U.S. patent application number 15/078840 was filed with the patent office on 2016-07-14 for solution mixer, fluidic device, and solution mixing method.
The applicant listed for this patent is Nikon Corporation, The University of Tokyo. Invention is credited to Takanori ICHIKI, Masashi KOBAYASHI, Kenji MIYAMOTO, Shoichi TSUCHIYA, Taro UENO.
Application Number | 20160199796 15/078840 |
Document ID | / |
Family ID | 52743398 |
Filed Date | 2016-07-14 |
United States Patent
Application |
20160199796 |
Kind Code |
A1 |
ICHIKI; Takanori ; et
al. |
July 14, 2016 |
SOLUTION MIXER, FLUIDIC DEVICE, AND SOLUTION MIXING METHOD
Abstract
A solution mixer comprising: a main flow path in which a
solution circulates; at least one solution introduction flow path
connected to the main flow path; and at least one solution
discharge flow path connected to the main flow path, wherein the
solution discharge flow path has at least one solution discharge
flow path valve, and wherein the main flow path has at least one
main flow path valve.
Inventors: |
ICHIKI; Takanori; (Tokyo,
JP) ; UENO; Taro; (Tokyo, JP) ; TSUCHIYA;
Shoichi; (Chigasaki-shi, JP) ; KOBAYASHI;
Masashi; (Tokyo, JP) ; MIYAMOTO; Kenji;
(Yokohama-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of Tokyo
Nikon Corporation |
Tokyo
Tokyo |
|
JP
JP |
|
|
Family ID: |
52743398 |
Appl. No.: |
15/078840 |
Filed: |
March 23, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/JP2014/075312 |
Sep 24, 2014 |
|
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15078840 |
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Current U.S.
Class: |
366/192 |
Current CPC
Class: |
G01N 2001/386 20130101;
G01N 2035/00158 20130101; B01L 2300/0867 20130101; B01F 5/102
20130101; B01L 3/5027 20130101; B01F 15/0292 20130101; B01F 5/108
20130101; B01F 13/0059 20130101; B01F 3/0865 20130101 |
International
Class: |
B01F 5/10 20060101
B01F005/10; B01F 15/02 20060101 B01F015/02; B01F 3/08 20060101
B01F003/08 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 25, 2013 |
JP |
2013-199071 |
Claims
1. A solution mixer comprising: a main flow path in which a
solution circulates; at least one solution introduction flow path
connected to the main flow path; and at least one solution
discharge flow path connected to the main flow path, wherein the
solution discharge flow path has at least one solution discharge
flow path valve, and wherein the main flow path has at least one
main flow path valve.
2. The solution mixer according to claim 1, which is configured to
quantitatively determine the volume of a plurality of solutions and
to circulate and mix the quantitatively determined plurality of
solutions, in the main flow path.
3. The solution mixer according to claim 1, wherein the main flow
path has at least two main flow path valves, each of which is
disposed such that at least one partial region of the main flow
path, which is defined by closing any of the two main flow path
valves, has a predetermined volume.
4. The solution mixer according to claim 1, wherein the main flow
path valve is disposed in the vicinity of the solution introduction
flow path and/or in the vicinity of the solution discharge flow
path.
5. The solution mixer according to claim 1, wherein the main flow
path has two main flow path valves, and wherein the solution
introduction flow path and the solution discharge flow path are
connected to the main flow path in a region other than a partial
region of the main flow path which is defined by closing the two
main flow path valves.
6. The solution mixer according to claim 1, wherein the main flow
path has two main flow path valves, and wherein two main flow path
valves are disposed in the vicinity of the solution discharge flow
path which is connected to the main flow path between the two main
flow path valves.
7. The solution mixer according to claim 1, wherein the main flow
path includes a first flow path, a second flow path, and first and
second connecting flow paths which respectively allow communication
with the first flow path and the second flow path, and wherein the
main flow path valve is disposed in the first connecting flow path
and/or the second connecting flow path.
8. The solution mixer according to claim 7, further comprising: two
or more of the main flow paths, wherein the adjacent main flow
paths share the first flow path or the second flow path, wherein at
least one solution introduction flow path is connected to each of
the main flow paths, wherein at least one solution discharge flow
path is connected to each of the main flow paths, wherein a
solution discharge flow path valve is disposed in each of the
solution discharge flow paths, and wherein at least one main flow
path valve is disposed in each of the connecting flow paths.
9. The solution mixer according to claim 1, further comprising a
pump which circulates a solution in the main flow path.
10. The solution mixer according to claim 9, wherein the pump
includes at least three pump valves.
11. The solution mixer according to claim 1, wherein the main flow
path includes a detection unit that detects a substance in a
solution.
12. The solution mixer according to claim 11, wherein the substance
in the solution is a biomolecule, and wherein the detection unit
includes a substrate to which a substance having affinity to the
biomolecule is immobilized.
13. A fluidic device comprising the solution mixer according to
claim 1.
14. A method of mixing two types of solutions with each other using
a solution mixer, wherein the solution mixer includes a main flow
path in which a solution circulates, a solution introduction flow
path connected to the main flow path, and a solution discharge flow
path connected to the main flow path, in which the solution
discharge flow path has at least one solution discharge flow path
valve, the main flow path has at least one main flow path valve,
and the at least one main flow path valve is disposed in the
vicinity of the solution discharge flow path, and wherein the
method comprises (A) sending a first solution to the main flow path
from the solution introduction flow path, while the main flow path
valve and the solution discharge flow path valve are open; (B)
closing the main flow path valve; (C) sending a second solution to
the main flow path from the solution introduction flow path; (D)
closing the solution discharge flow path valve; and (E) circulating
and mixing the first solution and the second solution by opening
the main flow path valve.
15. The method according to claim 14, wherein, in the solution
mixer, the main flow path has two main flow path valves, and the
solution introduction flow path and the solution discharge flow
path are connected to the main flow path in a region other than a
partial region of the main flow path which is defined by closing
two main flow path valves, wherein the step (A) is a step of
sending a first solution to the main flow path from the solution
introduction flow path, while the two main flow path valves and the
solution discharge flow path valve are open, wherein the step (B)
is a step of closing the two main flow path valves, and wherein the
step (E) is a step of circulating and mixing the first solution and
the second solution by opening the two main flow path valves.
16. A method of mixing a plurality of solutions with each other
using a solution mixer, wherein the solution mixer includes a first
flow path, a second flow path, and first and second connecting flow
paths which respectively allow communication with the first flow
path and the second flow path, first and second solution
introduction flow paths which are respectively connected to the
first and second flow paths, first and second solution discharge
flow paths which are respectively connected to the first and second
flow paths, first and second solution discharge flow path valves
which are respectively disposed in the first and second solution
discharge flow paths, and first and second main flow path valves
which are respectively disposed in the first and second connecting
flow paths, and wherein the method comprises (A) introducing a
first solution into the first flow path from the first solution
introduction flow path and introducing a second solution into the
second flow path from the second solution introduction flow path,
while the first and second main flow path valves are closed and the
first and second solution discharge flow path valves are open; and
(B) circulating and mixing the first and second solutions by
closing the first and second solution discharge flow path valves
and opening the first and second main flow path valves.
17. The method according to claim 16, wherein the solution mixer
further includes a third flow path, and third and fourth connecting
flow paths which respectively allow communication with the second
and third flow paths, a third solution introduction flow path which
is connected to the third flow path, a third solution discharge
flow path which is connected to the third flow path, a third
solution discharge flow path valve which is disposed in the third
solution discharge flow path, and third and fourth main flow path
valves which are respectively disposed in the third and fourth
connecting flow paths, and wherein the method further comprises (C)
introducing a third solution into the third flow path from the
third solution introduction flow path, while the third solution
discharge flow path valve is open, before or after the step (B);
and circulating and mixing the third solution with a mixed solution
of the first and second solutions by closing the third solution
discharge flow path valve and opening the third and fourth main
flow path valves, after the steps (B) and (C).
18. A method of mixing a plurality of solutions with each other
using a solution mixer, wherein the solution mixer includes two or
more main flow paths, in which a solution circulates, each of the
main flow paths including a first flow path, a second flow path,
and first and second connecting flow paths which allow
communication with the first flow path and the second flow path,
and the two adjacent main flow paths sharing the first flow path or
the second flow path, at least one solution introduction flow path
which is connected to each of the main flow paths, and at least one
solution discharge flow path which is connected to each of the main
flow paths, wherein each of the solution discharge flow paths has
at least one solution discharge flow path valve, wherein each of
the connecting flow paths has at least one main flow path valve,
wherein each of the valves is disposed such that each partial
region of the main flow paths which is defined by closing each of
the valves has a predetermined volume, and wherein the method
comprises respectively introducing a first solution and a second
solution into the first flow path and the second flow path after
closing the main flow path valves and the solution discharge flow
path valves such that the first flow path and the second flow path
of one main flow path are isolated from each other and from other
flow paths; circulating and mixing the first solution and the
second solution by opening the main flow path valves such that the
first flow path and the second flow path communicate with each
other; closing the main flow path valves and the solution discharge
flow path valves such that, in a main flow path next to the one
main flow path, a first or second flow path, which is not shared
with the one main flow path, is isolated from other flow paths, and
introducing a third solution into the isolated first or second flow
path, and circulating and mixing the third solution with a mixed
solution of the first solution and the second solution by opening
the main flow path valves such that the main flow path and the
neighboring main flow path are allowed to communicate with each
other.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] Priority is claimed on Japanese Patent Application No.
2013-199071, filed Sep. 25, 2013. This application is a
continuation application of International Patent Application No.
PCT/JP2014/075312, filed on Sep. 24, 2014. The contents of the
above-mentioned application are incorporated herein by
reference.
REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB
[0002] This application is being filed electronically via EFS-Web
and includes an electronically submitted sequence listing in .txt
format. The .txt file contains a sequence listing entitled
"OSP62359SequenceListing_v2.txt" created on Mar. 22, 2016 and is
7667 bytes in size. The sequence listing contained in this .txt
file is part of the specification and is incorporated herein by
reference in its entirety.
BACKGROUND
[0003] The present invention relates to a solution mixer, a fluidic
device, and a solution mixing method.
[0004] In recent years, development of micro-total analysis systems
(.mu.-TAS) or the like which aims at increasing speed, increasing
efficiency, and integration of experiments in the in vitro
diagnostic field, or ultra-miniaturization of a testing device has
attracted attention, and active research has been underway
globally.
[0005] It is possible to measure or analyze a small amount of
sample using the .mu.-TAS which is portable and is disposable at
low costs, and therefore, the .mu.-TAS is excellent compared to
testing devices in the related art.
[0006] Furthermore, in a case of using an expensive reagent or in a
case of testing a small amount of multiple specimens, the .mu.-TAS
has attracted attention as a method with high usefulness.
[0007] As a constituent of the a rotary mixer which includes a
loop-like flow path and a pump, which is disposed on the flow path,
is reported (Jong Wook Hong, Vincent Studer, Giao Hang, W French
Anderson and Stephen R Quake, Nature Biotechnology 22, 435-439
(2004)). In this rotary mixer, a plurality of solutions are
injected into the loop-like flow path, and are mixed together in
the loop-like flow path by operating the pump. The plurality of
solutions are loaded in an injection flow path which is connected
to the loop-like flow path, and are then injected into the
loop-like flow path. Valves are provided on the injection flow
path, and the volume of each of the solutions is quantitatively
determined within the flow path.
SUMMARY
[0008] In a method disclosed in Hong et al, a plurality of
solutions to be mixed in a loop-like flow path are first loaded and
quantitatively determined in an injection flow path, and are then
injected into the loop-like flow path.
[0009] In general, when injecting a solution into a flow path, if
trying to fill the flow path completely with the solution without
air being mixed, it is necessary to inject a larger amount of
solution than the volume within the flow path unless the injection
is stopped simultaneously with the completion of discharge of air.
The same principle applies to the rotary mixer disclosed in Hong et
al., and it is necessary to inject a larger amount of plurality of
solutions to be mixed in the above-described loop-like flow path
than the volume within the loop-like flow path. Accordingly, even
if the volume of solutions to be used for mixing is quantitatively
determined within an injection flow path, when actually mixing the
solutions within the loop-like flow path, there is a concern that
the quantitative determination may not always be accurate.
[0010] The present invention has been made in consideration of the
above-described circumstances, and an object of the present
invention is to provide a solution mixer which can accurately and
quantitatively determine the volume of each of the solutions to be
mixed in the mixer, a fluidic device including the solution mixer,
and a solution mixing method.
[0011] The present inventors have conducted extensive studies in
order to solve the above-described problems, and as a result, they
have found that it is possible to quantitatively determine and mix
solutions which have been injected into a main flow path by
quantitatively compartmentalizing the main flow path having an
arbitrary volume, using a valve. An embodiment of the present
invention provides the following (1) to (5).
[0012] (1) A solution mixer in an embodiment of the present
invention comprising:
a main flow path in which a solution circulates;
[0013] at least one solution introduction flow path connected to
the main flow path; and
[0014] at least one solution discharge flow path connected to the
main flow path,
[0015] wherein the solution discharge flow path has at least one
solution discharge flow path valve, and
[0016] wherein the main flow path has at least one main flow path
valve.
[0017] (2) A fluidic device in an embodiment of the present
invention comprising the above-described solution mixer.
[0018] (3) A method of mixing two types of solutions with each
other in an embodiment of the present invention using a solution
mixer,
[0019] wherein the solution mixer includes a main flow path in
which a solution circulates, a solution introduction flow path
connected to the main flow path, and a solution discharge flow path
connected to the main flow path, in which the solution discharge
flow path has at least one solution discharge flow path valve, the
main flow path has at least one main flow path valve, and the at
least one main flow path valve is disposed in the vicinity of the
solution discharge flow path, and
[0020] wherein the method comprises
[0021] a step A of sending a first solution to the main flow path
from the solution introduction flow path, while the main flow path
valve and the solution discharge flow path valve are open;
[0022] a step B of closing the main flow path valve;
[0023] a step C of sending a second solution to the main flow path
from the solution introduction flow path;
[0024] a step D of closing the solution discharge flow path valve;
and
[0025] a step E of circulating and mixing the first solution and
the second solution by opening the main flow path valve.
[0026] (4) A method of mixing a plurality of solutions with each
other in an embodiment of the present invention using a solution
mixer,
[0027] wherein the solution mixer includes
[0028] a first flow path, a second flow path, and first and second
connecting flow paths which respectively allow communication with
the first flow path and the second flow path,
[0029] first and second solution introduction flow paths which are
respectively connected to the first and second flow paths,
[0030] first and second solution discharge flow paths which are
respectively connected to the first and second flow paths,
[0031] first and second solution discharge flow path valves which
are respectively disposed in the first and second solution
discharge flow paths, and
[0032] first and second main flow path valves which are
respectively disposed in the first and second connecting flow
paths, and
[0033] wherein the method comprises
[0034] a step A of introducing a first solution into the first flow
path from the first solution introduction flow path and introducing
a second solution into the second flow path from the second
solution introduction flow path, while the first and second main
flow path valves are closed and the first and second solution
discharge flow path valves are open; and
[0035] a step B of circulating and mixing the first and second
solutions by closing the first and second solution discharge flow
path valves and opening the first and second main flow path
valves.
[0036] (5) A method of mixing a plurality of solutions with each
other in an embodiment of the present invention using a solution
mixer,
[0037] wherein the solution mixer includes
[0038] two or more main flow paths, in which a solution circulates,
each of the main flow paths including a first flow path, a second
flow path, and first and second connecting flow paths which allow
communication with the first flow path and the second flow path,
and the two adjacent main flow paths sharing the first flow path or
the second flow path,
[0039] at least one solution introduction flow path which is
connected to each of the main flow paths, and
[0040] at least one solution discharge flow path which is connected
to each of the main flow paths,
[0041] wherein each of the solution discharge flow paths has at
least one solution discharge flow path valve,
[0042] wherein each of the connecting flow paths has at least one
main flow path valve,
[0043] wherein each of the valves is disposed such that each
partial region of the main flow paths which is defined by closing
each of the valves has a predetermined volume, and
[0044] wherein the method comprises
[0045] a step of respectively introducing a first solution and a
second solution into the first flow path and the second flow path
after closing the main flow path valves and the solution discharge
flow path valves such that the first flow path and the second flow
path of one main flow path are isolated from each other and from
other flow paths;
[0046] a step of circulating and mixing the first solution and the
second solution by opening the main flow path valves such that the
first flow path and the second flow path communicate with each
other;
[0047] a step of closing the main flow path valves and the solution
discharge flow path valves such that, in a main flow path next to
the one main flow path, a first or second flow path, which is not
shared with the one main flow path, is isolated from other flow
paths, and introducing a third solution into the isolated first or
second flow path, and
[0048] a step of circulating and mixing the third solution with a
mixed solution of the first solution and the second solution by
opening the main flow path valves such that the main flow path and
the neighboring main flow path are allowed to communicate with each
other.
[0049] According to the present invention, it is possible to mix a
plurality of solutions with each other in a solution mixer in a
state in which the volume of the plurality of solutions are
accurately and quantitatively determined.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] FIG. 1 is a schematic view of an aspect of a solution mixer
in the present embodiment.
[0051] FIG. 2 is a schematic view of an aspect of a solution mixer
in the present embodiment.
[0052] FIG. 3 is a schematic view of an aspect of a solution mixer
in the present embodiment.
[0053] FIG. 4 is a schematic view of an aspect of a solution mixer
in the present embodiment.
[0054] FIG. 5 is a schematic view of an aspect of a solution mixer
in the present embodiment.
[0055] FIG. 6 is a schematic view of an aspect of a solution mixer
in the present embodiment.
[0056] FIG. 7 is a schematic view of an aspect of a solution mixer
in the present embodiment.
[0057] FIG. 8 is a schematic view of an aspect of a solution mixer
in the present embodiment.
[0058] FIG. 9 is a schematic view of an aspect of a fluidic device
in the present embodiment.
[0059] FIG. 10 is a schematic view of an aspect of the fluidic
device in the present embodiment.
[0060] FIG. 11 is a schematic view of an aspect of the fluidic
device in the present embodiment.
[0061] FIG. 12A is a schematic view of an aspect of a solution
mixing method in the present embodiment.
[0062] FIG. 12B is a schematic view of an aspect of the solution
mixing method in the present embodiment.
[0063] FIG. 12C is a schematic view of an aspect of the solution
mixing method in the present embodiment.
[0064] FIG. 12D is a schematic view of an aspect of the solution
mixing method in the present embodiment.
[0065] FIG. 12E is a schematic view of an aspect of the solution
mixing method in the present embodiment.
[0066] FIG. 13A is a schematic view of an aspect of a solution
mixing method (step A) in the present embodiment.
[0067] FIG. 13B is a schematic view of an aspect of a solution
mixing method (step B) in the present embodiment.
[0068] FIG. 14A is a schematic view of an aspect of a solution
mixing method (steps C and D) in the present embodiment.
[0069] FIG. 14B is a schematic view of an aspect of a solution
mixing method (step E) in the present embodiment.
[0070] FIG. 15A is a schematic view of an aspect of a solution
mixing method (step F) in the present embodiment.
[0071] FIG. 15B is a schematic view of an aspect of a solution
mixing method (step G) in the present embodiment.
[0072] FIG. 16A is a schematic view of an aspect of a solution
mixing method (step H) in the present embodiment.
[0073] FIG. 16B is a schematic view of an aspect of a solution
mixing method (step I) in the present embodiment.
[0074] FIG. 17 is a result of quantitative determination of an
exosome which is immobilized to a BAM substrate in Example.
[0075] FIG. 18 is a result of quantitative determination of miRNA
purified in Example.
[0076] FIG. 19 is a result of detection of miRNA using a fluidic
device which has a detection unit including a substrate to which a
probe complementary to miRNA is immobilized in Example.
[0077] FIG. 20 is an image showing a result of mixing solutions
using a solution mixer in Example.
[0078] FIG. 21A is a result showing the detail of controlling a
valve in a fluidic device in Example.
[0079] FIG. 21B is a result showing the detail of controlling a
valve in the fluidic device in Example.
[0080] FIG. 22 is a schematic view of an aspect of a substrate of
the fluidic device in the present embodiment.
DESCRIPTION OF EMBODIMENTS
Solution Mixer
First Embodiment
[0081] A solution mixer of the present embodiment includes: a main
flow path in which a solution circulates; a solution introduction
flow path connected to the main flow path; and a solution discharge
flow path connected to the main flow path. The solution discharge
flow path has a solution discharge flow path valve for opening and
closing the solution discharge flow path, the main flow path has a
first main flow path valve for quantitatively compartmentalizing
the main flow path, the first main flow path valve is disposed in
the vicinity of the solution discharge flow path, the main flow
path includes a second main flow path valve for quantitatively
compartmentalizing the main flow path, and the second main flow
path valve is disposed in the vicinity of the solution introduction
flow path.
[0082] FIG. 1 is a schematic view showing a basic configuration of
a solution mixer 20 of the present embodiment. The solution mixer
of the present embodiment includes: a main flow path 21 in which a
solution circulates; a solution introduction flow path 42 connected
to the main flow path; and a solution discharge flow path 32
connected to the main flow path. A solution discharge flow path
valve 33 for opening and closing the solution discharge flow path
32 is provided in the solution discharge flow path 32. A first main
flow path valve 23b for quantitatively compartmentalizing the main
flow path 21 is provided in the main flow path 21. The first main
flow path valve 23b is disposed in the vicinity of the solution
discharge flow path 32. The main flow path further includes a
second main flow path valve 23a for quantitatively
compartmentalizing the main flow path 21. The second main flow path
valve 23a is disposed in the vicinity of the solution introduction
flow path 42. The number of the solution introduction flow path 42
and the number of the solution discharge flow path 32 are not
particularly limited. However, the solution mixer 20 shown in FIG.
1 includes one solution introduction flow path 42 and one solution
discharge flow path 32.
[0083] The solution mixer 20 of the present embodiment has the main
flow path valves 23. The main flow path 21, which is
compartmentalized by putting the main flow path valves 23 into a
closed state, becomes flow paths each of which has an independent
volume. In addition, it is possible to control discharge of air or
the like in the main flow path 21 and filling with a solution, by
operating the opening and the closing of the solution discharge
flow path valve 33.
[0084] The solution mixer 20 of the present embodiment has the main
flow path 21 of which the volume within the flow path has already
been determined. Therefore, it is possible to mix solutions in a
state in which the volume of the solutions, with which the main
flow path 21 compartmentalized by the main flow path valves 23 is
filled, is accurately quantified. After the solutions are sent to
the flow paths and the volume of the solutions is quantitatively
determined, the compartmentalized flow paths communicate with each
other by opening the main flow path valves 23, and therefore, it
becomes possible to mix the solutions.
[0085] In this manner, as the main flow path 21 can be used in
mixing solutions as well as in quantitatively determining
solutions, it is possible to simultaneously perform injection of
solutions into mixer and quantitative determination of solutions,
and therefore, it is possible to promote efficiency of an
operation.
Second Embodiment
[0086] A solution mixer of the present embodiment further has
solution introduction flow path valves for opening and closing the
solution introduction flow paths, in the configuration of the
solution mixer of the above first embodiment. The solution mixer of
the present embodiment includes: a first introduction flow path
through which a first solution is introduced; and a second
introduction flow path through which a second solution is
introduced, as the solution introduction flow path. FIG. 2 is a
schematic view showing a basic configuration of a solution mixer 30
of the present embodiment. The solution mixer 30 further has
solution introduction flow path valves 43 for opening and closing
the solution introduction flow paths 42. The solution mixer
includes: a first introduction flow path 42a through which the
first solution is introduced, and a second introduction flow path
42b through which the second solution is introduced, as the
solution introduction flow paths. By providing the solution
introduction flow path valves 43, the main flow path 21 can be
completely compartmentalized by the solution introduction flow path
valves 43 and a solution discharge valve 33. Accordingly, in a case
where the solution introduction flow path valves 43 and the
solution discharge flow path valve 33 are closed and the main flow
path valves 23 are open, mixing of solutions is more efficiently
realized within the main flow path 21 which is closed by the
solution introduction flow path valves 43 and the solution
discharge flow path valve 33.
[0087] In addition, by providing the first introduction flow path
43a through which a first solution is introduced and the second
introduction flow path 43b through which a second solution is
introduced, as the solution introduction flow paths in the solution
mixer 30, it is possible to individually introduce solutions, which
are different from each other, into the main flow path 21 which is
compartmentalized by the main flow path valves 23. Accordingly, it
is preferable that the second main flow path valve 23a is disposed
between the first introduction flow path 42a, and the second
introduction flow path 42b through which the second solution is
introduced, as shown in FIG. 2.
Third Embodiment
[0088] The configuration of a solution mixer 30' of the present
embodiment is shown in FIG. 3. The solution mixer 30' of the
present embodiment includes: a first discharge flow path 32a
through which a first solution is discharged; and a second
discharge flow path 32b through which a second solution is
discharged, as the solution discharge flow paths 32, in the
configuration of the solution mixer 30 of the above second
embodiment. In addition, as shown in FIG. 3, it is preferable that
the first main flow path valve 23b is disposed between the first
discharge flow path 32a through which a first solution is
discharged and the second discharge flow path 32b through which a
second solution is discharged. The solution mixer 30' of the
present embodiment having such a configuration can individually
control the discharge of air or the like in the main flow path,
filling of the solutions, and the like with respect to each of the
first solution and the second solution which are introduced into
the main flow path 21 which is compartmentalized by the main flow
path valves 23, through an operation of opening and closing the
solution discharge flow path valve 33a or 33b.
Fourth Embodiment
[0089] A solution mixer of the present embodiment includes: a third
main flow path valve for quantitatively compartmentalizing the main
flow path. The third main flow path valve is in the vicinity of the
solution discharge flow path which is connected between the first
main flow path valve and the third main flow path valve. The view
schematically showing the solution mixer of the present embodiment
is shown in FIG. 4. A solution mixer 40 of the present embodiment
includes: a third main flow path valve 23b' for quantitatively
compartmentalizing the main flow path 21. The third main flow path
valve 23b' is in the vicinity of the solution discharge flow path
32 which is connected between the first main flow path valve 23b
and the third main flow path valve 23b'. The solution mixer 40 of
the present embodiment having such a configuration can individually
control the discharge of air or the like in the main flow path,
filling of the solutions, and the like with respect to the
solutions within the main flow path 21 which is compartmentalized
by the main flow path valves 23. For example, when the main flow
path 21 is in a state of being filled with solutions, it is
possible to discharge a solution in a flow path which is
compartmentalized by the main flow path valves 23a and 23b', out of
the solutions with which the main flow path 21 is filled, through
the solution discharge flow path 32 by closing the first main flow
path valve 23b and the second main flow path valve 23a and opening
the third main flow path valve 23b' and the solution discharge
valve 33.
Fifth Embodiment
[0090] In a solution mixer of the present embodiment, the main flow
path includes a first flow path, a second flow path, and a
connecting flow path which allows communication between the first
flow path and the second flow path. The connecting flow path has
the first main flow path valve.
[0091] The schematic view showing a basic configuration of the
solution mixer of the present embodiment is shown in FIG. 5. In a
solution mixer 50, the main flow path 21 includes a first flow path
21a, a second flow path 21b, and connecting flow paths 22 which
allows communication between the first flow path 21a and the second
flow path 21b. The connecting flow paths 22 have the first main
flow path valve 23b. In addition, it is preferable that the
solution mixer 50 of the present embodiment includes the second
main flow path valve 23a and the third main flow path valve 23b' as
shown in FIG. 5.
[0092] By allowing communication between the plurality of flow
paths represented by the first flow path 21a and the second flow
path 21b using the connecting flow paths 22, it becomes easy to
sequentially mix a plurality of solutions as will be described in
the second embodiment in the "Solution Mixing Method" to be
described below.
Sixth Embodiment
[0093] A solution mixer of the present embodiment further includes
a pump in the solution mixer 50 of the fifth embodiment which has
been described above. In addition, it is preferable that the pump
is a pump valve which can send a solution in accordance with
opening and closing of the valve. FIG. 6 is a view schematically
showing the solution mixer of the present embodiment. A solution
mixer 60 includes pump valves 24, and the pump is constituted of
three pump valves 24. The number of pump valves 24 may be greater
than or equal to four. By disposing the pumps in the main flow path
21, more efficient rotary mixing is realized. The main flow path
valves 23 may be used as the pump valves.
Seventh Embodiment
[0094] A solution mixer of the present embodiment includes a
detection unit of a mixed solution of the first solution and the
second solution. FIG. 7 is a view of schematically showing the
solution mixer of the present embodiment. A solution mixer 70
further includes: a detection unit 4c in the solution mixer 50 of
the fifth embodiment which has been described above.
[0095] The detection unit 4c increases the opportunity of contact
with a molecule contained in a solution by circulatory mixing the
solution within the main flow path 21 of the solution mixer 70.
[0096] It is preferable that the detection unit 4c included in the
fluidic device of the present embodiment includes a substrate to
which a substance having affinity to the molecule (biomolecule) is
immobilized. In a case where the biomolecule is a nucleic acid, it
is preferable that the detection unit 4c includes a substrate 136
to which a probe complementary to a target nucleic acid is
immobilized. In a case where the biomolecule is a miRNA, it is
preferable that the detection unit includes substrate 136 to which
a probe complementary to target miRNA is immobilized (refer to FIG.
22). In a case where the biomolecule is a protein, it is preferable
that the substrate 136 is a protein array. Examples of the
substrate to which a probe complementary to target miRNA is
immobilized include a DNA chip which is known in the related
art.
[0097] Furthermore, it is preferable that the detection unit 4c
includes the following configuration from the viewpoint of
specifically detecting target miRNA with high sensitivity.
[0098] In a case where target miRNA 133 includes a first section
131 and a second section 132 as shown in FIG. 22, it is preferable
that the detection unit 4c includes a substrate to which a capture
probe 134 including a sequence which can be hybridized with the
first section 131 is immobilized.
[0099] A detection probe 135 includes: two stem sections 135c and
135d forming a double stranded structure; a loop section 135e which
is a region between the two stem sections 135c and 135d and is
labeled using a labeling substance 135a; and a sequence 135b that
can be hybridized with the second section 132 in a case where the
target miRNA 133 includes the first section 131 and the second
section 132, and the detection probe has a 5'-protruding end or a
3'-protruding end.
[0100] The capture probe 134 and the detection probe 135 can
respectively be hybridized with the first section 131 and the
second section 132 of the miRNA 133. For this reason, the length of
the first section 131 and the length of the second section 132 are
preferably 5 bases to 17 bases, and more preferably 7 bases to 15
bases from the viewpoint of the number of bases in which miRNA
formed of about 22 bases is divided into two.
[0101] In the present embodiment, the section on the 5' side of the
miRNA 133 is regarded as the first section 131 and the section on
the 3' side of the miRNA 133 is regarded as the second section
132.
[0102] The expression "can be hybridized" in the present invention
and in the present specification means that a part of a capture
probe and a part of a detection probe which are used in the present
invention are hybridized with a target nucleic acid (target miRNA)
under stringent conditions, but are not hybridized with nucleic
acid molecule other than the target nucleic acid (target miRNA).
Examples of the "stringent conditions" include conditions disclosed
in Molecular Cloning-A Laboratory Manual, Third Edition (Sambrook
et al., Cold Spring Harbor Laboratory Press).
[0103] The capture probe 134 includes a sequence which can be
hybridized with the first section 131 of the miRNA 133 in a 5'-end
region.
[0104] It is preferable that the capture probe 134 does not include
a sequence complementary to the second section 132 of the miRNA 133
so as not to be hybridized with the second section 132 of the miRNA
133 from the viewpoint of quantitatively determining the miRNA 133
with high accuracy.
[0105] Molecular degrees of freedom are required in order for the
capture probe 134 which has been immobilized to the substrate 136
to be hybridized with the miRNA 133. Therefore, it is preferable
that the capture probe 134 has a spacer 134a, which is bound to the
substrate 136, at the 3'-end. The length of the spacer 134a is not
particularly limited, but is preferably 3 bases to 50 bases and
more preferably 5 bases to 25 bases. However, a base to be used for
the spacer can be replaced with a linker such as PEG which has the
same length and the same flexibility as that of the base. In that
case, the number of bases to be used for the spacer 134a may be
0.
[0106] The length of the capture probe 134 is not particularly
limited as long as the length is a length required for functioning
as a probe, but is preferably 3 bases to 50 bases and more
preferably 5 bases to 40 bases in consideration of the number of
bases of the first section 131 and the spacer 134a.
[0107] The capture probe 134 may be DNA or RNA. The capture probe
is not limited to be a natural one or a non-natural one as long as
the probe has the same function as that of DNA or RNA and may be
one containing an artificial nucleic acid such as a peptide nucleic
acid (PNA), a locked nucleic acid (LNA), and a bridged nucleic acid
(BNA). It is preferable that the capture probe 134 contains an LNA
or a BNA from the viewpoint of higher affinity to the target miRNA
133, being more hardly recognized by DNase or RNase, and being more
capable of becoming a substrate of DNA ligase such as T4 DNA
ligase, compared to DNA or RNA.
[0108] Examples of the substrate 136 used for immobilizing the
capture probe 134 include a glass substrate, a silicon substrate, a
plastic substrate, and a metal substrate. Examples of the method of
immobilizing the capture probe 134 on the substrate 136 include a
method of immobilizing a probe on a substrate at high density using
a photolithographic technology or a method of immobilizing a probe
on a glass substrate or the like through spotting.
[0109] In the present embodiment, the detection probe 135 includes
the sequence 135b which can be hybridized with the second section
132 of the miRNA 133 in a 3'-end region.
[0110] It is preferable that the detection probe 135 does not
contain a sequence complementary to the first section 131 of the
miRNA 133 so as not to be hybridized with the first section 131 of
the miRNA 133, from the viewpoint of quantitatively determining the
miRNA 133 with high accuracy.
[0111] The detection probe 135 forms a stem loop structure. The
stem loop structure refers to, when there are complementary
sequences at two regions which are distant from each other within a
single strand nucleic acid molecule, formation of a double stranded
structure (stem structure) through an interaction between base
pairs of nucleic acids and formation of a loop structure by a
sequence which is between the two regions. The stem loop structure
is also called a hairpin loop.
[0112] In the present embodiment, the detection probe 135 is
constituted of: the two stem sections 135c and 135d forming a
double stranded structure; the loop section 135e which is a region
between the two stem sections 135c and 135d; and the sequence 135b
that can be hybridized with the second section 132, from the 5'-end
side. That is, the detection probe 135 has a 3' protruding end. The
detection probe has a protruding end, and whether the protruding
end included in the detection probe is the 5'-protruding end or the
3'-protruding end depends on whether the capture probe and the
substrate bind to each other through the 5'-end of the capture
probe or through the 3'-end of the capture probe.
[0113] The length of a stem section in the detection probe 135 is
determined by a balance with the length of a loop section. The
length thereof is not particularly limited as long as the length
thereof is a length in which the detection probe 135 can stably
form a stem loop structure, and is preferably 3 bases to 50 bases
and more preferably 5 bases to 20 bases.
[0114] The length of a loop section in the detection probe 135 is
determined by a balance with the length of a stem section. The
length thereof is not particularly limited as long as the length
thereof is a length in which the detection probe 135 can stably
form a stem loop structure, and is preferably 3 bases to 200 bases
and more preferably 5 bases to 100 bases.
[0115] The length of the detection probe 135 is not particularly
limited as long as the length thereof is a length in which it is
possible to form a stem loop structure and which is required for
functioning as a probe, and is preferably 14 bases to 200 bases and
more preferably 24 bases to 150 bases in consideration of the
number of bases of the second section 132 and the number of bases
required for forming a stem loop structure.
[0116] The detection probe 135 may be DNA or RNA. The capture probe
is not limited to be a natural one or a non-natural one as long as
the probe has the same function as that of DNA or RNA and may be
one containing an artificial nucleic acid such as a peptide nucleic
acid (PNA), a locked nucleic acid (LNA), and a bridged nucleic acid
(BNA). It is preferable that the detection probe 135 contains an
LNA or a BNA from the viewpoint of higher affinity to the target
miRNA, being more hardly recognized by DNase or RNase, and being
more capable of becoming a substrate of DNA ligase such as T4 DNA
ligase, compared to DNA or RNA.
[0117] It is preferable that at least any one of the capture probe
134 and the detection probe 135 contains an LNA or a BNA and it is
more preferable that both of the capture probe 134 and the
detection probe 135 contain an LNA or a BNA.
[0118] The detection probe 135 is labeled by the labeling substance
135a. Examples of the labeling substance include fluorescent
pigments, fluorescent beads, quantum dots, biotin, antibodies,
antigens, energy absorption materials, radioisotopes,
chemiluminescent bodies, and enzymes.
[0119] Examples of the fluorescent pigments include
carboxyfluorescein (FAM), 6-carboxy-4',5'-dichloro-2', 7'-dimethoxy
fluorescein (JOE), fluorescein isothiocyanate (FITC), tetrachloro
fluorescein (TET), 5'-hexachloro-fluorescein-CE phosphoroamidite
(HEX), Cy3, Cy5, Alexa 568, and Alexa 647.
[0120] In the total RNA, there is only a minute amount of miRNA,
and therefore, it is difficult to label the miRNA at high
efficiency without fractionating the miRNA. In contrast, in the
present embodiment, a detection probe which has been previously
labeled is used, and therefore, it is possible to detect the miRNA
with high sensitivity.
[0121] According to the present embodiment, solutions which are
brought into contact with a detection unit are accurately and
quantitatively determined, and therefore, it is possible to realize
accurate analysis.
Eighth Embodiment
[0122] In a solution mixer of the present embodiment, the main flow
path includes an agitating structure. Examples of the agitating
structure include a structure having a curvature. In the structure
having a curvature, the flow velocity near the wall surface becomes
slow by the interaction (friction) between solutions and the wall
surface of the flow path within the flow path and the flow velocity
in the center of the flow path becomes fast. As a result, it is
possible to distribute solutions in accordance with the flow
velocity of a liquid, and therefore, the mixing of the solutions is
promoted. Examples of the inner diameter of the flow path include
0.01 mm to 3 mm or 0.5 mm to 1 mm.
[0123] In addition, the structure having a curvature may be
included in a folded structure. FIG. 8 is a view schematically
showing a solution mixer 80 of the present embodiment. In the
solution mixer 80, the main flow path includes a folded structure
31. Here, the "folded structure" refers to a structure in which a
flow path turns about 180 degrees to the direction perpendicular to
the major axis direction of the flow path which becomes a reference
line.
[0124] The number of times of the folding can be counted by the
number of times of the change in the direction in which the flow
path extends, and the number of times of the folding in the folded
structure 31 shown in FIG. 8 is eight. In the folded structure, the
difference in the above-described flow velocity is repeatedly
caused, and therefore, the mixing of solutions is further
promoted.
Fluidic Device
First Embodiment
[0125] The fluidic device of the present embodiment includes the
solution mixer which has been described above.
[0126] It is preferable that the fluidic device of the present
embodiment is a device which detects a biomolecule contained in an
exosome in a sample. The exosome is a small lipid vesicle having a
diameter of 30 nm to 100 nm, and is secreted as a fused body of an
endosome and a cell membrane in a body fluid such as blood, urine,
or saliva from various cells such as a tumor cell, a dendritic
cell, a T cell, or a B cell.
[0127] Abnormal cells such as cancer cells express a specific
protein, a specific nucleic acid, microRNA, or the like in the
inside of a cell membrane. An exosome secreted in a body fluid also
expresses a microRNA derived from a cell as a secretion source. For
this reason, it is expected that a technology which makes it
possible to examine an abnormality within a living body by
analyzing a biomolecule existing inside a membrane of an exosome in
a body fluid, even without performing a biopsy examination, is
established. The biopsy examination refers to a clinical
examination in which diagnosis or the like of a disease is examined
by observing a lesion site using a microscope after collecting a
tissue of the lesion site.
Second Embodiment
[0128] As shown in FIG. 9, a fluidic device 1 of the present
embodiment includes: an exosome purification unit 2 which has a
layer modified with a compound having a hydrophobic chain and a
hydrophilic chain; a biomolecule purification unit 3; a solution
mixer 4; a detection unit 4c; a first flow path 5 which connects
the exosome purification unit 2 to the biomolecule purification
unit 3; and a second flow path 6 which connects the biomolecule
purification unit 3 to the solution mixer 4.
[0129] It is preferable that the fluidic device 1 of the present
embodiment further includes a waste liquid tank from the viewpoint
of preventing a secondary infection due to a sample used in
analysis. For example, as shown in FIG. 10, a micro flow path
device (fluidic device 1) of the present embodiment includes a
first waste liquid tank 7, a second waste liquid tank 8, and a
third waste liquid tank 9, and preferably includes: a third flow
path 10 which connects the first waste liquid tank 7 and the
exosome purification unit 2; a fourth flow path 11 which connects
the second waste liquid tank 8 to the biomolecule purification unit
3; and a fifth flow path 12 which connects the third waste liquid
tank 9 to the solution mixer 4. There are three waste liquid tanks
shown in FIG. 10. However, the waste liquid tanks may be combined
in one or two waste liquid tanks.
[0130] As will be described below, a waste liquid from the exosome
purification unit 2 is sent to the first waste liquid tank 7
through the third flow path 10. A waste liquid from the biomolecule
purification unit 3 is sent to the second waste liquid tank 8
through the fourth flow path 11. A waste liquid from the solution
mixer 4 is sent to the third waste liquid tank 9 through the fifth
flow path 12.
[0131] An example of each configuration in the fluidic device 1 of
the present embodiment will be described using FIG. 11. The exosome
purification unit 2 includes an inlet, and an exosome
immobilization unit 2d which has the layer modified with the
compound having a hydrophobic chain and a hydrophilic chain. It is
preferable that the exosome purification unit 2 includes an inlet
for each reagent to be introduced, as shown in FIG. 11. That is, it
is preferable that the exosome purification unit 2 preferably
includes a sample introduction inlet 2b and a lysis buffer
introduction inlet 2c, and it is more preferable that the exosome
purification unit further includes a washing liquid introduction
inlet 2a.
[0132] The compound which has a hydrophobic chain and a hydrophilic
chain in the exosome immobilization unit 2d is a compound having a
hydrophobic chain in order to be bound to a lipid bilayer membrane,
and a hydrophilic chain in order to make dissolve this lipid chain
soluble. By using the compound, it is possible to immobilize an
exosome having a lipid bilayer membrane on the exosome
immobilization unit 2d.
[0133] In the present specification, the expression "immobilization
of an exosome on the exosome immobilization unit 2d" means
adsorption of an exosome onto the exosome immobilization unit.
[0134] The hydrophobic chain may be a single chain or a multiple
chain, and examples thereof include a saturated or unsaturated
hydrocarbon group which may have a substituent group.
[0135] As the saturated or unsaturated hydrocarbon group, a C6-C24
straight-chain or branched-chain alkyl group or alkenyl group is
preferable, and examples thereof include a hexyl group, a heptyl
group, an octyl group, a nonyl group, a decyl group, an undecyl
group, a dodecyl, a tridecyl group, a tetradecyl group, a
pentadecyl group, a hexadecyl group, a heptadecyl group, a stearyl
group (octadecyl group), a nonadecyl group, an icosyl group, a
heneicosyl group, a docosyl group, a tricosyl group, a tetracosyl
group, a myristoleyl group, a palmitoleyl group, an oleyl group, a
linoyl group, a linoleyl group, a ricinoleyl group, and an
isostearyl group.
[0136] Among these, a myristoleyl group, a palmitoleyl group, an
oleyl group, a linoyl group, and a linoleyl group are preferable,
and an oleyl group is more preferable.
[0137] Examples of the hydrophilic chain include proteins,
oligopeptides, polypeptides, polyacrylamide, polyethylene glycol
(PEG), and dextran, and PEG is preferable.
[0138] The hydrophilic chain is preferably modified chemically for
binding to a substrate, more preferably has an active ester group,
and particularly preferably has an N-hydroxysuccinimide group.
[0139] That is, as the compound having a hydrophobic chain and a
hydrophilic chain, a lipid-PEG derivative is preferable.
[0140] The lipid-PEG derivative is called a biocompatible anchor
for membrane (BAM). Examples of the BAM include a compound
represented by the following Formula (1).
##STR00001##
[In the formula, n represents an integer greater than or equal to
1.]
[0141] Examples of the substrate used as a layer of the exosome
immobilization unit 2d include a glass substrate, a silicon
substrate, a polymer substrate, and a metal substrate. The
substrate may bind to the compound having a hydrophobic chain and a
hydrophilic chain through a substance that binds to the hydrophilic
chain of the compound. Examples of the substance include a
substance having an amino group, a carboxyl group, a thiol group, a
hydroxyl group, or an aldehyde group, and
3-aminopropyltriethoxysilane is preferable.
[0142] Driving of a liquid in the fluidic device 1 of the present
embodiment is performed by an external suction pump, and the flow
of the liquid is controlled by opening and closing a pneumatic
valve. The opening and closing of a valve is driven and controlled
by an external pneumatic device which is connected to the fluidic
device 1.
[0143] As shown in FIG. 11, in the analysis of an exosome, a sample
is first injected into the sample introduction inlet 2b in the
above-described exosome purification unit, and the sample is
introduced into the exosome immobilization unit 2d through
suctioning, after opening a valve 2f of a flow path 2i.
[0144] As the amount of the sample used in the analysis is
preferably about 1 mL.
[0145] The sample is not particularly limited as long as the sample
can be obtained from an environment surrounding a cell to be
detected and contains an exosome secreted by the cell, and examples
thereof include blood, urine, breast milk, bronchoalveolar lavage
fluid, amniotic fluid, a malignant effusion, or saliva. Among
these, blood or urine from which it is easy to detect an exosome is
preferable. Furthermore, in blood, blood plasma is preferable in
view of ease of detection of an exosome.
[0146] In addition, the sample also includes a cell culture
solution which contains an exosome secreted by a culture cell.
[0147] Examples of the cell to be detected include a cancer cell, a
mast cell, a dendritic cell, a reticulocyte, an epithelial cell, a
B cell, and a neuron, which are known to produce an exosome.
[0148] The sample may be prepared through ultracentrifugation,
ultrafiltration, continuous flow electrophoresis, filtration using
a size filter, gel filtration chromatography, or the like. However,
in the present embodiment, the affinity between an exosome and a
compound having a hydrophobic chain and a hydrophilic chain in the
exosome immobilization unit 2d is significantly high, and
therefore, the sample may be a sample itself which has not been
prepared.
[0149] It is preferable to provide a non-specific adsorption
suppression unit to the exosome immobilization unit 2d from the
viewpoint of specifically binding an exosome to the exosome
immobilization unit 2d. Examples of the method thereof include a
method of modifying a substrate with a compound having a
hydrophobic chain and a hydrophilic chain, and then, treating a
site which is not modified with the compound having a hydrophobic
chain and a hydrophilic chain, with a compound having a hydrophilic
chain such as PEG.
[0150] An exosome in a sample which has been introduced into the
exosome immobilization unit 2d is captured by the above-described
compound having a hydrophobic chain and a hydrophilic chain. The
affinity between the exosome and the compound having a hydrophobic
chain and a hydrophilic chain is significantly high. Therefore,
exosomes in samples are captured on the exosome immobilization unit
2d at the same time when the samples continuously pass through the
top of the exosome immobilization unit 2d without allowing the
samples to stand in the exosome immobilization unit 2d.
[0151] For example, the suction pressure during the capturing of an
exosome is 1 kPa to 30 kPa and the time required for the capturing
is about 15 seconds. A waste liquid which has been passed through
the exosome immobilization unit 2d is sent to the first waste
liquid tank 7 after passing through the third flow path 10 via the
valve 10a.
[0152] In the fluidic device 1 of the present embodiment, it is
preferable to design the ceiling height of the exosome
immobilization unit 2d to be low. By way of this, the opportunity
of contact between an exosome and a compound having a hydrophobic
chain and a hydrophilic chain is increased, and therefore, it is
possible to improve the capturing efficiency of an exosome.
[0153] In blood, extracellular vesicles such as microvesicles or
apoptotic bodies are contained in addition to the exosome, and
there is a possibility that these extracellular vesicles will be
immobilized to the exosome immobilization unit 2d. From the
viewpoint of removing these extracellular vesicles from the exosome
immobilization unit 2d, it is preferable to wash an exosome on the
exosome immobilization unit 2d.
[0154] For example, as shown in FIG. 11, a washing liquid is
injected into the washing liquid introduction inlet 2a after
opening the valve 2e on the flow path 2h, and is introduced into
the exosome immobilization unit 2d.
[0155] In the present embodiment, the binding of the exosome to the
layer modified with the compound having a hydrophilic chain and a
hydrophobic chain is strong. Therefore, it is possible to adjust
the flow velocity to be fast and to perform washing in a short
period of time. For example, washing is performed by sending 500
.mu.L of a PBS washing liquid for about 15 seconds at a suction
pressure of 1 kPa to 30 kPa. A waste liquid which has been passed
through the exosome immobilization unit 2d is sent to the first
waste liquid tank 7 after passing through the third flow path 10
via the valve 10a.
[0156] Next, the exosome which has been immobilized on the exosome
immobilization unit 2d is lysed. As shown in FIG. 11, a lysis
buffer is injected into the lysis buffer introduction inlet 2c and
is introduced into the exosome immobilization unit 2d through
suctioning, after opening a valve 2g on a flow path 2j. Examples of
the lysis buffer include a known liquid in the related art which is
used in lysing a cell.
[0157] The exosome which has been captured on the exosome
immobilization unit 2d is lysed by the lysis buffer passing through
the exosome immobilization unit 2d, and a biomolecule contained in
the exosome is released.
[0158] For example, the suction pressure during the lysing of an
exosome is 1 kPa to 30 kPa and the time required for the lysing is
about 30 seconds. A waste liquid which has been passed through the
exosome immobilization unit 2d is sent to the first waste liquid
tank 7 after passing through the third flow path 10 via the valve
10a. The biomolecule which has been released from the exosome is
sent to the biomolecule purification unit 3 after passing through
the first flow path 5 via a valve 5a.
[0159] As shown in FIG. 11, the biomolecule purification unit 3
preferably includes a biomolecule recovery liquid introduction
inlet 3b and a biomolecule immobilization unit 3c, and more
preferably further includes a biomolecule washing liquid
introduction inlet 3a.
[0160] The biomolecule immobilization unit 3c is not particularly
limited as long as the biomolecule immobilization unit can fix a
biomolecule, and examples thereof include a silica membrane which
fixes a nucleic acid.
[0161] An exosome holds a protein or a nucleic acid which is
derived from a cell as a secretion source. Examples of the nucleic
acid include miRNA. In recent years, it has been reported that
miRNA which is non-code RNA with a short chain suppresses gene
expression within a living body, and the relationship between
abnormal expression of miRNA and various diseases including cancer
is becoming clear.
[0162] In the present embodiment, it is preferable that a
biomolecule which is immobilized by the biomolecule immobilization
unit 3c is miRNA. Examples of the biomolecule immobilization unit
3c include a silica membrane embedded on the flow path, as
described above.
[0163] A biomolecule is captured on the biomolecule immobilization
unit 3c by an exosome lysis buffer passing through the biomolecule
immobilization unit 3c.
[0164] For example, the suction pressure during the sending an
exosome lysis buffer is 50 kPa to 70 kPa and the time required for
the sending is about 1 minute. A waste liquid which has been passed
through the biomolecule immobilization unit 3c is sent to the
second waste liquid tank 8 after passing through the fourth flow
path 11 via a valve 11a.
[0165] After immobilizing a biomolecule on the biomolecule
immobilization unit 3c, it is preferable to remove impurities other
than the target biomolecule by washing the biomolecule
immobilization unit 3c.
[0166] As shown in FIG. 11, a valve 3d on a flow path 3e is opened,
a washing liquid is injected into the biomolecule washing liquid
introduction inlet 3a, and a washing liquid is introduced into the
biomolecule immobilization unit 3c through suctioning. Examples of
the washing liquid include ethanol at about 70% to 80%.
[0167] For example, the amount of washing liquid to be used during
washing is about 1 mL, the suction pressure is 50 kPa to 70 kPa,
and the time required for sending a washing liquid is about 1
minute. A waste liquid which has been passed through the
biomolecule immobilization unit 3c is sent to the second waste
liquid tank 8 after passing through the fourth flow path 11 via the
valve 11a. The biomolecule which has been released from the exosome
is sent to the biomolecule purification unit 3 after passing
through the first flow path 5 via the valve 5a.
[0168] In order to prevent the biomolecule washing liquid from
being brought into the solution mixer, it is preferable to dry the
biomolecule immobilization unit 3c after washing the biomolecule
immobilization unit 3c.
[0169] As shown in FIG. 11, drying of the biomolecule
immobilization unit is performed by suctioning air from the
biomolecule washing liquid introduction inlet 3a and passing the
air through the biomolecule immobilization unit 3c.
[0170] For example, the suction pressure during the drying of the
biomolecule immobilization unit 3c is 50 kPa to 70 kPa and the time
required for drying is about 2 minutes.
[0171] Next, the biomolecule which has been immobilized on the
biomolecule immobilization unit 3c is eluted. In order to improve
the recovery rate of the biomolecule, it is preferable to hold a
biomolecule recovery liquid for a certain time after introducing
the biomolecule recovery liquid into the biomolecule immobilization
unit 3c.
[0172] As shown in FIG. 11, the biomolecule recovery liquid is
injected into the biomolecule recovery liquid introduction inlet 3b
after opening a valve 3f of a flow path 3g, and is introduced into
the biomolecule immobilization unit 3c.
[0173] For example, the biomolecule recovery liquid is RNase-free
water, the amount of the recovery liquid used is 30 .mu.L, the
recovery liquid is suctioned at a suction pressure of 50 kPa to 70
kPa, the suctioning is stopped at a point in time at which the
recovery liquid has reached the biomolecule immobilization unit 3c,
and the recovery liquid is held for about 3 minutes.
[0174] Next, the biomolecule is recovered from the biomolecule
immobilization unit 3c. For example, the recovery liquid is
recovered for 30 seconds at a suction pressure of 50 kPa to 70
kPa.
[0175] The biomolecule is sent to the solution mixer 4 through the
second flow path 6. For example, the suction pressure of the
biomolecule into the solution mixer 4 is less than or equal to 6
kPa, and the biomolecule is sent to the solution mixer for about 30
seconds.
[0176] The sending of the biomolecule to the solution mixer 4 is
preferably performed after closing valves 4g and 4h in FIG. 11. By
doing this, the solution containing the biomolecule is
quantitatively determined within the flow path of the solution
mixer.
[0177] A flow path 6 is connected between the valves 4e and 4g. In
addition, the flow path 12 as a solution discharge flow path may be
set so as to be connected between the main flow path valve 23b and
the main flow path valve 23b' as shown in the fifth to seventh
embodiments in "Solution Mixer" which have been described
above.
[0178] After the biomolecule is sent to the solution mixer 4, a
detection probe dissolved liquid is injected into a detection probe
introduction inlet 4a after opening a valve 4d, and is sent to the
solution mixer 4. Transfer of the detection probe dissolved liquid
to the solution mixer 4 is performed after closing the valves 4g
and 4h in FIG. 11. By doing this, the detection probe dissolved
liquid is quantitatively determined within the flow path of the
solution mixer. The position of a flow path for discharge may be
set as shown in the fifth to seventh embodiments in
<<Solution Mixer>> which have been described above.
[0179] For example, the composition of the detection probe
dissolved liquid is a 100 nM to 200 nM detection probe, 100 mM to
200 mM Tris-HCl (pH 7.5), 200 mM to 400 mM NaCl, 10 mM to 30 mM
MgCl.sub.2, 0.5 mg/mL to 2 mg/mL BSA, 10 mM to 30 mM DTT, and 5
units/.mu.L to 20 units/.mu.L T4 DNA Ligase. The detection probe
dissolved liquid is sent to the solution mixer for about 30 seconds
at a suction pressure of less than or equal to 6 kPa.
[0180] Next, the biomolecule and the detection probe dissolved
liquid are circulated within the solution mixer after closing
valves 4d, 4e, 4f, and 12a and opening the valves 4g and 4h, and
are mixed with each other. For example, the opening and closing of
a pump valve which is not shown in the drawing is continuously
performed for about 10 minutes. A complex (miRNA 133-detection
probe 135-capture probe 134 complex) is efficiently formed on a
substrate within a short period of time through the circulation of
the liquid (refer to FIG. 22). In addition, the pump for
circulating a solution is constituted of at least three pump valves
including the valves 4g and 4h which are disposed within the flow
path of the solution mixer. For example, these three pump valves
include one valve 4g and two valves 4h. Alternately, these three
pump valves include two valves 4g and one valve 4h. The pump valves
include the valve 4g, the valve 4h, and a valve which is not shown
in the drawing.
[0181] Next, it is preferable to remove a non-specific adsorbed
material on the substrate by washing the substrate to which a
capture probe is immobilized. Accordingly, it is preferable that
the solution mixer 4 further includes a washing liquid introduction
inlet 4b as shown in FIG. 11. A washing liquid is injected into the
washing liquid introduction inlet 4b after opening the valve 4e,
and is introduced into the substrate.
[0182] For example, the washing liquid is a 0.2.times.SSC buffer of
which the amount used is 500 .mu.L. The washing is performed by
sending the washing liquid to the substrate for 1 minute at a
suction pressure of less than or equal to 6 kPa. It is preferable
that the washing liquid is circulated within the solution mixer.
The washing of the base is efficiently performed within a short
period of time through the circulation of the washing liquid. A
waste liquid which has been passed through the substrate is sent to
the third waste liquid tank 9 after passing through the fifth flow
path 12 via the valve 12a.
[0183] Next, the intensity of a labeling substance of the complex
which has been formed on the substrate is measured. The intensity
of a labeling substance reflects the amount of biomolecule
existing. Therefore, according to the present embodiment, it is
possible to quantitatively determine the amount of biomolecule
contained in a sample.
[0184] The measurement of the intensity of a labeling substance is
performed by, for example, a microscope, a light source, or a
control unit such as a personal computer, which is not shown in the
drawing.
[0185] According to the present embodiment, it is possible to
promptly perform analysis of an exosome only within about one hour
unlikely in the related art in which it has been taken one day or
longer. Furthermore, solutions which are brought into contact with
a detection unit are accurately quantitatively determined, and
therefore, it is possible to realize accurate analysis.
Solution Mixing Method
First Embodiment
[0186] A solution mixing method of the present embodiment which
uses the solution mixer that has been described above includes: a
step of sending a first solution from a solution introduction flow
path; a step of closing a main flow path valve so as to the first
solution being quantitatively delivered by quantitatively
compartmentalizing the main flow path; a step of sending a second
solution from the solution introduction flow path; a step of
closing a solution discharge valve; a step of obtaining a third
solution by subjecting the first solution and the second solution
to rotary mixing; a step of detecting the third solution; and a
step of washing a main flow path by sending a washing liquid to the
main flow path after the step of the detecting of the third
solution.
[0187] The solution mixing method of the present embodiment will be
described below while referring to FIG. 12. A mixer 20' shown in
FIG. 12 further includes a detection unit 4c, which is included in
the solution mixer 70 of the seventh embodiment in <<Solution
Mixer>> that has been described above, in the solution mixer
20 of the first embodiment in <<Solution Mixer>> that
has been described above. First, a first solution 91 is sent from a
solution introduction flow path (refer to FIG. 12B) while the main
flow path valves 23 and the discharge flow path valve 33 of the
solution mixer 20' are open (refer to FIG. 12A). Next, the main
flow path valves 23 are closed so as to quantitatively divide the
first solution by quantitatively compartmentalizing the main flow
path 21 (refer to FIG. 12C). Then, a second solution 92 is sent
from the solution introduction flow path 42 (refer to FIG. 12D). A
third solution 93 is obtained by circulatory mixing the first
solution 91 with the second solution 92 by closing the solution
discharge valve 33 and opening the main flow path valves 23 (refer
to FIG. 12E). Next, after discharging the third solution 93 by
opening the solution discharge flow path valve 33, a washing liquid
is sent to the main flow path 21 from the solution introduction
flow path 42 to wash the main flow path. The washing is efficiently
achieved by circulatory mixing the washing liquid through the same
method as that described above.
Second Embodiment
[0188] A solution mixing method of the present embodiment which
uses the solution mixer that has been described above includes: a
step A of selecting two adjacent flow paths (main flow paths) out
of a plurality of flow paths (main flow paths) included in the main
flow path; a step B of closing valves which are adjacent to the
flow paths (main flow paths) such that the two flow paths (main
flow paths) and the connecting flow path which is adjacent to the
two flow paths (main flow paths) are quantitatively
compartmentalized; a step C of sending the first solution to the
first flow path (main flow path) out of the two flow paths (main
flow paths); a step D of sending the second solution to the second
flow path (main flow path) out of the two flow paths (main flow
paths); and a step E of obtaining a third solution by circulatory
mixing the first solution with the second solution after opening
the main flow path valves for opening and closing the connecting
flow path which allows communication between the two flow paths
(main flow paths). The solution mixing method thereof further
includes: a step F of selecting a third flow path (main flow path)
which is adjacent to the two flow paths (main flow paths) after the
step E; a step G of closing valves which are adjacent to the third
flow path (main flow path) such that the third flow path (main flow
path) and the connecting flow path which is adjacent to the third
flow path (main flow path) are quantitatively compartmentalized; a
step H of sending a fourth solution to the third flow path (main
flow path); and a step I of obtaining a fifth solution by
circulatory mixing the third solution with the fourth solution
after opening the main flow path valves for opening and closing the
connecting flow path which allows communication between the three
flow paths (main flow paths).
[0189] The solution mixing method of the present embodiment will be
described below while referring to FIGS. 13 to 16. FIGS. 13 to 16
are modification examples of the solution mixer 30' which is shown
in the third embodiment and the solution mixer 50 which is shown in
the fifth embodiment in the above-described solution mixers. The
number of flow paths which communicates with each other using the
connecting flow path included in the solution mixer is two.
However, the solution mixer 50' in FIGS. 13 to 16 includes four
parallel flow paths 21a, 21b, 21c, and 21d as main flow paths. Each
of the flow paths includes each solution introduction flow path 42,
solution introduction flow path valves 43a, 43b, 43c, and 43d, each
solution discharge flow path 32, and solution discharge flow path
valves 33a, 33b, 33c, and 33d. In addition, the solution mixer 50'
includes each connecting flow path 22 which allows communication
between the flow paths, and main flow path valves 23a, 23b, 23c,
23d, 23e, and 23f which are arranged on the connecting flow path
22.
[0190] The solution mixing method using the solution mixer 50' will
be described below. FIGS. 13A and 13B may be referred to for the
steps A and B, FIGS. 14A and 14B may be referred to for the steps C
to E, FIGS. 15A and 15B may be referred to for the steps F and
FIGS. 16A and 16B may be referred to for the steps H and I.
[0191] (Step A): First, two adjacent flow paths (main flow paths)
21a and 21b out of main flow paths 21 are selected.
[0192] (Step B): Main flow path valves 23a, 23b, 23c, and 23d and
solution discharge flow path valves 33a and 33b which are adjacent
to the flow paths (main flow paths) are closed such that the two
flow paths (main flow paths) 21a and 21b and the connecting flow
path 22 which is adjacent to the two flow paths (main flow paths)
are quantitatively compartmentalized.
[0193] (Steps C and D): A first solution 91 and a second solution
92 are respectively sent to the first flow path (main flow path)
21a and the second flow path (main flow path) 21b.
[0194] (Step E): A third solution 93 is obtained by circulatory
mixing the first solution 91 with the second solution 92 after
opening the main flow path valves 23a and 23b for opening and
closing the connecting flow path which allows communication between
the flow path (main flow path) 21a and the flow path (main flow
path) 21b.
[0195] (Step F): A third flow path (main flow path) 21c which is
adjacent to the two flow paths (main flow paths) 21a and 21b is
selected.
[0196] (Step G): Main flow path valves 23e and 23f and a solution
discharge flow path valve 33c which are adjacent to the flow path
(main flow path) 21c are closed such that the flow path (main flow
path) 21c and the connecting flow path which is adjacent to the
flow path (main flow path) 21c are quantitatively
compartmentalized.
[0197] (Step H): A fourth solution 94 is sent to the flow path
(main flow path) 21c.
[0198] (Step I): A fifth solution 95 is obtained by circulatory
mixing the third solution with the fourth solution after opening
the main flow path valves 23a, 23b, 23c, and 23d for opening and
closing the connecting flow path which allows communication between
these three flow paths (main flow paths) 21a, 21b, and 21c.
[0199] As shown in the present embodiment, it is possible to
circulate mix desired solutions by sequentially selecting a
plurality of parallel flow paths included in the solution mixer
50'. In addition, the rotary mixing may be performed by similarly
repeating the step F to the step I.
[0200] Hereinafter, the present invention will be described using
Example, but is not limited to the following Example.
EXAMPLE
Purification of Exosome
[0201] A glass surface was modified with
3-aminopropyltriethoxysilane (hereinafter, also referred to as
APTES) and a terminal of APTES was then modified with a PEG-lipid
derivative, which captured an exosome to the terminal of APTES and
was represented by the Formula (1), and methoxy PEG which
suppresses non-specific adsorption. Next, a purification device was
produced by subjecting polymethacrylstyrene to cutting processing.
An exosome suspension, which was recovered through
ultracentrifugation of a culture supernatant of a breast cancer
cell strain MCF-7, and exosomes in human serum were immobilized to
the inside of the device. Then, the density of the immobilized
particles was measured by AFM.
[0202] AFM images and the immobilization density of exosomes which
have been immobilized to the inside of the device are shown in FIG.
17. First, it was confirmed that particles having diameters of 30
nm to 200 nm were immobilized thereto, from the AFM images.
[0203] Next, it was confirmed that the immobilization density was
exponentially decreased with respect to the distance from the
immobilization layer. In addition, the immobilized amount in a case
where exosomes were directly immobilized from human serum was 74%
of cases where purified exosomes were immobilized. Therefore, it
was considered that the methoxy PEG contributed to the suppression
of the non-specific adsorption.
[Purification of miRNA]
[0204] A device in which a miniaturized silica membrane was
immobilized to the inside of a flow path was produced to perform
purification of miRNA. miRNA was suspended in an exosome lysis
buffer which was then passed through the silica membrane through a
suction operation. Subsequently, washing and drying of the silica
membrane were performed, and then, miRNA was recovered by
introducing a miRNA elution liquid. The amount of miRNA recovered
was obtained through quantitative real-time PCR.
[0205] In addition, miRNeasy Mini Kit of QIAGEN was used for the
comparison with a general spin column method.
[0206] The recovery results of miRNA are shown in FIG. 18. In the
present unit, shortening of required time and reduction of the
amount of reagent used were achieved by reducing the size of the
silica membrane. In addition, it became possible to recover miRNA
using a small amount of elution liquid in accordance with the
reduction in the size, and therefore, it became possible to
concentrate the miRNA solution.
[Detection of miRNA]
[0207] RNA having a sequence of miR-141, miR-143, miR-1275,
miR-107, miR-181a-2*, miR-484, miR-21, let-7a, let-7b, let-7d,
let-7f, and miR-39 as target miRNAs was synthesized. In addition,
in total 12 kinds of nucleic acid probes of detection probes having
a sequence which is complementary to each target miRNA were
designed and synthesized. In contrast, capture probes having a
sequence complementary to each target miRNA were synthesized on a
glass substrate, and were arranged in a spot shape.
[0208] Used sequences of the target miRNA, the capture probes, and
the detection probes are shown below.
TABLE-US-00001 (1) Target miRNA 1: miR-141 (SEQ ID No: 1: 22-mer)
[Sequence: 5'-UAACACUGUCUGGUAAAGAUGG-3'] Target miRNA 2: miR-143
(SEQ ID No: 2: 21-mer) [Sequence: 5'-UGAGAUGAAGCACUGUAGCUC-3']
Target miRNA 3: miR-1275 (SEQ ID No: 3: 17-mer) [Sequence:
5'-GUGGGGGAGAGGCUGUC-3'] Target miRNA 4: miR-107 (SEQ ID No: 4:
23-mer) [Sequence: 5'-AGCAGCAUUGUACAGGGCUAUCA-3'] Target miRNA 5:
miR-181a-2* (SEQ ID No: 5: 22-mer) [Sequence:
5'-ACCACUGACCGUUGACUGUACC-3'] Target miRNA 6: miR-484 (SEQ ID No:
6: 22-mer) [Sequence: 5'-UCAGGCUCAGUCCCCUCCCGAU-3'] Target miRNA 7:
miR-21 (SEQ ID No: 7: 22-mer) [Sequence:
5'-UAGCUUAUCAGACUGAUGUUGA-3'] Target miRNA 8: let-7a (SEQ ID No: 8:
22-mer) [Sequence: 5'-UGAGGUAGUAGGUUGUAUAGUU-3'] Target miRNA 9:
let-7b (SEQ ID No: 9: 22-mer) [Sequence:
5'-UGAGGUAGUAGGUUGUGUGGUU-3'] Target miRNA 10: let-7d (SEQ ID No:
10: 22-mer) [Sequence: 5'-AGAGGUAGUAGGUUGCAUAGUU-3'] Target miRNA
11: let-7f (SEQ ID No: 11: 22-mer) [Sequence:
5'-UGAGGUAGUAGAUUGUAUAGUU-3'] Target miRNA 12: miR-39 (SEQ ID No:
12: 22-mer) [Sequence: 5'-UCACCGGGUGUAAAUCAGCUUG-3']
(2) Capture Probe 1
[0209] [Sequence: 5'-p-X1-fS-3']
[0210] X1 represents the following sequence, p represents a
phosphoric acid, S represents a thiol group, and f represents 6-FAM
(6-fluoroscein).
TABLE-US-00002 X1: (SEQ ID No: 13: 60-mer)
ACCAGACAGTGTTAACAACAACAACAACAACAACAACAACAACAACAACAA CAACAACAA
Capture probe 2 X1: (SEQ ID No: 14: 60-mer)
GTGCTTCATCTCAACAACAACAACAACAACAACAACAACAACAACAACAAC AACAACAAC
Capture probe 3 X1: (SEQ ID No: 15: 60-mer)
CTCCCCCACACAACAACAACAACAACAACAACAACAACAACAACAACAACA ACAACAACA
Capture probe 4 X1: (SEQ ID No: 16: 60-mer)
CTGTACAATGCTGCTACAACAACAACAACAACAACAACAACAACAACAACA ACAACAACA
Capture probe 5 X1: (SEQ ID No: 17: 60-mer)
CAACGGTCAGTGGTACAACAACAACAACAACAACAACAACAACAACAACAA CAACAACAA
Capture probe 6 X1: (SEQ ID No: 18: 60-mer)
GGGACTGAGCCTGAACAACAACAACAACAACAACAACAACAACAACAACAA CAACAACAA
Capture probe 7 X1: (SEQ ID No: 19: 60-mer)
AGTCTGATAAGCTAACAACAACAACAACAACAACAACAACAACAACAACAA CAACAACAA
Capture probe 8 X1: (SEQ ID No: 20: 60-mer)
AACCTACTACCTCAACAACAACAACAACAACAACAACAACAACAACAACAA CAACAACAA
Capture probe 9 X1: (SEQ ID No: 21: 60-mer)
ACCTACTACCTCAACAACAACAACAACAACAACAACAACAACAACAACAAC AACAACAAC
Capture probe 10 X1: (SEQ ID No: 22: 60-mer)
AACCTACTACCTCTACAACAACAACAACAACAACAACAACAACAACAACAA CAACAACAA
Capture probe 11 X1: (SEQ ID No: 23: 60-mer)
ATCTACTACCTCAACAACAACAACAACAACAACAACAACAACAACAACAAC AACAACAAC
Capture probe 12 X1: (SEQ ID No: 24: 60-mer)
TTTACACCCGGTGAACAACAACAACAACAACAACAACAACAACAACAACAA CAACAACAA
(3) Detection Probe 1
[0211] [Sequence: 5'-p-X2-Al-X3-3']
[0212] X2 and X3 represents the following sequences, p represents a
phosphoric acid, and Al represents Alexa647-AminoC6-dA.
TABLE-US-00003 X2: (SEQ ID No: 25: 17-mer) CTCAACTGGTGTCGTGG X3:
(SEQ ID No: 26: 26-mer) GTCGGCAATTCAGTTGAGCCATCTTT Detection probe
2 X2: (SEQ ID No: 25: 17-mer) CTCAACTGGTGTCGTGG X3: (SEQ ID NO: 27:
26-mer) GTCGGCAATTCAGTTGAGGAGCTACA Detection probe 3 X2: (SEQ ID
No: 25: 17-mer) CTCAACTGGTGTCGTGG X3: (SEQ ID NO: 28: 26-mer)
GTCGGCAATTCAGTTGAGGACAGCCT Detection probe 4 X2: (SEQ ID No: 25:
17-mer) CTCAACTGGTGTCGTGG X3: (SEQ ID NO: 29: 26-mer)
GTCGGCAATTCAGTTGAGTGATAGCC Detection probe 5 X2: (SEQ ID No: 25:
17-mer) CTCAACTGGTGTCGTGG X3: (SEQ ID NO: 30: 26-mer)
GTCGGCAATTCAGTTGAGGGTACAGT Detection probe 6 X2: (SEQ ID No: 25:
17-mer) CTCAACTGGTGTCGTGG X3: (SEQ ID NO: 31: 26-mer)
GTCGGCAATTCAGTTGAGATCGGGAG Detection probe 7 X2: (SEQ ID No: 25:
17-mer) CTCAACTGGTGTCGTGG X3: (SEQ ID NO: 32: 26-mer)
GTCGGCAATTCAGTTGAGTCAACATC Detection probe 8 X2: (SEQ ID No: 25:
17-mer) CTCAACTGGTGTCGTGG X3: (SEQ ID NO: 33: 26-mer)
GTCGGCAATTCAGTTGAGAACTATAC Detection probe 9 X2: (SEQ ID No: 25:
17-mer) CTCAACTGGTGTCGTGG X3: (SEQ ID NO: 34: 27-mer)
GTCGGCAATTCAGTTGAGAACCACACA Detection probe 10 X2: (SEQ ID No: 25:
17-mer) CTCAACTGGTGTCGTGG X3: (SEQ ID NO: 35: 26-mer)
GTCGGCAATTCAGTTGAGAACTATGC Detection probe 11 X2: (SEQ ID No: 25:
17-mer) CTCAACTGGTGTCGTGG X3: (SEQ ID NO: 36: 27-mer)
GTCGGCAATTCAGTTGAGAACTATACA Detection probe 12 X2: (SEQ ID No: 25:
17-mer) CTCAACTGGTGTCGTGG X3: (SEQ ID NO: 37: 26-mer)
GTCGGCAATTCAGTTGAGCAAGCTGA
[0213] A DNA micro array substrate to which one of the
above-described capture probes was immobilized was purchased from
Agilent Technologies, and was allowed to stand for 90 minutes at
room temperature while being brought into contact with a solution
in Table 1. After washing the DNA micro array substrate with
ultrapure water and drying the DNA micro array substrate, the DNA
micro array substrate was installed in a solution mixer.
[0214] In Table 1, the composition of Takara 10.times. buffer is
500 mM Tris-HCl (pH 7.5), 100 mM MgCl2, and 50 mM DTT.
TABLE-US-00004 TABLE 1 10 unit/.mu.l T4 Polynucleotide Kinase 25
.mu.l 5M betaine100 mM ATP 10 .mu.l Takara 10X buffer 100 .mu.l
Milli-QWater 865 .mu.l Total 1000 .mu.l
[0215] Furthermore, a miRNA solution with an arbitrary
concentration was adjusted as in Table 2, and a hybridization
reaction solution containing a detection probe was prepared as in
Table 3.
TABLE-US-00005 TABLE 2 1 .mu.M miR-141 1 .mu.l 1 .mu.M miR-143 1
.mu.l 1 nM miR-1275 1 .mu.l 1 .mu.M miR-107 1 .mu.l 1 .mu.M
miR-181a-2* 1 .mu.l 1 .mu.M miR-484 1 .mu.l 1 .mu.M miR-21 1 .mu.l
1 .mu.M let-7a 1 .mu.l 1 .mu.M let-7b 1 .mu.l 1 .mu.M let-7d 1
.mu.l 1 .mu.M let-7f 1 .mu.l 10 .mu.M miR-39 1 .mu.l RNase-free
water 88 .mu.l Total 100 .mu.l
TABLE-US-00006 TABLE 3 20 .mu.M Detect probe1 1 .mu.l 20 .mu.M
Detect probe2 1 .mu.l 20 .mu.M Detect probe3 1 .mu.l 20 .mu.M
Detect probe4 1 .mu.l 20 .mu.M Detect probe5 1 .mu.l 20 .mu.M
Detect probe6 1 .mu.l 20 .mu.M Detect probe7 1 .mu.l 20 .mu.M
Detect probe8 1 .mu.l 20 .mu.M Detect probe9 1 .mu.l 20 .mu.M
Detect probe10 1 .mu.l 20 .mu.M Detect probe11 1 .mu.l 20 .mu.M
Detect probe12 1 .mu.l 1M Tris-HCl (pH7.5) 13.3 .mu.l 1M MgCl.sub.2
2 .mu.l 100 mM ATP 2 .mu.l 10 mg/ml BSA 2 .mu.l 1M DTT 2 .mu.l 2.5M
NaCl 12 .mu.l 350 units/.mu.l T4 DNA ligase 2.9 .mu.l RNase-free
water 51.8 .mu.l Total 100 .mu.l
[0216] The prepared miRNA solution was introduced from an inlet 1
of a solution mixer and the hybridization reaction solution was
introduced from an inlet 2, and the solutions were hybridized by
being circulated for 10 minutes.
[0217] After the completion of the hybridization reaction, the DNA
micro array substrate was washed by sending 500 .mu.l of a washing
liquid, which contains 0.3 M NaCl and 30 mM sodium citrate, from
the inlet 3, and the fluorescence intensity was measured after
observing the substrate using a fluorescence microscope.
[0218] The results are shown in FIG. 19. FIG. 19(a) is an image of
the substrate showing miRNA analysis results.
[0219] FIG. 19(b) corresponds to FIG. 19(a), and a spot shown by
half-tone dot meshing is a spot which corresponds to target miRNA
and in which fluorescence is to be observed. Each letter
corresponds to the following miRNA.
A: 141, B: 143, C: 1275, D: 107, E: 181a-2*, F: 484, S: let-7a, T:
let-7b, U: let-7d
[0220] In each of the spots to which probes corresponding to the
introduced miRNA were immobilized, fluorescence images of the
detection probes which had been labeled with Alexa 647 were
observed. miR-1275 of "C" was put at a concentration of one
thousandth of the other miRNAs in order to check the detection
limit concentration, and therefore, the fluorescence becomes dark.
In addition, the difference in brightness for each of the sequences
of the probes is caused by the difference in affinity of
probes.
[0221] For this reason, it was confirmed that it was possible to
sequence-dependently detect miRNA using the solution mixer.
[Quantitative Determination of Solution and Rotary Mixing]
[0222] A first solution 91 was sent to a solution mixer by opening
a valve 43a in a state in which valves 23a and 23b on a main flow
path 21 of a solution mixer ((2) in FIG. 20) were closed. A second
solution 92 was sent to the solution mixer by opening a valve 43b
in a state in which valves 33 were closed ((3) in FIG. 20). Next, a
pump constituted of a pump valve (23a) was started by opening 23a
and 23b in a state in which the valves 43a, 43b, and 33 were
closed. Then, the first solution 91 was rotatably mixed with the
second solution 92 to obtain a third solution 93 ((4) in FIG. 20
and (5) in FIG. 20). The first solution 91 was sufficiently mixed
with the second solution 92.
[Opening and Closing of Valve in Fluidic Device]
[0223] A fluidic device shown in FIG. 21B was produced. It was
confirmed that it was possible to control the flow of a fluid
through the control of the opening and closing of valves in each
step shown in Table of FIG. 21A.
[0224] From the above-described results, according to the present
embodiments, it is possible to quantitatively mix a solution
containing target miRNA contained in an exosome and a solution
containing a detection probe, using a solution mixer which has a
detection unit on a flow path. Furthermore, the swift analysis of
exosomes can be automated.
REFERENCE SIGNS LIST
[0225] 1 . . . fluidic device, 2 . . . exosome purification unit,
2a . . . washing liquid introduction inlet, 2b . . . sample
introduction inlet, 2c . . . lysis buffer introduction inlet, 2d .
. . exosome immobilization unit, 2e, 2f, 2g, 3d, 3f, 4f, 4g, 4h,
5a, 10a, 11a . . . valve, 2h, 2i, 2j, 3e, 3g . . . flow path, 3 . .
. biomolecule purification unit, 3b . . . biomolecule recovery
liquid introduction inlet, 3c . . . biomolecule immobilization
unit, 4 . . . solution mixer, 4c . . . detection unit, 5 . . .
first flow path, 6 . . . second flow path, 7 . . . first waste
liquid tank, 8 . . . second waste liquid tank, 9 . . . third waste
liquid tank, 10 . . . third flow path, 11 . . . fourth flow path,
12 . . . fifth flow path, 20, 20', 30, 30', 40, 50, 50', 60, 70, 80
. . . solution mixer, 21a, 21b, 21c, 21d (21) . . . main flow path,
31 . . . folded structure, 22 . . . connecting flow path, 32 . . .
solution discharge flow path, 23a, 23b, 23c, 23d, 23e, 23f (23) . .
. main flow path valve, 24 . . . pump valve, 33a, 33b, 33c, 33d
(33) . . . solution discharge flow path valve, 43a, 43b, 43c, 43d
(43) . . . solution introduction flow path valve, 91 . . . first
solution, 92 . . . second solution, 93 . . . third solution, 94 . .
. fourth solution, 95 . . . fifth solution, 133 . . . miRNA, 131 .
. . first section, 132 . . . second section, 134 . . . capture
probe, 134a . . . spacer, 135 . . . detection probe, 135a . . .
labeling substance, 135b sequence, 135c, 135d . . . stem section,
136 substrate
* * * * *