U.S. patent application number 14/686366 was filed with the patent office on 2015-08-06 for process for analyzing sample by capillary electrophoresis method.
This patent application is currently assigned to NATIONAL INSTITUTE OF ADVANCED INDUSTRIAL SCIENCE AND TECHNOLOGY. The applicant listed for this patent is ARKRAY, Inc., NATIONAL INSTITUTE OF ADVANCED INDUSTRIAL SCIENCE AND TECHNOLOGY. Invention is credited to Yusuke Nakayama, Yoshihide Tanaka, Shinichi Wakida, Satoshi Yonehara.
Application Number | 20150219595 14/686366 |
Document ID | / |
Family ID | 39157116 |
Filed Date | 2015-08-06 |
United States Patent
Application |
20150219595 |
Kind Code |
A1 |
Tanaka; Yoshihide ; et
al. |
August 6, 2015 |
Process for Analyzing Sample by Capillary Electrophoresis
Method
Abstract
A process for analyzing a sample by a capillary electrophoresis
method is provided that allows for high analytic precision and
reduction in apparatus size, and can be readily carried out by
electrophoresing a complex of a sample and an anionic
group-containing compound in the capillary channel, wherein the
capillary channel includes an A layer that is coated on an inner
wall thereof and a B layer that is coated on the A layer, where the
A and B layers are as described.
Inventors: |
Tanaka; Yoshihide; (Osaka,
JP) ; Wakida; Shinichi; (Osaka, JP) ;
Nakayama; Yusuke; (Kyoto, JP) ; Yonehara;
Satoshi; (Kyoto, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NATIONAL INSTITUTE OF ADVANCED INDUSTRIAL SCIENCE AND
TECHNOLOGY
ARKRAY, Inc. |
Tokyo
Kyoto |
|
JP
JP |
|
|
Assignee: |
NATIONAL INSTITUTE OF ADVANCED
INDUSTRIAL SCIENCE AND TECHNOLOGY
Tokyo
JP
ARKRAY, Inc.
Kyoto
JP
|
Family ID: |
39157116 |
Appl. No.: |
14/686366 |
Filed: |
April 14, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12376739 |
Mar 20, 2009 |
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PCT/JP2007/066751 |
Aug 29, 2007 |
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14686366 |
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Current U.S.
Class: |
204/451 |
Current CPC
Class: |
G01N 27/447 20130101;
G01N 27/44756 20130101 |
International
Class: |
G01N 27/447 20060101
G01N027/447 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 4, 2006 |
JP |
2006-239640 |
Claims
1-18. (canceled)
19. A process for analyzing hemoglobin in a sample by a capillary
electrophoresis method comprising: providing a sample comprising
hemoglobin, preparing a capillary channel for capillary
electrophoresis, and performing electrophoretic separation of a
chemical complex comprising the hemoglobin and a first anionic
compound that is present in a running buffer solution at a pH in a
range of 4.5 to 6 in the capillary channel, wherein the capillary
channel comprises an inner wall coated with an A layer and a B
layer that coats the A layer, wherein the A layer is covalently
bonded to the inner wall and comprises at least one of a nonpolar
polymer and a cationic compound, and the B layer comprises a second
anionic compound, wherein the first anionic compound may be the
same or different from the second anionic compound.
20. The process of claim 19, wherein the nonpolar polymer of the A
layer is a silicone polymer.
21. The process of claim 20, wherein the silicone polymer is
selected from the group consisting of a polysiloxane and a
polysilazane.
22. The process of claim 20, wherein the silicone polymer is
selected from the group consisting of a polydialkylsiloxane, a
polydialkylsilazane, a polyarylsiloxane, a polyarylsilazane, a
polyalkylarylsiloxane, a polydiarylsiloxane, a cyclic siloxane and
a cyclic silazane.
23. The process of claim 19, wherein the cationic compound of the A
layer comprises a silylation agent containing at least one selected
from an amino group and an ammonium group.
24. The process of claim 23, wherein the silylation agent is
selected from the group consisting of
N-(2-diaminoethyl)-3-propyltrimethoxysilane,
aminophenoxydimethylvinylsilane,
3-aminopropyldiisopropylethoxysilane,
3-aminopropylmethylbis(trimethylsiloxy)silane,
3-aminopropylpentamethyldisiloxane, 3-aminopropylsilanetriol,
bis(p-aminophenoxy)dimethylsilane,
1,3-bis(3-aminopropyl)tetramethyldisiloxane,
bis(dimethylamino)dimethylsilane,
bis(dimethylamino)vinylmethylsilane,
bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane,
3-cyanopropyl(diisopropyl)dimethylaminosilane,
(aminoethylaminomethyl)phenethyltrimethoxysilane,
N-methylaminopropyltriethoxysilane, tetrakis(diethylamino)silane,
tris(dimethylamino)chlorosilane and tris(dimethylamino)silane.
25. The process of claim 19, wherein at least one of the first
anionic compound and the second anionic compound is an anionic
polysaccharide.
26. The process of claim 25, wherein the anionic polysaccharide is
at least one selected from the group consisting of a sulfated
polysaccharide, a carboxylated polysaccharide, a sulfonated
polysaccharide and a phosphorylated polysaccharide.
27. The process of claim 26, wherein the sulfated polysaccharide is
chondroitin sulfate.
28. The process of claim 19, wherein the first anionic compound is
chondroitin sulfate.
29. The process of claim 19, wherein the second anionic compound is
chondroitin sulfate.
30. The process of claim 19, wherein the A layer comprises a
nonpolar polymer.
31. The process of claim 19, wherein the A layer comprises a
cationic compound.
32. The process of claim 19, wherein the A layer consists of a
cationic compound.
33. The process of claim 19, wherein the hemoglobin is a glycated
hemoglobin.
34. The process of claim 19, wherein the hemoglobin is HbA1c.
Description
TECHNICAL FIELD
[0001] The present invention relates to a process for analyzing a
sample by a capillary electrophoresis method as well as a capillary
channel and a capillary electrophoresis apparatus that are used for
the process.
BACKGROUND ART
[0002] In the capillary electrophoresis method, ions that have
gathered on the inner wall of a capillary channel are transferred
upon voltage application to generate an electroosmotic flow, which
transfers the sample, and thus electrophoresis is performed. For
the capillary channel, one made of fused silica is used. In this
case, however, adsorption of the sample may prevent a good
electroosmotic flow from being obtained. Accordingly, techniques of
coating the inner walls of capillary channels have been proposed
(Patent Documents 1, 2, 3, and 4). On the other hand, hemoglobin
(Hb) in blood reacts with glucose in the blood to become glycated
Hb. The glycated Hb in the blood reflects the past history of the
blood glucose level in a biological body and therefore is
considered as an index in, for example, diagnosis and treatment of
diabetes. Particularly, glycated beta chain N-terminal valine is
called hemoglobin A1c (HbA1c) and is measured by, for example, a
laboratory test, as an especially important index. Examples of the
method of measuring hemoglobin in blood include an agarose
electrophoresis method, a capillary electrophoresis method, an HPLC
method, an immunization method, an enzymatic method, etc. Among
these, those allowing minute variations such as hemoglobin variants
to be detected are the capillary electrophoresis method and the
HPLC method. On the other hand, an apparatus for analyzing
hemoglobin is required to be reduced in size. With respect to this
point, it is difficult to reduce the size of the whole apparatus in
the HPLC method. On the other hand, the capillary electrophoresis
method allows the size of the whole apparatus to be reduced, with
the apparatus being formed into a microchip.
[0003] However, there is a problem in that the aforementioned
conventional capillary electrophoresis method does not allow
hemoglobin to be analyzed with high precision. In order to solve
this problem, there is a technique in which the inner wall of a
capillary channel is coated with a protein, which then is coated
with polysaccharide (Patent Document 5). However, in this
technique, an operation is required in which the inner wall of a
capillary channel is coated with a protein each time the analysis
is carried out, and therefore there is a problem in that the
analysis becomes complicated. On the other hand, there is a method
in which capillary electrophoresis is carried out with a
zwitterionic type of running buffer that is allowed to contain a
flow inhibitor such as aliphatic diamine, with the inner wall of
the capillary channel not being coated (Patent Document 6).
However, there is a problem in that this method allows variant
hemoglobin to be separated but does not allow hemoglobin A1c to be
separated. These problems apply to the general capillary
electrophoresis method with respect to not only hemoglobin but also
other samples.
[0004] [Patent Document 1] JP 2005-291926 A
[0005] [Patent Document 2] JP 4(1992)-320957 A
[0006] [Patent Document 3] JP 5(1993)-503989 A
[0007] [Patent Document 4] JP 8(1996)-504037 A
[0008] [Patent Document 5] JP 9(1997)-105739 A
[0009] [Patent Document 6] JP 2006-145537 A
DISCLOSURE OF THE INVENTION
[0010] Accordingly, the present invention is intended to provide a
process for analyzing a sample by a capillary electrophoresis
method that allows the apparatus to be reduced in size, allows a
high analytical precision to be obtained, and can be carried out
easily, as well as a capillary channel and a capillary
electrophoresis apparatus that are used for the process.
[0011] In order to achieve the aforementioned object, an analytical
process of the present invention is a process for analyzing a
sample by a capillary electrophoresis method. The process includes
a step of preparing a capillary channel to be used for the
capillary electrophoresis method and step of performing
electrophoretic separation of a complex of a sample and an anionic
group-containing compound that are bonded together, in the
capillary channel, wherein the capillary channel includes an A
layer that is coated on an inner wall thereof and a B layer that is
coated on the A layer.
A layer: a spacer layer formed of at least one selected from the
group consisting of polydiallyldimethylammoniumchloride, a nonpolar
polymer, and a cationic group-containing compound, wherein the
polydiallyldimethylammoniumchloride is coated on the inner wall of
the capillary channel by physical adsorption when the layer
includes the polydiallyldimethylammoniumchloride, and at least one
of the nonpolar polymer and the cationic group-containing compound
is coated on the inner wall of the capillary channel by covalent
bond when the layer includes at least one of the nonpolar polymer
and the cationic group-containing compound B layer: an anionic
layer formed of an anionic group-containing compound
[0012] A capillary channel of the present invention is a capillary
channel for capillary electrophoresis to be used for the analytical
process of the present invention, wherein an A layer is coated on
an inner wall of the capillary channel and a B layer is coated on
the A layer.
A layer: a spacer layer formed of at least one selected from the
group consisting of polydiallyldimethylammoniumchloride, a nonpolar
polymer, and a cationic group-containing compound, wherein the
polydiallyldimethylammoniumchloride is coated on the inner wall of
the capillary channel by physical adsorption when the layer
includes the polydiallyldimethylammoniumchloride, and at least one
of the nonpolar polymer and the cationic group-containing compound
is coated on the inner wall of the capillary channel by covalent
bond when the layer includes at least one of the nonpolar polymer
and the cationic group-containing compound B layer: an anionic
layer formed of an anionic group-containing compound
[0013] A capillary electrophoresis apparatus of the present
invention is a capillary electrophoresis apparatus to be used for
the analytical process of the present invention, wherein the
capillary channel of the present invention is included. The
capillary electrophoresis apparatus of the present invention may be
a microchip electrophoresis apparatus with a reduced size (formed
into a microchip) as described later.
[0014] In the analytical process of the present invention, the use
of a capillary channel including a B layer that is formed via an A
layer fixed to an inner wall thereof can prevent, for example, a
protein in a blood sample, such as hemoglobin, from being adsorbed
by the inner wall of the capillary channel. This makes it possible
to generate a good electroosmotic flow. Furthermore, in the
analytical process of the present invention, since a complex is
generated by bonding a sample and an anionic group-containing
compound, which then is performed electrophoretic separation, a
higher separation efficiency is obtained as compared to the case
where the sample alone is performed electrophoretic separation.
Thus, according to the analytical process of the present invention,
a sample such as hemoglobin can be analyzed in a short time with
high precision. Moreover, since the A layer is fixed firmly to the
inner wall of the capillary channel, once it is formed, it is not
detached therefrom easily, even when being washed, which allows it
to be used repeatedly. Accordingly, in the analytical process of
the present invention, once the A layer is formed, it is not
necessary to form the A layer every time an analysis is carried
out, and thereby the analysis can be carried out easily.
Furthermore, in the present invention, since the capillary
electrophoresis method is employed, it is possible to reduce the
size of the analysis apparatus.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is an electropherogram showing the result of analysis
of hemoglobin in an example of the present invention.
[0016] FIG. 2 is an electropherogram showing the result of analysis
of hemoglobin in another example of the present invention.
[0017] FIG. 3 is an electropherogram showing the result of analysis
of hemoglobin in still another example of the present
invention.
[0018] FIG. 4 is an electropherogram showing the result of analysis
of hemoglobin in yet another example of the present invention.
[0019] FIG. 5 is an electropherogram showing the result of analysis
of hemoglobin in further another example of the present
invention.
[0020] FIG. 6 shows diagrams illustrating the configuration of an
example of the capillary electrophoresis apparatus of the present
invention. FIG. 6(A) is a plan view of the capillary
electrophoresis apparatus of this example, FIG. 6(B) is a sectional
view taken on line I-I shown in FIG. 6(A), and FIG. 6(C) is a
sectional view taken on line II-II shown in FIG. 6(A).
[0021] FIG. 7 shows diagrams illustrating the configuration of
another example of the capillary electrophoresis apparatus of the
present invention.
[0022] FIG. 8 shows diagrams illustrating the configuration of
still another example of the capillary electrophoresis apparatus of
the present invention.
[0023] FIG. 9 shows diagrams illustrating the configuration of yet
another example of the capillary electrophoresis apparatus of the
present invention.
BEST MODE FOR CARRYING OUT THE INVENTION
[0024] In the present invention, the term "running buffer" denotes
a buffer solution (buffer) that is used in an actual separation
process. Preferably, in the analytical process of the present
invention, the B layer is formed on the A layer by contacting with
a solution containing an anionic group-containing compound. In this
state, it is preferable that the solution containing the anionic
group-containing compound is a running buffer containing an anionic
group-containing compound.
[0025] Preferably, in the analytical process of the present
invention, the A layer made of polydiallyldimethylammoniumchloride
is formed on the inner wall of the capillary channel by contacting
with a solution containing the polydiallyldimethylammoniumchloride
channel.
[0026] Preferably, in the capillary channel to be used in the
analytical process of the present invention, a sample is introduced
into the running buffer containing the anionic group-containing
compound, and voltage then is applied across both ends of the
capillary channel to perform electrophoretic separation of a
complex of the sample and the anionic group-containing
compound.
[0027] In the analytical process and the capillary channel of the
present invention, the anionic group-containing compound that forms
the B layer may be the same as or different from the anionic
group-containing compound that forms the complex together with the
sample. Preferably, the anionic group-containing compound is an
anionic group-containing polysaccharide. Examples of the anionic
group-containing polysaccharide include sulfated polysaccharide,
carboxylated polysaccharide, sulfonated polysaccharide, and
phosphorylated polysaccharide. Among them, sulfated polysaccharide
and carboxylated polysaccharide are preferable. The sulfated
polysaccharide is preferably, for example, chondroitin sulfate or
heparin, more preferably chondroitin sulfate. The carboxylated
polysaccharide is preferably alginic acid or a salt thereof (for
instance, sodium alginate). There are seven types of chondroitin
sulfates A, B, C, D, E, H, and K and any of them may be used.
[0028] In the analytical process and the capillary channel of the
present invention, it is preferable that the nonpolar polymer is a
silicone polymer and the cationic group is at least one of an amino
group and an ammonium group.
[0029] In the present invention, it is preferable that the sample
contains hemoglobin.
[0030] The capillary electrophoresis apparatus of the present
invention may include a substrate, a plurality of liquid
reservoirs, and a capillary channel, wherein the plurality of
liquid reservoirs may be formed in the substrate and may be allowed
to communicate with one another through the capillary channel, and
the capillary channel may be the capillary channel of the present
invention. In this case, the substrate has a maximum length, for
example, in the range of 10 to 100 mm, preferably in the range of
30 to 70 mm, a maximum width, for instance, in the range of 10 to
60 mm, and a maximum thickness, for example, in the range of 0.3 to
5 mm. The maximum length of the substrate is the length of the
portion that is longest in the longitudinal direction of the
substrate. The maximum width of the substrate is the length of the
portion that is longest in the direction (width direction)
perpendicular to the longitudinal direction of the substrate. The
maximum thickness of the substrate is the length of the portion
that is longest in the direction (thickness direction)
perpendicular to both the longitudinal direction and the width
direction of the substrate. As described above, the capillary
electrophoresis apparatus of the present invention may be a
microchip electrophoresis apparatus with a reduced size (formed
into a microchip).
[0031] Next, the present invention is described in detail.
[0032] As described above, the capillary channel of the present
invention is provided with the B layer that is formed on the inner
wall thereof via the A layer.
[0033] The material for the capillary channel is not particularly
limited. Examples thereof include glass, fused silica, and plastic.
The inner wall of a capillary channel made of glass or fused silica
usually has negative electric charges. The inner wall of a
capillary channel made of plastic has positive or negative electric
charges depending on the presence or absence and the type of the
polar group contained in the plastic, or is uncharged (nonpolar).
Even in the case of plastic having no polar group, introduction of
a polar group allows it to have electric charges. A commercial
product may be used as the capillary channel made of plastic.
Examples of the capillary channel include those formed of, for
example, polymethylmethacrylate, polycarbonate, polystyrene,
polyethylene, polytetrafluoroethylene (PTFE), and polyether ether
ketone (PEEK). The inner diameter of the capillary channel is, for
example, in the range of 10 to 200 .mu.m, preferably in the range
of 25 to 100 .mu.m. The length of the capillary channel is, for
example, in the range of 10 to 1000 mm.
[0034] The A layer may be formed of one of
polydiallyldimethylammoniumchloride, the nonpolar polymer, and the
cationic group-containing compound, or may be formed of two or more
of them.
[0035] When the A layer is formed on the inner wall of the
capillary channel using polydiallyldimethylammoniumchloride, for
example, a polydiallyldimethylammoniumchloride solution may be
passed through the capillary channel. In a case where the capillary
channel is made of glass or fused silica, the
polydiallyldimethylammoniumchloride is adsorbed firmly to the inner
wall of the capillary channel and thereby the A layer is formed.
The A layer is not detached easily even when being washed. The
concentration of the polydiallyldimethylammoniumchloride solution
is, for example, in the range of 1 to 20 wt % and preferably in the
range of 5 to 10 wt %. Preferably, the alkaline solution is passed
through the capillary channel and then the distilled water is
passed through the capillary channel to wash it before the
polydiallyldimethylammoniumchloride solution is passed
therethrough. An example of the alkaline solution includes, for
example, an aqueous sodium hydroxide. Further, after the
polydiallyldimethylammoniumchloride solution is passed through the
capillary channel, it is preferable that the distilled water is
passed through the capillary channel in order to remove residual
polydiallyldimethylammoniumchloride that was not involved in a
formation of the A layer.
[0036] As described above, the nonpolar polymer that forms the A
layer on the inner wall of the capillary channel is preferably a
silicone polymer. When the A layer is formed using the silicone
polymer, for example, a solution containing the silicone polymer
may be passed through the capillary channel. In a case where the
capillary channel is made of glass or fused silica, the silicone
polymer is fixed firmly to the inner wall of the capillary channel
by a covalent bond and thereby the A layer is formed. The A layer
is not detached easily even when being washed.
[0037] Examples of the silicone polymer include, for example,
polysiloxane and polysilazane. Examples of the polysiloxane and the
polysilazane include, for example, polydiorganosiloxane,
polydiorganosilazane, and polyorganohydrosiloxane. Specific
examples of the polysiloxane and the polysilazane include
polydialkylsiloxane, polydialkylsilazane, polyarylsiloxane,
polyarylsilazane, polyalkylarylsiloxane, polydiarylsiloxane, cyclic
siloxane, and cyclic silazane.
[0038] The solution containing the silicone polymer is, for
example, a dispersed solution containing the silicone polymer
dispersed in a solvent, or a dissolved solution containing the
silicone polymer dissolved in a solvent. After the dispersed
solution or the dissolved solution of the silicone polymer is
passed through the capillary channel, when the solvent is
evaporatively removed by drying, a film layer of the silicone
polymer is formed on the inner wall of the capillary channel. When
it is heated, the silicone polymer is bonded to the inner wall of
the capillary channel made of glass or fused silica by the covalent
bond. For example, the heating treatment preferably is carried out
as follows. First, inert gas is passed through the capillary
channel, in which the film layer of the silicone polymer is formed,
to remove oxygen. In this state, both ends of the capillary channel
are sealed by heating or the like. When the capillary channel in
this state is heated, for example, at 200 to 450.degree. C. for 10
minutes to 12 hours, the silicone polymer is bonded to the inner
wall of the capillary channel by the covalent bond.
[0039] Subsequently, the capillary channel is cooled and the both
ends thereof are opened by cutting or the like, and then unreacted
silicone polymer is removed by washing therein with the solvent. In
this manner, the A layer made of the silicone polymer is formed on
the inner wall of the capillary channel. The A layer made of the
silicone polymer has a thickness of, for example, in the range of
50 to 400 nm and preferably in the range of 100 to 400 nm. A
commercial product may be used as the capillary channel that
includes the A layer formed of the silicone polymer.
[0040] When the A layer is formed on the inner wall of the
capillary channel with the cationic group-containing compound, for
example, a compound containing the cationic group and a reaction
group may be used. In a case where the capillary channel is made of
glass or fused silica, a compound (a silylation agent) including
the cationic group and silicon may be used. Preferable examples of
the cationic group include an amino group and an ammonium group.
Further, a preferable example of the cationic group-containing
compound includes the silylation agent that contains at least one
of the cationic group of amino group and the ammonium group. The
amino group may be a primary amino group, a secondary amino group,
or a tertiary amino group.
[0041] Examples of the silylation agent include
N-(2-diaminoethyl)-3-propyltrimethoxysilane,
aminophenoxydimethylvinylsilane,
3-aminopropyldiisopropylethoxysilane,
3-aminopropylmethylbis(trimethylsiloxy) silane,
3-aminopropylpentamethyldisiloxane, 3-aminopropylsilanetriol,
bis(P-aminophenoxy)dimethylsilane,
1,3-bis(3-aminopropyl)tetramethyldisiloxane,
bis(dimethylamino)dimethylsilane, bis(dimethylamino)
vinylmethylsilane,
bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane,
3-cyanopropyl(diisopropyl)dimethylaminosilane,
(aminoethylaminomethyl)phenethyltrimethoxysilane,
N-methylaminopropyltriethoxysilane, tetrakis(diethylamino)silane,
tris(dimethylamino)chlorosilane, tris(dimethylamino)silane.
[0042] For the silylation agent, one obtained by substituting the
silicon atom with titanium or zirconium may be used. One silylation
agent may be used alone or two or more of them may be used in
combination.
[0043] The A layer is formed using the silylation agent, for
example, as follows. First, a silylation agent is dissolved or
dispersed in an organic solvent and thereby a treatment liquid is
prepared. The organic solvent to be used for preparing the
treatment liquid can be, for example, dichloromethane or toluene.
The concentration of the silylation agent of the treatment liquid
is not particularly limited. This treatment liquid is passed
through a capillary channel made of glass or fused silica and is
heated. This heating allows the silylation agent to be bonded to
the inner wall of the capillary channel by a covalent bond. As a
result, the cationic group is placed on the inner wall of the
capillary channel. Thereafter, it is washed with at least one of an
organic solvent (for instance, dichloromethane, methanol, or
acetone), an acid solution (for example, phosphoric acid), an
alkaline solution, and a surfactant solution (aftertreatment).
Preferably, this washing is carried out, although it is optional. A
commercial product may be used as the capillary channel that
includes the A layer formed of the silylation agent.
[0044] Next, the B layer is formed on the A layer with the anionic
group-containing compound. The B layer may be formed on the A layer
by contacting with the solution containing the anionic
group-containing compound. In this case, a solution for forming the
B layer may be prepared separately, however, from an aspect of the
operation efficiency, it is preferable that a running buffer
containing the anionic group-containing compound is prepared and is
passed through the capillary channel that is provided with the A
layer.
[0045] The running buffer is not particularly limited, but a buffer
containing acid used therein is preferred. Examples of the acid
include maleic acid, tartaric acid, succinic acid, fumaric acid,
phthalic acid, malonic acid, and malic acid. Preferably, the
running buffer contains a weak base. Examples of the weak base
include arginine, lysine, histidine, and tris. The running buffer
has a pH, for example, in the range of 4.5 to 6. The types of the
buffer of the running buffer include MES, ADA, ACES, BES, MOPS,
TES, and HEPES. In the running buffer, the anionic group-containing
compound has a concentration, for example, in the range of 0.01 to
5 wt %.
[0046] The analytical process of the present invention can be
carried out with respect to, for example, a sample containing
hemoglobin as follows.
[0047] The analytical process using the capillary channel that
includes the A layer formed of polydiallyldimethylammoniumchloride
is explained. First, a capillary channel made of glass or fused
silica is prepared. Next, the alkaline solution such as an aqueous
sodium hydroxide is passed through the capillary channel under
pressure applied by, for example, a pump.
[0048] Subsequently, the distilled water is passed through the
capillary channel and thereby it is washed. The time for which each
of the alkaline solution and the distilled water is passed
therethrough is respectively, for example, 1 to 10 minutes, and the
pressure when each of the alkaline solution and the distilled water
is passed therethrough is respectively, for example, 0.05 to 0.1
MPa. Next, a polydiallyldimethylammoniumchloride solution is passed
through the capillary channel under pressure applied by, for
example, a pump. The time for which the
polydiallyldimethylammoniumchloride solution is passed therethrough
is, for example, 5 to 30 minutes, and the pressure when the
polydiallyldimethylammoniumchloride solution is passed therethrough
is, for example, 0.05 to 0.1 MPa. Then, the distilled water is
passed through the capillary channel under pressure applied by, for
example, a pump to remove the residual
polydiallyldimethylammoniumchloride. The time for which the
distilled water is passed therethrough and the pressure when the
distilled water is passed therethrough is as same as in the case of
the aforementioned washing. In this manner, the A layer made of the
polydiallyldimethylammoniumchloride is formed on the inner wall of
the capillary channel. In this state, the time and the pressure are
determined suitably according to an inner diameter and a length of
the capillary channel. Each time and pressure mentioned above is an
example which is preferable for the capillary channel that has the
inner diameter of 50 .mu.m and the length of 320 mm. The same
applies to the following.
[0049] Next, a running buffer containing an anionic
group-containing compound such as chondroitin sulfate is passed
through the capillary channel under pressure applied by, for
example, a pump. The time for which the running buffer is passed
therethrough is, for example, 10 to 60 minutes, and the pressure
when the running buffer is passed therethrough is, for example,
0.05 to 0.1 MPa. As a result, a B layer formed of chondroitin
sulfate is coated on the A layer. In this state, a
hemoglobin-containing sample is introduced into the capillary
channel, and voltage then is applied across both ends of the
capillary channel to carry out electrophoresis. The
hemoglobin-containing sample is not particularly limited and is,
for example, a sample obtained by hemolyzing whole blood. This
sample may be diluted with distilled water or a running buffer. The
hemoglobin-containing sample is introduced from the anode side of
the capillary channel. The hemoglobin thus introduced forms a
complex by being bonded with the anionic group-containing compound
contained in the running buffer. The applied voltage generates an
electroosmotic flow in the running buffer contained in the
capillary channel and thereby the complex is transferred toward the
cathode side of the capillary channel. The voltage applied is, for
example, in the order of 5 to 30 kV. This transfer is detected by
an optical method. The detection made by the optical method is not
particularly limited. Preferably, it is carried out at a wavelength
of 415 nm.
[0050] The analytical process using the capillary channel that
includes the A layer formed of at least one of the nonpolar polymer
and the cationic group-containing compound can be carried out in
the same manner as described above except that the capillary
channel that includes the A layer formed in the aforementioned
manner is prepared.
[0051] In the present invention, the hemoglobin to be analyzed is
not particularly limited. Examples thereof include normal
hemoglobin, glycated hemoglobin (for instance, HbA1c, labile HbA1c,
and GHbLys), and hemoglobin variants. In the present invention, it
is possible to separate HbA1c and hemoglobin other than that from
each other to analyze them.
[0052] Next, the capillary electrophoresis apparatus of the present
invention is described using examples. However, the capillary
electrophoresis apparatus of the present invention is not limited
to the following examples.
[0053] FIG. 6 shows an example of the capillary electrophoresis
apparatus according to the present invention. FIG. 6(A) is a plan
view of the capillary electrophoresis apparatus of this example,
FIG. 6(B) is a sectional view taken on line I-I shown in FIG. 6(A),
and FIG. 6(C) is a sectional view taken on line II-II shown in FIG.
6(A). In those figures, for ease of understanding, for example, the
sizes and ratios of the respective components are different from
actual ones. The capillary electrophoresis apparatus of this
example is a microchip electrophoresis apparatus with a reduced
size (formed into a microchip). As shown in the figures, this
microchip electrophoresis apparatus includes a substrate 1, a
plurality (four in this example) of liquid reservoirs 2a to 2d, and
four capillary channels 3x1, 3x2, 3y1, and 3y2. All of the four
capillary channels are capillary channels of the present invention.
The four liquid reservoirs 2a to 2d include a first introduction
reservoir 2a, a first recovery reservoir 2b, a second introduction
reservoir 2c, and a second recovery reservoir 2d. In the four
capillary channels, first ends thereof meet at the central portion
c to be joined together in a cross shape. Accordingly, the four
capillary channels communicate with one another at their inner
parts. The substrate 1 is provided with a cavity for inserting the
four capillary channels thereinto (not shown in the figures). The
capillary channel 3x1 is inserted into the substrate 1 so that the
other end thereof is located at the bottom surface of the first
introduction reservoir 2a. The capillary channel 3x2 is inserted
into the substrate 1 so that the other end thereof is located at
the bottom surface of the first recovery reservoir 2b. The
capillary channels 3x1 and 3x2 form a capillary channel 3x for
sample analysis. The capillary channel 3y1 is inserted into the
substrate 1 so that the other end thereof is located at the bottom
surface of the second introduction reservoir 2c. The capillary
channel 3y2 is inserted into the substrate 1 so that the other end
thereof is located at the bottom surface of the second recovery
reservoir 2d. The capillary channels 3y1 and 3y2 form a capillary
channel 3y for sample introduction. The plurality of liquid
reservoirs 2a to 2d each are formed as a concave part in the
substrate 1. The substrate 1 has a rectangular parallelepiped
opening (window) 9 on the first recovery reservoir 2b side with
respect to the capillary channel 3y for sample introduction. The
microchip electrophoresis apparatus of this example is rectangular
parallelepiped. However, the present invention is not limited
thereto. The microchip electrophoresis apparatus of the present
invention may have any shape as long as it does not cause any
problems in the electrophoresis measurement. The planar shape of
the microchip electrophoresis apparatus of this example is
rectangular. However, the present invention is not limited thereto.
The planar shape of the microchip electrophoresis apparatus of the
present invention may be, for example, square or another shape. In
the microchip electrophoresis apparatus of this example, the
capillary channel 3x for sample analysis is different in maximum
length from the capillary channel 3y for sample introduction.
However, the present invention is not limited thereto. In the
microchip electrophoresis apparatus of the present invention, the
maximum length of the capillary channel 3x for sample analysis may
be identical to that of the capillary channel 3y for sample
introduction. Similarly with respect to items other than those
described above, the configuration of the microchip electrophoresis
apparatus of the present invention is not limited to this
example.
[0054] Next, the process for producing the microchip
electrophoresis apparatus of this example is described. However,
the microchip electrophoresis chip may be produced by a process
other than the production process described below.
[0055] In the microchip electrophoresis apparatus of this example,
the substrate 1 to be used can be one formed of, for example, a
glass or polymer material. Examples of the glass material include
synthetic silica glass, and borosilicate glass. Examples of the
polymer material include polymethylmethacrylate (PMMA), cycloolefin
polymer (COP), polycarbonate (PC), polydimethylsiloxane (PDMS),
polystyrene (PS), and polylactic acid.
[0056] In the microchip electrophoresis apparatus of this example,
the maximum length, maximum width, and maximum thickness of the
substrate 1 are as described above.
[0057] The inner diameters of the four capillary channels are the
same as that of the capillary channel of the present invention. The
capillary channel 3x for sample analysis and the capillary channel
3y for sample introduction each have a maximum length, for example,
in the range of 0.5 to 15 cm. The respective lengths of the four
capillary channels are determined according to the maximum lengths
of the capillary channel 3x for sample analysis and the capillary
channel 3y for sample introduction.
[0058] The volumes of the plurality of liquid reservoirs 2a to 2d
are not particularly limited. For example, each of them has a
volume of 1 to 1000 mm.sup.3, preferably in the range of 50 to 100
mm.sup.3. In FIG. 6, the shapes of the plurality of liquid
reservoirs 2a to 2d are cylindrical. However, the present invention
is not limited thereto. In the microchip electrophoresis apparatus
of the present invention, the shapes of the plurality of liquid
reservoirs are not particularly limited as long as they do not
cause any problems in introduction and recovery of the sample
described later. For example, each of them may have an arbitrary
shape, such as a quadrangular prism shape, a quadrangular pyramidal
shape, a conical shape, or a shape formed by combining them.
Furthermore, the volumes and shapes of the plurality of liquid
reservoirs may be identical to or different from one another.
[0059] An example of the process of producing a microchip
electrophoresis apparatus of this example is described below.
However, the microchip electrophoresis apparatus may be produced by
a process other than the production process described below.
[0060] First, the substrate 1 is produced. The methods of forming
the four liquid reservoirs 2a to 2d and the opening (window) 9 in
the substrate 1 are not particularly limited. For example, when the
material used for the substrate 1 is the aforementioned glass, the
formation method can be, for instance, ultrasonic machining. For
example, when the material used for the substrate 1 is the
aforementioned polymer material, the formation method can be, for
instance, a cutting method or a molding method such as injection
molding, cast molding, or press molding that employs a mold. The
four liquid reservoirs 2a to 2d and the opening (window) 9 each may
be formed independently, or all of them may be formed
simultaneously. When the four liquid reservoirs 2a to 2d and the
opening (window) 9 each are formed independently, they may be
formed in any order. It is preferable that all the four liquid
reservoirs 2a to 2d and the opening (window) 9 be formed
simultaneously by, for example, a method that employs a mold, since
the number of the steps is smaller in this case.
[0061] Next, the four capillary channels are inserted into the
substrate 1. Thus, a microchip electrophoresis apparatus of this
example can be obtained. The microchip electrophoresis apparatus
further may include a plurality of electrodes. FIG. 7 shows a
microchip electrophoresis apparatus of this example that includes
the plurality of electrodes. In FIG. 7, the identical parts to
those shown in FIG. 6 are indicated with identical numerals and
symbols. As shown in FIG. 7, this microchip electrophoresis
apparatus has four electrodes 6a to 6d. The four electrodes 6a to
6d are buried in the substrate 1 in such a manner that first ends
thereof are located inside the plurality of liquid reservoirs 2a to
2d, respectively. The four electrodes 6a to 6d can be disposed
easily when, for example, holes for introducing the four electrodes
6a to 6d are formed in the side faces of the substrate 1 in
producing the substrate 1. In the microchip electrophoresis
apparatus, the plurality of electrodes are optional components. For
example, the plurality of electrodes may be inserted into the
plurality of liquid reservoirs when the microchip electrophoresis
apparatus is used.
[0062] The plurality of electrodes 6a to 6d may be any electrodes,
as long as they can be used for the electrophoresis method. The
plurality of electrodes 6a to 6d each are, for example, an
electrode made of stainless steel (SUS), a platinum (Pt) electrode,
or a gold (Au) electrode.
[0063] The microchip electrophoresis apparatus further may include
a pretreatment reservoir for hemolyzing a sample containing
hemoglobin and diluting it. The treatment for hemolyzing the
hemoglobin-containing sample is not particularly limited. For
example, it may be a treatment for hemolyzing the
hemoglobin-containing sample with a hemolytic agent. The hemolytic
agent destroys, for example, a blood cell membrane of a blood cell
component in the hemoglobin-containing sample. Examples of the
hemolytic agent include the aforementioned running buffer, saponin,
and "Triton X-100" (trade name) manufactured by Nacalai Tesque,
Inc. Particularly preferable is the running buffer. Preferably, the
pretreatment reservoir communicates with, for example, the
introduction reservoirs. The pretreatment reservoir may be formed
in a suitable place such as a place near the liquid reservoir with
which it communicates, for example, the second introduction
reservoir 2c. When the pretreatment reservoir is provided, the
hemoglobin-containing sample is introduced into the pretreatment
reservoir. The hemoglobin-containing sample thus pretreated is
introduced into a liquid reservoir that communicates with the
pretreatment reservoir, for example, the second introduction
reservoir 2c through the channel connecting the pretreatment
reservoir and the second introduction reservoir 2c. The
pretreatment reservoir may have a configuration in which two
reservoirs, a reservoir for hemolyzing the hemoglobin-containing
sample and a reservoir for diluting the hemoglobin-containing
sample, are in communication with each other.
[0064] The microchip electrophoresis apparatus further may include
an analysis unit. FIG. 8 shows a microchip electrophoresis
apparatus of this example including the analysis unit. In FIG. 8,
identical parts to those shown in FIGS. 6 and 7 are indicated with
identical numerals and symbols. As shown in FIG. 8, this microchip
electrophoresis apparatus includes an analysis unit 7. In the
microchip electrophoresis apparatus of this example, the analysis
unit 7 is a detector (line detector). The line detector is disposed
directly on the capillary channel 3x2 in such a manner that it is
located on the first recovery reservoir 2b side with respect to the
intersection part between the capillary channel 3x for sample
analysis and the capillary channel 3y for sample introduction. In
this microchip electrophoresis apparatus, the substrate 1 is
provided with a cavity into which the analysis unit (line detector)
7 is to be inserted, in addition to the cavity into which the four
capillary channels are to be inserted (not shown in the figures).
The line detector has a light source and a detection unit built-in.
The line detector emits light from the light source towards the
sample to detect light reflected from the sample in the detection
unit, and thereby measures absorbance. The analysis unit 7 is not
limited to the line detector. It may be any analysis unit as long
as, for example, it can analyze a sample containing hemoglobin. For
example, the analysis unit 7 may be configured with a light source
disposed under the microchip electrophoresis apparatus and a
detection unit disposed in a place corresponding to the place where
the line detector is disposed. In this case, light is emitted from
the light source toward the sample, the transmitted light from the
sample is detected in the detection unit, and thus absorbance is
measured.
[0065] FIG. 9 shows still another example of the microchip
electrophoresis apparatus according to the present invention. In
FIG. 9, identical parts to those shown in FIG. 8 are indicated with
identical numerals and symbols. As shown in FIG. 9, the microchip
electrophoresis apparatus of this example has the same
configuration as that of the microchip electrophoresis apparatus
shown in FIG. 8 except that the analysis unit 7 is different. As in
this example, the analysis unit 7 may measure the absorbance at one
point.
[0066] The analytical processes of the present invention using the
microchip electrophoresis apparatuses shown in FIGS. 8 and 9 can be
carried out with respect to, for example, a sample containing
hemoglobin, as follows.
[0067] The analytical processes in a case in which the capillary
channel that includes the A layer formed of
polydiallyldimethylammoniumchloride is used for the aforementioned
four capillary channels is explained. First, an alkaline solution
such as an aqueous sodium hydroxide is passed through the capillary
channel 3x for sample analysis and the capillary channel 3y for
sample introduction under pressure applied by, for example, a pump.
Subsequently, distilled water is passed through the capillary
channel 3x for sample analysis and the capillary channel 3y for
sample introduction to wash them. The time for which each of the
alkaline solution and the distilled water is passed therethrough
and the pressure applied when each of them is passed therethrough
are, for example, as described above. Next, the
polydiallyldimethylammoniumchloride solution is passed through the
capillary channel 3x for sample analysis and the capillary channel
3y for sample introduction under pressure applied by, for example,
a pump. The time for which it is passed therethrough and the
pressure thereof are, for example, as described above. Then,
distilled water is passed through the capillary channel 3x for
sample analysis and the capillary channel 3y for sample
introduction under pressure applied by, for example, a pump to
remove residual polydiallyldimethylammoniumchloride. The time for
which it is passed therethrough and the pressure thereof are, for
example, as described above. In this manner, the A layer is formed
on the inner wall of the capillary channel 3x for sample analysis
and the capillary channel 3y for sample introduction with the
polydiallyldimethylammoniumchloride.
[0068] Next, a running buffer containing an anionic
group-containing polysaccharide such as chondroitin sulfate is
passed through the capillary channel 3x for sample analysis and the
capillary channel 3y for sample introduction under pressure applied
by, for example, a pump. The time for which it is passed
therethrough and the pressure thereof are, for example, as
described above. Thereby, the B layer made of such as chondroitin
sulfate is coated on the A layer. Thereafter, the capillary channel
3x for sample analysis and the capillary channel 3y for sample
introduction are filled with the running buffer by pressure or
capillary action.
[0069] It is preferable that when the microchip electrophoresis
apparatus is not in use (when no analysis is carried out), the step
of filling them with the running buffer be completed beforehand,
since it makes it possible to omit the respective steps described
above and to proceed directly to the following step.
[0070] Subsequently, a hemoglobin-containing sample is introduced
into the second introduction reservoir 2c. Examples of the
hemoglobin-containing sample are as described above. When the
microchip electrophoresis apparatus has the pretreatment reservoir
(not shown in the figures), the hemoglobin-containing sample is
introduced into the pretreatment reservoir and is pretreated there.
Subsequently, voltage is applied to the electrode 6c and the
electrode 6d to generate a potential difference between the ends of
the capillary channel 3y for sample introduction. Thus, the
hemoglobin-containing sample is introduced into the capillary
channel 3y for sample introduction. The hemoglobin thus introduced
is bonded with an anionic group-containing polysaccharide contained
in the running buffer to form a complex. Voltage is applied to
generate an electroosmotic flow in the running buffer contained in
the capillary channel 3y for sample introduction and thereby the
complex is transferred to the intersection part between the
capillary channel 3x for sample analysis and the capillary channel
3y for sample introduction.
[0071] The potential difference between the electrode 6c and the
electrode 6d is, for instance, in the range of 0.5 to 5 kV.
[0072] Next, voltage is applied to the electrode 6a and the
electrode 6b to generate a potential difference between the ends of
the capillary channel 3x for sample analysis. In this manner, the
capillary channel having a potential difference between the ends
thereof is changed momentarily from the capillary channel 3y for
sample introduction to the capillary channel 3x for sample
analysis, so that as shown with arrows in FIGS. 8 and 9, the sample
8 is transferred to the first recovery reservoir 2b side from the
intersection part between the capillary channel 3x for sample
analysis and the capillary channel 3y for sample introduction.
[0073] The potential difference between the electrode 6a and the
electrode 6b is, for example, in the range of 0.5 to 5 kV.
[0074] Subsequently, the respective components of the
hemoglobin-containing sample separated due to the difference in
transfer rate are detected with the detector 7. Thus, the
respective components of the hemoglobin-containing sample can be
separated to be analyzed.
[0075] The analytical processes in a case in which the capillary
channel that includes the A layer formed of at least one of the
nonpolar polymer and the cationic group-containing compound is used
for the aforementioned four capillary channels can be carried out
in the same manner as described above except that the capillary
channel that includes the A layer formed in the aforementioned
manner is used.
[0076] Next, examples of the present invention are described.
Example 1
[0077] A capillary channel (with an overall length of 32 cm, an
effective length of 8.5 cm, and an inner diameter of 50 .mu.m) made
of fused silica was prepared. An aqueous sodium hydroxide (1 mol/L)
was passed through this capillary channel at a pressure of 0.1 MPa
(1000 mbar) for 10 minutes. Subsequently, distilled water was
passed through this capillary channel at the same pressure as
described above for 20 minutes to wash it. Then, a
polydiallyldimethylammoniumchloride solution (10 wt %) was passed
through the capillary channel at the same pressure as described
above for 30 minutes. Subsequently, distilled water was passed
through the capillary channel at the same pressure as described
above for 20 minutes to form the A layer made of
polydiallyldimethylammoniumchloride on the inner wall of the
capillary channel. Then, a running buffer (pH 5.5) was prepared
that contains chondroitin sulfate added to 100 mM malic acid and an
arginine acid aqueous solution at a ratio of 0.5 wt %. This running
buffer was passed through the capillary channel, in which the A
layer is formed, at the same pressure as described above, and
thereby the B layer is formed on the A layer. With the capillary
channel being filled with the running buffer, a sample containing
hemoglobin dissolved in distilled water was injected into the
capillary channel. Thereafter, a voltage of 10 kV was applied
across both ends of the capillary channel, and thereby
electrophoresis was carried out. The hemoglobin-containing sample
was injected into the capillary channel from the anode side
thereof. The hemoglobin that had been transferred was detected at
an absorbance of 415 nm. This result is shown in the
electropherogram in FIG. 1. As shown in FIG. 1, in this example, it
was possible to detect normal hemoglobin (HbA0) and glycated
hemoglobin (HbA1c) separately. Furthermore, as for the capillary
channel used in this example, because the B layer was formed simply
by passing through the running buffer therein after being washed,
it was possible to carry out the analysis immediately.
Example 2
[0078] A capillary channel (with an overall length of 32 cm, an
effective length of 8.5 cm, and an inner diameter of 50 .mu.m) made
of fused silica was prepared. The capillary channel had an A layer
formed with a silylation agent having an amino group that was fixed
to the inner wall thereof by a covalent bond. Distilled water was
passed through this capillary channel at a pressure of 0.1 MPa
(1000 mbar) for 20 minutes to wash it. Then, a running buffer (pH
5.5) was prepared that contains chondroitin sulfate added to 100 mM
malic acid and an arginine acid aqueous solution at a ratio of 0.5
wt %. This running buffer was passed through the capillary channel
at the same pressure as described above, and thereby the B layer
was formed on the A layer. With the capillary channel being filled
with the running buffer, a sample containing hemoglobin dissolved
in distilled water was injected into the capillary channel.
Thereafter, a voltage of 10 kV was applied across both ends of the
capillary channel, and thereby electrophoresis was carried out. The
hemoglobin-containing sample was injected into the capillary
channel from the anode side thereof. The hemoglobin that had been
transferred was detected at an absorbance of 415 nm. This result is
shown in the electropherogram in FIG. 2. As shown in FIG. 2, in
this example, it was possible to detect normal hemoglobin (HbA0)
and glycated hemoglobin (HbA1c) separately. Furthermore, as for the
capillary channel used in this example, because the B layer was
formed simply by passing through the running buffer therein after
being washed, it was possible to carry out the analysis
immediately. In this state, the same analysis was carried out 10
times with the same sample as described above to evaluate
precision. This result is shown in the following Table 1. In Table
1, a relative area (%) denotes a ratio (%) of each peak area of the
normal hemoglobin (HbA0) and the glycated hemoglobin (HbA1c)
relative to a total peak area. As shown in Table 1, a value of
coefficient of variation (CV) is small in each of the normal
hemoglobin (HbA0) and the glycated hemoglobin (HbA1c). Thereby, it
can be said that the analytical processes of the present invention
is excellent in the repeatability.
TABLE-US-00001 TABLE 1 Relative Area (%) No. HbA1c HbA0 1 10.08
89.92 2 10.37 89.63 3 10.18 89.82 4 10.49 89.51 5 10.34 89.66 6
10.30 89.70 7 9.89 90.11 8 10.17 89.83 9 10.24 89.76 10 10.32 89.68
Average 10.24 89.76 Coefficient of Variation(CV) 1.7 0.2
Example 3
[0079] A capillary channel (with an overall length of 32 cm, an
effective length of 8.5 cm, and an inner diameter of 50 .mu.m) made
of fused silica was prepared. The capillary channel had an A layer
formed with a silylation agent having an amino group that was fixed
to the inner wall thereof by a covalent bond. Distilled water was
passed through this capillary channel at a pressure of 0.1 MPa
(1000 mbar) for 20 minutes to wash it. Then, a running buffer (pH
5.5) was prepared that contains sodium alginate added to 100 mM
malic acid and an arginine acid aqueous solution at a ratio of 0.8
wt %. This running buffer was passed through the capillary channel
at the same pressure as described above, and thereby the B layer is
formed on the A layer. With the capillary channel being filled with
the running buffer, a sample containing hemoglobin dissolved in
distilled water was injected into the capillary channel.
Thereafter, a voltage of 10 kV was applied across both ends of the
capillary channel, and thereby electrophoresis was carried out. The
hemoglobin-containing sample was injected into the capillary
channel from the anode side thereof. The hemoglobin that had been
transferred was detected at an absorbance of 415 nm. This result is
shown in the electropherogram in FIG. 3. As shown in FIG. 3, in
this example, it was possible to detect normal hemoglobin (HbA0)
and glycated hemoglobin (HbA1c) separately. Furthermore, as for the
capillary channel used in this example, because the B layer was
formed simply by passing through the running buffer therein after
being washed, it was possible to carry out the analysis
immediately.
Example 4
[0080] A capillary channel (with an overall length of 32 cm, an
effective length of 8.5 cm, and an inner diameter of 50 .mu.m) made
of fused silica was prepared. The capillary channel had an A layer
formed with a silylation agent having an amino group that was fixed
to the inner wall thereof by a covalent bond. Distilled water was
passed through this capillary channel at a pressure of 0.1 MPa
(1000 mbar) for 20 minutes to wash it. Then, a running buffer (pH
5.5) was prepared that contains heparin sodium added to 100 mM
malic acid and an arginine acid aqueous solution at a ratio of 0.5
wt %. This running buffer was passed through the capillary channel
at the same pressure as described above, and thereby the B layer
was formed on the A layer. With the capillary channel being filled
with the running buffer, a sample containing hemoglobin dissolved
in distilled water was injected into the capillary channel.
Thereafter, a voltage of 10 kV was applied across both ends of the
capillary channel, and thereby electrophoresis was carried out. The
hemoglobin-containing sample was injected into the capillary
channel from the anode side thereof. The hemoglobin that had been
transferred was detected at an absorbance of 415 nm. This result is
shown in the electropherogram in FIG. 4. As shown in FIG. 4, in
this example, it was possible to detect normal hemoglobin (HbA0)
and glycated hemoglobin (HbA1c) separately. Furthermore, as for the
capillary channel used in this example, because the B layer was
formed simply by passing through the running buffer therein after
being washed, it was possible to carry out the analysis
immediately.
Example 5
[0081] A capillary channel (with an overall length of 32 cm, an
effective length of 8.5 cm, and an inner diameter of 50 .mu.m) made
of fused silica was prepared. The capillary channel had an A layer
formed with poly(dimethylsiloxane) that was fixed to the inner wall
thereof by a covalent bond. Distilled water was passed through this
capillary channel at a pressure of 0.1 MPa (1000 mbar) for 20
minutes to wash it. Then, a running buffer (pH 5.5) was prepared
that contains chondroitin sulfate added to 100 mM malic acid and an
arginine acid aqueous solution at a ratio of 1.0 wt %. This running
buffer was passed through the capillary channel at the same
pressure as described above, and thereby the B layer was formed on
the A layer. With the capillary channel being filled with the
running buffer, a sample containing hemoglobin dissolved in
distilled water was injected into the capillary channel.
Thereafter, a voltage of 10 kV was applied across both ends of the
capillary channel, and thereby electrophoresis was carried out. The
hemoglobin-containing sample was injected into the capillary
channel from the anode side thereof. The hemoglobin that had been
transferred was detected at an absorbance of 415 nm. This result is
shown in the electropherogram in FIG. 5. As shown in FIG. 5, in
this example, it was possible to detect normal hemoglobin (HbA0)
and glycated hemoglobin (HbA1c) separately. Furthermore, as for the
capillary channel used in this example, because the B layer was
formed simply by passing through the running buffer therein after
being washed, it was possible to carry out the analysis
immediately. In this state, the same analysis was carried out 10
times with the same sample as described above to evaluate
repeatability. This result is shown in the following Table 2. In
Table 2, as same as in Table 1, a relative area (%) denotes a ratio
(%) of each peak area of the normal hemoglobin (HbA0) and the
glycated hemoglobin (HbA1c) relative to a total peak area. As shown
in Table 2, a value of coefficient of variation (CV) is small in
each of the normal hemoglobin (HbA0) and the glycated hemoglobin
(HbA1c). Thereby, it can be said that the analytical processes of
the present invention is excellent in the precision.
TABLE-US-00002 TABLE 2 Relative Area (%) No. HbA1c HbA0 1 10.06
89.94 2 10.87 89.13 3 9.68 90.32 4 10.08 89.92 5 9.45 90.55 6 10.48
89.52 7 10.87 89.13 8 10.88 89.12 9 9.20 90.80 10 10.17 89.83
Average 10.17 89.83 Coefficient of Variation(CV) 5.9 0.7
INDUSTRIAL APPLICABILITY
[0082] As described above, according to the present invention, a
sample such as hemoglobin can be analyzed easily with high
precision by the capillary electrophoresis method. Moreover, since
the present invention employs the capillary electrophoresis method,
it also is possible to reduce the size of the analysis apparatus.
The present invention is applicable to all the fields where a
sample such as hemoglobin is to be analyzed, such as laboratory
tests, biochemical examinations, and medical research. The intended
use thereof is not limited and it is applicable to a wide range of
fields.
* * * * *