A Digital Feedback Clamp Instrument for Neurophysiology

用于神经生理学的数字反馈钳仪器

• 批准号: 7909411
• 负责人: Andrew A Hill
• 金额: $ 53.44万
• 依托单位: ROKHAN, LLC
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7909411
• 关键词: Algorithms; Amplifiers; Animals; Automobile Driving; Back; Basic Science; Binding; Bite; Brain Stem; Cataloging; Catalogs; Cell membrane; Cells; Chills; Computer software; Computers and Advanced Instrumentation; Development; Devices; Digital Signal Processing; Electrodes; Equipment; Evaluation; Event; Exhibits; Feedback; Funding; Head; Health; Ion Channel; Ion Channel Protein; Ions; Lead; Legal patent; Libraries; Marketing; Mass Screening; Measurement; Measures; Membrane; Metabolism; Methods; Microelectrodes; Mus; Names; Neurons; Neurosciences; Noise; Outcome; Patch-Clamp Techniques; Pharmacologic Substance; Phase; Population Heterogeneity; Production; Property; Proteins; Publications; Research; Rhode Island; Role; Scheme; Signal Transduction; Small Business Innovation Research Grant; Societies; Speed; Staging; Suction; Surface; System; Techniques; Technology; Testing; The Sun; Therapeutic; Time; Tissues; Universities; Validation; Variant; Work; analog; base; conditioning; cost; design; digital; electric impedance; feeding; human disease; improved; instrument; interest; meetings; neurophysiology; novel; patch clamp; prototype; public health relevance; research study; response; seal; submicron; tool; voltage; voltage clamp; voltage gated channel; web site

DESCRIPTION (provided by applicant): The Patch Clamp technique has evolved in recent years from an esoteric method of basic electrophysiological research into properties of cell membranes, into a practical technique for mass screening compounds for potentially therapeutic pharmaceutical properties. The commercial market for such equipment is now in the region of $40M annually. Its role as a tool of basic research has also continued to expand, as a means of discovering types and functions of ion channels through cell membranes, and characterizing their response. Phase I SBIR funding has enabled us to develop a digital clamp amplifier that functions in whole cell clamping and produces waveforms identical to those from comparable analog instruments. We have demonstrated some novel and potentially useful applications, not available with analog devices, such as providing instant "feedback" to a second neuron (dynamic clamp), thus driving one neuron off another's firing rate, and voltage clamping using only a single electrode without an external switch, multiplexing the stimulation and recording stages. This project will lead to a new patch clamp amplifier based on a high-speed digital signal processor to interact with excitable tissues. The instrument integrates the traditional voltage clamp, current clamp, dynamic clamp, and patch clamp into one unit, allowing for transient-free switching among the clamping modes and interactive controls of the neurons. The focus for Phase II includes hardware improvements for patch clamping, software techniques for controls, and experimental validations with mammalian neurons. Patch clamping developed from whole cell voltage clamping, in which measured current is fed back through one of two electrodes in the cell in an amount to keep the voltage across the cell membrane constant, as measured on the second electrode. Ions cross the cell membrane through proteins on the surface which escort them through. These ion channels are frequently voltage gated, open to passing only at certain voltage ranges, or may be chemically gated, thermally gated, or other. Even different voltage gated channels for the same ion may open at different voltages. For patch clamping, the electrode tip is very small, commonly sub-micron, in contact with a part of the cell membrane surface, and a small suction is applied to seal the membrane to the pipette tip. This allows measurement and feedback over only a small bit of membrane, and recording of even single ion channel events. There is a continuing and growing need to catalog all the ion channels across cell membranes, characterize their gate controls, and their function in cell metabolism, and effects on whole animal function and health. A common application is to screen compounds for binding to ion channel proteins to open or close them. Ion channels have become a major target for development of new pharmaceuticals. Variations include voltage clamping, current clamping and conductance clamping. In present application and technology, these 3 techniques each require different analog instruments for the fast responsive feedback needed, and the different parameter measured. Single channel events occur at change rates of up 250K/sec. The high speeds of ion channel events have required analog circuitry. In previous work and publications, it has been shown that a digital throughput of at least 500 KHz would be required to allow a digital instrument to perform as well as the analog instruments in voltage clamping. Very recently, digital signal processing chips (DSP's) operating at as fast as 600 MHz have become commercially available. Digital clamping would save the added cost of A/D and signal conditioning units, and one instrument would be able to perform any of the 4 types of clamping. Other emergent properties allow new avenues of research with digital clamping amplifiers. This proposal is to develop low-noise bi-directional headstages and accessories needed to use this digital clamp amplifier in patch clamping applications, including a chilled headstage to minimize noise, and to develop a library of algorithms for adjustable feedback schemes for different applications, and test the suitability of the instrument for patch clamp experiments. The resulting product is expected to achieve a substantial share of a large market. PUBLIC HEALTH RELEVANCE: Patch clamp technique has emerged as a practical applied technique to test compounds for functional effects on ion channel proteins. Defective ion channels are implicated in several human diseases. The proposed instrument will replace 3 types of analog clamping instruments, and digitizers and signal conditioners with a single, versatile, more powerful digital clamping device capable of serving as a voltage, current or impedance clamp.

描述(由申请人提供)：近年来，膜片钳技术已经从一种深奥的细胞膜性质的基础电生理研究方法，发展成为一种用于大规模筛选具有潜在治疗药物性质的化合物的实用技术。目前，此类设备的商业市场规模约为每年4000万美元。它作为基础研究工具的作用也在继续扩大，成为通过细胞膜发现离子通道的类型和功能并表征其反应的手段。第一阶段SBIR资金使我们能够开发一种数字钳位放大器，该放大器可用于全细胞钳位，并能产生与同类模拟仪器相同的波形。我们已经展示了一些模拟设备所不具备的新颖且潜在有用的应用，例如向第二个神经元提供即时“反馈”(动态钳位)，从而使一个神经元偏离另一个神经元的放电频率，以及仅使用单个电极而不使用外部开关的电压钳制，多路传输刺激和记录阶段。该项目将导致一种新的基于高速数字信号处理器的膜片钳放大器，以与可兴奋的组织相互作用。该仪器将传统的电压钳位、电流钳位、动态钳位和膜片钳集成到一个单元中，允许在钳位模式和神经元交互控制之间进行瞬时无切换。第二阶段的重点包括膜片钳的硬件改进，对照的软件技术，以及哺乳动物神经元的实验验证。膜片钳技术是在全电池电压钳制的基础上发展起来的，测量的电流通过电池中的两个电极中的一个反馈到一定的量，以保持细胞膜上的电压恒定，就像在第二个电极上测得的那样。离子通过细胞膜表面的蛋白质穿过细胞膜，这些蛋白质护送它们通过。这些离子通道经常是电压门控的，仅在特定电压范围内开放通过，或者可以是化学门控、热门或其他。即使是同一离子的不同电压门控通道也可能在不同的电压下打开。对于膜片钳制，电极尖端非常小，通常为亚微米，与部分细胞膜表面接触，并应用小吸力将膜密封到移液管尖端。这只允许在一小块膜上进行测量和反馈，甚至可以记录单个离子通道事件。对所有跨细胞膜的离子通道进行分类，确定它们的门控制特性，它们在细胞新陈代谢中的作用，以及对整个动物功能和健康的影响，是一个持续且日益增长的需要。一种常见的应用是筛选与离子通道蛋白结合的化合物，以打开或关闭它们。离子通道已成为新药开发的主要靶点。变化包括电压钳位、电流钳位和电导钳位。在目前的应用和技术中，这三种技术都需要不同的模拟仪器来实现所需的快速响应反馈，并需要测量不同的参数。单通道事件以高达250K/秒的变化率发生。离子通道事件的高速需要模拟电路。在以前的工作和出版物中，已经表明，至少需要500 kHz的数字吞吐量才能使数字仪器在电压钳位方面与模拟仪器一样发挥作用。最近，运行速度高达600 MHz的数字信号处理芯片(DSP)已经商业化。数字钳位将节省A/D和信号调理单元的额外成本，一台仪器将能够执行四种钳位中的任何一种。其他新特性为研究数字钳位放大器开辟了新的途径。这项提议是开发在膜片钳应用中使用这种数字钳位放大器所需的低噪声双向前级和附件，包括将噪声降至最低的冷冻前级，并开发用于不同应用的可调反馈方案的算法库，并测试该仪器是否适用于膜片钳实验。由此产生的产品预计将在一个巨大的市场上占据相当大的份额。 与公众健康相关：膜片钳技术已经成为一种实际应用技术，用于测试化合物对离子通道蛋白的功能影响。有缺陷的离子通道与几种人类疾病有关。该仪器将取代三种类型的模拟钳位仪器、数字化仪和信号调节器，而是一种功能更强大、功能更强大的数字钳位装置，可以用作电压、电流或阻抗钳位。