WO2019131592A1 - Procédé pour supprimer l'apparition d'une détermination faussement négative dans la détection d'une molécule cible et dispositif de détection - Google Patents

Procédé pour supprimer l'apparition d'une détermination faussement négative dans la détection d'une molécule cible et dispositif de détection 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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English (en)
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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    • 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.

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Abstract

L'invention concerne un procédé permettant de supprimer l'apparition d'une détermination faussement négative dans la détection d'une molécule cible, le procédé comprenant : une étape consistant à préparer un dispositif sur lequel est disposé un réseau de puits comprenant une pluralité de puits et à introduire un liquide de réaction comprenant la molécule cible dans la pluralité de puits du réseau de puits ; une étape consistant à mettre le réseau de puits en contact avec un volume prédéfini d'un liquide d'isolement et isoler individuellement les puits ; une étape consistant à placer le liquide de réaction présent dans les puits individuellement isolés dans des conditions de réaction, un signal étant produit dans les puits lorsque la molécule cible est présente dans les puits ; et une étape consistant à déterminer si le signal a été produit dans les puits ; le liquide d'isolement étant introduit dans le dispositif pour que le volume du liquide d'isolement pendant la détection de la molécule cible satisfasse à la formule (1) : 0 < (volume du liquide d'isolement)/(volume total du réseau de puits) ≤ 12 000.
PCT/JP2018/047475 2017-12-26 2018-12-25 Procédé pour supprimer l'apparition d'une détermination faussement négative dans la détection d'une molécule cible et dispositif de détection WO2019131592A1 (fr)

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EP3933411A4 (fr) * 2019-02-27 2022-03-23 Toppan Printing Co., Ltd. Dispositif microfluidique et procédé d'analyse d'échantillon

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