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首页> 外文期刊>Cellular and Molecular Bioengineering >Whole-Cell Electrical Activity Under Direct Mechanical Stimulus by AFM Cantilever Using Planar Patch Clamp Chip Approach
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Whole-Cell Electrical Activity Under Direct Mechanical Stimulus by AFM Cantilever Using Planar Patch Clamp Chip Approach

机译：平面膜片钳芯片方法在AFM悬臂直接机械刺激下的全细胞电活动

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摘要

Patch clamp is a powerful tool for studying the properties of ion-channels and cellular membrane. In recent years, planar patch clamp chips have been fabricated from various materials including glass, quartz, silicon, silicon nitride, polydimethyl-siloxane (PDMS), and silicon dioxide. Planar patch clamps have made automation of patch clamp recordings possible. However, most planar patch clamp chips have limitations when used in combination with other techniques. Furthermore, the fabrication methods used are often expensive and require specialized equipments. An improved design as well as fabrication and characterization of a silicon-based planar patch clamp chip are described in this report. Fabrication involves true batch fabrication processes that can be performed in most common microfabrication facilities using well established MEMS techniques. Our planar patch clamp chips can form giga-ohm seals with the cell plasma membrane with success rate comparable to existing patch clamp techniques. The chip permits whole-cell voltage clamp recordings on variety of cell types including Chinese Hamster Ovary (CHO) cells and pheochromocytoma (PC12) cells, for times longer than most available patch clamp chips. When combined with a custom microfluidics chamber, we demonstrate that it is possible to perfuse the extra-cellular as well as intra-cellular buffers. The chamber design allows integration of planar patch clamp with atomic force microscope (AFM). Using our planar patch clamp chip and microfluidics chamber, we have recorded whole-cell mechanosensitive (MS) currents produced by directly stimulating human keratinocyte (HaCaT) cells using an AFM cantilever. Our results reveal the spatial distribution of MS ion channels and temporal details of the responses from MS channels. The results show that planar patch clamp chips have great potential for multi-parametric high throughput studies of ion channel proteins.
机译：膜片钳是研究离子通道和细胞膜特性的有力工具。近年来，平面膜片钳芯片已由多种材料制成，包括玻璃，石英，硅，氮化硅，聚二甲基硅氧烷（PDMS）和二氧化硅。平面膜片钳使膜片钳记录的自动化成为可能。但是，大多数平面膜片钳芯片在与其他技术结合使用时具有局限性。此外，所使用的制造方法通常很昂贵并且需要专门的设备。本报告介绍了一种改进的设计以及基于硅的平面膜片钳芯片的制造和表征。制造涉及真正的批量制造过程，可以使用成熟的MEMS技术在大多数常见的微型制造设施中执行该过程。我们的平面膜片钳芯片可以与细胞质膜形成千兆欧姆的密封，其成功率可与现有膜片钳技术相媲美。该芯片可以对包括中国仓鼠卵巢（CHO）细胞和嗜铬细胞瘤（PC12）细胞在内的各种细胞类型进行全细胞电压钳记录，其时间比大多数可用的膜片钳芯片长。当与定制的微流控室组合时，我们证明可以灌注细胞外和细胞内缓冲液。腔室设计允许将平面膜片钳与原子力显微镜（AFM）集成在一起。使用我们的平面膜片钳芯片和微流控室，我们记录了通过使用AFM悬臂直接刺激人角质形成细胞（HaCaT）细胞产生的全细胞机械敏感（MS）电流。我们的结果揭示了MS离子通道的空间分布以及MS通道响应的时间细节。结果表明，平面膜片钳芯片对离子通道蛋白的多参数高通量研究具有巨大的潜力。

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