JP7341492B2 - Reaction treatment container, reaction treatment container manufacturing method, and reaction treatment method - Google Patents

Description

The present invention relates to a reaction processing container used for polymerase chain reaction (PCR).

Genetic testing is widely used in various medical fields, identification of agricultural products and pathogenic microorganisms, food safety evaluation, and even testing for pathogenic viruses and various infectious diseases. In order to detect minute amounts of DNA with high sensitivity, a method is known in which a portion of DNA is amplified and the resultant product is analyzed. Among them, a method using PCR is a technique that is attracting attention for selectively amplifying a certain part of a very small amount of DNA collected from a living body or the like.

PCR is a mixture of a biological sample containing DNA and a PCR reagent consisting of primers, enzymes, etc., which is subjected to a predetermined thermal cycle to repeatedly cause denaturation, annealing, and elongation reactions to generate a specific portion of DNA. This is selective amplification.

In PCR, it is common to place a predetermined amount of the target sample into a reaction processing container such as a PCR tube or a microplate (microwell) in which multiple holes are formed. It has been put into practical use to use a reaction processing container (also called a reaction processing chip) equipped with fine flow channels (for example, Patent Documents 1 to 3).

JP2009-232700A Japanese Patent Application Publication No. 2007-51881 Japanese Patent Application Publication No. 2007-285777

It is industrially advantageous to manufacture a reaction treatment container made of a substrate with fine flow channels as described above by injection molding. However, the present inventor recognized that there are the following problems when manufacturing such a reaction treatment container by injection molding.

The cross section of the flow path is approximately 1 mm or less than 1 mm in both width and depth, and is a flow path composed of curved lines or straight lines. In particular, in the region of the flow path (appropriately referred to as the "reaction region") where the temperature is set to a predetermined temperature level (for example, approximately 95°C or 55°C) by an external heater, multiple It is advantageous to have a combination of straight channels and curved channels including turns.

In the injection molding method, a substrate is molded by pouring resin into a mold with a shape that corresponds to these flow channels, but the uneven structure that is a combination of straight and curved channels is continuous. In the complicated parts corresponding to the flow paths, the high-speed flow of resin becomes complicated, as there is a large time difference between resin filling and air removal inside the mold. A so-called weld line may occur in a portion of the substrate corresponding to such a portion. If such a weld line occurs near the flow path of the substrate, a recessed portion called a "pit" with a depth of several μm to several tens of μm may occur at the portion where the weld line and the flow path connect or touch. If such pits exist in the flow path, the movement of the sample may be hindered, and there is a risk that the sample may stop, stagnate, or remain.

The present invention was made in view of these circumstances, and its purpose is to provide a reaction processing container in which a sample can move smoothly in a flow path by suppressing the occurrence of inappropriate weld lines during molding. Our goal is to provide the following.

In order to solve the above problems, a reaction processing container according to an embodiment of the present invention includes a substrate made of resin and a groove-shaped flow path formed on the main surface of the substrate. The channel includes a bottom surface and side surfaces. In a part of the flow path, the bottom surface and the side surface are connected by a curved surface.

The channel may include a reaction channel for causing a predetermined reaction in a sample flowing through the channel, and the bottom surface and the side surface of the reaction channel may be connected by a curved surface.

The reaction channel may include a meandering channel that is a combination of a curved channel and a straight channel.

The opening width of the reaction channel may be 0.6 mm to 1.1 mm, and the radius of curvature of the curved surface in the reaction channel may be 0.2 mm to 0.38 mm.

The depth of the reaction channel may be 0.55 mm to 0.95 mm, and the taper angle of the reaction channel may be 10° to 30°.

The flow path may include a detection flow path that is irradiated with excitation light in order to detect fluorescence from a sample flowing through the flow path, and the bottom surface of the detection flow path may be formed in a plane parallel to the main surface of the substrate. good.

The detection channel may be formed as a linear channel.

The substrate may include a gate near the detection channel.

The substrate may include a gate near the intersection of an assumed vertical line perpendicular to the detection channel and an edge of the substrate.

The substrate may include a gate near the intersection of the hypothetical parallel line extending the linear detection channel and the end of the substrate.

The bottom width of the detection channel may be 0.5 mm to 0.8 mm.

The depth of the detection channel may be 0.8 mm to 1.25 mm, and the taper angle of the detection channel may be 10° to 30°.

According to the present invention, it is possible to provide a reaction processing container in which a sample can move smoothly in a flow path by suppressing the occurrence of inappropriate weld lines during molding.

FIG. 2 is a plan view of a substrate included in a reaction processing container according to an embodiment of the present invention. 1 is a diagram for explaining a cross-sectional structure of a reaction processing container according to an embodiment of the present invention. FIG. 2 is a schematic diagram for explaining a reaction processing apparatus that can utilize a reaction processing container. FIG. 3 is a diagram showing an example of weld lines generated on a substrate of a conventional reaction processing container. FIG. 3 is a diagram showing an example of a cross section of a reaction channel according to an embodiment of the present invention. FIG. 7 is a diagram showing another example of a cross section of a reaction channel according to an embodiment of the present invention. FIGS. 7A and 7B are diagrams for explaining the flow of resin in the mold. FIG. 3 is a diagram showing an example of a cross section of a detection channel. FIG. 3 is a plan view of a substrate included in a reaction processing container according to another embodiment of the present invention. FIG. 3 is an enlarged view of the vicinity of the flow path of the substrate of the reaction processing container according to the embodiment of the present invention.

Hereinafter, a reaction processing container according to an embodiment of the present invention will be described. Identical or equivalent components, members, and processes shown in each drawing are designated by the same reference numerals, and redundant explanations will be omitted as appropriate. Further, the embodiments are illustrative rather than limiting the invention, and all features and combinations thereof described in the embodiments are not necessarily essential to the invention.

A reaction processing container according to an embodiment of the present invention includes a substrate, a sealing film attached to the substrate, and a filter. FIG. 1 is a plan view of a substrate included in a reaction processing container. FIG. 2 is a diagram for explaining the cross-sectional structure of the reaction processing container. It should be noted that FIG. 2 is a diagram for explaining the positional relationship between a flow path formed on a substrate, a film, and a filter, and is different from a cross-sectional view of an actual reaction processing container.

The reaction processing container 10 includes a resin substrate 14 in which a groove-shaped channel 12 is formed on the upper surface 14a, a channel sealing film 16, a first sealing film 18, and a channel sealing film 16 pasted on the upper surface 14a of the substrate 14. A second sealing film 19, a third sealing film 20, a fourth sealing film 21, and a fifth film (not shown) pasted on the lower surface 14b of the substrate 14, and a 1 filter 28 and a second filter 30.

The substrate 14 is preferably formed of a material that is stable against temperature changes and is not easily attacked by the sample solution used. Further, the substrate 14 is preferably formed of a material that has good moldability, good transparency and barrier properties, and low autofluorescence. As such a material, resins such as acrylic, polypropylene, and silicone, among which cyclic polyolefin resins are suitable.

A groove-shaped channel 12 is formed on the upper surface 14a of the substrate 14. In the reaction processing container 10, most of the channel 12 is formed in the shape of a groove exposed on the upper surface 14a of the substrate 14. This is to enable easy molding by injection molding using a metal mold. In order to seal this groove and utilize it as a flow path, a flow path sealing film 16 is pasted on the upper surface 14a of the substrate 14. An example of the dimensions of the flow path 12 is a width of 0.7 mm and a depth of 0.7 mm. Further, in order to produce the substrate industrially more advantageously by the injection molding method, the structure of the channel may include a so-called "pull taper" side surface having a certain angle with respect to the main surface of the substrate.

The channel sealing film 16 may have adhesiveness on one main surface, or may have a functional layer on one main surface that exhibits adhesiveness or adhesive properties when pressed, irradiated with energy such as ultraviolet rays, heated, etc. It has the function of being easily integrated with the upper surface 14a of the substrate 14 in close contact with the upper surface 14a of the substrate 14. The channel sealing film 16, including the adhesive, is desirably made of a material with low autofluorescence. Transparent films made of resins such as, but not limited to, cycloolefin polymers, polyesters, polypropylene, polyethylene or acrylics are suitable in this regard. Moreover, the channel sealing film 16 may be formed from plate-shaped glass or resin. In this case, since rigidity can be expected, it is useful for preventing warpage and deformation of the reaction processing container 10.

A first filter 28 is provided at one end 12a of the flow path 12. A second filter 30 is provided at the other end 12b of the flow path 12. A pair of first filters 28 and second filters 30 provided at both ends of the flow path 12 are provided so as not to interfere with amplification or detection of the target DNA by PCR, or to prevent the quality of the target DNA from deteriorating. Prevent contamination. The dimensions of the first filter 28 and the second filter 30 are such that they can fit into the filter installation space formed on the substrate 14 without any gaps.

A first air communication port 24 is formed in the substrate 14 and communicates with one end 12a of the flow path 12 via an air introduction path 29 and a first filter 28. Similarly, a second air communication port 26 is formed in the substrate 14 and communicates with the other end 12b of the flow path 12 via the air introduction path 31 and the second filter 30. A pair of first air communication ports 24 and second air communication ports 26 are formed so as to be exposed on the upper surface 14a of the substrate 14.

In this embodiment, as the first filter 28 and the second filter 30, filters having good low impurity properties, air permeability, and water or oil repellency are used. As the first filter 28 and the second filter 30, for example, a porous resin or a sintered body of resin is preferable, but examples of fluorine-containing resin include PTFE (polytetrafluoroethylene) and PFA (permeable resin). Examples include fluoroalkoxyalkane), FEP (perfluoroethylene propene copolymer), and ETFE (ethylene tetrafluoroethylene copolymer). Furthermore, as a filter made of PTFE (polytetrafluoroethylene), PF020 (both manufactured by Advantech Group), etc. can be used, although the filter is not limited to these. Furthermore, as the first filter 28 and the second filter 30, filters whose surfaces are coated with a fluorine-containing resin to make them water-repellent can also be used. Other filter materials include polyethylene, polyamide, polypropylene, etc., as long as they can prevent contamination of samples to be subjected to PCR, do not interfere with PCR, and do not allow air to pass through. It is preferable that the material has properties that allow liquid to pass through it and that it does not allow liquid to pass through, and its performance and material do not matter as long as it satisfies some of these requirements for the desired performance.

The flow path 12 includes a reaction region between a pair of first filters 28 and second filters 30 in which temperature can be controlled at a plurality of levels by a reaction processing device to be described later. By continuously moving the sample back and forth through a reaction zone where multiple levels of temperature are maintained, the sample can be subjected to a thermal cycle.

In this embodiment, the reaction region includes a high temperature region 36 and a medium temperature region 38. When the reaction processing container 10 is installed in the reaction processing apparatus described below, the high temperature region 36 is maintained at a relatively high temperature (for example, about 95° C.), and the medium temperature region 38 is maintained at a temperature lower than the high temperature region 36 (for example, about 62° C.). ) will be maintained. One end of the high temperature region 36 communicates with the second air communication port 26 via the second filter 30 and the air introduction path 31, and the other end communicates with the medium temperature region 38 via the connection channel 40. One end of the medium temperature region 38 communicates with the high temperature region 36 via a connection channel 40, and the other end communicates with a buffer channel (preparation channel) 39. One end of the buffer flow path 39 communicates with the medium temperature region 38, and the other end communicates with the first air communication port 24 via the first filter 28 and the air introduction path 29.

The high-temperature region 36 and the medium-temperature region 38 each include a meandering flow path that is a combination of a curved flow path and a straight flow path and is continuously folded back. When creating a meandering flow path in this way, it is possible to effectively use the limited effective area of heaters, etc. that make up the temperature control system described later, and it is easy to reduce temperature variations within the reaction area. In addition, there is an advantage that the actual size of the reaction processing container can be reduced, contributing to miniaturization of the reaction processing apparatus. Further, the buffer flow path 39 is also a meandering flow path. On the other hand, the connecting flow path 40 between the high temperature region 36 and the medium temperature region 38 is a straight flow path. The connection flow path 40 includes a region (“fluorescence detection region”) that is irradiated with excitation light in order to detect fluorescence from a sample flowing in the flow path when the reaction processing container 10 is installed in a reaction processing apparatus to be described later. ) 86 is set.

A branch point is provided in a part of the buffer channel 39, and a branch channel 42 branches from the branch point. A sample introduction port 44 is formed at the tip of the branch channel 42 so as to be exposed on the lower surface 14b of the substrate 14. The buffer channel 39 can be used as a temporary sample standby channel when the reaction processing container 10 is introduced into the reaction processing apparatus after a predetermined amount of the sample is introduced from the sample introduction port 44.

As shown in FIG. 2, the first sealing film 18 is attached to the upper surface 14a of the substrate 14 so as to seal the first air communication port 24. The second sealing film 19 is attached to the upper surface 14a of the substrate 14 so as to seal the second air communication port 26. The third sealing film 20 is attached to the lower surface 14b of the substrate 14 so as to seal the air introduction path 29 and the first filter 28. The fourth sealing film 21 is attached to the lower surface 14b of the substrate 14 so as to seal the air introduction path 31 and the second filter 30. A fifth sealing film (not shown) is attached to the lower surface 14b of the substrate 14 so as to seal the sample introduction port 44. As these sealing films, transparent films based on resins such as cycloolefin polymer, polyester, polypropylene, polyethylene, or acrylic can be used. When all the sealing films including the channel sealing film 16 are pasted, all the channels are closed spaces.

When connecting a liquid feeding system to be described later to the reaction processing container 10, the first sealing film 18 and the second sealing film 19, which seal the first air communication port 24 and the second air communication port 26, are Peel it off and connect the tube provided in the liquid delivery system to the first air communication port 24 and the second air communication port 26. Alternatively, the first sealing film 18 and the second sealing film 19 may be perforated with a hollow needle (syringe needle with a pointed tip) provided in the liquid delivery system. In this case, the first sealing film 18 and the second sealing film 19 are preferably made of a material and having a thickness that allows easy perforation with a needle.

Introducing the sample into the flow path 12 through the sample introduction port 44 is performed by first peeling off the fifth sealing film from the substrate 14, and after introducing a predetermined amount of the sample, the fifth sealing film is again attached to the bottom surface of the substrate 14. Paste it back to 14b. At this time, since the air inside the channel is pushed by the introduction of the sample, the second sealing film may be peeled off in advance in order to release the air. Therefore, as the fifth sealing film, it is desirable to use a film with adhesiveness that can withstand several cycles of pasting and peeling. Further, the fifth sealing film may be such that a new film is pasted after the sample is introduced, and in this case, the importance of characteristics regarding repeated pasting/peeling can be alleviated.

The method for introducing the sample into the sample introduction port 44 is not particularly limited, but an appropriate amount of sample may be directly introduced from the sample introduction port 44 using, for example, a pipette, dropper, syringe, or the like. Alternatively, it may be introduced through a needle tip with a built-in filter made of porous PTFE or polyethylene while preventing contamination. Such needle tips are generally sold in many types and are easily available, and can be used by being attached to the tip of a pipette, dropper, syringe, or the like. Further, after discharging or introducing the sample using a pipette, dropper, syringe, etc., the sample may be moved to a predetermined location in the channel 12 by further applying pressure and pushing.

Examples of the sample include a mixture containing one or more types of DNA to which a thermostable enzyme and four types of deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP) are added as PCR reagents. It will be done. In addition, primers that specifically react with the DNA to be subjected to reaction treatment, and in some cases, fluorescent probes such as TaqMan (TaqMan is a registered trademark of Roche Diagnostics Gesellschaft mit Beschlenchtel Haftsung) or SYBR Green (SYBR is a molecular probe) (registered trademark of S.Incorporated). Commercially available real-time PCR reagent kits and the like can also be used.

FIG. 3 is a schematic diagram for explaining a reaction processing apparatus 100 in which the reaction processing container 10 can be used, and in particular only a portion directly related to the reaction processing container 10 is conceptually extracted.

The reaction processing apparatus 100 includes a container installation part (not shown) in which the reaction processing container 10 is installed, a high temperature heater 104 for heating the high temperature region 36 of the flow path 12, and a medium temperature region 38 of the flow path 12. A medium temperature heater 106 for heating and a temperature sensor (not shown) such as a thermocouple for measuring the actual temperature in each temperature range are provided. Each heater may be, for example, a resistance heating element, a Peltier element, or the like. These heaters, a suitable heater driver (not shown), and a control device such as a microcomputer (not shown) control the high temperature region 36 in the flow path 12 of the reaction processing vessel 10 to approximately 95° C., and the medium temperature region 38 to approximately 62° C. ℃, and the temperature of each temperature region of the thermal cycle region is set.

The reaction processing apparatus 100 further includes a fluorescence detector 140. As described above, a predetermined fluorescent probe is added to the sample S. Since the intensity of the fluorescent signal emitted from the sample S increases as the amplification of the DNA progresses, the intensity value of the fluorescent signal can be used as an index as a material for determining the progress of PCR and its termination.

The fluorescence detector 140 is an optical fiber type fluorescence detector manufactured by Nippon Sheet Glass Co., Ltd., which has a very compact optical system, can perform measurements quickly, and can detect fluorescence regardless of whether it is in a bright or dark place. A device FLE-510 can be used. This optical fiber type fluorescence detector can tune the wavelength characteristics of its excitation light/fluorescence to be suitable for the fluorescence characteristics emitted by the sample S, and can provide the optimal optical and detection system for samples with various characteristics. Furthermore, the small diameter of the light beam provided by the fiber optic fluorescence detector makes it suitable for detecting fluorescence from samples present in small or narrow areas such as flow channels. He also has good speed.

The optical fiber type fluorescence detector 140 includes an optical head 142, a fluorescence detector driver 144, and an optical fiber 146 connecting the optical head 142 and the fluorescence detector driver 144. The fluorescence detector driver 144 includes an excitation light source (LED, laser, or other light source adjusted to emit a specific wavelength), an optical fiber multiplexer/demultiplexer, and a photoelectric conversion element (such as a PD, APD, or photomultiplexer). (photodetector) (none of which are shown), etc., and a driver for controlling these. The optical head 142 is composed of an optical system such as a lens, and has the functions of directional irradiation of excitation light onto the sample and collection of fluorescence emitted from the sample. The collected fluorescence is separated from excitation light by an optical fiber multiplexer/demultiplexer in the fluorescence detector driver 144 through an optical fiber 146, and converted into an electrical signal by a photoelectric conversion element. As the optical fiber type fluorescence detector, the one described in JP-A No. 2010-271060 can be used. The optical fiber type fluorescence detector can also be modified to enable detection of multiple wavelengths in a coaxial manner using a single optical head or multiple optical heads. Regarding the fluorescence detector related to multiple wavelengths and its signal processing, the invention described in International Publication No. 2014/003714 can be utilized.

In the reaction processing apparatus 100, the optical head 142 is arranged so as to be able to detect the fluorescence from the sample S in the fluorescence detection region 86 in the connection channel 40 that connects the high temperature region 36 and the medium temperature region 38. As the sample S is repeatedly moved back and forth within the flow channel, the reaction progresses and the predetermined DNA contained in the sample S is amplified. By monitoring the fluctuation in the intensity value of the detected fluorescence signal, the amplification of the DNA can be carried out. You can check the progress in real time. Furthermore, in the reaction processing apparatus 100, the output value from the fluorescence detector 140 is used to control the movement of the sample S. For example, the output value from the fluorescence detector 140 may be sent to the control device and used as a parameter when controlling the liquid feeding system described below. The fluorescence detector is not limited to an optical fiber type fluorescence detector as long as it exhibits the function of detecting fluorescence from a sample.

The reaction processing apparatus 100 further includes a liquid feeding system (not shown) for moving and stopping the sample S within the channel 12 of the reaction processing container 10. Although the liquid feeding system is not limited to this, by feeding (blowing) air from either the first air communication port 24 or the second air communication port 26, the sample S is transferred into the flow path 12. can be moved in one direction. Furthermore, the liquid delivery system can be stopped at a predetermined position by stopping the air flow to the channel or by equalizing the pressure on both sides of the sample S in the channel 12.

In the liquid feeding system, a syringe pump, a diaphragm pump, a blower, or the like can be used as a means (air blowing means) having an air blowing or pressurizing function. Further, as a device having a function of stopping the sample S at a predetermined position, a combination of a blowing means and a three-way valve (3-port valve) or the like can be used. For example, a first three-way valve and a second three-way valve are provided, the first port (common port) of the first three-way valve is connected to the first air communication port, and the second port is connected to the above-mentioned ventilation means. and connect each port so that the third port is open to atmospheric pressure, and connect the second three-way valve with its first port (common port) to the second air communication port, A possible embodiment is such that the ports are connected so that the second port is connected to the above-mentioned air blowing means and the third port is opened to atmospheric pressure. Specific aspects of these are described, for example, in JP-A-4-325080 and JP-A-2007-285777. For example, if a three-way valve connected to one of the air communication ports is operated so that the air blowing means and the air communication port communicate with each other, and if the three-way valve is connected to the other air communication port, The sample S is moved in one direction by operating the three-way valve so that its air communication port communicates with atmospheric pressure. Subsequently, the sample S is stopped by operating both three-way valves so that both air communication ports communicate with atmospheric pressure.

Further, the three-way valve and the liquid feeding means can be operated by the control device through an appropriate driver. In particular, the fluorescence detector 140 arranged as described above transmits an output value based on the obtained fluorescence signal to the control device, so that the position of the sample S in the flow channel 12 and its passage can be controlled by the control device. By making the recognition, the liquid feeding system consisting of the three-way valve and the liquid feeding means is controlled.

Therefore, by sequentially and continuously operating the three-way valves connected to both sides of the flow path 12, the sample S is continuously moved back and forth between the high temperature region 36 and the medium temperature region 38 within the flow path 12. movement, thereby making it possible to subject the sample S to an appropriate thermal cycle.

FIG. 4 shows an example of a weld line generated on a substrate of a conventional reaction processing container. The reaction treatment container 10 according to this embodiment is manufactured by injection molding. In the injection molding method, the substrate 14 is molded by pouring resin into a mold having a convex shape corresponding to the groove-shaped flow path 12. In complex parts that correspond to channels with a continuous uneven structure such as a combination of channels and curved channels, high-speed The resin flow becomes complicated. A so-called weld line may occur in a portion of the substrate corresponding to such a region due to contact or collision between resins flowing from different directions, or due to differences in arrival times of resins to a certain position.

FIG. 4 shows a weld line 50 actually formed on the substrate 14. In the example shown in FIG. 4, the weld line 50 noticeably occurs near the channel 12 of the substrate 14, and a pit 52 (a recess with a depth of several tens of μm ) is occurring. If such a pit 52 exists in the flow path 12, movement of the sample may be hindered, and there is a possibility that the sample may stop or stagnate. Furthermore, when the sample passes through the region of the channel 12 where the pits 52 have occurred, there is a risk that air (bubbles) will be drawn in. During the movement of the sample dozens of times, the air trapped in the sample increases its volume, and eventually the sample is cut into pieces, making it impossible for the sample to move within the flow path 12, resulting in PCR failure. There is a possibility that the reaction cannot be continued. Further, part of the sample may be trapped in the pit 52 and released into the flow path again, and in this case as well, there is a risk that the sample may be shredded. In particular, pits that occur in the flow path 12 belonging to the high temperature region 36 are highly likely to cause air entrainment based on experience. Therefore, in the reaction processing container 10 according to the present embodiment, a structure of the flow path 12 that can suppress the generation of weld lines is adopted.

FIG. 5 shows an example of a cross section of a channel (hereinafter referred to as a "reaction channel") belonging to a reaction region such as a high temperature region or a medium temperature region. The reaction channel 60 shown in FIG. 5 includes a partially planar bottom surface 62 and side surfaces 64 located on both sides of the bottom surface 62. In the reaction channel 60, as shown in FIG. 5, a bottom surface 62 and a side surface 64 are connected by a curved surface 66. That is, the connecting portion between the bottom surface 62 and the side surface 64 has a curved shape. Parameters that define the shape and dimensions of the reaction channel 60 include the opening width W of the channel, the depth D of the channel, the taper angle Ta of the side surface 64, and the radius of curvature R of the curved surface 66. As described above, the opening of the reaction channel 60 is exposed to the main surface 14a of the substrate 14. The opening width W is the width of the flow path on the main surface 14a. The depth D of the flow path is the maximum depth of the flow path from the main surface 14a. The radius of curvature R of the curved surface 66 is the radius when the cross section of the curved surface that forms the connection between the bottom surface 62 and the side surface 64 is approximated by a circle. The taper angle Ta is the angle formed by the side surface 64. The taper of the side surface 64 can act as a punching taper when the substrate is molded by injection molding, and is advantageous in industrial production. The depth D of the channel is 0.55 mm to 0.95 mm, and the opening width W of the channel is 0.6 mm to 1.1 mm. These dimensions of the flow path are within the appropriate range of dimensions based on experience for performing thermal cycles on actual samples. The depth D of the channel is preferably 0.65 mm to 0.85 mm, and the opening width W of the channel is preferably 0.65 mm to 0.9 mm. The taper angle Ta is 10° to 30°. When the taper angle Ta is within this range, it becomes an appropriate draft angle when manufacturing the substrate 14 by the injection molding method. The taper angle Ta is preferably 15° to 25°.

FIG. 6 shows another example of the cross section of the reaction channel. The reaction channel 70 shown in FIG. 6 includes a curved bottom surface 72 and side surfaces 74 located on both sides of the bottom surface 72. The reaction channel 70 is a channel having a substantially U-shaped cross section, and as shown in FIG. 6, a bottom surface 72 and a side surface 74 are connected by a curved surface 76. Also in this example, the connecting portion between the bottom surface 72 and the side surface 74 is curved. In this example, the radius of curvature of the bottom surface 72 and the radius of curvature R of the curved surface 76 are the same, but they may be different. The parameters and ranges that define the shape and dimensions of the reaction channel 70 are similar to those of the reaction channel 60 shown in FIG.

FIGS. 7A and 7B are diagrams for explaining the flow of resin inside a mold in the injection molding method. This is a portion where a convex portion of a mold corresponding to a flow path crosses the direction in which resin flows, and is a schematic view taken from a direction representing a cross section of the flow path. FIG. 7A shows how resin flows into the mold 80 for molding the substrate of the reaction processing container 10 according to this embodiment. The mold 80 includes a convex portion 82 for molding the reaction channel of the substrate of the reaction processing container 10 . The convex portion 82 has a curved shape at the connection portion between the bottom surface and the side surface of the flow path, so that the convex portion 82 has a shape that changes gently in the corresponding portion. Therefore, the resin flows continuously without interruption, and as a result, the resin filling near the reaction channel becomes smooth with less resistance, reducing the probability of undesirable weld lines. or the probability of occurrence of noticeable or clear weld lines with recesses such as the aforementioned pits can be reduced. The radius of curvature R of the curved surface is 0.2 mm to 0.38 mm, preferably 0.3 mm to 0.35 mm, and the radius of curvature R of the curved surface is within these ranges, considering the width and depth of the flow path. Preferably, there is no sharp bend between the bottom and side surfaces.

Furthermore, the reaction channel 60 is a meandering channel consisting of a straight channel and a curved channel, as shown in FIG. It is also important here that the radius of curvature of the curved flow path is not too small. If the radius of curvature of the curved flow path is too small, resin will not be filled smoothly into the portion surrounded by such a small curve during injection molding of the substrate 14. The radius of curvature of the curved channel is one or more times the opening width W in the reaction channel 60. The radius of curvature of the curved channel is the radius of curvature of the channel centerline passing through the center of the channel in the width direction.

Further, as shown in FIG. 1, the radius of curvature of the curved flow path in the reaction flow path 60 may change or may remain the same as the distance from the center of the high temperature region 36 or the medium temperature region 38 increases. For example, in the reaction channel 60 shown in FIG. 1, when the radius of curvature of the curved channel is R1, R2, R3, and R4 from the inside to the outside, the curved channel is arranged so that R1<R2<R3<R4. may be configured. R1 to R4 may be 1 to 6 times the opening width W in the reaction channel 60. The radius of curvature of the curved channels other than the curved channels indicated by R1 to R4 in the reaction channel 60 may be equal to the radius of curvature of any one of R1 to R4. Increasing the radius of curvature of the curved channel appropriately relative to the opening width is advantageous from the viewpoint of resin filling during injection molding as described above, and also when the sample is being moved within the channel. , it is possible to suppress the deceleration of the sample at a bent part such as a curved channel, and it is possible to keep the moving speed of the sample constant. On the other hand, if the radius of curvature of the curved channel is too large, the volume of the channel in the reaction region will decrease, making it impossible to contain a predetermined amount of sample. For example, with respect to the opening width W of the reaction channel 60, R1 to R4 may be all or partially equal to 1×W to 6×W, R1=1×W to 3×W, R2 =1.5×W to 3.5×W, R3=2.5×W to 4.5×W, and R4=4×W to 6×W. Furthermore, clarify the difference between R1 to R4, R1 = 1 × W to 2 × W, R2 = 2 × W to 3 × W, R3 = 3 × W to 4 × W, R4 = 4.5 × W to 5 ×W may be used. In such a case, it is possible to suppress a clear weld line on the substrate 14, and it is expected that the moving speed of the sample when moving through the reaction channel 60 will be kept approximately constant, and it will also be possible to suppress the formation of a clear weld line on the substrate 14. It can contain as much sample as needed.

On the other hand, FIG. 7(b) shows how resin flows into a mold 85 for molding a substrate of a reaction processing container according to a comparative example. The substrate of the reaction processing container according to this comparative example includes a reaction channel having a trapezoidal cross section. The mold 85 includes a convex portion 88 for molding a trapezoidal reaction channel. The convex portion 88 has an angular shape at the connection portion between the bottom surface and the side surface of the flow path, so that the convex portion 88 has a shape that changes sharply at the corresponding portion. Therefore, it is suggested that a sudden change in the Reynolds number causes a change in the flow velocity during resin filling, resulting in a turbulent flow, increasing the probability that an undesirable weld line will occur in the vicinity of the reaction flow path.

As described above, according to the reaction processing container 10 according to the present embodiment, the connecting portion between the bottom surface and the side surface in the reaction channel is formed into a curved shape, so that when pouring the resin into the mold, the area near the reaction channel is Filling with resin becomes smooth. As a result, it is possible to reduce the probability that undesirable weld lines will occur near the reaction channel, thereby preventing pits and other recesses from forming in the area facing the reaction channel, and allowing the sample to flow inside the reaction channel. A reaction processing container that can be moved smoothly can be realized.

Further, according to the reaction processing container 10 according to the present embodiment, it is possible to prevent pits from forming in the reaction region, so that it is possible to avoid entrainment of air when the sample passes through the pits, and it is possible to suitably perform PCR. reactions can be carried out.

FIG. 8 shows an example of a cross section of a channel (hereinafter referred to as a "detection channel") belonging to the fluorescence detection region. The detection channel 90 includes a planar bottom surface 92 and side surfaces 94 located on both sides of the bottom surface 92. The side surfaces may have a tapered angle. A bottom surface 92 of the detection channel 90 is formed in a plane parallel to the main surface (upper surface 14a, lower surface 14b) of the substrate 14. Further, when the reaction processing container 10 is introduced into the reaction processing apparatus 100, the optical head 142 of the fluorescence detector 140 is arranged so that its optical axis is substantially perpendicular to the bottom surface 92 and the main surface of the substrate 14. . By arranging them in this way, it is possible to suppress undesirable refraction and reflection of the excitation light irradiated onto the sample from the optical head 142 or the fluorescence emitted from the sample, and it becomes possible to detect stable fluorescence intensity. .

Furthermore, in the detection channel 90, a planar bottom surface 92 and side surfaces 94 are directly connected. That is, the connecting portion 95 between the bottom surface 92 and the side surface 94 is not curved but has an angular shape. The intensity of fluorescence emitted from the sample and used for detection increases as the depth of the channel increases. Therefore, if the connecting portion 95 or part of the bottom surface 92 is curved, the depth of the channel becomes substantially smaller. Since the intensity is not constant, there is a possibility that the fluorescence intensity for detection may vary among individual reaction processing vessels 10. On the other hand, the cross-sectional area of the detection channel may be the same as or different from the cross-sectional area of the reaction channel described above. The cross-sectional area is determined by the width and depth of the channel. The speed of sample movement varies depending on the cross-sectional area of the channel, so this effect can be used to make the speed of the sample when moving through the reaction channel greater than the speed of the sample when moving through the detection channel. or vice versa.

Parameters that define the shape and dimensions of the detection channel 90 include the opening width W1, the channel depth Db, the bottom width W2, and the taper angle Tb of the side surface 94. The opening width W1 is the width of the flow path on the main surface 14a. The depth Db of the flow path is the maximum depth of the flow path from the main surface 14a. The bottom surface width W2 is the width of the bottom surface 92. The taper angle Tb is the angle formed by the side surface 94. The opening width W1 is 0.8 mm to 1.2 mm. This dimension is within a range of dimensions that are empirically appropriate for performing thermal cycles on actual samples. The opening width W1 of the detection channel 90 is preferably 0.9 mm to 1.1 mm. The depth Db of the detection channel 90 is 0.8 mm to 1.25 mm. This dimension is within a range of dimensions that are empirically appropriate for performing a thermal cycle on an actual sample, and the value of fluorescence intensity related to detection is sufficiently large, making it possible to improve the S/N ratio. The depth Db of the detection channel 90 is preferably 0.9 mm to 1.1 mm. The bottom width W2 of the detection channel 90 is 0.5 mm to 0.8 mm. This dimension is within a range of dimensions that are empirically appropriate for performing thermal cycles on actual samples. The bottom width W2 of the detection channel 90 is preferably 0.55 mm to 0.7 mm. The taper angle Tb of the detection channel 90 is 10° to 30°. When the taper angle Tb is within this range, it becomes an appropriate draft angle when manufacturing the substrate 14 by the injection molding method. The taper angle Tb of the detection channel 90 is preferably 15° to 25°.

It is preferable that the detection channel 90 is formed in a straight channel. By forming a straight channel, when pouring the resin into the mold, the filling of the resin is smoother than in the case of a meandering channel such as a reaction channel, so the flow path near the detection channel 90 is The occurrence of weld lines is suppressed, and the occurrence of pits in the detection channel 90 can be prevented.

Returning to FIG. 1, the substrate 14 of the reaction processing container 10 includes a gate 17 near the detection channel 90. A "gate" is an inlet for injecting resin into a mold when a substrate is manufactured by injection molding, and is an essential structure when obtaining a resin product using this type of method. As shown in FIG. 1, it is desirable that the gate 17 be arranged so that no other flow path exists between the gate 17 and the detection flow path 90. By arranging the gate 17 in this way, the resin injected from the gate 17 does not have to pass through a continuous portion of the uneven structure before reaching the portion corresponding to the detection flow path, and the resin injected from the gate 17 can be delivered in a relatively short time. Since it is possible to reach the detection channel 90 or its vicinity in a short period of time (or a short distance), the complexity of the resin flow can be avoided, and the occurrence of weld lines in the vicinity of the detection channel 90 can be further suppressed. can.

In FIG. 1, the gate 17 is formed directly above the detection channel 90 and near the upper side of the substrate on the paper. More specifically, when assuming a "hypothetical vertical line VV" perpendicular to the linear detection channel 90, the gate 17 is located near the intersection P1 of the hypothetical vertical line VV and the upper end of the substrate 14. is formed. Alternatively, the gate 17 may be formed directly below the detection channel 90 and near the lower side of the substrate on the paper. More specifically, the gate 17 (indicated by a broken line) may be formed near the intersection P2 between the hypothetical vertical line VV and the lower end of the substrate 14. By forming it at such a position, it is possible to reach the detection channel 90 or the vicinity thereof in a relatively short time (or short distance) as described above, so that the generation of weld lines can be effectively suppressed. I can do it. Although the gate 17 is located on the main surface of the substrate 14 in FIG. 1, the gate 17 may be located on the end surface of the substrate 14.

FIG. 9 is a plan view of the substrate 14 included in the reaction processing container 110 according to another embodiment of the present invention. As shown in FIG. 9, when assuming a "hypothetical parallel line HH" that is an extension of the linear detection flow path 90, a point near the intersection P3 of the hypothetical parallel line HH and the left end of the substrate 14. A gate 17 may also be formed. Alternatively, the gate 17 (indicated by a broken line) may be formed near the intersection P4 between the hypothetical parallel line HH and the right end of the substrate 14. In these cases, during injection molding, the direction toward the filling of the resin injected from the gate and the direction of the linear detection channel 90 substantially match, so that the resin flows into the metal corresponding to the detection channel 90. It does not cross the convex part of the mold. As a result, the resin is filled along the direction of the linear detection channel 90, so the probability of weld lines occurring can be reduced.

Here, analysis results of resin fluidity during injection molding of the substrate 14 will be explained. In analyzing the flow of the resin, Moldflow (manufactured by Autodesk) was used as software. A channel having a shape in which the bottom and side surfaces are connected by a curved surface as shown in FIG. 6 was used as an example, and the resin used was a cyclic polyolefin resin. The gate was set at the position shown in FIG. Further, the depth D of the channel was set to 0.75 mm, the taper angle Ta was set to 20°, the opening width W was set to 0.81 mm, and the radius of curvature R of the curved surface was set to 0.325 mm. Furthermore, as a comparative example, a trapezoidal channel as shown in FIG. 8 was also analyzed. The flow path of the comparative example had a depth D of 0.7 mm, a taper angle Ta of 20°, an opening width W1 of 0.75 mm, and a bottom width W2 of 0.50 mm.

As a result of the resin flow analysis, it was possible to fill the entire substrate in both the Example and the Comparative Example, but a difference was observed in the resin filling time in the vicinity of the reaction flow path. In the example, filling of the resin into the region near the reaction channel was smooth, and it did not take much time to fill the entire region near the reaction channel. In addition, the occurrence of voids and air pockets that cause weld lines was also suppressed.

On the other hand, in the comparative example, formation of voids and air pockets was observed in some areas from the time the resin reached the area near the reaction flow path. It was found that it became smaller as the filling time progressed, and that it took a relatively long time to fill the entire area near the reaction channel. Furthermore, in the comparative example, a boundary line between the voids and the resin that was being filled was clearly observed, and it was found that this could be a weld line. From this analysis result, the superiority of the channel shape according to the example was confirmed.

FIG. 10 is an enlarged view of the vicinity of the flow path 12 of the substrate 14 of the reaction processing container 10 according to the present embodiment, observed with a microscope. Although it can be seen that a slight weld line 50 has been formed, it is not a clear weld line like the one that occurs in the conventional substrate shown in FIG. could also be suppressed. In the substrate 14 of the reaction processing container 10 according to this embodiment, an improvement effect was clearly seen.

The present invention has been described above based on the embodiments. Those skilled in the art will understand that this embodiment is merely an example, and that various modifications can be made to the combinations of the constituent elements and processing processes, and that such modifications are also within the scope of the present invention. be.

10, 110 reaction treatment container, 12 channel, 14 substrate, 16 channel sealing film, 17 gate, 18 first sealing film, 19 second sealing film, 20 third sealing film, 21 fourth sealing film, 24 first air communication port, 26 second air communication port, 28 first filter, 29, 31 air introduction channel, 40 connection channel, 30 second filter, 36 high temperature region, 38 medium temperature region, 39 buffer channel , 42 branch channel, 44 sample introduction port, 50 weld line, 52 pit, 60, 70 reaction channel, 62, 72, 92 bottom surface, 64, 74, 94 side surface, 66, 76 curved surface, 80, 85 mold, 82, 88 convex portion, 86 fluorescence detection region, 90 detection channel, 95 connection portion, 100 reaction processing device, 104 high temperature heater, 106 medium temperature heater, 140 fluorescence detector, 142 optical head, 144 fluorescence detector driver, 146 Optical fiber.

Claims (13)

A substrate made of resin,
a groove-shaped channel formed on the main surface of the substrate;
a gate formed on the main surface of the substrate;
Equipped with
The flow path includes a meandering flow path that is a combination of a curved flow path and a straight flow path,
The meandering flow path includes a bottom surface, a side surface, and a curved surface connecting the bottom surface and the side surface,
A pair of air communication ports communicating with the flow path are formed in the substrate,
The reaction processing container is characterized in that the gate is formed between the pair of air communication ports . The substrate includes at least one side,
The reaction processing container according to claim 1, wherein the gate is formed near one side of the substrate. The flow path includes a linear detection flow path,
3. The reaction processing container according to claim 1 , wherein the gate is formed near an intersection between a hypothetical vertical line perpendicular to the linear detection channel and one side of the substrate. 4. The reaction processing container according to claim 3 , wherein a bottom surface of the linear detection channel is formed in a plane parallel to a main surface of the substrate. The flow path includes a reaction flow path in which a thermal cycle is applied to the sample by repeatedly reciprocating the sample to cause a predetermined reaction in the sample,
The reaction channel includes the meandering channel,
The reaction channel includes a first region and a second region maintained at different temperatures,
5. The reaction processing container according to claim 3 , wherein the first region and the second region are connected by the linear detection channel. 6. The reaction processing container according to claim 5 , wherein the substrate has a weld line near the reaction channel. 7. The reaction processing container according to claim 6 , wherein the weld line and a part of the reaction channel intersect. 8. The reaction processing container according to claim 7 , wherein there is no recess in the reaction channel near the intersection of the weld line and a part of the reaction channel. The depth of the reaction channel is 0.55 mm to 0.95 mm, and the taper angle of the reaction channel is 10° to 30°, according to any one of claims 5 to 8 . reaction treatment vessel. The reaction processing container according to any one of claims 3 to 9 , wherein the linear detection channel has a bottom width of 0.5 mm to 0.8 mm. 11. The linear detection channel has a depth of 0.8 mm to 1.25 mm, and a taper angle of the linear detection channel is 10° to 30°. The reaction treatment vessel described. A method for manufacturing a reaction treatment container according to any one of claims 1 to 11 , comprising:
A method for manufacturing a reaction processing container, comprising the step of injecting resin from the gate into a mold having a convex shape corresponding to the groove-like flow path. A reaction treatment method comprising using the reaction treatment container according to any one of claims 1 to 11 .

Images