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Abstract
The ambient signals derived from different soluble or physical cues trigger complicated cellular responses that, often, determine the destiny of cells. The task of understanding the mechanisms behind these cellular actions is crucial in the way of answering cell biology enquiries and designing new therapies. The aim of this dissertation was to study the cellular responses to various environmental cues, comprehensively. The cellular responses in the presence of adhesive and soluble cues occur regularly at multiple parts of the cell and vary in time duration and the place. Therefore, monitoring several aspects of cellular responses is not possible to recapitulate using a single cell-based technique. In this regard, optical microscopy and impedance spectroscopy on cell chips with molecularly engineered surfaces were coupled, simultaneously. This required the presence of adhesive cues in a controllable manner on an inert background on the surfaces. The underlying surfaces, therefore, should provide these possibilities as well as the compatibility with the measurement techniques.
Gold electrodes were modified with zwitterionic antifouling coatings that limited nonspecific proteins adsorption and had low-impedance, considering the advantages of the gold surface for electrochemical measurements. However, the fluorescence quenching property of the gold and the prerequisite of transparency for transmitted light microscopy applications highlighted the need of utilizing indium tin oxide (ITO) as a more appropriate candidate for developing the dual optical/electrical cell-based technique.
A well-controlled chemistry was achieved on interdigitated ITO surfaces by forming defined density of RGD molecules on an inert background using a multi-steps strategy. The simultaneous setup, then, was designed and developed on interdigitated ITO surfaces. The cell attachment and adhesion in the present of different expression of adhesive ligands were investigated using the simultaneous combination of phase contrast microscopy and impedance spectroscopy. Results indicated that live cells attach and spread with different rate on the surfaces with various RGD spatial distributions. The RGD spacing of 31 nm provided the fastest rate for adhesion of the HeLa cells. These results also were used to coordinate the impedance results with the fractional surface coverage of cells on the surface.
The coupling of fluorescence microscopy and impedance spectroscopy was used to investigate whether the presence of adhesive ligands affects the cellular responses to chemical cues. G-protein coupled cell receptors (GPCR) were used as the pathway influenced by, a model soluble cue, histamine on interdigitated ITO surfaces with various RGD spacing. The alteration of impedance readout resulted from the changes in cell shape and adhesion to the substratum. Whereas, the fluorescence microscopy was used to track intracellular Ca2+ signalling. Results illustrated that the cells on the surfaces with average RGD spacing of 31 nm displayed a faster histamine-induced release of Ca2+ and change in cell morphology and adhesion than cells on the other examined surfaces with less or more adhesive ligand spacing.
As a case study, the developed technique was used to screen the effect of potential antifibrotic compounds. The results of testing PXS64 prodrug on primary human Dupuytren`s cells detected the useful time window and illustrated the capability of this methodology in examining the potential antifibrotic compounds.
The developed dual detection highlighted the importance of controlling the cellular adhesive environment on cell response to drugs. This dissertation demonstrated the power of the developed dual optical/ electrical methodology in achieving a more comprehensive sigh on cell signalling process.