Dual-pore glass chips for cell-attached single-channel recordings.
While high-throughput planar patch-clamp instruments are now established to perform whole-cell recordings for drug screening, the conventional micropipette-based approach remains the gold standard for performing cell-attached single-channel recordings. Generally, planar platforms are not well-suited for such studies due to excess noise resulting from low seal resistances and the use of substrates with poor dielectric properties. Since these platforms tend to use the same pore to position a cell by suction and establish a seal, biological debris from the cell suspension can contaminate the pore surface prior to seal formation, reducing the seal resistance. Here, femtosecond laser ablation was used to fabricate dual-pore glass chips optimized for use in cell-attached single-channel recordings that circumvent this problem by using different pores to position a cell and to establish a seal. This dual-pore design also permitted the use of a relatively small patch aperture (D ~ 150 to 300 nm) that is better-suited for establishing high-resistance seals than the micropores used typically in planar patch-clamp setups (D ~ 1 to 2 μm) without compromising the ability of the device to position a cell. Taking advantage of the high seal resistances and low capacitive and dielectric noise realized using glass substrates, patch-clamp experiments with these dual-pore chips consistently achieved high seal resistances (rate of gigaseal formation = 61%, mean seal resistance = 53 GΩ), maintained gigaseals for prolonged durations (up to 6 hours), achieved RMS noise values as low as 0.46 pA at 5 kHz bandwidth, and enabled single-channel recordings in the cell-attached configuration that are comparable to those obtained by conventional patch-clamp.
Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA. mimayer@umich.edu.
Brandon R
BR
Bruhn
Haiyan
H
Liu
Stefan
S
Schuhladen
Alan J
AJ
Hunt
Aghapi
A
Mordovanakis
Michael
M
Mayer
D017097:Electric Impedance; D005898:Glass; D057809:HEK293 Cells; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods; D018408:Patch-Clamp Techniques / Q000295:instrumentation / Q000379:methods; D016062:Porosity