JP2005283553A - Patch clump device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a patch clamp device instantaneously varying a single biological cell temperature, and responsively measuring a temperature change of a single biological cell. <P>SOLUTION: A solution supply copper pipe to the single biological cell is heated or cooled by a Peltier element, and the single biological cell temperature is measured by a thermocouple wire of a diameter of 13 μm or below. The single biological cell temperature change is speeded up to 50°C/msec, while a temperature measurement response speed is speeded up to 70 msec. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

Detailed Description of the Invention

Industrial application fields

  The present invention mainly relates to a patch clamp apparatus for evaluating an in vivo information transmission mechanism of a living organism.

  As a conventional technique, a solution heated in a heating tank is supplied to a single living cell via a plastic tube or a glass tube. Usually, these pipe lengths are required to be at least about 50 cm. Further, a thermistor thermometer having a diameter of about 1 mm or more was inserted into a container containing cells.

Problems to be solved by the invention

  In the above-described conventional apparatus, when the solution supply rate is increased, biological cells may be flowed, and the cells are generally supplied at a low speed. However, it is possible to increase the temperature of a single biological cell by natural heat dissipation in the middle of the piping. It was extremely difficult, and it took time to raise the temperature. For example, only a heating rate of about 10 ° C./min was obtained. In addition, a thermistor is generally used as the thermometer, and the temperature sensitive part at the tip of the thermistor is approximately 1 mm in diameter, and the average temperature of the solution over a relatively long time can be measured. The temperature which changes every moment of the cell could not be measured accurately. That is, since the heat capacity of the thermistor temperature sensing part is large, the temperature measurement response speed is slow, and there is a drawback that a very instantaneous temperature change within several tens of milliseconds cannot be measured.

Means for solving the problem

  The present invention has been proposed to solve the above-mentioned problems. First, in order to realize a rapid temperature rise, which is the first problem, two copper tubes having excellent thermal conductivity are arranged and fixed, A copper plate is joined to each of the base portions of the two copper pipes, a micro Peltier element is mounted on each copper plate, and a heat pipe or a heat pipe with a heat sink is attached to the micro Peltier element. Further, the copper pipe part is covered with a heat insulating material. By controlling the temperature of the Peltier element with a constant current power source, the solution temperature in the copper tube is controlled through the copper plate. Furthermore, in order to improve the temperature measurement response speed, which is a second problem, a fine chromel alumel thermocouple wire with a diameter of 13 μm or less is used, and the tip temperature sensing portion is simply compared with the wire diameter without making a large ball roller shape. A thermosensitive part having a very small heat capacity, in which the tips of thermocouple wires are partially welded, is fixed in the vicinity of a single living cell.

Action

  As described above, the patch clamp apparatus of the present invention changes the temperature of a single living cell to 50 ° C./millisecond by the heating method of the solution supplied to the living cell, and the final temperature measurement response speed is achieved by a micro thermocouple wire having a diameter of 13 μm or less. The time for achieving 2/3 of the temperature reached 70 milliseconds.

  The attached sheet 1 shows an embodiment of the present invention. A single living cell 2 fixed to the tip of the ammeter probe 1 is installed in a state of being suspended in a solution in a chamber bath 18 in the chamber 3, and from the solution tank 5 and the solution tank 6 in the heating tank 4. Each solution enters the copper tubes 9 and 9 ′ via the tube 8 and is supplied to the single living cell 2 from the glass tubes 22 and 22 ′ having an inner diameter of 200 μm connected to the copper tubes 9 and 9 ′. . Each of the copper tubes 9 and 9 'is mounted with a Peltier element 11 through a copper plate 10. Further, a micro heat pipe 12 is installed on the back surface of the Peltier element 11, and a heat radiating plate 12' is attached to the micro heat pipe 12. It has been. Each of the copper tubes 9 and 9 'is wrapped with a heat insulating material 21 and kept warm. In order to change the solution temperature in the vicinity of the single living body cell 2, different currents including polarities are supplied to the two Peltier elements 11 from the DC power supply 13, and the two types of solution temperatures discharged from the copper tubes 9 and 9 ′ are changed. . When the double copper pipes 9 and 9 ′ are instantaneously moved by the reciprocating device 14 using the piezo element and the reciprocating clamp 15, for example, a single living cell 2 wrapped with a low temperature solution discharged from the low temperature copper pipe 9. Is wrapped in a high-temperature solution discharged from the high-temperature copper tube 9 'in an instant, and the temperature is rapidly raised. Further, the single living cell 2 can be instantaneously returned to the original low-temperature state by the reverse operation. Further, in order to measure the temperature of the single living cell 2, a fine chromel alumel thermocouple wire 16 having a diameter of 13 μm or less is used, and the tip temperature sensing part 17 is not made into a large ball roller shape compared to the wire diameter. The tip temperature sensing part 17 having a very small heat capacity, in which the tips of the wires are partly welded, is fixed in the very vicinity of the single living cell 2. A solution is poured from the chamber bath solution tank 7 through the tube 8 to the chamber bath 18 via the chamber bath temperature controller 19 to control the temperature of the chamber bath 18. FIG. 2 is a photograph using the ammeter 1, the tips of glass tubes 22 and 22 ′ having an inner diameter of about 200 μm, and the tip temperature sensing portion 17 of a fine chromel alumel thermocouple wire 16 having a diameter of 13 μm. FIG. 3 is an enlarged photograph of FIG. 2, showing a single living cell 17 having a diameter of about 7 μm, the tip of the ammeter 1, and the tip temperature sensing part 17 of a fine chromel alumel thermocouple wire 16 having a diameter of 13 μm. Yes. The microscope 20 is used for observation of a single living cell.

The invention's effect

With the above-described device, the temperature of the single living cell 2 can be changed instantaneously, that is, within 50 milliseconds, within a few milliseconds. Conventionally, the behavior of the single living cell 2 at 50 ° C. could not be evaluated because the single living cell 2 died at a high temperature. However, in general, the single living cell 2 does not die if it is instantaneous even at a high temperature of 50 ° C., and the present invention makes it possible to grasp the behavior of the single living cell 2 at a high temperature. In order to measure the temperature of the single living cell 2, the diameter of the single living cell 2 can be obtained by using a 13 μm fine thermocouple temperature sensing unit 17 and installing it at a position several μm from the single living cell 2. Since it is 10 micrometers-20 micrometers, the temperature of the single living cell 2 vicinity can fully be measured. The reciprocating device 14 using the piezo element, the reciprocating clamp 15 and the Peltier element 11 allow not only the temperature jump to a high temperature but also the jump to a low temperature instantaneously within a few milliseconds, and a free timing as many times as possible I was able to do it. By immediately returning to the original temperature even at a high temperature of 50 ° C., there was almost no damage to the single living cell 2 and it was possible to continue the experiment. In the case of the patch clamp method, since a minute current of picoampere (pA: ^10-12 A) is measured, it is indispensable to completely eliminate noise. Since the Peltier element 11 can be driven by the DC power source 13, commercial waves of 50 Hz and 60 Hz can be excluded. Other advantages of using the Peltier element 11 as a heating element or a cooling element can be combined with the heating element and the cooling element depending on the direction of current. Moreover, since it is compact and light, the load on the reciprocating device 14 using the piezo element and the reciprocating clamp 15 can be reduced.

It is a perspective view which shows the example of Claim 1 and Claim 2. It is a photograph which shows the double copper pipes 9 and 9 'inside diameter 200micrometer, and the fine thermocouple tip temperature sensing part 17. FIG. It is the photograph which shows the single biological cell 17 about 7 micrometers in diameter, the front-end | tip part of the ammeter 1, and the fine thermocouple front-end | tip temperature sensing part 17. FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Ammeter probe 2 Single biological cell 3 Chamber 4 Heating tank 5 Low temperature solution tank 6 High temperature solution tank 7 Chamber bath solution tank 8 Tube 9 Copper tube 9 'Copper tube 10 Copper plate 11 Peltier element 12 Micro heat pipe 12' Heat sink 13 DC power supply 14 Reciprocating device using piezo element 15 Reciprocating clamp 16 Chromel alumel thermocouple wire 17 Tip temperature sensing part 18 Chamber bath 19 Chamber bath temperature controller 20 Microscope 21 Heat insulating material 22 Glass tube 22 'inner diameter 200μm inner diameter 200μm Glass tube

Claims (2)

Each copper plate is joined to the base part of two copper tubes, each Peltier element is mounted on this copper plate, each radiator plate or heat pipe is attached to this minute Peltier element, and the copper tube part is further insulated with a heat insulating material. A patch clamp device characterized by controlling the temperature of a solution in a copper tube through a copper plate by coating and controlling the temperature of a Peltier element with a constant current power source. A patch clamp device characterized in that a thermosensitive wire tip temperature sensing portion having a diameter of 13 μm or less is installed in the vicinity of a living cell.

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