U.S. patent application number 16/782064 was filed with the patent office on 2020-09-10 for device for particle manipulation.
This patent application is currently assigned to SHIMADZU CORPORATION. The applicant listed for this patent is SHIMADZU CORPORATION. Invention is credited to Seiya FUJIWARA, Hiroyuki JIKUYA, Masaki KANAI, Ayaka MINAMIMOTO, Akira MURAMATSU, Tetsuo OHASHI, Masamitsu SHIKATA.
Application Number | 20200282407 16/782064 |
Document ID | / |
Family ID | 1000004807293 |
Filed Date | 2020-09-10 |
United States Patent
Application |
20200282407 |
Kind Code |
A1 |
SHIKATA; Masamitsu ; et
al. |
September 10, 2020 |
DEVICE FOR PARTICLE MANIPULATION
Abstract
An operation pipe and a device equipped with the operation pipe,
which use a gel to perform operations such as separation,
extraction, purification, elution, recovery, analysis and the like
of target components that are biological components such as nucleic
acids. More specifically, an operation pipe and a device, with
which it is possible to perform operations such as separation,
extraction, purification, elution, recovery, analysis and the like
of target components in a sealable pipe by operating magnetic
particles in the pipe under a magnetic field from outside of the
pipe.
Inventors: |
SHIKATA; Masamitsu; (Kyoto,
JP) ; MINAMIMOTO; Ayaka; (Kyoto, JP) ;
MURAMATSU; Akira; (Kyoto, JP) ; KANAI; Masaki;
(Kyoto, JP) ; FUJIWARA; Seiya; (Kyoto, JP)
; OHASHI; Tetsuo; (Kyoto, JP) ; JIKUYA;
Hiroyuki; (Kyoto, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SHIMADZU CORPORATION |
Kyoto |
|
JP |
|
|
Assignee: |
SHIMADZU CORPORATION
Kyoto
JP
|
Family ID: |
1000004807293 |
Appl. No.: |
16/782064 |
Filed: |
February 5, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B03C 2201/26 20130101;
B03C 2201/18 20130101; C12Q 1/6865 20130101; B01D 15/3819 20130101;
B01D 15/3885 20130101; C12Q 2563/107 20130101; B03C 1/12
20130101 |
International
Class: |
B03C 1/12 20060101
B03C001/12; C12Q 1/6865 20060101 C12Q001/6865; B01D 15/38 20060101
B01D015/38 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 9, 2019 |
JP |
2019-043228 |
Claims
1. An operation pipe for operating target components, comprising: a
hollow pipe, having a closable open end for supplying a sample
containing the target components on one side and a closed end on
the other side, and having an operation pipe portion on the open
end side and a recovery pipe portion on the closed end side; an
operation medium, which is filled in the operation pipe portion so
that gel layers and aqueous liquid layers are alternately
multi-layered in the longitudinal direction of the hollow pipe,
wherein a layer length of the gel layers and a layer length of the
aqueous liquid layers are determined by the length in the
longitudinal direction of the hollow pipe; a recovery medium, which
is filled in the recovery pipe portion so that a gel layer and an
aqueous liquid layer which is in contact with the closed end are
multi-layered, wherein the aqueous liquid layer in contact with the
closed end has a predetermined volume, and the layer length of the
gel layer is determined by the length in the longitudinal direction
of the hollow pipe; and magnetic particles for capturing and
transporting the target components; wherein the magnetic particles
pass through the gel layer in a gel state and move in the
longitudinal direction of the operation pipe due to application of
a magnetic field.
2. The operation pipe according to claim 1, wherein an inner
diameter of the hollow pipe is 0.1 mm-5 mm.
3. The operation pipe according to claim 1, wherein a volume of the
aqueous liquid layer in contact with the closed end is 1 .mu.L-1000
.mu.L.
4. The operation pipe according to claim 1, wherein the operation
pipe portion and the recovery pipe portion are separable.
5. The operation pipe according to claim 1, wherein the material of
the hollow pipe is selected from a group consisting of
polyethylene, polypropylene, fluororesin, polyvinyl chloride,
polystyrene, polycarbonate, acrylonitrile-butadiene-styrene
copolymer, acrylonitrile-styrene copolymer, acrylic resin,
polyvinyl acetate, polyethylene terephthalate, cyclic polyolefin,
and glass.
6. The operation pipe according to claim 1, wherein an inner
diameter of the open end is larger than an inner diameter of the
operation pipe portion and an inner diameter of the recovery pipe
portion.
7. The operation pipe according to claim 1, wherein the hollow pipe
has optical transparency.
8. The operation pipe according to claim 1, wherein surface
roughness of an inner surface of the hollow pipe is 0.1 Lm or
less.
9. The operation pipe according to claim 1, wherein a length of the
gel layer in the longitudinal direction of the hollow pipe is 1-20
mm, the gel layer being filled in the operation pipe portion and
the recovery pipe portion.
10. The operation pipe according to claim 1, wherein a length of
the aqueous liquid layer in the longitudinal direction of the
hollow pipe is 0.5-30 mm, the aqueous liquid layer being filled in
the operation pipe portion.
11. The operation pipe according to claim 1, wherein the magnetic
particles are particles having a binding force or adsorption force
to nucleic acids that are used as the target components, the
aqueous liquid layer in the operation medium is an aqueous liquid
layer containing a liquid that liberates nucleic acids and binds or
adsorbs the nucleic acids to the magnetic particles and/or an
aqueous liquid layer containing a cleaning liquid of the magnetic
particles, and the aqueous liquid layer in the recovery medium
which is in contact with the closed end is an aqueous liquid layer
containing a liquid that liberates nucleic acids.
12. The operation pipe according to claim 11, wherein the aqueous
liquid layer in the recovery medium which is in contact with the
closed end is an aqueous liquid layer further containing a reverse
transcription reaction liquid and/or a nucleic acid amplification
reaction liquid.
13. The operation pipe according to claim 12, wherein the aqueous
liquid layer in the recovery medium which is in contact with the
closed end is an aqueous liquid layer further containing a
fluorescent dye that is used to be specifically bound to the target
components and detect the target components by generating
fluorescence by light irradiation.
14. A device, comprising: the operation pipe according to claim 13;
a magnetic field applying part, which is capable of moving the
magnetic particles in the longitudinal direction of the operation
pipe by applying a magnetic field to the operation pipe; and an
optical detection part, which irradiates light to the recovery pipe
portion and detects fluorescence generated from the fluorescent dye
specifically bound to the target components.
15. A device comprising a plurality of operation pipes according to
claim 1, and further comprising a magnetic field applying part
capable of simultaneously moving, for the plurality of operation
pipes, the magnetic particles in the longitudinal direction of the
operation pipe by simultaneously applying a magnetic field to the
plurality of operation pipes.
16. The device according to claim 15, wherein the magnetic field
applying part comprises a movable substrate capable of moving in
the longitudinal direction of the operation pipe; a magnetic field
moving mechanism, which controls movement of the movable substrate
toward the longitudinal direction of the operation pipe; and a
plurality of magnetic sources, which corresponds to the plurality
of operation pipes and is held in the movable substrate.
17. A device comprising the operation pipe according to claim 1,
and a magnetic field applying part capable of moving the magnetic
particles in the longitudinal direction of the operation pipe by
applying a magnetic field to the operation pipe; wherein the
magnetic field applying part causes the magnetic field to perform
amplitude movement in the longitudinal direction of the operation
pipe or causes the magnetic field to perform rotational motion.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the priority benefit of Japan
application serial no. 2019-043228, filed on Mar. 9, 2019. The
entirety of the above-mentioned patent application is hereby
incorporated by reference herein and made a part of this
specification.
BACKGROUND OF THE DISCLOSURE
Technical Field
[0002] The disclosure relates to an operation pipe and a device
equipped with the operation pipe, which use a gel to perform
operations such as separation, extraction, purification, elution,
recovery, analysis and the like of target components that are
biological components such as nucleic acids. More specifically, the
disclosure relates to an operation pipe and a device, with which it
is possible to perform operations such as separation, extraction,
purification, elution, recovery, analysis and the like of target
components in a sealable pipe by operating magnetic particles in
the pipe under a magnetic field from outside of the pipe.
Related Art
[0003] By giving various chemical affinity to surfaces of
water-insoluble fine particles having a diameter of 0.5 am to
ten-odd micrometres, the target components can be separated,
extracted, purified, eluted, recovered and the like. In addition,
target cells can also be recovered by recognizing specific cell
surface molecules. Based on these findings, fine particles in which
functional molecules having affinity for the target components are
introduced onto the particle surface are commercially available.
Among these fine particles, ferromagnets of which the particle
material is iron oxide or the like can recover the target
components by a magnet, and have a feature advantageous for
automation of the extraction and purification of the target
components because centrifugation is not required.
[0004] For example, a system for continuously performing nucleic
acid extraction from cells to analysis performed by gene
amplification reaction in one device is commercially available. For
example, in GeneXpert System (non-patent literature 1) of Cepheid,
USA, the nucleic acid extraction to the analysis performed by gene
amplification reaction are performed in one cartridge-type device,
and the maximum number of specimens simultaneously processed is 16.
In addition, for example, in Simplexa (non-patent literature 2) of
3M Corporation, the nucleic acid extraction to PCR can be performed
in one disk-shaped device, and 12 specimens can be fixed on one
disk. However, these devices have a small number of specimens
simultaneously processed, and the devices themselves are
complicated and expensive to manufacture, making the devices
impractical. In addition, the device size is large and thus the
entire system is also large, making the devices impractical in
terms of mobility from an installation location.
[0005] Magnetic particles are commercially available as a part of
reagents that are extraction and purification kits. The kit is
configured by a plurality of reagents contained in separate
containers, and the user collects and dispenses the reagents by a
pipette or the like during use. Even in a case of an automated
device, in a currently marketed device, liquid collection is
mechanically performed by pipetting operations. For example, a
system (non-patent literature 3) for performing nucleic acid
extraction using magnetic particles is commercially available from
Precision System Science Co., Ltd. These pipetting operations are
accompanied by generation of aerosols. The generation of aerosol
increases the risk of contamination that hinders analysis. The same
also applies to a case in which the liquid collection is
mechanically performed by pipetting operations in an automated
device. In this case, since pollution source is accumulated in the
device due to the generation of aerosols, it is necessary to
periodically clean the device. However, in the device automated by
a pipette-type dispensing mechanism, the structure is complicated,
and it is difficult to completely remove the pollution source.
[0006] In general, the commercially available magnetic particles
enable separation and recovery of target components or specific
cells from a sample, but it is necessary to perform analysis of the
recovered material in another system such as a real-time PCR
device, a mass spectrometer, a flow cytometry or the like. In the
system which is commercially available from Precision System
Science Co., Ltd for using magnetic particles to perform nucleic
acid extraction, even the recovery of purified nucleic acids can be
performed, but it is necessary to perform the analysis performed by
gene amplification reaction and the like in another system such as
a real-time PCR device or the like. Furthermore, in this system,
the dispensing using a pipette-type dispenser is performed in an
open system, and thus is always accompanied by a risk of
contamination.
[0007] In order to solve the above problems, an operation pipe that
is small and has low running cost and a device equipped with the
operation pipe are reported, with which it is possible to perform
extraction and purification of target components in a completely
sealed container while avoiding contamination or possible to
analyze the target components in the same container while keeping
the sealed state following the extraction and purification (patent
literature 1). A sealable narrow pipe constituting the operation
pipe is filled with one or more liquid reagents partitioned by a
water-insoluble gel substance without using a dispenser accompanied
by the generation of aerosols, and the magnetic particles present
in the liquid reagents filled in the narrow pipe are moved by a
magnetic field applying part operable from outside of the narrow
pipe and pass through a water-insoluble gel substance layer.
LITERATURE OF RELATED ART
Patent Literature
[0008] [Patent literature 1] WO 2012/086243
Non-Patent Literature
[0009] [Non-patent literature 1] Clinical Chemistry 51: 882-890,
2005, Mar. 3, 2005 [0010] [Non-patent literature 2] "FDA Issues
Another Emergency Use Authorization for Commercial H1N1 Flu Test to
Quest Diagnostics' Focus Diagnostics", Focus Diagnostics Co., Ltd,
Oct. 17, 2009 [0011] [Non-patent literature 3] "GC series
Magtration Genomic DNA Whole Blood", Precision System Science Co.,
Ltd, December, 2008
SUMMARY
[0012] In the operation pipe disclosed in patent literature 1, a
sample containing magnetic particles is introduced from an open end
of a pipe constituting the operation pipe. The sample is obtained
by crushing or dissolving biological samples such as blood or
cells, and biological components such as nucleic acids is adsorbed
to the magnetic particles. The magnetic particles adsorbing the
biological components can be collected by an external magnetic
field. Furthermore, the magnetic particles adsorbing the biological
components can be moved in the pipe by movement of the external
magnetic field. Inside the pipe, a cleaning liquid layer for
washing away contaminants contained in the biological sample and an
elution liquid layer for liberating the biological components such
as nucleic acids are filled as liquid reagent layers, and the
cleaning liquid layer and the elution liquid layer are partitioned
by a water-insoluble gel layer so as not to be mixed. When the
external magnetic field is gently moved, the magnetic particles
adsorbing the biological components such as nucleic acids can
follow the movement and pass through the gel layer without mixing
the cleaning liquid and the elution liquid. On the other hand, when
the external magnetic field is rapidly reciprocated, the magnetic
particles in the liquid reagent layer cannot follow the movement of
the external magnetic field and are dispersed in the liquid. By
this operation, the contaminant components contained in the
biological sample are washed away in the cleaning liquid layer, and
the biological components such as nucleic acids are liberated from
the magnetic particles in the elution liquid layer.
[0013] The cleaning liquid layer, the eluent liquid layer, and the
water-insoluble gel layer filled in the pipe constituting the
operation pipe disclosed in patent literature 1 are filled based on
a distance in the longitudinal direction of the pipe. The reason is
that accurate setting of a movement position can be made based on
the distance in the setting of a driving program for moving the
external magnetic field. On the other hand, when an inner diameter
of the pipe varies, a volume of the liquid reagent in the liquid
reagent layer varies due to the variation. In particular, when the
liquid reagent is an elution liquid that liberates biological
components such as nucleic acids, variation in the volume of the
elution liquid causes variation in the concentration of the
biological components such as nucleic acids eluted in the elution
liquid. As a result, accurate recovery rate evaluation of the
biological components such as nucleic acids is hindered.
[0014] The disclosure provides an operation pipe and a device
equipped with the operation pipe, which enable accurate recovery
rate evaluation of biological components such as nucleic acids
recovered in an elution liquid.
[0015] That is, the disclosure includes the following aspects.
[1] An operation pipe for operating target components, including: a
hollow pipe, having a closable open end for supplying a sample
containing the target components on one side and a closed end on
the other side, and having an operation pipe portion a on the open
end side and a recovery pipe portion b on the closed end side; an
operation medium, which is filled in the operation pipe portion a
so that gel layers and aqueous liquid layers are alternately
multi-layered in the longitudinal direction of the hollow pipe,
wherein a layer length of the gel layers and a layer length of the
aqueous liquid layers are determined by the length in the
longitudinal direction of the hollow pipe; a recovery medium, which
is filled in the recovery pipe portion b so that a gel layer and an
aqueous liquid layer which is in contact with the closed end are
multi-layered, wherein the aqueous liquid layer in contact with the
closed end has a predetermined volume, and the layer length of the
gel layer is determined by the length in the longitudinal direction
of the hollow pipe; and magnetic particles for capturing and
transporting the target components; wherein the magnetic particles
pass through the gel layer in a gel state and move in the
longitudinal direction of the operation pipe due to application of
a magnetic field.
[0016] The open end is preferably closed so that all or a part of
the open end can be opened and closed. In FIGS. 1B and 1C, an
example of a preferable open end, an example of the hollow pipe
having the operation pipe portion a on the open end side and the
recovery pipe portion b on the closed end side, and an example of
the operation pipe are shown.
[0017] [2]
The operation pipe according to [1], wherein an inner diameter of
the hollow pipe is 0.1 mm-5 mm. [3]
[0018] The operation pipe according to [1] or [2], wherein a volume
of the aqueous liquid layer in contact with the closed end is 1
.mu.L-1000 .mu.L.
[4]
[0019] The operation pipe according to any one of [1] to [3],
wherein the operation pipe portion a and the recovery pipe portion
b are separable.
[5]
[0020] The operation pipe according to any one of [1] to [4],
wherein the material of the hollow pipe is selected from a group
consisting of polyethylene, polypropylene, fluororesin, polyvinyl
chloride, polystyrene, polycarbonate,
acrylonitrile-butadiene-styrene copolymer, acrylonitrile-styrene
copolymer, acrylic resin, polyvinyl acetate, polyethylene
terephthalate, cyclic polyolefin, and glass.
[6]
[0021] The operation pipe according to any one of [1] to [5],
wherein an inner diameter of the open end is larger than an inner
diameter of the operation pipe portion a and an inner diameter of
the recovery pipe portion b.
[7]
[0022] The operation pipe according to any one of [1] to [6],
wherein the hollow pipe has optical transparency.
[8]
[0023] The operation pipe according to any one of [1] to [7],
wherein surface roughness of an inner surface of the hollow pipe is
0.1 am or less.
[9]
[0024] The operation pipe according to any one of [1] to [8],
wherein a length of the gel layer in the longitudinal direction of
the hollow pipe is 1-20 mm, the gel layer being filled in the
operation pipe portion a and the recovery pipe portion b.
[10]
[0025] The operation pipe according to any one of [1] to [9],
wherein a length of the aqueous liquid layer in the longitudinal
direction of the hollow pipe is 0.5-30 mm, the aqueous liquid layer
being filled in the operation pipe portion a.
[11]
[0026] The operation pipe according to any one of [1] to [10],
wherein the magnetic particles are particles having a binding force
or adsorption force to nucleic acids that are used as target
components, the aqueous liquid layer in the operation medium is an
aqueous liquid layer containing a liquid that liberates nucleic
acids and binds or adsorbs the nucleic acids to the magnetic
particles and/or an aqueous liquid layer containing a cleaning
liquid of the magnetic particles, and the aqueous liquid layer in
the recovery medium which is in contact with the closed end is an
aqueous liquid layer containing a liquid that liberates nucleic
acids.
[12]
[0027] The operation pipe according to [11], wherein the aqueous
liquid layer in the recovery medium which is in contact with the
closed end is an aqueous liquid layer further containing a reverse
transcription reaction liquid and/or a nucleic acid amplification
reaction liquid.
[13]
[0028] The operation pipe according to [12], wherein the aqueous
liquid layer in the recovery medium which is in contact with the
closed end is an aqueous liquid layer further containing a
fluorescent dye that is used to be specifically bound to the target
components and detect the target components by generating
fluorescence by light irradiation.
[0029] [14]
[0030] A device, including: the operation pipe according to [13]; a
magnetic field applying part, which is capable of moving the
magnetic particles in the longitudinal direction of the operation
pipe by applying a magnetic field to the operation pipe; and an
optical detection part, which irradiates light to the recovery pipe
portion b and detects fluorescence generated from the fluorescent
dye specifically bound to the target components.
[15]
[0031] A device including a plurality of operation pipes according
to any one of [1] to [13], and further including a magnetic field
applying part capable of simultaneously moving, for the plurality
of operation pipes, the magnetic particles in the longitudinal
direction of the operation pipe by simultaneously applying a
magnetic field to the plurality of operation pipes.
[16]
[0032] The device according to [15], wherein the magnetic field
applying part includes: a movable substrate capable of moving in
the longitudinal direction of the operation pipe; a magnetic field
moving mechanism, which controls movement of the movable substrate
toward the longitudinal direction of the operation pipe; and a
plurality of magnetic sources, which corresponds to the plurality
of operation pipes and is held in the movable substrate.
[17]
[0033] A device including the operation pipe according to any one
of [1] to [13], and a magnetic field applying part capable of
moving the magnetic particles in the longitudinal direction of the
operation pipe by applying a magnetic field to the operation pipe,
wherein the magnetic field applying part causes the magnetic field
to perform amplitude movement in the longitudinal direction of the
operation pipe or causes the magnetic field to perform rotational
motion.
Effect
[0034] According to the disclosure, the volume of the elution
liquid that liberates biological components such as nucleic acids
is constant, the elution liquid being filled in the recovery pipe
portion b in a hollow pipe constituting an operation pipe and
constituting an aqueous liquid layer in contact with a closed end
of the hollow pipe, and thus accurate recovery rate evaluation of
the biological components such as nucleic acids recovered in the
elution liquid can be made. A gel layer and an aqueous liquid layer
other than the aqueous liquid layer in contact with the closed end
of the hollow pipe, which is the elution liquid for liberating the
biological components such as nucleic acids, are filled with a
thickness determined by the length in the longitudinal direction of
the hollow pipe, and thus it is unnecessary to change or modify a
drive program for moving an external magnetic field.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIGS. 1A, 1B, and 1C are longitudinal-sectional views of
examples of an operation pipe of the disclosure. A and B described
in FIG. 1C respectively represent an operation portion A and a
recovery portion B. The operation portion A includes an operation
pipe portion a corresponding in a hollow pipe and an operation
medium filled in the pipe portion a. The recovery portion B
includes a recovery pipe portion b corresponding in the hollow pipe
and a recovery medium filled in the pipe portion b.
[0036] FIGS. 2A to 2C show an example of a manufacturing method of
the operation pipe of the disclosure.
[0037] FIGS. 3A to 3O show a process in which the operation pipe of
the disclosure shown in FIGS. 1A to 1C is used to extract and
purify nucleic acids from a nucleic acid-containing sample.
[0038] FIGS. 4A to 4H show a process in which another example of
the operation pipe of the disclosure is used to extract and purify
nucleic acids from the nucleic acid-containing sample, and analysis
performed by a reverse transcription reaction and a PCR reaction
are further performed.
[0039] FIG. 5 is a perspective view showing an example of a device
that enables a simultaneous operation in the operation pipe by
using a plurality of operation pipes of the disclosure to make
multiple channels.
[0040] FIG. 6 is a cross-sectional view of a variation example of a
magnetic field applying part (movable magnet plate) shown in FIG.
5.
[0041] FIG. 7A is a longitudinal-sectional view of the variation
example of the magnetic field applying part (movable magnet plate)
shown in FIG. 5, FIG. 7B is a longitudinal-sectional view of a
variation example of a holding part (holding substrate), and FIG.
7C is a longitudinal-sectional view of a part including the
operation pipe held on the holding part.
[0042] FIG. 8 is a result obtained in Example 1 in which the
process shown in FIGS. 3A to 3O is performed.
DESCRIPTION OF THE EMBODIMENTS
[0043] [1. Operation of Target Components]
[0044] [1-1. Target Components]
[0045] Target components operated in the disclosure is not
particularly limited as long as the target components can be
operated in common aqueous liquids, emulsions, or hydrogels, and
may be any in vivo components and non-in vivo components. The in
vivo components include biomolecules such as nucleic acids
(including DNA and RNA), proteins, lipids, and sugars. The non-in
vivo components include non-biomolecules such as artificial (both
chemical and biochemical) modifiers, labelled bodies, mutants and
the like of the biomolecules, non-biomolecules derived from natural
products, and other components that can be operated in an aqueous
system.
[0046] The target components can usually be provided in the form of
a sample containing the target components. The sample include, for
example, biological samples such as animal and plant tissues, body
fluids and excreta, and biomolecule-containing bodies such as
cells, protozoa, fungi, bacteria and viruses. The body fluids
include blood, sputum, cerebrospinal fluid, saliva, and milk and
may be combinations thereof; and the excreta include feces, urine,
and sweat and may be combinations thereof. The cells include
leukocytes and platelets in blood or exfoliated cells of oral cells
and other mucosal cells and may be combinations thereof. These
samples can also be obtained as clinical swabs. In addition, the
above sample can also be prepared, for example, in the form of a
liquid mixture of a cell suspension, a homogenate, and a cell
lysate. In addition, the sample containing the target components
can also be obtained by applying treatments such as modification,
labelling, fragmentation, and mutation to the above sample.
[0047] The sample containing the target components may also be
prepared in advance by subjecting the above sample to
pre-treatment. The pre-treatment includes, for example, a treatment
for performing extraction, separation, and purification of the
target components or the target component-containing body from the
sample containing the target components, and the like. However,
since this pre-treatment can be performed in the operation pipe of
the disclosure, it is not always necessary to perform the
pre-treatment before the sample is added into the operation pipe.
By performing the pre-treatment in the operation pipe of the
disclosure, a contamination problem that is a concern in the
pre-treatment of the sample can be avoided.
[0048] [1-2. Operation]
[0049] [1-2-1. Operation Form]
[0050] In the disclosure, the sample containing the target
components is added to the operation pipe illustrated as 1 in FIGS.
1A to 1C, and the target components are operated in the operation
pipe. Operations of the target components in the disclosure include
supplying the target components to various treatments and
transporting the target components among a plurality of
environments for performing the various treatments. The operation
pipe is filled with gel layers and aqueous liquid layers. For
example, in the form illustrated in FIGS. 1A to 1C, the layers
represented by 2g and 3g consist of gels (gel plugs), and the layer
represented by 31 consists of an aqueous liquid. The layer
represented by 4 is an aqueous liquid layer in contact with the
closed end of a hollow pipe constituting the operation pipe of the
disclosure, and may also be a hydrogel as long as the aqueous
liquid can maintain a gel state. The aqueous liquid and the
hydrogel construct an environment for performing a treatment of the
target components. Accordingly, more specifically, the operations
of the target components in the disclosure include supplying the
target components to treatments in the aqueous liquid or hydrogel
and transporting the target components among a plurality of
environments for performing the treatments via a gel plug.
[0051] [1-2-2. Treatment of Target Components]
[0052] The treatments to which the target components are supplied
include the treatment accompanied by a substance change of the
target components and the treatment accompanied by a physical
change of the target components.
[0053] The treatment accompanied by a substance change of the
target components may be any treatment as long as a different
substance is newly generated by generating or breaking a bond
between substrates. More specifically, a chemical reaction and a
biochemical reaction are included. The chemical reaction may be any
reaction accompanied by compounding, decomposition, oxidation and
reduction. In the disclosure, generally, treatments that are
performed in an aqueous liquid are included. The biochemical
reaction may be any reaction accompanied by a change of biological
substances, and usually refers to an in vitro reaction. For
example, reactions based on a synthesis system, a metabolic system
and an immune system of biological substances such as nucleic
acids, proteins, lipids, sugars and the like are included.
[0054] The treatment accompanied by a physical change of the target
components may be any treatment not accompanied by the above
substance change. More specifically, denaturation (for example,
when the target components are biopolymers or other polymers
containing nucleic acids or proteins), dissolution, mixing,
emulsification, dilution, and the like of the target components are
included.
[0055] Accordingly, operations such as separation, extraction,
purification, elution, recovery, and analysis of the target
components can be performed by the treatment in the disclosure. By
these operations, isolation, detection, identification and the like
of the target components can be finally performed.
[0056] The treatments in the disclosure include not only the
desired treatments (treatments in a process in which effects of
isolation, detection, identification and the like of the target
components are directly obtained), but also a pre-treatment and/or
a post-treatment associated therewith as necessary. For example,
when the target components are nucleic acids, a nucleic acid
amplification reaction or a nucleic acid amplification reaction and
analysis of amplification products are performed, but extraction
(cell lysis) and/or purification (cleaning) of the nucleic acids
from a nucleic acid-containing sample and the like are essential as
the pre-treatments thereof. In addition, recovery and the like of
the amplification products may be performed as the
post-treatments.
[0057] [1-2-3. Transport of Target Components]
[0058] The transport of the target components is performed by
magnetic particles and a magnetic field applying part. The magnetic
particles are present in the operation pipe during operation, and
can transport the target components by moving the target components
in the operation pipe in a state that the target components are
captured by being bound and absorbed to the surface of the magnetic
particles. The magnetic particles can be dispersed in the aqueous
liquid layer in the operation pipe, and are usually aggregated in
the aqueous liquid layer due to generation of a magnetic field from
outside of the operation pipe by the magnetic field applying part.
The aggregated magnetic particles can move along with changes of
the magnetic field that is generated from outside of the operation
pipe by the magnetic field applying part. The aggregated magnetic
particles can move in the gel layer. By utilizing the thixotropic
property (thixotropic property) of the gel described in 3-2-3, the
aggregated magnetic particles can pass through the gel layer
without destroying the gel layer. In the gel, the aggregated
magnetic particles are accompanied by the target components by
binding or adsorption. Strictly speaking, the group of the
aggregated magnetic particles is coated with a very small amount of
aqueous liquid. Accordingly, components other than the target
components may be accompanied. However, since the amount of the
coated aqueous liquid is very small, almost no aqueous liquid is
contained. Therefore, the transport of the target components can be
performed very efficiently.
[0059] [2. Operation Pipe]
[0060] [2-1. Structure of Operation Pipe]
[0061] The structure of the operation pipe of the disclosure is
described with reference to FIGS. 1A-1C (in the following
description, the vertical direction uses FIGS. 1A-1C as a
reference). The hollow pipe constituting the operation pipe has an
upper end opened for sample feeding, and the open end is preferably
closable from the viewpoint of contamination. A lower end of the
hollow pipe is a closed end. Usually, the hollow pipe constituting
the operation pipe has a substantially circular cross section, but
pipes having other shapes of cross section are not excluded. The
pipe is filled with an operation medium in which aqueous liquid
layers 1 and gel layers g are alternately multi-layered in the
longitudinal direction of the pipe. FIGS. 1A-1C illustrate three
aspects FIGS. 1A-1C in which forms of an upper portion and a lower
portion of the operation pipe are different. However, the upper
portion and the lower portion can be arbitrarily combined and are
not limited to the combinations shown in FIGS. 1A-1C.
[0062] The upper open end of the pipe is a sample supply portion 5
for supplying the sample containing the target components, and the
sample supply portion 5 that is an open end may be temporarily
opened (see FIG. 1A), or all or a part of the sample supply portion
5 may be openably closed (shown in FIG. 1B). By using a septum
having a check valve function as an example that a part is openably
closed, it is possible to supply a sample by puncturing with an
injection needle that can substantially maintain a sealed state
(shown in FIG. 1C). It is preferable to close the sample supply
portion 5 that is an open end because a completely sealed system
can be constructed. By constructing a completely closed system,
contamination from outside during operation can be prevented, and
thus the operation pipe is very effective. The inner diameter of
the sample supply portion 5 may be the same as an inner diameter of
the pipe portion a filled with the gel layer and the aqueous liquid
layer which are operation mediums (shown in FIG. 1A), or may be
formed to have a wider inner diameter from the viewpoint of
operability during supply of the sample (shown in FIGS. 1B and
1C).
[0063] In the aspects illustrated in FIGS. 1A and 1B, the pipe is
integrally formed. In the aspect illustrated in FIG. 1C, the pipe
is configured by the operation pipe portion a and the recovery pipe
portion b. The upper end and the lower end of the operation pipe
portion a are open. The upper end of the recovery pipe portion b is
open and the lower end is closed. In the operation pipe portion a
and the recovery pipe portion b, one end of the pipe portion a and
the open end of the pipe portion b are connected. The operation
pipe portion a and the recovery pipe portion b may have a separable
shape, or may have a shape not considering separation (a shape that
cannot be separated).
[0064] The operation pipe portion a is filled with a gel layer 2g
that closes one end and a multi-layer that is multi-layered on the
gel layer 2g, that is, an operation medium 3. The operation medium
3 is configured so that aqueous liquid layers 31 and gel layers 3g
are alternately multi-layered. The part configured by the operation
pipe portion a and the operation medium that is the filling of the
operation pipe portion a is described as an operation portion A.
The recovery pipe portion b is filled so that a gel layer in
contact with the operation pipe portion a and an aqueous liquid
layer in contact with the closed end at the lower end are
multi-layered. The aqueous liquid layer in contact with the closed
end is represented by a recovery medium 4. The part configured by
the recovery pipe portion b, the recovery medium 4 that is the
filling of the recovery pipe portion b, and the gel layer in
contact with the operation pipe portion a is described as a
recovery portion B. The operation portion A and the recovery
portion B may be provided in a connected state or may be provided
in an independent state. In the recovery portion B, the gel layer
in contact with the operation pipe portion a has a function of
preventing the recovery medium 4 from flowing out in a state that
the recovery portion B is separated or independent. The recovery
portion B may be configured by a recovery pipe portion b that is
not filled with the gel layer and is filled only with the aqueous
liquid layer.
[0065] [2-2. Size of Operation Pipe]
[0066] The approximate inner diameter of the hollow pipe
constituting the operation pipe is, for example, 0.1 mm-5 mm,
preferably 1-2 mm. If the approximate inner diameter is in this
range, the operation pipe can have good operability. When the
substantially inner diameter is below the above range, a pipe wall
becomes thick to maintain the strength of the hollow pipe; as a
result, a distance between the magnetic particles and the magnet
increases, and a magnetic force reaching the magnetic particles
becomes weak, which may cause operational problems. On the other
hand, when the inner diameter of the hollow pipe exceeds the above
range, an interface between the multi-layer of the gel layer and
the aqueous liquid layer constituting the operation medium tends to
be easily disturbed due to an impact from outside or an influence
of gravity. Besides, in the disclosure, the pipe having an inner
diameter of 0.1 mm or less is not excluded as long as the capillary
material can withstand high-precision processing. The length in the
longitudinal direction of the operation pipe is, for example, 1-30
cm, preferably 5-15 cm.
[0067] Besides, as shown in FIGS. 1B and 1C, when the sample supply
portion 5 is formed in a manner that the inner diameter is wider,
the approximate inner diameter of the sample supply portion 5
exceeds the above range and may be 10 mm or less, preferably 5 mm
or less. It is preferable from the viewpoint of workability during
sample supply that the sample supply portion has a wider inner
diameter. When the wider inner diameter exceeds the above range,
for example, when a plurality of operation pipes are processed at
the same time, the operation pipes come into contact with each
other, and integration of the device tends to decrease.
[0068] [2-3. Material of Pipe]
[0069] The material of the hollow pipe constituting the operation
pipe is not particularly limited. For example, in order to reduce
movement resistance when the target components and a small amount
of liquid move together with the magnetic particles in the gel
layer, the inner wall which is a conveyance surface is smooth and
water-repellent. The material that gives such properties includes
resin materials such as polyethylene, polypropylene, fluororesin
(Teflon (registered trademark)), polyvinyl chloride, polystyrene,
polycarbonate, acrylonitrile-butadiene-styrene copolymer (ABS
resin), acrylonitrile-styrene copolymer (AS resin), acrylic resin,
polyvinyl acetate, polyethylene terephthalate, cyclic polyolefin
and the like. The resin materials are preferable in terms that the
layer in the operation pipe is unlikely to be disturbed even if the
operation pipe is dropped or bent and is highly robust. The
material of the pipe may be glass if necessary for transparency,
heat resistance and/or workability. The material of the sample
supply portion 5, the operation pipe portion a, and the recovery
pipe portion b may be the same or be different.
[0070] [2-4. Physical Properties of Hollow Pipe]
[0071] From the viewpoint of visibility during operation and from
the viewpoint of optical detection in a case of measurement of
absorbance, fluorescence, chemiluminescence, bioluminescence,
refractive index change and the like from outside of the pipe, the
material of the hollow pipe preferably has optical
transparency.
[0072] The conveyance surface constituting the inner wall of the
pipe is preferably a smooth surface in order to move a small amount
of liquid mass containing the target components together with the
magnetic particles in the gel layer; particularly, the surface
roughness is preferably Ra=0.1 .mu.m or less. For example, when a
permanent magnet is brought closer to the pipe from outside and the
small amount of liquid mass containing the target components moves
due to the changes of the magnetic field, the magnetic particles
move while being pressed against the conveyance surface, but by the
conveyance surface having surface roughness of Ra=0.1 .mu.m or
less, followability of the magnetic particles to the changed
magnetic field can be sufficiently provided.
[0073] [3. Filling in Operation Pipe]
[0074] [3-1. Operation Medium and Recovery Medium]
[0075] The operation pipe is at least filled with, as the operation
medium, the multi-layers in which the aqueous liquid layers and the
gel layers are alternately multi-layered. The uppermost layer may
be a gel layer (FIG. 1A) or an aqueous liquid layer (FIGS. 1B and
1C). When the uppermost layer is an aqueous liquid layer, the layer
may contain magnetic particles 6 (FIG. 1C) or may not contain the
magnetic particles 6 (FIGS. 1A and 1B). The lowermost layer may be
a gel layer or an aqueous liquid layer (FIGS. 1A-1C).
[0076] As shown in FIGS. 1A and 1B, when the hollow pipe
constituting the operation pipe is integrally formed, all layers
filled in the pipe may be in contact with each other. As shown in
FIG. 1C, when the hollow pipe constituting the operation pipe
consists of the operation pipe portion a and the recovery pipe
portion b, the recovery pipe portion b may be filled with an
aqueous liquid only as the recovery medium or may also be filled
with a gel layer on the aqueous liquid layer. The gel layer 2g
filled at the lowermost end of the operation pipe portion a and the
aqueous liquid layer or gel layer filled at the uppermost end of
the recovery pipe portion b may be in contact with each other, or
may not be in contact with a layer of gas interposed therebetween
(FIG. 1C).
[0077] The number and order of the layers filled in the hollow pipe
are not particularly limited, and can be appropriately determined
by those skilled in the art based on the number and order of the
operation processes for supplying the target components. Each of
the plurality of aqueous liquid layers filled in one hollow pipe
preferably consists of two or more different types of aqueous
liquids. As the aqueous liquid constituting each layer, liquids
that construct environment necessary for each of a treatment
process and a reaction process for supplying the target components
can be used in order from the upper end side of the hollow pipe.
Each of the plurality of gel layers filled in one hollow pipe may
consist of different types of gels or may consist of the same type
of gel. For example, when a heating treatment or reaction is
performed in a part of the plurality of aqueous liquid layers, a
gel having a high sol-gel transition point for which a gel state or
a gel-sol intermediate state can be maintained even at a
temperature necessary for the heating can be used in the gel layer
adjacent to the aqueous liquid layer only, and a gel having a
relatively low sol-gel transition point can be used in other gel
layers. In addition, anyone skilled in the art can appropriately
select a gel having proper characteristics corresponding to the
characteristics or volume of the aqueous liquid constituting
adjacent aqueous liquid layers.
[0078] The gel layer serves as a plug (gel plug) that partitions
the aqueous liquid layer on both sides in the longitudinal
direction of the hollow pipe in the operation pipe. As for the
layer length, those skilled in the art can appropriately determine
the length of the layer that functions as a plug in consideration
of the inner diameter and the length of the pipe, the amount of the
magnetic particles conveyed by the magnetic field applying part and
the like. For example, the layer length may be 1-20 mm, preferably
3-10 mm. When the layer length is below the range, the gel layer
tends to lack intensity as a plug. When the layer length is above
the range, the operation pipe becomes long, and operability,
durability and fillability of the operation pipe tend to
deteriorate.
[0079] The aqueous liquid layer filled in the operation pipe
portion a provides an environment of treatment, reaction or the
like in which the sample containing the target components is
supplied. As for the layer length of the aqueous liquid layer,
those skilled in the art can appropriately determine the layer
length that gives an aqueous liquid amount for achieving a desired
treatment or reaction for the target components, in consideration
of the inner diameter or length of the hollow pipe, the amount of
the target components, the type of the treatment or reaction in
which the target components are supplied and the like. For example,
the layer length is 0.5-30 mm, preferably 3-10 mm. When the layer
length is below the range, the treatment or reaction for the target
components may not be sufficiently achieved, and the plug may be
droplet-like and the magnetic particles may not be able to bind to
the reagents. When the layer length is above the range, the aqueous
liquid layer is often relatively much thicker compared with the gel
layer, which may cause the same problem as the gel plug, and the
interface of the multi-layer tends to be easily disturbed when the
specific gravity of the aqueous liquid is larger than that of the
gel.
[0080] The aqueous liquid layer filled in the recovery pipe portion
b is an elution liquid of the target components, and provides an
environment for liberating the target components from the magnetic
particles. The aqueous liquid layer is filled in the hollow pipe so
as to have a predetermined volume. Accordingly, the layer length
also varies with variations in the inner diameter or length of the
hollow pipe. The volume of the aqueous liquid layer can be
appropriately determined by those skilled in the art in
consideration of the amount of the target components, the type of
the treatment or reaction in which the target components are
supplied and the like so that the recovery rate of the target
components recovered in the elution liquid can be accurately
evaluated. For example, the volume of the aqueous liquid layer is 1
.mu.L-1000 .mu.L, preferably 50 .mu.L-300 .mu.L.
[0081] When the gel layer consists of a hydrogel, the hydrogel
layer can not only function to partition the reagents but also
provide an environment of the treatment or reaction or the like in
which a sample containing the target components is supplied in the
same manner as the aqueous liquid layer. In this case, the hydrogel
layer may also be longer than the aqueous liquid layer.
[0082] [3-2. Type of Gel]
[0083] The gel layer consists of a chemically inert substance that
is insoluble or poorly soluble in a liquid constituting the aqueous
liquid layer when multi-layered with the aqueous liquid in the
hollow pipe. Being insoluble or poor-soluble in liquid means that
the solubility in the liquid at 25.degree. C. is approximately 100
ppm or less. The chemically inert substance refers to substance
that has no chemical effect on the target components and the
aqueous liquid or the hydrogel during operation of the target
components (that is, the treatment of the target components in the
aqueous liquid or the hydrogel and transport of the target
components via the gel plug). The gel in the disclosure includes
both organogel and hydrogel.
[0084] [3-2-1. Organogel Gel]
[0085] Usually, the organogel can be obtained by adding a gelling
agent to a water-insoluble or poorly water-soluble liquid substance
for gelation.
[0086] [3-2-1-1. Water-Insoluble or Poorly Water-Soluble Liquid
Substance]
[0087] As the water-insoluble or poorly water-soluble liquid
substance, oil that has a solubility in water at 25.degree. C. of
approximately 100 ppm or less and that is liquid-like at room
temperature (20.degree. C..+-.15.degree. C.) can be used. For
example, one or a combination of two or more from a group
consisting of liquid oil, ester oil, hydrocarbon oil, and silicone
oil can be used.
[0088] The liquid oil includes linseed oil, camellia oil, mackerel
demia nut oil, corn oil, mink oil, olive oil, avocado oil, southern
power oil, castor oil, safflower oil, kyounin oil, cinnamon oil,
jojoba oil, grape oil, sunflower oil, almond oil, rapeseed oil,
sesame oil, wheat germ oil, rice germ oil, rice bran oil,
cottonseed oil, soybean oil, peanut oil, tea seed oil, evening
primrose oil, egg yolk oil, liver oil, palm oil, palm oil, palm
nuclear oil, and the like.
[0089] The ester oil includes octanoic esters such as cetyl
octanoate, lauric esters such as hexyl laurate, myristate esters
such as isopropyl myristate and octyldodecyl myristate, palmitate
esters such as octyl palmitate, stearic acid esters such as
isocetyl stearate, isostearic acid esters such as isopropyl
isostearate, isopalmitic acid esters such as octyl isopalmitate,
oleic acid esters such as isodecyl oleate, adipic acid esters such
as isopropyl adipate, sebacic acid esters such as ethyl sebacate,
malate esters such as isostearyl malate, glycerin trioctanoate,
glycerin triisopalmitate, and the like.
[0090] The hydrocarbon oil includes pentadecane, hexadecane,
octadecane, mineral oil, liquid paraffin, and the like. The
silicone oil includes dimethylpolysiloxane,
methylphenylpolysiloxane and other phenyl group-containing silicone
oil, methylhydrogenpolysiloxane, and the like.
[0091] [3-2-1-2. Gelling Agent]
[0092] As the gelling agent, an oil gelling agent selected from a
group consisting of hydroxy fatty acid, dextrin fatty acid ester,
and glycerin fatty acid ester can be used alone or in combination
of two or more.
[0093] The hydroxy fatty acid is not particularly limited as long
as it is a fatty acid having a hydroxyl group. Specifically, the
hydroxy fatty acid includes, for example, hydroxymyristic acid,
hydroxypalmitic acid, dihydroxypalmitic acid, hydroxystearic acid,
dihydroxystearic acid, hydroxymargaric acid, ricinoleic acid,
ricinaleic acid, linolenic acid, and the like. Among these,
hydroxystearic acid, dihydroxystearic acid, and ricinoleic acid are
particularly preferable. These hydroxy fatty acids may be used
alone or in combination of two or more. In addition, an animal and
vegetable oil fatty acid (for example, castor oil fatty acid,
hydrogenated castor oil fatty acid, and the like) that is a mixture
of these hydroxy fatty acids can also be used as the hydroxy fatty
acid.
[0094] The dextrin fatty acid esters include, for example, dextrin
myristate (trade name "Leopard MKL", manufactured by Chiba Flour
Milling Co., Ltd.), dextrin palmitate (trade names "Leopard KL",
"Leopard TL", both manufactured by Chiba Flour Milling Co., Ltd.),
dextrin (palmitate/2-ethylhexanoate) (trade name "Leopard TT",
manufactured by Chiba Flour Milling Co., Ltd.), and the like.
[0095] The glycerin fatty acid esters include glyceryl behenate,
glyceryl octastearate, glyceryl eicoate, and the like, and one or
more of these glycerin fatty acid esters may be used in
combination. Specifically, the glycerin fatty acid esters can
include trade name "TAISET 26" (manufactured by Taiyo Chemical Co.,
Ltd.) containing 20% of glyceryl behenate, 20% of glyceryl
octastearate and 60% of hydrogenated palm oil, trade name "TAISET
50" (manufactured by Taiyo Kagaku Co., Ltd.) containing 50% of
glyceryl behenate and 50% of glyceryl octastearate, and the
like.
[0096] The gelling agent can be used, of which the content added to
the water-insoluble or poorly water-soluble liquid substance is
equivalent to, for example, 0.1-0.5 weight %, 0.5-2 weight %, or
1-5 weight % of the total weight of the liquid substance. However,
the gelling agent is not limited hereto, and those skilled in the
art can appropriately determine the amount to a degree at which the
desired gel and sol state can be achieved.
[0097] The gelling method can be appropriately determined by those
skilled in the art. Specifically, a water-insoluble or poorly
water-soluble liquid substance is heated, a gelling agent is added
to the heated liquid substance, the gelling agent is completely
dissolved and then cooled, and thereby the liquid substance can be
gelled. A heating temperature may be determined in consideration of
the physical properties of the liquid substance and the gelling
agent that are used. For example, the heating temperature may be
preferably about 60-70.degree. C. The gelling agent is dissolved
for the liquid substance in a heated state; at this time, it is
preferable that the gelling agent is dissolved while being gently
mixed with the liquid substance. Cooling is preferably performed
slowly. For example, the cooling can be performed over a period of
about one hour to two hours. For example, the cooling can be
completed when the temperature is lowered to room temperature
(20.degree. C..+-.15.degree. C.) or less, preferably 4.degree. C.
An aspect to which a preferable aspect of the gelling method is
applied includes, for example, an aspect in which the
above-described TAISET 26 (manufactured by Taiyo Kagaku Co., Ltd.)
is used.
[0098] [3-2-2. Hydrogel]
[0099] As the hydrogel, for example, the hydrogel prepared by
equilibrating and swelling hydrogel materials in water or an
aqueous liquid can be used, the hydrogel materials including
gelatin, collagen, starch, pectin, hyaluronic acid, chitin,
chitosan or alginic acid and derivatives thereof. Among the
hydrogels, it is preferable to use a hydrogel prepared from
gelatin. In addition, the hydrogel can also be obtained by
chemically cross-linking the above hydrogel materials or processing
the above hydrogel materials with the gelling agents (for example,
salts of alkaline metals/alkaline earth metal such as lithium,
potassium and magnesium, or salts of transition metal such as
titanium, gold, silver and platinum, and silica, carbon, alumina
compound or the like). These chemical cross-linking and gelling
agents can be easily selected by those skilled in the art.
[0100] In particular, when the hydrogel provides an environment of
treatment or reaction or the like in which a sample containing the
target components is provided in the same manner as the aqueous
liquid, the hydrogel is appropriately prepared by those skilled in
the art so as to have a composition suitable for the treatment or
reaction. The hydrogel includes, for example, a DNA hydrogel
(P-gel) based on polydimethylsiloxane capable of synthesizing
proteins. This hydrogel is configured by DNA which is used as a
part of gel scaffold. When the target component is a substrate for
protein synthesis, this hydrogel can be supplied to a reaction for
obtaining a protein from the target component (the more specific
aspect can be appropriately determined by those skilled in the art
with reference to Nature Materials 8, 43 2-437 (2009), and Nature
Protocols 4: 1759-1770 (2009)). The produced protein can be
recovered, for example, by using the magnetic particles having an
antibody specific for the protein.
[0101] [3-2-3. Gel Characteristics]
[0102] The gel filled in the hollow pipe has a characteristic of
causing the sol-gel transition at a certain temperature. The
sol-gel transition point may be in a range of 25-70.degree. C. The
generation of the sol-gel transition point in this range is
desirable in a reaction system that requires fluidity obtained by
solification in recovery or the like. The sol-gel transition point
varies depending on conditions such as the type of organogel
material (oil) or hydrogel material, the type of gelling agent, the
added amount of gelling agent, and the like. Accordingly, each
condition is appropriately selected by those skilled in the art so
as to obtain a desired sol-gel transition point.
[0103] The gel plug can be fixed in a predetermined position in the
pipe by clamping the aqueous liquid in the hollow pipe from both
sides in the longitudinal direction of the pipe. On the other hand,
the magnetic particles can also be moved even in the gel by a
magnetic field operation from outside, and can pass through the gel
as a result. The reason is the thixotropic property of the gel
(thixotropy). That is, the magnetic particles inside the pipe give
a shearing force to the gel along the conveying surface due to the
magnet movement outside the pipe, and the gel in the forward
direction of the magnetic particles solates and fluidizes, and thus
the magnetic particles proceed directly. Moreover, the sol released
from the shearing force after the passage of the magnetic particles
returns quickly to the gel state, and thus no through hole caused
by the passage of the magnetic particles is formed in the gel. By
utilizing this phenomenon, the object can easily move using the
magnetic particles as a transporter, and thus various chemical
environments in which the object is supplied can be switched in a
very short time. For example, if the disclosure is used in a system
consisting of a plurality of chemical reactions using a plurality
of reagents, the treatment time of the object can be greatly
shortened. If the property of gelation at a temperature below room
temperature is utilized, even a reagent that exhibits a liquid
state at that temperature can also be immobilized by being
sandwiched by the gel plugs in the pipe. Therefore, the state in
which the narrow pipe is filled with the liquid reagents in advance
can be maintained from the time of device manufacture until the
delivery to the user, and the liquid reagents can be stably
supplied. Furthermore, reagent collecting and dispensing operations
for each work process are not necessary, labour reduction and time
saving can be achieved, and deterioration of analysis accuracy due
to contamination can be prevented. [0104] As for the physical
properties of the gel, storage modulus E' of the dynamic
viscoelasticity is preferably 10-100 kPa, more preferably 20-50 kPa
at room temperature (20.degree. C..+-.15.degree. C.). When the
storage modulus is below the range, the gel tends to lack the
intensity as a gel plug. When the storage modulus is above the
range, even magnetic particles having a particle size of about
several micrometres tend to be easily hindered in movement. In the
sol state, a kinematic viscosity may be 5 mm.sup.2/s-100
mm.sup.2/s, preferably 5 mm.sup.2/s-50 mm.sup.2/s, for example,
about 20 mm.sup.2/s (50.degree. C.).
[0105] [3-3. Type of Aqueous Liquid]
[0106] The aqueous liquid in the disclosure may be an aqueous
liquid that is insoluble or poorly soluble in the gel, and may be
provided in the form of water, an aqueous solution or a creamy
mixture of liquids called emulsion, or a suspension in which fine
particles are dispersed. The components of the aqueous liquid
include all components that provide the environment of reaction and
treatment in which the target components in the disclosure are
supplied.
[0107] More specific examples include a liquid for liberating
components to be operated in the disclosure into the aqueous liquid
layer and binding or adsorbing the components to the surfaces of
magnetic particles (that is, a liquid having an action of
separating the target components from contaminants and promoting
binding or adsorption to the surfaces of magnetic beads), a
cleaning liquid for removing the contaminants coexisting with the
target components, an elution liquid for separating the target
components adsorbed on the magnetic particles from the magnetic
particles, a reaction liquid for constructing a reaction system in
which the target components are supplied, and the like. For
example, when the target components are nucleic acids, the aqueous
liquid includes a reagent solution (cell lysate) for destroying
cells and liberating the nucleic acids, and adsorbing the nucleic
acids on the silica-coated surfaces of the magnetic particles, a
cleaning liquid for cleaning the magnetic particles and removing
components other than the nucleic acids, an elution liquid (nucleic
acid elution liquid) for separating the nucleic acids from the
magnetic particles, a nucleic acid amplification reaction liquid
for performing nucleic acid amplification reaction, and the like.
Hereinafter, a case in which the target components are nucleic
acids is illustrated, and the treatment liquid and reaction liquid
for the nucleic acids and the treatment and reaction in which the
nucleic acids are supplied are further described.
[0108] [3-3-1. Cell Lysate]
[0109] The cell lysate includes a buffer that contains chaotropic
substances. The buffer can further include EDTA and any other
chelating agent or TritonX-100 and any other surfactant. The buffer
is based on, for example, Tris-HCl and any other buffer. The
chaotropic substance includes guanidine hydrochloride, guanidine
isothiocyanate, potassium iodide, urea and the like.
[0110] The chaotropic substance is a powerful protein denaturant,
and has an action of pulling proteins such as histones bound to the
nucleic acids away from the nucleic acids and promoting adsorption
on the silica-coated surfaces of magnetic particles. The buffer
agent can be used as an auxiliary agent that adjusts a pH
environment in which the nucleic acids are easily adsorbed on the
surfaces of the magnetic particles. The chaotropic substance also
has an action of cell lysis (that is, an action of destroying cell
membranes). However, in the action of cell lysis, a surfactant
contributes more than the chaotropic substance. A chelating agent
can be used as an auxiliary agent that promote the cell lysis.
[0111] A specific protocol for extracting nucleic acids from a
sample containing nucleic acids can be appropriately determined by
those skilled in the art. In the disclosure, since the magnetic
particles are used for transporting the nucleic acids in a droplet
encapsulating medium, it is preferable to adopt a method using the
magnetic particles as the nucleic acid extraction method. For
example, with reference to Japanese Patent Laid-Open 2-289596, a
method for using magnetic particles to extract and purify nucleic
acids from a sample containing nucleic acids can be
implemented.
[0112] [3-3-2. Cleaning Liquid]
[0113] The cleaning liquid is preferably a solution capable of
dissolving components other than the nucleic acids contained in the
nucleic acid-containing sample (for example, proteins and sugars)
or the reagents and other components used for other treatments
performed in advance such as nucleic acid extraction while the
nucleic acids are adsorbed on the surfaces of the magnetic
particles. Specifically, the cleaning liquid includes high salt
concentration aqueous solutions such as sodium chloride, potassium
chloride and ammonium sulfate, alcohol aqueous solutions such as
ethanol and isopropanol, and the like. The cleaning of the nucleic
acids is cleaning of the magnetic particles on which the nucleic
acids are adsorbed. A specific protocol for this cleaning can be
appropriately determined by those skilled in the art. In addition,
the number of times of the cleaning of the magnetic particles on
which the nucleic acid are adsorbed can be appropriately selected
by those skilled in the art to a degree that undesired inhibition
does not occur during the nucleic acid amplification reaction. In
addition, when the effect of inhibitory components can be ignored
from the same viewpoint, the cleaning process can also be omitted.
The aqueous liquid layer consisting of the cleaning liquid is
prepared at least as many times as the number of times of the
cleaning.
[0114] [3-3-3. Nucleic Acid Elution Liquid]
[0115] A buffer containing water, salt or the like can be used as
the nucleic acid elution liquid. Specifically, a Tris buffer, a
phosphate buffer, distilled water and the like can be used. A
specific method for separating the nucleic acids from the magnetic
particles on which the nucleic acids are absorbed and eluting the
nucleic acids into the elution liquid can also be determined
appropriately by those skilled in the art.
[0116] [3-3-4. Nucleic Acid Amplification Reaction Liquid]
[0117] In the nucleic acid amplification reaction liquid of the
disclosure, various elements usually used in the nucleic acid
amplification reaction at least include nucleic acids containing
base sequences to be amplified and magnetic particles that adsorb
the nucleic acids on the surfaces thereof.
[0118] Since the nucleic acid amplification reaction is not
particularly limited as described later, the various elements used
in the nucleic acid amplification reaction can be appropriately
determined by those skilled in the art based on the known nucleic
acid amplification method illustrated later and the like. Usually,
the various elements include salts such as MgCl.sub.2, KCl,
primers, deoxyribonucleotides, nucleic acid synthases, and pH
buffers. In addition, the above salts may be appropriately changed
into other salts to use. In addition, substances for reducing
non-specific priming, such as dimethyl sulfoxide, betaine, glycerol
and the like, may be further added.
[0119] In addition to the above components, a blocking agent can be
added to the nucleic acid amplification reaction liquid in the
disclosure. The blocking agent can be used for the purpose of
preventing deactivation of DNA polymerase due to adsorption to the
inner wall of a reaction vessel, the surfaces of the magnetic
particles or the like. Specific examples of blocking agents include
bovine serum albumin (that is, BSA), other albumins, gelatin (that
is, denatured collagen), proteins such as casein and polylysine,
peptides (both natural and synthetic), ficoll, polyvinyl
pyrrolidone, polyethylene glycol, and the like.
[0120] The nucleic acid amplification reaction of the disclosure is
not particularly limited, and for example, PCR method (U.S. Pat.
No. 4,683,195, No. 4683202, No. 4800159, and No. 4965188), LCR
method (U.S. Pat. No. 5,494,810), Q.beta. method (U.S. Pat. No.
4,786,600), NASBA method (U.S. Pat. No. 5,409,818), LAMP method
(U.S. Pat. No. 3,313,358), SDA method (U.S. Pat. No. 5,455,166),
RCA method (U.S. Pat. No. 5,354,688), ICAN method (U.S. Pat. No.
3,433,929), TAS method (U.S. Pat. No. 2,843,586), and the like can
be used. In addition, a reverse transcription (RT) reaction can
also be performed prior to the above reaction. Those skilled in the
art can appropriately select the composition of reaction liquid and
the reaction temperature necessary for these nucleic acid
amplification reactions.
[0121] Besides, when the nucleic acid amplification reaction is
further performed after the reverse transcription (RT) reaction,
for example, when RT-PCR is performed, the RT reaction liquid layer
can be multi-layered on the PCR reaction liquid layer via the gel
layer in the recovery portion B (for example, as illustrated in
FIGS. 4A to 4H).
[0122] In a real-time nucleic acid amplification method,
fluorescence detection can be performed on amplification products
by a fluorescent dye capable of binding to a double-stranded DNA or
by a probe labelled with the fluorescent dye. Detection methods in
the real-time nucleic acid amplification method include the
following methods.
[0123] For example, when it is possible to amplify a desired target
only by a highly specific primer, an intercalator method using SYBR
(registered trademark) GREEN I or the like is used. An intercalator
that emits fluorescence by binding to a double-stranded DNA binds
to the double-stranded DNA synthesized by the nucleic acid
amplification reaction, and emits fluorescence of a specific
wavelength by irradiation of an excitation light. By detecting this
fluorescence, the generation amount of the amplification products
can be monitored. This method does not require design and synthesis
of a fluorescently labelled probe specific to the target, and can
be conveniently utilized in measurement of various targets.
[0124] In addition, when it is necessary to distinguish and detect
similar sequences or when SNPs are typed, a fluorescently labelled
probe method is used. As an example, there is a TaqMan (registered
trademark) probe method for using an oligonucleotide in which 5'
terminal is modified with a fluorescent substance and 3' terminal
is modified with a quencher substance as a probe. The TaqMan probe
is specifically hybridized to a template DNA in an annealing step,
but the quencher is present on the probe and thus fluorescence
emission is suppressed even when the excitation light is
irradiated. In an extension reaction step, when the TaqMan probe
hybridized to the template is decomposed by 5'.fwdarw.3'
exonuclease activity of the TaqDNA polymerase, the fluorescent dye
is liberated from the probe, the suppression caused by the quencher
is released and the fluorescence is emitted. By measuring the
fluorescence intensity, the generation amount of amplification
products can be monitored.
[0125] The principle of quantifying DNA in the real-time PCR by
this method is described below. First, a serially diluted standard
sample of known concentration is used as the template to perform
the PCR. Then, the number of cycles (threshold cycle; Ct value)
reaching a certain amount of amplification products is obtained. A
calibration curve is created by plotting the Ct value on the
horizontal axis and an initial DNA amount on the vertical axis. For
a sample of unknown concentration, the PCR reaction is also
performed in the same conditions to obtain the Ct value. From this
value and the above-described calibration curve, a desired DNA
amount in the sample can be measured.
[0126] Furthermore, in an intercalator method, when the temperature
of the liquid after the PCR reaction containing a fluorescent dye
is gradually increased from 40.degree. C. to about 95.degree. C.
and the fluorescence intensity is continuously monitored, a melting
curve of the amplification products can be obtained. The
double-stranded DNA generated by the nucleic acid amplification
reaction has a unique Tm value depending on the length of DNA and
the base sequence thereof. In other words, when the temperature of
droplets containing the DNA to which the fluorescent dye is bound
is gradually increased, a temperature at which the fluorescence
intensity rapidly decreases is observed. When the change amount of
change in fluorescence intensity is examined, the temperature peak
substantially coincides with the Tm value defined by the base
sequence and the length. As a result, for example, data which is
not the target gene and observed when a primer dimer occurs (that
is, false positive data) can be excluded from data regarded as
positives. In a genetic testing, non-specific reactions also often
occur due to contaminants in the sample, so it is important to
eliminate the false positives. Accordingly, a determination can
also be performed on whether the generated amplification products
are unique to the target genes.
[0127] [3-3-5. Other Aqueous Liquids]
[0128] For any reaction and treatment other than the above
reaction, the composition of each aqueous liquid can be easily
determined by those skilled in the art. In addition, even when the
target component is a component other than the above nucleic acid,
the composition of each aqueous liquid can be easily determined by
those skilled in the art.
[0129] [4. Manufacturing Method of Operation Pipe]
[0130] As a manufacturing method of operation pipe, the following
two methods are described according to an aspect in which a hollow
pipe to be filled with multilayers being an operation medium is
prepared.
[0131] [4-1. A Case in which One Hollow Pipe is Prepared for
Manufacturing One Operation Pipe]
[0132] The case in which this creation method is performed may be a
case in which the hollow pipe is prepared in a state of being
integrally formed, or a case in which the pipe is configured by the
operation pipe portion a and the recovery pipe portion b and
prepared in a state that the pipe portion a and the pipe portion b
are connected. In one hollow pipe, the necessary aqueous liquid and
gel are filled so as to be alternately multi-layered in a necessary
order from the lower closed end and thereby the operation medium
can be formed, and the operation pipe can be manufactured. In this
case, after a predetermined amount of volume of the aqueous liquid
in contact with the lower closed end is filled, filling is
performed while the vertical thickness of the aqueous liquid layer
and the gel layer multi-layered on the aqueous liquid layer, that
is, the length in the longitudinal direction of the hollow pipe is
specified. When the hollow pipe is configured by the operation pipe
portion a and the recovery pipe portion b, first, the recovery unit
B is completed when filling of the recovery medium necessary for
constituting the recovery portion B, that is, filling of the
aqueous liquid having a predetermined volume or formation of
multilayers of the aqueous liquid having a predetermined volume and
the gel layer having a predetermined thickness is completed.
Furthermore, the operation portion A is completed by filling of the
operation medium necessary for constituting the operation portion
A, that is, formation of multilayers of the aqueous liquid layer
and the gel layer that have a predetermined thickness is completed.
A more specific method for forming multilayers by alternately
multi-layering the aqueous liquid and the gel can be appropriately
performed by those skilled in the art according to the multi-layer
method in a case of 4-2 described later. Besides, after necessary
aqueous liquid and/or gel are/is filled, the sample supply portion
that is an upper open end may be appropriately closed.
[0133] [4-2. A Case in which a Plurality of Hollow Pipes are
Prepared for Manufacturing One Operation Pipe]
[0134] The case in which this creation method is performed may be a
case in which the pipe is configured by the operation pipe portion
a and the recovery pipe portion b and prepared in a state that the
pipe portion a is independent from the pipe portion b. In this
case, the operation portion A and the recovery portion B are
separately manufactured by filling necessary aqueous liquid and/or
gel in each of the pipe portion a and the pipe portion b, and the
manufactured operation portion A and recovery portion B are
connected to each other, and thereby the operation pipe can be
manufactured.
[0135] An outline of the manufacturing method of the operation
portion A is schematically shown in FIGS. 2A to 2C. An aqueous
liquid L (for example, a cleaning liquid) constituting the aqueous
liquid layer is filled in the container, and a gel G constituting
the gel layer is filled in another container in a sol state. In
FIGS. 2A to 2C, the sol state is maintained by heating in a
constant temperature bath 21 of 70.degree. C. for example. The
lower open end of the pipe portion a is prepared in a state of
being closed by being pressed against a holding mat 22.
[0136] A system for feeding liquid into the pipe portion a includes
tubes 23 and 23' that respectively extend from the container filled
with the aqueous liquid L and the sol-gel G and feed the aqueous
liquid L or the sol-gel G, a liquid feeding part 24 (peristaltic
pump in FIGS. 2A to 2C) to which the tube 23' is connected, and a
needle 25 for filling the pipe portion a with liquid substances
that are fed by the liquid feeding part. The needle 25 preferably
has a length enough to reach the bottommost portion of the pipe
portion a by being inserted into the pipe portion a.
[0137] In FIGS. 2A to 2C, the tube 23 extending from the container
filled with the aqueous liquid L and the tube 23' extending from
the container containing the sol-gel G are connected to a switching
valve 26. In this case, by switching the valve 26, different liquid
substances (the aqueous liquid L and the sol-gel G) can be
respectively fed to the same tube 23' and the same needle 25. This
aspect is preferably used when the inner diameter of the pipe
portion a is relatively small because only one needle is inserted
into the pipe portion a.
[0138] On the other hand, all liquid feeding paths from the
container to the needle may be made independent without using the
switching valve 26. For example, when the same operation portion A
as FIGS. 2A to 2C is manufactured, two liquid feeding paths can be
formed, one formed by a tube extending from the container filled
with the aqueous liquid and a needle connected to the tube and the
other formed by a tube extending from the container containing the
sol-gel and a needle connected to the tube. In this aspect, the
operation pipe is preferably used when the inner diameter of the
pipe portion a is relatively large because two needles can be
inserted into the pipe portion a.
[0139] As shown in order in FIGS. 2A-2C, the sol-gel G and the
aqueous liquid L are alternately fed and filled into the pipe
portion a in order from the sol-gel G. The leading end of the
needle 25 is raised as the liquid level in the pipe portion a
rises. When the aqueous liquid L is multi-layered as shown in FIG.
2B after the sol-gel is filled, the previously filled sol-gel may
be completely gelled or may not be completely gelled. Usually, the
liquid substances that are fed from the container filled with the
sol-gel G may be in an intermediate state of gel-sol with increased
viscoelasticity so as to be away from heat source (the constant
temperature bath 21 in FIGS. 2A to 2C) when discharged into the
pipe portion a from the needle 25 inserted into the pipe portion a.
Therefore, when the aqueous liquid is multi-layered, even if the
previous layer 2 is not completely gelled, the contact resistance
of the gel 2g against the inner wall of the pipe portion a works,
and the gel 2g having a low specific gravity does not float up.
Accordingly, by alternately feeding the sol-gel G and the aqueous
liquid L into the pipe portion a, the required number of layers can
be formed and the operation portion A can be obtained. Besides,
during the filling, the aqueous liquid and the gel are filled with
a prescribed thickness based on the thickness of the hollow pipe in
the vertical direction.
[0140] The recovery portion B can be obtained by filling necessary
aqueous liquid or gel. Alternatively, the recovery portion B can be
obtained by forming a single layer of the aqueous liquid layer or
multilayers of the aqueous liquid layer and the gel layer in an
appropriate and necessary order in the same manner as described
above except that a holding mat is not used. During the filling,
the aqueous liquid is filled with a predetermined amount on the
basis of volume, and the gel is filled with a prescribed thickness
on the basis of the thickness of the hollow pipe in the vertical
direction.
[0141] The operation portion A and the recovery portion B obtained
as described above are connected to each other. The operation
portion A may be connected to the recovery portion B in a state
that the pipe portion a is inclined and the holding mat is removed
and inclined or overturned so that the contents of the operation
portion A do not slide down. As a form of connection, the pipe
portion a and the pipe portion b may be wound around a tape or the
like, or the pipe portion a and the pipe portion b in which
connection portions that can be connected to each other are
respectively formed may be used to connect both of the connection
portions.
[0142] Besides, after necessary aqueous liquid and/or gel are/is
filled, the sample supply portion which is an upper open end of the
operation pipe portion a may be appropriately closed. The timing
for closing may be after the operation portion A is manufactured
and before the operation portion A and the recovery portion B are
connected, or after the operation portion A and the recovery
portion B are connected.
[0143] [5. Magnetic Particles]
[0144] The magnetic particles are used to move, by the variations
of the magnetic field from outside of the operation pipe, the
target components in the operation pipe by being accompanied by a
small amount of accompanying liquid mass. The magnetic particles
intended to enable separation, recovery and purification of
specific components by the above movement usually have chemical
functional groups on the surfaces thereof. The magnetic particles
may not be filled in the operation pipe in advance (FIGS. 1A and
1B) or may be filled in advance (FIG. 1C, FIGS. 3A to 3O and FIGS.
4A to 4H). When filled in the operation pipe in advance, the
magnetic particles can be added to the aqueous liquid constituting
the uppermost layer. When the magnetic particles are not added to
the operation pipe in advance, the magnetic particles are also
supplied to the operation pipe when the sample having the target
components is supplied to the operation pipe.
[0145] The magnetic particles are not particularly limited as long
as they are particles that respond to magnetism, and include, for
example, particles having a magnetic body such as magnetite,
.gamma.-iron oxide, manganese zinc ferrite or the like. In
addition, the magnetic particles have a chemical structure that
specifically binds to the target components supplied to the above
treatment or reaction, and may have a surface containing, for
example, amino group, carboxyl group, epoxy group, avidin, biotin,
digoxigenin, protein A, protein G, complex metal ion or antibody,
or may have a surface that specifically binds to the target
components by an electrostatic force or a van der Waals force.
Accordingly, the target components supplied to the reaction or
treatment can be selectively adsorbed to the magnetic particles.
Hydrophilic groups on the surfaces of the magnetic particles
include hydroxyl groups, amino groups, carboxyl groups, phosphoric
acid groups, sulfonic acid groups, and the like.
[0146] In addition to the above particles, the magnetic particles
can further include various elements appropriately selected by
those skilled in the art. For example, specific forms of the
magnetic particles having hydrophilic groups on the surfaces
preferably include particles consisting of mixture of magnetic
bodies and silica and/or an anion exchange resin, magnetic
particles of which the surfaces are covered by the silica and/or
the anion exchange resin, magnetic particles of which the surfaces
are covered by gold and which have hydrophilic groups via mercapto
groups, gold particles containing magnetic bodies and having
hydrophilic groups via mercapto groups on the surfaces, and the
like.
[0147] As for the size of the magnetic particles having hydrophilic
groups on the surfaces, the average particle size is about 0.1
.mu.m-500 .mu.m. When the average particle size is small, the
magnetic particles are easy to exist in a dispersed state when
released from the magnetic field in the aqueous liquid layer. An
example of commercially available magnetic particles includes
Magnetic Beads which are component reagents of Plasmid DNA
Purification Kit MagExtractor-Plasmid-sold by Toyo Tamotsu and are
silica-coated for nucleic acid extraction. When sold as a kit of
component reagents in this way, a product stock solution containing
magnetic particles contains a preservative solution and the like,
and thus is preferably cleaned by being suspended in pure water
(for example, about 10 times of the amount). The cleaning can be
performed by suspending in pure water and then removing the
supernatant by centrifugation or aggregation using a magnet, and
can be performed by repeating the suspension and supernatant
removal. Besides, the magnetic field applying part for giving
magnetic field variations to move the magnetic particles is
described in detail in item 8 below.
[0148] [6. Method for Operating Target Components in Pipe]
[0149] The operations of the target components in the operation
pipe are shown in FIGS. 3A-3O and FIGS. 4A-4H. Hereinafter,
description will be given with reference to FIGS. 3A to 3O and
FIGS. 4A-4H.
[0150] [6-1. Sample Supply to Operation Pipe]
[0151] When the operation pipe is used, a sample 32 containing the
target components is supplied from a reagent supply port 5 (FIG. 3B
and FIG. 4B). Usually, the sample is supplied in the form of
liquid. The sample supply may be performed manually by a syringe or
the like, or may be automatically controlled by a dispenser using a
pipetter or the like. The sample supply is performed in a state
that the operation pipe is held up by an appropriate holding part
(not shown; the holding part for holding the operation pipe is
described in detail in item 7 below).
[0152] In the uppermost layer in the operation pipe, an aqueous
liquid mixture 33 that contains the sample 32 containing the target
components, the magnetic particles 6 and the aqueous liquid are
obtained. More specifically, the aqueous liquid mixture can be
obtained as follows. For example, when the uppermost layer filled
in the operation pipe consists of an aqueous liquid, the sample may
be supplied into the operation pipe together with the magnetic
particles, or the sample may be supplied into the operation pipe
together with the aqueous liquid and suspended magnetic particles.
In this way, the aqueous liquid mixture can be obtained from the
aqueous liquid in the uppermost layer. In addition, for example,
when the uppermost layer filled in the operation pipe consists of
an aqueous liquid containing magnetic particles (the case
illustrated in FIGS. 3A to 3O and 4A to 4H is applicable), the
sample only may be supplied into the operation pipe, or the sample
may be supplied into the operation pipe together with the aqueous
liquid. In this way, the aqueous liquid mixture can be obtained
from the aqueous liquid containing magnetic particles in the
uppermost layer. Furthermore, for example, when the uppermost layer
filled in the operation pipe consists of gel, the sample may be
supplied into the operation pipe together with the aqueous liquid
and the magnetic particles. In this way, the aqueous liquid mixture
can be newly formed as the uppermost layer on the gel layer.
[0153] [6-2. Operations in Operation Pipe]
[0154] The operation pipe in which the sample is supplied and the
aqueous liquid mixture containing the sample and magnetic particles
is prepared in the uppermost layer is held up on the holding part
and directly set on a device or set on a device under the condition
of being transferred to a dedicated holding part in the device. In
the device, a magnetic field is generated by bringing a magnetic
field applying part (for example, a cylindrical neodymium magnet
having a diameter of 1 mm-5 mm and a length of 5 mm-30 mm) 31 from
outside close to an operation pipe 1, and magnetic particles 6
dispersed in an aqueous liquid mixture layer 31.sub.1 are
aggregated together with the target components (FIG. 3C and FIG.
4C). At this time, unnecessary components contained in the aqueous
liquid mixture layer 31.sub.1 can be also aggregated together. By
moving the magnetic field applying part 31 downward at a speed of
0.5 mm-10 mm per second, the magnetic particles accompanied by the
target components are transported from the aqueous liquid mixture
layer 31.sub.1 via a gel layer 3g.sub.1 located below and in
contact with the aqueous liquid mixture layer 31.sub.1 (see FIG. 3D
and FIG. 4D) to an aqueous liquid layer 31.sub.2 located below and
in contact with the gel layer 3g.sub.1 (FIG. 3E and FIG. 4E).
Besides, since the magnetic particles passing through the gel layer
3g.sub.1 are thinly coated on the aqueous liquid mixture of the
aqueous liquid mixture layer 31.sub.1 supplied before passage, the
magnetic particles are accompanied by concomitants in addition to
the target components and the concentration is low. The magnetic
particles are further transported to the aqueous liquid layer
31.sub.2. The size and movement speed of the magnet are
appropriately determined by those skilled in the art corresponding
to the amount of the magnetic particles, the inner and outer
diameters of the operation pipe, the state of the gel plug, and the
like.
[0155] Furthermore, transportation from the aqueous liquid layer
31.sub.2 to another aqueous liquid layer via the gel layer is
repeated by the magnetic field applying part 31 as necessary.
"Repeating as necessary" means that, as a general rule, the
transport operation may be performed as many as the number of times
corresponding to the layer number by moving the magnetic particles
only in one direction from the top to the bottom (FIGS. 3E-3N and
FIGS. 4E-4H), or the transport operation may be performed for the
number of times above the number corresponding to the layer number
by moving the magnetic particles not only in one direction from the
top to the bottom but also from the bottom back to the top as
appropriate. That is, other aqueous liquid layers of the transport
destination may be present above or below the aqueous liquid layer
of the transport source. By repeating this transport operation,
most of the contaminants transported by the magnetic particles
together with the target components are removed. Although the
magnetic particles accompanying the target components are
accompanied by a very small amount of cleaning liquid, the target
components on the surfaces of the particles are purified to such a
degree that a subsequent analysis process and the like are not
interfered. Accordingly, the purification of the target components
can be performed very efficiently by the magnetic field operation
only.
[0156] In addition, in the aqueous liquid layer, from the viewpoint
of improving the processing efficiency, it is preferable to operate
so that the magnetic particles with the target components
(specifically, including target components accompanied by
unnecessary components and target components from which unnecessary
components are removed) can be sufficiently brought into contact
with the aqueous liquid. As one of the methods for more efficiently
performing this operation, there is a method in which the magnetic
field applying part is moved up and down in a state that the
magnetic particles are aggregated due to application of the
magnetic field in the aqueous liquid layer. Other methods include
the method in which the magnetic particles that are aggregated due
to the application of the magnetic field are naturally diffused in
the aqueous liquid layer by opening the magnetic field from the
magnetic particles that are subjected to the application of the
magnetic field using the magnetic field applying part.
[0157] As a specific example, as shown in FIG. 3E, the magnetic
field is blocked or attenuated by temporarily bringing the magnetic
field applying part 31 away from the operation pipe 1, and the
magnetic particles are dispersed in a cleaning liquid layer
31.sub.2. In this way, the target components adsorbed on the
magnetic particles are cleaned by being sufficiently exposed in the
cleaning liquid 31.sub.2 together with the accompanying components.
As shown in FIG. 3F, the magnetic field applying part 31 is brought
closer to the operation pipe 1 again and thereby the magnetic
particles are aggregated together with the target components and
are in a transportable state. By further moving the magnetic field
applying part 31 downward, the magnetic particles are transported
to the gel layer 3g.sub.2 just below as shown in FIG. 3G. In the
magnetic particles and the target components in the gel layer
3g.sub.2 in FIG. 3G, compared with a case of the magnetic layer
particles and the target components in the gel layer 3g.sub.1 in
FIG. 3D, some or most of the accompanying components are removed by
the cleaning in FIG. 3E.
[0158] After separating a target substance from the magnetic
particles in the layer filled in the recovery portion B, the
magnetic particles from which the target substance is separated are
moved from the layer in which the target substance is separated to
another layer (for example, FIGS. 3N-3O), and thereby the target
substance can be recovered in a state of being eluted from the
magnetic particles in the recovery portion.
[0159] [6-3. Nucleic Acid Extraction]
[0160] For example, when the magnetic particle surfaces are coated
with silica, as shown in FIGS. 3A to 3O, the biological sample is
supplied to a cell lysate 31.sub.1 containing a surfactant and a
chaotropic salt such as guanidine thiocyanate, and thereby the
nucleic acids are liberated from the cells (FIG. 3B). The liberated
nucleic acids can be specifically adsorbed on the silica surfaces
of the particles. The adsorbed nucleic acids contain reaction
inhibition components in this state and thus cannot be utilized as
a template for gene amplification reaction. Therefore, the magnetic
particles are cleaned by the cleaning liquid 31.sub.2 while the
nucleic acids are adsorbed on the surfaces. At this time, in order
to prevent a large amount of reaction inhibition components from
being introduced into the cleaning liquid, the magnetic particles 6
are collected by a magnet 31 (FIG. 3C), and are made to pass
through a gel plug 3g.sub.1 that separates the cell lysate 31.sub.1
and the cleaning liquid 31.sub.2 (FIG. 3D). The magnetic particles
can reach the cleaning liquid 31.sub.2 with little liquid fraction
when passing through the gel plug 3g.sub.1 (FIG. 3E). Therefore,
the cleaning of the magnetic particles can be implemented with high
efficiency. By further repeating passage through gel plugs
(3g.sub.2, 3g.sub.3) and transport to cleaning liquids (31.sub.3,
31.sub.4) of the magnetic particles (FIGS. 3F-3K), purity of the
nucleic acids can be increased. The nucleic acids purified in a
state of being adsorbed on the magnetic particle surfaces are
collected again by a magnet (FIG. 3L), made to pass through a gel
plug 2g (FIG. 3M), and transported into an elution liquid 4 (FIG.
3N). In the elution liquid 4, the nucleic acids are separated from
the magnetic particles and eluted in the elution liquid. When it is
not desired to mix the magnetic particles, the magnetic particles
from which the nucleic acids are eluted are retained in the gel
plug 2g again, and the eluted purified nucleic acids remain in the
recovery portion B (FIG. 3O). The nucleic acids obtained in this
way are useful as template nucleic acids that can be analyzed by
nucleic acid amplification reaction. The obtained nucleic acids can
be used for the next operation (process for performing analysis by
nucleic acid amplification reaction) by removing the recovery
portion B of the operation pipe from the operation portion A.
[0161] [6-4. Nucleic Acid Synthesis and Analysis]
[0162] As shown in FIGS. 4A to 4H, when the operation pipe is used
in which the pipe portion a of the operation portion A and the pipe
portion b of the recovery portion B are formed integrally, and
which has the operation portion A similar to the operation portion
in FIGS. 3A to 3O and the recovery portion B filled with a RT
reaction liquid 411 and a PCR reaction liquid 412 separated by a
gel plug 4g, after the same operation (FIGS. 4B-4F) as that in
FIGS. 3B-3M is performed, the magnetic particles 6 are transported
to the RT reaction liquid 411 while adsorbing the purified nucleic
acid (RNA) and the RT reaction is performed (FIG. 4H). After
completion of the RT reaction, the magnetic particles also adsorb
the DNA (which is a template for PCR reaction) obtained by the RT
reaction, pass through the gel plug 4g to be transported to the PCR
reaction liquid 412, and the PCR reaction is performed (FIG. 4H).
The PCR product can be analyzed by a real-time detection method
using a fluorescent dye or a fluorescence detection method using an
endpoint detection method. Besides, in FIGS. 4A to 4H, 42 and 43
schematically show temperature control functions. A more specific
example of the temperature control function of 42 is shown in the
subsequent item 8-2-6, and a more specific example of the
temperature control function of 43 is shown in the subsequent item
7-3.
[0163] When the above operation is performed simultaneously in a
plurality of operation pipes, multiple channels can be made as
shown in FIG. 5. The device illustrated in FIG. 5 has a simple
configuration in which a magnetic field applying part (movable
magnet plate 53) having a magnet moving mechanism and a holding
substrate with temperature control function (temperature control
block 51) are main units. Each configuration is described in items
7 and 8 described later.
[0164] [6-5. Protein Synthesis, Separation and Analysis]
[0165] [6-5-1. Protein Synthesis Using Hydrogel (P-Gel)]
[0166] A cell-free protein synthesis system on the basis of
polydimethylsiloxane is published in the aforementioned reference
literature (Nature materials 8, 432-437, 2009). The cell-free
protein synthesis system is performed in a general-purpose sample
tube, but this cell-free protein synthesis system can also be
constructed in the operation pipe of the disclosure.
[0167] [6-5-2. Analysis Using Interaction Between Target Protein
and Other Proteins]
[0168] A separation and recovery part of protein already exists as
a commercially available purification kit, the separation and
recovery part utilizing an antigen-antibody reaction of a protein
and an antibody (also a protein) that is manufactured using the
protein as a target. The separation and recovery are implemented by
a protocol using a general-purpose tube and a centrifuge. In the
cell-free protein system described above, a spin column is also
used separately from the sample tube for separation of the
synthesized protein. In the disclosure, by adopting magnetic
particles in which the antibody of the target protein is
immobilized on the surface, the target protein can be separated and
acquired in one operation pipe without moving the target protein
between different devices.
[0169] [6-5-3. Mass Spectrometry in a State that Protein is
Adsorbed on Magnetic Particles]
[0170] A method for adsorbing a separately prepared protein to be
subjected to mass spectrometry to magnetic particles coated with
titanium oxide on the surface, mixing the protein with a matrix in
the above state, and analyzing the protein with a mass spectrometer
is described in the reference literature (Analytical Chemistry, 77,
5912-5919, 2005). In the disclosure, the preparation of the protein
to be subjected to mass spectrometry and the adsorption to the
magnetic particles can be performed in one operation pipe.
[0171] [7. Holding Part]
[0172] The operation pipe is usually installed in a substantially
vertical shape (that is, in a hold-up state) so that the sample
supply portion which is an opening portion is on the upper side
during use. An appropriate holding part can be used for
installation. In addition, the same holding part may be used during
sample supply and during operation of the target components, or
different holding parts may be used. When different holding parts
are used during sample supply and during operation of the target
components, the transfer of the operation pipe between the holding
parts may be manually performed or be automated.
[0173] [7-1. Holding Form]
[0174] The holding part is not particularly limited as long as it
can be held in a substantially vertical state (that is, in a
hold-up state) so that the sample supply portion which is an
opening portion of the operation pipe is generally on the upper
side. The holding part includes, for example, a rack which is
configured by combining one or two or more holding members formed
with holding holes that can hold the closed end of the operation
pipe by piercing of the closed end, or configured by assembling
linear members in a lattice shape to form lattice holes as holding
holes, but the disclosure is not limited hereto. In the former
case, the holding hole formed in the holding member may penetrate
or may not penetrate the holding member. The inner diameter of the
holding hole is determined based on the outer diameter of the
operation pipe to be held. Among the holding members, the one that
holds the closed end of the operation pipe is described as a
holding substrate. In the holding substrate, the holding hole can
be formed so that the closed end of the holding portion B does not
penetrate the holding substrate (that is, the holding hole itself
does not penetrate the holding substrate). The depth of the holding
hole is appropriately determined based on the range to be held in
the operation pipe.
[0175] [7-2. Holding of a Plurality of Operation Pipes]
[0176] Since the operation pipe of the disclosure is elongated and
has an extremely small installation area of one pipe during
hold-up, a plurality of operation pipes can be held up and
installed in a concentrated state even with a small installation
area. As a result, the plurality of operation pipes can be operated
simultaneously. That is, a multi-channel operation can be
achieved.
[0177] An example of this form is shown in FIG. 5. In the device of
FIG. 5, up to 20 operation pipes can be processed simultaneously.
More operation pipes can also be processed depending on the device
specifications. For example, if there is an installation area as
large as a standard 96-well plate, up to 96 operation pipes can be
held up, and thereby up to 96 specimens can be processed
simultaneously. In addition, since the operation pipes are
independent from each other, in the above example, the number of
operation pipes can be arbitrarily adjusted corresponding to the
number of specimens. This form is particularly useful in POCT
(Point Of Care Testing) applications in which the number of
specimens is small and the number is also not constant.
[0178] When a plurality of operation pipes are held up and
installed, for example, as shown by 51 in FIG. 5, a plurality of
holding holes 52 can be formed in the holding substrate. The
holding holes differ depending on a form of densifying the
operation pipes, and can be formed, for example, in an array shape
(that is, formed one-dimensionally in a row) or in a matrix shape
(that is, formed two-dimensionally) as shown in FIG. 5. An interval
between the holding holes 52 can be appropriately determined based
on the density of the operation pipes. When the operation pipe held
by the holding hole 52 has a larger inner diameter in the sample
supply portion, the interval between the holding holes 52 can be
appropriately determined based on the outer diameter of the sample
supply portion.
[0179] [7-3. Temperature Control Function]
[0180] The holding part may have a temperature control function.
More specifically, the holding part may have a temperature control
function in a portion that holds at least a part of the recovery
portion B. For example, in FIGS. 4A to 4H, the temperature control
function is schematically shown as 43. More specifically, when the
holding substrate holds the closed end of the recovery portion B in
the holding hole, the holding substrate can have the temperature
control function in the portion for holding. For example, a holding
substrate 51 shown in FIG. 5 holds the closed end of the recovery
portion B in a holding hole 52, but the holding substrate 51 itself
may be formed of a temperature control block. The temperature
control function makes it possible to perform a treatment or
reaction requiring temperature control in the aqueous liquid filled
at least at the lower end of the recovery portion B. In the
disclosure, this form is preferably used, for example, when the
nucleic acid amplification reaction is performed in the recovery
portion B.
[0181] [7-4. Optical Detection Port]
[0182] The holding substrate may have an optical detection port.
The optical detection port is arranged to be capable of irradiating
an excitation light into the recovery portion B, and detect a
signal derived from the target components or components related
thereto which is emitted in the treatment or reaction in the
recovery portion B. For example, as shown in FIGS. 7A to 7C, the
optical detection port 71 can be formed to penetrate the holding
substrate from the lower end of the holding hole 52 and has an
aperture smaller than the outer diameter of the pipe portion b held
by the holding hole 52. An optical detection part (including a
fluorescence detection lens 44 and an optical fiber cable 45 in
FIGS. 7A to 7C) may be arranged in the optical detection port 71.
The position of the optical detection port is not limited to the
position in FIGS. 7A to 7C, and for example, photometry from the
side surface of the recovery portion may be considered.
[0183] [8. Magnetic Field Applying Part]
[0184] The magnetic field applying part and the magnetic field
moving mechanism thereof that cause variations of the magnetic
field for moving the magnetic particles in the operation pipe
together with the target components are not particularly limited.
As the magnetic field applying part, a magnetic source such as a
permanent magnet (for example, a ferrite magnet or a neodymium
magnet) or an electromagnet can be used. Outside the operation
pipe, the magnetic field applying part can be disposed close to the
operation pipe to such a degree that it is possible to aggregate
the magnetic particles dispersed in the aqueous liquid layer in the
operation pipe on the transport surface side of the pipe and
transport the magnetic particles that are aggregated in the gel
layer in the operation pipe. Accordingly, the magnetic field
applying part can effectively generate a magnetic field for the
magnetic particles via the transport surface of the pipe, and
capture and transport the target components together with the
magnetic particle mass.
[0185] [8-1. Shape]
[0186] The shape of the magnetic field applying part is not
particularly limited. For example, it may be a massive magnetic
field applying part that can generate a magnetic field at one point
or a part of the operation pipe (for example, illustrated as the
magnet 31 in FIGS. 3A to 3O or FIGS. 4A to 4H). More specifically,
the magnetic field applying part may be cylindrical (for example, a
diameter of 1 mm-5 mm, a thickness of 5 mm-30 mm). In the case of
this shape, the magnetic field applying part can generating a
magnetic field inside the operation pipe by being attached to one
point or a part of the outer periphery of the operation pipe. On
the other hand, the magnetic field applying part may be a
ring-shaped magnet having a substantially-circular-centre hole and
capable of generating a magnetic field around the operation pipe
having a substantially circular cross section. In the case of this
shape, the magnetic field applying part can generate a magnetic
field inside the operation pipe by making the operation pipe pass
through the substantially-circular-centre hole of the ring. In this
case, since the magnetic field applying part having a ring shape
surrounds the operation pipe, the magnetic particles also have a
ring shape according to the shape of the magnetic field applying
part when the magnetic particles are aggregated. On the other hand,
if the shape of the magnetic field applying part is a massive
shape, the aggregation shape of the magnetic particles is also a
massive shape. In other words, when the magnetic field applying
part having a ring shape is used, it is preferable in terms that a
contact area between the magnetic particles and the aqueous liquid
is larger and thus the target components and the like adsorbed on
the magnetic particles can be more efficiently exposed in the
liquid constituting the aqueous liquid layer.
[0187] [8-2. Magnetic Field Moving Mechanism]
[0188] [8-2-1. Movement in Longitudinal Direction of Control
Pipe]
[0189] The magnetic field moving mechanism of the magnetic field
applying part can move, for example, the magnetic field in the
longitudinal direction (axial direction, at least downward
direction) of the operation pipe in a state that the aggregation
form of the magnetic particles can be maintained. When described as
a magnetic field moving mechanism below, the mechanism can
determine the stop position and control the moving speed, and the
control may be performed manually or may be performed automatically
by a computer and the like. The moving speed may be, for example,
0.5 mm-10 mm per second. The magnetic field moving mechanism is
preferably a mechanism that can physically move the magnetic field
applying part itself in the longitudinal direction of the operation
pipe. The magnetic field moving mechanism can move the magnetic
field applying part (permanent magnet 31 in FIGS. 3A to 3O and
FIGS. 4A to 4H) itself as shown in FIGS. 3A to 3O and FIGS. 4A to
4H in the vertical direction. In addition, even in a device capable
of concentrating a plurality of operation pipes as shown in FIG. 5,
the magnetic field applying part (movable magnet plate 53 in FIG.
5) can be moved in the vertical direction (the magnetic field
moving mechanism itself is not shown in any of the above
cases).
[0190] [8-2-2. Control of Magnetic Field Intensity]
[0191] The magnetic field moving mechanism of the magnetic field
applying part may be a mechanism that can variably control the
intensity of the magnetic field applied to the magnetic particles.
Specifically, the magnetic field can be blocked or attenuated. The
degree of blocking or attenuation of the magnetic field is
preferably a degree at which the aggregated magnetic particles can
be dispersed in the droplet (the above item 6-2). For example, in
the case of an electromagnet, an energization control part can be
used to block the magnetic field. In addition, for example, in the
case of a permanent magnet, a mechanism that can move a magnet
disposed outside the operation pipe away from the operation pipe
can be used. This mechanism may be controlled manually or
automatically. The magnetic particles can be naturally dispersed in
the aqueous liquid layer by attenuating the magnetic field applied
to the magnetic particles, preferably by releasing the magnetic
particles from the magnetic field. Accordingly, the target
components or the accompanying components adsorbed on the magnetic
particles can be sufficiently exposed in the liquid constituting
the aqueous liquid layer.
[0192] [8-2-3. A Case of Device in which a Plurality of Control
Pipes is Concentrated]
[0193] As illustrated in FIG. 5, in a device in which a plurality
of operation pipes 1 is concentrated, a plurality of magnetic
sources corresponding to the plurality of operation pipes can be
held by being unitized into one member that can move in the
longitudinal direction of the operation pipe. As illustrated in
FIG. 5, this unitized member can be embodied as the movable magnet
plate 53 which is a magnetic field applying part that can move in
the longitudinal direction of the operation pipe 1. As illustrated
in FIG. 6, the movable magnet plate 53 in FIG. 5 includes a movable
substrate that can move in the longitudinal direction of the
operation pipe and a magnetic source (magnet 31) held in the
movable substrate, and can be held in a state that a plurality of
magnets 31 corresponding to each of the operation pipes is
disposed. In addition, the member may or may not have a function of
holding the operation pipe as the holding part described above. In
the case illustrated in FIG. 5, a holding hole 54 corresponding to
the operation pipe 1 is formed and thereby the movable magnet plate
53 can also have a holding function. In the illustration of FIG. 6,
the magnetic field applying part is shown as a massive part, but
the magnetic field applying part may have a ring shape being hollow
corresponding to the holding hole 54.
[0194] As illustrated in FIG. 5, in the device in which the
plurality of operation pipes 1 is concentrated, the magnetic field
moving mechanism of the magnetic field applying part may be capable
of simultaneously controlling the intensity of the magnetic field
obtained by the magnetic field applying part in each of the
plurality of operation pipes. For example, when a plurality of
different magnetic field applying parts is used for each of the
plurality of operation pipes, the magnetic field moving mechanism
may be capable of simultaneously controlling the magnetic fields
generated by the plurality of magnetic field applying parts.
[0195] In this member, when an electromagnet is used as the
magnetic field applying part, the magnetic field can be controlled
by current control. On the other hand, when a permanent magnet is
used as the magnetic field applying part, in the above member, for
example, it is possible to provide a mechanism that brings the
member itself closer to or away from the operation pipe (for
example, the member itself is moved substantially perpendicular to
the longitudinal direction of the operation pipe), or inserts a
magnetic shield material therebetween, or brings the plurality of
magnetic field applying parts held in the member closer to or away
from the operation pipe at a time without moving the member
itself.
[0196] As illustrated in FIG. 6, the movable magnet plate 53 in
FIG. 5 can be filled in a magnet holding portion 61 in a state that
the magnet 31 corresponding to each operation pipe held in the
holding hole 54 is disposed. The magnet holding portion 61 is
formed in a size that allows the movement of the magnet 31 in the
movable magnet plate 53 (that is, a movement of bringing the magnet
31 closer to or away from the operation pipe). As illustrated in
FIG. 6, a plurality of magnets 31 can be connected to each other by
connection rods 62, and all the connection rods 62 can be coupled
to a handle member 63. By moving the handle member 63, as shown in
FIG. 6, it is possible to bring all the magnets closer to the
operation pipe (magnetic field applying state) and away from the
operation pipe (magnetic field release state).
[0197] When the magnet is ring-shaped and this magnet is used to
control the intensity of the magnetic field, for example, the
magnet that is configured by two or more arc-shaped magnet parts
and thereby formed into a ring shape can be used as the ring-shaped
magnet. This ring-shaped magnet can release the operation pipe from
the magnetic field by being divided substantially perpendicular to
the diameter direction.
[0198] [8-2-4. Movement of Magnetic Field Applying Part in Holding
Part Capable of Holding Recovery Portion B]
[0199] The holding part may have a recess in which the magnetic
field applying part can move in the longitudinal direction of the
pipe portion b. More specifically, the holding part may have, in a
portion holding the recovery portion B, a recess in which the
magnetic field applying part can move in the longitudinal direction
of the pipe portion b. The magnetic field applying part that moves
in the recess may be the same as or different from the magnetic
field applying part contributing to the operation in the operation
portion A. For example, as shown in FIG. 7A, a recess 72 is formed
in the holding substrate 51 (in FIGS. 7A to 7C, the holding
substrate 51 is configured by a temperature control block) equipped
with the holding hole 52, and the recess is filled with a magnet
31' in advance. The movable magnet plate 53 on which the magnet 31
is disposed descends, and as shown in FIG. 7B, the movable magnet
plate 53 is in contact with the holding substrate 51 and cannot
move further downward. That is, depending on the magnet 31, the
magnetic particles 6 cannot be transported further downward. At
this time, the magnet 31' filled in the recess 72 of the holding
substrate 51 is attracted to the magnet 31 by the magnetic field
exerted by the magnet 31 on the movable magnet plate 53. Then, the
magnetic particles 6 in the operation pipe 1 are attracted to both
the magnet 31 and the magnet 31'. Next, as shown in FIG. 7C, when
the magnet 31 in the movable magnet plate 53 is moved away from the
operation pipe 1, the magnet 31' is released from the magnetic
field generated by the magnet 31 and thus falls into the recess 72
due to gravity. At this time, the magnetic particles in the
operation pipe 1 can be transported into the aqueous liquid 412 in
the recovery portion B and be lowered near the bottom in the
recovery portion B due to effects of the magnetic field of the
magnet 31'. Accordingly, the magnetic particles can be delivered by
the magnet 31 and the magnet 31', and the magnetic particles
accompanied by the target components can be sufficiently exposed in
the lowermost layer in the operation pipe.
[0200] [8-2-5. Magnetic Field Fluctuations]
[0201] The magnetic field moving mechanism may include a mechanism
that enables a fluctuation motion such as an amplitude movement and
rotation of the magnetic field. For example, it is possible to
substitute a stirrer by providing a function that enables the
magnetic force source to perform an amplitude motion (vertical
motion) in the longitudinal direction of the operation pipe.
Thereby, mixing or stirring in the aqueous liquid is facilitated.
For example, in a case without the function of blocking or
attenuating the magnetic field, the magnetic field applying part is
made to reciprocate in the vertical direction for several times
within the width of the thickness of the aqueous liquid layer while
being kept close to the operation pipe (while the magnetic
particles are aggregated), and thereby the target components and
the like adsorbed on the magnetic particles in the aqueous liquid
can also be sufficiently exposed in the liquid constituting the
aqueous liquid layer.
[0202] [8-2-6. Temperature Control Function]
[0203] The magnetic field applying part may further have a
temperature control function. For example, in FIGS. 4A to 4H, the
temperature control function is schematically shown as 42.
Alternatively, a heater can be incorporated in the magnetic field
applying part. By the latter temperature control function, the
reagent temperature in the aqueous liquid layer at the position
where the magnetic particles are present can be arbitrarily
adjusted. For example, a case is described in which the operation
pipe shown in FIGS. 4A to 4H is held by the holding part (holding
substrate) as shown in FIGS. 7A to 7C and having the temperature
control function as described in the above 7-3. In the operation
pipe shown in FIGS. 4A to 4H, the recovery portion B fills
multilayers including the RT reaction liquid layer 411 and the PCR
reaction liquid layer 412 via the gel layer 4g as a recovery
medium. When the operation pipe of FIGS. 4A to 4H is held by the
holding substrate 51 as shown in FIGS. 7A to 7C, the portion
directly held in the holding hole 52 of the holding substrate 51
may be only approximately a portion corresponding to the lowest
layer of the operation pipe (PCR reaction liquid layer 412). In
this case, since the portion filled with the RT reaction liquid
layer 411 in which the reverse transcription reaction is performed
is separated from the PCR reaction liquid layer 412 held directly
on the holding substrate 51, it is difficult to add temperature
control using the holding substrate 51.
[0204] Therefore, the device of the disclosure can have a
temperature control function different from the temperature control
function in the holding part. For example, as illustrated as 42 in
FIGS. 4A to 4H, the temperature control function may not be
interlocked with the magnetic field applying part; alternatively,
as illustrated as 64 in FIG. 6, the temperature control function
may be incorporated in the movable magnet plate 53 which is a
magnetic field applying part and thereby be interlocked with the
magnetic field applying part. In a specific aspect of the movable
magnet plate 53 shown in FIG. 6 in which the temperature control
function (heater) is incorporated, a heater 64 has an annular shape
enclosing the holding hole 54. When the movable magnet plate 53 has
the temperature control function in this way, in a period in which
the movable magnet plate 53 is in a position filled with the RT
reaction liquid layer 411 (FIG. 7A), the RT reaction liquid layer
411 is heated by the heater 64 in the movable magnet plate 53 and
the optimum temperature (for example, 50.degree. C.) can be
achieved.
[0205] [9. Optical Detection Part]
[0206] The optical detection part is not particularly limited and
can be easily selected by those skilled in the art corresponding to
the analysis method in which the target components are supplied.
For example, a part can be used which appropriately includes a
light generation portion, a detection part, a light transmission
part, a personal computer and the like. For example, in a case of
the fluorescence detection part 41 shown in FIG. 4H, as shown more
specifically in FIGS. 7A to 7C, incidence from the light generation
portion (not shown) to the detection part (the light transmission
part attached to a detection lens 44 (the optical fiber cable 45))
is performed, and light irradiation to the reaction liquid 4 in the
operation pipe 1 through the detection lens 44 can be performed.
The optical signal detected by the detection lens 44 can be sent to
a light receiving element by the optical fiber cable 45, converted
into an electrical signal, and then transmitted in real time to a
personal computer (not shown), and changes in the fluorescence
intensity of the reaction liquid 4 can be monitored. This is
suitable when the disclosure performs a reaction or treatment such
as a real-time nucleic acid amplification reaction in which a
variable fluorescence intensity is detected.
[0207] An LED, a laser, a lamp or the like can be used as the light
generation portion. In addition, in the detection, various light
receiving elements from inexpensive photodiodes to photomultiplier
tubes aiming at higher sensitivity can be utilized without
particular limitation. For example, when a case in which a nucleic
acid-related reaction such as a real-time nucleic acid
amplification reaction or a nucleic acid-related treatment is
performed is used as an example, for example, when SYBR (registered
trademark) GREEN I is used, this dye is specifically bound to a
double-stranded DNA and generates fluorescence around 525 nm, and
thus the detection part can detect a light having a target
wavelength by cutting lights having wavelengths other than the
target wavelength with an optical filter. In addition, when a case
in which the nucleic acid amplification reaction using droplet
movement is performed in the operation pipe is used as an example,
the fluorescence observation of the droplet supplied to the nucleic
acid amplification reaction can be performed in a darkroom in a
state that excitation lights are irradiated to a temperature
position in which an extension reaction (usually about
68-74.degree. C.) using DNA polymerase is performed and the liquid
droplet is stopped in this position. Furthermore, when an
irradiation range of the excitation light is expanded from a
temperature position in which heat denaturation is performed to a
temperature position in which annealing is performed, the droplet
can be moved and a melting curve of the amplification products can
also be obtained.
EXAMPLE
[0208] Next, examples are given to describe the disclosure in more
detail, but the scope of the disclosure is not limited hereto.
Example 1
[0209] [Nucleic acid extraction and purification from blood]A
gelling agent (Taiyo Chemical Co., Ltd., TAISET 26) is added to
silicon oil (Shin-Etsu Silicone KF-56) to reach a ratio of 1.2%
(weight ratio) and heated to 70.degree. C. to be completely mixed
with the silicon oil. A required amount of the oil mixed into a sol
state and a required amount of necessary reagents are alternately
injected and multi-layered from the tip of the injection needle
into the operation pipe (consisting of a capillary (operation
portion A) and a sample tube (recovery portion B)) shown in FIG. 3A
in a manner that bubbles do not enter. When the capillary having an
inner diameter of 1.5 mm is used, respectively 10 .mu.L of the gel
plug, 15 .mu.L of the cleaning liquid (200 mM of KCl), and 20 .mu.L
of the elution liquid (10 mM of TrisHCl, 1 mM of EDTA pH 8.0)) are
filled as shown in FIG. 3A. The filled capillary is placed at room
temperature for 30 minutes to completely gel the gel plug. The
upper end of the capillary forms a funnel-shaped sample supply port
that is sealed by a film material and is sealed by a septum.
[0210] The uppermost layer in the capillary is made into 100 .mu.L
of a cell lysate (4M guanidine thiocyanate, 2% (w/v) of Triton
X-100, and 100 mM of Tris-HCl pH 6.3) and contains 500 .mu.g of
silica-coated magnetic particles (nucleic acid extraction kit,
MagExtractor-Plasmid-attached magnetic particles of Toyobo).
Besides, as a nucleic acid isolation method using silica particles
and chaotropic salts, a method disclosed by Boom et al. (Japanese
Patent Laid-Open No. 2-289596) is used.
[0211] FIGS. 3B-3O are diagrams showing a nucleic acid extraction
process from blood for each operation of the magnet. Finally, the
nucleic acid is recovered in the elution liquid in the sample tube
attached to the lower end of the capillary. In FIG. 3B, 200 .mu.L
of whole human blood is injected by an injection needle and is
gently mixed with the magnetic particles by pipetting. After five
minutes, as shown in FIGS. 3C and 3D, the magnetic particles are
collected by bringing the magnet close from one side of the
capillary, and the magnet is lowered at a speed of 0.5 mm per
second. After the magnetic particles pass through the gel plug, as
shown in FIG. 3E, the magnet is separated from the capillary. As
shown in FIGS. 3F-3M, the same cleaning is performed for three
times. Thereafter, the magnet is released as shown in FIG. 3N, and
the magnetic particles are dropped into the tube containing the
elution liquid. After one minute, the magnet is brought close again
to collect the magnetic particles, and as shown in FIG. 3O, the
magnet particles are retracted into the gel plug, and the nucleic
acid extraction and purification operations are completed. In this
example, 200 ng of DNA is obtained for 1 .mu.L of the elution
liquid.
[0212] The sample tube is removed from the capillary, 1 .mu.L of
the elution liquid obtained in the sample tube is used, and a PCR
reaction mixture (10 .mu.L of total reaction volume) containing
0.15 U of Taq DNA polymerase, 500 nM of human GAPDH gene detection
primer(5'-GCGCTGCCAAGGCTGTGGGCAAGG-3' (Sequence number 1) and
5'-GGCCCTCCGACGCCTGCTTCACCA-3' (Sequence number 2)) and 200 nM of
dNTP is used to perform PCR (temperature cycle: 95.degree. C., one
second, 60.degree. C., ten seconds, 72.degree. C., ten seconds, 40
cycles) by a thermal cycler (ABI9700, Applied Biosystems). As a
result, as shown in FIG. 8, a reaction product specific to the
human GAPDH gene (fragment size 171 bases) is confirmed by agarose
gel electrophoresis.
[0213] As described above, the disclosure is described according to
the embodiments of the disclosure, but it should not be considered
that the description and drawings constituting a part of this
disclosure limit the disclosure. From this disclosure, various
alternative embodiments, examples, and operational techniques are
apparent to those skilled in the art. The technical scope of the
disclosure is defined only by the invention specific matters of the
scope of claims reasonable from the above description, and can be
modified and embodied without departing from the scope in the
implementation stage.
[0214] Sequence numbers 1 and 2 are synthetic primers.
Sequence CWU 1
1
2124DNAArtificial Sequencesynthetic RT-PCR primer 1gcgctgccaa
ggctgtgggc aagg 24224DNAArtificial Sequencesynthetic RT-PCR primer
2ggccctccga cgcctgcttc acca 24
* * * * *