Nanoscale probes for electrochemical measurements on single cells and organelles.

摘要

New technologies have enabled the exploration of biological systems in ever increasing detail to better understand the fundamentals of cellular metabolism and function. Among these technologies, large contributions have been made especially in the area of silicon fabrication technology. It has emerged as a powerful tool that makes it possible to study revealing micro and nanometer-scale details of single cells and even sub-cellular organelles. Empowered by silicon fabrication technology, researchers have reported different types of methodologies as well as applications for analyzing biochemical activities and characteristics of cells. Current applications and methodologies allow for the examination of single cells both in vivo and in vitro.; To investigate the electrophysiological phenomena of single cells and organelles, ultra-sharp planar and bent probes with single or dual ultra-microelectrodes (UME) have been designed and fabricated, using a combination of micro-fabrication and focused ion beam (FIB) technology. Specifically, we have fabricated platinum, gold or silver microelectrodes (0.1 mum2 in surface area) embedded in silicon nitride layers that can be used to perform electrochemical experiments to detect REDOX (reduction and oxidation) reactions occurring inside single cells and organelles, at the nanometer scale. To validate the probes as an electrochemical sensing tool inside single cells and organelles, cyclic voltammetry has been conducted in 10 mM Ru(NH3)6Cl 3 and 0.1 M KCl solutions. The voltammogram provides information on electrochemical properties of the UME probes.; For characterizing electrochemical and electrophysiological properties of single cells and organelles, a unique platform has been developed to manipulate and visualize a probe that either touches or penetrates the cell (or organelle) surface while holding the cell and monitoring currents associated with transport or electron transfer processes; these currents were generally less than 1 pA. The functionalities required for cell capture, manipulation and visualization had to be performed at the nanometer scale and were achieved using atomic force microscopy (AFM), confocal laser scanning inverted microscopy, and open micro-fluidic channels.; After building the platform, the probes were used in biological experiments to characterize electrical currents generated by single cells and organelles. First, electrochemical properties of single mammalian cells, including impedance and open circuit voltage across cell membranes, were measured. Second, photosynthetic activities of single plant cells and chloroplasts were monitored by detecting oxygen evolution and electron transfer reactions with and without the addition of a mediator. Through this research, the electron transfer reactions occurring in photosynthetic algae, Chlamydomonas reinhardtii and chloroplasts have been elucidated experimentally at the single cell level.