Development of microfluidic devices for biomedical applications of synchrotron radiation infrared microspectroscopy

ABSTRACT DEVELOPMENT OF MICROFLUIDIC DEVICES FOR BIOMEDICAL APPLICATIONS OF SYNCHROTRON RADIATION INFRARED MICROSPECTROSCOPY by Birarda Giovanni The detection and measurement of biological processes in a complex living system is a discipline at the edge of Physics, Biology, and Engineering, with major scientific challenges, new technological applications and a great potential impact on dissection of phenomena occurring at tissue, cell, and sub cellular level. The present PhD Thesis dealt with the development of methodologies and technologies to transform InfraRed MicroSpectroscopy (IRMS) into a mature technique to observe in real time biological events, and improving its ability to perform in vitro bio-experiments under physiological conditions. This goal has been achieved through the exploitation of microfabrication techniques to realize lab-on-chip (LOCs) transparent both in the Infrared and Visible region (IR-Vis), which allows measuring living cells. Up to now, IRMS has been almost exclusively employed for studying fixed cellular samples or tissues, allowing acquiring only †œstill frames†� of the phenomena under investigation. The reason for that is to be ascribed both to the spectroscopic difficulties in working in water based environment and to the manufacturing constrains of the most common IR transparent materials, that limit the design flexibility of LOC devices suitable for IR analysis. We have overcome the so called †œwater absorption barrier†� by extending microfluidic concepts to calcium fluoride, implementing innovative fabrication solutions for the realization of custom devices for IRMS studies of living cells subjected to different chemical and physical stimuli. Exploiting the high brightness of Synchrotron Radiation (SR) IR sources, that allows sampling at diffraction limited spatial resolution, we demonstrated the feasibility of the detection of intra-cellular processes. In parallel, novel strategies for IR data acquisition and analysis have been developed, opening the possibility to execute novel original experiments. Our studies were focused on the immune system, and in particular in evaluating the biochemical rearrangements characterizing human circulating leukocytes during their deformation, either when induced by purely mechanical stimuli or in response to a chemical gradient. Thanks to the microfabrication approach, we were able to mimic the cellular microenvironment both for studying pressure-driven micro-capillary circulation and chemically-driven extravasations of white blood cells. The present Thesis demonstrates that the †œsynergy of micro-approaches†�, or rather the combination of micro-fabrication and IR micro-spectroscopy, can be exploited for extending the frontiers of Fourier Transform Infrared Spectroscopy (FTIR) to unexplored fields of life sciences. Through the careful control of the cellular microenvironment, crucial for an accurate data analysis as well as fundamental for the reliability of biological conclusions, some light could be shed on phenomena never investigated with IRMS, such as mechano-biology we directly explored, pulling down the water-barrier.