Patent Application
20250123207
The present invention relates to a detection device.
WO 2015/115635 A describes a digital measurement technique using a reaction container (also referred to as a fluidic device) having wells. The entire contents of this publication are incorporated herein by reference.
According to one aspect of the present invention, a detection device includes a temperature adjustment unit that adjusts a temperature of a fluidic device, an imaging unit that irradiates the fluidic device with excitation light and captures an image of fluorescence emitted from the fluidic device, and a conveying unit that conveys the fluidic device to the temperature adjustment unit and the imaging unit. The fluidic device accommodates a mixed aqueous solution of a liquid sample including a target substance and a detection reagent such that the detection reagent reacts with the target substance and generate a product that emits the fluorescence, and the temperature adjustment unit includes a heating unit that heats the fluidic device and a cooling unit that cools the fluidic device heated.
A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:
Embodiments will now be described with reference to the accompanying drawings, wherein like reference numerals designate corresponding or identical elements throughout the various drawings.
A detection device and a fluidic device according to the first embodiment of the present invention will be described below with reference to
In the following, an xyz orthogonal coordinate system is set, and the positional relationship of members will be described with reference to the xyz orthogonal coordinate system. Here, the x-axis direction is a predefined direction in a horizontal plane, the y-axis direction is a direction perpendicular to the x-axis direction in the horizontal plane, and the z-axis direction is a direction (i.e., vertical direction) perpendicular to each of the x-axis direction and the y-axis direction.
The temperature adjustment unit 10, the imaging unit 20, the conveying unit 30, and the control unit 40 are provided on a base 90.
The detection device 1 is used to detect a target substance contained in a liquid sample. Specifically, the detection device 1 is used when a target substance is detected using a fluidic device 100 based on the principle of digital ICA (hereinafter referred to as dICA). In the detection device 1, a product generated in the fluidic device 100 is excited by light, and an image of generated fluorescence is captured and analyzed to detect a target substance.
The detection device 1 will be described below
The temperature adjustment unit 10 includes a heating unit 11 that heats a fluidic device 100, and a cooling unit 12 that cools the heated fluidic device 100.
In the heating unit 11 in
Instead of the above configuration, for example, the heating unit 11 may be formed to have a cover to form an internal space for housing an object (i.e., fluidic device 100) and heat the internal space to indirectly heat the object.
The heating unit 11 is controlled to be 30° C. or higher and 99° C. or lower by the control unit 40.
The cooling unit 12 in
The cooling unit 12 is controlled to be 0.1° C. or higher and lower than 30° C. by the control unit 40.
Alternatively, the cooling unit 12 may be formed such that the upper surface is a heat exchanger plate made of a metal having high thermal conductivity and a water-cooled or air-cooled heat dissipation unit is provided on the lower surface. Alternatively, the cooling unit 12 may be simply a metal plate having high thermal conductivity.
The temperature adjustment unit 10 is provided on a mounting portion 50 bridging over the base 90 (in the +z direction with respect to the base 90). The mounting portion 50 has a stage 51 that has a rectangular shape in plan view and extends in the x direction, and a pair of bridge piers 52 that are provided on both ends of the stage 51 in the longitudinal direction. In the temperature adjustment unit 10, the heating unit 11 and the cooling unit 12 are positioned in this order in the +x direction from one end 51a of the stage 51.
At a position on the stage 51 further to the −x side than the heating unit 11 is, a standby unit 55 in which a conveying stage (described later) of the conveying unit 30 stands by may be provided.
The imaging unit 20 is provided at a position on the stage 51 further to the +x side than the cooling unit 12 is. The imaging unit 20 irradiates a fluidic device 100 with excitation light, and captures an image of fluorescence emitted from the fluidic device 100.
The light source unit 21 emits excitation light EL. The light source unit 21 includes a light source 211 that emits light L1 including excitation light EL, and an optical filter 212 that transmits part of the light L1 having a wavelength that is used as excitation light EL.
The light source 211 may be, for example, a mercury lamp. Alternatively, the light source 211 may be a LED light source or LD light source. The following will describe a case in which the light source 211 is a mercury lamp that is a white light source and the light L1 is white light.
The optical filter 212 transmits part (i.e., excitation light EL) of the light L1 as white light that has a wavelength required to excite a product (i.e., fluorescent material) generated in the fluidic device 100, and extracts the excitation light EL. The optical filter 212 is a bandpass filter having the property of transmitting excitation light EL and blocking light other than the excitation light EL.
Thus, the light source unit 21 can extract excitation light EL from the light L1 emitted from the light source 211, and emit the excitation light EL.
The light source unit 21 may include, at a position between the light source 211 and the optical filter 212 in the optical path, an external light introduction unit 215 that introduces external light L2 different from the light L1 emitted from the light source 211 and guides the external light L2 to the fluidic device 100. The external light introduction unit 215 includes a reflecting mirror 215a that can be freely placed on and removed from the optical path, and an introduction port 215b from which external light L2 is introduced.
In the light source unit 21, when light L1 emitted from the light source 211 is used, the reflecting mirror 215a of the external light introduction unit 215 is removed from the optical path to allow the light L1 to pass through. When external light L2 is used, the reflecting mirror 215a of the external light introduction unit 215 is placed in the optical path to block the light L1 and guide the external light L2 introduced from the introduction port 215b toward the optical filter 212.
The excitation light EL emitted from the light source unit 21 is incident on the dichroic mirror 22.
The dichroic mirror 22 has the property of reflecting excitation light EL and transmitting fluorescence FL. The dichroic mirror 22 is disposed at an angle of 45° (i.e., an incident angle of) 45° with respect to the incident direction of excitation light EL, and reflects excitation light EL to the direction with an angle of 90° with respect to the incident direction of the excitation light EL.
The excitation light EL reflected from the dichroic mirror 22 is incident on the projection lens 23, and is projected onto the fluidic device 100. In the fluidic device 100, fluorescence FL is emitted from a fluorescent material excited by the excitation light EL. The fluorescence FL is incident on the dichroic mirror 22 via the projection lens 23. The dichroic mirror 22 transmits the fluorescence FL. On the other hand, even when part of the excitation light EL is reflected from the fluidic device 100 and returned 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 from each other.
The fluorescence FL transmitted through the dichroic mirror 22 is incident on the absorption filter 26. The absorption filter 26 may be a dichroic mirror that transmits fluorescence FL and does not transmit light having a different wavelength from the fluorescence FL. When the absorption filter 26 is, for example, a dichroic mirror that has a higher wavelength resolution than the dichroic mirror 22 and that transmits light having a long wavelength including fluorescence FL and blocks (and reflects) light having a short wavelength including excitation light EL, it is possible to remove stray light and achieve detection with high accuracy.
The fluorescence FL transmitted through the absorption filter 26 is incident on the image forming lens 24 via the reflecting mirror 28, and forms an image.
The imaging measure 25 captures the image formed by the image forming lens 24. The imaging measure 25 may be a digital camera including an imaging element.
In the imaging unit 20 described above, the projection lens 23 that projects the excitation light EL emitted from the light source unit 21 onto the fluidic device 100 also serves as an objective lens that collects fluorescence FL generated by the fluidic device 100 and guides the fluorescence FL to the image forming lens 24. The projection lens 23 (also serving as an objective lens) may have an autofocus mechanism.
Furthermore, in the imaging unit 20, the projection lens 23 and the image forming lens 24 preferably form a confocal optical system. In the imaging unit 20, the spatial filter 27 having a pinhole P is provided between the image forming lens 24 and the imaging measure 25, and a confocus FP of the confocal optical system is located between the image forming lens 24 and the imaging measure 25. The other confocus of the confocal optical system overlaps with the fluidic device 100. The spatial filter 27 is disposed so that the pinhole P spatially overlaps with the confocus FP. This enables the imaging unit 20 to remove stray light and achieve detection with high accuracy. In order to allow the confocal optical system to function, the confocal optical system may be composed of a known member appropriately selected.
As shown in
The light source unit 21 may be configured such that switching can be performed between a first optical filter 212a that transmits part of the light L1 having a first wavelength that is used as excitation light EL and a second optical filter 212b that transmits part of the light L1 having a second wavelength that is used as excitation light EL.
Specifically, the light source unit 21 includes filter blocks 291 and 292 and a switching unit 295. The filter block 291 includes the first optical filter 212a. The filter block 292 includes the second optical filter 212b. The switching unit 295 performs switching between the filter block 291 and the filter block 292. The switching unit 295 includes a turntable 296 and a driving unit 297. The turntable 296 is provided with the filter blocks, and the driving unit 297 drives the turntable 296 in the circumferential direction. The filter blocks 29 and the switching unit 295 are supported by a support 299 provided on the base 90.
The switching unit 295 can switch the filter block 292 provided on the turntable 296 by causing the driving unit 297 to move the turntable 296 in the circumferential direction.
As shown in
The upper surface of the stage 323 is parallel to the xy plane, and a fluidic device 100 to be observed is placed on the stage 323. The stage 323 may have various configurations as long as a fluidic device 100 can be placed on the stage 323 and the fluidic device 100 can be observed from below (i.e., −z direction). The stage 323 may be, for example, a plate-shaped member that allows light to be transmitted, or a frame with a void in which a fluidic device 100 is observed. The stage 323 may be a holding member that holds a fluidic device 100 from the sides. In
The illumination unit 325 irradiates a fluidic device 100 placed on the stage 323 with white light to allow image capturing in a bright field.
The conveying unit 30 can move a fluidic device 100 placed on the stage 323 in the x-axis direction and the z-axis direction.
The control unit 40 shown in
The liquid sample is an aqueous solution containing a target substance, and contains, for example, a biological sample or an environmental sample. The biological sample is not specifically limited, and may be serum, plasma, urine, cell culture medium, or the like. For example, a PCR reaction solution that is obtained using a biological sample as a template and contains a dyeing reagent as a detection reagent may be used. Furthermore, the environmental sample may be, for example, river water, factory wastewater, or the like.
The target substance may be, for example, DNA, RNA, a protein, virus, cell, exosome, or the like. Examples of the RNA include miRNA and mRNA. Examples of the cell include a bacterium, a yeast, an animal cell, a plant cell, and an insect cell. In the fluidic device 100, a target substance contained in the sample as described above is reacted with a detection reagent to detect the target substance.
As shown in
The well plate 101 is a plate-shaped member having a rectangular shape or a narrow rectangular shape in plan view. Multiple wells (also referred to as microwells) 110 are provided in a center portion of an upper surface 101a of the well plate 101 in the longitudinal direction of the well plate 101.
The microwells 110 are recesses that are provided on the upper surface 101a of the well plate 101, and are open to the upper surface 101a. The microwells 110 each refer to a space surrounded by the recess and a virtual plane that is parallel to the upper surface 101a and is in contact with the upper surface 101a.
The microwells 110 house a sample housed in the internal space S, and function as a reaction site of a target substance contained in the sample and a detection reagent.
The material of the well plate 101 has electromagnetic wave transmission properties. An electromagnetic wave used to determine whether the material has electromagnetic wave transmission properties may be an X-ray, ultraviolet light, visible light, infrared radiation, or the like. When the well plate 101 has electromagnetic wave transmission properties, an electromagnetic wave can be used to analyze the results of an experiment conducted in the fluidic device 100 including the well plate 101. For example, fluorescence, phosphorescence, or the like generated by irradiation with an electromagnetic wave can be measured from the well plate 101 side.
Although the details will be described later, for example, when in the microwells 110, fluorescence having a peak in a wavelength range of 400 to 700 nm, which is the visible light range, is generated and used for sample detection, the well plate 101 may be made of a material having good transmission properties for at least light in the visible light range.
The material having electromagnetic wave transmission properties may be, for example, glass, resin, or the like. Examples of the resin include ABS resin, polycarbonate, cycloolefin copolymer (COC), cycloolefin polymer (COP), acrylic resin, polyvinyl chloride, polystyrene, polyethylene, polypropylene, polyvinyl acetate, polybutylene terephthalate (PET), and polyethylene naphthalate (PEN). These resins may contain various additives, and may be a polymer alloy in which multiple resins are mixed.
The material having electromagnetic wave transmission properties preferably has substantially no autofluorescence. The material having substantially no autofluorescence refers to a material having no autofluorescence having a wavelength used for sample detection, or a material having weak autofluorescence that does not affect sample detection. The intensity of the weak autofluorescence that does not affect sample detection may be, for example, approximately ½ or less the intensity of the fluorescence to be detected, and more preferably approximately 1/10 or less the intensity of the fluorescence to be detected. The well plate 101 made of such a material can achieve higher detection sensitivity in sample detection using an electromagnetic wave.
The material that has electromagnetic wave transmission properties and has no autofluorescence may be, for example, quartz glass. The material that has weak autofluorescence and does not interfere with sample detection using an electromagnetic wave may be low fluorescent glass, acrylic resin, COC, COP, or the like.
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 may be, for example, 5 mm or less, 2 mm or less, or 1.6 mm or less.
The well plate 101 may be a single layer made of only the above material, or may be a laminate made of multiple materials. When the well plate 101 is formed by processing a laminate, a first layer of the well plate 101 that has the microwells 110 and a second layer of the well plate 101 that supports the first layer may be made of different materials.
The microwells 110 may have various shapes. Examples of the shape of the microwells 110 include cylindrical shapes such as a cylindrical shape, an elliptical cylindrical shape, and a polygonal cylindrical shape, shapes such as a conical shape and a pyramidal shape, and frustum shapes such as a conical frustum shape and a pyramidal frustum shape. When the microwells 110 have a shape such as a conical or pyramidal shape, or a frustum shape, the opening size of the microwells 110 is preferably gradually reduced in the depth direction of the wells.
The microwells 110 may have a flat bottom, or may have a curved bottom with a convex or concave surface.
When the microwells 110 have a cylindrical shape, the maximum diameter of the microwells 110 in plan view is preferably, for example, 10 nm to 100 μm, more preferably 100 nm to 50 μm, and even more preferably 1 μm to 20 μm. Furthermore, the depth of the microwells 110 is preferably, for example, 10 nm to 100 μm, more preferably 100 nm to 50 μm, and even more preferably 1 μm to 20 μm.
The volume of the microwells 110 is preferably, for example, 1 fL to 6 nL, more preferably 1 fL to 5 pL, even more preferably 1 fL to 2 pL, and particularly preferably 1 fL to 300 fL. When the volume of each of the microwells 110 is in the above range, an enzymatic reaction in a minute space such as digital PCR or Invader reaction can be suitably performed. Digital PCR can detect, for example, a gene mutation or the like.
The well plate 101 has microwells 110 having the same shape and size. The microwells 110 having the same shape and size may have the same shape and volume to the extent required for digital measurement, and variation within the production error range is allowed.
The density of the microwells 110 is, for example, 100,000 to 10,000,000 microwells/cm2, preferably 100,000 to 5,000,000 microwells/cm2, and even more preferably 100,000 to 1,000,000 microwells/cm2. When the density of the microwells 110 is in the above range, it is easy to perform an operation in which a sample is enclosed in a predetermined number of microwells 110. Furthermore, observation of the wells to analyze the experimental results is easily performed.
For example, when the fluidic device 100 is used to measure cell-free DNA mutation, and the ratio of mutant cell-free DNA to be detected to wild-type cell-free DNA is approximately 0.01%, for example, the use of 1,000,000 to 2,000,000 microwells 110 is preferable.
The microwells 110 are regularly formed in a matrix. The state in which the microwells 110 are “regularly formed” refers to a state in which the centroids of the openings of the microwells 110 are formed in a certain pattern.
The centroids of the openings of the microwells 110 may be formed, for example, in a quadrilateral lattice. In such a case, a line connecting the centroids of the openings of four adjacent microwells 110 forms a rectangle, preferably, a square.
Alternatively, the centroids of the openings of the microwells 110 may be formed in a triangular lattice (hexagonal lattice). In such a case, a line connecting the centroids of the openings of three adjacent microwells 110 forms an equilateral triangle.
Of the microwells 110, a well (first well) A and a well (second well) B closest to the well A preferably satisfy Formula (1).
0.8≤Da/Dab<1 (1)
(Da is the equivalent circle diameter of the opening of the well A, and Dab is the distance between the centroid of the opening of the well A and the centroid of the opening of the well B.)
In the well plate 101, the lower limit of Da/Dab is 0.8, and may be 0.83 or more. Furthermore, the upper limit of Da/Dab is less than 1, and may be 0.92 or less, or may be approximately 0.9. These lower and upper limits can be used in any combination.
When the microwells 110 are formed in a triangular lattice and Formula (1) is satisfied, the distance between the centroid of the opening of a first microwell 110 and the centroid of the opening of a second microwell 110 closest to the first microwell 110 is uniform.
The microwells 110 shown in
In
In the well plate 101, the equivalent circle diameter of the openings of the microwells 110 is preferably 1 μm or more and 50 μm or less. That is, the lower limit of the equivalent circle diameter of the openings of the microwells 110 is preferably 1 μm. Furthermore, the upper limit of the equivalent circle diameter of the openings of the microwells 110 may be less than 20 μm, 19 μm or less, 18 μm or less, 17 μm or less, 16 μm or less, 15 μm or less, 14 μm or less, 13 μm or less, 12 μm or less, 11 μm or less, or 10 μm or less.
The upper and lower limits of the equivalent circle diameter of the openings of the microwells 110 can be used in any combination.
The cover member 102 has the same contour (i.e., narrow rectangular shape) as the well plate 101 in plan view. The cover member 102 is disposed to face the upper surface 101a of the well plate 101 with a gap between the upper surface 101a and the cover member 102.
The cover member 102 has two through holes passing through the cover member 102 in the thickness direction. The two through holes are provided on the respective end sides of the cover member 102 in the longitudinal direction. One of the through holes is an injection opening 121 used to inject a liquid into the internal space S of the fluidic device 100, and the other through hole is a discharge opening 122 used to discharge the liquid from the internal space S.
Other than a liquid sample, a detection reagent and a sealing liquid also correspond to the “liquid”.
The injection opening 121, the internal space S, and the discharge opening 122 communicate with each other in this order, and as a whole, form a channel FC. In the fluidic device 100, a liquid is caused to appropriately flow in the channel FC to perform a detection reaction for a target substance. The microwells 110 are disposed between the injection opening 121 and the discharge opening 122 in plan view.
An injection port 125 that has a cylindrical shape and surrounds the injection opening 121 is provided on an upper surface 102a of the cover member 102. The injection port 125 communicates with the injection opening 121. For example, when a liquid is filled in the internal space using a syringe filled with the liquid, the injection port 125 is used to connect the syringe.
Similarly, a discharge port 126 that has a cylindrical shape and surrounds the discharge opening 122 is provided on the upper surface 102a of the cover member 102. The discharge port 126 communicates with the discharge opening 122. For example, when the liquid is extracted from the internal space S, the discharge port 126 is used to connect a tube through which the liquid flows.
The material of the cover member 102 may be any of the materials described as examples of the material of the well plate 101. The material of the cover member 102 may be the same as or different from the material of the well plate 101.
The material of the cover member 102 may or may not have electromagnetic wave transmission properties.
The material of the cover member 102 is preferably hydrophobic. Specifically, the material of the surface (i.e., lower surface 102b) of the cover member 102 facing the internal space S preferably has a contact angle of 5° or more and 80° or less with a sealing liquid SL. When the cover member 102 is made of such a material, the lower surface 102b has a contact angle of 5° or more and 80° or less with the sealing liquid SL. The lower surface 102b having a contact angle in the above range tends to allow a sample to be easily isolated in the microwells 110 when a sealing liquid is introduced into the internal space S by a method described later.
The wall member 103 has a closed ring shape in plan view, and is provided along the outer edge of the upper surface 101a of the well plate 101. In
The wall member 103 is sandwiched between the well plate 101 and the cover member 102 and integrated with the well plate 101 and the cover member 102 to form the fluidic device 100. A space surrounded by the well plate 101, the cover member 102, and the wall member 103 is the internal space S in which a liquid sample is housed. The internal space S extends along the well plate 101 having a narrow rectangular shape in the longitudinal direction of the well plate 101.
The wall member 103 functions as a wall surface of the internal space S, and also functions as a spacer between the well 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.
The material of the wall member 103 is not specifically limited, and may be preferably, for example, a double-sided adhesive tape in which an acrylic adhesive is laminated on both surfaces of a core film. Examples of the material of the core film include silicone rubber and acrylic foam. The wall member 103 made of such a material enables the internal space S to be a liquid-tight space.
The material of the wall member 103 may be the same as the material of the well plate 101 described above. The wall member 103 made of such a material can be integrated with the well plate 101 and the cover member 102 by adhesion using an adhesive or welding such as thermal welding, ultrasonic welding, or laser welding.
The wall member 103 may be integrated with the well plate 101 and constitute part of the well plate 101. Similarly, the wall member 103 may be integrated with the cover member 102 and constitute part of the cover member 102.
The well plate 101 can be produced using injection molding, microimprinting technique, or nanoimprinting technique. Alternatively, the well plate 101 can be produced using a photolithography technique to form the microwells 110 by etching.
The cover member 102 and the wall member 103 can be produced by injection molding.
A detection reagent is reacted with a target substance and used to detect the target substance. Examples of the detection reagent include a buffer substance, an enzyme, a substrate, an antibody, and an antibody fragment.
For example, when the target substance is a nucleic acid, in order to perform a biochemical reaction such as an enzymatic reaction of a template nucleic acid associated with the target substance, an enzyme is selected according to the content of the biochemical reaction. The biochemical reaction of a template nucleic acid is, for example, a reaction in which signal amplification occurs under conditions in which a template nucleic acid is present.
The detection reagent is selected according to the detection reaction used. Specific examples of the detection reaction include an ICA method, a loop-mediated isothermal amplification (LAMP) method (registered trademark), a 5′ to 3′ nuclease method (TaqMan (registered trademark) method), and a fluorescent probe method.
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 types of detection reagent and detection reaction used.
The detection reagent may contain an adsorption inhibitor. Examples of the adsorption inhibitor include a surfactant and a protein.
In order to facilitate detection of a target substance, the sample may be pretreated prior to preparation of the mixed aqueous solution. Examples of the pretreatment include concentration adjustment (i.e., dilution or concentration), support by a carrier, and a binding reaction of two or more types of target substances.
Then, as shown in
After the internal space S and all the microwells 110 are filled with the mixed aqueous solution L, the mixed aqueous solution L passing through the channel FC is discharged from the discharge opening 122.
At this time, the concentration of the mixed aqueous solution Lis preferably adjusted in advance so that a single target substance is filled in a single microwell 110. For example, when after a sample detection method (described later) is performed, the results show that quantification of the target substance is difficult due to high concentration of the mixed aqueous solution L, the results are used as preliminary experiment results, and the mixed aqueous solution Lis diluted.
By performing the above operation, one or less target substance, that is, zero or one target substance, is filled in a single microwell 110. Thus, the number of microwells 110 in which a detection reaction (described later) is observed corresponds to the number of target substances, allowing target substance detection on a single-unit basis, that is, digital measurement. A target substance may not necessarily be introduced into all the microwells 110.
The measure for introducing a target substance into the microwells 110 is not specifically limited, and an appropriate measure can be selected according to the target substance to be detected. For example, target substances may be allowed to settle by their own weight in the fluidic device 100 (specifically, in the channel FC), and distributed to the microwells.
A substance (also referred to as a capture substance) that captures a target substance may be used and bound to a target substance which does not easily settle due to its own weight, to supply the target substance. Alternatively, a capture substance may be immobilized in the microwells in advance and used to capture a target substance supplied together with the mixed aqueous solution L to achieve higher efficiency of introducing the target substance into the microwells 110.
The reaction in which a capture substance and a target substance are bound together can be performed at any time point. For example, before preparation of the mixed aqueous solution, a target substance and a capture substance may be brought into contact with each other in a sample tube to perform the reaction.
Alternatively, after introduction of a capture substance into the microwells 110, a target substance may be introduced into the microwells 110 and brought into contact with the capture substance in the microwells 110.
A capture substance is a substance capable of capturing a target substance. The capture substance may be, for example, a bound body of a solid phase and a specific binding substance specifically binding to a target substance.
The solid phase constituting the capture substance may be particles, a film, a substrate, or the like. The capture substance may contain a single type or two or more types of specific binding substances to the target substance. The capture substance may contain, for example, three, four, five, or more types of specific binding substances.
The particles are not specifically limited, and may be polymer particles, magnetic particles, glass particles, or the like. In order to avoid nonspecific adsorption, the particles are preferably surface treated. Furthermore, in order to immobilize the specific binding substance, the particles preferably have a functional group such as a carboxyl group on the surface. More specifically, a product “Magnosphere LC300” manufactured by JSR Corporation or the like can be used.
For example, when a virus is used as a target substance, a cell to which the virus can attach (i.e., a cell having a virus receptor) may be used as a capture substance.
Then, as shown in
Thus, the microwells 110 housing the mixed aqueous solution L containing a target substance are individually sealed with the sealing liquid SL to be independent reaction spaces. When the channel FC is filled with the sealing liquid SL, excess sealing liquid SL is discharged from the discharge opening 122.
Then, the fluidic device 100 is heated to cause the target substance and the detection reagent to react with each other. A target substance may be detected using any detection method according to the characteristics of the target substance to be detected. A target substance can be detected by first performing a reaction (also referred to as a signal amplification reaction) in which a signal derived from the target substance is amplified to a detectable level, and then detecting the amplified signal using an appropriate measure.
The signal amplification reaction performed in the detection device 1 may be an isothermal signal amplification reaction, and may be an ICA reaction, a loop-mediated isothermal amplification (LAMP) method (registered trademark), a 5′ to 3′ nuclease method (TaqMan (registered trademark) method), or a fluorescent probe method. In an ICA reaction, signal amplification proceeds in two reaction cycles: (1) complementary binding of nucleic acids and (2) recognition and cleavage of a triple-stranded structure by an enzyme. In such a signal amplification reaction, the influence of inhibition of the reaction cycle by impurities other than the target substance is small. Thus, even when various components (e.g., impurities) other than the target substance are present in the microwells 110, the target substance can be accurately detected using an ICA reaction.
The mixed aqueous solution L contains a reaction reagent and a template nucleic acid required for the ICA reaction. When a target substance is present in a well, by an enzymatic reaction due to an isothermal reaction, a fluorescent material is released from a quencher, and emits a predetermined fluorescence signal corresponding to excitation light.
In the ICA reaction, the reaction temperature of the target substance and the detection reagent is preferably 50° C. or higher and 99° C. or lower. The specific reaction temperature is preferably determined by performing a preliminary experiment according to the reagent used and the sensitivity of the results required. The reaction temperature is preferably optimized by performing an ICA reaction as a preliminary experiment after determination of the composition of the reagent, and using, for example, the results such as a signal, a noise, a signal-to-noise ratio (also referred to as an S/N ratio), and a signal-to-noise difference after a certain time.
The ICA reaction will be described in more detail below.
Examples of the reaction reagent required for the ICA reaction include ICA reaction reagents such as a flap probe 810, a flap endonuclease FEN, a fluorescent substrate 820, and an invasive probe (Invader oligo) 830.
The flap probe 810 and the invasive probe 830 are each a nucleic acid fragment (also referred to as oligonucleotide) designed to be hybridized with DNA as a target substance (also referred to as target DNA) to form a flap structure with a double-stranded nucleic acid 140.
The fluorescent substrate 820 is a nucleic acid fragment that has a hairpin structure and in which a fluorescent material F and a quencher Q are bound together. In the fluorescent substrate 820 shown in
First, the flap probe 810 and the invasive probe 830 are hybridized with the target DNA. The flap probe 810 and the invasive probe 830 overlap by a single base at a SNP site of the target DNA to form an unstable tribasic structure. Thus, a first flap 811 is formed. The first flap 811 is a portion of the flap probe 810 that is not hybridized with the target DNA.
Then, FEN recognizes and reacts with the tribasic structure. Thus, the first flap 811 is cleaved to generate a nucleic acid fragment 811, and the nucleic acid fragment 811 is released in the mixed aqueous solution L.
The generated nucleic acid fragment 811 is hybridized with the fluorescent substrate 820. The nucleic acid fragment 811 enters the hairpin structure of the fluorescent substrate 820, and overlaps with the fluorescent substrate 820 by a single base at a SNP site to form an unstable tribasic structure. Thus, a second flap 821 is formed. The second flap 821 is a portion of the fluorescent substrate 820 that is not hybridized due to the entry of the nucleic acid fragment 811.
Then, FEN recognizes and reacts with the tribasic structure. Thus, the second flap 821 is cleaved to generate a nucleic acid fragment 821. In
Thus, the fluorescent material F is separated from the quencher Q, and generates fluorescence FL. The target DNA can be detected by detecting the fluorescence FL.
A target substance can also be detected by binding, to the target substance, a substance (also referred to as a specific binding substance) specifically bound to the target substance, and detecting the specific binding substance bound to the target substance. For example, when the target substance is a protein, the target substance can be detected using ELISA. More specifically, the target substance may also be detected, for example, by sandwich ELISA using the principle of FRET.
When the sandwich method using the principle of FRET is performed, first, a first specific binding substance (e.g., antibody) and a second specific binding substance are prepared. The first specific binding substance is labeled with a first fluorescent material (also referred to as a donor), and the second specific binding substance is labeled with a second fluorescent material (also referred to as an acceptor) having a light absorption wavelength that overlaps with the fluorescence wavelength of the first fluorescent material.
Then, a 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 composite. When the composite is formed, the donor and the acceptor are located closer to each other; thus, the fluorescence wavelength of the acceptor can be detected by irradiation with light having the excitation wavelength of the donor. Alternatively, a specific binding substance may be labeled with a nucleic acid fragment, and the nucleic acid fragment may be detected by an ICA reaction.
A specific binding substance similar to specific binding molecules for a structure (described later), for example, an antibody, an antibody fragment, an aptamer, or the like, can be used. In order to detect a specific binding substance bound to the target substance, the specific binding substance may be directly or indirectly labeled, for example, with an enzyme such as horseradish peroxidase (HRP). When two or more specific binding substances are used, specific binding molecules of each of the specific binding substances can be labeled in an identifiable manner.
A signal can be observed using an appropriate method selected according to the type of signal to be observed. For example, when bright field observation is performed, a substrate provided with a well array is irradiated with white light in the vertical direction. When a fluorescence signal is observed, the well is irradiated with excitation light corresponding to a fluorescent material from the bottom side of the well, and fluorescence emitted from the fluorescent material is observed. An image of the entire or part of the well array observed is captured and stored, followed by image processing using a computer system. The irradiation directions for light (i.e., white light and excitation light) in bright field observation and fluorescence signal observation described above are examples, and can be modified accordingly as long as intended observation can be performed. For example, in the fluorescence signal observation, the substrate may be irradiated with excitation light in the vertical direction as with the irradiation direction with white light in bright field observation.
First, as shown in
In this case, the control unit 40 preferably performs heating in advance (also referred to as preheating) of the heating unit 11 to a temperature higher than room temperature and lower than or equal to a reaction temperature set in advance. As described above, in an ICA reaction, the reaction temperature of the target substance and the detection reagent is preferably, for example, 50° C. or higher and 99° C. or lower. Under the above temperature conditions, the preheating temperature is preferably higher than room temperature and, for example, 90° C. or lower. “Room temperature” is, for example, 25° C.
Then, as shown in
As described above, preheating of the heating unit 11 prior to heating of the fluidic device allows quick heating of the fluidic device 100, and thus is preferable. In the case where the heating unit 11 is preheated, when the conveying unit 30 starts conveying the fluidic device 100 from the standby unit 55 to the heating unit 11, the control unit 40 preferably changes the heating temperature of the heating unit 11 from the set preheating temperature to the reaction temperature for the ICA reaction. Alternatively, while the conveying unit 30 is conveying the fluidic device 100 from the standby unit 55 to the heating unit 11, the control unit 40 preferably changes the heating temperature of the heating unit 11 from the set preheating temperature to the reaction temperature for the ICA reaction.
In this case, the control unit 40 preferably performs cooling in advance (precooling) of the cooling unit 12 to a temperature higher than or equal to a cooling temperature set in advance and lower than the reaction temperature in the heating unit 11. For example, when the reaction temperature for the ICA reaction is 60° C., the precooling temperature is preferably, for example, 0° C. or higher and lower than room temperature.
Then, as shown in
As described above, precooling of the cooling unit 12 prior to cooling of the fluidic device allows quick cooling of the fluidic device 100, and thus is preferable. In the case where the cooling unit 12 is precooled, when the conveying unit 30 starts conveying the fluidic device 100 from the heating unit 11 to the cooling unit 12, the control unit 40 preferably changes the cooling temperature of the cooling unit 12 from the set precooling temperature to the actual cooling temperature. Alternatively, while the conveying unit 30 is conveying the fluidic device 100 from the heating unit 11 to the cooling unit 12, the control unit 40 preferably changes the cooling temperature of the cooling unit 12 from the set precooling temperature to the actual cooling temperature.
Then, as shown in
Then, the captured image of the fluorescence is analyzed to measure the number of wells emitting fluorescence and quantify the target substance.
The detection device 1 described above provides the following effects.
When a detection reagent is continuously heated, a product that emits fluorescence is generated by a side reaction even if no target substance is present. In an image captured by the imaging unit 20, the product generated in this manner is indistinguishable from fluorescence derived from the target substance, and this may cause an error in detection of the target substance. Furthermore, in the fluidic device 100, the ICA reaction also proceeds during heating and cooling of the fluidic device 100. Thus, if time is required for heating and cooling of the fluidic device 100, the intensities of both the emission of signal light to be detected and the emission of noise light are assumed to be high, leading to low detection sensitivity.
On the other hand, in the detection device 1, after heating by the heating unit 11, the fluidic device 100 is cooled by the cooling unit 12 provided separately from the heating unit 11. Thus, the reaction of the target substance and the detection reagent accelerated by heating can be deactivated to prevent a side reaction. Therefore, the detection device 1 enables detection with high accuracy.
In a configuration in which the cooling unit is provided at the same position as the heating unit 11 so that both heating and cooling can be performed at the same position, when cooling is started after heating, together with the fluidic device 100, the heating unit 11 heated to the reaction temperature for a detection reaction such as an ICA reaction needs to be cooled. In such a configuration, the fluidic device 100 can be cooled, but additional time is required to cool the heating unit 11.
As described above, the fluidic device 100 is made of glass or resin. Furthermore, the fluidic device 100 stores liquids such as a liquid sample and a reagent. Furthermore, in order to increase the volume of the fluidic device 100, changes are assumed to be made, such as attachment of an additional member to the device or an increase in size of the device. All these factors lead to high specific heat of the fluidic device 100, interfering with quick cooling of the fluidic device 100.
On the other hand, in the detection device 1, the heating unit 11 and the cooling unit 12 are provided separately from each other; thus, after heating of the fluidic device 100, the fluidic device 100 can be quickly cooled as compared with the device configuration in which the cooling unit is provided at the same position as the heating unit 11. Furthermore, the detection device 1 in which the heating unit 11 and the cooling unit 12 are provided separately from each other allows preheating and precooling as described above. Thus, the detection device 1 enables detection in a short time, leading to higher work efficiency.
The detection device 1 having the above configuration provides a detection device enabling many detections based on the principle of digital ICA in a short time while ensuring high detection accuracy.
In the detection device 1 of the present embodiment, the member for cooling an object is provided at the position of the cooling unit 12; however, the present invention is not limited to this configuration. The cooling device may be configured such that cooling of the fluidic device 100 is completed before the fluidic device 100 heated by the heating unit 11 is conveyed to an imaging position of the imaging unit 20 and observation is started.
For example, the fluidic device 100 may be cooled by blowing air while the fluidic device 100 is conveyed from the heating unit 11 to the imaging position. In such a case, a measure for blowing air in the conveying path from the heating unit 11 to the imaging position corresponds to the cooling unit.
The fluidic device 100 may be cooled at the imaging position. The fluidic device 100 may be cooled by a measure having a known configuration. In such a case, the measure for cooling the fluidic device 100 at the imaging position corresponds to the cooling unit. In such a configuration, observation (image capturing) may be performed while the fluidic device 100 is cooled, or may be performed after the fluidic device 100 is cooled.
In the present embodiment, when the projection lens 23 (also serving as an objective lens) has an autofocus mechanism, prior to image capturing, the control unit 40 may control the autofocus mechanism to set the focus position of the objective lens to a microwell 110. The control performed by the control unit 40 allows the detection device 1 to accurately capture an image, achieving detection with high accuracy. Furthermore, by using a microwell 110 as an alignment mark and setting in advance the focus position to the microwell 110, even when warpage or distortion of the entire fluidic device 100 occurs, the focus position can be quickly readjusted.
When the imaging unit 20 has no autofocus mechanism, the focus position of the objective lens may be set to a microwell 110 in a different manner. For example, an alignment mark may be provided in the fluidic device 100, and in the detection device 1, after the focus is set to a microwell 110 using the alignment mark, an image of another microwell 110 may be captured. The alignment mark is preferably provided at the same focus position as that of the microwell 110.
Even when the focus position of an alignment mark is different from the focus position of a microwell 110 in the z-axis direction, the offset between the focus positions may be measured and corrected to set the focus position to the microwell 110 and capture an image of the microwell 110.
For example, when warpage or distortion occurs in the entire fluidic device 100, the focal length may be different for each microwell 110. In such a case, the amount of warpage of the fluidic device 100 may be approximated by measuring, for example, the focal length between one of microwells 110 (i.e., well array) that is located around the array and another one of the microwells 110 that is located in a center portion of the array, and using the correspondence between the obtained focal length and the positions of the microwells 110 for which the focal length has been measured. The obtained amount of warpage is preferably used as a correction value of the offset to set the focus position to the microwell 110 and capture an image of the microwell 110.
In the above method, the amount of warpage of the fluidic device 100 is approximated from the focal length of the microwells 110; however, an alignment mark of the fluidic device 100 may be used as a measurement object for obtaining the amount of warpage.
In such a case, as described above, some (e.g., one to four) of the wells may be used as alignment marks provided in the fluidic device 100, or an alignment mark may be separately provided around the region in which the microwells 110 are provided. Furthermore, an image of the fluidic device 100 may be captured at focus positions, and the obtained images may be used to composite a z-stack image to obtain a composite image in focus over the entire image.
The cover 60 is a box-shaped member that covers the entire components disposed on the stage 51. The cover 60 can be opened and closed by being rotated around a rotation axis that is set to pass through a point Pl and be parallel to the x-axis.
The extrusion units 71 each include a motor 711, a feed screw 712, a guide shaft 713, a nut 714, and a hook 715.
The motor 711 rotates the feed screw 712 via a deceleration gear (not shown).
The feed screw 712 extends in the x direction. Both ends of the feed screw 712 in the x direction are rotatably supported by the attaching portions 72. The motor 711 is driven to rotate the feed screw 712 around the center axis of the feed screw 712.
The guide shaft 713 extends in the x direction at a position in the −z direction with respect to the feed screw 712. The guide shaft 713 is inserted through a through hole (not shown) of the hook 715, and both ends of the guide shaft 713 in the x direction are fixed to the attaching portions 72.
The feed screw 712 is inserted into the nut 714. The hook 715 is coupled to the lower side (−z side) of the nut 714. The shape of the hook 715 is not specifically limited, as long as the hook 715 has an extrusion function (described later).
In the moving measure 70, when the motor 711 is rotated to rotate the feed screw 712, the nut 714 and the hook 715 are moved in the +x direction along the guide shaft 713.
The temperature control holder 15 includes a base portion 151 that has a rectangular shape in plan view, and multiple rails 152 (four rails in
When the temperature control holder 15 is a member separate from the heating unit 11, at least the base portion 151 is preferably made of a metal material having high thermal conductivity, such as aluminum or copper.
The conveying unit 35 includes the conveying body 36. The conveying body 36 includes the first member 321, the second member 322, and a measuring holder 330.
The measuring holder 330 includes a base portion 331 that has a rectangular shape in plan view, multiple rails 332 (four rails in
As with the stage 323 described above, the base portion 331 may have various configurations as long as a fluidic device 100 can be placed on the stage 323 and the fluidic device 100 can be observed from below (i.e., −z direction). The stage 323 may be, for example, a plate-shaped member that allows light to be transmitted, or a frame with a void in which a fluidic device 100 is observed.
The height position in the z direction of the upper surface of the base portion 331 of the measuring holder 330 is substantially the same as the height position in the z direction of the upper surface of the base portion 151 of the temperature control holder 15.
The interval between adjacent rails 332 is equivalent to the width of a fluidic device 100 in the transverse direction, and a groove (second housing portion) 332a into which a fluidic device 100 is inserted is provided between the rails 332. The grooves 332a extend in the conveying direction of the conveying body 36, that is, the x direction. The +x side of the grooves 332a is closed by the wall portion 333.
In
Furthermore, the number of grooves 152a of the temperature control holder 15 and the number of extrusion units 71 of the moving measure 70 shown in
First, as shown in
The conveying body 36 is moved in the −x direction to bring the end portion of the measuring holder 330 on the −x side closer to the end portion of the temperature control holder 15 on the +x side.
When the cover 60 shown in
Then, as shown in
The conveying unit 35 moves the conveying body 36 in the +x direction. In the detection device 2, the cooling unit 12 (see
Then, in the detection device 2, the remaining fluidic device 100B is subjected to the same operation as the fluidic device 100A, and the imaging unit 20 (see
Such a process performed to the fluidic devices 100 allows a detection process in which the fluidic device 100A and the fluidic device 100B are heated for different heating times.
The detection device 2 having the above configuration also provides a detection device enabling many detections based on the principle of digital ICA in a short time while ensuring high detection accuracy.
Embodiments of the present invention have been described with reference to the accompanying drawings; however, the present invention is not limited to the embodiments. The shapes, combinations, and the like of the components described in the above embodiments are examples, and various modifications can be made based on design, specifications, and the like without departing from the spirit of the present invention.
A detection device according to an embodiment of the present invention enables a large number of detections based on the principle of digital ICA in a short time while ensuring high detection accuracy.
In a DNA microarray technique, in some cases, biomolecules are introduced into micropores, and a reaction involving heating is performed to detect biomolecules. Techniques for single-molecule detection of biomolecules that can be used in that case include a digital measurement technique such as digital invasive cleavage assay (ICA).
In the method described in WO 2015/115635 A, an aqueous medium containing a target substance is supplied to a channel of a fluidic device and enclosed in wells provided on a wall surface of the channel. Furthermore, the fluidic device is heated to heat a reaction solution to cause a detection reaction. Thus, a specific fluorescence emission is caused which allows detection of the target substance.
This technical field requires detection devices capable of performing a large number of detections in a short time. On the other hand, digital ICA has heating in a detection reaction and requires prevention of a side reaction caused by excessive heating to achieve higher detection accuracy.
A detection device according to an embodiment of the present invention enables a large number of detections to be performed based on the principle of digital ICA in a short time while ensuring high detection accuracy.
A detection device according to an embodiment of the present invention includes a temperature adjustment unit that adjusts a temperature of a fluidic device, an imaging unit that irradiates the fluidic device with excitation light and captures an image of fluorescence emitted from the fluidic device, and a conveying unit that conveys the fluidic device to the temperature adjustment unit and the imaging unit. The fluidic device houses a mixed aqueous solution of a liquid sample that contains a target substance and a detection reagent that reacts with the target substance to generate a product that emits the fluorescence, and the temperature adjustment unit includes a heating unit that heats the fluidic device and a cooling unit that cools the fluidic device heated.
The detection device may include a control unit that controls operation of the temperature adjustment unit. The control unit controls the temperature of the heating unit to be 50° C. or higher and 99° C. or lower.
In the detection device, prior to heating of the fluidic device, the control unit may perform preheating in which the heating unit is heated to a temperature higher than room temperature and lower than or equal to a reaction temperature set in advance.
The detection device may include a control unit that controls operation of the temperature adjustment unit. The control unit controls the temperature of the cooling unit to be 0.1° C. or higher and lower than 30° C.
In the detection device, prior to cooling of the fluidic device, the control unit performs precooling in which the cooling unit is cooled to a temperature higher than or equal to a cooling temperature set in advance and lower than room temperature.
In the detection device, the imaging unit includes 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 from the dichroic mirror onto the fluidic device, an image forming lens that forms an image of the fluorescence transmitted through the dichroic mirror, and an imaging measure that captures the image formed by the image forming lens, and the projection lens also serves as an objective lens that collects the fluorescence generated by the fluidic device and guides the fluorescence to the image forming lens.
In the detection device, the light source unit includes a light source that emits light including the excitation light, and an optical filter that transmits part of the light having a wavelength that is used as the excitation light.
In the detection device, the light source unit includes a first optical filter that transmits part of the light having a first wavelength that is used as the excitation light, a second optical filter that transmits part of the light having a second wavelength that is used as the excitation light, and a switching unit that performs switching between the first optical filter and the second optical filter.
In the detection device, the light source unit includes an external light introduction unit that introduces external light and guides the external light to the fluidic device.
In the detection device, the projection lens and the image forming lens form a confocal optical system.
In the detection device, the imaging unit includes a spatial filter that has a pinhole and is provided between the image forming lens and the imaging measure, a confocal position of the confocal optical system is located between the image forming lens and the imaging measure, and the pinhole spatially overlaps with the confocal position.
The detection device may include a control unit. The fluidic device has a microwell that houses the target substance and the detection reagent and is used as a reaction site in which the product is generated, the objective lens has an autofocus mechanism, and prior to image capturing by the imaging measure, the control unit controls the autofocus mechanism to set a focus position of the objective lens to the microwell.
The detection device may further include a moving measure. The heating unit includes a temperature control holder that houses the fluidic devices, the conveying unit includes a conveying holder that houses the fluidic devices, the temperature control holder includes a first housing portion that extends in a conveying direction of the conveying unit and houses the fluidic devices, the conveying holder includes a second housing portion that extends in the conveying direction of the conveying unit and houses the fluidic devices, and the moving measure extrudes and moves fluidic devices housed in the first housing portion toward the second housing portion while the temperature control holder and the conveying holder are close to each other.
A detection device according to an embodiment of the present invention enables a large number of detections based on the principle of digital ICA in a short time while ensuring high detection accuracy.
Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2022-106963 | Jul 2022 | JP | national |
The present application is a continuation of and claims the benefit of priority to International Application No. PCT/JP2023/023317, filed Jun. 23, 2023, which is based upon and claims the benefit of priority to Japanese Application No. 2022-106963, filed Jul. 1, 2022. The entire contents of these applications are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP2023/023317 | Jun 2023 | WO |
| Child | 19002825 | US |
