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: 8136630
• 负责人: David Marshall Porterfield
• 金额: $ 18.45万
• 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/8136630
• 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; 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）：离子通道是一组多样的膜蛋白，在所有细胞的信号传导、转运和维持电生理电位中发挥核心和关键作用。缺乏离子通道会导致许多疾病，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，并作为基于新药的治疗靶点，这些新药可以解决以前误解的医学挑战。由于这一重大的范式转变，据估计，目前正在筛选的所有新药中约有15%通常被归类为“离子通道调节剂”。“迄今为止，在药物发现过程的早期阶段，用于高通量筛选（HTS）的新技术对于具有离子通道修饰剂活性的化合物已经取得了一定的成功。由于可用于筛选的化合物的大量、离子通道的快速动力学以及每天产生> 10，000个数据点的需要，需要动态测量来自这些离子通道的离子电流或通量的快速且自主的方法。此外，在哺乳动物细胞中，Na+、K+、Ca 2+和Cl-通道同步运行以维持不稳定的状态，因此需要监测多种离子活动以进一步理解。基于膜片钳方法的传统电生理技术提供了研究离子通道的最直接和详细的方法。然而，该方法确实受到一些基本限制，最显著的是低通量（< 100个数据点/天）和不能识别电生理事件的离子组成。尽管最近在自动化膜片钳和片上膜片钳方面取得了进展，但该技术的侵入性、对训练有素和技术熟练的科学家的需求以及其无法同时分辨和区分不同离子活性的能力，限制了该技术在HTS中的应用。其他离子通道HTS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具有与细胞毒性和有限的信息内容相关的缺点。目前，没有可用的技术可以提供特定离子通道活性的非侵入性、动态和自主记录作为微型HTS形式的体外离子通道表征测定。这项工作的长期目标是开发一种易于使用的HTS细胞电生理学技术，并在体外形式中筛选作为潜在药物靶点的离子通道调节剂。该提议的总体目标是能够与全细胞膜片钳集成，同时，定量和时间分辨，真实的时间测量来自单个细胞的细胞外Na+，K+，Cl-和Ca 2+。这将使得能够响应于各种离子通道治疗候选物对离子通道电生理学进行完整的动态分析。我们将通过开发“戈德曼芯片”来实现这一点;这是一种微加工平台，通过最大化从单个实验中获得的信息，可以在高分辨率、高通量系统中非侵入性地感测关键的细胞外离子活动。基于固态离子选择性传感器技术和平面贴片，Goldman芯片将提供同时细胞外Na+、K+、Cl-和Ca 2+测量能力以及全细胞电生理记录。全细胞电流的测量和控制将是可能的基础上集成的平面膜片钳技术，信号处理硬件/软件和自动控制的高通量实验。通过将标准膜片钳电生理接口与Na+、K+、Cl-和Ca 2 + ISE集成在单个平台上，将有可能直接识别全细胞电流记录的离子成分。这项技术是高度创新的，因为重点不完全是增加原始电生理实验吞吐量，而是通过将离子特异性结合到方法中来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学实验方法，因此预计总通量将增加，因为将消除多轮离子置换和通道阻断剂控制的要求。 公共卫生相关性（由申请人提供）：叙述离子通道是一组庞大且非常多样化的膜蛋白，是许多疾病的来源，包括：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。现在，基础生物医学研究人员和药理学家越来越多地关注离子通道，所有正在筛选的新药中约有15%通常被归类为“离子通道修饰剂”。“现在需要新技术用于离子通道修饰剂的高通量筛选（HTS），拟议的工作旨在通过使用bioMEMS方法将膜片钳电生理学与离子特异性微电极集成来推进这一领域。

项目成果

An aqueous media based approach for the preparation of a biosensor platform composed of graphene oxide and Pt-black.

• DOI: 10.1016/j.bios.2012.06.007
• 发表时间: 2012-10
• 期刊: BIOSENSORS & BIOELECTRONICS
• 影响因子: 12.6
• 作者: Shi, Jin;Zhang, Hangyu;Snyder, Alexandra;Wang, Mei-xian;Xie, Jian;Porterfield, D. Marshall;Stanciu, Lia A.
• 通讯作者: Stanciu, Lia A.

Nanomaterial based self-referencing microbiosensors for cell and tissue physiology research.

• DOI: 10.1016/j.bios.2012.06.059
• 发表时间: 2013-02-15
• 期刊: BIOSENSORS & BIOELECTRONICS
• 影响因子: 12.6
• 作者: Shi, Jin;McLamore, Eric S.;Porterfield, D. Marshall
• 通讯作者: Porterfield, D. Marshall