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WO2019230485A1 - Cartouche - Google Patents

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Publication number
WO2019230485A1
WO2019230485A1 PCT/JP2019/020003 JP2019020003W WO2019230485A1 WO 2019230485 A1 WO2019230485 A1 WO 2019230485A1 JP 2019020003 W JP2019020003 W JP 2019020003W WO 2019230485 A1 WO2019230485 A1 WO 2019230485A1
Authority
WO
WIPO (PCT)
Prior art keywords
droplet
cartridge
region
cartridge according
filling region
Prior art date
Application number
PCT/JP2019/020003
Other languages
English (en)
Japanese (ja)
Inventor
裕司 前原
南　昌人
淳 ▲高▼橋
久子 元木
西村　光夫
浩 城井
Original Assignee
キヤノン株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from JP2018105480A external-priority patent/JP2019211253A/ja
Priority claimed from JP2018105481A external-priority patent/JP2019211254A/ja
Priority claimed from JP2018105479A external-priority patent/JP7179494B2/ja
Priority claimed from JP2018105477A external-priority patent/JP2019208395A/ja
Priority claimed from JP2018125179A external-priority patent/JP2020003421A/ja
Application filed by キヤノン株式会社 filed Critical キヤノン株式会社
Publication of WO2019230485A1 publication Critical patent/WO2019230485A1/fr

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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
    • 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
    • G01N21/03—Cuvette constructions
    • G01N21/05—Flow-through cuvettes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/08—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N37/00—Details not covered by any other group of this subclass
    • 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

Definitions

  • the disclosure of this specification relates to a cartridge.
  • a nucleic acid sample digital PCR (dPCR: digital Polymer Chain Reaction) method using a water-in-oil emulsion as a reaction field has been proposed as a method for analyzing analytes such as compounds and particles contained in a sample.
  • dPCR digital Polymer Chain Reaction
  • a method of forming droplets of the reaction solution in oil that is, a water-in-oil emulsion (W / O)
  • W / O water-in-oil emulsion
  • each droplet in a water-in-oil emulsion is used as a reaction field (Patent Document 1).
  • the present invention is not limited to the above-described object, and is an operational effect derived from each configuration shown in an embodiment for carrying out the invention described later, and also has an operational effect that cannot be obtained by conventional techniques. It can be positioned as one of other purposes.
  • the cartridge disclosed in the present specification is installed on the bottom surface in a three-dimensional space formed by a first axis, a second axis, and a third axis perpendicular to the bottom surface defined by the two axes.
  • a cartridge used for analysis of an object contained in a droplet comprising a porous film that generates a plurality of the droplets, and a holding unit that holds the droplets .
  • loss of droplets can be reduced.
  • FIG. 3 is a diagram illustrating an example of an appearance of a cartridge according to the first embodiment.
  • FIG. 3 is a diagram illustrating an example of an appearance of a cartridge according to the first embodiment.
  • FIG. 3 is a diagram illustrating an example of an appearance of a cartridge according to the first embodiment.
  • the flowchart which shows an example of the whole process sequence in 1st Embodiment.
  • the figure which shows a series of analysis systems which concern on 1st Embodiment.
  • FIG. 10 is a diagram illustrating an example of an appearance of a cartridge according to a second embodiment.
  • FIG. 10 is a diagram illustrating an example of an appearance of a cartridge according to a second embodiment.
  • FIG. 10 is a diagram illustrating an example of an appearance of a cartridge according to a second embodiment.
  • the flowchart which shows an example of the whole process sequence in 2nd Embodiment The figure which shows an example of the cartridge which concerns on the modification of 2nd Embodiment. The figure which shows an example of the cartridge which concerns on the modification of 2nd Embodiment. The figure which shows an example of the cartridge which concerns on the modification of 2nd Embodiment. The figure which shows an example of the cartridge which concerns on the modification of 2nd Embodiment. The figure which shows an example of the cartridge which concerns on the modification of 2nd Embodiment. The figure which shows an example of the cartridge which concerns on the modification of 2nd Embodiment. The figure which shows an example of the cartridge which concerns on the modification of 2nd Embodiment. The figure which shows an example of the cartridge which concerns on the modification of 2nd Embodiment.
  • FIG. 6 is a view showing an example of a cartridge according to a modification of the first embodiment.
  • FIG. 6 is a view showing an example of a cartridge according to a modification of the first embodiment.
  • FIG. 6 is a view showing an example of a cartridge according to a modification of the first embodiment.
  • FIG. 6 is a view showing an example of a cartridge according to a modification of the first embodiment.
  • FIG. 6 is a view showing an example of a cartridge according to a modification of the first embodiment.
  • the bottom surface of the cartridge is indicated by the X axis and the Y axis, and the Z axis indicates the height direction of the cartridge (a direction orthogonal to the bottom surface of the cartridge). That is, the cartridge is a cartridge installed on the bottom surface in a three-dimensional space formed by a first axis, a second axis, and a third axis perpendicular to the bottom surface defined by the two axes. is there.
  • the cartridge according to this embodiment is a cartridge that can be used for analysis of a sample.
  • the sample here may be the specimen itself, or the specimen that has been subjected to pretreatment and adjustment for analysis, such as purification and concentration, chemical modification and fragmentation of the analyte. Also good.
  • the analysis object include nucleic acids, peptides, proteins, enzymes, and the like, and these may coexist.
  • the analysis target may be a molecule, a microparticle, a nanoparticle, or a virus, a bacterium, a cell, or the like to which at least one of a nucleic acid, a peptide, a protein, and an enzyme is bound or attached by a covalent bond.
  • a digital PCR method As an example of an analysis method, a digital PCR method is known.
  • a sample containing a nucleic acid to be analyzed is mixed with an amplification reagent for amplifying the nucleic acid, a fluorescent reagent for detecting the nucleic acid, etc., and diluted to obtain a number of physically independent reaction fields. Divide into Each droplet contained in the emulsion may be used as a reaction field in the digital PCR method. Then, PCR is performed independently in each of a large number of reaction fields (droplets) to amplify the nucleic acid.
  • the concentration of the nucleic acid in the sample can be estimated.
  • the cartridge according to the present embodiment is made of a transparent resin such as polycarbonate or glass. Specifically, the cartridge is formed by laminating a plurality of resin films or glasses having a predetermined thickness.
  • a cartridge is formed by laminating a plurality of resin films each having a predetermined thickness and having a channel shape punched out.
  • a method for bonding the films to be laminated surface activated bonding or molecular adhesion capable of bonding at a low processing temperature lower than the glass transition point of the resin can be used.
  • the glass transition point of these materials is preferably higher than 100 ° C.
  • the material of the cartridge, the flow path shape, the formation method, and the like are examples, and are not limited thereto.
  • FIG. 1 is a view showing an appearance of a cartridge 100 according to the present embodiment.
  • FIG. 1A is a plan view of the cartridge 100.
  • FIG. 1B is a cross-sectional view showing the AA cross section of the cartridge 100.
  • FIG. 1C is a cross-sectional view showing the BB cross section of the cartridge 100.
  • the black portion of the cartridge 100 indicates a portion formed of resin or the like.
  • the upper surface resin (the upper surface of the droplet filling region 104 in FIG. The black part) is shown for convenience.
  • the first member 101 is a member having an opening into which a first liquid (water phase) for generating droplets is injected.
  • a first liquid water phase
  • the first liquid (aqueous phase) injected into the first member 101 is sent to the generation unit 102.
  • the generation unit 102 has a plurality of holes, and a plurality of reaction fields that are physically independent from each other, that is, droplets, are generated when the injected first liquid passes through the plurality of holes.
  • the generation unit 102 is a member in which a plurality of holes are two-dimensionally arranged in a film-like member, and an open-cell porous body, a mesh in which fibers are arranged vertically and horizontally, and a through-hole in a single member Microchannels and emulsified membranes are used.
  • the pore diameter of the substance used as the generation unit 102 is preferably about 10 to 30 [ ⁇ m].
  • the droplet generated from the generation unit 102 preferably has a droplet diameter of 30 to 95 [ ⁇ m].
  • the CV (Coefficient of Variation) value of the generated droplet is preferably ⁇ 5 to 20%, and more preferably ⁇ 8 to 18%.
  • the generation unit 102 is provided at the end of the first member 101. Furthermore, it is desirable that the first member 101 is provided substantially parallel to the opening direction of the opening of the first member 101. Note that the position where the generation unit 102 is provided is not limited to the above as long as it is a position where droplets can be generated, and may be provided in the holding unit 103, for example.
  • the generation unit 102 is joined to the first member 101 with, for example, an adhesive. Note that the bonding method is not limited to the above.
  • the holding unit 103 holds the droplet generated by the generating unit 102 for performing the nucleic acid amplification process.
  • the height of the holding portion 103 in the Z-axis direction is desirably about 20 to 100 [ ⁇ m].
  • the droplet filling region 104 has a different height from the holding unit 103 in the Z-axis direction, and is filled with a droplet that has been subjected to nucleic acid amplification processing for concentration estimation.
  • the droplet filling region 104 preferably has a height in the Z-axis direction of approximately 20 to 200 [ ⁇ m].
  • the droplet damming portion 105 is provided at the end portion of the droplet filling region 104, and the liquid cross-sectional area of the end portion is made narrower than the cross-sectional area of the inlet where the droplet flows into the droplet filling region 104. Prevent spillage.
  • the second member 106 is a member into which the second liquid (oil phase) is injected and discharged.
  • the second member 106 has the same size as the first member 101.
  • the first member 101 and the second member 106 are provided so as to overlap the upper surface (Z-axis direction) of the holding unit 103 and the droplet filling region 104, respectively. It may be provided on the lower surface.
  • step S2000 the second liquid (oil phase) is injected by the oil phase injection means 306 shown in FIG. 3 to fill the cartridge.
  • the second liquid (oil phase) contains oil and a surfactant.
  • the oil phase consists of a solvent that is incompatible with the aqueous phase and separates, and typically consists of oils such as aliphatic hydrocarbons and silicone oils.
  • the oil phase injection means 306 may be a means for discharging a predetermined volume of liquid such as a syringe, a pump for pressurizing or depressurizing air in the flow path, and a combination of a pump and a valve.
  • the oil phase injection means 306 is realized by any means having a function capable of injecting the oil phase into the cartridge.
  • the second liquid (oil phase) is injected from the opening of the second member 106 and can be filled into the cartridge 100 without passing through the generation unit 102, the filling time is shortened.
  • the second liquid (oil phase) may be injected from the opening of the first member 101.
  • the second liquid (oil phase) injected by the oil phase injection means 306 is injected so as to fill the entire cartridge 100, and at least the amount of the generation unit 102 soaked in the second liquid (oil phase) is sufficient. It is desirable to be injected.
  • step S2010 the first liquid (aqueous phase) is injected by the aqueous phase injection means 305 and filled in the first member 101.
  • the first liquid (aqueous phase) is composed of a reaction solution, and the reaction solution contains water, a sample, an amplification reagent, and a fluorescent reagent.
  • the configuration of the reaction solution is not limited to this.
  • the water phase injection unit 305 may be a unit that discharges a predetermined volume of liquid such as a syringe, a pump that pressurizes or depressurizes air in the flow path, and a combination of a pump and a valve. That is, the water phase injection means 305 is realized by any means having a function capable of injecting the water phase into the cartridge.
  • step S2020 the driving unit pressurizes the inside of the cartridge 100 from the first member 101 or depressurizes the inside of the cartridge 100 from the second member 106, whereby the second liquid (oil phase) filled in the cartridge 100 is filled. ). Alternatively, by performing both, the second liquid (oil phase) filled in the cartridge 100 is driven.
  • the continuous phase is driven by reducing the pressure from the second member 106 by the driving means so as not to cause destruction or peeling. It is desirable.
  • the continuous phase may be driven by applying pressure from the first member 101.
  • pressure leakage can be prevented by attaching a pressure stopper to the member of the first member 101 and the second member 106 that uses the driving means. Note that a pressure stopper is not always necessary.
  • the drive means may use the water phase injection means 305 or the oil phase injection means 306, or a new means different from these may be used. That is, various means can be used as the driving means as long as the liquid can be driven by applying pressure.
  • the first liquid (aqueous phase) filled on the upper surface of the generation unit 102 passes through the generation unit 102 in step S2010, and droplets are generated. That is, an emulsion is formed in which the second liquid (oil phase) is a continuous phase and the droplets containing the first liquid (water phase) are dispersed phases.
  • the method for forming the emulsion is not limited to the above.
  • a mechanical emulsification method in which an emulsion is formed by applying mechanical energy by a conventionally known emulsification method such as a stirring device or an ultrasonic crushing device.
  • a conventionally known emulsification method such as a stirring device or an ultrasonic crushing device.
  • the method using microchannel devices such as the microchannel emulsification method and the microchannel branch emulsification method, the membrane emulsification method using an emulsification film, etc. are mentioned. These methods may be used alone or in combination.
  • the mechanical emulsification method and the membrane emulsification method tend to increase the dispersion (dispersion) of the droplet diameter of the droplet as compared with the method using the microchannel device, but can form an emulsion with high throughput.
  • the membrane emulsification method is particularly desirable because the apparatus configuration of the apparatus for forming the emulsion can be simplified and an emulsion having a relatively small variation in droplet diameter can be formed.
  • the generation unit 102 is preferably an emulsified film.
  • the membrane emulsification method a direct membrane emulsification method or a pumping emulsification method can be used.
  • the direct membrane emulsification method is a method of forming an emulsion in a continuous phase that is slowly flowing on the side to be extruded by extruding a dispersed phase at a constant pressure through the emulsion membrane.
  • the pumping emulsification method is a method of preparing an emulsion by sandwiching an emulsion film between a syringe that has collected a continuous phase and a syringe that has collected a dispersed phase, and by alternately extruding liquid from the two syringes and passing through the emulsion film. is there.
  • a mixture of a continuous phase and a dispersed phase may be collected in one of two syringes, and the other syringe may be empty.
  • a pumping type emulsification device in which an emulsification film is sandwiched between a pair of connectors each connectable to a syringe can be used.
  • step S2030 (The cartridge is tilted and the droplet is held in the holding portion)
  • the tilting means tilts the cartridge 100 to drive the second liquid (oil phase) as a continuous phase
  • the droplets generated in step S2020 are held in the holding unit 103.
  • a tilt stage can be used as the tilting means.
  • the tilt stage is an example of means for tilting the cartridge 100, and is not limited to this as long as it can drive the continuous phase by tilting the angle.
  • the angle of inclination for driving the continuous phase is not limited.
  • the cartridge 100 is tilted after the droplets are generated in step S2020.
  • the cartridge 100 may be tilted already in step S2000. That is, the tilting of the cartridge 100 by the tilting means performed in this step S2030 may be performed at any time prior to S2040.
  • the driving means may substitute for the function of the tilting means.
  • step S2040 a PCR process is performed on the nucleic acid sample contained in the droplet held in the holding unit 103 in step S2030 by a temperature adjusting unit (not shown).
  • the temperature adjusting means includes a Peltier element and a controller, and generates a nucleic acid amplification reaction in the droplet held in the holding unit 103 of the cartridge 100.
  • a thermal cycler can be used as the temperature adjusting means.
  • a nucleic acid amplification reaction a PCR method or a LCR (Ligase Chain Reaction) method in which the reaction is allowed to proceed by subjecting a droplet (reaction field) to a thermal cycle can be used.
  • the SDA (Strand Displacement Amplification) method, the ICAN (Isothermal and Primer Amplified of Nucleic acid), and the IDA (Isothermal and Primer Amplified of Nucleic acid) method are also used.
  • the Mediated Isometric Amplification) method or the like can also be used.
  • the apparatus used as the temperature adjusting means and the nucleic acid amplification method are not limited to the above. Further, when the thermal cycle is performed, it is preferable that the height of the holding portion 103 in the Z-axis direction is high in order to make it easier to keep away bubbles that cause droplet loss from the droplet. The higher the height of the holding portion 103 in the Z-axis direction, the easier the bubbles escape. However, since the heat capacity of the cartridge increases and a long processing time is required, it is desirable to keep the size within 0.2 to 0.8 mm. .
  • step S2050 The cartridge is tilted to fill the droplet filling region with the droplet
  • the tilting means tilts the cartridge 100 to drive the second liquid (oil phase) that is a continuous phase, and the droplets obtained by amplifying the internal nucleic acid are separated from the holder 103 to the holder 103.
  • the droplet filling region 104 is preferably lower in the Z-axis direction than the holding unit 103. This is because if the droplet filling region 104 is set to the same height as the holding unit 103, droplets that are likely to cause deterioration in reliability of analysis results are easily generated during droplet observation.
  • step S2060 the cartridge 100 is transferred to the observation stage and light such as fluorescence is measured. That is, the analysis object in the sample amplified in S2040 is analyzed. Fluorescence is measured by an illumination unit (not shown) and an observation unit (not shown), and the illumination unit irradiates a plurality of droplets filled in the droplet filling region 104 with light having a predetermined wavelength.
  • the illumination means an LED light, a halogen lamp, a fluorescent lamp, or the like can be used.
  • the observation means detects a signal emitted from each of the plurality of droplets irradiated with light, and observes the presence or absence of the amplification product in the droplet. In addition, the droplet diameter is measured.
  • a photodiode, a line sensor, an image sensor (imaging device), or the like can be used. Among them, it is preferable to use an image sensor because signals can be detected for a large number of droplets at once.
  • a CCD (Charge Coupled Device) or CMOS (Complementary Metal Oxide Semiconductor) image sensor can be used.
  • the observation means may be a digital camera provided with an image sensor.
  • the observation stage may have a tilting mechanism in order to suppress the flow of the liquid droplets during observation.
  • step S2070 the processor (not shown) estimates the concentration in the sample from the ratio of the volume of the droplet not containing the amplification product to the total volume of the sample from the result of the observation performed in S2060.
  • the calculation of the concentration of the analysis object can be performed by employing a concentration calculation method in digital analysis that is conventionally performed.
  • a concentration calculation method in digital analysis that is conventionally performed.
  • the number x of reaction fields (positive reaction fields) in which the analysis object is detected is equal to the number of analysis objects contained in the reaction solution of the volume Vs that is the object of detection of the analysis object by the observation means.
  • the concentration ⁇ r of the analyte in the reaction solution can be calculated by the following formula (1).
  • the concentration of the analyte is calculated by performing correction using the Poisson model. Can do.
  • the concentration of the analyte is calculated by estimating the average number C of analytes included in each reaction field before the reaction in the temperature adjusting means.
  • a reaction field that is an object to be detected by the observation means assuming that the average number of analysis objects included in one reaction field is C, n analysis objects are included in one reaction field.
  • the probability of including is expressed by the following equation (2) from the Poisson model equation.
  • P (n, C) (C n ⁇ e ⁇ C ) / n! ...
  • the concentration ⁇ r of the analyte in the reaction solution is obtained by multiplying the average number C of the analytes by the number of reaction fields, the average volume v of the reaction fields, and the number of reaction fields. You may calculate based on the total volume of the reaction field obtained by multiplying.
  • the concentration of the analyte in the reaction solution thus obtained is converted to the concentration of the analyte in the sample or sample by using the dilution factor when the reaction solution is prepared from the sample or sample. be able to.
  • the droplets to be analyzed are reduced by holding the droplets in two regions having different heights in the PCR process and the sample analysis process. Can be reduced.
  • the loss of droplets in the PCR process is reduced by separating the holding unit 103 and the droplet filling region 104.
  • the holding unit 103 and the droplet emphasis region 104 are not necessarily separated, and for example, a cartridge configuration as shown in FIG. 10 may be used. According to said structure, since a PCR process and an observation process are performed with respect to the droplet with which the same area
  • a cartridge 400 shown in FIG. 4 according to the present embodiment is a cartridge 400 that can be used for analyzing a sample as in the first embodiment.
  • the cartridge 400 according to the present embodiment has a flow channel shape in which the holding unit 103 has a first flow channel 403, a droplet holding region 404, and a second flow channel 405 communicated in a U-shape. It is formed with.
  • bubbles that cause droplet loss in the PCR process can be easily moved away from the droplet, and loss of the droplet can be further prevented.
  • the configuration of the cartridge 400 according to the present embodiment is the same as that of the first embodiment. However, since the first channel 403, the droplet holding region 404, the second channel 405, the droplet filling region 407, and the discharge unit 409 have different configurations from the first embodiment, the configurations will be described below. Other configurations are the same as those in the first embodiment, and thus the description thereof is omitted.
  • FIG. 4 is a view showing the appearance of the cartridge 400 according to this embodiment.
  • FIG. 4A is a plan view of the cartridge 400.
  • FIG. 4B is a cross-sectional view showing the A-A ′ cross section of the cartridge 400.
  • FIG. 4C is a cross-sectional view showing a B-B ′ cross section of the cartridge 400.
  • the hatched portion of the cartridge 400 indicates a portion formed of resin or the like.
  • the upper surface resin (the upper surface of the droplet filling region 407 in FIG. 4B) is shown to show the internal structure. The hatched portion is shown for convenience.
  • the first flow path 403 is connected to the first member 401 and the droplet holding region 404.
  • the droplet holding area 404 holds the droplet generated by the generating unit 402 for performing the nucleic acid amplification process.
  • the second flow path 405 is connected to the droplet holding region 404, the droplet filling region 407, and the second member 410.
  • the droplet filling region 407 is branched into a plurality of regions, and is filled with droplets having different droplet diameters.
  • the droplet filling area 407 may be one area as shown in the first embodiment.
  • the discharge unit 109 is a member that discharges the second liquid.
  • first flow path 403, the droplet holding region 404, and the second flow path 405 communicate with each other in a U shape.
  • the cartridge according to the present embodiment is made of a transparent resin such as polycarbonate or glass. Specifically, the cartridge is formed by laminating a plurality of resin films or glasses having a predetermined thickness.
  • a cartridge is formed by laminating a plurality of resin films each having a predetermined thickness and having a channel shape punched out.
  • a method for bonding the films to be laminated surface activated bonding or molecular adhesion capable of bonding at a low processing temperature lower than the glass transition point of the resin can be used.
  • resin or glass for forming a cartridge is formed into a plate shape.
  • the plate is punched into a channel shape including a U-shape.
  • the flow channel shape of each film is punched by a Thomson type or the like, and is processed so that all the flow channels punched when all the layers are laminated are connected.
  • the punched layers are stacked, and a cartridge having a continuous flow path is manufactured by bonding using surface activated bonding or molecular bonding that can be bonded at a low processing temperature lower than the glass transition point.
  • the material of the cartridge, the shape of the flow path, the formation method, and the like described above are examples, and the present invention is not limited thereto.
  • FIG. 5 shows a flowchart of the entire processing procedure performed using the cartridge 400. Steps S5000 to S5020, step S5040, step S5060, and step S5070 are the same as steps S2000 to S2020, step S2040, step S2060, and step S2070 of the first embodiment, and thus description thereof is omitted. Only the differences from the flowchart of FIG. 2 will be described below.
  • step S5030 (The cartridge is tilted to hold the droplet in the droplet holding area)
  • the tilting means tilts the cartridge 400 to drive the second liquid (oil phase) that is a continuous phase.
  • the droplet generated by the generation unit 402 in accordance with the driving of the continuous phase passes through the first channel 403 and is held in the droplet holding region 404.
  • a tilt stage can be used as the tilting means.
  • the tilt stage is an example of means for tilting the cartridge 400, and is not limited to this as long as it can drive the continuous phase by tilting the angle. Furthermore, the angle of inclination for driving the continuous phase is not limited.
  • the cartridge 400 is tilted after the droplets are generated in step S5020. However, the cartridge 400 may already be tilted in the step S4000. That is, the tilting of the cartridge 400 by the tilting means performed in this step S5030 may be performed at any time prior to S5040.
  • the driving means may substitute for the function of the tilting means.
  • step S5040 it is possible to prevent the loss of the droplet due to the expansion of the bubbles when the droplet is subjected to the thermal cycle. Note that the above effect is more effective by making the height of the first channel 403 and the second channel 405 in the Z-axis direction higher than the height of the droplet holding region 404 in the Z-axis direction.
  • the pressure in the direction in which bubbles are pushed out from the droplet holding region 404 to the first flow path 403 and the second flow path 405 causes the liquid to flow from the first flow path 403 and the second flow path 405.
  • the pressure is larger than the pressure at which the bubbles try to enter the droplet holding region 404. Therefore, bubbles easily escape toward the first channel 403 and the second channel 405.
  • the higher the height of the droplet holding region 404 in the Z-axis direction the easier the bubbles escape. It is desirable to fit in.
  • the height of the droplet holding region in the Z-axis direction is not limited to the above, and may be the same height as the first channel 403 and the second channel 405, for example.
  • the length of the first channel 403 in the Y-axis direction is preferably longer than the width of the second channel 405 in the Y-axis direction. That is, the width of the first flow path 403 is desirably equal to or greater than the width of the second flow path 405. According to the above, stable droplet generation is possible. Note that if the length of the width of the first channel in the Y-axis direction becomes too long, a long processing time is required in the PCR process, so it is desirable to keep the size within a range of 1 to 5 [mm].
  • step S5050 tilt the cartridge to fill the droplet filling area with droplets
  • the droplet subjected to the PCR process is filled in the droplet filling region 407.
  • the tilting unit tilts the cartridge 400 to drive the second liquid (oil phase) that is a continuous phase.
  • the droplets subjected to the PCR process in the droplet holding region 404 as the continuous phase is driven are filled into the droplet filling region 407 through the second channel 405.
  • the droplet filling region 407 through which the droplet flows through the second flow path 405 is branched into eight, and as shown in the BB cross-sectional view of FIG.
  • the height is 70 [ ⁇ m] and 90 [ ⁇ m].
  • the inlet portion 406 is rectangular, and the length in the X-axis direction is longer than the length in the Z-axis direction. That is, the height in the Z-axis direction corresponds to the short side of the rectangle. Note that the length in the X-axis direction may be shorter, in which case the width in the X-axis direction becomes the shorter side.
  • the inlet portion 406 is not necessarily rectangular and may be circular.
  • the short diameter corresponds to the short side
  • the circle is a perfect circle
  • the diameter corresponds to the short side.
  • the droplet filling region 407 is a region filled with droplets, and includes a first droplet filling region and a second droplet filling region where the short sides of the entrance where the droplets flow into the regions are different from each other. This corresponds to an example of a region to be included. If the height of the inlet portion 406 of the droplet filling region 407 in the Z-axis direction is too low due to the average droplet diameter of the dispersed phase to be handled and its dispersion, the shape of the dispersed phase, which is a sphere, is deformed, resulting in an estimated concentration accuracy Affect.
  • the number and height of the droplet filling region 407 are not limited to this, and the height of the inlet portion 406 of the droplet filling region 407 in the Z-axis direction is not necessarily changed for each droplet filling region and may be uniform. .
  • each droplet filling region in the Z-axis direction be lower as the upstream droplet filling region. If the height of the upstream droplet filling region in the Z-axis direction is made higher than that of the downstream droplet filling region, the upstream droplet filling region is also filled with small-diameter droplets. Therefore, when observing the presence or absence of the nucleic acid amplification product in the droplet in step S5060, the droplets appear to overlap and the accuracy of quantification is reduced.
  • the downstream droplet filling area has a large diameter. The droplets are filled, and the overlap of the droplets can be prevented.
  • the droplets filled by changing the width in the X-axis direction without changing the height in the Z-axis direction of the cross section at the inlet 406 may be divided for each droplet diameter.
  • the width in the X-axis direction is set to 70 [ ⁇ m] and 90 [ ⁇ m] without changing the height. According to the above, it is not necessary to increase the height of the droplet filling region 407 in the Z-axis direction when the droplets are distributed according to the droplet diameter and filled in the droplet filling region. It can prevent more overlapping. Note that both the height in the Z-axis direction and the width in the X-axis direction of the inlet portion 406 may be changed.
  • the droplet filling region 407 may have a configuration in which the cross-sectional area of the inlet portion 406 is different from the cross-sectional area of an arbitrary cross section inside the droplet filling region. For example, the internal cross-sectional area is increased with respect to the cross-sectional area of the inlet portion 406. Based on the above, it is possible to reduce the diameter of the droplets flowing into each droplet filling region with the cross-sectional area of the inlet portion 406 and to fill more droplets into the droplet filling region.
  • the height of the bottom surface of the droplet filling region 407 does not need to be common to each droplet filling region.
  • the short sides of the inlet portion 406 may all have different lengths. That is, the droplet filling region 407 includes three or more droplet filling regions, and the short sides of the inlets of the plurality of droplet filling regions may be different from one another.
  • the flow of the droplet filled in the droplet filling region 407 is suppressed by the droplet damming portion 408 at the end portion.
  • the droplet damming portion 408 is provided in such a size that the cross-sectional area at the end of the droplet filling region is smaller than the cross-sectional area of the droplet to be filled. For example, when the size of the cross-sectional area of the inlet portion 406 is 70 [ ⁇ m] and the average diameter of the droplets generated by the generating unit 102 is 60 [ ⁇ m], a droplet weir with a cross-sectional area of 40 [ ⁇ m] is used.
  • a stop 408 is provided at the end of the droplet filling region 407.
  • the droplet damming portion 408 By providing the droplet damming portion 408 as described above, the flow of the filled droplet to the outside of the cartridge 400 can be suppressed although the continuous phase flows. That is, by narrowing the range in which the droplets can move freely, the droplets can be prevented from flowing out, and the amount of observable droplets increases. Further, by providing the droplet damming portion 408, it is possible to reduce the artifacts caused by the flow of the droplet that hinders observation, so that the droplet can be observed without waste.
  • the droplet damming portion 408 is provided on the upper surface of the droplet filling region 407, but may be provided on the lower surface.
  • the region that holds the droplet in the PCR process and the region that is filled with the droplet in the observation step are separated, and the bubbles generated by changing the height in the Z-axis direction are heated to the droplet while further away. Since cycles can be added, loss of droplets can be prevented.
  • a concave suppressing portion 411 (hereinafter referred to as a concave portion) is provided on the lower surface of the second flow path 405 through which the droplets in the cartridge 400 pass.
  • a concave portion is provided on the lower surface of the second flow path 405 through which the droplets in the cartridge 400 pass.
  • the depth of the recess may be set according to the droplet diameter for which movement is desired to be suppressed. For example, when it is desired to prevent the droplet filling region 407 from being filled with droplets of 20 [ ⁇ m] or less, a recess having a depth of about 15 [ ⁇ m] is provided. That is, the recess preferably has a depth in the Z-axis direction that is greater than or equal to the radius of a droplet having a predetermined droplet diameter. This is because if the recess has a depth equal to or larger than the radius of the droplet diameter, the possibility that the droplet will escape from the recess can be reduced.
  • the recess has a depth that is less than half the height of the inlet portion 406 in the Z-axis direction, that is, a depth that is less than the radius of the droplet to be filled.
  • the plurality of recesses may have a small diameter droplet over the first recess and may flow into a droplet filling region provided between the recesses. It is preferable that they are provided continuously at intervals of. Specifically, it is preferable that the droplet filling region 407 is provided at an interval narrower than the interval between adjacent droplet filling regions.
  • each of the plurality of recesses may be made deeper in order in the direction in which the droplets flow in the second flow path 405. That is, the suppression part 411 may include two or more recesses, and the recesses may have different depths in the Z-axis direction. According to the above, among the generated droplets, a droplet having a small droplet diameter can be retained in the recess, so that the droplet filling region 107 is filled with a droplet having a droplet diameter equal to or smaller than a threshold value. Can be further reduced.
  • the suppression unit 411 is provided on the second channel 405, but the position to be provided may be on the first channel 403 or the droplet holding region 404. That is, it may be provided in a region upstream of the droplet filling region 407.
  • the depth and height of the suppressing portion 411 in the Z-axis direction can be arbitrarily determined, and are not limited to the above.
  • the suppressing portion 411 is provided in a concave shape inside the cartridge 400, but the suppressing portion 411 may be provided in a convex shape.
  • a convex suppressing portion 411 (hereinafter, convex portion) is provided on at least one of the upper surface and the lower surface of the second channel 405.
  • the suppressing portion 411 when the suppressing portion 411 is convex, it is more effective to provide a plurality. Specifically, a plurality of convex portions are provided on the upper surface of the second flow path 405, and the height of the convex portions in the Z-axis direction is increased in the direction in which the droplets flow. That is, the suppression unit 411 may include two or more convex portions, and the convex portions may have different heights in the Z-axis direction. According to the above, since the large-diameter droplet that could not be restrained from moving by the first convex portion can be suppressed by the second convex portion, the liquid droplet having a droplet diameter equal to or larger than the threshold value can be The probability of filling 407 can be further reduced.
  • a suppressing portion 411 that is two convex steps having different heights in the Z-axis direction is provided. That is, the suppressing portion 411 in the present modification is two or more convex portions, and the two or more convex portions have different heights in the direction orthogonal to the bottom surface of the cartridge 400.
  • the suppression part 411 does not need to be convex shape, and may be slope shape or curved shape.
  • the suppression unit 411 is desirably provided on the top surface in the droplet filling region 407, but may be provided on the bottom surface.
  • the convex portions are provided at intervals less than a predetermined droplet diameter to be filled. This is because when the interval between the convex portions is wide, there is a possibility that the droplets climb and overlap.
  • the suppressing portion 411 has two convex steps having different heights in the Z-axis direction, but may be a single convex step having two heights. That is, it is only necessary that the suppression unit 411 is provided so that a plurality of regions having different heights in the Z-axis direction are formed in the droplet filling region 407. As described above, it is possible to reduce variations in droplets when filling droplets with different droplet diameters into the droplet filling region 407.
  • a droplet having a small droplet diameter is filled in the droplet filling region provided with the high suppression unit 411.
  • droplets having different droplet diameters can be filled stepwise into droplet filling regions having different heights. That is, the two or more convex portions are provided at positions close to the inlet portion 406 where the droplets flow into the droplet filling region 407 in order of increasing height. According to the above, it is possible to fill the droplet filling region 407 with droplets having the same droplet diameter in regions having different heights, and the droplet diameter is uniform for each region in the droplet filling region 407. Therefore, variation in droplet diameter during analysis can be reduced.

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Abstract

Cette cartouche est utilisée pour analyser des objets à l'intérieur de gouttelettes placées sur une surface inférieure définie par un premier axe et un second axe dans un espace tridimensionnel formé par le premier axe, le second axe, et un troisième axe perpendiculaire à la surface inférieure, et est caractérisée en ce qu'elle comprend : une membrane poreuse pour générer une pluralité de gouttelettes et une unité de support pour supporter les gouttelettes.
PCT/JP2019/020003 2018-05-31 2019-05-21 Cartouche WO2019230485A1 (fr)

Applications Claiming Priority (10)

Application Number Priority Date Filing Date Title
JP2018105480A JP2019211253A (ja) 2018-05-31 2018-05-31 カートリッジ
JP2018-105480 2018-05-31
JP2018-105479 2018-05-31
JP2018105481A JP2019211254A (ja) 2018-05-31 2018-05-31 カートリッジ
JP2018-105481 2018-05-31
JP2018105479A JP7179494B2 (ja) 2018-05-31 2018-05-31 カートリッジ
JP2018105477A JP2019208395A (ja) 2018-05-31 2018-05-31 カートリッジ
JP2018-105477 2018-05-31
JP2018-125179 2018-06-29
JP2018125179A JP2020003421A (ja) 2018-06-29 2018-06-29 カートリッジ

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010532706A (ja) * 2007-05-18 2010-10-14 アプライド バイオシステムズ インコーポレイテッド 粒子を含む実質的に均一なエマルジョンを調製するための機器および方法
JP2012503773A (ja) * 2008-09-23 2012-02-09 クァンタライフ・インコーポレーテッド 液滴ベースの分析システム
JP2018007640A (ja) * 2016-07-15 2018-01-18 株式会社エンプラス 流体取扱装置

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010532706A (ja) * 2007-05-18 2010-10-14 アプライド バイオシステムズ インコーポレイテッド 粒子を含む実質的に均一なエマルジョンを調製するための機器および方法
JP2012503773A (ja) * 2008-09-23 2012-02-09 クァンタライフ・インコーポレーテッド 液滴ベースの分析システム
JP2018007640A (ja) * 2016-07-15 2018-01-18 株式会社エンプラス 流体取扱装置

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