ABSTRACT
During the past decade, details regarding the role of intracellular ions in signaling pathways have followed technological breakthroughs in biochemistry, genetics, and developmental biology. Yet the fundamental mechanisms by which the total intracellular ionic state influences the cell response to its chemical and mechanical environment are still undetermined. This deficiency is due in part to limitations of analytical methods for assessing intracellular ion flux in living cells. Many measurement methods rely on the addition of chemical reagents such as ion binding dyes that may alter cell metabolism and provide only limited spatial resolution. Other deficiencies in sensing technologies can be attributed to the difference in size between the molecular processes taking place and the sensor. Recent advances in nanofabrication allow for the creation of arrays of sensors, each with a tip radius of
tens of nanometers and a spacing of a few microns between tips. The tip arrays will form the core of massively parallel electrodes directly connected to field effect transistors and operational amplifiers. These probes introduce a new methodology for the direct assessment of intracellular metabolism in living cells. Following controlled insertion into the interior environment of living cells, the probes are designed to measure membrane permeability through impedance spectroscopy and changes in the intracellular pH in the cytosol and organelles. The distribution of the nanotips within the arrays will provide a spatial resolution of no more than 2 µm. Measurements will be made using 20 to 60 active probes almost simultaneously on a single cell crosssection. Sequential probing of cells at different depth levels will establish three-dimensional maps of internal cellular environments. Microelectromechanical systems (MEMS) and complementary metal oxide semiconductor (CMOS) fabrication can be used to advance current impedance spectroscopy technology towards a concept better referred to as impedance microscopy. This probe technology will provide a framework for investigating mechanisms underlying ionbased regulatory processes that modulate phenotypic expression. The data can be correlated to the cellular reaction to externally applied physical stimuli and soluble factors in the chemical environment.
