Fabrication of microfluidic devices for studying living cells responding to external stimuli by FTIR vibrational spectroscopy

The present PhD Thesis is about the development of new fabricative strategies to obtain microfluidic devices suitable for InfraRed MicroSpectroscopy (IRMS) studies on living cells in physiological environment and the demonstration of the screening and diagnostic capabilities of this technique for bio-medical applications. IRMS detects the vibrational pattern of molecules allowing the label-free characterization of the chemical profile of a biological specimen and its correlation with the sample morphology. Although powerful and versatile, this technique has been limited until recent years to the study of fixed or dried samples, in order to bypass the problem of water absorptions in the infrared spectral region. The use of microfabrication techniques for the production of Visible-Infrared (Vis-IR) transparent devices has recently opened an innovative approach, able to release some of the constrains encountered when dealing with living cells. Moreover, microfabrication is the best option to achieve long-range reproducibility of the optical path, which is mandatory for an accurate water subtraction in order to disclose cellular IR features. At first, we aimed to develop an IR-Vis transparent microfluidic chip with long-time stability in experimental conditions. The optical transparency was granted by the use of CaF2 or BaF2 as substrates, but their low surface energies imposed a challenge in order to establish reliable microfabrication protocols. With the introduction of a new strategy, that we refer to as †œsilicon-like†�, based on the sputtering of a thin silicon layer onto the IR materials, it was possible to modify the surface properties of the substrates without changing their optical properties. These new substrates allowed the use of several common photo-resists as structural materials. The epoxy-based negative tone SU-8 was chosen for its chemical properties (resistance to solvents and watery media) and its long-term stability in experimental conditions. We established a new sealing protocol exploiting the optical properties of SU-8, able to create a chemical bonding between two already patterned layers of the polymer. It was thus possible to produce a new generation of fluidic chips, characterized by broadband transparency from mid-IR to UV and long-term stability in continuous flow conditions. Subsequently, the devices were employed to perform IRMS measurements on both adherent and circulating cells. In particular, we characterize the spectroscopic features associated to each stage of B16 cell cycle, the changes undergone in living MCF-7 upon exposure to hypo-osmotic and thermal stress and the apoptosis progression of U-937 cells, induced by growth factors removal and CCCP (Carbonyl Cyanide m-Chloro Phenylhydrazone) stimulation. All the studies had the intent to further verify the effectiveness of the microfluidic approach for both circulating and adherent living cells analysis and to prove the capabilities of IRMS as tool for the observation of biochemical processes undergone by live beings. For this reason, to validate the achieved results, a parallel analysis with a well established analytical technique such as the flow-cytometry was performed. The present Thesis demonstrates the capabilities of IRMS coupled with microfluidic technologies, as a diagnostic tool for bio-medical investigation of bio-medical applications. Thanks to the precise control of the cellular microenvironment, as well as its flexibility in terms of experimental design, IRMS could be seen as a new promising frontier for modern biology.