DEVELOPMENT OF SELF REFERENCING ELECTROCHEMICAL MICROSENSORS NITRIC OXIDE

在构思了使用氧化还原势能签名来 通过自参照探针检测特定的化学物质 从氧气检测转移到 一氧化二氮的含量。我们之所以选择这个开发项目，是因为 认识到这种化合物在许多细胞和组织中的重要性 答复以及没有任何直接的测量方法 单细胞水平。自我指代的顺应性 测量一氧化氮的微电极系统已被要求 NO选择性微电极的研制。通过将和 我们现在正在修改文献中描述的现有协议 构建一种具有所需的电化学微探针 选择性。这个探头是由5米长的碳纤维制成的，斜面 控制最终尖端大小并提供更有效的几何图形。 这项技术基于这些元素的平移运动 在已知移动频率下的梯度中的微电极 在已知点之间。考虑了线性校准的影响 电极，则将差分电极输出转换为 用Fick方程定向测量通量。这个 此探头在自参考模式下的操作已在 使用人工一氧化氮供体创建的人工梯度 固定在琼脂基质中的S-亚硝基-N-乙酰青霉胺 并包含在一个微型移液管中。获取的一氧化氮通量值 自参比一氧化氮微电极的匹配衍生 通过静态测量获得的一氧化氮计算值 在人工污染源之外。用人工来源做的工作 不是告诉我们我们是否会达到所需的敏感性 测量单个细胞的释放量。为了测试这一点，我们选择了一个 与没有生理学经验的团队合作， 获得哺乳动物巨噬细胞，这是一种已知的排泄细胞系 大量的NO。正如亮点所报道的那样，我们非常 第一次成功地实现了无生产本地化 在单细胞水平上的直接测量。

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.