Chemistry: molecular biology and microbiology – Apparatus – Mutation or genetic engineering apparatus
Reexamination Certificate
2001-02-16
2004-11-09
Beisner, William H. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Mutation or genetic engineering apparatus
C435S285200, C435S287100, C435S286200, C435S288400
Reexamination Certificate
active
06815197
ABSTRACT:
The invention is related to an apparatus for conducting electrophysiological measurements on cells comprising a measuring head, the measuring head being provided with at least one electrode for impaling the cells.
An apparatus of the afore-mentioned type is disclosed in U.S. Pat. No. 6,048,722.
The prior art apparatus is used for conducting electrophysiological measurements on oocytes, in particular oocytes of Xenopus laevis, a South African claw frog. These oocytes are preferably used as expression systems for electrophysiological measurements. For that purpose the oocyte is placed and immobilized in a receptacle. The receptacle may, for example, be a funnel-type opening within a plate. Such plates are standardized and are conventionally used with 8×12=96, 16×24=384 or 32×48=1.536 receptacles (“wells”). The receptacles for the oocytes may, for example, be provided with an opening at their bottom side, enabling to attract the oocyte by means of vacuum and to immobilize same within the receptacle.
For conducting electrophysiological measurements a genetic information, namely a mRNA or a cDNA is injected into the oocyte. As a consequence, characteristic ion channels and/or receptors are incorporated on the surface and within the oocyte which may be measured by applying an electrical voltage or directing an electrical current therethrough or by application of a substance.
It is well known to conduct pharmacological measurements in this manner because the receptors or ion channels developed in the membrane of the oocytes are configured in a kind that is characteristic for certain properties of the substances under investigation.
Within the apparatus according to U.S. Pat. No. 6,048,722 mentioned at the outset, a measuring electrode and a reference electrode are impaled into the cell. In addition a perfusion apparatus is connected to the receptacle of the cell. By means of this perfusion apparatus various substances, in particular measuring substances, may be filled into the receptacle in a controlled manner, the receptacle having typically a volume of 100 &mgr;l.
Although this prior art apparatus is automatized for what concerns the supply of perfusion agents, it requires a considerable manual talent to bring the perfusion conduit into the area of the receptacle for the cell and to immobilize same at that location. Above all, the application of the electrodes to the cells, in particular impaling the electrodes into the cell, depends on the manual skill of the particular person conducting the experiment because these steps obviously have to be made manually. In case the application of the electrode or electrodes to the cell is unsuccessful, in particular, if the electrodes brake off, the entire experimental set up must be wholly re-assembled and re-adjusted. Finally, the prior art apparatus only allows to conduct individual measurements on one cell wherein only the sequence of different test fluids may be controlled automatically in a manner mentioned above.
It is, therefore, an object underlying the invention to improve an apparatus of the type specified at the outset such that the above-discussed disadvantages are avoided.
In particular it shall become possible to fully automatically conduct the experiments as well with respect to application of the electrodes as well as with respect to the application of the perfusion conduits. Further, the apparatus shall be adapted to be made ready for use with only a couple of manual operations according to the “plug-and play” principle and it shall be possible with the same principle that the apparatus can be put back to operation after a damage has happened.
Finally it shall become possible to execute a plurality of measurements on many different cells in a fully automatized mode, i.e. without supervision, in particular over night.
According to the apparatus specified at the outset this object is achieved by the present invention in that the electrodes are integrated into a common support.
This measure has the advantage that the carrier may be prefabricated on an industrial level and must, hence, only be plugged on a corresponding holder within the apparatus. The electrodes are already installed ready for operation, in particular with respect to their position relative to one another so that in contrast to conventional apparatuses there is no more need to delicately adjusting and orienting the electrodes relative to one another. By doing so the risk of damaging the electrodes during the setup of the apparatus is drastically minimized. Further, the measurements are clearly reproducible, because the electrodes are in a predetermined position relative to one another. Finally the integrated arrangement of the electrodes on the support allows that automatic displacement units for the measuring head thus obtained may be made, enabling to conduct the desired fully automatic measurements on a plurality of cells, for example in connection with a standardized multiwell-plate.
In preferred embodiments of the inventive apparatus the electrodes are inserted into recesses of the carrier, however, they may also be molded within the carrier.
This measure has the advantage that a stable and reproducible position of the electrodes within the carrier is achieved.
In other embodiments of the invention the electrodes consist of pulled glass tubes.
This measurement has the advantage that such glass electrodes may be configurated optimally for the specific application, as known per se. For example, it is possible to configurate the electrodes to have an electrical resistance of between 5 M&OHgr; and 100 M&OHgr;, i.e. as so-called “sharp electrodes”. As an alternative, the electrodes may be configurated as so-called “patch electrodes” having an electrical resistance of the order of 500 k&OHgr; through 5 M&OHgr;.
As an alternative, the invention may provide to configurate the electrodes as wire electrodes, preferably as silver wire electrodes which, further preferably, are surrounded by a chloride coating. Besides that also tungsten wires or the like may be used.
In preferred embodiments of the inventive apparatus at least one electrode is provided with a straight section.
This measure has the advantage that the electrode may particularly easily be attached to the support.
In another preferred embodiment of the invention two electrodes are arranged essentially symmetrical relative to a longitudinal axis of the carrier so that the distance between the free ends of the electrodes are in a range of between about 50 &mgr;m and 800 &mgr;pm, preferably between 200 &mgr;m and 500 &mgr;m.
This measure has the advantage that during the introduction of the electrodes into the cells individual impaling holes are generated and not a common big hole which would be the case if the free ends of the electrodes would be arranged too close to one another. This would have the disadvantage that important physiological functions of the cells would go lost. With the inventive approach this is avoided.
In this connection it is particularly preferred when a straight section of the electrodes encloses an acute angle with a longitudinal axis of the support, the angle being in particular between 3° and 10°, preferably 5°.
The inclined orientation of the electrodes with respect to one another has the advantage that the lower free ends of the electrodes may be positioned in a perfectly reproducible manner.
In a preferred group of embodiments of the invention at least one electrode is configured as a measuring electrode. The measuring electrode is preferably connected to a measuring amplifier which, further preferably, is adapted to be adjusted.
By doing so it is possible to conduct automatically con-trolled measurements of voltage and current signals.
This holds true in particular if the at least one measuring electrode is connected to a current source and the current source, in turn, is adapted to be adjusted.
This measure has the-advantage that if two different electrodes are used, the feeding of current on the one hand and the measuring of voltage on
Boven Karl-Heinz
Möller Andreas
Beisner William H.
Multi Channel System MCS GmbH
Weingarten Schurgin, Gagnebin & Lebovici LLP
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