Development of micropipette-based nanotools and their application in mechanobiology study

Abstract

Micropipette-based techniques have been extensively employed in biological research, encompassing vital applications such as in vitro fertilization (IVF) and micro-injection. Recent advancements in these techniques have focused specifically on exploring the mechanical properties of cells and subcellular biophysics. This thesis presents the significant findings and contributions derived from my intensive two-year MPhil study conducted under the guidance of A/Prof Lining Arnold Ju at the Mechanobiology Lab.

During the research period, a comprehensive and versatile micropipette-based biophysical platform was meticulously developed and its applications in biomedical research were demonstrated. At the single-cell level, a fluorescent micropipette aspiration assay (fMPA) was established to meticulously investigate the mechanosensing capabilities of live cells. Leveraging with a novel fabrication protocol, micropipettes with diverse geometries were utilised to mimick the intricate microenvironment within the human body and enabling comprehensive analysis of cellular adaptive behavior in complex microenvironments. Delving into the molecular level, a cutting-edge micropipette-based technology—biomembrane force probe (BFP), was devised to facilitate the study of in situ single molecule binding kinetics.

The outcomes of this study are twofold. Firstly, a robust platform for studying the mechanobiology of single live cells was successfully established, providing invaluable insights into the mechanisms by which cells perceive external mechanical stimulation through transmembrane proteins. Moreover, a comprehensive understanding of the cooperative interaction between mechanosensitive ion channels, such as PIEZO1, and the cytoskeleton in adapting to complex microenvironments was achieved. These findings not only illuminate the future directions of single cell mechanobiology studies but also introduce a new avenue for the engineering of cells within a mechanical context.