JP2008232939A - Microchip and its manufacturing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microchip made of a resin bondable easily even when a bonding surface is not smoothed strictly, the microchip which measures fluorescence intensity, and also to provide its manufacturing method. <P>SOLUTION: This microchip for measuring fluorescence intensity comprises: a resin substrate 1 having parts formed thereon, namely, a liquid introduction hole 2 penetrating in the thickness direction, a liquid storing hole 3 penetrating in the thickness direction, a channel 5 formed on one side in the thickness direction connecting the liquid introduction hole 2 to the liquid storing hole 3, and a measuring part 4 formed in the channel 5; the first coating resin film 6 bonded so as to cover the liquid storing hole 3, the liquid introduction hole 2, the channel 5 and the measuring part 4 on one surface side in the thickness direction of the resin substrate 1; and the second coating resin film 7 bonded so as to cover at least the liquid storing hole 3 on the other surface side in the thickness direction of the resin substrate. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a microchip for measuring fluorescence intensity and a manufacturing method thereof.

  In general, microchips used to mix, react, synthesize, extract, separate, and analyze a small amount of sample are made of a glass substrate with a fine channel, but it is difficult to precisely process the glass substrate. In recent years, various resin microchips have been proposed.

  The following Patent Document 1 discloses a plastic made by bonding a first plastic substrate having a fine flow path and a through-hole formed at the time of injection molding and a second plastic substrate having no flow path. A microchip is disclosed. The embodiment discloses a microchip including a slide glass-shaped first plastic substrate and a slide glass-shaped second plastic substrate.

  Patent Document 2 below discloses a homopolymer composed of 1,3-cyclohexadiene (CHD) or a CHD derivative, which exhibits excellent light transmittance with respect to a light source having a specific ultraviolet wavelength, and can be copolymerized therewith. A resin microchip formed from a polymer obtained by hydrogenating a polymer obtained by copolymerization with another monomer is disclosed.

Since general organic polymer materials have absorption in the ultraviolet region, accurate measurement values cannot be obtained by the absorbance method. Therefore, an object of the present invention is to provide an optical analysis microchip excellent in light transmittance in the ultraviolet wavelength region of 230 to 400 nm.
JP 2006-234600 A JP 2000-39420 A

  Patent Document 1 discloses a microchip in which a flow path is formed by bonding two plastic substrates. The plastic plates are joined by thermocompression bonding, solvent pressure bonding, laser welding, and ultrasonic welding. In this structure, there is a possibility that a gap is formed in the joint due to the surface roughness of each plastic plate. Accordingly, it is necessary to smooth the surface of the joint portion by polishing or the like so that no gap is formed in the joint portion of each plastic plate.

  The microchip described in Patent Document 2 is a microchip for optical analysis using a special resin excellent in light transmittance in the ultraviolet wavelength region. A general organic polymer material has absorption in the ultraviolet region. Therefore, it is disclosed that a special resin excellent in light transmittance in the ultraviolet wavelength region described in Patent Document 2 has been considered in order to directly detect a biological substance that only absorbs in the ultraviolet region without fluorescent labeling. . Therefore, the microchip described in Patent Document 2 is not used for fluorescence measurement.

  Further, the structure described in Patent Document 2 discloses a structure in which a plate-like member with grooves and a plate-like member without grooves are bonded together. The joining method here is also a joining between the plates, and it is necessary to smooth the surface of the joining portion by polishing or the like so that there is no gap in the joining portion of the plastic plate.

  The present invention has been made in view of the above situation, and is a microchip for measuring fluorescence intensity, which is a resin-made microchip that can be easily joined without strictly smoothing the joining surface of a resin substrate. It is an object to provide a chip and a manufacturing method thereof.

  As a result of intensive studies by the present inventors, a microchip suitable for fluorescence intensity measurement can be obtained simply by bonding a resin substrate on which a flow path or the like is formed and a resin film without strictly smoothing the bonding surface. I found what I could offer.

  That is, in order to solve the above problems, the microchip of the present invention is a liquid introduction hole penetrating in the thickness direction for introducing a liquid to be a measurement sample in the fluorescence intensity measurement microchip used for measuring the fluorescence intensity. A liquid reservoir hole penetrating in the thickness direction for accumulating the liquid after fluorescence intensity measurement, and a flow path through which the liquid is formed on one side in the thickness direction connecting the liquid introduction hole and the liquid reservoir hole And a measurement part for measuring the fluorescence intensity formed in the flow path, a resin substrate on which the liquid substrate is formed, the liquid introduction hole on the one side in the thickness direction of the resin substrate, the liquid reservoir hole, the measurement A first covering resin film joined so as to cover the portion and the flow path, and a second covering resin film joined so as to cover at least the liquid reservoir hole on the other surface side in the thickness direction of the resin substrate, And wherein the Ranaru.

  By bonding a resin film that easily follows the shape of the resin substrate on which the flow path and the like are formed, a microchip can be formed without precisely smoothing the surface of the bonded portion of the resin substrate. Moreover, even if it joins from both surfaces of the thickness direction of a resin substrate by using a resin film, the thickness and weight of a microchip do not differ greatly from the resin substrate itself. Moreover, the through-hole opened in the resin substrate by bonding the resin film from both surfaces in the thickness direction of the resin substrate can be used as a container closed on both surfaces in the thickness direction, for example, a liquid reservoir.

  The thickness of the resin substrate is preferably 1 mm or more and 5 mm or less. More preferably, it is 1.5 mm or more and 2.5 mm or less. By having such a thickness, the resin substrate becomes a resin substrate with less distortion. The depth of the through-hole formed in the resin substrate, that is, the capacity as a liquid reservoir can be adjusted by the thickness of the resin substrate. The thickness of the resin film is preferably 0.05 mm or more and 0.5 mm or less. More preferably, it is 0.05 mm or more and 0.2 mm or less. By having this thickness, the shape of the resin film is maintained, and it is easy to join the resin film to the resin substrate.

  The resin substrate has a liquid introduction hole penetrating in the thickness direction, a liquid reservoir hole penetrating in the thickness direction, a flow path formed on one side in the thickness direction connecting the liquid introduction hole and the liquid reservoir hole, and A measurement part for measuring the fluorescence intensity formed in the flow path is formed.

  The through hole and the flow path provided in the resin substrate become a line through which a liquid serving as a measurement sample flows by being joined to the first coating resin film and the second coating resin film. The liquid introduced into the liquid introduction hole passes through the flow path, passes through the measurement unit, and is guided to the liquid reservoir hole. If such a line through which the liquid flows can be formed, the liquid introduction hole, the liquid reservoir hole, the flow path, and the measurement unit are not particularly limited in shape or the like. The cross-sectional shape of the channel is not particularly limited. The cross-sectional shape of the channel may be any of a square shape, a triangle shape, a polygonal shape, a semicircular shape, a semielliptical shape, and the like. Further, the measurement unit may have a shape that can enclose a support material for fluorescence intensity measurement. In that case, a shape in which the width or height of the flow path is partially narrowed before and after the measurement unit so that the carrier substance does not flow may be taken. Further, the flow paths may be branched before and after the measurement unit to form an aggregate of a plurality of narrow flow paths.

  If the liquid reservoir hole is formed as one set of lines from the liquid introduction hole, a plurality of sets of lines may be formed on one microchip.

  The liquid as the measurement sample refers to a liquid sample containing, possibly including, or not including the measurement substance to be measured.

  The liquid may be of any origin as long as it has a viscosity that can move in the flow path of the microchip. For example, environmental samples, biopsy samples, water-soluble solvents, water and the like can be mentioned. Examples of environmental samples include soil collected from a factory site, water collected from a river, and the like. Biopsy samples include cells, cultures, body fluids, urine, blood and the like. Examples of the water-soluble solvent include buffer solutions such as phosphate buffered saline (PBS) and Tris buffer, and organic solvents such as alcohol and acetone.

  The measurement substance can be any substance such as a chemical substance, a polymer, a microorganism or virus and a fragment thereof, and a hormone. In particular, substances that can cause environmental pollution such as toxic substances (PCB, dioxins) and oily substances (heavy oil) contained in soil and insulating oil, etc., contained in foods of pathogenic E. coli contained in river water, and food Pesticides and the like that can be suitably exemplified.

  In order to measure the amount of the measurement substance, a method using a reaction via a substance having molecular recognition ability capable of selectively detecting the measurement substance is performed. Examples of reaction of a substance having molecular discrimination ability include antigen-antibody reaction, hybridization reaction between nucleic acids, biological response ability between enzyme and substrate, and the like.

  For example, there is a method of detecting the fluorescence intensity by labeling an antibody with a fluorescent substance using an antigen-antibody reaction. The fluorescent substance used for labeling is not particularly limited, and applicable fluorescent substances include fluorescein, rhodamine, phycocyanin and the like. The temperature at which the antigen-antibody reaction is carried out, the composition of the liquid in which the reaction is carried out and the pH are possible under the conditions used in ordinary immunoassay. For example, the temperature is preferably about 5 to 60 ° C. and the PH is preferably about 5 to 9. A temperature of 37 ° C. and a pH of around PH7 are particularly suitable.

  The fluorescence intensity can be measured by using a fluorescence intensity measuring machine of the principle generally used in the measurement unit. That is, the fluorescence intensity is measured by irradiating excitation light to the measurement unit and measuring the fluorescence light emitted from the measurement substance present in the measurement unit. Therefore, it is preferable that the first coating resin film covering the measurement part has a high transmittance for the wavelengths of excitation light and fluorescent light.

  A known method can be used to move the liquid in the flow path. For example, it is conceivable to send a liquid from a liquid introduction hole to a liquid reservoir hole by sucking the gas or pressing the gas using a gas. The resin substrate, the first coating resin film, and the second coating resin film may be provided with a small hole for venting air or a suction port connected to a suction pump for suction according to the purpose. For example, when a suction port for sucking and feeding liquid is provided on the resin substrate, the suction port is formed as a through hole connected to the liquid reservoir hole, and one side is covered with the first coating resin film or the second coating resin film. May be joined together. The suction port is connected to a suction pump provided outside to suck and feed the liquid. In this case, the amount of liquid feeding can be controlled by the suction amount of the pump.

  The resin substrate, the first coating resin film, and the second coating resin film are preferably formed of a low fluorescent resin. In that case, the noise of the fluorescence intensity of the measurement substance can be reduced.

  As the low fluorescent resin, acrylic resin, polyolefin resin, polyester resin, polycarbonate resin, polystyrene resin, polyamide resin, and polyvinylidene chloride resin can be used. Particularly preferred is a low-fluorescence grade type such as a type obtained by removing the fluorescent agent of the low-fluorescence resin. The first coating resin film is preferably a transparent resin having a high transmittance for wavelengths of excitation light and fluorescent light.

  The low-fluorescence resin may be added with known antioxidants, ultraviolet absorbers and the like as long as there is no hindrance in measuring the desired fluorescence intensity. The surface of the microchip formed of a low fluorescent resin may be coated with a known hard coat agent in order to improve chemical resistance, wear resistance, and the like. Further, the surface of the microchip formed of a low fluorescent resin may be subjected to a hydrophilic treatment (improvement of wettability) so that the measurement sample can easily flow.

  In particular, the low-fluorescence resin has a low-fluorescence property such that the fluorescence intensity of the low-fluorescence resin in the measurement unit is 1/5 or less, more preferably 1/10 or less, of the fluorescence intensity of the substance to be measured and detected. A resin is desirable. More preferably, it is desirable that the fluorescence intensity of the low fluorescence resin is negligible, that is, an intensity close to zero. Low noise due to the fluorescence intensity of the resin substrate, the first coating resin film, and the second coating resin film in the measurement unit being 1/5 or less, more preferably 1/10 or less of the fluorescence intensity of the substance to be measured and detected. Accurate fluorescence intensity can be measured.

  Examples of the bonding include thermocompression bonding, solvent welding, laser welding, ultrasonic welding, bonding via an adhesive and a pressure-sensitive adhesive, and bonding via an adhesive and pressure-sensitive adhesive is particularly desirable. In particular, in the case of non-heat bonding, the resin does not shrink and the dimensional accuracy is improved. A microcapsule type adhesive may be used as a simpler method. By applying a microcapsule type adhesive to the joint portion in advance and pressurizing the joint portion, the microcapsule is broken, and the adhesive inside can come out and adhere. Therefore, it is possible to apply the adhesive only to the joint portion without causing thermal contraction of the resin and without the adhesive protruding into the fine flow path.

  The microchip of the present invention can be produced by the following method.

A liquid introduction hole penetrating in the thickness direction for introducing a liquid as a measurement sample, a liquid reservoir hole penetrating in the thickness direction for accumulating the liquid after fluorescence intensity measurement, and the liquid introduction hole and the liquid reservoir hole A resin substrate preparation step of preparing a resin substrate on which a flow path in which the liquid formed on one side in the thickness direction to be connected flows and a measurement unit that measures fluorescence intensity formed in the flow path;
A resin film bonding step of pressure bonding the resin film to both surfaces in the thickness direction of the resin substrate via an adhesive or an adhesive.

  The resin substrate may be formed by cutting the cut resin substrate to form holes, flow paths, or the like, or having holes or flow paths by injection molding or the like using a molding die in which holes or flow paths are formed in advance. You may prepare by producing a resin substrate.

  The adhesive or pressure-sensitive adhesive may be applied to at least one of the resin substrate or the adhesive film. When applying an adhesive or pressure sensitive adhesive to a resin substrate or resin film, apply masking etc. on the other side of the bonding surface so that the adhesive or pressure sensitive adhesive is applied only to the bonding surface, or apply it with a spray or roller. Also good. Moreover, you may prepare the resin film beforehand apply | coated with the adhesive agent or the adhesive. The resin film and the resin substrate are bonded together by applying pressure.

  Depending on the method for measuring the fluorescence intensity, a supporting material disposing step of disposing a supporting material in the measurement part may be provided between the resin film joining step. In this case, it is desirable that the measurement part of the resin substrate has a shape that can enclose the support material.

  The supporting substance is not particularly limited as long as it is a water-insoluble substance capable of supporting the substance on its surface. For example, carriers made of polymers such as latex, polyethylene, polystyrene, polypropylene, inorganic silicate carriers (glass, silica gel, etc.), organic carriers (plastic, nitrocellulose, dextran, etc.), carbon materials such as activated carbon, metal particles / magnetite Examples thereof include porous materials such as magnetic nylon such as nylon, nitrocellulose, cellulose acetate, glass fiber, and porous polymer.

  The shape of the support material can take various forms such as particles (beads), sheets, and tubes. A preferable example is a bead shape having a diameter of 10 μm to 500 μm. More preferably, it is in the form of beads having a diameter of 50 μm to 200 μm.

  A substance having affinity for a substance having molecular recognition ability can be specifically detected by preliminarily supporting the substance having molecular recognition ability on the support substance. A method of supporting a substance having molecular recognition ability on the supporting substance can be performed by a known method. For example, there are a method of physically adsorbing an antibody, which is a substance having molecular recognition ability, on a support material, and a method of covalently bonding an amino group or the like of a substance having molecular recognition ability to a functional group provided on the surface of the support material. After immobilizing a molecule-recognizing substance on the support material, the surface of the support material and unreacted functional groups on the surface are blocked with an appropriate protein or surfactant to suppress nonspecific reactions. Is possible.

  The microchip for measuring fluorescence intensity of the present invention can be produced by the above method.

  The above-described microchip for measuring fluorescence intensity of the present invention has a precise bonding surface of a resin substrate by bonding a first coated resin film and a second coated resin film from both sides to a resin substrate on which a flow path or the like is formed. The first coated resin film and the second coated resin film can be easily bonded to the surface of the resin substrate without being smooth. Therefore, a microchip can be easily manufactured. Also, since the resin substrate is bonded from both sides in the thickness direction with the first coating resin film and the second coating resin film, the through-hole opened in the resin substrate can be used as a container closed on both sides in the thickness direction. can do.

  Further, if the resin substrate, the first coating resin film, and the second coating resin film are made of a low fluorescent resin, noise during measurement of the fluorescence intensity is reduced, and the fluorescence intensity of the measurement substance can be measured more accurately.

  Moreover, if the joining of the resin substrate, the first covering resin film, and the second covering resin film is joined via an adhesive or a pressure-sensitive adhesive, the adhesion of the joining surface is further increased. Further, when the bonding is non-heat bonding, there is no influence of the heat shrinkage of the resin substrate, the first coating resin film, and the second coating resin film, and the dimensional accuracy is improved. In particular, when a microcapsule type adhesive is used, the microcapsule is broken by pressurizing the joint portion to which the microcapsule type adhesive has been applied in advance, and the internal adhesive comes out and can be bonded. Therefore, heat shrinkage of the resin does not occur, and the adhesive can be applied only to the joining portion without the adhesive protruding into the fine flow path, so that more accurate joining can be performed.

  Further, according to the method for producing a fluorescence intensity measurement microchip, a fluorescence intensity measurement microchip can be easily produced.

  Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. It also serves as an explanation of the method for producing the fluorescence intensity measuring microchip of the present invention.

  FIG. 1 is a schematic view showing an embodiment of a microchip for measuring fluorescence intensity according to the present invention. FIG. 1A is a schematic plan view, and FIG. 1B is a schematic side view. FIG. 2 is a schematic cross-sectional explanatory diagram of the fluorescence intensity measurement microchip of FIG. FIG. 2 differs from FIG. 1B in that the size of each hole and the like is changed from the actual scale in order to explain the flow of the liquid. FIG. 3 is a perspective view of the fluorescence intensity measurement microchip of FIG. FIG. 4 is an explanatory diagram of the configuration of the microchip for measuring fluorescence intensity shown in FIG. FIG. 4A shows the configuration of the microchip before joining the resin film and the resin substrate, and FIG. 4B shows the microchip after joining.

  The resin substrate 1 shown in FIGS. 1, 2, 3 and 4 was manufactured by using an acrylic resin (Acrylite product number 000 (ultraviolet transmission type) manufactured by Mitsubishi Rayon Co., Ltd.) and then partially cutting it. The processed parts are through-holes 9a and 10a in the resin substrate thickness direction of the passage 9 and the gas passage 10 which will be described later.Other shapes are integrally manufactured in advance by injection molding. .

  The resin substrate 1 includes a resin plate having a thickness of 1.7 mm, a length of 26 mm, and a width of 40 mm, each having two liquid introduction holes 2, a liquid reservoir hole 3, a flow path 5, a measurement unit 4, a passage 9, a gas passage 10, and a suction port. 8 and an attachment hole 11 for attachment to a fluorescence intensity measuring machine. The upper surface of the resin substrate 1 is denoted by 1u and the lower surface is denoted by 1d.

  As shown in FIGS. 1 and 2, the liquid introduction hole 2 is a cylindrical through-hole having a diameter of 5 mm, and the height is 1.7 mm. Similarly, the liquid reservoir 3 is an elliptical or long cylindrical through hole having a width of 2 mm, a length of 9 mm and a height of 1.7 mm, and the mounting hole 11 is a cylindrical through hole having a diameter of 2.5 mm and a height of 1.7 mm. The passage 9 is a cylindrical through hole 9a having a diameter of 0.5 mm and a height of 1.7 mm. The diameter of the passage 9 is bent at right angles from the resin substrate upper surface 1 u side and extends parallel to the plane of the resin substrate 1 and reaches the upper surface side of the liquid reservoir hole 3. The gas passage 10 includes a cylindrical through hole 10a having a diameter of 0.5 mm and a height of 1.7 mm and is bent at a right angle from the resin substrate upper surface 1u side of the resin substrate 1 in the plane of the resin substrate 1. A recess-like gas passage 10b having a diameter of 0.5 mm that extends in parallel and communicates with the upper surface side of the liquid reservoir hole 3, and the suction port 8 is a hole having a diameter of 0.5 mm that is connected to the gas passage 10a.

  The flow path 5 is a recess having a width of 1 mm and a height of 130 μm so as to connect the liquid introduction hole 2 and the liquid reservoir hole 3 to the lower surface 1d side of the resin substrate 1 and having a sectional shape opened to the lower side 1d of the rectangle. There is no penetration in the thickness direction of the resin substrate 1. As shown in FIG. 2, the height h2 of the flow path 5 is about 1/10 of the height h1 of the liquid introduction hole 2 and the liquid reservoir hole 3 which are through holes.

  The measurement unit 4 is a cylindrical recess having a height of 130 μm and a diameter of 1 mm, which is the same as that of the flow channel 5, and the flow channel 51 in front of the measurement unit 4 and the flow channel 52 after the measurement unit 4 are lengthwise from the measurement unit 4 to the front and rear. The flow path 5 is branched into four narrow-width flow paths each having a width of 1 mm and a width of 50 μm flowing in parallel with each other by 1 mm. In other words, the flow path 5 has a structure in which a carrier material (to be described later) does not flow from the measurement unit 4 when the width of the flow path narrows before and after the measurement unit 4.

  As shown in FIG. 2, the flow path 5 is formed so that one end portion 5 f is connected to the space on the lower end 2 d side of the liquid introduction hole 2. As shown in FIG. 1A, the liquid reservoir hole 3 is not on the extension line of the flow path 5, but is disposed laterally with a certain space from the extension line. This is removed from the extension of the flow path 5 in order to increase the volume of the liquid reservoir 3 as necessary. Further, the flow path 5 is not directly connected to the liquid reservoir hole 3 as shown in FIG. 1 but is connected through a passage 9 leading to the liquid reservoir hole 3. Further, the liquid reservoir hole 3 is connected to the suction port 8 through the gas passage 10. Two mounting holes 11 are formed at a predetermined distance in a line in the longitudinal direction at a location where the liquid reservoir hole 3 or the like of the resin substrate 1 is not formed.

  A first coated resin film 6 having a vertical and horizontal width equivalent to that of the resin substrate 1 and having a vertical and horizontal width of 20 mm and a width of 40 mm is bonded to the lower surface 1 d of the resin substrate 1 except for a portion where the mounting holes 11 are formed. A hole 81 having a diameter equivalent to that of the suction port 8 and having a diameter of 0.5 mm is formed in the first coating resin film 6 at the position of the suction port 8.

  A polyolefin resin film having a thickness of 0.1 mm and having a low autofluorescence characteristic in which a microcapsule-type adhesive is applied in advance as the first coating resin film 6 and the second coating resin film 7 (manufactured by 3M, product number 9795, Microplate sealing tape) was used.

  The first encapsulating resin film 6 and the second encapsulating resin film 7 used here are preliminarily coated with a microcapsule type adhesive on one entire surface, but the microcapsule type adhesive is applied to a resin film not coated with an adhesive. May be applied. In that case, it is possible to prevent the adhesive from being applied to the place where the liquid comes into contact by masking or the like.

  The first coating resin film 6 is bonded to the resin substrate 1 by being pressed from the surface of the first coating resin film 6 on which the adhesive is not applied, and the first coating resin film 6 is bonded to the liquid introduction hole 2. The bottom surface of the lower side 2d opening, the lower side 5d opening of the flow channel 5 and the bottom surface of the lower side 4d opening of the measurement unit 4, and the bottom surface of the lower side 3d opening of the liquid reservoir hole 3 are configured.

  A second covering resin film 7 having a length of 20 mm and a width of 15 mm is bonded to the upper surface 1 u of the resin substrate 1 so as to cover a portion corresponding to the upper 3 u opening of the liquid reservoir hole 3. The second covering resin film 7 is not joined to the upper 2u opening of the liquid introduction hole 2, and the upper side is an opening. The second encapsulating resin film 7 is coated with the microcapsule adhesive on the entire surface of one side. The second coating resin film 7 is bonded to the resin substrate 1 by being pressed from the surface of the second coating resin film 7 where the adhesive is not applied, and the second coating resin film 7 is formed in the liquid reservoir hole 3. Configure the ceiling surface.

  A measurement unit 4 is formed between the liquid reservoir hole 3 and the liquid introduction hole 2 in the flow path 5. The measurement unit 4 contains 60 to 70 polystyrene beads having a diameter of 90 μm. As will be described later, a pseudoantigen is immobilized on the beads in advance. The flow path 51 in front of the measurement unit 4 and the flow path 52 after the measurement unit 4 are narrow-width flow paths 4 that flow in parallel with each other, with a width of 1 mm of the flow path 5 of 50 μm in width of 1 mm before and after the measurement unit 4. Branch to book. Since the beads encapsulated in the measurement unit 4 have a diameter of 90 μm, they cannot pass through the flow channel 51 and the flow channel 52 having a width of 50 μm and therefore do not flow out from the measurement unit 4 into the flow channel 5.

  In FIG. 1B, since the passage 9 and the gas passage 10 exist beyond the liquid reservoir hole 3 and cannot be described, FIG. 2 shows a schematic cross-sectional explanatory view of FIG. Since FIG. 2 is a diagram created for the purpose of explaining the flow of the liquid, the scales and positional relationships of the holes and the like are different from the actual ones. In FIG. 2, the upper surface of the resin substrate 1 is denoted by 1u and the lower surface is denoted by 1d. In FIG. 2, the passage 9 and the gas passage 10 are shown so as to be understood. Actually, as shown in FIGS. 1A and 1B, the passage 9 and the gas passage 10 extend from the side opposite to the liquid reservoir hole 3 toward the front of the drawing and are connected to the upper part of the liquid reservoir hole 3. .

  The microchip of one embodiment will be described in more detail by explaining the flow of liquid with reference to FIG.

  The passage 9 is connected to the cylindrical passage 9a having a diameter of 0.5 mm extending from the one end portion 5e of the flow passage 5 in the thickness direction of the resin substrate 1 and to the upper portion 3u side of the liquid reservoir hole 3 by being bent at right angles. And a cylindrical passage 9b extending in the plane direction of a resin substrate having a diameter of 0.5 mm. The passage 9b has a shape in which the resin substrate upper surface 1u side is open, and forms a sealed passage space when the second covering resin film 7 is joined. A gas passage 10 extending from the suction port 8 is connected to the upper end on the opposite side of the passage 9 on the downstream side of the liquid flow in the liquid reservoir 3. The gas passage 10 has a cylindrical gas passage 10b having a diameter of 0.5 mm extending from the upper 3u side of the liquid reservoir hole 3 in the plane direction of the resin substrate and a diameter of 0.5 mm extending from the liquid reservoir hole 3 in the thickness direction of the resin substrate 1 at a right angle. The gas passage 10a is connected to a suction port 8 formed in the lower surface 1d of the resin substrate. Further, the gas passage 10b has a shape in which the resin substrate upper surface 1u side is opened, and a sealed passage space is formed by joining the second covering resin film 7.

  In FIG. 2, the directions in which the liquid is sucked are indicated by arrows A1 to A6. A line is formed in which the liquid 21 flows from the liquid introduction hole 2 to the liquid reservoir hole 3 through the measurement unit 4 of the flow path 5. The liquid 21 as the sample introduced into the liquid introduction hole 2 is transferred to the liquid reservoir hole 3 by suction from the suction port 8.

  Since the flow path 5 extends in the plane direction of the resin substrate 1 from the lower end 2d of the liquid introduction hole 2, even if the liquid 21 includes bubbles 22, the bubbles 22 float on the liquid surface and enter the flow path 5. It has a difficult structure.

  Further, the flow path 5 and the liquid reservoir hole 3 are not directly connected to each other, but are once lifted to the upper surface 1 u side of the resin substrate 1 by the passage 9. Therefore, the liquid 21 is once lifted to the upper surface 1u of the resin substrate as indicated by the arrow A2, so that once the liquid 21 enters the liquid reservoir hole 3 (arrow A3), it is difficult to reversely flow until the liquid reservoir hole 3 is full. It has become. Further, if the size of the liquid reservoir hole 3 is set so as to have, for example, 1.5 times or more the volume of the liquid introduction hole 2, the liquid reservoir hole 3 does not first fill up. Further, since the gas passage 10 connected to the suction port 8 is connected to the upper part 3u side of the liquid reservoir hole 3, the liquid 21 is not sucked out from the suction port 8 until the liquid reservoir hole 3 is full. It is accumulated. The sucked gas passes through the gas passage 10 and is sucked in the directions of arrows A4, A5, and A6 and sucked out of the resin substrate 1 from the suction port 8.

  In addition, arrows B1 and B2 described below the resin substrate 1 in FIG. 2 indicate the incident direction B1 of excitation light and the emission direction B2 of fluorescence when measuring the fluorescence intensity. The incident and emission angles are not accurate. The excitation light passes through the first coated resin film 6 from the direction of the arrow B1 and hits the measuring unit 4 of the resin substrate 1, and the emission light of the fluorescence from the measuring unit 4 passes through the first coated resin film 6 and the direction of the arrow B2. To be released. The fluorescence intensity of the emitted fluorescence is measured.

  FIGS. 4A and 4B are explanatory diagrams of the configuration of the microchip for measuring fluorescence intensity. As shown in FIGS. 4A and 4B, the second coated resin film 7 and the first coated resin film 6 are joined from both sides in the thickness direction of the resin substrate 1 to thereby measure the fluorescence intensity measurement microchip. Is easily prepared.

  A test example in which fluorescence intensity measurement was performed using the microchip will be described below.

  In this test example, in order to measure the amount of PCB extracted from the insulating oil used in the transformer, fluorescence intensity measurement using an antigen-antibody reaction was performed.

  PCB pseudoantigen simulated by PCB was fixed to a plurality of polystyrene beads (particle size standard particles) having a diameter of 90 μm.

  0.125 g of beads were washed with ethanol and dried, 2.5 ml of a PCB pseudoantigen solution 20 μg / ml prepared with a phosphate buffer was added, and the mixture was stirred overnight at room temperature to physically adsorb the PCB pseudoantigen onto the bead surface. . The pseudoantigen-adsorbing beads stored at 4 ° C. were washed with pure water. After washing, 70 beads were packed in the measurement part 4 of the microchip, and the microchip was dried under reduced pressure for 5 minutes.

A fluorescent substance (registered trademark Qdot) is added to a PCB antibody having binding power to PCB. 655: manufactured by Invitrogen) was labeled. The labeling of the PCB antibody was carried out by mixing Qdot activated with SMCC (succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylate) with PCB antibody reduced with DTT (dithiothreitol), then β -Concentration and purification were performed after blocking with mercaptoethanol. The labeling method was performed according to the labeling kit manual of Invitrogen.

  The fluorescence intensity measurement was performed by irradiating the measurement unit 4 of the microchip with excitation light from the excitation light irradiating means, and detecting the optical signal emitted from the measurement unit with a fluorescence measurement device. FIG. 5 is a schematic perspective view showing the internal structure of the fluorescence intensity measuring machine used. The amount of PCB in the liquid sample was determined by comparison with the fluorescence intensity when a known amount of PCB was measured from the measured fluorescence intensity.

  FIG. 5 shows the internal structure of the fluorescence intensity measuring device 1000. The microchip 100 for fluorescence intensity is mounted and attached to the tray 130 such that the lower surface 1d side of the resin substrate 1 faces down. Although not shown in FIG. 5, mounting projections are formed on the tray 130, and the microchip 100 is positioned on the tray 130 by inserting the microchip mounting holes 11 into the projections. Although not shown in FIG. 5, the suction port 220 and the attachment port connected to the suction tube 230 are connected to the tray 130 at a location corresponding to the suction port 8 of the microchip 100.

  A suction path from the suction pump 200 and the suction pump 210 is formed by placing the mounting cover 110 on the positioned microchip 100. Each suction pump is connected to each one of the suction ports 8 and each feeds one line.

  The fluorescence intensity is detected by irradiating the microchip measuring unit 4 with laser light having a wavelength of 405 nm emitted from the laser 300 from the lower 4d side, and detecting the optical signal having a wavelength of 655 nm emitted from the measuring unit 4 by the fluorescence measuring device 310. The tray 130 is moved horizontally by the motor 120 and is controlled so that the laser beam strikes the measurement unit 4 of the microchip. Each control is performed by a plurality of control boards 400. The internal mechanism of the fluorescence intensity measuring device 1000 is housed in a case 500.

  The fluorescence intensity was measured using the fluorescence intensity measuring apparatus 1000 described above.

  30 μl of the sample solution to be inspected and 240 μl of the PCB antibody solution labeled with the fluorescent substance Qdot were mixed. At this time, the PCB antibody solution is mixed in a predetermined amount so as to be 10 times more than the PCB expected to be contained in the sample solution. 10 μl of the mixed solution was injected into the liquid introduction hole 2 of the microchip. In the test example, one sample was used, and the suction pump 200 was operated to feed the liquid. The suction speed of the suction pump 200 was 1 μl / min, and the solution was fed for 10 minutes. The liquid could be fed to the measuring unit 4 without any liquid bubbles.

  Before the liquid was fed to the measurement unit 4, the fluorescence intensity in the measurement unit 4 was measured. The measured fluorescence intensity at this time was 1/3 of the fluorescence intensity measured after the liquid was fed to the measurement unit 4. The fluorescence intensity of the measurement unit 4 before the liquid is fed is not less than 1/5 of the fluorescence intensity measured after the liquid is fed. .

  However, the fluorescence intensity before liquid feeding in the measurement unit 4 could be suppressed to be much lower than the fluorescence intensity after liquid feeding, and the fluorescence intensity could be measured with high accuracy.

  While the liquid mixture of the test sample liquid introduced into the liquid introduction hole 2 and the labeled PCB antibody solution is being sent, the test target sample liquid and the labeled PCB antibody cause an antigen-antibody reaction. All the PCBs in the sample liquid to be examined bound to the labeled PCB antibody. Since the labeled PCB antibody is in excess of the expected amount of PCB in the sample liquid to be examined, a labeled PCB antibody that has not reacted with PCB is present in the sample liquid to be examined.

  The labeled PCB antibody that has not reacted with the PCB in the sample liquid to be inspected sent to the measuring unit 4 is immobilized on the beads encapsulated in the measuring unit 4 in the measuring unit 4 and the antigen. An antibody reaction is caused, and the labeled PCB antibody is immobilized on the beads enclosed in the measurement unit 4. Substances other than the immobilized labeled PCB antibody are sent to the liquid reservoir 3, and the labeled PCB antibody immobilized on the beads remains in the measurement unit 4, and the fluorescence intensity of the labeled substance is measured. It was done. The fluorescence intensity derived only from the measurement sample was obtained by subtracting the fluorescence intensity before feeding the mixed liquid in the measurement unit 4 measured before from the fluorescence intensity measured here. Based on the obtained fluorescence intensity, the amount of PCB in the sample solution was calculated by comparison with a calibration curve similarly measured in advance using a known amount of PCB.

It is the schematic which shows one Embodiment of the microchip for fluorescence measurement of this invention. FIG. 2 is a schematic cross-sectional explanatory diagram of the fluorescence intensity measurement microchip in FIG. 1. It is a perspective view of the microchip for fluorescence measurement of FIG. FIG. 2 is a configuration explanatory view showing the fluorescence measurement microchip in FIG. 1. It is a schematic perspective view which shows the internal structure of a fluorescence intensity measuring machine.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Resin substrate, 2 ... Liquid introduction hole, 3 ... Liquid reservoir hole, 4 ... Measurement part,
5 ... channel, 6 ... first coated resin film, 7 ... second coated resin film,
8 ... suction port, 9 ... passage, 10 ... gas passage, 11 ... mounting hole,
100 ... microchip, 300 ... laser, 310 ... fluorescence measuring device,
200: suction pump, 1000: fluorescence intensity measuring machine.

Claims (8)

In the fluorescence intensity measurement microchip used to measure fluorescence intensity,
A liquid introduction hole penetrating in the thickness direction for introducing a liquid as a measurement sample, a liquid reservoir hole penetrating in the thickness direction for accumulating the liquid after fluorescence intensity measurement, and the liquid introduction hole and the liquid reservoir hole A resin substrate formed with a flow path through which the liquid formed on one side in the connecting thickness direction flows, and a measurement unit for measuring fluorescence intensity formed in the flow path;
A first covering resin film joined so as to cover the liquid introduction hole, the liquid reservoir hole, the measurement part, and the flow path on the one surface side in the thickness direction of the resin substrate;
A second covering resin film joined so as to cover at least the liquid reservoir hole on the other surface side in the thickness direction of the resin substrate;
A microchip for measuring fluorescence intensity, comprising:   The microchip for fluorescence intensity measurement according to claim 1, wherein the resin substrate, the first covering resin film, and the second covering resin film are formed of a low fluorescent resin.   3. The fluorescence intensity measurement micro of claim 2, wherein the low fluorescence resin is at least one resin selected from acrylic resin, polyolefin resin, polyester resin, polycarbonate resin, polystyrene resin, polyamide resin and polyvinylidene chloride resin. Chip.   The said chip | tip is a microchip for a fluorescence intensity measurement in any one of Claims 1-3 performed through an adhesive or an adhesive agent.   The microchip for measuring fluorescence intensity according to any one of claims 1 to 4, wherein the bonding is non-heat bonding.   The microchip for fluorescence intensity measurement according to claim 4 or 5, wherein the adhesive is a microcapsule type adhesive. A liquid introduction hole penetrating in the thickness direction for introducing a liquid as a measurement sample, a liquid reservoir hole penetrating in the thickness direction for accumulating the liquid after fluorescence intensity measurement, and the liquid introduction hole and the liquid reservoir hole A resin substrate preparation step of preparing a resin substrate on which a flow path in which the liquid formed on one side in the thickness direction to be connected flows and a measurement unit that measures fluorescence intensity formed in the flow path;
A resin film bonding step of pressure bonding the resin film to both surfaces in the thickness direction of the resin substrate via an adhesive or an adhesive;
A method for producing a microchip for measuring fluorescence intensity. The method for producing a microchip for measuring fluorescence intensity according to claim 7, further comprising a supporting material disposing step of disposing a supporting material in the measurement unit before the resin film joining step.

Images