la caractérisation et l'étude des surfaces sensibles au pH à l'aide de ces nouveaux FNP seront présentées (chapitre 3).

Deuxième partie:

Un problème majeur de santé publique est lié aux infections associées aux biofilms.

Les bactéries prolifèrent rapidement après avoir adhéré sur une surface, sécrétant une matrice extracellulaire et formant ainsi des biofilms (colonies). Plusieurs stratégies visant à éviter la fixation de bactéries sur les surfaces ont été proposées au cours de la dernière décennie. Parmi celles-ci, deux catégories générales peuvent être distinguées: premièrement, tuer des bactéries par des composés antimicrobiens présentes à la surface (par libération ou contact direct) et deuxièmement, empêcher l'adhésion bactérienne grâce à des interactions physico-chimiques ou à des approches morphologiques.

Nous avons exploré la voie consistant à « repousser » les bactéries et nous avons étudié l'interaction des pathogènes avec de nouveaux films polymères superhydrophobes et luminescents (chapitre 4), ceux-ci se sont révélés efficaces contre l'adhésion

General Introduction

The World Health Organization (WHO) recently reported that 'Antimicrobial resistance (AMR) represents a growing threat to global public health and security'. At least 50000 annual deaths, across Europe and the US alone, are due to antimicrobialresistant infections. As proposed by the Center for Disease Control and Prevention (CDC), four core actions are needed to prevent AMR: first, the prevention of infections and the spread of resistance; second, tracking and gathering data on antibiotic-resistant infections; third, improving antibiotic prescribing practices and use; and lastly, developing new drugs and diagnostic tests. In this manuscript two different strategies will be investigated that follow two of the four core actions proposed by the CDC: first, by the development and characterization of a sensor for bacterial detection; secondly, by the study and characterization of new antibacterial materials as repelling films or bacteria killing surfaces, to prevent biofilm-associated infections.

The manuscript is organized as follows:

First part:

Bacterial growth is often associated with a pH decrease of the growth medium due to a release of acidic metabolites such as acetic acid, lactic acid and CO2. Therefore, different kinds of pH-based sensors have been developed to measure the growth of bacteria. Taking advantage of formerly developed self-stabilized, water-soluble, ultrabright fluorescent nanoparticles (FNPs), we aimed to develop pH-sensitive surfaces, offering a more robust, transportable and easy to handle set up, able to detect bacteria within a few hours by a ratiometric fluorescence signal. The central part of the device will use immobilized nanosensors. The synthesis, characterization and pH-response of new kinds of FNPs is presented (Chapter 2). Later, as proof-of-concept, in an early step to our final goal of developing a point-of-care device: the preparation, characterization and study of pH-sensitive surfaces (Chapter 3) by using these new FNPs will be investigated.

Second part:

A major public health problem in nosocomial environments is due to biofilmassociated infections. Bacteria rapidly proliferate after adhering on surfaces, secreting an extracellular matrix and thus forming biofilms. Several strategies to avoid bacteria attachment on surfaces have been proposed over the last decade. Among these strategies, two general categories can be differentiated: first, surfaces that kill bacteria through antimicrobial compounds (by release of direct contact) and second, repelling bacterial attachment through physical-chemical interactions or morphological approaches.

Relying on the bacteria-repelling surface strategy, we studied the interaction of new, luminescent, superhydrophobic polymer films with bacteria (Chapter 5), which have proven to be effective against bacterial adhesion and thus, biofilm formation.

In addition, considering bacteria-killing surfaces, we determined the efficiency of killing bacteria of a new lithography-printed polymer. The well-organized surfaces consisted on a polymer containing a dye, which was able to release and allow the diffusion of singlet oxygen upon irradiation (Chapter 6).

Part I: Bacterial Growth Detection Chapter 1: State-of-art

Antimicrobial resistance

Bacterial infections have been dangerous for the human species since the beginning of human existence (1). After the discovery of the first antibiotic drug, penicillin, by Alexander Fleming, a huge decrease in the number of deaths caused by infectious diseases occurred (1). Nevertheless, for some antibiotics it took only a few years for bacteria to develop antimicrobial resistance (AMR). For example, methicillinresistant Staphylococcus aureus (MRSA) was reported just one year after methicillin was introduced (1). After almost nine decades of extended use of antibiotics, AMR has reemerged as a major and alarming threat to worldwide health.

In addition to AMR, very few new antibiotics molecules are approved each year for use in the clinic, and according to the World Health Organization (WHO), current clinical developments show that the drugs in the clinical pipeline are mainly modifications of existing antibiotics. This represents only short-term solutions and has been shown to be insufficient against the pathogens on the priority pathogens list of AMR (2).

AMR is caused by different factors, including poor sanitary conditions, inappropriate food handling, poor infection prevention and control practices in hospitals, but mainly it is due to the misuse and overuse of antibiotics in health care and for the breeding of crops and animals (3). AMR is caused by mutations in the DNA, which means that it is triggered by genetic alterations and is heritable (4). Some of the mechanisms bacteria use to resist antimicrobial agents are presented in Figure 1.1. A more detailed description of each mechanism has been described elsewhere (5,6).

As mentioned before, the Center for Disease Control and Prevention (CDC) has proposed four core actions needed to prevent AMR (3):

1. preventing the spread of resistance by the prevention of infections;

2. tracking and gathering data on antibiotic-resistant infections; 3. improving antibiotic prescription protocols and use; Herein, we approached two different strategies of the four core actions proposed by the CDC: first, we aimed to build a diagnostic test by the development and characterization of a sensor for early and sensitive bacterial detection and AMR screening. Secondly, to prevent the spread of the resistance by the study and characterization of new antibacterial materials as repelling films or killing bacteria surfaces, such surfaces might prove to be useful to prevent biofilm-associated infections.

developing new drugs and diagnostic tests

Next, the state-of-art concerning bacterial detection will be presented.

Bacterial Detection i. Traditional Methods

Fast, sensitive and accurate bacterial detection plays an important role for medical diagnostics and for the subsequent correct antibiotic prescription, reducing their misuse.

Ideally, an assay should be able to detect a few specific CFUs within one hour or less, with a low cost and easy manipulation by the user. Conventional techniques to identify bacterial pathogens and the effect of antibiotics against them rely on laboratory assays such as plate counting, which allows one to identify the number of growing cells in a sample by the colony-forming unit (CFU). Moreover, plate counting can be used to determine the minimum inhibitory concentrations (MIC) for the detection of AMR (4). Nevertheless, this approach is time consuming for the user and slow to obtain a result (requiring between 37-48 hours), also in cannot be used for the bacterial strains that cannot be cultured in agar plates (7).

The development of molecular methods such as Real-Time Quantitative Polymerase Chain Reaction (qPCR), has helped to overcome some limitations of the classical techniques to detect bacteria. PCR results for the specific detection of genes responsible for resistance can be obtained within hours (in between 5 to 24 hours), however it does not provide information on the viability of the bacteria (8,9). Viability is critical to monitor the efficacy of antibiotics as well as assessing the presence or absence of viable pathogens in the samples (10). qPCR therefore precisely detects gene expressions but is not reliable for an absolute quantification in microbiology (11).

Mass spectroscopy-based techniques like MALDI-TOF (Matrix-Assisted Laser

Desorption ionization-time of Flight), have emerged as a potential tool for bacterial detection (12). MALDI-TOF is currently used in clinical microbiology laboratories as routine technique for pathogens detection (13). The MALDI-TOF limit of detection (LOD) is as low as 10 5 CFU/ml, using few µL of sample in couple of hours (specimen dependent) (14). A new antibiotic susceptibility commercial test coupled with MALDI-TOF (Sepsityper®) normally requires 10-20 min to be completed but requires a preculturing step, which is adding extra hours to the analysis. Efforts are being done to improve the direct detection of proteins associated with antibiotic resistance, as this technique is being limited by a spectral database of mass fingerprints of bacterial strains (12,15).

Complementary to these techniques, immunological techniques rely on the specific binding of an antibody to an antigen, being widely used for medical diagnosis, such as Enzyme-Linked Immunosorbent Assay (ELISA) (16). Even though specificity is obtained, improvements in sensitivity remain necessary (̴ 10 6 CFU/ mL). While this approach sets the standard for all other assays for bacteria detection, the cost of antibodies remains quite high, in addition to the large reaction volumes required, and to being time consuming for the researcher or medical technician.

ii. Emerging Methods

With the advances in nanotechnology, new biosensors have been developed as an attempt to improve the commonly used techniques described above by implementing different types of nanoparticles. A biosensor must have a recognition element (such as enzymes, antibodies, nucleic acids, etc.) and a transduction element, which translates this interaction into an electrical, optical, thermometric signal (17) (Fig. 1.2). In the last decades, with the recent development in nanotechnology new solutions have been proposed to improve bacterial detection. For example, W. Chunglok, et al. (18), by coating with antibodies single walled carbon nanotubes, they improved detection from a LOD of 10 6 CFU(Traditional ELISA) to a LOD of 10 3 CFU for Salmonella enterica serovar Typhimurium detection.

Another example for an improvement to immunoassays tests was done by Joo et al (19). This was done by implementing magnetic beads (MNPs) conjugated with antibodies specific for Salmonella enterica. After capturing selectively Salmonella over a mixture of proteins, lipids and other bacterial strains, the MNPs-Salmonella complexes were efficiently separated by applying an external magnetic field. In the next step, the MNPs-Salmonella complexes were labeled with TiO2 nanocrystals (TNs), also conjugated with the same antibodies, in order to increase UV absorption. The sensitivity of the assay could thus be increased and has allowed the detection of low concentrations of Salmonella until reaching a detection limit of 100 CFU/ mL in milk. (Figure 1.3). Gold nanoparticles have also been used as biosensors (20,21). Shafiee et al., developed a paper-based biosensor by depositing by capillary effect gold nanoparticles which aggregate, within 10 min, only when E. coli is present in the solution. Specific detection was obtained by covalently grafting Lipopolysaccharide Binding Protein (LBP) on the gold nanoparticles. The presence of the bacteria resulted in a change of color (pink to dark blue) on the paper. LOD was as low as 8 CFU/mL (20).

Quantum Dots have been used as fluorescent markers for bacteria detection (22,23). Wang et al.prepared CdSe/ZnS QDs incorporated into SiO2 spheres to avoid toxicity.

A linear relationship was obtained for the concentration of Salmonella typhimurium and fluorescence intensity, with a final LOD of 100 CFU/mL (22). S. Agrawal et al. (23), developed a multiplexed system for waterborne pathogens (Escherichia coli and Salmonella typhimurium) in a microfluidic circular set-up (Fig. 1.4). First, using mobile magnets, antibodies conjugated with magnetic nanoparticles were entrapped by a magnetic field. In the next step, a mixture of bacteria and nonspecific components (such as other bacteria and proteins) were flowed through the channel. Finally, the captured bacteria were labeled with antibody-conjugated QDs (using different color for each bacterial strain), which were used to obtain an amplified signal.

The fluorescence signal from QDs was directly proportional to CFU/ mL of bacteria. The LOD was 10 3 , which is higher in comparison with Wang et al., but the implementation of a microfluidic system opened many possibilities of use of microchannels to screen different bacterial strains in addition to being reusable. Nevertheless, it is not possible with this technique to detect the viability of bacteria. A real-time measurement on a microfluidic device for single bacteria detection was presented by Yasaki et al. (24), combining ionic current sensing and fluorescence observation (with an epifluorescence microscope). This method could discriminate the bacteria by size (from polystyrene particles) and by using a fluorescent dye it could discriminate between gram positive and gram negative bacterial strains (B. subtilis and E. coli ) in 300 ms. This approach is interesting for real-time measurements, but it is still limited in having to stain the bacteria (however live/ dead test could be implemented).

Jalali et al. (25), proposed a microfluidic device obtained by modifying a gold microelectrodes surface and obtaining hierarchical 3D nano-/ micro protrusions, which were shown to attract bacteria. The introduction of these hierarchical 3D nano-/ micro protrusions improved greatly bacteria concentration and deposition, nevertheless, detection was done using dyes and antibodies to label the bacteria. LOD was 50 CFU/ mL for E. coli and Methicillin-resistant Staphylococcus aureus.

While these biosensors were designed to detect bacteria in a more accurate and faster way (as Shafiee et al., 8 CFU in 10 min), they left behind the measurement of the viability of the bacteria and most importantly, the screening of the resistance of bacteria to antibiotics, which remains one of the priorities to fight against AMR.

iii. Group expertise and approach Different acidic species such as acetic acid, lactic acid and other metabolites are released upon bacterial growth resulting in a decrease of the pH of the growth medium (26). Considering this principle, different pH-sensors have been developed to detect bacteria (as examples: Escherichia coli, Lactococcus lactis, Staphylococcus epidermidis…, 27,28,29). Our research group has been working over the last years on self-stabilized, water-soluble and ultra-bright fluorescent nanoparticles, which are easily synthesized and highly tunable (30,[START_REF] Grazon | Ultrabright Fluorescent Polymeric Nanoparticles Made from a New Family of BODIPY Monomers[END_REF][START_REF] Grazon | Fluorescent core-shell nanoparticles and nanocapsules using comb-like macromolecular RAFT agents: synthesis and functionalization thereof[END_REF]. This work started with the doctoral thesis Chloé Grazon [START_REF] Grazon | Élaboration de nanoparticules fluorescentes à base de BODIPY par polymérisation RAFT en miniémulsion : synthèse, caractérisation et fonctionnalisation de surface[END_REF], by optimizing the synthesis of polymer nanoparticles focusing on Green-BODIPY FNPs (Fig. 1.5). But also investigating the nanoparticles' versatility (fluorescent color core, polymer shell, monomers moiety, grafting of different chemical species on shell). As demonstrated by our group, organic nanoparticles can be smartly designed (besides having a tunable fluorescent core) their shell can provide functional groups to enable post-functionalization and facilitating the grafting over other nanoparticles. Nevertheless, organic nanoparticles remain scarcely investigated. By taking advantage of these FNPs, the following PhD. in our group, Yang Si [START_REF] Si | Fluorescent Nanomaterials for Bioimaging and Biosensing : Application on E.coli Bacteria. Other[END_REF], designed new FNPs without BODIPY in the core (only Styrene), but fluorescein in the shell as pH-sensitive FNPs. Y. Si et al. [START_REF] Si | Rapid and accurate detection of Escherichia coli growth by fluorescent pH-sensitive organic nanoparticles for high-throughput screening applications[END_REF], were able to detect bacterial growth with a low starting concentration (<10 3 CFU/mL, OD: 0.00001) and using only 20 µL. Figure 1.6 explains the central experiment. Assays were done in 96-well plates measuring E. coli bacteria growth incubated either with fluoresceinamine (FA) molecules or with nanoparticles grafted with Fluorescein on the shell (FANP1 and FNP2 correspond to higher and lower concentration of FA grafted on the shell respectively). Measurements were obtained using either optical density (600 nm, dotted lines) or the decrease of fluorescence intensity (%, full lines). The pH decreased from 7 to 5.8 in 300 min as the bacteria grew.

It is clearly seen in Fig. 1.6 that OD measurements are not sensitive enough in the early stages of bacterial growth, besides, the measurements are not reproducible during this phase (from 0 to 100 min). From Fig. 1.6 it can be seen that the slope obtained with FANP1 is higher than that of FANP2 and of the free FA. Quatitatively, the fluorescence intensity of the FANP1 decreased by almost 40 %, more than twice comparing to free FA. With this, FANPs proved to be more sensitive than the currently used molecular sensors (Free FA) but also more sensitive and reliable than typical optical density (OD) meters (Fig. 1.6). Besides bacterial growth detection, screening of bacterial growth inhibition by antibiotics was studied. The inhibitory effect of three antibiotics was measured and compared to OD measurement. pH-sensitive FNPs allow to continuously monitor bacterial growth via real-time detection over long time scales (up to several hours) in small volumes (from one mL to a few µL) and can thus be used for high-throughput applications such as screening for the presence of antibiotic resistant strains (Y. Si et al.

35).

Recently, another publication from our group by C. Grazon et al. [START_REF] Grazon | Core-shell polymeric nanoparticles comprising BODIPY and fluorescein as ultra-bright ratiometric fluorescent pH sensors[END_REF] As previously presented, LOD and time are crucial parameters since bacterial infections from most common pathogens (such as E. coli and S. aureus) pose a major threat even at low concentrations such as 10 to 1000 CFU/ mL in food poisoning, sepsis, peritonitis and skin infections (which may also lead to sepsis) (26,27). The faster and the more accurate (1 CFU/mL) the better. Point-of-care devices could offer a great potential concerning bacterial infections detection, where the common techniques such as ELISA, cell culturing and qPCR are limited. As told by Rajapaksha et al. [START_REF] Rajapaksha | A review of methods for the detection of pathogenic microorganisms[END_REF], a new bacterial detection method should: increase specificity; reliability; the availabity of use; rapid turnaround; low cost; be standardized and high throughput.

Motivated by these previous works, we aimed to develop a point-of-care device (long-term objective), which would offer a more robust, transportable and easy to handle set up, able to detect and identify specific bacteria strains within a few hours by ratiometric fluorescence signal to study antibiotic resistance. The central part of the device will use the immobilized nanosensors prepared from our FNPs. To achieve this, our first goal was to develop a surface sensitive to pH. Before explaining our methodology, we will expose some literature data to give an idea of the context in the following section.

iv. pH sensing surfaces

The development of surfaces to be used as pH-sensors has been subject of interest over the last few decades [START_REF] Lin | Recent development and applications of optical and fiber-optic pH sensors[END_REF]. Grant and Glass prepared a fiber-optic pH sensor by solgel encapsulation of a self-referencing dye, seminaphthorhodamine-1 carboxylate (SNARF-1C) [START_REF] Grant | A sol-gel based fiber optic sensor for local blood pH measurements[END_REF]. Calibration of the sensor was obtained with PBS buffer with a pH from 6.8 to 8 (average increment of 0.12 units), using 2 mL of sample and changing the pH by adding 0.1 M HCl or 0.1 M NH4OH (1-5 µL increments). The response of the sensor was linear in the pH range 6.8-8. Stability of the sensor was observed after 24h, the sensor showed some cracks, but this was improved by adding a coat of polyurethane.

Response of the sensor in blood was similar to PBS, but the intensity ratio decreased. The presence of high concentrations of proton acceptors found in the blood may perturb the ratio and apparent pH values as stated by Whitaker [START_REF] Whitaker | Spectral and photophysical studies of benzo[c]xanthene dyes: dual emission pH sensors[END_REF]. Reproducibility was compared by using 4 different sensors using the same formulation. The difficulty in preparing sensors reproducibly was evident, since the sensors should be calibrated individually before use.

Niu et al, prepared a new ratiometric pH-sensing surface by the attachment of a pH-sensitive porphyrin and a dye as reference (N-2(methacryloxyethyl) benzo[k,l] thioxanthene-3,4-dicaboximide, MBTD) [START_REF] Cg | Fluorescence ratiometric pH sensor prepared from covalently immobilized porphyrin and benzothioxanthene[END_REF]. The immobilization of the dyes was obtained by silanization of the glass surface, followed by the incorporation of a sensing photopolymerized membrane (40µm thick). This surface allowed a linear ratiometric measurement of the pH between 1.0 and 5.0 (pH increments around 0.25), in a reversible manner. Nevertheless, the fluorophores are not commercial and need to be synthesized.

The sensor detected the pH successfully even under the presence of certain ions simulating waste water (ions like Pb 2+ , Hg 2+ , Cd 2+ , Cd 2+ ).

Kateklum et al, compared the pH-sensitivity efficiency of two different aminosilane molecules, grafted with fluorescein [START_REF] Kateklum | Improving the sensitivity of amino-silanized sensors using selfstructured silane layers: Application to fluorescence pH measurement[END_REF]. The first method uses only 3-aminopropyl trimethoxysilane (APTMS), producing a flat available layer of amine. The second method uses first APTMS and then a second layer of 3-aminopropyl dimethoxymethylsilane (APDMS), producing a volume of brush-like structure resulting in more available amine groups (first time reported). Amine quantification was obtained using Fourier Transform Infrared Spectroscopy (FTIR). Buffers with various pH were tested in a range of 6.5 to 8.5 (6.5, 7.0, 7.2, 8.5). Few microliters of buffer were used, leaving about 10 min of reaction time to reach chemical equilibrium. The brush-like configuration proved to be more efficient than a single monolayer of silane by a factor of 5.

A different approach, using thiolene chemistry was done by Craciun et al., immobilizing polymer nanocompartments on glass surfaces [START_REF] Craciun | Surfaces Decorated with Polymeric Nanocompartments for pH Reporting[END_REF]. Glass cover slips were used as substrate and functionalized with a 3-(mercaptopropyl) trimethoxysilane linker according to previously published procedures (Fig. 1.8). Before attachment, the pH responsive dye (pyranine) was encapsulated within nanocompartments and the pH responsiveness of the particles in solution at pH 8 to pH 6 was established. A decrease in the fluorescent signal was observed upon addition of lactic acid (measured pH range 8-6, 0.5 increments) and an increase in fluorescence was observed upon addition of ethylenediamine, a biogenic amine (measured pH range 6 -8). These last two studies were only a proof of concept, without any application, and the measurements were carried out with only one sensing dye as a single-emitter, which can lead to a bias in the results, unlike with ratiometric detection. Rigo et al., used a different approach to immobilize fluorescent nanoparticles on glass, based on click-chemistry (DBCO, figure 1.9) [START_REF] Rigo | Surfaces with dual functionality through specific co-immobilization of self-assembled polymeric nanostructures[END_REF]. They used co-immobilized polymersomes micelle assemblies (polymersomes are made using amphiphilic synthetic block copolymers to form the vesicle membrane). The surface was activated by plasma, followed by silanization. No direct application was shown but the encapsulation of an hydrophobic dye was done as a proof of concept of the loading capability of the polymersomes. The DBCO molecule was covalently attached by peptide coupling. BODIPY (Figure 2.2) is a well-known fluorophore with very good photophysical properties, like usually high fluorescence quantum yields (ΦF ~ 0.7) and good photostability (3). Besides, if designed properly, the spectroscopic properties of BODIPY can be considered independent of the environment or can be designed to be sensitive.

Thus they can be used as tracer or internal reference (for instance in ratiometric systems) (3,4). The extension of the π conjugated system allows the shift of the maximum emission wavelength towards red. This is mainly achieved by substitution in positions 3 and 5 of the pyrrole. Substitutions in positions 1 and 7 would have the same consequences on the spectroscopic properties but are more difficult to achieve due to the conditions of pyrrole synthesis. The insertion of an aromatic group can be carried out by a synthesis of the corresponding pyrrole via a Trofimov reaction (5), like in the case of the 'red' BODIPY (Red BDP) in comparison with 'green' BODIPY (Green BDP), where the fluorescence emission is shifted from 540 to 600 nm (Figure 2.3). Red BDP l max,em : 600 nm l max,abs : 560 nm This methodology does not require the use of surfactants, nor ultra-hydrophobic additives, providing stable FNP over time (2). The inclusion of acrylic acid moieties on the shell polymer, provides carboxylic acid functions which allow post-functionalization and, in our case, the grafting of FNP on substrates by peptide coupling.

A (9,10). DIBO can be synthetized in a simple approach (described in materials section) and has shown nontoxicity and straightforward attachment in a variety of probes (9). Commercially available kits allow the specific-site azide labeling of monoclonal antibodies ensure their orientation while 'clicked'. A descriptive summary along with the abbreviated name used for each synthetized and characterized FNPs in this project are presented in Table 2.1.

Table 2.1 Abbreviated name for each type of FNPs along with their core and shell composition

Chronologically, do to their easy synthesis, FNPGreen were first synthesized and characterized. These FNP were were used to set all the protocols of characterization and surface modification (which will be introduced in the following chaper). 'Green' surfaces were obtained (data are not presented in this manuscript), but both bands of emission of BODIPY and FA are too close (515 and 547 nm, respectively). For this, we proceed to synthesize FNPRed although its synthesis is complex and time consuming. Anticipating future needs, FNPG-DIBO and FNPR-DIBO were synthesized in parallel.

The last part of this chapter is focused in the preparation of a new fluorescent ratiometric pH-nanosensor based on FNPred grafted by peptide coupling with a very wellknown pH-sensitive fluorophore: Fluoresceinamine (FA), obtaining FNPRed-FA. C.

Grazon et al. (2), have already developed a similar sensor based on 'green' FNP.

Nevertheless, the proximity of the emission bands of FNPGreen and FA makes difficult the analysis of the data obtained from the sensor. This proof-of-concept, by using 'red' BODIPY (FNPRed) and FA (grafted in the shell of the nanoparticles), will be translated to a solid substrate (glass slides) to aim at a more robust and transportable device for our final goal to obtain pH-sensing surfaces. Fluorescein in solution (11), exhibits different species depending on pH (Figure The pKa from anion to dianion is 6.4, the dianion being the most fluorescent species. This property makes fluorescein appropriate as pH-sensing fluorophore in a ratiometric measurement for bacterial growth detection. Next, the synthetic routes used will be described, as well as the techniques used to characterize the different FNP.

GREEN BODIPY (Green-BDP)

(+) (-) F F B OH N N 1 2,6-diethyl-4,4-difluoro-8-(4-hydroxyphenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a- diaza-s-indacene (1)
In a round flask, 4-hydroxylbenzaldehyde (1 eq., 20.30 mmol, 2.48 g) was dissolved into 500 mL of DCM at room temperature. It was added to the mixture Kryptopyrrole (2 eq., [START_REF] Whitaker | Spectral and photophysical studies of benzo[c]xanthene dyes: dual emission pH sensors[END_REF].60 mmol, 5 g) and 10 drops of TFA. Followed by TLC, after full consumption of pyrrole Chloranile (1 eq., 20.30 mmol, 5 g) was added, followed by the addition of N,Ndiisopropylethylamine (7 eq., 142.10 mmol, 18.36 g). After 15 minutes of stirring the mixture, Boron trifluoride diethyletherate (11 eq., 223.20 mmol, 31.68 g) was slowly added. After one hour the solvent was evaporated and the compound was purified by chromatography column (SiO2, Petroleum ether/DCM, 1:4). The final product was a green-orange powder obtained with 80 % yield. In a round flask, BODIPY-Phenol 1 (1 eq., 1.25 mmol, 0.5 g) was dissolved into 100 mL of DCM at room temperature. Followed by the addition of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 2 eq., 2.5 mmol, 0.38 g) and Methacryloyl chloride (1.5 eq., 2.0 mmol, 0.2 g). The mixture was stirred overnight, after the solvent was evaporated and the compound was purified by chromatography column (SiO2, Petroleum ether/DCM, 3:7). The final product was obtained with 78 % yield.

1-(8-Methylnaphthalene-1-yl)butane-1-one (3)

O 3

1-methylnaphthalene (1 eq., 35.2 mmol, 5 g), was dissolved in 100 mL of DCM in a round bottom flask and cooled down to 0 °C. Meanwhile, a suspension of butyryl chloride (1.1 eq., 38.7 mmol, 4.12 g) and aluminum trichloride (1.8 eq., 61.5 mmol, 8.2 g) dissolved in 50 mL of DCM was cooled down. This suspension was added to the initial mixture and stirred 2 h at room temperature. After, the mixture was poured over ice and mixed until it became colorless. The organic phase was recovered and washed with a basic solution of KHCO3, followed by washing with a saturated sodium chloride (NaCl) solution. The organic phase was dried over MgSO4, filtered and the solvent was evaporated. The compound was purified by chromatography column (SiO2, Petroleum ether/DCM, 2:1).

The final product was obtained with 86 % yield.

3-ethyl-2-(8-methylnaphthalene-1-yl)-1-vinyl-1H-pyrrole (6)

N 6

A mixture of the oxime 4 (1 eq., 38.1 mmol, 8.66 g), KOH (2 eq., 76.2 mmol, 4.28 g) and DMSO (175 mL) was heated until 110 °C. The mixture was fluxed with acetylene over 2 h, followed by TLC. After cooling down the mixture, the reaction was hydrolyzed by adding NH4Cl (2 eq., 76.2 mmol, 4.08 g). The organic phase was extracted with diethyl ether (5 x 100 mL), followed by washing with saturated sodium chlorine (NaCl) solution, dried, filtered and the solvent was evaporated. The compound was purified by chromatography column (SiO2, Petroleum ether/DCM, 3:7). A mixture of N-vinyl pyrrole and the desired pyrrole were obtained. The N-vinyl pyrrole was isolated and reconverted as the following step.

N-vinyl Pyrrole reconversion to Methylnaphthalene Pyrrole

3-ethyl-2-(8-methylnaphthalene-1-yl)-1H-pyrrole (5)

N H

5

The vinylpyrrole 6 (1 eq., 17.1 mmol, 4.47 g) and mercury acetate (3 eq., 51.3 mmol., 16.35 g), were separately dissolved in 60 mL of acetonitrile, each. The mercury acetate solution was dropwise added to the vinylpyrrole one. The mixture was warmed to 55 °C for 30 min. After cooling down the mixture, a solution of sodium borohydride (6 eq., 102.6 mmol, 3.88 g) was added portion-wise very slowly. The suspension was filtered and the liquid phase was diluted with 80 mL of diethyl ether. The organic phase was washed with brine (3 x 80 mL) and dried over K2CO3. The solution was filtered and the solvent was evaporated. The final product was obtained with 55 % yield. 7

Same protocol as green BDP 1 was used replacing kryptopyrrole by methylnaphthalene pyrrole 5. The final product was a pink-violet powder with (2.299 g) 28.0 % yield. Trimethylsilyl diazomethane (1.6 eq., 21.9 mmol, 10.5 mL) was dissolved in 20 mL of DCM. Meanwhile a solution of dibenzosuberenone (1 eq., 14.0 mmol, 2.88 g) and BF3•OEt2 (1.5 eq., 21.0 mmol, 2.59 mL) in 30 mL of DCM, was prepared. Both solutions were mixed dropwise at -10 °C over 1 h period. After the reaction was stirred over 2 h at -10 °C and then poured into ice water. The organic phase was extracted with DCM (3 x 100 mL), washed with brine, dried, filtered and the solvent was evaporated. The compound was purified by chromatography column (SiO2, Petroleum ether/DCM, 1:2).

The final product was obtained with 55 % yield. The previous compound 9 (1 eq., 4.5 mmol, 1 g) was dissolved in a mixture of EtOH/THF

(1:1, v/v, 54 mL). Sodium borohydride (2 eq., 9.08 mmol, 0.34 g) was added slowly and portion-wise to the mixture. The reaction mixture was stirred up to 7 h, followed by TLC.

The reaction was quenched by dropwise addition of 1 mL of acetic acid. The solvent was evaporated. The residue was dissolved in DCM, washed with brine and extracted with DCM (4 x 100 mL). The organic phase was dried, filtered and the solvent evaporated.

The obtained compound was used in the following reaction without further purification.

The final product was obtained with <98 % yield. The previous compound 10 (1 eq., 1.7 mmol, 0.38 g) was dissolved in 10 mL of DCM.

Bromine (1 eq., 1.7 mmol, 0.27 g) was added dropwise to the previous solution and stirred for 30 min, followed by TLC. The solvent was evaporated, and the compound was purified by chromatography column (SiO2, Petroleum ether/DCM, 1:2). The final product was obtained with 50 % yield. In a round bottom flask 9.7 mL of dioxane were mixed with the following reactants: acrylic acid (1 eq., 10 mmol, 0.72 g), poly(ethylene oxide) acrylate (PEOA, 1 eq., 10 mmol, 4.54 g), 2-methyl-2-[(dodecylsulfanyl-thiocarbonyl)sulfanyl] propanoic acid (TTCA, 0.08 eq., 0.8 mmol, 0.29g), 4,4'-azobis-4-cyanopentanoic acid (ACPA, 0.005 eq., 0.05 mmol, 0.015 g) and dimethylformamide (DMF, 0.4 eq., 4 mmol, 0.29 g). The mixture was degassed (Ar) 30 min in ice bath. Immediately after, the mixture was placed in oil bath at 75 °C and stirred for 140 min. The reaction was stopped by putting the flask in an ice bath. The copolymer was isolated after two consecutive precipitations in cold diethyl ether and dried under vacuum for 2 days. The conversion rate and polymer mass were estimated based on the total monomer conversion followed by NMR (≥87% yield).

Macro-RAFT Agent: (PEOA-co-AA)-TTCA-DIBO STEP 1: DIBO-TTCA (14)

A mixture was prepared in 5 mL of DCM by adding DIBO (1 eq., 0.7 mmol, 0.153 g), TTCA (2 eq., 1.39 mmol, 0.507 g) and 4-(dimethylamino) pyridine (DMAP, 0.24 eq., 0.17 mmol, 0.02 g). N-N'-dicyclohexylcarbodiimide (DCC, 2 eq., 1.39 mmol, 0.287 g) was dissolved in 5 mL of DCM and added dropwise to the previous mixture. The reaction mixture was stirred 10 h at room temperature. The compound was purified by chromatography column (SiO2, heptane/AcOEt, 4:1). The final product was a yellow oil with (139.4 mg) 75 % yield.

Nanoparticles Preparation

FNPGreen

In a 5 mL round bottom flask, the macroRAFT agent (PEOA-co-AA)-TTCA 13 (1 eq., 0.07 mmol, 0.42 g), the green-BDPMA 2 (3.2 eq., 0.21 mmol, 0.097 g), 2,2′-Azobisisobutyronitrile (AIBN, 0.5 eq., 0.03 mmol, 0.005 g) were dissolved in styrene (157 eq., 10.20 mmol, 1.06 g). The mixture was placed in ice bath and degassed with Ar flux for 30 min. Just after, the reaction was placed in an oil bath at 80 °C and stirred for 70 min. The reaction was quenched in an ice bath and 5 mL of NaOH (0.1 M) solution was added. The reaction was placed in an ice bath, under ultrasounds at 120 W for 10 min. The miniemulsion was placed in an ice bath and degassed for 30 min with Ar flux.

Subsequent, the reaction was placed in an oil bath at 80 °C and stirred overnight to restart the polymerization.

FNPRed (Figure 2.3)

In a 5 mL round bottom flask, the macroRAFT agent (PEOA-co-AA)-TTCA 13 (1 eq., 0.041 mmol, 0.261 g), the red-BDPMA 8 (3.2 eq., 0.131 mmol, 0.090 g), 2,2′-Azobisisobutyronitrile (AIBN, 0.5 eq., 0.02 mmol, 0.003 g) were dissolved in styrene (157 eq., 6.40 mmol, 0.666 g). The mixture was placed in ice bath and degassed with Ar flux for 30 min. Just after, the reaction was placed in an oil bath at 80 °C and stirred for 70 min. The reaction was quenched in an ice bath and 5 mL of NaOH (0.1 M) solution was added. The reaction was placed in an ice bath, under ultrasounds at 120 W for 10 min. The miniemulsion was placed in an ice bath and degassed for 30 min with Ar flux.

Subsequent, the reaction was placed in an oil bath at 80 °C and stirred overnight to restart the polymerization.

FNPG-DIBO

In a 5 mL round bottom flask, the macroRAFT agent (PEOA-co-AA)-TTCA 13 (1 eq., 0.35 mmol, 0.225 g), the macroRAFT agent DIBO-TTCA 15 (0.5 eq., 0.019 mmol, 0.113 g), the green-BDPMA 2 (4.9 eq., 0.17 mmol, 0.080 g), 2,2′-Azobisisobutyronitrile (AIBN, 0.7 eq., 0.027 mmol, 0.005 g) were dissolved in styrene (241 eq., 8.48 mmol, 0.882 g). The mixture was placed in ice bath and degassed with Ar flux for 30 min. Just after, the reaction was placed in an oil bath at 80 °C and stirred for 70 min. The reaction was quenched in an ice bath and 6.7 mL of NaOH (0.1 M) solution was added. The reaction was placed in an ice bath, under ultrasounds at 120 W for 10 min. The miniemulsion was placed in an ice bath and degassed for 30 min with Ar flux. Subsequent, the reaction was placed in an oil bath at 80 °C and stirred overnight to restart the polymerization.

FNPR-DIBO

In a 5 mL round bottom flask, the macroRAFT agent (PEOA-co-AA)-TTCA 13 (1 eq., 0.38 mmol, 0.242 g), the macroRAFT agent DIBO-TTCA 15 (0.5 eq., 0.020 mmol, 0.122 g), the Red-BDPMA 8 (4.9 eq., 0.187 mmol, 0.129 g), 2,2′-Azobisisobutyronitrile (AIBN, 0.7 eq., 0.029 mmol, 0.005 g) were dissolved in styrene (241 eq., 9.15 mmol, 0.952 g).

The mixture was placed in ice bath and degassed with Ar flux for 30 min. Just after, the reaction was placed in an oil bath at 80 °C and stirred for 70 min. The reaction was quenched in an ice bath and 7.2 mL of NaOH (0.1 M) solution was added. The reaction was placed in an ice bath, under ultrasounds at 120 W for 10 min. The miniemulsion was placed in an ice bath and degassed for 30 min with Ar flux. Subsequent, the reaction was placed in an oil bath at 80 °C and stirred overnight to restart the polymerization.

Fluorescein Grafting on FNP Red

FNPRed-FA 300 µL FNPRed (0.1 mg/mL) was diluted in 4 mL of water in the dark. In parallel, 1 equivalent of FA (6.7 mg) compared to the acrylic acid units (from synthesis) in FNPRed was dissolved in 0.4 mL of ethanol. The solution was added to the nanoparticles solution and the mixture was stirred at 4°C. Then, a solution of EDC (4 equiv., 15 mg) in water (1 mL) was added to the previous mixture. Lastly, ethanolamine (2 equiv., 2.3 µL) was added after 2 hours. The reaction solution was still stirred at 4°C overnight in the dark.

Finally, the mixture was purified with a tangential filtration cassette ready-to-use (Vivaflow 50 (230 V), Sartorius, France), for 8 hours. FluoroMax-4 Spectrofluorometer (Horiba Jobin-Yvon). Optical density of the samples was checked to be less than 0.1 to avoid reabsorption artifacts. Fluorescence quantum yields ΦF were determined using Rhodamine 6G (ΦF== 0.95 in ethanol) and Sulforhodamine 101 (ΦF = 0.90 in ethanol) as a references (12).

Fluorescence decay curves were obtained with a time-correlated single-photoncounting method using a titanium-sapphire laser (82 MHz, repetition rate lowered to 4

MHz thanks to a pulse-peaker, 1 ps pulse width, a doubling crystals is used to reach 485 nm excitation) pumped by an argon ion laser from Spectra Physics (Mountain View, CA USA). Decays were measured with a band-pass of 21 nm.

Average fluorescence lifetimes were calculated by global integration of the decays using the equation 1.1 ( 13):

(1.1)

where I (t) is the fluorescence intensity at time t of the decay.

Results & Discussion

Synthesis of Dyes

The synthesis of 'green' BODIPY 1 was done following a classic protocol (Figure 2.7), starting from the condensation of benzaldehyde with two kryptopyrrole units, catalyzed by TFA and then oxidized and complexed by BF3. Afterwards, the esterification with methacryloyl chloride was proceeded to obtain Green BDPMA 2, which can be easily polymerized.

    =  0 0 ) ( ) ( dt t I dt t tI 
For the synthesis of red BODIPY 7 , the synthesis of naphthalene-pyrrole was done first by following the Trofimov reaction (5), which requires the preparation of the oxime of the aromatic considered and then its reaction with acetylene (gas) (see

experimental part: 3-ethyl-2-(8-methylnaphthalene-1-yl)-1-vinyl-1H-pyrrole).
The scheme of the synthesis is detailed in Figure 2.8. At first, methylnaphthalene is functionalized with a ketone via a Friedel and Craft reaction using propionyl chloride.

Then, the ketone was converted into an oxime according to a classical method of synthesis. The synthesis of the pyrrole is then carried out by a Trofimov reaction. The oxime is placed in a very basic solution of KOH in DMSO and then the reaction is heated to 120 °C. Once the temperature is stabilized, an acetylene flow is installed for about 5 hours. At the end of the reaction, three products are generally collected after chromatography: pyrrole, N-vinyl pyrrole and the starting ketone. Vinyl-pyrrole can be deprotected in pyrrole by treatment with mercury acetate followed by reduction with sodium borohydride with almost quantitative yields. The synthesis of red BDPMA 8 (Figure 2.9) as well as the esterification to a phenyl methacrylate derivative are made under the same conditions as for green BDPMA 2.

FNP Synthesis

As previously described, FNP were synthesized with the general methodology described by C. Grazon et al. (1,2). After the preparation of the random copolymer, they were covalently linked to a BODIPY monomer and styrene. It is first block extended with a mixture of styrene and BODIPY monomers by partial bulk polymerization (approx. 20% conversion) to afford an amphiphilic copolymer. The mixture is then poured in basic water and submitted to ultrasound treatment to yield a mini-emulsion of nanoparticles that are brought to 80 °C to terminate the polymerization overnight.

FNP Size Characterization

Dynamic Light Scattering (DLS), alternatively called photon correlation spectroscopy, was the first technique used to determine the size of the nanoparticles in an aqueous solution. DLS measures the rate of diffusion of the particles using scattered light (14). During measurement, the Brownian motion of the particles, given by the bombardment with each other, is monitored and related to time-dependent fluctuations in scattering intensity (15). DLS measures the hydrodynamic diameter, which encompasses the diameter of the particle and a double layer thickness associated to the spread of the surrounding polymer chain. This is particularly important for these FNP, where the carboxylic terminals surrounding the core are hydrophilic. DLS correlates light scattering data to the size of moving particles in solution. However, if there are multiple populations of nanoparticles, also known as polydispersity, there is a possibility that large particles are overshadowing small ones affecting the accuracy of the recorded hydrodynamic diameter.

The DLS measurement device analyzes the data using 2 algorithms for polydisperse colloids. The cumulant algorithm provides a Gaussian distribution, which provides a good fit for samples with primarily one kind of nanoparticle. The Pade Laplace algorithm provides a discrete distribution that accounts for polydispersity among synthesized particles. The Pade Laplace algorithm is considered to be the most accurate since the cumulant algorithm is an average of all sizes. As it can be seen in Table 2.2, the average size (according to cumulant fit) of FNPGreen is 80 nm. FNPGreen average radio obtained is in good agreement with previous data from particles synthesized with the same conditions (77 nm from ref. 2, FANP2BOD). These are the only FNP which have been published. For DIBO-FNP there is not comparable data already published. FNPRed average hydrodynamic diameter was 110 nm, which is slightly larger in comparison with FNPGreen. However, only with this measurement is not possible to state FNPRed are larger than FNPGreen, since the value represents an average of all the populations present on the sample. The difference in size between green and red FNP could be explained by the red BODIPY dye being more sterically hindered than the green BODIPY and thus occupying a larger volume. Comparing to literature (2), From the Pade Laplace algorithm, it was shown that all these FNP are polydispersed. For FNPG-DIBO the average size was found to be 100 nm and for FNPR-DIBO 240 nm. As it can be seen from the Pade Laplace algorithm, the presence of a 'big' nanoparticle or aggregate increased the average value when using the Cumulant algorithm.

From Pade-Laplace two populations, with similar intensity, can be observed whatever the FNPs. As an example, FNPGreen: 54 ± 8 nm and 140 ± 40 nm, those values are very distant from the Cumulant (80 nm) and both standard deviations are big (~15-30% of the FNP size). This is general for all the types of nanoparticles, it confirms the large polydispersity of the FNP. Besides, it seems that the introduction of DIBO polymer in the nanoparticle structure, results in an increase of their diameter (from 54 nm for FNPGreen to 70 nm for FNPG-DIBO and from 100 nm for FNPRed to 150 nm for FNPR-DIBO).

From this data, it is easy to observe that DLS measurements are strongly dependent on the polydispersity of the nanoparticles and the algorithm used to determine their size. DLS analysis is an easy and fast technique to obtain the average hydrodynamic radio of a sample. However, for poly-disperse samples is not the best strategy to obtain reliable data. For this reason, other techniques used to evaluate the size of the nanoparticles will be discussed. Nanoparticle Tracking Analysis (NTA) is another technique used for particle characterization, which provides insights with respect to size distribution and concentration of particles with diameters ranging from 40 nm to 2000 nm. NTA obtains particle distribution and concentration measurements based on light scattering and Brownian motion (16). Nanoparticles exposed to a laser beam scatter light that can be visualized with the microscope embedded in the device. Since the sample flows underneath the laser, the camera of the microscope records a video that is immediately (16). The hydrodynamic diameter of the FNP is then calculated using the Stokes-Einstein Equation.

The size and concentration from the experimental results using NTA for each of FNPs are summarized in Table 2.3. On one side, NTA provided both the mean diameter of the flowing nanoparticles as well as the mode, which represents the most repeated nanoparticle size in the sample. The software of the device creates a plot showing the sizes in relation with their FNP concentration, which gives insights about the presence of multiple nanoparticle populations. Moreover, the polydispersity index (PDI) is calculated from the intensity level associated to each size, which represents the width of the overall size distribution and gives a quantitative measure of the appearance frequency of the average particle diameter. PDI is an indicator of polydisperse system (when higher than 0.5), and if it is closer to zero it denotes a monodisperse system. For all the samples the PDI was higher than 0.5, showing a polydisperse population. It seems consistent with the DLS analysis, Pade-Laplace fitting showing two populations.

On the other hand, the number of particles per mL of solution is automatically calculated by the software since the flowing rate throughout the recorded video is constant. Having the particle concentration of the diluted solution allows for the determination of the FNP concentration in the stock solution by using the dilution factors applied to each sample. Concerning "Green" particles, the concentration seems to be in the same order of magnitude (approx. 4 to 5.10 14 particles per mL). Surprisingly for "red" particles, stock concentration is 9.10 13 particles per mL for FNPR-DIBO and approximately 4.10 15 particles par mL for FNPRed. FNPRed 110 ± 1 80 ± 1 0.81 1.0x10 9 ± 3.6x10 7 3.6x10 15 ± 1.3x10 14 FNPG-DIBO 100 ± 1 80 ± 2 0.93 1.1x10 9 ± 3.0x10 7 3.7x10 14 ± 9.9x10 12 FNPR-DIBO 130 ± 2 110 ± 4 0.92 1.4x10 8 ± 6.2x10 6 9.0x10 13 ± 4.1x10 12 By comparing FNP average sizes from DLS and NanoSight NTA, it can be seen that the hydrodynamic diameters obtained throughout the characterization process are similar for the FNPGreen, FNPRed, and FNPG-DIBO (Table 2.4). DLS measurements for the remaining FNPR-DIBO are also close in magnitude. However, their hydrodynamic diameters seem to be much smaller according to NTA (average value), which could be explained by the underestimation of less frequent, yet present, larger FNP populations in the average diameter calculations (Table 2.2: Pade Laplace). from the histogram chart it can be observed there are many size distributions of the particles. This is in agreement with the PDI measured from NTA.

FNP Surface Charge Characterization

Zeta potential (ζ) of the different FNP were determined at different pH values (Figure 2.13). Seems that the macromolecular architecture of the hydrophilic block did not affect in great matter the surface charge. All values are negative, which is in accordance with values reported for pegylated polystyrene particles without PAA (17).

The protonation of the acrylic acid units was not detected (18). All particles were stable in the measured pH range. To summarize, after all the different size characterizations, the polydispersity of the FNPs is evident. The charge of the particles behaves similarly independently of the type of nanoparticle (color, shell structure), all particles zeta-potentials decrease with the increment of pH and the elongation of the PEG-Shell is also a similar value for all FNP (̴ 50-65 nm), Since our main objective is to build a sensor for bacterial detection, we decided to proceed with a polydisperse population of FNP without further size differentiation, expecting this will not affect the sensitivity of the sensor. Following, the spectroscopic properties of the FNP will be introduced.

FNP Spectroscopy Characterization

The absorption and fluorescent emission spectra for the FNP are presented in For red FPNs, a bathochromic shift in absorption and emission is observed on going from the monomer in an organic solvent to FNPs. Indeed in comparison with Red-BODIPY in DCM or toluene (558 nm), the maximum in absorption is 1 nm red-shifted for FNPRed, and 3 nm for FNPR-DIBO (1nm band-pass). Concerning emission, a red-shift is observed from DCM or toluene to FNPs: 6 or 5 nm for FNPRed and 2 nm for FNPR-DIBO.

Thus polymerization and polydispersity seem to have a weak impact on band-position.

Stokes shift in toluene is around 420 cm -1 for Green-BODIPY and 1255 cm -1 for the Red-BODIPY. Such increase from "green" to "red" monomer in an organic solvent, is connected to the change in electronic density between the ground-state and the excitedstate. Thus looking at Figure 2.2, the naphthalene groups are likely to rotate from groundto excited state inducing a larger Stokes shift than "green" dye (no aromatic ring present).

From table 2.6, it can be observed that 'green' (including DIBO) FNPs have a smaller Stokes shift in comparison with 'red' FNP. It is in agreement with monomers behavior (as discussed above). In any FNPs, Stokes shift is higher than for the monomer in a solvent (approx. +200 cm -1 for green-FNPs and +100 to 200 cm -1 for red FNPs). This is connected to the emission red-shift already discussed above.

Looking at Full Width at Half-Maximum (FWMH), for Green-BODIPY monomer in toluene, FWHM in absorption is around 830 cm -1 and 1000 cm -1 for the emission band.

For red-BODIPY monomer in toluene, FWHM in absorption is around 1340 cm -1 and 1520 cm -1 for the emission band. The increase in FWHM between the two different monomers may arise from the enlargement of conjugated system, which is also consistent with the absorption wavelength red shift (529 nm for green monomer and 548 nm for the red-one).

From monomer to polymer (Table 2.6), whatever the BODIPY involved is, absorption band FWHM is increased (830 to 1015 and 1030 cm -1 for Green FNPs, and 1340 to 1582 and 1916 cm -1 for Red-FNPs). As discussed above band-position is slightly affected by polymerization, whereas FWHM is. This might be consistent with aggregation of the fluorophore within the matrix. It might also be connected to polydispersity (different size may induce different environment for the dyes). Now looking at emission FWHM, polymerization induces a decrease, emission band is thinner, whatever the BODIPY and FNPs. Indeed for FNPGreen a decrease of more than 40 cm -1 is observed, and about 180 cm -1 FNPG-DIBO. For FNPRed a FWHM decrease of 180 cm -1 is observed, and about 50 cm -1 FNPR-DIBO. Such decrease in emission might be connected to both aggregation and confinement of the emitting species. If aggregates are present, we can suppose that they are not or very little emissive, they won't contribute to emission. If we make the assumption that emission is mainly coming from "monomer-like BODIPY" (isolated fluorophores i.e non-aggregated), such molecules shall have less degrees of freedom within the polymer, which leads to less accessible states which may explain the FWHM decrease from monomer in toluene to BODIPY in FNPs. Another hypothesis would be that only small particles will emit (low loading of fluorophore), whereas bigger won't (higher concentration and thus aggregation of fluorophore). fluorophore in their core. As previously mentioned, rhodamine 6G dye was used as reference for green FNP, while sulforhodamine was used for red FNP. From the slopes of the linear fits (area under the curve from the emission spectrum vs absorbance) and using the literature values of the reference dyes, ФF was calculated using Equation 1.1.

FNPG-DIBO FNPR-DIBO

For FNPGreen it was found ФF: 15 %, for FNPG-DIBO 13 %, which are lower in comparison to the one found for Green-BDPMA in toluene (ФF: 69 %). Similarly, for FNPRed it was found ФF: 35 %, for FNPR-DIBO 28 %, which are lower in comparison to the one found for Red-BDPMA in toluene (ФF: 53 %). These differences might come from the local environment of the dye in the core of the FNPs, in comparison with the free dye (2). Such decrease is consistent with fluorophore aggregated state. For green BODIPY, going from toluene to FNPs leads to almost a five-time decrease in fluorescence quantum yield. Whereas for Red BODIPY, the decrease is always less than twice. Thus the decrease in fluorescence quantum yield is more limited with the bulky red dye. Moreover, comparing red-FNPs (FNPRed and FNPR-DIBO) with green-FNPs (FNPGreen and FNPG-DIBO), ФF are around 2 times higher for red-FNPs than green ones. Again, this might come from the steric-hindrance coming from the more 'bulky' red-BDPMA, while in the green-FNPs BODIPY probably is more aggregated.

BODIPY concentration in the analyzed solutions was calculated from the Beer-Lambert Law, which relates concentration with maximum absorption 𝐴𝑏𝑠 𝑚𝑎𝑥 , extinction coefficient ε, and length L of quartz cuvettes where measurements were taken (21):

𝐶 𝐵𝑜𝑑𝑖𝑝𝑦 = 𝐴𝑏𝑠 𝑚𝑎𝑥 𝐿𝜀 × 1 𝐿 1000 𝑚𝐿 [=] 𝑚𝑜𝑙 𝑚𝐿 (1.2)
BODIPY concentration was converted to number of BODIPY molecules per mL using Avogadro's number, which was multiplied by the dilution factor of the analyzed solutions to obtain the magnitudes at the stock conditions:

𝑁𝑏 𝐵𝑂𝐷𝐼𝑃𝑌 = 𝐶 𝐵𝑜𝑑𝑖𝑝𝑦 × 6.022 𝑥10 23 × 1 1000 [=] 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐵𝐷𝑌 𝑝𝑒𝑟 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒 𝑚𝐿 (1.3)
From the ratio between the previously found FNP stock concentrations (NTA data, Table 2.3) and the number of BODIPY per mL of stock solution, the number of BODIPY molecules per FNP was found and presented in Table 2.7. The number of dyes per particle seems very high and may not reflect reality. This might come from unreliable concentration measurements from NTA. No correlation between quantum yield and BODIPY number is clear even though the number of BODIPY calculated per particle is consistent with the number of moles of BODIPY introduced during synthesis. Until now, different types of FNPs have been studied, each one with a different purpose in this project. Chronologically, FNP Green were first prepared and studied due to their simple and fast synthesis (one week, starting with a commercial reactant). They were used to implement all the protocols of characterization and to set the protocols of surface preparation (described in the following chaper). 'Green' surfaces were obtained (data are not presented in this manuscript), but similarly as C. Grazon et al (2), the emission band of BODIPY and FA are too close (515 and 547 nm, respectively). Subsequently, FNP Red were synthesized to improve the facility of ratiometric measurement when grafting FA.

FNP G-DIBO and FNP R-DIBO were prepared with the aim to improve selectivity of the sensor by targeting actively specific bacterial strains with the introduction of antibodies.

In the following part, FNP Red-FA photophysical properties in comparison with FNP Red upon pH will be described. The next chapter will focus in the preparation and characterization of surfaces grafted with these nanoparticles.

FNP

FNPRed-FA Synthesis and Spectroscopy Characterization

As previously described, in order to obtain ratiometric pH-sensitive nanoparticles, FluoresceinAmine (FA-was grafted on FNP Red-FA . Fluorescein has five acid-base forms (Figure 2.5). Its properties of absorption and fluorescence emission are completely dependent on the pH. In this work we focused mainly on forms c and d whose pKa is 6.4 (11). The spectroscopic characteristics of forms c, and d are summarized in Table 2.8 (11). The d-form has a strong absorption and fluorescent with a quantum yield of 0.93 and emits at 520, whereas the c-form is much less fluorescent and emits in the form of a wide band between 520 and 540 nm. These characteristics make it a molecule of interest for the elaboration of pH probes coupled with FNP Red , which absorbs at 560 nm. In our work, we chose to work with the commercial Fluoresceinamine (FA). FA has a quantum yield of 1.5% which is much lower than fluorescein or FITC (93%). This drop in quantum yield is due to the presence of the amine (photo-induced electron transfer). When the FA is converted to amide, the fluorescence quantum yield increases to 80%, depending on the nature of the group carried by the acid (22).

FA can be grafted onto FNP Red (AA monomers) by peptide coupling in water, activated with EDC (23,24). At first, FA was grafted on FNP Red choosing to initially have one FA for ten acrylic acid moieties (calculated from synthesis stoichiometry). (5 red-BODIPY per AA). FA was solubilized in ethanol and added to EDC activated FNP Red solution. Two hours after, an excess of ethanolamine was added to the solution in order to passivate the residual carboxylic acid functions. Thus, the FNP Red-FA should be stable whatever the pH. The FNP Red-FA were purified by a viva-flow membrane cassette system to remove the free FA in solution. Absorption spectra of supernatant were recorded till only a very weak signal from free FA can be seen (data not shown). BODIPY contribution seems to be slightly affected by pH changes, except when pH is below 6. At this stage we cannot find any reasonable explanation why.

The maximum in fluorescence emission of the FA band decreases with pH (Figure 2.16), which gets closer to the maximum intensity of 'red' BODIPY band. Such behavior is also consistent with fluorescein behavior in aqueous solutions as pH changes.

On the other hand the intensity of BODIPY emission seems to be much less affected by pH than FA-contribution. Thus we plotted the ratio of FA contribution and of BODIPY contribution as a function of pH (2.17). From Figure 2.17, which represents the variation of the ratio α 515/605 according to pH, it was possible to estimate by a Boltzmann fit a pKa of 6.11 ±0.05, which is similar to the pKa from anion to dianion of FA (6.4). From this we can observe it is possible to estimate the pH by the ratiometric change in fluorescence between FA in the shell and red-BODIPY in the core of the FNPs for the pH in between 5 and 8. This pH corresponds well to the pH of bacterial growth. As shown in Figure 2.17, the pH dependence is obtained no matter the conditions of the experiments (by fixing or not the dilution factor from stock solutions).

Föster radius of the donor-acceptor in basic conditions was estimated to 5 nm, while in acidic medium was estimated 7 nm. From this, it is possible that FRET occurs from FA to Red-BODIPY and might be modulated with pH changes (2).

To better observe the presence of energy transfer from the FA (donor) to the BODIPY (acceptor), excitation spectra were obtained at λ em = 620 nm and collected from 400 to 600 nm (Figure 2.18 and 2.19). At 620 nm, red-BODIPY is the only emitting specie (not FA), nevertheless, a second band around 500 nm corresponding to the FA is observed.

This means, there is energy transfer between red BODIPY in the core and FA from the shell, and it seems to be pH dependent (which is consistent with measured R 0 ). The ratio α500/560 (excitation maximums) vs pH are represented in Figure 2.18 and 2.19 and corresponds well to Figure 2.17. Next, the variation of quantum yield (ΦF) vs pH was analyzed. As previously mentioned, rhodamine 6G dye was used as reference for green FNP, it can be also used for FA contribution in grafted FNPs. Sulforhodamine was used as a reference for red BODIPY dye and red FNPs. From the slopes of the linear fits (Area under the curve from Emission spectrum vs Absorbance at the excitation wavelength) and using the literature values of the reference dyes, ФF was calculated using Equation 1.1. In FNPRed-FA there are two bands which correspond to a 'green part': FNPRed-FA-green which corresponds to FA emission (from 495 to 569) and 'red part': FNPRed-FA-red which belongs to 'red' BODIPY emission (from 570 to 750)]. These results are shown in table 2.9. From Figure 2.20 FNPRed-FA (green part) bottom image, pKa was estimated with Boltzmann fit, finding to be pka: 6.5, which is very close to the one found by the ratio α515/605 (pka: 6.11).

Nevertheless, the values of ΦF for FNPRed-FA are still very low compared to Fluorescein-amide (expected up to 80%). Even though we performed purification, still a weak signal from free FA was measured, thus we cannot rule out some free FA contribution, but it shall be weak and it shall not "shade" the Fluorescein-amide signal.

Since we have doubt about the NPs concentration, it is hard to estimate the number of From Figure 2.22, it can be observed that the average lifetime for FNPRed-FA (green-part) is decreasing with pH, which is totally consistent with ΦF evolution as seen in Figure 2.20. From Figure 2.22 FNPRed-FA (λem: 525 nm) bottom image, pKa was estimated with Boltzmann fit, finding to be pKa: 6.11, which remains consistent and still is alike to the one found by the ratio α515/605 (pKa: 6.11) and quantum yield (pKa: 6.18).

From table 2.10, it is possible to observe that mean lifetime (values and evolution vs pH) for FNPRed-FA in the 'red part' is somehow similar to the one in FNPRed.

Nevertheless the "red" decay time value is higher when FA is grafted on red FNPs (FNPRed-FA) than for FNPRed (except for pH 5). It could be eventually a clue of energy transfer from donor (FA) to acceptor (BODIPY). At this stage, we do not have NPFA which could allow us to investigate the "donor" alone behavior and measure fluorescence quantum yield and lifetime in absence of acceptor.

Looking closer at decays of red-BODIPY in any FNPs, lifetime decreases with pH. In FNPred (no grafted FA), the decrease is quite small, about 6% from pH=8 to pH=5.

In FNPRed-FA the decrease reaches 26% from pH=8 to pH=5. Going back to the 'red' fluorescence quantum yield issue raised above, 'red' decay time evolution with pH does not show the same trend as quantum yield. Since time-resolved experiments do not depend upon any reference, we believe quantum yields are indeed affected by experimental errors.

To summarize, in the 'red' band, pH effect on lifetime decrease is 4 times more visible in FA grafted FNPs compared to ungrafted. It seems that we have in FNPRed-FA decays a contribution of FA emission, this might be due to large slits used for the measurements.

The positive point is that decay time in FNPred is very slightly affected by pH, thus red-BODIPY may act as an internal reference for further ratiometric measurements. The 'green' contribution of FA, in FNPRed-FA, shows a sensitivity to pH, very similar to the fluorescence quantum yield evolution.

The plot of decay-time ratio as a function of pH is not informative and was not presented here.

Chapter 3: pH sensing surfaces Introduction

In the previous chapter, the synthesis and characterizations of different FNPs were described. In addition, we prepared a new ratiometric fluorescent pH nanosensor, following the same principle as the one described by C. Grazon et al. (1), but this time using FNPRed with 'red' emitting BODIPY (reference dye) in the core of the nanoparticle and fluorescein (pH sensing dye) covalently attached to the external surface. As described above, ratiometric measurements have the advantage over single-emission measurements of being independent of several experimental parameters.

This chapter is devoted to the development and characterization of pH-sensing surfaces by taking advantage of the ratiometric fluorescent pH nanosensor formed by FA and FNPRed. In this chapter, the study of pH-sensing with buffer solutions as proof-ofconcept will be shown. The study of the interactions of the surfaces with bacteria, in an aim to detect bacterial growth will be introduced.

Our initial trials to graft FNP on glass substrates, were done in suspension.

Nevertheless, the preliminary data shown a limited grafting of FNP and lack of stability over time. Thanks to our new collaborator at the time, Pr. Antoine Pallandre, a new protocol for gas-phase silanization proved to obtain good results.

After grafting and characterizing the surfaces grafted with the FNP, the introduction of FA is done in two different strategies as follows: this implies the previous grafting of FNPRed, followed by the grafting of FA in similar conditions (EDC coupling). 1 step: grafting by EDC coupling of FNPRed-FA nanoparticles characterized in the previous chapter.

First, 2 step approach was used since all the protocols and characterization techniques were set for FNPRed. Following, to introduce the pH-sensing molecule FA, same EDC coupling was done. However, as shown in the previous chapter, FNPRed-FA were synthesized and characterized (later in time). For this reason, 1 step protocol was introduced and used in the last steps of the pH study on surfaces. Nevertheless, both approaches provide the same characteristic fluorescence emission and sensitivity of pH, reason which they were used indiscriminately.

Next, the protocol used to prepare the surfaces sensitive to pH will be described, as well as the techniques used to characterize them. Lastly, pH analysis and bacterial interactions of the surfaces will be presented.

Materials & Methods

Materials

Reagents were used without further purification unless explicitly stated. Glass were purchased from Sigma Aldrich. For buffer preparation, sodium phosphate monobasic dehydrate, sodium phosphate dibasic dehydrate, and anhydrous citric acid were purchased from Sigma Aldrich.

Surface Functionalization

Gas-Phase Silanization

This protocol was adapted from a protocol belonging to Professor Alain Jonas (Louvain University-Belgium), given by Professor Antoine Pallandre. Glass slides were placed in a home-made Teflon slide-holder and immersed for 20 minutes in fresh piranha solution (1:1, H2O2 30% : H2SO4 95%). Slides were removed from the piranha solution and washed thoroughly with deionized water (D.I.). Subsequently, slides were immersed in absolute ethanol for 15 minutes, and dried one by one with nitrogen flux (N2). Slides were placed in a Schlenk tube previously warmed at 80°C. The slides were left under vacuum for two hours to ensure anhydrous conditions, followed by three to five cycles of N2-Vacuum and finalizing by Nitrogen. Then, 0.2 mL of silane were injected carefully in the Schlenk tube. The silanization process was left overnight at 80°C. Afterwards, the Schlenk tube was carefully opened under the fume hood letting the vapors to exit. The slide-holder was immersed in acetone and then placed in a Soxhlet (Figure 3.4). The Soxhlet system was run with acetone as solvent for a minimum of 3 cycles. When finished, the silanized-slides were dried with nitrogen flux and stored individually in small Petri dishes covered with aluminum foil to avoid contact with the plastic surface. The peaks were decomposed using a linear baseline, and a component shape defined by the product of a Gauss and a Lorentz function, in the 70:30 ratio. Molar concentration ratios were calculated using peak areas normalized according to Scofield factors. A minimum of 6 different zones from a minimum of 2 samples were analyzed for each type of surface.

Scanning Electron Microscopy

The observations were carried out using a scanning electron microscope ZEISS Sigma HD (FEG-SEM) operating at 1 kV (Field Electron Gun to be observe at low voltage to limit the destruction of organic molecules with low energy e-beam). The EBSD step size was 0.1 µm and the EBSD data acquisition and analysis were undertaken using the TSL Orientation Imaging Microscopy, OIMTM software. A minimum of 10 different zones from a minimum of 2 samples were analyzed for each kind of surface. Percentage of coverage was calculated by Image J software.

Atomic Force Microscopy

AFM studies were performed using a Nanowizard 3 AFM head (JPK Instruments AG, Germany) coupled to a commercial inverted microscope (Axio Observer Z1, Carl Zeiss, Germany). We used AFM cantilevers with nominal stiffness value in the range of 0.03 N/m, full tip cone angle around 40° and tip height larger than 3μm. Cantilever and tip are made from bulk n-type silicon recovered by a native silicon dioxide layer (HQ:CSC38 AFM probe, MikroMasch, Nanoworld AG), For each cantilever the sensitivity of the system deflection was measured by performing force spectroscopy (approach/retract curves) in air on a clean glass surface. For the spring constant calibration, the cantilever was retracted ~500μm away from the substrate and thermal oscillations were measured in air for few seconds. The resulting frequency curve (typical resonance frequency in air in the range of 10kHz) was then fitted using the JPK software to generate the correct spring constant value.

AFM data were acquired using a high-speed force spectroscopic mode (Quantitative Imaging mode, JPK). Under this mode which minimizes lateral interactions between tip and the surface, a complete force curve (approach and retract) was acquired at each pixel of the (128x128 pixels²) images whatever their lateral scan size.

For all the results that will be presented here, the approach and retraction speeds for every pixel were constant (100μm/s). The height extension of the AFM cantilever was fixed at 1μm. The digitization rate for every approach and retract curve was equal to 110kHz. The set-point force (maximum applied force during the tip approach) was chosen to fulfil the two next conditions: (i) reducing lateral interactions by minimizing the vertical force (to avoid sweeping away of the NP by AFM tip); (ii) getting stable and reproducible approach/retract curves. Thus typical optimal set-up forces were 1.5nN for silicon dioxide. Each AFM image was scanned line by line, starting from the bottom of the image to its top. For each of these lines, pixels are successively scanned from the left side to the right one. All the experiments were performed at a constant temperature of 22 12 bits. Laser (488 nm) was used as the excitation source and the corresponding fluorescence was collected in the 500-750 nm spectral range. Emission spectra were recorded using 5 nm bandwidth and an average of 2 frames.

Other than stated, images were taken using the confocal microscope. For those taken by epifluorescence, images were taken also in a Nikon ECLIPSE TI-E inverted epifluorescence microscope. Using an objective × 60 -0.7 numerical aperture (CFI S Plan Fluor ELWD). The microscope was set to take 6 x 6 images and merge in one big image of a final size of: 628.03 x 473.34 μm 2 (3784 x 2852 pixels). Images were excited at 482 nm and recorded using FITC-354C (λem: 530-560 nm) and TRITC-B (λem: 555-595) filters.

pH-Sensing

For acquisition, the samples were placed in a metallic holder (see Figure 3.6), and 60 µL of buffer solution was placed in the sample. To change buffer solution, the previous buffer was reabsorbed and then the surface was washed 3 times with the new buffer solution. After each acquisition, the pH of the buffer was checked with a pH paper. Three slides were analyzed (1-2 zones).

Bacteria Growth Detection

The same glass holder and methodology were used as the pH-sensing experiments.

For acquisition the samples were placed in a metallic holder (Figure 3.6), and 150 µL of bacterial solution (E. coli BW25113 and M9 minimal medium, OD0: 0.01) was placed onto the sample.

Modified M9 minimal medium was used with a lower concentration of phosphate salts in order to decrease the buffering capacity of the growth medium and thus obtain a more sensitive measure of bacterial growth from the response of the pH sensitive surface.

The modified M9 minimal medium contains 5.9 mM Na2HPO4 To decrease evaporation of the sample, a wet tissue was placed surrounding the sample avoiding contact, then the sample holder was covered with a transparent cover.

The temperature was set at 30 °C. A spectrum was acquired every 60 minutes. The pH of the bacterial culture was measured every 2 hours with a pH paper, and compared to a bulk bacteria solution incubated in the same conditions. A unique experiment was carried out.

Bacteria were quantified using Image J.

Results & Discussion

Surface Functionalization

The overall strategy for surface functionalization is schematically presented in Figure 3.8. Amino-silanized surfaces were prepared by gas-phase deposition. To start, glass slides were immersed in freshly prepared piranha solution: this step serves to clean the surfaces from any organic pollution, besides activating the surface by leaving hydroxylic groups available. Silanization was run overnight, allowing EDSA (3-(Ethoxydimethylsilyl) propylamine) to react from its ethoxy silane group with a silanol group of the surface by vapor deposition, as previously described by Pallandre et al. (23,24). This group studied different commonly used aminosilane molecules, showing that EDSA is a good candidate to form good quality monolayers (judging from the electron density and from the roughness of the monolayer/ air interface). In this study, it is shown how difficult it is to obtain monolayers from triethoxy-silanes, as commonly used (3-Aminopropyl) triethoxysilane (APTES), due to probable gel formation leading to thick layers of silane. Following silanization, a Soxhlet system was used to wash the slides with a minimum of 3 cycles of acetone. This resulted in removal of other subspecies, such as From the narrow windows on Nitrogen (Figure 3.9), it is easily observed that after silanization the concentration increased from the initial trace levels (glass after piranha).

After silanization, both amine and protonated amine components were clearly observed.

The N 1s peaks include two contributions: a component at 399.2 eV attributed to amine groups [C-NH2], a component at 401.3 eV due to protonated amines C-NH3 + , in good agreement with literature (25,26).

After grafting of particles, the component at 399.2 eV attributed to amine groups [C-NH2] was highly increased. For Carbon, after silanization it is also possible to see an increment of 2 times after silanization, and a higher increment of 2.5 times after nanoparticle grafting. From the narrow windows on Carbon (Figure 3.9), two main species are seen after silanization [C-(C,H); C-(O, N)], which can be found in the EDSA (probably not coupled ethoxy groups). After nanoparticle grafting the appearance of

[C=O], which can be found in the polymer on the shell, it is observed, besides the increment of the other two species. Besides, from AFM image (Figure 3.12) we can observe in the color scale bar the maximum height of the grafted FNP (dry conditions) is around 69 ± 3 nm, but this value is not conclusive since many of the particles are stacked to each other, thus the average diameter was not estimated.

Surface Spectroscopy Characterization

Absorption and emission spectra of dry FNPRed-FA surfaces were compared with FA and FNPRed (D.I., pH ̴ 5). As shown in figure 3.13, both absorption and emission spectra correspond quite well to the convolution of FA and FNPRed (with a shift to the red of FA, which might be due to a different environment than solution, or FA aggregates (27). These spectra allowed us to confirm the grafting of the FNPRed-FA on silanized surfaces. Spectroscopy properties are found in table 3.2.

Spectroscopy Characterization

Fluorescence emission spectra of FNPRed-FA surfaces were analyzed at different pH buffer solutions (citrate / phosphate 0.2 M), from pH 5 to pH 8 every pH 0.5 with a confocal microscope using zoom 3 (image size: 82 x 82 µm 2 , recording directly spectra).

The spectra are change from the one shown in Figure 3.13. In these spectra the red band is more emissive than the green band.

The response of the surfaces to the pH was later correlated with a linear model (better fit for the data), where the ratio (𝛼 520/600 ) of the intensity at 520 nm and 600 nm was related with a change in pH (Table 3.3 and 3.4). Three independent experiments from three different surfaces (from the same batch as in Figure 3.14) were analyzed, finding difficult to reproduce the results (obtain the same fit). On trial 2 a surface prepared with pre-grafted FNPRed-FA was used (same conditions of grafting as described before). This might be due to the heterogeneity of the surfaces that it is visible at microscopic level and might be related to the polydispersity of the FNPs (Figure 3.15). With this result, it is clear that it becomes necessary to calibrate the pH-sensing surface before biological measurements. As seen in figures 3.16 to 3.21, the 𝛼 520/600 decreases when the pH decreases. This is in congruence with the previous chapter, where it was demonstrated that the pH has little influence on the FNPs with BODIPY in the core, while spectroscopic properties of FNPs grafted with FA are dependent on pH (2). Thus, the properties of the grafted FNPs and post-functionalization with FA have proven to preserve the sensibility of the nanosensor. Data were fitted and correlated (linear fit, 28, 29, 30) the increase of 𝛼 520/600

with the pH (Table 3.4). However, as previously described, it remains necessary to calibrate the measurement on each sensing zone (28), independently if the FA is grafted before or after on FNPs (as shown before, 1 or 2 steps). Consequently, we aimed to detect bacterial growth using these surfaces. For this experiment FNPRed-FA were grafted on a surface. For this, we first quickly (̴ 30 min) calibrated the surface with the same three buffers at pH 5, 6.5 and 8, obtaining the linear model in table 3.5 by taking the data presented in figure 3.23. The R-square obtained shows a good fit.

In the next step, a bacteria solution of E. coli in M9 minimal medium was diluted to an OD: 0.01 and deposited on the functionalized surface. Then, images and spectra were obtained every hour to determine the 𝛼 520/600 and by brightfield channel to quantify the number of cells per image (to be considered for the bacterial growth) (Figure 3. As a control, bacteria were grown in bulk (cuvette, without shaking, under the microscope at the same temperature) in the same conditions as the surface. The pH and OD were measured for the control and compared to samples taken from the bacterial solution on the surface. The rate-limiting step was the small quantity of bacterial solution, which could only be measured by pH paper and just few times (3 times), to avoid the decrease of solution of the sample and finally drying it.

Afterwards, we estimated the pH with the linear model obtained from the calibration curve and it was found to be close to the measured one. Nevertheless, the calculated pH from spectral image analysis seemed to decrease faster than the measured pH. This could be related to the local pH of the bacteria, which are more concentrated on the surface (by their adhesion) than in the bulk solution. them that can precisely predict the growth of the bacteria by knowing the ratio. For this, further experiments should be carried out to obtain more data in shorter time intervals.

Conclusions & Perspectives

In conclusion, the preparation of pH sensitive surfaces was carried out successfully.

Firstly, the different steps to functionalize the glass substrates were presented. Then new pH sensitive surfaces were characterized by different techniques:

➢ By XPS analysis, the elementary composition of the surfaces after activation, silanization and grafting of the nanoparticles was by focusing mainly in the Nitrogen, since different states of Nitrogen and its different states we have obtained an evolution of the chemical composition at each step. Starting from the activated glass, only traces were found; after silanization it was observed an increment in the C-NH2, and also in the protonated amines C-NH3 + were in good agreement with the literature. After Nanoparticle grafting, the decrease of 1.5 times in the percentage of Nitrogen and an increment of 2.5 times in the percentage of Carbon corresponds well to the addition of the nanoparticles.

➢

A good coverage from the nanoparticles was determined by SEM and AFM on the surface, 8.1 ± 0.7 % by SEM and 13.1 ± 0.5 % by AFM. As described above, AFM images allowed to obtain a more reliable quantification of the coverage, but SEM images permit to observe larger areas which allowed to measure the homogeneity of the coverage.

➢

The photophysical properties were found to be in good agreement with the spectroscopic properties from the grafted elements in suspension (FNPRed and FA).

Besides, the response of the surface as a function of pH was investigated, obtaining a linear correlation with the ratio 𝛼 520/600 . The sensitivity of the sensitive surfaces remains lower than Grant et al. (28), (average slope: 0.05, range: 5.0 -8.0) but a larger pH range is considered.

➢

From bacterial growth study on the pH-sensitive surfaces, it can be observed the pH can be predicted efficiently. Nevertheless, to corroborate and evaluate the real accuracy it is needed to improve the method to measure the real pH, since only with pH paper is not very accurate. For this a micro-electrode could be used. Besides, with more data in between points, a better fit can be done and thus a correlation between ratio 𝛼 520/600 and bacterial growth will be obtained. This will be carried out in the coming weeks.

To the best of our knowledge, it is the first time pH-sensitive surfaces are used to effectively detect bacterial growth and also the first time thanks to a ratiometric measurement. Nevertheless, some critical aspects should be improved to obtain a more reliable and robust device. Herein some perspectives that are currently being evaluated to achieve this:

➢ Improve the homogeneity of grafting

It was clearly seen that grafting of the FNP is not homogeneous and some aggregates were observed. This could be improved by reducing the polydispersity of the FNP in solution, by using a filter with a size cut-off, allowing to separate the bigger particles from the smaller ones.

In addition, the improvement of the grafting of FA on the shell of FNP could be evaluated. It was tested to graft on the surface nanoparticles with FA already grafted on the surface, but the same sensitivity and heterogeneity was obtained as presented in this chapter (Trial 2 and bacteria test). Somehow this is expected, since the grafting of FA either in FNP or in grafted-FNP surfaces is done in both cases by peptide coupling, reaction which is not 100% efficient. This could be improved by using 'click-chemistry' instead of peptide-coupling, (by replacing FNPRed by FNPR-DIBO nanoparticles). FA could be 'clicked' on the nanoparticles before grafting them on the surface.

➢ Slow decrease in pH during bacterial growth

Different acidic species such as acetic acid, lactic acid, CO2 and other metabolites are released upon bacterial growth, resulting in a pH decrease of the growth medium.

Nevertheless, in an open system, the CO2 that is produced can escape from the growth medium. For this reason, a PDMS microfluidics circuit was designed and is currently being tested to slow down the diffusion of gases (PDMS could be replaced by a glass microfluidics circuit) (Figure 3.27). The design of the circuit allows to inject a sample and diffuse it in 5 individual channels, in addition, the flow can be stopped, and all the metabolites and gases generated from bacterial growth will remain without escaping. We hypothesized that in this case the pH will decrease faster allowing a faster detection.

Furthermore, the microfluidic circuit would also allow the addition of different antibiotics and screening the resistance of bacteria to them.

Part II: Antibacterial Surfaces Introduction

In the previous chapter, the synthesis and characterizations of pH-sensing surfaces were described. We have demonstrated that such surfaces could effectively detected bacterial growth by a ratiometric fluorescence measurement. In this part of the manuscript, the study and characterizations of new antibacterial materials as repelling films will be introduced.

Indeed, we focused on (super)hydrophobic surfaces due to their main advantages over other strategies to avoid bacteria. Among them are:

-easy manufacturing process in comparison with other surfaces which imply long synthetic steps or careful design of antimicrobial agent release;

-lack of antimicrobial agent, which in long term use can be a threat for the environment, we also avoid the issue of emptying reservoir with time;

-self-cleaning properties, which simplifies the elimination of the dead cells or prevents the attachment of biofilms and facilitates its removal.

Besides, the innovation of this project relies on the use of a luminescent surface. For the first time, our group and collaborators demonstrated the advantages of combining such properties : fluorescence and superhydrophobicity. This allowed to colocalize the bacteria and the substrate at the same time. More details about the properties of superhydrophobic surfaces and the results obtained with these new materials will be found in the publications below.

This project was done with significant the contribution of our collaborators. The preparation of the electropolymerized films was done by Thierry Darmanin and Gabriela Ramos-Chagas from NICE lab belonging to Université Côte d'Azur (Nice, France) with monomers from Institut Lavoisier de Versailles (Versailles, France). From our group, we contributed with the photophysical characterizations of different films, as well as the interaction of some of the coatings with different bacterial strains. This work was supported by LabEx CHARMMMAT.

The results are presented in the form of four publications as follows: In the following chapter another kind of surfaces will be presented, surfaces to kill bacteria.

Introduction

As described in the previous chapters, two general approaches are traditionally followed to obtain antibacterial properties: an antifouling route to prevent bacterial adhesion and antibacterial properties promoting bacteria death (1-6). In the previous chapter, the study and characterization of new antibacterial materials such as repellingbacteria coatings to prevent bacterial adhesion were shown (7-10). Among killingbacteria methods, antimicrobial photodynamic therapy (aPDT), has emerged as a promising approach since it relies on the use of coatings producing reactive oxygen species upon irradiation. This is done in the presence of a photosensitizer, avoiding the use of antibiotics and inorganic antimicrobial agents, which might require a delivery system or might be a threat to the environment (11)(12)(13)(14)(15)(16). Another advantage is that most biological entities (bacteria, viruses, parasites and yeast) are highly sensitive to reactive oxygen species, thus these surfaces can also be used against these organisms (15,17).

This last part of the manuscript will focus on the efficiency of photopolymerized surfaces to kill bacteria. They release singlet oxygen upon irradiation. Our input relies on the studies to characterize the antimicrobial effect of the coatings made by an innovative photochemical process to be used on material surfaces for medical applications. The photopolymerization process has many advantages such as a low energy consumption process (visible irradiation), room temperature operation, a very fast polymerization rate with high conversion rate and precise spatial control on where the polymerization takes place (13,18). The innovation of this project relies on the use of a natural dye: curcumin, which is used both as photosensitizer for the initiation of the polymerization and as a reactive oxygen species promoter for the synthesis of permanent antibacterial coatings (13,19,20).

This project was done with the contribution of two main collaborators, whose input and expertise will be described next:

In the present work, the study of different surfaces was done with the main objective to contribute in the long term to the fight of AMR. For this, three main projects were presented: a) Bacterial detection, b) Repelling bacteria surfaces and c) Killing bacteria surfaces. All these projects had a common methodology, which was the implementation of nanotechnology on their design and the use of fluorescence microscopy to determine their efficiencies on bacterial interaction studies. Fluorescence microscopy is a very powerful technique, very well-known by scientists over the world.

Such technic is now more and more available, and gives to the user many advantages over techniques like electron microscopies: it is easy to use, it has lower cost and allows the visualization of live microorganisms (like bacteria). Besides, the application of nanotechnology confers 'new' properties to materials, like the electropolymerized nanostructured films which prevent bacterial attachment or the micro grids killing bacteria presumably due to the diffusion of a singlet oxygen and finally the transfer of pH-sensitive nanoparticles to glass to detect cells growth. To conclude, a summary of the main results of each chapter will be done. (green or red-BODIPY). After grafting fluorescein amine (FA) on red FNPs, the spectroscopic characteristics were studied at various pH. The pKa of the grafted fluorescein was determined to be close to the tabulated one( pKa: 6.11). Following, fluorescence quantum yield and lifetime were measured and it was extremely difficult to quantify and probe energy transfer from them. Nevertheless the FNPRed-FA plot of intensity ratio ('green' vs 'red' contributions) shows a very nice pH sensitivity in between 5.5 and 7, which is the perfect window for biological applications such as bacterial growth. Thus, we proceed to our main objective.

Glass-substrates were functionalized by gas-phase silanization and further peptidecoupling was used to graft the different elements for the ratiometric sensor (FNPRed + FA). Then new pH sensitive surfaces were characterized by different techniques. XPS was mainly used to determine the elementary composition of the surfaces after piranha activation, silanization and grafting of the nanoparticles. Different states of Nitrogen allowed to obtain an evolution of the chemical composition at each step allowing to conclude about the presence of the chemical moieties grafted on the surface.

AFM (Atomic Force Microscopy) and SEM (Scanning Electronic Microscopy) allowed to determine the coverage of the FNP on the surface and to qualitatively evaluate the homogeneity of the coverage. The photophysical properties were found to be in good agreement with the spectroscopic properties from the grafted elements in suspension (FNPRed and FA). Similarly to FNPs in suspension, a good pH-sensitivity between 5 and 8 was obtained. From bacterial growth study, the pH could be predicted by knowing the ratio 𝛼 520/600 . Nevertheless, more data have to be acquired to correlate the ratio and the bacterial growth efficiently, moreover the methodology to measure pH should be improved. This will be carried out in the coming weeks. To the best of our knowledge, for the first time a pH-sensitive surface was used to detect bacterial growth with a ratiometric measurement.

Then, new micro and nanostructured fluorescent surfaces prepared by electropolymerization were introduced. By changing their polymerization parameters and monomers nature it was possible to tune their topography and thus their wettability.

Firstly, a review explaining the main concepts about superhydrophobicity was presented, including their application to medical environments. Different fluorinated and nonfluorinated surfaces with different hydrophobicity were characterized and their efficacy to prevent bacterial adhesion was studied. Besides and most importantly, for the first time, multifunctional (luminescent and superhydrophobic) films allowing to colocalize bacteria and substrate were published. A reduction between 30-70% in the early bacterial adhesion for non-fluorinated polypyrene and between 60-90% for fluorinated polypyrene was obtained for S. aureus and P. aeruginosa. Polypyrene surfaces demonstrated a high efficiency against biofilm attachment, reduced by 90-99%.

Finally, curcumin-grids prepared by photopolymerization were investigated and their efficiency to kill bacteria was studied. These curcumin-grids surfaces have proven to effectively kill up to 90 ± 9 % S. aureus upon irradiation (with a short time of 10 min) due to the assumption of the release of a singlet oxygen. Such coatings proved to be more efficient in term of irradiation time when compared to other studies.

To conclude, this manuscript presents the main results of different projects with the main objective to tune surface properties to either avoid / kill bacteria or detect their growth. In the long term, such surfaces shall be helpful to fight AMR. The complexity and richness of this project arises from the different interfaces studied: liquid-solid-gas, organic and physical-chemistry and microbiology. Besides this project represents the collaboration of many contributors who permitted to achieve our objectives and enriched the project with their scientific discussions and contributions. To conclude, this is an ongoing work, new improvements are being envisioned and further studies will be done.

For pH-sensitive surfaces, as mentioned before, the work will be continued by a

PhD student thanks to a CSC grant. Besides, a postdoctoral fellow will work on heavy metals detection using similar surfaces. Further work will be done to improve the sensitivity of the surfaces and to develop a microfluidic system. For DIBO FNPs, other applications such as specific cell targeting should be which are highly attractive for cancer research.

For the electropolymerized films, as mentioned before, new films are being studied (and another publication has been submitted) by changing pyrene core to another chemical moiety. These new films present nanostructures (like tubes) that could be used for gas-storage or another applications such as in drug delivery and catalysis. Besides, for polypyrene films, many questions remained unanswered (such as mechanical stability)

and should be investigated due to the antibacterial properties already proven of the materials.

And lastly, as stated before, the curcumin-grids are part of an ongoing project, and many of the remained questions will be further investigated. To ensure the singlet oxygen is diffused more experiments shall be conducted, such as labeling with a reactive oxygen species (ROS) reagents (Molecular Probes).

Figure 1 . 1

 11 Figure 1.1 Mechanisms of bacterial resistance. Adapted from Kenneth Todar, www.textbookofbacteriology.net

Figure 1 . 2

 12 Figure 1.2 Schematic representation of a biosensor. Optical transducing and antibody recognition are shown as examples.

Figure 1 . 3

 13 Figure 1.3 Schematic representation of the method using magnetic nanoparticles and optical nanoprobes (Ref. 19).

Figure 1 . 4

 14 Figure 1.4 Schematic representation of the method using magnetic nanoparticles and QDs (Ref. 23).

Figure 1 . 5

 15 Figure 1.5 Synthetic pathway employed for the synthesis of FNPs proposed by G. Clavier Group (PPSM) (Ref. 30). The general protocol for synthesis starts by a reversible addition-fragmentation transfer (RAFT) polymerization. Obtained from a hydrophilic Poly(ethylene oxide) and Poly( acrylic acid) macromolecular RAFT agent which is block-extended with styrene and fluorescent BODIPY monomer.Then, a mini-emulsion is formed, following bye the completion of the polymerization. (Please refer to abbreviations section for definitions).

Figure 1 . 6

 16 Figure 1.6 Growth of E. coli bacteria incubated with fluoresceinamine molecules (FA), Fluorescent nanoparticles with Fluorescein grafted on the shell (FANP1 and FNP2: higher and lower concentration of FA grafted on the shell respectively) using either the optical density (600 nm, dotted lines) or the decrease of fluorescence intensity (%, full lines) (Ref. 35).

  , introduced a work on a new ratiometric fluorescent pH nanosensor (Fig. 1.7). A ratiometric measurement uses two different fluorophores: one is not sensitive to the environment (reference dye) and the second one is (sensing dye). They used green-BODIPY FNPs with two spatially separated fluorophores: 4-difluoro-4-bora-3a,4a-diaza-s-indacene (Green-BODIPY) in the core of the nanoparticle and fluorescein covalently attached to the external surface. In this model, BODIPY was used as the reference dye and fluorescein as the sensing molecule. Meaning by this that the fluorescence of the reference dye (BODIPY) will remain stable upon the change in pH, while the fluorescence of the sensing molecule will change. Ratiometric measurements have the advantage over singleemission measurements of being independent of external fluctuations of the fluorescence signal, such as photobleaching or leakage of the dye. Ratiometric measurements are also insensitive to ambient or scattered light and any instrumental fluctuations.

Figure 1 . 7

 17 Figure 1.7 Variation of the ratio of fluorescence intensity at 515 nm (fluoresceinamide) and 542 nm (BODIPYinternal reference) of FNPs as a function of the pH. Adapted from ref. 36.

Figure 1 . 8

 18 Figure 1.8 Single-emitter pH sensitive surfaces prepared by Craciun et al. Adapted from 43.

Figure 1 . 9

 19 Figure 1.9 Scheme of DBCO molecule used by Rigo et al. (44), allowing both click-chemistry and peptide coupling.

Figure 1 . 10

 110 Figure 1.10 Working principle of the point-of-care device: upon bacterial growth, acidic species will be released to the medium resulting in a decrease of pH. The sensing dye (Fluorescein) will be quenched by this change, while the reference dye (BODIPY) in the core of the nanoparticles shall remain stable. The change of the ratio of fluorescence intensity at 525 and 600 nm of FNPs as a function of pH (an expected change of pH between 7 to 5.5) will be correlated with bacterial growth, allowing for further screening for AMR.

Figure 2 . 1

 21 Figure 2.1 Design of the core-shell FNP were: the core is made of a polymer block of styrene and BODIPY; the shell is made of a random copolymer of PEO and PAA.

Figure 2 . 2

 22 Figure 2.2 Structure and numbering scheme for BODIPY.

Figure 2 . 3

 23 Figure 2.3 Scheme of 'green' BODIPY (Green BDP) and 'red' BODIPY (Red BDP), used in this manuscript as fluorophores in the core of FNP. Maximum absorbance and emission wavelengths recorded in dichloromethane (DCM).

Figure 2 . 4

 24 Figure 2.4 General synthetic scheme for the synthesis of FNP, i.e. FNPRed: composed of 'red'-BDPMA in the core, stabilized by a hydrophilic random diblock copolymer shell.

Figure 2 . 5

 25 Figure 2.5 A new RAFT agent is prepared including DIBO moiety in its structure. Equal synthetic route as shown in Figure 2.4 is followed to obtain a new family of FNP including DIBO in the shell for click-chemistry reactions.

Figure 2 . 6

 26 Figure 2.6 Scheme of different species of fluorescein depending on pH.

Figure 2 . 7

 27 Figure 2.7 Scheme of synthesis of 'green' BODIPY (phenol) and polymerizable BODIPY (Methacrylate).

  diethyl-4,4-difluoro-8-(4-(methacryloyloxy)phenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (2)

Figure 2 . 8

 28 Figure 2.8 Scheme of Trofimov synthesis of MethylNaphthalene-Pyrrole starting from MethylNaphtalene.

Figure 2 . 9 8 (

 298 Figure 2.9 Scheme of synthesis of 'red' BODIPY 7 ans polymerizable BODIPY 8 (Methacrylate).

Figure 2 . 10

 210 Figure 2.10 Scheme of synthesis of 'DIBO'. (From Ref. 9)

STEP 2 :

 2 (PEOA-co-AA)-TTCA-DIBO (15)Same protocol as the MacroRAFT agent: (PEOA-co-AA)-TTCA was used replacing TTCA by DIBO-TTCA. The conversion rate and polymer mass were estimated based on the total monomer conversion followed by NMR (≥77%yield).

Figure 2 . 13

 213 Figure 2.13 Zeta potentials in mV recorded at room temperature in saline water ([NaCl] = 14mM) for different pH values (phosphate-Citrate buffers 1 mM) for FNP (a) FNPGreen, (b) FNPRed (c) FNPG-DIBO, (d) FNPR-DIBO.

Figure 2 . 14 .

 214 Figure 2.14. The absorption spectra showed the classical expected shape of the BODIPY dyes with one intense band in the visible range representing the transition energy betweenthe ground state and the first excited state and it is in good agreement with reported literature(2). The smaller band closer to UV of the absorption spectra represents the energy transition from the ground state to the second excited state.

Figure 2 .

 2 Figure2.14 shows green FNP spectra (FNPGreen and FNPG-DIBO), in comparison with green-BODIPY monomer in DCM. Data (not shown) were also recorded in toluene as this solvent has close properties with polystyrene. Green FNPs' absorption maximum (529 nm) is very close to the one in toluene (528nm) and is the same as in DCM. A redshift in the emission maximum is observed (compared to the monomer in toluene) : 7 nm for FNPGreen and 8 nm for FNPG-DIBO. Similar observation is seen in comparison with DCM : 9 nm for FNPGreen and 10 nm for FNPG-DIBO. Such bathochromic shift on emission maximum has already been observed by our group(19) and was previously reported byAlvarez et al. (20) for krypto-BODIPY copolymerized with MMA. Such behavior was attributed to the fluorophore confinement within the matrix.

Figure 2 . 14 FNP

 214 Figure 2.14 Normalized absorption and fluorescent emission spectra recorded in D.I. water at room temperature for FNPs (a) FNPGreen, (b) FNPRed (c) FNPG-DIBO, (d) FNPR-DIBO; compared with their respective BDPMA in DCM.

Figure 2 . 15 Figure 2 . 15 ,

 215215 Figure 2.15 Normalized absorption (Abs) and emission spectra (Em) (λexc: 485 nm) at pH 8 (Citrate/

Figure 2 . 18 Figure 2 .

 2182 Figure 2.18 Excitation spectra (λem: 620 nm) at different pH (Citrate/ Phosphate buffer) at room temperature for FNPRed (upper) and FNPRed-FA (bottom). Spectra were taken by adjusting the maximum of absorbance below 0.1.

Figure 2 .Figure 2 . 22

 2222 Figure 2.21 FNP decays vs pH (Citrate/ Phosphate buffer 0.2M) at room temperature found for FNPRed-FA and FNPRed, (OD < 0.1, λexc: 485 nm)

Figure 3 . 1

 31 Figure 3.1 Two different strategies to introduce FA on glass substrates for pH-sensing surfaces. 2 step:

slides of 19

 19 mm diameter and thickness #1.5 were purchased from Agar Scientific. EDSA (3-(Ethoxydimethylsilyl) propylamine) was purchased from Fluorochem Ltd. Absolute ethanol, Sulfuric acid 95 %, hydrogen peroxide 30 %, EDC (N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) and FA (Fluoresceinamine)

Figure 3 . 4

 34 Figure 3.4 From left to right: Schlenk tube, home-made Teflon sample holder and lastly, Soxhlet system used to clean the slides after silanization.

Figure 3 . 5

 35 Figure 3.5 Fluorescent nanoparticle grafting procedure on glass surfaces.

Figure 3 . 6

 36 Figure 3.6 Glass slides metallic holder with an internal diameter of 19 mm and external of 3.5 cm.

Figure 3 . 7

 37 Figure 3.7 Setting used for measurements of bacterial growth.

Figure 3 . 9 Figure 3 .

 393 Figure 3.9 XPS Windows of Carbon (left) and Nitrogen (right) after different surface treatments: top graph : after FNPRed grafting, middle graph : after glass silanization, bottom graph : after glass Piranha cleaning

Figure 3 . 13

 313 Figure 3.13 Normalized absorption (top) and Fluorescence emission (bottom) (λexc: 465 nm for FA and λexc: 485 nm for others). ( ̶ ̶ ) Dry FNPRed-FA surface, (--) FNPRed nanoparticles in suspension, (--) FA in suspension (pH ̴ 5).

Figure 3 . 14 Figure 3 . 15

 314315 Figure 3.14 Normalized absorption spectra: ( ̶ ̶ ) Dry FNPRed-FA surface, (--) FNPRed-FA nanoparticles in suspension at pH: 5.

Figure 3 . 16

 316 Figure 3.16 Normalized spectra at different pH for trial 1.

Figure 3 . 17

 317 Figure 3.17 Statistical box chart of the average ratio at different pH for trial 1 (𝛼 520/600 ). The box represents the interquartile range (25-75 %), the  is the mean value and the bars the data range.

Figure 3 . 19

 319 Figure 3.19 Statistical box chart of the average ratio at different pH for trial 2 (𝛼 520/600 ). The box represents the interquartile range (25-75 %), the  is the mean value and the bars the data range.

Figure 3 . 20 Figure 3 . 21

 320321 Figure 3.20 Normalized spectra at different pH for trial 3.

Figure 3 . 22

 322 Figure 3.22 Reproducibility and reversibility for the pH-sensor. The measurement was carried out by exposing the pH-sensitive surface to different buffer solutions in turn: (a) 5.0, (b) 6.5 and (c) 8.0.

24 and 3 .Figure 3 . 23

 3323 Figure 3.23 Normalized emission spectra at different pH to calibrate the FNPRed-FA grafted surface used for bacterial growth study.

Figure 3 . 24 Figure 3 . 25

 324325 Figure 3.24 Fluorescence imaging of E coli growth on a FNPRed-FA surface, in M9 minimal medium at different times. Fluorescence images corresponds to surface emission (red channel, λex: 488 nm). Bacterial growth can be observed in the brightfield images.

Figure 3 .

 3 Figure 3.26 Ratio 𝛼 520/600 , (red axis, on the right) and number of cells (black axis on the left) per image as a function of time.

Figure 3 .

 3 Figure 3.27 PDMS microfluidic circuit

4. 1 4 . 2 4 . 3

 14243 Photobiological Sciences, 2018 (doi: 10.1039/c8pp00043c), presenting the results about polypyrene electropolymerized films proving to be efficient to prevent bacterial adhesion. This work was also done in collaboration with Siewert Hugelier and Michel Sliwa from LASIR, Université de Lille, whom optimized the image analysis. Pages 137 to 157.

4. 4

 4 photophysical properties characterizations. Pages 171 to 183.

First, the synthesis

  and characterizations of different types of FNPs were carried out successfully. New types of particles were obtained for the first time (FNPRed -with high 'red' BODIPY per particle-, FNPG-DIBO and FNPR-DIBO). FNPRed were the main players in the manuscript, meanwhile FNPDIBO will lead to future studies. The surface charge of the different particles was estimated by ζ-potential. The hydrodynamic radius of the FNPs was characterized by DLS (Dynamic Light Scattering) and NTA (Nanoparticle Tracking Analysis). Concentration was estimated by NTA. TEM (Transmission Electron Microscopy) image analysis allowed for dried diameter estimation. In summary, all FNPs are polydispersed but a mean diameter could be estimated (FNPGreen: 80 nm, FNPRed: 110 nm, FNPG-DIBO: 100 nm and FNPR-DIBO: 130 nm). The absorption and emission of FNPs corresponded well to the dyes in the core

  A mixture of 1-methylnaphthyl propyl ketone 3 (1 eq., 37.7 mmol, 8 g), hydroxylamine hydrochloride (2 eq., 75.4 mmol, 5.24 g), 100 mL of pyridine and 100 mL of ethanol, were stirred at 80 °C overnight. After cooling down the mixture, this one was poured in

	1 H NMR (400 MHz, CDCl3): δ/ppm: 8.64 (m, 1H), 7.76 (d, 1H, J: 7.3 Hz), 7.58 (m, 2H), 7.34 (dd, 1H, J: 0.9 Hz), 3.02 (t, 2H, J: 7.3), 2.74 (s, 3H), 1.82 (m, 2H), 1.03 (t, 3H, J: 7.3 H N Hz). O 4

13

C NMR (100 MHz, CDCl3): δ/ppm: 205.0, 139.4, 134.9, 133.

1, 130.4, 127.5, 127.4, 126.5, 126.4, 125.4, 44, 1, 20.2, 18.5, 14.

0. C15H16O: 212.12.

Oxime of 1-(8-Methylnaphthalene-1-yl)butane-1-one (Z,E) (4)

water. The precipitated solid was recovered by filtration, washed and dried. If no precipitate was formed, the mixture was washed with HCl (1M), and extracted with diethyl ether. No further purification was done. The final product was obtained with 93 % yield.

1

H NMR (400 MHz, CDCl3): δ/ppm: 8.02-7.17 (m, 6H), 2.84 (m, 1H), 2.69 (s, 3H), 2.58 (m, 1H), 2.50 (m, 2H), 0.90 (m, 3H) .

13

C NMR (100 MHz, CDCl3): δ/ppm: 161.6, 159.4, 135.4, 135.4, 133.0, 132.9, 132.6, 131.6, 129.6, 126.3-126.0, 124.7, 124.6, 123.9, 38.8, 32.3, 20.0, 19.7, 19,7, 14.5, 14.0. C15H17NO: 227.13.

-diethyl-4,4-difluoro-8-(4-(methacryloyloxy)phenyl)-5,3-(9-methyl-1-naphtyl)-4- bora-3a,4a-diaza-s-indacene (referred as 8 or Red-BDPMA) Same

	C45H39BF2N2O2Na: 711.2992.
	1.1.2.2. Molecular Synthesis: DIBO
	N 2				Br	Br
	Si	NaBH 4	Br 2
	BF 3 .Et 2 O
	O	O			OH	OH
	9			10	11
	Br	Br
		iPr 2 NH, n-BuLi
		OH		O	12	OH
			O	DIBO
		N	(-)	N
			B	(+)
		F		F
			8
	2,61 H NMR (400 MHz, CDCl3): δ/ppm: 7.89 (d, 2H, J: 8.2 Hz), 7.83 (d, 2H, J: 7.8 Hz), 7.76
	(m, 2H), 7.71 (m, 1H), 7.47-7.19 (m, 8H), 7.21 (m, 1H), 6.69 (s,2H), 6.38 (s, 2H), 5.78
	(s, 1H), 2.64 (s, 6H), 2.14 (S, 3H), 2.02 (m, 2H), 1.88 (m, 2H), 0.88 (m, 6H, J: 7.6 Hz).

1

H NMR (400 MHz, CDCl3): δ/ppm: 7.96 (d, 2H, J: 8.2 Hz), 7.90 (d, 2H, J: 8.2 Hz), 7.6-7.1 (m, 12H), 6.75 (m, 2H), 2.64 (s, 6H), 2.03 (m, 2H), 1.91 (m, 2H), 0.90 (m, 6H).

13

C NMR (100 MHz, CDCl3): δ/ppm: 156.2, 137.6, 135.7, 134.4, 132.4, 130.0, 128.9, 128.3, 126.7, 126.0, 125.6, 124.5, 115.2, 19.9, 19,7, 14.4, 14.2.

19

F NMR (376 MHz, CDCl3): δ/ ppm: -125.9 (m, 0, 8F), -137.5 (q,2F, JF-B: 32.3 Hz), -147.7 (m, 0, 8F).

11

B NMR (128 MHz, CDCl3): δ/ ppm: -0.29 (t, JB-F: 29.5 Hz). HRMS m/z: 619.2732 [M + Na] + ; calc. for C41H34BF2N2O: 619.2749. RED BODIPY-NAPHTHALENE-METHACRYLATE 8 (Red-BDPMA, in Figure 2.3) protocol as green-BODIPY-methacrylate 2 was used replacing green BDP 1 by red BDP 7. The final product 8 was a pink-violet powder with (332 mg) 88.6 % yield. 13 C NMR (100 MHz, CDCl3): δ/ppm: 165.8, 156.4, 152.4, 137.7, 135.8, 135.7, 134.5, 132.45, 132.46, 132.1, 132.0, 131.8, 131.0, 128.9, 128.1, 127.0, 126.8, 126.0, 125.74, 125.66, 124.5, 121.8, 19.7, 19.4, 14.3. 19 F NMR (376 MHz, CDCl3): δ/ ppm: -125.8 (m, 0.8F), -137.6 (q, 2F, JF-B: 32.3 Hz), -147.7 (m, 0.8F). 11 B NMR (128 MHz, CDCl3): δ/ ppm: -0.25 (t, JB-F: 30.8 Hz). HRMS m/z: 711.2970 [M + Na] + ; calc. for

DIBO 5,6-Dihydro-11,12-didehydro-dibenzo[a,e]cycloocten-5-ol (12)

  The aqueous phase was extracted twice with AcOEt and the organic phase was dried, filtered and solvent was evaporated. The compound was purified by chromatography column (SiO2, heptane/AcOEt, 9:1). The final product was a white powder obtained with 50 % yield.

	1 H NMR (400 MHz, CDCl3): δ/ ppm: 7.67 (1 H, aromatics), 7.37-7.18 (7 H, aromatics),
	4.57 (dd, 1H, CHOH, J=2.1, 14.7 Hz), 3.04 (dd, 1H, CH2, J=2.1, 14.7 Hz), 2.86 (dd, 1H,
	CH2, J=2.1, 14.7 Hz); 13 C NMR (100 MHz, CDCl3): δ/ ppm: 154.5, 150.6, 128.6, 127.1,
	1127.0, 126.0, 125.8, 125.1, 124.7, 123.0, 122.7, 121.7, 111.9, 109.6, 74.2, 47.7.
	OH
	12
	Diisopropylamine (4.1 eq., 1.26 mmol, 0.18 mL) was disolved in dry THF (10 mL) at 0
	°C under Ar. Butyl Lithium (BuLi, 4 eq., 1.22 mmol, 0.77 mL) was added dropwise and

1

H NMR (400 MHz, CDCl3): δ/ ppm: 7.54-7.47 (2 H), 7.31-6.72 (6 H), 5.77 (d, 1H, CHBr, J=5.4 Hz), 5.22 (dd, 1H, CHOH, J=3.6, 5.9 Hz,), 5.19 (d, 1H, CHBr, J=5.4 Hz), 3.50 (dd, 1H, CH2, J=3.6, 15.9 Hz,), 2.75 (dd, 1H, CH2, J=3.6, 15.9 Hz,);

13

C NMR (100 MHz, CDCl3): δ/ ppm: 141.3, 140.0, 137.2, 134.0, 133.4, 131.5, 131.3, 130.9, 127.8, 126.2, 123.7, 121.3, 76.5, 70.0, 62.3, 32.2. HRMS m/z: 402.9313 [M + Na] + ; calc. for C16H14Br2NaO + : 402.9304. the mixture was stirred at 0 °C for 30 min. The previous bromide compound 11 was dissolved in 10 mL of dry THF and added dropwise to the Lithium diisopropylamide (LDA) solution. The reaction was stirred for 1.5 h and then quenched by adding a saturated solution of ammonium chloride.

1.1.2.3. Macromolecular Synthesis: Polymers

MacroRAFT Agent: (PEOA-co-AA)-TTCA

(13)

  Dilutedsamples were placed into dispensable 50 µL cuvette cells. Samples were inserted into the Particle Size Analyzer Vasco Flex by Corduran Technologies (from LAC -Laboratoire Aimé Cotton -UMR CNRS 9188). The laser of the Vasco Flex was calibrated by visually comparing it to the calibration laser of the equipment. Three measures of three scans were

	1.1.3.5. ABSORPTION & EMISSION
	UV-visible spectra were recorded on a UV-2600 UV-VIS Spectrometer by
	Shimadzu Scientific Instruments. Excitation and emission spectra were measured on a
	performed for each sample.
	1.1.3.2. TEM
	Transmission Electron Microscopy (TEM) was performed on a JEOL1400
	operating at 80kV. The samples were diluted in water prior to analysis and then deposited
	on a carbon-coated copper grid. Phosphotungstic acid (PTA) was used as contrast agent.
	each sample.
	1.1.3.4. NANOSIGHT
	Particle size, concentration and dispersion was determined by Nanoparticle
	Tracking Analysis (NTA) on a NanoSight NS300 by Malvern Analytical (PPSM -
	Laboratoire de Photophysique et Photochimie Supra-et Macromoléculaires -UMR
	CNRS 8531). For each sample, 1.2 µL of FNP stock solution were diluted to 2.0 mL using
	filtered D.I. water as solvent. A further dilution of 50 µL of already diluted FNP solution
	1.1.3. FNP Characterization Methods in 10 mL of water for green FNPs was performed. Three measurements of 60 seconds
	each were recorded to determine size and concentration for each type of FNPs. The FNP
	stock concentrations were found from the measured concentration of the diluted samples.
	1.1.3.1. DLS

For each type of FNPs, 1.2 µL of stock solution were diluted to 2.0 mL using filtered deionized (D.I.) water to achieve a concentration of 0.01% in weight.

The present work has benefited from the core facilities of Imagerie-Gif, (http://www.i2bc.paris-saclay.fr).

1.1.3.3. ZETA-POTENTIAL

Zeta potentials (ζ) were performed on a Zetasizer Nanoseries (Malvern) apparatus at the from LAC -Laboratoire Aimé Cotton -UMR CNRS 9188). Samples were prepared at concentration of 0.01 wt % diluted in buffered with 200 mM of phosphate/citrate salts (pH values varied from 4 to 8) at 25°C. Six measures of ten scans were performed for

Table 2 . 2

 22 Average hydrodynamic diameters (nm) for all FNP from DLS measurements using two different methods (Cumulant and Pade-Laplace).

Table 2 . 3

 23 Summary of results after NTA data analysis of all FNP

	FNP Type	Mean (nm)	Mode (nm)	PDI ( -)	Concentration (Particles/mL)		Stock Concentration (Particles/mL)
	FNPGreen	80 ± 1	70 ± 1	0.61	7.3x10 8 3.4x10 7	±	4.9x10 14 ± 2.3x10 13

Table 2 .

 2 4 FNP size comparison using different size characterization techniques.

	FNP	DLS Cumulant (nm)	NTA (nm)
	FNPGreen	80 ± 2	80 ± 1
	FNPRed	110 ± 2	110 ± 1
	FNPG-DIBO	100 ± 4	100 ± 1
	FNPR-DIBO	240 ± 3	130 ± 2

Transmission Electron Microscopy (TEM) was used to visualize the nanoparticles and determine their size in a "dry" state (which shall be different from the hydrodynamic radius), shape, and aggregation state. In this technique, electrons are emitted through a vacuum column until reaching the sample. Depending on the density of the surface, electrons either passed through it or get scattered. Since the nanoparticles are not good conductors, a contrast agent (phosphotungstic acid, PTA) to better visualize. PTA was used as negative stain, which means the background is stained and the sample is untouched. Due to short time, no further comparisons were done by changing or not using the contrast agent.

Table 2 .

 2 5 FNP size distribution found by TEM (Mean, Mode, Standard deviation of TEM analysis), and DLS (cumulant), NTA and the difference in comparison with TEM. FNP images from TEM using contrast agent and subsequent size analysis.In Figure2.10 and 2.11 different nanoparticles by TEM can be observed. FNPGreen and FNPG-DIBO aggregation state looks alike, only changing in size distribution, being FNPG-DIBO bigger than FNPGreen. Whereas for red particles, FNPR-DIBO are slightly smaller than FNPRed. As it can be seen in Table2.5 and Figure2.12, the average size of FNPGreen

		N	Mean	Mode	Standard	DLS Cumulant	NTA	Δ (DDLS-DTEM )	Δ (DNTA-DTEM )
		Total	(nm)	(nm)	Deviation	(nm)	(nm)	(nm)	(nm)
	FNPGreen	1595.0	29.0	20.1	± 7.7	80 ± 2	80 ± 1	51	51
	FNPRed	3549.0	44.6	22.3	± 16.2	110 ± 2	110 ± 1	65	65
	FNPG-DIBO	428.0	38.2	50.5	± 15.3	100 ± 4	100 ± 1	62	62
	FNPR-DIBO	63.0	31.7	22.0	± 10.3	240 ± 3	130 ± 2	207	67

elongated. In Table

2

.5 an estimated of the PEG-shell is shown comparing either DLS-TEM or NTA-TEM. PEG-Shell can be estimated between 51 to 67 nm. In Figure

2

.

12

Table 2 . 6

 26 Spectroscopy parameters for the different FNPs in D.I. and BODIPY dyes in DCM, at room temperature.

	/ Intensity (a.u)	0.6 0.8 1.0		Abs BODIPY Green Em BODIPY Green Abs FNP Green Em FNP Green	Intensity (a.u.)	1.0 0.6 0.8	Abs BODIPY Red Em BODIPY Red Abs FNP Red Em FNP Red
	Absorbance	0.2 0.4			Absorbance /	0.2 0.4
		400 0.0	500	600	700	0.0	500	600	700
			Wavelength (nm)			Wavelength (nm)
			FNPGreen			FNPRed
		1.0				1.0
	/ Intensity (a.u)	0.6 0.8		Abs BODIPY Green Em BODIPY Green Abs FNP G-DIBO Em FNP G-DIBO	Intensity (a.u.)	0.6 0.8	Abs BODIPY Red Em BODIPY Red Abs FNP R-DIBO Em FNP R-DIBO
	Absorbance	0.2 0.4			Absorbance /	0.2 0.4
		0.0				0.0
		400	500	600	700		500	600	700
			Wavelength (nm)			Wavelength (nm)

Table 2 . 7

 27 Fluorescent quantum yield and BODIPY per particles in D.I. water. As previously mentioned, reference dyes were used as follows: rhodamine 6G dye was used for green FNP, while sulforhodamine 101 was used for red FNP.

Table 2 .

 2

8 Spectroscopy parameters for fluorescein forms c and d water at room temperature (from 11).

Table 2 .

 2

	pH	ΦF FNPred (%)	ΦF FNPRed-FA-green (%)	ΦF FNPRed-FA-red (%)
	8	10.5	3.1	7.1
	7.5	10.9	3.2	10.3
	7	12.2	2.7	7.9
	6.5	11.3	2.3	10.1
	6	10.9	1.3	9.8
	5.5	12.0	1.1	11.1
	5	10.8	1.2	9.6

9

Fluorescence Quantum Yields for FNPRed-FA (Green part: FNPRed-FA-green and red part: FNPRed-FAred) and FNPRed at different pH (Citrate/ Phosphate buffer 0.2M) at room temperature.

Figure 2.20 Fluorescence

Quantum Yields vs pH (Citrate/ Phosphate buffer 0.2M) at room temperature found for FNPRed-FA (red part) and FNPRed, left image, and FNPRed-FA (green part) right image.

  °C. A minimum of 10 different zones from a minimum of 2 samples were analyzed for each kind of surface. Percentage of coverage was calculated by Image J software.

	3.1.3.3. Contact Angle
	Water contact angles were measured under ambient atmosphere with an EasyDrop
	Contact Angle Measuring Instrument by KRUSS GmbH. A drop of 1 µL MiliQ water
	was used each time. A minimum of 6 different zones from a minimum of 2 samples were
	analyzed for each type of surface.
	3.1.3.4. Absorption & Emission
	Absorption spectra were measured on a UV-2600 UV-Vis Spectrometer by
	Shimadzu Scientific Instruments. Emission spectra were measured on a FluoroMax-4
	Spectrofluorometer (Horiba Jobin-Yvon).
	Fluorescence images and spectra in solution were acquired using Leica TCS SP5-
	AOBS confocal laser scanning microscope. The surfaces were imaged using a × 63 -1.4
	numerical aperture plan apochromat oil immersion objective. The size of the xy image
	was either 246.03 x 246.03 μm 2 or 82.01 × 82.01 μm 2 , (1024 × 1024 pixels) recorded on

Table 3 . 2

 32 Spectroscopic data of FNPRed-FA surfaces compared to data of FA and FNPRed in D.I. water (pH ̴

	5)
		λabs,	max-1	λabs,	max-2	λabs,	max-3	λem,	max-1	λem,	max-2	FWHMem-1	FWHMem-2
		(nm)	(nm)	(nm)	(nm)	(nm)	(cm -1 )	(cm -1 )
	FA in water	454	478	--	511	--	1954	--
	FNPRed in water	--	--	559	--	600	--	1951
	FNPRed-FA surface	467	501	559	525	600	1426	1583

Table 3 . 3

 33 Average ratio 𝛼 520/600 at different pH, for three individual trials. Trial 1 and 3 are from FNPRed-FA surfaces and Trial 2 comes from pre-grafted FA on red FNP surface. All surfaces are from the same batch of silanization.

	Trial		pH 5			pH 5.5			pH 6			pH 6.5			pH 7			pH 7.5			pH 8
	1	0.09	±	0.04	-	-	-	-	-	-	0.14	±	0.06	-	-	-	-	-	-	0.28	±	0.04
	n=5
	2	0.04	±	0.00	0.05	±	0.00	0.05	±	0.2	0.07	±	0.02	0.08	±	0.01	0.13	±	0.01	0.17	±	0.01
	n=2
	3	0.05	±	0.00	0.06	±	0.00	0.07	±	0.00	0.09	±	0.00	0.12	±	0.00	0.15	±	0.02	0.17	±	0.00
	n=5

  Normalized spectra at different pH for trial 2 (only trial on pregrafted FA on FNP, before FNP grafting on surface).

	7.5a
	7.5b
	5a
	5b
	8a
	6.5a
	6a
	6b
	5.5a
	7a
	5.5b
	8c
	7b
	6.5b
	Figure 3.18

Table 3 . 4

 34 Linear fits for three individual trials, correlating pH and ratio of emission bands at 520 nm (FA) and 600 nm (Red BODIPY).Nevertheless, within the same zone, a good reproducibility and reversibility of the sensor is obtained, as it can be seen in figure3.22 (from Trial 3). Different cycles of pH 5, 6.5 and 8 were carried out in the same zone, obtaining each time a similar ratio. The pH-sensing surface can thus be used from acid to base and from base to acid in the range between pH 5 and 8, which are in the range of biological samples such as bacteria. In

		Slope	Intercept	R-square		Model
	Trial 1	0.06 ± 0.02	-0.24 ± 0.13	0.81	𝑝𝐻 =	𝛼 520/600 + 0.24 0.06
	Trial 2	0.04 ± 0.00	-0.17 ± 0.04	0.82	𝑝𝐻 =	𝛼 520/600 + 0.17 0.04
	Trial 3	0.05 ± 0.00	-0.25 ± 0.06	0.82	𝑝𝐻 =	𝛼 520/600 + 0.25 0.05

comparison with Niu et al.

(29)

with a slope of 0.11 and range in between: 6.6-7.6, the sensitivity of our sensor remains lower (average slope: 0.05, range: 5.0 -8.0) but a larger pH range is considered.

Table 3 . 5

 35 Linear fit of the sample used for bacterial growth study.

			pH 5	pH 6.5	pH 8
	Calibration α520/600	0.005	0.093	0.205
	Slope	Intercept	R-square		Model
	0.06 ± 0	-0.33 ± 0.02	0.99	𝑝𝐻 =	𝛼 520/600 + 0.33 0.06

Table 3 . 6

 36 Data obtained from bacterial growth study, showing the ratio 𝛼 520/600 ,calculated pH with the linear model obtained from the calibration, measured pH on surface and bulk, bulk OD and number of cells per image.

	T	Time (min)	α520/600	Calculated	Measured	Measured	Bulk OD	#cells/
				pH	pH	Bulk pH		image
	1	0	0.0998	6.5	6.5	6.5	0.01	4
	2	60	0.0692	6.0				9
	3	120	0.0568	5.8				44
	4	240	0.0668	6.0	6.5	6.5	0.10	652
	5	300	0.0525	5.8				1120
	6	360	0.0585	5.9	6	6	0.22	2208
	7	420	0.0456	5.7				2384

Abbreviations

grafted FA. Nevertheless one possible explanation for such a weak fluorescence quantum yield might be consistent with a high number of grafted FA, which then tend to aggregate through π-π stacking. This phenomenon, also called self-quenching, was described earlier by Charreyre et al. (20).

Another issue shall be arisen, if energy transfer occurs then Fluorescein-amide (donor) shall be quenched (as observed from excitation spectra, Figure 2. 19) at pH 8. The higher the pH is, the more quenched it shall be. When pH decreases, energy transfer shall be less efficient (see R0 value given p. 78 and see Figure 2. 19), thus ΦF should increase for FA but also decrease due to pH change. At this stage, we have difficulty to rationalize such opposite effects.From table 2.9 and Figure 2.20, it is possible to observe that ΦF for FNPRed-FA 'red part' remains very similar to ΦF FNPRed. Moreover we decided to perform time-resolved fluorescence experiments to check if such unexpected behavior occurred with decays as well.

The variation of fluorescence lifetime (τ) vs pH was investigated at a wavelength were mainly the donor is excited (FA, λexc: 485 nm) fixing the collection time to 10 min. Such experiment was also carried out for FNPs without FA (FNPRED). From Figure 2.21, and from a qualitative point of view, the decays, in the 'green part' of FNPRed-FA, decrease notably while pH decreases, meanwhile for the 'red part' this is much less evident. FNPRed decays seem to be weakly sensitive to pH (it shall be noted here that even though absorption is weak at the excitation wavelength, an emission signal is collected, thus when donor and acceptor are present, both shall be excited).

Looking at decays' shape, in the 'red' range, it seems that the presence of FA extends the decay time, for a same pH "red range" emission seem to decay less when FA is grafted. Unfortunately, due to a lack of time, we did not perform fits.

We evaluated the mean lifetime, <τ>, thanks to the equation 1.

Conclusions & Perspectives

In conclusion, the synthesis and characterization of different types of FNP were carried out successfully. Firstly, to our knowledge, new kinds of particles were obtained for the first time (FNPRed -with high 'red' BODIPY initial molarity-, FNPG-DIBO and FNPR-DIBO). The new family of DIBO particles containing 'DIBO' moiety may allow the postfunctionalization by the highly efficient 'click-chemistry'. Such coupling is bio-friendly since it doesn't require copper. Looking further for applications, these FNPDIBO could be used to target not only bacteria strains, but also different eukaryotic cell lines by changing the antibody grafted on them. Due to lack of time, the application of these FNP will not be studied, but they are being incorporated in a new project (heavy metals detection, with Labex Charmmmat fundings) and the continuation of this one (bacterial growth detection, with CSC Grant).

The surface charge of the different particles was estimated by ζ-potential. The size of all particles was characterized in different ways (DLS for hydrodynamic radius, NTA for size and concentration, TEM for dried diameter). Particles are highly dispersed.

Nevertheless, for our main objective (to produce a surface sensitive to pH for bacterial growth detection), we chose to proceed without further purification or size-classification (i.e. by Nanosep™ nanopore filters) of the particles.

Besides and most importantly, a new ratiometric fluorescent pH nanosensor was designed and characterized based on 'red' BODIPY and FA. The nanoparticles were stable down to pH=5 and contained a very large number of both fluorescent species. Their spectroscopic characteristics were studied in water at various pH and the pKa of the grafted fluorescein was determined to be close to the tabulated one. Even though excitation spectra showed that energy transfer occurs at pH ≥ 7, it was extremely difficult to quantify and probe it from quantum yield and fluorescence lifetime.

Nevertheless, the FNPRed-FA particles could be used as ratiometric pH-sensors. τF of the red-band of FNPRed-FA particles is weakly affected by pH, whereas ΦF and τF of greenband of FNPRed-FA is strongly affected. The plot of intensity ratio shows a very nice pH sensitivity in between 5.5 and 7, this is a perfect window for biological applications such as bacterial growth. The grafting efficiency of FA was not calculated since we were not able to measure properly the particles' concentration. oligomers, that could have been attached to the surface through the silanization process.

After silanization, glass slides were functionalized with FNPRed, following by fluorescein (FA) addition. Both grafting reactions were done by peptide coupling. 3) FNPRed grafting on silanized-glass slides by peptide coupling. 4) FA (yellow rectangle, fluorescein) grafting on FNPRed grafted on silanized-glass slides.

X-ray Photoelectron Spectroscopy

Surface elemental characterization was done by XPS analysis, considering only C, N, Si and O (Table 3.1). The relative atomic percentage of these four elements were obtained for analysis. Traces of Na, B, F and K were found in pristine glass; therefore, they were not considered for analysis. Nitrogen is only present in the aminosilane molecules, nevertheless, traces were found in activated glass, which are due to adventitious contamination (which can be expected. After activation, the negative surface charge of the glass attracts all the positive charged species). Nitrogen increased 4.5 times after silanization the decrease of 1.5 times in the percentage of Nitrogen after FNP grafting and an increment of 2.5 times in the percentage of Carbon corresponds well to the nanoparticle grafting.

Contact angle

Surface wettability was determined by measuring the contact angle of water drops on the different functionalized surfaces (Figure 3.10). The average water contact angle of glass after piranha was 4° ± 1°; for the silanized glass slide was 41° ± 2°; for FNPRed surfaces it was 68° ± 2° and for FNPRed-FA surfaces it was 68° ± 1°. For glass, after piranha-treatment, it is reasonable to obtain a very hydrophilic surface, later with the addition of organic silane and FNPRed the surface tends to become less and less hydrophilic as seen on the values of contact angles. These values are in good agreement with other silanized and organic-functionalized surfaces (23,24). It can be observed that after fluorescein (FA) coupling to nanoparticles, the contact angle value did not changed, probably because of a small amount of grafted organic molecule. Adding FA shall not tremendously change the composition and the nature of the shell thus hydrophobicity shall not be changed. Nevertheless changes in contact angle are directly connected to the different steps in surface functionalization and particles grafting. Such experiments allow to follow easily the surface chemistry.

After Piranha Silanized

FNPRed FNPRed-FA

Surface coverage: SEM & AFM

To quantify nanoparticle grafting, SEM and AFM images were taken (Figure 3.11 and 3.12). For SEM, controls were taken without EDC, to probe electrostatic interactions between FNPs and silanized glass. The coverage area for FNPRed was found to be 8.1 ± 0.7 % with EDC coupling, while without EDC was 0.8 ± 0.1 %. The surface coverage by EDC coupling was found to be 10 times higher than control (electrostatic interactions).

From this result we can observe that peptide coupling is necessary to ensure the anchoring of the FNP on the surface. Meanwhile, for AFM the coverage area was found to be 13.1

Conclusions & Perspectives

The use of electropolymerization for the elaboration of micro-and nanostructured surfaces with tunable wettability is an emerging field of investigation. The global aim of this work was to characterize new polymer films: their surface wettability, morphology, photophysical properties and for some of them the anti-bioadhesion properties were as well investigated.

Herein, we demonstrated the potential application of polypyrene films to serve as coatings to prevent bacterial adhesion. Studying fluorinated and nonfluorinated surfaces with different hydrophobicity, it was shown that it is not completely necessary to obtain highly superhydrophobic surfaces to prevent bacterial adhesion. More importantly, the combination between wettability and the topography (micro-and nanostructuration) of the film seemed to play a crucial role.

A reduction between 30-70% in the early bacterial adhesion for non-fluorinated polypyrene and between 60-90% for fluorinated polypyrene was obtained for S. aureus and P. aeruginosa strains. For longer incubation times, polypyrene surfaces demonstrated a high efficiency against biofilm attachment and reduced by 90-99% the bacteria coverage. These results demonstrate the capability of these surfaces to be used as coatings to prevent bacterial adhesion.

Yet, after participating and presenting this work to the scientific community some important questions to be investigated have arisen: what it is the average life and durability of these films? Are they reusable? Are they mechanically resistant? Is there any toxic effect due to their composition and could they be used for eukaryotic cells? Some of these questions shall be further investigated.

For new applications, through another collaboration with Universidad de Valle de