Electrolysis: processes – compositions used therein – and methods – Electrolytic analysis or testing – Involving enzyme or micro-organism
Reexamination Certificate
2000-10-13
2004-07-06
Olsen, Kaj K. (Department: 1753)
Electrolysis: processes, compositions used therein, and methods
Electrolytic analysis or testing
Involving enzyme or micro-organism
C204S403010, C204S403030, C435S173400, C435S173600
Reexamination Certificate
active
06758961
ABSTRACT:
TECHNICAL AREA
The present invention concerns a measuring arrangement, as well as positioning method for cells and vesicles or lipid membranes, which permits investigations on membranes, especially an electrophysiological method for the investigation of channel-forming proteins and receptors coupled through channel-forming proteins or to channel-forming proteins, by measuring the electrical properties of the channel-forming proteins. Especially, the measurement method according to the invention concerns a (multiarray) patch-clamp method which has the sensitivity and selectivity of the classical patch-clamp technique, but, simultaneously, because of the positioning of biological cells or vesicles on microstructured carriers, also a method according to the invention, simpler preparation of the patch-membranes as well as high signal-noise ratio are achieved. Furthermore, the present invention concerns a measuring arrangement which is suitable both for the positioning as well as for the electrophysiological measurement.
STATE OF THE ART
Many biologically important signal transduction processes, such as nerve conduction, occur on or in the cell membranes. Therefore, it is not surprising that the biological functions of membrane proteins in general and of neuroreceptors in particular are influenced by pharmacologically active compounds (J.-P. Changeux (1993), “Chemical signalling in the brain”,
Sci. Am. Nov
., pages 30 and following; A. G. Gilman (1995),
Angew. Chem. Int. Ed. Engl
. 34: 1406-1428; M. Rodbell (1995),
Angew. Chem. Int. Ed. Engl
. 34: 1420-1428).
The functional understanding of the molecular interactions on receptors, as well as the use of receptors in the screening of active compounds, play a central role in modern drug development. With increasing number of the known target receptors for active ingredients and the rapidly growing number of potential active ingredients from combinatorial chemistry, the demand is increasing for highly sensitive screening methods, which permits analysis of a large number of different substances at high time throughput (“high throughput screening”=HTS).
At the present time, in pharmacological active ingredient screening, still relatively traditional avenues are followed by carrying out time-consuming ligand binding tests and receptor function tests separately (J. Hodgson (1992)
Bio/Technology
9: 973). On the other hand, membrane proteins such as the receptors coupled to G-proteins and the channel-forming receptors are considered to belong among the most important target proteins for active ingredients (J. Knowles (1997) “Medicines for the new millenium hunting down diseases”
Odyssey
, Vol. 3 (1)). In this connection, still classical patch-clamp methods are used as functional receptor tests. The advantage of this electrophysiological method lies in the fact that the function of the corresponding channel-forming receptor or receptors coupled to channel-forming proteins is directly accessible through the measured electrical properties. The method is highly specific and extremely sensitive—in principle, the channel activity of individual receptor molecules can be measured. Glass micropipettes with an opening diameter typically 1-0.1 &mgr;m are placed on the surface of a biological cell. The membrane surface which is covered by the micropipette is called “patch”. When the contact between the glass electrode and the cell membrane surface is sufficiently insulating electrically, then, with the aid of microelectrodes, which are placed on the one hand in the glass pipette and, on the other hand, in the medium opposite the membrane, the ion current through the membrane patch is measured electrically (O. P. Hamill, A. Marty, et al., (1981), “Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches”,
Pflugers Arch
391 (2): 85-100).
In connection with active ingredients screening, the traditional patch-clamp technique also has decisive disadvantages. Patch-clamp measurements are extremely time-consuming, require specially trained personnel with long experience in this field, and in practice it cannot be used for HTS.
A method and a process have become known from U.S. Pat. No. 4,055,799 for the measurement of the elastic and dielectric properties of diaphragms of living cells. The disclosed device is a container, two electrodes for measuring voltage differences, 2 electrodes for sending out voltage and current pulses, a separating wall, which divides the container into two chambers and which contains one or more holes, a connection for physiological solution and a connection for the introduction of electrolyte solution. For the measurement, the cells are partially in the hole of the separating wall, so that no planar membrane is formed above the opening. EP-A-0 094 193 and WO-A-8 502 201 also disclose the attachment of cells in holes in a carrier, where the attachment is through electrical charging of the carrier, which cannot lead to an inherently accurate positioning of cells or vesicles above a previously defined point (the aperture) with a diameter smaller than the diameter of these objects.
Therefore, the goal of the present invention was to provide a measuring and positioning method which is simple to handle and permits rapid investigation, especially for a (multiarray) patch-clamp method which has the sensitivity and selectivity of the classical patch-clamp technique, but at the same time, eliminates its disadvantages because of the method of automatic positioning of biological cells or vesicles or corresponding lipid membranes by the method according to the invention, as well as the specific surface properties of the measuring arrangement. Furthermore, the present invention concerns a planar positioning and measuring arrangement, which is especially suitable for carrying out the method according to the invention.
DESCRIPTION OF THE INVENTION
The methods according to the invention excel by extreme simplicity in the production of electrically insulating patch membranes, as well as during the subsequent measurement; in combination with modern microtechnological methods, the new technology offers all the possibilities for use in “high throughput screening” (HTS). In addition, the positioning and measuring arrangement as well as the method according to the invention are suitable for combination of electrical and optical measurements, through which new important information about the investigated receptors can be obtained with the planar membrane, and, with the aid of the positioning method according to the invention, today new important information can be obtained on the receptors to be investigated.
The positioning method according to the invention for cells and vesicles or the corresponding lipid membranes is characterized by the fact that a separating wall of electrically insulating material, called carrier below, is arranged between the two electrodes. The carrier has an aperture as well as a surface onto which the membranes are attached. The carrier must not consist of a single piece, but it can be, for example, built up of a holder, onto which the material which is actually relevant for the membrane binding and membrane positioning is attached, or embedded in this material, and that this material has an aperture for the bonding or positioning of the membrane. The attachment of the membrane can be based, for example, on electrostatic interactions between, for example, a negatively charged membrane surface and a positively charged carrier surface. In case the carrier surface as such does not have the desired charge, it can be modified correspondingly. It was shown that cells and vesicles can be positioned very well when they are introduced into the apparatus through an inlet opening of usually 0.2-2 mm diameter, preferably 0.5-1 mm in one electrode or through a tube brought near the apparatus or with the aid of a pipette, where both electrodes, arranged above and below the carrier, have such an electrical potential difference that cells or vesicles are moved electrophoretica
Schmidt Christian
Vogel Horst
Ecole Polytechnique Federale de Lausanne
Kolisch & Hartwell, P.C.
Olsen Kaj K.
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