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Whole-Cell Electrical Activity Under Direct Mechanical Stimulus by AFM Cantilever Using Planar Patch Clamp Chip Approach

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Abstract

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.

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Acknowledgments

The authors would like to acknowledge the support provided by the John A. Swanson Center for Micro and Nano Systems and the Nanofabrication and Characterization Facility at the Petersen Institute of Nanoscience and Engineering for fabrication of the chip, John A. Swanson Center for Product Innovation for fabrication of the microfluidics and Center for Biological Imaging for electron microscopy. We would like to thank Prof. Ratneshwar Lal from University of California, San Diego and Prof. Sanjeev Shroff from University of Pittsburgh for their valuable advice and inputs. We are grateful to Prof. Elias Aizenman from the Department of Neurobiology of University of Pittsburgh School of Medicine for providing the CHO and PC12 cells as well as Prof. Chuanyue Wu from the Department of Pathology of University of Pittsburgh School of Medicine for providing the HaCaT cells. The research was supported by grants from NIH (R21-EB004474) and the Central Research Development Fund (CRDF) and startup fund from University of Pittsburgh.

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Authors and Affiliations

  1. Department of Bioengineering, University of Pittsburgh, Suite 306, 300 Technology Drive, Pittsburgh, PA, 15219, USA

    Kalpesh V. Upadhye

  2. Department of Bioengineering, University of Pittsburgh, Bioscience Tower 3-5059, 3501 Fifth Avenue, Pittsburgh, PA, 15260, USA

    Lance A. Davidson

  3. Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA

    Joseph E. Candiello & Hai Lin

Authors
  1. Kalpesh V. Upadhye
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  2. Joseph E. Candiello
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  3. Lance A. Davidson
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  4. Hai Lin
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Correspondence to Kalpesh V. Upadhye or Lance A. Davidson.

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Associate Editor Chwee Teck Lim oversaw the review of this article.

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Upadhye, K.V., Candiello, J.E., Davidson, L.A. et al. Whole-Cell Electrical Activity Under Direct Mechanical Stimulus by AFM Cantilever Using Planar Patch Clamp Chip Approach. Cel. Mol. Bioeng. 4, 270–280 (2011). https://doi.org/10.1007/s12195-011-0160-4

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  • DOI: https://doi.org/10.1007/s12195-011-0160-4

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