JP2006308419A - Microchip and analysis method using same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microchip that can sample a specimen liquid directly without using any sampling/weighing tools in a chemical analysis, can supply the specimen liquid to a reaction section, and can perform a simple and precise liquid weighting method and a quantitation method. <P>SOLUTION: In the microchip for analyzing a liquid sample, a porous substance film for wrapping a fixed amount of specimen liquid when it comes into contact with an excessive amount of specimen liquid is installed adjacent to a channel or a storage tank, an analysis element for analyzing a target substance in the inspection liquid is installed inside the microchip, and the inspection liquid in the channel or storage tank is introduced to the analysis element through the porous substance film. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention is tested using human or other animal blood, body fluids or urine, fresh water, seawater and soil extracts, agricultural products, marine products and processed food extracts, and liquid samples used in natural science research as specimens. The present invention relates to a microchip for analyzing components and an analysis method using the microchip.

  Usually, when analyzing by a chemical reaction, in order to obtain an accurate analysis result, it is necessary to quantitatively weigh a liquid such as a sample and a reaction reagent and perform a quantitative reaction. In particular, in microanalysis such as blood analysis, it is necessary to accurately measure a liquid that is a specimen or a reaction reagent and supply it to a reaction site.

  Conventionally, when quantifying a substance present in a liquid specimen, the liquid that is the specimen or the reaction reagent is weighed with a collection tool (for example, a pipette, micropipette, syringe, etc.), and a certain amount of the weighed liquid is supplied to the reaction container An operation is performed in which the reaction is performed and the target substance in the sample is quantified by the signal of the reaction.

  As an example of blood analysis, in the case of the conventional wet chemistry analysis method, a large number of reagent solutions necessary for measuring one item in blood and their handling are used in combination. And the procedure is not simple. For example, when measuring glycohemoglobin (HbA1c) in blood using the blood analyzer Hitachi 7170, first weigh out 240 μl of Reagent 1 solution and 8.0 μl of HbA1c, and mix them at 37 ° C. for 5 minutes. After the reaction, 80 μl of reagent 2 solution is weighed and added. After 5 minutes at 37 ° C., quantification is performed by the two-point end method (main wavelength 450 nm, subwavelength 800 nm) (Patent Document 1).

  As described above, in order to measure glycated hemoglobin (HbA1c) in blood, (1) a collection weighing instrument is required, a sample and reagent mixing container is required, and a sample liquid supply operation is also required. For example, there are problems that the configuration is complicated, the operation is complicated, and there are many waste substances (chips, mixing containers, cleaning solutions, etc.) associated with the measurement.

  On the other hand, a so-called dry chemistry analysis method in which reagents necessary for detecting a specific component are contained in a dry state has been developed (Non-patent Document 1). In the dry chemistry analysis method, all reagents necessary for qualitative / quantitative analysis are incorporated in analytical elements such as reagent paper, disposable electrodes, and magnetic materials. Basically, it is a disposable type capable of measuring one item per sample, and can perform blood analysis simply and quickly with a relatively small amount of blood (about 10 μl) (Patent Document 2). Numerous analyzers using dry chemistry analysis methods have been developed and commercialized, including Fuji Dry Chem (Fuji Photo Film Co., Ltd.), Ekutake Chem (USA, Eastman Kodak Co., Ltd.), Dry Lab (Konica Corp.) Spot Chem (Kyoto Daiichi Kagaku Co., Ltd.), Leflotron (Germany, Boehringer Mannheim), Cellarizer (USA, Miles Laboratory, Inc.), etc. are commercially available. However, in the dry chemistry analysis, the sample liquid supply and the mixing operation with the reagent are unnecessary, but the collection weighing instrument and the collection operation are still necessary.

  In addition, in recent years, home care has been advocated as a core of the future medical system in response to the rapid transition to an aging society and the rise in medical expenses due to the development of advanced treatment. A specific method is being studied. In the home care system corresponding to this, it is desirable to use a blood analysis method that can measure many items quickly and accurately using a small amount of blood in a small and simple manner.

  Therefore, in order to solve these problems, the application of μTAS (micro total analysis system) technology for reducing the size of a conventionally used analyzer and reacting a very small amount of liquid reagent is being studied. In μTAS, a groove is formed on the surface of a chip made of glass, silicon, or resin of about 10 cm to several centimeters in order to reduce the amount of a specimen, not limited to blood, and a reagent solution or specimen is placed in the groove. A small amount of sample is analyzed by flowing and separating and reacting (Patent Documents 3 to 6, Non-Patent Document 2, etc.). In this technique, it is necessary to sample a sample and a reagent necessary for detection in a chip. However, since the volume of the specimen to be handled is extremely small, it is difficult to quantitatively measure the liquid, and various complicated configurations are required for that purpose, and the operation for handling the configuration becomes complicated. there were.

  In Patent Document 7, a micro liquid weighing structure is provided in a flow path of a microchip, and a capillary phenomenon (capillary repulsion) caused by a liquid to the flow path is used. According to Patent Document 7, in various apparatuses that require conventional quantitative liquid handling, it is possible to reduce the dead volume of the sample, save the space of the entire apparatus, and reduce the cost. A micro liquid weighing structure is provided. However, the technique described in Patent Document 7 requires operations such as microchip channel design, channel hydrophilization treatment, hydrophobization treatment, and liquid feeding by air pressure. It is hard to say that it is a method to take.

International Publication WO02 / 018953 JP-A-8-122335 JP-A-2-245655 JP-A-3-226666 JP-A-8-233778 Japanese Patent Laid-Open No. 10-142177 JP 2004-163104 A Yuzo Iwata, “11. Other Analytical Methods (1) Dry Chemistry”, Clinical Chemistry Practice Manual, Medical School, 1993, Examination and Technology Extra Number, Vol. 21, No. 5, p. 328-333 Analytical Chem. 69, 2626-2630 (1997) Aclara Biosciences

  The first object of the present invention is to provide a simple and high-precision liquid weighing method that can directly weigh sample liquid and supply it to the reaction section without using a collection weighing instrument when performing chemical analysis. And providing a microchip capable of performing a quantitative method. In addition, a second object of the present invention is to provide a microchip that enables microweighing in a microchip that is difficult to accurately weigh by a conventional weighing method.

  As a result of intensive studies to solve the above problems, the present inventors can achieve the above object by joining the porous material and the analytical element by utilizing the saturated water absorption amount of the porous material. I found.

  That is, according to the present invention, the porous substance film that encloses a certain amount of the sample liquid when it comes into contact with the excess sample liquid is installed adjacent to the flow path or the reservoir, and the target in the sample liquid is The analysis element for analyzing the substance is installed inside the microchip, and the sample liquid in the flow path or the storage tank passes through the porous substance film and is introduced into the analysis element. A microchip for liquid sample analysis is provided.

Preferably, the porous material film is made of an organic porous material.
Preferably, the porous material film is made of an inorganic porous material.
Preferably, the porous material film is composed of one or more porous materials.

Preferably, the analytical element is a dry analytical element or an electrode.
Preferably, porous material films are installed at a plurality of locations in the microchip.

According to another aspect of the present invention, there is provided a test component analysis method for analyzing a test component in a sample liquid using the above-described microchip of the present invention.
Preferably, the specimen fluid is a body fluid or a processed body fluid.

  According to the microchip of the present invention, the sample liquid can be directly weighed and quickly supplied to the reaction unit without using a collection weighing instrument, and liquid weighing and quantification can be performed easily and with high accuracy. . In addition, it is possible to carry out microweighing in a microchip, which has been difficult to accurately weigh by a conventional weighing method.

Embodiments of the present invention will be described.
In the microchip for liquid sample analysis of the present invention, a porous material film that encloses a certain amount of sample liquid when it comes into contact with an excessive sample liquid is installed adjacent to the flow path or the storage tank. An analysis element for analyzing the target substance in the microchip is installed inside the microchip, and the sample liquid in the flow path or the reservoir is introduced into the analysis element through the porous substance film It has a structure. According to the microchip of the present invention, it is possible to join an organic or inorganic porous substance to an analysis element, and to measure the target liquid, supply it to the analysis unit, and detect the reaction in one step.

  As an example of the microchip referred to in the present invention, an inlet and an outlet of a specimen such as blood, and a flow path connecting between them are arranged, and an analysis element for detecting a test component in the specimen is arranged in the middle of the flow path. Can be mentioned. In order to accurately measure a test component in a sample using such a microchip, it is necessary to accurately collect a certain volume in the microchip from the initially supplied sample and supply it to the analysis element. is necessary. In the present invention, a minute amount of sample can be collected by using a porous substance film that encloses a certain amount of sample solution when contacted with an excessive amount of sample solution. In addition, by adopting a structure in which the sample liquid in the flow path or the storage tank passes through the porous material film and is introduced into the analytical element, it is possible to analyze a sample collected in a certain amount as it is. became.

  The porous material used in the present invention may be a fibrous material or a non-fibrous material as long as it has the property of containing a certain amount of the sample liquid when it comes into contact with an excess of the sample liquid. Examples of the fibrous material include filter paper, non-woven fabric, and woven fabric (for example, woven fabric described in JP-A-55-164356, JP-A-57-66359, etc.), and knitted fabric (for example, JP-A-60 Knitted fabrics described in JP-A-22222769, etc.), glass fiber filter paper, and the like can be used.

  As the non-fibrous material, the membrane filter (brush polymer layer), polymer microbeads, glass microbeads, and diatomaceous earth described in JP-B-53-21777, US Pat. No. 3,992,158 etc. are retained in the hydrophilic polymer binder. Non-fiber isotropic porous material layer such as a porous layer containing continuous microvoids, and polymer microbeads described in JP-A-55-90859 are bonded in a point contact manner with a polymer adhesive that does not swell with water It is possible to use a non-fiber isotropic porous material layer made of a continuous fine void-containing porous material layer (three-dimensional lattice-like granular structure layer).

  As the porous material film, a porous film made of an organic polymer can also be used. Specifically, 6,6-nylon, 6-nylon, acrylate copolymer, polyacrylate, polyacrylonitrile, polyacrylonitrile copolymer, polyamide, polyimide, polyamideimide, polyurethane, polyethersulfone, polysulfone, polyethersulfone And polysulfone mixtures, polyester, polyester carbonate, polyethylene, polyethylene chlorotrifluoroethylene copolymer, polyethylene tetrafluoroethylene copolymer, polyvinyl chloride, polyolefin, polycarbonate, polytetrafluoroethylene, polyvinylidene difluoride, polyphenylenesulfur Ferene sulfide, polyfluorocarbonate, polypropylene, polybenzimidazole, poly (methyl acrylate), styrene-acrylic Nitrile copolymer, styrene-butadiene copolymer, saponified product of ethylene-vinyl acetate copolymer, polyvinyl alcohol, saponified product of cellulose acetate butyrate, cellulose acetate butyrate butyrate, saponified product of cellulose acetate butyrate or these Mixtures and the like can be used.

  JP 61-4959 (corresponding US Patent 5,019,347; Corresponding European publication EP 0166365A), JP 62-116258, JP 62-138756 (corresponding European publication EP 0226465A), JP 62-138757 (corresponding European publication) EP 0226465A), JP-A 62-138758 (corresponding European publication EP 0226465A) and the like, and a laminate of a plurality of partially bonded porous layers is also suitable.

  In addition, polymer gel (natural polymer gel: gelatin, agar, agaropectin, starch, amylose, amylopectin, carrageenan, dielan gum, chitansan gum, curdlan, collagen, arginic acid, pectin, konjac mannan, methylcellulose, hydroxypropyl cellulose, Dextran; synthetic polymer gel: polyethylene, polystyrene, polyacrylate, polyacrylic acid, polymethacrylic acid, polyglutamic acid, polyvinylpyridine, polyvinylimidazole, acrylamide, vinylpyrrolidone, hydroxyethyl methacrylate, 0-benzyl-L-glutamate, polyethylene glycol , Hyaluronic acid), latex particles, inorganic materials (porous carbon porous silicon carbide, porous glass beads, silica gel, oxidized aluminum Na, zeolites, mesoporous) are also suitable.

  An analysis element for analyzing the target substance in the sample liquid is installed inside the microchip of the present invention, and the sample liquid in the flow path or the storage tank passes through the porous substance film and the analysis element. The structure is introduced into

  The analytical element used in the present invention may be a dry analytical element based on a colorimetric method or an electrode, but is particularly preferably a dry analytical element.

  The dry analytical element that can be used in the present invention refers to a dry analytical element in which all or a part of reagents necessary for the qualitative / quantitative analysis of a component to be measured is incorporated in one or more layers. This is an analytical element using so-called dry chemistry. Specifically, as an example of such a multilayer dry analysis element, Fuji Film Research Report No. 40 (Fuji Photo Film Co., Ltd., 1995) p. 83, clinical pathology, extra edition, special feature No. 106, new development of dry chemistry / simplified examination (Clinical Pathology Publishing Society, published in 1997), and the like.

  The multilayer dry analytical element described above usually includes at least one functional layer. The number of functional layers is not particularly limited as long as it is 1 or more, and may be one layer or a plurality of layers of two or more layers.

  Specific examples of the functional layer include an adhesive layer that bonds the spreading layer and the functional layer, a water absorbing layer that absorbs the liquid reagent, a mordant layer that prevents the diffusion of the dye generated by the chemical reaction, and a gas permeation that selectively permeates the gas. Layer, an intermediate layer for suppressing / promoting mass transfer between layers, a light shielding layer for stably performing reflection photometry, a color shielding layer for suppressing the influence of endogenous dye, and a reagent layer containing a reagent that reacts with an analyte And a coloring layer containing a coloring agent.

  As an example of the multilayer dry analysis element, for example, a hydrophilic polymer layer can be provided on the support as a functional layer via another layer such as an undercoat layer in some cases. The hydrophilic polymer layer is, for example, a non-porous, water-absorbing and water-permeable layer, basically a water-absorbing layer consisting only of a hydrophilic polymer, or a coloring reagent that directly participates in a coloring reaction using a hydrophilic polymer as a binder. A reagent layer including a part or all of it, and a detection layer containing a component (for example, mordant) that fixes and immobilizes the coloring dye in the hydrophilic polymer can be provided.

(Reagent layer)
The reagent layer is a water-absorbing material in which at least a portion of the reagent composition that reacts with a test component in an aqueous liquid to produce an optically detectable change is substantially uniformly dispersed in the hydrophilic polymer binder. It is a water-permeable layer. This reagent layer also includes an indicator layer, a coloring layer and the like.

  Hydrophilic polymers that can be used as a binder in the reagent layer generally have natural or synthetic hydrophilic properties with a water swell ratio of from about 150% to about 2000%, preferably from about 250% to about 1500% at 30 ° C. Polymer. Examples of such hydrophilic polymers include gelatin (eg, acid-treated gelatin, deionized gelatin, etc.) and gelatin derivatives (eg, phthalated gelatin, hydroxyacrylate) disclosed in JP-A-60-108753 and the like. Graft gelatin, etc.), agarose, pullulan, pullulan derivatives, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone and the like.

  The reagent layer can be a layer that is appropriately crosslinked and cured using a crosslinking agent. Examples of cross-linking agents include known vinyl sulfone cross-linking agents such as 1,2-bis (vinylsulfonylacetamido) ethane and bis (vinylsulfonylmethyl) ether for gelatin, aldehydes such as aldehydes for methallyl alcohol copolymers, Examples include glycidyl group-containing epoxy compounds.

  The dry thickness of the reagent layer is preferably in the range of about 1 μm to about 100 μm, more preferably in the range of about 3 μm to about 30 μm. The reagent layer is preferably substantially transparent.

  As a reagent to be included in the reagent layer and other layers of the dry multilayer analytical element, a reagent suitable for the detection can be selected according to the test substance.

(Light shielding layer)
A light shielding layer can be provided on the reagent layer as necessary. The light shielding layer has water permeability or water penetration in which fine particles having a light absorbing property or light reflecting property (collectively referred to as light shielding property) are dispersed and held in a hydrophilic polymer binder having a small amount of film forming ability. Is a sex layer. The light-shielding layer has a detectable change (color change, color development, etc.) generated in the reagent layer, and the color of the aqueous liquid spotted and supplied to the development layer, which will be described later, when the photometry is reflected from the support side having light permeability Especially, when the sample is whole blood, the red color of hemoglobin is shielded and also functions as a light emitting layer or a background layer.

  Examples of the light-reflecting fine particles include titanium dioxide fine particles (rutile type, anatase type or brookite type fine crystal particles having a particle diameter of about 0.1 μm to about 1.2 μm, etc.), barium sulfate fine particles, aluminum fine particles or Examples of the light-absorbing fine particles include carbon black, gas black, and carbon micro beads. Among these, titanium dioxide fine particles and barium sulfate fine particles are preferable. Particularly preferred are anatase-type titanium dioxide fine particles.

  Examples of the hydrophilic polymer binder having a film-forming ability include weakly hydrophilic regenerated cellulose, cellulose acetate, etc. in addition to the hydrophilic polymer similar to the hydrophilic polymer used in the production of the reagent layer described above. Of these, gelatin, gelatin derivatives, polyacrylamide and the like are preferable. In addition, gelatin and a gelatin derivative can be used by mixing a known hardening agent (crosslinking agent).

  The light shielding layer can be provided by applying an aqueous dispersion of light shielding fine particles and a hydrophilic polymer on the reagent layer by a known method and drying. Further, instead of providing the light shielding layer, light spreading fine particles may be contained in the above-described spreading layer.

(Detection layer)
The detection layer is generally a layer in which a dye or the like produced in the presence of a test component diffuses and can be optically detected through a light-transmitting support, and can be composed of a hydrophilic polymer. A mordant may be included, for example, a cationic polymer relative to the anionic dye. The water absorption layer generally refers to a layer in which the dye produced in the presence of the test component does not substantially diffuse, and is distinguished from the detection layer in this respect.

  The multilayer dry analytical element can be prepared by a known method, and can be used by cutting into small pieces such as a square having a side of about 5 mm to about 30 mm or a circle having the same size.

  Many such multi-layer dry analytical elements have already been developed and commercialized, and Fuji Dry Chem (manufactured by Fuji Photo Film Co., Ltd.) is one example. In the present invention, such a multilayer dry analytical element itself is used. Alternatively, a part thereof can be used.

The method for joining the porous material layer and the analytical element is not particularly limited, but it is preferable to join the multilayer dry element via an adhesive layer. When the adhesive layer is wet with water or swollen with water, the porous membrane is porously bonded to the dry multilayer film by pressing the porous membrane at room temperature under a pressure of 3 to 5 kg / cm ^2. The membrane can be adhered. It is preferable that it consists of a hydrophilic polymer which can integrate each layer by this. Examples of hydrophilic polymers that can be used in the production of the adhesive layer are preferably gelatin, gelatin derivatives, polyacrylamide and the like. The dry film thickness of the adhesive layer is generally in the range of about 0.5 μm to about 20 μm, preferably about 1 μm to about 10 μm.

  The porous material film (weighing layer) may be in contact with the analysis element unit as a single weighing layer, or may be mixed with the analysis element unit. For example, when the analytical element is a multilayer dry element, the porous material weighing layer may have the function of a spreading layer and a reagent layer at the same time. When the analytical element is an electrode, it is more convenient to use a porous material electrode (for example, carbon black) directly.

  In order to increase the accuracy of liquid weighing by the porous material and the supply speed, the wettability of the liquid to be weighed in the porous material is important. The amount of liquid weighed in a uniform porous material with good wettability develops in proportion to the area and thus has a metering action. At the same time, the developing speed of the liquid is greatly increased by the capillary force on the porous material layer. Therefore, the parent of the porous material layer and the hydrophobizing treatment are preferable. Hydrophilic treatment is required for aqueous samples, and hydrophobic treatment is required for oily samples. A conventional surface treatment method can be applied as the hydrophilic / hydrophobic treatment method. Broadly divided, there are chemical surface treatment methods and physical surface treatment methods. Chemical surface treatment methods include chemical treatment, treatment with a coupling agent, steam treatment, grafting, electrochemical methods, and surface modification with additives. Examples of physical surface treatment methods include UV irradiation, electron beam treatment, ion beam irradiation, low-frequency plasma treatment, CASING treatment, glow, corona discharge treatment, and oxygen plasma.

  The liquid weighed by the porous material can be controlled by controlling the porous saturated water absorption amount. Factors affecting the porous saturated water absorption include (1) the size of the porous material layer (membrane) and (2) the type of porous material. The amount of liquid in a uniform porous material with good wettability is approximately proportional to the area. By utilizing this property and changing the size of the porous material layer (membrane), the amount of liquid to be weighed can be controlled. Also, since the saturated water absorption of different types of porous materials is different (for example, the saturated water absorption of cloth is twice that of PS membrane (polysulfone membrane, manufactured by Fuji Photo Film)), weigh using this property. The amount of liquid can also be controlled.

  The liquid supply method to the porous material layer is as follows: liquid feeding by capillary tube (FIG. 1 (a)), liquid feeding by porous material (cloth, paper, hollow fiber, etc.) (FIG. 1 (b)), for weighing There is a system in which a porous substance is immersed in a liquid (FIG. 1 (c)) and a liquid is sent through a flow path (FIG. 1 (d)).

  The following examples further illustrate the present invention, but the present invention is not limited to the examples.

Example 1: Saturated water absorption and accuracy of liquid in various porous materials (1) Preparation method of dry multilayer analytical element using knitted fabric as porous material for weighing Gelatin-primed 180 μm polyethylene terephthalate colorless transparent film An aqueous solution having the following composition was applied to and dried.

Gelatin 16.6 g / m ²
Polyoxy (2-hydroxy) polopyrene noniphenyl ether 0.2 g / m ²

Next, a tricot knitted fabric (hereinafter referred to as a knitted fabric) knitted with 36-gauge polyethylene terephthalate spun yarn equivalent to 50 denier after the water was fully supplied to the film at a supply rate of about 30 g / m ² and moistened. Were laminated by applying light pressure almost uniformly to provide a porous layer (knitted layer). Dry and adhere. The dry multilayer analytical element was cut into a 12 mm (13 mm square chip) to obtain a slide 1 used for a weighing experiment.

(2) Method for Producing Dry Multilayer Analytical Element Using Polymer Porous Membrane as Porous Material for Weighing An aqueous solution having the following composition was applied to 180 μm polyethylene terephthalate colorless transparent film coated with gelatin and dried.

Gelatin 16.6 g / m ²
Polyoxy (2-hydroxy) polopyrene noniphenyl ether 0.2 g / m ²

Next, water was supplied to the film at a supply rate of about 15 g / m ² and moistened, and then the polysulfone membrane (SE-200: manufactured by Fuji Photo Film Co., Ltd., hereinafter referred to as PS membrane) was lightly pressurized. Laminated over, dried and bonded. Thus, a dry multilayer analytical element using a polysulfone membrane as a porous material for weighing was prepared. The above dry multilayer analytical element was cut into a 12 mm (13 mm square chip) to obtain a slide 2 used for a weighing experiment.

(3) Saturated water absorption with respect to liquid in various porous material materials and accuracy thereof Liquid (10 ppm dye aqueous solution) is supplied to slide 1 by the method shown in FIG. Measure the weight change of the slide with a precision balance (Mettler Toreto Co.). The slide weight increases with increasing liquid feed time, but reaches saturation after 2 minutes (FIG. 2). The saturated water absorption of the knitted fabric is about 55 μl. Furthermore, the accuracy of saturated water absorption by the knitted fabric (coefficient of variation CV (%), n = 10) was also determined (Table 1). It has been found that the weighing accuracy of the aqueous solution with the knitted fabric is 1% or less.

  The saturated water absorption and its accuracy were determined for the slide 2 in the same manner as described above. The results are shown in Table 1. The saturated water absorption of the PS membrane is about 27.5 μl, and the accuracy of the saturated water absorption by the PS membrane is about 1.4%.

Example 2: Quantitative experiment of dry multi-layer analytical element using porous material as weighing layer (1) Preparation method of CRP dry multi-layer analytical element as weighing layer (knitted fabric) 180 μm polyethylene terephthalate colorless coated with gelatin A cross-linking agent-containing reagent solution was applied to the transparent film so as to have the following coating amount, and dried to provide a reagent layer.
Alkali-treated gelatin 14.5 g / m2
Nonylphenoxypolyethoxyethanol (containing 9 to 10 average oxyethylene units) 0.2 g / m ²
Glucose oxidase 5000 U / m ²
Peroxidase 15000 U / m ²
Glucoamylase 5000 U / m ²
2- (4-Hydroxy-3,5-dimethoxyphenyl-4- [4- (dimethylamino)
Phenyl] -5-phenethylimidazole (leuco dye) acetate 0.38 g / m ²
Bis [(vinylsulfonylmethylcarbonyl) amino] methane 0.1 g / m ²

On this reagent layer, an adhesive layer was applied and dried so as to have the following coating amount.
Alkali-treated gelatin 14.5 g / m ²
Bis [(vinylsulfonylmethylcarbonyl) amino] methane 0.1 g / m ²

Next, the following reagent-containing aqueous solution was applied to the surface of the adhesive layer so as to have the following coating amount, the gelatin layer was swollen, and a PET spun yarn equivalent to 50 denier was knitted on a 36-gauge tricot knitted fabric with a thickness of about 250 μm. The fabric was laminated almost uniformly with light pressure to provide a porous spreading layer (knitted layer).
Nonylphenoxypolyethoxyethanol (containing 9 to 10 average oxyethylene units) 0.15 g / m ²
Bis [(vinylsulfonylmethyl carbonyl) amino] methane 0.4 g / m ²

Next, a multilayer analytical element for CRP analysis was prepared by applying and drying the substrate so as to have the following coating amount.
Carboxymethylated starch 5 g / m ²
Nonylphenoxypolyethoxyethanol (containing 9 to 10 average oxyethylene units) 0.2 g / m ²

Further, a tricot knitted fabric layer, which is a spreading layer and a weighing layer, is coated with an amylase-anti-CRP / IgG conjugate in an amount of 3 mg / m ² in an ethanol solution, impregnated and dried, and multilayered for CRP analysis. A dry analytical element was obtained.

  Next, the dry multilayer analytical element was cut into a 12 mm (13 mm square chip) and placed in a slide frame described in JP-A-57-63452 to obtain a multilayer dry slide for CRP analysis.

(Quantitative test)
Supply the saturated water absorption (55 μL) of 50 mM glycerophosphate buffer solution with pH 7 containing known amount of human CRP to the CRP slide in the method 4 (FIG. 1 (d)) and keep at 37 ° C. The reflection optical density was measured with visible light having a center wavelength of 650 nm. A calibration curve was prepared by determining the difference in reflection optical density (ΔOD5-3) 3 minutes and 5 minutes after the start of the reaction. As shown in FIG. 3, it is clear that CRP can be quantified with high sensitivity by the weighing method of the present invention.

Example 3: Quantitativeness of dry multi-layer analytical element as porous material for weighing porous material on microchip system (1) PDMS concave mold creation A thick photoresist SU-8 is spin coated on a silicon wafer The film thickness was 100 μm. After preheating at 90 ° C. for 1 hour, UV light was irradiated through a mask on which a flow path pattern I corresponding to FIG. 4 was drawn, and the light irradiated portion was cured at 90 ° C. for 1 hour. The uncured portion was dissolved and removed with propylene glycol monomethyl ether acetate (PGMEA), washed with water and dried, and used as a silicon wafer / SU8 convex shape.

On this silicon wafer convex mold, PDMS (DuPont Sylgard / curing liquid = 10/1 mixed liquid) was poured, cured at 80 ° C. for 3 hours, and then gently peeled off from the silicon wafer convex mold, and the PDMS shown in FIG. A concave pattern I was created. Furthermore, the specimen injection port 1 and the air vent 2 (diameter 1 mm) were created by the Seken trepan (manufactured by Ky Industries).
Next, a PDMS concave pattern II was created by the method described above.

(2) Preparation of dry multilayer analytical element for glucose analysis Type 1 (glucose dehydrogenase (GDH) method): An aqueous solution having the following composition on a smooth 180 μm colorless transparent PET film coated with gelatin, and having a thickness after drying Was applied to a thickness of 40 μm and dried to provide a reaction layer.
Alkali-treated gelatin 20.0 g / m ²
NTB 0.8g / m ²
Surfactant 0.8g / m ²
Diaphorase 0.75 KU / m ²
Acetone 2.4 g / m ²

  Here, as the surfactant, polyoxy (2-hydroxy) propylene nonylphenyl ether (Surfactant 10G, manufactured by Aurin) was used. NTB represents 3,3 ′-(3,3′-dimethoxy-4,4′-biphenylene) bis [2- (p-nitrophenyl) -5-phenyltetrazolium chloride].

Next, an aqueous solution having the following composition was applied so that each component had the following amount and dried to prepare a dry analytical element for glucose analysis of type 1 (GDH method).
Alkali-treated gelatin 14.5 g / m ²
Nonylphenoxypolyethoxyethanol (containing 9 to 10 average oxyethylene units) 0.2 g / m ²
Glucose dehydrogenase (GDH) 5000 U / m ²
NAD 0.25 g / m ²
Latex (40μm) suspension 25 mL / m ²
Bis [(vinylsulfonylmethylcarbonyl) amino] methane 0.1 g / m ²

Type 2 (glucose oxidase (GOD) method): An aqueous solution having the following composition was applied onto a smooth 180 μm colorless transparent PET film coated with gelatin so that the thickness after drying was 40 μm and dried. Then, a reaction layer was provided.
Alkali-treated gelatin 20.0 g / m ²
POD 15.2kU / m ²
Methanol 6.8 g / m ²
Monopotassium phosphate 0.46g / m ²
L-alanine 2.82 g / m ²
α-ketoglutaric acid · 2Na 0.48 g / m ²
20% MgCl ₂ 1.22 g / m ²
Pyruvate oxidase (POPG) 3.45kU / m ²
2- (3,5-dimethoxy-4-hydroxyphenyl)-
4- [4- (Dimethylamino) phenyl] -5-phenethylimidazole
0.29 g / m ²
1N NaOH 0.49g / m ²
(PH = 7.5)

Next, an aqueous solution having the following composition was applied so that each component had the following amount and dried to prepare a dry analytical element for glucose analysis of type 2 (GOD method).
Alkali-treated gelatin 14.5 g / m ²
Nonylphenoxypolyethoxyethanol (containing 9 to 10 average oxyethylene units) 0.2 g / m ²
Glucose oxidase (GOD) 5.0 kU / m ²

(3) Preparation of microchip for glucose determination PDMS concave patterns I and II prepared in (1) were processed 24 mm × 12 mm. Next, after cutting the two types of dry analytical elements of glucose prepared in (2) into (4mm x 4mm) and (2mm x 2mm), each is incorporated into a processed PDMS concave mold. A microchip for glucose determination was prepared (FIG. 5).

(Quantitative evaluation)
Glucose measurement with a gravimetric detection method using a porous layer in a microchip

(1) Preparation of glucose calibration curve by GDH method: (cell size larger (4mm x 4mm), weighed layer is latex particles / gelatin gel)
Glucose aqueous solution (50%; manufactured by Otsuka Pharmaceutical Co., Ltd.) was diluted with 20 mM Tris-HCl pH 7.5 buffer to prepare various concentrations of glucose aqueous solution (0 mg / dL to 400 mg / dL). Gently inject various concentrations of aqueous glucose solution from the inlet 1 of the PDMS concave pattern I until the cell at the center of the chip is filled, and keep the temperature at 37 ° C using the spectral radiance meter MCPD-2000 (Otsuka Electronics Co., Ltd.). 540 nm transmission photometry was performed, and after 5 minutes, the transmission OD was determined to prepare a calibration curve (FIG. 6). As shown in the calibration curve of FIG. 6, it is clear that glucose can be accurately quantified in the micro system by the weighing detection method of the present invention.

(2) Preparation of glucose calibration curve by GOD method: (cell size smaller (2mm x 2mm), weighing layer is gelatin gel)
Glucose aqueous solution (50%; manufactured by Otsuka Pharmaceutical Co., Ltd.) was diluted with 20 mM Tris-HCl pH 7.5 buffer to prepare various concentrations of glucose aqueous solution (0 mg / dL to 400 mg / dL). Gently inject various concentrations of aqueous glucose solution from the inlet 1 of the PDMS concave pattern II until the cell in the center of the chip is filled, and keep it at 37 ° C using the spectral radiance meter MCPD-2000 (Otsuka Electronics Co., Ltd.) 505 nm transmission photometry was performed, and after 5 minutes, the transmission OD was determined to prepare a calibration curve (FIG. 7). As shown in the calibration curve of FIG. 7, it is clear that glucose can be accurately quantified in the micro system by the weighing detection method of the present invention.

FIG. 1 is a view showing a liquid supply method to the porous layer for weighing. 1 is a storage tank, 2 is a sample solution, 3 is a capillary, 4 is a porous bridge, 5 is a PDMS cell, 6 is a flow path, 14 is a porous layer (weighing layer / development layer), and 12 is a reagent layer (reaction) Layer) 10 indicates a transparent support. FIG. 2 shows a change with time in weight due to water absorption when the porous layer for weighing is a knitted fabric. FIG. 3 shows a calibration curve of a CRP dry analytical element in which the weighing porous layer is a knitted fabric. FIG. 4 shows a PDMS concave pattern. FIG. 5 shows the creation of a microchip for glucose determination. 1 is a PDMS concave shape, 2 is a flow path, 3 is a dry analytical element for glucose determination, and 4 is a PDMS sheet. FIG. 6 shows a glucose calibration curve obtained by weighing the latex particles / gelatin layer in the microchip. FIG. 7 shows a glucose calibration curve obtained by weighing the gelatin layer in the microchip.

Claims (8)

An analysis element for analyzing a target substance in a sample liquid, in which a porous substance film that encloses a certain amount of the sample liquid in contact with an excess of the sample liquid is installed adjacent to the flow path or the storage tank A microchip for analyzing a liquid sample, having a structure in which a specimen liquid in a flow path or a storage tank is introduced into an analysis element through a porous material film. The microchip according to claim 1, wherein the porous material film is made of an organic porous material. The microchip according to claim 1, wherein the porous material film is made of an inorganic porous material. The microchip according to any one of claims 1 to 3, wherein the porous material film is made of one or more kinds of porous materials. The microchip according to any one of claims 1 to 4, wherein the analytical element is a dry analytical element or an electrode. The microchip according to any one of claims 1 to 5, wherein a porous material film is disposed at a plurality of locations in the microchip. A method for analyzing a test component, wherein the test component in a sample liquid is analyzed using the microchip according to claim 1. The analysis method according to claim 7, wherein the sample fluid is a body fluid or a processed product of the body fluid.

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