The present invention relates to an electrophoresis chip and an electrophoresis apparatus.
The degree of glycosylation of various proteins has been analyzed as an indicator that shows the condition of a living body. In particular, since the degree of glycosylation of hemoglobin (Hb), especially HbA1c, in blood cells reflects the history of glucose levels in a living body, it is regarded as an important indicator in the diagnosis, treatment or the like of diabetes. HbA1c is HbA(α2β2) whose β-chain N-terminal valine has been glycosylated.
HbA1c has been analyzed by, for example, immunological methods, enzymatic methods, high-performance liquid chromatography (HPLC) methods, and affinity methods, among others. Although immunological methods and enzymatic methods are generally used in processing and analyzing large numbers of specimens, they are of low accuracy when determining the risks due to complications. On the other hand, although HPLC methods have a poorer processing capability than immunological methods or enzymatic methods, they are useful in determining the risk of complications. However, due to the configuration of HPLC methods, the analysis apparatus is very large and costly. In affinity methods, types of glycosylated Hb other than HbA1c whose β-chain N-terminal have been glycosylated are also measured concurrently.
Furthermore, analysis of HbA1c using capillary electrophoresis has been attempted (see Non-Patent Document 1). This method, however, uses a fused silica capillary having an inner diameter of 25 μm, which is not advantageous in terms of sensitivity, and requires an analysis time of about 4 minutes for the analysis of HbA1c. Moreover, this method requires the use of a large electrophoresis apparatus. In addition, POC (point of care) testing using any of the aforementioned conventional methods is not sufficiently accurate to enable the management of the risks due to complications, and it has been used as no more than a screening test. These problems can be associated with glycosylated hemoglobin, including HbA1c, as a whole.
Non-Patent Document 1: Clinical Chemistry 43: 4, 644-648 (1997)
Therefore, an object of the present invention is to provide electrophoresis chips, for the analysis of glycosylated hemoglobin by capillary electrophoresis, that allow an apparatus to be small, analysis time to be short and glycosylated hemoglobin to be analyzed highly accurately.
To achieve the above object, electrophoresis chips of the present invention are electrophoresis chips for analyzing glycosylated hemoglobin;
electrophoresis chips include a substrate, a plurality of fluid reservoirs and a capillary channel;
the plurality of fluid reservoirs include a first introduction reservoir and a first recovery reservoir;
the capillary channel includes a capillary channel for sample analysis;
the first introduction reservoir and the first recovery reservoir are formed in the substrate; and
the first introduction reservoir and the first recovery reservoir are in communication with each other via the capillary channel for sample analysis.
Electrophoresis apparatuses of the present invention are electrophoresis apparatuses that include an electrophoresis chip and an analysis unit, with the electrophoresis chip being an electrophoresis chip of the present invention.
Electrophoresis chips of the present invention are chips wherein a first introduction reservoir and a first recovery reservoir are formed in a substrate, and the first introduction reservoir and the first recovery reservoir are in communication with each other via a capillary channel for sample analysis. Hence, in an analysis of glycosylated hemoglobin by capillary electrophoresis, the present invention allows an apparatus to be small and accordingly an analysis time to be short. Moreover, it is possible with electrophoresis chips of the present invention to analyze glycosylated hemoglobin highly accurately. Therefore, it is possible with electrophoresis chips of the present invention to accurately analyze glycosylated hemoglobin in, for example, POC testing, and thus, to manage the risks due to complications.
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An electrophoresis chip of the present invention may be configured such that:
the plurality of fluid reservoirs further include a second introduction reservoir and a second recovery reservoir,
the capillary channel further includes a capillary channel for sample introduction,
the second introduction reservoir and the second recovery reservoir are formed in the substrate,
the second introduction reservoir and the second recovery reservoir are in communication with each other via the capillary channel for sample introduction,
the capillary channel for sample analysis and the capillary channel for sample introduction intersect, and
the capillary channel for sample analysis and the capillary channel for sample introduction are in communication with each other at the intersection.
An electrophoresis chip of the present invention may be configured such that:
a first branching channel branches off from a part of the capillary channel for sample analysis,
the first branching channel is in communication with the second introduction reservoir,
a second branching channel branches off from a part of the capillary channel for sample analysis that is located on the downstream side relative to the first branching channel,
the second branching channel is in communication with the second recovery reservoir, and
the capillary channel for sample introduction is formed by the first branching channel, the second branching channel and the part of the capillary channel for sample analysis that connects the branching channels.
In an electrophoresis chip of the present invention, the maximum length of the whole chip is in a range of, for example, 10 to 100 mm and preferably in a range of 30 to 70 mm; the maximum width of the whole chip is in a range of, for example, 10 to 60 mm; and the maximum thickness of the whole chip is in a range of, for example, 0.3 to 5 mm. The maximum length of the whole chip refers to the dimension of the longest portion of the chip in the longitudinal direction; the maximum width of the whole chip refers to the dimension of the longest portion of the chip in a direction (width direction) perpendicular to the longitudinal direction; and the maximum thickness of the whole chip refers to the dimension of the longest portion of the chip in a direction (thickness direction) perpendicular to both the longitudinal direction and the width direction.
It is preferable that an electrophoresis chip of the present invention is such that, in analyzing glycosylated hemoglobin, a diluted sample in which a sample containing glycosylated hemoglobin is diluted with an electrophoresis running buffer is introduced into at least one fluid reservoir of the plurality of fluid reservoirs, and the volume ratio of the sample:the electrophoresis running buffer is in a range of 1:4 to 1:99. The volume ratio of the sample:the electrophoresis running buffer is more preferably in a range of 1:9 to 1:59 and still more preferably in a range of 1:19 to 1:29.
In an electrophoresis chip of the present invention, it is preferable that the capillary channel is filled with an electrophoresis running buffer.
In an electrophoresis chip of the present invention, the maximum diameter of the capillary channel is in a range of, for example, 10 to 200 μm and preferably in a range of 25 to 100 μm; and the maximum length thereof is in a range of, for example, 0.5 to 15 cm. When the shape of the cross section of the capillary channel is not circular, the maximum diameter of the capillary channel refers to the diameter of a circle having an area that corresponds to the cross sectional area of a portion having the largest cross-sectional area.
In an electrophoresis chip of the present invention, the inner wall of the capillary channel may be coated with a cationic group-containing compound. Examples of the cationic group-containing compound include compounds that contain cationic groups and reactive groups. Preferable examples of the cationic groups include amino groups and ammonium groups. A preferable example of the cationic group-containing compound is a silylating agent that contains at least an amino group or an ammonium group. The amino groups may be any of the primary, secondary and tertiary amino groups.
Examples of the silylating 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, and tris(dimethylamino)silane, among others.
Among such silylating agents, those in which silicon atom(s) are substituted with titanium or zirconium may be used. Such silylating agents may be used singly or may be used in a combination of two or more.
Coating of the inner wall of the capillary channel with the silylating agent is performed, for example, as follows. First, a silylating agent is dissolved or dispersed in an organic solvent to prepare a treatment fluid. Examples of the organic solvent for use in the preparation of the treatment fluid may be dichloromethane, toluene and the like. The concentration of the silylating agent in the treatment fluid is not particularly limited. This treatment fluid is passed through the capillary channel, and then heated. Due to this heating, the silylating agent is bonded to the inner wall of the capillary channel by covalent bonding, resulting in a cationic group being disposed on the inner wall of the capillary channel. Thereafter, washing (after-treatment) is performed with at least an organic solvent (dichloromethane, methanol, acetone or the like), an acid solution (phosphoric acid or the like), an alkaline solution, or a surfactant solution. Although this washing is optional, it is preferable to perform such washing. Moreover, as described below, when a capillary tube that is a member independent of the substrate serves as the capillary channel, a capillary tube whose inner wall is coated with a cationic group-containing compound through the use of a commercially available silylating agent of an aforementioned kind may be used.
It is preferable that an anionic layer formed from an anionic group-containing compound is further coated on the inner wall of the capillary channel that has been coated with a cationic group-containing compound. It is thus possible to prevent hemoglobin or the like present in a sample that will be described below from being adsorbed onto the inner wall of the capillary channel. Moreover, due to the formation of a complex between the sample and the anionic group-containing compound and due to the electrophoresis thereof, separation efficiency is enhanced compared with the electrophoresis of a sample alone. As a result of these, analysis of glycosylated hemoglobin or the like can be performed more accurately in a shorter period of time. An anionic group-containing polysaccharide is preferable as the anionic group-containing compound that forms a complex with the sample. Examples of anionic group-containing polysaccharides include sulfated polysaccharides, carboxylated polysaccharides, sulfonated polysaccharides and phosphorylated polysaccharides. Among these, sulfated polysaccharides and carboxylated polysaccharides are preferable. The sulfated polysaccharides are preferably chondroitin sulfate, or heparin, among others, with chondroitin sulfate being particularly preferable. The carboxylated polysaccharides are preferably alginic acid and salts thereof (for example, sodium alginate). There are seven types of chondroitin sulfate, for example, chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, chondroitin sulfate D, chondroitin sulfate E, chondroitin sulfate H, and chondroitin sulfate K, and any of these types may be used. The anionic layer can be formed by, for example, bringing a fluid that contains an anionic group-containing compound into contact with the inner wall of the capillary channel that has been coated with a cationic group-containing compound. In this case, although a fluid for forming an anionic layer may be prepared separately, it is preferable in terms of operation efficiency that an electrophoresis running buffer that contains an anionic group-containing compound is prepared and is passed through the capillary channel whose inner wall is coated with a cationic group-containing compound.
The electrophoresis running buffer is not particularly limited, and an electrophoresis running buffer that uses an organic acid is preferable. Examples of organic acids include maleic acid, tartaric acid, succinic acid, fumaric acid, phthalic acid, malonic acid, and malic acid, among others. Preferably, the electrophoresis running buffer contains a weak base. Examples of weak bases include arginine, lysine, histidine, and tris, among others. The pH of the electrophoresis running buffer is in a range of, for example, 4.5 to 6. In the electrophoresis running buffer, the concentration of an anionic group-containing compound is in a range of, for example, 0.001 to 10 wt %.
An electrophoresis chip of the present invention may further include a pretreatment reservoir for hemolyzing and diluting a sample containing glycosylated hemoglobin, and the pretreatment reservoir and at least one fluid reservoir of the plurality of fluid reservoirs may be in communication with each other. It is preferable that the pretreatment reservoir is in communication with at least one fluid reservoir of the first introduction reservoir and the second introduction reservoir, and it is more preferable that the pretreatment reservoir is only in communication with either the first introduction reservoir or the second introduction reservoir.
Glycosylated hemoglobin to be analyzed with an electrophoresis chip of the present invention is not particularly limited, and examples include HbA1c, labile HbA1c, and GHbLys, among others, with HbA1c being particularly preferable.
An electrophoresis chip of the present invention may be configured such that:
the substrate includes an upper substrate and a lower substrate,
a plurality of through-holes are formed in the upper substrate,
a groove is formed in the lower substrate,
the upper substrate is laminated onto the lower substrate,
spaces created by sealing the bottom parts of the plurality of through-holes formed in the upper substrate with the lower substrate serve as the plurality of fluid reservoirs, and
a space created by sealing the upper part of the groove formed in the lower substrate with the upper substrate serves as a capillary channel.
An electrophoresis chip of the present invention may be configured such that:
a plurality of concave portions and a groove are formed in a substrate,
a surface of the substrate is sealed with a sealing material that has openings at places corresponding to the plurality of concave portions,
the plurality of concave portions formed in the substrate serve as a plurality of fluid reservoirs, and
a space created by sealing the upper part of the groove formed in the substrate with the sealing material serves as a capillary channel.
An electrophoresis chip of the present invention may be configured such that:
the electrophoresis chip further includes a sealing material,
a plurality of through-holes are formed in a substrate,
a groove is formed in a bottom surface of the substrate,
the bottom surface of the substrate is sealed with the sealing material,
spaces created by sealing the bottom parts of the plurality of through-holes formed in the substrate with the sealing material serve as a plurality of fluid reservoirs; and
a space created by sealing the lower part of the groove formed in the bottom surface of the substrate with the sealing material serves as a capillary channel.
An electrophoresis chip of the present invention may be configured such that a plurality of fluid reservoirs are in communication with each other via a capillary tube that is a member independent of the substrate, and the capillary tube may serve as the capillary channel.
In an electrophoresis chip of the present invention, the volumes of the plurality of fluid reservoirs are not particularly limited, and are each in a range of, for example, 1 to 1000 mm3 and preferably in a range of 50 to 100 mm3.
An electrophoresis chip of the present invention may be configured such that the electrophoresis chip further includes a plurality of electrodes, and the plurality of electrodes may be disposed such that their first ends are placed in the plurality of fluid reservoirs.
Next, an electrophoresis chip of the present invention is described with reference to embodiments. The present invention, however, is not limited to the embodiments presented below.
Next, a method for producing an electrophoresis chip of this embodiment is described. An electrophoresis chip, however, may be produced according to methods other than the production method described below.
In an electrophoresis chip of this embodiment, a substrate formed from, for example, a glass material, a polymeric material or the like can be used as the lower substrate 1. Examples of a glass material include synthetic silica glass, and borosilicate glass, among others. Examples of a polymeric material include polymethylmethacrylate (PMMA), cycloolefin polymer (COP), polycarbonate (PC), polydimethylsiloxane (PDMS), polystyrene (PS), and polylactic acid, among others.
In an electrophoresis chip of this embodiment, the length and the width of the lower substrate 1 correspond to the maximum length and the maximum width of the whole chip described above, respectively. Therefore, the length and the width of the lower substrate 1 are arranged to be identical to the maximum length and the maximum width of the whole chip described above, respectively. The thickness of the lower substrate 1 in the electrophoresis chip of this embodiment is in a range of, for example, 0.1 to 3 mm and preferably in a range of 0.1 to 1 mm.
The material of the upper substrate 4 is not particularly limited insofar as it does not adversely affect an absorbance measurement that will be described below. For example, an upper substrate that is formed from the same material as the lower substrate 1 can be used as the upper substrate 4.
The length and the width of the upper substrate 4 are the same as the length and the width of the lower substrate 1, respectively. The thickness of the upper substrate 4 is suitably determined according to the volumes or like factors of the plurality of fluid reservoirs 2a to 2d and, for example, it is in a range of 0.1 to 3 mm and preferably in a range of 1 to 2 mm.
The width and the depth of the cross-shaped groove (the capillary channel for sample analysis 3x and the capillary channel for sample introduction 3y) are suitably determined according to the maximum diameter of the capillary channel and, for example, the width thereof is in a range of 25 to 200 μm and the depth thereof is in a range of 25 to 200 μm, and preferably the width thereof is in a range of 40 to 100 μm and the depth thereof is in a range of 40 to 100 μm. The maximum length of the capillary channel for sample analysis 3x and the maximum length of the capillary channel for sample introduction 3y are as described above.
The volumes of the plurality of fluid reservoirs 2a to 2d are as described above. In
In an electrophoresis chip of this embodiment, the maximum thickness of the whole chip is the sum of the thickness of the lower substrate 1 and the thickness of the upper substrate 4. The maximum thickness of the whole chip is as described above.
For example, when the material of the lower substrate 1 is glass, the electrophoresis chip can be produced as follows.
First, a surface of a glass plate 20 is masked with an alloy 21 of chromium and gold as shown in
Next, a photosensitive film on which a layout pattern for the capillary channel for sample analysis 3x and the capillary channel for sample introduction 3y is drawn is adhered to a surface of the photoresist 22 as shown in
Due to the exposure, the exposed portions of the photoresist 22 are solubilized as shown in
Next, the revealed portions of the alloy 21 are removed by aqua regia as shown in
The layout pattern is then etched with hydrogen fluoride into the glass plate 20 as shown in
Next, the photoresist 22 and the alloy 21 are removed to produce the lower substrate 1 as shown in
Next, the upper substrate 4 is prepared (not shown). A method for forming the four through-holes in the upper substrate 4 is not particularly limited. For example, when the material of the upper substrate 4 is glass, an example of the formation method is ultrasonic machining or the like. For example, when the material of the upper substrate 4 is polymeric material, examples of the formation method include a cutting method; a molding method such as injection molding, cast molding and press molding using a metal mold; and like methods. The four through-holes may each be formed separately or may all be formed simultaneously. When the four through-holes are formed separately, they may be formed in any order. Forming all four through-holes simultaneously according to an aforementioned method that uses a metal mold or a like method requires a small number of steps and is thus preferable.
Finally, by laminating the lower substrate 1 and the upper substrate 4, an electrophoresis chip of this embodiment can be produced. A method for laminating the lower substrate 1 and the upper substrate 4 is not particularly limited and, for example, thermal welding is preferable. Although a production process was described in reference to
For example, when the material of the lower substrate 1 is polymeric material, the electrophoresis chip can be produced as follows.
First, a surface of a silicon plate 31 is coated with a photoresist 32 as shown in
Next, a photosensitive film on which a layout pattern for the capillary channel for sample analysis 3x and the capillary channel for sample introduction 3y is drawn is adhered to a surface of the photoresist 32 as shown in
Due to the exposure, the exposed portions of the photoresist 32 are solubilized as shown in
Next, the layout pattern is etched into the silicon plate 31 to prepare a base mold 35 as shown in
Metallic nickel electrocasting is then performed on the base mold 35 to prepare a metal mold for injection molding 36 as shown in
Next, a lower substrate 1 composed of polymeric material is prepared by injection molding using the metal mold for injection molding 36 as shown in
Next, the upper substrate 4 is prepared (not shown). A method for preparing the upper substrate 4 is the same as the method used when the material of the lower substrate 1 is glass.
Finally, by laminating the lower substrate 1 and the upper substrate 4, an electrophoresis chip of this embodiment can be produced. A method for laminating the lower substrate 1 and the upper substrate 4 is the same as the method used when the material of the lower substrate 1 is glass. Although a production process was described in reference to
As described above, the electrophoresis chip of the present invention may further include a plurality of electrodes.
The plurality of electrodes 6a to 6d may be any electrodes insofar as they are functional with an electrophoresis method. The plurality of electrodes 6a to 6d are each, for example, a stainless steel (SUS) electrode, a platinum (Pt) electrode, a gold (Au) electrode or the like.
An electrophoresis chip of the present invention may further include a pretreatment reservoir for hemolyzing and diluting a sample containing glycosylated hemoglobin. A hemolysis treatment for a sample is not particularly limited and, for example, it may be a treatment in which a sample is hemolyzed with a hemolytic agent. The hemolytic agent destroys, for example, the blood cell membrane of a blood cell component present in a sample that will be described below. Examples of hemolytic agents include the aforementioned electrophoresis running buffer, saponin, and “Triton X-100” (trade name) manufactured by Nacalai Tesque, Inc., among others, with the electrophoresis running buffer being particularly preferable. It is preferable that the pretreatment reservoir is in communication with, for example, an aforementioned introduction reservoir. The pretreatment reservoir may be formed in a suitable place such as a place near an aforementioned fluid reservoir with which the pretreatment reservoir is in communication such as, for example, the second introduction reservoir 2c. When a pretreatment reservoir is provided, a sample that will be described below is introduced into the pretreatment reservoir. The sample thus pretreated is introduced, via a channel that connects the pretreatment reservoir and an aforementioned fluid reservoir that is in communication with the pretreatment reservoir such as, for example, the second introduction reservoir 2c, into the second introduction reservoir 2c. The pretreatment reservoir may be configured such that two reservoirs, i.e., a reservoir for hemolyzing the sample and a reservoir for diluting the sample, are in communication.
Next, a method for analyzing glycosylated hemoglobin in connection with the present invention is described using, as examples, the cases where the electrophoresis apparatuses shown in
First, the capillary channel for sample analysis 3x and the capillary channel for sample introduction 3y are filled with an electrophoresis running buffer by pressure or capillary action. The electrophoresis running buffer is as described above.
When the capillary channels are filled with an electrophoresis running buffer in advance, when the electrophoresis apparatus is not in use (when not in analysis), it is possible to omit the above-described step of filling with an electrophoresis running buffer and to immediately advance to the following steps, and it is thus preferable.
Next, a sample to be analyzed (a sample containing glycosylated hemoglobin) is introduced into the second introduction reservoir 2c. At this time, it is preferable to introduce a diluted sample that is diluted so as to have a volume ratio of the sample:the electrophoresis running buffer in a range of 1:4 to 1:99. That is, it is preferable that, in a method for analyzing glycosylated hemoglobin using an electrophoresis apparatus (electrophoresis chip) of the present invention, a diluted sample that is prepared by diluting a sample containing the glycosylated hemoglobin with an electrophoresis running buffer is introduced into at least one fluid reservoir of the plurality of fluid reservoirs, and the volume ratio of the sample:the electrophoresis running buffer is in a range of 1:4 to 1:99. However, the volume ratio is not limited to this. When an electrophoresis chip includes a pretreatment reservoir (not shown), the sample is introduced into the pretreatment reservoir and is pretreated therein. Next, a voltage is applied to the electrode 6c and the electrode 6d to generate a potential difference between both ends of the capillary channel for sample introduction 3y, thereby moving the sample to the intersection of the capillary channel for sample analysis 3x and the capillary channel for sample introduction 3y. The sample may be anything insofar as it contains hemoglobin (Hb), and examples include whole blood, hemolyzed samples prepared by subjecting whole blood to a hemolysis treatment, and like samples. Examples of hemolysis treatments include sonication treatment, freeze/thaw treatment, pressure treatment, osmotic pressure treatment, and surfactant treatment, among others. The hemolysis treatment may be performed in, for example, a pretreatment reservoir. Alternatively, a sample that has been subjected to a hemolysis treatment in advance in a separate apparatus or the like may be introduced into an electrophoresis apparatus (electrophoresis chip). The sample may be suitably diluted with, for example, water, physiological saline, or an electrophoresis running buffer, among others. This dilution may be performed in, for example, a pretreatment reservoir. Moreover, a sample that has been subjected to a dilution treatment in advance in a separate apparatus or the like may be introduced into an electrophoresis apparatus (electrophoresis chip).
The potential difference between the electrode 6c and the electrode 6d is in a range of, for example, 0.5 to 5 kV.
Next, a voltage is applied to the electrode 6a and the electrode 6b to generate a potential difference between both ends of the capillary channel for sample analysis 3x. In this manner, by instantly shifting a capillary channel having different potentials at both ends from the capillary channel for sample introduction 3y to the capillary channel for sample analysis 3x, the sample 8 is moved toward the first recovery reservoir 2b side from the intersection of the capillary channel for sample analysis 3x and the capillary channel for sample introduction 3y as indicated by the arrows in
The potential difference between the electrode 6a and the electrode 6b is in a range of, for example, 0.5 to 5 kV.
Next, each component of the sample that is separated due to the differences in migration speed is detected with the detector 7. It is thus possible to analyze (separate and measure) each component of the sample. According to the present invention, it is possible to analyze (separate and measure) with high accuracy glycosylated hemoglobin and other components of a sample that contains hemoglobin (Hb).
An electrophoresis chip of this embodiment can be produced, for example, as follows. However, the electrophoresis chip may be produced according to methods other than the production method described below.
For example, a substrate that is formed from the same material as the lower substrate 1 of the electrophoresis chip shown in
In an electrophoresis chip of this embodiment, the length and the width of the substrate (lower substrate) 1 correspond to the maximum length and the maximum width of the whole chip described above, respectively. Therefore, the length and the width of the substrate (lower substrate) 1 are arranged to be identical to the maximum length and the maximum width of the whole chip described above, respectively. The thickness of the substrate (lower substrate) 1 in an electrophoresis chip of this embodiment is in a range of, for example, 0.1 to 3 mm and preferably in a range of 1 to 2 mm.
The material of the sealing material (upper substrate) 4 is also not particularly limited and, for example, a substrate that is formed from the same material as the lower substrate 1 of the electrophoresis chip shown in
The length and the width of the sealing material (upper substrate) 4 are identical to the length and the width of the substrate (lower substrate) 1, respectively. The thickness of the sealing material (upper substrate) 4 is in a range of, for example, 50 to 1000 μm and preferably in a range of 100 to 300 μm.
For example, a commercially available sealing material may be used as the sealing material (upper substrate) 4 wherein holes are created in places corresponding to the four concave portions (the four fluid reservoirs 2a to 2d).
In an electrophoresis chip of this embodiment, the maximum thickness of the whole chip is the sum of the thickness of the substrate (lower substrate) 1 and the thickness of the sealing material (upper substrate) 4. The maximum thickness of the whole chip is as described above.
An example of a process for producing an electrophoresis chip of this embodiment is described below. However, an electrophoresis chip may be produced according to processes other than the production process described below.
First, the substrate (lower substrate) 1 is prepared. A method for forming the capillary channel for sample analysis 3x and the capillary channel for sample introduction 3y in the substrate (lower substrate) 1 is not particularly limited, and the capillary channels may be formed, for example, in the same manner as in Embodiment 1 above. A method for forming the four fluid reservoirs 2a to 2d in the substrate (lower substrate) 1 is also not particularly limited. For example, when the material of the substrate (lower substrate) 1 is glass, an example of the formation method is ultrasonic machining or the like. For example, when the material of the substrate (lower substrate) 1 is polymeric material, examples of the formation method include a cutting method; a molding method such as injection molding, cast molding and press molding using a metal mold; and like methods. The four fluid reservoirs 2a to 2d may each be formed separately or may all be formed simultaneously. When the four fluid reservoirs 2a to 2d are formed separately, they may be formed in any order. Forming all of the four through-holes simultaneously according to an aforementioned method that uses a metal mold or a like method requires a small number of steps and is thus preferable.
Next, by sealing a surface of the substrate (lower substrate) 1 with the sealing material (upper substrate) 4 in which holes are created in places corresponding to the four concave portions (the four fluid reservoirs 2a to 2d), an electrophoresis chip of this embodiment can be produced.
The configuration of an electrophoresis chip of this embodiment is not limited to that shown in
An electrophoresis chip of this embodiment can be produced, for example, as follows. However, an electrophoresis chip may be produced according to methods other than the production method described below.
For example, a substrate that is formed from the same material as the lower substrate 1 of the electrophoresis chip shown in
In an electrophoresis chip of this embodiment, the length and the width of the substrate (upper substrate) 4 correspond to the maximum length and the maximum width of the whole chip described above, respectively. Therefore, the length and the width of the substrate (upper substrate) 4 are arranged to be identical to the maximum length and the maximum width of 5 the whole chip described above, respectively. The thickness of the substrate (upper substrate) 4 in the electrophoresis chip of this embodiment is in a range of, for example, 0.1 to 3 mm and preferably in a range of 1 to 2 mm.
The material of the sealing material (lower substrate) 1 is also not particularly limited and, for example, a substrate that is formed from the same material as the lower substrate 1 of the electrophoresis chip shown in
The length and the width of the sealing material (lower substrate) 1 are identical to the length and the width of the substrate (upper substrate) 4, respectively. The thickness of the sealing material (upper substrate) 4 is in a range of, for example, 50 to 1000 μm and preferably in a range of 100 to 300 μm.
For example, a commercially available sealing material may be used for the sealing material (lower substrate) 1.
In an electrophoresis chip of this embodiment, the maximum thickness of the whole chip is the sum of the thickness of the substrate (upper substrate) 4 and the thickness of the sealing material (lower substrate) 1. The maximum thickness of the whole chip is as described above.
An example of a process for producing an electrophoresis chip of this embodiment is described below. However, an electrophoresis chip may be produced according to processes other than the production process described below.
First, the substrate (upper substrate) 4 is prepared. A method for forming the capillary channel for sample analysis 3x and the capillary channel for sample introduction 3y in the substrate (upper substrate) 4 is not particularly limited, and the capillary channels may be formed, for example, in the same manner as in Embodiment 1 above. A method for forming the four through-holes in the substrate (upper substrate) 4 is also not particularly limited, and the through-holes may be formed, for example, in the same manner as in Embodiment 1 above.
Next, by sealing the bottom surface of the substrate (upper substrate) 4 with the sealing material (lower substrate) 1, an electrophoresis chip of this embodiment can be produced.
The configuration of an electrophoresis chip of this embodiment is not limited to that shown in
An electrophoresis chip of this embodiment can be produced, for example, as follows. However, an electrophoresis chip may be produced according to methods other than the production method described below.
For example, a substrate that is formed from the same material as lower substrate 1 of the electrophoresis chip shown in
In an electrophoresis chip of this embodiment, the length, the width and the thickness of substrate 1 correspond to the maximum length, the maximum width and the maximum thickness of the whole chip described above, respectively. Therefore, the length, the width and the thickness of substrate 1 are arranged to be identical to the maximum length, the maximum width and the thickness of the whole chip described above, respectively.
The inner diameter of each of the four capillary tubes is the same as the maximum diameter of the above-described capillary channel. The length of each of the four capillary tubes is determined according to the maximum length of the capillary channel for sample analysis 3x and the maximum length of the capillary channel for sample introduction 3y.
An example of a process for producing an electrophoresis chip of this embodiment is described below. However, an electrophoresis chip may be produced according to processes other than the production process described below.
First, substrate 1 is prepared. A method for forming the four fluid reservoirs 2a to 2d and the opening (window) 9 in substrate 1 is not particularly limited and, for example, the fluid reservoirs can be formed according to the same method as used for the four fluid reservoirs 2a to 2d of an electrophoresis chip shown in
Next, the four capillary tubes are inserted into substrate 1. In this manner, the electrophoresis chip of this embodiment can be obtained.
The configuration of an electrophoresis chip of this embodiment is not limited to the configuration shown in
An analysis of HbA1c was carried out using an electrophoresis apparatus shown in
Next, a sample was introduced into the second introduction reservoir 2c. An Hb control sample was used as the sample. Next, a voltage of 0.60 kV was applied to the electrode 6c and no voltage was applied to the electrode 6d, thereby creating a potential difference between both ends of the capillary channel for sample introduction 3y. The sample was thereby moved to the intersection of the capillary channel for sample analysis 3x and the capillary channel for sample introduction 3y. In this case, a voltage of 0.30 kV was applied to both electrode 6a and electrode 6b.
Next, while a voltage of 0.40 kV was applied to both the electrode 6c and the electrode 6d, a voltage of 1.00 kV was applied to the electrode 6a and no voltage was applied to the electrode 6b, thereby creating a potential difference between both ends of the capillary channel for sample analysis 3x. The sample 8 was thereby moved from the intersection of the capillary channel for sample analysis 3x and the capillary channel for sample introduction 3y toward the first recovery reservoir 2b side.
Next, the relationship between the absorbance and the distance (migration distance) from the intersection of the capillary channel for sample analysis 3x and the capillary channel for sample introduction 3y was measured with the line detector 7. The results of the measurement are shown in the graph of
An electrophoresis chip of the present invention enables an apparatus to be small, analysis time to be short, and glycosylated hemoglobin to be analyzed with high accuracy. An electrophoresis chip of the present invention is applicable to all technical fields where glycosylated hemoglobin is analyzed, such as laboratory tests, biochemical examinations and medical research. The intended use of the electrophoresis chip is not limited and it is applicable to a broad range of technical fields.
Number | Date | Country | Kind |
---|---|---|---|
2007-119260 | Apr 2007 | JP | national |
Filing Document | Filing Date | Country | Kind | 371c Date |
---|---|---|---|---|
PCT/JP2008/057826 | 4/23/2008 | WO | 00 | 5/8/2009 |