Fort Worth, Texas (July 15, 2004)
There is an urgent need to determine the fundamental functions carried out by molecular machines inside biological cells at nanometer scale; however, the current technologies such as fluorescence are limited to 100nm resolution. Atomic force microscopy (AFM), even with its nanometer resolution, has not been used for monitoring of internal signals because of lack of nanoscale probe arrays with individual addressibiltiy. Electron Microscopy (EM), the other sub-nanometer resolution analytical technology, has also not been used for nanoscale probing of cells due to damage of cells by the electron beam and inability to maintain cells in vacuum.
An innovative approach called NanoBench was investigated during this project. NanoBench chip consists of an array of electrically conducting nanoelectrodes that can be inserted inside a cell to measure intra-cellular signalling pathways, both eletrically and optically.
During this project, (1) NanoBench chips with arrays of metallic tips were fabricated, (2) very sharp tungsten probes were fabricated, functionalized and used to demonstrate intra-cellular probing of cells, and (3) full range of experimental techniques have been developed to perform optical and electrical measurements if intracellular signals.
As a model system, a sharp tungsten probe was inserted into a lysosome that was swollen with sucrose (a sucrosome). Control over probe movement was sufficient to drive the probe into a sucrosome, and withdraw the probe. Upon withdrawl, the sucrosome collapsed. indicating that the vesicle was punctured by the probe tip. In a second model system, a lysosome labeled with the acidotropic dye LysoTracker Red was touched with a metal tip, which popped the lysosome and released the dye. Future work will concentrate on making sharper probes and coating them with biological molecules to sense analytes in cells.
