U.S. patent application number 14/273673 was filed with the patent office on 2014-09-04 for liquid reflux high-speed gene amplification device.
This patent application is currently assigned to KANAGAWA ACADEMY OF SCIENCE AND TECHNOLOGY. The applicant listed for this patent is KANAGAWA ACADEMY OF SCIENCE AND TECHNOLOGY, NATIONAL UNIVERSITY CORPORATION TOKYO MEDICAL AND DENTAL UNIVERSITY. Invention is credited to Hiroyuki Takei, Hideyuki TERAZONO, Kenji YASUDA.
Application Number | 20140248690 14/273673 |
Document ID | / |
Family ID | 42828279 |
Filed Date | 2014-09-04 |
United States Patent
Application |
20140248690 |
Kind Code |
A1 |
Takei; Hiroyuki ; et
al. |
September 4, 2014 |
LIQUID REFLUX HIGH-SPEED GENE AMPLIFICATION DEVICE
Abstract
One embodiment provides a liquid reflux reaction control device
comprising: a reaction vessel having one or a plurality of wells
configured to accommodate a sample; a heat exchange vessel provided
in contact with the reaction vessel so as to conduct heat to the
reaction vessel, and comprising an inlet and an outlet respectively
for introducing and draining a liquid of a predetermined
temperature; a plurality of liquid reservoir tanks provided with a
temperature-controllable heat source for maintaining liquids of
predetermined temperatures; a tubular flow channel that connects
the inlet and the outlet of the heat exchange vessel with the
liquid reservoir tanks; a pump disposed on the tubular flow
channel, and configured to circulate the liquid between the heat
exchange vessel and the liquid reservoir tank; and a switching
valve disposed on the tubular flow channel, and configured to
control the flow of the circulating liquid.
Inventors: |
Takei; Hiroyuki; (Kanagawa,
JP) ; TERAZONO; Hideyuki; (Kanagawa, JP) ;
YASUDA; Kenji; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KANAGAWA ACADEMY OF SCIENCE AND TECHNOLOGY
NATIONAL UNIVERSITY CORPORATION TOKYO MEDICAL AND DENTAL
UNIVERSITY |
Kanagawa,
Bunkyo-ku |
|
JP
JP |
|
|
Assignee: |
KANAGAWA ACADEMY OF SCIENCE AND
TECHNOLOGY
Kanagawa
JP
NATIONAL UNIVERSITY CORPORATION TOKYO MEDICAL AND DENTAL
UNIVERSITY
Bunkyo-ku,
JP
|
Family ID: |
42828279 |
Appl. No.: |
14/273673 |
Filed: |
May 9, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13138784 |
Dec 5, 2011 |
|
|
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PCT/JP2010/055787 |
Mar 31, 2010 |
|
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14273673 |
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Current U.S.
Class: |
435/286.1 ;
435/287.2; 435/303.1 |
Current CPC
Class: |
F28D 7/0008 20130101;
B01L 2400/0633 20130101; F28F 2280/02 20130101; B01L 3/50851
20130101; B01L 3/50855 20130101; F28D 2021/0077 20130101; F28F
27/02 20130101; B01L 2400/0644 20130101; B01L 7/52 20130101; C12Q
1/686 20130101; F28D 15/00 20130101; B01L 2300/185 20130101; B01L
2400/0622 20130101 |
Class at
Publication: |
435/286.1 ;
435/303.1; 435/287.2 |
International
Class: |
B01L 7/00 20060101
B01L007/00; C12Q 1/68 20060101 C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 31, 2009 |
JP |
2009-084450 |
Claims
1. A liquid reflux reaction control device comprising: a reaction
vessel having one or a plurality of wells configured to accommodate
a sample; a heat exchange vessel provided in contact with the
reaction vessel so as to conduct heat to the reaction vessel, and
comprising an inlet and an outlet respectively for introducing and
discharging a liquid of a predetermined temperature; a plurality of
liquid reservoir tanks provided with a temperature-controllable
heat source for maintaining liquids of predetermined temperatures;
a flow channel that connects the inlet and the outlet of the heat
exchange vessel with each of the plurality of the liquid reservoir
tanks; a pumping mechanism configured to circulate the liquid
between the heat exchange vessel and each of the plurality of the
liquid reservoir tanks; and a switching mechanism configured to
control the flow of the circulating liquid, which controls the
temperature of the reaction vessel to keep a desired temperature by
switching the flows of the liquids of the predetermined
temperatures from the plurality of liquid reservoir tanks into the
heat exchange vessel at a predetermined time interval, wherein the
amount of the sample is less than or equal to several .mu.L per
well.
2. The liquid reflux reaction control device according to claim 1,
which is used as a PCR device.
3. The liquid reflux reaction control device according to claim 1,
further comprising, where a fluorescent dye is added to the sample
solution in the reaction vessel, a fluorescent detector configured
to detect fluorescence emitted from the fluorescent dye and to
measure fluorescent intensity with time.
4. The liquid reflux reaction control device according to claim 3,
wherein the fluorescent detector is disposed in correspondence with
each of the wells of the reaction vessel.
5. The liquid reflux reaction control device according to claim 3,
further comprising: a means for estimating the change in a
temperature of a sample solution based on the change in the
fluorescent intensity of the sample solution in one or a plurality
of wells of the reaction vessel; and a means for rapidly shifting
the temperature of the reaction vessel based on the result
thereof.
6. The liquid reflux reaction control device according to claim 1,
wherein the number of the liquid reservoir tanks is the same as the
number of temperatures intended for the reaction vessel.
7. The liquid reflux reaction control device according to claim 6,
wherein the number of the liquid reservoir tanks is 2 or 3.
8. The liquid reflux reaction control device according to claim 1,
wherein the reaction vessel is formed of a metal selected from the
group consisting of aluminum, nickel, magnesium, titanium,
platinum, gold, silver and copper, or of silicon; has a thickness
of 1-100 microns.
9. The liquid reflux reaction control device according to claim 1,
wherein the shape of the bottom surface of the well is flat,
hemispherical, trigonal pyramid shape or spherical.
10. The liquid reflux reaction control device according to claim 1,
wherein a reagent necessary for the reaction is accommodated in
each of the wells in a dry form in advance such that it eluted and
brought into reaction upon contacting with the sample solution.
11. The liquid reflux reaction control device according to claim 1,
wherein the reaction vessel further comprises an aperture or an
optical window through which measurement of an optical signal from
the sample of the reaction vessel is made.
12. The liquid reflux reaction control device according to claim 1,
wherein the reaction vessel is detachably arranged on the heat
exchange vessel.
13. The liquid reflux reaction control device according to claim
12, wherein the reaction vessel is provided in a removable manner
with respect to the heat exchange vessel in one of the following
fashion: (a) a cylindrical casing is provided surrounding the
reaction vessel, and a cylindrical reaction vessel socket is
provided on the heat exchange vessel, while the outer surface of
the casing of the reaction vessel and the inner surface of the
reaction vessel socket of the heat exchange vessel are threaded so
that the reaction vessel is removably attached to the heat exchange
vessel through rotation movement along the thread; (b) a
cylindrical casing surrounding the reaction vessel and a
cylindrical reaction vessel socket of the heat exchange vessel are
tapered with respect to each other so as to be removably attached
to each other by pressing the reaction vessel against the reaction
vessel socket; (c) the reaction vessel is secured to a glass-slide
like reaction vessel casing while the reaction vessel socket of the
heat exchange vessel is provided with a guide rail so that the
glass-slide like reaction vessel casing is removably attached to
the socket along the guide rail; and (d) the glass-slide like
reaction vessel casing is inserted into a slide holder with a hinge
mechanism so that the glass-slide like reaction vessel casing is
removably attached to the reaction vessel socket of the heat
exchange vessel through rotation movement of the hinge
mechanism.
14. The liquid reflux reaction control device according to claim
12, further comprising a mechanism configured to allow the reaction
vessel to attach to or remove from the heat exchange vessel during
reflux of the liquid without leaking the liquid out from the liquid
reflux reaction control device.
15. The liquid reflux reaction control device according to claim 1,
wherein the plurality of the liquid reservoir tanks is provided
with a heat source, a thermometer and a liquid stirrer, wherein the
liquid stirrer is provided with a heat source controller configured
to control the temperature distribution of the liquid in the liquid
reservoir tank within 5.degree. C. by stirring the liquid in the
liquid reservoir tank continuously or at a duty cycle ratio of 10%
or higher.
16. The liquid reflux reaction control device according to claim 1,
further comprising a controller configured to control the switching
mechanism.
17. The liquid reflux reaction control system according to claim 1,
wherein the switching mechanism is configured to lead the liquid in
any liquid reservoir tank among the plurality of liquid reservoir
tanks to the heat exchange vessel and to return the liquid in the
heat exchange vessel to the original liquid reservoir tank.
18. The liquid reflux reaction control device according to claim
16, wherein, when the liquid in the heat exchange vessel is
replaced by controlling the switching mechanism, the switching
mechanism is controlled such that the liquid in the heat exchange
vessel is led to a liquid reservoir tank maintained at a
temperature closest to the temperature of the liquid.
19. The liquid reflux reaction control device according to claim 1,
further comprising an auxiliary temperature control mechanism
comprising a thermal insulator, a heater, and a cooling mechanism,
wherein the auxiliary temperature control mechanism is configured
to suppress the fluctuation of the temperature of the liquid in the
flow channel that connects the switching mechanism to the plurality
of liquid reservoir tanks.
20. The liquid reflux reaction control device according to claim 1,
further comprising in the switching mechanism a mechanism
configured to control the shift in the temperature by continuously
replacing the liquid from the liquid reservoir tank regardless of
whether or not the liquid in the flow channel connecting the
switching mechanism to the plurality of liquid reservoir tanks is
led to the heat exchange vessel.
21. The liquid reflux reaction control device according to claim 1,
wherein the switching mechanism comprises a piston configured to
slide in a hollow structure having a circular or polygonal
cross-section so as to control the temperature of the liquid that
is in contact with the reaction vessel according to the position of
the piston.
22. The liquid reflux reaction control device according to claim
21, wherein the piston in the switching mechanism is configured to
slide by: (a) mechanically applying external force to the piston
rod connected to the piston; (b) using a piston that is a magnetic
body itself or a piston mounted with a magnetic body inside to
utilize interaction between the piston and a magnetic field
generation mechanism including an electromagnetic coil arranged
outside the switching mechanism; or (c) generating difference in
pressure due to the flow of the liquids circulating at both ends of
the piston.
23. The liquid reflux reaction control device according to claim 1,
wherein the switching mechanism comprises: (I) a switching valve
disposed at each of the inlet and the outlet, wherein the one pair
of the inlet and the outlet is open, and alternately the other pair
of the inlet and the outlet is closed to switch between the liquids
while preventing each of the liquids from mixing together in the
heat exchange vessel; (II) a slide piston valve comprising a heat
exchange vessel and a piston which is inserted in the heat exchange
vessel and capable of occluding an opening when the reaction vessel
is displaced from the heat exchange vessel; (III) a heat exchange
vessel having a cylindrical inner space, a rotary valve comprised
of an oval plate defining the inner space in two parts, being
inserted in the heat exchange vessel and rotatable around an axis
that is generally perpendicular to the circular cross-section of
the heat exchange vessel and a rod inserted in the heat exchange
vessel generally perpendicularly to the circular cross-section of
the heat exchange vessel and connected to the rotary valve and
functioning as a rotating axis, wherein the liquids of the
predetermined temperatures are introduced into and discharged from
each of the divided inner spaces through the one pair of the inlet
and the outlet and the other pair of the inlet and the outlet,
respectively, such that the liquid whih contacts with the bottom
surface of the reaction vessel is replaced by the rotary valve by
rotating the rotating axis; (IV) a heat exchange vessel and a
membrane arranged to divide the inner space of the heat exchange
vessel in two parts such that the liquid introduced into and
discharged from the heat exchange vessel through the pari of the
inlet and the outlet does not mix with the liquid introduced into
and discharged from the heat exchange vessel hrough the other pair
of the inlet and the outlet; or (V) a cylindrical rotor rotatably
inserted into the heat exchange vessel, wherein said rotor
comprises a plurality of grooves formed in its outer surface as
flow channels for the liquid delivered from the liquid reservoir
tank, and a tunnel-like flow channel connected to each of the
grooves to allow fluid communication, wherein both ends of the
tunnel-like flow channel serve as an inlet or an outlet of the
switching mechanism, and wherein rotation of the rotor allows
liquids at different temperatures to be introduced into the inlet
to make contact with exterior of the reaction vessel upon passing
the groove part.
24. The liquid reflux reaction control device according to claim 1,
wherein the circulating liquid used is a liquid ammonia.
25. The liquid reflux reaction control device according to claim 1,
wherein the circulating liquid used is a liquid having a boiling
point higher than that of water.
26. The liquid reflux reaction control device according to claim 1,
wherein the circulating liquid used is a liquid having a freezing
point lower than that of water.
27. The liquid reflux reaction control device according to claim 1,
further comprising a mechanism configured to prevent the sample
from evaporating, the mechanism comprising: a member that sealingly
covers the surface of the reaction vessel having the well, such
that at least part of it is optically transparent so as to allow
optical observation of the sample solution in the well; and a
heating mechanism configured to heat a part of the optically
transparent part of the member.
28. The liquid reflux reaction control device according to claim
27, wherein the distance between the optically transparent part of
the member and the surface of the reaction vessel having the well
is less than or equal to 3 mm.
29. The liquid reflux reaction control device according to claim
27, wherein the temperature of the optically transparent part of
the member is heated with the heating mechanism in a range of
80.degree. C. to 110.degree. C.
30. The liquid reflux reaction control device according to claim 1,
wherein the sample solution contains nucleic acids and a DNA
polymerase.
31. The liquid reflux reaction control device according to claim 1,
wherein the total volume of the circulating liquid is more than or
equal to several tens of mL per liquid reservoir tank.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. application Ser.
No. 13/138,784, which is the U.S. National Stage application of
PCT/JP2010/055787, filed May 21, 2010, which claims priority from
Japanese application JP 2009-084450, filed Mar. 21, 2009, the
entire contents of which are incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to a gene analysis device
using a reaction container, which is suitable for rapidly
performing an analysis with a small amount of gene for studies or
clinical practice in basic bioscience, basic medical research and
medical fields, for example, to a gene analysis using a reaction
device for detecting a particular nucleotide sequence at high speed
from a nucleic-acid base sequence such as genomic DNA or messenger
RNA derived from an animal including human or a plant.
BACKGROUND ART
[0003] Polymerase chain reaction (hereinafter, abbreviated as PCR)
is a method for amplifying a particular nucleic acid sequence from
a mixture of various types of nucleic acids. A particular nucleic
acid sequence can be amplified by performing at least one cycle of
the following steps: adding into the mixture a DNA template such as
genomic DNA or complementary DNA obtained by reverse transcription
from messenger RNA, two or more types of primers, thermostable
enzymes, salt such as magnesium, and four types of
deoxyribonucleoside triphosphates (dATP, dCTP, dGTP and dTTP) and
splitting of the nucleic acids; and binding the primers to the
nucleic acids; and allowing hybridization using, as a template, the
nucleic acids bound by the primers and the thermostable enzymes.
Thermal cycling is performed by increasing and decreasing the
temperature of a reaction container used for DNA amplification
reaction. There are various mechanisms for changing the
temperature, including a mechanism in which the temperature of the
reaction container containing a sample is changed through heat
exchange using a heater, a Peltier element or hot air, a mechanism
in which the temperature is changed by alternately bringing the
reaction container into contact with heater blocks or liquid baths
at different temperatures, and a method in which the temperature is
changed by running a sample through a flow channel that has regions
of different temperatures. Currently, the fastest commercially
available device is, for example, Light Cycler from Roche, which
has a mechanism where a specimen, DNA polymerase, small sections of
DNA as primers and a fluorescent dye label for measurement are
placed into each of a plurality of glass capillary tubes, where the
temperatures of small amounts of droplets in the capillary tubes
are shifted by blowing hot air at a temperature intended for the
droplets, for example, at two temperatures, i.e., 55.degree. C. and
95.degree. C., while at the same time, the glass capillary tubes
are irradiated with light for exciting the fluorescent dye to
measure the resulting fluorescent intensity. According to these
methods, the temperature of the sample can be repeatedly
shifted.
[0004] Moreover, a fluid impingement thermal cycler device has been
reported that controls the temperature of a specimen by impingement
of fluid jet on the outer wall of the specimen-containing region
(Japanese Patent Publication No. 2001-519224 (Patent Document
1)).
PRIOR ART DOCUMENTS
Patent Documents
[0005] [Patent Document 1] Japanese Patent Publication No.
2001-519224
SUMMARY OF THE INVENTION
Problem to be Solved by the Invention
[0006] A temperature rate obtained with a heater or a Peltier
element is as slow as about a few .degree. C. per second, and they
have difficulty in shifting the temperature without overshoot in
the temperature. Basically, when conduction through a solid
substance is utilize, a heat gradient is generated between the heat
source and the surface thereof, rendering strict control of the
temperature impossible. Furthermore, since heat is lost as soon as
the sample touches the heater or the Peltier element, delay occurs
until the surface restores the predetermined temperature. Moreover,
bringing a reaction vessel into contact with a separate heater or
liquid bath is associated with complication in the transfer
mechanism and difficulty in controlling the temperature of the
heater or the liquid vessel. Further, with a method in which a
sample is fed into a flow channel having different temperature
regions, problems arise that the surface temperature of the flow
channel itself changes with the movement of the sample, and thus it
becomes difficult to control the temperature. When the temperature
is shifted by blowing hot air, the small heat capacity of the air
requires a large amount of air to be blown. Similarly, since the
heat capacity of the air is small, it is difficult to control the
eventual blowing temperature of the air in precise increments of
1.degree. C. using an electrically-heated wire or the like.
[0007] Thus, the present invention has an objective of providing a
reaction control device that is capable of conducting accurate
temperature control, temperature measurement and rapid increase and
decrease in the temperature. More specifically, the present
invention has an objective of providing a DNA amplification device
that is capable of conducting accurate temperature control,
temperature measurement and rapid increase and decrease in the
temperature, so as to carry out PCR reaction at high speed, with
high accuracy and high amplification rate.
Means for Solving the Problem
[0008] In order to accomplish the above-described objective, a
reaction control device of the present invention is characterized
by: using, as media for heat exchange, liquids having a large heat
capacity and maintaining respective temperatures for shifting the
temperature of a sample solution; using means for shifting the
plurality of liquids having large heat capacities and different
temperatures at high speed; and using a minute reaction vessel in
which rapid heat exchange is conducted between the liquids with
large heat capacities and the sample solution. Specifically, a
minute reaction vessel having a structure and a material suitable
for heat exchange; a heat exchange vessel for circulating the
liquids at temperatures appropriate for each reaction outside the
minute reaction vessel; a plurality of liquid reservoir tanks
including a heat source for maintaining the temperatures of the
liquids with high accuracy; a switching valve system for guiding a
liquid from any liquid reservoir tank to the exterior of the
reaction vessel so as to rapidly shift the temperature of the
minute reaction vessel; and a mechanism configured to prevent
liquids of different temperatures from being mixed upon switching
the valve system.
[0009] Thus, the present invention provides the following liquid
reflux reaction control device.
(1) A liquid reflux reaction control device comprising:
[0010] a reaction vessel having one or a plurality of wells
configured to accommodate a sample;
[0011] a heat exchange vessel provided in contact with the reaction
vessel so as to conduct heat to the reaction vessel, and comprising
an inlet and an outlet respectively for introducing and draining a
liquid of a predetermined temperature;
[0012] a plurality of liquid reservoir tanks provided with a
temperature-controllable heat source for maintaining liquids of
predetermined temperatures;
[0013] a tubular flow channel that connects the inlet and the
outlet of the heat exchange vessel with the liquid reservoir
tanks;
[0014] a pump disposed on the tubular flow channel, and configured
to circulate the liquid between the heat exchange vessel and the
liquid reservoir tank; and
[0015] a switching valve disposed on the tubular flow channel, and
configured to control the flow of the circulating liquid, wherein
said switching valve controls the temperature of the reaction
vessel to keep a desired temperature by switching the flows of the
liquids of the predetermined temperatures from the plurality of
liquid reservoir tanks into the heat exchange vessel at a
predetermined time interval,
[0016] wherein the amount of the sample is less than or equal to
several .mu.L per well, and the total volume of the circulating
liquid is more than or equal to several tens of mL per liquid
reservoir tank.
(2) The liquid reflux reaction control device according to (1)
above, which is used as a PCR device. (3) The liquid reflux
reaction control device according to either one of (1) and (2)
above, further comprising, where a fluorescent dye is added to the
sample, a fluorescent detector configured to detect fluorescence
emitted from the fluorescent dye in the well in conjunction with
switching the temperature of the reaction vessel and measure the
change in the fluorescent intensity with time. (4) The liquid
reflux reaction control device according to (3) above, wherein the
fluorescent detector is disposed in correspondence with each of the
wells of the reaction vessel. (5) The liquid reflux reaction
control device according to either one of (3) and (4) above,
further comprising:
[0017] means for estimating the change in a temperature of a sample
solution based on the change in the fluorescent intensity of the
sample solution in one or a plurality of wells of the reaction
vessel; and
[0018] means for rapidly shifting the temperature of the reaction
vessel based on the result thereof
(6) The liquid reflux reaction control device according to any one
of (1) to (5) above, wherein the number of the liquid reservoir
tanks is the same as the number of temperatures intended for the
reaction vessel. (7) The liquid reflux reaction control device
according to (6) above, wherein the number of the liquid reservoir
tanks is 2 or 3. (8) The liquid reflux reaction control device
according to any one of (1) to (7) above, wherein the bottom and
wall surfaces of the reaction vessel is formed of a metal including
aluminum, nickel, magnesium, titanium, platinum, gold, silver or
copper, or silicon having a thickness of 1-100 microns. (9) The
liquid reflux reaction control device according to any one of (1)
to (8) above, wherein the shape of the bottom surface of the well
is flat, hemispherical, trigonal pyramid shape or spherical. (10)
The liquid reflux reaction control device according to any one of
(1) to (9) above, wherein a reagent necessary for the reaction is
accommodated in each of the wells in a dry form in advance such
that it eluted and brought into reaction upon contacting with the
sample solution. (11) The liquid reflux reaction control device
according to any one of (1) to (10) above, wherein the reaction
vessel further comprises an aperture or an optical window that
facilitates measurement of an optical signal from the sample in the
reaction vessel. (12) The liquid reflux reaction control device
according to any one of (1) to (11) above, wherein the reaction
vessel is provided in a removable manner with respect to the heat
exchange vessel. (13) The liquid reflux reaction control device
according to (12) above, wherein the reaction vessel is provided in
a removable manner with respect to the heat exchange vessel in one
of the following fashion:
[0019] (a) a cylindrical casing is provided surrounding the
reaction vessel, and a cylindrical reaction vessel socket is
provided on the heat exchange vessel, while the outer surface of
the casing of the reaction vessel and the inner surface of the
reaction vessel socket of the heat exchange vessel are threaded so
that the reaction vessel is removably attached to the heat exchange
vessel through rotation movement along the thread;
[0020] (b) a cylindrical casing surrounding the reaction vessel and
a cylindrical reaction vessel socket of the heat exchange vessel
are tapered with respect to each other so as to be removably
attached to each other by pressing the reaction vessel against the
reaction vessel socket;
[0021] (c) the reaction vessel is secured to a glass-slide like
reaction vessel casing while the reaction vessel socket of the heat
exchange vessel is provided with a guide rail so that the
glass-slide like reaction vessel casing is removably attached to
the socket along the guide rail; and
[0022] (d) the glass-slide like reaction vessel casing is inserted
into a slide holder with a hinge mechanism so that the glass-slide
like reaction vessel casing is removably attached to the reaction
vessel socket of the heat exchange vessel through rotation movement
of the hinge mechanism.
(14) The liquid reflux reaction control device according to either
one of (12) or (13) above, further comprising a mechanism that
allows the reaction vessel to attach to or remove from the heat
exchange vessel during reflux of the liquid without leaking the
liquid out from the liquid reflux reaction control device. (15) The
liquid reflux reaction control device according to any one of (1)
to (14) above, wherein the liquid reservoir tank is provided with a
heat source, a thermometer and a liquid stirrer, wherein the liquid
stirrer is provided with a heat source controller that can control
the temperature distribution of the liquid in the liquid reservoir
tank within 5.degree. C. by stirring the liquid in the liquid
reservoir tank continuously or at a duty cycle ratio of 10% or
higher. (16) The liquid reflux reaction control device according to
any one of (1) to (15) above, further comprising a switching valve
control mechanism configured to control the switching valve. (17)
The liquid reflux reaction control system according to any one of
(1) to (16) above, wherein the switching valve can lead the liquid
in any liquid reservoir tank among the plurality of liquid
reservoir tanks to the heat exchange vessel, and return the liquid
in the heat exchange vessel to the original liquid reservoir tank.
(18) The liquid reflux reaction control device according to either
one of (16) and (17) above, wherein, when the liquid in the heat
exchange vessel is replaced by controlling the switching valve, the
switching valve is controlled such that the liquid in the heat
exchange vessel is led to a liquid reservoir tank maintained at a
temperature closest to the temperature of the liquid. (19) The
liquid reflux reaction control device according to any one of (1)
to (18) above, further comprising an auxiliary temperature control
mechanism comprising a thermal insulator, a heater and a cooling
mechanism, wherein the mechanism suppresses the fluctuation of the
temperature of the liquid in the flow channel that connects the
switching valve to the liquid reservoir tank. (20) The liquid
reflux reaction control device according to any one of (1) to (19)
above, further comprising in the switching valve a mechanism
configured to control the shift in the temperature by continuously
replacing the liquid from the liquid reservoir tank regardless of
whether or not the liquid in the flow channel connecting the
switching valve to the liquid reservoir tank is led to the heat
exchange vessel. (21) The liquid reflux reaction control device
according to any one of (1) to (20) above, wherein the switching
valve comprises a piston that slides in a hollow structure having a
circular or polygonal cross-section so as to control the
temperature of the liquid that is in contact with the reaction
vessel according to the position of the piston. (22) The liquid
reflux reaction control device according to (21) above, wherein the
piston in the switching valve slides by:
[0023] (a) mechanically applying external force to the piston rod
connected to the piston;
[0024] (b) using a piston that is a magnetic body itself or a
piston mounted with a magnetic body inside to utilize interaction
between the piston and a magnetic field generation mechanism
including an electromagnetic coil arranged outside the switching
valve; or
[0025] (c) generating difference in pressure due to the flow of the
liquids circulating at both ends of the piston.
(23) The liquid reflux reaction control device according to any one
of (1) to (20) above, wherein, in the switching valve,
[0026] a cylindrical, discoid or conical rotor that is rotatably
inserted into the heat exchange vessel, wherein said rotor
comprises a plurality of grooves formed in its outer surface as
flow channels for the liquid delivered from the liquid reservoir
tank, and a tunnel-like flow channel connected to each of the
grooves to allow fluid communication,
[0027] both ends of the tunnel-like flow channel serve as an inlet
or an outlet of the switching valve, and
[0028] rotation of the rotor allows liquids at different
temperatures to be introduced into the inlet to make contact with
exterior of the reaction vessel upon passing the groove part.
(24) The liquid reflux reaction control device according to any one
of (1) to (23) above, wherein the circulating liquid used is a
liquid with a large heat capacity and low viscosity. (25) The
liquid reflux reaction control device according to any one of (1)
to (24) above, wherein the circulating liquid used is a liquid
having a boiling point higher than that of water. (26) The liquid
reflux reaction control device according to any one of (1) to (25)
above, wherein the circulating liquid used is a liquid having a
freezing point lower than that of water. (27) The liquid reflux
reaction control device according to (1) above, further comprising
a mechanism configured to prevent the sample from evaporating, the
mechanism comprising:
[0029] a member that sealingly covers the surface of the reaction
vessel having the well, such that at least part of it is optically
transparent so as to allow optical observation of the sample
solution in the well; and
[0030] a heating mechanism configured to heat a part of the
optically transparent part of the member.
(28) The liquid reflux reaction control device according to (27)
above, wherein the distance between the optically transparent part
of the member and the surface of the reaction vessel having the
well is less than or equal to 3 mm. (29) The liquid reflux reaction
control device according to (27) above, wherein the temperature of
the optically transparent part of the member is heated with the
heating mechanism in a range of 80.degree. C. to 110.degree. C.
Advantageous Effects of the Invention
[0031] Examples of advantages of the present invention for
controlling the temperature of a reaction vessel with refluxing
liquids include the following. First, the problem of a temperature
overshoot can be solved. Specifically, since a temperature of a
constantly refluxing liquid is almost constant, the temperature of
the surface of a reaction vessel and the temperature of the liquid
can be equilibrated almost at once. According to the present
invention, heat capacities of the reaction vessel and the sample
are insignificant as compared to that of the refluxing liquid.
Moreover, even when some heat is lost from a part the liquid,
essentially no heat gradient is caused since the liquid is
continuously flowing. Of course, the temperature of the reaction
vessel does not exceed the temperature of the liquid. According to
a typical embodiment of the present invention, liquids of different
temperatures can sequentially be run into the heat exchange vessel
so as to shift the temperature by 30.degree. C. or higher within
0.5 seconds. Hence, according to the present invention, time
required for shifting the temperature can be made extremely short
and, for example, total time for conducting PCR reaction can be
made dramatically shorter than the time required with a
conventional device.
[0032] In a reaction control device according to the present
invention, a liquid kept at a constant temperature is brought into
contact with the exterior of a reaction vessel having good heat
conductivity, and thereafter the liquid is rapidly replaced with a
liquid at different temperature, so that rapid increase and
decrease in the temperature of a sample can be realized and
controlled with high accuracy. According to the present invention,
PCR reaction can be conducted at high speed, with high accuracy and
at high amplification rate.
[0033] In addition, since the present invention is capable of
preventing a sample solution from evaporating due to heating of the
sample solution, it is advantageous in PCR reaction that uses a
small amount of sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 A schematic view showing a general structure of a
reaction control device of the present invention.
[0035] FIG. 2 An outline view of a heat exchange vessel used in a
reaction control device of the present invention.
[0036] FIG. 3 A schematic view showing embodiments of a reaction
vessel used in a reaction control device of the present invention
and illustrating a method for dissolving a lyophilized reagent.
[0037] FIG. 4 A schematic view showing a cylindrical reaction
casing used in a reaction control device of the present invention
and illustrating methods for attaching it to a heat exchange
vessel.
[0038] FIG. 5 A schematic view showing a sequence of switching a
valve used for a reaction control device of the present
invention.
[0039] FIG. 6 Diagrams showing (A) data with respect to the shift
in the temperature and (B) results from PCR reaction, using a
reaction control device of the present invention.
[0040] FIG. 7 A schematic view showing methods for attaching a
glass-slide like reaction casing used in a reaction control device
of the present invention to the heat exchange vessel.
[0041] FIG. 8 A schematic view showing a driving mechanism for a
slide piston valve used in a reaction control device of the present
invention.
[0042] FIG. 9 A schematic view showing a driving mechanism for a
slide piston valve used in a reaction control device of the present
invention.
[0043] FIG. 10 A schematic view showing a driving mechanism for a
rotary valve used in a reaction control device of the present
invention.
[0044] FIG. 11 A schematic view showing a temperature shifting
mechanism with a membrane used in a reaction control device of the
present invention.
[0045] FIG. 12 A schematic view showing a driving mechanism for a
temperature-setting valve in a reaction control device of the
present invention.
[0046] FIG. 13 A schematic view showing an exemplary configuration
of a heat exchange vessel used in reaction control device of the
present invention.
EMBODIMENTS FOR CARRYING OUT THE INVENTION
[0047] Hereinafter, embodiments of the present invention will be
described with reference to the drawings although these embodiments
are provided for illustration only and do not limit the scope of
the present invention.
[0048] FIG. 1 is a schematic view showing a general structure of
one embodiment of a reaction control device of the present
invention. Typically, a reaction control device of the present
invention comprises a reaction vessel 1, a reaction vessel casing
2, a heat exchange vessel 3, liquid reservoir tanks 4, heat sources
5, stirring mechanisms 6, pumps 7, switching valves 8, bypass flow
channels 9 and auxiliary temperature control mechanisms 10.
Preferably, the reaction control device of the present invention
further comprises fluorescence detectors 201, a control analyzer
202 for transmitting a control signal 203 and an optical window (or
aperture) 204.
[0049] The reaction vessel 1 may typically be composed of a thin
plate of a metal such as aluminum, nickel, magnesium, titanium,
platinum, gold, silver and copper or silicon, with a plurality of
wells. It is, however, not limited to these materials as long as
the material has high heat conductivity and does not interfere with
PCR. Alternatively, the surface of a thin metal membrane may be
covered with a hydrophilic material such as plastic that prevents
corrosion of the metal. The thickness of the thin plate at the well
region is preferably thinner than the surrounding area in order to
increase the heat conductivity, and it is typically, but not
limited to, a thickness of about 10 to 30 microns. The region
between the adjacent wells is preferably thicker in order to
maintain the overall strength, and it is typically in a range of,
but not limited to, 100 microns to 500 microns. The reaction vessel
1 is typically secured to a square, circular or other bottom
surface of the reaction vessel casing 2 to be formed integrally.
Typically, the reaction vessel 1 and the reaction vessel casing 2
are removable with respect to the heat exchange vessel 3 (see FIG.
4).
[0050] A liquid used for heat exchange is introduced into the heat
exchange vessel 3. The temperature of the introduced liquid is
controlled by the heat source 5 disposed inside the liquid
reservoir tank 4. In order to even the temperature inside the
liquid reservoir tank 4 by rapidly conducting heat away from the
surface of the heat source 5, the stirring mechanism 6 is
preferably provided. The liquid in the liquid reservoir tank 4 is
led inside the flow channel with the pump 7. In accordance with the
switching valve 8, the liquid is led to the heat exchange vessel 3
or directly returns to the liquid reservoir tank 4 through the
bypass flow channel 9. If necessary, the auxiliary temperature
control mechanism 10 can delicately control the temperature of the
liquid so as to suppress the temperature fluctuation inside the
liquid reservoir tank 4.
[0051] A liquid introduced into the heat exchange vessel 3 may be,
but not limited to, water, and any liquid can be used as long as it
has large heat capacity and low viscosity (e.g., liquid ammonia).
For example, a liquid having a higher boiling point than water can
be used to ensure a sample solution of 100.degree. C., or a liquid
having a lower freezing point than water can be used to ensure
temperature shift to the freezing point of water while preventing
solidification of the liquid circulating within the device.
[0052] Preferably, as shown in FIG. 1, the reaction vessel casing 2
is provided with the optical window 204 that allows transmission of
light for exciting a fluorescent dye and fluorescence therefrom
such that change in the fluorescent intensity of the fluorescent
dye in a sample solution that alters according to the reaction of
the sample solution in the reaction vessel 1 can be measured for
one or each of the plurality of reaction vessels. In addition,
arrangement of the fluorescence detectors 201 allows measurement of
fluorescent intensity in the reaction vessel 1 with time. According
to the example shown in FIG. 1, an excitation light irradiation
mechanism and a fluorescence detecting mechanism are provided in
each of the plurality of fluorescence detectors 201. This structure
allows, for example, independent measurement of different PCR
amplification information of a plurality of reaction vessels 1
having different primers or different sample solutions upon PCR
reaction. In addition, the fluorescent intensity data acquired with
the fluorescence detector 201 is recorded with the control analyzer
202, which has a function of estimating the amount of DNA or mRNA
in the sample solution obtained by the PCR reaction. Moreover, the
control analyzer 202 also has a function of estimating whether or
not the temperature of the sample solution has reached the intended
temperature after valve switching based on the change in the
fluorescent intensity with time by acquiring switching information
of the switching valve 8, and also has a mechanism configured to
control the valve switching based on that result. This is estimated
by a decrease in the amount of change or elimination of the
fluorescent intensity with unit time while the mobility of water
molecules that are universally possessed by a fluorescent dye is
exploited in view that fluorescence quenching depends on the
temperature of a liquid, and this is particularly effective for
confirming achievement of a high temperature state that results in
DNA denaturation.
[0053] According to the example shown in FIG. 1, detectors are
provided one for each reaction vessel 1. Alternatively, a light
source for exciting fluorescence can be combined with a camera
capable of quantitating and detecting fluorescence such as a cooled
CCD camera to measure the change in the fluorescent intensity of
the plurality of reaction vessels 1. Alternatively, when the number
of detectors used is less than the number of the reaction vessels
1, a mechanical driving mechanism capable of travelling on the X-Y
plane at high speed can be combined with the detectors so as to
measure the fluorescent intensities of all of the reaction vessels
1.
[0054] The volume of the sample solution is usually less than or
equal to several .mu.L per well, but it may be used in a range of
0.1 .mu.L to 100 .mu.L per well, preferably 0.5 .mu.L to 10 .mu.L
per well, more preferably 1 .mu.L to 10 .mu.L per well, still more
preferably 1 .mu.L to 5 .mu.L per well and most preferably 1 .mu.L
to 2 .mu.L per well. Besides the sample solution, a mineral oil or
the like for preventing evaporation of the sample solution may also
be contained in the well. The volume of the mineral oil is
preferably, but not limited to, about several .mu.L (e.g., 3 to 4
.mu.L), and obviously appropriately changeable by those skilled in
the art according to the size of the well or the amount of the
sample.
[0055] FIG. 2 is an outline view of the heat exchange vessel 3 used
in the reaction control device of the present invention. As basic
configuration, the heat exchange vessel 3 is provided with inlets A
(11) and B (12) for introducing liquids at different temperatures.
The heat exchange vessel 3 is also provided with a plurality of
outlets, i.e., outlets A (13) and B (14), for returning the liquid
from the heat exchange vessel 3 to the liquid reservoir tank 4.
FIG. 2A schematically shows introduction and discharge of a liquid
at a certain temperature from the liquid reservoir tank 4 through
the inlet A (11) and the outlet A (13), respectively, while FIG. 2B
schematically shows introduction and discharge of a liquid at a
different temperature from the liquid reservoir tank 4 through the
inlet B (12) and the outlet B (14), respectively. The number of the
inlets is not limited to two, and multiple inlets may be provided
as much as the number that matches the number of the temperatures
of the sample solution to be shifted. For example, in order to
realize a three-temperature system, the number of inlets would be
three. Similar to the case of inlets, the number of the outlets is
also not limited to two. Here, the arrows in FIG. 2 briefly
indicate the flowing direction of the liquid introduced into or
discharged from the heat exchange vessel 3.
[0056] A total volume of a liquid circulating between the heat
exchange vessel 3 and the liquid reservoir tank 4 is generally more
than or equal to several tens of mL, preferably more than or equal
to 100 mL, more preferably more than or equal to 200 mL, and most
preferably more than or equal to 300 mL considering the heat
capacity and the temperature stability of the liquid. The upper
limit of the volume may appropriately be determined in
consideration of the portability of the device or the like.
[0057] The volume of the heat exchange vessel 3 is preferably about
10 times or more, more preferably about 100 times or more and most
preferably about 1000 times or more the amount of the sample per
well. Typically, the volume of the heat exchange vessel is about
0.01 mL to 10 mL, more preferably about 0.05 mL to 5 mL and most
preferably about 0.1 mL to 2 mL per well.
[0058] FIG. 3 is a schematic view showing an embodiment of a
reaction vessel used in a reaction control device of the present
invention and a method for dissolving a lyophilized reagent.
Reaction vessels or wells in various shapes can be used while FIG.
3A shows examples where the surface of the heat exchange vessel
that makes contact with a liquid is flat (reaction vessel A (21)),
hemispherical (reaction vessel B (22)), a trigonal pyramid shape
(reaction vessel C (23)) or spherical (reaction vessel D (24)). In
terms of the efficiency of heat conduction, those skilled in the
art would readily understand that the area of the surface of the
heat exchange vessel that makes contact with a liquid is preferably
larger for better efficiency.
[0059] Conveniently, the reagent necessary for reaction is
lyophilized. As can be appreciated from FIG. 3B, a lyophilized
reagent 25 can be prepared and placed in the bottom of the reaction
vessel 26. Alternatively, a plug-shaped lyophilized reagent 25 may
be provided inside a dispensing chip 27 used for dispensing the
sample so that the reagent can be dissolved in the sample by
shaking the sample solution 28 up and down. Alternatively, a
lyophilized reagent 25 can be provided on a surface of a fiber ball
29 made of a bundle of nylon fibers or the like so that the
lyophilized reagent is dissolved by inserting and stirring the
fiber ball in a sample 28 in the reaction vessel 26.
[0060] FIG. 4 is a schematic view showing a cylindrical reaction
casing 32 used in a reaction control device of the present
invention, and illustrating a method for attaching the cylindrical
reaction casing 32 to a heat exchange vessel 37. Since directly
handling a reaction vessel made from a thin membrane is
inconvenient, a reaction vessel 31 is conveniently secured to the
reaction vessel casing 32 as shown in FIG. 4A. The reaction vessel
casing 32 is preferably formed of a heat insulating material such
as polystyrene, polycarbonate, PEEK, acrylic or the like. In
addition, the area of the connection with the reaction vessel 31 is
desirably kept as small as possible (e.g., 5 mm.sup.2 or smaller)
for rapid and highly accurate increase and decrease in the
temperature of the reaction vessel 31.
[0061] FIG. 4B shows, as one embodiment for attaching the reaction
vessel 31 to the heat exchange vessel 37, a method in which a
thread 34 is formed in the surface of the reaction vessel casing 32
so as to screw the reaction vessel casing 32 in the reaction vessel
socket 33 of the heat exchange vessel 37. With reference to FIG.
4B, the opening is preferably provided with a seal 35 in order to
maintain water tightness. FIG. 4C shows other method for attaching
the reaction vessel 31 to the heat exchange vessel 37. Referring to
FIG. 4C, a tapered reaction vessel casing 36 can be employed for
attachment to the heat exchange vessel 38 by pressure only.
[0062] FIG. 5 shows specific examples of a switching mechanism of a
valve used in a reaction control device of the present invention,
where inlet valves A (41) and B (43) for introducing a liquid into
a reaction vessel and outlet valves A (42) and B (44) for leading
the liquid outside are shown. A liquid led in from the inlet valve
A (41) returns to the liquid reservoir tank 4 via the outlet valve
A (42) whereas a liquid led in from the inlet valve A (43) returns
to a different liquid reservoir tank 4 via the outlet valve B (44).
By alternately switching these two states, the sample in the
reaction vessel can be brought into reaction. According to a more
desirable valve switching method, other than the above-described
two states, the inlet valve B (43) and the outlet valve A (42) or
the inlet valve A (41) and the outlet valve B (44) are released at
the same time for a moment so that the liquids at different
temperatures can be prevented from mixing with each other, thereby
facilitating the temperature control of the liquid reservoir tank
in each system.
[0063] A circulating rate of a liquid is not particularly limited,
but generally about 1 mL/second to 100 mL/second, more preferably 5
mL/second to 50 mL/second and most preferably 7 mL/second to 15
mL/second.
[0064] FIG. 6A is a graph obtained from data of temperature control
realized by using the above-described mechanism. As can be
appreciated from FIG. 6A, the temperature can be increased from
60.degree. C. to 92.degree. C. and decreased to 60.degree. C.
within a short time of 1.5 seconds. FIG. 6B is a graph showing the
results from real-time PCR. The conditions of the solution upon
carrying out PCR were as follows. The followings were mixed in the
indicated proportion: 1.0 .mu.L of reaction buffer, 1 .mu.L of 2 mM
dNTP (dATP, dCTP, dGTP, dTTP), 1.2 .mu.L of 25 mM magnesium
sulfate, 0.125 .mu.L of 10% fetal bovine serum, 0.5 .mu.L of SYBR
Green I, 0.6 .mu.L each of two types of primers, 3.725 .mu.L of
sterile water, 0.25 .mu.L of KOD plus polymerase and 1.0 .mu.L of
genomic DNA. Temperature conditions were first 95.degree. C. for 10
seconds, then 40 cycles of temperature alteration at 95.degree. C.
for 1 second and 60.degree. C. for 3 seconds. The circulating rate
of the liquid was about 10 mL/second.
[0065] FIG. 7 shows variations of methods for attaching a reaction
vessel 59 and a reaction vessel casing 51 used in the reaction
control device of the present invention to the heat exchange
vessel. The reaction vessel 59 is held and attached to the
glass-slide like reaction vessel casing 51 (FIG. 7A). In order to
attach this glass-slide like reaction vessel casing 51 to the heat
exchange vessel, the reaction vessel casing 51 can transversely
slide along a guide rail 53 and pressed against a seal 54 for
fixation (FIG. 7B). Alternatively, the glass-slide like reaction
casing 51 can be inserted into a slide socket 55 and pressed
against a seal 57 utilizing a hinge 56 (FIG. 7C).
[0066] FIG. 8 gives schematic views showing variations of switching
mechanisms of a valve used in a reaction control device of the
present invention, having a driving mechanism with a slide piston
valve different from that shown in FIG. 5. A piston 65 that can
transversely slide is used as a valve mechanism for shifting the
temperature of a reaction vessel 66. On the left side of the piston
65, a liquid is introduced into a heat exchange vessel 67 via an
inlet A (61) and led outside via an outlet A (62). On the right
side of the piston 65, a liquid is introduced into the heat
exchange vessel 67 via an inlet B (63) and led outside via an
outlet B (64). When the piston 65 slides to the right with respect
to the reaction vessel 66, the temperature of the reaction vessel
66 comes to equilibrium with that of the liquid introduced via the
inlet A (61). On the other hand, when the piston 65 slides to the
left, the reaction vessel 66 comes to equilibrium with the
temperature of the liquid introduced via the inlet B (63). When the
piston 65 is positioned right below the reaction vessel 66, the
reaction vessel 66 can be removed without leakage of the liquid.
The piston 65 is preferably prepared from a material with good heat
insulation, or has a hollow inside filled with a gas or in vacuum
state. Here, the arrows in FIG. 8 briefly indicate flow directions
of the liquid.
[0067] FIG. 9 shows some variations of a driving mechanism of a
piston of a piston valve used in a reaction control device of the
present invention. According to one method, a piston 71 is
integrally formed with a piston rod 72 for direct driving from
outside (FIG. 9A). According to other method, a piston 73 is
prepared of a ferromagnetic material such as iron or nickel, or a
magnet 74 is incorporated inside the piston made of other material.
An electromagnetic coil 75 is externally provided to control the
current to slide the piston 73 from side to side (FIG. 9B).
According to other method, the pressure on the an inlet side or the
fluid resistance at the outlet can be controlled to slide the
piston 76 from side to side by utilizing difference in the
pressures between both sides of the piston 76 (FIG. 9C). Here, the
outlined arrows in FIG. 9 indicate the direction of the movement of
the piston whereas the solid arrows indicate the direction of the
flow of the fluid, where the pointings and the widths of the arrows
briefly show the flowing direction and flow rate of the fluid,
respectively.
[0068] FIG. 10 shows other embodiment of a switching mechanism of a
valve used in a reaction control device of the present invention. A
rotary valve 81 made from a slanted oval plate attached to a rod 82
as a rotation axis is inserted inside a circular cross-section heat
exchange vessel 83. The rotary valve 81 divides the heat exchange
vessel 83 to the right and left, and rotation of the rotation axis
82 can lead a liquid introduced from the right or left side of the
heat exchange vessel to the reaction vessel 84. The rotary valve 81
in FIG. 10 has a slanted flat plane shape, other shape such as a
spirally wound shape is also possible as long as it gives a similar
effect by rotating the rotation axis. Here, the solid arrow in FIG.
10 indicates the rotation direction of the rotation axis 82 while
the outlined arrows briefly indicate the flow of the liquids.
[0069] FIG. 11 shows a configuration in which a liquid is replaced
with a structure other than a valve. A heat exchange vessel 98 is
divided by a membrane A (95) and a membrane B (96). A liquid
introduced via an inlet A (91) is discharged outside via an outlet
A (92). The presence of the membranes prevents the liquid from
being discharged from an inlet B (93) or an outlet B (94) (FIG.
11A). When the pressure of the liquid introduced via the inlet A
(91) is higher than the pressure of the liquid introduced via the
inlet B (93), the membranes A (95) and B (96) are pushed toward the
left side so that the heat of the liquid introduced via the inlet A
(91) is conducted to the reaction vessel 97 (FIG. 11B). When the
pressure relationship between the liquids introduced from the
inlets A (91) and B (93) is reversed, the temperature of the
reaction vessel 97 comes to equilibrium with the temperature of the
liquid introduced via the inlet B (93) (FIG. 11C). The membranes
are preferably prepared from a thin membrane with good heat
resistance such as heat-resistant rubber. Here, arrows shown in
FIG. 11 briefly indicate the directions of the flow of the
liquids.
[0070] FIG. 12 is a schematic view showing other driving mechanism
of a temperature-setting valve used in a reaction control device of
the present invention. According to the present invention, the
number of temperatures to be set is not limited to two. FIG. 12
shows a configuration of setting three or more temperatures for the
reaction vessel. A rotary valve 101 with grooves 102 formed in the
side is inserted into a heat exchange vessel 103. Both sides of the
rotary valve 101 are provided with an inlet and an outlet. For
example, a liquid introduced via an inlet A (104) flows into the
grooves 102 via a flow channel 108 to conduct heat to a reaction
vessel 109 and then led outside via an outlet A (105). Meanwhile, a
liquid introduced via an inlet B (106) is led outside via an outlet
B (107) without making contact with a reaction vessel 109. However,
a liquid introduced via any inlet can be brought into contact with
the reaction vessel by rotating the rotary valve 101 (FIG. 12C).
The temperature 110 can be changed as represented by the graph
shown in FIG. 12C by rotating the rotary valve 101 with time 111.
The rotary valve 101 is preferably prepared from a heat insulating
material.
[0071] FIG. 13 is a schematic view showing an exemplary structure
of a heat exchange vessel used in a reaction control device of the
present invention. The upper panel A shows a lateral view and the
lower panel B shows a top view.
[0072] With reference to FIG. 13, a reaction vessel 1 is provided
with a plurality of concaved wells 306 arranged in arrays for
accommodating a sample. A heat exchange vessel 3 is arranged
beneath the reaction vessel 1 via an O-ring 305 in contact
therewith. The temperature of the reaction vessel 1 is adjusted by
a heat exchanging liquid introduced into the heat exchange vessel 3
via inlets A11 and A12. An anti-evaporation mechanism 301 is
arranged above the reaction vessel 1 in close contact therewith.
This anti-evaporation mechanism 301 prevents the sample solution
from evaporating and dissipating due to heating of the sample
solution with the heat from the heat exchange vessel 1. Typically,
the anti-evaporation mechanism 301 is provided with an adhering
member 302, an optically transparent member (e.g., glass heater)
303 and if necessary a polymeric sheet 304. The polymeric sheet 304
can enhance adhesion between the adhering member 302 and the
reaction vessel 1. A change in the fluorescent intensity of a
sample solution placed in the wells 306 can be detected via the
optically transparent member 303 with a fluorescence detector 202
whose operation is controlled with a control analyzer 201.
[0073] According to the example shown in FIG. 13, PCR reaction can
be carried out by repeating high-speed temperature shift of a small
amount of a reaction solution droplet of 1 to 10 microliters
mounted on each of the wells 306. First, a reaction vessel 1 having
PCR solution droplets mounted thereon is arranged in close contact
with a heat exchange vessel 3 via an O-ring 305. The heat exchange
vessel 3 is connected to a plurality of inlets for heat exchanging
liquids, where two or more heat exchanging liquids at different
temperatures are injected from the inlets. According to this
example, inlets A 11 and B 12 are shown as an example for heat
exchange between two temperatures, but the number of the inlets is
not limited thereto. If necessary, three or more inlets can be
provided in order to similarly realize three or more different
temperatures with the reaction vessel 1.
[0074] The optically transparent member 303 on the upper surface of
the anti-evaporation mechanism 301 is made from an optically
transmissive transparent material such as glass or plastic so that
optical characteristics such as change in the fluorescent intensity
of the reaction solution droplets in the wells 306 of the reaction
vessel 1 can be observed from outside with an optical device such
as a fluorescence detector 101. Furthermore, this optically
transparent member 303 may be a glass heater obtained by providing
a heat generating member made from an optically transparent
material such as ITO (Indium Tin Oxide) whose temperature can be
increased by running a current on the surface of the
above-mentioned optically transparent material to form an
integrated body of the optically transparent glass and the heat
generating member. Such a glass heater can be used to heat the
upper surface of the anti-evaporation mechanism to prevent the PCR
solution droplets in the reaction vessel 1 from evaporating.
[0075] Accordingly, one exemplary embodiment of the
anti-evaporation mechanism 301 comprises a glass heater 303 having
an integrated body of an optically transparent glass and a heat
generating mechanism, a sealing member 302, and a polymeric sheet
304 that adheres the reaction vessel 1 and the sealing member 302.
The reaction vessel 1 can be sandwiched by the anti-evaporation
mechanism 301 and the heat exchange vessel 3 so that even when a
small amount of reaction solution evaporates, saturated vapor
pressure is immediately restored in the space between the reaction
vessel 1 and the anti-evaporation mechanism 301. In order to
prevent moisture vapor from condensating on the inner wall of the
sealing member 302, the glass heater 303 or the like upon reaching
saturated vapor pressure, the temperature of the glass heater 303
that is accessible to the outer air can be heated to a range of
80.degree. C. to 110.degree. C., for example, thereby preventing
condensation of moisture vapor. In addition, a glass surface heated
by the glass heater 303 in this manner has a defogging effect, and
thus advantageous in that it does not interfere with detection of
the fluorescence intensity of the reaction solution with a
fluorescence detector 101.
[0076] The sealing performance of the sealing member 301 can be
enhanced with a polymeric sheet 304 or the like. Examples of
polymeric sheets that can be used include, but not limited to,
rubber and silicon.
[0077] Since a smaller volume of the space between the
anti-evaporation mechanism and the reaction vessel 1 can suppress
the total amount of the moisture vapor that reaches the saturated
moisture vapor pressure to a smaller amount, the distance between
the glass heater 303 and the surface of the reaction vessel 1 is
advantageously made as close as possible. The distance between the
glass heater 303 and the surface of the reaction vessel 1 is
preferably about 10 mm or less, more preferably about 7 mm or less,
still more preferably about 5 mm or less, and most preferably about
3 mm or less. According to this example, the glass heater 303 was
used as one example of the heating mechanism on the upper surface
of the anti-evaporation mechanism 301. Similarly, a metal plate or
the like having a heating mechanism or a heat conduction system can
be provided with an optically transparent window that allows
detection of fluorescence from the droplet with a fluorescence
detector 101, to be used in place of the glass heater 303. When an
anti-evaporation mechanism is used, evaporation of even a small
amount of droplet can be prevented and thus there is no need of
layering a liquid layer such as mineral oil above the droplet.
[0078] Thus, according to the example shown in FIG. 13, dissipation
of the sample solution due to evaporation of the liquid droplet of
the reaction vessel caused by heat from the heat exchange vessel 3
can be prevented.
INDUSTRIAL APPLICABILITY
[0079] The present invention is useful as a reaction device for
carrying out reaction that requires accurate control of the
temperature of a sample. The present invention is also useful as a
reaction device for carrying out reaction that requires rapid
shifting of the sample temperature.
[0080] In particular, the present invention is useful as a PCR
device capable of carrying out PCR reaction at high speed, high
accuracy and high amplification rate. Since a device of the present
invention can be downsized, it is also useful as a portable PCR
device.
[0081] In addition, since the present invention is capable of
preventing a sample solution from evaporating due to heating
thereof, it is useful for PCR reaction that uses a small amount of
sample.
DESCRIPTION OF REFERENCE NUMERALS
[0082] 1 Reaction vessel [0083] 2 Reaction vessel casing [0084] 3
Heat exchange vessel [0085] 4 Liquid reservoir tank [0086] 5 Heat
source [0087] 6 Stirring mechanism [0088] 7 Pump [0089] 8 Switching
valve [0090] 9 Bypass flow channel [0091] 10 Auxiliary temperature
control mechanism [0092] 11 Inlet A [0093] 12 Inlet B [0094] 13
Outlet A [0095] 14 Outlet B [0096] 21, 22, 23, 24, 26 Reaction
vessel [0097] 25 Lyophilized reagent [0098] 27 Dispensing chip
[0099] 28 Sample [0100] 29 Fiber ball [0101] 31 Reaction vessel
[0102] 32 Reaction vessel casing [0103] 33 Reaction vessel socket
[0104] 34 Thread [0105] 35 Seal [0106] 36 Tapered reaction vessel
casing [0107] 37, 38 Heat exchange vessel [0108] 41 Inlet valve A
[0109] 42 Outlet valve A [0110] 43 Inlet valve B [0111] 44 Outlet
valve B [0112] 51 Glass-slide like reaction vessel casing [0113]
52, 58 Reaction vessel socket of heat exchange vessel [0114] 53
Guide rail [0115] 54, 57 Seal [0116] 55 Slide socket [0117] 56
Hinge [0118] 59 Reaction vessel [0119] 61 Inlet A [0120] 62 Outlet
A [0121] 63 Inlet B [0122] 64 Outlet B [0123] 65 Piston [0124] 66
Reaction vessel [0125] 67 Heat exchange vessel [0126] 71 Piston
[0127] 72 Piston rod [0128] 73 Piston [0129] 74 Magnet [0130] 75
Electromagnetic coil [0131] 76 Piston [0132] 81 Rotary valve [0133]
82 Rotation axis [0134] 83 Heat exchange vessel [0135] 84 Reaction
vessel [0136] 91 Inlet A [0137] 92 Outlet A [0138] 93 Inlet B
[0139] 94 Outlet B [0140] 95 Membrane A [0141] 96 Membrane B [0142]
97 Reaction vessel [0143] 98 Heat exchange vessel [0144] 101 Rotary
valve [0145] 102 Grooves [0146] 103 Heat exchange vessel [0147] 104
Inlet A [0148] 105 Outlet A [0149] 106 Inlet B [0150] 107 Outlet B
[0151] 108 Flow channel [0152] 109 Reaction vessel [0153] 110
Temperature [0154] 111 Elapsed time [0155] 201 Fluorescence
detector [0156] 202 Control analyzer [0157] 203 Control signal
[0158] 204 Optical window [0159] 301 Anti-evaporation mechanism
[0160] 302 Sealing member [0161] 303 Glass heater [0162] 304
Polymeric sheet [0163] 305 O-ring [0164] 306 Well
* * * * *