JP6281945B2 - Assay device using porous media - Google Patents

Description

  The present invention relates to an assay device, and more particularly to a microfluidic device using a porous medium.

  A microfluidic device that performs biological and chemical screening using a small amount of liquid is generally manufactured using an expensive semiconductor manufacturing apparatus and requires complicated operations and external devices such as a pump. Durability and ease of use are obstacles to practical use. On the other hand, lateral flow and flow-through paper assay devices manufactured using a porous medium move the detection target in the porous medium due to capillary action, and interpret the presence or absence of the target. The above obstacles can be overcome without requiring complicated operations and external devices such as pumps. For this reason, it has attracted a great deal of attention in point of care testing (POCT) for medical diagnosis and low cost environments such as developing countries.

  So far, there has been disclosed a method (Patent Document 1) in which a fluid-impermeable barrier formed from a polymerized photoresist or a curable polymer is provided inside a hydrophilic porous medium to realize a complicated flow path pattern and reaction. .

JP 2010-515877

  In Patent Document 1, it is realized that complex fluid passages are integrated on a porous medium by patterning and laminating the porous medium, but complicated and expensive operations such as polymerization photoresist at the time of production. Need. In addition, since the fluid passage leading to the assay region is long, a large amount of reagent is required, the risk of non-specific adsorption increases, and problems such as the time required for effect determination occur. Furthermore, the method for increasing the sensitivity is not specified.

  In the first place, in most paper assay devices, the specimen moves on the hydrophilic porous medium and reacts, discolors, emits light, etc. on the medium, so compared to glass and plastic devices that are made of hard microspace on a transparent substrate. There is a problem of low sensitivity, large variation, and difficulty in quantification. In addition, it is extremely difficult to repeatedly flow and stop a plurality of liquids continuously in a fluid passage on the same hydrophilic porous medium, so that a multi-step assay is difficult, and most of them are single-step assays. Limited.

  Accordingly, one object of the present invention is to provide an assay device equipped with a microchannel that uses a porous medium and realizes high-sensitivity and high-accuracy in a general purpose.

  Another object of the present invention is to prevent drying of the fluid in the microchannel.

  Another object of the present invention is to maintain the analyte concentration in the microchannel at a high concentration.

  Another further object of the present invention is to provide an assay device that allows a multi-stage assay.

  The present inventors have intensively studied to solve the above problems, and by providing a side path in the middle of the micro flow path, the fluid is divided into two flow path portions of the micro flow path, and in particular, the second flow on the downstream side. The present invention has been completed by adopting a configuration in which it is detained in the road.

  That is, the present invention is as follows.

Item 1. An assay device comprising:
A microchannel having a tip,
A porous medium housed in a housing space arranged near the tip of the microchannel;
A space portion disposed between the microchannel and the accommodation space;
A side path that branches off from the micro-channel in the middle of the micro-channel and extends to the accommodation space,
The microchannel is divided into a first channel portion and a second channel portion including the tip portion through the branch portion of the microchannel,
The fluid that has moved in the micro flow path based on the lateral flow is separated into the first flow path section and the second flow path section, and is separated into the second flow path section and the absorbent paper by the space section. The assay device is configured to be placed in the second flow path portion after being performed.

  Item 2. Item 2. The assay device according to Item 1, wherein a hydrophobic member is disposed on the bottom surface of the side passage.

  Item 3. Item 3. The assay device according to Item 1 or 2, wherein the position of the bottom surface of the branching portion is lower than the height of the bottom surface of the first channel portion and the height of the bottom surface of the second channel portion.

  Item 4. Item 4. The assay device according to any one of Items 1 to 3, further comprising an assay reagent in the second flow path part.

  Item 5. Item 5. The assay device according to any one of Items 1 to 4, wherein the space portion is branched into a plurality of space portions toward the accommodation space.

  Item 6. The space portion has a linear main space portion extending between the microchannel and the accommodation space, and a sub branch extending from the main space portion to both sides and extending to a position of the accommodation space different from the main space portion. Item 5. The flow path width of the main space part and the sub space part is smaller than the flow path width of the second flow path part, and the flow path width of the sub space part is smaller than the width of the side path. The assay device according to 1.

  Item 7. The flow width of the main space portion and the sub space portion is ½ or less of the flow width of the second flow passage portion, and the sub space portion and the porous medium at the branch point with the main space portion of the sub space portion Item 7. The assay device according to Item 5 or 6, wherein the distance between the two is less than or equal to the channel width of the second channel part.

  Item 8. Item 8. The assay device according to any one of Items 3 to 7, further comprising a recess for accommodating the assay reagent that extends from the second channel portion in the middle of the second channel portion.

  Item 9. The width of the portion of the first flow passage portion adjacent to the branch portion with the side passage in the micro flow passage is narrower than the width of the first flow passage portion and the width of the branch portion on both sides of the portion. Item 9. The assay device according to any one of Items 1 to 8.

Item 10. The fluid is a first liquid and a second liquid;
The first liquid that has moved from the microchannel to the space is separated by the space, one is placed in the microchannel, and the other is absorbed by the porous medium,
Next, the second liquid moved in the microchannel pushes the first liquid retained in the microchannel to the porous medium, and the first liquid and the second liquid in the microchannel are exchanged. Item 10. The assay device according to any one of Items 1 to 9.

  Item 11. Item 11. The assay device according to any one of Items 1 to 10, wherein the space is subjected to a hydrophilic treatment.

  Item 12. Item 12. The assay device according to any one of Items 1 to 11, further comprising a second porous medium disposed in the fluid introduction part of the microchannel.

  ADVANTAGE OF THE INVENTION According to this invention, the general purpose and reliable high sensitivity and high precision of the measurement of the test substance in the sample liquid with the assay apparatus using a porous medium are attained. Further, since the sample solution is placed in the second flow path, the sample solution is prevented from being dried at the sample measurement location.

1 is an exploded perspective view of a microfluidic device according to a first embodiment of the present invention. (A) Schematic sectional view of the microfluidic device of FIG. 1, (b) Schematic plan view. (A)-(E) The photograph which showed the state when flowing blue ink to the microfluidic device of 1st Embodiment. The schematic plan view of the microfluidic device of 2nd Embodiment of this invention. (A)-(F) The photograph which showed the state when blue ink was poured into the microfluidic device of 2nd Embodiment. (A) The schematic plan view of the microfluidic device of 3rd Embodiment of this invention. (B) A substantially enlarged plan view of 6BC surrounded by a dotted-line square in FIG. (C) The substantially expanded sectional view in 6BC enclosed with the dotted-line square of FIG. 6 (A). (D) The substantially enlarged plan view in 6DE enclosed with the dotted-line square of FIG. 6 (A). FIG. 6E is a schematic enlarged cross-sectional view at 6DE surrounded by a dotted-line square in FIG. (A)-(G) The photograph which showed the state when blue ink was poured into the microfluidic device of 3rd Embodiment. (H) An enlarged photograph showing how the meniscus is reduced. The schematic plan view of the microfluidic device of 4th Embodiment of this invention. (A)-(D) The photograph which showed the state when a phosphate buffer was poured into the microfluidic device of 4th Embodiment. FIG. 4 is a schematic cross-sectional view of another embodiment of the microfluidic device of the present invention. (A) The schematic plan view explaining the movement of the sample liquid in the microfluidic device in Example 1. FIG. (B) A photograph of the microfluidic device in Example 1. (C) A schematic plan view illustrating movement of a sample solution in the microfluidic device in Comparative Example 1. (D) Photograph of the microfluidic device in Comparative Example 1. The graph which shows the example of solution exchange. The horizontal axis represents time, and the vertical axis represents fluorescence intensity. The graph which showed the average of five data of the assay which compared the device of this invention and the device (negative control) of a comparative example by relative standard deviation. The left side is the device of the present invention, and the right side is the device of the comparative example. The vertical axis represents the intensity of chemiluminescence.

(First embodiment)
The assay device of the first embodiment of the present invention will be described with reference to FIGS. In the present specification, “upper”, “lower”, “right”, and “left” correspond to “upper”, “lower”, “right”, and “left” in each drawing.

  In the present specification, “lateral flow” refers to a flow of fluid that moves by gravity settling as a driving force. The movement of the fluid based on the lateral flow means that the driving force of the fluid due to gravity settling is dominant, which is different from the movement of the fluid due to the capillary phenomenon in which the interfacial tension is dominant (dominant).

  In the present specification, the “microfluidic device” refers to an apparatus that performs chemical or biochemical reaction in a channel capable of detecting or measuring an analyte with a fluid of the order of μl, that is, 1 μl or more and less than 1,000 μl. .

  In the present specification, “microchannel” refers to a channel space in a microfluidic device that enables detection or measurement of an analyte with a fluid of the order of μl, that is, 1 μl or more and less than 1,000 μl.

  As used herein, “liquid sample” refers not only to a chemically pure liquid sample but also to a sample in which a gas, another liquid or solid is dissolved, dispersed or suspended in the liquid.

  As used herein, “analyte” refers to a compound or composition that is detected or measured from a liquid sample, such as a saccharide (eg, glucose), protein or peptide (eg, serum protein, hormone, enzyme, immunomodulator, lymphokine). Monokines, cytokines, glycoproteins, vaccine antigens, antibodies, growth factors, or growth factors), fats, amino acids, nucleic acids, steroids, vitamins, pathogens and their antigens, natural or synthetic chemicals, pollutants, for therapeutic purposes Or illegal drugs, as well as metabolites or antibodies of these substances.

  In the present specification, “plastic” refers to a material that can be polymerized or polymerized or molded using a polymer material as an essential component, and becomes a plastic material when heated to a temperature above the softening temperature. The plastic includes a polymer alloy in which two or more kinds of polymers are combined.

  In the present specification, “film” refers to a film-like material having a thickness of 200 μm or less, and “sheet” refers to a film thicker than that.

  In this specification, the term “soft” means that the fluid-impermeable member is hard enough to be deformed by the pressure of the liquid sample when the liquid sample is passed through.

  FIG. 1 is an exploded perspective view of a microfluidic device 10 as an assay device according to a first embodiment of the present invention. The microfluidic device 10 includes a first fluid impermeable member 12 and a second fluid impermeable member 20. The second fluid impervious member 20 includes two members, an upper member 21 and a lower member 22.

  In the present embodiment, the first fluid impermeable member 12 and the second fluid impermeable member 20 (the upper member 21 and the lower member 22) are transparent and soft sheets or films. The material of the first and second fluid-impermeable members 12 and 20 is, for example, plastic, and is preferably plastic having a contact angle of 90 degrees or less that promotes lateral flow (described later) of a hydrophilic liquid sample. .

  Such plastics include polyethylene (PE), polypropylene (PP), polyvinylidene chloride (PVDC), polystyrene (PS), polyethylene terephthalate (PET), polyvinyl chloride (PVC), polyolefin (PO), nylon, poly Biodegradability including methyl methacrylate (PMMA), cycloolefin copolymer (COC), cycloolefin polymer (COP), polycarbonate (PC), polydimethylsiloxane (PDMS), polyacrylonitrile (PAN), polylactic acid (PLA) Plastics or other polymers or combinations thereof can be mentioned.

  While not wishing to be bound by theory, if the first fluid impermeable member 12 and the second fluid impermeable member 20, which are soft plastic sheets or films, face each other, they will partially adhere. Even so, water molecules are attracted to the surfaces of the fluid-impermeable members 12 and 20 by being peeled off by the lateral flow and causing peeling charges on the opposing surfaces of the fluid-impermeable members 12 and 20. With this and the surface tension of the liquid sample, the liquid sample flows without greatly reducing the moving speed, and it is considered that the liquid sample can move by an autonomous lateral flow without using an external device such as a pump. An adhesive tape 28 as an interposed member is disposed between the first fluid impermeable member 12 and the second fluid impermeable member 20. The adhesive tape 28 has adhesive surfaces on the upper and lower surfaces, and the fluid impermeable members 12 and 20 are respectively bonded to the adhesive tape 28 on the upper and lower surfaces of the adhesive tape 28 so as to be substantially parallel and substantially horizontal to each other. The

  The first fluid impermeable member 12 has a proximal end portion 14 and a distal end portion 16, the adhesive tape 28 has a proximal end portion 29 and a distal end portion 30, and the second fluid impervious member 20 has a proximal end portion. It has a part 23 and a tip part 24. In the present embodiment, the three members 12, 20, and 28 are configured to have shapes and dimensions corresponding to the base end portions 14, 23, and 29 and the distal end portions 16, 24, and 30 stacked on each other.

  The proximal end portion 29 of the adhesive tape 28 is provided with an opening 32 into which a second porous medium, in particular a developing paper 42 which is a hydrophilic porous medium, is fitted, and the distal end portion 30 is made of the first porous medium. An opening 34 into which an absorbent paper 44 is fitted is provided, and the opening 32 and the opening 34 are connected to each other through a linearly extending gap 35 that is narrower than the openings 32 and 34. The gap 35 is branched to the side gaps 36 and 37 on both sides in the middle (in FIG. 1, approximately in the middle of the gap 35), and each of the side gaps 36 and 37 extends from the gap 35 to the length of the gap 35. After branching perpendicularly to the direction, it bends vertically and extends to the opening 34 parallel to the longitudinal direction of the gap 35.

  A U-shaped groove 75 is provided at a position corresponding to the branch position of the gap 35 of the second fluid impermeable member 20 and the side gaps 36 and 37 of the second fluid impermeable member 20. The bottom surface of the groove 75 is provided with hydrophobic members 38 and 39 (see also FIG. 2B) such as a hydrophobic adhesive, film or sheet. When the microfluidic device 10 is assembled, the hydrophobic members 38 and 39 are disposed on the bottom surface of a side path 75 (see FIGS. 2A and 2B) described later.

  The tip 24 of the second fluid-impermeable member 20 is provided with a recess 25 for accommodating the absorbent paper 44, and even when the thickness (height) of the absorbent paper 44 is greater than the height of the adhesive tape 28 Allows the flow path 74 to remain horizontal. Further, a proximal end recessed portion 26 having a width that matches the width of the gap 35 is provided on the proximal end side of the recessed portion 25, and when the microfluidic device 10 is assembled, the proximal end recessed portion 26 is a space portion 82 (FIG. 2) described later. (See (a)).

  The developing paper 42 acts to permeate and spread the liquid sample. A projecting portion is provided on the leading end side of the developing paper 42, the projecting portion is disposed in the gap 35, and the liquid sample that has been immersed in the developing paper 42 and developed is guided to the micro flow channel 74. In addition, the absorbent paper 44 that absorbs the developed liquid sample is provided at a position separated from the microchannel 74 by a space portion 82 (see FIG. 2A), which will be described later, in the opening 34 into which the absorbent paper 44 is fitted. .

  The material of the spread paper 42 and the absorbent paper 44 may be the same or different. For example, the developing paper 42 and the absorbent paper 44 are preferably a hydrophobic material when the liquid sample is hydrophobic, and are preferably a hydrophilic material when the liquid sample is hydrophilic. When the developing paper 42 and the absorbent paper 44 are hydrophilic porous media, for example, cellulose, nitrocellulose, cellulose acetate, filter paper, tissue paper, toilet paper, paper towel, or cloth, or a hydrophilic porous polymer that transmits water It may be one of

  The developing paper 42 is preferably formed of a material that hardly or hardly adsorbs the test substance or assay reagent in the liquid sample. Even if the developing paper 42 is not provided, it is only necessary to have a shape in which the liquid sample flows from the injection port to the microchannel 74 and a state in which hydrophilic treatment / hydrophobic treatment is performed. Such a shape means that in the structure of the assay device from the injection port to the microchannel 74, the space to the microchannel 74 is not completely blocked so as to prevent the flow of the liquid sample. .

  Further, the flow of the liquid sample to the micro flow path 74 is mainly caused by a lateral flow in the substantially horizontal direction in the micro flow path 74 of the liquid sample, so that the primary side pressure before the liquid sample reaches the developing paper 42, It is desirable that the differential pressure of the secondary side pressure after the liquid sample flows through the developing paper 42 is small, and the differential pressure is such that the differential pressure is less than the lateral flow of the liquid in the microchannel is stopped. Paper 42 needs to be selected.

  Further, the amount of the liquid sample penetrating the developing paper 42 needs to be smaller than the total amount of liquid introduced from the inlet. In order to avoid the effect of slowing the flow rate, it is desirable that the developed paper 42 has a small amount of liquid that can penetrate.

  The amount of the liquid sample is usually in micro units, that is, about 1 μl or more and less than about 1 ml, preferably about 1.5 μl, more preferably about 3.0 μl or more, the detection sensitivity becomes stable and the detection becomes easy. The upper limit amount of the liquid sample is usually several μl to several hundred μl, for example. Thus, in many cases, even a single drop of liquid sample can be detected. The amount of the liquid sample may be less than or equal to the volume of the microchannel 74, but when the amount of the liquid sample is larger than the volume of the microchannel 74, the space portion 82 (FIG. )) Works well as a valve mechanism.

  Liquid samples are in particular hydrophilic liquid samples, including human or animal whole blood, serum, plasma, urine, stool dilution, saliva, or cerebrospinal fluid. It is. In this case, a diagnostically effective sample in a liquid sample can be measured for uses such as pregnancy test, urine test, stool test, adult disease test, allergy test, infectious disease test, drug test, or cancer test. Liquid samples also include food suspensions, drinking water, river water, soil suspensions, etc., to measure pathogens in foods and drinking water, and in river water and soil. It is also possible to measure contaminants.

  The first fluid impermeable member 12, the adhesive tape 28, and the second fluid impermeable member 20 have the spread paper 42 disposed in the opening 32, the absorbent paper 44 disposed in the opening 34, and absorption. The paper 44 and the first fluid impermeable member 12 are connected to each other with an optional pair of double-sided adhesive tapes 46 and 48 provided.

  As shown in FIG. 2A, the first fluid impermeable member 12, the second fluid impermeable member 20, and the adhesive tape 28 form a flow path 72 through which the liquid sample passes. . Further, the first fluid impermeable member 12, the second fluid impermeable member 20, and the pair of laterally extending portions 36 and 38 define a linear micro flow path 74, and the developing paper Reference numeral 42 denotes a proximal end portion 78 of the microchannel 74 serving as a fluid or liquid sample introduction portion and the vicinity thereof. The tip 80 of the microchannel 74 is connected to the space 82, and the absorbent paper 44 is disposed at a position sandwiching the space 82 from the tip 80 of the microchannel 74. In the present embodiment, the sectional area of the space 82 is larger than the sectional area of the microchannel 74. The width of the space portion 82 and the microchannel 74 is the same, and the height of the space portion 82 is lower than the height of the bottom surface of the microchannel 74. The volume of the space 82 is not particularly limited, but is usually 0.001 μl or more and 10,000 μl or less. Further, the volume ratio of the space 82 to the microchannel 74 is not particularly limited, but is, for example, 0.01 or more.

  In the present embodiment, the configuration of the flow path 72 is such that the base end space for accommodating the developing paper 42, the micro flow path 74, the side path 75 in the middle of the micro flow path 74, and the valve mechanism continuously from right to left. And a space 82 at the distal end for accommodating the absorbent paper 44. When the microfluidic device 10 is assembled, the space at the proximal end for accommodating the developing paper 42 and the distal end for accommodating the absorbent paper 44 are accommodated. The space 84 is almost closed. Further, as shown in FIG. 2B, the micro flow path 74 has a first flow path section 74 a on the base end section 78 side and a first flow path section on the distal end section 80 side by a branch section 74 c with the side path 75. It is divided into two flow path parts 74b.

  In the present embodiment, the height of the bottom surface of the branch portion 74c and the bottom surface of the side path 75 is the same, and the positions of the bottom surface c of the branch portion 74 and the bottom surface of the side path 75 are the height of the bottom surface of the first flow path portion 74a. And it is lower than the height of the bottom face of the 2nd channel part 74b.

  The space 82 and the microchannel 74 are preferably subjected to a hydrophilic treatment in order to increase the hydrophilicity of the surface in contact with the liquid sample. The hydrophilic treatment of the space 82 is indicated by reference numeral 83 in particular. By applying the hydrophilic treatment 83 to the space portion 82, separation of the liquid sample between the micro flow path 74 and the absorbent paper 44 in the space portion 82 is promoted. Further, by applying hydrophilic treatment to the microchannel 74 and providing the hydrophobic members 38 and 39 on the bottom surface of the side channel 75, the liquid sample flowing from the upstream to the downstream in the microchannel 74 is accumulated in the branch portion 74c which is a recess. After that, the liquid sample flows into the second flow path portion 74 b that is more hydrophilic than the side path 75 and is difficult to flow into the side path 75.

  Hydrophilic treatment includes treatment with a blocking agent that prevents non-specific adsorption of specific conjugates in the liquid sample to the channel, including commercially available blocking agents such as Block Ace, bovine serum albumin, Examples include casein, skim milk, gelatin, surfactant, polyvinyl alcohol, globulin, serum (eg, fetal bovine serum or normal rabbit serum), ethanol, and MPC polymer. Such blocking agents can be used alone or in admixture of two or more.

  The height of the microchannel 74 is, for example, about 15 μm or more and 1,000 μm (1 mm) or less. The width of the microchannel 74 is, for example, not less than about 100 μm and not more than about 10,000 μm (1 cm). The length of the microchannel 74 is, for example, not less than about 10 μm and not more than about 10 cm. The volume of the microchannel 74 is 0.1 μl or more and 1,000 μl or less, preferably 1 μl or more and less than 500 μl.

  In the second flow path portion 74b of the micro flow path 74, an assay region 76 including an assay reagent that reacts with a specimen in a liquid sample to produce a detectable result is provided. This detectable result may be observable with the naked eye (for example, a color change) or may be detectable only with a spectrometer or other measuring means.

  The assay reagent may be a chemical or color reagent such as an iron (III) ion that develops color upon reaction with the sample, or reacts with an enzyme, antibody, epitope, or sample to produce a detectable result. It may be any other substance to be made. The assay reagent is fixed to the assay region 76 by a known immobilization technique such as a physical adsorption method or a chemical adsorption method, preferably the first fluid impermeable member 12 or the second fluid impermeable member 20, particularly preferably. The first fluid impermeable member 12 may be fixed. The assay reagent may be bound with an arbitrary labeling substance such as a radioisotope, an enzyme, a colored molecule such as gold colloid, latex, a dye, a fluorescent substance, or a luminescent substance in order to amplify a detection signal.

  As shown in FIG. 2 (a), when two or more kinds of assay reagents are present, the assay region 76 includes a first assay reagent fixing position 86 and a second assay reagent fixing position 88. The sample reacts with the second assay reagent, and after the complex of the sample and the second assay reagent moves, the complex reacts with the first assay reagent. The complex of the second assay reagent and the first assay reagent form a further complex, and the signal of the complex is detected by known methods.

  When the specimen is an antigen, generally the first assay reagent is a primary antibody, the second assay reagent is a secondary antibody, and the primary and secondary antibodies bind to two different epitopes of the specimen, or the secondary antibody is The primary antibody binds to the epitope of the analyte and binds to the secondary antibody, at least one of the primary antibody and the secondary antibody is labeled, and the primary antibody is fixed at the fixing position 86, The secondary antibody moves from the fixed position 88 according to the flow of the liquid sample, and a complex of the antigen, the primary antibody, and the secondary antibody is detected at the fixed position 86. When the specimen is an antibody, the assay reagent contains an antigen, and the signal of the complex produced by the antigen-antibody reaction is detected in the same manner. When the sample is an enzyme, detection or measurement can be performed in the same manner using the specificity of the enzyme substrate reaction using the assay reagent as a substrate. When the sample is a substrate, the first assay reagent may be an enzyme and optionally the second assay reagent may be a color reagent.

  Optionally, a control region is provided on the downstream side (tip side) of the microchannel 74 where the assay reagent is provided to confirm that a sufficient amount of sample has reached the assay region. May be. The control region is provided with a control reagent that can bind to an analyte in the liquid sample and move with the flow of the liquid sample while moving with the flow of the liquid sample (for example, the second assay reagent described above), but does not bind to the analyte. The control reagent can be any molecule or composition known in the art. By using, as a control reagent, a reagent that causes a color change or the like when a reaction with the assay reagent occurs, the observer can confirm that the assay was performed in a reliable state.

  Returning to FIG. 1, a cover member 50 serving as a first casing for providing robustness or rigidity to the microfluidic device 10 is provided on the first fluid-impermeable member 12. Also, below the second fluid-impermeable member 20, a base member 51 as a second casing for providing the strength or rigidity of the microfluidic device 10 is provided. The cover member 50 and the base member 51 are connected to the fluid-impermeable member 12 and the fluid-impermeable member 20 using the same adhesive tape as the adhesive tape 28 or a similar member.

  The cover member 50 includes a proximal end portion 54 and a distal end portion 56, and a pair of laterally extending portions 58, 60 extending in the longitudinal direction of the proximal end side of the distal end portion 56 and extending perpendicularly to the longitudinal direction. They are arranged side by side in the width direction. Therefore, a slit 62 as an opening for observing the assay region 76 is formed between most of the longitudinal direction of the cover member 50 between the pair of laterally extending portions 58 and 60. Since the slit 62 is at a position corresponding to the assay region 76, as shown in FIG. 2B, when the specimen in the liquid sample is analyzed by the microfluidic device 10 of the present invention, the slit 62 and the transparent second The observer can visually observe the assay region 76 through one fluid-impermeable member 12.

  An adhesive tape 64 is disposed on the base end side of the base end portion 54 of the cover member 50, and a plasma separation paper 67 that separates plasma components from whole blood is bonded onto the adhesive tape 64. An adhesive tape 68 is further adhered on the plasma separation paper 67. Circular holes 66 and 69 are formed in the adhesive tapes 64 and 68. The circular holes 66 and 69 align with the circular holes 18 of the first fluid impermeable member 12 when the microfluidic device 10 is assembled. When a liquid sample such as whole blood is applied to the plasma separation paper 67 through the hole 69, the applied liquid sample moves downward through the plasma separation paper 67 and passes through the hole 66 and the hole 18 to the developing paper 42. It arrives.

  The adhesive tapes 64 and 68 are hydrophobic and function to prevent the liquid sample from leaking out from the circular hole 66, and the plasma separation paper 67 is sandwiched between the adhesive tapes 64 and 68, so that the plasma separation paper 67 The degree of sealing is increased, drying of the liquid sample that wets the plasma separation paper 67 can be reduced, and the liquid sample in the flow path 72 is less likely to volatilize. Further, since the adhesive tape 68 is hydrophobic, the liquid sample placed on the hole 69 is prevented from leaking out of the hole 69 and can flow to the developing paper 42 with high reproducibility. .

  The cover member 50 and the base member 51 may be formed of any material such as metal, plastic, and paper, but are preferably formed of paper, particularly cardboard, in terms of cost reduction and manufacturability of the microfluidic device 10. The When the specimen is detected based on a color change, if the base member 51 is white, the color contrast due to the color change can be clearly read.

  As shown in FIGS. 1 and 2 (b), the cover member 50 and the first fluid-impermeable member 12 are formed with air vent holes 70 (70a and 70b) penetrating them, and a liquid sample is used during the assay. Acts to draw air from the channel 72 out of the microfluidic device 10 when the channel 72 flows from the proximal side to the distal side.

  Next, the operation of the microfluidic device 10 according to the first embodiment of the present invention will be described with reference to FIG.

  3A to E show an experiment using a blue ink solution to confirm the action of the valve mechanism of the microfluidic device 10. The absorbent paper 44 was disposed on the tip side of the microchannel 74, and a blue ink solution as a liquid sample was injected into the microchannel 74. Then, the ink solution that has flowed into the micro flow path 74 flows through the branch portion 74c near the center of the micro flow path 74 that has been subjected to hydrophilic treatment by lateral flow, and hardly or not flows into the side path 75 (FIG. 3A). Next, the ink solution moves in the downstream direction in a lateral flow (FIG. 3B) and is sucked by the absorbent paper 44 on the tip side of the microchannel 74 (FIG. 3C). Although the ink solution is pulled further downstream by the absorbent paper 44, since the air is supplied from the side path 75, the ink solution is in the vicinity of the branch portion 74 c of the microchannel 74 and the first channel portion 74 a and the second channel. Separation into part 74b begins (FIG. 3D). In the space portion 82, the ink solution droplets that have moved from the microchannel 74 grow into, for example, a spherical shape or the like, and contact with the absorbent paper 44 to be destroyed. That is, the space part 82 acts as a valve mechanism, and the ink solution is separated into the second flow path part 74b and the absorbent paper 44, that is, one of the ink solutions is absorbed into the absorbent paper 44 and the other is the micro flow path. It is detained in 74 2nd flow-path part 74b (FIG. 3E). In this experiment, the space 82 is designed to have the same width as the microchannel 74 and a lower bottom than the microchannel 74, and is subjected to a hydrophilic treatment 83 using a blocking agent.

  As described above, the side path 75 is provided from the micro flow path 74, and the micro flow path 74 and the absorbent paper 44 are separated by the space portion 82, whereby the liquid sample is detained in the second flow path portion 74b. . If the ink solution is not separated by the first flow path part 74a and the second flow path part 74b, the ink solution evaporates from the inlet 66 side with time, but according to the above configuration, the liquid sample is the second flow path part 74b. It becomes difficult to receive the influence of drying by being left inside. In addition, since the concentration of the liquid sample containing the assay reagent can be maintained in the second flow path portion 74b, highly accurate measurement is possible.

  In the microfluidic device 10, two or more different solutions can be passed and the solutions in the device 10 can be exchanged.

  First, the first liquid that is a liquid sample is applied to the inlet 66 of the microfluidic device 10. Then, the first liquid penetrates into the developing paper 42 by the capillary force, flows along the tip of the developing paper 42, and flows into the micro flow path 74. The first liquid flows downstream in the microchannel 74, and after the flow of the first liquid is absorbed by the porous medium 44 by the space portion 82 which is a valve mechanism in front of the absorbent paper 44 installed in the downstream portion. The first liquid placed in the microchannel 74 by the action of the side passage 75 is separated into two, one in the absorbent paper 44, the other in the microchannel 74, and the first liquid in the microchannel 74. The first flow path portion 74b and the second flow path portion 74b are separated and placed in each. The first liquid flows stably until there is no excess permeate on the developing paper 42 (on the plasma separating paper 67 when the plasma separating paper 67 is present, or on the upper surface of the inlet when there is no developing paper 42), The entire amount of the first liquid is drawn into the microchannel 74.

  Next, when the second liquid, which is a solution containing a reagent necessary for the assay, is dropped, the second liquid again moves in the microchannel 74 by the lateral flow, and the first liquid that has been filled in advance to the absorbent paper 44. Extruded and exchanged with second liquid. When the second liquid is applied to the microfluidic device 10, the space 82 separates the flow of the second liquid into two, and the second liquid placed in the microchannel 74 is almost entirely inside the microchannel 74. The second liquid is placed in the microchannel 74 so as to satisfy the product, and the first liquid and the second liquid are exchanged. Therefore, a multi-step antigen-antibody reaction such as ELISA can be easily performed. Also in this case, the second liquid retained in the micro flow path 74 by the action of the side path 75 is separated into the first flow path portion 74b and the second flow path portion 74b, and is retained in each.

  Although not limited, in order to ensure solution exchange, the amount of the second liquid is set to be equal to or greater than the amount of the first liquid filled in the microchannel 74. If the amount of the second liquid is smaller than the amount of the first liquid filled in the microchannel 74, the first liquid will not reach the tip 80 of the microchannel 74 even if the second liquid flows through the microchannel. The first liquid and the second liquid remaining as droplets of a sphere or the like are not well separated. Alternatively, even if the sphere made of the first liquid breaks at the tip 80 and is absorbed by the absorbent paper 44, the total amount of the first liquid filled in the microchannel 74 cannot be reliably exchanged.

  In this way, it is possible to exchange the solution in the microchannel while stably flowing the liquid sample to the microchannel without requiring a complicated operation or an external device such as a pump. In the movement of fluid by capillary force, the interfacial tension acting between the wall surface of the microchannel and the liquid acts predominantly, and the fluid moves so as to pull up the liquid according to the surface tension of the liquid. In the process of flowing, the tip of the fluid is concavely curved with respect to the space, and the surface tension of the liquid acts to reduce the surface area, so that a force acts to flatten the concave surface. Since the front end of the fluid needs to be horizontal in order to become flat, the liquid flows as a result. When the liquid flows by this capillary force, the liquid does not form a sphere convexly with respect to the space at the tip of the microchannel. Therefore, when the capillary force is dominantly acting on the fluid rather than the lateral flow, the phenomenon that the fluid leaks from the tip portion does not occur.

Next, the effect of the first embodiment will be described.
(1) A side path 75 branched from the micro flow path 74, a capillary force of the absorbent paper 44, and a space portion 82 as a valve mechanism are provided. In the space portion 82, the fluid that has moved in the micro flow path 74 mainly based on the lateral flow grows into a droplet in the space portion 82 at the tip portion 80, and the droplet that has contacted the absorbent paper 44 beyond the space portion is destroyed. And absorbed by the porous medium 44. For this reason, the operation of “flowing / stopping” the liquid sample into the microchannel can be autonomously performed. Further, since air is supplied from the side path 75, the fluid in the micro flow path 74 is separated by the branching section 74c and is left in the second flow path section 74b. Therefore, the liquid sample is not easily affected by drying, and high-precision measurement can be performed in the second flow path portion 74b.
(2) A plurality of liquid samples are continuously and repeatedly flowed to place the first liquid sample in the second flow path portion 74b, and then the second liquid sample is flowed to the micro flow path 74 and the second liquid. Since the sample can be placed in the second flow path portion 74b, multi-item measurement can also be performed with high accuracy.
(3) Since the branching portion 74c is a hydrophilic recess and the hydrophobic members 38 and 39 are disposed in the side passage 75, the side passage 75 is in gas communication with the microfluid 74 and the accommodation space 84, so that the liquid sample The liquid sample is prevented or suppressed from flowing into the side passage 75 while promoting the separation.
(4) Since the assay region 76 provided with the assay reagent is in the second flow path portion 74b, highly accurate measurement in the second flow path portion 74b is possible.
(5) Since the space portion 82 is subjected to the hydrophilic treatment 83, the separation of the liquid sample between the micro flow path 74 and the absorbent paper 44 in the space portion 82 is promoted.
(6) Since the developing paper 42, which is a hydrophilic porous medium, is provided at the base end of the flow path 72, the liquid that has passed through the plasma separation paper 67 is reliably reproduced from the developing paper 42 into the micro flow path 74. Is sucked in and fluidized. In general, plasma separation using a separation membrane or a fine channel often causes problems such as blood flow aggregation or clogging, or the separation flow stops, or hemolysis or blood cell components are mixed by strong pressure from a pump or the like. However, by realizing the optimization of this structure, only the plasma component from the whole blood can flow into the microchannel 74 with a reliable reproducibility by the plasma separation paper 67 and the lateral flow that is a soft pressure. Can do.
(7) Since the side wall of the microchannel 74 is made of the adhesive tape 28, the height of the microchannel 74 can be easily maintained uniformly. In addition, the microchannel 74 can be configured at a lower cost and more easily than thermocompression bonding.
(8) Since the first fluid impermeable member 12 and the second fluid impermeable member 20 are transparent sheets or films, the assay region 76 in the microchannel 74 can be visually observed. (9) Since the whole blood can be diagnosed on the spot by providing the plasma separation paper 67, dramatic speed-up and efficiency can be expected for early detection of diseases and development of new drugs.
(10) The spread paper and / or the porous medium is cellulose, nitrocellulose, cellulose acetate, filter paper, tissue paper, toilet paper, paper towel, fabric, porous polymer, etc. Can be used easily.
(11) Since the microfluidic device 10 can be manufactured very inexpensively and easily using commercially available inexpensive porous media, transparent films, adhesive tapes, and paper, it can be purchased so far in hospitals in developing countries. In addition to being able to expand to new customers, it can also be expanded to OTC markets in Japan and developed countries, contributing to improving the quality of life (QOL) of people around the world.
(12) The material of the component of the microfluidic device 10 is, in particular, the material of the first and second fluid-impermeable members 12 and 20 is plastic, the interposed member is the adhesive tape 28, the cover member 50 and the base member 51 are paper. Since all of the materials are soft as described above, the sample in the microchannel 74 can be collected by cutting with a scissors or a cutter. Therefore, it can be used as a life science research support tool for the next phase such as gene analysis.

(Second Embodiment)
Next, an assay device according to a second embodiment of the present invention will be described with reference to FIGS. In addition, a different point from 1st Embodiment is demonstrated and description about the same structure as 1st Embodiment is abbreviate | omitted.

  FIG. 4 is a schematic plan view of the microfluidic device 10 as the assay device according to the second embodiment of the present invention. In the second embodiment, only one side path 75 is provided in the middle of the micro flow path 74. Also in this case, the micro flow path 74 is divided into a first flow path section 74a and a second flow path section 74b by a branch section 74c with the side path 75. Although the position of the bottom face of the branch part 74 c and the side path 75 is lower than the height of the bottom face of the first flow path part 74 a and the bottom face of the second flow path part 74 b, the hydrophobic member 39 is located on the bottom face of the side path 75. Since the side passage 75, the microfluidic fluid 74, and the accommodating space 84 are in gas communication with each other, the liquid sample that flows through the microchannel 74 is configured not to easily flow into the side passage 75.

  5A to 5F are photographs showing a state when blue ink is allowed to flow through the microfluidic device of the second embodiment. The behavior of the blue ink in the microfluidic device is similar to that described in FIGS. 3A-E.

  That is, the ink solution that has flowed into the micro flow path 74 flows through the branch portion 74c near the center of the micro flow path 74 that has been subjected to hydrophilic treatment by lateral flow, and hardly or not flows into the side path 75 (FIG. 5A). Next, the ink solution moves in the downstream direction in a lateral flow (FIG. 5B), and is sucked by the absorbent paper 44 on the tip side of the microchannel 74 (FIG. 5C). Although the ink solution is pulled further downstream by the absorbent paper 44, since the air is supplied from the side path 75, the ink solution is in the vicinity of the branch portion 74 c of the microchannel 74 and the first channel portion 74 a and the second channel. The ink solution begins to be separated into the portion 74b (FIG. 5D), and the ink solution is further pulled downstream by the absorbent paper 44, and the ink solution is separated into the first flow passage portion 74a and the second flow passage portion 74b at the branch portion 74c (FIG. 5D). 5E). In the space portion 82, the ink solution droplets that have moved from the microchannel 74 grow and contact the absorbent paper 44 to be destroyed. That is, the space part 82 acts as a valve mechanism, and the ink solution is separated into the second flow path part 74b and the absorbent paper 44, that is, one of the ink solutions is absorbed into the absorbent paper 44 and the other is the micro flow path. 74 is placed in the second flow path portion 74b (FIG. 5F). In this experiment, the space 82 is designed to have the same width as the microchannel 74 and a lower bottom than the microchannel 74, and is subjected to a hydrophilic treatment 83 using a blocking agent.

  As described above, in the second embodiment as well, the side channel 75 is provided from the microchannel 74, and the space between the microchannel 74 and the absorbent paper 44 is separated by the space portion 82. 74b. If the ink solution is not separated by the first flow path part 74a and the second flow path part 74b, the ink solution evaporates from the inlet 66 side with time, but according to the above configuration, the liquid sample is the second flow path part 74b. It becomes difficult to receive the influence of drying by being left inside. In addition, since the concentration of the liquid sample in the second flow path portion 74b can be maintained, highly accurate measurement can be performed.

(Third embodiment)
Next, an assay device according to a third embodiment of the present invention will be described with reference to FIGS. In addition, a different point from 1st and 2nd embodiment is demonstrated, and the description about the same structure as 1st and 2nd embodiment is abbreviate | omitted.

  6A is a schematic plan view of a microfluidic device according to a third embodiment of the present invention, FIGS. 6B and 6C are a schematic enlarged plan view and a schematic enlarged cross-sectional view, respectively, of 6BC surrounded by a dotted square in FIG. 6A, FIG. 6E is a schematic enlarged plan view and a schematic enlarged cross-sectional view of 6DE surrounded by a dotted-line square in FIG. 6A, respectively.

  6A and 6B, in the microfluidic device 10 as the assay device of the third embodiment, the width of the first channel portion 74c adjacent to the branch portion 74c with the side channel 75 in the microchannel 74 is wide. The narrow part 74d is provided, and the width e of the narrow part 74d is narrower than the width a of the first flow path part 74a and the width a of the branch part 74c on both sides of the narrow part 74d. 6A and 6D, in the third embodiment, the space portion 82 is branched into a plurality of space portions toward the accommodation space 84. That is, the space portion 82 includes a linear main space portion 82a extending between the micro flow path 74 and the storage space 84, and a storage space 84 that branches from the main space portion 82a to both sides and is different from the main space portion 82a. And a pair of sub space portions 82b extending to the position. The space 82 is subjected to a hydrophilic treatment 83 with a blocking agent.

  6B and 6D, the width c of the side path 75 is smaller than the channel width a of the first channel part 74a, and the channel width b of the main space part 82a and the channel width d of the sub space part 82b are the first. The flow path width d of the two flow path parts 74 b is smaller than the flow path width a of the sub-space part 82 b, and the width c of the side path 75 is smaller. Further, the channel width b of the main space portion 82a and the channel width d of the sub space portion 82b are equal to or less than ½ of the channel width a of the second channel portion 74b, and the main space portion of the sub space portion 82b. The distance g between the sub space portion 82b and the absorbent paper 44 at the branch point with respect to 82a is equal to or less than the channel width a of the second channel portion 74b. With such a configuration, in the third embodiment, when the liquid sample is caused to flow through the micro flow path 74, the liquid sample can be placed in the second flow path portion 74b with little influence of the meniscus. This will be described below with reference to FIGS. 7A-G.

  7A-G are photographs showing a state when blue ink is allowed to flow through the microfluidic device of the third embodiment. The ink solution that has flowed into the micro flow path 74 flows through the branch portion 74c near the center of the micro flow path 74 that has been hydrophilically treated by lateral flow, and hardly or not flows into the side path 75 (FIG. 7A). Next, the ink solution moves in the downstream direction in a lateral flow (FIGS. 7B and C), and is sucked by the absorbent paper 44 on the tip side of the microchannel 74 (FIG. 7D). Although the ink solution is pulled further downstream by the absorbent paper 44, since the air is supplied from the side path 75, the ink solution is in the vicinity of the branch portion 74 c of the microchannel 74 and the first channel portion 74 a and the second channel. The ink solution is separated into the second flow path portion 74b and the absorbent paper 44 at the positions of the main space portion 82a of the space portion 82 and the T-shaped branch portion of the sub space portion 82b. Separation begins (FIG. 7F). At the T-shaped branch portion of the space 82, the ink solution is pulled toward the absorbent paper 44, and at the same time, air is supplied from the side, and the ink solution in the second flow path portion 74b is almost affected by the meniscus. Without separating from the second flow path portion 74b, the second flow path portion 74b stays in the second flow path portion 74b (FIGS. 7G and 7H). Thus, in the third embodiment, in addition to the space portion 82 acting as a valve mechanism, it is possible to reduce the meniscus of the ink solution exiting from the second flow path portion 74b.

(Fourth embodiment)
Next, an assay device according to a fourth embodiment of the present invention will be described with reference to FIGS. In addition, a different point from 1st-3rd embodiment is demonstrated and description about the same structure as 1st-3rd embodiment is abbreviate | omitted.

  The microfluidic device 10 as the assay device of the fourth embodiment includes a concave portion 79 for accommodating an assay reagent that protrudes laterally from the second flow path portion 74b in the middle of the second flow path portion 74b. Yes. The recess 79 normally extends in a direction substantially perpendicular to the longitudinal direction of the second flow path portion 74b from the second flow path portion 74b. In addition, the assay reagent is fixed to the recess 79 with, for example, a water-soluble gel. With such a configuration, in the fourth embodiment, since the assay region 76 provided with the assay reagent is provided in the recess 79, the analyte concentration in the measurement can be maintained at a high concentration. This will be described below with reference to FIGS. 9A-D.

  FIGS. 9A to 9D are photographs showing states when a phosphate buffer, which is a transparent liquid sample, is passed through the microfluidic device of the fourth embodiment. In the concave portion 79, a water-soluble gel mixed with blue ink as a reagent is arranged in advance. A state in which two kinds of reagents are mixed and flowed by a one-step assay using a phosphate buffer solution of a transparent liquid sample and a trehalose gel containing blue methylene blue supported in the chamber of the recess 79 will be described.

  The transparent liquid sample 101 flowing from the inlet by the lateral flow flows toward the downstream absorbent paper 44 and is mixed with the water-soluble gel in the recess 79. At this time, since the water-soluble gel is in the recess 79, it is hardly affected by the movement in the longitudinal direction of the liquid that is continuously flowing from the upstream side to the downstream side of the microchannel 74. Therefore, the reagent in the gel mixed with the transparent liquid sample hardly moves to the absorption paper 44 in the downstream portion (FIG. 9A). After the liquid is absorbed by the downstream absorbent paper 44, the liquid sample in the micro flow path 74 is separated into the first flow path section 74a and the second flow path section 74b, and the liquid is stored in the second flow path section 74b. The sample is placed, and the reagent (blue ink) in the water-soluble gel in the recess 79 diffuses into the second flow path portion 74b (FIG. 9B). As the liquid in the first flow path part 74a is shown by the movement of the front end part 101 of the liquid to the upper side of the drawing (FIGS. 9B-C), the liquid amount decreases due to drying from the flow path inlet. In the second flow path portion 74b, the reagent (blue ink) continues to diffuse (FIG. 9C). The liquid in the first flow path portion 74a is further dried to reduce the amount of liquid, but the liquid in the second flow path portion 74b is hardly affected by the drying. The reagent (blue ink) continues to diffuse in the second flow path portion 74b until it becomes substantially uniform (FIG. 9D). Thus, in the fourth embodiment, the reagent concentration in the assay region 76 can be maintained at a high concentration.

Up to this point, the present invention has been described with reference to the first to fourth embodiments. However, the present invention is not limited to this, and various modifications such as the following are possible.
In the first to fourth embodiments, the width of the first flow path portion 74a and the width of the second flow path portion 74b are the same, but they are not necessarily the same.
In the first to fourth embodiments, the height of the bottom surface of the space portion 82 is lower than the height of the bottom surface of the microchannel 74, but the height of the bottom surface of the space portion 82 and the bottom surface of the microchannel 74 The height may be the same. When the height of the bottom surface of the space portion 82 and the height of the bottom surface of the microchannel 74 are the same, in order to make the cross-sectional area of the space portion 82 larger than the cross-sectional area of the microchannel 74, It is preferable to make it larger than the width of the path 74.
In the first to fourth embodiments, the positions of the bottom surfaces of the branch portion 74c and the side passage 75 are set lower than the height of the bottom surface of the first flow path portion 74a and the height of the bottom surface of the second flow path portion 74b. However, as long as the side channel 75 is in gas communication with the microfluid 74 and the accommodation space 84 to promote separation of the liquid sample, the liquid sample is prevented from flowing into the side channel 75 as long as the liquid sample is prevented or suppressed. The height of the bottom surface of the first flow path part 74a is the same as the height of the bottom surface of the first flow path part 74a and the second flow path part 74b. The position may be the same as or higher than.
-The material which comprises the hydrophobic members 38 and 39 may be arbitrary materials which provide hydrophobicity, such as a commercially available adhesive agent, an adhesive tape, a film, a sheet | seat.
○ At least one of the first and second fluid-impermeable members 12, 20 and the interposition member may be translucent or opaque, and in addition to plastic, fluid such as resin, glass or metal It may be formed from other materials that do not penetrate. The materials of the first and second fluid-impermeable members 12, 20 and the interposition member may be the same or different. When the materials of the first and second fluid-impermeable members 12, 20 and the interposition member are different, only the first fluid-impermeable member 12 may be a transparent material, or the first fluid-impermeable member 12 Only the permeable member 12 may be a soft sheet or film.
The shape and dimensions of the hole 18 of the first fluid-impermeable member 12 and the hole 66 of the adhesive tape 64 are not particularly limited as long as they communicate with the developing paper 42.
In order to adjust the height of the microchannel 74, the interposition members including the adhesive tape 28 may be stacked with a plurality of interposition members. Further, the interposition member is not limited to a single member, and may be composed of a plurality of members connected in the horizontal direction so that there is no leakage of the liquid sample. Further, the interposition member is not limited to the adhesive tape 28, and may be other members of plastic or film as long as the space portion in which the fluid including the micro flow path 74 and the space portion 82 flows can be sealed.
○ The liquid sample is not limited to blood, and various specimens in various liquid samples can be detected.
○ If the liquid sample is not blood, the adhesive tape 68 and the plasma separation paper 67 may be omitted, and a filter for removing substances in the liquid sample which is not preferable for the assay is provided instead of the plasma separation paper 67. Also good.
A hydrophilic film as the second porous medium may be provided at or near the base end of the flow path 72 in order to perform hydrophilic treatment instead of or in addition to the development paper 42. Good. A hydrophilic membrane means a blocking agent that prevents non-specific adsorption of a specific conjugate in a liquid sample to a channel, including commercially available blocking agents such as Block Ace, bovine serum albumin, casein, and skim milk. Gelatin, surfactant, polyvinyl alcohol, globulin, serum (eg, fetal bovine serum or normal rabbit serum), ethanol, MPC polymer, and the like. Such blocking agents can be used alone or in admixture of two or more.
The cover member 50 and / or the base member 51 may be formed from a porous medium other than paper, plastic, resin, glass, or metal.
A slit may be provided in the base member 51. For example, when a specimen is detected by light emission or fluorescence measurement, the base member 51 is provided with a slit such as the slit 62 in FIG. 1, and the microfluidic device is mounted on a mirror surface reflecting aluminum or a metal plate made of aluminum and stainless steel. Measurements may be made in
The recess 25 at the tip 52 of the base member 51 may be omitted when the thickness of the absorbent paper 44 is equal to or smaller than the adhesive tape 28.
If the first fluid impermeable member 12 and / or the second fluid impermeable member 20 itself provides sufficient strength or rigidity to the configuration of the microfluidic device 10, the cover member 50 and / or the base The member 51 may be omitted.
The first fluid impermeable member 12, the second fluid impermeable member 20, the adhesive tape 28, the cover member 50, and the base member 51 have the same shape and dimensions, but the micro flow path 74 is secured. As long as the cover member 50 and the base member 51 are made larger in size than the other members and the cover member 50 and the base member 51 are connected to each other, for example, the shape and dimensions of each member may be changed as appropriate.
The hole 70 for venting the flow path 72 may be provided anywhere on the absorbent paper 44 or in the vicinity thereof so long as it is in air communication with the flow path 72, or even if the hole 70 is not provided. It may be omitted as long as the fluidic device 10 operates.
The microfluidic device 10 may include a plurality of flow paths as shown in FIG. For example, the microfluidic device 10 includes four flow paths 72, and the four flow paths 72 are aligned with each other at the base end, and are arranged radially so as to form a substantially vertical angle with the adjacent flow paths 72. In this case, the liquid sample applied to the plasma separation paper 67 flows radially through the four microchannels 74, and the analyte in the liquid sample is analyzed or detected in the assay region 76 of each microchannel 74, and an extra liquid sample is obtained. Is absorbed by the absorbent paper 44. If different assay reagents are arranged in the assay region 76, up to four items can be analyzed or detected simultaneously in one liquid sample.

  Hereinafter, the present invention will be described more specifically with reference to examples, but it goes without saying that the present invention is not limited thereto.

  The disclosures of all patent applications and documents cited herein are hereby incorporated by reference in their entirety.

Example 1-Confirmation of Influence of Microfluidic Device Side Path on Liquid Sample Movement (Materials and Methods)
The microfluidic device of Example 1 (FIG. 11A) provided with one side passage 75 similar to the assay device of the second embodiment of the present invention, and a linear microchannel 74 without the side passage 75 were provided. A microfluidic device (FIG. 11C) of Comparative Example 1 was produced. The dimensions and materials of the components of the microfluidic device were as follows. The blue ink was poured into the produced microfluidic device, and the length from the absorbent paper 44 to the tip of the blue ink liquid after flowing the blue ink was measured over time (after 5, 10, 15, 20 minutes) ( Example 1-FIG. 11B, Comparative Example 1-11D).

    ・ Microchannel width 500μm, length 5-10mm. Channel height is 50μm. Diameter of entrance circle: 300 μm.

  The position of the side path 75 was set to be approximately the center in the longitudinal direction of the microchannel. The depth of the branch part 74c and the hydrophobic parts 38 and 39 was 65 to 300 μm.

First fluid impermeable member 10 (transparent film)
Material: PVDC, Dimensions: 35mm x 12mm x 0.01mm (length x width x thickness), source: Asahi KASEI
Second fluid impermeable member 20 (transparent film)
Material: PET, Dimensions: 35mm x 12mm x 0.13mm (length x width x thickness), source: EPSON
・ Intervening member (adhesive tape) Material: A4 adhesive transfer sheet (adhesive layer 25μm, film layer 15μm, adhesive layer 25μm stacked in order), source: Pland industry, hydrophobic member of side 75 Material: Adhesive tape Adhesive surface, dimensions: 20 mm x 0.5 mm x 0.2 mm (length x width x thickness), source: Pland industry, hydrophilic treatment 83 in space 82 Material: Adhesive surface of adhesive tape, dimensions: 10 mm x 12 mm x 0.4mm (length x width x thickness), source: Planned Industry, surface treatment: Block Ace, source: DS Pharma Biomedical Co., Ltd.

(result)
As shown in Table 1, it was found that in Example 1 with the side passage 75, the ink liquid hardly moved even after 20 minutes, and drying from the inlet of the microfluidic was prevented. On the other hand, in Comparative Example 1, the ink liquid moved greatly with time.

Example 2-Confirmation of Solution Exchange in Microchannel by Valve Mechanism (Materials and Methods)
The specifications of the microfluidic device were the same as those of the microfluidic device of Example 1. In the assay device of the present invention of the second embodiment having a valve mechanism space 82 and a side passage 75, buffer solution 5 μl (0.1 M phosphate buffer pH 7.4) and fluorescent reagent 5 μl (FITC solution 10 nM) Are alternately flown, and an optical probe (No. 4040 (Spot dia .: 0.4 mm, Nippon Sheet Glass Co., Ltd.) is applied to a point 1 mm from the tip of the microchannel, and an optical fiber type fluorescence detector (FLE1100B, Nippon Sheet Glass Co., Ltd.) Fluorescence intensity was measured at a company).

(result)
As a result, it was confirmed that the buffer solution and the fluorescent reagent flow alternately, and that the solution exchange in the microchannel is performed well (FIG. 12). In particular, the solution is exchanged, and the height of the fluorescence intensity peak is kept flat over time. Even when the solution is exchanged, the liquid sample is well placed in the second flow path portion 74b, so It was shown that the concentration of the reagent was kept constant in the second flow path part and was measured with high accuracy.

Example 3-Antibody-based assay (materials and methods)
Whole blood collected from a human subject was used as a liquid sample to detect adiponectin in the blood. The specifications of the assay device of the present invention were as follows. The assay device is the specification of the assay device of the second embodiment including the valve mechanism space 82 and the side passage 75.
・ Microchannel width 500μm, length 5-10mm. Channel height is 50μm. Diameter of entrance circle: 300 μm. The position of the side path 75 was set to be approximately the center in the longitudinal direction of the microchannel. The depth of the branch part 74c and the hydrophobic parts 38 and 39 was 65 to 300 μm.
・ First fluid-impermeable member (transparent film)
Material: PVDC, Dimensions: 35mm x 12mm x 0.01mm (length x width x thickness), source: Asahi KASEI
・ Second fluid-impermeable member (transparent film)
Material: PET, Dimensions: 35mm x 12mm x 0.13mm (length x width x thickness), source: EPSON
・ Intervening member (adhesive tape) Material: A4 adhesive transfer sheet (adhesive layer 25μm, film layer 15μm, adhesive layer 25μm stacked in order), source: Pland industry ・ Cover member Material: Pro paper (cellulose), dimensions : 35mm x 12mm x 0.33mm (length x width x thickness), source: Aoyagi Co., Ltd., a base material with three sheets of 0.33mm thick paper laminated. The two sheets have an opening having the same shape as the opening 34 and have a space in which the absorbent paper 44 having a thickness of about 0.5 mm can be sufficiently accommodated. The third sheet has no space for the opening 34. Material: Pro paper (cellulose), dimensions: 35mm x 12mm x 0.99mm (length x width x thickness), source: Aoyagi, plasma separation paper Co., Ltd. dimensions: 5mm x 5mm (length x width), source: Nippon Pole Absorbent paper Co., Ltd. Dimensions: 8mm x 10mm (length x width), source: Nippon Paper Crecia Co., Ltd., hydrophobic member of side 75 Material: Adhesive surface of adhesive tape, dimensions: 20mm x 0.5mm x 0.2mm (Vertical x Horizontal x Thickness), Obtained from: Pland industry, hydrophilic treatment 83 of space part 82 Material: Adhesive surface of adhesive tape, Dimensions: 10 mm x 12 mm x 0.4 mm (Vertical x width x thickness), Obtained from: Pland Industry, surface treatment: Block Ace, source: DS Pharma Biomedical Co., Ltd.

  The method for producing the interface for recognizing the specimen was as follows. The first fluid impervious member 12 is PVDC, the second fluid impervious member 20 is PET, and a masking tape (rubber adhesive) is placed at the position where the antibody of the second fluid impervious member 20 should be fixed. ), 4% Block Ace solution (pH 7.4 phosphate buffer) was applied, allowed to stand for 1 hour to dry, and then the masking tape was peeled off. For detection of adiponectin in blood as a specimen, a human adiponectin ELISA kit (96 assay, CY-8050) manufactured by Circulex was used, and antibodies and antigens were prepared by methods recommended for the kit. That is, 1% trehalose was mixed with the primary antibody solution prepared by the method recommended in the kit, 1 μL of the droplet was placed at the position where the masking tape was peeled off, and left standing for 1 hour, so that the first fluid impermeability The primary antibody was fixed to the member 12. The amount of antibody and the concentration of trehalose vary depending on the type of antibody and the size of the microchannel 76.

  In the assay device of the present invention, in the assay region 76, the primary antibody was fixed to the circular region of 1 mm in diameter of the second fluid-impermeable member 20 by physical adsorption, and the interface was prepared. The intensity of chemiluminescence was measured with a comparative assay device in which nitrocellulose paper was placed in a circular area. Nitrocellulose paper used was PROTRAN Nitrocellulose Transfer Membrane manufactured by Whatman.

  The order of solution exchange was as follows. First, it was washed with 10 μL of physiological saline, reacted with 10 μL of 20 ng / ml antigen for 10 minutes, then washed with 10 μL of physiological saline, reacted with 10 μL of 200-fold diluted HRP-labeled secondary antibody solution for 10 minutes, The plate was washed with 10 μL of saline and further reacted with 5 μL of SuperSignal West Femto (Thermo Scientific) luminescent substrate solution for 30 minutes. As the measuring device, ImageQuant LAS4000 / 4010 manufactured by GE Healthcare was used.

(result)
As a result, the assay device of the present invention (Positive control) has a larger luminescence value in the liquid sample than the assay device of the comparative example (Negative control), and the difference in the results of the antibody reaction can be obtained significantly and sharply. It was confirmed (FIG. 13).

  The assay device using the porous medium of the present invention can measure a specimen in a liquid sample in a micro flow channel that is a space, and can achieve high sensitivity and high accuracy in general-purpose measurement. Useful. Further, by providing a side path in the middle of the micro flow path, the fluid that has moved in the micro flow path is separated into the first flow path portion and the second flow path portion, and the second flow is generated by the space portion. After being separated into the path part and the absorbent paper, it is placed in the second flow path part. For this reason, the liquid sample containing the assay reagent is not easily affected by drying, and high-precision measurement can be performed in the second flow path portion 74b.

DESCRIPTION OF SYMBOLS 10 ... Microfluidic device as an assay apparatus, 38, 39 ... Hydrophobic member, 42 ... Development paper as 2nd porous medium, 44 ... Absorption paper as porous medium, 74 ... Micro flow path 74a ... first flow path part, 74b ... second flow path part, 74c ... branch part, 74d ... narrow part, 75 ... side path, 79 ... concave part, 80 ... tip of microchannel, 82 ... space, 82a ... main space, 82b ... subspace, 83 ... hydrophilic treatment, 84 ... housing space, a ... second channel , B..., The channel width of the main space, c... The width of the side path, d... The channel width of the sub space, g. The distance between the subspace and the porous medium at the bifurcation point.

Claims (12)

An assay device comprising:
A microchannel having a tip,
A porous medium housed in a housing space arranged near the tip of the microchannel;
A space portion disposed between the microchannel and the accommodation space;
And a side path that branches off from the micro flow path in the middle of the micro flow path and extends to the accommodation space, and the micro flow path includes a first flow path portion and a tip end portion via the branch portion of the micro flow path. Divided into the flow channel section
The fluid that has moved in the micro flow path based on the lateral flow is separated into the first flow path portion and the second flow path portion, and the second flow path portion and the porous medium are separated by the space portion. After being separated into two, the assay device is configured to be detained in the second flow path section.   The assay device according to claim 1, wherein a hydrophobic member is disposed on a bottom surface of the side passage.   The assay device according to claim 1 or 2, wherein the position of the bottom surface of the branch portion is lower than the height of the bottom surface of the first flow channel portion and the height of the bottom surface of the second flow channel portion.   The assay device according to any one of claims 1 to 3, further comprising an assay reagent in the second flow path section.   The assay device according to any one of claims 1 to 4, wherein the space portion is branched into a plurality of space portions toward the accommodation space.   The space portion has a linear main space portion extending between the microchannel and the accommodation space, and a sub branch extending from the main space portion to both sides and extending to a position of the accommodation space different from the main space portion. A flow path width of the main space section and the sub space section is smaller than a flow path width of the second flow path section, and a flow path width of the sub space section is smaller than a width of the side path. 6. The assay device according to 5.   The flow width of the main space portion and the sub space portion is ½ or less of the flow width of the second flow passage portion, and the sub space portion and the porous medium at the branch point with the main space portion of the sub space portion The assay device according to claim 5 or 6, wherein the distance between the two is less than or equal to the channel width of the second channel section. The assay device according to any one of claims 3 to 7, further comprising a concave portion for accommodating the assay reagent, which protrudes laterally from the second flow channel portion in the middle of the second flow channel portion.   The width of the portion of the first flow passage portion adjacent to the branch portion with the side passage in the micro flow passage is narrower than the width of the first flow passage portion and the width of the branch portion on both sides of the portion. The assay device according to any one of claims 1 to 8. The fluid is a first liquid and a second liquid;
The first liquid that has moved from the microchannel to the space is separated by the space, one is placed in the microchannel, and the other is absorbed by the porous medium,
Next, the second liquid moved in the microchannel pushes the first liquid retained in the microchannel to the porous medium, and the first liquid and the second liquid in the microchannel are exchanged. The assay device according to any one of claims 1 to 9.   The assay device according to any one of claims 1 to 10, wherein the space portion is subjected to a hydrophilic treatment.   The assay device according to any one of claims 1 to 11, further comprising a second porous medium disposed in the fluid introduction part of the microchannel.