JP2005265627A - Microplate temperature controller - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a temperature control device for a microplate capable of accurate dispensation without hindrance of dispensation operation caused by deformation of the microplate. <P>SOLUTION: This temperature control device has a well block equipped with a temperature control function and a thermal insulation lid, and controls a sample temperature in the microplate. The temperature control device for the microplate is provided with a plate pressing member for allowing a part other than a hole part of the microplate to adhere to the well block. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a temperature control device for a microplate that promotes a reaction by controlling the temperature of a sample in the microplate in nucleic acid hybridization and subsequent B / F separation.

When performing nucleic acid hybridization, a hybridization apparatus as shown in FIG. 1 is used. In such a hybridization apparatus, in order to perform denaturation (around 95 ° C.) and annealing (40 to 70 ° C.), it is possible to control the sample temperature in the microplate using a well block equipped with a heater and a chiller. (For example, refer to Patent Document 1).
In general, a thermal insulation lid is provided on the upper part of the reaction part b of such an apparatus to prevent evaporation of the sample at the time of temperature rise. Then, at the time of washing after hybridization, a dispensing head that opens this lid and sucks the supernatant from the reaction part using the dispensing head a and discards it to the waste liquid part c, or dispenses the washing liquid from the washing liquid part d to the reaction part. Dispensing work is performed.

  However, in such a conventional apparatus, the microplate is often deformed and warped due to heat, which causes a difference in the height of each well. In some cases, the tip of the tip does not reach the bottom of the well, and accurate suction and discharge cannot be performed.

Also, in known PCR devices, there is a thermal insulation lid that presses a heater or a seal member on the heater against the top of the microplate, but in this case the microplate will continue to be pressed when the lid is opened for dispensing work. It is impossible to solve the above problems during dispensing.
JP 2003-164279 A

  An object of this invention is to provide the temperature control apparatus for microplates which can perform exact dispensing, without causing the trouble of dispensing operation resulting from a deformation | transformation of a microplate.

  That is, the present invention is a temperature control device that has a well block having a temperature control function and a thermal insulation lid, and controls the sample temperature in the microplate, and closely contacts the well block except for the hole of the microplate. A temperature control device for a microplate provided with plate pressing means is provided.

According to the apparatus of the present invention, 1) Dispensing can be performed while the microplate is pressed, and accurate and accurate dispensing can be performed. 2) By attaching a member and a leaf spring for guiding the presser means to the heat shield cover, the presser means can be closed by the operation of closing the heat shield cover without closing the presser means in advance. 3) Even when the thermal insulation lid is opened, the plate presser keeps holding the microplate so that the microplate will not warp even if the thermal insulation lid is opened, and the dispensing operation is performed reliably. . 4) After the thermal insulation lid is completely closed, by further generating a moment with a spring, the thermal insulation lid and the base surface are brought into close contact with each other, and the outside air can be surely shut off. Furthermore, since the position of the thermal insulation lid is reliably secured in a close contact state, the seal on the heater can uniformly hold the upper part of the microplate, and evaporation from the well can be suppressed.
Moreover, if the device of the present invention in which a part of the thermal insulation lid is made of transparent glass or resin is used, it is not necessary to open the thermal insulation lid when measuring the light intensity of the solution in the container, and the thermal insulation lid is opened. Since the time can be shortened and the solution does not evaporate when the thermal insulation lid is opened, measurement with little data error is possible.

Hereinafter, embodiments of the apparatus of the present invention will be described with reference to FIGS. 2 to 9, the same reference numerals denote the same parts.
2 and 3 are perspective views of the entire apparatus. FIG. 2 shows a state in which the plate presser is opened, and FIG. 3 shows a state in which the plate presser is closed. FIG. 4 is a left side view showing a state where the thermal insulation lid is closed and an opened state. FIG. 5 is a top view in the vicinity of the thermal insulation lid fulcrum shaft, and FIG. 6 is a schematic left side view of the connecting portion between the thermal insulation lid fulcrum shaft and the thermal insulation lid drive shaft.

A well block 2 is installed on the base surface 1. The well block 2 is provided with a temperature control device (not shown) such as a Peltier element for controlling the temperature of the sample in the microplate 3 placed thereon. On the base surface 1, there is a rotatable plate presser fulcrum shaft 5 supported by a bracket 4, and a plate presser 6 serving as a plate presser is fixed on the shaft. The plate holding means is for tightly fixing other than the hole portion of the microplate to the well block, and if it can hold the microplate to the well block without blocking the hole portion of the microplate, the material and The shape is not particularly limited. For example, a frame-shaped plate (plate presser 6) that cuts through all the parts corresponding to the holes of the microplate and presses only the periphery of the plate, or a large number of individual holes in each plate. Examples include a plate having a hole.
When the plate presser 6 is closed, the latch member 7 and the tip of the plate presser 6 are engaged, and the microplate 3 is locked onto the well block 2. That is, the plate presser 6 is locked by the latch, and even when the thermal insulation lid 10 is opened, the microplate presser continues and the microplate does not warp.

  On the other hand, on the base surface 1, there is a temperature-insulating lid fulcrum shaft 9 supported by another bracket 8, which can be rotated together with the temperature-insulating lid 10 fastened. A temperature control device such as a heater 12 that can be moved in the plate thickness direction by a guide member 11 is mounted on the thermal insulation lid 10, and is biased in a direction of pressing the microplate 3 by a compression spring 13. As the temperature control device, for example, a heater plate can be mentioned, and if necessary, a sealing material can be stuck on it to prevent evaporation of the sample from the well more completely when the thermal insulation lid is closed. It becomes possible. A guide plate 14 is provided beside the heater 12 so that the heat shield lid and the plate presser come into proper contact with each other when the plate presser 6 is pushed by the heat shield lid 10 and closed. In a state where both the thermal insulation lid 10 and the plate presser 6 are opened, the tip of the plate presser 6 is in contact with the guide plate 14. Leaf springs 15 are provided on the left and right sides of the guide plate 14 on the side far from the temperature-insulating lid rotation fulcrum. In other words, the plate presser 6 does not have a unique drive source and can be closed in conjunction with the thermal insulation lid.

On the outer periphery of the thermal insulation lid 10, a thermal insulation seal 16 is attached to the surface that abuts on the base surface 1, and a convex portion 17 that abuts on the base surface 1 is provided on the tip of the thermal insulation lid 10.
A slit is provided on the end face of the thermal insulation lid fulcrum shaft 9, and one end of an elastic body that twists in the rotational direction, such as a damper spring 18, is inserted therein. Further, the outer periphery of the damper spring 18 is covered with a collar 19 provided with a convex portion 20 in the axial direction attached integrally to the thermal insulation lid fulcrum shaft 9. The other end of the damper spring 18 is engaged with a thermal insulation lid drive shaft 21 having a slit. The temperature-insulating lid driving shaft 21 is rotatably supported by another bracket 23 and supported at a position that is coaxial with the temperature-insulating lid fulcrum shaft 9, and includes a pulley 24, a timing belt 25, which are integrally attached to the shaft, And it is driven by a motor (not shown).

A flange portion having a convex portion 22 in the circumferential direction is formed in the vicinity of the damper spring 18 of the thermal insulation lid driving shaft, and the damper spring 18 stores a sufficient moment with the thermal insulation lid 10 opened. The temperature-insulating lid driving shaft convex portion 22 and the collar convex portion 20 are assembled in such a relationship as to contact each other. This moment is biased in the direction in which the thermal insulation lid fulcrum shaft closes when the thermal insulation lid drive shaft is fixed.
Therefore, from the state in which the thermal insulation lid 10 is opened until the convex portion 17 of the thermal insulation lid contacts the base surface 1, the thermal insulation lid drive shaft 21 and the thermal insulation lid fulcrum shaft 9 rotate together. That is, when the thermal insulation lid is closed, the lid is further twisted to increase the pressing force of the lid, the thermal insulation lid and the base surface are brought into close contact with each other, and the outside air can be surely shut off.

  A sensor detection plate 26 is attached to the other end of the heat shield lid drive shaft 21, and an appropriate position of the drive shaft can be known by a sensor 27 attached to the bracket 23. In addition, a sensor detection plate 28 is attached to the other end of the thermal insulation lid fulcrum shaft 9, and an appropriate position of the thermal insulation lid can be known by a sensor 29 attached to the bracket 8.

FIG. 7 is a perspective view of the device of the present invention when detecting the fluorescence intensity of a sample, FIG. 8 is a cross-sectional view seen from the front, and FIG. 9 shows an example of a heater attached on a glass plate.
When detecting the fluorescence intensity or the like of the sample, the thermal barrier 10 is made of glass or resin that is partially or entirely transparent. A row of wells of the microplate 3 is located under the hole 31 opened in the thermal insulation lid 10, and a glass plate 32 with a heater 33 is sandwiched between the hole 31 of the thermal insulation lid 10 and the microplate 3. It is. The heater is arranged avoiding the microplate hole. The fluorescence detection unit 30 is moved onto a target well by an XY direction drive device (not shown) or a Z direction drive device, and the fluorescence intensity of the sample in the well is measured from the top of the lid.

Next, an example in which nucleic acid hybridization is performed using the apparatus of the present invention will be described.
Nucleic acid hybridization is performed, for example, by placing a reaction vessel into which a nucleic acid probe-immobilized magnetic particle and a labeled sample nucleic acid are injected and mixed in a reaction section, and setting the temperature in the reaction vessel with a heating / cooling device. The temperature is set and the temperature is maintained for a predetermined time, and the sample nucleic acid is single-stranded (for example, Japanese Patent Application Laid-Open No. 2003-164279). Can be done.
That is, after placing the microplate 3 (the reaction container) on the well block 2 of the present invention, the thermal insulation lid 10 is automatically closed by a motor (not shown) via the pulley 24 and the timing belt 25. At this time, the plate presser 6 is slid by the guide plate 14 of the thermal insulation lid 10 and rotates in a closing direction around the plate presser fulcrum shaft 5. Therefore, it is pushed until it is located on the microplate without touching the heater 12 attached to the thermal insulation lid 10 or the sealing material that prevents evaporation of the sample from the microplate (and thus damaging the heater or the sealing material). go. When the thermal insulation lid 10 is further closed, this time, it is pushed by the leaf spring 15 of the thermal insulation lid 10, and the tip of the plate presser 6 is locked by the latch member 7. When the motor is further rotated, the tip convex portion 17 of the thermal insulation lid 10 hits the base surface 1, the thermal insulation seal 16 comes into close contact with the base surface 1, and the inside of the thermal insulation lid 10 is sealed. When the motor is further rotated from this state, the thermal insulation lid driving shaft convex portion 22 and the collar convex portion 20 are separated from each other, and the damper spring between the thermal insulation lid driving shaft and the thermal insulation lid fulcrum shaft is rotated in the tightening direction. Without changing the position of the heat shield, the heat insulating cover 10 is given an appropriate moment to overcome the reaction force of the plate spring 15 for locking the compression spring 13 of the heater plate 12 and the plate presser 6. The sensor 27 detects the sensor detection plate 26 of the thermal insulation lid drive shaft 21 at a position where the motor is stopped. In this state, the sample nucleic acid is made into a single strand at 70 ° C., for example.
In the subsequent dispensing operation such as suction / discharge, the thermal insulation lid 10 closed in the above-described stage is opened by a motor (not shown) via the pulley 24 and the belt 25. At this initial stage, the damper spring 18 is only loosely tightened between the thermal insulation lid drive shaft 21 and the thermal insulation lid fulcrum shaft 9, and even if the thermal insulation lid drive shaft 21 is rotated, the thermal insulation lid fulcrum shaft 9 does not yet rotate. . If the thermal insulation lid drive shaft 21 continues to rotate as it is, the thermal insulation lid drive shaft convex portion 22 and the collar convex portion 20 come into contact with each other, and this time, the rotation of the thermal insulation lid drive shaft 21 is transmitted to the thermal insulation lid fulcrum shaft 9 as it is. The warm lid 10 begins to open. When the thermal insulation lid 10 is opened to the specified position, the sensor 29 detects the sensor detection plate 28 and stops the rotation of the motor.
Thus, the device of the present invention can be used as a temperature control device in nucleic acid hybridization using a microplate.

Example 1
Using the apparatus of the present invention shown in FIG. 9, genotyping of single nucleotide polymorphisms (SNPs) present in exon 12 of the ALDH2 gene (chromosome 12) that determines the presence or absence of the enzyme activity of aldehyde dehydrogenase was performed.
That is, two primers (5′-biotin-GGAGT TGGGC GAGTA CGG-3 ′ and 5′−) were prepared using genomic DNA extracted from blood by a commercial kit QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany) as a template. A 63 bp DNA fragment was PCR amplified using GCAGG TCCAC AATCA CAG-3 ′). 50 μl of this PCR fragment and 100 μg of streptavidin-immobilized magnetic particles are mixed in 50 μl of TEN buffer (10 mM Tris-HCl, 1 mM EDTA and 1 M NaCl, pH 7.4), and this container is set in the well block 2 of the present invention. did. After incubation at room temperature for 15 minutes, the conjugate of DNA and magnetic microparticles was collected at the bottom of the microplate 3 with a magnet (not shown), and the supernatant was removed with a dispensing head (not shown) (solid phase separation). Thereafter, a 50 mM sodium hydroxide solution was dispensed to denature the DNA on the magnetic fine particles into single strands, and the supernatant was removed again by solid phase separation. Furthermore, 200pmol detection probe (Cy5-labeled ALDH2 * 1 type detection probe: 5'-Cy5-TTCAC TTCAG TGTAT-3 'and Cy3-labeled ALDH2 * 2 type detection probe: 5'-Cy3-TTTCA CTTTA GTGTAT-3') , Closed the temperature-insulated lid 10 controlled at 90 ° C., heat-denatured at 72 ° C. for 3 minutes, slowly cooled to 25 ° C. and incubated for 10 minutes. Double strands were formed. Thereafter, the thermal insulation lid 10 was opened, and solid phase separation / washing was performed 3 times with a washing buffer, thereby removing unreacted detection probes. Further, after closing the thermal insulation lid 10, the fluorescence detection unit 30 was moved to the upper surface of the target well of the microplate 3, and the fluorescence intensity of the solution in the container at 25 ° C. was measured for two detection probes (Cy5: Ex645nm, Em675nm). , Cy3: Ex540nm, Em570nm). After the measurement, the container temperature was raised by 2 ° C., and the fluorescence intensity of the solution was measured again. In the same manner, the solution fluorescence intensity was sequentially measured while raising the container temperature by 2 ° C. to 80 ° C. After all measurements are completed, plot the difference in fluorescence intensity between the two detection probes at each temperature (ALDH2 * 1 probe minus ALDH2 * 2 probe) in the figure, and see if this curve expands to the positive side (ALDH2 * 1 Homotype), genotype was determined by observing whether it bulges to the negative side (ALDH2 * 2 homotype) or flat (near heterotype) near zero.
According to the device of the present invention, since the fluorescence intensity can be measured with the thermal insulation lid closed, the time for opening the thermal insulation lid can be shortened, and the solution does not evaporate when the thermal insulation lid is opened, Measurement with little data error was possible.

It is a perspective view of the conventional hybridization apparatus. FIG. 2 is a perspective view of the entire device of the present invention (plate presser opened). FIG. 3 is a perspective view of the entire apparatus of the present invention (with the plate presser closed). FIG. 4 is a left side view of the device of the present invention. FIG. 5 is a top view in the vicinity of the temperature-insulating lid fulcrum shaft. FIG. 6 is a schematic left side view of the connecting portion between the thermal insulation lid fulcrum shaft and the thermal insulation lid drive shaft. FIG. 7 is a perspective view of the device of the present invention when detecting the fluorescence intensity. FIG. 8 is a cross-sectional view seen from the front of the device of the present invention when detecting fluorescence intensity. FIG. 9 is a view showing the arrangement of the heaters affixed on the glass plate.

Explanation of symbols

1. Base surface 2. 2. Well block Microplate 4. Bracket 5. Plate presser fulcrum shaft 6. Plate holding means 7. Latch member 8. Bracket 9. 10. Thermal insulation lid fulcrum shaft Thermal barrier 11. Guide member 12. Heater 13. Compression spring 14. Guide plate 15. Leaf spring 16. Thermal barrier seal 17. Convex part 18. Damper spring 19. Color 20. Convex part 21. Thermal insulation lid drive shaft 22. Convex part 23. Bracket 24. Pulley 25. Timing belt 26. Sensor detection plate 27. Sensor 28. Sensor detection plate 29. Sensor 30. Fluorescence detection unit 31. Hole 32. Glass plate 33. heater

Claims (9)

  A temperature control device that has a well block having a temperature control function and a thermal insulation lid and controls the sample temperature in the microplate, and includes a plate pressing means for closely contacting the well block except the hole portion of the microplate. A temperature control device for a microplate.   2. The apparatus according to claim 1, wherein the plate pressing means does not have its own drive source but closes in conjunction with the thermal insulation lid.   3. An apparatus according to claim 2, wherein a guide member is provided for properly contacting the heat shield cover and the presser means when the plate presser means is closed by being pressed by the heat shield cover.   The apparatus according to any one of claims 1 to 3, wherein an elastic body is provided so that the plate pressing means can reliably hold the microplate when the thermal insulation lid is closed.   The apparatus according to any one of claims 1 to 4, wherein the plate pressing means continues to hold the microplate when the thermal insulation lid is opened.   The apparatus according to any one of claims 1 to 5, wherein all or part of the pivot point of the thermal insulation lid is formed of an elastic body that twists in the pivot direction.   7. The apparatus according to claim 6, wherein the thermal insulation lid opens and closes when the elastic body is twisted to a predetermined position, and when the lid is closed, the thermal insulation lid is further twisted to increase the pressing force of the lid.   The apparatus according to any one of claims 1 to 7, wherein all or a part of the heat insulating lid is made of transparent glass or resin.   9. The apparatus according to claim 8, wherein a heater is disposed in the transparent glass or resin part of the heat insulation cover so as to avoid the microplate hole.

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