JP5207231B2 - Nucleic acid amplification apparatus, method and cell culture / nucleic acid amplification method - Google Patents

Description

  The present invention provides a real-time nucleic acid amplification apparatus and method for amplifying a small amount of nucleic acid, and a cell culture / real-time nucleic acid amplification method capable of executing the process from cell culture to real-time nucleic acid amplification in the same apparatus.

  Various new technologies have been proposed for realizing microdevices that perform chemical reactions in extremely small amounts and systems that handle cells one by one and perform various reactions and observations on cells. For example, Japanese Patent Laid-Open No. 2006-162264 by the inventors of the present application discloses a specific plan in which a plurality of types of liquid droplets containing a reaction medium are prepared and reacted by selectively colliding them.

Conventional chemical reactions such as biochemical reactions are generally performed in a container having a certain size, but if such droplets can be operated, literally minute reaction can be realized. Realization of such a small amount of reaction not only makes it possible to reduce the amount of sample to be prepared, but also is advantageous not only in terms of operability such as improving the reaction rate, but also because reaction products can be minimized. It is also useful in that there is no waste.
JP 2006-162264 A

  The present invention intends to realize nucleic acid amplification in a droplet as a minute reaction. That is, the present invention relates to a real-time nucleic acid amplification apparatus that stores a single cell in a droplet and amplifies the nucleic acid of the cell in cell units. Further, the present invention relates to a real-time nucleic acid amplification method for amplifying cells contained in a droplet that is a target of nucleic acid amplification within the droplet, and amplifying the nucleic acid of the cell using the amplified cell.

  The present invention provides the following nucleic acid amplification apparatus, nucleic acid amplification method, nucleic acid amplification substrate, cell culture / nucleic acid amplification method, and cell culture / nucleic acid amplification substrate.

(1) PCR amplification of a cell and a nucleic acid contained in the cell in a tub defined by the side wall and the upper surface of the substrate, having a side wall provided so as to surround a predetermined region on the upper surface of the substrate A thermally conductive substrate for holding a droplet containing a PCR reaction solution containing a primer and a polymerase, and a solvent layer having a specific gravity smaller than that of the droplet and substantially not mixed with the droplet,
Photodetecting means comprising a plurality of photodetectors that detect the light from the droplets in a one-to-one correspondence with the plurality of droplets arranged in the solvent layer on the thermally conductive substrate;
A heat source provided on the lower surface of the thermally conductive substrate;
And controlling the heat energy transmitted from the heat source to the droplets via the heat conducting substrate according to a predetermined program so that the temperature of the droplets changes with time in a predetermined pattern. Temperature control means for controlling the relative position of the drop,
A real-time nucleic acid amplification apparatus comprising:
(2) a first heat source plate having a predetermined amount of heat, maintained at a temperature for denaturation of nucleic acid into a single strand, and having a surface having substantially the same area as the predetermined region; ,
A second heat source plate having a predetermined amount of heat, maintained at a temperature at which the primer binds to the nucleic acid denatured into a single strand, and having a surface having substantially the same area as the predetermined region;
As well as
The real-time nucleic acid amplification device according to (1), wherein the first heat source plate and the second heat source plate are brought into contact with the lower surface of the thermally conductive substrate according to a predetermined program.
(3) The real-time nucleic acid amplification according to (2), wherein the first heat source plate includes protrusions of a heat conductive material at positions corresponding to the droplets arranged on the heat conductive substrate on a one-to-one basis. apparatus.
(4) PCR amplification of a cell and a nucleic acid contained in the cell in a tub defined by the side wall and the upper surface of the substrate, having a side wall provided so as to surround a predetermined region on the upper surface of the substrate Thermally conductive substrate for holding a plurality of arranged droplets containing a PCR reaction solution containing a primer and a polymerase, and a solvent layer having a specific gravity smaller than that of the droplets and substantially not mixed with the droplets ,
Provided on the lower surface of the thermally conductive substrate;
A first heat source plate having a predetermined amount of heat, maintained at a temperature for denaturation of nucleic acid into a single strand, and having a surface equivalent to an array of droplets arranged in the predetermined region;
A second heat source having a predetermined amount of heat, maintained at a temperature at which a primer binds to a nucleic acid denatured into a single strand, and having a surface having an area equivalent to an array of droplets arranged in the predetermined region; A heat source plate;
A heat source comprising,
A plurality of droplets provided on the lower surface of the thermally conductive substrate and corresponding to each droplet of the plurality of droplet rows arranged on the thermally conductive substrate, and detecting light from the droplets A light detection means comprising a light detector;
And the thermal energy propagated from the first heat source plate and the second heat source plate to the droplets via the heat conducting substrate is controlled according to a predetermined program so that the temperature of the droplets changes over time in a predetermined pattern. So that the relative positions of the first heat source plate, the second heat source plate, and the droplet row are controlled, and the light detection means is connected to the first heat source plate and the first heat source plate. Control means for controlling the position corresponding to the heat source plate of 2;
A real-time nucleic acid amplification apparatus comprising:
(5) The real-time nucleic acid amplification according to (4), wherein the first heat source plate includes protrusions of a heat conductive material at positions corresponding to the droplets arranged on the heat conductive substrate on a one-to-one basis. apparatus.
(6) A PCR reaction solution containing a plurality of cells, a primer for PCR amplification of a nucleic acid contained in the cells, and a polymerase can be held, and has a tip opening diameter of a predetermined size suitable for passage of one of the cells. Means to hold the pipette,
Means for extruding the solution from inside the pipette to form a droplet containing one cell at the pipette tip;
Means for confirming that a single cell is contained in the droplet formed at the tip of the pipette to form a droplet of a predetermined size;
A thermally conductive substrate having a side wall provided to receive the droplet dropped from the tip of the pipette and to surround the region holding the droplet, the thermal conductive substrate being defined by the side wall and the upper surface of the substrate A thermally conductive substrate for holding a solvent layer having a specific gravity smaller than that of the droplets and substantially not mixed with the droplets,
A substrate mounting means for mounting the thermally conductive substrate;
Means for operating the thermally conductive substrate mounting means to arrange the dropped droplets on the thermally conductive substrate at predetermined intervals; and thermal energy propagated to the droplets via the thermally conductive substrate Temperature control means for controlling the droplet according to a predetermined program so that the temperature of the droplet changes with time in a predetermined pattern,
A photodetecting means comprising a plurality of photodetectors for detecting light from the droplets in a one-to-one correspondence with the droplets arranged on the thermally conductive substrate;
A real-time nucleic acid amplification device comprising:
(7) PCR amplification of a cell and a nucleic acid contained in the cell in a tub defined by the side wall and the upper surface of the substrate, having a side wall provided so as to surround a predetermined region on the upper surface of the substrate A thermally conductive substrate for holding a plurality of droplets containing a PCR reaction solution containing a primer and a polymerase, and a solvent layer having a specific gravity smaller than that of the droplets and substantially not mixed with the droplets,
Photodetecting means comprising a plurality of photodetectors that correspond to the plurality of droplets arranged on the heat conducting substrate in a one-to-one correspondence and detect light from the droplets;
A laser light source provided on the lower surface of the thermally conductive substrate,
And the laser light irradiation position is controlled in a predetermined pattern so that the temperature of the liquid droplet changes over time in a predetermined pattern by controlling the laser light applied to the liquid droplet from the laser light source through the thermal conductive substrate. Temperature control means controlled according to the program of
A real-time nucleic acid amplification apparatus comprising:
(8) The real-time nucleic acid amplification device according to (7), wherein the means for controlling the laser light applied to the droplet from the laser light source is a means for controlling a galvanometer mirror and its reflection angle.

  In the present invention, a plurality of droplets containing cells arranged on a heat conducting substrate and a PCR reaction solution containing a primer and a polymerase for PCR amplification of nucleic acids encapsulated in the cells, the droplets and a heat source By controlling the relative position of the droplets so that the temperature of the droplet changes with time in a predetermined pattern, the nucleic acid by PCR of the cells in the droplet independently in a short time Perform amplification.

  A droplet of an appropriate size containing one cell is formed at the tip of a pipette that sucks up a cell suspension that has been separated in advance by paying attention to the characteristics of the cell. At this time, the cell suspension is composed of a primer for amplification of nucleic acid expected to be possessed by the cell, a polymerase which is an enzyme for extending a DNA strand, and an intercalator fluorescence for evaluating diffusion amplification. It can be a PCR solution containing the dye. Alternatively, the polymerase may be a polymerase having hexokinease and a PCR solution containing a reporter fragment (probe) instead of an intercalator fluorescent dye. The droplet formed at the tip of the pipette is monitored by an optical system, and the size of the droplet is monitored and controlled.

  The droplets are dropped on a transparent substrate, and the droplets are appropriately arranged. The temperature with respect to the time of the droplet on the transparent substrate is changed to a predetermined pattern, and nucleic acid amplification by PCR of the cells in the droplet is performed independently for each droplet.

  If necessary, prior to the nucleic acid amplification of the cells, the cells are cultured using the solution in each droplet as a cell culture solution. Thereafter, the culture solution in the droplet is replaced with a PCR solution, and then nucleic acid amplification of the cultured cells is performed.

Typically, in the nucleic acid amplification method using the apparatus of the present invention, droplets obtained by mixing a sample and a PCR reaction solution are arranged in an XY direction at an appropriate cycle on a mineral oil layer formed on a chip. Furthermore, it implements like the following (1)-(3).
(1) While maintaining the mineral oil layer at the annealing temperature (50 ° C. to 70 ° C.), the heating plate maintained at the denaturation temperature (90 ° C. to 95 ° C.) is repeatedly approached and separated from the lower surface of the chip at a predetermined time period. . On the other hand, a large number of photodetectors (for example, CCD elements) corresponding to the liquid droplets are arranged on the upper surface of the chip.
(2) The heating plate A that is stretched in the Y-axis direction on the lower surface of the chip and that is maintained at a denaturing temperature (90 ° C. to 95 ° C.), the heating plate B that is maintained at an annealing temperature (50 ° C. to 70 ° C.), and the Y axis A plurality of photodetectors corresponding to one-to-one are arranged as a set on the droplets arranged in the direction, and the heating plate A and the heating plate B are alternately approached and separated from the lower surface of the chip at predetermined time intervals.
(3) Two galvanometer mirrors are arranged on the lower surface of the chip, and temperature control is performed by scanning all droplets at a predetermined cycle.

  According to the present invention, nucleic acid amplification can be performed in a short time independently in units of cells.

  According to the present invention, a plurality of droplets holding a cell arranged on the upper surface of a heat conductive substrate and a PCR reaction solution containing a primer and a polymerase for PCR amplification of a nucleic acid contained in the cell are separated from the droplet. It is held in a solvent layer having a small specific gravity and substantially not mixed with the droplets, and heat energy is propagated from the heat source provided on the lower surface of the heat conducting substrate to the droplets, so that the temperature of the droplets has a predetermined pattern We propose a real-time nucleic acid amplifying device that is controlled so as to change with time.

  Before describing the real-time nucleic acid amplification apparatus according to the embodiment of the present invention, a specific example in which droplets are arranged on the upper surface of a heat conductive substrate suitable for the embodiment will be described. The specific examples described below are merely shown as examples of the present invention, and the present invention is not limited to these embodiments.

FIG. 1A is a plan view showing the relationship between the thermally conductive substrate 1, the droplet 21, and the wall 2 that defines the region holding the droplet 21, and FIG. 1B is the AA position of the plan view. It is sectional drawing when it sees in the arrow direction. Here, 38 is a solvent layer having a specific gravity smaller than that of the droplets and substantially not mixed with the droplets, for example, a mineral oil layer. The thermally conductive substrate 1 is optically transparent and is, for example, a silicon substrate, and has a thickness of 1 mm and a size of 20 mm × 20 mm, for example. The wall 2 is made of, for example, a silicon substrate, and has a thickness of, for example, 1 mm and a height of 10 mm. (C), (d), and (e) are schematic examples of devices for stably holding the droplets 21 arranged on the upper surface of the thermally conductive substrate 1 on the thermally conductive substrate 1, respectively. FIG. (C) is an example in which protrusions 41 ₁ and 41 ₂ for holding by SU-8 are formed on the heat conductive substrate 1. Since the figure is a cross section, only the protrusions on both sides are shown, but it is preferable to form protrusions in the length direction of the thermally conductive substrate 1. (D) is an example in which the hydrophilic region 42 is formed on the thermally conductive substrate 1. In order to form the hydrophilic region 42 on the silicon substrate, for example, plasma ions generated by alternating current discharge may be lightly implanted into the upper surface of the heat conductive substrate 1 only in the hydrophilic region 42. (E) is an example in which a recess 43 is formed instead of the formation of the holding projections 41 ₁ and 41 ₂ . Reference numeral 5 denotes a positioning marker, which is formed on one surface of the silicon substrate 1. In the figure, it is provided inside the wall 2, but it may be provided outside.

  As shown in FIG. 1B, droplets 21 including cells 12 and nucleic acid amplification target cells 12 and PCR solutions 13 are periodically arranged on the heat conductive substrate 1 in the XY directions. An example of the arrangement of the droplets will be described later.

  FIG. 2 is a conceptual diagram illustrating an example of a system configuration in which droplets 21 including cells 12 to be subjected to nucleic acid amplification are placed on the heat conductive substrate 1. In this example, a first pipette 11 for placing a droplet 21 containing cells 12 on a thermally conductive substrate 1 and a second pipette 20 for adjusting the size of the droplet 21 are provided. As described. Prior to the operation of placing the droplets 21, the mineral oil 38 is stretched to a depth of 8 mm in the region surrounded by the wall 2. The operation of applying the mineral oil 38 may be performed after the operation of placing the droplet 21.

  Cells 12 to be subjected to nucleic acid amplification are classified in advance with a cell sorter or the like. The cells 12 to be subjected to nucleic acid amplification in DNA replication include primers for amplification of nucleic acids expected for the cells 12, polymerases that are enzymes that extend DNA strands, and intercalator fluorescent dyes for evaluating diffusion amplification. Or a polymerase having a hexokinase, and prepared in a state suspended in a PCR solution 13 containing a reporter fragment (probe) instead of an intercalator fluorescent dye.

In the lower part of FIG. 2, test tubes 40 ₁ , 40 ₂ , 40 ₃ and 40 ₄ containing samples prepared for each classified cell are shown. The first pipette 11 sucks a sample from the test tubes 40 ₁ , 40 ₂ , 40 _3, and 40 ₄ and optically monitors a droplet formed at the tip of the first pipette 11 while monitoring a real-time nucleic acid amplification device. The droplets of the PCR solution 13 containing the cells 12 are distributed on the heat conductive substrate 1 for use. At this time, if the first pipette 11 including the tube 30 connecting the first pipette 11 and the syringe pump 31 is replaced for each cell, the cells 12 placed on the heat conductive substrate 1 are replaced for each row. Even when they are different, it is possible to prevent them from being mixed.

  Reference numeral 19 denotes an optically transparent stage driven in the XY directions, and 27 denotes a drive device for the stage 19. On the upper surface of the stage 19, the heat conductive substrate 1 for the real-time nucleic acid amplification device is placed. A first pipette 11 on which a PCR solution 13 containing cells 12 to be distributed is sucked and held in advance is disposed on the upper part of the heat conductive substrate 1. A syringe pump 31 is provided at the base of the first pipette 11 via a tube 30, and a drive device 32 is attached to the syringe pump 31. In addition, the PCR solution 13 is sucked into the syringe pump 31 in advance. When the syringe pump 31 is driven by the driving device 32, the PCR solution 13 in the first pipette 11 is pushed out together with the cells 12. The reason why the base of the first pipette 11 and the connection portion of the tube 30 are separated is that the first pipette 11 is enlarged and displayed, and is not separated. Absent.

  On the other hand, in order to adjust the size of the droplet formed at the tip of the first pipette 11, the second pipette 20 for supplying a solution to the droplet formed at the tip of the first pipette 11. The tip of is placed. A syringe pump 35 is provided at the base of the second pipette 20 via a tube 34, and a drive device 36 is attached to the syringe pump 35. In addition, the PCR solution 13 is sucked into the syringe pump 35 in advance. When the syringe pump 35 is driven by the drive device 36, the PCR solution in the syringe pump 35 is pushed out from the second pipette 20. The size of the droplet can be increased by supplying a PCR solution to the droplet formed at the tip of the first pipette 11.

A light source 16 and a condensing lens 17 constituting an optical system for monitoring the size of the droplet 21 formed inside and near the tip of the first pipette 11 are provided and face each other. At the position, a collimating lens 18 and a monitor 25 are provided below the heat conductive substrate 1. Therefore, the heat conductive substrate 1 and the stage 19 need to be optically transparent. A so-called personal computer 26 is a control signal obtained from a predetermined program stored in advance according to an input signal from the monitor 25, and an operation input signal given by the user while viewing the display screen of the monitor 25. 28, the necessary signals are given to the driving devices 27, 32, 36 and 37. Although not shown here, it is convenient to display the same display on the monitor of the personal computer 26 as the screen detected by the monitor 25. Then, the monitor 25 can be a small CCD camera. The operation input signal 28 is given via an input device of the personal computer 26. Further, as illustrated in FIGS. 1C and 1E, in order to stably maintain the position of the droplet, the protrusions 41 ₁ and 41 ₂ are formed on the heat conductive substrate 1, or The formation of the recess 43 instead has an effect that can be used as positioning information for the droplet 21.

  Here, the size of the first pipette 11 is considered as follows. The first pipette 11 needs to be able to form an appropriately sized droplet containing only one cell at its tip. The first pipette 11 is used after the PCR solution 13 containing the cells 12 is sucked up by the pipette, but when the droplet 21 is formed, the cells passing through the tip of the first pipette 11 are displayed on the monitor 25 without error. It must be detectable. Thus, the diameter of the tip of the first pipette 11 allows one cell to pass through, but not more than one cell at a time. That is, it is transparent and the diameter of the tip is preferably about 20 to 100 μm for general animal cells and about 5 μm for microorganisms such as bacteria.

  A pipette vertical movement drive device 37 is provided for transferring the droplet 21 formed on the tip of the first pipette 11 onto the heat conductive substrate 1. Here, it is assumed that the vertical movement drive device 37 is linked to the first pipette 11. When an operation signal 28 for lowering the first pipette 11 is given to the vertical movement drive device 37 by the user, the first pipette 11 moves downward, and a droplet formed at the tip of the first pipette 11. Is moved to a predetermined position on the heat conductive substrate 1. When the operation signal 28 for restoring the first pipette 11 is given to the vertical movement drive device 37 by the user, the first pipette 11 returns to the position shown in the figure. The restoration of the first pipette 11 to the position shown in the drawing may be performed in a time sequential manner by the personal computer 26 from the lowering operation. A one-point difference line 39 means linkage between the vertical movement drive device 37 and the first pipette 11.

The overall operation for distributing the cells 12 on the thermally conductive substrate 1 will be described below. First, when the system is activated, the user pays attention to the marker 5 described with reference to FIG. 1A and positions the thermally conductive substrate 1 so as to be in a predetermined activation position. Next, the stage 19 is operated by the driving device 27 in response to an operation input signal 28 for moving the initial distribution position of the droplet 21 to a position corresponding to the tip of the first pipette 11 and the second pipette 20. . When the thermally conductive substrate 1 reaches the first distribution position of the droplet 21, an operation of discharging the PCR solution 13 including the cells 12 inside the first pipette 11 together with the cells 12 is performed. For this positioning, the position information of the protrusions 41 ₁ and 41 ₂ formed on the heat conductive substrate 1 or the recesses 43 instead of the protrusions 41 ₁ and 41 can be used as auxiliary information. At this time, the outside of the distal end portion of the first pipette 11 and the interior in the vicinity of the distal end portion are monitored by an optical system including the light source 16 and the monitor 25. The output of the monitor 25 can be taken into the personal computer 26 and the drive device 32 can be operated based on the image calculation result of the personal computer 26 to control the liquid feeding of the syringe pump 31.

  While monitoring the tip of the first pipette 11 with the monitor 25, the driving device 32 is operated to move the syringe pump 31, and the PCR solution 13 containing the cells 12 is discharged from the tip of the first pipette 11. A droplet 21 is formed at the tip. At this time, the personal computer 26 recognizes that one cell has been inserted into the droplet 21 through the monitor 25, issues a stop command to the drive device 32, and stops the syringe pump 31.

  The cell 12 may be recognized by directly detecting the cell 12 present in the droplet 21 at the tip of the first pipette 11, but more efficiently, the cell moving inside the first pipette 11. 12 is monitored by the monitor 25, the position and moving speed of the cells in the pipette are calculated by the personal computer 26, and the syringe pump 31 is operated by predicting when the cells are discharged from the tip of the first pipette 11 into the droplet 21. You may control. If the latter recognition method is used, for example, when a plurality of cells are moving in the pipette 11 at short intervals, it is advantageous when only one cell is put in the droplet.

  Here, when the cell concentration of the PCR solution 13 containing the cells 12 is low, if the cells start to form the droplets 21 immediately before exiting from the tip of the first pipette 11, and the droplet formation is stopped after a predetermined time, The size of the droplet 21 can be made constant. When it is not desired to form droplets, for example, the liquid coming out from the tip of the first pipette 11 may be blown off with a blower. Alternatively, a drain may be provided outside the substrate 1 and discharged there.

  On the other hand, when the cell concentration of the PCR solution 13 containing the cells 12 is high, the amount of liquid discharged from the first pipette 11 varies. That is, since the frequency of discharge of the cells 12 discharged from the first pipette 11 increases, if the time for discharging the liquid is fixed at a predetermined time, the next cell 12 will drop the droplet 21 within that time. There is a possibility of getting inside. In such a case, the second pipette 20 is used. A PCR solution is placed in the second pipette 20 and the syringe pump 35 connected thereto. That is, when the personal computer 26 confirms that the cell 12 enters the droplet 21 through the monitor 25, a stop command is issued to the driving device 32 to stop the syringe pump 31, and the droplet 21 is formed by this time. The volume of the droplet 21 at that time is determined from the feed amount of the syringe pump 31 that is driven. The personal computer 26 calculates the difference between this volume and the desired volume of the droplet 21. In accordance with this calculation result, an operation signal is sent from the personal computer 26 to the driving device 36 so that the solution is added to the droplet 21 formed at that time by the second pipette 20, and the syringe pump 35 is driven to The PCR solution is added using the second pipette 20 until the volume of the droplet 21 reaches a predetermined value.

  When the PCR solution 13 is added to the droplet 21 using the second pipette 20, the tip of the second pipette 20 does not pass through the cell so that the cells 12 in the droplet do not flow back to the second pipette 20. For example, 0.2 μmφ is preferable. Alternatively, it is preferable to have a filter structure with a tip of 0.2 μm.

  The droplet 21 containing one cell prepared in this manner is brought into contact with the thermally conductive substrate 1 placed on the stage 19 by the vertical movement drive device 37 of the first pipette 11, and the droplet 21 21 moves onto the thermally conductive substrate 1. When it is confirmed that the droplet 21 including the cell 12 has moved to a predetermined position on the heat conductive substrate 1, the user gives an operation signal 28 to move the stage driving device 27, and the next droplet is dropped. The thermally conductive substrate 1 is moved so that the pipette tip is located at the position to be placed. This movement can be automatically performed by the personal computer 26 if information on the arrangement of the droplets with respect to the heat conductive substrate 1 is given to the personal computer 26. Then, at this new position, a new droplet is formed at the tip of the first pipette 11 as described above and moved onto the thermally conductive substrate 1. By repeating this, a droplet is placed on a necessary portion of the heat conductive substrate 1.

  In this manner, as described with reference to FIG. 1B, the droplets 21 made of the cells 12 and the PCR solution 13 are distributed on the heat conductive substrate 1. A droplet 21 in the form of wrapping the cell 12 and the PCR solution 13 is disposed in a region surrounded by the wall 2 on the silicon substrate 1. Mineral oil 38 is stretched over the entire area surrounded by the wall 2. Since the droplet 21 is about 0.2 to 2 μl, if the mineral oil 38 is put inside the wall 2 having a height of 10 mm, the droplet 21 is protected from evaporation due to temperature rise during nucleic acid amplification.

  Here, the reason why mineral oil is used is that the stability of the droplets 21 is good because the specific gravity of the mineral oil 38 is as small as about 0.9. Thereby, the droplet 21 always remains stably on the heat conductive substrate 1 during nucleic acid amplification. Since mineral oil is mainly used to prevent evaporation, it is sufficient that the droplet 21 is sufficiently buried in the mineral oil. For example, the mineral oil 38 is gently poured so as to have a depth of 8 mm.

  Next, real-time nucleic acid amplification according to an embodiment of the present invention will be described. PCR repeats the temperature cycle of (1) denaturation of DNA into a single strand (eg, 94 ° C.), (2) primer binding and synthesis of a complementary strand using a thermostable polymerase (eg, 60 ° C.) many times. Thus, nucleic acid amplification is performed. In this example, first, the temperature of the droplet 21 is raised to 94 ° C. to cause denaturation of DNA into a single strand, but in the first stage, the cells 12 are crushed by this high temperature, The nucleic acid is dispersed in the droplet and becomes a nucleic acid as a template. Next, the temperature of the droplet 21 is lowered to 60 ° C. to bind the primer to the nucleic acid as a template, and the complementary strand of the nucleic acid as a template is synthesized by a polymerase. In this case, when the PCR solution is a polymerase that extends a normal DNA strand, an intercalator fluorescent dye for evaluating diffusion amplification is incorporated into the double strand in conjunction with the synthesis of the complementary strand. On the other hand, when the PCR solution is a polymerase having a hexokinease and includes a reporter fragment (probe) instead of an intercalator fluorescent dye, the reporter fragment (probe) is incorporated into a double strand. .

  FIG. 3 shows the temperature control (temperature cycle) of the droplet 21 by causing the thermal energy of the heat source to act on all the droplets 21 arranged on the upper surface of the thermally conductive substrate 1 from the lower surface of the thermally conductive substrate 1. FIG. In this embodiment, the heat source is maintained at a predetermined temperature, and the two heat source plates 45 and the heat source plates 46 holding a sufficiently large amount of heat are alternately substantially in contact with the lower surface of the heat conductive substrate 1. Heat is applied to the droplet. In this case, not only the temperature of the droplet 21 becomes the temperature of the heat source plate 45 and the heat source plate 46 but also the mineral oil 38 becomes the temperature of the heat source plate 45 and the heat source plate 46 at the same time. Therefore, for example, a heat source plate 45 maintained at 94 ° C. and a heat source plate 46 maintained at 60 ° C., for example, are provided, and the period of denaturation of DNA into a single strand is maintained at 94 ° C. The existing heat source plate 45 is brought into contact with the lower surface of the thermally conductive substrate 1. On the other hand, the heat source plate 46 maintained at 60 ° C. is brought into contact with the lower surface of the heat conductive substrate 1 during the period of primer binding and synthesis of the complementary strand by the heat-resistant polymerase. By controlling the personal computer 48 with a predetermined program, the heat source plate 45 and the heat source plate 46 are alternately brought into contact with the lower surface of the heat conductive substrate 1 to complete the temperature cycle of the droplet 21. The amount of heat of the droplet 21 and the mineral oil 38 is sufficiently smaller than the amount of heat of each of the heat source plate 45 and the heat source plate 46, so the temperature of the droplets 21 arranged on the upper surface of the heat conductive substrate 1 in a very short time. Reaches the respective temperatures of the heat source plate 45 and the heat source plate 46 in contact with the lower surface of the heat conductive substrate 1. As a material of the heat source plate 45 and the heat source plate 46, copper (Cu) having a large specific heat is practically advantageous.

  The heat source plate 45 and the heat source plate 46 are alternately formed on the lower surface of the heat conductive substrate 1 in accordance with a signal given to the drive device 47 by the personal computer 48 in response to an operation input signal 50 given by the user while viewing the display screen of the personal computer 48. To be contacted. This will be described in more detail later with reference to FIG.

A CCD holding plate 49 is provided on the upper end of the wall 2. On the lower surface of the CCD holding plate 49, a CCD element is provided which is partitioned at a position corresponding to the droplet 21 arranged in the region of the wall 2 on a one-to-one basis. The CCD elements 51 ₁₁ , 51 ₁₂ ,..., 51 ₁₇ are provided in the region of the wall 2 so as to correspond to the X-axis direction liquid droplets 21 arranged at the top of the display in the figure. Although figure omitted appropriately display the reference numerals since complicated, CCD elements _{51 21, 51 22, ---,} X -axis 51 ₂₇ is arranged at the top immediately preceding stage of the display of FIG. It is provided so as to correspond to the droplet 21 in the direction. Hereinafter, CCD elements 51 _31, 51 _32, ---, 51 _37, CCD elements 51 _41, 51 _42, ---, 51 ₄₇ CCD elements ₅₁ 51, 51 _52, --- is the same for 51 ₅₇ . Signals detected by these CCD elements are transmitted to a personal computer 48 through a communication line 56 represented by reference numeral 56 and used.

  For fluorescence observation, the light from the light sources 57 and 57 'provided on both sides of the region of the wall 2 is transmitted through only the excitation light wavelength by the band-pass filters 58 and 58', respectively, and the shutter is only used for observation. 59 and 59 ′ irradiate the droplet 21 with the excitation light as shown by the broken line. The shutters 59 and 59 ′ are controlled by the personal computer 48 in synchronization with the control of the heat source plate 45 and the heat source plate 46. For the shutter 59 'on the left side of the figure, the illustration of the signal line for control from the personal computer 48 is omitted because the figure becomes complicated.

  4 (a) to 4 (c), the heat source plate 45 and the heat source plate 46 are alternately brought into contact with the lower surface of the heat conductive substrate 1 under the embodiment shown in FIG. It is a conceptual diagram explaining the procedure made to do. FIG. 4A shows an initial stage where the heat source plate 45 and the heat source plate 46 are waiting at a position away from the thermally conductive substrate 1 on which the droplets 21 are arranged. In this state, the heat source plate 45 is disposed below the heat conductive substrate 1 (Z-axis direction) at a distance corresponding to the thickness of the heat source plate 46 from the lower surface of the heat conductive substrate 1, and the heat source plate 46 is thermally conductive. If it is moved leftward (X-axis direction) on the lower surface of the conductive substrate 1, it is shifted to the right side of the thermal conductive substrate 1 at a position in contact with the lower surface of the thermal conductive substrate 1. It is assumed that the heat source plate 45 and the heat source plate 46 are disposed at a position that coincides with the heat conductive substrate 1 in the Y-axis direction. FIG. 4B is a diagram showing the positional relationship between the heat source plate 45 and the heat source plate 46 with respect to the heat conductive substrate 1 in the step of denaturing DNA into single strands. For example, the heat source plate 45 held at 94 ° C. is moved upward (Z-axis direction) to a position where the heat source plate 45 substantially contacts the lower surface of the heat conductive substrate 1. Waiting at the initial position away from 1. FIG. 4C is a view showing the positional relationship between the heat source plate 45 and the heat source plate 46 with respect to the heat conductive substrate 1 in the step of primer binding and the synthesis of complementary strands by heat-resistant polymerase. The heat source plate 45 is retracted downward (Z-axis direction) from the heat conductive substrate 1 and is waiting at the initial position. On the other hand, for example, the heat source plate 46 held at 60 ° C. is moved to the left (X-axis direction) of the thermally conductive substrate 1 and is substantially in contact with the lower surface of the thermally conductive substrate 1. In this way, the vertical movement of the heat source plate 45 (Z-axis direction) and the horizontal movement of the heat source plate 46 (X-axis direction) are repeated at predetermined time intervals as shown in FIGS. 4B and 4C. As a result, the temperature cycle can be repeated many times, and nucleic acid amplification is performed in the droplet 21.

  Instead of moving the heat source plate, the heat conductive substrate 1 may be moved left and right (X-axis direction). That is, the heat source plate 45 and the heat source plate 46 are arranged side by side at the same position (Z-axis direction) at the lower surface position of the heat conductive substrate 1, and the heat conductive substrate 1 moves left and right (X axis direction). The cycle can be repeated many times, and nucleic acid amplification can be performed in the droplet 21.

  Here, if the heat quantity held by the heat source plate 45 and the heat source plate 46 is sufficiently larger than that of the droplets and mineral oil on the heat conductive substrate 1, the nucleic acid amplification of the droplets 21 is executed. The necessary temperature cycle can be executed without controlling the temperature of the heat source plate 45 and the heat source plate 46. If it is necessary to control the temperature of the heat source plate 45 and the heat source plate 46, the heater and the temperature sensor are embedded in the heat source plate 45 and the heat source plate 46, and the temperature sensor signal is taken into the personal computer 48 to control the temperature of the heater. And it is sufficient.

FIG. 5 is a diagram showing a temperature change pattern of the droplet 21 due to the temperature cycle described in FIG. T ₁ is the period of the denaturation step of DNA into a single strand by the heat source plate 45, and T ₂ is the period of the primer binding by the heat source plate 46 and the complementary strand synthesis step by the heat-resistant polymerase. T ₃ is one period of the temperature cycle. Here, T ₁ is 1 second, T ₂ is 3 seconds, and T ₃ is 4 seconds. In about 1 second of the denaturation step, 94 ° C. required for the denaturation step is reached in about 0.4 seconds, and the first 0.4 seconds out of 3 seconds in the step of primer binding and complementary strand synthesis by the thermostable polymerase. It can be seen that the temperature reached 60 ° C., which is necessary for primer binding and the step of synthesizing a complementary strand by a thermostable polymerase.

  In this stage, the synthesis of the complementary strand proceeds in 2 seconds after the transition time from 94 ° C. to 60 ° C. (0.4 seconds) within 3 seconds of the primer binding and the complementary strand synthesis step by the thermostable polymerase. In the case where the PCR solution is a polymerase that elongates a normal DNA strand, an intercalator fluorescent dye for evaluating diffusion amplification is incorporated into the double strand together with the synthesis of the complementary strand. On the other hand, when the PCR solution is a polymerase having a hexokinease and includes a reporter fragment (probe) instead of an intercalator fluorescent dye, the reporter fragment (probe) is incorporated into a double strand. .

  The light from the light sources 57 and 57 'is transmitted only through the excitation light wavelength by the band-pass filters 58 and 58', and the excitation light is applied to the droplet 21 by the shutters 59 and 59 'only for fluorescence observation. The shutters 59 and 59 ′ are controlled by the personal computer 48 in synchronization with the control of the heat source plate 45 and the heat source plate 46. As a result, the fluorescence from the intercalator fluorescent dye or reporter fragment incorporated into the double strands by the excitation light from the light sources 57 and 57 ′ in accordance with the progress speed of the PCR in the droplet 21 is changed on the CCD holding plate 49. It is detected by a CCD element provided on the lower surface and sent to a personal computer 48 via a signal line 56.

When the timing of this excitation light irradiation and the detection by the CCD is entered in FIG. 5, the timing of T _{4 in} the first half of the last 1 second of the 3 seconds of the primer binding and the complementary strand synthesis stage by the thermostable polymerase is good. At this point, the incorporation of the intercalator fluorescent dye or reporter fragment (probe) into the double strand is almost complete, and the PCR results in one temperature cycle can be detected correctly.

  FIG. 6 shows the temperature control (temperature cycle) of the droplet 21 by causing the thermal energy of the heat source to act on all the droplets 21 arranged on the upper surface of the thermally conductive substrate 1 from the lower surface of the thermally conductive substrate 1. It is a perspective view which shows typically the other Example performed by this. Compared with the embodiment of FIG. 3, two heat source plates and a CCD element for detecting the PCR result of the droplet 21 are arranged as a set on the lower surface of the thermally conductive substrate 1 and excited for the droplet 21. The difference is that a light source 57 for irradiating light, a band pass filter 58 and a shutter 59 are provided at the center of the upper portion of the wall 2 region. Further, as a result of the CCD element for detecting the PCR result of the droplet 21 being disposed on the lower surface of the thermally conductive substrate 1, the light source 57, the band-pass filter 58 and the shutter 59 for observing the fluorescence of the droplet 21 are arranged on the wall 2. It can be provided immediately above the area.

61 ₁ , 61 ₂ , and 61 ₃ are heat source plates each maintained at a temperature of 94 ° C., and have a sufficiently large amount of heat as compared with the amount of heat in the region of the wall 2. Reference numerals 62 ₁ , 62 ₂ , and 62 ₃ denote heat source plates each having a temperature maintained at 60 ° C., and have a sufficiently large amount of heat as compared with the amount of heat in the region of the wall 2. The heat source plates 61 ₁ , 61 ₂ , 61 ₃ correspond to the heat source plate 45 of the embodiment shown in FIG. The heat source plates 62 ₁ , 62 ₂ and 62 ₃ correspond to the heat source plate 46 of the embodiment shown in FIG. Reference numerals 63 ₁ , 63 ₂ , and 63 ₃ denote CCD holding plates, which are divided on the upper surface into positions corresponding to one row and one row of droplets 21 arranged in the Y-axis direction in the region of the wall 2. A CCD element is provided. Since FIG becomes complicated, the reference numeral is omitted, for example, the CCD holding plate 63 _1, corresponding to the CCD element 51 _11, 51 _21, 51 _31, 51 ₄₁ and 51 ₅₁ of the embodiment shown in FIG. 3 A CCD element is provided in an integral structure. The other reference numerals in FIG. 6 that are the same as those shown in FIG. 3 have the same or corresponding functions.

The heat source plates 61 ₁ , 61 ₂ , and 61 ₃ and the heat source plates 62 ₁ , 62 ₂ , and 62 ₃ are given to the drive device 47 by the personal computer 48 in response to an operation input signal 50 given by the user while viewing the display screen of the personal computer 48. In response to the signal, the lower surface of the heat conductive substrate 1 is alternately brought into contact. Further, the CCD elements held on the CCD holding plates 63 ₁ , 63 ₂ , 63 ₃ are brought into contact with the lower surface of the thermally conductive substrate 1. This will be described in more detail with reference to FIG. Incidentally, the heat source plate 61 _1, 61 _2, 61 _3, the heat source plate 62 _1, 62 _2, 62 ₃ and CCD holding plate 63 _1, 63 _2, 63 ₃ are linked to move integrally, respectively.

FIGS. 7A to 7D show heat source plates 61 ₁ , 61 ₂ , 61 ₃ , heat source plates 62 ₁ , 62 ₂ , 62 ₃ and CCD holding plates 63 ₁ , 63 under the embodiment shown in FIG. _2, 63 ₃ alternately in contact with the lower surface of the thermally conductive substrate 1, with to complete the temperature cycle of the droplet 21 is a conceptual diagram illustrating a procedure for detecting the results of PCR in the droplet 21.

FIG. 7A is an initial stage, and the heat source plates 61 ₁ , 61 ₂ , 61 ₃ , the heat source plates 62 ₁ , 62 ₂ , 62 ₃ and the positions away from the thermally conductive substrate 1 in which the droplets 21 are arranged are shown. The CCD holding plates 63 ₁ , 63 ₂ , and 63 ₃ are waiting at an appropriate distance from the lower surface of the thermally conductive substrate 1. In this state, each of the heat source plates 61 ₁ , 61 ₂ , 61 ₃ corresponds to each column in the Y-axis direction of the droplets 21 arranged in the region of the wall 2 on the upper surface of the heat conductive substrate 1. It is supposed to be. FIG. 7B shows the heat source plates 61 ₁ , 61 ₂ , 61 ₃ , the heat source plates 62 ₁ , 62 ₂ , 62 ₃ and the CCD holding plates 63 ₁ , 63 ₂ , 63 _{3 in} the step of denaturing DNA into single strands. It is a figure which shows the positional relationship with the heat conductive substrate. For example, the heat source plates 61 ₁ , 61 ₂ , 61 ₃ held at 94 ° C. are moved upward (Z-axis direction) to a position where they substantially contact the lower surface of the heat conductive substrate 1. plates 62 _1, 62 _2, 62 ₃ and CCD holding plate 63 _1, 63 _2, 63 ₃ is held in a standby position away from the heat conductive substrate 1. FIG. 7 (c) shows the heat source plates 61 ₁ , 61 ₂ , 61 ₃ , the heat source plates 62 ₁ , 62 ₂ , 62 ₃ and the CCD holding plates 63 ₁ , 63 _{2 at the} stage of primer binding and complementary strand synthesis by heat-resistant polymerase. is a diagram showing the positional relationship between the thermally conductive substrate 1 of 63 _3. The heat source plates 61 ₁ , 61 ₂ , 61 ₃ are moved to the left (X-axis direction) of the heat conductive substrate 1 and retracted downward (Z-axis direction) from the heat conductive substrate 1. At the same time, for example, the heat source plates 62 ₁ , 62 ₂ , 62 ₃ and the CCD holding plates 63 ₁ , 63 ₂ , 63 ₃ held at 60 ° C. are also moved to the left (X-axis direction) of the heat conductive substrate 1. At the same time, the heat source plates 62 ₁ , 62 ₂ , and 62 ₃ are moved upward (in the Z-axis direction) and are substantially brought into contact with the lower surface of the thermally conductive substrate 1. FIG. 7D shows heat source plates 61 ₁ , 61 ₂ , 61 ₃ , heat source plates 62 ₁ , 62 ₂ , 62 ₃ and CCD holding plates 63 ₁ , 63 ₂ , 63 at the stage of detecting the PCR result of the droplet 21. It is a figure which shows the positional relationship with the ₃ thermal conductive board | substrate 1. FIG. Heat plate 61 _1, 61 _2, 61 ₃ and the heat source plate 62 _1, 62 _2, 62 ₃ are both downward from a thermally conductive substrate 1 with is moved to the left side of the thermally conductive substrate 1 (X-axis direction) It is retracted in the (Z-axis direction). The CCD holding plates 63 ₁ , 63 ₂ , and 63 ₃ are substantially in contact with the lower surface of the heat conductive substrate 1. That is, the fluorescence from the intercalator fluorescent dye or reporter fragment incorporated in the double strand by the excitation light from the light source 57 in accordance with the progress speed of PCR in the droplet 21 is converted into the CCD holding plates 63 ₁ , 63 ₂ , detected by the CCD element provided 63 ₃ of the upper surface, are sent to the computer 48 via a signal line 56.

A temperature cycle necessary for nucleic acid amplification can be realized by repeating the operations of FIGS. 7B to 7D many times. Also in this example, the amount of heat held by the heat source plates 61 ₁ , 61 ₂ , 61 ₃ and the heat source plates 62 ₁ , 62 ₂ , 62 ₃ is compared with that of the droplets 21 and the mineral oil 38 on the heat conductive substrate 1. If it is sufficiently large, the necessary temperature cycle for executing nucleic acid amplification of the droplet 21 is not controlled by the temperature control of the heat source plates 61 ₁ , 61 ₂ , 61 ₃ and the heat source plates 62 ₁ , 62 ₂ , 62 ₃ . But it is feasible. Heat plate 61 _1, 61 _2, 61 ₃ and if a heat source plate 62 _1, 62 _2, 62 requires _three temperature control, is embedded a heater and a temperature sensor in each of the heat source plate, PC a signal of the temperature sensor 48 The temperature of the heater may be controlled by taking it into

FIG. 8 is a diagram showing a temperature change pattern of the droplet 21 due to the temperature cycle described in FIG. This is essentially the same as the temperature change pattern described with reference to FIG. T ₁ is the period of denaturation of DNA into single strands by the heat source plates 61 ₁ , 61 ₂ , 61 ₃ , and T ₂ is due to primer binding by the heat source plates 62 ₁ , 62 ₂ , 62 ₃ and heat resistant polymerase. This is the period of the complementary strand synthesis stage. T ₃ is one period of the temperature cycle. Here, T ₁ is 1 second, T ₂ is 2 seconds + α, and T ₃ is 4 seconds. Within 1 second of the denaturation step, 94 ° C. necessary for the denaturation step is reached in about 0.4 seconds, and the first 0. It can be seen that the temperature reached 60 ° C. necessary for the primer binding and the complementary strand synthesis step with the thermostable polymerase in about 4 seconds. In the temperature cycle described with reference to FIG. 7, the heat source plates 62 ₁ , 62 ₂ , and 62 ₃ are withdrawn during the period T _{4 in} the latter half of one cycle T ₃ of the temperature cycle, and PCR measurement is performed using a CCD element. This is different from the temperature cycle described in FIG. During the period T ₄ , the heat source plates 62 ₁ , 62 ₂ , 62 ₃ are retracted, so that the temperature of the droplets 21 is slightly lowered, but there is no substantial influence.

  FIG. 9 shows the temperature control (temperature cycle) of the droplet 21 by causing the thermal energy of the heat source to act on all the droplets 21 arranged on the upper surface of the thermally conductive substrate 1 from the lower surface of the thermally conductive substrate 1. It is a perspective view which shows typically the other Example performed by this. In this embodiment, the two heat source plates holding a sufficiently large amount of heat maintained at a predetermined temperature in the two embodiments are alternately brought into contact with the lower surface of the heat conductive substrate 1 alternately. It is supposed that heat is applied to the droplet, but it is greatly different in that a monochromatic laser beam having a wavelength of 1480 nm having a large absorption is added to the water in the droplet while scanning. In this case, the temperature of the droplet 21 rises due to the scanning of the laser beam, but the mineral oil 38 basically does not absorb the laser beam, so the temperature does not rise. Therefore, it is assumed that the mineral oil 38 is controlled and maintained in advance at the temperature of the step of synthesizing the complementary strand by the primer binding and the heat-resistant polymerase, for example, 60 degrees.

In FIG. 9, reference numerals 65 ₁ and 65 ₂ denote galvanometer mirrors, which scan the laser beam emitted from the monochromatic laser source 66 in the XY plane to form droplets 21 on the optically transparent thermally conductive substrate 1. Irradiate. The personal computer 48 controls the laser beam by controlling a control signal to the monochromatic laser source 66 according to a predetermined program stored in advance according to the laser beam power and irradiation time data that has been studied and input in advance. . The galvanometer mirrors 65 ₁ and 65 ₂ are also controlled by the personal computer 48 corresponding to the predetermined program. In this embodiment, since each of the individual droplets 21 is individually scanned and controlled, it is not necessary to control the mechanical movement of the heat source plate as in the two embodiments described above. In FIG. 9, the same reference numerals as those in FIG. 3 denote the same or equivalent functions.

FIG. 10 is a diagram showing a temperature change pattern of the droplet 21 focusing on one cycle of the temperature cycle described with reference to FIG. 5 in the case of being controlled by the embodiment shown in FIG. T ₁ , T ₂ , T ₃ and T ₄ are the same as described in FIG. As is clear from a comparison between FIG. 10 and FIG. 5, in the embodiment shown in FIG. 9, the droplet 21 is irradiated with a laser beam only during the period T ₁ of the denaturation stage of DNA into a single strand. However, since the irradiation is performed for each scan (1 / number of droplets) instead of continuous irradiation, the temperature rise is stepwise. The dotted line shows this stepwise increase in temperature as an envelope.

Figure 11, for example of changing the galvanometer mirror 65 _1, 65 ₂ of the embodiment shown in FIG. 9 to the polygon mirror 67 _1, 67 _2, an example of a configuration of the polygon mirror 67 _1, 67 ₂ and monochromatic laser source 66 It is the shown schematic diagram. Here, the polygon mirror is a regular octagon. Polygon mirror 67 ₁ is assumed to rotate in the direction of the rotation arrows shown in figure XY plane about the Z axis. Polygon mirror 67 ₂ is assumed to rotate in the direction of the rotation arrows shown in figure XZ plane around the Y axis. If each polygon mirror and rotation speed are set appropriately, the area of the wall 2 can be repeatedly scanned. According to the polygon mirror, since the laser beam moves largely with respect to the mirror surface as compared with the scan by the galvanometer mirror, damage to the mirror surface by the laser beam can be reduced.

  FIG. 12 is a cross-sectional view showing the thermally conductive substrate 1 and the droplet 21 in the example in which the CCD element in the above-described embodiment is disposed below the droplet 21. On the upper surface of the thermally conductive substrate 1, a CCD holding plate 49 and a CCD element 51 supported by the holding plate 49 are provided in the region of the wall 2, and the droplet 21 is disposed on each CCD element 51. Has been. In this example, since the CCD element 51 is placed in the mineral oil 38, although not shown in the drawing, the entire CCD element 51 including the CCD holding plate 49 is made of, for example, polydimethylsiloxane (PDMS). It is necessary to cover and protect the entire surface with a thin layer.

  13 (a)-(c) shows the heat conductivity of the heat source plate 45 and the heat source plate 46 alternately described with reference to FIGS. 4 (a)-(c) under the embodiment shown in FIG. It is a conceptual diagram explaining the control by the same procedure as the procedure which contacts the lower surface of the board | substrate 1 and completes the temperature cycle of the droplet 21. FIG. 13 (a)-(c), unlike FIG. 4 (a)-(c), the heat source plate 45 is provided with a protrusion 45 'at a position corresponding to the droplet 21 of the thermally conductive substrate 1. In FIG. Only points differ from FIGS. 4A to 4C. Here, since the protrusion 45 ′ is for the purpose of transferring the heat of the heat source plate 45 to the droplet 21, it is natural that the protrusion 45 ′ has good heat conduction. The heat source plate 45 may be made of the same material.

  FIG. 13A shows an initial stage in which the heat source plate 45 and the heat source plate 46 are waiting at a position away from the thermally conductive substrate 1 on which the droplets 21 are arranged. In this state, the heat source plate 45 is disposed below the heat conductive substrate 1 (Z-axis direction) at a distance corresponding to the thickness of the heat source plate 46 from the lower surface of the heat conductive substrate 1, and the heat source plate 46 is thermally conductive. If it is moved leftward (X-axis direction) on the lower surface of the conductive substrate 1, it is shifted to the right side of the thermal conductive substrate 1 at a position in contact with the lower surface of the thermal conductive substrate 1. It is assumed that the heat source plate 45 and the heat source plate 46 are disposed at a position that coincides with the heat conductive substrate 1 in the Y-axis direction. Here, on the upper surface of the heat source plate 45, a protrusion 45 'corresponding to the droplet 21 disposed on the heat conductive substrate 1 is formed. FIG. 13B is a diagram showing the positional relationship between the heat source plate 45 and the heat source plate 46 with respect to the heat conductive substrate 1 at the stage of denaturation of DNA into single strands. For example, the heat source plate 45 held at 94 ° C. is moved upward (Z-axis direction) to a position where the protrusion 45 ′ substantially contacts the lower surface of the heat conductive substrate 1. Stands by at an initial position away from the heat conductive substrate 1. FIG. 13C is a diagram showing the positional relationship between the heat source plate 45 and the heat source plate 46 with respect to the heat conductive substrate 1 in the step of primer binding and the synthesis of complementary strands with a heat-resistant polymerase. The heat source plate 45 is retracted downward (Z-axis direction) from the heat conductive substrate 1 and is waiting at the initial position. On the other hand, for example, the heat source plate 46 held at 60 ° C. is moved to the left (X-axis direction) of the thermally conductive substrate 1 and is substantially in contact with the lower surface of the thermally conductive substrate 1. In this way, the vertical movement of the heat source plate 45 (Z-axis direction) and the horizontal movement of the heat source plate 46 (X-axis direction) are repeated at predetermined time intervals as shown in FIGS. 4B and 4C. As a result, the temperature cycle can be repeated many times, and nucleic acid amplification is performed in the droplet 21.

  FIG. 14 is a perspective view for explaining the relationship between the heat source plate 45 and the protrusion 45 '. The protrusion 45 ′ is provided on the upper surface of the heat source plate 45 at a position corresponding to the droplet 21 of the heat conductive substrate 1 on a one-to-one basis. Therefore, as shown in FIG. 13B, when the heat source plate 45 is brought close to the lower surface of the heat conductive substrate 1, the protrusion 45 ′ appears as if the heat source plate 45 is in direct contact with the droplet 21. Heat of 94 ° C. is transmitted to the droplet 21. As a result, the heat transfer to the mineral oil 38 is smaller than when the heat source plate 45 is in direct contact with the heat conductive substrate 1 on the entire surface. This is because, as shown in FIG. 13C, when the heat source plate 46 is brought close to the lower surface of the heat conductive substrate 1 and the temperature is shifted to 60 ° C. in the complementary strand synthesis stage, the temperature of the mineral oil 38 increases. Means that the temperature of the mineral oil 38 can be recovered to 60 ° C. in a shorter time.

  The meaning of providing the protrusion 45 ′ on the heat source plate 45 has been described with reference to FIGS. 13 and 14. On the other hand, as for the heat source plate 46, it is possible to provide protrusions in the same manner as the heat source plate 45, but it is not necessarily effective. This is because it is advantageous as a thermal cycle that the temperature of the mineral oil 38 is maintained at a temperature of 60 ° C. in the step of synthesizing the complementary strand. This is because it can be said that it is better to move the droplet 21 and the mineral oil 38 together to 60 ° C. than to move to 60 ° C.

  In the above-described embodiment, the nucleic acid of the cell in the droplet is amplified. Prior to the amplification of the nucleic acid of the cell, the cell culture solution is placed in the droplet containing the cell, and the cell is cultured. Then, nucleic acid amplification of the cultured cells can be performed effectively by nucleic acid amplification. FIG. 15 (a) is a perspective view showing an example of a pipette suitable for such use and suitable for exchanging the culture medium in the droplet 21 during cell culture, and FIG. 15 (b) is a perspective view of FIG. 15 (a). It is sectional drawing which shows typically the state which replaces | exchanges a culture solution using the pipette shown to). Reference numeral 71 denotes a pipette, and a cell blocking piece 72 is attached to the opening at the tip by attachment parts 73 and 74. A gap through which cells cannot pass is provided between the periphery of the opening at the tip of the pipette 71 and the cell blocking piece 72. As shown in FIG. 15 (b), the pipette 71 and the pipette 20 are inserted into the droplet 21, and a new culture solution is injected by the pipette 20 while sucking the culture solution in the droplet 21 from the pipette 71. The culture solution can be exchanged without accidentally sucking and losing the cells in the droplet 21. In the configuration shown in FIG. 2, if the pipette 71 is attached instead of the pipette 11 and the culture liquid in the droplet 21 is sucked by the syringe pump 31, and the culture liquid is supplied from the pipette 20, FIG. The culture solution can be exchanged in the configuration shown in b). After culturing proceeds in such a form, the process proceeds to nucleic acid amplification. At this stage, it is necessary to remove the culture solution in the droplet 21 and replace it with a solution for PCR. Also at this time, the liquid may be exchanged using the pipette shown in FIG.

  The minor modifications of the above embodiment are briefly described as follows.

  In the embodiment shown in FIG. 3, the light source 57, the band pass filter 58 and the shutter 59 can be arranged around the CCD element of the CCD holding plate 49. In the embodiment shown in FIG. 3, the intensity of the excitation light with respect to the droplet 21 is different between the droplets on both sides close to the wall 2 and the droplet at the center position. For this reason, the fluorescence intensity which a droplet emits differs according to the result of PCR. Therefore, it is preferable to determine the coefficient for correcting the intensity at the center position in advance by comparing the fluorescence intensity emitted by the droplets at both positions with the fluorescence intensity emitted by the droplet at the center position. Correction using this can be easily performed by the personal computer 48.

  In the embodiment shown in FIG. 6, the number of droplets that can be processed is smaller than that in the embodiment shown in FIG. Therefore, in order to increase the throughput, providing a plurality of the configurations shown in FIG. 6 is one solution.

  Although the invention has been described in some detail with its most preferred embodiments, the invention can be embodied in many forms without departing from its essential characteristics. The preferred embodiments described above are illustrative only and not restrictive. Further, since the scope of the present invention is defined by the claims rather than by the detailed description of the invention, any changes within the requirements of the claims, or equivalents thereof, are claimed. It is included within the range.

(A) is a top view which shows the relationship of the heat conductive substrate 1, the droplet 21, and the wall 2 which prescribes | regulates the area | region which hold | maintains the droplet 21, (b) is an arrow direction in the AA position of a top view. It is sectional drawing when it sees in. It is a conceptual diagram explaining the example of the system configuration which mounts the droplet 21 containing the cell 12 used as the object of nucleic acid amplification on the heat conductive substrate 1. FIG. Embodiment in which the temperature control (temperature cycle) of the droplet 21 is performed by causing the thermal energy of the heat source to act on all the droplets 21 arranged on the upper surface of the thermally conductive substrate 1 from the lower surface of the thermally conductive substrate 1. It is a perspective view which shows typically. (A)-(c) is a procedure in which the heat source plate 45 and the heat source plate 46 are alternately brought into contact with the lower surface of the thermally conductive substrate 1 under the embodiment shown in FIG. FIG. FIG. 5 is a diagram showing a temperature change pattern of droplets 21 due to the temperature cycle described in FIG. 4. Other temperature control (temperature cycle) of the droplet 21 is performed by applying the thermal energy of the heat source to all the droplets 21 arranged on the upper surface of the thermally conductive substrate 1 from the lower surface of the thermally conductive substrate 1. It is a perspective view which shows an Example typically. (A)-(d) are the heat source plates 61 ₁ , 61 ₂ , 61 ₃ , the heat source plates 62 ₁ , 62 ₂ , 62 ₃ and the CCD holding plates 63 ₁ , 63 ₂ , under the embodiment shown in FIG. 63 ₃ alternately in contact with the lower surface of the thermally conductive substrate 1, with to complete the temperature cycle of the droplet 21 is a conceptual diagram illustrating a procedure for detecting the results of PCR in the droplet 21. It is a figure which shows the pattern of the temperature change of the droplet 21 by the temperature cycle demonstrated in FIG. Other temperature control (temperature cycle) of the droplet 21 is performed by applying the thermal energy of the heat source to all the droplets 21 arranged on the upper surface of the thermally conductive substrate 1 from the lower surface of the thermally conductive substrate 1. It is a perspective view which shows an Example typically. FIG. 10 is a diagram showing a temperature change pattern of a droplet 21 focusing on one cycle of the temperature cycle described in FIG. 5 when controlled by the embodiment shown in FIG. 9. For example changing the galvanometer mirror 65 _1, 65 ₂ of the embodiment shown in FIG. 9 to the polygon mirror 67 _1, 67 _2, schematic diagram showing an example of the configuration of the polygon mirror 67 _1, 67 ₂ and monochromatic laser source 66 It is. FIG. 3 is a cross-sectional view showing the thermally conductive substrate 1 and the droplet 21 in an example in which the CCD element in the above-described embodiment is disposed under the droplet 21. (A)-(c) shows the heat conductive substrate 1 alternately with the heat source plate 45 and the heat source plate 46 described with reference to FIGS. 4 (a)-(c) under the embodiment shown in FIG. It is a conceptual diagram explaining the control by the same procedure as the procedure which is made to contact the lower surface of this and completes the temperature cycle of the droplet 21. FIG. It is a perspective view explaining the relationship between the heat source plate 45 and protrusion 45 '. (A) is a perspective view showing an example of a pipette suitable for exchanging the culture solution in the droplet 21 during cell culture. (B) is a diagram of the culture solution using the pipette shown in FIG. It is sectional drawing which shows typically the state which performs exchange.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Thermally conductive substrate, 2 ... Wall, 5 ... Positioning marker, 12 ... Cell, 11, 20 ... Pipette, 13 ... PCR solution containing cell 12, 16 ... Light source, 17 ... Condensing lens, 18 ... Collimate Lens, 19 ... Stage, 21 ... Droplet, 25 ... Monitor, 26 ... Personal computer, 27, 32, 36, 37 ... Drive device, 28 ... Operation input signal, 30, 34 ... Tube, 31, 35 ... Syringe pump, 38 ... mineral oil, 39 ... line indicating the linkage between the driving device and the pipette, 40 ... test tube, 45, 46 ... heat source plate, projection 45 '... projection on the upper surface of the heat source plate 45, 47 ... driving device, 48 ... personal computer, 49 ... CCD holding plate, 50 ... operation input signal, 51 ... CCD element, 56 ... communication line, 57, 57 '... light source, 58, 58' ... band pass filter, 59, 59 '... shutter, 61 ₁ , 61 ₂ , 1 ₃ ... heat plate, 62 _1, 62 _2, 62 ₃ ... heat plate, 63 _1, 63 _2, 63 ₃ ... CCD holding plate, 65 ... galvanomirror 66 ... monochromatic laser source, 67 ... a polygon mirror.

Claims (8)

Having side walls provided so as to surround a predetermined region of the upper surface of the substrate;
A plurality of droplets containing a cell and a PCR reaction solution containing a primer and a polymerase for PCR amplification of a nucleic acid contained in the cell in a tub defined by the side wall and the upper surface of the substrate; A thermally conductive substrate for holding a solvent layer having a lower specific gravity and substantially not mixed with the droplets;
Photodetecting means comprising a plurality of photodetectors that detect the light from the droplets in a one-to-one correspondence with the plurality of droplets arranged in the solvent layer on the thermally conductive substrate;
A heat source provided on the lower surface of the thermally conductive substrate;
And the thermal energy transmitted from the heat source to the droplets via the heat conductive substrate is controlled according to a predetermined program, and the temperature of the droplets is denatured into single strands of nucleic acids and denatured into single strands. has been to vary over time in the temperature at which the primer binds to a nucleic acid, the temperature control means for controlling the heat source,
A real-time nucleic acid amplification apparatus comprising: A first heat source plate having a predetermined amount of heat, maintained at a temperature for denaturation of nucleic acid into a single strand, and having a surface having substantially the same area as the predetermined region;
A second heat source plate having a predetermined amount of heat, maintained at a temperature at which the primer binds to the nucleic acid denatured into a single strand, and having a surface having substantially the same area as the predetermined region;
As well as
The real-time nucleic acid amplification device according to claim 1, wherein the first heat source plate and the second heat source plate are brought into contact with the lower surface of the thermally conductive substrate according to a predetermined program.   The real-time nucleic acid amplification device according to claim 2, wherein the first heat source plate includes protrusions of a heat conductive material at positions corresponding to the droplets arranged on the heat conductive substrate on a one-to-one basis. A primer having a side wall provided so as to surround a predetermined region on the upper surface of the substrate, and a PCR for amplifying a cell and a nucleic acid contained in the cell in a cage defined by the side wall and the upper surface of the substrate; A thermally conductive substrate for holding a plurality of arranged droplets containing a PCR reaction solution containing a polymerase and a solvent layer having a specific gravity smaller than that of the droplets and substantially not mixed with the droplets;
Provided on the lower surface of the thermally conductive substrate;
A first heat source plate having a predetermined amount of heat, maintained at a temperature for denaturation of nucleic acid into a single strand, and having a surface equivalent to an array of droplets arranged in the predetermined region;
A second heat source having a predetermined amount of heat, maintained at a temperature at which a primer binds to a nucleic acid denatured into a single strand, and having a surface having an area equivalent to an array of droplets arranged in the predetermined region; A heat source plate;
A heat source comprising,
A plurality of droplets provided on the lower surface of the thermally conductive substrate and corresponding to each droplet of the plurality of droplet rows arranged on the thermally conductive substrate, and detecting light from the droplets A light detection means comprising a light detector;
And the thermal energy propagated from the first heat source plate and the second heat source plate to the droplets via the heat conducting substrate is controlled according to a predetermined program so that the temperature of the droplets changes over time in a predetermined pattern. So that the relative positions of the first heat source plate, the second heat source plate, and the droplet row are controlled, and the light detection means is connected to the first heat source plate and the first heat source plate. Control means for controlling the position corresponding to the heat source plate of 2;
A real-time nucleic acid amplification apparatus comprising:   5. The real-time nucleic acid amplification device according to claim 4, wherein the first heat source plate includes protrusions of a heat conductive material at a position corresponding to the droplets arranged on the heat conductive substrate on a one-to-one basis. Holds a PCR reaction solution containing a plurality of cells and a primer and polymerase for PCR amplification of nucleic acids contained in the cells, and holds a pipette having a tip opening diameter of a predetermined size suitable for passage of one of the cells. Means to
Means for extruding the solution from inside the pipette to form a droplet containing one cell at the pipette tip;
Means for confirming that a single cell is contained in the droplet formed at the tip of the pipette to form a droplet of a predetermined size;
A thermally conductive substrate having a side wall provided to receive the droplet dropped from the tip of the pipette and to surround the region holding the droplet, the thermal conductive substrate being defined by the side wall and the upper surface of the substrate A thermally conductive substrate for holding a solvent layer having a specific gravity smaller than that of the droplets and substantially not mixed with the droplets,
A substrate mounting means for mounting the thermally conductive substrate;
Means for operating the thermally conductive substrate mounting means to arrange the dropped droplets on the thermally conductive substrate at predetermined intervals; and thermal energy propagated to the droplets via the thermally conductive substrate Is controlled according to a predetermined program so that the temperature of the droplet changes over time to the temperature of denaturation of nucleic acids into single strands and the temperature at which primers bind to nucleic acids denatured into single strands. Temperature control means,
A photodetecting means comprising a plurality of photodetectors for detecting light from the droplets in a one-to-one correspondence with the droplets arranged on the thermally conductive substrate;
A real-time nucleic acid amplification device comprising: Having side walls provided so as to surround a predetermined region of the upper surface of the substrate;
A plurality of droplets containing a cell and a PCR reaction solution containing a primer and a polymerase for PCR amplification of a nucleic acid contained in the cell in a tub defined by the side wall and the upper surface of the substrate; A thermally conductive substrate for holding a solvent layer having a lower specific gravity and substantially not mixed with droplets;
Photodetecting means comprising a plurality of photodetectors that correspond to the plurality of droplets arranged on the heat conducting substrate in a one-to-one correspondence and detect light from the droplets;
A laser light source provided on the lower surface of the thermally conductive substrate,
And a laser beam irradiated onto the droplet from the laser light source through the heat conducting substrate, and the temperature of the droplet is denatured into a single strand of nucleic acid, and the nucleic acid denatured into a single strand Temperature control means for controlling the irradiation position of the laser beam according to a predetermined program so as to change over time with the temperature at which the primer binds to
A real-time nucleic acid amplification apparatus comprising: 8. The real-time nucleic acid amplification apparatus according to claim 7, wherein the means for controlling the laser light emitted to the droplet from the laser light source is a means for controlling a galvanomirror and a reflection angle thereof.

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