Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Cell membrane or cell surface is target
Reexamination Certificate
2001-02-12
2004-03-02
Redding, David A. (Department: 1744)
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Cell membrane or cell surface is target
C435S173500, C435S173600, C435S287100, C435S288400, C204S280000, C204S403100
Reexamination Certificate
active
06699697
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to biosensors, including methods of making biosensors and methods of measuring cellular signal transduction processes using biosensors. More specifically, the present invention relates to ionic electrodes, particularly microelectrodes and electrode arrays, and also relates to fabrication methods for such electrodes and to methods of using such electrodes. More particularly, the present invention relates to planar silicone polymer electrodes for making patch clamp measurements of ionic currents through biological membranes, such as the plasma membranes of living cells, as well as to methods of making and utilizing such electrodes. The biosensor apparatus and methodology of the present invention provide for the high-throughput measurement of ionic currents through biological membranes. The biosensors of the present invention are particularly useful for screening new drugs that act on cell-membrane ion channels and transporters.
BACKGROUND OF THE INVENTION
All publications, patents and patent applications herein are incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.
Many cellular processes are controlled by changes in cell membrane potential due to the action of ion channels. Action potentials trigger the release of hormones and neurotransmitters in secretory cells and neurons; they trigger contractions in muscle cells and influence biochemical events and levels of gene expression. Action potentials and other changes of membrane potential are in turn triggered by the opening of ion channels that are coupled to receptors for neurotransmitters or intracellular messengers, or by ion channels that are mechanosensitive or voltage-sensitive. The wide variety of ion channels arises from large families of genes and represents a rich collection of new targets for pharmaceutical agents.
The patch clamp technique is the central technique for studying ion channels. It provides a “voltage clamp” measurement of ionic current in either a small “patch” of cell membrane, or the entire membrane of a small cell. Because it is a measurement of current, it directly monitors the number of active channels in the membrane, and is therefore the assay of choice for agents that block or modulate channel activity. It is also the only reliable way to make electrical recording from small cells, such as neurons and most other cells in vertebrate animals. Its development was recognized by the Nobel Prize in 1991, which was awarded to E. Neher and B. Sakmann. For general introductions, see Boulton et al.(Editors),
Patch Clamp Applications and Protocols
, Humana Press (1995); Neher and Sakmann (Editors),
Single
-
Channel Recording
, Plenum Press (1995), and DeFelice,
Electrical Properties of Cells: Patch Clamp for Biologists
(The Language of Science), Plenum Pub. Corp. (1997), each of which is specifically incorporated by reference in its entirety.
Fundamentally, an electrode for patch clamp recording consists of an insulating partition that separates the cell and its surrounding “bath” solution, on one side, from a compartment filled with ionic solution and containing a non-polarizable electrically conducting contact, typically a silver wire with a silver-chloride (AgCl) surface coating. A small aperture in this partition defines a region from which current is measured. A conventional patch clamp electrode consists of a saline-filled glass micropipette, having a tip opening of roughly one micron, which is pressed against the cell membrane. The pipette's tip comprises the electrode partition; with the AgCl coated silver wire inserted into the back end of the pipette, the pipette collects ionic current passing through the membrane patch covered by the tip, allowing “patch” recording. In the alternative, “whole-cell recording” configuration, the membrane patch is ruptured, giving direct electrical access to the cell interior. The current collected by the patch electrode ranges in magnitude from roughly one picoampere (for currents of individual channels in the membrane patch) to tens of nanoamperes (in the case of large whole-cell currents). The current is monitored by a sensitive preamplifier that is located close to the pipette and is connected to the AgCl coated silver wire.
Conventionally, glass micropipettes act as electrode partitions and are fabricated in the user's laboratory within a few hours of their use. The fabrication consists of three steps. First, a glass or quartz capillary tube (about 1.5 mm diameter) is heated at its center while the ends are pulled slowly by carefully moving it until the tube breaks into two pieces. Each has a tapered shank leading to a tip opening of a few microns. In a second, optional, step, the shank of the pipette is coated with a hydrophobic material such as wax or a silicone elastomer to decrease the total electrical capacitance of the patch electrode; care is taken so that the coating does not approach the tip opening, where it may prevent sealing to the cell membrane. Lastly, the tip is “heat-polished” by carefully moving it closely to a heated filament to round the edges and establish the final tip diameter of roughly one micron.
The patch electrode is filled with an ionic solution that is typically chosen to mimic the extracellular fluid, and is mounted onto a holder that is attached directly to a preamplifier, which in turn is mounted on a three-axis micromanipulator. Viewing a petri dish of cells through an inverted microscope, the operator uses the micromanipulator to position the pipette tip to touch the membrane of a chosen cell. Gentle suction is applied to the pipette interior to draw the cell membrane toward the tip, and under favorable conditions a mechanically stable, high-resistance seal (>1 gigaohm) spontaneously occurs between the membrane and the clean glass surface of the tip. Once the seal is established, the voltage of the membrane can be set according to the experimental goals and currents are recorded through the use of the preamplifier and its associated main amplifier. Examples of commercial patch clamp amplifiers include the AxoPatch 200 series by Axon Instruments, Inc. and the EPC series by HEKA Elektronik.
The present invention improves the instrumentation used to study membrane proteins, especially the study of ion channels. This is important for the advancement of basic research in this area as well as for high-throughput screening of pharmacological agents acting on these proteins.
Several attempts have been made to improve the reliability, reproducibility and overall throughput capacity of conventional glass patch clamp readings. For example, WO 98/50791 discloses a fully automated pipette patch clamp technique which provides computer aided guiding of the pipette to the cell. While this method is useful, it only provides for measuring one cell at a time and connecting the cell and the pipette is difficult. WO 99/31503 discloses a glass patch electrode with an electrically charged surface which provides for more precise positioning of biological membranes on the electrode surface. U.S. Pat. No. 6,048,722 combines a glass patch electrode with an automatic perfusion system connected to a recording chamber, permitting cells to be perfused with a plurality of solutions containing different concentrations of one or more agents to be tested. JP 4338240 discloses treating the surface of a glass micro-pipette in oxygen plasma to improve the ability of the glass surface to seal against biological membranes.
The present invention greatly improves the “ease of use” of patch clamp recording of membrane ion currents and increases the overall throughput of data collection, especially for drug discovery and research purposes. In addition, the present invention permits simple exchange of the electrode chamber solution. The novel patch electrodes of the present invention allow a better signal-to-noise ratio for high-resolution recording of membra
Klemic James F.
Klemic Kathryn G.
Reed Mark A.
Sigworth Frederick J.
Morgan & Lewis & Bockius, LLP
Redding David A.
Yale University
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