U.S. patent application number 10/464674 was filed with the patent office on 2004-07-29 for analytical chip and analyzer.
Invention is credited to Inami, Hisao, Miyake, Ryo, Sasaki, Yasuhiko.
Application Number | 20040146874 10/464674 |
Document ID | / |
Family ID | 31177774 |
Filed Date | 2004-07-29 |
United States Patent
Application |
20040146874 |
Kind Code |
A1 |
Inami, Hisao ; et
al. |
July 29, 2004 |
Analytical chip and analyzer
Abstract
An analytical chip comprising a substrate, a reaction cell(s)
formed in said substrate, into which a sample collected from a
living body is introduced, a reagent storage portion(s) in which
reagents to be supplied to said reaction cell(s) are stored, and a
reagent nozzle portion(s) constituting a flow path(s) which
connects said reaction cell(s) to said reagent storage portion(s)
and along which said reagents stored in said reagent storage
portion(s) flow into said reaction cell(s).
Inventors: |
Inami, Hisao; (Matsudo,
JP) ; Sasaki, Yasuhiko; (Chiyoda, JP) ;
Miyake, Ryo; (Tsukuba, JP) |
Correspondence
Address: |
ANTONELLI, TERRY, STOUT & KRAUS, LLP
1300 NORTH SEVENTEENTH STREET
SUITE 1800
ARLINGTON
VA
22209-9889
US
|
Family ID: |
31177774 |
Appl. No.: |
10/464674 |
Filed: |
June 19, 2003 |
Current U.S.
Class: |
435/6.12 ;
435/287.2; 435/288.5; 435/288.7 |
Current CPC
Class: |
B01L 2300/1822 20130101;
B01L 2200/16 20130101; B01L 2400/0677 20130101; B01L 3/5027
20130101; B01L 2400/0481 20130101; B01L 2300/0867 20130101; G01N
35/00871 20130101; B01L 2300/023 20130101; B01L 2300/1894
20130101 |
Class at
Publication: |
435/006 ;
435/287.2; 435/288.5; 435/288.7 |
International
Class: |
C12M 001/34; C12Q
001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 21, 2002 |
JP |
2002-180746 |
Claims
What is claimed is:
1. An analytical chip comprising a substrate, a reaction cell(s)
formed in said substrate, into which a sample collected from a
living body is introduced, a reagent storage portion(s) in which
reagents to be supplied to said reaction cell(s) are stored, and a
reagent nozzle portion(s) constituting a flow path(s) which
connects said reaction cell(s) to said reagent storage portion(s)
and along which said reagents stored in said reagent storage
portion(s) flow into said reaction cell(s).
2. An analytical chip comprising a substrate, a reaction cell(s)
formed in said substrate, into which a sample collected from a
living body is introduced, a reagent storage portion(s) in which
reagents to be supplied to said reaction cell(s) are stored, a
reagent nozzle portion(s) constituting a flow path(s) which
connects said reaction cell(s) to said reagent storage portion(s)
and along which said reagents stored in said reagent storage
portion(s) flow into said reaction cell(s), and a fluid-supplying
path(s) which leads to said reagent storage portion(s) and supplies
a fluid for allowing said reagents to flow from said reagent
storage portion(s) to said reagent nozzle portion(s), to said
reagent storage portion(s).
3. An analytical chip comprising a substrate, a reaction cell(s)
formed in said substrate, into which a sample collected from a
living body is introduced, a reagent storage portion(s) in which
reagents to be supplied to said reaction cell(s) are stored, and a
reagent nozzle portion(s) constituting a flow path(s) which
connects said reaction cell(s) to said reagent storage portion(s)
and along which said reagents stored in said reagent storage
portion(s) flow into said reaction cell(s), wherein said flow
path(s) is formed by the formation of a gap(s) extending from said
reagent storage portion(s) to said reaction cell, at least before
the introduction of said sample.
4. An analytical chip according to claim 3, wherein the fine flow
path(s) has a region where the sectional area of the flow path is
15,000 .mu.m.sup.2 or less.
5. An analytical chip according to claim 3, wherein said reagent
storage portion(s) has a region which is deformed by an external
force so as to reduce the capacity of said reagent storage
portion(s).
6. An analytical chip according to claim 2, wherein said
fluid-supplying path is such that its connecting portion to said
reagent storage portion is formed in a region present at a distance
of 80% or more of the distance between the connecting portion of
said reagent nozzle to said reagent storage portion and the
farthest region of the inner wall of said reagent storage
portion.
7. An analytical chip according to claim 2, wherein said substrate
comprises a first substrate and a second substrate formed on one
principal surface of the first substrate; said reagent storage
portion(s) is formed as a region(s) for storing said reagents,
between said first substrate and said second substrate; and at
least a part of said reagent nozzle(s) is formed in a region(s)
outside the region(s) where said reagent storage portion(s) is
formed, in relation to the direction perpendicular to said
principal surface.
8. An analytical chip according to claim 2, wherein said
fluid-supplying path(s) is formed so as to have a capacity larger
than that of said reagent nozzle(s).
9. An analytical chip according to claim 2, wherein said
fluid-supplying path(s) is formed so as to have a minimum flow path
sectional area larger than that of the said reagent nozzle(s).
10. An analytical chip according to claim 2, which comprises a
plurality of said reagent storage portions and a plurality of said
reagent nozzles connecting said reagent storage portions to the
reaction cell(s).
11. An analytical chip according to claim 2, wherein in at least
said reaction cell(s), a reflective plate capable of reflecting
light due to the reaction of the sample introduced into the
reaction cell(s) with each reagent is set in said substrate.
12. An analytical chip according to claim 2, wherein said substrate
comprises an organic material as its main constituent.
13. An analytical chip comprising a substrate, a reaction cell(s)
formed in said substrate, into which a sample collected from a
living body is introduced, a reagent storage portion(s) in which
reagents to be supplied to said reaction cell(s) are stored, a
reagent nozzle portion(s) constituting a flow path(s) which
connects said reaction cell(s) to said reagent storage portion(s)
and along which said reagents stored in said reagent storage
portion(s) flow into said reaction cell(s), and a fluid-supplying
path(s) which leads to said reagent storage portion(s) and supplies
a fluid for allowing said reagents to flow from said reagent
storage portion(s) to said reagent nozzle portion(s), to said
reagent storage portion(s), wherein a plurality of said reagent
storage portions are formed in said substrate and the shortest flow
path among the flow paths from said reagent storage portions to
said reaction cell(s) has a length of 95% or more of the length of
the longest flow path.
14. An analyzer comprising an analytical chip setting portion for
setting therein an analytical chip comprising a reaction cell(s)
into which a sample collected from a living body is introduced, a
reagent storage portion(s) in which reagents to be supplied to said
reaction cell(s) are stored, and a reagent nozzle portion(s)
connecting said reaction cell(s) to said reagent storage
portion(s), a supplying mechanism for supplying a fluid for
discharging said reagents in said reagent storage portion(s) into
said reagent nozzle portion(s), to said reagent storage portion(s)
of said analytical chip, and a detection portion which faces said
reaction cell(s) and detects light emitted in said reaction
cell(s).
15. An analyzer according to claim 14, wherein said analytical chip
setting portion is equipped with a fixing mechanism for fixing said
analytical chip in said analytical chip setting portion.
16. An analytical method comprising a first supplying step in which
a first nucleotide is supplied to a sample containing a
single-stranded DNA, a second supplying step in which a second
nucleotide is supplied to said sample, a first heating step in
which said first nucleotide and said sample are adjusted to a first
temperature higher than room temperature, a step of lowering the
temperature from said first temperature to a second temperature
lower than said first temperature, and a step of adjusting said
second nucleotide and said sample to a third temperature higher
than said second temperature.
17. A method for using an analytical chip comprising a step of
providing an analytical chip comprising a reaction cell(s) into
which a sample collected from a living body is introduced, a
reagent storage portion(s) in which reagents to be supplied to said
reaction cell(s) have been stored, and a reagent nozzle portion(s)
constituting a flow path(s) which connects said reaction cell(s) to
said reagent storage portion(s) and along which said reagents
stored in said reagent storage portion(s) flow into said reaction
cell(s), a step of solidifying the reagents in said reagent storage
portion(s) of said analytical chip provided, by cooling, a step of
conveying said analytical chip in which said reagents solidified
have been stored, a step of heating said analytical chip conveyed,
to melt said reagents, a step of introducing the sample collected
from a living body, into said reaction cell(s) to mix the same with
each of said reagents, and a step of detecting the reaction caused
by this mixing.
18. A method for using an analytical chip comprising a step of
providing an analytical chip comprising a reaction cell(s) into
which a sample collected from a living body is introduced, a
reagent storage portion(s) in which reagents to be supplied to said
reaction cell(s) have been stored, and a reagent nozzle portion(s)
constituting a flow path(s) which connects said reaction cell(s) to
said reagent storage portion(s) and along which said reagents
stored in said reagent storage portion(s) flow into said reaction
cell(s), a step of solidifying the reagents in said reagent storage
portion(s) of said analytical chip provided, by cooling, and a step
of conveying said analytical chip in which said reagents solidified
have been stored.
19. A method for using an analytical chip comprising a step of
providing an analytical chip comprising a reaction cell(s) into
which a sample collected from a living body is introduced, a
reagent storage portion(s) in which reagents to be supplied to said
reaction cell(s) have been stored, and a reagent nozzle portion(s)
constituting a flow path(s) which connects said reaction cell(s) to
said reagent storage portion(s) and along which said reagents
stored in said reagent storage portion(s) flow into said reaction
cell(s), a step of introducing said analytical chip into a cooling
unit to cool the same to a temperature higher than a temperature at
which the reagents in said reagent storage portion(s) of said
analytical chip are solidified, a step of conveying said analytical
chip at a temperature which is lower than the temperature outside
said cooling unit and is higher than a temperature at which said
reagents are solidified, a step of heating said analytical chip
conveyed, a step of introducing the sample collected from a living
body, into said reaction cell(s) to mix the same with each of said
reagents, and a step of detecting the reaction caused by this
mixing.
20. A method for using an analytical chip comprising a step of
providing an analytical chip comprising a reaction cell(s) into
which a sample collected from a living body is introduced, a
reagent storage portion(s) in which reagents to be supplied to said
reaction cell(s) have been stored, and a reagent nozzle portion(s)
constituting a flow path(s) which connects said reaction cell(s) to
said reagent storage portion(s) and along which said reagents
stored in said reagent storage portion(s) flow into said reaction
cell(s), a step of introducing said analytical chip provided, into
a cooling unit to cool the reagents in said reagent storage
portion(s) to a temperature higher than the solidifying points of
said reagents, and a step of conveying said analytical chip at a
temperature which is lower than the temperature outside said
cooling unit and is higher than a temperature at which said
reagents are solidified.
21. An information management center having a database concerning
genetic information which is connected through a network to a
plurality of users who carry out an analysis on the genes of a
sample, which information management center receives information on
the results of said analysis and the ID of each of said users from
the user, transmits evaluation information obtained by evaluating
the thus received analysis result information on the basis of
corresponding information in said database, to the user, and
incorporates said analysis result information into said
database.
22. An information management center having a database concerning
genetic information which is connected through a network to a
plurality of users who carry out an analysis on the genes of a
sample, which information management center has receiving equipment
for receiving information on the results of said analysis, the ID
of each of said users and information on use of an apparatus for
said analysis from the user, and transmitting equipment for
transmitting evaluation information obtained by evaluating the thus
received analysis result information on the basis of corresponding
information in said database, to the user, and which information
management center is connected through a network to a service
provider which utilizes the above use information, transmits said
use information to said service provider, and receives information
on information in the database from said service provider.
23. A service provider that is connected through a network to an
information management center having a database concerning genetic
information which is connected through a network to a plurality of
users who carry out an analysis on the genes of a sample, and that
gives services regarding an apparatus for said analysis, which
service provider receives information on use of the apparatus for
said analysis from said information management center which
receives information on the results of said analysis, the ID of
each of said users and information on use of the apparatus for said
analysis from the user and transmits evaluation information
obtained by evaluating the thus received analysis result
information on the basis of corresponding information in said
database, to the user, and which service provider transmits
information on information in said database to said information
management center.
Description
BACKGROUND OF THE INVENTION
[0001] (1) Field of the Invention
[0002] The present invention relates to an analytical chip for
subjecting a subject to analysis.
[0003] (2) Description of the Related Art
[0004] JP-A-2000-266759 discloses, as an analyzer usable for POC
testing, an analyzer comprising a chip comprising an organic
polymer flat plate having fine grooves for flow of fluids,
respectively, on its surface, and a photothermic conversion
detector for measuring a physical quantity change accompanying a
partial temperature change of a liquid, by irradiating at least a
portion of the liquid in the groove on the chip with exciting
light. In this analyzer, at first, 200 .mu.L of a sample is poured
into a reservoir for sample and 200 .mu.L of a reagent into a
reservoir for reagent. Then, electro-osmosis flows from the
aforesaid reservoir for sample and reservoir for reagent to a
reservoir for waste liquid are caused to send the sample to the
reservoir for waste liquid through a groove connecting the
reservoir for sample to the reservoir for waste liquid and send the
reagent to the reservoir for waste liquid through a groove
connecting the reservoir for reagent to the reservoir for waste
liquid. In this case, since the above-mentioned two kinds of the
grooves meet each other on the upstream side of the reservoir for
waste liquid, the reagent and the sample are mixed in the single
groove formed by the meeting and the reaction goes to completion in
3 to 5 minutes. The sample after completion of the reaction is
irradiated with laser beams, followed by detection by a
photothermic conversion method.
[0005] JP-A-6-197751 discloses a magnifying detector for a nucleic
acid sample containing reagents. JP-A-2001-527220 discloses a
disposable cartridge containing reagents.
[0006] However, in the analyzer disclosed in the above reference,
the reduction of the amount of the reagent supplied for the
reaction is limited because the sample and the reagent react with
each other while flowing in the grooves of the analytical chip.
Moreover, the reaction time is not sufficiently reduced because the
sample and the reagent are mixed while flowing.
[0007] Carrying out a rapid analysis is important in an analyzer
used as follows: a sample is fed to a container (e.g. a substrate)
in which reagents necessary for reaction have been previously
stored, to be reacted with the reagents in the container, and the
container is disposed of after the analysis. In addition, carrying
out the analysis easily with high precision is required in such an
analyzer.
[0008] The present invention provides an analytical chip in which
reagents for reaction are previously stored and which can be
disposed of together with the reagents after analysis, and an
analyzer. The analytical chip and analyzer provided solve the above
problems. The present invention provides, in particular, a
disposable analytical chip that is easy to handle and permits rapid
analysis, and an analyzer equipped with the analytical chip.
SUMMARY OF THE INVENTION
[0009] For the solution of the above problems, the analytical chip
of the present invention may be a disposable analytical chip
comprising a reaction cell(s) in which a sample is stored and the
reaction of said sample with each of reagents individually
introduced into the reaction cell(s) is allowed to proceed; one or
more reagent storage portions for storing the reagents which are
connected to said reaction cell; and a connecting tube(s) (a
nozzle(s)) connecting said reaction cell to said reagent storage
portion(s). The reaction cell(s) may be of either an open type or a
closed type. The number of the reaction cell(s) may be either one
or more.
[0010] Specifically, the analytical chip of the present invention
may have the following structure.
[0011] (1) The analytical chip is formed so that the reaction of a
sample with each reagent is carried out in a sample feed
portion.
[0012] Specifically, the analytical chip is that characterized by
comprising a substrate; a reaction cell(s) formed in said
substrate, into which a sample collected from a living body is
introduced; a reagent storage portion(s) in which reagents to be
supplied to said reaction cell(s) are stored; and a reagent nozzle
portion(s) constituting a flow path(s) which connects said reaction
cell(s) to said reagent storage portion(s) and along which said
reagents stored in said reagent storage portion(s) flow into said
reaction cell(s).
[0013] When as described above, the reaction is carried out in the
portion into which the sample is fed, the amounts of the reagents
can be reduced as compared with the case where a reaction portion
is formed downstream to a portion where each reagent is mixed with
the sample. Furthermore, the reaction can be rapidly carried out.
Therefore, the analytical chip is suitable as a disposal analytical
chip.
[0014] (2) The analytical chip is that having a mechanism for
controlled feed of a flow of each reagent by the use of an external
fluid.
[0015] The analytical chip is, for example, that characterized by
comprising a substrate; a reaction cell(s) formed in said
substrate, into which a sample collected from a living body is
introduced; a reagent storage portion(s) in which reagents to be
supplied to said reaction cell(s) are stored; a reagent nozzle
portion(s) constituting a flow path(s) which connects said reaction
cell(s) to said reagent storage portion(s) and along which said
reagents stored in said reagent storage portion(s) flow into said
reaction cell(s); and a fluid-supplying path(s) which leads to said
reagent storage portion(s) and supplies a fluid for allowing said
reagents to flow from said reagent storage portion(s) to said
reagent nozzle portion(s), to said reagent storage portion(s).
[0016] For example, said fluid-supplying path(s) is equipped with
an introduction portion for introducing said fluid from the
outside.
[0017] (3) The analytical chip is such that reagent storage
portion(s) is connected to a reaction cell(s) by a fine flow
path(s).
[0018] The analytical chip is, for example, that characterized by
comprising a substrate; a reaction cell(s) formed in said
substrate, into which a sample collected from a living body is
introduced; a reagent storage portion(s) in which reagents to be
supplied to said reaction cell(s) are stored; and a reagent nozzle
portion(s) constituting a flow path(s) which connects said reaction
cell(s) to said reagent storage portion(s) and along which said
reagents stored in said reagent storage portion(s) flow into said
reaction cell(s), wherein said flow path(s) is formed by the
formation of a gap(s) extending from said reagent storage
portion(s) to said reaction cell, at least before the introduction
of said sample.
[0019] In particular, the fine flow path(s) preferably has a region
where the sectional area of the flow path is 15,000 .mu.m.sup.2 or
less.
[0020] (4) In any of the above items (1) to (3), the
above-mentioned reagent storage portion(s) may have a region which
is deformed by an external force so as to reduce the capacity of
the reagent storage portion(s), in place of the above-mentioned
mechanism for supplying a fluid from the outside.
[0021] (5) In any of the above items (1) to (4), the
above-mentioned fluid-supplying path is characterized in that its
connecting portion to said reagent storage portion is formed in a
region present at a distance of 80% or more of the distance between
the connecting portion of said reagent nozzle to said reagent
storage portion and the farthest region of the inner wall of said
reagent storage portion.
[0022] (6) In any of the above items (1) to (5), the
above-mentioned substrate comprises a first substrate and a second
substrate formed on one principal surface of the first substrate;
the above-mentioned reagent storage portion(s) is formed as a
region(s) for storing said reagents, between said first substrate
and said second substrate; and at least a part of the
above-mentioned reagent nozzle(s) is formed in a region(s) outside
the region(s) where said reagent storage portion(s) is formed, in
relation to the direction perpendicular to said principal
surface.
[0023] (7) In any of the above items (1) to (6), the
above-mentioned fluid-supplying path(s) is formed so as to have a
capacity larger than that of the above-mentioned reagent
nozzle(s).
[0024] (8) In any of the above items (1) to (7), the
above-mentioned fluid-supplying path(s) is formed so as to have a
minimum flow path sectional area larger than that of the
above-mentioned reagent nozzle(s).
[0025] (9) In any of the above items (1) to (8), the
above-mentioned reagent storage portions and the above-mentioned
reagent nozzles connecting the reagent storage portions to the
reaction cell(s) are provided in numbers, respectively, of two or
more.
[0026] (10) In any of the above items (1) to (9), in at least the
above-mentioned reaction cell(s), a reflective plate capable of
reflecting light due to the reaction of the sample introduced into
the reaction cell(s) with each reagent is set in the
above-mentioned substrate.
[0027] (11) In any of the above items (1) to (10), the
above-mentioned substrate comprises an organic material as its main
constituent.
[0028] (12) In any of the above items (1) to (11), the flow paths
connecting the reaction cell(s) and reagent storage portion(s) of
the analytical chip are preferably similar in length as much as
possible. For example, a plurality of said reagent storage portions
are formed in said substrate and the shortest flow path among the
flow paths from said reagent storage portions to said reaction
cell(s) has a length of 95% or more of the length of the longest
flow path. Owing to such lengths of the flow paths, the control of
the feed rate of a liquid is facilitated, so that a chip can be
provided which permits high-precision detection. The analytical
chip is suitable, for example, in the case of causing a luminous or
fluorescent reaction by the use of a reagent, such as the case of
feeding a sample containing genetic information.
[0029] Alternatively, the analyzer of the present invention
preferably comprises a member for supporting an analytical chip
having at least any of the characteristics described above (this
member preferably has a flow path(s) capable of leading to the flow
path(s) of the analytical chip, and a groove for adsorption); a
fixing mechanism for fixing the analytical chip to said supporting
member so that the chip can be detached; a mechanism (a pump) for
pushing out the reagents in the reagent storage portion(s) into the
reaction cell(s) in the analytical chip by sending a fluid to the
reagent storage portion(s) in the analytical chip through the flow
path(s) of the substrate (i.e., the aforesaid supporting member)
and the flow path(s) of the analytical chip; and a detection
portion for detecting the reaction of the sample stored and each
reagent introduced (the reaction may be detected by employing any
of light emission, fluorescence emission and colorimetry).
[0030] (13) The analyzer is, for example, that comprising an
analytical chip setting portion for setting therein an analytical
chip comprising a reaction cell(s) into which a sample collected
from a living body is introduced, a reagent storage portion(s) in
which reagents to be supplied to said reaction cell(s) are stored,
and a reagent nozzle portion(s) connecting said reaction cell(s) to
said reagent storage portion(s); a supplying mechanism for
supplying a fluid for discharging said reagents in said reagent
storage portion(s) into said reagent nozzle portion(s), to the
reagent storage portion(s) of said analytical chip; and a detection
portion which faces said reaction cell(s) and detects light emitted
in said reaction cell(s).
[0031] (14) In the above item (13), the analytical chip is
preferably held by so-called vacuum chuck.
[0032] For example, said analytical chip setting portion is
equipped with a fixing mechanism for fixing said analytical chip in
said analytical chip setting portion. In addition, it is equipped
with a path leading to a vacuum pump.
[0033] Owing to the analyzer of the present invention, it is
possible to provide a small and portable apparatus that can give
analysis results rapidly and is suitable as an analyzer used in a
medical treatment site, such as an analyzer used in POC testing.
Moreover, an apparatus easy to handle can be formed which is
suitable for use in a routine examination carried out in a place
near a patient, such as a clinic.
[0034] Furthermore, the analyzer is suitable also for a manner of
use in which the chip is replaced with fresh chip and disposed of
for each sample, because the amounts of the reagents used are
slight.
[0035] The analytical method of the present invention preferably
comprises a procedure of heating a reaction cell(s) accommodating a
sample, by means of a temperature control mechanism to heat said
sample to a certain temperature in a short time; a procedure of
detecting light emission caused by the reaction of the sample with
each of reagents supplied, in said reaction cell(s); and a
procedure of cooling the reaction cell(s) to cool said sample to
room temperature in a short time.
[0036] (15) The analytical method comprises a step of lowering the
temperature and then raising the temperature, between steps of
supplying a plurality of nucleotides, respectively.
[0037] Specifically, the analytical method is that characterized by
comprising a first supplying step in which a first nucleotide is
supplied to a sample containing a single-stranded DNA; a second
supplying step in which a second nucleotide is supplied to said
sample; a first heating step in which said first nucleotide and
said sample are adjusted to a first temperature higher than room
temperature; a step of lowering the temperature from said first
temperature to a second temperature lower than said first
temperature; and a step of adjusting said second nucleotide and
said sample to a third temperature higher than said second
temperature.
[0038] It is preferable to add a first detection step in which the
interaction between said first nucleotide and said sample is
detected, after said first supplying step. In addition, it is
preferable to add a second detection step in which the interaction
between said second nucleotide and said sample is detected, after
said second supplying step.
[0039] Said sample may be a sample containing a single-stranded DNA
having a primer bonded thereto. The above-mentioned nucleotides
include nucleotides complementary to the bases constituting said
DNA. Nucleotides of A, T, G and C are preferably supplied in order.
The above-mentioned first or third temperature is preferably
45.degree. C. or lower. As said temperature, a temperature of
25.degree. C. or higher is effective.
[0040] (16) A method for using an analytical chip, characterized by
comprising a step of providing an analytical chip comprising a
reaction cell(s) into which a sample collected from a living body
is introduced, a reagent storage portion(s) in which reagents to be
supplied to said reaction cell(s) have been stored, and a reagent
nozzle portion(s) constituting a flow path(s) which connects said
reaction cell(s) to said reagent storage portion(s) and along which
said reagents stored in said reagent storage portion(s) flow into
said reaction cell(s); a step of solidifying the reagents in said
reagent storage portion(s) of said analytical chip provided, by
cooling; a step of conveying said analytical chip in which said
reagents solidified have been stored; a step of heating said
analytical chip conveyed, to melt said reagents; a step of
introducing the sample collected from a living body, into said
reaction cell(s) to mix the same with each of said reagents; and a
step of detecting the reaction caused by this mixing.
[0041] According to this method, the deterioration of the reagents
can be suppressed and damage to the analytical chip and the flow of
the stored reagents can be reduced even when an external force is
applied during handling (e.g. conveyance) of the analytical chip.
In addition, since an analytical chip can be provided which permits
easy and high-precision analysis, highly reliable analysis can be
carried out. Even in the case of reagents that are rapidly
inactivated, such as many reagents for biochemical analysis,
analysis can be carried out while keeping the reagents highly
active. Moreover, as compared with the case where a user dispenses
the reagents into reagent reservoirs from reagent tanks just before
examination in order to prevent the inactivation of the reagents,
the burden of the dispensation on the user and the possibility of
contamination of the reagents can be easily reduced.
[0042] For example, in the analytical chip, said flow path
preferably has such a shape that a gap extending from said reagent
storage portion to said reaction cell is formed, at least before
the introduction of said sample.
[0043] (17) As to handling of the analytical chip, a method for
using an analytical chip is preferably adopted which is
characterized by comprising a step of providing an analytical chip
comprising a reaction cell(s) into which a sample collected from a
living body is introduced, a reagent storage portion(s) in which
reagents to be supplied to said reaction cell(s) have been stored,
and a reagent nozzle portion(s) constituting a flow path(s) which
connects said reaction cell(s) to said reagent storage portion (s)
and along which said reagents stored in said reagent storage
portion(s) flow into said reaction cell(s); a step of solidifying
the reagents in said reagent storage portion(s) of said analytical
chip provided, by cooling; and a step of conveying said analytical
chip in which said reagents solidified have been stored.
[0044] (18) A method for using an analytical chip may also be
adopted which is characterized in the same manner as in the above
item (16) except for comprising a step of introducing the provided
analytical chip into a cooling unit to cool the same to a
temperature higher than a temperature at which the reagents in said
reagent storage portion(s) of said analytical chip are solidified,
instead of solidifying the reagents; a step of conveying said
analytical chip at a temperature which is lower than the
temperature outside said cooling unit and is higher than a
temperature at which said reagents are solidified; a step of
heating said analytical chip conveyed; a step of introducing a
sample collected from a living body, into said reaction cell(s) to
mix the same with each of said reagents; and a step of detecting
the reaction caused by this mixing.
[0045] Owing to this characteristic, the lowering of activity of
the reagents can be suppressed, so that high-precision and
efficient analysis can be carried out by the use of the analytical
chip containing such reagents stored therein. Particularly when the
reagents include an enzyme, the lowering of activity of the enzyme
can be suppressed by cooling. In addition, it is possible to
suppress a lowering of the activity caused by, for example, a pH
change due to contact with, for instance, a cleaning fluid which
adheres to a dispensing unit in dispensation with the dispensing
unit during examination, by heating the analytical chip containing
the reagents previously stored therein, and mixing the sample with
each of said reagents in the analytical chip.
[0046] In particular, such a method is preferable to the method
involving the solidification because the deformation of the
above-mentioned flow path(s) by cubical expansion at the time of
solidification can be prevented because the former method involves
a step in which the reagents in a liquid state are kept in cold
storage. For example, the temperature at the above-mentioned
cooling can be adjusted to a temperature of not higher than about
10.degree. C. and higher than a temperature at which the reagents
are solidified.
[0047] For example, in the analytical chip, said flow path
preferably has such a shape that a gap extending from said reagent
storage portion to said reaction cell is formed, at least before
the introduction of said sample.
[0048] (19) As to handling of the analytical chip, a method for
using an analytical chip can be adopted which is characterized by
comprising a step of providing an analytical chip comprising a
reaction cell(s) into which a sample collected from a living body
is introduced, a reagent storage portion(s) in which reagents to be
supplied to said reaction cell(s) have been stored, and a reagent
nozzle portion(s) constituting a flow path(s) which connects said
reaction cell(s) to said reagent storage portion (s) and along
which said reagents stored in said reagent storage portion(s) flow
into said reaction cell(s); a step of introducing said analytical
chip provided, into a cooling unit to cool the reagents in said
reagent storage portion(s) to a temperature higher than the
solidifying points of said reagents; and a step of conveying said
analytical chip at a temperature which is lower than the
temperature outside said cooling unit and is higher than a
temperature at which said reagents are solidified.
[0049] (20) An information management center having a database
concerning genetic information which is connected through a network
to a plurality of users who carry out an analysis on the genes of a
sample, said information management center being characterized by
receiving information on the results of said analysis and the ID of
each of said users from the user, transmitting evaluation
informa-tion obtained by evaluating the thus received analysis
result information on the basis of corresponding information in
said database, to the user, and incorporating said analysis result
information transmitted from the user into said database through
the network connecting the user and said information management
center to each other. Transmitting equipment may be installed for
the above transmission. Receiving equipment may be installed for
the reception. Controlling equipment may be installed for the
incorporation.
[0050] (21) In the above item (20), said information management
center receives information on use of an apparatus for the above
analysis, and transmits this use information to a service provider
which utilizes said use information and is connected to the
information management center through a network. Said information
management center receives information on information in the
database from said service provider.
[0051] (22) In at least one of the above items (20) and (21), the
service provider receives information on use of an apparatus for
the above analysis from said information management center, and
transmits information on information in the database to said
service provider.
[0052] By using the analytical chip, analyzer and the like
described above, there can be suitably carried out so-called POC
(Point-of-Care) testing, such as home or bedside examination, or
rapid examination in a place near a patient, such as a common
clinic.
[0053] Specifically, a small and portable analyzer that requires an
analysis time shorter than before can be provided, for example, by
combining the above-mentioned analytical chip having a very small
reaction cell(s) with a simple photodetector. Optimum circumstances
for POC examination can be created by providing a user with a
disposable analytical chip in which reagents have been previously
stored in amounts required for only one run of examination and
which is in a frozen state. Suitable medical services utilizing
genetic information and clinical data can be provided by making
preparations for examination services for POC by using the
analytical chip of the present invention as an essential.
[0054] The present invention makes it possible to provide an
analytical chip which is easy to handle, permits rapid analysis,
permits previous storage of reagents for reaction therein, and can
be disposed of together with the reagents after analysis, and an
analyzer equipped with the analytical chip.
[0055] Other objects, features and advantages of the invention will
become apparent from the following description of the embodiments
of the invention taken in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0056] FIG. 1 is a perspective view showing one example of the
structure of the apparatus described in Example 1.
[0057] FIG. 2 is an enlarged view showing the structure of the
analytical chip shown in FIG. 1.
[0058] FIG. 3 is a cross-sectional view showing the structure of
the pump shown in FIG. 1.
[0059] FIG. 4 is a flow chart showing a procedure for producing the
analytical chip shown in FIG. 1.
[0060] FIG. 5 is a cross-sectional view showing the structure of
the analytical chip shown in FIG. 1.
[0061] FIG. 6 is a graph showing the relationship between a
material for the base plate of an analytical chip and the amount of
emitted light detected.
[0062] FIG. 7 is a flow chart showing a procedure for the analysis
described in Example 1.
[0063] FIG. 8 is a cross-sectional view showing the structure of
the apparatus described in Example 1.
[0064] FIG. 9 is a graph showing the effect of temperature control
on the amount of light emitted.
[0065] FIG. 10 is a schematic view illustrating the combinations of
a DNA sample with a dNTP.
[0066] FIG. 11 is a cross-sectional view showing the structure of
the analytical chip shown in FIG. 2.
[0067] FIG. 12 is an illustration showing POC examination service
using the analytical chip of the present invention as an
essential.
DESCRIPTION OF REFERENCE NUMERALS
[0068] 100 - - - substrate, 101-1, 101-2, 101-3 and 101-4 - - -
pumps, 102-1, 102-2, 102-3 and 102-4 - - - flow paths of substrate,
103 - - - temperature control mechanism, 104 - - - adsorption
groove, 105 - - - pump for vacuum exhaustion, 106 - - - base plate
of analytical chip, 110 - - - analytical chip, 111-1 - - - dATP
vessel, 111-2 - - - dCTP vessel, 111-3 - - - dTTP vessel, 111-4 - -
- dGTP vessel, 112-1, 112-2, 112-3 and 112-4 - - - flow paths of
analytical chip, 113 - - - reaction cell, 114-1 - - - dATP nozzle,
114-2 - - - dCTP nozzle, 114-3 - - - dTTP nozzle, 114-4 - - - dGTP
nozzle, 115 - - - PDMS first layer, 116 - - - PDMS second layer,
120 - - - photodetector, 121 - - - electric source, 122 - - -
recorder, 131 - - - suction opening, 132 - - - discharge opening,
133 - - - diaphragm, 134 - - - pump chamber, 135 - - - suction
valve, 136 - - - discharge valve, 137 - - - actuator, 207 - - -
photosensitive thick-film resist, 208 - - - silicon wafer, 209 - -
- photomask, 210 - - - PDMS, 301 - - - DNA sample, 302 - - -
single-stranded DNA sample, 303 - - - primer, 310 - - - mixed
reagent, 311 - - - dATP, 312 - - - dCTP, 313 - - - dTTP, 314 - - -
dGTP, 400 - - - analytical chip, 401 - - - reaction cell, 402-1,
402-2, 402-3 and 402-4 - - - reagent vessels, 403-1, 403-2, 403-3
and 403-4 - - - reagent nozzles, 404-1, 404-2, 404-3 and 404-4 - -
- actuators, 405 - - - PDMS first layer, 406 - - - PDMS second
layer, 500 - - - service provider, 501 - - - user, 502 - - -
information management center, 503 - - - analyzer, 504 - - -
analytical chip, 505 - - - internet, 506 - - - examination data,
507 - - - diagnosis information, 508 - - - database, 509 - - -
consumption information.
PREFERRED EMBODIMENT OF THE INVENTION
[0069] Examples of analysis of the base sequence of DNA are
explained below as examples of the present invention.
EXAMPLE 1
[0070] (Principle of the Analysis)
[0071] PCTWO No. 98/28440 discloses a method for analyzing the base
sequence beyond a primer of a DNA sample having the primer bonded
thereto, as a method for analyzing the base sequence of DNA.
Specifically, four kinds of deoxynucleotide triphosphates (dNTPs),
i.e., deoxythymine triphosphate (dTTP), deoxyguanidine triphosphate
(dGTP), deoxycytosine triphosphate (dCTP) and deoxyadenine
triphosphate (dATP) which have complementarity (i.e., attraction
between bases A and T or between bases C and G by hydrogen bonding)
to four bases constituting the DNA sample, i.e., adenine (A),
cytosine (C), guanine (G) and thymine (T), respectively, are added
to the DNA sample in regular order. When the dNTP added is bonded
to the DNA sample, pyrophosphate is produced as a by-product.
Therefore, this pyrophosphate is detected by a luminous reaction.
The base sequence of the DNA sample can be determined by adding the
four kinds of dNTPs in regular order to know which dNTP causes
light emission when added. The reaction procedure described above
is broadly divided into the following three stages.
[0072] First Stage
[0073] When the dNTP added is bonded to the DNA sample in the
presence of DNA polymerase (DNA synthetase), the primer is extended
and pyrophosphate (PPi) is liberated from the dNTP.
(DNA)n+dNTP.fwdarw.(DNA)n+1+PPi (1)
[0074] Second Stage
[0075] PPi reacts with adenosine 5'-phosphosulfate (APS) in the
presence of adenosine triphosphate sulfurylase (ATP sulfurylase) to
be converted to adenosine triphosphate (ATP).
PPi+APS.fwdarw.ATP+SO.sub.4.sup.2- (2)
[0076] Third Stage
[0077] The ATP produced reacts with luciferin in the presence of
luciferase to produce oxyluciferin, a luminous substance. The
extension of the primer is analyzed by detecting light emitted by
the oxyluciferin. That is, whether the dNTP added and the DNA
sample are combined or not is judged.
ATP+luciferin+O.sub.2.fwdarw.AMP+PPi+oxyluciferin+CO.sub.2+light
(3)
[0078] The unreacted dNTP and the excess ATP are decomposed by
nucleases such as apyrase.
dNTP.fwdarw.dNDP+PPi.fwdarw.dNMP+PPi (4)
ATP.fwdarw.ADP+PPi.fwdarw.AMP+PPi (5)
[0079] (Structure of an Analyzer)
[0080] The structure of an analyzer is explained with reference to
FIG. 1, FIG. 2 and FIG. 3. FIG. 1 is a perspective view of the
analyzer. FIG. 2 is a detail view of an analytical chip. FIG. 3 is
a cross-sectional view of a pump.
[0081] The analyzer is composed of three constituents, i.e., a
substrate 100, an analytical chip 110 and a photodetector 120 when
broadly divided. The substrate 100 comprises four pumps 101-i (i=1,
2, 3 or 4) for sending a slight volume of a fluid (the volume of
the fluid sent per sending operation is 0.1 .mu.L in the present
example), four flow paths of substrate 102-i (i=1, 2, 3 or 4) along
which the fluid discharged from the pumps flows, a temperature
control mechanism 103 for optimizing the temperature of the
analytical chip 110 which is formed in a region corresponding to a
region where the reaction cell 113 of the analytical chip 110 is
located, and an adsorption groove 104 for adsorbing the analytical
chip 110. In the present example, the numbers of the pumps 101-i
and the flow paths of substrate 102-i are 4 because four kinds of
dNTPs (dATP, dTTP, dGTP and dCTP) are used in the present example,
though these numbers may be changed depending on purpose of the
analysis.
[0082] When Pertier effect is utilized in the temperature control
mechanism 103, either heating or cooling of the analytical chip 110
can easily be conducted merely by changing the direction of an
electric current applied.
[0083] The shape of the pumps 101-i is not particularly limited. In
the present example, the pumps are silicone-made positive
displacement pumps of about 1.5 mm in thickness as shown in FIG. 3.
In each of the pumps, a suction opening 131, a discharge opening
132, a diaphragm 133, a pump chamber 134, a suction valve 135 and a
discharge valve 136 are formed by micro-fabrication. The pumps are
driven by deforming the diaphragm 133 by the use of an external
actuator 137. That is, when the diaphragm 133 is swollen upward,
the pressure inside the pump chamber 134 is reduced. Therefore, the
discharge valve 136 is closed and the suction valve 135 is opened,
so that a liquid is sucked into the pump chamber 134 through the
suction opening 131. When the diaphragm 133 is then depressed
downward, the pressure inside the pump chamber 134 is increased.
Therefore, the suction valve 135 is closed and the discharge valve
136 is opened, so that the liquid is discharged from the pump
chamber 134 through the discharge opening 132.
[0084] The analytical chip 110 to be placed on the substrate 100
has four dNTP vessels 111-i (i=1, 2, 3 or 4) for storing therein
four kinds of dNTPs (dATP, dTTP, dGTP and dCTP), respectively,
i.e., a dATP vessel 111-1, a dCTP vessel 111-2, a dTTP vessel 111-3
and a dGTP vessel 111-4, and four flow paths of analytical chip
112-i (i=1, 2, 3 or 4) for conducting the liquid sent through the
flow paths of the substrate 100 to the dNTP vessels 111-i,
respectively. The analytical chip 110 comprises regions to be
connected to the flow paths of substrate 102-i, a reaction cell
113, and four dNTP nozzles 114-i (i=1, 2, 3 or 4) connecting the
reaction cell 113 to the four dNTP vessels 111-i, i.e., a dATP
nozzle 114-1, a dCTP nozzle 114-2, a dTTP nozzle 114-3 and a dGTP
nozzle 114-4. As described above, the numbers of the flow paths of
analytical chip 112-i, the dNTP vessels 111-i, the dNTP nozzles
114-i and the reaction cell 113 may be changed depending on purpose
of the analysis. Said dNTP nozzles are preferably similar to one
another in length. For example, the shortest nozzle among the dNTP
nozzles preferably has a length of 95% or more of the length of the
longest nozzle. Owing to such lengths of the dNTP nozzles, the
control of the feed rate of the liquid is facilitated, so that the
analysis can be carried out with high precision.
[0085] The analytical chip 110 is placed on the substrate 100 and
the substrate 100 is fixed. The flow paths of analytical chip 112-i
are connected to the flow paths of substrate 102-i. Specifically,
the analytical chip 110 can be adsorbed on the substrate 100 by
evacuating the adsorption groove 104 of the substrate 100 with a
pump for vacuum exhaustion 105. By thus carrying out vacuum chuck,
the flow paths of substrate 102-i and the flow paths of analytical
chip 112-i are certainly connected together to prevent the leakage
of the liquid, and the analytical chip 110 is made easily
detachable from the substrate 100. For making the analytical chip
110 disposable in its use in POC, a method for fixing the
analytical chip 110 by vacuum chuck is very practical.
[0086] The photodetector 120 is located so that the light-receiving
surface of the photodetector 120 faces the reaction cell 113 (for
example, the photodetector 120 is located over the reaction cell
113). As the photodetector 120, a CCD camera, a photomultiplier
tube, a photodiode and the like can be used. The photodiode is
preferable for miniaturizing the apparatus.
[0087] In the embodiment of the present invention, a small portable
analyzer can be provided by placing the analytical chip 110 on the
substrate 100 and combining the simple photodetector 120 with them,
without using a large laser for excitation.
[0088] (Production of the Analytical Chip)
[0089] As a material for the analytical chip 110, a resin excellent
in discardability is preferable to glass that entails high
processing costs and is brittle. Although the kind of the resin is
not particularly limited, a poly(dimethylsiloxane) (PDMS) (Silpot
184, mfd. by Dow Corning Asia Inc.) having the following excellent
characteristics was used in the present example.
[0090] Good biocompatibility (conventional silicone rubber is
physiologically inactive).
[0091] A pattern can be transferred with a precision of submicron
(PDMS has a low viscosity and a high fluidity before curing and
hence infiltrates small parts of complicated shape
satisfactorily).
[0092] Low cost (PDMS is more inexpensive than pyrex, a material
for micro-device).
[0093] Easily discardable by incineration.
[0094] FIG. 4 shows steps for the production of the analytical chip
110 by the use of a resin substrate (the case of using PDMS is
described below as an example). The analytical chip can be shaped
by forming a pattern in conformity with the constituents of the
analytical chip by photolithography, and transferring the pattern
to a resin.
[0095] When broadly divided, a process of the production comprises
[1] formation of each pattern to be transferred to PDMS, [2]
transfer of the pattern to PDMS, and [3] joining of the resulting
PDMS layers to each other.
[0096] [1] Formation of Each Pattern to be Transferred to PDMS
[0097] A micro-pattern is formed through the following steps: a
step of applying a photosensitive thick-film resist 207 (NANOSU-8,
mfd. by Micro. Chem. Inc.) as a material for pattern on a silicon
wafer 208 (step 201), a step of placing a photomask 209 on the
photosensitive thick-film resist 207, followed by light exposure
(step 202), and a step of development (step 3). A process for the
formation is not limited to the above process. The above process is
advantageous in that a curvilinear shape can be formed while
maintaining a rectangular section, by photofabrication by wet
etching.
[0098] [2] Transfer of the Pattern to PDMS
[0099] PDMS 210 is applied on the pattern and heated to be cured
(step 204). A PDMS layer 210 having a depressed pattern is obtained
by the use of the raised micro-pattern (step 205).
[0100] [3] Joining of the PDMS Layers to Each Other
[0101] The surfaces of the thus obtained PDMS layers 210 having the
patterns, respectively, transferred thereto are subjected to oxygen
plasma treatment, and the two PDMS layers 210 are placed one upon
another to be joined together. The strength of joining is so
sufficient that the PDMS layers 210 are broken when joining
portions are tried to be peeled. The PDMS layer may be joined to a
silicon plate or a glass plate. FIG. 5 shows a section of the
analytical chip 110 obtained by the above process. As shown in FIG.
5, the analytical chip 110 is a joined product of a PDMS first
layer 115 (25 mm long, 25 mm wide and 1 mm thick) having the dNTP
nozzles 114-i and reaction cell 113 formed thereon according to a
pattern, and a PDMS second layer 116 (25 mm long, 25 mm wide and 1
mm thick) having the dNTP vessels 111-i, flow paths of analytical
chip 112-i and reaction cell 113 formed thereon according to
another pattern. Needless to say, the analytical chip 110 can be
produced by joining a PDMS layer having the flow paths of
analytical chip 112-i, dNTP vessels 111-i, dNTP nozzles 114-i and
reaction cell 113 formed thereon according to a pattern to a flat
PDMS plate having nothing formed thereon according to a
pattern.
[0102] When the reaction cell 113 formed by the above method is a
through-hole, a plate as base is necessary. As described above, the
base plate of the analytical chip 110 is preferably a material with
a high thermal conductivity because it serves also as a medium
capable of conducting heat from the temperature control mechanism
103 to the reaction cell 113. In addition, when the surface of the
base plate is specular, emitted light due to a reagent in the
reaction cell 113 is reflected from the base plate, so that the
light emission intensity detected by the photodetector 120 over the
reaction cell 113 is increased. FIG. 6 shows the results of
carrying out the luminous reaction of ATP with luciferin in the
presence of luciferase (formula (3)), a final and typical reaction
in DNA analysis, after attaching each of three kinds of materials,
i.e., material A, material B and material C which are different in
reflectance, as the base plate of the analytical chip 110 to the
bottom of the analytical chip 110. When a slice of silicon wafer
was used as material C, the amount of light emitted was
considerably larger than that attained when material A (glass) was
used, owing to the effect of reflection by the specular surface of
the slice. Therefore, a slice of silicon wafer which was good in
thermal conductivity and can easily be joined to PDMS merely by
oxygen plasma treatment was attached as the base plate of the
analytical chip 110. For example, the above-mentioned base plate or
the bottom of the reaction cell 113 is formed so as to have a
reflectance higher than that of the surface of the aforesaid
analytical chip.
[0103] A method for shaping PDMS is not limited to the above
method. PDMS may be processed also by, for example, extrusion.
[0104] The dimensions of the constituents of the analytical chip
110 are as follows. The dNTP vessels 111-i have a length of 10 mm,
a width of 10 mm, a depth of 0.1 mm and a capacity of 10 .mu.L. The
shape of the dNTP vessels 111-i is not particularly limited.
[0105] The flow paths of analytical chip 112-i have a shape of
section with a length of 1 mm and a width of 0.1 mm, and a length
of 2 mm and lead to the dNTP vessels 111-i, respectively. The ends
of the flow paths of analytical chip 112-i are preferably formed
near regions farthest from the edge faces of the dNTP nozzles
114-i, respectively. The ends are formed in regions present at a
distance of 80% or more of the distance to the farthest positions,
respectively, in the dNTP vessels 111-i, respectively. The
formation in such a manner is preferable because it permits smooth
flow and effective use of a liquid. The dNTP nozzles 14-i have a
shape of section with a length of 40 .mu.m and a width 20 .mu.m,
and a length of 4 mm. The leakage of reagents into the reaction
cell 113 from the dNTP vessels 111-i can be effectively prevented
by adjusting the sectional area of the dNTP nozzles 114-i to a
sufficiently low value of 1,000 .mu.m.sup.2 or less. The sectional
area of the dNTP nozzles 114-i is more preferably adjusted to 800
.mu.m.sup.2 or less, still more preferably 500 .mu.m.sup.2 or less.
By thus reducing the sectional area of the dNTP nozzles 114-i
sufficiently, a simple structure can be formed without using a
valve for handling a liquid. As to the lower limit of the sectional
area, the dNTP nozzles 114-i preferably have a sectional area of 80
.mu.m.sup.2 or more for the prevention of pulsation during the
supply of a liquid. Alternatively, the dNTP nozzles 114-i
preferably have a sectional area of 300 .mu.m.sup.2 or more for the
prevention of swelling of air bubbles in the liquid which hinders
sending of the liquid. Considering the conveyance of the analytical
chip in a frozen state, the dNTP nozzles 114-i preferably have a
sectional area of 300 .mu.m.sup.2 or more for the prevention of,
for example, damage to the analytical chip. On the other hand, as
to the upper limit of the sectional area, the dNTP nozzles 114-i
have a sectional area of 15,000 .mu.m.sup.2 or less so that the
liquid can be properly held without a valve. The above-mentioned
values were calculated according to the equation
Pw-Pa+mg/R=.gamma.(2/R) wherein Pw: liquid pressure, Pa: air
pressure, mg/R: the pressure of a drop of water, .gamma.: surface
tension between the liquid and air, and R: the radius of the flow
path. Considering the influence of a change in the volume of the
analytical chip caused by the conveyance of the analytical chip in
a frozen state, the sectional area is preferably 8,000 .mu.m.sup.2
or less. Considering the application of an external force during
the conveyance, the sectional area is preferably 3,000 .mu.m.sup.2
or less. As to the shape of the flow paths, the upper limit or the
lower limit is preferably chosen depending on the purpose from the
above point of view.
[0106] For example, the reaction cell 113 connected to a plurality
of the dNTP vessels 111-i by the dNTP nozzles 114-i, respectively,
has a diameter of 2 mm and a capacity of about 5 .mu.L. The
adjustment of the capacity of the reaction cell 113 to a very low
value of 1 to 5 .mu.L improves the thermal response and permits
rapid control of temperature of the reaction cell 113. Thus, the
reaction can be allowed to proceed under optimum conditions while
varying the temperature of the reaction cell 113 at intervals of
one or more seconds. Moreover, owing to the adjustment of the
capacity of the reaction cell 113 to a very low value, each dNTP
introduced into the reaction cell 113 from the dNTP vessel 111-i is
diffused throughout the reaction cell 113 in a short time (for
example, about 1 second), resulting in an easy mixing operation.
For simplification, employment of no mixing operation is thought
of. In prior art (JP-A-2000-266759), the reaction is detected in 3
to 5 minutes while mixing a sample with a reagent in a flow path.
On the other hand, the reaction can be detected in about 1 second
by batchwise mixing of a sample with a reagent in the reaction cell
113 having a very small capacity of 1 to 5 .mu.L. Adjusting the
capacity of the reaction cell 113 to 1 to 7 .mu.L is also
effective. Also when the capacity is set to 1 to 10 .mu.L, the
adjustment to such a very small value is effective
[0107] (Procedure for the Analysis)
[0108] A procedure for the analysis using the analytical chip 110
is explained below with reference to FIG. 7 and FIG. 8. FIG. 7 is a
flow chart showing the procedure for the analysis. FIG. 8 is a
cross-sectional view of the analyzer.
[0109] At first, a DNA sample is prepared. A primer having 15 to 20
bases is added to a single-stranded DNA sample whose base sequence
is desired to be analyzed, followed by annealing at 95.degree. C.
for 5 minutes and then standing at room temperature for 30 minutes.
Thus, the single-stranded DNA sample and the primer are combined.
In this case, the unbound primer is washed away. Then, a reagent is
prepared by mixing a primer-extending enzyme with a
dNTP-decomposing reagent. As the primer-extending enzyme, Taq DNA
polymerase, Tth DNA polymerase, Vent DNA polymerase or
Thermosequenase may be used. As the dNTP-decomposing reagent, ATP
diphosphatase or ATP pyrophosphatase may be used. A buffer solution
is preferably added to the mixed reagent for the smooth progress of
extension of the primer. As the buffer solution, for example,
Tris-hydrochloric acid as pH buffer solution is preferably used
after mixing with MgCl.sub.2 for forming a complex (dNTP and
Mg.sup.2+) as a substrate for the extension reaction of the primer,
and dithiothreitol for protecting the enzyme protein against
oxidative denaturation (step 211).
[0110] After completion of the preparation of the DNA sample, the
analytical chip 110 which has four kinds of dNTPs previously stored
in the four dNTP vessels 111-i, respectively, and has been
preserved in a frozen state, is thawed at room temperature. When
the analytical chip 110 is provided for a user after previous
storage therein of the dNTPs in amounts required for only one run
of examination, these reagents are not wasted, resulting in an
increased economical benefit, even if the analytical chip 110 is
disposed of after the one run of the examination. Moreover, the
analytical chip 110 saves the user the trouble of dispensing the
dNTPs into the dNTP vessels 111-i, respectively, resulting in not
only time reduction but also the prevention of contamination. In
addition, when the analytical chip 110 is provided in a frozen
state for the user and the user preserves the analytical chip 110
in a frozen state at 0.degree. C., the activity of the dNTPs can be
maintained for half a month. When the analytical chip 110 is
preserved in a frozen state at -20.degree. C., the activity of the
dNTPs can be maintained for half a year or more. Optimum
circumstances for POC examination can be created by thus storing
the reagents in amounts required for only one run of examination in
the disposable analytical chip 110 previously and providing the
analytical chip 110 in a frozen state for the user (step 212).
[0111] Next, the analytical chip 110 is placed on the substrate 100
and it is confirmed that the flow paths of analytical chip 112-i
and the flow paths of substrate 102-i, respectively, communicate
with each other. Thereafter, the adsorption groove 104 is evacuated
to fix the analytical chip 110 to the substrate 100 by vacuum chuck
(step 213).
[0112] The four pumps 101-i are driven to fill the four flow paths
of substrate 102-i and the four flow paths of analytical chip
112-i, respectively, communicating therewith, with a fluid. As the
fluid used, any fluid such as water, an alcohol or air may be used
so long as it does not lower the activity of the dNTPs when brought
into contact with the dNTPs (step 214).
[0113] Then, 5 .mu.L of the DNA sample previously prepared is
poured into the reaction cell 113 from above (step 215).
[0114] Subsequently, the temperature control mechanism 103 is
driven to raise the temperature of the DNA sample in the reaction
cell 113 in a short time through the base plate 106 of the
analytical chip. Several enzymes are contained in the DNA sample
and their activity is increased with a rise of the temperature.
However, when the enzymes are allowed to stand above a certain
temperature for a long period of time, they are denatured to lose
their activity rapidly. Therefore, in the reaction of ATP with
luciferin in the presence of luciferase (formula (3)), i.e., a
final and typical reaction in DNA analysis, temperature control was
carried out which consisted of heating the enzymes to each of
various preset temperatures in a short time, detecting fluorescence
and then cooling the enzymes to room temperature rapidly. FIG. 9
shows the results obtained. When the enzymes were heated to
43.degree. C. in a short time, the amount of light emitted became
twice that attained at 20.degree. C. That is, by raising the
temperature of the DNA sample to about 43.degree. C. in a short
time, the amount of light emitted which is detected in the analysis
can be increased to twice that attained at 20.degree. C. The amount
of light emitted is sufficiently increased also when the enzymes
are heated to 40.degree. C. An amount of light emitted which is
larger than that attained at 20.degree. C. can be attained by
heating the enzymes to 25.degree. C. or higher.
[0115] Since the amount of light emitted is thus increased by
raising the temperature of the DNA sample in a short time, the
value of a luminous signal is increased as compared with background
noises, so that the emitted light can be detected with high
sensitivity (step 216). The temperature of the DNA sample is raised
by at least 5.degree. C. in view of, for example, the activity of
the enzymes. The temperature is raised to at most 45.degree. C.
from the same viewpoint.
[0116] Then, one of the four pumps 101-i, for example, the pump
101-1 is driven to send 0.1 .mu.L of a fluid filling up the flow
path of substrate 102-1 and the flow path of analytical chip 112-1.
By this sending, 0.1 .mu.L of dATP is introduced into the reaction
cell 113 from the dATP vessel 111-1 connected to the flow path of
analytical chip 112-1, through the dATP nozzle 114-1 (step
217).
[0117] When the dATP introduced binds to the DNA sample, light is
emitted within 1 second. Therefore, the combination of the DNA
sample with the dATP can be detected by measuring the emitted light
with a photodetector 120 (step 218).
[0118] Thereafter, the temperature inside the reaction cell 113 is
lowered to room temperature in a short time by operating the
temperature control mechanism 103. This is because the
decomposition of the unbound dATP by the mixed reagent requires
about 50 seconds and hence the enzymes lose their activity if the
temperature inside the reaction cell 113 is maintained at
43.degree. C. during the decomposition (step 219).
[0119] The base sequence of the DNA sample 201 is analyzed by
carrying out the same operations as above for each of dCTP 212,
dTTP 213 and dGTP 214. That is, the temperature inside the reaction
cell 113 is raised to 43.degree. C. in a short time by operating
the temperature control mechanism 103 (step 220).
[0120] Then, 0.1 .mu.L of dCTP is introduced into the reaction cell
113 from the dCTP vessel 111-2 through the dCTP nozzle 114-2 by
driving the pump 101-2 (step 221).
[0121] The combination of the DNA sample with the dCTP is detected
by measuring emitted light with a photodetector 120 (step 222).
[0122] Thereafter, the temperature inside the reaction cell 113 is
lowered to room temperature in a short time by operating the
temperature control mechanism 103 during the decomposition of the
unreacted dCTP by the mixed reagent (step 223).
[0123] The temperature inside the reaction cell 113 is raised to
43.degree. C. in a short time by operating the temperature control
mechanism 103 again (step 224).
[0124] Then, 0.1 .mu.L of dTTP is introduced into the reaction cell
113 from the dTTP vessel 111-3 through the dTTP nozzle 114-3 by
driving the pump 101-3 (step 225).
[0125] The combination of the DNA sample with the dTTP is detected
by measuring emitted light with a photodetector 120 (step 226).
[0126] Thereafter, the temperature inside the reaction cell 113 is
lowered to room temperature in a short time by operating the
temperature control mechanism 103 during the decomposition of the
unreacted dTTP by the mixed reagent (step 227).
[0127] Lastly, the temperature inside the reaction cell 113 is
raised to 43.degree. C. in a short time by operating the
temperature control mechanism 103 again (step 228).
[0128] Then, 0.1 .mu.L of dGTP is introduced into the reaction cell
113 from the dGTP vessel 111-4 through the dGTP nozzle 114-4 by
driving the pump 101-4 (step 229).
[0129] The combination of the DNA sample with the dGTP is detected
by measuring emitted light with a photodetector 120 (step 230).
[0130] Thereafter, the temperature inside the reaction cell 113 is
lowered to room temperature in a short time by operating the
temperature control mechanism 103 during the decomposition of the
unreacted dGTP by the mixed reagent (step 231).
[0131] The above process is repeated for the four kinds of dNTPs to
determine the base sequence of the DNA sample, base by base (step
232).
[0132] Since there is a possibility of combination of dATP with
dTTP or combination of dCTP with dGTP, the dNTPs are preferably
reacted in such an order that dATP and dTTP, or dCTP and dGTP, do
not succeed one another as described above. By employing this
order, contamination of the dNTPs with one another can be
prevented, so that each dNTP can be certainly tried to be combined
with the DNA sample.
[0133] Actual combination of the DNA sample with the dNTPs in the
case of analyzing the base sequence of the DNA sample according to
the above procedure is explained with reference to FIG. 10. FIG. 10
is a schematic view showing the combination of the DNA sample with
the dNTPs.
[0134] A DNA sample 301 is composed of a single-stranded DNA sample
302 and a primer 303 bonded to the single-stranded DNA sample 302.
As shown in FIG. 10(a), the initial state of the DNA sample 301 is
that in the case where the primer 303 is bonded to the
single-stranded DNA sample 302 and the base sequence beyond the
primer 303 of the single-stranded DNA sample 302 is unknown. The
symbols used for expressing the base sequence are as follows; A:
adenine, G: guanine, C: cytosine, and T: thymine.
[0135] When dATP 311 is added to the DNA sample 301 in the
above-mentioned initial state in the presence of the mixed reagent,
dATP 311 is bonded to the DNA sample 301 as shown in FIG. 10(b). By
detecting light emitted in this case, a change in the amount of
light emitted is measured which corresponds to a single base.
Thereafter, the unreacted dATP 311 is decomposed with the mixed
reagent.
[0136] When dCTP 312 is then added in the presence of the mixed
reagent, dCTP 312 is not bonded to the DNA sample 301 as shown in
FIG. 10(c). Therefore, no change in the amount of light emitted is
measured by detecting emitted light. Thereafter, the unreacted dCTP
312 is decomposed with the mixed reagent.
[0137] When dTTP 313 is then added in the presence of the mixed
reagent, two molecules of dTTP 313 are bonded to the DNA sample 301
one after another as shown in FIG. 10(d). By detecting light
emitted in this case, a change in the amount of light emitted is
measured which corresponds to two bases. Thereafter, the unreacted
dTTP 313 is decomposed with the mixed reagent.
[0138] When dGTP 314 is lastly added in the presence of the mixed
reagent, dGTP 314 is bonded to the DNA sample 301 as shown in FIG.
10(e). By detecting light emitted in this case, a change in the
amount of light emitted is measured which corresponds to a single
base. Thereafter, the unreacted dGTP 314 is decomposed with the
mixed reagent.
[0139] The profile of the light emission intensity measured by the
above process indicates the order of one base due to dATP 311, two
bases due to dTTP 313, and one base due to dGTP 314. Therefore, the
base sequence beyond the primer 303 of the single-stranded DNA
sample 301 is found to be TAAC by the above analysis.
[0140] Although the base sequence of DNA is analyzed in the present
example, a wide variety of subjects such as RNA, proteins, allergy,
various antigens, etc. can be analyzed.
[0141] For example, analysis for the infection of a sample
(containing an antigen) with an infectious disease can be carried
out by filling the reaction cell of the analytical chip with the
sample, introducing a reagent (for example, HBs antibody for the
diagnosis of hepatitis B virus disease, or HCV antibody for the
diagnosis of hepatitis C virus disease) into the reaction cell from
the reagent vessel, and detecting the agglutination of the antigen
and the antibody in the reaction cell by means of a
photodetector.
[0142] In addition, analysis for a protein, enzyme or the like in
blood can be carried out by filling the reaction cell with serum,
and introducing the reagent for blood analysis disclosed in
Japanese Patent Application Kohyo No. 9-504732 into the reaction
cell from the reaction vessel, followed by colorimetry with an
absorptiometer.
[0143] As described above, as compared with a method comprising
allowing a sample and a reagent to flow down along a flow path
while mixing them in the flow path, and detecting their reaction in
3 to 5 minutes, the present invention makes it possible to detect
the reaction of a sample with a reagent in a short time (for
example, about 1 second) by batchwise mixing of the sample and the
reagent in the reaction cell 113 having a very small capacity of 5
.mu.L. Moreover, according to the present invention, a small and
portable analyzer can be provided which requires no large laser for
excitation and is obtained merely by placing the analytical chip
110 on the substrate 100 and combining the simple photodetector 120
therewith. Furthermore, optimum circumstances for POC examination
can be created by providing a user with the disposable analytical
chip 110 in which reagents have been previously stored in amounts
required for only one run of examination and which is in a frozen
state.
EXAMPLE 2
[0144] Although in Example 1, the reagents are sent by the use of
the pumps 101-i because the reagents are individually introduced
into the reaction cell 113 many times, the analysis can be easily
carried out by utilizing the change in capacity of the reagent
vessels shown in FIG. 11, when the reagents are individually
introduced once or twice. That is, a reaction cell 401, reagent
vessels 402-i and reagent nozzles 403-i are provided in an
analytical chip 400. Actuators 404-i are located over the reagent
vessels 402-i, respectively. In the production of the analytical
chip 400, reagents are previously introduced into the reagent
vessels before joining a PDMS first layer 405 having the reaction
cell 401 and reagent nozzles 403-i formed therein according to a
pattern, to a PDMS second layer 406 having the reaction cell 401
and reagent vessels 402-i formed therein according to another
pattern, and just after joining the two PDMS layer, the resulting
analytical chip 400 is quickly frozen, whereby the reagents can be
enclosed in the reagent vessels 402-i without being decreased in
activity.
[0145] The reagents are individually introduced into the reaction
cell 401 by depressing the reagent vessels 402-i downward by
driving the actuators 404-i, respectively, located over the reagent
vessels 402-i. Since PDMS is a soft material, the reagent vessels
402-i can be deformed when the thickness of the PDMS first layer
405 is 0.1 to 1 mm.
[0146] Although such an analytical chip is somewhat inferior to
that described in Example 1 in the precision of introduction of the
reagents, an analyzer can be miniaturized because a pump for vacuum
exhaustion required for fixing the analytical chip to a substrate
by vacuum chuck and pumps for sending the reagents are
unnecessary.
EXAMPLE 3
[0147] FIG. 12 shows a service for POC (Point-of Care) using the
analytical chip and analyzer of the present invention. A service
provider 500 provides a user 501 with an analyzer 503 suitable for
POC and a disposable analytical chip 504 to be set in the analyzer
503. In this case, the analytical chip 504 is in a frozen state.
The user 501 can exchange data with an information management
center 502 through an internet 505. That is, the user 501 has
access to the information management center 502 to register user
ID. The user 501 transmits examination data 506 together with the
user ID to the information management center 502 after examination,
whereby the user 501 can obtain diagnosis information 507 based on
the examination data 505 from the information management center
502. On the other hand, the information management center 502 and
the service provider 500 exchange data with each other through the
internet 505. That is, the information management center 502
transmits consumption data 509 concerning the analytical chip 504
consumed by the user 501 and the user ID to the service provider
500 in exchange for a database 508 including genetic analysis
information and clinical data, which the information management
center 502 receives from the service provider 500. After receiving
the above information transmitted, the service provider 500 can
prepare reagents suitable for use situation for each user 501 and
can provide the user 501 rapidly with the analytical chip 504 in
which these reagents have been stored in a frozen state. The user
501 can receive the analytical chip 504 having the prepared
reagents instantly stored therein in a frozen state, and hence can
carry out examination without spending his or her labor for
maintaining the activity of the reagents.
[0148] A specific example of the case where the information
management center 502 has a database is described below.
[0149] The user 501 transmits analysis result information such as
the examination data 506 and the previously registered ID of the
user 501 to the information management center 502. The ID is that
certified, for example, by the user and the information management
center. The information management center 502 transmits the
diagnosis information 507, i.e., evaluation information obtained by
evaluation based on information in said database which corresponds
to the examination data 506 received, to the user 501. The
examination data 506 is incorporated into the database and utilized
for higher-precision diagnoses. Transmitting equipment may be
installed for the above transmission. Receiving equipment may be
installed for the reception. Controlling equipment may be installed
for the incorporation.
[0150] Thus, the information management center 502 can construct a
database consisting of many data samples, by providing evaluation
of the examination data 506 transmitted which is based on the
original database. Therefore, the information management center 502
can carry out higher-precision evaluation. Particularly when a
subject for analysis is such that the tendency of a disease can be
apprehended by accumulation of information on a sample as in the
case of a genetic sequence, etc., the precision can be effectively
improved.
[0151] The above-mentioned analysis regarding genes may be, for
example, analysis regarding a genetic sequence in a specific site.
The database concerning genetic information may be a database
concerning a disease due to a genetic sequence in a specific site.
The evaluation described above may be, for example, the rate of
onset of a disease such as cancer which is determined based on
genetic polymorphism information.
[0152] The analysis described above can be carried out as not only
analysis regarding genes but also analysis regarding other items on
a sample. In the analysis regarding other items, the aforesaid
database concerning genetic information is replaced with a database
concerning, for example, the degree of allergy of an individual
subjected to the analysis and the intensity of the adverse effects
of a drug.
[0153] In addition, the information management center 502 receives
information on use of an apparatus for the above analysis from the
user 501. This use information preferably includes, for example,
information on consumption of the expendable supplies of the
apparatus. Owing to this information, the information management
center 502 can know the degree of consumption of the expendable
supplies of the apparatus for the analysis and the like and hence
can provide the user 501 with effective services.
[0154] The aforesaid information management center receives the
information on use of the apparatus, such as the consumption data
and transmits this use information to the service provider 500
which utilizes said use information. The service provider 500
provides the information management center with a genetic and
clinic database 508.
[0155] Owing to the above previous information exchange between the
information management center 502 and the aforesaid specific
service provider 500, the information management center 502 can
increase information accumulated in its database effectively and
hence can carry out high-precision evaluation.
[0156] Owing to the above previous information exchange between the
service provider 500 and the aforesaid specific information
management center 502, the service provider 500 can collect the
information on use of the apparatus by the user and hence can
provide effective services.
[0157] When preparations for examination services for POC are thus
made by using the disposable analytical chip 504 as an essential,
the user 501 can obtain the diagnosis information 507 while saving
time and labor, and the analytical chip 504 is rapidly
supplied.
[0158] It should be further understood by those skilled in the art
that the foregoing description has been made on embodiments of the
invention and that various changes and modifications may be made in
the invention without departing from the spirit of the invention
and the scope of the appended claims.
* * * * *