Chemistry: molecular biology and microbiology – Apparatus – Mutation or genetic engineering apparatus
Reexamination Certificate
1999-05-27
2002-04-09
Redding, David A. (Department: 1641)
Chemistry: molecular biology and microbiology
Apparatus
Mutation or genetic engineering apparatus
C435S287200, C435S288300, C435S288700, C204S403060
Reexamination Certificate
active
06368851
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention involves a method for measuring at least one state variable of a biological cell located in a nutrient medium, the cell being supported on and adhering to a support area, wherein at least one opening is made in the membrane of the cell for measuring the state variable. The invention further involves an apparatus for measuring at least one state variable of at least one biological cell located in a nutrient medium, the apparatus including an object carrier (specimen slide) having a support area, on which the cell can be supported in an adherent manner, and at least one measuring probe that can be brought into contact with the cell liquid located inside the cell for measuring the state variable, wherein the measuring probe is connected or combined with a measurement amplifier.
From the book,
Human Physiology,
Schmidt; Thews (publisher), 23
rd
edition (1987), pages 20-21, an apparatus of this type is already known, which has a suction device connected to a hollow needle with an inner cavity that has an opening on the open end of the hollow needle. In the inner cavity of the hollow needle, a measuring probe is arranged for measuring the cell potential of the cell. With this device, operating according to the so-called patch-clamp process, the hollow needle is applied on the outside of the cell membrane in order to bring the measuring electrode into contact with the cell liquid by the opening located on the free end of the hollow needle, in order to then generate a partial vacuum in the inner cavity of the hollow needle using the suction device. With this partial vacuum a piece of the cell membrane, located in front of the opening of the hollow needle, is torn out of the membrane structure. Via the resulting opening in the cell membrane, the ions located in the cell liquid get into an electrolyte located in the hollow needle and from there to the measuring probe. A reference electrode functions for determining a reference potential.
The previously known method and associated apparatus have the disadvantage that, for positioning of the hollow needle on the cell, a micromanipulator is necessary. This results in a comparatively complicated and expensive device. Moreover, the accessibility of the cells located on the specimen slide is greatly restricted by the micromanipulator. The process and apparatus are thus suitable only for an investigation of individual, or at most for a simultaneous investigation of a small number of, cells located on the specimen slide.
From U.S. Pat. No. 4,461,304 an apparatus is further known, which has a needle-shaped tip for making an opening in a cell membrane. On the tip a plurality of sensors is arranged for neurophysiological investigations. Even with this device, for positioning of the tip on the cell, a micromanipulator is necessary.
From published European and German patent applications EP 0 689 051 A2; DE 197 12 309 A1; and EP 0 585 933 A2 and German patent DE 195 29 371 C2, apparatus are already known for measuring a cell potential, which have a specimen slide having a plurality of microelectrodes arranged in matrix form, which can be brought into connection with the outside of the membrane of a cell to be investigated. These devices make possible, however, only an extracellular measurement of the cell potential, since no opening is made in the cell membrane.
From published German patent applications DE 195 36 389 A1 and DE 195 36 384 A1 methods are already known for measuring a state variable, in which a biological component is contacted. Also with this method, an opening is not made in the biological component.
In German published patent application DE 38 16 458 A1 a microelectrode is further described, which can be used for a potentiometric or amperometric measurement in the biochemical and medical fields.
German patent DE 44 22 049 C2 discloses an ultra-microelectrode array for chemical and biochemical analyses, which has several pyramids or cone-shaped electrode tips on a substrate. According to statements of the patent document, the ultra-microelectrode array can be inserted into electrode structures for the measurement of oxygen according to Clark.
From U.S. Pat. No. 5,173,158, moreover, a process outside of the generic concept is known for generating new cells, in which cells of a first type located in a liquid are supported by a partial vacuum or hydrostatic pressure on a support area of a porous layer, such that the cells engage with a component in the pores of the porous layer. The porous layer with the cells is arranged between electrode plates bordering on the liquid to which an electric voltage is applied, which opens the cell membrane of the cells in the component that engages with the pores. Thereafter, cells of a second type are brought into the insides of the cells of the first type through these openings.
SUMMARY OF THE INVENTION
Thus, an object of the present invention is to create a method and apparatus of the type mentioned at the outset, which makes possible a simple measurement of a state variable of the cells. In particular, a costly manual positioning of a hollow needle on the cells to be investigated should be avoided.
This object is achieved with respect to the method in that the opening in the cell membrane is made within the support area of the cell and spaced from the support edge, and through this opening the state variable is measured.
In this way it is possible to arrange a poration agent or a poration tool, which is used for making the cell membrane opening, in the support area of the cells on the specimen slide so that the cell is also positioned simultaneously on the poration agent or poration tool when it is supported on the support area. In this way, a costly manual positioning of a poration tool can be omitted. Since the opening is formed in the cell membrane within the support area of the cells and at a distance from the edge of the support area, the membrane area of the cells, which surrounds the opening and is adheringly attached to the support area, seals off the opening from the nutrient liquid. The cell liquid located inside the cell is thus electrically insulated to the greatest extent possible from nutrient liquid. The measured state variable of the cells can, for example, be an ion concentration, a gas quantity, a temperature or any other desired physical, chemical or biological characteristic of a cell.
In order to measure the cell potential of the cells, the electrical voltage between the cell liquid and the nutrient medium can be measured through the opening formed in the cell membrane. Using this process, for example, electrical signals transmitted between nerve cells can be tested. Using this process, electric d.c. and/or a.c. voltage potentials, especially potentials that change quickly in time, can be measured.
In an especially advantageous embodiment of the invention, the opening can be made using electroporation of the cell membrane. For the performance of the process, for example, an electroporation-electrode can be arranged in the support area for the cell, on which the cell is supported in an adherent manner. In order to form the opening in the cell membrane, an electric voltage then only needs to be applied between the electroporation electrode and the nutrient medium, which causes an electrical current flow that opens the cell membrane. After switching off the electroporation voltage, the electric potential can be measured through the opening of the cell membrane between the cell liquid and the nutrient medium. For this purpose, optionally, the electrode used for electroporation can also be used to measure the cell potential, so that the electrode performs a double function.
In another embodiment of the invention, at least one mechanical impulse is applied in order to form the opening in the cell membrane on a portion of the cell membrane. In this process, this portion of the cell membrane detaches from the membrane structure. Optionally , an impulse sequence can also be applied with several individual impulses.
It is especially advantageou
Baumann Werner
Brischwein Martin
Ehret Ralf
Freund Ingo
Gahle Hans-Jürgen
Akin Gump Strauss Hauer & Feld L.L.P.
Micronas GmbH
Redding David A.
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