Our objective is to develop a technology that will allow us to probe, stimulate, and modify nanometer-scale areas of the cell membrane surface on large numbers of cultured cells simultaneously. The novelty of our approach, and its very engineering challenge, is to build an array of addressable submicron-sized fluidic channels which can be sealed against the cell membrane. The cells will rest on or be attached to a surface containing nanometric apertures, and each aperture will communicate with a dedicated microfluidic channel embedded under the cell culture surface. The cell membrane will be sealed against the aperture(s) by applying gentle suction to the channels. Thus, the microchannels will provide fluidic access only to the area of the cell membrane sealed against the aperture, thereby forming a """"""""nanofluidic probe"""""""" that can be used to sample and stimulate the area just above the aperture. Optionally, stronger suction may be used to rupture the cell membrane while maintaining the seal and thus create a mechanically stable, fluidically-addressable window for sampling or modifying the intracellular milieu. Our approach represents a radical paradigm shift from presently-available technology for probing small areas of cell membranes, which is based on glass micropipettes. These """"""""patch clamp"""""""" pipettes can indeed be sealed against the cell membrane surface and are routinely used to sample the activity of ion channels and stimulate receptors on a local scale. However, bringing the pipette in contact with the cell membrane involves the careful manipulation of a three-axis micropositioner by a skilled human operator under a microscope's visual field. Clearly, this procedure cannot be used to probe many cells simultaneously nor be repeated rapidly, limiting it to low-throughput, statistically-weak studies on a small number of cells. While the technique's throughput can be improved by increasing the number of resources (personnel, time, equipment), the number of cells that can be probed simultaneously remains fundamentally limited. Essentially, we propose to reverse the geometrical configuration of present electrophysiology setups, i.e. to embed the recording probes under the cell culture surface. Since the probes can be manufactured in an array format, the technology we propose to develop will allow for high-throughput, simultaneous probing of large numbers of cells. Consequently, it could have a significant impact in problems ranging from basic biology to drug discovery.
| Kosar, T Fettah; Tourovskaia, Anna; Figueroa-Masot, Xavier et al. (2006) A nanofabricated planar aperture as a mimic of the nerve-muscle contact during synaptogenesis. Lab Chip 6:632-8 |
