The need for potable water is increasing with the ever-increasing world population. Further development of fast, portable, and cost effective analytical tools is necessary in order to create diagnostic techniques capable of supporting the water needs of the world’s population. Within the last decade microfluidics and Lab-on-a-Chip (LOC) technologies have increased the portability and speed of detection for aqueous samples. Photolithography techniques serve as a cost effective fabrication tool to create LOC electrodes on the micron scale. An in-depth look at the fabrication process is undertaken in this paper in order to further the development of micro-scale detection techniques. An electrode array capable of detecting multiple targets within one aqueous sample was designed and fabricated. The electrode array was assessed for performance characteristics to determine if reproducibility is possible. The fabrication process was also detailed for a new chemical separation technique, traveling-wave electrophoresis (TWE). TWE could serve as a separation tool capable of separating out specific charged molecules for biological and chemical samples. The TWE device was assessed on the capabilities to move charged molecules.

The aim to provide preventive healthcare and high quality medical diagnostics and treatment to an increasingly ageing population caused a rapidly increasing demand for point-of-care diagnostic devices. Disposables have an advantage over re-usable units as cross-contamination is avoided, no cleaning and sterilising of equipment is required and devices can be used out of centralised laboratories. To remain cost-effective, costs for disposables should be kept low. This makes polymer materials an obvious choice. One method for the realisation of fluidic micro devices is the stacking of several layers of microstructured polymer films. Reel-to-reel manufacturing is a promising technique for high-volume manufacturing of disposable polymer bio-analytical devices. Polyethylene terephthalate (PET) and cycloolefin copolymer (COC) were selected as suitable polymer substrate materials and polydimethyl siloxane (PDMS) as membrane layer. Bonding of polymer films with the help of adhesives carries the risk of channel blocking. Despite this drawback, no other method of bonding PDMS to a structural layer could be identified. Bonding with solvents avoids channel blocking issues, but adversely affects biocompatibility. Thermal diffusion processes enable bonding of COC and PET without the use of any auxiliary material. The extensive process times requires for thermal diffusion bonding can be considerably shortened by pre-treating the material with plasma or UV exposure. Welding with the use of a laser energy absorbing dye was demonstrated to be particularly suitable for selective bonding around channels and reservoirs. None of the assessed bonding methods provide a generic solution to all bonding applications. Instead, the selection of an appropriate technique depends on the intended application and the required level of biocompatibility. Since this selection has implications on the feasibility and reliability of microfluidic structures on the device, design rules which ensure design for production have to be established and followed.

L'étude des plantes utilisées en médecine traditionnelle au Togo est compliquée à cause de l’absence de matériel de pointe. L'identification assistée par ordinateur de produits basés sur des utilisations en médecine traditionnelle (CAPITURE) a été évaluée dans le cadre d'une enquête ethnobotanique sur le traitement traditionnel des maladies fongiques dans la Préfecture de Tchamba (Togo). Cette méthode a prédit et identifié les plantes les plus biologiquement actives parmi les 43 espèces recensées au cours de l’enquête : Pterocarpus erinaceus prédit être plus actif contre les champignons, et Daniellia oliveri contre les bactéries. Les plantes ont ensuite été testées contre les champignons, les bactéries et les cellules cancéreuses. Comme prédit par CAPITURE, P. erinaceus était plus actif contre les champignons et D. oliveri contre les bactéries. Fait intéressant, les deux plantes ont présenté une activité sur les cellules cancéreuses sans être toxique pour les cellules humaines normales. Dans une troisième étape, en utilisant la chimie analytique, les composés responsables des activités biologiques ont été identifiés. La plupart de ces composés n'ont jamais été signalés dans les espèces végétales ou dans la nature, avec une activité biologique dans la gamme micro-molaire. Enfin, en réduisant la poudre des organes végétaux à la taille de nano-particules, une meilleure activité biologique a été observée par rapport à celle de l'extrait organique. En conclusion, cette recherche a mené à la découverte de nouvelles molécules avec une activité biologique intéressante, molécules qui nécessiteront une étude approfondie et détaillée / The investigation of plants used for traditional medicine in Togo is complicated as modern techniques are not available. Computer-aided product identification from traditional usage records (CAPITURE) was evaluated in the context of an ethnobotanical survey on the traditional treatment of fungal diseases in Tchamba District (Togo). This method predicted and identified the most biologically active plants out of the 43 species survey-recorded: Pterocarpus erinaceus predicted to be more active against fungi and Daniellia oliveri against bacteria. The plants were then tested against fungi, bacteria and cancer cells. As predicted with CAPITURE, P. erinaceus was more active against fungi and D. oliveri against bacteria. Interestingly, both plants presented activity on cancer cells without being toxic to normal human cells. In a third step, using analytical chemistry, the compounds responsible for the biological activities were identified. Most of those compounds have never been reported in the plant species or in nature at all, with biological activity in the micromolar range. Finally, pharmaceutical technology was used: by nanosizing the powder of the plant organs, a better biological activity was observed in comparison to that of the organic extract. In conclusion, this research led to the discovery of new molecules with an interesting biological activity that will need further and more detailed investigation

Dans ce travail, nous avons étudié le mécanisme d'émission de la partie optiquement active du complexe luciférine-luciférase. Ce système bioluminescent est largement utilisé dans un très grand nombre d'approches bioanalytiques. Ce phénomène naturel résulte en l'émission de lumière visible (vert-jaune-rouge) à partir du photoproduit : l’oxyluciférine. Une des hypothèses couramment admise pour expliquer le mécanisme d’émission de l’oxyluciféreine fait intervenir un équilibre complexe entre six espèces chimiques, mais le détail exact du mécanisme reste à élucider. Les principales conclusions présentées ici repose sur l'identification des six formes chimiques de l’oxyluciférine impliquées dans le mécanisme d'émission de fluorescence et la caractérisation d’un point de vu dynamique du transfert de proton à l’état excité. Ces résultats ont été obtenus par l'étude des propriétés optiques de différents analogues structuraux de l’oxyluciférine dans un tampon aqueux. Différent techniques de spectroscopie (état stable et résolue en temps) et des approches chimiométriques ont été appliquées pour étudier ce mécanisme d'émission. En outre, les propriétés photophysiques de l’oxyluciférine en complexe avec l'enzyme luciférase (Luciola cruciata) ont été étudiées également en milieu aqueux. En parallèle, les dérivés présentant des propriétés d’émission sensibles à l’environnement ont été utilisés pour visualiser l'interaction entre biomolécules. En particulier, nous avons démontré que l’oxyluciférine peut être utilisée pour cartographier le pH intracellulaire à l'aide de la microscopie de fluorescence dans des cellules vivantes. Avec l'aide d'un autre dérivé de l'oxyluciférine nous avons été en mesure de caractériser l'interaction entre une protéine du VIH-1 et des séquences d'oligonucléotide au moyen de mesures ratiométriques. Enfin, nous avons développé une approche basée sur le marquage de résidus cystéine pour suivre, in vitro, l'interaction protéine-protéine. / In this work, we investigated the emission mechanism of the optically active part of the firefly luciferin-luciferase complex. This bioluminescent system is widely used in bioanalytical assay. This amazing natural phenomenon results in the emission of visible light (yellow-green-red) from the photoproduct Oxyluciferin. This color tuning mechanism involves six chemical species, but their active involvement in the excited state proton transfer (ESPT) mechanism was poorly understood so far. One of the main finding presented here relies on the identification of six chemical forms of Oxyluciferin involved in the color tuning fluorescence emission mechanism. This result was obtained by studying the optical properties of different structural analogues of firefly Oxyluciferin in aqueous buffer. Different spectroscopic (steady state and time-resolved) and chemometric approaches have been applied to reveal the emission mechanism. In addition, the photophysical properties of Oxyluciferin in complex with the Luciferase enzyme Luciola cruciata have been studied in aqueous buffer as well. In parallel, derivatives displaying environment sensitive emission were used to monitor biomolecular interactions. In particular, we demonstrated that Oxyluciferin can be employed to map intracellular pH by using fluorescence microscopy within living cells. With the help of another Oxyluciferin derivative we were able to monitor the interaction between a HIV-1 protein and different oligonucleotide sequences by means of ratiometric measurements. Finally we develop an approach based on cysteine labeling to monitor in vitro protein-protein interaction.

Summary: Many primary human tumors and tumor cell lines highly express human L-type amino acid transporter 1 (hLAT1); cancerous cells in vivo are strongly linked to LAT1 expression. Synthetic chemistry and in vitro screening efforts have afforded a variety of novel and highly hLAT1 selective compounds, such as JPH203 1. In a recent report, we demonstrated that 1 has potent in vitro and in vivo activity. JPH203 was intravenously administered to produce significant growth inhibition against HT-29 tumors transplanted in nude mice. The current work develops a robust LC/MS-MS method to monitor 1 and its major Phase II metabolite N-acetyl-JPH203 2 from biological samples. We have conducted in vitro and in vivo experiments and the major scientific findings are: i) the major route of biotransformation of 1 is Phase II metabolism to produce 2; ii) metabolite 2 is formed in various organs/tissues (i.e. blood, liver, kidney); and iii) as dogs, which are deficient in NAT genes, do not produce 2, the dog will not be an appropriate toxicological model to evaluate 1.