The present project proposes an innovative, multidisciplinary approach to the investigation of cell communication processes at the molecular level. In this project, novel, innovative and interdisciplinary research is emphasized with a focus on the application of microfabricated integrated scanning nanoprobe and nanobiosensing systems. Scanning probe microscopy (SPM) techniques provide powerful means for obtaining chemical, topographical and optical information with high spatial resolution. Each technique - atomic force microscopy (AFM), scanning nearfield optical microscopy (SNOM) and scanning electrochemical microscopy (SECM) - is designed to provide specific information based on interaction with the sample surface in the nearfield regime. SECM is especially interesting for biochemical/biological investigation, since this technique provides information on electrochemical, chemical and biochemical (re)activity at sample surfaces. The proposed multifunctional tool based on combination of scanning probe techniques will provide simultaneous information with a high selectivity for individual transmitters, high temporal resolution of changes in transmitter concentration, and high spatial resolution to distinguish which cells are releasing transmitter molecules, information required for in-situ investigations of complex biological systems and heterogeneous matrices. Many pathological events involve disruption of chemical communication between cells, most notably in the central nervous system, lungs, and kidneys. An important component of cell communication is the exocytotic release of transmitters from a variety of cells (e.g., the presynaptic terminal), their diffusion across between cells (e.g., a cross the synaptic cleft to the postsynaptic membrane) and the selective binding to suitable receptors, which lead to the initiation of cellular signalling events specific to the transmitter and the receptor. Little is known about the formation of secretory granules and the characteristics of transmitter release, which is frequently altered during physiological or pathophysiological conditions. Hence, detailed knowledge and multiparametric analytical assessment of exocytotic events will help understand the underlying mechanisms of cellular communication. Multifunctional microfabricated SECM-AFM, SECM-SNOM and SECM-AFM-SNOM with integrated nanobiosensors will provide simultaneous topographical, optical/fluorescence and electrochemical information at nanometer resolution for the first time. We will apply these combined multifunctional techniques in combination with tip-integrated nanobiosensors to a relatively simple model biological system to demonstrate the feasibility of the approach for measuring biologically relevant transmitter molecules. The model system will involve the exocytotic release of ATP from a monolayer of epithelial cells in culture. This system is a good model for a variety of epithelial tissues in mammals and the question of the amount and regulation of ATP release is a contemporary issue following the hypothesis that abnormalities in ATP release could play a significant role in the pathophysiology of cystic fibrosis (CF). Quantitative theoretical models and simulations of multifunctional scanning probes in order to facilitate interpretation of electrochemical results and imaging data will complement the proposed tasks.
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