DEVELOPMENT OF SELF REFERENCING ELECTROCHEMICAL MICROSENSORS NITRIC OXIDE

构思了使用氧化还原电位特征进行 通过自参照探针检测特定化学物质， 从氧气检测到 一氧化氮 我们选择这个项目是因为 公认的重要性，这种化合物在许多细胞和组织 答复和缺乏任何直接的测量方法， 单细胞水平。 自我参照的适应性 微电极系统的测量一氧化氮已要求 NO选择性微电极的研制。 通过组合和 修改现有的协议中描述的文献中，我们现在 构建一个电化学微探针， 选择性 这个探测器是由一个5？米碳纤维，斜面， 控制最终尖端尺寸并提供更有效的几何形状。 这种技术是基于这些的平移运动 微电极以已知的运动频率呈梯度， 已知点之间。 考虑到线性校准， 电极，差分电极输出被转换为 使用Fick方程的通量的定向测量。 的 该探头在自参考模式下的操作已在 使用人工一氧化氮供体产生的人工梯度 S-亚硝基-N-乙酰青霉胺（SNAP），固定在琼脂基质中 并包含在微量移液管中。 获得的一氧化氮通量值 从自参比一氧化氮微电极匹配导出 从一氧化氮的静态测量值计算得到 在人造源之外。 使用人造源的工作 不告诉我们是否能达到所需的灵敏度， 测量单个细胞的NO释放。 为了测试这一点，我们选择了一个 与一个在NO生理学方面经验丰富的小组合作， 进入哺乳动物巨噬细胞，一种已知能分泌 大量的否。正如亮点中所报道的那样，我们非常 成功的，能够，第一次，本地化NO生产， 在单细胞水平上直接测量。

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.