Electrolysis: processes – compositions used therein – and methods – Electrolytic analysis or testing – Involving enzyme or micro-organism
Reexamination Certificate
2000-10-02
2004-01-27
Olsen, Kaj K. (Department: 1753)
Electrolysis: processes, compositions used therein, and methods
Electrolytic analysis or testing
Involving enzyme or micro-organism
C204S403010, C422S082010
Reexamination Certificate
active
06682649
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a substrate and a method for determining and/or monitoring electrophysiological properties of ion channels of ion channel-containing structures, typically lipid membrane-containing structures such as cells, by establishing an electrophysiological measuring configuration in which a cell membrane forms a high resistive seal around a measuring electrode, making it possible to determine and monitor a current flow through the cell membrane. The substrate is typically part of an apparatus for studying electrical events in cell membranes, such as an apparatus for carrying out patch clamp techniques utilised to study ion transfer channels in biological membranes. More particularly, the invention relates to a substrate for such patch clamp apparatus having high through-put and utilising only small amounts of test compounds, only small amounts of liquid carrier, and being capable of carrying out many tests in a short period of time by performing parallel tests on a number of cells simultaneously and independently.
BACKGROUND ART
The general idea of electrically insulating a patch of membrane and studying the ion channels in that patch under voltage-clamp conditions was outlined by Neher, Sakmann, and Steinback in “The Extracellular Patch Clamp, A Method For Resolving Currents Through Individual Open Channels In Biological Membranes”, Pflueger Arch. 375; 219-278,1978. They found that, by pressing a pipette containing acetylcholine (ACh) against the surface of a muscle cell membrane, they could see discrete jumps in electrical current that were attributable to the opening and closing of ACh-activated ion channels. However, they were limited in their work by the fact that the resistance of the seal between the glass of the pipette and the membrane (10-50 M&OHgr;) was very small relative to the resistance of the channel (10 G&OHgr;). The electrical noise resulting from such a seal is inversely related to the resistance and was large enough to obscure the currents flowing through ion channels, the conductance of which are smaller than that of the ACh channel. It also prohibited the clamping of the voltage in the pipette to values different from that of the bath due to the large currents through the seal that would result.
It was then discovered that by fire polishing the glass pipettes and by applying suction to the interior of the pipette a seal of very high resistance (1-100 G&OHgr;) could be obtained with the surface of the cell. This Giga-seal reduced the noise by an order of magnitude to levels at which most channels of biological interest can be studied and greatly extended the voltage range over which these studies could be made. This improved seal has been termed a “giga-seal”, and the pipette has been termed a “patch pipette”. A more detailed description of the giga-seal may be found in O. P. Hamill, A. Marty, E. Neher, B. Sakmann & F. J. Sigworth: Improved patch-clamp techniques for high resolution current recordings from cells and cell-free membrane patches. Pflügers Arch. 391, 85-100, 1981. For their work in developing the patch clamp technique, Neher and Sakmann were awarded the 1991 Nobel Prize in Physiology and Medicine.
Ion channels are transmembrane proteins which catalyse transport of inorganic ions across cell membranes. The ion channels participate in processes as diverse as gener-ating and timing action potentials, synaptic transmission, secretion of hormones, contraction of muscles, etc. Many drugs exert their specific effects via modulation of ion channels. Examples are antiepileptic compounds like phenytoin and lamotrigine which block voltage-dependent Na
+
-channels in the brain, antihypertensive drugs like nifedipine and diltiazem which block voltage dependent Ca
2+
-channels in smooth muscle cells, and stimulators of insulin release like glibenclamide and tolbutamide which block an ATP-regulated K
+
-channel in the pancreas. In addition to chemically induced modulation of ion-channel activity, the patch clamp technique has enabled scientists to perform manipulations with voltage dependent channels. These techniques include adjusting the polarity of the electrode in the patch pipette and altering the saline composition to moderate the free ion levels in the bath solution.
The patch clamp technique represents a major development in biology and medicine, since this technique allows measurement of ion flow through single ion channel proteins, and also allows the study of the single ion channel responses to drugs. Briefly, in standard patch clamp technique, a thin (app. 0.5-2 &mgr;m in diameter) glass pipette is used. The tip of this patch pipette is pressed against the surface of the cell membrane. The pipette tip seals tightly to the cell and isolates a few ion channel proteins in a tiny patch of membrane. The activity of these channels can be measured individually (single channel recording) or, alternatively, the patch can be ruptured allowing measurements of the channel activity of the entire cell membrane (whole cell recording). High-conductance access to the cell interior for performing measurements can be obtained, e.g., by rupturing the membrane by applying subatmospheric pressure in the pipette.
During both single channel recording and whole-cell recording, the activity of individual channel subtypes can be characterised by imposing a “voltage clamp” across the membrane. In the voltage clamp technique the membrane current is recorded at a constant membrane potential. Or—to be more precise—the amplifier supplies exactly the current, which is necessary to keep the membrane potential at a level determined by the experimenter. Hence, currents resulting from opening and closing of ion channels are not allowed to recharge the membrane.
FIG. 1
shows a simplified diagram of the basic operation of a standard prior art voltage clamp amplifier such as the EPC-9 amplifier from HEKA Elektronik. An electrode
6
inside a pipette
4
is connected to the negative terminal of a feedback amplifier, while the clamping voltage (referred to a grounded bath electrode (
8
)) is connected to a positive terminal (from Stim. In.) and made available at a voltage monitor output. Since the measured pipette voltage and the clamp voltage are supposed to be identical, a correction potential is constantly supplied at the pipette electrode as a current forced through the large feedback resistor. After inversion, the current is made available as an analogue voltage at the Current Monitor output.
The time resolution and voltage control in such experiments are impressive, often in the msec or even &mgr;sec range. However, a major obstacle of the patch clamp technique as a general method in pharmacological screening has been the limited number of compounds that could be tested per day (typically no more than 1 or 2). Also, the very slow rate of solution change that can be accomplished around cells and patches may constitute a major obstacle.
A major limitation determining the throughput of the patch clamp technique is localisation and clamping of cells and pipette, and the nature of the feeding system, which leads the dissolved compound to cells and patches.
In usual patch clamp setups, cells are placed in experimental chambers which are continuously perfused with a physiological salt solution. The establishment of the cell-pipette connection in these chambers is time-consuming and troublesome. Compounds are applied by changing the inlet to a valve connected to a small number of feeding bottles. The required volumes of the supporting liquid and the sample to be tested are high. High throughput systems for performing patch clamp measurements have been proposed, which typically consist of a substrate with a plurality of sites adapted to hold cells in a measuring configuration where the electrical properties of the cell membrane can be determined.
U.S. Pat. No. 5,187,096, Rensselaer, discloses an apparatus for monitoring cell-substrate impedance of cells. Cells are cultured directly on the electrodes which are then covered with a plural
Bech Morten
Christophersen Palle
Due Jorgen
Hansen Ole
Olesen Soren Peter
Olsen Kaj K.
Sophion Bioscience A/S
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