MEASURING FASTER PHYSIOLOGICAL EVENTS WITH ELECTROCHEMICAL PROBES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 细胞通过获取和排出从代谢废物到化学信使的无机离子、气体和有机化合物来维持与环境的动态相互作用。化合物的释放导致表面浓度较高，当化合物扩散离开细胞时会产生梯度；摄取会导致相反的梯度。这些梯度是使用提高信噪比的电化学探针的调制来测量的，但到目前为止仅限于相对稳定的应用。 在BRC进行的研究回顾了电化学传感器的预期响应时间，并注意到几种电位测量设计可以在不到20毫秒的时间内实现90%的响应。这一速度将我们带到了衡量渠道活动的范围内。我们使用K+选择性微电极来监测在非洲爪哇卵母细胞和中国仓鼠卵巢细胞中表达的外源[K+]通过人工通道外排后和通过钙激活的K+通道外排后的变化。已使用建模和数据分析方案的组合来确认单通道检测并确定该系统的优点和缺点。 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洞察，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。 离子选择电极(ISES)自参照法已被非侵入性地用于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，包括通过通道或运输器的运输。用于测量稳定梯度的数据收集和平均方案使单个事件变得模糊，导致丢失了有关离子梯度性质的有用信息。通过使用快速响应电极和信号分析方法，我们希望能够表征正常和致病状态下的离子通道和转运体，以便用非侵入性的方法研究疾病状态。

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.