Towards a Fully Automated Planar Patch Clamp Based Dose

TOWARDS A FULLY AUTOMATED PLANAR PATCH CLAMP BASED DOSE-RESPONSE MEASU~MENT SYSTEM B. Matthews and J. W. Judy Hectrical

Engineering

University of Callfor-nia, Los Angeles, Calzyomiu, USA Abstract- We have designed, fabricated, integrated, and tested microfabricated planar patch-clamp substrates and poly(dimethylsiloxane) (PDMS) microfluidic dose-response components suitable for use in an automated planar patch-clamp measurement system. Substrates with cell-patch-site apertures ranging from 300 nm to 12 pm were fabricated using standard MEMS techniques. The resistance of the cell-patch sites ranged from 0.2 to 47 MC!, for apertures ranging from 12 pm to 0.75 pm. The substrate capacitance was 17.2 pF/mm’ of fluid contact. In addition, testing shows that the fluidic components are appropriate for driving human embryonic kidney cells (HEK 293) to patch apertures, for trapping cells on patch apertures, and for rapidly exchanging the extracellular fluid environment. Keywords - Patch-Clamp,

Department,

Micromachining,

MEMS, Electrophysiology

INTRODUCTION The patch-clamp technique, which has been used extensively to investigate electrophysiological cell properties, measures the ionic current passing through a cellular membrane (Fig. la). Ion-channel proteins, which are embedded within cell membranes, control the flow of ions through the membrane and are gated by electrical, mechanical, or chemical stimuli. The patch-clamp technique has become an indispensable tool in cellular physiology because it provides a method to directly measure a signaling pathway for many cellular processes. Although microfabricated structures have been used to patch to cells with gigaohm seals in academic and commercial laboratories, these devices do not patch with high reliability, have not been integrated into miniaturized systems, or have solely targeted high-throughput screening [l-9].

Fig. 1 (a) Schematic representation of a traditional patch-clamp system using a pulled glass pipette and a close-up of the cell and patch pipette tip. (b) Schematic representation of a planar patch-clamp based dose-response measurement system.

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Our initial system focuses on dose-response measurements that quantify the activity of ion channels in a range of chemical environments and are thus useful for drug-dosing studies and fundamental studies of cellular physiology. Through the integration of microfluidics with a planar patch-clamp substrate, our completed system presents a new tool specifically engineered to enable a complex electrophysiological experiment on individual cells [lo-l l] (Fig. lb). RESULTS Patch-clamp substrates were fabricated using standard microfabrication techniques with patch apertures from 300 nm to 12 pm (Fig. 2). In order to achieve nanometer scale hole diameters with out resorting to e-beam lithography, the conformal nature of chemical vapor deposition was used to reduce micron scale holes to nanometer scale holes. The fabrication process for the patch-clamp substrates is shown in Fig. 3.

Fig. 2. Examples

of a 2.5~pm-diameter

and a 300-nm diameter patch apertures

Step 1: Deposit 200 nm of silicon nitride

Step 4: Etch front-side silicon nitride

SteD 2: Etch back-side silicon nitride

Step 5: Etch single-crawl silicon to form 3-urn-diameter holes

-pm-diam&r

ho

Step 3: Etch single-crysfaf silicon to form

Fig. 3. Planar patch-clamp

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Fig. 4. Patch-aperture

resistance

and substrate capacitance

In order to extract the series resistance and capacitance of our substrates, a potentiostat was used to measure their impedance spectra with a 100 mM KC1 electrolyte solution. The series resistance of the aperture and the substrate capacitance are shown in Fig. 4. The microfluidics were fabricated by molding PDMS onto a two level SU-8 mold master: a 30-pm-thick mold of SU-8 forms the fluidic channels and a 2-mm-thick layer of SU-8 forms the macroscale fluid-access ports (Fig. 5ab). The macroscale fluid-access ports provide an easily operated and robust fhtidic connection technology that seals to glass capillary tubing without glue. The patch-clamp substrate and PDMS microfluidics were aligned and bonded using a mask aligneribonder. Prior to bonding the patch-clamp substrates and PDMS microfluidics are exposed to an oxygen plasma to modify the PDMS surface to form a permanent bond 1111.

(ai

(b)

(cl

w

Fig. 5. (a) Schematic representation of the microfluidic system. (b) Layout of the molded and integrated prototype microfluidic system. (c) Cell in microfluidic channel that has been trapped on cell-patch site. (d) Test of fluid exchange in a PDMS molded microfluidic system.

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The microfluidic operation of the patch-clamp system was tested using HEK 293 cells and dyes. Cells were driven through the fluidic system and trapped on a patch aperture by suction (Fig 5~). Dyes representing ion-channel-gating compounds were flowed through the system simulating extracellular fluid exchange (Fig. 5d). This work represents the first integration of a micromachined patch-clamp substrate with microfluidics and has the potential to simplify difficult electrophysiology experiments. CONCLUSION Specifically engineered patch-clamp systems present an opportunity to simplify currently difficult electrophysiology experiments. In a first step toward an automated single-cell dose-response curve measurement system, we have fabricated substrates containing cell-patch sites, fabricated simple PDMS microfluidic components, and integrated the two. Once complete, our patch-clamp system holds the promise of parallelism, microfluidic manipulation, uniformity, optical access, and automation that is currently impossible to obtain with traditional patch-clamp systems. RElFERENCES [I]

R. Pantoja, D. Sigg, R. Blunck, F. Bezanilla, and J. R. Heath, “Bilayer reconstitution of voltage-dependent ion channels using a microfabricated silicon chip,” Biophys. J., vol. 81, pp. 2389-2394,200l. [2] C. Schmidt, M. Mayer, and J. Vogel, “A chip-based biosensor for the functional analysis of single ion channels,” Angew. Chem. hat., vol. 39, pp. 3 137-3 140,200O. [3] N. Fertig, C. Meyer, R. Blick, and J. Behrends, “A microstructured chip electrode for low noise single channel recording,” Biophys. J., vol. 80, pp. 337a, 2001. [4] K. Cheung, T. Kubow, and L. P. Lee, “Individually addressable planar patch clamp array,” 2nd Annual International IEEE-EMBS Special Topic Conference on Microtechnologies in Medicine and Biology, pp. 71-75,2002. [5] K. G. Klemic, J. F. Klemic, M. A. Reed, and F. J. Sigworth, “Micromolded PDMS planar electrode allows patch clamp electrical recordings from cells,” Biosensors & Bioelectronics, vol. 17, pp. 597~604,2002. [6] PatchXpress, Axon Instruments, www.axon.com. [7] IonWorksHT, Molecular Devices, www.moleculardevices.com. [8] Nanion Technologies, wwwnanionde. [9] Sophion Biosciences, h~://~.sophion.dk. [IO] J. McDonald, D Duff, J. Anderson, D. Chiu, H. Wu, 0. Schueller, G. Whitesides, “Fabrication of microfluidic systems in poly(dimethylsiloxane),” Electrophoresis, vol. 21 pp. 27-40,200O. [ 1 I] K. Hosokawa, K. Hanada, and R. Maeda, “A polydimethylsiloxane (PDMS) deformable diffraction grating for monitoring of local pressure in microfluidic devices,” J. Micromech, Microeng, vol. 12, pp.l-6,2002. [ 121 B. Matthews and J. Judy, “Characterization of a Micromachined Planar Patch Clamp for Cellular Electrophysiology,” IEEE-EMBS International Conference on Neural Engineering, pp. 648-65 1,2003.

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