Patent Application
20240278250
The present invention relates to a flow path plate for thermal cycle and a thermal cycle device.
Flow path plates for thermal cycle internally including a flow path of a micron order (a micro flow path) are used in genetic testing and the like in which a very small quantity part of a DNA, which constitutes a gene, is amplified and analyzed, because such flow path plates generally require only small quantities of a measurement-target sample and a reagent for the analysis.
As a flow path plate for thermal cycle, for example, a PCR reaction container including: a resin substrate including a groove-like flow path in a lower surface; a flow path sealing film for sealing the flow path, which is pasted on the lower surface of the substrate; and a sealing film pasted on the upper surface of the substrate is disclosed (for example, see Japanese Patent No. 6803030).
According to Japanese Patent No. 6803030, the PCR reaction container is placed on a first heater and a second heater such that two reaction regions in a thermal cycle region of the flow path in the PCR reaction container are positioned on the first heater and the second heater. A sample obtained by mixing a biological sample containing DNA with a PCR reagent containing a primer, an enzyme, and the like is supplied into the flow path, the thermal cycle region of the flow path is heated, and the sample flowing through the flow path is moved back and forth in the thermal cycle region. A predetermined thermal cycle is applied to the sample to make the sample repeatedly undergo modification, annealing, and elongation. In this way, a specific part of the DNA is selectively amplified, and the amplified product is analyzed.
According to Japanese Patent No. 6803030, as the first heater and the second heater are both set under the PCR reaction container, the sample passing through the flow path in the reaction regions positioned above the first heater and the second heater are heated from under in both of the reaction regions. Hence, a convection flow of the sample flowing from a lower side to an upper side occurs in the flow path positioned above the first heater. However, when the sample having been heated by the first heater is to be cooled to a temperature lower than that by the first heater in the flow path positioned above the second heater, the sample to pass through the flow path positioned above the second heater has a temperature higher than the temperature of the second heater. Hence, the sample passing through the flow path positioned above the second heater does not convect. As a result, a long time is taken until the temperature distribution of the sample passing through the flow path positioned above the second heater becomes uniform, giving rise to a problem that the sample processing time in the PCR reaction container is long.
An object of an embodiment of the present invention is to provide a flow path plate for thermal cycle that can shorten the sample processing time in a flow path.
An embodiment of a flow path plate for thermal cycle according to the present invention is a flow path plate for thermal cycle including a plate-shaped member extending in a horizontal direction, a heating flow path extending in a horizontal direction in the plate-shaped member, a cooling flow path extending in a horizontal direction in the plate-shaped member, a connecting flow path connecting the heating flow path and the cooling flow path, a heating-target surface contacted by a first temperature adjuster configured to heat the heating flow path, and a cooling-target surface contacted by a second temperature adjuster configured to cool the cooling flow path at a temperature lower than that by the first temperature adjuster, wherein a sample containing a nucleic acid is conveyed from the heating flow path to the cooling flow path, the heating-target surface is situated under the heating flow path, and the cooling-target surface is situated above the cooling flow path.
An embodiment of the flow path plate for thermal cycle according to the present invention can shorten the sample processing time in the flow path.
Embodiments of the present invention will be described below in detail. To facilitate understanding of the description, the same components in the drawings will be denoted by the same reference numerals, and overlapping descriptions of the same components are omitted. The components in the drawings may not be to scale. In the present specification, the term “through” indicating numerical ranges is meant to include the values specified before and after “through” as the lower limit and the upper limit, unless otherwise particularly specified.
A thermal cycle device including a flow path plate for thermal cycle according to a first embodiment will be described.
The thermal cycle device 1A can bring efficiency to an analysis by shortening the processing time taken for an amplification reaction of a nucleic acid, when promoting the amplification reaction of the nucleic acid contained in a sample conveyed into the flow path plate 10A for thermal cycle by the conveying member 20.
The sample is a nucleic acid-containing sample, and an example of the sample is an analyte obtained by mixing a DNA-containing biological sample such as blood, nasal mucus, saliva, urine, and the like with a PCR reagent containing a primer, an enzyme, and the like. In the present embodiment, a case in which the sample is an analyte obtained by mixing a biological sample with a PCR reagent will be described.
Nucleic acid amplification reactions are utilized in, for example, a method of detecting a trace DNA constituting a gene with a high sensitivity by amplifying a part of DNA, and analyzing the detected DNA. Among nucleic acid amplification reactions, a Polymerase Chain Reaction (PCR) is effectively utilized as a method for selectively amplifying a specific part of a very small quantity of a DNA collected from a living body and the like to identify the gene polymorphism (SNP) of the living organism, or a method for inspecting the amount of expression of a gene introduced into a cell.
The flow path plate 10A for thermal cycle will be described.
In
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The center base material 111, the lower thin plate member 112, and the upper thin plate member 113 may be made of a material through which the measurement light used for an analysis has a high transmittance. Examples of the material include olefin-based resins, acrylic-based resins, styrene-based resins, vinyl-based resins, fluorine-based resins, engineering plastics, super engineering plastics, thermosetting resins, glass, and the like. These materials may be used alone or in combination of two or more.
Examples of the olefin-based resins include: polyethylene resins such as polyethylene (PE), high-density polyethylene, low-density polyethylene, and the like; polypropylene resins such as polypropylene (PP), propylene-ethylene copolymers, and the like: cycloolefin-based resins such as cycloolefin polymers (COP), cycloolefin copolymers (COC), ethylene-cyclic olefin copolymers, and the like. Among these olefin-based resins, it is preferable to use cycloolefin-based resins in terms of ease of production, wideness of the range of wavelengths of light that can be transmitted, chemical resistance, and the like, and COPs and COCs are particularly preferable among cycloolefin-based resins.
The COPs are resins obtained by polymerizing a monomer component containing a cycloolefin monomer. The cycloolefin monomer that forms the COPs is not particularly limited, yet norbornene-based monomers are preferable. The norbornene-based monomers are not particularly limited so long as they have a norbornene ring. The COPs may contain any other monomer that is copolymerizable with the cycloolefin monomer in addition to the cycloolefin monomer. Examples of the any other monomer include straight-chained or branched alkene monomers, examples of which include α-olefins such as ethylene, propylene, 1-butene, isobutene, 1-hexene, and the like.
The COCs are copolymers in which two or more types of the cycloolefin monomers are combined.
An example of the acrylic-based resins is polymethyl methacrylate (PMMA).
Examples of the styrene-based resins include polystyrene (PS), acrylonitrile-styrene resins, and acrylonitrile-butadiene-styrene (ABS) resins.
Examples of the vinyl-based resins include polyvinyl chloride (PVC) resins, vinylidene chloride resins, polyacrylonitrile, polyvinyl acetate, acrylic acid copolymers, and polyvinyl alcohols.
Examples of the fluorine-based resins include polytetrafluoroethylene (PTFE), polychlorotrifluoroethylene (PCTFE), polyvinyl fluoride resins, and polyvinylidene fluoride.
Examples of the engineering plastics include: polycarbonate (PC) resins; polyacetal (POM) resins; polyester resins such as polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polycyclohexylene dimethyl terephthalate, and the like; polyphenylene ether (PPE) resins; polyphenylene oxide; polyamide (PA) resins such as nylon 6, nylon 66, aromatic polyamides, and the like.
Examples of the super engineering plastics include polyphenylene sulfide (PPS) resins, polysulfone (PSF) resins, polyether sulfone (PES), polyether ether ketone (PEEK), polyallylate resins, aromatic polyester resins, polyimide (PI) resins, polyamide imide (PAI) resins, polyether imide (PEI) resins, and aramid resins.
Examples of the thermosetting resins include epoxy resins, silicone resins, phenol resins, unsaturated polyester resins, polyurethane resins, and the like.
As the center base material 111, the lower thin plate member 112, and the upper thin plate member 113, the materials specified above may be used alone or in combinations of two or more.
The center base material 111, the lower thin plate member 112, and the upper thin plate member 113 may be made of the same material, or may be made of different materials. In terms of inhibiting transmission of the measurement light to the outside halfway while passing through the flow path in the plate-shaped member 11A, the lower thin plate member 112 and the upper thin plate member 113 may be made of a material through which the measurement light used for an analysis has a high transmittance, and the center base material 111 may be made of a material through which the measurement light used for an analysis has a lower transmittance than through the lower thin plate member 112 and the upper thin plate member 113.
In this case, from among the materials specified above, a material through which the measurement light has a lower transmittance than through the material used to form the lower thin plate member 112 and the upper thin plate member 113 may be used as the material used to form the center base material 111. The center base material 111 needs only for the measurement light to have a lower transmittance therethrough than through the lower thin plate member 112 and the upper thin plate member 113, and it is preferable that the center base material 111 does not transmit the measurement light. The center base material 111 may be colored so as not to transmit the measurement light.
The materials used to form the center base material 111, the lower thin plate member 112, and the upper thin plate member 113 are appropriately selected in accordance with the wavelength of the measurement light used. For example, in a case of forming all of the center base material 111, the lower thin plate member 112, and the upper thin plate member 113 using COCs, a COC through which the measurement light has a lower transmittance than through the COCs used to form the lower thin plate member 112 and the upper thin plate member 113 is used as the COC used to form the center base material 111.
In a case of forming the center base material 111, the lower thin plate member 112, and the upper thin plate member 113 using olefin-based resins, acrylic-based resins, styrene-based resins, vinyl-based resins, fluorine-based resins, engineering plastics, super engineering plastics, or thermosetting resins from among the materials specified above, it is optional to obtain the members by molding resin materials containing these materials as a main component (a base resin).
The center base material 111, the lower thin plate member 112, and the upper thin plate member 113 may further contain one, or two or more selected from a group of reinforcing materials, release agents, antioxidants, and the like, as sub components. In a case of forming the center base material 111, the lower thin plate member 112, and the upper thin plate member 113 using COCs or COPs as super engineering plastics, it is possible to adjust the transmittance by adjusting the contents of any additives to be added in addition to COCs or COPs serving as the main component. Hence, by adjusting the amount of any additive to be used in the lower thin plate member 112 and the upper thin plate member 113 and the amount of any additive to be used in the center base material 111, it is possible to make the transmittance through the center base material 111 lower than the transmittance through the lower thin plate member 112 and the upper thin plate member 113.
The thickness of each of the center base material 111, the lower thin plate member 112, and the upper thin plate member 113 may be appropriately designed in accordance with the size of the plate-shaped member 11A and the like. For example, the thickness of the center base material 111 is preferably from 2 mm through 5 mm. When the thickness of the center base material 111 is from 2 mm through 5 mm, the plate-shaped member 11A can have a sufficient strength even when a groove is formed in the center base material 111.
The thickness of the upper thin plate member 113 is preferably from 0.1 mm through 0.2 mm. When the thickness of the upper thin plate member 113 is from 0.1 mm through 0.2 mm, the upper thin plate member 113 can easily conduct heat from the second temperature adjuster 40 to the interior of a cooling flow path 143, and can have a strength enough to stay unbroken when contacted by the second temperature adjuster 40.
The thickness of the lower thin plate member 112 is preferably from 0.1 mm through 0.2 mm. When the thickness of the lower thin plate member 112 is from 0.1 mm through 0.2 mm, the lower thin plate member 112 can easily conduct heat from the first temperature adjuster 30 to the interior of a heating flow path 141, and can have a strength enough to stay unbroken when contacted by the first temperature adjuster 30.
In a case where the center base material 111, the lower thin plate member 112, and the upper thin plate member 113 are made of, for example, a synthetic resin or glass, they may be joined by thermocompression bonding, or may be joined using an adhesive such as an ultraviolet curable resin and the like. In a case where the upper thin plate member 113 and the center base material 111 are made of glass, they may be joined using an adhesive.
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A lower groove 111c in the center base material 111 is formed in the lower surface of the center base material 111 as illustrated in
An upper groove 111d in the center base material 111 is formed in the upper surface of the center base material 111 as illustrated in
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The flow path 12A is formed as a result of the center base material 111, the lower thin plate member 112, and the upper thin plate member 113 being joined. In this way, the flow path 12A is formed in the interior of the plate-shaped member 11A as illustrated in
The diameter the guiding flow path 13 and the micro flow path 14 is designed to be, for example, from some nanometers through some hundreds of micrometers. The diameter (inner diameter) of the guiding flow path 13 and the micro flow path 14 is defined as the diameter of a hole that can be imaginarily regarded as a circle in a cross-section obtained by imaginarily cutting the guiding flow path 13 and the micro flow path 14 to pass a center of the guiding flow path 13 and the micro flow path 14 perpendicularly to their longer direction. When the guiding flow path 13 and the micro flow path 14 have a circular shape, the size of the diameter of the guiding flow path 13 and the micro flow path 14 is defined as the length of the diameter. When the guiding flow path 13 and the micro flow path 14 have an approximately rectangular shape, the size of the diameter is defined as the diameter of a circle when they are assumed as a circle that has the same area as the approximately rectangular shape.
As illustrated in
It is preferable that a cross-section of the guiding flow path 13 is formed to be larger than a cross-section of the micro flow path 14. When suppling a sample, a supply tube for supplying the sample is inserted into the inlet opening 15 of the guiding flow path 13. Hence, the supply tube can be easily inserted into the guiding flow path 13 when the cross-section of the guiding flow path 13 is formed to be larger.
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A cross-section of the heating flow path 141 is formed in an approximately rectangular shape when seen in a direction orthogonal to the flow of a sample.
The connecting flow path 142 is formed to penetrate the center base material 111 such that light can be transmitted in the upper-lower direction of the plate-shaped member 11A, and connects the heating flow path 141 and the cooling flow path 143 to each other. The connecting flow path 142 is a space that is constituted by a hole 111b formed in the principal surfaces of the center base material 111, and through which the lower groove 111c and the upper groove 111d communicate with each other. The connecting flow path 142 is a space into which the measurement light is emitted, and can be used as a flow path for optical detection. As illustrated in
The connecting flow path 142 is formed in a circular shape when seen in the axial direction thereof as illustrated in
The connecting flow path 142 is formed in a size through which a reagent and the measurement light can pass when seen in the axial direction thereof, and has a cross-sectional area approximately equal to or smaller than the heating flow path 141 and the cooling flow path 143 when seen in the axial direction thereof as illustrated in
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A cross-section of the cooling flow path 143 is formed in an approximately rectangular shape when seen in a direction orthogonal to the flow of a sample, like the heating flow path 141.
The plate-shaped member 11A has a heating-target surface contacted by the first temperature adjuster 30 configured to heat the heating flow path 141, and a cooling-target surface contacted by the second temperature adjuster 40 configured to cool the cooling flow path 143 at a temperature lower than that by the first temperature adjuster 30.
The heating-target surface is the principal surface (lower surface) 11b of the plate-shaped member 11A, and is positioned under the heating flow path 141. The cooling-target surface is the principal surface (upper surface) 11a of the plate-shaped member 11A, and is positioned above the cooling flow path 143.
Next, an example of the method for producing the flow path plate 10A for thermal cycle will be described. First, a groove or holes that constitute(s) the flow path 12A, or the inlet opening 15 and the outlet opening 16 of the flow path plate 10A for thermal cycle are formed in the junction surface sides of two plates from among three rectangular plates. As a result, the center base material 111 and the upper thin plate member 113 are obtained. The remaining one plate is used as the lower thin plate member 112.
The groove and the holes between the center base material 111 and the upper thin plate member 113 may be formed when molding the center base material 111 and the upper thin plate member 113 by injection molding, transfer molding, or the like, or may be machining-formed by mechanical machining, a laser, or the like. Alternatively, the groove may be formed in the surfaces of the center base material 111 and the upper thin plate member 113 by press machining or the like.
Next, the junction surfaces of the center base material 111 are sandwiched between the lower thin plate member 112 and the upper thin plate member 113, and these members are overlaid on each other such that the members are not misaligned. Subsequently, the center base material 111, the lower thin plate member 112, and the upper thin plate member 113 are joined integrally by, for example, heating and welding (thermocompression bonding) or the like. For example, an adhesive is applied to the center base material 111, the lower thin plate member 112, and the upper thin plate member 113 on portions other than the portions corresponding to the grooves and the holes, and then the members are pasted to each other and joined integrally. In this way, the flow path plate 10A for thermal cycle illustrated in
As illustrated in
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The first temperature adjuster 30 needs only to be able to heat the plate-shaped member 11A. As the first temperature adjuster 30, a common heating device such as a hot plate, an aluminum block heater, and the like may be used.
The first temperature adjuster 30 can appropriately heat the heating flow path 141 to a desirably selected temperature in accordance with the type of a sample, and the temperature is preferably, for example, from 85° C. through 98° C., and particularly preferably around 94° C. In a case where a sample is an analyte obtained by mixing a DNA and a PCR reagent, a double-stranded DNA can be separated into single-stranded DNA in the specified temperature range.
There is a case where the first temperature adjuster 30 cannot conduct heat sufficiently to a sample when heating the sample in the heating flow path 141. Hence, the setting temperature of the first temperature adjuster 30 may be set to be higher by some degrees Celsius (e.g., by from 2° C. through 3° C.) in accordance with the temperature of the sample.
The installation area of the first temperature adjuster 30 needs only to be in a range in which the heating flow path 141 is included in a plan view of the plate-shaped member 11A.
The thermal cycle device 1A may include a non-illustrated temperature sensor or the like on the heating-target surface (principal surface 11b) of the plate-shaped member 11A, and may control the temperature of the heating flow path 141.
As illustrated in
The second temperature adjuster 40 needs only to be able to cool the sample heated through the heating flow path 141. As the second temperature adjuster 40, a common heating device such as a heater and the like, or a cooling device may be used.
The second temperature adjuster 40 can appropriately cool the cooling flow path 143 to a desirably selected temperature in accordance with the type of a sample, and the temperature is preferably, for example, from 55° C. through 65° C., and particularly preferably around 60° C. In a case where a sample is an analyte obtained by mixing a DNA and a PCR reagent, the amplification reaction of the DNA contained in the sample can be promoted in the specified temperature range through annealing of single-stranded DNA and the PCR reagent and elongation of the DNA.
There is a case where the second temperature adjuster 40 cannot absorb heat sufficiently from a sample when cooling the sample in the cooling flow path 143. Hence, the setting temperature of the second temperature adjuster 40 may be set to be lower by some degrees Celsius (e.g., by from 2° C. through 3° C.) in accordance with the temperature of the sample.
The installation area of the second temperature adjuster 40 needs only to be in a range in which the cooling flow path 143 is included in a plan view of the plate-shaped member 11A.
The thermal cycle device 1A may include a non-illustrated temperature sensor or the like on the cooling-target surface of the plate-shaped member 11A, and may control the temperature of the cooling flow path 143.
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The light-receiving member 60 needs only to be able to detect the measurement light, and a publicly-known detector may be used. The light-receiving member 60 is connected to the control unit 70 through a wiring 61.
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An example of how to use the thermal cycle device 1A according to the present embodiment will be described.
In response to the flow path plate 10A for thermal cycle being inserted into the device main body of the thermal cycle device 1A, the flow path plate 10A for thermal cycle is positionally fixed in the device main body. Subsequently, as illustrated in
The conveying member 20 pressure-feeds a sample in the sample storage 21, to be supplied into the inlet opening 15 through the supply tube 91. As illustrated in
The sample that flows through the heating flow path 141 is heated to a high temperature (e.g., 94° C.) by the first temperature adjuster 30 while passing through the heating flow path 141 (thermal denaturation). By the sample in the heating flow path 141 being heated, double-stranded DNA contained in the sample is separated into single-stranded DNA.
Here, the sample flowing through the heating flow path 141 is heated from under. Hence, the sample positioned at a lower side in the heating flow path 141 is rapidly heated to a high temperature, and the sample positioned at an upper side in the heating flow path 141 is slowly heated to a high temperature. Hence, as illustrated in
The conveying speed of the sample flowing through the heating flow path 141 may be adjusted such that the sample is heated to a predetermined temperature (e.g., 94° C.).
As the period of time for which the sample flows through the heating flow path 141, it is preferable to retain the sample at the predetermined temperature (e.g., 94° C.) for from 20 seconds through 2 minutes after the sample has reached the predetermined temperature (e.g., 94° C.).
The sample having passed throughout the heating flow path 141 is supplied into the connecting flow path 142. The sample flows in the thickness direction (+Z-axis direction) of the flow path plate 10A for thermal cycle by passing through the connecting flow path 142.
Before the sample comes flowing through the connecting flow path 142 or in a state in which the sample is flowing through the connecting flow path 142, the measurement light is emitted from the light-emitting member 50 into the connecting flow path 142 such that the measurement light passes through the connecting flow path 142. The light emitted from the light-emitting member 50 passes through the connecting flow path 142, and is then received and detected by the light-receiving member 60. As a result, the components in the sample that has passed through the connecting flow path 142 become ready to be analyzed. As the measurement light, visible light, ultraviolet light, infrared light, and the like may be used.
The detection result detected by the light-receiving member 60 is sent to the control unit 70 through the wiring 61, and the control unit 70 analyzes each component in the sample that has passed through the connecting flow path 142. The control unit 70 sends the analysis result to the display unit 80, and the analysis result is displayed on the display unit 80. From the displayed result on the display unit 80, it is possible to confirm that the sample has passed through the connecting flow path 142 from the heating flow path 141.
After having passed through the connecting flow path 142, the sample from the connecting flow path 142 flows through the cooling flow path 143 formed in the upper surface of the center base material 111.
The sample flowing through the cooling flow path 143 is cooled to a low temperature (e.g., 60° C.) by the second temperature adjuster 40 (annealing and elongation). By the sample in the cooling flow path 143 being cooled, the amplification reaction of the DNA contained in the sample is promoted through annealing of single-stranded DNA and the primer contained in the sample and elongation of the DNA.
Here, the sample flowing through the cooling flow path 143 is cooled from above in the cooling flow path 143. Hence, the sample positioned at an upper side in the cooling flow path 143 is rapidly cooled to a low temperature, and the sample positioned at a lower side in the cooling flow path 143 is slowly cooled to a low temperature. Hence, as illustrated in
The sample passing through the cooling flow path 143 is expelled into the expelling tube 92 from the outlet opening 16. The sample expelled into the expelling tube 92 from the outlet opening 16 is analyzed (observed) by the control unit 70.
The sample supplied into the flow path 12A may be moved to the heating flow path 141 and to the cooling flow path 143 in the micro flow path 14 in an alternating manner back and forth a plurality of times, to form a thermal cycle in the plate-shaped member 11A. For example, by being pressure-fed and suctioned by the conveying member 20, the sample can be moved between the heating flow path 141 and the cooling flow path 143 in the micro flow path 14 in an alternating manner back and forth a plurality of times. After the sample is pressure-fed by the conveying member 20 to be moved from the heating flow path 141 to the cooling flow path 143, the sample is then suctioned by the conveying member 20 to be moved from the cooling flow path 143 to the heating flow path 141. By moving the sample supplied into the flow path 12A to the heating flow path 141 and to the cooling flow path 143 in the micro flow path 14 in an alternating manner back and forth a plurality of times, it is possible to reliably promote the nucleic acid amplification reaction, particularly, PCR of the sample.
The number of times to move the sample in the flow path 12A back and forth between the heating flow path 141 and the cooling flow path 143 is appropriately determined in accordance with the combination of the target nucleic acid, the primer, the enzyme, and the like.
Hence, the flow path plate 10A for thermal cycle according to the present embodiment includes the plate-shaped member 11A, the heating flow path 141, the connecting flow path 142, the cooling flow path 143, the heating-target surface (principal surface 11b), and the cooling-target surface (principal surface 11a), with the heating-target surface situated under the heating flow path 141 and the cooling-target surface situated above the cooling flow path 143. The flow path plate 10A for thermal cycle can easily make the sample temperature uniform, because it can induce a convection flow of the sample in the heating flow path 141 and in the cooling flow path 143 both when the sample is heated by the first temperature adjuster 30 in the heating flow path 141 and when the sample is cooled by the second temperature adjuster 40 in the cooling flow path 143. The flow path plate 10A for thermal cycle can shorten the sample processing time in the heating flow path 141 and the cooling flow path 143, because it can shorten the time taken until the temperature distribution in the heating flow path 141 and the cooling flow path 143 becomes uniform.
The flow path plate 10A for thermal cycle can inhibit variation in the temperature of the reagent while being heated, because it can heat the reagent to a uniform temperature in the heating flow path 141. Hence, the flow path plate 10A for thermal cycle can inhibit what is generally referred to as an overshoot, in which the heating temperature rises to equal to or higher than the setting temperature, from occurring while the reagent is heated in the heating flow path 141. Raising the temperature of the reagent by setting the heating temperature to be higher than a predetermined temperature in order to shorten the time taken to raise the temperature of the reagent is likely to cause variation in the temperature of the reagent, inclining to an overshoot in which the reagent becomes equal to or higher than the setting temperature. The flow path plate 10A for thermal cycle can inhibit an overshoot, because it can heat the reagent to a uniform temperature in the heating flow path 141, keeping the reagent from becoming equal to or higher than the setting temperature.
In the flow path plate 10A for thermal cycle, the plate-shaped member 11A may be formed of the center base material 111, the lower thin plate member 112, and the upper thin plate member 113, the heating flow path 141 may be constituted by a space enclosed by the lower groove 111c in the center base material 111 and by the lower thin plate member 112, the cooling flow path 143 may be constituted by a space enclosed by the upper groove 111d formed in the upper surface of the center base material 111 and by the upper thin plate member 113, and the connecting flow path 142 may be constituted by a space through which the lower groove 111c and the upper groove 111d communicate with each other. The lower thin plate member 112 and the upper thin plate member 113 both having a small thickness can increase the thermal transfer efficiency. The flow path plate 10A for thermal cycle can further shorten the sample processing time in the heating flow path 141 and the cooling flow path 143, because it can increase the sample heating or cooling efficiency in the heating flow path 141 and the cooling flow path 143 via the lower thin plate member 112 and the upper thin plate member 113.
In the flow path plate 10A for thermal cycle, the lower thin plate member 112 and the upper thin plate member 113 may be made of a material having light transmissivity, and the connecting flow path 142 may be formed to penetrate the center base material 111 in the upper-lower direction of the plate-shaped member 11A. Hence, the flow path plate 10A for thermal cycle can inhibit the measurement light, which comes incident into the connecting flow path 142 through the upper thin plate member 113, from being transmitted to outside the connecting flow path 142 when passing through the center base material 111, making it possible to increase the detection amount of the measurement light to go outside through the lower thin plate member 112. With the flow path plate 10A for thermal cycle, it is possible to perform a high-accuracy optical detection by detecting the measurement light that is to go outside through the lower thin plate member 112.
In a case where a sample contains a nucleic acid, the flow path plate 10A for thermal cycle can induce the nucleic acid amplification reaction in the flow path 12A including the heating flow path 141 and the cooling flow path 143. Hence, the flow path plate 10A for thermal cycle can be effectively used as an analysis plate in which PCR or the like is induced.
The thermal cycle device 1A according to the present embodiment includes the flow path plate 10A for thermal cycle, the conveying member 20 the micro flow path 14, the first temperature adjuster 30, and the second temperature adjuster 40, wherein the micro flow path 14 includes the heating flow path 141, the connecting flow path 142, and the cooling flow path 143, the first temperature adjuster 30 is situated under the heating flow path 141, and the second temperature adjuster 40 is situated above the cooling flow path 143. The thermal cycle device 1A can induce a convection flow of a reagent through heating or cooling in the flow path 12A both when heating a sample in the heating flow path 141 by the first temperature adjuster 30 and when cooling the sample in the cooling flow path 143 by the second temperature adjuster 40. Hence, the thermal cycle device 1A can shorten the sample processing time in the flow path 12A, because it can shorten the time taken until the temperature distribution in the flow path 12A becomes uniform.
The flow path plate 10A for thermal cycle and the thermal cycle device 1A according to the present embodiment, which can shorten the sample processing time as described above, can be effectively used for the nucleic acid amplification reaction such as PCR and the like. The nucleic acid amplification reaction can be effectively used in a method for keeping track of gene expression patterns in cells that are being in unusual states such as iPS cells, ES cells, cancer cells, and the like, for identifying pathogens, and for inspecting the gene polymorphism (SNP) of living organisms, a method for inspecting the amount of expression of a gene introduced into a cell, and the like. Moreover, the nucleic acid amplification reaction, which can amplify a trace nucleic acid to a quantity in which the nucleic acid can be visually precepted, is also useful for very small quantity detection of microorganisms, and can be effectively used in a method for quick detection of microorganisms and the like. Furthermore, the nucleic acid amplification reaction, which can amplify a complementary DNA (cDNA) transformed from RNA with a reverse transcriptase, can be effectively used for small quantity detection of RNA, and the like.
The flow path plate 10A for thermal cycle and the thermal cycle device 1A according to the present embodiment, which can shorten the processing time taken to promote the nucleic acid amplification reaction such as PCR and the like, can easily increase the accuracy of analyses and the like of trace substances such as blood components including proteins and nucleic acids contained in the blood. Hence, the flow path plate 10A for thermal cycle and the thermal cycle device 1A can be suitably used for, for example, genetic testing such as medical testing, identification of agricultural produce and pathogenic microorganisms, food safety evaluation, pathogenic virus and infectious disease testing, and the like.
A modified example of the thermal cycle device 1A will be described.
In the present embodiment, the thermal cycle device 1A may include a non-illustrated first pressure feeder connected to the inlet opening 15, and a non-illustrated second pressure feeder (e.g., a pressurizing pump or the like) connected to the outlet opening 16. In this case, after a sample is introduced into the inlet opening 15, a first nozzle connected to the non-illustrated first pressure feeder (e.g., a pressurizing pump or the like) is connected to the inlet opening 15 with the supply tube 91 removed from the plate-shaped member 11A, and a second nozzle connected to the non-illustrated second pressure feeder (e.g., a pressurizing pump or the like) is connected to the outlet opening 16. Then, the non-illustrated first pressure feeder is actuated to supply air into the flow path 12A from the non-illustrated first pressure feeder to move the sample from the heating flow path 141 to the cooling flow path 143, followed by the non-illustrated first pressure feeder being stopped and the non-illustrated second pressure feeder being actuated to supply air into the flow path 12A from the non-illustrated second pressure feeder, to move the sample from the cooling flow path 143 to the heating flow path 141. In this way, the flow path plate 10A for thermal cycle can reliably promote the nucleic acid amplification reaction, particularly, PCR of the sample, through moving the sample supplied into the flow path 12A to the heating flow path 141 and to the cooling flow path 143 in the micro flow path 14 in an alternating manner back and forth a plurality of times and thereby forming a thermal cycle in the plate-shaped member 11A.
A thermal cycle device including a flow path plate for thermal cycle according to a second embodiment will be described. The thermal cycle device according to the present embodiment includes two of the heating flow path 141 of the micro flow path 14A included in the flow path 12A of the flow path plate 10A for thermal cycle according to the first embodiment described above, with the second heating flow path situated at the succeeding stage of the cooling flow path 143. The thermal cycle device according to the present embodiment also includes a third temperature adjuster configured to adjust a sample to an intermediate temperature between the adjusting-target temperature of the first temperature adjuster 30 and the adjusting-target temperature of the second temperature adjuster 40 of the thermal cycle device 1A according to the first embodiment described above.
As illustrated in
The flow path 12B includes a guiding flow path 13 and a micro flow path 14B. The flow path 12B has openings 17A to 17C in the principal surface 11a of the plate-shaped member 11B in the +Z-axis direction, and includes membrane valves 18 in halfway portions of the micro flow path 14B.
As illustrated in
Holes 111e to 111g in the center base material 111 are formed in an approximately circular shape when seen in a center line of the holes 111e to 111g. The guiding flow path 13 and parts of the micro flow path 14B (connecting flow paths 142A and 142B described below), which are illustrated in
Lower grooves 111h and 111j of the center base material 111 are formed in the lower surface of the center base material 111, and are formed in an approximately rectangular shape like the lower groove 111c. By the center base material 111 and the lower thin plate member 112 being pasted to each other, parts of the micro flow path 14B (heating flow paths 141A and 141B described below) illustrated in
An upper groove 111i of the center base material 111 is formed in the upper surface of the center base material 111, and is formed in an approximately rectangular shape like the upper groove 111d. By the center base material 111 and the upper thin plate member 113 being pasted to each other, a part of the micro flow path 14B (the cooling flow path 143 escribed below) illustrated in
Holes having a shape conforming to the openings 17A to 17C are formed in the upper thin plate member 113. The holes in the upper thin plate member 113 are formed in an approximately circular shape when seen in a center line of the holes. That is, the openings 17A to 17C are formed in the upper thin plate member 113, and are formed in an approximately circular shape in a plan view of the plate-shaped member 11B.
As illustrated in
By the center base material 111, the lower thin plate member 112, and the upper thin plate member 113 being joined with each other, the flow path 12B is formed. As illustrated in
As illustrated in
As illustrated in
As illustrated in
As illustrated in
A cross-section of the heating flow path 141A has an approximately rectangular shape when seen in a direction orthogonal to the flow of a sample.
The connecting flow path 142A is formed to penetrate the center base material 111 such that light can be transmitted in the upper-lower direction of the plate-shaped member 11B, and connects the heating flow path 141A and the cooling flow path 143 to each other. The connecting flow path 142A is a space that is constituted by a hole 111f formed in the principal surfaces of the center base material 111, and through which the lower groove 111h and the upper groove 111i communicate with each other as illustrated in
As illustrated in
The connecting flow path 142B is formed to penetrate the center base material 111 such that light can be transmitted in the upper-lower direction of the plate-shaped member 11B like the connecting flow path 142A, and connects the cooling flow path 143 and the heating flow path 141B to each other. The connecting flow path 142B is a space that is constituted by a hole 111g formed in the principal surfaces of the center base material 111, and through which the upper groove 111i and the lower groove 111j communicate with each other as illustrated in
As illustrated in
As illustrated in
As illustrated in
The membrane valve 18A is attached in a horizontal direction to the internal wall of a joining portion of the heating flow path 141A joining a larger cross-sectional area region of the heating flow path 141A to a smaller cross-sectional area region thereof. As illustrated in
The membrane valve 18B is attached in a horizontal direction to the internal wall of a joining portion of the cooling flow path 143 joining a larger cross-sectional area region of the cooling flow path 143 to a smaller cross-sectional area region thereof. As illustrated in
The membrane valve 18C is attached in a horizontal direction to the internal wall of a joining portion of the heating flow path 141B joining a larger cross-sectional area region of the heating flow path 141B to a smaller cross-sectional area region thereof. As illustrated in
The membrane valves 18A to 18C are tubular moldings made of an elastic material such as rubber, and, in a free state, have an opening shape gradually narrowing from the upstream ends 181A to 181C to the downstream ends 182A to 182C as illustrated in
At the downstream ends 182A to 182C, the opening is closed in an openable/closable manner, such that the opening is maintained in the closed state while no sample is coming flowing into the membrane valves 18A to 18C, and becomes opened due to a supplying pressure (positive pressure) of a sample when the sample comes flowing into the membrane valves 18A to 18C, or due to a negative pressure.
When the flow path 12B is at a negative pressure, a pulling force acts on the membrane valves 18A to 18C, and the closed downstream ends 182A to 182C become open as illustrated in
The membrane valves 18A to 18C are not particularly limited to the membrane valve having the configuration illustrated in
Like the plate-shaped member 11A, the plate-shaped member 11B includes a heating-target surface contacted by first temperature adjusters 30 and 45 configured to heat the heating flow paths 141A and 141B, and a cooling-target surface contacted by the second temperature adjuster 40 configured to cool the cooling flow path 143 at a temperature lower than that by the first temperature adjuster 30.
The heating-target surface is the principal surface (lower surface) 11b of the plate-shaped member 11B, and is positioned under the heating flow paths 141A and 141B. The cooling-target surface is a principal surface (upper surface) 11a of the plate-shaped member 11B, and is positioned above the cooling flow path 143.
As illustrated in
The third temperature adjuster 45 needs only to be able to heat a sample in the heating flow path 141B via the plate-shaped member 11B by adjusting the temperature of the sample to an intermediate temperature between the adjusting-target temperatures of the first temperature adjuster 30 and the second temperature adjuster 40. As the third temperature adjuster 45, the same heating device as the first temperature adjuster 30 may be used.
The third temperature adjuster 45 can appropriately heat the heating flow path 141B to a desirably selected temperature in accordance with the type of the sample, and the temperature is preferably, for example, from 60° C. through 72° C., and particularly preferably around 70° C. In a case where a sample is an analyte obtained by mixing a DNA and a PCR reagent, the temperature of the sample can be maintained at an optimal temperature for the activity of a DNA polymerase without separation of a primer contained in the sample in the specified temperature range.
There is a case where the third temperature adjuster 45 cannot conduct heat sufficiently to a sample when heating the sample in the heating flow path 141B. Hence, the setting temperature of the third temperature adjuster 45 may be set to be higher by some degrees Celsius (e.g., by from 2° C. through 3° C.) in accordance with the temperature of the sample.
The installation area of the third temperature adjuster 45 needs only to be in a range in which the heating flow path 141B is included in a plan view of the plate-shaped member 11B.
The thermal cycle device 1B may include a non-illustrated temperature sensor or the like on the heating-target surface (principal surface 11b) of the plate-shaped member 11B, and may control the temperature of the heating flow path 141B.
An example of how to use the thermal cycle device 1B according to the present embodiment will be described.
After the flow path plate 10B for thermal cycle is inserted into the device main body of the non-illustrated thermal cycle device and positionally fixed in the device main body, the syringes 93 are automatically inserted into the openings 17A to 17C as illustrated in
The sample flowing through the heating flow path 141A is heated to a high temperature (e.g., 94° C.) by the first temperature adjuster 30 while passing through the heating flow path 141A (thermal denaturation). By the sample in the heating flow path 141A being heated, double-stranded DNA contained in the sample is separated into single-stranded DNA.
Here, because the sample that flows through the heating flow path 141A is heated from under, a convection flow of the sample flowing through the heating flow path 141A is easily induced as described above. Hence, the sample flowing through the heating flow path 141A can be easily heated to a high temperature owing to the convection flow thereof.
The sample may be retained in the heating flow path 141A until it is heated to a high temperature (e.g., 94° C.), by supplying of the sample from the opening 17A being stopped and by the membrane valves 18A to 18C being controlled to be opened or closed by stopping suctioning of air in the flow path 12B through the syringes provided into the openings 17B and 17C.
The sample that has passed the heating flow path 141A then passes the membrane valve 18B, and then flows in the thickness direction (+Z-axis direction) of the flow path plate 10B for thermal cycle by passing through the connecting flow path 142A.
Before the sample comes flowing through the connecting flow path 142A or in a state in which the sample is flowing through the connecting flow path 142A, the measurement light may be emitted from the light-emitting member 50 (see
Next, by suctioning of air in the flow path 12B through the syringe 93 inserted into the opening 17B being stopped, the sample goes flowing from the connecting flow path 142A through the cooling flow path 143 formed in the upper surface of the center base material 111.
The sample flowing through the cooling flow path 143 is cooled to a low temperature (e.g., 60° C.) by the second temperature adjuster 40 (annealing and elongation). By the sample in the cooling flow path 143 being cooled, the amplification reaction of the DNA contained in the sample is promoted through annealing of single-stranded DNA and the primer contained in the sample and elongation of the DNA.
Here, because the sample flowing through the cooling flow path 143 is cooled from above, a convection flow of the sample flowing through the cooling flow path 143 is easily induced as described above. Hence, the sample flowing through the cooling flow path 143 can be easily cooled to a low temperature owing to the convection flow thereof.
The sample may be retained in the cooling flow path 143 until it is cooled to a low temperature (e.g., 60° C.), by supplying of the sample from the opening 17A being stopped and by the membrane valves 18A to 18C being controlled to be opened or closed by stopping supplying of air in the flow path 12B through the syringes provided into the openings 17B and 17C.
The sample that has passed the cooling flow path 143 then passes the membrane valve 18C, and then flows in the thickness direction (−Z-axis direction) of the flow path plate 10B for thermal cycle by passing through the connecting flow path 142B.
Before the sample comes flowing through the connecting flow path 142B or in a state in which the sample is flowing through the connecting flow path 142B, the measurement light may be emitted from the light-emitting member 50 (see
Next, supplying of the sample through the syringe 93 provided into the opening 17A is stopped to stop air from being pressure-fed into the flow path 12B, and air is supplied into the flow path 12B through the syringe provided into the opening 17C to close the membrane valve 18C. This causes the sample from the connecting flow path 142B to flow through the heating flow path 141B formed in the lower surface of the center base material 111.
The sample flowing through the heating flow path 141B is heated to an intermediate temperature (e.g., from 60° C. through 72° C.) by the third temperature adjuster 45 while passing through the heating flow path 141B. By the sample in the heating flow path 141B being heated to an intermediate temperature, the temperature of the sample can be maintained at an optimal temperature for the activity of a DNA polymerase without separation of a primer contained in the sample.
Here, because the sample that flow through the heating flow path 141B is heated from under, a convection flow of the sample flowing through the heating flow path 141B is easily induced as described above. Hence, the sample flowing through the heating flow path 141B can be easily cooled to an intermediate temperature owing to the convection flow thereof.
The sample may be retained in the heating flow path 141B until it is heated to an intermediate temperature (e.g., from 60° C. through 72° C.), by supplying of the sample from the opening 17A being stopped and by the membrane valves 18A to 18C being controlled to be opened or closed by stopping supplying of air in the flow path 12B through the syringes 93 provided into the openings 17B and 17C.
The sample that has passed the heating flow path 141B then passes the membrane valve 18A, and then flows into the guiding flow path 13. The process from the sample being supplied into the heating flow path 141A until the sample being expelled from the heating flow path 141B is defined as one cycle. The sample that has passed the heating flow path 141B, then passes the membrane valve 18A, and then passes through the guiding flow path 13, to be flowed again into the heating flow path 141A and circulated by one cycle or more in the same manner as described above. This causes the sample temperature to repeat reaching 90° C., 50° C., and 70° C. as illustrated in
After circulating through the flow path 12B by one cycle or more, the sample is expelled through the guiding flow path 13 into the syringe 93 from the opening 17A. The sample expelled into the syringe 93 (see
Hence, the flow path plate 10B for thermal cycle according to the present embodiment includes the two heating flow paths 141A and 141B, with the heating flow path 141B situated at the succeeding stage of the cooling flow path 143. The heating-target surface is situated under the heating flow paths 141A and 141B, and the cooling-target surface is situated above the cooling flow path 143. The flow path plate 10B for thermal cycle can induce a convection flow of a reagent in any of the heating flow paths 141A and 141B and the cooling flow path 143, not only when heating the sample in the heating flow path 141A or cooling the sample in the cooling flow path 143, but also when heating the sample in the heating flow path 141B by the third temperature adjuster 45. Hence, the flow path plate 10B for thermal cycle can easily make the reagent temperature uniform in any of these flow paths. Hence, the flow path plate 10B for thermal cycle, which can shorten the time taken until the temperature distribution in the heating flow paths 141A and 141B and the cooling flow path 143 becomes uniform, can shorten the sample processing time in the heating flow paths 141A and 141B and the cooling flow path 143.
The thermal cycle device 1B according to the present embodiment includes the flow path plate 10B for thermal cycle, the conveying member 20, the micro flow path 14B, the first temperature adjuster 30, the second temperature adjuster 40, and the third temperature adjuster 45. The micro flow path 14B includes the heating flow paths 141A and 141B, the connecting flow paths 142A and 142B, and the cooling flow path 143, with the first temperature adjuster 30 situated under the heating flow path 141A, the second temperature adjuster 40 situated above the cooling flow path 143, and the third temperature adjuster 45 situated under the heating flow path 141B. The thermal cycle device 1B can induce convection flows of a sample in the flow path 12B by heating or cooling, when heating the sample in the heating flow path 141A by the first temperature adjuster 30, cooling the sample in the cooling flow path 143 by the second temperature adjuster 40, and heating the sample in the heating flow path 141B by the third temperature adjuster 45. Hence, the thermal cycle device 1B, which can shorten the time taken until the temperature distribution in the flow path 12B becomes uniform, can shorten the sample processing time in the flow path 12B, like the thermal cycle device 1A.
The embodiments will be described more specifically below by way of Examples and Comparative Examples. The embodiments should not be construed as being limited to these Examples and Comparative Examples.
Three plate-shaped plates molded into a plate shape were prepared using a cycloolefin polymer (COP). Among them, one plate-shaped plate was prepared to have a thickness of 4 mm, and the remaining two plate-shaped plates were prepared to have a thickness of 0.188 mm. A lower groove was formed in the lower surface of the one plate-shaped plate, to produce a center base material. The lower groove was formed to have a depth of 1 mm, but to have a depth smaller than 1 mm at both ends of the groove and in the surrounding portions of the ends. The remaining two plate-shaped plates were used as a lower thin plate member and an upper thin plate member, respectively. Subsequently, an adhesive was applied to the junction surfaces between the center base material and each of the lower thin plate member and the upper thin plate member, and then thermocompression bonding was performed with the center base material sandwiched between the lower thin plate member and the upper thin plate member, to join the center base material and the lower thin plate member with each other, and join the center base material and the upper thin plate member with each other. In this way, a flow path plate for thermal cycle including: a flow path having a height of 1 mm in the lower surface of the center base material; and an inlet opening and an outlet opening on both ends of the flow path was produced. The flow path was formed to have a chamber shape in a cross-section taken in its longer axis direction.
The flow path plate for thermal cycle was placed on a hot plate to make the lower surface of the flow path plate for thermal cycle contact the hot plate, and, as illustrated in
A flow path plate for thermal cycle was produced in the same manner as in Example 1 except that a center base material was produced by forming the lower groove formed in the lower surface of the one plate-shaped plate in Example 1 in the upper surface of a plate-shaped plate as an upper groove.
The same as in Example 1 was performed except that unlike in Example 1, an aluminum block heater was placed on the upper surface of the flow path plate for thermal cycle to make the aluminum block heater contact the upper surface of the flow path plate for thermal cycle, and, as illustrated in
A flow path plate for thermal cycle was produced in the same manner as in Comparative Example 1.
In the same manner as in Comparative Example 1, an aluminum block heater to be used as the second temperature adjuster was placed on the upper surface of the flow path plate for thermal cycle to make the aluminum block heater contact the upper surface of the flow path plate for thermal cycle. The flow path plate for thermal cycle was placed on a hot plate. Then, as illustrated in
The aluminum block heater was heated to 50° C. to heat the upper surface of the flow path plate for thermal cycle to 50° C., and pure water (70° C.) was supplied into the flow path, to cool the pure water in the flow path. In order that the flow path plate for thermal cycle would not be cooled at anywhere other than the upper surface, the hot plate was heated to 70° C., to heat the lower surface of the flow path plate for thermal cycle to 70° C. The temperature measurements from the upper surface (the internal surface of the flow path on the aluminum block heater side) (the point B in
A flow path plate for thermal cycle was produced in the same manner as in Example 1.
In the same manner as in Example 1, the flow path plate for thermal cycle was placed on a hot plate to be used as the second temperature adjuster to make the lower surface of the flow path plate for thermal cycle contact the hot plate. An aluminum block heater was placed on the upper surface of the flow path plate for thermal cycle. Then, as illustrated in
The hot plate was heated to 50C to heat the lower surface of the flow path plate for thermal cycle to 50° C., and pure water (70° C.) was supplied into the flow path, to cool the pure water in the flow path. In order that the flow path plate for thermal cycle would not be cooled at anywhere other than the lower surface, the aluminum block heater was heated to 70° C., to heat the upper surface of the flow path plate for thermal cycle to 70° C. The temperature measurements from the lower surface (the internal surface of the flow path on the hot plate side) (the point B in
As indicated in
As indicated in
The embodiments have been described above. However, the embodiments described above have been presented as examples, and the present invention is not to be limited by the embodiments described above. The embodiments described above can be carried out in various other modes, and various combinations, omissions, replacements, modifications, and the like are applicable within the scope of the spirit of the invention. The embodiments and modifications thereof are included in the scope and spirit of the invention, and are included in the scope of the invention described in the claims and equivalents thereof.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2021-186649 | Nov 2021 | JP | national |
This application is a continuation application of International Application No. PCT/JP2022/038090, filed on Oct. 12, 2022, and designating the U.S., which is based upon and claims priority to Japanese Patent Application No. 2021-186649, filed on Nov. 16, 2021, the entire contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP2022/038090 | Oct 2022 | WO |
| Child | 18639578 | US |
