U.S. patent application number 13/573138 was filed with the patent office on 2012-12-20 for reaction chip, reaction method, temperature controlling unit for gene treating apparatus and gene treating apparatus.
This patent application is currently assigned to Toppan Printing Co., Ltd.. Invention is credited to Shuichi AKASHI, Masahiko AMANO, Masaaki CHINO, Sayaka GOMI, Ryoko IMAGAWA, Eiji KAWATA, Daisuke NUMAI.
Application Number | 20120322110 13/573138 |
Document ID | / |
Family ID | 40579575 |
Filed Date | 2012-12-20 |
United States Patent
Application |
20120322110 |
Kind Code |
A1 |
GOMI; Sayaka ; et
al. |
December 20, 2012 |
Reaction chip, reaction method, temperature controlling unit for
gene treating apparatus and gene treating apparatus
Abstract
The reaction chip of the present invention has a plurality of
recesses 6 constituting a part of a reaction container and a groove
constituting a part of a channel formed on at least one of one face
of a first base material (resin base material 2) and one face of a
second base material (metallic base material) and a notch 15
showing a gradual increase in width and a gradual increase in depth
from one face 2d of the base material toward an inner wall surface
6d of the recess is formed on an edge of at least one recess in an
extending direction of the groove. One face of the first base
material and one face of the second base material are stuck
together opposite to each other to form the plurality of reaction
containers and the channel.
Inventors: |
GOMI; Sayaka; (Tokyo,
JP) ; AKASHI; Shuichi; (Tokyo, JP) ; NUMAI;
Daisuke; (Tokyo, JP) ; IMAGAWA; Ryoko; (Tokyo,
JP) ; CHINO; Masaaki; (Tokyo, JP) ; KAWATA;
Eiji; (Tokyo, JP) ; AMANO; Masahiko; (Tokyo,
JP) |
Assignee: |
Toppan Printing Co., Ltd.
Tokyo
JP
|
Family ID: |
40579575 |
Appl. No.: |
13/573138 |
Filed: |
August 23, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12799410 |
Apr 22, 2010 |
|
|
|
13573138 |
|
|
|
|
PCT/JP08/69273 |
Oct 23, 2008 |
|
|
|
12799410 |
|
|
|
|
Current U.S.
Class: |
435/91.2 |
Current CPC
Class: |
G01N 2035/00366
20130101; B01L 7/52 20130101; B01L 2300/0816 20130101; B01L
2200/0642 20130101; B01L 3/502723 20130101; B01L 3/527 20130101;
B01L 2400/084 20130101; B01L 2300/087 20130101; G01N 2035/00158
20130101; B01L 3/50851 20130101; B01L 2400/0677 20130101; B01L
2200/16 20130101; B01L 2200/0684 20130101; B01L 3/5025 20130101;
B01L 2300/123 20130101; B01L 2400/0655 20130101 |
Class at
Publication: |
435/91.2 |
International
Class: |
C12P 19/34 20060101
C12P019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 26, 2007 |
JP |
2007-279107 |
Oct 26, 2007 |
JP |
2007-279108 |
Oct 26, 2007 |
JP |
2007-279109 |
Claims
1. A reaction method using a reaction chip having a plurality of
reaction containers constituted by a pair of base materials and a
channel that mutually communicates with the plurality of reaction
containers, the method comprising the steps of: arranging a reagent
inside a recess of a first base material of the pair of base
materials, the recess constituting a part of one of the reaction
containers formed in the first base material; sealing the reagent
with a hot-melt sealing compound; producing the reaction chip,
having the reaction containers in which the reagent is arranged and
the channel, by sticking together the first base material and a
second base material of the pair of base materials, the second base
material constituted by a material whose thermal conductivity is
higher than that of the first base material; feeding a reagent
solution into the reaction containers through the channel; and
causing a reaction of the reagent and the reagent solution to
proceed while heat is added from a side of the second base
material, after the reagent and the reagent solution are brought
into contact with each other by heating the reaction chip from the
side of the second base material to melt the sealing compound.
2. The reaction method according to claim 1, wherein the first base
material and the second base material are stuck together through
thermal welding of a sealant layer provided on at least one side of
the first base material and the second base material by adding heat
from the side of the second base material.
3. The reaction method according to claim 1, wherein the recess in
the first base material and a recess in the second base material
constitute the one reaction container.
4. The reaction method according to claim 1, wherein a resin
material is used as the first base material and a metallic material
is used as the second base material.
5. The reaction method according to claim 1, wherein the sealing
compound is constituted by a material that is soluble in neither
the reagent nor the reagent solution.
6. The reaction method according to claim 1, wherein the one
reaction container is a reaction container for enzyme reaction.
Description
CROSS REFERENCE
[0001] This is a divisional of application Ser. No. 12/799,410,
filed Apr. 22, 2010, which is a continuation of PCT/JP2008/069273
filed on Oct. 23, 2008, which claims priority to Japanese
application numbers 2007-279107, 2007-279108 and 2007-279109, which
were filed on Oct. 26, 2007, all of which are incorporated by
reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a reaction chip and a
reaction method suitably used for a biochemical reaction such as a
chemical reaction, DNA reaction, and protein reaction and a
temperature controlling unit and a gene treating apparatus
including the temperature controlling unit for treatment such as
amplification on genes contained in a biological sample.
[0004] 2. Description of the Related Art
[0005] In recent years, in the field of, for example, a biochemical
reaction such as a chemical reaction, DNA reaction, and protein
reaction, a technology called a .mu.-TAS (Total Analysis System) or
Lab-on-Chip is studied and put to practical use as a technique to
treat a very small quantity of sample solution on a chip. This
enables a reaction experiment, which required large-scale
laboratory equipment and a large quantity of reaction reagents in
the past, to perform with a small quantity of reaction reagents
using a reaction chip measuring several mm or less pre side.
[0006] Examples of this kind of biochemical reaction include a DNA
amplification reaction by an enzyme reaction, hybridization
reaction to detect a sequence of specimen DNA by using a probe DNA
having a known sequence, and detection reaction of SNP (monobasic
polymorphism) in a DNA sequence. The invader (registered trademark)
method and the TaqMan PCR method are known as SNP detection methods
(see, for example, Patent Document 1).
[0007] When these reactions are caused using a chip, for example,
to decide the sequence of a gene or DNA, a method by which a probe
DNA is fixed onto slide glass to allow a hybridization reaction
thereon is known.
[0008] Further, a method by which a microscopic hole or dent called
a well is formed on a chip to use the well as a reaction field is
known.
[0009] A plurality of well-shaped reaction containers is mutually
connected by a reagent solution channel installed from a reagent
reservoir part (see, for example, Patent Document 2). When a
reagent solution is fed by using such a channel, it is important to
fill a reaction container with the reagent solution to prevent
bubbles from being left behind. If bubbles remain inside the
reaction container, quantities and concentrations of the reagent
solution in each reaction container fluctuate, leading to
fluctuations of reaction states. Moreover, even if reaction states
do not fluctuate, it is extremely probable that an error of
photometric intensity is caused by the bubbles.
[0010] Thus, several methods are proposed to remove bubbles from
the reaction container.
[0011] As a method thereof, a liquid circuit having at least one
channel inside a layered product formed by laminating a plurality
of substrates in which a communication hole communicating the
channel and outside bypassing through at least one substrate is
proposed (see, for example, Patent Document 3). Patent Document 3
discloses that the communication hole is a hole formed in a
single-crystal silicon substrate or glass substrate by using
photolithography and has a tapered inner circumferential surface
with an increasingly smaller opening area from the channel toward
the outside to remove bubbles to the outside through the
communication hole. Further, the communication hole preferably has
at least hydrophobicity and Patent Document 3 describes for this
purpose that the substrate itself has hydrophobicity or adding
hydrophobicity to a substrate having no hydrophobicity
afterwards.
[0012] Further, a reaction chip having a channel passing through a
first surface and a second surface and including a bubble trap to
separate bubbles fed together with a sample in each sample hole is
proposed (see, for example, Patent Document 4).
[0013] Incidentally, a reagent solution fed to a chip used for
reaction analysis is frequently a reagent solution having high
viscosity such as organic substance for a chemical reaction and
extracted DNA, synthetic DNA, and enzyme for a biochemical
reaction. When using such a reagent solution, according to the
method described in Patent Document 2 or 3, there is a possibility
that bubbles are not sufficiently removed or separated so that
bubbles remain in the reaction container. Moreover, the method
described in Patent Document 2 or 3 is a technology applicable only
to a so-called open reaction container that is open to the outer
space and is not applicable to a closed reaction container whose
outer circumference is completely enclosed by walls.
[0014] In addition, a method of making a reaction container
hydrophobic with surface treatment such as corona treatment and
plasma treatment is frequently used to remove bubbles. In this
case, however, surface modification of the reaction container may
occur to result in different batch reaction conditions such as a
change in pH, posing a problem of a possibility of an intended
desired reaction from being blocked. Moreover, if plasma treatment
is applied, surface modification of the reaction container occurs,
but the surface modification is hard to persist so that there is a
problem that the state immediately after treatment cannot be
maintained.
[0015] When a reaction is caused using these analysis chips, a
reaction reagent is first arranged inside a plurality of
well-shaped reaction containers. Next, a reaction reagent solution
is fed to the plurality of well-shaped reaction containers via a
channel by infusing the reaction reagent solution into the analysis
chip. Accordingly, the fixing reagent and the reaction reagent
solution come into contact to start a reaction. The well-shaped
reaction containers are heated during reaction if necessary.
[0016] However, according to the above reaction method, when the
reaction reagent solution is fed into the well-shaped reaction
container via the channel, there is a possibility that the fixing
reagent prearranged inside the well-shaped reaction container flows
out to adjacent well-shaped reaction containers. Accordingly, there
is a problem that contamination may be caused. There is also a
possibility that the fixing reagent, reaction reagent solution, or
fluorescent substance for detection is diffused into the adjacent
well-shaped reaction containers during reaction in each well-shaped
reaction container. Accordingly, there is a problem that it becomes
impossible to measure accurate reaction data.
[0017] Thus, a reaction chip and a reaction method capable of
preventing an occurrence of such contamination and measuring
accurate reaction data are disclosed (see, for example, Patent
Document 5).
[0018] The reaction chip described in Patent Document 5 is
constituted by a substrate forming a well-shaped reaction container
and a cover material covering the substrate. The reaction method
using the reaction chip is to cover a reaction reagent arranged in
the well-shaped reaction container with a hot-melt sealing compound
and feed the reaction reagent solution on top of the sealing
compound before the sealing compound being melted by heating to
bring the reaction reagent and the reaction reagent solution into
contact. According to the method, the reaction reagent will not
flow out to adjacent well-shaped reaction containers, so that
contamination can be prevented from occurring.
[0019] For a reaction chip used for biochemical reaction such as an
enzyme reaction, it is advantageous to use a substrate with a high
thermal conductivity because such a reaction frequently requires
heating a reagent. However, if a reaction reagent is fixed onto a
substrate with a high thermal conductivity, heat when the substrate
and cover material were stuck together may be conducted to the
reaction reagent, posing a problem that activity of the reaction
reagent is lowered or devitalized. Speaking of, for example, the
reaction method described in Patent Document 5, it is desirable to
use a substrate with a high thermal conductivity as a substrate on
the side on which a well-shaped reaction container is formed, but
in such a case, the above problem is caused.
[0020] If a reaction reagent is fixed onto a substrate with a high
thermal conductivity and the reaction reagent is covered, like the
method in Patent Document 5, with a hot-melt sealing compound, the
sealing compound is melted by heat when the substrate and cover
material were stuck together, posing a problem that the sealing
compound flows out to a channel of the reaction chip to block the
channel or the shape of the sealing compound when re-solidified
becomes unstable, leading to incomplete sealing of the reaction
reagent. Because of this problem, there is a possibility that
contamination cannot be sufficiently prevented from occurring.
[0021] In these genetic tests, the amount of nucleic acid (DNA)
contained in a sample is amplified by the polymerase chain reaction
(PCR) for the test and an attempt is being made to make the test
faster by reducing the time necessary for the PCR.
[0022] As a method of executing the PCR in a shorter time, an
attempt is being made to execute the PCR with a smaller amount of
sample and a reaction container and a reaction apparatus
(temperature controlling unit) therefor are devised.
[0023] Most reaction containers are made of synthetic resin that
does not inhibit a biological reaction and a reaction is caused by
reducing a reaction volume to several tens microliter. In addition,
in some instances, the reaction container is formed from
aluminum.
[0024] In such reaction containers, a PCR reaction is allowed to
occur without a minimum amount of sample being evaporated by a
heating unit being brought into contact from above and below by a
reaction apparatus described in Patent Document 6 or 7.
[0025] The reaction apparatus described in Patent Document 6 or 7
causes no big problem if the reaction container is formed from a
single material. However, if a PCR reaction is allowed to occur by
using a container constructed by separate materials having
different thermal conductivities in an upper part and a lower part
of the reaction container for the purpose of improving performance
of the reaction container, the temperature distribution of the
sample inside the reaction container becomes inhomogeneous due to a
difference in thermal conductivity, posing a problem that the PCR
reaction does not proceed smoothly. [0026] Patent Document 1:
Japanese Patent Application Laid-Open No. 2002-300894 [0027] Patent
Document 2: Japanese Patent Application National Publication No.
2002-503336 [0028] Patent Document 3: Japanese Patent Application
Laid-Open No. 9-257748 [0029] Patent Document 4: Japanese Patent
No. 2955229 [0030] Patent Document 5: Japanese Patent Application
Laid-Open No. 2007-090290 [0031] Patent Document 6: Japanese Patent
No. 3661112 [0032] Patent Document 7: Japanese Patent No.
3686917
SUMMARY OF THE INVENTION
[0033] The present invention has been made to solve the above
problems.
[0034] A first object of the present invention is to provide a
reaction chip capable of easily feeding a liquid without surface
modification of a reaction container and leaving bubbles behind and
performing accurate detection and measurement of a desired
reaction.
[0035] A second object of the present invention is to provide a
reaction method capable of reliably preventing an occurrence of
contamination and measuring accurate reaction data without lowering
or devitalizing activity of a reaction reagent.
[0036] A third object of the present invention is to provide a
temperature controlling unit for a gene treating apparatus and a
gene treating apparatus capable of treating genes contained in a
gene sample filled in a reaction container swiftly and
appropriately even if the reaction container constructed from a
plurality of materials having different thermal conductivities is
used.
[0037] A thorough examination by the present inventors to attain
the first object shows, as a result, that a conventional reaction
container composed of a recess having a steep inner wall surface
traps bubbles particularly in a space sandwiched between the inner
wall surface present in a flow direction of a reagent solution and
a bottom. Therefore, the present inventors conceive the
constitution of the present invention by realizing that it becomes
harder for bubbles to remain in a recess by making such an inner
wall surface smaller and adopting a configuration that allows a
reagent solution to flow smoothly near the recess.
[0038] A reaction chip in the present invention is a reaction chip
having a plurality of reaction containers constituted by a pair of
base materials to cause a reaction between a reagent and a reagent
solution and a channel that mutually communicates the plurality of
reaction containers to feed the reagent solution to the plurality
of reaction containers, including forming a plurality of recesses
constituting a part of the reaction container on at least one of
one face of a first base material and one face of a second base
material of the pair of base materials, forming a groove
constituting a part of the channel at a position corresponding to
between the recess and the recess on at least one of one face of
the first base material and one face of the second base material,
forming a notch showing a gradual increase in width and a gradual
increase in depth from one face of the base material where the
recess is formed toward an inner wall surface of the recess on an
edge of at least one recess of the recesses in an extending
direction of the groove, and forming the plurality of reaction
containers and the channel by one face of the first base material
and one face of the second base material being stuck together
facing each other.
[0039] In the reaction chip of the present invention, an angle
formed by one face of the base material where the recess is formed
and the inner wall surface of the notch is smaller than the angle
formed by one face of the base material where the recess is formed
and the inner wall surface of the recess.
[0040] In the reaction chip of the present invention, the notch is
formed on at least an inflow side of the reagent solution flowing
through the groove on the edge of the recess in the extending
direction of the groove.
[0041] In the reaction chip of the present invention, a
configuration may be adopted in which the notch is formed on at
least an outflow side of the reagent solution flowing through the
groove on the edge of the recess in the extending direction of the
groove so that the notch formed on the inflow side of the reagent
solution and the notch formed on the outflow side of the reagent
solution form a line symmetric shape.
[0042] In the reaction chip of the present invention, the recess
has a columnar space having the inner wall surface at substantially
right angles to one face of the base material where the recess is
formed on at least an opening side, and the maximum depth on the
edge of the notch is shallower than the depth of the columnar
space.
[0043] An outer shape of the recess is circular in plane view and a
plane shape of the notch is defined, when two tangents to a circle
forming an outer edge of the recess are drawn from one point on one
face of the base material where the recess is formed in the
extending direction of the groove, by an area inside the two
tangents.
[0044] In the reaction chip of the present invention, a center line
in the extending direction of the groove of the notch is aligned
with the center line of the groove on a same straight line.
[0045] To achieve the second object, a reaction method of the
present invention is a reaction method using a reaction chip having
a channel that mutually communicates a plurality of reaction
containers constituted by a pair of base materials and the
plurality of reaction containers, including the steps of arranging
a reagent inside a recess by using, of the pair of base materials,
the first base material having the recess constituting a part of
the reaction container formed therein, sealing the reagent with a
hot-melt sealing compound, producing the reaction chip having the
reaction containers in which the reagent is arranged and the
channel by sticking the second base material constituted by a
material whose thermal conductivity is higher than that of the
first base material and the first base material together, feeding a
reagent solution into the reaction containers through the channel,
and causing a reaction of the reagent and the reagent solution to
proceed while heat being added from a side of the second base
material after the reagent and the reagent solution being brought
into contact by heating the reaction chip from the side of the
second base material to melt the sealing compound.
[0046] In the reaction method of the present invention, the first
base material and the second base material are stuck together
through thermal welding of a sealant layer provided on at least one
side of the first base material and the second base material by
adding heat from the side of the second base material.
[0047] In the reaction method of the present invention, the recess
corresponding to the recess in the first base material is also
formed in the second base material to constitute the reaction
container by both the recess of the first base material and the
recess of the second base material.
[0048] In the reaction method of the present invention, a resin
material is used as the first base material and a metallic material
is used as the second base material.
[0049] In the reaction method of the present invention, the sealing
compound is constituted by a material that is insoluble in neither
the reaction reagent nor the reagent solution.
[0050] In the reaction method of the present invention, the
reaction container is a reaction container for enzyme reaction.
[0051] To achieve the third object, a temperature controlling unit
for gene treating apparatus of the present invention is a
temperature controlling unit for gene treating apparatus that
treats a gene inside a gene sample by heating/cooling the gene
sample filled in a reaction container composed of a first member
arranged in an upper part and a second member having a different
thermal conductivity from that of the first member and arranged in
a lower part, including a first temperature controlling unit
arranged in such a way to allow contact with a top face of the
reaction container, a second temperature controlling unit arranged
in such a way to allow contact with an undersurface of the reaction
container and also arranged in such a way to be able to sandwich
the reaction container between the first temperature controlling
unit and the second temperature controlling unit, a pair of
metallic plates arranged on surfaces where the first temperature
controlling unit or the second temperature controlling unit is in
contact with the reaction container, a pair of heat conduction
members arranged on surfaces of the pair of metallic plates facing
the reaction container and also arranged in such a way to allow
contact with the top face and the undersurface of the reaction
container, a first heat dissipation unit provided in contact with
the first temperature controlling unit, and a second heat
dissipation unit provided in contact with the second temperature
controlling unit.
[0052] According to a temperature controlling unit for gene
treating apparatus of the present invention, heat of the first
temperature controlling unit or the second temperature controlling
unit is dissipated uniformly by a pair of metallic plates and
transmitted efficiently to a reaction container by a pair of heat
conduction members.
[0053] The temperature controlling unit for gene treating apparatus
of the present invention further includes a control unit connected
to the first temperature controlling unit and the second
temperature controlling unit to control temperatures of the first
temperature controlling unit and the second temperature controlling
unit, wherein the control unit may exercise temperature control of
the first temperature controlling unit and the second temperature
controlling unit independently based on the thermal conductivities
of the first member and the second member.
[0054] In this case, the temperature difference of a gene sample
between the first member and the second member becomes smaller, so
that the gene can be treated more suitably.
[0055] A gene treating apparatus of the present invention includes
a temperature controlling unit for gene treating apparatus of the
present invention.
[0056] According to a gene treating apparatus of the present
invention, a gene can be treated quickly and suitably even if a
reaction container composed of a first member and a second member
is used.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing (s) will be provided by the Office
upon request and payment of the necessary fee.
[0058] FIG. 1 is a perspective view of a reaction chip according to
an embodiment of the present invention.
[0059] FIG. 2 is a plan view of a resin base material constituting
the reaction chip.
[0060] FIG. 3 is a plan view of a metallic base material
constituting the reaction chip.
[0061] FIG. 4 is an enlarged view of a recess of the resin base
material, and FIG. 4A is a perspective view thereof, FIG. 4B is a
plan view thereof, and FIG. 4C is a side sectional view
thereof.
[0062] FIG. 5 is a diagram showing another example of the recess,
and
[0063] FIG. 5A is a perspective view thereof and FIG. 5B is a side
sectional view thereof.
[0064] FIGS. 6A, 6B and 6C are process sectional views showing a
reaction detection method using the reaction chip following
procedures thereof.
[0065] FIGS. 7A, 7B and 7C are process sectional views when the
reaction chip of another type is used.
[0066] FIG. 8 is a perspective view of a reaction chip in an
embodiment of the present invention.
[0067] FIG. 9A is a plan view of the reaction chip and FIG. 9B is a
sectional view along a line A-A' in FIG. 8.
[0068] FIGS. 10A, 10B and 10C are process sectional views showing a
reaction method using the reaction chip following procedures
thereof.
[0069] FIG. 11 is a sectional view showing another example of the
reaction chip.
[0070] FIG. 12 is a perspective view showing the configuration of a
gene amplifying apparatus including a temperature controlling unit
for gene amplifying apparatus according to an embodiment of the
present invention.
[0071] FIG. 13 is a schematic sectional view showing a state where
a reaction container is sandwiched by the temperature controlling
unit for gene amplifying apparatus.
[0072] FIG. 14 is an enlarged sectional view in the vicinity of the
reaction container in FIG. 13.
[0073] FIG. 15 is a perspective view showing the reaction
container.
[0074] FIG. 16 is a sectional view along a line A-A' in FIG.
15.
[0075] FIG. 17 is a sectional view along a line B-B' in FIG.
15.
[0076] FIG. 18 is a graph showing a temperature cycle by the PCR
method, temperature control by the temperature controlling unit for
gene amplifying apparatus, and temperature changes of each unit of
the reaction container.
[0077] FIG. 19 is a graph showing temperature changes when the
reaction container 100 is used in a conventional gene amplifying
apparatus.
[0078] FIG. 20 is an enlarged view of the recess of the reaction
chip in Comparative Example 1, and FIG. 20A is a plan view thereof
and FIG. 20B is a side sectional view thereof.
[0079] FIGS. 21A, 21B and 21C are process sectional views when the
reaction chip of Comparative Example 1 is used.
[0080] FIG. 22 is a photo shooting conditions inside the reaction
container of the reaction chip of Example 1.
[0081] FIG. 23 is a photo shooting conditions inside the reaction
container of the reaction chip of Comparative Example 1.
[0082] FIG. 24A is a plan view showing reagent arrangement of
Example 2 and Comparative Example 2 of the present invention, FIG.
24B is a sectional view of the reaction chip of Example 2, and FIG.
24C is a sectional view of the reaction chip of Comparative Example
2.
[0083] FIGS. 25A and 25B are graphs showing reaction results of
Example 2.
[0084] FIG. 26 is a graph showing reaction results of Comparative
Example 2.
[0085] 1: Reaction chip, 2: Resin base material (first base
material), 3: Metallic base material (second base material), 4:
Reaction container, 5: Channel, 6: Recess (of a resin base
material), 6a: Columnar space, 6b: Truncated cone shaped space, 11:
Recess (of a metallic basematerial), 12: Groove, 15, 16: Notch, 21:
Reaction chip, 22: Cover material (first base material), 23:
Substrate (second base material), 24: Reaction container, 25:
Channel, 26: Recess (of a cover material), 27: Recess (of a
substrate), 28: Groove, 30: Reagent solution injecting hole, 31:
Through hole, 41: Temperature controlling unit for gene amplifying
apparatus, 42: Gene amplifying apparatus (gene treating apparatus),
43: Movable carriage, 44: Measuring unit, 45: Moving unit, 46:
Rail, 47: Emission detection unit, 48: Measuring unit moving unit,
49: First unit, 50: Second unit, 51: Support arm, 52: First
temperature controlling unit, 53: First heat sink (first heat
dissipation unit), 54: Second temperature controlling unit, 55:
Second heat sink (second heat dissipation unit), 56: First heat
conduction layer, 58: Control unit, 59: Metallic plate, 60: Second
heat conduction layer (heat conduction member), 61: Heat insulating
material, 100: Reaction container, 101: First member, 102: Second
member, 103: Well, 104: Reagent, 105: Channel, 106: Injecting hole,
107: Deaeration port, S: Reagent, W: Sealing compound, L: Reagent
solution
DETAILED DESCRIPTION OF THE INVENTION
First Embodiment
[0086] An embodiment of the present invention will be described
below with reference to FIGS. 1 to 7.
[0087] The present embodiment shows an example of a reaction chip
for biochemical reaction analysis.
[0088] FIG. 1 is a perspective view of a reaction chip in the
present embodiment. FIG. 2 is a plan view of a resin base material
(first base material) constituting the reaction chip. FIG. 3 is a
plan view of a metallic base material (second base material)
constituting the reaction chip. FIGS. 4A to 4C are enlarged views
of a recess of the resin base material and FIG. 4A is a perspective
view thereof, FIG. 4B is a plan view thereof, and FIG. 4C is a side
sectional view thereof. FIGS. 5A and 5B are diagrams showing
another example of the recess and FIG. 5A is a perspective view
thereof and FIG. 5B is aside sectional view thereof. FIGS. 6A to 6C
are process sectional views showing a reaction detection method
using the reaction chip following procedures thereof. FIG. 7 is a
process sectional view when the reaction chip of another type is
used.
[0089] For convenience of description, it is assumed below that the
side of resin base material positioned on the upper side, when a
fluorescent reaction is detected or measured, is the "upper side"
and the side of metallic base material positioned on the lower side
is the "lower side".
[0090] A reaction chip 1 in the present embodiment is a small chip
that has, as shown in FIG. 1, a rectangular shape whose plane shape
has about several tens mm both in length and width and a thickness
of about several mm. The reaction chip 1 is constituted by a resin
base material 2 (first base material) and a metallic base material
3 (second base material) arranged on the lower side of the resin
base material 2. In the reaction chip 1 in the present embodiment,
the resin base material 2 has recesses constituting reaction
containers 4 formed therein and the metallic base material 3 has
recesses constituting the reaction containers 4 and grooves
constituting channels 5 formed therein.
[0091] A plate material of polypropylene superior in terms of light
transmission, heat resistance, chemical resistance, molding
workability, and strength may be used as the resin base material 2.
In addition to this, a resin material such as polycarbonate,
acryl(polymethyl methacrylate), polyethylene terephthalate,
polyethylene, polyvinyl chloride, and polystyrene as materials
having similar characteristics.
[0092] The thickness of the resin base material 2 is preferably
such that the resin base material 2 should not easily be bent while
being used. Moreover, the resin base material 2 may be formed by
two types of resin or more being bonded. In such a case, various
base materials in accordance with physical properties of the
reaction reagent or sample can be produced by preparing base
materials making the most of characteristics of each resin so that
different base materials can be used for different purposes. For
example, the material for the upper part and that for the lower
part of the base material may be separated. Further, the material
of base material is not limited to resin and quartz glass may also
be used.
[0093] The resin base material 2 has, as shown in FIG. 2, a
plurality (in the present embodiment 36 recesses, 6 rows.times.6
columns) of recesses 6 constituting a part of the reaction
container 4 formed on the undersurface thereof. These recesses 6 do
not mutually communicate and thus is isolated. The plane shape of
the recess 6 is circular and the sectional shape thereof is, as
shown in FIG. 4C, a columnar space 6a on the side closer to the
undersurface of the resin base material 2 and a truncated cone
shaped space 6b on the side farther from the undersurface. The
shape of the recess 6 (together with the plane shape and sectional
shape) can appropriately be designed such that the whole reagent
can reliably be accommodated at the bottom of the recess 6 in
accordance with the amount of reagents or reagent solutions
necessary for a reaction. However, if, like the present embodiment,
a configuration in which the recess has a truncated cone shaped
space on the bottom side of a columnar space is adopted, a liquid
settles down in a portion of the truncated cone shaped space with
stability so that a reagent or a fixing agent can reliably be
accommodated. Particularly, if the base material on the side on
which a recess is formed is constituted by a material having light
transmission, the flat bottom of the truncated cone shaped space is
suitable for detection of a fluorescent reaction so that
fluorescence can be detected accurately.
[0094] The recess 6 is formed by methods of cutting a resin plate
constituting the resin base material 2, injection-molding a resin
material constituting the base material or the like. In view of
miniaturization of the reaction chip, the diameter of the recess 6
(the reaction container 4) is preferably about 0.01 mm or more and
10 mm or less. This makes feeding of a reagent solution described
later relatively easier and can reduce the amount of a fixing
reagent or reagent solution to a minimum. As described later, a
reagent necessary for an incipient reaction of a sample containing
DNA added when used is arranged in each of the recesses 6 of the
resin base material 2. Alternatively, the reagent may be arranged
only in a portion of the recesses 6 so that plural types of
reactions can be caused in one reaction chip.
[0095] The configuration of the recess 6 will be described
later.
[0096] As shown in FIGS. 1 and 2, a plurality (in the present
embodiment, six) of reagent solution injecting holes 7 is provided
at one end on the top face (the surface on the opposite side of the
surface where the recesses 6 are formed) of the resin base material
2. The reagent solution injecting hole 7 is communicatively
connected to a through hole (not shown) passing through a top plate
part 2a of the resin base material 2 and is formed in a cylindrical
shape protruding upward. Air discharge holes 8 are provided at the
other end of the resin base material 2 on the opposite side of the
side where the reagent solution injecting holes 7 are provided. The
air discharge hole 8 has a cylindrical shape, has a through hole in
the center, and has a filter (not shown) filled in the through
hole. The filter has a function to smoothly pass a reagent solution
by allowing air to pass through while the reagent solution flows.
On the other hand, when the reagent solution that has flown through
the channel reaches the air discharge hole 8, the filter has a
function to prevent the reagent solution from flowing out by
holding back the reagent solution. A frame part 2b that hangs down
from the top plate part 2a is provided on the edge of the top plate
part 2a of the resin base material 2, and the metallic base
material 3 is arranged and fixed inside the frame part 2b.
[0097] An aluminum sheet, for example, can be used as the metallic
base material 3 and a resin sealant layer (not shown) is formed on
one side of the aluminum sheet. The resin sealant layer is made of
polypropylene as a main material and is a bonding layer that can
thermally be welded with the metallic base material 3 and the resin
base material 2.
[0098] In addition to aluminum, copper, silver, nickel, brass, or
gold may be used as the material of the metallic base material
3.
[0099] The metallic base material 3 has, as shown in FIG. 3, a
plurality (in the present embodiment, 36) of recesses 11
constituting a part of the reaction container 4 formed on the top
face of the metallic base material 3. These recesses 11 are formed
at a position corresponding to the recesses 6 of the resin base
material 2 when the metallic base material 3 and the resin base
material 2 are aligned.
[0100] In contrast to the recess 6 of the resin base material 2,
the sectional shape of the recess 11 has, as shown in FIG. 6, a
substantially hemispheric shape. While the recesses 6 and 11
correspond one-to-one between the resin base material 2 and the
metallic base material 3 in the present embodiment, one-to-one
correspondence may not necessarily be realized or recesses of
different sizes may be formed depending on purposes of use. In the
present embodiment, the volume of the recess 6 and that of the
recess 11 are substantially the same on the resin base material 2
side and the metallic base material 3 side.
[0101] Moreover, a groove 12 constituting a part of the channel 5
is formed between the recesses 11 on the top face of the metallic
base material 3. The reaction chip 1 in the present embodiment has,
as shown in FIGS. 1 and 3, six sets of the channel 5 and the six
recesses 11 (reaction containers 4) are serially connected in one
set of the channel 5. A slight recess 13 is formed at a position
corresponding to each of the reagent solution injecting holes 7 and
each of the air discharge holes 8, and the groove 12 is also formed
between the recess 13 and the recess 11. Thus, a reagent solution
injected from each of the reagent solution injecting holes 7 flows
through the channel 5 and, after the six reaction containers 4
being filled successively, is held back by the filter of the air
discharge holes 8.
[0102] The configuration of the recess 6 of the resin base material
2 will be described in detail below using FIGS. 4A to 4C.
Incidentally, FIG. 4A alone is depicted by inverting vertically so
that the shape of the recess 6 can be made easier to view.
[0103] The recess 6 has a circular shape with a diameter T1 as a
plane shape, the columnar space 6a with the diameter T1 and a depth
D1 as a sectional shape on the side closer to the undersurface of
the resin base material 2, and the truncated cone shaped space 6b
with a diameter T2 of the circle at the bottom and a depth D2 as a
sectional shape on the side farther from the undersurface (bottom
side of the recess). A notch 15 showing a gradual increase in width
and a gradual increase in depth from an undersurface 2d of the
resin base material 2 toward an inner wall surface 6d of the recess
6 is formed on edges of both an inflow side and an outflow side of
a reagent of the recesses 6 along an extending direction of the
groove 12 (the channel 5). The notch 15 on the inflow side and that
on the outflow side of a reagent have the same shape and are
arranged, as shown in FIG. 4B, symmetrically with respect to a
center line C extending in a direction perpendicular to the
extending direction of the groove 12. The notch 15 on the inflow
side and that on the outflow side of a reagent may have different
shapes and may not necessarily be symmetric with respect to the
center line C.
[0104] As shown in FIG. 4A, the notch 15 has a shape cut out like a
triangular pyramid shape from the undersurface 2d of the resin base
material 2 toward the inner wall surface 6d of the recess 6 in the
extending direction of the groove 12. Therefore, the bottom of the
notch 15 forms an acute valley line 15b. In the columnar space 6a
of the recess 6, the inner wall surface 6d rises steeply at
substantially right angles to the undersurface 2d of the resin base
material 2 and, as shown in FIG. 4C, an angle .theta.1 formed by
the undersurface 2d of the resin base material 2 and the valley
line 15b of the notch 15 becomes sufficiently smaller than an angle
.theta.2 (.theta.2.apprxeq.90.degree. formed by the undersurface 2d
of the resin base material 2 and the inner wall surface 6d of the
recess 6 due to formation of the notch 15. The bottom of the notch
15 need not necessarily be an acute valley line and may be, for
example, a gently curved surface.
[0105] The plane shape of the notch 15 is defined, when two
tangents L1 and L2 to a circle forming an outer edge of the recess
6 are drawn from any point A on the undersurface 2d of the resin
base material 2 in the extending direction of the groove 12, as
shown in FIG. 4B, by an internal area enclosed by the two tangents
L1 and L2 and the circle. If the angle formed with the two tangents
L1 and L2 is .theta., .theta. is preferably 5.degree. or more. This
is because .theta. of 5.degree. or less makes processing harder and
also an effect of bubble removal is hardly achieved. A distance T3
from the point A to an intersection of the valley line 15b of the
notch 15 and the circle can be appropriately decided in accordance
with an interval between the adjacent recesses 6 of the resin base
material 2. The recess 6 is designed such that the center line (the
valley line 15b) along the extending direction of the groove 12 of
the notch 15 is aligned with the center line of the groove 12 of
the metallic base material 3 on the same straight line. With the
above plane shape, the plane shape of each of the recesses 6 in the
present embodiment has a shape that has sufficiently small flow
resistance so that a reagent solution flows extremely smoothly.
[0106] On the other hand, the sectional shape when the notch 15 is
cut along the center line extending in the extending direction of
the groove 12 of the notch 15 is as shown in FIG. 4C. Lines
represented as solid lines are what visually appears of the notch
15 and those represented as chain double-dashed lines are a
(virtual) triangle for design when the recess 6 is designed so that
the notch 15 is cut out in a triangular shape as the sectional
shape. The virtual depth at a vertex positioned inside the columnar
space 6a of the triangle is set as D3. Consequently, the point
where the valley line 15b of the notch 15 and the inner wall
surface 6d of the recess 6 (the columnar space 6a) intersect is a
point where the distance of the intersection point from the
undersurface 2d of the resin base material 2 becomes a maximum
depth D4 of the notch 15.
[0107] Examples of dimensions in the present embodiment are: the
diameter T1 of the recess (columnar space) is 3 mm, the diameter T2
of the truncated cone shaped space is 2 mm, the distance T3 from
the point A to an intersection of the valley line 15b of the notch
15 and the circle is 1 mm, the depth D1 of the columnar space 6a is
0.8 mm, the depth D2 of the truncated cone shaped space 6b is 0.7
mm, and the virtual depth D3 at a vertex positioned inside the
columnar space 6a of the virtual triangle is 0.6 mm. These
dimensions are only examples and the design can be changed when
appropriately.
[0108] In the example shown in FIG. 4C, the depth D4 to be the
maximum depth of the notch 15 is shallower than the depth D1 of the
columnar space 6a of the recess 6. Thus, even if the notch 15 is
formed on the edge of the columnar space 6a, the vertical inner
wall surface 6d in the columnar space 6a of the recess 6 remains at
the position of the valley line 15b of the notch 15. Therefore, in
this example, a volume capable of accommodating reagents or wax
described later can be ensured and also reagents or wax can
reliably be accommodated inside the columnar space 6a and the
truncated cone shaped space 6b so that the top face of the wax is
positioned in a part of the columnar space 6a in which the vertical
inner wall surface 6d remains.
[0109] Alternatively, as shown in FIGS. 5A and 5B, a design in
which a dimension D4' to be the maximum depth of a notch 16 is made
equal to the depth D1 of the columnar space 6a of the recess 6 may
be adopted. In this case, the size of the notch 16 becomes
substantially larger than the configuration shown in FIG. 4B and no
vertical inner wall surface in the columnar space 6a of the recess
6 remains at the position of a valley line 16b of the notch 16. In
this example, while the volume of the space to accommodate reagents
or wax is slightly reduced when compared with the configuration
shown in FIG. 4B, reagents are made easier to flow, so that bubbles
can be prevented from remaining.
[0110] A manufacturing method of a reaction chip in the present
embodiment will be described below using FIGS. 6 and 7.
[0111] As shown in FIG. 6A, after a resin sealant layer is formed
on one side of an aluminum sheet to produce a base material sheet,
the metallic base material 3 including a plurality of recesses 11
and a plurality of grooves 12 is produced by a method of drawing on
the base material sheet or the like. On the other hand, the resin
base material 2 having a plurality of recesses 6 is formed by a
method of injection molding or the like. Then, the notch 15 is
formed on the edge of the recess 6 by a method of cutting or the
like. Any cutting method can be selected. The resin base material 2
including the plurality of recesses 6 having the notch 15 from the
start may be produced by the method of injection molding or the
like.
[0112] Next, the opening of the recess 6 is directed upward and a
reagent S is put into the plurality of recesses 6 of the resin base
material 2 before being fixed. Further, the reagent S is covered
with wax W before being solidified. The wax (fixing material)
herein is a material that covers the reagent arranged inside the
recess 6 and remains in a solid state until a reaction between a
reagent and a reagent solution begins. The wax may be a single
material or made up of a plurality of materials (for example, a
mixture). In embodiments of the present embodiment, hot-melt wax is
used, but wax that is melted by a factor other than heat or breaks
so that a reagent and a reagent solution mix may be adopted. Any
kind of wax that does not prevent a reaction between a reagent and
a reagent solution but melts at a necessary temperature may be
selected.
[0113] The reagent used in the present invention may be in a solid
state or a liquid state.
[0114] Next, as shown in FIG. 6B, the resin base material 2 to
which the reagent S is fixed is placed on top of the metallic base
material 3 such that surfaces on which the mutual recesses 6 and 11
are formed face each other and then heat is added. The resin
sealant layer on the surface of the metallic base material 3 melts
and the resin base material 2 and the metallic base material 3 are
welded. With the above processes, a reaction chip including a
plurality of reaction containers 4 and a plurality of channels 5 is
completed.
[0115] The method of thermal welding can be appropriately selected
from methods such as the heat sealer, laser welding, and ultrasonic
welding. Alternatively, instead of welding, the resin base material
2 and the metallic base material 3 may be stuck together. In such a
case, an adhesive used for pasting can appropriately be selected
from any adhesive on the market that does not inhibit the target
reaction. Alternatively, a method of mechanical crimp by a roller
or the like with involvement of an adhesive material between the
resin base material 2 and the metallic base material 3 may be
adopted.
[0116] Next, as shown in FIG. 6C, a reagent solution L is fed into
each of the reaction containers 4 of the completed reaction chip.
After the reagent solution L being fed, the channel 5 is blocked by
plastically deforming a part of the groove 12 of the metallic base
material 3 to isolate each of the reaction containers 4. By
isolating each of the reaction containers 4, mixing of unnecessary
reagents between the adjacent reaction containers 4 can be
prevented. As a means for plastic deformation of the groove 12 of
the metallic base material 3, an external force may mechanically be
applied to a part of the groove 12 from outside by using a device,
or an external force may be applied by hand. Then, when the
temperature of the reaction chip 1 is controlled to a predetermined
temperature (the melting point of the wax W or higher), the
solidified wax W is melted and the reagent S and the reagent
solution L are mixed inside the reaction container 4, initiating a
reaction. In the present embodiment, the resin base material 2 made
of polypropylene has greater transparency, so that fluorescence
during reaction can be detected from outside on the side of the
resin base material 2.
[0117] In the foregoing, the manufacturing method of a reaction
chip in the present embodiment has been described by using an
example of a reaction chip having the recess 11 and the groove 12
formed on the side of the metallic base material 3, but a
configuration in which the recesses and grooves are all formed on
the side of the resin base material and a flat plate material or a
film 3A (a material other than metal is also allowed) is used on
the side of the metallic base material to cover the recesses of the
resin base material with the flat plate material or the film 3A may
be adopted. An example thereof is shown in FIGS. 7A to 7C. This
example is different from the above example only in which base
material to form the recesses and grooves and the basic
manufacturing processes are the same and thus, the same reference
numerals are attached to components in FIGS. 7A to 7C that are
common to those in FIGS. 6A to 6C and a description thereof is
omitted.
[0118] The reaction chip 1 in the present embodiment has the notch
15 formed on the edges on the inflow side and outflow side of the
reagent L of the recesses 6 formed on the resin base material 2 and
thus, the reagent solution L flows smoothly near the recess 6 so
that the inflow of the reagent solution L from the channel 5 into
the reaction container 4 and the outflow of the reagent solution L
from the reaction container 4 to the channel 5 become smooth. Thus,
even if the reagent solution L containing bubbles flows in, bubbles
pass through the reaction container 4, so that the frequency of
bubbles remaining the recess 6 can significantly be decreased. As a
result, the desired reaction can accurately be detected or measured
by using the reaction chip 1 in the present embodiment. Moreover,
there is no need of hydrophobic/hydrophilic treatment and surface
treatment such as corona treatment and plasma treatment because
bubbles can be removed from inside the recess 6 only by forming the
notch 15 on the edge of the recess 6.
[0119] The technical scope of the present invention is not limited
to the above embodiment and various modifications can be made
without deviating from the spirit of the present invention. For
example, while grooves constituting a channel are formed only in
the metallic base material in the above embodiment, grooves may
also be formed in the resin base material in accordance with the
volume of a reagent solution so that the channel is constituted by
both the metallic base material and resin base material. Further,
an example in which the reaction contains have all the same size is
described in the above embodiment, but instead thereof, a plurality
of reaction containers having different sizes may be included. In
this case, the shape or dimensions of the notch may be optimized by
adjusting to the size of each reaction container. Moreover,
concrete configurations such as the shape, number, and arrangement
of the reaction containers and channel, materials and dimensions of
each base material, various methods used in each manufacturing
process exemplified in the above embodiment are only examples and
may be changed when appropriate.
Second Embodiment
[0120] An embodiment of the present invention will be described
below with reference to FIGS. 8 to 11.
[0121] FIG. 8 is a perspective view of a reaction chip in the
present embodiment. FIG. 9A is a plan view of the reaction chip and
FIG. 9B is a sectional view along a line A-A' in FIG. 8. FIG. 10 is
a process sectional view showing a reaction method using the
reaction chip following procedures thereof. FIG. 11 is a sectional
view showing another example of the reaction chip.
[0122] For convenience of description, it is assumed below that
side of the resin base material positioned on the upper side, when
a fluorescent reaction is detected or measured, is the "upper side"
and the side of metallic base material positioned on the lower side
is the "lower side".
[0123] A reaction chip 21 in the present embodiment is a small chip
that has, as shown in FIG. 8, a rectangular shape and a thickness
of about several mm. The reaction chip 21 is constituted by a cover
material 22 (first base material) and a substrate 23 (second base
material) embedded on the side of the undersurface of the cover
material 22. The reaction chip 21 in the present embodiment has, as
shown in FIG. 9B, recesses 26 constituting reaction containers 24
formed in the cover material 22 and grooves 28 constituting
recesses 27 and channels 25 formed in the substrate 23. The
reaction chip 21 in the present embodiment includes three sets of
the channels 25 having the 12 reaction containers 24.
(Cover Material)
[0124] The cover material 22 presents a rectangular shape as a
whole and is formed to such a thickness that the cover material 22
is not easily bent while being used. The cover material 22 is
constituted by a resin material such as PP (polypropylene), PC
(polycarbonate), acryl resin (polymethyl methacrylate), PET
(polyethylene terephthalate), PE (polyethylene), PV (polyvinyl
chloride), and PS (polystyrene). The cover material 22 produced by
using such a synthetic resin is preferable due to superiority in
heat resistance, chemical resistance, and molding workability.
Further the cover material 22 produced by two types of resin or
more being bonded may be used. In such a case, various kinds of the
cover materials 22 in accordance with properties of the reaction
reagent or reagent solution can be produced by preparing the cover
material 22 making the most of characteristics of each resin, so
that the different cover materials 22 can be used for different
purposes. For example, the material for the upper part and that for
the lower part of the cover material 22 may be separated.
Incidentally, in addition to resin materials, quartz glass or the
like may be used as the material of the cover material 22.
[0125] The cover material 22 has, as shown in FIGS. 9A and 9B, a
plurality (in the present embodiment, 36) of recesses 26 in which a
reagent is arranged and a reagent solution injecting hole 30
communicatively connected to the reaction container 24 and the
channel 25 to feed a reagent solution provided therein. In one set
of the channel 25, a minute through hole 31 is provided at the end
on the opposite side of the reagent solution injecting hole 30 and
a high-density filter (not shown) is filled inside the through hole
31. Accordingly, a fed reagent solution can be prevented from
overflowing from an outlet. Alternatively, a similar reagent
solution injecting hole may be provided at the end on the opposite
side of the reagent solution injecting hole 30 so that a reagent
solution can be injected through whichever of the reagent solution
injecting holes of the channel 25. The inner side of the reagent
solution injecting hole 30 is preferably tapered so that the tip of
a dispensing chip for general PIPETMAN fits in halfway through the
injecting hole. Accordingly, feeding of a reagent solution is made
easier and the mixing of bubbles can be prevented. Moreover,
contamination of the apparatus due to scattering of a reagent
solution during reaction can be prevented by providing a lid
covering the reagent solution injecting hole 30 and a structure on
the outlet side.
(Substrate)
[0126] The substrate 23 presents a rectangular shape as a whole.
The substrate 23 is constituted by materials containing metal such
as gold, silver, copper, aluminum, zinc, tin, platinum, nickel,
brass, or alloys of at least two of these metals. Producing the
substrate 23 using materials containing such metals makes the
thermal conductivity to a reaction liquid in the reaction container
24 higher so that the reaction can preferably be caused to occur
efficiently in a short time. Moreover, a sealant layer (not shown)
is provided in an upper part of the metallic layer of the substrate
23 to stick the cover material 22 and the substrate 23 together by
thermal welding. According to this configuration, a metal that
inhibits a reaction can also be used because the metal constituting
the substrate 23 does not come directly into contact with a
reaction liquid.
[0127] The substrate 23 has a plurality (in the present embodiment,
36) of recesses 27 formed at positions corresponding to the
recesses 26 of the cover material 22 and the grooves 28 to feed a
reagent solution to allow communicative connection between the
adjacent recesses 27. The diameter of the recess 27 is preferably
almost the same as that of the recess 26 of the cover material 22.
Accordingly, a reaction reagent solution can be fed equally to the
recesses 26 and the recesses 27 and also the mixing of bubbles can
be prevented. The width and depth of the channel 25 are preferably
0.5 mm or more and 5 mm or less. If the width and depth are within
these dimensions, channel blockage caused by extrusion of the
sealant layer into the channel when the cover material 22 and the
substrate 23 are stuck together can be prevented and also the
mixing of bubbles can be prevented.
(Reaction Container)
[0128] As shown in FIG. 10A, a lower area facing the substrate 23
of the recess 26 on the cover material 22 side is a columnar space
and an upper (bottom side) area is a truncated cone shaped space.
As shown in this case, the bottom of the recess 26 is preferably
flat. Accordingly, when a reaction result is obtained by
fluorescence detection through the transparent cover material 22,
diffusion of light is reduced when compared with a case where the
bottom is not flat and fluorescence can efficiently be detected.
The diameter of the recess 26 is preferably 0.5 mm or more and 10
mm or less. Accordingly, feeding of a reagent solution to the
recess 26 is made easier and the mixing of bubbles can be
prevented.
[0129] The recess 27 on the substrate 23 side has, as shown in FIG.
10A, a hemispheric shape. If the recess 27 in a lower part of the
reaction container 24 is formed in a hemispheric shape, convection
is efficiently caused inside the reaction container 24 when heat is
added for a reaction after a reagent solution being filled so that
the reaction can be made to proceed more smoothly. If the shape of
the reaction container 24 is formed into a shape similar to that of
a tube made of PP generally used for PCR, adhesion property to a
heat block that adds heat for a reaction is increased, so that heat
can be transmitted efficiently to a reaction liquid, allowing the
reaction to proceed in a short time.
[0130] The recess 26 is formed by a method of cutting the cover
material 22 made of resin material or a method of injection-molding
a resin material inside a die. If the cover material 22 is
constituted by a hard resin material such as PC (polycarbonate),
the recess 26 can be formed using the cutting method. If the cover
material 22 is constituted by a soft resin material such as PP
(polypropylene), the recess 26 is preferably formed using the
molding method. The recess 26 can also be formed from PC using the
molding method.
[0131] On the other hand, the recess 27 and the groove 28 are
formed by a method of performing drawing using a die on the
substrate 23 in which a metallic layer and a sealant layer are
stuck together by an adhesive or the like.
(Sealing Compound)
[0132] As shown in FIG. 10A, a fixing reagent S such as a nucleic
acid probe is arranged inside the recess 26 of the cover material
22. The fixing reagent S is covered with a hot-melt sealing
compound W arranged inside the recess 26. The hot-melt sealing
compound W is a sealing compound that is in a solid state at
ordinary temperature and melts near a starting temperature of a
reaction of the fixing reagent and a reagent solution (hereinafter,
referred to as a "main reaction"). The melting point thereof is
preferably near 35 to 90.degree. so that sealing compound W melts
at least near 80 to 90.degree.. It is preferable to adopt a sealing
compound whose specific gravity is smaller than that of the fixing
reagent and also that of the reagent solution as the sealing
compound W. However, the premise is that the main reaction is not
inhibited. As a concrete sealing compound, AmpliWax (registered
trademark) PCR Gem 100 manufactured by Applied Biosystems can be
adopted. This is a product invented as a replacement of mineral oil
so that evaporation of a reaction reagent solution is prevented by
forming a layer after melting when a PCR amplification reaction
occurs. This product is in a solid state at ordinary temperature
and melts at 55 to 58.degree.. The fixing reagent S covered with
the sealing compound W may be in a liquid state or a solid state.
If the fixing reagent S is in a liquid state whose specific gravity
is larger than that of the reagent solution, or the melted sealing
compound W has a specific gravity larger than that of the fixing
reagent S and that of the reagent solution, it is easier and more
advantageous to mix the fixing reagent S and the reagent
solution.
[0133] To arrange the sealing compound W inside the recess 26, a
method of injecting a proper amount of the solid sealing compound W
into the recess 26 in which the fixing reagent S is pre-arranged
and heating the sealing compound W can be used.
[0134] Accordingly, the melted sealing compound W spreads at the
bottom of the recess 26 while wetting the bottom and, if the
sealing compound W is cooled thereafter, the sealing compound W can
be arranged inside the recess 26 while the fixing reagent S being
covered therewith. Alternatively, a method of dispensing the
pre-melted sealing compound W into the recess 26 in which the
fixing reagent S is pre-arranged using PIPETMAN may be used. This
method is more advantageous because the amount of the sealing
compound W can be defined more accurately.
[0135] Further, it is preferable to perform a centrifugal operation
before cooling the melted sealing compound W while wetting and
spreading at the bottom of the recess 26. Accordingly, the sealing
compound W can hide the fixing reagent S more reliably. Moreover,
the sealing compound W on the wall surface of the recess 26 moves
to the bottom of the recess 26 and thus, when the cover material 22
and the substrate 23 are stuck together by thermal welding, the
outflow of the sealing compound W to the channel 25 due to
re-melting can be prevented.
[0136] In this manner, the sealing compound W is arranged inside
the recess 26 before the substrate 23 being stuck together.
(Reaction Method)
[0137] Next, the reaction method using the above reaction chip will
be described using FIGS. 8 to 10.
[0138] First, the reagent solution L is injected through the
reagent solution injecting hole 30 shown in FIGS. 8 and 9. In this
manner, as shown in FIG. 10B, the reagent solution L is passed from
the reagent solution injecting hole 30 into the channel 25. Then,
the reagent solution L passes through the channel 25 before being
fed into a plurality of the reaction containers 24 one by one. The
feeding of the reagent solution L occurs at ordinary temperature or
a lower temperature below the ordinary temperature at which the
reagent solution L can be fed.
[0139] Here, as shown in FIG. 10B, the solid sealing compound W in
a state covering the fixing reagent S is arranged inside the recess
26 on the cover material 22 side. Thus, the reagent solution L fed
to the reaction container 24 is arranged on the surface of the
sealing compound W without coming into contact with the fixing
reagent S.
[0140] Thus, in the reaction chip 21 in the present embodiment, the
reagent solution L is fed below the sealing compound W covering the
fixing reagent S, so that the fixing reagent S will not flow out to
the adjacent reaction containers 24. Therefore, contamination can
be prevented from occurring.
[0141] After the reagent solution L being fed into the reaction
container 24, the channel 25 is blocked by plastically deforming a
part of the groove 28 between the adjacent recesses 27 of the
substrate 23 to isolate each of the reaction containers 24. By
isolating each of the reaction containers 24, mixing of unnecessary
reagents between the adjacent reaction containers 24 can be
prevented.
[0142] As means for plastic deformation of the groove 28 of the
substrate 23, an external force may mechanically be applied to
apart of the groove 28 from outside by using a device or an
external force may be applied by hand.
[0143] Next, as shown in FIG. 10C, the reaction container 24 is
heated to melt the sealing compound W. At this point, heat is added
not from the cover material 22 side where the fixing reagent S is
arranged, but from the substrate 23 side. If the specific gravity
of the sealing compound W is smaller than that of the fixing
reagent S and also that of the reagent solution L, the sealing
compound W changes places with the fixing reagent S vertically when
melted and the fixing reagent S comes into contact with the reagent
solution L. If the specific gravity of the sealing compound W is
larger than that of the fixing reagent S and also that of the
reagent solution L, the sealing compound W changes places with the
reagent solution L vertically when melted and the reagent solution
L comes into contact with the fixing reagent S. If the reaction
initiation temperature of the main reaction is equal to the melting
point of the sealing compound W or higher than the melting point of
the sealing compound W, the fixing reagent S and the reagent
solution L come into contact while being heated to the reaction
initiation temperature and the main reaction is initiated when the
reaction initiation temperature is reached.
[0144] If the reaction initiation temperature of the main reaction
is lower than the melting point of the sealing compound W, the
reaction container 24 is further heated to bring the fixing reagent
S into contact with the reagent solution L and then, the
temperature is lowered to the reaction initiation temperature to
initiate the main reaction. In the present embodiment, heat is
added during reaction from the substrate 23 side.
[0145] According to the reaction method in the present embodiment,
the reaction chip 21 is constituted by the cover material 22 made
of resin having a relatively low thermal conductivity and the
substrate 23 made of metal having a relatively high thermal
conductivity and the fixing reagent S is arranged inside the recess
26 of the cover material 22. When a reaction is caused, heat is
added from the substrate 23 side with a high thermal conductivity
and the sealing compound W is melted to bring the fixing reagent S
and the reagent solution L into contact before causing the reaction
to proceed. Therefore, when the reagent solution L is fed, the
reagent S is covered with the sealing compound W so that
contamination can be prevented from occurring. While thermal
efficiency for the whole reaction container 24 can be made better
by adding heat from the substrate 23 side, the reagent S is
arranged on the cover material 22 side with a low thermal
conductivity and thus, it is hard for heat during chip
manufacturing to conduct to the reagent S so that activity of the
reagent S is neither lowered nor devitalized. Accordingly, accurate
reaction data can be measured.
[0146] The technical scope of the present invention is not limited
to the above embodiment and various modifications can be made
without deviating from the spirit of the present invention. For
example, the recess 27 constituting the reaction container 24 and
the groove 28 constituting the channel 25 are formed on the
substrate 23 side in the above embodiment, but as shown in FIG. 11,
a groove 33 may be formed on a cover material 22A side so that a
flat plate is used as a substrate 23A in accordance with the volume
necessary for the reaction container. Alternatively, the grooves 28
constituting the channel 25 are formed only in the substrate 23 in
the above embodiment, grooves may also be formed on the cover
material 22 side in accordance with the volume of the reagent
solution L so that the channel may be constituted by both the cover
material 22 and the substrate 23. Moreover, concrete configurations
such as the shape, number, and arrangement of the reaction
containers and channels, materials and dimensions of each base
material, various methods used in each manufacturing process
exemplified in the above embodiment are only examples and may be
changed when appropriate.
Third Embodiment
[0147] A temperature controlling unit for a gene treating apparatus
(hereinafter, referred to as a "temperature controlling unit")
according to an embodiment of the present invention will be
described below with reference to FIGS. 12 to 19.
[0148] FIG. 12 is a perspective view showing principal parts of a
gene amplifying apparatus (gene treating apparatus) 42 including a
temperature controlling unit 41 in the present embodiment. The gene
amplifying apparatus 42 includes a movable carriage 43 on which a
reaction container is placed, the temperature controlling unit 41
that heats or cools the reaction container, and a measuring unit 44
that measures a reaction in the reaction container.
[0149] The movable carriage 43 is formed in a frame shape and the
reaction container described later is mounted thereon with the
undersurface thereof exposed. The movable carriage 43 is configured
to be able to move above the temperature controlling unit 41 along
a rail 46 set up on the top face of the gene amplifying apparatus
42 by a moving unit 45 composed of a publicly known configuration
such as a stepping motor and a servo motor.
[0150] In addition to the above configuration, the moving unit 45
can be used by appropriately selecting from configurations of
publicly known moving units, for example, a combination of a
stepping motor and a belt or a configuration in which the rail 46
and the movable carriage 43 are moved in a non-contact fashion by
using a magnetic force or the like.
[0151] The measuring unit 44 is constituted by an emission
detection unit 47 that introduces excitation light and measures
fluorescence and a measuring unit moving unit 48 that moves the
emission detection unit 47, and carries out an inspection of a gene
sample after being amplified. The measuring unit 44 is not
indispensable for the gene amplifying apparatus 42 in the present
invention and may not be provided if the configuration is intended
for amplification only.
[0152] The temperature controlling unit 41 is constituted by a
first unit 49 arranged above the movable carriage 43 and a second
unit 50 arranged below the movable carriage 43. The first unit 49
is vertically movably supported by a pair of support arms 51. The
second unit 50 is also vertically movably supported by a moving
unit (not shown).
[0153] With the above configuration, the temperature controlling
unit 41 is configured so that the first unit 49 and the second unit
50 moves toward the movable carriage 43 stopped between the first
unit 49 and the second unit 50 by moving on the rail 46 to be able
to sandwich the movable carriage 43 and the reaction container
placed on the movable carriage 43 for heating/cooling.
[0154] FIG. 13 is a schematic sectional view showing a state where
a reaction container 100 is sandwiched by the temperature
controlling unit 41 and FIG. 14 is an enlarged sectional view
showing details near the reaction container 100 in FIG. 13. In
FIGS. 13 and 14, units such as the movable carriage 43 and the rail
46 are omitted to make the configuration of the temperature
controlling unit 41 easier to understand.
[0155] As shown in FIG. 13, the first unit 49 includes a first
temperature controlling unit 52 that heats/cools the top face side
of the reaction container 100 and a first heat sink (first heat
dissipation unit) 53 provided above the first temperature
controlling unit 52 in contact with the first temperature
controlling unit 52. Similarly, the second unit 50 includes a
second temperature controlling unit 54 that heats/cools the
undersurface of the reaction container 100 and a second heat sink
(second heat dissipation unit) 55, and the first unit 49 and the
second unit 50 have the temperature controlling units 52, 54
arranged opposite to each other respectively.
[0156] Each of the temperature controlling units 52, 54 is composed
of a Peltier module and heats/cools the reaction container 100 by
being energized by a power supply (not shown). As shown in FIG. 14,
each of the temperature controlling units 52, 54 has a first heat
conduction layer 56 made of carbon graphite to improve thermal
conductivity provided on upper and lower sides thereof.
[0157] Each of the heat sinks 53, 55 is a publicly known air-cooled
heat sink closely provided with a fan 57 and dissipates heat
generated by each of the temperature controlling units 52, 54 out
of the apparatus. Instead of an air-cooled heat sink, a
water-cooled heat sink may be provided.
[0158] Each of the units 49, 50 is connected to a control unit 58
that sets and controls the temperature each of the temperature
controlling units 52, 54. The control unit 58 may be embedded in
the gene amplifying apparatus 42 or accommodated in a device such
as an external personal computer connected to the gene amplifying
apparatus 42. The mode of temperature control of the control unit
58 will be described later.
[0159] As shown in FIG. 14, a pair of metallic plates 59 to
uniformly dissipate heat generated by each of the temperature
controlling units 52, 54 in a surface direction are arranged on the
first heat conduction layer 56 on the side of each of the
temperature controlling units 52, 54 in contact with the reaction
container 100. Silver, aluminum or the like can be adopted as the
material of the metallic plate 59.
[0160] The above first heat conduction layer 56 is provided on the
side of the metallic plate 59 facing the reaction container 100 and
a pair of second heat conduction layers (heat conduction members)
60 made of thermal conductive material having elasticity. A silicon
rubber sheet (trade name: Sarcon, manufactured by Fuji Polymer)
having a high thermal conductivity, a silicone gel sheet (trade
name: .lamda.GEL, manufactured by Geltec) having a high thermal
conductivity or the like can be adopted as the sheet material
constituting the second heat conduction layer 60.
[0161] The second heat conduction layer 60 mounted on the second
unit 50 is preferably made thicker slightly so that the second heat
conduction layer 60 is able to be in contact with the entire
surface of the reaction container 100 even if the lower part of the
reaction container 100 is uneven. In the present embodiment, the
thickness of the second heat conduction layer 60 mounted on the
first unit 49 to 0.5 mm and that of the second unit 50 to 2.0 mm.
If the upper part of the reaction container 100 is uneven,
countermeasures can be taken by making the second heat conduction
layer 60 mounted on the first unit 49 thicker.
[0162] With the above configuration, each of the temperature
controlling units 52, 54 heats and cools the entire top face and
undersurface of the reaction container 100 via the first heat
conduction layer 56, the metallic plates 59, and the second heat
conduction layer 60.
[0163] Outer circumferences of each of the temperature controlling
units 52, 54 and the reaction container 100 are covered with a heat
insulating material 61. Resin, Styrofoam or the like can be adopted
as the heat insulating material 61.
[0164] FIG. 15 is a perspective view exemplifying the reaction
container 100 used in the gene amplifying apparatus 42, FIG. 16 is
a sectional view along a line A-A' in FIG. 15, and FIG. 17 is a
sectional view along a line B-B' in FIG. 15.
[0165] As shown in FIGS. 15 and 16, the reaction container 100 is
constituted by a first member 101 made of resin and arranged in the
upper part and a second member 102 made of metal and arranged in
the lower part.
[0166] Polypropylene or the like can be adopted as the first member
101 and aluminum, copper or the like can be adopted as the second
member 102.
[0167] As shown in FIG. 15, the reaction container 100 has a
plurality of wells 103 filled with a gene sample containing genes
(nucleic acid). That is, the upper part of each of the wells 103 is
formed of the first member 101 and the lower part thereof is formed
of the second member 102 and thus, thermal conductivity is
different in the upper part and the lower part of each of the wells
103, with the upper part having a lower thermal conductivity.
[0168] A reagent 104 used for PCR reaction is arranged on the inner
surface of the first member constituting the upper part of the well
103. Incidentally, instead of the reagent 104 being arranged inside
the well, the well may be filled with the reagent 104 together with
a gene sample described later.
[0169] As shown in FIGS. 15 and 17, the wells 103 are
communicatively connected by a channel 105 in groups of any number
and a injecting hole 106 to inject a gene sample and a deaeration
port 107 are provided at both ends of each of the channels 105.
When a gene sample is injected through the injecting hole 106, the
air inside the channel 105 is exhausted through the deaeration port
107, and the gene sample passes through the channel 105 before each
of the communicatively connected wells 103 being filled
therewith.
[0170] The operation when the gene amplifying apparatus 42
configured as described above is used will be described below.
[0171] First, the channel 105 of the reaction container 100 filled
with a gene sample is by a jig or the like to make each of the
wells 103 an independent space. Then, the reaction container 100 is
placed on the movable carriage 43 and the gene amplifying apparatus
42 is started.
[0172] The reaction container 100 on the movable carriage 43 is
moved on the rail 46 by the moving unit 45 before being stopped
between the first unit 49 and the second unit 50 of the temperature
controlling unit 41. After the movable carriage 43 being stopped,
the first unit 49 falls and the second unit 50 rises before the
reaction container 100 being sandwiched from above and from below
by the temperature controlling unit 41 so that the first unit 49
and the second unit 50 comes into contact with the entire top face
and undersurface of the reaction container 100 respectively.
[0173] After the reaction container 100 being sandwiched by the
temperature controlling unit 41, the first temperature controlling
unit 52 and the second temperature controlling unit 54 are
energized by a power supply (not shown). Then, the reaction
container 100 is heated and cooled to reach a predetermined
temperature cycle under the control of the control unit 58 to
amplify a gene contained in the gene sample inside the reaction
container 100 by the PCR method.
[0174] FIG. 18 is a graph showing a temperature cycle by the PCR
method, temperature control by the temperature controlling unit 41,
and temperatures of each unit of the reaction container. The
control unit 58 exercises independent temperature control for the
first temperature controlling unit 52 and the second temperature
controlling unit 54.
[0175] In the present embodiment, as shown in FIG. 18, gene
amplification by the PCR method is carried out, as indicated by a
thick solid line, in a temperature cycle in which temperatures near
95.degree. and 68.degree. are mutually repeated. Therefore, this
temperature cycle becomes a target temperature for the gene sample
and the reference for temperature control by each of the
temperature controlling units 52, 54.
[0176] The lower part of the reaction container 100 is formed of
the second member 102 having a high thermal conductivity and thus,
by heating the second temperature controlling unit 54, as indicated
by an alternate long and short dash line, up to about 95.degree.
C., the temperature inside the well 103 (near the central part in
the vertical direction) also rises, as indicated by a broken line,
up to close to 95.degree. C. However, the upper part of the
reaction container 100 is formed of the first member 101 whose
thermal conductivity is lower than that of the second member 102
and therefore, the temperature of the gene sample near the first
member 101 may not rise up to close to 95.degree. C. necessary for
PCR reaction. In such a case, the PCR reaction may not proceed or
proceeds only insufficiently.
[0177] Thus, as indicated by a chain double-dashed line, the preset
temperature of the first temperature controlling unit 52 is set to
about 105.degree. C., which is higher than the target temperature
95.degree. C. Accordingly, as indicated by a thin solid line, the
surface temperature of the first member 101 rises to 95.degree. C.
or higher so that it is supposed that the temperature inside the
well 103 becomes uniform as a whole and also the fact that
temperature changes proceed quickly along the preset temperature
cycle was confirmed.
[0178] Actually, with the above temperature settings, the PCR
reaction inside the reaction container 100 proceeded satisfactorily
so that 35 cycles could be completed in about 41 minutes, which is
about half the time that was needed with a conventional
apparatus.
[0179] FIG. 19 is a graph showing, as an example of a conventional
apparatus, temperature changes when the reaction container 100 is
used in a gene amplifying apparatus that heats/cools the reaction
container 100 by a Peltier module being brought into contact with
only the lower part thereof. While the temperature inside the well
rises up to close to 95.degree. C. by the Peltier module, the
surface temperature of the first member 101 rises only to close to
80.degree. C. so that it is supposed that the temperature inside
the well is non-uniform. Actually, cases where the PCR reaction did
not proceed in this gene amplifying apparatus were confirmed.
[0180] In this apparatus, it seems that quite a long time will be
needed to bring the surface temperature of the first member 101
closer to the target temperature as far as a temperature profile is
concerned and it is assumed that the total time necessary for PCR
reaction will be very long.
[0181] On the other hand, even if an attempt is made to reduce the
PCR time by rapid heating and rapid cooling by setting the control
temperature of the Peltier module higher than the target
temperature when the temperature rises and lower when the
temperature falls, a region where the temperature inside the well
(particularly near the lower part) follows behavior of the control
temperature arises because the undersurface of the reaction
container 100 is composed of the second member 102 having a high
thermal conductivity so that a case where the reagent inside the
well is devitalized can be considered.
[0182] Actually, a case where the PCR reaction did not proceed when
such control was exercised is confirmed and devitalization of the
reagent was suggested as one of possible causes why the PCR
reaction did not proceed.
[0183] The temperature control of the temperature controlling unit
41 by the control unit 58 described above is only an example and
setting parameters such as the actual preset temperature and
lengths of the heating/cooling time (the time during which the
preset temperature is maintained) are independently decided for the
first temperature controlling unit 52 and the second temperature
controlling unit 54 depending on reaction container parameters such
as the thermal conductivity, thickness and the like for each
material of the first member 101 and the second member 102.
[0184] Thus, not only the preset temperature, but also the
heating/cooling time may be different for the first temperature
controlling unit 52 and the second temperature controlling unit 54.
Therefore, setting parameters for each reaction container parameter
may be stored in the control unit 58 as a table in advance so that,
based on user input or the like, the control unit 58 exercises
temperature control of the first temperature controlling unit 52
and the second temperature controlling unit 54 by referring to
corresponding setting parameters in the table when appropriate.
[0185] The reaction container 100 whose amplification is completed
moves up to the measuring unit 44 along the rail 46. Then, various
measurements such as fluorescence intensity of the sample in each
of the wells 103 are made by the measuring unit 44. A sample
amplified by the gene amplifying apparatus 42 of the present
invention can be offered to various genetic tests such as a base
sequence test of a gene, polymorphism test based on a repetitive
sequence with a certain base sequence set as a unit, and test of
single nucleotide polymorphism (SNPs or SNP).
[0186] According to the temperature controlling unit 41 and the
gene amplifying apparatus 42 in the present embodiment, heat
generated by the first temperature controlling unit 52 and the
second temperature controlling unit 54 is efficiently transmitted
to the reaction container 100 by the first heat conduction layer
56, the metallic plates 59, and the second heat conduction layer
60. Thus, even if the reaction container 100 is formed by including
the first member 101 and the second member 102 having different
thermal conductivities, a filled gene sample is appropriately
heated/cooled and a time required to realize a temperature cycle
necessary for the PCR method is reduced so that the gene can be
amplified quickly and suitably.
[0187] Moreover, the control unit 58 exercises temperature control
of the first temperature controlling unit 52 and the second
temperature controlling unit 54 independently in accordance with
various parameters including thermal conductivities of the members
101, 102 of the reaction container 100 so that each of the first
temperature controlling unit 52 and the second temperature
controlling unit 54 is controlled to the optimum preset
temperatures and heating/cooling times to set the gene sample at
temperatures in keeping with the temperature cycle of PCR.
Therefore, the gene sample filled inside the reaction container 100
can be PCR-treated more suitably.
[0188] Moreover, the circumference of each of the temperature
controlling units 52, 54 sandwiching the reaction container 100 is
covered with the heat insulating material 61 and thus, heat
exchanged between each of the temperature controlling units 52, 54
and the reaction container 100 does not escape to the outside so
that the temperature of the reaction container 100 can be
controlled more efficiently.
[0189] Further, the first heat sink 53 and the second heat sink 55
are provided in contact with the first temperature controlling unit
52 and the second temperature controlling unit 54 respectively and
thus, when the reaction container 100 is cooled, heat transmitted
to each of the temperature controlling units 52, 54 can efficiently
be dissipated out of the temperature controlling unit 41.
[0190] In the foregoing, an embodiment of the present invention has
been described, but the technical scope of the present invention is
not limited to the above embodiment and various modifications can
be made without deviating from the spirit of the present
invention.
[0191] For example, an example in which the first heat conduction
layer 56 is provided in contact with each of the temperature
controlling units 52, 54 and the metallic plates 59 is described in
the above embodiment, but the arrangement position of the first
heat conduction layer 56 is not limited to this. Other examples may
include providing the first heat conduction layer 56 in contact
with only each of the temperature controlling units 52, 54 and in
contact with only the metallic plates 59.
[0192] Moreover, if the temperature is controlled satisfactorily,
the first heat conduction layer 56 need not necessarily be
provided.
[0193] The reaction container to be used is not limited to, as
described above, a reaction container in which the thermal
conductivity of the upper part is lower than that of the lower part
and, for example, a reaction container in which the thermal
conductivity of the upper part is higher than that of the lower
part may also be adopted. In such a case, the PCR reaction can be
caused to proceed more suitably by changing the control mode of the
control unit.
[0194] In a temperature controlling unit and a gene treating
apparatus of the present invention, the control unit is not
required. For example, if the difference in thermal conductivity
between first and second members is relatively small and the PCR
reaction can be caused to proceed without independent temperature
control of first and second temperature controlling units, the
temperature controlling unit and the gene treating apparatus may be
configured without providing a control unit.
[0195] Further, units such as a movable carriage and a rail to move
a reaction container are not required in a gene treating apparatus
of the present invention. For example, the gene treating apparatus
may be configured in such a way a reaction container is directly
set up between the first unit 49 and the second unit 50 by the user
and the temperature is controlled for PCR reaction at the setup
position.
[0196] In addition to an amplification reaction by the PCR method,
a temperature controlling unit and a gene treating apparatus of the
present invention can also be used when a predetermined temperature
is maintained for a fixed time like, for example, a reaction by the
invader method. In such a case, the temperature of a gene sample in
a reaction container can suitably be controlled without being
affected by a difference in thermal conductivity of members
constituting the reaction container.
[0197] According to a reaction chip of the present invention, a
notch showing a gradual increase in width and a gradual increase in
depth from one face of the base material toward an inner wall
surface of the recess is formed on an edge of at least one recess
of the recesses in an extending direction of the groove and
therefore, the flow of a reagent solution becomes smooth near the
recess so that the reagent solution smoothly flows into the recess
constituting a reaction container from a groove constituting a
channel or flows out to the groove from the recess. Thus, even a
reagent solution containing bubbles comes flowing, the frequency
with which bubbles remain inside the recess by being entrapped by
the inner wall surface of the recess can significantly be
decreased. Therefore, the desired reaction can accurately be
detected and measured by using a reaction chip of the present
invention. Moreover, bubbles can be removed from inside the recess
simply by molding a notch on an edge of the recess and therefore,
there is no need of hydrophobic/hydrophilic treatment and surface
treatment such as corona treatment and plasma treatment.
[0198] If the configuration is adopted in which the angle formed by
one face of the base material and the notch is smaller than that
formed by one face of the base material and the inner wall surface
of the recess, the inclination of the inner wall surface on the
inflow side or outflow side of the recess becomes gentle, which
makes the flow of the reagent solution in a sectional direction of
the base material smooth, so that bubbles can effectively be
prevented from remaining.
[0199] If a notch is formed only on one side of the extending
direction of the groove and the configuration is adopted in which
the notch is formed on the inflow side of the reagent solution, the
reagent solution flows into the recess from the groove smoothly so
that bubbles can effectively be prevented from remaining.
[0200] If a notch is formed also on the outflow side of the reagent
solution and the configuration in which the notch formed on the
inflow side of the reagent solution and the notch formed on the
outflow side of the reagent solution form a line symmetric shape is
adopted, two notches can be formed easily and also the flow of the
reagent solution becomes smooth, so that bubbles can more
effectively be prevented from remaining.
[0201] If the recess has a columnar space having the inner wall
surface at substantially right angles to one face of the base
material on an opening side and the maximum depth on the edge of
the notch is shallower than the depth of the columnar space, the
inner wall surface at substantially right angles in the columnar
space will remain on the edge of the recess on the side on which
the notch is formed. By using this inner wall surface, a reagent or
a fixing agent that temporarily fixes the reagent can reliably be
accommodated, so that reaction products can be prevented from
leaking to adjacent reaction containers.
[0202] If an outer shape of the recess is circular in plane view
and the configuration is adopted in which a plane shape of the
notch is defined, when two tangents to a circle forming an outer
edge of the recess are drawn from one point on one face of the base
material in the extending direction of the groove, by an area
inside the two tangents, an overall shape of the recess including
the notch has a shape with the least flow resistance and the flow
of the reagent solution near the recess becomes extremely smooth,
so that bubbles can more reliably be prevented from remaining.
[0203] If a center line in the extending direction of the groove of
the notch is aligned with the center line of the groove on the same
straight line, the flow of the reagent solution becomes smooth
without being deviated inside the recess, so that bubbles can more
reliably be prevented from remaining.
[0204] According to a reaction method of the present invention, a
reaction chip is constituted by a first base material with a
relatively low thermal conductivity, and a second base material
with a relatively high thermal conductivity and a reagent is
arranged inside a recess of the first base material. Then, when a
reaction is caused, heat is added from the side of the second base
material with a higher thermal conductivity and the reagent and a
reagent solution are brought into contact by melting a sealing
compound to cause the reaction to proceed. Therefore, when the
reagent solution is fed, the reagent is covered with the sealing
compound so that contamination can be prevented from occurring.
While thermal efficiency for the whole reaction container is
excellent by adding heat from the side of the second base material,
the reagent is arranged on the side of the first base material with
a lower thermal conductivity and therefore, it is hard for heat
added during chip manufacturing to be transmitted to the reagent so
that activity of the reagent will be neither lowered nor
devitalized. Accordingly, accurate reaction data can be
measured.
[0205] If the configuration in which heat is added from the side of
the second base material and the first base material and the second
base material are stuck together by thermal welding of a sealant
layer provided on at least one of the first base material and the
second base material is adopted, it is hard for the sealing
compound to melt by heat when the base materials are stuck
together, malfunctions such as blockage of the channel due to
outflow of the sealing compound and incomplete sealing of the
reagent can be prevented so that a reaction chip can be produced
with stability and also contamination can reliably be prevented
from occurring. If a sealant layer that does not inhibit a reaction
is used, the material of each base material can freely be selected.
Accordingly, the material capable of realizing a chip having high
heat resistance, barrier property, chemical resistance, and reagent
preservability and superior in reactivity (thermal conductivity)
can be selected.
[0206] If a recess corresponding to the recess of the first base
material is formed also on the second base material to configure
the reaction container by both the recess of the first base
material and that of the second base material, a sufficient volume
of the reaction container can be ensured and also flexibility of
design for the volume and shape of the reaction container can be
increased. Moreover, the surface area of the second base material
with a higher thermal conductivity increases, which increases the
thermal conductivity for the whole reaction container, so that a
reaction that proceeds by heating such as an enzyme reaction can be
caused more efficiently in a short time.
[0207] If the configuration in which a resin material is used as
the first base material and a metallic material is used as the
second base material is adopted, a reaction container or channel
having the above excellent characteristics can easily be worked
on.
[0208] If the sealing compound is constituted by a material soluble
in neither a reagent nor a reagent solution, the reagent and the
reagent solution can come into contact and react without any change
in composition thereof so that accurate reaction data can be
measured.
[0209] If the reaction container is a reaction container for enzyme
reaction, a DNA amplification reaction by an enzyme reaction, DNA
detection reaction by hybridization, and detection reaction of SNP,
which are general biochemical reactions, can be realized on a
reaction chip.
[0210] According to a temperature controlling unit for gene
treating apparatus and a gene treating apparatus of the present
invention, even if a reaction container constituted by plural types
of materials having different thermal conductivities, a gene
contained in a gene sample filled in the reaction container can
suitably be treated.
EXAMPLES
[0211] The present inventors carried out experiments below to
demonstrate effects of the first embodiment of the present
invention.
Example 1
[0212] First, the resin base material 2 including the recess 6
having the shape and layout shown in FIGS. 2 and 4 according to the
above embodiments is produced by injection molding. Polypropylene
determined not to inhibit a reaction is used as a material. As for
the notch 15, after the resin base material 2 including the recess
6 being produced by using injection molding, the notch 15 is formed
by cutting at upstream and downstream positions of the channel 5 on
the edge of the recess 6. On the other hand, what is produced by
performing drawing on an aluminum original sheet to which a
polypropylene sealant is applied is used as the metallic base
material 3 with the intention of improving thermal efficiency
during reaction.
[0213] While the reaction chip has a reagent arranged in each of
the reaction containers 4 in a normal case, the present experiment
is intended to check the state of bubbles during feeding and thus,
instead of a reagent, 5 .mu.l of AmpliWax (trade name, manufactured
by ABI) is each put into the recess 6 of the resin base material 2.
The resin base material 2 having AmpliWax arranged inside the
recess 6 and the metallic base material 3 are welded by thermal
welding to produce a reaction chip in the present embodiment.
Comparative Example 1
[0214] A resin base material 2A including the recess 6 having no
notch as shown in FIGS. 20A and 20B is produced and then, a
reaction chip of Comparative Example 1 is produced by following the
same method as that of Example 1 described above (see FIGS. 21A and
21B).
[0215] As reagent solutions to be fed, a reagent solution A
obtained by diluting a PCR product 10 times and a reagent solution
B composed of a 10 mg/ml protein solution (BSA solution) are
prepared. These reagent solutions A and B are fed to three reaction
chips each for Example 1 and Comparative Example 1 by an electric
pipette (Finnpipette Novas 30-300 .mu.l (trade name), manufactured
by Thermo Fisher Scientific) at the flow rate of 200 .mu.l/21
sec.
[0216] If a reagent solution is fed, bubbles contained in the
reagent solution are introduced into each reaction container, but
in reaction chips of Example 1, bubbles once introduced flow out
together with the reagent solution and no bubble remains inside the
reaction container. FIG. 22 shows a photo shooting conditions
inside the reaction container through a transparent resin base
material.
[0217] In reaction chips of Comparative Example 1, on the other
hand, bubbles once introduced do not flow out of the reaction
container and, as shown in FIG. 21C, a bubble B remaining inside
the reaction container 4 is observed. FIG. 23 shows a photo
shooting conditions inside the reaction container.
[0218] Numbers of reaction containers in which bubbles remain
during feeding in each of reaction chips of Example 1 and
Comparative Example 1 are listed in [Table 1]. The total number of
reaction containers is 108 because three reaction chips each with
36 reaction containers are fed. While the number of reaction
containers in which bubbles remain is 0 for both the reagent
solution A and the reagent solution B in reaction chips of Example
1, the number of reaction containers in which bubbles remain is 8
for the reagent solution A and 18 for the reagent solution B in
reaction chips of Comparative Example 1.
TABLE-US-00001 TABLE 1 Reagent solution Reagent solution Reaction A
PCR diluted B Protein container shape solution solution Example 1
With notch 0 0 Comparative Without notch 8 18 Example 1
[0219] From the above result, an effect of the reaction chip of the
present invention having a recess with notch is proved.
[0220] The detection method of SNP using the invader (registered
trademark) method executed by the present inventors as Example 2 of
a reaction chip and a reaction method according to the second
embodiment of the present invention will be described below. FIG.
24A is a plan view showing reagent arrangement of Example 2 and
Comparative Example 2, FIG. 24B is a sectional view of the reaction
chip of Example 2, and FIG. 24C is a sectional view of the reaction
chip of and Comparative Example 2.
Example 2
[0221] As shown in FIG. 24A, a reaction chip described in the above
embodiment is produced. The cover material 22 is produced by the
method of injection-molding polypropylene resin inside a die and a
plurality of recesses 26 and a plurality of reagent solution
injecting holes 30 are formed on a resin base material with a
thickness of 2 mm. The opening diameter of the recess 26 is 3 mm,
the diameter thereof at the bottom is 2 mm, and the depth thereof
is 1.5 mm. The volume of the recess 26 is theoretically about 9
.mu.L and the distance between the adjacent recesses 26 is 6 mm.
The outside diameter of the reagent solution injecting hole 30 is 4
mm and the inside diameter of the hole is 1.5 mm to 2 mm, creating
a tapered shape. The height thereof is 6 mm from the top face of
the cover material 22.
[0222] A material in which a sealant layer made of polypropylene of
70 .mu.m is stacked on an aluminum plate of 0.1 mm via an adhesive
is used as the substrate 23 and the channel 25 communicatively
connecting the recess 27 and the recess 27 is formed by drawing.
The opening diameter of the recess 27 is 3 mm and the depth thereof
is 1.5 mm. The volume of the recess 27 is theoretically about 7
.mu.L and the distance between the adjacent recesses 27 is 6 mm.
The width of the channel 25 is 1 mm and the depth thereof is 0.3
mm.
[0223] As shown in FIG. 24B, the fixing reagent S is arranged in
the recess 26 of the cover material 22 corresponding to shaded
reaction containers 4S in FIG. 24A. An allele probe 1, an allele
probe 2, and an invader probe used for an invader (registered
trademark) reaction and an FRET probe 1, an FRET probe 2, and
Cleavase (registered trademark) are arranged as the fixing reagents
S before heating/drying.
[0224] Next, AmpliWax (registered trademark) PCR Gem 100
manufactured by Applied Biosystems is put into all the recesses 26
as the sealing compound W. The amount of the sealing compound W for
one recess 26 is 4.5 .mu.L. The sealing compound W is heated to 80
to 100.degree. C. to be dispensed to the recess 26 while being
melted and, after a centrifugal operation being performed by a
plate centrifuge, the sealing compound W is solidified again at
ordinary temperature. Accordingly, the fixing reagent S is covered
with the sealing compound W in the shaded reaction containers 4S in
FIG. 24A.
[0225] Next, the cover material 22 and the substrate 23 produced
above are stuck together by a heat seal under the conditions of
250.degree. C., 0.5 MPa, 1.4 s, and one-sided heating from the
substrate 23 side using a heat seal tester manufactured by Tester
Sangyo.
[0226] Next, a mixed solution of a PCR product amplified from
purified genome DNA, an invader buffer, and water for dilution
thereof is fed as a reaction reagent solution. The reaction reagent
solution L is caused to reach all the reaction containers 24
through the channel 25 from the reagent solution injecting hole 30
of the cover material 22. The amount of feeding of the reaction
reagent solution L to one reaction container 24 is 12 to 15
.mu.L.
[0227] Then, the reaction chip 21 is set to a developed analysis
chip dedicated apparatus. In the apparatus, a part of the channel
25 is crushed by an external force for sealing between the reaction
containers 24 and 4S so that a reaction liquid during reaction
should not be exchanged between the reaction containers 24. At this
point, heat is added simultaneously with the external force for
thermal welding of the sealant layer of the cover material 22 and
the substrate 23 to strengthen sealing. The sealing occurs under
the conditions of 190.degree. C., 120 kgf, and 1.5 s.
[0228] Next, the reaction chip 21 is heated from the substrate 23
side inside the apparatus to cause a reaction. First, while the
sealing compound W is melted under the conditions of 95.degree. C.
and 5 minutes to bring the fixing reagent S and the reaction
reagent solution L into contact, the PCR product in the reaction
reagent solution is altered in quality. Then, the temperature is
lowered to 63.degree. C. and a fluorescent substance is detected
inside the reaction containers 4S are detected once in every 30
seconds while causing an invader reaction to observe reaction
conditions at regular intervals.
Comparative Example 2
[0229] The cover material 22 and the substrate 23 are produced in
the same manner as the above Example 2. Then, in Comparative
Example 2, as shown in FIG. 24C, the fixing reagent S and the
sealing compound W are arranged in the recess 27 of the substrate
23. After the cover material 22 and the substrate 23 being stuck
together by a heat seal, the reaction reagent solution L is input
through the reagent solution injecting hole 30 at ordinary
temperature. Next, after being sealed between the reaction
containers 24 and 4S in the apparatus, the reaction chip 21 is
heated from the substrate 23 side to melt the sealing compound and
bring the fixing reagent and a reaction reagent solution into
contact and then, a fluorescent substance is detected while an
invader reaction being caused.
(Experiment Results)
[0230] FIGS. 25A and 25B show results of reactions caused in
Example 2, and FIG. 25A shows luminescence intensity in an adjacent
reaction container of a reaction container in which a fixing
reagent is arranged and FIG. 25B shows luminescence intensity in a
reaction container in which a fixing reagent is arranged. The
horizontal axis of the graph represents a reaction time and the
vertical axis thereof represents fluorescence intensity. As is
evident from these graphs, only the graph of the reaction container
in which the fixing reagent is arranged in FIG. 25B shows that the
reaction proceeded without any problems. Moreover, no proceeding
reaction is observed in the adjacent reaction container in which no
fixing reagent is arranged of the reaction container in which the
fixing reagent is arranged, which shows that the fixing reagent can
be concealed by the sealing compound without causing any
problem.
[0231] On the other hand, FIGS. 26A and 26B show results of
reactions performed in Comparative Example 2, and FIG. 26A shows
luminescence intensity in an adjacent reaction container of a
reaction container in which a fixing reagent is arranged and FIG.
26B shows luminescence intensity in a reaction container in which a
fixing reagent is arranged. These results show that the reaction
did not proceed at all in the reaction container in which the
fixing reagent is arranged, either. This can be considered that
because the fixing reagent S is arranged in the recess 27 on the
substrate 23 side having an aluminum plate, heat during heat
sealing of the cover material 22 and the substrate 23 is
transmitted through aluminum with a high thermal conductivity to
add more heat to the fixing reagent S, resulting in lowering or
devitalization of activity of the reagent.
[0232] The above demonstrates that, according to the reaction
method of the present invention, reaction data can be measured
accurately without causing lowering or devitalization of activity
of a reagent.
* * * * *