JP2005114433A - Chip for electrophoresis and sample analyzing method using it - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a chip for electrophresis enhanced in the resolving power of the separation/detection of a sample, excellent in the reproducibility and determination properties of the analysis of the sample in the analysis of the sample such as an extremely small amount of protein, nucleic acid or the like, and suppressing applied voltage at the time of separation of the sample low, and also to provide a sample analyzing method using it. <P>SOLUTION: The chip 1 for electrophoresis is equipped with a port 2 for introducing the sample, a flow channel 3 of the introduced sample, and at least two electrodes 4 connected to the flow channel. Two or more air permeation parts 56 are connected side by side in the vicinity of either one of the electrodes 4 and the respective fine pipe bundles 6 are connected to air ports 8 for introducing/discharging air to air passages through air passages 7. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention reproduces biological components such as minute amounts of proteins, peptides, amino acids and nucleic acids, drugs such as vitamins and steroids, optical isomers, and inorganic compounds at high resolution and low voltage. The present invention relates to an electrophoresis chip capable of quantitatively separating and detecting with good quality and a sample analysis method using the same.

  In recent years, active research has been conducted to produce microchips with a microchannel structure using micromachine technology and to reduce the size and integration of chemical and bio-related devices. These research fields are called microchemical analysis systems, and are expected to improve sample analysis accuracy, reduce sample / reagent / waste solution, increase processing speed and efficiency, and reduce system size and automation. Many microchips having functions such as electrophoresis and PCR have been announced so far.

A cross injection method is known as an electrophoresis method using a microchip (see Non-Patent Document 1).
An example of an electrophoresis chip using the cross injection method is shown in FIG. In the electrophoresis chip 11 of this example, the cross flow path is formed by bonding a polymethylsiloxane (PDMS) chip having a cross groove on a glass substrate. Ports for introducing liquid are provided at four ends of the cross-shaped flow path, and electrodes (not shown) are connected thereto. In this example, the polymer solution is filled from the port 24 into the cross flow path, and then the liquid sample is injected into the port 21, and the ports 22 and 23 are filled with the buffer solution. First, a sample is filled in the short-side channel 31 by applying a voltage to the short-side channel 31 for introducing the sample. Subsequently, a voltage is applied to the long side channel 32 of the cross for sample separation, and a micro sample piece (plug) at the intersection IS of the short side channel 31 and the long side channel 32 is inserted into the long side of the cross. It introduces into the flow path. Thereafter, the sample is separated into bands for each molecular weight of the mixed substance.
Effenhauser, et.al., “Integrated Capillary Electrophoresis on Flexible Silicone Microdevices: Analysis of DNA Resriction Fragments and Detection of Single DNA Molecules on Microchips,” Anal. Chem, 1997, 69, 3451-3457

In the above technique, in order to increase the resolution of the sample separation / detection, the liquid levels of the ports 22 and 24 provided at both ends of the flow path 32 having the long side of the cross are adjusted to be equal. There is a need to. However, in practice, it is difficult to adjust the liquid levels of the ports 22 and 24 to be equal. Therefore, a difference occurs in the height of both liquid levels of the ports 22 and 24, and the long side flow path is caused by the difference in pressure applied to both ends of the long side flow path 32 due to the difference in height between the two liquid levels. An internal flow is likely to occur in the liquid in the liquid 32. As a result, the sample analysis method using this technique has a problem that the resolution of sample separation and detection is relatively low, and the reproducibility of sample analysis is not good.
In this technique, an electrical plug is generated by controlling the applied voltage. Depending on the time of voltage application at the time of introduction, the ratio of the components in the generated sample plug is the configuration of the original sample at the port 21. There is a problem that it is different from the ratio of components. Therefore, in this technique, it is difficult to compensate the quantitativeness when the voltage application time for sample introduction is not sufficient.
In this technique, an applied voltage for introducing the sample and an applied voltage for separating the sample are required to be about 100 to 300 volts, and a large-scale apparatus is required.

  The present invention has been made in view of such a situation, and in the analysis of a sample such as a very small amount of protein or nucleic acid, 1) the resolution of separation and detection of the sample is high, and 2) the reproducibility of the analysis of the sample is excellent. 3) It is an object of the present invention to provide an electrophoresis chip capable of quantitative analysis of a sample and 4) keeping an applied voltage low when separating a sample. It is another object of the present invention to provide a sample analysis method using the electrophoresis chip.

  As a result of extensive research, the present inventors have connected two or more gas permeable portions side by side and connected each gas permeable portion to this gas permeable portion as a mechanism for introducing a liquid sample into the flow channel. If an electrophoresis chip incorporating a mechanism for introducing a sample or a buffer into a flow path is used by connecting a gas port via a gas passage and introducing and discharging gas from the gas port, Has been found to be resolved. Here, the gas permeation section allows gas to permeate but does not allow liquid or the like to permeate.

In this electrophoresis chip, unlike the conventional technique, 1) it is not necessary to provide ports at both ends of the flow path. Therefore, in the analysis of the sample using this electrophoresis chip, as in the conventional technique, It is not necessary to adjust the liquid level difference between the two ports located at both ends of the flow path, and 2) the sample having the same component as the original sample because no electrical operation is involved in generating the sample plug. This is because a plug can be generated.
Moreover, according to the sample detection method using this electrophoresis chip, it discovered that the said subject could be solved, and completed this invention based on these knowledge.

  That is, the first invention of the present invention is an electrophoresis chip comprising a port for introducing a sample, a flow path of the introduced sample, and two or more electrodes connected to the flow path. In the vicinity of any one of the electrodes, two or more gas permeable portions are connected in parallel, and each gas permeable portion is a gas port for introducing and discharging gas into the gas passage through the gas passage. It is a chip | tip for electrophoresis characterized by being connected with.

  The second invention of the present invention is the electrophoresis according to the first invention, characterized in that the gas permeable portion is a bundle of thin tubes composed of a plurality of thin tubes or a pore portion having a large number of pores. Chip.

According to a third aspect of the present invention, in the first or second aspect, the edge of the electrode protruding into the flow path is a straight line perpendicular to the longitudinal direction of the flow path. It is the chip | tip for electrophoresis as described.
Use of this electrophoresis chip is preferable because, when a sample is separated, the band shape of the separated substance is not deformed and remains rectangular, so that the resolution of separation and detection of the sample is further improved.

A fourth invention of the present invention is the electrophoresis chip according to the second invention or the third invention, wherein the wall surface of the capillary tube is subjected to hydrophobic processing or hydrophilic processing.
In this electrophoresis chip, if the wall surface of the thin tube bundle is hydrophobic, it is preferable to analyze a hydrophilic sample because a hydrophilic substance is prevented from entering the thin tube bundle. In this electrophoresis chip, if the wall surface of the thin tube bundle is hydrophilic, it is suitable for analyzing a hydrophobic sample because a hydrophobic substance is prevented from entering the thin tube bundle.

  According to a fifth aspect of the present invention, in any one of the second to fourth aspects, the cross-sectional shape of the thin tube is rectangular, and the width and height are 0.1 μm or more and 20 μm or less. The electrophoresis chip as described in 1. above.

A sixth invention of the present invention is the electrophoresis chip according to any one of the first to fifth inventions, wherein a plurality of the flow paths are arranged in parallel.
Use of this electrophoresis chip is preferable in that it is not necessary to provide a different gas port for each flow path, and the apparatus can be simplified. Furthermore, according to this electrophoresis chip, since a plurality of channels are provided, a marker whose molecular weight is known is introduced into one of the channels, and a sample whose molecular weight is to be measured is introduced into the other channel, By performing electrophoresis with the same power supply device, the molecular weight of the sample can be measured.

According to a seventh aspect of the present invention, there is provided a glass substrate having an upper surface on which the electrode is formed and a resin substrate having a processed surface that has been grooved in advance, and the processed surface of the resin substrate is provided on the upper surface of the glass substrate. By facing and bonding,
According to any one of the first to sixth inventions, wherein the flow path, the gas permeable portion, and the gas passage are formed by a groove on a processed surface of the resin substrate. It is the chip | tip for electrophoresis as described.

In an eighth aspect of the present invention, two or more gas permeable portions are arranged in the vicinity of an electrophoresis start electrode provided at an end of the flow path,
After introducing the sample into a part or the whole of the flow path by discharging the gas from the first gas port leading to the gas permeation portion closest to the electrode for starting electrophoresis,
A part of the sample is measured into the flow path by introducing a gas from a second gas port connected to any one gas permeable part other than the gas permeable part closest to the electrode for starting electrophoresis. Leave, drain the rest of the sample from the flow path,
Next, a buffer solution is introduced into the flow path by discharging gas from the second gas port,
The electrophoresis chip according to any one of the first to seventh inventions, wherein a sample is separated by applying a voltage to an electrode.

According to a ninth aspect of the present invention, there is provided an electrophoresis chip array in which two or more electrophoresis chips according to any one of the first to eighth aspects are arranged in parallel.
According to this electrophoresis chip array, since a plurality of flow paths are provided, a marker whose molecular weight is known is introduced into one of the flow paths, and a sample whose molecular weight is to be measured is introduced into the other flow path. By performing electrophoresis with the power supply device, the molecular weight of the sample can be measured, and the component amount of the sample can be quantified.

  According to a tenth aspect of the present invention, there is provided a sample using the electrophoresis chip according to any one of the first to eighth aspects or the electrophoresis chip array according to the ninth aspect. This is an analysis method.

In an eleventh aspect of the present invention, two or more gas permeable portions are arranged in the vicinity of an electrophoresis start electrode provided at an end of the flow path,
After introducing the first sample into a part or the whole of the flow path by discharging the gas from the first gas port leading to the gas permeation part closest to the electrode for starting electrophoresis,
A part of the first sample is measured in the flow path by introducing a gas from the second gas port connected to any one gas permeable part other than the gas permeable part closest to the electrode for starting electrophoresis. Leaving the other part of the first sample out of the flow path,
Then, after introducing the second sample into the flow path by discharging the gas from the second gas port,
Gas is introduced from a third gas port connected to any one gas permeable portion located on the opposite side of the electrophoresis start electrode through a gas permeable portion connected to the second gas port. Thereby leaving a part of the second sample in the flow path, leaving the other part of the second sample out of the flow path,
Next, a buffer solution is introduced into the flow path by discharging gas from the third gas port,
The method for analyzing a sample according to the tenth invention, wherein a voltage is applied to the electrode to analyze a reaction between the first sample and the second sample.
According to this sample analysis method, genes can be identified by detecting the reaction between two types of samples, for example, the binding between single-stranded DNAs or the reaction between DNA and a restriction enzyme.

When the electrophoresis chip of the present invention is used, in analysis of a sample such as a very small amount of protein or nucleic acid, analysis with high resolution of sample separation / detection and excellent reproducibility / quantitativeness can be performed. In addition, since the applied voltage for separating the sample is low, the electrophoresis chip of the present invention does not require a large-scale power supply device and is easy to use. Therefore, it can be used in various fields and places and is useful.
Moreover, the sample analysis method of the present invention has the same effects as described above and is useful.

Hereinafter, the present invention will be described in detail with reference to the drawings.
Schematic diagrams of an example of an electrophoresis chip according to the present invention are shown in FIGS. FIG. 1B is an enlarged schematic view of the vicinity of the end of the flow path 3 when the electrophoresis chip 12 shown in FIG. The electrophoresis chip 12 in this example has two main parts, a glass substrate 11 and a resin substrate 13. The glass substrate 11 has a size of 50 mm × 76 mm × 1 mm. The resin substrate 13 has a size of 25 mm × 40 mm × 3 mm and is bonded onto the glass substrate 11. Two electrodes 4 are provided on the upper surface 11 a of the glass substrate 11.

  A port 2 is provided at the end of the resin substrate 13, from which a sample can be introduced. The port 2 is substantially cylindrical and has a diameter of 1 mm. A flow path 3 is provided in a straight line from the port 2. The length of the flow path 3 in the longitudinal direction is 15 mm, the cross section is substantially rectangular, the width is 90 μm, and the height is 25 μm. The channel 3 is closed at its end 33. One of the two electrodes 4 provided on the upper surface 11 a of the glass substrate 11 is connected to the end 33 of the flow path 3. Since this electrode is located at the starting point of electrophoresis, it is hereinafter referred to as an electrophoresis starting electrode 41 as appropriate. The end of the other electrode 4 is located in the flow path 3 about 3 mm from the end 33. A part of each electrode protrudes into the flow path 3. The electrode 4 has a thickness of several tens of nm.

  In the vicinity of the electrophoresis start electrode 41 connected to the end 33 of the flow path 3, two gas permeable portions 56 are arranged side by side and connected to the flow path 3. The gas permeation section 56 communicates the flow path 3 and the gas passage 7 so as to allow gas to permeate but not liquid or the like. In this example, the gas permeable portion 56 is composed of a bundle of thin tubes 6 composed of a plurality of thin tubes 5, and the thin tubes 5 communicate with the flow path 3. One thin tube bundle 6 has 1 to 100 thin tubes 5 arranged in parallel, and one thin tube 5 has a rectangular cross section, a width of 2 μm, and a height of 3 μm. The interval between the thin tubes is 12.5 μm. In this example, the two thin tube bundles 6 are provided at right angles to the flow path 3. A gas passage 7 is connected to each thin tube bundle 6, and the thin tube 5 communicates with the gas passage 7. The cross-sectional shape of the gas passage 7 is rectangular, the width is 60 μm, and the height is 25 μm.

FIG. 3 shows a CD cross-sectional view of the electrophoresis chip 12 shown in FIG. This clearly shows that the flow path 3, the narrow tube 5 and the gas passage 7 communicate with each other.
A gas port 8 is connected to one end of the gas passage 7 not communicating with the narrow tube, and the gas passage 7 and the gas port 8 are communicated with each other. The gas port is substantially cylindrical and has a diameter of 1 mm. A gas tube 9 is inserted into the gas port 8. The gas tube is connected to an air pressure control device (not shown). By controlling the device, the gas tube can introduce gas into the gas port 8, the gas passage 7, the thin tube bundle 6, and the flow path 3, and can discharge the gas. It has become. The pneumatic control device may be an automated machine or a manual instrument such as a syringe.
The size and shape of each member of the electrophoresis chip 1 of the present invention and the positional relationship between the members are not limited to this example. In addition, by providing three or more electrodes 4, two or more measurement lengths of electrophoresis can be set.

  In the electrophoresis chip 12 of this example, the edge 4a protruding into the flow path 3 of the electrode 4 connected to the flow path 3 is orthogonal to the longitudinal direction of the flow path 3 as shown in FIG. A straight line is preferred. In this case, when the sample is separated, the band shape of the separated substance is not deformed and remains rectangular, so that the resolution of separation and detection of the sample is further improved. This is presumed to be because the electric lines of force generated between the electrodes at the time of electrophoresis can easily take the longitudinal direction of the flow path 3 by making the edge 4a a straight line orthogonal to the longitudinal direction of the flow path 3. Is done.

  For the resin substrate 13 used in the electrophoresis chip 12 of this example, polydimethylsiloxane (hereinafter referred to as “PDMS”), which is a kind of silicone elastomer, is used. The electrode 4 is a laminate of chromium and gold. The gas tube 9 is a normal silicone tube.

  In this example, the electrophoresis chip is manufactured by etching the electrode 4 on the upper surface 11a of the glass substrate 11, forming the resin substrate 13 by molding a groove 13b in the resin, and the upper surface of the glass substrate 11. 11a and the process of making the process surface 13a of the resin substrate 13 face each other and bonding as shown in FIG. When the glass substrate 11 and the resin substrate 13 are bonded together, the flow path 3, the thin tube 5 and the thin tube bundle 6, and the gas passage 7 are formed by the molded grooves.

  The electrode structure of the glass substrate is formed by vapor deposition of chromium and gold, patterning of a photoresist as an etching protective film, and etching of chromium.

  In order to mold the groove in the resin, first, an inversion mold for molding the resin is manufactured. For example, “Hosokawa, et. Al.,“ Handling of Picoliter Liquid Samples in a Poly (dimethylsiloxane) -Based Microfluidic Device, ”Anal. Chem. 1999, 71, 4781-4785” (reference 1). ) Can be used. In this example, a mask for forming a groove for the flow path 3 and the gas passage 7 and a mold for forming a groove for the thin tube bundle 6 are manufactured by a photolithography method using a photoresist. On the other hand, the grooves can be molded in the resin by using the method described in the above-mentioned reference 1, that is, the replica molding method. The resin substrate on which the groove is molded is drilled in a necessary portion with a metal drill to create the port 2 and the gas port 8.

  In this manufacturing method, since the resin substrate 13 has self-adhesiveness with respect to a flat surface such as the glass substrate 11, the flow path 3, the narrow tube 5 and the narrow tube bundle 6 are not subjected to a special joining process. The gas passage 7 can be formed.

  A gas tube 9 is inserted into the gas port 8 and, if necessary, the joint of the gas tube 9 is closed to the gas port 8 with an adhesive so as to prevent gas leakage.

  In the present invention, if PDMS is used as the resin, the wall surface 5a of the capillary tube becomes hydrophobic, and thus the electrophoresis chip thus produced is convenient for analyzing a sample in an aqueous solution containing DNA or protein. Good. On the other hand, a hydrophilic polymer (for example, polyethylene glycol) or the like is applied to the grooves for forming the thin tubes 5 and the flow paths 3, or a glass substrate having the same shape is used instead of the resin substrate 13. As a result, the walls of the thin tube 5 and the flow path 3 become hydrophilic, and the thus prepared electrophoresis chip can analyze a sample in a hydrophobic solution containing lipids, organic substances, and the like. The glass substrate can also be manufactured by manufacturing a reversal mold for molding glass in the same manner as the resin substrate 13 is manufactured. Alternatively, the glass substrate may be formed by forming the thin tube 5 and the flow path 3 in the glass by wet etching.

In the present invention, the gas permeation part 56 may have a structure of the porous body 51 having many pores instead of the structure of the thin tube bundle 6.
FIG. 5 shows an example in which the gas permeable portion 56 has a porous body 51 structure. FIG. 5A is an enlarged schematic view of the vicinity of the end of the flow path 3 when the electrophoresis chip of this example is viewed from directly above, and FIG. 5B is a diagram of the electrophoresis chip of this example. It is sectional drawing when cut | disconnecting in CD cross section shown to Fig.1 (a). As is apparent from this figure, the electrophoresis chip of this example is different from the structure of the porous body 51 in place of the structure of the narrow tube 6 in the portion connecting the flow path 3 and the gas passage 7. 1 has the same configuration as the electrophoresis chip 12 shown in FIG.

The electrophoresis chip of this example can be manufactured by molding a resin or the like by the same method as described above and forming the porous body 51 by insert molding. That is, in insert molding, a porous material that is an insert product is loaded into an inversion mold, and then a resin is injected and the porous material is wrapped and solidified with a molten resin, so that the resin and the porous body 51 are integrated. Make a member.
Substances used for the porous body 51 include 1) porous glass, porous silicon, activated carbon and ceramics having an irregular pore structure, and 2) zeolite having a regular pore structure, layered There are structures, etc.

A method of using the electrophoresis chip 12 of this example will be described with reference to FIG. In this example, the gas permeable portion 56 is composed of thin tube bundles 61 and 62.
Sample S is injected into port 2 (not shown). When the gas is discharged from the gas port 81 by the gas control device (not shown) and the pressures of the thin tube bundle 61 and the gas passage 71 are made negative with respect to the flow channel 3, the sample S is partly in the flow channel 3. It is introduced (FIG. 6 (a)). Here, one end of the sample S is placed near the end 33 (or the electrophoresis start electrode 41), and the other end is placed between the bundle of thin tubes 62 and the port 2. As shown in FIG. 6B, when gas is introduced from the gas port 82 and the pressures of the thin tube bundle 62 and the gas passage 72 are made positive with respect to the flow passage 3, a part SP of the sample remains, This is for forming a small sample piece (plug). In this way, compared with the case where the sample S is introduced into the entire flow path 3, analysis can be performed with fewer samples, and valuable samples can be used effectively.
Next, when the gas is introduced from the gas port 82 by the gas control device and the pressures of the thin tube bundle 62 and the gas passage 72 are made positive with respect to the flow passage 3, a part of the sample SP remains, and a small sample piece (plug) ) Is formed (FIG. 6B).

Next, buffer B is injected into port 2. When the gas is discharged from the gas port 82 and the pressure of the thin tube bundle 62 and the gas passage 72 is made negative with respect to the flow passage 3 by the gas control device, the buffer B is introduced into the flow passage 3 (FIG. 6). (C)).
Thereafter, by applying a voltage to the electrode 4, the sample moves toward the port 2, but a substance having a low molecular weight has a high mobility, and a substance having a high molecular weight has a low mobility, so that the sample can be separated and detected. it can. Here, since the electrode has a thickness of several tens of nm, the structure of the flow path 3 is substantially uniform in the longitudinal direction, and the influence on the analysis of the sample can be ignored. In addition, the time required for sample separation is about 3 minutes, and the performance is equivalent to that of the conventional technique. However, since the component ratio does not change when the sample plug is generated, the sample is quantitative. Can be analyzed.

  The applied voltage required to separate and detect the sample using the electrophoresis chip 12 of this example may be several V (5 to 6 V). Therefore, the power source used may be a normal DC power supply device, and a dry battery. You may use what connected in series. In any case, no large-scale equipment is required as in conventional techniques.

  Regarding the detection of the sample, in addition to detecting the sample dyed with a fluorescent dye with a fluorescence microscope or the like, a laser excitation fluorescence method or the like can be used, or a sample labeled with a radioactive element can be detected with a counter. Further, according to infrared spectroscopy or surface plasmon analysis, it is not necessary to stain or label the sample, so that it can be detected in the original state of the sample. In addition, electrochemical detection, conductivity detection, etc. can be considered.

A schematic diagram of another example of the electrophoresis chip according to the present invention is shown in FIG. In this example, the gas permeable portion 56 is composed of a thin tube bundle 6 composed of a plurality of thin tubes 5, and the thin tubes 5 communicate with the flow path 3. The description of the same members as those used in FIG. 1 is omitted. In the electrophoresis chip 14 of this example, four thin tube bundles 6 are aligned and connected to the flow path 3, and accordingly, the gas passage 7 and the four gas ports 8 are provided. The configuration is the same as that of the electrophoresis chip 12. The electrophoresis chip 14 in this example can be manufactured by the same method as described above.
In this example, similarly to the above, the gas permeable portion 56 may have the structure of the porous body 51 instead of the structure of the narrow tube 6.

A method of using the electrophoresis chip 14 of this example will be described with reference to FIG. In this example, the gas permeable portion 56 is composed of thin tube bundles 61, 62 and 63.
The first sample S1 is injected into the port 2 (not shown). When gas is discharged from the gas port 81 by a gas control device (not shown) and the pressure of the thin tube bundle 61 and the gas passage 71 is made negative with respect to the flow passage 3, the sample S1 is introduced into the entire flow passage 3. (FIG. 7A). Here, one end of the sample S1 is placed near the end 33 (or the electrophoresis start electrode 41), and the other end is placed between the bundle of thin tubes 62 and the port 2. As shown in FIG. 7B, when a gas is introduced from the gas port 82 and the pressure of the thin tube bundle 62 and the gas passage 72 is made positive with respect to the flow passage 3, a part of the sample S1P is left, This is for forming a small sample piece (plug). In this way, it is possible to analyze with a smaller number of samples as compared with the case where the sample S1 is introduced into the entire flow path 3, which is economical.
Next, when the gas is introduced from the gas port 82 by the gas control device and the pressures of the thin tube bundle 62 and the gas passage 72 are made positive with respect to the flow passage 3, a part S1P of the first sample remains, and the first A small sample piece (plug) of the sample is formed (FIG. 7B).

Next, the second sample S2 is injected into the port 2. When the gas is discharged from the gas port 82 and the pressure of the thin tube bundle 62 and the gas passage 72 is made negative with respect to the flow path 3 by the gas control device, the second sample S2 is introduced into the flow path 3 ( FIG. 6 (c)). Here, one end of the sample S2 is placed near the thin tube bundle 62, and the other end is placed between the thin tube bundle 63 and the port 2. As shown in FIG. 7D, when gas is introduced from the gas port 83 and the pressure of the thin tube bundle 63 and the gas passage 73 is made positive with respect to the flow passage 3, a part of the sample S2P is left, This is for forming a small sample piece (plug). In this way, it is possible to analyze with a smaller number of samples as compared with the case where the sample S2 is introduced into the entire flow path 3, which is economical.
Next, when the gas is introduced from the gas port 83 by the gas control device and the pressure of the thin tube bundle 63 and the gas passage 73 is made positive with respect to the flow passage 3, a part S2P of the second sample remains, and the second A small sample piece (plug) of the sample is formed. In this way, the reaction can be performed by bringing together the micro sample piece (plug) of the first sample and the micro sample piece (plug) of the second sample.

Next, the second buffer B is injected into the port 2. When the gas is discharged from the gas port 83 and the pressure of the thin tube bundle 63 and the gas passage 73 is made negative with respect to the flow path 3 by the gas control device, the buffer B is introduced into the flow path 3.
Thereafter, by applying a voltage to the electrode 4, the sample moves toward the port 2, but a substance having a low molecular weight has a high mobility, and a substance having a high molecular weight has a low mobility, so that the sample can be separated and detected. it can.

  As described above, when the electrophoresis chip 14 is used, by introducing two or more kinds of samples, these samples can be reacted and their states can be examined. For example, if a single-stranded RNA and a complementary single-stranded RNA are used as a sample, these two types bind to each other, so the molecular weight increases and a separate band is formed with a single RNA. By detecting this, it is confirmed that single-stranded RNAs are bound to each other. Even when DNA and DNA-binding protein are used as a sample, the binding between the two can be confirmed in the same manner.

  Further, when this electrophoresis chip 14 is used, the sample volume of the sample piece is increased by using the port 81 and the port 83 or the port 81 and the port 84 for a relatively low concentration sample. Can be analyzed.

  In the electrophoresis chip of the present invention, the number of thin tube bundles 6 composed of a plurality of thin tubes 5 in the vicinity of the electrophoresis start electrode 41 connected to the end 33 of the flow path 3 may be two or more. In addition, the greater the number, the greater the degree of freedom of the combination of samples to be separated and detected, but in reality, it may be about 7-8.

  In the electrophoresis chip of the present invention, as shown in FIGS. 8 and 9, a plurality of flow paths 3 can be provided in one chip. Use of this electrophoresis chip is preferable in that it is not necessary to provide different gas ports 8 for each flow path, and the apparatus can be simplified. Furthermore, according to this electrophoresis chip, since a plurality of channels are provided, a marker whose molecular weight is known is introduced into one of the channels, and a sample whose molecular weight is to be measured is introduced into the other channel, By performing electrophoresis with the same power supply device, the molecular weight of the sample can be measured.

  A plurality of electrophoresis chips 12 or 14 of the present invention can be arranged in parallel to form an electrophoresis chip array 10 (see FIG. 10). According to this, since a plurality of flow paths 3 are provided, a marker having a known molecular weight is introduced into one of the flow paths 3, a sample whose molecular weight is to be measured is introduced into the other flow path, and the same power supply device is used. By performing electrophoresis, there is an advantage that the molecular weight of the sample can be measured.

  Using the electrophoresis chip of the present invention, separation and detection of a DNA marker (100 bp Molecular Ruler; BIO-RAD, USA) in which 10 types of DNA that are 100 bases long from 100 bases to 1000 bases are mixed is used. went. This DNA marker was stained with a fluorescent label (SYGRGreen I; Molecular Probes, USA). Hydroxymethyl cellulose (HEC) solution (Cellulose, Hydroxyethyl ether (molecular weight 90000-105000); Polyscience, Inc., USA) (1.2% concentration in TBE buffer) was used as a sieve for separating DNA samples. In this example, the electrophoresis chip 14 was used. As the gas control device, an automatic air pressure control device was used, and each silicone tube was connected to the automatic air pressure control device to control the gas.

  Since the sample used is only one of the DNA markers, the sample introduction, separation and detection methods are the same as those described for the electrophoresis chip 12. That is, the DNA marker was injected into port 2. Gas was discharged from the gas port 81 by an automatic air pressure control device, the pressure of the thin tube bundle 61 and the gas passage 71 was made negative with respect to the flow channel 3, and the DNA marker was introduced into the entire flow channel 3 ( FIG. 8B). Next, gas is introduced from the gas port 82 by a syringe, and the pressure of the thin tube bundle 62 and the gas passage 72 is made positive with respect to the flow passage 3, leaving a part SP of the DNA marker, and a micro sample piece (plug). Formed. The liquid volume was about 315 pL (FIG. 8 (c)).

  Next, the HEC solution was injected into port 2 as a buffer. Gas is discharged from the gas port 82 by an automatic pneumatic control device, and the HEC solution is introduced into the flow path 3 by making the pressure of the thin tube bundle 62 and the gas path 72 negative with respect to the flow path 3 (FIG. 8 (d)). Thereafter, the sample was separated and detected by applying a voltage of 5 V to the electrode 4. The applied voltage was performed by a direct current power supply (PAK20-18A; Kikusui, Japan). For the detection of the DNA sample, a fluorescence microscope connected with a CCD camera was used. The detector was installed at a point 2.64 mm from the electrophoresis start electrode.

An embodiment of the present invention is shown in FIGS. (E) is after 1 second from the start of electrophoresis, (f) is after 10 seconds, (g) is after 25 seconds, and (h) is after 90 seconds. It can be seen that the separation is complete in 90 seconds. It can also be seen that the individual bands remain rectangular.
Moreover, the result of the detection of the DNA marker of a present Example is shown in FIG. In this example, no overlapping of undesired peaks of 10 types of DNA was observed.

  Thus, in this example, it was confirmed that the resolution of sample separation / detection was high. In addition, the electrophoresis chip of this embodiment has a low applied voltage and is suitable for downsizing the apparatus. This electrophoresis chip may also be applied to gene hybridization and analysis of gene cleavage by restriction enzymes.

It is a schematic diagram which shows an example of the chip | tip for electrophoresis of this invention. It is a schematic diagram which shows an example of the chip | tip for electrophoresis of this invention. It is sectional drawing of an example of the chip | tip for electrophoresis of this invention. It is a schematic diagram which shows the state of preparation of the chip | tip for electrophoresis of this invention. It is the enlarged view and sectional drawing of the principal part of an example of the chip | tip for electrophoresis of this invention. It is a schematic diagram which shows an example of the sample introduction method by the chip | tip for electrophoresis of this invention. It is a schematic diagram which shows another example of the sample introduction method by the chip | tip for electrophoresis of this invention. It is a schematic diagram which shows an example of the chip | tip for electrophoresis of this invention. It is a schematic diagram which shows an example of the chip | tip for electrophoresis of this invention. It is a schematic diagram which shows an example of the chip | tip array for electrophoresis of this invention. It is a figure which shows an example of the analysis of the sample by the chip | tip for electrophoresis of this invention. It is a figure which shows an example of a sample separation and detection by the chip | tip for electrophoresis of this invention. It is a schematic diagram which shows an example of the chip | tip for the conventional electrophoresis.

Explanation of symbols

1 .. Electrophoresis chip 2.. Port 3.. Flow path 4.. Electrode 4 a ... End edge 5 ... Capillary tube 5 a ... Wall surface of the capillary tube 6. · Gas passage, 8 ·· Gas port, 10 ·· Electrophoresis chip array, 11 ·· Glass substrate, 11a ·· Upper surface, 12 ·· Electrophoresis chip, 13 ·· Resin substrate, 13a ·· Processing surface, 13b..Groove 33 ... Terminal 41.Electrophoresis start electrode 51..Porous body 56..Gas permeation part 81..First gas port 82..Second gas Port, 83 ... third gas port

Claims (11)

  An electrophoresis chip comprising a port for introducing a sample, a flow path of the introduced sample, and two or more electrodes connected to the flow path, and in the vicinity of any one of the electrodes Two or more gas permeation portions are connected side by side, and each gas permeation portion is connected to a gas port for introducing / extracting gas into / from the gas passage via a gas passage. Electrophoresis chip.   The electrophoresis chip according to claim 1, wherein the gas permeable portion is a thin tube bundle composed of a plurality of thin tubes or a porous body.   3. The electrophoresis chip according to claim 1, wherein an edge of the electrode protruding into the flow path is a straight line perpendicular to the longitudinal direction of the flow path.   4. The electrophoresis chip according to claim 2, wherein a wall surface of the thin tube is subjected to hydrophobic processing or hydrophilic processing.   5. The electrophoresis chip according to claim 2, wherein a cross-sectional shape of the thin tube is a rectangle, and a width and a height are 0.1 μm or more and 20 μm or less.   6. The electrophoresis chip according to claim 1, wherein a plurality of the flow paths are arranged in parallel. By laminating a glass substrate having an upper surface on which the electrodes are formed and a resin substrate having a processed surface that has been grooved in advance, with the processed surface of the resin substrate facing the upper surface of the glass substrate,
7. The electrophoresis according to claim 1, wherein the flow path, the gas permeable portion, and the gas passage are formed by a groove on a processed surface of the resin substrate. Chip. Two or more gas permeable portions are arranged in the vicinity of the electrophoresis start electrode provided at the end of the flow path,
After introducing the sample into a part or the whole of the flow path by discharging the gas from the first gas port leading to the gas permeation portion closest to the electrode for starting electrophoresis,
A part of the sample is measured into the flow path by introducing a gas from a second gas port connected to any one gas permeable part other than the gas permeable part closest to the electrode for starting electrophoresis. Leave, drain the rest of the sample from the flow path,
Next, a buffer solution is introduced into the flow path by discharging gas from the second gas port,
The electrophoresis chip according to claim 1, wherein the sample is separated by applying a voltage to the electrode.   9. An electrophoresis chip array comprising two or more electrophoresis chips according to claim 1 arranged in parallel.   A sample analysis method using the electrophoresis chip according to any one of claims 1 to 8 or the electrophoresis chip array according to claim 9. Two or more gas permeable portions are arranged in the vicinity of the electrophoresis start electrode provided at the end of the flow path,
After introducing the first sample into a part or the whole of the flow path by discharging the gas from the first gas port leading to the gas permeation part closest to the electrode for starting electrophoresis,
A part of the first sample is measured in the flow path by introducing a gas from the second gas port connected to any one gas permeable part other than the gas permeable part closest to the electrode for starting electrophoresis. Leaving the other part of the first sample out of the flow path,
Then, after introducing the second sample into the flow path by discharging the gas from the second gas port,
Gas is introduced from a third gas port connected to any one gas permeable portion located on the opposite side of the electrophoresis start electrode through a gas permeable portion connected to the second gas port. Thereby leaving a part of the second sample in the flow path, leaving the other part of the second sample out of the flow path,
Next, a buffer solution is introduced into the flow path by discharging gas from the third gas port,
11. The sample analysis method according to claim 10, wherein a voltage is applied to the electrode to analyze a reaction between the first sample and the second sample.

Images