JP2008039541A - Microchannel chip and biopolymer processing method using the same - Google Patents

Abstract

The present invention provides a microchannel chip and a method for treating a biopolymer using the same, which can perform pretreatment or genetic diagnosis of a polymer, for example, a gene at high speed and in situ.
A microchannel chip 1 includes a microchannel 2 having solution introduction ports 2A and 2B and a solution discharge port 4A, a micropillar 2P provided in the microchannel 2, a micropillar 2P and a solution discharge port. The micropillar 5 is covered with a material whose zeta potential changes depending on the pH of the solution introduced from the solution introduction port 2A. The hydrogel valve 5 is made of a polymer material. It opens and closes depending on the pH of the solution.
[Selection] Figure 1

Description

  The present invention relates to a microchannel chip and a method for performing treatments such as purification and washing of biopolymers using the microchannel chip.

  Patent Document 1 discloses a gene purification and extraction kit, which is commercially available and widely used (for example, Geneclean, manufactured by Qbiogene). For example, there is a product in which a gene is fixed to fibrous silica (silicon oxide) in a cartridge and impurities are removed by introducing a cleaning solution. These are tasks indispensable for pretreatment of genetic diagnosis, for example. For diagnosis of infectious diseases, in order to prevent infection of medical staff such as doctors, and to realize simple and quick diagnosis, a chip composed of a single micro-channel that integrates functions from blood collection to diagnosis, that is, There is a need to do so in a closed system within the biochip.

  Non-patent document 1 reports a biochip for gene diagnosis. After pretreatment of genes using the above-described kit or the like, a sample is introduced into the chip for diagnosis. In order to integrate these devices on a single chip and continuously perform gene pretreatment and diagnosis within the biochip, it is necessary to install a device that performs the pretreatment step in a fine flow path.

US Patent 6,027,750 Eiichi Minutani, “Biomolecular Devices”, Applied Physics, Vol. 74, No. 7, pp. 903-909, 2005

  In order to connect the biochip gene pretreatment device and the diagnostic device flow path, and to prevent the gene before purification / extraction from entering the diagnostic device, the gene is fixed during the pretreatment process and connected to the diagnostic device. Although it is necessary to construct a system that closes the part with a valve or the like and removes the fixed gene after processing and opens the valve at the connection part, it has not been obtained at present.

  In view of the above problems, an object of the present invention is to install a micropillar for fixing a polymer such as DNA in a microchannel of a microchannel chip and a hydrogel valve that can be opened and closed by changing pH. Accordingly, it is an object of the present invention to provide a microchannel chip and a method for treating a biopolymer using the same, which can perform pretreatment and gene diagnosis of a polymer such as a gene at high speed and on the spot.

  The inventors of the present invention are intensively developing a system in which a gene pretreatment device is installed in a biochip consisting of microchannels, and the zeta potential of alumina (aluminum oxide) is acidic and positive, and alkaline and negative. Attention, since the gene has a negative charge, the gene can be detached by fixing the gene to an alumina-coated micropillar during the pretreatment of the gene with an acidic solution, and introducing an alkaline solution after the treatment, Attention is also focused on hydrogels that change in volume due to changes in pH. During the pretreatment with the gene fixed in an acidic solution, the hydrogel expands in volume and closes the flow path to confine the gene, and the gene is placed in an alkaline solution. Upon desorption, the hydrogel contracted to open the flow path, confirming that the gene can be passed, and the present invention was completed.

  In order to achieve the above object, a microchannel chip of the present invention includes a microchannel having a solution introduction port and a solution discharge port, a micropillar provided in the microchannel, a micropillar, and a solution discharge port. The micropillar is covered with a material whose zeta potential changes depending on the pH of the solution introduced from the solution introduction port, the hydrogel valve is made of a polymer material, It is characterized by being opened and closed by a change in pH. According to this configuration, a solution containing a polymer or the like is introduced into the microchannel, and the polymer is fixed to the micropillar using the zeta potential of the material covering the surface of the micropillar. By opening and closing the hydrogel valve depending on the pH, it is possible to perform cleaning or purification treatment of polymers in the microchannel chip.

  The second micro-channel chip of the present invention includes a micro-channel having a solution introduction port and a solution discharge port, a micro-pillar provided in the micro-channel, and a hydro-channel provided between the micro-pillar and the solution discharge port. A cleaning part comprising a gel valve, an inspection object introduction part connected to the solution introduction port of the washing part, a solution introduction part connected to the solution introduction port of the washing part, and a solution discharge port of the washing part The micropillar is coated with a material whose zeta potential changes depending on the pH of the solution introduced from the solution introduction port, the hydrogel valve is made of a polymer material, and the pH of the solution is It is characterized by opening and closing by a change. According to this configuration, since the diagnosis unit connected to the solution outlet of the microchannel is further provided, the diagnosis and inspection of the polymer processed in the microchannel can be performed in the chip. it can.

  In the above configuration, preferably, when the solution is acidic, the zeta potential of the material covering the micropillar is a positive potential, and when the hydrogel valve is closed and the solution is alkaline, the micropillar is The zeta potential of the material that coats is negative and the hydrogel valve is open. The material for coating the micropillar is preferably alumina. The micro pillar preferably has a shape and a size for fixing the biopolymer. When the solution is acidic, since the zeta potential of the material covering the micropillar is a positive potential, a substance having a negative charge contained in the solution can be fixed to the micropillar. In this state, since the hydrogel valve is in a closed state, cleaning can be performed by introducing a cleaning solution of the above substance into this solution. If the solution is acidified, the zeta potential of the material covering the micropillar becomes a negative potential, so the substance is detached and the hydrogel valve is open, so that the washed substance is recovered from the solution outlet. can do.

  The biopolymer processing method using the microchannel chip of the present invention uses the above-described microchannel chip, flows a solution containing the biopolymer into the microchannel, and fixes the biopolymer to the micropillar. The biopolymer treated with the solution is discharged from the solution outlet, and the biopolymer is purified and washed. By using the microchannel chip of the present invention, for example, DNA can be purified and washed at high speed by introducing a biopolymer into the field without introducing complicated equipment.

  The biopolymer processing method using the microchannel chip of the present invention uses the microchannel chip equipped with the above-described diagnostic substance, introduces the test object and the solution into the microchannel, The biopolymer treated with the solution is discharged from the solution outlet and diagnosed by the treated biopolymer diagnostic unit. By using the microchannel chip of the present invention, by introducing biopolymers in place without introducing complicated equipment, for example, after purifying and washing DNA, genetic diagnosis and testing are performed. Can be implemented at high speed.

In the above configuration, preferably, the hydrogel valve is closed, the biopolymer gene is extracted in the microchannel, the gene is fixed to the micropillar and purified, and the hydrogel valve is opened. Drain the purified gene to the solution outlet. Preferably, the biopolymer comprises plasma and the gene is a gene derived from a virus. According to the said structure, the washing | cleaning and refinement | purification of the gene derived from the virus in biopolymers, for example, DNA, and also diagnosis and test | inspection, such as gene diagnosis, can be performed in a microchannel chip. Therefore, even if the virus is derived from an infectious disease, the operator can safely perform various genetic tests and diagnoses.

According to the microchannel chip of the present invention and the biopolymer processing method using the chip, pretreatment such as gene purification and washing can be performed.
According to the microchannel chip including the diagnosis unit of the present invention and the biopolymer processing method using the chip, for example, pretreatment such as gene purification and washing can be performed, and the extracted gene can be diagnosed. it can. In particular, the method of the present invention fixes genes extracted from a whole blood sample such as peripheral blood to micropillars in a microchannel and purifies them on the spot by injecting a washing solution. Only a pretreated gene can be transported to a diagnostic device for genetic diagnosis. Therefore, it is possible to make a single chip from blood collection to diagnosis. For this reason, it is possible to realize a system for performing gene diagnosis easily by the patient's own hand.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In each figure, the same or corresponding members are denoted by the same reference numerals.
A microchannel chip according to a first embodiment of the present invention will be described.
1A and 1B schematically show the configuration of a microchannel chip according to a first embodiment of the present invention, where FIG. 1A is a plan view and FIG. 1B is a cross-sectional view along the XX direction of FIG. is a sectional view taken along the _Y 2 -Y ₂ direction (C) is a sectional view taken along the _Y 1 -Y ₁ direction (a), (D) is (a). As shown in FIG. 1A, a microchannel chip 1 of the present invention is composed of a substrate 3 on which a microchannel 2 is formed and a substrate 4 provided on the lower side facing the substrate 3. Yes. The substrate 4 is a substrate that does not form the microchannel 2, and is provided with a through hole 4 </ b> A connected to the discharge port 2 </ b> C of the microchannel 2, and a region other than the through hole 4 </ b> A is introduced into the microchannel 2. The solution containing a biopolymer or the like is closely fixed so as not to leak.
As shown in FIG. 1B, in the substrate 3, the microchannel 2 having a depth of d is formed on the surface facing the substrate 4. The micro flow path 2 includes a micro pillar 2P, a solution introduction port 2A provided at the right end portion thereof, and a solution discharge port 2D provided at the left end portion thereof. A hydrogel valve 5 is provided between the micropillar 2P in the microchannel 2 and the solution outlet 2D. Furthermore, a plurality of solution inlets 2B may be provided in the microchannel 2 of the substrate 3 as necessary. FIG. 1 shows a case where a second solution introduction port 2B is provided and a capillary valve 6 is provided for taking a solution into and out of the solution introduction port 2B.

  The microchannel chip 1 of the present invention can be used as a microreactor that performs various chemical reactions using a solution or a so-called biochip. For example, a solution containing a biopolymer such as DNA (deoxyribonucleic acid) can be purified, or used for pretreatment when performing genetic diagnosis. In the following description, unless otherwise specified, the microchannel chip 1 of the present invention will be described as performing gene pretreatment.

As shown in FIG. 1C, the micro pillar 2P includes a plurality of pillars having a predetermined dimension in a lattice shape within a range of a width W of the micro flow path 2 and a length L in the flow path direction at a predetermined interval Sp. Arranged. The micro pillar 2P is not particularly limited in its shape and dimensions, but can be, for example, a cylindrical shape. In addition to the cylindrical shape, it can also be a polygonal column (for example, quadrangular column, hexagonal column, octagonal column, etc.) shape composed of a plurality of planes, an elliptical column shape, and a combination of two or more of these shapes You can also For example, a part may be cylindrical and the rest may be a polygonal column. The bottom of the microchannel 2 is usually flat, but may be a curved surface (convex surface or concave surface). As will be described later, the micro pillar 2 </ b> P can be formed simultaneously by etching a substrate region that is not a pillar when the micro flow path 2 is formed on the substrate 3. In this case, the height of each column constituting the micro pillar 2 </ b> P is the depth d of the micro flow path 2.

The shape and dimensions of the micropillar 2P may be determined by the substance that is contained in the solution and is to be fixed to the micropillar 2P. When the substance contained in the solution is a gene, the gene is fixed on the side wall of the micropillar 2P in consideration of the type, shape, size, etc. of the gene, and is appropriately determined. In order to allow the gene to be fixed during pretreatment and to be detached after treatment, for example, on the side wall of the micropillar 2P, the zeta potential shows a positive value in an acidic solution and negative in an alkaline solution. A material having the characteristic of exhibiting the value of may be used. When the micro pillar 2P itself does not have this property, the micro pillar 2P may be covered with a material that can obtain the zeta potential. Examples of such a material include an alumina film (Al ₂ O ₃ ). Examples of the substance capable of obtaining the same reaction as alumina include silica (silicon oxide) which has a smaller response range of zeta potential to pH value than alumina but is also used in commercially available kits. Here, the zeta potential will be described. When a solid such as alumina is present in the electrolyte solution, the potential at a position called a slip plane near the solid surface when the potential at infinity in the liquid from the solid surface is zero is called a zeta potential. The slip surface is located outside the electric double layer formed on the solid surface, but the thickness of the electric double layer is usually very thin, 10 nm or less. For this reason, in the case of the alumina film formed on the side wall of the micro pillar 2P as in the present invention, it may be considered as the potential of the surface of the micro pillar 2P.

  When the micro pillar 2P has a cylindrical shape, for example, the diameter Dp may be about 1 to 30 μm, and the interval between the micro pillars 2P may be about 1 to 30 μm. The height of the micro pillar 2P can be set to about 10 to 300 μm according to the depth d of the micro flow path 2. However, as described above, the dimensions of the micropillar 2P are preferably determined in consideration of a suitable ratio with respect to the inertia radius of the gene to be preprocessed and the micropillar spacing Sp, as described above. .

  When the number of micro-channels 2 is one, the number of micro-pillars 2P is not particularly limited. For example, when columnar pillars having a diameter of 10 μm, an interval of 10 μm, and a height of 60 μm are arranged vertically and horizontally, the width W is 200 to 1000 μm. When the length L is 500 to 2000 μm, the number of columns can be 250 to 5000. When a plurality of microchannels 2 are formed in the microchannel chip 1, the micropillar 2 </ b> P having the above arrangement can be arranged in each microchannel 2.

As shown in FIG. 1D, a plurality of hydrogel valves having a predetermined dimension in a direction (Y ₂ -Y ₂ direction, appropriately referred to as a longitudinal direction) perpendicular to the flow direction of the micro flow channel 2 5 are arranged at a predetermined interval Sh. The hydrogel valve 5 is a valve using a polymer material that expands and contracts due to a change in pH. There is no restriction | limiting in particular in the shape and dimension of each hydrogel valve | bulb 5. FIG. The shape of the hydrogel valve 5 can be, for example, a cylindrical shape, and in addition to the cylindrical shape, a polygonal column shape (for example, a quadrangular column, a hexagonal column, an octagonal column, etc.) configured by a plurality of planes, an elliptical column shape, You can also The shape may be a combination of two or more of these shapes, part of which may be a cylindrical shape, and the rest may be a polygonal column shape.

When the gene is contained in the solution and the gene is blocked or passed through the hydrogel valve 5, it is appropriately determined in consideration of the state of the gene. In order to close the hydrogel valve 5 at the time of gene pretreatment and to open the hydrogel valve 5 after the treatment, for example, it has a feature of volume expansion in an acidic solution and volume contraction in an alkaline solution. The hydrogel valve 5 may be manufactured using a molecular material.

  In the case of a cylindrical shape in which a plurality of hydrogel valves 5 are arranged in the vertical direction, the size of one hydrogel valve 5 is, for example, a diameter Dg of about 50 to 200 μm, and an interval Sh between the hydrogel valves 5 is 10 to 10. What is necessary is just to be about 100 micrometers. The height d of the hydrogel valve 5 can be set to about 10 to 300 μm according to the depth of the microchannel 2. However, as described above, the dimensions of the hydrogel valve 5 can be appropriately determined in consideration of a suitable ratio to a gene that is blocked or opened by the hydrogel valve.

  The number of hydrogel valves 5 included in one microchannel 2 is not particularly limited. For example, when a plurality of cylindrical hydrogel valves 5 having a diameter of 80 μm, a distance of 10 μm, and a height of 60 μm are arranged in the vertical direction, 2 to 10 pieces can be arranged in the microchannel 2 having a width W of 200 to 1000 μm. When a plurality of microchannels 2 are formed in the microchannel chip 1, the hydrogel valves 5 having the above arrangement can be disposed in each microchannel 2.

  Examples of materials used for the substrates 3 and 4 of the microchannel chip 1 of the present invention include substrates made of quartz, silicon, glass, and polymer. By processing these substrates, the microchannel 2 and the microchannel chip 1 are processed. The pillar 2P can be manufactured. Alternatively, a single-layer film or multilayer film of insulators deposited on the substrates 3 and 4 or a combination of them can be used.

  As described above, according to the microchannel chip 1 of the present invention, the microchannel 2 is provided inside the substrates 3 and 4, and the micropillar 2 </ b> P, the hydrogel valve 5, As will be described later, a biopolymer such as DNA contained in the acidic solution is fixed to the side wall of the micro pillar 2P, and the hydrogel valve can be closed in this state. Examples of the biomolecule include oligonucleotides, RNA, antibodies, peptides and the like other than DNA. If the solution is made alkaline from this state, the DNA can be removed from the side wall of the micropillar 2P, and the hydrogel valve 5 can be opened to recover the DNA from the solution outlet.

Next, the manufacturing method of the microchannel chip 1 of the present invention will be described.
First, the substrate 3 is covered with a mask material from Si or the like. Next, the mask material is opened only in regions where the microchannels 2 and the micropillars 5 are formed by a known photolithography method. In this case, ultraviolet exposure etc. can be used for exposure.
Next, selective etching is performed using the mask so that only the regions of the substrate 3 where the microchannels 2 and the micropillars 5 are formed have a predetermined depth d of the microchannels 2. Subsequently, the hydrogel valve 5 is formed into a predetermined shape using a polymer material in the microchannel 2. As the polymer material, a material that is polymerized by light irradiation can be used. Next, a through hole is formed through the substrate 3 and a predetermined portion of the substrate 4 made of glass or the like.
Finally, the microchannel chip 1 can be manufactured by bonding a glass substrate 4 that does not form the microchannel 2 to the Si substrate 3. As described above, the microchannel chip 1 can be manufactured by using a photolithography technique and a dry etching technique, which are semiconductor manufacturing process techniques.

Next, a biopolymer processing method using the microchannel chip of the present invention will be described.
The surface of the micro pillar 2P is covered with an alumina film, and the solution touching the hydrogel valve 5 is acidic and closed, and is alkaline and open.
An acidic solution containing a biopolymer flows into the microchannel 2, the biopolymer is fixed to the micropillar 2P, the hydrogel valve 5 is closed, and the biopolymer is washed and gene extracted in the microchannel 2 Etc. can be performed.
Next, if the solution is an alkaline solution, the washed biopolymer and the extracted gene fixed to the micropillar 2P can be detached from the micropillar 2P. By discharging the detached biopolymer from the solution discharge port 4A, it is possible to perform treatments such as washing of the biopolymer and gene extraction. In this case, an acidic solution containing a biopolymer can be introduced into the solution introduction port 2B via the capillary valve 6. A solution for washing or gene extraction or an alkaline solution can be introduced from the solution introduction port 2A.

  The biopolymer may be plasma extracted from whole blood. Here, the whole blood is blood and consists of plasma and blood cells. A gene may be appropriately selected according to the purpose. Such a gene may be a gene derived from a virus. The virus may be an infectious disease virus, and examples thereof include viruses such as HIV, hepatitis, and influenza.

Next, a microchannel chip according to a second embodiment of the present invention will be described.
FIG. 2 is a schematic cross-sectional view showing the configuration of the microchannel chip according to the second embodiment of the present invention. As shown in FIG. 2, the microchannel chip 10 of the present invention uses the microchannel chip 1 shown in FIG. 1 as the cleaning unit 1 and is connected to the first solution introduction port 2 </ b> A of the cleaning unit 1. An inspection object introduction unit 12, a solution introduction unit 14 connected to the second solution introduction port 2B of the cleaning unit 1, and a diagnosis unit 16 connected to the solution discharge port 4A of the cleaning unit 1 are provided. Has been. From the test substance introduction unit 14, a biopolymer such as whole blood or plasma 15 of a living body can be introduced. The biological whole blood 15 or the like introduced from the test substance introduction unit 14 can be washed with the solution 13 introduced from the solution introduction unit 12 or can be pretreated. The diagnosis unit 16 is connected to a diagnosis kit for performing various diagnoses and a test kit for performing tests. Other configurations are the same as those of the microchannel chip shown in FIG.

  According to the microchannel chip 10 of the present invention, the solution 15 introduced from the solution introduction part 12 connected to the second solution introduction port 2 </ b> A of the washing part 1 is introduced into the washing part 1. Or the gene 18 can be extracted and diagnosed by the diagnosis unit 16. Therefore, when the inspected object 15 is a living tissue, a series of steps in which the living tissue is pre-processed by the cleaning unit and diagnosed by the diagnosis unit can be performed in one chip.

Next, a method for manufacturing the microchannel chip 10 of the present invention will be described.
The microchannel chip 10 of the present invention can be manufactured by connecting the separately-inspected object introducing unit 12, the solution introducing unit 14, and the diagnostic unit 16 after the microchannel chip 1 is manufactured. . The inspection object introduction part 12, the solution introduction part 14, and the diagnosis part 16 to be added can be produced by using a semiconductor production process technique such as a photolithography technique and a dry etching technique.

Next, a biopolymer processing method using the microchannel chip according to the second embodiment of the present invention will be described.
The surface of the micro pillar 2P is covered with an alumina film, and the hydrogel valve 5 is closed in an acidic state and open in an alkaline state. The test object 15 and the acidic solution 13 are introduced into the microchannel 2, the biopolymer is fixed to the micropillar 2P, and purification and gene extraction are performed. Next, the solution 13 is changed to the alkaline solution 13 so that the processed biopolymer and the extracted gene are detached from the micropillar, discharged from the solution outlet 4A, and processed by the diagnostic unit 16 and extracted. The diagnosed gene can be diagnosed.

As a method of treating the biopolymer, a case where the test object 15 is plasma contained in whole blood, a gene is extracted from the plasma 15 by the washing unit 1 and the extracted gene is subjected to genetic diagnosis by the diagnostic unit This will be described in more detail.
FIG. 3 is a schematic diagram for explaining a genetic diagnosis method using the microchannel chip 10 according to the second embodiment of the present invention. A blood cell / plasma separation device is connected to the inspection object introduction section 12, and plasma separated from whole blood is introduced into the first solution introduction port 2 </ b> B of the microchannel 2 via the capillary valve 6. The The virus gene extract 13A is introduced into the first solution introduction port 2B of the microchannel 2 from the inspection object introduction unit 12 via the capillary valve 2A provided at the first solution introduction port. 2 to extract gene 18 from virus 20.

  Subsequently, the gene fixing solution 13B is introduced from the capillary valve 2A, and the unpurified gene 18 is fixed on the micropillar 2P. Since the solutions used in the above steps are all acidic, the hydrogel valve 5 that is acidic and volume-expands blocks the microchannel 2 and prevents the inflow of the unpurified gene 18 into the downstream genetic diagnostic device 16. be able to.

  Subsequently, the gene purification solution 13C is introduced from the capillary valve 2A, and impurities attached to the unpurified gene 18 are removed. Finally, after the purification is completed, the purified gene 24 is eluted from the micropillar 2P using the gene eluent 13D. Since the gene eluent 13D is alkaline, the hydrogel valve 5 contracts due to alkalinity and the microchannel 2 opens, and the purified gene 24 can be transported to the genetic test device 16, that is, recovered. By the above method, plasma separation from whole blood and genetic testing can be performed in one microchannel chip 10.

  According to the biopolymer processing method using the microchannel chip 10 of the present invention, the plasma 15 extracted from a whole blood sample such as peripheral blood of a living body is extracted, and the gene 20 is extracted from the plasma in the microchannel 2. Then, the extracted gene 20 is fixed to the micropillar 2P in the microchannel 2, and only the purified gene 24 can be diagnosed.

Hereinafter, the present invention will be described in detail with reference to examples.
As Example 1, the microchannel chip 1 according to the first embodiment of the present invention was manufactured. 4A to 4F are cross-sectional views schematically showing a manufacturing process when the microchannel chip 1 of Example 1 is manufactured. As shown in FIG. 4A, a chromium (Cr) film 30 having a thickness of about 100 nm was deposited on the silicon substrate 3 by a sputtering method. On the surface of the chromium film 30, a positive photoresist (manufactured by Tokyo Ohka Kogyo Co., Ltd., OFPR800) was applied. Next, a microchannel photoresist pattern 32 including a micropillar pattern was formed by ultraviolet exposure.
As shown in FIG. 4B, the exposed portion of the chromium film 30 was selectively etched by a wet etching technique using a ceric ammonium nitrate etchant using the photoresist pattern 32 as a mask.

  Next, as shown in FIG. 4C, a portion where the silicon substrate 3 was exposed was selectively etched using a dry etching technique by a Bosch etching method, thereby forming the microchannel 2 and the micropillar 2P. The photoresist pattern 32 was removed using a resist stripping solution, and the chromium film 30 was completely removed using a wet etching technique or the like. The height of the micro pillar 2P manufactured in this process is about 100 μm, and the interval between the micro pillars 2P is about 15 μm. Here, the Bosch etching method is a dry etching method in which a deep groove is formed by repeatedly performing a gas discharge for silicon etching and a gas discharge for protecting a silicon side wall serving as a micro pillar. The polymer serving as a protective film for the silicon side wall is formed by discharge of a CF-based gas.

Next, as shown in FIG. 4D, a through hole for introducing a solution was formed from the back surface of the silicon substrate 3 by using a sandblast method or the like. An alumina film 34 having a thickness of 40 nm was deposited on the surfaces of the microchannel 2 and the micropillar 2P thus formed by an atomic layer deposition method using trimethylaluminum and hydrogen peroxide.

  Next, as shown in FIG. 4E, the glass substrate 4 was bonded to the surface of the silicon substrate 3 on which the microchannels and micropillars were formed using an anodic bonding technique.

  Finally, as shown in FIG. 4F, a hydrogel valve 5 was formed in the microchannel 2. Specifically, the hydrogel valve is prepared by adding an alkylphenone photopolymerization initiator (Irgacure 651, manufactured by Ciba) to a mixed solution composed of 2- (dimethylamino) ethyl methacrylate, 2-hydroxyethylene methacrylate, and ethylene glycol methacrylate. ) Was added into the microchannel, and a bulb pattern was formed by ultraviolet exposure to form a hydrogel bulb 5. In the hydrogel valve 5, cylinders having a diameter of about 80 μm were arranged in the vertical direction with respect to the flow path direction of the micro flow path 2 with an interval of about 10 μm. The microchannel chip 1 of Example 1 was manufactured through the above steps.

  FIG. 5 is a diagram showing an electron microscope image obtained by observing a cross section of the micropillar 2P in the microchannel chip 1 of the first embodiment. As can be seen from FIG. 5, a micro pillar 2P having a height of about 100 μm and a width of about 15 μm is formed in the silicon substrate 3 by the Bosch etching method. Further, from the enlarged view indicated by the arrow in the region surrounded by the square (□) of the micro pillar 2P, it can be seen that the alumina film 34 having a thickness of 40 nm is uniformly deposited on the surface of the micro pillar 2P. From this, it can be seen that the side wall of the micro pillar can be uniformly coated with an alumina film, and for example, a large amount of genes can be immobilized on a wide area of the micro pillar 2P.

  FIG. 6 is a diagram showing the results of measuring the zeta potential of the alumina film 34 coated on the micro pillar 2P in the microchannel chip 1 of Example 1. In FIG. 6, the horizontal axis represents the pH value, and the vertical axis represents the zeta potential. As is apparent from FIG. 6, it was confirmed that the zeta potential showed a positive potential when the pH value was low, ie, acidic, and the negative potential tended to show a negative potential when the pH value was high, ie, alkaline.

  Using the microchannel chip 1, fluorescent modification of the DNA gene was performed as a treatment of the biopolymer. In the microchannel chip, the microchannel chip 1 is the same as in Example 1 except that the depth of the microchannel 2 is about 60 μm, the diameter of the micro pillar 2P is about 10 μm, and the interval is about 10 μm. Was used.

In the microchannel chip, DNA of about 50 μm in length (T4 DNA, manufactured by Nippon Gene Co., Ltd., 166 kbps (bp is the base number)) is immersed in an acidic or alkaline solution, and a phosphor (YOYOGEN Co., Ltd., YOYO) is immersed. -1).

  When the acidic or alkaline solution containing the DNA and the phosphor are introduced into the microchannel chip 1, when the solution is acidic, the DNA is fixed to the alumina film 34 coated on the surface of the micropillar 2P, and It was confirmed that the gel valve 5 expanded and the valve was closed. On the other hand, when the solution is alkaline, it can be confirmed that the DNA is detached from the alumina film 34 coated on the surface of the micropillar 2P, and the hydrogel valve 5 is contracted and the valve is moved to the open state. It was.

The degree of DNA adhesion to the micropillar 2P coated with an alumina film was examined. After introducing each acidic and alkaline solution into the microchannel 2 of the microchannel chip 1 and rotating the entire microchannel chip 1, the DNA remaining on the micropillar 2P is observed with a fluorescence microscope, The degree of DNA adhesion to the micropillar 2P was evaluated based on the fluorescence intensity. Here, the rotation of the microchannel chip 1 was performed as follows. A device in which an electric motor was connected to a Teflon (registered trademark) dish was prepared, and the microchannel chip 1 was fixed on the dish and rotated at about 4000 revolutions per minute.
FIG. 7 is a diagram showing a fluorescence microscope image obtained by observing the plane of the micro flow path 2 provided with the micro pillar 2P with a fluorescence microscope in Example 2, and (A) shows the case of an acidic (pH value 5) solution. (B) shows the case of an alkaline (pH value 10) solution, and (C) shows the fluorescence intensity. 7A and 7B, the left side shows after solution introduction, and the right side of the arrow (→) shows after rotation. In each figure, the lattice part on the left side of the micro flow path 2 is a place where the micro pillar 2P is provided.
As is clear from FIG. 7A, when the solution containing DNA is acidic, fluorescence can be confirmed after the solution is introduced and rotated, and it is confirmed that the DNA is fixed on the micropillar 2P. On the other hand, as shown in FIG. 5B, when the solution containing DNA is alkaline, it can be seen that the fluorescence intensity is almost lost after rotation, and that the DNA is detached from the micropillar 2P. found.

FIG. 7 (C) shows the results of measuring the fluorescence intensity of the micropillar 2P region after the same test as described above using solutions having different pH values containing DNA, where the horizontal axis represents the pH value, The vertical axis represents the fluorescence intensity. From the figure, it was confirmed that the fluorescence intensity after rotation decreased as the pH value increased.
As a result, it was found that the DNA fixing and desorption from the micropillar 2P covered with the alumina film 34 can be controlled by selecting the pH value of the solution in the fluorescence modification treatment of DNA.

FIG. 8 shows an optical microscope image of the hydrogel bulb 5 in Example 2, in which (A) is before introduction of the solution, (B) is after introduction of the acidic standard solution having a pH value of 4, (C) shows after introduction of an alkaline standard solution having a pH value of 12.
As can be seen from FIG. 8A, when the solution is not introduced, the hydrogel valves are arranged such that cylinders having a diameter of about 80 μm are vertically arranged at intervals of about 10 μm. As is clear from FIG. 8B, it can be seen that the introduction of the acidic standard solution causes the hydrogel valve to expand in volume, its diameter becomes about 100 μm, and the hydrogel valve is closed. On the other hand, as is apparent from FIG. 8C, it was found that when the alkaline standard solution was introduced, the hydrogel valve contracted in volume to a diameter of about 90 μm, and the valve was opened. From this, it was found that the opening and closing of the hydrogel valve can be controlled by adjusting the pH value of the solution used in the DNA washing step.

  Thus, in the gene pretreatment step in which the inside of the microchannel 2 of the microchannel chip 1 is exposed to the acidic solution, the gene is fixed on the micropillar coated with alumina, and the gene is eluted by the gene eluent that is an alkaline solution after the processing. Was found to detach.

  The microchannel chip 1 of Example 1 is used as a cleaning unit, and a device for separating plasma from whole blood is connected to the test object introduction unit connected to the first solution introduction port of the cleaning unit 1, and the cleaning unit The microfluidic chip 10 of Example 3 is manufactured by connecting a solution introducing part comprising a microchannel to the second solution introducing port and connecting a biochip for genetic diagnosis to the solution discharging port of the cleaning unit. did. Whole blood consists of plasma and blood cells. The device for separating plasma from whole blood is a chip having a microchannel, and a plurality of fine chambers for taking blood cells are arranged in the middle of the microchannel. Whole blood was introduced into the microchannel, rotated at high speed, and only plasma was extracted from the whole blood due to the difference in specific gravity.

Using the microchannel chip 10 of Example 3, plasma was extracted from whole blood, genes contained therein were extracted, and gene diagnosis was performed by the method described in FIG. Using the same device for separating plasma from whole blood as in Example 3, the plasma separated from whole blood was introduced into the first solution inlet 2B of the microchannel 2 through the capillary valve 6.
From the solution introduction part 12, the virus gene extract 13A (manufactured by Shimadzu Corporation, Ampdirect) is supplied to the first solution introduction port 2A of the microchannel 2 through the capillary valve 2A provided at the first solution introduction port. The gene 18 was extracted from the virus 20 in the microchannel 2.

  Subsequently, a gene fixing solution 13B (manufactured by Qbiogene, GENECLEAN Turbo Salt Solution) was introduced from the capillary valve 2A, and the unpurified gene 18 was fixed on the micropillar 2P. Since each of the above solutions is acidic, the hydrogel valve 5 that is acidic and volume-expands closes the microchannel 2 and prevents the inflow of the unpurified gene 18 into the genetic diagnosis section connected to the microchannel 2. I was able to.

  Subsequently, a gene purification solution 13C (manufactured by Qbiogene, GENECLEAN Turbo Wash Solution) was introduced from the capillary valve 2A to remove impurities attached to the unpurified gene 18. After completion of purification, finally, the purified gene 24 was eluted from the micropillar 2P using a gene eluent 13D (manufactured by Qbiogene, GENECLEAN Turbo Elution Solution). Since the gene eluent is alkaline, the hydrogel valve 5 is alkaline, the volume of the hydrogel valve 5 is shrunk, the microchannel 2 is opened, and the purified gene 24 can be transported to the genetic test device 16, that is, recovered. By the above method, plasma separation from whole blood and genetic testing could be performed in one microchannel chip 10.

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the invention described in the claims, and it goes without saying that these are also included in the scope of the present invention. For example, in the present embodiment, the extraction of a gene, purification thereof, and diagnosis of the extracted gene have been described as a microchannel chip and a biopolymer processing method using the microchannel chip. Needless to say, the reaction and purification, and further use in diagnosis and testing are also included in the scope of the present invention.

  The gene washing device of the present invention is expected to increase in demand in the future as part of a medical instrument such as a DNA chip that can rapidly diagnose an infectious disease at a gene level. In addition, since manufacturing uses semiconductor microfabrication technology such as photolithography of the prior art, mass production is easy, and it is expected to be an inexpensive disposable chip, which makes industrialization easy. .

BRIEF DESCRIPTION OF THE DRAWINGS The structure of the microchannel chip concerning the 1st Embodiment of this invention is shown typically, (A) is a top view, (B) is sectional drawing in alignment with the XX direction of (A), (C) is a sectional view taken along the _Y 2 -Y ₂ direction sectional view taken along the _Y 1 -Y ₁ direction (a), (D) is (a). It is a cross-sectional schematic diagram which shows the structure of the microchannel chip concerning the 2nd Embodiment of this invention. It is a schematic diagram explaining the gene diagnostic method using the microchannel chip concerning the 2nd Embodiment of the present invention. (A)-(F) is sectional drawing which shows typically the manufacturing process at the time of manufacturing the microchannel chip of Example 1. FIG. In the microchannel chip | tip of Example 1, it is a figure which shows the electron microscope image which observed the cross section of the micro pillar. In the microchannel chip of Example 1, it is a figure which shows the result of having measured the zeta potential of the alumina film | membrane coat | covered with the micro pillar. In Example 2, it is the figure of the fluorescence-microscope image which observed the plane of the microchannel which provided the micro pillar with the fluorescence microscope, (A) is the case of an acidic (pH value 5) solution, (B) is alkaline ( In the case of a solution having a pH value of 10), (C) shows the fluorescence intensity. It is a figure which shows the optical microscope image of the hydrogel valve | bulb in Example 2, (A) is before introduction | transduction of a solution, (B) is after introduction | transduction of the acidic standard solution whose pH value is 4, (C) is respectively. It shows after the introduction of an alkaline standard solution having a pH value of 12.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1,10: Microchannel chip | tip 2: Microchannel 2A, 2B: Solution inlet 2C: Outlet 2P: Micro pillar 3, 4: Substrate 5: Hydrogel valve 6: Capillary valve 12: Inspecting object introduction part 13 : Solution 13A: Virus gene extract 13B: Gene immobilization solution 13C: Gene purification solution 14: Solution introduction part 15: Whole blood (plasma)
16: Diagnostic unit 18: Gene (unpurified gene)
24: Purified gene 30: Chromium film 32: Photoresist pattern 34: Alumina film

Claims (9)

A microchannel having a solution inlet and a solution outlet, a micropillar provided in the microchannel, and a hydrogel valve provided between the micropillar and the solution outlet,
The micro pillar is coated with a material whose zeta potential changes depending on the pH of the solution introduced from the solution introduction port,
The micro-channel chip, wherein the hydrogel valve is made of a polymer material, and opens and closes according to a change in pH of the solution. A cleaning section comprising: a microchannel having a solution inlet and a solution outlet; a micropillar provided in the microchannel; and a hydrogel valve provided between the micropillar and the solution outlet. ,
An inspection object introduction part connected to the solution introduction port of the cleaning part;
A solution introduction unit connected to the solution introduction port of the cleaning unit;
A diagnostic unit connected to the solution outlet of the cleaning unit,
The micro pillar is coated with a material whose zeta potential changes depending on the pH of the solution introduced from the solution introduction port,
The micro-channel chip, wherein the hydrogel valve is made of a polymer material, and opens and closes according to a change in pH of the solution. When the solution is acidic, the zeta potential of the material covering the micropillar is a positive potential, and the hydrogel valve is closed,
When the solution is alkaline, the zeta potential of the material covering the micropillar is a negative potential, and the hydrogel valve is in an open state. Microchannel chip.   The microchannel chip according to claim 3, wherein the material covering the micropillar is alumina.   The microchannel chip according to claim 1 or 2, wherein the micropillar has a shape and a size for fixing a biopolymer. A microchannel having a solution inlet and a solution outlet; a micropillar provided in the microchannel; and a hydrogel valve provided between the micropillar and the solution outlet. The micro-channel is formed so that the pillar is covered with a material whose zeta potential changes depending on the pH of the solution introduced from the solution introduction port, the hydrogel valve is formed of a polymer material, and is opened and closed according to the change in the pH of the solution. A method of processing a biopolymer using the microchannel chip while configuring a chip,
Purify the biopolymer by flowing a solution containing the biopolymer into the microchannel, fixing the biopolymer to the micropillar, and discharging the biopolymer treated with the solution from the solution outlet. A method for treating a biopolymer, characterized by performing a treatment such as washing. A cleaning section comprising a microchannel having a solution inlet and a solution outlet, a micropillar provided in the microchannel, a hydrogel valve provided between the micropillar and the solution outlet, and the cleaning An inspection object introduction part connected to the solution introduction port of the cleaning part, a solution introduction part connected to the solution introduction port of the cleaning part, and a diagnosis part connected to the solution discharge port of the cleaning part, The micropillar is coated with a material whose zeta potential changes depending on the pH of the solution introduced from the solution introduction port, the hydrogel valve is formed of a polymer material, and the micropillar is opened and closed by changing the pH of the solution. A method of processing a biopolymer using the microchannel chip while configuring the channel chip,
The test object and the solution are introduced into the microchannel, the biopolymer is fixed to the micropillar, the biopolymer treated with the solution is discharged from the solution discharge port, and the treated biopolymer A method for treating a biopolymer, characterized in that: Closing the hydrogel valve, extracting the biopolymer gene in the microchannel, immobilizing the gene on the micropillar and performing a purification treatment,
The method for treating a biopolymer using a microchannel chip according to claim 6 or 7, wherein the hydrogel valve is opened and the purified gene is discharged to the solution outlet.   The method for treating a biopolymer using a microchannel chip according to claim 6 or 7, wherein the biopolymer comprises plasma and the gene is a gene derived from a virus.