JP3799998B2 - Electrophysiological automatic measuring device and electrophysiological automatic measuring method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an automatic electrophysiology measurement apparatus and an electrophysiology automatic measurement method using an oocyte.
[0002]
[Prior art]
Science related to the electrical properties of living organisms has a history of about 200 years as “electrophysiology” and has developed as one of the major fields in physiology. Especially in nerves and muscles, action potential is the most essential phenomenon of excitement, and it has been clarified that excitatory conduction is mediated by electric current, and electrophysiology has become the most important research field.
[0003]
The voltage clamp method was proposed by Cole, USA in the 1940s. This potential fixing method is a method in which the membrane potential is always kept constant by using a feedback circuit that allows current to flow in the direction of suppressing the membrane potential at the moment when the membrane potential changes. This potential fixation method enables quantitative measurement of the relationship between membrane potential and ion permeability. The UK's Hodgkinkin, Huxley and Katz et al. Use the potential fixation method to characterize the membrane conductance of the squid giant nerve. And has made many achievements that will serve as the basis for subsequent neurobiological research.
[0004]
These results are described in Journal of Physiology, 116 (1952), pages 424 to 448 (Hodgikin, A, L., Huxley, A.F. and Katz, B., J. Physiol. 116 (1952), pp. 424-448).
[0005]
The method was then further refined by Neher and Sackman. They succeeded in measuring the current flowing from a living cell to a single channel molecule in real time. This technique is called the patch clamp method. The technique of the patch clamp method is described by Sackman et al. In “Single Channel Recording” (1995) (Sackman, B., Neher, E. (eds.): Single-Channel Recording (2nd ed.), Plenum Press, New. (York 1995)).
[0006]
As an electrode for recording an electrical signal, a central portion of a hard glass capillary tube having a diameter of about 1 mm is softened by heating with a heater, and it is rapidly stretched in the long axis direction and prepared. Select the one with a stretched tip open and a diameter of 1 μm or less, fill the capillary tube with a 3M potassium chloride (KCl) solution, and manually puncture the cell as an electrode. Thus, it is possible to measure the membrane potential.
[0007]
Many hormones and neurotransmitters transmit information to cells via receptors called 7-transmembrane receptors or G protein-coupled receptors. In recent years, the human genome project has advanced rapidly, and a large amount of information on the base sequence of genes has been accumulated. It is estimated that there are many so-called “orphan” receptor genes whose ligands are unknown.
[0008]
The seven-transmembrane receptor ligands are diverse, including hormones, signal transmitters, cytokines, and enzymes, and molecular species include amines, amino acids, peptides, proteins, lipids, nucleic acids, ions, and the like. Furthermore, sensory receptors for light, smell, taste, and the like are a kind of seven-transmembrane receptors and play an important role in the regulation of biological functions. For this reason, 7-transmembrane receptors have often been an important target for drug discovery because they are often deeply involved in diseases. In fact, many commercially available drugs exhibit a medicinal effect by binding to a 7-transmembrane receptor. Electrophysiology is also important as a means of screening and determining such orphan receptor ligands.
[0009]
Electrophysiology is one of the few methods that can measure the function of membrane protein molecules in real time, and is a central research method for receptor proteins. Therefore, electrophysiology is an indispensable technology in drug development, and its importance will increase in the future.
[0010]
JP-A-11-083785 describes a technique for expressing an histamine receptor in an oocyte, measuring the response of the oocyte to histamine, and detecting an allergic reaction in a tissue-specific manner.
[0011]
[Problems to be solved by the invention]
As mentioned above, electrophysiology is indispensable for ion channel research and drug development. However, in order to conduct electrophysiological experiments, there is a problem that many complicated manual operations are required. First, the measurer must heat and stretch a thin glass tube and manually create a glass microelectrode with a tip diameter of 1 μm or less. Furthermore, in order to insert the glass electrode into the cell, it is necessary to manually operate the micromanipulator.
[0012]
A micromanipulator is a device that holds a glass electrode and can manually adjust a small amount of displacement of the glass electrode, but there is a problem that a skilled technique is required for the operation. In general, a micromanipulator is manually used to insert a glass electrode into a cell. For this reason, from the 1940's when the glass electrode was devised to the present, measurements related to electrophysiology have relied heavily on the manual work of the measurer.
[0013]
When automating insertion of glass electrodes into oocytes using existing technology, application of image recognition can be considered. It is conceivable that the position of the oocyte membrane surface is measured by a CCD camera from above, and the glass electrode is driven and moved by a motor or the like. However, since the membrane surface of the oocyte is elastically deformed when the glass electrode is inserted, it is extremely difficult to determine whether the glass electrode has been correctly inserted into the membrane by image recognition from above. Further, glass electrode insertion control by image recognition is extremely expensive and cannot be said to be a realistic control means.
[0014]
Since the current change response in electrophysiological experiments using oocytes or cultured cells varies greatly from cell to cell, it is necessary to increase the reliability of the data by averaging the current responses from many cells. Therefore, in order to obtain sufficiently reliable data, it is necessary for each measurer to conduct electrophysiological experiments in which a glass electrode is manually inserted into a cell many times. Therefore, there is a problem that it takes a long time and much labor to obtain reliable data.
[0015]
An object of the present invention is to automatically measure electrophysiology using an oocyte that inserts a glass electrode into an oocyte membrane based on an electrical signal from the glass electrode, automatically measures the reaction when the membrane potential is fixed and a drug is administered. It is to provide an apparatus and an electrophysiological automatic measurement method.
[0016]
[Means for Solving the Problems]
In the electrophysiology automatic measuring device of the present invention, an oocyte is held in each cell of a container in which a plurality of cells (for example, 8 × 12 = 96) in which earth electrodes are arranged or formed are regularly arranged and molded. Then, the container is held and moved to position the oocyte in the predetermined cell by moving the XY stage, and one or two glass electrodes are inserted into the oocyte in the predetermined cell by the inserting means. Then, the electric signal emitted from the glass electrode is detected by the detecting means, the membrane potential of the oocyte is fixed to a predetermined value by the fixing means, the chemical substance is administered to the oocyte by the microsyringe, and the oocyte The electrophysiological measurement is automatically performed.
[0017]
In order to automatically perform electrophysiological measurement, the XY stage, insertion means, fixing means, and microsyringe are controlled by the control means. The control means detects and identifies the contact of the glass electrode with the solution surface in the cell, the contact of the oocyte with the membrane surface, and the insertion of the oocyte into the membrane based on the electrical signal emitted from the glass electrode. It controls the insertion of glass electrodes into the oocytes and controls the administration of chemical substances to the oocytes using a microsyringe, making it possible to automate the automatic electrophysiology measurement device. That is, automatic measurement is performed by measuring the resistance of the glass electrode, adjusting the zero potential of the glass electrode, vibrating the glass electrode, fixing the membrane potential, and controlling the administration of the chemical substance.
[0018]
Each cell has a shape having a hemisphere at the tip of the cone, and efficiently holds the oocyte in each cell.
[0019]
More preferably, the electrophysiology automatic measuring device is shielded from electromagnetic waves by the shielding means. Further, an optical microscope for optically monitoring the oocyte held in the cell with the naked eye may be arranged.
[0020]
The ground electrode is formed of a conductive metal, the surface is coated with silver chloride, and a ground wire is individually attached to the cell. A plurality of cells can be uniformly grounded by molding the container with agar and bringing a ground electrode into contact with a part of the agar. Alternatively, the ground electrode may be moved within the solution held in the cell on one axis and grounded independently for each cell.
[0021]
More specifically, an oocyte holding plate in which 8 × 12 = 96 oocytes are regularly arranged in each cell is mounted on the XY stage. A ground electrode is independently added to each cell holding one oocyte. Two glass electrodes are automatically inserted into one oocyte positioned by an XY stage in the center of the field of view of a stereomicroscope by movement driven by a motor. The XY stage, the motor that drives the glass electrode, the motor that drives the micro-syringe for administering the ligand, and the electrophysiological measurement device are collectively controlled from the outside by the control computer based on the electrical signal from the glass electrode, and multiple eggs Electrophysiological measurement of mother cells can be performed automatically. The measured current response of the oocyte is recorded by a measurement recording computer.
[0022]
In the electrophysiology automatic measurement method of the present invention, a predetermined cell is obtained by holding an oocyte, holding a container having a plurality of cells on which an earth electrode is disposed or formed on an XY stage, and moving the XY stage. The oocyte in the cell is positioned, one or two glass electrodes are inserted into the oocyte in a given cell, the electrical signal emitted from the glass electrode is detected, and the membrane potential of the oocyte is determined. It is fixed at a predetermined value, a chemical substance is administered to the oocyte by a microsyringe, and electrophysiological measurement of the oocyte is automatically performed. In order to perform electrophysiology measurement automatically, control of movement of XY stage, control of insertion of glass electrode into oocyte, control of fixation of membrane potential of oocyte, administration of chemical substance to oocyte Each control is performed based on an electrical signal emitted from the glass electrode. Each of the above controls is performed by detecting and identifying the contact of the glass electrode with the solution surface in the cell, the contact of the oocyte with the membrane surface, and the insertion of the oocyte into the membrane. Enables automation.
[0023]
The present invention provides an automatic electrophysiology measuring apparatus that automatically inserts a glass electrode into an oocyte membrane to maintain a membrane potential and automatically measures a reaction when a drug is administered. Figure 5 Hereinafter, the principle of the present invention will be described. First, the glass electrode 103 in the air is moved toward the oocyte 601 held in the cell. When the glass electrode 103 lands on the solution surface 603 and the earth electrode 604 is energized, the potential of the glass electrode shows around 0 mV. When the glass electrode 103 is moved and the glass electrode 103 contacts the membrane surface of the oocyte 601, a potential of about −5 mV is exhibited. Furthermore, when the glass electrode 102 is moved and the glass electrode 102 is inserted into the film, a potential of about −20 mV is exhibited. The above-described potential state of the glass electrode is captured by a control computer, the relative positions of the glass electrode 102 and the oocyte 601 are identified, the glass electrode 102 is inserted into the oocyte 601, the membrane potential is maintained, and a drug is administered. Automate a series of electrophysiological measurements.
[0024]
In the electrophysiology automatic measurement apparatus using the oocyte of the present invention, it is possible to improve the reliability of data by automating electrophysiology measurement, reducing the burden on the measurer, and increasing the number of measurements. Moreover, it is possible to speed up the measurement of a large number of samples by automation and to process a large number of electrophysiological measurements in a short time. Furthermore, it is possible to screen the function of a gene whose base sequence is known but whose function is unknown.
[0025]
DETAILED DESCRIPTION OF THE INVENTION
FIG. 1 is a diagram showing a configuration example of an electrophysiology automatic measuring apparatus according to an embodiment of the present invention. In the electrophysiology automatic measurement apparatus according to the embodiment of the present invention, a plurality of cells are continuously and automatically measured using an electrophysiology method.
[0026]
As shown in FIG. 1, the automatic electrophysiology measuring apparatus of the present invention includes a stereomicroscope 116 for observing an oocyte; glass electrodes 102 and 103 inserted into the oocyte; a micro electric signal amplifier circuit for amplifying a micro electric signal. Part 122, 123; microsyringe 126 for administering ligand to the oocyte; XY stage 101 for holding the stereomicroscope 116 and the glass electrodes 102, 103; 96 (cell) holes for holding the oocyte 96-well oocyte holding plate 111; earth electrode 124 for 96-well plate; electrophysiological measurement circuit unit 109 for measuring an electric signal indicating the potential state and resistance state of glass electrodes 102, 103; XY stage 101, and glass A motor drive controller 107 for controlling the drive of the motor for moving the electrodes 102 and 103; Measuring records the measurement result recording computer 121; electrophysiological measurement circuit section 109, and a control computer 108 for controlling the motor driving controller 107 and the measurement recording computer 121,.
[0027]
The stereomicroscope 116 is arranged on the XY stage 101 so that its optical axis is vertical. The glass electrodes 102 and 103 respectively driven by the motors 104 and 105 are arranged symmetrically at a position inclined by 30 degrees from the vertical axis on which the stereomicroscope 116 is arranged. In order to reduce noise and conduct electrophysiological experiments efficiently, the entire device is covered with a shield to shield electromagnetic waves in the air.
[0028]
In this example, an oocyte that has been cultured for several days after injection of the human histamine receptor gene is used. These oocytes are held on a 96-well oocyte holding plate 111. 96 oocytes expressing the human histamine receptor are regularly arranged on an 8 × 12 = 96 matrix.
[0029]
The positions of the glass electrodes 102 and 103 are adjusted so that they can be inserted into one oocyte in the cell positioned at the center of the visual field of the stereomicroscope 116.
[0030]
Under the control of the control computer 108, the glass electrode driving motors 104 and 105 are driven on one axis (indicated by the arrows shown in FIG. 1), and the oocytes positioned in the center of the visual field of the stereomicroscope 116 are moved to the glass electrode 102. , 103 can be inserted.
[0031]
The process of inserting the glass electrodes 102 and 103 into the cell membrane can be observed with the naked eye by the stereomicroscope 116. It is also possible to add a CCD camera to the stereomicroscope 116, monitor the insertion state of the glass electrodes 102 and 103 into the oocyte, and control the movement of the glass electrodes 102 and 103 from image processing.
[0032]
Note that the stereomicroscope 116 is not necessarily an essential configuration for inserting the glass electrodes 102 and 103 into the oocyte, but only for visually confirming the operation of the glass electrodes 102 and 103. Therefore, the stereomicroscope 116 can be removed from the apparatus by increasing the accuracy of the moving mechanism of the glass electrodes 102 and 103 and making the glass electrodes 102 and 103 insertable into the oocyte without visual confirmation. Is possible.
[0033]
Since the ligand administration microsyringe 126 is held in the minute electric signal amplification circuit unit 122 through the support 127, it can be moved together with the glass electrode 102 by the glass electrode driving motor 105. When the glass electrode 102 is inserted into the oocyte membrane, the tip of the ligand administration microsyringe 126 is adjusted to a position sufficiently close to the vicinity of the membrane surface of the oocyte, so that histamine can be administered efficiently.
[0034]
The electrophysiological measurement circuit unit 109 measures electrical signals indicating the potential state and resistance state of the glass electrodes 102 and 103. An electrical signal indicating that the glass electrodes 102 and 103 are inserted into the cell membrane is taken in by the electrophysiological measurement circuit unit 109, and the control computer 108, which has identified this, fixes the membrane potential to −60 mV in the electrophysiological measurement circuit unit 109. Signal is sent back, and the membrane potential of the oocyte is fixed.
[0035]
After confirming that the membrane potential of the cell is fixed, the control computer 108 drives the motor 125 for driving the microsyringe so that the histamine that is a ligand for the histamine receptor expressed on the membrane surface of the oocyte is the microsyringe. 126.
[0036]
The receptors that can be expressed on the membrane surface of the oocyte are not limited to histamine receptors, and various receptors can be expressed. The histamine receptor present on the membrane surface of the oocyte is activated by the binding of histamine, a series of signal transduction proceeds, and finally chloride ions flow out of the oocyte chloride channel.
[0037]
The measurement recording computer 121 captures and records this chloride ion flow as a current change. After the measurement of one cell is completed, the control computer 108 releases the fixation of the membrane potential through the electrophysiological measurement circuit unit 109, moves the glass electrode driving motors 104 and 105 to the initial position, and 2 The glass electrodes 102 and 103 of the book are extracted from the oocyte. Next, the XY stage 101 is moved by the interval between adjacent cells, and the oocyte for the next measurement is positioned in the center of the field of view of the stereomicroscope 116. By repeating the above operations, it is possible to automate electrophysiological experiments efficiently and continuously for a plurality of oocytes. In general, in an electrophysiological measurement method using an oocyte, a two-electrode membrane potential fixing method using two glass electrodes 102 and 103 is widely used. On the other hand, the one-electrode membrane potential fixing method using only one glass electrode is widely used for neurons smaller than oocytes. This is because the neuron measures the amount of ionic current at the picoampere level, whereas in the case of an oocyte, the cell body is large, and thus a microampere current is generated. This is because it is difficult to use the fixing method as it is. However, in principle, the one-electrode membrane potential fixing method can be applied to oocyte measurement by designing an electrical circuit that can measure the current of several microamperes, which is expected to occur in an oocyte. Is possible. This simplifies the insertion of a plurality of glass electrodes in the two-electrode membrane potential fixing method, and can reduce the occurrence of errors associated with the insertion of the glass electrodes. In addition, in order to simplify the measurement procedure as well, the 1-electrode membrane potential fixation method can increase the number of oocytes that can be measured per unit time compared to the 2-electrode membrane potential fixation method. , Throughput is improved. There is also an advantage that the manufacturing cost of the apparatus can be reduced.
[0038]
In addition, a microinjection method in which a target gene is manually injected into a cell using a glass microtubule has been one of the mainstream methods for gene introduction into a cell. By applying a method of inserting cells into a cell and adding a mechanism to inject a certain amount of gene into the cell, a glass microtubule is automatically inserted into each cell of a plurality of cells, and then a desired amount of gene is injected. Can be made.
[0039]
FIG. 2 is a diagram illustrating a configuration example of an automatic electrophysiology measuring apparatus that performs allergy diagnosis according to an embodiment of the present invention. A plurality of 96-hole oocyte holding plates 111 are conveyed by a belt conveyor 809 and mounted on a plate stacker 803. By using the plate stacker 803, a plurality of 96-hole oocyte holding plates 111 conveyed to the belt conveyor by 809 are efficiently arranged, and the 96-hole oocyte holding plate 111 is smoothly placed on the XY stage 101. Can be sent sequentially. The histamine receptor is already expressed in the oocytes on each plate.
[0040]
Specific test items for allergy diagnosis include cedar pollen, house dust, house dust mites and the like. Allergen is added to blood collected from a specimen in advance, and the one incubated for 30 to 90 minutes is centrifuged to collect plasma components. This pre-processing is performed in advance. Plasma components from each of these specimens are placed in a reaction container 805 in the reaction disk 806.
[0041]
The plasma component in the reaction container 805 is collected in the ligand administration microsyringe 126, and the microsyringe 126 is rotated by the motor 810 to administer the plasma component to the oocyte held in the 96-well oocyte holding cell 111. To do. Thereby, it is possible to test about many items about the blood collected from many samples. The above operation is integrated by the control computer 108 '. The control computer 108 'controls the motor drive controller 107 and the electrophysiological measurement circuit unit 109 shown in FIG. 1, which are not shown in FIG. 2, and also serves as the measurement recording computer 121 shown in FIG. Data acquisition and management are performed collectively.
[0042]
FIG. 3 is a plan view showing a configuration example of a 96-hole oocyte holding plate used in the embodiment of the present invention, and FIG. 4 is a cross-sectional view of the 96-hole oocyte holding plate shown in FIG. is there. The oocyte holding plate 111 has 8 × 12 = 96 independent cells 202. The cell 202 is open upward at an angle of 90 degrees, and has a hemispherical portion 203 at the lower portion of the cell. Note that the number of cells on the plate need not be limited to 96, and can be expanded to 384,864,1536 cells depending on the size of the cells used in the electrophysiological experiment.
[0043]
Each of the cells 202 holds and can hold one oocyte. The oocyte holding plate 111 has the same size and hole (cell) position as a commercially available 96-well titer plate. The size of the oocyte holding plate 111 is 85 mm long, 127 mm wide, and 10 mm thick. The diameter of the cells 202 is 9 mm, and the interval between the cells 202 is 9.5 mm.
[0044]
The cell 202 can hold the oocyte in the hemispherical part 203 at the lower part of the cell. If the diameter of the hemispherical portion 203 is too large, the oocytes are not stably held in the cell 202 when the glass electrodes 102 and 103 are inserted, and thus move, so that the glass electrodes 102 and 103 are stably held. Cannot insert into. On the other hand, if the diameter of the hemispherical portion 203 is too small, the oocyte cannot fully enter the hemispherical portion 203, and thus the oocyte cannot be stably held. When the two glass electrodes 102 and 103 are inserted into the oocyte, the optimum diameter for stably holding the oocyte in the hemispherical portion 203 is 1.2 mm ± 0.1 mm.
[0045]
In addition, when the insertion of the glass electrodes 102 and 103 into the oocytes is visually observed using the stereomicroscope 116, the glass electrodes 102 and 103 are inserted into the oocytes from the vertical direction of the oocyte holding plate 111. I can't. In this case, the glass electrodes 102 and 103 need to hold a fixed angle with respect to the vertical axis from the oocyte holding plate 111.
[0046]
FIG. 5 is a diagram showing the insertion control of the glass electrode into the oocyte in the embodiment of the present invention. In the present invention, control for inserting the glass electrode into the oocyte is performed based on the potential signal of the glass electrode.
[0047]
An oocyte 601 is held in one cell of the oocyte holding plate. The oocyte 601 is located on the axis on which the glass electrodes 102 and 103 move. The ligand administration syringe 126 is adjusted and installed at a position slightly retracted from the glass electrode 103 (therefore, not shown in FIG. 5). The graph shown in FIG. 5 shows the transition of the potential of the glass electrodes 102 and 103 changing with time, and shows different potential states corresponding to different positions of the glass electrodes shown in (a) to (e).
[0048]
As shown in (a), the glass electrode 102 is positioned above the solution surface 603 and is not energized with the ground electrode 604, and therefore the potential is infinite. The control computer recognizes that the glass electrode 102 is in the air because the potential is infinite.
[0049]
As shown in (b), when the glass electrode 102 that has started moving toward the oocyte 601 reaches the solution surface 603, the glass electrode 102 and the ground electrode 604 come into contact with each other through the solution 603 and are energized. Around 0 ± 3 mV. The control computer that recognizes the arrival of the glass electrode 102 on the solution surface 603 measures and determines whether or not the resistance of the glass electrode 102 falls within the allowable range of 0.8 MΩ to 2.4 MΩ. If the resistance of the glass electrode 102 does not fall within the allowable value, the glass electrode 102 is pulled up by the motor and the glass electrode 102 is replaced. Furthermore, the control computer automatically removes the offset potential of about ± 3 mV and adjusts the potential to 0 mV.
[0050]
As shown in (c), the control computer moves the glass electrode 102 toward the oocyte 601 by driving the motor. When the glass electrode 102 contacts the oocyte 601, the oocyte is elastically deformed. At this time, the potential of the glass electrode further drops by about 5 mV. The control computer detects this potential drop and recognizes that the glass electrode 102 is in contact with the membrane surface of the oocyte 601.
[0051]
As shown in (d), the control computer vibrates the glass electrode 102 on the drive shaft of the motor while pressure is applied to the membrane surface of the oocyte 102. Thereby, it is possible to smoothly insert the glass electrode 102 into the membrane without damaging the oocyte 601. Since the membrane potential of the normal oocyte 102 is about −20 mV, the control computer recognizes that the glass electrode 102 has been inserted into the oocyte 601 by detecting a potential of about −20 mV.
[0052]
As shown in (e), the glass electrode 103 is also inserted into the oocyte 601 membrane by repeating the operations (a) to (d). After the two glass electrodes 102 and 103 are inserted into the oocyte 601, the control computer sends a signal for fixing the membrane potential to −60 mV to the electrophysiological measurement circuit unit 109, thereby setting the membrane potential to −60 mV. Fix it.
[0053]
As described above in detail, in this embodiment, the insertion of the two glass electrodes 102 and 103 into the oocyte 601 can be performed using only the electrical signal obtained from the glass electrodes 102 and 103, and histamine administration is further performed. It is possible to detect a current change in response to.
[0054]
Next, three methods of grounding when performing electrophysiological measurement using the 96-hole oocyte holding plate 111 will be described below.
[0055]
6 is a plan view showing the shape of a 96-hole plate ground electrode used in the embodiment of the present invention, and FIG. 7 is a partial perspective view of the 96-hole oocyte plate ground electrode shown in FIG. . FIG. 6 shows a 96-hole plate ground electrode 301 to be combined with the 96-hole oocyte holding plate 111. The material of the ground electrode 301 for the 96-hole plate is copper or aluminum, and silver plating is applied after it is formed. Further, the 96-well plate ground electrode 301 plated with silver is immersed in a 150 mM sodium chloride solution and left to stand for 24 hours or more in a state where a direct current of about 1.5 V flows, so that silver and chloride ions are removed. A silver chloride film is formed on the surface of the 96-hole plate ground electrode 301 by reaction. The main component of extracellular fluid in the body is NaCl, and if there is a chemical substance (in this case, silver chloride) that has common ions between the silver electrode and the NaCl solution, the generation of polarization voltage can be prevented and measured. The above electrical noise can be reduced.
[0056]
FIG. 7 is a diagram of the 96-hole plate ground electrode 301 as viewed obliquely from above. The 96-hole plate ground electrode 301 has a ground portion 302 that extends independently of each cell 202, and the ground portion 302 is folded along a side surface of the cell 202 at an angle of 45 degrees. For this reason, it is possible to ground all 96 cells 202 by grounding the 96-hole plate ground electrode 301 at one location. In addition to the above method, the ground electrode can be directly patterned on the 96-hole oocyte holding plate 111 to be grounded.
[0057]
FIG. 8 is a diagram showing a method of creating a 96-hole oocyte holding plate made of agar used in the embodiment of the present invention and grounding. In the method described below, an oocyte holding plate 405 made of agar is prepared and grounded. Agar 402 that has been heated and made into sol in advance is poured into the container 403. Next, a 96-well oocyte holding cell mold 401 is immersed in a sol agar 402 and fixed. After standing for a certain period of time, the sol agar 402 is cooled to room temperature and gelled. When the mold 401 is removed from the gelled agar 404, the gelled agar 404 is molded into a shape capable of holding an oocyte, and a 96-well oocyte holding cell 405 made of agar can be created. Since the gelled agar 404 contains an aqueous solution sufficient for energization in the gel, it is possible to ground the 96 individual cells at once by arranging the ground electrode 406 at any location on the agar. is there. Therefore, by using the 96-well oocyte holding cell 405 made of agar, electrophysiological measurement can be easily and continuously performed without adding a complicated ground electrode. Disposable agar containers are much cheaper than plastic containers.
[0058]
FIG. 9 is a diagram showing a configuration example in which each cell is grounded by moving the ground electrode in the embodiment of the present invention. As shown in FIG. 9, the earth electrode is moved along with the glass electrode, whereby the earth is taken in the electrophysiological measurement in each cell. The ground electrode 503 is fixed through the support 502 near the tip of the glass electrode 103. The ligand administration microsyringe 126 is fixed to the glass electrode 102 while maintaining an offset.
[0059]
When the glass electrode 103 is moved toward the oocyte 601, the ground electrode 503 comes into contact with the solution surface 603. When the ground electrode 503 contacts the solution 603, the glass electrode 103 is energized and the offset potential is removed. Similarly, the glass electrode 102 can be grounded. By attaching the ground electrode 503 to the glass electrode 103, it is possible to stably ground a plurality of cells without adding a complicated ground electrode. Also, instead of attaching the ground electrode 503 to the glass electrode 103, a ground electrode driving motor for newly moving the ground electrode 503 on one axis is prepared. This solves the problem that measurement errors are likely to occur because the two glass electrodes, ground electrode, and syringe for ligand administration on the membrane surface of the oocyte during histamine administration are very close together. It is also possible to ground in a stable state.
[0060]
FIG. 10 is experimental data showing a reaction when histamine is administered to an oocyte using the electrophysiology automatic measuring apparatus according to the embodiment of the present invention. FIG. 10 shows an example of the result of performing automatic measurement after administration of histamine by fully fixing the membrane potential to an oocyte expressing a histamine receptor. The membrane potential of the cell is fixed at the time indicated by the arrow (e) through the state of the different positions (a) to (e) of the glass electrode described in FIG. 5, and 1 μM at the time indicated by the arrow (f). This is a result of automatically measuring the current response emitted from the oocyte by administering 5 μL (liter) of histamine.
[0061]
In the measurement shown in FIG. 10, it takes about 180 seconds to measure one oocyte. However, the measurement time can be greatly shortened by speeding up the movement of the glass electrode and the stage, and by inserting two glass electrodes into the oocyte at the same time. Further, in the example shown in FIG. 10, confirmation of the membrane potential is confirmed in 20 seconds, but this can be omitted. Furthermore, if only the histamine concentration is measured, it is sufficient to detect only the peak value of the generated inward current (current peak at the point of about 130 seconds in the example shown in FIG. 10). Can be omitted. Therefore, measurement of one oocyte within 60 seconds can be easily achieved.
[0062]
According to the electrophysiology automatic measuring apparatus of the present invention, a glass electrode is inserted fully automatically into each of a plurality of oocytes expressing a receptor by gene transfer, the potential is fixed, and the corresponding ligand is added. Current response when administered can be measured. As a result, for example, allergy-related diagnosis can be accelerated, and a large number of samples can be diagnosed in a short time. Furthermore, by introducing the 7-transmembrane receptor gene whose function is unknown into the oocyte and automating the screening of the corresponding ligand against the oocyte expressing the unknown receptor, Efficiently perform functional analysis search for large-scale genes.
[0063]
【The invention's effect】
According to the electrophysiology automatic measuring apparatus of the present invention, the burden on the measurer can be reduced by automating routine work, the measurement can be speeded up, and a large number of measurements can be performed in a short time. There is an effect that high data can be acquired.
[Brief description of the drawings]
FIG. 1 is a diagram showing a configuration example of an electrophysiology automatic measurement apparatus according to an embodiment of the present invention.
FIG. 2 shows a configuration example of an electrophysiology automatic measuring apparatus for performing allergy diagnosis according to an embodiment of the present invention.
FIG. 3 is a plan view showing a configuration example of a 96-hole oocyte holding plate used in an embodiment of the present invention.
4 is a cross-sectional view of the 96-hole oocyte holding plate shown in FIG.
FIG. 5 is a diagram showing a method for controlling insertion of a glass electrode into an oocyte in an embodiment of the present invention.
FIG. 6 is a plan view showing the shape of a 96-hole plate ground electrode used in an embodiment of the present invention.
7 is a partial perspective view of the 96-hole oocyte plate ground electrode shown in FIG. 6. FIG.
FIG. 8 is a diagram showing a method for preparing a 96-well oocyte holding plate made of agar used in an embodiment of the present invention and grounding it.
FIG. 9 is a diagram showing a configuration example for grounding each cell by moving a ground electrode in the embodiment of the present invention.
FIG. 10 is experimental data showing a reaction when histamine is administered to an oocyte using the electrophysiology automatic measuring apparatus according to an embodiment of the present invention.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 101 ... XY stage, 102,103 ... Glass electrode, 104,105 ... Glass electrode drive motor, 107 ... Motor drive controller, 108,108 '... Control computer, 109 ... Electrophysiological measurement circuit part, 111 ... 96 holes Plate for holding oocyte, 116 ... stereo microscope, 121 ... computer for measurement recording, 125 ... motor for driving microsyringe, 126 ... microsyringe for ligand administration, 202 ... cell, 203 ... hemisphere, 301 ... for 96-well plate Earth electrode, 302 ... Earth part, 401 ... Mold, 402 ... Soled agar, 404 ... Gelled agar, 405 ... Oocyte holding plate made of agar, 127, 502 ... Support, 406 ... Inserted into agar Earth electrode, 503... Earth electrode fixed to the support, 604. Earth Electrode, 603 ... Solution surface, 601 ... Oocyte, 803 ... Plate stacker, 805 ... Reaction vessel, 806 ... Reaction disk, 809 ... Belt conveyor, 810 ... Motor.

Claims (3)

A step of positioning a first oocyte among the oocytes held in the plurality of cells by moving a stage holding a container having a plurality of cells holding the oocytes expressing the gene; When,
The insertion of the glass electrode to the first oocyte, based on Hassu that electrical signals from the glass electrode, the contact of the solution surface in the cell, contacting the membrane surface of the first oocyte Detecting the insertion of the first oocyte into the membrane; and
After detecting insertion into the first oocyte membrane, and fixing the membrane potential of the first oocyte to a predetermined value,
Administering a chemical to the first oocyte after detecting fixation of the membrane potential;
And a step of measuring a response of the first oocyte to the chemical substance by measuring a current value . 2. The automatic electrophysiology measurement method according to claim 1, wherein a glass electrode is vibrated in a state where pressure is applied to a membrane surface of the first oocyte, and the glass electrode is inserted into the first oocyte. A characteristic electrophysiology automatic measuring method.   2. The electrophysiology automatic measurement method according to claim 1, further comprising a step of removing an offset potential after contact with the solution surface in the cell.

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