The present thesis work deals with the development of a novel manufacturing protocol for the realization of excimer laser micro-patterned freestanding hydrogel layers (50 to 300 ??m thickness) based on thermo-responsive poly-(N-isopropyl)acrylamide (PNIPAAm) which can operate as temperature-triggered actuators for cells-on-chip applications. PNIPAAm based thin films were synthesized in house and manufactured by an injection/compression moulding based technique in order to obtain flat hydrogels attached to rigid polyvinyl chloride (PVC) substrates to facilitate laser focusing. Laser machining parameters were empirically optimized to fabricate arrays of through-holes with entrance diameter ranging from 30 ??m to 150 ??m and having different exit diameter (from 10 to 20 ??m) on the PNIPAAm employing a stencil aluminum mask. After laser processing, the microstructured layers were detached from the PVC using a chemical treatment and then left to swoll in pure water. The KrF excimer laser machined through-holes could be reversibly modulated in terms of size as a consequence of the polymer volumetric phase transition induced by a temperature change above the critical value of 32 ??C. Thermo-responsiveness characterization was carried out on the detached water swollen freestanding layers using a thermostat bath, by changing the temperature from 18 ??C to 39 ??C and each sample could undergo multiple cycles. As a result of the polymer water loss, the shrinkage of the layer caused the holes to shrink homogeneously, thus reducing their original size of about the 50% in the polymer collapsed state. To prove the functionality of these stimuli-responsive smart surfaces in the frame of cells-on-chip systems, they were integrated in a multilayer microfluidic device to operate as self-regulating cell sorting actuators for single cell assays applications. Using mechanical fastening as the packaging strategy, the hydrated hydrogel was sealed between two micro-milled poly-methyl methacrylate (PMMA) components, which provided the fluid accesses and ducts to the cell suspension to be flown over the thermoresponsive actuator (top layer) and the well to collect the sorted sample (bottom layer). The device is also equipped with a thin transparent heater to control the microfluidic chip temperature. When the system is assembled, the temperature-triggered actuation mechanism was exploited to trap a cellular sample in the shrunken exit hole on the top of the hydrogel layer by applying a negative pressure across the film via the bottom PMMA component when the system is kept at 37 ??C. Subsequently, the sorting of the trapped cell took place through the micro-capillary when the polymer natural relaxation at room temperature towards its initial state occurred; the operational principle of the device was proved using MG63 cells as a model cell line by monitoring the sorting through the size-modulating structures using optical microscopy.

Dans son ensemble, les techniques de pointe actuelles procurent l'information nécessaire à une analyse approfondie de la cellule, ce qui nécessite cependant l’utilisation d’instruments et de plateformes analytiques différentes. Les biopuces à cellule permettent l’analyse des cellules vivantes en temps réel et constituent donc un outil important pour de nombreuses applications dans la recherche biomédicale telles que la toxicologie et la pharmaceutique.En effet, le suivi en temps réel de la réponse non-seulement physique mais aussi chimique des cellules, obtenue suite à des stimuli externes spécifiques et en utilisant un système d'imagerie cellulaire, peut fournir une meilleure compréhension des mécanismes et des voies de signalisation impliquées dans la réaction toxicologique.Le développement de tels dispositifs multianalytiques pour l'analyse biologique repose essentiellement sur la capacité de produire des surfaces fonctionnelles de pointe permettant une interaction et organisation contrôlée des cellules et d'autres entités telles que par exemple des anticorps ou des nanoparticules. Par conséquent, un grand effort technologique repose sur le développement des techniques permettant la création de motifs fonctionnels sur une surface de nature souvent inerte. Dans cette thèse, nous proposons deux techniques de micro- et nanofabrication permettant la création de motifs de cellules et d’anticorps sur un revêtement non-adhésif composé de poly (oxyde d'éthylène) (« PEO-like ») déposé par plasma. La première approche consiste à immobiliser par physisorption un micro-réseau de molécules adhésives de la matrice extracellulaire (par exemple la fibronectine) en utilisant des techniques d’impression par microcontact et par non-contact. La deuxième approche permet la création de motifs adhésifs sur la surface constitués de nanoparticules d'or (Au NPs) en utilisant des techniques d’impression similaire. L'immobilisation des Au NPs sur le revêtement « PEO-like » ne nécessite pas de modifications chimiques et est réalisé par une technique d'autoassemblage simple et irréversible. Ces surfaces d'or nanostructurées ont été testées pour l’analyse du phénomène de reconnaissance biomoléculaire et en tant que plateforme de culture cellulaire. Finalement, cette plateforme a été intégrée à un microscope plasmonique qui a permis, de façon préliminaire, la surveillance et la visualisation de la motilité d’une cellule unique, cela en temps réel et sans marquage, ainsi que la détection spécifique et sensible de protéines tests / State of the art techniques give as a whole the required information needed for the complete cell analysis but require different instruments and different types of platforms. The concept of cells on-a-chip allowing real-time analysis of living cells is, therefore, an important tool for many biomedical research applications such as toxicology and drug discovery. Monitoring in real-time the physical but also chemical response of live cells to specific external stimuli using live-cell imaging can provide a better understanding of the mechanisms and pathways involved in the toxicological reaction. The development of such multianalytical devices for biological analysis relies essentially on the ability to design advanced functional surfaces enabling a controlled interaction and organisation of cells and other nanostructures (e.g antibodies and nanoparticles). Therefore, a large technological effort is related on the development of advanced patterning techniques. In this thesis, we propose two simple and direct micro- and nano-fabrication techniques enabling the creation of cellular and sensing patterns on a non-adhesive and cell repellent plasma-deposited poly (ethyleneoxide) (PEO-like) coating. The first approach consists in immobilising a microarray of ECM molecules (cell-adhesive proteins, e.g fibronectin) on the cell repellent PEO-like surface by physisorption using microspotting or microcontact printing techniques. The second approach enables the creation of Gold nanoparticles (Au NPs) adhesive patterns on the surface using similar spotting techniques. The immobilization of Au NPs on PEO-like coatings does not require any prior chemical modifications and is achieved by a straightforward and irreversible self-assembly technique. These gold nanostructured surfaces have been tested for protein bio-recognition analysis and as a cell culture platform. Ultimately, this platform was integrated to a novel plasmonic microscope which enabled, preliminarily, the label-free monitoring and visualisation of a single cell attachment and detachment in real time, as well as the specific and sensitive detection of test proteins in a cell-free environment