U.S. patent application number 13/733684 was filed with the patent office on 2013-08-01 for microchip and method of using the same.
This patent application is currently assigned to Rohm Co., Ltd.. The applicant listed for this patent is Rohm Co., Ltd.. Invention is credited to Shun Momose, Toshihiro Mori, Akinori Yokogawa.
Application Number | 20130195719 13/733684 |
Document ID | / |
Family ID | 40534625 |
Filed Date | 2013-08-01 |
United States Patent
Application |
20130195719 |
Kind Code |
A1 |
Momose; Shun ; et
al. |
August 1, 2013 |
Microchip and Method of Using the Same
Abstract
A microchip includes fluid circuits therein, formed by uniting
together at least a first substrate that is a transparent substrate
and a second substrate having grooves provided at the substrate
surface and/or through holes penetrating in a thickness direction.
The fluid circuits include a liquid reagent receptacle unit to
store a liquid reagent, a quantification unit to quantify the
liquid reagent or specimen, and an overflow liquid storage unit
connected to the quantification unit to store the liquid reagent or
specimen overflowing from the quantification unit during
quantification. There is also provided a method of using the
microchip.
Inventors: |
Momose; Shun; (Kyoto,
JP) ; Yokogawa; Akinori; (Kyoto, JP) ; Mori;
Toshihiro; (Kyoto, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Rohm Co., Ltd.; |
Kyoto |
|
JP |
|
|
Assignee: |
Rohm Co., Ltd.
Kyoto
JP
|
Family ID: |
40534625 |
Appl. No.: |
13/733684 |
Filed: |
January 3, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12250592 |
Oct 14, 2008 |
8367424 |
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13733684 |
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Current U.S.
Class: |
422/68.1 ;
422/502 |
Current CPC
Class: |
G01N 33/5002 20130101;
B01L 2200/12 20130101; G01N 21/07 20130101; B01L 3/50273 20130101;
B01L 2300/168 20130101; B01L 2200/16 20130101; B01L 2300/0887
20130101; B01L 2300/0829 20130101; B01L 3/5027 20130101; B01L
2300/0819 20130101; B01L 3/5025 20130101; G01N 2021/0325
20130101 |
Class at
Publication: |
422/68.1 ;
422/502 |
International
Class: |
B01L 3/00 20060101
B01L003/00; G01N 33/50 20060101 G01N033/50 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 15, 2007 |
JP |
2007267833 |
Oct 31, 2007 |
JP |
2007284212 |
Oct 31, 2007 |
JP |
2007284213 |
Jan 17, 2008 |
JP |
2008008154 |
Claims
1-14. (canceled)
15. A microchip formed by uniting together a first substrate, a
second substrate having grooves provided at both surfaces of the
substrate and a plurality of through holes penetrating in a
thickness direction, and a third substrate in the cited order, said
microchip comprising: a first fluid circuit constituted of grooves
provided at a surface of said first substrate facing said second
substrate and at a surface of said second substrate facing said
first substrate, and a second fluid circuit constituted of grooves
provided at a surface of said third substrate facing said second
substrate and at a surface of said second substrate facing said
third substrate.
16. The microchip according to claim 15, wherein said first fluid
circuit communicates with said second fluid circuit by at least one
of said plurality of through holes.
17. The microchip according to claim 15, wherein each of said first
and second fluid circuits includes at least one site selected from
the group consisting of a liquid reagent receptacle unit to store a
liquid reagent, a liquid reagent quantification unit to quantify
said liquid reagent, a specimen quantification unit to quantify a
specimen, and a mixing unit to mix said specimen and said liquid
reagent.
18. The microchip according to claim 17, wherein only said first
fluid circuit includes one or a plurality of liquid reagent
receptacle units.
19. The microchip according to claim 17, wherein only said second
fluid circuit includes one or a plurality of liquid reagent
quantification units and one or a plurality of specimen
quantification units.
20. The microchip according to claim 15, wherein the grooves
provided at the surface of said second substrate facing said first
substrate is deeper than the grooves provided at the surface of
said second substrate facing said third substrate.
21. The microchip according to claim 15, further including one or
more detection unit connected to said first fluid circuit or said
second fluid circuit, formed of a cavity constituted of at least
one of said plurality of through holes, the surface of said first
substrate facing said second substrate, and the surface of said
third substrate facing said second substrate.
22. The microchip according to claim 15, wherein said first
substrate, said second substrate, and said third substrate include
styrene-butadiene copolymer.
23. The microchip according to claim 15, wherein said first
substrate and said third substrate are transparent substrates.
24. The microchip according to claim 15, wherein said second
substrate is an opaque substrate.
25. The microchip according to claim 24, wherein said second
substrate is a black substrate.
26-29. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a microchip valid for a
.mu.-TAS (Micro Total Analysis System) and the like, suitable for
use in environmental analysis, chemical synthesis and biochemical
assays of DNA, protein, cells, immunity, blood and the like.
[0003] 2. Description of the Background Art
[0004] In line with the recent increase in the importance of
sensing, detecting and determining the quantity of chemical
substances and biological material such as DNA (Deoxyribo Nucleic
Acid), enzyme, antigen, antibody, protein, virus, and cells in the
field of medical care, health, food product, development of
medicine and the like, various biochips and microchemical chips
(hereinafter, generically referred to as a microchip) that allow
relevant measurement conveniently have been proposed (for example,
Japanese Patent Laying-Open No. 2007-017342). A microchip is
characterized in that the series of experiments and analytical
operations carried out at laboratories can be performed within a
chip that is several to 10 cm square and several millimeters to
several centimeters in thickness. Accordingly, only a small amount
of specimen and reagent is required, leading to reduction in cost.
Assays can be performed with a high response rate and high
throughput. Another advantage is that the examination result can be
obtained immediately at the site where the specimen has been
collected. A microchip can be used conveniently in biochemical
examination such as a blood test.
[0005] A microchip has fluid circuits therein. The fluid circuits
are mainly constituted of a plurality of sites such as a liquid
reagent receptacle unit to store a liquid reagent directed to
mixing or reacting with, or treating a specimen (for example,
blood), a quantification unit to quantify a specimen and/or liquid
reagent, a mixing unit to mix the specimen and liquid reagent, and
a cuvette (detection unit) for optical measurement to examine
and/or analyze the mixture, as well as minute channels
appropriately connecting the sites. In use, a microchip is
typically mounted on an apparatus (a centrifuge) that can apply
centrifugal force to the chip. By applying centrifugal force to the
microchip in an appropriate direction, measurement and mixture of a
specimen and/or liquid reagent, in addition to introduction of the
mixture to the optical measurement cuvette, can be effected. The
examination and analysis of the mixture introduced into the optical
measurement cuvette (for example, detection of a certain component
in the mixture) can be implemented by directing light to the
cuvette in which the mixture is stored and measure the
transmissivity or the like.
[0006] In the examination and/or analysis based on the
above-described microchip, a guarantee that the liquid treatment in
the fluid circuits effected by application of centrifugal force is
extremely critical. This is because, if such guarantee cannot be
afforded, evaluation as to whether the result of the examination
and analysis is reliable or not cannot be made. Unreliable
situations include, for example, the case where the liquid reagent
that should be stored in the liquid reagent receptacle unit at the
time of using the microchip is not present at a predetermined
location or is insufficient due to evaporation or drop off during
transportation, the case where the introduced amount of specimen
into the fluid circuits is insufficient, and the case where liquid
leakage has occurred due to a defect in the fabrication of the
microchip. In such cases, the quantified amount of liquid reagent
and specimen will not be accurate, leading to incorrect resultant
data from the examination and analysis. There is also the
possibility of erroneous operation at the centrifuge equipment,
which may impede appropriate fluid transfer. Therefore, a guarantee
that the fluid in the fluid circuits is transferred to an
appropriate site, fluid treatment is carried out appropriately, the
amount of the specimen and/or liquid reagent is sufficient, without
erroneous operation of the centrifuge equipment, is critical.
However, the approach to actually introducing a specimen into the
fluid circuits to conduct fluid treatment, and checking whether
there is the aforementioned error, prior to the examination and
analysis based on a microchip (actual usage), cannot be employed
since a microchip is generally not reusable.
[0007] U.S. Pat. No. 5,590,052 discloses a method for confirming
that the fluid introduced into a blood analysis system has flown to
a predetermined site. This method includes the steps of directing
light to a predetermined site, and detecting the passing light.
[0008] The microchip disclosed in the aforementioned Japanese
Patent Laying-Open No. 2007-017342 is formed by uniting together a
first substrate with grooves that correspond to fluid circuits, and
a second substrate. The first and second substrates are united such
that the surface of the first substrate where grooves are provided
corresponds to the joining face. The microchip includes one layer
of fluid circuits therein. As used herein, "one layer" implies that
the microchip includes only one fluid circuit layer in the
thickness direction of the microchip.
[0009] In a microchip directed to a blood test, various types of
examinations are often performed using the blood plasma component
in the whole blood. Therefore, fluid circuits in such a microchip
generally includes a blood plasma separator unit to remove
hematocytes from the whole blood introduced into the fluid circuits
to extract and separate the plasma component.
SUMMARY OF THE INVENTION
[0010] In the conventional method of confirming that the fluid
introduced into the blood analyzer has flown to a predetermined
site, the blood analyzer must be filled with the fluid along the
entire width at the predetermined site to identify the absence or
presence of the fluid by measuring transmitting light. This means
that the amount of fluid introduced into the blood analyzer must be
increased if the blood analyzer is thick. Therefore, the benefit of
allowing examination and/or analysis with a minute amount of fluid
at the microchip, if the aforementioned conventional method is
applied thereto, will be degraded.
[0011] Furthermore, when the transmitting light is to be measured
to confirm that fluid has flown to a predetermined site in a
microchip, a transparent substrate with respect to light must be
employed for at least the light channel region of the detected
light. This imposes the problem that the configuration of the
microchip is rendered complex, and the degree of freedom in
designing a microchip is degraded.
[0012] Moreover, when light is directed to a predetermined site in
order to confirm arrival of the fluid at the predetermined site,
there was a problem that, although the presence of the fluid at the
predetermined site can be confirmed, the amount of the fluid at
that predetermined site cannot be detected. In other words, the
event of an insufficient amount of the specimen and/or liquid
reagent caused by some error as described above cannot be
detected.
[0013] In view of the foregoing, an object of the present invention
is to provide a microchip improved in reliability, allowing
detection of an insufficient amount of a specimen and/or liquid
reagent as well as a fault such as an erroneous operation at a
centrifuge, requiring only a minute amount of liquid, to guarantee,
immediately before and during use of a microchip, that the fluid
treatment in fluid circuits according to application of centrifugal
force has been carried out appropriately, for every microchip, and
a method of using the microchip.
[0014] The present invention is directed to a microchip including
fluid circuits therein, formed by uniting together at least a first
substrate that is a transparent substrate, and a second substrate
having grooves provided at a substrate surface and/or through holes
penetrating in a thickness direction. The fluid circuits include a
liquid reagent receptacle unit storing a liquid reagent, at least
one quantification unit to quantify the liquid reagent or a
specimen, and at least one overflow liquid storage unit to store
the liquid reagent or specimen overflowing from the quantification
unit during quantification.
[0015] The microchip of the present invention may be a microchip
including two layers of fluid circuits therein, formed by uniting
together a first substrate that is a transparent substrate, a
second substrate having grooves provided at both surfaces of the
substrate and through holes penetrating in a thickness direction,
and a third substrate. The overflow liquid storage unit is the site
irradiated with light to detect the presence or absence of the
liquid reagent or specimen overflowing from the quantification
unit.
[0016] In the present invention, the microchip including two layers
of fluid circuits therein may include a plurality of overflow
liquid storage units. In this case, the fluid circuits at the side
of the first substrate, of the two layers of fluid circuits,
preferably includes all the overflow liquid storage units.
[0017] In the case where the microchip of the present invention
includes a plurality of overflow liquid storage units, the
plurality of overflow liquid storage units preferably are disposed
on the circumference of the same circle at the surface of the
second substrate. The second substrate is preferably an opaque
substrate, and more preferably a black substrate. In addition, the
fluid circuits of the present invention may include at least one
liquid reagent quantification unit to quantify the liquid reagent,
and at least one specimen quantification unit to quantify the
specimen. In this case, an overflow liquid storage unit is
connected to at least one of the quantification units. Preferably,
an overflow liquid storage unit is connected to each quantification
unit. The fluid circuits may further include a mixing unit to mix
the quantified specimen and the quantified liquid reagent, and a
detection unit to examine and analyze the obtained mixture.
[0018] The present invention also provides a method of using the
microchip set forth above, including the steps of introducing a
liquid reagent or specimen into the quantification unit by applying
centrifugal force, and detecting absence or presence of the liquid
reagent or specimen in the overflow liquid storage unit by
directing light from the first substrate side to the overflow
liquid storage unit and measuring an intensity of light reflected
therefrom. The detection of the absence or presence of the liquid
reagent or specimen can be carried out by obtaining a ratio of the
intensity of reflected light obtained by directing light to the
overflow liquid storage unit from the first substrate side before
the liquid reagent or specimen is introduced into a quantification
unit to the intensity of reflected light obtained by directing
light to the overflow liquid storage unit from the first substrate
side after the liquid reagent or specimen is introduced into a
quantification unit.
[0019] The method of using the microchip of the present invention
may further include the step of detecting the absence or presence
of a liquid reagent in the liquid reagent receptacle unit by
directing light from the first substrate side to the liquid reagent
receptacle unit to measure the intensity of light reflected
therefrom. The detection of absence or presence of a liquid reagent
can be carried out by obtaining the ratio of the intensity of
reflected light obtained by directing light from the first
substrate side to the liquid reagent receptacle unit, before the
liquid reagent is introduced to the liquid reagent receptacle unit,
to the intensity of reflected light obtained by directing light
from the first substrate side to the liquid reagent receptacle
unit, after the liquid reagent is introduced to the liquid reagent
receptacle unit. The method of using the microchip of the present
invention may further include the step of detecting absence or
presence of a liquid reagent or specimen in at least one site
selected from the liquid reagent quantification unit, the mixing
unit, and the detection unit by directing light from the first
substrate side to the at least one site and measuring the intensity
of light reflected therefrom.
[0020] According to the present invention, detection can be made of
an insufficient amount of a specimen and liquid reagent as well as
a fault such as an erroneous operation at a centrifuge, requiring
only a minute amount of liquid, during actual use of a microchip
(during examination, analysis, and the like of a specimen) to
guarantee that the fluid treatment in fluid circuits according to
application of centrifugal force has been carried out
appropriately. Therefore, a microchip improved in reliability can
be provided by the present invention.
[0021] The aforementioned conventional "one layer type" microchip
has the following problems (1) to (3). There is a need for a
microchip that can overcome such problems.
[0022] (1) A one-layer type microchip must have the area of the
substrate increased in order to form desired fluid circuits.
Therefore, (i) the uniting area in affixing two substrates together
is increased, leading to difficulty in obtaining flatness at the
two substrates. Accordingly, welding failure will readily occur in
welding the substrates together such as by laser welding, thermal
welding, ultrasonic welding, and welding employing an adhesive.
(ii) The uniting area in affixing two substrates together is
increased, leading to difficulty in obtaining pressure evenness at
the time of substrate welding. Accordingly, welding failure will
readily occur in welding the substrates together. (iii) If the
pressure at the time of welding is increased and/or a large amount
of adhesive is employed in view of alleviating such welding
failure, leakage of the resin constituting the substrate and/or run
off of the adhesive may occur to block the minute pattern (grooves)
formed at the surface of the substrate. (iv) In the case where
leakage of the resin constituting the substrate and/or run off of
the adhesive occurs, uniformity in the shape of the fluid circuits
among microchips cannot be achieved. In addition, the volume of the
quantification unit in the fluid circuits will vary, disallowing
accurate measurement. In view of such problems, it was difficult to
form intricate channel patterns and increase the scale of
integration and density of fluid circuits in a one-layer type
microchip.
[0023] (2) A one-layer type microchip will have a mixture of deep
grooves and shallow grooves formed within one fluid circuit layer.
(i) The width of the rib constituting the grooves is increased to
maintain the aspect ratio of the mold directed to forming grooves
in the substrate. Therefore, the likelihood of deviation in the
dimension accuracy and variation in the dimension among microchips,
due to leakage of an adhesive or resin during welding, increases.
(ii) When the substrate is fabricated by injection molding or
imprinting, the region of shallow grooves will correspond to the
bottom region of a mold. Therefore, fabrication of a mold
accommodating the minute channel region is rendered difficult,
resulting in poor mass production. (iii) In the case where there is
a mixture of deep grooves and shallow grooves in one fluid circuit
layer, the occupying ratio of the fluid circuits to the microchip
cannot be increased. It is difficult to increase the scale of
integration and density of fluid circuits.
[0024] The above problem (2) will be described in further detail
with reference to the drawings. FIG. 23 is a schematic sectional
view of a configuration of a mold to form a substrate with grooves
constituting fluid circuits, employed for a conventional microchip.
FIG. 24 is a schematic sectional view of a microchip formed using
the substrate obtained from the mold of FIG. 23. For a substrate
constituting a microchip, a plastic substrate, for example, can be
employed. A substrate with grooves constituting fluid circuits can
be produced by injection molding employing a mold with a transfer
configuration. As shown in FIG. 23, the recess and projection in a
mold 1701 (the transfer configuration of the grooves in a
substrate) can be cut out using an end mill or the like. Consider
the case where a substrate including both deep grooves and shallow
grooves are to be formed. When a shape corresponding to shallow
grooves is to be provided in the mold, a long end mill blade 1702
is required since cutting must be effected down to a deep region in
the mold. End mill blade 1702 must have a diameter corresponding to
the length. In other words, when a substrate including both deep
grooves and shallow grooves is to be produced, a long and thick end
mill blade must be employed to produce a companion mold.
[0025] As a result, a width W of a rib 1803 constituting shallow
grooves at an upper substrate 1801 obtained from the mold of FIG.
23 is increased due to the diameter of the end mill blade.
Accordingly, the area of contact between upper substrate 1801 and a
lower substrate 1802 to be united is increased. Such increase in
the area of contact will cause more leakage of the substrate
material when the substrates are united together by, for example,
fusing and welding the uniting faces. This induces deviation in the
dimension accuracy of the fluid circuits and/or variation in the
dimension among microchips.
[0026] Moreover, in the case where deep grooves and shallow grooves
are formed in a mixed manner in one fluid circuit layer, there will
be a space S that cannot be used for the fluid circuits, as shown
in FIG. 24. Thus, it will be difficult to increase the scale of
integration and density of fluid circuits
[0027] (3) Application of centrifugal force towards a microchip can
be implemented by mounting a microchip on a rotatable stage of a
centrifuge, and spinning the stage. The rotating diameter of a
stage must be increased since the area of the one-layer type
microchip is large. This results in the increase of the size and
power consumption of the centrifuge.
[0028] The present invention is directed to overcoming these
problems, and an object according to another aspect is to provide a
microchip with a sufficiently small substrate area (microchip
area), increased in the scale of integration and density of fluid
circuits.
[0029] According to the present invention, a microchip is provided,
formed by uniting together a first substrate, a second substrate
having grooves provided at both surfaces of the substrate and a
plurality of through holes penetrating in a thickness direction,
and a third substrate in the cited order. The microchip includes a
first fluid circuit constituted of grooves provided at a surface of
the first substrate facing the second substrate and at a surface of
the second substrate facing the first substrate, and a second fluid
circuit constituted of grooves provided at a surface of the third
substrate facing the second substrate and at a surface of the
second substrate facing the third substrate.
[0030] The first and second fluid circuits preferably communicate
via at least one of the plurality of through holes.
[0031] Each of the first and second fluid circuits preferably
includes at least one site selected from the group consisting of a
liquid reagent receptacle unit to store a liquid reagent, a liquid
reagent quantification unit to quantify the liquid reagent, a
specimen quantification unit to quantify a specimen, and a mixing
unit to mix the specimen and liquid reagent.
[0032] The grooves provided at the surface of the second substrate
facing the first substrate is preferably deeper than the grooves
provided at the surface of the second substrate facing the third
substrate.
[0033] In the microchip of the present invention, preferably only
the first fluid circuit includes one or a plurality of liquid
reagent receptacle units, and preferably only the second fluid
circuit includes one or a plurality of liquid reagent
quantification units and one or a plurality of specimen
quantification units.
[0034] The microchip of the present invention preferably further
includes at least one detection unit, formed of a cavity
constituted of at least one of the plurality of through holes, the
surface of the first substrate facing the second substrate, and the
surface of the third substrate facing the second substrate, and
connected to the first or second fluid circuit.
[0035] The first substrate, the second substrate, and the third
substrate are preferably formed of styrene-butadiene copolymer.
[0036] The first substrate and the third substrate are preferably
transparent substrates. The second substrate is preferably a
transparent substrate, more preferably a black substrate.
[0037] Since the microchip of the present invention includes fluid
circuits of two layers, increase in the scale of integration and
density of fluid circuits is allowed. Thus, fluid circuits that
allows fluid treatment of a more complex level can be formed.
Moreover, the substrate area (microchip area) can be reduced by
employing fluid circuits of two layers. Thus, the flatness of each
substrate at the time of uniting the substrates can be ensured,
leading to the likelihood of obtaining pressure evenness over the
entire substrate. Therefore, welding failure can be prevented or
suppressed. Preventing or suppressing welding failure allows
improvement in the mass production of microchips.
[0038] In the case where blood collected from a person with
hyperlipidemia or with symptoms thereof is to be examined using a
microchip directed to a blood test, the separated plasma component
will include a component such as lipid that is insoluble with
respect to the plasma component, impeding an accurate examination
and analysis on the plasma component. Namely, if the mixture
introduced into the detection unit includes an insoluble matter
such as lipid, the directed light in optical measurement, when
employed in the detection of the property component in the mixture,
will be disturbed by the presence of such insoluble matter. There
was a problem that accurate measurement data could not be obtained.
Furthermore, the presence of such insoluble matter disallows
accurate quantification of the plasma component, leading to the
problem that proper measurement data cannot be obtained.
[0039] The present invention is directed to solving the problem set
forth above. An object of the present invention is to provide a
microchip for a blood test that can remove a component that may
disturb the examination and analysis from a sample of whole blood,
prior to mixture with a liquid reagent, allowing accurate
examination and analysis.
[0040] In addition, the present invention provides a microchip for
a blood test, including a blood plasma separation unit to separate
a blood plasma component from a sample including whole blood
introduced into the microchip. The blood plasma separation unit
includes a suspension removal unit to remove suspensions present in
proximity to the surface of the plasma blood component. The
suspension removal unit includes one or a plurality of discrete
columnar structures, and a suspension storage unit to store the
removed suspensions. The columnar structure preferably has a
triangular cross section.
[0041] The suspension removal unit may includes a plurality of
discrete columnar structures, disposed in a plurality of rows. The
suspension is, for example, lipid.
[0042] Since lipid and the like included in the whole blood sample
can be removed in advance by the microchip of the present
invention, a blood test can be carried out accurately without being
disturbed by the lipid and the like.
[0043] The foregoing and other objects, features, aspects and
advantages of the present invention will become more apparent from
the following detailed description of the present invention when
taken in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] FIGS. 1A, 1B and 1C represent the outer shape of an example
of a microchip according to the present invention.
[0045] FIGS. 2A and 2B are perspective view of an example of fluid
circuits formed at a second substrate of a microchip of the present
invention.
[0046] FIGS. 3 and 4 are a top view and a bottom view,
respectively, of an example of a second substrate of a microchip of
the present invention.
[0047] FIGS. 5A and 5B are diagrams to describe the relationship
between the intensity of incident light and the intensity of
reflected light before and after introduction of the overflow
liquid in the overflow liquid storage unit.
[0048] FIGS. 6A and 6B represent the state of the liquid at the top
face of the second substrate (the surface facing the first
substrate) and at the bottom face of the second substrate (the
surface facing the third substrate), respectively, in a hematocyte
separation and liquid reagent quantification procedure.
[0049] FIGS. 7A and 7B represent the state of the liquid at the top
face of the second substrate (the surface facing the first
substrate) and at the bottom face of the second substrate (the
surface facing the third substrate), respectively, in a specimen
quantification procedure.
[0050] FIGS. 8A and 8B represent the state of the liquid at the top
face of the second substrate (the surface facing the first
substrate) and at the bottom face of the second substrate (the
surface facing the third substrate), respectively, in a first step
of a first mixture procedure.
[0051] FIGS. 9A and 9B represent the state of the liquid at the top
face of the second substrate (the surface facing the first
substrate) and at the bottom face of the second substrate (the
surface facing the third substrate), respectively, in a second step
of the first mixture procedure.
[0052] FIGS. 10A and 10B represent the state of the liquid at the
top face of the second substrate (the surface facing the first
substrate) and at the bottom face of the second substrate (the
surface facing the third substrate), respectively, in a first step
of a second mixture procedure.
[0053] FIGS. 11A and 11B represent the state of the liquid at the
top face of the second substrate (the surface facing the first
substrate) and at the bottom face of the second substrate (the
surface facing the third substrate), respectively, in a second step
of the second mixture procedure.
[0054] FIGS. 12A and 12B represent the state of the liquid at the
top face of the second substrate (the surface facing the first
substrate) and at the bottom face of the second substrate (the
surface facing the third substrate), respectively, in a detection
unit introduction procedure.
[0055] FIGS. 13 and 14 are a top view and a bottom view,
respectively, of another example of a second substrate of the
microchip of the present invention.
[0056] FIG. 15 is a schematic sectional view of a shape of a mold
used to form a second substrate having a plurality of shallow
grooves at one surface and deep grooves at another surface.
[0057] FIG. 16 is a schematic sectional view of a microchip of the
present invention, produced based on a second substrate obtained
from the mold of FIG. 15, a first substrate, and a third
substrate.
[0058] FIG. 17 is a schematic top view of an example of fluid
circuits configuration of a microchip for a blood test of the
present invention.
[0059] FIG. 18 is a schematic enlarged top view of a blood plasma
separation unit in the microchip of FIG. 17.
[0060] FIG. 19 is a schematic top view representing the separated
state of the blood plasma component and hematocyte component in the
whole blood sample as a result of introduction of the whole blood
sample into a blood plasma separation unit 1902 of FIG. 18 and
application of centrifugal force.
[0061] FIGS. 20, 21, and 22 are schematic enlarged top views of
examples of a blood plasma separation unit of the present
invention.
[0062] FIG. 23 is a schematic sectional view of a configuration of
a mold used to form a substrate having grooves constituting fluid
circuits, employed in a conventional microchip.
[0063] FIG. 24 is a schematic sectional view of a microchip
produced using the substrate obtained from the mold of FIG. 23.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0064] Embodiments of the present invention will be described
hereinafter with reference to the drawings. In the drawings, the
same or corresponding elements have the same reference characters
allotted, and description thereof will not be repeated. The scales
in dimension such as the length, size, and width in the drawings
are modified appropriately for the sake of simplification, and do
not represent the actual scale dimension.
First Embodiment
[0065] The present invention will be described in detail based on
FIGS. 1A, 1B and 1C that are a top view, side view, and bottom
view, respectively, of an example of a microchip of the present
invention. Referring to FIGS. 1A, 1B and 1C, a microchip 100
according to a first embodiment of the present invention is formed
by uniting together a first substrate 101 that is a transparent
substrate, a second substrate 102 that is a black substrate, and a
third substrate 103 that is a transparent substrate, in the cited
order (refer to FIG. 1B). The length of these substrates is, though
not particularly limited to, approximately 62 mm in the lateral
direction (L1 in FIG. 1) and approximately 30 mm in the vertical
direction (L2 in FIG. 1) in the present embodiment. The thickness
of first substrate 101 (L3 in FIG. 1), second substrate 102 (L4 in
FIG. 1) and third substrate 103 (L5 in FIG. 1) is, though not
particularly limited to, approximately 1.6 mm, approximately 9 mm,
and approximately 1.6 mm, respectively.
[0066] First substrate 101 includes a liquid reagent inlet 110 (a
total of 11 inlets in the present embodiment) penetrating in the
thickness direction, and a specimen inlet 120 to introduce a
specimen (for example, whole blood) into the microchip fluid
circuits. As used herein, "specimen" refers to a sample (for
example, whole blood) that is the subject of various chemical
synthesis, examination, analysis, and the like, introduced into the
fluid circuits, or a certain component separated from the sample in
microchip 100 (for example, plasma component separated from the
whole blood). A liquid reagent is already stored in a liquid
reagent receptacle unit in the fluid circuits, prior to actual
usage (examination, analysis, and the like of a specimen) of
microchip 100, and is a substance used for mixture with or reaction
with the specimen, or treatment of the specimen. Microchip 100
generally has a liquid reagent introduced through liquid reagent
inlet 110, which is then sealed by a label or the like to be
presented for actual use.
[0067] Second substrate 102 includes grooves formed at opposite
surfaces, and a plurality of through holes penetrating in the
thickness direction. By uniting first substrate 101 and third
substrate 103 with second substrate 102, two layers of fluid
circuits are formed in microchip 100. Hereinafter, the fluid
circuits constituted of grooves provided at the surface of first
substrate 101 facing second substrate 102 and at the surface of
second substrate 102 facing first substrate 101 is referred to as
"first fluid circuit". The fluid circuits constituted of grooves
provided at the surface of third substrate 103 facing second
substrate 102 and at the surface of second substrate 102 facing
third substrate 103 is referred to as "second fluid circuit". The
two layers of fluid circuits may communicate with each other via
through holes penetrating in the thickness direction, formed in
second substrate 102. Detection units 311, 312, 313, 314, 315 and
316 that will be described afterwards are formed at second
substrate 102. The configuration of the fluid circuits (grooves)
formed at opposite sides of second substrate 102 will be described
in detail hereinafter.
[0068] As used herein, "two layers" implies that fluid circuits are
provided at two different positions in the thickness direction of
the microchip. The first and second fluid circuits may be
communicated with each other via one or more through holes
penetrating in the thickness direction, formed in the second
substrate.
[0069] The microchip includes fluid circuits of two layers,
increase in the scale of integration and density of fluid circuits
is allowed. Thus, fluid circuits that allows fluid treatment of a
more complex level can be formed. Moreover, the substrate area
(microchip area) can be reduced by employing fluid circuits of two
layers. Thus, the flatness of each substrate at the time of uniting
the substrates can be ensured, leading to the likelihood of
obtaining pressure evenness over the entire substrate. Therefore,
welding failure can be prevented or suppressed.
[0070] FIGS. 2A and 2B are perspective views of fluid circuits
(grooves) formed at second substrate 102, the former representing
the fluid circuits formed at the surface of second substrate 102
facing first substrate 101 (hereinafter, also simply referred to as
"upper side"), and the later representing the fluid circuits formed
at the surface of second substrate 102 facing third substrate 103
(hereinafter, also simply referred to as "lower side"). Namely,
FIGS. 2A and 2B represent the first fluid circuit and the second
fluid circuit, respectively. As shown in FIGS. 2A and 2B, second
substrate 102 has grooves formed at the surface, and through holes
penetrating in the thickness direction, which constitute respective
sites where a specimen, liquid reagent, or mixture thereof is
treated, and minute channels appropriately connecting these
sites.
[0071] FIGS. 3 and 4 represent a top view and bottom view,
respectively, of second substrate 102, the former corresponding to
the upper side fluid circuits and the latter corresponding to the
lower side fluid circuits, of second substrate 102. In FIG. 4, the
lower side fluid circuits of the second substrate is illustrated in
a mirror-reversed manner for the sake of convenience to identify
the corresponding relationship with the upper side fluid circuits
of FIG. 3. Microchip 100 of the present embodiment is a multi-test
chip that allows examination and/or analysis of six items for one
specimen. The fluid circuits are divided into six sections
(sections 1-6 in FIG. 3) to allow examination and analysis of six
items. It is to be noted that the six sections are connected with
each other at the region where the specimen quantification unit is
located (upper region of lower side fluid circuits). One or two
liquid reagent receptacle units in which a liquid reagent is stored
are provided for each section. In FIG. 3, a total of eleven liquid
reagent receptacle units 301a, 301b, 302a, 302b, 303a, 303b, 304a,
304b, 305a, 305b and 306a are shown. The specimen introduced
through specimen inlet 120 shown in FIG. 1 has the hematocyte
component separated and removed, distributed to each section and
quantified, mixed with one or two types of liquid reagents that is
quantified individually in each section, and then guided to
detection units 311, 312, 313, 314, 315 and 316. The mixture
introduced into the detection unit of each section is subjected to
optical measurement such as being irradiated with light at the
detection unit from, for example, a direction substantially
perpendicular to the surface of microchip 100, and measuring the
transmissivity of the transmitted light, which is used for the
detection of a certain component in the mixture. The series of
treatments is effected by applying centrifugal force with respect
to microchip 100 in an appropriate direction so that a liquid
reagent, specimen or mixture thereof is appropriately and
sequentially distributed to each site in the two layers of fluid
circuits located at each section. Application of centrifugal force
to microchip 100 can be effected by, for example, placing microchip
100 in a centrifuge that has a mounting portion for microchip
100.
[0072] At each of the aforementioned sections, a specimen
quantification unit for quantifying the specimen (a total of six
specimen quantification units 401, 402, 403, 404, 405 and 406 in
FIG. 4) and a liquid reagent quantification unit for quantifying
the liquid reagent (a total of eleven liquid reagent quantification
units 411a, 411b, 412a, 412b, 413a, 413b, 414a, 414b, 415a, 415b
and 416a in FIG. 4) are provided in the lower side fluid circuits.
Each specimen quantification unit is connected in series by a
channel (refer to FIG. 4).
[0073] As shown in FIG. 3, microchip 100 of the present invention
includes an overflow specimen storage unit 330 to store a specimen
overflowing from the specimen quantification unit during
quantification, and overflow reagent storage units 331a, 331b,
332a, 332b, 333a, 333b, 334a, 334b, 335a, 335b and 336a to store a
liquid reagent overflowing from the liquid reagent quantification
unit. Overflow specimen storage unit 330 is connected with specimen
quantification unit 406 via a channel 16a (refer to FIG. 4), a
through hole 26a penetrating in the thickness direction, and a
channel 16b (refer to FIG. 3). Each overflow reagent storage unit
is connected with a corresponding liquid reagent quantification
unit via a channel and a through hole. In section 1, for example,
liquid reagent quantification unit 411a to quantify the liquid
reagent stored in liquid reagent receptacle unit 301a is connected
with overflow reagent storage unit 331a that stores the overflowing
liquid reagent via a channel 11a (refer to FIG. 4), a through hole
21a penetrating in the thickness direction, and a channel 11b
(refer to FIG. 3). The same applies to other overflow reagent
storage units.
[0074] By detecting the absence or presence of overflowing liquid
at a relevant overflow specimen storage unit and overflow reagent
storage unit (hereinafter, also generically referred to as
"overflow liquid storage unit") in microchip 100, identification
can be readily made as to whether a specimen and liquid reagent is
reliably distributed to a specimen quantification unit and liquid
reagent quantification unit, representatively by means of
centrifugal operation, and the relevant specimen quantification
unit or liquid reagent quantification unit is filled with a
specimen or liquid reagent. Namely, detection of the presence of
overflow liquid at the overflow liquid storage unit guarantees that
the specimen or liquid reagent at the specimen quantification unit
or liquid reagent quantification unit has been properly quantified.
Accordingly, the reliability of the examination and/or analysis on
a specimen is improved. If an error in quantification is detected,
determination can be made to not employ the obtained examination
and/or analysis data. An error in quantification includes the case
where a specimen or liquid reagent is not introduced into a
specimen quantification unit or liquid reagent quantification unit
due to an erroneous operation at the device, the case where a
specimen or liquid reagent that should be quantified is not
quantified due to evaporation of the liquid reagent, insufficient
introduction of the specimen due to an erroneous manipulation by
the user, a defect in uniting substrates together in the microchip
fabrication procedure, and the like.
[0075] The method of detecting whether an overflow specimen or
liquid reagent is present or not in an overflow liquid storage unit
preferably includes, though not particularly limited to, the method
of directing light from the side of first substrate 101 that is a
transparent substrate towards the relevant overflow liquid storage
unit, and measuring the intensity of reflected light thereof. The
light to be employed is not particularly limited, and may be
monochromatic light (for example, laser beam) having a wavelength
of 400 to 1000 nm, for example, or mixed light of white light. The
intensity of reflected light can be measured using a
commercially-available reflection sensor.
[0076] The method of detecting the absence or presence of overflow
liquid according to measurement of the intensity of reflected light
basically includes the steps of obtaining the ratio of the
intensity of reflected light identified by directing light from the
first substrate side to the overflow liquid storage unit before
overflow liquid is introduced into the overflow liquid storage unit
to the intensity of reflected light identified by directing light
to the overflow liquid storage unit from the first substrate side
after a specimen or liquid reagent is introduced into the specimen
quantification unit or liquid reagent quantification unit. When the
ratio (reflected light intensity after introduction/reflected light
intensity before introduction) is lower than 1 (i.e. the intensity
of reflected light after introduction is lower), determination is
made that overflow liquid is present in the overflow liquid storage
unit. The measurement of the intensity of reflected light before
introduction of overflow liquid may be skipped in the case where
fabrication variation among microchips is small so that the
intensity of reflected light before introduction of overflow liquid
is assumed to be substantially constant among microchips.
[0077] FIGS. 5A and 5B are diagrams to describe the relationship
between the intensity of incident light and reflected light before
and after overflow liquid is introduced into the overflow liquid
storage unit. FIGS. 5A and 5B corresponds to the case before
introduction and after introduction, respectively. The intensity of
reflected light is represented by the following equation (1):
Reflected light intensity = R 1 I + T 1 2 R 2 1 - R 1 R 2 I + T 1 2
T 2 2 1 - R 1 R 2 R 3 I ( 1 ) ##EQU00001##
[0078] where R.sub.1, R.sub.2 and R.sub.3 are the light
reflectivity and T.sub.1, T.sub.2 and T.sub.3 are the light
transmissivity at the surface of a first substrate 501, at the
interface between first substrate 501 and an overflow liquid
storage unit 510, and at the interface between overflow liquid
storage unit 510 and a second substrate 502, and T.sub.4 is the
light transmissivity of the liquid (or air) in overflow liquid
storage unit 510, when the refractive index of first and second
substrates 501 and 502 is 1.57, and the light beam of incident
intensity I is applied from the side of first substrate 501.
[0079] The first term at the right side in equation (1) corresponds
to the intensity originating from reflected light X shown in FIGS.
5A and 5B (light reflected from the surface of first substrate
501), the second term corresponds to the intensity originating from
reflected light Y (reflection from the interface between first
substrate 501 and overflow liquid storage unit 510), and the third
term corresponds to the intensity originating from reflected light
Z (reflection from the interface between overflow liquid storage
unit 510 and second substrate 502).
[0080] In the case where there is no overflow liquid and only air
is present in overflow liquid storage unit 510 (the event of FIG.
5A), R.sub.1 is calculated to be approximately 0.05. Moreover,
R.sub.1=R.sub.2=R.sub.3 is established, and therefore
T.sub.1=T.sub.2=0.95. By inserting this into the right side of
equation (1), reflected light intensity=2.72.times.R.sub.1I is
obtained.
[0081] In the case where water (refractive index 1.33), for
example, is introduced in overflow liquid storage unit 510 (the
event of FIG. 5B), R.sub.2 is calculated to be 0.0068 (therefore,
T.sub.2=0.9932). By inserting this into the right side of equation
(1) similarly, reflected light intensity=1.24.times.R.sub.1I is
obtained. The aforementioned calculation results indicate that the
ratio of the reflected light intensity after
introduction/reflective light intensity before introduction is
0.45. In view of the reflected light intensity being degraded by
the introduction of overflow liquid in overflow liquid storage unit
510 (in the present example of calculation, a reduction of 55%),
identification of the absence or presence of overflow liquid can be
made readily by identifying such reduction in the reflected light
intensity. In practice, a microchip based on a thermoplastic resin
having the refractive index set forth above was produced and
monochromatic light having a wavelength of 800 nm was applied,
resulting in the ratio of 0.425 with respect to the reflected light
intensity before and after introduction of water.
[0082] In the case where an opaque substrate (for example, black
substrate) is employed for second substrate 502, the third term in
equation (1) (reflectivity at the interface between overflow liquid
storage unit 510 and second substrate 502) is substantially 0.
Therefore, the difference in the reflected light intensity value
before and after overflow liquid introduction will depend only on
the difference in the intensity of the second term (reflectivity
from the interface between first substrate 501 and overflow liquid
storage unit 510). Since the intensity of the second term does not
depend upon the transparency/non-transparency of the overflow
liquid, the absence or presence of overflow liquid can be
identified regardless of whether the overflow liquid is opaque or
not in the case where an opaque substrate (for example, black
substrate) is employed for second substrate 502. In contrast, the
usage of a transparent substrate for second substrate 502 will
afford a contribution to the third term in equation (1), rendering
the measurement of the reflected light intensity complicated. This
will induce the events set forth below. Firstly, there may be a
case where a constant reflected light intensity cannot be obtained
for a plurality of overflow liquid storage units since the
reflected light intensity will vary depending upon the
transmissivity of the fluid (overflow liquid) stored in an overflow
liquid storage unit (the reflected light intensity will depend upon
T.sub.4 as a result of the contribution of the third term, as
indicated in equation (1)). Therefore, a threshold value for
determining the absence or presence of overflow liquid must be
defined for each type of liquid stored in the overflow liquid
storage unit.
[0083] Furthermore, when a transparent substrate is employed for
second substrate 502, the reflected light intensity may vary
depending upon the thickness (depth) of the overflow liquid storage
unit even if the liquid that is the target of detection is opaque
having a constant transmissivity. Therefore, the threshold value
used for determining the absence or presence of fluid liquid must
be determined according to, not only the type of the stored liquid,
but also the thickness (depth) of the overflow liquid storage unit.
In the case where an opaque substrate (for example, black
substrate) is employed for second substrate 502, a constant
reflected light intensity value can be obtained even if an opaque
liquid is stored in the overflow liquid storage unit. Therefore,
the absence or presence of liquid in an overflow liquid storage
unit can be identified using a threshold value identical to that
used in the case of determining the absence or presence of the
overflow liquid for a transparent liquid.
[0084] The technique of identifying whether liquid is present or
not (or absent or not) based on measurement of the reflected light
intensity set forth above can be applied to other sites of
microchip 100, in addition to the overflow liquid storage unit. For
example, light can be directed to a liquid reagent receptacle unit
before actual usage of microchip 100 and measure the reflected
light intensity to allow identification of whether a liquid reagent
is present or not in a liquid reagent receptacle unit. Accordingly,
the failure of a liquid reagent not being stored in the liquid
reagent receptacle unit due to flow out by shock or evaporation
during transportation can be identified. Moreover, light can be
directed to a specimen quantification unit, a liquid reagent
quantification unit, a mixing unit in which a specimen and liquid
reagent are mixed, or the like, and measure the intensity of
reflected light therefrom to reliably identify whether a specimen,
liquid reagent, or mixture thereof is present in a relevant
quantification unit or mixing unit. This can guarantee that a
predetermined treatment through application of centrifugal force is
reliably carried out. In addition, light can be directed to a
liquid reagent quantification unit, mixing unit, and detection unit
during the stage prior to the blood plasma separation and liquid
reagent quantification procedure (for example, immediately before
actual use of microchip 100), and measure the intensity of
reflected light therefrom. Accordingly, identification can be made
as to whether liquid reagent or a specimen is present at these
sites. Thus, the event of an error in which a liquid reagent or
specimen has run to an undesired site due to liquid leakage caused
by a fall during transportation or fabrication failure can be
detected.
[0085] As another method of detecting whether an overflow specimen
or liquid reagent is present in an overflow liquid storage unit,
there is known the method of directing light to the overflow liquid
storage unit and measuring the transmitting light thereof, as
disclosed in the aforementioned U.S. Pat. No. 5,590,052. The
above-described method of measuring reflected light is preferable,
as compared to the method of measuring transmitted light, as will
be described hereinafter.
[0086] (i) An amount of liquid corresponding to the thickness of
the microchip is not required. Therefore, detection with a minute
amount is allowed.
[0087] (ii) The method of measuring reflected light allows the
usage of an opaque substrate, since transparency is required only
at the substrate of the light incident side (for example, the
second substrate in the embodiment set forth above may be an opaque
substrate).
[0088] (iii) Fabrication of a region through which light passes
(optical region) is facilitated, and the configuration of the
optical region can be rendered simple. In other words, the optical
region does not have to be formed spanning over both the layers of
fluid circuits, when provided in the microchip, as in the present
embodiment. Accordingly, the degree of freedom in designing can be
increased. In addition, the area occupied by the optical region can
be reduced. In contrast, if the method of measurement based on
transmitted light is employed, the optical region must be formed at
both layers. Therefore, the area occupied by the optical regions
will be increased, and an additional step of design registration
will be required for the positioning of the optical regions.
[0089] (iv) In the case where an opaque substrate (for example,
black substrate) is employed for second substrate 502 as set forth
above, a constant reflected light intensity value can be obtained
even in the case where opaque liquid is stored in the overflow
liquid storage unit. Therefore, the absence or presence of liquid
in the overflow liquid storage unit can be detected using a
threshold value identical to that used in the case of determining
the absence or presence of the overflow liquid for a transparent
liquid.
[0090] The total of eleven overflow reagent storage units
corresponding to respective liquid reagents and one overflow
specimen storage unit in microchip 100 of the present embodiment
are all preferably formed in the first fluid circuit (upper side
fluid circuits) (refer to FIG. 3). By forming all overflow liquid
storage units at one side fluid circuits, microchip 100 does not
have to be turned over at the time of measuring the reflected light
intensity, allowing the detection of the absence or presence of
overflow liquid at all the storage units readily and rapidly.
Moreover, the overflow liquid storage units are preferably arranged
on the circumference of the same circle in one side fluid circuits
formed at the surface of the second substrate (refer to FIG. 3).
This circle is preferably a circle about the center of the circular
path along with microchip 100 moves such that centrifugal force is
exerted to microchip 100. Specifically, since microchip 100 is
generally mounted on a rotatable circular stage of a centrifuge and
subjected to centrifugal force, it can be said that the circle
about the center of the circular path is a circle about the
revolution center of the circular stage. The arrangement of all the
overflow liquid storage units on the circumference of the same
circle is advantageous in that the light reflected intensity can be
measured by directing light from a fixed light source (an apparatus
having the light source and reflected light intensity measurement
means integrally formed) while the circular stage on which
microchip 100 is mounted is rotated to sequentially locate an
overflow liquid storage unit on the optical axis of the reflected
light. Thus, measurement of reflected light intensity can be
carried out conveniently and rapidly.
[0091] Referring to FIG. 1, a recess 130 (a total of 12 recesses)
is formed on the surface of first substrate 101, at a location
immediately above an overflow liquid storage unit in the first
fluid circuit (upper side fluid circuits). The formation of such a
recess can prevent reduction in the intensity of reflected light,
caused by attachment of a fingerprint, before the overflow liquid
is introduced into the overflow liquid storage unit. Although the
measurement of the reflected light intensity prior to introduction
of overflow liquid can be skipped in the case where the intensity
of reflected light before introduction of overflow liquid can be
assumed to be substantially constant among microchips 100, there is
a possibility of erroneous determination of the absence or presence
of overflow liquid when the reflected light intensity is actually
reduced due to attachment of a finger print. The depth of recess
130 is, but not particularly limited to, approximately 1.1 mm at
most, for example, when the thickness of first substrate 101 is 1.6
mm. In microchip 100 of the present embodiment, a recess is
similarly formed (a total of six) on the surface of first substrate
101 at a location immediately above the optical measurement cuvette
in the first fluid circuit from the standpoint of the same reason
set forth above. However, such a recess is dispensable in the
present invention.
[0092] An example of fluid treatment based on microchip 100 of the
present embodiment will be described hereinafter with reference to
FIGS. 6A to 12B. These drawings represent the state of the liquid
(specimen, liquid reagent, and mixture thereof) at the top face of
second substrate 102 (the surface facing a first substrate) and at
the bottom face (the surface facing the third substrate) of second
substrate 102 during respective procedures in the fluid treatment.
FIGS. 6A, 7A, 8A, 9A, 10A, 11A and 12A represent the state of the
liquid at the top face of the second substrate (first fluid
circuit) whereas FIGS. 6B, 7B, 8B, 9B, 10B, 11B and 12B represent
the state of the liquid at the bottom face of the second substrate
(second fluid circuit). Likewise with FIG. 4, the lower side fluid
circuits of second substrate 102 in FIGS. 6B, 7B, 8B, 9B, 10B, 11B
and 12B is illustrated in a mirror-reversed manner to readily
identify the corresponding relationship with the upper side fluid
circuits in FIGS. 6A, 7A, 8A, 9A, 10A, 11A and 12A. Although the
description set forth below is based on the fluid treatment at the
fluid circuits in section 1, it is to be understood that a similar
treatment is carried out at other sections by referring to the
drawings. Further, although the following description is based on
the case where the specimen is whole blood (as defined before, the
blood plasma component separated from whole blood may also be
referred to as "specimen" hereinafter), the type of specimen is not
limited thereto.
[0093] (1) Hematocyte Separation and Liquid Reagent Quantification
Procedure
[0094] In the present procedure, centrifugal force is applied in
the downward direction in FIGS. 6A and 6B (hereinafter, simply
referred to "downward"; the same applies for FIGS. 7A, 7B to FIGS.
12A, 12B, as well as to other directions) with respect to the
microchip in the state indicated in FIGS. 3 and 4. Accordingly, the
whole blood introduced through specimen inlet 120 (refer to FIG. 1)
of first substrate 101 is delivered to the lower side fluid
circuits via a through hole 20a to enter hematocyte separation unit
420 (refer to FIG. 6B). Whole blood 600 introduced into hematocyte
separation unit 420 is subjected to centrifugation thereat to be
divided into blood plasma components (upper layer) and hematocyte
components (lower layer). The whole blood overflowing from
hematocyte separation unit 420 moves to the upper side fluid
circuits via a through hole 20b to be stored in a waste reservoir
430 (refer to FIG. 6A). By this downward application of centrifugal
force, the liquid reagents in liquid reagent receptacle units 301a
and 301b are shifted via through holes 21b and 21c to reach liquid
reagent quantification units 411a and 411b for quantification
(refer to FIG. 6B). The liquid reagent overflowing from each liquid
reagent quantification unit runs via through holes 21a and 21b to
be stored in overflow reagent storage units 331a and 331b in the
upper side fluid circuits (refer to FIG. 6A). At this stage, a
liquid reagent is present in all overflow reagent storage units
except for overflow reagent storage unit 332b in the case where
there is no fault in the liquid amount in association with the
liquid reagent. The presence of a liquid reagent may be confirmed
by directing light to the liquid reagent receptacle unit, prior to
the present procedure, and measuring the intensity of reflected
light therefrom. In addition, by directing light to the liquid
reagent quantification unit, mixing unit, and detection unit, and
measure the intensity of reflected light therefrom at the stage
prior to the hematocyte separation and liquid reagent measurement
procedure, the absence or presence of a liquid reagent and/or
specimen at respective sites may be identified.
[0095] (2) Specimen Quantification Procedure
[0096] Then, leftward centrifugal force is applied. In response,
the blood plasma component separated at hematocyte separation unit
420 is introduced into specimen quantification unit 401 (also
introduced simultaneously to specimen quantification units 402,
403, 404 as well as to 405 and 406) to be quantified (refer to FIG.
7B). The plasma component overflowing from the quantification unit
is delivered to the upper side fluid circuits via a through hole
26a (refer to FIG. 7A). This leftward centrifugal force causes the
liquid reagent in liquid reagent quantification unit 411a to move
to mixing unit 441a, and the liquid reagent in liquid reagent
quantification unit 411b to channel 12a. At this stage, the
presence of blood plasma components at the specimen quantification
unit may be identified by directing light to each specimen
quantification unit and measuring the intensity of reflected light
therefrom.
[0097] (3) First Mixing Procedure
[0098] Then, downward centrifugal force is applied. In response,
the quantified liquid reagent (liquid reagent stored in liquid
reagent receptacle unit 301a) and the blood plasma component
quantified at specimen quantification unit 401 are mixed at liquid
reagent quantification unit 411a (refer to the first step in the
first mixing procedure of FIG. 8B). At this stage, a liquid reagent
remains in mixing unit 441a at the lower side fluid circuits.
Presence of a mixture at the liquid reagent quantification unit may
be identified at this stage by directing light to each liquid
reagent quantification unit to measure the intensity of light
reflected therefrom. Measurement of the intensity of reflected
light from the overflow specimen storage unit at this stage allows
early detection of a defect such as insufficient introduction of a
specimen. Then, by applying leftward centrifugal force, the mixture
is further mixed with the liquid reagent remaining in mixing unit
441a (refer to the second step in the first mixing procedure in
FIG. 9B). Mixture is ensured by carrying out the first and second
steps for a plurality of times, as necessary. Eventually, a state
similar to that shown in FIGS. 9A and 9B is obtained.
[0099] (4) Second Mixing Procedure
[0100] Then, upward centrifugal force is applied. In response, the
mixture in mixing unit 441a reaches mixing unit 441b via a through
hole 21e, whereas the other quantified liquid reagent (liquid
reagent stored in liquid reagent receptacle unit 301b) reaches
mixing unit 441b via a through hole 21e to be mixed together (refer
to the first step in the second mixing procedure in FIG. 10A).
Confirmation of the presence of mixture at the mixing unit can be
made at this stage by directing light to each mixing unit and
measuring the intensity of reflected light therefrom. Then, by
applying rightward centrifugal force, the mixture is moved within
mixing unit 441b, as shown in FIG. 11A, to promote the mixing
(refer to the second step in the second mixing procedure in FIG.
11A). This rightward centrifugal force also causes the liquid
reagent to be stored in overflow reagent storage unit 332b (refer
to FIG. 11A). Mixture is ensured by carrying out these first and
second steps a plurality of times, as necessary. Eventually, a
state similar to that shown in FIGS. 11A and 11B is obtained.
[0101] (5) Detection Unit Introduction Procedure
[0102] Lastly, downward centrifugal force is applied. In response,
the mixture is introduced into a cuvette (detection unit) 311 for
optical measurement. The same applies to the other mixture (refer
to FIGS. 12A and 12B). In addition, overflow reagent storage units
331a and 331b as well as overflow specimen storage unit 330 has a
liquid reagent or specimen (blood plasma component) stored therein.
The same applies to other overflow reagent storage units. The
mixture in the optical measurement cuvette (detection unit) is
subjected to optical measurement for the examination and analysis
of the specimen (plasma component). Detection and the like of a
certain component in the mixture is carried out by, for example,
directing light from a direction substantially perpendicular to the
surface of microchip 100 and measuring the transmitted light
thereof. Furthermore, light is directed to the overflow specimen
storage unit and each overflow reagent storage unit to measure the
intensity of reflected light therefrom at this stage to confirm the
absence or presence of the specimen or liquid reagent. Although
this confirmation of the presence/absence of the specimen or liquid
reagent does not have to be necessarily carried out at this stage,
it is preferable to confirm the presence/absence of a specimen or
liquid reagent after the detection unit introduction procedure for
the sake of simplifying the operation since it is this stage when
the specimen or liquid reagent should be stored in all the overflow
specimen storage unit and overflow reagent storage units.
[0103] The method of uniting substrates together is not
particularly limited. For example, the method of fusing at least
one of the uniting faces of the substrates for welding (welding
method), the method of attaching using an adhesive, and the like
are known. The welding method includes the method of heating a
substrate for welding, the method of directing a laser beam or the
like to effect welding by the heat generated during light
absorption, and the method of welding based on an ultrasonic wave
can be cited.
[0104] The size of the microchip of the present invention may be
set to, though not particularly limited to, several cm to
approximately 10 cm in the vertical and horizontal directions, and
several millimeters to several centimeters in thickness.
[0105] The material of each substrate set forth above constituting
a microchip of the present invention includes, though not
particularly limited to, an organic material such as polyethylene
terephthalate (PET), polybutylene terephthalate (PBT),
polymethylmethacrylate (PMMA), polycarbonate (PC), polystyrene
(PS), polypropylene (PP), polyethylene (PE),
polyethylenenaphthalate (PEN), polyalylate resin (PAR),
acrylonitrile butadiene styrene resin (ABS), styrene-butadiene
resin (styrene-butadiene copolymer), polyvinyl chloride resin
(PVC), polymethyl pentene resin (PMP), polybutadiene resin (PBD),
biodegradable polymer (BP), cycloolefin (COP), and poly dimethyl
siloxane (PDMS), as well as an inorganic material such as silicon,
glass, and quartz. In consideration of facilitating formation of
fluid circuits, resin is preferable, and styrene-butadiene
copolymer is more preferable. Styrene-butadiene copolymer has the
property of both favorable transparency based on styrene, and
favorable viscosity based on butadiene, and is advantageous in that
the resin can be readily detached from the mold without breakage
while maintaining the shape even in the case where the area of
contact between the resin and mold is extremely large in order to
form minute patterns.
[0106] In the case where the first substrate, second substrate and
third substrate are to be united by welding such as laser welding,
thermal welding, or ultrasonic welding, the melting point or glass
transition point of the resin or resin composition constituting the
second substrate is preferably higher than the melting point or
glass transition point of the resin or resin composition
constituting the second and third substrates. Accordingly,
deformation of grooves on the second substrate during the uniting
procedure can be prevented effectively.
[0107] Each of the first, second, and third substrates may be a
transparent substrate, or an opaque substrate (colored substrate)
such as a black substrate having the substrate base formed of
resin, and adding black pigment such as carbon black into the
resin. Preferably, an opaque substrate such as a black substrate is
used for the second substrate that is located in the middle, and a
transparent substrate is used for the first and third substrates
that sandwich the second substrate. This allows optical measurement
such as by directing light from a direction substantially
perpendicular to the microchip surface to a site where a mixture of
specimen and liquid reagent that is to be subjected to examination
and/or analysis is stored (for example, optical measurement cuvette
(detection unit)) and detecting the intensity of the transmitting
light (transmissivity), as will be described afterwards.
[0108] The method of forming grooves (pattern grooves) constituting
fluid circuits at the surface of the second substrate includes,
though not particularly limited to, injection molding employing a
mold of a transfer configuration, imprinting, and the like. In the
case where the substrate is to be formed using an inorganic
material, an etching method or the like can be employed.
[0109] The sites constituting the fluid circuits are not
particularly limited to those in the microchip of FIG. 1. Each of
the sites such as a liquid reagent receptacle unit to store a
liquid reagent, a separation unit to extract a certain component
from the specimen introduced into the fluid circuits, a specimen
quantification unit to quantify a specimen, a liquid reagent
quantification unit to quantify a liquid reagent, a mixing unit to
mix a specimen and liquid reagent, an optical measurement cuvette
(detection unit) to carry out examination and analysis (for
example, detect or quantify a certain component in the mixture) on
the obtained mixture may be one or more in number. The microchip of
the present invention may include all or at least one of these
exemplified sites. Furthermore, a site other than that set forth
above may be included.
[0110] Although each of the sites set forth above may be arranged
in either the first fluid circuit or the second fluid circuit, it
is preferable to gather the sites formed of deep grooves at one of
the first and second fluid circuits, and the sites formed of
shallow grooves at the other of the first and second fluid
circuits. FIG. 15 is a schematic sectional view of a configuration
of a mold to form a second substrate having a plurality of shallow
grooves at one surface and deep grooves at the other surface. FIG.
16 is a schematic sectional view of a microchip of the present
invention produced based on a second substrate 1602, obtained from
molds 1501a and 1501b of FIG. 15, a first substrate 1601, and a
third substrate 1603. By gathering sites formed of shallow grooves
at one of the first and second fluid circuits, a thin and short end
mill blade can be employed in cutting out a recess in mold 1501a
directed to forming shallow grooves of the second substrate since
the cutting depth of the recess may be shallow, as shown in FIG.
15. Accordingly, the width W of rib 1604 constituting shallow
grooves can be made smaller (refer to FIG. 16). This is
advantageous in that the running out amount of the substrate
material is reduced, so that of deviation in the dimension accuracy
the fluid circuits and variation in the dimension among microchips
can be suppressed. Moreover, by gathering the sites formed of deep
grooves at one of the fluid circuits and the sites formed of
shallow grooves at the other of the fluid circuits,
microfabrication on the mold to be used for producing the second
substrate is facilitated. In addition, the fabrication of the mold
per se is facilitated. Thus, the productivity of the microchip is
improved. Furthermore, since a dead space S that cannot be used for
the fluid circuits will not be generated, the scale of integration
and density of fluid circuits can be increased, as shown in FIG.
17. In the microchip of FIG. 16, the grooves constituting the first
fluid circuit are deeper than the grooves constituting the second
fluid circuit.
[0111] Among the sites set forth above, the specimen quantification
unit and liquid reagent quantification unit are preferably gathered
at the second fluid circuit formed of shallow grooves since they do
not necessarily require a large volume while dimension accuracy of
a more critical level is required. In contrast, the liquid reagent
receptacle unit is a site that requires a large volume, and is
preferably gathered at the first fluid circuit, differing from the
fluid circuits where the specimen quantification unit and liquid
reagent quantification unit are disposed.
[0112] In the case where the microchip of the present invention
includes a liquid reagent receptacle unit, a liquid reagent inlet
that is through holes penetrating to the internal liquid reagent
receptacle unit is generally formed at the microchip surface
(typically, the first substrate surface). Such a microchip
generally has a liquid reagent introduced through the liquid
reagent inlet, and then a label or seal is attached to the surface
of the microchip to close the opening for actual use.
[0113] A microchip has various treatments carried out on the fluid
such as extraction of a certain component from the specimen
(separation of unnecessary component), quantification of a specimen
and/or liquid reagent, mixture of the specimen and liquid reagent,
introduction of the obtained mixture into the optical measurement
cuvette (detection unit) and the like by the sequential application
of centrifugal force in an appropriate direction towards the
microchip. Application of centrifugal force towards a microchip can
be implemented with the microchip mounted on an apparatus (a
centrifuge) that can apply centrifugal force. A centrifuge
includes, for example, a rotatable stage on which a microchip is
placed. Centrifugal force is applied by the rotation of the stage.
The mixture eventually obtained by mixing the specimen and liquid
reagent is subjected to optical measurement, for example, based on
the method of directing light to a site where the mixture is stored
(typically, an optical measurement cuvette (detection unit)) and
detecting the intensity of the transmitting light (transmissivity)
for examination and analysis.
[0114] The optical measurement cuvette can be configured as, though
not particularly limited to, a columnar cavity having a circular or
rectangular cross section extending in, for example, the thickness
direction of the microchip. An optical measurement cuvette formed
of a cavity is connected to one or both of first and second fluid
circuits.
[0115] Since the present invention has two layers of fluid
circuits, there can be provided a microchip that allows increase in
the scale of integration and density of fluid circuits, and that
allows examination and analysis of multiple items despite the
relatively small area.
[0116] Each liquid reagent receptacle unit is connected with a
liquid reagent quantification unit via through holes penetrating
second substrate 102. For example, liquid reagent receptacle unit
301a (refer to FIG. 3) and liquid reagent quantification unit 411a
at section 1 are connected via a through hole 21b. The same applies
to other liquid reagent receptacle units and liquid reagent
quantification units. By providing two layers of fluid circuits
communicating with each other via through holes, the fluid circuits
can be used effectively by the shift between the first and second
fluid circuits, despite the relatively small area for a microchip.
Control of intricate liquid shifting and the like are also
allowed.
[0117] In microchip 100 of the present embodiment, the grooves
provided at second substrate 102 facing first substrate 101 (the
grooves constituting the first fluid circuit) is basically made
deeper than the grooves provided at the surface of second substrate
102 facing third substrate 103 (grooves constituting the second
fluid circuit). Namely, a site and channel where a greater depth is
required is provided at the first fluid circuit whereas a site or
channel where dimension accuracy of a critical level is more
important than the requirement of depth is provided at the second
fluid circuit. Accordingly, mixture of deep grooves and shallow
grooves in one side fluid circuits can be avoided, allowing a
smaller rib width in the formation of a substrate using a mold.
Therefore, leakage of resin at the time of substrate welding can be
prevented, leading to improvement in the dimension accuracy of the
fluid circuits as well as eliminating dimension variation among
microchips. By avoiding the mixture of deep grooves and shallow
grooves within one side fluid circuits, microfabrication on the
mold can be carried out relatively easily, leading to improvement
in the mass production of microchips.
[0118] The site formed of relatively shallow grooves, accommodating
the requirement of preventing/suppressing variation in dimension
accuracy and dimension variation among products, are gathered at
the second fluid circuit. Thus, as compared to the case where both
deep grooves and shallow grooves are provided within one side fluid
circuits, microfabrication on the mold can be carried out easily.
Fabrication of fluid circuits satisfying the required dimension
accuracy is facilitated.
[0119] In the present embodiment, liquid reagent receptacle units
that require a relatively large capacity (relatively large depth)
are gathered at the first fluid circuit, whereas a specimen
quantification unit and liquid reagent quantification unit with the
requirement of preventing/suppressing variation in dimension
accuracy and dimension variation among products are gathered at the
second fluid circuit. By improving the dimension accuracy in the
specimen quantification unit and liquid reagent quantification unit
and preventing/suppressing dimension variation among products, the
quantification accuracy is improved and variation in the
quantification can be suppressed. Therefore, the performance and
reliability of the microchip can be improved. This gathering based
on a configuration accommodating the requirements of such sites and
channels provides equalization of the depth in each fluid circuits.
Accordingly, the occupying ratio of the fluid circuits to the
microchip can be increased, allowing a high scale of integration
and density of the fluid circuits.
[0120] Although a microchip of the present invention and a method
of using the microchip have been described based on a microchip
having two layers of fluid circuits, the microchip of the present
invention may be based having fluid circuits of one layer. Namely,
the microchip may be formed by uniting together a first substrate
that is a transparent substrate and a second substrate having
grooves and/or through holes constituting fluid circuits formed at
one side. The microchip of the present invention does not
necessarily have to be a multi-test chip, and may be a single test
chip that carries out only one type of examination and analysis.
Although the present invention requires only at least one of an
overflow specimen storage unit and overflow reagent storage unit,
preferably both are provided for the purpose of further improving
the reliability of the microchip. The number of the overflow
specimen storage units and overflow reagent storage units are not
particularly limited, and at least one of either the overflow
specimen storage unit or overflow reagent storage unit is to be
provided. However, a storage unit for storing each liquid reagent
and specimen is preferably provided in order to further improve the
reliability of the microchip.
[0121] In a microchip including two layers of the fluid circuits,
the third substrate does not necessarily have to be a transparent
substrate. However, at least the surface region constituting the
detection unit is preferably transparent to allow measurement of
the transmitting light corresponding to the incident light. In the
case where the uniting welding method of directing light to the
uniting faces of substrates for fusion is to be employed as the
method of uniting the first, second and third substrates, the
second substrate is preferably an opaque substrate (preferably, a
black substrate) and the third substrate is preferably a
transparent substrate such that the incident light can be absorbed
more efficiently. This facilitates the uniting of the second and
third substrates by directing light from the third substrate side
to fuse the uniting face of the second substrate. The same applies
to the uniting of the first and second substrates.
[0122] Although the microchip of the present invention has been
described based on a preferable example, the microchip of the
present invention is not limited to the embodiment set forth above.
For example, the microchip of the present invention does not
necessarily have to be a multi-test chip, and may be a single test
chip that carries out only one type of examination and analysis.
Furthermore, all the sites set forth above do not necessarily have
to be included in the present invention. Moreover, one or more of
the type of the sites set forth above may be absent. In addition,
another site not set forth above may be provided. Further, the
number of sites in the microchip is not particularly limited.
[0123] The fluid circuits of the microchip in the present invention
(the first fluid circuit and second fluid circuit) is not limited
to the configuration of the embodiment set forth above, and may
take various configurations. FIGS. 13 and 14 are a top view and a
bottom view, respectively, of a second substrate according to
another example of a microchip of the present invention. FIG. 13
represents the upper side fluid circuits (first fluid circuit) of
the second substrate whereas FIG. 14 represents the lower side
fluid circuits (second fluid circuit).
[0124] The microchip of FIGS. 13 and 14 is a multi-test chip.
Attention is now focused on one section. The first fluid circuit
includes liquid reagent receptacle units 1301a and 1301b, and a
mixing unit 1302a (refer to FIG. 13). The second fluid circuit
includes a specimen quantification unit 1303, liquid reagent
quantification units 1304a and 1304b, a hematocyte separation unit
1305, and a mixing unit 1302b. This microchip also includes an
optical measurement cuvette (detection unit) 1306. As shown in
FIGS. 13 and 14, the microchip of the present invention may have a
configuration and shape of fluid circuits differing from that
described above.
[0125] In the present invention, the third substrate does not
necessarily have to be a transparent substrate. However, at least
the surface region constituting the detection unit is preferably
transparent to allow measurement of the transmitting light
corresponding to the incident light. In the case where the uniting
welding method of directing light to the uniting faces of
substrates for fusion is to be employed as the method of uniting
the first, second and third substrates, the second substrate is
preferably an opaque substrate (preferably, a black substrate) and
the third substrate is preferably a transparent substrate such that
the incident light can be absorbed more efficiently. This
facilitates the uniting of the second and third substrates by
directing light from the third substrate side to fuse the uniting
face of the second substrate. The same applies to the uniting of
the first and second substrates.
Second Embodiment
[0126] The present invention relates to a microchip for a blood
test, including a site for separating a component such as lipid
that is insoluble to the blood plasma component included in the
whole blood sample. The size of the microchip of the present
invention is, though not particularly limited to, several
centimeters in the horizontal and vertical length, and several
millimeters in thickness. The microchip is typically mounted on a
device that can apply centrifugal force thereto for use. By
applying centrifugal force in an appropriate direction to the
microchip, the blood plasma component having lipid and the like
removed from the whole blood sample is extracted, followed by
quantification, mixture, and the like of the blood plasma component
and liquid reagent to detect a certain component in the mixture at
the detection unit.
[0127] The microchip for a blood test of the present invention has
a fluid circuit structure therein. The fluid circuits include,
though not particularly limited to, a blood plasma separation unit
removing hematocytes from the whole blood sample and also removing
suspensions such as lipids to obtain a blood plasma component, a
liquid reagent receptacle unit to store a liquid reagent,
quantification units to quantify each of a liquid reagent and
extracted blood plasma component, a mixing unit to mix the
quantified liquid reagent and blood plasma component, and a
detection unit to analyze and/or examine the obtained mixture.
Other sites are provided, as necessary. There may be two or more
sites in one microchip.
[0128] Each of the sites constituting fluid circuits are disposed
at appropriate positions and connected through minute channels
(hereinafter, also simply referred to as "channel") to sequentially
allow quantification of the blood plasma component and liquid
reagent, mixing of the blood plasma component and liquid reagent,
introduction of the mixture to the detection unit, or the like,
based on externally applied centrifugal force. Examination and
analysis of the mixture at the detection unit (for example,
detection of a certain component in the mixture) is carried out
generally by, but not particularly limited to, optical measurement
such as measuring the absorption spectrum for a mixture stored in a
detection unit, including the steps of directing light to the
detection unit and identifying the intensity of the output
light.
[0129] FIG. 17 is a schematic top view of an example of fluid
circuits configuration of a microchip for a blood test of the
present invention. The microchip of FIG. 17 includes a sample tube
mount unit 1901 for fitting in a sample tube such as a capillary in
which whole blood is collected, a blood plasma separation unit 1902
removing hematocytes and also suspensions such as lipids from the
whole blood extracted from the sample tube to obtain blood plasma
component, a first quantification unit 1903 to quantify the
separated blood plasma component, two liquid reagent receptacle
units 1904a and 1904b to store a liquid reagent, a second
quantification unit 1905a to quantify a liquid reagent, a third
quantification unit 1905b, mixing units 1906a-1906d to mix the
blood plasma component and liquid reagent, and a detection unit
1907 for carrying out examination and/or analysis on the obtained
mixture. The number of liquid reagent receptacle units and mixing
units is not limited to those shown in FIG. 17. In the microchip of
the present invention, the blood plasma separation unit includes a
suspension removal unit to remove suspensions such as lipids.
[0130] FIG. 18 is a schematic enlarged top view of blood plasma
separation unit 1902 in the microchip of FIG. 17. Referring to FIG.
18, blood plasma separation unit 1902 includes a suspension removal
unit 1910 formed of a plurality of columnar structures 1911
arranged in a row in a discrete manner and a suspension storage
unit 1912 to store the removed suspensions such as lipids, a blood
plasma reservoir 1920 to store mainly the separated blood plasma
component, and a hematocyte reservoir 1930 communicating with blood
plasma reservoir 1920 to store mainly the separated hematocytes. A
first channel 1940 to introduce the separated blood plasma
component to first quantification unit 1903 is connected with blood
plasma reservoir 1920. The other end of hematocyte reservoir 1930
is connected to a second channel 1950 to introduce the separated
component mainly constituted of hematocytes to the waste reservoir
unit (waste reservoir 1908 in FIG. 17) that stores the same as
waste.
[0131] The inclusion of suspension removal unit 1910 in blood
plasma separation unit 1902 allows suspensions such as lipids
floating in proximity to the surface of the blood plasma component
(liquid level) to be removed at blood plasma separation unit 1902
during separation of the whole blood sample introduced into blood
plasma separation unit 1902 into the blood plasma component and
hematocyte component by application of centrifugal force.
Therefore, examination and analysis on the mixture can be carried
out at the detection unit without being marred by suspensions,
allowing an accurate examination and analysis on the plasma
component.
[0132] FIG. 19 is a schematic top view of the separated state of
the blood plasma component and hematocyte component from the whole
blood sample, after introduction of the whole blood sample into
blood plasma separation unit 1902 of FIG. 18 and application of
centrifugal force. As shown in FIG. 19, the whole blood sample
introduced into the fluid circuits is guided into blood plasma
separation unit 1902 by the application of the centrifugal force in
the direction of arrow 10 of FIG. 19, further subjected to
centrifugation by the application of centrifugal force in the same
direction, resulting in the separation of the blood plasma
component and hematocyte component. Since the specific gravity of
the hematocyte component is larger than the blood plasma component,
the hematocyte component is stored in hematocyte reservoir 1930,
whereas the blood plasma component is stored mainly in blood plasma
reservoir 1920, as the upper layer to the hematocyte component
layer. The interface between the plasma component layer and the
hematocyte component layer may vary depending upon the content of
the hematocyte component in the whole blood sample. In the case
where the whole blood sample introduced into blood plasma
separation unit 1902 includes suspensions such as lipids, the
centrifugation on the whole blood sample thereat will result in
suspensions 1960 separated by the application of the centrifugal
force to be located in proximity to the surface of the blood plasma
component layer (interface). As used herein, "suspension" is the
substance having a lower specific gravity than the blood plasma
component, insoluble or not readily soluble with respect to the
plasma component, such as lipid.
[0133] The microchip of the present invention is configured such
that the surface of the separated blood plasma component layer at
the side where suspensions are present (interface) is located upper
than the line corresponding to the alignment of columnar structures
1911 (in other words, such that the blood plasma component will
overflow from the alignment line of columnar structures 1911), by
appropriately setting the capacity of blood plasma reservoir 1920
and hematocyte reservoir 1930, and the location of the plurality of
columnar structures 1911 in one row. Accordingly, suspensions 1960
present in proximity to the surface of the blood plasma component
layer separated by the application of centrifugal force in the
direction of arrow 10 in FIG. 19 will be located upper than the
line corresponding to the alignment of one row of columnar
structures 1911.
[0134] The separated suspensions 1960 then move to suspension
storage unit 1912 to be stored therein as a result of application
of centrifugal force in the direction of arrow 20 in FIG. 19. By
the centrifugal force in the direction of arrow 20, the blood
plasma component having the hematocyte component and suspensions
1960 removed are introduced into first quantification unit 1903
(not shown in FIG. 19) via first channel 1940, and the separated
hematocyte component located in hematocyte reservoir 1930 is
introduced into waste reservoir 1908 (not shown in FIG. 19) via
second channel 1950.
[0135] The number of columnar structures 1911 is, though not
particularly limited to, one or more. As shown in FIG. 18, in the
case where a plurality of columnar structures are disposed apart
from each other, the width of the gap therebetween can be set to 50
to 500 gm, for example. The cross section of columnar structure
1911 is not particularly limited, and may be a polygonal cross
section such as a triangle or rectangle, or a circular or optical
cross section. The diameter of the cross section of columnar
structure 1911 can be set to 50 to 500 pm, for example. The
orientation of the cross section of columnar structure 1911, when
taking a triangular shape, is not particularly limited. For
example, columnar structure 1911 may be oriented such that the
width of the opening formed between columnar structures becomes
wider in the downward direction, as shown in FIG. 18, or such that
the width of the opening becomes wider in the upward direction, as
shown in blood plasma separation unit 2002 of FIG. 20. The former
is advantageous in that the flow of the separated suspensions 1960
into first quantification unit 1903 through the gaps of columnar
structures 1911 can be prevented more effectively during the
passage of the blood plasma component to first quantification unit
1903 by application of centrifugal force. The latter is
advantageous in that the flow of the blood plasma component located
lower than the line corresponding to one row of columnar structures
2011 into suspension storage unit 1912 through the gaps of columnar
structures 2011 can be prevented more effectively.
[0136] Further, as shown in blood plasma separation unit 3002 of
FIG. 21, a plurality of discrete columnar structures 3011 may be
arranged to form a plurality of rows. Accordingly, the passage of
the separated suspensions to the first quantification unit and/or
the passage of the blood plasma component located lower than
columnar structures 3011 towards suspension storage unit 1912 can
be prevented more effectively. The cross section of the columnar
structure is not particularly limited in this case.
[0137] The suspension storage unit will be described hereinafter.
The suspension storage unit of the present invention is arranged
upper than the line corresponding to the columnar structures, i.e.
at the surface side where the suspensions of the blood plasma
component are present based on the line of the columnar structures
as the reference, so as to store suspensions present in proximity
to the surface of the blood plasma component. Referring to FIG. 18,
suspension storage unit 1912 is preferably connected to a region
located upper than the line of columnar structures at blood plasma
reservoir 1920.
[0138] First channel 1940 is a path for introduction of the blood
plasma component having suspensions 1960 and the hematocyte
component removed to first quantification unit 1903. First channel
1940 is connected to a region located lower than the line
constituted of columnar structures at blood plasma reservoir
1920.
[0139] Second channel 1950 is a path for introduction of the
separated hematocyte component to the waste reservoir (waste
reservoir 1908 in FIG. 17). The separated hematocyte component in
the hematocyte reservoir 1930 flows into waste reservoir 1908 by
the application of centrifugal force in the direction of arrow 20
in FIG. 19. As shown in blood plasma separation unit 4002 of FIG.
22, hematocyte detection unit 4051 to identify the ratio of the
hematocyte to the whole cell may be provided at second channel
1950. The configuration of hematocyte detection unit 4051 has,
though not particularly limited to, a circular or tubular cross
section. The depth direction of the tube corresponds to the
thickness direction of the microchip. The amount of the hematocyte
component in the whole blood can be identified, taking advantage of
the fact that the transmissivity of light directed to hematocyte
detection unit 4051 differs depending upon the liquid (blood plasma
component or hematocyte component) in hematocyte detection unit
4051. The direction of the radiation light may be parallel with the
thickness direction of the microchip, or parallel with the surface
of the microchip, though not particularly limited.
[0140] An example of an operating method of the microchip of FIG.
17 will be described hereinafter. The operation method that will be
described hereinafter is only a way of example, and not of
limitation. First, a sample tube in which whole blood sample is
collected is fitted in sample tube mount unit 1901. Centrifugal
force is applied to the microchip in the leftward direction in FIG.
17 (hereinafter, simply referred to as leftward; the same applies
to other directions). The whole blood sample in the sample tube is
taken out, and then introduced to blood plasma separation unit 1902
to be subjected to centrifugation by downward centrifugal force,
resulting in the separation into the blood plasma component and
hematocyte component. In the case where suspensions such as lipids
are included in the whole blood sample, such suspensions will also
be separated. Moreover, by the downward centrifugal force, a liquid
reagent X in liquid reagent receptacle unit 1904a is quantified at
second quantification unit 1905a.
[0141] The separated blood plasma component is introduced into
first quantification unit 1903 by the rightward centrifugal force.
At this stage, the separated suspensions move to the suspension
storage unit whereas the separated hematocyte component moves to
waste reservoir 1908. Liquid reagent X subjected to quantification
moves to mixing unit 1906b and a liquid reagent Y in liquid reagent
receptacle unit 1904b is output therefrom.
[0142] In response to the downward centrifugal force, the
quantified blood plasma component and liquid reagent X are mixed at
mixing unit 1906a, and liquid reagent Y is quantified at third
quantification unit 1905b. Then, the centrifugal force is
sequentially applied rightward, downward, and rightward to cause
the mixture to run between mixing units 1906a and 1906b to effect
sufficient mixing of the mixture. Next, in response to the upward
centrifugal force, the mixture of liquid reagent X and the blood
plasma component as well as quantified liquid reagent Y are mixed
at mixing unit 1906c. The centrifugal force is sequentially applied
leftward, upward, leftward and upward to cause the mixture to run
between mixing units 1906c and 1906d to effect sufficient mixing of
the mixture.
[0143] Lastly, by the rightward centrifugal force, the mixture in
mixing unit 1906c is introduced into detection unit 1907 to be
subjected to examination and analysis based on the optical scheme.
This examination and analysis can be carried out accurately since
suspensions such as lipids are removed at blood plasma separation
unit 1902.
[0144] Although the present invention has been described and
illustrated in detail, it is clearly understood that the same is by
way of illustration and example only and is not to be taken by way
of limitation, the scope of the present invention being interpreted
by the terms of the appended claims.
* * * * *