Surgery – Diagnostic testing – Measuring or detecting nonradioactive constituent of body...
Reexamination Certificate
2000-12-21
2003-03-25
Hindenburg, Max F. (Department: 3736)
Surgery
Diagnostic testing
Measuring or detecting nonradioactive constituent of body...
C435S004000, C435S007100, C435S287100, C204S400000, C204S408000, C204S403010
Reexamination Certificate
active
06539244
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to an automatic electrophysiological measuring apparatus and method using Xenopus Oocytes.
The science of inquiring into the electrical properties of living organisms is known as “electrophysiology,” which has some 200 years' history and has developed as one of the main areas of physiology. Especially in nerves and muscles, the action potential is the most essential event. As it has been revealed that the conduction of excitement is mediated by an electric current, electrophysiology has become one of the most important fields of study for physiologists.
The voltage clamp method was proposed by Cole of the United States in the 1940s. This is a method to keep the membrane potential constant all the time by using at the moment of fluctuation of the membrane potential a feedback circuit which flows an electric current in the direction of suppressing the membrane potential. This voltage clamp method made possible quantitative measurement of the relationship between the membrane potential and ion permeability, and Hodgikin, Huxley and Katz of the United Kingdom used this voltage clamp method to analyze the nature of the membrane conductance of giant axons of Loligo, and made many achievements which would constitute the basis of subsequent studies in neurobiology.
These achievements are reported by Hodgikin, A. L., Huxley, A. F. and Katz in the
Journal of Physiology
, No. 116(1952), pp. 424-44.
This method was further sophisticated by Neher and Sackman subsequently. They succeeded in measuring on a real time basis a current flowing from a live cell to a single-channel molecule. This technique, known as the patch clamp method, is described in detail by Sackman, B. and Neher, E. (eds.),
Single
-
Channel Recording
(2nd ed.), Plenum Press, New York (1995).
The central part of a hard glass capillary tube of about 1 mm in diameter is softened by heating with a heater, quickly extended in its lengthwise direction and pulled off to prepare an electrode for recording electric signals. A piece whose pulled-off tip is open and whose diameter is no greater than 1 &mgr;m is selected, filled inside with a 3M potassium chloride (KCl) solution by injection, and used as the electrode. By manually penetrating this electrode into each cell, the membrane potential of the cell can be measured.
Most of hormones and other nerve-carried substances convey information to cells via receptors known as seven transmembrane receptors or G protein-coupled receptors. The rapid progress of the Human Genome Project in recent years has resulted in a vast accumulation of information on the base sequences of genes, and it is presumed that there are many so-called “orphan” receptor genes, whose ligands are unidentified.
The ligands of seven transmembrane receptors are diverse, including hormones, signal transducing substances, cytokine and enzymes, and their molecular variety includes amines, amino acids, peptides, proteins, lipids, nucleic acids and ions. Furthermore, sensory receptors for light, smell and taste are a sort of seven transmembrane receptors, which play an important role in controlling the functions of living organisms. For this reason and because of their deep involvement in diseases, seven transmembrane receptors have been made a major target of powerful medicines. Actually, many of commercially available pharmaceuticals manifest their intended effects when combined with seven transmembrane receptors. Electrophysiometry also provides an important means for screening and determining the ligands of such orphan receptors.
Electrophysiometry is one of the few techniques available for real time measurement of the functions of membrane protein molecules, providing a central approach to receptor proteins. Therefore, electrophysiometry also is an indispensable tool for the development of pharmaceuticals, and its importance is expected to further increase in the future.
The Japanese Published Unexamined Patent Application No. Hei 11-083785 discloses a technique by which Xenopus Oocytes are caused to express histamine receptors, the response of Xenopus Oocytes to histamine is measured, and allergic reactions are detected tissue-specifically.
As stated above, electrophysiometry is indispensable for research on ion channels and the development of pharmaceuticals. However, electrophysiological experiments involve the problem of many troublesome procedures that have to be done manually. First, the worker should prepare glass microelectrodes each with a pulled-off tip whose diameter is no greater than 1 &mgr;m by heating and stretching a glass capillary tube. Furthermore, in order to penetrate the glass electrodes into a cell, a micromanipulator should be operated manually.
The micromanipulator, which is an apparatus to hold a glass electrode and manually control minute displacements of the glass electrode, involves the problem of requiring a high level skill to operate. Usually, penetration of a glass electrode into a cell is accomplished by manual operation with a micromanipulator. For this reason, since the glass electrode was devised in the 1940s until even today, electrophysiological measurement has depended heavily on the worker's craftsmanship.
A breakthrough in the automation of the penetration of Xenopus Oocytes by a glass electrode according to the prior art might be found in the application of image recognition. It is conceivable to determine the position of the membrane surface of a Xenopus Oocyte is determined with a CCD camera from above, and move the glass electrode, driven by a motor or otherwise, to penetrate the oocyte. However, when it is penetrated by the glass electrode, as the membrane surface of the Xenopus Oocyte would be subject to elastic deformation, it would be extremely difficult to check by image recognition from above whether or not the membrane has been accurately penetrated by the glass electrode. Moreover, control of the penetration of the glass electrode by image recognition is an extremely expensive and accordingly unrealistic means of control.
Since current variation responses in an electrophysiological experiment using Xenopus Oocytes or cultured cells may greatly fluctuate from cell to cell, it is necessary to increase the reliability of the data thereby obtained by averaging current responses from many cells. Therefore, in order to obtain fully reliable data, in the electrophysiological experiments each worker should carry out the penetration of a glass electrode into cells many times, resulting in the problem that acquisition of reliable data has to take a long time and a great amount of labor.
SUMMARY OF THE INVENTION
An object of the present invention, therefore, is to provide an automatic electrophysiological measuring apparatus and method using Xenopus Oocytes for automatically measuring responses of the cells to the administration of a medicine according to electric signals from a glass electrode penetrating the membranes of the Xenopus Oocytes membrane whose potential is fixed.
The automatic electrophysiological measuring apparatus according to the invention automatically carries out electrophysiological measurement regarding Xenopus Oocytes held in each of a plurality of cells (e.g. 8×12=96 cells) of a container in which a grounding electrode is arranged or formed by holding and moving the container to position the Xenopus Oocyte in each cell by shifting an XY stage, penetrating the Xenopus Oocyte in each cell with one or two glass electrodes with an inserting means, detecting electric signals emitted from the glass electrodes) with a detecting means, fixing the membrane potential of the Xenopus Oocytes to a prescribed value with a fixing means, and administering a chemical substance to the Xenopus Oocytes with a microsyringe.
In order to accomplish electrophysiological measurement automatically, the XY stage, inserting means, fixing means and microsyringe are controlled by a control means. The control means detects and distinguishes the contact of the glass electrode(s) with the solution
Kato Hirokazu
Matsunami Shokichi
Moriya Noboru
Nomura Sayuri
Otomo Jun
Hindenburg Max F.
Hitachi , Ltd.
Natnithithadha Navin
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