WO2019131592A1 - Method for suppressing occurrence of false-negative determination in detection of target molecule, and detection device - Google Patents

Method for suppressing occurrence of false-negative determination in detection of target molecule, and detection device Download PDF

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Publication number
WO2019131592A1
WO2019131592A1 PCT/JP2018/047475 JP2018047475W WO2019131592A1 WO 2019131592 A1 WO2019131592 A1 WO 2019131592A1 JP 2018047475 W JP2018047475 W JP 2018047475W WO 2019131592 A1 WO2019131592 A1 WO 2019131592A1
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Prior art keywords
volume
well
target molecule
detection device
sealing
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PCT/JP2018/047475
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French (fr)
Japanese (ja)
Inventor
匠 平瀬
牧野　洋一
雅之 荻野
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凸版印刷株式会社
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Publication of WO2019131592A1 publication Critical patent/WO2019131592A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/11—Filling or emptying of cuvettes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator

Definitions

  • the present invention relates to a method and detection device for suppressing the occurrence of false negative determination in detection of a target molecule.
  • Patent Document 1 Patent Document 2
  • Non-Patent Document 1 describe a technique for performing an enzyme reaction in a large number of minute sections. These techniques are called digital measurement.
  • the sample solution is divided into a large number of micro solutions. Then, the signal from each micro solution is binarized, and only the presence or absence of the target molecule is determined to measure the number of target molecules. According to digital measurement, detection sensitivity and quantitativeness can be remarkably improved as compared with conventional ELISA, real time PCR method and the like.
  • a mixture of PCR reaction reagent and nucleic acid is diluted such that there are no or one template nucleic acid present in one microdroplet.
  • the volume of each microdroplet is preferably small in order to increase the sensitivity of nucleic acid amplification and to perform nucleic acid amplification simultaneously on a large number of microdroplets.
  • Patent Document 3 discloses a microarray-like reaction container formed such that the volume of each well is 6 nL (nanoliter).
  • Patent Document 1 after a sample is made to flow in the flow channel with respect to a microarray in which a large number of wells with a depth of 3 ⁇ m and a diameter of 5 ⁇ m are formed in the flow channel, the sample is introduced into each well.
  • a method is disclosed for introducing a sample into each well by extruding the reagent with oil.
  • an object of this invention is to provide the technique which suppresses generation
  • a method of suppressing the occurrence of false negative determination in detection of a target molecule comprises preparing a device provided with a well array having a plurality of wells, and selecting a plurality of the plurality of wells in the well array.
  • the steps of: introducing a reaction solution containing the target molecule into the wells; contacting the well array with a predetermined volume of sealing solution to individually seal the wells; and the individually sealed wells By setting the reaction solution in the reaction solution to the step of generating a signal in the well when the target molecule is present in the well, and determining whether the signal is generated in the well And, in the detection of the target molecule, the volume of the sealing solution is expressed by the equation (1): 0 ⁇ volume of the sealing solution / total of well volumes of the well array ⁇ 12,000. Meet The sea urchin the sealing liquid is introduced into the device.
  • the sealing solution may be introduced into the device so as to satisfy 1 ⁇ total volume of the sealing solution / well volume of the well array ⁇ 12,000.
  • the reaction condition may be 50 to 80 ° C.
  • the reaction conditions may be maintained for 1 hour or less.
  • the reaction solution may be an Invasive Cleavage Assay reaction reagent.
  • the maximum water content of the sealing solution at 25 ° C. may be 1 to 1000 mass ppm.
  • the target molecule detection device is a well array having a plurality of wells into which a reaction solution containing the target molecule is introduced and a reaction for detecting the target molecule occurs.
  • a lid member provided so as to form an inner space into which a sealing solution for sealing the well array is introduced together with the base material, and the inner space is (1): 0 ⁇ sum of volume of sealing solution / well volume of well array ⁇ 12,000. [8] In the formula (1), the sum of 1 ⁇ volume of the sealing solution / well volume of the well array may satisfy 12,000. [9]
  • the volume of the sealing solution may be equal to the volume of the internal space.
  • the lid may further include an inlet and an outlet, and the volume of the sealing liquid may be equal to the volume of the inlet and the volume of the outlet and the volume of the internal space. .
  • the system further comprises a drainage storage connected to the outlet, wherein the volume of the sealing liquid is the volume of the inlet, the volume of the outlet, the volume of the internal space, and the drainage storage. May be equal to the sum of the volumes of
  • (A) to (d) are representative fluorescence micrographs showing the results of Experimental Example 1. It is a cross-sectional schematic diagram which shows generation
  • a method for suppressing the occurrence of false negative determination in detection of a target molecule prepares a device provided with a well array having a plurality of wells, and uses the plurality of wells in the well array. The steps of: introducing a reaction solution containing the target molecule; contacting the well array with a predetermined volume of sealing solution to individually seal the wells; and individually sealing the wells.
  • the volume of the sealing solution satisfies the following formula (1): 0 ⁇ volume of the sealing solution / well volume of the well array ⁇ 12,000.
  • the sea urchin the sealing liquid is introduced into the device.
  • the false negative means that although a test actually shows a positive reaction, it is detected as negative for some reason.
  • false negative determination may occur, for example, that the signal generation reaction for detection of the target molecule is not normally performed, and the signal generation reaction is normally performed. It is conceivable that the signal can not be detected normally.
  • the signal can not be detected normally, for example, as described in the examples, the case where a signal (for example, fluorescence) is generated only in the peripheral part of the well, that is, near the wall of the well Be
  • a signal for example, fluorescence
  • the method according to this embodiment can suppress the occurrence of false negative determination in the detection of a target molecule.
  • the method according to the present embodiment can be suitably applied to digital measurement.
  • the target molecule detection device includes a substrate provided with a well array having a plurality of wells into which a reaction solution containing the target molecule is introduced and a reaction for detecting the target molecule occurs. And a lid member provided to form an internal space into which a sealing liquid for sealing the well array is introduced, together with the base material, wherein the internal space is expressed by the equation (1): 0 ⁇ the sealing The sum of volume of stop solution / well volume of the well array is configured to satisfy ⁇ 12,000. FIG.
  • the detection device 100 is a schematic cross-sectional view showing an example of a detection device according to the first embodiment for detecting a target molecule in a sample by digital measurement.
  • the detection device 100 according to the present embodiment includes a base 110 and a lid 120.
  • the detection device 100 can be used to detect target molecules in a sample by digital measurement.
  • a well array 112 in which a plurality of wells 111 of the same shape and size are arranged is formed.
  • the well 111 is open to the surface of the substrate 110.
  • the shape, size and arrangement of the well 111 are not particularly limited, but the shape and size of the well 111 may be designed according to the size of the target molecule introduced into the well 111. In addition, it is possible to control the number of target molecules introduced into the well by controlling the total volume of the well and the like.
  • microbeads can be used for detection of target molecules.
  • an antibody against a target molecule may be used to bind the target molecule to the surface of the microbead, and the microbead to which the target molecule is bound may be introduced into the well 111.
  • the well 111 may have a shape and size that can accommodate only one microbead, or may have a shape and size that can accommodate a plurality of microbeads.
  • the diameter of the well 111 may be 1 ⁇ m to 15 ⁇ m, preferably 2 ⁇ m to 12 ⁇ m, and more preferably 3 ⁇ m to 10 ⁇ m.
  • the diameter of the well 111 may be, for example, about 3 ⁇ m, about 5 ⁇ m, or about 10 ⁇ m.
  • the depth of the well 111 may be 1 ⁇ m to 20 ⁇ m, preferably 2 ⁇ m to 17 ⁇ m, and more preferably 3 ⁇ m to 15 ⁇ m.
  • the depth of the well 111 may be, for example, about 3 ⁇ m, about 4.5 ⁇ m, or about 15 ⁇ m.
  • the diameter of the well 111 may be about 3 ⁇ m, and the depth of the well 111 may be about 4.5 ⁇ m.
  • the plurality of wells 111 are aligned to form a well array 112.
  • the wells 111 may be aligned, for example, in a triangular lattice, for example, may be aligned in a square lattice.
  • the lid member 120 is welded or adhered to the base 110.
  • a space (internal space) between the lid 120 and the base 110 forms a flow path 130 into which the fluid 160 is introduced.
  • the lid member 120 includes an inlet 140 for introducing the fluid 160 into the flow channel 130 and an outlet 150 for discharging the fluid 160 from the flow channel 130.
  • the fluid 160 introduced from the inlet 140 flows through the surface of the well array 112 and is then discharged from the outlet 150.
  • the material of the lid member 120 is not particularly limited, and examples thereof include thermoplastic resins such as cycloolefin polymers and cycloolefin copolymers.
  • the lid member 120 can be formed by molding a fluid of a thermoplastic resin using a molding die.
  • the material of the substrate 110 is preferably a material resistant to the fluid 160 fed to the flow channel 130.
  • the fluid 160 include a reaction liquid 170 containing a target molecule, a sealing liquid 180, and the like, which will be described later.
  • the material of the substrate 110 is preferably a light transmitting resin capable of observing the fluorescence signal generated inside the well 111, and a resin with less autofluorescence Is preferred.
  • Examples of the material of the substrate 110 include cycloolefin polymers, cycloolefin copolymers, silicon, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluorocarbon resin, and amorphous fluorocarbon resin.
  • a plurality of wells 111 are formed on one surface (first surface) in the thickness direction of the substrate 110.
  • the formation of the well 111 can be performed by injection molding, thermal imprint, optical imprint, or the like.
  • the well 111 can be formed on the surface of the base 110 by using a fluorine resin.
  • a fluorocarbon resin such as CYTOP (registered trademark) (Asahi Glass) is disposed on the base material 110, and further micropores are formed in the layer of CYTOP (registered trademark). It can be done.
  • the lid member 120 is formed to have a convex portion 121 on the surface directed to the substrate 110 at the time of assembly. Further, an inlet 140 and an outlet 150 are also formed in the lid member 120. Subsequently, the lid member 120 and the base member 110 are overlapped so that the convex portion 121 of the lid member 120 is in contact with the surface (first surface) where the well 111 is opened in the base member 110. Subsequently, the detection device 100 can be manufactured by welding the lid member 120 and the base 110 by laser welding or the like. Thereby, the flow path 130 into which the fluid 160 is introduced between the lid 120 and the base 110 is formed.
  • the height of the flow path (internal space) 130 formed between the lid 120 (the inner surface of the lid and the lower surface of the lid) and the base 110 (the inner surface of the base and the upper surface of the base) is Although not particularly limited, for example, it may be 1 ⁇ m to 150 ⁇ m, preferably 5 ⁇ m to 120 ⁇ m, more preferably 7.5 ⁇ m to 100 ⁇ m, still more preferably 8 ⁇ m to 60 ⁇ m, and more preferably 9 ⁇ m to 50 ⁇ m.
  • the thickness is more preferably 10 ⁇ m to 40 ⁇ m, and more preferably 10 ⁇ m to 30 ⁇ m.
  • Target molecules include DNA, RNA, miRNA, mRNA, proteins and the like.
  • the reaction solution 170 contains an enzyme and other reagents that generate a signal when the target molecule is present.
  • the target molecule is a nucleic acid
  • Invasive (registered trademark) Invasive (registered trademark) or other Invasive (registered trademark) Invasive (registered trademark) or other Invasive (registered trademark) method LAMP method (registered trademark), TaqMan (registered trademark), fluorescence probe method Etc.
  • the reaction solution may contain an allele probe, an ICA oligo, an enzyme Flap endonuclease-1 (FEN-1), a fluorescent substrate and the like.
  • the target molecule when the target molecule is a protein, ELISA or the like can be used to detect the target molecule.
  • the reaction solution may contain an antibody-modified carrier (eg, beads), an enzyme-modified antibody, a substrate, and the like.
  • a reaction solution containing an allele probe, an ICA oligo, FEN-1, a fluorescent substrate and the like used in the ICA method is an ICA reaction reagent (Invasive Cleavage Assay reaction reagent) Sometimes called.
  • the introduced reaction liquid 170 flows through the flow path 130 and is introduced into the wells 111 of the well array 112.
  • the target molecule contained in the reaction solution 170 is preferably adjusted to a concentration at which zero or one target molecule is introduced into the well 111. This enables digital measurement.
  • the sealing liquid 180 is introduced from the introduction port 140 of the lid member 120.
  • the introduced sealing solution 180 flows through the flow path 130 and contacts the well array 112.
  • the sealing liquid 180 displaces the reaction liquid 170 which is not contained in the well 111 among the reaction liquid 170 which is fed to the flow path 130.
  • the reaction liquid 170 which is not accommodated in the well 111 is discharged from the discharge port 150.
  • the wells 111 are individually sealed with the reaction liquid 170 containing the target molecule contained therein, and each well 111 becomes an independent reaction space.
  • the sealing liquid 180 include fluorine-based oil, silicon-based oil, hydrocarbon-based oil, and mixtures thereof.
  • reaction liquid 170 in the well 111 is subjected to reaction conditions. Thereby, when the target molecule is present in the well 111, a signal is generated in the well. Signals include fluorescence, color development, potential change, pH change and the like.
  • the generation of the signal is a signal amplification reaction. That is, the signal is amplified to an observable level so that when the target molecule is contained in the well 111, the signal is detected.
  • the reaction conditions of the reaction solution 170 differ depending on the reaction system and enzyme employed as a method for detecting a target molecule.
  • the enzyme may be Flap endonuclease-1 (FEN-1), and the temperature (temperature as reaction conditions) may be 50 to 80 ° C., preferably It may be 65 to 75 ° C.
  • the signal generated may be fluorescence.
  • the above reaction conditions are maintained for 1 hour or less, preferably 30 minutes or less, more preferably 20 minutes or less, and particularly preferably 15 minutes or less.
  • FIG. 4 is a schematic cross-sectional view showing the detection device 100 after generating a signal.
  • each well 111 constituting the well array 112 may include the reaction liquid 171 that has generated a signal or the reaction liquid 170 that has not generated a signal.
  • a signal is generated in the well 111. For example, first, bright field observation using white light that is irradiated in a direction perpendicular to the base 110 in the detection device 100 is performed. As a result, since the well 111 formed on the substrate 110 can be identified, the position of the well 111 can be confirmed.
  • fluorescence images of sections of all or part of the well array 112 including the plurality of wells 111 are taken.
  • the captured image may be analyzed by image processing by a computer system.
  • the excitation light corresponding to the fluorescent substance to be generated is irradiated from the base 110 side to the lid 120 side into the well 111 through the base 110 and generated in the well 111
  • the fluorescence emitted from the fluorescent substance is observed from the substrate 110 side. Since the substrate 110 is substantially transparent, fluorescence observation can be performed with the same sensitivity as that of the known detection device 100 used for fluorescence observation.
  • the detection device 100 can be used to detect target molecules.
  • the signal generated by the presence of the target molecule can be performed by bright field observation and fluorescence observation, but may be performed only by fluorescence observation.
  • sealing liquid 180 examples include fluorine-based oil, silicon-based oil, hydrocarbon-based oil, and mixtures thereof.
  • Sealing solution 180 is hydrophobic but has some water solubility. Therefore, when the reaction liquid 170 containing the target molecule is introduced into the wells 111 of the well array 112 and the wells 111 are individually sealed with the sealing liquid 180, the water of the reaction liquid 170 contained in each well 111 is sealed. It may be absorbed by the stop solution 180. In particular, when the reaction liquid 170 in the well 111 is heated, absorption of water is likely to occur.
  • the amount of water absorbed by the sealing solution 180 is the type of the sealing solution 180, the maximum amount of water absorbed by the sealing solution 180 (maximum water content of the sealing solution 180), the volume of the sealing solution 180, the enzyme reaction
  • the reaction temperature at which the reaction is performed, the reaction time at which the enzyme reaction is performed, and the total volume of the wells 111 constituting the well array 112 are influenced.
  • the maximum water content at 25 ° C. of Fluorinert® FC-40 (manufactured by Sigma), which is a fluorine-based oil, is about 7 mass ppm, and the maximum water content at 25 ° C. of silicone oil is about 200 ppm.
  • the detection device has a flow path (internal space), and the sealing liquid is introduced into the flow path (internal space).
  • the sealing liquid introduced into the flow path (internal space) has a value of Vo / Vsd in that the flow path (internal space) has a certain volume to facilitate manufacture of the detection device.
  • the volume of the sealing solution is preferably as small as possible with respect to the sum of the volumes of the wells of the well array.
  • the volume of the sealing liquid may be equal to the volume of the flow path (internal space) of the detection device. In other words, the volume of the flow path (inner space) of the detection device may be equal to the sum of the well volumes of the well array.
  • the volume of the sealing liquid when the lid has the inlet and the outlet, the volume of the sealing liquid is the volume of the inlet and the volume and the flow of the outlet. It may be equal to the sum of the volumes of the channels (internal space). In other words, the sum of the volume of the inlet and the volume of the outlet and the volume of the flow path (internal space) may be equal to the total of the well volumes of the well array.
  • the volume of the sealing liquid is the volume of the inlet and the volume of the outlet and the internal space thereof. It may be equal to the sum of the volume and the volume of the drainage reservoir. In other words, the sum of the volume of the inlet and the volume of the outlet and the volume of the internal space and the volume of the drainage reservoir may be equal to the total of the well volumes of the well array.
  • the moisture of the reaction liquid 170 contained in each well 111 is the sealing liquid
  • the volume (Vo) of the sealing liquid exists in the channel 130, the inlet 140, and the outlet 150, and is in spatial contact with the well 111. It is defined as the sum of the volumes of the sealing solution 180.
  • the volume (Vo) of the sealing solution may be referred to as “volume of the sealing solution 180”.
  • the volume of the sealing solution 180 is small, the presence of the lid member 120 can effectively cover the upper surface of the well array 112 (well 111) formed on the base 110.
  • the volume of the sealing solution 180 is about 100 ⁇ L or less, if the lid 120 is present, it is easy to effectively cover the upper surface of the well array 112 (well 111).
  • the lid material 120 is not necessarily required to cover the top surface of the well array 112 (well 111) formed on the substrate 110.
  • the lid material 120 is necessarily required as long as the top surface of the well array 112 (well 111) formed on the substrate 110 can be covered. is not.
  • reaction conditions such as reaction temperature and reaction time are determined by the efficiency of the signal amplification reaction by the reaction liquid 170.
  • the heating reaction is performed within the range of 50 ° C. to 75 ° C. for 1 hour or less.
  • the reaction conditions are preferably 70 ° C. or less. However, if the temperature is too low, the enzyme reaction rate will decrease. Therefore, the reaction conditions are preferably 55 ° C. or higher.
  • the reaction time is preferably 1 hour or less, more preferably 30 minutes or less, still more preferably 20 minutes or less, and particularly preferably 15 minutes or less.
  • the maximum water content of the sealing liquid at 25 ° C. is 1 to 1000 mass ppm, preferably 10 mass ppm or less, more preferably 8 mass ppm or less, and 7 mass ppm or less Is more preferred.
  • the preferable maximum water content of the sealing solution at 25 ° C. is considered to be about 1 mass ppm or so.
  • the total well volume (Vsd) of the well array can be determined by the product of the volume (Vd) of the well 111 and the total number (S) of the wells 111 present in the well array 112.
  • Vd is large, even if water is absorbed in the sealing solution 180, it is considered that the influence of the water becomes difficult.
  • V sd is large, the water in the sealing solution 180 is likely to be saturated, so it is considered that the rate of absorption of water from the reaction solution 170 contained in the well 111 into the sealing solution 180 is reduced.
  • the ratio (Vo / Vsd) of the volume (Vo) of the sealing solution to the total of the well volumes of the well array (Vsd) is 10 in the case of reacting at 66 ° C. for 30 minutes. It is preferable that it is 1,000 or less.
  • sealing solution 180 Even if water is absorbed in the sealing solution 180 and reaches the maximum water content (when the water is saturated), even if the sealing solution 180 releases water into the atmosphere, the sealing solution It is considered that 180 can absorb water again, and water is further absorbed from the reaction liquid 170 stored in the well 111 to the sealing liquid 180.
  • the sealing liquid 180 may be covered with a hydrophilic liquid so that the sealing liquid 180 is not in direct contact with the atmosphere.
  • a hydrophilic liquid such as water
  • the method according to the present embodiment can also be said to be a method for suppressing the reduction of the liquid contained in the well and sealed with the sealing liquid. Further, according to the detection device according to the present embodiment, the method according to the present embodiment can be suitably implemented.
  • FIG. 8 is a cross-sectional view schematically showing a detection device according to a second embodiment of the present invention.
  • the same components as the components shown in the first embodiment are given the same reference numerals as in the first embodiment, and the detailed description thereof will be omitted.
  • the method of suppressing the occurrence of false negative determination in detection of a target molecule using the detection device 200 according to the present embodiment is omitted for the same parts as the method according to the first embodiment.
  • the detection device 200 includes a base 110 and a lid 220 provided on the base 110.
  • the detection device 200 can be used to detect target molecules in a sample by digital measurement.
  • the detection device 200 according to the present embodiment has a configuration suitable for a method of suppressing the occurrence of false negative determination in detection of a target molecule.
  • the base 110 is the same as that of the first embodiment, and thus will not be described.
  • the lid 220 is welded or adhered to the substrate 110.
  • a space between the lid 220 and the base 110 forms an internal space 230 into which the fluid 160 is introduced.
  • the internal space 230 may be the flow channel 130 as in the first embodiment.
  • a convex portion which is a side surface of the lid is formed to be longer than illustrated in FIG. 8 so that the lid 220 has a certain length (distance) from the upper surface of the base to the inner surface of the lid.
  • the internal space 230 may be formed in the detection device 200 so that a large amount of sealing liquid 180 is poured.
  • the height of the internal space (flow path) 230 in the present embodiment may be the same as that in the first embodiment.
  • the shape of the internal space 230 formed in the detection device 200 may be any shape that can seal the well array 112, and is not particularly limited.
  • the lid 220 may have a bottomed cylindrical shape or a box shape without an upper surface. In that case, the inlet 240 and the outlet 250 are formed on the bottom of the lid 220. Also, the lid 220 may be much thicker than the height of the internal space (flow path) 230.
  • the internal space 230 formed in the detection device 200 may be formed as a shape that can seal the well array 112, and is not particularly limited.
  • the lid 220 includes an inlet 240 for introducing the fluid 160 into the inner space 230 and an outlet 250 for discharging the fluid 160 from the inner space 230.
  • the fluid 160 introduced from the inlet 240 flows through the surface of the well array 112 and is then discharged from the outlet 250.
  • the space configured to retain the sealing liquid 180 mainly includes the internal space 230 formed by the base material 110 and the lid member 220.
  • the inlet 240 is formed at one end (first end) of the upper surface of the lid member 220 so as to have a size capable of injecting the fluid 160.
  • the other end (second end) on the upper surface of the lid 220 may be formed with the discharge port 250 so as to have a size capable of discharging the fluid 160.
  • the lid 220 can be formed by directly forming the inlet 240 and the outlet 250 in a member to be a lid, the detection device 200 can be easily produced. Further, since the shape of the lid member 220 can be simply designed, there is an advantage in that the shape and configuration of the detection device 200 can be easily changed so that the internal space 230 has the intended capacity according to the application.
  • the material of the lid 220 is not particularly limited, and examples thereof include thermoplastic resins such as cycloolefin polymers and cycloolefin copolymers.
  • the lid member 220 can be formed by molding a fluid of a thermoplastic resin using a molding die.
  • the material of the substrate 110 is the same as that of the first embodiment, and is preferably a material resistant to the fluid 160 sent to the internal space 230.
  • the fluid 160 the reaction liquid 170 containing a target molecule, the sealing liquid 180, etc. are mentioned similarly to the said 1st Embodiment.
  • the material of the substrate 110 is preferably a light transmitting resin capable of observing the fluorescence signal generated inside the well 111, and a resin with less autofluorescence Is preferred.
  • cycloolefin polymer for example, cycloolefin polymer, cycloolefin copolymer, silicon, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluorocarbon resin, amorphous fluorocarbon resin, etc. may be mentioned as in the first embodiment.
  • a plurality of wells 111 are formed on one surface (first surface) in the thickness direction of the substrate 110.
  • the formation of the well 111 can be performed by injection molding, thermal imprint, optical imprint, or the like.
  • the well 111 can be formed on the surface of the base 110 by using a fluorine resin.
  • a fluorocarbon resin such as CYTOP (registered trademark) (Asahi Glass) is disposed on the base material 110, and further micropores are formed in the layer of CYTOP (registered trademark). It can be done.
  • the lid 220 is shaped so as to have a projection 221 on the surface directed to the base 110 during assembly. Further, an inlet 240 and an outlet 250 are also formed in the lid 220. Subsequently, the lid member 220 and the base member 110 are overlapped so that the convex portion 221 of the lid member 220 is in contact with the surface (first surface) where the well 111 is opened in the base member 110. Subsequently, the detection device 200 can be manufactured by welding the lid 220 and the base 110 by laser welding or the like. Thereby, an internal space 230 in which the fluid 160 flows or the fluid 160 is stored is formed between the lid 220 and the base 110.
  • the volume (Vo) of the sealing liquid exists in the internal space 230, the inlet 240, and the outlet 250, and , Defined as the sum of the volumes of the sealing liquid 180 in spatial contact with the well 111.
  • the volume (Vo) of the sealing liquid in the above equation (1) exists in the flow channel 230, and It may be defined as the volume of the sealing liquid 180 in spatial contact with the well 111.
  • FIG. 9 is a cross-sectional view schematically showing a detection device according to a third embodiment of the present invention.
  • the same components as the components shown in the first embodiment are given the same reference numerals as in the first embodiment, and the detailed description thereof will be omitted.
  • the method for suppressing the occurrence of false negative determination in detection of a target molecule using the detection device 300 according to the present embodiment is omitted for the same part as the method according to the first embodiment.
  • the detection device 300 includes a base 110 and a lid 320 provided on the base 110.
  • the detection device 300 can be used to detect target molecules in a sample by digital measurement.
  • the detection device 300 according to the present embodiment has a configuration suitable for a method of suppressing the occurrence of false negative determination in detection of a target molecule.
  • the substrate 110 is the same as that of the first embodiment, and thus will not be described.
  • the lid member 320 is welded or adhered to the base 110.
  • a space between the lid 320 and the base 110 forms an internal space 330 into which the fluid 160 is introduced.
  • the internal space 330 may be the flow passage 130 as in the first embodiment.
  • a convex portion which is a side surface of the lid is formed longer than that illustrated in FIG. 9 so that the lid 320 has a certain length (distance) from the upper surface of the base to the inner surface of the lid.
  • the internal space 330 may be formed in the detection device 300 so that a large amount of sealing solution 180 is poured.
  • the height of the internal space (flow path) 330 in the present embodiment may be the same as that in the first embodiment.
  • the shape of the internal space 330 formed in the detection device 300 may be any shape that can seal the well array 112, and is not particularly limited.
  • the lid member 320 may have a bottomed cylindrical shape or a box shape without an upper surface. In that case, the inlet 340 and the outlet 350 are formed on the bottom of the lid 320. Also, the lid 320 may be much thicker than the height of the internal space (flow path) 330.
  • the lid 320 includes an inlet (inlet) 340 for introducing the fluid 160 into the inner space 330 and an outlet 350 for discharging the fluid 160 from the inner space 330.
  • the fluid 160 introduced from the inlet 340 flows on the surface of the well array 112 and then is discharged from the outlet 350.
  • the detection device 300 according to the present embodiment is configured to be introduced with the reaction liquid 170 and the sealing liquid 180, and has a predetermined length in the thickness direction of the detection device 300. And a discharge port 350 having a predetermined length in the thickness direction of the detection device 300, and the seal liquid 180 is formed.
  • the places to stay are the internal space (flow path) 330, the inlet 340 and the outlet 350.
  • the fixed length at the inlet 340 may have a non-negligible length between the inner surface ceiling of the lid 320 and the top of the inlet 340 inside the detection device 300.
  • the fixed length at the discharge port 350 may have a non-negligible length from the inner surface ceiling of the lid 320 to the top of the injection port 340 inside the detection device 300.
  • the length which can not be disregarded means the length beyond the length which can be visually recognized, when observing detection device 300 with the naked eye.
  • the inlet 340 and the outlet 350 may have a projecting shape as shown in FIG. 9, and may form the projecting inlet 340 and the projecting outlet 350.
  • the lid upper member constituting the upper surface of the lid 320 has a structure thicker than the lid shown in FIG.
  • the inlet 340 and the outlet 350 may be formed as a simple hole in the lid material if they have a fixed length (distance) up to the top of the and the top of the outlet 350.
  • the inlet 340 may have a cylindrical shape, or may have a conical or inverted conical shape such as a funnel shape so that it is easy to introduce the reaction solution and the sealing solution with a pipette or the like. Good.
  • the volume may be calculated only from the volume of the internal space 330.
  • the material of the lid member 320 is not particularly limited, and examples thereof include thermoplastic resins such as cycloolefin polymers and cycloolefin copolymers.
  • the lid member 320 can be formed by molding a thermoplastic resin fluid using a molding die.
  • the material of the substrate 110 is the same as that of the first embodiment, and is preferably a material resistant to the fluid 160 fed to the internal space 330.
  • the fluid 160 the reaction liquid 170 containing a target molecule, the sealing liquid 180, etc. are mentioned similarly to the said 1st Embodiment.
  • the material of the substrate 110 is preferably a light transmitting resin capable of observing the fluorescence signal generated inside the well 111, and a resin with less autofluorescence Is preferred.
  • cycloolefin polymer for example, cycloolefin polymer, cycloolefin copolymer, silicon, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluorocarbon resin, amorphous fluorocarbon resin, etc. may be mentioned as in the first embodiment.
  • a plurality of wells 111 are formed on one surface (first surface) in the thickness direction of the substrate 110.
  • the formation of the well 111 can be performed by injection molding, thermal imprint, optical imprint, or the like.
  • the well 111 can be formed on the surface of the base 110 by using a fluorine resin.
  • a fluorocarbon resin such as CYTOP (registered trademark) (Asahi Glass) is disposed on the base material 110, and further micropores are formed in the layer of CYTOP (registered trademark). It can be done.
  • the lid member 320 is formed to have a convex portion 321 on the surface directed to the substrate 110 at the time of assembly. Further, an inlet 340 and an outlet 350 are also formed in the lid member 320. Subsequently, the lid material 320 and the base material 110 are overlapped so that the convex portion 321 of the lid material 320 is in contact with the surface (first surface) where the well 111 is opened in the base material 110. Subsequently, the detection device 300 can be manufactured by welding the lid member 320 and the base 110 by laser welding or the like. Thereby, an internal space 330 in which the fluid 160 flows or the fluid 160 is stored is formed between the lid 320 and the base 110.
  • the volume (Vo) of the sealing liquid is present in the internal space 330, the inlet 340, and the outlet 350, and , Defined as the sum of the volumes of the sealing liquid 180 in spatial contact with the well 111.
  • the second embodiment is the same as the first embodiment, and thus will not be described.
  • FIG. 10 is a cross-sectional view schematically showing a detection device according to a fourth embodiment of the present invention.
  • the same components as the components shown in the first embodiment are indicated by the same reference numerals as in the first embodiment, and the detailed description thereof will be omitted.
  • a method for suppressing the occurrence of false negative determination in detection of a target molecule using the detection device 400 according to the present embodiment is omitted for the same part as the method according to the first embodiment.
  • the detection device 400 includes a base 110 and a lid 420 provided on the base 110.
  • the detection device 400 can be used to detect target molecules in a sample by digital measurement.
  • the substrate 110 is the same as that of the first embodiment, and thus will not be described.
  • the lid member 420 is welded or adhered to the substrate 110.
  • a space between the lid 420 and the base 110 forms an internal space 430 through which the fluid 160 flows.
  • the internal space 430 may be the flow channel 130 as in the first embodiment.
  • the convex portion which is the side surface of the lid is formed longer than illustrated in FIG. 10 so that the lid 420 has a certain length (distance) from the upper surface of the base to the inner surface of the lid.
  • the convex portion which is the side surface of the lid is formed to have a predetermined length so that the distance from the upper surface to the lid inner surface ceiling is constant, and a large amount of the sealing liquid 180 is
  • the inner space 430 may be formed in the detection device 400 to be poured.
  • the height of the internal space (flow path) 430 in the present embodiment may be the same as that in the first embodiment.
  • the shape of the inner space 430 formed in the detection device 400 may be any shape that can seal the well array 112, and is not particularly limited.
  • the lid 420 may have a bottomed cylindrical shape or a box shape without an upper surface. In that case, the inlet 440 and the outlet 450 are formed on the bottom of the lid 420. Also, the lid 420 may be much thicker than the height of the internal space (flow path) 430.
  • the lid 420 includes an inlet (inlet) 440 for introducing the fluid 160 into the inner space 430 and an outlet 450 for discharging the fluid 160 from the inner space 430.
  • the fluid 160 introduced from the inlet 440 flows on the surface of the well array 112 and then is discharged from the outlet 450.
  • the detection device 400 according to the present embodiment is configured to be introduced with the reaction liquid 170 and the sealing liquid 180, and has a predetermined length in the thickness direction of the detection device 400.
  • an outlet port 450 connected to the drainage storage unit 451.
  • the place where the sealing liquid 180 stays is the internal space (flow path) 430, the inlet 440, the outlet 450, and the drainage storage portion 451.
  • the drainage storage unit 451 may be integrally formed with the lid 420, and may have a removable configuration. In the case where the drainage storage unit 451 has a removable configuration, the discharge port 450 and the drainage storage unit 451 may have a configuration that is easy to attach and detach, and the shape is not particularly limited.
  • the disposition of the drainage storage unit 451 may be provided on the top of the lid 420 as shown in FIG. 10, and the false negative determination in the operation of the detection device 400 and the detection of the target molecule according to the present embodiment In the range which does not inhibit the method of suppressing generation
  • the shape and length of the discharge port 450 can be changed as appropriate, for example, by changing the length of the discharge port 450 according to the arrangement of the drainage storage section 451.
  • the fixed length at the inlet 440 may have a non-negligible length between the inner surface ceiling of the lid 420 and the top of the inlet 440 inside the detection device 400.
  • the length that can not be ignored refers to a length that is longer than a visible length when the detection device 400 is observed with the naked eye.
  • the inlet 440 may have a projecting shape as shown in FIG. 10, and may form the projecting inlet 440.
  • the lid upper member constituting the upper surface of the lid 420 has a structure thicker than the lid shown in FIG.
  • the inlet 440 and the outlet 450 are formed as simple holes in the lid material. It may be done.
  • the inlet 440 may have a cylindrical shape, or may have a conical or inverted conical shape such as a funnel shape so that the reaction solution and the sealing solution can be easily introduced by a pipette or the like. Good.
  • the volume Vo of the existing sealing liquid 180 may be calculated from the volumes of the internal space 430 and the drainage reservoir 451.
  • the material of the lid 420 is not particularly limited, and examples thereof include thermoplastic resins such as cycloolefin polymers and cycloolefin copolymers.
  • the lid member 420 can be formed by molding a fluid of a thermoplastic resin using a molding die.
  • the material of the substrate 110 is the same as that of the first embodiment, and is preferably a material resistant to the fluid 160 sent to the internal space 430.
  • the fluid 160 the reaction liquid 170 containing a target molecule, the sealing liquid 180, etc. are mentioned similarly to the said 1st Embodiment.
  • the material of the substrate 110 is preferably a light transmitting resin capable of observing the fluorescence signal generated inside the well 111, and a resin with less autofluorescence Is preferred.
  • cycloolefin polymer for example, cycloolefin polymer, cycloolefin copolymer, silicon, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluorocarbon resin, amorphous fluorocarbon resin, etc. may be mentioned as in the first embodiment.
  • a plurality of wells 111 are formed on one surface (first surface) in the thickness direction of the substrate 110.
  • the formation of the well 111 can be performed by injection molding, thermal imprint, optical imprint, or the like.
  • the well 111 can be formed on the surface of the base 110 by using a fluorine resin.
  • a fluorocarbon resin such as CYTOP (registered trademark) (Asahi Glass) is disposed on the base material 110, and further micropores are formed in the layer of CYTOP (registered trademark). It can be done.
  • the lid member 420 is formed to have a convex portion 421 on the surface directed to the substrate 110 at the time of assembly. Further, an inlet 440 and an outlet 450 are also formed in the lid 420. The discharge port 450 and the drainage storage portion 451 may be integrally formed or may be detachably formed. Subsequently, the lid 420 and the base 110 are overlapped so that the convex portion 421 of the lid 420 is in contact with the surface (first surface) where the well 111 is opened in the base 110. Subsequently, the detection device 400 can be manufactured by welding the lid member 420 and the base 110 by laser welding or the like. Thereby, an internal space 430 in which the fluid 160 flows or the fluid 160 is stored is formed between the lid 420 and the base 110.
  • the volume (Vo) of the sealing liquid means the internal space 430, the inlet 440, the outlet 450, and the drainage storage part 451 and is defined as the sum of the volumes of the sealing solution 180 in spatial contact with the well 111.
  • the volume (Vo) of the sealing liquid exists in the internal space 430 and the drainage storage portion 451, and It may be defined as the sum of the volumes of the sealing solution 180 in spatial contact with the well 111.
  • the substrate 110 As the substrate 110, six types of substrates (substrates 1A to 6A) having different volumes and numbers of wells 111 were prepared.
  • the volume Vd of the wells 111 was 93 fL, and the total (Vsd) of the well volumes of the well array 112 was 0.00005859 mL.
  • the substrate 2A had a volume Vd of the wells 111 of 1,860 fL, and the total of the well volumes of the well array 112 (Vsd) was 0.002604 mL.
  • the volume Vd of the wells 111 was 59 fL, and the total (Vsd) of well volumes of the well array 112 was 0.000054 mL.
  • the substrate 4A had a volume Vd of the wells 111 of 59 fL and a total of the well volumes of the well array 112 (Vsd) was 0.000084 mL.
  • the substrate 5A had a volume Vd of well 111 of 1,178 fL, and the total well volume (Vsd) of the well array 112 was 0.0011 mL.
  • the base 6A had a volume Vd of well 111 of 1,178 fL, and the total well volume (Vsd) of the well array 112 was 0.0017 mL.
  • Each of the substrates 110 was adhered to each of the lids with a double-sided tape to form a flow path, thereby manufacturing a detection device.
  • an ICA reaction reagent (reaction solution 170) containing a DNA fragment as a target molecule was injected into the flow channel of each detection device.
  • the target oligo DNA (SEQ ID NO: 1) was used as a target molecule.
  • the concentration of the target oligo DNA was adjusted to a concentration such that zero or one target oligo DNA was introduced into the well 111 when the reaction solution 170 was introduced into the detection device.
  • the composition of the ICA reaction reagent is shown in Table 1.
  • a sealing solution was introduced to seal the wells 111 individually.
  • a sealing solution Fluorinert (registered trademark) FC-40 (manufactured by Sigma), which is a fluorine-based oil, was used.
  • the amount of the sealing solution to be introduced is 1.150, 0.650, 0.350, 0.150, 0.024 mL, 0.010 mL, 0.008 mL, 0.006 mL, 0.004 mL, 0.1.
  • the examination was conducted by changing each to 002 mL.
  • capacitance of an inlet (inlet) and an outlet could be disregarded was used.
  • the detection device adjusted so that the amount of the sealing liquid is 0.004 mL and 0.002 mL is an example in the case where the volume of the sealing liquid Vo is substantially the same as the volume of the internal space (flow path) .
  • the sealing solution After introducing the sealing solution, it was heated at 66 ° C. for 30 minutes. As a result, when the well 111 contains the target molecule, an ICA reaction occurs and a fluorescence signal is generated. Subsequently, the fluorescence signal emitted from each well 111 was photographed with a fluorescence microscope BZ-710 (manufactured by KEYENCE). A 10 ⁇ magnification lens was used as an objective lens. The exposure time was 3000 milliseconds. A filter for observing green fluorescent protein (GFP) was used as a fluorescence filter.
  • GFP green fluorescent protein
  • 6 (a) to 6 (d) show that 0.150, 0.350, 0.650 and 1.150 mL of sealing solution is used when the total well volume (Vsd) of the well array is 0.00005859 mL. It is a representative photograph which shows the result of having observed fluorescence after introducing and sealing each well and making it react at 66 ° C for 30 minutes.
  • the fluorescence is different unlike in the cases where the sealing solution is 0.150, 0.350, and 0.650 mL.
  • the emitting wells were observed in a ring shape. That is, no fluorescence was observed in the central part of the well, but fluorescence was observed in the peripheral part of the well.
  • Such a well is easy to make a false negative determination (it is easy to make a false negative determination), particularly when determining whether or not a signal is generated by image analysis using a computer.
  • the reason why the fluorescence signal is ring-shaped is not clear, for example, as shown in FIG. 7, the moisture in the well 111 is absorbed into the sealing solution 180 and the sealing solution 180 enters the well 111, etc. Is considered.

Abstract

A method for suppressing the occurrence of false-negative determination in detection of a target molecule, the method including: a step for preparing a device to which a well array having a plurality of wells is provided, and introducing a reaction liquid including the target molecule to the plurality of wells in the well array; a step for bringing the well array into contact with a predetermined volume of a sealing liquid and individually sealing the wells; a step for placing the reaction liquid in the individually sealed wells in a reaction condition, whereby a signal is generated in the wells when the target molecule is present in the wells; and a step for determining whether the signal was generated in the wells; the sealing liquid being introduced to the device so that the volume of the sealing liquid during detection of the target molecule satisfies formula (1): 0 < Volume of the sealing liquid/Total well volume of the well array ≤ 12,000.

Description

標的分子の検出における偽陰性判定の発生を抑制する方法および検出デバイスMethod and detection device for suppressing occurrence of false negative judgment in detection of target molecule
　本発明は、標的分子の検出における偽陰性判定の発生を抑制する方法および検出デバイスに関する。
　本願は、２０１７年１２月２６日に日本に出願された特願２０１７－２４９２２４号に基づき優先権を主張し、その内容をここに援用する。 The present invention relates to a method and detection device for suppressing the occurrence of false negative determination in detection of a target molecule.
Priority is claimed on Japanese Patent Application No. 2017-249224, filed Dec. 26, 2017, the content of which is incorporated herein by reference.
　生体試料中の標的分子を定量的に検出することにより、疾患の早期発見や投薬の効果予測が行われている。従来、タンパク質の定量は酵素結合免疫吸着アッセイ（ＥＬＩＳＡ）等により行われ、核酸の定量はリアルタイムＰＣＲ法等により行われてきた。 By detecting target molecules in biological samples quantitatively, early detection of diseases and prediction of the effects of medication are performed. Conventionally, quantification of proteins has been performed by enzyme-linked immunosorbent assay (ELISA) or the like, and quantification of nucleic acids has been performed by real-time PCR or the like.
　近年、疾患をより早期に発見する等の目的で、標的分子をより精度よく検出するニーズが高まっている。標的分子を精度よく検出する手法として、例えば、特許文献１、特許文献２、非特許文献１等には、多数の微小区画内で酵素反応を行う技術が記載されている。
　これらの手法はデジタル計測と呼ばれている。 In recent years, there is an increasing need to detect target molecules more accurately for the purpose of detecting diseases earlier. As a method for detecting a target molecule with high accuracy, for example, Patent Document 1, Patent Document 2, Non-Patent Document 1, etc. describe a technique for performing an enzyme reaction in a large number of minute sections.
These techniques are called digital measurement.
　デジタル計測では、試料溶液を極めて多数の微小溶液に分割する。そして、各微小溶液からの信号を２値化し、標的分子が存在するか否かのみを判別して、標的分子数を計測する。デジタル計測によれば、従来のＥＬＩＳＡやリアルタイムＰＣＲ法等と比較して、検出感度及び定量性を格段に向上させることができる。 In digital measurements, the sample solution is divided into a large number of micro solutions. Then, the signal from each micro solution is binarized, and only the presence or absence of the target molecule is determined to measure the number of target molecules. According to digital measurement, detection sensitivity and quantitativeness can be remarkably improved as compared with conventional ELISA, real time PCR method and the like.
　デジタルＰＣＲでは、１つの微小液滴に存在する鋳型となる核酸が０個又は１個になるように、ＰＣＲ反応試薬と核酸との混合物が希釈されている。デジタルＰＣＲでは、核酸増幅の感度を高めるために、また多数の微小液滴に対して同時に核酸増幅を行うために、各微小液滴の体積は小さい方が好ましい。例えば、特許文献３には、各ウェルの容積が６ｎＬ（ナノリットル）となるように形成されたマイクロアレイ状の反応容器が開示されている。また、特許文献１には、深さ３μｍ、直径５μｍのウェルが流路内に多数形成されたマイクロアレイに対して流路に試料を流して各ウェルに試料を導入した後、流路内の余剰試薬をオイルで押し出すことによって、各ウェル内に試料を導入する方法が開示されている。 In digital PCR, a mixture of PCR reaction reagent and nucleic acid is diluted such that there are no or one template nucleic acid present in one microdroplet. In digital PCR, the volume of each microdroplet is preferably small in order to increase the sensitivity of nucleic acid amplification and to perform nucleic acid amplification simultaneously on a large number of microdroplets. For example, Patent Document 3 discloses a microarray-like reaction container formed such that the volume of each well is 6 nL (nanoliter). Further, in Patent Document 1, after a sample is made to flow in the flow channel with respect to a microarray in which a large number of wells with a depth of 3 μm and a diameter of 5 μm are formed in the flow channel, the sample is introduced into each well. A method is disclosed for introducing a sample into each well by extruding the reagent with oil.
日本国特許第６１８３４７１号公報Japanese Patent No. 6183471 日本国特表２０１４－５０３８３１号公報Japanese Patent Application Publication No. 2014-503831 国際公開第２０１３－１５１１３５号International Publication No. 2013-151135
　デジタル計測においては、オイル（封止液）中に親水性の微小液滴を形成し、酵素反応や免疫反応等により微小液滴内に標的分子が存在する場合にシグナルを発生させ、標的分子の存在を検出する場合がある。今回、発明者らは、このようなデジタル計測において、偽陰性判定が発生する場合があることを見出した。そこで、本発明は、デジタル計測における偽陰性判定の発生を抑制する技術を提供することを目的とする。 In digital measurement, hydrophilic microdroplets are formed in oil (sealing liquid), and when a target molecule is present in the microdroplet by an enzyme reaction or an immune reaction, a signal is generated. May detect presence. The inventors have now found that false negative determinations can occur in such digital measurements. Then, an object of this invention is to provide the technique which suppresses generation | occurrence | production of the false negative determination in digital measurement.
　本発明は以下の態様を含む。
[１]本発明の第一態様に係る標的分子の検出における偽陰性判定の発生を抑制する方法は、複数のウェルを有するウェルアレイが設けられたデバイスを準備し、前記ウェルアレイにおける前記複数のウェルに前記標的分子を含む反応液を導入する工程と、前記ウェルアレイを所定の体積の封止液に接触させて、前記ウェルを個別に封止する工程と、個別に封止された前記ウェル内の前記反応液を反応条件にすることによって、前記ウェル内に前記標的分子が存在する場合に前記ウェル内でシグナルが生成される工程と、前記ウェル内でシグナルが生成されたか否かを判定する工程と、を含み、前記標的分子の検出の際に、前記封止液の体積を、式（１）：０＜前記封止液の体積／前記ウェルアレイのウェル容積の合計≦１２，０００を満たすように前記封止液を前記デバイスに導入する。
[２]　 前記式（１）において、１≦前記封止液の体積／前記ウェルアレイのウェル容積の合計≦１２，０００を満たすように前記封止液を前記デバイスに導入してもよい。
[３]　前記反応条件が５０～８０℃であってもよい。
[４]　前記反応条件を１時間以下維持してもよい。
[５]　前記反応液がＩｎｖａｓｉｖｅ　Ｃｌｅａｖａｇｅ　Ａｓｓａｙ反応試薬であってもよい。
[６]　前記封止液の２５℃における最大含水量が１～１０００質量ｐｐｍであってもよい。
[７]　本発明の第二態様に係る標的分子の検出デバイスは、前記標的分子を含有する反応液が導入され、かつ、前記標的分子を検出する反応が起こるように複数のウェルを有するウェルアレイが設けられた基材と、前記基材とともに、前記ウェルアレイを封止する封止液が導入される内部空間を形成するように設けられた蓋材と、を備え、前記内部空間は、式（１）：０＜前記封止液の体積／前記ウェルアレイのウェル容積の合計≦１２，０００を満たす。
[８]　前記式（１）において、１≦前記封止液の体積／前記ウェルアレイのウェル容積の合計≦１２，０００を満たしてもよい。
[９]　前記封止液の体積が前記内部空間の容積と等しくてもよい。
[１０]　前記蓋材に注入口と排出口とをさらに有し、前記封止液の体積が、前記注入口の容積および前記排出口の容積および前記内部空間の容積の合計と等しくてもよい。
[１１]　前記排出口に接続された排液貯蔵部をさらに有し、前記封止液の体積が、前記注入口の容積および前記排出口の容積および前記内部空間の容積および前記排液貯蔵部の容積の合計と等しくてもよい。 The present invention includes the following aspects.
[1] A method of suppressing the occurrence of false negative determination in detection of a target molecule according to the first aspect of the present invention comprises preparing a device provided with a well array having a plurality of wells, and selecting a plurality of the plurality of wells in the well array. The steps of: introducing a reaction solution containing the target molecule into the wells; contacting the well array with a predetermined volume of sealing solution to individually seal the wells; and the individually sealed wells By setting the reaction solution in the reaction solution to the step of generating a signal in the well when the target molecule is present in the well, and determining whether the signal is generated in the well And, in the detection of the target molecule, the volume of the sealing solution is expressed by the equation (1): 0 <volume of the sealing solution / total of well volumes of the well array ≦ 12,000. Meet The sea urchin the sealing liquid is introduced into the device.
[2] In the formula (1), the sealing solution may be introduced into the device so as to satisfy 1 ≦ total volume of the sealing solution / well volume of the well array ≦ 12,000.
[3] The reaction condition may be 50 to 80 ° C.
[4] The reaction conditions may be maintained for 1 hour or less.
[5] The reaction solution may be an Invasive Cleavage Assay reaction reagent.
[6] The maximum water content of the sealing solution at 25 ° C. may be 1 to 1000 mass ppm.
[7] The target molecule detection device according to the second aspect of the present invention is a well array having a plurality of wells into which a reaction solution containing the target molecule is introduced and a reaction for detecting the target molecule occurs. And a lid member provided so as to form an inner space into which a sealing solution for sealing the well array is introduced together with the base material, and the inner space is (1): 0 <sum of volume of sealing solution / well volume of well array ≦ 12,000.
[8] In the formula (1), the sum of 1 ≦ volume of the sealing solution / well volume of the well array may satisfy 12,000.
[9] The volume of the sealing solution may be equal to the volume of the internal space.
[10] The lid may further include an inlet and an outlet, and the volume of the sealing liquid may be equal to the volume of the inlet and the volume of the outlet and the volume of the internal space. .
[11] The system further comprises a drainage storage connected to the outlet, wherein the volume of the sealing liquid is the volume of the inlet, the volume of the outlet, the volume of the internal space, and the drainage storage. May be equal to the sum of the volumes of
　本発明によれば、デジタル計測における偽陰性判定の発生を抑制する技術を提供することができる。 According to the present invention, it is possible to provide a technique for suppressing the occurrence of false negative determination in digital measurement.
第１実施形態に係る検出デバイスの一例を示す断面模式図である。It is a cross section showing an example of a detection device concerning a 1st embodiment. 第１実施形態に係る検出デバイスへの封止液の導入を説明する模式断面図である。It is a schematic cross section explaining the introduction of the sealing liquid to the detection device concerning a 1st embodiment. 第１実施形態に係る検出デバイスへの封止液の導入を説明する模式断面図である。It is a schematic cross section explaining the introduction of the sealing liquid to the detection device concerning a 1st embodiment. シグナルを生成させた後の第１実施形態に係る検出デバイス１００を示す断面模式図である。It is a cross section showing a detection device 100 concerning a 1st embodiment after generating a signal. 封止液から大気中への水分の放出を防ぐ方法の一例を説明する模式断面図である。It is a schematic cross section explaining an example of the method of preventing the discharge | release of the water | moisture content from the sealing liquid to air | atmosphere. （ａ）～（ｄ）は、実験例１の結果を示す代表的な蛍光顕微鏡写真である。(A) to (d) are representative fluorescence micrographs showing the results of Experimental Example 1. 偽陰性判定の発生を示す断面模式図である。It is a cross-sectional schematic diagram which shows generation | occurrence | production of a false negative determination. 第２実施形態に係る検出デバイスを示す断面模式図である。It is a cross-sectional schematic diagram which shows the detection device which concerns on 2nd Embodiment. 第３実施形態に係る検出デバイスを示す断面模式図である。It is a cross section showing a detection device concerning a 3rd embodiment. 第４実施形態に係る検出デバイスを示す断面模式図である。It is a cross section showing a detection device concerning a 4th embodiment.
　以下、場合により図面を参照しつつ、本発明の実施形態について詳細に説明する。なお、図面中、同一又は相当部分には同一符号を付し、重複する説明は省略する。なお、各図における寸法比は、説明のため誇張している部分があり、必ずしも実際の寸法比とは一致しない。 Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as the case may be. In the drawings, the same or corresponding parts are denoted by the same reference numerals, and overlapping descriptions will be omitted. In addition, the dimensional ratio in each figure has the part which exaggerates for description, and does not necessarily correspond with an actual dimensional ratio.
［偽陰性判定の発生を抑制する方法（第１実施形態）］
　本発明の第１実施形態に係る標的分子の検出における偽陰性判定の発生を抑制する方法は、複数のウェルを有するウェルアレイが設けられたデバイスを準備し、前記ウェルアレイにおける前記複数のウェルに前記標的分子を含む反応液を導入する工程と、前記ウェルアレイを所定の体積の封止液に接触させて、前記ウェルを個別に封止する工程と、個別に封止された前記ウェル内の前記反応液を反応条件にすることによって、前記ウェル内に前記標的分子が存在する場合に前記ウェル内でシグナルが生成される工程と、前記ウェル内でシグナルが生成されたか否かを判定する工程と、を含み、前記標的分子の検出の際に、前記封止液の体積を、式（１）：０＜前記封止液の体積／前記ウェルアレイのウェル容積の合計≦１２，０００を満たすように前記封止液を前記デバイスに導入する。 [Method for suppressing occurrence of false negative determination (first embodiment)]
A method for suppressing the occurrence of false negative determination in detection of a target molecule according to a first embodiment of the present invention prepares a device provided with a well array having a plurality of wells, and uses the plurality of wells in the well array. The steps of: introducing a reaction solution containing the target molecule; contacting the well array with a predetermined volume of sealing solution to individually seal the wells; and individually sealing the wells. A step of generating a signal in the well when the target molecule is present in the well by setting the reaction solution as a reaction condition, and a step of determining whether a signal is generated in the well And, at the time of detection of the target molecule, the volume of the sealing solution satisfies the following formula (1): 0 <volume of the sealing solution / well volume of the well array ≦ 12,000. The sea urchin the sealing liquid is introduced into the device.
　偽陰性とは、ある試験において、実際には陽性の反応を示しているにもかかわらず、何らかの原因で陰性として検出されることをいう。標的分子の検出において、偽陰性判定が発生する原因としては、例えば、標的分子の検出のためのシグナル生成反応が正常に行われないこと、シグナル生成反応が正常に行われたにもかかわらず、シグナルを正常に検出できないこと等が考えられる。 The false negative means that although a test actually shows a positive reaction, it is detected as negative for some reason. In the detection of a target molecule, false negative determination may occur, for example, that the signal generation reaction for detection of the target molecule is not normally performed, and the signal generation reaction is normally performed. It is conceivable that the signal can not be detected normally.
　シグナルを正常に検出できない場合の具体的な例としては、例えば、実施例において述べるように、シグナル（例えば、蛍光）がウェルの周辺部分、すなわちウェルの壁付近のみに生じてしまう場合等が挙げられる。 As a specific example of the case where the signal can not be detected normally, for example, as described in the examples, the case where a signal (for example, fluorescence) is generated only in the peripheral part of the well, that is, near the wall of the well Be
　偽陰性判定が発生すると、治療判断ミスにつながったり、再度の試験が必要となり、検体採取の再実施が必要になる等の問題が生じる場合がある。 If a false negative judgment occurs, it may lead to a treatment judgment error or require another test, which may cause problems such as re-execution of sample collection.
　実施例において述べるように、本実施形態に係る方法により、標的分子の検出における偽陰性判定の発生を抑制することができる。本実施形態に係る方法は、デジタル計測に好適に適用することができる。 As described in the examples, the method according to this embodiment can suppress the occurrence of false negative determination in the detection of a target molecule. The method according to the present embodiment can be suitably applied to digital measurement.
（検出デバイス（第１実施形態））
　まず、本実施形態に係る方法に用いる検出デバイスについて説明する。
　本実施形態に係る標的分子の検出デバイスは、前記標的分子を含有する反応液が導入され、かつ、前記標的分子を検出する反応が起こる複数のウェルを有するウェルアレイが設けられた基材と、前記基材とともに、前記ウェルアレイを封止する封止液が導入される内部空間を形成するように設けられた蓋材と、を備え、前記内部空間は、式（１）：０＜前記封止液の体積／前記ウェルアレイのウェル容積の合計≦１２，０００を満たすように構成されている。
　図１はデジタル計測により、試料中の標的分子を検出する、第１実施形態に係る検出デバイスの一例を示す断面模式図である。図１に示すように、本実施形態に係る検出デバイス１００は、基材１１０と蓋材１２０とを備えている。検出デバイス１００を用いて、試料中の標的分子をデジタル計測により検出することができる。 (Detection Device (First Embodiment))
First, the detection device used in the method according to the present embodiment will be described.
The target molecule detection device according to the present embodiment includes a substrate provided with a well array having a plurality of wells into which a reaction solution containing the target molecule is introduced and a reaction for detecting the target molecule occurs. And a lid member provided to form an internal space into which a sealing liquid for sealing the well array is introduced, together with the base material, wherein the internal space is expressed by the equation (1): 0 <the sealing The sum of volume of stop solution / well volume of the well array is configured to satisfy ≦ 12,000.
FIG. 1 is a schematic cross-sectional view showing an example of a detection device according to the first embodiment for detecting a target molecule in a sample by digital measurement. As shown in FIG. 1, the detection device 100 according to the present embodiment includes a base 110 and a lid 120. The detection device 100 can be used to detect target molecules in a sample by digital measurement.
　基材１１０の表面には、同形同大の複数のウェル１１１が配置されたウェルアレイ１１２が形成されている。ウェル１１１は、基材１１０の表面に開口している。ウェル１１１の形状、寸法及び配置は特に限定されないが、ウェル１１１内に導入される標的分子のサイズに応じて、ウェル１１１の形状および寸法を設計してもよい。また、ウェルの容積の合計等を制御することにより、ウェルに導入される標的分子の分子数を制御することが可能である。 On the surface of the base 110, a well array 112 in which a plurality of wells 111 of the same shape and size are arranged is formed. The well 111 is open to the surface of the substrate 110. The shape, size and arrangement of the well 111 are not particularly limited, but the shape and size of the well 111 may be designed according to the size of the target molecule introduced into the well 111. In addition, it is possible to control the number of target molecules introduced into the well by controlling the total volume of the well and the like.
　例えば、標的分子の検出にマイクロビーズを用いることができる。例えば、標的分子に対する抗体等を用いて、マイクロビーズの表面に標的分子を結合させ、標的分子が結合したマイクロビーズをウェル１１１に導入してもよい。この場合、ウェル１１１は、マイクロビーズを１個のみ収容可能な形状及び寸法を有していてもよいし、マイクロビーズを複数個収容可能な形状及び寸法を有していてもよい。 For example, microbeads can be used for detection of target molecules. For example, an antibody against a target molecule may be used to bind the target molecule to the surface of the microbead, and the microbead to which the target molecule is bound may be introduced into the well 111. In this case, the well 111 may have a shape and size that can accommodate only one microbead, or may have a shape and size that can accommodate a plurality of microbeads.
　ウェル１１１の直径は、１μｍ～１５μｍであってよく、２μｍ～１２μｍであることが好ましく、３μｍ～１０μｍであることがより好ましい。ウェル１１１の直径は、例えば、３μｍ程度であってよく、５μｍ程度であってよく、１０μｍ程度であってもよい。また、ウェル１１１の深さは、１μｍ～２０μｍであってよく、２μｍ～１７μｍであることが好ましく、３μｍ～１５μｍであることがより好ましい。ウェル１１１の深さは、例えば、３μｍ程度であってよく、４．５μｍ程度であってよく、１５μｍ程度であってもよい。
　本実施形態において、例えば、ウェル１１１の直径が３μｍ程度であり、かつ、ウェル１１１の深さが４．５μｍ程度であってもよい。
　複数のウェル１１１が整列してウェルアレイ１１２を形成する。ウェル１１１は例えば三角格子状に整列していてもよく、例えば正方格子状に整列していてもよい。 The diameter of the well 111 may be 1 μm to 15 μm, preferably 2 μm to 12 μm, and more preferably 3 μm to 10 μm. The diameter of the well 111 may be, for example, about 3 μm, about 5 μm, or about 10 μm. The depth of the well 111 may be 1 μm to 20 μm, preferably 2 μm to 17 μm, and more preferably 3 μm to 15 μm. The depth of the well 111 may be, for example, about 3 μm, about 4.5 μm, or about 15 μm.
In the present embodiment, for example, the diameter of the well 111 may be about 3 μm, and the depth of the well 111 may be about 4.5 μm.
The plurality of wells 111 are aligned to form a well array 112. The wells 111 may be aligned, for example, in a triangular lattice, for example, may be aligned in a square lattice.
　蓋材１２０は、基材１１０に溶着又は接着されている。蓋材１２０と基材１１０との間の空間（内部空間）は、流体１６０が導入される流路１３０を形成している。蓋材１２０は、流路１３０に流体１６０を導入する導入口１４０及び流路１３０から流体１６０を排出する排出口１５０を備えている。導入口１４０から導入された流体１６０は、ウェルアレイ１１２の表面を流れた後、排出口１５０から排出される。 The lid member 120 is welded or adhered to the base 110. A space (internal space) between the lid 120 and the base 110 forms a flow path 130 into which the fluid 160 is introduced. The lid member 120 includes an inlet 140 for introducing the fluid 160 into the flow channel 130 and an outlet 150 for discharging the fluid 160 from the flow channel 130. The fluid 160 introduced from the inlet 140 flows through the surface of the well array 112 and is then discharged from the outlet 150.
　蓋材１２０の材質は特に限定されないが、シクロオレフィンポリマー、シクロオレフィンコポリマー等の熱可塑性樹脂が挙げられる。蓋材１２０の形成は、熱可塑性樹脂の流動体を、成形型を用いて成形すること等により行うことができる。 The material of the lid member 120 is not particularly limited, and examples thereof include thermoplastic resins such as cycloolefin polymers and cycloolefin copolymers. The lid member 120 can be formed by molding a fluid of a thermoplastic resin using a molding die.
　基材１１０の材質は、流路１３０に送液される流体１６０に対し耐性のある材質であることが好ましい。流体１６０としては、後述する、標的分子を含む反応液１７０、封止液１８０等が挙げられる。また、検出するシグナルが蛍光である場合、基材１１０の材質は、ウェル１１１の内部に発生した蛍光シグナルを観察することが可能な光透過性樹脂であることが好ましく、更に自家蛍光の少ない樹脂であることが好ましい。 The material of the substrate 110 is preferably a material resistant to the fluid 160 fed to the flow channel 130. Examples of the fluid 160 include a reaction liquid 170 containing a target molecule, a sealing liquid 180, and the like, which will be described later. When the signal to be detected is fluorescence, the material of the substrate 110 is preferably a light transmitting resin capable of observing the fluorescence signal generated inside the well 111, and a resin with less autofluorescence Is preferred.
　基材１１０の材質としては、例えば、シクロオレフィンポリマー、シクロオレフィンコポリマー、シリコン、ポリプロピレン、ポリカーボネート、ポリスチレン、ポリエチレン、ポリ酢酸ビニル、フッ素樹脂、アモルファスフッ素樹脂等が挙げられる。 Examples of the material of the substrate 110 include cycloolefin polymers, cycloolefin copolymers, silicon, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluorocarbon resin, and amorphous fluorocarbon resin.
　基材１１０の、板厚方向の一方の面（第一面）に複数のウェル１１１が形成される。ウェル１１１の形成は、射出成形、熱インプリント、光インプリント等により行うことができる。 A plurality of wells 111 are formed on one surface (first surface) in the thickness direction of the substrate 110. The formation of the well 111 can be performed by injection molding, thermal imprint, optical imprint, or the like.
　また、フッ素樹脂を用いて基材１１０の表面にウェル１１１を形成することもできる。
　例えば、基材１１０の上にＣＹＴＯＰ（登録商標）（旭硝子）等のフッ素樹脂で形成された層を配置し、更にＣＹＴＯＰ（登録商標）の層に微小な孔を形成し、当該孔をウェル１１１とすることができる。 Alternatively, the well 111 can be formed on the surface of the base 110 by using a fluorine resin.
For example, a layer formed of a fluorocarbon resin such as CYTOP (registered trademark) (Asahi Glass) is disposed on the base material 110, and further micropores are formed in the layer of CYTOP (registered trademark). It can be done.
　蓋材１２０は、組立時に基材１１０に向けられる面に凸部１２１を有するように成形される。また、蓋材１２０には導入口１４０及び排出口１５０も形成される。続いて、基材１１０においてウェル１１１が開口する面（第一面）に蓋材１２０の凸部１２１が接するように、蓋材１２０と基材１１０とが重ねられる。続いて、レーザー溶着等により蓋材１２０と基材１１０とを溶着し、検出デバイス１００を製造することができる。これにより、蓋材１２０と基材１１０との間に流体１６０が導入される流路１３０が形成される。
　なお、蓋材１２０（蓋材の内面、蓋材の下面）と基材１１０（基材の内面、基材の上面）との間に形成された流路（内部空間）１３０の高さは、特に限定されないが、例えば、１μｍ～１５０μｍであってよく、５μｍ～１２０μｍであることが好ましく、７．５μｍ～１００μｍであることがより好ましく、８μｍ～６０μｍであることがより好ましく、９μｍ～５０μｍであることがより好ましく、１０μｍ～４０μｍであることがより好ましく、１０μｍ～３０μｍであることがより好ましい。 The lid member 120 is formed to have a convex portion 121 on the surface directed to the substrate 110 at the time of assembly. Further, an inlet 140 and an outlet 150 are also formed in the lid member 120. Subsequently, the lid member 120 and the base member 110 are overlapped so that the convex portion 121 of the lid member 120 is in contact with the surface (first surface) where the well 111 is opened in the base member 110. Subsequently, the detection device 100 can be manufactured by welding the lid member 120 and the base 110 by laser welding or the like. Thereby, the flow path 130 into which the fluid 160 is introduced between the lid 120 and the base 110 is formed.
The height of the flow path (internal space) 130 formed between the lid 120 (the inner surface of the lid and the lower surface of the lid) and the base 110 (the inner surface of the base and the upper surface of the base) is Although not particularly limited, for example, it may be 1 μm to 150 μm, preferably 5 μm to 120 μm, more preferably 7.5 μm to 100 μm, still more preferably 8 μm to 60 μm, and more preferably 9 μm to 50 μm. The thickness is more preferably 10 μm to 40 μm, and more preferably 10 μm to 30 μm.
（標的分子の検出）
　続いて、本実施形態に係る検出デバイス１００を用いた標的分子の検出について説明する。図１に示すように、まず、検出デバイス１００の導入口１４０に、標的分子を含む反応液１７０を導入する。標的分子としては、ＤＮＡ、ＲＮＡ、ｍｉＲＮＡ、ｍＲＮＡ、タンパク質等が挙げられる。 (Detection of target molecule)
Subsequently, detection of a target molecule using the detection device 100 according to the present embodiment will be described. As shown in FIG. 1, first, a reaction liquid 170 containing a target molecule is introduced into the inlet 140 of the detection device 100. Target molecules include DNA, RNA, miRNA, mRNA, proteins and the like.
　反応液１７０は、標的分子が存在する場合にシグナルを生成する酵素及びその他の試薬を含んでいる。例えば、標的分子が核酸である場合には、標的分子の検出にインベーダー（登録商標）法等のＩｎｖａｓｉｖｅ　Ｃｌｅａｖａｇｅ　Ａｓｓａｙ（ＩＣＡ）法、ＬＡＭＰ法（商標登録）、ＴａｑＭａｎ（登録商標）法、蛍光プローブ法等を用いることができる。例えばＩＣＡ法により標的分子を検出する場合、反応液は、アレルプローブ、ＩＣＡオリゴ、酵素であるＦｌａｐ　ｅｎｄｏｎｕｃｌｅａｓｅ－１（ＦＥＮ－１）、蛍光基質等を含んでいてもよい。また、例えば、標的分子がタンパク質である場合には、標的分子の検出にＥＬＩＳＡ法等を用いることができる。例えばＥＬＩＳＡ法により標的分子を検出する場合、反応液は、抗体が修飾された担持体（例えば、ビーズ）、酵素が修飾された抗体、基質等を含んでいてもよい。
　なお、本実施形態および以下の実施形態、ならびに、実施例において、ＩＣＡ法に用いる、アレルプローブ、ＩＣＡオリゴ、ＦＥＮ－１、蛍光基質等を含む反応液をＩＣＡ反応試薬（Ｉｎｖａｓｉｖｅ　Ｃｌｅａｖａｇｅ　Ａｓｓａｙ反応試薬）と呼ぶことがある。 The reaction solution 170 contains an enzyme and other reagents that generate a signal when the target molecule is present. For example, when the target molecule is a nucleic acid, for detecting the target molecule, Invasive (registered trademark) Invasive (registered trademark) or other Invasive (registered trademark) Invasive (registered trademark) or other Invasive (registered trademark) method, LAMP method (registered trademark), TaqMan (registered trademark), fluorescence probe method Etc. can be used. For example, when the target molecule is detected by the ICA method, the reaction solution may contain an allele probe, an ICA oligo, an enzyme Flap endonuclease-1 (FEN-1), a fluorescent substrate and the like. Also, for example, when the target molecule is a protein, ELISA or the like can be used to detect the target molecule. For example, in the case of detecting a target molecule by ELISA, the reaction solution may contain an antibody-modified carrier (eg, beads), an enzyme-modified antibody, a substrate, and the like.
In addition, in this embodiment and the following embodiments and examples, a reaction solution containing an allele probe, an ICA oligo, FEN-1, a fluorescent substrate and the like used in the ICA method is an ICA reaction reagent (Invasive Cleavage Assay reaction reagent) Sometimes called.
　導入された反応液１７０は、流路１３０を流れ、ウェルアレイ１１２のウェル１１１に導入される。 The introduced reaction liquid 170 flows through the flow path 130 and is introduced into the wells 111 of the well array 112.
　反応液１７０に含まれる標的分子は、ウェル１１１に０個又は１個の標的分子が導入される濃度に調整されていることが好ましい。これによりデジタル計測が可能になる。 The target molecule contained in the reaction solution 170 is preferably adjusted to a concentration at which zero or one target molecule is introduced into the well 111. This enables digital measurement.
　続いて、図２に示すように、蓋材１２０の導入口１４０から、封止液１８０を導入する。導入された封止液１８０は、流路１３０を流れ、ウェルアレイ１１２に接触する。封止液１８０は、流路１３０に送液された反応液１７０のうち、ウェル１１１に収容されなかった反応液１７０を置換していく。最終的には図３に示すように、ウェル１１１に収容されなかった反応液１７０は排出口１５０から排出される。 Subsequently, as shown in FIG. 2, the sealing liquid 180 is introduced from the introduction port 140 of the lid member 120. The introduced sealing solution 180 flows through the flow path 130 and contacts the well array 112. The sealing liquid 180 displaces the reaction liquid 170 which is not contained in the well 111 among the reaction liquid 170 which is fed to the flow path 130. Finally, as shown in FIG. 3, the reaction liquid 170 which is not accommodated in the well 111 is discharged from the discharge port 150.
　その結果、ウェル１１１は、内部に、標的分子を含む反応液１７０を収容したまま個別に封止され、各ウェル１１１はそれぞれ独立した反応空間となる。封止液１８０としては、フッ素系オイル、シリコン系オイル、炭化水素系オイル、これらの混合物等が挙げられる。 As a result, the wells 111 are individually sealed with the reaction liquid 170 containing the target molecule contained therein, and each well 111 becomes an independent reaction space. Examples of the sealing liquid 180 include fluorine-based oil, silicon-based oil, hydrocarbon-based oil, and mixtures thereof.
　続いて、ウェル１１１内の反応液１７０を反応条件にする。これにより、ウェル１１１内に標的分子が存在する場合に、当該ウェル内でシグナルが生成される。シグナルとしては、蛍光、発色、電位変化、ｐＨ変化等が挙げられる。 Subsequently, the reaction liquid 170 in the well 111 is subjected to reaction conditions. Thereby, when the target molecule is present in the well 111, a signal is generated in the well. Signals include fluorescence, color development, potential change, pH change and the like.
　本実施形態に係る方法において、シグナルの生成はシグナル増幅反応である。すなわち、ウェル１１１内に標的分子が含まれる場合にシグナルが検出されるように、シグナルを観察可能なレベルまで増幅させる。 In the method according to the present embodiment, the generation of the signal is a signal amplification reaction. That is, the signal is amplified to an observable level so that when the target molecule is contained in the well 111, the signal is detected.
　反応液１７０の反応条件は、標的分子の検出方法として採用した反応系及び酵素により異なる。例えば、標的分子の検出をＩＣＡ法により行う場合、酵素はＦｌａｐ　ｅｎｄｏｎｕｃｌｅａｓｅ－１（ＦＥＮ－１）であってもよく、温度（反応条件としての温度）は５０～８０℃であってよく、好ましくは６５～７５℃であってよい。また、生成されるシグナルは蛍光であってもよい。 The reaction conditions of the reaction solution 170 differ depending on the reaction system and enzyme employed as a method for detecting a target molecule. For example, when detection of a target molecule is performed by the ICA method, the enzyme may be Flap endonuclease-1 (FEN-1), and the temperature (temperature as reaction conditions) may be 50 to 80 ° C., preferably It may be 65 to 75 ° C. Also, the signal generated may be fluorescence.
　また、例えば、標的分子の検出をＩＣＡ法により行う場合、上記の反応条件を１時間以下、好ましくは３０分以下、より好ましくは２０分以下、特に好ましくは１５分以下維持する。 Also, for example, when detection of a target molecule is performed by the ICA method, the above reaction conditions are maintained for 1 hour or less, preferably 30 minutes or less, more preferably 20 minutes or less, and particularly preferably 15 minutes or less.
　図４は、シグナルを生成させた後の検出デバイス１００を示す断面模式図である。図４に示すように、ウェルアレイ１１２を構成する各ウェル１１１は、シグナルを生成した反応液１７１を含む場合と、シグナルを生成しなかった反応液１７０を含む場合がある。 FIG. 4 is a schematic cross-sectional view showing the detection device 100 after generating a signal. As shown in FIG. 4, each well 111 constituting the well array 112 may include the reaction liquid 171 that has generated a signal or the reaction liquid 170 that has not generated a signal.
　続いて、ウェル１１１内でシグナルが生成されたか否かを判定する。例えば、まず、検出デバイス１００における基材１１０に対して垂直な方向に照射する白色光を用いた明視野観察を行う。これによって、基材１１０上に形成されたウェル１１１を特定することができるので、ウェル１１１の位置を確認することができる。 Subsequently, it is determined whether or not a signal is generated in the well 111. For example, first, bright field observation using white light that is irradiated in a direction perpendicular to the base 110 in the detection device 100 is performed. As a result, since the well 111 formed on the substrate 110 can be identified, the position of the well 111 can be confirmed.
　続いて、複数のウェル１１１を含むウェルアレイ１１２の全て又は一部の区画の蛍光画像を撮影する。撮影した画像は、コンピューターシステムによる画像処理により解析してもよい。 Subsequently, fluorescence images of sections of all or part of the well array 112 including the plurality of wells 111 are taken. The captured image may be analyzed by image processing by a computer system.
　例えばＩＣＡ反応を行った場合には、生成される蛍光物質に対応する励起光を、基材１１０側から蓋材１２０側へ、基材１１０を通じてウェル１１１内へ照射し、ウェル１１１内で生成された蛍光物質が発する蛍光を基材１１０側から観察する。基材１１０は実質的に透明であるので、蛍光観察に使用される公知の検出デバイス１００と同等の感度で蛍光観察を行うことができる。 For example, when ICA reaction is performed, the excitation light corresponding to the fluorescent substance to be generated is irradiated from the base 110 side to the lid 120 side into the well 111 through the base 110 and generated in the well 111 The fluorescence emitted from the fluorescent substance is observed from the substrate 110 side. Since the substrate 110 is substantially transparent, fluorescence observation can be performed with the same sensitivity as that of the known detection device 100 used for fluorescence observation.
　このようにして、検出デバイス１００を用いて標的分子を検出することができる。標的分子の存在により生成されたシグナルは明視野観察及び蛍光観察により行うことができるが、蛍光観察のみにより行ってもよい。 In this manner, the detection device 100 can be used to detect target molecules. The signal generated by the presence of the target molecule can be performed by bright field observation and fluorescence observation, but may be performed only by fluorescence observation.
（封止液）
　封止液１８０としては、フッ素系オイル、シリコン系オイル、炭化水素系オイル、これらの混合物等が挙げられる。 (Sealing liquid)
Examples of the sealing liquid 180 include fluorine-based oil, silicon-based oil, hydrocarbon-based oil, and mixtures thereof.
　封止液１８０は疎水性であるが、若干の水の溶解性を有する。このため、ウェルアレイ１１２のウェル１１１に標的分子を含む反応液１７０を導入後、封止液１８０でウェル１１１を個別に封止した場合、各ウェル１１１に収容された反応液１７０の水分が封止液１８０に吸収される場合がある。特に、ウェル１１１中の反応液１７０を加熱した際には、水分の吸収が起こりやすい。 Sealing solution 180 is hydrophobic but has some water solubility. Therefore, when the reaction liquid 170 containing the target molecule is introduced into the wells 111 of the well array 112 and the wells 111 are individually sealed with the sealing liquid 180, the water of the reaction liquid 170 contained in each well 111 is sealed. It may be absorbed by the stop solution 180. In particular, when the reaction liquid 170 in the well 111 is heated, absorption of water is likely to occur.
　封止液１８０が吸収する水分の量は、封止液１８０の種類、封止液１８０が吸収する水の最大量（封止液１８０の最大含水量）、封止液１８０の体積、酵素反応を行う反応温度、酵素反応を行う反応時間、ウェルアレイ１１２を構成するウェル１１１の容積の合計等の影響を受ける。 The amount of water absorbed by the sealing solution 180 is the type of the sealing solution 180, the maximum amount of water absorbed by the sealing solution 180 (maximum water content of the sealing solution 180), the volume of the sealing solution 180, the enzyme reaction The reaction temperature at which the reaction is performed, the reaction time at which the enzyme reaction is performed, and the total volume of the wells 111 constituting the well array 112 are influenced.
　例えば、フッ素系オイルであるフロリナート（商標）ＦＣ－４０（シグマ社製）の２５℃における最大含水量は約７質量ｐｐｍであり、シリコーンオイルの２５℃における最大含水量は約２００ｐｐｍである。 For example, the maximum water content at 25 ° C. of Fluorinert® FC-40 (manufactured by Sigma), which is a fluorine-based oil, is about 7 mass ppm, and the maximum water content at 25 ° C. of silicone oil is about 200 ppm.
（偽陰性判定の発生の抑制）
　実施例において後述するように、発明者らは、標的分子の検出における偽陰性判定の発生と、封止液の体積（Ｖｏ）と、ウェルアレイを構成するウェルの容積（Ｖｄ）（ウェルアレイのウェル容積）の合計（Ｖｓｄ）と、の間に関連があることを見出した。そして、これらが下記式（１）を満たす条件であれば、偽陰性判定の発生が抑制される傾向にあることを明らかにした。
　０＜封止液の体積（Ｖｏ）／ウェルアレイのウェル容積の合計（Ｖｓｄ）≦１２，０００…（１） (Suppressing the occurrence of false negative judgment)
As will be described later in the Examples, the inventors have determined that occurrence of false negative determination in detection of a target molecule, volume of sealing solution (Vo), volume of wells constituting well array (Vd) (well array It was found that there was an association between the well volume) and the sum (Vsd). And it clarified that it was in the tendency for generation | occurrence | production of a false negative determination to be suppressed if these were the conditions with which following formula (1) is satisfy | filled.
0 <volume of sealing solution (Vo) / well volume of well array (Vsd) ≦ 12,000 (1)
　すなわち、上記式（１）を満たす範囲で、Ｖｏ／Ｖｓｄの値が小さいほど、偽陰性判定の発生が抑制される傾向にある。Ｖｏ／Ｖｓｄの値は、例えば１０，０００以下であってもよく、６，０００以下であってもよく、３，０００以下であってもよく、１，０００以下であってもよく、５００以下であってもよい。
　後述する本実施形態、および、以下の実施形態においては、検出デバイスが流路（内部空間）を有し、流路（内部空間）に封止液が導入される。流路（内部空間）はある程度の容積を有している方が検出デバイスの製造が容易であるという観点においては、流路（内部空間）に導入される封止液は、Ｖｏ／Ｖｓｄの値が１以上であることを満たすこと（換言すれば、式（１）において、１≦前記封止液の体積／前記ウェルアレイのウェル容積の合計≦１２，０００を満たすこと）が好ましい。しかしながら、本発明においては、封止液の体積は、ウェルアレイのウェルの容積の合計に対してできるだけ小さいことが好ましい。 That is, as the value of Vo / Vsd is smaller within the range satisfying the above equation (1), the occurrence of false negative determination tends to be suppressed. The value of Vo / Vsd may be, for example, 10,000 or less, 6,000 or less, 3,000 or less, 1,000 or less, or 500 or less. It may be
In the embodiment described below and the embodiments below, the detection device has a flow path (internal space), and the sealing liquid is introduced into the flow path (internal space). The sealing liquid introduced into the flow path (internal space) has a value of Vo / Vsd in that the flow path (internal space) has a certain volume to facilitate manufacture of the detection device. Preferably satisfies 1 or more (in other words, in the formula (1), 1 ≦ volume of the sealing solution / total of well volumes of the well array ≦ 12,000). However, in the present invention, the volume of the sealing solution is preferably as small as possible with respect to the sum of the volumes of the wells of the well array.
　なお、本実施形態、および、以下の実施形態においては、封止液の体積が検出デバイスの流路（内部空間）の容積と等しくてもよい。
　換言すれば、検出デバイスの流路(内部空間)の容積とウェルアレイのウェル容積の合計とが等しくてもよい。
　また、本実施形態、および、以下の実施形態においては、蓋材が注入口と排出口とを有している場合に、封止液の体積が、注入口の容積および排出口の容積および流路（内部空間）の容積の合計と等しくてもよい。
　換言すれば、注入口の容積および排出口の容積および流路（内部空間）の容積の合計が、ウェルアレイのウェル容積の合計と等しくてもよい。
　また、後述する実施形態に示すような排出口に接続された排液貯蔵部をさらに有している場合には、封止液の体積が、注入口の容積および排出口の容積および内部空間の容積および排液貯蔵部の容積の合計と等しくてもよい。
　換言すれば、注入口の容積および排出口の容積および内部空間の容積および排液貯蔵部の容積の合計が、ウェルアレイのウェル容積の合計と等しくてもよい。 In the present embodiment and the following embodiments, the volume of the sealing liquid may be equal to the volume of the flow path (internal space) of the detection device.
In other words, the volume of the flow path (inner space) of the detection device may be equal to the sum of the well volumes of the well array.
In the present embodiment and the following embodiments, when the lid has the inlet and the outlet, the volume of the sealing liquid is the volume of the inlet and the volume and the flow of the outlet. It may be equal to the sum of the volumes of the channels (internal space).
In other words, the sum of the volume of the inlet and the volume of the outlet and the volume of the flow path (internal space) may be equal to the total of the well volumes of the well array.
Further, in the case of further having a drainage storage part connected to the outlet as shown in the embodiment described later, the volume of the sealing liquid is the volume of the inlet and the volume of the outlet and the internal space thereof. It may be equal to the sum of the volume and the volume of the drainage reservoir.
In other words, the sum of the volume of the inlet and the volume of the outlet and the volume of the internal space and the volume of the drainage reservoir may be equal to the total of the well volumes of the well array.
　本実施形態に係る方法により、偽陰性判定の発生が抑制される理由は明らかではないが、上記式（１）を満たさない場合、各ウェル１１１に収容された反応液１７０の水分が封止液１８０に吸収され、酵素反応が抑制されること、あるいは、各ウェル１１１に収容された反応液１７０の水分が封止液１８０に吸収された結果、生成されたシグナルを正確に検出できなくなること等が考えられる。 Although the reason why the occurrence of false negative determination is suppressed by the method according to the present embodiment is not clear, when the above formula (1) is not satisfied, the moisture of the reaction liquid 170 contained in each well 111 is the sealing liquid The absorption of the enzyme reaction by 180 and the inhibition of the enzyme reaction, or the absorption of the water of the reaction solution 170 contained in each well 111 into the sealing solution 180, and thus the generated signal can not be accurately detected, etc. Is considered.
　本実施形態においては、上記式（１）において、封止液の体積（Ｖｏ）とは、流路１３０、導入口１４０及び排出口１５０に存在し、かつ、ウェル１１１と空間的に接している封止液１８０の体積の合計であると定義される。
　なお、以降、封止液の体積（Ｖｏ）を「封止液１８０の体積」と呼ぶことがある。
　封止液１８０の体積が少ない場合には、蓋材１２０が存在することにより、基材１１０に形成されたウェルアレイ１１２（ウェル１１１）の上面を効果的に覆うことができる。例えば、封止液１８０の体積が約１００μＬ以下の場合には、蓋材１２０が存在するとウェルアレイ１１２（ウェル１１１）の上面を効果的に覆うことが容易である。しかしながら、封止液１８０の体積が約１００μＬ以上の場合には、基材１１０に形成されたウェルアレイ１１２（ウェル１１１）の上面を覆うために蓋材１２０は必ずしも必要ではない。また、封止液１８０の体積が約１００μＬ以下の場合であっても、基材１１０に形成されたウェルアレイ１１２（ウェル１１１）の上面を覆うことができるのであれば、必ずしも蓋材１２０は必要ではない。 In the present embodiment, in the above formula (1), the volume (Vo) of the sealing liquid exists in the channel 130, the inlet 140, and the outlet 150, and is in spatial contact with the well 111. It is defined as the sum of the volumes of the sealing solution 180.
Hereinafter, the volume (Vo) of the sealing solution may be referred to as “volume of the sealing solution 180”.
When the volume of the sealing solution 180 is small, the presence of the lid member 120 can effectively cover the upper surface of the well array 112 (well 111) formed on the base 110. For example, in the case where the volume of the sealing solution 180 is about 100 μL or less, if the lid 120 is present, it is easy to effectively cover the upper surface of the well array 112 (well 111). However, when the volume of the sealing solution 180 is about 100 μL or more, the lid material 120 is not necessarily required to cover the top surface of the well array 112 (well 111) formed on the substrate 110. In addition, even if the volume of the sealing solution 180 is about 100 μL or less, the lid material 120 is necessarily required as long as the top surface of the well array 112 (well 111) formed on the substrate 110 can be covered. is not.
　本実施形態に係る方法のシグナルを生成する工程において、反応温度、反応時間等の反応条件は、反応液１７０によるシグナル増幅反応の効率によって決定される。例えば、ＩＣＡ法を利用する場合には５０℃～７５℃の範囲で１時間以内の加熱反応を行う。 In the step of generating a signal of the method according to the present embodiment, reaction conditions such as reaction temperature and reaction time are determined by the efficiency of the signal amplification reaction by the reaction liquid 170. For example, when using the ICA method, the heating reaction is performed within the range of 50 ° C. to 75 ° C. for 1 hour or less.
　温度が高くなると、ウェル１１１に収容された反応液１７０から封止液１８０への水分の溶け込み量が増えると考えられる。そこで、反応条件は７０℃以下であることが好ましい。しかしながら温度が低くなりすぎると酵素反応速度が低下する。このため、反応条件は５５℃以上であることが好ましい。 It is considered that the amount of dissolution of water from the reaction solution 170 contained in the well 111 into the sealing solution 180 increases as the temperature rises. Therefore, the reaction conditions are preferably 70 ° C. or less. However, if the temperature is too low, the enzyme reaction rate will decrease. Therefore, the reaction conditions are preferably 55 ° C. or higher.
　また、反応を行う加熱時間が長くなると、ウェル１１１に収容された反応液１７０から封止液１８０への水分の溶け込み量が増えると考えられる。そこで、反応時間は、１時間以下が好ましく、３０分以下がより好ましく、２０分以下が更に好ましく、１５分以下が特に好ましい。 In addition, it is considered that the amount of dissolution of water from the reaction liquid 170 contained in the well 111 into the sealing liquid 180 increases as the heating time for performing the reaction becomes longer. Therefore, the reaction time is preferably 1 hour or less, more preferably 30 minutes or less, still more preferably 20 minutes or less, and particularly preferably 15 minutes or less.
　また、封止液の２５℃における最大含水量は、１～１０００質量ｐｐｍであり、１０質量ｐｐｍ以下であることが好ましく、８質量ｐｐｍ以下であることがより好ましく、７質量ｐｐｍ以下であることが更に好ましい。封止液の２５℃における好ましい最大含水量には下限は存在しないが、入手可能な封止液の最大含水量の下限は約１質量ｐｐｍ程度であると考えられる。 Further, the maximum water content of the sealing liquid at 25 ° C. is 1 to 1000 mass ppm, preferably 10 mass ppm or less, more preferably 8 mass ppm or less, and 7 mass ppm or less Is more preferred. There is no lower limit to the preferable maximum water content of the sealing solution at 25 ° C., but the lower limit of the maximum water content of the available sealing solution is considered to be about 1 mass ppm or so.
　上記式（１）において、ウェルアレイのウェル容積の合計（Ｖｓｄ）は、ウェル１１１の容積（Ｖｄ）とウェルアレイ１１２中に存在するウェル１１１の総数（Ｓ）との積により求めることができる。 In the above equation (1), the total well volume (Vsd) of the well array can be determined by the product of the volume (Vd) of the well 111 and the total number (S) of the wells 111 present in the well array 112.
　Ｖｄが大きいと、封止液１８０中に水分が吸収されても水分の影響を受けにくくなると考えられる。また、Ｖｓｄが大きいと、封止液１８０中の水分が飽和しやすくなるため、ウェル１１１に収容された反応液１７０から封止液１８０への水分の吸収速度が低下すると考えられる。 If Vd is large, even if water is absorbed in the sealing solution 180, it is considered that the influence of the water becomes difficult. In addition, when V sd is large, the water in the sealing solution 180 is likely to be saturated, so it is considered that the rate of absorption of water from the reaction solution 170 contained in the well 111 into the sealing solution 180 is reduced.
　実施例において後述するように、例えば、封止液の体積（Ｖｏ）とウェルアレイのウェル容積の合計（Ｖｓｄ）との比（Ｖｏ／Ｖｓｄ）は、６６℃で３０分間反応させる場合においては１０，０００以下であることが好ましい。 As described later in the Examples, for example, the ratio (Vo / Vsd) of the volume (Vo) of the sealing solution to the total of the well volumes of the well array (Vsd) is 10 in the case of reacting at 66 ° C. for 30 minutes. It is preferable that it is 1,000 or less.
　封止液１８０中に水分が吸収されて最大含水量に達した場合（水分が飽和した場合）であっても、封止液１８０から大気中に水分が放出された場合には、封止液１８０は再び水分を吸収することが可能となり、ウェル１１１に収容された反応液１７０から封止液１８０へと更に水分が吸収されると考えられる。 Even if water is absorbed in the sealing solution 180 and reaches the maximum water content (when the water is saturated), even if the sealing solution 180 releases water into the atmosphere, the sealing solution It is considered that 180 can absorb water again, and water is further absorbed from the reaction liquid 170 stored in the well 111 to the sealing liquid 180.
　これを抑制するために、封止液１８０が大気に直接接しないように、親水性の液体で封止液１８０に蓋をしてもよい。例えば、図５に示すように、導入口１４０及び排出口１５０に反応液１７０を残しておくことにより、封止液１８０から大気中への水分の放出を防ぐことができる。排出口１５０のみに反応液１７０を残しておくだけでも、同等ではないものの同様の効果を得ることができるので、構わない。 In order to suppress this, the sealing liquid 180 may be covered with a hydrophilic liquid so that the sealing liquid 180 is not in direct contact with the atmosphere. For example, as shown in FIG. 5, by leaving the reaction liquid 170 in the inlet 140 and the outlet 150, the release of water from the sealing liquid 180 into the atmosphere can be prevented. Only leaving the reaction liquid 170 only at the discharge port 150 is acceptable because similar effects can be obtained although they are not equivalent.
　本実施形態に係る方法は、ウェル内に収容され、封止液で封止された液体の減少を抑制する方法であるということもできる。
　また、本実施形態に係る検出デバイスによれば、本実施形態に係る方法が好適に実施できる。 The method according to the present embodiment can also be said to be a method for suppressing the reduction of the liquid contained in the well and sealed with the sealing liquid.
Further, according to the detection device according to the present embodiment, the method according to the present embodiment can be suitably implemented.
（検出デバイス、および、標的分子の検出における偽陰性判定の発生を抑制する方法（第２実施形態））
　以下には、本発明の第２実施形態に係る検出デバイス、および、第２実施形態に係る検出デバイスを用いた標的分子の検出における偽陰性判定の発生を抑制する方法を説明する。
　図８は、本発明の第２実施形態に係る検出デバイスを模式的に示す断面図である。
　なお、図８において、上記第１実施形態に示す構成要素と同じ構成要素には、第１実施形態の場合と同じ符号を付し、その詳細な説明は省略する。
　また、本実施形態に係る検出デバイス２００を用いた標的分子の検出における偽陰性判定の発生を抑制する方法は、上記第１実施形態に係る方法と同様である部分については、以下、省略する。 (Detection device and method for suppressing occurrence of false negative determination in detection of target molecule (second embodiment))
Hereinafter, a detection device according to the second embodiment of the present invention and a method for suppressing the occurrence of false negative determination in detection of a target molecule using the detection device according to the second embodiment will be described.
FIG. 8 is a cross-sectional view schematically showing a detection device according to a second embodiment of the present invention.
In FIG. 8, the same components as the components shown in the first embodiment are given the same reference numerals as in the first embodiment, and the detailed description thereof will be omitted.
Moreover, the method of suppressing the occurrence of false negative determination in detection of a target molecule using the detection device 200 according to the present embodiment is omitted for the same parts as the method according to the first embodiment.
（検出デバイス（第２実施形態））
　本実施形態に係る検出デバイス２００は、基材１１０と、基材１１０上に設けられた蓋材２２０と、を備えている。検出デバイス２００を用いて、試料中の標的分子をデジタル計測により検出することができる。
　本実施形態に係る検出デバイス２００は、標的分子の検出における偽陰性判定の発生を抑制する方法に適した構成を有している。 (Detection Device (Second Embodiment))
The detection device 200 according to the present embodiment includes a base 110 and a lid 220 provided on the base 110. The detection device 200 can be used to detect target molecules in a sample by digital measurement.
The detection device 200 according to the present embodiment has a configuration suitable for a method of suppressing the occurrence of false negative determination in detection of a target molecule.
　基材１１０は、上記第１実施形態と同様の基材であるため、以下省略する。 The base 110 is the same as that of the first embodiment, and thus will not be described.
　蓋材２２０は、基材１１０に溶着又は接着されている。蓋材２２０と基材１１０との間の空間は、流体１６０が導入される内部空間２３０を形成している。
　なお、本実施形態に係る内部空間２３０は、上記第１実施形態のような流路１３０であってもよい。
　例えば、蓋材２２０が、基材上面から蓋材内面天井までが一定の長さ（距離）を有するように蓋材側面である凸部が図８に図示されるよりも長く形成されており、多量の封止液１８０が注がれるように検出デバイス２００に内部空間２３０が形成される場合であってもよい。
　なお、本実施形態における内部空間（流路）２３０の高さは、上記第１実施形態と同様であってもよい。
　検出デバイス２００に形成される内部空間２３０の形状は、ウェルアレイ１１２を封止可能な形状として形成されていればよく、特に限定されない。
　例えば、蓋材２２０が、有底円筒状であってもよく、上面のない箱状であってもよい。その場合、蓋材２２０の底面に導入口２４０及び排出口２５０が形成される。また、蓋材２２０が、内部空間（流路）２３０の高さと比べて大幅に厚くてもよい。 The lid 220 is welded or adhered to the substrate 110. A space between the lid 220 and the base 110 forms an internal space 230 into which the fluid 160 is introduced.
The internal space 230 according to the present embodiment may be the flow channel 130 as in the first embodiment.
For example, a convex portion which is a side surface of the lid is formed to be longer than illustrated in FIG. 8 so that the lid 220 has a certain length (distance) from the upper surface of the base to the inner surface of the lid. The internal space 230 may be formed in the detection device 200 so that a large amount of sealing liquid 180 is poured.
In addition, the height of the internal space (flow path) 230 in the present embodiment may be the same as that in the first embodiment.
The shape of the internal space 230 formed in the detection device 200 may be any shape that can seal the well array 112, and is not particularly limited.
For example, the lid 220 may have a bottomed cylindrical shape or a box shape without an upper surface. In that case, the inlet 240 and the outlet 250 are formed on the bottom of the lid 220. Also, the lid 220 may be much thicker than the height of the internal space (flow path) 230.
　検出デバイス２００に形成される内部空間２３０は、ウェルアレイ１１２を封止可能な形状として形成されていればよく、特に限定されない。
　蓋材２２０は、内部空間２３０に流体１６０を導入する導入口２４０及び内部空間２３０から流体１６０を排出する排出口２５０を備えている。導入口２４０から導入された流体１６０は、ウェルアレイ１１２の表面を流れた後、排出口２５０から排出される。
　なお、本実施形態に係る検出デバイス２００は、図８に示すように、封止液１８０が留まるように構成された場所が、主に基材１１０と蓋材２２０とで形成された内部空間２３０になるように形成されている。
　すなわち、本実施形態では、上記第１実施形態のように、蓋材１２０上部の導入口１４０に近い位置に封止液が貯留される空間を有していなくともよい。
　換言すれば、本実施形態においては、図８に示すように蓋材２２０の上面における一端（第一端）に、流体１６０の注液が可能な大きさを有するように導入口２４０が形成され、かつ、図８に示すように蓋材２２０の上面における他端（第二端）に、流体１６０の排液が可能な大きさを有するように排出口２５０が形成されていればよい。
　本実施形態においては、蓋となる部材に、導入口２４０、および、排出口２５０を直接形成することにより蓋材２２０を形成できるため、簡易に検出デバイス２００を作成することができる。
　また、蓋材２２０の形状を簡易に設計できるため、用途に応じて内部空間２３０が意図した容量を有するように、検出デバイス２００の形状、構成を変更しやすい点で利点がある。 The internal space 230 formed in the detection device 200 may be formed as a shape that can seal the well array 112, and is not particularly limited.
The lid 220 includes an inlet 240 for introducing the fluid 160 into the inner space 230 and an outlet 250 for discharging the fluid 160 from the inner space 230. The fluid 160 introduced from the inlet 240 flows through the surface of the well array 112 and is then discharged from the outlet 250.
In the detection device 200 according to the present embodiment, as shown in FIG. 8, the space configured to retain the sealing liquid 180 mainly includes the internal space 230 formed by the base material 110 and the lid member 220. It is formed to be
That is, in the present embodiment, as in the first embodiment, it is not necessary to have a space where the sealing liquid is stored at a position close to the inlet 140 at the top of the lid member 120.
In other words, in the present embodiment, as shown in FIG. 8, the inlet 240 is formed at one end (first end) of the upper surface of the lid member 220 so as to have a size capable of injecting the fluid 160. And, as shown in FIG. 8, the other end (second end) on the upper surface of the lid 220 may be formed with the discharge port 250 so as to have a size capable of discharging the fluid 160.
In the present embodiment, since the lid 220 can be formed by directly forming the inlet 240 and the outlet 250 in a member to be a lid, the detection device 200 can be easily produced.
Further, since the shape of the lid member 220 can be simply designed, there is an advantage in that the shape and configuration of the detection device 200 can be easily changed so that the internal space 230 has the intended capacity according to the application.
　蓋材２２０の材質は特に限定されないが、シクロオレフィンポリマー、シクロオレフィンコポリマー等の熱可塑性樹脂が挙げられる。蓋材２２０の形成は、熱可塑性樹脂の流動体を、成形型を用いて成形すること等により行うことができる。 The material of the lid 220 is not particularly limited, and examples thereof include thermoplastic resins such as cycloolefin polymers and cycloolefin copolymers. The lid member 220 can be formed by molding a fluid of a thermoplastic resin using a molding die.
　基材１１０の材質は、上記第１実施形態と同様であり、内部空間２３０に送液される流体１６０に対し耐性のある材質であることが好ましい。
　流体１６０としては、上記第１実施形態と同様に、標的分子を含む反応液１７０、封止液１８０等が挙げられる。
　また、検出するシグナルが蛍光である場合、基材１１０の材質は、ウェル１１１の内部に発生した蛍光シグナルを観察することが可能な光透過性樹脂であることが好ましく、更に自家蛍光の少ない樹脂であることが好ましい。 The material of the substrate 110 is the same as that of the first embodiment, and is preferably a material resistant to the fluid 160 sent to the internal space 230.
As the fluid 160, the reaction liquid 170 containing a target molecule, the sealing liquid 180, etc. are mentioned similarly to the said 1st Embodiment.
When the signal to be detected is fluorescence, the material of the substrate 110 is preferably a light transmitting resin capable of observing the fluorescence signal generated inside the well 111, and a resin with less autofluorescence Is preferred.
　基材１１０の材質としては、上記第１実施形態と同様に、例えば、シクロオレフィンポリマー、シクロオレフィンコポリマー、シリコン、ポリプロピレン、ポリカーボネート、ポリスチレン、ポリエチレン、ポリ酢酸ビニル、フッ素樹脂、アモルファスフッ素樹脂等が挙げられる。 As a material of the substrate 110, for example, cycloolefin polymer, cycloolefin copolymer, silicon, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluorocarbon resin, amorphous fluorocarbon resin, etc. may be mentioned as in the first embodiment. Be
　基材１１０の、板厚方向の一方の面（第一面）に複数のウェル１１１が形成される。ウェル１１１の形成は、射出成形、熱インプリント、光インプリント等により行うことができる。 A plurality of wells 111 are formed on one surface (first surface) in the thickness direction of the substrate 110. The formation of the well 111 can be performed by injection molding, thermal imprint, optical imprint, or the like.
　また、フッ素樹脂を用いて基材１１０の表面にウェル１１１を形成することもできる。
　例えば、基材１１０の上にＣＹＴＯＰ（登録商標）（旭硝子）等のフッ素樹脂で形成された層を配置し、更にＣＹＴＯＰ（登録商標）の層に微小な孔を形成し、当該孔をウェル１１１とすることができる。 Alternatively, the well 111 can be formed on the surface of the base 110 by using a fluorine resin.
For example, a layer formed of a fluorocarbon resin such as CYTOP (registered trademark) (Asahi Glass) is disposed on the base material 110, and further micropores are formed in the layer of CYTOP (registered trademark). It can be done.
　蓋材２２０は、組立時に基材１１０に向けられる面に凸部２２１を有するように成形される。また、蓋材２２０には導入口２４０及び排出口２５０も形成される。続いて、基材１１０においてウェル１１１が開口する面（第一面）に蓋材２２０の凸部２２１が接するように、蓋材２２０と基材１１０とが重ねられる。続いて、レーザー溶着等により蓋材２２０と基材１１０とを溶着し、検出デバイス２００を製造することができる。これにより、蓋材２２０と基材１１０との間に流体１６０が流れる、または、流体１６０が貯留される内部空間２３０が形成される。 The lid 220 is shaped so as to have a projection 221 on the surface directed to the base 110 during assembly. Further, an inlet 240 and an outlet 250 are also formed in the lid 220. Subsequently, the lid member 220 and the base member 110 are overlapped so that the convex portion 221 of the lid member 220 is in contact with the surface (first surface) where the well 111 is opened in the base member 110. Subsequently, the detection device 200 can be manufactured by welding the lid 220 and the base 110 by laser welding or the like. Thereby, an internal space 230 in which the fluid 160 flows or the fluid 160 is stored is formed between the lid 220 and the base 110.
（標的分子の検出における偽陰性判定の発生を抑制する方法（第２実施形態））
　本実施形態においては、第１実施形態の項に記載した上記式（１）において、封止液の体積（Ｖｏ）とは、内部空間２３０、導入口２４０、および排出口２５０に存在し、かつ、ウェル１１１と空間的に接している封止液１８０の体積の合計であると定義される。
　なお、本実施形態において、導入口２４０および排出口２５０の容積が無視できるほど小さい場合には、上記式（１）における封止液の体積（Ｖｏ）は、流路２３０に存在し、かつ、ウェル１１１と空間的に接している封止液１８０の体積であると定義してもよい。
　なお、検出デバイス２００を用いた本実施形態に係る標的分子の検出における偽陰性判定の発生を抑制する方法における（標的分子の検出）、（封止液）、および（偽陰性判定の発生の抑制）などの項目の説明については、上記第１実施形態と同様であるため、省略する。 (Method of suppressing occurrence of false negative determination in detection of target molecule (second embodiment))
In the present embodiment, in the formula (1) described in the section of the first embodiment, the volume (Vo) of the sealing liquid exists in the internal space 230, the inlet 240, and the outlet 250, and , Defined as the sum of the volumes of the sealing liquid 180 in spatial contact with the well 111.
In the present embodiment, when the volumes of the inlet 240 and the outlet 250 are so small that they can be ignored, the volume (Vo) of the sealing liquid in the above equation (1) exists in the flow channel 230, and It may be defined as the volume of the sealing liquid 180 in spatial contact with the well 111.
In the method of suppressing the occurrence of false negative determination in detection of a target molecule according to the present embodiment using the detection device 200 (detection of target molecule), (sealing liquid), and (suppressing occurrence of false negative determination) Description of items such as) is the same as that of the first embodiment, and thus will not be repeated.
（検出デバイス、および、標的分子の検出における偽陰性判定の発生を抑制する方法（第３実施形態））
　以下には、本発明の第３実施形態に係る検出デバイス、および、第３実施形態に係る検出デバイスを用いた標的分子の検出における偽陰性判定の発生を抑制する方法を説明する。
　図９は、本発明の第３実施形態に係る検出デバイスを模式的に示す断面図である。
　なお、図９において、上記第１実施形態に示す構成要素と同じ構成要素には、第１実施形態の場合と同じ符号を付し、その詳細な説明は省略する。
　また、本実施形態に係る検出デバイス３００を用いた標的分子の検出における偽陰性判定の発生を抑制する方法は、上記第１実施形態に係る方法と同様である部分については、以下、省略する。 (Detection device and method for suppressing occurrence of false negative determination in detection of target molecule (third embodiment))
Hereinafter, a detection device according to the third embodiment of the present invention and a method for suppressing the occurrence of false negative determination in detection of a target molecule using the detection device according to the third embodiment will be described.
FIG. 9 is a cross-sectional view schematically showing a detection device according to a third embodiment of the present invention.
In FIG. 9, the same components as the components shown in the first embodiment are given the same reference numerals as in the first embodiment, and the detailed description thereof will be omitted.
Moreover, the method for suppressing the occurrence of false negative determination in detection of a target molecule using the detection device 300 according to the present embodiment is omitted for the same part as the method according to the first embodiment.
（検出デバイス（第３実施形態））
　本実施形態に係る検出デバイス３００は、基材１１０と、基材１１０上に設けられた蓋材３２０と、を備えている。検出デバイス３００を用いて、試料中の標的分子をデジタル計測により検出することができる。
　本実施形態に係る検出デバイス３００は、標的分子の検出における偽陰性判定の発生を抑制する方法に適した構成を有している。 (Detection Device (Third Embodiment))
The detection device 300 according to the present embodiment includes a base 110 and a lid 320 provided on the base 110. The detection device 300 can be used to detect target molecules in a sample by digital measurement.
The detection device 300 according to the present embodiment has a configuration suitable for a method of suppressing the occurrence of false negative determination in detection of a target molecule.
　基材１１０は、上記第一実施形態と同様の基材であるため、以下省略する。 The substrate 110 is the same as that of the first embodiment, and thus will not be described.
　蓋材３２０は、基材１１０に溶着又は接着されている。蓋材３２０と基材１１０との間の空間は、流体１６０が導入される内部空間３３０を形成している。
　なお、本実施形態に係る内部空間３３０は、上記第１実施形態のような流路１３０であってもよい。
　例えば、蓋材３２０が、基材上面から蓋材内面天井までが一定の長さ（距離）を有するように蓋材側面である凸部が図９に図示されるよりも長く形成されており、多量の封止液１８０が注がれるように検出デバイス３００に内部空間３３０が形成される場合であってもよい。
　なお、本実施形態における内部空間（流路）３３０の高さは、上記第１実施形態と同様であってもよい。
　検出デバイス３００に形成される内部空間３３０の形状は、ウェルアレイ１１２を封止可能な形状として形成されていればよく、特に限定されない。
　例えば、蓋材３２０が、有底円筒状であってもよく、上面のない箱状であってもよい。その場合、蓋材３２０の底面に注入口３４０及び排出口３５０が形成される。また、蓋材３２０が、内部空間（流路）３３０の高さと比べて大幅に厚くてもよい。 The lid member 320 is welded or adhered to the base 110. A space between the lid 320 and the base 110 forms an internal space 330 into which the fluid 160 is introduced.
The internal space 330 according to the present embodiment may be the flow passage 130 as in the first embodiment.
For example, a convex portion which is a side surface of the lid is formed longer than that illustrated in FIG. 9 so that the lid 320 has a certain length (distance) from the upper surface of the base to the inner surface of the lid. The internal space 330 may be formed in the detection device 300 so that a large amount of sealing solution 180 is poured.
The height of the internal space (flow path) 330 in the present embodiment may be the same as that in the first embodiment.
The shape of the internal space 330 formed in the detection device 300 may be any shape that can seal the well array 112, and is not particularly limited.
For example, the lid member 320 may have a bottomed cylindrical shape or a box shape without an upper surface. In that case, the inlet 340 and the outlet 350 are formed on the bottom of the lid 320. Also, the lid 320 may be much thicker than the height of the internal space (flow path) 330.
　蓋材３２０は、内部空間３３０に流体１６０を導入する注入口（導入口）３４０及び内部空間３３０から流体１６０を排出する排出口３５０を備えている。注入口３４０から導入された流体１６０は、ウェルアレイ１１２の表面を流れた後、排出口３５０から排出される。
　なお、本実施形態に係る検出デバイス３００は、図９に示すように、反応液１７０と封止液１８０が導入されるように構成され、かつ、検出デバイス３００の厚み方向に一定の長さを有する注入口３４０と、余剰の封止液などが排出されるように構成され、かつ、検出デバイス３００の厚み方向に一定の長さを有する排出口３５０を有しており、封止液１８０が留まる場所は内部空間（流路）３３０と注入口３４０と排出口３５０である。
　注入口３４０における一定の長さとは、検出デバイス３００の内部で蓋材３２０の内面天井から注入口３４０の最上部までの間に無視できない程度の長さを有していればよい。
　同様に、排出口３５０における一定の長さとは、検出デバイス３００の内部で蓋材３２０の内面天井から注入口３４０の最上部までの間に無視できない程度の長さを有していればよい。
　なお、「無視できない程度の長さ」とは、検出デバイス３００を肉眼で観察した際に、視認できる長さ以上の長さを言う。
　例えば、本実施形態においては、注入口３４０と排出口３５０は、図９のように突出した形状を有し、突出した注入口３４０、および、突出した排出口３５０を形成していてもよい。
　また、本実施形態においては、蓋材３２０の上面を構成する蓋材上部部材が、図９に示す蓋材よりも厚みのある構造を有しており、蓋材３２０の内面天井から注入口３４０の最上部および排出口３５０の最上部までに一定の長さ（距離）を有する場合には、注入口３４０と排出口３５０は、蓋材に単なる穴として形成されていてもよい。
　注入口３４０は、円筒状であってもよく、ピペット等で反応液と封止液を導入することが容易になるように、すり鉢状、ロート形状のような逆円錐状を有していてもよい。
　また、注入口３４０と排出口３５０の容積が、無視できるくらい小さい場合（例えば、検出デバイス３００の厚み方向における注入口３４０および排出口３５０の長さが短い場合）には、封止液１８０の体積は内部空間３３０の容積のみから算出してもよい。 The lid 320 includes an inlet (inlet) 340 for introducing the fluid 160 into the inner space 330 and an outlet 350 for discharging the fluid 160 from the inner space 330. The fluid 160 introduced from the inlet 340 flows on the surface of the well array 112 and then is discharged from the outlet 350.
In addition, as shown in FIG. 9, the detection device 300 according to the present embodiment is configured to be introduced with the reaction liquid 170 and the sealing liquid 180, and has a predetermined length in the thickness direction of the detection device 300. And a discharge port 350 having a predetermined length in the thickness direction of the detection device 300, and the seal liquid 180 is formed. The places to stay are the internal space (flow path) 330, the inlet 340 and the outlet 350.
The fixed length at the inlet 340 may have a non-negligible length between the inner surface ceiling of the lid 320 and the top of the inlet 340 inside the detection device 300.
Similarly, the fixed length at the discharge port 350 may have a non-negligible length from the inner surface ceiling of the lid 320 to the top of the injection port 340 inside the detection device 300.
In addition, "the length which can not be disregarded" means the length beyond the length which can be visually recognized, when observing detection device 300 with the naked eye.
For example, in the present embodiment, the inlet 340 and the outlet 350 may have a projecting shape as shown in FIG. 9, and may form the projecting inlet 340 and the projecting outlet 350.
Further, in the present embodiment, the lid upper member constituting the upper surface of the lid 320 has a structure thicker than the lid shown in FIG. The inlet 340 and the outlet 350 may be formed as a simple hole in the lid material if they have a fixed length (distance) up to the top of the and the top of the outlet 350.
The inlet 340 may have a cylindrical shape, or may have a conical or inverted conical shape such as a funnel shape so that it is easy to introduce the reaction solution and the sealing solution with a pipette or the like. Good.
In addition, when the volumes of the inlet 340 and the outlet 350 are negligibly small (for example, when the lengths of the inlet 340 and the outlet 350 in the thickness direction of the detection device 300 are short), The volume may be calculated only from the volume of the internal space 330.
　蓋材３２０の材質は特に限定されないが、シクロオレフィンポリマー、シクロオレフィンコポリマー等の熱可塑性樹脂が挙げられる。蓋材３２０の形成は、熱可塑性樹脂の流動体を、成形型を用いて成形すること等により行うことができる。 The material of the lid member 320 is not particularly limited, and examples thereof include thermoplastic resins such as cycloolefin polymers and cycloolefin copolymers. The lid member 320 can be formed by molding a thermoplastic resin fluid using a molding die.
　基材１１０の材質は、上記第１実施形態と同様であり、内部空間３３０に送液される流体１６０に対し耐性のある材質であることが好ましい。
　流体１６０としては、上記第１実施形態と同様に、標的分子を含む反応液１７０、封止液１８０等が挙げられる。
　また、検出するシグナルが蛍光である場合、基材１１０の材質は、ウェル１１１の内部に発生した蛍光シグナルを観察することが可能な光透過性樹脂であることが好ましく、更に自家蛍光の少ない樹脂であることが好ましい。 The material of the substrate 110 is the same as that of the first embodiment, and is preferably a material resistant to the fluid 160 fed to the internal space 330.
As the fluid 160, the reaction liquid 170 containing a target molecule, the sealing liquid 180, etc. are mentioned similarly to the said 1st Embodiment.
When the signal to be detected is fluorescence, the material of the substrate 110 is preferably a light transmitting resin capable of observing the fluorescence signal generated inside the well 111, and a resin with less autofluorescence Is preferred.
　基材１１０の材質としては、上記第１実施形態と同様に、例えば、シクロオレフィンポリマー、シクロオレフィンコポリマー、シリコン、ポリプロピレン、ポリカーボネート、ポリスチレン、ポリエチレン、ポリ酢酸ビニル、フッ素樹脂、アモルファスフッ素樹脂等が挙げられる。 As a material of the substrate 110, for example, cycloolefin polymer, cycloolefin copolymer, silicon, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluorocarbon resin, amorphous fluorocarbon resin, etc. may be mentioned as in the first embodiment. Be
　基材１１０の、板厚方向の一方の面（第一面）に複数のウェル１１１が形成される。ウェル１１１の形成は、射出成形、熱インプリント、光インプリント等により行うことができる。 A plurality of wells 111 are formed on one surface (first surface) in the thickness direction of the substrate 110. The formation of the well 111 can be performed by injection molding, thermal imprint, optical imprint, or the like.
　また、フッ素樹脂を用いて基材１１０の表面にウェル１１１を形成することもできる。
　例えば、基材１１０の上にＣＹＴＯＰ（登録商標）（旭硝子）等のフッ素樹脂で形成された層を配置し、更にＣＹＴＯＰ（登録商標）の層に微小な孔を形成し、当該孔をウェル１１１とすることができる。 Alternatively, the well 111 can be formed on the surface of the base 110 by using a fluorine resin.
For example, a layer formed of a fluorocarbon resin such as CYTOP (registered trademark) (Asahi Glass) is disposed on the base material 110, and further micropores are formed in the layer of CYTOP (registered trademark). It can be done.
　蓋材３２０は、組立時に基材１１０に向けられる面に凸部３２１を有するように成形される。また、蓋材３２０には導入口３４０及び排出口３５０も形成される。続いて、基材１１０においてウェル１１１が開口する面（第一面）に蓋材３２０の凸部３２１が接するように、蓋材３２０と基材１１０とが重ねられる。続いて、レーザー溶着等により蓋材３２０と基材１１０とを溶着し、検出デバイス３００を製造することができる。これにより、蓋材３２０と基材１１０との間に流体１６０が流れる、または、流体１６０が貯留される内部空間３３０が形成される。 The lid member 320 is formed to have a convex portion 321 on the surface directed to the substrate 110 at the time of assembly. Further, an inlet 340 and an outlet 350 are also formed in the lid member 320. Subsequently, the lid material 320 and the base material 110 are overlapped so that the convex portion 321 of the lid material 320 is in contact with the surface (first surface) where the well 111 is opened in the base material 110. Subsequently, the detection device 300 can be manufactured by welding the lid member 320 and the base 110 by laser welding or the like. Thereby, an internal space 330 in which the fluid 160 flows or the fluid 160 is stored is formed between the lid 320 and the base 110.
（標的分子の検出における偽陰性判定の発生を抑制する方法（第３実施形態））
　本実施形態においては、第１実施形態の項に記載した上記式（１）において、封止液の体積（Ｖｏ）とは、内部空間３３０、導入口３４０、および排出口３５０に存在し、かつ、ウェル１１１と空間的に接している封止液１８０の体積の合計であると定義される。
　なお、本実施形態に係る標的分子の検出における偽陰性判定の発生を抑制する方法における（標的分子の検出）、（封止液）、および（偽陰性判定の発生の抑制）などの項目の説明については、上記第１実施形態と同様であるため、省略する。 (Method of suppressing occurrence of false negative determination in detection of target molecule (third embodiment))
In the present embodiment, in the formula (1) described in the section of the first embodiment, the volume (Vo) of the sealing liquid is present in the internal space 330, the inlet 340, and the outlet 350, and , Defined as the sum of the volumes of the sealing liquid 180 in spatial contact with the well 111.
Description of items such as (detection of target molecule), (sealing liquid), and (suppression of generation of false negative determination) in the method of suppressing occurrence of false negative determination in detection of a target molecule according to the present embodiment The second embodiment is the same as the first embodiment, and thus will not be described.
（検出デバイス、および、標的分子の検出における偽陰性判定の発生を抑制する方法（第４実施形態））
　以下には、本発明の第４実施形態に係る検出デバイス、および、第４実施形態に係る検出デバイスを用いた標的分子の検出における偽陰性判定の発生を抑制する方法を説明する。
　図１０は、本発明の第４実施形態に係る検出デバイスを模式的に示す断面図である。
　なお、図１０において、上記第１実施形態に示す構成要素と同じ構成要素には、第１実施形態の場合と同じ符号を付し、その詳細な説明は省略する。
　また、本実施形態に係る検出デバイス４００を用いた標的分子の検出における偽陰性判定の発生を抑制する方法は、上記第１実施形態に係る方法と同様である部分については、以下、省略する。 (Detection device and method for suppressing occurrence of false negative determination in detection of target molecule (fourth embodiment))
Hereinafter, a detection device according to the fourth embodiment of the present invention and a method for suppressing the occurrence of false negative determination in detection of a target molecule using the detection device according to the fourth embodiment will be described.
FIG. 10 is a cross-sectional view schematically showing a detection device according to a fourth embodiment of the present invention.
In FIG. 10, the same components as the components shown in the first embodiment are indicated by the same reference numerals as in the first embodiment, and the detailed description thereof will be omitted.
In addition, a method for suppressing the occurrence of false negative determination in detection of a target molecule using the detection device 400 according to the present embodiment is omitted for the same part as the method according to the first embodiment.
（検出デバイス（第４実施形態））
　本実施形態に係る検出デバイス４００は、基材１１０と、基材１１０上に設けられた蓋材４２０と、を備えている。検出デバイス４００を用いて、試料中の標的分子をデジタル計測により検出することができる。 (Detection Device (Fourth Embodiment))
The detection device 400 according to the present embodiment includes a base 110 and a lid 420 provided on the base 110. The detection device 400 can be used to detect target molecules in a sample by digital measurement.
　基材１１０は、上記第一実施形態と同様の基材であるため、以下省略する。 The substrate 110 is the same as that of the first embodiment, and thus will not be described.
　蓋材４２０は、基材１１０に溶着又は接着されている。蓋材４２０と基材１１０との間の空間は、流体１６０が流れる内部空間４３０を形成している。
　なお、本実施形態に係る内部空間４３０は、上記第１実施形態のような流路１３０であってもよい。
　例えば、蓋材４２０が、基材上面から蓋材内面天井までが一定の長さ（距離）を有するように、蓋材側面である凸部が、図１０に図示されるよりも長く形成されており、上面から蓋材内面天井までが一定の長さ（距離）を有するように、蓋材側面である凸部が一定の長さを有して形成されており、多量の封止液１８０が注がれるように検出デバイス４００に内部空間４３０が形成される場合であってもよい。
　なお、本実施形態における内部空間（流路）４３０の高さは、上記第１実施形態と同様であってもよい。
　検出デバイス４００に形成される内部空間４３０の形状は、ウェルアレイ１１２を封止可能な形状として形成されていればよく、特に限定されない。
　例えば、蓋材４２０が、有底円筒状であってもよく、上面のない箱状であってもよい。その場合、蓋材４２０の底面に注入口４４０及び排出口４５０が形成される。また、蓋材４２０が、内部空間（流路）４３０の高さと比べて大幅に厚くてもよい。 The lid member 420 is welded or adhered to the substrate 110. A space between the lid 420 and the base 110 forms an internal space 430 through which the fluid 160 flows.
The internal space 430 according to the present embodiment may be the flow channel 130 as in the first embodiment.
For example, the convex portion which is the side surface of the lid is formed longer than illustrated in FIG. 10 so that the lid 420 has a certain length (distance) from the upper surface of the base to the inner surface of the lid. The convex portion which is the side surface of the lid is formed to have a predetermined length so that the distance from the upper surface to the lid inner surface ceiling is constant, and a large amount of the sealing liquid 180 is The inner space 430 may be formed in the detection device 400 to be poured.
The height of the internal space (flow path) 430 in the present embodiment may be the same as that in the first embodiment.
The shape of the inner space 430 formed in the detection device 400 may be any shape that can seal the well array 112, and is not particularly limited.
For example, the lid 420 may have a bottomed cylindrical shape or a box shape without an upper surface. In that case, the inlet 440 and the outlet 450 are formed on the bottom of the lid 420. Also, the lid 420 may be much thicker than the height of the internal space (flow path) 430.
　蓋材４２０は、内部空間４３０に流体１６０を導入する注入口（導入口）４４０及び内部空間４３０から流体１６０を排出する排出口４５０を備えている。注入口４４０から導入された流体１６０は、ウェルアレイ１１２の表面を流れた後、排出口４５０から排出される。
　なお、本実施形態に係る検出デバイス４００は、図１０に示すように、反応液１７０と封止液１８０が導入されるように構成され、かつ、検出デバイス４００の厚み方向に一定の長さを有する注入口４４０と、余剰の封止液などが排出されるように構成され、かつ、排液貯蔵部４５１に接続された排出口４５０と、を有している。
　なお、封止液１８０が留まる場所は内部空間（流路）４３０と注入口４４０と排出口４５０と排液貯蔵部４５１である。 The lid 420 includes an inlet (inlet) 440 for introducing the fluid 160 into the inner space 430 and an outlet 450 for discharging the fluid 160 from the inner space 430. The fluid 160 introduced from the inlet 440 flows on the surface of the well array 112 and then is discharged from the outlet 450.
In addition, as shown in FIG. 10, the detection device 400 according to the present embodiment is configured to be introduced with the reaction liquid 170 and the sealing liquid 180, and has a predetermined length in the thickness direction of the detection device 400. And an outlet port 450 connected to the drainage storage unit 451.
Here, the place where the sealing liquid 180 stays is the internal space (flow path) 430, the inlet 440, the outlet 450, and the drainage storage portion 451.
　排出口４５０が接続された排液貯蔵部４５１には、排出口から出てきた余剰の封止液が蓄えられる。
　排液貯蔵部４５１は、蓋材４２０と一体的に形成されたものでもよく、取り外し可能な構成を有していてもよい。排液貯蔵部４５１が取り外し可能な構成を有している場合には、排出口４５０と排液貯蔵部４５１とは、着脱が容易な構成を有していればよく、特に形状は限定されない。
　なお、排液貯蔵部４５１の配置は、図１０に示すように、蓋材４２０の上部に設けられていてもよく、検出デバイス４００の操作および本実施形態に係る標的分子の検出における偽陰性判定の発生を抑制する方法を阻害しない範囲で、別の場所に設けられていてもよい。排液貯蔵部４５１の配置に応じて、排出口４５０の長さを変更するなど、排出口４５０の形状および長さは適宜変更可能である。 In the drainage storage unit 451 to which the discharge port 450 is connected, the excess sealing liquid coming out of the discharge port is stored.
The drainage storage unit 451 may be integrally formed with the lid 420, and may have a removable configuration. In the case where the drainage storage unit 451 has a removable configuration, the discharge port 450 and the drainage storage unit 451 may have a configuration that is easy to attach and detach, and the shape is not particularly limited.
The disposition of the drainage storage unit 451 may be provided on the top of the lid 420 as shown in FIG. 10, and the false negative determination in the operation of the detection device 400 and the detection of the target molecule according to the present embodiment In the range which does not inhibit the method of suppressing generation | occurence | production of this, it may be provided in another place. The shape and length of the discharge port 450 can be changed as appropriate, for example, by changing the length of the discharge port 450 according to the arrangement of the drainage storage section 451.
　注入口４４０における一定の長さとは、検出デバイス４００の内部で蓋材４２０の内面天井から注入口４４０の最上部までの間に無視できない程度の長さを有していればよい。
　なお、「無視できない程度の長さ」とは、検出デバイス４００を肉眼で観察した際に、視認できる長さ以上の長さを言う。
　例えば、本実施形態においては、注入口４４０は、図１０のように突出した形状を有し、突出した注入口４４０を形成していてもよい。
　また、本実施形態においては、上記第３実施形態と同様に、蓋材４２０の上面を構成する蓋材上部部材が、図１０に示す蓋材よりも厚みのある構造を有しており、蓋材４２０の内面天井から注入口４４０の最上部および排出口４５０の最上部までに一定の長さ（距離）を有する場合には、注入口４４０と排出口４５０は、蓋材に単なる穴として形成されていてもよい。
　注入口４４０は、円筒状であってもよく、ピペット等で反応液と封止液を導入することが容易になるように、すり鉢状、ロート形状のような逆円錐状を有していてもよい。
　また、注入口４４０と排出口４５０の容積が、無視できるくらい小さい場合（例えば、検出デバイス４００の厚み方向における注入口４４０および排出口４５０の長さが短い場合）には、検出デバイス４００内部に存在する封止液１８０の体積Ｖｏは内部空間４３０および排液貯蔵部４５１の容積から算出してもよい。 The fixed length at the inlet 440 may have a non-negligible length between the inner surface ceiling of the lid 420 and the top of the inlet 440 inside the detection device 400.
Note that “the length that can not be ignored” refers to a length that is longer than a visible length when the detection device 400 is observed with the naked eye.
For example, in the present embodiment, the inlet 440 may have a projecting shape as shown in FIG. 10, and may form the projecting inlet 440.
Moreover, in the present embodiment, as in the third embodiment, the lid upper member constituting the upper surface of the lid 420 has a structure thicker than the lid shown in FIG. In the case of having a fixed length (distance) from the inner surface ceiling of the material 420 to the top of the inlet 440 and the top of the outlet 450, the inlet 440 and the outlet 450 are formed as simple holes in the lid material. It may be done.
The inlet 440 may have a cylindrical shape, or may have a conical or inverted conical shape such as a funnel shape so that the reaction solution and the sealing solution can be easily introduced by a pipette or the like. Good.
In addition, when the volumes of the inlet 440 and the outlet 450 are negligibly small (for example, when the lengths of the inlet 440 and the outlet 450 in the thickness direction of the detection device 400 are short), The volume Vo of the existing sealing liquid 180 may be calculated from the volumes of the internal space 430 and the drainage reservoir 451.
　蓋材４２０の材質は特に限定されないが、シクロオレフィンポリマー、シクロオレフィンコポリマー等の熱可塑性樹脂が挙げられる。蓋材４２０の形成は、熱可塑性樹脂の流動体を、成形型を用いて成形すること等により行うことができる。 The material of the lid 420 is not particularly limited, and examples thereof include thermoplastic resins such as cycloolefin polymers and cycloolefin copolymers. The lid member 420 can be formed by molding a fluid of a thermoplastic resin using a molding die.
　基材１１０の材質は、上記第１実施形態と同様であり、内部空間４３０に送液される流体１６０に対し耐性のある材質であることが好ましい。
　流体１６０としては、上記第１実施形態と同様に、標的分子を含む反応液１７０、封止液１８０等が挙げられる。
　また、検出するシグナルが蛍光である場合、基材１１０の材質は、ウェル１１１の内部に発生した蛍光シグナルを観察することが可能な光透過性樹脂であることが好ましく、更に自家蛍光の少ない樹脂であることが好ましい。 The material of the substrate 110 is the same as that of the first embodiment, and is preferably a material resistant to the fluid 160 sent to the internal space 430.
As the fluid 160, the reaction liquid 170 containing a target molecule, the sealing liquid 180, etc. are mentioned similarly to the said 1st Embodiment.
When the signal to be detected is fluorescence, the material of the substrate 110 is preferably a light transmitting resin capable of observing the fluorescence signal generated inside the well 111, and a resin with less autofluorescence Is preferred.
　基材１１０の材質としては、上記第１実施形態と同様に、例えば、シクロオレフィンポリマー、シクロオレフィンコポリマー、シリコン、ポリプロピレン、ポリカーボネート、ポリスチレン、ポリエチレン、ポリ酢酸ビニル、フッ素樹脂、アモルファスフッ素樹脂等が挙げられる。 As a material of the substrate 110, for example, cycloolefin polymer, cycloolefin copolymer, silicon, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluorocarbon resin, amorphous fluorocarbon resin, etc. may be mentioned as in the first embodiment. Be
　基材１１０の、板厚方向の一方の面（第一面）に複数のウェル１１１が形成される。ウェル１１１の形成は、射出成形、熱インプリント、光インプリント等により行うことができる。 A plurality of wells 111 are formed on one surface (first surface) in the thickness direction of the substrate 110. The formation of the well 111 can be performed by injection molding, thermal imprint, optical imprint, or the like.
　また、フッ素樹脂を用いて基材１１０の表面にウェル１１１を形成することもできる。
　例えば、基材１１０の上にＣＹＴＯＰ（登録商標）（旭硝子）等のフッ素樹脂で形成された層を配置し、更にＣＹＴＯＰ（登録商標）の層に微小な孔を形成し、当該孔をウェル１１１とすることができる。 Alternatively, the well 111 can be formed on the surface of the base 110 by using a fluorine resin.
For example, a layer formed of a fluorocarbon resin such as CYTOP (registered trademark) (Asahi Glass) is disposed on the base material 110, and further micropores are formed in the layer of CYTOP (registered trademark). It can be done.
　蓋材４２０は、組立時に基材１１０に向けられる面に凸部４２１を有するように成形される。
　また、蓋材４２０には導入口４４０及び排出口４５０も形成される。
　排出口４５０と排液貯蔵部４５１とは一体形成してもよく、着脱可能に形成してもよい。
　続いて、基材１１０においてウェル１１１が開口する面（第一面）に蓋材４２０の凸部４２１が接するように、蓋材４２０と基材１１０とが重ねられる。
　続いて、レーザー溶着等により蓋材４２０と基材１１０とを溶着し、検出デバイス４００を製造することができる。
　これにより、蓋材４２０と基材１１０との間に流体１６０が流れる、または、流体１６０が貯留される内部空間４３０が形成される。 The lid member 420 is formed to have a convex portion 421 on the surface directed to the substrate 110 at the time of assembly.
Further, an inlet 440 and an outlet 450 are also formed in the lid 420.
The discharge port 450 and the drainage storage portion 451 may be integrally formed or may be detachably formed.
Subsequently, the lid 420 and the base 110 are overlapped so that the convex portion 421 of the lid 420 is in contact with the surface (first surface) where the well 111 is opened in the base 110.
Subsequently, the detection device 400 can be manufactured by welding the lid member 420 and the base 110 by laser welding or the like.
Thereby, an internal space 430 in which the fluid 160 flows or the fluid 160 is stored is formed between the lid 420 and the base 110.
（標的分子の検出における偽陰性判定の発生を抑制する方法（第４実施形態））
　本実施形態においては、第１実施形態の項に記載した上記式（１）において、封止液の体積（Ｖｏ）とは、内部空間４３０、導入口４４０、排出口４５０、および排液貯蔵部４５１に存在し、かつ、ウェル１１１と空間的に接している封止液１８０の体積の合計であると定義される。
　なお、本実施形態において、注入口４４０と排出口４５０の容積が、無視できるくらい小さい場合には、封止液の体積（Ｖｏ）は、内部空間４３０および排液貯蔵部４５１に存在し、かつ、ウェル１１１と空間的に接している封止液１８０の体積の合計であると定義してもよい。
　なお、本実施形態に係る標的分子の検出における偽陰性判定の発生を抑制する方法における（標的分子の検出）、（封止液）、および（偽陰性判定の発生の抑制）などの項目の説明については、上記第１実施形態と同様であるため、省略する。 (Method of suppressing occurrence of false negative determination in detection of target molecule (fourth embodiment))
In the present embodiment, in the above-mentioned formula (1) described in the section of the first embodiment, the volume (Vo) of the sealing liquid means the internal space 430, the inlet 440, the outlet 450, and the drainage storage part 451 and is defined as the sum of the volumes of the sealing solution 180 in spatial contact with the well 111.
In the present embodiment, when the volumes of the inlet 440 and the outlet 450 are negligibly small, the volume (Vo) of the sealing liquid exists in the internal space 430 and the drainage storage portion 451, and It may be defined as the sum of the volumes of the sealing solution 180 in spatial contact with the well 111.
Description of items such as (detection of target molecule), (sealing liquid), and (suppression of generation of false negative determination) in the method of suppressing occurrence of false negative determination in detection of a target molecule according to the present embodiment The second embodiment is the same as the first embodiment, and thus will not be described.
　次に実施例を示して本発明を更に詳細に説明するが、本発明は以下の実施例に限定されない。 EXAMPLES The present invention will next be described in more detail by way of examples, which should not be construed as limiting the invention thereto.
［実験例１］
　封止液の体積（Ｖｏ）とウェルアレイのウェル容積の合計（Ｖｓｄ）の比が標的分子の検出における偽陰性判定の発生に及ぼす影響を検討した。 [Experimental Example 1]
The effect of the ratio of the volume of the sealing solution (Vo) to the sum of the well volume of the well array (Vsd) on the occurrence of false negative determination in the detection of the target molecule was investigated.
　まず、射出成形で形成したシクロオレフィンポリマー（ＣＯＰ）製の基材１１０とＣＯＰ製の蓋材の２つの樹脂製の部材を使用した。蓋材はカーボンブラック添加により着色されており、導入口及び排出口を有していた。 First, two resin members, a base 110 made of cycloolefin polymer (COP) formed by injection molding and a lid made of COP, were used. The lid was colored by the addition of carbon black and had an inlet and an outlet.
　基材１１０としては、ウェル１１１の容積及び数が異なる６種類の基材（基材１Ａ～６Ａ）を用意した。
　基材１Ａは、ウェル１１１の容積Ｖｄが９３ｆＬであり、ウェルアレイ１１２のウェル容積の合計（Ｖｓｄ）が０．００００５８５９ｍＬであった。
　基材２Ａは、ウェル１１１の容積Ｖｄが１，８６０ｆＬであり、ウェルアレイ１１２のウェル容積の合計（Ｖｓｄ）が０．００２６０４ｍＬであった。
　基材３Ａは、ウェル１１１の容積Ｖｄが５９ｆＬであり、ウェルアレイ１１２のウェル容積の合計（Ｖｓｄ）が０．００００５４ｍＬであった。
　基材４Ａは、ウェル１１１の容積Ｖｄが５９ｆＬであり、ウェルアレイ１１２のウェル容積の合計（Ｖｓｄ）が０．００００８４ｍＬであった。
　基材５Ａは、ウェル１１１の容積Ｖｄが１，１７８ｆＬであり、ウェルアレイ１１２のウェル容積の合計（Ｖｓｄ）が０．００１１ｍＬであった。
　基材６Ａは、ウェル１１１の容積Ｖｄが１，１７８ｆＬであり、ウェルアレイ１１２のウェル容積の合計（Ｖｓｄ）が０．００１７ｍＬであった。
　なお、上記第１～第４実施形態に対応するように、かつ、検出デバイスに導入される封止液の体積（Ｖｏ）を調整できるような内部空間（流路）の容量を有するように、凸部の高さ、導入口（注入口）の容量、および排出口の容量を変化させた蓋材を準備した。 As the substrate 110, six types of substrates (substrates 1A to 6A) having different volumes and numbers of wells 111 were prepared.
In the substrate 1A, the volume Vd of the wells 111 was 93 fL, and the total (Vsd) of the well volumes of the well array 112 was 0.00005859 mL.
The substrate 2A had a volume Vd of the wells 111 of 1,860 fL, and the total of the well volumes of the well array 112 (Vsd) was 0.002604 mL.
In the substrate 3A, the volume Vd of the wells 111 was 59 fL, and the total (Vsd) of well volumes of the well array 112 was 0.000054 mL.
The substrate 4A had a volume Vd of the wells 111 of 59 fL and a total of the well volumes of the well array 112 (Vsd) was 0.000084 mL.
The substrate 5A had a volume Vd of well 111 of 1,178 fL, and the total well volume (Vsd) of the well array 112 was 0.0011 mL.
The base 6A had a volume Vd of well 111 of 1,178 fL, and the total well volume (Vsd) of the well array 112 was 0.0017 mL.
In order to correspond to the first to fourth embodiments, and to have a capacity of an internal space (flow path) which can adjust the volume (Vo) of the sealing solution introduced into the detection device, The lid material in which the height of the convex part, the volume of the inlet (inlet), and the volume of the outlet were changed was prepared.
　これらの各基材１１０を各蓋材にそれぞれ両面テープで接着して流路を形成し、検出デバイスを製造した。 Each of the substrates 110 was adhered to each of the lids with a double-sided tape to form a flow path, thereby manufacturing a detection device.
　続いて、各検出デバイスの流路に、標的分子としてのＤＮＡ断片を含むＩＣＡ反応試薬（反応液１７０）を注入した。標的分子としては、標的オリゴＤＮＡ（配列番号１）を使用した。標的オリゴＤＮＡの濃度は、反応液１７０を検出デバイスに導入した場合に、ウェル１１１に標的オリゴＤＮＡが０個又は１個導入される濃度に調製した。ＩＣＡ反応試薬の組成を表１に示す。 Subsequently, an ICA reaction reagent (reaction solution 170) containing a DNA fragment as a target molecule was injected into the flow channel of each detection device. The target oligo DNA (SEQ ID NO: 1) was used as a target molecule. The concentration of the target oligo DNA was adjusted to a concentration such that zero or one target oligo DNA was introduced into the well 111 when the reaction solution 170 was introduced into the detection device. The composition of the ICA reaction reagent is shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　表１中、アレルプローブ及びＩＣＡオリゴはファスマック社製であり、ＦＲＥＴカセット（Ａｌｅｘａ４８８－ＢＨＱ）は日本バイオサービス社製であった。 In Table 1, the allele probe and the ICA oligo were manufactured by Fasmac, and the FRET cassette (Alexa 488-BHQ) was manufactured by Japan Bioservices.
　続いて封止液を導入し、ウェル１１１を個別に封止した。封止液としてはフッ素系オイルであるフロリナート（商標）ＦＣ－４０（シグマ社製）を使用した。ここで、導入する封止液の量を、１．１５０、０．６５０、０．３５０、０．１５０、０．０２４ｍＬ、０．０１０ｍＬ、０．００８ｍＬ、０．００６ｍＬ、０．００４ｍＬ、０．００２ｍＬにそれぞれ変化させて検討を行った。
　なお、封止液の量が０．００４ｍＬ、０．００２ｍＬとなるように調整した検出デバイスにおいては、導入口（注入口）および排出口の容量が無視できるほど小さく形成した蓋材を用いた。換言すれば、封止液の量が０．００４ｍＬ、０．００２ｍＬとなるように調整した検出デバイスは、封止液体積Ｖｏが内部空間（流路）の容積とほぼ同一の場合の例である。 Subsequently, a sealing solution was introduced to seal the wells 111 individually. As a sealing solution, Fluorinert (registered trademark) FC-40 (manufactured by Sigma), which is a fluorine-based oil, was used. Here, the amount of the sealing solution to be introduced is 1.150, 0.650, 0.350, 0.150, 0.024 mL, 0.010 mL, 0.008 mL, 0.006 mL, 0.004 mL, 0.1. The examination was conducted by changing each to 002 mL.
In addition, in the detection device adjusted so that the quantity of sealing liquid might be 0.004 mL and 0.002 mL, the lid material formed so small that the capacity | capacitance of an inlet (inlet) and an outlet could be disregarded was used. In other words, the detection device adjusted so that the amount of the sealing liquid is 0.004 mL and 0.002 mL is an example in the case where the volume of the sealing liquid Vo is substantially the same as the volume of the internal space (flow path) .
　封止液を導入した後、６６℃で３０分間加熱した。この結果、ウェル１１１が標的分子を含む場合にはでＩＣＡ反応が起こり、蛍光シグナルが生成された。続いて、各ウェル１１１から発せられる蛍光シグナルを、蛍光顕微鏡ＢＺ－７１０（ＫＥＹＥＮＣＥ社製）で撮影した。対物レンズとして倍率１０倍のレンズを使用した。また、露光時間は、３０００ミリ秒とした。蛍光フィルターとしては、緑色蛍光タンパク質（ＧＦＰ）観察用のフィルターを使用した。 After introducing the sealing solution, it was heated at 66 ° C. for 30 minutes. As a result, when the well 111 contains the target molecule, an ICA reaction occurs and a fluorescence signal is generated. Subsequently, the fluorescence signal emitted from each well 111 was photographed with a fluorescence microscope BZ-710 (manufactured by KEYENCE). A 10 × magnification lens was used as an objective lens. The exposure time was 3000 milliseconds. A filter for observing green fluorescent protein (GFP) was used as a fluorescence filter.
　図６（ａ）～（ｄ）は、ウェルアレイのウェル容積の合計（Ｖｓｄ）が０．００００５８５９ｍＬである場合に、０．１５０、０．３５０、０．６５０及び１．１５０ｍＬの封止液を導入して各ウェルを封止し、６６℃で３０分間反応させた後に蛍光観察した結果を示す代表的な写真である。 6 (a) to 6 (d) show that 0.150, 0.350, 0.650 and 1.150 mL of sealing solution is used when the total well volume (Vsd) of the well array is 0.00005859 mL. It is a representative photograph which shows the result of having observed fluorescence after introducing and sealing each well and making it react at 66 ° C for 30 minutes.
　図６に示すように、封止液が１．１５０ｍＬの場合（Ｖｏ／Ｖｓｄ＝１９，６２８）には、封止液が０．１５０、０．３５０、０．６５０ｍＬの場合と異なり、蛍光を発するウェルがリング形状に観察された。すなわち、ウェルの中央部分には蛍光が観察されず、ウェルの周辺部分に蛍光が観察された。このようなウェルは、特にコンピュータを用いた画像解析でシグナルが生成されたか否かを判定する場合に、偽陰性判定を行いやすい（偽陰性判定されやすい）。 As shown in FIG. 6, in the case where the sealing solution is 1.150 mL (Vo / Vsd = 19,628), the fluorescence is different unlike in the cases where the sealing solution is 0.150, 0.350, and 0.650 mL. The emitting wells were observed in a ring shape. That is, no fluorescence was observed in the central part of the well, but fluorescence was observed in the peripheral part of the well. Such a well is easy to make a false negative determination (it is easy to make a false negative determination), particularly when determining whether or not a signal is generated by image analysis using a computer.
　下記表２に、本実験例の結果をまとめた。表２中、「Ｂ」は蛍光シグナルがリング状になったことを示し、偽陰性判定を起こす可能性があり、「Ａ」は蛍光シグナルを正しく測定可能であったことを示す。 The results of this experiment are summarized in Table 2 below. In Table 2, "B" indicates that the fluorescent signal is in the form of a ring, which may cause a false negative judgment, and "A" indicates that the fluorescent signal could be measured correctly.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　この結果、下記式（１）が満たされる場合に、偽陰性判定の発生が抑制される傾向が認められた。
０＜封止液の体積／ウェルアレイのウェル容積の合計≦１２，０００…（１） As a result, when the following formula (1) was satisfied, a tendency was observed that the occurrence of false negative determination was suppressed.
0 <total volume of sealing solution / well volume of well array ≦ 12,000 (1)
　蛍光シグナルがリング状になる理由は明らかではないが、例えば、図７に示すように、ウェル１１１中の水分が封止液１８０中に吸収され、ウェル１１１中に封止液１８０が入り込むこと等が考えられる。 Although the reason why the fluorescence signal is ring-shaped is not clear, for example, as shown in FIG. 7, the moisture in the well 111 is absorbed into the sealing solution 180 and the sealing solution 180 enters the well 111, etc. Is considered.
　以上に、本発明の実施形態を説明したが、実施形態における各構成及びそれらの組み合わせ等は一例であり、本発明の趣旨から逸脱しない範囲内で、構成の付加、省略、置換、及びその他の変更が可能である。また、本発明は実施形態によって限定されない。 The embodiment of the present invention has been described above, but each configuration and combination thereof in the embodiment are an example, and addition, omission, replacement, and other configurations can be made without departing from the spirit of the present invention. Changes are possible. Moreover, the present invention is not limited by the embodiments.
　本発明によれば、デジタル計測における偽陰性判定の発生を抑制する技術を提供することができる。 According to the present invention, it is possible to provide a technique for suppressing the occurrence of false negative determination in digital measurement.
　１００、２００、３００、４００…検出デバイス
　１１０…基材
　１１１…ウェル
　１１２…ウェルアレイ
　１２０、２２０、３２０、４２０…蓋材
　１２１、２２１、３２１、４２１…凸部
　１３０、２３０、３３０、４３０…流路
　１４０、２４０、３４０、４４０…導入口（注入口）
　１５０、２５０、３５０、４５０…排出口
　１６０…流体
　１７０…反応液
　１７１…シグナルを生成した反応液
　１８０…封止液
　５５０…排液貯蔵部 100, 200, 300, 400 ... detection device 110 ... base material 111 ... well 112 ... well array 120, 220, 320, 420 ... lid material 121, 221, 321, 421 ... convex portion 130, 230, 330, 430 ... flow Route 140, 240, 340, 440 ... inlet (inlet)
150, 250, 350, 450: Discharge port 160: Fluid 170: Reaction liquid 171: Reaction liquid that generated a signal 180: Sealing liquid 550: Drain storage part

Claims (11)

  1. 　標的分子の検出における偽陰性判定の発生を抑制する方法であって、
    　複数のウェルを有するウェルアレイが設けられたデバイスを準備し、前記ウェルアレイにおける前記複数のウェルに前記標的分子を含む反応液を導入する工程と、
    　前記ウェルアレイを所定の体積の封止液に接触させて、前記ウェルを個別に封止する工程と、
    　個別に封止された前記ウェル内の前記反応液を反応条件にすることによって、前記ウェル内に前記標的分子が存在する場合に前記ウェル内でシグナルが生成される工程と、
    　前記ウェル内でシグナルが生成されたか否かを判定する工程と、を含み、
    　前記標的分子の検出の際に、前記封止液の体積を、式（１）：０＜前記封止液の体積／前記ウェルアレイのウェル容積の合計≦１２，０００を満たすように前記封止液を前記デバイスに導入する、方法。     A method of suppressing the occurrence of false negative judgment in detection of a target molecule, comprising
    Preparing a device provided with a well array having a plurality of wells, and introducing a reaction solution containing the target molecule into the plurality of wells in the well array;
    Bringing the well array into contact with a predetermined volume of sealing solution to individually seal the wells;
    Placing the reaction solution in the individually sealed wells under reaction conditions to generate a signal in the wells when the target molecule is present in the wells;
    Determining whether a signal is generated in the well.
    At the time of detection of the target molecule, the sealing is performed such that the volume of the sealing solution satisfies Formula 1: 0 <volume of the sealing solution / well volume of the well array ≦ 12,000. Introducing a fluid into the device.
  2. 　前記式（１）において、１≦前記封止液の体積／前記ウェルアレイのウェル容積の合計≦１２，０００を満たすように前記封止液を前記デバイスに導入する、請求項１に記載の方法。 The method according to claim 1, wherein the sealing solution is introduced into the device so as to satisfy 1 ≦ volume of the sealing solution / total of well volumes of the well array ≦ 12,000 in the formula (1). .
  3. 　前記反応条件が５０～８０℃である、請求項１または２に記載の方法。 The method according to claim 1 or 2, wherein the reaction conditions are 50 to 80 属 C.
  4. 　前記反応条件を１時間以下維持する、請求項３に記載の方法。 The method according to claim 3, wherein the reaction conditions are maintained for 1 hour or less.
  5. 　前記反応液がＩｎｖａｓｉｖｅ　Ｃｌｅａｖａｇｅ　Ａｓｓａｙ反応試薬である、請求項１～４のいずれか一項に記載の方法。 The method according to any one of claims 1 to 4, wherein the reaction solution is an Invasive Cleavage Assay reaction reagent.
  6. 　前記封止液の２５℃における最大含水量が１～１０００質量ｐｐｍである、請求項１～５のいずれか一項に記載の方法。 The method according to any one of claims 1 to 5, wherein the maximum water content at 25 属 C of the sealing solution is 1 to 1000 mass ppm.
  7. 　標的分子の検出デバイスであって、
    　前記標的分子を含有する反応液が導入され、かつ、前記標的分子を検出する反応が起こるように複数のウェルを有するウェルアレイが設けられた基材と、
    　前記基材とともに、前記ウェルアレイを封止する封止液が導入される内部空間を形成するように設けられた蓋材と、を備え、
    　前記内部空間は、式（１）：０＜前記封止液の体積／前記ウェルアレイのウェル容積の合計≦１２，０００を満たす、
    　検出デバイス。     A target molecule detection device,
    A substrate provided with a well array having a plurality of wells into which a reaction solution containing the target molecule is introduced, and a reaction for detecting the target molecule occurs;
    And a lid provided so as to form an internal space into which a sealing liquid for sealing the well array is introduced together with the base material,
    The internal space satisfies the following formula (1): 0 <volume of the sealing solution / well volume of the well array ≦ 12,000.
    Detection device.
  8. 　前記式（１）において、１≦前記封止液の体積／前記ウェルアレイのウェル容積の合計≦１２，０００を満たす、請求項７に記載の検出デバイス。 The detection device according to claim 7, wherein in the formula (1), a total of 1 ≦ volume of the sealing solution / well volume of the well array ≦ 12,000 is satisfied.
  9. 　前記封止液の体積が前記内部空間の容積と等しい、請求項７に記載の検出デバイス。 The detection device according to claim 7, wherein a volume of the sealing solution is equal to a volume of the internal space.
  10. 　前記蓋材に注入口と排出口とをさらに有し、
    　前記封止液の体積が、前記注入口の容積および前記排出口の容積および前記内部空間の容積の合計と等しい、
    　請求項７に記載の検出デバイス。     The lid further includes an inlet and an outlet,
    The volume of the sealing solution is equal to the sum of the volume of the inlet and the volume of the outlet and the volume of the internal space,
    A detection device according to claim 7.
  11. 　前記排出口に接続された排液貯蔵部をさらに有し、
    　前記封止液の体積が、前記注入口の容積および前記排出口の容積および前記内部空間の容積および前記排液貯蔵部の容積の合計と等しい、
    　請求項１０に記載の検出デバイス。     It further comprises a drainage reservoir connected to the outlet,
    The volume of the sealing solution is equal to the sum of the volume of the inlet and the volume of the outlet and the volume of the internal space and the volume of the drainage reservoir.
    A detection device according to claim 10.
PCT/JP2018/047475 2017-12-26 2018-12-25 Method for suppressing occurrence of false-negative determination in detection of target molecule, and detection device WO2019131592A1 (en)

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