Py Christophe, Martina Marzia, Monette Robert, Comas Tanya, Denhoff Mike W, Luk Collin, Syed Naweed I, Mealing Geoff
Institute for Microstructural Sciences, National Research Council of Canada.
J Vis Exp. 2012 Feb 7(60):3288. doi: 10.3791/3288.
Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons; recently, we have also reported progress towards interrogating mammalian neurons. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel .The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.
膜片钳技术由于其极高的灵敏度以及在离子通道水平上监测和控制单个细胞的能力，成为应用于疾病模型和药物筛选的电生理学黄金标准。传统方法是用一根充满生理溶液的玻璃微吸管轻轻接触细胞，以分离其顶端下方的一小片细胞膜。插入微吸管的电极可捕获膜片内的离子通道活动，若微吸管尖端破裂，还能捕获整个细胞的离子通道活动。在过去十年中，膜片钳芯片被提出来作为一种替代方案：一片悬浮膜将生理介质与培养基隔开，膜上微加工的小孔取代了微吸管的尖端。膜片钳芯片已集成到自动化系统中，并实现了商业化以用于高通量筛选。为了提高通量，它们包括从悬浮液中流体输送细胞、通过抽吸将细胞定位在小孔上，以及检测细胞与探针密封并进入全细胞模式的自动化程序。我们已报道了一种具有优化阻抗和孔口形状的硅膜片钳芯片的制造，该芯片能够高质量记录培养的蜗牛神经元的动作电位；最近，我们也报道了在研究哺乳动物神经元方面取得的进展。我们的膜片钳芯片是在加拿大光子学制造中心（一家商业代工企业）制造的，并且有大量现货。我们渴望与电生理学家合作，以验证NRCC技术在不同模型中的应用。芯片的使用遵循图1所示的总体方案：硅芯片位于有机玻璃培养瓶底部，小孔的背面连接到封装两端装有管子的地下通道。细胞在培养瓶中培养，通过插入通道的测量电极监测探针顶部的细胞。两个外部流体端口便于溶液交换，对细胞的干扰最小；与用于细胞内灌流的玻璃微吸管相比，这是一个优势。
