ELECTRICAL, CHEMICAL, AND MECHANICAL MANIPULATION OF THE ENDOTHELIAL CELL SURFACE WITH A MULTIFUNCTIONAL NANOPIPETTE
Open Access
- Author:
- Bae, Chilman
- Graduate Program:
- Bioengineering
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 03, 2009
- Committee Members:
- Peter J Butler, Dissertation Advisor/Co-Advisor
Peter J Butler, Committee Chair/Co-Chair
Ahmed A Heikal, Committee Member
Ryan Clement, Committee Member
Venkatraman Gopalan, Committee Member - Keywords:
- electroporation
mechanotransduction
micropipette
Cell membrane
endothelial cell - Abstract:
- An integrated and automated micropipette electrode (ME)-based single-cell electroporation (SCE) system was developed for rapid, efficient, cell selective and non-invasive delivery of fluorescent compounds into adherent endothelial cells. Key features include software-controlled pulse waveform generation, continuous monitoring of electroporation events, feedback-controlled motorized micromanipulation, image capture, and data acquisition. The main innovations of automation are image-based cell selection, 40nm-precision feedback control of ME approach, and a method to prescribe the applied membrane potential. Comparisons of experiment measurements and finite element analysis of cleft resistance enabled identification of three distinct phases of pipette positioning: positioning phase, approach phase, and indenting phase. Computational simulations were also performed to determine the quantitative relationships of cleft size and resistance, transmembrane potential (TP) and cleft size, and TP and electrode axial distance. The simulation also shows that pores are focused on the membrane area corresponding to the inner pipette lumen. In addition, we quantitatively determined membrane tension, stress, and strain distributions in the vicinity of a nanoelectrode using finite element analysis of a multiscale electro-mechanical model which consisted of pipette, media, membrane, actin cortex, and cytoplasm. Results suggest that nanopipette electrodes (nE) provide a new non-contact method to deliver physiological stresses directly to membranes in a focused and controlled manner, thus providing the quantitative foundation for micreoelectrotension. Finally, we developed a new simple method for dynamic deformation spectroscopy with a functionalized nE. The electrical and mechanical characteristics of the system and the system resolution were tested. The results suggest that the adhesion of a nanopipette electrode tip onto the cell surface is dependent on the nanopipette electrode tip size, the fibronectin concentration for coating the nanopipette electrode tip, and the reaction time. This system has high versatility because of its simplicity, easy of interpretation, and non-contact analysis of surface properties.