The Goldman Chip: A bioMEMS Platform for Integrating Patch Clamp with Ion-Selecti

Goldman 芯片：将膜片钳与离子选择集成的生物 MEMS 平台

• 批准号: 7945084
• 负责人: David Marshall Porterfield
• 金额: $ 18.66万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7945084
• 关键词: Address; Agonist; Area; Biological Assay; Biological Sciences; Biosensor; Blindness; Cardiovascular system; Cell Culture Techniques; Cell membrane; Cell physiology; Cells; Cellular Structures; Chemistry; Computer software; Core Facility; Coupling; Custom; Cytometry; Data; Disease; Dopamine; Drug Delivery Systems; Electrodes; Electrophysiology (science); Epilepsy; Equipment; Event; Functional disorder; Glass; Goals; Housing; In Vitro; Industry; Integral Membrane Protein; Ion Channel; Ion-Selective Electrodes; Ions; Kinetics; Lead; Long QT Syndrome; Mammalian Cell; Measurement; Measures; Medical; Membrane Potentials; Methodology; Methods; Microbial Biofilms; Microelectrodes; Microfabrication; Microfluidics; Mission; Monitor; Multiple Sclerosis; NBL1 gene; Nanotechnology; Nature; Noise; Optics; Pattern; Pharmaceutical Preparations; Physiological; Physiology; Play; Process; Qualifying; Radiolabeled; Research; Research Personnel; Resolution; Role; Scientist; Screening procedure; Signal Transduction; Silicon; Source; Specificity; Spectrum Analysis; Staging; Suction; System; Techniques; Technology; Testing; Therapeutic; Time; Tissues; Training; Universities; Work; ascorbate; base; biochip; channel blockers; computerized data processing; cytotoxicity; deafness; design; drug discovery; experience; extracellular; high throughput screening; holistic approach; innovation; metabolomics; miniaturize; nervous system disorder; new technology; novel; patch clamp; public health relevance; radiotracer; research study; response; sensor; solid state

DESCRIPTION (provided by applicant): Ion channels are a large diverse group of integral membrane proteins, which play a central and key role in signaling, transport, and maintaining electrophysiological potential in all cells. Abnormalities in ion channels cause many diseases such as: cardiovascular dysfunction (Long QT syndrome), neurological disorders (multiple sclerosis, epilepsy), deafness and blindness. Biomedical researchers and pharmacologists are increasingly looking at ion channels as the source of disease and as targets for therapies based on novel drugs which can address previously misunderstood medical challenges. Because of this major paradigm shift, it is now estimated that approximately 15% of all new drugs being screened are generally classified as "ion channel modifiers." To date new technologies for high throughput screening (HTS) at an early stage of the drug discovery process for compounds with activities as ion channel modifiers have been marginally successful. Because of the large number of compounds available for screening, the fast kinetics of ion channels and the need to generate > 10,000 data points per day, rapid and autonomous methods to dynamically measure ionic current or fluxes from these ion channels are needed. Additionally, in mammalian cells Na+, K+, Ca2+ and Cl- channels operate synchronously to maintain a precarious status, thus requiring the monitoring of multiple ionic activities to further our understanding. Conventional electrophysiology technology, based on the patch clamp approach, provides the most direct and detailed method of studying ion channels. However the approach does suffer from some basic limitations, most notably low throughput (< 100 data points/day) and inability to identify ionic composition of electrophysiological events. In spite of recent progress towards automated patch clamp and patch-on-a-chip, the invasive nature of this technique, the need of highly trained and skilled scientists and its inability to resolve and distinguish between different ionic activities simultaneously, has limited the utility of this technique for HTS. Other ion channel HTS techniques, fluorescent/potentiometric probes, radiolabelled ion flux assays, and automated spectrometric assays have drawbacks related to cytotoxicity and limited information content. Currently there is no technology available which can provide non invasive, dynamic, and autonomous recordings of specific ion channel activities as an in vitro ion channel characterization assay in a miniaturized HTS format. The long term goal of this work is to develop an easy-to-use technology for HTS cell electrophysiology, and screening of ion channel modulators as potential drug targets in an in vitro format. The overall objective of this proposal is to enable simultaneous, quantitative and temporally resolved, real time measurement of extracellular Na+, K+, Cl- and Ca2+ from single cells integrated with whole cell patch clamp. This will enable complete dynamic analysis of ion channel electrophysiology in response to various ion channel therapeutic candidates. We will do this by developing the "Goldman chip"; a microfabricated platform which will enable non- invasive sensing of key extracellular ionic activities in a high resolution, high throughput system by maximizing the information gained from a single experiment. Based on solid state ion- selective sensor technology and planar patch, the Goldman chip will provide simultaneous extracellular Na+, K+, Cl- and Ca2+ measurement capabilities with whole cell electrophysiological recordings. Whole cell current measurement and control will be possible based on integration of planar patch clamp technology, signal processing hardware/software and automated control for high throughput experimentation. By integrating a standard patch clamp electrophysiological interface with Na+, K+, Cl- and Ca2+ ISEs on a single platform, it will be possible to directly identify the ionic component of a whole cell current recording. This technology is highly innovative because the focus is not entirely on increasing raw electrophysiological experimental throughput, but instead offers to qualitatively change the value and scope of the information obtained from a single experiment by incorporating ion-specificity into the approach. Because this is a more holistic approach to electrophysiology experimentation, the overall throughput is expected to increase as the requirement for multiple rounds of ion-replacement, and channel blocker controls will be eliminated. PUBLIC HEALTH RELEVANCE (provided by the applicant): Narrative Ion channels are a large and very diverse group of integral membrane proteins, which are the source of many diseases including: cardiovascular dysfunction (Long QT syndrome), neurological disorders (multiple sclerosis, epilepsy), deafness and blindness. Now basic biomedical researchers and pharmacologists are increasingly looking at ion channels and approximately 15% of all new drugs being screened are generally classified as "ion channel modifiers." New technologies are now needed for High Throughput Screening (HTS) for ion channel modifiers, and the proposed work seeks to advance this area by using a bioMEMS approach to integrate patch-clamp electrophysiology with ion-specific microelectrodes.

描述（由申请人提供）：离子通道是一大类多样化的整体膜蛋白，在所有细胞的信号传导、运输和维持电生理电位中起着核心和关键作用。离子通道异常可引起许多疾病，如：心血管功能障碍（长QT综合征）、神经系统疾病（多发性硬化症、癫痫）、耳聋和失明。生物医学研究人员和药理学家越来越多地将离子通道视为疾病的来源和基于新型药物的治疗靶点，这些药物可以解决以前被误解的医学挑战。由于这一重大的范式转变，现在估计所有正在筛选的新药中约有15%通常被归类为“离子通道调节剂”。到目前为止，在药物发现过程的早期阶段，用于高通量筛选（HTS）具有离子通道修饰活性的化合物的新技术已经取得了一定的成功。由于可用于筛选的化合物数量众多，离子通道的快速动力学以及每天需要生成100万个数据点，因此需要快速自主地动态测量这些离子通道的离子电流或通量的方法。此外，在哺乳动物细胞中，Na+, K+， Ca2+和Cl-通道同步运行以保持不稳定状态，因此需要监测多种离子活动以进一步了解。基于膜片钳方法的传统电生理技术提供了研究离子通道最直接、最详细的方法。然而，该方法确实存在一些基本的局限性，最明显的是低通量（< 100个数据点/天）和无法识别电生理事件的离子组成。尽管最近在自动膜片钳和片上贴片方面取得了进展，但这种技术的侵入性、对训练有素和技术熟练的科学家的需求以及它无法同时解决和区分不同的离子活性，限制了这种技术在HTS中的应用。其他离子通道高温超导技术、荧光/电位探针、放射性标记离子通量测定和自动光谱测定都存在与细胞毒性和有限的信息含量相关的缺点。目前还没有一种技术可以提供非侵入性的、动态的、自主的特定离子通道活性记录，作为一种小型HTS格式的体外离子通道表征分析。这项工作的长期目标是开发一种易于使用的HTS细胞电生理技术，并在体外筛选离子通道调节剂作为潜在的药物靶点。本提案的总体目标是实现与全细胞膜片钳结合的单细胞同时，定量和暂时解决的细胞外Na+， K+， Cl-和Ca2+的实时测量。这将使离子通道电生理响应于各种离子通道治疗候选物的完整动态分析成为可能。我们将通过开发“高盛芯片”来实现这一点；一个微加工平台，通过最大限度地从单个实验中获得信息，可以在高分辨率，高通量系统中实现关键细胞外离子活性的非侵入式传感。基于固态离子选择传感器技术和平面贴片，高盛芯片将提供同时的细胞外Na+， K+， Cl-和Ca2+测量能力，并具有全细胞电生理记录。基于平面膜片钳技术、信号处理硬件/软件和高通量实验自动化控制的集成，全细胞电流测量和控制将成为可能。通过在单一平台上集成标准膜片钳电生理接口与Na+， K+， Cl-和Ca2+ ISEs，将有可能直接识别整个细胞电流记录的离子成分。这项技术具有高度创新性，因为其重点不完全在于增加原始电生理实验的吞吐量，而是通过将离子特异性纳入该方法，从定性地改变从单个实验中获得的信息的价值和范围。由于这是一种更全面的电生理实验方法，因此随着对多轮离子置换的需求，预计总通量将增加，并且通道阻断剂控制将被消除。