Detection device

Abstract

The detection device is provided with: a temperature adjustment unit for adjusting the temperature of the fluid equipment; an imaging unit that irradiates the fluid device with excitation light and captures an image of fluorescence emitted from the fluid device; and a transport unit that transports the fluid device to the temperature adjustment unit and the imaging unit, the fluid device containing a mixed aqueous solution of a liquid sample containing a target substance and a detection reagent that reacts with the target substance to generate a fluorescent product, the temperature adjustment unit having: a heating unit that heats the fluid device; and a cooling unit that cools the heated fluid device.

Description

Technical Field

[0001] The invention relates to a detection device.

[0002] This application claims the benefit of priority based on Tokugan Application No. 2022-106963 filed in Japan on July 1, 2022, the contents of which are incorporated herein by reference. Background Art

[0003] Technologies for detecting biomolecules in fluidic devices are known. For example, in DNA microarray technology, biomolecules are sometimes detected by introducing biomolecules into micropores and performing a reaction accompanied by heating. In this case, digital measurement technologies such as digital invasive cleavage assay (ICA) are known as technologies capable of detecting single-molecule biomolecules.

[0004] The inventors have previously developed a digital measurement technology using a reaction container (also called a fluid device) having a plurality of wells (see Patent Document 1). In the method described in Patent Document 1, an aqueous medium containing a target substance is transported to a flow path of the fluid device and sealed in a plurality of wells provided on the wall surface of the flow path. Furthermore, the fluid device is heated to heat the reaction solution, thereby causing a detection reaction to proceed, generating specific fluorescence, and detecting the target substance.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: International Publication No. 2015 / 115635 Summary of the invention

[0008] Technical problem to be solved by the invention

[0009] In this technical field, there is a demand for detection devices that can perform a large number of detections in a short time. On the other hand, although digital ICA requires heating during the detection reaction, it is also required to suppress side reactions caused by overheating and improve detection accuracy.

[0010] The present invention has been made in view of such circumstances, and an object of the present invention is to provide a detection device that can ensure high detection accuracy in detection based on the principle of digital ICA and can perform a large amount of detection in a short time.

[0011] Unit for solving technical problems

[0012] In order to solve the above technical problems, one aspect of the present invention includes the following solutions.

[0013] [1] A detection device comprises: a temperature adjustment unit for adjusting the temperature of a fluid device; a photographing unit for irradiating the fluid device with excitation light and photographing an image of fluorescence emitted from the fluid device; and a transport unit for transporting the fluid device to the temperature adjustment unit and the photographing unit, wherein the fluid device contains a mixed aqueous solution of a liquid sample and a detection reagent, wherein the liquid sample contains a target substance, and the detection reagent reacts with the target substance to produce a product that emits the fluorescence, and the temperature adjustment unit comprises: a heating unit for heating the fluid device; and a cooling unit for cooling the heated fluid device.

[0014] [2] The detection device according to [1], further comprising a control unit that controls the operation of the temperature adjustment unit, wherein the control unit controls the heating unit to a temperature of 50° C. or higher and 99° C. or lower.

[0015] [3] The detection device according to [2], wherein the control unit performs preheating of the heating unit to a temperature higher than room temperature and lower than a predetermined reaction temperature before heating the fluid device.

[0016] [4] The detection device according to any one of [1] to [3], further comprising a control unit that controls the operation of the temperature adjustment unit, wherein the control unit controls the cooling unit to a temperature of 0.1°C or higher and lower than 30°C.

[0017] [5] The detection device according to [4], wherein the control unit performs pre-cooling of the cooling unit to a temperature not lower than a preset cooling temperature and lower than room temperature before cooling the fluid device.

[0018] [6] A detection device according to any one of [1] to [5], wherein the photographing unit comprises: a light source unit that emits the excitation light; a dichroic mirror that reflects the excitation light and transmits the fluorescence; a projection lens that projects the excitation light reflected by the dichroic mirror toward the fluid device; an imaging lens that images the fluorescence transmitted from the dichroic mirror; and a photographing unit that photographs the image of the image formed by the imaging lens, the projection lens also serving as an objective lens that focuses the fluorescence generated from the fluid device and guides the fluorescence to the imaging lens.

[0019] [7] The detection device according to [6], wherein the light source unit comprises: a light source that emits light including the excitation light; and an optical filter that transmits light of a wavelength used as the excitation light in the light.

[0020] [8] The detection device according to [7], wherein the light source unit comprises: a first optical filter that transmits light of a first wavelength used as the excitation light in the light; a second optical filter that transmits light of a second wavelength used as the excitation light in the light; and a switching unit that switches between the first optical filter and the second optical filter.

[0021] [9] The detection device according to [6], wherein the light source unit has an external light introduction unit that introduces external light and guides the light to the fluid device.

[0022]

[10] The detection device according to any one of [6] to [9], wherein the projection lens and the objective lens form a confocal optical system.

[0023]

[11] The detection device according to

[10] , wherein the shooting unit has a spatial filter with a pinhole between the imaging lens and the shooting unit, the confocal point position of the confocal optical system is located between the imaging lens and the shooting unit, and the pinhole and the confocal point position overlap in space.

[0024]

[12] A detection device according to any one of [6] to

[11] , wherein the fluid device has a micropore that contains the target substance and the detection reagent and is used as a reaction site for generating the product, the objective lens has an autofocus mechanism, and the detection device has a control unit, and before using the shooting unit to shoot, the control unit controls the autofocus mechanism to align the focal position of the objective lens with the micropore.

[0025]

[13] A detection device according to any one of [1] to

[12] , wherein the heating section has a temperature control rack for accommodating a plurality of the fluid devices, the conveying section has a conveying rack for accommodating a plurality of the fluid devices, the temperature control rack has a first accommodation section extending along the conveying direction of the conveying section and accommodating the fluid devices, the temperature control rack has a second accommodation section extending along the conveying direction of the conveying section and accommodating the fluid devices, and the detection device further has a moving unit, which pushes out and moves the fluid devices accommodated in the first accommodation section toward the second accommodation section when the temperature control rack and the conveying rack are close to each other.

[0026] Effects of the Invention

[0027] According to the present invention, it is possible to provide a detection device that can ensure high detection accuracy in detection based on the principle of digital ICA and can perform a large amount of detection in a short time. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic perspective view showing the detection device 1 .

[0029] Figure 2 It is a schematic diagram showing the configuration of the imaging unit 20 .

[0030] Figure 3 It is a schematic diagram of the transport unit 30 included in the detection device 1 .

[0031] Figure 4 It is a schematic perspective view showing the fluid device 100 .

[0032] Figure 5 is a cross-sectional view of the fluid device 100 .

[0033] Figure 6 1 is a plan view showing a portion of the plurality of microholes 110 .

[0034] Figure 7 1 is an explanatory diagram of a method for detecting a sample using the above-mentioned fluid device 100 .

[0035] Figure 8 1 is an explanatory diagram of a method for detecting a sample using the above-mentioned fluid device 100 .

[0036] Fig. 9 1 is an explanatory diagram of a method for detecting a sample using the above-mentioned fluid device 100 .

[0037] Fig.10 is a schematic diagram illustrating an example of the ICA method.

[0038] Fig.11 It is an explanatory diagram for explaining the operation of the detection device 1.

[0039] Fig.12 It is an explanatory diagram for explaining the operation of the detection device 1.

[0040] Fig.13 It is an explanatory diagram for explaining the operation of the detection device 1.

[0041] Fig.14 It is an explanatory diagram for explaining the operation of the detection device 1.

[0042] Fig.15 It is a schematic perspective view showing the detection device 2 .

[0043] Fig.16 It is a schematic perspective view of the moving unit 70 .

[0044] Fig.17 It is a schematic perspective view showing the heating unit 11 and the conveying unit 35 .

[0045] Fig.18 It is an explanatory diagram for explaining the operation of the detection device 2.

[0046] Fig.19 It is an explanatory diagram for explaining the operation of the detection device 2. DETAILED DESCRIPTION

[0047] [First embodiment]

[0048] Below, refer to Figure 1 to Figure 14 A detection device and a fluid apparatus according to a first embodiment of the present invention will be described. It should be noted that in all the following drawings, the dimensions and ratios of the components are appropriately varied to make the drawings easier to read.

[0049] In the following description, an xyz rectangular coordinate system is set, and the positional relationship of each component is described with reference to the xyz rectangular coordinate system. Among them, the prescribed direction in the horizontal plane is set as the x-axis direction, the direction in the horizontal plane perpendicular to the x-axis direction is set as the y-axis direction, and the direction perpendicular to the x-axis direction and the y-axis direction (i.e., the vertical direction) is set as the z-axis direction.

[0050] 《Detection device》

[0051] Figure 1 1 is a schematic perspective view showing a detection device 1 according to the present embodiment. The detection device 1 includes a temperature adjustment unit 10, an imaging unit 20, and a transport unit 30. Furthermore, the detection device 1 includes a control unit 40 for controlling the operation of the detection device 1.

[0052] The temperature adjustment unit 10 , the imaging unit 20 , the transport unit 30 , and the control unit 40 are disposed on the base 90 .

[0053] The detection device 1 is used to detect a target substance contained in a liquid sample. Specifically, the detection device 1 is used when the target substance is detected using the fluid device 100 based on the principle of digital ICA (hereinafter, dICA). In the detection device 1, the product generated inside the fluid device 100 is excited by light, and the generated fluorescence is photographed and analyzed, thereby detecting the target substance.

[0054] The following will explain them one by one.

[0055] [Temperature Control Unit]

[0056] The temperature adjustment unit 10 includes a heating unit 11 for heating the fluid device 100 and a cooling unit 12 for cooling the heated fluid device 100 .

[0057] Figure 1 The upper surface (surface on the +z side) of the heating unit 11 generates heat to directly heat the object in contact with the upper surface. The heating unit 11 can be a heater using a known heating method, and examples thereof include resistance heating and induction heating. Figure 1In the figure, the heating unit 11 is shown as a rectangular member in a top view. The heating unit 11 is preferably provided with a known temperature sensor such as a thermocouple, and is capable of controlling the temperature while measuring the temperature. "Top view" refers to the field of view from the +z side to the -z side along the z axis.

[0058] In addition to the above-described configuration, the heating unit 11 may also have a cover having an internal space for accommodating an object (ie, the fluid device 100 ) and may indirectly heat the object by heating the internal space.

[0059] The heating unit 11 is controlled by the control unit 40 to be 30° C. or higher and 99° C. or lower.

[0060] Figure 1 The cooling unit 12 cools the object in contact with the upper surface. The cooling unit 12 can adopt a unit using a known cooling method. For example, the cooling unit 12 can also be a structure that uses a known Peltier element to cool the upper surface by energizing. Figure 1 In FIG. 1 , the cooling unit 12 is shown as a rectangular member in plan view. The cooling unit 12 is preferably provided with a known temperature sensor such as a thermocouple, and is configured to be able to perform temperature control while measuring the temperature.

[0061] The cooling unit 12 is controlled by the control unit 40 to be 0.1°C or higher and lower than 30°C.

[0062] In addition, the cooling unit 12 may also be a heat transfer plate made of metal with good heat transfer characteristics on the upper surface and a known water-cooled or air-cooled heat dissipation unit on the lower surface. Furthermore, the cooling unit 12 may also be provided with only a metal plate with good heat transfer characteristics.

[0063] The temperature control unit 10 is provided on a mounting unit 50 mounted above the base 90 (in the +z direction). The mounting unit 50 includes a rectangular stage 51 extending in the x direction and a pair of bridge piers 52 provided at both ends of the length direction of the stage 51. The temperature control unit 10 is provided with a heating unit 11 and a cooling unit 12 in order from one end 51a of the stage 51 in the +x direction.

[0064] In the stage 51 , a standby section 55 for a transport stage (described later) included in the transport section 30 to stand by may be provided on the −x side of the heating section 11 .

[0065] [Photography Department]

[0066] The imaging unit 20 is provided on the stage 51 at the +x side relative to the cooling unit 12. The imaging unit 20 irradiates the fluid device 100 with excitation light and captures an image of fluorescence emitted from the fluid device 100.

[0067] Figure 22 is a schematic diagram showing the configuration of the imaging unit 20. The imaging unit 20 includes a light source unit 21, a dichroic mirror 22, a projection lens 23, an imaging lens 24, an imaging unit 25, an absorption filter 26, a spatial filter 27, and a reflection mirror 28.

[0068] The light source unit 21 emits the excitation light EL. The light source unit 21 includes a light source 211 that emits the light L1 including the excitation light EL, and an optical filter 212 that transmits light of a wavelength used as the excitation light EL in the light L1.

[0069] The light source 211 can be, for example, a known mercury lamp. Alternatively, the light source 211 may be a known LED light source or LD light source. In the following description, the light source 211 is assumed to be a mercury lamp that is a white light source, and the light L1 is assumed to be white light.

[0070] The optical filter 212 transmits light of a wavelength required for exciting the product (i.e., fluorescent substance) generated inside the fluid device 100 (i.e., excitation light EL) in the light L1 which is white light, and extracts the excitation light EL. The optical filter 212 is a bandpass filter having the property of transmitting the excitation light EL and shielding other light.

[0071] Such a light source unit 21 can extract the excitation light EL from the light L1 emitted by the light source 211 and emit the excitation light EL.

[0072] It should be noted that the light source unit 21 may also include an external light introduction unit 215 for introducing external light L2 different from the light L1 emitted from the light source 211 and guiding the light to the fluid device 100 at a position between the light source 211 and the optical filter 212 on the optical path. The external light introduction unit 215 includes a reflector 215a that is freely provided in and out of the optical path, and an introduction port 215b for introducing the external light L2.

[0073] In the light source unit 21, when the light L1 emitted from the light source 211 is used, the reflector 215a of the external light introduction unit 215 is retracted from the optical path to allow the light L1 to pass. In addition, when the external light L2 is used, the reflector 215a of the external light introduction unit 215 is arranged on the optical path to shield the light L1 and guide the external light L2 introduced from the introduction port 215b to the direction of the optical filter 212.

[0074] The excitation light EL emitted from the light source section 21 enters the dichroic mirror 22 .

[0075] The dichroic mirror 22 has the property of reflecting the excitation light EL and transmitting the fluorescent light FL. The dichroic mirror 22 is arranged at an angle of 45° to the incident direction of the excitation light EL (i.e., incident angle 45°), and reflects the excitation light EL in a direction of 90° to the incident direction of the excitation light EL.

[0076] The excitation light EL reflected by the dichroic mirror 22 enters the projection lens 23 and is projected onto the fluid device 100. In the fluid device 100, fluorescent light FL is emitted from the fluorescent substance excited by the excitation light EL.

[0077] The fluorescence FL enters the dichroic mirror 22 via the projection lens 23. The dichroic mirror 22 transmits the fluorescence FL. On the other hand, even if a part of the excitation light EL is reflected in the fluid device 100 and returns to the dichroic mirror 22, the dichroic mirror 22 reflects the excitation light EL. Thus, the excitation light EL and the fluorescence FL are separated.

[0078] The fluorescence FL transmitted from the dichroic mirror 22 is incident on the absorption filter 26. As the absorption filter 26, a dichroic mirror that transmits the fluorescence FL and does not transmit light of other wavelengths can be used. For example, as the absorption filter 26, by using a dichroic mirror that has a better wavelength separation ability than the dichroic mirror 22, transmits the long-wavelength light including the fluorescence FL, and shields (and reflects) the short-wavelength light including the excitation light EL, it is possible to remove stray light and perform high-precision detection.

[0079] The fluorescence FL transmitted through the absorption filter 26 is incident on the imaging lens 24 via the reflection mirror 28 and is imaged.

[0080] The imaging unit 25 captures an image formed by the imaging lens 24. As the imaging unit 25, a well-known digital camera having an imaging element can be adopted.

[0081] In the above-mentioned imaging unit 20, the projection lens 23 that projects the excitation light EL emitted from the light source unit 21 to the fluid device 100 also serves as an objective lens that collects the fluorescence FL generated from the fluid device 100 and guides it to the imaging lens 24. The projection lens 23 (also the objective lens) may also have an autofocus mechanism.

[0082] Furthermore, in the photographing unit 20, the projection lens 23 and the imaging lens 24 preferably form a confocal optical system. The photographing unit 20 is provided with a spatial filter 27 having a pinhole P between the imaging lens 24 and the photographing unit 25, and the confocal point FP of the confocal optical system is located between the imaging lens 24 and the photographing unit 25. Another confocal point in the confocal optical system is located at a position overlapping with the fluid device 100. The spatial filter 27 is arranged at a position where the pinhole P and the confocal point FP overlap in space. Thus, the photographing unit 20 can remove stray light and perform high-precision detection. It should be noted that in order to make the confocal optical system function, known components constituting the confocal optical system can be appropriately adopted.

[0083] It should be noted that if Figure 1 and Figure 2As shown, the imaging unit 20 may further include a filter block 29 in which the optical filter 212 , the dichroic mirror 22 , and the absorption filter 26 are integrated.

[0084] In addition, the light source unit 21 can also be constructed as described below: it has a first optical filter 212a that transmits light of a first wavelength in the light L1 used as the excitation light EL, and a second optical filter 212b that transmits light of a second wavelength in the light L1 used as the excitation light EL, and the first optical filter 212a and the second optical filter 212b are switchable.

[0085] Specifically, the light source unit 21 has filter blocks 291 and 292, and a switching unit 295. The filter block 291 has a first optical filter 212a. The filter block 292 has a second optical filter 212b. The switching unit 295 switches the filter block 291 and the filter block 292. The switching unit 295 has a turntable 296 and a driving unit 297. The turntable 296 is a turntable 296 on which the filter block is provided, and the driving unit 297 drives the turntable 296 in the circumferential direction. The filter block 29 and the switching unit 295 are supported by a support 299 provided on the base 90.

[0086] The switch unit 295 can switch the filter block 292 installed on the turntable 296 by moving the turntable 296 in the circumferential direction using the drive unit 297 .

[0087] [Conveying Department]

[0088] like Figure 1 As shown, the conveying unit 30 includes a guide rail 31 extending in the x-axis direction, a conveying body 32 moving in the ±x-axis directions along the guide rail 31 , and a driving unit 33 driving the conveying body 32 .

[0089] Figure 3 Schematic diagram of the conveying unit 30 of the detection device 1. The conveying body 32 has a first member 321, a second member 322, a stage 323, and an illumination unit 325. The first member 321 is connected to the guide rail 31 and moves in the ±x-axis direction. The second member 322 is connected to the first member 321 and moves in the ±z-axis direction. The stage 323 is connected to the second member 322. The illumination unit 325 illuminates the stage 323.

[0090] The upper surface of the stage 323 is a surface parallel to the xy plane, and the fluid device 100 as the observation object is placed. The stage 323 can adopt various structures as long as it can place the fluid device 100 and can observe the fluid device 100 from below (i.e., the -z direction). For example, the stage 323 can be a plate-like member with light transmittance, or a frame with a gap in the observation part of the fluid device 100. In addition, the stage 323 can also be a holding member for holding the fluid device 100 from the side. Figure 3 In the figure, three fluid devices 100 are placed, but the number of fluid devices 100 placed on the stage 323 can be set according to the size of the stage 323.

[0091] The illumination unit 325 illuminates the fluid device 100 placed on the stage 323 by irradiating white light, and enables imaging in a bright field.

[0092] Such a transport unit 30 can move the fluid device 100 placed on the stage 323 in the x-axis direction and the z-axis direction.

[0093] [Control Department]

[0094] Figure 1 The control unit 40 shown in FIG. 4 controls the operations of the temperature adjustment unit 10, the imaging unit 20, and the transport unit 30. The control unit 40 may be a dedicated device provided in the detection device 1, or a general-purpose computer installed with control software may be used.

[0095] [Fluid Equipment]

[0096] Figure 4 It is a schematic perspective view showing the fluid device 100 . Figure 5 is a cross-sectional view of the fluid device 100, Figure 4 The fluidic device 100 is used for detecting a target substance contained in a liquid sample (liquid sample).

[0097] Liquid samples are aqueous solutions containing target substances, such as biological samples or environmental samples. As biological samples, there are no particular limitations, and examples include serum, plasma, urine, and cell culture fluid. In addition, it can also be a PCR reaction solution that uses a biological sample as a template and contains a staining reagent as a detection reagent. Furthermore, as environmental samples, for example, river water and factory drainage can be listed.

[0098] As target substances, for example, DNA, RNA, protein, virus, cell, exosome, etc. can be listed. Among them, as RNA, miRNA and mRNA, etc. can be listed. In addition, as cells, bacteria, yeast, animal cells, plant cells, insect cells, etc. can be listed.

[0099] In the fluidic device 100 , the target substance contained in the sample as described above is reacted with the detection reagent to detect the target substance.

[0100] [Fluid Equipment]

[0101] like Figure 4 and Figure 5As shown, the fluid device 100 includes a well plate 101, a cover member 102, and a wall member 103. The fluid device 100 contains a sample in an internal space S and is used as a reaction container for performing a detection reaction of a target substance contained in the sample.

[0102] (Orifice Plate)

[0103] The orifice plate 101 is a plate-like member that is rectangular or strip-shaped in a plan view. A plurality of holes (also referred to as microholes) 110 are provided on an upper surface 101 a of the orifice plate 101 at the center in the longitudinal direction of the orifice plate 101 .

[0104] The micropore 110 is a recessed portion provided on the upper surface 101a of the orifice plate 101 and is open on the upper surface 101a. The micropore 110 is a space surrounded by the recessed portion and a virtual plane parallel to and in contact with the upper surface 101a.

[0105] The microwell 110 holds the sample contained in the internal space S inside, and functions as a reaction site between the target substance contained in the sample and the detection reagent.

[0106] The material of the orifice plate 101 has electromagnetic wave permeability. Among them, the electromagnetic waves as the object of permeability judgment can be listed as X-rays, ultraviolet rays, visible light, infrared rays, etc. When the orifice plate 101 has electromagnetic wave permeability, the test results of the fluid device 100 having the orifice plate 101 can be analyzed using electromagnetic waves. For example, fluorescence or phosphorescence generated as a result of irradiation with electromagnetic waves can be measured from the orifice plate 101 side.

[0107] The details will be described later. For example, when fluorescence having a peak in the visible light range, that is, the wavelength range of 400 to 700 nm is generated in the micropore 110 to detect the sample, a material having good light transmittance at least in the visible light range may be used.

[0108] As materials having electromagnetic wave permeability, for example, glass and resin can be cited. As resins, for example, ABS resin, polycarbonate, COC (cycloolefin copolymer), COP (cycloolefin polymer), acrylic resin, polyvinyl chloride, polystyrene, polyethylene, polypropylene, polyvinyl acetate, PET (polyethylene terephthalate) and PEN (polyethylene naphthalate) can be cited. These resins may contain various additives, or may be polymer alloys mixed with a plurality of resins.

[0109] The material having electromagnetic wave permeability preferably has substantially no autofluorescence. Substantially having no autofluorescence means that the material has no autofluorescence at the wavelength used for sample detection, or even if it has autofluorescence, it is so weak that it does not affect the sample detection. For example, if the autofluorescence is less than 1 / 2, more preferably less than 1 / 10 of the fluorescence of the detection object, it can be said that it is so weak that it does not affect the sample detection. If such a material is used to form the orifice plate 101, the sensitivity of detection can be improved in sample detection using electromagnetic waves.

[0110] Examples of materials that are electromagnetic wave-transmissive and do not emit autofluorescence include quartz glass, and examples of materials that have weak autofluorescence and do not hinder sample detection using electromagnetic waves include low-fluorescence glass, acrylic resin, COC, and COP.

[0111] The thickness of the well plate 101 can be appropriately determined. When fluorescence is observed from the well plate 101 side using a fluorescence microscope, the thickness of the well plate 101 can be, for example, 5 mm or less, 2 mm or less, or 1.6 mm or less.

[0112] The orifice plate 101 may be a single layer of the above-mentioned material or a laminate of a plurality of materials. When the laminate is processed to form the orifice plate 101, the layer having the micropores 110 and the layer supporting the layer may be made of different materials.

[0113] (Micropores)

[0114] The micropore 110 can adopt various shapes. Examples of the shape of the micropore 110 include cylindrical shapes such as a cylinder, an elliptical cylinder, and a polygonal cylinder, conical shapes such as a cone and a pyramid, and truncated cone shapes such as a truncated cone and a truncated cone. When the micropore 110 is a cone or a truncated cone, it is preferably a shape in which the opening diameter gradually decreases in the depth direction of the hole.

[0115] The bottom of the microwell 110 may be flat or may be a curved surface with a convex surface or a concave surface.

[0116] When the micropore 110 is cylindrical, the maximum diameter of the micropore 110 in a plan view is, for example, preferably 10 nm to 100 μm, more preferably 100 nm to 50 μm, and further preferably 1 μm to 20 μm. In addition, the depth of the micropore 110 is, for example, preferably 10 nm to 100 μm, more preferably 100 nm to 50 μm, and further preferably 1 μm to 20 μm.

[0117] The capacity of the microwell 110 is preferably 1fL to 6nL, more preferably 1fL to 5pL, further preferably 1fL to 2pL, and particularly preferably 1fL to 300fL. If the capacity of each microwell 110 is within such a range, enzyme reactions such as digital PCR and invader reactions in a microscopic space can be well performed. Through digital PCR, for example, gene mutation detection can be performed.

[0118] The well plate 101 has a plurality of microwells 110 of the same shape and size. The so-called same shape and same size only need to be the same shape and the same capacity to the extent required for digital measurement, and variations of the degree of manufacturing error are acceptable.

[0119] The density of the micropores 110 is, for example, 100,000 to 10 million per cm 2 , preferably 100,000 to 5 million / cm 2 , more preferably 100,000 to 1,000,000 / cm 2 When the density of the micropores 110 is within such a range, the operation of sealing the sample in a predetermined number of micropores 110 is easy. In addition, observation of the pores for analyzing the test results is also easy.

[0120] For example, when measuring mutations in cell-free DNA using the fluidic device 100 , when the presence ratio of the mutation to be detected relative to the wild type is approximately 0.01%, for example, 1 to 2 million microwells 110 are preferably used.

[0121] Figure 6 1 is a plan view showing a portion of the plurality of micropores 110. Figure 6 As shown, the plurality of micro-holes 110 in the well plate 101 have the same shape in a plan view. It should be noted that a small number (eg, 1 to 4) of the plurality of micro-holes may have different shapes in order to be used as alignment marks in imaging or device manufacturing.

[0122] Furthermore, the plurality of micropores 110 are regularly arranged in a matrix. Here, the micropores 110 are “regularly arranged” means that the centers of gravity of the openings of the plurality of micropores 110 are arranged in a certain pattern.

[0123] For example, the centroids of the openings of the micropores 110 may be arranged in a four-sided grid. In this case, a line connecting the centroids of the openings of four adjacent micropores 110 forms a rectangle, preferably a square.

[0124] Alternatively, the centroids of the openings of the micropores 110 may be arranged in a triangular lattice (hexagonal lattice). In this case, a line connecting the centroids of the openings of three adjacent micropores 110 forms an equilateral triangle.

[0125] In the plurality of micropores 110 , an arbitrary pore (first pore) A and a pore (second pore) B closest to the pore A preferably satisfy the following formula (1).

[0126] 0.8≤Da / Dab<1…(1)

[0127] (Da is the equivalent circle diameter of the opening of hole A, and Dab is the distance between the center of gravity of the opening of hole A and the center of gravity of the opening of hole B.)

[0128] In the orifice plate 101, the lower limit of the value of Da / Dab is 0.8, and may be 0.83 or more. In addition, the upper limit of the value of Da / Dab is less than 1, and may be 0.92 or less, and may be about 0.9. These lower limits and upper limits may be arbitrarily combined.

[0129] When the above formula (1) is satisfied, if the plurality of micropores 110 are arranged in a triangular lattice, the distance between the centroid of the opening of any micropore 110 and the centroid of the opening of the micropore 110 closest to the micropore 110 is constant.

[0130] Figure 6 The micropores 110 shown are arranged in a triangular lattice. Figure 6 As shown, the centroids Ca, Cb, and Cc of the openings of an arbitrary hole A, a hole B closest to hole A, and a hole C closest to both holes A and B form equilateral triangles with the centroids as vertices.

[0131] Figure 6 In FIG. 1 , the line connecting the centroids Ca, Cb, and Cc of the openings of the holes A, B, and C, respectively, forms an equilateral triangle.

[0132] In the orifice plate 101 , the equivalent circle diameter of the opening of the micropore 110 is preferably 1 μm or more and 50 μm or less. That is, the lower limit of the equivalent circle diameter of the opening of the micropore 110 is preferably 1 μm.

[0133] In addition, the upper limit of the equivalent circular diameter of the opening of the micropore 110 can be lower than 20 μm, can be less than 19 μm, can be less than 18 μm, can be less than 17 μm, can be less than 16 μm, can be less than 15 μm, can be less than 14 μm, can be less than 13 μm, can be less than 12 μm, can be less than 11 μm, and can also be less than 10 μm.

[0134] The upper limit value and the lower limit value of the circle-equivalent diameter of the opening of the micropore 110 can be arbitrarily combined.

[0135] (Cover member)

[0136] The cover member 102 has the same outline shape (ie, a strip shape) as the orifice plate 101 in a plan view. The cover member 102 is disposed opposite to the upper surface 101a of the orifice plate 101 with a gap therebetween.

[0137] The cover member 102 has two through holes penetrating in the thickness direction. The two through holes are respectively provided at one end side and the other end side in the length direction of the cover member 102. One through hole is an injection port 121 used when injecting a liquid into the internal space S of the fluid device 100, and the other through hole is an outlet port 122 used when discharging a liquid from the internal space S.

[0138] Here, in addition to liquid samples, detection reagents and sealing liquids also belong to "liquid substances".

[0139] The injection port 121, the internal space S and the discharge port 122 are connected in sequence to form a flow path FC. In the fluid device 100, the liquid is appropriately flowed in the flow path FC to perform a detection reaction of the target substance. In a plan view, a plurality of micropores 110 are arranged between the injection port 121 and the discharge port 122.

[0140] A cylindrical injection port 125 surrounding the injection port 121 is provided on the upper surface 102a of the cover member 102. The injection port 125 is connected to the injection port 121. For example, when a syringe filled with a liquid is used to fill the internal space with a liquid, the injection port 125 is used to connect the syringe.

[0141] Likewise, a cylindrical discharge port 126 surrounding the discharge port 122 is provided on the upper surface 102a of the cover member 102. The discharge port 126 communicates with the discharge port 122. For example, when a liquid is extracted from the internal space S, the discharge port 126 is used to connect a pipe through which the liquid flows.

[0142] The material of the cover member 102 can be the material exemplified above as the material of the orifice plate 101. The material of the cover member 102 may be the same as or different from the material of the orifice plate 101.

[0143] In addition, the material of the cover member 102 may or may not be electromagnetic wave transparent.

[0144] The material of the cover member 102 is preferably hydrophobic. Specifically, the material constituting the surface of the cover member 102 facing the internal space S (i.e., the lower surface 102b) preferably has a contact angle with the sealing liquid SL of not less than 5° and not more than 80°. If the cover member 102 is formed with such a material, the contact angle between the lower surface 102b and the sealing liquid SL is not less than 5° and not more than 80°. If the contact angle of the lower surface 102b is within the above range, when the sealing liquid is introduced into the internal space S by the method described later, there is a tendency for the sample to be easily isolated in the micropores 110.

[0145] (Wall component)

[0146] The wall member 103 forms a closed loop shape in a plan view, and is disposed along the outer edge of the upper surface 101 a of the orifice plate 101 . Figure 4 In the embodiment, the wall surface of the wall member 103 facing the internal space S is generally rectangular in a plan view, and its width gradually decreases on the injection port 121 side.

[0147] The wall member 103 is sandwiched between the orifice plate 101 and the cover member 102 to form a fluid device 100. The space surrounded by the orifice plate 101, the cover member 102 and the wall member 103 is an internal space S for accommodating a liquid sample. The internal space S extends along the short strip of the orifice plate 101 in the longitudinal direction of the orifice plate 101.

[0148] The wall member 103 functions as a wall surface of the internal space S and also functions as a spacer between the orifice plate 101 and the cover member 102. The height of the wall member 103, that is, the height of the internal space S, may be, for example, 100 μm or less.

[0149] The material of the wall member 103 is not particularly limited, and for example, a double-sided adhesive tape in which acrylic adhesive is laminated on both sides of a core film can be preferably used. As the material of the core film, silicone rubber and acrylic foam can be exemplified. By using such a material to form the wall member 103, the internal space S can be formed liquid-tightly.

[0150] The wall member 103 can be made of the same material as the orifice plate 101. The wall member 103 made of such a material can be integrated with the orifice plate 101 and the cover member 102 by bonding with an adhesive or welding such as heat welding, ultrasonic welding or laser welding.

[0151] It should be noted that the wall member 103 may be formed integrally with the orifice plate 101 to constitute a part of the orifice plate 101. Similarly, the wall member 103 may be formed integrally with the cover member 102 to constitute a part of the cover member 102.

[0152] The above-mentioned orifice plate 101 can be manufactured by using known injection molding, micro-imprinting technology or nano-imprinting technology. In addition, the orifice plate 101 can also be manufactured by using known photolithography technology to form micro-holes 110 by etching.

[0153] The above-mentioned cover member 102 and wall member 103 can be manufactured by known injection molding.

[0154] [Detection reagents]

[0155] The detection reagent reacts with the target substance and is used for detecting the target substance. Examples of the detection reagent include buffer substances, enzymes, substrates, antibodies, and antibody fragments.

[0156] For example, when the target substance is a nucleic acid, an enzyme is selected according to the content of the biochemical reaction in order to perform a biochemical reaction such as an enzyme reaction on a template nucleic acid related to the target substance. The biochemical reaction on the template nucleic acid is, for example, a reaction that amplifies a signal in the presence of the template nucleic acid.

[0157] The detection reagent is selected according to the detection reaction to be used. Specific detection reactions include ICA method, loop-mediated isothermal amplification (LAMP) method (registered trademark), 5'→3' nuclease method (TaqMan (registered trademark) method), and fluorescent probe method.

[0158] Figures 7 to 9 1 is an explanatory diagram of a method for detecting a sample using the above-mentioned fluid device 100 .

[0159] First, a liquid sample and a detection reagent are mixed to prepare a mixed aqueous solution. The concentration of the detection reagent in the mixed aqueous solution is appropriately adjusted according to the type of detection reagent used and the type of detection reaction.

[0160] The detection reagent used may also contain an adsorption inhibitor. Examples of the adsorption inhibitor include surfactants and proteins.

[0161] In order to facilitate the detection of target substances, the sample may be pretreated before preparing the mixed aqueous solution. Examples of pretreatment include concentration adjustment (i.e., dilution or concentration), loading on a carrier, and binding reaction of two or more target substances.

[0162] Then, if Figure 7 As shown, a mixed aqueous solution L containing a detection reagent is injected from the injection port 121 into the internal space S. When the mixed aqueous solution L flows in the internal space S (ie, the flow path FC), the micropores 110 opening into the internal space S are also filled with the mixed aqueous solution L.

[0163] After the internal space S and all the micropores 110 are filled with the mixed aqueous solution L, the mixed aqueous solution L flowing through the flow path FC is discharged from the discharge port 122 .

[0164] At this time, it is preferred to adjust the concentration of the mixed aqueous solution L in advance so that one target substance is filled inside one micropore 110. For example, after the sample detection method described later is implemented, if the result shows that it is difficult to quantify the target substance due to the high concentration of the mixed aqueous solution L, the result is used as a preliminary test result and the mixed aqueous solution L is diluted.

[0165] Through the above operation, one or less target substance is filled in one micropore 110. Thus, the number of micropores 110 in which the detection reaction described later is confirmed corresponds to the number of target substances, and the target substance can be detected in units of one, that is, digital measurement can be performed. It should be noted that it is not necessary to introduce the target substance into all micropores 110.

[0166] There is no particular limitation on the means for introducing the target substance into the microwell 110, and an appropriate means may be selected according to the target substance to be detected. For example, a method may be cited in which the target substance is allowed to settle in the fluid device 100 (specifically, in the flow path FC) by its own weight and then be distributed into the microwell.

[0167] In addition, a substance that captures the target substance (also referred to as a capture) can be used to combine the capture with the target substance that is difficult to settle by its own weight and then deliver the liquid. Furthermore, by pre-immobilizing the capture with the micropore, the target substance that is delivered together with the mixed aqueous solution L is captured by the capture, which can also improve the efficiency of introducing the target substance into the micropore 110.

[0168] The reaction of binding the captured substance to the target substance can be performed at any time point. For example, the binding reaction can be performed by bringing the target substance into contact with the captured substance in a sample tube before preparing the mixed aqueous solution.

[0169] Alternatively, after the captured object is introduced into the microwell 110 , the target substance may be introduced into the microwell 110 , so that the captured object and the target substance come into contact with each other in the microwell 110 .

[0170] The captured substance is a substance that can capture the target substance. For example, the captured substance may be a combination of a solid phase and a substance that specifically binds to the target substance.

[0171] As the solid phase constituting the captured object, particles, membranes, substrates, etc. can be listed. In addition, the specific binding substances for the target substance can be one, or two or more. For example, it can be three, four, or five or more.

[0172] As particles, there is no particular limitation, and polymer particles, magnetic particles and glass particles can be cited. Particles are preferably particles that have been surface treated to avoid nonspecific adsorption. In addition, in order to immobilize the specific binding substance, particles having functional groups such as carboxyl groups on the surface are preferred. More specifically, the trade name "Magnosphere LC300" manufactured by JSR Corporation can be used.

[0173] Furthermore, for example, when a virus is used as a target substance, cells to which the virus can attach (ie, cells having a virus receptor) may be used as captured objects.

[0174] Then, if Figure 8 As shown, sealing liquid SL is supplied to flow path FC from injection port 121. Sealing liquid SL supplied to flow path FC flows along the surface direction of upper surface 101a in internal space S, and washes away the mixed aqueous solution L not received by micropores 110 in the mixed aqueous solution L supplied to flow path FC, thereby replacing the mixed aqueous solution L.

[0175] Thus, the sealing liquid SL individually seals the plurality of microwells 110 containing the mixed aqueous solution L containing the target substance, and the microwells 110 become independent reaction spaces. After the flow path FC is filled with the sealing liquid SL, the excess sealing liquid SL is discharged from the discharge port 122 . Fig. 9 This shows a state in which all of the plurality of micropores 110 are sealed with the sealing liquid SL and the flow path FC is completely replaced with the sealing liquid SL.

[0176] Next, the fluid device 100 is heated to react the target substance with the detection reagent. The method for detecting the target substance can be combined with the characteristics of the target substance to be detected, and any known detection method can be used. First, a reaction is performed to amplify the signal from the target substance to a detectable level (also called a signal amplification reaction), and then the amplified signal is detected using appropriate means.

[0177] In the detection device 1, as a signal amplification reaction, an isothermal signal amplification reaction can be used, and an ICA reaction, a loop-mediated isothermal amplification (LAMP) method (registered trademark), a 5'→3' nuclease method (TaqMan (registered trademark) method) or a fluorescent probe method can be used. The ICA reaction amplifies the signal through a cycle of two reactions: (1) complementary binding between nucleic acids and (2) recognition and cleavage of the triple chain structure by the enzyme. In such a signal amplification reaction, the influence of inclusions other than the target substance on the reaction cycle is small. Therefore, even in the case where various components other than the target substance (such as inclusions) are present in the micropore 110, the target substance can be detected with good accuracy by using the ICA reaction.

[0178] The mixed aqueous solution L contains the reaction reagents and template nucleic acid required for the ICA reaction. When the target substance is present in the well, the fluorescent substance is released from the quenching substance by an isothermal enzyme reaction, and a predetermined fluorescent signal is emitted in response to the excitation light.

[0179] In the ICA reaction, the reaction temperature of the target substance and the detection reagent is preferably above 50°C and below 99°C. The specific reaction temperature can be determined by preliminary tests based on the reagents used and the sensitivity of the required results. After determining the composition of the reagents used, an ICA reaction is performed as a preliminary test, and the reaction temperature can be optimized based on the results of the signal, noise, signal-to-noise ratio (also called S / N ratio), signal-to-noise difference, etc. after a specified time.

[0180] Hereinafter, the ICA reaction will be described in further detail.

[0181] Fig.10 is a schematic diagram illustrating an example of the ICA method. Fig.10 Detection of DNA as a target substance by the ICA method is shown in FIG.

[0182] Examples of the reaction reagents required for the ICA reaction include a flanking (Flap) probe 810, a flanking (Flap) endonuclease FEN, a fluorescent substrate 820, and an invader probe (invader oligomer) 830 and the like.

[0183] The flanking probe 810 and the invasive probe 830 are nucleic acid fragments (also called oligonucleotides) designed to hybridize with target DNA (also called target DNA) to form a double-stranded nucleic acid 140 and a flanking (Flap) structure.

[0184] The fluorescent substrate 820 has a hairpin structure and is a nucleic acid fragment formed by the combination of a fluorescent substance F and an extinguishing substance Q. Fig.10 In the fluorescent substrate 820 shown, a fluorescent substance F is bound to the 5' end of the nucleic acid fragment, and an extinguishing substance Q is bound to the 3' side several bases away from the 5' end. The extinguishing substance Q inhibits the emission of the fluorescent substance F.

[0185] First, the flanking probe 810 and the invader probe 830 are hybridized with the target DNA. The flanking probe 810 and the invader probe 830 overlap one base at the SNP site of the target DNA, forming an unstable three-base structure. As a result, a first flanking site 811 is formed. The first flanking site 811 is a portion of the flanking probe 810 that is not hybridized with the target DNA.

[0186] Next, FEN recognizes the three-base-like structure and reacts. As a result, the first flanking site 811 is cleaved, generating a nucleic acid fragment 811, which is released into the mixed aqueous solution L.

[0187] The generated nucleic acid fragment 811 hybridizes with the fluorescent substrate 820. The nucleic acid fragment 811 invades the hairpin structure of the fluorescent substrate 820, overlaps one base at the SNP site, and forms an unstable 3-base-like structure. As a result, a second flanking site 821 is formed. The second flanking site 821 is a portion of the fluorescent substrate 820 that is not hybridized due to the invasion of the nucleic acid fragment 811.

[0188] Next, FEN recognizes the three-base-like structure and reacts, thereby cleaving the second flanking site 821 and generating a nucleic acid fragment 821 . Fig.10 In the figure, the remaining portion after the nucleic acid fragment 821 is cut from the fluorescent substrate 820 is represented by symbol 820'.

[0189] As a result, the fluorescent substance F is separated from the quenching substance Q, and fluorescence FL is emitted. By detecting the fluorescence FL, the target DNA can be detected.

[0190] In addition, the detection of the target substance can also be performed by binding a substance that specifically binds to the target substance (also referred to as a specific binding substance) to the target substance and detecting the bound specific binding substance. For example, when the target substance is a protein, ELISA can be used for detection. More specifically, detection can also be performed, for example, by sandwich ELISA using the principle of FRET.

[0191] In the case of a sandwich method utilizing the FRET principle, first, a first specific binding substance (e.g., an antibody) labeled with a first fluorescent substance (also called a donor) and a second specific binding substance labeled with a second fluorescent substance (also called an acceptor) having an absorption wavelength that overlaps the fluorescence wavelength of the first fluorescent substance are prepared.

[0192] Next, the target substance (e.g., antigen) is brought into contact with both the first specific binding substance and the second specific binding substance to form a complex. After the complex is formed, the distance between the donor and the receptor becomes closer, so that the fluorescence wavelength of the receptor can be detected by irradiating the excitation wavelength of the donor. Alternatively, the specific binding substance can be labeled with a nucleic acid fragment and the nucleic acid fragment can be detected by an ICA reaction.

[0193] As specific binding substances, substances similar to the specific binding molecules for structures described later can be used, such as antibodies, antibody fragments or aptamers. In order to detect specific binding substances that have been bound to target substances, enzymes such as horseradish peroxidase (HRP) can be used to directly or indirectly mark specific binding substances. When using more than two specific binding substances, each specific binding molecule can be marked in a manner that can recognize each other.

[0194] The signal observation method can select a known appropriate method according to the type of signal to be observed. For example, in the case of bright field observation, white light is irradiated in a direction perpendicular to the substrate on which the hole array is provided. In the case of observing fluorescent signals, excitation light corresponding to the fluorescent substance is irradiated into the hole from the bottom side of the hole, and the fluorescence emitted by the fluorescent substance is observed. The image of the entire or a part of the observed hole array is photographed and saved, and the image is processed using a computer system. It should be noted that the irradiation direction of light (i.e., white light and excitation light) in the above-mentioned bright field observation and fluorescent signal observation is only an example, and can be appropriately changed as long as the purpose of observation can be achieved. For example, in the fluorescent signal observation, the excitation light can also be irradiated in a direction perpendicular to the substrate, just like the irradiation direction of white light in the bright field observation.

[0195] [action]

[0196] Figures 11 to 14 It is an explanatory diagram for explaining the operation of the detection device 1.

[0197] First, if Fig.11 As shown, a mixed aqueous solution containing a liquid sample of a target substance and a detection reagent that reacts with the target substance to generate a fluorescent product is contained in the fluid device 100 and placed on the stage 323 of the transport body 32. This operation can be performed in the standby section 55 of the fluid device 100.

[0198] At this time, the control unit 40 preheats the heating unit 11 (also called preheating) to a temperature higher than room temperature and below a preset reaction temperature for standby use. As described above, in the ICA reaction, as an example, the reaction temperature of the target substance and the detection reagent is preferably above 50°C and below 99°C. Under this temperature condition, the preheating temperature is preferably higher than room temperature, for example, below 90°C. "Room temperature" refers to, for example, 25°C.

[0199] Then, if Fig.12 As shown, the transport unit 30 is driven to move the fluid device 100 placed on the stage 323 to the heating unit 11. As a result, the fluid device 100 is heated in the heating unit 11.

[0200] As described above, if the heating unit 11 is preheated before the fluid device is heated, it is preferable because the fluid device 100 can be heated quickly. In the case of preheating the heating unit 11, when the conveying unit 30 starts to convey the fluid device 100 from the standby unit 55 to the heating unit 11, the control unit 40 may change the heating temperature of the heating unit 11 from the preheating set temperature to the reaction temperature of the ICA reaction. In addition, at any time during the period from when the conveying unit 30 conveys the fluid device 100 from the standby unit 55 to the heating unit 11, the control unit 40 may change the heating temperature of the heating unit 11 from the preheating set temperature to the reaction temperature of the ICA reaction.

[0201] At this time, the control unit 40 cools the cooling unit 12 (precooling) in advance to a temperature higher than a preset cooling temperature and lower than the reaction temperature in the heating unit 11. For example, when the reaction temperature of the ICA reaction is 60°C, the precooling temperature is preferably, for example, higher than 0°C and lower than room temperature.

[0202] Then, if Fig.13 As shown, the transport unit 30 is driven to move the fluid device 100 placed on the stage 323 from the heating unit 11 to the cooling unit 12. Thus, the fluid device 100 is cooled in the cooling unit 12.

[0203] As described above, it is preferable to pre-cool the cooling unit 12 before cooling the fluid device, because the fluid device 100 can be quickly cooled. In the case of pre-cooling the cooling unit 12, when the conveying unit 30 starts to convey the fluid device 100 from the heating unit 11 to the cooling unit 12, the control unit 40 may change the cooling temperature of the cooling unit 12 from the pre-cooling set temperature to the actual cooling temperature. In addition, at any time during the period from when the conveying unit 30 conveys the fluid device 100 from the heating unit 11 to the cooling unit 12, the control unit 40 may change the cooling temperature of the cooling unit 12 from the pre-cooling set temperature to the actual cooling temperature.

[0204] Then, if Fig.14 As shown, the transport unit 30 is driven to move the fluid device 100 placed on the stage 323 from the cooling unit 12 to above the projection lens 23 of the imaging unit 20. The imaging unit 20 irradiates the fluid device 100 with excitation light and captures an image of the generated fluorescence.

[0205] Then, by performing image analysis on the captured fluorescence image, the number of holes emitting fluorescence can be measured, thereby quantifying the target substance.

[0206] The detection device 1 described above can obtain the following effects.

[0207] If the detection reagent is continuously heated, a product that emits fluorescence will be generated due to a side reaction even if the target substance is not present. In the image captured by the imaging unit 20, the product generated in this way cannot be distinguished from the fluorescence caused by the target substance, which becomes a cause of error in the detection of the target substance. In addition, in the fluid device 100, the ICA reaction is also carried out during the period from heating to cooling. Therefore, it takes time from heating to cooling, and both the luminescence of the signal and the luminescence of the noise as the detection object become stronger, and it can be expected that the detection sensitivity will decrease.

[0208] In contrast, in the detection device 1, after the heating by the heating unit 11, the fluid device 100 is cooled in the cooling unit 12 provided separately from the heating unit 11. Therefore, the reaction between the target substance and the detection reagent accelerated by the heating is inactivated, and the side reaction can be suppressed. Thus, the detection device 1 can perform high-precision detection.

[0209] Furthermore, if the cooling unit is also provided at the location where the heating unit 11 is provided, so that heating and cooling can be performed at the same location, when cooling starts after heating, the heating unit 11 heated to the reaction temperature of the detection reaction such as the ICA reaction needs to be cooled together with the fluid device 100. In the case of such a configuration, although the fluid device 100 can be cooled, the time required for cooling the heating unit 11 will be unnecessary.

[0210] In addition, as described above, the fluid device 100 is made of glass and resin. In addition, liquids such as liquid samples and reagents are stored inside the fluid device 100. Furthermore, in order to increase the volume of the fluid device 100, it is foreseeable that additional components are installed in the device, or the device itself is enlarged. These changes will increase the specific heat of the fluid device 100 and hinder the rapid cooling of the fluid device 100.

[0211] In contrast, in the detection device 1, since the heating unit 11 and the cooling unit 12 are provided separately, the fluid device 100 can be quickly cooled after heating the fluid device 100, compared with a device configuration in which the cooling unit is also provided at the location where the heating unit 11 is provided. Furthermore, in the detection device 1, since the heating unit 11 and the cooling unit 12 are provided separately, the above-mentioned preheating and precooling can be performed. As a result, the detection device 1 can perform detection in a short time, and the operation efficiency is improved.

[0212] According to the detection device 1 having the above-described configuration, it is possible to provide a detection device that can ensure high detection accuracy in detection based on the principle of digital ICA and can perform a large amount of detection in a short time.

[0213] It should be noted that, in the detection device 1 of this embodiment, a member for cooling the object is provided at the position of the cooling unit 12, but the present invention is not limited thereto. In the cooling device, any configuration is sufficient as long as the fluid device 100 heated by the heating unit 11 is transported to the imaging position of the imaging unit 20 and the fluid device 100 is cooled during the period until the observation starts.

[0214] For example, the fluid device 100 may be cooled by blowing air while being transported from the heating unit 11 to the imaging position. In this case, the unit that blows air in the transport path from the heating unit 11 to the imaging position corresponds to the cooling unit.

[0215] In addition, the fluid device 100 may be cooled at the imaging position. The unit for cooling the fluid device 100 may adopt a known structure. In this case, the unit for cooling the fluid device 100 at the imaging position corresponds to a cooling unit. In such a structure, observation (or imaging) may be performed while cooling, or observation (or imaging) may be performed after cooling.

[0216] In addition, in the present embodiment, when the projection lens 23 (also an objective lens) has an autofocus mechanism, the control unit 40 can also control the autofocus mechanism before shooting so that the focus position of the objective lens is aligned with the microhole 110. By performing such control by the control unit 40, the detection device 1 can accurately shoot and perform high-precision detection. In addition, by using the microhole 110 as an alignment mark and aligning the focus position with the microhole 110 in advance, even if the fluid device 100 as a whole is warped or deformed, the focus position can be quickly readjusted.

[0217] When the photographing unit 20 does not have an autofocus mechanism, the focal position of the objective lens can be aligned with the microhole 110 by other methods. For example, an alignment mark can be set in the fluid device 100, and in the detection device 1, the alignment mark is used to align the focus and then photograph other microholes 110. The alignment mark can be set at the focal position just like the microhole 110.

[0218] Even if the focus position of the alignment mark and the focus position of the microhole 110 are different in the z-axis direction, the deviation between the focus positions of the two may be measured, the deviation may be corrected to align the focus positions, and the microhole 110 may be photographed.

[0219] For example, when the fluid device 100 as a whole is warped or deformed, it can be considered that the focal length relative to the microhole 110 varies depending on the microhole 110. In this case, for a plurality of microholes (i.e., a hole array), for example, the focal lengths of the microholes 110 located at the periphery and the center of the array can be measured, and the warpage of the fluid device 100 can be estimated based on the correspondence between the obtained focal lengths and the positions of the microholes 110 at which the focal lengths are measured. The obtained warpage can be used as a correction value for the offset, and the microhole 110 can be photographed at the focus position.

[0220] It should be noted that in the above method, the warpage amount of the fluid device 100 is estimated based on the focal length of the microhole 110 , and the alignment mark of the fluid device 100 can also be used as a measurement object when calculating the warpage amount.

[0221] In this case, the alignment marks provided in the fluid device 100 may use a portion (eg, 1 to 4) of the plurality of holes as described above, or may be additionally provided around the region where the microhole 110 is provided.

[0222] Alternatively, the fluid device 100 may be photographed at a plurality of focal positions, and a known z-stack image may be synthesized using the plurality of images obtained, thereby obtaining a synthesized image in which the entire image is focused.

[0223] [Second embodiment]

[0224] Figures 15 to 19 This is an explanatory diagram of a detection device according to a second embodiment of the present invention. The same components as those of the first embodiment are denoted by the same reference numerals, and detailed description thereof will be omitted.

[0225] Fig.15 2 is a schematic perspective view showing a detection device 2 of this embodiment, which is different from that of the first embodiment. Figure 1 The detection device 2 includes a temperature adjustment unit 10 , an imaging unit 20 , a transport unit 35 , and a control unit 40 . The detection device 2 also includes a cover 60 and a moving unit 70 .

[0226] The cover 60 is a box-shaped member that covers all components arranged on the stage 51. The cover 60 is provided to be openable and closable by rotating about a rotation axis that passes through the point P1 and is set parallel to the x-axis.

[0227] Fig.16 2 is a schematic perspective view of the moving unit 70. The moving unit 70 is provided on the inner upper surface of the cover 60 (see Fig.15 The moving unit 70 includes a plurality of (three in the figure) push-out portions 71 arranged in the y direction, and a pair of mounting portions 72 that support the push-out portions 71 on both sides in the x direction. Fig.16In FIG. 1 , the mounting portion 72 is shown as a plate-shaped member that supports the plurality of push-out portions 71 together, but the present invention is not particularly limited thereto.

[0228] The push-out portion 71 includes a motor 711 , a feed screw 712 , a guide shaft 713 , a nut 714 , and a hook 715 .

[0229] The motor 711 rotates the feed screw 712 via a reduction gear (not shown).

[0230] The feed screw 712 is provided to extend in the x direction. Both ends of the feed screw 712 in the x direction are rotatably supported by the mounting portion 72. The feed screw 712 is driven by the motor 711 to rotate around the central axis of the feed screw 712.

[0231] The guide shaft 713 is provided to extend in the x direction at a position in the −z direction of the feed screw 712 . The guide shaft 713 is inserted through a through hole (not shown) provided in the hook 715 , and both ends in the x direction are fixed to the mounting portion 72 .

[0232] The feed screw 712 is inserted into the nut 714. The lower side (-z side) of the nut 714 is connected to the hook 715. The shape of the hook 715 is not particularly limited as long as it has a push-out function described later.

[0233] In such a moving unit 70 , the feed screw 712 is rotated by rotating the motor 711 , so that the nut 714 and the hook 715 are moved in the ±x directions along the guide shaft 713 .

[0234] Fig.17 1 is a schematic perspective view showing the heating unit 11 and the conveying unit 35. The heating unit 11 has a temperature control frame 15. The temperature control frame 15 may be integrally formed on the upper surface of the heating unit 11, or may be formed as a member different from the heating unit 11 and fixed on the upper surface of the heating unit 11.

[0235] The temperature control rack 15 has a rectangular base 151 in a plan view, and a plurality of (four in the figure) guide rails 152 extending in the x direction on the upper surface of the base 151. The interval between adjacent guide rails 152 is the same as the width of the short side direction of the fluid device 100, and a groove (also referred to as a first receiving portion) 152a for inserting the fluid device 100 is formed between the guide rails 152. The groove 152a is formed to extend in the conveying direction of the conveying body 36, that is, the x direction.

[0236] When the temperature control frame 15 is a separate member from the heating unit 11 , it is preferable that at least the base 151 is formed of a metal material having high thermal conductivity, such as aluminum or copper.

[0237] The transport unit 35 includes a transport body 36 . The transport body 36 includes a first member 321 , a second member 322 , and a measurement frame 330 .

[0238] The measurement frame 330 includes a rectangular base 331 in plan view, a plurality of (four in the figure) guide rails 332 extending in the x direction on the upper surface of the base 331 , and a wall 333 provided at the +x side end of the guide rail 332 .

[0239] The base 331, like the stage 323, can have various configurations as long as the fluid device 100 is placed thereon and the fluid device 100 can be observed from below (i.e., in the -z direction). For example, the stage 323 can be a light-transmissive plate-shaped member or a frame in which the observation portion of the fluid device 100 is a gap.

[0240] The upper surface of the base 331 of the measurement frame 330 and the upper surface of the base 151 of the temperature control frame 15 are at substantially the same height position in the z direction.

[0241] The interval between adjacent guide rails 332 is the same as the width of the short side direction of the fluid device 100, and a groove (second receiving portion) 332a for inserting the fluid device 100 is formed between the guide rails 332. The groove 332a is formed to extend along the conveying direction of the conveying body 36, that is, the x direction. In addition, the +x side of the groove 332a is closed by the wall portion 333.

[0242] Fig.17 The illustrated temperature control rack 15 has the same number of slots 152a as the slots 332a of the measurement rack 330, but they may be different in number. The slots 152a and the slots 332a are arranged in the x direction.

[0243] In addition, the groove 152a of the temperature control rack 15 is Fig.16 The pushing parts 71 of the illustrated moving unit 70 are, for example, the same in number, but may also be different in number.

[0244] Fig.18 , 19 It is an explanatory diagram for explaining the operation of the detection device 2. Fig.18 , 19 In order to make the drawing easier to see, the guide shaft of the pusher 71 is omitted from the drawing. In the detection device 2, a plurality of fluid devices 100 can be heated and cooled at different times for observation.

[0245] First, if Fig.18 As shown, the fluid device 100 is placed in the groove 152a of the temperature control rack 15 (see Fig.17 ), in the heating unit 11 , the fluid device 100 is heated for each temperature control rack 15 .

[0246] Furthermore, the transport body 36 is moved in the −x direction so that the end of the −x side of the measurement rack 330 is brought close to the end of the +x side of the temperature control rack 15 .

[0247] By closing Fig.15 In the illustrated cover 60 , the lower end of the hook 715 of the push-out portion 71 is located closer to the −z side than the upper surface of the fluid device 100 .

[0248] Then, if Fig.19 As shown in FIG. 1 , the motor 711 of the push-out unit 71 is driven to move the nut 714 and the hook 715 in the +x direction. The hook 715 contacts the fluid device 100A when moving in the +x direction. As the hook 715 moves, the fluid device 100A is pushed out in the +x direction and moved to the measurement rack 330. The fluid device 100 moved to the measurement rack 330 is accommodated in the groove 332a. The remaining fluid devices 100B remain in the state of being arranged on the temperature control rack 15.

[0249] The conveying unit 35 moves the conveying body 36 in the +x direction. Fig.15 ) in the process, the fluid device 100A is appropriately cooled and photographed and detected by the photographing unit 20.

[0250] After that, the detection device 2 performs the same operation as the fluid device 100A on the remaining fluid devices 100B, using the imaging unit 20 (see Fig.15 ) for shooting and testing.

[0251] By performing these processes in the fluid device 100 , it is possible to perform the detection process by making the heating times of the fluid device 100A and the fluid device 100B different from each other.

[0252] The detection device 2 having the above-described configuration can also provide a detection device that can ensure high detection accuracy in detection based on the principle of digital ICA and can perform a large amount of detection in a short time.

[0253] The preferred embodiments of the present invention are described above with reference to the accompanying drawings, but the present invention is not limited to these examples. The various shapes and combinations of the components shown in the above examples are examples, and various changes can be made according to the design, specifications, etc. without departing from the scope of the present invention.

[0254] Industrial Applicability

[0255] According to the present invention, it is possible to provide a detection device that can ensure high detection accuracy in detection based on the principle of digital ICA and can perform a large amount of detection in a short time.

[0256] Reference numerals

[0257] 1, 2…detection device, 10…temperature adjustment unit, 11…heating unit, 12…cooling unit, 15…temperature control rack, 20…photographing unit, 21…light source unit, 22…dichroic mirror, 23…projection lens, 24…imaging lens, 25…photographing unit, 27…spatial filter, 30, 35…transport unit, 40…control unit, 70…moving unit, 100, 100A, 100B…fluid device, 110…hole (microhole), 152a…groove (first receiving unit), 211…light source, 212…optical filter, 212a…first optical filter, 212b…second optical filter, 215…external light introduction unit, 295…switching unit, 327a…groove (second receiving unit), A, B, C…hole, DNA…target, EL…excitation light, FL…fluorescence, FP…confocal point, L…mixed aqueous solution, L1…light, L2…external light, P…pinhole

Claims

1. A detection device, comprising: A temperature regulating unit, which regulates the temperature of the fluid equipment; an imaging unit that irradiates the fluid device with excitation light and captures an image of fluorescence emitted from the fluid device; and a transporting unit, which transports the fluid device to the temperature regulating unit and the photographing unit, The fluid device contains a mixed aqueous solution of a liquid sample and a detection reagent, the liquid sample contains a target substance, and the detection reagent reacts with the target substance to produce a product that emits the fluorescence. The temperature adjustment unit has: a heating unit for heating the fluid device; and The cooling unit cools the heated fluid device.

2. The detection device according to claim 1, wherein: A control unit is provided for controlling the operation of the temperature adjustment unit. The control unit controls the heating unit to be at least 50° C. and no more than 99° C.

3. The detection device according to claim 2, wherein: The control unit performs preheating of heating the heating unit to a temperature higher than room temperature and lower than a predetermined reaction temperature before heating the fluid device.

4. The detection device according to any one of claims 1 to 3, wherein: A control unit is provided for controlling the operation of the temperature adjustment unit. The control unit controls the cooling unit to be at least 0.1° C. and less than 30° C.

5. The detection device according to claim 4, wherein: The control unit performs pre-cooling of cooling the cooling unit to a temperature not lower than a preset cooling temperature and lower than a room temperature before cooling the fluid device.

6. The detection device according to claim 1, wherein: The photographing unit comprises: A light source unit, emitting the excitation light; A dichroic mirror, reflecting the excitation light and transmitting the fluorescence; A projection lens, for projecting the excitation light reflected by the dichroic mirror toward the fluid device; An imaging lens for imaging the fluorescence transmitted from the dichroic mirror; as well as a photographing unit for photographing the image formed by the imaging lens, The projection lens also serves as an objective lens for collecting the fluorescence generated from the fluid device and guiding the fluorescence to the imaging lens.

7. The detection device according to claim 6, wherein: The light source unit comprises: a light source that emits light including the excitation light; and The optical filter transmits light of a wavelength used as the excitation light among the light.

8. The detection device according to claim 7, wherein: The light source unit comprises: a first optical filter that transmits light of a first wavelength used as the excitation light among the light; a second optical filter that transmits light of a second wavelength used as the excitation light in the light; as well as The switching unit switches between the first optical filter and the second optical filter.

9. The detection device according to claim 6, wherein: The light source unit includes an external light introduction unit for introducing external light and guiding the light to the fluid device.

10. The detection device according to claim 6, wherein: The projection lens and the objective lens form a confocal optical system.

11. The detection device according to claim 10, wherein: The imaging unit includes a spatial filter having a pinhole between the imaging lens and the imaging unit. The confocal position of the confocal optical system is located between the imaging lens and the shooting unit. The pinhole spatially overlaps with the confocal point position.

12. The detection device according to claim 6, wherein: The fluid device has a microwell that contains the target substance and the detection reagent and is used as a reaction site for generating the product. The objective lens has an automatic focusing mechanism, The detection device includes a control unit, and before the imaging unit performs imaging, the control unit controls the autofocus mechanism so that the focal position of the objective lens is aligned with the micro-hole.

13. The detection device according to claim 1, wherein: The heating unit has a temperature control rack for accommodating a plurality of the fluid devices. The conveying unit has a conveying rack for accommodating a plurality of the fluid devices. The temperature control rack has a first receiving portion extending along the conveying direction of the conveying portion and receiving the fluid device. The temperature control rack has a second receiving portion extending along the conveying direction of the conveying portion and receiving the fluid device. The detection device further includes a moving unit configured to push out and move the fluid device accommodated in the first accommodation portion toward the second accommodation portion in a state where the temperature control rack and the transport rack are brought close to each other.

Images