JP3965806B2 - Electrophoresis chip - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an electrophoresis chip used when analyzing, chemical synthesis, or chemical reaction of a very small amount of sample by electrophoresis, and in particular, a flow path is formed inside a transparent plate member, and one of the transparent plate members is provided. The present invention relates to an electrophoresis chip in which holes reaching the flow path are formed on the surface at positions corresponding to end portions of the flow path.
[0002]
[Prior art]
In the case of analyzing a very small amount of protein, nucleic acid, or the like, an electrophoresis method has been conventionally used, and a capillary electrophoresis apparatus is an example of the apparatus technology. A capillary electrophoresis apparatus is a device in which an electrophoresis buffer is filled in a glass capillary having an inner diameter of 100 μm or less, a sample is introduced into one end side, and then a high voltage is applied between both ends to develop an analyte in the capillary. is there. Since the inside surface of the capillary has a large surface area relative to the volume, that is, the cooling efficiency is high, a high voltage can be applied, and a very small sample such as DNA can be analyzed at high speed and with high resolution.
[0003]
In recent years, DJ Harrison et al./Science 261 (1993) 895-897 and Anal. Chim. Acta 283 (1993) can be expected to replace analysis of glass capillaries, which are complicated to handle. As shown in 361-366, an electrophoresis chip (also referred to as a microchip) used for capillary electrophoresis formed by joining two substrates is proposed. An example of the electrophoresis chip is shown in FIG. A pair of transparent substrates (generally glass, quartz, resin, etc.) 31, 32 is formed, and a sample channel 34 and an analysis channel 35 are formed on the surface of one substrate 32. A reservoir 33 is provided as a through hole at a position corresponding to both ends of the flow path 34 and the analysis flow path 35.
[0004]
When this electrophoresis chip is used, as shown in (C), the substrates 31 and 32 are overlapped so that the flow paths 34 and 35 are inside, and the buffer solution is supplied from one of the reservoirs 33 to the sample flow path 34. And into the analysis channel 35. Thereafter, after injecting the sample into the reservoir 33 at one end of the sample channel 34, electrodes are inserted into the respective reservoirs 33, and a predetermined high voltage is applied to both ends of the sample channel 34 for a predetermined time. The sample is guided to the intersection 36 of the sample channel 34 and the analysis channel 35 by electrophoresis (electrophoresis in a narrow sense and electroosmotic flow, hereinafter simply referred to as electrophoresis). Next, a predetermined voltage for electrophoresis is applied to both ends of the analysis channel 35, and the sample existing at the intersection 36 is guided into the analysis channel 35 by electrophoresis and separated. By disposing a detector at an appropriate position in the analysis flow path 35, the separation component is detected.
In addition to separating and analyzing samples, electrophoresis chips are used for chemical synthesis and chemical reactions.
[0005]
[Problems to be solved by the invention]
Since the buffer stored in the reservoir is in contact with the atmosphere on the surface of the reservoir, it is concentrated by evaporation with time. When the concentrated buffer comes into contact with the electrode or flows into the sample channel or the analysis channel, the electrical conductivity changes and the amount of flowing current is not constant. For this reason, the mobility of the substance by electrophoresis is not constant, the reproducibility of the analysis may be reduced, and the expected chemical reaction may not occur.
[0006]
Furthermore, when using a buffer solution, sample, reagent, etc. that easily oxidizes due to contact with air, if it is electrophoresed in the atmosphere, the air-oxidized buffer solution will flow into the flow path, thus blocking the air. Therefore, the experiment had to be performed under an inert atmosphere such as nitrogen, which was very complicated.
In view of the above, the present invention has an object to eliminate problems caused by evaporation or oxidation of a buffer solution or a sample in electrophoresis or chemical synthesis using an electrophoresis chip.
[0007]
[Means for Solving the Problems]
In the present invention, a flow path is formed inside a transparent plate-shaped member, and a hole reaching the flow path is formed on one surface of the transparent plate-shaped member at a position corresponding to the end of the flow path. Is an electrophoresis chip in which an electrode is arranged, and at least one of the end portions of the flow path where the liquid flows in is provided with an electrode at a position away from the hole in the flow path.
[0008]
In the part where the electrode is arranged in the flow path away from the reservoir, the buffer solution is in contact with the electrode inside the flow path at a position sufficiently away from the interface contacting the atmosphere in the reservoir. The concentrated buffer does not reach the electrode within the experiment time, and the electrode reaction can be prevented from being affected by the concentrated buffer. Moreover, only the buffer solution and sample which are separated from the interface with air | atmosphere and are not oxidized can be flowed in in a flow path.
[0009]
The buffer solution near the electrode undergoes a change in composition due to electrolysis. If a buffer solution having a changed composition is present in the flow path, the electrical resistance of the flow path changes, causing a problem.
Therefore, the flow path of an electrophoresis chip of the present invention comprises at least one extension part of is larger at the upstream side and downstream side of the flow of the negative electrode of the liquid.
By increasing the cross-sectional area of the upstream and downstream sides of the electrode, the capacity of the buffer solution near the electrode is increased, and the buffer solution whose composition has been changed by electrolysis is analyzed within the analysis time. Reaching the flow path can be suppressed. Since the electrical resistance of the flow path is determined by the flow path portion in which the analysis or the like is performed, a slight change in the electrical resistance is caused by a change in the composition of a part of the buffer solution, but hardly affects the analysis or the like.
[0010]
【Example】
FIG. 2 is a plan view illustrating an embodiment. FIGS. 3A to 3E are cross-sectional views partially showing this embodiment. FIG. 3A is a cross-sectional view taken along the line AA ′ of FIG. 2, and FIG. A sectional view taken along line -B ', (C) is a sectional view taken along line CC' in FIG. 2, (D) is a sectional view taken along line DD 'in FIG. 2, and (E). FIG. 3 is a cross-sectional view taken along the line EE ′ of FIG. 2.
This embodiment is composed of a base plate 1 and a cover plate 3 made of, for example, glass, and the base plate 1 and the cover plate 3 are joined by thermal fusion.
[0011]
A groove for the analysis channel 5 having a width of 50 μm, a depth of 20 μm, and a length of 60 mm is formed in the base plate 1, and the base end side of the groove for the analysis channel 5 has a width of 1000 μm and a depth of An L-shaped groove having a length of 20 μm and a length of 30 mm is connected for the capillary reservoir 7 containing a buffer solution.
A groove for the sample flow channel 9 having a width of 50 μm, a depth of 20 μm, and a length of 30 mm is formed perpendicular to the analysis flow channel 5, and on the proximal end side of the groove for the sample flow channel 9, A groove having a width of 500 μm, a depth of 20 μm, and a length of 5 mm is formed for the capillary reservoir 11 containing a sample and a buffer solution. Each groove is covered with a cover plate 3 to form an analysis channel 5, capillary reservoirs 7 and 11, or a sample channel 9.
[0012]
Wirings 12a, 12b, 12c and 12d made of Au / Cr (a gold film laminated on a chromium film) are patterned on the surface of the base plate 1 on the side bonded to the cover plate 3, and these wirings Are connected to the other ends of the capillary reservoirs 7 and 11 and the sample flow path 9 or the analysis flow path 5 to form electrodes 13a, 13b, 13c and 13d, respectively, and the other end is a base plate. The terminals 14a, 14b, 14c, and 14d are respectively guided to one side. The electrodes 13 a and 13 b are formed at positions away from both ends of the capillary reservoirs 7 and 11.
[0013]
In the cover plate 3, through holes having a diameter of 1 mm are formed as reservoirs 15a, 15b, 15c, and 15d at positions corresponding to the end portions of the capillary reservoirs 7 and 11, the sample channel 9, and the analysis channel 5, respectively. ing.
The width of the cover plate 3 is smaller than that of the base plate 1 so that the terminals 14a, 14b, 14c, and 14d are exposed. As a result, the electrodes 13a, 13b, 13c, and 13d can be energized by connecting the respective terminals to the power source.
[0014]
When heat-resistant glass such as Pyrex having a high temperature required for bonding is used as the base plate 1 and the cover plate 3, it is preferable to use a refractory metal such as tungsten or molybdenum as the electrode material.
The base plate 1 and the cover plate 3 can also be made of a plastic material.
[0015]
When this electrophoresis chip is used, a buffer solution is injected from the reservoir 15a, and the buffer solution is applied to the capillary reservoir 7, the analysis channel 5, the sample channel 9, the capillary reservoir 11, and the reservoirs 15a, 15b, 15c, and 15d. After filling, the sample is injected into the reservoir 15b. Furthermore, the terminals 14a, 14b, 14c, and 14d are connected to a power source, and a detector that detects a separation component at an appropriate position in the analysis flow path 5 is disposed.
[0016]
A predetermined voltage is applied to the electrodes 13a, 13b, 13c, and 13d so that electrophoresis occurs in the capillary reservoir 11 and the sample channel 9 in the direction from the reservoir 15b to the reservoir 15c, and the sample is connected to the analysis channel 5 and Guide to the intersection of the sample flow path 9.
Next, the voltage application is switched, and a predetermined voltage is applied to the electrodes 13a, 13b, 13c, and 13d so that electrophoresis occurs in the capillary reservoir 7 and the analysis channel 5 in the direction from the reservoir 15a to the reservoir 15d. The sample at the intersection of the analysis channel 5 and the sample channel 9 is guided in the direction of the reservoir 15d of the analysis channel 5 and separated in the analysis channel 5.
Thereafter, a predetermined voltage is continuously applied to the electrodes 13a, 13b, 13c, and 13d, and the separation component is detected at the detection position.
[0017]
The buffer solution stored in the vicinity of the surfaces of the reservoirs 15a, 15b, 15c, and 15d is concentrated over time by evaporation. At the time of analysis, the concentrated buffer solutions in the reservoirs 15a and 15b are guided to the capillary reservoirs 7 and 11 by electrophoresis, respectively. However, since the electrodes 13a and 13b are separated from the reservoirs 15a and 15b, the concentrated buffer solution does not reach the electrodes 13a and 13b during the analysis. As a result, a change in electrical conductivity due to the concentrated buffer can be prevented, and analysis reproducibility can be improved.
[0018]
When a buffer solution or sample that easily oxidizes due to contact with air is used, the buffer solution or sample denatured in the reservoirs 15a and 15b is part of the vicinity of the interface with the air. The denatured buffer in 15b does not reach the electrodes 13a and 13b, and the denatured sample near the surface of the reservoir 15b does not reach the analysis flow path 5. As a result, even in the case of using a buffer solution or sample that easily oxidizes due to contact with air, analysis can be performed in the atmosphere.
[0019]
In addition, the buffer solution in contact with the electrodes 13a and 13b undergoes a change in composition due to electrolysis. However, the capillary reservoirs 7 and 11 are sufficiently provided between the electrode 13a and the analysis flow path 5 and between the electrode 13b and the sample flow path 9. Thus, the buffer solution having the composition change does not reach the analysis channel 5 and the sample channel 9 within the analysis time. The change in the electrical resistance of the flow path is caused by the buffer solution that causes the change in the composition. However, since the electrical resistance of the flow path is determined by the analysis flow path 5 and the sample flow path 9, the change in the electrical resistance is slight. It does not affect the analysis.
[0020]
4A and 4B are diagrams illustrating another embodiment, in which FIG. 4A is a plan view, and FIG. 4B is a cross-sectional view taken along the line AA ′ in FIG.
Through holes 17 are formed in the vicinity of the center of the capillary reservoir 7a of the base plate 1, in the channels on both ends of the sample channel 9a, and in the channels on the other end of the analysis channel 5, respectively. Electrodes 19a, 19b, 19c, 19d made of Pt lines are inserted into the holes 17 with their tips protruding into the grooves. The electrodes 19 a, 19 b, 19 c, 19 d are fixed to the base plate 1 by filling a filler such as epoxy resin or solder glass into the through holes 17. The other ends of the electrodes 19a, 19b, 19c, and 19d are connected to a power source.
[0021]
In the cover plate 3, through holes are formed as reservoirs 21a, 21b, 21c, and 21d at positions corresponding to the end portions of the capillary reservoir 7a, the sample channel 9a, and the analysis channel 5.
Also in this embodiment, since the electrode 19a is arranged inside the capillary reservoir 7a at a position away from the reservoir 21a, the same effect as in the embodiment of FIG. 2 can be obtained.
[0022]
5A and 5B are diagrams showing still another embodiment, in which FIG. 5A is a plan view, FIG. 5B is a cross-sectional view taken along the line AA ′ in FIG. It is an expanded sectional view of a mark, and (D) is a sectional view which met a BB 'line of (A).
Similar to the embodiment of FIG. 4, grooves for forming the analysis channel 5, capillary reservoir 7 a and sample channel 9 a are formed in the base plate 1, and reservoirs 21 a, 21 b, 21 c and 21 d are formed in the cover plate 3. ing.
The strip-like electrodes 23a, 23b, 23c, and 23d made of Au / Cr are disposed on the reservoir 21a side from the bottom of the grooves forming the capillary reservoir 7a and the bottoms of the reservoirs 21b, 21c, and 21d into the flow path. It is arranged from the end to the vicinity of the center of the groove.
[0023]
On the surface of the cover plate 3 opposite to the surface joined to the base plate 1, wirings 25a, 25b, 25c, 25d made of Au / Cr are patterned, one end of which is the reservoirs 21a, 21b, 21c, 21d. The electrodes 27a, 27b, 27c, and 27d are guided to the inner wall, and the other end is a terminal for energization connected to a power source. The electrodes 23a, 23b, 23c, and 23d are electrically connected to the electrodes 27a, 27b, 27c, and 27d.
[0024]
The manufacturing process of this embodiment will be briefly described.
After the grooves for forming the analysis flow path 5, the capillary reservoir 7a, and the sample flow path 9a are formed in the base plate 1, the capillary reservoir 7a, the sample flow path 9a, and the analysis flow path 5 are formed by sputtering and photolithography. Electrodes 23a, 23b, 23c, and 23d are formed on a part of the bottom of the groove to be formed. After the base plate 1 is fused to the cover plate 3 in which the through holes for the reservoirs 21a, 21b, 21c, and 21d are formed, the surface of the cover plate 3 and the reservoirs 21a, 21b, 21c, and 21d are formed by sputtering and photolithography. On the inner wall, wirings 25a, 25b, 25c, 25d made of Au / Cr and electrodes 27a, 27b, 27c, 27d are patterned.
Also in this embodiment, since the electrodes 23a, 23b, 23c, and 23d are arranged to reach the inside of the capillary reservoir 7a at a position away from the reservoir 21a, the same effect as in the embodiment of FIG. 2 can be obtained.
[0025]
The embodiment of FIGS. 2, 4 and 5 is applied to an analysis technique using separation by electrophoresis, but these embodiments are chemical synthesis techniques in which a substance is moved by electrophoresis to cause a chemical reaction. It can also be applied to.
Furthermore, the numerical values and shapes in the above-described embodiments represent examples, and the present invention is not limited to these embodiments, and various modifications are made within the scope of the gist of the claims. be able to.
[0026]
【The invention's effect】
In the electrophoresis chip of the present invention, since the electrode is provided at a position away from the reservoir inside the channel on the side where the buffer solution or sample flows in the channel, the buffer solution near the reservoir surface concentrated by evaporation remains within the experiment time. In addition, since only the buffer solution and the sample that are away from the interface with the atmosphere flow into the flow path without reaching the electrode, problems due to evaporation and oxidation of the buffer solution and the sample can be solved.
Furthermore, by providing a capillary reservoir with a large channel cross-sectional area in the vicinity of the buffer solution in the channel and the electrode on the side where the sample flows, the buffer solution whose composition has changed due to electrolysis can be analyzed within the analysis time. Reaching the flow channel can be suppressed, and the change in the electrical resistance of the flow channel can be suppressed.
[Brief description of the drawings]
1A and 1B are diagrams showing an electrophoresis chip, wherein FIGS. 1A and 1B are plan views showing a transparent plate-like member constituting the electrophoresis chip, and FIG. 1C is a front view of the electrophoresis chip.
FIG. 2 is a plan view illustrating an embodiment.
3 is a diagram showing a partial cross section of the embodiment, (A) is a cross-sectional view taken along line AA ′ of FIG. 2, and (B) is a line BB ′ of FIG. (C) is a cross-sectional view taken along the line CC ′ of FIG. 2, (D) is a cross-sectional view taken along the line DD ′ of FIG. 2, and (E) is E of FIG. It is sectional drawing along line -E '.
4A and 4B are diagrams illustrating another embodiment, in which FIG. 4A is a plan view, and FIG. 4B is a cross-sectional view taken along line AA ′ in FIG.
5A is a plan view, FIG. 5B is a cross-sectional view taken along the line AA ′ of FIG. 5A, and FIG. It is an expanded sectional view of a mark, and (D) is a sectional view which met a BB 'line of (A).
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Base plate 3 Cover plate 5 Analysis flow path 7, 11 Capillary reservoir 9 Sample flow paths 12a, 12b, 12c, 12d Wirings 13a, 13b, 13c, 13d Electrodes 14a, 14b, 14c, 14d Terminals 15a, 15b, 15c, 15d reservoir

Claims (1)

A flow path is formed inside the transparent plate-shaped member, and a hole reaching the flow path is formed on one surface of the transparent plate-shaped member at a position corresponding to the end of the flow path, and an electrode is disposed at each end. In the prepared electrophoresis chip,
An electrode is provided at a position away from the hole in the flow path at at least one end of the flow path where the liquid flows , and the cross-sectional area of the flow path is upstream and downstream of the electrode. An electrophoretic chip characterized by an increase in the size .

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