Having conceived the idea of using a redox potential signature for the detection of specific chemicals by self-referencing probes it has been a comparatively simple task to move from oxygen detection to that of nitric oxide. We chose this development because of the acknowledged importance of this compound in many cellular and tissue responses and the absence of any direct method of measurement at the single cell level. The adaptation of the self-referencing microelectrode system for the measurement of nitric oxide has required the development of an NO selective microelectrode. By combining and modifying existing protocols described in the literature we are now building an electrochemical microprobe that has the required selectivity. This probe is built from a 5?m carbon fiber, beveled to control the final tip size and to provide a more effective geometry. This technique is based on the translational movement of these microelectrodes in a gradient at a known frequency of movement and between known points. Accounting for the linear calibration of the electrode, the differential electrode output is converted into a directional measurement of flux using the Fick equation. The operation of this probe in self-referencing mode has been validated in artificial gradients created using the artificial nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP), immobilized in an agar matrix and contained in a micropipette. Nitric oxide flux values obtained from the self-referencing nitric oxide microelectrode matched derived values calculated from static measurements of nitric oxide obtained outside of the artificial source. Work with an artificial source did not tell us whether we would achieve the sensitivity required to measure NO release from a single cell. To test this we chose a collaboration with a group experienced in NO physiology and with access to mammalian macrophage cells, a cell line known to excrete large amounts of NO. As reported in the highlights we were very successful, able, for the first time, to localize NO production by direct measurement at the single cell level.
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