A novel silicon patch‐clamp chip permits high‐fidelity recording of ion channel activity from functionally defined neurons

Abstract

We report on a simple and high‐yield manufacturing process for silicon planar patch‐clamp chips, which allow low capacitance and series resistance from individually identified cultured neurons. Apertures are etched in a high‐quality silicon nitride film on a silicon wafer; wells are opened on the backside of the wafer by wet etching and passivated by a thick deposited silicon dioxide film to reduce the capacitance of the chip and to facilitate the formation of a high‐impedance cell to aperture seal. The chip surface is suitable for culture of neurons over a small orifice in the substrate with minimal leak current. Collectively, these features enable high‐fidelity electrophysiological recording of transmembrane currents resulting from ion channel activity in cultured neurons. Using cultured Lymnaea neurons we demonstrate whole‐cell current recordings obtained from a voltage‐clamp stimulation protocol, and in current‐clamp mode we report action potentials stimulated by membrane depolarization steps. Despite the relatively large size of these neurons, good temporal and spatial control of cell membrane voltage was evident. To our knowledge this is the first report of recording of ion channel activity and action potentials from neurons cultured directly on a planar patch‐clamp chip. This interrogation platform has enormous potential as a novel tool to readily provide high‐information content during pharmaceutical assays to investigate in vitro models of disease, as well as neuronal physiology and synaptic plasticity. Biotechnol. Bioeng. 2010;107:593–600. © 2010 Wiley Periodicals, Inc.

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

“…The process described in 6 results in a 3 μm thick self-standing film, a low 1 Mohm access resistance and a smooth-surfaced funnel shaped aperture that facilitates an intimate seal with the cell 9 , see Figure 2. The chips are singulated and glued in Plexiglas packages with the aperture facing a hole connecting the chip to a subterranean channel.…”

“…An impedance meter is used to confirm that the access resistance is between 300kohm and 3Mohm and the shunt capacitance is between 10pF and 25pF. A chip with a lower resistance is likely to have a leak, and a higher capacitance is considered improper for use as it may not capture the fastest dynamics of the cell′s electrophysiology 6 . A chip with a lower capacitance or higher resistance is considered plugged, though it could be simply that a bubble is trapped in the orifice.…”

“…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 6 ; recently, we have also reported progress towards interrogating mammalian neurons 7 . Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre 8 , a commercial foundry, and are available in large series.…”

Development of Patch-Clamp Chips for Mammalian Cell Applications

“…(3b). The fabrication procedure was amended from [12]. Apertures were etched in a 1 μm thick silicon nitride film, and inverted pyramid-shaped wells were etched anisotropically in a KOH solution from the back of the wafer.…”

“…(2a). Detailed fabrication procedures have been presented previously [12] and follow the procedure described above for the sieve chip. Single-aperture neurochips were made functional, i.e.…”