Electrophysiology using a microfluidic array for high-throughput patch clamp measurements

The fact that cellular ion channels are effective drug targets, coupled with the laboriousness of traditional patch clamp techniques, has created a need for hi-throughput electrophysiology platforms[1]. Patch clamp based drug screening technology has been recently implemented by using microfabricated patch clamp designs that replace the traditional patch pipette with a pore in a silicon substrate [2, 3]. While successful at high-throughput measurements of channel activity, current devices have yet to achieve high densities of patched cells per unit volume and rely on robotically operated pipettes for reagent and cell delivery.

We are working on a device that is based on elastomer micromolding and takes advantage of integrated fluidic networks in order to achieve high patch site density and very small reagent volumes per experiment. Integration and close placement of individually addressed patch sites is enabled by the replacement of the patch pore with a microfluidic channel junction (Fig. 1, 2), where cells can be trapped from the main channel by applying negative pressure to the side channels[4]. We have fabricated devices that range from a lower density of 2.5x102 sites/µl (Fig. 1), to a high density of 3.3x104 sites/µl (Fig. 2). By comparison, current technology utilizes a single patch site at the bottom a 500 µl well. Even if additional reagent has to be used to fill the dead volume for both on-chip channels and exterior tubing, one application is sufficient for recording the response of a large number of cells (for current designs n=12).

We show the current recordings for high resistance seals (500M to 1G ) between the poly-dimethylsiloxane (PDMS) microchannel and the cell membrane and the attainment of a whole cell patch configuration (Fig. 3). Whole cell currents show increased capacitance of the whole membrane (C = 28 pF, red line in Fig. 3) and decreased overall resistance (R = 200 MO). Whole cell currents from a Chinese hamster ovary (CHO) cell line expressing the potassium channel Kv2.1 [5] were recorded using this device.

An additional advantage of using transparent PDMS as the molded material and glass as the bottom support is the possibility for optical observation of cell deformation into the patch pore and fluorescent measurements. The deformation of a HeLa (human tumor cell line) cell under the application of negative pressure and subsequent membrane break and cytoplasmic dye leak into the patch channels are shown in Figure 4. Thus, fluorescent binding assays can then be correlated easily with electrophysiology data.

In the near future, we plan to control cell trapping pressure and use PDMS valves for on-chip reagent delivery. This will reduce solution exchange times and reagent dead volume per experiment dramatically. This will also allow dynamic control of applied pressure in accordance with the multiplexed current recording setup of Figure 1.

References:

1. Bennett, P.B. and H.R.E. Guthrie, Trends in ion channel drug discovery: advances in screening technologies. Journal of Biomolecular Screening, 2003. 8(6): p. 660-667.
2. Schroeder, K., et al., IonWorks (TM) HT: A new high-throughput electrophysiology measurement platform. Journal of Biomolecular Screening, 2003. 8(1): p. 50-64.
3. Xu, J., et al., A benchmark study with SealChip (TM) planar patch-clamp technology. Assay and Drug Development Technologies, 2003. 1(5): p. 675-684.
4. Seo, J., et al., Integrated Multiple Patch-Clamp Array Chip via Lateral Cell Trapping Junctions. Applied Physics Letters, 2004. 84(11): p. 1973-1975.
5. Trapani, J.G. and S.J. Korn, Control of ion channel expression for patch clamp recordings using an inducible expression system in mammalian cell lines. Bmc Neuroscience, 2003. 4.

Experimental setup for a multiplexed patch clamp array for hi-thoughput measurements.

SEM image of a high density patch array (a) and phase contrast image of cells trapped at the patch pore.

Current traces recorded for three different configurations. The open channel (green) has a resistance of 14 MO, and after cell trapping the resistance is increased to 1 GO (black). After the membrane patch is broken, a whole cell configuration is achieved (red, R = 200 MO, C = 28 pF).

Optical observation of the cell membrane break. The cell interior has been loaded with a fluorescent cytoplasmic dye. The cell is first trapped (a) then protrudes inside the channel (b), and is broken by the application of a pressure pulse (c).

For additional information concerning this project, contact: Cristian Ionescu-Zanetti,