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Biophys J, June 2002, p. 2831-2832, Vol. 82, No. 6
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520 USA
To use one of the new benchtop patch-clamp
systems, you first harvest the cells from a tissue-culture dish and
wash by pelleting them in a microcentrifuge. Then you open the lid of
the patch clamp box and pipette a few microliters of the cell
suspension into the recording well. After a few minutes the cells
settle onto the electrode array at the bottom of the well, the computer selects an electrode that has made a good seal, and the recording starts.
This "biochemist's patch clamp" is still a few years in the
future, but in this issue of Biophysical Journal
Fertig et al. (2002) In conventional patch-clamp recording, a glass or fused-quartz
micropipette is used as the ionic electrode: it is filled with an ionic
solution which electrically connects a silver-silver chloride electrode
wire to a small "patch" area of cell membrane at the small opening
of the pipette tip. Critical to the method is the formation of a high
resistance (gigohm) seal between the pipette and the cell membrane.
For about a decade, workers in various laboratories have sought ways to
replace the pipette with "planar" patch-clamp electrodes, in which
a micrometer-sized hole is made in a suitable, thin insulating partition. The idea is then to place a cell over this hole (which corresponds to the pipette tip) and to fill the underside of the partition with electrode solution. A metal contact on the underside completes the patch electrode. Early attempts to make planar electrodes involved the formation of silicon oxide or silicon nitride membranes suspended over pits etched in silicon wafers. The oxide or nitride membranes were typically 0.1-1 µm thick; into them were introduced micrometer-sized holes by photolithography and etching. These structures have proven useful for recording from liposomes
(Schmidt et al., 2000 For their successful planar electrodes Fertig et al.,
have turned to a proven material and to an electrode geometry very
similar to that of conventional pipettes. Starting with fused-quartz
wafers, they formed round, gradually tapering holes by ion-track
etching. In this amazing process, a single high-energy gold ion is shot through a 20-µm-thick region of a wafer, leaving behind a track of
molecular damage that is highly susceptible to etching by hydrofluoric acid. Exposing the inner surface of the wafer to an HF solution therefore produces a narrowly tapered cavity. When etching is stopped
at the correct moment, the cavity breaks through the outer surface to
produce a micrometer-sized hole.
The result of this process is an electrode with quite good properties.
The authors report a very respectable 30% success rate in obtaining
"blind" recordings from cell suspensions, and the mechanical
stability of the system allows whole-cell recordings to be made by
mechanically rupturing the cell membrane after sealing. The capacitance
and access resistance of the planar electrodes is potentially superior
to those properties of conventional pipettes, but the seal resistances
are somewhat lower The practical application of these planar electrodes will require some
further development. First, a means for fabricating arrays of identical
electrodes will need to be developed. In principle this is a matter of
careful process engineering like that employed in the microelectronics
industry. Second, assuming that the electrodes will not be inexpensive
to make, a reliable method for cleaning electrodes for re-use will need
to be worked out. Finally, techniques for preparing clean cell
suspensions and methods for guiding cells to the electrode apertures
may need to be developed. In this regard Fertig et al.
have obtained surprisingly good results with a simple washing procedure
involving centrifugation of cells and a simple cell-positioning process
that makes use of gentle suction through the electrode aperture.
Meanwhile in various laboratories other materials and technologies are
being pursued. Although gigohm seals on cell membranes have not to date
been reported with them, thin silicon nitride membranes are still under
active investigation for patch electrodes. The structures can be
fabricated with relative ease, and a particularly elegant property of
this electrode geometry is the high electrostatic field that appears
near the hole when a potential is applied to an "open" electrode.
Schmidt et al. (2000) A promising alternative material for planar patch electrodes is
polydimethylsiloxane (PDMS), a silicone elastomer that is well known to
the patch-clamp community under Dow Corning's trade name Sylgard. PDMS
can readily be molded with sub-micrometer features; its surface can
also be modified by plasma oxidation to make it hydrophilic. It turns
out that the modified surface forms gigohm seals with cell membranes.
Macro-patch recordings from Xenopus oocytes have recently
been made from micromolded PDMS electrodes and arrays (Klemic et
al. 2002 While much activity has focused on planar electrode arrays, the
automation of patch-clamp recording is also being pursued with the
traditional glass-pipette technology. The company Sophion in Denmark
has developed a robotic patch-clamp system in which machine vision is
used to locate cells under a microscope, position the pipette and
establish recordings. In the instrument developed by the company CeNeS,
cells are suspended in a drop of solution, and a pipette blindly
approaching the drop from below encounters cells concentrated near the
air-water interface.
The paper by Fertig et al. in this issue represents an
important step in the race to develop a high-throughput patch-clamp system for the pharmaceutical industry (Xu et al.,
2001
ARTICLE
describe an important milestone
toward its development, namely the first successful demonstration of
patch-clamp recordings using a micromachined glass chip. The strong
economic driving force behind this and related technical developments
is not necessarily to make life easier for researchers at the bench,
but rather to provide a high-throughput screening tool for drug
discovery efforts (Xu et al., 2001). Suppose one had
electrodes at the bottom of each well of a 384-well plate, each
connected to an amplifier and able to make a whole-cell patch
recording. Then massively parallel screens for ion channel activity
could be carried out. But we predict that a useful spin-off of this
effort will also be simple benchtop patch-clamp systems.
) and painted artificial
lipid bilayers (Pantoja et al., 2001), but no reports of
gigohm seals or successful recordings from cells have appeared. We also
failed to obtain gigohm seals with structures made in our laboratory by
anisotropic etching of single-crystal quartz. It is not certain what is
the difficulty with seal formation, but we speculate that the narrow
sidewalls or sharp corners of the etched apertures do not provide
sufficient contact area with the cell membrane to allow a seal
to be formed.
by about a factor of 3than those obtained with
pipettes. This last property might reflect roughness of the surface
after etching.
have exploited the field to guide
liposomes to the electrode aperture, and one imagines that small cells
might be guided in the same way.
).
). Such a system should be useful also in proteomics, for
example in screening for new ion channel subunits in expression
libraries. We expect that the benefits of the new technologies will
also trickle down to the biophysical community. Planar electrodes from
materials like quartz and PDMS promise to provide higher-resolution
recordings, due to their potentially lower capacitance, access
resistance, and dielectric noise. Transparent planar electrodes will
also be well suited for optical measurements, making possible for
example simultaneous single-molecule electrical and optical recordings. And in the not-too-distant future the new electrode technologies may
greatly simplify the technique of routine patch-clamp recording.
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FOOTNOTES |
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.
Address reprint requests to Fred J. Sigworth, Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520. Tel.: 203-785-5773; Fax: 203-785-5773; E-mail: fred.sigworth{at}yale.edu.
Submitted February 14, 2002, and accepted for publication February 19, 2002.
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REFERENCES |
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Biophys J, June 2002, p. 2831-2832, Vol. 82, No. 6
© 2002 by the Biophysical Society 0006-3495/02/06/2831/02 $2.00
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