MEASURING FASTER PHYSIOLOGICAL EVENTS WITH ELECTROCHEMICAL PROBES

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。细胞通过获取和排出无机离子、气体和有机化合物（从代谢废物到化学信使）来保持与环境的动态相互作用。化合物的释放导致表面高浓度，当化合物扩散离开细胞时产生梯度;摄取导致相反的梯度。这些梯度使用增强信噪比的电化学探针的调制来测量，但迄今为止仅限于相对稳定状态的应用。 在BRC进行的研究审查了电化学传感器的预期响应时间，并指出几种电位计设计可以在不到20毫秒的时间内实现90%的响应。这个速度使我们在测量渠道活动的范围内。我们已经使用K+选择性微电极监测外部[K +]流出后，通过人工通道在平面脂双层和流出后，通过Ca2+激活的K+通道表达在非洲爪蟾卵母细胞和中国卵巢细胞的变化。建模和数据分析方案的组合已被用来确认单通道检测和识别系统的优点和缺点。 将这些非侵入性传感器和分析方法与其他地方描述的扫描技术相结合，将提供对细胞组织的独特见解，揭示细胞周围化学梯度对细胞过程的空间和时间调节的更精细细节。 离子选择性电极（ISE）的自参考已被用于非侵入性地测量细胞和组织附近相对稳定的离子梯度。然而，这些相对稳定的梯度是许多离散事件的平均值，包括通过通道或转运蛋白的转运。用于测量稳定梯度的数据收集和平均方案模糊了各个事件，导致丢失关于离子梯度性质的有用信息。通过使用具有信号分析方法的快速响应电极，我们希望表征正常和致病条件下的离子通道和转运蛋白，以便用非侵入性方法研究疾病状态。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cells maintain a dynamic interaction with their environment by acquiring and expelling inorganic ions, gases and organic compounds ranging from metabolic wastes to chemical messengers. Release of a compound results in a surface high concentration that produces a gradient as the compound diffuses away from the cell; uptake results in an inverse gradient. These gradients are measured using modulation of electrochemical probes that enhance the signal to noise ratio but to date have been restricted to relatively steady state applications. Studies conducted at the BRC reviewed the expected response time for electrochemical sensors and noted that several of the potentiometric design can achieve 90% response in less than 20msec. This speed brings us to within the scope of measuring channel activities. We have used K+-selective microelectrodes to monitor changes in external [K+] after efflux through artificial channels in a planar lipid bilayer and after efflux through Ca2+-activated K+ channels expressed in Xenopus oocytes and Chinese Hamster Ovary cells. A combination of modeling and data analysis schemes has been used to confirm single channel detection and identify the strengths and weaknesses of the system. Combining these non-invasive sensors and analysis approaches with a scanning technique described elsewhere will provide a unique insight into cellular organization, revealing finer details of spatial and temporal regulation of cellular processes from chemical gradients surrounding cells. Self-referencing with ion-selective electrodes (ISEs) has been used noninvasively, to measure relatively steady ionic gradients near cells and tissues. However, these relatively steady gradients are the average of many discrete events including transport through channels or transporters. The data collection and averaging scheme used for measuring the steady gradients blurs the individual events, leading to the loss of useful information regarding the nature of the ionic gradient. By using fast responding electrodes with signal analysis methods we hope to characterize ion channels and transporters under normal and pathogenic conditions in order to study the diseased state with a non-invasive approach.